Molecular Mechanisms Of Hydrogen And Melatonin In Alleviating Inhibition Of Seed Germination And Root Elongation Of Rice And Arabidopsis Under Aluminum Stress

Aluminum stress is a major factor limiting growth of crops in acid soils,root elongation and growth inhibition are major symptoms of aluminum toxicity.Germination is an essential step during plant morphogenesis.This process is susceptible to many environmental factors during which plant hormones GA/ABA play a key role.In recent years,Similar to nitric oxide(NO),hydrogen(H2)has emerged as a signaling molecule and its biological effects have been widely studied,research in animals uncovered its antioxidant,anti-inflammatory and anti-apoptotic properties.Melatonin is a small molecule with pleiotropic effects.Within animal kingdom melatonin was found to regulate physiological rhythm,affect mood,body temperature,sleep and other physiological activities.Hydrogen and melatonin research in plant has just begun.Whether hydrogen can regulate plant hormones such as GA/ABA and the detailed mechanisms need further reserch.Similarly,the effect of melatonin in Arabidopsis aluminum tolerance and its relationship with NO remains to be further elucidated.Take rice and Arabidopsis as model,we looked into the role of hydrogen in rice seed germination,and molecular mechanism of melatonin in alleviation Arabidopsis root elongation inhibition under aluminum stress.The results showed that 0.39 mmol·L-1 H2 increased amylase activity,sugar content,ameliorated lipid peroxidation,increased the activity of antioxidant enzyme system in germinating rice seeds,thus significantly alleviated germination inhibition;further study revealed that H2 could increase expression of GA biosynthesis genes GA20ox1 and GA20ox2 and ABA catabolism genes ABA8ox1 and ABA8ox2,with increased ratio of GA/ABA.This effect of H2 could be reversed by GA biosynthesis inhibitor paclobutrazol,implying GA as the potential downstream phytohormone of H2 effect.The expression of aluminum tolerant gene OsSTAR1/2 and OsFRDL4 can also be up-regulated by H2,increasing citrate efflux in roots of germinating seeds.Moreover,H2 could regulate abundance of stress related miR398a,miR159a,miR528 and miR160a together with their correspondent target gene OsSOD2,OsGAMYB,OsAO and OsARF10,thus improve the tolerance of germinating rice seeds under aluminum stress.Further study revealed that exogenous NO exacerbated root elongation inhibition of Arabidopsis under aluminum stress,this can be alleviated by the NO scavenger cPTIO.On the contrary,melatonin pretreatment reduced NO content in roots of Arabidopsis under Al stress,thus alleviated root inhibition,this effect could be reversed by NO donor sodium nitroprusside(SNP)plus melatonin pretreatment,while cPTIO plus melatonin reinforced this effect.Genetic evidence showed that nitrate reductase(NR)double mutant nial/2 and nitric oxide associated-1(noal)mutant displayed less sensitivity to aluminum,which is consistent with reduced NO content in their roots,melatonin pretreatment further decreased their sensitivity to Al.Subsequent studies revealed that reduced NO content by melatonin or cPTIO pretreatment and concomitant alleviation of root elongation brought no changes to early citrate efflux in roots of three lines,but significantly reduced cell wall component hemicellulosel(HC1)content in aluminum stressed Arabidopsis roots,consequently decreased aluminum accumulation in the root cell wall,while two mutant lines exhibited less HC1 and Al content compared with wild type plants.Moreover,melatonin or cPTIO pretreatment significantly decreased pectin methylesterase(PME)activity,which was significantly lower in two mutant lines.This further reduced aluminum content in pectin,with reduced aluminum accumulation in cell wall,consequently alleviated root elongation inhibition,while SNP exerted the opposite effect.Taken together,our research showed that H2 alleviated germination inhibition of rice seed under aluminum is due to following reasons:increased GA/ABA ratio,regulated miRNA and their target gene expression,enhanced activity of antioxidant system and citrate efflux.Meanwhile,melatonin could alleviate Arabidopsis root elongation inhibition upon aluminum stress via reducing NO content,HC1 content and PME activity.Our results shed light on the application of H2 and melatonin in agricultural production on acid soil contaminated by aluminum.