OsCPK12 phosphorylates OsCATA and OsCATC to regulate H2O2 homeostasis and improve oxidative stress tolerance in rice.

Calcium-dependent protein kinases (CPKs), the best-characterized calcium sensors in plants, regulate many aspects of plant growth and development as well as plant adaptation to biotic and abiotic stresses. However, how CPKs regulate the antioxidant defense system remains largely unknown. We previously found that impaired function of OsCPK12 leads to oxidative stress in rice, with more H2O2, lower catalase (CAT) activity, and lower yield. Here, we explored the roles of OsCPK12 in oxidative stress tolerance in rice. Our results show that OsCPK12 interacts with and phosphorylates OsCATA and OsCATC at Ser11. Knockout of either OsCATA or OsCATC leads to an oxidative stress phenotype accompanied by higher accumulation of H2O2. Overexpression of the phosphomimetic proteins OsCATAS11D and OsCATCS11D in oscpk12-cr reduced the level of H2O2 accumulation. Moreover, OsCATAS11D and OsCATCS11D showed enhanced catalase activity in vivo and in vitro. OsCPK12-overexpressing plants exhibited higher CAT activity as well as higher tolerance to oxidative stress. Our findings demonstrate that OsCPK12 affects CAT enzyme activity by phosphorylating OsCATA and OsCATC at Ser11 to regulate H2O2 homeostasis, thereby mediating oxidative stress tolerance in rice.

Photoperiod and gravistimulation-associated Tiller Angle Control 1 modulates dynamic changes in rice plant architecture.

KEY MESSAGE: TAC1 is involved in photoperiodic and gravitropic responses to modulate rice dynamic plant architecture likely by affecting endogenous auxin distribution, which could explain TAC1 widespread distribution in indica rice. Plants experience a changing environment throughout their growth, which requires dynamic adjustments of plant architecture in response to these environmental cues. Our previous study demonstrated that Tiller Angle Control 1 (TAC1) modulates dynamic changes in plant architecture in rice; however, the underlying regulatory mechanisms remain largely unknown. In this study, we show that TAC1 regulates plant architecture in an expression dose-dependent manner, is highly expressed in stems, and exhibits dynamic expression in tiller bases during the growth period. Photoperiodic treatments revealed that TAC1 expression shows circadian rhythm and is more abundant during the dark period than during the light period and under short-day conditions than under long-day conditions. Therefore, it contributes to dynamic plant architecture under long-day conditions and loose plant architecture under short-day conditions. Gravity treatments showed that TAC1 is induced by gravistimulation and negatively regulates shoot gravitropism, likely by affecting auxin distribution. Notably, the tested indica rice containing TAC1 displayed dynamic plant architecture under natural long-day conditions, likely explaining the widespread distribution of TAC1 in indica rice. Our results provide new insights into TAC1-mediated regulatory mechanisms for dynamic changes in rice plant architecture.

Associations Between Body Composition and Sensorineural Hearing Loss Among Adults Based on the UK Biobank.

OBJECTIVE: To explore the association between body composition and sensorineural hearing loss (SNHL). STUDY DESIGN: Cross-sectional study, prospective study and Mendelian randomization (MR) analyses. SETTING: UK Biobank. METHODS: This cross-sectional study included 147,296 adult participants with complete data on body composition and the speech-reception-threshold (SRT) test. We further conducted a prospective study with 129,905 participants without SNHL at baseline and followed up to 15 years to explore the association between body composition and new-onset SNHL. Multivariable logistic regression and Cox regression models were used. Subgroup analyses stratified by age and sex were performed. We further assessed the causal association between body composition and SNHL using two-sample MR analyses. RESULTS: Our cross-sectional study revealed that fat percentage, especially leg (odds ratio [OR] 1.46, p = .029) and arm (OR 1.43, p = .004), were significant risk factors for SNHL. However, fat-free mass, especially in the arm (OR 0.27, p < .001) and leg (OR 0.58, p < .001) showed significant protective effects against SNHL, which was substantially consistent with the results of the prospective study. In addition, we found that young women with SNHL were more susceptible to body composition indicators. However, MR analyses revealed no evidence of significant causal association. CONCLUSION: Fat percentage, especially in the leg and arm, was a significant risk factor for SNHL, whereas fat-free mass, especially in the leg and arm, had significant protective effects against SNHL, however, these associations may not be causal.

