Application of a Sensitive and Reproducible Label-Free Proteomic Approach to Explore the Proteome of Individual Meiotic-Phase Barley Anthers

Abstract

Meiosis is a highly dynamic and precisely regulated process of cell division, leading to the production of haploid gametes from one diploid parental cell. In the crop plant barley (Hordeum vulgare), male meiosis occurs in anthers, in specialized cells called meiocytes. Barley meiotic tissue is scarce and not easily accessible, making meiosis study a challenging task. We describe here a new micro-proteomics workflow that allows sensitive and reproducible genome-wide label-free proteomic analysis of individual staged barley anthers. This micro-proteomic approach detects more than 4,000 proteins from such small amounts of material as two individual anthers, covering a dynamic range of protein relative abundance levels across five orders of magnitude. We applied our micro-proteomics workflow to investigate the proteome of the developing barley anther containing pollen mother cells in the early stages of meiosis and we successfully identified 57 known and putative meiosis-related proteins. Meiotic proteins identified in our study were found to be key players of many steps and processes in early prophase such as: chromosome condensation, synapsis, DNA double-strand breaks or crossover formation. Considering the small amount of starting material, this work demonstrates an important technological advance in plant proteomics and can be applied for proteomic examination of many size-limited plant specimens. Moreover, it is the first insight into the proteome of individual barley anther at early meiosis. The proteomic data have been deposited to the ProteomeXchange with the accession number PXD010887.

Keywords: anthers; label-free micro-proteomics; mass spectrometry LC-MS/MS; meiocytes; meiosis; plants.

FIGURE 1

Barley anthers and meiocytes. (A) Mature barley anthers at late meiosis stage. (B) Anther’s Cross-section showing 4 pollen sacs (asterisk). (C) Meiocytes at zygotene stage within pollen sac after an anther squash.

FIGURE 2

Pearson correlations (R²) among biological triplicates (1, 2, and 3) and among samples prepared from 1, 2, or 5 anthers (B1, B2, and B5).

FIGURE 3

Summary of micro-proteomics results. (A) Venn diagram comparing proteins identified from three biological replicates of two pooled barley anthers. (B) Distribution of the protein intensities in the sample of two pooled anthers.

FIGURE 4

Subcellular localization of paired barley anther proteins predicted by GO annotation (Singular Enrichment Analysis, SEA).

FIGURE 5

Enriched molecular functions of paired barley anther proteins predicted by GO annotation (Singular Enrichment Analysis, SEA).

FIGURE 6

Interaction network analysis of 57 meiosis-associated proteins identified in the barley anther proteome.