Malate Synthesis And Nodule Growth Is Regulated By Malate Dehydrogenase In Soybean(Glycine Max)

Phosphorus is one of the essential elements,which is required for plant growth and development,and plays an important role in plants.Because of its physical and chemical properties,phosphate?Pi?fertilizer is easily fixed by soil particles,and thus results in low Pi availability on soils.It has been documented that secreted malate is involved in insoluble inorganic-P activation in plants,which might be regulated by malate dehydrogenase?MDH?.Therefore,it is important to study the MDH functions in order to further understand adaptive strategies in plants to phosphorus?P?deficiency.In the study,using soybean as the material,the method for malate extraction and analysis was firstly optimized.Subsequently,effects of P deficiency on endogenous malate concentrations in different soybean tissues and its secretion rate were investigated.At the same time,expression patterns of soybean malate dehydrogenase?GmMDH?members in responses to P deficiency were examined.Two Pi-starvation responsive GmMDH members?GmMDH3and GmMDH12?were selected,comparatively analyzed for their biochemical characteristics.Finally,GmMDH12 functions in malate synthesis and nodule growth were studied through analyzing its overexpression transgenic soybean plants.The main results were as follows:1.Based on high performance liquid chromatography?HPLC?analysis,endogenous malate concentration in both soybean leaves and roots were analyzed using two extraction buffer(i.e.,deionized water and 0.25 mol·L^-1 hydrochloric acid),combined with water bath at two temperatures.The results showed that deionized water extraction is more stable to determine soybean malate concentration through HPLC analysis.2.Dynamic effects of P deficiency on malate concentration and exudation rate were studied in soybean.The results showed that malate concentration in soybean young leaves was not affected by 7,14 and 21 d of P deficiency.However,malate concentration in old leaves was significantly reduced by P deficiency,especially at 21 d,as reflected by 62%decreases.On the contrary,root malate concentration was significantly increased at 14 and21 d of P deficiency,which was increased by 1.6 and 1.7 folds,respectively.Furthermore,root malate secretion rate was significantly increased by P deficiency,especial at 14 d,as reflected a 2.9-fold increase.3.Bioinformatics analysis showed that a total of 16 GmMDH members presents in soybean genome.Among them,increased transcripts were observed for 9 members in young leaves,7 in old leaves,and 3 in roots at 14 d of P deficiency.Subsequently,dynamic expression patterns of GmMDH3 and GmMDH12 were assayed in soybean nodules at two P levels.The results showed that the expression patterns of GmMDH3 and GmMDH12 in the nodules were significantly up-regulated at 20 and 30 d of P deficiency,especially at 30d,as reflected by 2-fold increases for GmMDH3 and 7-fold increases for GmMDH12.4.Through heterologous expression in E.coli,fused GmMDH3 and GmMDH12proteins were separately purified,and their several enzymatic properties were analyzed.The results showed that both GmMDH3 and GmMDH12 exhibited the ability to catalyze the synthesis of malate using oxaloacetate?OAA?as a substrate in vitro.The optimal temperature for the GmMDH3 enzymatic reaction was 60?and the optimal substrate concentration was 1 mmol·L^-1 oxaloacetate.However,the optimal reaction temperature for GmMDH12 was 40?,and the optimal substrate concentration was 1.5 mmol·L^-1oxaloacetate.Furthermore,different metal ions had different effects on their enzymatic activities.Among them,Fe²⁺significantly enhanced GmMDH3 activity,but inhibited GmMDH12 activity.Mg²⁺and Zn²⁺had no effect on GmMDH3 activity,but significantly inhibited GmMDH12 activity.5.Functions of GmMDH12 were further investigated in soybean transgenic plants with its overexpression.Results showed that GmMDH12 overexpression not only significantly increased nodule malate concentration and number,but also significantly increased hemoglobin concentration,both total nitrogen and phosphorus content in nodules,strongly suggesting that GmMDH12 might regulate nodule malate synthesis,and thus affect nodule growth in soybean.