OsLDDT1, encoding a transmembrane structural DUF726 family protein, is essential for tapetum degradation and pollen formation in rice.

Formation of the pollen wall, which is mainly composed of lipid substances secreted by tapetal cells, is important to ensure pollen development in rice. Although several regulatory factors related to lipid biosynthesis during pollen wall formation have been identified in rice, the molecular mechanisms controlling lipid biosynthesis are unclear. In this study, we isolated the male-sterile rice mutant oslddt1 (leaked and delayed degraded tapetum 1). oslddt1 plants show complete pollen abortion resulting from delayed degradation of the tapetum and blocked formation of Ubisch bodies and pollen walls. OsLDDT1 (LOC_Os03g02170) encodes a DUF726 containing protein of unknown function with highly conserved transmembrane and α/ß Hydrolase domains. OsLDDT1 localizes to the endoplasmic reticulum and the gene is highly expressed in rice panicles. Genes involved in regulating fatty acid synthesis and formation of sporopollenin and pollen exine during anther development showed significantly different expression patterns in oslddt1 plants. Interestingly, the wax and cutin contents in mature oslddt1-1 anthers were decreased by 74.07 % and 72.22 % compared to WT, indicating that OsLDDT1 is involved in fatty acid synthesis and affects formation of the anther epidermis. Our results provide as deeper understanding of the role of OsLDDT1 in regulating male sterility and also provide materials for hybrid rice breeding.

The clock component OsLUX regulates rice heading through recruiting OsELF3-1 and OsELF4s to repress Hd1 and Ghd7.

INTRODUCTION: Circadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination. OBJECTIVES: In this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s-OsELF3-1-OsLUX complex directly regulates flowering-related genes to mediate rice heading. METHODS: We identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s-OsELF3-1-OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s-OsELF3-1-OsLUX) complex. RESULTS: The circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s-OsELF3-1-OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element. CONCLUSION: Our findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s-OsELF3-1-OsLUX)-Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.

Disruption of OsPHD1, Encoding a UDP-Glucose Epimerase, Causes JA Accumulation and Enhanced Bacterial Blight Resistance in Rice.

Lesion mimic mutants (LMMs) have been widely used in experiments in recent years for studying plant physiological mechanisms underlying programmed cell death (PCD) and defense responses. Here, we identified a lesion mimic mutant, lm212-1, which cloned the causal gene by a map-based cloning strategy, and verified this by complementation. The causal gene, OsPHD1, encodes a UDP-glucose epimerase (UGE), and the OsPHD1 was located in the chloroplast. OsPHD1 was constitutively expressed in all organs, with higher expression in leaves and other green tissues. lm212-1 exhibited decreased chlorophyll content, and the chloroplast structure was destroyed. Histochemistry results indicated that H2O2 is highly accumulated and cell death is occurred around the lesions in lm212-1. Compared to the wild type, expression levels of defense-related genes were up-regulated, and resistance to bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) was enhanced, indicating that the defense response was activated in lm212-1, ROS production was induced by flg22, and chitin treatment also showed the same result. Jasmonic acid (JA) and methyl jasmonate (MeJA) increased, and the JA signaling pathways appeared to be disordered in lm212-1. Additionally, the overexpression lines showed the same phenotype as the wild type. Overall, our findings demonstrate that OsPHD1 is involved in the regulation of PCD and defense response in rice.

OsPG1 Encodes a Polygalacturonase that Determines Cell Wall Architecture and Affects Resistance to Bacterial Blight Pathogen in Rice.

