Exploration of plant genomes in the FLAGdb++ environment

Background : In the contexts of genomics, post-genomics and systems biology approaches, data integration presents a major concern. Databases provide crucial solutions: they store, organize and allow information to be queried, they enhance the visibility of newly produced data by comparing them with previously published results, and facilitate the exploration and development of both existing hypotheses and new ideas. Results : The FLAGdb++ information system was developed with the aim of using whole plant genomes as physical references in order to gather and merge available genomic data from in silico or experimental approaches. Available through a JAVA application, original interfaces and tools assist the functional study of plant genes by considering them in their specific context: chromosome, gene family, orthology group, co-expression cluster and functional network. FLAGdb++ is mainly dedicated to the exploration of large gene groups in order to decipher functional connections, to highlight shared or specific structural or functional features, and to facilitate translational tasks between plant species (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Vitis vinifera). Conclusion : Combining original data with the output of experts and graphical displays that differ from classical plant genome browsers, FLAGdb++ presents a powerful complementary tool for exploring plant genomes and exploiting structural and functional resources, without the need for computer programming knowledge. First launched in 2002, a 15th version of FLAGdb++ is now available and comprises four model plant genomes and over eight million genomic features

Transcriptional Regulation of Ribosome Components Are Determined by Stress According to Cellular Compartments in Arabidopsis thaliana

Plants have to coordinate eukaryotic ribosomes (cytoribosomes) and prokaryotic ribosomes (plastoribosomes and mitoribosomes) production to balance cellular protein synthesis in response to environmental variations. We identified 429 genes encoding potential ribosomal proteins (RP) in Arabidopsis thaliana. Because cytoribosome proteins are encoded by small nuclear gene families, plastid RP by nuclear and plastid genes and mitochondrial RP by nuclear and mitochondrial genes, several transcriptional pathways were attempted to control ribosome amounts. Examining two independent genomic expression datasets, we found two groups of RP genes showing very different and specific expression patterns in response to environmental stress. The first group represents the nuclear genes coding for plastid RP whereas the second group is composed of a subset of cytoribosome genes coding for RP isoforms. By contrast, the other cytoribosome genes and mitochondrial RP genes show less constraint in their response to stress conditions. The two subsets of cytoribosome genes code for different RP isoforms. During stress, the response of the intensively regulated subset leads to dramatic variation in ribosome diversity. Most of RP genes have same promoter structure with two motifs at conserved positions. The stress-response of the nuclear genes coding plastid RP is related with the absence of an interstitial telomere motif known as telo box in their promoters. We proposed a model for the “ribosome code” that influences the ribosome biogenesis by three main transcriptional pathways. The first pathway controls the basal program of cytoribosome and mitoribosome biogenesis. The second pathway involves a subset of cytoRP genes that are co-regulated under stress condition. The third independent pathway is devoted to the control of plastoribosome biosynthesis by regulating both nuclear and plastid genes

Expression variation in connected recombinant populations of Arabidopsis thaliana highlights distinct transcriptome architectures

<p>Abstract</p> <p>Background</p> <p>Expression traits can vary quantitatively between individuals and have a complex inheritance. Identification of the genetics underlying transcript variation can help in the understanding of phenotypic variation due to genetic factors regulating transcript abundance and shed light into divergence patterns. So far, only a limited number of studies have addressed this subject in Arabidopsis, with contrasting results due to dissimilar statistical power. Here, we present the transcriptome architecture in leaf tissue of two RIL sets obtained from a connected-cross design involving 3 commonly used accessions. We also present the transcriptome architecture observed in developing seeds of a third independent cross.</p> <p>Results</p> <p>The utilisation of the novel R/eqtl package (which goal is to automatize and extend functions from the R/qtl package) allowed us to map 4,290 and 6,534 eQTLs in the Cvi-0 × Col-0 and Bur-0 × Col-0 recombinant populations respectively. In agreement with previous studies, we observed a larger phenotypic variance explained by eQTLs in linkage with the controlled gene (potentially <it>cis</it>-acting), compared to distant loci (acting necessarily indirectly or in <it>trans</it>). Distant eQTLs hotspots were essentially not conserved between crosses, but instead, cross-specific. Accounting for confounding factors using a probabilistic approach (VBQTL) increased the mapping resolution and the number of significant associations. Moreover, using local eQTLs obtained from this approach, we detected evidence for a directional allelic effect in genes with related function, where significantly more eQTLs than expected by chance were up-regulated from one of the accessions. Primary experimental data, analysis parameters, eQTL results and visualisation of LOD score curves presented here are stored and accessible through the QTLstore service database <url>http://qtlstore.versailles.inra.fr/</url>.</p> <p>Conclusions</p> <p>Our results demonstrate the extensive diversity and moderately conserved eQTL landscape between crosses and validate the utilisation of expression traits to explore for candidates behind phenotypic variation among accessions. Furthermore, this stresses the need for a wider spectrum of diversity to fully understand expression trait variation within a species.</p

