Chromatin dynamics associated with seed desiccation tolerance/sensitivity at early germination in Medicago truncatula.

Desiccation tolerance (DT) has contributed greatly to the adaptation of land plants to severe water-deficient conditions. DT is mostly observed in reproductive parts in flowering plants such as seeds. The seed DT is lost at early post germination stage but is temporally re-inducible in 1 mm radicles during the so-called DT window following a PEG treatment before being permanently silenced in 5 mm radicles of germinating seeds. The molecular mechanisms that activate/reactivate/silence DT in developing and germinating seeds have not yet been elucidated. Here, we analyzed chromatin dynamics related to re-inducibility of DT before and after the DT window at early germination in Medicago truncatula radicles to determine if DT-associated genes were transcriptionally regulated at the chromatin levels. Comparative transcriptome analysis of these radicles identified 948 genes as DT re-induction-related genes, positively correlated with DT re-induction. ATAC-Seq analyses revealed that the chromatin state of genomic regions containing these genes was clearly modulated by PEG treatment and affected by growth stages with opened chromatin in 1 mm radicles with PEG (R1P); intermediate openness in 1 mm radicles without PEG (R1); and condensed chromatin in 5 mm radicles without PEG (R5). In contrast, we also showed that the 103 genes negatively correlated with the re-induction of DT did not show any transcriptional regulation at the chromatin level. Additionally, ChIP-Seq analyses for repressive marks H2AK119ub and H3K27me3 detected a prominent signal of H3K27me3 on the DT re-induction-related gene sequences at R5 but not in R1 and R1P. Moreover, no clear H2AK119ub marks was observed on the DT re-induction-related gene sequences at both developmental radicle stages, suggesting that silencing of DT process after germination will be mainly due to H3K27me3 marks by the action of the PRC2 complex, without involvement of PRC1 complex. The dynamic of chromatin changes associated with H3K27me3 were also confirmed on seed-specific genes encoding potential DT-related proteins such as LEAs, oleosins and transcriptional factors. However, several transcriptional factors did not show a clear link between their decrease of chromatin openness and H3K27me3 levels, suggesting that their accessibility may also be regulated by additional factors, such as other histone modifications. Finally, in order to make these comprehensive genome-wide analyses of transcript and chromatin dynamics useful to the scientific community working on early germination and DT, we generated a dedicated genome browser containing all these data and publicly available at https://iris.angers.inrae.fr/mtseedepiatlas/jbrowse/?data=Mtruncatula.

Chromatin Immunoprecipitation dataset of H3ac and H3K27me3 histone marks followed by DNA sequencing of Medicago truncatula embryos during control and heat stress conditions to decipher epigenetic regulation of desiccation tolerance acquisition.

Desiccation tolerance (DT) is one of the most important processes that seeds need to acquire during seed maturation because it will ensure survival until seeds have favourable conditions for germinating. Moreover, in the current climate warming context, heat stress and its impact on seed maturation and quality has been increasingly studied by the scientific community. Even if the transcriptomic changes enrolled in DT acquisition and seed heat stress response are fairly known, its epigenetic control has not yet been investigated. Medicago truncatula is a model legume for studying seed molecular mechanisms, which is known to display a delay in the acquisition of seed maturation mechanisms under heat conditions, except for desiccation acquisition. Our aim was to evaluate the role of two histone marks during embryo development under control and heat stress conditions on seed maturation processes, including the DT acquisition. These histone marks have either repressive (H3K27me3) or inducible (H3ac) effects on gene transcription, respectively corresponding to markers of packed and accessible chromatins. We identified all genomic regions bound to the H3K27me3 histones at four developmental stages and to the H3ac histones at the two earlier developmental stages during seed maturation, from seed filling to mature dry seeds, collected under optimal and heat stress conditions in the model legume, Medicago truncatula (reference genotype A17). A list of genes and promoters potentially linked to these two histone marks is reported and could provide clues about the epigenetic regulation of seed maturation between control and heat stress conditions, including the desiccation tolerance acquisition.

Regulation of DNA (de)Methylation Positively Impacts Seed Germination during Seed Development under Heat Stress.

