H2O2-Responsive Hormonal Status Involves Oxidative Burst Signaling and Proline Metabolism in Rapeseed Leaves

Abstract

Drought alters the level of endogenous reactive oxygen species (ROS) and hormonal status, which are both involved in the regulation of stress responses. To investigate the interplay between ROS and hormones in proline metabolism, rapeseed (Brassica napus L.) plants were exposed to drought or exogenous H₂O₂ (Exo-H₂O₂) treatment for 10 days. During the first 5 days, the enhanced H₂O₂ concentrations in drought treatment were associated with the activation of superoxide dismutase (SOD) and NADPH oxidase, with enhanced ABA and SA levels, while that in Exo-H₂O₂ treatment was mainly associated with SA-responsive POX. During the latter 5 days, ABA-dependent ROS accumulation was predominant with an upregulated oxidative signal-inducible gene (OXI1) and MAPK6, leading to the activation of ABA synthesis and the signaling genes (NCED3 and MYC2). During the first 5 days, the enhanced levels of P5C and proline were concomitant with SA-dependent NDR1-mediated signaling in both drought and Exo-H₂O₂ treatments. In the latter 5 days of drought treatment, a distinct enhancement in P5CR and ProDH expression led to higher proline accumulation compared to Exo-H₂O₂ treatment. These results indicate that SA-mediated P5C synthesis is highly activated under lower endogenous H₂O₂ levels, and ABA-mediated OXI1-dependent proline accumulation mainly occurs with an increasing ROS level, leading to ProDH activation as a hypersensitive response to ROS and proline overproduction under severe stress.

Keywords: Brassica napus; abscisic acid; drought; hydrogen peroxide; proline; salicylic acid.

Figure 1

Changes in the concentration of ROS and in the activity of antioxidative enzymes in the leaves of control, drought−, or exogenous H₂O₂ (Exo−H₂O₂) −treated plants for 10 days. (A) O₂^•– and (B) H₂O₂ concentration, (C) peroxidase (POX), (D) superoxide dismutase (SOD), and (E) catalase (CAT) activity. Results are represented as mean ± SE for n = 3. Different letters indicate values that are significantly different at p < 0.05 according to Duncan’s multiple range test.

Figure 2

Changes in the expression of (A) ABA synthesis-related gene NCED3, (B) ABA receptor gene PYL1, (C) ABA signaling-related gene MYC2, (D) SA synthesis-related gene ICS1, (E) NDR1, and (F) SA signaling-related gene NPR1 in the leaves of control, drought-, or exogenous H₂O₂ (Exo-H₂O₂)-treated plants for 10 days. Results are represented as mean ± SE for n = 3. Different letters indicate values that are significantly different at p < 0.05 according to Duncan’s multiple range test.

Figure 3

Changes in the expression of (A) NADPH oxidase, (B) transcription factor MAPK6, and (C) oxidative signal-inducible (OXI1) gene in the leaves of control, drought-, or exogenous H₂O₂ (Exo-H₂O₂)-treated plants for 10 days. Results are represented as mean ± SE for n = 3. Different letters indicate values that are significantly different at p < 0.05 according to Duncan’s multiple range test.

Figure 4

Changes in proline metabolism in the leaves of control, drought-, or exogenous H₂O₂ (Exo-H₂O₂)-treated plants for 10 days. (A) Pyrroline-5-carboxylate (P5C) and (B) proline content, and expression of (C) P5C synthase 1 (P5CS1), (D) P5CS2, (E) P5C reductase (P5CR), (F) proline dehydrogenase (ProDH), and (G) P5C dehydrogenase (P5CDH). Results are represented as mean ± SE for n = 3. Different letters indicate values that are significantly different at p < 0.05 according to Duncan’s multiple range test.

Figure 5

Heatmap analysis of the treatment effect and correlations among the variables measured for 10 days. (A) Heatmap comparing the changes of the identified metabolites or gene expression levels in the leaves of control, drought-, or exogenous H₂O₂ (Exo-H₂O₂)-treated plants for 10 days. The normalization procedure consisted of mean row centering with color scales. (B) Heatmap showing the correlations among the identified metabolites or gene expression levels. Correlation coefficients were calculated based on Pearson’s correlation. Red indicates a positive effect, whereas blue indicates a negative effect. Color intensity is proportional to the correlation coefficients.

Figure 6

Proposed model of crosstalk between ROS signaling, hormones, and proline metabolism in response to endogenous H₂O₂ level. Green and red arrows represent the SA- and ABA-dependent pathways, respectively.