Cadmium effects in Nitzschia Palea frustule proteins

Universidade de Aveiro 2010

Departamento de Biologia

José António da Costa

Santos

Efeitos do cádmio nas proteínas da frústula de

Nitzschia palea

Universidade de Aveiro 2010

Departamento de Biologia

José António da Costa

Santos

Efeitos do cádmio nas proteínas da frústula de

Nitzschia palea

Cadmium effects in Nitzschia palea frustule proteins

Ecotoxicologia, realizada sob a orientação científica da Doutora Etelvina Maria de Almeida Paula Figueira, Professora auxiliar do Departamento de Biologia da Universidade de Aveiro e co-orientação científica da Doutora Salomé

Fernandes Pinheiro de Almeida, Professora auxiliar do Departamento de Biologia da Universidade de Aveiro

Este texto é dedicado a todos aqueles que directa e indirectamente, no contexto pessoal e académico, permitiram a concretização deste trabalho. Obrigado.

o júri

presidente Prof. Doutor António José Arsénia Nogueira

Professor Associado c/ Agregação do Departamento de Biologia da Universidade de Aveiro

Prof. Doutora Helena Guasch Padro

Professora Auxiliar Institut d'Ecologia Aquàtica, Campus Montilivi Girona, Espanha

Prof. Doutora Etelvina Maria de Almeida Paula Figueira Professora Auxiliar do Departamento de Biologia da Universidade de Aveiro

Prof. Doutora Salomé Fernandes Pinheiro de Almeida Professor Auxiliar do Departamento de Biologia da Universidade de Aveiro

agradecimentos

Agradeço às minhas orientadoras, Professora Doutora Etelvina Figueira e Professora Doutora Salomé Almeida, o terem incentivado a realização deste trabalho, a constante disponibilidade e dedicação, o aconselhamento e orientação científica, a paciência e o auxílio no decorrer de todo o processo.

Agradeço a todos os colegas de laboratório o excelente ambiente criado, o espírito de entre ajuda, muito especialmente à Ana Lima e à Diana Branco, pelo extraordinário contributo ensinamentos e empenho, em todas as fases deste trabalho, possibilitando a conclusão do mesmo.

Agradeço á minha família o apoio a todos os níveis, a quem devo a motivação para esta dissertação.

Agradeço aos meus amigos, a boa disposição e companhia nas ocasiões mais difíceis.

palavras-chave diatomáceas, cádmio, teratologia, contaminação, metais, proteinas, frústula

resumo As diatomáceas são organismos eucarióticos fotossintéticos, cuja relevância

como espécies bioindicadoras foi há muito estabelecida, por via de índices ecológicos, ou por via de testes de toxicidade baseados em características ecológicas. A parede celular silicificada (frustula) é a característica mais distintiva destes organismos, permitindo uma identificação da espécie, e fornecendo a indicação de stresses ambientais, devido à indução de formações anormais da frústula. Estas teratologias são a consequência de perturbações no processo de biosilicificação, e podem ocorrer em culturas laboratoriais, ou devido a contaminação por metais, ou pesticidas. De entre os indutores de teratologias, os metais, como o cádmio, são a classe mais

relevante devido à sua ocorrência natural ou antropogénica na natureza, e pela sua alta toxicidade relativa às pequenas quantidades presentes. Embora o processo de formação da frustula não esteja ainda completamente esclarecido, nos últimos anos tem sido publicada informação que revela a existência de proteínas na frústula, algumas delas contribuindo para a biosilicificação. O estudo das alterações induzidas pelo cádmio na quantidade, variedade, e relação das proteínas presentes na frústula, foram os objectivos deste trabalho, juntamente com a quantificação de cádmio nas fracções da frústula. Os resultados deste trabalho mostraram que a exposição ao cádmio aumentou o conteúdo proteico da frústula. Cerca de 80% dos peptideos aumentou a expressão na presença de Cd. Este foi sobretudo retido extracelularmente, encontrando-se 85% do Cd ligado a frustulinas. O presente trabalho

demonstrou que as frustulinas são extremamente importantes para a defesa da célula dos efeitos do cádmio, contribuindo com dois novos supostos mecanismos de tolerância ao cádmio: o de reforço da frústula, e a protecção celular contra a entrada de Cd, através da quelação extracelular dos iões metálicos. Estes resultados mostram que as frustulinas podem ter um papel importante na tolerância das diatomáceas a metais.

keywords diatoms, cadmium, teratology, contamination, metals, proteins, frustules

abstract Diatoms are unicellular eukaryotic photosynthetic organisms whose relevance

as biomonitor species have long been established, either by ecological indexes, or by tolerance and other toxicity tests, based on ecological

properties. The silicified cell wall of diatoms (frustule), is the most visible and distinguished characteristic of these organisms, providing species identification, and indication of environmental stressors, due to the induction of abnormal frustule formation. These teratologies are the consequence of perturbation in the biosilicification process, and can occur either by artificial growth, heavy metal contamination, or pesticides. Amongst the frustule abnormality inductors, metals such as cadmium are the most relevant class due to both anthropogenic and natural occurrence in nature and by the high toxicity relative to the small amounts present in the habitat. Although the process of frustule formation is not completely understood, in the last years it has been published data that show the existence of proteins in the frustules, some of them contributing to the biosilicification. The study of alterations induced by cadmium to the quantity, variety, and ratio of proteins present in frustules were the objectives of this work, along with cadmium quantification in the frustule fractions. Results showed that Cd increased frustule protein content. About 80% of the peptides increased their expression in the presence of Cd . Cadmium was mostly retained extracellularly, and 85% was bound to frustulines. Frustulins were found to be extremely important to the cell defense against cadmium stress, providing two putative novel mechanisms of cadmium tolerance: strengthening of frustules, and protection against Cd, through extracellular metal chelation. These results show that frustulines can play a leading role in the tolerance of diatoms to metals.

