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Linked article: This paper is the subject of a Research Highlight article. To view this Research Highlight article visit https://doi.org/10.1111/tpj.15118, Members of the plant specific RAV family of transcription factors regulate several developmental and physiological processes. In the model plant Arabidopsis thaliana, the RAV TEMPRANILLO 1 (TEM1) and TEM2 control important phase changes such as the juvenile to adult and the vegetative to reproductive transitions. Besides their known regulatory function in plant development, a transcriptomics analysis of transgenic plants overexpressing TEM1 also revealed overrepresentation of Gene Ontology (GO) categories related to abiotic stress responses. Therefore, to investigate the biological relevance of these TEM-dependent transcriptomic changes and elucidate whether TEMs contribute to the modulation of plant growth in response to salinity, we analyzed the behavior of TEM gain and loss of function mutants subjected to mild and high salt stresses at different development stages. With respect to increasing salinity, TEM overexpressing plants were hypersensitive whereas the tem1 tem2 double mutants were more tolerant. Precisely, tem1 tem2 mutants germinated and flowered faster than the wild-type plants under salt stress conditions. Also, tem1 tem2 plants showed a delay in salt-induced leaf senescence, possibly as a consequence of downregulation of jasmonic acid biosynthesis genes. Besides a shorter life cycle and delayed senescence, tem1 tem2 mutants appeared to be better suited to withstand oxidative stress as they accumulated higher levels of α-tocopherol (an important antioxidant metabolite) and displayed a slower degradation of photosynthetic pigments. Taken together, our studies suggest novel and crucial roles for TEM in adaptive growth as they modulate plant development in response to environmental changes such as increasing soil salinity., We thank Luca Morelli for advice with PAM imaging and our funders: MINECO-MICIU/FEDER (BFU2015-64409-P, PCG2018-095804-B-I00) to SP and the CERCA Programme/Generalitat de Catalunya (2017 SGR 718) and ‘Severo Ochoa Programme for Centres of Excellence in R&D’ 2016-2019 (SEV-2015-0533) to CRAG. Work in the JLR laboratory was supported by MINECO/FEDER grant BFU2014-58289-P. AEA-J and UC performed this work within the frame of a PhD Program of the Universitat Autònoma de Barcelona with an Investigator Training Program PhD fellowship from Generalitat de Catalunya to AEA-J and an FPI fellowship from the Spanish Ministry to UC. Sequentia Biotech SL (Barcelona, Spain) helped with RNA-Seq analysis.

Special Issue: From Model Systems to Crops ‐ Challenges for a New Era in Plant Biology.

Floral organs are specified by the activities of a small group of transcriptional regulators, the floral organ identity factors. Extensive genetic and molecular analyses have shown that these proteins act as master regulators of flower development, and function not only in organ identity determination but also during organ morphogenesis. Although it is now well established that these transcription factors act in higher order protein complexes in the regulation of transcription, the gene expression programmes controlled by them have remained largely elusive. Only recently, detailed insights into their functions have been obtained through the combination of a wide range of experimental methods, including transcriptomic and proteomic approaches. Here, we review the progress that has been made in the characterization of the floral organ identity factors from the main model plant Arabidopsis thaliana, and we discuss what is known about the processes acting downstream of these regulators. We further outline open questions, which we believe need to be addressed to obtain a more complete view of the molecular processes that govern floral organ development and specification., Work in our laboratories is funded by grants from the Science Foundation Ireland (to EG and FW) and from the Spanish Ministerio de Economía y Competitividad (to JLR).

Shoot apical meristem activity is controlled by complex regulatory networks in which components such as transcription factors, miRNAs, small peptides, hormones, enzymes and epigenetic marks all participate. Many key genes that determine the inherent characteristics of the shoot apical meristem have been identified through genetic approaches. Recent advances in genome-wide studies generating extensive transcriptomic and DNA-binding datasets have increased our understanding of the interactions within the regulatory networks that control the activity of the meristem, identifying new regulators and uncovering connections between previously unlinked network components. In this review, we focus on recent studies that illustrate the contribution of whole genome analyses to understand meristem function., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2014-58289-P) and from the Agencia de Gestió d’Ajuts Universitaris I de Recerca (2014 SGR 1406) to JLR.

