Transcriptional Regulation: a Genomic Overview

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

Intracompartmental and Intercompartmental Transcriptional Networks Coordinate the Expression of Genes for Organellar Functions

Genes for mitochondrial and chloroplast proteins are distributed between the nuclear and organellar genomes. Organelle biogenesis and metabolism, therefore, require appropriate coordination of gene expression in the different compartments to ensure efficient synthesis of essential multiprotein complexes of mixed genetic origin. Whereas organelle-to-nucleus signaling influences nuclear gene expression at the transcriptional level, organellar gene expression (OGE) is thought to be primarily regulated posttranscriptionally. Here, we show that intracompartmental and intercompartmental transcriptional networks coordinate the expression of genes for organellar functions. Nearly 1,300 ATH1 microarray-based transcriptional profiles of nuclear and organellar genes for mitochondrial and chloroplast proteins in the model plant Arabidopsis (Arabidopsis thaliana) were analyzed. The activity of genes involved in organellar energy production (OEP) or OGE in each of the organelles and in the nucleus is highly coordinated. Intracompartmental networks that link the OEP and OGE gene sets serve to synchronize the expression of nucleus- and organelle-encoded proteins. At a higher regulatory level, coexpression of organellar and nuclear OEP/OGE genes typically modulates chloroplast functions but affects mitochondria only when chloroplast functions are perturbed. Under conditions that induce energy shortage, the intercompartmental coregulation of photosynthesis genes can even override intracompartmental networks. We conclude that dynamic intracompartmental and intercompartmental transcriptional networks for OEP and OGE genes adjust the activity of organelles in response to the cellular energy state and environmental stresses, and we identify candidate cis-elements involved in the transcriptional coregulation of nuclear genes. Regarding the transcriptional regulation of chloroplast genes, novel tentative target genes of σ factors are identified

Diverse biological effects of glycosyltransferase genes from Tartary buckwheat

Background: Tartary buckwheat (Fagopyrum tataricum) is an edible cereal crop whose sprouts have been marketed and commercialized for their higher levels of anti-oxidants, including rutin and anthocyanin. UDP-glucose flavonoid glycosyltransferases (UFGTs) play an important role in the biosynthesis of flavonoids in plants. So far, few studies are available on UFGT genes that may play a role in tartary buckwheat flavonoids biosynthesis. Here, we report on the identification and functional characterization of seven UFGTs from tartary buckwheat that are potentially involved in flavonoid biosynthesis (and have varying effects on plant growth and development when overexpressed in Arabidopsis thaliana.) Results: Phylogenetic analysis indicated that the potential function of the seven FtUFGT proteins, FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, could be divided into three Arabidopsis thaliana functional subgroups that are involved in flavonoid biosynthesis of and anthocyanin accumulation. A significant positive correlation between FtUFGT8 and FtUFGT15 expression and anthocyanin accumulation capacity was observed in the tartary buckwheat seedlings after cold stress. Overexpression in Arabidopsis thaliana showed that FtUFGT8, FtUFGT15, and FtUFGT41 significantly increased the anthocyanin content in transgenic plants. Unexpectedly, overexpression of FtUFGT6, while not leading to enhanced anthocyanin accumulation, significantly enhanced the growth yield of transgenic plants. When wild-type plants have only cotyledons, most of the transgenic plants of FtUFGT6 had grown true leaves. Moreover, the growth speed of the oxFtUFGT6 transgenic plant root was also significantly faster than that of the wild type. At later growth, FtUFGT6 transgenic plants showed larger leaves, earlier twitching times and more tillers than wild type, whereas FtUFGT15 showed opposite results. Conclusions: Seven FtUFGTs were isolated from tartary buckwheat. FtUFGT8, FtUFGT15, and FtUFGT41 can significantly increase the accumulation of total anthocyanins in transgenic plants. Furthermore, overexpression of FtUFGT6 increased the overall yield of Arabidopsis transgenic plants at all growth stages. However, FtUFGT15 shows the opposite trend at later growth stage and delays the growth speed of plants. These results suggested that the biological function of FtUFGT genes in tartary buckwheat is diverse

Development and Application of Comparative Gene Co-expression Network Methods in Brachypodium distachyon

