@article{fernandez-moreno_yaschenko_neubauer_marchi_zhao_ascencio-ibanez_alonso_stepanova_2024, title={A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters}, volume={2}, ISSN={["1467-7652"]}, url={https://doi.org/10.1111/pbi.14313}, DOI={10.1111/pbi.14313}, abstractNote={Advancement of DNA-synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high-complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single-tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single-tube digestion-ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone-responsive, synthetic, repetitive proximal promoters were generated and tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene-responsive promoter 10x2EBS-S10 fused to the GUS reporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution.}, journal={PLANT BIOTECHNOLOGY JOURNAL}, author={Fernandez-Moreno, Josefina-Patricia and Yaschenko, Anna E. and Neubauer, Matthew and Marchi, Alex J. and Zhao, Chengsong and Ascencio-Ibanez, Jose T. and Alonso, Jose M. and Stepanova, Anna N.}, year={2024}, month={Feb} }
 @article{yaschenko_alonso_stepanova_2024, title={Arabidopsis as a model for translational research}, volume={2}, ISSN={["1532-298X"]}, url={https://doi.org/10.1093/plcell/koae065}, DOI={10.1093/plcell/koae065}, abstractNote={Abstract
               Arabidopsis thaliana is currently the most-studied plant species on earth, with an unprecedented number of genetic, genomic, and molecular resources having been generated in this plant model. In the era of translating foundational discoveries to crops and beyond, we aimed to highlight the utility and challenges of using Arabidopsis as a reference for applied plant biology research, agricultural innovation, biotechnology, and medicine. We hope that this review will inspire the next generation of plant biologists to continue leveraging Arabidopsis as a robust and convenient experimental system to address fundamental and applied questions in biology. We aim to encourage laboratory and field scientists alike to take advantage of the vast Arabidopsis datasets, annotations, germplasm, constructs, methods, and molecular and computational tools in our pursuit to advance understanding of plant biology and help feed the world's growing population. We envision that the power of Arabidopsis-inspired biotechnologies and foundational discoveries will continue to fuel the development of resilient, high-yielding, nutritious plants for the betterment of plant and animal health and greater environmental sustainability.}, journal={PLANT CELL}, author={Yaschenko, Anna E. and Alonso, Jose M. and Stepanova, Anna N.}, year={2024}, month={Feb} }
 @article{neubauer_vollen_ascencio-ibanez_hanley-bowdoin_stepanova_alonso_2024, title={Development of modular geminivirus-based vectors for high cargo expression and gene targeting in plants}, url={https://doi.org/10.1101/2024.06.29.601216}, DOI={10.1101/2024.06.29.601216}, abstractNote={Viral vectors can be useful tools for expressing recombinant proteins as well as delivering gene-editing machinery. Despite their utility, the development and subsequent optimization of these tools is often a difficult and tedious process. Thus, although considerable work has been done to create useful viral vectors for gene editing and protein expression, there is a lack of understanding of how best to design these vectors for specific applications. For instance, it is often unclear whether the inclusion of heterologous promoter sequences or different viral components will improve cargo expression or replicon accumulation. To address some of these hurdles, we designed a GoldenBraid (GB) compatible viral vector system based on the geminivirus Beet curly top virus (BCTV). This system allows for simple, modular cloning of a variety of reporter constructs. Making use of this modular cloning strategy, we compared a variety of alternative viral vector architectures. Interestingly, native BCTV promoters outperformed the constitutive 35S promoter, while the removal of the BCTV virion-sense genes promoted reporter expression. Intriguingly, these modifications had no effect on total replicon accumulation. These results show the utility of the new modular BCTV-based viral vectors for protein expression and gene targeting applications, as well as uncover design principles that may inform future geminivirus-based viral vector architectures. We anticipate that the availability of this new modular system will spark the broad adoption of replicon-based strategies in protein expression and gene editing experiments in plants.}, author={Neubauer, Matthew and Vollen, Katie and Ascencio-Ibanez, Jose T. and Hanley-Bowdoin, Linda and Stepanova, Anna N. and Alonso, Jose M.}, year={2024}, month={Jun} }
 @misc{vollen_zhao_alonso_stepanova_2024, title={Sourcing DNA parts for synthetic biology applications in plants}, volume={87}, ISSN={["1879-0429"]}, DOI={10.1016/j.copbio.2024.103140}, abstractNote={Transgenic approaches are now standard in plant biology research aiming to characterize gene function or improve crops. Recent advances in DNA synthesis and assembly make constructing transgenes a routine task. What remains nontrivial is the selection of the DNA parts and optimization of the transgene design. Early career researchers and seasoned molecular biologists alike often face difficult decisions on what promoter or terminator to use, what tag to include, and where to place it. This review aims to inform about the current approaches being employed to identify and characterize DNA parts with the desired functionalities and give general advice on basic construct design. Furthermore, we hope to share the excitement about new experimental and computational tools being developed in this field.}, journal={CURRENT OPINION IN BIOTECHNOLOGY}, author={Vollen, Katie and Zhao, Chengsong and Alonso, Jose M. and Stepanova, Anna N.}, year={2024}, month={Jun} }
 @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{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} }
