| Drought and soil salinity have been the major environmental stresses severely restricting the agricultural productivity in northern China.Most of crops and forages have grown in favorable farming conditions for a long time,thus their genetic potential of stress resistance is very limited.The xerophytes and halophytes distributed in extremely harsh environments have evolved unique stress resistance mechanisms and therefore harbored abundant gene resources;the analysis of stress resistance mechanisms of these wild plant species would provide a theoretical foundation for the genetical improvement of stress resistance in crops and forages.Pugionium cornutum is one kind of xerophyte distributed throughout the desert areas of northwestern China,with prominent drought resistance and salt tolerance.Previous studies have found that under salt and drought stresses,P.cornutum could absorb a large amount of Cl~-and transport it into leaves as a beneficial osmoticum,which is an important physiological strategy of P.cornutum adapting to saline and drought environments.However,under saline conditions,the function of Cl~-in leaf turgor generation and leaf cell growth of P.cornutum is not clear,and the molecular mechanisms of Cl~-transport in P.cornutum still remain elusive.Therefore,in order to further investigate the Cl~-tolerance of P.cornutum,this project firstly compared the effects of different Cl~-salts(KCl and Na Cl)and NO3~-salts(KNO3 and Na NO3)on leaf water potential,osmotic potential and turgor pressure of P.cornutum seedlings,as well as the morphological difference of leaf cells under these treatments;then on this basis,cloned the key genes involved in vacuole Cl~-sequestration,Cl~-long-distance transport from roots to shoots,and root Cl~-uptake,analyzed the expression patterns,tissue and subcellular locations,as well as ion transport characteristics of these genes by the methods of real-time quantative PCR,in situ hybridization,transient expression in the leaf epidermal cells of tobacco(Nicotiana tabacum),yeast heterologous expression system,and verified their functions in the model plant Arabidopsis thaliana.The main findings are as follows:1.The growth of P.cornutum seedlings was significantly improved by 50 m M KCl treatment,but unaffected by 50 m M KNO3 treatment;the growth of P.cornutum under 50 m M Na Cl treatment was slightly inhibited,but was much better than that under 50 m M Na NO3 treatment;meanwhile,under KCl and Na Cl treatments,the amounts of Cl~-accumulation in shoots of P.cornutum were far higher than the tolerance threshold of most other plant species,indicating that P.cornutum should be a typical Cl~-tolerant plant.2.Tissue K~+and Na~+concentrations were the same between KCl treatment and KNO3 treatment,as well as between Na Cl treatment and Na NO3 treatment,however,under KCl and Na Cl treatments,leaf turgor pressure,area and thickness,as well as leaf epidermal and mesophyll cell volume were all significantly higher than those under KNO3 and Na NO3 treatments,respectively,indicating that the large accumulation of Cl~-in shoots could enhance leaf growth of P.cornutum by improving osmotic adjustment ability of leaf cells.3.The expression of P.cornutum tonoplast chloride channel gene Pc CLCg in shoots was significantly higher than in roots,this gene was highly expressed in both leaf epidermal and mesophyll cells,and its expression in shoots was significantly induced by Na Cl and KCl treatments.Pc CLCg was located on the cell tonoplast,and in yeast heterologous expression system,it mediated Cl~-sequestration into vacuole,and showed the same activity in Cl~-transport as At CLCg from A.thaliana.After the overexpression or complementation of Pc CLCg in A.thaliana wild-type or atclcg mutant,respectively,under high concentrations of salt treatment,the shoot biomass,water content and Cl~-concentration in transgenic lines were significantly higher than those in WT or atclcg mutant,suggesting that the expression of Pc CLCg could enhance salt tolerance of plant by enhancing the ability of vacuolar Cl~-sequestration in shoots.4.The expression of P.cornutum slow type anion channel gene Pc SLAH1 in roots was far higher than in shoots,and this gene was specifically expressed in root stelar cells;different with the expression pattern of At SLAH1 from A.thaliana,the expression of Pc SLAH1 was significantly induced by Cl~-salt treatments.After the stelar-specific expression of Pc SLAH1 in A.thaliana wild-type or atslah1 mutant,the shoot Cl~-concentration in transgenic lines under Na Cl and KCl treatments were significantly higher than that in WT or atslah1 mutant,while tissue NO3~-concentrations in transgenic lines under Na NO3 treatment were the same as those in WT or atslah1 mutant,indicating that Pc SLAH1 is involved in the long-distance transport of Cl~-from roots to shoots under salt stress.5.After the expression of Pc SLAH1 and At SLAH1 respectively in atslah1 mutant,when their expression levels were the same,the former transformed lines accumulated significantly higher Cl~-in shoots than the latter transformed lines under Na Cl treatment,suggesting that Pc SLAH1 should be more efficient than At SLAH1 in regulating Cl~-long-distance transport from roots to shoots.6.The P.cornutum nitrate transporter 1/peptide transporter gene Pc NPF6.4a was only expressed in shoots of P.cornutum,and its expression was highly induced by Na Cl,KCl and Na NO3 treatments.The expression of Pc NPF6.4b in roots was significantly higher than in shoots,and only in roots,its expression was remarkably induced by Cl~-salt treatments.Both Pc NPF6.4a and Pc NPF6.4b were located on the plasma membrane.In yeast heterologous expression system,Pc NPF6.4b mediated cell Cl~-uptake,presuming that Pc NPF6.4b is involved in root Cl~-uptake of P.counutum under saline conditions.In conclusion,P.counutum possesses a prominent adaptability to external high concentrations of Cl~-,and it can accumulate a large quantity of Cl~-in shoots and use Cl~-as a beneficial osmoticum to improve the water status of leaves and enhance the growth of leaf cells;meanwhile,Pc CLCg,Pc SLAH1 and Pc NPF6.4b are key proteins respectively mediating vacuole Cl~-sequestration in shoot cells,Cl~-long-distance transport from roots to shoots,and root Cl~-uptake.Above results provide a theoretical foundation to the further understanding of the molecular mechanisms in P.counutum adapting saline environments. |
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