Molecular Cloning and Functional Characterization of the Dehydrin (IpDHN) Gene From Ipomoea pes-caprae

Abstract

Dehydrin (DHN) genes can be rapidly induced to offset water deficit stresses in plants. Here, we reported on a dehydrin gene (IpDHN) related to salt tolerance isolated from Ipomoea pes-caprae L. (Convolvulaceae). The IpDHN protein shares a relatively high homology with Arabidopsis dehydrin ERD14 (At1g76180). IpDHN was shown to have a cytoplasmic localization pattern. Quantitative RT-PCR analyses indicated that IpDHN was differentially expressed in most organs of I. pes-caprae plants, and its expression level increased after salt, osmotic stress, oxidative stress, cold stress and ABA treatments. Analysis of the 974-bp promoter of IpDHN identified distinct cis-acting regulatory elements, including an MYB binding site (MBS), ABRE (ABA responding)-elements, Skn-1 motif, and TC-rich repeats. The induced expression of IpDHN in Escherichia coli indicated that IpDHN might be involved in salt, drought, osmotic, and oxidative stresses. We also generated transgenic Arabidopsis lines that over-expressed IpDHN. The transgenic Arabidopsis plants showed a significant enhancement in tolerance to salt/drought stresses, as well as less accumulation of hydrogen peroxide (H₂O₂) and the superoxide radical (O₂ ^-), accompanied by increasing activity of the antioxidant enzyme system in vivo. Under osmotic stresses, the overexpression of IpDHN in Arabidopsis can elevate the expression of ROS-related and stress-responsive genes and can improve the ROS-scavenging ability. Our results indicated that IpDHN is involved in cellular responses to salt and drought through a series of pleiotropic effects that are likely involved in ROS scavenging and therefore influence the physiological processes of microorganisms and plants exposed to many abiotic stresses.

Keywords: Ipomoea pes-caprae L.; dehydrin; drought; promoter; salt.

FIGURE 1

Phylogenetic relationships between the IpDHN protein and dehydrins from other plant species. The molecular phylogeny was constructed from a complete protein sequence alignment of DHNs by the neighbor-joining method with ClustalW. The numbers beside the branches indicate bootstrap values. The symbol “” shows the position of IpDHN in the phylogenetic tree.

FIGURE 2

Nucleotide sequence of the IpDHN promoter (974 bp). Nucleotides are numbered on the left. The putative translation start sites, TATA box, and other important cis-regulatory elements are boxed and labeled.

FIGURE 3

Functional analysis of IpDHN induced-expression for salt, drought, and H₂O₂ tolerance in Escherichia coli. (A) Induced expression of the GST-IpDHN (IpDHN- pGEX 6p-1) fusion protein and single GST (pGEX 6p-1 empty vector) protein in E. coli. 0 and 2 h: the IPTG induction time. (B) The growth performance of E. coli BL21 (pGEX 6p-1, upper)/(IpDHN-pGEX 6p-1) on LB plates containing stress factors. Control (top): LB medium; 5% NaCl: LB medium containing 5% NaCl; 6% NaCl: LB medium containing 6% NaCl; 2 M Sorbitol: LB medium containing 2 M sorbitol; 5 mM H₂O₂: LB medium containing 5 mM H₂O₂; 8 mM H₂O₂: LB medium containing 8 mM H₂O₂. The cell cultures were adjusted to OD₆₀₀ to 1 and were then diluted serially (1:10, 1:100, and 1:1000, respectively). Two microliters of each sample was spotted onto the LB plates containing 0.2 mM IPTG.

FIGURE 4

Subcellular localization of IpDHN. Arabidopsis protoplasts expressing the 35S:IpDHN-GFP fusion protein (upper) and 35S:GFP (lower) observed under a laser scanning confocal microscope. The blue color indicates the nucleus using mCherry as the nuclear marker. The red color indicated autofluorescence emitted by chloroplasts. Bar represents 5 μm.

