Proteomics Identification of Differentially Expressed Proteins Associated with Pollen Germination and Tube Growth Reveals Characteristics of Germinated Oryza sativa Pollen*□S

Mature pollen from most plant species is metabolically quiescent; however, after pollination, it germinates quickly and gives rise to a pollen tube to transport sperms into the embryo sac. Because methods for collecting a large amount of in vitro germinated pollen grains for transcriptomics and proteomics studies from model plants of Arabidopsis and rice are not available, molecular information about the germination developmental process is lacking. Here we describe a method for obtaining a large quantity of in vitro germinating rice pollen for proteomics study. Two-dimensional electrophoresis of 2300 protein spots revealed 186 that were differentially expressed in mature and germinated pollen. Most showed a changed level of expression, and only 66 appeared to be specific to developmental stages. Furthermore 160 differentially expressed protein spots were identified on mass spectrometry to match 120 diverse protein species. These proteins involve different cellular and metabolic processes with obvious functional skew toward wall metabolism, protein synthesis and degradation, cytoskeleton dynamics, and carbohydrate/energy metabolism. Wall metabolism-related proteins are prominently featured in the differentially expressed proteins and the pollen proteome as compared with rice sporophytic proteomes. Our study also revealed multiple isoforms and differential expression patterns between isoforms of a protein. These results provide novel insights into pollen function specialization. Molecular & Cellular Proteomics 6:207–230, 2007. Pollen of flowering plants, generated in diploid sporophytic plants via meiosis followed by two cycles of mitosis, contains three haploid genomes and is a highly reduced organism. Mature pollen grains from most plant species are metabolically quiescent. However, during pollination, they can quickly germinate and give rise to a polarly growing pollen tube whereby the pollen interacts with pistils and then delivers two sperms into the embryo sac to initiate double fertilization. Besides having biological importance, pollen germination and tube growth have been considered unique developmental processes for studying cell polar establishment, cell differentiation, cell fate determination, and cell-to-cell recognition. Thus, the molecular mechanisms underlining the specific cellular programs have been the focus of investigation over the past 50 years (1). However, until now, only a limited number of genes encoding coat/wall proteins or signal molecules have been shown to be essential for pollen germination, tube growth, and interaction of the tube and stigma (1–7). Recent analyses of mature pollen of Arabidopsis revealed the transcriptome to have reduced complexity and a higher proportion of selectively expressed transcripts than sporophytic tissues (8–10). As well, about one-third of the genes expressed in vegetative tissues are not expressed in the pollen (8). The observation suggests that the transcriptional characteristics involve pollen function specialization. Furthermore these studies determined that pollen transcriptome has a functional skew toward transcripts implicated in cell wall metabolism, signaling, and cytoskeletal dynamics. These results also support the early notion that mature pollen has stored presynthesized mRNAs (11) and present a novel insight into pollen function specialization at the whole genome level. However, the transcriptomic data of Arabidopsis pollen showed that transcripts related to translation and glycolysis/ energy metabolism were underrepresented in pollen (8–9). All transcripts encoding putative or known ribosomal proteins seem to be undetectable in the transcriptome (9). Given the ability of pollen to initiate protein synthesis rapidly upon germination and the carbon skeleton/energy needs for active From the ‡Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China, §Graduate School of Chinese Academy of Sciences, Beijing 100049, China, ¶Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China, and Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 101300, China Received, April 19, 2006, and in revised form, October 10, 2006 Published, MCP Papers in Press, November 27, 2006, DOI 10.1074/mcp.M600146-MCP200 Research