Cellulose: Biogenesis and Degradation

Bruce Stone, La Trobe University, Bundoora, Victoria, Australia

Cellulose is synthesized by plasma membrane-bound cellulose synthase complexes found in bacteria, slime moulds, algae and embryophytes. Cellulases produced by many bacteria, protistans and fungi break down cellulose to soluble saccharides for use in growth and energy production. Plant pathogens use cellulases to gain access to the contents of plant cells by digesting their walls, and in embryophytes cellulases have a role in cell wall development and differentiation.

Secondary article
Article Contents
. Introduction . Biogenesis: Biochemistry, Molecular Genetics and Cytology . Enzymic Depolymerization

Introduction
Cellulose biogenesis by land plants and marine algae, from photosynthetically derived carbohydrate, occurs at the prodigious rate of 0.85 1011 tonnes per year. The cellulose so formed is turned over, in part by combustion, and more slowly by a select group of organisms possessing enzymes capable of its breakdown. and c-di-GMP, but has no absolute requirement for the nucleotide. In the slime mould Dictyostelium discoideum, synthase is stimulated by cellobiose, but not by c-di-GMP. Membrane preparations from embryophytes incorporate glucose from UDPGlc into (1!4)-b-glucans although at rates far below those in vivo. The same preparations also actively synthesize the (1!3)-b-glucan callose. The two synthases have been resolved from bean (Vigna radiata) membranes Kudlicka and Brown, 1997). The product of (1!4)-b-glucan synthase is microbrillar in form with a diraction pattern of the cellulose II allomorph. Genes concerned with cellulose production in A. xylinum (Ross et al., 1991) have been identied in an operon (acsA/bcsA). The acsA/bcsA gene encodes the UDPGlc-binding catalytic polypeptide and acsB/bcsB encodes a presumptive regulatory polypeptide that binds the activator c-di-GMP. Two others, acsC/bcsC and acsD/ bcsD, are involved in regulation of the cellulose synthase, and their expression is required for maximal cellulose synthesis. It has been suggested that the acsC/bcsC gene encodes a protein that putatively forms a pore in the outer membrane through which the nascent cellulose molecule is secreted. Mutants in acsD/BcsD form reduced amounts of cellulose in vivo, but at similar levels to the wild type in vitro. The cells of the mutant, when agitated, produce the cellulose II allomorph, whereas wild type cells under the same conditions synthesize exclusively cellulose I. This suggests that the acsD/bcsD encoded polypeptide is involved in crystallization. In Agrobacterium tumefaciens there are two operons required for cellulose production, celABC and celDE (Matthysse et al., 1995a, b). The products of the celABC operon are active in membrane fractions, whereas the products of the celDE operon are active in soluble fractions. The celA gene is believed to encode the cellulose synthase polypeptide. The celB, celD and celE gene products synthesize lipid-linked glucosyl intermediates, suggesting the existence of a signicantly dierent mechanism of cellulose synthesis from that present in A. xylinum.
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Biogenesis: Biochemistry, Molecular Genetics and Cytology

Biochemistry and molecular genetics
Cellulose synthases (EC 2.4.1.12) whether from bacterial, protistan, cellular slime mould or embryophyte sources are associated with cell surface membranes. Generally, the synthases use nucleoside diphospho-monosaccharides, commonly uridine diphospho-a-d -glucose (UDPaGlc), directly as the glucosyl donor in a transglycosylation reaction in which the a-anomeric conguration of the glucose in the donor is inverted in the product (eqn [1]).
UDPaGlc bGlcn UDP bGlcn1 1     glycosyl glycosyl donor acceptor where bGlc(n) 5 (1!4)-b-glucan molecule. The synthase acts by repetitively attaching glucosyl residues to the end of the growing cellulose molecule. How chain growth is initiated or terminated is not understood. Suitably stabilized membrane preparations of Acetobacter xylinum are capable of incorporating glucose from UDPGlc into cellulose at a rate comparable with that found in living cells. Mg2 1 ions are required, and there is an absolute requirement for the cofactor, (3,5)-cyclic diguanylic acid (c-di-GMP), that binds and activates a synthase-activating protein. In the water mould Saprolegnia monoica, cellulose synthase is activated by cellobiose