To obtain detailed information about gene expression during stamen development in Arabidopsis (Arabidopsis thaliana), we compared, by microarray analysis, the gene expression profile of wild-type inflorescences to those of the floral mutants apetala3, sporocyteless/nozzle, and male sterile1 (ms1), in which different aspects of stamen formation are disrupted. These experiments led to the identification of groups of genes with predicted expression at early, intermediate, and late stages of stamen development. Validation experiments using in situ hybridization confirmed the predicted expression patterns. Additional experiments aimed at characterizing gene expression specifically during microspore formation. To this end, we compared the gene expression profiles of wild-type flowers of distinct developmental stages to those of the ms1 mutant. Computational analysis of the datasets derived from this experiment led to the identification of genes that are likely involved in the control of key developmental processes during microsporogenesis. We also identified a large number of genes whose expression is prolonged in ms1 mutant flowers compared to the wild type. This result suggests that MS1, which encodes a putative transcriptional regulator, is involved in the stage-specific repression of these genes. Lastly, we applied reverse genetics to characterize several of the genes identified in the microarray experiments and uncovered novel regulators of microsporogenesis, including the transcription factor MYB99 and a putative phosphatidylinositol 4-kinase.

The analysis of the gene regulatory networks underlying development is of central importance for a better understanding of the mechanisms that control the formation of the different cell-types, tissues or organs of an organism. The recent invention of genomic technologies has opened the possibility of studying these networks at a global level. In this paper, we summarize some of the recent advances that have been made in the understanding of plant development by the application of genomic technologies. We focus on a few specific processes, namely flower and root development and the control of the cell cycle, but we also highlight landmark studies in other areas that opened new avenues of experimentation or analysis. We describe the methods and the strategies that are currently used for the analysis of plant development by genomic technologies, as well as some of the problems and limitations that hamper their application. Since many genomic technologies and concepts were first developed and tested in organisms other than plants, we make reference to work in non-plant species and compare the current state of network analysis in plants to that in other multicellular organisms.

We have developed a versatile floral induction system that is based on ectopic overexpression of the transcription factor LEAFY (LFY) in callus. During shoot regeneration, flowers or floral organs are formed directly from root explants without prior formation of rosette leaves. Morphological and reporter gene analyses show that leaf-like structures are converted to floral organs in response to LFY activity. Thus, increased levels of LFY activity are sufficient to bypass normal vegetative development and to direct formation of flowers in tissue culture. We found that about half of the cultured cells respond to inducible LFY activity with a rapid upregulation of the known direct target gene of LFY, APETALA1 (AP1). This dramatic increase in the number of LFY-responsive cells compared to whole plants suggested that the tissue culture system could greatly facilitate the analysis of LFY-dependent gene regulation by genomic approaches. To test this, we monitored the gene expression changes that occur in tissue culture after activation of LFY using a flower-specific cDNA microarray. Induction of known LFY target genes was readily detected in these experiments. In addition, several other genes were identified that had not been implicated in signaling downstream of LFY before. Thus, the floral induction system is suitable for the detection of low abundance transcripts whose expression is controlled in an LFY-dependent manner.

Epicuticular wax forms a layer of hydrophobic material on plant aerial organs, which constitutes a protective barrier between the plant and its environment. We report here the identification of WIN1, anArabidopsis thalianaethylene response factor-type transcription factor, which can activate wax deposition in overexpressing plants. We constitutively expressedWIN1in transgenicArabidopsisplants, and found that leaf epidermal wax accumulation was up to 4.5-fold higher in these plants than in control plants. A significant increase was also found in stems. Interestingly, ≈50% of the additional wax could only be released by complete lipid extractions, suggesting that not all of the wax is superficial. Gene expression analysis indicated that a number of genes, such asCER1, KCS1, andCER2, which are known to be involved in wax biosynthesis, were induced inWIN1overexpressors. This observation indicates that induction of wax accumulation in transgenic plants is probably mediated through an increase in the expression of genes encoding enzymes of the wax biosynthesis pathway.

The Arabidopsis FLOWERING LOCUS C (FLC) gene is a key floral repressor in the maintenance of a vernalization response. In vernalization-sensitive genetic backgrounds, FLC levels are high, and they decline after exposure to long cold periods. Four FLC paralogs (MAF2 [MADS AFFECTING FLOWERING2] to MAF5) are arranged in a tandem array on the bottom of Arabidopsis chromosome V. We used a reverse genetics approach to analyze their functions. Loss-of-function and gain-of-function studies indicate that MAF2 acts as a floral repressor. In particular, maf2 mutant plants display a pronounced vernalization response when subjected to relatively short cold periods, which are insufficient to elicit a strong flowering response in the wild type, despite producing a large reduction in FLC levels. MAF2 expression is less sensitive to vernalization than that of FLC, and its repressor activity is exerted independently or downstream of FLC transcription. Thus, MAF2 can prevent premature vernalization in response to brief cold spells. Overexpression of MAF3 or MAF4 produces alterations in flowering time that suggest that these genes also act as floral repressors and might contribute to the maintenance of a vernalization requirement. However, the final gene in the cluster, MAF5, is upregulated by vernalization. Therefore, MAF5 could play an opposite role to FLC in the vernalization response.

