Effects of copper enrichment on survival,growth and photosynthetic pigment of seedlings and young plants of the eelgrass Zostera marina

Copper (Cu²⁺) is an essential nutrient for plants but toxic at high concentrations. We subjected seedlings and young plants of eelgrass Zostera marina to different seawater Cu concentrations (3, 4, 5, 10, 30 and 50?µg?l^?1) for over 30 days under controlled laboratory conditions. Natural seawater without added Cu (3?µg?l^?1) was used as reference seawater. We measured plant response in terms of survivorship, morphology, growth, productivity and leaf pigment concentration. Survival analysis combined with morphological, dynamic and productive assessment suggested that the optimum seawater Cu concentration for the establishment of Z. marina seedlings and young plants is 4?μg?l^?1. The photosynthetic response of young plants to copper enrichment, including an increase in chlorophyll content under low Cu concentration treatment but significant decrease when treated with high concentrations of Cu, is similar to those reported for other seagrass species. NOEC (no observed effect concentration), LOEC (lowest observed effect concentration) and LC₅₀ (lethal concentration that caused an increase in mortality to 50% of that of the control) values of seedlings were significantly lower than those of young plants, implying a reduced Cu tolerance to high concentrations (>10?μg?l^?1). This study provides data that could prove helpful in the development of successful eelgrass restoration and conservation.     

Response of two tomato cultivars to field-applied proline under irrigation with saline water: Growth,chlorophyll fluorescence and nutritional aspects

The response of tomato (Solanum lycopersicum L.) to abiotic stress has been widely investigated. Recent physiological studies focus on the use of osmoprotectants to ameliorate stress damage, but experiments at a field level are scarce. Two tomato cultivars were used for an experiment with saline water (6.57 dS m^?1) and subsurface drip irrigation (SDI) in a silty clay soil. Rio Grande is a salinity-tolerant cultivar, while Heinz-2274 is the salt-sensitive cultivar. Exogenous application of proline was done by foliar spray at two concentrations (10 and 20 mg L^?1) during the flowering stage. Control plants were treated with saline water without proline. Proline at the lower concentration (10 mg L^?1) increased dry mass of different plant organs (leaves, stems, and roots) and it improved various chlorophyll a fluorescence parameters compared with controls. Regarding mineral nutrition, K⁺ and P were higher in different organs, while low accumulation of Na⁺ occurred. However, Mg²⁺ was very high in all tissues of Rio Grande at the higher concentration of proline applied. Thus, the foliar spray of proline at 10 mg L^?1 increased the tolerance of both cultivars. The growth of aboveground biomass of Heinz-2274 was enhanced by 63.5%, while Rio Grande improved only by 38.9%.     

Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation

Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵¹-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.     

Glutamine synthetase activity and free amino acid pools of eelgrass (Zostera marina L.) roots

Activity of the enzyme glutamine synthetase (GS, EC 6.3.1.2) was determined in vitro for roots of the marine angiosperm Zostera marina L. (eelgrass) collected from a population in Great Harbor, Woods Hole, Massachusetts, U.S.A. The GS synthetase activity was lowest in roots of plants collected from the shallow region of the eelgrass bed (12.0 μmol·g⁻¹ (fresh wt)· h⁻¹) and increased in the mid (3.0 m, 40.3 μmol·g⁻¹ (fresh wt)·h⁻¹) and deep (5.0 m, 72.3 μmol·g⁻¹ (fresh wt)·h⁻¹) plant collection depths. GS transferase activity increased with collection depth in a similar manner: shallow, 28.6 μmol·g⁻¹ (fresh wt)·h⁻¹; mid, 52.0 μmol·g⁻¹ (fresh wt)·h⁻¹; deep, 92.8 μmol·g⁻¹ (fresh wt)·h⁻¹. When sediment-embedded plants were held in continuous darkness for 2 days to create extended root anoxia, root GS activities nearly doubled. In contrast, in vivo incorporation of ¹⁴C-glutamate into glutamine and protein residue remained constant or declined under short-term hypoxia and anoxia. During aerobic recovery from anoxia, root labelling of glutamine and protein increased markedly. Free amino acid patterns of eelgrass roots growing in situ were determined over a diurnal cycle. Total free amino acid content was maximal at dawn and decreased 50% by noon. In contrast, the proportion of glutamine was lowest at dawn and maximal at noon for both shallow and deep-growing plants. Despite differences in depth-specific plant sizes, root/rhizome/shoot ratios, and relative growth rates, the daily whole plant nitrogen demand of shallow and deep growing plants were equivalent. When corrected for assay temperature response, the enzyme synthetase activities measured in vitro suggest that all of the plant nitrogen assimilation requirements can be met within daylight hours during the period of peak summer biomass.     

