Induction Of Aleurone ?-amylase Production By GABA In Seeds Of Barley And Rice

The aleurone layer is a prevalent form in cereal seed,and it is composed of aleurone cells surrounding the endosperm.Germinating embryo produces GAs and then GAs move to the aleurone layer,where in aleurone cells GAs induce the synthesis and secretion of ?-amylase for the hydrolysis of Endosperm.This process is a typical GAs response event in the cereal aleurone cells,which can provide energy and substrate for growth of young seedlings.The synthesis and secretion of ?-amylases is induced by GAs and repressed by ABA.Because aleurone cells response differently to plant hormones,they have been regarded as one of the model systems for the study of phytohormones action and signaling.Gamma-aminobutyric acid?GABA?is a four-carbon non-protein amino acid presented in plants just as a metabolite or a signalling molecule.In this study,the inductive effect of GABA on the activity of ?-amylase was studied by using the seeds of barley and rice.The main results are shown as follows:The content of endogenous GABA in barley seeds increased gradually during germination,suggesting that endogenous GABA is involved in barley seed germination.Then we measured the degradation of starch under different treatments and found that,GABA promoted the degradation of starch in seeds with a concentration effect.The optimal concentration of GABA was 16 mM.This shows that exogenous GABA could consume more endosperm for the germination.The activity of ?-amylase was determined by agar plate assay and test tube assay.We found that,?-amylase activity was higher in barley seeds treated by GABA or GA3 compared with that of the control.In agar plate assay,GABA induced bigger halos than control after 24 h and 16 mM GABA has the best effect.In addition,the inhibitors of GA3 biosynthesis S-3307 and PP333,as well as ABA,cannot suppress inductive effect of GABA on ?-amylase activity.The results suggest that the effect of GABA on induction of ?-amylase secretion in aleurone cells is independent on GA or ABA.Molecular evidence suggested that GABA up-regulated ?-amylase gene expression.We used real-time PCR to measure the expression levels of members of the ?-amylase gene family;GABA increased transcription of these ?-amylase genes.These results suggested that GABA might be involved in the induction of ?-amylase gene expression in the aleurone layer.The content of soluble protein and free amino acids was deteced using barley and rice seeds,we found they all increased with the seed germination and Glu content was higher than the other 16 kinds of free amino acids.Meanwhile the value of GABA yield,GAD activity and PAO activity in grain embryo were exceeding the endosperm greatly.Relative expression of OsGAD1 gene was detected in the rice embryo.The result suggested that GABA was synthesized mainly in the embryo during germination.With the increase concentration of exogenous GABA,the weighted germination percentage increased gradually in barley,and reached the peak at the concentration of 16 mM.The biomass of shoots and roots also showed a rising trend,and the roots biomass treated by 16 mM GABA has significant difference compared to the control.We then detected respiratory rate,GDH activity and content of ?-ketoglutarate and found that,they both increased with increasing concentration of GABA up to 16 mM.The results indicate that the seedlings treated by GABA might accumulate more organic matters.Using rice as experimental materials to verify the effect of GABA can explore whether GABA-induced ?-amylase on cereal is universal.Further analyses showed that GABA-treated can induce ?-amylase production,promote the seed germination and the seedling growth.Rice is not sensitive to low concentration GABA treatment,and there is no significant difference between control and 0.25 mM GABA treatment on weighted germination percent and biomass.Higher concentrations of GABA have inductive effect on rice seeds respiratory rate as well as rice seedlings growth and the effect on rice root is better than GA3.All results suggested that GABA has its own unique effect relative to the GAs effect on germination.The activity of APX,CAT and SOD as well the rate of oxygen consumption and the percentage of live cells in barley aleurone layer were tested,we found that the activities of these enzymes as well the rate of oxygen consumption and the percent live cells treated by GABA declined sharply.Meanwhile H₂O₂ and O2·-accumulation was up-regulated by GABA.The result suggested that GABA might be involved in programmed cell death.Our results show that GABA can induce aleurone cells to synthesis and secrete ?-amylase,and promote of seed germination.To our knowledge,this is the first report about the inductive effect of GABA on seed germination.The effect of GABA was affirmed by analyzing ?-amylase activity and gene expression and the relation to GA.