Iron-induced behavioural and biochemical responses of charophytes in consequence of phosphates coagulant addition: Threats to lake ecosystems restoration.

The study aimed to evaluate the impact of iron (Fe) on the physiological and behavioural reaction of Chara tomentosa L. Fe was introduced into the environment in the form of iron chloride, the most common coagulants used in the restoration of water bodies. The investigations concerned the oxidative stress comprising phenolic compounds content, antioxidant activity and photosynthetic pigments concentration. Research was conducted as a laboratory microcosm experiment with one-off application of Fe at the level of 26.8 mg dm-3. Coagulant application caused short-term acidification, increased salinity and deterioration of light conditions. The shading resulted initially from the increase of water colour and turbidity and was followed by covering of the charophytes with a precipitated suspension. C. tomentosa did not activate defensive mechanisms to prevent the shading effect such as intensive elongation and elevated concentration of chlorophylls. Neither oxidative stress nor production of stress-specific phenolic metabolites was found. It was a result of iron coagulant toxicity, which led to cell membrane damage and leakage of cell contents to the water environment. Charophyte growth was significantly impaired, and thalli suffered numerous chlorotic and necrotic spots which extended gradually during experiment and finally caused death of specimens.

Primitive Auxin Response without TIR1 and Aux/IAA in the Charophyte Alga Klebsormidium nitens.

The phytohormone auxin regulates many aspects of growth and development in land plants, but the origin and evolution of auxin signaling and response mechanisms remain largely unknown. Indeed, it remains to be investigated whether auxin-related pathways diverged before the emergence of land plants. To address this knowledge deficit, we analyzed auxin responses in the charophyte alga Klebsormidium nitens NIES-2285, whose ancestor diverged from a green algal ancestor during the evolution of land plants. This strain is the same as Klebsormidium flaccidum NIES-2285, for which the draft genome was sequenced in 2014, and was taxonomically reclassified as K. nitens This genome sequence revealed genes involved in auxin responses. Furthermore, the auxin indole-3-acetic acid (IAA) was detected in cultures of K. nitens, but K. nitens lacks the central regulators of the canonical auxin-signaling pathway found in land plants. Exogenous IAA inhibited cell division and cell elongation in K. nitens Inhibitors of auxin biosynthesis and of polar auxin transport also inhibited cell division and elongation. Moreover, exogenous IAA rapidly induced expression of a LATERAL ORGAN BOUNDARIES-DOMAIN transcription factor. These results suggest that K. nitens has acquired the part of the auxin system that regulates transcription and cell growth without the requirement for the central players that govern auxin signaling in land plants.

Accumulation of copper in the cell compartments of charophyte Nitellopsis obtusa after its exposure to copper oxide nanoparticle suspension.

Cu accumulation in the internodal cell of charophyte Nitellopsis obtusa or its compartments was investigated after 3-h-exposure to lethal effective concentrations (8-day LC50) of CuO nanoparticle (nCuO) suspension or CuSO4 solution, i.e. 100 mg/L nCuO or 3.18 mg Cu/L as CuSO4. In both cases, the major part of Cu accumulated in the cell walls. The presence of CuO NPs in the cell wall and within the cell was visualized by scanning electron microscope images as well as confirmed by energy dispersive X-ray spectrum data. Although a threefold higher intracellular concentration of Cu was found after treatment with nCuO suspension, 3.18 mg Cu/L as CuSO4 induced fast and substantial depolarization of cell membrane potential contrary to that of 100 mg/L nCuO. A delayed effect of nCuO on the survival of the cells was also observed. This suggests that internally accumulated Cu was far less active and further supports the hypothesis of delayed toxicity of internalized nCuO NPs to charophyte cells.

Pectin metabolism and assembly in the cell wall of the charophyte green alga Penium margaritaceum.

The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.

The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 µM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

Allelopathic effects of microcystin-LR on the germination, growth and metabolism of five charophyte species and a submerged angiosperm.

