This report describes part of the signaling pathway and some of the molecules involved in the auxin-induced adventitious root formation in cucumber (Cucumis sativus). Previous results showed that nitric oxide (NO) mediates the auxin response during adventitious root formation (Pagnussat et al., 2002). To determine the order of action of indole acetic acid (IAA) and NO within the signal transduction pathway and to elucidate the target molecules that are downstream of NO action, cucumber hypocotyl cuttings were submitted to a pretreatment leading to endogenous auxin depletion. The auxin depletion treatment provoked a 3-fold reduction of the root number in comparison to the nondepleted explants. The NO-donor sodium nitroprusside was able to promote adventitious rooting in auxin-depleted explants, whereas the specific NO scavenger cPTIO prevented the effect of sodium nitroprusside. The endogenous NO level was monitored in both control and auxin-depleted explants using a NO-specific fluorescent probe. The NO level was 3.5-fold higher in control (nondepleted) explants than in auxin-depleted ones. The exogenous application of IAA restored the NO concentration to the level found in nondepleted explants. Because NO activates the enzyme guanylate cyclase (GC), we analyzed the involvement of the messenger cGMP in the adventitious root development mediated by IAA and NO. The GC inhibitor LY83583 reduced root development induced by IAA and NO, whereas the cell-permeable cGMP derivative 8-Br-cGMP reversed this effect. The endogenous level of cGMP is regulated by both the synthesis via GC and its degradation by the phosphodiesterase activity. When assayed, the phosphodiesterase inhibitor sildenafil citrate was able to induce adventitious rooting in both nondepleted and auxin-depleted explants. Results indicate that NO operates downstream of IAA promoting adventitious root development through the GC-catalyzed synthesis of cGMP.The plant hormone auxin is involved in the regulation of most aspects of plant growth and development, including cell division, elongation, and differentiation. The role of auxins in adventitious rooting is particularly important because the hormone initiates this process-which involves cell division and primordium formation-inducing the dedifferentiation of the cells to form the apical meristem. Although genetic approaches have revealed some discrete aspects of auxin transport, signaling, and response, the understanding of the molecular mechanism of auxin action in the establishment of new root meristems remains preliminary (Doerner, 2000; Berleth and Sachs, 2001).Nitric oxide (NO) is a diffusible multifunctional molecule involved in numerous physiological processes in phylogenetically distant species (Gow and Ischiropoulos, 2001). It was first described in mammals, where it plays variable functions ranging from dilation of blood vessels to neurotransmission and immune response. In comparison with animal studies, relative little is known about NO biological functions in plants. However, the presence of...

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002Pagnussat et al., , 2003. However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)-and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA-and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogenactivated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA 1 cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process induced by IAA in a NO-mediated but cGMP-independent pathway. The activation of MAPKs is discussed in relation to the cell responses modulating mitotic process.Auxins play a central role in numerous developmental processes functioning as a signal for cell division, elongation and differentiation. Among auxin actions, an indole acetic acid (IAA) dependence of root formation, apical dominance, and tropic responses have been described (Theologis, 1986;Estelle, 1992;Davies, 1995). The molecular basis of auxin action is an area of intense study. Genetic approaches to dissect how this hormone triggers a variety of effects in plants rely on the analysis of mutants modified in their response to auxin (Walden and Lubenow, 1996). The available mutants display an array of phenotypes that indicates changes in auxin action or response including altered growth, changes in root length and apical dominance, and reduced gravitropism (Lincoln et al., 1990;Pickett et al., 1990;Wilson et al., 1990;Hobbie and Estelle, 1995;Leyser et al., 1996).Adventitious root for...

A few years ago it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during adventitious root (AR) formation in cucumber (Cucumis sativus). More recently, a mitogen-activated protein kinase cascade was shown to be induced by IAA in a NO-dependent, but cGMP-independent, pathway. In the present study, the involvement of Ca2+ and the regulation of Ca2+-dependent protein kinase (CDPK) activity during IAA- and NO-induced AR formation was evaluated in cucumber explants. The effectiveness of several broad-spectrum Ca2+ channel inhibitors and Ca2+ chelators in affecting AR formation induced by IAA or NO was also examined. Results indicate that the explants response to IAA and NO depends on the availability of both intracellular and extracellular Ca2+ pools. Protein extracts from cucumber hypocotyls were assayed for CDPK activity by using histone IIIS or syntide 2 as substrates for in-gel or in vitro assays, respectively. The activity of a 50 kDa CDPK was detected after 1 d of either NO or IAA treatments and it extended up to the third day of treatment. This CDPK activity was affected in both extracts from NO- and IAA-treated explants in the presence of the specific NO-scavenger cPTIO, suggesting that NO is required for its maximal and sustained activity. The in-gel and the in vitro CDPK activity, as well as the NO- or IAA-induced AR formation, were inhibited by calmodulin antagonists. Furthermore, the induction of CDPK activity by NO and IAA was shown to be reliant on the activity of the enzyme guanylate cyclase.