Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 enhances salt and drought tolerance in transgenic Lotus corniculatus by increasing cations accumulation

Abstract

Lotus corniculatus L. is an important legume for forage, but is sensitive to salinity and drought. To develop salt- and drought-resistant L. corniculatus, ZxNHX and ZxVP1-1 genes encoding tonoplast Na+/H+ antiporter and H+-pyrophosphatase (H+-PPase) from a succulent xerophyte Zygophyllum xanthoxylum L., which is well adapted to arid environments through accumulating Na+ in its leaves, were transferred into this forage. We obtained the transgenic lines co-expressing ZxNHX and ZxVP1-1 genes (VX) as well as expressing ZxVP1-1 gene alone (VP). Compared with wild-type, both VX and VP transgenic lines grew better at 200 mM NaCl, and also exhibited higher tolerance and faster recovery from water-deficit stress: these performances were associated with more Na+, K+ and Ca2+ accumulation in their leaves and roots, which caused lower leaf solute potential and thus retained more water. Moreover, the transgenic lines maintained lower relative membrane permeability and higher net photosynthesis rate under salt or water-deficit stress. These results indicate that expression of tonoplast Na+/H+ antiporter and H+-PPase genes from xerophyte enhanced salt and drought tolerance of L. corniculatus. Furthermore, compared with VP, VX showed higher shoot biomass, more cations accumulation, higher water retention, lesser cell membrane damage and higher photosynthesis capacity under salt or water-deficit condition, suggesting that co-expression of ZxVP1-1 and ZxNHX confers even greater performance to transgenic L. corniculatus than expression of the single ZxVP1-1.

Assessment of Stress Tolerance, Productivity, and Forage Quality in T1 Transgenic Alfalfa Co-overexpressing ZxNHX and ZxVP1-1 from Zygophyllum xanthoxylum

“…In recent years, the development of molecular biology and transgenic engineering provides tools for creation of new cultivars with enhanced stress tolerance (Hasegawa et al, 2000; Herrera-Estrella, 2000; Yang et al, 2010; Tang et al, 2015; Zhang et al, 2016). Numerous studies have demonstrated that (co-)overexpression tonoplast NHX or/and H + -PPase genes is one of the most effective strategies to create transgenic species with enhanced salt and drought tolerance through genetic engineering technology (e.g., Apse et al, 1999; Gaxiola et al, 2001, 2007; Zhao et al, 2006; Liu et al, 2010; Bhaskaran and Savithramma, 2011; Pasapula et al, 2011; Gouiaa et al, 2012; Bao et al, 2014; Bassil and Blumwald, 2014). To develop salt- and drought-resistant forage cultivar, we have co-transferred the tonoplast NHX and H + -PPase genes ( ZxNHX and ZxVP1-1 , respectively) from a xerophyte Z. xanthoxylum into the important legume forage alfalfa and successfully improved the salt and drought tolerance in T 0 generation transgenic plants (Bao et al, 2016).…”

“…The better salt and drought tolerance of NHX and H + -PPase transgenic plants could be explained as a consequence of increased ion compartmentation into vacuole resulting from increased expression of NHX and H + -PPase (Gaxiola et al, 2001, 2007; Leidi et al, 2010; Bao et al, 2014; Yang et al, 2015). Because vacuolar compartmentation of cations (such as, Na + and K + ) is mediated by NHXs and H + -PPase provides the proton motive force for this process as a tonoplast H + pump (Apse et al, 1999; Zhang et al, 2001; Gaxiola et al, 2002; Kronzucker and Britto, 2011; Yamaguchi et al, 2013).…”

“…The PCR analysis was conducted using genomic DNA isolated from leaf of putative T 1 transgenic plants and WT plants according to the method as described by Bao et al (2014).…”

“…To perform further molecular and physiological assays, the first PCR positive plant and WT were propagated from stem cuttings as described by Bao et al (2014, 2016). Then total RNA was extracted from root, stem and leaf of propagated transgenic plants (GM) and WT with a Trizol Kit (Sangon Biotech, Shanghai, China) following manufacturer’s instructions.…”

“…Overexpression of NHXs or H + -PPase genes significantly improved growth performance and the tolerance to multiple abiotic stresses in various transgenic plants (e.g., Apse et al, 1999; Gaxiola et al, 2001; Zhang and Blumwald, 2001; Park et al, 2005; Schilling et al, 2014; Yang et al, 2014). Of significance, co-overexpression of both NHXs and H + -PPase conferred transgenic plants greater tolerance and higher biomass accumulation than expression of the single gene (Zhao et al, 2006; Liu et al, 2010; Bhaskaran and Savithramma, 2011; Gouiaa et al, 2012; Bao et al, 2014). These findings indicated that tonoplast NHXs and H + -PPase genes have the potential in the development of crop cultivars with stronger stress tolerance, higher yield and better quality.…”