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Drought poses a significant constraint on rice production, and, in this study, we have discovered a novel drought-sensitive mutant, designated as dsm3, arising from the progenies of indica rice variety Zhonghui8015 treated with ethyl methane sulphonate (EMS). Under drought stress conditions, dsm3 exhibited characteristic withered leaf tips, accompanied by increased levels of malondialdehyde (MDA) and H2O2, a reduced net photosynthetic rate (Pn), and decreased activity of peroxidase (POD) and superoxide dismutase (SOD). Genetic analysis revealed that the withered leaf tip phenotype was governed by a single recessive gene, designated as Osdsm3. To begin with, Osdsm3 was initially mapped to the short arm of chromosome 1 through a cross involving dsm3 and 02428. Subsequently, utilizing a population of 2591 F2 individuals, we narrowed down the location of Osdsm3 to a 78 Kb interval, encompassing 13 open reading frames (ORFs). Sequencing analysis unveiled a mutation (1275G → A) in the exon of the candidate gene (LOC_Os01g10680), leading to premature translation termination. Moreover, a quantitative RT-PCR assay demonstrated a high expression of OsDSM3 in the panicle and sheath, with a significant upregulation of drought-stress-related genes under drought conditions. Phylogenetic analyses indicated that Osdsm3 shares evolutionary homology with UNE1, an intracellular transport protein found in Arabidopsis thaliana. Subcellular studies further confirmed that OsDSM3 resides in the cytoplasm. In conclusion, the forthcoming cloning of Osdsm3 holds promise for delving deeper into the molecular mechanisms governing rice drought resistance. [ABSTRACT FROM AUTHOR]

Grain shape improvement, which determines grain yield, quality traits and commercial value, is an extremely important aspect of rice breeding. Grain size is controlled by multiple genes, and Maker Assistant Selection (MAS) breeding is effective for breeders in developing stable and efficient markers to aggregate these genes in order to speed up the selection of new lines with desirable traits during the breeding process. In this study, functional markers were developed based on the sequence differences of five grain-shaped genes (GL7, GW6a, GS6, GW5 and TGW6) between the long-grain japonica rice variety Zhendao and the indica-japonica restorer R2027. We then constructed a population of recombinant inbred lines (RILs) based on their cross. The newly designed functional markers were used to genotype grain-size genes, and a genetic effect analysis was conducted to screen high-quality long-grain restorers. Our results reveal diverse effects of different genes on grain size, and the five genotypes were distributed in the 36 selected BC1F8 lines. Specifically, gw5 positively regulates grain width and 1000-grain weight, gl7 and gs6 positively regulate grain length but negatively regulate grain width and 1000-grain weight, tgw6 positively regulates grain length and gw6a positively regulates 1000-grain weight. The most outstanding outcome is that 5 of the 36 lines achieved in this study showing an excellent performance of long grain and yield characters are ideal materials not only for studying the interaction and genetic effects between polygenes but also as restorers or donors for dominant genes in indica-japonica hybrid rice breeding. [ABSTRACT FROM AUTHOR]

Heading date (HD) is one of the agronomic traits that influence maturity, regional adaptability, and grain yield. The present study was a follow-up of a previous quantitative trait loci (QTL) mapping study conducted on three populations, which uncovered a total of 62 QTLs associated with 10 agronomic traits. Two of the QTLs for HD on chromosome 7 (qHD7a and qHD7b) had a common flanking marker (RM3670) that may be due to tight linkage, and/or weakness of the statistical method. The objectives of the present study were to map QTLs associated with HD in a set of 76 chromosome segment substitution lines (CSSLs), fine map and validate one of the QTLs (qHD7b) using 2997 BC5F2:3 plants, and identify candidate genes using sequencing and expression analysis. Using the CSSLs genotyped with 120 markers and evaluated under two short-day and two long-day growing conditions, we uncovered a total of fourteen QTLs (qHD2a, qHD4a, qHD4b, qHD5a, qHD6a, qHD6b, qHD7b, qHD7c, qHD8a, qHD10a, qHD10b, qHD11a, qHD12a, and qHD12b). However, only qHD6a and qHD7b were consistently detected in all four environments. The phenotypic variance explained by qHD6a and qHD7b varied from 10.1% to 36.1% (mean 23.1%) and from 8.1% to 32.8% (mean 20.5%), respectively. One of the CSSL lines (CSSL52), which harbored a segment from the early heading XieqingzaoB (XQZB) parent at the qHD7b locus, was then used to develop a BC5F2:3 population for fine mapping and validation. Using a backcross population evaluated for four seasons under different day lengths and temperatures, the qHD7b interval was delimited to a 912.7-kb region, which is located between RM5436 and RM5499. Sequencing and expression analysis revealed a total of 29 candidate genes, of which Ghd7 (Os07g0261200) is a well-known gene that affects heading date, plant height, and grain yield in rice. The ghd7 mutants generated through CRISPR/Cas9 gene editing exhibited early heading. Taken together, the results from both the previous and present study revealed a consistent QTL for heading date on chromosome 7, which coincided not only with the physical position of a known gene, but also with two major effect QTLs that controlled the stigma exertion rate and the number of spikelets in rice. The results provide contributions to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Mutation of CS3 leads to rice seedling chlorotic lethal phenotype. • CS3 interacts with YGL8 and form the MgPME-cyclase complex. • S136 F of CS3 affects its chloroplast localization and the interaction with YGL8. 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 H 2 O 2 content measurement showed that there was excessive H 2 O 2 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). [ABSTRACT FROM AUTHOR]

Abstract: Excess zinc harms the growth of rice plants and zinc toxicity can easily occur in acid soils. The aim of the study was to map quantitative trait loci (QTLs) in rice for tolerance to zinc toxicity, using a recombinant inbred (RI) population derived from the cross of a japonica variety (Asominori: relatively tolerant to Zn2+ toxicity) with an indica variety (IR24, relatively susceptible), through 289 RFLP markers. The index scores of damage (representing Zn2+ toxicity tolerance), after irrigating rice seedlings with a 1000-ppm Zn2+ solution for 20 successive days, were examined for each RI line and its parental varieties. Continuous distributions and transgressive segregations of the index scores were observed in the RI population, suggesting that Zn2+ toxicity tolerance was a quantitatively inherited trait. Three QTLs for Zn2+ toxicity tolerance were detected on chromosomes 1, 3 and 10 and explained 21.9, 8.9 and 7.6%, respectively, of the total phenotypic variation. The results and the tightly linked molecular markers that flank the QTLs, detected in this study, will be useful in improving Zn2+ tolerance in rice. In addition, the genomic positions between QTLs for Zn2+ toxicity tolerance and the QTLs for other metal (Fe2+, Mn2+, Al3+) toxicity tolerances, from previous studies, are discussed. [Copyright &y& Elsevier]

• The mutated OsCUL3a can cause cell death related lesion mimic formation with dynamic chloroplast degradation and signal molecule SA and H 2 O 2 accumulation in different leaf positions. • Proteomic approach identified differentially expressed proteins that mainly located in the chloroplast and cytoplasm, with enhanced lipid metabolism, but suppressed carbon/nitrogen metabolism. • Some new identified natural chemicals in secondary metabolic pathways can be used for disease control in crop plants. 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 H 2 O 2 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. [ABSTRACT FROM AUTHOR]