Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes

Abstract

The pollen tube is an excellent single-cell model system for studying cellular processes in plant cell biology. This protocol describes a detailed step-by-step procedure with optimized conditions for introducing various fluorescent reporter proteins into lily, tobacco and Arabidopsis pollen grains by means of biolistics for their transient expression and subsequent analysis in germinating pollen tubes. The whole experiment consists of four major stages: coating gold microcarriers with DNA constructs, preparation of pollen grains, transformation of plasmid DNA into pollen grains by particle delivery system and germination of bombarded pollen grains in optimized germination media to obtain pollen tubes for protein trafficking, protein localization, drug treatment and organelle dynamics analysis. This protocol takes about 4-12 h from pollen preparation to protein detection.

Tomato Pistil Factor STIG1 Promotes in Vivo Pollen Tube Growth by Binding to Phosphatidylinositol 3-Phosphate and the Extracellular Domain of the Pollen Receptor Kinase LePRK2

“…Transient expression of fluorescent reporter proteins in fastgrowing pollen tubes by microprojectile bombardment (Twell et al, 1989) is a convenient and effective way to study protein localization (Cheung and Wu, 2007;Wang and Jiang, 2011). When transiently expressed in pollen tubes, STIG1-mRFP localized to numerous vesicular structures (Supplemental Figure 6), resembling the localization of PI(3)P in pollen tubes (Vermeer et al, 2006;Zhang et al, 2011).…”

Rice Morphology Determinant-Mediated Actin Filament Organization Contributes to Pollen Tube Growth

“…Mature tobacco pollen grains were used for transient expression with a particle bombardment procedure as described previously (Fu et al, 2001;Wang and Jiang, 2011). Briefly, tobacco pollen grains were collected immediately before each experiment and suspended in a pollen germination medium containing 0.01% (w/v) boric acid, 1 mM CaCl 2 , 1 mM Ca(NO 3 ) 2 $4H 2 O, 1 mM MgSO 4 $7H 2 O, and 10% (w/v) Suc (pH 6.5).…”

A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation

“…They were vortexed vigorously to release pollen grains into the germination medium. Pollen grain preparation and subsequent transient expression of proteins in the growing pollen tube via particle bombardment were carried out as previously described (Wang and Jiang, 2011a). For BY2 cells, in order to increase the expression efficiency in dividing BY2 cells, cell synchronization was first performed as previously described (Kumagai-Sano et al, 2006).…”