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Cellulose: Biogenesis and Degradation

However, the presumptive synthase polypeptides show high sequence homology with the A. xylinum synthase. Genes encoding cellulose synthase (CesA) catalytic polypeptides have been cloned from cottonseed hairs, and an homologous gene has been identied in cellulosedecient mutants of Arabidopsis thaliana (Richmond and Somerville, 2000). In addition, cellulose synthase-like (Csl) homologues are present in A. thaliana, poplar (Populus alba P. tremula hybrid) and rice (Oryza sativa) (Richmond and Somerville, 2000) (Figure 1). The cellulose synthases belong to a family of repetitive glycosyl transferases with sequence and structural characteristics showing homology with synthases for the bacterial (1!3)-b-glucan (curdlan), hyaluronan, and the NodC chitin oligosaccharide. They are classied in Family 2 and are among the 43 families of glycosyl transferases (Coutinho and Henrissat, 1999). Based on predicted protein sequences, the embryophyte CesA-like sequences in the Arabidopsis database have been grouped into 7 subfamilies: the CesA sub-family and six sub-families of structurally related, cellulose synthase-like (Csl) genes of unknown function. The relationships between members of these sub-families (that together form the CesA superfamily) are shown in the unrooted tree in Figure 1 (Richmond and Somerville, 2000). It is possible that synthases for non-cellulosic wall polysaccharides such as the heteroglucans, heteroxylans, mannans and galactans

are represented in the Csl gene families, but functional relationships are yet to be established. All cellulose synthases are integral plasma membrane proteins, and consistent with this, topological modelling shows that they all have multiple transmembrane domains. The embryophyte synthases are predicted to have three to six transmembrane domains in the carboxy terminus of the protein and one or two in the amino terminal region. These may form a pore as shown in the hypothetical topological model of a CesA protein in an embryophyte plasma membrane (Figure 2). In addition to having domains homologous to the prokaryote cellulose synthases, the embryophyte cellulose synthases have embryophyte-specic, conserved regions separating these domains from one another, as well as two embryophyte specic hypervariable regions (Figures 2 and 3). The function of these domains is unknown but the conserved domains may code for portions of the synthase protein that interact with other polypeptides in the cellulose synthase complex to form the intramembranous particles making up rosettes (see Cytology) or with ancillary cytoskeletal proteins. All CesA family members
Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc C-ter HVR N-ter CR-P Zn2+ Zn2+ D DD

AtCesA7 AtCesA5 OsCesA01 AtCesA6 AtCslD2 AtCesA2 AtCesA1 OsCslD2 AtCslD3 AtCesA10 AtCesA9 OsCslD1 AtCesA3 AtCslD4 GhCesA3 OsCesA07 AtCslD1 AtCesA4 GhCesA2 AtCslD5 Pt/PaCesA01 AtCesA8 PtCesA02 AtCslC8 GhCesA1 AtCslC5 LeCslC1 AtCslC4 AtCslE1

Plasma membrane

QXXRW Active site UDP-Glc HVR

0.1

AtCslG3

AtCslC12 AtCslC6 MtCslA3 AtCslA2 OsCslA1 AtCslA9 AtCslA7 AtCslA14 AtCslA3 AtCslB2 AtCslB3 AtCslB4 AtCslB5 AtCslB6 AtCslG1 AtCslG2 AtCslB1 OsCslA2

CR-P

AtCslA1 AtCslA15 AtCslA10 AtCslA11

Figure 1 Unrooted tree of the CesA superfamily. The tree was constructed on the basis of alignment of full-length protein sequences of Arabidopsis and cotton CesA polypeptides with available protein sequences in the database. Forty one CesA-like sequences were revealed. Subfamilies are boxed. At, Arabidopsis; Gh, cotton; Le, tomato; Mt, Medicago trunculata; Os, rice; ble Pt, Populus tremuloides; Pt/Pa, Populus tremula Populus alba. Reproduced from Richmond TA and Somerville CR (2000).

