Integrating ecology, physiology and transcriptomics reveals the response of Zostera marina to rusting of iron transplantation frame.

Frame Transplantation System (FTS) is considered an efficient method for seagrass restoration, but the effect of the rusting of iron frame on seagrass restoration remains unclear. We transplanted Zostera marina plants using iron FTS treated with fluorocarbon paint (painted treatment, PT) and traditional unpainted iron FTS (unpainted treatment, UT) under controlled mesocosm conditions for 24 days. Our results showed that the survival rate of Z. marina under the UT was significantly 31.2 % lower than that of the plants under the PT. Soluble sugar content in Z. marina rhizomes under the UT was significantly 2.19 times higher than that of the plants under the PT. Transcriptome analysis revealed differentially expressed genes (DEGs) involved in photosynthesis, metabolism and signal transduction functions. The results provide valuable data that could prove helpful in the development of efficient restoration techniques for Z. marina beds.

Detrimental impact of sulfide on the seagrass Zostera marina in dark hypoxia.

Sulfide poisoning, hypoxia events, and reduced light availability pose threats to marine ecosystems such as seagrass meadows. These threats are projected to intensify globally, largely due to accelerating eutrophication of estuaries and coastal environments. Despite the urgency, our current comprehension of the metabolic pathways that underlie the deleterious effects of sulfide toxicity and hypoxia on seagrasses remains inadequate. To address this knowledge gap, I conducted metabolomic analyses to investigate the impact of sulfide poisoning under dark-hypoxia in vitro conditions on Zostera marina, a vital habitat-forming marine plant. During the initial 45 minutes of dark-hypoxia exposure, I detected an acclimation phase characterized by the activation of anaerobic metabolic pathways and specific biochemical routes that mitigated hypoxia and sulfide toxicity. These pathways served to offset energy imbalances, cytosolic acidosis, and sulfide toxicity. Notably, one such route facilitated the transformation of toxic sulfide into non-toxic organic sulfur compounds, including cysteine and glutathione. However, this sulfide tolerance mechanism exhibited exhaustion post the initial 45-minute acclimation phase. Consequently, after 60 minutes of continuous sulfide exposure, the sulfide toxicity began to inhibit the hypoxia-mitigating pathways, culminating in leaf senescence and tissue degradation. Utilizing metabolomic approaches, I elucidated the intricate metabolic responses of seagrasses to sulfide toxicity under in vitro dark-hypoxic conditions. My findings suggest that future increases in coastal eutrophication will compromise the resilience of seagrass ecosystems to hypoxia, primarily due to the exacerbating influence of sulfide.

Molecular Characterization of ZosmaNRT2, the Putative Sodium Dependent High-Affinity Nitrate Transporter of Zostera marina L.

One of the most important adaptations of seagrasses during sea colonization was the capacity to grow at the low micromolar nitrate concentrations present in the sea. In contrast to terrestrial plants that use H+ symporters for high-affinity NO3- uptake, seagrasses such as Zostera marina L. use a Na+-dependent high-affinity nitrate transporter. Interestingly, in the Z. marina genome, only one gene (Zosma70g00300.1; NRT2.1) is annotated to this function. Analysis of this sequence predicts the presence of 12 transmembrane domains, including the MFS domains of the NNP transporter family and the "nitrate signature" that appears in all members of the NNP family. Phylogenetic analysis shows that this sequence is more related to NRT2.5 than to NRT2.1, sharing a common ancestor with both monocot and dicot plants. Heterologous expression of ZosmaNRT2-GFP together with the high-affinity nitrate transporter accessory protein ZosmaNAR2 (Zosma63g00220.1) in Nicotiana benthamiana leaves displayed four-fold higher fluorescence intensity than single expression of ZosmaNRT2-GFP suggesting the stabilization of NRT2 by NAR2. ZosmaNRT2-GFP signal was present on the Hechtian-strands in the plasmolyzed cells, pointing that ZosmaNRT2 is localized on the plasma membrane and that would be stabilized by ZosmaNAR2. Taken together, these results suggest that Zosma70g00300.1 would encode a high-affinity nitrate transporter located at the plasma membrane, equivalent to NRT2.5 transporters. These molecular data, together with our previous electrophysiological results support that ZosmaNRT2 would have evolved to use Na+ as a driving ion, which might be an essential adaptation of seagrasses to colonize marine environments.

Cadmium and/or copper excess induce interdependent metal accumulation, DNA methylation, induction of metal chelators and antioxidant defences in the seagrass Zostera marina.