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Index

1. Introduction ... 3

1.1- General description of Diatoms ... 3

1.2- Ecology and environmental relevance of diatoms ... 4

1.3- Teratologies ... 5

1.4- Metal contamination ... 5

1.4.1- Cadmium ... 6

1.5- Frustule formation and cell cycle ... 7

1.6- Silica uptake and transport... 9

1.7- Biosilicification and morphogenesis ... 11

1.8- Biomolecules associated with diatom frustules ... 12

1.8.1 Frustulins ... 12 1.8.2- Pleuralins ... 13 1.8.3- Silaffins ... 14 1.8.4- Long-Chain Polyamines ... 16 1.8.5- Silacidins ... 17 2. Objectives ... 19

3. Materials and methods ... 20

3.1- Microorganism and culture conditions ... 20

3.2- Analysis of frustules proteins ... 20

3.2.1- Cell lysis ... 20

3.2.2- Sequential protein extraction ... 21

3.2.3- Protein content ... 21

3.2.4- Protein separation by SDS-PAGE ... 21

3.3- Cadmium quantification in the frustule protein fraction ... 22

4. Results ... 23

4.1- Cadmium effects on frustule proteins ... 23

4.2- Cadmium effects in protein expression ... 24

4.3- Results from cadmium quantification ... 25

5. Discussion ... 27

5.1- Protein extraction ... 27

2 6. Conclusions ... 30 7. References ... 31

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1. Introduction

1.1- General description of Diatoms

Diatoms are mostly unicellular or colonial C4 based photosynthetic

eukaryotes belonging to the class Bacillariophyceae from the Heterokontophyta division, usually of a golden-brown colour due to the masked chlorophyll by fucoxanthin, surrounded by four membrane plastids. A major characteristic of diatoms is a silicified cell wall called frustule, composed of two overlapping thecas (epitheca and hypotheca), that are connected by girdle bands. The silica cell walls are ornamented with hereditary specific patterns and structures (Van Den Hoek et al., 1995).

There are nearly 12000 identified species ( Norton et al., 1996) out of an estimated number of 100000, distributed over more than 250 genera of living diatoms, ranging from 5 µm to 5 mm in size (Van Den Hoek et al., 1995).

Diatoms are traditionally organized in two major morphologic groups: centric and pennate, according to cell symmetry and ornamentation. Centric diatoms have radial symmetry and are usually found in marine habitats, while pennate diatoms are elongated with bilateral symmetry and are usually found in freshwater habitats (Van Den Hoek et al., 1995).

Fig.1-Schematic overview of the siliceous components of diatom cell walls. Drawing by Ian Nettleton. Extracted from Zurzolo et. al, (2001)

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1.2- Ecology and environmental relevance of diatoms

Diatoms are a widespread group and can be found in the oceans, in freshwater, in soils and on damp surfaces. Some live pelagically in open water, some are benthic, living as surface films at the water-sediment interface, and some are epilithic, epiphytic and epizoic living respectively on stone, plants, and animal surfaces(Van Den Hoek et al., 1995).

The ubiquitous nature of diatom distribution, species richness, short generation time, fast reaction times to environmental changes, specific sensitivity, tolerance, intolerance and preference range to a number of environmental parameters or stressors such as salinity, metals, pH, saprobity, turbidity, water current and depth, and substrate availability, make these organisms particularly useful for environmental quality studies (Dixit et al. 1992; Stoermer and Smol, 1999), whose implementation is facilitated by a well documented taxonomy and ecology, and the possibility of permanent storage of diatom slides.

A particularly useful characteristic of the diatoms for biomonitoring purposes is the species-specific ornamentation of the frustule, which is faithfully reproduced through the generations. However, if diatom cells during the reproductive phase are exposed in vivo to physical, chemical or mechanical environmental stress, the frustule morphology can be modified in different ways leading, to abnormal forms (Falasco et al., 2009).

Recording both normal and teratological cells in a diatom population or community in sediment samples, can give both a temporal and spatial indication of contamination of water bodies and its past assemblages (Falasco et al., 2009).

Nitzschia palea was previously considered a tolerant diatom species to

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1.3- Teratologies

Teratological forms differ from polymorphisms. While polymorphisms are an adaptative genetic response driven by selective pressure to environmental factors, teratological forms of diatoms are non-adaptive phenotypic

abnormalities caused by exposure to chronical or acute stresses (Cox et al., 2006).

Diatom teratological forms present several deformed valvar characteristics such as outline, striation pattern, or symmetry, resulting from modifications in physiological processes (Falasco et al., 2009).

According to Falasco et al. (2009), teratological forms can be organized in seven types, with type 1 and type 2 being the most common (table 1).

Table 1.:Types of teratological forms. Adapted from Falasco et. al, (2009)

Type 1: deformed valve outline (loss of symmetry relative to both axes; pentagonal or trilobate shapes; abnormal

outline: bent, jagged, incised, kidney-shaped, swollen, twinned, wiggly, showing bulbous protrusions, or ‘boomerang’-shaped).

Type 2: changes in striation pattern, costae and septae (multiple orientation centres for the striation; branched striae;

loss of transverse costae; arrangement and shape of areolae; loss of the areolae or striae).

Type 3: changes in the shape and size of the longitudinal and central area (e.g. displaced, doubled, abnormally

enlarged, absent).

Type 4: modifications of the raphe (split, sinuate or fragmented, sometimes turned down, double or triple, angular,

bifurcate, orientated toward the centre of the valve, completely absent, with portions associated with and occasionally connected to the areolae).

Type 5: modifications of the raphe canal system (distorted, curved and occasionally doubled back or displaced,

disordered and stretched out fibulae).

Type 6: unusual arrangement of the cells forming colonies.

Type 7: mixed type in which one valve shows more than one kind of teratology, each independent of the others.

