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Article

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

by
Bok-Rye Lee
1,†,
Van Hien La
1,2,†,
Sang-Hyun Park
1,
Md Al Mamun
1,
Dong-Won Bae
3 and
Tae-Hwan Kim
1,*
1
Department of Animal Science, Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University, Gwangju 61186, Korea
2
Department of Biotechnology and Food Technology, Thai Nguyen University of Agriculture and Forestry, Thai Nguyen 24000, Vietnam
3
Central Instruments Facility, Gyeongsang National University, Jinju 52828, Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Antioxidants 2022, 11(3), 566; https://0-doi-org.brum.beds.ac.uk/10.3390/antiox11030566
Submission received: 15 February 2022 / Revised: 14 March 2022 / Accepted: 14 March 2022 / Published: 16 March 2022
(This article belongs to the Section Antioxidant Enzyme Systems)
Browse Figures
Versions Notes

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.

1. Introduction

Reactive oxygen species (ROS) are generated due to the univalent reduction of oxygen in the metabolic pathway as one of the earliest responses of plant cells to drought [1,2,3,4,5,6] and pathogen infection [7,8,9]. Excess of ROS causes oxidative stress that can damage proteins, lipids, and DNA [10,11,12]. ROS also function as secondary messengers in the regulation of stress responses in plants [13,14,15]. Thus, the steady-state level of ROS in cells needs to be tightly regulated by ROS-scavenging and ROS-producing proteins, such as peroxidases (POXs), NADPH oxidase, superoxide dismutase (SOD), and catalase (CAT) [16,17,18], as well as by non-enzymatic metabolic pathways (e.g., glutathione-ascorbate cycle) [19,20]. As the most stable among the ROS, H2O2 is appropriate to play this function [2,17,21]. H2O2 produced by cytosolic membrane-bound NADPH oxidase is the key player associated with the ROS-related signal transduction [14,20,21]. Oxidative burst-mediated signaling is required for the induction of the oxidative signal-inducible gene (OXI1). The OXI1 encoding a serine/threonine kinase is induced in response to a wide range of H2O2-generating stimuli [13]. Activation of OXI1 results in the activation of a mitogen-activated protein kinase (MAPK) cascade (MAPK3/6) and the induction or activation of different transcription factors that regulate the ROS-scavenging and ROS-producing pathways [2]. In addition, plants exposed to stress stimuli often upregulate ROS (especially H2O2) and phytohormone signaling [5,8,22]. In this regard, the interaction between H2O2 and hormones has been widely studied under different environmental stresses in various plants [6,20,23,24].
Another common response to drought stress is the accumulation of proline along with enhanced H2O2 levels. The H2O2 produced by NADPH oxidase increases proline accumulation to scavenge ROS [25,26,27], whereas overproduced proline leads to increase in endogenous ROS [28,29,30]. Numerous studies have shown that ROS and proline accumulation are regulated by stress-responsive hormones, of which the best studied are abscisic acid (ABA) and salicylic acid (SA). Drought, in general, increases levels of both endogenous ABA and SA, as well as their signaling along with an enhanced H2O2 level [5,6,31]. ROS (particularly H2O2) is thought to be a part of ABA signaling. For instance, drought-enhanced H2O2 from NADPH oxidase [27,32] induces proline accumulation via upregulation of pyrroline-5-carboxylate synthetase (P5CS) and downregulation of proline dehydrogenase (ProDH) [5,33] in an ABA-dependent manner [31,34]. Our previous studies have shown that severe drought symptoms, characterized by the ABA-responsive proline and H2O2 accumulation leading to the oxidized state of redox, are alleviated by a SA-mediated antagonistic depression of ABA responses [5,31]. Despite the increasing evidence of a close relationship between ROS and proline metabolism linked to hormonal interaction, the hormonal regulation of proline metabolism in relation to endogenous H2O2 levels and different H2O2 sources (e.g., drought-induced and exogenous H2O2), which are partially associated with the discrepancies observed in their regulatory roles in stress response and resistance processes, has rarely been studied.
In the present study, we hypothesized that (1) the regulatory actions of drought-induced H2O2 (as the internal H2O2 trigger) and of exogenous H2O2 are different in ROS signal transduction and (2) the altered H2O2 levels and their signaling modulate proline metabolism with hormonal interaction. To test these hypotheses, antioxidant activity, ABA and SA responses, and proline metabolism were interpreted with respect to the altered H2O2 levels and ROS signaling, in response to drought or exogenous-H2O2 treatment.