In plants, low temperature and dehydration activate a set of genes containing C-repeat/dehydration-responsive elements in their promoter. It has been shown previously that the Arabidopsis CBF/DREB1 transcription activators are critical regulators of gene expression in the signal transduction of cold acclimation. Here, we report the isolation of an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. Overexpression of CBF4 in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation. As a result, the transgenic plants are more tolerant to freezing and drought stress. Because of the physiological similarity between freezing and drought stress, and the sequence and structural similarity of the CBF/DREB1 and the CBF4 proteins, we propose that the plant's response to cold and drought evolved from a common CBF-like transcription factor, first through gene duplication and then through promoter evolution.

[Background] Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis., [Results] Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species., [Conclusion] Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants., P. Baldrich is a recipient of a Ph.D grant from the “Ministerio de Ciencia e Innovación, Formación de Personal Investigador-FPI, ref. BES-2010-032879). This work was supported by grants BIO2009-08719 and BIO2012-32838 to BSS, AGL2010-14949 to JJLM, and BFU2008-04251 to JLR, from the Spanish Ministry of Economy and Competitiveness (MINECO), and grant 2010–0520193 to JLR from the National Science Foundation (NSF). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Background Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. Results We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Conclusions Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility., This work was supported by an NWO-VIDI grant to KK, and a Marie-Curie-ITN network grant SYSFLO (FP7/2007-2011, grant agreement no. 237909) to AP, PM, PK, and GCA. KK wishes to thank the Alexander-von-Humboldt foundation for support. This work was also supported by grants from Spanish Ministerio de Ciencia e Innovación (BFU2011-22734 to JLR). JTM and JJ were recipients of EMBO postdoctoral fellowships.

The completion of theArabidopsis thalianagenome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms.Arabidopsisdedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants.Arabidopsistranscription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

Data from the Arabidopsis genome project suggest that more than 5% of the genes of this plant encode transcription factors. The necessity for the use of genomic analytical approaches becomes clear when it is considered that less than 10% of these factors have been genetically characterized. A variety of tools for functional genomic analyses in plants have been developed over the past few years. The availability of the full complement of Arabidopsis transcription factors, together with the results of recent studies that illustrate some of the challenges to their functional characterization, now provides the basic framework for future analyses of transcriptional regulation in plants.

PISTILLATA (PI) is a floral homeotic B function gene in Arabidopsis and together with the other B function gene, APETALA3 (AP3), is involved in specifying petal and stamen identities. The expression of PI and AP3 is under similar developmental control. The initiation of AP3 and PI expression is at least partly caused by the floral meristem identity gene LEAFY, but the maintenance of AP3 and PI expression involves an autoregulatory loop requiring the activity of both genes. PI and AP3 are MADS domain proteins that form, and appear to function as, a heterodimer. AP3/PI binds in vitro to a sequence motif, CC(A/T)6GG, a MADS domain protein consensus binding site also known as the CArG box. We identified a 481-bp PI promoter region that confers both the initiation and the maintenance of PI expression patterns. We further dissected the promoter and identified minimal regions responsible for the AP3/PI-dependent expression. No CArG box is present in these minimal regions, suggesting that either AP3/PI does not bind directly to the PI promoter for the maintenance control, or that it requires additional factors to bind to the PI promoter. Our results suggest that the mechanisms of regulation of the two B function genes, AP3 and PI, are different, because CArG boxes are present in the AP3 promoter and are necessary for the AP3 feedback control.

&p.1: The MADS domain proteins AP1, AP3, PI, and AG are required to specify the four classes of organs in an Arabidopsis flower. Each of these proteins is involved in specifying the identity of two different organs in two adjacent floral whorls. They all share a 56-amino acid MADS domain required for DNA binding and dimerization, a region (I or L) involved in dimerization specificity, the K domain named for its sequence similarity to the coiled-coil of keratin, and a variable carboxy terminal sequence. The abilities of these four related proteins to specify distinct organs presumably result from differential effects on transcriptional regulation. We have previously used chimeric MADS box genes, expressed under the constitutive 35S promoter, to map the regions of these proteins that are responsible for their different organ identity activities. In this paper, we extend these studies by characterizing the phenotypes of plants ectopically expressing chimeric genes under the control of the endogenous AP1 promoter. Similar results are obtained with the 35S and AP1 promoters, although the endogenous promoter does provide a more rigorous test of function. We also describe results from new chimeric gene constructs that show new in vivo functions for the K domain and the amino-terminal portion of the MADS domain.

