Download Coupling of Silicon, Carbon and Nitrogen Metabolisms in Marine

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
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 .H 20)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
69
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.
71
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.
72
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).
74
Œl
V
ET
,
--- --
-GB
-
-- -
HT
V
RAPHE
RAPHE
-
silicified structures
c:=J
unsilicified/ organic structures
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)
75
A
E
H
B
c
D
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)
76
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)
77
-...J
00
1
K2
-VESICLES OF
TRANSPORT
-ENDOPLASMIC
RETICULUM
- GOLGI
Solubles Pools
silicic acid &
derivatives
6 fm Si/ceU
Figure 5. Proposed representation of sequential chemical transformation during silicic acid
metabolism in Navicula saprophilia (from Sullivan, 1986)
Si(OH)4
1 -500 J.lmol/litre
Ki'
SILICON
DEPOSITION
VESICLE (SDV)
Biogenic Silica
(Opal)
46 fm Si/cell
80
CELLS
•
- 1.60
':'
- 1.40
'"Q
0
70
ri:)
- 1.00
u
1
,
........
r:.l
- 1.20
1
1
,
5
....
1
60
0
- 0.80
,
1
'"~
~
,,
50
,
1
S
PCA Insoluble
S
p- -0- -
iJi
ri 40
Q
S
_0 - - 0- -
=
/
o
6
0'
_ -0 - -O-
//
/
c!
_0---0- '
ç:5
30
/
Hp Insoluble
p
p
20
Hp Soluble
10
PCA Soluble
0
0
1
1
1
1
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
10
11
HOURS
Figure 6. Kinetics of Si(OH)4 uptake, incorporation and soluble Si pool formation in Navicula
pelliculosa, during synchronized division cycle (from Sullivan, 1979).
79
o
00
Cbloropbyll
Lipids
Carbobydrates
Protéins
ADN
Orgaoic carbon
Figure 7. Changes in cellular composition, photosynthesis and respiration in Navicula
pelliculosa, during exponentiaI growth, Si(OH)4 starvation, and cel! separation
(Volcani, 1978)
Photosyntbesis (COz)
Photosyntbesis (Oz)
Phosphate uptake
ATP/cell
Respiration
Silicon uptake
% complete pairs
% binucleate cells
% single cells
cell number
~
RuP
~
RuBP ...
CALVIN Cycle
~
Triose P
~
~
Succinate
~
~
U
e
~
GL YCOLATE
~
r
Isocitrate _ ...._--I.~ Glyoxylate
~
~s=
02
PGA
C
~
~;:c:
GL YCOLATE
CO 2
~
U
~
~
~
Malate
..J
GLYCOLATE
Oxaloacetate "
1
Citrate
~
Malate
'\
'- Isocitrate
~
~
Succinate
~
Glyoxylate
Acetyl
CoA
"8
SERINE
~
GLYCINE
Figure 9. G1ycolate pathway in diatoms, according to the data obtained from three different
organisms (Winkler and Stabenau, 1995)
82
IO' - - r - - - - - - - - - , - - - - - - - - - - - , - - - - - - - ,
DARK
LIGnT
1&2
3&4
1' -" .
IO'+-__r--r-,-,-__r_---r-.-,~-,--J.__,____r~,..___,___r--r-r+-__r____r--t
o
2
4
6
8
10
12
14
16
18
20
22
24 26
28
30
TIMEh ..urs
SER
e- - - eGLY
[}-{J
1&2
T
8
10
12
14
16
18
20
o------a
22
24
26
3&4
DARK
LIGHT
12
30
SER
...- - - eGLY
10
28
14
16
lB
20
22
24
26
28
30
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)
83