The evolution of glycogen and starch metabolism in eukaryotes gives molecular clues to understand the establishment of plastid endosymbiosis

Solid semi-crystalline starch and hydrosoluble glycogen define two distinct physical states of the same type of storage polysaccharide. Appearance of semi-crystalline storage polysaccharides appears linked to the requirement of unicellular diazotrophic cyanobacteria to fuel nitrogenase and protect it from oxygen through respiration of vast amounts of stored carbon. Starch metabolism itself resulted from the merging of the bacterial and eukaryote pathways of storage polysaccharide metabolism after endosymbiosis of the plastid. This generated the three Archaeplastida lineages: the green algae and land plants (Chloroplastida), the red algae (Rhodophyceae), and the glaucophytes (Glaucophyta). Reconstruction of starch metabolism in the common ancestor of Archaeplastida suggests that polysaccharide synthesis was ancestrally cytosolic. In addition, the synthesis of cytosolic starch from the ADP-glucose exported from the cyanobacterial symbiont possibly defined the original metabolic flux by which the cyanobiont provided photosynthate to its host. Additional evidence supporting this scenario include the monophyletic origin of the major carbon translocators of the inner membrane of eukaryote plastids which are sisters to nucleotide-sugar transporters of the eukaryote endomembrane system. It also includes the extent of enzyme subfunctionalization that came as a consequence of the rewiring of this pathway to the chloroplasts in the green algae. Recent evidence suggests that, at the time of endosymbiosis, obligate intracellular energy parasites related to extant Chlamydia have donated important genes to the ancestral starch metabolism network.     

Early gene duplication within chloroplastida and its correspondence with relocation of starch metabolism to chloroplasts

The endosymbiosis event resulting in the plastid of photosynthetic eukaryotes was accompanied by the appearance of a novel form of storage polysaccharide in Rhodophyceae, Glaucophyta, and Chloroplastida. Previous analyses indicated that starch synthesis resulted from the merging of the cyanobacterial and the eukaryotic storage polysaccharide metabolism pathways. We performed a comparative bioinformatic analysis of six algal genome sequences to investigate this merger. Specifically, we analyzed two Chlorophyceae, Chlamydomonas reinhardtii and Volvox carterii, and four Prasinophytae, two Ostreococcus strains and two Micromonas pusilla strains. Our analyses revealed a complex metabolic pathway whose intricacies and function seem conserved throughout the green lineage. Comparison of this pathway to that recently proposed for the Rhodophyceae suggests that the complexity that we observed is unique to the green lineage and was generated when the latter diverged from the red algae. This finding corresponds well with the plastidial location of starch metabolism in Chloroplastidae. In contrast, Rhodophyceae and Glaucophyta produce and store starch in the cytoplasm and have a lower complexity pathway. Cytoplasmic starch synthesis is currently hypothesized to represent the ancestral state of storage polysaccharide metabolism in Archaeplastida. The retargeting of components of the cytoplasmic pathway to plastids likely required a complex stepwise process involving several rounds of gene duplications. We propose that this relocation of glucan synthesis to the plastid facilitated evolution of chlorophyll-containing light-harvesting complex antennae by playing a protective role within the chloroplast.     

The guanine nucleotide exchange factors Sec2 and PRONE: candidate synapomorphies for the Opisthokonta and the Archaeplastida

Although recent multigene phylogenetic analyses support close relationship of Metazoa and Fungi (the eukaryotic supergroup Opisthokonta) and monophyly of eukaryotes with the primary plastid, that is, Chloroplastida, Rhodophyta, and Glaucophyta (the supergroup Archaeplastida or Plantae), some authors still challenge this scheme. I found that 2 particular types of guanine nucleotide exchange factors (GEFs, i.e., cofactors of GTPases) might provide a new piece of evidence to resolve this controversy. An exhaustive analysis of available sequence data revealed that Sec2-related proteins, known to serve as GEF for exocytic GTPases of the Rab8/Sec4 subfamily, are restricted to opisthokonts, whereas proteins with the PRONE domain, recently described as novel plant-specific GEFs for RHO family GTPases, occur only in Chloroplastida and Rhodophyta. The results thus point to possible evolutionary innovations in the exocytic apparatus of the ancestral opisthokonts and reveal the probably first plastid-independent trait (i.e., a unique mode of RHO GTPase regulation) exclusive for Chloroplastida + Rhodophyta, further supporting monophyly of these 2 groups.     

