EFFECTS OF COPPER TOXICITY ON SILICIC ACID UPTAKE AND GROWTH IN THALASSIOSIRA PSEUDONANA11

The toxicity of Cu to Thalassiosira pseudonana (Hustedt) Hasle and Heimdal was investigated by examining both short and long term effects of Cu on cellular processes. Toxic levels of Cu (pCu* < 13) were found to inhibit short term Si(OH)₄ uptake rates with kinetics characteristic of irreversible inhibition at a hypothetical Cu-sensitive Si(OH)₄ transport site. Residual Si(OH)₄ concentrations (those below which no uptake could occur) were found to increase with increasing levels of Cu, and the toxic effects of Cu could be reversed by increasing the concentrations of Si(OH)₄ in the medium. The actual uptake of Cu by the cells was found to vary inversely with the ambient Si(OH)₄ concentration. Copper did not inhibit the uptake of NO₃^? or PO₄^3-. The long term inhibition of growth rate by Cu in this species was shown not to be a result of Si deficiency caused by the inhibition of Si(OH)₄ uptake. Cu inhibited cells were found to have higher Si cell quotas (including a sizable soluble pool) than the control cultures. They were, however, observed to have aberrant frustules, significantly larger than the control cells, suggesting interference with the silification process as a possible mechanism for inhibition of growth by Cu. A conceptual model is proposed for the Cu-Si(OH)₄-growth relationship. It includes a Cu sensitive Si(OH)₄ transport site that may also serve to transport Cu into the cell, and growth inhibition mediated by intracellular Cu concentrations which may block cell division or cause general cellular disfunction.     

Conserved motif CMLD in silicic acid transport proteins of diatoms

Sequenced fragments of genes coding for silicon transporters (SITs) were analyzed for diatoms of evolutionarily distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kützing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate Cylindrotheca fusiformis Reimann et Lewin with a keeled raphe system). SITs were found to contain a conserved motif, CMLD. Hydropathy profiles showed that the motif CMLD is between two transmembrane domains lacking Lys and Arg, and the domains were consequently assumed to play a role in the formation of a channel mediating silicic acid transport. The motif CMLD proved to be rare. Since Zn²⁺ is necessary for silica incorporation into diatom cells, a hypothesis was advanced that the motif CMLD acts as a Zn-binding site. Diatom growth suppression was observed in the presence of the alkylating agent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonic acid (AEDANS), which does not penetrate into the cell. Cys of the motif CMLD was assumed to act as a target for AEDANS. Zinc ions inhibited Cys alkylation in the synthetic peptide NCMLDY, testifying to the above hypothesis.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 303–316.Original Russian Text Copyright © 2005 by Sherbakova, Masyukova, Safonova, Petrova, Vereshagin, Minaeva, Adelshin, Triboy, Stonik, Aizdaitcher, Kozlov, Likhoshway, Grachev.     

DIATOM MINERALIZATION OF SILICIC ACID IV. KINETICS OF SOLUBLE Si POOL FORMATION IN EXPONENTIALLY GROWING AND SYNCHRONIZED NAVICULA PELLICULOSA1

Silicic acid taken up from the growth medium by Navicula pelliculosa (Bréb.) Hilse was shown to enter at least two compartments: i) soluble pools; ii) insoluble fraction comprised predominantly of the silica frustule. Soluble Si pools were extracted by a variety of agents from cells uniformly labeled for ten generations in medium containing ⁶⁸Ge-Si(OH)₄. 100 C water soluble and 0 C perchloric acid (PCA) soluble Si pools of 680 mM Si·l^?1 and 490 mM Si·l^?1 cell water represented 13 and 9%, respectively, of total, cell Si in exponential growth phase cells. Uniformly labeled cells synchronized by the combined synchronization technique accumulate at the cell cycle stage where silica frustule development is initiated. These cells contain water and PCA soluble pools of 10 nmol Si·10⁶ cells^?1 and-8.8 nmol Si·10⁶ cells^?1, respectively. On addition of Si(OH)₄, a rapid uptake ensues allowing the Si pool to expand 2.5-fold, apparently to provide precursors of the silica frustule.     