Seed development needs the coordination of multiple molecular mechanisms to promote correct tissue development, seed filling, and the acquisition of germination capacity, desiccation tolerance, longevity, and dormancy. Heat stress can negatively impact these processes and upon the increase of global mean temperatures, global food security is threatened. Here, we explored the impact of heat stress on seed physiology, morphology, gene expression, and methylation on three stages of seed development. Notably, Arabidopsis Col-0 plants under heat stress presented a decrease in germination capacity as well as a decrease in longevity. We observed that upon mild stress, gene expression and DNA methylation were moderately affected. Nevertheless, upon severe heat stress during seed development, gene expression was intensively modified, promoting heat stress response mechanisms including the activation of the ABA pathway. By analyzing candidate epigenetic markers using the mutants' physiological assays, we observed that the lack of DNA demethylation by the ROS1 gene impaired seed germination by affecting germination-related gene expression. On the other hand, we also observed that upon severe stress, a large proportion of differentially methylated regions (DMRs) were located in the promoters and gene sequences of germination-related genes. To conclude, our results indicate that DNA (de)methylation could be a key regulatory process to ensure proper seed germination of seeds produced under heat stress.

The 5'-3' Exoribonuclease XRN4 Regulates Auxin Response via the Degradation of Auxin Receptor Transcripts.

Auxin is a major hormone which plays crucial roles in instructing virtually all developmental programs of plants. Its signaling depends primarily on its perception by four partially redundant receptors of the TIR1/AFB2 clade (TAARs), which subsequently mediate the specific degradation of AUX/IAA transcriptional repressors to modulate the expression of primary auxin-responsive genes. Auxin homeostasis depends on complex regulations at the level of synthesis, conjugation, and transport. However, the mechanisms and principles involved in the homeostasis of its signaling are just starting to emerge. We report that xrn4 mutants exhibit pleiotropic developmental defects and strong auxin hypersensitivity phenotypes. We provide compelling evidences that these phenotypes are directly caused by improper regulation of TAAR transcript degradation. We show that the cytoplasmic 5'-3' exoribonuclease XRN4 is required for auxin response. Thus, our work identifies new targets of XRN4 and a new level of regulation for TAAR transcripts important for auxin response and for plant development.

HISTONE DEACETYLASE6 Controls Gene Expression Patterning and DNA Methylation-Independent Euchromatic Silencing.

To investigate the role of chromatin regulators in patterning gene expression, we employed a unique epigenetically controlled and highly tissue-specific green fluorescent protein reporter line in Arabidopsis (Arabidopsis thaliana). Using a combination of forward and reverse genetic approaches on this line, we show here that distinct epigenetic regulators are involved in silencing the transgene in different tissues. The forward genetic screen led to the identification of a novel HISTONE DEACETYLASE6 (HDA6) mutant allele (epigenetic control1, hda6-8). This allele differs from the previously reported alleles, as it did not affect DNA methylation and only had a very modest effect on the release of transposable elements and other heterochromatic transcripts. Overall, our data shows that HDA6 has at least two clearly separable activities in different genomic regions. In addition, we present an unexpected role for HDA6 in the control of DNA methylation at CG dinucleotides.

MiR393 regulation of auxin signaling and redox-related components during acclimation to salinity in Arabidopsis.

One of the most striking aspects of plant plasticity is the modulation of development in response to environmental changes. Plant growth and development largely depend on the phytohormone auxin that exerts its function through a partially redundant family of F-box receptors, the TIR1-AFBs. We have previously reported that the Arabidopsis double mutant tir1 afb2 is more tolerant to salt stress than wild-type plants and we hypothesized that down-regulation of auxin signaling might be part of Arabidopsis acclimation to salinity. In this work, we show that NaCl-mediated salt stress induces miR393 expression by enhancing the transcription of AtMIR393A and leads to a concomitant reduction in the levels of the TIR1 and AFB2 receptors. Consequently, NaCl triggers stabilization of Aux/IAA repressors leading to down-regulation of auxin signaling. Further, we report that miR393 is likely involved in repression of lateral root (LR) initiation, emergence and elongation during salinity, since the mir393ab mutant shows reduced inhibition of emergent and mature LR number and length upon NaCl-treatment. Additionally, mir393ab mutant plants have increased levels of reactive oxygen species (ROS) in LRs, and reduced ascorbate peroxidase (APX) enzymatic activity compared with wild-type plants during salinity. Thus, miR393 regulation of the TIR1 and AFB2 receptors could be a critical checkpoint between auxin signaling and specfic redox-associated components in order to coordinate tissue and time-specific growth responses and tolerance during acclimation to salinity in Arabidopsis.

miR393 is required for production of proper auxin signalling outputs.

