Comparative proteomics illustrates the complexity of drought resistance mechanisms in two wheat (Triticum aestivum L.) cultivars under dehydration and rehydration

Abstract

Background: Drought stress is one of the most adverse environmental constraints to plant growth and productivity. Comparative proteomics of drought-tolerant and sensitive wheat genotypes is a strategy to understand the complexity of molecular mechanism of wheat in response to drought. This study attempted to extend findings regarding the potential proteomic dynamics in wheat under drought stress and to enrich the research content of drought tolerance mechanism.

Results: A comparative proteomics approach was applied to analyze proteome change of Xihan No. 2 (a drought-tolerant cultivar) and Longchun 23 (a drought-sensitive cultivar) subjected to a range of dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h) using 2-DE, respectively. Quantitative image analysis showed a total of 172 protein spots in Xihan No. 2 and 215 spots from Longchun 23 with their abundance significantly altered (p < 0.05) more than 2.5-fold. Out of these spots, a total of 84 and 64 differentially abundant proteins were identified by MALDI-TOF/TOF MS in Xihan No. 2 and Longchun 23, respectively. Most of these identified proteins were involved in metabolism, photosynthesis, defence and protein translation/processing/degradation in both two cultivars. In addition, the proteins involved in redox homeostasis, energy, transcription, cellular structure, signalling and transport were also identified. Furthermore, the comparative analysis of drought-responsive proteome allowed for the general elucidation of the major mechanisms associated with differential responses to drought of both two cultivars. These cellular processes work more cooperatively to re-establish homeostasis in Xihan No. 2 than Longchun 23. The resistance mechanisms of Xihan No. 2 mainly included changes in the metabolism of carbohydrates and amino acids as well as in the activation of more antioxidation and defense systems and in the levels of proteins involved in ATP synthesis and protein degradation/refolding.

Conclusions: This study revealed that the levels of a number of proteins involved in various cellular processes were affected by drought stress in two wheat cultivars with different drought tolerance. The results showed that there exist specific responses to drought in Xihan No. 2 and Longchun 23. The proposed hypothetical model would explain the interaction of these identified proteins that are associated with drought-responses in two cultivars, and help in developing strategies to improve drought tolerance in wheat.

Keywords: 2-DE; Differentially abundant proteins; Drought stress; MALDI-TOF/TOF; Proteomics; Wheat.

Fig. 1

The drought-induced morphological responses in wheat seedlings. The wheat seeds of Xihan No. 2 and Longchun 23 were sown in glass plates containing expanded perlite in an environmentally controlled growth room with 25 ± 2 °C, 70 % relative humidity and 16 h photoperiod (300 μmol m⁻² · s⁻¹ light intensity). One-week-old seedlings were subjected to progressive drought stress up to 48 h. Then, the glass plates were re-watered for the recovery of dehydrated seedlings. The photographs of two wheat cultivars were taken from 0 h, dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h), respectively

Fig. 2

The drought-induced physiological responses in wheat seedlings. The RWC (a), free proline content (b), MDA content (c), electrolyte leakage (d), chlorophyll content (e), carotenoid content (f), H₂O₂ content (g), SOD activity (h) and CAT activity (i) were measured from control, dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h) of Xihan No. 2 and Longchun 23, respectively. Each value is represented as means ± SE for three independent experiments. Means followed by different small letters are significantly different at p < 0.05 according to Duncan’s multiple range test

Fig. 3

2-DE gel analysis of proteins extracted from leaves of Xihan No. 2 during dehydration and rehydration. Equal amounts (900 μg) of proteins were separated on pH 3–10 IPG strips (17 cm, linear) in the first dimension and by SDS-PAGE on 12 % polyacrylamide gels in the second dimension. The gels were visualized by CBB staining. Three replicate CBB-stained gels for control, dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h) (Additional file 1: Figure S3) were computationally combined using PDQuest v8.0.1 software, respectively. Protein spots indicated with numbers were identified by MALDI-TOF/TOF MS. The identified spots were numbered in accordance with Additional file 6: Table S4. a 2-DE protein profile for control; (b-e) 2-DE protein profile for dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h), respectively