THE CHEMICAL FORM OF DISSOLVED SI TAKEN UP BY MARINE DIATOMS

Results of past studies of the pH-dependent Si uptake kinetics of Phaeodactylum tricornutum Bohlin suggested that the anion SiO(OH)     is the chemical form of dissolved Si taken up by marine diatoms. We determined the chemical form of Si taken up by three other marine diatom species and P. tricornutum by examining the kinetics of Si use under two dramatically different SiO(OH)     :Si(OH)₄ ratios in seawater by varying pH from ≈8 to ≈9.6. Uptake rates were determined using a precise and sensitive ³²Si tracer methodology. The pH-dependent uptake kinetics obtained for all species except P. tricornutum suggest that marine diatoms transport Si(OH)₄. The half-saturation constant (K _m ) varies strongly as a function of pH for all species when the substrate of transport is assumed to be SiO(OH)     . Kinetic curves for Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, Thalassiosira weissflogii (Grunow) G. Fryxell et Hasle, and Cylindrotheca fusiformis Reimann et Lewin have statistically identical values of K _m at each pH when the substrate for transport is assumed to be Si(OH)₄ ( T. pseudonana and T. weissflogii ) or total dissolved silicon ( C. fusiformis ). In contrast, P. tricornutum exhibits unusual biphasic uptake kinetics: uptake conforms to Michaelis–Menten kinetics up to 15 to 25 μM, above which uptake increases linearly. This enigmatic response may have biased conclusions drawn from past experiments using this species. However, based on the consistency of the results for the three other species, a new model of Si transport in marine diatoms is proposed on the basis of the direct formation of a complex between the Si-transport protein and Si(OH)₄.     

DIATOM MINERALIZATION OF SILICIC ACID. II. REGULATION OF Si (OH)4 TRANSPORT RATES DURING THE CELL CYCLE OF NAVICULA PELLICULOSA1

Synchronized populations of Navicula pelliculosa (Bréb.) Hilse show a 10-fold increase in Si(OH)₄ transport rate during traverse through the cell division cycle. The transport activity pattern is similar to a “peak enzyme.” Kinetic analysis showed there was a significant change in K_s values, indicating increased “affinity” for Si(OH)₄ as cells neared maximal uptake rates. However, the dramatic changes in transport rate at various cell cycle stages were also reflected by alterations in the V_max, values of the transport process, suggesting a change in the number of functional transport “sites” in the plasma membrane. Cells in the wall forming stage, arrested from further development by Si(OH)₄ deprivation, maintained high transport rates for as long as 7 h. The rates decreased rapidly if protein synthesis were blocked or if Si(OH)₄ was added, the latter allowing the cells to traverse the rest of the cycle. The half-life of the transport activity ranged from 1.0 to 2.2 h when protein synthesis was inhibited at various cell cycle stages and during the natural decline of activity late in the cycle. The transport system appears to be metabolically unstable as is typical for a “peak protein.” The rise in transport rate through the cell cycle did not depend on the presence of Si(OH)₄ in the medium; therefore, the transport system does not appear to be induced by its substrate. The rise in transport is also observed in L:D synchronized cells developing in the presence of Si(OH)₄; neither does the transport system appear to be derepressed. The transport rate was strongly cell cycle-stage dependent; the data appeared to fit the “dependent pathway” model proposed by Hart-well to explain oscillations in enzyme synthesis during the cell cycle.     

