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Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase

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Abstract

l-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative l-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco (Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content.

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Abbreviations

AAO :

Ascorbate oxidase

APX :

Ascorbate peroxidase

AsA :

l-Ascorbic acid

BY-2 :

Bright Yellow 2

cAPX :

Cytosolic ascorbate peroxidase

DHAR :

Dehydroascorbate reductase

DIG :

Digoxigenin

FDA :

Fluorescein diacetate

GalL :

l-Galactono-1,4-lactone

GalLDH :

l-Galactono-1,4-lactone dehydrogenase

GMEase :

GDP-d-Mannose-3,5-epimerase

GMPase :

GDP-d-Mannose pyrophosphorylase

GR :

Glutathione reductase

sAPX :

Stromal ascorbate peroxidase

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Acknowledgments

We thank Prof. K. Nakamura (Nagoya University, Japan) and Dr. K. Matsuoka (Plant Science Center, RIKEN, Japan) for the generous gift of pMAT037. We are also thankful to Prof. Oba (Nagoya Women’s University, Japan) for the generous gift of GalLDH-specific antibody. Mr. A.A. Badejo (Hiroshima University, Japan) is thanked for critically reading and correcting this paper.

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Correspondence to Muneharu Esaka.

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Tokunaga, T., Miyahara, K., Tabata, K. et al. Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase. Planta 220, 854–863 (2005). https://doi.org/10.1007/s00425-004-1406-3

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