Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobacco

Abstract

The mitogen-activated protein kinase (MAPK) cascade plays pivotal roles in diverse signalling pathways related to plant development and stress responses. In this study, the cloning and functional characterization of a group-I MAPK gene, PtrMAPK, in Poncirus trifoliata (L.) Raf are reported. PtrMAPK contains 11 highly conserved kinase domains and a phosphorylation motif (TEY), and is localized in the nucleus of transformed onion epidermal cells. The PtrMAPK transcript level was increased by dehydration and cold, but was unaffected by salt. Transgenic overexpression of PtrMAPK in tobacco confers dehydration and drought tolerance. The transgenic plants exhibited better water status, less reactive oxygen species (ROS) generation, and higher levels of antioxidant enzyme activity and metabolites than the wild type. Interestingly, the stress tolerance capacity of the transgenic plants was compromised by inhibitors of antioxidant enzymes. In addition, overexpression of PtrMAPK enhanced the expression of ROS-related and stress-responsive genes under normal or drought conditions. Taken together, these data demonstrate that PtrMAPK acts as a positive regulator in dehydration/drought stress responses by either regulating ROS homeostasis through activation of the cellular antioxidant systems or modulating transcriptional levels of a variety of stress-associated genes.

Fig. 1.

Multiple alignments of the predicted protein sequence of PtrMAPK and MAPKs from other plants, including Arabidopsis thaliana (AtMPK3, At3g45640; AtMPK4, NP_192046.1), Brassica napus (BnMPK4, ABB69023), Malus hupehensis (MhMAPK, ABR10070), Nicotiana tabacum (NtMAPK, BAE46985), Petroselinum crispum (PcMPK4, AAN65180), Prunus armeniaca (PaMAPK, AF134730), Solanum lycopersicum (SlMPK4, ADH43227), and Zea mays (ZmMPK4, ACG31917). Identical and similar amino acid residues among the aligned sequences are indicated by black and grey shading, respectively. The 11 major conserved subdomains of a protein kinase are marked by Roman numerals. The dual phosphorylation motif (TEY) is shown by a double line, while a common docking (CD) motif at the C-terminus is indicated by a single line.

Fig. 2.

A phylogenetic tree created based on PtrMAPK and its homologous sequence from different plant species. Information about the proteins included in the tree (GenBank accession numbers) is given in Supplementary Table S1 available at JXB online.

Fig. 3.

Expression patterns of PtrMAPK in trifoliate orange under dehydration (A), low temperature (B), and salt (C), as analysed by qRT-PCR. For each stress, the expression level at time point 0 (the beginning of the relevant treatment) was defined as 1.0, and the expression level at other time points was normalized accordingly. Error bars show standard deviations for four independent replicates.

Fig. 4.

Cellular localization of PtrMAPK in onion epidermal cells. PtrMAPK was fused to the N-terminus of green fluorescence protein (PtrMAPK–GFP), which was transformed into onion epidermal cells through Agrobacterium-mediated infection, using GFP as a control. The expression of PtrMAPK–GFP or GFP alone was examined under a universal fluorescence microscope. Bright-field images (A and D), fluorescence images (B and E), and the overlapped images (C and F) of representative cells expressing PtrMAPK–GFP fusion protein (upper panels) or GFP (lower panels) are shown.

Fig. 5.

Analysis of kinase activity in PtrMAPK. (A) SDS–PAGE separation of protein extracted from E. coli transformed with pGEX-PtrMAPK induced (+) or not (–) with 1 mM IPTG. (B) SDS–PAGE separation of the purified protein derived from the IPTG-induced extract in A. The predicted protein in A and B is shown by an open arrow. M, a molecular weight marker (kDa). (C) Detection of protein phosphorylation. The purified PtrMAPK protein was incubated in a buffer to which MBP for kinase reaction was added (+) or not (–), followed by detection of phosphorylation with SDS–PAGE and autoradiography. The positions of the proteins are shown by the filled arrows.

Fig. 6.

