A 3-bp deletion of WLS5 gene leads to weak growth and early leaf senescence in rice.
ABSTRACT: BACKGROUND:In rice (Oryza sativa) and other grains, weak growth (dwarfism, short panicle length, and low seed-setting rate) and early senescence lead to reduced yield. The molecular mechanisms behind these processes have been widely studied; however, the complex genetic regulatory networks controlling growth and senescence require further elucidation. RESULTS:We isolated a mutant exhibiting weak growth throughout development and early senescence of leaf tips, and designated this mutant weakness and leaf senescence5 (wls5). Histological analysis showed that the poor growth of wls5 plants involved a reduction in cell length and number. Physiological analysis and transmission electron microscopy revealed that the wls5 cells had abnormal chloroplasts, and the mutants underwent chlorophyll degradation triggered by accumulation of reactive oxygen species. Consistent with this, RNA sequencing revealed changes in senescence-related gene expression in wls5 plants. The wls5 mutants also exhibited significantly higher stomatal density and altered phytohormone contents compared with wild-type plants. Fine mapping delimited WLS5 to a 29-kb region on chromosome 5. DNA sequencing of wls5 identified a 3-bp deletion in the first exon of LOC_Os05g04900, resulting in a deletion of a lysine in the predicted protein. Knockout of LOC_Os05g04900 in Nipponbare plants caused leaf senescence, confirming this locus as the causal gene for WLS5. CONCLUSIONS:We identified a novel mutant (wls5) that affects plant development and leaf senescence in rice. LOC_Os05g04900, encoding a protein of unknown function, is the causal gene for wls5. Further molecular study of WLS5 will uncover the roles of this gene in plant growth and leaf senescence.
Project description:Leaf senescence is one of the most common factors that affects the growth and yield of rice. Although numerous genes affecting leaf senescence have been identified, few involved in cuticular wax synthesis have been described for rice premature leaf senescence. Here, we cloned and characterized Premature Leaf Senescence 4 (PLS4) in rice (Oryza sativa), which encodes a putative 3-oxoacyl-reductase in the fatty acid biosynthetic pathway. Subcellular localization of OsPLS4 was observed in the chloroplast. A single nucleotide substitution in OsPLS4 reduced leaf cuticular wax, and the expression levels of most wax biosynthesis-associated genes were downregulated. TEM showed chloroplast development were defective in the pls4 mutant. Further investigation revealed that the chlorophyll (Chl) content was reduced in the pls4 mutant compared with the WT and that the photosynthesis rate was lower, which caused ROS dramatic accumulation at the heading stage. These results confirmed premature leaf senescence in pls4 plants. Cold treatment indicated that the mutant was more sensitive than the WT was to cold stress. Together, all the above results indicate that the OsPLS4 mutation affects cuticular wax biosynthesis and chloroplast development in rice, causing reduced cuticular wax and premature leaf senescence.
Project description:Leaf senescence is the last period of leaf growth and a dynamic procedure associated with its death. The adaptability of the plants to changing environments occurs thanks to leaf senescence. Hence, transcriptional profiling is important to figure out the exact mechanisms of inducing leaf senescence in a particular crop, such as rice. From this perspective, leaf samples of two different rice genotypes, the brown midrib leaf (bml) mutant and its wild type (WT) were sampled for transcriptional profiling to identify differentially-expressed genes (DEGs). We identified 2670 DEGs, among which 1657 genes were up- and 1013 genes were down-regulated. These DEGs were enriched in binding and catalytic activity, followed by the single organism process and metabolic process through gene ontology (GO), while the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the DEGs were related to the plant hormone signal transduction and photosynthetic pathway enrichment. The expression pattern and the clustering of DEGs revealed that the WRKY and NAC family, as well as zinc finger transcription factors, had greater effects on early-senescence of leaf compared to other transcription factors. These findings will help to elucidate the precise functional role of bml rice mutant in the early-leaf senescence.
Project description:Plant leaf senescence and defence responses are important biological processes, but the molecular mechanisms involved are not well understood. This study identified a new rice mutant, spotted leaf 29 (spl29). The SPL29 gene was identified by map-based cloning, and SPL29 was confirmed as UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) by enzymatic analysis. The mutant spl29 lacks UAP activity. The biological phenotypes for which UAP is responsible have not previously been reported in plants. The spl29 mutant displayed early leaf senescence, confirmed by chlorophyll loss and photosystem II decline as physiological indicators, chloroplast degradation as a cellular characteristic, and both upregulation of senescence transcription factors and senescence-associated genes, and downregulation of photosynthesis-related genes, as molecular evidence. Defence responses were induced in the spl29 mutant, shown by enhanced resistance to bacterial blight inoculation and upregulation of defence response genes. Reactive oxygen species, including O2 (-) and H2O2, accumulated in spl29 plants; there was also increased malondialdehyde content. Enhanced superoxide dismutase activity combined with normal catalase activity in spl29 could be responsible for H2O2 accumulation. The plant hormones jasmonic acid and abscisic acid also accumulated in spl29 plants. ROS and plant hormones probably play important roles in early leaf senescence and defence responses in the spl29 mutant. Based on these findings, it is suggested that UAP1 is involved in regulating leaf senescence and defence responses in rice.
