During crop cultivation, water-deficit conditions retard growth, thus reducing crop productivity. Therefore, uncovering the mechanisms behind drought tolerance is a critical task for crop improvement. Here, we show that the rice (Oryza sativa) WRKY transcription factor OsWRKY5 negatively regulates drought tolerance. We determined that OsWRKY5 was mainly expressed in developing leaves at the seedling and heading stages, and that its expression was reduced by drought stress and by treatment with NaCl, mannitol, and abscisic acid (ABA). Notably, the genome-edited loss-of-function alleles oswrky5-2 and oswrky5-3 conferred enhanced drought tolerance, measured as plant growth under water-deficit conditions. Conversely, the overexpression of OsWRKY5 in the activation-tagged line oswrky5-D resulted in higher susceptibility under the same conditions. The loss of OsWRKY5 activity increased sensitivity to ABA, thus promoting ABA-dependent stomatal closure. Transcriptome deep sequencing and reverse transcription quantitative polymerase chain reaction analyses demonstrated that the expression of abiotic stress-related genes including rice MYB2 (OsMYB2) was upregulated in oswrky5 knockout mutants and downregulated in oswrky5-D mutants. Moreover, dual-luciferase, yeast one-hybrid, and chromatin immunoprecipitation assays showed that OsWRKY5 directly binds to the W-box sequences in the promoter region of OsMYB2 and represses OsMYB2 expression, thus downregulating genes downstream of OsMYB2 in the ABA signaling pathways. Our results demonstrate that OsWRKY5 functions as a negative regulator of ABA-induced drought stress tolerance, strongly suggesting that inactivation of OsWRKY5 or manipulation of key OsWRKY5 targets could be useful to improve drought tolerance in rice cultivars.

Leaf senescence is the final stage of leaf development and an important step that relocates nutrients for grain filling in cereal crops. Senescence occurs in an agedependent manner and under unfavorable environmental conditions such as deep shade, water deficit, and high salinity stresses. Although many transcription factors that modulate leaf senescence have been identified, the mechanisms that regulate leaf senescence in response to environmental conditions remain elusive. Here, we show that rice (Oryza sativa) ETHYLENE RESPONSE FACTOR 101 (OsERF101) promotes the onset and progression of leaf senescence. OsERF101 encodes a predicted transcription factor and OsERF101 transcript levels rapidly increased in rice leaves during dark-induced senescence (DIS), indicating that OsERF101 is a senescence-associated transcription factor. Compared with wild type, the oserf101 T-DNA knockout mutant showed delayed leaf yellowing and higher chlorophyll contents during DIS and natural senescence. Consistent with its delayed-yellowing phenotype, the oserf101 mutant exhibited downregulation of genes involved in chlorophyll degradation, including rice NAM, ATAF1/2, and CUC2 (OsNAP), STAY-GREEN (SGR), NON-YELLOW COLORING 1 (NYC1), and NYC3 during DIS. After methyl jasmonate treatment to induce rapid leaf de-greening, the oserf101 leaves retained more chlorophyll compared with wild type, indicating that OsERF101 is involved in promoting jasmonic acid (JA)-induced leaf senescence. Consistent with the involvement of JA, the expression of the JA signaling genes OsMYC2/JA INSENSITIVE 1 (OsJAI1) and CORONATINE INSENSITIVE 1a (OsCOI1a), was downregulated in the oserf101 leaves during DIS. Transient transactivation and chromatin immunoprecipitation assays revealed that OsERF101 directly binds to the promoter regions of OsNAP and OsMYC2, which activate genes involved in chlorophyll degradation and JA signaling-mediated leaf senescence. These

Lesion mimic mutants (LMMs) are usually controlled by single recessive mutations that cause the formation of necrotic lesions without pathogen invasion. These genetic defects are useful to reveal the regulatory mechanisms of defense-related programmed cell death in plants. Molecular evidence has been suggested that some of LMMs are closely associated with the regulation of leaf senescence in rice (Oryza sativa). Here, we characterized the mutation underlying spotted leaf4 (spl4), which results in lesion formation and also affects leaf senescence in rice. Map-based cloning revealed that the γ ray-induced spl4-1 mutant has a single base substitution in the splicing site of the SPL4 locus, resulting in a 13-bp deletion within the encoded microtubule-interacting-and-transport (MIT) spastin protein containing an AAA-type ATPase domain. The T-DNA insertion spl4-2 mutant exhibited spontaneous lesions similar to those of the spl4-1 mutant, confirming that SPL4 is responsible for the LMM phenotype. In addition, both spl4 mutants exhibited delayed leaf yellowing during dark-induced or natural senescence. Western blot analysis of spl4 mutant leaves suggested possible roles for SPL4 in the degradation of photosynthetic proteins. Punctate signals of SPL4-fused fluorescent proteins were detected in the cytoplasm, similar to the cellular localization of animal spastin. Based on these findings, we propose that SPL4 is a plant spastin that is involved in multiple aspects of leaf development, including senescence.

The vegetative-to-reproductive transition requires the complex, coordinated activities of many transcriptional regulators. Rice (Oryza sativa), a facultative short-day (SD) plant, flowers early under SD (≤10 h light/day) and late under long-day (LD; ≥14 h light/day) conditions. Here, we demonstrate that rice LATE FLOWERING SEMI-DWARF (LFS) encodes an APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factor that promotes flowering under non-inductive LD conditions. LFS showed diurnal expression peaking at dawn, and transcript levels increased gradually until heading. Mutation of LFS delayed flowering under LD but not SD conditions. Expression of the LD-specific floral repressor gene LEAFY COTYLEDON2 AND FUSCA3-LIKE 1 (OsLFL1) was upregulated in lfs knockout mutants, and LFS bound directly to the GCC-rich motif in the OsLFL1 promoter, repressing OsLFL1 expression. This suggests that increased LFS activity during vegetative growth gradually attenuates OsLFL1 activity. Subsequent increases in Early heading date 1, Heading date 3a, and RICE FLOWERING LOCUS T 1 expression result in flowering under noninductive LD conditions. LFS did not affect the expression of other OsLFL1 regulators, including OsMADS50, OsMADS56, VERNALIZATION INSENSITIVE3-LIKE 2, and GERMINATION DEFECTIVE 1, or interact with them. Our results demonstrate the novel roles of LFS in inducing flowering under natural LD conditions.

No abstract