The Roles Of ZxNHX In Controlling Na~+and K~+Homeostasis In The Succulent Xerophyte Zygophyllum Xanthoxylum

Drought and salinity are two major abiotic stresses constraining agricultural expansion onto uncultivated land and limiting crop productivity worldwide. Most crops are very sensitive to drought and salinity:both generate osmotic stress and a disequilibrium in intracellular ion homeostasis, resulting in cell membrane dysfunction and attenuation of metabolic activity, leading to growth inhibition and even plant death. Desert xerophytes grown in extremely arid regions, however, have developed various mechanisms to adapt to harsh environments during the process of long-term evolution. Absorbing a great quantity of Na+from culture medium which is efficiently transported to the leaves, and maintaining the stability of K+concentration in those leaves are two major adaptive strategies used by the succulent xerophytes native to the desert areas of northwest China, such as Zygophyllum xanthoxylum, to adapt arid and salt environment. These results suggest that Z. xanthoxylum has a strong ability to regulate Na+and K+homeostasis thus maintaining plant growth when subjected to drought and salinity. The tonoplast Na+/H+antiporters, NHXs, have been suggested to play an important role in sequestration of Na+into vacuoles and improvement plant drought resistance or salt resistance. However, so far the function of NHX proteins was identified and characterized mainly at a cell or tissue level; its role in regulating Na+and K+homeostasis at the whole-plant level remained unclear.In the current study we have evaluated, using post-transcriptional gene silencing, the physiological and molecular mechanisms of ZxNHX in feedback regulating the expression of genes sunch as ZxSOS1, ZxHKT1;1, ZxAKTl and ZxSKOR which involved in Na+and K+uptake and transport, thereby maintaining Na+and K+homeostasis at the whole-plant level. We also analyzed the effects of ZxNHX silencing on Na+accumulation, space distribution and the expression of genes involved in Na+uptake and transport under-0.5MPa osmotic stress conditions. The main results of this study are as following:(1) Plant siRNA expression vector pARX with inverted repeats of ZxNHX target fragment was constructed based on the vectors pART27. Two ZxNHX-silenced lines (L2and L7) with different (reduced) expression levels of ZxNHX were chosen for further analysis. The expression level of ZxNHX in leaves was lower than that of WT by8%for L2and45%for L7under50mM NaCl treatment, meanwhile, lower by17%for L2and45%for L7under-0.5MPa osmotic stress conditions.(2) The silencing of ZxNHX triggered a significant inhibition on plant growth under salt treatment,-0.5MPa osmotic stress treatment and even control conditions.(3)50mM NaCl induced an620%increase in transcripts of ZxSOS1but only an80%increase in transcripts of ZxHKT1;1in roots of wild-type (WT). Consequently, the transport ability of ZxSOSl for Na+exceeded that of ZxHKT1;1and Na+was loaded into the xylem by ZxSOS1and delivered to the shoots. However, in a ZxNHX-silenced line (L7), the capacity to sequester Na+into vacuoles of leaves was weakened, which in turn regulated long-distance Na+transport from roots to shoots. In roots of L7, NaCl (50mM) increased transcripts of ZxSOS1by only10%whereas transcripts of ZxHKT1;1increased by53%. Thus in L7, the transport ability of ZxHKT1;1for Na+outweighed that of ZxSOS1. Na+was unloaded from the xylem stream, consequently reducing Na+accumulation and relative distribution in leaves but increasing the relative distribution of Na+in roots and the net selective transport capacity for K+over Na+from roots to shoots compared to WT.(4) Silencing of ZxNHX also triggered a down-regulation of ZxAKT1and ZxSKOR in roots, resulting in a significant decrease in K+accumulation in all the tissues in plant grown in50mM NaCl.(5) Under-0.5MPa osmotic stress treatment, the transcription increase of ZxSOS1(130%) was higher than ZxHKT1;1(no increase) in roots of WT compared to control condition. However, in roots of L7, osmotic stress treatment only induced an51%increase in transcripts of ZxSOS1but an756%increase in transcripts of ZxHKT1;1. Consequently, Na+accumulation of L7was significant increase in roots, but decrease in leaves.Therefore, ZxNHX is not only essential for spatial distribution of Na+, but also vital for controlling Na+, K+uptake and their long-distance transport systems thereby maintaining the characteristic of salt-accumulation in Z. xanthoxylum and regulating its normal growth.