LIKE EARLY STARVATION 1 interacts with amylopectin during starch biosynthesis.

Starch is the major energy storage compound in plants. Both transient starch and long-lasting storage starch accumulate in the form of insoluble, partly crystalline granules. The structure of these granules is related to the structure of the branched polymer amylopectin: linear chains of glucose units organized in double helices that align to form semi-crystalline lamellae, with branch points located in amorphous regions between them. EARLY STARVATION 1 (ESV1) and LIKE EARLY STARVATION 1 (LESV) proteins are involved in the maintenance of starch granule structure and in the phase transition of amylopectin, respectively, in Arabidopsis (Arabidopsis thaliana). These proteins contain a conserved tryptophan-rich C-terminal domain folded into an antiparallel ß-sheet, likely responsible for binding of the proteins to starch, and different N-terminal domains whose structure and function are unknown. In this work, we combined biochemical and biophysical approaches to analyze the structures of LESV and ESV1 and their interactions with the different starch polyglucans. We determined that both proteins interact with amylopectin but not with amylose and that only LESV is capable of interacting with amylopectin during starch biosynthesis. While the C-terminal domain interacts with amylopectin in its semi-crystalline form, the N-terminal domain of LESV undergoes induced conformational changes that are probably involved in its specific function of mediating glucan phase transition. These results clarify the specific mechanism of action of these two proteins in the biosynthesis of starch granules.

BE3 is the major branching enzyme isoform required for amylopectin synthesis in Chlamydomonas reinhardtii.

Starch-branching enzymes (BEs) are essential for starch synthesis in both plants and algae where they influence the architecture and physical properties of starch granules. Within Embryophytes, BEs are classified as type 1 and type 2 depending on their substrate preference. In this article, we report the characterization of the three BE isoforms encoded in the genome of the starch producing green algae Chlamydomonas reinhardtii: two type 2 BEs (BE2 and BE3) and a single type 1 BE (BE1). Using single mutant strains, we analyzed the consequences of the lack of each isoform on both transitory and storage starches. The transferred glucan substrate and the chain length specificities of each isoform were also determined. We show that only BE2 and BE3 isoforms are involved in starch synthesis and that, although both isoforms possess similar enzymatic properties, BE3 is critical for both transitory and storage starch metabolism. Finally, we propose putative explanations for the strong phenotype differences evidenced between the C. reinhardtii be2 and be3 mutants, including functional redundancy, enzymatic regulation or alterations in the composition of multimeric enzyme complexes.

Further insight into the involvement of PII1 in starch granule initiation in Arabidopsis leaf chloroplasts.

The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.

Deletion of BSG1 in Chlamydomonas reinhardtii leads to abnormal starch granule size and morphology.

Chlamydomonas reinhardtii represents an ideal model microbial system to decipher starch metabolism. In this green algae, in cells growing in photosynthetic conditions, starch mainly accumulates as a sheath surrounding the pyrenoid while in cells subjected to a nutrient starvation, numerous starch granules are filling up the plastid stroma. The mechanisms underlying and regulating this switch from photosynthetic to storage starch metabolisms are not known. In this work, we have isolated a Chlamydomonas mutant strain containing a deletion in chromosome 2 which displays abnormal starch granule distribution. Under nitrogen starvation, this strain contains an additional starch granules population. These granules are twice as big as the wild-type granules and display characteristics of photosynthetic starch. Genetic and functional complementation analyses allowed us to identify the gene responsible for this original phenotype which was called BSG1 for "Bimodal Starch Granule". Possible roles of BSG1 in starch metabolism modifications during the transition from photosynthetic to starved growth conditions are discussed.

PII1: a protein involved in starch initiation that determines granule number and size in Arabidopsis chloroplast.

The initiation of starch granule formation is still poorly understood. However, the soluble starch synthase 4 (SS4) appears to be a major component of this process since it is required to synthesize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana plants. A yeast two-hybrid screen allowed the identification of several putative SS4 interacting partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 display an alteration of the starch initiation process and accumulate, on average, one starch granule per plastid instead of the five to seven granules found in plastids of wild-type plants. These granules are larger than in wild-type, and they remain flat and lenticular. pii1 mutants display wild-type growth rates and accumulate standard starch amounts. Moreover, starch characteristics, such as amylopectin chain length distribution, remain unchanged. Our results reveal the involvement of PII1 in the starch priming process in Arabidopsis leaves through interaction with SS4.

The Chlamydomonas mex1 mutant shows impaired starch mobilization without maltose accumulation.

