Damage of heavy metals to Vallisneria natans (V. natans) and characterization of microbial community in biofilm.

Heavy metals can cause a significant damage to submerged macrophytes and affect its periphyton biofilms in aquatic environments. This study investigated the effects of heavy metals such as copper (Cu), lead (Pb), cadmium (Cd) and their mixture on physiological and biochemical responses and ultrastructure characteristics of Vallisneria natans (V. natans). Furthermore, differences in structures of microbial communities were observed in biofilms. The results showed that Cu2+, Pb2+, Cd2+ and their mixture could destroy cell structure and photosynthetic system, and directly caused oxidative damage to submerged macrophyte and induced antioxidant enzyme system. In general, biomass and total chlorophyll content of V. natans noticeably decreased, while the activities of superoxide dismutase, peroxidase and catalase were enhanced by heavy metal stress inducement in restricted range, and the malondialdehyde content increased with the aggravation of the damage. The single heavy metal stress played a negative impact, however, the combined stress was not always synergistic effects on plants. High-throughput sequencing analysis suggested that heavy metals changed the abundance and structure of the microbial biofilm community. Proteobacteria and Bacteroidete were the dominant bacteria under heavy metal stress and other species and abundance of bacteria such as Firmicute, Cyanobacteria, Chloroflexi, Actinobacteria, Verrucomicrobia, Acidobacteria, Deinococcus-Thermus, Chlamydiae were also present. These findings provided useful information for further understanding about submerged macrophytes and periphyton biofilms responsed to heavy metal stress in aquatic environments in the future.

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.

Uranium biosorption from aqueous solution by the submerged aquatic plant Hydrilla verticillata.

The biosorption characteristics of U(VI) from aqueous solution onto a nonliving aquatic macrophyte, Hydrilla verticillata (dry powder), were investigated under various experimental conditions by using batch methods. Results showed that the adsorption reached equilibrium within 60 min and the experimental data were well fitted by the pseudo-first-order kinetic model. U(VI) adsorption was strongly pH dependent, and the optimum pH for U(VI) removal was 5.5. Isotherm adsorption data displayed good correlation with the Langmuir model, with a maximum monolayer adsorption capacity of 171.52 mg/g. Thermodynamic studies suggested that U(VI) adsorption onto H. verticillata was an exothermic and spontaneous process in nature. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy indicated that the amino and hydroxyl groups on the algal surface played an important role in U(VI) adsorption. The mechanisms responsible for U(VI) adsorption could involve electrostatic attraction and ion exchange. In conclusion, H. verticillata biomass showed good potential as an adsorption material for the removal of uranium contaminants in aqueous solution.

Circadian rhythm of a red-tide dinoflagellate Peridinium quadridentatum in the port of Veracruz, Gulf of Mexico, its thecal morphology, nomenclature and geographical distribution.

A circadian rhythm of the dinoflagellate Peridinium quadridentatum was studied at a time-series station in the southwestern Gulf of Mexico, in May 2007. Different substrates (water column, the seagrass Thalassia testudinum, macroalgae, coral rubble and sandy sediment surface) were sampled at the site at 1.5-3.5m depth. In the samples of coral rubble, P. quadridentatum was scarce. In the water column, the species showed an abundance peak at 15:00. The cell abundance of P. quadridentatum in Thalassia samples increased from 15:00 until 18:00 (1.81×10(4)cells/gsubstratewet weight), and then continuously decreased until 06:00. Changes in P. quadridentatum cell abundance on macroalgae followed the same trend as on Thalassia, with the maximal value at 18:00. The higher abundance of P. quadridentatum (up to 1.40×10(4)cells/gSWW) in macroalgae samples showed the preference for seaweeds. P. quadridentatum has a neritic tropical-boreal distribution. A new combination is proposed: Peridinium quadridentatum var. trispiniferum.

Effects of water turbulence on variations in cell ultrastructure and metabolism of amino acids in the submersed macrophyte, Elodea nuttallii (Planch.) H. St. John.