1.4- Metal contamination

Metal contamination has been identified as one of the chemical sources responsible for teratological effects in Nitzschia palea (Morin et al., 2008b). Toxic metals in the environment occur via either natural weathering, or anthropogenic processes (De Filippis and Pallaghy, 1994). The later becoming

the predominant contributor for increased metal concentrations in aquatic ecosystems.

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In uncontaminated aquatic ecosystems, metals are present in trace concentrations, and some like Cu, Fe, Zn, are essential oligoelements performing key metabolic functions, as cofactors of enzimes involved in many biological processes (De Filippis and Pallaghy, 1994), while others considered nonessential, like Pb, As, and Hg, even at equally low concentrations, can disrupt cellular homeostasis by inhibiting metabolic processes (Morin, 2003).

Although the uptake of dissolved metals by diatoms is limited by environmental factors that reduce the free ionic concentration, such as water hardness, salinity, chelating agents, or high organic content of water, (Dobson, 1992) cellular defense mechanisms must be present to avoid or reduce the effects of metals (Tripathi et al., 2006, Branco et al., 2010). Some of those mechanisms involve phytochelatins (Morelli and Scarano, 2004) or ROS scavenging (Jin et al., 2008; Qiu et al., 2008), thus conferring tolerance or resistance to the cell.

1.4.1- Cadmium

Until recently, cadmium was not considered an essential element, however Lane et al. (2000) found that Thalassiosira weissflogii expresses a Cd-specific carbonic anhydrase, which, particularly under conditions of Zn limitation, can replace Zn by Cd (Lane et al., 2000), but this still remains the only known biological function of Cd. Despite this function, cadmium is still considered by USA Occupational Safety and Health Administration, one of the most toxic elements, and ranked the seventh most dangerous substance byThe Agency for Toxic Substances and Disease Registry (ATSDR).

Cadmium is a chemical element with the symbol Cd and atomic number 48 belonging to the 12th group on the periodic table of the elements together with zinc and mercury. It has a density of 8.6g cm3, a molecular weight of 112.41 g mol-1, its fusion temperature is 321ºC, its boiling temperature is 767.2 ºC and is insoluble in water and organic solvents.

Cadmium shares some chemical and physical properties with elements of the same group or period. It has a strong affinity to sulfhydryl groups (SH)

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(Lehninger, 1970) and is linked to Zn and Cu displacement in metalloenzymes (Price and Morel, 1990), leading to inhibition of their functions, thus further increasing the ROS concentration already induced by cadmium presence, causing damage to cellular components due to oxidative stress (Price et al., 1990; Sunda et al.,1989; Torres et al., 2000).

Information related to modifications of physiological processes by cadmium in freshwater diatoms is practically nonexistent (Branco et al., 2010), but modifications during the reproductive phase were pointed out as responsible for teratological cell formation (Falasco et al. 2009).

1.5- Frustule formation and cell cycle

New frustules are formed from a parental cell during cell division. Each parental cell forms two complete daughter cells by using each theca as an epitheca for the daughter cells. Daughter cells must develop their own hypotheca while still inside the parental cell. This process occurs inside silica deposition vesicles (SDV) (Fig2). These are specialized intracellular compartments bound by a membrane called the silicalemma formed just under/bellow the cell membrane (Pickett-Heaps et al., 1990).

Two types of SDV´s, (valvar SDV and girdle band SDV), at different stages of the cell cycle, are required for the hypotheca formation (Pickett-Heaps et al. 1990).

During cell division, each daughter cell produces a valvar SVD where silica is deposited, being continuously expanded in a bidimensional plane, until the optimal size of the new hypovalve is reached and then thickened in the third dimension(Schmid and Schulze, 1979). When valve formation is complete, it is deposited on the cell surface by exocytosis of the SDV, which fuses with the cell membrane (Van de Poll et. al., 1999).

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Due to the rigid nature of the silicified cell wall, growth is only possible by parental theca separation synchronized with formation of new girdle bands, each formed inside an individual SDV, thus preventing any gaps in the cell wall during the process (Pickett-Heaps et al., 1990).

The formation of a valve is a rapid process. It generally requires the daughter cell 6 to 8 hours to form a new hypovalve and to separate from each other, and only takes 15 minutes for the two dimensional expansion of the valve in the SDV (Hazelaar et al., 2005).

Silica transport and formation of cell wall are processes strongly associated to the cell cycle, (Thamatrakoln et al., 2007) resulting in an interdependence between biosilicification and cell growth rate (Martin-Jézéquel et al., 2000).

Silica uptake and deposition occurs during one continuous segment beginning just before cell division and ending just before daughter cell separation. This uptake appears to be confined to the G2 and M phase between cytokinesis and daughter cell separation (Martin-Jézéquel et al., 2000).

Fig 2. Schematic overview of mitotic cell division and hypovalve and girdle band formation. N, Nucleus; MC, microtubule center; SDV, silica deposition vesicle. Drawing by Ian Nettleton. Extracted from Zurzolo et. al, (2001).

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The strict requirement of silica in frustules formation has led to the evolution of silica-dependent checkpoints in diatom mitosis, ensuring that cell division does not occur unless there is sufficient bio-available silica for frustule formation. Checkpoints have been found to occur in diatoms at both G1/S and G2/M transitions (Martin-Jézéquel et al., 2000).

1.6- Silica uptake and transport

In it’s soluble form, silicon (Si) exists predominantly as monosilicic acid Si(OH)4, a monomer formed by a silicon atom coordinated by four hydroxyl

groups, being the preferred source for biogenic conversion into the biosilica (SiO2) of the frustula (Lewin, 1955b; Del Amo and Brzezinski, 1999; Iler,1979).

Silicic acid is found universally in water at concentrations of a few ppm, and is stable until 100 ppm, when it starts to exceed the solubility of the amorphous phase, undergoing autopolycondensation reactions, by nucleation processes (Iler, 1979; Vrieling et al., 2003; Sumper, 2004; Martin-Jézéquel et al., 2000).

