Isolation and Functional Analysis of ZmLTP3, a Homologue to Arabidopsis LTP3

Abstract

Plant lipid transfer proteins (LTPs) are encoded by multigene families and play important roles in plant physiology. One full-length cDNA encoding an Arabidopsis LTP3 homologue was isolated from maize by RT-PCR and named as ZmLTP3. RT-PCR analysis indicated that the ZmLTP3 expression is induced by salicylic acid (SA), mannitol and salt. Furthermore, in different tissues the ZmLTP3 displayed different expression patterns, indicating that ZmLTP3 may play multiple roles in stress resistance. Over-expression of ZmLTP3 in wild-type Arabidopsis resulted in the increased salt tolerance. Under salt stress condition, compared to wild-type (WT) plants, transgenic Arabidopsis grew better, had higher seedling fresh (FW), dry weight (DW), seed yields, proline content and lower MDA content and relative electric conductivity level. Our results suggest that maize ZmLTP3 might encode a member of LTPs family and play roles in salt resistance.

Figure 1

Sequence analysis of ZmLTP3. The predicted transmembrane regions are shown within gray boxes. Two sequences in frames indicate two consensus pentapeptides, and the underlined amino acids indicate the eight conserved cysteine residues.

Figure 2

A phylogenetic tree of maize ZmLTP3 with other plant LTP genes. This tree was constructed using the neighbor-joining method.

Figure 3

Expression of the ZmLTP3 gene in various tissues and in response to various treatments. (A) Expression of ZmLTP3 in various tissues. 1–5 shows different tissues: (1) roots, (2) coleoptiles, (3) leaves, (4) silks and (5) ovaries; (B) Expression of ZmLTP3 under various treatments: (1) control, (2) mannitol, (3) salt, (4) 4 °C and (5) SA.

Figure 4

Western blot analysis of the ZmLTP3 gene expression in wild type and homozygous transgenic lines. M, protein molecular weight marker; WT, wild type Arabidopsis; OE3, OE5, OE10, OE14 and OE33, transgenic lines.

Figure 5

Effects of salt stress on growth of transgenic plants. WT, wild type Arabidopsis; OE14 and OE33, transgenic lines.

Figure 6

Morphological analysis of transgenic and WT plants under normal or salt treatment. (A) FW of plants treated with 0 mM NaCl or 300 mM NaCl; (B) DW of plants treated with 0 mM NaCl or 300 mM NaCl; (C) seeds weight of plants treated with 0 mM NaCl or 300 mM NaCl. Error bars indicate ± SE (n = 3). * and **, Significantly different from the WT at p < 0.05 and < 0.01, respectively, by Student’s t test.

Figure 7

Physiological analysis of transgenic and WT plants under normal or salt treatment. (A) MDA content in plants treated with 0 mM NaCl or 300 mM NaCl; (B) Ion leakage ration of plants treated with 0 mM NaCl or 300 mM NaCl; (C) Proline content in plants treated with 0 mM NaCl or 300 mM NaCl. Error bars indicate ± SE (n = 3). * and **, Significantly different from the WT at p < 0.05 and < 0.01, respectively, by Student’s t test.