Comparative proteomic analysis of generative and sperm cells reveals molecular characteristics associated with sperm...

In flowering plants, two sperm cells (SCs) are generated from a generative cell (GC) in the developing pollen grain or growing pollen tube and are then delivered to the embryo sac to initiate double fertilization. SC development and function specialization involve the strict control of the protein (gene) expression program and coordination of diverse cellular processes. However, because methods for collecting a large amount of highly purified GCs and SCs for proteomic and transcriptomic studies from a plant are not available, molecular information about the program and the interconnections is lacking. Here, we describe a method for obtaining a large quantity of highly purified GCs and SCs from just-germinated lily pollen grains and growing pollen tubes for proteomic analysis. Our observation showed that SCs had less condensed chromatin and more vacuole-like structures than GCs and that mature SCs were arrested at the G2 phase. Comparison of SC and GC proteomes revealed 101 proteins differentially expressed in the two proteomes. These proteins are involved in diverse cellular and metabolic processes, with preferential involvement in metabolism, the cell cycle, signaling, the ubiquitin/proteasome pathway, and chromatin remodeling. Impressively, almost all proteins in SCF complex-mediated proteolysis and the cell cycle were up-regulated in SCs, whereas those in chromatin remodeling and stress response were down-regulated. Our data also reveal the coordination of SCF complex-mediated proteolysis, cell cycle progression, and DNA repair in SC development and function specialization. This study revealed for the first time a difference in protein profiles between GCs and SCs.