Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.

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

Functional Specialization of the Plant miR396 Regulatory Network through Distinct MicroRNA–Target Interactions

MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5′ portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant

To preserve or to destroy, that is the question: the role of the cell wall integrity pathway in pollen tube growth

In plants, cell-shape is defined by the cell wall, a complex network of polymers located outside the plasma membrane. During cell growth, cell wall properties have to be adjusted, assuring cell expansion without compromising cell integrity. Plasma membrane-located receptors sense cell wall properties, transducing extracellular signals into intracellular cascades through the cell wall integrity (CWI) pathway that, in turn, leads to adjustments in the regulation and composition of the cell wall. Using pollen tube growth as a single celled model system, we describe the importance of RAPID ALKALINIZATION FACTOR (RALF) peptides as sensors of cell wall integrity. RALF peptides can mediate the communication between cell wall components and plasma membrane-localized receptor-like kinases (RLKs) of the CrRLK1L family. The subsequent activation of intracellular pathways regulates H+, Ca2+, and ROS levels in the cell and apoplast, thereby modulating cell wall integrity. Interestingly, the RALF-CrRLK1L module and some of the components working up- and downstream of the RLK is conserved in many other developmental and physiological signaling processes

Characterization of the single FERONIA homolog in Marchantia polymorpha reveals an ancestral role of CrRLK1L receptor kinases in regulating cell expansion and morphological integrity

Plant cells are surrounded by a cell wall, a rigid structure rich in polysaccharides and glycoproteins. The cell wall is not only important for cell and organ shape, but crucial for intercellular communication, plant-microbe interactions, and as a barrier to the environment. In the flowering plant Arabidopsis thaliana, the 17 members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase subfamily are involved in a multitude of physiological and developmental processes involving the cell wall, including reproduction, hormone signaling, cell expansion, innate immunity, and various stress responses. Due to genetic redundancy and the fact that individual CrRLK1Ls can have distinct and sometimes opposing functions, it is difficult to assess the primary or ancestral function of CrRLK1Ls. To reduce genetic complexity, we characterized the single CrRLK1L gene of Marchantia polymorpha, MpFERONIA (MpFER). Plants with reduced MpFER levels show defects in vegetative development, i.e., rhizoid formation and cell expansion, but also affect male fertility. In contrast, Mpfer null mutants and overexpression lines severely affect cell integrity and morphogenesis of the gametophyte. Thus, the CrRLK1L gene family originated from a single gene with an ancestral function in cell expansion and the maintenance of cellular integrity. During land plant evolution, this ancestral gene diversified and was recruited to fulfil a multitude of specialized physiological and developmental and roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants

Delving into the ancient stem cell niche

Resumen del póster presentado al Congreso 'At the Forefront of Plant Research', celebrado en Barcelona (España) del 6 al 8 de mayo de 2019.Plant growth and development is sustained by a pool of undifferentiated and pluripotent stem cells. These cells have the ability to self-renew and give rise to all type of tissues. Genes in the WUSCHEL-RELATED HOMEOBOX(WOX) family constitute the master regulators that maintain a stable stem cell population in superior plants. The WOX family consists of three clades: the ancient clade, present in the earliest diverging green plants; the intermediate clade that emerged in vascular plants; and the WUSCHEL(WUS) clade, which appears specifically in ferns and seed plants. Therefore, the complexity of WOX protein family has increased during plant evolution coupled with a tighter regulation and organization of stem cells. To date, the major studies to understand stem cell regulation were carried out in angiosperms models. However, the knowledge about stem cell control in early divergent land plants is still limited. Focusing our efforts in this direction may help to clarify how the activity and regulation pathway of WOXs members evolved. In this sense, the bryophyte Marchantia polymorpha constitute a suitable model due to its critical evolutionary position and genome simplicity. Here, we identified one WOX protein(MpWOX) closely related with members from the WOX ancient clade. Overexpressor, amiRNA and reporter lines of MpWOX were generated in Marchantia and a phenotypic analysis of these lines in meristematic and stem cell function is undergoing. In addition, we are carrying a complementation of Arabidopsis thaliana wox mutants to investigate the ancestral role of these proteins. Our preliminary results indicate a possible conservation of the WOX function on stem cells maintenance.Peer reviewe

Activity of endogenous miR396 towards different substrates in <i>Arabidopsis thaliana</i>.

<p>(A) GUS stainings of typical transgenic plants harboring <i>wtGRF2</i> and <i>pGRF2</i> reporters (15-day old seedlings). Sensors were built by fusing the upstream regulatory regions of <i>GRF2</i> and its first 4 exons to <i>GUS</i>. The miR396 target site was modified as indicated below the pictures. Interaction energy values for miR396b are indicated below each miRNA-target pair. Scale Bar: 2 mm. (B) Expression levels of <i>GRF2-GUS</i> RNA in leaves #1 and #2 (15-day old seedlings) of the different sensors. Two representative lines for each vector out of a total of 20 independent lines were selected. Expression levels were normalized to <i>wtGRF2-GUS</i> line #3. Data shown are mean ± SEM of 4 biological replicates. Asterisks indicate significant differences between plants harboring different transgenes, as determined by ANOVA (P<0.05). (C) GUS staining in developing leaves #4 (right) and #5 (left) of transgenic plants harboring miR396, <i>rGRF2</i>, <i>wtGRF2</i>, <i>pGRF2</i> and <i>CYCLIN B1;1</i> reporters (14-day old seedlings). Scale Bar: 1 mm.</p

Variations among miR396 family members in plants.

