J. Appl. Glycosci.: Advance Publication doi:10.5458/jag.jag.JAG-2012_016 Review (Received October 24, 2012; Accepted December 3, 2012) J-STAGE Advance Published Date: January 25, 2013

Special Issue " Metabolism, Structure and Properties"

Review

Variation of Storage Polysaccharides in Phototrophic Microorganisms

Eiji Suzuki1,* and Ryuichiro Suzuki1

1Department of Biological Production, Faculty of Bioresource Sciences, Akita

Prefectural University (Akita 010-0195, Japan)

*Corresponding Author

(Tel.: +81-18-872-1653, Fax: +81-18-872-1681, E-mail: [email protected])

List of Abbreviations:

AGPase, ADP- pyrophosphorylase; DP, degree of polymerization; FACE, fluorophore-assisted carbohydrate electrophoresis; GBSS, granule-bound starch synthase; ISA, isoamylase; SS, starch synthase

Running Title: Starch in Various Phototrophic Organisms

J. Appl. Glycosci.: Advance Publication

Abstract

Phototrophic eukaryotes were established by the engulfment of oxygenic phototrophic prokaryotes () by a heterotrophic host. This process, called primary endosymbiosis, gave rise to the taxon , which comprises green , rhodophytes, and . Further rounds of endosymbiotic events produced a variety of phototrophic organisms, which could accumulate

-1,4-/-1,6- (including starch) or -1,3-/-1,6-glucans. In this article, we review the recent progress in the study of the intracellular localization and molecular forms of storage , especially starch, among the known phototrophs and related organisms.

Key words: Archaeplastida, cyanobacteria, endosymbiosis, semi-, starch

2 Suzuki et al.: Starch in Various Phototrophic Organisms

INTRODUCTION

Starch is produced and stored in of tissues ( in leaves and amyloplasts in seeds and tubers). It is widely accepted that the organelle was derived from an independent organism, which was closely related to the extant cyanobacteria, through an event known as endosymbiosis.1) During the course of evolution of photosynthetic organisms, the “primary” endosymbiosis gave rise to plastids in green plants (including land plants and ), , and glaucophytes,2) which are collectively designated as Archaeplastida.3,4) Moreover, some green and red algae were thought to be subjected to secondary endosymbiosis, where they were engulfed by heterotrophic eukaryotes, resulting in their diversification into euglenophytes and chlorarachniophytes, which acquired plastids from green algae, and stramenopiles, haptophytes, cryptophytes, and alveolates ( and apicomplexans), which acquired plastids from a red alga.2,5) These organisms have plastids enveloped by 3 or

4 membranes, which are the relics of multiple rounds of previous endosymbiotic events.6) Furthermore, some of these organisms subsequently lost their ability to perform , while they retained plant-like genes (Trypanosoma related to euglenophytes)7) or even the remnants of plastids ( protozoa related to dinoflagellates).8,9) The complex evolutionary histories of these organisms are reflected in the diversity of their metabolism. The molecular form of storage polysaccharides (- or -glucans) and their intracellular localization vary greatly depending on the lineage of the organism. In this mini-review, the variation in the forms of storage polysaccharide among photosynthetic organisms is summarized. The characteristics of these polysaccharides, especially starch-like -glucans in representative organisms from each lineage, as revealed by recent studies are discussed.

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The form of storage polysaccharide and the site of its accumulation in various phototrophic organisms.

1. Cyanobacteria.

In the bacterial domain, photosynthetic organisms are distributed in 6 phyla, but the members of only 1 phylum, cyanobacteria, are capable of oxygenic photosynthesis, which can be achieved by the operation of 2 distinct types of reaction centers (type

II/quinone type, and type I/FeS type).10) All the other photosynthetic have only

1 type of reaction center (either quinone- or FeS-type) and carry out anoxygenic

(non-oxygen-evolving) photosynthesis, which is confined to the bacterial domain.10)

Cyanobacteria are thus regarded as the progenitors of all photosynthetic eukaryotes.

