@article{broeck_bhosale_song_lima_ashley_zhu_zhu_cotte_neyt_ortiz_et al._2023, title={Functional annotation of proteins for signaling network inference in non-model species}, volume={14}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-023-40365-z}, abstractNote={Molecular biology aims to understand cellular responses and regulatory dynamics in complex biological systems. However, these studies remain challenging in non-model species due to poor functional annotation of regulatory proteins. To overcome this limitation, we develop a multi-layer neural network that determines protein functionality directly from the protein sequence. We annotate kinases and phosphatases in Glycine max. We use the functional annotations from our neural network, Bayesian inference principles, and high resolution phosphoproteomics to infer phosphorylation signaling cascades in soybean exposed to cold, and identify Glyma.10G173000 (TOI5) and Glyma.19G007300 (TOT3) as key temperature regulators. Importantly, the signaling cascade inference does not rely upon known kinase motifs or interaction data, enabling de novo identification of kinase-substrate interactions. Conclusively, our neural network shows generalization and scalability, as such we extend our predictions to Oryza sativa, Zea mays, Sorghum bicolor, and Triticum aestivum. Taken together, we develop a signaling inference approach for non-model species leveraging our predicted kinases and phosphatases.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Broeck, Lisa and Bhosale, Dinesh Kiran and Song, Kuncheng and Lima, Cassio Flavio and Ashley, Michael and Zhu, Tingting and Zhu, Shanshuo and Cotte, Brigitte and Neyt, Pia and Ortiz, Anna C. and et al.}, year={2023}, month={Aug} }
 @article{madison_gillan_peace_gabrieli_broeck_jones_sozzani_2023, title={Phosphate starvation: response mechanisms and solutions}, volume={8}, ISSN={["1460-2431"]}, DOI={10.1093/jxb/erad326}, abstractNote={Phosphorus is essential to plant growth and agricultural crop yields, yet the challenges associated with phosphorus fertilization in agriculture, such as aquatic runoff pollution and poor phosphorus bioavailability, are increasingly difficult to manage. Comprehensively understanding the dynamics of phosphorus uptake and signaling mechanisms will inform the development of strategies to address these issues. This review describes regulatory mechanisms used by specific tissues in the root apical meristem to sense and uptake phosphate from the rhizosphere. The major regulatory mechanisms and related hormone crosstalk underpinning phosphate starvation responses, cellular phosphate homeostasis, and plant adaptations to phosphate starvation are also discussed in this review. In addition, this review overviews the major mechanism of plant systemic phosphate starvation responses. Finally, this review discusses recent promising genetic engineering strategies for improving crop phosphorus use and computational approaches that may help further design strategies for improved plant phosphate acquisition. The mechanisms and approaches presented in this review include a wide variety of species not only including Arabidopsis thaliana but also including crop species such as Oryza sativa (rice), Glycine max (soybean), and Triticum aestivum (wheat) to address both general and species-specific mechanisms and strategies. The aspects of phosphorus deficiency responses and recently employed strategies of improving phosphate acquisition that are detailed in this review may provide insights on the mechanisms or phenotypes that may be targeted in efforts to improve crop phosphorus content and plant growth in low phosphorus soils.}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={Madison, Imani and Gillan, Lydia and Peace, Jasmine and Gabrieli, Flavio and Broeck, Lisa and Jones, Jacob L. and Sozzani, Rosangela}, year={2023}, month={Aug} }
 @article{beretta_franchini_din_lacchini_broeck_sozzani_orozco-arroyo_caporali_adam_jouannic_et al._2023, title={The ALOG family members OsG1L1 and OsG1L2 regulate inflorescence branching in rice}, volume={4}, ISSN={["1365-313X"]}, DOI={10.1111/tpj.16229}, abstractNote={SUMMARY The architecture of the rice inflorescence is an important determinant of crop yield. The length of the inflorescence and the number of branches are among the key factors determining the number of spikelets, and thus grains, that a plant will develop. In particular, the timing of the identity transition from indeterminate branch meristem to determinate spikelet meristem governs the complexity of the inflorescence. In this context, the ALOG gene TAWAWA1 ( TAW1 ) has been shown to delay the transition to determinate spikelet development in Oryza sativa (rice). Recently, by combining precise laser microdissection of inflorescence meristems with RNA‐seq, we observed that two ALOG genes, OsG1‐like 1 ( OsG1L1 ) and OsG1L2 , have expression profiles similar to that of TAW1 . Here, we report that osg1l1 and osg1l2 loss‐of‐function CRISPR mutants have similar phenotypes to the phenotype of the previously published taw1 mutant, suggesting that these genes might act on related pathways during inflorescence development. Transcriptome analysis of the osg1l2 mutant suggested interactions of OsG1L2 with other known inflorescence architecture regulators and the data sets were used for the construction of a gene regulatory network (GRN), proposing interactions among genes potentially involved in controlling inflorescence development in rice. In this GRN, we selected the homeodomain‐leucine zipper transcription factor encoding the gene OsHOX14 for further characterization. The spatiotemporal expression profiling and phenotypical analysis of CRISPR loss‐of‐function mutants of OsHOX14 suggests that the proposed GRN indeed serves as a valuable resource for the identification of new proteins involved in rice inflorescence development.}, journal={PLANT JOURNAL}, author={Beretta, Veronica M. and Franchini, Emanuela and Din, Israr Ud and Lacchini, Elia and Broeck, Lisa and Sozzani, Rosangela and Orozco-Arroyo, Gregorio and Caporali, Elisabetta and Adam, Helene and Jouannic, Stefan and et al.}, year={2023}, month={Apr} }
 @article{ortiz_de smet_sozzani_locke_2022, title={Field-grown soybean shows genotypic variation in physiological and seed composition responses to heat stress during seed development}, volume={195}, ISSN={["1873-7307"]}, DOI={10.1016/j.envexpbot.2021.104768}, abstractNote={An average temperature increase between 2.6 and 4.8 °C, along with more frequent extreme temperatures, will challenge crop productivity by the end of the century. To investigate genotypic variation in soybean response to elevated temperature, six soybean (Glycine max) genotypes were subjected to elevated air temperature of + 4.5 °C above ambient for 28 days in open-top field chambers. Gas exchange and chlorophyll fluorescence were measured before and during heating and yield as well as seed composition were evaluated at maturity. Results show that long-term elevated air temperature increased nighttime respiration, increased the maximum velocity of carboxylation by Rubisco, impacted seed protein concentration, and reduced seed oil concentration across genotypes. The genotypes in this study varied in temperature responses for photosynthetic CO2 assimilation, stomatal conductance, photosystem II operating efficiency, quantum efficiency of CO2 assimilation, and seed protein concentration at maturity. These diverse responses among genotypes to elevated air temperature during seed development in the field, reveal the potential for soybean heat tolerance to be improved through breeding and underlines the importance of identifying efficient selection strategies for stress-tolerant crops.}, journal={ENVIRONMENTAL AND EXPERIMENTAL BOTANY}, author={Ortiz, Anna C. and De Smet, Ive and Sozzani, Rosangela and Locke, Anna M.}, year={2022}, month={Mar} }
 @article{thomas_broeck_spurney_sozzani_frank_2022, title={Gene regulatory networks for compatible versus incompatible grafts identify a role for SlWOX4 during junction formation}, volume={34}, ISSN={["1532-298X"]}, url={https://doi.org/10.1093/plcell/koab246}, DOI={10.1093/plcell/koab246}, abstractNote={Grafting has been adopted for a wide range of crops to enhance productivity and resilience; for example, grafting of Solanaceous crops couples disease-resistant rootstocks with scions that produce high-quality fruit. However, incompatibility severely limits the application of grafting and graft incompatibility remains poorly understood. In grafts, immediate incompatibility results in rapid death, but delayed incompatibility can take months or even years to manifest, creating a significant economic burden for perennial crop production. To gain insight into the genetic mechanisms underlying this phenomenon, we developed a model system using heterografting of tomato (Solanum lycopersicum) and pepper (Capsicum annuum). These grafted plants express signs of anatomical junction failure within the first week of grafting. By generating a detailed timeline for junction formation, we were able to pinpoint the cellular basis for this delayed incompatibility. Furthermore, we inferred gene regulatory networks for compatible self-grafts and incompatible heterografts based on these key anatomical events, which predict core regulators for grafting. Finally, we examined the role of vascular development in graft formation and uncovered SlWOX4 as a potential regulator of graft compatibility. Following this predicted regulator up with functional analysis, we show that Slwox4 homografts fail to form xylem bridges across the junction, demonstrating that indeed, SlWOX4 is essential for vascular reconnection during grafting, and may function as an early indicator of graft failure.}, number={1}, journal={PLANT CELL}, publisher={Oxford University Press (OUP)}, author={Thomas, Hannah and Broeck, Lisa and Spurney, Ryan and Sozzani, Rosangela and Frank, Margaret}, year={2022}, month={Jan}, pages={535–556} }
 @article{muhammad_clark_haque_williams_sozzani_long_2022, title={POPEYE intercellular localization mediates cell-specific iron deficiency responses}, volume={8}, ISSN={["1532-2548"]}, url={https://doi.org/10.1093/plphys/kiac357}, DOI={10.1093/plphys/kiac357}, abstractNote={Plants must tightly regulate iron (Fe) sensing, acquisition, transport, mobilization, and storage to ensure sufficient levels of this essential micronutrient. POPEYE (PYE) is an iron responsive transcription factor that positively regulates the iron deficiency response, while also repressing genes essential for maintaining iron homeostasis. However, little is known about how PYE plays such contradictory roles. Under iron-deficient conditions, pPYE:GFP accumulates in the root pericycle while pPYE:PYE-GFP is localized to the nucleus in all Arabidopsis (Arabidopsis thaliana) root cells, suggesting that PYE may have cell-specific dynamics and functions. Using scanning fluorescence correlation spectroscopy and cell-specific promoters, we found that PYE-GFP moves between different cells and that the tendency for movement corresponds with transcript abundance. While localization to the cortex, endodermis, and vasculature is required to manage changes in iron availability, vasculature and endodermis localization of PYE-GFP protein exacerbated pye-1 defects and elicited a host of transcriptional changes that are detrimental to iron mobilization. Our findings indicate that PYE acts as a positive regulator of iron deficiency response by regulating iron bioavailability differentially across cells, which may trigger iron uptake from the surrounding rhizosphere and impact root energy metabolism.}, journal={PLANT PHYSIOLOGY}, publisher={Oxford University Press (OUP)}, author={Muhammad, DurreShahwar and Clark, Natalie M. and Haque, Samiul and Williams, Cranos M. and Sozzani, Rosangela and Long, Terri A.}, year={2022}, month={Aug} }
 @article{adhikari_aryal_redpath_broeck_ashrafi_philbrick_jacobs_sozzani_louws_2022, title={RNA-Seq and Gene Regulatory Network Analyses Uncover Candidate Genes in the Early Defense to Two Hemibiotrophic Colletorichum spp. in Strawberry}, volume={12}, ISSN={["1664-8021"]}, DOI={10.3389/fgene.2021.805771}, abstractNote={Two hemibiotrophic pathogens, Colletotrichum acutatum (Ca) and C. gloeosporioides (Cg), cause anthracnose fruit rot and anthracnose crown rot in strawberry (Fragaria × ananassa Duchesne), respectively. Both Ca and Cg can initially infect through a brief biotrophic phase, which is associated with the production of intracellular primary hyphae that can infect host cells without causing cell death and establishing hemibiotrophic infection (HBI) or quiescent (latent infections) in leaf tissues. The Ca and Cg HBI in nurseries and subsequent distribution of asymptomatic infected transplants to fruit production fields is the major source of anthracnose epidemics in North Carolina. In the absence of complete resistance, strawberry varieties with good fruit quality showing rate-reducing resistance have frequently been used as a source of resistance to Ca and Cg. However, the molecular mechanisms underlying the rate-reducing resistance or susceptibility to Ca and Cg are still unknown. We performed comparative transcriptome analyses to examine how rate-reducing resistant genotype NCS 10-147 and susceptible genotype 'Chandler' respond to Ca and Cg and identify molecular events between 0 and 48 h after the pathogen-inoculated and mock-inoculated leaf tissues. Although plant response to both Ca and Cg at the same timepoint was not similar, more genes in the resistant interaction were upregulated at 24 hpi with Ca compared with those at 48 hpi. In contrast, a few genes were upregulated in the resistant interaction at 48 hpi with Cg. Resistance response to both Ca and Cg was associated with upregulation of MLP-like protein 44, LRR receptor-like serine/threonine-protein kinase, and auxin signaling pathway, whereas susceptibility was linked to modulation of the phenylpropanoid pathway. Gene regulatory network inference analysis revealed candidate transcription factors (TFs) such as GATA5 and MYB-10, and their downstream targets were upregulated in resistant interactions. Our results provide valuable insights into transcriptional changes during resistant and susceptible interactions, which can further facilitate assessing candidate genes necessary for resistance to two hemibiotrophic Colletotrichum spp. in strawberry.}, journal={FRONTIERS IN GENETICS}, author={Adhikari, Tika B. and Aryal, Rishi and Redpath, Lauren E. and Broeck, Lisa and Ashrafi, Hamid and Philbrick, Ashley N. and Jacobs, Raymond L. and Sozzani, Rosangela and Louws, Frank J.}, year={2022}, month={Mar} }
 @article{roszak_heo_blob_toyokura_sugiyama_balaguer_lau_hamey_cirrone_madej_et al._2021, title={Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization}, volume={374}, ISSN={["1095-9203"]}, DOI={10.1126/science.aba5531}, abstractNote={In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.}, number={6575}, journal={SCIENCE}, author={Roszak, Pawel and Heo, Jung-Ok and Blob, Bernhard and Toyokura, Koichi and Sugiyama, Yuki and Balaguer, Maria Angels de Luis and Lau, Winnie W. Y. and Hamey, Fiona and Cirrone, Jacopo and Madej, Ewelina and et al.}, year={2021}, month={Dec}, pages={1577-+} }
