Martin A. Mecchia

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.

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

BackgroundDevelopment 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.ResultsWe 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.ConclusionsOur 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.

Post-transcriptional control of GRF transcription factors by microRNA miR396 and GIF co-activator affects leaf size and longevity

The growth-regulating factors (GRFs) are plant-specific transcription factors. They form complexes with GRF-interacting factors (GIFs), a small family of transcriptional co-activators. In Arabidopsis thaliana, seven out of the nine GRFs are controlled by microRNA miR396. Analysis of Arabidopsis plants carrying a GRF3 allele insensitive to miR396 revealed a strong boost in the number of cells in leaves, which was further enhanced synergistically by an additional increase of GIF1 levels. Genetic experiments revealed that GRF3 can still increase cell number in gif1 mutants, albeit to a much lesser extent. Genome-wide transcript profiling indicated that the simultaneous increase of GRF3 and GIF1 levels causes additional effects in gene expression compared to either of the transgenes alone. We observed that GIF1 interacts in vivo with GRF3, as well as with chromatin-remodeling complexes, providing a mechanistic explanation for the synergistic activities of a GRF3-GIF1 complex. Interestingly, we found that, in addition to the leaf size, the GRF system also affects the organ longevity. Genetic and molecular analysis revealed that the functions of GRFs in leaf growth and senescence can be uncoupled, demonstrating that the miR396-GRF-GIF network impinges on different stages of leaf development. Our results integrate the post-transcriptional control of the GRF transcription factors with the progression of leaf development.

MicroRNA miR396 and RDR6 synergistically regulate leaf development

The microRNA (miRNA) miR396 regulates GROWTH-REGULATING FACTORs (GRFs), a plant specific family of transcription factors. Overexpression of miR396 causes a decrease in the GRFs that has been shown to affect cell proliferation in the meristem and developing leaves. To bring further insights into the function of the miR396 regulatory network we performed a mutant enhancer screen of a stable Arabidopsis transgenic line expressing 35S:miR396b, which has a reduction in leaf size. From this screen we recovered several mutants enhancing this phenotype and displaying organs with lotus- or needle-like shape. Analysis of these plants revealed mutations in as2 and rdr6. While 35S:miR396b in an as2 context generated organs with lotus-like shape, the overexpression of the miRNA in an rdr6 mutant background caused more important developmental defects, including pin-like organs and lobed leaves. Combination of miR396 overexpressors, and rdr6 and as2 mutants show additional organ defects, suggesting that the three pathways act in concert. Genetic interactions during leaf development were observed in a similar way between miR396 overexpression and mutants in RDR6, SGS3 or AGO7, which are known to participate in trans-acting siRNA (ta-siRNA) biogenesis. Furthermore, we found that miR396 can cause lotus- and pin-like organs per se, once a certain expression threshold is overcome. In good agreement, mutants accumulating high levels of TCP4, which induces miR396, interacted with the AS1/AS2 pathway to generate lotus-like organs. The results indicate that the miR396 regulatory network and the ta-siRNA biogenesis pathway synergistically interact during leaf development and morphogenesis.

MicroRNA miR396 Regulates the Switch between Stem Cells and Transit-Amplifying Cells in Arabidopsis Roots

Regulatory interactions between microRNA miR396 and PLT and GRF transcription factors affect the boundary between root stem cells and the rapidly dividing transit-amplifying cells. To ensure an adequate organ mass, the daughters of stem cells progress through a transit-amplifying phase displaying rapid cell division cycles before differentiating. Here, we show that Arabidopsis thaliana microRNA miR396 regulates the transition of root stem cells into transit-amplifying cells by interacting with GROWTH-REGULATING FACTORs (GRFs). The GRFs are expressed in transit-amplifying cells but are excluded from the stem cells through inhibition by miR396. Inactivation of the GRFs increases the meristem size and induces periclinal formative divisions in transit-amplifying cells. The GRFs repress PLETHORA (PLT) genes, regulating their spatial expression gradient. Conversely, PLT activates MIR396 in the stem cells to repress the GRFs. We identified a pathway regulated by GRF transcription factors that represses stem cell-promoting genes in actively proliferating cells, which is essential for the progression of the cell cycle and the orientation of the cell division plane. If unchecked, the expression of the GRFs in the stem cell niche suppresses formative cell divisions and distorts the organization of the quiescent center. We propose that the interactions identified here between miR396 and GRF and PLT transcription factors are necessary to establish the boundary between the stem cell niche and the transit-amplifying region.

Pollen-Specific Aquaporins NIP4;1 and NIP4;2 Are Required for Pollen Development and Pollination in Arabidopsis thaliana

NIP4;1 and NIP4;2, two pollen-specific aquaporins, are paralogous genes displayed in tandem that are critical for appropriate pollen development and pollen tube growth. In flowers with dry stigmas, pollen development, pollination, and pollen tube growth require spatial and temporal regulation of water and nutrient transport. To better understand the molecular mechanisms involved in reproductive processes, we characterized NIP4;1 and NIP4;2, two pollen-specific aquaporins of Arabidopsis thaliana. NIP4;1 and NIP4;2 are paralogs found exclusively in the angiosperm lineage. Although they have 84% amino acid identity, they displayed different expression patterns. NIP4;1 has low expression levels in mature pollen, while NIP4;2 expression peaks during pollen tube growth. Additionally, NIP4;1pro:GUS flowers showed GUS activity in mature pollen and pollen tubes, whereas NIP4;2pro:GUS flowers only in pollen tubes. Single T-DNA mutants and double artificial microRNA knockdowns had fewer seeds per silique and reduced pollen germination and pollen tube length. Transport assays in oocytes showed NIP4;1 and NIP4;2 function as water and nonionic channels. We also found that NIP4;1 and NIP4;2 C termini are phosphorylated by a pollen-specific CPK that modifies their water permeability. Survival assays in yeast indicated that NIP4;1 also transports ammonia, urea, boric acid, and H2O2. Thus, we propose that aquaporins NIP4;1 and NIP4;2 are exclusive components of the reproductive apparatus of angiosperms with partially redundant roles in pollen development and pollination.

An update on cell surface proteins containing extensin-motifs

In recent years it has become clear that there are several molecular links that interconnect the plant cell surface continuum, which is highly important in many biological processes such as plant growth, development, and interaction with the environment. The plant cell surface continuum can be defined as the space that contains and interlinks the cell wall, plasma membrane and cytoskeleton compartments. In this review, we provide an updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs (for proline-rich extensin-like receptor kinases); with an EXT-motif and an actin binding domain, known as formins; and with extracellular hybrid-EXTs. We focus our attention on the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role. A closer understanding of the dynamic regulation of plant cell surface continuum and its relationship with the downstream signalling cascade is a crucial forthcoming challenge.