TC-motifs at the TATA-box expected position in plant genes: a novel class of motifs involved in the transcription regulation

<p>Abstract</p> <p>Background</p> <p>The TATA-box and TATA-variants are regulatory elements involved in the formation of a transcription initiation complex. Both have been conserved throughout evolution in a restricted region close to the Transcription Start Site (TSS). However, less than half of the genes in model organisms studied so far have been found to contain either one of these elements. Indeed different core-promoter elements are involved in the recruitment of the TATA-box-binding protein. Here we assessed the possibility of identifying novel functional motifs in plant genes, sharing the TATA-box topological constraints.</p> <p>Results</p> <p>We developed an <it>ab-initio </it>approach considering the preferential location of motifs relative to the TSS. We identified motifs observed at the TATA-box expected location and conserved in both <it>Arabidopsis thaliana </it>and <it>Oryza sativa </it>promoters. We identified TC-elements within non-TA-rich promoters 30 bases upstream of the TSS. As with the TATA-box and TATA-variant sequences, it was possible to construct a unique distance graph with the TC-element sequences. The structural and functional features of TC-element-containing genes were distinct from those of TATA-box- or TATA-variant-containing genes. <it>Arabidopsis thaliana </it>transcriptome analysis revealed that TATA-box-containing genes were generally those showing relatively high levels of expression and that TC-element-containing genes were generally those expressed in specific conditions.</p> <p>Conclusions</p> <p>Our observations suggest that the TC-elements might constitute a class of novel regulatory elements participating towards the complex modulation of gene expression in plants.</p

A strategy for the identification of new abiotic stress determinants in arabidopsis using web-based data mining and reverse genetics

Since the sequencing of the Arabidopsis thaliana genome in 2000, plant researchers have faced the complex challenge of assigning function to thousands of genes. Functional discovery by in silico prediction or homology search resolved a significant number of genes, but only a minor part has been experimentally validated. Arabidopsis entry into the post-genomic era signified a massive increase in high-throughput approaches to functional discovery, which have since become available through publicly-available web-based resources. The present work focuses on an easy and straightforward strategy that couples data-mining to reverse genetics principles, to allow for the identification of new abiotic stress determinant genes. The strategy explores systematic microarray-based transcriptomics experiments, involving Arabidopsis abiotic stress responses. An overview of the most significant resources and databases for functional discovery in Arabidopsis is presented. The successful application of the outlined strategy is illustrated by the identification of a new abiotic stress determinant gene, HRR, which displays a heat stress-related phenotype after a loss-of-function reverse genetics approach.No competing financial interests exist. The present work was supported by Foundation for Science and Technology (POCTI/AGR/45462/2002). H. Azevedo (SFRH/BPD/17198/2004), J. Correia (SFRH/BD/16663/2004), J. Oliveira (SFRH/BD/38379/2007), S. Laranjeira (SFRH/BD/29778/2006), C. Barbeta (SFRH/BD/12081/2003) and V. Amorim-Silva (SFRH/BD/29778/2006) were supported by Foundation for Science and Technology

ATM-Mediated Transcriptional and Developmental Responses to γ-rays in Arabidopsis

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of γ-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed

Conservation of Salmonella Infection Mechanisms in Plants and Animals

Salmonella virulence in animals depends on effectors injected by Type III Secretion Systems (T3SSs). In this report we demonstrate that Salmonella mutants that are unable to deliver effectors are also compromised in infection of Arabidopsis thaliana plants. Transcriptome analysis revealed that in contrast to wild type bacteria, T3SS mutants of Salmonella are compromised in suppressing highly conserved Arabidopsis genes that play a prominent role during Salmonella infection of animals. We also found that Salmonella originating from infected plants are equally virulent for human cells and mice. These results indicate a high degree of conservation in the defense and infection mechanism of animal and plant hosts during Salmonella infection

Gene expression variation and buffering mechanisms in Arabidopsis thaliana

Non-genetic variability in gene expression is an inevitable consequence of the stochastic nature of processes driving transcription and translation, as well as the epigenetic modifications of the genome. This phenomenon has been observed in both unicellular and multicellular organisms. Largely thought to be deleterious to cell fitness, it is not uniform across the transcriptome. This implies the existence of mechanisms regulating expression variability, although they, and the role played by inter-individual expression variability, remain poorly researched in multicellular systems. I utilised multiple Arabidopsis thaliana time series expression datasets to identify variable genes and analyse their cellular functions. I show that variable genes are enriched for Gene Ontology terms related to biotic and abiotic stress response and that, inversely, low variability genes are enriched for housekeeping terms. Moreover, I also investigated DNA methylation as a potential mechanism buffering expression variability by analysing methylation of Arabidopsis genes and promoters, and by comparing wild type plants with CG methylation reduction and CG methylation loss methyltransferase mutant specimens. I found that variable genes are less methylated in the CG context in wild type Arabidopsis. Loss of CG methylation alters expression variability of some genes. Of those, significantly greater portion of genes gained variability, compared to those that lost it. These results are an important step towards greater understanding of these processes in multicellular organisms, and their role

Protein subcellular trafficking during the oxidative stress response in plants

Due to their sessile lifestyle, plants are permanently exposed to a variety of adverse environmental conditions leading to the accumulation of reactive oxygen species (ROS). Although initially considered as harmful byproducts of aerobic metabolism reacting in high concentrations with all cellular components, ROS are generally accepted as key signaling molecules that coordinate a broad range of environmental and developmental processes. At the beginning of my Ph.D., significant progress has been made in the description of oxidative stressdependent gene expression, but the regulation of the complex ROS signal transduction network remains largely unknown. The functional analysis of proteins that are encoded by genes that rapidly respond to ROS can therefore give novel insights into the early signaling steps triggered by a sudden increase in intracellular ROS. During adverse environmental conditions, the regulation of dynamic protein trafficking is an important intracellular signaling strategy to elicit a fast defense response. The aim of this project was to identify and characterize proteins that relocalize during oxidative stress. Therefore, we first made a comprehensive inventory of hydrogen peroxide (H2O2)-induced genes by comparing H2O2-related expression data sets and selected in a well-considered manner 85 candidate genes for further functional studies. We focussed our selection on genes encoding transcription factors and proteins of unknown function. To identify proteins with a potential dynamic behaviour during oxidative stress, we employed two medium-throughput localization screens of green fluorescent protein (GFP)-tagged proteins by transient expression in Nicotiana benthamiana and by stable expression in transgenic Arabidopsis thaliana lines. Transgenic Arabidopsis plants with perturbed levels of interesting candidate genes were assayed for altered tolerance to abiotic and biotic stress. The identification of proteins that dynamically relocalize during stress conditions, together with a detailed understanding of the mechanisms behind the identified oxidative stress-induced relocalizations, will provide a better understanding of stress response signaling