FIGURE 5

Expression profiles of the IpDHN gene among Ipomoea pes-caprae tissues. (A) Differential expression of IpDHN in various tissues (young root, young leaf, shoot bud, mature root, vine, mature leaf, flower bud, petal, young seed). (B–F) Time-course expression patterns of IpDHN in response to different abiotic stresses: NaCl (B), Mannitol (C), MV (D), freeze (E), and ABA treatment (F). Error bars indicate the SD based on three replicates. Asterisks indicate significant differences from the control (Student’s t-test P-values, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 6

The overexpression analyses of IpDHN in transgenic Arabidopsis lines (IpDHN OX1 and IpDHN OX2). (A) Quantitative RT-PCR analysis of IpDHN in transgenic Arabidopsis lines and WT Arabidopsis (WT). AtActin2 was used as an internal control. Error bars indicate the SD based on three replicates. (B) RT-PCR analysis of IpDHN in transgenic Arabidopsis lines and two WT lines. (C) RT-PCR analysis of AtActin2 in transgenic Arabidopsis lines and two WT lines as a control.

FIGURE 7

Osmotic and salt stress analyses of transgenic plants with IpDHN with respect to the seed germination rate. (A) Photographs of transgenic lines (IpDHN OX1 and IpDHN OX2) and WT seeds germinated on MS medium or MS medium with mannitol (left, 200, 300, and 400 mM) or NaCl (right, 100, 125, 150, 175, and 200 mM) for 7 days. Seed germination rates were calculated for the WT and transgenic lines under NaCl (B) and mannitol (C) stress after 7 days. Error bars indicate the SD based on three replicates. Asterisks indicate significant differences from the WT (control, Student’s t-test P-values, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 8

Osmotic and salt stresses analyses of transgenic plants with IpDHN with respect to seedling root length. Four-day-old seedlings were transplanted to MS medium containing NaCl or mannitol and were then grown for 7 days before measuring the root length. (A) Photographs of transgenic lines (IpDHN OX1 and IpDHN OX2) and WT seedlings on MS medium or MS medium with NaCl (right, 100, 125, and 150 mM) or mannitol (200, 300, and 400 mM). (B,C) Seedling root length of WT and transgenic lines under NaCl (B) and mannitol (C) stress after 7 days. Error bars indicate the SD based on three replicates. Asterisks indicate significant differences from the WT (control, Student’s t-test P-values, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 9

Photographs and survival rate of the transgenic lines (IpDHN OX1 and IpDHN OX2) and WT plants grown in pots under normal and salt/drought conditions. (A) The effects of 150 mM NaCl on transgenic lines and WT. (B) The effects of 200 mM NaCl on transgenic lines and WT. (C) The effects of water withholding on transgenic lines and WT. (D) The statistics for the survival rate of transgenic lines and WT Arabidopsis after salt/drought stresses. Thirty plants from the WT and from the two transgenic lines (IpDHN OX1 and IpDHN OX2) were treated with various concentrations of NaCl or drought.

FIGURE 10

Changes in physiological parameters of IpDHN overexpressing Arabidopsis and WT seedlings (4 weeks) under 200 mM NaCl and 200 mM mannitol treatments for 24 h. (A) RWC; (B) free proline content; (C) IL; (D) MDA. All determinations were carried out on three biological replicates. Error bars indicate the SD based on three replicates. Asterisks indicate significant differences from the WT (control, Student’s t-test P-values, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 11

Oxidative stress analyses of transgenic lines and WT plants. Histochemical staining assays were used to detect H₂O₂ and O₂⁻ by DAB (A) or NBT (B) staining, respectively. (C) Analysis of CAT activities in the WT and transgenic lines under normal conditions and osmotic stresses. (D) Analysis of SOD activities in the WT and transgenic lines under normal conditions and osmotic stresses. Error bars indicate the SD based on three replicates. Asterisks indicate significant differences from the WT (control, Student’s t-test P-values, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 12

Analysis of expression levels of ROS-related and stress-responsive genes in the WT and the transgenic line by qRT-PCR under normal and osmotic conditions. (A) CAT1, FSD1, CSD1, and APX2; (B) NCED3, HAI2, RD29A, RD29B, HVA22D, ANAC19, RD22, and RD26. Error bars indicate the SD based on three replicates. Asterisks indicate significant differences from the WT (control, Student’s t-test P-values, ^∗p < 0.05, ^∗∗p < 0.01).