The Role Of Populus Euphratica PeREMs And PeJRL In The Mediation Of Salt Tolerance In Higher Plants

Soil salinization is a serious agricultural and ecological problem in China.Therefore,improving salt tolerance of tree species holds a great promise for tree plantations and afforestation in sodic and salt-affected land areas.P.euphratica has widely been considered as a model species to elucidate stress molecular and physiological mechanisms in terrestrial woody plants.P.euphratica maintains a greater capacity to regulate ionic and ROS homeostasis under saline environment.due to sodium(Na+)extrusion and reduction of potassium(K+)loss which mainly relies on the higher activity of PM H+-ATPases.Previous studies have shown that the PM H+-ATPases act as an ionic sensor and contribute to H2O2 signaling in P.euphratica cells.In addition.recent studies revealed that PeRIN4 could increase the activity of PM H+-ATPases to maintain K+/Na+ homeostasis,and thus enhanced salt tolerance in transgenic Arabidopsis.In Arabidopsis thaliana,the mass spectrometry result indicated that the RIN4 protein complex contains remorin and jacalin-related lectin.It has been reported that remorin and jacalin lectins play an important role in improving plant resistance to biotic stress;however,their role in abiotic stress has rarely been reported,especially in woody plants.The object of this study was to elucidate the molecular mechanism underlying salt stress signaling that mediated by P.euphratica remorin and jacalin-related lectin protein.This study evaluated the interaction between remorin protein PeREM6.5,PeREM 1.3,jacalin-related lectin protein PeJRL and PeRIN4 protein by yeast two-hybrid technique.The strategy of expressing target genes were heterologously expressed in model plants to analyze the role of PeREM6.5,PeREM1.3 and PeJRL in salt tolerance.The main results were listed as below,briefly,1.PeREM6.5,a protein of the remorin family in Populus euphratica,was induced by salt stress.The 1130 bp full-length of open reading frame(ORF)of PeREM6.5 encoded a putative protein of 370 amino acids.Using Y2H the interaction between PeREM6.5 and PeRIN4 was identified in this study.In vitro enzyme experiments showed that PeREM6.5 and PeRIN4 were both positive regulators of PM H+-ATPases under salt environment.Moreover,the activity of PM H+-ATPases from P.euphratica was regulated by PeREM6.5 and enhanced by PeRIN4.In order to identify in the role of PeREM6.5-mediated signal transduction in salinity tolerance,the full length of PeREM6.5 was cloned from P.euphratica and transformed it into Arabidopsis plants.The overexpression of PeREM6.5 significantly improved the salt tolerance of transgenic Arabidopsis plants,in terms of survival rate,root growth,electrolyte leakage,and MDA content.Compared to wild type,transgenic plants retained higher activity of PM H+-ATPases.The activated H+-pumps enabled transgenic plants to maintain ionic and ROS homeostasis under NaCl stress.NMT flux data showed that transgenic plants retained K+and reduced the accumulation of Na+,resulting from(i)upward regulation of Na+/H+ antiport system(PM H+-ATPases,and Na+/H+antiport),and(ii)reduced K+loss through depolarization-activated outward rectifying K channels(KORCs)in the PM.Compared with WT,activities of the antioxidant enzymes were typically higher in PeREM6.5-transgenic Arabidopsis.This increased the capacity of transgenic plants to retain ROS homeostasis under salt treatment.Taken together,these results revealed that PeREM6.5 enhanced salt tolerance through increasing activity of PM H+-ATPases in transgenic plants.2.PeREM1.3,another protein of the Remorin family in Populus euphratica,was also induced by salt and osmotic stress.PeREM1.3 was similar to the remorin protein At3G61260(REM1.2)belonging to the RIN4 complex in Arabidopsis plants.However,the Y2H result indicated that there was no direct interaction between PeREM1.3 and PeRIN4.In order to explore in the role of PeREM1.3 in salt signal transduction,PeREM1.3 gene was cloned from Populus euphratica and transformed to tobacco plants.The 600 bp ORF of PeREM1.3 encoded a putative protein of 199 amino acids.PeREM1.3 protein localized to the plasma membrane and overexpression of PeREM1.3 increased the salt tolerance of tobacco.The activities of antioxidant enzymes such as SOD,POD and CAT significantly increased to maintain ROS homeostasis in transgenic lines under salt stress.Moreover,the transcriptional levels of stress-resistance related genes,such as SOS1,HAK,NHA1,VAG1 and PMA 4,were significantly increased,which benefited transgenic plants to maintain K+/Na+ homeostasis under salt stress.The above experimental data indicate that the remorin protein PeREM1,3,was similar to PeREM6.5,could enhance plant salt tolerance through enhanced capacity to maintain ROS and K+/Na+ homeostasis under salinity stress.3.NaCl induced expression of jacalin-related mannose-binding lectin(JRL)gene in leaves,roots and callus cultures of Populus euphratica.The 1338 bp ORP of PeJRL encoded a putative protein of 445 amino acids.Our Y2H result indicated that there was no direct interaction between PeJRL and PeRIN4.To explore the mechanism of the PeJRL in salinity tolerance,full length of PeJRL was cloned from the P.euphratica,and transformed it into Arabidopsis.PeJRL was localized to the cytoplasm.Overexpression of PeJRL in Arabidopsis significantly improved the salt tolerance of transgenic plants,in term of survival rate,root growth,and electrolyte leakage.Under NaCl stress,transgenic plants retained K+ and limited the accumulation of Na+.PeJRL-transgenic lines increased Na+ extrusion,which was associated with the upward regulation of SOS1,AHA1,and AHA2 genes encoding plasma membrane Na+/proton(H+)antiporter and H+-pumps.The activated H+-ATPases in PeJRL-overexpressed plants restricted the channel-mediated loss of K+ that activated by NaCl-induced depolarization.PeJRL-transgenic Arabidopsis maintained ROS homeostasis by activating the antioxidant enzymes and reducing the production of O2·-through down-regulation of PM NADPH oxidases.In addition,PeJRL-transgenic Arabidopsis repressed ABA biosynthesis,thus reduced the ABA-elicited ROS production and the oxidative damage during the period of salt stress.A schematic model was proposed to show the mediation of PeJRL on ionic and ROS homeostasis under NaCl stress.