Gene discovery and characterization is a long and labor-intensive process. Gene co-expression network analysis is a long-standing powerful approach that can strongly enrich signals within gene expression datasets to predict genes critical for many cellular functions. Leveraging this approach with a large number of transcriptome datasets does not yield a concomitant increase in network granularity. Independently generated datasets that describe gene expression in various tissues, developmental stages, times of day, and environments can carry conflicting co-expression signals. The gene expression responses of the model C3 grass Brachypodium distachyon to abiotic stress is characterized by a co-expression-based analysis, identifying 22 modules of genes, annotated with putative DNA regulatory elements and functional terms. A great deal of co-expression elasticity is found among the genes characterized therein. An algorithm, dGCNA, designed to determine statistically significant changes in gene-gene co-expression relationships is presented. The algorithm is demonstrated on the very well-characterized circadian system of Arabidopsis thaliana, and identifies potential strong signals of molecular interactions between a specific transcription factor and putative target gene loci. Lastly, this network comparison approach based on edge-wise similarities is demonstrated on many pairwise comparisons of independent microarray datasets, to demonstrate the utility of fine-grained network comparison, rather than amassing as large a dataset as possible. This approach identifies a set of 182 gene loci which are differentially expressed under drought stress, change their co-expression strongly under loss of thermocycles or high-salinity stress, and are associated with cell-cycle and DNA replication functions. This set of genes provides excellent candidates for the generation of rhythmic growth under thermocycles in Brachypodium distachyon

Fonctions génomiques de la famille des facteurs de transcription AP2 chez les céréales

Les plantes sont exposées, à chaque étape de leur cycle de vie, à différents stress abiotiques notamment la sécheresse, le froid et la salinité. Bien que ces différents types de stress aient des effets spécifiques pour chacun d'entre eux, ils ont tous la capacité d'induire un effet commun soit le déficit hydrique qui affecte la croissance et le développement des plantes. Certaines plantes s'adaptent au déficit hydrique en modifiant leurs métabolismes ainsi que l'expression de leurs gènes afin de synchroniser leur développement avec les conditions de stress. Cette modification se traduit par la synthèse de nouvelles protéines, particulièrement régulée par le déficit hydrique, notamment les protéines de type Ap2/EREBP. Chez Arabidopsis thaliana, ces protéines forment une famille de facteurs de transcription de 145 membres. Certains membres jouent des rôles essentiels de régulateur dans plusieurs processus de développement tandis que d'autres sont impliqués dans les mécanismes de réponses des plantes aux stress. Chez les céréales, très peu de gènes Ap2/EREBP avec des fonctions connues ont été identifiés. Ce projet de recherche vise à identifier cette famille de gènes chez le blé hexaploid (Triticum aestivum L.). Le choix de ce type de blé comme modèle d'étude réside dans sa capacité de tolérance au gel et sa grande variabilité de réponses aux stress abiotiques. Le blé est la céréale la plus importante au monde puisqu'il représente environ 73% de la production céréalière mondiale. Il est cultivé dans divers types d'environnements climatiques, de productions et de fermes. De plus, il représente la principale source d'énergie alimentaire, d'emploi et de revenu dans plusieurs pays en voie de développement. Le blé agronomique est plus nutritif et économique qu'Arabidopsis. Cependant, le choix entre les deux espèces est influencé par la taille et la complexité de leurs génomes. Le premier chapitre décrit l'identification, la phylogénie et la localisation chromosomique des gènes Ap2/EREBP du blé. Une approche génomique combinant une recherche de bases de données de séquences suivie d'une analyse bioinformatique, un criblage de banques d'ADNc et des amplifications par réaction de polymérisation en chaîne a permis d'identifier 107 ADNc du blé qui codent potentiellement pour des facteurs de transcription de type Ap2/EREBP. Leur localisation chromosomique montre que ces gènes sont dispersés sur tout le génome du blé. Cependant, quelques gènes de cette famille, appelés CBFs (C-repeat Binding Factors), sont regroupés principalement sur le long bras du groupe de chromosomes 5 et ont été associés directement à des déterminants génétiques (QTLs) contrôlant les réponses aux stress et divers aspects du développement floral. Le deuxième chapitre présente la caractérisation de la sous-famille de gènes CBFs. L'analyse phylogénétique a indiqué que les espèces de blé contiennent au moins 23 gènes différents de type CBF. Chez les Poaceae, les CBFs sont classés dans 10 groupes qui partagent une origine phylogénétique commune et des caractéristiques structurales semblables. Six de ces groupes (3C, 3D, 4A, 4B, 4C et 4D) sont trouvés seulement chez les Pooideae, suggérant ainsi qu'ils représentent les mécanismes de réponse de CBF qui ont évolué récemment pendant la colonisation des habitats tempérés. Les études menées sur le profil d'expression de ces gènes démontrent que 5 des groupes spécifiques aux Pooideae montrent une expression constitutive élevée et une expression inductible par les basses températures chez le cultivar d'hiver. Par contre à des températures modérées, la régulation de l'expression varie en fonction de la période de la journée. L'expression inductible et non héritée au sein des groupes de CBF a possiblement joué un rôle prédominant dans l'aptitude des cultivars d'hiver à tolérer les basses températures et elle est probablement à la base de la variabilité génétique de la tolérance au gel chez les Pooideae. Le troisième article décrit l'identification de deux gènes chez le blé, ICE1-like (Inducteur de l'expression de CBF 1) codant pour un facteur de transcription qui régule l'expression des gènes CBF. TaICE87 et TaICE41 codent pour un activateur de transcription de type MYC-like bHLH. TaICE87 et TaICE41 se lient spécifiquement à différentes séquences de reconnaissance MYC du promoteur de TaCBFIVd-B9. TaICE87 and TaICE41 sont constitutivement exprimés chez le blé et leur surexpression chez Arabidopsis mène à l'augmentation du niveau d'expression des gènes AtCOR et AtCBF3 et de la tolérance des plantes au gel. Ces résultats suggèrent que TaICE87 and TaICE41 sont des orthologues fonctionnels d'AtICE1 et peuvent réguler la transcription d'AtCBF3. La structure complexe des éléments MYC au niveau des promoteurs CBF et la différence d'affinité entre TalCE87 et TalCE41 pour les éléments MYC suggèrent que ces 2 protéines peuvent différentiellement activer CBF chez le blé. En résumé, le clonage de la famille de gènes Ap2/EREBP chez le blé a permis d'identifier et de caractériser les membres CBF régulés par les basses températures. De plus, les résultats de cette étude démontrent que l'expression des gènes CBF est amplifiée chez les Pooideae, suggérant ainsi un rôle plausible dans la tolérance au froid des cultivars d'hiver. L'analyse des gènes CBFs pourrait nous conduire à améliorer la tolérance au gel chez le blé, et par conséquent faciliter l'établissement de nouvelles stratégies visant à améliorer la productivité des céréales et leur adaptation aux changements climatiques. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Blé, Acclimatation au froid, Tolérance au gel, Facteurs de transcription, Ap2/EREBP

