V. Martin-Jézéquel* ,N. Daoud, B. Quéguiner UMR CNR Flux ...

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Coupling of Silicon, Carbon and Nitrogen Metabolisms in Marine Diatoms. V. Martin-Jézéquel* ,N. Daoud, B. Quéguiner UMR CNR 6539 « Flux de Matière et Réponse du Vivant », Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, BP 809, F-29285, Brest Cédex, France. Fax: (33) 02 98016636 e-mail: vmartin@ univ-brestjr * corresponding author Abstract The siliceous structures of diatom walls have been studied both as models of biomineralization and for the purpose of taxonomic identification. The frustule is composed of hydrated silica and organic constituents, and the knowledge of the chemical composition of this coating may allow insight in the mechanisms of silicification. Silicon is taken up as orthosilicic acid (Si(OH)4) which then polymerizes inside the cell within a silicon-deposition vesicle (SDV). Heterogeneous nucleation and growth via autopolymerization may be subsequently induced by the surface of the SDV to forrn amorphous biogenic silica (opal). Orthosilicic acid uptake as weil as silica deposition are mai nI y confined to one part of the cell cycle and the new valves are made up during cell division. Silicon incorporation is then closely related to the cell growth. The metabolic and physicochemical dependencies of the polymerization process have not yet been elucidated. The energy required for silicon metabolism is mainly originating from respiration processes (oxidative phosphorylation). Silicification is controlled at the cellular level by an organic template which is mainly composed by proteins enriched in serine and glycine. These amino acids are obligatory metabolites from photorespiration The glycolate pathway via serine and glycine appears to be rudimentary in diatoms when compared to the green algae, and the activation of the serine/glycine pathways must be original in diatoms. Preliminary experiments have revealed close relationships between the serine and glycine synthesis, and the cell division. Thus, we 65

Transcript of V. Martin-Jézéquel* ,N. Daoud, B. Quéguiner UMR CNR Flux ...

Page 1: V. Martin-Jézéquel* ,N. Daoud, B. Quéguiner UMR CNR Flux ...

Coupling of Silicon, Carbon and Nitrogen Metabolisms in Marine Diatoms.

V. Martin-Jézéquel* ,N. Daoud, B. Quéguiner

UMR CNR 6539 « Flux de Matière et Réponse du Vivant », Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, BP 809, F-29285, Brest Cédex, France. Fax: (33) 02 98016636 e-mail: vmartin@ univ-brestjr

* corresponding author

Abstract

The siliceous structures of diatom walls have been studied both as models of

biomineralization and for the purpose of taxonomic identification. The frustule is composed

of hydrated silica and organic constituents, and the knowledge of the chemical composition of

this coating may allow insight in the mechanisms of silicification. Silicon is taken up as

orthosilicic acid (Si(OH)4) which then polymerizes inside the cell within a silicon-deposition

vesicle (SDV). Heterogeneous nucleation and growth via autopolymerization may be

subsequently induced by the surface of the SDV to forrn amorphous biogenic silica (opal).

Orthosilicic acid uptake as weil as silica deposition are mai nI y confined to one part of the cell

cycle and the new valves are made up during cell division. Silicon incorporation is then

closely related to the cell growth. The metabolic and physicochemical dependencies of the

polymerization process have not yet been elucidated. The energy required for silicon

metabolism is mainly originating from respiration processes (oxidative phosphorylation).

Silicification is controlled at the cellular level by an organic template which is mainly

composed by proteins enriched in serine and glycine. These amino acids are obligatory

metabolites from photorespiration The glycolate pathway via serine and glycine appears to be

rudimentary in diatoms when compared to the green algae, and the activation of the

serine/glycine pathways must be original in diatoms. Preliminary experiments have revealed

close relationships between the serine and glycine synthesis, and the cell division. Thus, we

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can postulate a true and obligate relationship between photorespiration and respiration, and

silicification process in diatoms.

Keywords : silicon, diatom , frustule, silicification, photorespiration, serine

1. Introduction

Despite the good taxonomie and morphological knowledge of diatoms, due to systematic

studies of their frustules (Pickett-Heaps et al., 1990; Gordon and Drum, 1994), the silicon

metabolism and the building of their walls is not yet elucidated. ln the sea water, because of

the basic pH and because of low concentrations, dissolved silicon is mainly in the form of

monomeric orthosilicic acid. The leve\s vary from 0 to 180 IlM, with a mean value of 70 IlM

for the World Ocean. Thus, diatoms have developed specific mechanisms to take up silicon

from very diluted solutions.