BACKGROUND: Plant cell walls are the main physical barrier encountered by pathogens colonizing plant tissues. Alteration of cell wall integrity (CWI) can activate specific defenses by impairing proteins involved in cell wall biosynthesis, degradation and remodeling, or cell wall damage due to biotic or abiotic stress. Polygalacturonase (PG) depolymerize pectin by hydrolysis, thereby altering pectin composition and structures and activating cell wall defense. Although many studies of CWI have been reported, the mechanism of how PGs regulate cell wall immune response is not well understood. RESULTS: Necrosis appeared in leaf tips at the tillering stage, finally resulting in 3-5 cm of dark brown necrotic tissue. ltn-212 showed obvious cell death and accumulation of H2O2 in leaf tips. The defense responses were activated in ltn-212 to resist bacterial blight pathogen of rice. Map based cloning revealed that a single base substitution (G-A) in the first intron caused incorrect splicing of OsPG1, resulting in a necrotic phenotype. OsPG1 is constitutively expressed in all organs, and the wild-type phenotype was restored in complementation individuals and knockout of wild-type lines resulted in necrosis as in ltn-212. Transmission electron microscopy showed that thicknesses of cell walls were significantly reduced and cell size and shape were significantly diminished in ltn-212. CONCLUSION: These results demonstrate that OsPG1 encodes a PG in response to the leaf tip necrosis phenotype of ltn-212. Loss-of-function mutation of ltn-212 destroyed CWI, resulting in spontaneous cell death and an auto-activated defense response including reactive oxygen species (ROS) burst and pathogenesis-related (PR) gene expression, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). These findings promote our understanding of the CWI mediated defense response.

Reduction of OsMPK6 activity by a R89K mutation induces cell death and bacterial blight resistance in rice.

KEY MESSAGE: The R89 is essential for the kinase activity of OsMPK6 which negatively regulates cell death and defense response in rice. Mitogen-activated protein kinase cascade plays critical roles in various vital activities, including the plant immune response, but the mechanisms remain elusive. Here, we identified and characterized a rice lesion mimic mutant osmpk6 which displayed hypersensitive response-like lesions in company with cell death and hydrogen peroxide hyperaccumulation. Map-based cloning and complementation demonstrated that a G702A single-base substitution in the second exon of OsMPK6 led to the lesion mimic phenotype of the osmpk6 mutant. OsMPK6 encodes a cytoplasm and nucleus-targeted mitogen-activated protein kinase and is expressed in the various organs. Compared with wild type, the osmpk6 mutant exhibited high resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), likely due to the increased ROS production induced by flg22 and chitin and up-regulated expression of genes involved in pathogenesis, as well as activation of SA and JA signaling pathways after inoculation. By contrast, the OsMPK6-overexpression line (OE-1) was found to be susceptible to the bacterial pathogens, indicating that OsMPK6 negatively regulated Xoo resistance. Furthermore, the G702A single-base substitution caused a R89K mutation at both polypeptide substrate-binding site and active site of OsMPK6, and kinase activity assay revealed that the R89K mutation led to reduction of OsMPK6 activity, suggesting that the R89 is essential for the function of OsMPK6. Our findings provide insight into a vital role of the R89 of OsMPK6 in regulating cell death and defense response in rice.

Exploiting Genic Male Sterility in Rice: From Molecular Dissection to Breeding Applications.

