Functional Characterization Of Na~+/N~+ Antiporter In Wheat (Triticum Aestivum)

Salinity is one of the important abiotic stresses and becoming a worldwide problem to constraint of crop productivity. As a non-halophyte, wheat production is affected by salt stress and its growth is severely inhibited under salinity condition. Na+ can enter the cytosol by non-selective cation channels under salt stress. To tolerate high levels of sodium ions, plants cells must transport Na+ from cytoplasm to the external medium by the operation of plasma membrane Na+/H+ antiporter or to compartmentalize it into vacuoles by the tonoplast-bound Na+/H+ antiporter, thus maintaining the cytosolic ion homeostasis. Here we cloned the plasma membrane Na+/H+ antiporter (TaSOS1) and vacuolar Na+/H+ antiporter (TaNHX2) from wheat (Triticum aestivum). The functional of these two genes to salt tolerance and the Na+/H+ exchange activity were characterized using Saccharomyces cerevisiae nhx mutant strain. The main results are as following:1. The predicted protein of TaSOS1 shares higher identity in amino acid sequences with similar proterins from Arabidopsis thaliana (AtSOS1) and Oryza sativa (OsSOS1) by bioinformatics methods. The data indicated that TaSOS1 is a putative plasma membrane Na+/H+ antiporter that effluxes of Na+ from cytosol as AtSOS1 and OsSOS1. The expression level to different stress of TaSOS1 including NaCl salt stress, low tempture stress at 4℃, ABA ion stress and PEG6000 drought stress was analysized in the roots and leaves of wheat plants by real-time PCR. The steady-state mRNA level in roots was higher than in leaves in control plants which suggested that TaSOS1 transcript was abundant in roots. Upon slat treatment, the expression of TaSOSl was induced and up regulated abundant in roots of plants. While there are no obvious difference on the mRNA expression level in leaves of plants. It showed time-dependant and organ-dependent changes in wheat plants. No clearly up-regulation of TaSOS1 was found under ABA and PEG6000 treatment. It appears that TaSOS1 is involved in ABA-independent pathway as AtSOS1.2. The yeast expression plasmid pYPGE15-TaSOS1 was constructed and transformed it into yeast mutant strain AXT3K by the polyethylene glycol-lithium acetate method. When TaSOS1 was expressed in AXT3K from vector pYPGE15, the wheat gene could partly compensate the sensitivity of the yeast strain and grow on AP plates containing 80 mM NaCl. While the control that strain only expressed vector pYPGE15 could not grow on the same stress condition. The Na+ content of cells expressing TaSOS1 was significantly lower than that of control cells after exposure to 20 mM NaCl stress. The results suggested that TaSOS1 functions as Na+/H+ antiporter that efflux the Na+ out of the cells and decrease the Na+ content in the cytosol to keep the ion homeostasis.3. Yeast plasma membrane that expressing TaSOS1 or empty vector (control) was isolated from yeast cells by aqueous two-phase system. The purity of vesicle preparations was tested by measuring hydrolytic activities of ATPase in the presence of inhibitors. Vanadate reduced ATPase activity about 60% in the plasma membrane-enriched fraction. However, inhibitors of azide, nitrate and molybdate had no obvious effect on ATPase activity. These results indicated that the vesicle isolated from yeast was enriched in plasma membrane without significant contamination from other cellular membranes of mitochondrial, vacuolar and non-specific phosphatases.4. The fluorescent quenching of quinacrine was used to detect the pH gradient over the plasma membrane vesicle from untransformed and transgenic yeast cells overexpressing TaSOS1. The formation ofΔpH was established by the activity of the plasma membrane H+-ATPase and Na+/H+ exchange activity was measured as a Na+-induced dissipation ofΔpH with the pH value sensitive fluorescent probe. Vesicles from yeast cells expressing TaSOS1 exhibited a higher Na+/H+ exchange activity than control cells with empty vector. The difference in Na+/H+ exchange activity is in agreement with the greater tolerance to NaCl and the lower intracellular Na+ content of the transformant relative to control cells. It deduced that TaSOS1 characters as Na+/H+ exchange activity and may have an important role in the salt tolerance of wheat.5. The wheat vacuolar Na+/H+ antiporter TaNHX2 was cloned and the phylogenetic tree shows that TaNHX2 is most closely related to AtNHX1 and AtNHX2. It indicated that TaNHX2 may catalyze the exchange of Na+ or K+ across vacuolar membranes and contribute to salt tolerance of wheat. Western blot analysis shows that the TaNHX2 could be expressed well in transgenic yeast cells. The expressed protein of TaNHX2 could partly compensate the sensitivity of AXT3K to salt stress so that cells can grow well in the AP plates containing 30 and 60 mM NaCl, albeit they did not reach the salt tolerance of wild-type yeast cells. Interestingly, TaNHX2 could also increase the tolerance of transgenic yeast to potassium. This is the first report that identifies the vacuolar NHX proteins function as a K-tolerant target. These demonstrated that TaNHX2 antiporter might not only catalyze Na+ but also K+ transport. The Na+ and K+ ion contents of transgenic and untransformed yeast mutant strains were measured under NaCl stress. Sodium content did not change in transgenic cells as compared to control cells under both normal growth and mild salt stress. On the contrary, the TaNHX2 protein caused the significant increase in potassium content in transgenic cells exposed to 20 mM NaCl treatment. These suggest that the enhanced salt tolerance for transgenic cells is not involved in sodium transport, but potassium accumulation is more important for the tolerance of cells to salt stress.6. To test whether TaNHX2 functions as cation/H+ exchanger and has the ability to sequestrate cation into vacuolar, we isolated intact vacuoles from transgenic yeast mutant strain OC02 and control cells. OC02 are almost devoid of background of K+/H+ and Na+/H+ exchange at vacuolar membrane. Transport assays with intact vacuoles indicated that TaNHX2 could transport K+ and Na+ into vacuoles, but also had a greater capacity for K+/H+ exchange compared to minor Na+/H+ exchange, even the Na+/H+ exchange of TaNHX2 could not be induced with the concentrations of less than 37.5 mM Na+ in reaction medium. This is in agreement with ion contents in yeast cells exposed to mild salt stress, K+ content significantly increased but Na+ content did not change in transgenic yeast cells treated with 20 mM NaCl as compared to control cells. So the increased accumulation of K+ is likely to be consequence of the activity of TaNHX2. It is speculated that the TaNHX2 antiporter facilitates K+ transport into vacuoles in exchange for H+ into the cytosol. The K+ ions compartmentalized into vacuoles can act as osmoticum helping to maintain an osmotic potential that drives water into the cell. So the compartmentalization may prevent Na+ toxicity by facilitating cellular K+ uptake. The prevent novel findings can help to understand the relationship between NHX proteins and potassium nutrition in plants.