Determining gene function through reverse genetics has been an important experimental approach in the field of flower development. The method largely relies on the availability of knockout lines for the gene of interest. Insertional mutagenesis can be performed using either T-DNA or transposable elements, but the former has been more frequently employed in Arabidopsis. A primary concern for working with insertional mutant lines is whether the respective insertion results in a complete or rather a partial loss of gene function. The effect of the insertion largely depends on its position with respect to the structure of the gene. In order to quickly identify and obtain knockout lines for genes of interest in Arabidopsis, more than 325,000 mapped insertion lines have been catalogued on indexed libraries and made publicly available to researchers. Online accessible databases provide information regarding the site of insertion, whether a mutant line is available in a homozygous or hemizygous state, and outline technical aspects for plant identification, such as primer design tools used for genotyping. In this chapter, we describe the procedure for isolating knockout lines for genes of interest in Arabidopsis.

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler(®) 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment.

Almost three decades of genetic and molecular analyses have resulted in detailed insights into many of the processes that take place during flower development and in the identification of a large number of key regulatory genes that control these processes. Despite this impressive progress, many questions about how flower development is controlled in different angiosperm species remain unanswered. In this chapter, we discuss some of these open questions and the experimental strategies with which they could be addressed. Specifically, we focus on the areas of floral meristem development and patterning, floral organ specification and differentiation, as well as on the molecular mechanisms underlying the evolutionary changes that have led to the astounding variations in flower size and architecture among extant and extinct angiosperms.

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler® 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment., Work in the authors’ laboratory is funded by grants from Spanish Ministerio de Economía y Competividad (BFU2011-22734; and Programa Consolider-Ingenio, CSD2007-00036), and the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (grant SGR2009-GRC476). J. J. was supported by a fellowship from the European Molecular Biology Organization (EMBO).

Determining gene function through reverse genetics has been an important experimental approach in the field of flower development. The method largely relies on the availability of knockout lines for the gene of interest. Insertional mutagenesis can be performed using either T-DNA or transposable elements, but the former has been more frequently employed in Arabidopsis. A primary concern for working with insertional mutant lines is whether the respective insertion results in a complete or rather a partial loss of gene function. The effect of the insertion largely depends on its position with respect to the structure of the gene. In order to quickly identify and obtain knockout lines for genes of interest in Arabidopsis, more than 325,000 mapped insertion lines have been catalogued on indexed libraries and made publicly available to researchers. Online accessible databases provide information regarding the site of insertion, whether a mutant line is available in a homozygous or hemizygous state, and outline technical aspects for plant identification, such as primer design tools used for genotyping. In this chapter, we describe the procedure for isolating knockout lines for genes of interest in Arabidopsis.

The MADS domain proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) specify the identity of Arabidopsis floral organs. AP1 and AG homocomplexes and AP3-PI heterocomplexes bind to CArG-box sequences. The DNA-binding properties of these complexes were investigated. We find that AP1, AG and AP3-PI are all capable of recognizing the same DNA-binding sites, although with somewhat different affinities. In addition, the three complexes induce similar conformational changes on a CArG-box sequence. Phasing analysis reveals that the induced distortion is DNA bending, oriented toward the minor groove. The molecular dissection of AP1, AP3, PI and AG indicates that the boundaries of the dimerization domains of these proteins vary. The regions required to form a DNA-binding complex include, in addition to the MADS box, the entire L region (which follows the MADS box) and the first putative amphipathic helix of the K box in the case of AP3-PI, while for AP1 and AG only a part of the L region is needed. The similarity of the DNA-binding properties of AP1, AP3-PI and AG is discussed with regard to the biological specificity that these proteins exhibit.

Gene transcripts are under extensive posttranscriptional regulation, including the regulation of their stability. A major route for mRNA degradation produces uncapped mRNAs, which can be generated by decapping enzymes, endonucleases, and small RNAs. Profiling uncapped mRNA molecules is important for the understanding of the transcriptome, whose composition is determined by a balance between mRNA synthesis and degradation. In this chapter, we describe a method to profile these uncapped mRNAs at the genome scale.

