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.

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.

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.

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.

CS3, a Ycf54 domain-containing protein, affects chlorophyll biosynthesis in rice (Oryza sativa L.).

Chlorophyll plays a vital role in harvesting light and turning it into chemical energy. In this study, we isolated and characterized a chlorophyll-deficient mutant, which we named cs3 (chlorotic seedling 3). The cs3 mutant seedlings exhibit a yellowish phenotype at germination, and they do not survive at the seedling stage. In addition, brown necrotic spots appear on the surface of the leaves and leaf sheaths during development. DAB staining and H2O2 content measurement showed that there was excessive H2O2 accumulation in the cs3 mutant leaf. Accompanying the chlorophyll deficiency, the chloroplasts in cs3 leaf cells were abnormal. Using a map-based cloning strategy, we mapped the CS3 gene, which encodes a Ycf54 domain-containing protein, to a locus on chromosome 3. CS3 is mainly expressed in green tissues and the S136 F would influence CS3 interacting with YGL8 and its chloroplast localization. qRT-PCR analysis revealed the changes in the expression of genes involved in chlorophyll biosynthesis and degradation, chloroplast development, senescence, and photosynthesis in the cs3 mutant. In addition, our study also supports the notion that the mutation in the CS3/Ycf54 gene arrests chlorophyll biosynthesis by negatively affecting the activity of magnesium protoporphyrin IX monomethylester cyclase (MgPME-cyclase).

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.

RDWN6XB, a major quantitative trait locus positively enhances root system architecture under nitrogen deficiency in rice.

BACKGROUND: Nitrogen (N) is a major input cost in rice production, in addition to causing severe pollution to agricultural and ecological environments. Root dry weight has been considered the most important component related to crop yields than the other root traits. Therefore, development of rice varieties/lines with low input of N fertilizer and higher root traits are essential for sustainable rice production. RESULTS: In this context, a main effect quantitative trait locus qRDWN6XB on the long arm of chromosome 6 which positively confers tolerance to N deficiency in the Indica rice variety XieqingzaoB, was identified using a chromosomal segment substitution line (CSSL) population. qRDWN6XB was determined to be located near marker InD90 on chromosome 6 based on association analysis of phenotype data from three N levels and 120 polymorphic molecular markers. The target chromosomal segment substitution line CSSL45, which has the higher root dry weight (RDW) than indica cultivar Zhonghui9308 and carry qRDWN6XB, was selected for further study. A BC5F2:3 population derived from a cross between CSSL45 and Zhonghui9308 was constructed. To fine-map qRDWN6XB, we used the homozygous recombinant plants and ultimately this locus was narrowed to a 52.3-kb between markers ND-4 and RM19771, which contains nine candidate genes in this region. One of these genes, LOC_Os06g15910 as a potassium transporter was considered a strong candidate gene for the RDWN6XB locus. CONCLUSIONS: The identification of qRDWN6XB provides a new genetic resource for breeding rice varieties and a starting point to improve grain yield despite the decreased input of N fertilizers. The newly developed and tightly linked InDel marker ND-4 will be useful to improve the root system architecture under low N by marker-assisted selection (MAS) in rice breeding programs.

qSE7 is a major quantitative trait locus (QTL) influencing stigma exsertion rate in rice (Oryza sativa L.).

Stigma exsertion is a key determinant to increase the efficiency of commercial hybrid rice seed production. The major quantitative trait locus (QTL) qSE7 for stigma exsertion rate was previously detected on the chromosome 7 using 75 Chromosome Segment Substitution Lines (CSSLs) derived from a cross between the high stigma exsertion indica maintainer XieqingzaoB (XQZB) and low stigma exsertion indica restorer Zhonghui9308 (ZH9308). The C51 line, a CSSL population with an introgression from XQZB, was backcrossed with ZH9308 to produce the secondary F2 (BC5F2) and F2:3 (BC5F2:3) populations. As a result, the Near Isogenic Line (NIL qSE7XB) was developed. Analysis indicated qSE7 acted as a single Mendelian factor and decreased the stigma exsertion. We hypothesized qSE7 regulates single, dual, and total stigma exsertion rate, provided experimental support. qSE7 was mapped and localized between RM5436 and RM5499 markers, within a physical distance of 1000-kb. With use of new insertion-deletion (InDel) markers and analysis of the heterozygous and phenotypic data, it was ultimately dissected to a 322.9-kb region between InDel SER4-1 and RM5436. The results are useful for additional identification and isolation of this candidate gene controlling stigma exsertion rate, and provide a basis for further fine mapping, gene cloning, and Marker Assisted Selection (MAS) breeding later.

Genetic Dissection of qPCG1 for a Quantitative Trait Locus for Percentage of Chalky Grain in Rice (Oryza sativa L.).

Rice is a pivotal cereal crop that provides the staple food for more than half of the world's population. Along with improvements in the standard of living, people not only pay attention to the grain yield but also to the grain quality. Chalkiness is one of the most important index of grain quality. In this study, qPCG1, a QTL for percentage of chalky grain, was mapped in an interval with a physical distance about 139 kb on chromosome 1 by residual heterozygous line (RHL) method. qPCG1 was incomplete dominant and the additive effect plays a major role and explained 6.8-21.9% of phenotypic variance within the heterogeneous region on chromosome 1. The effect of allele from Zhonghui9308 was decreasing the percentage of chalky grains (PCG). Microscope observation results indicated that there are great differences in the shape, structure and arrangement of starch granule between the chalky part and transparent part. Analysis of starch physicochemical properties showed that the total starch content, amylose content and chain length distribution of amylopectin changed while the protein contents were not apparently affected with the changed chalkiness. qPCG1 had little influence on main agronomic traits and it might be useful in rice breeding for it did not bring negative effect on grain yield while reducing the chalkiness.