Identification of proteins using iTRAQ and VIGS revealed that three proteins were involved into cold response in bread wheat

iTRAQ-based quantitative proteomic and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were applied to identify protein spots involved in cold responses in wheat. By comparing the differentially accumulated proteins from the derivatives (UC1110 x PI 610750), as well as the F10 recombinant inbred line (RIL) population differing in cold-tolerance, a total of 223 proteins with significantly altered abundance were identified, respectively. These proteins were classified into several main groups, as follows: protein metabolism, stress/defense, carbohydrate metabolism, lipid metabolism, RNA metabolism, energy production, cell-wall and cytoskeleton metabolism, membrane and transportation, signal transduction, other metabolic processes, and unknown biological processes. The 22 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 10 genes were consistent with their protein expression models. Furthermore, quantitative real-time PCR (qRT-PCR) results implied that abiotic stresses increased the expression of four candidate protein genes Heat shock protein 90 (TaHsp90.2), Bowman-Birk type protease inhibitor (Bowman), REP14, and VER2 in wheat leaves. VIGS (virus-induced gene silencing)-treated plants generated for TaHsp90.2, Bowman, and REP14 were subjected to cold limitation, and had more serious droop and wilt, increased rate of relative electrolyte leakage and reduced relative water content (RWC) when compared to viral control plants. These results indicated that TaHsp90.2, Bowman, and REP14 possibly played the important roles in conferring cold tolerance in wheat. This study could provide useful information for dissection of molecular and genetics basis of cold stress in bread wheat.