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.

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Rapid activation of phospholipase A (PLA) by auxin or plant-pathogen interaction suggests a function in signal transduction for this enzyme, but the molecular identification of a cytosolic PLA carrying out this function remains open. We isolated four cDNA sequences from Arabidopsis (ecotype Columbia), AtPLA I, AtPLA IIA, AtPLA IVA, and AtPLA IVC, which are members of the patatin-related PLA gene family in plants and which are homologous to the animal Ca 2ϩ -independent PLA 2 gene family. Expression was measured by reverse transcriptase-polymerase chain reaction, and AtPLA I transcripts were found preferentially in shoots, AtPLA IIA and AtPLA IVA in roots, and AtPLA IVC in flowers. Transient expression of the four PLA-green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) leaves showed they were located in the cytosol and not in the vacuoles. Surprisingly, AtPLA::green fluorescent protein was also localized to chloroplasts. The enzymatic activity of the purified recombinant AtPLA IVA toward phosphatidylcholine was dependent on Ca 2ϩ , saturated at 0.5 mm, and had a pH optimum of about 7.0. It had both PLA 1 and PLA 2 specificity. The enzyme showed in vitro highest sensitivity toward the PLA 2 inhibitors palmitoyltrifluoromethyl ketone (PACOCF 3 , K i approximately 30 nm), arachidonyltrifluoromethyl ketone (AACOCF 3 , K i approximately 25 m), and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (K i approximately 200 nm) and was also sensitive to other previously used inhibitors 5,8,11,14-eicosatetraynoic acid (K i approximately 3 m) and nordihydroguajaretic acid (K i approximately 15 m). The influence of these PLA 2 inhibitors on elongation in etiolated Arabidopsis seedlings was tested, and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one and 5,8,11,14-eicosatetraynoic acid inhibited hypocotyl elongation maximally at concentrations close to their K i in vitro.

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