 @misc{schwartz_peters_hunt_abdul-matin_broeck_sozzani_2021, title={Divide and Conquer: The Initiation and Proliferation of Meristems}, volume={40}, ISSN={["1549-7836"]}, DOI={10.1080/07352689.2021.1915228}, abstractNote={In contrast to animals, which complete organogenesis early in their development, plants continuously produce organs, and structures throughout their entire lifecycle. Plants achieve the continuous growth of organs through the initiation and maintenance of meristems that populate the plant body. Plants contain two apical meristems, one at the shoot and one root, to produce the lateral organs of the shoot and the cell files of the root, respectively. Additional meristems within the plant produce branches while others produce the cell types within the vasculature system. Throughout development, plants must balance producing organs and maintaining their meristems, which requires tightly controlled regulations. This review focuses on the various plant meristems, how cells within these meristems maintain their identity, and particularly the molecular players that regulate stem cell maintenance. In addition, we summarize cell types which share molecular features with meristems, but do not follow the same rules regarding maintenance, including pericycle and rachis founder cells. Together, these populations of cells contribute to the entire organogenesis of plants.}, number={2}, journal={CRITICAL REVIEWS IN PLANT SCIENCES}, author={Schwartz, Michael F. and Peters, Rachel and Hunt, Aitch M. and Abdul-Matin, Abdul-Khaliq and Broeck, Lisa and Sozzani, Rosangela}, year={2021}, month={Mar}, pages={147–156} }
 @article{orozco-navarrete_song_casanal_sozzani_flors_sanchez-sevilla_trinkl_hoffmann_merchante_schwab_et al._2021, title={Down-regulation of Fra a 1.02 in strawberry fruits causes transcriptomic and metabolic changes compatible with an altered defense response}, volume={8}, ISSN={["2052-7276"]}, DOI={10.1038/s41438-021-00492-4}, abstractNote={Abstract The strawberry Fra a 1 proteins belong to the class 10 Pathogenesis-Related (PR-10) superfamily. In strawberry, a large number of members have been identified, but only a limited number is expressed in the fruits. In this organ, Fra a 1.01 and Fra a 1.02 are the most abundant Fra proteins in the green and red fruits, respectively, however, their function remains unknown. To know the function of Fra a 1.02 we have generated transgenic lines that silence this gene, and performed metabolomics, RNA-Seq, and hormonal assays. Previous studies associated Fra a 1.02 to strawberry fruit color, but the analysis of anthocyanins in the ripe fruits showed no diminution in their content in the silenced lines. Gene ontology (GO) analysis of the genes differentially expressed indicated that oxidation/reduction was the most represented biological process. Redox state was not apparently altered since no changes were found in ascorbic acid and glutathione (GSH) reduced/oxidized ratio, but GSH content was reduced in the silenced fruits. In addition, a number of glutathione-S-transferases (GST) were down-regulated as result of Fra a 1.02-silencing. Another highly represented GO category was transport which included a number of ABC and MATE transporters. Among the regulatory genes differentially expressed WRKY33.1 and WRKY33.2 were down-regulated, which had previously been assigned a role in strawberry plant defense. A reduced expression of the VQ23 gene and a diminished content of the hormones JA, SA, and IAA were also found. These data might indicate that Fra a 1.02 participates in the defense against pathogens in the ripe strawberry fruits.}, number={1}, journal={HORTICULTURE RESEARCH}, author={Orozco-Navarrete, Begona and Song, Jina and Casanal, Ana and Sozzani, Rosangela and Flors, Victor and Sanchez-Sevilla, Jose F. and Trinkl, Johanna and Hoffmann, Thomas and Merchante, Catharina and Schwab, Wilfried and et al.}, year={2021}, month={Mar} }
 @article{clark_nolan_wang_song_montes_valentine_guo_sozzani_yin_walley_2021, title={Integrated omics networks reveal the temporal signaling events of brassinosteroid response in Arabidopsis}, volume={12}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-021-26165-3}, abstractNote={Abstract Brassinosteroids (BRs) are plant steroid hormones that regulate cell division and stress response. Here we use a systems biology approach to integrate multi-omic datasets and unravel the molecular signaling events of BR response in Arabidopsis . We profile the levels of 26,669 transcripts, 9,533 protein groups, and 26,617 phosphorylation sites from Arabidopsis seedlings treated with brassinolide (BL) for six different lengths of time. We then construct a network inference pipeline called Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) to integrate these data. We use our network predictions to identify putative phosphorylation sites on BES1 and experimentally validate their importance. Additionally, we identify BRONTOSAURUS (BRON) as a transcription factor that regulates cell division, and we show that BRON expression is modulated by BR-responsive kinases and transcription factors. This work demonstrates the power of integrative network analysis applied to multi-omic data and provides fundamental insights into the molecular signaling events occurring during BR response.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Clark, Natalie M. and Nolan, Trevor M. and Wang, Ping and Song, Gaoyuan and Montes, Christian and Valentine, Conner T. and Guo, Hongqing and Sozzani, Rosangela and Yin, Yanhai and Walley, Justin W.}, year={2021}, month={Oct} }
 @article{betegon-putze_mercadal_bosch_planas-riverola_marques-bueno_vilarrasa-blasi_frigola_burkart_martinez_conesa_et al._2021, title={Precise transcriptional control of cellular quiescence by BRAVO/WOX5 complex in Arabidopsis roots}, volume={17}, ISSN={["1744-4292"]}, DOI={10.15252/msb.20209864}, abstractNote={Article16 June 2021Open Access Transparent process Precise transcriptional control of cellular quiescence by BRAVO/WOX5 complex in Arabidopsis roots Isabel Betegón-Putze Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, SpainThese authors contributed equally to this work as first authors Search for more papers by this author Josep Mercadal Departament de Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain Universitat de Barcelona Institute of Complex Systems (UBICS), Barcelona, SpainThese authors contributed equally to this work as first authors Search for more papers by this author Nadja Bosch Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author Ainoa Planas-Riverola orcid.org/0000-0001-8679-7812 Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author Mar Marquès-Bueno Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author Josep Vilarrasa-Blasi Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author David Frigola Departament de Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Rebecca C Burkart Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Search for more papers by this author Cristina Martínez Department of Plant Molecular Genetics, Centro Nacional de Biotecnologı́a (CNB), Madrid, Spain Search for more papers by this author Ana Conesa Microbiology and Cell Science, Institute for Food and Agricultural Research, Genetics Institute, University of Florida, Gainesville, FL, USA Search for more papers by this author Rosangela Sozzani orcid.org/0000-0003-3316-2367 Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA Search for more papers by this author Yvonne Stahl Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Search for more papers by this author Salomé Prat Department of Plant Molecular Genetics, Centro Nacional de Biotecnologı́a (CNB), Madrid, Spain Search for more papers by this author Marta Ibañes Corresponding Author [email protected] orcid.org/0000-0002-7913-7936 Departament de Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain Universitat de Barcelona Institute of Complex Systems (UBICS), Barcelona, SpainThese authors contributed equally to this work as senior authors Search for more papers by this author Ana I Caño-Delgado Corresponding Author [email protected] orcid.org/0000-0002-8071-6724 Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, SpainThese authors contributed equally to this work as senior authors Search for more papers by this author Isabel Betegón-Putze Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, SpainThese authors contributed equally to this work as first authors Search for more papers by this author Josep Mercadal Departament de Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain Universitat de Barcelona Institute of Complex Systems (UBICS), Barcelona, SpainThese authors contributed equally to this work as first authors Search for more papers by this author Nadja Bosch Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author Ainoa Planas-Riverola orcid.org/0000-0001-8679-7812 Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author Mar Marquès-Bueno Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author Josep Vilarrasa-Blasi Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain Search for more papers by this author David Frigola Departament de Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Rebecca C Burkart Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Search for more papers by this author Cristina Martínez Department of Plant Molecular Genetics, Centro Nacional de Biotecnologı́a (CNB), Madrid, Spain Search for more papers by this author Ana Conesa Microbiology and Cell Science, Institute for Food and Agricultural Research, Genetics Institute, University of Florida, Gainesville, FL, USA Search for more papers by this author Rosangela Sozzani orcid.org/0000-0003-3316-2367 Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA Search for more papers by this author Yvonne Stahl Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany Search for more papers by this author Salomé Prat Department of Plant Molecular Genetics, Centro Nacional de Biotecnologı́a (CNB), Madrid, Spain Search for more papers by this author Marta Ibañes Corresponding Author [email protected] orcid.org/0000-0002-7913-7936 Departament de Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain Universitat de Barcelona Institute of Complex Systems (UBICS), Barcelona, SpainThese authors contributed equally to this work as senior authors Search for more papers by this author Ana I Caño-Delgado Corresponding Author [email protected] orcid.org/0000-0002-8071-6724 Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, SpainThese authors contributed equally to this work as senior authors Search for more papers by this author Author Information Isabel Betegón-Putze1, Josep Mercadal2,3, Nadja Bosch1, Ainoa Planas-Riverola1, Mar Marquès-Bueno1, Josep Vilarrasa-Blasi1,†, David Frigola2, Rebecca C Burkart4, Cristina Martínez5, Ana Conesa6, Rosangela Sozzani7, Yvonne Stahl4, Salomé Prat5, Marta Ibañes *,2,3 and Ana I Caño-Delgado *,1 1Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), Barcelona, Spain 2Departament de Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain 3Universitat de Barcelona Institute of Complex Systems (UBICS), Barcelona, Spain 4Institute for Developmental Genetics, Heinrich-Heine University, Düsseldorf, Germany 5Department of Plant Molecular Genetics, Centro Nacional de Biotecnologı́a (CNB), Madrid, Spain 6Microbiology and Cell Science, Institute for Food and Agricultural Research, Genetics Institute, University of Florida, Gainesville, FL, USA 7Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA †Present address: Department of Biology, Stanford University, Stanford, CA, USA * Corresponding author. Tel: +34 935636600 ext 3210; E-mail: [email protected] Corresponding author. E-mail: [email protected] Mol Syst Biol (2021)17:e9864https://doi.org/10.15252/msb.20209864 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Understanding stem cell regulatory circuits is the next challenge in plant biology, as these cells are essential for tissue growth and organ regeneration in response to stress. In the Arabidopsis primary root apex, stem cell-specific transcription factors BRAVO and WOX5 co-localize in the quiescent centre (QC) cells, where they commonly repress cell division so that these cells can act as a reservoir to replenish surrounding stem cells, yet their molecular connection remains unknown. Genetic and biochemical analysis indicates that BRAVO and WOX5 form a transcription factor complex that modulates gene expression in the QC cells to preserve overall root growth and architecture. Furthermore, by using mathematical modelling we establish that BRAVO uses the WOX5/BRAVO complex to promote WOX5 activity in the stem cells. Our results unveil the importance of transcriptional regulatory circuits in plant stem cell development. SYNOPSIS The transcription factors BRAVO and WOX5 are essential regulators of stem cell division in Arabidopsis roots. Genetic analysis and mathematical modeling reveal BRAVO/WOX5 regulatory interactions and indicate mechanisms of stem cell control. The stem cell transcription factors BRAVO and WOX5 jointly promote quiescent center (QC) division in the Arabidopsis root apex. BRAVO and WOX5 mutually regulate their expression and form a protein complex that preserves quiescence at the root stem cell niche. Mathematical modelling uncovers precise regulatory circuits between BRAVO and WOX5 in plant stem cells. Introduction Roots are indispensable organs to preserve plant life and terrestrial ecosystems under normal and adverse environmental conditions. In Arabidopsis thaliana (Arabidopsis), the primary root derives from the activity of the stem cells located at the base of the meristem in the root apex (Dolan et al, 1993; van den Berg et al, 1995). The root stem cell niche (SCN) is composed of a set of proliferative stem cells that surround the mitotically less active cells, named the quiescent centre (QC; Scheres, 2007). Proximally to the QC, the vascular stem cells (VSC, also called vascular initial cells) and the cortical endodermal initials give rise to functional procambial, xylem and phloem conductive vessels and the ground tissue, respectively. Distally and laterally to the QC, the columella stem cells (CSC) and the lateral root cap and epidermal initials give rise to the most outer layer root tissues (Appendix Fig S1; Stahl et al, 2009; Gonzalez-Garcia et al, 2011; De Rybel et al, 2016). The QC prevents differentiation of all these surrounding stem cells (van den Berg et al, 1997), and its low proliferation rate provides a way to preserve the genome from replication errors (Cheung & Rando, 2013). It also acts as a root stem cells reservoir, having the ability of promoting its own division rate to replenish the stem cells when they are damaged (Fulcher & Sablowski, 2009; Lozano-Elena et al, 2018). BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTER (BRAVO) and WUSCHEL RELATED HOMEOBOX 5 (WOX5) are two transcription factors (TFs) that are expressed in the QC and control its quiescence, as mutation of either BRAVO or WOX5 promotes QC cell division (Forzani et al, 2014; Vilarrasa-Blasi et al, 2014; Pi et al, 2015). BRAVO is an R2R3-MYB transcription factor and besides being expressed in the QC, it is also present at the vascular initials (Vilarrasa-Blasi et al, 2014). It was identified as a target of Brassinosteroid (BR) signalling, being directly repressed by BRI1-EMS-SUPPRESSOR 1 (BES1), one of the main effectors of the BR signalling pathway, altogether with its co-repressor TOPLESS (TPL; Vilarrasa-Blasi