The abundance,biomass and acetylene reduction activity of bacteria associated with decomposing rhizomes of two seagrasses,Zostera marina and Thalassia testudinum

Bacteria growing on and in close association with the rhizome detritus of two seagrasses, Zostera marina L. and Thalassia testudinum Banks ex König, were examined using epifluorescence and scanning electron microscopy. The microbial community consisted of a diverse assemblage of bacteria dominated in biomass by large rod-shaped and filamentous cells. The large size of cells and the occurrence of measurable acetylene reduction activity suggested that a healthy, growing population of bacteria was associated with the rhizome detritus. Bacteria carbon biomass ranged betwee 5.2×10⁻⁵ and 1.7×10⁻³ g C gdw⁻¹ of rhizome detritus. Depending on cell doubling times, bacterial metabolism could account for a substantial portion of the turnover of rhizome detritus. Estimates of potential microbial production, nitrogen fixation and the physico-chemical nature of rhizome detritus are discussed and we propose hypotheses for the disposition of this detrital organic matter.     

Effects of filamentous algal mats on sulfide invasion in eelgrass (Zostera marina)

The effect of filamentous algae invasion into Zostera marina meadows on water quality, sediment sulfur pools and sulfide invasion into plant tissues was studied experimentally. Sulfide invasion was assessed through analysis of sulfur isotopic composition (δ³⁴S) and total sulfur (TS) concentrations in plant tissues. The algal mats (5 and 10 cm thickness) depleted oxygen in the mats and increased the pools of sulfides in the sediments. Plants exposed to algal mats had δ³⁴S signals closer to the δ³⁴S of sediment sulfide, whereas plants with no mats present had δ³⁴S signals closer to the δ³⁴S of seawater sulfate, indicating a higher sulfide invasion in plants exposed to algal mats. The δ³⁴S varied between the plant tissues with the leaves having more positive δ³⁴S signals than roots and rhizomes, indicating that sulfide was invading into the roots and moved to the other tissues through the lacunae. TS concentrations were higher in plants exposed to algal mats suggesting that sulfur derived from sediment sulfide accumulated in the plants. F_sulfide showed that up to 50% of the sulfides in the plants were derived from sedimentary sulfides. The combined effect of water column anoxia in the lower parts of the meadow and high sulfide invasion into the plants lead to significantly reduced growth rates after 3 weeks and the below-ground tissues showed signs of degradation suggesting that algal mats invasion in to Zostera marina meadows can result in seagrass decline.     

Nonrecirculating hydroponic system suitable for uptake studies at very low nutrient concentrations

Seedlings of maize (Zea mays L. cv WF9 × Mo 17) growing at low water potentials in vermiculite contained greatly increased proline concentrations in the primary root growth zone. Proline levels were particularly high toward the apex, where elongation rates have been shown to be completely maintained over a wide range of water potentials. Proline concentration increased even in quite mild treatments and reached 120 millimolal in the apical millimeter of roots growing at a water potential of −1.6 megapascal. This accounted for almost half of the osmotic adjustment in this region. Increases in concentration of other amino acids and glycinebetaine were comparatively small. We have assessed the relative contributions of increased rates of proline deposition and decreased tissue volume expansion to the increases in proline concentration. Proline content profiles were combined with published growth velocity distributions to calculate net proline deposition rate profiles using the continuity equation. At low water potential, proline deposition per unit length increased by up to 10-fold in the apical region of the growth zone compared to roots at high water potential. This response accounted for most of the increase in proline concentration in this region. The results suggest that osmotic adjustment due to increased proline deposition plays an important role in the maintenance of root elongation at low water potentials.     