Fig. 4

2-DE gel analysis of proteins extracted from leaves of Longchun 23 during dehydration and rehydration. Equal amounts (900 μg) of proteins were separated on pH 3–10 IPG strips (17 cm, linear) in the first dimension and by SDS-PAGE on 12 % polyacrylamide gels in the second dimension. The gels were visualized by CBB staining. Three replicate CBB-stained gels for control, dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h) (Additional file 2: Figure S4) were computationally combined using PDQuest v8.0.1 software, respectively. Protein spots indicated with numbers were identified by MALDI-TOF/TOF MS. The identified spots were numbered in accordance with Additional file 7: Table S5. a 2-DE protein profile for control; (b-e). 2-DE protein profile for dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h), respectively

Fig. 5

The total number of protein spots detected from the 2-DE gel of Xihan No. 2 and Longchun 23 during dehydration and rehydration

Fig. 6

Venn diagrams of the number of up- (a) and down-regulated (b) proteins in Xihan No. 2 and Longchun 23 under drought stress. Overlapping regions of the circles indicate the number of proteins regulated in either the same manner in both two wheat cultivars, whereas non-overlapping circles indicated proteins regulated in only that cultivar

Fig. 7

Functional classification of the differentially abundant proteins in Xihan No. 2 (a) and Longchun 23 (b) during dehydration and rehydration. The protein function classification was conducted according to the putative functions assigned to each of the candidate proteins using the protein functional database and displayed in the pie chart

Fig. 8

Clustering analysis of the expression profiles of differentially abundant proteins in Xihan No. 2 during dehydration and rehydration. The hierarchical cluster tree is shown at the top, and the expression profiles are shown below. The five rows of hierarchical cluster tree represent control, dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h), respectively. Each individual protein is represented by a single column of colour boxes. The up- and down-regulated proteins are indicated in red and green, respectively. The colours intensity is increased with the expression differences increasing, as shown in the bar. The expression profile of each individual protein in the cluster is depicted by gray lines, while the mean expression profile is marked in pink for each cluster. The number of proteins in each cluster is given in the left upper corner, and the cluster number is given in the right lower corner. Only the clusters with n > 6 were taken to investigate the co-expression patterns for functionally similar proteins. The detailed information on proteins within each cluster is presented in Additional file 8: Figure S1

Fig. 9

Clustering analysis of the expression profiles of differentially abundant proteins in Longchun 23 during dehydration and rehydration. The hierarchical cluster tree is shown at the top, and the expression profiles are shown below. The five rows of hierarchical cluster tree represent control, dehydration treatments (18 h, 24 h and 48 h) and rehydration treatment (R24 h), respectively. Each individual protein is represented by a single column of colour boxes. The up- and down-regulated proteins are indicated in red and green, respectively. The colours intensity is increased with the expression differences increasing, as shown in the bar. The expression profile of each individual protein in the cluster is depicted by gray lines, while the mean expression profile is marked in pink for each cluster. The number of proteins in each cluster is given in the left upper corner, and the cluster number is given in the right lower corner. Only the clusters with n > 6 were taken to investigate the co-expression patterns for functionally similar proteins. The detailed information on proteins within each cluster is presented in Additional file 9: Figure S2

Fig. 10

The representative models for summarizing the functional and regulatory networks activated by drought stress in Xihan No. 2. The identified proteins are displayed on the corresponding metabolic pathways. The colour boxes are representative of expression profiles of individual protein during dehydration and rehydration. The up- and down-regulation of proteins are indicated in red and green, respectively. The colours intensity is increased with the expression differences increasing. The number given on the left side of each colour boxes indicates the protein identification number in accordance with Additional file 6: Table S4

Fig. 11

The representative models for summarizing the functional and regulatory networks activated by drought stress in Longchun 23. The identified proteins are displayed on the corresponding metabolic pathways. The colour boxes are representative of expression profiles of individual protein during dehydration and rehydration. The up- and down-regulation of proteins are indicated in red and green, respectively. The colours intensity is increased with the expression differences increasing. The number given on the left side of each colour boxes indicates the protein identification number in accordance with Additional file 7: Table S5