Wide genetic diversity of picoplanktonic green algae (Chloroplastida) in the Mediterranean Sea uncovered by a phylum-biased PCR approach

The genetic diversity of picoplanktonic (i.e. cells that can pass through a 3 μm pore-size filter) green algae was investigated in the Mediterranean Sea in late summer by a culture-independent approach. Genetic libraries of the 18S rRNA gene were constructed using two different primer sets. The first set is commonly used to amplify the majority of eukaryotic lineages, while the second was composed of a general eukaryotic forward primer and a reverse primer biased towards the phylum Chloroplastida. A total of 3980 partial environmental sequences were obtained: 1668 using the general eukaryotic primer set and 2312 using the Chloroplastida-biased primer set. Of these sequences, 65 (4%) and 594 (26%) belonged to the Chloroplastida respectively. A 99.5% sequence similarity cut-off value allowed classification of these 659 Chloroplastida sequences into 74 different operational taxonomic units. A majority of the Chloroplastida sequences (99%) belonged to the prasinophytes. In addition to the seven independent prasinophyte lineages previously described, we discovered two new clades (clades VIII and IX), as well as a significant genetic diversity at the species and subspecies levels, notably among the genera Crustomastix , Dolichomastix and Mamiella (Mamiellales), but also within Pyramimonas and Halosphaera (Pyramimonadales). Such diversity within prasinophytes has not previously been observed by cloning approaches, illustrating the power of using targeted primers for clone library construction. Prasinophyte assemblages differed especially in relation to nutrient levels. Micromonas and Ostreococcus were mainly recovered from mesotrophic areas, whereas Mamiella , Crustomastix and Dolichomastix were mostly detected in oligotrophic surface waters. Within genera such as Ostreococcus or Crustomastix for which several clades were observed, depth seemed to be the main factor controlling differential distribution of genotypes.     

Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants

It has been generally assumed that the [alpha]-(1->4)-linked and [alpha]-(1->6)-branched glucans of starch are generated by the coordinated action of elongation (starch synthases) and branching enzymes. We have identified a novel Chlamydomonas locus (STA7) that when defective leads to a wipeout of starch and its replacement by a small amount of glycogen-like material. Our efforts to understand the enzymological basis of this phenotype have led us to determine the selective disappearance of an 88-kD starch hydrolytic activity. We further demonstrate that this enzyme is a debranching enzyme. Cleavage of the [alpha]-(1->6) linkage in a branched precursor of amylopectin (preamylopectin) has provided us with the ground rules for understanding starch biosynthesis in plants. Therefore, we propose that amylopectin clusters are synthesized by a discontinuous mechanism involving a highly specific glucan trimming mechanism.     

Starch granule initiation in Arabidopsis thaliana chloroplasts

The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.     

转基因技术改良稻米淀粉品质的研究进展

淀粉是稻米胚乳的主要组成成分, 而淀粉含量和性质的差异又直接决定稻米的品质。早期关于稻米淀粉的研究局限于其组成成分、品种间含量的差异以及稻米淀粉合成的经典遗传学方面。随着现代分子生物学和转基因技术的发展, 稻米品质、淀粉的生物合成及其分子调控成为研究热点。随着淀粉合成相关基因的克隆、分子特性及其表达调控的研究取得了较大进展, 人们也开始尝试利用现代基因工程技术, 高效地改良稻米品质, 如提高或降低淀粉含量及改变支链淀粉的结构。本文从综述稻米淀粉的组成、结构和淀粉合成相关酶的研究进展入手, 探讨了转基因技术改良稻米品质的研究进展和发展前景。     

