Combined transcriptome and proteome analysis reveal the key physiological processes in seed germination stimulated by decreased salinity in the seagrass Zostera marina L.

BACKGROUND: Zostera marina L., or eelgrass, is the most widespread seagrass species throughout the temperate northern hemisphere. Unlike the dry seeds of terrestrial plants, eelgrass seeds must survive in water, and salinity is the key factor influencing eelgrass seed germination. In the present study, transcriptome and proteome analysis were combined to investigate the mechanisms via which eelgrass seed germination was stimulated by low salinity, in addition to the dynamics of key metabolic pathways under germination. RESULTS: According to the results, low salinity stimulated the activation of Ca2+ signaling and phosphatidylinositol signaling, and further initiated various germination-related physiological processes through the MAPK transduction cascade. Starch, lipids, and storage proteins were mobilized actively to provide the energy and material basis for germination; abscisic acid synthesis and signal transduction were inhibited whereas gibberellin synthesis and signal transduction were activated, weakening seed dormancy and preparing for germination; cell wall weakening and remodeling processes were activated to provide protection for cotyledon protrusion; in addition, multiple antioxidant systems were activated to alleviate oxidative stress generated during the germination process; ERF transcription factor has the highest number in both stages suggested an active role in eelgrass seed germination. CONCLUSION: In summary, for the first time, the present study investigated the mechanisms by which eelgrass seed germination was stimulated by low salinity and analyzed the transcriptomic and proteomic features during eelgrass seed germination comprehensively. The results of the present study enhanced our understanding of seagrass seed germination, especially the molecular ecology of seagrass seeds.

Comparison of metals in eelgrass (Zostera marina L.) and the environment across the North Pacific Ocean: Environmental processes drive source delivery.

Seagrass beds play a critical role in biodiversity maintenance, serving as nursery habitats for fisheries, and aiding in carbon and sediment sequestration in the ecosystem. These habitats receive dissolved and particulate material inputs, like nutrients and heavy metals, affecting both plant health and the ecosystem. Eelgrass (Zostera marina L.), sediments, and water were randomly collected at twenty sites along the temperate North Pacific coasts of Asia and North America to assess heavy metals concentrations (Cr, Cu, Zn, Cd, and Pb). This aimed to understand heavy metal distribution and accumulation patterns in eelgrass tissues, revealing crucial factors influencing metal accumulation. The sampling included various areas, from pristine marine reserves to human-influenced zones, covering industrial, agricultural, and aquaculture regions, enabling a thorough analysis. This study's uniqueness lies in comparing heavy metal distributions in eelgrass tissues with sediments, uncovering unique accumulation patterns. Aboveground eelgrass tissues mainly accumulated Cd, Zn, and Cu, while belowground tissues stored Cr and Pb. Aboveground eelgrass tissues proved reliable in indicating Cd and Pb concentrations in sediments. However, the correlation between Cu, Zn, and Cr in eelgrass tissues and environmental concentrations seemed less direct, requiring further investigation into factors affecting metal accumulation in seagrass. Human activities are probable major contributors to heavy metal presence in Asian marine environments, whereas oceanographic processes serve as primary metal sources in North American Pacific estuaries. Critical discoveries emphasize the necessity for ongoing research on phytotoxic thresholds and in-depth studies on the complex connections between seagrass physiology and environmental metal concentrations. Understanding these dynamics is crucial for evaluating the broader impact of heavy metal pollution on coastal ecosystems and developing effective conservation measures.

Population genetic patterns across the native and invasive range of a widely distributed seagrass: Phylogeographic structure, invasive history and conservation implications.

