Effect of arsenic stress on 5-methylcytosine, photosynthetic parameters and nutrient content in Pteris cretica var. Albo-lineata hyperaccumulator

Arsenic (As) toxicity induces a range of metabolic responses in plants, including DNA methylation. The focus of this paper was on the relationship between As-induced long-term stress and plant senescence in the hyperaccumulator Pteris cretica var. Albo-lineata ( Pc -Al).Results We showed that Pc -Al grown in pots of haplic chernozem contaminated with 100 mg As kg -1 (As 100 ) for 122 days could accumulate more than 2000 and 2800 mg As kg -1 dry matter in old and young fronds, respectively. Analysis of 5-methylcytosine (5mC) in Pc -Al confirmed that the overall DNA methylation status in fronds of As 100 ferns was reduced in contrast to control treatment. Compared with controls, the overall DNA methylation status in fronds of As 100 ferns was reduced (by 6% in young and 10% in old fronds); however, the decrease was significant only in old fronds. The significant correlations for 5mC, in contrast to direct As toxicity, showed that decreases of chlorophylls, fluorescence and photosynthetic rate could be affected by epigenetic changes. Photosynthetic processes were determined in As 100 treatment and showed a reduction of gas-exchange parameters, and a decrease in carotenoids and chlorophylls (by 5% and 26%, respectively). Hyperaccumulation of As resulted in a significant elevation of all analysed nutrients (Cu, Mn, Zn, Mg, S) in old fronds, but not in young fronds.Conclusions The results of this paper point to complex changes in the metabolism of the hyperaccumulator plant Pc -Al, upon exposure to As contamination. The most significant impact was found in young fronds. The physiological parameters correlated more significantly with a decrease of DNA methylation than with direct As toxicity. Our analysis of the very low water potential values and lignification of cell walls in roots showed that transports of assimilated metabolites and water between roots and fronds were reduced.

4 the degradation of photosynthetic pigments and remobilises the basic nutrients C, N, P, and S. Modified stress metabolism due to extended reversible senescence releases nutrients from catabolic processes and transports them from old leaves into young leaves more efficiently.
Studies have also revealed that stress-inducing abiotic factors, including As, can trigger epigenetic changes (in particular DNA methylation/demethylation), which may contribute to the regulation of gene expression in chronic stress conditions [10]. Phenotypic manifestations of epigenetic changes include reduced plant growth (dwarfism) and development (in particular seed germination [11]), flowering period [12,13] and male fertility/sterility of anthers and/or pollens [14].
The primary detoxification of As in the cells of terrestrial plants relies on rapid reduction of As V to As III and the formation of As III -glutathione or As III -phytochelatin complexes, which are eventually transported to the vacuole. Disturbances in cellular processes, caused by a toxic excess of As, induce oxidative stress responses [15][16][17], methylation of both As forms in P. cretica [18], and epigenetic changes in DNA [19]. In this study, we aimed to gain insight into the context of As hyperaccumulation and plant senescence in Pteris cretica var. Albo-lineata. In addition, we examined related changes in selected physiological parameters and DNA methylation status as potentially indicative of epigenetic modification. The degree of senescence was evaluated with respect to different changes found in young and old fronds of P. cretica var. Albo-lineata.

Results
Growth and elemental content of As-exposed P. cretica var. Albo-lineata The effect of As 100 soil was observed only on young fronds. The dry biomass of Pc-Al young fronds was decreased by 43% (Fig. 1). Differences between young and old fronds of Pc-Al were not statistically significant. Symptoms of As toxicity were not observed.
The analysis of the concentration of elements in dried fronds of Pc-Al revealed that young fronds had approximately 1.5 times higher concentrations of As than old ones, both in the control and As 100 soil conditions (Table 1). Compared with controls, ferns grown in the As 100 soils increased As concentrations 150-and 170-fold in young and old fronds, respectively. Irrespective of the presence of added As in the As 100 soils, old fronds tended to accumulate higher concentrations of Cu, Mg, Mn, S and Zn than young fronds (Table 1, Fig. 2). While the effect of high soil As on the Cu accumulation showed an increase in both young and old fronds (by 17% and 27%, respectively), increases in S (44%) and Zn (28.5%) concentration were only observed in old fronds. When grown in As 100 soils, the concentrations of Mg and Mn increased in old fronds of Pc-AlMg by 33% and Mn by 49%) but decreased in young fronds (Mg by 6% and Mn by 18%). DNA methylation status of As-exposed P. cretica var. Albo-lineataSince As and senescence might affect the methylation of DNA at cytosines in plants, the 5-methylcytosine content 6 (5mC, %) of Pc-AlDNA was analysed ( Fig. 2 and 3). Compared with controls, the overall DNA methylation status in fronds of ferns grown in As 100 soils was reduced (by 6% in young and 10% in old fronds); however, the decrease was only proved in old fronds. The effect of frond senescence on 5mC content was not statistically significant, but the trend, in terms of average 5mC content, was lower in old fronds.
Pigment content, fluorescence, WP and GEP of As-exposed P. cretica var.

