Regulatory Roles Of γ-aminobutyric Acid (GABA)in Growth And Acclimation In Poplar Under Salt Stress

Soil salinization not only influences the growth,production,and distribution of trees,but also threatens the land ecological security.Thus,the study on the mechanisms of tree salt tolerance has important guiding significances for the enhancement of the ecological economic benefits of trees under salt stress and promotion of the ecological restoration of saline-alkali land.γ-Aminobutyric acid（GABA）is a small molecular metabolite of four carbon non-proteinogenic amino acid,acting as an important inhibitory neurotransmitter in animals.It has been proved to participate in many processes as a signal molecule and metabolite in plants response to abiotic stresses as well as growth and development.But the mechanism of GABA on plant adaptation and growth under stresses is still unclear.Here,the possible mechanism of GABA in regulating poplar growth and acclimation was explored by the ways of morphology and anatomy,physiological and biochemical indexes,transcriptomics and metabonomics as well as ¹³C labeling technology in poplar seedlings under short-term（hydroponic culture）and long-term（soil culture）salt stress after Na Cl treatments.1.Responses of GABA shunt coupled with carbon and nitrogen metabolism in poplar seedlings under Na Cl stressSix-week-old poplar seedlings（Populus alba×Populus glandulosa‘84K’）were treated with 0,50,100,200 and 300 m M Na Cl for 30 h under the hydroponic conditions.The results showed that the contents of H2O2,MDA and chlorophyll in leaves and the accumulation of sodium in roots and leaves,were obviously increased.The activities of glutamate decarboxylase（GAD）and GABA-transaminase（GABA-T）in GABA shunt,as well asα-ketoglutarate dehydrogenase（α-KGDH）（except 300-m M Na Cl）and succinate dehydrogenase（SDH）activities in TCA cycle,were enhanced 27.2%-301.3%by different concentrations of Na Cl stresses.Meanwhile,the expression levels of GADs,GABA-Ts,SDHs,succinyl-Co A ligases（SCSs）,and succinic acid aldehyde dehydrogenases（SSADHs）were also increased.Notably,significant increases 30.0%-200.0%in the key components of GABA shunt,Glu and GABA,were observed under salt stress（except 300-m M Na Cl）.Soluble sugars and free amino acids were enhanced,whereas malate and succinate were almost inhibited.Thus,these results suggested that the carbon-nitrogen balance could be altered by activating the GABA shunt when main TCA-cycle intermediates were inhibited,improving salt tolerance in poplar.2.Roles of GABA on regulating ROS homeostasis and hormone（ABA and ethylene）signalling in poplar seedlings under Na Cl stressSix-week-old poplar seedlings（Populus tomentosa）were treated with 0,0.25,and 10 m M GABA under 200 m M Na Cl for 0,6 and 24 h under the hydroponic conditions.The results showed that both 0.25 m M and 10 m M GABA affected the accumulation of hydrogen peroxide and hormones（ABA and ethylene）in the different manners,compared with 0 m M GABA under salt stress,and even 0.1 m M GABA significantly made endogenous ABA levels decrease 44.8%in the salinity condition.At the transcription level,exogenous GABA regulated the expression of ABA related genes（ABAG,ABAHs and PYLs）and ethylene related genes（ACOs,ACSs and ERFs）under salt stress.In addition,the elevated endogenous GABA levels,resulting from overexpression of GAD2 and blockage the activities of GABA-T andα-KGDH with inhibitors of vigabatrin and succinyl phosphonate respectively,altered the expression of ABA levels and ethylene related genes.Therefore,these results indicated that GABA may act as a signal molecule involved in regulating the homeostasis of H2O2 and hormones（ABA and ethylene）signalling pathways in poplar under salt stress.3.GABA regulates carbon metabolism of poplar and promotes growth under salt stressNine-week-old 84K poplar seedlings were treated with 0 and 0.4%GABA under low（1.0%）,medium（1.5%）and high（2.0%）doses of Na Cl treatments for 45 day under soil conditions.The results showed that exogenous GABA enhanced the leaf carbon fixation ability under medium-dose salt stress by increasing leaf area,leaf number,relative chlorophyll content and photosynthetic capacity,which changed height and base diameter,and the poplar biomass increased 29.0%compared with medium-dose salt stress.Meanwhile,exogenous GABA changed carbohydrate levels caused by medium-dose salt stress,and alleviated the inhibition of Glu related amino acids and TCA-organic acids levels caused by salt stress,accompanied by the expression changes of carbon and nitrogen metabolism related genes.In addition,exogenous GABA changed the proportion of cell wall components by a significant reduction of hemicellulose（6.4%）and a significant incerease of acid soluble lignin（28.6%）in dry weight per gram under medium-dose salt stress,whereas a significant increase in total lignin（20.8%）and slight increases in total hemicellulose（6.5%）and cellulose and（5.1%）at the biomass level under salt stress,coupled with the expression changes of structural carbohydrates related genes.Thus,these results indicated that exogenous GABA could promote poplar growth by regulating carbon allocation in poplar stem under salt stress.4.GABA regulates carbon allocation to xylem in poplar under salt stress based on ¹³C labeling experimentNine-week-old 84K poplar seedlings were treated with 0 and 0.4%GABA under medium（1.5%）doses of Na Cl treatments for 21 day under soil conditions.The results showed that exogenous GABA indeed significantly enhanced poplar growth and altered the contents of cellulose and hemicellulose as the aforementioned results by salt treated 45 day.Stem anatomical observation demonstrated that exogenous GABA significantly increased the xylem width and vessel diameter（>15.5%）.Further result by ¹³C labeling experiment demonstrated that exogenous GABA significantly increased photosynthetic carbon fixation in leaves（>24.2%）,and prompted the labeled photosynthates into the bark,xylem and roots（>25.1%）under salt stress.Compared with 24 h after the labeling,exogenous GABA increased the ¹³C allocation in bark and roots under salt stress at 48 h after the labeling,whereas there was no change in xylem.Thus,our results suggested that exogenous GABA played a positive role in regulating the poplar growth by directly affecting the carbon fixation ability and regulating carbon allocation under salt stress.