Aim: The seagrass Zostera japonica is a dramatically declined endemic species in the Northwestern Pacific from the (sub)tropical to temperate areas, however, it is also an introduced species along the Pacific coast of North America from British Columbia to northern California. Understanding the population's genetic patterns can inform the conservation and management of this species. Location: North Pacific. Methods: We used sequences of the nuclear rDNA internal transcribed spacer (ITS) and chloroplast trnK intron maturase (matK), and 24 microsatellite loci to survey 34 native and nonnative populations (>1000 individuals) of Z. japonica throughout the entire biogeographic range. We analysed the phylogeographic relationship, population genetic structure and genetic diversity of all populations and inferred possible origins and invasion pathways of the nonnative ones. Results: All markers revealed a surprising and significant deep divergence between northern and southern populations of Z. japonica in the native region separated by a well-established biogeographical boundary. A secondary contact zone was found along the coasts of South Korea and Japan. Nonnative populations were found to originate from the central Pacific coast of Japan with multiple introductions from at least two different source populations, and secondary spread was likely aided by waterfowl. Main Conclusions: The divergence of the two distinct clades was likely due to the combined effects of historical isolation, adaptation to distinct environments and a contemporary physical barrier created by the Yangtze River, and the warm northward Kuroshio Current led to secondary contact after glacial separation. Existing exchanges among the nonnative populations indicate the potential for persistence and further expansion. This study not only helps to understand the underlying evolutionary potential of a widespread seagrass species following global climate change but also provides valuable insights for conservation and restoration.

The potential for large-scale kelp aquaculture to counteract marine eutrophication by nutrient removal.

Large-scale kelp aquaculture offers several opportunities to counteract eutrophication. Here, six sites from Ailian Bay to its offshore waters in Rongcheng, northern China, were selected to investigate the spatiotemporal variations of kelp (Saccharina japonica) and seawater concentrations of C, N, and P in 2017 and 2019 in order to assess nutrient removal by kelp farming. Variations in biological parameters and elemental nutrient concentrations of whole thalli were also quantified in 2015 and 2016. We found that kelp farming in Ailian Bay can remove 1309 tons of C, 104 tons of N, and 12 tons of P. The increase of total suspended matter and nutrient concentrations from the coast to offshore waters supports the hypothesis of nutrient removal by farmed kelp. This study provides evidence of the bioremediation potential of farmed kelp.

Warming northward shifting southern limits of the iconic temperate seagrass (Zostera marina).

Global warming can shift the range edges of numerous species poleward. Here, eelgrass distribution was reinvestigated at its southern limits on the eastern coast of China, which indicated that there has been a northward shift in the southern limit of Z. marina. To determine if regional warming resulted in a northward shift in suitable eelgrass habitats, sixteen transplantations of adult eelgrass shoots and seeds at the historical southern distribution limit of eelgrass were conducted between 2016 and 2021. The results showed that high water temperatures in summer had negative effects on eelgrass growth, and directly triggered shoot mortality during 2016-2018. Under heat stress, antioxidant enzyme activity was initially increased, but then decreased under more stressful heat conditions; and the HSP70 protein and its molecular chaperone protein were highly expressed under heat stress. These results demonstrated that suitable eelgrass habitat was now located further north along the eastern coast of China.

Multi-leveled insights into the response of the eelgrass Zostera marina L to Cu than Cd exposure.

Seagrass beds are recognized as critical and among the most vulnerable habitats on the planet; seagrass colonize the coastal waters where heavy metal pollution is a serious problem. In this study, the toxic effects of copper and cadmium in the eelgrass Zostera marina L. were observed at the individual, subcellular, physiologically biochemical, and molecular levels. Both Cu and Cd stress significantly inhibited the growth and the maximal quantum yield of photosystem II (Fv/Fm); and high temperature increased the degree of heavy metal damage, while low temperatures inhibited damage. The half-effect concentration (EC50) of eelgrass was 28.9 µM for Cu and 2246.8 µM for Cd, indicating Cu was much more toxic to eelgrass than Cd. The effect of Cu and Cd on photosynthesis was synergistic. After 14 days of enrichment, the concentration of Cu in leaves and roots of Z. marina was 48 and 37 times higher than that in leaf sheath, and 14 and 11 times higher than that in rhizome; and the order of Cd concentration in the organs was root > leaf > rhizome > sheath. Heavy metal uptake mainly occurred in the organelles, and Cd enrichment also occurred to a certain extent in the cytoplasm. Transcriptome results showed that a number of photosynthesis-related KEGG enrichment pathways and GO terms were significantly down-regulated under Cd stress, suggesting that the photosynthetic system of eelgrass was severely damaged at the transcriptome level, which was consistent with the significant inhibition of Fv/Fm and leaf yellowing. Under Cu stress, the genes related to glutathione metabolic pathway were significantly up-regulated, together with the increased autioxidant enzyme activity of GSH-PX. In addition, the results of recovery experiment indicated that the damage caused by short-term Cd and Cu stress under EC50 was reversible. These results provide heavy metal toxic effects at multiple levels and information relating to the heavy metal resistance strategies evolved by Z. marina to absorb and isolate heavy metals, and highlight the phytoremediation potential of this species especially for Cd.