Overexpression of PtrMAPK enhances dehydration tolerance in transgenic tobacco. (A) Relative water loss rate in the wild type (WT) and transgenic lines (OE-2, OE-19) under dehydration for 90 min in an ambient environment, as measured by reduction of fresh weight every 10 min. * (P <0.05) and ** (P <0.01) indicate that the water loss rate in the two transgenic lines is significantly lower than that of the WT at the same time point. (B) A representative photograph showing the dehydrated seedlings. (C and D) Ion leakage (C) and MDA level (D) in the WT, OE-2, and OE-19 after 90 min dehydration. Asterisks indicate significant difference between the WT and the two transgenic lines (*P <0.05; **P <0.01). (This figure is available in colour at JXB online.)

Fig. 7.

The transgenic lines exhibited a higher survival rate under drought and recovery when compared with the WT. (A) The photographs are of representative plants after drought for 20 d and a subsequent recovery for 3 d. (B) The survival rate in OE-2, OE-19, and the WT after the 3 d re-watering following the drought. Data are means ±SD calculated from three replicates. * (P <0.05) and ** (P <0.01) indicate that the value in the transgenic lines is significantly different from that of the WT. (This figure is available in colour at JXB online.)

Fig. 8.

Overexpression of PtrMAPK enhances drought tolerance in transgenic tobacco. (A) The photographs are of representative WT, OE-2, and OE-19 plants after drought for 21 d. (B and C) Ion leakage (B) and total chlorophyll content (C) of the WT, OE-2, and OE-19 after drought stress for 21 d. A significant difference from the WT is indicated by asterisks (*P <0.05; **P <0.01). (This figure is available in colour at JXB online.)

Fig. 9.

Relative water content (RWC) and cell death staining in the WT and transgenic lines (OE-2 and OE-19) under drought. (A) The RWC in the leaves of the three lines before and after 7 d drought treatment. (B) Trypan blue staining of the leaves from the three lines after 7 d drought treatment.

Fig. 10.

Accumulation of O₂⁻ and H₂O₂ in the WT and transgenic lines (OE-2 and OE-19) under dehydration or drought, as measured by histochemical staining with NBT and DAB, respectively. (A and B) Representative photographs showing staining of O₂⁻ (A) and H₂O₂ (B) in the leaves before (0, upper panel) and after dehydration (90, lower panel). (C) Representative photographs showing accumulation of O₂⁻ (upper panel) and H₂O₂ (lower panel) in leaves that have been subjected to drought for 21 d. (This figure is available in colour at JXB online.)

Fig. 11.

Analysis of enzyme activity and metabolite levels in the WT and the transgenic lines (OE-2 and OE-19) before and after drought treatment for 7 d. (A–C) Activity of SOD (A), POD (B), and CAT (C) in the three lines. (D–F) Content of ascorbic acid (D), GSH (E), and proline (F) in the three lines. Asterisks show that the values are significantly different between the transgenic lines and the WT at the same time point (*P <0.05; **P <0.01; ***P <0.001).

Fig. 12.

Effect of a SOD inhibitor (DDC) and a POD inhibitor (NaN₃) on the dehydration response of the transgenic lines. (A and D) Representative photographs showing the morphology of leaves from WT, OE-2, OE-19, and DDC- (A) or NaN₃- (D) treated OE-2 and OE-19 plants (from the left to the right). (B and E) Ion leakage of the samples indicated in A and D, respectively. Asterisks show that the value is significantly different between the transgenic line treated or not with the enzyme inhibitor. (C and F) NBT (C) and DAB (F) staining of the leaves from WT, OE-2, OE-19, and DDC- or NaN₃-treated OE-2 and OE-19 plants. (This figure is available in colour at JXB online.)

Fig. 13.

Quantitative real-time PCR analysis of expression levels of ROS-related and stress-responsive genes in the WT and the transgenic lines (OE-2 and OE-19) under normal and drought conditions. Data represent the means ±SE of four replicates. Asterisks show that the values are significantly different between the transgenic lines and the WT under the same growth conditions (*P <0.05; **P <0.01).