et al, 2014; Espinosa-Ruiz et al, 2017). WOX5 is a member of the WUSCHEL homeodomain transcription factor family, and it is localized mainly in the QC and to a lesser extent at the surrounding CSC and vascular initials (Sarkar et al, 2007; Pi et al, 2015). WOX5 can repress QC divisions by repressing CYCLIN-D3;3 (Forzani et al, 2014) and, in contrast to BRAVO, is also involved in CSC differentiation, as in the wox5 mutant CSC differentiate prematurely (Sarkar et al, 2007). Although BRAVO and WOX5 are well-studied plant cell-specific repressors of QC division, their molecular connection and the biological relevance in SCN proper functioning has not yet been established. In this study, we set the regulatory and molecular interactions between BRAVO and WOX5 at the SCN and disclose a common role as regulators of primary and lateral root development. Our results show that BRAVO and WOX5 promote each other’s expressions and can directly bind to form a protein regulatory complex with common downstream regulators in the QC cells. BRAVO/WOX5 protein interaction underlies their functions as QC repressors to maintain stem cell development, that is essential for root growth. Results BRAVO and WOX5 control QC division and lateral root density We have previously shown that bravo mutants have increased divisions at the QC compared to the wild type (WT; Vilarrasa-Blasi et al, 2014; Fig 1A and B), which resemble the ones described for wox5 mutant (Sarkar et al, 2007; Bennett et al, 2014; Forzani et al, 2014; Fig 1C). To address whether BRAVO and WOX5 together play a repressing function for QC divisions, we generated the double bravo wox5 mutants (Materials and Methods, Appendix Table S1). The bravo wox5 double mutants also exhibited increased cell division compared to the WT (Fig 1A and D). Importantly, the frequency of divided QC was similar to that of bravo and wox5 single mutants (Fig 1E). The mutual epistatic effect of these mutations suggests that BRAVO and WOX5 function interdependently at the WT primary root apex to suppress QC divisions. Figure 1. BRAVO and WOX5 are required for the QC identity and stem cells maintenance A–D. Confocal images of mPS-PI-stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells, and right white arrows indicate CSC. Scale bar: 50 µm. E. Quantification of the QC divisions in 6-day-old roots expressed in percentage (n > 50, 3 replicates). D: QC divided; ND: QC non-divided. F. Quantification of CSC layers in 6-day-old roots expressed in percentage (n > 50, 3 replicates). G. Lateral root density (number of lateral roots per mm of root length) of 7-day-olf WT, bravo-2, wox5-1 and bravo-2 wox5-1 mutants (n > 40, 2 replicates). Different letters indicate statistically significant differences (P-value < 0.05 Student’s t-test). In the boxplot, box width represents the interquartile range (IQR = Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers. Download figure Download PowerPoint Previous studies proposed that WOX5 represses CSC differentiation in a non-cell autonomous manner (Sarkar et al, 2007; Bennett et al, 2014), whereas no link was reported between this process and BRAVO, since the bravo mutants are not defective in CSC differentiation (Fig 1A, B and F). Genetic analysis showed that bravo wox5 mutants display the same CSC differentiation as wox5 single mutant (Fig 1A, C, D and F), corroborating that BRAVO does not control CSC differentiation (Vilarrasa-Blasi et al, 2014). To address whether these stem cell-specific defects account for overall alterations in root growth and development, root architecture was analysed. The bravo wox5 double mutant shows slightly but significantly shorter roots than the WT (Appendix Fig S2A) and lower lateral root density (Fig 1G). In the case of the lateral root density, 7-day-old bravo wox5 seedlings show the same phenotype as the single mutants (Fig 1G), in agreement with previous reports for wox5 (Tian et al, 2014a). Root growth defects become more exaggerated in the bravo wox5 double mutant than WT and single mutants in 10-day-old seedlings (Appendix Fig S2B), therefore supporting their joint contribution to overall root growth and architecture. BRAVO and WOX5 control each other’s expression at the root stem cell niche We have previously shown that WOX5 expression is reduced in the bravo mutant (Vilarrasa-Blasi et al, 2014), indicating that BRAVO regulates WOX5 expression. To gain insight on the mutual regulatory activity of these two transcription factors, we investigated BRAVO and WOX5 expressions at the SCN in the single mutant and in the double bravo wox5 mutant backgrounds. In the WT primary root, BRAVO expression, reported by the pBRAVO:GFP line, is specifically located in the QC and the vascular initials (Vilarrasa-Blasi et al, 2014; Fig 2A). The pBRAVO signal was increased in the bravo mutant extending shootwards (Fig 2B and H), suggesting that BRAVO negatively regulates its own expression. In contrast, in the wox5 mutant, pBRAVO expression was strongly reduced, especially at the QC, suggesting that WOX5 promotes BRAVO expression (Fig 2C and H). Inducible expression of WOX5 under the 35S promoter (35S:WOX5-GR) resulted in an increased total BRAVO expression, as measured by RT–qPCR of root tips (Appendix Fig S3A). Together, these results support that WOX5 activates BRAVO expression. Moreover, pBRAVO expression was equally reduced in the double bravo wox5 mutant (Appendix Fig S4), as in the wox5 mutant (Fig 2C and H), suggesting that the BRAVO induction by WOX5 is stronger than the BRAVO self-repression. Figure 2. BRAVO and WOX5 reinforce each other at the root stem cell niche A–G. Confocal images of PI-stained 6-day-old roots. GFP-tagged expression is shown in yellow. A-C) pBRAVO:GFP in WT (A), bravo-2 (B) and wox5-1 (C) knockout backgrounds. D-G) pWOX5:GFP in the WT (D), bravo-2 (E), wox5-1 (F) and bravo-2 wox5-1 (G) knockout backgrounds. Scale bar: 50 µm. H, I. Quantification of the GFP fluorescent signal of the roots in A-C (H) and D-G (I). Boxplot indicating the average pixel intensity of the GFP in the stem cell niche (n > 25, 3 biological replicates, *P-value < 0.05 Student’s t-test for each genotype versus the WT in the same condition). Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN (Appendix Fig S7). In the boxplot, box width represents the interquartile range (IQR = Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers, and black dots are the experimental observations. Download figure Download PowerPoint In the primary root, WOX5 expression, as reported by the pWOX5:GFP line, is enriched in the QC, with some expression detected in the vascular initials (Pi et al, 2015; Clark et al, 2020; Fig 2D). We found that bravo mutant displayed a reduction of WOX5 expression (Fig 2E and I; Vilarrasa-Blasi et al, 2014), supporting that BRAVO in turn is able to control the expression of the WOX5 gene. Further analysis of WOX5 expression upon overexpressing BRAVO under an inducible 35S promoter (35S:BRAVO-Ei) showed that when BRAVO levels were induced, pWOX5 levels remained similar to the WT, indicating that BRAVO is not sufficient to induce an increase of WOX5 expression (Appendix Fig S3C–G). Together, these results support that BRAVO is necessary to maintain proper WOX5 levels in the QC. Subsequently, an increased pWOX5:GFP expression towards the provascular cells and columella stem cells was observed in the bravo wox5 double mutant (Fig 2G), similar to wox5 mutant (Fig 2F and I). These findings suggest that WOX5 regulates its own expression and restricts it to the QC, while BRAVO helps to maintain WOX5 expression. Two possible scenarios for BRAVO/WOX5 cross-regulations To provide a comprehensive scheme of BRAVO and WOX5 cross-regulation in the SCN able to account for the changes in expression levels observed in the various mutant backgrounds and overexpression lines, we turned to mathematical modelling. We formulated two models for these regulations, differing by the way BRAVO regulates the activity of WOX5 (Fig 3A and B, Materials and Methods). In both cases, the modelling approach took as variables the total BRAVO and total WOX5 expressions at the root tip (mimicking the total pBRAVO:GFP and pWOX5:GFP expressions integrated across the SCN; Fig 2H and I) and modelled effective cross-regulations between them that can drive all experimental observations on the promoter fold-changes between WT and mutants, and overexpressing lines. These effective regulations are expected to encompass several possible transcriptional and post-transcriptional mechanisms, which may take place across the whole SCN, within or between cells. Figure 3. Two different models for the cross-regulations between BRAVO and WOX5 in the SCN A, B. Schematic representation of the effective regulations in the SCN between BRAVO and WOX5 in the Alleviation (A) and Activation (B) models. (A) In the alleviation model, BRAVO feeds back on its own expression by reducing it and is activated by WOX5. WOX5 also feeds back on its own expression by reducing it, a regulation that becomes partially impaired by BRAVO. (B) In the activation model, BRAVO is also able to activate WOX5 which negatively feedbacks on its own expression. BRAVO is regulated by WOX5 as in the alleviation model. C, D. Parameter space exploration of (C) the alleviation and the (D) activation models. Boxplots showing the distribution of stationary promoter pBRAVO (pB) and pWOX5 (pW) fold-changes in each mutant and overexpression lines over the WT obtained for different parameter values. Superindexes denote the genotype, standing bravo for bravo mutant, wox5 for wox5 mutant, dm for the bravo wox5 double mutant, Woe for WOX5 overexpression and Boe for BRAVO overexpression. Box width represents the interquartile range (IQR = Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. The results are obtained by solving N = 1,000 different runs of each model at the stationary state (red and blue stripplots) for the WT, mutants and overexpressor scenarios. These N runs differ in the parameter values, which are chosen at random from a uniform distribution between P0/2 and 2P0, where P0 is the default set of non-dimensional parameter values (see Appendix Table S1). For each run, the WT, mutants and overexpressor scenarios are computed all with the same parameter values. Circles denote the fold-change obtained in each run, which indicates the fold-change predicted by the model for that parameter set. These are to be compared with the experimental values of the fold-changes of the mean expressions in the mutants and overexpressor lines over the mean WT expression, which are represented by the red and blue squares overlaying the box plots, with error bars denoting ± standard deviations. The experimental values are computed from the same data as in Fig 2H and I, Appendix Figs S3 and S4). The horizontal grey line at promoter fold-change = 1 is indicated to visually separate the region of fold-change < 1 (i.e. the promoter activity is reduced in the mutant or in the overexpressor line) from the region where the fold-change > 1 (i.e. the promoter activity is increased in the mutant or in the overexpressor line). Download figure Download PowerPoint Because BRAVO is induced in the WOX5 overexpression line (Appendix Fig S3A) and pBRAVO:GFP expression decreases in the wox5 mutant (Fig 2C), both models considered that WOX5 expression induces (either directly and/or through intermediate molecules) the expression of BRAVO (Fig 3A and B). To account for the increased total pBRAVO:GFP expression in the bravo background (Fig 2B), the models assumed that BRAVO is able to inhibit its own total expression (Fig 3A and B), a regulation that is probably indirect. When the induction by WOX5 is stronger than the BRAVO self-inhibition, these two regulations can account for a decrease in BRAVO expression in the bravo wox5 double mutant, as found by the GFP expression data (Appendix Fig S4). Therefore, these two regulations on BRAVO are expected to be sufficient to account for all the changes of its expression that we observed in the single and double mutants as well as in the inducible overexpression of WOX5 with respect to WT. Because pWOX5:GFP expression in the SCN increases in the wox5 mutant (Fig 2F), both models consider that WOX5 represses (directly and/or indirectly) its own promoter activity (Fig 3A and B). In contrast, two different regulations of WOX5 by BRAVO were hypothesized to account for both the decreased pWOX5:GFP expression in the bravo mutant (Fig 2E and I) and the absence of change in WOX5 expression upon BRAVO overexpression (Appendix Fig S3C–G). In one model, hereafter named “alleviation model”, BRAVO inhibits partially WOX5 self-repression (Fig 3A). Therefore, this model proposes that BRAVO promotes WOX5 expression by alleviating WOX5 self-inhibition. In the other model, hereafter named “activation model”, BRAVO activates WOX5 (Fig 3B). With these interactions, the two models precisely capture all changes in BRAVO and WOX5 expression in the bravo, wox5 and bravo wox5 mutants as well as in the overexpressing lines (Appendix Fig S8). Parameter values (Appendix Table S1) were chosen such that the fold-changes between expression levels in the single mutants compared to the WT matched the fold-changes in the GFP and transcript levels of our empirical data in mutants and overexpressing lines (Appendix Fig S8, Materials and Methods). Nonetheless, the GFP fold-change values have to be taken as qualitative trends rather than specific quantitative fold-changes in mRNA levels. In addition, the parameter values were restricted such that under control conditions BRAVO expression is lower than WOX5 expression in the WT (Appendix Fig S8A and B), as suggested by pBRAVO:GFP and pWOX5:GFP GFP expression (Materials and Methods) and RNA sequencing (RNA-seq) of the root tip (Clark et al, 2019). Moreover, the two models indicated that the trends in the changes of expression levels between each mutant and the WT (i.e. whether the fold-change is above or below 1) are maintained when the rate of BRAVO promoter activity decreases and/or the rate of WOX5 promoter activity is increased (Appendix Fig S8C–F), mimicking the results obtained upon Brassinolide (BL, the most active BR hormone compound) treatment (Appendix Figs S4 and S5). Exploration of the parameter space around the default parameter set (Appendix Table S1) indicated that both models can reproduce the trends of changes in WOX5 promoter expression in the mutants and in overexpression lines in a large parameter space (Fig 3C and D, Appendix Fig S9). Yet, the alleviation model performs better than the activation model. In larger parameter regions, this latter model can predict fold-changes that are opposite to those found in the experiments, especially in the bravo wox5 mutant (Fig 3C and D, Appendix Fig S9). Taken together, the alleviation and the activation models reflect two, non-exclusive hypotheses for the mutual regulations between BRAVO and WOX5 which can reproduce the changes of their expressions we found in the mutants and overexpressing lines. The alleviation model is more robust than the activation model at recapitulating these trends. BRAVO and WOX5 directly interact in a heterodimeric complex Our results so far support that BRAVO and WOX5 reinforce each other at the SCN. To further decipher how BRAVO and WOX5 interplay, we next evaluated the possible physical interaction between the BRAVO and WOX5 proteins. Using Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM; Fig 4A–K) and yeast two-hybrid assays (Fig 4L, Appendix Fig S6A), we observed that BRAVO can interact with WOX5 (Fig 4B, G, K and L), indicating that BRAVO and WOX5 form a transcriptional complex. Figure 4. BRAVO interacts with WOX5 A–J. Interaction of BRAVO with WOX5 (B), BES1 (C), BES1-D (D) and TPL (E); and interaction of WOX5 with BRAVO (G), BES1 (H), BES1-D (I) and TPL (J) measured by FRET-FLIM. GFP fluorescence lifetime τ [ns] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells. GFP fluorescence lifetime fitted pixel-wise with a mono-exponential model of BRAVO and WOX5 interactions. mV, mVenus; mCh, mCherry. Scale bar: 5 µm. K. Fluorescence-weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double-exponential model of the indicated samples are summarized in box plots. Statistical significance was tested by one-way ANOVA with a Sidakholm post hoc test. Different letters indicate statistically significant differences (P-value < 0.01). For each combination, two to three independent experiments were carried out, and in total, more than 20 biological replicates were measured. In the boxplot, box width represents the interquartile range (IQR = Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers. L. Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column, yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins. Download figure}, number={6}, journal={MOLECULAR SYSTEMS BIOLOGY}, author={Betegon-Putze, Isabel and Mercadal, Josep and Bosch, Nadja and Planas-Riverola, Ainoa and Marques-Bueno, Mar and Vilarrasa-Blasi, Josep and Frigola, David and Burkart, Rebecca C. and Martinez, Cristina and Conesa, Ana and et al.}, year={2021}, month={Jun} }