Do adult eelgrass shoots rule seedling fate in a large seagrass meadow in a eutrophic bay in northern China?

We conducted field sampling over 19 months to investigate eelgrass population reproduction status and ecological interactions in a large seagrass meadow in a eutrophic bay in northern China. The results showed asexual growth played an important role in the maintenance of existing meadows, and sexual reproduction played a critical role in the colonization of new areas. We conclude that adult eelgrass shoots do rule the fate of seedlings in the large seagrass meadow. Additionally, nutrient resources (N and P) at this location were found to meet eelgrass growth demand. The N/P ratios of seawater and seagrass indicated N limitation relative to P in the eutrophic bay based on the seagrass Redfield ratio (25-30). Nutrient uptake by seagrass might be an important factor in reducing the probability of a red tide in the study area. The results of this study provide fundamental information for eelgrass restoration and conservation.

Reciprocal Field Transplant Experiment and Comparative Transcriptome Analysis Provide Insights Into Differences in Seed Germination Time of Two Populations From Different Geographic Regions of Zostera marina L.

Seagrasses are the only submerged marine higher plants, which can colonize the sea through sexual (via seeds) reproduction. The transition between seed dormancy and germination is an important ecological trait and a key stage in the life cycle of higher plants. According to our observations, the seeds of Zostera marina L. (eelgrass) in Swan Lake (SL) and Qingdao Bay (QB) in northern China have the same maturation time (summer) but different germination time. To investigate this phenomenon, we further carried out reciprocal transplantation experiment and transcriptome analysis. Results revealed that differences in the seed germination time between the two sites do exist and are determined by internal molecular mechanisms as opposed to environmental factors. Furthermore, we conducted comparative transcriptome analysis of seeds at the mature and early germination stages in both locations. The results that the number of genes related to energy, hormone and cell changes was higher in SL than in QB, could account for that the dormancy depth of seeds in SL was deeper than that in QB; consequently, the seeds in SL needed to mobilize more related genes to break dormancy and start germination. The results could have important practical implications for seagrass meadow restoration via seeds and provide in-depth and comprehensive data for understanding the molecular mechanisms related to seagrass seed germination.

Programmed responses of different life-stages of the seagrass Ruppia sinensis to copper and cadmium exposure.

Seagrass meadows are recognized as crucial and are among the most vulnerable habitats worldwide. The aquatic plant genus Ruppia is tolerant of a wide salinity range, and high concentrations of trace metals. However, the tolerance of its early life stages to such trace metal exposure is unclear. Thus, the current study investigated the trace metal-absorbing capacity of three different life-history stages of Ruppia sinensis, a species that is widely distributed in China, by observing toxic symptoms at the individual, subcellular, and transcription levels. The seedling period was the most vulnerable, with visible toxic effects at the individual level in response to 50 µM copper and 500 µM cadmium after 4 days of exposure. The highest concentrations of trace metals occurred in the vacuoles and cytoplasmic structures of aboveground tissues. Genes related to signal identification and protein processing were significantly downregulated after 4 days of exposure to copper and cadmium. These results provide information relating to the strategies evolved by R. sinensis to absorb and isolate trace elements, and highlight the phytoremediation potential of this species.

Can the Non-native Salt Marsh Halophyte Spartina alterniflora Threaten Native Seagrass (Zostera japonica) Habitats? A Case Study in the Yellow River Delta, China.