Starch biosynthesis: the primer nonreducing-end mechanism versus the nonprimer reducing-end two-site insertion mechanism

Two mechanisms are recognized for polysaccharide chain elongation: (a) the nonreducing-end, primer-dependent mechanism and (b) the reducing-end, two-site insertion mechanism. We recently demonstrated the latter mechanism for starch biosynthesis by pulsing starch granules with ADP-[14C]Glc and chasing with ADPGlc for eight varieties of starch granules. Others have reported the addition of glucose from ADPGlc to the nonreducing ends of maltose, maltotriose, and maltopentaose and a branched maltopentasaccharide. It was concluded that starch chains are biosynthesized by the addition of glucose to the nonreducing ends of maltodextrin primers. In this study, we reinvestigated the maltodextrin reactions by reacting three kinds of starch granules from maize, wheat, and rice with ADP-[14C]Glc in the absence and presence of maltose (G2), maltotriose (G3), and maltodextrin (d.p.12) and found that they inhibited starch biosynthesis rather than stimulating it, as would be expected for primers. The major product in the presence of G2 was G3 with decreasing amounts of G4-G9 and the major products in the presence of G3 was G4 and G5, with decreasing amounts of G6-G9. It was concluded that maltodextrins are acceptors rather than primers. This was confirmed by pulsing the starch granules with ADP-[14C]Glc and chasing with G2, G3, and G6, which gave release of 14C-label from the pulsed granules in the absence of ADPGlc, further demonstrating that maltodextrins are acceptors that inhibit starch biosynthesis by releasing glucose from starch synthase, rather than acting as primers and stimulating biosynthesis.     

Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

Background  

Glutamine synthetase (GS) is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII) are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria) to the Chloroplastida.     

植物体内淀粉磷酸化的生物学研究进展

植物中淀粉是主要储存碳水化合物形式,是食品和工业应用中最重要的植物原材料之一。而植物淀粉中惟一的取代是磷酸化作用,更体现了淀粉的独特性能。淀粉的品质和理化性质影响其应用。因此对淀粉的研究是有重要意义的。主要介绍了淀粉磷酸化作用机制,概述GWD功能与磷酸化作用和淀粉代谢的关系的生物学研究进展,并在此基础上讨论利用基因工程改良淀粉品质的可能途径以及今后的研究任务。     

Sucrose-Starch Conversion in Heterotrophic Tissues of Plants

The historical progress in recent years pertaining to the sucrose-starch conversion in heterotrophic tissues of plants has been described. Special attention has been focused on the enzymatic breakdown of sucrose to produce hexose units that are transported to the amyloplast compartment by means of specific translocator molecules and act as glucose donors for starch biosynthesis. Although the current prevailing view is that variable mechanisms operate in different plant tissues and organs, it is often argued that the following enzymic steps are essential in the overall step of sucrose to starch conversion: sucrose + UDP -> UDPGlc + Fru(sucrose synthase-SS) (UDPGlc UDPGlc + PPi -» G1P + UTPpyrophosphorylase-UGPase) (ADPGlc GlP + ATP -» ADPGlc + PPipyrophosphorylase-AGPase) (starch ADPGlc -> starchsynthase) The presence of an ADPGlc-specific translocator in the amyloplast envelope has been demonstrated in a number of plant sources, which indicates the potential role of ADPGlc-synthesizing machineries located in the cytosol of starch-storing cells. Although it was initially believed that AGPase is present exclusively in the amyloplast compartment, the presence of a cytosolic enzyme has been shown in some cereals. The SS has a potential to produce ADPGlc, but the general belief is that this is not a dominant reaction in the mechanism of starch biosynthesis. Numerous experimental trials have been reported by many scientists employing transgenic plants transformed with cDNAs either in antisense- or sense- orientation encoding enzymes which are presumably involved in the process of sucrose-starch conversion. Although great caution is needed to interpret the data obtained, the general picture is contradictory to the mechanism presented above. It now appears that serious reconsideration is needed for the possible mechanism of SS-catalyzed ADPGlc formation and its subsequent link to starch formation. In the newly proposed mechanistic scheme, which appears to be consistent with the results by other scientists as well, hexokinase, phosphoglucomutase (PGM), and ADPGlc formation by AGPase are components in the cyclic turnover of starch molecules in the amyloplast compartment.     

