Molecular Mechanism Of Receptor Kinase GSO2 Underlying Shoot Regeneration Capacity In Arabidopsis

Plant cell has a powerful capacity with which highly differentiated tissues or organs can produce shoots in vitro under appreciate culture conditions.This process is termed as shoot regeneration.Shoot regeneration not only provided an essential system to study important biological questions,but also is widely applied in plant tissue culture and rapid propagation and genetic transformation.Natural variation of shoot regeneration capacity commonly existed among genotypes within the same plant species,and mechanisms underlying this genotypic variation were largely unknown.Here,we used Arabidopsis as material to explore candidate causal genes by high throughput sequencing,and tried to demonstrate the mechanism in regulation of shoot regeneration capacity in different genotypes.We found that the ecotypes Col-0 and Gu-0 of Arabidopsis possessed extreme phenotypes in shoot regeneration capacity among different genotypes.After culture for 20 days on shoot-induction medium(SIM),the shoot regeneration frequency of Col-0 genotype reached 100%,whereas that of Gu-0 genotype was only 5%,suggesting that shoot regeneration was highly repressed in Gu-0 genotype.We crossed Col-0 with Gu-0 plant;the F2 population was used to map the candidate genes controlling shoot regeneration capacity difference by BSA high throughput sequencing strategy.By analyses the phenotypes of shoot regeneration of the mutants of candidate genes,GASSHO2(GSO2)was identified as a most likely causal gene to be responsible for shoot regeneration.Loss-of-function of GSO2 resulted in the repression of shoot regeneration.Sequence analysis showed that 32 amino acid residues of GSO2 protein were different between Col-0 and Gu-0.GSO2 encoded a receptor kinase,which was identified to be localized the cell membrane.GSO2 was mainly expressed in the callus cells at early stages and the de novo shoot meristem.By yeast hybrid screen,we found GSO2 interacted with RHIP1 protein,which played crucial function in glucose-mediated signaling.Moreover,we used pull-down assay to confirm this interaction.Finally,mutation in GSO2 resulted in the decreased sensitivity to glucose in SIM,suggesting GSO2 was involved in the regulation of shoot regeneration by modulation of glucose signaling.The expression of glucose-related gene CA2 and DIN1,shoot apical meristem related genes WUS,STM and CLV3 and auxin synthesis key gene YUC4,YUC7 and YUC8 in gso2 and rhip1 mutant was decreased by quantitative PCR.This study demonstrated that GSO2 may regulate the synthesis of auxin through RHIP1-mediated glucose signal,affect the activity of shoot apical meristem and regulate the shoot regeneration.Our findings provided new information regarding the mechanism of shoot regeneration capacity difference among Arabidopsis genotypes.