Rice (Oryza sativa L.) occupies a very salient and indispensable status among cereal crops, as its vast production is used to feed nearly half of the world's population. Male sterile plants are the fundamental breeding materials needed for specific propagation in order to meet the elevated current food demands. The development of the rice varieties with desired traits has become the ultimate need of the time. Genic male sterility is a predominant system that is vastly deployed and exploited for crop improvement. Hence, the identification of new genetic elements and the cognizance of the underlying regulatory networks affecting male sterility in rice are crucial to harness heterosis and ensure global food security. Over the years, a variety of genomics studies have uncovered numerous mechanisms regulating male sterility in rice, which provided a deeper and wider understanding on the complex molecular basis of anther and pollen development. The recent advances in genomics and the emergence of multiple biotechnological methods have revolutionized the field of rice breeding. In this review, we have briefly documented the recent evolution, exploration, and exploitation of genic male sterility to the improvement of rice crop production. Furthermore, this review describes future perspectives with focus on state-of-the-art developments in the engineering of male sterility to overcome issues associated with male sterility-mediated rice breeding to address the current challenges. Finally, we provide our perspectives on diversified studies regarding the identification and characterization of genic male sterility genes, the development of new biotechnology-based male sterility systems, and their integrated applications for hybrid rice breeding.

The MYB transcription factor Baymax1 plays a critical role in rice male fertility.

Key message Rice male fertility gene Baymax1, isolated through map-based cloning, encodes a MYB transcription factor and is essential for rice tapetum and microspore development.Abstract The mining and characterization of male fertility gene will provide theoretical and material basis for future rice production. In Arabidopsis, the development of male organ (namely anther), usually involves the coordination between MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic helix-loop-helix) members. However, the role of MYB proteins in rice anther development remains poorly understood. In this study, we isolated and characterized a male sterile mutant (with normal vegetative growth) of Baymax1 (BM1), which encodes a MYB protein. The bm1 mutant exhibited slightly lagging meiosis, aborted transition of the tapetum to a secretory type, premature tapetal degeneration, and abnormal pollen exine formation, leading to ultimately lacks of visible pollens in the mature white anthers. Map-based cloning, complementation and targeted mutagenesis using CRISPR/Cas9 technology demonstrated that the mutated LOC_Os04g39470 is the causal gene in bm1. BM1 is preferentially expressed in rice anthers from stage 5 to stage 10. Phylogenetic analysis indicated that rice BM1 and its homologs in millet, maize, rape, cabbage, and pigeonpea are evolutionarily conserved. BM1 can physically interacts with bHLH protein TIP2, EAT1, and PHD (plant homeodomain)-finger member TIP3, respectively. Moreover, BM1 affects the expression of several known genes related to tapetum and microspore development. Collectively, our results suggest that BM1 is one of key regulators for rice male fertility and may serve as a potential target for rice male-sterile line breeding and hybrid seed production.

Rice dwarf and low tillering 10 (OsDLT10) regulates tiller number by monitoring auxin homeostasis.

Tiller number is a crucial agronomic trait that directly affects the number of effective panicles and yield formation in rice. Here, we report a semi-dwarf and low tillering mutant Osdlt10 (dwarf and low tillering 10) that exhibited reduced tiller number, semi-dwarfism, increased grain width, low seed-setting rate, curled leaf tip and a series of abnormalities of agronomic traits. Phenotypic observations showed that Osdlt10 mutants had defects in tiller bud formation and grew slowly at the tillering stage. Map-based cloning revealed that LOC_Os10g41310 was the responsible gene for OsDLT10, which was subsequently demonstrated using the CRISPR/Cas9 system and a complementary experiment. Expression pattern analysis indicated that OsDLT10 was primarily expressed in the stem node, the basic part of axillary bud and leaf sheath, pulvinus. The hormone treatment investigation indicated that extremely high of exogenous auxin concentrations can inhibit the expression of OsDLT10. Endogenous auxin content decreased significantly at the base of stem node and axillary bud in Osdlt10 mutants. The results showed that OsDLT10 was related to auxin. qPCR analysis results further showed that the expression levels of auxin transport genes (PINs) and early response genes (IAAs) were significantly increased. The expression levels of WUS-like and FON1 were substantially decreased in the Osdlt10 mutants. These results revealed that OsDLT10 played a critical role in influencing tiller number, likely in association with hormone signals and the WUS-CLV pathway, to regulate axillary bud development in rice.

Characterization and genetic analysis of the oshpl3 rice lesion mimic mutant showing spontaneous cell death and enhanced bacterial blight resistance.