Amyloplast Size and Number in Gravity-compensated Oat Seedlings

Gravity compensation by the horizontal clinostat increases the diameter of amyloplast starch grains of oat (Avena sativa cv. Victory) coleoptile parenchyma cells, as compared to vertically rotated and stationary controls. In dark-grown coleoptile tip parenchyma cells, measured starch grain sizes exhibit a wide distribution of diameters, from approximately 1.5 to approximately 8.0 mum, but fall into three prominent diameter classes. The compensated tissues from both the tip and the subapical region have more starch grains in the larger, and fewer in the smaller size classes, compared to controls. The total number of starch grains per cell, the total plastid number per cell, and cell volume are unaffected by gravity compensation. Amyloplasts with large starch grains are denser, as well as larger in diameter, than those with smaller starch grains. The amyloplast is considered as a geosensor with an active metabolic role in the geotropic transduction mechanism.     

Distinct Functional Properties of Isoamylase-Type Starch Debranching Enzymes in Monocot and Dicot Leaves

Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.Semicrystalline starch enables photosynthetic eukaryotes to store large quantities of Glc over extended time periods compared with other species, in which the soluble polymer glycogen functions to store carbohydrate reserves (Ball and Morell, 2003). Eukaryotes gained the capacity to photosynthesize after the capture of a cyanobacterial endosymbiont by a glycogen-metabolizing host cell. In the lineage that evolved subsequently, known as the Archaeplastida, select glucan-storage enzymes encoded within the host nucleus, the endosymbiont, and potentially a prokaryotic parasite located within the host cell developed so as to generate the branched glucan polymer amylopectin (Ball et al., 2011, 2013). Such molecules are highly similar to glycogen in terms of chemical structure, but the molecular architecture of amylopectin enables the formation of semicrystalline structures (Buléon et al., 1998). These latter then assemble into higher order structures leading to starch granule formation. The advent of starch granules is likely to have been critical for the evolution of chloroplast-containing organisms, including the spread of land plants on the Earth’s surface, because they enable the storage of photosynthetically generated Glc for many hours in tissues such as leaves during diurnal cycles or for months to years in seeds.An important aspect of the evolutionary change from glycogen to starch is the use of particular α(1→6)-glucosidases, referred to as isoamylase-type starch debranching enzymes (ISA), in the production of amylopectin (Ball et al., 1996; Myers et al., 2000; Hennen-Bierwagen et al., 2012). A suite of genes encoding the enzymes that accomplish starch biosynthesis was established early in the evolution of chloroplast-containing organisms (i.e. the Chloroplastida) prior to the divergence of distantly related groups including green algae and land plants. Included in this gene set are three paralogs that encode the proteins ISA1, ISA2, and ISA3, each of which is highly conserved in chloroplast-containing species. ISA1 of vascular plants and bryophytes, for example, are approximately 70% identical over more than 600 residues, and between land plants and prasinophyte algae this value is about 60%. ISA1 or ISA2 deficiencies in potato (Solanum tuberosum) tuber, Arabidopsis (Arabidopsis thaliana) leaf, Chlamydomonas reinhardtii cells, and cereal endosperms result in reduced starch content, altered amylopectin structure, and the appearance of soluble, branched glucans similar to native glycogen (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Bustos et al., 2004; Delatte et al., 2005; Wattebled et al., 2005). Such soluble polymers, referred to as phytoglycogen, have not been observed in wild-type plants. Thus, ISA1 and ISA2 functions are