Screening And Identification Of Cotyledon Albino Mutant Sca1 In Arabidopsis

Chloroplast development during seed germination and seedling photomorphogenesis is crucial for the smooth transition of seedlings from heterotrophic to photoautotrophic.From the initial protoplast to the mature thylakoid structure of chloroplast,chloroplast development is regulated by the synthesis of photosynthetic genes,chlorophyll synthesis,and the exchange of nuclear and cytoplasmic signals.Chloroplasts are responsible for plant carbon dioxide fixation,carbon skeleton production,fatty acid,pigmentation and amino acid synthesis.Sugar not only serves as an energy source and structural material for plant metabolism,but also acts as a signaling molecule involved in many metabolic processes in plants.Recent studies have also confirmed that sugar as a signal molecule affects the expression of nuclear genes,especially the expression of photosynthetic genes that inhibit nuclear encoding,such as the small subunit gene of rubisco of the chlorophyll binding protein gene.At present,through a variety of screening methods,although has been isolated from plants to destroy different stages of chloroplast development of a variety of mutants,and the regulation effect of corresponding mutant genes on chloroplast development has been studied.However,the research on the regulation mechanism of chloroplast development is not complete,and it is necessary to select an appropriate angle to explore and find new mutant.Sugar was used as a key factor in the regulation of chloroplast development.Sugar was used as screening condition to search for abnormal mutant of chloroplast development and study the role of mutant gene in chloroplast development.It will provide new clues to understand the mechanism of chloroplast development.In this study,the mutant population of arabidopsis M2 induced by EMS(ethylmethylsulfone)was cultured in sucrose deficient medium to screen the mutant with abnormal chloroplast development.We obtained a seedling cotyledon albino mutant called sca1(seedling cotyledon albino 1).Phenotypic analysis showed that mutant sca1 seedlings showed albinism in cotyledon on sugar-free medium,and exogenous application of various sugars could not restore their albinism phenotype.Compared with the wild type,the mutant sca1 showed small chloroplast and less grana lamella,decreased chlorophyll fluorescence parameters F0,Fm and NPQ,and decreased photosynthetic pigment content.With the prolongation of growth period,the true leaf of mutant sca1 was regreenened to some extent,but showed slow growth,multiple branches,deformity of flowers,low seed setting rate and long growth cycle.It has been reported that photooxidative damage caused by the lack of carotenoid protection under high light can cause chlorophyll damage and cause leaf albino.The cotyledons of sca1 mutants were treated with high and low light,Found that the cotyledon of sca1 mutant was obviously green under low light condition.Therefore,it was speculated that sca1 cotyledon albinism might be the chlorophyll photooxidation damage caused by the synthesis defect of carotenoids.Genetic analysis showed that the phenotype of mutant sca1 was caused by single recessive mutation.The map-based cloning showed that the mutant gene was located on BAC of D between F14M13 and T19L18 on chromosome 2,The 26 effective genes on the BAC were analyzed and sequenced,and three genes were eventually found to have an effective mutation.The three genes were named D1,D2 and D3.After searching and reading literature,it was found that these three genes were unknown genes.At the same time of ordering the corresponding mutant seeds,in order to determine the genes,we constructed the 35S::GFP vector of three genes.Using gene gun transformation and agrobacterium to infect sca1 mutants,it is found that both transient materials and screened permanent materials,D1 gene can restore the mutant leaf color to green.Therefore,we speculated that D1 gene might be the mutant gene we were looking for.Subcellular localization analysis showed that D1 candidate gene coding protein was located in the nucleus,D2 candidate gene coding protein was located in the cytoplasm,and D3 candidate gene coding protein was located in the cytoplasm.In summary,we screened the cotyledon albino mutant sca1,which was treated with strong and weak light,and proved that the cotyledon albino of sca1 may be the chlorophyll photooxidation damage caused by the synthesis defect of carotenoids.Three candidate genes,D1,D2 and D3,were located by map-based cloning of mutant sca1.It was proved that D1 gene may contribute to cotyledon albinism of mutants by transient transformation and screening of hyperexpression materials.Subsequently,the mutant gene will be identified by homozygous identification and phenotype analysis of the ordered mutant seeds,and its biological function will be studied,which is expected to make a breakthrough in explaining the regulation of chloroplast development.