ROS-Scavenging Enzymes as an Antioxidant Response to High Concentration of Anthracene in the Liverwort Marchantia polymorpha L.

plants Article ROS-Scavenging Enzymes as an Antioxidant Response to High Concentration of Anthracene in the Liverwort Marchantia polymorpha L. Nahuel Spinedi 1,†, Romina Storb 1,†, Elisabet Aranda 2, Facundo Romani 3,4 , Maya Svriz 5, Santiago A. Varela 6, Javier E. Moreno 3,* , Sebastian Fracchia 7, Juan Cabrera 1, Ramón Alberto Batista-García8, Inés Ponce de León9and J. Martín Scervino 1,*,†   Citation: Spinedi, N.; Storb, R.; Aranda, E.; Romani, F.; Svriz, M.; Varela, S.A.; Moreno, J.E.; Fracchia, S.; Cabrera, J.; Batista-García, R.A.; et al. ROS-Scavenging Enzymes as an Antioxidant Response to High Concentration of Anthracene in the Liverwort Marchantia polymorpha L. Plants 2021,10, 1478. https:// doi.org/10.3390/plants10071478 Academic Editors: Pedro Piedras and Gregorio Gálvez-Valdivieso Received: 9 June 2021 Accepted: 15 July 2021 Published: 19 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Instituto de Investigaciones en Biodiversidad y Medioambiente (INIBIOMA), CONICET-UNCo, SC Bariloche, Río Negro 8400, Argentina; [email protected] (N.S.); [email protected] (R.S.); [email protected] (J.C.) 2 Institute of Water Research, University of Granada, Ramón y Cajal, 4, Bldg. Fray Luís, 18071 Granada, Spain; [email protected] 3Instituto de Agrobiotecnología del Litoral, UNL-Conicet, Facultad de Bioquímica y Ciencias Biológicas, Santa Fe 3000, Argentina; r[email protected] or [email protected] 4Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK 5Instituto de Investigaciones en Recursos Naturales, Agroecología y Desarrollo Rural (IRNAD), Universidad Nacional de Río Negro, CONICET, SC Bariloche, Río Negro 8400, Argentina; [email protected] 6Grupo de Ecología Forestal, Instituto Nacional de Tecnología Agropecuaria (INTA) EEA Bariloche, CC 277, Bariloche 8400, Argentina; santiago.var[email protected] 7Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja (CRILAR), UNLAR, SEGEMAR, UNCa, CONICET, Entre Ríos y Mendoza, 530 Anillaco, La Rioja 5300, Argentina; [email protected] 8Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Ave Universidad 1001, Col. Chamilpa, CP 62209 Cuernavaca, Morelos, Mexico; [email protected] 9Departamento de Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, CP 11600 Montevideo, Uruguay; [email protected] *Correspondence: javier.mor[email protected].ar (J.E.M.); [email protected] (J.M.S.) † Both authors contributed equally to this study. Abstract: Marchantia polymorpha L. responds to environmental changes using a myriad set of physiological responses, some unique to the lineage related to the lack of a vascularand root-system. This study investigates the physiological response of M. polymorpha to high doses of anthracene analysing the antioxidant enzymes and their relationship with the photosynthetic processes, as well as their transcriptomic response. We found an anthracene dose-dependent response reducing plant biomass and associated to an alteration of the ultrastructure of a 23.6% of chloroplasts. Despite a reduction in total thallus-chlorophyll of 31.6% of Chl aand 38.4% of Chl b, this was not accompanied by a significant change in the net photosynthesis rate and maximum quantum efficiency (Fv/Fm). However, we found an increase in the activity of main ROS-detoxifying enzymes of 34.09% of peroxidase and 692% of ascorbate peroxidase, supported at transcriptional level with the upregulation of ROS-related detoxifying responses. Finally, we found that M. polymorpha tolerated anthracene-stress under the lowest concentration used and can suffer physiological alterations under higher concentrations tested related to the accumulation of anthracene within plant tissues. Our results show that M. polymorpha under PAH stress condition activated two complementary physiological responses including the activation of antioxidant mechanisms and the accumulation of the pollutant within plant tissues to mitigate the damage to the photosynthetic apparatus. Keywords: antioxidant enzymes; peroxidases; liverwort; polycyclic aromatic hydrocarbons; anthracene; photosynthesis; RNA-sequencing Plants 2021,10, 1478. https://doi.org/10.3390/plants10071478 https://www.mdpi.com/journal/plants Plants 2021,10, 1478 2 of 18 1. Introduction Anthropogenic pollution increasingly provokes