| Maize is the largest crop in the world and in China. Because of high photosynthetic efficiency and high yield potential, there is a trend of further expansion in the planting area and production. However, abiotic stress such as drought is the main limiting factor of maize for its yield-increasing potential. Under drought and other abiotic stress, the activity of the endogenous abscisic acid increases and regulates the expression of downstream resistant genes through signal transduction. This is one of the significant mechanisms for plants to adapt to environmental stress. In recent years, pyrabactin-like protein (PYL), serine/threonine protein phosphatase type 2C (PP2C), and sucrose non-fermenting protein kinase 2 (SnRK2) have been confirmed to be the direct receptor, the second messenger, and the third messenger of ABA signal transduction, one after another. However, both PP2C and SnRK2 are large families. Different members of the families may have different mechanisms to transduce ABA signal in response to environmental stress. Each member catalyzes dephosphorylation or phosphorylation of various downstream proteins, and then regulate various resistant response mechanisms. Therefore, the phosphoproteomic differentiation under drought stress and ABA induction can be used for genome-wide analysis of the downstream substrate phosphoproteins involved in ABA signal transduction in response to abiotic stress, and provides information for study on molecular mechanisms of plant resistance to abiotic stress.In this study, quantitative phophoproteomic analysis based on liquid chromatography tandem-mass spectrometry (LC-MS/MS) was employed to identify the differential phosphoproteins of maize seedlings in response to drought stress and ABA induction, screen probable dephosphorylated or phosphorylated substrates of PP2C and SnRK.2 in the downstream of ABA signaling pathway. The main results are as below:(1) After drought stress and ABA induction,2750 phosphoproteins were identified by high performance liquid chromatography tandem mass spectrometry analysis.517 of them were screened in response to drought stress and ABA induction by label-free quantitative phosphoproteomics, and 96 of them had the same direction of upregulation or downregulation. The 517 had similar subcellular location and functional classification. Thirteen of them were receptor proteins, and eleven of the thirteen were kinase.(2) By STRING software, interaction was found among 42 of the 266 phosphoproteins in response to drought stress. Fructose kinase 2, phosphoglycerate kinase, serine/threonine protein kinase SNT7, salt-induced MAP kinase 1, and Ca2+-dependent protein kinase are located at the intersections of the interaction network, and interact with multiple proteins. Interaction was found among 49 of the 352 phosphoproteins in response to ABA induction, including serine/threonine protein kinase NAK and SNT7, and a member of PP2C family. This PP2C member is located at the intersection of the interaction network, and interacts with six proteins.(3) By Motif-X software, phosphorylated motifs [pSP], [pSD], [pTP], [RXXpS], and [LXRXXpS] were identified from degraded peptides of the 517 phosphoproteins in response to drought stress and ABA induction. Motif [pSP] was found in the substrate of the mitogen activated protein kinase. Motif [pTP] is the phosphorylation site of the substrate of the cell cycle-dependent kinase. Both [RXXpS] and [LXRXXpS] are the phosphorylation sites of SnRK2 substrate proteins. They were found from 18 of the 238, and 23 of the 171 phosphoporteins with upregulated phosphorylation in response to drought and ABA induction. These proteins are likely to be SnRK2 substrates, and the downstream components of ABA signaling in response of maize to abiotic stress. Further verification is needed.In summary, drought stress and ABA induction caused significant changes in phosphorylation of many proteins, including many phosphatases and kinases catalyzing reversible protein phosphorylation, as well as their substrates. Many of them function in ABA signaling of maize in response to abiotic stress. |