Contrary to ionic calcium biominerals, silicon forms polymerie material with covalent bonds,

which implies control at every production step. In the organisms biogenic silicon does not

show crystalline form but rather appears to consist of amorphous material with, however

regions of local order at the atomic level or in extremely small crystals (Pickett-Heaps et al.,

1990). The organization of the silicification allows specific structures to be found in the living

matter, from sponges to higher plants. The control of that morphogenesis is still a matter of

debate.

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2. Discussion

In diatoms (Bacillariophyceae), the frustule is composed of two parts which overlap : the

epitheca and the hypotheca. Each of them is coated in an organic casing both at the outer and

inner surfaces of the silicified structures: one valve and several girdle bands (Fig. 1).

Oiatoms are subdivided into two main groups: pennate and centric, which are characterized

by their symmetry, but also by their sexual reproduction (Pickett-Heaps et al., 1990). The

girdle bands are often smooth and display little ornamentation . The valves of pennates

diatoms show a bilateral symmetry. They possess a silicified rib, the raphe, which ex tend

along the valve median. The centric diatoms are (tri or omni)radialy, or bipolary symmetric ,

and display a complex pattern, composed of ribs, marginal spines or processes, tubular

extensions.

Oepending of the species, the frustules are more or less silicified, and organic components are

complex : organic coat around the silica, organic matrix penetrating the silica, organic layer on

the cytoplasmic side of the wall in many diatoms (Schmid, 1994). In Cylindrothecafusiformis

for example, the valve area between the raphe and the girdle bands is totally unsilicified,

consisting of pure organic material (Kroger et al., 1994) (Fig. 2).

The valve formation has been studied in detail by man y workers (see reviews by Crawford,

1981, VoIcani, 1981, Pickett-Heaps et al., 1990, Gordon and Orum, 1994). Mitosis and

cytokinesis occur in the parental cel1 , with a coincident movement of the nucleus (Fig. 3 A

and B ) which migrates from the inside of the epivalve face to the midgirdle position during

the expansion of the protoplasm (Fig. 3C). At the same time new girdle bands are built,

attached to the parental hypovalve ; they protect the protoplasm during its expansion through

the cell cycle. Then the valves of the new cells are formed as hypolvalves, the parental

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valves becoming the new epivalves of the daughter cells (Fig. 3D). Deposition of the silicon

to build up the new valves occur in a large vesicle which extend during the silicification

processes : the silicon deposition vesicle (SDV) which appears beneath the plasmalemma.

Within that vesicle silica is polymerized and forms the pattern characteristic of the species.

Sorne authors have suggested that the membrane of the SDV, the silicalemma, is lost when the

valves reach maturity, but others consider that it is incorporated into the organic coating

(Crawford, 1981). When the new valves are formed each nucleus migrates again to the

epivalve face of the new protoplasms (Fig. 3D) and new organic material is deposited on the

inside and the outside of the new valve after the formation of the new hypovalve.

Ali the silicification processes in diatoms are closely linked to the cell division and growth

(Pickett-Heaps, 1991). Silicon deposition into the valves starts during the mitosis for centric

diatoms or G 1 phase for the pennate diatoms while additional and girdle bands can be added

during the Gl and G2 phases (Fig. 4) (Schmid, 1994).

From the external medium to the formation of the frustules silicification involved sequential

steps of soluble silicon pools (silicic acid : Si(OH)4 ; or the monosilicate anion: SiO(OH)3) in

interaction with the cell metabo1ism (Sullivan and Volcani, 1981 ; Sullivan, 1986). The rigid

valve is cornposed of hydrated, amorphous silica (Si02.H20)n, also called biogenic silica. The

silicic acid transport is made by a carrier-mediated system with Michaelis-Menten type

saturation kinetics. That transmembrane transport system is an energy dependent process

promoted by Na+ or K+, or both. After entering the cell, the regulation of Si metabolism is not

clearly defined (Fig. 5). Soluble Si pools are involved with chemical modification of the Si

species to polymerized forms or organosilicon compounds. The intracellular location of these

pools are not well known except the site of the SDY. Small vesicles, derived from Golgi

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apparatus or the endoplasmic reticulum, are probably involved in the Si transport, with

subsequent coalescence at the site of wall formation. Direct transport of silicon into the

silicalemma is also proposed (Pickett-Heaps et al., 1990 ; Lee and Li, 1992 ; Gordon and

Drum, 1994; Schmid, 1994). Moreover, sorne authors measured silicon into the mitochondria

and chloroplasts (Azam and Volcani, 1981 ; Sullivan and Volcani , 1981).

The regulation of each of these events is linked to the cell division, and lead to a precise

timing of the evolution of Si pools during the cell growth (Fig. 6). The soluble pools are

formed very rapidly, increasing during the first hours of the uptake phase. The complex

polymerization occurs later, leading to the diatom frustule building up which allows the

completion of daughter cells and their separation. Photosynthetic energy does not seem to be

required for these processes, which are rather c10sely linked to the energy provided by the

oxidative phosphorylation (Fig. 7), (Volcani, 1978 ; Sullivan and Volcani, 1981 , Sullivan

1986).