2. Materials and Methods

2.1. Plant Materials, Growth Conditions, and Stress Treatments

Plants of the rapeseed (Brassica napus L.) cultivar Capitol were used for this study (Gwangju, Korea) and cultivated as previously described by Lee. et al. [35]. The seedlings at four-leaf stage were transferred to soil-filled 2 L pots and irrigated continuously in complete nutrient solution for 6 weeks. Then, plants were divided into three groups according to morphological similarity. The first group was irrigated with 200 mL of water for the well-watered plants (control), the second with 20 mL of water (drought), and the third was daily foliar-sprayed with 20 mL of 50 µM H2O2 under well-watered conditions (exogenous-H2O2 (Exo-H2O2)) for 10 days. Sampling was performed at 0, 5, and 10 days after treatment. In this study, the mature leaves ranked 4–12 (i.e., rank 1 for the oldest leaf) were considered. After sampling, leaf tissues were cut and frozen immediately in liquid nitrogen and stored in a deep-freezer (−80 °C) until further analysis.

2.2. Measurement of Leaf Water Potential (Ψw) and Chlorophyll Content

For measurement of leaf water potential (Ψw), the seventh leaf was cut and then inserted the pressure chamber (PMS Instruments, Corvallis, OR, USA) to expose the cut end of the petiole on the outside. Afterwards, pressure was applied to the chamber until liquid was observed at the end of petiole, which corresponds to the Ψw. For total chlorophyll, approximately 100 mg of fresh-cut leaves were extracted with 10 mL of 99% dimethyl sulfoxide [36]. After 48 h, the absorbance of the supernatants was read at 645 and 663 nm and calculated using the following formula: total chlorophyll (µg/mL) = 20.2 A645 + 8.02 A663.

2.3. Dtermination of Phytohormones

For the quantification of phytohormones, 50 mg of finely ground fresh leaves was extracted with 500 µL of the extraction solvent (2-propanol/H2O/concentrated HCl (2:1:0.002, v/v/v)) containing d6-ABA and d6-SA as the internal standard (50 ng) for ABA and SA, respectively, for 24 h at 4 °C [37]. The supernatant was mixed with 1 mL of dichloromethane and at 13,000× g for 5 min at 4 °C. After centrifugation, two phases were formed. The supernatant in the lower phase was transferred to clean screw-cap glass vial and dried using a nitrogen evaporator with nitrogen flow. Then, samples were re-suspended in 1 mL of methanol and further purified with filtering through 0.22 μm organic membrane filters. The extracted solution transferred to vials with a glass insert and stored at −80 °C until high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis. Ten microliters of plant extracts were injected onto an Agilent 1100 HPLC system, equipped with a Waters C18 column (150 × 2.1 mm, 5µm) and API3000 MS-MRM (Applied Biosystems, Waltham, MA, USA).

2.4. Antioxidant Enzyme Activities

For extraction of antioxidant enzymes, approximately 500 mg of finely ground fresh samples were extracted with 1.5 mL of 100 mM potassium phosphate buffer pH 7.5 containing 2 mM phenylmethylsulfonyl fluoride. After centrifugation at 14,000× g for 20 min at 4 °C, the supernatants were used as enzyme sources [38]. Protein concentration was determined using Bradford reagent with bovine serum albumin as a proteins standard. For cell wall POX activity, the oxidation of guaiacol was evaluated by monitoring the increase in absorbance at 470 nm for 1 min (coefficient of absorbance, ε = 26.6 mM−1 cm−1) [39]. One unit of enzyme activity was defined as the amount of enzyme causing the formation of 1 M tetraguaiacol per min. SOD activity was measured by its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) [11]. One unit of enzyme activity was defined as the amount of enzyme causing 50% inhibition of NBT photoreduction in comparison with tubes lacking the plant extract. CAT activity was monitored by following the decrease in absorbance at 240 nm due to H2O2 consumption (ε = 36 mM−1 cm−1). One unit of enzyme activity was defined as the amount of enzyme causing the degradation of 1 µmol of H2O2 per min [38].