deleterious impacts in all ecosystems, and particularly in soils, agriculture, mining, or domestic and industrial activities release a myriad of organic and inorganic pollutants, such as heavy metals, pesticides, pharmaceutical compounds and hydrocarbons, among others [1]. These substances are xenobiotic compounds that are not easily degraded by natural mechanisms, causing their accumulation in soils and the consequent radical increase of abiotic stresses in natural ecosystems [ 2 ]. Numerous investigations have approached this problematic issue from different points of view, such as using phytoremediation to reduce the phytoavailability of pollutants and to contribute to their reduced chance to get into the food chain, microbial remediation or the use of soil amendments to reduce their impact [ 3 , 4 ]. Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants that enter into the soil ecosystem primarily via atmospheric deposition. The concentration of these substances in the environment varies according to several parameters, such as pH, soil particle size, and presence of organic matter or lipophilic material, which tends to absorb PAHs into the soil. Anthracene, the structurally simplest PAH, can be found in nature (up to 504 µM on coal tar) [5–7]. PAH toxicity has been thoroughly studied in vascular plants, including some species of amaranth, poplar, pines and soybeans; there are a number of strategies used by plants to absorb, accumulate and detoxify chemical pollutants, showing different tolerance thresholds to PAHs [ 8 – 10 ]. Among the plant strategies used to cope with PAHs are: (1) accumulation and isolation of the pollutant within specialised organelles to limit toxicity or (2) activation of a detoxification response [ 11 , 12 ]. However, regardless of the strategy, the toxicity on vascular plants of different PAH concentrations causes different physiological responses and effects on organs depending on the duration, intensity and type of exposure. For example, in vitro experiments using Arabidopsis thaliana show the existence of a rapid oxidative stress response when exposed to 250 µ M and 1250 µ M of phenanthrene [ 13 ]. Among the reported effects to this exposure are root necrosis, leaf chlorosis, altered trichome formation, delayed flowering, inhibition of photosynthesis, oxidative stress and stunted growth [11,13–17]. The PAHs are widespread and accumulate at high levels in coastal areas, wetlands, and riverbanks. These habitats can be occupied by liverwort plants, such as Marchantia polymorpha L. whose ability to accumulate and detoxify these compounds is largely unknown. Unlike vascular plants, bryophytes are short creeping plants with intimate contact with the soil. Therefore, in the case of the thalloid liverwort M. polymorpha, the cells absorb water, nutrients and pollutants directly from the soil, which means a strong selection for tolerance and plasticity to different stressors [18]. Hence, it is important to investigate the physiological responses of liverwort grown in such habitats, to understand better the biochemical and molecular mechanisms by which liverwort counteracts the presence of high levels of PAH in the environment. It has been proved that M. polymorpha plants complete their life-cycle growth in concentrations of anthracene ranging from 50 to 280 µ M [ 19 ]. Anthracene toxicity induced apparent phenotypes such as stunted growth with a marked reduction in dry weight and chlorophyll content index. Higher concentrations than 280 µ M proved to be lethal for this plant. However, this was the first approach to understand the potential capacity of liverworts to accumulate and detoxify anthracene; at the biochemical and molecular level there are still several questions that remain unresolved. A natural cytotoxic by-product of photosynthetic activity is the production of reactive oxygen species (ROS), including H 2 O 2 , O 2− and HO · that paradoxically serve as signalling molecules [ 20 , 21 ]. Given the alteration of the CO 2 fixation or the inhibition of the Calvin cycle, the ROS in the chloroplast tend to increase, and upon certain conditions become toxic to the cell since they can oxidize cellular components that the antioxidant machinery cannot repair. Because of this, plant cells respond by means of highly controlled mechanisms to neutralize these compounds, including enzymatic and buffering