Cloning and Characterisation of Two H+ Translocating Organic Pyrophos-phatase Genes in Salix and Their Expression Differences in Two Willow Varieties with Different Salt Tolerances

Abstract

Willows are one of the most important tree species for landscaping, biofuel and raw timber. Screening salt-tolerant willow varieties is an effective approach to balance wood supply and demand. However, more salt-tolerant willow varieties are required and little is known regarding the mechanism of salt tolerance at the gene expression level. In this paper, two willow varieties were studies in terms of their differences in salt-tolerances and mechanism of salt tolerance at the level of VP1 gene expression. The results showed that Salix L0911 (L0911) had higher biomass than Salix matsudana (SM), and salt injuries were less severe in L0911 than in SM. The activities of peroxidase and superoxide dismutase, as well as the contents of soluble protein and proline, were higher in L0911 than in SM, whereas the contents of Na(+) and K(+), as well as the Na(+)/K(+) ratio, were lower in L0911 than in SM. Two VP1 genes (VP1.1 and VP1.2) cloned in L0911 and SM had similar sequences and structures. VP1.1 and VP1.2 belonged to different subgroups. Total expression levels of the VP1.1 gene in both roots and leaves of L0911 were higher than that in SM under normal conditions. Under salt stress, expression of VP1 in SM roots initially increased and then decreased, whereas the expression of VP1 in leaves of L0911 and SM, as well as in roots of L0911, decreased with increasing salt concentrations. This study increased our understanding of the salt-tolerance mechanism of willow and may facilitate the selection of salt-tolerant willow resources.

Keywords: Gene expression; Phylogenetic analysis.; Salt tolerance; VP1 gene; Willow.

Fig. (1)

Multiple alignments of VP1 amino acid residues in Arabidopsis (At1G15690), Vigna radiate (VrVP1), Populus triocarpa (potri) and two Salix VP1 genes (VP1.1 and VP1.2) obtained in this study. Sequences shown in black bold fonts are membrane region related.

Fig. (2)

Phylogenetic tree of VP1 sequences. Amino acid sequences were searched from website www.phytozome.com using Arabidopsis AVP1 as probe. Bold line and “▲”show the two Salix VP1 genes cloned in this study. AT, Glyma, GSVIVT, Medtr, Potri., Solyc, and Vr stand for Arabidopsis thaliana, Glycine max, Vitis vinifera, Medicago truncatula, Populus triocarpa, Solanum lycopersicum and Vigna radiate (PDB: 4A01), respectively.

Fig. (3)

Expression of two Salix VP1 genes under salt stress. A. Root expressions of VP1 genes in 100, 200, 300 mM NaCl solution. B. Leaf expressions of VP1 genes in 100, 200, 300 mM NaCl solution for 48 hours. C. Root expressions of VP1 genes treated with 200 mM NaCl solution for 12, 24, 48, 72 and 96 hours. D. expressions of VP1 genes treated with 200 mM NaCl solution for 12, 24, 48, 72 and 96 hours. The Ubiquitin gene was used as an internal control, and all samples were compared with the untreated controls.