Molecular mechanisms associated to thermotolerance in plants

Tese de doutoramento em CiênciasOne of the most typical abiotic stresses encountered by plants is extreme temperatures. High temperature leads to a series of morphological, physiological and molecular alterations that adversely affect plant growth and productivity. Acquisition of thermotolerance is largely controlled through molecular mechanisms based on the activation and regulation of specific stress‐related genes. The elucidation of these gene/protein functions will give insights on the various mechanisms of plant response to heat stress, providing useful information to improve plant thermotolerance. The present work aims to contribute for the understanding of the molecular mechanisms that are responsible for plant adaptation to heat stress. Two species, Populus euphratica Oliv. and Arabidopsis thaliana L., were used as models due to the latest development of genomic and molecular biology resources and tools for both plants. P. euphratica is naturally found under severe conditions such as extreme temperatures (‐45°C to +54°C), high soil salinity and drought. The physiological response of P. euphratica cultured cells was evaluated at different temperatures. Contrasting with its innate enhanced tolerance to extreme temperatures, the in vitro system did not present an outstanding tolerance capacity. P. euphratica suspended cells heat‐shocked for 20 min were able to tolerate temperatures up to 45°C. Heat‐associated events as PCD and ROS production were suggested not to be implicated in the occurring cell death. Supported by the use of publicly available A. thaliana expression data and other webbased tools and resources, a reverse genetics strategy was followed for the identification of novel determinants for heat stress tolerance (HZF and HRR). In silico analysis revealed that both genes putatively encode effector proteins involved in different stages of the heat stress response. Moreover, HZF and HRR were found to be co‐regulated with genes already implicated in the regulation of heat responses. Functional characterization of HZF was primarily supported by the use of several web‐based tools and resources specifically created for Arabidopsis functional analysis. HZF was found to be a zinc finger family protein containing a conserved C3H2C3‐type RING domain and its possible role as E3 ubiquitin ligase was suggested. To pursue with reverse genetics approaches for identifying heat stress‐associated mutations, a phenotypic analysis based on germination and seedling survival assays was proposed. Temperatures and periods of treatment were diversely combined to test basal thermotolerance in either seeds or 7‐day‐old seedlings, or acquired thermotolerance only in 7‐ day‐old seedlings. The effectiveness of the proposed protocols was illustrated by detection of heat‐associated phenotypes in two mutants (hot1‐3 and atrbohD) previously identified to be thermotolerance defective. Regarding germination assays, special attention should be given to the time‐course evaluation of the number of germinated seeds for an accurate phenotypic detection. A delayed germination was observed in hzf mutant seeds in the following days after heat treatment when compared to wild‐type seeds, suggesting a role for HZF in the transition from dormant to germinating state. HZF was then suggested to mediate the ubiquitination of a regulator protein implicated in promoting seed dormancy or repressing germination upon heat stress. This function seems to be mainly assured by a redundant gene product (L‐HZF) under standard conditions, since similar germination timing was observed for hzf and wild‐type seeds. Maximum HZF transcript accumulation in heat‐treated (38°C for 1 h) wild‐type seedlings was achieved 15 min after heat treatment, suggesting also the HZF involvement in the initial phase of heat stress response. Expression vectors suitable for overexpression studies and in situ analysis were constructed and used to transform wild‐type Arabidopsis plants. The transgenic T3 plants will be soon available for further experiments that will contribute to elucidate the specific role of HZF in thermotolerance. The complete functional characterization of HZF, currently in progress, will provide novel information that would contribute to the dissection of its particular role in plant thermotolerance.A temperatura extrema constitui um dos principais factores de stresse abiótico a que as plantas estão frequentemente sujeitas. A exposição a temperaturas elevadas resulta em alterações profundas do metabolismo, executadas a nível morfológico, fisiológico e molecular, que prejudicam o desenvolvimento e produtividade vegetal. A aquisição de termotolerância é essencialmente controlada por mecanismos moleculares, os quais incluem a activação e regulação de genes específicos