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In Situ Localisation of Superoxide Generated in Leaves of Alocasia macrorrhiza (L.) Shott under Various Stresses

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Abstract

Leaf discs of Alocasia macrorrhiza were treated with various stress factors, including two photo-oxidants, methyl viologen (MV) or riboflavin (RB); three pollutants, sodium bisulphite (NaHSO3), or the heavy metals lead or cadmium; or an osmotic medium, polyethylene glycol 6000. The in situ localisation sites for O2 − generation were identified using specific dye nitro blue tetrazolium as a probe. The level of superoxide production was determined by scanning the blue-stained formazan area and was defined as the percentage of pixels from the stained portion versus the total number of pixels in the entire leaf disc area. All stress factors induced the generation of O2 − in a time- or concentration-dependent pattern. Although superoxide production also was enhanced by longer time periods in untreated discs (control), the degree to which this occurred was less than that measured in leaves treated with either MV or RB. Generation sites were primarily found in the chloroplasts of stomatal guard cells and in the plasma membrane of the epidermis and mesophyll cells, indicating that they were most responsive to stress conditions. Nevertheless, the site of O2 − generation varied among these stress factors.

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Abbreviations

DDC:

diethyldithiocarbamic acid

MV:

methyl viologen

NBT:

nitroblue tetrazolium

O2 − :

superoxide anion

RB:

riboflavin

Tiron:

4,5-dihydroxy-1,3-benzene disulfonic acid

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Acknowledgements

We thank Prof. Fred Chow (Research School of Biological Sciences, the Australian National University) for critical reading of the manuscript and helpful suggestions and Ms. X.L. Xu for preparation of semi-thin leaf sections. This research was financially supported by the National Natural Science Foundation of China (30770173).

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Authors and Affiliations

  1. South China Botanical Garden, Chinese Academy of Sciences, 510650, Guangzhou, China

    Zhi-Fang Lin, Nan Liu & Gui-Zhu Lin

  2. College of Life Science, South China Normal University, 510631, Guangzhou, China

    Chang-Lian Peng

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  1. Zhi-Fang Lin
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  2. Nan Liu
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  3. Gui-Zhu Lin
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  4. Chang-Lian Peng
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Correspondence to Zhi-Fang Lin or Nan Liu.

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Lin, ZF., Liu, N., Lin, GZ. et al. In Situ Localisation of Superoxide Generated in Leaves of Alocasia macrorrhiza (L.) Shott under Various Stresses. J. Plant Biol. 52, 340–347 (2009). https://doi.org/10.1007/s12374-009-9044-8

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