@misc{yaschenko_fenech_mazzoni-putman_alonso_stepanova_2022, title={Deciphering the molecular basis of tissue-specific gene expression in plants: Can synthetic biology help?}, volume={68}, ISSN={["1879-0356"]}, url={https://doi.org/10.1016/j.pbi.2022.102241}, DOI={10.1016/j.pbi.2022.102241}, abstractNote={Gene expression differences between distinct cell types are orchestrated by specific sets of transcription factors and epigenetic regulators acting upon the genome. In plants, the mechanisms underlying tissue-specific gene activity remain largely unexplored. Although transcriptional and epigenetic profiling of individual organs, tissues, and more recently, of single cells can easily detect the molecular signatures of different biological samples, how these unique cell identities are established at the mechanistic level is only beginning to be decoded. Computational methods, including machine learning, used in combination with experimental approaches, enable the identification and validation of candidate cis-regulatory elements driving cell-specific expression. Synthetic biology shows great promise not only as a means of testing candidate DNA motifs but also for establishing the general rules of nature driving promoter architecture and for the rational design of genetic circuits in research and agriculture to confer tissue-specific expression to genes or molecular pathways of interest.}, journal={CURRENT OPINION IN PLANT BIOLOGY}, publisher={Elsevier BV}, author={Yaschenko, Anna E. and Fenech, Mario and Mazzoni-Putman, Serina and Alonso, Jose M. and Stepanova, Anna N.}, year={2022}, month={Aug} }
 @article{mallery_yanagisawa_zhang_lee_robles_alonso_szymanski_2022, title={Tandem C2 domains mediate dynamic organelle targeting of a DOCK family guanine nucleotide exchange factor}, volume={135}, ISSN={["1477-9137"]}, url={https://doi.org/10.1242/jcs.259825}, DOI={10.1242/jcs.259825}, abstractNote={Multicellular organisms use dedicator of cytokinesis (DOCK) family guanine nucleotide exchange factors (GEFs) to activate Rac/Rho-of-plants small GTPases and coordinate cell shape change. In developing tissues, DOCK signals integrate cell-cell interactions with cytoskeleton remodeling, and the GEFs cluster reversibly at specific organelle surfaces to orchestrate cytoskeletal reorganization. The domain organizations among DOCK orthologs are diverse, and the mechanisms of localization control are poorly understood. Here, we use combinations of transgene complementation and live-cell imaging assays to uncover an evolutionarily conserved and essential localization determinant in the DOCK-GEF named SPIKE1. The SPIKE1-DHR3 domain is sufficient for organelle association in vivo, and displays a complicated lipid-binding selectivity for both phospholipid head groups and fatty acid chain saturation. SPIKE1-DHR3 is predicted to adopt a C2-domain structure and functions as part of a tandem C2 array that enables reversible clustering at the cell apex. This work provides mechanistic insight into how DOCK GEFs sense compositional and biophysical membrane properties at the interface of two organelle systems.}, number={7}, journal={JOURNAL OF CELL SCIENCE}, publisher={The Company of Biologists}, author={Mallery, Eileen L. and Yanagisawa, Makoto and Zhang, Chunhua and Lee, Youngwoo and Robles, Linda M. and Alonso, Jose M. and Szymanski, Daniel B.}, year={2022}, month={Apr} }
 @article{chen_bullock_alonso_stepanova_2022, title={To Fight or to Grow: The Balancing Role of Ethylene in Plant Abiotic Stress Responses}, volume={11}, ISSN={["2223-7747"]}, url={https://doi.org/10.3390/plants11010033}, DOI={10.3390/plants11010033}, abstractNote={Plants often live in adverse environmental conditions and are exposed to various stresses, such as heat, cold, heavy metals, salt, radiation, poor lighting, nutrient deficiency, drought, or flooding. To adapt to unfavorable environments, plants have evolved specialized molecular mechanisms that serve to balance the trade-off between abiotic stress responses and growth. These mechanisms enable plants to continue to develop and reproduce even under adverse conditions. Ethylene, as a key growth regulator, is leveraged by plants to mitigate the negative effects of some of these stresses on plant development and growth. By cooperating with other hormones, such as jasmonic acid (JA), abscisic acid (ABA), brassinosteroids (BR), auxin, gibberellic acid (GA), salicylic acid (SA), and cytokinin (CK), ethylene triggers defense and survival mechanisms thereby coordinating plant growth and development in response to abiotic stresses. This review describes the crosstalk between ethylene and other plant hormones in tipping the balance between plant growth and abiotic stress responses.}, number={1}, journal={PLANTS-BASEL}, author={Chen, Hao and Bullock, David A., Jr. and Alonso, Jose M. and Stepanova, Anna N.}, year={2022}, month={Jan} }
 @article{mcfarlane_mutwil-anderwald_verbancic_picard_gookin_froehlich_chakravorty_trindade_alonso_assmann_et al._2021, title={A G protein-coupled receptor-like module regulates cellulose synthase secretion from the endomembrane system in Arabidopsis}, volume={56}, ISSN={["1878-1551"]}, DOI={10.1016/j.devcel.2021.03.031}, abstractNote={Cellulose is produced at the plasma membrane of plant cells by cellulose synthase (CESA) complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked to the plasma membrane. Because CESAs are only active in the plasma membrane, control of CSC secretion regulates cellulose synthesis. We identified members of a family of seven transmembrane domain-containing proteins (7TMs) that are important for cellulose production during cell wall integrity stress. 7TMs are often associated with guanine nucleotide-binding (G) protein signaling and we found that mutants affecting the Gβγ dimer phenocopied the 7tm mutants. Unexpectedly, the 7TMs localized to the Golgi/trans-Golgi network where they interacted with G protein components. Here, the 7TMs and Gβγ regulated CESA trafficking but did not affect general protein secretion. Our results outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses to cell wall integrity stress.}, number={10}, journal={DEVELOPMENTAL CELL}, author={McFarlane, Heather E. and Mutwil-Anderwald, Daniela and Verbancic, Jana and Picard, Kelsey L. and Gookin, Timothy E. and Froehlich, Anja and Chakravorty, David and Trindade, Luisa M. and Alonso, Jose M. and Assmann, Sarah M. and et al.}, year={2021}, month={May}, pages={1484-+} }
 @article{mazzoni-putman_brumos_zhao_alonso_stepanova_2021, title={Auxin Interactions with Other Hormones in Plant Development}, volume={13}, ISSN={1943-0264}, url={http://dx.doi.org/10.1101/cshperspect.a039990}, DOI={10.1101/cshperspect.a039990}, abstractNote={Auxin is a crucial growth regulator that governs plant development and responses to environmental perturbations. It functions at the heart of many developmental processes, from embryogenesis to organ senescence, and is key to plant interactions with the environment, including responses to biotic and abiotic stimuli. As remarkable as auxin is, it does not act alone, but rather solicits the help of, or is solicited by, other endogenous signals, including the plant hormones abscisic acid, brassinosteroids, cytokinins, ethylene, gibberellic acid, jasmonates, salicylic acid, and strigolactones. The interactions between auxin and other hormones occur at multiple levels: hormones regulate one another's synthesis, transport, and/or response; hormone-specific transcriptional regulators for different pathways physically interact and/or converge on common target genes; etc. However, our understanding of this crosstalk is still fragmentary, with only a few pieces of the gigantic puzzle firmly established. In this review, we provide a glimpse into the complexity of hormone interactions that involve auxin, underscoring how patchy our current understanding is.}, number={10}, journal={Cold Spring Harbor Perspectives in Biology}, publisher={Cold Spring Harbor Laboratory}, author={Mazzoni-Putman, Serina M. and Brumos, Javier and Zhao, Chengsong and Alonso, Jose M. and Stepanova, Anna N.}, year={2021}, month={Apr}, pages={a039990} }
 @misc{zhao_yaschenko_alonso_stepanova_2021, title={Leveraging synthetic biology approaches in plant hormone research}, volume={60}, ISSN={["1879-0356"]}, DOI={10.1016/j.pbi.2020.10998}, journal={CURRENT OPINION IN PLANT BIOLOGY}, author={Zhao, Chengsong and Yaschenko, Anna and Alonso, Jose M. and Stepanova, Anna N.}, year={2021}, month={Apr} }
 @article{brumos_zhao_gong_soriano_patel_perez-amador_stepanova_alonso_2020, title={An Improved Recombineering Toolset for Plants}, url={https://doi.org/10.1105/tpc.19.00431}, DOI={10.1105/tpc.19.00431}, abstractNote={Abstract Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing have made it possible to directly modify the target genes in their native chromosomal locations, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the results obtained by direct chromosomal tagging using genome-editing strategies. These simple, scalable, customizable recombineering toolsets and protocols allow a variety of genetic modifications to be generated. In addition, we developed a highly efficient recombinase-mediated cassette exchange system to facilitate the transfer of the desired sequences from a bacterial artificial chromosome clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis (Arabidopsis thaliana). We demonstrated the utility of this system by generating more than 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes and characterizing the translational reporter expression patterns for 14 auxin biosynthesis genes.}, journal={The Plant Cell}, author={Brumos, Javier and Zhao, Chengsong and Gong, Yan and Soriano, David and Patel, Arjun P. and Perez-Amador, Miguel A. and Stepanova, Anna N. and Alonso, Jose M.}, year={2020}, month={Jan} }
 @article{bagley_stepanova_ekelof_alonso_muddiman_2020, title={Development of a relative quantification method for infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging of Arabidopsis seedlings}, volume={34}, ISSN={["1097-0231"]}, DOI={10.1002/rcm.8616}, abstractNote={Mass spectrometry imaging of young seedlings is an invaluable tool in understanding how mutations affect metabolite accumulation in plant development. However, due to numerous biological considerations, established methods for the relative quantification of analytes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging are not viable options. In this study, we report a method for the quantification of auxin-related compounds using stable-isotope-labelled (SIL) indole-3-acetic acid (IAA) doped into agarose substrate.Wild-type Arabidopsis thaliana seedlings, sur2 and wei8 tar2 loss-of-function mutants, and YUC1 gain-of-function line were grown for 3 days in the dark in standard growth medium. SIL-IAA was doped into a 1% low-melting-point agarose gel and seedlings were gently laid on top for IR-MALDESI imaging with Orbitrap mass spectrometry analysis. Relative quantification was performed post-acquisition by normalization of auxin-related compounds to SIL-IAA in the agarose. Amounts of auxin-related compounds were compared between genotypes to distinguish the effects of the mutations on the accumulation of indolic metabolites of interest.IAA added to agarose was found to remain stable, with repeatability and abundance features of IAA comparable with those of other compounds used in other methods for relative quantification in IR-MALDESI analyses. Indole-3-acetaldoxime was increased in sur2 mutants compared with wild-type and other mutants. Other auxin-related metabolites were either below the limits of quantification or successfully quantified but showing little difference among mutants.Agarose was shown to be an appropriate sampling surface for IR-MALDESI mass spectrometry imaging of Arabidopsis seedlings. SIL-IAA doping of agarose was demonstrated as a viable technique for relative quantification of metabolites in live seedlings or tissues with similar biological considerations.}, number={6}, journal={RAPID COMMUNICATIONS IN MASS SPECTROMETRY}, author={Bagley, M. Caleb and Stepanova, Anna N. and Ekelof, Mans and Alonso, Jose M. and Muddiman, David C.}, year={2020}, month={Mar} }
 @article{dolores gomez_barro-trastoy_fuster-almunia_tornero_alonso_perez-amador_2020, title={Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis}, volume={71}, ISSN={["1460-2431"]}, DOI={10.1093/jxb/eraa395}, abstractNote={Abstract Ovule development is essential for plant survival, as it allows correct embryo and seed development upon fertilization. The female gametophyte is formed in the central area of the nucellus during ovule development, in a complex developmental programme that involves key regulatory genes and the plant hormones auxins and brassinosteroids. Here we provide novel evidence of the role of gibberellins (GAs) in the control of megagametogenesis and embryo sac development, via the GA-dependent degradation of RGA-LIKE1 (RGL1) in the ovule primordia. YPet-rgl1Δ17 plants, which express a dominant version of RGL1, showed reduced fertility, mainly due to altered embryo sac formation that varied from partial to total ablation. YPet-rgl1Δ17 ovules followed normal development of the megaspore mother cell, meiosis, and formation of the functional megaspore, but YPet-rgl1Δ17 plants had impaired mitotic divisions of the functional megaspore. This phenotype is RGL1-specific, as it is not observed in any other dominant mutants of the DELLA proteins. Expression analysis of YPet-rgl1Δ17 coupled to in situ localization of bioactive GAs in ovule primordia led us to propose a mechanism of GA-mediated RGL1 degradation that allows proper embryo sac development. Taken together, our data unravel a novel specific role of GAs in the control of female gametophyte development.}, number={22}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={Dolores Gomez, Maria and Barro-Trastoy, Daniela and Fuster-Almunia, Clara and Tornero, Pablo and Alonso, Jose M. and Perez-Amador, Miguel A.}, year={2020}, month={Dec}, pages={7059–7072} }
 @article{barro‐trastoy_carrera_baños_palau‐rodríguez_ruiz‐rivero_tornero_alonso_lópez‐díaz_gómez_pérez‐amador_2020, title={Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms}, url={https://doi.org/10.1111/tpj.14684}, DOI={10.1111/tpj.14684}, abstractNote={Summary Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co‐regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.}, journal={The Plant Journal}, author={Barro‐Trastoy, Daniela and Carrera, Esther and Baños, Jorge and Palau‐Rodríguez, Julia and Ruiz‐Rivero, Omar and Tornero, Pablo and Alonso, José M. and López‐Díaz, Isabel and Gómez, María Dolores and Pérez‐Amador, Miguel A.}, year={2020}, month={Jun} }
 @inbook{perkins_stepanova_alonso_heber_2020, place={Cham, Switzerland}, series={Lecture Notes in Computer Science}, title={RiboSimR: A Tool for Simulation and Power Analysis of Ribo-seq Data}, ISBN={9783030461645 9783030461652}, ISSN={0302-9743 1611-3349}, url={http://dx.doi.org/10.1007/978-3-030-46165-2_10}, DOI={10.1007/978-3-030-46165-2_10}, abstractNote={RNA-seq and Ribo-seq are widespread quantitative methods for assessing transcription and translation. They can be used to detect differential expression, differential translation, and differential translation efficiency between conditions. The statistical power to detect differential genes is affected by multiple factors, such as the number of replicates, sequencing depth, magnitude of differential expression and translation, distribution of gene counts, and method for estimating biological variance. As power estimation of translational efficiency involves the combination of both RNA-seq measurements and Ribo-seq measurements, this task is particularly challenging. Here we propose a power assessment tool, called RiboSimR, based purely on data simulation. RiboSimR, produces semi-parametric simulations that generate data based on real RNA and Ribo-seq experiments, with customizable choices on baseline parameters and tool configurations. We demonstrate the usefulness of our tool by simulating data based on two published Ribo-seq datasets and analyzing various aspects of experimental design.}, booktitle={Computational Advances in Bio and Medical Sciences}, publisher={Springer International Publishing}, author={Perkins, Patrick and Stepanova, Anna and Alonso, Jose and Heber, Steffen}, editor={Măndoiu, I. and Murali, T. and Narasimhan, G. and Rajasekaran, S. and Skums, P. and Zelikovsky, A.Editors}, year={2020}, pages={121–133}, collection={Lecture Notes in Computer Science} }
 @article{brumos_bobay_clark_alonso_stepanova_2020, title={Structure–Function Analysis of Interallelic Complementation in ROOTY Transheterozygotes}, volume={183}, url={https://doi.org/10.1104/pp.20.00310}, DOI={10.1104/pp.20.00310}, abstractNote={Auxin is a crucial plant growth regulator. Forward genetic screens for auxin-related mutants have led to the identification of key genes involved in auxin biosynthesis, transport, and signaling. Loss-of-function mutations in genes involved in glucosinolate biosynthesis, a metabolically related route that produces defense compounds, result in auxin overproduction. We identified an allelic series of fertile, hypomorphic Arabidopsis (Arabidopsis thaliana) mutants for the essential glucosinolate biosynthetic gene ROOTY (RTY) that exhibit a range of phenotypic defects characteristic of enhanced auxin production. Genetic characterization of these lines uncovered phenotypic suppression by cyp79b2 cyp79b3, wei2, and wei7 mutations and revealed the phenomenon of interallelic complementation in several RTY transheterozygotes. Structural modeling of RTY elucidated the relationships between structure and function in the RTY homo- and heterodimers, and unveiled the likely structural basis of interallelic complementation. This work underscores the importance of employing true null mutants in genetic complementation studies.}, number={3}, journal={Plant Physiology}, publisher={American Society of Plant Biologists (ASPB)}, author={Brumos, Javier and Bobay, Benjamin G. and Clark, Cierra A. and Alonso, Jose M. and Stepanova, Anna N.}, year={2020}, month={Jul}, pages={1110–1125} }
 @article{brumos_zhao_gong_soriano_patel_perez-amador_stepanova_alonso_2019, title={An improved plant toolset for high-throughput recombineering}, volume={6}, url={https://doi.org/10.1101/659276}, DOI={10.1101/659276}, abstractNote={Abstract Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing make it possible to directly modify the target genes in their native chromosomal location, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the obtained results provided by the direct chromosomal tagging achievable by genome editing strategies. These simple and customizable recombineering toolsets and protocols allow for high-throughput generation of a variety of genetic modifications. In addition, a highly efficient recombinase-mediated cassette exchange system has been developed to facilitate the transfer of the desired sequences from a BAC clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis . The utility of this system is demonstrated by the generation of over 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes, and the characterization of the translational reporter expression patterns for 14 auxin biosynthesis genes.}, publisher={Cold Spring Harbor Laboratory}, author={Brumos, J. and Zhao, C. and Gong, Y. and Soriano, D. and Patel, A.P. and Perez-Amador, M.A. and Stepanova, A.N. and Alonso, J.M}, year={2019}, month={Jun} }
 @article{stepanova_alonso_2019, title={From Ethylene-Auxin Interactions to Auxin Biosynthesis and Signal Integration}, volume={31}, ISSN={["1532-298X"]}, url={https://doi.org/10.1105/tpc.19.00339}, DOI={10.1105/tpc.19.00339}, abstractNote={The field of plant hormone biology, like many other research areas in plant sciences, has benefited tremendously from the adoption of Arabidopsis ( Arabidopsis thaliana ) as a model system. The development of a great genetic toolbox in this species led to the identification of many of the core}, number={7}, journal={PLANT CELL}, publisher={American Society of Plant Biologists (ASPB)}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2019}, month={Jul}, pages={1393–1394} }
 @article{dolores gomez_fuster-almunia_ocana-cuesta_alonso_perez-amador_2019, title={RGL2 controls flower development, ovule number and fertility in Arabidopsis}, volume={281}, ISSN={["0168-9452"]}, DOI={10.1016/j.plantsci.2019.01.014}, abstractNote={DELLA proteins are a group of plant specific GRAS proteins of transcriptional regulators that have a key role in gibberellin (GA) signaling. In Arabidopsis, the DELLA family is formed by five members. The complexity of this gene family raises the question on whether single DELLA proteins have specific or overlapping functions in the control of several GA-dependent developmental processes. To better understand the roles played by RGL2, one of the DELLA proteins in Arabidopsis, two transgenic lines that express fusion proteins of Venus-RGL2 and a dominant version of RGL2, YPet-rgl2Δ17, were generated by recombineering strategy using a genomic clone that contained the RGL2 gene. The dominant YPet-rgl2Δ17 protein is not degraded by GAs, and therefore it blocks the RGL2-dependent GA signaling and hence RGL2-dependent development. The RGL2 role in seed germination was further confirmed using these genetic tools, while new functions of RGL2 in plant development were uncovered. RGL2 has a clear function in the regulation of flower development, particularly stamen growth and anther dehiscence, which has a great impact in fertility. Moreover, the increased ovule number in the YPet-rgl2Δ17 line points out the role of RGL2 in the determination of ovule number.}, journal={PLANT SCIENCE}, author={Dolores Gomez, Maria and Fuster-Almunia, Clara and Ocana-Cuesta, Javier and Alonso, Jose M. and Perez-Amador, Miguel A.}, year={2019}, month={Apr}, pages={82–92} }
 @article{perkins_mazzoni-putman_stepanova_alonso_heber_2019, title={RiboStreamR: a web application for quality control, analysis, and visualization of Ribo-seq data}, volume={20}, ISSN={["1471-2164"]}, DOI={10.1186/s12864-019-5700-7}, abstractNote={Ribo-seq is a popular technique for studying translation and its regulation. A Ribo-seq experiment produces a snap-shot of the location and abundance of actively translating ribosomes within a cell’s transcriptome. In practice, Ribo-seq data analysis can be sensitive to quality issues such as read length variation, low read periodicities, and contaminations with ribosomal and transfer RNA. Various software tools for data preprocessing, quality assessment, analysis, and visualization of Ribo-seq data have been developed. However, many of these tools require considerable practical knowledge of software applications, and often multiple different tools have to be used in combination with each other. We present riboStreamR, a comprehensive Ribo-seq quality control (QC) platform in the form of an R Shiny web application. RiboStreamR provides visualization and analysis tools for various Ribo-seq QC metrics, including read length distribution, read periodicity, and translational efficiency. Our platform is focused on providing a user-friendly experience, and includes various options for graphical customization, report generation, and anomaly detection within Ribo-seq datasets. RiboStreamR takes advantage of the vast resources provided by the R and Bioconductor environments, and utilizes the Shiny R package to ensure a high level of usability. Our goal is to develop a tool which facilitates in-depth quality assessment of Ribo-seq data by providing reference datasets and automatically highlighting quality issues and anomalies within datasets.}, journal={BMC GENOMICS}, author={Perkins, Patrick and Mazzoni-Putman, Serina and Stepanova, Anna and Alonso, Jose and Heber, Steffen}, year={2019}, month={Jun} }
 @article{bhosale_giri_pandey_giehl_hartmann_traini_truskina_leftley_hanlon_swarup_et al._2018, title={A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate}, volume={9}, ISSN={2041-1723}, url={http://dx.doi.org/10.1038/S41467-018-03851-3}, DOI={10.1038/S41467-018-03851-3}, abstractNote={Abstract Phosphate (P) is an essential macronutrient for plant growth. Roots employ adaptive mechanisms to forage for P in soil. Root hair elongation is particularly important since P is immobile. Here we report that auxin plays a critical role promoting root hair growth in Arabidopsis in response to low external P. Mutants disrupting auxin synthesis ( taa1 ) and transport ( aux1 ) attenuate the low P root hair response. Conversely, targeting AUX1 expression in lateral root cap and epidermal cells rescues this low P response in aux1 . Hence auxin transport from the root apex to differentiation zone promotes auxin-dependent hair response to low P. Low external P results in induction of root hair expressed auxin-inducible transcription factors ARF19, RSL2, and RSL4. Mutants lacking these genes disrupt the low P root hair response. We conclude auxin synthesis, transport and response pathway components play critical roles regulating this low P root adaptive response.}, number={1}, journal={Nature Communications}, publisher={Springer Science and Business Media LLC}, author={Bhosale, Rahul and Giri, Jitender and Pandey, Bipin K. and Giehl, Ricardo F. H. and Hartmann, Anja and Traini, Richard and Truskina, Jekaterina and Leftley, Nicola and Hanlon, Meredith and Swarup, Kamal and et al.}, year={2018}, month={Apr} }
 @article{bhosale_giri_pandey_giehl_hartmann_traini_truskina_leftley_hanlon_swarup_et al._2018, title={Author Correction: A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate}, volume={9}, ISSN={2041-1723}, url={http://dx.doi.org/10.1038/S41467-018-04281-X}, DOI={10.1038/S41467-018-04281-X}, abstractNote={The original version of this Article omitted the following from the Acknowledgements: ‘We also thank DBT-CREST BT/HRD/03/01/2002.’ This has been corrected in both the PDF and HTML versions of the Article.}, number={1}, journal={Nature Communications}, publisher={Springer Science and Business Media LLC}, author={Bhosale, Rahul and Giri, Jitender and Pandey, Bipin K. and Giehl, Ricardo F. H. and Hartmann, Anja and Traini, Richard and Truskina, Jekaterina and Leftley, Nicola and Hanlon, Meredith and Swarup, Kamal and et al.}, year={2018}, month={May}, pages={1818} }
 @article{gomez_barro-trastoy_escoms_saura-sánchez_sánchez_briones-moreno_vera-sirera_carrera_ripoll_yanofsky_et al._2018, title={Gibberellins negatively modulate ovule number in plants}, volume={145}, ISSN={1477-9129 0950-1991}, url={http://dx.doi.org/10.1242/dev.163865}, DOI={10.1242/dev.163865}, abstractNote={Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed.}, number={13}, journal={Development}, publisher={The Company of Biologists}, author={Gomez, M. D. and Barro-Trastoy, D. and Escoms, E. and Saura-Sánchez, M. and Sánchez, I. and Briones-Moreno, A. and Vera-Sirera, F. and Carrera, E. and Ripoll, J. J. and Yanofsky, M. F. and et al.}, year={2018}, month={Jan}, pages={dev163865} }
