Mechanisms Underlying A Phosphate-starvation Responsive Gene,GmMDH3 Controlling Malate Synthesis In Soybean Roots

Soybean（Glycine max）is an important grain and oil crop in China,and plays an important role in our daily lives.Phosphorus（P）is one of macronutrients,which is required for plant growth and development.However,phosphate（Pi）fertilizer is easily fixed by soil components into unavailable forms,and thus results in low Pi availability on soils,especially on acid soils.It has been documented that secreted malate is involved in the activation and utilization of insoluble P in plants.Therefore,it is very important to study the physiological and molecular mechanisms of malate involved in soybean adaptation of low phosphate starvation.This research provided important theoretical significance in screening soybean varieties with high P efficieny.In the study,endogenous malate concentration in soybean roots were analyzed among 28 soybean varieties under two P levels.Subsequently,the phosphorus efficient soybean variety YC03-3 was further selected to analyze the expression patterns of malate dehydrogenase（GmMDH）members,which might be involved in malate synthesis.Furthermore,a Pi-starvation responsive GmMDH member,GmMDH3,was selected for functional analysis.In this text,GmMDH3controlled malate synthesis in soybean roots.The main results were listed as follows:1.The effects of low Pi availability on soybean plant growth among 28 soybean varieties were comparatively analyzed.The results showed that the shoot fresh weight was reduced under the low phosphate treatment（-P）than that under high phosphate treatment（+P）;meanwhile,the root fresh weight,primary root length,total root number,and root surface area were significantly increased.The malate concentration in 17 of 28 soybean genotypes was significantly higher than that under normal P conditions.2.A total of 16 GmMDH members were identified in soybean genome.Among them,transcripts of 9 members in leaves and 4 in roots at 14 d was upregulatd under low phosphate starvation.Among them,GmMDH3 was significantly up-regulated in both leaves and roots.GmMDH3 was selected as the candidate gene for further function analysis.Subsequently,expression patterns of GmMDH3 were assayed in soybean at two P levels.The results showed that the expression levels of GmMDH3 were increased by 1.4,0.4 and2.0 folds in young leaves,old leaves and roots at 14 d of P deficiency,respectively.Beside,the transcription levels were increased by 0.4,0.4 and 4.5 folds at 21 d of P deficiency in young leaves,old leaves and roots,respectively.3.The staining results of Pro GmMDH3:GUS transgenic hairy roots showed that the expression levels of GmMDH3 was enhanced under low P conditions.The GUS staining was mainly detected in the root tip,lateral root primordia and pericycle.Furhtemrore,the results of subcellular localization showed that GmMDH3 was mainly localized on cell membrane and cytoplasms.4.Enzymatic properties analysis of recombinant protein showed the K_mand V_maxof GmMDH3-GST were 0.23 mM,21.56 nmol^-1min^-1mg^-1,and the K_cat/K_mwas 2.812 mmol^-1min^-1when OAA and NADH was used as the substrate and cofactor,respectively.However,no catalytic reaction was observed when malate and NAD⁺was used as the substrate and cofactor,respectively.The optimal p H value of GmMDH3-GST was 8.0,respectively when1 mmol.L^-1oxaloacetate and 0.5 mmol.L^-1NADH was used as the substrate and cofactor.5.The endogenous malate concentration of transgenic yeast cells with overexpressing GmMDH3 was significantly higher than empty vector treatment（Control）under the calcium-P conditions,as reflected by 12.4%,11.7%and 12%increases,respectively.Furthermore,soluble Pi concentration in the growth media was increased in overexpressing GmMDH3 yeast cells,suggesting that GmMDH3 resulted in an increase of calcium-P utilization.6.The endogenous malate concentration of transgenic Arabidopsis with overexpressing GmMDH3 was significantly higher than that in wild-type（WT）.And total P content was also higher than that in wild-type under low P conditions.When supplemented calcium-P as the sole P source,fresh weight,total phosphorus content and endogenous malate of transgenic Arabidopsis were significantly increased by 52.1%,22.4%and 32.1%compared to WT,indicating that GmMDH3 was involved in the activation and utilization of exogenous calcium-P.In summary,The study indicated that GmMDH3 could improve the tolearance of soybean in low Pi availability by controlling malate synthesis in roots.This study laid a foundation for further understanding the mechanism of soybean in adaptation to low phosphorus stress,and provided a candidate gene for developing soybean varieties with high P efficiency.