Effect Of Ca²⁺ On Molecular And Physiological Mechanism Of GABA Accumulation In Kandelia Candel?L.? Druce Under High Salt Stress

Soil salinization is one of the most important abiotic stress factors that affect agricultural production and ecological environment in the world.With the increase of population,the development and utilization of the existing salinized land is urgently needed.Kandelia candel(L.)Druce,which is a secrete salt xylophyte that mainly distributes in tropical and subtropical coast intertidal zone,is a model plant for studying molecular mechanism of salt-tolerant xylophyte based on that it has evolved a salt tolerance mechanism in the salt environment through suffering from sea water perennially.?-aminobutyric acid(GABA),is a four-carbon non-protein amino acid,when the plant is in stress,the GABA content will accumulate.The K.candel seedlings of 60 d in the Hoagland solution were treated with 500 mM NaCl as well as Ca2+chelating agent EGTA or Ca2+carrier agent(sugar alcohol calcium)for 3 d as experiment materials.(1)High Performance Liquid Chromatography(HPLC)was used to determine the GABA content of K.candel leaves in different treatments.The results showed that GABA content under 4 mM Ca2+ treatment was the highest in all treatments,and lowest under 10 mM EGTA treatment.(2)The proteins of K.candel leaves under 4 mM Ca2+treatment and 10 mM EGTA treatment were separated by two-dimensional electrophoresis(2-DE),and the differentially expressed proteins were analyzed using ImageMaster 7.0 software.More than 600 proteins were detected in reprolineducibly 2-D gels,of which 35 proteins showed more than 2-fold change in abundance.Total 31 proteins were successfully identified by MALDI-TOF/TOF-MS.According to their functions,these proteins were involved in photosynthesis(39%),molecular regulation(16%),GABA synthesis(4%),TCA cycle(3%),detoxification antioxidant(13%),molecular chaperone(6%),transcription and translation(3%),ATP synthesis(4%)and other physiological processes.Among them,the most of proteins in Calvin cycle were up-regulated under Ca2+ treatment;the proteins in GABA synthesis pathway were up-regulated under Ca2+ treatment and down-regulated under 10mM EGTA.(3)RT-PCR and RT-qPCR were used to investigate mRNA expressions of GAD,GABA-T,SSADH,MDH,DAO,P5CS,APX,POD.The results showed that the mRNA expressions of GAD,MDH,APX,POD were up-regulated under Ca2+ treatment,the mRNA expression of GABA-T was not changed,the mRNA expression of SSADHwas down-regulated,while the results under EGTA were the opposite;the mRNA expression of DAO was down-regulated under Ca2+ and EGTA treatments;and the mRNA expression of P5CS wasn't changed significantly under both Ca2+ and EGTA treatments.(4)The enzyme activity experiments of GAD,DAO,P5CS,POD,SOD were performed,showing that the activities of GAD,POD,SOD increased under Ca2+ treatment and decreased under EGTA;the DAO activity decreased under both Ca2+ treatment and EGTA;and P5CS activity was not changed.The essays of proline,H2O2,and O2-,ASA contents were carried out,finding that the proline content was not changed significantly under both Ca2+ treatment and EGTA;the contents of H2O2 and O2-decreased under Ca2+ treatment and increased under EGTA,while the result of the ASA content was the opposite.To sum up,according to the protein levels,physiological levels and transcription level of GAD and P5CS together with the content of GABA and proline under different Ca2+ treatment,GABA could reduce the damage of K.candel seedlings under salt stress by osmotic adjustment rather than proline.The results of the mRNA expressions of GAD,GABA-T,SSADH,MDH and DAO as well as the activities of GAD and DAO under different Ca2+treatment suggested that the accumulation of GABA in K.candel under salt stress was mainly due to the enhancement of GABA shunt by Ca2+.The data of the contents of reactive oxygen species(ROS),the activities of antioxidant enzymes,as well as the ASA content indicated that Ca2+ contributed to oxidation resistance and salt tolerance of K.candel leaves in order to maintain plant growth under salt stress.In conclusion,the protein levels,mRNA levels and enzymatic activity of GAD were regulated by Ca2+ to accumulate GABA through the enhancement of GABA shunt in order to reduce the damage of K.candel,meanwhile,the increased activities of antioxidant enzymes and upregulated contents of antioxidants might improve salt tolerance.These findings will provide a new theoretical point of molecular mechanisms of salt-tolerant xylophyte under protein,mRNA and physiological levels.