ADP-glucose synthesis through ADP-glucose pyrophosphorylase defines the major rate-controlling step of storage polysaccharide synthesis in both bacteria and plants. We have isolated mutant strains defective in the STA6 locus of the monocellular green alga Chlamydomonas reinhardtii that fail to accumulate starch and lack ADP-glucose pyrophosphorylase activity. We show that this locus encodes a 514-amino-acid polypeptide corresponding to a mature 50-kDa protein with homology to vascular plant ADP-glucose pyrophosphorylase small-subunit sequences. This gene segregates independently from the previously characterized STA1 locus that encodes the large 53-kDa subunit of the same heterotetramer enzyme. Because STA1 locus mutants have retained an AGPase but exhibit lower sensitivity to 3-phosphoglyceric acid activation, we suggest that the small and large subunits of the enzyme define, respectively, the catalytic and regulatory subunits of AGPase in unicellular green algae. We provide preliminary evidence that both the small-subunit mRNA abundance and enzyme activity, and therefore also starch metabolism, may be controlled by the circadian clock.

In Chlamydomonas reinhardtii, the presence of a defective STA11 locus results in significantly reduced granular starch deposition displaying major modifications in shape and structure. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides (MOS). The mutants of STA11 were showed to lack d-enzyme, a plant ␣-1,4 glucanotransferase analogous to the Escherichia coli amylomaltase. We have cloned and characterized both the cDNA and gDNA corresponding to the C. reinhardtii d-enzyme. We now report allele-specific modifications of the d-enzyme gene in the mutants of STA11. These allele-specific modifications cosegregate with the corresponding sta11 mutations, thereby demonstrating that STA11 encodes d-enzyme. MOS production and starch accumulation were investigated during day and night cycles in wild-type and mutant C. reinhardtii cells. We demonstrate that in the algae MOS are produced during starch biosynthesis and degraded during the phases of net polysaccharide catabolism.Until recently plant starch was believed to be synthesized from ADP-Glc solely through a combination of starch synthases and branching enzymes. However the finding of low-starch or starchless mutants defective for a particular form of debranching enzyme in four different plant systems established that other enzymes of starch metabolism are equally important in ensuring normal starch granule biogenesis (James et al., 1995;Mouille et al., 1996;Nakamura et al., 1996;Zeeman et al., 1998). This came as a surprise because debranching enzymes were initially thought to be enzymes involved solely in starch breakdown. Mutants of the corresponding activities in yeast (Teste et al., 2000) or Escherichia coli (I. Kinderf, Z. Li, M.S. Samuel, B. Koshar-Hashezmi, S. Ball, L. Rampling, and M. Morell, unpublished data) are clearly glycogen over-producers, confirming the initial suspicion. Although the detailed interpretation of the results obtained with the plant mutants vary somewhat, there is now a general agreement that isoamylases, the particular form of debranching enzyme affected in these studies, are enzymes required during starch biosynthesis exclusively. It has been comforting to realize that all mutants affected in starch metabolism behaved in a similar fashion in plants as different as Chlamydomonas reinhardtii, Arabidopsis, pea (Pisum sativum), maize (Zea mays), or rice (Oryza sativa). Some discrepancies in expressivity of mutant phenotypes could be easily explained most of the time by subtle differences in the pathways. For instance, the presence of extraplastidial and plastidial ADP-Glc pyrophosphorylases in cereals easily explains why mutants of cereals lacking the major enzyme form displayed reduced expressivity in their starch accumulation phenotype (for review, see Kossmann and Lloyd, 2000). The starch accumulation phenotypes of the Arabidopsis or C. reinhardtii mutants defective for the catalytic subunit of their sole plastidial ADP-Glc pyrophosphorylase were much more severe (Lin et al., 1988;Zabawinski et al., 2001...

The chloroplast H(+)-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO(2) concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H(+)-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.

In a synchronously grown Chlamydomonas reinhardtii (Chlorophyceae) culture the CO2-concentrating mechanism (CCM) was induced by lowering the CO2 level from 4% to 0.036% CO2 (culture HL). The effects of the reduced carbon supply on starch levels were studied over a period of up to 100 h and compared with control cultures kept either at 4% CO2 (culture H) or continuously at ambient air (0.036% CO2, culture L). Lowering the CO2 supply reduced culture growth as estimated by chlorophyll, protein and cell density. The starch level continued to show diurnal variations with an initially reduced rate of starch synthesis at reduced or abolished culture growth. Subsequently, starch maxima and minima increased. After 4 days the resulting pattern for culture HL was similar to that of culture L, which possessed higher minima but identical maxima to culture H. The intracellular starch localisation was examined on electron micrographs. Cell extracts were assayed for ADP-glucose pyrophosphorylase (EC 2.7.7.27) and starch phosphorylase (EC 2.4.1.1) activities. Over the assayed period of 2 days, there was a good correlation between the observed changes in the starch levels and the measured enzyme activities. The rate of CO2-dependent oxygen evolution of culture HL declined from 100% to 60% of the control over the day. This indicates that the diminished or abolished growth and the impairment of starch accumulation upon CO2 depletion are not simply consequences of the lowered level of the substrate CO2. The diminished growth and the peculiar starch accumulation pattern with higher positions of the starch minima in low-CO2 cells are interpreted as economised starch utilisation as long-term aspects of induction of the CCM.

The flagellate Cyanophora paradoxa contains blue-greenish, organelle-like inclusions termed cyanelles which perform photosynthetic C02-fixation in place of chloroplasts. By the use of the HPLC-technique, Cyanophora was shown to form glucose, sucrose, maltose, mannitol, ribose, glycerol and trehalose. Extracts from the whole organism and from the eucaryotic host, but not from the cyanelles, convert 14C-labelled UDP-glucose to polyglucan. Synthesis of sucrose from UDP-glucose and fructose-6-P or fructose could not be demonstrated in any extract from Cyanophora. The transfer of metabolites into cyanelles was monitored by the silicone oil filtering technique. The solute spaces for 14Clabelled sorbitol and 3H20 were the same indicating that sorbitol freely penetrated the plasma membrane of cyanelles in contrast to the situation found in chloroplasts. The measurements ofthe solute spaces for the different compounds showed that maltose and sucrose were not accumulated by isolated cyanelles. Other compounds like fructose, fucose, glutamine or glycine had intermediate sizes of their solute spaces. Cyanelles apparently possess a rapidly transporting glucose carrier and not a malate/oxaloacetate shuttle and also not an ATP/ ADP translocator. The carrier composition at the plasma membrane of cyanelles and at the inner envelope membrane of chloroplasts seems to be totally different.