Co-expression of tonoplast Cation/H(+) antiporter and H(+)-pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions

Abstract

Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H(+)-PPase genes, ZxNHX and ZxVP1-1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens-mediated transformation. Compared with wild-type (WT) plants, transgenic alfalfa plants co-expressing ZxNHX and ZxVP1-1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water-deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na(+), K(+) and Ca(2+) accumulation in leaves and roots, as a result of co-expression of ZxNHX and ZxVP1-1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water-deficit stress. Furthermore, the transgenic alfalfa co-expressing ZxNHX and ZxVP1-1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water-use efficiency than WT plants. Our results indicate that co-expression of tonoplast NHX and H(+)-PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China.

Keywords: H+-PPase; co-expression; stress tolerance; tonoplast NHXs; transgenic alfalfa.

Figure 1

Expression analysis of ZxNHX and ZxVP1‐1 in transgenic alfalfa plants. RT‐PCR was performed using total RNA from leaves of wild‐type and PCR‐positive transgenic lines. ACTIN gene fragment was amplified as an internal control. WT, wild‐type plants; 1‐25, PCR‐positive transgenic lines.

Figure 2

Growth of transgenic alfalfa and wild‐type plants under normal and saline conditions. Representative photographs show 4‐week‐old plants were treated for 30 days without (a) and with 200 mm NaCl (b). WT, wild‐type plants; L9, transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1.

Figure 3

Response of transgenic alfalfa and wild‐type plants to water‐deficit. Representative photographs show 4‐week‐old plants were withheld water for 5 days (a) and 8 days (b), then were rewatered to field water capacity for 2 day (c), and 7 days (d). WT, wild‐type plants; L9, transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1.

Figure 4

Cation concentration in wild‐type and transgenic alfalfa in response to salt treatment. The Na⁺ (a, b), K⁺ (c, d) and Ca²⁺ (e, f) concentrations were measured in leaf (left) and root (right) of alfalfa plants treated with different NaCl concentrations for 30 days. Values are the means ± SE (n = 6). Different letters indicate significant differences (P < 0.05) among columns. WT, wild‐type plants; L9, transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1.

Figure 5

Cation concentration in wild‐type and transgenic alfalfa under water‐deficit. The Na⁺ (a, b), K⁺ (c, d) and Ca²⁺ (e, f) concentrations were measured in leaf (left) and root (right) of alfalfa plants treated with withholding water for 0, 1, 3, 5 and 7 days. Values are the means ± SE (n = 6). Different letters indicate significant differences (P < 0.05) among columns. WT, wild‐type plants; L9, transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1.

Figure 6

Leaf relative water content of wild‐type and transgenic alfalfa under salt and water‐deficit. Plants were treated with different NaCl concentrations for 30 days (a) or withholding water for 0, 1, 3, 5 and 7 days (b), respectively. Values are the means ± SE (n = 6). Different letters indicate significant differences (P < 0.05) among columns, and asterisks indicate there is a significant difference (P < 0.05) between WT and L9. WT, wild‐type plants; L9, transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1.

Figure 7

Net photosynthetic rate (Pn) of wild‐type and transgenic alfalfa under salt and water‐deficit. Plants were treated with different NaCl concentrations for 30 days (a) or withholding water for 0, 1, 3, 5 and 7 days (b), respectively. Values are the means ± SE (n = 6). Different letters indicate significant differences (P < 0.05) among columns, and asterisks indicate there is a significant difference (P < 0.05) between WT and L9. WT, wild‐type plants; L9, transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1.

Figure 8

Photosynthetic responses of wild‐type and transgenic alfalfa under field conditions. Stomatal conductance (Gs) (a), net photosynthesis rate (Pn) (b) and water‐use efficiency (WUE) (c) were determined after plants transplanting into field conditions for 30–90 days. Values are the means ± SE (n = 15). Asterisks indicate there is a significant difference (P < 0.05) between WT and L9. WT, wild‐type plants; L9, transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1.