Isolation And Characterization Of Downstream Regulator Of PHOT2-mediated Hypocotyl Phototropism In Arabidopsis

Plants are photoautotroph sessile organisms,unable to choose their living environment,usually through regulating their behavior and developmental processes in response to the environment changes.Among the many environmental conditions,plants as photosynthetic autotrophs,are particularly sensitive to light.Theeffect of light on plants is mainly reflected in energy and signal molecules.As energy,plants convert light energy into the chemical energy through photosynthesis to provide growth and development.As environmental signaling molecules,plants have evolved different types of photoreceptor that mediate the perception of light signals,such as phototropin,phytochrome,and cryptochrome.Phototropism is decided by the asymmetric growth of hypocotyl,which depends on the asymmetric localization of auxin in hypocotyl under unilateral blue light.Blue light receptor photoreceptor phototropins?PHOT1 and PHOT2?regulate phototropism in Arabidopsis thaliana.Phototropin is a photoreceptor protein kinase containing flavonoid mononucleotide?FMN?related to plasma membrane.Under dark conditions,the activities of the kinase are suppressed,because of combining LOV2?Light-Oxygen-Voltage 2?domain.Upon blue light irradiation,LOV2 domain self-repression was released to enhance the activities of phototropin.Activated phototropin can interact with PKS1,PKS2 and PKS4,that regulate the asymmetric localization of auxin,which leads to the difference in plant growth.It has been reported that PHOT1 and PHOT2 mediate stomatal opening,chloroplast accumulation,phototropism,cotyledon extension and development in the form of functional redundancy.In terms of phototropism,PHOT1 regulates a wide range of blue light response,while PHOT2and PHOT1 mediate high-intensity blue light in the form of functional redundancy.In order to analyze the mechanism of PHOT2 regulating plant phototropism.In the early stage of our research group phot1 single mutant was used as the material for EMS mutation,screening phototropic insensitive mutant.And the non-bending mutant to strong blue light,named P2SA1?PHOT2 Signaling Associated 1?,has been screened out.The mutant showed the phenotype of cotyledon vitrification and root growth stagnation in the culture condition without sucrose.It suggested that the regulators of early development of Arabidopsis seedlings might be involved in the regulation of plant photoadaptation.Therefore,this study cultured the M₂ population of phot1 single mutant mutagenized EMS mutation without sucrose.For screening mutants with high-intensity blue light curvature loss and dysplastic seedling development.It is expected to elucidate the mechanism of early seedling development involved in regulating phototropism.A total of 16 stable genetic mutants were screened out,of which 7 mutant traits were caused by recessive single gene mutation.Phenotypic analysis showed that p2sa3 and p2sa4 mutants could not complete photomorphogenesis under sucrose-free culture conditions,and the hypocotyls lacked high-intensity blue light-induced bending.Mutant p2sa6 and p2sa7 showed vitrification and root growth stagnation under sucrose-free culture conditions,and the hypocotyls appear high-intensity blue light-induced bending.Mutant sdcg1,sdcg2 and sdcg3 showed the cotyledon yellow and albino phenotype under sucrose-free culture,and supplied exogenous sucrose can restore cotyledons to green.The result of coarse positioning by map-based cloning show that mutant p2sa3 and p2sa4 were located on upstream of chromosomes 2 and bottom of chromosomes 4 of Arabidopsis thaliana.The mutant sdcg1,sdcg2 and sdcg3 were located on bottom of chromosomes 2,middle-lower of chromosomes 4 and middle-upper of chromosomes 3 of Arabidopsis thaliana.After fine mapping and the analysis of mutation site,combined with possible gene prediction showed that the 142 and 314 sites of the 19 gene of middle-lower of chromosomes 4 of sdcg2 mutant were both mutated from base G to base A,while the amino acids were mutated from serine to asparagine and glutamic acid to lysine,respectively.The 1076 base of middle-upper part of chromosomes 3 of sdcg3 mutant 11 gene was mutated from G to A,and the amino acid was mutated from glycine to glutamic acid.Currently,we have ordered the T-DNA insertion mutant seeds of 19 gene corresponding to sdcg2 and 11 gene corresponding to sdcg3.And two functional complement vectors have been constructed and transformed into mutants sdcg2and sdcg3.Subcellular localization of SDCG2 and SDCG3 were located in chloroplast and cell membrane,respectively.The ordering of corresponding mutants and the acquisition of related transgenic plants will provide an important basis for studying the basic biological functions of SDCG2 and SDCG3.In conclusion,this study established a screening system of mutants with high-intensity blue light curvature loss and dysplastic seedling development in the culture condition without sucrose for Arabidopsis thaliana and successfully screened out 16 stable mutant materials,of which 7 mutant traits were caused by recessive single gene mutation.By forward genetics and map-based cloning,five mutants were coarse located,five mutants identified mutation intervals and two mutants obtained possible mutation genes.Analysis of the genetic phenotype of the above mutants and prediction of the function of the possible mutant genes.We present our preliminary evidence of the regulators of early development of Arabidopsis seedlings might be involved in the regulation of plant phototropism.Next mutant p2sa3 and p2sa4 are cloned and functionally analyzed,which will provide an important basis for understanding the mechanism of early seedling development involved in regulating phototropism.