Are cysteine, glutathione and phytochelatins responses of Myriophyllum alterniflorum to copper and arsenic stress affected by trophic conditions?

The aim of this article is to study the impact of both copper (Cu2+) and arsenic (As (V)) at 100 µg/L, with each element being combined with trophic conditions at the level of glutathione, cysteine and phytochelatins in the aquatic macrophyte Myriophyllum alterniflorum, whose potential for bioindication and phytoremediation of metal/metalloid pollution has already been demonstrated. To achieve this goal, a synthetic medium, of a composition similar to the water found in the Vienne River in France's Limousin Region and modified for eutrophic or oligotrophic conditions, is prepared. The analysis of cysteine, glutathione and phytochelatins is performed at 0, 3, 7, 14 and 21 days. Our results indicate that the eutrophic medium without contaminant only induces a significant increase in the glutathione level when compared to the oligotrophic medium. However, the joint presence of As (V) and Cu is able to increase the synthesis of cysteine, glutathione and phytochelatins (PC2 and PC3) under both eutrophic and oligotrophic conditions, with a significant increase in the eutrophic medium compared to the oligotrophic one. Phytochelatins (PC2 and PC3) are induced after as little as 3 days of exposure to copper and arsenic under both trophic conditions. Copper induces the synthesis of more PC3 than PC2, unlike arsenic. Our results confirm the potential use of phytochelatins as a specific biochemical biomarker for metal/metalloid stress. In conclusion, the eutrophic condition combined with copper or arsenic does change the response of Myriophyllum alterniflorum by enhancing its antioxidative defense. Thus, M. alterniflorum phytochelatins represent a potential dedicated biomarker to monitor water quality in terms of metal/metalloid stress regardless of the trophic level.

In search for potential biomarkers of copper stress in aquatic plants.

Over the last few decades, the use of pesticides and discharge of industrial and domestic wastewater on water surfaces have increased. Especially, Copper (Cu) pollution in aquatic ecosystems could constitute a major health problem, not only for flora and fauna but also for humans. To cope with this challenge, environmental monitoring studies have sought to find Cu-specific biomarkers in terrestrial and aquatic flora and/or fauna. This review discusses the toxic effects caused by Cu on the growth and development of plants, with a special focus on aquatic plants. While copper is considered as an essential metal involved in vital mechanisms for plants, when in excess it becomes toxic and causes alterations on biomarkers: biochemical (oxidative stress, pigment content, phytochelatins, polyamines), physiological (photosynthesis, respiration, osmotic potential), and morphological. In addition, Cu has a detrimental effect on DNA and hormonal balance. An overview of Cu toxicity and detoxification in plants is provided, along with information regarding Cu bioaccumulation and transport. Awareness of the potential use of these reactions as specific biomarkers for copper contamination has indeed become essential.

Structural features in tension wood and distribution of wall polymers in the G-layer of in vitro grown poplars.

Under the effect of disturbances, like unbalanced stem, but also during normal development, poplar trees can develop a specific secondary xylem, called "tension wood" (TW), which is easily identifiable by the presence of a gelatinous layer in the secondary cell walls (SCW) of the xylem fibers. Since TW formation was mainly performed on 2-year-old poplar models, an in vitro poplar that produces gelatinous fibers (G-fibers) while offering the same experimental advantages as herbaceous plants has been developed. Using specific cell wall staining techniques, wood structural features and lignin/cellulose distribution were both detailed in cross-sections obtained from the curved stem part of in vitro poplars. A supposed delay in the SCW lignification process in the G-fibers, along with the presence of a G-layer, could be observed in the juvenile plants. Moreover, in this G-layer, the immunolabeling of various polymers carried out in the SCW of TW has allowed detecting crystalline cellulose, arabinogalactans proteins, and rhamnogalacturonans I; however, homogalacturonans, xylans, and xyloglucans could not be found. Interestingly, extensins were detected in this typical adaptative or stress-induced structure. These observations were corroborated by a quantitation of the immunorecognized polymer distribution using gold particle labeling. In conclusion, the in vitro poplar model seems highly convenient for TW studies focusing on the implementation of wall polymers that provide the cell wall with greater plasticity in adapting to the environment.