 @article{friesner_colon-carmona_schnoes_stepanova_mason_macintosh_ullah_baxter_callis_sierra-cajas_et al._2021, title={Broadening the impact of plant science through innovative, integrative, and inclusive outreach}, volume={5}, ISSN={["2475-4455"]}, url={http://dx.doi.org/10.1002/pld3.316}, DOI={10.1002/pld3.316}, abstractNote={AbstractPopulation growth and climate change will impact food security and potentially exacerbate the environmental toll that agriculture has taken on our planet. These existential concerns demand that a passionate, interdisciplinary, and diverse community of plant science professionals is trained during the 21st century. Furthermore, societal trends that question the importance of science and expert knowledge highlight the need to better communicate the value of rigorous fundamental scientific exploration. Engaging students and the general public in the wonder of plants, and science in general, requires renewed efforts that take advantage of advances in technology and new models of funding and knowledge dissemination. In November 2018, funded by the National Science Foundation through the Arabidopsis Research and Training for the 21st century (ART 21) research coordination network, a symposium and workshop were held that included a diverse panel of students, scientists, educators, and administrators from across the US. The purpose of the workshop was to re‐envision how outreach programs are funded, evaluated, acknowledged, and shared within the plant science community. One key objective was to generate a roadmap for future efforts. We hope that this document will serve as such, by providing a comprehensive resource for students and young faculty interested in developing effective outreach programs. We also anticipate that this document will guide the formation of community partnerships to scale up currently successful outreach programs, and lead to the design of future programs that effectively engage with a more diverse student body and citizenry.}, number={4}, journal={PLANT DIRECT}, publisher={Wiley}, author={Friesner, Joanna and Colon-Carmona, Adan and Schnoes, Alexandra M. and Stepanova, Anna and Mason, Grace Alex and Macintosh, Gustavo C. and Ullah, Hemayat and Baxter, Ivan and Callis, Judy and Sierra-Cajas, Kimberly and et al.}, year={2021}, month={Apr} }
 @article{qiao_stepanova_2021, title={Editorial overview: Toward deciphering the molecular basis of plant phenotypic plasticity}, volume={63}, ISSN={["1879-0356"]}, DOI={10.1016/j.pbi.2021.102107}, journal={CURRENT OPINION IN PLANT BIOLOGY}, author={Qiao, Hong and Stepanova, Anna N.}, year={2021}, month={Oct} }
 @article{zhao_yaschenko_alonso_stepanova_2021, title={Leveraging synthetic biology approaches in plant hormone research}, url={https://doi.org/10.1016/j.pbi.2020.101998}, DOI={10.1016/j.pbi.2020.101998}, abstractNote={The study of plant hormones is critical to understanding development, physiology and interactions of plants with their environment. Synthetic biology holds promise to provide a new perspective and shed fresh light on the molecular mechanisms of plant hormone action and propel the design of novel biotechnologies. With the recent adoption of synthetic biology in plant sciences, exciting first examples of successful tool development and their applications in the area of plant hormone research are emerging, paving the way for new cadres to enter this promising field of science.}, journal={Current Opinion in Plant Biology}, author={Zhao, Chengsong and Yaschenko, Anna and Alonso, Jose M and Stepanova, Anna N}, year={2021}, month={Apr} }
 @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{stepanova_2021, title={Plant Biology Research: What Is Next?}, volume={12}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2021.749104}, abstractNote={Plant biology is a key area of science that bears major weight in the mankind’s ongoing and future efforts to combat the consequences of global warming, climate change, pollution, and population growth. An in-depth understanding of plant physiology is paramount to our ability to optimize current agricultural practices, to develop new crop varieties, or to implement biotechnological innovations in agriculture. The next-generation cultivars would have to withstand environmental contamination and a wider range of growth temperatures, soil nutrients and moisture levels and effectively deal with growing pathogen pressures to continue to yield well in even suboptimal conditions. What are the next big questions in plant physiology, and plant biology in general, and what avenues of research should we be investigating and training students in for the next decade? As a plant scientist surrounded by like-minded individuals, I hear a lot of ideas that over time turn into buzz words, such as plant resilience, genotype-to-phenotype, data science, systems biology, biosensing, synthetic biology, neural networks, robustness, interdisciplinary training, new tool development, modeling, etc. What does it all mean and what are the main challenges that we should all be working on solving? Herein, I present my personal perspective on what the immediate questions and the biggest longer-term issues in plant science are. I suggest some themes and directions for future research in plant biology, some relatively obvious and some potentially unique, having been shaped by my own professional interests, experiences and the background in plant molecular genetics and physiology.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Stepanova, Anna N.}, year={2021}, month={Sep} }
 @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{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={Structure–function modeling explores the basis of interallelic complementation in multiple combinations of rooty alleles. 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_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{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={AbstractGene 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{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={RationaleMass 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.MethodsWild‐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.ResultsIAA 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.ConclusionsAgarose 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{zander_willige_he_nguyen_langford_nehring_howell_mcgrath_bartlett_castanon_et al._2019, title={Epigenetic silencing of a multifunctional plant stress regulator}, volume={8}, ISSN={["2050-084X"]}, DOI={10.7554/eLife.47835}, abstractNote={The central regulator of the ethylene (ET) signaling pathway, which controls a plethora of developmental programs and responses to environmental cues in plants, is ETHYLENE-INSENSITIVE2 (EIN2). Here we identify a chromatin-dependent regulatory mechanism at EIN2 requiring two genes: ETHYLENE-INSENSITIVE6 (EIN6), which is a H3K27me3 demethylase also known as RELATIVE OF EARLY FLOWERING6 (REF6), and EIN6 ENHANCER (EEN), the Arabidopsis homolog of the yeast INO80 chromatin remodeling complex subunit IES6 (INO EIGHTY SUBUNIT). Strikingly, EIN6 (REF6) and the INO80 complex redundantly control the level and the localization of the repressive histone modification H3K27me3 and the histone variant H2A.Z at the 5’ untranslated region (5’UTR) intron of EIN2. Concomitant loss of EIN6 (REF6) and the INO80 complex shifts the chromatin landscape at EIN2 to a repressive state causing a dramatic reduction of EIN2 expression. These results uncover a unique type of chromatin regulation which safeguards the expression of an essential multifunctional plant stress regulator.}, journal={ELIFE}, author={Zander, Mark and Willige, Bjorn C. and He, Yupeng and Nguyen, Thu A. and Langford, Amber E. and Nehring, Ramlah and Howell, Elizabeth and McGrath, Robert and Bartlett, Anna and Castanon, Rosa and et al.}, year={2019}, month={Aug} }