Project description:Leaf senescence is a programmed developmental process orchestrated by many factors, but its molecular regulation is not yet fully understood. In this study, a novel Oryza sativa premature leaf senescence mutant (ospls1) was examined. Despite normal development in early seedlings, the ospls1 mutant leaves displayed lesion-mimics and early senescence, and a high transpiration rate after tillering. The mutant also showed seed dormancy attributable to physical (defect of micropyle structure) and physiological (abscisic acid sensitivity) factors. Using a map-based cloning approach, we determined that a cytosine deletion in the OsPLS1 gene encoding vacuolar H(+)-ATPase subunit A1 (VHA-A1) underlies the phenotypic abnormalities in the ospls1 mutant. The OsPSL1/VHA-A1 transcript levels progressively declined with the age-dependent leaf senescence in both the ospls1 mutant and its wild type. The significant decrease in both OsPSL1/VHA-A1 gene expression and VHA enzyme activity in the ospls1 mutant strongly suggests a negative regulatory role for the normal OsPLS1/VHA-A1 gene in the onset of rice leaf senescence. The ospls1 mutant featured higher salicylic acid (SA) levels and reactive oxygen species (ROS) accumulation, and activation of signal transduction by up-regulation of WRKY genes in leaves. Consistent with this, the ospls1 mutant exhibited hypersensitivity to exogenous SA and/or H2O2 Collectively, these results indicated that the OsPSL1/VAH-A1 mutation played a causal role in premature leaf senescence through a combination of ROS and SA signals. To conclude, OsPLS1 is implicated in leaf senescence and seed dormancy in rice.
Project description:BACKGROUND:Spotted-leaf mutants are important to reveal programmed cell death and defense-related pathways in rice. We previously characterized the phenotype performance of a rice spotted-leaf mutant spl21 and narrowed down the causal gene locus spl21(t) to an 87-kb region in chromosome 12 by map-based cloning. RESULT:We showed that a single base substitution from A to G at position 836 in the coding sequence of Oryza sativa beta-1,6-N-acetylglucosaminyl transferase (OsGCNT), effectively mutating Tyr to Cys at position 279 in the translated protein sequence, was responsible for the spotted-leaf phenotype as it could be rescued by functional complementation. Compared to the wild type IR64, the spotted-leaf mutant spl21 exhibited loss of chlorophyll, breakdown of chloroplasts, down-regulation of photosynthesis-related genes, and up-regulation of senescence associated genes, which indicated that OsGCNT regulates premature leaf senescence. Moreover, the enhanced resistance to the bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae, up-regulation of pathogenesis-related genes and increased level of jasmonate which suggested that OsGCNT is a negative regulator of defense response in rice. OsGCNT was expressed constitutively in the leaves, sheaths, stems, roots, and panicles, and OsGCNT-GFP was localized to the Golgi apparatus. High throughput RNA sequencing analysis provided further evidence for the biological effects of loss of OsGCNT function on cell death, premature leaf senescence and enhanced disease resistance in rice. Thus, we demonstrated that the novel OsGCNT regulated rice innate immunity and immunity-associated leaf senescence probably by changing the jasmonate metabolic pathway. CONCLUSIONS:These results reveal that a novel gene Oryza sativa beta-1,6-N-acetylglucosaminyl transferase (OsGCNT) is responsible for the spotted-leaf mutant spl21, and OsGCNT acts as a negative-regulator mediating defense response and immunity-associated premature leaf senescence probably by activating jasmonate signaling pathway.
Project description: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.
Project description:Lesion-mimic mutants are useful to dissect programmed cell death and defense-related pathways in plants. Here we identified a new rice lesion-mimic mutant, spotted leaf 33 (spl33) and cloned the causal gene by a map-based cloning strategy. SPL33 encodes a eukaryotic translation elongation factor 1 alpha (eEF1A)-like protein consisting of a non-functional zinc finger domain and three functional EF-Tu domains. spl33 exhibited programmed cell death-mediated cell death and early leaf senescence, as evidenced by analyses of four histochemical markers, namely H2O2 accumulation, cell death, callose accumulation and TUNEL-positive nuclei, and by four indicators, namely loss of chlorophyll, breakdown of chloroplasts, down-regulation of photosynthesis-related genes, and up-regulation of senescence-associated genes. Defense responses were induced in the spl33 mutant, as shown by enhanced resistance to both the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae and by up-regulation of defense response genes. Transcriptome analysis of the spl33 mutant and its wild type provided further evidence for the biological effects of loss of SPL33 function in cell death, leaf senescence and defense responses in rice. Detailed analyses showed that reactive oxygen species accumulation may be the cause of cell death in the spl33 mutant, whereas uncontrolled activation of multiple innate immunity-related receptor genes and signaling molecules may be responsible for the enhanced disease resistance observed in spl33. Thus, we have demonstrated involvement of an eEF1A-like protein in programmed cell death and provided a link to defense responses in rice.
