MicroRNAs are 19- to 22-nucleotide small noncoding RNAs that have been implicated in abiotic stress responses. In this study, we found that knockdown of microRNA166, using the Short Tandem Target Mimic (STTM) system, resulted in morphological changes that confer drought resistance in rice (). From a large-scale screen for miRNA knockdown lines in rice, we identified miR166 knockdown lines (STTM166); these plants exhibit a rolled-leaf phenotype, which is normally displayed by rice plants under drought stress. The leaves of STTM166 rice plants had smaller bulliform cells and abnormal sclerenchymatous cells, likely causing the rolled-leaf phenotype. The STTM166 plants had reduced stomatal conductance and showed decreased transpiration rates. The STTM166 lines also exhibited altered stem xylem and decreased hydraulic conductivity, likely due to the reduced diameter of the xylem vessels. Molecular analyses identified rice (), a member of HD-Zip III gene family, as a major target of miR166; moreover, rice plants overexpressing a miR166-resistant form of resembled the STTM166 plants, including leaf rolling and higher drought resistance. The genes downstream of miR166- consisted of polysaccharide synthesis-related genes that may contribute to cell wall formation and vascular development. Our results suggest that drought resistance in rice can be increased by manipulating miRNAs, which leads to developmental changes, such as leaf rolling and reduced diameter of the xylem, that mimic plants' natural responses to water-deficit stress.

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SUMMARYHormones play pivotal roles in regulating plant development, growth, and stress responses, and cross-talk among different hormones fine-tunes various aspects of plant physiology. Jasmonic acid (JA) is important for plant defense against herbivores and necrotic fungi and also regulates flower development; in addition, Arabidopsis mutants over-producing JA usually have stunted stems and wound-induced jasmonates suppress Arabidopsis growth, suggesting that JA is also involved in stem elongation. Gibberellins (GAs) promote stem and leaf growth and modulate seed germination, flowering time, and the development of flowers, fruits, and seeds. However, little is known about the interaction between the JA and GA pathways. Two calcium-dependent protein kinases, CDPK4 and CDPK5, are important suppressors of JA accumulation in a wild tobacco species, Nicotiana attenuata. The stems of N. attenuata silenced in CDPK4 and CDPK5 (irCDPK4/5 plants) had dramatically increased levels of JA and exhibited stunted elongation and had very high contents of secondary metabolites. Genetic analysis indicated that the high JA levels in irCDPK4/5 stems accounted for the suppressed stem elongation and the accumulation of secondary metabolites. Supplementation of GA 3 to irCDPK4/5 plants largely restored normal stem growth to wild-type levels. Measures of GA levels indicated that over-accumulation of JA in irCDPK4/5 stems inhibited the biosynthesis of GAs. Finally, we show that JA antagonizes GA biosynthesis by strongly inhibiting the transcript accumulation of GA20ox and possibly GA13ox, the key genes in GA production, demonstrating that high JA levels antagonize GA biosynthesis in stems.

Summary Degeneration of apical spikelets and reduced panicle fertility are common reasons for low seed‐setting rate in rice (Oryza sativa). However, little is known about the underlying molecular mechanisms. Here, we report a novel degenerated panicle and partial sterility 1 (dps1) mutant that showed panicle apical degeneration and reduced fertility in middle spikelets. dps1 plants were characterized by small whitish anthers with altered cuticle morphology and absence of pollen grains. Amounts of cuticular wax and cutin were significantly reduced in dps1 anthers. Panicles of dps1 plants showed an accumulation of reactive oxygen species (ROS), lower antioxidant activity, and increased programmed cell death. Map‐based cloning revealed that DPS1 encodes a mitochondrial‐localized protein containing a cystathionine β‐synthase domain that showed the highest expression in panicles and anthers. DPS1 physically interacted with mitochondrial thioredoxin proteins Trx1 and Trx20, and it participated in ROS scavenging. Global gene expression analysis in dps1 revealed that biological processes related to fatty acid metabolism and ROS homeostasis were significantly affected, and the expression of key genes involved in wax and cutin biosynthesis were downregulated. These results suggest that DPS1 plays a vital role in regulating ROS homeostasis, anther cuticle formation, and panicle development in rice.

Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1) show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1), nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogenous expression of NAL1 in fission yeast (Schizosaccharomyces pombe) further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division.

Drought causes osmotic stress and rapidly triggers abscisic acid (ABA) accumulation in plants. The roles of various ABA receptors in drought tolerance and molecular mechanisms regulating ABA receptor stability needs to be elucidated. Here, we report that Arabidopsis plants overexpressing PYL9, one of the 14 pyrabactin resistance (PYR)/pyrabactin resistance-like (PYL)/regulatory component of ABA receptors (RCAR) family ABA receptors, gained drought tolerance trait. Osmotic stress induced accumulation of the PYL9 protein, which was regulated by the 26S proteasome. PYL9 interacted with two highly homologous plant U-box E3 ubiquitin ligases PUB22 and PUB23. In the cell-free degradation assay, the degradation of GST-PYL9 was accelerated in protein extract from plants overexpressing PUB22 but slowed down in protein extract from the pub22 pub23 double mutant. The in vivo decay of Myc-PYL9 was significantly reduced in the pub22 pub23 double mutant as compared with the wild-type. Additionally, PUB22 also interacted with other ABA receptors such as PYL5, PYL7 and PYL8. Considering the improved drought tolerance in the pub22 pub23 double mutant in previous studies, our results suggest that PUB22 and PUB23 negatively regulate drought tolerance in part by facilitating ABA receptors degradation.

Summary Shoot branching is regulated by multiple signals. Previous studies have indicated that sucrose may promote shoot branching through suppressing the inhibitory effect of the hormone strigolactone (SL). However, the molecular mechanisms underlying this effect are unknown. Here, we used molecular and genetic tools to identify the molecular targets underlying the antagonistic interaction between sucrose and SL. We showed that sucrose antagonizes the suppressive action of SL on tillering in rice and on the degradation of D53, a major target of SL signalling. Sucrose inhibits the gene expression of D3, the orthologue of the Arabidopsis F‐box MAX2 required for SL signalling. Overexpression of D3 antagonizes sucrose inhibition of D53 degradation and enables the SL inhibition of tillering under high sucrose. Sucrose prevents SL‐induced degradation of D14, the SL receptor involved in D53 degradation. In contrast to D3, D14 overexpression enhances D53 protein levels and sucrose‐induced tillering, even in the presence of SL. Our results show that sucrose inhibits SL response by affecting key components of SL signalling and, together with previous studies reporting the inhibition of SL synthesis by nitrate and phosphate, demonstrate the central role played by SLs in the regulation of plant architecture by nutrients.

Leaf morphology influences photosynthesis, transpiration and ultimately crop yield. However, the molecular mechanism of leaf development is still not fully understood. Here, we identified and characterized the narrow leaf21 (nal21) mutant in rice (Oryza sativa), showing a significant reduction in leaf width, leaf length and plant height, and increased tiller number. Microscopic observation revealed defects in the vascular system and reduced epidermal cell size and number in the nal21 leaf blade. Map-based cloning revealed that NAL21 encodes a ribosomal small subunit protein RPS3A. Ribosome-targeting antibiotics resistance assay and ribosome profiling showed a significant reduction in the free 40S ribosome subunit in the nal21 mutant. The nal21 mutant showed aberrant auxin responses in which multiple auxin response factors (ARFs) harboring upstream open reading frames (uORFs) in their 5′-untranslated region (5’-UTR) were repressed at the translational level. The WUSCHEL-related homeobox 3A (OsWOX3A) gene, a key transcription factor involved in leaf blade lateral outgrowth, is also under the translational regulation by RPS3A. Transformation with modified OsARF11, OsARF16 and OsWOX3A genomic DNA lacking uORFs rescued the narrow leaf phenotype of nal21 to a better extent than transformation with their native genomic DNA, implying that RPS3A could regulate translation of ARFs and WOX3A through uORFs. Our results demonstrate that proper translational regulation of key factors involved in leaf development is essential to maintain normal leaf morphology.