associados ao stresse. A elucidação da função desses genes/proteínas irá contribuir para a compreensão dos mecanismos de resposta das plantas que permitem a aquisição de tolerância a temperaturas elevadas. O conhecimento desses mecanismos poderá ainda ser utilizado na implementação de estratégias para o aumento da termotolerância em plantas. O presente trabalho pretende contribuir para o conhecimento dos mecanismos moleculares responsáveis pela adaptação ao stresse térmico em plantas. Duas espécies, Populus euphratica Oliv. e Arabidopsis thaliana L., foram utilizadas como modelos, devido ao recente desenvolvimento de recursos e ferramentas genómicas e de biologia molecular. P. euphratica encontra‐se distribuída em ambientes adversos, designadamente apresentando temperaturas extremas (‐45°C a +54°C), elevada salinidade e secura. Neste trabalho, procedeu‐se à caracterização fisiológica de células em suspensão de P. euphratica a diferentes temperaturas. Contrariamente à elevada termotolerância inata, o sistema in vitro não apresentou termotolerância significativa. As células em suspensão de P. euphratica, sujeitas a stresse térmico durante 20 min, atingiram um máximo de tolerância para temperaturas inferiores a 45°C. A avaliação de processos associados à resposta a temperaturas elevadas, como a morte celular programada ou a produção de espécies reactivas de oxigénio, sugeriu que estes não se encontram implicados na morte celular observada. Fazendo uso de informação sobre expressão genética e outras ferramentas e recursos disponibilizados publicamente para a espécie A. thaliana, foi efectuada a identificação de novos determinantes para a tolerância ao stresse térmico (HZF e HRR). A análise in silico revelou que ambos os genes possivelmente codificam proteínas efectoras envolvidas em diferentes estádios da resposta ao stresse térmico. A análise de genes que apresentam co-expressão com HZF e HRR permitiu identificar outros genes implicados na regulação de respostas a temperaturas elevadas. A caracterização funcional do gene HZF foi inicialmente suportada pelo uso de ferramentas e recursos disponíveis on‐line especialmente concebidos para análise funcional em Arabidopsis. HZF foi identificado como sendo uma proteína da família zinc finger, que possui um domínio conservado do tipo RING C3H2C3, tendo sido sugerida a sua possível função como E3 ubiquitina ligase. De forma a prosseguir a metodologia de genética inversa, para a identificação de mutações associadas ao stresse térmico, foi proposta uma análise fenotípica baseada em ensaios de germinação e viabilidade de plântulas de A. thaliana. Os protocolos utilizados abrangeram uma combinação de temperaturas e tempos de tratamento térmico, para avaliar a termotolerância basal em sementes e plântulas com 7 dias, ou a termotolerância adquirida em plântulas com 7 dias. A eficácia dos protocolos foi demonstrada através da identificação de fenótipos associados ao calor em dois mutantes (hot1‐3 e atrbohD), cuja termotolerância reduzida tinha sido previamente descrita. No que se refere aos ensaios de germinação, foi sugerida a avaliação do número de sementes germinadas ao longo do tempo para uma detecção fenotípica mais precisa. Quando comparadas com as sementes da estirpe selvagem, as sementes do mutante hzf exibiram atraso na germinação nos dias seguintes ao tratamento térmico, sugerindo o envolvimento de HZF na transição na fase de dormência para o estado germinativo. A proteína HZF foi então sugerida como mediadora da ubiquitinação de uma proteína reguladora implicada na manutenção da dormência ou repressão da germinação em condições de stresse térmico. Esta função parece ser principalmente assegurada por uma outra proteína com função redundante (L‐HZF) em condições normais, dado que o tempo de germinação observado para as sementes hzf e selvagem foi semelhante. A máxima acumulação de transcriptos de HZF em plântulas selvagem sujeitas a stress térmico (38°C durante 1 h) foi observada 15 min após tratamento, o que sugeriu também o envolvimento de HZF na fase inicial da resposta ao stresse térmico. Vectores de expressão apropriados para estudos de sobre‐expressão e análise in situ foram concebidos e utilizados na transformação de plântulas selvagem de A. thaliana. As plantas transgénicas T3 estarão brevemente disponíveis para ensaios suplementares que irão contribuir para esclarecer a função específica de HZF na termotolerância. A caracterização funcional completa do gene HZF, actualmente em curso, irá fornecer informação adicional que poderá contribuir para a elucidação do seu papel particular na termotolerância de plantas.Fundação para a Ciência e a Tecnologia (FCT) – Bolsa de Doutoramento SFRH/BD/16663/2004 e POCTI/AGR/45462/2002 “Isolamento e clonagem de genes associados à tolerância de Populus euphratica a condições de temperaturas extremas