Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development

Abstract

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., 2002, 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 mitogen-activated 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 + 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.

Figure 1.

Primordia formation during adventitious root development in cucumber explants. Primary roots were removed and the explants were treated with water or 10 μm of the NO-donor SNP or 10 μm IAA for 3 d. Hypocotyl transverse sections were stained with toluidine blue and photographed using a digital camera attached to the microscope. For photographs and insets, magnifications of ×100 (bar indicates 0.2 mm) and ×400 (bar indicates 0.05 mm) were respectively used. MC, meristematic cells; P, parenchyma; PH, phloem; RP, root primordium; VP, vascular parenchyma; X, xylem. Arrows indicate the MC or RP magnified in the insets.

Figure 2.

MBP-kinase activity during adventitious root development induced by NO. A, Cucumber explants were incubated for different times with water or 10 μm of the NO-donor SNP or with SNP plus 200 μm of the specific NO-scavenger cPTIO. In vitro MBP-kinase activity was measured in total soluble extracts from those explants. Data are means of two independent experiments with three repetitions for each point. The sd of water and SNP plus cPTIO-treatments are not shown for the sake of clarity of the figure. B, In-gel protein kinase assays. The arrow heads indicate an apparent molecular mass of approximately 48 kD.

Figure 3.

MBP-kinase activity during the IAA-induced adventitious root development requires endogenous NO. A, Cucumber explants were incubated for different times either with 10 μm IAA or with IAA plus 200 μm of the specific NO-scavenger cPTIO. In vitro MBP-kinase activity was measured in total soluble extracts from those explants. Values are expressed as mean ± sd from two independent experiments with three repetitions for each point. B, Protein kinase activity was measured by in-gel assays as described. The arrow heads indicate an apparent molecular mass of approximately 48 kD.

Figure 4.

MBP-kinase activity induced by IAA and NO is inhibited by the MAPKK inhibitor PD098059. In vitro MBP-kinase activity was measured in total soluble extracts from cucumber explants treated with 10 μm SNP or 10 μm IAA, or with SNP or IAA in the presence of 50 μm of PD098059 for different times as indicated. Values are expressed as mean ± sd from two independent experiments with three repetitions for each point.

Figure 5.

MAPK activity is required for adventitious root formation induced by IAA and NO. A, Cucumber explants were treated for 5 d as indicated. Root number values are expressed as mean ± se (n = 10 explants from at least 3 independent experiments). Bars with different letters are significantly different with P < 0.05 (t test). The cell-permeable MAPKK inhibitor PD098059 was used at 50 μm and SNP and IAA were used at 10 μm. The inset shows the effect of different concentrations of PD098059 on the NO-induced adventitious root formation. Cucumber explants were treated with water (control) or 10 μm SNP in the presence or absence of 10 μm, 50 μm, or 100 μm of the MAPKK inhibitor PD098059 for 4 d. Inhibition values are expressed as %, where 100% means root number values per explant similar to those obtained in control explants. B, Photographs were taken after 4 d of treatment. Bar indicates 5 mm.

Figure 6.

The MBP-kinase activity is induced by NO in a cGMP-independent pathway. Cucumber explants were incubated for different times with 10 μm of the NO-donor SNP or with SNP plus 50 μm of the guanylate cyclase inhibitor LY83583 in the presence or absence of 50 μm of the MAPKK inhibitor PD098059. In vitro MBP-kinase activity was measured in total soluble extracts from those explants. Data are expressed as mean ± sd from two independent experiments with three repetitions for each point.

Figure 7.

Schematic illustration of the signaling networks involving IAA, NO, and cellular messengers that regulate ARF in cucumber. The auxin IAA triggers a transient NO accumulation (Pagnussat et al., 2002). In turn, NO regulates the activation of a MAPK signaling cascade. This pathway could be mediating both mitotic process and the expression of auxin-responsive genes (Mockaitis and Howell, 2000). In parallel, a cGMP-dependent pathway is also present leading to ARF (Pagnussat et al., 2003). NO-mediated signaling pathways that regulate ARF in cucumber: light gray, cGMP-independent; dark gray, cGMP-dependent.