Diatoms have mechanisms to transport silica (in the form of Si(OH)4

silicilic acid) from the environment, even at low concentrations, through the plasma membrane, leading to an intracellular concentration of silica about 100 times higher than in the outside (Hildebrand et al., 1997,1998). In 1954, Lewin wrote that silicic acid uptake in diatoms is regulated by sulphydryl groups on the cell surface (Lewin,1954). Recently, silicon transporters (SITs) where found in the plasmalema of diatoms (Hildebrand et al., 1997). SIT’s are a novel class of membrane proteins. These specific silica carriers were found in all diatoms studied to date, and are thus considered ubiquitous in diatoms (Hildebrand et al., 1997, 1998; Thamatrakoln et al., 2006).

Under certain conditions, silicic acid can be captured by a mechanism that does not involve SITs (Vrieling et al., 2007). Macropinocitosis is a non-specific mechanism proposed by Vrieling et al. (2007), by which silica is internalized via part of plasmalema invagination. This mechanism helps to explain why salinity levels in the environment affect the nanostructure pattern

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inside the SDV, fact that would be hard to explain if salt wasn’t in direct contact with monosilicic acid.

The relationship between monosilicic acid and metal ions has been studied but it’s not completely understood. Zinc and copper were found to affect diatom monosilicic acid uptake (Rueter and Morel, 1981), but it was not clear if the transporter was directly affected. De la Rocha et al. (2000), observed changes in the monosilicic acid uptake kinetics due to Fe2+/3+ and Zn2+ stress, showing uptake rates two to three times lower than monosilicic acid depleted cells.

Falasco et al. (2009), related the silicon uptake decline in the presence of sulfhydryl group binders, like Cadmium, with the inhibition of ATPases containing sulfhydryl groups in their active sites, leading to the development of teratological forms (Falasco et al., 2009).

Monosilicic acid uptake must be regulated for the cell to define and maintain a sufficient amount for each phase of the biosilicification process. It was shown that this control is mainly affected by the intracellular rate of incorporation, rather than the extracellular availability of silica (Conway et al., 1976, 1977; Hildebrand, 2000). SITs determine silicon uptake, specially at low concentrations of monosilicic acid, whether at high concentrations uptake occurs passively (Thamatrakoln and Hildebrand, 2008).

Once inside the cell, monosilicic acid must then be transferred to the SDV where the conversion of silica from Si(OH)4 to biosilica (SiO2) takes place.

Several papers report the existence of intracellular silicon storage pools that might represent up to 50% of the total cellular silica, (Chisholm et al., 1978, Martin-Jézéquel and Lopez, 2003). These storage pools are believed to contain sufficient silica for the production of a new valve (Hildebrand, 2000).

The reported concentrations of the intracellular silicon within these storage pools strongly exceed the levels necessary for stability of monosilicic acid, but somehow these over saturated silicic acid values are maintained stable (Iler 1979; Martin-Jézéquel et al., 2000). Azam et al. (1974), have suggested that in these storage pools, silicic acid is associated with some kind of organic molecule or specific proteins, thus forming a stable silicic acid pool

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inside the cell. This concept of organic stabilization is shared by Sumper et al. (2004), proving that polyamine stabilization of silicic acid is possible. Schmid and Schultz (1979), proposed that these pools were located inside special compartments called silicon transport vesicles (STV) after observations of citoplasmatic vesicles fusing with the developing SDV. This opinion was corroborated by Pickett-Heaps et al. (1990), who observed clouds of vesicles surrounding Golgi complex moving to and fusing with the silicalema of the SDV, thus suggesting that the role of these STVs was to supply silicic acid to the SDV. Hildebrand and Wetherbee (2003), based on SEM observations challenged this theory, stating that these vesicles didn’t have enough electronic dense silicia to be detected, and instead proposed that silicic acid transport to the SDV might also occur via a SIT process, like the proposed for the silica uptake into the cell (Hildebrand, 2000).

Until now, there is no evidence for the presence of silicon inside these STVs, however, the intracellular silicon processing, transport, and transfer into the silica deposition vesicle are still poorly understood (Hildebrand, 2000; Sumper and Brunner, 2008).

1.7- Biosilicification and morphogenesis

The formation of biosilica (SiO2) starts only after sufficient Si(OH)4 is

uptaken and transported inside the SDV, the organelle responsible for deposition and formation of the cell wall.

By definition, amorphous minerals do not possess structural order at the molecular level, which implies that there is no preferential growth direction (Addadi and Weiner, 1992). In the case of silica, its polymerization can be induced relatively easily (Iler, 1979), but a form of specific shape requires control at a different level.

Biomineralisation is the process of formation and patterning of inorganic materials by living organisms, and in diatoms, due to silicon being used it’s called biosilicification. Biosilicification is one of the processes involved in the complete frustule formation, named morphogenesis.

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Micromorphogenesis comprises all biological or physical-chemical cellular processes leading to the formation of small-scale biosilicification of structures up to 50 nm, like the conversion of monosilicic acid into biogenic silica by specific biomolecules forming the specific cell patterns (Iler, 1979; Hildebrand, 2003; Vrieling et al., 2003).

Macromorphogenesis encompasses mechanisms responsible for the control of a larger-scale structure of diatom like movements of the SDV, to mold the form of polymerizing silica. These movements can either be externally directed by the cytoskeleton, internally driven by proteins intrinsic to the SDV, or by passive molding around external structures like organelles (Pickett-Heaps et al., 1990; Crawford et al., 2001).

The processes resulting in the formation of biosilica are only partly understood, remaining challenging subjects of fundamental research.

Isolation of a functional SDV or its specialized components would allow for detailed study of the processes, and biomolecules involved in diatom silica biogenesis and patterning. However, there are currently no methods available that allow for such isolation. Nonetheless, important insights into diatom biosilicification have been made by isolating and characterizing specific biomolecules entrapped within the silica of the frustule.