The interactions between macrophytes and water movement are not yet fully understood, and the causes responsible for the metabolic and ultrastructural variations in plant cells as a consequence of turbulence are largely unknown. In the present study, growth, metabolism and ultrastructural changes were evaluated in the aquatic macrophyte Elodea nuttallii, after exposure to turbulence for 30 days. The turbulence was generated with a vertically oscillating horizontal grid. The turbulence reduced plant growth, plasmolysed leaf cells and strengthened cell walls, and plants exposed to turbulence accumulated starch granules in stem chloroplasts. The size of the starch granules increased with the magnitude of the turbulence. Using capillary electrophoresis-mass spectrometry (CE-MS), analysis of the metabolome found metabolite accumulation in response to the turbulence. Asparagine was the dominant amino acid that was concentrated in stressed plants, and organic acids such as citrate, ascorbate, oxalate and γ-amino butyric acid (GABA) also accumulated in response to turbulence. These results indicate that turbulence caused severe stress that affected plant growth, cell ultrastructure and some metabolic functions of E. nuttallii. Our findings offer insights to explain the effects of water movement on the functions of aquatic plants.

Direct contribution of the seagrass Thalassia testudinum to lime mud production.

Seagrass beds contribute to oceanic carbonate lime mud production by providing a habitat for a wide variety of calcifying organisms and acting as efficient sediment traps. Here we provide evidence for the direct implication of Thalassia testudinum in the precipitation of aragonite needles. The crystals are located internally in the cell walls, and as external deposits on the blade, and are similar in size and shape to the aragonite needles reported for modern tropical carbonate factories. Seagrass calcification is a biological, light-enhanced process controlled by the leaf, and estimates of seagrass annual carbonate production in a Caribbean reef lagoon are as significant as values reported for Halimeda incrassata. Thus, we conclude that seagrass calcification is another biological source for the aragonite lime mud deposits found in tropical banks, and that tropical seagrass habitats may play a more important role in the oceanic carbon cycle than previously considered.

Copper ultrastructural localization, subcellular distribution, and phytotoxicity in Hydrilla verticillata (L.f.) Royle.

Laboratory experiments were conducted to investigate copper (Cu) subcellular distribution and toxicity in Hydrilla verticillata. Fronds were subjected to different concentrations (15, 75, and 150 µM) of Cu for 7 days. Cu grains were found in cell walls, plasmodesmata, and within the nuclei and chloroplasts using the autometallographic technique. Subcellular fractionation of Cu-containing tissues indicated that in leaves subjected to high Cu concentrations, 59-65 % of the element was located in the cell wall fraction, followed by cell organelles (21-30 %) and the soluble fraction (10-14 %). The levels of K, P, Zn, and Mg declined under all Cu concentrations, but Ca, Mn, and Fe contents reached their peak at 15 µM Cu and decreased thereafter. F v/F m, F 0, and F m fell significantly in line with the decrease in pigment content. Cu exposure also caused significant damage to the chloroplasts, mitochondria, and nuclei, including disintegration of the chloroplasts and vacuolization of the mitochondria and nuclei, all of which suggested that Cu hastened plant senescence. The Cu maximum permissible concentration for H. verticillata was 10 µM, which was less than the existing general water quality standard. This suggested that H. verticillata could be used to assess Cu phytotoxicity.

Laboratory assessment of uptake and toxicity of lanthanum (La) in the leaves of Hydrocharis dubia (Bl.) Backer.

Increasing amounts of lanthanum (La) is released into aquatic environments. However, little information is available on the influence of La on aquatic plants. In this study, physiological and ultrastructural responses of Hydrocharis dubia (Bl.) Backer leaves to elevated concentrations of La (up to 160 µM) were investigated. The accumulation of La was found to be increased in a concentration-dependent manner. La disturbed the intrinsic balance of nutrient elements (P, Mg, Ca, Fe, K, and Zn). Pigment content decreased with the rise of the La concentrations and the EC(50) value for chlorophyll was 20 µM on day 7. The antioxidants (superoxide dismutase, peroxidase, catalase, reduced ascorbate, and reduced glutathione) exhibited varied response to the La treatments. Malondialdehyde content enhanced gradually at all La concentrations. The enhancement in proline content was found in a concentration-dependent manner. The amounts of three polypeptides with apparent molecular weights of 61.9, 51.5, and 16.7 kDa, respectively, were gradually diminished, as well as one existing polypeptides with apparent molecular weight of 22.3 kDa, elevating in response to increasing La concentrations. Significant damage to the chloroplast, mitochondrion, and nucleus was imposed by La indicated a general disarray in the cellular functions. The negative effects of La on H. dubia unequivocally indicate that La could exert an adverse influence on aquatic ecosystem and should lead to a more careful discharge of such elements into water environment.

