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Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low

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Abstract

Arabidopsis chloroplasts have a multi-layered defense against hydrogen peroxide (H2O2) that includes a stromal and thylakoid ascorbate peroxidase (sAPX and tAPX). Single and double null mutants in SAPX and TAPX (sapx and tapx) were each crossed with ascorbate deficient vtc2. The single, double and triple mutants did not show visual light stress phenotypes when grown at control or high light intensities (CL and HL; 120 and 1,000 μmol photons m−2 s−1). Upon shift from CL to HL, mesophyll of expanded leaves of the triple mutant bleached within hours, with exclusion of the major vein areas; this contrasts to reported patterns of cell death under ozone treatment and calatase deficiency. tapx–vtc2 and sapx–vtc2, but not tapx–sapx or single mutants, showed limited bleaching. Bleaching and necrosis were accompanied by accumulation of H2O2. Cellular concentrations of α-tocopherol, ascorbate and glutathione showed dramatic increase in response to HL in all eight genotypes and the four vtc2 genotypes accumulated more glutathione under CL than the others. Transcript analysis of other ROS responsive genes in vtc2 and the triple mutant showed up to 20-fold induction after transition to HL, generally irrespective of genotype. We conclude that chloroplast APX proteins in Arabidopsis can be effectively compensated by other endogenous H2O2 detoxification systems, but that low cellular ascorbate levels in absence of chloroplast APX activity are detrimental to the cell during excess light.

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Abbreviations

APX:

Ascorbate peroxidase

CL:

Control light (120 μmol photons m−2 s−1)

HL:

High light (1,000 μmol photons m−2 s−1)

AsA:

Ascorbate

Glutathione:

GSH

SOD:

Superoxide dismutase

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Acknowledgments

We thank Dr. P. Conklin for donation of vtc2-2 mutant seeds and the antisera against CDS2 and FeSOD. We thank Dr. A. Jimmy Ytterberg for identification of sAPX protein by tandem mass spectrometry and Dr. Hong-Gu Kang for help with the Typan blue staining. We also thank numerous colleagues for generous gifts of antisera, namely Dr. A. Sokolenko for anti-SppA; Dr. K.K. Niyogi for anti-PsbS, Dr. P. Rey for anti-CDSP34, Drs. I. Murgia and C. Soave for anti-tAPX and we thank Dr. D. DellaPenna for seeds of vte2. This work was supported by funding from the USDA (#2003-00786) and New York Science Technology and Research to KVW. AM was supported by a Fulbright fellowship and MJL was supported by a fellowship from the South Korean Government. DRR was supported by resources of the Computational Biology Service Unit from Cornell University which is partially funded by the Microsoft Corporation.

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  1. Lisa Giacomelli

    Present address: Consorcio CSIC-IRTA, Laboratorio de Genética Molecular Vegetal, Instituto de Biología Molecular de Barcelona (IBMB-CSIC), C/Jordi Girona 18-26, Barcelona, 08034, Spain

  2. Antonio Masi

    Present address: Dipartimento di Biotecnologie Agrarie, I 35020, Legnaro, Padova, Italy

  3. Mi Ja Lee

    Present address: Honam Agricultural Research Institute, Iksan, Jeonbuk, 570-080, South Korea

Authors and Affiliations

  1. Department of Plant Biology, Cornell University, Emerson Hall 332, Tower Road, Ithaca, NY, 14853, USA

    Lisa Giacomelli, Antonio Masi, Mi Ja Lee & Klaas J. van Wijk

  2. Computation Biology Service Unit, Cornell Theory Center, Cornell University, Ithaca, NY, 14853, USA

    Daniel R. Ripoll

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  1. Lisa Giacomelli
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Correspondence to Klaas J. van Wijk.

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Giacomelli, L., Masi, A., Ripoll, D.R. et al. Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low. Plant Mol Biol 65, 627–644 (2007). https://doi.org/10.1007/s11103-007-9227-y

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