1.8- Biomolecules associated with diatom frustules

Although previously established by Volcani et al. (1981) that removing the mineral content of the frustules with fluoridric acid vapors leaves behind an organic mesh with the frustules outline (Volcani et al., 1981), it was not until the work of Kröger et al. (1994) that isolation and characterization of these biomolecules was possible.

1.8.1 Frustulins

Frustulins where the first family of biomolecules isolated from diatom frustules, and compose the outer protein coat of the frustule, thus its name.

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They have been identified from both pennate and centric diatoms, and can be extracted from the cell wall by EDTA (Kröger et al., 1996).

Frustulins consist of five Ca2C-binding glycoproteins with molecular

weights ranging from 15 to 200 kDa, presenting multiple acidic and cysteine-rich domains (ACR) that confer a specific affinity for Ca2+ ions. These ACR domains are either directly adjacent to each other, or spaced by hydrophobic polyglycine and or proline-rich domains (Fig. 3) (Kröger et al.,1994, 1996; Armbrust et al., 2004; Kröger and Poulsen, 2008; Bowler et al., 2008).

Frustulins become associated with the cell wall only after silica formation is complete. Based on structural and chemical properties, they are thought to maintain the structural and mechanical integrity of the cell wall (Hildebrand and Wetherbee, 2003; Van de Poll et al. 1999).

1.8.2- Pleuralins

Pleuralins, formerly known as HF-extractable proteins (HEPs), were the second family of frustule associated proteins identified (Fig. 3) (Kröger et al., 1997). Pleuralins are localized on the frustule’s pleural bands (Fig. 5), and are tightly bound to the cell wall, as they can be only extracted by treatment with anhydrous HF.

They are involved in functional conversion of the parental hipoteca into their daughter cell’s epitheca during formation of a new theca, but are absent from girdle band SDVs, being instead secreted and attached to the newly deposited pleural bands of the corresponding theca (Kröger et al., 1997).

Three members of this protein family have been identified, pleuralin 1 (200 kDa), pleuralin 2 (180 kDa) and pleuralin 3 (150 kDa). All containing multiple repeats of the PSCD domain named after the aminoacids that together constitute more than 50% of the domain (proline, 22%; serine, 11%; cysteine, 11%; aspartate, 9%) (Hildebrand and Wetherbee., 2003).

Frustulins and pleuralins, due to the fact of neither being signaled to the inside of an SDV nor being entrapped in biosilica, where considered not having silica precipitation activity.

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1.8.3- Silaffins

Silaffins are a family of phosphoproteins, that become solubilized by an acidified ammonium fluoride solution (10 M NH4F, pH 4-5) which distinguishes

them from pleuralins, as the latter can only be solubilized by anhydrous HF (Kröger and Poulsen, 2008).

Fig.3 Schematic primary structures of diatom cell wall proteins. Each diatom cell wall protein is synthesized as a precursor protein containing an N-terminal signal peptide for cotranslational import into the endoplasmic reticulum (ER). In most cases additional polypeptide stretches ( gray ovals) are removed from the N terminus during maturation of the protein. Adapted from Kröger and Poulsen (2008).

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Silaffins have atomic masses from 4 to 17 KDa and where respectively named silaffin-1A (4 kDa), silaffin-1B (8 kDa) and silaffin-2 (17 kDa) after their strong affinity with silica (Kröger et al., 1999). Silaffins are highly homologous polycationic peptides, rich in alkaline serine, lysine, and glycine residues and are generated through the enzymatic cleavage of a single protopolypeptide (i.e., sil- 1p) (Fig.3) (Kröger et al., 1999). The lysine and serine residues of these peptides are highly modified. The serine residues are phosphorylated, a modification that adds negative charges to these peptides (Kröger et al., 1999), making silaffins zwitterionic molecules, capable of self assembly, promoted by ionic interactions (Poulsen et al., 2003). Silaffins carrying their full complement of lysine and serine modifications are known as native silaffins (abbreviated natSil). Native silaffins (natSil) including natSil-1A (6.5 kDa), natSil-1B (10 kDa), and natSil-2 (40 kDa), are highly post-translationally modified, conserving functional groups such as phosphate, sulfate and carbohydrate, resulting in higher molecular weight proteins (Kröger et al., 2002).

Silaffins appear to be entrapped inside the silica because they are not extracted from diatom cell wall even under harsh extraction conditions (eg. 2% SDS at 95ºC, 8M urea) (Kröger and Poulsen, 2008).

The extensive post-translational modifications of natsil-1A1, 1B and their zwitterionic character are thought to perform a role on silica formation in vivo.

Under in vitro conditions mimicking the environment of the SDV, Kröger et al. (2002), found that the phosphorylated natsil-1A was capable of inducing silica condensation, while silaffins lacking these native phosphate groups were unable to precipitate silica until phosphate anions were added to the solution.

However silaffin-2 was found to lack silica precipitation activity, but instead was observed to act as a modifier of the in vitro silica-precipitation activity of Long Chain Polyamines, regulating not only the amount of material precipitated by LCPAs or natSil-1A, but also the morphology of the silica produced by these cationic biomolecules (Kröger et al., 2004). In fact, when combined in different ratios, the silica formed in the presence of natSil-2 and natSil-1A presented a pear-shaped or porous sheet morphology (Poulsen et al. 2003).

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1.8.4- Long-Chain Polyamines

Long-chain polyamines (LCPA) have been found associated with silaffins upon dissolution of silica with HF, and thus are thought to be embedded within the diatom frustules, and signalized to the interior of the SDV (Kröger et al., 2000).

LCPAs are the only biomolecules associated with silica that are not proteins. Its basic structure consist of repeated units of N-methylpropylamine attached to putrescine or a putrescine derivative, varying according to the species, in length (i.e., number of propylamine repeat units), position of the secondary amine functionalities, and degree of methylation, LCPAs have atomic masses ranging from 600 to 1500 Da (Fig. 4) (Kröger et al., 2000).