Algicidal and growth-inhibiting bacteria associated with seagrass and macroalgae beds in Puget Sound,WA, USA

The algicidal and growth-inhibiting bacteria associated with seagrasses and macroalgae were characterized during the summer of 2012 and 2013 throughout Puget Sound, WA, USA. In 2012, Heterosigma akashiwo-killing bacteria were observed in concentrations of 2.8 × 10⁶ CFU g⁻¹ wet in the outer organic layer (biofilm) on the common eelgrass (Zostera marina) in north Padilla Bay. Bacteria that inhibited the growth of Alexandrium tamarense were detected within the biofilm formed on the eelgrass canopy at Dumas Bay and North Bay at densities of ∼10⁸ CFU g⁻¹ wet weight. Additionally, up to 4100 CFU mL⁻¹ of algicidal and growth-inhibiting bacteria affecting both A. tamarense and H. akashiwo were detected in seawater adjacent to seven different eelgrass beds. In 2013, H. akashiwo-killing bacteria were found on Z. marina and Ulva lactuca with the highest densities of ∼10⁸ CFU g⁻¹ wet weight at Shallow Bay, Sucia Island. Bacteria that inhibited the growth of H. akashiwo and A. tamarense were also detected on Z. marina and Z. japonica at central Padilla Bay. Heterosigma akashiwo cysts were detected at a concentration of 3400 cysts g⁻¹ wet weight in the sediment from Westcott Bay (northern San Juan Island), a location where eelgrass disappeared in 2002. These findings provide new insights on the ecology of algicidal and growth-inhibiting bacteria, and suggest that seagrass and macroalgae provide an environment that may influence the abundance of harmful algae in this region. This work highlights the importance of protection and restoration of native seagrasses and macroalgae in nearshore environments, in particular those regions where shellfish restoration initiatives are in place to satisfy a growing demand for seafood.     

Pyrroline-5-Carboxylate Reductase Is in Pea (Pisum sativum L.) Leaf Chloroplasts

Proline accumulation is a well-known response to water deficits in leaves. The primary cause of accumulation is proline synthesis. Δ¹-Pyrroline-5-carboxylate reductase (PCR) catalyzes the final reaction of proline synthesis. To determine the subcellular location of PCR, protoplasts were made from leaves of Pisum sativum L., lysed, and fractionated by differential and Percoll density gradient centrifugation. PCR activity comigrated on the gradient with the activity of the chloroplast stromal marker NADPH-dependent triose phosphate dehydrogenase. We conclude that PCR is located in chloroplasts, and therefore that chloroplasts can synthesize proline. PCR activities from chloroplasts and etiolated shoots were compared. PCR activity from both extracts is stimulated at least twofold by 100 millimolar KCl or 10 millimolar MgCl₂. The pH profiles of PCR activity from both extracts reveal two separate optima at pH 6.5 and 7.5. Native isoelectric focusing gels of sampies from etiolated tissue reveal a single band of PCR activity with a pl of 7.8.     