 @article{brumos_robles_yun_vu_jackson_alonso_stepanova_2018, title={Local Auxin Biosynthesis Is a Key Regulator of Plant Development}, volume={47}, ISSN={["1878-1551"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85055097260&partnerID=MN8TOARS}, DOI={10.1016/j.devcel.2018.09.022}, abstractNote={•Local auxin production in roots is required for maintaining functional root meristems•Local biosynthesis and transport of auxin cooperate at generating robust auxin maxima•Auxin produced in the root quiescent center is sufficient for root meristem viability Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima. Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima. Nearly every aspect of a plant’s life is influenced by auxin. Not only does this phytohormone govern many developmental programs of the plant but also is able to tune plant growth and development according to the ever-changing external conditions surrounding the plant. In the meristems, auxin regulates cell division, elongation, and differentiation leading to downstream organogenesis that shapes shoot and root architecture. The vital roles of auxin gradients in plant growth and development have been established and interpreted mainly as the product of the combined action of auxin transport, signaling and response. Conversely, little is known about the contribution of the de novo auxin biosynthesis to the generation and maintenance of the morphogenic auxin maxima. A thorough understanding of how and where auxin is produced is nonetheless critical to our ability to precisely define auxin sources and sinks and thereby to establish a more refined picture of the polar transport system and auxin activity. It is only in the past 20 years that a combination of genetic, biochemical, and pharmacological approaches (Zhao et al., 2001Zhao Y. Christensen S.K. Fankhauser C. Cashman J.R. Cohen J.D. Weigel D. Chory J. A role for flavin monooxygenase-like enzymes in auxin biosynthesis.Science. 2001; 291: 306-309Crossref PubMed Scopus (868) Google Scholar, Cheng et al., 2006Cheng Y. Dai X. Zhao Y. Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis.Genes Dev. 2006; 20: 1790-1799Crossref PubMed Scopus (808) Google Scholar, Cheng et al., 2007Cheng Y. Dai X. Zhao Y. Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis.Plant Cell. 2007; 19: 2430-2439Crossref PubMed Scopus (474) Google Scholar, Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, Tao et al., 2008Tao Y. Ferrer J. Ljung K. Pojer F. Hong F. Long J.A. Li L. Moreno J.E. Bowman M.E. Ivans L.J. et al.Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants.Cell. 2008; 133: 164-176Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar, Mashiguchi et al., 2011Mashiguchi K. Tanaka K. Sakai T. Sugawara S. Kawaide H. Natsume M. Hanada A. Yaeno T. Shirasu K. Yao H. et al.The main auxin biosynthesis pathway in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2011; 108: 18512-18517Crossref PubMed Scopus (629) Google Scholar, Stepanova et al., 2011Stepanova A.N. Yun J. Robles L.M. Novak O. He W. Guo H. Ljung K. Alonso J.M. The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis.Plant Cell. 2011; 23: 3961-3973Crossref PubMed Scopus (260) Google Scholar), together with the development of more sensitive methodologies for auxin metabolite quantification (Novák et al., 2012Novák O. Hényková E. Sairanen I. Kowalczyk M. Pospíšil T. Ljung K. Tissue-specific profiling of the Arabidopsis thaliana auxin metabolome.Plant J. 2012; 72: 523-536Crossref PubMed Scopus (207) Google Scholar), have led to the identification of the first complete pathway of auxin biosynthesis in plants. The best characterized auxin in plants, indole-3-acetic acid (IAA), is predominantly produced from the aromatic amino acid L-tryptophan (Trp) via indole-3-pyruvic acid (IPyA) in a two-step pathway (reviewed in Brumos et al., 2014Brumos J. Alonso J.M. Stepanova A.N. Genetic aspects of auxin biosynthesis and its regulation.Physiol. Plant. 2014; 151: 3-12Crossref PubMed Scopus (62) Google Scholar). Trp is first converted to IPyA by a small family of tryptophan aminotransferases that in Arabidopsis is represented by TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) (also known as WEAK ETHYLENE INSENSITIVE8 [WEI8] [Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar], SHADE AVOIDANCE3 [SAV3] [Tao et al., 2008Tao Y. Ferrer J. Ljung K. Pojer F. Hong F. Long J.A. Li L. Moreno J.E. Bowman M.E. Ivans L.J. et al.Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants.Cell. 2008; 133: 164-176Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar], TRANSPORT INHIBITOR RESPONSE2 [TIR2] [Yamada et al., 2009Yamada M. Greenham K. Prigge M.J. Jensen P.J. Estelle M. The TRANSPORT INHIBITOR RESPONSE2 gene is required for auxin synthesis and diverse aspects of plant development.Plant Physiol. 2009; 151: 168-179Crossref PubMed Scopus (144) Google Scholar], and CYTOKININ INDUCED ROOT CURLING1 [CKRC1] [Zhou et al., 2011Zhou Z.Y. Zhang C.G. Wu L. Zhang C.G. Chai J. Wang M. Jha A. Jia P.F. Cui S.J. Yang M. et al.Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root.Plant J. 2011; 66: 516-527Crossref PubMed Scopus (54) Google Scholar]) and its paralogs TRYPTOPHAN AMINOTRANSFERASE RELATED1 (TAR1) and TAR2 (Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). IPyA is then converted into IAA by the YUCCA (YUC) family of flavin monooxygenases that in Arabidopsis consists of 11 members, YUC1 through YUC11 (reviewed in Zhao, 2014Zhao Y. Auxin biosynthesis.in: The Arabidopsis Book. American Society of Plant Biologists, 2014: e0173Crossref Google Scholar). According to the classical view in the auxin field, a majority of auxin in plants is produced in the shoot apical meristems, young leaves, and flower buds and is rapidly distributed throughout the plant via the phloem (reviewed in Teale et al., 2006Teale W.D. Paponov I.A. Palme K. Auxin in action: signalling, transport and the control of plant growth and development.Nat. Rev. Mol. Cell Biol. 2006; 7: 847-859Crossref PubMed Scopus (848) Google Scholar). In addition, auxin can move more slowly cell to cell via polar auxin transport with the help of auxin influx carriers AUXIN1 (AUX1)/LIKE-AUXs (LAXs) and auxin efflux transporters PIN FORMED (PINs) and ABCB/MULTIDRUG RESISTANCE (MDR)/PHOSPHOGLYCOPROTEIN (PGPs) (reviewed in Adamowski and Friml, 2015Adamowski M. Friml J. PIN-dependent auxin transport: action, regulation, and evolution.Plant Cell. 2015; 27: 20-32Crossref PubMed Scopus (460) Google Scholar). It is the polar distribution of PINs within cells that is thought to enable directional flow of auxin to generate robust morphogenic auxin gradients that instruct plant development. The classical view has however been challenged by the finding that auxin can also be synthesized locally in roots (Ljun et al., 2002Ljung K. Hul A.K. Kowalczyk M. Marchant A. Celenza J. Cohen J.D. Sandberg G. Biosynthesis, conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana.Plant Mol. Biol. 2002; 50: 309-332Crossref PubMed Scopus (126) Google Scholar, Ljung et al., 2005Ljung K. Hull A.K. Celenza J. Yamada M. Estelle M. Normanly J. Sandberg G. Sites and regulation of auxin biosynthesis in Arabidopsis roots.Plant Cell. 2005; 17: 1090-1104Crossref PubMed Scopus (409) Google Scholar, Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). Cloning of the auxin biosynthesis genes WEI8/TARs and YUCs uncovered that they are expressed not only in shoots but also in roots, and the domains of expression of several of these genes coincide with the auxin response maxima in root tips (Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, Chen et al., 2014Chen Q. Dai X. De-Paoli H. Cheng Y. Takebayashi Y. Kasahara H. Kamiya Y. Zhao Y. Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots.Plant Cell Physiol. 2014; 55: 1072-1079Crossref PubMed Scopus (142) Google Scholar), as defined by the auxin activity reporters DR5:GFP or DII-VENUS (Ulmasov et al., 1997Ulmasov T. Murfett J. Hagen G. Guilfoyle T.J. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements.Plant Cell. 1997; 9: 1963-1971Crossref PubMed Scopus (1591) Google Scholar, Brunoud et al., 2012Brunoud G. Wells D.M. Oliva M. Larrieu A. Mirabet V. Burrow A.H. Beeckman T. Kepinski S. Traas J. Bennett M.J. et al.A novel sensor to map auxin response and distribution at high spatio-temporal resolution.Nature. 2012; 482: 103-106Crossref PubMed Scopus (528) Google Scholar, Liao et al., 2015Liao C.Y. Smet W. Brunoud G. Yoshida S. Vernoux T. Weijers D. Reporters for sensitive and quantitative measurement of auxin response.Nat. Methods. 2015; 12: 207-210Crossref PubMed Scopus (245) Google Scholar). Multiple higher order wei8/tar and yuc mutants show dramatic developmental effects (Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, Tao et al., 2008Tao Y. Ferrer J. Ljung K. Pojer F. Hong F. Long J.A. Li L. Moreno J.E. Bowman M.E. Ivans L.J. et al.Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants.Cell. 2008; 133: 164-176Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar, Cheng et al., 2007Cheng Y. Dai X. Zhao Y. Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis.Plant Cell. 2007; 19: 2430-2439Crossref PubMed Scopus (474) Google Scholar, Cheng et al., 2006Cheng Y. Dai X. Zhao Y. Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis.Genes Dev. 2006; 20: 1790-1799Crossref PubMed Scopus (808) Google Scholar), suggesting that local activity of the biosynthesis genes may have a direct role in the generation and/or maintenance of auxin maxima. Supporting the key role of local biosynthesis is the finding that auxin produced in the aerial parts of plants is unable to compensate for an auxin biosynthesis deficiency of yucQ mutants in roots, resulting in the degeneration of root meristems (Chen et al., 2014Chen Q. Dai X. De-Paoli H. Cheng Y. Takebayashi Y. Kasahara H. Kamiya Y. Zhao Y. Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots.Plant Cell Physiol. 2014; 55: 1072-1079Crossref PubMed Scopus (142) Google Scholar). The discovery of an essential contribution of locally made auxin to PIN polarization and to the establishment of the apical-basal axis in young embryos further implicates tissue-specific auxin production in the control of plant development (Robert et al., 2013Robert H.S. Grones P. Stepanova A.N. Robles L.M. Lokerse A.S. Alonso J.M. Weijers D. Friml J. Local auxin sources orient the apical-basal axis in Arabidopsis embryos.Curr. Biol. 2013; 23: 2506-2512Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Thus, in some developmental contexts, the location of auxin sources is of critical physiological importance, and long-distance transport may not suffice for the generation of morphogenetic auxin gradients in all tissues. On the other hand, a uniform supply of IAA in the growth medium can partially suppress the root meristem defects of strong auxin biosynthetic mutants (Stepanova et al., 2011Stepanova A.N. Yun J. Robles L.M. Novak O. He W. Guo H. Ljung K. Alonso J.M. The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis.Plant Cell. 2011; 23: 3961-3973Crossref PubMed Scopus (260) Google Scholar), suggesting that a non-localized source of auxin readily available to roots may be all that is needed for the transport machinery to generate the auxin maxima required for root development. Consistent with the latter argument are the conclusions of the first experimental and mathematical modeling approaches that showed how the auxin transport system alone could generate robust auxin gradients in roots with any (i.e., non-local) sources of auxin (Grieneisen et al., 2007Grieneisen V.A. Xu J. Marée A.F.M. Hogeweg P. Scheres B. Auxin transport is sufficient to generate a maximum and gradient guiding root growth.Nature. 2007; 449: 1008-1013Crossref PubMed Scopus (626) Google Scholar). Alternative models were later built under the assumption that minor production and degradation of auxin do take place in every root cell, but higher auxin production rates occur in the quiescent center (QC) and columella initials to account for the local synthesis of auxin in the root meristem (Band et al., 2014Band L.R. Wells D.M. Fozard J.A. Ghetiu T. French A.P. Pound M.P. Wilson M.H. Yu L. Li W. Hijazi H.I. et al.Systems analysis of auxin transport in the Arabidopsis root apex.Plant Cell. 2014; 26: 862-875Crossref PubMed Scopus (147) Google Scholar). If locally made auxin was removed from these models, the gradient was not qualitatively affected (Band et al., 2014Band L.R. Wells D.M. Fozard J.A. Ghetiu T. French A.P. Pound M.P. Wilson M.H. Yu L. Li W. Hijazi H.I. et al.Systems analysis of auxin transport in the Arabidopsis root apex.Plant Cell. 2014; 26: 862-875Crossref PubMed Scopus (147) Google Scholar), arguing against the crucial role of local sources of auxin. These modeling experiments, however, made an assumption that all root cells make some auxin, which is not in agreement with the highly restricted expression patterns of the WEI8/TAR family in roots (Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). Thus, the relative contributions of local auxin production and transport, both long and short distance, remain largely undefined, warranting the need for further inquiry into the potentially overlapping roles of the biosynthesis and transport of this hormone. Both auxin biosynthesis and transport regulate plant development not only under optimal laboratory conditions but also in response to a stress hormone ethylene (Růzicka et al., 2007Růzicka K. Ljung K. Vanneste S. Podhorská R. Beeckman T. Friml J. Benková E. Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution.Plant Cell. 2007; 19: 2197-2212Crossref PubMed Scopus (575) Google Scholar, Swarup et al., 2007Swarup R. Perry P. Hagenbeek D. Van Der Straeten D. Beemster G.T. Sandberg G. Bhalerao R.P. Ljung K. Bennett M.J. Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation.Plant Cell. 2007; 19: 2186-2196Crossref PubMed Scopus (457) Google Scholar, Stepanova et al., 2007Stepanova A.N. Yun J. Likhacheva A.V. Alonso J.M. Multilevel interactions between ethylene and auxin in Arabidopsis roots.Plant Cell. 2007; 19: 2169-2185Crossref PubMed Scopus (410) Google Scholar, Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). Exposure of plants to ethylene or its precursor ACC is known to trigger specific patterns of local auxin production, transport, and response in roots of Arabidopsis seedlings by stimulating transcription of auxin biosynthesis and transport genes (Růzicka et al., 2007Růzicka K. Ljung K. Vanneste S. Podhorská R. Beeckman T. Friml J. Benková E. Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution.Plant Cell. 2007; 19: 2197-2212Crossref PubMed Scopus (575) Google Scholar, Swarup et al., 2007Swarup R. Perry P. Hagenbeek D. Van Der Straeten D. Beemster G.T. Sandberg G. Bhalerao R.P. Ljung K. Bennett M.J. Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation.Plant Cell. 2007; 19: 2186-2196Crossref PubMed Scopus (457) Google Scholar, Stepanova et al., 2007Stepanova A.N. Yun J. Likhacheva A.V. Alonso J.M. Multilevel interactions between ethylene and auxin in Arabidopsis roots.Plant Cell. 2007; 19: 2169-2185Crossref PubMed Scopus (410) Google Scholar, Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). Not surprisingly, genetic defects in the numerous components of the auxin biosynthesis, transport, perception, or response machineries (Merchante and Stepanova, 2017Merchante C. Stepanova A.N. The triple response assay and its use to characterize ethylene mutants in Arabidopsis.Methods Mol. Biol. 2017; 1573: 163-209Crossref Scopus (14) Google Scholar) lead to root-specific ethylene insensitivity. These findings suggest that auxin production and transport and, consequently, proper levels of auxin signaling and response are prerequisites for normal responses of Arabidopsis roots to ethylene and provide a convenient platform for elucidating the respective roles of auxin biosynthesis and transport in a physiologically relevant context. Herein, we evaluated the contribution of localized auxin production versus polar transport to specific growth and development programs in Arabidopsis. Our data indicate that root-produced local sources of auxin are required for root stem cell niche maintenance, whereas shoot-derived auxin alone cannot keep the root meristems alive. Local auxin biosynthesis and auxin transport in roots act redundantly in the establishment and maintenance of robust morphogenetic maxima critical for root meristem activity. In contrast, local auxin biosynthesis is essential (and cannot be fully compensated for by transport) for root responses to hormone ethylene, as well as for flower development. Thus, local auxin production and transport in plants represent partially redundant physiological mechanisms that work together to confer greater robustness and tunability to instructional auxin gradients, enabling developmental plasticity and adaptation of plants to changing environmental conditions. Prior studies (Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar) demonstrated that in wei8 tar2 a deficiency in auxin synthesized via the IPyA pathway results in a loss of robust auxin gradients and leads to root meristem degeneration. Herein, to dissect the role of shoot- versus root-derived auxin in root meristem maintenance, we performed reciprocal grafting of shoots and roots of wild-type (WT) and wei8 tar2 seedlings introgressed with an auxin-responsive reporter DR5:GFP at 3 days post germination, i.e., prior to mutant seedlings displaying any obvious signs of root stem cell loss (Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar), and examined DR5:GFP activity 1 week and 3 weeks after the surgery (Figures 1A and S1A). WT shoots grafted onto the mutant roots (WT/wei8 tar2) did not prevent root meristem degeneration, suggesting that WT shoot sources of auxin could not compensate for the auxin deficiency in roots and that auxin produced locally in roots is critical for root meristem health. One can argue that in these plants the vasculature may reconnect too late for the shoot-derived auxin to reach the root tip on time and prevent wei8 tar2 root meristem degeneration. Indeed, although the phloem of grafted WT plants reconnects within 3 days of grafting (Melnyk et al., 2015Melnyk C.W. Schuster C. Leyser O. Meyerowitz E.M. A developmental framework for graft formation and vascular reconnection in Arabidopsis thaliana.Curr. Biol. 2015; 25: 1306-1318Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), the process may take longer in grafts involving the compromised wei8 tar2 vasculature, and the meristematic potential of wei8 tar2 roots does decline over time (see below). Nonetheless, WT roots that received mutant shoots (wei8 tar2/WT) maintained healthy root meristems with normal patterns of DR5:GFP (Figure 1A), implying that local supply of auxin is necessary for root stem cell niche maintenance. One caveat of this experiment is that the shoots of wei8 tar2 are not completely devoid of auxin (Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jürgens G. Alonso J.M. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar), and thus, the possibility that in these grafted plants some shoot auxin did travel to the roots cannot be discarded. On the other hand, auxin locally produced in WT roots of wei8 tar2/WT grafted plants was not sufficient to reverse the morphological defects of wei8 tar2 shoots (Figure S1B). Control experiments with WT/WT grafts resulted in normal DR5:GFP expression in roots, whereas wei8 tar2/wei8 tar2 grafts lost their DR5:GFP and root meristem activity (Figure 1A). Additional controls using detached roots showed root degeneration and loss of DR5:GFP activity (Figure S1A). These results are consistent with the notion that local auxin produced in roots is required for root meristem activity. Likewise, shoots but not necessarily shoot-derived auxin are needed for root survival. To differentiate between the roles of shoot versus root sources of auxin in root meristem maintenance, we next employed a heat shock-inducible Cre/Lox auxin production system (Figure S1C) (Dubrovsky et al., 2008Dubrovsky J.G. Sauer M. Napsucialy-Mendivil S. Ivanchenko M.G. Friml J. Shishkova S. Celenza J. Benková E. Auxin acts as a local morphogenetic trigger to specify lateral root founder cells.Proc. Natl. Acad. Sci. USA. 2008; 105: 8790-8794Crossref PubMed Scopus (444) Google Scholar). This system turns on a bacterial auxin biosynthesis enzyme, iaaM, upon exposure of plants to heat stress, and the activation of the system can be monitored by GUS staining. We amended the system by introducing another bacterial auxin biosynthesis gene, iaaH, into the plants that harbor the Cre≫iaaM/GUS system to insure rapid and efficient auxin production independent of the endogenous plant pathways (Figure S1C). We triggered auxin synthesis in shoots or in roots by exposing the above- or below-ground parts of seedlings to high temperatures and monitored the effectiveness of the treatment by relying on GUS activity (Figures 1B and S1D). To eliminate the effect of the major endogenous pathway of auxin biosynthesis, we applied a combination of kynurenine (an inhibitor of TAA1/TARs) and yucasin (an inhibitor of YUCs) to block the IPyA route of auxin biosynthesis and to ensure that most if not all auxin in the heat-treated plants is made via the transgenic bacterial iaaM/iaaH pathway (Figure 1B). In the presence of the inhibitors, the roots of transgenic plants that received no heat shock or had auxin biosynthesis induced only in the aerial parts, like WT plants receiving the root heat shock but not harboring the auxin inducible system, lost their root meristematic activity and degenerated, whereas the heat-treated roots of transgenic plants retained healthy meristems (Figures 1B and S1D). Thus, shoot-derived auxin alone cannot support the root stem cell niche, and root sources of auxin are critical for maintaining the root meristem identity, consistent with prior findings (Chen et al., 2014Chen Q. Dai X. De-Paoli H. Cheng Y. Takebayashi Y. Kasahara H. Kamiya Y. Zhao Y. Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots.Plant Cell Physiol. 2014; 55: 1072-1079Crossref PubMed Scopus (142) Google Scholar). Next, we applied exogenous IAA to the shoots of wei8 tar2 seedlings and examined the fate of the root meristems (Figure 1C). Unlike mock-treated plants, double mutants treated on the shoot with IAA formed several lateral and adventitious roots (Figures 1C and S1E), indicating that externally supplied auxin did move down the hypocotyl and the root. Nonetheless, the primary root meristems of plants treated with IAA on the shoot lost their stem cell identity and differentiated (Figure 1C) despite repeated application of IAA. Importantly, not only the primary roots but also the later emerging lateral and adventitious root meristems degenerated (Figures 1C and S1E), again confirming that auxin production in the root is necessary for supporting a healthy root meristem and that shoot-derived auxin, although capable of inducing new roots, is not sufficient to keep root stem cells functional. Thus, the long-distance phloem-based auxin transport from the shoot previously implicated in adventitious and lateral root emergence (Swarup et al., 2008Swarup K. Benková E. Swarup R. Casimiro I. Péret B. Yang Y. Parry G. Nielsen E. De Smet I. Vanneste S. et al.The auxin influx carrier LAX3 promotes lateral root emergence.Nat. Cell Biol. 2008; 10: 946-954Crossref PubMed Scopus (567) Google Scholar, Overvoorde et al., 2010Overvoorde P. Fukaki H. Beeckman T. Auxin control of root development.Cold Spring Harb. Perspect. Biol. 2010; 2: a001537Crossref PubMed Scopus (520) Google Scholar) and root growth (Fu and Harberd, 2003Fu X. Harberd N.P. Auxin promotes Arabidopsis root growth by modulating gibberellin response.Nature. 2003; 421: 740-743Crossref PubMed Scopus (604) Google Scholar) may not be a critical player in the generation and maintenance of a local auxin maximum in the root meristem. Root meristems of wei8 tar2 grown in media supplemented with IAA do not degenerate (Figure 2A) (Stepano}, number={3}, journal={DEVELOPMENTAL CELL}, author={Brumos, Javier and Robles, Linda M. and Yun, Jeonga and Vu, Thien C. and Jackson, Savannah and Alonso, Jose M. and Stepanova, Anna N.}, year={2018}, month={Nov}, pages={306-+} }
 @article{yanagisawa_alonso_szymanski_2018, title={Microtubule-Dependent Confinement of a Cell Signaling and Actin Polymerization Control Module Regulates Polarized Cell Growth}, volume={28}, ISSN={0960-9822}, url={http://dx.doi.org/10.1016/J.CUB.2018.05.076}, DOI={10.1016/J.CUB.2018.05.076}, abstractNote={•The ROP/Rac GEF SPIKE1 emits signals from plasma membrane-associated nodules•SPIKE1 patterns the actin cytoskeleton by clustering and activating the WAVE complex•Microtubules confine SPIKE1 signaling nodules within an apical microtubule-free zone•Feedback control from the cell wall modulates cytoskeletal organization Cell types with wildly varying shapes use many of the same signaling and cytoskeletal proteins to dynamically pattern their geometry [1Panteris E. Galatis B. The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments.New Phytol. 2005; 167: 721-732Crossref PubMed Scopus (107) Google Scholar, 2Murphy D.A. Courtneidge S.A. The ‘ins’ and ‘outs’ of podosomes and invadopodia: characteristics, formation and function.Nat. Rev. Mol. Cell Biol. 2011; 12: 413-426Crossref PubMed Scopus (756) Google Scholar, 3Coles C.H. Bradke F. Coordinating neuronal actin-microtubule dynamics.Curr. Biol. 2015; 25: R677-R691Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar]. Plant cells are encased in a tough outer cell wall, and growth patterns are indirectly controlled by the cytoskeleton and its ability to locally specify the material properties of the wall [4Baskin T.I. Anisotropic expansion of the plant cell wall.Annu. Rev. Cell Dev. Biol. 2005; 21: 203-222Crossref PubMed Scopus (409) Google Scholar, 5Szymanski D.B. Plant cells taking shape: new insights into cytoplasmic control.Curr. Opin. Plant Biol. 2009; 12: 735-744Crossref PubMed Scopus (38) Google Scholar]. Broad and non-overlapping domains of actin and microtubules are predicted to create sharp cell-wall boundaries with distinct mechanical properties [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar] that are often proposed to direct growth patterns and cell shape [1Panteris E. Galatis B. The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments.New Phytol. 2005; 167: 721-732Crossref PubMed Scopus (107) Google Scholar, 6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar, 7Xu T. Wen M. Nagawa S. Fu Y. Chen J.G. Wu M.J. Perrot-Rechenmann C. Friml J. Jones A.M. Yang Z. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.Cell. 2010; 143: 99-110Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar]. However, mechanisms by which the cytoskeleton is patterned at the spatial and temporal scales that dictate cell morphology are not known. Here, we used combinations of live-cell imaging probes and unique morphology mutants in Arabidopsis to discover how the microtubule and actin systems are spatially coordinated to pattern polarized growth in leaf epidermal cells. The DOCK family guanine nucleotide exchange factor (GEF) SPIKE1 [8Basu D. Le J. Zakharova T. Mallery E.L. Szymanski D.B. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.Proc. Natl. Acad. Sci. USA. 2008; 105: 4044-4049Crossref PubMed Scopus (110) Google Scholar, 9Qiu J.L. Jilk R. Marks M.D. Szymanski D.B. The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development.Plant Cell. 2002; 14: 101-118Crossref PubMed Scopus (166) Google Scholar] clusters and activates conserved heteromeric WAVE/SCAR and ARP2/3 complexes at the cell apex to generate organized actin networks that define general cytoplasmic flow patterns. Cortical microtubules corral punctate SPIKE1 signaling nodules and restrict actin polymerization within a broad microtubule-depletion zone at the cell apex. Our data provide a useful model for cell-shape control, in which a GEF, actin filament nucleation complexes, microtubules, and the cell wall function as interacting systems that dynamically pattern polarized growth. Cell types with wildly varying shapes use many of the same signaling and cytoskeletal proteins to dynamically pattern their geometry [1Panteris E. Galatis B. The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments.New Phytol. 2005; 167: 721-732Crossref PubMed Scopus (107) Google Scholar, 2Murphy D.A. Courtneidge S.A. The ‘ins’ and ‘outs’ of podosomes and invadopodia: characteristics, formation and function.Nat. Rev. Mol. Cell Biol. 2011; 12: 413-426Crossref PubMed Scopus (756) Google Scholar, 3Coles C.H. Bradke F. Coordinating neuronal actin-microtubule dynamics.Curr. Biol. 2015; 25: R677-R691Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar]. Plant cells are encased in a tough outer cell wall, and growth patterns are indirectly controlled by the cytoskeleton and its ability to locally specify the material properties of the wall [4Baskin T.I. Anisotropic expansion of the plant cell wall.Annu. Rev. Cell Dev. Biol. 2005; 21: 203-222Crossref PubMed Scopus (409) Google Scholar, 5Szymanski D.B. Plant cells taking shape: new insights into cytoplasmic control.Curr. Opin. Plant Biol. 2009; 12: 735-744Crossref PubMed Scopus (38) Google Scholar]. Broad and non-overlapping domains of actin and microtubules are predicted to create sharp cell-wall boundaries with distinct mechanical properties [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar] that are often proposed to direct growth patterns and cell shape [1Panteris E. Galatis B. The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments.New Phytol. 2005; 167: 721-732Crossref PubMed Scopus (107) Google Scholar, 6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar, 7Xu T. Wen M. Nagawa S. Fu Y. Chen J.G. Wu M.J. Perrot-Rechenmann C. Friml J. Jones A.M. Yang Z. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.Cell. 2010; 143: 99-110Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar]. However, mechanisms by which the cytoskeleton is patterned at the spatial and temporal scales that dictate cell morphology are not known. Here, we used combinations of live-cell imaging probes and unique morphology mutants in Arabidopsis to discover how the microtubule and actin systems are spatially coordinated to pattern polarized growth in leaf epidermal cells. The DOCK family guanine nucleotide exchange factor (GEF) SPIKE1 [8Basu D. Le J. Zakharova T. Mallery E.L. Szymanski D.B. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.Proc. Natl. Acad. Sci. USA. 2008; 105: 4044-4049Crossref PubMed Scopus (110) Google Scholar, 9Qiu J.L. Jilk R. Marks M.D. Szymanski D.B. The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development.Plant Cell. 2002; 14: 101-118Crossref PubMed Scopus (166) Google Scholar] clusters and activates conserved heteromeric WAVE/SCAR and ARP2/3 complexes at the cell apex to generate organized actin networks that define general cytoplasmic flow patterns. Cortical microtubules corral punctate SPIKE1 signaling nodules and restrict actin polymerization within a broad microtubule-depletion zone at the cell apex. Our data provide a useful model for cell-shape control, in which a GEF, actin filament nucleation complexes, microtubules, and the cell wall function as interacting systems that dynamically pattern polarized growth. In general, the cellular control of ARP2/3 is poorly understood; most genomes contain gene families that encode many types of ARP2/3 activators and in nearly all cases the specific guanine nucleotide exchange factors (GEFs) that transmit small GTPase signals to ARP2/3 are unknown [10Stradal T.E. Scita G. Protein complexes regulating Arp2/3-mediated actin assembly.Curr. Opin. Cell Biol. 2006; 18: 4-10Crossref PubMed Scopus (203) Google Scholar]. However, in plants the situation is simplified, because ARP2/3 appears to be activated solely by the evolutionarily conserved WAVE/SCAR regulatory complex (W/SRC) [11Zhang C. Mallery E.L. Schlueter J. Huang S. Fan Y. Brankle S. Staiger C.J. Szymanski D.B. Arabidopsis SCARs function interchangeably to meet actin-related protein 2/3 activation thresholds during morphogenesis.Plant Cell. 2008; 20: 995-1011Crossref PubMed Scopus (56) Google Scholar, 12Yanagisawa M. Zhang C. Szymanski D.B. ARP2/3-dependent growth in the plant kingdom: SCARs for life.Front. Plant Sci. 2013; 4: 166Crossref PubMed Scopus (45) Google Scholar], and there is biochemical and genetic evidence that the ROP/Rac GEF SPIKE1 may transmit activating signals to W/SRC [8Basu D. Le J. Zakharova T. Mallery E.L. Szymanski D.B. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.Proc. Natl. Acad. Sci. USA. 2008; 105: 4044-4049Crossref PubMed Scopus (110) Google Scholar, 13Uhrig J.F. Mutondo M. Zimmermann I. Deeks M.J. Machesky L.M. Thomas P. Uhrig S. Rambke C. Hussey P.J. Hülskamp M. The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis.Development. 2007; 134: 967-977Crossref PubMed Scopus (76) Google Scholar]. SPK1 was also involved in ARP2/3-dependent cell tapering [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar] because, like w/src and arp2/3 trichomes [11Zhang C. Mallery E.L. Schlueter J. Huang S. Fan Y. Brankle S. Staiger C.J. Szymanski D.B. Arabidopsis SCARs function interchangeably to meet actin-related protein 2/3 activation thresholds during morphogenesis.Plant Cell. 2008; 20: 995-1011Crossref PubMed Scopus (56) Google Scholar], those of spk1 had blunt branch tips that failed to taper normally during elongation (Figures 1A and 1B ). To analyze the function and dynamics of SPK1 in vivo, we generated stable yellow fluorescent protein (3X-YFP):SPK1 lines in the context of a 64 kb genomic fragment using recombineering (Figure S1A) [14Zhou R. Benavente L.M. Stepanova A.N. Alonso J.M. A recombineering-based gene tagging system for Arabidopsis.Plant J. 2011; 66: 712-723Crossref PubMed Scopus (47) Google Scholar]. The YFP:SPK1 fusion protein was functional because in four of four independent transformed lines with a complete T-DNA, it dominantly rescued all null spk1 mutant phenotypes (Figures S1B–S1E), and the fusion protein was intact (Figure S1F). SPK1 localized at the apex of all young trichome branches, and the geometry of the SPK1 domain scaled closely with the changing tip morphology (Figures 1C and 1D). In rare instances (∼5%), a relatively broad domain of SPK1 signal was observed closer to the branch base. The branch tip localization pattern resembles that of the W/SRC and ARP2/3 complexes, which reside in an apical microtubule-depletion zone (MDZ) [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. SPK1 has GEF activity in vitro [8Basu D. Le J. Zakharova T. Mallery E.L. Szymanski D.B. