Molecular Mechanism Of Thioredoxin DCC1-Mediated ROS Homeostasis Underlying Genotypic Variation Of Shoot Regeneration Capacity In Arabidopsis

Shoot regeneration is the essential prerequisite for genetic transformation and thus crucial for biotechnological breeding and gene function analysis.However,a lot of plant species display shoot regeneration recalcitrance.Even within a particular species,the capacity of shoot regeneration also varies significantly among genotypes such as cultivars or ecotypes.Up to date,little is known about the causal gene(s)and molecular mechanism(s)underlying genotypic variation of shoot regeneration capacity.In this study,we identified DCC1 as the candidate causal gene responsible for natural variation of shoot regeneration capacity in Arabidopsis thaliana by a map-based strategy.Further,we analyzed the function of DCC1 in shoot regeneration and attempted to uncover the molecular mechanism through the genetic approaches combined with molecular biology and biochemistry methods.The main results are as follows:We analyzed the shoot regeneration capacities of 48 ecotypes using a highly efficient shoot regeneration system and found that the regeneration capacities among different ecotypes were highly variable.For example,ecotypes Col-0,Ws-0,Pu2-7,Lan-0 and Bu-0 could completely regenerate into new shoots on SIM for 32 days,with the 100%frequencies.In contrast,the frequencies of the ecotypes Nc-1,Wa-1,Gu-0 and Kelsterbach-4 were still very low at this time.Further,we chose two ecotypes Col-0 and Gu-0 with the very significant difference in regeneration capacity,to map the causal gene,and DCC1 was identified as the candidate factor.Linkage disequilibrium analysis with haplotype structure in DCC1 sequences among alleles of different ecotypes revealed that six SNPs were associated with the regeneration frequencies.During shoot regeneration,DCC1 was mainly expressed in the inner tissues of the callus.The dccl mutant in Col-0 background showed severe defects in the capacity of shoot regeneration.Complementation analysis revealed that the DCC1 allele of Col-0 rescued the dccl phenotypes,indicating that the defects of dccl in shoot regeneration were caused by mutation in DCC1.On the other hand,the failure of DCC1 allele from Gu-0 in restoration of dccl defects suggested natural variations in Gu-0 allele resulted in low capacity of shoot regeneration.DCC1 encodes a thioredoxin targeted to mitochondria and chloroplasts.We assumed that the thioredoxin was involved in Reactive Oxygen Species(ROS)homeostasis.During shoot regeneration,loss of function of DCC1 resulted in the increased ROS level measured by DAB staining.And the exogenous H2O2 repressed shoot regeneration significantly.The higher levels of H2O2 were correlated with the lower capacities of shoot regeneration.These findings suggested that DCC1 mediates ROS homeostasis and then,regulates shoot regeneration.To reveal the mechanism of DCC1-mediated ROS homeostasis,we performed yeast two hybrid,pull-down and BiFC assay.The results indicated that DCC1 protein interacted with CA2,a subunit of mitochondrial respiratory chain complex I.CA2 encodes a carbonic anhydrase and functions in assembling of respiratory chain complex I,which contributes to the ROS production.Similar to DCC1,mutation of CA2 led to the increased ROS level and the decreased capacity of shoot regeneration.Furthoremore,the mutants dcc1,ca2 and dcc1ca2 showed significant decrease in activity of respiratory chain complex I and shoot regeneration.These findings suggest that DCC1 regulates the activity of respiratory chain complex I and ROS homeostasis in combination with CA2.Thus,we made a hypothesis that the capacity of shoot regeneration might be enhanced by decreasing the ROS level.Indeed,during shoot regeneration,the exogenous GSH promoted the shoot regeneration.The phenotypes of the single mutant dccl or ca1 and the double mutant dccl ca1 were completely rescued by GSH.To find out the correlation between ROS and shoot regeneration in different ecotypes,we used DAB staining to detect the ROS level of different ecotypes during shoot regeneration.The low level of ROS was correlated with the high capacity of shoot regeneration among different ecotypes.ROS homeostasis is a critical factor to determine the variations of shoot regeneration capacities in different ecotypes.Our findings uncovered a close correlation between the polymorphism of the thioredoxin gene DCC1 and the variation of shoot regeneration capacities of different ecotypes.DCC1 regulated the capacity of shoot regeneration through modulating the ROS level.Our study provided new perspective and approaches for revealing the mechanism of shoot regeneration variation in different ecotypes,and might be helpful for overcoming the recalcitrance of shoot regeneration in grain crops and horticultural crops,as well as the genetic transformation.