The biomineralization processes cannot occur without new organic synthesis, first to develop

the transport system, and then to encase the new valve into a complex organic coating

(Sullivan and Volcani, 1981, Sullivan 1986). Synthesis of proteins is increased at the

beginning of the silicification phase, and synthesis of carbohydrates at the end, after the valve

complet ion (Coombs and Volcani , 1968).

But the mineralization itself is probably a pure che mie al reaction, in which

autopolycondensation and colloidal assembly are not coupled to the cellular metabolism. This

hypothesis implies the filling of the SDV (and/or small vesicles) to sorne threshold level of

internai Si, allowing initiation of the polycondensation (Sullivan, 1986 ; Lobel et al. , 1996).

But the morphological design of the frustule may involved specifie control of the silicon

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deposition. It seems that the composition of the organic matrix of the valve, probably derived

from the silicalemma, could act as a template for that structure. Moreover, that organic casing,

enriched in specifie molecules, allows the first bonds with Si, and the further nucleation and

growth of the minerai part of the valve. Based on the organic content of the cells walls

(Table 1) of the diatoms analysed by Hecky et al. (1973), Lobel ~. (1996) have studied the

potential role of the proteic structure of the valve casing for silicon nucleation and

mineralization. Their model completes the first structural model proposed by Hecky et al.

(1973), (Fig. 8) and supports the proposition that the polymerization is mediated by a p-sheet

protein that contains hydroxyl-rich amino acids su ch as serine and threonine.

Data of Hecky et al. show also the enrichment of the proteic template of the cell walls with

glycine (Table 1). Both glycine and serine are metabolized through a specifie pathway of the

carbine metabolism: photorespiration. Furtherrnore, the glycolate pathway via serine and

glycine appears to be rudimentary in diatoms (Winkler and Stabenau, 1995) (Fig 9), when

compared to green algae (Raven and Beardall, 1981 ; Beardall , 1989; Beardall and Raven,

1990). Under growth conditions favouring the synthesis of glycolate, only the enzymes of

glycolate metabolism via malate were found to increase (Fig.9) (Winkler and Stabenau, 1995).

Therefore, the activation of serine and glycine metabolism must be specifie in diatoms. That

activation was shown in synchronized culture of Thalassiosira weissflogii (Martin-Jézéquel,

1992). During exponential growth, the free pools of serine and glycine decreased during the

cell division (8 to 12 hours). Under stationary phase, when diatom cells were not supplied

with silicon (Fig. 10), these pools still increased during the corresponding period of the cycle.

Thus, it appears in these preliminary experiments that the metabolism of these amino acids is

probably activated in relation to specifie periods of the cells cycle and silicon metabolism in

diatoms.

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3. Concluding remarks

Recent papers have reviewed the cell biology of diatom valve formation (Pickett-Heaps et al. ,

1990), diatom cell division (Pickett-Heaps 1991), and the chemical basis of diatom

morphogenesis (Gordon and Drum, 1994). But in spite of the extensive knowledge on these

subjects, a lot of questions are still opened : on the transport system and its regulation ; on the

silicalemma, its origin and control; on the valve morphology control. Also, the nature and the

level of the silicon free pool is not elucidated. A very recent study using molecular biology

has given preliminary answers on the proteic involvement during silicon uptake (Hildebrand

et al., 1993; 1997). In the future, combination of cellular biology, molecular biology and

biochemistry should allow the description of every step of the silicon metabolism in diatoms,

from the Si uptake to the valve completion

References

Azam, F. and Volcani, B .E., 1981. Germanium- silicon interactions in biological systems. In :

T.L. Simpson and B.E. Volcani (Editors), Silicon and siliceous structures in biological

systems. Springer Verlag, New York, pp 43-67.

Beardall, J., 1989. Photosynthesis and photorespiration in marine phytoplankton. Aquatic

Botany, 34 : 105-130

Beardall, J. and Raven, J .A., 1990. Pathways and mechanisms of respiration in microalgae. Marine

Microbial Food Webs, 4: 7-30

Coombs, J. and Volcani, B.E. , 1968. Studies on the Biochemistry and fine structure of silica shell

formation in diatoms. Chemical changes in the wall of Navicula pelliculosa during its

formation. Planta (Berl), 82 : 280-292.

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Crawford, R.M., 1981. The siliceous components of the diatom cell wall and their morphological

variation. In : T.L. Simpson and B.E. Vo\cani (Editors), Silicon and siliceous structures in

biological systems. Springer VerIag, New York, pp 129-156.