mechanisms [ 22 , 23 ]. The Plants 2021,10, 1478 3 of 18 principal abiotic stress triggered by PAHs is an oxidative stress induced by high levels of ROS resulting in programmed defence responses [ 24 , 25 ]. The accumulation of PAHs in the membrane activates the generation of antioxidant enzymes, principally SOD, to mitigate stress [ 24 , 25 ]. Plants have evolved ROS-detoxifying enzymatic machineries to minimize ROS-deleterious impacts. Mainly, there are three enzymes that work in tandem in the process of detoxification: superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). If the production of antioxidants is not enough, the excess of ROS usually reduces photosynthesis and respiration [ 13 ]. On the other hand, a plant with a robust antioxidant enzymatic system can likely sustain the rates of these central metabolic processes. In this study, M. polymorpha L. plants exposed to anthracene were used to understand the physiological and molecular responses of the plant. In addition, the activity of the enzymes (SOD, CAT, POD and APX) related to the stress induced on the photosynthesis process were studied. Finally, we performed a transcriptomic study to analyse the transcriptomic landscape under an anthracene exposure. 2. Results 2.1. Effect of Anthracene on M. polymorpha Biomass and Anthracene Accumulation on the Tissues M. polymorpha plants showed a clear sensitivity to increasing concentrations of anthracene (Figure 1). When the plants were exposed to the pollutant, a decrease in the final biomass was observed in the presence of 100 and 280 µ M (0.056 mg ± 0.008 and 0.017 mg ±0.014 , respectively) of anthracene with respect to the control (0.284 mg ± 0.024), resulting in a significant decrease of plant dry weight (80% and 94%, respectively) (Figure 1) . The accumulation of anthracene in plant thallus was also analysed. The absorption on plant tissue increased when increasing anthracene concentration. No significant differences were observed at 50 and 100 µ M of anthracene, and 1.4 µ mol anthracene mg −1 plant dry weight was detected at 280 µ M of anthracene (Figure 1). Our results support a high coefficient of regression confirming that anthracene absorption within the thallus depends on the media concentration of the pollutant (R2= 0.191, (p≤0.001)). Plants 2021, 10, x FOR PEER REVIEW 3 of 18 mechanisms to neutralize these compounds, including enzymatic and buffering mechanisms [22,23]. The principal abiotic stress triggered by PAHs is an oxidative stress induced by high levels of ROS resulting in programmed defence responses [24,25]. The accumulation of PAHs in the membrane activates the generation of antioxidant enzymes, principally SOD, to mitigate stress [24,25]. Plants have evolved ROS-detoxifying enzymatic machineries to minimize ROS-deleterious impacts. Mainly, there are three enzymes that work in tandem in the process of detoxification: superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). If the production of antioxidants is not enough, the excess of ROS usually reduces photosynthesis and respiration [13]. On the other hand, a plant with a robust antioxidant enzymatic system can likely sustain the rates of these central metabolic processes. In this study, M. polymorpha L. plants exposed to anthracene were used to understand the physiological and molecular responses of the plant. In addition, the activity of the enzymes (SOD, CAT, POD and APX) related to the stress induced on the photosynthesis process were studied. Finally, we performed a transcriptomic study to analyse the transcriptomic landscape under an anthracene exposure. 2. Results 2.1. Effect of Anthracene on M. polymorpha Biomass and Anthracene Accumulation on the Tissues M. polymorpha plants showed a clear sensitivity to increasing concentrations of anthracene (Figure 1). When the plants were exposed to the pollutant, a decrease in the final biomass was observed in the presence of 100 and 280 µM (0.056 mg ± 0.008 and 0.017 mg ± 0.014, respectively) of anthracene with respect to the control (0.284 mg ± 0.024), resulting in a significant decrease of plant dry weight (80% and 94%, respectively) (Figure 1). The accumulation of anthracene in plant thallus was also analysed. The absorption on plant tissue increased when increasing anthracene concentration. No significant differences were observed at 50 and 100 µM of anthracene, and 1.4 µmol