COPPER TOXICITY TO SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1,2

Copper toxicity to Skeletonema costatum (Grev.) Cleve has been studied in batch cultures of chemically defined culture media. The alga is relatively insensitive to cupric ion activity, demonstrating no effect on growth up to (Cu²⁺) = 10^?8.5 M. Cultures inoculated from stationary phase stocks exhibit a prolongation of the lag phase with increasing copper concentrations near and above the point of precipitation of the copper. The toxicity of copper is a function of the silicic acid concentration in the medium. This effect is observed in a range of Si(OH)₄ concentrations (10^?5 M to 10^?4 M) above known values for the saturation of silicon uptake kinetics, thus suggesting an influence of copper on silicate metabolism.     

INTERACTIVE EFFECTS OF COPPER AND SILICIC ACID ON RESTING SPORE FORMATION AND VIABILITY IN A MARINE DIATOM1

The toxic effects of copper on resting spore formation and viability in the marine diatom Chaetoceros protuberans Lauder were determined both with and without silicic acid added to the medium. With silicic acid available, partial inhibition of resting spore formation occurred only at the highest cupric ion activity (pCu 8.6), while the percentage of cells forming spores at pCu's 10.2 and 11.3 was nearly the same as in the controls. Without silicic acid added to the medium, sporulation was completely inhibited at pCu 8.6 and greatly inhibited, at pCu 10.2. At pCu 11.3 and in the controls, the rate of spore formation was less than 50%. The results indicate that the inhibition of resting spore formation by copper is related to the concentration of silicic acid available to cells of C protuberans. This is consistent with previous studies which show that copper toxicity during vegetative growth involves interference with silicification in diatoms and is a Junction of the silicic acid concentration of the medium. Viable resting spores of C. protuberans were still present in cultures following exposure to elevated copper concentrations during a 100-day incubation period. This indicates that resting spores can serve to enhance diatom survival in areas polluted by heavy metals.     

INTERACTIVE EFFECTS OF COPPER AND SILICIC ACID ON RESTING SPORE FORMATION AND VIABILITY IN A MARINE DIATOM1

The toxic effects of copper on resting spore formation and viability in the marine diatom Chaetoceros protuberans Lauder were determined both with and without silicic acid added to the medium. With silicic acid available, partial inhibition of resting spore formation occurred only at the highest cupric ion activity (pCu 8.6), while the percentage of cells forming spores at pCu's 10.2 and 11.3 was nearly the same as in the controls. Without silicic acid added to the medium, sporulation was completely inhibited at pCu 8.6 and greatly inhibited at pCu 10.2. At pCu 11.3 and in the controls, the rate of spore formation was less than 50%. The results indicate that the inhibition of resting spore formation by copper is related to the concentration of silicic acid available to cells of C. protuberans. This is consistent with previous studies which show that copper toxicity during vegetative growth involves interference with silicification in diatoms and is a function of the silicic acid concentration of the medium. Viable resting spores of C. protuberans were still present in cultures following exposure to elevated copper concentrations during a 100-day incubation period. This indicates that resting spores can serve to enhance diatom survival in areas polluted by heavy metals.     

Diatom immigration drives biofilm recovery after chronic copper exposure

1. The impact of immigration on the recovery of diatom assemblages after chronic exposure to copper was investigated in laboratory microcosms. 2. We examined the recovery trajectories of copper‐contaminated biofilms after reducing copper stress and with or without the possibility of immigration from unimpaired communities. 3. The biofilms mixed with unimpaired communities returned to a ‘control’ assemblage structure within 6 weeks, with recovery patterns depending on the endpoint considered (i.e. 2 weeks for relative abundances of diatom species but 6 weeks for total diatom biomass). In contrast, no recovery was observed in assemblages placed under control conditions without external immigrants. 4. Immigration has important effects on the recovery of quantitative and qualitative characteristics of biofilms.     