Darley, W.M., 1977. Biochemical composition .. In : . D. Werner (Editor), The biology of diatorns.

University of California Press. Botanical monograph, vol 13., pp 198-223

Gordon, R.and Drum R.W., 1994. The chemical basis of diatom morphogenesis. International

Reviewof Cytology, 150 : 243-372

Hecky, R.E., Mopper, K., Kilham, P. and Degens, E.T., 1973. The amine acids and sugar

composition of diatom cell-walls. Marine Biology, 19 : 323-331

Hildebrand, M., Higgins, D.R., Busser, K. and Volcani, B.E., 1993. Silicon-responsive cDNA

clones isolated from the marine diatom Cylindrothecafusifonnis. Gene, 132: 213-218

Hildebrand, M., Vo\cani, B.E., Gassman, W. and Schroeder, J.1. , 1997. A gene family of silicon

transporters. Nature, 385 : 688-689.

Kroger, N., Bergsdorf, C. and Sumper, M., 1994. A new calcium binding glycoprotein family

constitutes a major diatom cell wall component. The EMBO Journal, 13: 4676-4683

Lee, M. and Li, c.w., 1992. The origin of the silica deposition vesicle of diatoms. Botanical

Bulletin of Academy Sin., 33, 317-325

Lobel, K.D., West, J.K. and Hench, L.L., 1996. Computation al model for protein-mediated

biomineralization of the diatom frustule. Marine Biology, 126 : 353-360

Martin-Jézéquel, v., 1992. Effect of Si-status on diel variation of intracellular free amino acids in

Thalassiosira weissflogii under low-light intensity. In: T. Berman, H.J . Gons and L.R. Mur

(Editors), The daily growth cycle of Phytoplankton. Hydrobiologia, 238: 159-167.

Pickett-Heaps, J., 1991. Cell division in diatoms. International Review ofCytology, 128: 63-\08.

Pickett-Heaps, J., Schmid, A.M.M. and Edgar, L.A., 1990. The cell biology of diatom valve

formation. Progress in Phycological Research,7: 1-168.

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v

Si

Figure 1. A. Schematic representation of a cell wall of Navicula pel/iculosa .V :valve; G: girdle bands; R: raphe slit; P: pore; Si: silica shell . B. Portion of the valve showing the silica shell (Si) interlocked with the casing (C) (redrawn from Volcani, 1981).

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Œl V

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Figure 2. Schematic drawing of a diatom cell wall . A. Diatom cell wall in section: ET, epitheca; HT, hypotheca : V : valve; GB : girdle bands. B. Cross section through a cell wall of Cylindrotheca fusiformis (redrawn from Kr5ger et al. , 1994)

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A H E

B

c

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Figure 3. Schematic view of four stages of cell division. A. Non dividing cell with nucleus (N) beneath epivalve (E) face; H, hypovalve ; G, girdle band. B. Extension of protoplast and star! of hypocyngulum formation . Nucleus begins to migrate. C. Nucleus undergoes mitosis and protoplast invaginates. D. Cytokinesis complete and followed by new hypovalve formation. Nuclei begin to migrate back to epivalve (redrawn from Crawford, 1981)

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PENNATE

Figure 4. Silicon deposition phases during the cell cycle of diatoms. G 1 : biosynthetic phase, S : DNA replication ; G2 : biosynthetic phase; M : mitosis ; g (1 to z) : girdle bands; va : valve (redrawn from Schmid, (994)

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Figure 6. Kinetics of Si(OH)4 uptake, incorporation and soluble Si pool formation in Navicula pelliculosa, during synchronized division cycle (from Sullivan, 1979).

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~ RuP RuBP ... ~ GL YCOLATE

~ CALVIN Cycle ~ 02 ~ CO2

~ U Triose P PGA

~ Succinate GL YCOLA TE

~ r ~ ~ ~ Isocitrate _ .... _--I.~ Glyoxylate

C ..J ~ ~ Malate

Oxaloacetate " GLYCOLATE

~ 1 Malate ;:c: Citrate '\

~ ~ s= ~ '- Isocitrate U e ~ ~ ~ Succinate

~ Acetyl Glyoxylate CoA

"8 SERINE ~ GLYCINE

Figure 9. G1ycolate pathway in diatoms, according to the data obtained from three different organisms (Winkler and Stabenau, 1995)

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IO'--r---------,-----------,-------, LIGnT DARK

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Figure 10. Evolution of serine and glycine free pools, during the cell division of synchronized culture of Thalassiosira weisflogii. 1 and 2 : stationnary phase obtained by silicon limitation; 3 and 4: exponential phase (Martin-Jézéquel , 1992)

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