Annals of Botany 87: 567-574, 2001
© 2001 Annals of Botany Company
Mechanical Stress Elicits Nitric Oxide Formation and DNA Fragmentation in Arabidopsis thaliana
Centro de Biotecnologia Vegetal, Dept. Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, Bloco C2, Piso 1, Campo Grande, P-1749-016, Lisboa, Portugal Dept. Environmental Horticulture, University of California, Davis, CA 95616-8587, USA
Received: 11 October 2000 ; Returned for revision: 6 November 2000 . Accepted: 7 December 2000
The effect of mechanical stress (centrifugation) on the induction of nitric oxide (NO) formation and DNA fragmentation was investigated in leaf cells of Arabidopsis thaliana. Centrifuged and non-centrifuged leaves from wild-type and nitrate reductase (NR)nia1, nia2 double mutant, defective in the assimilation of nitrate, were labelled with 4,5-diaminofluorescein diacetate (DAF-2 DA) to visualize in vivo NO production. After these treatments, DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labelling (TUNEL) method. Exposure to an NO-releasing compound, sodium nitroprusside (SNP) mimicked the cell response to centrifugation (20 g). The involvement of endogenous NO as a signal in mechanical stress and in DNA fragmentation was confirmed by inhibition of NO production using a nitric oxide synthase (NOS) inhibitor viz. NG-monomethyl- L -arginine (L -NMMA). These results indicate that NOS-like activity was present in A. thaliana leaves and was increased by mechanical stress. The effect of leaf-wounding on nitric oxide production was identical to that of centrifugation. Experiments with A. thaliana NR mutant also showed that NO bursts were induced by mechanical and wounding stresses and that NO was not a by-product of NR activity. A positive and significant correlation between NO production and DNA fragmentation was recorded for both centrifuged and non-centrifuged cells. Our results suggest that factors other than NO contribute to DNA damage and cell death, and furthermore, that an inducible form of NOS is present in A. thaliana. Copyright 2001 Annals of Botany Company
Arabidopsis thaliana, cell death, DNA fragmentation, NO, plant stress, wounding
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