Plant lesion mimic mutants have been used as ideal materials for studying pathogen defense mechanisms due to their spontaneous activation of defense responses in plants. Here, we report the identification and characterization of a rice lesion mimic mutant, oshpl3. The oshpl3 mutant initially displayed white spots on leaves of 7-day-old seedlings, and the white spots gradually turned into large brown spots during plant development, accompanied by poor metrics of major agronomic traits. Histochemical analysis showed that spontaneous cell death and H2O2 hyperaccumulation occurred in oshpl3. Defense responses were induced in the oshpl3 mutant, such as enhanced ROS signaling activated by recognition of pathogen-associated molecular patterns, and also upregulated expression of genes involved in pathogenesis and JA metabolism. These defense responses enhanced resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae. The mutated gene was identified as OsHPL3 (LOC_Os02g02000) by map-based cloning. A G1006A mutation occurred in OsHPL3, causing a G-to-D mutation of the 295th amino acid in the transmembrane region of OsHPL3. OsHPL3 localized to the chloroplast, cytoplasm, and another unknown organelle, while the mutated protein OsHPL3G295D was not obviously observed in the chloroplast, suggesting that the G295D mutation affected its chloroplast localization. Based on our findings, the G295D mutation in OsHPL3 is most likely responsible for the phenotypes of the oshpl3 mutant. Our results provide new clues for studying the function of the OsHPL3 protein.

OsCUL3a-Associated Molecular Switches Have Functions in Cell Metabolism, Cell Death, and Disease Resistance.

This study applies parallel reaction monitoring (PRM) proteomics and CRISPR-Cas9 mutagenesis to identify relationships between cell metabolism, cell death, and disease resistance. In oscul3a (oscullin3a) mutants, OsCUL3a-associated molecular switches are responsible for disrupted cell metabolism that leads to increased total lipid content in rice grain, a late accumulation of H2O2 in leaves, enhanced Xanthomonas oryzae pv. oryzae disease resistance, and suppressed panicle and first internode growth. In oscul3a mutants, PRM-confirmed upregulated molecular switch proteins include lipoxygenases (CM-LOX1 and CM-LOX2), suggesting a novel connection between ferroptosis and rice lesion mimic formation. Rice immunity-associated proteins OsNPR1 and OsNPR3 were shown to interact with each other and have opposing regulatory effects based on the cell death phenotype of osnpr1/oscul3a and osnpr3/oscul3a double mutants. Together, these results describe a network that regulates plant growth, disease resistance, and grain quality that includes the E3 ligase OsCUL3a, cell metabolism-associated molecular switches, and immunity switches OsNPR1 and OsNPR3.

The OsMPK15 Negatively Regulates Magnaporthe oryza and Xoo Disease Resistance via SA and JA Signaling Pathway in Rice.

Mitogen-activated protein kinase (MAPK) cascades play central roles in response to biotic and abiotic stresses. However, the mechanisms by which various MAPK members regulate the plant immune response in rice remain elusive. In this article, to characterize the mechanisms, the knock-out and overexpression mutants of OsMPK15 were constructed and the disease resistance was investigated under the various fungal and bacterial inoculations. The knock-out mutant of OsMPK15 resulted in the constitutive expression of pathogenesis-related (PR) genes, increased accumulation of reactive oxygen species (ROS) triggered by the pathogen-associated molecular pattern (PAMP) elicitor chitin, and significantly enhanced the disease resistance to different races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo), which cause the rice blast and bacterial blight diseases, respectively. On contrary, the expression of PR genes and ROS were down-regulated in the OsMPK15-overexpressing (OsMPK15-OE) lines. Meanwhile, phytohormones such as salicylic acid (SA) and jasmonic acid (JA) were accumulated in the mpk15 mutant lines but decreased in the OsMPK15-OE lines. The expression of SA- and JA-pathway associated genes were significantly upregulated in the mpk15 mutant, whereas it was down regulated in the OsMPK15-OE lines. We conclude that OsMPK15 may negatively regulate the disease resistance through modulating SA- and JA-mediated signaling pathway.

