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.

Since we are living in the ‘age of transcription’, awareness of aspects other than transcription in auxin signal transduction seems to have faded. One purpose of this review is to recall these other aspects. The focus will also be on the time scales of auxin responses and their potential or known dependence on either AUXIN BINDING PROTEIN 1 (ABP1) or on TRANSPORT-INHIBITOR-RESISTANT1 (TIR1) as a receptor. Furthermore, both direct and indirect evidence for the function of ABP1 as a receptor will be reviewed. Finally, the potential functions of a two-receptor system for auxin and similarities to other two-receptor signalling systems in plants will be discussed. It is suggested that such a functional arrangement is a property of plants which strengthens tissue autonomy and overcomes the lack of nerves or blood circulation which are responsible for rapid signal transport in animals.

• Here, cytokinin-induced nitric oxide (NO) biosynthesis and cytokinin responses were investigated in Arabidopsis thaliana wild type and mutants defective in NO biosynthesis or cytokinin signaling components. • NO release from seedlings was quantified by a fluorometric method and, by microscopy, observed NO biosynthesis as fluorescence increase of DAR-4M AM (diaminorhodamine 4M acetoxymethyl ester) in different tissues. • Atnoa1 seedlings were indistinguishable in NO tissue distribution pattern and morphological responses, induced by zeatin, from wild-type seedlings. Wild-type and nia1,2 seedlings, lacking nitrate reductase (NR), responded to zeatin with an increase within 3 min in NO biosynthesis so that NR does not seem relevant for rapid NO induction, which was mediated by an unknown 2-(2-aminoethyl)2-thiopseudourea (AET)-sensitive enzyme and was quenched by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO). Long-term morphological responses to zeatin were severely altered and NO biosynthesis was increased in nia1,2 seedlings. As cytokinin signaling mutants we used the single-receptor knockout cre1/ahk4, three double-receptor knockouts (ahk2,3, ahk2,4, ahk3,4) and triple-knockout ahp1,2,3 plants. All cytokinin-signaling mutants showed aberrant tissue patterns of NO accumulation in response to zeatin and altered morphological responses to zeatin. • Because aberrant NO biosynthesis correlated with aberrant morphological responses to zeatin the hypothesis was put forward that NO is an intermediate in cytokinin signaling.