2.5. Chemical Analysis

Fresh samples (0.5 g) were mixed with 1.5 mL of 50 mM KPO4− buffer (pH 7.0) and centrifuged at 10,000× g for 25 min at 4 °C. After centrifugation, the supernatants were used to determine the superoxide anion radical (O2•–) and H2O2 concentration. The O2•– concentration was conducted according to the method of Lee et al. [11] by O2•– oxidation of hydroxylamine. Briefly, the supernatants were mixed with hydroxylamine solution, incubated for 1 h at 25 °C, and then reacted with 17 mM sulfanilic acid and 7 mM a-naphthylamine for 20 min. The absorbance was read at 530 nm and calculated using the NO2 standard. The H2O2 concentration was measured by the method of Lin and Kao [40]. The supernatants were mixed with 0.1% titanium chloride in 20% H2O2. The absorbance was immediately read at 410 nm. The H2O2 concentration was determined by measuring the absorbance at 410 nm and calculated using the extinction coefficient of 0.28 mM−1 cm−1. The lipid peroxidation level was determined by measuring the concentration of malondialdehyde (MDA), as described previously [3]. Fresh samples (0.5 g) were extracted with 0.1% trichloroacetic acid, centrifuged, mixed with 0.5% tribabutric acid in 20% TCA, and then boiled for 30 min at 95 °C. The absorbance was measured at 532 nm and calculated using the extinction coefficient of 155 mM−1 cm−1. The concentration of proline and pyrroline-5-carboxylate (P5C) was measured using the method described by Bates et al. [41] and Deuschle et al. [42], respectively. Proline and P5C contents in the eluate were quantified using ninhydrin assay and the o-aminobenzaldehyde method, respectively.

2.6. RNA Isolation and Expression Quantification

Fresh leaves (200 mg) were mixed with RNAiso Plus reagent (Takara, Nojihigashi 7-4-38 Kusatsu, Shiga, Japan) for total RNA isolation. First-strand complementary DNA (cDNA) was synthesized with a GoScript Reverse Transcription System (Takara). RT-qPCR reactions were carried out on a BioRad CFX96 qPCR System using the TB Green Premix Ex Taq (Takara). Three biological replications were carried out for each treatment, each with two technical replicates. The relative expression levels of were normalized to actin and calculated using the 2−ΔΔCt method [43]. Supplementary Table S1 provides the gene-specific primer used for qRT-PCR.

2.7. Statistical Analysis

All measurements were performed with three replicates per treatment. The experimental results are presented as mean ± SE. Duncan’s multiple range test was performed to compare the means of separate replicates. Statistical significance was established at p < 0.05. Statistical analysis of all measurements was performed using SAS 9.1.3 software (SAS Institute Inc., Cary, NC, USA). Heatmap and Pearson correlation analyses were conducted using MetaboAnalyst 4.0 (http://www.metaboanalyst.ca, assessed on 20 September 2021).

3. Results

3.1. Leaf Water Potential, Chlorophyll Content, and Lipid Peroxidation Level

Drought and Exo-H2O2 treatments for 10 days significantly decreased the leaf water potential (Ψw) (Table 1). The drought-responsive decrease in Ψw was earlier and higher than that observed in Exo-H2O2 treatment. The chlorophyll content on day 10 also tended to decrease in both drought and Exo-H2O2 treatments compared to the control (Table 1). The concentration of MDA, as a marker of lipid peroxidation caused by oxidative stress, significantly increased to 2.0- and 1.3-fold in drought and Exo-H2O2 treatments, respectively, compared to the control on day 10 (Table 1).

3.2. ROS Status and Antioxidative Enzymes Activity

To quantify the oxidative responses of plants to drought or Exo-H2O2 treatment, we determined the O2•– and H2O2 contents and enzyme activities of POX, SOD, and CAT. The O2•– concentration significantly increased in both the treatments throughout the experimental period (except on day 5 of Exo-H2O2 treatment). On day 10, the accumulation of O2•– was much higher in drought treatment as it showed a 5.2-fold increase, while Exo-H2O2 treatment showed a 2.4-fold increase when compared to the control on day 0 (40.6 pmol g−1 FW) (Figure 1A). A significant increase in H2O2 concentration and accumulation was also observed in both treatments. Drought-responsive H2O2 accumulation was higher than that in Exo-H2O2 treatment (Figure 1B). The enzymatic activity of cell wall POX also increased in both the treatments. The activation of POX in both drought and Exo-H2O2 treatments was not significant on day 5, whereas it was 1.4-fold higher in drought treatment than that in Exo-H2O2 treatment on day 10 (Figure 1C). The activity of SOD progressively increased with time in both the treatments. The SOD activity on day 10 was 46.2 and 33.8 unit mg−1 protein in drought and Exo-H2O2 treatments, respectively (Figure 1D). The CAT activity was not significantly different between treatments throughout the experimental period (Figure 1E). These results indicated that the endogenous H2O2 level affects the ROS status and activities of antioxidant enzymes.

