Physiological, structural and ultrastructural impacts of silver nanoparticles on the seagrass Cymodocea nodosa.

Silver nanoparticles (AgNPs) are an emerging contaminant, currently considered to be a significant potential risk to the coastal environment. To further test potential risk, and to determine effect concentrations and sensitive response parameters, toxic effects of environmentally relevant AgNP concentrations on the seagrass Cymodocea nodosa were evaluated. Alterations of the cytoskeleton, endoplasmic reticulum, ultrastructure, photosystem II function, oxidative stress markers, cell viability, and leaf, rhizome and root elongation in C. nodosa exposed to AgNP concentrations (0.0002-0.2 mg L-1) under laboratory conditions for 8 days were examined. An increase in H2O2 level, indicating oxidative stress, occurred after the 4th day even at 0.0002 mg L-1. Increased antioxidant enzyme activity, potentially contributing to H2O2 level decline at the end of the experiment, and reduced protein content were also observed. Actin filaments started to diminish on the 6th day at 0.02 mg L-1; microtubule, endoplasmic reticulum, chloroplast and mitochondrion disturbance appeared after 8 days at 0.02 mg L-1, while toxic effects were generally more acute at 0.2 mg L-1. A dose-dependent leaf elongation inhibition was also observed; as for juvenile leaves, toxicity index increased from 2.8 to 40.7% with concentration. Hydrogen peroxide (H2O2) overproduction and actin filament disruption appeared to be the most sensitive response parameters, and thus could be utilized as early warning indicators of risk to seagrass meadows. A risk quotient of 1.33 was calculated, confirming previous findings, that AgNPs may pose a significant risk to the coastal environment.

Floral development of petaloid Alismatales as an insight into the origin of the trimerous Bauplan in monocot flowers.

Monocots are remarkably homogeneous in sharing a common trimerous pentacyclic floral Bauplan. A major factor affecting monocot evolution is the unique origin of the clade from basal angiosperms. The origin of the floral Bauplan of monocots remains controversial, as no immediate sister groups with similar structure can be identified among basal angiosperms, and there are several possibilities for an ancestral floral structure, including more complex flowers with higher stamen and carpel numbers, or strongly reduced flowers. Additionally, a stable Bauplan is only established beyond the divergence of Alismatales. Here, we observed the floral development of five members of the three 'petaloid' Alismatales families Butomaceae, Hydrocharitaceae, and Alismataceae. Outer stamen pairs can be recognized in mature flowers of Alismataceae and Butomaceae. Paired stamens always arise independently, and are either shifted opposite the sepals or close to the petals. The position of stamen pairs is related to the early development of the petals. In Butomaceae, the perianth is not differentiated and the development of the inner tepals is not delayed; the larger inner tepals (petals) only permit the initiation of stamens in antesepalous pairs. Alismataceae has delayed petals and the stamens are shifted close to the petals, leading to an association of stamen pairs with petals in so-called stamen-petal complexes. In the studied Hydrocharitaceae species, which have the monocot floral Bauplan, paired stamens are replaced by larger single stamens and the petals are not delayed. These results indicate that the origin of the floral Bauplan, at least in petaloid Alismatales, is closely linked to the position of stamen pairs and the rate of petal development. Although the petaloid Alismatales are not immediately at the base of monocot divergence, the floral evolution inferred from the results should be a key to elucidate the origin of the floral Bauplan of monocots.

Structural and physiological responses of Halodule wrightii to ocean acidification.

Coastal areas face high variability of seawater pH. Ocean acidification (OA) and local stressors are enhancing this variability, which poses a threat to marine life. However, these organisms present potential phenotypic plasticity that can offer physiological and structural tools to survive in these extreme conditions. In this study, we evaluated the effects of elevated CO2 levels and consequent pH reduction on the physiology, anatomy and ultrastructure of the seagrass Halodule wrightii. A mesocosm study was conducted in an open system during a 30-day experiment, where different concentrations of CO2 were simulated following the natural variability observed in coastal reef systems. This resulted in four experimental conditions simulating the (i) environmental pH (control condition, without CO2 addition) and (ii) reduced pH by - 0.3 units, (iii) - 0.6 units and (iv) - 0.9 units, in relation to the field condition. The evaluated population only suffered reduced optimum quantum yield (Y(II)), leaf width and cross-section area under the lowest CO2 addition (- 0.3 pH units) after 30 days of experiment. This fitness commitment should be related to carbon concentration mechanisms present in the evaluated species. For the highest CO2 level, H. wrightii demonstrated a capacity to compensate any negative effect of the lowest pH. Our results suggest that the physiological behaviour of this primary producer is driven by the interactions among OA and environmental factors, like irradiance and nutrient availability. The observed behaviour highlights that high-frequency pH variability and multifactorial approaches should be applied, and when investigating the impact of OA, factors like irradiance, nutrient availability and temperature must be considered as well.