Myriophyllum alterniflorum biochemical changes during in vitro Cu/Cd metal stress: Focusing on cell detoxifying enzymes.

Given the toxicity of trace metals, their concentration, speciation and bioavailability serve to induce various plant detoxification processes, which themselves are specific to several parameters like plant species, tissue type and developmental stage. In this study, Myriophyllum alterniflorum (or alternate watermilfoil) enzyme activities (ascorbate peroxidase, catalase, glutathione peroxidase and superoxide dismutase) from in vitro cultures was measured over 27 days in response to copper (Cu) or cadmium (Cd) stress. These enzymes are unique to reactive oxygen species (ROS) scavenging (mainly hydrogen peroxide H2O2 and superoxide anion O2-) and moreover showed specific or unspecific activity profiles, depending on the metal concentrations used. Our results suggest a higher-priority protection of chloroplasts during the initial days of exposure to both metals. At the same time, the increased catalase activity could indicate an H2O2 diffusion in peroxisome in order to protect other organelles from ROS accumulation. However, as opposed to the Cd effects, high Cu concentrations appear to induce a "limited oxidative threshold" for some antioxidant enzymes, which could suggest an ion absorption competition between Cu2+ and Fe2+. In spite of an overall analysis conducted of the scavenging processes occurring in plant cells, biochemical analyses still yielded relevant indications regarding the watermilfoil strategies used for ROS management.

Are Myriophyllum alterniflorum biomarker responses to arsenic stress differentially affected by hydrodynamic conditions?

Arsenic (As) is a significant contaminant in the environment and its detection through macrophytes can provide a powerful tool. Myriophyllum alterniflorum constitutes a good candidate by virtue of its ability to accumulate contaminants, and moreover its biomarkers can respond to the presence of trace metals and metalloids. The objective of this study therefore is to evaluate the watermilfoil response to As exposure under several hydrodynamic conditions since it is well known that hydrodynamics affect plant functioning. For this purpose, fresh watermilfoil plants are subjected to three hydrodynamic conditions, namely laminar, turbulent and calm, in a synthetic medium either enriched or not by 100 µg.L-1 arsenic for 21 days. Growth, pigment content (chlorophyll a, b and carotenoids), respiratory and photosynthetic activities, osmotic potential and hydrogen peroxide concentration are all monitored. Arsenic accumulation is measured separately in the roots and shoots of Myriophyllum alterniflorum. On the one hand, it should be noted that arsenic induces: (i) a significant increase in H2O2 content; (ii) a decrease in osmotic potential, pigment content, photosynthesis and respiration rates, shoot and root growth; and (iii) an inhibition of shoot branching. Moreover, a higher accumulation of this metalloid in roots than in shoots, regardless of the hydrodynamic condition, is witnessed. While on the other hand, hydrodynamic conditions only affect watermilfoil morphology and arsenic accumulation. Also, the younger and older parts have experienced differential toxic effects. Overall, our results suggest the effective use of M. alterniflorum in both water quality biomonitoring and phytoremediation studies.

Comparative in vitro/in situ approaches to three biomarker responses of Myriophyllum alterniflorum exposed to metal stress.

Surface water pollution by trace metal elements constitutes problems for both public and terrestrial/aquatic ecosystem health. Myriophyllum alterniflorum (alternate watermilfoil), an aquatic macrophyte known for bioaccumulating this type of pollutant, is an attractive species for plant biomonitoring within the scope of environmental research. The two metal elements copper (Cu) and cadmium (Cd) are considered in the present study. Cu is essential for plant development at low concentrations, while very high Cu concentrations are detrimental or even lethal to most plants. On the other hand, Cd is usually toxic even at low concentrations since it adversely affects the physiological plant functions. In order to check whether watermilfoil could be used for the in situ biomonitoring of Cu or Cd pollution in rivers, the plant biomarker sensitivity is first tested during long-term in vitro assays. Three markers specific to oxidative stress (glucose-6-phosphate dehydrogenase, malondialdehyde and α-tocopherol) are evaluated by varying the pollutant concentration levels. Given the absence of effective correlations between Cu and all biomarkers, the response profiles actually reveal a dependency between Cd concentration and malondialdehyde or α-tocopherol biomarkers. Conversely, preliminary in situ assays performed at 14 different localities demonstrate some clear correlations between all biomarkers and Cu, whereas the scarcity of Cd-contaminated rivers prevents using the statistical data. Consequently, the three indicated biomarkers appear to be effective for purposes of metal exposure analyses; moreover, the in situ approach, although preliminary, proves to be paramount in developing water biomonitoring bases.