In many organisms, the circadian clock is composed of functionally coupled morning and evening oscillators. In Arabidopsis, oscillator coupling relies on a core loop in which the evening oscillator component TOC1 (TIMING OF CAB EXPRESSION1) was proposed to activate a subset of morning-expressed oscillator genes. Here, we show that TOC1 does not function as an activator but as a general repressor of oscillator gene expression. Repression occurs through TOC1 rhythmic association to the promoters of the oscillator genes. Hormone-dependent induction of TOC1 and analysis of RNAi plants show that TOC1 prevents the activation of morning-expressed genes at night. Our study overturns the prevailing model of the Arabidopsis circadian clock showing that the morning and evening oscillator loops are connected through the repressing activity of TOC1., This work was supported by grants to P.M. from the Ramón Areces Foundation, the Spanish Ministry of Science and Innovation (MICINN), the EMBO YIP program and from the European Heads of Research Councils and the European Science Foundation through the EURYI Award; and to J.L.R. from the EC Marie Curie program and MICINN; and by the European Commission FP7 Collaborative Project TiMet to A.J.M. and others. The Centre for Systems Biology at Edinburgh is a Centre for Integrative and Systems Biology supported by BBSRC and EPSRC award D019621. W.H. is supported by a Juan de la Cierva contract (MICINN) and P.P.G. by a FPI fellowship (MICINN). Sequencing data have been deposited with the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE35952.

The availability of the Arabidopsis thaliana genome sequence allows a comprehensive analysis of transcriptional regulation in plants using novel genomic approaches and methodologies. Such a genomic view of transcription first necessitates the compilation of lists of elements. Transcription factors are the most numerous of the different types of proteins involved in transcription in eukaryotes, and the Arabidopsis genome codes for more than 1,500 of them, or approximately 6% of its total number of genes. A genome-wide comparison of transcription factors across the three eukaryotic kingdoms reveals the evolutionary generation of diversity in the components of the regulatory machinery of transcription. However, as illustrated by Arabidopsis, transcription in plants follows similar basic principles and logic to those in animals and fungi. A global view and understanding of transcription at a cellular and organismal level requires the characterization of the Arabidopsis transcriptome and promoterome, as well as of the interactome, the localizome, and the phenome of the proteins involved in transcription.

Gene transcripts are under extensive posttranscriptional regulation, including the regulation of their stability. A major route for mRNA degradation produces uncapped mRNAs, which can be generated by decapping enzymes, endonucleases, and small RNAs. Profiling uncapped mRNA molecules is important for the understanding of the transcriptome, whose composition is determined by a balance between mRNA synthesis and degradation. In this chapter, we describe a method to profile these uncapped mRNAs at the genome scale.

We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e., real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation, washing artifacts, microarray spot-to-spot variations, and other intensity-affecting impediments. We demonstrate in both theory and practice that the time-constant of target capturing is inversely proportional to the concentration of the target analyte, which we take advantage of as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to experimentally validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e., real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation, washing artifacts, microarray spot-to-spot variations, and other intensity-affecting impediments. We demonstrate in both theory and practice that the time-constant of target capturing is inversely proportional to the concentration of the target analyte, which we take advantage of as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to experimentally validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

The effect of different protease inhibitors on the proteolytic processing of the plum pox potyvirus (PPV) polyprotein has been analyzed. Human cystatin C, an inhibitor of cysteine proteases, interfered with the outoprocessing of the viral papain-like cysteine protease HCPro. Unexpectedly, it also had an inhibitory effect on the autocatalytic cleavage of the Nla protease which, although it has a Cys residue in its active center, has been described as structurally related to serine proteases. Other protease inhibitors tested had no effect on any of the cleavage events analyzed.

The onset of flower formation is a key regulatory event during the life cycle of angiosperm plants, which marks the beginning of the reproductive phase of development. It has been shown that floral initiation is under tight genetic control, and deciphering the underlying molecular mechanisms has been a main area of interest in plant biology for the past two decades. Here, we provide an overview of the developmental and genetic processes that occur during floral initiation. We further review recent studies that have led to the genome-wide identification of target genes of key floral regulators and discuss how they have contributed to an in-depth understanding of the gene regulatory networks controlling early flower development. We focus especially on a master regulator of floral initiation in Arabidopsis thaliana APETALA1 (AP1), but also outline what is known about the AP1 network in other plant species and the evolutionary implications. © 2010 Elsevier Ltd., Our work on flower development is supported by grants from Science Foundation Ireland (06/IN.1/B851 to F.W.), the Spanish Ministerio de Ciencia e Innovación (BFU2008-04251 to J.L.R., and Programa Consolider-Ingenio, CSD2007-00036), the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (SGR2009-GRC476 to J.L.R.) and the EU-Marie Curie program (IRG-224864 to J.L.R.).

The recently cloned INTERFASCICULAR FIBERLESS1 ( IFL1 ) gene encodes a homeodomain–leucine zipper protein (HD-ZIP) that spatially regulates fiber differentiation in Arabidopsis ([Zhong and Ye, 1999][1]). Mutations of the IFL1 gene are recessive and highly pleiotropic. In ifl1 mutants, normal

We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays. © The Author(s) 2009. Published by Oxford University Press., Grubstake grant from Caltech; Millard and Muriel Jacobs Genetics Laboratory at the California Institute of Technology; David and Lucille Packard Foundation. Funding for open access charge: University of Texas at Austin New Faculty Startup Fund.

