Stigma-Specific Comparative Proteomic Analysis Reveals the Distyly Response to Self-Incompatibility in Plumbago auriculata Lam

In plants, heteromorphic self-incompatibility (HetSI) is a strategy for avoiding self-pollination and promoting outcrossing, and during this process, numerous protein-protein interaction events occur between the pistil and pollen. Previous studies in Primula and Fagopyrum that focused on HetSI systems have provided interesting insights; however, the molecular mechanism underlying HetSI remains largely unknown. In this study, we profiled the proteome of Plumbago auriculata stigmas before and after self-incompatible (SI) and self-compatible (SC) pollination. Comparative analyses were conducted by 4D-DIA (Four-dimensional data independent acquisition), a promising technology that increases the sensitivity and reduces the spectral complexity of proteomic analysis by adding a fourth dimension, ion mobility. The results revealed 33387 peptides and 5311 proteins in all samples. The pathways in which the differentially expressed proteins (DEPs) identified in the P × P (Pin style self-pollinated with pin pollen) vs. PS (Pin style) and T × T (Thrum style self-pollinated with thrum pollen) vs. TS (Thrum style) comparisons were significantly enriched were biosynthesis of secondary metabolites and pentose and glucuronate interconversions. In the P × T (Pin style cross-pollinated with thrum pollen) vs. PS and T × P (Thrum style cross-pollinated with pin pollen) vs. TS comparison, the top three pathways were biosynthesis of secondary metabolites, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis. The phenylpropanoid biosynthesis, cutin, suberine and wax biosynthesis, and flavonoid biosynthesis pathways were enriched in the P × T vs. P × P comparison, and starch and sucrose metabolism, glycerophospholipid metabolism, and alpha-linolenic acid metabolism were abundant in the T × T vs. T × P comparison. The enriched pathways between PS and TS were the biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and pentose and glucuronate interconversion. Self-incompatibility protein S1 (SI S1), Mitogen-activated protein kinase 3/4 (MPK3/4), Mitogen-activated protein kinase kinase 2/3 (M2K2/3), Exocyst complex component EXO70A1 (E70A1) and Thioredoxin H1/2 (TRXH1/2) were found to be HetSI-related candidates, and O-fucosyltransferase 23 (OFT23), 3-ketoacyl-CoA synthase 6 (KCS6), Receptor-like protein kinase FERONIA (FERON), Fimbrin-5 (FIMB5), Pollen-specific leucine-rich repeat extensin-like protein 4 (PLRX4), Transcription initiation factor IIB-2 (TF2B2) and Pectinesterase 1 (AL11A), etc., were identified as other regulatory transducers. These findings combined with our morphological and reactive oxygen species (ROS) intensity analyses indicate that P. auriculata has typical dry-stigmas and that the HetSI mechanism might differ between the pin and thrum. SI S1 might be the key factor in HetSI, and ROS are overexpressed during SC pollination to rapidly activate the mitogen-activated protein kinase (MAPK)-mediated phosphorylation of E70A1 to maintain stigma receptivity in plants with HetSI.