 @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{fernandez‐moreno_stepanova_2020, title={Monitoring Ethylene in Plants: Genetically Encoded Reporters and Biosensors}, url={https://doi.org/10.1002/smtd.201900260}, DOI={10.1002/smtd.201900260}, abstractNote={AbstractPhytohormone ethylene regulates numerous aspects of plant physiology, from fruit ripening to pathogen responses. The molecular basis of ethylene biosynthesis and action has been investigated for over 40 years, and a combination of biochemistry, genetics, cell, and molecular biology have proven successful at uncovering the core machinery of the ethylene pathway. A number of molecular tools have been developed over the years that enable visualization of the sites of ethylene production and response in the plant. Genetically encoded biosensors take advantage of reporter proteins, i.e., fluorescent, luminescent, or colorimetric markers, to highlight the tissues that make, perceive, or respond to the hormone. This review describes the different types of biosensors currently available to the ethylene community and discusses potential new strategies for developing the next generation of genetically encoded ethylene reporters.}, journal={Small Methods}, author={Fernandez‐Moreno, Josefina‐Patricia and Stepanova, Anna N.}, year={2020}, month={Aug} }
 @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} }
 @misc{mazzoni-putman_stepanova_2018, title={A Plant Biologist's Toolbox to Study Translation}, volume={9}, ISSN={["1664-462X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85049512067&partnerID=MN8TOARS}, DOI={10.3389/fpls.2018.00873}, abstractNote={Across a broad range of species and biological questions, more and more studies are incorporating translation data to better assess how gene regulation occurs at the level of protein synthesis. The inclusion of translation data improves upon, and has been shown to be more accurate than, transcriptional studies alone. However, there are many different techniques available to measure translation and it can be difficult, especially for young or aspiring scientists, to determine which methods are best applied in specific situations. We have assembled this review in order to enhance the understanding and promote the utilization of translational methods in plant biology. We cover a broad range of methods to measure changes in global translation (e.g., radiolabeling, polysome profiling, or puromycylation), translation of single genes (e.g., fluorescent reporter constructs, toeprinting, or ribosome density mapping), sequencing-based methods to uncover the entire translatome (e.g., Ribo-seq or translating ribosome affinity purification), and mass spectrometry-based methods to identify changes in the proteome (e.g., stable isotope labeling by amino acids in cell culture or bioorthogonal noncanonical amino acid tagging). The benefits and limitations of each method are discussed with a particular note of how applications from other model systems might be extended for use in plants. In order to make this burgeoning field more accessible to students and newer scientists, our review includes an extensive glossary to define key terms.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Mazzoni-Putman, Serina M. and Stepanova, Anna N.}, year={2018}, month={Jul} }
 @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={AbstractPhosphate (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{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={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.}, 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{ferrando_castellano_lisón_leister_stepanova_hanson_2017, title={Editorial: Relevance of translational regulation on plant growth and environmental responses}, volume={8}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85040509996&partnerID=MN8TOARS}, DOI={10.3389/fpls.2017.02170}, abstractNote={Editorial : Relevance of Translational Regulation on Plant Growth and Environmental Responses}, journal={Frontiers in Plant Science}, author={Ferrando, A. and Castellano, M.M. and Lisón, P. and Leister, D. and Stepanova, A.N. and Hanson, J.}, year={2017} }
 @article{merchante_stepanova_2017, title={The triple response assay and its use to characterize ethylene mutants in Arabidopsis}, volume={1573}, journal={Ethylene signaling: methods and protocols}, author={Merchante, C. and Stepanova, A. N.}, year={2017}, pages={163–209} }
 @inbook{merchante_stepanova_2017, title={The triple response assay and its use to characterize ethylene mutants in arabidopsis}, volume={1573}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85015705453&partnerID=MN8TOARS}, DOI={10.1007/978-1-4939-6854-1_13}, abstractNote={Exposure of plants to ethylene results in drastic morphological changes. Seedlings germinated in the dark in the presence of saturating concentrations of ethylene display a characteristic phenotype known as the triple response. This phenotype is robust and easy to score. In Arabidopsis the triple response is usually evaluated at 3 days post germination in seedlings grown in the dark in rich media supplemented with 10 μM of the ethylene precursor ACC in air or in unsupplemented media in the presence of 10 ppm ethylene. The triple response in Arabidopsis consists of shortening and thickening of hypocotyls and roots and exaggeration of the curvature of apical hooks. The search for Arabidopsis mutants that fail to show this phenotype in ethylene or, vice versa, display the triple response in the absence of exogenously supplied hormone has allowed the identification of the key components of the ethylene biosynthesis and signaling pathways. Herein, we describe a simple protocol for assaying the triple response in Arabidopsis. The method can also be employed in many other dicot species, with minor modifications to account for species-specific differences in germination. We also compiled a comprehensive table of ethylene-related mutants of Arabidopsis, including many lines with auxin-related defects, as wild-type levels of auxin biosynthesis, transport, signaling, and response are necessary for the normal response of plants to ethylene.}, booktitle={Methods in Molecular Biology}, author={Merchante, C. and Stepanova, A.N.}, year={2017}, pages={163–209} }
