Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics

Abstract

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

Autophagy and its mediated mitochondrial quality control maintain pollen tube growth and male fertility in Arabidopsis

“…Tobacco pollen germination: fresh and mature tobacco pollens were selected and harvested before use as described previously [ 69 ]. For in vitro pollen tube germination, the pollen grains were suspended in tobacco pollen-specific germination medium containing 10% sucrose (Fluka, 84,097), 0.01% boric acid (Sigma-Aldrich, 202,878), 1 mM CaCl 2 (Ajax Chemicals, 960), 1 mM Ca(NO 4 ) 2 ∙4 H 2 O (Sigma-Aldrich, C1396) and 1 mM MgSO 4 ∙7H 2 O (Sigma-Aldrich, M1880) at pH 6.5.…”

“…Arabidopsis pollen germination: i) in vitro pollen germination and collection of Arabidopsis flowers at the proper growth stage were conducted as previously described [ 69 ]. Arabidopsis pollen grains were released from anthers by vigorous vortex in Arabidopsis pollen germination medium containing 0.01% boric acid, 1 mM Ca(NO 3 ) 2 , 1 mM MgSO 4 , 5 mM CaCl 2 and 18% (w:v) sucrose at pH 7.0.…”

ATG5- and ATG7-mediated autophagy regulates pollen tube guidance and male fertility in Arabidopsis thaliana

“…Pollens were released into the medium from anthers by vigorous agitation with a vortex for 2 min. Subsequent preparation of pollen grains and transient expression of proteins in growing pollen tubes via biolistic bombardment were performed as previously described (Wang and Jiang, 2011; Zhong et al, 2017).…”

“…For in vitro pollen germination: mature pollens at the specific developmental stage were collected as previously described (Mori et al, 2006; Zhong et al, 2017). Mature Arabidopsis pollen grains were transferred from anthers by spreading them on Arabidopsis solid pollen germination medium containing 0.01% boric acid, 1 mM Ca(NO 3 )2, 1 mM MgSO 4 , 5 mM CaCl 2 , 18% (w/v) sucrose and 0.9% agarose at pH 7.0 on an standard glass microscopy slide.…”

“…For in vitro pollen germination: mature pollens at the specific developmental stage were collected as previously described (Mori et al, 2006;Zhong et al, 2017). Mature Arabidopsis pollen grains were transferred from anthers by spreading them on Arabidopsis solid pollen germination medium containing (which was not certified by peer review) is the author/funder.…”