 @article{spurney_schwartz_gobble_sozzani_broeck_2021, title={Spatiotemporal Gene Expression Profiling and Network Inference: A Roadmap for Analysis, Visualization, and Key Gene Identification}, volume={2328}, ISBN={["978-1-0716-1533-1"]}, ISSN={["1940-6029"]}, DOI={10.1007/978-1-0716-1534-8_4}, abstractNote={Gene expression data analysis and the prediction of causal relationships within gene regulatory networks (GRNs) have guided the identification of key regulatory factors and unraveled the dynamic properties of biological systems. However, drawing accurate and unbiased conclusions requires a comprehensive understanding of relevant tools, computational methods, and their workflows. The topics covered in this chapter encompass the entire workflow for GRN inference including: (1) experimental design; (2) RNA sequencing data processing; (3) differentially expressed gene (DEG) selection; (4) clustering prior to inference; (5) network inference techniques; and (6) network visualization and analysis. Moreover, this chapter aims to present a workflow feasible and accessible for plant biologists without a bioinformatics or computer science background. To address this need, TuxNet, a user-friendly graphical user interface that integrates RNA sequencing data analysis with GRN inference, is chosen for the purpose of providing a detailed tutorial.}, journal={MODELING TRANSCRIPTIONAL REGULATION}, author={Spurney, Ryan and Schwartz, Michael and Gobble, Mariah and Sozzani, Rosangela and Broeck, Lisa}, year={2021}, pages={47–65} }
 @article{crook_willoughby_hazak_okuda_vandermolen_soyars_cattaneo_clark_sozzani_hothorn_et al._2020, title={BAM1/2 receptor kinase signaling drives CLE peptide-mediated formative cell divisions in Arabidopsis roots}, volume={117}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.2018565117}, abstractNote={Significance Proper elaboration of the plant body plan requires that cell division patterns are coordinated during development in complex tissues. Activation of cell cycle machinery is critical for this process, but it is not clear how or if this links to cell-to-cell communication networks that are important during development. Here we show that key cell divisions that generate the plant root are controlled by cell-to-cell signaling peptides which act through plant-specific receptor kinases to control expression of a specific cyclinD cell cycle regulatory gene. We show that cyclinD gene expression depends on both receptor signaling and the SHORT-ROOT transcription factor to ensure timely and robust cell division patterns.}, number={51}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Crook, Ashley D. and Willoughby, Andrew C. and Hazak, Ora and Okuda, Satohiro and VanDerMolen, Kylie R. and Soyars, Cara L. and Cattaneo, Pietro and Clark, Natalie M. and Sozzani, Rosangela and Hothorn, Michael and et al.}, year={2020}, month={Dec}, pages={32750–32756} }
 @inbook{buckner_madison_melvin_long_sozzani_williams_2020, title={BioVision Tracker: A semi-automated image analysis software for spatiotemporal gene expression tracking in Arabidopsis thaliana}, volume={160}, ISBN={9780128215333}, ISSN={0091-679X}, url={http://dx.doi.org/10.1016/bs.mcb.2020.04.017}, DOI={10.1016/bs.mcb.2020.04.017}, abstractNote={Fluorescence microscopy can produce large quantities of data that reveal the spatiotemporal behavior of gene expression at the cellular level in plants. Automated or semi-automated image analysis methods are required to extract data from these images. These data are helpful in revealing spatial and/or temporal-dependent processes that influence development in the meristematic region of plant roots. Tracking spatiotemporal gene expression in the meristem requires the processing of multiple microscopy imaging channels (one channel used to image root geometry which serves as a reference for relating locations within the root, and one or more channels used to image fluorescent gene expression signals). Many automated image analysis methods rely on the staining of cell walls with fluorescent dyes to capture cellular geometry and overall root geometry. However, in long time-course imaging experiments, dyes may fade which hinders spatial assessment in image analysis. Here, we describe a procedure for analyzing 3D microscopy images to track spatiotemporal gene expression signals using the MATLAB-based BioVision Tracker software. This software requires either a fluorescence image or a brightfield image to analyze root geometry and a fluorescence image to capture and track temporal changes in gene expression.}, booktitle={Methods in Cell Biology}, publisher={Elsevier}, author={Buckner, Eli and Madison, Imani and Melvin, Charles and Long, Terri and Sozzani, Rosangela and Williams, Cranos}, year={2020}, pages={419–436} }
 @article{van norman_strader_sozzani_2020, title={Editorial overview: Directionality and precision - how signaling and gene regulation drive plant development and growth}, volume={57}, ISSN={["1879-0356"]}, DOI={10.1016/j.pbi.2020.11.001}, journal={CURRENT OPINION IN PLANT BIOLOGY}, author={Van Norman, Jaimie M. and Strader, Lucia C. and Sozzani, Rosangela}, year={2020}, month={Oct}, pages={A1–A3} }
 @article{van den broeck_gordon_inzé_williams_sozzani_2020, title={Gene Regulatory Network Inference: Connecting Plant Biology and Mathematical Modeling}, volume={11}, ISSN={1664-8021}, url={http://dx.doi.org/10.3389/fgene.2020.00457}, DOI={10.3389/fgene.2020.00457}, abstractNote={Plant responses to environmental and intrinsic signals are tightly controlled by multiple transcription factors (TFs). These TFs and their regulatory connections form gene regulatory networks (GRNs), which provide a blueprint of the transcriptional regulations underlying plant development and environmental responses. This review provides examples of experimental methodologies commonly used to identify regulatory interactions and generate GRNs. Additionally, this review describes network inference techniques that leverage gene expression data to predict regulatory interactions. These computational and experimental methodologies yield complex networks that can identify new regulatory interactions, driving novel hypotheses. Biological properties that contribute to the complexity of GRNs are also described in this review. These include network topology, network size, transient binding of TFs to DNA, and competition between multiple upstream regulators. Finally, this review highlights the potential of machine learning approaches to leverage gene expression data to predict phenotypic outputs.}, journal={Frontiers in Genetics}, publisher={Frontiers Media SA}, author={Van den Broeck, Lisa and Gordon, Max and Inzé, Dirk and Williams, Cranos and Sozzani, Rosangela}, year={2020}, month={May} }
 @inbook{madison_melvin_buckner_williams_sozzani_long_2020, title={MAGIC: Live imaging of cellular division in plant seedlings using lightsheet microscopy}, volume={160}, ISBN={9780128215333}, ISSN={0091-679X}, url={http://dx.doi.org/10.1016/bs.mcb.2020.04.004}, DOI={10.1016/bs.mcb.2020.04.004}, abstractNote={Imaging technologies have been used to understand plant genetic and developmental processes, from the dynamics of gene expression to tissue and organ morphogenesis. Although the field has advanced incredibly in recent years, gaps remain in identifying fine and dynamic spatiotemporal intervals of target processes, such as changes to gene expression in response to abiotic stresses. Lightsheet microscopy is a valuable tool for such studies due to its ability to perform long-term imaging at fine intervals of time and at low photo-toxicity of live vertically oriented seedlings. In this chapter, we describe a detailed method for preparing and imaging Arabidopsis thaliana seedlings for lightsheet microscopy via a Multi-Sample Imaging Growth Chamber (MAGIC), which allows simultaneous imaging of at least four samples. This method opens new avenues for acquiring imaging data at a high temporal resolution, which can be eventually probed to identify key regulatory time points and any spatial dependencies of target developmental processes.}, booktitle={Methods in Cell Biology}, publisher={Elsevier}, author={Madison, Imani and Melvin, Charles and Buckner, Eli and Williams, Cranos and Sozzani, Rosangela and Long, Terri}, year={2020}, pages={405–418} }
 @article{clark_van den broeck_guichard_stager_tanner_blilou_grossmann_iyer-pascuzzi_maizel_sparks_et al._2020, title={Novel Imaging Modalities Shedding Light on Plant Biology: Start Small and Grow Big}, volume={71}, ISSN={1543-5008 1545-2123}, url={http://dx.doi.org/10.1146/annurev-arplant-050718-100038}, DOI={10.1146/annurev-arplant-050718-100038}, abstractNote={The acquisition of quantitative information on plant development across a range of temporal and spatial scales is essential to understand the mechanisms of plant growth. Recent years have shown the emergence of imaging methodologies that enable the capture and analysis of plant growth, from the dynamics of molecules within cells to the measurement of morphometricand physiological traits in field-grown plants. In some instances, these imaging methods can be parallelized across multiple samples to increase throughput. When high throughput is combined with high temporal and spatial resolution, the resulting image-derived data sets could be combined with molecular large-scale data sets to enable unprecedented systems-level computational modeling. Such image-driven functional genomics studies may be expected to appear at an accelerating rate in the near future given the early success of the foundational efforts reviewed here. We present new imaging modalities and review how they have enabled a better understanding of plant growth from the microscopic to the macroscopic scale.}, number={1}, journal={Annual Review of Plant Biology}, publisher={Annual Reviews}, author={Clark, Natalie M. and Van den Broeck, Lisa and Guichard, Marjorie and Stager, Adam and Tanner, Herbert G. and Blilou, Ikram and Grossmann, Guido and Iyer-Pascuzzi, Anjali S. and Maizel, Alexis and Sparks, Erin E. and et al.}, year={2020}, month={Apr}, pages={789–816} }
 @article{clark_fisher_berckmans_van den broeck_nelson_nguyen_bustillo-avendaño_zebell_moreno-risueno_simon_et al._2020, title={Protein complex stoichiometry and expression dynamics of transcription factors modulate stem cell division}, volume={117}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.2002166117}, DOI={10.1073/pnas.2002166117}, abstractNote={Stem cells divide and differentiate to form all of the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the quiescent center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here we gained insight into the differences between the QC and the cortex endodermis initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an ordinary differential equation model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction, coupled with experimental validation, showed that high levels of the SHR-SCR complex are associated with more CEI division but less QC division. Furthermore, our model prediction allowed us to propose the putative upstream SHR regulators SEUSS and WUSCHEL-RELATED HOMEOBOX 5 and to experimentally validate their roles in QC and CEI division. In addition, our model established the timing of QC and CEI division and suggests that SHR repression of QC division depends on formation of the SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.}, number={26}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Clark, Natalie M. and Fisher, Adam P. and Berckmans, Barbara and Van den Broeck, Lisa and Nelson, Emily C. and Nguyen, Thomas T. and Bustillo-Avendaño, Estefano and Zebell, Sophia G. and Moreno-Risueno, Miguel A. and Simon, Rüdiger and et al.}, year={2020}, month={Jun}, pages={15332–15342} }
 @article{spurney_broeck_clark_fisher_balaguer_sozzani_2020, title={tuxnet: a simple interface to process RNA sequencing data and infer gene regulatory networks}, volume={101}, ISSN={["1365-313X"]}, url={https://doi.org/10.1111/tpj.14558}, DOI={10.1111/tpj.14558}, abstractNote={Summary Predicting gene regulatory networks (GRNs) from expression profiles is a common approach for identifying important biological regulators. Despite the increased use of inference methods, existing computational approaches often do not integrate RNA‐sequencing data analysis, are not automated or are restricted to users with bioinformatics backgrounds. To address these limitations, we developed tuxnet , a user‐friendly platform that can process raw RNA‐sequencing data from any organism with an existing reference genome using a modified tuxedo pipeline ( hisat 2 + cufflinks package) and infer GRNs from these processed data. tuxnet is implemented as a graphical user interface and can mine gene regulations, either by applying a dynamic Bayesian network (DBN) inference algorithm, genist , or a regression tree‐based pipeline, rtp‐star . We obtained time‐course expression data of a PERIANTHIA ( PAN ) inducible line and inferred a GRN using genist to illustrate the use of tuxnet while gaining insight into the regulations downstream of the Arabidopsis root stem cell regulator PAN . Using rtp‐star , we inferred the network of ATHB13 , a downstream gene of PAN, for which we obtained wild‐type and mutant expression profiles. Additionally, we generated two networks using temporal data from developmental leaf data and spatial data from root cell‐type data to highlight the use of tuxnet to form new testable hypotheses from previously explored data. Our case studies feature the versatility of tuxnet when using different types of gene expression data to infer networks and its accessibility as a pipeline for non‐bioinformaticians to analyze transcriptome data, predict causal regulations, assess network topology and identify key regulators.}, number={3}, journal={PLANT JOURNAL}, publisher={Wiley}, author={Spurney, Ryan J. and Broeck, Lisa and Clark, Natalie M. and Fisher, Adam P. and Balaguer, Maria A. de Luis and Sozzani, Rosangela}, year={2020}, month={Feb}, pages={716–730} }