 @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={SummaryChanges 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} }
 @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., 2016aShi H. Shen X. Liu R. Xue C. Wei N. Deng X.W. Zhong W. Dev. Cell. 2016; 39 (this issue): 597-610Abstract Full Text Full Text PDF Scopus (18) Google Scholar 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. In this issue of Developmental Cell, Shi et al., 2016aShi H. Shen X. Liu R. Xue C. Wei N. Deng X.W. Zhong W. Dev. Cell. 2016; 39 (this issue): 597-610Abstract Full Text Full Text PDF Scopus (18) Google Scholar 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. One of the most dramatic environmental changes that a plant experiences during germination is the transition from growing in a dark underground environment to the bright sun of the soil surface. Prior to emergence, dark-germinated seedlings are under the developmental control of a program called skotomorphogenesis, which guides the young seedlings toward their energy source, the light, while protecting their valuable and delicate meristems: dark-grown seedlings develop long hypocotyls (embryonic stems), and their apices are bent to form apical hooks that enable efficient soil penetration. Once the seedling reaches the soil surface, its growth pattern changes to slow down hypocotyl elongation and to induce apical hook opening and expansion of the cotyledons, a program collectively known as photomorphogenesis. It is then that the different components of the photosynthetic machinery are produced and assembled to enable the capture of the energy of light. The transition from skoto- to photomorphogenesis must be precisely timed because, for example, excess accumulation of photosynthetic precursors such as protochlorophyllide can cause extensive oxidative damage once the seedlings are exposed to light (Reinbothe et al., 1996Reinbothe S. Reinbothe C. Apel K. Lebedev N. Cell. 1996; 86: 703-705Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), whereas a delay in the initiation of this program would deprive young seedlings of the valuable energy source at a critical moment in their development. The identification and characterization of mutants affected in this transition has uncovered a series of genes that repress or promote photomorphogenesis, including the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) (Seo et al., 2003Seo H.S. Yang J.Y. Ishikawa M. Bolle C. Ballesteros M.L. Chua N.H. Nature. 2003; 423: 995-999Crossref PubMed Scopus (397) Google Scholar) and the red light photoreceptors phytochromes (Quail et al., 1995Quail P.H. Boylan M.T. Parks B.M. Short T.W. Xu Y. Wagner D. Science. 1995; 268: 675-680Crossref PubMed Scopus (654) Google Scholar), respectively. Although these genes have been known for a long time, only recently have we started to understand the molecular mechanisms by which they trigger dramatic morphological and physiological changes. Earlier studies showed that the plant hormone ethylene plays a key role in repressing photomorphogenesis in dark-grown seedlings and preparing the plants for their transition to the light environment (Shi et al., 2016bShi H. Liu R. Xue C. Shen X. Wei N. Deng X.W. Zhong S. Curr. Biol. 2016; 26: 139-149Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, Zhong et al., 2009Zhong S. Zhao M. Shi T. Shi H. An F. Zhao Q. Guo H. Proc. Natl. Acad. Sci. USA. 2009; 106: 21431-21436Crossref PubMed Scopus (179) Google Scholar, Zhong et al., 2014Zhong S. Shi H. Xue C. Wei N. Guo H. Deng X.W. Proc. Natl. Acad. Sci. USA. 2014; 111: 3913-3920Crossref PubMed Scopus (115) Google Scholar). COP1 was found to promote ethylene activity by stimulating the degradation of two negative regulators of the ethylene signaling pathway, EIN3-BINDING F-BOX1 (EBF1) and EBF2. Gradual inactivation of COP1 as the plant approaches the soil surface results in the attenuation of ethylene responses. Shi et al., 2016aShi H. Shen X. Liu R. Xue C. Wei N. Deng X.W. Zhong W. Dev. Cell. 2016; 39 (this issue): 597-610Abstract Full Text Full Text PDF Scopus (18) Google Scholar now show that ethylene plays an additional role in modulating the rapid changes that take place upon seeding emergence from the soil. Interestingly, these responses are triggered by the direct binding of the photo-activated light receptor, phytochrome B (phyB), to the transcriptional master regulator of ethylene responses, ETHYLENE INSENSITIVE3 (EIN3). Upon seedling emergence from the soil, light responses get activated, and ethylene responses get turned off. In fact, Shi et al., 2016aShi H. Shen X. Liu R. Xue C. Wei N. Deng X.W. Zhong W. Dev. Cell. 2016; 39 (this issue): 597-610Abstract Full Text Full Text PDF Scopus (18) Google Scholar found that the hook opening and cotyledon expansion phenotypes typically observed under red-light conditions could be effectively counteracted by the activation of the ethylene signaling pathway, suggesting the existence of a mechanism by which ethylene responses are actively repressed at the time of seedling emergence from the soil. A clue for such an active mechanism comes from our understanding of the modes of action of phytochromes and the hormone ethylene themselves. Red light activates phytochromes, promoting their translocation from the cytosol to the nucleus, where they interact with the PHYTOCHROME-INTERACTING FACTOR (PIF) family of transcription factors. These interactions trigger the degradation of the PIFs and thus a series of transcriptional changes associated with the plant responses to red light (Ni et al., 2014Ni W. Xu S.L. Tepperman J.M. Stanley D.J. Maltby D.A. Gross J.D. Burlingame A.L. Wang Z.Y. Quail P.H. Science. 