important determinants of whether storage glucans are semicrystalline or soluble. ISA3, in contrast, functions primarily in starch catabolism (Wattebled et al., 2005; Delatte et al., 2006).ISA1 and ISA2 appear to function together in Arabidopsis leaf as a single entity, because essentially identical phenotypes are observed in single mutants lacking either protein or double mutants lacking both of them (Zeeman et al., 1998; Delatte et al., 2005; Wattebled et al., 2005). Biochemical analysis of native and recombinant proteins has shown directly that ISA1 and ISA2 function together in a complex. ISA activity was first purified from potato tuber and found to contain two distinct polypeptides identified as ISA1 and ISA2 (Ishizaki et al., 1983; Hussain et al., 2003). Heteromultimers containing these two proteins were also purified from rice (Oryza sativa) and maize (Zea mays) endosperm (Utsumi and Nakamura, 2006; Kubo et al., 2010). Finally, a mixture of native and recombinant rice proteins demonstrated directly that specific enzymatic activities are provided by ISA1 and ISA2 functioning together in a heteromultimeric complex (Utsumi and Nakamura, 2006). ISA1 is the catalytic subunit within this complex, whereas ISA2 is noncatalytic, owing to amino acid substitutions at residues that are essentially invariant in the GH13 family of glycoside hydrolases (i.e. the α-amylase superfamily), several of which participate in the catalytic mechanism (Hussain et al., 2003; Utsumi and Nakamura, 2006). Despite lacking catalytic activity, ISA2 proteins are conserved in all chloroplast-containing species that have been examined, which rules out recently evolved mutations and, to the contrary, suggests a functional selective advantage.The necessity for the ISA1/ISA2 heteromultimer is not obvious in light of the fact that, in some instances, ISA1 by itself can condition normal levels of starch and the suppression of phytoglycogen accumulation. Cyanidioschyzon merolae, a species within the Rhodophyta lineage of the Archaeplastida family, contains semicrystalline starch and amylopectin with physical characteristics similar to that of Chloroplastida species (Hirabaru et al., 2010). The C. merolae genome contains elements that encode ISA1 and ISA3 yet lacks a homolog encoding ISA2 (Coppin et al., 2005). Thus, in some instances, starch can be generated, and phytoglycogen accumulation suppressed, without an ISA2 protein. Cereal endosperms provide additional evidence that ISA2 is not strictly required for normal starch levels and the suppression of phytoglycogen accumulation. Mutants or transgenic lines lacking ISA2 are known in rice (Utsumi et al., 2011) and maize (Kubo et al., 2010). Endosperm from these plants exhibits normal starch levels, with amylopectin structure essentially the same as the wild type, and lacks phytoglycogen. ISA activity presumably is provided in the endosperm of these mutants by a homomultimeric enzyme containing only ISA1.The reason why ISA2 is strictly conserved in the Chloroplastida is not understood yet. Two explanations can be considered. One possibility is that the inherent structure of ISA1 in cereals, resulting from mutations accumulated specifically in this evolutionary lineage, allows it to act without ISA2. Another possibility is that metabolic differences in specific tissues (e.g. leaf versus endosperm) require specialized enzymatic properties of the ISA1/ISA2 heteromer that ISA1 by itself does not provide. To test these hypotheses, this study combined maize and Arabidopsis ISA1 and ISA2 isoforms both in vitro and in vivo. Maize ISA1 was found to be active without any ISA2 protein, either in vitro or in Arabidopsis leaves, whereas Arabidopsis ISA1 required an ISA2 partner in all instances. Thus, ISA1 appears to have evolved in the cereal lineage so that it no longer requires ISA2 for enzymatic activity or metabolic function in the generation of starch and the suppression of phytoglycogen accumulation.     

