Screening And Identification Of The Suppressors Of Arabidopsis Shb1-D Mutant

Light is one of the most important environmental factors that affect the development of plants,almost affecting all life aspects of plants.Photosynthesis provides energy for plant growth and development.At the same time,as a signal,it triggers various responses of plants to the ambient environments.As the storage organ of nutrients,seeds provide food and raw materials for humans and meet people's daily needs.Seed size is one of the key factors affecting crop yield,but the molecular mechanism of regulating seed growth and development is still not well understood.SHB1(SHORT HYPOCOTYL UNDER BLUE 1)is involved in the regulation of plant light signal transduction and regulates the expression of PIF4 and HFR1.SHB1 specifically induces the expression of PIF4 gene under red light and HFR1 gene under red and blue light.At the same time,SHB1 also regulates seed development.SHB1 associates with the promoters of MINISEED3(MINI3)and HAIKU2(IKU2),regulates their expression and endosperm development,and affects the final seed size.The shb1-D mutant has a gain-of-function mutation in Ws background.In this study,we used EMS to mutagenize shb1-D seeds,and then screened suppressors based on their hypocotyl phenotype and seed size.Three mutants,b2 shb1-D,r4 shb1-D and r175 shb1-D,were isolated by screening the mutated M2 population.In this study,phenotypic analysis of these shb1-D suppressor mutants was performed,and the mutated genes were identified through map-based cloning.The specific research results are as follows:(1)We performed phenotypic analysis on the three suppressor mutants.Under red and blue light,the hypocotyls of the three mutants were significantly shorter than those of the shb1-D mutant,and the hypocotyls of the three mutants were also shorter than those of the Ws wild type.After these mutants were crossed to Ws wild type to remove the shb1-D mutation,the hypocotyls of the mutants in Ws background were still significantly shorter than that of the Ws wild type.(2)Compared with shb1-D,the cotyledon area of b2 shb1-D,r4 shb1-D and r175 shb1-D were significantly reduced.The chlorophyll content and the expression of CAB3 were significantly increased.(3)After the three mutants were crossed to shb1-D,genetic analysis showed that the three mutants were recessive.We next crossed the three mutants to each other for allelic test,and b2 shb1-D and r4 shb1-D were allelic.r175 shb1-D was an independent mutant.(4)To map the chromosomal location of the three mutants,we used rough mapping and genome sequencing technology.Mutations in b2 shb1-D and r4 shb1-D mutants were mapped to chromosome II,and there are nine candidate genes.Phenotypic rescue experiments were performed by transforming each of the candidate genes to b2 shb1-D and r4 shb1-D mutants.The mutant gene was identified as AT2G252 XX and it was localized in the nucleus.Yeast twohybrid and Bi FC experiments revealed that SHB1 and AT2G252 XX do not interact directly.