Abstract
Proteomic approaches were applied to identify protein spots involved in cold responses in wheat. By comparing the differentially accumulated proteins from two cultivars (UC1110 and PI 610750) and their derivatives, as well as the F10 recombinant inbred line population differing in cold-tolerance, a total of 20 common protein spots representing 16 unique proteins were successfully identified using 2-DE method. Of these, 14 spots had significantly enhanced abundance in the cold-sensitive parental cultivar UC1110 and its 20 descendant lines when compared with the cold-tolerant parental cultivar PI 610750 and its 20 descendant lines. Six protein spots with reduced abundance were also detected. The identified protein spots are involved in stress/defense, carbohydrate metabolism, protein metabolism, nitrogen metabolism, energy metabolism, and photosynthesis. The 20 differentially expressed protein spots were chosen for quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression changes at the RNA level. The results indicated that the transcriptional expression patterns of 11 genes were consistent with their protein expression models. Among the three unknown proteins, Spot 20 (PAP6-like) showed high sequence similarities with PAP6. qRT-PCR results implied that cold and salt stresses increased the expression of PAP6-like in wheat leaves. Furthermore, VIGS (virus-induced gene silencing)-treated plants generated for PAP6-like were subjected to freezing stress, these plants had more serious droop and wilt, an increased rate of relative electrolyte leakage, reduced relative water content (RWC) and decreased tocopherol levels when compared with viral control plants. However, the plants that were silenced for the other two unknown proteins had no significant differences in comparison to the BSMV0-inoculated plants under freezing conditions. These results indicate that PAP6-like possibly plays an important role in conferring cold tolerance in wheat.
Highlights
From the ‡Agronomy College/National Key Laboratory of Wheat and Corn Crop/Collaborative Innovation Center of Henan Grain Crops, Henan Agricultural University, Zhengzhou 450002, China
The phenotypes of the cold-tolerant cultivar PI610750 (Fig. 1A), cold-sensitive cultivar UC1110 (Fig. 1B), and partial lines composed of cold-tolerant pools (CTPs; Fig. 1C) and coldsensitive pools (CSPs; Fig. 1D) were shown
According to the differential functions, the 20 identified protein spots were classified into six main groups: stress/defense (5%, 1), carbohydrate metabolism (5%, 1), protein metabolism (10%, 2), nitrogen metabolism (10%, 2), energy production and transportation (15%, 3), photosynthesis (40%, 8), and unknown function (15%, 3) (Fig. 3)
Summary
From the ‡Agronomy College/National Key Laboratory of Wheat and Corn Crop/Collaborative Innovation Center of Henan Grain Crops, Henan Agricultural University, Zhengzhou 450002, China. The comparison of differentially abundant proteins during cold treatment is mostly performed by conventional two-dimensional electrophoresis (2-DE) or two-dimensional differential gel electrophoresis (2D-DIGE) via MS analysis, as seen in specific tissues or organelles: for example, Arabidopsis cold-and salt-tolerant relative of Thellungiella halophila rosette leaves [15]; pea roots, stems and leaves [23]; rice seedlings [24]; rice leaves [25]; rice roots [18]; and wheat leaves [4, 22] These studies have observed that changes in protein abundance during cold treatment were related to abiotic stress responses [26], (e.g. pathogenesis-related protein, cold-regulated protein, coldresponsive LEA/RAB-related COR protein, oxygen-evolving enhancer protein, and oxalate oxidase) [4]. Better understanding of the cold-tolerance mechanisms in bread wheat using these candidate winter-responsive proteins would be beneficial and further studies could be conducted to maximize the potential applications in crop breeding
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