Molecular cloning and functional characterisation of an H+-pyrophosphatase from Iris lactea

Abstract

Tonoplast H⁺-pyrophosphatases (VPs) mediate vacuolar Na⁺ sequestration, a process important for salt tolerance of plants. The function of VP in the highly drought- and salt-tolerant perennial Iris lactea under salt stress is unclear. Here, we isolated IlVP from I. lactea and investigated its function in transgenic tobacco. IlVP was found to comprise 771 amino acid residues and showed 88% similarity with Arabidopsis AtVP1. IlVP was mainly expressed in shoots and was up-regulated by salt stress. Overexpression of IlVP enhanced growth of transgenic tobacco plants compared with wild-type (WT) plants exposed to salt stress. Transgenic plants accumulated higher quantities of Na⁺ and K⁺ in leaves, stems, and roots under salt stress, which caused higher leaf relative water content and decreased cell membrane damage compared with WT plants. Overall, IlVP encoding a tonoplast H⁺-pyrophosphatase can reduce Na⁺ toxicity in plant cells through increased sequestration of ions into vacuoles by enhanced H⁺-pyrophosphatase activity.

Figure 1

(a) Alignment of amino acid sequences of H⁺-PPase genes from Iris lactea var. chinensis (IlVP) with those from Phoenix dactylifera (PdVP), Oryza sativa (OsVP), and Arabidopsis thaliana (AtVP). Amino acid sequences enclosed in red frames represent the PPi binding sites and activity domains of H⁺-PPase. (b) Phylogenetic tree of H⁺-PPase genes from Iris lactea and other plant species. Genes and GenBank accession numbers are as follows: AtVP (Arabidopsis thaliana, NM_101437), BdVP (Brachypodium distachyon, XM_003564169), CrVP (Chenopodium rubrum, AF533336), CsVP (Citrus sinensis, XM_006474322), EgVP (Eucalyptus grandis, XM_010035677), GmVP (Glycine max, XM_003528254), HbVP (Hevea brasiliensis, AY514019), HvVP (Hordeum vulgare, AK360389), IlVP (Iris lactea, KY406740), MaVP (Musa acuminata, XM_009386846), NtVP (Nicotiana tomentosiformis, XM_009630002), NnVP (Nelumbo nucifera, XM_010246610), OsVP (Oryza sativa, D45383), PdVP (Phoenix dactylifera, XM_008790581), PpVP (Prunus persica, AF367446), PtVP (Populus trichocarpa, XM_006381029), RcVP (Ricinus communis, XM_002530709), SbVP (Sorghum bicolor, HM143921), SiVP (Setaria italic, XM_004964638), SlVP (Solanum lycopersicum, NM_001278976), TcVP (Theobroma cacao, XM_007023235), VvVP (Vitis vinifera, XM_002273171), and ZmVP (Zea mays, BT086232).

Figure 2

Expression analysis of IlVP in shoots and roots of Iris lactea var. chinensis under different NaCl treatments. The expression levels of IlVP in shoot and root under control (0 mM) and 200 mM were normalized with that in shoot in control. (a) IlVP expression in roots and shoots under control and 200 mM NaCl treatment for 24 h as indicated by quantitative real-time PCR (qRT-PCR); (b) IlVP expression in shoots after treatment with different concentrations of NaCl (0, 25, 50, 100, and 200 mM) for 0, 6, 12, 24, and 48 h as indicated by qRT-PCR. Each bar represents the mean (n = 3), and bars indicate the standard deviation (SD).

Figure 3

(a) Growth of wild-type and IlVP-transgenic tobacco plants in response to 200 mM NaCl treatment for 7 days. WT: wild type; T4, T18: transgenic tobacco. (b–d) Root, stem and leaf dry weight of wild-type and IlVP-transgenic tobacco plants in response to salt stress, respectively. Each bar represents the mean (n = 7), and error bars indicate the standard deviation (SD). Columns with different letters indicate a significant difference at P < 0.05 (Duncan’s multiple range test).

Figure 4

Leaf relative water content (a) and relative membrane permeability (b) of wild-type and IlVP-transgenic tobacco plants in response to salt stress for 7 days. Each bar represents the mean (n = 7), and error bars indicate the standard deviation (SD). Columns with different letters indicate a significant difference at P < 0.05 (Duncan’s multiple range test).

Figure 5

Cation concentration in tissues of wild-type and IlVP-transgenic tobacco plants in response to salt stress. Na⁺ (a–c) and K⁺ (d–f) concentrations were measured after treatment for 7 days with different NaCl concentrations (0, 50, 100, and 200 mM). Each bar represents the mean (n = 7), and error bars indicate the standard deviation (SD). Columns with different letters indicate a significant difference at P < 0.05 (Duncan’s multiple range test).