Seagrass meadows are critical ecosystems, and they are among the most threatened habitats on the planet. As an anthropogenic biotic invader, Spartina alterniflora Loisel. competes with native plants, threatens native ecosystems and coastal aquaculture, and may cause local biodiversity to decline. The distribution area of the exotic species S. alterniflora in the Yellow River Delta had been expanding to ca.4,000 ha from 1990 to 2018. In this study, we reported, for the first time, the competitive effects of the exotic plant (S. alterniflora) on seagrass (Zostera japonica Asch. & Graebn.) by field investigation and a transplant experiment in the Yellow River Delta. Within the first 3 months of the field experiment, S. alterniflora had pushed forward 14 m into the Z. japonica distribution region. In the study region, the area of S. alterniflora in 2019 increased by 516 times compared with its initial area in 2015. Inhibition of Z. japonica growth increased with the invasion of S. alterniflora. Z. japonica had been degrading significantly under the pressure of S. alterniflora invasion. S. alterniflora propagates sexually via seeds for long distance invasion and asexually by tillers and rhizomes for short distance invasion. Our results describe the invasion pattern of S. alterniflora and can be used to develop strategies for prevention and control of S. alterniflora invasion.

The super typhoon Lekima (2019) resulted in massive losses in large seagrass (Zostera japonica) meadows, soil organic carbon and nitrogen pools in the intertidal Yellow River Delta, China.

Seagrass meadows are key ecosystems, and they are among the most threatened habitats on the planet. Increased numbers of extreme climate events, such as hurricanes and marine heatwaves have caused severe damage to global seagrass meadows. The largest Zostera japonica meadows in China are located in the Yellow River Delta. It had a distribution area of 1031.8 ha prior to August 2019 when the Yellow River Delta was severely impacted by the passage of typhoon Lekima. In this study, we compared field data collected before and after the typhoon to determine its impact on seagrass beds in the Yellow River Delta. The super typhoon caused dramatic changes in Z. japonica in the Yellow River Delta, resulting in a greater than 100-fold decrease in distribution area, a greater than 35% loss of soil organic carbon, and a greater than 65% loss of soil total nitrogen in the top 35 cm sediments. Owing to the lack of seeds and overwintering shoots, as well as the small remaining distribution area, recovery was impossible, even though environmental factors were still suitable for species growth. Thus, restoration efforts are required for seagrass meadow recovery. Additionally, the long-term monitoring of this meadow will provide new information on the ecosystem's status and will be useful for future protection.

Relationships Between Annual and Perennial Seagrass (Ruppia sinensis) Populations and Their Sediment Geochemical Characteristics in the Yellow River Delta.

Annual and perennial populations commonly occur for the same submerged aquatic angiosperm species, yet relationships between population types and sediment characteristics are poorly understood. In the current study two Ruppia sinensis habitats with annual and perennial populations were surveyed in the Yellow River Delta (YRD). Biomass and seasonal seed bank size were used to evaluate population status and potential recruitment capacity. Sediment geochemical parameters including moisture, sulfide, Chl a, carbohydrate, OM, TOC, TN, and TP were measured to compare sediment nutrient composition and variability. The results revealed a higher biomass and larger seed bank in the annual R. sinensis population compared with the perennial population. The P levels in sediments between the two R. sinensis populations were similar; while the N level in the sediment of the annual population was significantly higher than the perennial population, which might support the recruitment of vegetative shoots when a large amount of seeds germinated during wet periods. The annual population exhibited greater resilience after habitat desiccation, with the population recovering rapidly once water appeared. The results of this study add to the knowledge of R. sinensis populations and their sediment geochemical characteristics, and can be used as a reference for Ruppia population conservation and management.

New insights into physiological effects of anoxia under darkness on the iconic seagrass Zostera marina based on a combined analysis of transcriptomics and metabolomics.