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.Proc. Natl. Acad. Sci. USA. 2008; 105: 4044-4049Crossref PubMed Scopus (110) Google Scholar, 9Qiu J.L. Jilk R. Marks M.D. Szymanski D.B. The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development.Plant Cell. 2002; 14: 101-118Crossref PubMed Scopus (166) Google Scholar]. To test for SPK1 GEF activity in trichome apices, we created a biosensor for active ROP by fusing the Cdc42/Rac-interactive binding domain of RIC1 with green fluorescent protein (CRIB:GFP). 45% of the branch apices (n = 56) had elevated levels of active ROP (Figure 1C; Table S1), and no such localized accumulation was observed with GFP alone (Figure S1G). These data suggest that SPK1 GEF activity at the cell apex is latent or variable, because SPK1 is a permanent resident at the apex whereas active ROP was present in only a fraction of the branches. SPK1 contributed to the apical pool of active ROP because both molecules localized to the tapering tip (Figure 1D), and the percentage of branches showing tip-concentrated CRIB:GFP signal was reduced to 24% in spk1 (Table S1). This result also indicates that other GEFs, probably plant-specific PRONE domain-containing family members [15Berken A. Thomas C. Wittinghofer A. A new family of RhoGEFs activates the Rop molecular switch in plants.Nature. 2005; 436: 1176-1180Crossref PubMed Scopus (201) Google Scholar], are active in the trichome branch tip. The localization of SPK1 to the branch apex is consistent with the GEF having a role in the recruitment and/or activation of the W/SRC and ARP2/3 complexes. The localization of SPK1 was compared with those of BRK1/HSPC300:YFP and ARPC5:GFP, which are validated live-cell probes for the functional W/SRC and ARP2/3 complexes, respectively [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar, 16Le J. Mallery E.L. Zhang C. Brankle S. Szymanski D.B. Arabidopsis BRICK1/HSPC300 is an essential WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2.Curr. Biol. 2006; 16: 895-901Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 17Djakovic S. Dyachok J. Burke M. Frank M.J. Smith L.G. BRICK1/HSPC300 functions with SCAR and the ARP2/3 complex to regulate epidermal cell shape in Arabidopsis.Development. 2006; 133: 1091-1100Crossref PubMed Scopus (80) Google Scholar, 18Dyachok J. Shao M.R. Vaughn K. Bowling A. Facette M. Djakovic S. Clark L. Smith L. Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots.Mol. Plant. 2008; 1: 990-1006Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. Like SPK1, the W/SRC and ARP2/3 complexes were clearly tip localized (Figure 2A). A previous analysis reported BRK1 and the ARP2/3 complex at the apex in 66% and 28% of the trichome branches, respectively [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar] (Table S1). A punctate distribution of SPK1 was more apparent in face-on views of the branch apex (Figure 2B), and the W/SRC and ARP2/3 complexes also had punctate distributions that were indistinguishable from SPK1 in terms of both their size and density (Figures 2C and 2D). Unfortunately, we could not conduct two-color live-cell imaging of SPK1 and BRK1 or ARP2/3, because mCherry-fusion proteins of BRK1 and ARPC5 were too dim to resolve at the trichome apex. Given the localization data above, and that SPK1 genetically and physically interacts with W/SRC [8Basu D. Le J. Zakharova T. Mallery E.L. Szymanski D.B. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.Proc. Natl. Acad. Sci. USA. 2008; 105: 4044-4049Crossref PubMed Scopus (110) Google Scholar, 13Uhrig J.F. Mutondo M. Zimmermann I. Deeks M.J. Machesky L.M. Thomas P. Uhrig S. Rambke C. Hussey P.J. Hülskamp M. The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis.Development. 2007; 134: 967-977Crossref PubMed Scopus (76) Google Scholar], it is very likely, but not proven directly, that all of these proteins are functioning at the same subcellular location. Nonetheless, it was clear that SPK1 was required to cluster W/SRC normally, because in the spk1 background only 17% of the branches had a normal distribution of BRK1 at the apex (Figure 2E; Table S2). The majority of spk1 branches had no cortical BRK1 signal, and in the remaining ∼30% of spk1 branches BRK1 signal was clearly off the center axis of the cell or mislocalized to the flanking region of the apex. Therefore, Arabidopsis W/SRC has some ability to cluster near the cell apex independent of SPK1. However, in spk1 cells, W/SRC was not effective in terms of ARP2/3 complex clustering and activation. None of the branches analyzed had a normal distribution of apical ARP2/3 complex, and among the ∼15% that had any tip-localized ARP2/3 complex the pattern was altered in a way that mirrored the defective distribution of BRK1 (Figure 2E; Table S2). In tip-growing moss cells [19Perroud P.F. Quatrano R.S. BRICK1 is required for apical cell growth in filaments of the moss Physcomitrella patens but not for gametophore morphology.Plant Cell. 2008; 20: 411-422Crossref PubMed Scopus (44) Google Scholar] and in the trichome apex [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar], the polarized localization of the ARP2/3 complex depends on a functional W/SRC. In the spk1 background W/SRC is assembled [8Basu D. Le J. Zakharova T. Mallery E.L. Szymanski D.B. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.Proc. Natl. Acad. Sci. USA. 2008; 105: 4044-4049Crossref PubMed Scopus (110) Google Scholar] but apparently inefficient in terms of recruiting the ARP2/3 complex, because the localization defects of the ARP2/3 complex are more severe compared to BRK1 and the W/SRC (Table S2). It has previously been shown that arp2/3 mutants completely lack an apical cortical actin meshwork [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar], and that null mutants for arp2/3, w/src, and spk1 all have very similar actin phenotypes in early-stage trichomes [8Basu D. Le J. Zakharova T. Mallery E.L. Szymanski D.B. A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.Proc. Natl. Acad. Sci. USA. 2008; 105: 4044-4049Crossref PubMed Scopus (110) Google Scholar, 11Zhang C. Mallery E.L. Schlueter J. Huang S. Fan Y. Brankle S. Staiger C.J. Szymanski D.B. Arabidopsis SCARs function interchangeably to meet actin-related protein 2/3 activation thresholds during morphogenesis.Plant Cell. 2008; 20: 995-1011Crossref PubMed Scopus (56) Google Scholar]. Therefore, our data indicate that SPK1 recruits and channels activating signals to W/SRC to promote localized actin polymerization at the apex as the cell tapers. We found no evidence for direct feedback control of the W/SRC or ARP2/3 complexes on the ability of SPK1 to be clustered in the cell apex, because it was tip localized in nearly every early-stage branch when either of the above complexes was mutated (Figure S2A; Table S1). However, the size of the SPK1 domain became more random in the w/src and arp2/3 mutants (Figure S2B) compared to the wild-type (Figure 1D) when graphed as a function of the changing tip radius of curvature. This is consistent with the previously reported randomized size of the MDZ in arp2/3 mutants [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar], and suggests some form of feedback control between cell-wall geometry and the size and location of the MDZ. The geometries of the MDZ and ARP2/3 complex activation domains are tightly regulated during branch elongation to enable normal cell tapering. The ARP2/3-generated apical patch of cortical actin orients cytoplasmic actin bundle networks to enable highly organized long-distance transport and the maintenance of cell-wall thickness gradients that enable polarized expansion [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. As the branch elongates the apical MDZ becomes progressively constricted, and this is predicted to generate a specialized patch of cell wall at the apex with randomized cellulose fibers and isotropic mechanical properties that can mediate cell tapering [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. Our data suggest that actin-based outputs of the W/SRC-ARP2/3 complex pathway are required to properly couple the geometries of the cell wall to the size of the SPK1-positive domain and the MDZ. The SPK1 live-cell probe allowed us to analyze the spatial and temporal dynamics of the GEF as a function of a changing cell morphology. Knowledge on the precise origin of DOCK GEF signals is limited. They may function at the plasma membrane [18Dyachok J. Shao M.R. Vaughn K. Bowling A. Facette M. Djakovic S. Clark L. Smith L. Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots.Mol. Plant. 2008; 1: 990-1006Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 20Kunisaki Y. Nishikimi A. Tanaka Y. Takii R. Noda M. Inayoshi A. Watanabe K. Sanematsu F. Sasazuki T. Sasaki T. Fukui Y. DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis.J. Cell Biol. 2006; 174: 647-652Crossref PubMed Scopus (163) Google Scholar] or at sub-domains of the endoplasmic reticulum [21Zhang C. Kotchoni S.O. Samuels A.L. Szymanski D.B. SPIKE1 signals originate from and assemble specialized domains of the endoplasmic reticulum.Curr. Biol. 2010; 20: 2144-2149Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22Zhang C. Mallery E. Reagan S. Boyko V.P. Kotchoni S.O. Szymanski D.B. The endoplasmic reticulum is a reservoir for WAVE/SCAR regulatory complex signaling in the Arabidopsis leaf.Plant Physiol. 2013; 162: 689-706Crossref PubMed Scopus (29) Google Scholar]. The SPK1 and BRK1 punctae were not obviously localized to sub-domains of the plasma membrane, because the peak signal intensities of YFP:SPK1 and BRK1:YFP were not in the plane of the plasma membrane but almost always were in contact with the plasma membrane (Figures 2F–2I, S2C, and S2D). These nodule-like structures could be a localized invagination of the plasma membrane; however, such structures are not common in electron microscopy analyses of the plant cortex. Our leading hypothesis is that nodules correspond to a distinct endomembrane compartment that is associated with the plasma membrane. An ultrastructural analysis using electron microscopy is needed to better define the membrane topology of these striking SPK1-positive structures. The SPK1 punctae appeared to be anchored stably to a particular domain of the plasma membrane, because time-lapsed analyses showed that SPK1 punctae were relatively immobile, with most having very slow average speeds of 228 ± 156 nm/min (Figure S2E). There were maturation and destabilization phases to SPK1 punctae formation, because they often became progressively bright prior to their disappearance, and punctae size was correlated with mean particle brightness (Figures S2F–S2H). On rare occasions, punctae would detach and stream in the subcortical cytoplasm (Figures S2G and S2I; Video S1). The image data indicate that SPK1 signals originate primarily from a distributed network of nodules that are clustered within the specialized apical domain of the cell. https://www.cell.com/cms/asset/31966408-365a-4c7d-b69d-79b476c802fe/mmc2.mp4Loading ... Download .mp4 (1.42 MB) Help with .mp4 files Video S1. Dynamics of Cytoplasmic SPK1 Punctae in a Developing Trichome, Related to Figure 3 We were not able to precisely identify the organelles that form SPK1 nodules. In epidermal pavement cells, SPK1 is a peripheral endoplasmic reticulum (ER)-associated protein, and a sub-pool colocalizes with ER-exit site markers [21Zhang C. Kotchoni S.O. Samuels A.L. Szymanski D.B. SPIKE1 signals originate from and assemble specialized domains of the endoplasmic reticulum.Curr. Biol. 2010; 20: 2144-2149Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Using the general ER marker GFP:HDEL and a validated ER-exit site marker (YFP:SEC24A), it was clear that the ER was widely distributed in young trichomes (Figure S3A). The ER tended to occupy the extreme branch apex, compared to an array of other organelle markers, which were excluded from the apex (Figures S3B and S3C). We tested for tip gradients of the known ER-PM (plasma membrane) contact site markers SYT1 and VAP27 (Figure S3D). Both had a broader subcellular distribution compared to SPK1. However, sub-pools of these proteins were present in the cortex at the branch apex. It remains possible that sub-pools of SYT1 and VAP27, perhaps in combination with additional proteins and lipid modifications, have specialized functions that enable SPK1 to be efficiently clustered at specific organelle domains. For a better understanding of how individual SPK1 punctae and the apical SPK1 signaling domain as a whole are reshaped as a function of morphogenesis, the dynamics of SPK1 nodules were analyzed across wide temporal and spatial scales. In emerging branch buds, SPK1 nodules were enriched at the apex compared to the cell flank (Figure 3A). As the branch elongated, the tip radius of curvature and the cortical domain-containing SPK1 nodules were constricted at similar rates (Figure 3B; Video S2). Particle tracking analyses indicated that the total number of trackable punctae in the branch decreased in both apex and flanking regions of the cell; however, because the area of the SPK1 signaling domain was decreasing, the average density of SPK1 nodules was maintained at the apex, but not in the flank (Figures 3C and S3E–S3H). The cortex of the branch apex was specialized in terms of forming SPK1 punctae, as they formed 3.5 times more frequently in the apex compared to the flank (Figure S3I). This result suggests that a specialized endomembrane organization that efficiently promotes SPK1 clustering is present at the branch apex. https://www.cell.com/cms/asset/aca5a32f-4024-4eb7-97a0-26cc9efde7f5/mmc3.mp4Loading ... Download .mp4 (0.93 MB) Help with .mp4 files Video S2. Dynamics of Cortical SPK1 Punctae in a Developing Trichome, Related to Figure 3 Although SPK1 was always present in the apex of developing branches (Table S1), time-lapsed analyses consistently revealed unstable SPK1 punctae appearing and disappearing at variable rates (Video S2). The lifetimes of SPK1 nodules were compared between the extreme apical region and the region labeled “interface” that was adjacent to the cell flank (Figures 3A and 3D). At both locales, the punctae varied greatly in their persistence; most appeared and disappeared within ∼2 min, but others were trackable for over 30 min. However, at the interface region, punctae lifetimes were significantly shorter, suggesting that SPK1 nodules are destabilized near the proximal boundary of the signaling domain. The directionality of SPK1 nodule movement also differed as a function of their location: those within the apical domain tended to move slowly toward the cell tip, but the particle trajectories along the cell flank were more random (Figures 3E and S3D). This important result suggested a potential control mechanism for SPK1 nodule localization based on the known localization of cortical microtubules in this cell type. Cortical microtubules are tightly associated with the PM [23Ambrose J.C. Shoji T. Kotzer A.M. Pighin J.A. Wasteneys G.O. The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division.Plant Cell. 2007; 19: 2763-2775Crossref PubMed Scopus (164) Google Scholar] and have the potential to act as a barrier to lateral diffusion for PM-localized proteins [24Paredez A.R. Somerville C.R. Ehrhardt D.W. Visualization of cellulose synthase demonstrates functional association with microtubules.Science. 2006; 312: 1491-1495Crossref PubMed Scopus (953) Google Scholar, 25Oda Y. Fukuda H. Initiation of cell wall pattern by a Rho- and microtubule-driven symmetry breaking.Science. 2012; 337: 1333-1336Crossref PubMed Scopus (178) Google Scholar, 26Sugiyama Y. Wakazaki M. Toyooka K. Fukuda H. Oda Y. A novel plasma membrane-anchored protein regulates xylem cell-wall deposition through microtubule-dependent lateral inhibition of Rho GTPase domains.Curr. Biol. 2017; 27: 2522-2528.e4Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. In early-stage trichomes, microtubules surround an apical domain where ARP2/3 is activated, and this apical MDZ is a location at which multiple cytoskeletal and cell-wall parameters are integrated to dictate the geometry of cell expansion [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. We wanted to directly determine whether the SPK1-positive cortical domain resided within the MDZ and whether this localization was influenced by cortical microtubules. Most live-cell probes for microtubules polymerize poorly in young trichomes (Figure S4A). Therefore, we constructed dim trichome-specific mRUBY2:MBD reporter lines that labeled microtubules but did not have a noticeable effect on trichome morphology. Two independent lines were crossed into the YFP:SPK1-expressing line and yielded identical results. Two-color live-cell imaging showed that the cortical domain of SPK1 nodules was always positioned within the apical MDZ (n = 47) (Figure 4A). This result suggests that cortical microtubules influence the distribution of SPK1 signaling nodules at the cell apex. We next quantified SPK1 localization patterns after treating young trichomes with the microtubule-depolymerizing drug oryzalin, which is known to randomize microtubules and lead to isotropic cell expansion in this cell type [27Szymanski D.B. Marks M.D. Wick S.M. Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis.Plant Cell. 1999; 11: 2331-2347Crossref PubMed Scopus (157) Google Scholar]. As expected, oryzalin caused isotropic cell swelling (Figure S4B), and its effects on microtubules were reversible (Figures 4B–4E). Following oryzalin treatment, the SPK1 punctae remained associated with the PM, but over time the SPK1-positive cortical domain spread slowly from the apex to more distal regions (Figures 4C, 4D, and S4C). The SPK1-positive cortical domain became progressively dim and the size of individual punctae appeared smaller as the domain spread down the cell flank (Figures 4C–4E). This suggests that the degree of SPK1 clustering is related to the spatial distribution and quality of the endomembrane domain from which they originate. Additional tip-localized parameters are likely to be involved, as SPK1 clustering at the apex as bright and localized SPK1 signals was again detected upon oryzalin removal (Figure 4C). The distribution of BRK1 punctae also progressively broadened in an identical way in response to oryzalin treatment (Figures S4D–S4F). The altered geometries of the BRK1 and SPK1 domains were not driven by changes in cell shape or PM curvature, because the geometry of the cell apex changed very little at the 2 hr and 5 hr time points following oryzalin treatment (Figures 4C and S4D). However, the size of both the SPK1- and BRK1-positive cortical domains became uncoupled from cell shape in the absence of microtubules (Figures S4C and S4E). Because the timescales of the inhibitor treatment exceeded the lifetime of SPK1 nodules by more than an order of magnitude, our results are consistent with a model in which microtubules restrict the distal movement of a specialized PM-anchored organelle system that promotes SPK1-nodule formation. If cortical microtubules simply blocked SPK1 punctae from forming in association with the PM, one would expect new punctae to form anywhere at the cell cortex upon microtubule depolymerization, and that was not observed. The actin cytoskeleton and acto-myosin transport were not directly required to generate an SPK1 signaling domain, because its localization was not affected even after 3 hr of treatment with the actin-polymerization inhibitor latrunculin B (LatB) (Figures S4G and S4H). Only after 5 hr of actin depolymerization were there subtle and random effects on the size and location of the SPK1 domain (Figure S4I). We confirmed that 5 hr treatments with LatB do not eliminate the MDZ; instead, its size and position are altered after extended LatB exposure (Figures S4I–S4K). These types of changes in the SPK1 domain are very similar to those reported for arp2/3 mutants that lack an apical actin meshwork but still retain an MDZ, albeit with a more random size and position compared to the wild-type [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. Together, these results argue against a simple cortical competition model in which cortical actin is required to exclude microtubules from a region of the PM. Instead, the results suggest that the absence of an organized actin cytoskeleton indirectly influences the distributions of the MDZ and SPK1. In arp2/3 mutants the spatial patterns of cell-wall assembly are not properly coordinated with growth [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. Perhaps the effects of actin depolymerization on SPK1 localization are delayed because its mis-localization is caused by aberrant cell-wall assembly and growth that occur at the timescale of hours. Defective cell-wall mechanical properties could lead to altered feedback between the cell wall and microtubule systems that also disrupts the geometry of the SPK1 domain. Specialized cell types assemble spatially distinct actin and microtubule networks to generate predictable shapes [1Panteris E. Galatis B. The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments.New Phytol. 2005; 167: 721-732Crossref PubMed Scopus (107) Google Scholar, 2Murphy D.A. Courtneidge S.A. The ‘ins’ and ‘outs’ of podosomes and invadopodia: characteristics, formation and function.Nat. Rev. Mol. Cell Biol. 2011; 12: 413-426Crossref PubMed Scopus (756) Google Scholar, 3Coles C.H. Bradke F. Coordinating neuronal actin-microtubule dynamics.Curr. Biol. 2015; 25: R677-R691Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 28Kobayashi H. Fukuda H. Shibaoka H. Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in cultured Zinnia cells.Protoplasma. 1988; 143: 29-37Crossref Scopus (91) Google Scholar], and in plants cortical actin meshworks within MDZs coordinate long-distance transport and cell-wall assembly to pattern growth at the cellular scale [1Panteris E. Galatis B. The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments.New Phytol. 2005; 167: 721-732Crossref PubMed Scopus (107) Google Scholar, 5Szymanski D.B. Plant cells taking shape: new insights into cytoplasmic control.Curr. Opin. Plant Biol. 2009; 12: 735-744Crossref PubMed Scopus (38) Google Scholar, 6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. Here, we show that a broad apical zone of microtubule depletion is a cellular hub for cell-shape control where the ROP GEF SPK1 is a permanent resident. When fully activated, SPK1 recruits W/SRC to a distributed network of PM-associated organelle domains, and the resulting ARP2/3-dependent actin meshwork organizes cytoplasmic flow that has been shown to maintain cell-wall thickness gradients that enable polarized growth cellular scales [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. Activating SPK1 signals are transmitted from a concentrated population of unstable nodules that appear to be physically linked to the PM. Importantly, the network of SPK1 nodules is confined to the cell apex by a negative feedback loop in which cortical microtubules restrict the movement of the SPK1 punctae. Septins have a general role as diffusion barriers to confine signaling molecules to a specific subcellular domain [29Mostowy S. Cossart P. Septins: the fourth component of the cytoskeleton.Nat. Rev. Mol. Cell Biol. 2012; 13: 183-194Crossref PubMed Scopus (480) Google Scholar]. However, septins are absent in plants, and we show here that microtubules most likely provide a similar function to confine SPK1 signaling within the MDZ boundary. During cell tapering, there are additional forms of feedback control in which the geometry of the ARP2/3 complex activation domain is adaptively tuned to a decreasing cell radius of curvature, and this feedback may occur at the MDZ boundary, where wall stress is maximal and microtubules are stabilized [6Yanagisawa M. Desyatova A.S. Belteton S.A. Mallery E.L. Turner J.A. Szymanski D.B. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.Nat. Plant. 2015; 1: 15014Crossref PubMed Scopus (76) Google Scholar]. We propose that the slow process of actin-dependent secretion and cell-wall assembly enables accurate information flow between the cell wall and the MDZ. We anticipate this new knowledge about the cellular-scale controls of actin, microtubules, and morphogenesis will serve as a useful comparative model for other cell types that use a diffuse growth mechanism, and accelerate the engineering of improved traits for economically important plant cell types such as cotton fibers.}, number={15}, journal={Current Biology}, publisher={Elsevier BV}, author={Yanagisawa, Makoto and Alonso, Jose M. and Szymanski, Daniel B.}, year={2018}, month={Aug}, pages={2459–2466.e4} }
 @article{estrada-johnson_csukasi_pizarro_vallarino_kiryakova_vioque_merchante_brumos_medina-escobar_botella_et al._2017, title={Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle (vol 8, pg 889, 2017)}, volume={8}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2017.01305}, abstractNote={[Catharina Merchante] was not included as an author in the published article. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Estrada-Johnson, Elizabeth and Csukasi, Fabiana and Pizarro, Carmen M. and Vallarino, Jose G. and Kiryakova, Yulia and Vioque, Amalia and Merchante, Catharina and Brumos, Javier and Medina-Escobar, Nieves and Botella, Miguel A. and et al.}, year={2017}, month={Jul} }
 @article{estrada-johnson_csukasi_pizarro_vallarino_kiryakova_vioque_brumos_medina-escobar_botella_alonso_et al._2017, title={Transcriptomic analysis in strawberry fruits reveals active auxin biosynthesis and signaling in the ripe receptacle}, volume={8}, journal={Frontiers in Plant Science}, author={Estrada-Johnson, E. and Csukasi, F. and Pizarro, C. M. and Vallarino, J. G. and Kiryakova, Y. and Vioque, A. and Brumos, J. and Medina-Escobar, N. and Botella, M. A. and Alonso, J. M. and et al.}, year={2017} }
 @article{merchante_stepanova_alonso_2017, title={Translation regulation in plants: an interesting past, an exciting present and a promising future}, volume={90}, ISSN={["1365-313X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85017002891&partnerID=MN8TOARS}, DOI={10.1111/tpj.13520}, abstractNote={Changes in gene expression are at the core of most biological processes, from cell differentiation to organ development, including the adaptation of the whole organism to the ever-changing environment. Although the central role of transcriptional regulation is solidly established and the general mechanisms involved in this type of regulation are relatively well understood, it is clear that regulation at a translational level also plays an essential role in modulating gene expression. Despite the large number of examples illustrating the critical role played by translational regulation in determining the expression levels of a gene, our understanding of the molecular mechanisms behind such types of regulation has been slow to emerge. With the recent development of high-throughput approaches to map and quantify different critical parameters affecting translation, such as RNA structure, protein–RNA interactions and ribosome occupancy at the genome level, a renewed enthusiasm toward studying translation regulation is warranted. The use of these new powerful technologies in well-established and uncharacterized translation-dependent processes holds the promise to decipher the likely complex and diverse, but also fascinating, mechanisms behind the regulation of translation.}, number={4}, journal={PLANT JOURNAL}, author={Merchante, Catharina and Stepanova, Anna N. and Alonso, Jose M.}, year={2017}, month={May}, pages={628–653} }
 @book{merchante_yun_valpuesta-fernandez_stepanova_alonso_2017, title={Translation regulation of uORFs-containing genes in Arabidopsis}, author={Merchante, C. and Yun, J. and Valpuesta-Fernandez, V. and Stepanova, A. and Alonso, J.}, year={2017} }
 @misc{provart_alonso_assmann_bergmann_brady_brkljacic_browse_chapple_colot_cutler_et al._2016, title={50 years of Arabidopsis research: Highlights and future directions}, volume={209}, number={3}, journal={New Phytologist}, author={Provart, N. J. and Alonso, J. and Assmann, S. M. and Bergmann, D. and Brady, S. M. and Brkljacic, J. and Browse, J. and Chapple, C. and Colot, V. and Cutler, S. and et al.}, year={2016}, pages={921–944} }
 @article{merchante_hu_heber_alonso_stepanova_2016, title={A Ribosome Footprinting Protocol for Plants}, volume={6}, ISSN={2331-8325}, url={http://dx.doi.org/10.21769/bioprotoc.1985}, DOI={10.21769/bioprotoc.1985}, number={21}, journal={BIO-PROTOCOL}, publisher={Bio-Protocol, LLC}, author={Merchante, Catharina and Hu, Qiwen and Heber, Steffen and Alonso, Jose and Stepanova, Anna}, year={2016} }
 @article{stepanova_alonso_2016, title={Auxin catabolism unplugged: Role of IAA oxidation in auxin homeostasis}, volume={113}, ISSN={["0027-8424"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84989870534&partnerID=MN8TOARS}, DOI={10.1073/pnas.1613506113}, abstractNote={Plants rely on the levels and concentration gradients of the hormone auxin as crucial information cues to trigger and modulate almost every aspect of their life cycle, from establishing embryo polarity to promoting phototropic and gravitropic responses (1). Thus, it is critical for plants to finely control the levels of the bioactive form of this hormone, both at spatial and temporal levels. Local production, transport, conjugation, storage, and catabolism are all well-known processes participating in the dynamic regulation of auxin homeostasis (2). It is, however, unknown whether or how these different auxin homeostasis mechanisms talk to each other. In the past 30 y, the identification of the genes coding for the key components of the auxin biosynthetic, transport, and conjugation machineries has proven instrumental in assessing the contribution of each of these processes to concrete developmental pathways, as well as to specific plant responses to environmental changes. Although much less is known about the ways the predominant auxin, indole-3-acetic acid (IAA), is catabolized, classical labeling and metabolite quantification experiments indicate that the oxidation of IAA into 2-oxindole-3-acetic acid (oxIAA) is one of the most prevalent mechanisms to inactivate this hormone (3). Identifying the specific enzymes catalyzing this reaction in vivo has proved to be more challenging than anticipated, as different plant peroxidases and oxygenases, the original suspects for catalyzing this reaction, were found not to play a significant physiological role in the production of oxIAA (3). The recent identification of a rice 2-oxoglutarate-dependent-Fe(II) dioxygenase, DIOXYGENASE FOR AUXIN OXIDATION ( DAO ), originally linked to male fertility in rice and capable of oxidizing IAA into oxIAA (1⇓⇓–4), has opened new avenues for addressing the physiological significance of auxin catabolism in plant growth and development. In PNAS, Zhang et al. (5), Porco et al. (6), and Mellor et al. (7 … 

[↵][1]1To whom correspondence should be addressed. Email: jmalonso{at}ncsu.edu.