anthracene mg−1 plant dry weight was detected at 280 µM of anthracene (Figure 1). Our results support a high coefficient of regression confirming that anthracene absorption within the thallus depends on the media concentration of the pollutant (R2 = 0.191, (p ≤ 0.001)). Figure 1. Biomass (bar plot), anthracene content (line plot) in plant tissues of M. polymorpha in vitro experiments in minimum medium supplemented with 50, 100 and 280 µM of anthracene. The data represent the mean ± SE (standard error, n = 3). Values with the same letter are not significantly different between treatments (p ≤ 0.05), as determined by Tukey’s test. Figure 1. Biomass (bar plot), anthracene content (line plot) in plant tissues of M. polymorpha in vitro experiments in minimum medium supplemented with 50, 100 and 280 µ M of anthracene. The data represent the mean ± SE (standard error, n= 3). Values with the same letter are not significantly different between treatments (p≤0.05), as determined by Tukey’s test. 2.2. Effect of Anthracene on the Photosynthetic Apparatus and Chloroplast Ultrastructure The content of chlorophyll aand bfollowed a monophasic behaviour typical of a dose-dependent response of biological systems. Whereas 50 µM slightly induced, but not Plants 2021,10, 1478 4 of 18 always significantly, both pigments’ concentrations in plant tissues, higher concentrations of the pollutant (100 µ M and 280 µ M) strongly repressed their concentration in plant thallus (Figure 2A–C). Although changes in chlorophyll concentrations were observed, no statistically significant changes were measured for Chl a/b ratio. Only at 280 µ M anthracene, we observed 23.6% of the chloroplast showed changes in their ultrastructure compared to the control treatment with membrane damage and starch granule’s presence. The abnormal organization of the chloroplast anatomy was was associated to the development of large starch granules with thylakoid-granum morphology lacking the smooth elongated shape found in control plants (Figure 3). These ultrastructure changes in the chloroplast were not observed at 50 and 100 µM concentrations. Plants 2021, 10, x FOR PEER REVIEW 4 of 18 2.2. Effect of Anthracene on the Photosynthetic Apparatus and Chloroplast Ultrastructure The content of chlorophyll a and b followed a monophasic behaviour typical of a dose-dependent response of biological systems. Whereas 50 µM slightly induced, but not always significantly, both pigments’ concentrations in plant tissues, higher concentrations of the pollutant (100 µM and 280 µM) strongly repressed their concentration in plant thallus (Figure 2a–c). Although changes in chlorophyll concentrations were observed, no statistically significant changes were measured for Chl a/b ratio. Only at 280 µM anthracene, we observed 23.6% of the chloroplast showed changes in their ultrastructure compared to the control treatment with membrane damage and starch granule’s presence. The abnormal organization of the chloroplast anatomy was was associated to the development of large starch granules with thylakoid-granum morphology lacking the smooth elongated shape found in control plants (Figure 3). These ultrastructure changes in the chloroplast were not observed at 50 and 100 µM concentrations. Figure 2. Chlorophyll a content (A); chlorophyll b content (B); Chlorophyll a/b ratio (C) of M. polymorpha in the presence of the anthracene (0, 50, 100, 280 µM). The data represent the mean ± SE (standard error, n = 3). Values with the same letter are not significantly different between treatments (p ≤ 0.05), as determined by Tukey’s test. Figure 3. Transmission electron microscopy (TEM) of M. polymorpha chloroplast growing in the absence (A) and in the presence (B) of 280 µM of anthracene. Arrows indicate chloroplasts in the control and chloroplast membrane alteration in 280 µM treatment. The net photosynthetic rate did not significantly differ between treatments (Supplementary Table S1). Although the differences detected in the photosynthesis rates were not Figure 2. Chlorophyll a content ( A ); chlorophyll b content ( B ); Chlorophyll a/b ratio ( C ) of M. polymorpha in the presence of the anthracene (0, 50, 100, 280 µ M). The data represent the mean ± SE (standard error, n= 3). Values with the same letter are not significantly different between treatments (p≤0.05), as determined by Tukey’s test. Plants 2021, 10, x