Coastal hypoxia/anoxia is a major emerging threat to global coastal ecosystems. Macroalgae blooms of tens of kilometers are often observed in open waters. These blooms not only cause a lack of oxygen, but also benthic light limitation. We explored the physiological responses of Zostera marina L. to anoxia under darkness. After exposing Z. marina to anoxia under darkness for 72 h, we measured the elongation of leaves and the decrease in maximal quantum yield of photosystem II (Fv/Fm), and investigated the transcriptomic and metabolomic responses to anoxic stress based on RNA-sequencing and liquid chromatography-mass spectrometry (LC-MS) technology. The results showed that anoxic stress significantly reduced the leaf Fv/Fm, and had a significant negative effect on the photosynthesis and growth of Z. marina. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of up-regulated differentially expressed genes (DEGs) showed that glycolysis was the most significant enrichment pathway (p < 0.001), and most of the important products in glycolysis were significantly up-regulated. This indicated that the glycolysis process of anaerobic respiration is promoted under anoxia. The metabolite results also showed that glyceraldehyde 3-phosphate in the glycolysis pathway was significantly up-regulated. Moreover, three genes encoding sucrose synthase (gene-ZOSMA_310G00150, gene-ZOSMA_81G00980, and gene-ZOSMA_8G00730) and one gene encoding alpha-amylase (gene-ZOSMA_95G00270) were significantly up-regulated, providing the sugar basis for the subsequent increase in glycolysis. Furthermore, gene-encoding oxoglutarate dehydrogenase, the rate-limiting step of the tricarboxylic acid (TCA) cycle, was significantly down-regulated, indicating that this cycle was inhibited under anoxia. Metabolomic results showed that L-tryptophan, L-phenylalanine, and DL-leucine were significantly up-regulated. Only significantly decreased glutamate and non-significantly decreased glutamine, substances consumed in alanine and γ-aminobutyric acid (GABA) shunt mechanisms, were detected in the leaves, while GABA and alanine were not detected. The results of this study show that anoxic stress induces a programmed transcriptomic and metabolomic response in seagrass, most likely reflecting a complex strategy of acclimation and adaptation in seagrass to resist anoxic stress.

Diversity, distribution and conservation of seagrass in coastal waters of the Liaodong Peninsula, North Yellow Sea, northern China: Implications for seagrass conservation.

Seagrass beds are highly productive coastal ecosystems that are widely distributed along temperate and tropical coastlines globally. Although seagrass distribution and diversity have been widely reported on a global scale, there have been few reports on seagrass distribution and diversity in northern China, especially for coastal waters of the Liaodong Peninsula in the North Yellow Sea. In the present study, we investigated the distribution and diversity of seagrass in coastal waters of the Liaodong Peninsula in the North Yellow Sea, northern China. Field surveys of seagrass wrack were conducted along shorelines, to identify whether seagrass beds occurred in nearby waters, and sonar methods were then used to collect data relating to seagrass bed extent. Also, we analyzed the major threats facing seagrass beds. The results of the study revealed that four species (Zostera marina L., Z. japonica Aschers. & Graebn., Z. caespitosa M., and Phyllospadix iwatensis M.) were found in study area, covering a total area of 1253.47 ha. Seagrass bed area significantly decreased with increasing water depth, and most seagrass was recorded at depths of 2-5 m. Due to the steep slope of the seabed, seagrass beds exhibited a zonal distribution in most of the study areas. In addition, the amount of seagrass wrack along shorelines could be used to infer the size and distance of seagrass beds. Human activities, such as clam harvesting, land reclamation, coastal aquaculture pose a threat to the seagrass beds. This study provides new information to fill knowledge gaps regarding seagrass distribution in northern China and it provides a baseline for further monitoring of these seagrass beds.

Sonar and in situ surveys of eelgrass distribution, reproductive effort, and sexual recruitment contribution in a eutrophic bay with intensive human activities: Implication for seagrass conservation.

Seagrass beds are recognized as pivotal and among the most vulnerable coastal marine ecosystems globally. The eelgrass Zostera marina L. is the most widely distributed seagrass species and dominates the temperate northern hemisphere. However, an alarming decline in seagrass has been occurring worldwide due to multiple stressors. Seagrass meadow degradation is particularly serious in the Bohai Sea, in temperate China; however, large areas (> 500 ha) of seagrass meadows and population recruitment have rarely been reported in this area. In the present study, we report on a large eelgrass bed in a eutrophic bay of the Bohai Sea. Sonar and field survey methods were used to investigate the distribution of seagrass and its population recruitment. We also analyzed the major threats to this large seagrass bed. Results showed that a large Z. marina bed with an area of 694.36 ha occurred in this area of the Bohai Sea, with a peripheral area of ~25 km2. Seagrass canopy height and plant coverage had a significant correlation with water depth. Asexual reproduction principally occurred in autumn and played a dominant role in population recruitment in vegetated areas, where no seedlings successfully colonized. In contrast, a considerable number of seedlings survived in the seagrass meadow gaps, and thus played a critical role in the recruitment in these areas. The maximum reproductive shoot densities were about 100 and 70 shoots m-2 at sampling site (S)-1 and S-2 in 2018, respectively, which was about two times more than in 2019 (50 and 20 reproductive shoots m-2 at S-1 and S-2, respectively). The potential seed output per unit area in 2019 was about 1020 seeds m-2 at S-1 and 830 seeds m-2 at S-2, and the seed output in the study area was at a low level compared with global values. Overall, high spring and summer water temperature appeared to induce sexual reproduction of Z. marina in the study area, including reproductive effort, reproductive investment, and seedling development. Furthermore, eelgrass height, aboveground biomass, and density were significantly related to water temperature. Among the potential threatening factors to seagrass in this area, the activities of clam harvesting were intense with daily clam catches >2000 kg, leading to patchy seagrass meadows, especially in the fringe areas. The seagrass bed was also threatened by marine pollution (nutrient loading) and land reclamation. Therefore, the protection and restoration of this seagrass bed are strongly recommended. Our study will provide fundamental information for the conservation and management strategies of large eelgrass beds in the Bohai Sea.

