DWARF AND LESS TILLERS ON CHROMOSOME 3 Promotes Tillering in Rice by Sustaining FLORAL ORGAN NUMBER 1 Expression.

Three key factors determine yield in rice (Oryza sativa): panicle number, grain number, and grain weight. Panicle number is strongly associated with tiller number. Although many genes regulating tillering have been identified, whether Dof proteins are involved in controlling plant architecture remains unknown. The dwarf and less tillers on chromosome 3 (dlt3) rice mutant produces fewer tillers than the wild type. We cloned DLT3, which encodes a Dof protein that interacts with MONOCULM 3 (MOC3) in vivo and in vitro and recruits MOC1, forming a DLT3-MOC3-MOC1 complex. DLT3 binds to the promoter of FLORAL ORGAN NUMBER 1 (FON1) to activate its transcription and positively regulate tiller number. The overexpression of MOC1, MOC3, or FON1 in the dlt3 mutant increased tiller number. Collectively, these results suggest a model in which DLT3 regulates tiller number by maintaining the expression of MOC1, MOC3, and FON1. We discovered that DLT3 underwent directional selection in the Xian/indica and Geng/japonica populations during rice domestication. To provide genetic resources for breeding varieties with optimal panicle numbers, we performed large-scale diversity sequencing of the 1080-bp DLT3 coding region of 531 accessions from different countries and regions. Haplotype analysis showed that the superior haplotype, DLT3H1, produced the most tillers, while haplotype DLT3H6 produced the fewest tillers. Our study provides important germplasm resources for breeding super high-yielding rice varieties with combinations of superior haplotypes in different target genes, which will help overcome the challenge of food and nutritional security in the future.

OsCOL5 suppresses heading through modulation of Ghd7 and Ehd2, enhancing rice yield.

KEY MESSAGE: OsCOL5, an ortholog of Arabidopsis COL5, is involved in photoperiodic flowering and enhances rice yield through modulation of Ghd7 and Ehd2 and interactions with OsELF3-1 and OsELF3-2. Heading date, also known as flowering time, plays a crucial role in determining the adaptability and yield potential of rice (Oryza sativa L.). CONSTANS (CO)-like is one of the most critical flowering-associated gene families, members of which are evolutionarily conserved. Here, we report the molecular functional characterization of OsCOL5, an ortholog of Arabidopsis COL5, which is involved in photoperiodic flowering and influences rice yield. Structural analysis revealed that OsCOL5 is a typical member of CO-like family, containing two B-box domains and one CCT domain. Rice plants overexpressing OsCOL5 showed delayed heading and increases in plant height, main spike number, total grain number per plant, and yield per plant under both long-day (LD) and short-day (SD) conditions. Gene expression analysis indicated that OsCOL5 was primarily expressed in the leaves and stems with a diurnal rhythm expression pattern. RT-qPCR analysis of heading date genes showed that OsCOL5 suppressed flowering by up-regulating Ghd7 and down-regulating Ehd2, consequently reducing the expression of Ehd1, Hd3a, RFT1, OsMADS14, and OsMADS15. Yeast two-hybrid experiments showed direct interactions of OsCOL5 with OsELF3-1 and OsELF3-2. Further verification showed specific interactions between the zinc finger/B-box domain of OsCOL5 and the middle region of OsELF3-1 and OsELF3-2. Yeast one-hybrid assays revealed that OsCOL5 may bind to the CCACA motif. The results suggest that OsCOL5 functions as a floral repressor, playing a vital role in rice's photoperiodic flowering regulation. This gene shows potential in breeding programs aimed at improving rice yield by influencing the timing of flowering, which directly impacts crop productivity.

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.

Mutation of DEFECTIVE EMBRYO SAC1 results in a low seed-setting rate in rice by regulating embryo sac development.

The seed-setting rate has a significant effect on grain yield in rice (Oryza sativa L.). Embryo sac development is essential for seed setting; however, the molecular mechanism underlying this process remains unclear. Here, we isolated defective embryo sac1 (des1), a rice mutant with a low seed-setting rate. Cytological examination showed degenerated embryo sacs and reduced fertilization capacity in des1. Map-based cloning revealed a nonsense mutation in OsDES1, a gene that encodes a putative nuclear envelope membrane protein (NEMP)-domain-containing protein that is preferentially expressed in pistils. The OsDES1 mutation disrupts the normal formation of functional megaspores, which ultimately results in a degenerated embryo sac in des1. Reciprocal crosses showed that fertilization is abnormal and that the female reproductive organ is defective in des1. OsDES1 interacts with LONELY GUY (LOG), a cytokinin-activating enzyme that acts in the final step of cytokinin synthesis; mutation of LOG led to defective female reproductive organ development. These results demonstrate that OsDES1 functions in determining the rice seed-setting rate by regulating embryo sac development and fertilization. Our study sheds light on the function of NEMP-type proteins in rice reproductive development.