3.3. Endogenous ABA and SA Status, and ABA and SA Synthesis and Signaling Genes Expression

In order to effects of drought or Exo-H2O2 treatment on phytohormone metabolism, ABA and SA contents and their synthesis- and signaling-related gene expressions were measured. Endogenous ABA level on day 5 was significantly increased only in drought-treated leaves. The accumulation of ABA occurred in both treatments, with 22- and 6.4-fold increase in drought and Exo-H2O2 treatments, respectively, compared to the control on day 0 (Table 2). Levels of SA were significantly higher on day 5, then decreased on day 10 in both treatments. The highest SA level was recorded on day 5 in the Exo-H2O2 treatment, with a 1.9-fold higher level than that measured in the drought treatment (Table 2). The resulting ratio of ABA/SA on day 10 was 27.55 and 6.12 in drought and Exo-H2O2 treatments, respectively (Table 2).
The expression of the ABA synthesis-related gene, 9-cis-epoxycarotenoid dioxygenase (NCED3), was enhanced from day 5 (e.g., 4.2- and 2.3-fold in drought and Exo-H2O2 treatments, respectively) and continued until day 10. The enhancement of NCED3 was much higher in the drought treatment (7.6-fold) (Figure 2A). The expressions of the ABA receptor gene (PYL1) and ABA signaling gene (MYC2) were also highly enhanced in the drought treatment compared to that in Exo-H2O2 treatment throughout the experimental period (Figure 2B,C). Higher expression of the SA synthesis gene, iso-chorismate synthase 1 (ICS1), was observed in both drought (4.6-fold) and Exo-H2O2 treatments (5.1-fold) on day 5, which then decreased up until 10 days, but the expression of ICS1 was significantly higher in Exo-H2O2 than in the drought treatment (Figure 2D). The expression of the non-race-specific-disease resistance 1 gene (NDR1) on day 5 was significantly higher in drought treatment than in Exo-H2O2 treatment, but it reversed on day 10 (Figure 2E). The expression of the SA signaling gene, non-expressor of pathogenesis-related gene (NPR1), followed a similar pattern of endogenous SA level throughout the experimental period (Figure 2F). Therefore, it appears that the SA synthesis- and signaling-related pathway is promoted at a lower endogenous H2O2 level, whereas the ABA synthesis- and signaling-related pathway is enhanced at a higher endogenous H2O2 level.

3.4. Production of ROS and Expression of ROS Signaling Genes

The expression of NADPH oxidase-encoding gene was significantly upregulated only in the drought treatment (5.1-fold) on day 5, but then was continuously enhanced in both treatments. The enhancement was much higher in drought treatment on day 10, as shown by a 10.2- and 4.4-fold increase in the drought and Exo-H2O2 treatments, respectively (Figure 3A). One of the ROS-responsive signaling genes, MAPK6, was significantly enhanced only in the drought treatment (3.2-fold) on day 5, and highly enhanced in both treatments on day 10 (9.8- and 5.0-fold in drought and Exo-H2O2 treatments, respectively) (Figure 3B). The expression of OXI1 showed responses similar to those of MAPK6, representing a 8.1- and 5.9-fold higher expression in drought and Exo-H2O2 treatments, respectively, on day 10 (Figure 3C).

3.5. Proline and P5C Concentration, and Proline Metabolism-Related Gene Expression

To evaluate the effects of altered H2O2 levels on proline metabolism, P5C and proline concentrations and proline metabolism-related gene expression were evaluated. Drought Exo-H2O2 treatments showed a progressive increase in P5C concentration. Drought-responsive enhancement of P5C was significantly higher than Exo-H2O2-responsive enhancement, as shown by 5.8- and 2.4-fold increased levels after 10 days in drought and Exo-H2O2 treatments, respectively, compared to the control (Figure 4A). The increase in proline concentration and its accumulation was also significant in both the treatments. The increase in proline concentration was much higher in drought treatment than in Exo-H2O2 treatment, with a 14.1- and 2.7-fold increase on day 10 in drought and Exo-H2O2 treatments, respectively, compared to the control on day 0 (Figure 4B). The resulting ratio of proline/P5C on day 10 was 3.9 and 1.8 in drought and Exo-H2O2 treatments, respectively.
The expression of P5CS-encoding genes (P5CS1 and P5CS2) enhanced in a pattern with a much higher expression in drought treatment during the first 5 days and then a significant decrease in both treatments on day 10 (Figure 4C,D). The expression of P5C reductase-encoding gene (P5CR) enhanced to 4.4- and 3.3-fold in drought and Exo-H2O2 treatments, respectively, on day 5. The P5CR expression in drought continued to enhance (7.7-fold), while it remained restricted in Exo-H2O2 (2.2-fold) treatment (Figure 4E). The expression of ProDH reduced in both treatments on day 5, while it was enhanced strongly in drought (6.4-fold) and slightly in Exo-H2O2 (2.4-fold) treatments on day 10 (Figure 4F). The expression of the P5C dehydrogenase-encoding gene (P5CDH) was continuously reduced along with the progression of drought, whereas it was remarkably enhanced on 10 days after Exo-H2O2 treatment (Figure 4G). Thus, drought-induced H2O2 leads to high upregulation of P5CR and ProDH expression, resulting in increase of the proline/P5C ratio, compared to Exo-H2O2 treatment.