In this investigation, we assessed the effects of Cu and/or Cd excess on physiological and metabolic processes of the widespread seagrass Zostera marina. Adult were exposed to low Cd and Cu (0.89 and 0.8 µM, respectively) and high Cd and Cu (8.9 and 2.4 µM, respectively) for 6 d at: Control conditions; low Cu; high Cu; low Cd; high Cd; low Cd and low Cu; and high Cd and high Cu. Photosynthetic performance decreased under single and combined treatments, although effects were more negative under Cu than Cd. Total Cu accumulation was higher than Cd, under single and combined treatments; however, their accumulation was generally lower when applied together, suggesting competition among them. Levels of glutathione (GSH) and phytochelatins (PCs) followed patterns similar to metal accumulation, with up to PC5, displaying adaptations in tolerance. A metallothionein (MET) gene showed upregulation only at high Cd, low Cu, and high Cu. The expression of the enzymes glutathione reductase (GR), ascorbate peroxidase (APX), and catalase (CAT) was greatest at high Cu, and at high Cd and Cu together; the highest expression was under Cu, alone and combined. Both metals induced upregulation of the DNA methyltransferases CMT3 and DRM2, with the highest expression at single Cu. The DNA demethylation ROS1 was overexpressed in treatments containing high Cu, suggesting epigenetic modifications. The results show that under copper and/or cadmium, Z. marina was still biologically viable; certainly based, at least in part, on the induction of metal chelators, antioxidant defences and methylation/demethylation pathways of gene regulation.

Plant Life without Ethylene.

We propose that the ability to synthesize ethylene was selectively lost in evolution when the ancestors of fully aquatic higher plants lost their terrestrial lifestyle. We suggest that there has been negative selection on ethylene in these submerged species because it might interfere with growth in permanently deluged environments.

Genome-wide survey of the seagrass Zostera muelleri suggests modification of the ethylene signalling network.

Seagrasses are flowering plants which grow fully submerged in the marine environment. They have evolved a range of adaptations to environmental challenges including light attenuation through water, the physical stress of wave action and tidal currents, high concentrations of salt, oxygen deficiency in marine sediment, and water-borne pollination. Although, seagrasses are a key stone species of the costal ecosystems, many questions regarding seagrass biology and evolution remain unanswered. Genome sequence data for the widespread Australian seagrass species Zostera muelleri were generated and the unassembled data were compared with the annotated genes of five sequenced plant species (Arabidopsis thaliana, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Genes which are conserved between Z. muelleri and the five plant species were identified, together with genes that have been lost in Z. muelleri. The effect of gene loss on biological processes was assessed on the gene ontology classification level. Gene loss in Z. muelleri appears to influence some core biological processes such as ethylene biosynthesis. This study provides a foundation for further studies of seagrass evolution as well as the hormonal regulation of plant growth and development.

De novo assembly and characterization of the transcriptome of seagrass Zostera marina using Illumina paired-end sequencing.

BACKGROUND: The seagrass Zostera marina is a monocotyledonous angiosperm belonging to a polyphyletic group of plants that can live submerged in marine habitats. Zostera marina L. is one of the most common seagrasses and is considered a cornerstone of marine plant molecular ecology research and comparative studies. However, the mechanisms underlying its adaptation to the marine environment still remain poorly understood due to limited transcriptomic and genomic data. PRINCIPAL FINDINGS: Here we explored the transcriptome of Z. marina leaves under different environmental conditions using Illumina paired-end sequencing. Approximately 55 million sequencing reads were obtained, representing 58,457 transcripts that correspond to 24,216 unigenes. A total of 14,389 (59.41%) unigenes were annotated by blast searches against the NCBI non-redundant protein database. 45.18% and 46.91% of the unigenes had significant similarity with proteins in the Swiss-Prot database and Pfam database, respectively. Among these, 13,897 unigenes were assigned to 57 Gene Ontology (GO) terms and 4,745 unigenes were identified and mapped to 233 pathways via functional annotation against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). We compared the orthologous gene family of the Z. marina transcriptome to Oryza sativa and Pyropia yezoensis and 11,667 orthologous gene families are specific to Z. marina. Furthermore, we identified the photoreceptors sensing red/far-red light and blue light. Also, we identified a large number of genes that are involved in ion transporters and channels including Na+ efflux, K+ uptake, Cl- channels, and H+ pumping. CONCLUSIONS: Our study contains an extensive sequencing and gene-annotation analysis of Z. marina. This information represents a genetic resource for the discovery of genes related to light sensing and salt tolerance in this species. Our transcriptome can be further utilized in future studies on molecular adaptation to abiotic stress in Z. marina.

Photosynthetic utilization of bicarbonate in Zostera marina is reduced by inhibitors of mitochondrial ATPase and electron transport.

When Zostera marina was irradiated after a period of darkness, initiation of photosynthetic O2 evolution occurred in two phases. During a lag phase, lasting 4 to 5 min, photosynthesis was supported by a diffusive entry of CO2. Photosynthesis then rapidly increased to its full rate. Tris buffer, at a concentration of 50 mm, completely inhibited this increase without affecting CO2-supported photosynthesis during the lag phase. These results verify that the increase in photosynthesis after the lag phase depended on an activation of bicarbonate (HCO3-) utilization through acid zones generated by proton pumps located to the outer cell membrane. In similar experiments, 6.25 microm of the mitochondrial ATPase blocker oligomycin inhibited photosynthetic HCO3(-) utilization by more than 60%. Antimycin A, a selective blocker of mitochondrial electron transport, caused a similar inhibition of HCO3(-) utilization. Measurements at elevated CO2 concentrations verified that neither oligomycin nor antimycin interfered with linear photosynthetic electron transport or with CO2 fixation. Thus, a major part of the ATP used for the generation of acid zones involved in HCO3(-) utilization in Z. marina was derived from mitochondrial respiration.