The composition of the transcriptome is determined by a balance between mRNA synthesis and degradation. An important route for mRNA degradation produces uncapped mRNAs, and this decay process can be initiated by decapping enzymes, endonucleases, and small RNAs. Although uncapped mRNAs are an important intermediate for mRNA decay, their identity and abundance have never been studied on a large scale until recently. Here, we present an experimental method for transcriptome-wide profiling of uncapped mRNAs that can be used in any eukaryotic system. We applied the method to study the prevalence of uncapped transcripts during the early stages of Arabidopsis thaliana flower development. Uncapped transcripts were identified for the majority of expressed genes, although at different levels. By comparing uncapped RNA levels with steady state overall transcript levels, our study provides evidence for widespread mRNA degradation control in numerous biological processes involving genes of varied molecular functions, implying that uncapped mRNA levels are dynamically regulated. Sequence analyses identified structural features of transcripts and ciselements that were associated with different levels of uncapping. These transcriptome-wide profiles of uncapped mRNAs will aid in illuminating new regulatory mechanisms of eukaryotic transcriptional networks. © 2008 American Society of Plant Biologists., This work was supported by National Science Foundation 2010 Project Grant 0520193 to J.L.R. and E.M.M. and by the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology.

A binary Escherichia coli expression system has been used to study the pathway for proteolytic processing of the plum pox potyvirus (PPV) polyprotein. Trans cleavage at the carboxyl end of the cylindrical inclusion protein occurred, although with lower efficiency than that at the large nuclear inclusion protein-capsid protein junction. No trans cleavage at the carboxyl end of the small nuclear inclusion protein (NIa) was detected. The proteolytic activities at different cleavage sites of several deletion and point mutations of NIa protein have been analysed. The large delta SX deletion and two different point mutations at His 239 abolished proteolytic activity at all sites. The effect of other mutations, particularly a Glu substitution for Asp 274, depended on the particular cleavage site analysed. The results obtained with the PPV NIa protein mutants were similar to those reported for comparable mutations in the tobacco etch virus 49K protease, despite differences in the sequences recognized for processing. No evident competitive inhibition of the proteolytic activity of PPV NIa protease by the presence of an excess of the different protease mutants could be demonstrated.

Most positive strand RNA viruses infecting plants and animals encode proteins containing the so-called nucleotide binding motif (NTBM) (1) in their amino acid sequences (2). As suggested from the high level of sequence similarity of these viral proteins with the recently described superfamilies of helicase-like proteins (3-5), the NTBM-containing cylindrical inclusion (CI) protein from plum pox virus (PPV), which belongs to the potyvirus group of positive strand RNA viruses, is shown to be able to unwind RNA duplexes. This activity was found to be dependent on the hydrolysis of NTP to NDP and Pi, and thus it can be considered as an RNA helicase activity. In the in vitro assay used, the PPV CI protein was only able to unwind double strand RNA substrates with 3' single strand overhangs. This result indicates that the helicase activity of the PPV CI protein functions in the 3' to 5' direction (6). To our knowledge, this is the first report on a helicase activity associated with a protein encoded by an RNA virus.

The sections in this article are: Introduction - Arabidopsis and Rice Genomes: The Angiosperm Complement of Transcription Factors Plant Promoters.

The composition and permeability of the cuticle has a large influence on its ability to protect the plant against various forms of biotic and abiotic stress. WAX INDUCER1 (WIN1) and related transcription factors have recently been shown to trigger wax production, enhance drought tolerance, and modulate cuticular permeability when overexpressed in Arabidopsis thaliana. We found that WIN1 influences the composition of cutin, a polyester that forms the backbone of the cuticle. WIN1 overexpression induces compositional changes and an overall increase in cutin production in vegetative and reproductive organs, while its downregulation has the opposite effect. Changes in cutin composition are preceded by the rapid and coordinated induction of several genes known or likely to be involved in cutin biosynthesis. This transcriptional response is followed after a delay by the induction of genes associated with wax biosynthesis, suggesting that the regulation of cutin and wax production by WIN1 is a two-step process. We demonstrate that at least one of the cutin pathway genes, which encodes long-chain acyl-CoA synthetase LACS2, is likely to be directly targeted by WIN1. Overall, our results suggest that WIN1 modulates cuticle permeability in Arabidopsis by regulating genes encoding cutin pathway enzymes.