 @article{buckner_madison_chou_matthiadis_melvin_sozzani_williams_long_2019, title={Automated Imaging, Tracking, and Analytics Pipeline for Differentiating Environmental Effects on Root Meristematic Cell Division}, volume={10}, ISSN={1664-462X}, url={http://dx.doi.org/10.3389/fpls.2019.01487}, DOI={10.3389/fpls.2019.01487}, abstractNote={Exposure of plants to abiotic stresses, whether individually or in combination, triggers dynamic changes to gene regulation. These responses induce distinct changes in phenotypic characteristics, enabling the plant to adapt to changing environments. For example, iron deficiency and heat stress have been shown to alter root development by reducing primary root growth and reducing cell proliferation, respectively. Currently, identifying the dynamic temporal coordination of genetic responses to combined abiotic stresses remains a bottleneck. This is, in part, due to an inability to isolate specific intervals in developmental time where differential activity in plant stress responses plays a critical role. Here, we observed that iron deficiency, in combination with temporary heat stress, suppresses the expression of iron deficiency-responsive pPYE::LUC (POPEYE::luciferase) and pBTS::LUC (BRUTUS::luciferase) reporter genes. Moreover, root growth was suppressed less under combined iron deficiency and heat stress than under either single stress condition. To further explore the interaction between pathways, we also created a computer vision pipeline to extract, analyze, and compare high-dimensional dynamic spatial and temporal cellular data in response to heat and iron deficiency stress conditions at high temporal resolution. Specifically, we used fluorescence light sheet microscopy to image Arabidopsis thaliana roots expressing CYCB1;1:GFP, a marker for cell entry into mitosis, every 20 min for 24 h exposed to either iron sufficiency, iron deficiency, heat stress, or combined iron deficiency and heat stress. Our pipeline extracted spatiotemporal metrics from these time-course data. These metrics showed that the persistency and timing of CYCB1;1:GFP signal were uniquely different under combined iron deficiency and heat stress conditions versus the single stress conditions. These metrics also indicated that the spatiotemporal characteristics of the CYCB1;1:GFP signal under combined stress were more dissimilar to the control response than the response seen under iron deficiency for the majority of the 24-h experiment. Moreover, the combined stress response was less dissimilar to the control than the response seen under heat stress. This indicated that pathways activated when the plant is exposed to both iron deficiency and heat stress affected CYCB1;1:GFP spatiotemporal function antagonistically.}, journal={Frontiers in Plant Science}, publisher={Frontiers Media SA}, author={Buckner, Eli and Madison, Imani and Chou, Hsuan and Matthiadis, Anna and Melvin, Charles E. and Sozzani, Rosangela and Williams, Cranos and Long, Terri A.}, year={2019}, month={Nov} }
 @article{vallarino_merchante_sánchez‐sevilla_luis balaguer_pott_ariza_casañal_posé_vioque_amaya_et al._2019, title={Characterizing the involvement of
            FaMADS9
            in the regulation of strawberry fruit receptacle development}, volume={18}, ISBN={1467-7652}, ISSN={1467-7644 1467-7652}, url={http://dx.doi.org/10.1111/pbi.13257}, DOI={10.1111/pbi.13257}, abstractNote={Abstract FaMADS9 is the strawberry ( Fragaria x ananassa ) gene that exhibits the highest homology to the tomato ( Solanum lycopersicum ) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid‐1,4‐lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10 , DIV , ZFN1 , ZFN2 , GT2 , and GT5 , or associated with the action of hormones, such as abscisic acid, SHP , ASR , GTE7 and SnRK2.7 . The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1 , DIV , ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9 ‐specific CArG box was identified in the promoters of these genes.}, number={4}, journal={Plant Biotechnology Journal}, publisher={Wiley}, author={Vallarino, José G. and Merchante, Catharina and Sánchez‐Sevilla, José F. and Luis Balaguer, María Angels and Pott, Delphine M. and Ariza, María T. and Casañal, Ana and Posé, David and Vioque, Amalia and Amaya, Iraida and et al.}, year={2019}, month={Oct}, pages={929–943} }
 @article{haque_ahmad_clark_williams_sozzani_2019, title={Computational prediction of gene regulatory networks in plant growth and development}, volume={47}, ISSN={1369-5266}, url={http://www.sciencedirect.com/science/article/pii/S1369526618300839}, DOI={10.1016/j.pbi.2018.10.005}, abstractNote={Plants integrate a wide range of cellular, developmental, and environmental signals to regulate complex patterns of gene expression. Recent advances in genomic technologies enable differential gene expression analysis at a systems level, allowing for improved inference of the network of regulatory interactions between genes. These gene regulatory networks, or GRNs, are used to visualize the causal regulatory relationships between regulators and their downstream target genes. Accordingly, these GRNs can represent spatial, temporal, and/or environmental regulations and can identify functional genes. This review summarizes recent computational approaches applied to different types of gene expression data to infer GRNs in the context of plant growth and development. Three stages of GRN inference are described: first, data collection and analysis based on the dataset type; second, network inference application based on data availability and proposed hypotheses; and third, validation based on in silico, in vivo, and in planta methods. In addition, this review relates data collection strategies to biological questions, organizes inference algorithms based on statistical methods and data types, discusses experimental design considerations, and provides guidelines for GRN inference with an emphasis on the benefits of integrative approaches, especially when a priori information is limited. Finally, this review concludes that computational frameworks integrating large-scale heterogeneous datasets are needed for a more accurate (e.g. fewer false interactions), detailed (e.g. discrimination between direct versus indirect interactions), and comprehensive (e.g. genetic regulation under various conditions and spatial locations) inference of GRNs.}, journal={Current Opinion in Plant Biology}, publisher={Elsevier BV}, author={Haque, Samiul and Ahmad, Jabeen S. and Clark, Natalie M. and Williams, Cranos M. and Sozzani, Rosangela}, year={2019}, month={Feb}, pages={96–105} }
 @article{smet_sevilem_luis balaguer_wybouw_mor_miyashima_blob_roszak_jacobs_boekschoten_et al._2019, title={DOF2.1 Controls Cytokinin-Dependent Vascular Cell Proliferation Downstream of TMO5/LHW}, volume={29}, ISSN={["1879-0445"]}, DOI={10.1016/j.cub.2018.12.041}, abstractNote={To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [1, 2]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [3-7]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 (LOG3) and LOG4 [8, 9] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [3, 6]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.}, number={3}, journal={CURRENT BIOLOGY}, author={Smet, Wouter and Sevilem, Iris and Luis Balaguer, Maria Angels and Wybouw, Brecht and Mor, Eliana and Miyashima, Shunsuke and Blob, Bernhard and Roszak, Pawel and Jacobs, Thomas B. and Boekschoten, Mark and et al.}, year={2019}, month={Feb}, pages={520-+} }
 @article{miyashima_roszak_sevilem_toyokura_blob_heo_mellor_help-rinta-rahko_otero_smet_et al._2019, title={Mobile PEAR transcription factors integrate hormone and miRNA cues to prime cambial growth}, volume={565}, ISSN={0028-0836, 1476-4687}, url={http://www.nature.com/articles/s41586-018-0839-y}, DOI={10.1038/s41586-018-0839-y}, abstractNote={Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors—PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)—and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors4—the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth. Radial growth in the roots of Arabidopsis, which is mediated by gene expression activated by the mobile PEAR1 and PEAR2 transcription factors, is initiated around protophloem-sieve-element cell files of procambial tissue.}, number={7740}, journal={Nature}, author={Miyashima, Shunsuke and Roszak, Pawel and Sevilem, Iris and Toyokura, Koichi and Blob, Bernhard and Heo, Jung-ok and Mellor, Nathan and Help-Rinta-Rahko, Hanna and Otero, Sofia and Smet, Wouter and et al.}, year={2019}, month={Jan}, pages={490–494} }
 @article{powers_holehouse_korasick_schreiber_clark_jing_emenecker_han_tycksen_hwang_et al._2019, title={Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana}, volume={76}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2019.06.044}, abstractNote={The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.}, number={1}, journal={MOLECULAR CELL}, author={Powers, Samantha K. and Holehouse, Alex S. and Korasick, David A. and Schreiber, Katherine H. and Clark, Natalie M. and Jing, Hongwei and Emenecker, Ryan and Han, Soeun and Tycksen, Eric and Hwang, Ildoo and et al.}, year={2019}, month={Oct}, pages={177-+} }
 @article{clark_buckner_fisher_nelson_nguyen_simmons_balaguer_butler-smith_sheldon_bergmann_et al._2019, title={Stem-cell-ubiquitous genes spatiotemporally coordinate division through regulation of stem-cell-specific gene networks}, volume={10}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-019-13132-2}, abstractNote={Abstract Stem cells are responsible for generating all of the differentiated cells, tissues, and organs in a multicellular organism and, thus, play a crucial role in cell renewal, regeneration, and organization. A number of stem cell type-specific genes have a known role in stem cell maintenance, identity, and/or division. Yet, how genes expressed across different stem cell types, referred to here as stem-cell-ubiquitous genes, contribute to stem cell regulation is less understood. Here, we find that, in the Arabidopsis root, a stem-cell-ubiquitous gene, TESMIN-LIKE CXC2 (TCX2), controls stem cell division by regulating stem cell-type specific networks. Development of a mathematical model of TCX2 expression allows us to show that TCX2 orchestrates the coordinated division of different stem cell types. Our results highlight that genes expressed across different stem cell types ensure cross-communication among cells, allowing them to divide and develop harmonically together.}, journal={NATURE COMMUNICATIONS}, author={Clark, Natalie M. and Buckner, Eli and Fisher, Adam P. and Nelson, Emily C. and Nguyen, Thomas T. and Simmons, Abigail R. and Balaguer, Maria A. de Luis and Butler-Smith, Tiara and Sheldon, Parnell J. and Bergmann, Dominique C. and et al.}, year={2019}, month={Dec} }
 @article{di mambro_svolacchia_dello ioio_pierdonati_salvi_pedrazzini_vitale_perilli_sozzani_benfey_et al._2019, title={The Lateral Root Cap Acts as an Auxin Sink that Controls Meristem Size}, volume={29}, ISSN={["1879-0445"]}, DOI={10.1016/j.cub.2019.02.022}, abstractNote={Plant developmental plasticity relies on the activities of meristems, regions where stem cells continuously produce new cells [1]. The lateral root cap (LRC) is the outermost tissue of the root meristem [1], and it is known to play an important role during root development [2-6]. In particular, it has been shown that mechanical or genetic ablation of LRC cells affect meristem size [7, 8]; however, the molecular mechanisms involved are unknown. Root meristem size and, consequently, root growth depend on the position of the transition zone (TZ), a boundary that separates dividing from differentiating cells [9, 10]. The interaction of two phytohormones, cytokinin and auxin, is fundamental in controlling the position of the TZ [9, 10]. Cytokinin via the ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) control auxin distribution within the meristem, generating an instructive auxin minimum that positions the TZ [10]. We identify a cytokinin-dependent molecular mechanism that acts in the LRC to control the position of the TZ and meristem size. We show that auxin levels within the LRC cells depends on PIN-FORMED 5 (PIN5), a cytokinin-activated intracellular transporter that pumps auxin from the cytoplasm into the endoplasmic reticulum, and on irreversible auxin conjugation mediated by the IAA-amino synthase GRETCHEN HAGEN 3.17 (GH3.17). By titrating auxin in the LRC, the PIN5 and the GH3.17 genes control auxin levels in the entire root meristem. Overall, our results indicate that the LRC serves as an auxin sink that, under the control of cytokinin, regulates meristem size and root growth.}, number={7}, journal={CURRENT BIOLOGY}, author={Di Mambro, Riccardo and Svolacchia, Noemi and Dello Ioio, Raffaele and Pierdonati, Emanuela and Salvi, Elena and Pedrazzini, Emanuela and Vitale, Alessandro and Perilli, Serena and Sozzani, Rosangela and Benfey, Philip N. and et al.}, year={2019}, month={Apr}, pages={1199-+} }
 @article{o'lexy_kasai_clark_fujiwara_sozzani_gallagher_2018, title={Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose}, volume={69}, ISSN={["1460-2431"]}, DOI={10.1093/jxb/ery171}, abstractNote={Both plants and animals must contend with changes in their environment. The ability to respond appropriately to these changes often underlies the ability of the individual to survive. In plants, an early response to environmental stress is an alteration in plasmodesmatal permeability with accompanying changes in cell to cell signaling. However, the ways in which plasmodesmata are modified, the molecular players involved in this regulation, and the biological significance of these responses are not well understood. Here, we examine the effects of nutrient scarcity and excess on plasmodesmata-mediated transport in the Arabidopsis thaliana root and identify two CALLOSE SYNTHASES and two β-1,3-GLUCANASES as key regulators of these processes. Our results suggest that modification of plasmodesmata-mediated signaling underlies the ability of the plant to maintain root growth and properly partition nutrients when grown under conditions of excess nutrients.}, number={15}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={O'Lexy, Ruthsabel and Kasai, Koji and Clark, Natalie and Fujiwara, Toru and Sozzani, Rosangela and Gallagher, Kimberly L.}, year={2018}, month={Jul}, pages={3715–3728} }
 @article{shibata_breuer_kawamura_clark_rymen_braidwood_morohashi_busch_benfey_sozzani_et al._2018, title={GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis}, volume={145}, ISSN={["1477-9129"]}, DOI={10.1242/dev.159707}, abstractNote={ABSTRACT How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis. Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors.}, number={3}, journal={DEVELOPMENT}, author={Shibata, Michitaro and Breuer, Christian and Kawamura, Ayako and Clark, Natalie M. and Rymen, Bart and Braidwood, Luke and Morohashi, Kengo and Busch, Wolfgang and Benfey, Philip N. and Sozzani, Rosangela and et al.}, year={2018}, month={Feb} }