3.6. Heatmap Visualization and Pearson Correlation Analysis among the Metabolites or Gene Expression

To further examine the functional implications and correlations of the measured metabolites and gene expression levels in drought and Exo-H2O2 treatments, a heatmap and Pearson’s correlation coefficients were adapted (Figure 5). Drought had a more positive influence on ABA, ROS, P5C, and proline levels, as well as on the ABA/SA ratio, when compared with that of Exo-H2O2 (Figure 5A). Altered ROS level, which positively regulated the expression of MAPK6 and OXI1, was closely correlated with ABA and the ABA/SA ratio. The close relationships between ROS and ABA were also found to be positively correlated with P5C and proline, which had a strong correlation with P5CR and ProDH, as well as with ABA-regulated genes (NCED3, PYL1, and MYC2) (Figure 5B).

4. Discussion

4.1. ROS and Hormone Responses to Drought-Induced H2O2 and Exogenous H2O2

Droughts reduce leaf water potential, which is used as an index of the water statues [3,35]. In the present study, a significant decrease of leaf water potential was observed in drought-stressed plants, accompanied by the loss of chlorophyll and enhanced lipid peroxidation level (Table 1). The accumulation of O2•– and H2O2, an early response to various stress stimuli [6,20], occurred in both drought and Exo-H2O2 treatments. The endogenous H2O2 levels in Exo-H2O2 (68.2 nmol g−1 FW) on day 10 corresponded to that of 76% of drought treatment (Figure 1B), which is similar to the results obtained on day 5 of drought treatment in a previous study [6]. During the first 5 days of treatments, the increase in H2O2 concentration was concomitant with the enhanced SOD activity (Figure 1D) and NADPH oxidase expression (Figure 3A) in drought treatment and with POX activity in Exo-H2O2 treatment (Figure 1C). Wang et al. [44] reported that endogenous H2O2 accumulation in the apoplast was triggered by both cell wall peroxidase and membrane-linked NADPH oxidase. It has been documented that SA is involved in the production of H2O2, leading to SA-induced abiotic and biotic stress resistance [20,45]. In the present study, during the first 5 days when the increase in SA levels was predominant, the increased endogenous H2O2 level coincided with the increased activation of SA-dependent POX in the Exo-H2O2 treatment, and was mainly due to ABA- and/or SA-dependent SOD activity and NADPH oxidase expression in the drought treatment (Table 2, Figure 1C,D and Figure 3A), in accordance with SA-mediated H2O2 production by activating membrane-linked NADPH oxidase [46] and cell wall peroxidase [44,47]. During the latter 5 days, when ABA accumulation with an antagonistic depression of SA was remarked, the accumulation of H2O2 occurred with a proportional enhancement of POX and SOD activity and NADPH oxidase expression in an ABA-dependent manner (Table 2, Figure 1C,D and Figure 3A). Ample evidence has shown that H2O2 generated by SA-mediated POX and NADPH oxidases acts downstream of ABA signaling in mediating drought-induced stress responses [47,48,49,50]. Our recent study reported that SA-stimulated H2O2 accumulation and SA responses during the early drought phase are part of upstream H2O2-stimulated ABA accumulation, which causes ABA signaling and responses, leading to severe drought symptoms during the late phase [6]. The present data indicate that during the first 5 days, H2O2 is produced mainly from SA-mediated activation of NADPH oxidase in drought treatment and POX in the Exo-H2O2 treatment, whereas ROS accumulation at day 10 was due to the increase in SOD activity (an H2O2-producing enzyme), and in NADPH induction (superoxide-producing enzyme), respectively, in an ABA-dependent manner.