 [1]: #xref-corresp-1-1}, number={39}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2016}, month={Sep}, pages={10742–10744} }
 @article{stepanova_alonso_2016, title={Cutting Out the Middle Man in Light-Hormone Interactions}, volume={39}, ISSN={["1878-1551"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85000977812&partnerID=MN8TOARS}, DOI={10.1016/j.devcel.2016.11.013}, abstractNote={In this issue of Developmental Cell, Shi et al. (2016a) show that red-light-activated phytochrome B interacts with transcriptional regulators of ethylene signaling, EIN3/EIL1, triggering their degradation by bringing the F-box proteins EBF1 and 2 to the complex. These findings provide a paradigm for crosstalk between light and hormone signaling pathways.}, number={5}, journal={DEVELOPMENTAL CELL}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2016}, month={Dec}, pages={524–526} }
 @article{hu_merchante_stepanova_alonso_heber_2016, title={Genome-wide search for translated upstream open reading frames in Arabidopsis thaliana}, volume={15}, number={2}, journal={IEEE Transactions on Nanobioscience}, author={Hu, Q. W. and Merchante, C. and Stepanova, A. N. and Alonso, J. M. and Heber, S.}, year={2016}, pages={150–159} }
 @book{merchante_brumós_yun_stepanova_alonso_2016, title={Hormone-Mediated Gene-Specific Translation Regulation}, url={http://hdl.handle.net/10630/11885}, author={Merchante, C. and Brumós, J. and Yun, J. and Stepanova, A. and Alonso, J.}, year={2016} }
 @article{alonso_stepanova_2015, title={A Recombineering-based gene tagging system for Arabidopsis}, volume={1227}, journal={Bacterial artificial chromosomes, 2nd edition}, author={Alonso, J. M. and Stepanova, A. N.}, year={2015}, pages={233–243} }
 @inbook{hu_merchante_stepanova_alonso_heber_2015, title={A Stacking-Based Approach to Identify Translated Upstream Open Reading Frames in Arabidopsis Thaliana}, volume={9096}, ISBN={9783319190471 9783319190488}, ISSN={0302-9743 1611-3349}, url={http://dx.doi.org/10.1007/978-3-319-19048-8_12}, DOI={10.1007/978-3-319-19048-8_12}, abstractNote={Upstream open reading frames (uORFs) are open reading frames located within the 5’ UTR of an mRNA. It is believed that translated uORFs reduce the translational efficiency of the main coding region, and play an important role in gene regulation. However, only few uORFs are experimentally characterized. In this paper, we use ribosome footprinting together with a stacking-based classification approach to identify translated uORFs in Arabidopsis thaliana. Our approach resulted in a set of 5360 potentially translated uORFs in 2051 genes. GO terms enriched in uORF-containing genes include gene regulation, signal transduction and metabolic pathway. The identified uORFs occur with a higher frequency in multi-isoform genes, and many uORFs are affected by alternative transcript start sites or alternative splicing events.}, booktitle={Bioinformatics Research and Applications}, publisher={Springer International Publishing}, author={Hu, Qiwen and Merchante, Catharina and Stepanova, Anna N. and Alonso, Jose M. and Heber, Steffen}, year={2015}, pages={138–149} }
 @inproceedings{hu_merchante_stepanova_alonso_heber_2015, title={A stacking-based approach to identify translated upstream open reading frames in Arabidopsis thaliana}, volume={9096}, booktitle={Bioinformatics research and applications (isbra 2015)}, author={Hu, Q. W. and Merchante, C. and Stepanova, A. N. and Alonso, J. M. and Heber, S.}, year={2015}, pages={138–149} }
 @article{fabregas_formosa-jordan_confraria_siligato_alonso_swarup_bennett_mahonen_cano-delgado_ibanes_2015, title={Auxin Influx Carriers Control Vascular Patterning and Xylem Differentiation in Arabidopsis thaliana}, volume={11}, ISSN={["1553-7404"]}, DOI={10.1371/journal.pgen.1005183}, abstractNote={Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.}, number={4}, journal={PLOS GENETICS}, author={Fabregas, Norma and Formosa-Jordan, Pau and Confraria, Ana and Siligato, Riccardo and Alonso, Jose M. and Swarup, Ranjan and Bennett, Malcolm J. and Mahonen, Ari Pekka and Cano-Delgado, Ana I. and Ibanes, Marta}, year={2015}, month={Apr} }
 @article{moreno-risueno_sozzani_yardimici_petricka_vernoux_blilou_alonso_winter_ohler_scheres_et al._2015, title={Bird and Scarecrow proteins organize tissue formation in Arabidopsis roots}, volume={350}, ISSN={["1095-9203"]}, DOI={10.1126/science.aad1171}, abstractNote={Tissue patterns are dynamically maintained. Continuous formation of plant tissues during postembryonic growth requires asymmetric divisions and the specification of cell lineages. We show that the BIRDs and SCARECROW regulate lineage identity, positional signals, patterning, and formative divisions throughout Arabidopsis root growth. These transcription factors are postembryonic determinants of the ground tissue stem cells and their lineage. Upon further activation by the positional signal SHORT-ROOT (a mobile transcription factor), they direct asymmetric cell divisions and patterning of cell types. The BIRDs and SCARECROW with SHORT-ROOT organize tissue patterns at all formative steps during growth, ensuring developmental plasticity.}, number={6259}, journal={Science}, author={Moreno-Risueno, MA. and Sozzani, R. and Yardimici, GG. and Petricka, JJ. and Vernoux, T. and Blilou, I. and Alonso, J. and Winter, CM. and Ohler, U. and Scheres, B. and et al.}, year={2015}, pages={426–430} }
 @article{worden_wilkop_esteve_jeannotte_lathe_vernhettes_weimer_hicks_alonso_labavitch_et al._2015, title={CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN}, volume={167}, ISSN={0032-0889 1532-2548}, url={http://dx.doi.org/10.1104/pp.114.249003}, DOI={10.1104/pp.114.249003}, abstractNote={Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.}, number={2}, journal={Plant Physiology}, publisher={Oxford University Press (OUP)}, author={Worden, Natasha and Wilkop, Thomas E. and Esteve, Victor Esteva and Jeannotte, Richard and Lathe, Rahul and Vernhettes, Samantha and Weimer, Bart and Hicks, Glenn and Alonso, Jose and Labavitch, John and et al.}, year={2015}, month={Feb}, pages={381–393} }
 @article{merchante_brumos_yun_hu_spencer_enriquez_binder_heber_stepanova_alonso_2015, title={Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2}, volume={163}, ISSN={["1097-4172"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84948814371&partnerID=MN8TOARS}, DOI={10.1016/j.cell.2015.09.036}, abstractNote={The central role of translation in modulating gene activity has long been recognized, yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to a specific stimulus has only recently become technically feasible. Using the well-characterized signaling pathway of the phytohormone ethylene and plant-optimized genome-wide ribosome footprinting, we have uncovered a molecular mechanism linking this hormone’s perception to the activation of a gene-specific translational control mechanism. Characterization of one of the targets of this translation regulatory machinery, the ethylene signaling component EBF2, indicates that the signaling molecule EIN2 and the nonsense-mediated decay proteins UPFs play a central role in this ethylene-induced translational response. Furthermore, the 3′UTR of EBF2 is sufficient to confer translational regulation and required for the proper activation of ethylene responses. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator.}, number={3}, journal={CELL}, author={Merchante, Catharina and Brumos, Javier and Yun, Jeonga and Hu, Qiwen and Spencer, Kristina R. and Enriquez, Paul and Binder, Brad M. and Heber, Steffen and Stepanova, Anna N. and Alonso, Jose M.}, year={2015}, month={Oct}, pages={684–697} }
 @inproceedings{hu_merchante_stepanova_alonso_heber_2015, title={Mining transcript features related to translation in Arabidopsis using LASSO and random forest}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84960900444&partnerID=MN8TOARS}, DOI={10.1109/iccabs.2015.7344713}, abstractNote={Translation is an important process for all living organisms. During translation, messenger RNA is rewritten into protein. Multiple control mechanisms determine how much protein is generated during translation. In particular, several regulatory elements located on mRNA transcripts are known to affect translation. In this study, a genome-wide analysis was performed to mine features related to translation in the genome of Arabidopsis thaliana. We used ribosome footprinting data to measure translation and constructed a predictive model using LASSO and random forest to select features that likely affect translation. We identified multiple transcript features and measured their influence on translation in different transcript regions. We found that features related to different translation stages may have a different impact on translation; often, features relevant to the elongation step were playing a stronger role. Interestingly, we found that the contribution of features may be different for transcripts belonging to different functional groups, suggesting that transcripts might employ different mechanisms for the regulation of translation.}, booktitle={International conference on computational advances in bio and medical}, author={Hu, Q. W. and Merchante, C. and Stepanova, A. N. and Alonso, Jose and Heber, S.}, year={2015} }
 @article{alonso_stepanova_2015, title={Plant functional genomics methods and protocols second edition preface}, volume={1284}, journal={Plant functional genomics: methods and protocols, 2nd edition}, author={Alonso, J. M. and Stepanova, A. N.}, year={2015}, pages={V-} }
 @book{alonso_stepanova_2015, place={Humana Press}, edition={2nd}, series={Methods in Molecular Biology}, title={Plant functional genomics: Methods and protocols}, ISBN={9781493924431 9781493924448}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84954619611&partnerID=MN8TOARS}, DOI={10.1007/978-1-4939-2444-8}, abstractNote={This second edition volume discusses the revolutionary development of faster and less expensive DNA sequencing technologies from the past 10 years and focuses on general technologies that can be utili}, journal={Plant Functional Genomics: Methods and Protocols: Second Edition}, publisher={New York}, author={Alonso, Jose and STEPANOVA, ANNA}, editor={Alonso, J. M. and Stepanova, A. N.Editors}, year={2015}, pages={1–526}, collection={Methods in Molecular Biology} }
 @article{han_alonso_rojas-pierce_2015, title={Regulator of bulb biogenesis1 (RBB1) is involved in vacuole bulb formation in arabidopsis}, volume={10}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84928576948&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0125621}, abstractNote={Vacuoles are dynamic compartments with constant fluctuations and transient structures such as trans-vacuolar strands and bulbs. Bulbs are highly dynamic spherical structures inside vacuoles that are formed by multiple layers of membranes and are continuous with the main tonoplast. We recently carried out a screen for mutants with abnormal trafficking to the vacuole or aberrant vacuole morphology. We characterized regulator of bulb biogenesis1-1 (rbb1-1), a mutant in Arabidopsis that contains increased numbers of bulbs when compared to the parental control. rbb1-1 mutants also contain fewer transvacuolar strands than the parental control, and we propose the hypothesis that the formation of transvacuolar strands and bulbs is functionally related. We propose that the bulbs may function transiently to accommodate membranes and proteins when transvacuolar strands fail to elongate. We show that RBB1 corresponds to a very large protein of unknown function that is specific to plants, is present in the cytosol, and may associate with cellular membranes. RBB1 is involved in the regulation of vacuole morphology and may be involved in the establishment or stability of trans-vacuolar strands and bulbs.}, number={4}, journal={PLoS One}, publisher={Public Library of Science (PLoS)}, author={Han, S. W. and Alonso, Jose and Rojas-Pierce, Marcela}, editor={Bassham, DianeEditor}, year={2015}, pages={e0125621} }
 @inbook{alonso_stepanova_2014, title={Arabidopsis Transformation with Large Bacterial Artificial Chromosomes}, booktitle={Arabidopsis Protocols}, author={Alonso, Jose M. and Stepanova, Anna N.}, year={2014}, pages={271–283} }
 @article{brumos_alonso_stepanova_2014, title={Genetic aspects of auxin biosynthesis and its regulation}, volume={151}, ISSN={["1399-3054"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84898468296&partnerID=MN8TOARS}, DOI={10.1111/ppl.12098}, abstractNote={Auxin is an essential plant hormone that controls nearly every aspect of a plant's life, from embryo development to organ senescence. In the last decade the key genes involved in auxin transport, perception, signaling and response have been identified and characterized, but the elucidation of auxin biosynthesis has proven to be especially challenging. In plants, a significant amount of indole-3-acetic acid (IAA), the predominant biologically active form of auxin, is synthesized via a simple two-step route where indole-3-pyruvic acid (IPyA) produced from l-tryptophan by tryptophan aminotransferases (TAA1/TAR) is converted to IAA by the YUC family of flavin monooxygenases. The TAA1/TAR and YUC gene families constitute the first complete auxin biosynthetic pathway described in plants. Detailed characterization of these genes' expression patterns suggested a key role of local auxin biosynthesis in plant development. This has prompted an active search for the molecular mechanisms that regulate the spatiotemporal activity of the IPyA route. In addition to the TAA1/TAR and YUC-mediated auxin biosynthesis, several alternative routes of IAA production have been postulated to function in plants, but their biological significance is yet to be demonstrated. Herein, we take a genetic perspective to describe the current view of auxin biosynthesis and its regulation in plants, focusing primarily on Arabidopsis.}, number={1}, journal={PHYSIOLOGIA PLANTARUM}, author={Brumos, Javier and Alonso, Jose M. and Stepanova, Anna N.}, year={2014}, month={May}, pages={3–12} }
 @article{band_wells_fozard_ghetiu_french_pound_wilson_yu_li_hijazi_et al._2014, title={Systems analysis of auxin transport in the arabidopsis root apex}, volume={26}, number={3}, journal={Plant Cell}, author={Band, L. R. and Wells, D. M. and Fozard, J. A. and Ghetiu, T. and French, A. P. and Pound, M. P. and Wilson, M. H. and Yu, L. and Li, W. D. and Hijazi, H. I. and et al.}, year={2014}, pages={862–875} }
 @article{catalá_lópez-cobollo_mar castellano_angosto_alonso_ecker_salinas_2014, title={The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation
 }, volume={26}, ISSN={1532-298X 1040-4651}, url={http://dx.doi.org/10.1105/tpc.114.127605}, DOI={10.1105/tpc.114.127605}, abstractNote={Abstract In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis.}, number={8}, journal={The Plant Cell}, publisher={Oxford University Press (OUP)}, author={Catalá, Rafael and López-Cobollo, Rosa and Mar Castellano, M. and Angosto, Trinidad and Alonso, José M. and Ecker, Joseph R. and Salinas, Julio}, year={2014}, month={Aug}, pages={3326–3342} }
 @article{pietra_gustavsson_kiefer_kalmbach_horstedt_ikeda_stepanova_alonso_grebe_2013, title={Arabidopsis SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity}, volume={4}, ISSN={["2041-1723"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84889562507&partnerID=MN8TOARS}, DOI={10.1038/ncomms3779}, abstractNote={The orientation of cell division and the coordination of cell polarity within the plane of the tissue layer (planar polarity) contribute to shape diverse multicellular organisms. The root of Arabidopsis thaliana displays regularly oriented cell divisions, cell elongation and planar polarity providing a plant model system to study these processes. Here we report that the SABRE protein, which shares similarity with proteins of unknown function throughout eukaryotes, has important roles in orienting cell division and planar polarity. SABRE localizes at the plasma membrane, endomembranes, mitotic spindle and cell plate. SABRE stabilizes the orientation of CLASP-labelled preprophase band microtubules predicting the cell division plane, and of cortical microtubules driving cell elongation. During planar polarity establishment, sabre is epistatic to clasp at directing polar membrane domains of Rho-of-plant GTPases. Our findings mechanistically link SABRE to CLASP-dependent microtubule organization, shedding new light on the function of SABRE-related proteins in eukaryotes.}, journal={NATURE COMMUNICATIONS}, author={Pietra, Stefano and Gustavsson, Anna and Kiefer, Christian and Kalmbach, Lothar and Horstedt, Per and Ikeda, Yoshihisa and Stepanova, Anna N. and Alonso, Jose M. and Grebe, Markus}, year={2013}, month={Nov} }
 @misc{merchante_alonso_stepanova_2013, title={Ethylene signaling: simple ligand, complex regulation}, volume={16}, ISSN={["1879-0356"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84885368622&partnerID=MN8TOARS}, DOI={10.1016/j.pbi.2013.08.001}, abstractNote={The hormone ethylene plays numerous roles in plant development. In the last few years the model of ethylene signaling has evolved from an initially largely linear route to a much more complex pathway with multiple feedback loops. Identification of key transcriptional and post-transcriptional regulatory modules controlling expression and/or stability of the core pathway components revealed that ethylene perception and signaling are tightly regulated at multiple levels. This review describes the most current outlook on ethylene signal transduction and emphasizes the latest discoveries in the ethylene field that shed light on the mechanistic mode of action of the central pathway components CTR1 and EIN2, as well as on the post-transcriptional regulatory steps that modulate the signaling flow through the pathway.}, number={5}, journal={CURRENT OPINION IN PLANT BIOLOGY}, author={Merchante, Catharina and Alonso, Jose M. and Stepanova, Anna N.}, year={2013}, month={Oct}, pages={554–560} }
 @article{hill_mathews_kim_street_wildes_chiang_mason_alonso_ecker_kieber_et al._2013, title={Functional Characterization of Type-B Response Regulators in the Arabidopsis Cytokinin Response
 
 }, volume={162}, ISSN={1532-2548}, url={http://dx.doi.org/10.1104/pp.112.208736}, DOI={10.1104/pp.112.208736}, abstractNote={Cytokinins play critical roles in plant growth and development, with the transcriptional response to cytokinin being mediated by the type-B response regulators. In Arabidopsis (Arabidopsis thaliana), type-B response regulators (ARABIDOPSIS RESPONSE REGULATORS [ARRs]) form three subfamilies based on phylogenic analysis, with subfamily 1 having seven members and subfamilies 2 and 3 each having two members. Cytokinin responses are predominantly mediated by subfamily 1 members, with cytokinin-mediated effects on root growth and root meristem size correlating with type-B ARR expression levels. To determine which type-B ARRs can functionally substitute for the subfamily 1 members ARR1 or ARR12, we expressed different type-B ARRs from the ARR1 promoter and assayed their ability to rescue arr1 arr12 double mutant phenotypes. ARR1, as well as a subset of other subfamily 1 type-B ARRs, restore the cytokinin sensitivity to arr1 arr12. Expression of ARR10 from the ARR1 promoter results in cytokinin hypersensitivity and enhances shoot regeneration from callus tissue, correlating with enhanced stability of the ARR10 protein compared with the ARR1 protein. Examination of transfer DNA insertion mutants in subfamilies 2 and 3 revealed little effect on several well-characterized cytokinin responses. However, a member of subfamily 2, ARR21, restores cytokinin sensitivity to arr1 arr12 roots when expressed from the ARR1 promoter, indicating functional conservation of this divergent family member. Our results indicate that the type-B ARRs have diverged in function, such that some, but not all, can complement the arr1 arr12 mutant. In addition, our results indicate that type-B ARR expression profiles in the plant, along with posttranscriptional regulation, play significant roles in modulating their contribution to cytokinin signaling.}, number={1}, journal={Plant Physiology}, publisher={Oxford University Press (OUP)}, author={Hill, Kristine and Mathews, Dennis E. and Kim, Hyo Jung and Street, Ian H. and Wildes, Sarah L. and Chiang, Yi-Hsuan and Mason, Michael G. and Alonso, Jose M. and Ecker, Joseph R. and Kieber, Joseph J. and et al.}, year={2013}, month={May}, pages={212–224} }
 @article{kopycki_wieduwild_kohlschmidt_brandt_stepanova_alonso_pedras_abel_grubb_2013, title={Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition}, volume={450}, ISSN={["1470-8728"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84873043425&partnerID=MN8TOARS}, DOI={10.1042/bj20121403}, abstractNote={Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios.}, number={1}, journal={BIOCHEMICAL JOURNAL}, author={Kopycki, Jakub and Wieduwild, Elizabeth and Kohlschmidt, Janine and Brandt, Wolfgang and Stepanova, Anna N. and Alonso, Jose M. and Pedras, M. Soledade C. and Abel, Steffen and Grubb, C. Douglas}, year={2013}, month={Feb}, pages={37–46} }
 @article{robert_grones_stepanova_robles_lokerse_alonso_weijers_friml_2013, title={Local Auxin Sources Orient the Apical-Basal Axis in Arabidopsis Embryos}, volume={23}, ISSN={["1879-0445"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84890790080&partnerID=MN8TOARS}, DOI={10.1016/j.cub.2013.09.039}, abstractNote={Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin to generate an asymmetric auxin response that specifies the embryonic apical-basal axis. The auxin flow directionality depends on the polarized subcellular localization of PIN-FORMED (PIN) auxin transporters. It remains unknown which mechanisms and spatial cues guide cell polarization and axis orientation in early embryos. Herein, we provide conceptually novel insights into the formation of embryonic axis in Arabidopsis by identifying a crucial role of localized tryptophan-dependent auxin biosynthesis. Local auxin production at the base of young embryos and the accompanying PIN7-mediated auxin flow toward the proembryo are required for the apical auxin response maximum and the specification of apical embryonic structures. Later in embryogenesis, the precisely timed onset of localized apical auxin biosynthesis mediates PIN1 polarization, basal auxin response maximum, and specification of the root pole. Thus, the tight spatiotemporal control of distinct local auxin sources provides a necessary, non-cell-autonomous trigger for the coordinated cell polarization and subsequent apical-basal axis orientation during embryogenesis and, presumably, also for other polarization events during postembryonic plant life.}, number={24}, journal={CURRENT BIOLOGY}, author={Robert, Helene S. and Grones, Peter and Stepanova, Anna N. and Robles, Linda M. and Lokerse, Annemarie S. and Alonso, Jose M. and Weijers, Dolf and Friml, Jiri}, year={2013}, month={Dec}, pages={2506–2512} }
 @article{robles_stepanova_alonso_2013, title={Molecular Mechanisms of EthyleneAuxin Interaction}, volume={6}, ISSN={["1752-9867"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84891933044&partnerID=MN8TOARS}, DOI={10.1093/mp/sst113}, abstractNote={During the century-long history of plant hormone research, the focus in this area has shifted from the original physiological experiments to molecular, genetic, biochemical, and, more recently, genomic approaches. During this journey, we have learned about the many effects these natural compounds have on plant growth and development at the morphological and molecular levels. We have also uncovered how these molecules are sensed by the plant cell and how they trigger signaling cascades that relay information to the nucleus, ultimately culminating in a transcriptional cascade. With this fundamental knowledge at hand, the main efforts in the field have turned towards a more integrative systems approach, in which the function of a hormone is considered in the context of the complex and dynamic net of interactions with other hormones or, more broadly, with other developmental and environmental signals. Initially, due to the complexity of the problem, most crosstalk studies have focused on pairwise interactions between two or few hormones. Special attention has been given to the interactions between ethylene and auxin, perhaps in part because of the experimental tractability of some of the processes in which the interaction between these two hormones is most manifested. An excellent review has been recently written describing the many different processes in which these two hormones are known to interact, either synergistically, such as root growth inhibition, or antagonistically, as is the case for lateral root formation (Muday et al., 2012Muday G.K. Rahman A. Binder B.M Auxin and ethylene: collaborators or competitors?.Trends Plant Sci. 2012; 17: 181-195Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Rather than following this process-centered approach, herein we decided to focus on the molecular mechanisms involved in the interaction between ethylene and auxin. We take a closer look at the best-known transcriptional and posttranscriptional points of interaction, as well as revisit the most recent experimental findings that suggested additional levels of ethylene–auxin crosstalk. The examples illustrating these types of interactions are by far the most abundant in recent literature, although this is probably not only due to the prevalence of the transcriptional-level control in inter-hormone relationships, but also due to the technical ease of monitoring transcriptomic changes. For example, both auxin and ethylene have been shown to reciprocally regulate the transcriptional activity of key biosynthetic genes creating an interlocked feedback loop (Figure 1A). ACC synthase (ACS), the enzyme that catalyzes the rate-limiting step of ethylene biosynthesis, is transcriptionally up-regulated by auxin (Tsuchisaka and Theologis, 2004Tsuchisaka A. Theologis A Unique and overlapping expression patterns among the Arabidopsis 1-amino- cyclopropane-1-carboxylate synthase gene family members.Plant Physiol. 2004; 136: 2982-3000Crossref PubMed Scopus (289) Google Scholar). In fact, three auxin-regulated ACS genes possess putative auxin-response-factor binding sites in the 500-bp upstream promoter regions, suggesting a possible direct link (Stepanova et al., 2007Stepanova A.N. Yun J. Likhacheva A.V. Alonso J.M Multilevel interactions between ethylene and auxin in Arabidopsis roots.Plant Cell. 2007; 19: 2169-2185Crossref PubMed Scopus (410) Google Scholar). On the other hand, ethylene has been shown to up-regulate auxin biosynthesis in a tissue-specific manner mediating processes such as inhibition of growth in roots or differential cell elongation in apical hooks (Stepanova et al., 2005Stepanova A.N. Hoyt J.M. Hamilton A.A. Alonso J.M A link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis.Plant Cell. 2005; 17: 2230-2242Crossref PubMed Scopus (368) Google Scholar, Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jurgens G. Alonso J.M TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar; Vandenbussche et al., 2010Vandenbussche F. Petrasek J. Zadnikova P. Hoyerova K. Pesek B. Raz V. Swarup R. Bennett M. Zazimalova E. Benkova E. et al.The auxin influx carriers AUX1 and LAX3 are involved in auxin–ethylene interactions during apical hook development in Arabidopsis thaliana seedlings.Development. 2010; 137: 597-606Crossref PubMed Scopus (196) Google Scholar; Zadnikova et al., 2010Zadnikova P. Petrasek J. Marhavy P. Raz V. Vandenbussche F. Ding Z. Schwarzerova K. Morita M.T. Tasaka M. Hejatko J. et al.Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana.Development. 2010; 137: 607-617Crossref PubMed Scopus (236) Google Scholar; Gallego-Bartolome et al., 2011Gallego-Bartolome J. Arana M.V. Vandenbussche F. Zadnikova P. Minguet E.G. Guardiola V. Van Der Straeten D. Benkova E. Alabadi D. Blazquez M.A Hierarchy of hormone action controlling apical hook development in Arabidopsis.Plant J. 2011; 67: 622-634Crossref PubMed Scopus (75) Google Scholar). The first evidence that this regulation is, at least in part, mediated by an ethylene-dependent induction of auxin biosynthetic genes came from forward genetic screens aimed at identifying mutants impaired in ethylene responses in roots (Stepanova et al., 2005Stepanova A.N. Hoyt J.M. Hamilton A.A. Alonso J.M A link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis.Plant Cell. 2005; 17: 2230-2242Crossref PubMed Scopus (368) Google Scholar, Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jurgens G. Alonso J.M TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). Multiple alleles of auxin biosynthetic genes, including anthranilate synthases weak ethylene insensitive2 (wei2) and wei7 and a tryptophan aminotransferase of Arabidopsis taa1/wei8, were isolated in these screens. Importantly, all three WEI genes were found to be transcriptionally up-regulated by ethylene specifically in roots leading to increased auxin production in the presence of ethylene and, consequently, to root growth inhibition, thus providing a mechanistic explanation for the root-specific ethylene insensitivity of the corresponding mutants (Stepanova et al., 2005Stepanova A.N. Hoyt J.M. Hamilton A.A. Alonso J.M A link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis.Plant Cell. 2005; 17: 2230-2242Crossref PubMed Scopus (368) Google Scholar, Stepanova et al., 2008Stepanova A.N. Robertson-Hoyt J. Yun J. Benavente L.M. Xie D.Y. Dolezal K. Schlereth A. Jurgens G. Alonso J.M TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.Cell. 2008; 133: 177-191Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). The transcriptional interaction between ethylene and auxin is not restricted to biosynthetic genes. For example, ethylene has been shown to promote auxin transport from the meristem towards the root elongation zone, where the resulting auxin increase triggers the well-known ethylene-mediated root growth inhibition (Lewis et al., 2011Lewis D.R. Negi S. Sukumar P. Muday G.K Ethylene inhibits lateral root development, increases IAA transport and expression of PIN3 and PIN7 auxin efflux carriers.Development. 2011; 138: 3485-3495Crossref PubMed Scopus (180) Google Scholar). In fact, ethylene was found to up-regulate the expression of several auxin efflux transporters (PINFORMED1 (PIN1), PIN2, and PIN4) and the auxin influx carrier AUXIN RESISTANT1 (AUX1) (Vandenbussche et al., 2010Vandenbussche F. Petrasek J. Zadnikova P. Hoyerova K. Pesek B. Raz V. Swarup R. Bennett M. Zazimalova E. Benkova E. et al.The auxin influx carriers AUX1 and LAX3 are involved in auxin–ethylene interactions during apical hook development in Arabidopsis thaliana seedlings.Development. 2010; 137: 597-606Crossref PubMed Scopus (196) Google Scholar; Lewis et al., 2011Lewis D.R. Negi S. Sukumar P. Muday G.K Ethylene inhibits lateral root development, increases IAA transport and expression of PIN3 and PIN7 auxin efflux carriers.Development. 2011; 138: 3485-3495Crossref PubMed Scopus (180) Google Scholar; Muday et al., 2012Muday G.K. Rahman A. Binder B.M Auxin and ethylene: collaborators or competitors?.Trends Plant Sci. 2012; 17: 181-195Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). It remains to be determined whether or not this regulation is directly mediated by the key ethylene transcription factor ETHYLENE INSENSITIVE3 (EIN3) or represents a more indirect downstream ethylene response. In addition to the specific examples discussed above, a more comprehensive picture of the transcriptional interaction between these two hormones has been obtained from several whole-genome transcriptomic analyses (reviewed in Muday et al., 2012Muday G.K. Rahman A. Binder B.M Auxin and ethylene: collaborators or competitors?.Trends Plant Sci. 2012; 17: 181-195Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). These high-throughput studies revealed that, although ethylene and auxin trigger similar phenotypic responses, such as root growth inhibition, the two hormones share relatively few common target genes, suggesting that, in general, different plant hormones use unique routes to regulate similar processes (Muday et al., 2012Muday G.K. Rahman A. Binder B.M Auxin and ethylene: collaborators or competitors?.Trends Plant Sci. 2012; 17: 181-195Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar; Zheng et al., 2013Zheng Z. Guo Y. Novak O. Dai X. Zhao Y. Ljung K. Noel J.P. Chory J Coordination of auxin and ethylene biosynthesis by the aminotransferase VAS1.Nat. Chem. Biol. 2013; 9: 244-246Crossref PubMed Scopus (79) Google Scholar). A more detailed approach involving auxin- and ethylene-insensitive mutants in combination with ethylene and auxin treatments, respectively, was employed to further elucidate the mechanisms that auxin and ethylene utilize to control gene expression (Stepanova et al., 2007Stepanova A.N. Yun J. Likhacheva A.V. Alonso J.M Multilevel interactions between ethylene and auxin in Arabidopsis roots.Plant Cell. 2007; 19: 2169-2185Crossref PubMed Scopus (410) Google Scholar). Again, the vast majority of genes were found to be regulated independently by each hormone and a much smaller subset of genes was shown to be co-regulated by both auxin and ethylene. Using an elegant design, additional categories of genes were identified including ethylene-regulated genes controlled via auxin-dependent or auxin-mediated pathways, as well as auxin-regulated genes controlled in an ethylene-dependent or ethylene-mediated manner (Stepanova et al., 2007Stepanova A.N. Yun J. Likhacheva A.V. Alonso J.M Multilevel interactions between ethylene and auxin in Arabidopsis roots.Plant Cell. 2007; 19: 2169-2185Crossref PubMed Scopus (410) Google Scholar). Although not as abundant and, perhaps, not as well characterized in comparison to the transcriptional interactions described above, several examples of the ethylene–auxin crosstalk at the posttranscriptional level can be found in recent literature (Vandenbussche et al., 2010Vandenbussche F. Petrasek J. Zadnikova P. Hoyerova K. Pesek B. Raz V. Swarup R. Bennett M. Zazimalova E. Benkova E. et al.The auxin influx carriers AUX1 and LAX3 are involved in auxin–ethylene interactions during apical hook development in Arabidopsis thaliana seedlings.Development. 2010; 137: 597-606Crossref PubMed Scopus (196) Google Scholar; He et al., 2011He W. Brumos J. Li H. Ji Y. Ke M. Gong X. Zeng Q. Li W. Zhang X. An F. et al.A small-molecule screen identifies L-kynurenine as a competitive inhibitor of TAA1/TAR activity in ethylene-directed auxin biosynthesis and root growth in Arabidopsis.Plant Cell. 2011; 23: 3944-3960Crossref PubMed Scopus (250) Google Scholar; Schepetilnikov et al., 2013Schepetilnikov M. Dimitrova M. Mancera-Martinez E. Geldreich A. Keller M. Ryabova L.A TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.EMBO J. 2013; 32: 1087-1102Crossref PubMed Scopus (200) Google Scholar). Vandenbussche et al. reported that the protein turnover of the auxin influx carrier AUX1 in response to ethylene is stimulated specifically on the inner side of the seedling apical hook (Vandenbussche et al., 2010Vandenbussche F. Petrasek J. Zadnikova P. Hoyerova K. Pesek B. Raz V. Swarup R. Bennett M. Zazimalova E. Benkova E. et al.The auxin influx carriers AUX1 and LAX3 are involved in auxin–ethylene interactions during apical hook development in Arabidopsis thaliana seedlings.Development. 2010; 137: 597-606Crossref PubMed Scopus (196) Google Scholar). This tissue-specific effect on the rates of AUX1 turnover indicates the existence of an ethylene-mediated posttranscriptional regulation and suggests involvement of an additional molecular mechanism in the interaction between auxin and ethylene. Another example of posttranscriptional interaction between these two hormones comes from a detailed analysis of modulation of the ethylene response by auxin. Initially, Stepanova et al. showed that the ethylene-induced EIN3-mediated response in the root transition zone requires high levels of auxin activity in this region (Stepanova et al., 2007Stepanova A.N. Yun J. Likhacheva A.V. Alonso J.M Multilevel interactions between ethylene and auxin in Arabidopsis roots.Plant Cell. 2007; 19: 2169-2185Crossref PubMed Scopus (410) Google Scholar). Later, a more detailed analysis by He et al. discovered that the protein levels of the master transcriptional regulator of the ethylene response, EIN3, are induced by auxin (He et al., 2011He W. Brumos J. Li H. Ji Y. Ke M. Gong X. Zeng Q. Li W. Zhang X. An F. et al.A small-molecule screen identifies L-kynurenine as a competitive inhibitor of TAA1/TAR activity in ethylene-directed auxin biosynthesis and root growth in Arabidopsis.Plant Cell. 2011; 23: 3944-3960Crossref PubMed Scopus (250) Google Scholar) (Figure 1B). This auxin-mediated effect required the function of two F-box proteins, EIN3-BINDING F-BOX PROTEIN1 (EBF1) and EBF2, responsible for targeting EIN3 for proteasomal degradation. Of key relevance to this discussion is the finding that, unlike ethylene, auxin does not alter the levels of EBF1 and EBF2; thus the molecular mechanism behind this auxin-mediated posttranscriptional regulation of the EIN3 levels is yet to be uncovered. Besides the illustrative examples described above, there have been additional reports that may unveil, in the future, novel posttranscriptional nodes of interaction between ethylene and auxin (Skottke et al., 2011Skottke K.R. Yoon G.M. Kieber J.J. DeLong A Protein phosphatase 2A controls ethylene biosynthesis by differentially regulating the turnover of ACC synthase isoforms.PLoS Genet. 2011; 7: e1001370Crossref PubMed Scopus (111) Google Scholar; Schepetilnikov et al., 2013Schepetilnikov M. Dimitrova M. Mancera-Martinez E. Geldreich A. Keller M. Ryabova L.A TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.EMBO J. 2013; 32: 1087-1102Crossref PubMed Scopus (200) Google Scholar). Thus, for example, in a recent report, Schepetilnikov et al. showed that auxin activates the translational regulator TARGET-OF-RAPAMYCIN (TOR) (Schepetilnikov et al., 2013Schepetilnikov M. Dimitrova M. Mancera-Martinez E. Geldreich A. Keller M. Ryabova L.A TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.EMBO J. 2013; 32: 1087-1102Crossref PubMed Scopus (200) Google Scholar). Importantly, this activation has a selective effect on the translation of mRNAs that contain upstream open reading frames (uORFs) in their 5’ untranslated regions. Although these results per se do not provide clear evidence for the interaction between ethylene and auxin at the translational level, the abundance of uORFs in the transcripts of several critical ethylene signaling components, including ETHYLENE INSENSITIVE2 (EIN2) and EIN3, makes this possibility plausible (Yamada et al., 2003Yamada K. Lim J. Dale J.M. Chen H. Shinn P. Palm C.J. Southwick A.M. Wu H.C. Kim C. Nguyen M. et al.Empirical analysis of transcriptional activity in the Arabidopsis genome.Science. 2003; 302: 842-846Crossref PubMed Scopus (749) Google Scholar; Schepetilnikov et al., 2013Schepetilnikov M. Dimitrova M. Mancera-Martinez E. Geldreich A. Keller M. Ryabova L.A TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.EMBO J. 2013; 32: 1087-1102Crossref PubMed Scopus (200) Google Scholar). One of the most striking examples of interaction between ethylene and auxin was recently uncovered during the characterization of a genetic suppressor of the auxin biosynthetic mutant shade avoidance3 (sav3, an allele of taa1/wei8) named vas1 (Zheng et al., 2013Zheng Z. Guo Y. Novak O. Dai X. Zhao Y. Ljung K. Noel J.P. Chory J Coordination of auxin and ethylene biosynthesis by the aminotransferase VAS1.Nat. Chem. Biol. 2013; 9: 244-246Crossref PubMed Scopus (79) Google Scholar). The VAS1 aminotransferase was found to catalyze the transamination of the auxin precursor indole-3-pyruvic acid (IPyA) into the ubiquitous amino acid tryptophan (Trp) (Figure 1C). Importantly, for this reaction, VAS1 was shown to use the ethylene precursor methionine (Met) as the preferred amino group donor, thus linking together the biosyntheses of these two important plant hormones. In other words, VAS1 appears to inhibit both auxin and ethylene biosynthesis by simultaneously down-regulating the levels of IPyA and Met, the precursors of auxin and ethylene biosyntheses, respectively. Thus, VAS1 represents a novel mechanism of coordinating the levels of auxin and ethylene by linking their biosynthetic pathways at the metabolic level. Although we have focused this brief review exclusively on the crosstalk between auxin and ethylene, it must be noted that similar types of interactions are being discovered among other plant hormones. From the first studies focused on the effects of single hormones, the hormone biology field has progressed to new heights and is now embarking on an ambitious journey of disentangling the complex net of interactions between multiple plant hormones. Herein, with just a few representative examples, we have sampled the steadily increasing number of molecular mechanisms involved in hormonal crosstalk. We anticipate that, with the development of new and more powerful tools for exploring the many levels of posttranscriptional gene regulation, this area of research will continue to grow and bring many exciting new discoveries in the years to come. Work in the Alonso-Stepanova lab is supported by NSF-MCB0923727 grant to J.M.A. and A.N.S. and NSF-MCB 1158181 grant to J.M.A.}, number={6}, journal={MOLECULAR PLANT}, author={Robles, Linda and Stepanova, Anna and Alonso, Jose}, year={2013}, month={Nov}, pages={1734–1737} }
 @article{robles_stepanova_alonso_2013, title={Molecular mechanisms of ethylene–auxin interactions}, journal={Molecular plant}, author={Robles, Linda and Stepanova, Anna and Alonso, Jose}, year={2013}, pages={sst113} }
 @article{péret_swarup_ferguson_seth_yang_dhondt_james_casimiro_perry_syed_2012, title={AUX/LAX genes encode a family of auxin influx transporters that perform distinct functions during Arabidopsis development}, volume={24}, DOI={10.1105/tpc.112.097766}, abstractNote={Auxin transport, which is mediated by specialized influx and efflux carriers, plays a major role in many aspects of plant growth and development. AUXIN1 (AUX1) has been demonstrated to encode a high-affinity auxin influx carrier. In Arabidopsis thaliana, AUX1 belongs to a small multigene family comprising four highly conserved genes (i.e., AUX1 and LIKE AUX1 [LAX] genes LAX1, LAX2, and LAX3). We report that all four members of this AUX/LAX family display auxin uptake functions. Despite the conservation of their biochemical function, AUX1, LAX1, and LAX3 have been described to regulate distinct auxin-dependent developmental processes. Here, we report that LAX2 regulates vascular patterning in cotyledons. We also describe how regulatory and coding sequences of AUX/LAX genes have undergone subfunctionalization based on their distinct patterns of spatial expression and the inability of LAX sequences to rescue aux1 mutant phenotypes, respectively. Despite their high sequence similarity at the protein level, transgenic studies reveal that LAX proteins are not correctly targeted in the AUX1 expression domain. Domain swapping studies suggest that the N-terminal half of AUX1 is essential for correct LAX localization. We conclude that Arabidopsis AUX/LAX genes encode a family of auxin influx transporters that perform distinct developmental functions and have evolved distinct regulatory mechanisms.}, number={7}, journal={The Plant Cell Online}, author={Péret, Benjamin and Swarup, Kamal and Ferguson, Alison and Seth, Malvika and Yang, Yaodong and Dhondt, Stijn and James, Nicholas and Casimiro, Ilda and Perry, Paula and Syed, Adnan}, year={2012}, pages={2874–2885} }
 @article{peret_swarup_ferguson_seth_yang_dhondt_james_casimiro_perry_syed_et al._2012, title={AUX/LAX genes encode a family of auxin influx transporters that perform distinct functions during arabidopsis development}, volume={24}, number={7}, journal={Plant Cell}, author={Peret, B. and Swarup, K. and Ferguson, A. and Seth, M. and Yang, Y. D. and Dhondt, S. and James, N. and Casimiro, I. and Perry, P. and Syed, A. and et al.}, year={2012}, pages={2874–2885} }
 @article{he_brumos_li_ji_ke_gong_zeng_li_zhang_an_et al._2011, title={A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis}, volume={23}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.111.089029}, DOI={10.1105/tpc.111.089029}, abstractNote={The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.}, number={11}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={He, Wenrong and Brumos, Javier and Li, Hongjiang and Ji, Yusi and Ke, Meng and Gong, Xinqi and Zeng, Qinglong and Li, Wenyang and Zhang, Xinyan and An, Fengying and et al.}, year={2011}, month={Nov}, pages={3944–3960} }
 @article{zhou_benavente_stepanova_alonso_2011, title={A recombineering-based gene tagging system for Arabidopsis}, volume={66}, ISSN={["0960-7412"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79955809131&partnerID=MN8TOARS}, DOI={10.1111/j.1365-313x.2011.04524.x}, abstractNote={One of the most information-rich aspects of gene functional studies is characterization of gene expression profiles at cellular resolution, and subcellular localization of the corresponding proteins. These studies require visualization of the endogenous gene products using specific antibodies, or, more commonly, generation of whole-gene translational fusions with a reporter gene such as a fluorescent protein. To facilitate the generation of such translational fusions and to ensure that all cis-regulatory sequences are included, we have used a bacterial homologous recombination system (recombineering) to insert fluorescent protein tags into genes of interest harbored by transformation-competent bacterial artificial chromosomes (TACs). This approach has several advantages compared to other classical strategies. First, the researcher does not have to guess what the regulatory sequences of a gene are, as tens of thousands of base pairs flanking the gene of interest can be included in the construct. Second, because the genes of interest are not amplified by PCR, there are practically no limits to the size of a gene that can be tagged. Third, there are no restrictions on the location in which the fluorescent protein can be inserted, as the position is determined by sequence homology with the recombination primers. Finally, all of the required strains and TAC clones are publically available, and the experimental procedures described here are simple and robust. Thus, we suggest that recombineering-based gene tagging should be the gold standard for gene expression studies in Arabidopsis.}, number={4}, journal={PLANT JOURNAL}, author={Zhou, Rongrong and Benavente, Larissa M. and Stepanova, Anna N. and Alonso, Jose M.}, year={2011}, month={May}, pages={712–723} }
 @article{stepanova_alonso_2011, title={Bypassing Transcription: A Shortcut in Cytokinin-Auxin Interactions}, volume={21}, ISSN={["1534-5807"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-80054765937&partnerID=MN8TOARS}, DOI={10.1016/j.devcel.2011.09.016}, abstractNote={In this issue of Developmental Cell, Marhavý et al., 2011Marhavý P. Bielach A. Abas L. Abuzeineh A. Duclercq J. Tanaka H. Pařezová M. Petrášek J. Friml J. Kleine-Vehn J. Benková E. Dev. Cell. 2011; 21 (this issue): 796-804Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar uncover a transcription-independent molecular mechanism of interaction between auxin and cytokinin in the regulation of plant meristem function. By modulating endocytic trafficking of PIN1, cytokinin controls auxin flux and, therefore, auxin gradients. In this issue of Developmental Cell, Marhavý et al., 2011Marhavý P. Bielach A. Abas L. Abuzeineh A. Duclercq J. Tanaka H. Pařezová M. Petrášek J. Friml J. Kleine-Vehn J. Benková E. Dev. Cell. 2011; 21 (this issue): 796-804Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar uncover a transcription-independent molecular mechanism of interaction between auxin and cytokinin in the regulation of plant meristem function. By modulating endocytic trafficking of PIN1, cytokinin controls auxin flux and, therefore, auxin gradients. Unlike animals, whose basic body architecture is defined during embryogenesis, plants have the ability to constantly generate new organs from pools of stem cells set aside during embryogenesis, primarily at the shoot apical and root meristems. Additional new organs can also form from specialized cells, as in the case of lateral root primordia that originate from the pericycle cells. Both the maintenance of existing meristems and the generation of new ones are finely tuned by several plant hormones. Among these hormones, auxin and cytokinin have been shown to play critical roles in the establishment and maintenance of these stem cell niches. Since the classical physiological studies of the 1950s, the prevailing idea has been that the interaction between these two hormones is critical for the correct balance of cell proliferation and differentiation required for proper meristem function. However, only recently has the molecular mechanism behind the interaction between these two hormones started to emerge. Recent studies have pointed to a role for cytokinin in controlling the expression of the auxin efflux carrier PIN1 (reviewed in Moubayidin et al., 2009Moubayidin L. Di Mambro R. Sabatini S. Trends Plant Sci. 2009; 14: 557-562Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). The findings of Marhavý et al., 2011Marhavý P. Bielach A. Abas L. Abuzeineh A. Duclercq J. Tanaka H. Pařezová M. Petrášek J. Friml J. Kleine-Vehn J. Benková E. Dev. Cell. 2011; 21 (this issue): 796-804Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, reported in this issue of Developmental Cell, now add another dimension to our understanding of cytokinin-mediated auxin regulation. Auxin or, more specifically, auxin gradients are conclusively linked to the proliferative properties of root meristems. Auxin, either locally synthesized or produced in more distant tissues, is actively transported to generate an auxin maximum in the root quiescent center. Key to active hormone transport is the PIN family of auxin efflux carriers, whose polar localization at the plasma membrane confers directionality to auxin transport. Continuous endocytic trafficking of PINs between the plasma membrane and the endomembrane compartments or the lytic vacuole allows for rapid changes in the distribution or levels of PINs at the cell surface. Auxin maxima generated by the direct activity of PIN1 and other PIN family members in the QC cells are then interpreted by the auxin signaling machinery that feeds back into the regulation of auxin transport. Cytokinins, unlike auxin, stimulate cell differentiation. Consequently, activation of cytokinin responses results in a reduction in the size of the root meristem. At the molecular level, all cytokinin responses were thought to be mediated by the transcriptional regulation of cytokinin-controlled genes. This transcriptional regulation is initiated by a family of three cytokinin receptor histidine kinases, AHK2, AHK3, and CRE1 (Figure 1). The interaction between the receptors and the hormone initiates a phosphorelay cascade, starting with phosphorylation by the receptors of histidine phosphotransfer proteins (AHPs) and the translocation of these proteins to the nucleus, where the cascade ends with the phosphorylation of the ARR protein family. Two distinct classes of ARRs have been identified. A-type ARRs are transcriptionally induced by cytokinins but lack a DNA-binding domain and negatively affect cytokinin responses. In contrast, B-type ARRs positively regulate cytokinin responses and are able to bind DNA (reviewed in Argueso et al., 2010Argueso C.T. Raines T. Kieber J.J. Curr. Opin. Plant Biol. 2010; 13: 533-539Crossref PubMed Scopus (92) Google Scholar). Previously, several studies have highlighted the importance of the auxin-cytokinin interactions at the transcriptional level (Dello Ioio et al., 2008Dello Ioio R. Nakamura K. Moubayidin L. Perilli S. Taniguchi M. Morita M.T. Aoyama T. Costantino P. Sabatini S. Science. 2008; 322: 1380-1384Crossref PubMed Scopus (585) Google Scholar, Müller and Sheen, 2008Müller B. Sheen J. Nature. 2008; 453: 1094-1097Crossref PubMed Scopus (449) Google Scholar, Zhao et al., 2010Zhao Z. Andersen S.U. Ljung K. Dolezal K. Miotk A. Schultheiss S.J. Lohmann J.U. Nature. 2010; 465: 1089-1092Crossref PubMed Scopus (314) Google Scholar). In one elegant and illustrative study, the cytokinin effects on the root meristem function were shown to be in part mediated by the downregulation of PIN1 mRNA levels (Dello Ioio et al., 2008Dello Ioio R. Nakamura K. Moubayidin L. Perilli S. Taniguchi M. Morita M.T. Aoyama T. Costantino P. Sabatini S. Science. 2008; 322: 1380-1384Crossref PubMed Scopus (585) Google Scholar). Mechanistically, this was achieved by the activation of the B-type ARR1 by cytokinins; this activation directly promotes the expression of SHY2/IAA3, a negative regulator of auxin responses, and thus impinges on PIN1 mRNA levels. The resulting changes in auxin distribution could, therefore, explain the well-known cytokinin-induced reduction of meristem size. Although conceptually elegant and experimentally well supported, this discovery now appears to be only part of the story. Marhavý et al., 2011Marhavý P. Bielach A. Abas L. Abuzeineh A. Duclercq J. Tanaka H. Pařezová M. Petrášek J. Friml J. Kleine-Vehn J. Benková E. Dev. Cell. 2011; 21 (this issue): 796-804Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar convincingly show that cytokinin can quickly regulate PIN1 levels by a mechanism that does not involve transcriptional regulation. These findings have several important implications. Not only do they provide an additional and potentially much faster mechanism of regulating auxin gradients in response to cytokinins, but they also reveal a distinct branch of the canonical cytokinin signaling cascade. Using the development of the lateral root primordia (LRP), another well-defined developmental process that is strictly dependent on auxin gradients, Marhavý et al., 2011Marhavý P. Bielach A. Abas L. Abuzeineh A. Duclercq J. Tanaka H. Pařezová M. Petrášek J. Friml J. Kleine-Vehn J. Benková E. Dev. Cell. 2011; 21 (this issue): 796-804Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar showed that the cytokinin-mediated arrest of the LRP organogenesis was accompanied by a quick and dose-dependent decrease in the levels of PIN1 in the plasma membrane. These rapid cytokinin responses required the functional cytokinin receptors and, importantly, could still be observed even when PIN1 was expressed under the control of the 35S cauliflower mosaic virus promoter or when global new protein synthesis or RNA transcription were drastically reduced. Together, these results support the existence of a transcription-independent cytokinin signaling cascade that operates downstream of the CRE1 cytokinin receptor. Furthermore, Marhavý et al., 2011Marhavý P. Bielach A. Abas L. Abuzeineh A. Duclercq J. Tanaka H. Pařezová M. Petrášek J. Friml J. Kleine-Vehn J. Benková E. Dev. Cell. 2011; 21 (this issue): 796-804Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar found that either chemical inhibitors or genetic mutations that alter specific steps of endomembrane trafficking negatively affect cytokinin-mediated arrest of LRP development, pointing to the regulation of endocytic trafficking of PIN1 as the target of this cytokinin response pathway. The authors showed that the cytokinin-induced endocytosis was fast and specific. After as little as 90 min of cytokinin treatment, PIN1 (but not PIN2) levels were dramatically reduced. This could be indicative of a direct and specific interaction between a still-undetermined component of the cytokinin signaling pathway and PIN1. In this regard, the finding that single knockouts of two different B-type ARRs dramatically reduce this fast cytokinin effect on PIN1 levels would suggest that the studied interaction between these two hormones takes place at the level of ARRs or further downstream. This is quite surprising because the B-type ARRs were believed to function as transcriptional regulators, whereas the aforementioned results imply that these genes are required for modulating a process that is transcription independent. Importantly, another recent report suggests that the cytokinin-dependent posttranslational regulation of several PINs is also key to the maintenance of primary root meristems (Zhang et al., 2011Zhang W. To J.P. Cheng C.Y. Eric Schaller G. Kieber J.J. Plant J. 2011; 68: 1-10Crossref PubMed Scopus (75) Google Scholar). This posttranscriptional regulation of PIN protein levels could be mimicked by the loss of function of several A-type ARRs, suggesting that this could be the branching point of this cytokinin signaling pathway. The differences in the timing of events and the specific PINs affected in these two studies suggest the existence of at least two different posttranscriptional mechanisms by which cytokinins regulate PIN protein levels. With the wide arsenal of genetic tools available in the reference plant Arabidopsis, the exact node where this cytokinin signaling module branches out from the canonical phosphorelay cascade should soon be revealed. In contrast, the identification of the molecular elements that link the receptor-generated signal with the degradation of the PIN1 transporter may prove more challenging. The study by Marhavý et al., 2011Marhavý P. Bielach A. Abas L. Abuzeineh A. Duclercq J. Tanaka H. Pařezová M. Petrášek J. Friml J. Kleine-Vehn J. Benková E. Dev. Cell. 2011; 21 (this issue): 796-804Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar represents a major step toward deciphering the mechanisms behind the phytohormone crosstalk that drives the remarkable plant phenotypic plasticity. Cytokinin Modulates Endocytic Trafficking of PIN1 Auxin Efflux Carrier to Control Plant OrganogenesisMarhavý et al.Developmental CellSeptember 29, 2011In BriefCytokinin is an important regulator of plant growth and development. In Arabidopsis thaliana, the two-component phosphorelay mediated through a family of histidine kinases and response regulators is recognized as the principal cytokinin signal transduction mechanism activating the complex transcriptional response to control various developmental processes. Here, we identified an alternative mode of cytokinin action that uses endocytic trafficking as a means to direct plant organogenesis. This activity occurs downstream of known cytokinin receptors but through a branch of the cytokinin signaling pathway that does not involve transcriptional regulation. Full-Text PDF Open Archive}, number={4}, journal={DEVELOPMENTAL CELL}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2011}, month={Oct}, pages={608–610} }