In the cyanobacterial cells, photosynthetic carbon assimilation and accumulation of storage polysaccharide takes place in the cytoplasmic compartment (Fig. 1). While most species of cyanobacteria produce soluble , several unicellular, diazotrophic (nitrogen-fixing) species synthesize insoluble -glucan, which is closely analogous to amylopectin found in cereal plants.11-14) The latter polysaccharide was termed as semi-amylopectin, based on its lower content of long glucan chains, as compared to that of amylopectin.11) A few cyanobacterial species even contain in addition to semi-amylopectin.12,14) In the organisms containing semi-amylopectin

(plus amylose), the starch granules have been observed as electron-transparent particles in the cells.15-17) The occurrence of a starch-like substance in photosynthetic prokaryotes is highly intriguing in the light of evolution of starch metabolism.13,18,19)

2. Archaeplastida: organisms derived by primary endosymbiosis.

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In many cases (if not all), accumulation of storage polysaccharide in various algae appears to be associated, structurally and hence metabolically, with the distinct protein body in the , the pyrenoid,20,21) which is primarily composed of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) protein.22,23) In a model green alga Chlamydomonas reinhardtii, starch was observed as “the sheath” around the pyrenoid.20) Distribution of starch granules within the of green algae may also differ depending on the growth conditions. For example, starch granules were dispersed in the stroma of the chloroplast in Chlorella kessleri strain 11h grown with an elevated CO2 concentration (3%), while starch accumulated around the developed pyrenoid in the organism exposed to a lower CO2 concentration (0.04%, or

24) air-level). During the exposure to low CO2 concentration, relative amylose content increased from 9–14% to 28%.25) A study with Chloromonas lacking pyrenoids showed that several granules of starch were distributed in the stroma between the thylakoid membranes.26) Ostreococcus tauri is an ancient green alga belonging to

Prasinophyceae and is the smallest free-living eukaryote.27) The organism has only 1 chloroplast, apparently lacks pyrenoids and retains a single starch granule at the chloroplast center.28) During plastid division, the starch granule constricts in the middle and then partitions into 2 plastids that belong to the daughter cells.28)

Unlike in green plants, the storage polysaccharide in red algae and glaucophytes is accumulated in the (Fig. 1). The storage polysaccharide in the red algae is referred to as floridean starch, because the color of the iodine complex is not blue, but varies from deep violet to brown.29,30) As described below in detail, recent studies have shown that the polysaccharide species in red algae are diverse, ranging from glycogen to semi-amylopectin with or without amylose.31-35) In a unicellular red alga

Porphyridium cruentum, starch granules lacking a bounding membrane were

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distributed throughout the cytosol, while the pyrenoid was located at the center of the chloroplast.20) In another unicellular red alga, Rhodosorus marinus, the pyrenoid was found to be bounded by peripheral thylakoid and chloroplast envelope membranes, protruding into the cytosol, and was surrounded by starch granules.36) In the latter case, although starch granules and the pyrenoid seem to be physically associated, they are separated by multiple membranes.

Glaucophytes are a small group of freshwater microalgae retaining apparently ancient traits in their cellular structure, such as a peptidoglycan layer around the chloroplast (termed as “cyanelle”), and a carboxysome (also called central body) at the center of the cyanelle, both reminiscent of those observed in extant cyanobacteria.37,38)

In the cells of a model Cyanophora paradoxa, starch granules were observed in the cytosol, apart from the central body in the cyanelles.37,38)

As in cyanobacteria, storage-polysaccharide is biosynthesized in green plants by the

ADP-glucose pathway, which operates in the plastids,39) while the UDP-glucose pathway is functional in the cytosol of red algae and glaucophytes.40-42) However, in both of the green and the red lineages, the pathway of starch (-glucan) biosynthesis is composed of enzymes derived from the , as well as from the host.43)

Only in the green plants, it is postulated that the biosynthetic pathway for starch was transiently established in the cytosol and subsequently rewired into the plastids.12,19)

Although the accumulation of starch in the stroma of plastids is regarded as a common trait, it is rather exceptional among photosynthetic organisms exclusive to green plants

(Fig. 1).

3. Organisms with secondary plastids.

Among organisms with secondary plastids, starch-like -glucans have been found in

6 Suzuki et al.: Starch in Various Phototrophic Organisms

cryptophytes and alveolates (dinoflagellates and Apicomplexa protozoa), whose plastids are of red algal origin.

In cryptophytes, starch granules accumulate in the periplastidal (also spelled as periplastidial) compartment (corresponding to the cytosol of the red algal ) between 2 inner and 2 outer envelope membranes of the chloroplast

(Fig. 1).44) Some of the starch granules are closely associated with the pyrenoid protruding into the periplastidal compartment, even though starch and the pyrenoid are separated by 2 inner envelope membranes.44,45) Cryptophytes and chlorarachniophytes are known to retain a degenerated nucleus of the eukaryotic endosymbiont, termed as nucleomorph46,47) in the periplastidal compartment and are therefore intriguing models for investigating the mechanism of secondary endosymbiosis.

