Genomic basis of multiphase evolution driving divergent selection of zinc-finger homeodomain genes.

Gene families divergently evolve and become adapted as different genes with specific structures and functions in living organisms. We performed comprehensive structural and functional analyses of Zinc-finger homeodomain genes (ZF-HDs), including Mini zinc-finger genes (MIFs) and Zinc-finger with homeodomain genes (ZHDs), displaying competitive functions each other. Intensive annotation updates for 90 plant genomes verified that most MIFs (MIF-Is) exhibited distinct motif compositions from ZHDs, although some MIFs (MIF-Zs) contained ZHD-specific motifs. Phylogenetic analyses suggested that MIF-Zs and ZHDs originated from the same ancestral gene, whereas MIF-Is emerged from a distinct progenitor. We used a gene-editing system to identify a novel function of MIF-Is in rice: regulating the surface material patterns in anthers and pollen through transcriptional regulation by interacting ZHDs. Kingdom-wide investigations determined that (i) ancestral MIFs diverged into MIF-Is and MIF-Zs in the last universal common ancestor, (ii) integration of HD into the C-terminal of MIF-Zs created ZHDs after emergence of green plants and (iii) MIF-Is and ZHDs subsequently expanded independently into specific plant lineages, with additional formation of MIF-Zs from ZHDs. Our comprehensive analysis provides genomic evidence for multiphase evolution driving divergent selection of ZF-HDs.

Cytokinin increases vegetative growth period by suppressing florigen expression in rice and maize.

Increasing the vegetative growth period of crops can increase biomass and grain yield. In rice (Oryza sativa), the concentration of trans -zeatin, an active cytokinin, was high in the leaves during vegetative growth and decreased rapidly upon induction of florigen expression, suggesting that this hormone is involved in the regulation of the vegetative phase. To elucidate whether exogenous cytokinin application influences the length of the vegetative phase, we applied 6-benzylaminopurine (BAP) to rice plants at various developmental stages. Our treatment delayed flowering time by 8-9 days when compared with mock-treated rice plants, but only at the transition stage when the flowering signals were produced. Our observations also showed that flowering in the paddy field is delayed by thidiazuron, a stable chemical that mimics the effects of cytokinin. The transcript levels of florigen genes Heading date 3a (Hd3a) and Rice Flowering locus T1 (RFT1) were significantly reduced by the treatment, but the expression of Early heading date 1 (Ehd1), a gene found directly upstream of the florigen genes, was not altered. In maize (Zea mays), similarly, BAP treatment increased the vegetative phage by inhibiting the expression of ZCN8, an ortholog of Hd3a. We showed that cytokinin treatment induced the expression of two type-A response regulators (OsRR1 and OsRR2) which interacted with Ehd1, a type-B response regulator. We also observed that cytokinin did not affect flowering time in ehd1 knockout mutants. Our study indicates that cytokinin application increases the duration of the vegetative phase by delaying the expression of florigen genes in rice and maize by inhibiting Ehd1.

A myosin XI adaptor, TAPE, is essential for pollen tube elongation in rice.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

Recurrent mutations promote widespread structural and functional divergence of MULE-derived genes in plants.

Transposable element (TE)-derived genes are increasingly recognized as major sources conferring essential traits in agriculturally important crops but underlying evolutionary mechanisms remain obscure. We updated previous annotations and constructed 18,744 FAR-RED IMPAIRED RESPONSE1 (FAR1) genes, a transcription factor family derived from Mutator-like elements (MULEs), from 80 plant species, including 15,546 genes omitted in previous annotations. In-depth sequence comparison of the updated gene repertoire revealed that FAR1 genes underwent continuous structural divergence via frameshift and nonsense mutations that caused premature translation termination or specific domain truncations. CRISPR/Cas9-based genome editing and transcriptome analysis determined a novel gene involved in fertility-regulating transcription of rice pollen, denoting the functional capacity of our re-annotated gene models especially in monocots which had the highest copy numbers. Genomic evidence showed that the functional gene adapted by obtaining a shortened form through a frameshift mutation caused by a tandem duplication of a 79-bp sequence resulting in premature translation termination. Our findings provide improved resources for comprehensive studies of FAR1 genes with beneficial agricultural traits and unveil novel evolutionary mechanisms generating structural divergence and subsequent adaptation of TE-derived genes in plants.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice.

