The Molecular Physiology And Regulatory Network Of Mangrove Kandelia Candel In Response To Salt Stress

Kandelia candel(L.)Druce is a non-salt-secreting mangrove species,which is found to be growing in the intertidal areas of tropical and subtropical coastal areas.It is not only a major green vegetation to maintain the ecological balance of the coasts,but also it is considered as a model plant for studying molecular mechanisms of salt-tolerant xylophyte.The regulation network of K.candel in response to salt stress at mRNA and protein levels was investigated.In addition,the results of transcriptome and proteome of the chloroplast were verified through the physiological and biochemical determination and ultrastructure observation.This is the first systematic study of molecular physiological mechanisms and regulatory network on salt-tolerant mangrove K.candel.Several different physiological characteristics of salt tolerance from the herbaceous plant were found in this species.1.Chloroplast proteome of K.candel in response to salt stressChloroplasts are not only the site of photosynthesis but also are thought to be one of the most sensitive organelles to salinity.In this subcellular level study,we systemically provided an overview of the characteristic proteome dynamics of light-dependent reactions and Calvin cycle of K.candel under salt stress by using iTRAQ technique.Seventy six proteins showed more than 1.5-fold change in abundance,including the light-dependent reactions(52%),the Calvin cycle(29%),ATP synthesis(2%),amino acid transport(2%),protein synthesis(2%),antioxidative proteins(7%)and other proteins(5%).Among them,light-dependent reaction proteins were up-regulated under all NaCl treatments(200-600 mM).The expression of most carbon reaction-related proteins showed no significantly changes under 200-400 mM NaCl stress.However,they were found to be down-regulated under high salinity(600 mM NaCl)stress.We found that the stability of photosynthetic activity in K.candel under salt stress is closely related to the up-regulated proteins of light reactions.Meanwhile,decreasing of photosynthetic rate in K.candel was due to down-regulated enzymes at the carboxylation and reduction stages in Calvin cycle under the high salt stress,which was caused by non-stomatal factor.2.Changes of photosynthetic rate(Pn),photosynthetical fluorescence parameter and chloroplast ultrastructure in the leaves of K.candel in response to salt stressThe Pn and PS ? maximum photochemical efficiency(Fv/Fm)determinations and chloroplast ultrastructure observation in K.candel under different NaCl concentration treatment were conducted.The study showed that Fv/Fm did not change significantly,indicating that K.candel was able to maintain the normal PS ? photochemical activity andphotosynthetic electron transfer under 200-600 mM NaCl treatment.However,Pn decreased significantly and the change of Pn was opposite to that of Ci in K.candel leaves under high salt concentration,which conformed our previous chloroplast proteome result that declined photosynthesis under high salt stress was due to non-stomatal factor.The observation of chloroplast ultrastructure showed that K.candel thylakoid membrane structure was only slightly swelled,but grana thylakoid structure was not damaged under high salt stress.In addition,the study also found that plastoglobuli increased in the stroma,which are regarded to serve as lipid reservoirs for thylakoid membranes for preventing oxidative damage of the membrane,decreasing osmotic potential and keeping the cells absorbing water and inorganic nutrients efficiently.3.The changes of transcriptome and proteome in leaves of K.candel in response to salt stressRNA-seq and iTRAQ techniques were used to investigate the molecular mechanism at transcriptome and proteome levels in response to salt stress in leaves of K.candel.Furthermore,qRT-PCR,RT-PCR and Western blot approaches were applied to validate the expressed patterns of the differential genes and proteins,respectively.Six thousand two hundred and eighty-five differentially expressed genes were identified using RNA-seq technique.Two hundred and seventy-five out of the 3,676 identified proteins showed greater than 1.5-fold change(Unique Peptide ? 2).Genes involved in phenylpropanoid pathway,amino acid metabolism.DNA replication and repair and IP3-Ca2+ signal transduction pathway were found to be significantly up-regulated.Nevertheless,proteins encoded by these genes did not significantly change,or a few of them were up-regulated,suggesting that transcription regulation plays an important role in response to salt-stress in leaves of K.candel during short term(3 days).This result could explain the poor correlation between mRNA and protein in K.candel leaves under satt stress.The differentially expressed genes patterns confirmed by RT-PCR and qRT-PCR were consistent with the transcriptome data.4.GABA accumulation mediated by IP3-Ca2+ signal pathway in leaves of K.candel inresponse to salt stressIP3-Ca2+ signaling pathway in plants can mediate the biosynthesis of gamma-aminobutyric acid(GABA)and proline(Pro).Accumulation of GABA and Pro is one of crucial physiological mechanisms in response to stress in plant.Most plants accumulate Pro under different type of stress,but this study found that the three key genes involved in IP3-Ca2+mediated GABA accumulation pathway was significantly up-regulated and GABA content increased in K.candel,indicating the GABA accumulation was mediated by IP3-Ca2+signaling pathway.Meanwhile,the content of Pro and the expression of P5CS,a key gene related to Pro synthesis,did not change significantly,suggesting that GABA is the osmotic adjustment and it is an antioxidant substance in K.candel under salt stress rather than Pro.The results implied that GABA accumulation may be one of the physiological characteristics of salt tolerance in woody mangrove K.candel.5.The changes of physiological and biochemical index in K.candel leaves in response to salt stressThe contents of reactive oxygen species(ROS),ions,GSH,soluble sugar,starch,amino acids and the activities of antioxidant enzymes,as well as the enzyme activities and product contents related to phenylpropanoid pathway in leaves of K.candel under salt stress were analyzed.The toxicity of ions was reduced by balancing regulation of ions including Na+ and K+ in K.candel.ROS,mainly H2O2,were induced by salt stress.However,salt tolerance will be improved by increasing antioxidant enzymes,ROS scavengers,in K.candel leaves.Soluble sugars were actively involved in the cell osmotic adjustment,in which mannitol might be a major osmotic regulator in K.candel leaves under high salt stress.Glutamic acid(Glu),asparatic acid(Asp),serine(Ser)and threonine(Thr)contents were increased in K.candel under salt stress,especially Glu accounting for 50%of total free amino acids.In addition,Thr and histidine(His)were significantly increased along with the increase of salt concentration,which showed 5-fold and 34-fold change,respectively.The products of secondary metabolites were increased through enhancing phenylpropanoid metabolism in K.candel under salt stress.The accumulation of anthocyanins,flavonoids and lignins could play an important role in improving salt resistance in K.candel.These results verified the transcriptome and proteome data at physiological and biochemical level.This study has an important academic value for providing a new theoretical knowledge about the molecular mechanisms of salt-tolerant xylophyte;in addition,it is of great significance in the ecosystem restoration,exploitation and utilization of the coastal shoreline wetland,as well as in breeding of salt-tolerant trees and crops.