 @article{stepanova_yun_robles_novak_he_guo_ljung_alonso_2011, title={The Arabidopsis YUCCA1 Flavin Monooxygenase Functions in the Indole-3-Pyruvic Acid Branch of Auxin Biosynthesis}, volume={23}, ISSN={["1532-298X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84855168181&partnerID=MN8TOARS}, DOI={10.1105/tpc.111.088047}, abstractNote={Abstract The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.}, number={11}, journal={PLANT CELL}, author={Stepanova, Anna N. and Yun, Jeonga and Robles, Linda M. and Novak, Ondrej and He, Wenrong and Guo, Hongwei and Ljung, Karin and Alonso, Jose M.}, year={2011}, month={Nov}, pages={3961–3973} }
 @article{tsuchisaka_yu_jin_alonso_ecker_zhang_gao_theologis_2009, title={A combinatorial interplay among the 1-aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana}, volume={183}, DOI={10.1534/genetics.109.107102}, abstractNote={Abstract Ethylene (C2H4) is a unique plant-signaling molecule that regulates numerous developmental processes. The key enzyme in the two-step biosynthetic pathway of ethylene is 1-aminocyclopropane-1-carboxylate synthase (ACS), which catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC, the precursor of ethylene. To understand the function of this important enzyme, we analyzed the entire family of nine ACS isoforms (ACS1, ACS2, ACS4-9, and ACS11) encoded in the Arabidopsis genome. Our analysis reveals that members of this protein family share an essential function, because individual ACS genes are not essential for Arabidopsis viability, whereas elimination of the entire gene family results in embryonic lethality. Phenotypic characterization of single and multiple mutants unmasks unique but overlapping functions of the various ACS members in plant developmental events, including multiple growth characteristics, flowering time, response to gravity, disease resistance, and ethylene production. Ethylene acts as a repressor of flowering by regulating the transcription of the FLOWERING LOCUS C. Each single and high order mutant has a characteristic molecular phenotype with unique and overlapping gene expression patterns. The expression of several genes involved in light perception and signaling is altered in the high order mutants. These results, together with the in planta ACS interaction map, suggest that ethylene-mediated processes are orchestrated by a combinatorial interplay among ACS isoforms that determines the relative ratio of homo- and heterodimers (active or inactive) in a spatial and temporal manner. These subunit isoforms comprise a combinatorial code that is a central regulator of ethylene production during plant development. The lethality of the null ACS mutant contrasts with the viability of null mutations in key components of the ethylene signaling apparatus, strongly supporting the view that ACC, the precursor of ethylene, is a primary regulator of plant growth and development.}, number={3}, journal={Genetics}, author={Tsuchisaka, Atsunari and Yu, Guixia and Jin, Hailing and Alonso, Jose M. and Ecker, Joseph R. and Zhang, Xiaoming and Gao, Shang and Theologis, Athanasios}, year={2009}, pages={979–1003} }
 @misc{stepanova_alonso_2009, title={Ethylene signaling and response: where different regulatory modules meet}, volume={12}, ISSN={["1879-0356"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-70349554136&partnerID=MN8TOARS}, DOI={10.1016/j.pbi.2009.07.009}, abstractNote={The structural simplicity of the gaseous hormone ethylene stands in contrast with the complexity of the physiological processes ethylene regulates. Initial studies suggested a simple linear arrangement of signaling molecules leading from the ethylene receptors to the EIN3 family of transcription factors. Recent discoveries have substantially changed this view. Current models suggest existence of a complex signaling pathway composed of several phosphorylation cascades, feedback-regulated transcriptional networks, and protein and mRNA turnover regulatory modules. Interactions between ethylene and other signals determine which of the ethylene-mediated responses get activated in a particular cell at a particular time. Tissue-specific regulation of auxin biosynthesis, transport, and response by ethylene is emerging as a key element in this signal integration process.}, number={5}, journal={CURRENT OPINION IN PLANT BIOLOGY}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2009}, month={Oct}, pages={548–555} }
 @article{ikeda_men_fischer_stepanova_alonso_ljung_grebe_2009, title={Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis}, volume={11}, ISSN={["1476-4679"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-67349090679&partnerID=MN8TOARS}, DOI={10.1038/ncb1879}, number={6}, journal={NATURE CELL BIOLOGY}, author={Ikeda, Yoshihisa and Men, Shuzhen and Fischer, Urs and Stepanova, Anna N. and Alonso, Jose M. and Ljung, Karin and Grebe, Markus}, year={2009}, month={Jun}, pages={731–U70} }
 @article{hu_mitchum_barnaby_ayele_ogawa_nam_lai_hanada_alonso_ecker_2008, title={Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis}, volume={20}, DOI={10.1105/tpc.107.057752}, abstractNote={Abstract Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter–β-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.}, number={2}, journal={The Plant Cell Online}, author={Hu, Jianhong and Mitchum, Melissa G. and Barnaby, Neel and Ayele, Belay T. and Ogawa, Mikihiro and Nam, Edward and Lai, Wei-Chu and Hanada, Atsushi and Alonso, Jose M. and Ecker, Joseph R.}, year={2008}, pages={320–336} }
 @article{stepanova_robertson-hoyt_yun_benavente_xie_doležal_schlereth_jürgens_alonso_2008, title={TAA1-Mediated Auxin Biosynthesis Is Essential for Hormone Crosstalk and Plant Development}, volume={133}, ISSN={0092-8674}, url={http://dx.doi.org/10.1016/j.cell.2008.01.047}, DOI={10.1016/j.cell.2008.01.047}, abstractNote={Plants have evolved a tremendous ability to respond to environmental changes by adapting their growth and development. The interaction between hormonal and developmental signals is a critical mechanism in the generation of this enormous plasticity. A good example is the response to the hormone ethylene that depends on tissue type, developmental stage, and environmental conditions. By characterizing the Arabidopsis wei8 mutant, we have found that a small family of genes mediates tissue-specific responses to ethylene. Biochemical studies revealed that WEI8 encodes a long-anticipated tryptophan aminotransferase, TAA1, in the essential, yet genetically uncharacterized, indole-3-pyruvic acid (IPA) branch of the auxin biosynthetic pathway. Analysis of TAA1 and its paralogues revealed a link between local auxin production, tissue-specific ethylene effects, and organ development. Thus, the IPA route of auxin production is key to generating robust auxin gradients in response to environmental and developmental cues.}, number={1}, journal={Cell}, publisher={Elsevier BV}, author={Stepanova, Anna N. and Robertson-Hoyt, Joyce and Yun, Jeonga and Benavente, Larissa M. and Xie, De-Yu and Doležal, Karel and Schlereth, Alexandra and Jürgens, Gerd and Alonso, Jose M.}, year={2008}, month={Apr}, pages={177–191} }
 @article{leivar_monte_al-sady_carle_storer_alonso_ecker_quail_2008, title={The Arabidopsis phytochrome-interacting factor PIF7, together with PIF3 and PIF4, regulates responses to prolonged red light by modulating phyB levels}, volume={20}, DOI={10.1105/tpc.107.052142}, abstractNote={Abstract We show that a previously uncharacterized Arabidopsis thaliana basic helix-loop-helix (bHLH) phytochrome interacting factor (PIF), designated PIF7, interacts specifically with the far-red light–absorbing Pfr form of phyB through a conserved domain called the active phyB binding motif. Similar to PIF3, upon light exposure, PIF7 rapidly migrates to intranuclear speckles, where it colocalizes with phyB. However, in striking contrast to PIF3, this process is not accompanied by detectable light-induced phosphorylation or degradation of PIF7, suggesting that the consequences of interaction with photoactivated phyB may differ among PIFs. Nevertheless, PIF7 acts similarly to PIF3 in prolonged red light as a weak negative regulator of phyB-mediated seedling deetiolation. Examination of pif3, pif4, and pif7 double mutant combinations shows that their moderate hypersensitivity to extended red light is additive. We provide evidence that the mechanism by which these PIFs operate on the phyB signaling pathway under prolonged red light is through maintaining low phyB protein levels, in an additive or synergistic manner, via a process likely involving the proteasome pathway. These data suggest that the role of these phyB-interacting bHLH factors in modulating seedling deetiolation in prolonged red light may not be as phy-activated signaling intermediates, as proposed previously, but as direct modulators of the abundance of the photoreceptor.}, number={2}, journal={The Plant Cell Online}, author={Leivar, Pablo and Monte, Elena and Al-Sady, Bassem and Carle, Christine and Storer, Alyssa and Alonso, Jose M. and Ecker, Joseph R. and Quail, Peter H.}, year={2008}, pages={337–352} }
 @article{li_he_lu_lee_alonso_ecker_luan_2007, title={A WD40 domain cyclophilin interacts with histone H3 and functions in gene repression and organogenesis in Arabidopsis}, volume={19}, ISSN={["1040-4651"]}, DOI={10.1105/tpc.107.053579}, abstractNote={Chromatin-based silencing provides a crucial mechanism for the regulation of gene expression. We have identified a WD40 domain cyclophilin, CYCLOPHILIN71 (CYP71), which functions in gene repression and organogenesis in Arabidopsis thaliana. Disruption of CYP71 resulted in ectopic activation of homeotic genes that regulate meristem development. The cyp71 mutant plants displayed dramatic defects, including reduced apical meristem activity, delayed and abnormal lateral organ formation, and arrested root growth. CYP71 was associated with the chromatin of target gene loci and physically interacted with histone H3. The cyp71 mutant showed reduced methylation of H3K27 at target loci, consistent with the derepression of these genes in the mutant. As CYP71 has close homologs in eukaryotes ranging from fission yeast to human, we propose that it serves as a highly conserved histone remodeling factor involved in chromatin-based gene silencing in eukaryotic organisms.}, number={8}, journal={PLANT CELL}, author={Li, Hong and He, Zengyong and Lu, Guihua and Lee, Sung Chul and Alonso, Jose and Ecker, Joseph R. and Luan, Sheng}, year={2007}, month={Aug}, pages={2403–2416} }
 @article{ronan c. o'malley_alonso_kim_leisse_ecker_2007, title={An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome}, volume={2}, ISSN={["1754-2189"]}, DOI={10.1038/nprot.2007.425}, number={11}, journal={NATURE PROTOCOLS}, author={Ronan C. O'Malley and Alonso, Jose M. and Kim, Christopher J. and Leisse, Thomas J. and Ecker, Joseph R.}, year={2007}, pages={2910–2917} }
 @article{chekanova_gregory_reverdatto_chen_kumar_hooker_yazaki_li_skiba_peng_2007, title={Genome-Wide High-Resolution Mapping of Exosome Substrates Reveals Hidden Features in the< i> Arabidopsis</i> Transcriptome}, volume={131}, DOI={10.1016/j.cell.2007.10.056}, abstractNote={The exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes. The exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes. The exosome is an evolutionarily conserved macromolecular complex that mediates numerous reactions of 3′–5′ RNA processing and degradation and is essential for viability (Estevez et al., 2003Estevez A.M. Lehner B. Sanderson C.M. Ruppert T. Clayton C. The roles of intersubunit interactions in exosome stability.J. Biol. Chem. 2003; 278: 34943-34951Crossref PubMed Scopus (75) Google Scholar, Mitchell et al., 1997Mitchell P. Petfalski E. Shevchenko A. Mann M. Tollervey D. The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′→5′ exoribonucleases.Cell. 1997; 91: 457-466Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar). Loss of any individual subunit of its nine-component core is lethal in S. cerevisiae and causes near-identical profiles of RNA-processing defects (Allmang et al., 1999aAllmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. Functions of the exosome in rRNA, snoRNA and snRNA synthesis.EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (470) Google Scholar, Allmang et al., 1999bAllmang C. Petfalski E. Podtelejnikov A. Mann M. Tollervey D. Mitchell P. The yeast exosome and human PM-Scl are related complexes of 3′–> 5′ exonucleases.Genes Dev. 1999; 13: 2148-2158Crossref PubMed Scopus (366) Google Scholar). Moreover, X-ray crystallographic analysis of the human exosome indicates that all nine core subunits are required for its integrity (Liu et al., 2006Liu Q. Greimann J.C. Lima C.D. Reconstitution, activities, and structure of the eukaryotic RNA exosome.Cell. 2006; 127: 1223-1237Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). The salient feature of the exosome core is the hexameric ring defined by heterodimers of the RNase PH domain-type proteins RRP41-RRP45, MTR3-RRP42, and RRP43-RRP46. These heterodimers are bridged on one side by three subunits containing S1 and KH domains: RRP40 links RRP45 and RRP46, RRP4 interacts with RRP41 and RRP42, and CSL4 contacts MTR3 and RRP43. Surprisingly, all six RNase PH-type proteins in yeast and human complexes are catalytically inactive and serve to mediate interactions with RRP44 (Dis3), a 3′–5′ hydrolytic RNase responsible for most if not all of the catalytic activity of the yeast exosome (Dziembowski et al., 2007Dziembowski A. Lorentzen E. Conti E. Seraphin B. A single subunit, Dis3, is essentially responsible for yeast exosome core activity.Nat. Struct. Mol. Biol. 2007; 14: 15-22Crossref PubMed Scopus (310) Google Scholar, Liu et al., 2007Liu Q. Greimann J.C. Lima C.D. Erratum: Reconstitution, activities, and structure of the eukaryotic RNA exosome.Cell. 2007; 131: 188-190Abstract Full Text Full Text PDF Scopus (13) Google Scholar). In contrast, the RRP41 exosome subunit in the plant lineage retained its catalytic competence (Chekanova et al., 2000Chekanova J.A. Shaw R.J. Wills M.A. Belostotsky D.A. Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells.J. Biol. Chem. 2000; 275: 33158-33166Crossref PubMed Scopus (117) Google Scholar). Furthermore, RRP44 is stably associated with the core complex in yeast and Drosophila but not in human and T. brucei (Chen et al., 2001Chen C.Y. Gherzi R. Ong S.E. Chan E.L. Raijmakers R. Pruijn G.J. Stoecklin G. Moroni C. Mann M. Karin M. AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.Cell. 2001; 107: 451-464Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, Estevez et al., 2001Estevez A.M. Kempf T. Clayton C. The exosome of Trypanosoma brucei.EMBO J. 2001; 20: 3831-3839Crossref PubMed Scopus (163) Google Scholar, Estevez et al., 2003Estevez A.M. Lehner B. Sanderson C.M. Ruppert T. Clayton C. The roles of intersubunit interactions in exosome stability.J. Biol. Chem. 2003; 278: 34943-34951Crossref PubMed Scopus (75) Google Scholar). These observations hint at a yet to be explored diversity of structure-function relationships in the exosome complex. Many auxiliary factors interact with the exosome and facilitate its functions. Most of its cytoplasmic activities, such as homeostatic mRNA turnover, decay of unstable mRNAs, nonsense-mediated mRNA decay, as well as the degradation of the mRNA fragments derived from endonucleolytic cleavage by RISC or from no-go decay, are mediated by the SKI2/SKI3/SKI8 complex and the SKI7 protein (reviewed in Houseley et al., 2006Houseley J. LaCava J. Tollervey D. RNA-quality control by the exosome.Nat. Rev. Mol. Cell Biol. 2006; 7: 529-539Crossref PubMed Scopus (487) Google Scholar). The exosome also has numerous targets in the nucleus. The nuclear exosome is remarkably versatile and is able to carry out precise 3′ end processing of the 5.8S rRNA precursor (Allmang et al., 1999aAllmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. Functions of the exosome in rRNA, snoRNA and snRNA synthesis.EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (470) Google Scholar) but also completely degrades the external transcribed rRNA spacer (Allmang et al., 2000Allmang C. Mitchell P. Petfalski E. Tollervey D. Degradation of ribosomal RNA precursors by the exosome.Nucleic Acids Res. 2000; 28: 1684-1691Crossref PubMed Scopus (191) Google Scholar), aberrant pre-rRNAs, pre-mRNAs, and pre-tRNAs (Bousquet-Antonelli et al., 2000Bousquet-Antonelli C. Presutti C. Tollervey D. Identification of a regulated pathway for nuclear pre-mRNA turnover.Cell. 2000; 102: 765-775Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, Kadaba et al., 2004Kadaba S. Krueger A. Trice T. Krecic A.M. Hinnebusch A.G. Anderson J. 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Furthermore, exosome substrates have yet to be comprehensively identified in any system, as even the most extensive datasets available only address its nuclear-specific functions and/or are based on microarray platforms that are not genome wide and/or not strand specific (Davis and Ares, 2006Davis C.A. Ares Jr., M. Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. USA. 2006; 103: 3262-3267Crossref PubMed Scopus (182) Google Scholar, Houalla et al., 2006Houalla R. Devaux F. Fatica A. Kufel J. Barrass D. Torchet C. Tollervey D. Microarray detection of novel nuclear RNA substrates for the exosome.Yeast. 2006; 23: 439-454Crossref PubMed Scopus (59) Google Scholar, Wyers et al., 2005Wyers F. Rougemaille M. Badis G. Rousselle J.C. Dufour M.E. Boulay J. Regnault B. Devaux F. Namane A. Seraphin B. et al.Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase.Cell. 2005; 121: 725-737Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). Here, we present evidence for a unique subfunctionalization of the individual subunits of the plant exosome core and widespread oligoadenylation- and exosome-mediated RNA quality-control pathways in plants. Further, we report the first high-resolution genome-wide map of exosome targets. These targets include multiple classes of stable structural RNAs, a select subset of mRNAs, primary microRNA (pri-miRNA) processing intermediates, tandem repeat-associated siRNA precursor species, as well as numerous noncoding RNAs, many of which can only be revealed through repressing the exosome. Previously, we demonstrated that Arabidopsis thaliana RRP4 and RRP41 proteins physically interact and reside in a high-molecular-weight complex in planta (Chekanova et al., 2000Chekanova J.A. Shaw R.J. Wills M.A. Belostotsky D.A. Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells.J. Biol. Chem. 2000; 275: 33158-33166Crossref PubMed Scopus (117) Google Scholar, Chekanova et al., 2002Chekanova J.A. Dutko J.A. Mian I.S. Belostotsky D.A. Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3′→5′ exonuclease containing S1 and KH RNA-binding domains.Nucleic Acids Res. 2002; 30: 695-700Crossref PubMed Scopus (68) Google Scholar). To elucidate its composition, we generated transgenic plants expressing either TAP-tagged RRP4 or RRP41 at physiological levels in rrp4-1 or rrp41-1 mutant plants, respectively. TAP-tagged RRP4 and RRP41 fully rescued the lethal phenotypes of their corresponding null alleles. TAP-tagged complexes were purified, and polypeptides shared between RRP4-TAP and RRP41-TAP samples but absent from the wild-type (WT) sample were subjected to MALDI and MS/MS analyses. Nine polypeptides corresponding to known subunits of the exosome core were identified: S1 and/or KH domain-containing subunits RRP4, RRP40A, and CSL4 as well as the RNase PH-type subunits RRP41, RRP42, RRP43, RRP45B, RRP46, and MTR3 (Figure 1 and Table S1 available online). In the case of subunits encoded by duplicated genes, only RRP40A and RRP45B were identified. This may be due to differences in the expression patterns and/or levels between the members of these gene pairs (Hooker et al., 2007Hooker T.S. Lam P. Zheng H. Kunst L. A core subunit of the RNA-processing/degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis.Plant Cell. 2007; 19: 904-913Crossref PubMed Scopus (87) Google Scholar). RRP6, which is restricted to a nuclear form of the exosome (Allmang et al., 1999bAllmang C. Petfalski E. Podtelejnikov A. Mann M. Tollervey D. Mitchell P. The yeast exosome and human PM-Scl are related complexes of 3′–> 5′ exonucleases.Genes Dev. 1999; 13: 2148-2158Crossref PubMed Scopus (366) Google Scholar, Brouwer et al., 2001Brouwer R. Allmang C. Raijmakers R. van Aarssen Y. Egberts W.V. Petfalski E. van Venrooij W.J. Tollervey D. Pruijn G.J. Three novel components of the human exosome.J. Biol. Chem. 2001; 276: 6177-6184Crossref PubMed Scopus (88) Google Scholar, Graham et al., 2006Graham A.C. Kiss D.L. Andrulis E.D. Differential distribution of exosome subunits at the nuclear lamina and in cytoplasmic foci.Mol. Biol. Cell. 2006; 17: 1399-1409Crossref PubMed Scopus (64) Google Scholar) and is likely underrepresented in our preparations, was also absent. On the other hand, the absence of RRP44, which is responsible for most if not all of the catalytic activity of the core exosome in yeast and humans, may reflect a genuine species-specific difference in the functional architecture of the exosome since the Arabidopsis RRP41 subunit is unique in retaining its full catalytic activity (Chekanova et al., 2000Chekanova J.A. Shaw R.J. Wills M.A. Belostotsky D.A. Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells.J. Biol. Chem. 2000; 275: 33158-33166Crossref PubMed Scopus (117) Google Scholar). To determine the consequences of losing specific exosome components on plant development, we characterized transfer-DNA (T-DNA) insertional alleles in several core subunits of the Arabidopsis exosome. In yeast, the CSL4 subunit is essential for viability (Allmang et al., 1999bAllmang C. Petfalski E. Podtelejnikov A. Mann M. Tollervey D. Mitchell P. The yeast exosome and human PM-Scl are related complexes of 3′–> 5′ exonucleases.Genes Dev. 1999; 13: 2148-2158Crossref PubMed Scopus (366) Google Scholar, Baker et al., 1998Baker R.E. Harris K. Zhang K. Mutations synthetically lethal with cep1 target S. cerevisiae kinetochore components.Genetics. 1998; 149: 73-85Crossref PubMed Google Scholar), and X-ray crystallographic analysis of the human exosome predicts that all of its core subunits are critical to maintaining structural integrity and functionality of the complex (Liu et al., 2006Liu Q. Greimann J.C. Lima C.D. Reconstitution, activities, and structure of the eukaryotic RNA exosome.Cell. 2006; 127: 1223-1237Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). In marked contrast, we found that neither integrity nor function of the Arabidopsis exosome was significantly compromised by loss of CSL4. First, neither csl4-1 nor csl4-2 (a confirmed null allele) mutant plants manifested any discernible phenotype (Figure S1). Second, size fractionation demonstrated that the Arabidopsis exosome complex lacking CSL4 remained nearly intact (Figure 1C). Furthermore, tiling microarray analyses (below) revealed that loss of CSL4 affects only a subset of exosome targets (Figure S2 and Tables S2 and S3). In contrast, Arabidopsis RRP41 was essential for development of the female gametophyte, an eight-celled haploid structure derived from the primary product of female meiosis. While the rrp41-1 mutant allele was normally transmitted through the male parent, it was not transmitted through the female (n = 194), and selfed rrp41/RRP41 heterozygotes produced seeds and aborted ovules in a 1:1 ratio (Figures 2A and S3; three independent rrp41 alleles showed identical phenotypes). Furthermore, the resulting progeny segregated 1:1 for WT and heterozygous plants. The mutant female gametophytes arrested (n = 422) after the first mitosis (two-nucleate stage, 43.1%; Figures 2B and 2C) and less frequently at one-nucleate (1.4%), four-nucleate (3.3%), or later stages (3.0%). Finally, loss of RRP4 resulted in an additional unique phenotype. Specifically, rrp4-1 mutant seeds arrested at early stages of embryogenesis (Figures 2D and S4A–S4E). By the time WT progeny seeds of rrp4-1/RRP4 plants reached the heart or torpedo stages of embryogenesis, 30% of the rrp4-1/rrp4-1 progeny contained two-cell embryos, 0.5% undivided zygotes, and 3% had embryos at the early globular stage (n = 393). Analysis of stage-specific markers confirmed that rrp4-1 seed morphology faithfully reflects their developmental timing (Figures S4F and S4G). The rrp4-1 mutant endosperm developed to varying degrees but never past the cellularization stage (Figures S4B–S4D). These phenotypes cosegregated with the T-DNA genetic lesion, which was confirmed to be a null mutation using an SNP-based assay (Figure 2E), and were fully rescued by the WT and TAP-tagged RRP4 transgenes. In light of the recent findings that loss of the Arabidopsis mRNA decapping complex results in seedling lethality (Goeres et al., 2007Goeres D.C. Van Norman J.M. Zhang W. Fauver N.A. Spencer M.L. Sieburth L.E. Components of the Arabidopsis mRNA decapping complex are required for early seedling development.Plant Cell. 2007; 19: 1549-1564Crossref PubMed Scopus (93) Google Scholar, Xu et al., 2006Xu J. Yang J.Y. Niu Q.W. Chua N.H. Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development.Plant Cell. 2006; 18: 3386-3398Crossref PubMed Scopus (184) Google Scholar), the phenotype of rrp4-1 mutant seeds suggests a more general function for RRP4 in postzygotic development, which is consistent with its broad substrate range revealed by tiling microarray analyses (below). In summary, the distinctiveness of the phenotypes of csl4, rrp4, and rrp41 mutant plants and their associated molecular signatures (below) indicate that the individual subunits in the Arabidopsis exosome core make nonequivalent contributions to its integrity and function. These findings set the plant exosome complex apart from those analyzed so far in other systems. To address the functions of RRP4 and RRP41 during vegetative growth, we engineered an estradiol-inducible RNAi (iRNAi) system (see Experimental Procedures). Growing these transgenic plants on estradiol-containing medium induced the RNAi-mediated knockdown of RRP4 (rrp4iRNAi) or RRP41 (rrp41iRNAi) mRNA, resulting in growth arrest (Figure 2F) and subsequent death of seedlings. Importantly, arrest was preceded by accumulation of 3′-underprocessed 5.8S rRNA species (Figure 2G). This molecular phenotype is indicative of exosome malfunction (Mitchell et al., 1997Mitchell P. Petfalski E. Shevchenko A. Mann M. Tollervey D. The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′→5′ exoribonucleases.Cell. 1997; 91: 457-466Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar) and was never observed in WT plants exposed to estradiol (neither is growth inhibition). These results show that Arabidopsis RRP4 and RRP41 are essential for postembryonic growth and validate the conditional iRNAi knockdown system as a useful approach for investigating their functions in vivo. To comprehensively identify exosome targets in Arabidopsis and gain additional insights into the apparent subfunctionalization of its core subunits, we implemented iRNAi in conjunction with whole-genome tiling microarrays. To minimize changes in gene expression that did not result directly from exosome depletion, we selected the earliest time point of estradiol treatment corresponding to the accumulation of underprocessed 5.8S rRNA species, but before growth retardation. Oligo(dT)-primed targets prepared from RNA samples from plants containing empty vector, rrp4iRNAi, or rrp41iRNAi constructs grown with or without estradiol were used to interrogate oligonucleotide tiling arrays. Therefore, the array signals should correspond exclusively to polyadenylated RNA species. Moreover, to rule out the possibility of spurious internal priming events, we employed 3′-rapid amplification of cDNA ends (3′-RACE) to map the polyadenylation sites in a subset of targets (Figure S9) as well as compared the relative change in expression between poly(A)+ and total RNA fractions for selected targets (Figures 3C, S5B, S5C, S5H, and S8). We used the TileMap algorithm, which utilizes a two-state hidden Markov model based on probe-level t statistics (Ji and Wong, 2005Ji H. Wong W.H. TileMap: create chromosomal map of tiling array hybridizations.Bioinformatics. 2005; 21: 3629-3636Crossref PubMed Scopus (191) Google Scholar), to identify genomic regions showing statistically significant changes. Expression data from arrays hybridized with targets from rrp4iRNAi and rrp41iRNAi plants that had been estradiol-treated were compared against the corresponding mock (DMSO)-treated samples, as well as against the empty-vector line treated with estradiol. We identified a total of 1612 genomic regions exhibiting increased levels of polyadenylated RNA upon the depletion of RRP4 (rrp4iRNAi) and RRP41 (rrp41iRNAi), while only about 1/10 as many regions showed downregulation (Figures 2H and 2I; Tables S2 and S3). Depleting an exoribonucleolytic complex should cause increased accumulation of its target RNAs, thus the overwhelming majority of expression changes in RRP4- and RRP41-depleted seedlings most likely represent direct effects. In contrast, when we conducted a similar analysis of csl4-2 mutant versus wild-type (Ws) plants, upregulation was no longer a predominant trend (Figure S2). Thus, the constitutive absence of CSL4 likely results in many secondary effects. This observation raises a general concern applicable to transcriptome studies using constitutive loss-of-function mutants and, conversely, emphasizes the value of conditional alleles like rrp4iRNAi and rrp41iRNAi. At the same time, it is notable that the overlap in the upregulated RNA targets among the csl4-2, rrp4iRNAi, and rrp41iRNA samples is highly significant, while the overlap in spectra of downregulated RNAs is negligible (Figure S2). Hence, the majority of upregulated RNA targets in csl4-2 seedlings constitute a direct molecular signature of the CSL4-less exosome. Remarkably, many of the exosome targets upregulated in rrp4iRNAi and rrp41iRNAi samples were unaffected in the csl4-2 seedlings, including both nuclear-confined species (e.g., miRNA precursors, Figure 5) as well as cytoplasmic RNAs (e.g., spliced mRNAs, Figure 4). Therefore, the CSL4-less exosome is fully active on some of the exosome substrates in both cellular compartments. In addition, these data represent a valuable resource for narrowing down which of the exosome targets are essential for viability, via subtracting the csl4-2 upregulated dataset from the rrp4iRNAi and rrp41iRNA analyses. For example, the 7S pre-rRNA processing defect in the csl4-2 seedlings was as severe as in the RRP4- and RRP41-depleted seedlings (Figure 2G), and yet csl4-2 mutant plants are phenotypically indistinguishable from WT. A global comparative overview of similarities and differences in the expression changes among the csl4-2, rrp4iRNAi, and rrp41iRNA lines can be found in Figure S2 and Tables S2–S11 and on the accompanying website (http://signal.salk.edu/cgi-bin/exosome). The following major classes of Arabidopsis exosome direct targets were defined by tiling microarray analysis of rrp4iRNAi and rrp41iRNAi plants: (1) small nuclear RNAs (snRNAs; 9 snRNA genes from both rrp4iRNAi and rrp41iRNAi; Table S4); (2) the majority of small nucleolar RNAs (snoRNAs) encoded in the genome (83 and 96 snoRNA genes from rrp4iRNAi and rrp41iRNAi samples, respectively; Table S5); (3) a select subset of tRNA genes (20 and 14, respectively; Table S6); (4) an upregulated subset of Arabidopsis mRNAs (205 and 266 mRNAs, respectively; Table S7); (5) a subset of mRNAs that extend beyond their annotated 3′ end, indicative of 3′-processing defects (29 from both rrp4iRNAi and rrp41iRNAi; Table S8); (6) a subset of specific pri-miRNA genes (12 from rrp4iRNAi and 11 from rrp41iRNAi; Table S9); (7) a large class of previously uncharacterized noncoding RNAs (ncRNAs); many of these ncRNAs overlap with repetitive elements and small RNA (smRNA)-generating loci (210 and 156 ncRNAs, respectively; Table S10); and (8) a distinct class of previously undetected polyadenylated transcripts that map exclusively to the 5′ ends of known protein-coding mRNAs and hence may possess regulatory potential (52 from both rrp4iRNAi and rrp41iRNAi; Table S11). Notably, while the overlap in the spectra of upregulated target RNAs revealed by the depletions of RRP4 and RRP41 was highly significant (∼64%), the extent of differences between them corroborates the notion of subfunctionalization of the subunits in the Arabidopsis exosome core (for example see Figure 4). Taken together, these results circumscribe a complex spectrum of Arabidopsis exosome targets that spans RNAP I, II, III, and possibly RNAP IV transcripts and includes nuclear-restricted RNAs (e.g., pri-miRNAs), cytoplasmic RNAs (e.g., spliced mRNAs), as well as RNAs distributed between the two compartments. Interestingly, our array analyses identified appreciable amounts of polyadenylated RNA signals across the rDNA repeat unit even under normal conditions, which were dramatically upregulated upon depletion of RRP4 or RRP41 (Figure 3). This increased and expanded signal corresponded largely to polyadenylated pre-rRNA precursors. For example, northern analysis targeting the sequences just downstream of 5.8S rRNA revealed an increase in a polyadenylated, 3′-underprocessed species of ∼2.4 kb (Figure 3C, top) that included both the 18S and 5.8S mature rRNA regions, while neither the nonpolyadenylated precursor (Figure 3C, bottom) nor the levels of mature rRNAs (data not shown) were affected. Furthermore, two major clusters of polyadenylation sites identified by 3′-RACE were both located outside of the boundaries of the mature rRNA (Figure S9). These findings parallel observations in yeast, where targeting of pre-rRNA species for degradation by the exosome is mechanistically linked to their oligoadenylation by the TRAMP complex (Kadaba et al., 2004Kadaba S. Krueger A. Trice T. Krecic A.M. Hinnebusch A.G. Anderson J. Nuclear surveillance and degradation of hypomodified initiator tRNAMet in S. cerevisiae.Genes Dev. 2004; 18: 1227-1240Crossref PubMed Scopus (365) Google Scholar, Vanacova et al., 2005Vanacova S. Wolf J. Martin G. Blank D. Dettwiler S. Friedlein A. Langen H. Keith G. Keller W. A new yeast poly(A) polymerase complex involved in RNA quality control.PLoS Biol. 2005; 3: e189https://doi.org/10.1371/journal.pbio.0030189Crossref PubMed Scopus (452) Google Scholar). In addition, both tiling array and qPCR analyses revealed increased accumulation of poly(A)+ RNAs in the intergenic spacer region (IGS, Figures 3A and 3B). Notably, in mouse cells the IGS-derived RNA regulates the activity of the main rDNA promoter in an epigenetically stable}, number={7}, journal={Cell}, author={Chekanova, Julia A. and Gregory, Brian D. and Reverdatto, Sergei V. and Chen, Huaming and Kumar, Ravi and Hooker, Tanya and Yazaki, Junshi and Li, Pinghua and Skiba, Nikolai and Peng, Qian}, year={2007}, pages={1340–1353} }
 @article{stepanova_yun_likhacheva_alonso_2007, title={Multilevel interactions between ethylene and auxin in Arabidopsis roots}, volume={19}, ISSN={["1532-298X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-34548310412&partnerID=MN8TOARS}, DOI={10.1105/tpc.107.052068}, abstractNote={Hormones play a central role in the coordination of internal developmental processes with environmental signals. Herein, a combination of physiological, genetic, cellular, and whole-genome expression profiling approaches has been employed to investigate the mechanisms of interaction between two key plant hormones: ethylene and auxin. Quantification of the morphological effects of ethylene and auxin in a variety of mutant backgrounds indicates that auxin biosynthesis, transport, signaling, and response are required for the ethylene-induced growth inhibition in roots but not in hypocotyls of dark-grown seedlings. Analysis of the activation of early auxin and ethylene responses at the cellular level, as well as of global changes in gene expression in the wild type versus auxin and ethylene mutants, suggests a simple mechanistic model for the interaction between these two hormones in roots, according to which ethylene and auxin can reciprocally regulate each other's biosyntheses, influence each other's response pathways, and/or act independently on the same target genes. This model not only implies existence of several levels of interaction but also provides a likely explanation for the strong ethylene response defects observed in auxin mutants.}, number={7}, journal={PLANT CELL}, author={Stepanova, Anna N. and Yun, Jeonga and Likhacheva, Alla V. and Alonso, Jose M.}, year={2007}, month={Jul}, pages={2169–2185} }
 @article{mori_murata_yang_munemasa_wang_andreoli_tiriac_alonso_harper_ecker_2006, title={CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion-and Ca2+-permeable channels and stomatal closure}, volume={4}, number={10}, journal={PLoS biology}, author={Mori, Izumi C. and Murata, Yoshiyuki and Yang, Yingzhen and Munemasa, Shintaro and Wang, Yong-Fei and Andreoli, Shannon and Tiriac, Hervé and Alonso, Jose M. and Harper, Jeffery F. and Ecker, Joseph R.}, year={2006}, pages={e327} }
 @article{rudella_friso_alonso_ecker_wijk_2006, title={Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis}, volume={18}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.106.042861}, abstractNote={Abstract Plastids contain tetradecameric Clp protease core complexes, with five ClpP Ser-type proteases, four nonproteolytic ClpR, and two associated ClpS proteins. Accumulation of total ClpPRS complex decreased twofold to threefold in an Arabidopsis thaliana T-DNA insertion mutant in CLPR2 designated clpr2-1. Differential stable isotope labeling of the ClpPRS complex with iTRAQ revealed a fivefold reduction in assembled ClpR2 accumulation and twofold to fivefold reductions in the other subunits. A ClpR2:(his)6 fusion protein that incorporated into the chloroplast ClpPRS complex fully complemented clpr2-1. The reduced accumulation of the ClpPRS protease complex led to a pale-green phenotype with delayed shoot development, smaller chloroplasts, decreased thylakoid accumulation, and increased plastoglobule accumulation. Stromal ClpC1 and 2 were both recruited to the thylakoid surface in clpr2-1. The thylakoid membrane of clpr2-1 showed increased carotenoid content, partial inactivation of photosystem II, and upregulated thylakoid proteases and stromal chaperones, suggesting an imbalance in chloroplast protein homeostasis and a well-coordinated network of proteolysis and chaperone activities. Interestingly, a subpopulation of PsaF and several light-harvesting complex II proteins accumulated in the thylakoid with unprocessed chloroplast transit peptides. We conclude that ClpR2 cannot be functionally replaced by other ClpP/R homologues and that the ClpPRS complex is central to chloroplast biogenesis, thylakoid protein homeostasis, and plant development.}, number={7}, journal={PLANT CELL}, author={Rudella, Andrea and Friso, Giulia and Alonso, Jose M. and Ecker, Joseph R. and Wijk, Klaas J.}, year={2006}, month={Jul}, pages={1704–1721} }
 @article{kim_punshon_lanzirotti_li_alonso_ecker_kaplan_guerinot_2006, title={Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1}, volume={314}, ISSN={["1095-9203"]}, DOI={10.1126/science.1132563}, abstractNote={Iron deficiency is a major human nutritional problem wherever plant-based diets are common. Using synchrotron x-ray fluorescence microtomography to directly visualize iron in Arabidopsis seeds, we show that iron is localized primarily to the provascular strands of the embryo. This localization is completely abolished when the vacuolar iron uptake transporter VIT1 is disrupted. Vacuolar iron storage is also critical for seedling development because vit1-1 seedlings grow poorly when iron is limiting. We have uncovered a fundamental aspect of seed biology that will ultimately aid the development of nutrient-rich seed, benefiting both human health and agricultural productivity.}, number={5803}, journal={SCIENCE}, author={Kim, Sun A. and Punshon, Tracy and Lanzirotti, Antonio and Li, Liangtao and Alonso, Jose M. and Ecker, Joseph R. and Kaplan, Jerry and Guerinot, Mary Lou}, year={2006}, month={Nov}, pages={1295–1298} }
 @article{benavente_alonso_2006, title={Molecular mechanisms of ethylene signaling in Arabidopsis}, volume={2}, ISSN={1742-206X 1742-2051}, url={http://dx.doi.org/10.1039/b513874d}, DOI={10.1039/b513874d}, abstractNote={Ethylene is a gaseous plant hormone involved in several important physiological processes throughout a plant's life cycle. Decades of scientific research devoted to deciphering how plants are able to sense and respond to this key molecule have culminated in the establishment of one of the best characterized signal transduction pathways in plants. The ethylene signaling pathway starts with the perception of this gaseous hormone by a family of membrane-anchored receptors followed by a Raf-like kinase CTR1 that is physically associated with the receptors and actively inhibits downstream components of the pathway. A major gap is represented by the mysterious plant protein EIN2 that genetically works downstream of CTR1 and upstream of the key transcription factor EIN3. Transcriptional regulation by EIN3 and EIN3-family members has emerged as a key aspect of ethylene responses. The major components of this transcriptional cascade have been characterized and the involvement of post-transcriptional control by ubiquitination has been determined. Nevertheless, many aspects of this pathway still remain unknown. Recent genomic studies aiming to provide a more comprehensive view of modulation of gene expression have further emphasized the ample role of ethylene in a myriad of cellular processes and particularly in its crosstalk with other important plant hormones. This review aims to serve as a guide to the main scientific discoveries that have shaped the field of ethylene biology in the recent years.}, number={3-4}, journal={Molecular BioSystems}, publisher={Royal Society of Chemistry (RSC)}, author={Benavente, Larissa M. and Alonso, Jose M.}, year={2006}, pages={165} }
 @article{alonso_ecker_2006, title={Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis}, volume={7}, DOI={10.1038/nrg1893}, number={7}, journal={Nature Reviews Genetics}, author={Alonso, Jose and Ecker, Joseph R.}, year={2006}, pages={524–536} }
 @inbook{stepanova_alonso_2006, title={PCR-based screening for insertional mutants}, booktitle={Arabidopsis Protocols}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2006}, pages={163–172} }
 @article{lariguet_schepens_hodgson_pedmale_trevisan_kami_carbonnel_alonso_ecker_liscum_2006, title={PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism}, volume={103}, DOI={10.1073/pnas.0603799103}, abstractNote={Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis . Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis ( PKS1 – PKS4 ). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro , indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.}, number={26}, journal={Proceedings of the National Academy of Sciences}, author={Lariguet, Patricia and Schepens, Isabelle and Hodgson, Daniel and Pedmale, Ullas V. and Trevisan, Martine and Kami, Chitose and Carbonnel, Matthieu and Alonso, José M. and Ecker, Joseph R. and Liscum, Emmanuel}, year={2006}, pages={10134–10139} }
 @article{chen_ullah_temple_liang_guo_alonso_ecker_jones_2006, title={RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis}, volume={57}, DOI={10.1093/jxb/erl035}, abstractNote={The scaffold protein RACK1 (Receptor for Activated C Kinase 1) serves as an integrative point for diverse signal transduction pathways. The Arabidopsis genome contains three RACK1 orthologues, however, little is known about their functions. It is reported here that one member of this gene family, RACK1A, previously identified as the Arabidopsis homologue of the tobacco arcA gene, mediates hormone responses and plays a regulatory role in multiple developmental processes. RACK1A expresses ubiquitously in Arabidopsis. Loss-of-function mutations in RACK1A confer defects in multiple developmental processes including seed germination, leaf production, and flowering. rack1a mutants displayed reduced sensitivity to gibberellin and brassinosteroid in seed germination, hypersensitivity to abscisic acid in seed germination and early seedling development, and hyposensitivity to auxin in adventitious and lateral root formation. These results provide the first genetic evidence that RACK1A is involved in multiple signal transduction pathways.}, number={11}, journal={Journal of experimental botany}, author={Chen, Jin-Gui and Ullah, Hemayet and Temple, Brenda and Liang, Jiansheng and Guo, Jianjun and Alonso, José M. and Ecker, Joseph R. and Jones, Alan M.}, year={2006}, pages={2697–2708} }
 @article{hutchison_li_argueso_gonzalez_lee_lewis_maxwell_perdue_schaller_alonso_2006, title={The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling}, volume={18}, DOI={10.1105/tpc.106.045674}, abstractNote={Abstract Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.}, number={11}, journal={The Plant Cell Online}, author={Hutchison, Claire E. and Li, Jie and Argueso, Cristiana and Gonzalez, Monica and Lee, Eurie and Lewis, Michael W. and Maxwell, Bridey B. and Perdue, Tony D. and Schaller, G. Eric and Alonso, Jose M.}, year={2006}, pages={3073–3087} }
 @article{pfund_tans-kersten_dunning_alonso_ecker_allen_bent_2005, title={" Flagellin is not a major defense elicitor in Ralstonia solanacearum cells (vol 17, pg 696, 2005)}, volume={18}, number={9}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Pfund, C. and Tans-Kersten, J. and Dunning, F. M. and Alonso, J. M. and Ecker, J. R. and Allen, C. and Bent, A. F.}, year={2005}, pages={1024} }
 @article{stepanova_hoyt_hamilton_alonso_2005, title={A link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis}, volume={17}, ISSN={["1532-298X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-27744445823&partnerID=MN8TOARS}, DOI={10.1105/tpc.105.033365}, abstractNote={The plant hormone ethylene participates in the regulation of a variety of developmental processes and serves as a key mediator of plant responses to biotic and abiotic stress factors. The diversity of ethylene functions is achieved, at least in part, by combinatorial interactions with other hormonal signals. Here, we show that ethylene-triggered inhibition of root growth, one of the classical effects of ethylene in Arabidopsis thaliana seedlings, is mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE alpha1 (WEI2/ASA1) and WEI7/ANTHRANILATE SYNTHASE beta1 (ASB1) genes that encode alpha- and beta-subunits of a rate-limiting enzyme of Trp biosynthesis, anthranilate synthase. Upregulation of WEI2/ASA1 and WEI7/ASB1 by ethylene results in the accumulation of auxin in the tip of primary root, whereas loss-of-function mutations in these genes prevent the ethylene-mediated auxin increase. Furthermore, wei2 and wei7 suppress the high-auxin phenotypes of superroot1 (sur1) and sur2, two auxin-overproducing mutants, suggesting that the roles of WEI2 and WEI7 in the regulation of auxin biosynthesis are not restricted to the ethylene response. Together, these findings reveal that ASA1 and ASB1 are key elements in the regulation of auxin production and an unexpected node of interaction between ethylene responses and auxin biosynthesis in Arabidopsis. This study provides a mechanistic explanation for the root-specific ethylene insensitivity of wei2 and wei7, illustrating how interactions between hormones can be used to achieve response specificity.}, number={8}, journal={PLANT CELL}, author={Stepanova, AN and Hoyt, JM and Hamilton, AA and Alonso, JM}, year={2005}, month={Aug}, pages={2230–2242} }
 @article{liu_holub_alonso_ecker_fobert_2005, title={An Arabidopsis NPR1-like gene, NPR4, is required for disease resistance}, volume={41}, ISSN={["1365-313X"]}, DOI={10.1111/j.1365-313x.2004.02296.x}, abstractNote={The Arabidopsis genome contains six NPR1-related genes. Given the pivotal role played by NPR1 in controlling salicylic acid (SA)-mediated gene expression and disease resistance, functional characterization of other family members appears to be justified. Reverse genetics was used to analyze the role of one NPR1-like gene, which we called NPR4. The NPR4 protein shares 36% identity with NPR1 and interacts with the same spectrum of TGA transcription factors in yeast two-hybrid assays. Plants with T-DNA insertions in NPR4 are more susceptible to the virulent bacterial pathogen Pseudomonas syringe pv. tomato DC3000. This phenotype is complemented by expression of the wild type NPR4 coding region. As determined by the parasite reproduction, the npr4-1 mutant is more susceptible to the fungal pathogen Erysiphe cichoracearum, but does not differ markedly from wild type in its interaction with virulent and avirulent strains of the oomycete Peronospora parasitica. In leaves of wild-type plants, NPR4 mRNA levels increase following pathogen challenge or SA treatment, and decrease rapidly following methyl jasmonic acid (MeJA) treatment. Transcripts of the pathogenesis-related (PR) genes PR-1, PR-2, and PR-5 are only marginally reduced in the npr4-1 mutant following pathogen challenge or SA treatment. This reduction of PR gene expression is more pronounced when leaves are challenged with the bacterial pathogen following SA treatment. Expression of the jasmonic acid-dependent pathway marker gene PDF1.2 is compromised in npr4-1 leaves following application of MeJA or a combination of SA and MeJA. These results indicate that NPR4 is required for basal defense against pathogens, and that it may be implicated in the cross-talk between the SA- and JA-dependent signaling pathways.}, number={2}, journal={PLANT JOURNAL}, author={Liu, GS and Holub, EB and Alonso, JM and Ecker, JR and Fobert, PR}, year={2005}, month={Jan}, pages={304–318} }
 @article{stepanova_alonso_2005, title={Arabidopsis ethylene signaling pathway}, volume={2005}, number={276}, journal={Science Signaling}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2005}, pages={cm4} }
 @article{nagpal_ellis_weber_ploense_barkawi_guilfoyle_hagen_alonso_cohen_farmer_2005, title={Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation}, volume={132}, number={18}, journal={Development}, author={Nagpal, Punita and Ellis, Christine M. and Weber, Hans and Ploense, Sara E. and Barkawi, Lana S. and Guilfoyle, Thomas J. and Hagen, Gretchen and Alonso, José M. and Cohen, Jerry D. and Farmer, Edward E.}, year={2005}, pages={4107–4118} }
 @article{prigge_otsuga_alonso_ecker_drews_clark_2005, title={Class III homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development}, volume={17}, number={1}, journal={The Plant Cell Online}, author={Prigge, Michael J. and Otsuga, Denichiro and Alonso, Jose M. and Ecker, Joseph R. and Drews, Gary N. and Clark, Steven E.}, year={2005}, pages={61–76} }
 @article{stepanova_alonso_2005, title={Ethylene signalling and response pathway: A unique signalling cascade with a multitude of inputs and outputs}, volume={123}, DOI={10.1111/j/1399-3054.2004.00447}, number={2}, journal={Physiologia Plantarum}, author={STEPANOVA, ANNA and Alonso, Jose}, year={2005}, pages={195–206} }
 @article{okushima_overvoorde_arima_alonso_chan_chang_ecker_hughes_lui_nguyen_2005, title={Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19}, volume={17}, number={2}, journal={The Plant Cell Online}, author={Okushima, Yoko and Overvoorde, Paul J. and Arima, Kazunari and Alonso, Jose M. and Chan, April and Chang, Charlie and Ecker, Joseph R. and Hughes, Beth and Lui, Amy and Nguyen, Diana}, year={2005}, pages={444–463} }
