Carbon monoxide regulates the expression of the wound-inducible gene ipomoelin through antioxidation and MAPK phosphorylation in sweet potato

Abstract

Carbon monoxide (CO), one of the haem oxygenase (HO) products, plays important roles in plant development and stress adaptation. However, the function of CO involved in wounding responses is seldom studied. A wound-inducible gene, ipomoelin (IPO), of sweet potato (Ipomoea batatas cv. Tainung 57) was used as a target to study the regulation of CO in wounding responses. After wounding for 1h, the endogenous CO content and IbHO expression level were significantly reduced in leaves. IPO expression upon wounding was prohibited by the HO activator hemin, whereas the HO inhibitor zinc protoporphyrin IX elevated IPO expression. The IPO expression induced by wounding, H2O2, or methyl jasmonate was inhibited by CO. CO also affected the activities of ascorbate peroxidase, catalase, and peroxidase, and largely decreased H2O2 content in leaves. CO inhibited the extracellular signal-regulated kinase (ERK) phosphorylation induced by wounding. IbMAPK, the ERK of sweet potato, was identified by immunoblotting, and the interaction with its upstream activator, IbMEK1, was further confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Conclusively, wounding in leaves repressed IbHO expression and CO production, induced H2O2 generation and ERK phosphorylation, and then stimulated IPO expression.

Keywords: Carbon monoxide; ERK phosphorylation; H2O2; ipomoelin; sweet potato; wounding..

Fig. 1.

Levels of endogenous CO and haem oxygenase transcripts in sweet potato upon wounding. The third fully expanded leaves of sweet potato were wounded by forceps and collected at 0, 0.5, 1, 3, and 6h later. These leaves were used to analyse the endogenous CO contents (A) and the expression levels of the haem oxygenase gene (IbHO) (B). CO contents were detected by haemoglobin binding. IbHO expression levels were analysed by qRT-PCR. IbActin expression was used as an internal control. Statistic differences between unwounded and wounded sweet potato plants are marked with asterisk when P<0.01 according to Student’s t-test. The error bars are indicated as SD for at least three biological assays.

Fig. 2.

Effects of HO on IPO expression upon wounding. (A) Effects of the HO activator Hm on IPO expression. Leaves with petiole cuts of sweet potato were immersed in water for 12h and then treated with various concentrations (0, 0.1, 1, and 10 μM) of the HO activator Hm for another 12h. These leaves were unwounded or wounded (W+) by tweezers. After 6h, the total RNAs from these leaves were analysed by qRT-PCR to detect IPO expression. (B) Effects of the HO repressor ZnPP on IPO expression. The petiole cuts of leaves were immersed in water for 24h. Then, the total RNAs of leaves were isolated at 6h after wounding (W+) or the addition of various concentrations (0, 1, 5, and 10 μM) of HO repressor ZnPP. The IPO expression levels were analysed by qRT-PCR. The IbActin expression was used as an internal control. The error bars are indicated as SD for at least three biological assays.

Fig. 3.

Effects of CO on IPO expression induced by wounding, H₂O₂, or MJ. (A) Effects of CO on the IPO expression upon wounding. Leaves with petiole cuts of sweet potato were immersed in water for 12h, and then treated with various concentrations (0, 3, 5, and 10%) of CO solution for another 12h. These leaves were then unwounded (H₂O) or wounded (W+) by tweezers. After 6h, the total RNAs from these leaves were analysed by qRT-PCR to detect IPO expression. (B) Effects of CO on IPO expression induced by H₂O₂ or MJ. Leaves with petiole cuts were immersed in water for 12h and then some of these leaves were treated with 5% CO solution for another 12h. These leaves were then treated with 20mM H₂O₂ or 50 μM MJ for 6h. The IPO expression levels of these leaves were analysed by qRT-PCR. The IbActin expression level was used as an internal control. The error bars are indicated as SD for at least three biological assays.

Fig. 4.

Effects of CO on the production of H₂O₂ and the activities of APX, CAT, and POX. Leaves with petiole cuts of sweet potato were immersed in water for 12h and then treated with water or 5% CO solution for another 12h. These leaves were then wounded by tweezers. The leaves at the times indicated were collected to extract H₂O₂ and total proteins. H₂O₂ content was detected by TiCl₄ method (A). Total proteins were used for the activity assays of APX (B), CAT (C), and POX (D). The error bars are indicated as SD for at least three biological assays.

Fig. 5.

Effects of CO on ERK phosphorylation. (A) Effects of CO on IPO expression induced by STA, an ERK1/2 phosphorylation inducer. Leaves with petiole cuts of sweet potato were immersed in water for 12h and then treated with water or 5% CO solution for another 12h. Some of them were treated with 1 μM STA for 2h. Total RNAs from these leaves were analysed by qRT-PCR to detect IPO expression. (B) Effects of PD98059, an ERK1/2 phosphorylation inhibitor, on the IPO expression induced by ZnPP. Leaves with petiole cuts were immersed in water for 12h and then treated with water or 0.1 μM PD980559 (PD) for another 12h. Some of these leaves were then treated with 10 μM ZnPP for 6h. The IPO expression levels of these leaves were analysed by qRT-PCR. IbActin expression was used as an internal control. The error bars are indicated as SD for at least three biological assays for both (A) and (B). (C) Effects of CO on the phosphorylation of ERK (p-ERK) upon wounding. Leaves with petiole cuts were immersed in water for 12h and then treated with water, 5% CO solution, or 0.1 μM PD980559 for another 12h. The leaves treated with water and CO were then wounded for 0, 1, 2, and 3h. Leaves treated with PD980559 were further wounded for 6h. Some leaves treated with water were incubated in 1 μM STA for 2h. The total proteins were analysed by western blot assays for the detection of p-ERK. Rubisco from the same amounts of total protein was separated by SDS-PAGE, and stained by Coomassie blue as a loading control.

Fig. 6.

In vitro phosphorylation of IbMAPK by anti-p-ERK antibody. Leaves with petiole cuts were immersed in water for 12h, and then treated with water, 5% CO solution, or 0.1 μM PD98059 for another 12h. The leaves were then left unwounded (W-) or wounded (W+) by tweezers. The total proteins extracted from these leaves were incubated with recombinant IbMAPK–His at 30 °C for 1h and purified by His resin. The bound proteins were eluted from the resin and detected by anti-p-ERK and anti-His antibody. Immunoblots using anti-His antibody were used as controls.

Fig. 7.

IbMEK1 interacts with IbMAPK both in vivo and in vitro. (A) BiFc assays in Arabidopsis protoplasts for interaction between IbMEK1 and IbMAPK. Protoplasts were co-transformed with plasmids encoding IbMEK1 and IbMAPK fused with the YC and YN of YFP. These protoplasts were then visualized using a confocal microscope. Column 1 shows signals from YFP, column 2 shows chlorophyll autofluorescence, column 3 shows signals from NLS–mCherry as a nuclear maker, column 4 shows bright-field images, and column 5 shows merged images of columns 1–4. (B) GST pull-down assays for interaction between IbMEK1 and IbMAPK. GST–IbMEK1 or GST was incubated with IbMAPK–His and GST resin, and the bound proteins were then eluted from resin. These eluted proteins were detected with an anti-His antibody. (C) Co-immunoprecipitation assays in N. benthamiana leaves for interaction between IbMEK1 and IbMAPK. Tobacco leaves were infiltrated with agrobacteria carrying vectors containing 35S:GST (GST), 35S:GST-IbMEK1 (GST–IbMEK1), or 35S:His-IbMAPK (His–IbMAPK). After 4 d, total proteins extracted from these infiltrated leaves were incubated with GST resin, and the bound proteins were then eluted from the resin. These eluted proteins were detected with an anti-His antibody.

Fig. 8.

Schematic representation of the role of CO in the signal transduction pathway inducing IPO expression. The signal transducers, including H₂O₂ and MAPK cascades, induced by wounding for stimulating IPO expression have been reported previously (Chen et al., 2003, 2008). In sweet potato, CO could increase the activities of antioxidants APX, CAT, and POX and decrease the accumulation of H₂O₂ and the phosphorylation of ERK. Upon wounding, the expression of IbHO was repressed, and further decreased the contents of CO. Subsequently, the content of H₂O₂ and the phosphorylation of ERK were induced, and the wound-inducible gene IPO was then expressed.