Molecular Mechanism Of Melatonin-and Heme Oxygenase 1-induced Salinity Tolerance In Arabidopsis

Melatonin(N-acetyl-5-methoxytryptamine,Mel)isolated from the bovine pineal gland in organisms,is an indoleamine.Ample evidence showed that it is involved in the regulation of physiological processes in animals,including anti-inflammation,innate immunity,and Alzheimer's disease.Heme oxygenase(HY1/HO1),the rate-limiting enzyme that degrades heme to carbon monoxide(CO),iron(Fe2+),and biliverdin(BV),plays a protective role against inflammatory,oxidative,apoptotic,and proliferative actions as well.Recent studies in plants have confirmed the vital functions of melatonin and heme oxygenase 1(HY1/HO1)in growth and development,the enhancement of the resistance and tolerance against biotic and abiotic stresses.However,the signal transduction pathways in salinity-stress response are not well understood,and thus required detailed research.To solve the above questions,we used pharmacological and genetic approaches,and acquired results were shown as follows:In this research,it was found that the Arabidopsis melatonin synthesis mutant(atsnat)showed salinity hypersensitivity,which can be rescued by melatonin or hydrogen peroxide(H2O2).This was evidenced by alleviation of the inhibition of primary root elongation,and the degradation of chlorophyll content.Meanwhile,melatonin-induced salinity tolerance in wild-type was suppressed by the addition of diphenyleneiodonium(DPI),an NADPH oxidase inhibitor.Interestingly,the rapid stimulated AtrbohF transcripts and production of reactive oxygen species(ROS)elicited by exogenous melatonin in stressed wild-type plants,were largely abolished in mutant atrbohF.Afterwards,salinity sensitivity of atrbohF was not altered by melatonin.Further results showed melatonin up-regulated the SOS1,SOS2,and SOS3 transcripts,reduced the sodium(Na+)content.Above changes were not found in melatonin-treated atrbohF mutant,compared to NaCl stress alone.The addition of melatonin could intensify the increased antioxidant defence in stressed wild-type,but not in atrbohF mutant,both of which were confirmed by the changes of the activities and transcripts of genes encoding representative antioxidant enzymes,and the histochemical staining for ROS production and lipid peroxidation,during the later period of stress.Together,above results indicated that the AtrbohF-dependent ROS signaling was required for melatonin-mediated salinity tolerance via the reestablishment of ion and redox homeostasis.Further study found that the application of carbon oxide(CO),the catalytic product of HO1,alleviated the salinity sensitivity in melatonin synthesis mutants atsnat and atcomt,and the tolerance of transgentic 35S:SNAT seedlings to salinity stress was inhibited by zinc protoporphyrin IX(ZnPP),the potent HO1 inhibitor.Besides,NaCl-induced expression of HY1 was strengthened by melatonin.Afterwards,the effect of melatonin on the salinity sensitivity of mutant hy1-100 was analyzed.Our results found that unlike the response in wild-type,salinity hypersensitivity in hy1-100 was failed to be improved by melatonin,which was rescued in CORM-2-treated hy1-100.Subsequent analysis of miR398a,miR398b,miR408,and miR393 transcripts mediated by melatonin in wild-type was found,but not in hy1-100 mutant.Moreover,the expression levels of representative antioxidant enzyme transcripts(CSD1,CSD2,and GST-U25)and the ion-channel gene(SOS1),were regulated by the above miRNAs.Above changes were consistent with the analyses for ROS and ion levels.Collectively,these findings suggested that HY1 operates downstream of melatonin promoting salt tolerance via reestablishment of redox and ion homeostasis mediated by salt-related miRNAs.In response to salt stress,plants have evolved various mechanisms to modulate the ion and ROS homeostasis.Autophagy is also involved in salinity stress,and which contributes to maintain the intracellular homeostasis.Compared to the wild-type,NaCl treatment induced more autophagosome in 35S:HY1-4,and which was not obviously altered in hy1-100.In addition,the formation of autophagy was induced by exogenous CORM-2,while inhibited by ZnPP.Upon NaCl stress,the changes of ATG2,ATG5,ATG8e,and ATG18 transcripts,exhibited a similar trend in the changes of autophagosome of various genotypes.Additionally,mutants of atg2,atg5,and atg18 showed more sensitivity than wild-type plants upon salinity stress.Representative phenotypes,including the inhibition of primary root elongation and fresh weight,and loss of chlorophyll of mutants,were aggravated.Above results preliminarily revealed that HY1-induced salinity tolerance was probably relied on the autophagy,at least partly.In summary,above results preliminarily revealed the possible molecular mechanisms of salinity tolerance induced by melatonin and HY1 in Arabidopsis.Relevant results,thus,not only contributed to understand the functions of melatonin and HY1,but also provided a theoretical basis for enhancing plant salinity tolerance in agriculture.