Christoph Giersch

Starchless Mutants of Chlamydomonas reinhardtii Lack the Small Subunit of a Heterotetrameric ADP-Glucose Pyrophosphorylase

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.

STA11, a Chlamydomonas reinhardtii Locus Required for Normal Starch Granule Biogenesis, Encodes Disproportionating Enzyme. Further Evidence for a Function of α-1,4 Glucanotransferases during Starch Granule Biosynthesis in Green Algae

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 ofSTA11 were showed to lack d-enzyme, a plant α-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 encodesd-enzyme. MOS production and starch accumulation were investigated during day and night cycles in wild-type and mutantC. reinhardtii cells. We demonstrate that in the algae MOS are produced during starch biosynthesis and degraded during the phases of net polysaccharide catabolism.

The CO2-concentrating mechanism in the physiological context: lowering the CO2 supply diminishes culture growth and economises starch utilisation in Chlamydomonas reinhardtii

Abstract. 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 100xa0h 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 4xa0days 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 2xa0days, 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.

A simple dynamic model of photosynthesis in oak leaves: coupling leaf conductance and photosynthetic carbon fixation by a variable intracellular CO2 pool

Modelling the diurnal course of photosynthesis in oak leaves (Quercus robur L.) requires appropriate description of the dynamics of leaf photosynthesis of which diurnal variations in leaf conductance and in CO2 assimilation are essential components. We propose and analyse a simple photosynthesis model with three variables: leaf conductance (gs), the CO2 partial pressure inside the leaf (pi), and a pool of Calvin cycle intermediates (aps). The environmental factors light (I) and vapour pressure deficit (VPD) are used to formulate a target function G(I, VPD) from which the actual leaf conductance is calculated. Using this gs value and a CO2 consumption term representing CO2 fixation, a differential equation for pi is derived. Carboxylation corresponds to the sink term of the pi pool and is assumed to be feedback-inhibited by aps. This simple model is shown to produce reasonable to excellent fits to data on the diurnal time courses of photosythesis, pi and gs sampled for oak leaves.

Control analysis of biochemical pathways: A novel procedure for calculating control coefficients, and an additional theorem for branched pathways

A novel method for calculating control coefficients of individual enzymes on fluxes and concentrations in metabolic pathways is presented. This method is derived by applying the theorem on implicit functions to the equations defining the steady state metabolite concentrations; it allows verification of the existing summation theorems and connectivity relations, and leads to a novel theorem for flux control coefficients in branched pathways. The method and the novel theorem are illustrated by several examples.

Stationary diffusion gradients associated with photosynthetic carbon flux-a study of compartmental versus diffusion-reaction models.

Metabolic processes usually involve diffusion of compounds in addition to their metabolic reactions. Such processes are adequately described by reaction-diffusion models (in the form partial differential equations) which are usually difficult and tedious to solve. Compartmental models (ordinary differential equations) are much easier to analyse but may be inadequate since they do not allow for spatial gradients. However, a compartmental model can be considered as the limit of a reaction-diffusion model for very fast diffusion (all diffusion coefficients D(j)--> infinity ). A compartmental model m(c) is termed associated to the reaction-diffusion model m(rd) if m(c) is the limit of m(rd) for all D(j)--> infinity. From the analytical solutions of a reaction-diffusion model and its associated compartmental model the extent of a diffusion gradient of m(rd) can be estimated by means of parameters from both m(c) and m(rd). This approach is extended to more complicated models that cannot be solved analytically. Gradients can be neglected and, consequently, the compartmental description be used, if the characteristic length s of the diffusion path is small compared with the distance a particle travels in time T(e), where T(e) is the characteristic time for the compartmental model. This ratio of lengths can also be expressed as the ratio of two times, namely the residence time s(2)/D(X) and the turnover time X(C)/v, where X(C) and v are the steady-state concentration of X and its import rate, respectively, for the associated compartmental model. Characteristic times are given for several simple reaction-diffusion systems in rectangular and spherical geometries. Intracellular gradients of HCO(3)(-) and CO(2) are calculated for some flux situations relevant to photosynthetic carbon fixation in green microalgae.

Determining Elasticities in Situ

Knowledge of in situ enzyme kinetics is an important aspect of metabolic regulation. Reaction kinetics is usually studied in vitro using enzymes that have been isolated from the organism by biochemical methods, even though it is known that such in vitro data may have little relevance in situ. Recently, this old and famous dilemma of enzyme kinetics has won new relevance by the needs of metabolic engineers for advice as to how the kinetics should be modified in order to reach a specific goal, such as overproduction of a compound. However, it is not only for the applied aspects that knowledge of the in situ kinetics of metabolic systems is essential. In situ kinetics is also an important and interesting topic in its own right. Among others, it helps us to find out whether our present understanding of enzyme kinetics is adequate to describe the in vivo situation.