 @inbook{clark_fisher_sozzani_2018, place={New York, NY}, series={Methods in Molecular Biology}, title={Identifying Differentially Expressed Genes Using Fluorescence-Activated Cell Sorting (FACS) and RNA Sequencing from Low Input Samples}, volume={1819}, ISBN={978-1-4939-8617-0 978-1-4939-8618-7}, url={http://link.springer.com/10.1007/978-1-4939-8618-7_6}, DOI={10.1007/978-1-4939-8618-7_6}, abstractNote={Cell type-specific gene expression profiles are useful for understanding genes that are important for the development of different tissues and organs. Here, we describe how to perform fluorescence-activated cell sorting (FACS) on Arabidopsis root protoplasts to isolate specific cell types in the root. We then detail how to extract and process RNA from a very low number of cells (≥40 cells) for RNA sequencing (RNA seq). Finally, we describe how to process RNA seq data using TopHat and how to identify differentially expressed genes using PoissonSeq.}, booktitle={Computational Cell Biology}, publisher={Springer New York}, author={Clark, Natalie M. and Fisher, Adam P. and Sozzani, Rosangela}, editor={Stechow, Louise von and Santos Delgado, AlbertoEditors}, year={2018}, pages={139–151}, collection={Methods in Molecular Biology} }
 @inproceedings{buckner_ottley_williams_luis balaguer_melvin_sozzani_2018, place={Honolulu, HI}, title={Tracking Gene Expression via Light Sheet Microscopy and Computer Vision in Living Organisms}, ISBN={978-1-5386-3646-6}, url={https://ieeexplore.ieee.org/document/8512416/}, DOI={10.1109/EMBC.2018.8512416}, abstractNote={Automated tracking of spatiotemporal gene expression using in vivo microscopy images have given great insight into understanding developmental processes in multicellular organisms. Many existing analysis tools rely on the fluorescent tagging of cell wall or cell nuclei localized proteins to assess position, orientation, and overall shape of an organism; information necessary for determining locations of gene expression activity. Particularly in plants, organism lines that have fluorescent tags can take months to develop, which can be time consuming and costly. We propose an automated solution for analyzing spatial characteristics of gene expression without the necessity of fluorescent tagged cell walls or cell nuclei. Our solution indicates, segments, and tracks gene expression using a fluorescent imaging channel of a light sheet microscope while determining gene expression location within an organism from a Brightfield (non-fluorescent) imaging channel. We use the images obtained from the Arabidopsis thaliana root as a proof of concept for our solution by studying the effects of heat shock stress on CYCLIN B1 protein production.}, booktitle={2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC)}, publisher={IEEE}, author={Buckner, Eli and Ottley, Chanae and Williams, Cranos and Luis Balaguer, Angels de and Melvin, Charles E. and Sozzani, Rosangela}, year={2018}, month={Jul}, pages={818–821} }
 @article{di mambro_de ruvo_pacifici_salvi_sozzani_benfey_busch_novak_ljung_di paola_et al._2017, title={Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root}, volume={114}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1705833114}, abstractNote={In multicellular organisms, a stringent control of the transition between cell division and differentiation is crucial for correct tissue and organ development. In the Arabidopsis root, the boundary between dividing and differentiating cells is positioned by the antagonistic interaction of the hormones auxin and cytokinin. Cytokinin affects polar auxin transport, but how this impacts the positional information required to establish this tissue boundary, is still unknown. By combining computational modeling with molecular genetics, we show that boundary formation is dependent on cytokinin's control on auxin polar transport and degradation. The regulation of both processes shapes the auxin profile in a well-defined auxin minimum. This auxin minimum positions the boundary between dividing and differentiating cells, acting as a trigger for this developmental transition, thus controlling meristem size.}, number={36}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Di Mambro, Riccardo and De Ruvo, Micol and Pacifici, Elena and Salvi, Elena and Sozzani, Rosangela and Benfey, Philip N. and Busch, Wolfgang and Novak, Ondrej and Ljung, Karin and Di Paola, Luisa and et al.}, year={2017}, month={Sep}, pages={E7641–E7649} }
 @article{liao_melvin_sozzani_jones_elston_jones_2017, title={Dose-Duration Reciprocity for G protein activation: Modulation of kinase to substrate ratio alters cell signaling}, volume={12}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0190000}, abstractNote={In animal cells, activation of heterotrimeric G protein signaling generally occurs when the system’s cognate signal exceeds a threshold, whereas in plant cells, both the amount and the exposure time of at least one signal, D-glucose, are used toward activation. This unusual signaling property called Dose-Duration Reciprocity, first elucidated in the genetic model Arabidopsis thaliana, is achieved by a complex that is comprised of a 7-transmembrane REGULATOR OF G SIGNALING (RGS) protein (AtRGS1), a Gα subunit that binds and hydrolyzes nucleotide, a Gβγ dimer, and three WITH NO LYSINE (WNK) kinases. D-glucose is one of several signals such as salt and pathogen-derived molecular patterns that operates through this protein complex to activate G protein signaling by WNK kinase transphosphorylation of AtRGS1. Because WNK kinases compete for the same substrate, AtRGS1, we hypothesize that activation is sensitive to the AtRGS1 amount and that modulation of the AtRGS1 pool affects the response to the stimulant. Mathematical simulation revealed that the ratio of AtRGS1 to the kinase affects system sensitivity to D-glucose, and therefore illustrates how modulation of the cellular AtRGS1 level is a means to change signal-induced activation. AtRGS1 levels change under tested conditions that mimic physiological conditions therefore, we propose a previously-unknown mechanism by which plants react to changes in their environment.}, number={12}, journal={PLOS ONE}, author={Liao, Kang-Ling and Melvin, Charles E. and Sozzani, Rosangela and Jones, Roger D. and Elston, Timothy C. and Jones, Alan M.}, year={2017}, month={Dec} }
 @article{wendrich_moller_li_saiga_sozzani_benfey_de rybel_weijers_2017, title={Framework for gradual progression of cell ontogeny in the Arabidopsis root meristem}, volume={114}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1707400114}, abstractNote={In plants, apical meristems allow continuous growth along the body axis. Within the root apical meristem, a group of slowly dividing quiescent center cells is thought to limit stem cell activity to directly neighboring cells, thus endowing them with unique properties, distinct from displaced daughters. This binary identity of the stem cells stands in apparent contradiction to the more gradual changes in cell division potential and differentiation that occur as cells move further away from the quiescent center. To address this paradox and to infer molecular organization of the root meristem, we used a whole-genome approach to determine dominant transcriptional patterns along root ontogeny zones. We found that the prevalent patterns are expressed in two opposing gradients. One is characterized by genes associated with development, the other enriched in differentiation genes. We confirmed these transcript gradients, and demonstrate that these translate to gradients in protein accumulation and gradual changes in cellular properties. We also show that gradients are genetically controlled through multiple pathways. Based on these findings, we propose that cells in the Arabidopsis root meristem gradually transition from stem cell activity toward differentiation.}, number={42}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Wendrich, Jos R. and Moller, Barbara K. and Li, Song and Saiga, Shunsuke and Sozzani, Rosangela and Benfey, Philip N. and De Rybel, Bert and Weijers, Dolf}, year={2017}, month={Oct}, pages={E8922–E8929} }
 @article{coneva_frank_balaguer_li_sozzani_chitwood_2017, title={Genetic Architecture and Molecular Networks Underlying Leaf Thickness in Desert-Adapted Tomato Solanum pennellii}, volume={175}, ISSN={["1532-2548"]}, DOI={10.1104/pp.17.00790}, abstractNote={Thicker leaves allow plants to grow in water-limited conditions. However, our understanding of the genetic underpinnings of this highly functional leaf shape trait is poor. We used a custom-built confocal profilometer to directly measure leaf thickness in a set of introgression lines (ILs) derived from the desert tomato Solanum pennellii and identified quantitative trait loci. We report evidence of a complex genetic architecture of this trait and roles for both genetic and environmental factors. Several ILs with thick leaves have dramatically elongated palisade mesophyll cells and, in some cases, increased leaf ploidy. We characterized the thick IL2-5 and IL4-3 in detail and found increased mesophyll cell size and leaf ploidy levels, suggesting that endoreduplication underpins leaf thickness in tomato. Next, we queried the transcriptomes and inferred dynamic Bayesian networks of gene expression across early leaf ontogeny in these lines to compare the molecular networks that pattern leaf thickness. We show that thick ILs share S. pennellii-like expression profiles for putative regulators of cell shape and meristem determinacy as well as a general signature of cell cycle-related gene expression. However, our network data suggest that leaf thickness in these two lines is patterned at least partially by distinct mechanisms. Consistent with this hypothesis, double homozygote lines combining introgression segments from these two ILs show additive phenotypes, including thick leaves, higher ploidy levels, and larger palisade mesophyll cells. Collectively, these data establish a framework of genetic, anatomical, and molecular mechanisms that pattern leaf thickness in desert-adapted tomato.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Coneva, Viktoriya and Frank, Margaret H. and Balaguer, Maria A. de Luis and Li, Mao and Sozzani, Rosangela and Chitwood, Daniel H.}, year={2017}, month={Sep}, pages={376–391} }
 @inbook{de luis balaguer_sozzani_2017, place={New York, NY}, series={Methods in Molecular Biology}, title={Inferring Gene Regulatory Networks in the Arabidopsis Root Using a Dynamic Bayesian Network Approach}, volume={1629}, ISBN={978-1-4939-7124-4 978-1-4939-7125-1}, url={http://link.springer.com/10.1007/978-1-4939-7125-1_21}, DOI={10.1007/978-1-4939-7125-1_21}, abstractNote={Gene regulatory network (GRN) models have been shown to predict and represent interactions among sets of genes. Here, we first show the basic steps to implement a simple but computationally efficient algorithm to infer GRNs based on dynamic Bayesian networks (DBNs), and we then explain how to approximate DBN-based GRN models with continuous models. In addition, we show a MATLAB implementation of the key steps of this method, which we use to infer an Arabidopsis root GRN.}, booktitle={Plant Gene Regulatory Networks}, publisher={Springer New York}, author={Luis Balaguer, Maria Angels de and Sozzani, Rosangela}, editor={Kaufmann, Kerstin and Mueller-Roeber, BerndEditors}, year={2017}, pages={331–348}, collection={Methods in Molecular Biology} }
 @inbook{clark_sozzani_2017, place={New York, NY}, series={Methods in Molecular Biology}, title={Measuring Protein Movement, Oligomerization State, and Protein–Protein Interaction in Arabidopsis Roots Using Scanning Fluorescence Correlation Spectroscopy (Scanning FCS)}, volume={1610}, ISBN={978-1-4939-7001-8 978-1-4939-7003-2}, url={http://link.springer.com/10.1007/978-1-4939-7003-2_16}, DOI={10.1007/978-1-4939-7003-2_16}, abstractNote={Scanning fluorescence correlation spectroscopy (scanning FCS) can be used to determine protein movement, oligomerization state, and protein–protein interaction. Here, we describe how to use the scanning FCS techniques of raster image correlation spectroscopy (RICS) and pair correlation function (pCF) to determine the rate and direction of protein movement. In addition, we detail how number and brightness (N&B) and cross-correlation analyses can be used to determine oligomerization state and binding ratios of protein complexes. We specifically describe how to acquire suitable images for scanning FCS analysis using the model plant Arabidopsis and how to perform the various analyses using the SimFCS software.}, booktitle={Plant Genomics}, publisher={Springer New York}, author={Clark, Natalie M. and Sozzani, Rosangela}, editor={Busch, WolfgangEditor}, year={2017}, pages={251–266}, collection={Methods in Molecular Biology} }
 @article{balaguer_fisher_clark_fernandez-espinosa_moller_weijers_lohmann_williams_lorenzo_sozzani_et al._2017, title={Predicting gene regulatory networks by combining spatial and temporal gene expression data in Arabidopsis root stem cells}, volume={114}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1707566114}, abstractNote={Significance We developed a computational pipeline that uses gene expression datasets for inferring relationships among genes and predicting their importance. We showed that the capacity of our pipeline to integrate spatial and temporal transcriptional datasets improves the performance of inference algorithms. The combination of this pipeline with Arabidopsis stem cell-specific data resulted in networks that capture the regulations of stem cell-enriched genes in the stem cells and throughout root development. Our combined approach of molecular biology, computational biology, and mathematical biology, led to successful findings of factors that could play important roles in stem cell regulation and, in particular, quiescent center function.}, number={36}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Balaguer, M. A. D. and Fisher, A. P. and Clark, N. M. and Fernandez-Espinosa, M. G. and Moller, B. K. and Weijers, D. and Lohmann, J. U. and Williams, C. and Lorenzo, O. and Sozzani, Rosangela and et al.}, year={2017}, month={Sep}, pages={E7632–E7640} }
 @article{fisher_sozzani_2016, title={Gene and networks regulating the root stem cell niche of Arabidopsis}, volume={29}, ISSN={["1879-0356"]}, DOI={10.1016/j.pbi.2015.11.002}, abstractNote={Stem cells are the source of different cell types and tissues in all multicellular organisms. In plants, the balance between stem cell self-renewal and differentiation of their progeny is crucial for correct tissue and organ formation. How transcriptional programs precisely control stem cell maintenance and identity, and what are the regulatory programs influencing stem cell asymmetric cell division (ACD), are key questions that researchers have sought to address for the past decade. Successful efforts in genetic, molecular, and developmental biology, along with mathematical modeling, have identified some of the players involved in stem cell regulation. In this review, we will discuss several studies that characterized many of the genetic programs and molecular mechanisms regulating stem cell ACD and their identity in the Arabidopsis root. We will also highlight how the growing use of mathematical modeling provides a comprehensive and quantitative perspective on the design rules governing stem cell ACDs.}, journal={Curr Opin Plant Biol}, author={Fisher, A.P. and Sozzani, R.}, year={2016}, month={Feb}, pages={38–43} }
