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Manipulation of l-ascorbic acid biosynthesis pathways in Solanum lycopersicum: elevated GDP-mannose pyrophosphorylase activity enhances l-ascorbate levels in red fruit

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Abstract

Ascorbate (AsA) plays a fundamental role in redox homeostasis in plants and animals, primarily by scavenging reactive oxygen species. Three genes, representing diverse steps putatively involved in plant AsA biosynthesis pathways, were cloned and independently expressed in Solanum lycopersicum (tomato) under the control of the CaMV 35S promoter. Yeast-derived GDP-mannose pyrophosphorylase (GMPase) and arabinono-1,4-lactone oxidase (ALO), as well as myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana, were targeted. Increases in GMPase activity were concomitant with increased AsA levels of up to 70% in leaves, 50% in green fruit, and 35% in red fruit. Expression of ALO significantly pulled biosynthetic flux towards AsA in leaves and green fruit by up to 54 and 25%, respectively. Changes in AsA content in plants transcribing the MIOX2 gene were inconsistent in different tissue. On the other hand, MIOX activity was strongly correlated with cell wall uronic acid levels, suggesting that MIOX may be a useful tool for the manipulation of cell wall composition. In conclusion, the Smirnoff–Wheeler pathway showed great promise as a target for biotechnological manipulation of ascorbate levels in tomato.

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Abbreviations

GMPase:

Guanidine-diphosphate mannose pyrophosphorylase

ALO:

Arabinono-1,4-lactone oxidase

MIOX:

Myo-inositol oxygenase

MI:

Myo-inositol

L-GulL:

l-Gulono-1,4-lactone

GlucA:

d-Glucuronic acid

DHA:

Dehydroascorbate

L-Asc:

l-Ascorbate

AsA:

Total ascorbate

GalUR:

Galacturonic acid reductase

L-GalLDH:

l-Galactono-1,4-lactone dehydrogenase

GME:

GDP-d-mannose 3,5-epimerase

O/N:

Over night

GDP:

Guanidine-diphosphate

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Acknowledgments

Technical support from Ilse Balbo and scientific discussions with Prof. Adriano Nunes-Nesi (Max Planck Institute for Molecular Plant Physiology, Golm, Germany) are much appreciated. Dr Bénédicte A Lebouteiller (Institute for Plant Biotechnology; Stellenbosch University; South Africa) is thanked for her assistance as is funding from the National Research Foundation; South Africa.

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  1. Rolene Bauer

    Present address: Department of Biotechnology, Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, Private Bag X17, Cape Town, 7535, South Africa

Authors and Affiliations

  1. Genetics Department, Institute for Plant Biotechnology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

    Christelle Cronje, Gavin M. George, Jan Bekker & Jens Kossmann

  2. Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany

    Alisdair R. Fernie

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  1. Christelle Cronje
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Cronje, C., George, G.M., Fernie, A.R. et al. Manipulation of l-ascorbic acid biosynthesis pathways in Solanum lycopersicum: elevated GDP-mannose pyrophosphorylase activity enhances l-ascorbate levels in red fruit. Planta 235, 553–564 (2012). https://doi.org/10.1007/s00425-011-1525-6

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