FOR PEER REVIEW 4 of 18 2.2. Effect of Anthracene on the Photosynthetic Apparatus and Chloroplast Ultrastructure The content of chlorophyll a and b followed a monophasic behaviour typical of a dose-dependent response of biological systems. Whereas 50 µM slightly induced, but not always significantly, both pigments’ concentrations in plant tissues, higher concentrations of the pollutant (100 µM and 280 µM) strongly repressed their concentration in plant thallus (Figure 2a–c). Although changes in chlorophyll concentrations were observed, no statistically significant changes were measured for Chl a/b ratio. Only at 280 µM anthracene, we observed 23.6% of the chloroplast showed changes in their ultrastructure compared to the control treatment with membrane damage and starch granule’s presence. The abnormal organization of the chloroplast anatomy was was associated to the development of large starch granules with thylakoid-granum morphology lacking the smooth elongated shape found in control plants (Figure 3). These ultrastructure changes in the chloroplast were not observed at 50 and 100 µM concentrations. Figure 2. Chlorophyll a content (A); chlorophyll b content (B); Chlorophyll a/b ratio (C) of M. polymorpha in the presence of the anthracene (0, 50, 100, 280 µM). The data represent the mean ± SE (standard error, n = 3). Values with the same letter are not significantly different between treatments (p ≤ 0.05), as determined by Tukey’s test. Figure 3. Transmission electron microscopy (TEM) of M. polymorpha chloroplast growing in the absence (A) and in the presence (B) of 280 µM of anthracene. Arrows indicate chloroplasts in the control and chloroplast membrane alteration in 280 µM treatment. The net photosynthetic rate did not significantly differ between treatments (Supplementary Table S1). Although the differences detected in the photosynthesis rates were not Figure 3. Transmission electron microscopy (TEM) of M. polymorpha chloroplast growing in the absence ( A ) and in the presence ( B ) of 280 µ M of anthracene. Arrows indicate chloroplasts in the control and chloroplast membrane alteration in 280 µM treatment. The net photosynthetic rate did not significantly differ between treatments (Supplementary Table S1). Although the differences detected in the photosynthesis rates were not significant, a strong reduction in the photosynthesis values of the plants treated with anthracene was detected, reducing at least 50%. In addition, the Fv/Fm ratio remained constant between treatments (Supplementary Table S1). Plants 2021,10, 1478 5 of 18 2.3. Anthracene Treatment Induced ROS-Scavenging Genes RNA-sequencing (RNA-seq) analyses provided a genome-wide view of anthracene toxicity on M. polymorpha thallus. We identified 349 differentially expressed genes: 92 downregulated (pvalue < 0.05, log2(FC) < − 1) and 257 (pvalue < 0.05, log2(FC) > 1) upregulated (Supplementary Table S2) upon exposure to anthracene. We identified several protein families within each group of up and downregulated genes (Supplementary Figure S1c). The group of upregulated genes included several late-embryogenesis abundant proteins (LEA) and dehydrin (DHN) genes. To evaluate the overlap of the molecular response induced by anthracene and other abiotic stresses we compared the fold change of differentially expressed genes (DEG) of M. polymorpha plants treated with anthracene, ABA [ 26 ] and NaCl [ 27 ]. We observed a high correlation of the anthracene treatment with NaClinduced genes (pvalue = 1.3 × 10 −24 ) and ABA-induced genes (pvalue = 2.9 × 10 −84 ) (Supplementary Figure S1a). Among anthracene-induced genes, we found an enrichment of polyphenol oxidase (PPO) and dirigent (DIR) protein families involved in secondary metabolism. We detected only a few transcription factors among DEGs, including MpERF20 and the MYCtype MpBHLH4, also induced by the defence related hormone 12-oxo-phytodienoic acid (OPDA) [28,29]. As ROS-scavenging enzymes play a central role in the physiological response to oxidative stress, we studied them in the RNA-seq experiment. First, we annotated all DHAR, MDHAR, APX, CAT, SOD, NOX, GR, PRX and GST enzyme genes in the M. polymorpha genome (Supplementary Table S3) and predicted their respective subcellular localization using LOCALIZER [ 30 ]. From 246 genes involved in ROS scavenging, only 10 were DEGs (Supplementary Figure S1b). Within