2014; 344: 1160-1164Crossref PubMed Scopus (183) Google Scholar). On the other hand, ethylene responses are initiated by the stabilization and activation of the transcription factors EIN3 and ETHYLENE INSENSITIVE3-LIKE1 (EIL1). This transcriptional induction is, at least in part, mediated by the inactivation of two F-box proteins, EBF1 and EBF2, that, in the absence of ethylene, target EIN3 and EIL1 to ubiquitin-mediated degradation (Guo and Ecker, 2003Guo H. Ecker J.R. Cell. 2003; 115: 667-677Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). Surprisingly, Shi et al., 2016aShi H. Shen X. Liu R. Xue C. Wei N. Deng X.W. Zhong W. Dev. Cell. 2016; 39 (this issue): 597-610Abstract Full Text Full Text PDF Scopus (18) Google Scholar found that EIN3 levels rapidly decline during the first 30 min of seedling exposure to red light, thus questioning the role of the previously reported, inherently slower, COP1-dependent downregulation of EIN3 levels via the gradual accumulation of EBFs under prolonged light-growth conditions (Shi et al., 2016bShi H. Liu R. Xue C. Shen X. Wei N. Deng X.W. Zhong S. Curr. Biol. 2016; 26: 139-149Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Furthermore, although both EBF1 and EBF2 are required for the rapid degradation of EIN3 in response to red light, the accumulation of these two F-box proteins did not significantly change over the course of the light treatment carried out by Shi et al., 2016aShi H. Shen X. Liu R. Xue C. Wei N. Deng X.W. Zhong W. Dev. Cell. 2016; 39 (this issue): 597-610Abstract Full Text Full Text PDF Scopus (18) Google Scholar. Thus, the observed light-triggered disappearance of EIN3 implied the existence of a different mechanism that enabled the rapid degradation of EIN3 in response to red light. The first critical clue for the mechanism behind this rapid response to red light came from the observation that phyB interacts, both in vitro and in vivo, with EIN3; importantly, this interaction preferentially occurs between the red-light-activated form of phyB and EIN3. In addition, EBF1 and EBF2 were found to also interact with phyB, although in this case with both its active and inactive forms. Taken together, these results suggest an elegant, mechanistic model in which, upon emergence from the soil, phyB is induced by red light, promoting the phyB-EBF1/2-EIN3 interactions, thus triggering the rapid degradation of EIN3—the transcriptional master regulator of ethylene responses—and thereby turning off the ethylene-mediated repression of photomorphogenesis (Figure 1). Consistent with this model, a short exposure of dark-grown seedlings to red light promoted the interaction between EIN3 and EBFs. In fact, immunoprecipitation experiments, both in vivo and in vitro, showed that EIN3, EBFs, and phyB form a tripartite protein complex, and the formation of this complex is promoted by the red-light-mediated activation of phyB. The study by Shi et al., 2016aShi H. Shen X. Liu R. Xue C. Wei N. Deng X.W. Zhong W. Dev. Cell. 2016; 39 (this issue): 597-610Abstract Full Text Full Text PDF Scopus (18) Google Scholar thus builds on earlier findings implicating phytochrome-ethylene crosstalk in critical aspects of the developmental transition from dark to light, revealing a complex picture of multiple mechanisms of interaction between these two important signals in plants. The surprising discovery that activated phyB functions as the "molecular glue" between the key ethylene transcription factors and their negative regulators opens an exciting possibility of similar, strikingly direct mechanisms functioning in the interaction between light and other hormone signaling pathways. The Red Light Receptor Phytochrome B Directly Enhances Substrate-E3 Ligase Interactions to Attenuate Ethylene ResponsesShi et al.Developmental CellNovember 23, 2016In BriefExposure of plants to sunshine upon emergence from the soil causes dramatic developmental changes, including inhibition of ethylene signaling. Shi, Shen et al. now show that the light-activated photoreceptor phyB contributes to this acute transition by mediating an interaction between the ethylene signaling regulator EIN3 and an E3 ubiquitin ligase. Full-Text PDF Open Archive}, 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}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84970016925&partnerID=MN8TOARS}, DOI={10.1109/TNB.2016.2516950}, abstractNote={Upstream open reading frames (uORFs) are open reading frames that occur within the 5' UTR of an mRNA. uORFs have been found in many organisms. They play an important role in gene regulation, cell development, and in various metabolic processes. It is believed that translated uORFs reduce the translational efficiency of the main coding region. However, only few uORFs are experimentally characterized. In this paper, we use ribosome footprinting together with a semi-supervised approach based on stacking classification models to identify translated uORFs in Arabidopsis thaliana. Our approach identified 5360 potentially translated uORFs in 2051 genes. GO terms enriched in genes with translated uORFs include catalytic activity, binding, transferase activity, phosphotransferase activity, kinase activity, and transcription regulator activity. The reported uORFs occur with a higher frequency in multi-isoform genes, and some uORFs are affected by alternative transcript start sites or alternative splicing events. Association rule mining revealed sequence features associated with the translation status of the uORFs. We hypothesize that uORF translation is a complex process that might be regulated by multiple factors. The identified uORFs are available online at: https://www.dropbox.com/sh/zdutupedxafhly8/AABFsdNR5zDfiozB7B4igFcja?dl=0. This paper is the extended version of our research presented at ISBRA 2015.}, number={2}, journal={IEEE Transactions on Nanobioscience}, author={Hu, Q. and Merchante, C. and STEPANOVA, ANNA and Alonso, Jose and Heber, S.}, year={2016}, pages={150–159} }
 @article{villarino_hu_manrique_flores-vergara_sehra_robles_brumos_stepanova_colombo_sundberg_et al._2016, title={Transcriptomic Signature of the SHATTERPROOF2 Expression Domain Reveals the Meristematic Nature of Arabidopsis Gynoecial Medial Domain}, volume={171}, ISSN={["1532-2548"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84964798633&partnerID=MN8TOARS}, DOI={10.1104/pp.15.01845}, abstractNote={Transcriptional profiles of spatially and temporally restricted cell populations from the Arabidopsis gynoecium reveals the meristematic nature of the gynoecial medial domain. Plant meristems, like animal stem cell niches, maintain a pool of multipotent, undifferentiated cells that divide and differentiate to give rise to organs. In Arabidopsis (Arabidopsis thaliana), the carpel margin meristem is a vital meristematic structure that generates ovules from the medial domain of the gynoecium, the female floral reproductive structure. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present a novel approach to analyze the transcriptional signature of the medial domain of the Arabidopsis gynoecium, highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Using a floral synchronization system and a SHATTERPROOF2 (SHP2) domain-specific reporter, paired with FACS and RNA sequencing, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. This analysis reveals a set of genes that are differentially expressed within the SHP2 expression domain, including genes that have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Global analyses of the transcriptomic data set indicate a similarity of the pSHP2-expressing cell population to previously characterized meristematic domains, further supporting the meristematic nature of this gynoecial tissue. Our method identifies additional genes including novel isoforms, cis-natural antisense transcripts, and a previously unrecognized member of the REPRODUCTIVE MERISTEM family of transcriptional regulators that are potential novel regulators of medial domain development. This data set provides genome-wide transcriptional insight into the development of the carpel margin meristem in Arabidopsis.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Villarino, Gonzalo H. and Hu, Qiwen and Manrique, Silvia and Flores-Vergara, Miguel and Sehra, Bhupinder and Robles, Linda and Brumos, Javier and Stepanova, Anna N. and Colombo, Lucia and Sundberg, Eva and et al.}, year={2016}, month={May}, pages={42–61} }
