Physiological And Molecular Mechanisms Of Soybean Nodulation Affected By Magnesium

Nodule is a symbiotic organ formed by legume plants and rhizobia,which could fix atmospheric dinitrogen（N₂）into ammonia,and plays important roles in agro-ecosystems.Given that magnesium（Mg）is an essential nutrient for both plant and rhizobium growth,Mg is of great importance to both nodule growth and symbiotic nitrogen fixation（SNF）in legumes.However,the physiological and molecular mechanisms underlying how Mg regulates nodulation in legumes remain largely unknown.In this study,phenotypic and transcriptomic analysis,as well as molecular biology approaches were carried out,in order to reveal the underlying mechanisms of Mg on soybean nodulation and nitrogen fixation in soybean.Furthermore,two Mg transporter genes Gm MGT4 and Gm MGT5 related to nodulation and SNF were functionally characterized in soybean.The main results were shown as follows:（1）Both pot and hydroponic experiments revealed that external Mg supply significantly improved nodule growth and SNF.Compared with low Mg treatment（5μM）,the fresh weight and number of big nodules but not small nodules,and nitrogenase activity and ureide concentrations in xylem sap were increased by 8.9 and 5.7,and 31.2 and 3.7 folds in normal Mg treatment,respectively.Number of big nodules treated with 10,50 and 100μM Mg concentration showed no significant difference compared with normal Mg treatment.Toluidine blue staining showed that the development of nodule structure was unaffected by Mg supply.The results from elemental analysis revealed that Mg concentration in nodules under normal Mg was increased by 22.8%compared with that under low Mg.All above indicated that Mg supply mainly affected nodule enlargement and SNF,but not nodule primordia initiation and early nodule development.（2）The concentrations of carbohydrates in roots and nodules were determined.The results revealed that Mg deficiency remarkably decreased the starch and sucrose accumulations in roots,and sucrose concentration in both the cortex and infection zone by31.4%and 54.8%,55.4%and 40.6%compared with that from normal Mg treatment,respectively.Lugol’s staining showed that Mg deficiency significantly reduced starch grain accumulations in the parenchymatous tissue and infection zone,further proving that carbohydrate transport from shoots to nodules in soybean was blocked under low Mg.Those results together revealed that Mg-promoted nodule growth and SNF was indirectly derived from regulation of Mg-facilitated carbohydrate allocation to nodules.（3）Transcriptomic analysis of roots treated with low N and Mg deficiency revealed that the expression of genes relative to nod factor perception,signal transduction,and infection thread formation and nodule primordium development were not affected by Mg deficiency.Transcriptomic analysis of nodules treated with Mg deficiency revealed that Mg deficiency mainly affected the glycolysis/gluconeogenesis,carbon metabolism and biosynthesis of amino acids pathways,verifying the important roles of carbon metabolism in nodule responses to Mg deficiency.（4）Expression pattern analysis showed that Gm MGT4 had the highest expression in roots and nodules among the 17 Gm MGTs,followed by its paralog Gm MGT5（similarity88.4%）.The transcripts of Gm MGT4/5 were accumulated in nodules throughout the growth stage and reached to the highest in the big nodules,suggesting that Gm MGT4/5 may be involved in Mg uptake and nodule development.Gm MGT4 was located to the plasma membrane of the cortex of both roots and nodules,as well as the nodule infection zones,indicating that it might play the roles as the Mg transporters in Mg uptake of roots and nodules.（5）Functional analysis of Gm MGT4/5 involved in Mg uptake was performed using the transgenic hairy roots and double RNAi transgenic lines.Results showed that single RNAi of neither Gm MGT4 nor Gm MGT5 affected Mg uptake in roots,while double RNAi of Gm MGT4/5 significantly decreased the Mg concentration in roots.Double RNAi of Gm MGT4/5 remarkably reduced the Mg concentration in nodules.The Mg concentration in the double RNAi nodules from normal and low Mg treatment decreased by 57.0%and 67.7%compared with that in WT.The non-invasive micro-test revealed that the Mg²⁺influx in root and nodule epidermis were significantly decreased in double RNAi lines.Those results indicate that Gm MGT4/5 are responsible for Mg entry into roots and nodules,and have functional redundancy.（6）Functional analysis of Gm MGT4/5 involved in nodule development and SNF revealed that double RNAi of Gm MGT4/5 significantly reduced the dry weight and number of big nodules,decreased nitrogenase activity and ureide content in xylem sap.Lugol’s staining showed that starch grain accumulation in the parenchymatous tissue and infection zone of double RNAi nodule was significantly reduced under normal Mg supply,indicating the disrupt carbon allocation.These results together demonstrated Gm MGT4/5 were involved in nodule growth and SNF through mediating Mg transport and subsequently affecting carbohydrate allocation.（7）Functional analysis was performed using transgenic nodules from hairy roots,which were double RNAi Gm MGT4/5 as driven by p N23（nodule-specific promoter）.The results showed that double RNAi of Gm MGT4/5 directly reduced Mg concentration in the cell sap of nodules,as well as the individual nodule dry weight and nitrogenase activity,further verifying that Gm MGT4/5 play critical roles in Mg entry into the infection zone of nodules.Taken together,our study revealed that Mg-promoted nodule growth and SNF were indirectly derived from Mg-facilitated alteration of carbohydrate allocation into nodules.Functional analysis of two Mg transporter genes Gm MGT4 and Gm MGT5 indicated that Gm MGT4/5-mediated Mg transport affected carbohydrate allocation within nodules,which is critical for nodule growth and SNF.Our study partially explored the physiological and molecular mechanisms involved in soybean nodulation affected by Mg,which may provide theoretical basis and candidate genes for genetic breeding of soybean varieties with higher SNF capacity.