Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function

Abstract

Pollen, the male gametophyte of flowering plants, represents an ideal biological system to study developmental processes, such as cell polarity, tip growth, and morphogenesis. Upon hydration, the metabolically quiescent pollen rapidly switches to an active state, exhibiting extremely fast growth. This rapid switch requires relevant proteins to be stored in the mature pollen, where they have to retain functionality in a desiccated environment. Using a shotgun proteomics approach, we unambiguously identified ∼3500 proteins in Arabidopsis pollen, including 537 proteins that were not identified in genetic or transcriptomic studies. To generate this comprehensive reference data set, which extends the previously reported pollen proteome by a factor of 13, we developed a novel deterministic peptide classification scheme for protein inference. This generally applicable approach considers the gene model–protein sequence–protein accession relationships. It allowed us to classify and eliminate ambiguities inherently associated with any shotgun proteomics data set, to report a conservative list of protein identifications, and to seamlessly integrate data from previous transcriptomics studies. Manual validation of proteins unambiguously identified by a single, information-rich peptide enabled us to significantly reduce the false discovery rate, while keeping valuable identifications of shorter and lower abundant proteins. Bioinformatic analyses revealed a higher stability of pollen proteins compared to those of other tissues and implied a protein family of previously unknown function in vesicle trafficking. Interestingly, the pollen proteome is most similar to that of seeds, indicating physiological similarities between these developmentally distinct tissues.

Disruption of the pollen-expressedFERONIAhomologsANXUR1andANXUR2triggers pollen tube discharge

“…Based on their complementary expression pattern, At3g04690 and At5g28680 were renamed ANXUR1 (ANX1) and ANXUR2 (ANX2), respectively, after the male consort of the goddess Feronia in ancient Italy (Funke, 1801). Consistent with this, ANX1 and ANX2 proteins were both identified in the pollen proteome (Grobei et al, 2009). …”

Inference and Validation of Protein Identifications

“…single hit protein identifications. There has been considerable debate about whether such post-processing enhances protein inference (40,41). Latter approaches might miss protein identifications that are falsely discarded by the a priori grouping scheme or the parsimony constraint.…”

Dynamic Adaption of Metabolic Pathways during Germination and Growth of Lily Pollen Tubes after Inhibition of the Electron Transport Chain

“…The majority of studies including proteomic studies focused on differences between the expression levels of pollen and sporophytic tissue or of different states in pollen development (Becker et al, 2003;Honys and Twell, 2003;Pina et al, 2005;Bock et al, 2006). Only few studies focused on pollen tube growth (Dai et al, 2006;Wang et al, 2008;Grobei et al, 2009;Qin et al, 2009). Although metabolic pathway networks were reconstructed from these data (Boavida et al, 2005;Colaço et al, 2012) using the MAPMAN tool (Thimm et al, 2004), the correlation between the presence of mRNA, the amount of protein, and its specific activity remains uncertain (Gry et al, 2009;Taniguchi et al, 2010).…”