4.2. H2O2-Responsive Interaction between ROS and Hormonal Signaling

The altered endogenous H2O2 level was strongly correlated (p < 0.001) to the expression of two protein kinases (MAPK6 and OXI1), which are an essential part of the signal transduction pathway linking oxidative burst signaling to diverse downstream responses [2,13,34,51]. MAPKs, which are downstream of OXI1 [13], are known to be involved in H2O2 signaling ability for regulating hormonal and metabolic responses [6,22,29,50]. With respect to ROS signaling, a preponderance of evidence supports ABA as a key regulator of stress responses [5,6,34]. Indeed, in the present study, the endogenous ABA level (Table 2) and the expression of NCED3 and MYC2 (Figure 2A,C) in drought treatment were consistent with a progressive increase in the endogenous H2O2 level, leading to a proportional upregulation of MAPK6 (Figure 3B) and OXI1 (Figure 3C), while the expression of these genes was not significantly activated in Exo-H2O2 treatment during the first 5 days when the ABA level did not change significantly (Figure 3). Moreover, the overall pattern of MAPK6 and OXI1 (Figure 3B,C) indicated that they are ROS level-responsive (Figure 1A,B) ABA-regulated genes (Figure 2A–C), in accordance with our previous results obtained from a time course of drought intensity [6]. Similarly, in Arabidopsis, overexpression of AtMPK6 enhanced the ABA-dependent H2O2 production, which is blocked in the mpk6 mutant [52,53]. In addition, the inhibition of MAPK signaling by PD98059 decreases sensitivity to the response of ABA under drought conditions [54]. The endogenous SA level and the expression of SA-related genes (ICS1 and NPR1) in drought treatment (Figure 2D,F) progressively decreased with an antagonistic increase in the ABA level (Table 2) and ABA-related gene expression (Figure 2A–C), along with ROS accumulation (Figure 1A,B). The SA-signaling genes (NDR1 and NPR1) were highly developed in Exo-H2O2 on day 5 (Figure 2E,F), in which the endogenous H2O2 level was relatively lower (≤20 nmol g−1 FW) (Figure 1B). These enhanced SA-signaling genes did not significantly activate MAPK6 and OXI1 (Figure 3B,C). The time-course analysis showed that the crosstalk between H2O2 and SA has a much earlier peak than in ABA [6,55]. In the present study, an altered endogenous H2O2 level was highly correlated with increased ABA and ABA-regulated genes, but not with SA responses (Figure 5B). Therefore, a positive feedback loop between H2O2- and ABA-mediated pathways might lead to upregulation of MAPK6 via OXI1, thereby activating ABA synthesis and signaling genes (NCED3 and MYC2). Thus, the actions of this core pathway in the control of proline metabolism under drought stress needs to be discussed further.