Light-induced degradation of starch granules in turions of Spirodela polyrhiza studied by electron microscopy

Spirodela polyrhiza forms turions, starch-storing perennial organs. The light-induced process of starch degradation starts with an erosion of the surface of starch grains. The grain size decreases over a period of red irradiation and the surface becomes rougher. The existence of funnel-shaped erosion structures demonstrates that starch degradation is also possible inside the grains. Neither etioplasts nor clues as to their transition into chloroplasts were found in the storage tissue by transmission electron microscopy. Juvenile chloroplasts always contained the starch grains which remained from amyloplasts. No chloroplasts were found which developed independently of starch grains. Amyloplasts are therefore the only source of chloroplasts in the cells of irradiated turions. The intactness of amyloplast envelope membranes could not be directly proved by electron microscopy. However, the light-induced transition of amyloplasts into chloroplasts provides indirect evidence for the integrity of the envelope membranes throughout the whole process. The starch grains are sequestered from the cytosolic enzymes, and only plastid-localized enzymes, which have access to the starch grains, can carry out starch degradation. In this respect the turion system resembles transitory starch degradation as known from Arabidopsis leaves. On the other hand, with α-amylase playing the dominant role, it resembles the mechanism operating in the endosperm of cereals. Thus, turions appear to possess a unique system of starch degradation in plants combining elements from both known starch-storing systems.     

Starch Granule Initiation in Arabidopsis Requires the Presence of Either Class IV or Class III Starch Synthases

The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.     

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO₂ concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO₂-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO₂ conditions. These growth conditions also partly inhibit the induction of the CO₂-concentrating mechanism. When cells are grown with acetate in the dark, the CO₂-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO₂-concentrating mechanism under low-CO₂ conditions. We suggest that this ultrastructural reorganization under lowCO₂ conditions plays a role in the CO₂-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.     

Starch division and partitioning. A mechanism for granule propagation and maintenance in the picophytoplanktonic green alga Ostreococcus tauri

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery.     

Redox regulation of carbon storage and partitioning in response to light and sugars

Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin-thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid synthesis in response to physiological and environmental inputs.     

A possible structure of retrograded maize starch speculated by UV and IR spectra of it and its components

“Retrogradation” has been used to describe the changes that occur in starch after gelatinization, from an initially amorphous state to a more ordered or crystalline state, which has a significant impact on starch application in food, textiles and materials fields. But mechanism of starch retrogradation is still unclear until now and there is no breakthrough in this area. Here we are speculating a possible structure of retrograded maize starch by UV (binding with iodine) and IR spectra of it and its compositions. We speculate that nucleation of retrograded starch origins from combination of reducing end of amylopectin and non-reducing end of amylose, and retrogradation terminates at combining of non-reducing end of amylopectin and reducing end of amylose. The chain length of resistant digestion retrograded starch should be nearly same. The hydroxyl associated with sixth carbon atoms of glucan must form hydrogen bond with other hydroxyl of starch.     

Starch biosynthesis: further evidence against the primer nonreducing-end mechanism and evidence for the reducing-end two-site insertion mechanism

Two reactions were studied with three varieties of starch granules from maize, wheat, and rice. In Reaction-I, the granules were reacted with 1 mM ADP-[(14)C]Glc and in Reaction-II, a portion of the granules from Reaction-I was reacted with 1 mM ADP-Glc. The starch granules were solubilized and reacted with the exo-acting glucoamylase and beta-amylase to an extent of 50% or less of the (14)C-label. The amounts of (14)C-labeled products from glucoamylase and beta-amylase were nearly equal for Reaction-I and Reaction-II. If the addition had been to the nonreducing ends of primers, Reaction-II would not have given any labeled products from the hydrolysis of glucoamylase and beta-amylase. These results indicate that the elongation of the starch chain is the addition of D-glucose to the reducing end by a de novo two-site insertion mechanism and not by the addition of D-glucose to the nonreducing end of a primer. This is in conformity with previous results in which starch granules were pulsed with ADP-[(14)C]Glc and chased with nonlabeled ADP-Glc, giving (14)C-labeled D-glucitol from the pulsed starch and a significant decrease in (14)C-labeled D-glucitol from the chased starch on reducing with NaBH(4) and hydrolyzing with glucoamylase [Carbohydr. Res.2002, 337, 1015-1022]. It also is in conformity with the inhibition of starch synthesis that occurs when putative primers are added to starch granule-ADP-Glc digests, indicating that the elongation is not by the nonreducing-end primer mechanism [Carbohydr. Res.2005, 340, 245-255].