Michael Geiger

Regulation of sucrose and starch metabolism in potato tubers in response to short-term water deficit

Abstract. To investigate the effect of water stress on carbon metabolism in growing potato tubers (Solanum tuberosum L.), freshly cut and washed discs were incubated in a range of mannitol concentrations corresponding to external water potential between 0 and −1.2 MPa. (i) Incorporation of [14C]glucose into starch was inhibited in water-stressed discs, and labeling of sucrose was increased. High glucose overrode the changes at low water stress (up to −0.5 MPa) but not at high water stress. (ii) Although [14C]sucrose uptake increased in water-stressed discs, less of the absorbed [14C]sucrose was metabolised. (iii) Analysis of the sucrose content of the discs confirmed that increasing water deficit leads to a switch, from net sucrose degradation to net sucrose synthesis. (iv) In parallel incubations containing identical concentrations of sugars but differing in which sugar was labeled, degradation of [14C]sucrose and labeling of sucrose from [14C]glucose and fructose was found at each mannitol concentration. This shows that there is a cycle of sucrose degradation and resynthesis in these tuber discs. Increasing the extent of water stress changed the relation between sucrose breakdown and sucrose synthesis, in favour of synthesis. (v) Analysis of metabolites showed a biphasic response to increasing water deficit. Moderate water stress (0–200 mM mannitol) led to a decrease of the phosphorylated intermediates, especially 3-phosphoglycerate (3PGA). The decrease of metabolites at moderate water stress was not seen when high concentrations of glucose were supplied to the discs. More extreme water stress (300–500 mM mannitol) was accompanied by an accumulation of metabolites at low and high glucose. (vi) Moderate water stress led to an activation of sucrose phosphate synthase (SPS) in discs, and in intact tubers. The stimulation involved a change in the kinetic properties of SPS, and was blocked␣by protein phosphatase inhibitors. (vii) The amount of ADP-glucose (ADPGlc) decreased when discs were incubated on 100 or 200 mM mannitol. There was a strong correlation between the in vivo levels of ADPGlc and 3PGA when discs were subjected to moderate water stress, and when the sugar supply was varied. (viii) The level of ADPGlc increased and starch synthesis was further inhibited when discs were incubated in 300–500 mM mannitol. (ix) It is proposed that moderate water stress leads to an activation of SPS and stimulates sucrose synthesis. The resulting decline of 3PGA leads to a partial inhibition of ADP-glucose pyrophosphorylase and starch synthesis. More-extreme water stress leads to a further alteration of partitioning, because it inhibits the activities of one or more of the enzymes involved in the terminal reactions of starch synthesis.

RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants.

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers

Abstract. Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571–581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers. Unexpectedly, the rate of degradation of [14C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed.

The role of transient starch in acclimation to elevated atmospheric CO2

Although increased concentrations of CO2 stimulate photosynthesis, this stimulation is often lost during prolonged exposure to elevated carbon dioxide, leading to an attenuation of the potential gain in yield. Under these conditions, a wide variety of species accumulates non‐structural carbohydrates in leaves. It has been proposed that starch accumulation directly inhibits photosynthesis, that the rate of sucrose and starch synthesis limits photosynthesis, or that accumulation of sugars triggers changes in gene expression resulting in lower activities of Rubisco and inhibition of photosynthesis. To distinguish these explanations, transgenic plants unable to accumulate transient starch due to leaf mesophyll‐specific antisense expression of AGP B were grown at ambient and elevated carbon dioxide. There was a positive correlation between the capacity for starch synthesis and the rate of photosynthesis at elevated CO2 concentrations, showing that the capability to synthesize leaf starch is essential for photosynthesis in elevated carbon dioxide. The results show that in elevated carbon dioxide, photosynthesis is restricted by the rate of end product synthesis. Accumulation of starch is not responsible for inhibition of photosynthesis. Although transgenic plants contained increased levels of hexoses, transcripts of photosynthetic genes were not downregulated and Rubisco activity was not decreased arguing against a role of sugar sensing in acclimation to high CO2.

Metabolism in slices from growing potato tubers responds differently to addition of sucrose and glucose

Abstract. The short-term changes in metabolism that occurred after adding glucose or sucrose to freshly cut discs from growing potato (Solanum tuberosum L.) tubers were investigated. (i) When glucose was supplied, there was a marked increase in glycolytic metabolites, and respiration was stimulated. When sucrose was supplied, amounts of glycolytic metabolites including hexose phosphates and 3-phosphoglycerate (3PGA) were similar to or lower than in control discs incubated without sugars, and respiration did not rise initially above that in control discs. This different response to sucrose and glucose was found across the concentration range 5–200 mM. A larger proportion of the metabolised 14C was converted to starch when [14C] sucrose was supplied than when [14C] glucose was supplied. The different effect on metabolite levels, respiration and starch synthesis was largest after 20–30 min, and decreased in longer incubations. (ii) When 5 or 25 mM sucrose was added in the presence of [14C] glucose, it led to a decrease in hexose phosphates and 3PGA, and a small increase in the rate of starch synthesis compared to discs incubated with glucose in the absence of sucrose. These differences were seen in a 30-min pulse and a 2-h pulse. Whereas ADP-glucose levels after adding sucrose resembled those in control discs, glucose led to a decrease in ADP-glucose. This decrease did not occur when 5 or 25 mM sucrose was added with the glucose. (iii) To check the relevance of these experiments for intact tubers, water or 100 mM mannitol, sucrose or glucose were supplied through the stolon to intact tubers for 24 h. A 0.2 mM solution of [14C] glucose was then introduced into the tubers, and its metabolism investigated during the next 30 min. Labelling of starch was increased after preincubation with sucrose, and significantly inhibited after preincubation with glucose. (iv) It is concluded that glucose and sucrose have different effects on tuber metabolism. Whereas glucose leads to a preferential stimulation of respiration, sucrose preferentially stimulates starch synthesis via a novel mechanism that allows stimulation of ADP-glucose pyrophosphorylase even though the levels of hexose phosphates and the allosteric activator 3PGA decrease.

Indirect Multiple Shooting for Nonlinear Parabolic Optimal Control Problems with Control Constraints

We discuss the indirect multiple shooting approach for the solution of PDE-based parabolic optimal control problems with control constraints. The method is formulated within an abstract function space setting and uses a space-time Galerkin finite element discretization. The emphasis is on the embedding of indirect multiple shooting into the optimal control framework as well as the detailed description of the discretization within the PDE context. Numerical results for linear and nonlinear model problems with and without control constraints illustrate the efficient use of indirect multiple shooting particularly in cases where other standard methods fail.

Direct and Indirect Multiple Shooting for Parabolic Optimal Control Problems

We present two multiple shooting approaches for optimal control problems (OCP) governed by parabolic partial differential equations (PDE). In the context of ordinary differential equations, shooting techniques have become a state-of-the-art solver component, whereas their application in the PDE case is still in an early phase of development. We derive both direct (DMS) and indirect (IMS) multiple shooting for PDE optimal control from the same extended problem formulation. This approach shows that they are algebraically equivalent on an abstract function space level. However, discussing their respective algorithmic realizations, we underline differences between DMS and IMS. In the numerical examples, we cover both linear and nonlinear parabolic side conditions.