In situ Responses of the Eelgrass Zostera marina L. to Water Depth and Light Availability in the Context of Increasing Coastal Water Turbidity: Implications for Conservation and Restoration.

Accelerating losses of seagrass meadows has led to efforts to restore these highly productive and beneficial ecosystems globally. Depth and light availability are critical determinants of seagrass restoration success. Eelgrass (Zostera marina L.) is the dominant seagrass species in the temperate northern hemisphere, but its global distribution has reduced dramatically. The main aims of this study were to determine: (1) the depth limit for Z. marina survival in Ailian Bay, north China, and (2) how light availability affects the growth and recruitment of Z. marina as a basis for identifying a suitable depth range for successful restoration. To achieve these aims, Z. marina shoots were transplanted from a nearby donor site, Swan Lake, to an experimental site, Ailian Bay, and the temporal responses of Z. marina shoots to light availability at water depths ranging from 1 to 8 m were investigated using in situ suspended cultures. Four suspended shoot transplantation experiments were conducted in 4 years. The results showed that the transplanted Z. marina shoots could survive and branch during an annual growth cycle, permanently underwater, at a depth ≤3 m. Due to the local turbidity of the waters in Ailian Bay, a depth of 4 m led to sufficient light deprivation (reduced to 6.48-10.08% of surface irradiance) to negatively affect seagrass shoot density and clonal reproduction. In addition, reproductive shoot density also tended to decline with water depth and light deprivation. Our results indicated that Z. marina population recruitment, through sexual and asexual (clonal growth) reproduction, were negatively affected by increasing water depth and light deprivation. These findings may provide a suitable depth range for the successful restoration of Z. marina in local coastal waters. They may also be applied to the management and restoration of Z. marina globally.

Plant morphology and seed germination responses of seagrass (Zostera japonica) to water depth and light availability in Ailian Bay, northern China.

Poor water quality and light reduction owing to anthropogenic impacts are the most widespread causes behind marine submerged angiosperm (seagrass) declines, worldwide. Seagrasses could respond to sustained environmental stresses, such as increasing water depth and light reduction, through morphological changes, particularly shoot density and/or biomass reductions. The seagrass Zostera japonica Asch. and Graebn. has been introduced to the Pacific Coast of North America, but it is widely threatened in its native northwestern Pacific Coast range alongside the east coast of China. The main aims of this study were to determine: 1) the depth limit of Z. japonica growth in its native range, and 2) how light availability affects the growth and recruitment of Z. japonica. To achieve these aims, we investigated the temporal responses of Z. japonica shoots and seeds from an intertidal donor site, Swan Lake, to light availability at water depths ranging from 1 to 6 m using in situ suspended cultures deployed in the experimental site, Ailian Bay, off the coast of Weihai City, China. The results showed that the transplanted Z. japonica shoots and seeds could survive for the duration of their annual growth cycle, permanently underwater, at a depth ≤2 m. There was a significant inverse relationship between water depth and time to complete shoot loss, despite temporally varying water clarity levels. Due to the local turbidity of the waters in Ailian Bay, a depth of 2 m yielded sufficient light deprivation (5%-37% surface irradiance) to negatively affect the seagrass shoot density. Our results suggest that this intertidal species can potentially persist in shallow subtidal areas following transplantation with shoots and seeds. The findings may also serve as useful information for local seagrass distribution limits, and will facilitate their habitat establishment and restoration efforts.

