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

Exploring genetic methods to improve yield in grain crops such as rice (Oryza sativa) is essential to help meet the needs of the increasing population. Here, we report that rice ONAC096 affects grain yield by regulating leaf senescence and panicle number. ONAC096 expression increased rapidly in rice leaves upon the initiation of aging- and dark-induced senescence. Two independent T-DNA insertion mutants (onac096-1 and onac096-2) with downregulated ONAC096 expression retained their green leaf color during natural senescence in the field, thus extending their photosynthetic capacity. Reverse-transcription quantitative PCR analysis showed that ONAC096 upregulated genes controlling chlorophyll degradation and leaf senescence. Repressed OsCKX2 (encoding cytokinin oxidase/dehydrogenase) expression in the onac096 mutants led to a 15% increase in panicle number without affecting grain weight or fertility. ONAC096 mediates abscisic acid (ABA)-induced leaf senescence by upregulating the ABA signaling genes ABA INSENSITIVE5 and ENHANCED EM LEVEL. The onac096 mutants showed a 16% increase in grain yield, highlighting the potential for using this gene to increase grain production.

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Leaf senescence is the final stage of leaf development and in cereal crops, the timing of senescence relative to grain filling has major effects on agronomic traits such as yield. Although many genetic factors are involved in the regulation of leaf senescence in cereals, the key regulators remain to be determined. Plant transcription factors with a conserved DOF (DNA-binding one zinc finger) domain play roles in multiple physiological processes. Here, we show a novel function for OsDOF24 as a repressor of leaf senescence in rice (Oryza sativa). In wild-type leaves, OsDOF24 expression rapidly decreased during natural senescence (NS) and dark-induced senescence (DIS). The gain-of-function mutant osdof24-D, which contains an enhancer-trap T-DNA in the OsDOF24 promoter, exhibited delayed leaf yellowing during NS and DIS. Transgenic plants overexpressing OsDOF24 showed the same phenotype during DIS. Reverse-transcription quantitative real-time PCR analysis revealed that senescence-associated genes (Osl85, Osl57 and OsNAP) and chlorophyll degradation genes (NYC1, NYC3 and SGR) were downregulated in the osdof24-D mutant during dark incubation. Among the phytohormones, only methyl jasmonate induced OsDOF24 expression. Furthermore, the reduced expression of jasmonate biosynthesis-related genes (OsLOX2, OsLOX8, OsHI-LOX, OsAOS1 and OsAOS2) in osdof24-D decreased endogenous jasmonate levels, resulting in delayed leaf senescence under DIS conditions. Yeast one-hybrid assays showed that OsDOF24 binds to the promoter region of OsAOS1. Taken together, our results demonstrate that OsDOF24 suppresses the induction of leaf senescence during vegetative growth by deactivating jasmonate biosynthetic pathways.