In plants, members of microRNA (miRNA) families are often predicted to target the same or overlapping sets of genes. It has thus been hypothesized that these miRNAs may act in a functionally redundant manner. This hypothesis is tested here by studying the effects of elimination of all three members of the MIR164 family from Arabidopsis. It was found that a loss of miR164 activity leads to a severe disruption of shoot development,in contrast to the effect of mutation in any single MIR164 gene. This indicates that these miRNAs are indeed functionally redundant. Differences in the expression patterns of the individual MIR164 genes imply,however, that redundancy among them is not complete, and that these miRNAs show functional specialization. Furthermore, the results of molecular and genetic analyses of miR164-mediated target regulation indicate that miR164 miRNAs function to control the transcript levels, as well as the expression patterns, of their targets, suggesting that they might contribute to developmental robustness. For two of the miR164targets, namely CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, we provide evidence for their involvement in the regulation of growth and show that their derepression in miR164 loss-of-function mutants is likely to account for most of the mutant phenotype.

Detailed information about stage-specific changes in gene expression is crucial for the understanding of the gene regulatory networks underlying development. Here, we describe the global gene expression dynamics during early flower development, a key process in the life cycle of a plant, during which floral patterning and the specification of floral organs is established. We used a novel floral induction system in Arabidopsis, which allows the isolation of a large number of synchronized floral buds, in conjunction with whole-genome microarray analysis to identify genes with differential expression at distinct stages of flower development. We found that the onset of flower formation is characterized by a massive downregulation of genes in incipient floral primordia, which is followed by a predominance of gene activation during the differentiation of floral organs. Among the genes we identified as differentially expressed in the experiment, we detected a significant enrichment of closely related members of gene families. The expression profiles of these related genes were often highly correlated, indicating similar temporal expression patterns. Moreover, we found that the majority of these genes is specifically up-regulated during certain developmental stages. Because co-expressed members of gene families in Arabidopsis frequently act in a redundant manner, these results suggest a high degree of functional redundancy during early flower development, but also that its extent may vary in a stage-specific manner., Synopsis The development of flowers is one of the characteristic features of higher plants. In an effort to gain detailed insights into the molecular processes underlying flower development, the authors have analyzed the expression of the genes of the small plant Arabidopsis thaliana, which is widely used by biologists for the study of plant development, during the early stages of flower formation. To this end, they used DNA microarray analysis, a technology that allows the simultaneous detection of thousands of gene transcripts in a single experiment. Because young floral buds of Arabidopsis are minute and are difficult to dissect, the authors established a system that allows the simultaneous induction of a large number of flowers on a single plant. Using this system, they identified groups of genes, many of them novel or uncharacterized, that are highly active during distinct stages of flower development. These genes are likely involved in controlling the various developmental changes that take place during the formation of flowers. The authors also found that many of these genes are closely related in sequence, suggesting that they might be involved in similar or identical processes, and thus uncovering a large degree of potential functional redundancy during flower development.

The Arabidopsis homeotic gene AGAMOUS (AG) is necessary for the specification of reproductive organs (stamens and carpels) during the early steps of flower development. AG encodes a transcription factor of the MADS-box family that is expressed in stamen and carpel primordia. At later stages of development, AG is expressed in distinct regions of the reproductive organs. This suggests that AG might function during the maturation of stamens and carpels, as well as in their early development. However, the developmental processes that AG might control during organogenesis and the genes that are regulated by this factor are largely unknown. Here we show that microsporogenesis, the process leading to pollen formation, is induced by AG through activation of the SPOROCYTELESS gene (SPL, also known as NOZZLE,NZZ), a regulator of sporogenesis. Furthermore, we demonstrate that SPL can induce microsporogenesis in the absence of AG function, suggesting that AG controls a specific process during organogenesis by activating another regulator that performs a subset of its functions.

The availability of the Arabidopsis genome sequence allows for novel approaches in the analysis of many aspects of plant biology. Approximately 6% of Arabidopsis genes code for transcription factors, which can be grouped into different families according to similarities within the DNA binding domains. Transcription factors are critical regulatory components of the pathways that underpin many aspects of plant growth, development, and physiology. In particular, a substantial number of them are emerging as having crucial roles in controlling one of the most important, but complex, steps in the plant life cycle: the transition to flowering. Genome-wide studies offer the opportunity to gain a comprehensive understanding of this polygenic process, making it possible to appreciate both the large number of genes involved, as well as the complex regulatory networks into which those genes are integrated.

The Arabidopsis FLC gene encodes a MADS domain protein that acts as a repressor of flowering. Late-flowering vernalization-responsive ecotypes and mutants have high steady-state levels of FLC transcript, which decrease during the promotion of flowering by vernalization. Therefore, FLC has a central role in regulating the response to vernalization. We have isolated an Arabidopsis gene, MAF1, which encodes a protein that is closely related to FLC. Overexpression studies demonstrate thatMAF1 produces comparable effects to FLC, and likely has a similar function in the regulation of flowering. In contrast to FLC, however, MAF1 expression shows a less clear correlation with the vernalization response. In addition, MAF1 overexpression does not influenceFLC transcript levels. Thus, MAF1 likely acts downstream or independently of FLC transcription. We further report identification of a cluster of four additionalFLC-like genes in the Arabidopsis genome.