Comparison of Zostera marina and maerl community metabolism

The photosynthetic and repiratory metabolism of Zostera marina and maerl communities was compared, in the same area of the Bay of Brest in March–April, using benthic chambers. P–E curves for both oxygen and carbon were established for bottom irradiances between 0 and 525 μmol m⁻² s⁻¹. An exponential function was fitted to calculate daily production. Community metabolic quotients did not differ for maerl and seagrass beds. Community photosynthetic quotients were significantly higher (1.19) whereas community respiratory quotients were lower (0.70) than 1. Maerl and seagrass bed P–E curves mainly differed by the minimum saturating irradiance (E_k). Net community production was estimated to 26.8 mmol C m⁻² d⁻¹ for Z. marina meadows and 8.6 mmol C m⁻² d⁻¹ for maerl beds. The two communities can, therefore, be considered as autotrophic during the March–April period. Community respiration did not differ between Z. marina meadows and maerl beds, with an average value of 53.8 mmol C m⁻² d⁻¹ during a day. In similar environmental conditions, the production of maerl beds corresponds to approximately one third that of seagrass meadows. The maerl communities, therefore, form productive ecosystems, relevant to temperate coastal ecosystems functioning.     

Littoral benthos of the Northern Baltic Sea. IV. Pattern and dynamics of macrobenthos in a sandy-bottom Zostera marina community in Tvärminne

The composition and abundance of the macroscopic biota of a sandy-bottom Zostera marina community at a depth of 3–5 m were studied in Tvärminne, S Finland, in 1968–1971. Zostera occurred contagiously and very sparsely, 6 ± 2 shoots · m⁻², ash-free dry weight (AFDW) 2 ± 1 g · m⁻² (95% confidence interval). Totals of 45 plant and 70 animal taxa were found. Fucus vesiculosus (80%) and Zostera marina (11%) dominated the plant biomass, 16.5 ± 17.8 g · m⁻² in June 1971. At this time animal density was 8540 ± 830 ind. · m⁻² (0.6-mm mesh) and AFDW 17.3 ± 5.0 g · m⁻², 80–90% of the latter consisting of Macoma baltica, Mya arenaria, Corophium volutator, Nereis diversicolor and Hydrobia spp. The same percentage of animal density was made up by Hydrobia spp., M. baltica, C. volutator and Pygospio elegans. Normal analysis by classification of the samples suggested that the community was spatially rather homogeneous in 1971. Inverse analysis, based on interspecific correlations, revealed three distributional groups of species. These groups corresponded to the compositional subdivisions suggested by normal analysis. The community was subdivided into strata characterized by (D) deposit feeders, (Z) Zostera marina and (F) Fucus vesiculosus. The periodicity of the biota in the Zostera-characterized stratum was investigated. The principal dominants, especially Fucus, Zostera and Macoma, showed no distinct seasonal or interannual differences in 1968–1971, which made the community seem very stable. Yet, the species diversity of plants varied with the abundance of filamentous algae. Animal species diversity and mean animal density reached their seasonal minima in late summer, when the parental stocks of many invertebrates were dying and the summer's offspring were often still too small to be obtained by our methods. The recruitment of many important animal species failed in 1968 as compared with the other years.     

Factors controlling Zostera marina L. growth in the eastern and western Pacific Ocean: Comparisons between Korea and Oregon,USA

Zostera marina distribution is circum-global and tolerates a wide range of environmental conditions. Consequently, it is likely that populations have adapted to local environmental conditions of light, temperature and nutrient supply. We compared Z. marina growth dynamics over a 2-year period in relation to environmental characters at Jindong Bay, South Korea and Yaquina Bay, Oregon, USA. Water temperature in Jindong Bay showed stronger seasonal variation (summer–winter ΔT = 20 °C) than in Yaquina Bay (summer–winter ΔT < 5 °C). Underwater irradiance in Jindong Bay exhibited a winter maximum, while in Yaquina Bay underwater light exhibited a summer maximum. Integrated annual underwater irradiance during 2003 was 2200 and 1200 mol photons m⁻² year⁻¹ in Korea and Oregon, respectively. Z. marina shoot density, biomass and integrated production were not significantly different between the two study sites. Seasonal Z. marina growth in Jindong Bay appeared to be controlled by temperature and light, while the growth pattern in Yaquina Bay suggested light regulation. Several seagrass parameters were correlated to phosphate concentrations, even though nutrients did not appear limiting. Despite differences in environmental factors, relative growth rates and temporal growth dynamics between study sites, integrated annual leaf production was quite similar at 335 and 353 g DW m⁻² year⁻¹ in the Jindong and Yaquina Bay study sites. We suggest that Z. marina net productivity is acclimated to the local environmental conditions and may be a general characteristic of temperate seagrass populations.     