 @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{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={2015 IEEE 5th International Conference on Computational Advances in Bio and Medical Sciences, ICCABS 2015}, author={Hu, Q. and Merchante, C. and STEPANOVA, ANNA 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{alonso_stepanova_2015, title={A recombineering-based gene tagging system for Arabidopsis}, volume={1227}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84921711835&partnerID=MN8TOARS}, DOI={10.1007/978-1-4939-1652-8_11}, abstractNote={Many of the experimental approaches aimed at studying gene function heavily rely on the ability to make precise modifications in the gene's DNA sequence. Homologous recombination (HR)-based strategies provide a convenient way to create such types of modifications. HR-based DNA sequence manipulations can be enormously facilitated by expressing in E. coli a small set of bacteriophage proteins that make the exchange of DNA between a linear donor and the target DNA molecules extremely efficient. These in vivo recombineering techniques have been incorporated as essential components of the molecular toolbox in many model organisms. In this chapter, we describe the experimental procedures involved in recombineering-based tagging of an Arabidopsis gene contained in a plant transformation-ready bacterial artificial chromosome (TAC).}, journal={Methods in molecular biology (Clifton, N.J.)}, author={Alonso, Jose and STEPANOVA, ANNA}, year={2015}, pages={233–243} }
 @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. Cell and planar polarity are important for the organization of cells within organisms. Pietra et al. demonstrate in Arabidopsisthat the SABRE protein is important for mediating cell and planar polarity by stabilizing the orientation of microtubules during cell division and cell elongation.}, 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} }
 @book{alonso_stepanova_2014, title={Arabidopsis transformation with large bacterial artificial chromosomes}, volume={1062}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84934439816&partnerID=MN8TOARS}, DOI={10.1007/978-1-62703-580-4_15}, abstractNote={The study of a gene’s function requires, in many cases, the ability to reintroduce the gene of interest or its modified version back into the organism of choice. One potential caveat of this approach is that not only the coding region but also the regulatory sequences of a gene should be included in the corresponding transgenic construct. Even in species with well-annotated genomes, such as Arabidopsis, it is nearly impossible to predict which sequences are responsible for the proper expression of a gene. One way to circumvent this problem is to utilize a large fragment of genomic DNA that contains the coding region of the gene of interest and at least 5–10 kb of flanking genomic sequences. To facilitate these types of experiments, libraries harboring large genomic DNA fragments in binary vectors have been constructed for Arabidopsis and several other plant species. Working with these large clones, however, requires some special precautions. In this chapter, we describe the experimental procedures and extra cautionary measures involved in the identification of the clone containing the gene of interest, its transfer from E. coli to Agrobacterium, and the generation, verification, and analysis of the corresponding transgenic plants.}, journal={Methods in Molecular Biology}, author={Alonso, J.M. and Stepanova, A.N.}, year={2014}, pages={271–283} }
 @inproceedings{merchante_hu_stepanova_eonso_heber_2013, title={Deep sequencing of ribosomal footprints for studying genome-wide mRNA translation in plants}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84889042453&partnerID=MN8TOARS}, DOI={10.1109/ICCABS.2013.6629221}, abstractNote={Ribosome footprinting measures mRNA translation via deep sequencing of ribosome-protected mRNA fragments (footprints). Ribosome footprinting has been successfully applied to various model systems, including yeast, human, mouse, and worm; and it has been demonstrated that the technology is capable to produce global translation measurements with up to a single-codon resolution. Here we present our first experiences with a footprinting experiment that probes ethylene-induced translational changes in the reference plant Arabidopsis thaliana. The goal of our project is to develop genomic tools for the qualitative and quantitative characterization of the plant translatome. We hope ribosome footprinting will allow researchers to correlate precisely mRNA features with translation efficiency, and open the way to investigate globally the mechanism of protein synthesis and its regulation at an unprecedented level of detail.}, booktitle={2013 IEEE 3rd International Conference on Computational Advances in Bio and Medical Sciences, ICCABS 2013}, author={Merchante, K. and Hu, Q. and Stepanova, A.N. and Eonso, J.M. and Heber, S.}, year={2013} }
 @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{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{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.}, number={6}, journal={MOLECULAR PLANT}, author={Robles, Linda and Stepanova, Anna and Alonso, Jose}, year={2013}, month={Nov}, pages={1734–1737} }
 @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{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={AbstractThe 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={SummaryOne 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} }