UMP Kinase Regulates Chloroplast Development and Cold Response in Rice.

Pyrimidine nucleotides are important metabolites that are building blocks of nucleic acids, which participate in various aspects of plant development. Only a few genes involved in pyrimidine metabolism have been identified in rice and the majority of their functions remain unclear. In this study, we used a map-based cloning strategy to isolate a UMPK gene in rice, encoding the UMP kinase that phosphorylates UMP to form UDP, from a recessive mutant with pale-green leaves. In the mutant, UDP content always decreased, while UTP content fluctuated with the development of leaves. Mutation of UMPK reduced chlorophyll contents and decreased photosynthetic capacity. In the mutant, transcription of plastid-encoded RNA polymerase-dependent genes, including psaA, psbB, psbC and petB, was significantly reduced, whereas transcription of nuclear-encoded RNA polymerase-dependent genes, including rpoA, rpoB, rpoC1, and rpl23, was elevated. The expression of UMPK was significantly induced by various stresses, including cold, heat, and drought. Increased sensitivity to cold stress was observed in the mutant, based on the survival rate and malondialdehyde content. High accumulation of hydrogen peroxide was found in the mutant, which was enhanced by cold treatment. Our results indicate that the UMP kinase gene plays important roles in regulating chloroplast development and stress response in rice.

Impaired Function of the Calcium-Dependent Protein Kinase, OsCPK12, Leads to Early Senescence in Rice (Oryza sativa L.).

Premature leaf senescence affects plant yield and quality, and numerous researches about it have been conducted until now. In this study, we identified an early senescent mutant es4 in rice (Oryza sativa L.); early senescence appeared approximately at 60 dps and became increasingly senescent with the growth of es4 mutant. We detected that content of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as activity of superoxide dismutase (SOD) were elevated, while chlorophyll content, soluble protein content, activity of catalase (CAT), activity of peroxidase (POD) and photosynthetic rate were reduced in the es4 mutant leaves. We mapped es4 in a 33.5 Kb physical distance on chromosome 4 by map-based cloning. Sequencing analysis in target interval indicated there was an eight bases deletion mutation in OsCPK12 which encoded a calcium-dependent protein kinase. Functional complementation of OsCPK12 in es4 completely restored the normal phenotype. We used CRISPR/Cas9 for targeted disruption of OsCPK12 in ZH8015 and all the mutants exhibited the premature senescence. All the results indicated that the phenotype of es4 was caused by the mutation of OsCPK12. Overexpression of OsCPK12 in ZH8015 enhanced the net photosynthetic rate (P n) and chlorophyll content. OsCPK12 was mainly expressed in green organs. The results of qRT-PCR analysis showed that the expression levels of some key genes involved in senescence, chlorophyll biosynthesis, and photosynthesis were significantly altered in the es4 mutant. Our results demonstrate that the mutant of OsCPK12 triggers the premature leaf senescence; however, the overexpression of OsCPK12 may delay its growth period and provide the potentially positive effect on productivity in rice.

WB1, a Regulator of Endosperm Development in Rice, Is Identified by a Modified MutMap Method.