The Requirement for Low-Intensity Light in the Accumulation of Proline as a Response to Water Deficit

The effect of light on proline accumulation has been examinedin excised segments from the first leaf of barley (cv. Prior)that were subjected to water deficit by exposure to polyethyleneglycol solutions. Illumination either of the intact plant priorto leaf excision or of the leaf segments after excision promotedproline accumulation. This promotion was not due to a changein tissue water potential and the response was saturated ata low irradiance (15–75 µE m ²s ¹ depending uponprevious illumination of the intact plant). Saturation was notdue to a finite capacity of the leaf segments to accumulateproline, but was apparently associated with a finite rate ofaccumulation. The stimulation of proline accumulation by lightwas apparent at all levels of external water potential imposedbut the magnitude of accumulation increased as water potentialwas decreased both in light and darkness. Proline. or its precursor,was readily translocated within the leaf, since leaves thatwere partially shaded whilst subjected to water deficit containedthe same proline concentration throughout. It is concluded thatproline accumulation is not evoked by light through a triggeringmechanism but rather is stimulated by the continuous presenceof light. Key words: Light intensity, Barley, Water deficit, Proline accumulation     

Seasonal and year-to-year variations in the growth of Zostera marina L. (eelgrass) in the lower Chesapeake Bay

Seasonal and year-to-year variations in the growth of Zostera marina L. were measured at three sites in two locations in the lower Chesapeake Bay between 1978 and 1980. The maximum values for the 1979 above- and belowground standing crop ranged from 161–336 g dry wt m⁻² and 61–155 g dry wt m⁻², respectively, leaf length was 19.6–59.7 cm and shoot density 1418–2576 shoot m⁻². Values for 1980 tended to be greater and may be related to climatical differences between the two years. Maximum values were usually recorded in the months of June and July when water temperatures were between 20 and 25°C. Significant loss of leaves occurred in July and August, when water temperatures ranged between 25 and 30°C, while new shoots began to appear more rapidly in late September as water temperatures dropped below 20°C. The greatest increase in all growth parameters occurred from April to June during which time reproductive shoots were present, and accounted for up to 25% of the total number of shoots.     

Effect of salinity on osmotic adjustment,proline accumulation and possible role of ornithine-δ-aminotransferase in proline biosynthesis in <Emphasis Type="Italic">Cakile maritima</Emphasis>

The short time response to salt stress was studied in Cakile maritima. Plants were exposed to different salt concentrations (0, 100, 200 and 400 mM NaCl) and harvested after 4, 24, 72 and 168 h of treatment. Before harvesting plants, tissue hydration, osmotic potential, inorganic and organic solute contents, and ornithine-δ-aminotransferase activity were measured. Plants of C. maritima maintained turgor and tissue hydration at low osmotic potential mainly at 400 mM NaCl. The results showed that, in leaves and stems, Na⁺ content increased significantly after the first 4 h of treatment. However, in roots, the increase of Na⁺ content remained relatively unchanged with increasing salt. The K⁺ content decreased sharply at 200 and 400 mM NaCl with treatment duration. This decrease was more pronounced in roots. The content of proline and amino acids increased with increasing salinity and treatment duration. These results indicated that the accumulation of inorganic and organic compounds was a central adaptive mechanism by which C. maritima maintained intracellular ionic balance under saline conditions. However, their percentage contribution to total osmotic adjustment varies from organ to organ; for example, Na⁺ accumulation mainly contributes in osmotic adjustment of stem tissue (60%). Proline contribution to osmotic adjustment reached 36% in roots. In all organs, proline as well as δ-OAT activity increased with salt concentration and treatment duration. Under normal growth conditions, δ-OAT is mainly involved in the mobilization of nitrogen required for plant growth. However, the highly significant positive correlation between proline and δ-OAT activity under salt-stress conditions suggests that ornithine pathway contributed to proline synthesis.     

