RESUMEN
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⯵M, respectively) and high Cd and Cu (8.9 and 2.4⯵M, respectively) for 6â¯dâ¯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.
Asunto(s)
Antioxidantes/metabolismo , Cadmio/farmacología , Cobre/farmacología , Metilación de ADN/efectos de los fármacos , Metales/metabolismo , Fitoquelatinas/metabolismo , Zosteraceae/efectos de los fármacos , Redes Reguladoras de Genes , Zosteraceae/enzimología , Zosteraceae/metabolismoRESUMEN
Copper (Cu) is an essential micronutrient for plants and as such is vital to many metabolic processes. Nevertheless, when present at elevated concentrations, Cu can exert toxic effects on plants by disrupting protein functions and promoting oxidative stress. Due to their proximity to the urbanised estuaries, seagrasses are vulnerable to chemical contamination via industrial runoff, waste discharges and leachates. Zostera muelleri is a common seagrass species that forms habitats in the intertidal areas along the temperate coast of Australia. Previous studies have shown the detrimental effects of Cu exposure on photosynthetic efficiency of Z. muelleri. The present study focuses on the impacts of sublethal Cu exposure on the physiological and molecular responses. By means of a single addition, plants were exposed to 250 and 500 µg Cu L-1 (corresponding to 3.9 and 7.8 µM, respectively) as well as uncontaminated artificial seawater (control) for 7 days. Chlorophyll fluorescence parameters, measured as the effective quantum yield (ÏPSII), the maximum quantum yield (Fv/Fm) and non-photochemical quenching (NPQ) were assessed daily, while Cu accumulation in leaf tissue, total reactive oxygen species (ROS) and the expression of genes involved in antioxidant activities and trace metal binding were determined after 1, 3 and 7 days of exposure. Z. muelleri accumulated Cu in the leaf tissue in a concentration-dependent manner and the bioaccumulation was saturated by day 3. Cu exposure resulted in an acute suppression of ÏPSII and Fv/Fm. These two parameters also showed a concentration- and time-dependent decline. NPQ increased sharply during the first few days before subsequently decreasing towards the end of the experiment. Cu accumulation induced oxidative stress in Z. muelleri as an elevated level of ROS was detected on day 7. Lower Cu concentration promoted an up-regulation of genes encoding Cu/Zn-superoxide dismutase (Cu/Zn-sod), ascorbate peroxidase (apx), catalase (cat) and glutathione peroxidase (gpx), whereas no significant change was detected with higher Cu concentration. Exposure to Cu at any concentration failed to induce regulation in the expression level of genes encoding metallothionein type 2 (mt2), metallothionein type 3 (mt3) and cytochrome c oxidase copper chaperone (cox17). It is concluded that chlorophyll fluorescence parameters provide timely probe of the status of photosynthetic machinery under Cu stress. In addition, when exposed to a moderate level of Cu, Z. muelleri mitigates any induced oxidative stress by up-regulating transcripts coding for antioxidant enzymes.
Asunto(s)
Antioxidantes/metabolismo , Cobre/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Genes de Plantas , Luz , Zosteraceae/genética , Ascorbato Peroxidasas/metabolismo , Australia , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Fotosíntesis/efectos de los fármacos , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Contaminantes Químicos del Agua/toxicidad , Zosteraceae/efectos de los fármacos , Zosteraceae/enzimologíaRESUMEN
Worldwide seagrass declines have been observed due to multiple stressors. One of them is the mixture of pesticides used in intensive agriculture and boat antifouling paints in coastal areas. Effects of mixture toxicity are complex and poorly understood. However, consideration of mixture toxicity is more realistic and ecologically relevant for environmental risk assessment (ERA). The first aim of this study was to determine short-term effects of realistic herbicide mixture exposure on physiological endpoints of Zostera noltei. The second aim was to assess the environmental risks of this mixture, by comparing the results to previously published data. Z. noltei was exposed to a mixture of four herbicides: atrazine, diuron, irgarol and S-metolachlor, simulating the composition of typical cocktail of contaminants in the Arcachon bay (Atlantic coast, France). Three stress biomarkers were measured: enzymatic activity of glutathione reductase, effective quantum yield (EQY) and photosynthetic pigment composition after 6, 24 and 96 h. Short term exposure to realistic herbicide mixtures affected EQY, with almost 100% inhibition for the two highest concentrations, and photosynthetic pigments. Effect on pigment composition was detected after 6 h with a no observed effect concentration (NOEC) of 1 µg/L total mixture concentration. The lowest EQY effect concentration at 10% (EC10) (2 µg/L) and pigment composition NOEC with an assessment factor of 10 were above the maximal field concentrations along the French Atlantic coast, suggesting that there are no potential short term adverse effects of this particular mixture on Z. noltei. However, chronic effects on photosynthesis may lead to reduced energy reserves, which could thus lead to effects at whole plant and population level. Understanding the consequences of chemical mixtures could help to improve ERA and enhance management strategies to prevent further declines of seagrass meadows worldwide.
