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Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for "omics" analysis.

ABSTRACT: The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput "omics" approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs), from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative cell nuclei (VN) from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants.

SUBMITTER: Lu Y

PROVIDER: S-EPMC4451641 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for "omics" analysis.

Lu Yunlong Y Wei Liqin L Wang Tai T

Frontiers in plant science 20150602

The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput "omics" approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cel ...[more]

PMID: 26082789

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Project description:In plants, the cell fates of a vegetative cell (VC) and generative cell (GC) are determined after the asymmetric division of the haploid microspore. The VC exits the cell cycle and grows a pollen tube, while the GC undergoes further mitosis to produce two sperm cells for double fertilization. However, our understanding of the mechanisms underlying their fate differentiation remains limited. One major advantage of the nuclear proteome analysis is that it is the only method currently able to uncover the systemic differences between VC and GC due to GC being engulfed within the cytoplasm of VC, limiting the use of transcriptome. Here, we obtained pure preparations of the vegetative cell nuclei (VNs) and generative cell nuclei (GNs) from germinating lily pollens. Utilizing these high-purity VNs and GNs, we compared the differential nucleoproteins between them using state-of-the-art quantitative proteomic techniques. We identified 720 different amount proteins (DAPs) and grouped the results in 11 fate differentiation categories. Among them, we identified 29 transcription factors (TFs) and 10 cell fate determinants. Significant differences were found in the molecular activities of vegetative and reproductive nuclei. The TFs in VN mainly participate in pollen tube development. In comparison, the TFs in GN are mainly involved in cell differentiation and male gametogenesis. The identified novel TFs may play an important role in cell fate differentiation. Our data also indicate differences in nuclear pore complexes and epigenetic modifications: more nucleoporins synthesized in VN; more histone variants and chaperones; and structural maintenance of chromosome (SMC) proteins, chromatin remodelers, and DNA methylation-related proteins expressed in GN. The VC has active macromolecular metabolism and mRNA processing, while GC has active nucleic acid metabolism and translation. Moreover, the members of unfolded protein response (UPR) and programmed cell death accumulate in VN, and DNA damage repair is active in GN. Differences in the stress response of DAPs in VN vs. GN were also found. This study provides a further understanding of pollen cell differentiation mechanisms and also a sound basis for future studies of the molecular mechanisms behind cell fate differentiation.

Dataset Information

Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for "omics" analysis.

Publications

Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for "omics" analysis.

Similar Datasets

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