Temporally regulated microRNAs have been identified as master regulators of developmental timing in both animals and plants. In plants, vegetative development is regulated by a temporal decrease in miR156 level, but how this decreased expression is initiated and then maintained during shoot development remains elusive. Here, we show that miR159 is required for the correct timing of vegetative development in Loss of miR159 increases miR156 level throughout shoot development and delays vegetative development, whereas overexpression of miR159 slightly accelerated vegetative development. The repression of miR156 by miR159 is predominantly mediated by MYB33, an R2R3 MYB domain transcription factor targeted by miR159. Loss of led to subtle precocious vegetative phase change phenotypes in spite of the significant downregulation of miR156. MYB33 simultaneously promotes the transcription of and, as well as their target, , by directly binding to the promoters of these three genes. Rather than acting as major players in vegetative phase change in Arabidopsis, our results suggest that miR159 and MYB33 function as modifiers of vegetative phase change; i.e., miR159 facilitates vegetative phase change by repressing MYB33 expression, thus preventing MYB33 from hyperactivating miR156 expression throughout shoot development to ensure correct timing of the juvenile-to-adult transition in Arabidopsis.

Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.

After germination, plants progress through juvenile and adult phases of vegetative development before entering the reproductive phase. The character and timing of these phases vary significantly between different plant species, which makes it difficult to know whether temporal variations in various vegetative traits represent the same, or different, developmental processes. miR156 has been shown to be the master regulator of vegetative development in plants. Overexpression of miR156 prolongs the juvenile phase of development, whereas knocking-down the level of miR156 promotes the adult phase of development. Therefore, artificial modulation of miR156 expression is expected to cause corresponding changes in vegetative-specific traits in different plant species, particularly in those showing no substantial difference in morphology during vegetative development. To identify specific traits associated with the juvenile-to-adult transition in tobacco, we examined the phenotype of transgenic tobacco plants with elevated or reduced levels of miR156. We found that leaf shape, the density of abaxial trichomes, the number of leaf veins, the number of stomata, the size and density of epidermal cells, patterns of epidermal cell staining, the content of chlorophyll and the rate of photosynthesis, are all affected by miR156. These newly identified miR156-regulated traits therefore can be used to distinguish between juvenile and adult phases of development in tobacco, and provide a starting point for future studies of vegetative phase change in the family Solanaceae.

Silencing of miR156 in rice confers enhanced resistance to brown planthopper through reducing JA and JA-Ile biosynthesis. Rice brown planthopper (BPH, Nilaparvata lugens Stål) threatens the sustainability of rice production and global food security. Due to the rapid adaptation of BPH to current germplasms in rice, development of novel types of resistant germplasms becomes increasingly important. Plant ontogenetic defense against pathogen and herbivores offers a broad spectrum and durable resistance, and has been experimentally tested in many plants; however, the underlying molecular mechanism remains unclear. miR156 is the master regulator of ontogeny in plants; modulation of miR156 is, therefore, expected to cause corresponding changes in BPH resistance. To test this hypothesis, we silenced miR156 using a target mimicry method in rice, and analyzed the resistance of miR156-silenced plants (MIM156) to BPH. MIM156 plants exhibited enhanced resistance to BPH based on analyses of honeydew excretion, nymph survival, fecundity of BPH, and the survival ratio of rice plants after BPH infestation. Molecular analysis indicated that the expression of MPK3, MPK6, and WRKY70, three genes involved in BPH resistance and jasmonic acid (JA) signaling, was altered in MIM156 plants. The JA and bioactive jasmonoyl-isoleucine levels and the expression of genes involved in JA biosynthesis were significantly reduced in MIM156 plants. Restoration of JA level by exogenous application increased the number of BPH feeding on MIM156 plants and reduced its resistance to BPH. Our findings suggest that miR156 negatively regulates BPH resistance by increasing JA level in rice; therefore, modulation of miR156-SPLs' pathway may offer a promising way to breed rice varieties with enhanced resistance against BPH and elite agronomically important traits.

Drought is one of the critical factors limiting reproductive yields of rice and other crops globally. However, little is known about the molecular mechanism underlying reproductive development under drought stress in rice. To explore the potential gene function for improving rice reproductive development under drought, a drought induced gene, Oryza sativa Drought-Induced LTP (OsDIL) encoding a lipid transfer protein, was identified from our microarray data and selected for further study. OsDIL was primarily expressed in the anther and mainly responsive to abiotic stresses, including drought, cold, NaCl, and stress-related plant hormone abscisic acid (ABA). Compared with wild type, the OsDIL-overexpressing transgenic rice plants were more tolerant to drought stress during vegetative development and showed less severe tapetal defects and fewer defective anther sacs when treated with drought at the reproductive stage. The expression levels of the drought-responsive genes RD22, SODA1, bZIP46 and POD, as well as the ABA synthetic gene ZEP1 were up-regulated in the OsDIL-overexpression lines but the ABA degradation gene ABAOX3 was down-regulated. Moreover, overexpression of OsDIL lessened the down-regulation by drought of anther developmental genes (OsC4, CYP704B2 and OsCP1), providing a mechanism supporting pollen fertility under drought. Overexpression of OsDIL significantly enhanced drought resistance in transgenic rice during reproductive development, while showing no phenotypic changes or yield penalty under normal conditions. Therefore, OsDIL is an excellent candidate gene for genetic improvement of crop yield in adaption to unfavorable environments.