Proteomic analysis of ubiquitinated proteins in 'Xiangshui' lemon [Citrus limon (L.)] pistils after self- and cross-pollination

Abstract

'Xiangshui' lemon exhibits gametophyte self-incompatibility: after self-pollination, the growth of a pollen tube in the style is inhibited. The aim of this work was to study the effect of pollination on the ubiquitylome and proteome in 'Xiangshui' lemon pistil. Here, by using label-free quantification, enrichment by anti-diglycine lysine antibody conjugated agarose beads, high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) and the method of shotgun proteomics, we investigated the quantitative proteome, ubiquitylome, and crosstalk between the two datasets in the 'Xiangshui' lemon pistil after pollination. In total, 4682 protein species, 1956 ubiquitinated protein species and 6922 ubiquitination sites were quantified. A principal component analysis revealed that there were some differences in the ubiquitylome and proteome of CK, self-pollination and cross-pollination. The results of the crosstalk showed that the proteome and ubiquitylome were negatively correlated. After self-pollination, the abundance of the ubiquitinated protein species related to programmed cell death, cell aging and sphingolipid metabolic pathways was changed. Furthermore, we analyzed the proteasome pathway in the pistil after self-pollination and further discussed the mechanism by which ubiquitination events affect S-RNase in the style. Overall, pollination greatly changed the ubiquitinated protein species abundance in the pistils of 'Xiangshui' lemon. Accordingly, our work provides a systematic analysis of the proteome and ubiquitylome in 'Xiangshui' lemon pistils under self-incompatibility and sheds light for future studies on the function and mechanism of ubiquitination during self-incompatibility in 'Xiangshui' lemon. SIGNIFICANCE: As a mechanism to prevent self-pollination, self-incompatibility (SI) exists widely in plant species. Although a large number of SI-related genes have been found in fruit trees, there are few studies on the PTMs that affect and are involved in fruit tree responses to SI. This study highlights the fact that the effects of pollination on proteome and ubiquitylome in the 'Xiangshui' lemon pistil, we discussed the correlation between transcriptome and proteome, ubiquitylome and proteome, and we analyzed the expression and the changes of ubiquitination levels of SI related proteins and pathway after self- and cross pollination, and the changes of ubiquitination level of 26S proteasome pathway after cross-pollination. This study provides new insights into the ubiquitination pathway of SI in 'Xiangshui' lemon.

Keywords: Citrus; Lysine ubiquitination; Posttranslational modification; Proteomics; Self-incompatibility; Ubiquitinated protein.