Microcystins (MCs) are produced by cyanobacteria in aquatic environments and adversely affect macrophytes at very high concentrations. However, the effects of MC on macrophytes at concentrations of environmental relevance are largely unknown. The main objective of this study was to analyze the allelopathic effects of MC-LR at natural concentrations (1, 8 and 16 µg MC-LR/L) on five charophyte species (Chara aspera, C. baltica, C. hispida, C. vulgaris and Nitella hyalina) and the angiosperm Myriophyllum spicatum. Macrophyte specimens were obtained from a restored area located in Albufera de València Natural Park, a protected coastal Mediterranean wetland. Two different experiments were conducted involving (i) the addition of MC-LR to natural sediment to evaluate its effects on seed germination and (ii) the addition of MC-LR to water cultures of macrophytes to evaluate its effects on growth and metabolic functions. In water, the MC-LR concentration decreased by 84% in two weeks; the loss was not significant in sediment. The first seedlings (all C. hispida) emerged from the wetland sediment following a delay of a few days in the presence of MC-LR. The germination rates in 8 and 16 µg MC-LR/L treatments were 44% and 11% of that occurring in the absence of MC, but these differences disappeared over time. The final density was 6-7 germlings/dm(3). Final germling length was unaffected by MC-LR. Rotifers (Lecane spp.) emerging from the natural sediment during the experiment were favored by MC-LR; the opposite pattern was observed in the cladoceran Daphnia magna. The growth rates of C. vulgaris, C. baltica and N. hyalina were unaffected by MC exposure, whereas those of C. hispida and C. aspera were reduced in the MC treatments relative to the control treatment. The concentration of chlorophyll-a and the in vivo net photosynthetic rate were lower in the presence of MC-LR, even at the lowest concentration, for all of the characeans tested. M. spicatum was sensitive to the presence of MC-LR in the culture medium; the growth and chlorophyll-a concentrations were reduced. Therefore, environmentally relevant concentrations of MC might induce important changes in macrophyte meadows and the structure of the associated plankton community. Synchrony or delay in the processes evaluated here in response to environmentally relevant concentrations of cyanobacteria MC exudates can enhance understanding of the turning point to alternative states and the point of no return in eutrophicated shallow lakes.

Phytoremediation potential of charophytes: bioaccumulation and toxicity studies of cadmium, lead and zinc.

The ability for usage of common freshwater charophytes, Chara aculeolata and Nitella opaca in removal of cadmium (Cd), lead (Pb) and zinc (Zn) from wastewater was examined. C. aculeolata and N. opaca were exposed to various concentrations of Cd (0.25 and 0.5 mg/L), Pb (5 and 10 mg/L) and Zn (5 and 10 mg/L) solutions under hydroponic conditions for 6 days. C. aculeolata was more tolerant of Cd and Pb than N. opaca. The relative growth rate of N. opaca was drastically reduced at high concentrations of Cd and Pb although both were tolerant of Zn. Both macroalgae showed a reduction in chloroplast, chlorophyll and carotenoid content after Cd and Pb exposure, while Zn exposure had little effects. The bioaccumulation of both Cd and Pb was higher in N. opaca (1544.3 microg/g at 0.5 mg/L Cd, 21657.0 microg/g at 10 mg/L Pb) whereas higher Zn accumulation was observed in C. aculeolata (6703.5 microg/g at 10 mg/L Zn). In addition, high bioconcentration factor values (> 1000) for Cd and Pb were observed in both species. C. aculeolata showed higher percentage of Cd and Pb removal (> 95%) than N. opaca and seemed to be a better choice for Cd and Pb removal from wastewater due to its tolerance to these metals.

Water quality as a threat to aquatic plants: discriminating between the effects of nitrate, phosphate, boron and heavy metals on charophytes.

• Eutrophication is a threat to wetlands worldwide. Elevated phosphorus concentration is often the main driver of loss of biodiversity and ecosystem function, but effects of phosphorus and nitrogen have proved largely inseparable, because they vary colinearly. Charophytes, aquatic algae that are very close to the evolutionary link with the land-plant lineage, provide a sensitive system for disentangling complex pollutant threats. • Here, we investigated aquatic vegetation and water quality at the principal sites for charophyte biodiversity in the UK and used hierarchical partitioning to discriminate independent effects of pollutants on their occurrence. A laboratory experiment examined the growth responses of a representative species (Chara globularis) to nitrate. • Nitrate-N exerted the greatest detrimental effect on charophyte occurrence in the field. Furthermore, growth of C. globularis in the laboratory was inhibited above very low concentrations. Smaller independent effects of copper (Cu), cadmium (Cd), cobalt (Co), phosphate-P, nickel (Ni), boron (B) and manganese (Mn) on charophyte occurrence were discriminated. • It is possible to separate the deleterious effects of phosphorus and nitrogen on aquatic organisms in the field. Nitrate is a critical factor and a mean annual average concentration limit of c. 2 mg l⁻¹ nitrate-N is necessary to protect charophytes and their services within wetland ecosystems.