Expansins are key regulators of cell-wall extension and are also involved in the abiotic stress response. In this study, we evaluated the function of OsEXPA7 involved in salt stress tolerance. Phenotypic analysis showed that OsEXPA7 overexpression remarkably enhanced tolerance to salt stress. OsEXPA7 was highly expressed in the shoot apical meristem, root, and the leaf sheath. Promoter activity of OsEXPA7:GUS was mainly observed in vascular tissues of roots and leaves. Morphological analysis revealed structural alterations in the root and leaf vasculature of OsEXPA7 overexpressing (OX) lines. OsEXPA7 overexpression resulted in decreased sodium ion (Na⁺) and accumulated potassium ion (K⁺) in the leaves and roots. Under salt stress, higher antioxidant activity was also observed in the OsEXPA7-OX lines, as indicated by lower reactive oxygen species (ROS) accumulation and increased antioxidant activity, when compared with the wild-type (WT) plants. In addition, transcriptional analysis using RNA-seq and RT-PCR revealed that genes involved in cation exchange, auxin signaling, cell-wall modification, and transcription were differentially expressed between the OX and WT lines. Notably, salt overly sensitive 1, which is a sodium transporter, was highly upregulated in the OX lines. These results suggest that OsEXPA7 plays an important role in increasing salt stress tolerance by coordinating sodium transport, ROS scavenging, and cell-wall loosening.

He onset of leaf senescence is triggered by external cues and internal factors such as phytohormones and signaling pathways involving transcription factors (TFs). Abscisic acid (ABA) strongly induces senescence and endogenous ABA levels are finely tuned by many senescence-associated TFs. Here, we report on the regulatory function of the senescence-induced TF OsWRKY5 TF in rice (Oryza sativa). OsWRKY5 expression was rapidly upregulated in senescing leaves, especially in yellowing sectors initiated by aging or dark treatment. A T-DNA insertion activation-tagged OsWRKY5-overexpressing mutant (termed oswrky5-D) promoted leaf senescence under natural and dark-induced senescence (DIS) conditions. By contrast, a T-DNA insertion oswrky5-knockdown mutant (termed oswrky5) retained leaf greenness during DIS. Reverse-transcription quantitative PCR (RT-qPCR) showed that OsWRKY5 upregulates the expression of genes controlling chlorophyll degradation and leaf senescence. Furthermore, RT-qPCR and yeast one-hybrid analysis demonstrated that OsWRKY5 indirectly upregulates the expression of senescence-associated NAM/ATAF1/2/CUC2 (NAC) genes including OsNAP and OsNAC2. Precocious leaf yellowing in the oswrky5-D mutant might be caused by elevated endogenous ABA concentrations resulting from upregulated expression of ABA biosynthesis genes OsNCED3, OsNCED4, and OsNCED5, indicating that OsWRKY is a positive regulator of ABA biosynthesis during leaf senescence. Furthermore, OsWRKY5 expression was suppressed by ABA treatment. Taken together, OsWRKY5 is a positive regulator of leaf senescence that upregulates senescence-induced NAC, ABA biosynthesis, and chlorophyll degradation genes.

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.

Chlorophyll breakdown is a vital catabolic process of leaf senescence as it allows the recycling of nitrogen and other nutrients. In the present study, we isolated rice senescence-induced receptor-like kinase (OsSRLK), whose transcription was upregulated in senescing rice leaves. The detached leaves of ossrlk mutant (ossrlk) contained more green pigment than those of the wild type (WT) during dark-induced senescence (DIS). HPLC and immunoblot assay revealed that degradation of chlorophyll and photosystem II proteins was repressed in ossrlk during DIS. Furthermore, ultrastructural analysis revealed that ossrlk leaves maintained the chloroplast structure with intact grana stacks during dark incubation; however, the retained green color and preserved chloroplast structures of ossrlk did not enhance the photosynthetic competence during age-dependent senescence in autumn. In ossrlk, the panicles per plant was increased and the spikelets per panicle were reduced, resulting in similar grain productivity between WT and ossrlk. By transcriptome analysis using RNA sequencing, genes related to phytohormone, senescence, and chlorophyll biogenesis were significantly altered in ossrlk compared to those in WT during DIS. Collectively, our findings indicate that OsSRLK may degrade chlorophyll by participating in a phytohormone-mediated pathway.

In rice (Oryza sativa), moderate leaf rolling increases photosynthetic competence and raises grain yield; therefore, this important agronomic trait has attracted much attention from plant biologists and breeders. However, the relevant molecular mechanism remains unclear. Here, we isolated and characterized Rolled Fine Striped (RFS), a key gene affecting rice leaf rolling, chloroplast development, and reactive oxygen species (ROS) scavenging. The rfs-1 gamma-ray allele and the rfs-2 T-DNA insertion allele of RFS failed to complement each other and their mutants had similar phenotypes, producing extremely incurved leaves due to defective development of vascular cells on the adaxial side. Map-based cloning showed that the rfs-1 mutant harbors a 9-bp deletion in a gene encoding a predicted CHD3/Mi-2 chromatin remodeling factor belonging to the SNF2-ATP-dependent chromatin remodeling family. RFS was expressed in various tissues and accumulated mainly in the vascular cells throughout leaf development. Furthermore, RFS deficiency resulted in a cell death phenotype that was caused by ROS accumulation in developing leaves. We found that expression of five ROS-scavenging genes [encoding catalase C, ascorbate peroxidase 8, a putative copper/zinc superoxide dismutase (SOD), a putative SOD, and peroxiredoxin IIE2] decreased in rfs-2 mutants. Western-blot and chromatin immunoprecipitation (ChIP) assays demonstrated that rfs-2 mutants have reduced H3K4me3 levels in ROS-related genes. Loss-of-function in RFS also led to multiple developmental defects, affecting pollen development, grain filling, and root development. Our results suggest that RFS is required for many aspects of plant development and its function is closely associated with epigenetic regulation of genes that modulate ROS homeostasis.

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.