Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (18)

MAPK Pathway Under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms

Affiliations

MAPK Pathway Under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms

Fernanda Rodríguez-Rojas et al. Int J Mol Sci.

Abstract

Following the physiological complementary/parallel Celis-Plá et al., by inhibiting extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and cytokinin specific binding protein (p38), we assessed the role of the mitogen-activated protein kinases (MAPK) pathway in detoxification responses mediated by chronic copper (10 µM) in U. compressa. Parameters were taken at 6, 24, and 48 h, and 6 days (d). H2O2 and lipid peroxidation under copper and inhibition of ERK, JNK, or p38 alone increased but recovered by the sixth day. By blocking two or more MAPKs under copper, H2O2 and lipid peroxidation decayed even below controls. Inhibition of more than one MAPK (at 6 d) caused a decrease in total glutathione (reduced glutathione (GSH) + oxidised glutathione (GSSG)) and ascorbate (reduced ascorbate (ASC) + dehydroascorbate (DHA)), although in the latter it did not occur when the whole MAPK was blocked. Catalase (CAT), superoxide dismutase (SOD), thioredoxin (TRX) ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione synthase (GS), were downregulated when blocking more than one MAPK pathway. When one MAPK pathway was blocked under copper, a recovery and even enhancement of detoxification mechanisms was observed, likely due to crosstalk within the MAPKs and/or other signalling processes. In contrast, when more than one MAPK pathway were blocked under copper, impairment of detoxification defences occurred, demonstrating that MAPKs were key signalling mechanisms for detoxification in macroalgae.