Figure 2 Hypothetical topology model of a CesA protein subunit in an embryophyte plasma membrane. The eight transmembrane helices are predicted to interact to form a pore through which the nascent cellulose molecule is secreted into the wall. The large central domain protrudes from the cytoplasmic face of the plasma membrane and would be folded in such a way as to bring together the conserved central regions containing the three Asp residues and the QXXRW motif that are believed to be important for catalysis and the repetitive elongation of the growing cellulose molecule, respectively. The two hypervariable (HVR) and two conserved (CR-P) embryophyte-specific regions are shown. Zn2+ represents a zinc finger domain. Reproduced from Delmer DP (2000).

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Cellulose: Biogenesis and Degradation

Zinc fingers

D QXXRW Plant CesAs

O H

HO

H-1

H-2

H-3

Plant ancestor of CesAs Csl genes of plants Bacterial CesAs

O O M O
2

O O

Homologous regions Plant-specific and conserved (CR-P) Plant-specific hypervariable (HVR) No obvious conservation
Figure 3 Domain structures of CesA related proteins showing regions homologous in prokaryote and eukaryote cellulose synthase proteins with their conserved aspartate and QXXRW motifs. The embryophyte-specific and conserved domains and the embryophyte hypervariable regions are shown. Reproduced from Delmer DP (2000).

NDP

NDP

Figure 4 Possible catalytic mechanism for an inverting, NDP-dependent glycosyl transferase. The leaving group (-O-NDP) departure is assisted by an aspartate residue in the catalytic site (not shown). A second aspartate acts as the general base, by abstracting a proton from the terminal glycosyl residue of the nascent polysaccharide chain (H-O-R). Reproduced from Charnock SJ and Davies GJ (1999).

have Zn nger domains in their N-terminal region. These domains are known to mediate proteinprotein interactions. This extension is absent in Csl encoded proteins (Richmond and Somerville, 2000). All cellulose synthase topological models show a large extramembranous domain, probably facing the cytoplasm (Figure 2). This domain bears a number of well-conserved motifs that are putatively involved in UDPGlc-binding and in the repetitive action of the synthases. Conserved aspartic acid residues are probably involved in metal ion and nucleotide binding, and in the acid-base mediated transglycosylation reaction mechanism. This is supported by evidence from the crystal structure of Bacillus subtilis SpsA protein, in association with UDP-Mg2+. This Family 2 inverting synthase catalyses a reaction in the formation of a bacterial cell wall polysaccharide (Charnock and Davies, 1999). A mechanistic scheme for the transglycosylation reaction is shown in Figure 4. Maize and Arabidopsis cellulose synthases are found to be expressed in unique cell types engaged in either primary or secondary cell wall synthesis (Holland et al., 2000).

Cytology of cellulose deposition

The polymerization of the (1!4)-b-glucan chains is the rst step in cellulose biogenesis; in the second step, cellulose chains are crystallized into microbrils. In A. xylinum, freeze-fracture images of the inner cell membrane reveal a linear array of intramembranous particles (IMPs) that appear to feed growing cellulose chains through an extrusion pore in the outer membrane. The IMPs traverse the inner membrane, periplasmic space and the outer membrane. The externalized cellulose chains associate

(crystallize) into 2-nm microbrils which further aggregate as bundles (ribbons) of 10100 microbrils. When Calcouor White, a uorescent dye that is known to associate with the surface of cellulose microbrils, is present during cellulose synthesis, ordered but not crystalline cellulose is produced in place of the normal bundles. Washing the dye from the cellulose allows formation of cellulose I microbrils. The rate of cellulose synthesis is increased 4-fold in the presence of Calcouor, suggesting that crystallization is rate-limiting. The carboxymethyl ether of cellulose (CMcellulose) also increases the rate of synthesis, possibly by preventing microbril bundling (Haigler, 1991). Freeze-fracture images of plasma membranes from algal, slime mould and embryophyte cells producing cellulose microbrils also reveal aggregates of IMPs. These are designated terminal complexes (TC) to reect their presence at the end of microbril impressions. In some red and brown algae and in the tunicate Metandrocarpa uedai, the TCs, as in A. xylinum, consist of single linear arrays of particles. In the chlorococcalean green algae Oocystis and Glaucocystis, the siphonocladalean Valonia, and the slime mould, Dictyostelium, the TCs comprise characteristic multiple transverse or diagonal rows of particles, whereas in some red and brown algae, in certain green algae (Micrasterias and Nitella) and in all embryophytes, the particles in the TCs form rosettes. The number of particles in the rosettes is four in some red algae, ve in some brown algae and six in the zygnematalean and charalean green algae and embryophytes. In Coleochaete, a green alga believed to be an ancestor of land plants, the rosettes have eight particles. The IMPs are believed to contain multiple cellulose synthases and the size and arrangement of the particles in the TC appears to determine the overall size and shape of the microbrils produced. Also, those algae with linear TCs produce the most highly crystalline microbrils. Organisms with linear TCs produce a higher proportion of cellulose Ia whereas those with rosettes produce more cellulose Ib.
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Cellulose: Biogenesis and Degradation