The mercury binding activity of pectin isolated from the seagrass Zostera marina

The kinetics of mercury binding by pectin isolated from the seagrass Zostera marina was described, and its maximum mercury binding activity at pH from 2.0 up to 6.0 was determined. It was shown that mercury binding by pectin in in vitro conditions did not depend on concentration of hydrogen ions in the environment. The maximum mercury binding activity estimated from the Langmuir equation was 2.64 mmol/g of dry mass of the pectin.     

Metal binding activity of pectin isolated from seagrass Zostera marina and its derivatives

Low esterified pectin was isolated from the seagrass Zostera marina. Dynamics of the isolation process for the pectin from this seagrass was investigated. Two pectin derivatives: galacturonide (A) with molecular weight 30.55 kDa and galacturonide (B) with molecular weight 3.94 kDa were obtained using acidic hydrolysis of the native pectin from Zostera marina. Molecular weight parameters (Mw, Mn, Mw/Mn) of this pectin and its derivatives as well as of commercial apple pectin were determined using gel-filtration chromatography method. Comparative assessment of Cd²⁺, Pb²⁺, Y³⁺-binding activity of the native Zostera pectin, galacturonides A and B, and commercial apple pectin was performed. The results showed that galacturonide A with molecular weight 30.55 kDa possesses highest metal-binding capacity and may be considered as a candidate for development of medicines with metal-binding activity.     

Low-salinity stress in the American lobster,Homarus americanus,after chronic sublethal exposure to cadmium: Biochemical effects

Lobsters(Homarus americanus) were exposed to cadmium (6μg l⁻¹, 30 days) in flowing seawater, then held for 7 days in aerated “clean ” seawater at either ambient (27 ‰) or low (17 ‰) salinity. Cadmium exposure alone (ambient salinity) induced a general elevation of enzyme activity (heart, antennal gland, and muscle MDH; heart LDH and GPI), despite the probability of some clearance of cadmium from body tissues during the “clean ” seawater holding period. Low-salinity alone (non-exposed lobsters) caused a decrease of enzyme activity (AAT, LDH, GPI, PK) in most tissues examined, except for tail muscle IDH, the activity of which was increased, and MDH, which was significantly elevated above ambient controls in all tissues except heart. Most low-salinity effects were observed in tail muscle, and most cadmium effects, in heart; low-salinity effects outnumbered cadmium stress by nine to four. In heart and tail muscle of cadmium-exposed lobsters held at low salinity, each of the two stresses apparently operated to nullify the other's effects. The most prominent single biochemical response to these sublethal stresses was the elevation of MDH activity. The ratio MDH: LDH gave the clearest indication of overall relative stress.     

Congener comparison of native (Zostera marina) and introduced (Z. japonica) eelgrass at multiple scales within a Pacific Northwest estuary

A congener comparison of native (Zostera marina) and introduced (Zostera japonica) eelgrasses was conducted in Willapa Bay, Washington, USA. Along intertidal transects, Z. japonica (0.1–1.5 m above mean lower low water [MLLW]) occurred above Z. marina (<0.6 m MLLW). Both species declined in shoot length at higher elevation, but Z. japonica was always shorter (20 vs. 100 cm) and occurred at higher shoot density (>3,800 vs. <360 m^?2 in Z. marina). Z. japonica exhibited greater seasonal variation in biomass, with increases supported by both sustained asexual reproduction (rhizome branching) and recruitment from seeds (30 vs. <5% in Z. marina). Z. japonica’s successful invasion appears related to small size and high reproductive output, allowing it to spread quickly in a variable and stressful intertidal environment where competitive effects are low. Based on interannual changes in abundance, the native eelgrass has also recently increased in Willapa Bay, and one hypothesis involves “engineering” of suitable habitat at higher tidal elevations by Z. japonica.