Study On Melatonin And Vacuolar Na~+(K~+)/H~+ Antiporter In The Regulatory Mechanisms Of Salt Tolerance In Switchgrass

Switchgrass(Panicum virgatum L.)has been increasingly recognized as one of the most valuable bioenergy crops.Genetic improvements on improving salt tolerance is of great significance in achieving large-scale cultivation of switchgrass on marginal land and promote the biomass energy industry's growth in China.Melatonin and vacuolar Na+(K+)/H+ antiporter(NHX protein)play an important role in plant responses to salt stress.In this study,we explored the physiological functions and molecular mechanism of melatonin and PvNHXl gene on the regulation of salt tolerance in switchgrass.These results are summarized as following:(1)We obtained transgenic switchgrass with increased melatonin levels by overexpressing ovine oAANAT and oHIOMT genes,respectively.Transgenic switchgrass exhibited promoted growth performance and earlier flowering compared with wild-type(WT)plants,suggesting melatonin may functions as a promoter in the regulation of switchgrass growth and flowering;Transgenic switchgrass displayed better growth performance,increased biomass and physiological parameter,as well as the ability to maintain lower Na+ and higher K+ contents under high salinity stress,indicating the enhanced salt tolerance in transgenic switchgrass may be associated with Na+ and K+ homeostasis.In addition,the stomatal opening and chlorophyll content in the transgenic switchgrass were significantly higher than those in the WT,suggesting a role for melatonin in maintaining high photosynthetic efficiency.(2)To obtain greater insights into the molecular mechanism underlying the function of melatonin in plant growth and salt tolerance,we performed the high throughput proteome and transcriptome analysis of overexpressing oAANAT and oHIOMT switchgrass,respectively.We found that overexpressing oAANAT mainly affected the genes in the photosynthesis pathway,while overexpressing oHIOMT significantly altered the secondary metabolite synthesis process.The melatonin potentially took part in the regulation of flowering shown by the significant up-regulation of multiple flowering related genes(APL3,SL1,FT1)in both oAANAT and oHIOMT transgenic switchgrass,and the expression of FT1 gene is regulated by CO and other photoperiodic related genes,indicating that melatonin regulates the formation of flower organs and promotes flowering by the regulation of photoperiodic pathways.Besides,the expression levels of genes enriched to sodium ion transmembrane transporter activity and potassium channel activity were significantly altered,and the overrepresented GO terms were associated with vacuolar.Furthermore,the PvNHX1 gene was significantly up-regulated in transgenic oAANAT and oHIOMT switchgrass,implying that the effect of melatonin on enhancing plant salt tolerance is possible to be the result of induced NHX1 expression.(3)To study the regulatory mechanism of NHX1 on Na+ and K+,we isolated the PvNHX1 gene from switchgrass.Subcellular localization suggested the PvNHXl protein was localized on the vacuole membrane.The PvNHX1 gene was detected throughout the entire growth period of switchgrass,and was highly induced by salt stress.Transgenic switchgrass exhibited obvious advantages in plant growth,development and salt tolerance comparing with WT.The decreased Na+ and increased K+ contents were also observed in transgenic PvNHX1 switchgrass.Above result suggested that the PvNHXl protein mainly functioned as a K+/H+ antiporter in switchgrass.(4)We performed the trans criptome analysis of trans genic PvNHX1 switchgrass and found that the significant up-regulated genes associated with K+ transport,such as HKT,KC02,HAK27 and HAK5 etc,exhibited that the PvNHX1 increasing the affinity of K+ and conveying salt tolerance by enhancing K+sequestration into vacuoles;PvNHX1 protein had an important role in plant growth through its regulation of photosynthetic activity and cell expansion;PvNHXl protein was potentially involved in the pollen development by significant up-regulation of multiple pollen development related genes.In addition,we also found that transgenic switchgrass exhibited increased expression level of disease resistance related genes,involving in various pathways,such as Ca2+ signaling,protein kinase,and phosphatidylinositol signaling to improve the disease resistance of plants.The function roles and molecular mechanism of oAANAT,oHIOMT and PvNHX1 in switchgrass were firstly revealed in our study,and the results contributed to the further understanding of the molecular mechanism and regulatory networks of NHX protein underlying the salinity tolerance,and also provide the theoretical base and valuable genetic resources for molecular breeding of the enhanced salt tolerant switchgrass plants.