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Ascorbic acid toxicity is related to oxidative stress and enhanced by high light and knockdown of chloroplast ascorbate peroxidases in rice plants

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Abstract

Toxicity caused by high concentrations of ascorbic acid (AA) has been widely reported in animal cells but is scarcely described in plants. In this study, rice plants deficient (knockdown) in two chloroplast ascorbate peroxidases (APX7/8) and non-transformed (NT) were exposed to wide exogenous AA concentrations in the presence of low light and high light (HL). Reduced (ASC) and oxidized (DHA) ascorbate reached much higher concentrations in symplast compared to the apoplastic space, and high redox states were found in both cellular compartments. Exogenous AA concentrations above 30 mM caused strong cellular and oxidative damage indicated by decreased cell integrity and increased lipid peroxidation in leaves. These toxic effects were strongly enhanced by HL and, to a small extent, by deficiency of both chloroplastic proteins APX7/8. The combination of HL and high AA concentration induced a strong increase in H2O2, associated with decrease in the content of chlorophylls and carotenoids. High AA concentrations strongly induced stomatal closure and impairment in CO2 assimilation, in combination with decreased quantum efficiency of photosystem II (PSII) and PSI. We postulate that oxidative stress caused by AA toxicity in the presence of HL was induced by over-production of reactive oxygen species due to an imbalance between excess energy in the photosystems and low CO2 assimilation, which was related closely to strong decrease in stomatal conductance. In addition, high ASC levels might have acted as a pro-oxidant in the presence of high H2O2 concentrations, stimulating the Fenton reaction and contributing to the intensification of oxidative stress in rice leaves.

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Abbreviations

APX7/8:

Knockdown plants in both chloroplast ascorbate peroxidases APX7 and APX8

NT:

Non-transformed plants

HL:

High light

LL:

Low light

ASC:

Reduced ascorbate

MDHA:

Monodehydroascorbate

DHA:

Dehydroascorbate

ROS:

Reactive oxygen species

O −•2 :

Superoxide

PPFD:

Photosynthetic photons flux density

PSII:

Photosystem II

PSI:

Photosystem I

Fv/Fm:

Indicator of maximum quantum yield of PSII

ΦPSII:

Effective quantum yield of PSII

TBARS:

Thiobarbituric acid-reactive substances

MD:

Membrane damage

FW:

Fresh weight

DTT:

Dithiothreitol

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Acknowledgements

The authors acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq): Proc. 460214/2014-4 for financial support.

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Authors and Affiliations

  1. Laboratório de Metabolismo de Plantas, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Av. Humberto Monte 2825, Campus do Pici, Bl 907., CP 6020, Fortaleza, CE, CEP 60451-970, Brazil

    Jamyla L. S. Castro, Yugo Lima-Melo, Fabricio E. L. Carvalho, Antonio G. S. Feitosa & Joaquim A. G. Silveira

  2. Molecular Plant Biology, Department of Biochemistry, University of Turku, Turku, Finland

    Yugo Lima-Melo

  3. State University of São Paulo, UNESP-CLP, Experimental Campus of São Paulo Coast, São Vicente, SP, Brazil

    Milton C. Lima Neto

  4. Department of Genetics, Federal University of Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brazil

    Andréia Caverzan & Marcia Margis-Pinheiro

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  1. Jamyla L. S. Castro
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  2. Yugo Lima-Melo
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  3. Fabricio E. L. Carvalho
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  7. Marcia Margis-Pinheiro
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  8. Joaquim A. G. Silveira
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Contributions

JLSC performed all the experiments and contributed to manuscript writing; YLM performed gas-exchange measurements and contributed to manuscript writing; FELC contributed to data interpretation and manuscript writing; AGSF contributed to biochemical analysis; MCLN performed photochemical measurements; AC obtained the silenced rice plants; MM-P designed and obtained the plant mutants; JAGS was the research mastermind and contributed to manuscript writing.

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Correspondence to Joaquim A. G. Silveira.

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Castro, J.L.S., Lima-Melo, Y., Carvalho, F.E.L. et al. Ascorbic acid toxicity is related to oxidative stress and enhanced by high light and knockdown of chloroplast ascorbate peroxidases in rice plants. Theor. Exp. Plant Physiol. 30, 41–55 (2018). https://doi.org/10.1007/s40626-018-0100-y

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