this selected group, the anthracene treatment induced MpDHAR1, MpPOD161/159 and MpGST27 and repressed MpCAT4 and several MpPOD including MpPOD26/112/114/115/117/122 (see Supplementary Figure S1d). We questioned how similar the transcriptional responses to PAHs of M. polymorpha and the angiosperm Arabidopsis thaliana were. Therefore, we analysed the group of ortholog genes with a similar response to PAHs comparing the transcriptomic response of anthracene-treated M. polymorpha to phenanthrene-treated A. thaliana plants [ 31 ]. We found a modest overlap (10 genes) of both transcriptomes (p value = 0.00015), including orthologs of GST, DHAR and LEA genes that were positively regulated in both PAH treatments (Supplementary Figure S1B). We also compared the anthracene transcriptional response with DEGs reported in M. polymorpha upon UV-B treatment [ 32 ] or exogenous application of OPDA [ 29 ]. We found a modest overlap with upregulated genes in UV-B (pvalue = 0.0064) and a significant enrichment with OPDA (pvalue = 2.1 × 10 −55 ), suggesting a mild cross-talk of anthracene response with other stresses (Supplementary Figure S2). Altogether, the results show a significant impact of anthracene in the transcriptomic landscape related to ROS-detoxifying genes of M. polymorpha. 2.4. Anthracene Induces Oxidative Damage: Histochemical Staining and Fluorescence Test Determinations The qualitative analysis to test oxidative damage by H 2 O 2 in the plant using histochemical DAB staining showed strong ROS-related precipitates in anthracene-treated plants (280 µ M) (Figure 4). Concomitantly, plants exposed to anthracene showed increased DCF oxidation by the action of intracellular ROS (Figure 5A–D) and a strong green fluorophore emission was observed in comparison to the control in the 280 µ M treatments (Figure 5C,D, respectively). The emitted intensity was greater in plants exposed to anthracene compared to the control; 364.5 ±154.2 and 641 ±140 DCF fluorescence (relative units), respectively (p= 0.009) (see Bar graph Figure 5Hk). In addition, the arrows in the overlap of autofluorescence and DCFDA fluorescence micrographies (Figure 5H,HI) show that ROS are specifically located in the chloroplast aggrupation and around them. Plants 2021,10, 1478 6 of 18 Plants 2021, 10, x FOR PEER REVIEW 6 of 18 (Figure 5c,d, respectively). The emitted intensity was greater in plants exposed to anthracene compared to the control; 364.5 ± 154.2 and 641 ± 140 DCF fluorescence (relative units), respectively (p = 0.009) (see Bar graph Figure 5 Hk). In addition, the arrows in the overlap of autofluorescence and DCFDA fluorescence micrographies (Figure 5H–HI) show that ROS are specifically located in the chloroplast aggrupation and around them. Figure 4. Histochemical analysis with 3,3′-Diaminobenzidine (DAB) as ROS indicator. Thallus of M. polymorpha grown in the absence and presence of anthracene. (A)–(D) are treatments without DAB staining, and (E)–(H) are the treatments with the DAB staining. Arrows indicate assays positive in the polymerisation of DAB in the presence of H2O2. Figure 4. Histochemical analysis with 3,3 0 -Diaminobenzidine (DAB) as ROS indicator. Thallus of M. polymorpha grown in the absence and presence of anthracene. ( A – D ) are treatments without DAB staining, and ( E – H ) are the treatments with the DAB staining. Arrows indicate assays positive in the polymerisation of DAB in the presence of H2O2. Plants 2021,10, 1478 7 of 18 Plants 2021, 10, x FOR PEER REVIEW 7 of 18 Figure 5. ROS presence in thallus of M. polymorpha.. Thallus of M. polymorpha grown in the absence (A) and presence (B) of 280 µM of anthracene; (C,D) are the same treatments after DCFDA staining. Green signals indicate DCF fluorescence. Thallus of M. polymorpha in presence of 280 µM of anthracene (E), chlorophyll autofluorescence (F), DCFDA fluorescence (G) overlap of autofluorescence and DCFDA fluorescence micrographies (H), green florescence around chloroplast under zoom in H (HI–HJ) and quantitative oxidation measured as relative units of fluorescence (Hk). Red signals indicate chlorophyll fluorescence; green signals indicate DCF fluorescence. The data represent the mean ± SE (standard error, n = 4). Values with the same letter are not significantly different between treatments (p ≤ 0.05), as determined by Tukey’s test. 2.5. Analysis of Oxidative Damage at the