The Roles Of ZxSKOR And ZxAKT1 In Drought Resistance And Salt Tolerance Of Succulent Xerophyte Zygophyllum Xanthoxylum

Abiotic stresses such as drought and soil salinity are serious threats to agricultural and animal-husbandry sustainable development worldwide.Zygopyllum xantylum,,a shrub species with excellent drought and salt resistance,is a typical salt-accumulating succulent plant.Our previous researches discovered that Z.xanthoxylum has ability to absorb a large amount of Na+ from low salinity soil and efficiently compartmentalize Na+ into leaf vacuoles as an important inorganic osmo-regulator to reduce osmotic potential of cells,thereby increasing water uptake under drought stress,which is one key mechanism of enhancing its drought tolerance.Na+ concentration in leaves was significantly increased under salt and drought conditions,while K+ maintained stable in Z.xanthoxylum.Therefore,maintaining K+ homeostasis in leaves is another important strategy of Z.xanthoxylum to survive well in adverse environment.However,the molecular mechanism concerning K+,Na+ uptake and transport is still unclear.The stelar K+ outward rectifying channel(SKOR)was proved to be a K+channel involved in loading K+ into xylem for distribution from root to shoot,and play a key role in long-distance K+ transport.Inward rectifying K&channel AKT1(Arabidopsis K+ transporter 1)not only played important roles of K+ uptake but also was concerned with Na+ accumulation.Therefore,in order to elucidate the mechanism of K+ homeostasis during salt and drought conditions in Z.xanthoxylum,a K+ outward rectifying channel gene ZxSKOR was isolated and characterized,and the gene expression patterns and K+,Na+ accumulation under different concentration of KCl,NaCl,osmotic stress(-0.5 MPa)or osmotic stress plus NaCl(-0.5 MPa+50 mM NaCl)were investigated,respectively.Using posttranscriptional gene silencing(PTGS),we evaluated the role played by ZxAKT1 in K+,Na+ uptake and accumulation among plants,and the expression level of genes involved in K+ and Na+ uptake and transport of ZxAKT1-silenced lines and WT were also studied.The results are as follows:(1)ZxSKOR consisted of 847 amino acid residues and shared high similarity(>66%)with the identified stelar K+ outward rectifying channel from other plant species.Structure analysis showed that the ZxSKOR has a six-segment(S1-S6)membrane-spanning topology,a putative cyclic nucleotide binding domain,and an ankyrin domain,S4 contains positively charged amino acids.The P(pore)domain located in S5 and S6 comprised the universal GYGD motif.There were additional residues in this channel just upstream from the P domain,which is the most distinctive difference from the other plant K+ channels.Phylogenetic analysis showed that ZxSKOR was grouped with outward rectifying K+ channels SKOR.These results supported that ZxSKOR encoded an outward rectifying K+ channel SKOR.(2)Semi-quantitative RT-PCR showed that the mRNA level of ZxSKOR was significantly higher in root and stem than leaf.The expression levels of ZxSKOR in root and stem increased significantly when plants were exposed to 5-10 mM KCl.A positive correlation was not only found between ZxSKOR expression in root and K+accumulation in shoot,but also between Zx-SKOR expression in stem and K+accumulation in leaf under 0-10 mM KCl,indicating that ZxSKOR in root and stem was finely coordinated to mediate long-distance K+ transport in Z.xanthoxylum,and is vital for accumulating K+ in leaf.(3)Under 5-150 mM NaCl treatment,the expression level of ZxSKOR in root and stem was increased continuously.Moreover,the induction of ZxSKOR mRNA in plants exposed to 150 mM NaCl was far stronger than that of 50 mM NaCl.These results demonstrate that Z.xanthoxylum has the ability to efficiently transport K+ to leaves and maintain K+ homeostasis by up-regulating ZxSKOR,thus avoid K+deficiency caused by the competition of the high external Na+ concentration.Therefore,it is proposed that the strong transportation of K+ to leaf mediated by ZxSKOR is one of the strategies for Z.xanthoxylum to adapt to salt stress.(4)The transcript level in root and stem was higher than in leaf under normal condition and-0.5 MPa osmotic stress,obviously induced the expression of ZxSKOR in root and stem.The expression level of ZxSKOR in root under-0.5 MPa osmotic stress plus 50 mM NaCl was significantly higher than that under-0.5 MPa osmotic stress during 12-48 h.Therefore,it is strong evident that up-regulating the transcript level of ZxSKOR is essential for K+ homeostasis in leaf and mitigating the deleterious impacts of drought stress on Z.xanthoxylum(5)A high efficient silencing of plant RNAi vectors was constructed,and a total of 14 transgenic lines carrying the RNAi construct were identified by PCR amplification.Under control and 50 mM NaCl for 7 days,the ZxAKT1-silenced lines displayed a decrease in fresh weight,dry weight,shoot height and root length.This result indicated that ZxAKTl was a candidate gene for regulating the growth and development of Z.xanthoxylum.(6)Under 2.5 mM K+ conditions,ZxAKT1-silenced plants accumulated less K+ in root,stem and leaf than do wild Z.xanthoxylum,these results above support this hypothesis that ZxAKT1 mediated K+ uptake.(7)Under control treatment,Na+ concentration in all tissues and net uptake rate were no significant difference of both wide type(WT)and interference lines,but when addition 50 mM NaCl,Na+ concentration and net uptake rate of 7 lines were obvious lower than WT,indicating that ZxAKT1 related to Na+ uptake in high Na+concentration.(8)We found that under normal condition,the whole plant K+/Na+ ratio of line 7 was significantly lower than WT,but the expression level of ZxNHX,ZxSKOR,ZxSOS1 and ZxHKT1;1 were not affected.Nevertheless,the difference in whole plant K+/Na+ ratio between line 7 and WT was not detected when exposed to 50 mM NaCl,interestingly,50 mM NaCl down-regulated the transcript level of ZxNHX in leaf and ZxSKOR,ZxSOS1 and ZxHKT1;1 in root significantly in line 7 compared to WT.From the data available on these,it is becoming likely that ZxAKT1 regulation of K+and Na+ transport and homeostasis by altering the expression of K+ and Na+ channels and transporters.(9)With the increase of the degree of silence,osmotic potential(?s)of ZxAKT1-silenced lines increased significantly under-0.5 MPa,the net photosynthetic rate(Pn),transpiration rate(Tr),stomatal conductance(Gs),K+/Na+ ratio decreased significantly bothunder normal conditions and-0.5 MPa treatments compared with WT.These results demonstrate that ZxAKT1 is a key gene which plays important roles in drought resistance by improving photosynthesis and decreasing osmotic potential.In summary,these results demonstrate that ZxSKOR in Z.xanthoxylum not only has the function in long-distance transport of K+ from root to shoot,but also plays an essential role in improving the drought and salt tolerance of Z.xanthoxylum by maintaining K+ homeostasis.Moreover,ZxAKTl contributes to regulating of the normal growth and K+ and Na+ uptake,transport and homeostasis of Z.xanthoxylum,and plays important role in Z.xanthoxylumto adapt arid environment.