The Regulatory Role Of H₂S In Response To Aluminum Stress In Soybean

Aluminum?Al?stress is a main constraint on plant growth in acid soils.Improving plant resistance to Al toxicity is an important strategy to increase plant production.Plants develop various mechanisms to cope with Al toxicity,such as organic acids secretion?citrate,malate,oxalate?from roots and enhancement of antioxidant defenses.However,it is still poorly understood how this process is regulated under Al stress.Hydrogen sulfide?H₂S?,newly identified as the third signaling gasotransmitter following NO and CO,has been reported to be involved in responses to various environmental stresses such as drought,salt and heavy metals.However,the role of H2S in alleviating Al toxicity and the underlying mechanism are poorly known.In this study,the effects of H₂S on citrate secretion and antioxidant capacity under Al stress were investigated in soybean roots.Furthermore,the relationship between H₂S and NO in Al stress signaling was also explored.The main results are as follows:1.H₂S production in root tips,labeled by fluorescent probe WSP-1,was significantly induced by Al treatment and showed a time-dependent manner,suggesting that H2S participates in response to Al stress.Further investigation showed that Al treatment increased the activities of L-CD,D-CD and CAS,while the activity of OAS-TL,mainly responsible for H₂S degradation,decreased significantly under Al stress.These results indicate that both biosynthesis and degradation of H₂S pathways contribute to H₂S accumulation in Al-treated root tips.2.Application of H₂S donor NaHS alleviated Al-induced inhibition of root elongation and reduced MDA and reactive oxygen species?ROS?contents as well as cell death in root tips,while treatment with H₂S inhibitor HA had the opposite effect,suggesting that H₂S enhanced soybean resistance to Al toxicity.Furthermore,H2S could stimulate the activities of antioxidant enzymes?POD,CAT,APX?to remove excess ROS accumulation in root tips under Al stress,thus avoiding oxidative damage.3.NaHS treatment increased Al-induced citrate secretion and reduced Al accumulation in root tips,while HA had the opposite effect under Al stress,suggesting that Al-induced exudation of citrate contributes to the reduction of Al accumulation in root tips.In addition,H₂S increased citrate content in root tips under Al stress.Further investigation showed that H₂S positively regulated critrate synthetase?CS?activity and expression but negatively regulated aconitase activity and expression under Al stress.These results indicate that H₂S mediates the activation of citrate synthesis through regulating citrate metabolism under Al stress,thus increasing the exudation of citrate from roots.4.H₂S upregulated the gene expression of plasma membrane H⁺-ATPase and citrate transporter?GmMATE13 and GmMATE47?.Overexpression of GmMATE13 or GmMATE47 in Arabidopsis confers citrate efflux and Al tolerance.These results indicate that H₂S mediates the activation of plasma membrane H⁺-ATPase-coupled MATE transporter co-transport systems,thus conferring plant resistance to Al toxicity.5.Application of NO donor caused a significant increase in H₂S generation in root tips with or without Al,whereas PTIO treatment significantly reduced Al-induced increase in H₂S accumulation.Conversely,NO production in root tips was insensitive to NaHS or HA,both in the presence and absence of Al.Furthermore,NO elevated the activities of L-CD,D-CD and CAS,but inhibited the activity of OAS-TL.These findings suggest that NO promotes H₂S production in root tips and acts upstream of H₂S in Al stress signaling pathway.In summary,H₂S reduced ROS accumulation in root tips to avoid oxidative damage through enhancement of antioxidant enzymes?POD,CAT,APX?activities.Furthermore,H₂S-dependent upregulation of citrate synthesis and activation of PM H⁺-ATPase-coupled MATE transporter co-transport systems participates in Al-activated citrate exudation,thus reducing Al accumulation in root tips and conferring plant resistance to Al toxicity,and NO may act upstream of H₂S in this process.