Regulation Of ?-aminobutyric Acid Metabolism On Endoreplication In Arabidopsis Thaliana

?-Aminobutyric acid?GABA?is an important non-protein amino acid in plants and plays an important role in regulating the growth and development of plants.Under stress conditions,GABA accumulated a lot,and scavenging reactive oxygen?ROS?,slowed down the damage caused by high concentration of ROS.Endoreplication is a special form of mitosis,which refers to the process in which DNA replicates but cells do not divide in a cell cycle.The cells that replicate within the cell usually have greater growth potential and stronger stress resistance.In order to study the relationship between GABA anabolism and endoreplication,we used GABA synthetic mutants?gad1,gad2,gad1/gad2?as experimental materials and analyzed the expression changes of cell cycle regulatory factors from cell ploidy analysis in order to explore the molecular mechanism of GABA metabolism and endoreplication.The experimental results show that:1.Image J was used to measure the leaf area of each mutant.The results showed that under normal growth conditions,the leaf area of GABA synthetic mutants was larger than that of wild-type Arabidopsis thaliana.At the eighth day of leaf age,the first pair of true leaf area of gad1 and gad2mutants was 1.3 times of that in wild-type,and the leaf area of gad1/gad2double mutants was 1.8 times of that of wild-type,This indicated that GABA synthesis mutation would lead to the increase of leaf area of A r a b i d o p s i s t h a l i a n a,and the effect of difference in double mutants was more obvious;2.The average cell area of leaf epidermis of GABA synthetic mutants was larger than that of wild type.At the 8th day of leaf age,the average cell area of the first pair of true leaf epidermis in gad1 and gad2 mutants was1.2 times of that in wild type,and the average cell area of epidermis in the first pair of the true leaf in gad1/gad2 double mutants was 1.4 times of that in wild type,this result indicated that GABA synthesis mutation would lead to the increase of the epidermal cell area of Arabidopsis thaliana leaves,and the effect of difference in double mutants was more obvious;3.Flow cytometry analysis of mutant leaves showed that in the early stage of leaf development?the first pair of true leaves at 8th d of foliar age?,octoploidy cells appeared earlier in the leaves of GABA synthetic mutants than those in wild type;in the late stage of leaf development(at 21^{s t} d of foliar age in the first pair of true leaves),the proportion of high ploidy cells in mutant leaves was higher than that in wild type,this indicated that GABA synthesis mutatants could promote the endoreplication in Arabidopsis t h a l i a n a leaf cells;4.CDC6 is an important component in the pre-RC wich navigates G1/S phase transition in cell cycle.Its activity regulation affects cell proliferation and differentiation.The results of qRT-PCR showed that the expression of CDC6 in gad1/gad2 double mutants was up-regulated about 8folds,this result indicated that GABA synthesis mutations could promote DNA synthesis relevant with endoreplication in the early stage of cell cycle;5.Quantitative analysis of the expression of SIAMESE?SIM?and SMR?SIAMESE-RELATED?,the inhibitors of mitotic CDK activity,showed that the genes of SIM/SMR family were generally up-regulated.The expression of SIM/SMR was up-regulated 2-5 folds in gad1 mutants,4-6 folds in gad2mutants,and 6-17 folds in gad1/gad2 double mutants,this result indicated that GABA synthesis mutation coud promote the expression of cyclin-related protein inhibiting genes,and the effect was more obvious in the double mutatans;6.Quantitative analysis of CYCD3;1 and CDKA;1 which directly regulate the occurrence of endoreplication,showed that the expression of these two genes was down-regulated in mutants,The expression level of C Y C D 3;1 in g a d 1 and g a d 2 mutants was about 0.2 folds of that in wild type,about 0.1 folds of that in gad1/gad2 double mutants.The expression level of C D K A;1 in g a d 1 mutants was about 0.4 folds of that in wild type,about 0.2folds of that in gad2 mutants,and about 0.1 times in gad1/gad2 double mutants.These results indicated that the absence of GABA endogenous synthesis could affect the occurrence of endoreplication in the early stage of cell cycle.This meant that GABA negatively controls the endoreplication and plays a crucial regulatory role in the normal development of plants.