The MYST histone acetyltransferases are essential for gametophyte development in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>Histone acetyltransferases (HATs) play critical roles in the regulation of chromatin structure and gene expression. Arabidopsis genome contains 12 HAT genes, but the biological functions of many of them are still unknown. In this work, we studied the evolutionary relationship and cellular functions of the two Arabidopsis HAT genes homologous to the MYST family members.</p> <p>Results</p> <p>An extensive phylogenetic analysis of 105 MYST proteins revealed that they can be divided into 5 classes, each of which contains a specific combination of protein modules. The two Arabidopsis MYST proteins, HAM1 and HAM2, belong to a "green clade", clearly separated from other families of HATs. Using a reverse genetic approach, we show that <it>HAM1 </it>and <it>HAM2 </it>are a functionally redundant pair of genes, as single Arabidopsis <it>ham1 </it>and <it>ham2 </it>mutants displayed a wild-type phenotype, while no double mutant seedling could be recovered. Genetic analysis and cytological study revealed that <it>ham1ham2 </it>double mutation induced severe defects in the formation of male and female gametophyte, resulting in an arrest of mitotic cell cycle at early stages of gametogenesis. RT-PCR experiments and the analysis of transgenic plants expressing the <it>GUS </it>reporter gene under the <it>HAM1 </it>or the <it>HAM2 </it>promoter showed that both genes displayed an overlapping expression pattern, mainly in growing organs such as shoots and flower buds.</p> <p>Conclusion</p> <p>The work presented here reveals novel properties for MYST HATs in Arabidopsis. In addition to providing an evolutionary relationship of this large protein family, we show the evidence of a link between MYST and gamete formation as previously suggested in mammalian cells. A possible function of the Arabidopsis MYST protein-mediated histone acetylation during cell division is suggested.</p

Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis

Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23 C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172- overexpressing plants showed a temperatureindependent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions

Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis

Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions

Regulation of High-Temperature Stress Response by Small RNAs

Temperature extremes constitute one of the most common environmental stresses that adversely affect the growth and development of plants. Transcriptional regulation of temperature stress responses, particularly involving protein-coding gene networks, has been intensively studied in recent years. High-throughput sequencing technologies enabled the detection of a great number of small RNAs that have been found to change during and following temperature stress. The precise molecular action of some of these has been elucidated in detail. In the present chapter, we summarize the current understanding of small RNA-mediated modulation of high- temperature stress-regulatory pathways including basal stress responses, acclimation, and thermo-memory. We gather evidence that suggests that small RNA network changes, involving multiple upregulated and downregulated small RNAs, balance the trade-off between growth/development and stress responses, in order to ensure successful adaptation. We highlight specific characteristics of small RNA-based tem- perature stress regulation in crop plants. Finally, we explore the perspectives of the use of small RNAs in breeding to improve stress tolerance, which may be relevant for agriculture in the near future

Intelligent Joystick Sensing the User's Emotion and Providing Biofeedback

Development of an intelligent joystick is proposed which senses the user’s bio-signals and recognises the user’s emotion. It provides biofeedback to the user as well as the user’s emotional state information to the computer allowing human-computer interaction over sensitive environment. While the user is interacting with a computer via a joystick the bio-signals can be collected through the user’s fingers touching it. The collected bio-signals information is mapped on a two-dimensional space to find out the quality and intensity of emotion continuously and in a real-time manner. The intelligent joystick has application within several fields such as healthcare, sport and game industries. In such cases, the user can be influenced, or suffer from medical problems while under stress during interaction with the machines. The intelligent joystick will provide feedback to the user and alert alarm about unhealthy conditions through the embedded actuators and allow the machine to adapt with the users’ emotional state

Rôle de l'acétylation des histones dans le contrôle de l'expression du génome d'Arabidopsis thaliana