LCPA also showed sílica precipitating activity but only if polianions such as phosphate ions or phosphorylated silaffins were present to allow electrostatic interactions between the components (Kröger et al., 2000, 2002, 2004; Sumper et al., 2003; Brunner et al., 2004). Addition of LCPA to a silicic acid solution led to a fast silica precipitation, incorporating the LCPA (Kröger et al., 2000).

Fig.4 Chemical structures of polyamines and modified lysines. (a) Modified lysine residues present in silaffins. In each structure the lysine moiety is depicted in gray. (b) Natural polyamines. (c) Long-chain polyamines (LCPA) from diatoms. Propyldiamine, putrescine, and spermidine moieties are depicted in red, blue and green, respectively. The n and m values indicate the numbers of propyleneimine repeats. Extracted from Kröger and Poulsen (2008).

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Long-chain polyamines do not directly catalyze silica polycondensation (Behrens et al. 2007), instead they accelerate silica precipitation if present in solution. It is, therefore, assumed that the LCPA’s exert two functions in cell wall biogenesis: The induction/acceleration of silica precipitation and involvement in the pattern formation, as self-assembling structure-directing templates (Gröger et al. 2008a).

1.8.5- Silacidins

The most recently discovered examples of proteins isolated from within the diatom cell wall and associated with SDV are the silacidins (Wenzl et al., 2008), named for their acidic nature, and residence in the biosilica.

Silacidins are isolated from an ammonium fluoride extract, followed by an HF treatment necessary to further isolate and identify the amino acid sequence.

Silacidins are polyanionic polypeptides, enriched in aspartic acid, glutamic acid, and phosphoserine residues. Three silacidins (A, B, and C) where identified, with a molecular mass of 2920 Da for silacidin A. The silacidins (A, B, and C) contain no lysine residues.

Silacidins do not present the extensive post-translational modifications found in native silaffins and lack silica-precipitation activity, but are able to modify the in vitro silica precipitation activity of LCPAs, inducing LCPA-mediated silica formation in a concentration dependent manner. Unlike natSil-2, the silacidins were not observed to inhibit the silica precipitation activity of the LCPAs or modify precipitate morphology (Wenzl et al., 2008).

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Fig.5 Schematic view of diatoms proteins and of their interaction with silicon. Frustulines are located around the frustules. Pleuralines are linked to the pleural bands. Silaffins and polyamines interact by electrostatic links between positively charged groups of polyamines (amines) and negatively charged groups of silaffins (phosphates, sulphates).Extracted from Hatte et al. (2008).

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2. Objectives

Given the importance of diatoms as biomonitors in water quality assessments, and the finding that metals cause frustule abnormalities, the present work was designed to understand the alterations induced by cadmium in the protein fraction of the frustules, some of them reported to have a role in biosilicification and morphogenesis.

Our main goals where to:

1. Efficiently extract frustule proteins, adapting a sequential extraction protocol.

2. Identify qualitative and quantitative alterations induced by cadmium in frustule proteins.

3. Analyze cadmium distribution throughout protein fractions.

4. Understand how these alterations can be involved with cadmium tolerance and frustule abnormalities induced by metal stress.

The expected results may bring novel insights to cadmium tolerance in diatoms.

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3. Materials and methods

3.1- Microorganism and culture conditions

A superficial sediment sample was collected in Pero Bonito, a stream near a pyrite mine, in Aljustrel, Portugal, by Ana Teresa Luis, whom we thank, and was microscopically examined to determine the species composition. The diatom Nitzschia palea (Kützing) W. Smith, was isolated from the sample and grown in 100 ml Erlenmeyer flasks containing 50 ml of Chu nº 10 medium (Chu, 1942). Unialgal cultures were grown under 75 µmol photons m−2 s−1 photosynthetically available radiation in 12 h:12 h light/dark cycle at 18±1 ◦C.

After an initial inoculum from a stock culture at the exponential growth phase (4 days of growth), approximately 2×104 cells ml−1 were exposed to media containing 0.2 mg Cd l−1 (in the form of CdCl2) while a same number of

control cells remained unexposed. At the end of the log phase (after 5 days), cell density of both groups was measured by direct counting in 1ml chambers using an inverted light microscope (Leitz Labovert SF). Cells where then harvested at 16000 x g for 5 min and stored at -80 ºC until further treatment.

All glassware was washed with 10% HCl and rinsed thoroughly with distilled water prior to use, to prevent unwanted metal binding.

3.2- Analysis of frustules proteins 3.2.1- Cell lysis

Frozen samples of diatoms (2,4x108 cells) grown in the absence and presence (0.2 mg Cd l-1) of cadmium were resuspended in 1mM CaCl2, and

washed twice by centrifugation at 16000 x g for 2 minutes. Cells were lysed by maceration in liquid nitrogen, resuspended in HEPES (25 mM pH 7.4) and pelleted by centrifugation at 4200 x g for 4 min at 4ºC. This procedure was repeated three times to minimize contamination of cell walls with intracellular proteins. The resulting cell wall fraction was stored at -80ºC until further use.

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3.2.2- Sequential protein extraction

Sequential fractionation of frustule proteins was based on the method described by Frigeri et al. (2006). Frozen samples were resuspended in 3 ml of 0.1 M EDTA pH 7.8 overnight (12–16 h) at 4ºC with constant rotation to extract Ca2+ binding cell wall proteins. After centrifugation at 9000 x g for 2 min, the supernatant was transferred to a new tube and stored at -80 ºC. The pellet was washed twice with distilled water and resuspended in 3 ml 1 M NaCl, extracted with rotation for 30 min at 4ºC, and then centrifuged. The supernatant was removed and stored, and the pellet was washed. The residual pellet was then extracted in 3 ml of 8 M urea for 30 min at 4ºC and treated as above. From the pellets, membrane-associated proteins were extracted in 3 ml 2% (w/v) SDS by boiling for 15 min, the supernatant was removed and stored, and the pellet was washed twice with water following the previously described process. The pellet from the SDS extract was dried, then resuspended in 3 ml of 40% concentrated hydrofluoric acid, and incubated at 4°C for 45 min. The HF extract was centrifuged at 16,000 x g for 2 min at 4°C to pellet insoluble material, and the supernatant was removed and evaporated to dryness. The resulting residual pellet was stored at -80 ºC.

3.2.3- Protein content

In each extract, protein content was determined by the BCA method (BCATM Protein Assay Kit, PIERCE), with BSA as standard.

3.2.4- Protein separation by SDS-PAGE

The EDTA fractions were concentrated using vivaspin 10000 MW concentrators at 6000 x g 4 ºC until a final volume of 200 µl was obtained. The

22

NaCl, urea, and SDS fractions were treated overnight with 7% (w/v) TCA, and 0.014% (w/v) deoxycholate, to precipitate proteins from the extracting agent, followed by a cold acetone wash to remove residual TCA. The HF fraction residual pellets were dissolved in a small volume of water, and neutralized with 0.2 M NaOH. All extracts were dissolved in an appropriate amount of 4 x SDS sample buffer (Hames,1981) with 5 mM β-mercaptoethanol and heated in a water bath for 3 min at 95ºC. Equivalent amounts, based on starting cell numbers, were used for protein separation by sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS-PAGE), carried out in bis-Tris precast gels (4–12%) of 0.75mm (Bio-Rad) following the system of Laemmli (1970). Molecular weight standards used were purchased from Bio-Rad. Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad). Densitometric readings were performed by a Bio-Rad densitometer (model GS 710). The molecular weight and relative amount of proteins corresponding to each band were calculated using Quantity-One Software (Bio-Rad) (Figueira et al., 2005).

3.3- Cadmium quantification in the frustule protein fraction

Samples of diatoms (3,6x107) grown as described previously, were harvested at 9000 × g for 5 min following the procedure described above. Pelleted cells were immediately washed with destiled water at 55 ºC for 30 min followed by CaCl2 5mM to remove cell wall associated polysaccharides. Cells

were then resuspended in 2 ml of 0.1 M EDTA pH 7.8 overnight (12–16 h) at 4ºC with constant rotation to extract Ca2+ binding cell wall proteins. After centrifugation at 9000 x g for 2 min, the supernatants were transferred to a new tube, acidified with 100µl of concentrated HCl and stored at 4 ºC. Cells were then resuspended in HEPES 25mM pH 7.4 buffer, and lysed by vigorous sonication. From this point sequential protein extraction was performed using the same procedure described in 3.2.3.

The fractions obtained were acidified as described above and stored at 4º C. In each fraction, cadmium was quantified by Inductively coupled plasma mass spectrometry (ICP-MS) analysis.

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4. Results

4.1- Cadmium effects on frustule proteins

Frustules have different proteins, with different degrees of association with the mineral fraction, and for that reason a sequential extraction procedure had to be used so that proteins were extracted efficiently. Fig. 6 shows the total protein content obtained in the different sequential extractions.

Fig 6. Proteins in N. palea grown 5 days in medium not supplemented (control), or supplemented (Cd) with cadmium (0,2 mg l-1). (a) Protein content (µg 106 cells-1) in each fraction. (b) Relative protein content in each fraction.

(a)

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Cadmium induced a steady increase in the amount of proteins in each fraction. The protein content in the control cells, was determined to be 5,8 µg per million cells, while in the cadmium treated group was 33,9 per million cells, indicating that cadmium induced the synthesis of frustule proteins near six times. Nonetheless, the relative abundance was not altered. Proteins extracted by EDTA where the more abundant, constituting half of the proteins. Proteins in the SDS and NaCl were the less abundant.

4.2- Cadmium effects in protein expression

The proteins obtained in each extract were further analyzed by SDS-page. Fig 7a shows an example of an electrophoretic separation of each of the extracts, in the presence or absence of Cd and in Fig 7b the relative abundance in the number of protein bands identified in each fraction is represented.

Fig.7 Electroforetic separation of frustule proteins from different fractions (EDTA, SDS, NaCl, urea, and HF) in control (C) and cadmium (Cd) exposed N. palea cells. (a) Electrophoretic patterns. Bands which showed the highest expression increases are marked in the gel. (b) Relative abundance of bands in each fraction.

(b) (a)

(b) (a)

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When comparing the profiles between fractions, it is evident that they have different patterns, which shows that different proteins are associated to each fraction. The number of proteins identified in each fraction also differed, being most of them from the EDTA, urea and NaCl fractions, and fewer from the SDS (Fig 7b).

The electrophoretic separation made possible to identify a total of 32 peptide bands. Globally, polypeptide expression was differently affected by Cd, 5 polypeptides were not affected, 10 increased, and 17 showed a high expression increase (more than 5 times) (Fig. 8a). Analyzing the polypeptide expression in each fraction, the most affected were those extracted with urea and EDTA, where 70% and 63% respectively were highly increased (Fig. 8b).

4.3- Results from cadmium quantification

Cadmium content was analyzed both extracellular and intracellularly. Since most of the Cd (97%) was allocated outside of the cell, this association

Fig.8. Cadmium effect on protein expression compared to control in frustule proteins. (a) Total polypeptides. (b) Polypeptide in each fraction

(a)

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was further analyzed, and Cd associated to polysaccharides and to the different fractions of the frustules were determined (Fig.9).

The extracellular Cd was practically all bonded either to polysaccharides (11%), or to the EDTA fraction (85%). Intracellular cadmium represented near 3% of total, and Cd associated with the other frustule fractions (NaCl, urea, SDS and HF fractions) is negligible (0.8%).