 @article{slattery_grennan_sivaguru_sozzani_ort_2016, title={Light sheet microscopy reveals more gradual light attenuation in light-green versus dark-green soybean leaves}, volume={67}, ISSN={["1460-2431"]}, DOI={10.1093/jxb/erw246}, abstractNote={Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This over-saturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light. Reducing chl content could create a more even leaf light distribution and thereby increase leaf light-use efficiency and overall canopy photosynthesis. This was tested on soybean cultivar 'Clark' (WT) and a near-isogenic chl b deficient mutant, Y11y11, grown in controlled environment chambers and in the field. Light attenuation was quantified using a novel approach involving light sheet microscopy. Leaf adaxial and abaxial surfaces were illuminated separately with blue, red, and green wavelengths, and chl fluorescence was detected orthogonally to the illumination plane. Relative fluorescence was significantly greater in deeper layers of the Y11y11 mesophyll than in WT, with the greatest differences in blue, then red, and finally green light when illuminated from the adaxial surface. Modeled relative photosynthesis based on chlorophyll profiles and Beer's Law predicted less steep gradients in mutant relative photosynthesis rates compared to WT. Although photosynthetic light-use efficiency was greater in the field-grown mutant with ~50% lower chl, light-use efficiency was lower in the mutant when grown in chambers where chl was ~80% reduced. This difference is probably due to pleiotropic effects of the mutation that accompany very severe reductions in chlorophyll and may warrant further testing in other low-chl lines.}, number={15}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={Slattery, Rebecca A. and Grennan, Aleel K. and Sivaguru, Mayandi and Sozzani, Rosangela and Ort, Donald R.}, year={2016}, month={Aug}, pages={4697–4709} }
 @article{de luis balaguer_ramos-pezzotti_rahhal_melvin_johannes_horn_sozzani_2016, title={Multi-sample Arabidopsis Growth and Imaging Chamber (MAGIC) for long term imaging in the ZEISS Lightsheet Z.1}, volume={419}, ISSN={1095-564X}, DOI={10.1016/j.ydbio.2016.05.029}, abstractNote={Time-course imaging experiments on live organisms are critical for understanding the dynamics of growth and development. Light-sheet microscopy has advanced the field of long-term imaging of live specimens by significantly reducing photo-toxicity and allowing fast acquisition of three-dimensional data over time. However, current light-sheet technology does not allow the imaging of multiple plant specimens in parallel. To achieve higher throughput, we have developed a Multi-sample Arabidopsis Growth and Imaging Chamber (MAGIC) that provides near-physiological imaging conditions and allows high-throughput time-course imaging experiments in the ZEISS Lightsheet Z.1. Here, we illustrate MAGIC's imaging capabilities by following cell divisions, as an indicator of plant growth and development, over prolonged time periods. To automatically quantify the number of cell divisions in long-term experiments, we present a FIJI-based image processing pipeline. We demonstrate that plants imaged with our chamber undergo cell divisions for >16 times longer than those with the glass capillary system supplied by the ZEISS Z1.}, number={1}, journal={Developmental Biology}, author={Luis Balaguer, Maria Angels de and Ramos-Pezzotti, Marina and Rahhal, Morjan B. and Melvin, Charles E. and Johannes, Eva and Horn, Timothy J. and Sozzani, Rosangela}, year={2016}, month={Jan}, pages={19–25} }
 @article{clark_hinde_winter_fisher_crosti_blilou_gratton_benfey_sozzani_2016, title={Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy}, volume={5}, journal={Elife}, author={Clark, N. M. and Hinde, E. and Winter, C. M. and Fisher, A. P. and Crosti, G. and Blilou, I. and Gratton, E. and Benfey, P. N. and Sozzani, R.}, year={2016} }
 @article{moreno-risueno_sozzani_yardimici_petricka_vernoux_blilou_alonso_winter_ohler_scheres_et al._2015, title={Bird and Scarecrow proteins organize tissue formation in Arabidopsis roots}, volume={350}, ISSN={["1095-9203"]}, DOI={10.1126/science.aad1171}, abstractNote={Tissue patterns are dynamically maintained. Continuous formation of plant tissues during postembryonic growth requires asymmetric divisions and the specification of cell lineages. We show that the BIRDs and SCARECROW regulate lineage identity, positional signals, patterning, and formative divisions throughout Arabidopsis root growth. These transcription factors are postembryonic determinants of the ground tissue stem cells and their lineage. Upon further activation by the positional signal SHORT-ROOT (a mobile transcription factor), they direct asymmetric cell divisions and patterning of cell types. The BIRDs and SCARECROW with SHORT-ROOT organize tissue patterns at all formative steps during growth, ensuring developmental plasticity.}, number={6259}, journal={Science}, author={Moreno-Risueno, MA. and Sozzani, R. and Yardimici, GG. and Petricka, JJ. and Vernoux, T. and Blilou, I. and Alonso, J. and Winter, CM. and Ohler, U. and Scheres, B. and et al.}, year={2015}, pages={426–430} }
 @misc{sozzani_busch_spalding_benfey_2014, title={Advanced imaging techniques for the study of plant growth and development}, volume={19}, ISSN={["1878-4372"]}, DOI={10.1016/j.tplants.2013.12.003}, abstractNote={A variety of imaging methodologies are being used to collect data for quantitative studies of plant growth and development from living plants. Multi-level data, from macroscopic to molecular, and from weeks to seconds, can be acquired. Furthermore, advances in parallelized and automated image acquisition enable the throughput to capture images from large populations of plants under specific growth conditions. Image-processing capabilities allow for 3D or 4D reconstruction of image data and automated quantification of biological features. These advances facilitate the integration of imaging data with genome-wide molecular data to enable systems-level modeling.}, number={5}, journal={TRENDS IN PLANT SCIENCE}, author={Sozzani, Rosangela and Busch, Wolfgang and Spalding, Edgar P. and Benfey, Philip N.}, year={2014}, month={May}, pages={304–310} }
 @misc{clark_balaguer_sozzani_2014, title={Experimental data and computational modeling link auxin gradient and development in the Arabidopsis root}, volume={5}, journal={Frontiers in Plant Science}, author={Clark, N. M. and Balaguer, M. A. D. and Sozzani, R.}, year={2014} }
 @article{gallagher_sozzani_lee_2014, title={Intercellular Protein Movement: Deciphering the Language of Development}, volume={30}, ISSN={["1530-8995"]}, DOI={10.1146/annurev-cellbio-100913-012915}, abstractNote={Development in multicellular organisms requires the coordinated production of a large number of specialized cell types through sophisticated signaling mechanisms. Non-cell-autonomous signals are one of the key mechanisms by which organisms coordinate development. In plants, intercellular movement of transcription factors and other mobile signals, such as hormones and peptides, is essential for normal development. Through a combination of different approaches, a large number of non-cell-autonomous signals that control plant development have been identified. We review some of the transcriptional regulators that traffic between cells, as well as how changes in symplasmic continuity affect and are affected by development. We also review current models for how mobile signals move via plasmodesmata and how movement is inhibited. Finally, we consider challenges in and new tools for studying protein movement.}, journal={ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 30}, author={Gallagher, Kimberly L. and Sozzani, Rosangela and Lee, Chin-Mei}, year={2014}, pages={207–233} }
 @misc{kajala_ramakrishna_fisher_bergmann_de smet_sozzani_weijers_brady_2014, title={Omics and modelling approaches for understanding regulation of asymmetric cell divisions in arabidopsis and other angiosperm plants}, volume={113}, ISSN={["1095-8290"]}, DOI={10.1093/aob/mcu065}, abstractNote={Asymmetric cell divisions are formative divisions that generate daughter cells of distinct identity. These divisions are coordinated by either extrinsic (‘niche-controlled’) or intrinsic regulatory mechanisms and are fundamentally important in plant development. This review describes how asymmetric cell divisions are regulated during development and in different cell types in both the root and the shoot of plants. It further highlights ways in which omics and modelling approaches have been used to elucidate these regulatory mechanisms. For example, the regulation of embryonic asymmetric divisions is described, including the first divisions of the zygote, formative vascular divisions and divisions that give rise to the root stem cell niche. Asymmetric divisions of the root cortex endodermis initial, pericycle cells that give rise to the lateral root primordium, procambium, cambium and stomatal cells are also discussed. Finally, a perspective is provided regarding the role of other hormones or regulatory molecules in asymmetric divisions, the presence of segregated determinants and the usefulness of modelling approaches in understanding network dynamics within these very special cells. Asymmetric cell divisions define plant development. High-throughput genomic and modelling approaches can elucidate their regulation, which in turn could enable the engineering of plant traits such as stomatal density, lateral root development and wood formation.}, number={7}, journal={ANNALS OF BOTANY}, author={Kajala, Kaisa and Ramakrishna, Priya and Fisher, Adam and Bergmann, Dominique C. and De Smet, Ive and Sozzani, Rosangela and Weijers, Dolf and Brady, Siobhan M.}, year={2014}, month={Jun}, pages={1083–1105} }
 @misc{sozzani_iyer-pascuzzi_2014, title={Postembryonic control of root meristem growth and development}, volume={17}, ISSN={["1879-0356"]}, DOI={10.1016/j.pbi.2013.10.005}, abstractNote={Organ development in multicellular organisms is dependent on the proper balance between cell proliferation and differentiation. In the Arabidopsis root apical meristem, meristem growth is the result of cell divisions in the proximal meristem and cell differentiation in the elongation and differentiation zones. Hormones, transcription factors and small peptides underpin the molecular mechanisms governing these processes. Computer modeling has aided our understanding of the dynamic interactions involved in stem cell maintenance and meristem activity. Here we review recent advances in our understanding of postembryonic root stem cell maintenance and control of meristem size.}, journal={CURRENT OPINION IN PLANT BIOLOGY}, author={Sozzani, Rosangela and Iyer-Pascuzzi, Anjali}, year={2014}, month={Feb}, pages={7–12} }
 @article{moubayidin_di mambro_sozzani_pacifici_salvi_terpstra_bao_van dijken_dello ioio_perilli_et al._2013, title={Spatial Coordination between Stem Cell Activity and Cell Differentiation in the Root Meristem}, volume={26}, ISSN={15345807}, url={https://linkinghub.elsevier.com/retrieve/pii/S1534580713003882}, DOI={10.1016/j.devcel.2013.06.025}, abstractNote={A critical issue in development is the coordination of the activity of stem cell niches with differentiation of their progeny to ensure coherent organ growth. In the plant root, these processes take place at opposite ends of the meristem and must be coordinated with each other at a distance. Here, we show that in Arabidopsis, the gene SCR presides over this spatial coordination. In the organizing center of the root stem cell niche, SCR directly represses the expression of the cytokinin-response transcription factor ARR1, which promotes cell differentiation, controlling auxin production via the ASB1 gene and sustaining stem cell activity. This allows SCR to regulate, via auxin, the level of ARR1 expression in the transition zone where the stem cell progeny leaves the meristem, thus controlling the rate of differentiation. In this way, SCR simultaneously controls stem cell division and differentiation, ensuring coherent root growth.}, number={4}, journal={Developmental Cell}, author={Moubayidin, Laila and Di Mambro, Riccardo and Sozzani, Rosangela and Pacifici, Elena and Salvi, Elena and Terpstra, Inez and Bao, Dongping and van Dijken, Anja and Dello Ioio, Raffaele and Perilli, Serena and et al.}, year={2013}, month={Aug}, pages={405–415} }
 @article{cruz-ramírez_díaz-triviño_blilou_grieneisen_sozzani_zamioudis_miskolczi_nieuwland_benjamins_dhonukshe_et al._2012, title={A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetric stem cell division}, volume={150}, ISSN={1097-4172}, DOI={10.1016/j.cell.2012.07.017}, abstractNote={In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.}, number={5}, journal={Cell}, author={Cruz-Ramírez, Alfredo and Díaz-Triviño, Sara and Blilou, Ikram and Grieneisen, Verônica A. and Sozzani, Rosangela and Zamioudis, Christos and Miskolczi, Pál and Nieuwland, Jeroen and Benjamins, René and Dhonukshe, Pankaj and et al.}, year={2012}, month={Aug}, pages={1002–1015} }
 @article{liberman_sozzani_benfey_2012, title={Integrative systems biology: an attempt to describe a simple weed}, volume={15}, ISSN={1879-0356}, DOI={10.1016/j.pbi.2012.01.004}, abstractNote={Genome-scale studies hold great promise for revealing novel plant biology. Because of the complexity of these techniques, numerous considerations need to be made before embarking on a study. Here we focus on the Arabidopsis model system because of the wealth of available genome-scale data. Many approaches are available that provide genome-scale information regarding the state of a given organism (e.g. genomics, epigenomics, transcriptomics, proteomics, metabolomics interactomics, ionomics, phenomics, etc.). Integration of all of these types of data will be necessary for a comprehensive description of Arabidopsis. In this review we propose that ‘triangulation’ among transcriptomics, proteomics and metabolomics is a meaningful approach for beginning this integrative analysis and uncovering a systems level perspective of Arabidopsis biology.}, number={2}, journal={Current Opinion in Plant Biology}, author={Liberman, Louisa M. and Sozzani, Rosangela and Benfey, Philip N.}, year={2012}, month={Apr}, pages={162–167} }