Successful delivery of sperm cells to the embryo sac in higher plants is mediated by pollen tube growth. The molecular mechanisms underlying pollen germination and tube growth in crop plants remain rather unclear, although these mechanisms are crucial to plant reproduction and seed formation. By screening pollen-specific gene mutants in rice (Oryza sativa), we identified a T-DNA insertional mutant of Germinating modulator of rice pollen (GORI) that showed a one-to-one segregation ratio for wild type (WT) to heterozygous. GORI encodes a seven-WD40-motif protein that is homologous to JINGUBANG/REN4 in Arabidopsis. GORI is specifically expressed in rice pollen, and its protein is localized in the nucleus, cytosol and plasma membrane. Furthermore, a homozygous mutant, gori-2, created through CRISPR-Cas9 clearly exhibited male sterility with disruption of pollen tube germination and elongation. The germinated pollen tube of gori-2 exhibited decreased actin filaments and altered pectin distribution. Transcriptome analysis revealed that 852 pollen-specific genes were downregulated in gori-2 compared with the WT, and Gene Ontology enrichment analysis indicated that these genes are strongly associated with cell wall modification and clathrin coat assembly. Based on the molecular features of GORI, phenotypical observation of the gori mutant and its interaction with endocytic proteins and Rac GTPase, we propose that GORI plays key roles in forming endo-/exocytosis complexes that could mediate pollen tube growth in rice.

Comparative transcriptome analysis of pollen and anther wall reveals novel insights into the regulatory mechanisms underlying anther wall development and its dehiscence in rice.

To further understand the regulatory mechanism for anther dehiscence in rice, we carried out transcriptome analysis for the following two tissues: the anther wall and pollen at the anthesis stage. With the anatomical meta-expression data, in addition to these tissues, the differentially expressed genes (DEGs) between the two tissues were further refined to identify 1,717 pollen-preferred genes and 534 anther wall-preferred genes. A GUS transgenic line and RT-qPCR analysis for anther wall-preferred genes supported the fidelity of our gene candidates for further analysis. The refined DEGs were functionally classified through Gene Ontology (GO) enrichment and MapMan analyses. Through the analysis of cis-acting elements and alternative splicing variants, we also suggest the feature of regulatory sequences in promoter regions for anther wall-preferred expression and provide information of the unique splicing variants in anther wall. Subsequently, it was found that hormone signaling and the resulting transcriptional regulation pathways may play an important role in anther dehiscence and anther wall development. Our results could provide useful insights into future research to broaden the molecular mechanism of anther dehiscence or anther wall development in rice.

CAFRI-Rice: CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice.

Rice (Oryza sativa L.) is a staple crop with agricultural traits that have been intensively investigated. However, despite the variety of mutant population and multi-omics data that have been generated, rice functional genomic research has been bottlenecked due to the functional redundancy in the genome. This phenomenon has masked the phenotypes of knockout mutants by functional compensation and redundancy. Here, we present an intuitive tool, CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice (CAFRI-Rice; cafri-rice.khu.ac.kr). To create this tool, we generated a phylogenetic heatmap that can estimate the similarity between protein sequences and expression patterns, based on 2,617 phylogenetic trees and eight tissue RNA-sequencing datasets. In this study, 33,483 genes were sorted into 2,617 families, and about 24,980 genes were tested for functional redundancy using a phylogenetic heatmap approach. It was predicted that 7,075 genes would have functional redundancy, according to the threshold value validated by an analysis of 111 known genes functionally characterized using knockout mutants and 5,170 duplicated genes. In addition, our analysis demonstrated that an anther/pollen-preferred gene cluster has more functional redundancy than other clusters. Finally, we showed the usefulness of the CAFRI-Rice-based approach by overcoming the functional redundancy between two root-preferred genes via loss-of-function analyses as well as confirming the functional dominancy of three genes through a literature search. This CAFRI-Rice-based target selection for CRISPR/Cas9-mediated mutagenesis will not only accelerate functional genomic studies in rice but can also be straightforwardly expanded to other plant species.

CTP synthase is essential for early endosperm development by regulating nuclei spacing.

Cereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild-type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10-16 clumped nuclei while wild-type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild-type nuclei were evenly distributed by microtubules while the enl2-2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.

Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa).

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

OsPP2C09 Is a Bifunctional Regulator in Both ABA-Dependent and Independent Abiotic Stress Signaling Pathways.

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.

Genome-wide analysis of RopGEF gene family to identify genes contributing to pollen tube growth in rice (Oryza sativa).

