| Leaf is the most important source organ of crops,chloroplast is the place of photosynthesis,its development directly affects the growth and development of plants,and ultimately affects biomass,yield and quality.Leaf color variation in rice is a kind of mutation character which is easy to identify.It can not only be used as morphological marker in rice molecular breeding,but also be an important material for studying rice functional genome,especially light system,chlorophyll synthesis and metabolism,and genetic differentiation and development of chloroplast.In our previous study,the rice oslcd1(Oryza sativa low-temperature-sensitive chlorophyll-deficient 1)mutate with spontaneous chlorophyll-deficient leaf phenotype was obtained through EMS-mutagenesis of cultivar indica HuaZhan.And the mutant material oslcd1 with chlorotic leaves appeared at the seedling stage was selected from the offspring.In this study,oslcd1 was used as the material to determine the reasons for the phenotype from physiological,cellular,and molecular levels.The main results are as follows:1.The leaves of the oslcd1 were always white and green under the field condition,,which resulted in a significantly lower chlorophyll content and net photosynthetic rate than the wild type plants.Finally,the plant height,tiller number and total grain number per panicle of the mutant were significantly lower than the wild type plants.It was found that there were more macrohair and microhair on the leaf surface than wild type by SEM;Furthermore,the observations by TEM revealed that many chloroplasts in mesophyll cells of oslcd1 were vacuolated.2.The leaves of oslcd1 were basically white at 20? and were not different from the wild type at 28?.And the total chlorophyll content of oslcd1 was only 24.72% compared with the wild type plants at 20?,but was not different from the wild type plants at 28?.The fluorescence parameters showed that there was no significant difference in the potential maximum photosynthetic efficiency between oslcd1 and its wild-type at different temperatures,but with the decrease of temperature,the actual photosynthetic efficiency of oslcd1 was significantly lower,which might be caused by the low relative electron transfer efficiency;the light damage of oslcd1 under high temperature was significantly higher.Furthermore,TEM results showed that the chloroplasts of oslcd1 were few and incomplete at 20?.3.The results of RNA-seq showed that a large number of chloroplast related genes were significantly differentially expressed at 20?,among which 389 genes were up-regulated and 178 genes were down regulated.The analysis of chlorophyll synthesis and chloroplast development related genes showed that GluR remarkably lower expression in oslcd1 leaves,GSA1,CAO2,HEMC,HEME1,OsCHLD,OsCHLH,OsRpoTp,rpoA,rpoB were significantly higher,so were the expression of some temperature sensitive genes determined.4.The mutant plants oslcd1 was sensitive to salt stress and simulated drought stress.Compared with the control,the plant height and root length of the mutant under salt stress were significantly lower than that of the wild type plants,while the root growth of oslcd1 was significantly inhibited by simulated drought stress.5.Genetic analysis showed that the trait of oslcd1 was regulated by a single recessive nuclear gene.Using the F2 mapping population derived from oslcd1/02428 and oslcd1/Zhonghua11,bulked segregant analysis mapped this gene to a 710 kb interval between the RM19441 and R6M37 markers in rice chromosome 6 which contained 35 candidate genes according to RAP-DB Database.Sequencing of these ORFs confirmed that the thing happened in oslcd1 was two GG substitued by TT in the first exon of LOC_Os06g08060 encoding a leucoanthocyanidin Dioxygenase(LDOX),which led to early termination.Furthermore,it was confirmed that LOC_Os06g08060 was the OsLCD1 by genetic complementation.6.Two-line thermo-sensitive chlorotic leaf mutant oslcd1-2 was obtained by gene editing. |
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