 @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}, abstractNote={Arabidopsis root-hair position and orientation in epithelial cells is directed by an auxin gradient maximum at the root tip. The Raf-like kinase CTR1 negatively regulates local auxin biosynthesis in the root to determine root hair position. The coordination of cell polarity within the plane of a single tissue layer (planar polarity) is a crucial task during development of multicellular organisms. Mechanisms underlying establishment of planar polarity, however, differ substantially between plants and animals1,2,3. In Arabidopsis thaliana, planar polarity of root-hair positioning along epidermal cells is coordinated towards maximum concentration of an auxin gradient in the root tip3,4,5. This gradient has been hypothesized to be sink-driven6 and computational modelling suggests that auxin efflux carrier activity may be sufficient to generate the gradient in the absence of auxin biosynthesis in the root7. Here, we demonstrate that the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1; Refs 8, 9) acts as a concentration-dependent repressor of a biosynthesis-dependent auxin gradient that modulates planar polarity in the root tip. We analysed auxin biosynthesis and concentration gradients in a variety of root-hair-position mutants affected in CTR1 activity, auxin biosynthesis and transport. Our results reveal that planar polarity relies on influx- and efflux-carrier-mediated auxin redistribution from a local biosynthesis maximum. Thus, a local source of auxin biosynthesis contributes to gradient homeostasis during long-range coordination of cellular morphogenesis.}, 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{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{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={AbstractHormones 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{stepanova_alonso_2006, title={PCR-based screening for insertional mutants.}, volume={323}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33745265233&partnerID=MN8TOARS}, journal={Methods in molecular biology (Clifton, N.J.)}, author={Stepanova, A.N. and Alonso, J.M.}, year={2006}, pages={163–172} }
 @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 α1 (WEI2/ASA1) and WEI7/ANTHRANILATE SYNTHASE β1 (ASB1) genes that encode α- and β-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{stepanova_alonso_2005, title={Arabidopsis ethylene signaling pathway.}, volume={2005}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33644876995&partnerID=MN8TOARS}, number={276}, journal={Science's STKE : signal transduction knowledge environment}, author={Stepanova, A.N. and Alonso, J.M.}, year={2005} }
 @article{stepanova_alonso_2005, title={Ethylene signaling pathway.}, volume={2005}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33644877967&partnerID=MN8TOARS}, number={276}, journal={Science's STKE : signal transduction knowledge environment}, author={Stepanova, A.N. and Alonso, J.M.}, year={2005} }
 @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{stepanova_alonso_2005, title={Ethylene signalling and response pathway: A unique signalling cascade with a multitude of inputs and outputs}, volume={123}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-14644388900&partnerID=MN8TOARS}, DOI={10.1111/j.1399-3054.2005.00447.x}, abstractNote={ Plants as immobile organisms need to constantly monitor the changes in the environment to modify and adjust developmental and metabolic pathways accordingly. The responses to these environmental cues require an integrative mechanism where external and internal signals are detected and processed to trigger an appropriate ‘reaction’ in the plant. Hormones play a key role in mediating some of these integrative processes and in generating the response reactions. The identification and characterization of the basic hormone signalling components and their interactions represent the first step towards comprehensive understanding of plant responses to intrinsic and extrinsic cues. A relatively well‐characterized ethylene signalling and response pathway, together with numerous evidences of its interactions with other signalling/response pathways, provide an excellent example to illustrate our current knowledge and perspective on how signal integration occurs in plants. }, number={2}, journal={Physiologia Plantarum}, author={Stepanova, A.N. and Alonso, J.M.}, year={2005}, pages={195–206} }
 @article{li_johnson_stepanova_alonso_ecker_2004, title={Convergence of signaling pathways in the control of differential cell growth 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={<h2>Abstract</h2> 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 <i>Arabidopsis</i> ethylene-regulated <i>HOOKLESS1</i> (<i>HLS1</i>) 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 <i>hls1</i> were identified as mutations in <i>AUXIN RESPONSE FACTOR 2</i> (<i>ARF2</i>). 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, Joseph R.}, year={2004}, pages={193–204} }
 @article{binder_mortimore_stepanova_ecker_bleecker_2004, title={Short-term growth responses to ethylene in arabidopsis seedlings are EIN3/EIL1 independent}, volume={136}, ISSN={["1532-2548"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-14844327480&partnerID=MN8TOARS}, DOI={10.1104/pp.104.050393}, abstractNote={Abstract
               Kinetic studies indicate there are two phases to growth inhibition by ethylene for the hypocotyls of etiolated Arabidopsis seedlings. Phase I is transient, while phase II results in sustained growth inhibition. The EIN2 membrane protein is required for both the first and second phases of growth inhibition by ethylene, while the transcription factors EIN3 and EIL1 are required for the second phase but not the first phase. The first phase lasts no more than 2 h. It is less sensitive to the ethylene response inhibitor 1-methylcyclopropene and more sensitive to ethylene than the second phase. The first phase shows adaptation at low concentrations of ethylene (≤0.01 μL L−1) with a relative refractory period of 5 h after ethylene is added. A modified signal transduction model is proposed that accounts for the two phases of growth inhibition.}, number={2}, journal={PLANT PHYSIOLOGY}, author={Binder, BM and Mortimore, LA and Stepanova, AN and Ecker, JR and Bleecker, AB}, year={2004}, month={Oct}, pages={2921–2927} }
 @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{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}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037418331&partnerID=MN8TOARS}, DOI={10.1073/pnas.0438070100}, abstractNote={
            Five ethylene-insensitive loci (
            wei1
            –
            wei5
            ) were identified by using a low-dose screen for “weak” ethylene-insensitive mutants.
