Mechanical stress elicits nitric oxide formation and DNA fragmentation in Arabidopsis thaliana
2001
Garces, H. | Durzan, D. | Pedroso, M.C.
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. N(G)-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.
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