Sheaths of Zostera marina L. as ecological indicators of shoot length and the elemental stoichiometry of aboveground tissues.

Given a large quantity of epiphytes and other material attached on eelgrass leaf blades, we explored the relationship between eelgrass sheaths and different-aged leaf blades (1st, 2nd, 3rd, and 4th leaf blade) on nutrient content and their ratios (C, N, P, C/N, C/P, and N/P) to identify whether eelgrass sheaths could be used to instead of leaf blades in terms of nutrient content. In addition, we explored the relationship between eelgrass sheath length and shoot length. Results showed that there were significant relationships between the sheath and leaf blades in terms of N and P content and their ratios. For length analysis, there was a significant relationship between sheath length and shoot length, and shoot length was approximately four to five times (mean 4.4659) longer than sheath length, such that shoot length can be estimated by sheath length. These significant relationships suggest that eelgrass sheath could be used as a suitable predictor of leaf blade in length and nutrient stoichiometry, thus eelgrass sheath could be used as an indicator for further eelgrass nutrient monitoring and research.

Seed selection and storage with nano-silver and copper as potential antibacterial agents for the seagrass Zostera marina: implications for habitat restoration.

Globally, seagrass meadows are extremely important marine ecosystems that are disappearing at an alarming rate. Therefore, research into seagrass restoration has become increasingly important. Various strategies have been used in Zostera marina L. (eelgrass) restoration, including planting seeds. To improve the efficiency of restoration by planting seeds, it is necessary to select high-quality seeds. In addition, a suitable antibacterial agent is necessary for wet storage of desiccation sensitive seeds to reduce or inhibit microorganism infection and seed decay. In the present study, an efficient method for selecting for high-quality eelgrass seeds using different specific gravities of salt water was developed, and potential antibacterial agents (nano-silver and copper sulfate) for seed storage were assessed. The results showed that the highest proportion of intact seeds (72.91 ± 0.50%) was recorded at specific gravities greater than 1.20. Therefore, specific gravities greater than 1.20 can be used for selecting high-quality eelgrass seeds. During seed storage at 0 °C, the proportion of intact seeds after storage with nano-silver agent was over 90%, and also higher than 80% with copper sulfate agent, which was significantly higher than control treatments. The findings revealed a potential selection method for high-quality seeds and long-term seed storage conditions for Z. marina, which could facilitate conservation and habitat restoration.

Optimal long-term seed storage conditions for the endangered seagrass Zostera japonica: implications for habitat conservation and restoration.

BACKGROUND: Seagrass meadows are recognized as critical and among the most vulnerable habitats on the planet. The alarming rates of decline in seagrass meadows have attracted the attention globally. There is an urgent need to develop techniques to restore and preserve these vital coastal ecosystems. So far little work has been done to develop effective long-term storage method for seagrass seeds. The seagrass Zostera japonica Asch. & Graebn is an endangered species in its native range. Here we utilized combinations of different storage times, salinities, and temperature to determine the most appropriate conditions for optimal seed storage. RESULTS: Zostera japonica seeds were strongly desiccation sensitive, with a complete loss of viability after 24 h of desiccation. Therefore, long periods of exposure to air should be avoided to minimize seed mortality. In addition, Z. japonica seeds could not endure freezing conditions such as - 5 °C. However, our results indicated that reduced storage temperature to 0 °C could effectively prolong the duration of dormancy of Z. japonica seeds. Seeds stored at 0 °C under a salinity of 40-60 psu showed relatively low seed loss, high seed vigor and fast seed germination, suggesting these to be optimal seed storage conditions. For example, after storage for 540 days (ca. 600 days since the seed collection from reproductive shoots in early October, 2016) at 0 °C under a salinity of 50 psu, seeds still had a considerable vigor, i.e. 57.8 ± 16.8%. CONCLUSION: Our experiments demonstrated that seeds stored at 0 °C under a salinity of 40-60 psu could effectively prolong the duration of dormancy of Z. japonica seeds. The proposed technique is a simple and effective long-term storage method for Z. japonica seeds, which can then be used to aid future conservation, restoration and management of these sensitive and ecologically important habitat formers. The findings may also serve as useful reference for seed storage of other threatened seagrass species and facilitate their ex situ conservation and habitat restoration.