            wei1
            ,
            wei2
            , and
            wei3
            seedlings showed hormone insensitivity only in roots, whereas
            wei4
            and
            wei5
            displayed insensitivity in both roots and hypocotyls. The genes corresponding to
            wei1
            ,
            wei4
            , and
            wei5
            were isolated using a positional cloning approach. The
            wei1
            mutant harbored a recessive mutation in
            TIR1
            , which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response.
            wei4
            , a dominant mutant, resulted from a mutation in the ethylene receptor
            ERS
            , whereas
            wei5
            , a semidominant mutant, was caused by a mutation in the
            EIN3
            -related transcription factor gene
            EIL1
            . The simultaneous loss of functional
            WEI5
            /
            EIL1
            and
            EIN3
            nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen
            Botrytis cinerea
            . Moreover,
            wei5
            /
            eil1 ein3
            double mutants were able to fully suppress constitutive signaling caused by
            ctr1
            , suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants,
            wei2
            and
            wei3
            were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that
            wei2
            and
            wei3
            correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.
          }, number={5}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Alonso, J.M. and Stepanova, A.N. and Solano, R. and Wisman, E. and Ferrari, S. and Ausubel, F.M. and Ecker, J.R.}, year={2003}, pages={2992–2997} }
 @article{alonso_stepanova_leisse_kim_chen_shinn_stevenson_zimmerman_barajas_cheuk_et al._2003, title={Genome-wide insertional mutagenesis of Arabidopsis thaliana}, volume={301}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0042768158&partnerID=MN8TOARS}, DOI={10.1126/science.1086391}, abstractNote={
            Over 225,000 independent
            Agrobacterium
            transferred DNA (T-DNA) insertion events in the genome of the reference plant
            Arabidopsis thaliana
            have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the ∼29,454 predicted
            Arabidopsis
            genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.
          }, number={5633}, journal={Science}, author={Alonso, J.M. and Stepanova, A.N. and Leisse, T.J. and Kim, C.J. and Chen, H. and Shinn, P. and Stevenson, D.K. and Zimmerman, J. and Barajas, P. and Cheuk, R. and et al.}, year={2003}, pages={653–657} }
 @inbook{stepanova_alonso_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}, publisher={Totowa, NJ: Humana Press}, author={Stepanova, A. N. and Alonso, J. M.}, year={2003} }
 @article{alonso_stepanova_2003, title={T-DNA mutagenesis in Arabidopsis.}, volume={236}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-1542548233&partnerID=MN8TOARS}, journal={Methods in molecular biology (Clifton, N.J.)}, author={Alonso, J.M. and Stepanova, A.N.}, year={2003}, pages={177–188} }
 @article{stepanova_ecker_2000, title={Ethylene signaling: From mutants to molecules}, volume={3}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033832308&partnerID=MN8TOARS}, DOI={10.1016/S1369-5266(00)00096-0}, abstractNote={The past decade has been incredibly productive for ethylene researchers. Major components in the ethylene signaling pathway in plants have been identified and characterized. The past year’s contributions include the crystallographic analysis of the Arabidopsis ETR1 receiver domain, antisense studies of the tomato ethylene receptor genes LeETR4 and NR, and the cloning and functional characterization of several Arabidopsis EREBP-related transcription activators and repressors, and of an EIN3-ortholog of tobacco. Additional evidence for the interconnection of the ethylene and auxin responses was provided by the cloning and characterization of Arabidopsis NPH4. Finally, the first discovery of ethylene responsiveness in an animal species implied a more universal role for ethylene than previously thought.}, number={5}, journal={Current Opinion in Plant Biology}, author={Stepanova, A.N. and Ecker, J.R.}, year={2000}, pages={353–360} }
 @article{solano_stepanova_chao_ecker_1998, title={Nuclear events in ethylene signaling: A transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1}, volume={12}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0032417391&partnerID=MN8TOARS}, DOI={10.1101/gad.12.23.3703}, abstractNote={Response to the gaseous plant hormone ethylene inArabidopsis requires the EIN3/EIL family of nuclear proteins. The biochemical function(s) of EIN3/EIL proteins, however, has remained unknown. In this study, we show that EIN3 and EILs comprise a family of novel sequence-specific DNA-binding proteins that regulate gene expression by binding directly to a primary ethylene response element (PERE) related to the tomato E4-element. Moreover, we identified an immediate target of EIN3,ETHYLENE-RESPONSE-FACTOR1 (ERF1), which contains this element in its promoter. EIN3 is necessary and sufficient forERF1 expression, and, like EIN3-overexpression in transgenic plants, constitutive expression of ERF1 results in the activation of a variety of ethylene response genes and phenotypes. Evidence is also provided that ERF1 acts downstream of EIN3 and all other components of the ethylene signaling pathway. The results demonstrate that the nuclear proteins EIN3 and ERF1 act sequentially in a cascade of transcriptional regulation initiated by ethylene gas.}, number={23}, journal={Genes and Development}, author={Solano, R. and Stepanova, A. and Chao, Q. and Ecker, J.R.}, year={1998}, pages={3703–3714} }