Germin and germin-like proteins: evolution, structure, and function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily

Isolation and functional characterization of cold-regulated promoters, by digitally identifying peach fruit cold-induced genes from a large EST dataset

<p>Abstract</p> <p>Background</p> <p>Cold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset.</p> <p>Results</p> <p>Digital expression analyses of EST datasets, revealed 164 cold-induced peach genes, several of which show similarities to genes associated with cold acclimation and cold stress responses. The promoters of three of these cold-inducible genes (<it>Ppbec1</it>, <it>Ppxero2 </it>and <it>Pptha1</it>) were fused to the GUS reporter gene and characterized for cold-inducibility using both transient transformation assays in peach fruits (<it>in fruta</it>) and stable transformation in <it>Arabidopsis thaliana</it>. These assays demonstrate that the promoter <it>Pptha1 </it>is not cold-inducible, whereas the <it>Ppbec1 and Ppxero2 </it>promoter constructs are cold-inducible.</p> <p>Conclusion</p> <p>This work demonstrates that during cold storage, peach fruits differentially express genes that are associated with cold acclimation. Functional characterization of these promoters in transient transformation assays <it>in fruta </it>as well as stable transformation in Arabidopsis, demonstrate that the isolated <it>Ppbec1 </it>and <it>Ppxero2 </it>promoters are cold-inducible promoters, whereas the isolated <it>Pptha1 </it>promoter is not cold-inducible. Additionally, the cold-inducible activity of the <it>Ppbec1 </it>and <it>Ppxero2 </it>promoters suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. These results reveal that digital expression analyses may be used in non-model species to identify candidate genes whose promoters are differentially expressed in response to exogenous stimuli.</p

RAP2.4c and RAP2.4d in the regulation of cold stress and cold priming in Arabidopsis thaliana