The influence of salinity on growth, morphology, leaf ultrastructure, and cell viability of the seagrass Halodule wrightii Ascherson.

Halodule wrightii is an ecologically important seagrass; however, little is known about the adaptation of this species in the context of environmental change, particularly changes arising from alterations in salinity of coastal ecosystems. This study aimed to determine the effects of different salinities on growth, morphology, leaf ultrastructure, and cell viability of H. wrightii. To accomplish this, plants were cultivated for 21 days in salinities of 25, 35, and 45. More hydropotens were observed in samples exposed to salinity of 45 with increased invagination of the plasma membrane and cell wall. These invaginations were also observed in other epidermal cells of the leaf blade. In particular, a significant retraction of plasma membrane was seen in samples exposed to salinity of 45, with possible deposition of compounds between the membrane and cell wall. Osmotic stress in samples exposed to salinity of 45 affected the chloroplasts through an increase in plastoglobules and thylakoids by granum in the epidermal chloroplasts of the leaf and decrease in the number of chloroplasts. Overall, this study showed that H. wrightii can survive within salinities that range between 25 and 45 without changing growth rate. However, the plant did have higher cell viability at salinity of 35. Salt stress in mesocosms, at both salinity of 25 and 45, decreased cell viability in this species. H . wrightii had greater changes in salinity of 45; this showed that the species is more tolerant of salinities below this value.

Microtubule integrity and cell viability under metal (Cu, Ni and Cr) stress in the seagrass Cymodocea nodosa.

The effects of increasing Cu, Ni and Cr concentrations (0.5, 5, 10, 20 and 40 mg L(-1)) on microtubule organization and the viability of leaf cells of the seagrass Cymodocea nodosa for 13 consecutive days were investigated under laboratory conditions. Increased oblique microtubule orientation, microtubule depolymerization at the 5-40 mg L(-1) Ni treatments after 3 d of exposure, and a complete microtubule depolymerization at all Ni treatments after 5 d were observed. Cu depolymerised microtubules after three to 7 d of exposure, while Cr caused an extensive microtubule bundling after 9 or 11 d of exposure, depending on metal dosage. Fluorescence intensity measurements further consolidated the above phenomena. Cell death, occurring at later time than microtubule disturbance, was also observed at all Cu and Ni treatments and at the 10-40 mg L(-1) Cr treatments and adding to the above quantification of the number of dead cells clearly showed that only a portion of the cell population studied died. The data presented, being the first assessment of microtubule disturbance in seagrasses, indicate that microtubules in seagrass leaf cells could be used as a valuable and early marker of metal-induced stress in biomonitoring programmes.

Flowers and inflorescences of the seagrass Posidonia (Posidoniaceae, Alismatales).

PREMISE OF THE STUDY: The predominantly aquatic order Alismatales displays a highly variable flower groundplan associated with a diverse range of developmental patterns. We present the first detailed description of flower anatomy and development in Posidonia, the sole genus of the seagrass family Posidoniaceae. Existing accounts provide conflicting interpretations of floral and inflorescence structure, so this investigation is important in clarifying morphological evolution within this early-divergent monocot order. • METHODS: We investigated two species of Posidonia using light microscopy and scanning electron microscopy. Our observations are interpreted in the framework of a recent molecular phylogeny. • KEY RESULTS: Partial inflorescences are bracteate spikes, which are arranged into a botryoid or a panicle. The flowers are perianthless. The gynoecium is monomerous with the ventral carpel side oriented abaxially. The carpel contains a single pendent bitegmic ovule with a nucellus and long chalaza, both extending along the carpel wall. The ovule develops an integumentary outgrowth. Each flower is supplied by a vascular bundle, whereas the flower-subtending bracts are nonvascularized. • CONCLUSIONS: Our data support a racemose interpretation for the partial inflorescence of Posidonia and the presence of flower-subtending bracts. In common with some other Alismatales, Posidonia has simultaneous development of the flower and its subtending bract and loss of the bract vascular supply accompanied by innervation of the flower by a single vascular strand. The unusual carpel orientation could be an evolutionary reduction of a formerly tricarpellate gynoecium. The ovule of Posidonia is campylotropous and unusual within Alismatales in possessing an integumentary outgrowth.

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning.

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 µM) and high (50 µM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.