L acétylation des histones apparaît comme un commutateur central de l inter-conversion entre l état permissif et répressif des domaines de la chromatine. L homéostasie de l acétylation des histones est assurée par les histones acétyltransférases (HAT) et les histones déacétylases (HDAC). Mais leurs rôles aux niveaux de la chromatine et de la régulation transcriptionnelle ou post-transcriptionnelle ne sont pas éclairci. Les objectifs de ce travail étaient d étudier le rôle de l acétylation des histones dans le contrôle de l expression de gènes chez Arabidopsis thaliana. Pour cela, des mutants HDACs et HATs ont été identifié et caractérisé. Tout d abord, AtGCN5 intervient, avec la voie des miARNs, aux niveaux transcriptionnel et post-transcriptionnel. Ceci indique que l acétylation/déacétylation des histones est un mécanisme épigénétique impliqué dans la régulation de la production de miARN. La caractérisation des mutations HDACs révèle que la mutation de AtHDA9 induit un phénotype de floraison précoce en conditions de jours courts. Ces caractéristiques s accompagnent d une sur-expression de gènes activateurs dans la voie de la transition florale. La mutation de AtHDA9 et AtSRT2 affecte également la méthylation de l ADN de la séquence péricentromérique répétée de 180 pb et du transposon AtSN1. Ces résultats révèlent les différents rôles joués par des HATs et HDACs individus dans le contrôle de l expression du génome d Arabidopsis.Histone acetylation appears as a central switch for interconversion between permissive and repressive state of chromatin domains. Homeostasis of histone acetylation is made sure by histones acetyltransferases (HAT) and histones desacetylases (HDAC). But their role on chromatin and transcriptional/posttranscriptional regulation was not clear. The objective of my work was to study the role of histone acetylation in the control of gene expression in Arabidopsis thaliana. For this reason, HAT and HDAC mutants had been identified and characterized. First of all, we show that AtGCN5 interfere, in the pathway of miRNA, on transcriptional and posttranscriptional regulation of gene. It indicates that histone acetylation/desacetylation is an epigenetic mechanism involved in the regulation of miRNA production. Characterization of the mutants reveled that AtHDA9 mutation induces a phenotype of early flowering in short days. This characteristics is associated with overexpression of activator genes in the pathway of flowering. AtHDA9 and AtSRT2 mutation affect also DNA méthylation of pericentromeric sequence repeat 180 bp and retroelement AtSN1. Our results reveal the different role of individual HAT and HDAC in the control of genome expression of Arabidopsis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

A fast, efficient chromatin immunoprecipitation method for studying protein-DNA binding in Arabidopsis mesophyll protoplasts

Abstract Background Binding of transcription factors to their target sequences is a primary step in the regulation of gene expression and largely determines gene regulatory networks. Chromatin immunoprecipitation (ChIP) is an indispensable tool used to investigate the binding of DNA-binding proteins (e.g., transcription factors) to their target sequences in vivo. ChIP assays require specific antibodies that recognize endogenous target transcription factors; however, in most cases, such specific antibodies are unavailable. To overcome this problem, many ChIP assays use transgenic plants that express epitope-tagged transcription factors and immunoprecipitate the protein with a tag-specific antibody. However, generating transgenic plants that stably express epitope-tagged proteins is difficult and time-consuming. Results Here, we present a rapid, efficient ChIP protocol using transient expression in Arabidopsis mesophyll protoplasts that can be completed in 4 days. We provide optimized experimental conditions, including the amount of transfected DNA and the number of protoplasts. We also show that the efficiency of our ChIP protocol using protoplasts is comparable to that obtained using transgenic Arabidopsis plants. We propose that our ChIP method can be used to analyze in vivo interactions between tissue-specific transcription factors and their target sequences, to test the effect of genotype on the binding of a transcription factor within a protein complex to its target sequences, and to measure temperature-dependent binding of a transcription factor to its target sequence. Conclusions The rapid and simple nature of our ChIP assay using Arabidopsis mesophyll protoplasts facilitates the investigation of in vivo interactions between transcription factors and their target genes

MOESM1 of A fast, efficient chromatin immunoprecipitation method for studying protein-DNA binding in Arabidopsis mesophyll protoplasts

Additional file 1: Figure S1. A full image of the western blot shown in Fig. 6b

The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 Module Regulates Ambient Temperature-Responsive Flowering via FLOWERING LOCUS T in Arabidopsis1[C][W][OA]

The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(−)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(−). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature