HYL1 regulates the balance between adaxial and abaxial identity for leaf flattening via miRNA-mediated pathways

HYPONASTIC LEAVES1 (HYL1) is an important regulator of microRNA (miRNA) biogenesis. Incurvature of rosette leaves in loss-of-function mutants of HYL1 implicates the regulation of leaf flatness by HYL1 via miRNA pathways. Recent studies have identified jba-1D, jaw-1D, and oe-160c, the dominant mutants of MIR166g, MIR319a, and MIR160c genes, respectively, which display three types of leaf curvature. However, it remains unclear whether or how HYL1 controls leaf flatness through the pathways mediated by these miRNAs. To define which miRNAs and target genes are relevant to the hyl1 phenotype in terms of leaf incurvature, the effects of three mutated MIRNA genes and their targets on the direction and extent of leaf curvature in hyl1 mutants were examined. The genetic analysis shows that the hyl1 phenotype is strongly rescued by jba-1D, but not by jaw-1D or oe-160c, whereas the mutant phenotypes of jba-1D, jaw-1D, or oe-160c leaves are compromised by the hyl1 allele. Expression analysis indicates that reduced accumulation of miR166, rather than of miR319a or miR160, causes incurvature of hyl1 leaves, and that miR319a-targeted TCP3 positively regulates the adaxial identity gene PHABULOSA while miR160-targeted ARF16 negatively regulates the abaxial identity gene FILAMENTOUS FLOWER. In these cases, the direction and extent of leaf incurvature are associated with the expression ratio of adaxial to abaxial genes (adaxial to abaxial ratio). HYL1 regulates the balance between adaxial and abaxial identity and modulates leaf flatness by preventing leaf incurvature, wavy margins, and downward curvature. It is concluded that HYL1 monitors the roles of miR165/166, miR319a, and miR160 in leaf flattening through the relative activities of adaxial and abaxial identity genes, thus playing an essential role in leaf development.     

The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis

It has been reported that some double-stranded RNA (dsRNA) binding proteins interact with small RNA biogenesis-related RNase III enzymes. However, their biological significance is poorly understood. Here we examine the relationship between the Arabidopsis microRNA- (miRNA) producing enzyme DCL1 and the dsRNA binding protein HYL1. In the hyl1-2 mutant, the processing steps of miR163 biogenesis were partially impaired; increased accumulation of pri-miR163 and reduced accumulation of short pre-miR163 and mature miR163 as well as misplaced cleavages in the stem structure of pri-miR163 were detected. These misplaced cleavages were similar to those previously observed in the dcl1-9 mutant, in which the second double-stranded RNA binding domain of the protein was disrupted. An immunoprecipitation assay using Agrobacterium-mediated transient expression in Nicotiana benthamiana showed that HYL1 was able to form a complex with wild-type DCL1 protein, but not with the dcl1-9 mutant protein. We also examined miR164b and miR166a biogenesis in hyl1-2 and dcl1-9. Increased accumulation of pri-miRNAs and reduced accumulation of pre-miRNAs and mature miRNAs were detected. Misplaced cleavage on pri-miR164b was observed only in dcl1-9 but not in hyl1-2, whereas not on pri-miR166a in either mutant. These results indicate that HYL1 has a function in assisting efficient and precise cleavage of pri-miRNA through interaction with DCL1.     

MicroRNA gene regulation cascades during early stages of plant development

MicroRNAs (miRNAs) regulate various developmental programs of plants. This review focuses on miRNA involvement in early events of plant development, such as seed germination, seedling development and the juvenile to adult phase transition. miR159 and miR160 are involved in the regulation of seed germination through their effects on the sensitivity of seeds to ABA. miR156 and miR172 play critical roles in the emergence of vegetative leaves at post-germinative stages, which is important for the transition to autotrophic growth. The phase transition from the juvenile to adult stage in both monocots and dicots is also regulated by miR156 and miR172. In these early developmental processes, there are miRNA gene regulation cascades where the miR156 pathway acts upstream of the miR172 pathway. Moreover, targets of miR156 and miR172 exert positive feedback on the expression of MIR genes that suppress themselves. The early events of plant development appear to be controlled by complex mechanisms involving sequential expression of different miRNA pathways and feedback loops among miRNAs and their target genes.     

miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions

In plants, developmental timing is coordinately regulated by a complex signaling network that integrates diverse intrinsic and extrinsic signals. miR172 promotes photoperiodic flowering. It also regulates adult development along with miR156, although the molecular mechanisms underlying this regulation are not fully understood. Here, we demonstrate that miR172 modulates the developmental transitions by regulating the expression of a subset of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are also regulated by miR156. The SPL3/4/5 genes were upregulated in the miR172-overproducing plants (35S:172) and its target gene mutants that exhibit early flowering. In contrast, expression of other SPL genes was not altered to a discernible level. Kinetic measurements of miR172 abundance in the transgenic plants expressing the MIR156a gene driven by a β-estradiol-inducible promoter revealed that expressions of miR172 and miR156 are not directly interrelated. Instead, the 2 miRNA signals are integrated at the SPL3/4/5 genes. Notably, analysis of developmental patterns in the 156?×?172 plants overproducing both miR172 and miR156 showed that whereas vegetative phase change was delayed as observed in the miR156-overproducing plants (35S:156), flowering initiation was accelerated as observed in the 35S:172 transgenic plants. Together, these observations indicate that although miR172 and miR156 play distinct roles in the timing of developmental phase transitions, there is a signaling crosstalk mediated by the SPL3/4/5 genes.     

miRNA control of vegetative phase change in trees

After germination, plants enter juvenile vegetative phase and then transition to an adult vegetative phase before producing reproductive structures. The character and timing of the juvenile-to-adult transition vary widely between species. In annual plants, this transition occurs soon after germination and usually involves relatively minor morphological changes, whereas in trees and other perennial woody plants it occurs after months or years and can involve major changes in shoot architecture. Whether this transition is controlled by the same mechanism in annual and perennial plants is unknown. In the annual forb Arabidopsis thaliana and in maize (Zea mays), vegetative phase change is controlled by the sequential activity of microRNAs miR156 and miR172. miR156 is highly abundant in seedlings and decreases during the juvenile-to-adult transition, while miR172 has an opposite expression pattern. We observed similar changes in the expression of these genes in woody species with highly differentiated, well-characterized juvenile and adult phases (Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima), as well as in the tree Populus x canadensis, where vegetative phase change is marked by relatively minor changes in leaf morphology and internode length. Overexpression of miR156 in transgenic P. x canadensis reduced the expression of miR156-targeted SPL genes and miR172, and it drastically prolonged the juvenile phase. Our results indicate that miR156 is an evolutionarily conserved regulator of vegetative phase change in both annual herbaceous plants and perennial trees.     

Cloning and characterization of micro-RNAs from moss

Micro-RNAs (miRNAs) are one class of endogenous tiny RNAs that play important regulatory roles in plant development and responses to external stimuli. To date, miRNAs have been cloned from higher plants such as Arabidopsis, rice and pumpkin, and there is limited information on their identity in lower plants including Bryophytes. Bryophytes are among the oldest groups of land plants among the earth's flora, and are important for our understanding of the transition to life on land. To identify miRNAs that might have played a role early in land plant evolution, we constructed a library of small RNAs from the juvenile gametophyte (protonema) of the moss Physcomitrella patens. Sequence analysis revealed five higher plant miRNA homologues, including three members of the miR319 family, previously shown to be involved in the regulation of leaf morphogenesis, and miR156, which has been suggested to regulate several members of the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family in Arabidopsis. We have cloned PpSBP3, a moss SPL homologue that contains an miR156 complementary site, and demonstrated that its mRNA is cleaved within that site suggesting that it is an miR156 target in moss. Six additional candidate moss miRNAs were identified and shown to be expressed in the gametophyte, some of which were developmentally regulated or upregulated by auxin. Our observations suggest that miRNAs play important regulatory roles in mosses.     

Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) are an important gene regulator, controlling almost all biological and metabolic processes, in both plants and animals. In this study, we investigated the effect of drought and salinity stress on the expression of miRNAs and their targets in cotton (Gossypium hirsutum L.). Our results show that the expression change of miRNAs and their targets were dose-dependent and tissue-dependent under salinity and drought conditions. The expression of miRNAs in leaf was down-regulated under higher salinity stress while shows variable patterns in other conditions. The highest fold-changes of miRNAs were miR398 in roots with 28.9 fold down-regulation under 0.25% NaCl treatment and miR395 in leaves with 7.6 fold down-regulation under 1% PEG treatment. The highest up-regulation of miRNA targets was AST in roots with 4.7 fold-change under 2.5% PEG and the gene with highest down-regulation was CUC1 in leaves with 25.6 fold-change under 0.25% NaCl treatment. Among seven miRNA-target pairs we studied, five pairs, miR156–SPL2, miR162–DCL1, miR159–TCP3, miR395–APS1 and miR396–GRF1, show significant regulation relationship in roots and leaves under salinity stress concentration.