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.

Transcriptomics analyses reveal the molecular roadmap and long non‐coding RNA landscape of sperm cell lineage development

“…Cells were washed three times in PBS and collected by centrifugation at 500 g for 2 min. For hybridization with GCs and SCs, germinated pollen grains and pollen tubes were harvested as described (Lu et al ., ) and incubated in a 15.3% sucrose solution for 10 min at 28°C to release GCs and SCs, which were filtered through an 11‐μm nylon mesh and collected by centrifugation at 850 g for 4 min. Cell pellets were suspended in ice cold fixation buffer [10 m m 2‐( N ‐ morpholine)‐ethanesulfonic acid (MES)‐KOH, 10 m m NaCl, 18% sucrose, 3.7% formaldehyde, pH 6.0] and incubated for 2 h at 4°C.…”

“…Mature tomato pollen is bicellular at anthesis, which enables isolation of GCs from mature pollen and SCs from cultured pollen tubes where SCs are generated. We have reported efficient methods for isolating GCs and SCs in tomato (Lu et al, 2015). Here, we prepared high-purity SC lineage cells for transcriptomic study (Figure 1a).…”

“…The MPGs were collected from plants grown in a greenhouse. Generative cells were isolated from just-germinated pollen cultured in vitro for 20 min and SCs from 10-h-cultured pollen tubes (Lu et al, 2015). For microspore isolation, 7-mm long flowers were excised and 5-mm long anthers were squashed in icecold 0.6 M mannitol solution.…”

Bioinformatics resources for pollen

“…Indeed, gene expression studies by RNA-seq permitted to identify around 150 different pollen-expressed genes in different plant species associated with a range of different functions, including cell wall metabolism, cytoskeleton and signaling (Honys and Twell 2003;Palanivelu and Tsukamoto 2012;Rutley and Twell 2015). Moreover, RNA-seq also shed light on the composition and expression profile of pollen small RNAs (Bokszczanin et al 2015;Borges et al 2011;GrantDownton et al 2009;Li et al 2013;Lu et al 2015;Slotkin et al 2009;Zhang et al 2009). …”

“…In the case of pollen, the short developmental time resulted in few (Lu et al 2015) and outdated studies (Simel et al 1997) aiming to unravel the molecular biology of pollen. However, in order to partially overcome the lack of resources specifically dedicated to pollen, it may be useful to query the general reference platforms searching for pollen information, at the cost of the specificity of the results.…”