Albo-lineata
Growth in As 100 soil resulted in a decrease of chlorophyll contents (Chl A, Chl B and Σ Chl) of Pc-Al ( Table 2). The content of carotenoids (Crt) was reduced by As, but not significantly. Irrespective of the presence of added As in the soil, contents of all analysed pigments were higher in young fronds than in old fronds. Compared with controls, pigment content of ferns grown in the As 100 soils was higher in young fronds, especially Chl A and Crt contents (5-fold and 6-fold higher than those in old fronds, respectively). While the average value of Chl A and Chl B ratio remained unaffected by As in old fronds, it increased in young fronds of ferns grown in As 100 soils ( Table 2). Water potential (WP) was decreased in young and old fronds of the plants grown in As 100 7 soil (by 36% and 118%, respectively). Higher values of WP were observed in young fronds, irrespective of added As. To explore potential changes in roots, cross-section analysis through adventitious roots was performed. The roots of As 100 treated plants showed thinning of the sclerenchymatous inner cortex and a reduction in average tracheid metaxylem in the vascular cylinder, compared to controls (Fig. 4).  The P N and the rate of transpiration (E) were determined to gain further insight into the photosynthetic performance in Pc-Alfronds ( Table 2). The P N and E data indicated a higher photosynthetic activity in the young fronds of the control ferns ( Table 2, Fig. 2), and a decrease in the photosynthesis rate under As 100 conditions (by 5% in young fronds and 8 6.5% in old fronds). Application of As reduced transpiration in old fronds by 12.5%, while a 28.5% increase was observed in young fronds. Compared with controls, added As decreased WUE only in young fronds (by 29%).

Principal component analysis of physiological parameters
The first axis of the PCA analysis explained 74% of the variability of all analysed data, the first two axes explained 94% of the variability, and the first four axes together explained 99% of the variability. Diagramming PCA analysis was used for visualisation of all relationships between Pc-Alparameters (Fig. 2). In the PCA diagram, the first ordination axis divided the young fronds group on the left side from old fronds on the right side. This division indicated a large effect of frond senescence on all studied parameters. For young and old fronds, marks for treatments (control, As 100 ) were located in the different parts of the diagram, which indicated a high effect of the treatments on all the recorded data. As observed with primary data, PCA confirmed that the accumulation of Cu, S, Zn, Mg and Mn was more pronounced in old fronds of Pc-Algrown in As 100 soils and that ChlB, P N , Fv/Fm, as well as 5mC were higher in young fronds of control plants. Arsenic content was negatively correlated with relative 5mC content of DNA as the angle between the vectors for As and 5mC was > 90°. Relationships visualised in the PCA diagram were confirmed by linear correlations (Table 3). The results in Table 3 showed an effect of As and 5mC on other measured parameters. Correlations of As effect and 5mC on other parameters were calculated in different old fronds, where senescence was evaluated as a difference of tested parameters between young and old fronds ( Table 3). The content of As in Pc-Al fronds significantly correlated with 5mC, Cu and E. Negative relationships of As were confirmed for F v /F m , P N and WP. By comparison, these parameters were positively correlated with 5mC. Negative relationships were found between 5mC and Mn, S and Zn. 9 Notably, a significant, positive correlation was determined for 5mC and WP, while the correlation with As content was negative. The same trend was observed for Chl B.

