N-3-oxo-decanoyl-L-homoserine-lactone activates auxin-induced adventitious root formation via hydrogen peroxide- and nitric oxide-dependent cyclic GMP signaling in mung bean

Abstract

N-Acyl-homoserine-lactones (AHLs) are bacterial quorum-sensing signaling molecules that regulate population density. Recent evidence demonstrates their roles in plant defense responses and root development. Hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and cyclic GMP (cGMP) are essential messengers that participate in various plant physiological processes, but how these messengers modulate the plant response to N-acyl-homoserine-lactone signals remains poorly understood. Here, we show that the N-3-oxo-decanoyl-homoserine-lactone (3-O-C10-HL), in contrast to its analog with an unsubstituted branch chain at the C3 position, efficiently stimulated the formation of adventitious roots and the expression of auxin-response genes in explants of mung bean (Vigna radiata) seedlings. This response was mimicked by the exogenous application of auxin, H(2)O(2), NO, or cGMP homologs but suppressed by treatment with scavengers or inhibitors of H(2)O(2), NO, or cGMP metabolism. The 3-O-C10-HL treatment enhanced auxin basipetal transport; this effect could be reversed by treatment with H(2)O(2) or NO scavengers but not by inhibitors of cGMP synthesis. Inhibiting 3-O-C10-HL-induced H(2)O(2) or NO accumulation impaired auxin- or 3-O-C10-HL-induced cGMP synthesis; however, blocking cGMP synthesis did not affect auxin- or 3-O-C10-HL-induced H(2)O(2) or NO generation. Additionally, cGMP partially rescued the inhibitory effect of H(2)O(2) or NO scavengers on 3-O-C10-HL-induced adventitious root development and auxin-response gene expression. These results suggest that 3-O-C10-HL, unlike its analog with an unmodified branch chain at the C3 position, can accelerate auxin-dependent adventitious root formation, possibly via H(2)O(2)- and NO-dependent cGMP signaling in mung bean seedlings.

Figure 1.

Dose effects of AHL signal molecules on adventitious root formation in mung bean explants. A, Dose-response effects of a series of AHLs on adventitious root formation. The AHLs were applied to the mung bean explants at the indicated concentrations, and the adventitious root number was calculated after 1 week of treatment. The root number values are expressed as means ± se (n = 30 explants) from at least three independent experiments. Single asterisks indicate values significantly different at P < 0.05, and double asterisks indicate values significantly different at P < 0.01 (Student’s t test) compared with the control.. B, Photographs were taken of mung bean explants after 4 d of treatment with the indicated AHLs at 100 nm. The chemical structures of the individual AHLs are shown below the photographs. [See online article for color version of this figure.]

Figure 2.

Detection of 3-O-C10-HL-induced H₂O₂ (A) and NO (B) by fluorescence staining. Three-day-old mung bean explants were treated with water as the control (a), 100 nm 3-O-C10-HL for 6 h (b) and 24 h (c), or 100 nm IAA for 6 h (d). After treatment, the stems of the explants were loaded with H₂DCF-DA (A) and DAF-FM DA (B) for 30 min, and the fluorescence intensity of the dissected stem sections was determined using confocal fluorescence microscopy. For inhibitor treatment, the explants were pretreated with 10 μm Ly83083 (e), 1 μm DPI (f), 10 μm BHT (g), or 50 μm cPTIO (h) for 2 h, followed with 100 nm 3-O-C10-HL for 24 h, and the fluorescence intensity was observed as above.

Figure 3.

The effects of different inhibitors or scavengers and donors on 3-O-C10-HL-induced accumulation of H₂O₂ and NO in mung bean explants. Three-day-old mung bean explants were subjected to 100 nm 3-O-C10-HL or a donor (100 nm C10-HL, 10 μm IAA, 100 μm H₂O₂, 50 μm GSNO, or 1 μm 8-Br-cGMP) treatment for 3 d or were pretreated with different inhibitors or scavengers (10 μm NPA, 1 μm DPI, 1 mm BHT, 1 mm AsA, 50 μm cPTIO, 10 μm LY83583, or 50 μm ODQ) for 2 h. This treatment was followed by the addition of 100 nm 3-O-C10-HL for 24 h, and H₂O₂ (A) and NO (B) contents were determined. The experiments were performed in triplicate and repeated at least three times (n = 20). The vertical bars represent means ± se, and bars with different letters are significantly different at P < 0.05 (Tukey’s test). FW, Fresh weight.