Fig. 9. Cadmium distribuition in N. palea grown 5 days in medium supplemented with cadmium (0,2 mg l-1).

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5. Discussion

In the present work, we aimed to extract the different peptide fractions of the frustules. The adaptation of the procedures developed by Kröger et al. (1994), and Frigeri et al. (2006) to N. palea have proven to be a challenging task. Previous works used marine planktonic species that yielded large amounts of cells in culture (Kröger et al., 1994, 1999, 2000). In contrast, N. palea, is a freshwater benthic species, and the number of cells per volume of culture is much lower, thus requiring more resources to obtain equivalent amounts of cells. Furthermore, freshwater diatoms contain approximately one order of magnitude more silica in their cell walls than marine diatoms (Alverson, 2007), being more resistant, and requiring a different method, from the one used by Kröger et al. (1994), to brake the cell frustules. Liquid nitrogen not only efficiently broke the frustule, but also maintained the temperature low during the process which is an important aspect to maintain the integrity of the proteins. Adaptations to the duration and speed of centrifugation were also required. The inclusion of more steps for diatom cell wall preparation to help the sequential removal of nonspecifically associated proteins, added by Frigeri et al. (2006) to the Kröger et al. (1994) method, not only allowed the recovery of more protein, but also produced different protein banding patterns in each fraction. As a result, a successful separation of the EDTA, NaCl, urea and SDS extracts, was obtained, yielding higher amounts of protein than those obtained by Kröger et al. (1994) and Frigeri et al (2006).

A direct observation of the gel shows an increase of the protein content in the fractions from the Cd grown cells, suggesting a general effect of cadmium on the proteins associated with frustules. In fact, 84% of the polypeptides separated showed an increase or a high increase of expression, corroborating the 6 fold increase of the total frustule protein. Although the EDTA extract was responsible for only 25% of the number of polypeptides isolated, it contained

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50% of the total protein content, allegedly frustulins (Kröger and Pulsen, 2008), showing that these proteins are the most abundant in the frustule.

Culture experiments have demonstrated that diatoms grown under limiting silicon concentrations present thinner frustules (Alverson, 2007). The same result was obtained in N.palea grown under cadmium stress (unpublished data). Falasco et al. (2009) reported that silicon uptake declined in the presence of metals, which was related with the inhibition of ATPases containing sulfhydryl groups in their active sites. It is then plausible that in the presence of an excess of Cd, thinner frustules are formed. To compensate for thinner frustules, caused by lower intracellular silica amounts, diatoms might strengthen cell wall by increasing frustule proteins. In particular, frustulines are thought to maintain the structural and mechanical integrity of the cell wall (Wong Po Foo et al., 2004) and the high amount of these proteins in Cd exposed cells (Fig. 6a), may support a role of this sort. However, since the peptides present in the other fractions that interact electrostatically (NaCl), or via hydrogen bonding (urea), with the cell (Frigeri et al.,2006), also show similar increases ( four to six fold), they could also play a similar role to that already described for frustulins, that is to maintain the structural and mechanical strength of the cell wall, weakened by a lower incorporation of the biomineral component.

5.2- Cadmium quantification

The present work also aimed to analyze the cadmium distribution throughout the frustule fractions. In a vast number of organisms extracellular metal binding is the most efficient mechanism of metal tolerance. For diatoms the exudation of carbohydrates in the presence of metals, such as Cd and Cu, as an extracellular mechanism of metallic tolerance was described (Pistochi et al., 1997).

Analysis of the cadmium associated with the different fractions of the cell, revealed that cadmium is mostly retained extracellularly, and accumulated mainly (85%) in the EDTA fraction (frustulins) of the cell wall, rather than being associated with polysaccharides (11%). Although it was found previously that in

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location remained unknown, up until now. Frustulins, constitute a continuous protein coat that cover all parts of the cell wall, and contain multiple acidic and cysteine-rich domains (Kröger et al.,1997). Sulfhydryl groups, are the functional group of cysteines, and have high affinity for Cd (Lehninger, 1970), thus the high number of cysteine residues in frustulines make them excellent candidates for Cd binding. In fact, the presence of the majority of cadmium in the EDTA fraction, corroborates that Cd must be primarily associated with frustulins.

These results are of significant importance since they suggest that cadmium binding to frustulins is an important cadmium tolerance mechanism, described for the first time in N. palea. By forming an enlarged frustulin barrier (Fig. 6a), cells may increase their competence to bind Cd, and possibly other metals. This process would decrease metal bioavailability and uptake, thus reducing its toxic effects where metallic ions are more detrimental, inside the cell.

Frustulins definitely must play an important role in protecting the cell from cadmium effects, either by preventing its entrance, or by compensating the effects of a thinner frustule. Nonetheless, the fact that frustulins are induced by cadmium, and are the principal cadmium chelator in this species, strongly support the hypothesis of a cadmium induced mechanism unknown until now.

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6. Conclusions

Exposure of N. palea to 0.2 mg Cd l−1 globally increases frustule proteins expression and quantity. Particularly the EDTA extract, were frustulins are found, EDTA extract contains 50% of the proteins although it only represents 25% of the polypeptides extracted, making these proteins the most abundant in the frustule.

Cadmium is mostly retained extracellularly, and accumulated mainly (85%) in the frustulin fraction of the cell wall, rather than being associated with polysaccharides.

There are evidences of two mechanisms that may increment N. palea cells tolerance to cadmium effects involving frustulins. One mechanism would be the protection of the structural and mechanical integrity of the frustule, compromised by the thinner frustules with lower incorporation of biosilica. The other preventing the entrance of cadmium by complexation of Cd ions with sulfhydryl groups, present in the acidic and cysteine-rich domains of frustulins which would be aided by the proteins present in the other fractions.

Our results point out to the identification of a novel defense mechanism in diatoms in which frustulins play the role of the major cadmium chelator in the cell.

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