Discussion
Our results show that when grown in chernozem soils spiked with 100 mg As kg -1 , As couldaccumulate in the fronds of P. cretica var. Albo-lineata to > 2000 mg As kg -1 dry mass, and the amount of As that accumulated in young fronds was higher than in old fronds. These data confirmed the As hyperaccumulation status of Pc-Al and were consistent with results reported by Zhao et al. [3] for this fern and by Tu et al. [5] for P.

vittata.
Arsenic stress induces epigenetic changes in organisms, resulting in a decrease or increase in DNA methylation [19]. Analysis of 5mC in Pc-Al showed that As reduced the extent of DNA methylation. Similar results for heavy metals were published by Aina et al. [20]. The first paper focused on the effect of As on DNA methylation in plants was published by Erturk et al. [21]. Their results showed DNA hypermethylation of some genes in germinating maize seeds exposed to low As levels.
An increase of DNA methylation increases plant growth and transcriptionally represses genes involved in flavonoid biosynthesis [22]. A decrease of DNA methylation reduces plant growth and stimulates flowering, formation and growth of buds [13,23,24]. This finding was confirmed by our results. Dry biomass of Pc-Al young fronds was decreased by 43% ( Fig. 1). As revealed by PCA analysis (Fig. 2), physiological parameters of the plant are affected more strongly by the methylation status of Pc-Al DNA than by direct As toxicity.
Some publications suggest that parts of DNA are sensitive to epigenetic changes [25].
However, in plants, the conservative parts of DNA without changes in DNA methylation were observed. Little information about the epigenetic activation of transcription of silenced plant genes of primary and secondary metabolites is known. Cazzonelli [26] described epigenetic changes linked to the regulation of metabolic pathways leading to carotenoid biosynthesis in relation to abscisic acid (control of carotenogenesis). According to Zhang et al. [27] epigenetic changes are linked with the biosynthesis of chlorophylls and tocopherols whose precursor is phytyl diphosphate. Lushchak and Semchuk [28] were also interested inthese epigenetic changes.According to these authors, plants can increase photosynthesis by chlorophylls biosynthesis or by synthesizing the antioxidant metabolites tocopherols. We showed the continuity of changes in methylation/demethylation of cytosine DNA in relation to the photosynthetic pigments carotenoids and chlorophylls (primary relationship) and also to gas-exchange parameters (GEP) or to Fv/Fm, which are indicators of plant photosynthetic activity.
It has been well documented that stress-related senescence processes involve the degradation of photosynthetic pigments [9], accompanied by a reduction in photosynthetic efficiency. Our results indicated that excess As reduced the level of chlorophylls and 11 affected photosynthetic processes in Pc-Al fronds (Fig. 2, Table 2, 3). Farooq et al. [29] reported that As decreased GEP and pigment content in Brassica napus and, according to Wang et al. [30] this toxic element significantly affected Fv/Fm in P. vittata within 60 days of exposure. An association between epigenetic changes in old fronds, resulting from As stress, and a reduced chlorophyll content might be indicated by the correlation between 5mC and Chl A and Chl B levels ( Table 3). The decline in the levels of carotenoids and chlorophylls in young fronds of As 100 treated plants was less than that seen in older fronds. A significant increase of the Chl A/Chl B ratio was confirmed for young fronds of As 100 plants. These changes, together with the values of P N , E and Fv/Fm, pointed to a senescence in As 100 plants. During senescence, plant metabolites are remobilised from old leaves to young leaves after expression of genes typical for senescence [31]. We found that As toxicity slightly increased leaf senescence (Fig. 2, Tables 1, 2, 3). The more significant correlations for 5mC, in contrast to direct As toxicity ( Table 3), showed that decreases of chlorophylls, fluorescence and P N could be affected by epigenetic changes.
Ay et al. [32] published similar conclusions for the effects of epigenetic changes on physiological processes in plants.
A decrease of 5mC content in As 100 plants led to a significant negative correlation with increased amounts of Cu, Zn and Mn, cofactors of superoxide dismutases, and with S, a key element in the biosynthesis of cysteine and methionine. Increased accumulation of tested elements could be affected by epigenetic changes as showed by the significant correlations for 5mC, as opposed to direct As stress (Table 3). While there was an elevation in the concentrations of Cu, Mn, and Zn, in P. vittata exposed to As contamination [5], in Pc-Alit was more pronounced in old fronds as compared to young ones. These authors observed a same trend as we did-higher Mg content in old fronds compared to young ones in P. vittata. It was reasonable to assume that these elements were significant as cofactors of antioxidative metalloenzymes [33][34][35] as a part of chlorophylls (Mg), and as non-enzymatic antioxidants, protecting against As-induced stress. Increased concentrations of S can be linked to the accumulation of glutathione and phytochelatins involved in detoxification of cellular As in P. cretica var. Mayii [36]. Based on this, the observation that the concentration of S and Zn remained unaffected and the concentrations of Mn and Mg were reduced in young fronds of Pc-Al grown in As 100 soil was surprising. We hypothesised that changes in Mn and Mg content were linked with N metabolism.
Water consumption by ferns is directly proportional to As contamination. One of the causes of changes in water content in fronds is the lignification of the conducting tissues in the roots. We found very low WP values for As 100 plants as a result of stress attributable to As contamination ( Table 2). Induced stress in cell walls leads primarily to the lack of water in fronds and secondarily to osmotic stress, which is a limiting factor for the growth and development of these plants. Leaf senescence together with the effect of As resulted in lignification of conducting tissue ( Figure 3). Similar reports of WP reduction as a result of the lignification of conducting tissue in plants exposed to stress conditions were published by Hare and Cress [37] and Yamaguchi et al. [38]. If the photosynthetic membrane system is protected by flavonoids, ascorbate and tocopherols [28,[39][40][41], then cell walls are protected by lignin [38]. Reduced DNA methylation in Pc-Al increases biosynthesis of sterols, tocopherols, flavonoids, isoflavonoids and lignins because these plant metabolites are epigenetically regulated by silencing genes [40,42,43].Crosssections through adventitious roots of As 100 plants showed the deformation of root cell walls as a result of lignification ( Figure 3). Zanella et al. [44] observed morphological 13 changes in tobacco roots growing in As and Cd contaminated solutions. They found increased cell wall thickness due to lignin over-deposition in the rhizodermal and external cortical parenchyma cells of the primary structure zone, which led to premature exodermis formation. Similar changes in root, lignification upon exposure to metals were confirmed by Piršelová et al. [45]. According to cited works, the lignification-induced cellular changes result in a reduction in water uptake by plants. This finding is in line with our results: changes in WP values and morphological changes in the roots. Reduction of water content and metabolites subsequently limits the ability of the plant to overcome As toxicity.

