Overexpression of the Zygophyllum xanthoxylum Aquaporin, ZxPIP1;3, Promotes Plant Growth and Stress Tolerance

Abstract

Drought and salinity can result in cell dehydration and water unbalance in plants, which seriously diminish plant growth and development. Cellular water homeostasis maintained by aquaporin is one of the important strategies for plants to cope with these two stresses. In this study, a stress-induced aquaporin, ZxPIP1;3, belonging to the PIP1 subgroup, was identified from the succulent xerophyte Zygophyllum xanthoxylum. The subcellular localization showed that ZxPIP1;3-GFP was located in the plasma membrane. The overexpression of ZxPIP1;3 in Arabidopsis prompted plant growth under favorable condition. In addition, it also conferred salt and drought tolerance with better water status as well as less ion toxicity and membrane injury, which led to more efficient photosynthesis and improved growth vigor via inducing stress-related responsive genes. This study reveals the molecular mechanisms of xerophytes' stress tolerance and provides a valuable candidate that could be used in genetic engineering to improve crop growth and stress tolerance.

Keywords: Zygophyllum xanthoxylum; abiotic stress; aquaporin; plant growth.

Figure 1

ZxPIP1;3 encodes a PIP1 protein whose expression level is related with osmotic and salt treatments. (A) qRT-PCR validation of RNA sequencing data in Z. xanthoxylum roots under osmotic stress or salt treatment for 6 h. (B,C), alignment (B) and phylogenetic analysis (C) of ZxPIP1;3 with other known PIP1 proteins. Dark blue, pink and aqua indicate that the homology levels of these amino acids are 100%, more than 75% and more than 50% respectively. (D) Subcellular localization of ZxPIP1;3-GFP in epidermal cells of tobacco leaves. GFP driven by CaMV35S promoter served as control. PM, plasma membrane. N, nucleus. Green fluorescence represents GFP. Bar = 10 μm. For (A), asterisks indicate significant differences from control condition. Data shown are means of three independent biological replicates (* p < 0.05, one way ANOVA).

Figure 2

ZxPIP1;3 plays positive roles in plant growth. (A) Phenotypes of wild-type (Col-0) and ZxPIP1;3 overexpression lines (OE2 and OE3) grow vertically on 1/2 Murashige and Skoog (MS) medium for 7 days after germination. Bar = 1 cm. (B–D) Primary root length (B), number of emerged lateral roots and lateral root primordia (C) as well as cotyledon diameter (D) of plants treated as described in the legend of (A). (E) Phenotypes of 4-week-old seedlings grown on soil culture. Bar = 5 cm. (F) Rosette leaves diameter of plants treated as described in the legend of (E). (G) Phenotypes of 7-week-old seedlings on soil culture. Bar = 5 cm. (H) Height of plants treated as described in the legend of (G). For (B–D), (F,H), asterisks indicate significant differences from Col-0 (n = 13 per column. * p < 0.05, one way ANOVA).

Figure 3

Overexpression of ZxPIP1;3 improves growth vigor under salt treatment. (A) Phenotypes of soil-cultured seedlings treated with (Salt) or without (Mock) 100 mM NaCl for 20 days. Bar = 10 cm. (B) Phenotypes of shoot apices of plants under salt treatment as described in (A). Bar = 1 cm. (C–E) Dry weight of stems (C), number of branches (D) and plant height (E) of plants described in the legend of (A). For (C–E), asterisks indicate significant differences from Col-0 (n = 13 per column. * p < 0.05, ** p < 0.01, one way ANOVA).

Figure 4

Several physiological parameters of the salt-treated wild-type and transgenic plants. Soluble sugar content (A), proline content (B), relative water content (C), chlorophyll A content (D), chlorophyll B content (E), chlorophyll content (F), net photosynthetic rate (Pn) (G), water-use efficiency (WUE) (H), content of malondialdehyde (MDA) (I), K⁺ content (J), Na⁺ content (K), and K⁺/Na⁺ (L) were tested. Asterisks indicate significant differences from Col-0 (Values are mean ± SE of three replicates. * p < 0.05, one way ANOVA).

Figure 5

Overexpression of ZxPIP1;3 improves growth vigor under drought treatment. (A) Phenotypes of soil-cultured seedlings treated with (Drought) or without (Mock) period dehydration. Bar = 10 cm. (B,C), Dry weight of stems (B) and number of branches (C) of plants treated as described in the legend of (A). For (B,C), asterisks indicate significant differences from Col-0 (n = 13 per column. * p < 0.05, one way ANOVA).

Figure 6

Several physiological parameters of the drought-treated wild-type and transgenic plants. Soluble sugar content (A), proline content (B), relative water content (C), chlorophyll A content (D), chlorophyll B content (E), chlorophyll content (F), Pn (G), WUE (H), and content of MDA (I) were tested. Asterisks indicate significant differences from Col-0 (Values are mean ± SE of three replicates. * p < 0.05, one way ANOVA).

Figure 7

Relative expression levels of stress-related genes in wild-type and transgenic plants. The expression levels of P5CS1 (A), RD29A (B), and DREB1A (C) were tested in 7-day-old wild-type (Col-0) and transgenic plants (OE2, OE3) under normal (Control), salt (NaCl), and osmotic (Mannitol) treatments. Asterisks indicate significant differences from Col-0. Data shown are means of three independent biological replicates. * p < 0.05, one way ANOVA.