UMR BGPI Equipe 6 Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; A full-length genomic cDNA clone of a plum pox potyvirus (PPV) isolate belonging to the M strain (PPV-PS) has been cloned downstream from a bacteriophage T7 polymerase promoter and sequenced. Transcripts from the resulting plasmid, pGPPVPS, were infectious and, in herbaceous hosts, produced symptoms that differed from those of virus progeny of pGPPV, a full-length genomic cDNA clone of the D strain PPV-R. Viable PPV-R/-PS chimeric viruses were constructed by recombination of the cDNA clones in vitro. Analysis of plants infected with the different chimeras indicated that sequences encoding the most variable regions of the potyvirus genome, the P1 and capsid protein coding sequences, were not responsible for symptom differences between the two PPV isolates in herbaceous hosts. On the contrary, complex symptomatology determinants seem to be located in the central region of the PPV genome. The results indicate that a genomic fragment that encodes 173 aa from the C-terminal part of the P3+6K1 coding region is enough to confer, on a PPV-R background, a PS phenotype in Nicotiana clevelandii. This pathogenicity determinant also participates in symptom induction in Pisum sativum, although the region defining the PS phenotype in this host is probably restricted to 74 aa

The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation codon, and the actual amino terminus of the AG protein remains uncharacterized. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, are present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experiments by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiation site does not affect complementation of the ag-3 mutation in transgenic plants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced immediately 5' to the disrupted ACG codon, the resulting construct fully complements the ag-3 mutation. The AG protein synthesized in vitro by initiating translation at the ACG position is active in DNA binding and is of the same size as the AG protein detected from floral tissues, whereas AG polypeptides with additional amino-terminal residues do not appear to bind DNA. These results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the sole translation initiation site in some plant cellular mRNAs.

The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) combinatorially specify the identity of Arabidopsis floral organs. AP1/AP1, AG/AG, and AP3/PI dimers bind to similar CArG box sequences; thus, differences in DNA-binding specificity among these proteins do not seem to be the origin of their distinct organ identity properties. To assess the overall contribution that specific DNA binding could make to their biological specificity, we have generated chimeric genes in which the amino-terminal half of the MADS domain of AP1, AP3, PI, and AG was substituted by the corresponding sequences of human SRF and MEF2A proteins. In vitro DNA-binding assays reveal that the chimeric proteins acquired the respective, and distinct, DNA-binding specificity of SRF or MEF2A. However, ectopic expression of the chimeric genes reproduces the dominant gain-of-function phenotypes exhibited by plants ectopically expressing the corresponding Arabidopsis wild-type genes. In addition, both the SRF and MEF2 chimeric genes can complement the pertinent ap1-1, ap3-3, pi-1, or ag-3 mutations to a degree similar to that of AP1, AP3, PI, and AG when expressed under the control of the same promoter. These results indicate that determination of floral organ identity by the MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity. In addition, the DNA-binding experiments show that either one of the two MADS domains of a dimer can be sufficient to confer a particular DNA-binding specificity to the complex and that sequences outside the amino-terminal basic region of the MADS domain can, in some cases, contribute to the DNA-binding specificity of the proteins.

Proteolytic processing of the potyvirus polyprotein is mainly performed by the virus-encoded NIa protease, whose cleavage sites are characterized by conserved heptapeptide sequences. Partial processing at the cleavage site present between the P3 and 6K1 cistrons by the plum pox potyvirus (PPV) NIa protease has been previously shown to occur in vitro. We have now studied the role of polyprotein processing at the P3-6K1 junction in vivo, using a full-length PPV cDNA clone. PPV mutant transcripts containing a histidine for glutamine substitution in the cleavage site sequence (a change that abolishes in vitro processability) are able to infect Nicotiana clevelandii plants, indicating that normal processing at the P3-6K1 junction is not required for virus viability. However, disease symptoms were not detected and virus accumulation occurred after a second site mutation was introduced into the 6K1 cistron during replication. This additional change did not restore the in vitro processability of the mutant heptapeptide. Changes at other positions in the heptapeptide (that only slightly altered the in vitro processability of this NIa site) were also engineered and it was found that these mutations affected the time course and severity of the symptom induction process. A possible regulatory effect on the function of the potyvirus P3 + 6K1 protein by processing at the P3-6K1 junction is discussed in light of our present results with PPV.