Keywords: Ulva compressa; antioxidant; metal chelator; mitogen-activated protein kinases; oxidative stress.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Hydroxide peroxide (H2O2) levels in U. compressa under copper and/or exposure to mitogen-activated protein kinases (MAPK) inhibitors. Treatments consisted in: T1) control only with seawater; T2) solely copper exposure as 10 µM of CuSO4 (Cu); T3) copper + 5 µM MAPK extracellular signal regulated kinases (ERK) inhibitor PD98059 (Cu + ERKi); T4) copper + 5 µM MAPK c-Jun N-terminal kinases (JNK) inhibitor SP600125 (Cu + JNKi); T5) copper + MAPK cytokinin specific binding protein (p38) inhibitor SB203580 (Cu + p38i); T6) Cu + ERKi + JNKi; T7) Cu + ERKi + p38i; T8) Cu + p38i + JNKi; and T9) Cu + ERKi + JNKi + p38i. Samples were analysed after 6 h (A), 24 h (B), 48 h (C), and 6 d (D) treatments. Different letters represent significant difference at 95% confidence interval (p < 0.05). Plots are represented as mean ± SE (n = 3).
Figure 2
Figure 2
Thiobarbituric acid reactive substances (TBARS) accumulation in U. compressa under copper and/or exposure to MAPK inhibitors. Treatments consisted in: T1) control only with seawater; T2) solely copper exposure as 10 µM of CuSO4 (Cu); T3) copper + 5 µM MAPK ERK inhibitor PD98059 (Cu + ERKi); T4) copper + 5 µM MAPK JNK inhibitor SP600125 (Cu + JNKi); T5) copper + MAPK p38 inhibitor SB203580 (Cu + p38i); T6) Cu + ERKi + JNKi; T7) Cu + ERKi + p38i; T8) Cu + p38i + JNKi; and T9) Cu + ERKi + JNKi + p38i. Samples were analyzed after 6 h (A), 24 h (B), 48 h (C) and 6 d (D) treatments. Different letters represent significant difference at 95% confidence interval (p < 0.05). Plots are represented as mean ± SE (n = 3).
Figure 3
Figure 3
Total reduced (GSH) and oxidised glutathione (GSSG) content in U. compressa under copper and/or exposure to MAPK inhibitors. Treatments consisted in: T1) control only with seawater; T2) Solely copper exposure as 10 µM CuSO4 (Cu); T3) copper + 5 µM MAPK ERK inhibitor PD98059 (Cu + ERKi); T4) copper + 5 µM MAPK JNK inhibitor SP600125 (Cu + JNKi); T5) copper + MAPK p38 inhibitor SB203580 (Cu + p38i); T6) Cu + ERKi + JNKi; T7) Cu + ERKi + p38i; T8) Cu + p38i + JNKi; and T9) Cu + ERKi + JNKi + p38i. Samples were analysed after 6 h (A), 24 h (B), 48 h (C), and 6 d (D) treatments. Different letters represent significant difference at 95% confidence interval (p < 0.05). Plots are represented as mean ± SE (n = 3).
Figure 4
Figure 4
Total reduced ascorbate (ASC) and dehydroascorbate (DHA) concentrations in U. compressa under copper and/or exposure to MAPK inhibitors. Treatments consisted in: T1) control only with seawater; T2) solely copper exposure as 10 µM of CuSO4 (Cu); T3) copper + 5 µM MAPK ERK inhibitor PD98059 (Cu + ERKi); T4) copper + 5 µM MAPK JNK inhibitor SP600125 (Cu + JNKi); T5) copper + MAPK p38 inhibitor SB203580 (Cu + p38i); T6) Cu + ERKi + JNKi; T7) Cu + ERKi + p38i; T8) Cu + p38i + JNKi; and T9) Cu + ERKi + JNKi + p38i. Samples were analysed after 6 h (A), 24 h (B), 48 h (C), and 6 d (D) treatments. Different letters represent significant difference at 95% confidence interval (p < 0.05). Plots are represented as mean ± SE (n = 3).
Figure 5
Figure 5
Relative expression of the genes catalase (CAT; A), superoxide dismutase (SOD; B), dehydroascorbate reductase (DHAR; C) in U. compressa under copper and/or exposure to MAPK inhibitors. Expression was relative to the levels of transcripts in U. compressa under control conditions. Treatments consisted in: T1) solely copper exposure as 10 µM of CuSO4 (Cu); T2) copper + 5 µM MAPK ERK inhibitor PD98059 (Cu + ERKi); T3) copper + 5 µM MAPK JNK inhibitor SP600125 (Cu + JNKi); T4) copper + MAPK p38 inhibitor SB203580 (Cu + p38i); T5) Cu + ERKi + JNKi; T6) Cu + ERKi + p38i; T7) Cu + p38i + JNKi; and T8) Cu + ERKi + JNKi + p38i. Samples were analysed after 6 h (A), 24 h (B), 48 h (C), and 6 d (D) treatments. Different letters, in italics and/or with asterisk represent significant difference at 95% confidence interval (p < 0.05) within each experimental time. Plots are represented as mean ± SE (n = 3).
Figure 6
Figure 6
Relative expression of the genes glutathione synthase (GS; A), thioredoxin (TRX; B), and dehydroascorbate reductase (DHAR; C) in U. compressa under copper and/or exposure to MAPK inhibitors. Expression was relative to the levels of transcripts in U. compressa under control conditions. Treatments consisted in: T1) solely copper exposure as 10 µM of CuSO4 (Cu); T2) copper + 5 µM MAPK ERK inhibitor PD98059 (Cu + ERKi); T3) copper + 5 µM MAPK JNK inhibitor SP600125 (Cu + JNKi); T4) copper + MAPK p38 inhibitor SB203580 (Cu + p38i); T5) Cu + ERKi + JNKi; T6) Cu + ERKi + p38i; T7) Cu + p38i + JNKi; and T8) Cu + ERKi + JNKi + p38i. Samples were analysed after 6 h (A), 24 h (B), 48 h (C), and 6 d (D) treatments. Different letters, in italics and/or with asterisk represent significant difference at 95% confidence interval (p < 0.05) within each experimental time. Plots are represented as mean ± SE (n = 3).
Figure 7
Figure 7
Principal components ordination (PCO) analysis diagrams in relation with time (a): T1: 6 h, T2: 24 h, T3: 48 h, and T4: 6 d; and treatments (b): t1: Control only with seawater, t2: Solely copper exposure as 10 µM of CuSO4 (Cu), t3: copper + 5 µM MAPK ERK inhibitor PD98059 (Cu + ERKi), t4: Copper + 5 µM MAPK JNK inhibitor SP600125 (Cu + JNKi), t5: Copper + MAPK p38 inhibitor SB203580 (Cu + p38i), t6: Cu + ERKi + JNKi, t7: Cu + ERKi + p38i, t8: Cu + p38i + JNKi, and t9: Cu + ERKi + JNKi + p38i. Vectors overlay (Sperman rank correlation) indicate the relationship between the PCO axes and the parameters H2O2, TBARS (thiobarbituric acid reactive substance), GSH (reduced glutathione), GSSG (oxidised glutathione), ASC (reduced ascorbate), DHA (dehydroascorbate), and relative gene expression catalase (CAT), superoxide dismutase (SOD), dehydroascorbate reductase (DHAR), thioredoxin (TRX), ascorbate peroxidase (APX), and glutathione synthase (GS).
Figure 8
Figure 8
Principal Components Ordination (PCO) analysis diagrams considering the data covered in this article and in the parallel/complementary Celis-Plá et al. [1]. PCOs are related with time (a): T1: 6 h, T2: 24 h, T3: 48 h, and T4: 6 d; and treatments (b): t1: control only with seawater, t2: solely copper exposure as 10 µM CuSO4 (Cu), t3: copper + 5 µM MAPK ERK inhibitor PD98059 (Cu + ERKi), t4: copper + 5 µM MAPK JNK inhibitor SP600125 (Cu + JNKi), T5: copper + MAPK p38 inhibitor SB203580 (Cu + p38i), t6: Cu + ERKi + JNKi, t7: Cu + ERKi + p38i, t8: Cu + p38i + JNKi, and t9: Cu + ERKi + JNKi + p38i. Vectors overlay (Sperman rank correlation) indicate the relationship between the PCO axes and the parameters H2O2, TBARS (thiobarbituric acid reactive substance), GSH (reduced glutathione), GSSG (oxidised glutathione), ASC (reduced ascorbate), DHA (dehydroascorbate), and relative gene expression catalase (CAT), superoxide dismutase (SOD), dehydroascorbate reductase (DHAR), thioredoxin (TRX), ascorbate peroxidase (APX), and glutathione synthase (GS). Furthermore, the physiological variables intracellular copper accumulation (copper photoinhibition (Fv/Fm) productivity (ETRmax), efficiency (αETR) and saturation of irradiance (EkETR), accompanied by higher non-photochemical quenching (NPQmax) [1].