Evaluation of the Relevance of Myriophyllum alterniflorum (Haloragaceae) Cadmium-Sensitive Biomarkers for Ecotoxicological Surveys.

Toxicity caused by trace metal elements in water is a major concern, leading to environmental disturbances and public health problems. The effect of cadmium on clonal macrophyte populations is poorly documented despite its high level of toxicity among aquatic organisms. Our aim here is to highlight the strong relationship existing between the physiological responses of Myriophyllum alterniflorum and the cadmium level over a long exposure period. Nine potential biomarkers of cadmium stress are tested, with three of them appearing to be highly sensitive: free proline, Hsp70, and malondialdehyde. Long-term follow-up analysis after metal exposure (27 days) also proves to be quite beneficial by providing a detailed overview of ecotoxicological events that is more complete and extensive than data recordings conducted over a few days. Taken together, these results support our initial hypothesis that leads to recommending biomarker analyses over at least 2 weeks of metal exposure.

Combined effect of copper and hydrodynamic conditions on Myriophyllum alterniflorum biomarkers.

The aim of this study is to determine the combined effect of copper and hydrodynamic conditions on the response of certain biomarkers of an aquatic macrophyte, namely Myriophyllum alterniflorum. Watermilfoil biomarkers are monitored in a synthetic medium enriched or not with copper (100 µg.L-1) for 21 days in aquarium systems (150 L), under three hydrodynamic conditions: laminar, turbulent, and calm. The studied biomarkers are: respiratory and photosynthetic activities; concentrations of chlorophyll a, b and carotenoids; osmotic potential; hydrogen peroxide content; and growth. In addition, Cu contents in water and in Myriophyllum alterniflorum (roots and shoots) are investigated. The hydrodynamic conditions only affect watermilfoil morphology. Copper accumulates less in turbulent zones; moreover, it is more likely to accumulate in shoots than in roots, except within the calm zone. Cu leads to: i) a significant increase in H2O2 content, ii) a decrease in root growth, pigment content, osmotic potential, photosynthesis and respiration rates, and iii) an inhibition of shoot branching. Differential effects are also observed between younger and older parts, thus indicating the benefit of considering these two plant parts separately in water quality biomonitoring.

Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress.

Proline plays an important role in plant response to various environmental stresses. However, its involvement in mitigation of heavy metal stress in plants remains elusive. In this study, we examined the effectiveness of exogenous proline (10 and 20 mM) in alleviating cadmium induced inhibitory effects in young olive plants (Olea europaea L. cv. Chemlali) exposed to two Cd levels (10 and 30 mg CdCl2 kg(-1) soil). The Cd treatment induced substantial accumulation of Cd in both root and leaf tissues and a decrease in gas exchange, photosynthetic pigments contents, uptake of essential elements (Ca, Mg and K) and plant biomass. Furthermore, an elevation of antioxidant enzymes activities (superoxide dismutase, catalase, glutathione peroxydase) and proline content in association with relatively high amounts of hydrogen peroxide, thiobarbituric acid reactive substances and electrolyte leakage were observed. Interestingly, the application of exogenous proline alleviated the oxidative damage induced by Cd accumulation. In fact, Cd-stressed olive plants treated with proline showed an increase of antioxidant enzymes activities, photosynthetic activity, nutritional status, plant growth and oil content of olive fruit. Generally, it seems that proline supplementation alleviated the deleterious effects of young olive plants exposed to Cd stress.

Absorption and translocation of copper and arsenic in an aquatic macrophyte Myriophyllum alterniflorum DC. in oligotrophic and eutrophic conditions.

