Decreased arginine and nitrite levels in nitrate reductase-deficient Arabidopsis thaliana plants impair nitric oxide synthesis and the hypersensitive response to Pseudomonas syringae

Nitric oxide (NO) produced in plants is implicated in defense responses against pathogens. NO synthesis in such conditions has been attributed to a nitric oxide synthase (NOS)-like enzyme and, more recently, to a mitochondrial-dependent NO2−-reducing activity. In this work, we used an NR-deficient double mutant (nia1 nia2) of Arabidopsis thaliana that is deficient in endogenous NO2− to analyse the hypersensitive response (HR) against an avirulent strain of Pseudomonas syringae pv. maculicola (Psm). The inoculation of Psm into nia1 nia2 A. thaliana caused leaf chlorosis whereas the HR was induced in wild-type plants. NO production in situ was substantially increased in wild-type but not in nia1 nia2 leaves following inoculation of Psm, as measured with the fluorescent NO indicator 4,5-diaminofluorescein diacetate. However, the infiltration of l-arginine or NO2− into nia1 nia2 leaves triggered NO production in situ. Moreover, co-infiltration of NO2− and Psm restored the HR in the leaves of nia1 nia2 plants. The total content of free amino acids, particularly l-arginine, was much lower in nia1 nia2 leaves compared to wild-type leaves. Overall, these results suggest that the HR is affected in NR-deficient plants because these plants lack l-arginine and NO2−, two important endogenous substrates for NO synthesis.