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.

The timing of flowering is a crucial factor for successful grain production at a wide range of latitudes. Domestication of rice (Oryza sativa) included selection for natural alleles of flowering-time genes that allow rice plants to adapt to broad geographic areas. Here, we describe the role of natural alleles of CIRCADIAN CLOCK ASSOCIATED1 (OsCCA1) in cultivated rice based on analysis of single-nucleotide polymorphisms deposited in the International Rice Genebank Collection Information System database. Rice varieties harboring japonica-type OsCCA1 alleles (OsCCA1a haplotype) flowered earlier than those harboring indica-type OsCCA1 alleles (OsCCA1d haplotype). In the japonica cultivar “Dongjin”, a T-DNA insertion in OsCCA1a resulted in late flowering under long-day and short-day conditions, indicating that OsCCA1 is a floral inducer. Reverse transcription quantitative PCR analysis showed that the loss of OsCCA1a function induces the expression of the floral repressors PSEUDO-RESPONSE REGULATOR 37 (OsPRR37) and Days to Heading 8 (DTH8), followed by repression of the Early heading date 1 (Ehd1)–Heading date 3a (Hd3a)–RICE FLOWERING LOCUS T 1 (RFT1) pathway. Binding affinity assays indicated that OsCCA1 binds to the promoter regions of OsPRR37 and DTH8. Naturally occurring OsCCA1 alleles are evolutionarily conserved in cultivated rice (O. sativa). Oryza rufipogon-I (Or-I) and Or-III type accessions, representing the ancestors of O. sativa indica and japonica, harbored indica- and japonica-type OsCCA1 alleles, respectively. Taken together, our results demonstrate that OsCCA1 is a likely domestication locus that has contributed to the geographic adaptation and expansion of cultivated rice.

Light is an important factor influencing melatonin synthesis in response to cadmium treatment in rice. However, the effects of light quality on, and the involvement of phytochrome light receptors in, melatonin production have not been explored. In this study, we used light-emitting diodes (LEDs) to investigate the effect of light wavelength on melatonin synthesis, and the role of phytochromes in light-dependent melatonin induction in rice. Upon cadmium treatment, peak melatonin production was observed under combined red and blue (R + B) light, followed by red (R) and blue light (B). However, both far-red (FR) LED light and dark treatment (D) failed to induce melatonin production. Similarly, rice seedlings grown under the R + B treatment showed the highest melatonin synthesis, followed by those grown under B and R. These findings were consistent with the results of our cadmium treatment experiment. To further confirm the effects of light quality on melatonin synthesis, we employed rice photoreceptor mutants lacking functional phytochrome genes. Melatonin induction was most inhibited in the phytochrome A mutant (phyA) followed by the phyB mutant under R + B treatment, whereas phyB produced the least amount of melatonin under R treatment. These results indicate that PhyB is an R light receptor. Expression analyses of genes involved in melatonin biosynthesis clearly demonstrated that tryptophan decarboxylase (TDC) played a key role in phytochrome-mediated melatonin induction when rice seedlings were challenged with cadmium.

Flowering time is a crucial adaptive response to seasonal variation in plants and is regulated by environmental cues such as photoperiod and temperature. In this study, we demonstrated the regulatory function of rice CRYPTOCHROME‐INTERACTING BASIC HELIX‐LOOP‐HELIX 1‐LIKE (OsCIBL1) in flowering time. Overexpression of OsCIB1L promoted flowering, whereas the oscib1l knockout mutation did not alter flowering time independent of photoperiodic conditions. Cryptochromes (CRYs) are blue light photoreceptors that enable plants to sense photoperiodic changes. OsCIBL1 interacted with OsCRY2, a member of the rice CRY family (OsCRY1a, OsCRY1b, and OsCRY2), and bound to the Early heading date 1 (Ehd1) promoter, activating the rice‐specific Ehd1‐Heading date 3a/RICE FLOWERING LOCUS T 1 pathway for flowering induction. Dual‐luciferase reporter assays showed that the OsCIBL1‐OsCRY2 complex required blue light to induce Ehd1 transcription. Natural alleles resulting from nonsynonymous single nucleotide polymorphisms in OsCIB1L and OsCRY2 may contribute to the adaptive expansion of rice cultivation areas. These results expand our understanding of the molecular mechanisms controlling rice flowering and highlight the importance of blue light‐responsive genes in the geographic distribution of rice.

SUMMARY: Salt stress impairs photosynthetic efficiency and consequently reduces the growth, development, and grain yield of crop plants. The formation of hydrophobic barriers in the root endodermis, including the suberin lamellae and Casparian strips, is a key adaptive strategy for salt stress tolerance. In this study, we identified the role of the rice NAC transcription factor, ONAC005, in salt stress tolerance. ONAC005 expression was induced by NaCl and abscisic acid (ABA). Expression analysis using the β‐glucuronidase reporter gene driven by the ONAC005 promoter revealed that ONAC005 is predominantly expressed in the stele and endodermis of rice roots. The null mutation of ONAC005 increased sodium ion levels in the shoots and roots, indicating susceptibility to salt stress, whereas ONAC005 overexpression enhanced tolerance to salt stress by reducing sodium ion accumulation. Yeast one‐hybrid, chromatin immunoprecipitation, and dual‐luciferase assays demonstrated that ONAC005 upregulates the expression of trehalose‐6‐phosphate synthase 8 (OsTPS8) by directly binding to its promoter region, leading to increased trehalose accumulation. ONAC005 enhances the formation of the root hydrophobic barrier by upregulating OsTPS8 expression under salt stress. Furthermore, considering the altered expression of ABA signaling and responsive genes, ONAC005 regulates the expression of genes in specific stress‐responsive pathways that are independent of OsTPS8‐mediated signaling. These results indicate that ONAC005 positively regulates hydrophobic barrier formation in the roots, thereby enhancing salt stress tolerance in rice.