 @article{overvoorde_okushima_alonso_chan_chang_ecker_hughes_liu_onodera_quach_2005, title={Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana}, volume={17}, number={12}, journal={The Plant Cell Online}, author={Overvoorde, Paul J. and Okushima, Yoko and Alonso, José M. and Chan, April and Chang, Charlie and Ecker, Joseph R. and Hughes, Beth and Liu, Amy and Onodera, Courtney and Quach, Hong}, year={2005}, pages={3282–3300} }
 @article{mason_mathews_argyros_maxwell_kieber_alonso_ecker_schaller_2005, title={Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis}, volume={17}, number={11}, journal={The Plant Cell Online}, author={Mason, Michael G. and Mathews, Dennis E. and Argyros, D. Aaron and Maxwell, Bridey B. and Kieber, Joseph J. and Alonso, Jose M. and Ecker, Joseph R. and Schaller, G. Eric}, year={2005}, pages={3007–3018} }
 @article{wilmoth_wang_tiwari_joshi_hagen_guilfoyle_alonso_ecker_reed_2005, title={NPH4/ARF7 and ARF19 promote leaf expansion and auxin‐induced lateral root formation}, volume={43}, number={1}, journal={The Plant Journal}, author={Wilmoth, Jill C. and Wang, Shucai and Tiwari, Shiv B. and Joshi, Atul D. and Hagen, Gretchen and Guilfoyle, Thomas J. and Alonso, Jose M. and Ecker, Joseph R. and Reed, Jason W.}, year={2005}, pages={118–130} }
 @article{ryu_kim_kunkel_kim_cho_hong_kim_fernández_kim_alonso_et al._2005, title={Phytochrome-Specific Type 5 Phosphatase Controls Light Signal Flux by Enhancing Phytochrome Stability and Affinity for a Signal Transducer}, volume={120}, ISSN={0092-8674}, url={http://dx.doi.org/10.1016/j.cell.2004.12.019}, DOI={10.1016/j.cell.2004.12.019}, abstractNote={Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.}, number={3}, journal={Cell}, publisher={Elsevier BV}, author={Ryu, Jong Sang and Kim, Jeong-Il and Kunkel, Tim and Kim, Byung Chul and Cho, Dae Shik and Hong, Sung Hyun and Kim, Seong-Hee and Fernández, Aurora Piñas and Kim, Yumi and Alonso, Jose M. and et al.}, year={2005}, month={Feb}, pages={395–406} }
 @article{ryu_kim_kunkel_kim_cho_hong_kim_fernández_kim_alonso_2005, title={Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer}, volume={120}, number={3}, journal={Cell}, author={Ryu, Jong Sang and Kim, Jeong-Il and Kunkel, Tim and Kim, Byung Chul and Cho, Dae Shik and Hong, Sung Hyun and Kim, Seong-Hee and Fernández, Aurora Piñas and Kim, Yumi and Alonso, Jose M.}, year={2005}, pages={395–406} }
 @article{page_hamel_gabilly_zegzouti_perea_alonso_ecker_theg_christensen_merchant_2004, title={A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA Is Required for the Assembly of the Cytochrome b6f Complex in Arabidopsis Chloroplasts}, volume={279}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M404285200}, DOI={10.1074/JBC.M404285200}, abstractNote={The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b(6)f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.}, number={31}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Page, M. L. Dudley and Hamel, Patrice P. and Gabilly, Stéphane T. and Zegzouti, Hicham and Perea, John V. and Alonso, José M. and Ecker, Joseph R. and Theg, Steven M. and Christensen, Sioux K. and Merchant, Sabeeha}, year={2004}, month={May}, pages={32474–32482} }
 @article{novillo_alonso_ecker_salinas_2004, title={CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis}, volume={101}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.0303029101}, DOI={10.1073/PNAS.0303029101}, abstractNote={CBF/DREB1 (C-repeat-binding factor/dehydration responsive element-binding factor 1) genes encode a small family of transcriptional activators that have been described as playing an important role in freezing tolerance and cold acclimation in Arabidopsis. To specify this role, we used a reverse genetic approach and identified a mutant, cbf2, in which the CBF2/DREB1C gene was disrupted. Here, we show that cbf2 plants have higher capacity to tolerate freezing than WT ones before and after cold acclimation and are more tolerant to dehydration and salt stress. All these phenotypes correlate with a stronger and more sustained expression of CBF/DREB1-regulated genes, which results from an increased expression of CBF1/DREB1B and CBF3/DREB1A in the mutant. In addition, we show that the expression of CBF1/DREB1B and CBF3/DREB1A in response to low temperature precedes that of CBF2/DREB1C. These results indicate that CBF2/DREB1C negatively regulates CBF1/DREB1B and CBF3/DREB1A, ensuring that their expression is transient and tightly controlled, which, in turn, guarantees the proper induction of downstream genes and the accurate development of Arabidopsis tolerance to freezing and related stresses.}, number={11}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Novillo, F. and Alonso, J. M. and Ecker, J. R. and Salinas, J.}, year={2004}, month={Mar}, pages={3985–3990} }
 @article{li_johnson_stepanova_alonso_ecker_2004, title={Convergence of Signaling Pathways in the Control of Differential Cell Growth in< i> Arabidopsis</i>}, volume={7}, number={2}, journal={Developmental cell}, author={Li, Hai and Johnson, Phoebe and Stepanova, Anna and Alonso, Jose M. and Ecker, Joseph R.}, year={2004}, pages={193–204} }
 @article{li_johnson_stepanova_alonso_ecker_2004, title={Convergence of signaling of differential cell growth pathways in the control in Arabidopsis}, volume={7}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-4344648019&partnerID=MN8TOARS}, DOI={10.1016/j.devcel.2004.07.002}, abstractNote={Seedling apical hook development involves a complex interplay of hormones and light in the regulation of differential cell growth. However, the underlying molecular mechanisms that integrate these diverse signals to control bending of the embryonic stem are poorly understood. The Arabidopsis ethylene-regulated HOOKLESS1 (HLS1) gene is essential for apical hook formation. Herein, we identify two auxin response regulators that act downstream of HLS1 to control cell elongation in the hypocotyl. Extragenic suppressors of hls1 were identified as mutations in AUXIN RESPONSE FACTOR 2 (ARF2). The level of ARF2 protein was decreased by ethylene, and this response required HLS1. Exposure to light decreased HLS1 protein levels and evoked a concomitant increase in ARF2 accumulation. These studies demonstrate that both ethylene and light signals affect differential cell growth by acting through HLS1 to modulate the auxin response factors, pinpointing HLS1 as a key integrator of the signaling pathways that control hypocotyl bending.}, number={2}, journal={Developmental Cell}, author={Li, H. and Johnson, P. and Stepanova, A. and Alonso, J. M. and Ecker, J. R.}, year={2004}, pages={193–204} }
 @article{tyler_thomas_hu_dill_alonso_ecker_sun_2004, title={DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis}, volume={135}, number={2}, journal={Plant Physiology}, author={Tyler, Ludmila and Thomas, Stephen G. and Hu, Jianhong and Dill, Alyssa and Alonso, Jose M. and Ecker, Joseph R. and Sun, Tai-ping}, year={2004}, pages={1008–1019} }
 @article{pfund_tans-kersten_dunning_alonso_ecker_allen_bent_2004, title={Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana}, volume={17}, ISSN={["1943-7706"]}, DOI={10.1094/MPMI.2004.17.6.696}, abstractNote={The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts.}, number={6}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Pfund, C and Tans-Kersten, J and Dunning, FM and Alonso, JM and Ecker, JR and Allen, C and Bent, AF}, year={2004}, month={Jun}, pages={696–706} }
 @article{chen_pandey_huang_alonso_ecker_assmann_jones_2004, title={GCR1 can act independently of heterotrimeric G-protein in response to brassinosteroids and gibberellins in Arabidopsis seed germination}, volume={135}, number={2}, journal={Plant Physiology}, author={Chen, Jin-Gui and Pandey, Sona and Huang, Jirong and Alonso, José M. and Ecker, Joseph R. and Assmann, Sarah M. and Jones, Alan M.}, year={2004}, pages={907–915} }
 @article{mockler_yu_shalitin_parikh_michael_liou_huang_smith_alonso_ecker_et al._2004, title={Regulation of flowering time in Arabidopsis by K homology domain proteins}, volume={101}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.0404552101}, DOI={10.1073/PNAS.0404552101}, abstractNote={The transition from vegetative growth to reproductive development in Arabidopsis is regulated by multiple floral induction pathways, including the photoperiodic, the autonomous, the vernalization, and the hormonal pathways. These pathways converge to regulate the expression of a small set of genes critical for floral initiation and different signal transduction pathways can interact to govern the time to flower. One important regulator of floral initiation is the MADS-box transcription factor FLC, which acts as a negative regulator of flowering in response to both endogenous and environmental signals. In this report, we describe a study of the flowering-time gene, FLK [flowering locus K homology (KH) domain] that encodes a putative RNA-binding protein with three KH domains. The flk mutations cause delayed flowering without a significant effect on the photoperiodic or vernalization responses. FLK functions primarily as a repressor of FLC expression, although it also modestly affects expression of genes associated with the photoperiodic pathway. In addition to FLK , the expression of two other KH domain genes are modestly affected by the flk mutation, suggesting a possible involvement of more than one KH domain protein in the regulation of flowering time in Arabidopsis .}, number={34}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Mockler, T. C. and Yu, X. and Shalitin, D. and Parikh, D. and Michael, T. P. and Liou, J. and Huang, J. and Smith, Z. and Alonso, J. M. and Ecker, J. R. and et al.}, year={2004}, month={Aug}, pages={12759–12764} }
 @article{stepanova_alonso_2004, title={The canonical ethylene signaling pathway}, volume={2004}, number={221}, journal={Science Signaling}, author={Stepanova, Anna N. and Alonso, Jose M.}, year={2004}, pages={cm1} }
 @article{alonso_stepanova_2004, title={The ethylene signaling pathway}, volume={306}, ISSN={["0036-8075"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-9444243287&partnerID=MN8TOARS}, DOI={10.1126/science.1104812}, abstractNote={Plants use a structurally very simple gas molecule, the hydrocarbon ethylene, to modulate various developmental programs and coordinate responses to a multitude of external stress factors. How this simple molecule generates such a diverse array of effects has been the subject of intense research for the past two decades. A fascinating signaling pathway, with classical as well as novel plant-specific signaling elements, is emerging from these studies. We describe the four main modules that constitute this signaling pathway: a phosphotransfer relay, an EIN2-based unit, a ubiquitin-mediated protein degradation component, and a transcriptional cascade. The canonical and Arabidopsis ethylene signaling pathways in the Signal Transduction Knowledge Environment Connections Maps provide a complete panoramic view of these signaling events in plants.}, number={5701}, journal={SCIENCE}, author={Alonso, JM and Stepanova, AN}, year={2004}, month={Nov}, pages={1513–1515} }
 @article{monte_tepperman_al-sady_kaczorowski_alonso_ecker_li_zhang_quail_2004, title={The phytochrome-interacting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development}, volume={101}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0407107101}, DOI={10.1073/pnas.0407107101}, abstractNote={The phytochrome (phy) family of sensory photoreceptors transduce informational light signals to selected nuclear genes, inducing plant growth and developmental responses appropriate to the environment. Existing data suggest that one signaling pathway by which this occurs involves direct, intranuclear interaction of the photoactivated phy molecule with PIF3, a basic helix-loop-helix transcription factor. Here, we provide evidence from recently identified pif3 mutant alleles that PIF3 is necessary for early chloroplast greening and rapid phy-induced expression of nuclear genes encoding chloroplast components upon first exposure of seedlings to light. Therefore, these data indicate that PIF3 functions to transduce phy signals to genes involved in a critical facet of the early seedling deetiolation process, the generation of a functional photosynthetic apparatus. When transgenically expressed GUS:PIF3 fusion protein constructs were used, we found that PIF3 protein levels are rapidly and reversibly modulated by the photoreceptor over diurnal cycles in Arabidopsis seedlings. The PIF3 protein declines rapidly to a basal steady-state level upon initial light exposure, but reaccumulates to preirradiation levels in darkness during the subsequent night period. These data suggest that PIF3 may function in early phy signaling at the dark-to-light transition, not only during initial seedling deetiolation, but daily at dawn under diurnal light-dark cycles.}, number={46}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Monte, E. and Tepperman, J. M. and Al-Sady, B. and Kaczorowski, K. A. and Alonso, J. M. and Ecker, J. R. and Li, X. and Zhang, Y. and Quail, P. H.}, year={2004}, month={Oct}, pages={16091–16098} }
 @article{to_haberer_ferreira_deruère_mason_schaller_alonso_ecker_kieber_2004, title={Type-A Arabidopsis response regulators are partially redundant negative regulators of cytokinin signaling}, volume={16}, number={3}, journal={The Plant Cell Online}, author={To, Jennifer P. C. and Haberer, Georg and Ferreira, Fernando J. and Deruère, Jean and Mason, Michael G. and Schaller, G. Eric and Alonso, Jose M. and Ecker, Joseph R. and Kieber, Joseph J.}, year={2004}, pages={658–671} }
 @article{mackey_belkhadir_alonso_ecker_dangl_2003, title={< i> Arabidopsis</i> RIN4 Is a Target of the Type III Virulence Effector AvrRpt2 and Modulates RPS2-Mediated Resistance}, volume={112}, number={3}, journal={Cell}, author={Mackey, David and Belkhadir, Youssef and Alonso, Jose M. and Ecker, Joseph R. and Dangl, Jeffery L.}, year={2003}, pages={379–389} }
 @article{lariguet_boccalandro_alonso_ecker_chory_casal_fankhauser_2003, title={A growth regulatory loop that provides homeostasis to phytochrome A signaling}, volume={15}, number={12}, journal={The Plant Cell Online}, author={Lariguet, Patricia and Boccalandro, Hernan E. and Alonso, José M. and Ecker, Joseph R. and Chory, Joanne and Casal, Jorge J. and Fankhauser, Christian}, year={2003}, pages={2966–2978} }
 @article{strand_asami_alonso_ecker_chory_2003, title={Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX}, volume={421}, number={6918}, journal={Nature}, author={Strand, Åsa and Asami, Tadao and Alonso, Jose and Ecker, Joseph R. and Chory, Joanne}, year={2003}, pages={79–83} }
 @article{michael_salome_hannah_spencer_sharp_mcpeek_alonso_ecker_mcclung_2003, title={Enhanced fitness conferred by naturally occurring variation in the circadian clock}, volume={302}, number={5647}, journal={Science}, author={Michael, Todd P. and Salome, Patrice A. and Hannah, J. Yu and Spencer, Taylor R. and Sharp, Emily L. and McPeek, Mark A. and Alonso, Jose M. and Ecker, Joseph R. and McClung, C. Robertson}, year={2003}, pages={1049–1053} }
 @article{alonso_stepanova_solano_wisman_ferrari_ausubel_ecker_2003, title={Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis}, volume={100}, number={5}, journal={Proceedings of the National Academy of Sciences}, author={Alonso, Jose M. and Stepanova, Anna N. and Solano, Roberto and Wisman, Ellen and Ferrari, Simone and Ausubel, Frederick M. and Ecker, Joseph R.}, year={2003}, pages={2992–2997} }
 @article{larkin_alonso_ecker_chory_2003, title={GUN4, a regulator of chlorophyll synthesis and intracellular signaling}, volume={299}, number={5608}, journal={Science}, author={Larkin, Robert M. and Alonso, Jose M. and Ecker, Joseph R. and Chory, Joanne}, year={2003}, pages={902–906} }
 @article{alonso_stepanova_leisse_kim_chen_shinn_stevenson_zimmerman_barajas_cheuk_2003, title={Genome-wide insertional mutagenesis of Arabidopsis thaliana}, volume={301}, number={5633}, journal={Science}, author={Alonso, Jose M. and Stepanova, Anna N. and Leisse, Thomas J. and Kim, Christopher J. and Chen, Huaming and Shinn, Paul and Stevenson, Denise K. and Zimmerman, Justin and Barajas, Pascual and Cheuk, Rosa}, year={2003}, pages={653–657} }
 @article{alonso_2003, title={Genome-wide insertional mutagenesis of Arabidopsis thaliana (vol 301, pg 653, 2003)}, volume={301}, number={5641}, journal={Science}, author={Alonso, J. M.}, year={2003}, pages={1849} }
 @article{monte_alonso_ecker_zhang_li_young_austin-phillips_quail_2003, title={Isolation and characterization of phyC mutants in Arabidopsis reveals complex crosstalk between phytochrome signaling pathways}, volume={15}, number={9}, journal={The Plant Cell Online}, author={Monte, Elena and Alonso, José M. and Ecker, Joseph R. and Zhang, Yuelin and Li, Xin and Young, Jeff and Austin-Phillips, Sandra and Quail, Peter H.}, year={2003}, pages={1962–1980} }
 @article{catalá_santos_alonso_ecker_martínez-zapater_salinas_2003, title={Mutations in the Ca2+/H+ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis}, volume={15}, number={12}, journal={The Plant Cell Online}, author={Catalá, Rafael and Santos, Elisa and Alonso, José M. and Ecker, Joseph R. and Martínez-Zapater, José M. and Salinas, Julio}, year={2003}, pages={2940–2951} }
 @inbook{alonso_stepanova_2003, title={T-DNA mutagenesis in Arabidopsis}, ISBN={1588291456}, DOI={10.1385/1-59259-413-1:177}, abstractNote={Insertional mutagenesis is a basic genetic tool that allows for a rapid identification of the tagged genes responsible for a particular phenotype. Transposon and Agrobacterium-mediated DNA integration are the most commonly used biological mutagens in plants. The main drawback of these technologies is the relatively low frequency of mutations, as compared to those induced by conventional chemical or physical agents, thus limiting the use of insertional mutagens to the generation of large mutant populations in few genetic backgrounds. Recent improvements in Agrobacterium-mediated transformation efficiency and an increasing repertoire of transformation vectors available to the research community is making this type of mutagen very attractive for individual laboratories interested in the studies of mutations in particular genetic backgrounds. Herein, we describe a simple yet robust Arabidopsis transformation procedure that can be used to generate large numbers of insertional mutants in Arabidopsis thaliana. Using this protocol, transformation efficiencies of up to 5% can be achieved.}, booktitle={Plant Functional Genomics}, author={Alonso, Jose M. and Stepanova, Anna N.}, year={2003}, pages={177–187} }
 @article{lariguet_boccalandro_alonso_ecker_chory_casal_fankhausera_2003, title={The Balance between phytochrome kinase substrate1 and PKS2 Provides Homeostasis for Phytochrome A Signaling in Arabidopsis}, journal={The Plant Cell Online}, author={Lariguet, Patricia and Boccalandro, Hernan E. and Alonso, José M. and Ecker, Joseph R. and Chory, Joanne and Casal, Jorge J. and Fankhausera, Christian}, year={2003} }
 @article{ullah_chen_temple_boyes_alonso_davis_ecker_jones_2003, title={The β-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes}, volume={15}, number={2}, journal={The Plant Cell Online}, author={Ullah, Hemayet and Chen, Jin-Gui and Temple, Brenda and Boyes, Douglas C. and Alonso, José M. and Davis, Keith R. and Ecker, Joseph R. and Jones, Alan M.}, year={2003}, pages={393–409} }
 @article{hu_aguirre_peto_alonso_ecker_chory_2002, title={A role for peroxisomes in photomorphogenesis and development of Arabidopsis}, volume={297}, number={5580}, journal={Science}, author={Hu, Jianping and Aguirre, Maria and Peto, Charles and Alonso, José and Ecker, Joseph and Chory, Joanne}, year={2002}, pages={405–409} }
 @article{schroeder_gahrtz_maxwell_cook_kan_alonso_ecker_chory_2002, title={De-Etiolated 1 and Damaged DNA Binding Protein 1 Interact to Regulate< i> Arabidopsis</i> Photomorphogenesis}, volume={12}, number={17}, journal={Current Biology}, author={Schroeder, Dana F. and Gahrtz, Manfred and Maxwell, Bridey B. and Cook, R. Kimberley and Kan, Jack M. and Alonso, José M. and Ecker, Joseph R. and Chory, Joanne}, year={2002}, pages={1462–1472} }
 @article{zheng_bednarek_sanderfoot_alonso_ecker_raikhel_2002, title={NPSN11 is a cell plate-associated SNARE protein that interacts with the syntaxin KNOLLE}, volume={129}, number={2}, journal={Plant physiology}, author={Zheng, Haiyan and Bednarek, Sebastian Y. and Sanderfoot, Anton A. and Alonso, Jose and Ecker, Joseph R. and Raikhel, Natasha V.}, year={2002}, pages={530–539} }
 @article{friedrichsen_nemhauser_muramitsu_maloof_alonso_ecker_furuya_chory_2002, title={Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth}, volume={162}, number={3}, journal={Genetics}, author={Friedrichsen, Danielle M. and Nemhauser, Jennifer and Muramitsu, Takamichi and Maloof, Julin N. and Alonso, José and Ecker, Joseph R. and Furuya, Masaki and Chory, Joanne}, year={2002}, pages={1445–1456} }
 @article{zhao_hull_gupta_goss_alonso_ecker_normanly_chory_celenza_2002, title={Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3}, volume={16}, number={23}, journal={Genes & Development}, author={Zhao, Yunde and Hull, Anna K. and Gupta, Neeru R. and Goss, Kendrick A. and Alonso, José and Ecker, Joseph R. and Normanly, Jennifer and Chory, Joanne and Celenza, John L.}, year={2002}, pages={3100–3112} }
 @article{jarillo_capel_tang_yang_alonso_ecker_cashmore_2001, title={An Arabidopsis circadian clock component interacts with both CRY1 and phyB}, volume={410}, number={6827}, journal={Nature}, author={Jarillo, Jose A. and Capel, Juan and Tang, Ru-Hang and Yang, Hong-Quan and Alonso, Jose M. and Ecker, Joseph R. and Cashmore, Anthony R.}, year={2001}, pages={487–490} }
 @article{jarillo_gabrys_capel_alonso_ecker_cashmore_2001, title={Phototropin-related NPL1 controls chloroplast relocation induced by blue light}, volume={410}, number={6831}, journal={Nature}, author={Jarillo, Jose A. and Gabrys, Halina and Capel, Juan and Alonso, Jose M. and Ecker, Joseph R. and Cashmore, Anthony R.}, year={2001}, pages={952–954} }
 @article{alonso_ecker_2001, title={The ethylene pathway: a paradigm for plant hormone signaling and interaction}, volume={2001}, number={70}, journal={Science Signaling}, author={Alonso, Jose M. and Ecker, Joseph R.}, year={2001}, pages={re1} }
 @article{hirayama_alonso_2000, title={Ethylene captures a metal! Metal ions are involved in ethylene perception and signal transduction}, volume={41}, number={5}, journal={Plant and Cell Physiology}, author={Hirayama, Takashi and Alonso, Jose M.}, year={2000}, pages={548–555} }
 @article{curie_alonso_le jean_ecker_briat_2000, title={Involvement of NRAMP1 from Arabidopsis thaliana in iron transport}, volume={347}, DOI={10.1042/0264-6021:3470749}, abstractNote={Nramp genes code for a widely distributed class of proteins involved in a variety of processes, ranging from the control of susceptibility to bacterial infection in mammalian cells and taste behaviour in Drosophila to manganese uptake in yeast. Some of the NRAMP proteins in mammals and in yeast are capable of transporting metal ions, including iron. In plants, iron transport was shown to require a reduction/Fe(II) transport system. In Arabidopsis thaliana this process involves the IRT1 and Fro2 genes. Here we report the sequence of five NRAMP proteins from A. thaliana. Sequence comparison suggests that there are two classes of NRAMP proteins in plants: A. thaliana (At) NRAMP1 and Oriza sativa (Os) NRAMP1 and 3 (two rice isologues) represent one class, and AtNRAMP2-5 and OsNRAMP2 the other. AtNramp1 and OsNramp1 are able to complement the fet3fet4 yeast mutant defective both in low- and high-affinity iron transports, whereas AtNramp2 and OsNramp2 fail to do so. In addition, AtNramp1 transcript, but not AtNramp2 transcript, accumulates in response to iron deficiency in roots but not in leaves. Finally, overexpression of AtNramp1 in transgenic A. thaliana plants leads to an increase in plant resistance to toxic iron concentration. Taken together, these results demonstrate that AtNramp1 participates in the control of iron homoeostasis in plants.}, journal={Biochem. J}, author={Curie, Catherine and Alonso, J. and Le Jean, Marie and Ecker, J. and Briat, J.}, year={2000}, pages={749–755} }
 @article{theologis_ecker_palm_federspiel_kaul_white_alonso_altafi_araujo_bowman_2000, title={Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana}, volume={408}, number={6814}, journal={Nature}, author={Theologis, Athanasios and Ecker, Joseph R. and Palm, Curtis J. and Federspiel, Nancy A. and Kaul, Samir and White, Owen and Alonso, Jose and Altafi, Hootan and Araujo, Rina and Bowman, Cheryl L.}, year={2000}, pages={816–820} }
 @article{alonso_hirayama_roman_nourizadeh_ecker_1999, title={EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis}, volume={284}, number={5423}, journal={Science}, author={Alonso, Jose M. and Hirayama, Takashi and Roman, Gregg and Nourizadeh, Saeid and Ecker, Joseph R.}, year={1999}, pages={2148–2152} }
 @inbook{cubells-martinez_alonso_sanchez-ballesta_granell_1999, title={Ethylene Perception and Response in Citrus Fruit}, booktitle={Biology and Biotechnology of the Plant Hormone Ethylene II}, author={Cubells-Martinez, X. and Alonso, J. M. and Sanchez-Ballesta, M. T. and Granell, A.}, year={1999}, pages={137–143} }
 @article{hirayama_kieber_hirayama_kogan_guzman_nourizadeh_alonso_dailey_dancis_ecker_1999, title={RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson Disease–Related Copper Transporter, Is Required for Ethylene Signaling in< i> Arabidopsis</i>}, volume={97}, number={3}, journal={Cell}, author={Hirayama, Takashi and Kieber, Joseph J. and Hirayama, Noriko and Kogan, Mikhail and Guzman, Plinio and Nourizadeh, Saeid and Alonso, Jose M. and Dailey, William P. and Dancis, Andrew and Ecker, Joseph R.}, year={1999}, pages={383–393} }
 @article{alonso_chamarro_granell_1995, title={A non-photosynthetic ferredoxin gene is induced by ethylene in Citrus organs}, volume={29}, number={6}, journal={Plant molecular biology}, author={Alonso, José Miguel and Chamarro, Jesús and Granell, Antonio}, year={1995}, pages={1211–1221} }
 @article{alonso_granell_1995, title={A putative vacuolar processing protease is regulated by ethylene and also during fruit ripening in Citrus fruit}, volume={109}, number={2}, journal={Plant Physiology}, author={Alonso, Jose Miguel and Granell, Antonio}, year={1995}, pages={541–547} }
 @article{alonso_chamarro_granell_1995, title={Evidence for the involvement of ethylene in the expression of specific RNAs during maturation of the orange, a non-climacteric fruit}, volume={29}, number={2}, journal={Plant molecular biology}, author={Alonso, Jose Miguel and Chamarro, Jesús and Granell, Antonio}, year={1995}, pages={385–390} }
 @article{alonso_garcía‐martínez_chamarro_1992, title={Two dimensional gel electrophoresis patterns of total, in vivo labelled and in vitro translated polypeptides from orange flavedo during maturation and following ethylene treatment}, volume={85}, number={2}, journal={Physiologia Plantarum}, author={Alonso, José Miguel and García‐Martínez, José Luis and Chamarro, Jesús}, year={1992}, pages={147–156} }
 @inproceedings{merchante_hu_stepanova_alonso_heber, title={Deep sequencing of ribosomal footprints for studying genome-wide mRNA translation in plants}, author={Merchante, Karen and Hu, Qiwen and Stepanova, Anna N. and Alonso, Jose M. and Heber, Steffen} }