The predicted outcome of secondary endosymbiosis would be that the plastids should be surrounded by 4 membranes. Therefore, how or why dinoflagellates (as well as euglenophytes) have plastids bounded by 3 membranes, while apicomplexans, relatives of dinoflagellates, have plastids bounded by 4 membranes remains unresolved.5,6,21) Following secondary endosymbiosis, starch metabolism appears to be further relocated in dinoflagellates and apicomplexans. In a heterotrophic Crypthecodinium cohnii and an apicomplexan parasite Toxoplasma gondii, starch granules have been shown to be accumulated in the cytosol (Fig. 1).48)

Starch granules in cryptophytes and alveolates are synthesized by the UDP-glucose pathway, which originated from red algae.45,48,49)

In euglenophytes, chlorarachniophytes, stramenopiles, and haptophytes, the storage polysaccharide is -1,3-glucan (with or without -1,6 branching linkages), which is localized in vacuoles. The storage polysaccharide in euglenophytes is an unbranched

-1,3-glucan called paramylon,50) which forms highly crystalline, fibrillar granules

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bounded by a membrane in the cytosol (Fig. 1).51-54) It is not clear whether the membrane surrounding the paramylon granule constitutes an organelle, that is comparable to the vacuole. A study with 2 species of Chlorarachnion showed that

-1,3-glucan, with an approximate degree of polymerization (DP) of 23 was accumulated in a vacuole (Fig. 1), which encapsulated the pyrenoid protruding from the chloroplast.55) While there appeared to be a close association between the stored carbohydrate and the pyrenoid in Chlorarachnion, they were separated by 5 membranes (the vacuole membrane and 4 layers of the chloroplast envelope).55)

Stramenopiles (meaning hollow hairs attached to 1 of the 2 flagella) or heterokonts

(meaning 2 differently shaped flagella) are the most flourishing phototrophic eukaryotes on this planet,56) and include brown algae, diatoms, and other phototrophic and non-phototrophic microorganisms.3,4) Water-soluble -D-glucan in brown algal macrophytes (Laminaria and related species) is called laminaran (or laminarin57)), which was shown to comprise -1,3-linked chains with occasional -1,6-linked branches.58,59) Laminaran exhibited DP of approximately 25, and a substantial proportion of the molecules contained a mannitol moiety at their reducing end through

-1,1 linkage.58-61)

The storage polysaccharide in diatoms and haptophytes is chrysolaminaran

(originally termed as chrysolaminarin62)), despite a distant phylogenetic relationship between the 2 algal groups. The chrysolaminaran of diatoms consists of -1,3-linked glucan chains with the presence of -1,6-linked chains,58) or branching at C-2 and C-6 positions and DP values of 24–80.63,64) The storage glucan of the haptophyte,

Phaeocystis globosa was reported to consist of about 20 glucose residues, linked mainly by -1,3-glucosidic bonds, with branching at C-6 positions.65) In contrast, studies with 2 other haptophyte species showed that chrysolaminaran comprised

8 Suzuki et al.: Starch in Various Phototrophic Organisms

primarily -1,6-linked glucan with a smaller proportion of -1,3-linkages and had a mean DP of 106 (Emiliania huxleyi) or 203 (Pleurochrysis haptonemofera).66,67)

As described above, the molecular structure of laminaran and chrysolaminaran seems to be quite diverse and may differ depending on the source organisms. In addition, although it has been reported (in studies with a limited number of species) that chrysolaminaran is stored in vacuoles in diatoms68,69) and haptophytes70,71) (Fig. 1), extensive examination of the intracellular distribution of these -glucans remains to be carried out.

Variation in the properties of -glucans in phototrophic microorganisms.