BACKGROUND: In plants, the key roles played by RopGEF-mediated ROP signaling in diverse processes, including polar tip growth, have been identified. Despite their important roles in reproduction, a comprehensive analysis of RopGEF members has not yet been performed in rice (Oryza sativa). To determine whether RopGEF regulators are involved in rice pollen tube growth, we performed genome-wide analysis of this family in rice. RESULTS: Phylogenomic and meta-expression analysis of eleven RopGEFs in rice showed that four genes were preferentially expressed in mature pollen. These four genes contain the plant-specific Rop nucleotide exchanger (PRONE) domain and possible phosphorylated residues, suggesting a conserved role in polar tip growth with Arabidopsis thaliana. In subcellular localization analysis of the four RopGEFs through tobacco (Nicotiana benthamiana) infiltration, four proteins were predominantly identified in plasma membrane. Moreover, double mutants of RopGEF2/8 exhibited reduced pollen germination, causing partial male sterility. These genes possess unique cis-acting elements in their promoters compared with the other RopGEF genes. CONCLUSIONS: In this study, four RopGEF genes were identified as pollen-specific gene in eleven members of rice, and the expression pattern, promoter analysis, and evolutionary relationship of the RopGEF family were studied compared with Arabidopsis. Our study indicated that four RopGEF genes might function during pollen germination in distinct subcellular localization. Our study could provide valuable information on the functional study of RopGEF in rice.

RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development.

Root hairs are important for absorption of nutrients and water from the rhizosphere. The Root Hair Defective-Six Like (RSL) Class II family of transcription factors is expressed preferentially in root hairs and has a conserved role in root hair development in land plants. We functionally characterized the seven members of the RSL Class II subfamily in the rice (Oryza sativa) genome. In root hairs, six of these genes were preferentially expressed and four were strongly expressed. Phenotypic analysis of each mutant revealed that Os07g39940 plays a major role in root hair formation, based on observations of a short root hair phenotype in those mutants. Overexpression (OX) for each of four family members in rice resulted in an increase in the density and length of root hairs. These four members contain a transcription activation domain and are targeted to the nucleus. They interact with rice Root Hairless1 (OsRHL1), a key regulator of root hair development. When heterologously expressed in epidermal cells of Nicotiana benthamiana leaves, OsRHL1 was predominantly localized to the cytoplasm. When coexpressed with each of the four RSL Class II members, however, OsRLH1 was translocated to the nucleus. Transcriptome analysis using Os07g39940-OX plants revealed that 86 genes, including Class III peroxidases, were highly up-regulated. Furthermore, reactive oxygen species levels in the root hairs were increased in Os07g39940-OX plants but were drastically reduced in the os07g39940 and rhl1 mutants. Our results demonstrate that RSL Class II members function as essential regulators of root hair development in rice.

Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Systematic Analysis of Cold Stress Response and Diurnal Rhythm Using Transcriptome Data in Rice Reveals the Molecular Networks Related to Various Biological Processes.

Rice (Oryza sativa L.), a staple crop plant that is a major source of calories for approximately 50% of the human population, exhibits various physiological responses against temperature stress. These responses are known mechanisms of flexible adaptation through crosstalk with the intrinsic circadian clock. However, the molecular regulatory network underlining this crosstalk remains poorly understood. Therefore, we performed systematic transcriptome data analyses to identify the genes involved in both cold stress responses and diurnal rhythmic patterns. Here, we first identified cold-regulated genes and then identified diurnal rhythmic genes from those (119 cold-upregulated and 346 cold-downregulated genes). We defined cold-responsive diurnal rhythmic genes as CD genes. We further analyzed the functional features of these CD genes through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses and performed a literature search to identify functionally characterized CD genes. Subsequently, we found that light-harvesting complex proteins involved in photosynthesis strongly associate with the crosstalk. Furthermore, we constructed a protein-protein interaction network encompassing four hub genes and analyzed the roles of the Stay-Green (SGR) gene in regulating crosstalk with sgr mutants. We predict that these findings will provide new insights in understanding the environmental stress response of crop plants against climate change.

A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement.

Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist's mind, as it allows genome editing in multiple biological systems.

Chromatin interacting factor OsVIL2 increases biomass and rice grain yield.

Grain number is an important agronomic trait. We investigated the roles of chromatin interacting factor Oryza sativa VIN3-LIKE 2 (OsVIL2), which controls plant biomass and yield in rice. Mutations in OsVIL2 led to shorter plants and fewer grains whereas its overexpression (OX) enhanced biomass production and grain numbers when compared with the wild type. RNA-sequencing analyses revealed that 1958 genes were up-regulated and 2096 genes were down-regulated in the region of active division within the first internodes of OX plants. Chromatin immunoprecipitation analysis showed that, among the downregulated genes, OsVIL2 was directly associated with chromatins in the promoter region of CYTOKININ OXIDASE/DEHYDROGENASE2 (OsCKX2), a gene responsible for cytokinin degradation. Likewise, active cytokinin levels were increased in the OX plants. We conclude that OsVIL2 improves the production of biomass and grain by suppressing OsCKX2 chromatin.

Rice Senescence-Induced Receptor-Like Kinase (OsSRLK) Is Involved in Phytohormone-Mediated Chlorophyll Degradation.

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.