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.

Deletion of Diterpenoid Biosynthetic Genes CYP76M7 and CYP76M8 Induces Cell Death and Enhances Bacterial Blight Resistance in Indica Rice '9311'.

Lesion mimic mutants (LMMs) are ideal materials for studying cell death and resistance mechanisms. Here, we identified and mapped a novel rice LMM, g380. The g380 exhibits a spontaneous hypersensitive response-like cell death phenotype accompanied by excessive accumulation of reactive oxygen species (ROS) and upregulated expression of pathogenesis-related genes, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). Using a map-based cloning strategy, a 184,916 bp deletion on chromosome 2 that overlaps with the diterpenoid biosynthetic gene cluster was identified in g380. Accordingly, the content of diterpenoids decreased in g380. In addition, lignin, one of the physical lines of plant defense, was increased in g380. RNA-seq analysis showed 590 significantly differentially expressed genes (DEG) between the wild-type 9311 and g380, 585 of which were upregulated in g380. Upregulated genes in g380 were mainly enriched in the monolignol biosynthesis branches of the phenylpropanoid biosynthesis pathway, the plant-pathogen interaction pathway and the phytoalexin-specialized diterpenoid biosynthesis pathway. Taken together, our results indicate that the diterpenoid biosynthetic gene cluster on chromosome 2 is involved in immune reprogramming, which in turn regulates cell death in rice.

STRIPE3, encoding a human dNTPase SAMHD1 homolog, regulates chloroplast development in rice.

Chloroplast is an important organelle for photosynthesis and numerous essential metabolic processes, thus ensuring plant fitness or survival. Although many genes involved in chloroplast development have been identified, mechanisms underlying such development are not fully understood. Here, we isolated and characterized the stripe3 (st3) mutant which exhibited white-striped leaves with reduced chlorophyll content and abnormal chloroplast development during the seedling stage, but gradually produced nearly normal green leaves as it developed. Map-based cloning and transgenic tests demonstrated that a splicing mutation in ST3, encoding a human deoxynucleoside triphosphate triphosphohydrolase (dNTPase) SAMHD1 homolog, was responsible for st3 phenotypes. ST3 is highly expressed in the third leaf at three-leaf stage and expressed constitutively in root, stem, leaf, sheath, and panicle, and the encoded protein, OsSAMHD1, is localized to the cytoplasm. The st3 mutant showed more severe albino leaf phenotype under exogenous 1-mM dATP/dA, dCTP/dC, and dGTP/dG treatments compared with the control conditions, indicating that ST3 is involved in dNTP metabolism. This study reveals a gene associated with dNTP catabolism, and propose a model in which chloroplast development in rice is regulated by the dNTP pool, providing a potential application of these results to hybrid rice breeding.

Tiller Angle Control 1 Is Essential for the Dynamic Changes in Plant Architecture in Rice.

Plant architecture is dynamic as plants develop. Although many genes associated with specific plant architecture components have been identified in rice, genes related to underlying dynamic changes in plant architecture remain largely unknown. Here, we identified two highly similar recombinant inbred lines (RILs) with different plant architecture: RIL-Dynamic (D) and RIL-Compact (C). The dynamic plant architecture of RIL-D is characterized by 'loosetiller angle (tillering stage)-compact (heading stage)-loosecurved stem (maturing stage)' under natural long-day (NLD) conditions, and 'loosetiller angle (tillering and heading stages)-loosetiller angle and curved stem (maturing stage)' under natural short-day (NSD) conditions, while RIL-C exhibits a compact plant architecture both under NLD and NSD conditions throughout growth. The candidate locus was mapped to the chromosome 9 tail via the rice 8K chip assay and map-based cloning. Sequencing, complementary tests, and gene knockout tests demonstrated that Tiller Angle Control 1 (TAC1) is responsible for dynamic plant architecture in RIL-D. Moreover, TAC1 positively regulates loose plant architecture, and high TAC1 expression cannot influence the expression of tested tiller-angle-related genes. Our results reveal that TAC1 is necessary for the dynamic changes in plant architecture, which can guide improvements in plant architecture during the modern super rice breeding.

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.

DCET1 Controls Male Sterility Through Callose Regulation, Exine Formation, and Tapetal Programmed Cell Death in Rice.