4.3. H2O2-Responsive Hormonal Regulation of Proline Metabolism

Along with ROS accumulation, proline is the most common free amino acid to accumulate in the plants exposed to drought stress [5,30,35,56]. Based on the data obtained during the entire experimental period, the correlation between endogenous H2O2 level, P5C, and proline concentration was highly positive, and these parameters were also positively correlated with ROS- and ABA-signaling genes’ expression (Figure 5B). Indeed, it has been documented that H2O2 causes proline accumulation or promotes ABA-induced proline accumulation [23,57]. Previous results reveal that H2O2 produced by NADPH oxidase increases proline accumulation under drought or osmotic stress [5,27], with a strong correlation with ABA accumulation [34,56]. However, in the present study, the data collected in Exo-H2O2, especially during the first 5 days, did not directly match these linear relationships. For instance, the enhanced H2O2 level in Exo-H2O2 treatment led to a significant increase in P5C and proline levels (Figure 4A,B), although the ABA level and NADPH oxidase gene expression did not change during the first 5 days (Table 2, Figure 3A). The Exo-H2O2-responsive increases in P5C and proline levels were found to be associated with SA-dependent proline synthesis-related genes (Figure 4C–E), possibly related to SA-mediated activation of POX and not with the NADPH oxidase-dependent process (Figure 1C). Apart from these early responses to Exo-H2O2, the enhanced P5C and proline levels and their accumulation under drought stress were found to be parallel with upregulated NADPH oxidase (Figure 3A) in an ABA-related pattern, with an increasing endogenous-H2O2 concentration (Figure 1B). These results clearly indicate that drought-induced H2O2 (often considered the internal H2O2 trigger) might be directly involved in triggering the NADPH oxidase-dependent proline synthesis, but not in Exo-H2O2. In drought treatment, P5C and proline levels increased (Figure 4A,B) along with H2O2 accumulation, with proportional enhancements of ROS-signaling genes (OXI1 and MAPK6) (Figure 3B,C) and ABA-regulated genes (NCED3, PYL1, and MYC2) (Figure 2A–C), suggesting a greater ABA-dependent proline accumulation in higher H2O2 level. This observation further indicates that higher proline accumulation does not directly contribute to the scavenging of cellular ROS, although studies show that proline metabolism has a function as ROS scavenger [30]. The proline levels in plant cells depend on tight regulation of its biosynthesis and degradation catabolism. Proline accumulation under stress is accompanied by the upregulation of proline biosynthesis (P5CS and P5CR) and downregulation of proline catabolism-related genes (ProDH and P5CDH) [5,6,33,35], in an ABA-dependent manner [31,34]. Indeed, in drought treatment, P5C and proline accumulation occurred with highly enhanced expression of P5CS1, P5CS1, and P5CR (Figure 4C–E), accompanied with a progressive enhancement of NADPH oxidase (Figure 3A), which coincided with the enhanced H2O2-responsive increases in ABA level (Table 2) and ABA-regulated genes’ expressions (Figure 2A–C). This observation confirmed the given hypothesis that H2O2 generated by NADPH oxidases acts downstream of ABA signaling [48,49,58], and involves in proline accumulation by upregulating P5CS [27]. However, in Exo-H2O2 treatment, the pattern of P5C and proline was not directly associated with those of H2O2- and ABA-mediated NADPH oxidase, as shown lower activation of P5CS, P5CR (Figure 4C–E), and NADPH oxidase (Figure 3A), even though endogenous H2O2 was enhanced up to 68.2 nmol g−1 FW on day 10 (Figure 1B). Besides the interaction between H2O2- and ABA-signaling in proline biosynthesis, it was noteworthy that significant enhancements of P5CS and P5CR also coincided with the increased SA level and upregulation of SA-related genes (Figure 2D–F), especially when endogenous H2O2 was less than 42 nmol g−1 FW (e.g., during the first 5 days after both treatments). This observation suggests that SA signaling is also involved in the activation of proline synthesis as part of early stress response, confirming the role of SA as a signal of different types of stresses [20,45,59]. In our previous studies, leaf spraying with 30 mL of 0.5 mM SA enhanced enzymatic activity of SOD and their encoding genes, and induced proline accumulation with enhanced synthesis-related genes (P5CS and P5CR) [5].
Proline is oxidized to glutamate by the sequential action of ProDH and P5CDH [30,60]. In the present study, the expression of ProDH tended to decrease with increasing endogenous proline levels, except in the data of 10 days after drought treatment (Figure 4B,F). The enhanced activity of ProDH leads to ROS formation in mitochondria by coupling proline oxidation to reduction of the respiratory electron transport chain [30,57]. Indeed, the highest activation of ProDH (on 10 days after drought treatment) coincided with the most accumulated proline and ROS level in an ABA-dependent manner (Figure 1B and Figure 4B,F). Mani et al. [60] reported that altered levels of ProDH cause hypersensitivity to proline and its analog. Moreover, an exceptional enhancement of P5CDH, the second enzyme for proline catabolism, was observed on day 10 following Exo-H2O2 treatment, which showed a considerable accumulation (68.2 nmol g−1 FW) of endogenous H2O2 (Figure 1B). Thus, an overexpression of ProDH and P5CDH might be part of the hypertensive response to over-produce H2O2 and/or proline, predicting an increase in proline-P5C cycling, leading to ROS accumulation [25,57,61].

5. Conclusions

The present data indicate that hormonal interaction with proline metabolism is governed by endogenous levels of ROS (especially H2O2), as shown by SA-mediated activation of proline synthesis at lower endogenous H2O2 levels, and a predominant ABA-dependent proline accumulation along with ROS accumulation. Furthermore, to the best of our knowledge, the present data are the first to report that H2O2-responsive SA and ABA involves in ROS signaling and proline metabolism in rapeseed leaves (Figure 6), representing two distinct phases characterized by the following: (1) an active NDR1-mediated SA-dependent proline synthesis with upregulation of P5CS and P5CR, and depression of ProDH as an acclamatory process at lower level of endogenous H2O2 produced by either Exo-H2O2 or drought treatment; and (2) drought-induced proline accumulation with ABA-dependent MAPK6 activation via OXI1, leading to upregulation of ProDH as hypersensitive responses to a higher H2O2 level. Future studies are necessary (1) to define the threshold at which proline level switches from inducing cellular protection to hypersensitivity to over-produced ROS and/or proline, and (2) to elucidate hormonal interaction with proline in redox regulation.

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/antiox11030566/s1, Supplementary Table S1: Primer sequences used for qRT-PCR analysis.