Enzymatic and Lipid Peroxidation Levels SOD and CAT enzymes did not show significant differences with respect to the control treatments. On the other hand, POD and APX enzymatic activity increased compared to control plants. POD activity was significantly higher in the presence of 50 µM and 100 Figure 5. ROS presence in thallus of M. polymorpha. Thallus of M. polymorpha grown in the absence ( A ) and presence ( B ) of 280 µ M of anthracene; ( C , D ) are the same treatments after DCFDA staining. Green signals indicate DCF fluorescence. Thallus of M. polymorpha in presence of 280 µ M of anthracene ( E ), chlorophyll autofluorescence ( F ), DCFDA fluorescence ( G ) overlap of autofluorescence and DCFDA fluorescence micrographies ( H ), green florescence around chloroplast under zoom in H ( HI , HJ ) and quantitative oxidation measured as relative units of fluorescence ( Hk ). Red signals indicate chlorophyll fluorescence; green signals indicate DCF fluorescence. The data represent the mean ± SE (standard error, n= 4). Values with the same letter are not significantly different between treatments (p≤0.05), as determined by Tukey’s test. 2.5. Analysis of Oxidative Damage at the Enzymatic and Lipid Peroxidation Levels SOD and CAT enzymes did not show significant differences with respect to the control treatments. On the other hand, POD and APX enzymatic activity increased compared Plants 2021,10, 1478 8 of 18 to control plants. POD activity was significantly higher in the presence of 50 µ M and 100 µ M of anthracene than in control plants (Figure 6A). In addition, a greater activity of APX was registered in plants grown in 280 µ M of anthracene (Figure 6B). Related to MDA content, plants grown in 280 µ M of anthracene reached values almost three times greater (Figure 6E). Plants 2021, 10, x FOR PEER REVIEW 8 of 18 µM of anthracene than in control plants (Figure 6a). In addition, a greater activity of APX was registered in plants grown in 280 µM of anthracene (Figure 6b). Related to MDA content, plants grown in 280 µM of anthracene reached values almost three times greater (Figure 6e). Figure 6. Effect of various anthracene concentrations on the activity of (A) Peroxidase (POD); (B) Superoxide dismutase (SOD); (C) ascorbate peroxidase (APX); (D) Catalase and (E) malondialdehyde (MDA) content. The data represent the mean ± SE (standard error, n = 6). Values with the same letter are not significantly different between treatments (p ≤ 0.05), as determined by Tukey’s test. 3. Discussion In this study, we documented the physiological effects at the biochemical and molecular level of high doses of anthracene on M. polymorpha L. plants. The ability of this plant to tolerate adverse environmental conditions and to colonize contaminated soils and wetlands could be related to the physiological response described in this study. As indicated above, concentrations of more than 500 µM of anthracene have been recorded in nature. In this study, M. polymorpha L. grew within the entire range of tested concentrations, although a loss of biomass was recorded. Previous studies with a different PAH using smaller concentrations in the order of 10 µM, showed that the liverwort Riccia fluitans L., when exposed to phenanthrene, was also affected [33]. Thus, this indicates, that M. polymorpha has a high resistance to this type of contaminant when compared to R. fluitans, other liverwort plants, and this characteristic could be related to the ability of this Figure 6. Effect of various anthracene concentrations on the activity of ( A ) Peroxidase (POD); ( B ) Superoxide dismutase (SOD); ( C ) ascorbate peroxidase (APX); ( D ) Catalase and ( E ) malondialdehyde (MDA) content. The data represent the mean ± SE (standard error, n= 6). Values with the same letter are not significantly different between treatments (p≤0.05), as determined by Tukey’s test. 3. Discussion In this study, we documented the physiological effects at the biochemical and molecular level of high doses of anthracene on M. polymorpha L. plants. The ability of this plant to tolerate adverse environmental conditions and to colonize contaminated soils and wetlands could be related to the physiological response described in this study. As indicated above, concentrations of more than 500 µ M of anthracene have been recorded in nature. In this study, M. polymorpha L. grew within the entire range of