Cold stress is a major factor restricting plant performance and fitness. Depending on the length and intensity of the cold stimulus, plants respond to the stress event by alleviating immediately occurring adverse effects of cold stress and by inducing cold acclimation. In the case of a timely limited cold stimulus, Arabidopsis thaliana responds with the formation of a cold memory that modifies its response to a future cold stimulus, a phenomenon which is called priming. Short-term cold priming was previously shown to attenuate chloroplast to nucleus signalling in the regulation of cold-induced ZAT10 expression. In the present study, this effect was mimicked by transient overexpression of tAPX, but not of sAPX at 20 °C, while counteracting priming-induced tAPX accumulation during the lag-phase abolished the priming effect. This demonstrated that cold priming is mediated at the thylakoid membrane and is regulated by post-priming tAPX expression. Electrolyte leakage assays demonstrated that the AP2/ERF-Ib transcription factors RAP2.4c and RAP2.4d, that have been proposed to regulate chloroplast APX gene expression, negatively regulate the direct cold response and cold acclimation. RNAseq analysis of rap2.4c and rap2.4d KO plants one hour after transfer to 4 °C revealed stronger induction of genes that are involved in JA/ET, JA and SA signalling pathways than in wild type Col-0 pants. Subsequent analyses did not show differences in hormone contents and in sensitivity to hormone signals in rap2.4c and rap2.4d plants, demonstrating that misregulation of gene expression in the knock-out lines is independent of hormone availability and sensing. Transient overexpression of RAP2.4c and RAP2.4d also did not influence hormone-related transcript levels. Additionally, the lack of RAP2.4c and RAP2.4d did not affect cold priming-dependent attenuation of ZAT10 regulation. The similarities of the expression patterns of rap2.4c and rap2.4d with that of an npr1 mutant in the cold indicate an upstream function of both transcription factors in NPR1-mediated gene expression regulation. A putative target is TRXH5, which is involved in quaternary structure regulation and, consequently, nuclear translocation of NPR1. This gene was strongly upregulated in the rap2.4d line. Besides a DRE-motif, which is a known RAP2.4d binding site, the TRXH5 promoter contains a RAP2.4d binding motif, which was identified in the present study based on a Yeast-One-Hybrid screen with RAP2.4d and random genomic DNA fragments of Arabidopsis thaliana. The present study highlights RAP2.4d, and to a lesser extent RAP2.4c, as cold-inducible inhibitors of TRXH5 expression attenuating the NPR1-mediated induction of JA/ET, JA and SA responses in the early cold response

Characterization of six Arabidopsis AROGENATE DEHYDRATASE promoters

Phenylalanine is an important aromatic amino acid synthesized by higher plants, and is a major component of numerous specialized metabolites including structural components, pigments, and defense compounds. The last step in the synthesis of phenylalanine is catalyzed by an enzyme called AROGENATE DEHYDRATASE, of which there are six different isoenzymes encoded by the Arabidopsis genome. All six have specialized roles within the plant, and are differentially expressed during development and under stressful conditions. To deduce the specialized role of each ADT, unique patterns of regulatory motifs were identified for all six ADT promoters, as well as corresponding transcription factors with similar expression profiles to each enzyme. Seven stable transgenic Arabidopsis lines were also generated using ADT promoter-eGFP/GUS constructs to test expression in all tissues during development, and under stressful conditions

TRANSCRIPTIONAL REGULATION OF SPECIALIZED METABOLITES IN \u3cem\u3eARABIDOPSIS THALIANA\u3c/em\u3e AND \u3cem\u3eCATHARANTHUS ROSEUS\u3c/em\u3e

For millennia humans have utilized plant specialized metabolites for health benefits, fragrances, poisons, spices, and medicine. Valued metabolites are often produced in small quantities and may command high prices. Understanding when and how the plant synthesizes these compounds is important for improving their production. Phytohormone signaling cascades, such as jasmonate (JA) activate or repress transcription factors (TF) controlling expression of metabolite biosynthetic genes. TFs regulating specialized metabolite biosynthetic genes can be manipulated to engineer plants with increased metabolite production. WRKY transcription factor are known components of both JA signaling cascades and regulation of specialized metabolism. The presence of WRKY binding sites in promoters of several terpene indole alkaloids suggested their involvement in regulating biosynthesis of these compounds. A phylogenetic analysis was used to compare Arabidopsis and Catharanthus WRKY TFs families. Gene expression analysis identified WRKY TFs induced by JA in both Arabidopsis and Catharanthus, providing candidates for future characterization. WRKY TFs suggest a possible conserved regulatory network of TFs downstream of JA signaling cascades. The origin and conservation of JA signaling in plants remain ambiguous. Identification of the first algal TIFY factor helped determine when JA signaling appeared. The charophyte, Klebsormidium flaccidum does not possess genes encoding key green-plant JA signaling components, including CORONATINE INSENSTIVE1, JASMONATE-ZIM DOMAIN, NOVEL INTERACTOR OF JAZ, and the JAZ-interacting bHLH factors, yet their orthologs are present in the moss. A molecular clock analysis dated the evolution of JA signaling evolution to during the early Ediacaran to late Cambrian periods 628 to 491 million years ago – a time corresponding to rapid diversification of animal predators. The plant Mediator complex is a core component of gene expression. Conservation of the MED25 subunit in plants, and its known involvement in JA signaling implicates this factor in regulation of specialized metabolism. MED25 is involved in anthocyanin accumulation, but how it functions remains unknown. Characterization of MED25 in Arabidopsis revealed it interacts with the transcription factor GL3 as well as the JAZ1 repressor. Importantly, the interaction of JAZ1 with MED25 reveals a new mechanism by which JAZ proteins regulate gene expression, improving our understanding of JA signaling