Project description:BACKGROUND:Early leaf senescence influences yield and yield quality by affecting plant growth and development. A series of leaf senescence-associated molecular mechanisms have been reported in rice. However, the complex genetic regulatory networks that control leaf senescence need to be elucidated. RESULTS:In this study, an early senescence 2 (es2) mutant was obtained from ethyl methanesulfonate mutagenesis (EMS)-induced mutational library for the Japonica rice cultivar Wuyugeng 7 (WYG7). Leaves of es2 showed early senescence at the seedling stage and became severe at the tillering stage. The contents of reactive oxygen species (ROS) significantly increased, while chlorophyll content, photosynthetic rate, catalase (CAT) activity significantly decreased in the es2 mutant. Moreover, genes which related to senescence, ROS and chlorophyll degradation were up-regulated, while those associated with photosynthesis and chlorophyll synthesis were down-regulated in es2 mutant compared to WYG7. The ES2 gene, which encodes an inositol polyphosphate kinase (OsIPK2), was fine mapped to a 116.73-kb region on chromosome 2. DNA sequencing of ES2 in the mutant revealed a missense mutation, ES2 was localized to nucleus and plasma membrane of cells, and expressed in various tissues of rice. Complementation test and overexpression experiment confirmed that ES2 completely restored the normal phenotype, with chlorophyll contents and photosynthetic rate increased comparable with the wild type. These results reveal the new role of OsIPK2 in regulating leaf senescence in rice and therefore will provide additional genetic evidence on the molecular mechanisms controlling early leaf senescence. CONCLUSIONS:The ES2 gene, encoding an inositol polyphosphate kinase localized in the nucleus and plasma membrane of cells, is essential for leaf senescence in rice. Further study of ES2 will facilitate the dissection of the genetic mechanisms underlying early leaf senescence and plant growth.
Project description:CONSTANS (CO)-like genes have been intensively investigated for their roles in the regulation of photoperiodic flowering, but very limited information has been reported on their functions in other biological processes. Here, we found that a CO-like gene, Ghd2 (Grain number, plant height, and heading date2), which can increase the yield potential under normal growth condition just like its homologue Ghd7, is involved in the regulation of leaf senescence and drought resistance. Ghd2 is expressed mainly in the rice (Oryza sativa) leaf with the highest level detected at the grain-filling stage, and it is down-regulated by drought stress conditions. Overexpression of Ghd2 resulted in significantly reduced drought resistance, while its knockout mutant showed the opposite phenotype. The earlier senescence symptoms and the transcript up-regulation of many senescence-associated genes (SAGs) in Ghd2-overexpressing transgenic rice plants under drought stress conditions indicate that Ghd2 plays essential roles in accelerating drought-induced leaf senescence in rice. Moreover, developmental and dark-induced leaf senescence was accelerated in the Ghd2-overexpressing rice and delayed in the ghd2 mutant. Several SAGs were confirmed to be regulated by Ghd2 using a transient expression system in rice protoplasts. Ghd2 interacted with several regulatory proteins, including OsARID3, OsPUR?, and three 14-3-3 proteins. OsARID3 and OsPUR? showed expression patterns similar to Ghd2 in rice leaves, with the highest levels at the grain-filling stage, whereas OsARID3 and the 14-3-3 genes responded differently to drought stress conditions. These results indicate that Ghd2 functions as a regulator by integrating environmental signals with the senescence process into a developmental programme through interaction with different proteins.
Project description:Leaf senescence is a complex biological process and defense responses play vital role for rice development, their molecular mechanisms, however, remain elusive in rice. We herein reported a rice mutant spotted leaf 32 (spl32) derived from a rice cultivar 9311 by radiation. The spl32 plants displayed early leaf senescence, identified by disintegration of chloroplasts as cellular evidence, dramatically decreased contents of chlorophyll, up-regulation of superoxide dismutase enzyme activity and malondialdehyde, as physiological characteristic, and both up-regulation of senescence-induced STAY GREEN gene and senescence-associated transcription factors, and down-regulation of photosynthesis-associated genes, as molecular indicators. Positional cloning revealed that SPL32 encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). Compared to wild type, enzyme activity of GOGAT was significantly decreased, and free amino acid contents, particularly for glutamate and glutamine, were altered in spl32 leaves. Moreover, the mutant was subjected to uncontrolled oxidative stress due to over-produced reactive oxygen species and damaged scavenging pathways, in accordance with decreased photorespiration rate. Besides, the mutant showed higher resistance to Xanthomonas oryzae pv. Oryzae than its wild type, coupled with up-regulation of four pathogenesis-related marker genes. Taken together, our results highlight Fd-GOGAT is associated with the regulation of leaf senescence and defense responses in rice.