Abnormally developed endosperm strongly affects rice (Oryza sativa) appearance quality and grain weight. Endosperm formation is a complex process, and although many enzymes and related regulators have been identified, many other related factors remain largely unknown. Here, we report the isolation and characterization of a recessive mutation of White Belly 1 (WB1), which regulates rice endosperm development, using a modified MutMap method in the rice mutant wb1. The wb1 mutant develops a white-belly endosperm and abnormal starch granules in the inner portion of white grains. Representative of the white-belly phenotype, grains of wb1 showed a higher grain chalkiness rate and degree and a lower 1000-grain weight (decreased by ~34%), in comparison with that of Wild Type (WT). The contents of amylose and amylopectin in wb1 significantly decreased, and its physical properties were also altered. We adopted the modified MutMap method to identify 2.52 Mb candidate regions with a high specificity, where we detected 275 SNPs in chromosome 4. Finally, we identified 19 SNPs at 12 candidate genes. Transcript levels analysis of all candidate genes showed that WB1 (Os04t0413500), encoding a cell-wall invertase, was the most probable cause of white-belly endosperm phenotype. Switching off WB1 with the CRISPR/cas9 system in Japonica cv. Nipponbare demonstrates that WB1 regulates endosperm development and that different mutations of WB1 disrupt its biological function. All of these results taken together suggest that the wb1 mutant is controlled by the mutation of WB1, and that the modified MutMap method is feasible to identify mutant genes, and could promote genetic improvement in rice.

Premature leaf senescence 3, encoding a methyltransferase, is required for melatonin biosynthesis in rice.

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H2 O2 ) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence-associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O-methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild-type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild-type rice. Moreover, overexpression of OsMTS1 in the wild-type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.

The rice CONSTANS-like protein OsCOL15 suppresses flowering by promoting Ghd7 and repressing RID1.

The photoperiodic flowering pathway is one of the most important regulatory networks controlling flowering time in rice (Oryza sativa L.). Rice is a facultative short-day (SD) plant; flowering is promoted under inductive SD conditions and delayed under non-inductive long-day (LD) conditions. In rice, flowering inhibitor genes play an important role in maintaining the trade-off between reproduction and yield. In this study, we identified a novel floral inhibitor, OsCOL15, which encodes a CONSTANS-like transcription factor. Consistent with a function in transcriptional regulation, OsCOL15 localized to the nucleus. Moreover, OsCOL15 had transcriptional activation activity, and the central region of the protein between the B-box and CCT domains was required for this activity. We determined that OsCOL15 is most highly expressed in young organs and exhibits a diurnal expression pattern typical of other floral regulators. Overexpression of OsCOL15 resulted in a delayed flowering phenotype under both SD and LD conditions. Real-time quantitative RT-PCR analysis of flowering regulator gene expression suggested that OsCOL15 suppresses flowering by up-regulating the flowering repressor Grain number, plant height and heading date 7 (Ghd7) and down-regulating the flowering activator Rice Indeterminate 1 (RID1), thus leading to the down-regulation of the flowering activators Early heading date 1, Heading date 3a, and RICE FLOWERING LOCUS T1. These results demonstrate that OsCOL15 is an important floral regulator acting upstream of Ghd7 and RID1 in the rice photoperiodic flowering-time regulatory network.

ES7, encoding a ferredoxin-dependent glutamate synthase, functions in nitrogen metabolism and impacts leaf senescence in rice.

Glutamate synthase (GOGAT) is a key enzyme for nitrogen metabolism and ammonium assimilation in plants. In this study, an early senescence 7 (es7) mutant was identified and characterized. The leaves of the es7 mutant begin to senesce at the tillering stage about 60day after sowing, and become increasingly senescent as the plants develop at the heading stage. When es7 plants are grown under photorespiration-suppressed conditions (high CO2), the senescence phenotype and chlorophyll content are rescued. qRT-PCR analysis showed that senescence- associated genes were up-regulated significantly in es7. A map-based cloning strategy was used to identify ES7, which encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). ES7 was expressed constitutively, and the ES7 protein was localized in chloroplast. qRT-PCR analysis indicated that several genes related to nitrogen metabolism were differentially expressed in es7. Further, we also demonstrated that chlorophyll synthesis-associated genes were significantly down-regulated in es7. In addition, when seedlings are grown under increasing nitrogen concentrations (NH4NO3) for 15days, the contents of chlorophyll a, chlorophyll b and total chlorophyll were significantly lower in es7. Our results demonstrated that ES7 is involved in nitrogen metabolism, effects chlorophyll synthesis, and may also associated with photorespiration, impacting leaf senescence in rice.