The MEX1 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that affects starch metabolism. Mutation of MEX1 causes a slow-down in the mobilization of storage polysaccharide. Cosegregation and functional complementation analyses were used to assess the involvement of the Mex1 protein in starch degradation. Heterologous expression experiments performed in Escherichia coli and Arabidopsis thaliana allowed us to test the capacity of the algal protein in maltose export. In contrast to the A. thaliana mex1 mutant, the mutation in C. reinhardtii does not lead to maltose accumulation and growth impairment. Although localized in the plastid envelope, the algal protein does not transport maltose efficiently across the envelope, but partly complements the higher plant mutant. Both Mex orthologs restore the growth of the E. coli ptsG mutant strain on glucose-containing medium, revealing the capacity of these proteins to transport this hexose. These findings suggest that Mex1 is essential for starch mobilization in both Chlamydomonas and Arabidopsis, and that this protein family may support several functions and not only be restricted to maltose export across the plastidial envelope.

Hyper-accumulation of starch and oil in a Chlamydomonas mutant affected in a plant-specific DYRK kinase.

BACKGROUND: Because of their high biomass productivity and their ability to accumulate high levels of energy-rich reserve compounds such as oils or starch, microalgae represent a promising feedstock for the production of biofuel. Accumulation of reserve compounds takes place when microalgae face adverse situations such as nutrient shortage, conditions which also provoke a stop in cell division, and down-regulation of photosynthesis. Despite growing interest in microalgal biofuels, little is known about molecular mechanisms controlling carbon reserve formation. In order to discover new regulatory mechanisms, and identify genes of interest to boost the potential of microalgae for biofuel production, we developed a forward genetic approach in the model microalga Chlamydomonas reinhardtii. RESULTS: By screening an insertional mutant library on the ability of mutants to accumulate and re-mobilize reserve compounds, we isolated a Chlamydomonas mutant (starch degradation 1, std1) deficient for a dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK). The std1 mutant accumulates higher levels of starch and oil than wild-type and maintains a higher photosynthetic activity under nitrogen starvation. Phylogenetic analysis revealed that this kinase (named DYRKP) belongs to a plant-specific subgroup of the evolutionarily conserved DYRK kinase family. Furthermore, hyper-accumulation of storage compounds occurs in std1 mostly under low light in photoautotrophic condition, suggesting that the kinase normally acts under conditions of low energy status to limit reserve accumulation. CONCLUSIONS: The DYRKP kinase is proposed to act as a negative regulator of the sink capacity of photosynthetic cells that integrates nutrient and energy signals. Inactivation of the kinase strongly boosts accumulation of reserve compounds under photoautotrophic nitrogen deprivation and allows maintaining high photosynthetic activity. The DYRKP kinase therefore represents an attractive target for improving the energy density of microalgae or crop plants.

Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids.

When green algae are exposed to physiological stresses such as nutrient deprivation, growth is arrested and the cells channel fixed carbon instead into storage compounds, accumulating first starch granules and then lipid bodies containing triacylglycerides. In recent years there has been significant interest in the commercial exploitation of algal lipids as a sustainable source of biodiesel. Since starch and lipid biosynthesis involves the same C3 precursor pool, it has been proposed that mutations blocking starch accumulation should result in increased lipid yields, and indeed several studies have supported this. The fast-growing, thermotolerant alga Chlorella sorokiniana represents an attractive strain for industrial cultivation. We have therefore generated and characterized starch-deficient mutants of C. sorokiniana and determined whether lipid levels are increased in these strains under stress conditions. One mutant (ST68) is shown to lack isoamylase, whilst two others (ST3 and ST12) are defective in starch phosphorylase. However, we find no significant change in the accumulation or profile of fatty acids in these mutants compared to the wild-type, suggesting that a failure to accumulate starch per se is not sufficient for the hyper-accumulation of lipid, and that more subtle regulatory steps underlie the partitioning of carbon to the two storage products.

Crystal structure of the Chlamydomonas starch debranching enzyme isoamylase ISA1 reveals insights into the mechanism of branch trimming and complex assembly.

The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites.

A forward genetic approach in Chlamydomonas reinhardtii as a strategy for exploring starch catabolism.

A screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding ß-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae.

Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis?

Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydia-controlled assimilation pathway.

Microarray data can predict diurnal changes of starch content in the picoalga Ostreococcus.

BACKGROUND: The storage of photosynthetic carbohydrate products such as starch is subject to complex regulation, effected at both transcriptional and post-translational levels. The relevant genes in plants show pronounced daily regulation. Their temporal RNA expression profiles, however, do not predict the dynamics of metabolite levels, due to the divergence of enzyme activity from the RNA profiles.Unicellular phytoplankton retains the complexity of plant carbohydrate metabolism, and recent transcriptomic profiling suggests a major input of transcriptional regulation. RESULTS: We used a quasi-steady-state, constraint-based modelling approach to infer the dynamics of starch content during the 12 h light/12 h dark cycle in the model alga Ostreococcus tauri. Measured RNA expression datasets from microarray analysis were integrated with a detailed stoichiometric reconstruction of starch metabolism in O. tauri in order to predict the optimal flux distribution and the dynamics of the starch content in the light/dark cycle. The predicted starch profile was validated by experimental data over the 24 h cycle. The main genetic regulatory targets within the pathway were predicted by in silico analysis. CONCLUSIONS: A single-reaction description of starch production is not able to account for the observed variability of diurnal activity profiles of starch-related enzymes. We developed a detailed reaction model of starch metabolism, which, to our knowledge, is the first attempt to describe this polysaccharide polymerization while preserving the mass balance relationships. Our model and method demonstrate the utility of a quasi-steady-state approach for inferring dynamic metabolic information in O. tauri directly from time-series gene expression data.

Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules.

BACKGROUND: Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii. METHODS AND FINDINGS: We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species. CONCLUSION: This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production of edible vaccines can be genetically produced in Chlamydomonas.

Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol.

Many microalgae and plants have the ability to synthesize large amounts of triacylglycerol (TAG) that can be used to produce biofuels. Presently, TAG-based biofuel production is limited by the feedstock supply. Metabolic engineering of lipid synthesis pathways to overproduce TAGs in oleaginous microalgae and oil crop plants has achieved only modest success. We demonstrate that inactivation of ADP-glucose pyrophosphorylase in a Chlamydomonas starchless mutant led to a 10-fold increase in TAG, suggesting that shunting of photosynthetic carbon partitioning from starch to TAG synthesis may represent a more effective strategy than direct manipulation of the lipid synthesis pathway to overproduce TAG.

Genetic dissection of floridean starch synthesis in the cytosol of the model dinoflagellate Crypthecodinium cohnii.

Starch defines an insoluble semicrystalline form of storage polysaccharides restricted to Archaeplastida (red and green algae, land plants, and glaucophytes) and some secondary endosymbiosis derivatives of the latter. While green algae and land-plants store starch in plastids by using an ADP-glucose-based pathway related to that of cyanobacteria, red algae, glaucophytes, cryptophytes, dinoflagellates, and apicomplexa parasites store a similar type of polysaccharide named floridean starch in their cytosol or periplast. These organisms are suspected to store their floridean starch from UDP-glucose in a fashion similar to heterotrophic eukaryotes. However, experimental proof of this suspicion has never been produced. Dinoflagellates define an important group of both photoautotrophic and heterotrophic protists. We now report the selection and characterization of a low starch mutant of the heterotrophic dinoflagellate Crypthecodinium cohnii. We show that the sta1-1 mutation of C. cohnii leads to a modification of the UDP-glucose-specific soluble starch synthase activity that correlates with a decrease in starch content and an alteration of amylopectin structure. These experimental results validate the UDP-glucose-based pathway proposed for floridean starch synthesis.

Hydrogen production in Chlamydomonas: photosystem II-dependent and -independent pathways differ in their requirement for starch metabolism.

Under sulfur deprivation conditions, the green alga Chlamydomonas reinhardtii produces hydrogen in the light in a sustainable manner thanks to the contribution of two pathways, direct and indirect. In the direct pathway, photosystem II (PSII) supplies electrons to hydrogenase through the photosynthetic electron transport chain, while in the indirect pathway, hydrogen is produced in the absence of PSII through a photosystem I-dependent process. Starch metabolism has been proposed to contribute to both pathways by feeding respiration and maintaining anoxia during the direct pathway and by supplying reductants to the plastoquinone pool during the indirect pathway. At variance with this scheme, we report that a mutant lacking starch (defective for sta6) produces similar hydrogen amounts as the parental strain in conditions of sulfur deprivation. However, when PSII is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, conditions where hydrogen is produced by the indirect pathway, hydrogen production is strongly reduced in the starch-deficient mutant. We conclude that starch breakdown contributes to the indirect pathway by feeding electrons to the plastoquinone pool but is dispensable for operation of the direct pathway that prevails in the absence of DCMU. While hydrogenase induction was strongly impaired in the starch-deficient mutant under dark anaerobic conditions, wild-type-like induction was observed in the light. Because this light-driven hydrogenase induction is DCMU insensitive and strongly inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, we conclude that this process is regulated by the proton gradient generated by cyclic electron flow around PSI.

The heterotrophic dinoflagellate Crypthecodinium cohnii defines a model genetic system to investigate cytoplasmic starch synthesis.

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model heterotrophic dinoflagellate Crypthecodinium cohnii. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of green algae and land plant starch. Preliminary characterization of the starch pathway demonstrated that C. cohnii contains multiple forms of soluble starch synthases and one major 110-kDa granule-bound starch synthase. All purified enzymes displayed a marked substrate preference for UDP-glucose. At variance with most other microorganisms, the accumulation of starch in the dinoflagellate occurs during early and mid-log phase, with little or no synthesis witnessed when approaching stationary phase. In order to establish a genetic system allowing the study of cytoplasmic starch metabolism in eukaryotes, we describe the isolation of marker mutations and the successful selection of random recombinant populations after homothallic crosses.

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.

Metabolic symbiosis and the birth of the plant kingdom.

Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria.

Pathway of cytosolic starch synthesis in the model glaucophyte Cyanophora paradoxa.

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.