In algae and embryophytes, the complexes are an integral part of the plasma membrane and are believed to move in the plane of the membrane as the cellulose is synthesized. The direction of movement of the TC determines the pattern of microbril deposition. In embryophytes, this direction is often controlled by plasma membrane-bound cortical microtubules; in algae, the formation of parallel bands of brils appears to depend on other mechanisms. Cellulose production in embryophytes is inhibited by the herbicides 2,6-dichlorobenzonitrile (DCB), isoxaben, phthoxazolin, triazofenamide and a thiatriazine derivative. Application of the thiatriazine derivative causes rosettes to fall apart, and in Arabidopsis causes the accumulation of non-crystalline glucan, part of which may be linked to protein. DCB prevents deposition of cellulose on tobacco protoplasts and in the developing cell plate at the stage after its fusion with the parent cell wall. In suspension-cultured cells of tomato and tobacco that have been adapted to DCB, cellulose (and heteroglucan) deposition is inhibited. The mechanism of the action of DCB is, however, dierent from the thiatriazine derivative, since it does not cause the accumulation of non-crystalline (1!4)-b-glucan (Delmer, 1999).

Enzymic Depolymerization
Cellulose molecules are cleaved by specic glucoside hydrolysing enzymes that are conventionally classied on the basis of their action pattern as (1!4)-b-glucan exo-or endohydrolases. The endohydrolases (EC 3.2.1.4) act by preferentially cleaving internal linkages in (1!4)-b-glucan chains, ultimately producing a mixture of short cellooligosaccharides. The exohydrolases generally act from the nonreducing end of the chain, releasing either glucose or cellobiose. A third class of hydrolases, the b-glucosidases (EC 3.2.1.21), cleave glucose residues from the nonreducing ends of cello-oligosaccharides. The action of endocellulases creates new chain ends for attack by exohydrolases and in the presence of b-glucosidases this synergy results in the complete digestion of cellulose chains to glucose. Depending on the origin of the cellulase, the hydrolysis of the glycosidic linkages in cellulose proceeds with either retention or inversion of conguration at the newly exposed anomeric centre in the products. In each case the catalytic cleavage of the glycosidic linkage involves an acidbase mechanism requiring principally two carboxyl groups located at the active site, but following dierent reaction pathways (Davies and Henrissat, 1995). A large number of cellulases have been isolated, puried, and their cDNAs cloned. In some cases they have been crystallized, allowing their three-dimensional structures to be deduced from X-ray diraction patterns. Cellulases can
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be classied into a number of families on the basis of the folding patterns of their polypeptide chains. Their catalytic domains are typically globular with the substrate-binding site and catalytic residues situated in shallow grooves or troughs or in deep clefts or tunnels, allowing binding anywhere along the cellulose chain (Davies and Henrissat, 1995). The tunnel-type active site of the cellobiohydrolase 1 (CBH1), from soil fungus Trichoderma reesei, is shown in Figure 5a. Many (1!4)-b-glucan endohydrolases readily depolymerize swollen cellulose or soluble cellulose derivatives such as CM-cellulose, but not crystalline cellulose. On the other hand, the cellobio-exohydrolases and some (1!4)-bglucan endohydrolases are also able to depolymerize crystalline cellulose. These cellulases have a catalytic domain connected by a linker peptide to a functionally independent carbohydrate-binding module (Coutinho and Henrissat, 1999) known as the cellulose-binding domain (CBD), which associates tightly, but not irreversibly, with cellulose microbril surfaces. The CBD anchors the associated catalytic domain to the solid substrate and so maintains a high local concentration of the enzyme (Figure 5b). In the anaerobic, mesophilic bacteria Clostridium thermocellum and C. cellulolyticum, cellulases that actively depolymerize crystalline cellulose are organized into a high-molecular-weight (2.02.5 106 Da) complex, the cellulosome, that is anchored to the cell surface (Bayer et al., 1998). Cellulosomes are also found in anaerobic rumen bacteria, several anaerobic pseudofungi and one aerobic bacterium. The core of the cellulosome is a multidomain, nonenzymic scaolding protein (210 kDa in C. thermocellum) that binds eight endocellulases through the interaction of domains on the scaold protein with noncatalytic, binding domains on the cellulases. Cellulose arising from plant debris in soil or water is digested extracellularly by bacterial and fungal cellulases to provide a source of assimilable carbohydrate for growth and energy production. In the specialized digestive tracts of ruminants, cellulose is digested by commensal anaerobic bacteria, protozoa and fungi and the hydrolysis products fermented to volatile fatty acids, methane and carbon dioxide. The fatty acids are absorbed by the rumen epithelium and transferred to the bloodstream for use as energy sources. In the digestive tracts of herbivorous mammals, birds, reptiles and some insects (termites, cockroaches), cellulose digestion and fermentation is also largely achieved by commensal bacteria and/or protozoa. Limited bacterial digestion of cellulose occurs in the colons of humans and other monogastric animals. Autocthonous cellulases have been reported from microbe-free insects (silversh, silkworms, cockroaches, termites), molluscs, earthworms and nematodes. cDNAs for (1!4)-b-endoglucanases have been cloned from the oesophageal glands of two nematodes and their high sequence identity with (1!4)-b-endoglucanases from several bacteria suggests