青藏高原人工草地土壤可溶性氮组分与植被生产力动态变化过程

氮作为人工草地最为重要的限制性因子, 在时间、空间上分布不均匀, 且在形态上存在差异, 与种植方式及地上净初级生产力(ANPP)存在相关关系。该研究以青海省同德牧场的无芒雀麦(Bromus inermis)、老芒麦(Elymus sibiricus)、垂穗披碱草(E. nutans)、西北羊茅(Festuca ryloviana)、中华羊茅(F. sinensis)、青海扁茎早熟禾(Poa pratensis var. anceps ‘Qinghai’)、冷地早熟禾(P. crymophila)、星星草(Puccinellia tenuiflora) 8种牧草单播人工草地为研究对象, 分析人工草地土壤可溶性氮库季节和年际动态变化过程及与ANPP之间的相互关系。该人工草地种植于2013年, 在2014-2016年(二龄、三龄和四龄)生长季6-9月测定土壤铵态氮(NH₄⁺-N)、硝态氮(NO₃^--N)、可溶性有机氮(SON)和可溶性总氮(STN)含量, 每年9月初测定ANPP, 所有样地没有施肥, 每年9月中旬刈割, 留茬5 cm。研究发现: (1) 8种禾本科牧草的ANPP在329.67-794.67 g·m^-2之间, 其中垂穗披碱草为794.67 g·m^-2, 显著高于其他牧草。(2)在二至四龄人工草地中, 土壤NO₃^--N、SON和STN含量均显著下降, 但NH₄⁺-N含量却显著增加。(3)土壤可溶性氮以SON为主, 占STN的45.11%-88.76% (0-10 cm)和47.75%-88.18% (10-20 cm); 其次为NO₃^--N, 占STN的5.81%-34.85% (0-10 cm)和6.08%-40.42% (10-20 cm); NH₄⁺-N最少仅3.41%-22.18% (0-10 cm)和3.09%-19.56% (10-20 cm)。(4)非度量多维尺度分析(NMDS)结果显示, 随种植年限的增加, 不同禾本科牧草对0-10 cm土壤可溶性氮影响趋于离散, 而对10-20 cm土壤的影响则相反, 且牧草对土壤可溶性氮含量的影响程度与土壤深度有关。(5)相关性分析表明, 土壤SON、STN含量与人工草地ANPP呈正相关关系, 与无机氮(IN)含量呈负相关关系。综上所述, 三至四龄人工草地增施氮肥是维持草地生产力的关键因素。以上结果为更深入了解青藏高原人工草地土壤可溶性氮动态变化及维持人工草地生产力和稳定性提供了科学依据。     

Increased production of extra- and intracellular metal-ligands in phytoplankton exposed to copper and cadmium

Several diatom and dinoflagellate species isolatedfrom the Adriatic Sea, grown in batch cultures in f/2medium, were exposed to two different concentrationsof Cu and Cd. The concentrations chosen caused areduction of growth, but were not lethal. Thespecies able to tolerate the highest Cu concentrations(in the order of 0.2–0.5 mg L^-1) were diatoms,while some diatoms and all the dinoflagellates wereinhibited by concentrations between 0.01 and 0.05 mgCu L^-1. Cadmium was less toxic in that it did notaffect diatom or dinoflagellate growth up to aconcentration of 0.2 mg L^-1. Increasedproduction of extracellular polysaccharides was foundto be a general response to the presence of thepollutant. Cylindrotheca fusiformis, Achnanthesbrevipes and Prorocentrum micans wereinvestigated for their production of intracellularmetal-ligands. Molecules with Cd-binding propertiesand having a molecular weight comparable with that ofphytochelatins were found only in C.fusiformis, and their presence showed both adose- and time-dependent response. The presence orabsence of intracellular ligands in these algae isconsistent with their ability to exclude the metalextracellularly.     

