In this study, we examined the regulation by putrescine, spermidine and spermine of nitric oxide (NO) biosynthesis in Arabidopsis thaliana seedlings. Using a fluorimetric method employing the cell-impermeable NO-binding dye diaminorhodamine-4M (DAR-4M), we observed that the polyamines (PAs) spermidine and spermine greatly increased NO release in the seedlings, whereas arginine and putrescine had little or no effect. Spermine, the most active PA, stimulated NO release with no apparent lag phase. The response was quenched by addition of 2-aminoethyl-2-thiopseudourea (AET), an inhibitor of the animal nitric oxide synthase (NOS) and plant NO biosynthesis, and by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO), an NO scavenger. By fluorescence microscopy, using the cell-permeable NO-binding dye diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), we observed that PAs induced NO biosynthesis in specific tissues in Arabidopsis seedlings. Spermine and spermidine increased NO biosynthesis in the elongation zone of the Arabidopsis root tip and in primary leaves, especially in the veins and trichomes, while in cotyledons little or no effect of PAs beyond the endogenous levels of NO-induced fluorescence was observed. We conclude that PAs induce NO biosynthesis in plants.

Polyamines (PA), polyamine oxidases, copper amine oxidases, and nitric oxide (NO) play important roles in physiology and stress responses in plants. NO biosynthesis as a result of catabolism of PA by polyamine oxidases and copper amine oxidases may explain in part PA-mediated responses. Involvement of a copper amine oxidase gene, COPPER AMINE OXIDASE1 (CuAO1), of Arabidopsis was tested for its role in stress responses using the knockouts cuao1-1 and cuao1-2. PA-induced and ABA-induced NO production investigated by fluorometry and fluorescence microscopy showed that the cuao1-1 and cuao1-2 are impaired in NO production, suggesting a function of CuAO1 in PA and ABA-mediated NO production. Furthermore, we found a PA-dependent increase in protein S-nitrosylation. The addition of PA and ABA also resulted in H(2)O(2) increases. cuao1-1 and cuao1-2 showed less sensitivity to exogenous ABA supplementation during germination, seedling establishment, and root growth inhibition as compared to wild-type. In response to ABA treatment, expression levels of the stress-responsive genes RD29A and ADH1 were significantly lower in the knockouts. These observations characterize cuao1-1 and cuao1-2 as ABA-insensitive mutants. Taken together, our findings extend the ABA signal transduction network to include CuAO1 as one potential contributor to enhanced NO production by ABA.

The functions of plant class B-heat shock factors (Hsfs) are not well understood. Hsfs belonging to this group differ from class A-Hsfs in structural features of the oligomerization domain and by the absence of a typical AHA motif for transcriptional activation. AtHsfB4 is expressed in different parts of the plants with highest levels in root tissue. Transgenic Arabidopsis plants overexpressing (OE) HsfB4 by CaMV-35S-promoter showed massively enhanced levels of Hsf mRNAs. The root surface of OE-plants was rough and cells became detached. Crossings with cell type specific root marker lines and confocal laser scanning microscopy provided clear evidence for a duplication of cells in the ground tissue and ectopic layers of lateral root cap (LRC) cells in HsfB4-OE plants. A duplication of endodermis cells occurs already during embryonic development, while the ectopic LRC cells are only detected during postembryonic growth. The mutant phenotypes of Hsf-OE plants are without precedence and indicate that class B-Hsfs may play an important role in root development.