Figure 4.

The effects of different inhibitors or scavengers and donors on 3-O-C10-HL-induced adventitious root formation. A, Three-day-old mung bean explants (black bars, nondepleted; gray bars, IAA depleted) were subjected to 100 nm 3-O-C10-HL or donor (100 nm C10-HL, 10 μm IAA, 100 μm H₂O₂, or 50 μm GSNO) treatment for 6 d or were pretreated with different inhibitors or scavengers (concentrations are as shown in Fig. 2) for 2 h. This was followed by treatment with 100 nm 3-O-C10-HL for 7 d, and the adventitious root numbers were calculated. The experiments were performed in triplicate and repeated at least three times (n = 20). The vertical bars represent means ± se, and bars with different letters are significantly different at P < 0.05 (Tukey’s test). B, Photographs of mung bean seedlings following the treatments as in A were taken after 4 d of treatment. [See online article for color version of this figure.]

Figure 5.

The effects of different inhibitors or scavengers and donors on 3-O-C10-HL-induced accumulations of cGMP in mung bean explants. Three-day-old mung bean explants were subjected to 100 nm 3-O-C10-HL or donor (100 nm C10-HL, 10 μm IAA, 100 μm H₂O₂, or 50 μm GSNO) treatment for 3 d or were pretreated with different inhibitors or scavengers (concentrations are as shown in Fig. 2; 1 mg mL⁻¹ sildenafil) for 2 h. This was followed by treatment with 100 nm 3-O-C10-HL for 3 d, and the content of cGMP was determined. The experiments were performed in triplicate and repeated at least three times (n = 20). The vertical bars represent means ± se, and bars with different letters are significantly different at P < 0.05 (Tukey’s test). FW, Fresh weight.

Figure 6.

The effects of different inhibitors or scavengers and donors on 3-O-C10-HL-induced adventitious root formation. A, Three-day-old mung bean explants (black bars, nondepleted; gray bars, IAA depleted) were subjected to 100 nm 3-O-C10-HL or 10 μm IAA treatment for 6 d or were pretreated with different inhibitors or scavengers (concentrations are as shown in Fig. 2) for 2 h. This was followed by treatment with 100 nm 3-O-C10-HL for 7 d, and the adventitious root numbers were calculated. The experiments were performed in triplicate and repeated at least three times (n = 20). The vertical bars represent means ± se, and bars with different letters are significantly different at P < 0.05 (Tukey’s test). B, Photographs of seedlings from the above treatments were taken after 4 d. [See online article for color version of this figure.]

Figure 7.

The effects of different inhibitors or scavengers and donors on 3-O-C10-HL-induced polar auxin transport. Three-day-old mung bean explants were subjected to 100 nm 3-O-C10-HL or 10 μm IAA treatment for 6 d or were pretreated with different inhibitors or scavengers (the concentrations are as shown in Fig. 2) for 2 h. This was followed by treatment with 100 nm 3-O-C10-HL for 12 h, and stem basipetal auxin transport was measured immediately. The experiments were performed in triplicate and repeated at least three times (n = 30). The vertical bars represent means ± se, and bars with different letters are significantly different at P < 0.05 (one-way ANOVA with Tukey’s test).

Figure 8.

The effects of different inhibitors or scavengers and donors on the 3-O-C10-HL-induced transcript levels of auxin-response and cell division-related genes. Three-day-old mung bean explants were subjected to 100 nm 3-O-C10-HL or different donor treatments for 24 h or pretreated with different inhibitors or scavengers (the concentrations are as shown in Fig. 2) for 2 h. This was followed by treatment with 100 nm 3-O-C10-HL for 24 h, and the stem basipetal auxin transport was measured immediately. RNA from the stem portion of the explants was extracted for use in the RT-PCR assay.

Figure 9.

Schematic illustration of a proposed model for regulating adventitious root formation by 3-O-C10-HL in mung bean. In this model, IAA is synthesized in the apical bud and basipetally transported to the base of the hypocotyls. 3-O-C10-HL promotes the polar auxin transport to induce the generation of H₂O₂ and NO and subsequently induce the synthesis of cGMP to activate adventitious root formation. During this process, H₂O₂ and NO can provide positive feedback by regulating polar auxin transport to enhance H₂O₂ and NO accumulation, and H₂O₂ can also positively affect NO synthesis. [See online article for color version of this figure.]