Through the examination of 26 species of cyanobacteria, Nakamura et al.11) found that a few species accumulated a distinct -glucan (named as semi-amylopectin) instead of glycogen. The relative amount of long chains (DP ≥ 37) of -1,4-glucan in semi-amylopectin was 2–6% (depending on the species), which was an amount intermediate to that of amylopectin in rice endosperm (6.7%) and glycogen in cyanobacteria (<1%).11,14) The most abundant -1,4-glucan chains in semi-amylopectin were found to have DP of 11–12, which was similar to that of cereal amylopectin, while those in glycogen of cyanobacteria were in the range of DP 6–8.11,14) The molecular mass of semi-amylopectin was much greater than that of glycogen and virtually indistinguishable from that of amylopectin.11,12,14) The water-insoluble polysaccharide preparations purified from 3 strains of cyanobacteria exhibited thermal properties and crystallinity with A-type diffraction pattern (Table 1).14) Gel filtration analysis after debranching treatment indicated the presence of B2 chains in semi-amylopectin: evidence supporting that the polysaccharide has tandem-cluster

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structure.14) Furthermore, one of these strains, Cyanobacterium sp. CLg1 had amylose in addition to semi-amylopectin.12,14) Based on these properties, we propose that semi-amylopectin with or without amylose in cyanobacteria should be called as

“cyanobacterial starch.”

The chain length profile essential to distinguish semi-amylopectin from glycogen can be determined by the fluorophore-assisted carbohydrate electrophoresis (FACE) method.72) Since the method is applicable for the analysis of either purified polysaccharide granules or crude methanol extracts of the cells,73) it is suitable for examination of a small amount of material as well as a large number of samples simultaneously.

The unicellular green alga Chlamydomonas reinhardtii served as a model system to study of the mechanism of starch metabolism. Mutants with defects or alterations in starch metabolism were characterized, and the genetic loci encoding the responsible enzymes were identified, including STA1 (large subunit of ADP-glucose pyrophosphorylase, AGPase),74) STA2 (granule-bound starch synthase I, GBSSI),75)

STA3 (soluble starch synthase III, SS III),76,77) STA6 (small subunit of AGPase),78)

STA7 (isoamylase, ISA),79) STA8 (a polypeptide in multimeric ISA complex),80) and

STA11 (-1,4-glucanotransferase).81) Starch from the wild type and sta2 mutant of

Chlamydomonas displayed the A-type allomorph (Table 1): starch in sta3 and sta8 mutants exhibited B-type: and starch in sta2/sta3 double mutant showed C-type diffraction pattern, a mixture of A- and B-type.80,82) The ability of starch production was completely or partially lost in sta7 and sta8 mutants, respectively, and substituted by the accumulation of phytoglycogen.79,80) Analysis of purified starch granules from several green algae, including Chlorella vulgaris, Scenedesmus basilensis, and

Chlamydomonas sp., showed that all the starch preparations consisted of amylopectin

10 Suzuki et al.: Starch in Various Phototrophic Organisms

and amylose and displayed A-type diffraction pattern.83) The A-type pattern of the starch granules remained unchanged whether the Chlorella vulgaris cells were grown at 20°C, 30°C or 40°C.84) Examination of the genome sequence of a prasinophyte

Ostreococcus taurii indicated the presence of multiple forms of enzymes for each step of starch metabolism, including AGPase, SS, branching enzyme, and ISA.28) The metabolism of starch in green plants is therefore virtually complete in the very early stage of their evolution. The starch of O. taurii showed the B-type allomorph (Table

1).28)

Recent studies with unicellular red algae demonstrated a considerable variability in the physicochemical property of the storage -glucans in these organisms. Among species belonging to the most ancient , Cyanidium caldarium and

Galdieria sulphuraria produced glycogen, while the model rhodophyte

Cyanidioschyzon merolae synthesized semi-amylopectin and lacked amylose (Table

1).33-35) Four other species of Porphyridiales, Rhodosorus marinus, Porphyridium purpureum, P. sordidum, and Rhodella violacea contained semi-amylopectin plus amylose.33,34) The floridean starch of R. violacea displayed B-type allomorph, whereas that of the other unicellular species showed A-type diffraction pattern, with varying degrees of crystallinity (Table 1).33-35) These observations are in contrast to the findings of an earlier study, in which A-type floridean starch was not reported to be found.85) It has been reported that a red algal macrophyte, Gracilariopsis lemaneiformis, has floridean starch which consists of semi-amylopectin (inferred from the chain length profile), lacks amylose, and shows C-type diffraction pattern (Table

1).32) It should be noted that semi-amylopectin from any rhodophyte species so far examined by gel filtration invariably has demonstrated high molecular mass, comparable to that of amylopectin in green plants.33-35)

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Alpha-glucan-type storage polysaccharides have been characterized in 4 representative organisms, 1 from each of the 4 distinct taxa. A glaucophyte

Cyanophora paradoxa produces starch, which showes high amylose content (ranging from 30% to 60% depending on the growth conditions) and B-type diffraction pattern