tested concentrations, although a loss of biomass was recorded. Previous studies with a different PAH using smaller concentrations in the order of 10 µ M, showed that the liverwort Riccia fluitans L., when exposed to phenanthrene, was also affected [ 33 ]. Thus, this indicates, that M. polymorpha has a high resistance to this type of contaminant when compared to Plants 2021,10, 1478 9 of 18 R. fluitans, other liverwort plants, and this characteristic could be related to the ability of this plant to colonize contaminated or altered places [ 33 ]. Although the experimental conditions of our tests are not the same as other studies carried out with plants and PAHs, M. polymorpha tolerated higher anthracene concentrations than some vascular plants. For example, with respect to anthracene, [ 34 ] tested concentrations of anthracene up to 0.04 µ M in leaves of lettuce and radish plants, while [ 35 ] tested concentrations of up to 240 µ M in carrot roots. The authors of [ 13 ] tested concentrations between 40 µ M to 1 mM in Arabidopsis using a different PAH such as phenanthrene. This indicates that the tolerance is intrinsic to the plant, the compound used and the study system. The presence of anthracene in the medium strongly inhibits the growth of M. polymorpha and decreases the plant biomass (Figure 1), which is a typical stress response-like symptom observed in plants. Thereby, the detection of anthracene in the tissues correlates with the amount of pollutant in the medium, showing that anthracene accumulates in the cells. This fact supports the hypothesis of previous studies that until now have not been tested [ 19 ], confirming that M. polymorpha is able to accumulate, maybe passively, anthracene in their tissues. Although we cannot establish whether the internalization of the contaminant is passive or active, our microscopic assays indicate that anthracene or part of it accumulates in the cell walls of M. polymorpha and in the chloroplast. In bryophytes, the transport route of nutrients and contaminants is apoplastic and follow the same route as the circulation of water [ 36 ]. In vascular plants, simple diffusion and aquaglyceroporins may be involved in the passive uptake of PAH, and active uptake is mediated by a phenanthrene/H+symporter [37]. Although these processes are unknown in liverworts, the internalization mechanisms of contaminants could be similar to those described for vascular plants [ 19 ]. Interestingly, some transporter-encoding genes are upregulated in our transcriptomic analysis, although further studies are needed to confirm their involvement in anthracene uptake. A previous report hypothesized that the presence of anthracene could induce variations in the total content of photosynthetic pigments causing a decrease in plant biomass [ 19 ]. In the present study, it was also observed that, although the amount of chlorophylls (aand b) decreased, the rate between both types of chlorophylls remained constant. These results are in agreement with previous studies in vascular plants that show an alteration in the concentration of chlorophylls in the presence of PAHs [ 13 , 38 ]. PSI and PSII exclusively contain chlorophyll a, while the light harvesting complex (LHC) has both chlorophylls a and b, plus some accessory pigments. Therefore, any variation in the a to b chlorophyll ratio indicates a decrease in the efficiency of light collection [39]. Interestingly, our results suggest that the chlorophylls ratio found is at least adequate or sufficient for a functional photosynthesis in our experimental conditions, which was accompanied by values of net photosynthetic rates and Fv/Fm comparable to those found in control plants. These results are opposed to those found in vascular plants, where those variations produced a decrease in the rate of photosynthesis [ 38 , 40 ]. The differences in results between the studies could be due to two principal reasons. On the one hand, the measurements were conducted at single time points and longer periods of exposure to the contaminant could generate effects on the photosynthetic apparatus or, on the contrary, acclimatization and recovery of the Fv/Fm was not tested in our experiments. It has even been observed that Riccia sp. plants exposed to 0.5 µ M of phenanthrene showed almost a full recovery of Fv/Fm in time [ 33 ]. 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