Mechanisms Of Melatonin-mediated Dehydration Responses In Nicotiana Tabacum

1.Plant adaptation to water deficiency is the consequence of diverse phenotypic,physiological,and molecular variations.To gain insight into the molecular mechanisms underpinning tobacco(Nicotiana tabacum)drought stress tolerance,we integrated anatomical,physiological,and proteomic analyses of drought-tolerant(Yuyan6,[Y6])and drought-sensitive(Yunyan87[Y87])varieties.Our data showed that Y87 suffered greater negative impacts than Y6 under dehydration stress,as evidenced by the higher water retention capability,improved photosynthetic performance,delayed leaf-senescence,stable leaf ultrastructure,a stronger antioxidant defense,and less ROS accumulation detected in Y6 after polyethylene glycol(PEG)treatment.Using an iTRAQ-based proteomics approach,405 and 1,560 differentially accumulated proteins(DAPs)were identified from Y6 and Y87 plants,respectively,of which 114 were found to be shared by both cultivars.A subsequent functional characterization analysis revealed that these DAPs were enriched in biological processes,molecular functions,and cellular components and displayed differential expression patterns in Y6 and Y87,suggesting the occurrence of dehydration stress response divergence between the two varieties at the proteomic level.In addition,qPCR analysis indicated that the majority of genes encoding selected proteins showed consistency between mRNA levels and their corresponding protein expression levels.Collectively,the results of this study provide new insights into the genetic regulatory mechanisms associated with drought adaptation and may help in the identification of potential targets for further molecular breeding of drought-resistant plants.2.Melatonin(MEL)is a pleiotropic agent with crucial functions reported in a variety of stress responses and developmental processes.Although MEL involvement in plant defense against natural leaf senescence has been widely reported,the precise regulatory mechanisms by which it delays stress-induced senescence remain unclear.In this study,we found that foliar spraying of melatonin markedly ameliorated dehydration-induced leaf senescence in Nicotiana tabacum,accompanied by attenuated oxidative damage.Gene expression analysis revealed that the delayed senescence of stressed plants achieved by melatonin treatment might be partially ascribed to the upregulated expression of genes involved in ROS scavenging,photosynthesis,and carbon/nitrogen balances,and downregulated expression of senescence-associated genes.Metabolite profiling indicated that melatonin-treated plants accumulated higher concentrations of sugars,sugar alcohol,and organic acids,but fewer concentrations of amino acids in the leaves,than untreated plants after exposure to dehydration.Moreover,the carbohydrate levels and metabolites of the tricarboxylic acid cycle were reduced following dehydration treatment.Among them,12 metabolites were increased in the MEL_D group relative to those in D.These findings may provide more comprehensive insights into the role of melatonin in alleviating leaf senescence and enhancing dehydration resistance.3.To further elucidate the underlying mechanism by which melatonin regulates the dehydration response,we analyzed the transcriptional regulatory networks.Transcriptomic analysis identified more than 30,000 differentially expressed genes(DEGs)in MEL-,D-,and MEL_D-treated leaves versus CK.Compared to the transcripts of untreated leaves,the number of genes commonly downregulated in D was greater than that in MEL_D.The qPCR analysis of six randomly selected genes(NtBH0283,NtARR6,NtCAO,NtCIPK1,NtGDCSP,and NtLOGI)confirmed the accuracy of RNA-seq.In the presence of MEL,MEL regulates the transcription of TFs,such as NAC,bHLH,AP2,bZIP,MYB,WRKY,and ABF,which in turn modulates the expression of genes responsible for ROS scavenging and chlorophyll degradation.Functional characterization analysis revealed that the expression of genes involved in diverse biological functions,such as photosynthesis,oxidationreduction,stress responses,starch and sucrose metabolism,autophagy,and carbohydrate/nitrogen metabolism,are modified by MEL,resulting in delayed leaf senescence and elevated endurance to dehydration stress.Our comparative transcriptomic analyses will be helpful for illuminating the mechanisms of water stress tolerance in plants,and highlights the exciting potential of MEL for crop improvement.4.To explore the roles of exogenous addition of melatonin on the plant hormones and carotenoid accumulation in flue-cured tobacco leaves under dehydration stress,we analyzed the hormone and carotenoid profiles with respect to dehydration responses in tobacco leaves.In this study,we identified 15 phytohormones and 21 carotenoids in tobacco leaves and performed PC A to analyze their responses.Hormone responses suggested that melatonin played a prominent role in the regulation of phytohormone levels in tobacco seedlings under dehydration conditions.Melatonin regulates leaf senescence via modulation of phytohormonal biosynthesis and signaling.Our findings demonstrate that melatonin supplementation decreases ABA accumulation and maintains high IAA levels by simultaneously inhibiting and activating ABA and IAA biosynthetic genes,respectively,under dehydration conditions.Furthermore,carotenoid responses showed an extensively modulated expression,complemented by carotenoid biosynthesis regulation to achieve growth acceleration in melatonin-treated plants upon exposure to dehydration stress.Accordingly,expression profiling revealed that MEL selectively induced a different set of carotenoid biosynthesis genes from those mediated by dehydration.These results provide new information for melatonin-induced manipulation of hormone and carotenoid accumulation in plants under drought conditions.