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Studies of mitochondrial morphology and DNA amount in the rice egg cell

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Abstract

In plant vegetative cells, mitochondria are usually small and grain-shaped. In contrast, unusually shaped giant mitochondria (large cup-shaped or long stretched-rod-shaped) appear in the egg cells of geranium, maize, Ipomoea nil, and bracken. In this study, to characterize egg cell mitochondria in rice, we used nonenzymatic manual dissection to isolate unfertilized egg cells of rice and observed the egg cell mitochondria and mitochondrial DNA (mtDNA) simultaneously. These observations showed that the mitochondria in the rice egg cell are small and grain-shaped, unlike the mitochondria in geranium, maize, I. nil, and bracken. Double staining of mitochondria by MitoTracker and mtDNA by SYBR Green I showed that mitochondria in the rice egg cell have a large amount of mtDNA compared with the rice root protoplast. We also used real-time PCR analysis to quantify the mtDNA amount in the rice egg cell. We quantified the copy numbers of four mitochondrial genes per single rice egg cell and rice leaf protoplast. Real-time PCR analysis revealed that the egg cell has more than ten times more copy numbers of all of four genes encoded in the mitochondrial genome compared with the leaf protoplast.

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Acknowledgments

This research was supported by Grants-in-Aid for Scientific Research on Priority Area (Grant 18075005) and Scientific Research (A) (Grant 18208002) to N. T. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Authors and Affiliations

  1. Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Hideki Takanashi, Takayuki Ohnishi, Mirai Mogi, Shin-ichi Arimura & Nobuhiro Tsutsumi

  2. Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-osawa, Hachioji, 192-0397, Tokyo, Japan

    Takashi Okamoto

Authors
  1. Hideki Takanashi
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  2. Takayuki Ohnishi
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  3. Mirai Mogi
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  4. Takashi Okamoto
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  5. Shin-ichi Arimura
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  6. Nobuhiro Tsutsumi
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Corresponding author

Correspondence to Nobuhiro Tsutsumi.

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Communicated by R. Bock.

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Supplemental Figure 1 Burst tests of the egg cell and leaf protoplast and their mitochondria.

Images show the rice egg cell (a) and the rice leaf protoplast (b) stained with MitoTracker Green FM (Invitrogen, 500 nM, 20 min) after being transferred from 370 mM mannitol into distilled water (times are given in min:sec). The inserted squares in a at 00:30 and b at 03:00 show high magnification images. The cell membranes burst within 30 sec and signals of MitoTracker Green FM disappeared within 3 min in both the egg cell and the leaf protoplast. Inserted images in a at 00:30 show ballooning mitochondria. These results indicate that the cells and their mitochondria had been ruptured within 3 min by placing the cells into distilled water. Bars in a = 40 μm and in b = 20 μm. (PSD 15,485 kb)

Supplemental Figure 2 Schematic of real-time PCR analysis and result of a control experiment. Figure 2a

shows how we prepared the lysates for real-time PCR. We used Y μl fresh mannitol because X < Y. b shows an experimental design to check whether cell extracts affected the PCR amplification efficiency. The premix contained approximately 3 pg lambda DNA (λ DNA; Nippon Gene, Japan) and primers for λ DNA (forward, 5′-CATCAAACGCACGGGTAATG-3′; reverse, 5′-CAGAACTGGCAGACGACATG-3′): 19 μl each amplification. The PCR reactions were performed in a total volume of 20 μl, and 1 μl of lysate was used as the template for Fig. 2a. A dilution series of λ DNA was used as the standard. Real-time PCR was performed for three sets of independent lysates. Figure 2c shows the results of control analysis of the data for 2b. The results were normalized to the copy number of “Water”, set to 1 (bars indicate standard error, n = 3). There was no significant difference in relative copy numbers of λ DNA between samples. Thus the extracts of both egg cells and leaf protoplasts had no inhibitor (or accelerator) of PCR amplification. (PSD 3,869 kb)

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Takanashi, H., Ohnishi, T., Mogi, M. et al. Studies of mitochondrial morphology and DNA amount in the rice egg cell. Curr Genet 56, 33–41 (2010). https://doi.org/10.1007/s00294-009-0277-3

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  • DOI: https://doi.org/10.1007/s00294-009-0277-3

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