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis.

BACKGROUND AND AIMS: Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells. METHODS: Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. KEY RESULTS: The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls. CONCLUSIONS: The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.

Nitric oxide supplementation alleviates ammonium toxicity in the submerged macrophyte Hydrilla verticillata (L.f.) Royle.

The likely protective effects of nitric oxide (NO) against ammonium toxicity were investigated in the submerged macrophyte Hydrilla verticillata. The plants were subjected to ammonium stress (3mM ammonium chloride) in the presence of sodium nitroprusside (SNP, 10 µM), an NO donor. Treatment with SNP significantly increased the NO content and partially reversed the ammonium-induced negative effects, including membrane damage and the decrease in levels of chlorophyll, malondialdehyde, glutathione and ascorbic acid. Further, SNP application increased the catalytic activities of ascorbate peroxidase, superoxide dismutase, guaiacol peroxidase, catalase and glutathione S-transferase, but decreased that of NADH-oxidase. Histochemical staining showed that SNP application caused a significant decrease in the levels of superoxides and hydrogen peroxide. In contrast, application of other breakdown products of SNP (10 µM sodium ferrocyanide, 10 µM sodium nitrite and 10 µM sodium nitrate) failed to show any protective effect. The results suggest that the increased intracellular NO, resulting from SNP application, improved the antioxidant capacity of H. verticillata plants in coping with ammonium-induced oxidative stress.

Chromatically resolved optical microscope (CROMoscope): a grating-based instrument for spectral imaging.

The chromatically resolved optical microscope (CROMoscope) is capable of spectral imaging with tunable spectral and spatial resolutions. Because of its remarkably simple design, the CROMoscope can be easily assembled and aligned. Spectral resolution as low as 2.5 nm full width at half maximum (fwhm) was measured using an atomic emission line of Hg. Absorption spectra of different parts of a micrograph can readily be compiled using white-light illumination. Chloroplast absorption from an Elodea plant leaf was used to demonstrate this capability. Spectral imaging is widely applicable to many areas of science, and the CROMoscope is particularly simple to adapt to conventional microscopes and should enable detailed spectroscopic information to be obtained from microscopy.

Effects of chitosan on growth of an aquatic plant (Hydrilla verticillata) in polluted waters with different chemical oxygen demands.

Effects of chitosan on a submersed plant, Hydrilla verticillata, were investigated. Results indicated that H. verticillata could prevent ultrastructure phytotoxicities and oxidativereaction from polluted water with high chemical oxygen demand (COD). Superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents in H. verticillata treated with 0.1% chitosan in wastewater increased with high COD (980 mg/L) and decreased with low COD (63 mg/L), respectively. Ultrastructural analysis showed that the stroma and grana of chloroplast basically remained normal. However, plant cells from the control experiment (untreated with chitosan) were vacuolated and the cell interval increased. The relict of protoplast moved to the center, with cells tending to disjoint. Our findings indicate that wastewater with high COD concentration can cause a substantial damage to submersed plant, nevertheless, chitosan probably could alleviate the membrane lipid peroxidization and ultrastructure phytotoxicities, and protect plant cells from stress of high COD concentration polluted water.

Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light.

In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15-20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells.

Correlation between pollen morphology and pollination mechanisms in the Hydrocharitaceae.

