Abundant protein phosphorylation potentially regulates Arabidopsis anther development

Abstract

As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development.

Keywords: Anther development; Arabidopsis; mass spectrometry; phosphoproteomics.

Fig. 1.

An overview of the proteins and phosphoproteins identified in the proteome and phosphoproteome of developing anthers. (A) Overlap of the identified proteins from proteomic and phosphoproteomic analyses of two phases. (B) Overlap of the phosphorylation sites identified from two phases. (C) Overlap of the phosphoproteins identified from two phases. (D) Distribution of the identified phosphorylation sites per protein.

Fig. 2.

Enrichment analysis of low-abundance and regulatory proteins in the proteome and phosphoproteome of anthers in phases I and II. (A) Average abundance of proteins identified in three datasets (proteins identified only in the phosphoproteomic analysis, proteins identified in both analyses and proteins identified only in the proteomic analysis) in phases I and II. (B) Distribution of TFs and kinases/phosphatases in three datasets, the phosphoproteome only, both the proteome and phosphoproteome and the proteome only. (C) Over/under-represented gene ontology (GO) molecular function categories in the proteome and phosphoproteome of two phases. Regulatory proteins are dominantly phosphorylated, especially in anther phase I.

Fig. 3.

Characteristics of proteins identified in the phosphoproteome of anther phases I and II. (A) Mapman analysis of the phosphoproteins identified in anther phases I and II. Only the significantly over/under-represented bins (primary Mapman bins with P<1.0×10^–2) are included. (B) Mapman analysis of the phosphoproteins only identified in phase I. (C) Mapman analysis of the phosphoproteins identified in both phases. (D) Mapman analysis of the phosphoproteins only identified in phase II. Only the bins (secondary Mapman bins) with P<1.0×10^–2 are included.

Fig. 4.

Analysis of identified phosphorylation sites. (A) Positional distribution of the identified phosphorylation sites in protein sequences. In this analysis, each protein sequence was evenly divided into 100 fractions and every five fractions were set as one unit, and then the number of phosphorylation sites within each unit was calculated. Phosphorylation preferentially occurred in the protein terminus (N-terminus or C-terminus). (B) Comparison of the phosphorylation sites located inside and outside the region of the functional domains (predicted by Pfam) of the phosphoproteins. Phosphorylation preferentially occurred in the unconserved linker regions. (C) Conservative analysis of phophorylation sites across eudicots and angiosperm.

Fig. 5.

An overview of the TFs and kinases/phosphatases identified in proteome and phosphoproteome of developing anthers at phase I and II. (A) The over-represented TF families in the proteome and phosphoproteome of anther phases I and II, compared with the whole Arabidopsis genome (value >1.33 indicates P<0.05). The results showed that the phosphorylation patterns differ for TF families in different phases. (B) The enriched kinase or phosphatase families in proteome and phosphoproteome of anther phase I and II, compared with the whole Arabidopsis genome (value >1.33 indicates P<0.05).

Fig. 6.

An overview of the 493 novel phosphoproteins from our developing anther phosphoproteome. (A) Overlap of the identified phosphoproteins in our developing anther phosphoproteome with phosphoproteins in the PhosPhAt database, the P³DB database and the mature pollen phosphoproteome (Mayank et al., 2012). The 493 phosphoproteins were absent from pervious results. (B) Relative expression values across different tissues (data were retrieved from Ma et al., 2012) (anther, inflorescent, stem, leaves and root) for 493 gene encoding phosphoproteins that are absent from the PhosPhAt database, the P³DB database and the mature pollen phosphoproteome. The 493 novel phosphoproteins are largely anther-enriched.

Fig. 7.

The kinase–substrate network extracted from the phosphoproteome of anther phase I. The results are color-coded to indicate the kinase and its substrate where the putative substrates of MAPK6 were found to be over-represented. Triangular arrowhead represents phosphorylation regulation, T-arrowhead indicates dephosphorylation regulation, and circular arrowhead indicates autophosphorylation of kinase.