Auxins are crucial for plant growth and development. Auxin signalling primarily depends on four partially redundant F-box proteins of the TIR1/AFB2 Auxin Receptor (TAAR) clade to trigger the degradation of AUX/IAA transcriptional repressors. Auxin signalling is a balanced system which involves complex feedback regulations. miR393 regulation of TAAR genes is important for different developmental programs and for responses to environment. However, so far, the relevance of the two MIR393 genes for Arabidopsis leaf development and their significance for auxin signalling homeostasis have not been evaluated. First, our analyses of mir393a-1 and mir393b-1 mutants and of mir393ab double mutant show that the two genes have only partially redundant functions for leaf development. Expression analyses of typical auxin-induced reporter genes have shown that the loss of miR393 lead to several unanticipated changes in auxin signalling. The expression of DR5pro:GUS is decreased, the expression of primary AUX/IAA auxin-responsive genes is slightly increased and the degradation of the AXR3-NT:GUS reporter protein is delayed in mir393ab mutants. Additional analyses using synthetic auxin and auxin antagonists indicated that miR393 deficient mutants have higher levels of endogenous AUX/IAA proteins, which in turn create a competition for degradation. We propose that the counter-intuitive changes in the expression of AUX/IAA genes and in the accumulation of AUX/IAA proteins are explained by the intrinsic nature of AUX/IAA genes which are feedback regulated by the AUX/IAA proteins which they produce. Altogether our experiments provide an additional highlight of the complexity of auxin signaling homeostasis and show that miR393 is an important component of this homeostasis.

Introns of plant pri-miRNAs enhance miRNA biogenesis.

Plant MIR genes are independent transcription units that encode long primary miRNA precursors, which usually contain introns. For two miRNA genes, MIR163 and MIR161, we show that introns are crucial for the accumulation of proper levels of mature miRNA. Removal of the intron in both cases led to a drop-off in the level of mature miRNAs. We demonstrate that the stimulating effects of the intron mostly reside in the 5'ss rather than on a genuine splicing event. Our findings are biologically significant as the presence of functional splice sites in the MIR163 gene appears mandatory for pathogen-triggered accumulation of miR163 and proper regulation of at least one of its targets.

Primary and secondary siRNAs in geminivirus-induced gene silencing.

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.

Role of microRNAs and other sRNAs of plants in their changing environments.

Plants constantly face a complex array of environmental biotic and abiotic stimuli. Recent studies in various plants have highlighted the key roles of microRNAs and of different siRNA classes in the post-transcriptional regulation of plant genes essential for conserved responses of plants to individual stress conditions. It is not yet clear how these different signals and responses are integrated in nature. In the present review, we summarize current knowledge on sRNA-mediated responses to stress, and highlight possible directions of future research.

Pretreatments, conditioned medium and co-culture increase the incidence of somatic embryogenesis of different Cichorium species.

Somatic embryogenesis (SE) in Cichorium involves dedifferentiation and redifferentiation of single cells and can be induced by specific in vitro culture conditions. We have tested the effect of various treatments on the incidence of SE (ISE) of an interspecific embryogenic hybrid (C. endivia x C. intybus) and of different commercial chicories (C. endivia and C. intybus) that are typically recalcitrant to SE in standard culture conditions. We found that the ISE of the hybrid is significantly increased by pretreatment of tissues by submersion in solutions of glycerol, abscisic acid, spermine, putrescine or of combinations of these compounds. Interestingly, the most efficient of these pretreatments also had an unexpectedly high effect on the ISE of the C. intybus cultivars. The ISE of the hybrid and of the commercial chicories were increased when explants were co-cultured with highly embryogenic chicory explants or when they were cultured in conditioned medium. These observations established that unidentified SE-promoting factors are released in the culture medium. HPLC analyses of secreted Arabino-Galactan Proteins (AGPs), which are known to stimulate SE, did not allow identifying a fraction containing differentially abundant AGP candidates. However, pointing to their role in promoting SE, we found that the hybrid had a drastically higher ISE when amino sugars and L-Proline, the putative precursors of secreted AGPs, were both added to the medium.

miR393: integrator of environmental cues in auxin signaling?

Auxin signalling and plant development depend on a family of partially redundant F-box receptors of the TIR1/AFB2 Auxin Receptor (TAAR) clade. We have recently shown that the post-transcriptional regulation of the Arabidopsis thaliana TAAR gene family invokes complex sRNA regulations during development. In leaves, the microRNA miR393 appears (1) to be primarily generated from one of the two genes, AtMIR393B, (2) to regulate the expression of all four members of the clade and (3) to initiate the formation of functional secondary siRNAs, named siTAARs, from TAAR transcripts themselves. Strikingly, mir393b-1 mutants, which are impaired in the biogenesis of miR393b and siTAARs, exhibit rather mild developmental defects. The known roles of miR393 in nitrate response, in defence against pathogenic bacteria and in plant development lead us to hypothesize that miR393 plays an important role to integrate complex environmental stimuli.  

miR393 and secondary siRNAs regulate expression of the TIR1/AFB2 auxin receptor clade and auxin-related development of Arabidopsis leaves.