Author Contributions

Conceptualization, B.-R.L., V.H.L. and T.-H.K.; methodology, B.-R.L., V.H.L. and T.-H.K.; formal analysis B.-R.L., V.H.L., S.-H.P., M.A.M. and D.-W.B.; investigation, B.-R.L., V.H.L. and T.-H.K.; writing—original draft preparation, B.-R.L., V.H.L. and T.-H.K.; review and editing, B.-R.L., V.H.L., S.-H.P., M.A.M., D.-W.B. and T.-H.K.; supervision, T.-H.K.; project administration, T.-H.K.; funding acquisition, T.-H.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a grant from the National Research Foundation of South Korea under project NRF-2021R1A4A1031220.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and its Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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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 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.
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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 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.
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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 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.
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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 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.
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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 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.
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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.
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.
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Table
Table 1. The changes in leaf water potential (Ψw), chlorophyll content, and lipid peroxidation level under control, exogenous H2O2 (Exo-H2O2), or drought treatments for 10 days.
Table 1. The changes in leaf water potential (Ψw), chlorophyll content, and lipid peroxidation level under control, exogenous H2O2 (Exo-H2O2), or drought treatments for 10 days.
Days after Treatment
0510
Leaf water potential (Ψw, MPa)
Control−0.44 ± 0.02 a−0.44 ± 0.02 a−0.48 ± 0.02 a
Drought −0.66 ± 0.03 b−1.20 ± 0.02 d
Exo-H2 O2 −0.47 ± 0.02 a−1.07 ± 0.04 c
Chlorophyll (mg g−1 FW)
Control1.61 ± 0.08 ab1.73 ± 0.10 a1.62 ± 0.09 ab
Drought 1.45 ± 0.07 abc1.22 ± 0.06 c
Exo-H2 O2 1.63 ± 0.09 ab1.34 ± 0.05 bc
Lipid peroxidation (MDA, nmol g−1 FW)
Control4.55 ± 0.24 d4.46 ± 0.32 d4.84 ± 0.23 cd
Drought 5.85 ± 0.24 bc9.45 ± 0.33 a
Exo-H2O2 4.80 ± 0.35 cd6.33 ± 0.43 b
Results are represented as mean ± SE for n = 3. Different letters in a vertical column or a horizontal row indicate values that are significantly different at p < 0.05 according to Duncan’s multiple range test.
Table
Table 2. The changes in levels of endogenous abscisic acid (ABA) and salicylic acid (SA), and in the ratio of ABA to SA under control, drought, or exogenous H2O2 (Exo-H2O2) treatments for 10 days.
Table 2. The changes in levels of endogenous abscisic acid (ABA) and salicylic acid (SA), and in the ratio of ABA to SA under control, drought, or exogenous H2O2 (Exo-H2O2) treatments for 10 days.
Days after Treatment
0510
ABA (ng g−1 FW)
Control5.24 ± 0.29 c6.44 ± 0.36 c5.93 ± 0.11 c
Drought 32.66 ± 0.04 b115.09 ± 5.56 a
Exo-H2O2 7.24 ± 0.14 c33.45 ± 0.34 b
SA (ng g−1 FW)
Control2.21 ± 0.20 f2.69 ± 0.36 ef3.21 ± 0.11 e
Drought 7.09 ± 0.13 b4.18 ± 0.19 d
Exo-H2O2 13.45 ± 0.37 a5.49 ± 0.24 c
ABA/SA ratio
Control2.40 ± 0.13 d2.48 ± 0.20 d1.85 ± 0.03 d
Drought 4.61 ± 0.08 c27.55 ± 0.74 a
Exo-H2O2 0.54 ± 0.01 e6.12 ± 0.23 b
Results are represented as mean ± SE for n = 3. Different letters in a vertical column or a horizontal row indicate values that are significantly different at p < 0.05 according to Duncan’s multiple range test.
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Lee, B.-R.; La, V.H.; Park, S.-H.; Mamun, M.A.; Bae, D.-W.; Kim, T.-H. H2O2-Responsive Hormonal Status Involves Oxidative Burst Signaling and Proline Metabolism in Rapeseed Leaves. Antioxidants 2022, 11, 566. https://0-doi-org.brum.beds.ac.uk/10.3390/antiox11030566

AMA Style

Lee B-R, La VH, Park S-H, Mamun MA, Bae D-W, Kim T-H. H2O2-Responsive Hormonal Status Involves Oxidative Burst Signaling and Proline Metabolism in Rapeseed Leaves. Antioxidants. 2022; 11(3):566. https://0-doi-org.brum.beds.ac.uk/10.3390/antiox11030566

Chicago/Turabian Style

Lee, Bok-Rye, Van Hien La, Sang-Hyun Park, Md Al Mamun, Dong-Won Bae, and Tae-Hwan Kim. 2022. "H2O2-Responsive Hormonal Status Involves Oxidative Burst Signaling and Proline Metabolism in Rapeseed Leaves" Antioxidants 11, no. 3: 566. https://0-doi-org.brum.beds.ac.uk/10.3390/antiox11030566

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