Conclusion
The results of this paper point to complex changes in the metabolism of the hyperaccumulator plant Pteris cretica var. Albo-lineata, on exposure to arsenic contamination. Compared with controls, ferns grown in the As 100 soils increased As concentrations in young and old fronds, 150-and 170-fold, respectively. Higher As content was found in the young fronds of control and As 100 plants, in comparison to old fronds (1.5-fold higher on average). Analysis of 5mC content showed that accumulation of As was associated with reduced DNA methylation (by 10%). As revealed by PCA, physiological parameters of the plant are more strongly affected by the methylation status of Pteris cretica var. Albo-lineataDNA than by direct As toxicity.
The more significant correlations for 5mC, in contrast to As toxicity, showed that  (Table 4) was collected from a nonpolluted area in Prague-Suchdol, Czech Republic (50º8ˊ8˝ N, 14º22ˊ43˝ E). Ferns were grown in this soil without As supplement (controls) and with 100 mg As per kg soil (As 100 ).
Arsenic was added as a solution of Na 2 HAsO 4 and was thoroughly mixed with the soil; maturation period of spiked soil was ten days. Each treatment was replicated three times.
After being harvested, the young and old fronds (Fig. 4) were treated as described below.
Cross-sections through an adventitious root were inspected using a Nikon E 200 microscope equipped with DS camera head and the NIS-Elements application (Nikon Instruments, Inc., Melville, NY, USA). Determination of arsenic and other elements PCA was used to draw correlations from the complex data set. The results were visualised in the form of bi-plot ordination diagrams using the CanoDraw program [47]. Correlations 18 were confirmed using a linear correlation (r, p < 0.05, p < 0.01, p < 0.001) by Statistica 12.0. funding bodies provided the financial support to the research projects, but did not involve in study design, data collection, analysis, or preparation of the manuscript.

Abbreviations
Authors' contributions MP and DP conceived and designed the experiments. MP and VZ calculated the relative DNA methylation status.FH calculated selected photosynthesis parameters. JČ performed cross-sections through the roots. VZ, DP, PK, MP analysed the data and wrote the paper.
All authors have read and approved the final version of the manuscript.
Ethics approval and consent to participate Not applicable.

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