In angiosperms, anther development comprises of various complex and interrelated biological processes, critically needed for pollen viability. The transitory callose layer serves to separate the meiocytes. It helps in primexine formation, while the timely degradation of tapetal cells is essential for the timely callose wall dissolution and pollen wall formation by providing nutrients for pollen growth. In rice, many genes have been reported and functionally characterized that are involved in callose regulation and pollen wall patterning, including timely programmed cell death (PCD) of the tapetum, but the mechanism of pollen development largely remains ambiguous. We identified and functionally characterized a rice mutant dcet1, having a complete male-sterile phenotype caused by defects in anther callose wall, exine patterning, and tapetal PCD. DCET1 belongs to the RNA recognition motif (RRM)-containing family also called as the ribonucleoprotein (RNP) domain or RNA-binding domain (RBD) protein, having single-nucleotide polymorphism (SNP) substitution from G (threonine-192) to A (isoleucine-192) located at the fifth exon of LOC_Os08g02330, was responsible for the male sterile phenotype in mutant dcet1. Our cytological analysis suggested that DCET1 regulates callose biosynthesis and degradation, pollen exine formation by affecting exine wall patterning, including abnormal nexine, collapsed bacula, and irregular tectum, and timely PCD by delaying the tapetal cell degeneration. As a result, the microspore of dcet1 was swollen and abnormally bursted and even collapsed within the anther locule characterizing complete male sterility. GUS and qRT-PCR analysis indicated that DCET1 is specifically expressed in the anther till the developmental stage 9, consistent with the observed phenotype. The characterization of DCET1 in callose regulation, pollen wall patterning, and tapetal cell PCD strengthens our knowledge for knowing the regulatory pathways involved in rice male reproductive development and has future prospects in hybrid rice breeding.

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.

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.

ES5 is involved in the regulation of phosphatidylserine synthesis and impacts on early senescence in rice (Oryza sativa L.).

Leaf senescence, which affects plant growth and yield in rice, is an ideal target for crop improvement and remarkable advances have been made to identify the mechanism underlying this process. We have characterized an early senile mutant es5 (early leaf senescence 5) in rice exhibiting leaf yellowing phenotype after the 4-leaf stage. This phenotype was confirmed by the higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the disintegration of chloroplasts, reduction in chlorophyll content and photosynthetic rate and up-regulation of senescence-associated genes (SAGs) like Osh36, OsI57, and OsI85. Positional cloning revealed that the es5 phenotype is the result of one base substitution in ES5, encoding phosphatidylserine synthase (PSS) family protein, which is involved in the base-exchange type reaction to synthesize the minor membrane phospholipid phosphatidylserine. Functional complementation of ES5 in the es5 plants completely restored the wild-type phenotype. Ultra-high-performance liquid chromatography (UHPLC) analysis showed that es5 plants had increased levels of phosphatidylserine (PS) and decreased level of phosphatidylcholine (PC). These results provide evidence about the role of PS in rice leaf senescence.

A proteomic approach identifies novel proteins and metabolites for lesion mimic formation and disease resistance enhancement in rice.

Lesion mimic mutants are ideal genetic materials to study programmed cell death and defense signaling in plants. However, the molecular basis of lesion mimic formation remains largely unknown. Here, we first used a proteomic approach to identify differentially expressed proteins during dynamic lesion mimic formation in the rice oscul3a mutant, then electron microscope observation and physiological assays were used to analyze the mutant. The oscul3a mutant had disrupted cell metabolism balance, and the identified differentially expressed proteins were mainly located in the chloroplast and cytoplasm, which caused enhanced lipid metabolism, but suppressed carbon/nitrogen metabolism with reduced growth and grain quality. The oscul3a mutant had higher salicylic acid (SA) concentration in leaves, and H2O2 was shown to accumulate late in the formation of lesions. The secondary metabolite coumarin induced reactive oxygen species (ROS) and had rice blast resistance activity. Moreover, the cell death initiated lesion mimic formation of oscul3a mutant was light-sensitive, which might be associated with metabolite biosynthesis and accumulation. This study sheds light on the metabolic transition associated with cell death and defense response, which is under tight regulation by OsCUL3a and metabolism-related proteins, and the newly identified chemicals in the secondary metabolic pathway can potentially be used to control disease in crop plants.

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.

LMM24 Encodes Receptor-Like Cytoplasmic Kinase 109, Which Regulates Cell Death and Defense Responses in Rice.

Lesion mimic mutants are excellent models for research on molecular mechanisms of cell death and defense responses in rice. We identified a new rice lesion mimic mutant lmm24 from a mutant pool of indica rice cultivar "ZhongHui8015". The LMM24 gene was identified by MutMap, and LMM24 was confirmed as a receptor-like cytoplasmic kinase 109 by amino acid sequence analysis. The lmm24 mutant displayed dark brown lesions in leaves and growth retardation that were not observed in wild-type ZH8015. The results of histochemical staining and TUNEL assays showed enhanced ROS accumulation and cell death in lmm24. Chloroplast degradation was observed in lmm24 leaves, with decreased expression of photosynthesis-related genes and increased expression of the senescence-induced STAYGREEN (SGR) gene and other senescence-associated genes. Furthermore, lmm24 exhibited enhanced resistance to rice blast fungus Magnaporthe oryzae (M. oryzae) and up-regulation of defense response genes. Our data demonstrate that LMM24 regulates cell death and defense responses in rice.

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.