The aim of this study is to evaluate copper and arsenic accumulation and translocation at a concentration of 100 µg/L of a submersed macrophyte Myriophyllum alterniflorum. The trophic level (eutrophic and oligotrophic conditions) of the medium was also considered. To achieve this goal, plants were incubated for 21 days in the presence of 100 µg/L of Cu or AsV. The heavy metal transfers from the contaminated medium to plants and into plant tissues was discussed in terms of the bioconcentration factor (BCF) and the translocation factor (TF). Malondialdehyde (MDA) content in tissues was analyzed in order to study the toxicity of these two contaminants. Our results show that copper was more accumulated in shoots, than roots, whereas the opposite trend was observed for arsenic. In addition, the two contaminants were more accumulated in oligotrophic than eutrophic medium. The BCF of copper in shoots was 1356 in oligotrophic condition, while that of arsenic was higher in roots about 620 in the same condition. The TF was less than 1 for arsenic, and higher than 1 for copper, indicating that watermilfoil restrains the translocation of arsenic to shoots, while it has a low capacity to control the translocation of an essential micronutrient like copper. An increase in MDA content was observed under Cu and As stress. On the basis of this experiment, M. alterniflorum has a higher accumulation potential of copper and arsenic, and therefore, it can be a good candidate for the phytofiltration of these two contaminants from water.

Micropropagation of Myriophyllum alterniflorum (Haloragaceae) for stream rehabilitation: first in vitro culture and reintroduction assays of a heavy-metal hyperaccumulator immersed macrophyte.

Nowadays, submersed aquatic macrophytes play a key role in stream ecology and they are often used as biomonitors of freshwater quality. So, these plants appear as natural candidates to stream rehabilitation experiments. Among them, the stream macrophyte Myriophyllum alterniflorum is used recently as biomonitor and is potentially useful for the restoration of heavy-metal contaminated localities. The best way to obtain a mass production of watermilfoil plants is micropropagation. We developed in vitro culture of M. alterniflorum and the effects of five media on the plant development were assessed. Five morphological and four physiological endpoints were examined leading to the recommendation of the Murashige and Skoog medium for ecotoxicological studies on chlorophyllous parts, and of the Gaudet medium for root cytotoxicity and phytoremediation studies. Micropropagated clones were acclimatized in a synthetic medium and in situ reintroduction was performed efficiently. This is the first report of micropropagated plants transplantation in streams. The successful establishment of watermilfoil beds even in polluted areas strongly suggested that ecological restoration using micropropagated watermilfoil is a promising biotechnology for phytoremediation and rehabilitation of degraded areas. Moreover, high bioconcentration factors evidenced that watermilfoil hyperaccumulates Cd and Cu, and could be potentially used in phytoremediation studies.

In vitro establishment and multiplication of the Normania triphylla (Lowe) Lowe

Normania triphylla is an endemic species from Madeira island (Portugal) extinct in the wild since 1991. The aim of this work was to culture the meristems of this species in vitro and to multiply its shoots in order to preserve this endangered species. The best results in terms of multiplication were obtained in Murashige and Skoog medium supplemented with 10 µM 6‑benzylaminopurine (BAP). The number of shoots, the number of nodes and the number of leaves were the most important in this medium. However, the best results concerning the total shoot length were obtained when BAP was not supplemented into the medium and in the presence of 5 or 7.5 µM 1-naphtalene acetic acid (NAA). This is the first report on the in vitro culture of N. triphylla which could brings new avenues for the development of this species.

Nemesia root hair response to paper pulp substrate for micropropagation.

Agar substrates for in vitro culture are well adapted to plant micropropagation, but not to plant rooting and acclimatization. Conversely, paper-pulp-based substrates appear as potentially well adapted for in vitro culture and functional root production. To reinforce this hypothesis, this study compares in vitro development of nemesia on several substrates. Strong differences between nemesia roots growing in agar or in paper-pulp substrates were evidenced through scanning electron microscopy. Roots developed in agar have shorter hairs, larger rhizodermal cells, and less organized root caps than those growing on paper pulp. In conclusion, it should be noted that in this study, in vitro microporous substrates such as paper pulp lead to the production of similar root hairs to those found in greenhouse peat substrates. Consequently, if agar could be used for micropropagation, rooting, and plant acclimatization, enhancement could be achieved if rooting stage was performed on micro-porous substrates such as paper pulp.