Silica in plants: biological, biochemical and chemical studies

BACKGROUND: The incorporation of silica within the plant cell wall has been well documented by botanists and materials scientists; however, the means by which plants are able to transport silicon and control its polymerization, together with the roles of silica in situ, are not fully understood. RECENT PROGRESS: Recent studies into the mechanisms by which silicification proceeds have identified the following: an energy-dependent Si transporter; Si as a biologically active element triggering natural defence mechanisms; and the means by which abiotic toxicities are alleviated by silica. A full understanding of silica formation in vivo still requires an elucidation of the role played by the environment in which silica formation occurs. Results from in-vitro studies of the effects of cell-wall components associated with polymerized silica on mineral formation illustrate the interactions occurring between the biomolecules and silica, and the effects their presence has on the mineralized structures so formed. SCOPE: This Botanical Briefing describes the uptake, storage and function of Si, and discusses the role biomolecules play when incorporated into model systems of silica polymerization as well as future directions for research in this field.     

DIATOM MINERALIZATION OK SILICIC ACID. I Si(OH)4 TRANSPORT CHARACTERISTICS IN NAVICULA PELLICULOSA

Silicic acid transport was studied in the photosynthetic diatom Navicula pelliculosa (Bréb.) Hilse using [⁶⁸Ge] germanic acid (⁶⁸Ge(OH)₄) as a tracer of silicic acid (Si(OH)₄). The initial uptake rate of Si(OH)₄ was dependent on cell number, pH, temperature, light and was promoted by certain monovalent cations in the medium. Na⁺ was more effective than K⁺, whereas Li⁺ and NH⁺₄ were ineffective at promoting uptake. Uncouplers and inhibitors of oxidative phosphorylation and of photophosphorylation reduced uptake by 40–99% of control values. Uptake was also especially sensitive to the sulfhydryl blocking agents at 10^?5 M and to the ionophorous compound valinomycin (10^?7 M) which inhibited uptake by 82%. The Si(OH)₄ transport system displayed Michaelis-Menten-type saturation kinetics with kinetic parameters of K_S= 4.4 p. mol Si(OH)₄· 1^?1, V_max= 334 pmol Si(OH)₄· 10⁶ cells^?1· min^?1. Calculations of the acid soluble silicic acid pool size based on 60 s uptake at 20 μM Si(OH)₄ suggested that intracellular levels of Si could reach 20 mM and as much as 5 mM could exist as free silicic acid, representing maintenance of a 250-fold concentration gradient compared with the medium. Efflux from preloaded cells was dependent on temperature and the Si(OH)₄ concentration of the external medium. In the presence of 100 μMM “cold” Si(OH)₄, approximately 30% of the Si(OH)₄ in preloaded cells was exchanged in 20 min. The initial uptake rate of Si(OH)₄ in logarithmic phase cells was constant, but the uptake rate increased in a linear fashion for 6 h in stationary phase cells. These results suggest that the first step in silica mineralization by diatoms is the active transmembrane transport of Si(OH)₄ by an energy dependent, saturable, membrane-carrier mechanism which requires the monovalent cations Na⁺ and K⁺ and is sensitive to sulfhydryl blocking agents. Silicic acid transport activity also appears to be regulated during different growth stages of the diatom.     

A review of diatoms found in highly acidic environments

A review is presented of 28 studies in the literature of diatoms in environments at pH 3.5, including natural and anthropogenic acid sources. A total of 124 diatom taxa have been reported, but many are likely to have been accidental occurrences, because only a few specimens were found. Approximately 19 taxa abundant in at least one study or common in several studies are considered true inhabitants of highly acidic waters. These include: Achnanthes minutissima, Eunotia exigua, E. tenella, E. septentrionalis, E. osoresanensis, E. arcus, E. glacialis, E. pectinalis, Frustulia rhomboides, F. rhomboides var. saxonica, Nitzschia capitellata/subcapitellata, Nitzschia communis, Nitzschia pusilla, Nitzschia vasta, Pinnularia acoricola, P. obscura, P. braunii var. amphicephala, P. subcapitata and P. terminitina. There are inconsistencies in the taxonomy of several of these species and possible synonymies could lower the number of taxa to less than 9. Compared to diatom species richness in environments at pH 4.5–5.0, there are many fewer taxa in environments pH 3.5, suggesting a threshold between pH 4.5 and 3.5 below which many species are unable to maintain a population.