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Celis-Plá P.S.M., Rodríguez-Rojas F., Méndez L., Moenne F., Muñoz P., Lobos M., Díaz P., Sánchez-Lizaso J.L., Brown M., Moenne A., et al. MAPK pathway under chronic copper excess in green macroalgae (Chlorophyta): Influence on metal exclusion/extrusion mechanisms and photosynthesis. Int. J. Mol. Sci. 2019 in press. - PMC - PubMed
    1. Moenne A., González A., Sáez C.A. Mechanisms of metal tolerance in marine macroalgae, with emphasis on copper tolerance in Chlorophyta and Rhodophyta. Aquat. Toxicol. 2016;176:30–37. doi: 10.1016/j.aquatox.2016.04.015. - DOI - PubMed
    1. Navarrete A., González A., Gómez M., Contreras R.A., Díaz P., Lobos G., Brown M.T., Sáez C.A., Moenne A. Copper excess detoxification is mediated by a coordinated and complementary induction of glutathione, phytochelatins and metallothioneins in the green seaweed Ulva compressa. Plant Physiol. Biochem. 2019;135:423–431. doi: 10.1016/j.plaphy.2018.11.019. - DOI - PubMed
    1. Foyer C.H., Noctor G. Ascorbate and glutathione: The heart of the redox hub. Plant Physiol. 2011;155:2–18. doi: 10.1104/pp.110.167569. - DOI - PMC - PubMed
    1. Sáez C.A., Roncarati F., Moenne A., Moody J.A., Brown M.T. Copper-induced intra-specific oxidative damage and antioxidant responses in strains of the brown alga Ectocarpus siliculosus with different pollution histories. Aquat. Toxicol. 2015;159:81–89. doi: 10.1016/j.aquatox.2014.11.019. - DOI - PubMed

MeSH terms

Feedback