TaSnRK2.4, an SNF1-type serine/threonine protein kinase of wheat (Triticum aestivum L.), confers enhanced multistress tolerance in Arabidopsis

Abstract

Osmotic stresses such as drought, salinity, and cold are major environmental factors that limit agricultural productivity worldwide. Protein phosphorylation/dephosphorylation are major signalling events induced by osmotic stress in higher plants. Sucrose non-fermenting 1-related protein kinase2 family members play essential roles in response to hyperosmotic stresses in Arabidopsis, rice, and maize. In this study, the function of TaSnRK2.4 in drought, salt, and freezing stresses in Arabidopsis was characterized. A translational fusion protein of TaSnRK2.4 with green fluorescent protein showed subcellular localization in the cell membrane, cytoplasm, and nucleus. To examine the role of TaSnRK2.4 under various environmental stresses, transgenic Arabidopsis plants overexpressing wheat TaSnRK2.4 under control of the cauliflower mosaic virus 35S promoter were generated. Overexpression of TaSnRK2.4 resulted in delayed seedling establishment, longer primary roots, and higher yield under normal growing conditions. Transgenic Arabidopsis overexpressing TaSnRK2.4 had enhanced tolerance to drought, salt, and freezing stresses, which were simultaneously supported by physiological results, including decreased rate of water loss, enhanced higher relative water content, strengthened cell membrane stability, improved photosynthesis potential, and significantly increased osmotic potential. The results show that TaSnRK2.4 is involved in the regulation of enhanced osmotic potential, growth, and development under both normal and stress conditions, and imply that TaSnRK2.4 is a multifunctional regulatory factor in Arabidopsis. Since the overexpression of TaSnRK2.4 can significantly strengthen tolerance to drought, salt, and freezing stresses and does not retard the growth of transgenic Arabidopsis plants under well-watered conditions, TaSnRK2.4 could be utilized in transgenic breeding to improve abiotic stresses in crops.

Fig. 1.

Sequence alignment of TaSnRK2.4 and SnRK2s in other plant species. (A) Amino acid alignment of TaSnRK2.4 and other SnRK2 family members from selected plant species. The numbers on the left indicate the amino acid position. Identical amino acid residues are shown with a black background. Gaps, indicated by dashed lines, are introduced for optimal alignment. The box indicated by a solid triangle is the ATP-binding region signature. The box indicated by an asterisk is the serine/threonine protein kinase-activating signature. The region underlined indicates the divergent C-terminus. Alignments were performed using the Megalign program of DNAStar. (B) Phylogenetic tree of TaSnRK2.4 and SnRK2 members from other plant species. At, Arabidopsis thaliana; Fs, Fagus sylvatica; Gm, Glycine max; Os, Oryza sativa; Zm, Zea mays. The phylogenetic tree was constructed with the PHYLIP 3.68 package; bootstrap values are in percentages. (This figure is available in colour at JXB online.)

Fig. 2.

Expression patterns of TaSnRK2.4. (A) Expression patterns of TaSnRK2.4 in wheat tissues at different developmental stages. SL, seedling leaf; SR, seedling root; BS, booting spindle; HS, heading spike. The 2^–ΔΔCT method was used to measure the relative expression level of the target gene, and the expression of TaSnRK2.4 in seedling leaves was regarded as the standard for its lower level. (B) Expression patterns of TaSnRK2.4 under ABA, salt (NaCl), PEG, and low temperature (LT) treatments. Two-leaf seedlings of common wheat cv. Hanxuan 10 were exposed to abiotic stresses as described in the Materials and methods. The 2^–ΔΔCT method was used to measure the relative expression level of the target gene, and the expression of TaSnRK2.4 in non-stressed seedling leaves was regarded as the standard. Means were generated from three independent measurements; bars indicate standard errors.

Fig. 3.

Subcellular localization of TaSnRK2.4 in onion epidermal cells. Cells were bombarded with constructs carrying GFP or TaSnRK2.4–GFP as described in the Materials and methods. GFP and TaSnRK2.4–GFP fusion proteins were transiently expressed under control of the CaMV 35S promoter in onion epidermal cells and observed with a laser scanning confocal microscope. Images were taken in the dark field for green fluorescence (1, 4), while the outline of the cell (2, 5) and the combination (3, 6) were photographed in a bright field. (This figure is available in colour at JXB online.)

Fig. 4.

Expression levels of TaSnRK2.4 in different transgenic Arabidopsis lines. Gene expression level of TaSnRK2.4 in different transgenic Arabidopsis lines. L1–L6, six individual TaSnRK2.4 transgenic lines. The expression of TaSnRK2.4 in L4 was regarded as the standard due to its lower level.

Fig. 5.

Morphological characterization of TaSnRK2.4 plants. (A) Comparison of primary root lengths. Because of the prolonged SET for transgenic lines, WT seeds were planted 1 d later than the transgenic lines, and root lengths were compared on the seventh day. (B) The silique sizes of TaSnRK2.4 plants were larger than those of the WT under well-watered conditions. Plants of the same size and siliques at the same stem location were selected to measure silique length, and 10 plants were used for each line in triplicate (F-test **P <0.01). (C) TaSnRK2.4 plants had higher yields than the WT. The seeds of transgenic TaSnRK2.4 and WT plants cultured under well-watered conditions were harvested separately, and the yield of each plant was measured after complete dehydration. Thirty plants were used for each line; values are the mean ±SE (F-test **P <0.01). (This figure is available in colour at JXB online.)

Fig. 6.

Transgenic TaSnRK2.4 plants had significantly higher osmotic potential. Six TaSnRK2.4 transgenic lines, as well as WT and GFP plants, cultured under well-watered conditions, were selected to perform osmotic potential assays as described in the Materials and methods. L1–L6, six individual TaSnRK2.4 transgenic lines; WT, wild type; GFP, GFP transgenic line.

Fig. 7.

TaSnRK2.4 plants have stronger water retention ability. (A) Comparison of water loss rates for detached rosettes between transgenic plants and WT and GFP controls. Values are the mean ±SE (n=10 plants). (B) Comparison of relative water contents of detached rosettes of transformed plants and controls 7 h after treatment. Values are mean ±SE (n=10 plants).

Fig. 8.

Transgenic TaSnRK2.4 Arabidopsis has enhanced drought tolerance. (A) Survival rates of TaSnRK2.4 transformants and controls following severe and moderate drought stress conditions at two developmental stages. Values are the mean ±SE (n=20 plants). (B) Phenotypes of selected TaSnRK2.4 lines and WT and GFP controls, following severe drought stress at the seedling stage. (C) Phenotypes of selected TaSnRK2.4 lines and WT and GFP controls following moderate drought stress at the mature growth stage. (This figure is available in colour at JXB online.)

Fig. 9.

Transgenic TaSnRK2.4 Arabidopsis has enhanced salt tolerance. Comparison of survival rates of TaSnRK2.4 lines and WT and GFP controls treated with 350 mM NaCl. Twenty plants of each line were used in each of three experiments. (This figure is available in colour at JXB online.)

Fig. 10.

Comparison of freezing tolerance for TaSnRK2.4 and control plants. Normally cultured transgenic seedlings at 4 weeks were stressed at –10 °C for 1.5 h. Twenty plants were used in each of three experiments. Survival rates were determined 2 weeks after freezing. (This figure is available in colour at JXB online.)