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Cellulose: Biogenesis and Degradation

Figure 5 (a) Schematic representation of Trichoderma reesei cellobiohydrolase 1(CBH1) catalytic domain with a cello-nonosaccharide (reducing end on the right) bound in the tunnel-shaped active site. The catalytic pair of glutamic acid residues (not shown) lie above and below the site of chain cleavage of the substrate chain between the second and third residues from the reducing end. The secondary-structure elements are coloured as follows: b strands, blue arrows; a helices, red spirals; loop regions, yellow coils. The cello-oligosaccharide is shown in pink as the ball-and-stick object. Reproduced from Divne et al. 1998. (b) Model of intact cellobiohydrolase 1 (CBH1) from Trichoderma reesei bound to a cellulose microfibril surface. The model is based on the crystal structure of CBH1 in complex with cellohexaose, the NMR structure of the cellulose binding domain (CBD) and a modelled linker region. CBH1 is believed to act repetitively from the reducing end towards the non-reducing end of the cellulose chain. Reproduced from Munoz, et al., 1998.

that the genes may have been acquired by horizontal transfer from a prokaryote (Smant et al., 1998). Bacterial, fungal and nematode pathogens use cellulases and other polysaccharide-degrading enzymes to gain access to plant cells by digesting their walls. In embryophytes, (1!4)-b-glucan endohydrolases, that hydrolyse CM-cellulose, but not crystalline cellulose, are found associated within specic cell structures in dierentiating laticifers, aerenchyma, root nodules, xylem and phloem, in lateral root initiation, and in tissues undergoing cell division and expansion. The endocellulases are also found in senescent tissues, e.g. in abscission zones of leaves, owers, pods and fruits, dehiscence zones of fruits,

softening fruits (avocado, Persea americana; raspberry, Rubus ideaus) and in apical buds. Their physiological substrates may be non-crystalline and/or cellulose-like wall polysaccharides such as the heteroglucans and (1!3;1!4)-b-glucans (Brummell et al., 1994). An Arabidopsis gene encoding a membrane-anchored member of the endo-cellulase family has been shown to have a central role in the assembly of the cellulose-noncellulosic polysaccharide network in expanding cell walls (Nicol et al., 1998). Genes encoding endo-cellulases have also been detected in A. xylinum and A. tumefaciens. The specic functions of the endo-cellulases are unknown but they may have an editing role in cellulose synthesis.
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Cellulose: Biogenesis and Degradation

