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Plant and Cell Physiology Advance Access originally published online on June 3, 2008
Plant and Cell Physiology 2008 49(7):1074-1083; doi:10.1093/pcp/pcn084
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Mitochondrial Dynamics in Plant Male Gametophyte Visualized by Fluorescent Live Imaging

Ryo Matsushima1, Yuki Hamamura2,3, Tetsuya Higashiyama3, Shin-ichi Arimura4, Sodmergen5, Nobuhiro Tsutsumi4 and Wataru Sakamoto1,*

1 Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan
2 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo, 113-0033 Japan
3 Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
4 Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan
5 College of Life Sciences, Peking University, Beijing 100871, PR China

*Corresponding author: E-mail, saka{at}rib.okayama-u.ac.jp; Fax, +81-86-434-1206.


  Abstract

Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F1 plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.

Keywords: Arabidopsis thaliana - Fertilization - Fluorescent protein - Mitochondria - Pollen grain - Sperm cell

Abbreviations: CaMV, cauliflower mosaic virus; DAPI, 4',6-diamidino-2-phenylindole; GC, generative cell; GFP, green fluorescent protein; mtDNA, mitochondrial DNA; PMI, pollen mitosis I; PMII, pollen mitosis II; RFP, red fluorescent protein; SC, sperm cell; VC, vegetative cell.

(Received April 13, 2008; Accepted May 29, 2008)
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