Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana
In animals, aconitase is a bifunctional protein. When an iron-sulfur cluster is present in its catalytic center, aconitase displays enzymatic activity; when this cluster is lost, it switches to an RNA-binding protein that regulates the translatability or stability of certain transcripts. To investigate the role of aconitase in plants, we assessed its ability to bind mRNA. Recombinant aconitase failed to bind an iron responsive element (IRE) from the human ferritin gene. However, it bound the 5′ UTR of the Arabidopsis chloroplastic CuZn superoxide dismutase 2 (CSD2) mRNA, and this binding was specific. Arabidopsis aconitase knockout (KO) plants were found to have significantly less chlorosis after treatment with the superoxide-generating compound, paraquat. This phenotype correlated with delayed induction of the antioxidant gene GST1, suggesting that these KO lines are more tolerant to oxidative stress. Increased levels of CSD2 mRNAs were observed in the KO lines, although the level of CSD2 protein was not affected. Virus-induced gene silencing (VIGS) of aconitase in Nicotiana benthamiana caused a 90% reduction in aconitase activity, stunting, spontaneous necrotic lesions, and increased resistance to paraquat. The silenced plants also had less cell death after transient co-expression of the AvrPto and Pto proteins or the pro-apoptotic protein Bax. Following inoculation with Pseudomonas syringae pv. tabaci carrying avrPto, aconitase-silenced N. benthamiana plants expressing the Pto transgene displayed a delayed hypersensitive response (HR) and supported higher levels of bacterial growth. Disease-associated cell death in N. benthamiana inoculated with P. s. pv. tabaci was also reduced. Taken together, these results suggest that aconitase plays a role in mediating oxidative stress and regulating cell death.
KeywordsAconitase Arabidopsis Cell death Nicotiana benthamiana Oxidative stress Superoxide dismutase RNA-binding VIGS
CuZn superoxide dismutase 2
Gel mobility shift assay
Iron responsive element
Iron regulatory protein 1
Virus-induced gene silencing
We thank Dr. P. Perez for the Arabidopsis aconitase clone, Dr. Kühn for the IRP-1 clone and the pSPT-fer plasmid, Dr. D. Kliebenstein for the CSD2 antibodies, and D’Maris Dempsey for critical reading of the manuscript. This work was supported by grant MCB-0110404 (D.F.K.) and grant DBI-116076 (G.B.M.) from the National Science Foundation and by a fellowship from the Deutsche Forschungsgemeinschaft (MO 955/1-1) to W.M.
- Ahlfors R, Lang S, Overmyer K, Jaspers P, Brosche M, Tauriainen A, Kollist H, Tuominen H, Belles-Boix E, Piippo M, Inze D, Palva ET, Kangasjarvi J (2004) Arabidopsis radical-induced cell death1 belongs to the WWE protein–protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses. Plant Cell 16:1925–1937PubMedCrossRefGoogle Scholar
- Gangloff SP, Marguet D, Lauquin GJ (1990) Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate. Mol Cell Biol 10:3552–3561Google Scholar
- Heazlewood JL, Tonti-Filippini JS, Gout AM, Day DA, Whelan J, Millar AH (2004) Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins. Plant Cell 16:241–256PubMedCrossRefGoogle Scholar
- Overmyer K, Tuominen H, Kettunen R, Betz C, Langebartels C, Sandermann H, Kangasjarvi J (2000) Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death. Plant Cell 12:1849–1862PubMedCrossRefGoogle Scholar
- Slaymaker DH, Navarre DA, Clark D, del Pozo O, Martin GB, Klessig DF (2002) The tobacco salicylic acid-binding protein (SABP) 3 is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proc Natl Acad Sci USA 99:11640–11645PubMedCrossRefGoogle Scholar