Molecular Mechanism Of ?-aminobutyric Acid Metabolism Regulating Arabidopsis Thaliana Leaf Development

Leaf is an important organ for plants to synthesize organic products during photosynthesis,and stomata are the main channels for CO₂and water vapor exchange between plants and the environment.Stomatal development is affected by internal factors and external signals,and is regulated by a complex regulatory network.?-aminobutyric acid(GABA)is a metabolic signaling molecule,and plays a dual role in metabolic regulation and signal regulation in plant development.GABA has the function of eliminating reactive oxygen species(ROS),and ROS can regulate leaf development by regulating the cell cycle.In this study,from the aspects of leaf stomatal development and ROS signal-mediated leaf development,Arabidopsis GABA synthetic mutants(gad1,gad2,and gad1/gad2 double mutants)were used as materials,aiming to decipher the regulating signal pathways of GABA metabolism on leaf development.The main results are as following:1.The leaf area of Arabidopsis thaliana was firstly measured.The results showed that the leaf area of the GABA synthetic mutant was significantly larger than that of the wild type,and the area of the gad1/gad2 double mutant was significantly different from that of the wild type.It shows that GABA synthesis mutation can result in the normal growth of leave;2.Observation of the stomata in epidermal cells was conducted.Results showed that the stomatal density(SD)in the leaf epidermis of GABA synthetic mutants were significantly lower than those of wild type.It showed that GABA synthesis mutation inhibited the production of stomata;3.In order to clarify the regulatory mechanism of GABA metabolism and stomatal development,the genes related regulating stomatal development were conducted q RT-PCR analysis.The results showed that the expression of MUTE?BASL and phy B,which promoted the development of stomata,was significantly down-regulated in GABA synthetic mutants;4.Furthermore,the expression of signal components regulating stomatal lineage was analyzed.The results showed that in GABA synthetic mutants,the expression of COP1?YDA?MKK4 and MKK5 was significantly up-regulated.COP1 can inhibit the generation of stomata,and YDA and MKK4/5 play a consistent role in promoting the differentiation of paving cells and inhibiting the formation of stomata.GABA synthesis mutation can cause the differentiation of PC and inhibit the production of stomata by promoting the expression of COP1?TMM,?MKK4 and MKK5;5.To explore the correlation between ROS and stomatal development,the reactive oxygen species(ROS)fluorescent probe H2DCFDA was used to analyze ROS levels in leaves and epidermal cells.The results showed that the total content of ROS in leaves and epidermal cells in GABA synthetic mutants was significantly higher than that of wild type.ROS in epidermal cells was concentrated in guard cells of stomata,and there was no fluorescent signal in the PC cells.It indicates that GABA synthesis mutation will affect the accumulation of ROS in leaves and epidermal cells,thus inhibiting cell proliferation;6.NBT and DAB were also used to detect and quantify the content of O₂^-and H₂O₂in Arabidopsis leaves.In the leaves of GABA synthetic mutants,the levels of O₂^-and H₂O₂were significantly higher than those of the wild type.It shows that inhibition of GABA synthesis will promote the accumulation of O₂^-and H₂O₂in leaves,which may be the reason for the increase of leaf area;7.TARGET OF RAPAMYCIN(TOR)is a kinase whose expression level is positively related with the growth of Arabidopsis thaliana,and is a hub of growth and development regulation.The q RT-PCR results showed that the expression of TOR in the gad1/gad2 mutant was significantly up-regulated;8.In order to elucidate the signal pathways of GABA metabolism and accumulation of ROS activity,q RT-PCR analysis was performed on genes related to ROS accumulation.The results showed that the relative expressions of MPK9 and MPK12 in the GABA synthetic mutant were significantly upregulated.This indicates that endogenous GABA may indirectly inhibit the expression of MPK9 and MPK12and participate in the signaling pathway regulated by ROS.In this paper,through the phenotype analysis of leaf area and epidermal stomata density,the expression analysis of signal pathways involved in stomata development is analyzed.A combing study of ROS levels in the leaves and the key components of ROS accumulation confirmed the possible mechanism of GABA metabolism on leaf development.