In the present study, we investigated the involvement of the mitochondrial permeability transition pore (PTP) in nitric oxide (NO)-induced plant cell death. NO donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine inhibited growth and caused death in suspension-cultured cells of Citrus sinensis. Cells treated with SNP showed chromatin condensation and fragmentation, characteristic of apoptosis. SNP caused loss of the mitochondrial membrane electrical potential, which was prevented by cyclosporin A (CsA), a specific inhibitor of PTP formation. CsA also prevented the nuclear apoptosis and subsequent Citrus cell death induced by NO. These findings indicate that mitochondrial PTP formation is involved in the signaling pathway by which NO induces apoptosis in cultured Citrus cells. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOR(OE)) or antisense (GSNOR(AS)) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOR(OE) had the lowest SNO and NO levels and GSNOR(AS) the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOR(OE) cells grown under nutritional stress. Moreover, GSNOR(OE) could not increase alternative oxidase (AOX) activity under nutritional stress. GSNOR(AS) showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells.

Nitrate reductase (NR) was recently shown to play an important role during phytopathogenic interactions by providing substrates for the synthesis of nitric oxide (NO), a key signal for plant defense responses. In order to give additional support to this hypothesis, we compared NO-mediated defense responses of wild-type and NR double-deficient (nia1 nia2) Arabidopsis thaliana plants inoculated with the IBSBF-1115 (ibs) strain of Pseudomonas syringae pv. maculicola (Psm) and with genetically characterized avirulent (avr) or virulent (vir) strains of Psm. Inoculation of wild-type leaves with avr or ibs, but not vir, stimulated NO emission, as measured by the indicator 4,5-diaminofluorescein. NO emission induced by avr was higher than that induced by ibs. Wild-type plants displayed the hypersensitive response (HR) when infiltrated with the strains avr or ibs, although a stronger HR was induced by avr. The vir strain did not induce HR in wild-type plants, and leaves developed severe infection symptoms. nia1 nia2 plants did not show significantly increased NO emission nor did they develop HR to any of the analyzed strains of Psm, but displayed clorotic lesions and higher bacterial growth in their leaves. Overall, these results highlight the importance of NR-dependent NO synthesis for plant defenses against pathogen attack. Keywords: Pseudomonas syringae pv. maculicola, avirulent strain, hypersensitive response, nia1 nia2 mutant. RESUMO Síntese de óxido nítrico dependente da nitrato redutase na resposta de defesa de Arabidopsis thaliana contra Pseudomonas syringaeA nitrato redutase (NR) possui importante papel na interação fitopatogênica por prover os substratos para a síntese do óxido nítrico (NO), um sinalizador essencial para a defesa vegetal. A fim de fornecer suporte adicional a essa hipótese, neste trabalho foram comparadas as respostas de defesa mediadas por NO de plantas de Arabidopsis thaliana selvagens e deficientes para a NR (nia1 nia2) quando inoculadas com a linhagem IBSBF-1115 (ibs) de Pseudomonas syringae pv. maculicola (Psm) e com as linhagens geneticamente caracterizadas como avirulenta (avr) ou virulenta (vir) de Psm. A inoculação de folhas selvagens com avr ou ibs, mas não vir, estimulou a emissão de NO, medida pelo indicador 4,5-diaminofluoresceína. A emissão de NO induzida por avr foi maior que aquela induzida por ibs. Plantas selvagens apresentaram resposta hipersensitiva (RH) quando infiltradas com avr ou ibs, mas uma RH mais forte foi induzida por avr. A linhagem vir não induziu RH nas plantas selvagens, e as folhas desenvolveram sintomas severos da infecção. Plantas nia1 nia2 não tiveram um aumento significativo da emissão de NO nem desenvolveram RH após inoculação com as linhagens analisadas de Psm, apresentando intenso crescimento bacteriano e clorose foliar. Em suma, esses resultados evidenciam a importância da síntese de NO dependente da NR na defesa vegetal ao ataque de patógenos. Palavras-chave: Pseudomonas syringae pv. maculicola, linhagem avirulenta, mutante nia1 nia2, resposta ...

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