<p>(A) MiR396 family composition of selected species. Differences in the 5′ region are highlighted with colored boxes, while variations at the 3′ end for each case are indicated in parentheses. (B) Diagrams showing the relative abundance of miR396 variants in pine, poplar, Arabidopsis and monocot libraries according to deep sequencing data (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419.s012" target="_blank">Table S5</a>).</p

Characterization of transgenic plants expressing a miR396-resistant <i>bHLH74</i>.

<p>(A) Schematic representation of <i>bHLH74</i> and <i>rbHLH74</i> constructs. (B) <i>bHLH74</i> transcript levels estimated by RT-qPCR in mature leaves of plants expressing a wild-type (<i>bHLH74:wtbHLH74</i>) or miR396-resistant (<i>bHLH74:rbHLH74</i>) form of the gene encoding the transcription factor. Data shown are mean ± SEM of 3 biological replicates. Asterisks indicate significant differences between transgenic and wild-type plants, as determined by ANOVA (P<0.05). (C) Angle determination at the distal tip of leaf #5 in transgenics depicted on panel (B). Asterisks indicate significant differences between transgenics and wild-type plants, as determined by ANOVA (P<0.05). (D) Morphology of fully-expanded leaf #5. The different angles at the distal edge of the leaf are highlighted in yellow for wild-type and <i>bHLH74:rbHLH74</i> leaves. An inset on the right shows the difference in venation (secondary veins are highlighted in red), with the number of branching points (NBP) indicated below. Data shown are mean ± SEM of 8 biological replicates. Scale Bar: 1 cm. (E) Scheme highlighting differences in leaf edges of wild-type and miR396-resistant <i>bHLH74</i> plants.</p

Analysis of <i>bhlh74</i> mutants.

<p>(A) Scheme of the <i>bHLH74</i> locus showing the T-DNA insertion corresponding to the GABI-Kat 720G11 line. Arrows indicate the pairs of primers (1–2 or 3–4, see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419.s014" target="_blank">Table S7</a>) used to quantify <i>bHLH74</i> transcript levels by RT-qPCR. (B) <i>bHLH74</i> transcript levels in wt and <i>bhlh74-1</i> (GABI-Kat 720G11) seedlings (12–day old), using pairs of primers shown in (A). Data shown are mean ± SEM of 3 biological replicates; n.d.: not detected. Asterisks indicate significant differences between mutant and wild-type plants, as determined by ANOVA (P<0.05). (C) Number of branching points (NBP) in fully-expanded first leaves of wt, <i>bhlh74-1</i> and <i>bHLH74:rbHLH74</i> (lines #18 and #8) plants. Bars marked with different letters are significantly different as determined by ANOVA and Duncan's multiple range test (P<0.05). (D) Fully-expanded leaves (#1) from wt, <i>bhlh74-1</i> and <i>bHLH74:rbHLH74</i> (lines #18 and #8) plants. Red dots highlight branching points.</p

Analysis of potential miR396 targets in <i>Arabidopsis thaliana</i>.

<p>(A) Scheme representing a typical <i>GRF</i> gene and the conservation of the target site in selected angiosperm and gymnosperm species. Conserved positions across all species are indicated by asterisks. Note that the number of exons might vary among <i>GRF</i> genes. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419.s011" target="_blank">Table S4</a> for accession numbers of sequences used. (B) Scheme representing the strategy used to identify new miR396 targets of potential biological significance. Target search was performed over the TAIR9 database using the WMD3 target search tool (<a href="http://wmd3.weigelworld.org/" target="_blank">http://wmd3.weigelworld.org/</a>), allowing 5 mismatches and gaps in the miRNA-target pairs. Predicted targets are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419.s008" target="_blank">Table S1</a>. (C) Diagram showing putative miR396 targets that are up-regulated at least 30% in miRNA mutants <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419-Allen1" target="_blank">[32]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419-Lobbes1" target="_blank">[35]</a>. Expression levels were obtained from Genevestigator (<a href="http://www.genevestigator.com" target="_blank">www.genevestigator.com</a>). (D,E) Modified RACE-PCR mapping of At1g10120 and At5g24660 mRNA cleavage sites. Red arrows indicate predicted miR396 cleavage sites. (F) At1g10120 and At5g24660 transcript levels in different small RNA mutant plants estimated by RT-qPCR. Data shown are mean ± SEM of 3 biological replicates. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419.s014" target="_blank">Table S7</a> for a list of mutant alleles used. (G) At1g10120 and At5g24660 transcript levels in plants expressing an artificial target-mimic against miR396 (<i>MIM396</i>) estimated by RT-qPCR. Data shown are mean ± SEM of 3 biological replicates. (H) Scheme representing the At1g10120 locus. The miR396 target site is formed after the splicing of the first two exons. Target site conservation in several species is indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002419#pgen.1002419.s011" target="_blank">Table S4</a> for accession numbers of sequences used). Conserved positions across all species are indicated by asterisks.</p