References
Bayer EA, Chanzy H, Lamed R and Shoham Y (1998) Cellulose, cellulases and cellulosomes. Current Opinion in Structural Biology 8:548-557. Brummell DA, Lashbrook CC and Bennett A (1994) Plant endo-1,4-a -d glucanases. Structure, properties, and physiological function. American Chemical Society Symposium Series 566: 100129. Charnock SJ and Davies GJ (1999) Structure of the nucleotidediphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry 38: 6380-6385. Coutinho PM and Henrissat B (1999) Carbohydrate-active enzymes server at URL: [http://afmb.cnrs-mrs.fr/ $ pedro/CAZY/db.html] Davies G and Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3: 853859. Delmer DP (2000) Cellulose biosynthesis: Exciting times for a dicult eld of study. Annual Review of Plant Physiology and Plant Molecular Biology 50: 245276. Divne C, Stahlberg J, Teeri TT and Jones TA (1998) High-resolution long crystal structures reveal how a cellulose chain is bound in the 50 A tunnel of cellobiohydrolase I from Trichoderma reesei. Journal of Molecular Biology 275: 309325. Haigler CH (1991) Relationship between polymerization and crystallization in microbril biogenesis. In: Haigler CH and Weimer PJ (eds) Biosynthesis and Biodegradation of Cellulose, pp. 99124. New York: Dekker. Holland N, Holland D, Helentjaris T et al. (2000) A comparative analysis of the plant cellulose synthase (CesA) gene family. Plant Physiology 123: 13131323. Kudlicka K and Brown RM Jr (1997) Cellulose and callose biosynthesis in higher plants. I. Solubilization and separation of (1!3)-and (1!4)b-glucan synthase activities from mung bean. Plant Physiology 115: 643656. Matthysse AG, Thomas DL and White AR (1995a) Mechanism of cellulose synthesis in Agrobacterium tumefaciens. Journal of Bacteriology 177: 10761081.

Matthysse AG, White S and Lightfoot R (1995b) Genes required for cellulose synthesis in Agrobacterium tumefaciens. Journal of Bacteriology 177: 10691075. Munoz IG, Stahlberg J, Divne C et al. (1999) 3rd Carbohydrate Bioengineering Conference. Newcastle-upon-Tyne p 4.7. Nicol F, His I, Jauneau A et al. (1998) A plasma membrane-bound putative endo-1,4-a -d -glucanase is required for normal wall assembly and cell elongation in Arabidopsis. EMBO Journal 17: 55635576. Richmond TA and Somerville CR (2000) The cellulose synthase superfamily. Plant Physiology 124: 495498. Ross P, Meyer R and Benziman M (1991) Cellulose biosynthesis and function in bacteria. Microbiological Reviews 55: 3558. Smant G, Stokkermans JPWG, Yan Y et al. (1998) Endogenous cellulases in animals: Isolation of b-1,4-endoglucanase genes from two species of plant-parasitic nematodes. Proceedings of the National Academy of Sciences of the USA 95: 49064911.

Further Reading
Delmer DP (2000) Structure and biosynthesis of cellulose. Part II: Biosynthesis. In: S-D Kung and S-F Yang (eds) The Discoveries in Plant Biology Series, Vol. III., Hong Kong: World Scientic Publishing Co, pp. 199216. Emons AMC (1991) Role of particle rosettes and terminal globules in cellulose synthesis. In: Haigler CH and Weimer PJ (eds) Biosynthesis and Biodegradation of Cellulose, pp. 523. New York: Dekker. Fry SC (1995) Polysaccharide-modifying enzymes in the plant cell. Annual Review of Plant Physiology and Plant Molecular Biology 46: 497520. Okuda K and Mizuta S (1993) Diversity and evolution of putative cellulose-synthesizing enzyme complexes in green plants. Japanese Journal of Phycology 41: 151173.

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