The phytohormone auxin is a key regulator of plant growth and development that exerts its functions through F-box receptors. Arabidopsis (Arabidopsis thaliana) has four partially redundant of these receptors that comprise the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX1 auxin receptor (TAAR) clade. Recent studies have shown that the microRNA miR393 regulates the expression of different sets of TAAR genes following pathogen infection or nitrate treatment. Here we report that miR393 helps regulate auxin-related development of leaves. We found that AtMIR393B is the predominant source for miR393 in all aerial organs and that miR393 down-regulates all four TAAR genes by guiding the cleavage of their mRNAs. A mutant unable to produce miR393 shows developmental abnormalities of leaves and cotyledons reminiscent of enhanced auxin perception by TAARs. Interestingly, miR393 initiates the biogenesis of secondary siRNAs from the transcripts of at least two of the four TAAR genes. Our results indicate that these siRNAs, which we call siTAARs, help regulate the expression of TAAR genes as well as several unrelated genes by guiding the cleavage of their mRNAs. Thus, miR393 and possibly siTAARs regulate auxin perception and certain auxin-related aspects of leaf development.

Specific impact of tobamovirus infection on the Arabidopsis small RNA profile.

Tobamoviruses encode a silencing suppressor that binds small RNA (sRNA) duplexes in vitro and supposedly in vivo to counteract antiviral silencing. Here, we used sRNA deep-sequencing combined with transcriptome profiling to determine the global impact of tobamovirus infection on Arabidopsis sRNAs and their mRNA targets. We found that infection of Arabidopsis plants with Oilseed rape mosaic tobamovirus causes a global size-specific enrichment of miRNAs, ta-siRNAs, and other phased siRNAs. The observed patterns of sRNA enrichment suggest that in addition to a role of the viral silencing suppressor, the stabilization of sRNAs might also occur through association with unknown host effector complexes induced upon infection. Indeed, sRNA enrichment concerns primarily 21-nucleotide RNAs with a 5'-terminal guanine. Interestingly, ORMV infection also leads to accumulation of novel miRNA-like sRNAs from miRNA precursors. Thus, in addition to canonical miRNAs and miRNA*s, miRNA precursors can encode additional sRNAs that may be functional under specific conditions like pathogen infection. Virus-induced sRNA enrichment does not correlate with defects in miRNA-dependent ta-siRNA biogenesis nor with global changes in the levels of mRNA and ta-siRNA targets suggesting that the enriched sRNAs may not be able to significantly contribute to the normal activity of pre-loaded RISC complexes. We conclude that tobamovirus infection induces the stabilization of a specific sRNA pool by yet unknown effector complexes. These complexes may sequester viral and host sRNAs to engage them in yet unknown mechanisms involved in plant:virus interactions.

The biosynthetic pathways and biological scopes of plant small RNAs.

RNA silencing plays a crucial role in coordinating the expression, stability, protection and inheritance of eukaryotic genomes. It comprises several mechanisms that invariably depend on core small RNA molecules (sRNAs) and that achieve dedicated sequence-specific functions. Biogenesis and function of sRNAs generally follow a framework that involves main protein families conserved among eukaryotes. The most recent studies have provided structural insights into the function of RNA silencing components, identified novel players of these pathways and highlighted possible emerging classes of sRNA regulators. In this review, we integrate these studies to provide updated views on the biosynthetic pathways and dedicated functions of the endogenous sRNA classes of plants, emphasizing their specialized scopes.

cDNA-AFLP display for the isolation of differentially expressed genes during chicory root development.

To identify genes expressed during root enlargment during the early vegetative growth of chicory (Cichorium intybus L.), we used the cDNA-AFLP technology. The radial pattern of chicory roots was investigated by histological analysis to determine the spatial vascular cambium setting. In young plantlets, serial root sections showed that differentiation of secondary tissues occurred along a gradient extending from the apex to the crown. The cDNA-AFLP technique was carried out on total mRNAs extracted from root tissues producing secondary structures and root tissues developing primary structures only. This study reports on the isolation of two transcript-derived fragments (TDFs) referred to as Y-16 and Y-21. Sequence analysis at the protein level showed that Y-16 carries a sequence highly homologous (93% identities) to the amino acid transporter-like protein 1 (AATL 1) from Arabidopsis and Y-21 presents 72% identity to AAD25141.1 Arabidopsis protein. The expression pattern of both these TDFs was analysed by northern blot and showed an over-expression during early development of chicory roots.