Asunto(s)
Herbicidas/toxicidad , Fotosíntesis/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Zosteraceae/efectos de los fármacos , Bahías , Francia , Glutatión Reductasa/metabolismo , Insecticidas/toxicidad , Zosteraceae/enzimología , Zosteraceae/metabolismoRESUMEN
Superoxide dismutase (SOD) is an essential enzyme playing a pivotal role in the protection mechanism against oxidative stress by reducing superoxide radicals. In the present study, the full-length cDNA sequence of manganese superoxide dismutase was identified from Zostera marina (ZmMnSOD) via raid amplification of cDNA ends (RACE) technique and expressed sequence tags (ESTs) analysis. The open reading frame (ORF) encoded a polypeptide of 254 amino acid residues, which shared 69%-77% similarity with previous identified SODs. Analysis of the deduced amino acid revealed conserved features, including functional domains, signature motifs and metal binding sites. Phylogenetic analysis revealed that ZmMnSOD was closer to the SODs from angiosperm than those from other organisms. The mRNA expression level of ZmMnSOD at different temperatures was investigated using real-time PCR and it was significantly up-regulated from 5°C to 15°C, and then dramatically down-regulated. The recombinant ZmMnSOD protein was purified and exhibited Mn(2+) ions dependency specific enzymatic activity and strong antioxidant activity over a wide temperature range. All these results indicate that ZmMnSOD is an authentic member of the plant SOD family and may play important roles in minimizing the effect of oxidative damage in Z. marina against temperature stress and affect the adaptability of Z. marina to global warming.
Asunto(s)
Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Zosteraceae/enzimología , Sitios de Unión , Clonación Molecular , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Estrés Oxidativo , Filogeografía , Proteínas de Plantas/química , Estrés Fisiológico , Superóxido Dismutasa/química , TemperaturaRESUMEN
The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T1 to T4 generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice.
Asunto(s)
Glucosiltransferasas/genética , Glucosiltransferasas/fisiología , Oryza/genética , Transformación Genética , Zosteraceae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Conductividad Eléctrica , Glucosiltransferasas/metabolismo , Datos de Secuencia Molecular , Oryza/metabolismo , Filogenia , Plantas Modificadas Genéticamente , Tolerancia a la Sal/genética , Transformación Genética/fisiología , Trehalosa/metabolismo , Zosteraceae/enzimologíaRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Bicarbonatos/metabolismo , Inhibidores Enzimáticos/farmacología , Mitocondrias/efectos de los fármacos , Fotosíntesis , Zosteraceae/metabolismo , Adenosina Trifosfatasas/metabolismo , Transporte de Electrón , Mitocondrias/enzimología , Zosteraceae/enzimologíaRESUMEN
A vacuolar H(+)-ATPase (VHA) gene (ZMVHA-B1) was isolated from an eelgrass (Zostera marina) leaf cDNA library and was characterized to be approximately 1.4 kbp in length and to encode the B subunit protein of VHA comprising 488 amino acids. ZMVHA-B1 was highly expressed in all organs of eelgrass; the expression level was highest in the leaves. On transformation of a yeast vma2 null mutant with ZMVHA-B1, yeast cells became able to grow at pH 7.5, accompanied by the vesicular accumulation of LysoSensor green DND-189. Thus, ZMVHA-B1 expressed in yeast cells produced a functional B subunit that was efficiently incorporated into the VHA complex and eventually restored vacuolar morphology and activity. This success expedites the application of heterologous expression in yeast mutant cells to the screening of eelgrass genes involved in salt-resistance mechanisms, which are to be utilized in improving important crops.
Asunto(s)
Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares/genética , Zosteraceae/enzimología , Clonación Molecular , Ecología , Prueba de Complementación Genética , Isoenzimas/genética , Mutación , Subunidades de Proteína , Cloruro de Sodio/farmacología , ATPasas de Translocación de Protón Vacuolares/fisiologíaRESUMEN
Phylogenetic analysis of the plastid (chloroplast) DNA matK gene of Zosteraceae species was undertaken. A molecular phylogenetic tree based on matK sequence data showed the monophyly of Heterozostera tasmanica and subgenus Zosterella and did not support the separation of Heterozostera from the genus Zostera. The tree based on matK supported the monophyly of the subgenus Zostera, and showed that Zosteraceae consist of three main groups: Phyllospadix, which is clearly defined by being dioecious; the subgenus Zosterella and Heterozostera; and the subgenus Zostera. Character-state reconstruction of chromosome number and geographic distribution for our molecular phylogenetic tree showed that 2 n=12 is a plesiomorphic character for Zostera and Heterozostera, that the chromosome number was doubled or tripled in two lineages, and that the initial speciation of Zostera and Heterozostera occurred in the Northern Hemisphere. The matK tree showed the close affinity of Z. noltii and Z. japonica, which have disjunct distributions. Zostera marina, which is the only widely distributed species in the subgenus Zostera, also occurring in the northern Atlantic, was shown to be embedded within other subgenus Zostera species.
Asunto(s)
Cloroplastos/genética , Endorribonucleasas/genética , Nucleotidiltransferasas/genética , Zosteraceae/clasificación , Evolución Biológica , ADN de Plantas , Filogenia , Zosteraceae/enzimología , Zosteraceae/genéticaRESUMEN
Plasma membrane (PM) H(+)-ATPase and H(+) transport activity were detected in PM fractions prepared from Zostera marina (a seagrass), Vallisneria gigantea (a freshwater grass) and Oryza sativa (rice, a terrestrial plant). The properties of Z. marina PM H(+)-ATPase, specifically, the optimal pH for ATPase activity and the result of trypsin treatment, were similar to those of authentic PM H(+)-ATPases in higher plants. In V. gigantea and O. sativa PM fractions, vanadate-sensitive (P-type) ATPase activities were inhibited by the addition of NaCl. In contrast, activity in the Z. marina PM fraction was not inhibited. The nitrate-sensitive (V-type) and azide-sensitive (F-type) ATPase activities in the Z. marina crude microsomal fraction and the cytoplasmic phosphoenolpyruvate carboxylase activity, however, were inhibited by NaCl, indicating that not all enzyme activities in Z. marina are insensitive to salt. Although the ratio of Na(+) to K(+) (Na(+)/K(+)) in seawater is about 30, Na(+)/K(+) in the Z. marina cells was about 1.0. The salt-tolerant ATPase activity in the plasma membrane must play an important role in maintaining a low Na(+) concentration in the seagrass cells.