Regulatory Mechanism Of Nitric Oxide On The Secretion Of Citrate In Soybean Root Under Aluminum Stress

In acid soils?pH<5.0?,aluminum?Al?stress is the main limiting factor for plant growth and crop yield.The secretion of organic acids,such as citric acid,oxalic acid and malic acid,can effectively alleviate the Al toxicity of some plant species.Nitric oxide?NO?not only participates in the regulation of plant growth and development,but also plays an important role in responses to environmental stresses.Our previous study showed that NO was involved in the process of Al-induced citrate secretion.However,the mechanism of how NO regulates citrate secretion under Al stress remains unclear.In this study,we first investigated whether the metabolism and transport of citrate were associated with Al-induced citrate secretion in soybean roots.Furthermore,the role of NO in regulation of metabolism and transport of citrate was further explored under Al stress.The main results are as follows:1.Exposure of soybean seedlings to Al stress induced citrate secretion from roots.Generally,the citrate exudation from roots increased with increasing Al concentration?0,10,25,50,100?M?and treatment time?0,3,6,9,12,24 h?.The spatial distribution of citrate exudation was explored using excised root segments?0-5,5-10,10-15,15-20 mm?.Al-activated citrate exudation was mainly attributed to the apical 0–5 mm region.2.Al stress enhanced citrate synthase?CS?activity and inhibited aconitase?ACO?activity in root apices.qRT-PCR assay showed that Al treatment increased the expression level of CS and ACO genes.Under Al stress,application of CS inhibitor?Sur?significantly reduced citrate secretion from roots,and treatment with ACO inhibitor?FCA?significantly increased citrate secretion from roots.These results indicated that citrate metabolism was involved in Al-induced citrate secretion from roots.3.Al treatment enhanced the activity and gene expression level of plasma membrane?PM?H⁺-ATPase.Under Al stress,treatment with PM H⁺-ATPase activator?Fus?enhanced enzyme activity and citrate secretion,while PM H⁺-ATPase inhibitor?Va?inhibited enzyme activity and citrate secretion.These findings suggested that PM H⁺-ATPase was involved in the process of Al-induced citrate secretion.4.Two candidate genes of citrate transporter,MATE13?Glyma.02G181800.1?and MATE47?Glyma.09G102800.1?,were screened from the 117 MATE genes of soybean using bioinformatics method.The qRT-PCR assay showed that Al stress mainly enhanced the expression level of MATE13 and MATE47 in roots.Subcellular localization showed that the expression products of MATE13 and MATE47 were located at plasma membrane.Expressing MATE13 and MATE47 and microinjecting with C¹⁴-labeled citrate in Xenopus oocytes showed that MATE13 and MATE47 could mediate the citrate efflux.These results indicated that MATE13 and MATE47 are two Al-induced citrate transporter genes responsible for citrate exudation in soybean roots.5.Al stress enhanced the production of NO in root apices and the secretion of citrate from roots.Furthermore,exogenous NO treatment further increased Al-induced secretion of citrate,while NO scavenger inhibited the secretion of citrate induced by Al,indicating that NO mediated the secretion of citrate in soybean root under Al stress.Under Al stress,exogenous NO treatment could reduce the accumulation of Al in the root apices,while the NO scavenger treatment increased the accumulation of Al in the root apices.6.Under Al stress,exogenous NO increased the activities of CS and PM H⁺-ATPase and decreased ACO activity,while NO scavengers inhibited the activities of CS and PM H⁺-ATPase and increased ACO activity.qRT-PCR assay showed that NO enhanced the expression of PM H⁺-ATPase and CS genes,but had no significant effect on the expression of ACO gene.In addition,NO upregulated the expression of MATE13 and MATE47 under Al stress.These observations suggested that NO regulates the metabolism and transport of citrate under Al stress.To sum up,our findings suggested that NO-dependent upregulation of citrate synthesis?stimulation of CS activity and inhibition of ACO activity?and transport?activation of PM H⁺-ATPase and citrate transporter?participated in Al-activated citrate exudation from soybean roots,thus conferring plant resistance to Al toxicity.