(Table 1).42) Starch granules from a cryptophyte Guillardia theta and a heterotrophic dinoflagellate Crypthecodinium cohnii comprise amylopectin and amylose and display

A-type allomorph (Table 1).45,49) An apicomplexan protozoon Toxoplasma gondii accumulates starch which apparently lacks amylose, exhibits a monomodal profile of chain-length distribution, and B-type pattern (Table 1).48)

The advent and revolutionary improvement of novel sequencing technologies will further accelerate the elucidation of the genomic sequences and the overall metabolic system in the phototrophic microorganisms. The precise nature of the storage glucan, however, cannot be predicted from the genome-based enzyme repertoires. Thus, the physicochemical analyses of the purified polysaccharide will continue to be essential for the detailed characterization of the substance. Significant opportunities therefore remain to explore and manipulate these polysaccharide resources for future research and applications.

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Table 1. Property of -glucans in cyanobacteria, microalgae and related organisms.

Organism Type of -glucan Allomorph a Reference

Cyanobacteria

Synechococcus elongatus PCC 7942 b Glycogen n/a c 11),14),86)

Cyanobacterium sp. NBRC 102756 Semi-amylopectin A-type 11),14)

Cyanobacterium sp. CLg1 Semi-amylopectin + amylose A-type 12),14)

Cyanothece sp. ATCC 51142 Semi-amylopectin A-type 12),14)

Chlorophytes (green algae)

Ostreococcus tauri Amylopectin + amylose B-type 28)

Chlorella vulgaris Amylopectin + amylose A-type 83)

Chlamydomonas reinhardtii Amylopectin + amylose A-type 82)

Rhodophytes (red algae)

Cyanidium caldarium Glycogen n/a 33)

Galdieria sulphuraria Glycogen n/a 34)

Cyanidioschyzon merolae Semi-amylopectin A-type 35)

Rhodosorus marinus Semi-amylopectin + amylose weak A-type 34)

Porphyridium purupureum Semi-amylopectin + amylose A-type 33)

Porphyridium sordidum Semi-amylopectin + amylose weak A-type 34)

Rhodella violacea Semi-amylopectin + amylose B-type 34)

Gracilariopsis lemaneiformis Semi-amylopectin C-type 32)

Glaucophyte

Cyanophora paradoxa Amylopectin + amylose B-type 42)

Cryptophyte

Guillardia theta Amylopectin + amylose A-type 45)

Dinoflagellate

Crypthecodinium cohnii Amylopectin + amylose A-type 49)

Apicomplexa

Toxoplasma gondii Amylopectin B-type 48) a X-ray diffraction pattern b Shown as a representative of most cyanobacterial species c Not applicable

28 Suzuki et al.: Starch in Various Phototrophic Organisms

Figure Legends

Figure 1. Intracellular localization and the molecular form of storage polysaccharide in various phototrophic organisms.

Tabular (upper) and schematic (lower) representations are indicated together to assist comprehension of the variety of polysaccharide species and localization.

Upper panel: Green plants consist of chlorophytes (green algae) and land plants, and are shaded in green, while rhodophytes (red algae) are shaded in red. Organisms whose plastids are derived from a red alga (cryptophytes, dinoflagellates, stramenopiles, and haptophytes) or green algae (euglenophytes and chlorarachniophytes) are indicated as

“red” or “green,” respectively. As the storage carbohydrates, -glucans (comprising

-1,4 glucan chains with branches at -1,6 linkages) or -glucans (including -1,3 glucans with branches at -1,6 linkages or -1,6 glucans with branches at -1,3 linkages) are shaded in green or yellow, respectively.

Lower panel: Green and yellow ovals represent -1,4 (-1,6) and -1,3 (-1,6) glucans, respectively. Oval lines indicate lipid membranes. For cyanobacteria

(Gram-negative bacteria), plasma membrane and outer membrane are shown. For eukaryotic algae, plasma membrane, plastid envelope membranes, periplastidal endoplasmic reticulum membranes, and glucan storage vesicle or vacuole membrane are shown. Nucleus, mitochondria, nucleomorph, other organelles, thylakoid membranes, and pyrenoid within the plastids are not shown for simplicity. Red and green lines indicate that the plastids were derived from rhodophyte and chlorophyte algae through secondary endosymbiosis, respectively. For dinoflagellates and euglenophytes, it remains to be elucidated whether the intermediate membrane of the triple plastid envelope was derived from endosymbiont or host.

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Figure 1

30