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

Abstract

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 NO 2 −-reducing activity. In this work, we used an NR-deficient double mutant ( nia1 nia2) of Arabidopsis thaliana that is deficient in endogenous NO 2 − 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 NO 2 − into nia1 nia2 leaves triggered NO production in situ. Moreover, co-infiltration of NO 2 − 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 NO 2 −, two important endogenous substrates for NO synthesis.