The pollen morphology of 11 genera and 11 species of the Hydrocharitaceae and one species of the Najadaceae was studied using scanning and transmission electron microscopies, and the exine structures and sculptures are discussed in relation to pollination mechanisms and the molecular phylogeny. The pollen grains of the Hydrocharitaceae are spherical, inaperturate, and form monads or tetrads, while those of the Najadaceae are elliptical, inaperturate, and form monads. The entomophilous genera Egeria, Blyxa, Ottelia, Stratiotes, and Hydrocharis share pollen grains that have projections like spines or bacula. The anemophilous genus Limnobium has reticulate pollen grains. The hypohydrophilous genera Thalassia and Najas are characterized by pollen grains with reduced exine structures. The pollen-epihydrophilous genera Elodea and Hydrilla have tightly arranged small spinous pollen grains, and the male flower-epihydrophilous genera Enhalus and Vallisneria have reduced reticulate or gemmate exines. Character state reconstruction of the exine structures and sculptures using a molecular phylogenetic tree suggests that variation in the exine is generally correlated with the pollination mechanism; the selective pressures acting on the pollination mechanisms have reduced the exine structure in hypohydrophilous plants and resulted in various exine sculptures that are adapted to the different pollination mechanisms in entomophilous, anemophilous, and pollen-epihydrophilous plants.

[Distribution and toxicity of cadmium in Hydrilla verticillata (L.f.) Royle].

Aquatic plants are known to accumulate and bioconcentrate heavy metals. In this study, aquatic vascular plant Hydrilla verticillata (L.f.) Royle was cultivated in water containing elevated concentrations of cadmium (up to 10 mg/L) for 7 d, the accumulation, subcellular distribution, ultrastructural localization, chemical form, toxic effects on mineral nutrient absorption of cadmium, photosynthesis rate and respiration rate were studied. It was found that H. verticillata fronds was able to accumulate cadmium, the bioconcentration factor was 193-307; subcellular fraction analysis revealed that cadmium major accumulated in cell wall (61.66%-52.00%) with decrease trends, the content of Cd enhanced in soluble fraction and remained stable in organelles, with the augment of pollutant concentration. The Cd levels occurred in different parts of leaf cell with the following sequence: cell wall > soluble fraction > organelles. Ultrastructural localization of cadmium with sulfide-silver method showed that Cd appeared in cell wall, chloroplast, nucleus and vacuole. Sequential extraction indicated that the ratio of different cadmium chemical form was different markedly, of which NaCl extractable Cd was predominated as compared with the other 5 forms; which could be seen in the following order: F(NaCl) > F(HAc) >F(Water) > F(Ethanol) > F(HCl) > F(Residue). The mineral nutrient absorption was also affected by cadmium stress, it increased the absorption of Ca, Mn, Cu, and Fe; but reduced that of P and K. Cd had a strong inhibitive effect on photosynthesis rate and respiration rate. The results suggested that the toxic symptoms of plant showed an evident correlation between dose and effect; the ultrastructural damage was closely related to the distribution of Cd. The conclusion could be reached that the death of plant was resulted from destruction of structure foundation of physiological function, unbalance of ion equilibrium and disorder of physiological metabolism.

Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability.

In this report, we describe a highly reproducible femtosecond near-infrared (NIR) laser-based nanoprocessing technique that can be used both for non-invasive intra-tissue nanodissection of plant cell walls as well as selective destruction of a single plastid or part thereof without compromising the viability of the cells. The ultra-precise intra-tissue nanoprocessing is achieved by the generation of high light intensity (10(12)W cm(-2)) by diffraction-limited focusing of the radiation of an NIR (lambda = 740 and 800 nm) femtosecond titanium-sapphire laser to a sub-femtolitre volume and subsequent highly localized instantaneous plasma formation. Following nanosurgery, electron microscopical analysis of the corresponding cellular target areas revealed clean non-staggering lesions across the cell wall with a cut width measuring less than 400 nm. To our knowledge, this is the smallest cut made non-invasively within a plant tissue. Further evidence, including two-photon imaging of chlorophyll fluorescence, revealed that a single target chloroplast or part thereof can be completely knocked out using intense ultra-fast NIR pulses without any visible deleterious effect on the adjacent plastids. The vitality of the cells after nanoprocessing has been ascertained by exclusion of propidium iodide from the cells as well as by the presence of cytoplasmic streaming. The potential applications of this technical advance include developmental biology applications, particularly studies addressing spatio-temporal control of ontogenetic events and cell-cell interactions, and gravitational biology applications.