Nitrate reductase-dependent nitric oxide synthesis in the defense response of Arabidopsis thaliana against Pseudomonas syringae

Abstract

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 ...

Long-term copper (Cu2+) exposure impacts on auxin, nitric oxide (NO) metabolism and morphology of Arabidopsis thaliana L.

“…Author's Personal copy osmotic stress or pathogen attack (Wang et al 2010;Kolbert et al 2010;Oliveira et al 2010). Therefore, we investigated the possible involvement of this enzyme in Cu 2?…”

How Biology Handles Nitrite

“…In accordance with its key role in the nitrogen metabolism, C-NaR is highly regulated by complex transcriptional, translational, and posttranslational mechanisms that respond to nitrogen, carbon dioxide, and dioxygen availabilities, pH, temperature, and light. ,− ,, Noteworthy, C-NaR is rapidly degraded in darkness (half-life of 6 h). Besides this well-established role in the reduction of nitrate, C-NaR from different species were shown to also catalyze the subsequent nitrite reduction to NO (eq ), not only in vitro, ,− but also in vivo. The in vivo evidences for C-NaR-dependent NO generation were provided by studies with (i) transgenic plants expressing a permanently active C-NaR, ,,− (ii) C-NaR knockout mutants ( nia 1 and nia 2 genes), ,− (iii) inactive C-NaR (e.g., plants with tungstate supply instead of molybdate), ,,− and (iv) others. ,,,− The nitrite reduction by C-NaR was also studied in silico, and it was found that both nitrate and nitrite are easily reduced (to nitrite and NO, respectively), although, as expected, nitrate is the preferred substrate These two C-NaR activities, nitrate reductase and nitrite reductase, seem to be controlled by the dioxygen concentration.…”

“…The C-NaR-dependent NO formation has been suggested to be involved in (i) stomatal closure, ,,,, (ii) onset of germination, (iii) phenylpropanoid metabolism, or (iv) immune defense mechanisms, because pathogen signals induce the C-NaR and increase the NO formation (strik­ingly similar to the mammalian inducible NOS). ,,,,, This enzyme is also suited to play a role as a cytoplasmatic nitrite sensor, to “signalize” the presence of toxic nitrite concentrations . In addition, C-NaR/nitrite may be acting not only as a NO source, but also as an oxygen sensor: it is intriguing that an enzyme that is rapidly degraded in darkness to avoid nitrite accumulation is increased during hypoxia, which also leads to nitrite accumulation.…”

Nitrate–Nitrite–Nitric Oxide Pathway: A Mechanism of Hypoxia and Anoxia Tolerance in Plants

“…Interestingly, this occurs not only during O 2 deficiency but many other biotic- and abiotic-stress-induced reductive pathways of NO formation. For example, salinity stress, water deficiency, UV radiation, freezing, pathogen attacks, and wounding can trigger NO production in plants [ 38 , 39 , 40 , 41 , 42 , 43 ], which could be due to the fact that these stresses could lead to hypoxia and anoxia in plant tissues, while its formation could be a defense strategy to survive harsh conditions.…”