 @article{engstrom_andersen_gumulak-smith_hu_orlova_sozzani_bowman_2011, title={Arabidopsis homologs of the petunia hairy meristem gene are required for maintenance of shoot and root indeterminacy}, volume={155}, ISSN={1532-2548}, DOI={10.1104/pp.110.168757}, abstractNote={Abstract Maintenance of indeterminacy is fundamental to the generation of plant architecture and a central component of the plant life strategy. Indeterminacy in plants is a characteristic of shoot and root meristems, which must balance maintenance of indeterminacy with organogenesis. The Petunia hybrida HAIRY MERISTEM (HAM) gene, a member of the GRAS family of transcriptional regulators, promotes shoot indeterminacy by an undefined non-cell-autonomous signaling mechanism(s). Here, we report that Arabidopsis (Arabidopsis thaliana) mutants triply homozygous for knockout alleles in three Arabidopsis HAM orthologs (Atham1,2,3 mutants) exhibit loss of indeterminacy in both the shoot and root. In the shoot, the degree of penetrance of the loss-of-indeterminacy phenotype of Atham1,2,3 mutants varies among shoot systems, with arrest of the primary vegetative shoot meristem occurring rarely or never, secondary shoot meristems typically arresting prior to initiating organogenesis, and inflorescence and flower meristems exhibiting a phenotypic range extending from wild type (flowers) to meristem arrest preempting organogenesis (flowers and inflorescence). Atham1,2,3 mutants also exhibit aberrant shoot phyllotaxis, lateral organ abnormalities, and altered meristem morphology in functioning meristems of both rosette and inflorescence. Root meristems of Atham1,2,3 mutants are significantly smaller than in the wild type in both longitudinal and radial axes, a consequence of reduced rates of meristem cell division that culminate in root meristem arrest. Atham1,2,3 phenotypes are unlikely to reflect complete loss of HAM function, as a fourth, more distantly related Arabidopsis HAM homolog, AtHAM4, exhibits overlapping function with AtHAM1 and AtHAM2 in promoting shoot indeterminacy.}, number={2}, journal={Plant Physiology}, author={Engstrom, Eric M. and Andersen, Carl M. and Gumulak-Smith, Juliann and Hu, John and Orlova, Evguenia and Sozzani, Rosangela and Bowman, John L.}, year={2011}, month={Feb}, pages={735–750} }
 @article{sozzani_benfey_2011, title={High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype}, volume={12}, ISSN={1474-760X}, url={https://doi.org/10.1186/gb-2011-12-3-219}, DOI={10.1186/gb-2011-12-3-219}, abstractNote={High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms.}, number={3}, journal={Genome Biology}, author={Sozzani, Rosangela and Benfey, Philip N.}, year={2011}, month={Mar}, pages={219} }
 @article{sozzani_cui_moreno-risueno_busch_van norman_vernoux_brady_dewitte_murray_benfey_2010, title={Spatiotemporal regulation of cell-cycle genes by SHORTROOT links patterning and growth}, volume={466}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/nature09143}, DOI={10.1038/nature09143}, abstractNote={In higher animals and plants the processes of growth and patterning are coordinated. Much is known about the genes regulating patterning and many genes have been identified that are involved in cell-cycle control, but there are few instances in which a connection has been made between the two. A study of patterning in Arabidopsis root now shows that two key regulators of root-organ patterning directly control the transcription of specific components of the cell-cycle machinery. This demonstrates a direct link between developmental regulators, components of the cell-cycle machinery and organ patterning. In higher animals and plants, the processes of growth and patterning are coordinated. In this study, the authors study patterning in Arabidopsis root and show that two key regulators of root organ patterning directly control the transcription of specific components of the cell-cycle machinery. This study provides a direct link between developmental regulators, components of the cell-cycle machinery and organ patterning. The development of multicellular organisms relies on the coordinated control of cell divisions leading to proper patterning and growth1,2,3. The molecular mechanisms underlying pattern formation, particularly the regulation of formative cell divisions, remain poorly understood. In Arabidopsis, formative divisions generating the root ground tissue are controlled by SHORTROOT (SHR) and SCARECROW (SCR)4,5,6. Here we show, using cell-type-specific transcriptional effects of SHR and SCR combined with data from chromatin immunoprecipitation-based microarray experiments, that SHR regulates the spatiotemporal activation of specific genes involved in cell division. Coincident with the onset of a specific formative division, SHR and SCR directly activate a D-type cyclin; furthermore, altering the expression of this cyclin resulted in formative division defects. Our results indicate that proper pattern formation is achieved through transcriptional regulation of specific cell-cycle genes in a cell-type- and developmental-stage-specific context. Taken together, we provide evidence for a direct link between developmental regulators, specific components of the cell-cycle machinery and organ patterning.}, number={7302}, journal={Nature}, publisher={Springer Science and Business Media LLC}, author={Sozzani, R. and Cui, H. and Moreno-Risueno, M. A. and Busch, W. and Van Norman, J. M. and Vernoux, T. and Brady, S. M. and Dewitte, W. and Murray, J. A. H. and Benfey, P. N.}, year={2010}, month={Jul}, pages={128–132} }
 @article{sozzani_maggio_giordo_umana_ascencio-ibañez_hanley-bowdoin_bergounioux_cella_albani_2010, title={The E2FD/DEL2 factor is a component of a regulatory network controlling cell proliferation and development in Arabidopsis}, volume={72}, ISSN={1573-5028}, DOI={10.1007/s11103-009-9577-8}, number={4-5}, journal={Plant Molecular Biology}, author={Sozzani, Rosangela and Maggio, Caterina and Giordo, Roberta and Umana, Elisabetta and Ascencio-Ibañez, Jose Trinidad and Hanley-Bowdoin, Linda and Bergounioux, Catherine and Cella, Rino and Albani, Diego}, year={2010}, month={Mar}, pages={381–395} }
 @article{sozzani_cui_moreno-risueno_busch_van norman_vernoux_brady_dewitte_murray_benfey_2010, title={The SHR/SCR pathway directly activates genes involved in asymmetric cell division in the Arabidopsis root}, volume={466}, number={7302}, journal={Nature}, author={Sozzani, R. and Cui, H. and Moreno-Risueno, M.A. and Busch, W. and Van Norman, J.M. and Vernoux, T. and Brady, S.M. and Dewitte, W. and Murray, J.A. and Benfey, P.N.}, year={2010}, pages={128–132} }
 @article{benfey_cui_twigg_long_iyer-pascuzzi_tsukagoshi_sozzani_jackson_van norman_moreno-risueno_2009, title={Development rooted in interwoven networks}, volume={331}, ISSN={0012-1606}, url={http://dx.doi.org/10.1016/j.ydbio.2009.05.012}, DOI={10.1016/j.ydbio.2009.05.012}, abstractNote={Freshwater planarians appear to utilize inductive signals to specify their germ cell lineage: germ cells are believed to form post-embryonically from the pluripotent somatic stem cells, known as neoblasts. Previously, we identified a planarian homolog of nanos (Smed-nanos) and demonstrated by RNA interference (RNAi) that this gene is required for the development, maintenance, and regeneration of planarian germ cells. We have performed microarray analyses to compare gene expression profiles between planarians with early germ cells and those without them. We identified ∼300 genes that are significantly down-regulated in animals lacking early germ cells. This data set contains genes implicated in germ cell development in other organisms, conserved genes not yet reported to have germ cell-related functions, and novel genes. Analysis using putative domain functions (Clusters of Orthologous Groups) suggested diverse molecular functions, including cytoskeletal components, metabolism, RNA processing and modification, transcription, as well as signal transduction. Top hits have been validated by in situ hybridization. Functional analyses of these genes via RNA interference are being carried out. Thus far, we have identified several genes that, when knocked down by RNAi, cause various defects in germ cell development, including: impaired testes development; loss of spermatogonial stem cells; meiotic failure; and defects in sperm elongation. This work will contribute to our knowledge of conserved regulators of germ cell differentiation. (Supported by NIH-NICHD R01-HD043403.)}, number={2}, journal={Developmental Biology}, publisher={Elsevier BV}, author={Benfey, Philip N. and Cui, Hongchang and Twigg, Richard and Long, Terri and Iyer-Pascuzzi, Anjali and Tsukagoshi, Hironaka and Sozzani, Rosangela and Jackson, Terry and Van Norman, Jaimie and Moreno-Risueno, Miguel}, year={2009}, month={Jul}, pages={386} }
 @article{ni_sozzani_blanchet_domenichini_reuzeau_cella_bergounioux_raynaud_2009, title={The Arabidopsis MCM2 gene is essential to embryo development and its over-expression alters root meristem function}, volume={184}, ISSN={1469-8137}, DOI={10.1111/j.1469-8137.2009.02961.x}, abstractNote={• Minichromosome maintenance (MCM) proteins are subunits of the pre-replication complex that probably function as DNA helicases during the S phase of the cell cycle. Here, we investigated the function of AtMCM2 in Arabidopsis. • To gain an insight into the function of AtMCM2, we combined loss- and gain-of-function approaches. To this end, we analysed two null alleles of AtMCM2, and generated transgenic plants expressing AtMCM2 downstream of the constitutive 35S promoter. • Disruption of AtMCM2 is lethal at a very early stage of embryogenesis, whereas its over-expression results in reduced growth and inhibition of endoreduplication. In addition, over-expression of AtMCM2 induces the formation of additional initials in the columella root cap. In the plt1,2 mutant, defective for root apical meristem maintenance, over-expression of AtMCM2 induces lateral root initiation close to the root tip, a phenotype not reported in the wild-type or in plt1,2 mutants, even when cell cycle regulators, such as AtCYCD3;1, were over-expressed. • Taken together, our results provide evidence for the involvement of AtMCM2 in DNA replication, and suggest that it plays a crucial role in root meristem function.}, number={2}, journal={The New Phytologist}, author={Ni, Di An and Sozzani, Rosangela and Blanchet, Sophie and Domenichini, Séverine and Reuzeau, Christophe and Cella, Rino and Bergounioux, Catherine and Raynaud, Cécile}, year={2009}, month={Oct}, pages={311–322} }
 @article{ascencio-ibáñez_sozzani_lee_chu_wolfinger_cella_hanley-bowdoin_2008, title={Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection}, volume={148}, ISSN={0032-0889}, DOI={10.1104/pp.108.121038}, abstractNote={Geminiviruses are small DNA viruses that use plant replication machinery to amplify their genomes. Microarray analysis of the Arabidopsis (Arabidopsis thaliana) transcriptome in response to cabbage leaf curl virus (CaLCuV) infection uncovered 5,365 genes (false discovery rate <0.005) differentially expressed in infected rosette leaves at 12 d postinoculation. Data mining revealed that CaLCuV triggers a pathogen response via the salicylic acid pathway and induces expression of genes involved in programmed cell death, genotoxic stress, and DNA repair. CaLCuV also altered expression of cell cycle-associated genes, preferentially activating genes expressed during S and G2 and inhibiting genes active in G1 and M. A limited set of core cell cycle genes associated with cell cycle reentry, late G1, S, and early G2 had increased RNA levels, while core cell cycle genes linked to early G1 and late G2 had reduced transcripts. Fluorescence-activated cell sorting of nuclei from infected leaves revealed a depletion of the 4C population and an increase in 8C, 16C, and 32C nuclei. Infectivity studies of transgenic Arabidopsis showed that overexpression of CYCD3;1 or E2FB, both of which promote the mitotic cell cycle, strongly impaired CaLCuV infection. In contrast, overexpression of E2FA or E2FC, which can facilitate the endocycle, had no apparent effect. These results showed that geminiviruses and RNA viruses interface with the host pathogen response via a common mechanism, and that geminiviruses modulate plant cell cycle status by differentially impacting the CYCD/retinoblastoma-related protein/E2F regulatory network and facilitating progression into the endocycle.}, number={1}, journal={Plant Physiology}, author={Ascencio-Ibáñez, José Trinidad and Sozzani, Rosangela and Lee, Tae-Jin and Chu, Tzu-Ming and Wolfinger, Russell D. and Cella, Rino and Hanley-Bowdoin, Linda}, year={2008}, month={Sep}, pages={436–454} }
 @article{sozzani_maggio_varotto_canova_bergounioux_albani_cella_2006, title={Interplay between Arabidopsis Activating Factors E2Fb and E2Fa in Cell Cycle Progression and Development}, volume={140}, ISSN={0032-0889, 1532-2548}, url={http://www.plantphysiol.org/content/140/4/1355}, DOI={10.1104/pp.106.077990}, abstractNote={Abstract Eukaryotic E2Fs are conserved transcription factors playing crucial and antagonistic roles in several pathways related to cell division, DNA repair, and differentiation. In plants, these processes are strictly intermingled at the growing zone to produce postembryonic development in response to internal signals and environmental cues. Of the six AtE2F proteins found in Arabidopsis (Arabidopsis thaliana), only AtE2Fa and AtE2Fb have been clearly indicated as activators of E2F-responsive genes. AtE2Fa activity was shown to induce S phase and endoreduplication, whereas the function of AtE2Fb and the interrelationship between these two transcription factors was unclear. We have investigated the role played by the AtE2Fb gene during cell cycle and development performing in situ RNA hybridization, immunolocalization of the AtE2Fb protein in planta, and analysis of AtE2Fb promoter activity in transgenic plants. Overexpression of AtE2Fb in transgenic Arabidopsis plants led to striking modifications of the morphology of roots, cotyledons, and leaves that can be ascribed to stimulation of cell division. The accumulation of the AtE2Fb protein in these lines was paralleled by an increased expression of E2F-responsive G1/S and G2/M marker genes. These results suggest that AtE2Fa and AtE2Fb have specific expression patterns and play similar but distinct roles during cell cycle progression.}, number={4}, journal={Plant Physiology}, author={Sozzani, Rosangela and Maggio, Caterina and Varotto, Serena and Canova, Sabrina and Bergounioux, Catherine and Albani, Diego and Cella, Rino}, year={2006}, month={Apr}, pages={1355–1366} }
 @article{raynaud_sozzani_glab_domenichini_perennes_cella_kondorosi_bergounioux_2006, title={Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis}, volume={47}, ISSN={0960-7412}, DOI={10.1111/j.1365-313X.2006.02799.x}, abstractNote={Summary The proliferating cell nuclear antigen (PCNA) functions as a sliding clamp for DNA polymerase, and is thus a key actor in DNA replication. It is also involved in DNA repair, maintenance of heterochromatic regions throughout replication, cell cycle regulation and programmed cell death. Identification of PCNA partners is therefore necessary for understanding these processes. Here we identify two Arabidopsis SET‐domain proteins that interact with PCNA: ATXR5 and ATXR6. A truncated ATXR5Δex2, incapable of interacting with PCNA, also occurs in planta . ATXR6 , upregulated during the S phase, is upregulated by AtE2F transcription factors, suggesting that it is required for S‐phase progression. The two proteins differ in their subcellular localization: ATXR5 has a dual localization in plastids and in the nucleus, whereas ATXR6 is solely nuclear. This indicates that the two proteins may play different roles in plant cells. However, overexpression of either ATXR5 or ATXR6 causes male sterility because of the degeneration of defined cell types. Taken together, our results suggest that both proteins may play a role in the cell cycle or DNA replication, and that the activity of ATXR5 may be regulated via its subcellular localization.}, number={3}, journal={The Plant Journal: For Cell and Molecular Biology}, author={Raynaud, Cécile and Sozzani, Rosangela and Glab, Nathalie and Domenichini, Séverine and Perennes, Claudette and Cella, Rino and Kondorosi, Eva and Bergounioux, Catherine}, year={2006}, month={Aug}, pages={395–407} }