Enzymes of starch metabolism in developing grains of high lysine barley mutant

The absolute activities of ADPG(UDPG)-pyrophosphorylase, starch phosphorylase, ADPG(UDPG)-starch synthetase, NDP-kinase and inorganic pyrophosphatase have been studied in high lysine mutant barley Notch-2 and its parent NP 113 grains during development. In general, mutant Notch-2 grains had higher average activities of UDPG-pyrophosphorylase and starch phosphorylase and lower activity of ADPG(UDPG)-starch synthetase per grain than the parent NP 113 during grain development. Activities of NDP-kinase, ADPG-pyrophosphorylase and inorganic pyrophosphatase differed only to a small extent between the mutant Notch-2 and NP 113. It is suggested that the lower activity of ADPG(UDPG)-starch synthetase might be responsible for the reduced accumulation of starch in the mutant Notch-2 grain as compared with parent NP 113 during development.     

An Escherichia coli mutant producing a truncated inactive form of GlgC synthesizes glycogen: further evidences for the occurrence of various important sources of ADPglucose in enterobacteria

AC70R1-504 Escherichia coli mutants possess a glgC* gene with a nucleotide change resulting in a premature stop codon that renders a truncated, inactive form of GlgC. Cells over-expressing the wild type glgC, but not those over-expressing the AC70R1-504 glgC*, accumulated high ADPglucose and glycogen levels. AC70R1-504 mutants accumulated glycogen, whereas DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery displayed a glycogen-less phenotype. AC70R1-504 cells with enhanced glycogen synthase activity accumulated high glycogen levels. By contrast, AC70R1-504 cells with high ADPG hydrolase activity accumulated low glycogen. These data further confirm that enterobacteria possess various sources of ADPglucose linked to glycogen biosynthesis.     

Escherichia coli AspP activity is enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars

Escherichia coli ADP-sugar pyrophosphatase (AspP) is a "Nudix" hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to glycogen biosynthesis. Moderate increases of AspP activity in the cell are accompanied by significant reductions of the glycogen content. In vitro analyses showed that AspP activity is strongly enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars, providing a first set of indicative evidences that AspP is a highly regulated enzyme. To our knowledge, AspP is the sole bacterial enzyme described to date which is activated by both G1,6P(2) and nucleotide-sugars.     

Phosphorylase activity in relation to starch synthesis in developing Hordeum distichum grain

Activity, control and primer requirements of starch phosphorylase in developing barley endosperm were investigated. Phosphorylase was detected in endosperm extracts from 3 days after anthesis. Unprimed activity was predominant between 2 and 10 days after anthesis, when it constituted 70–80% of total activity, but this proportion declined rapidly as the grain developed. The existence of at least 2 isoenzymes was indicated by studies of pH dependence and phosphate inhibition, and was further supported by acrylamide gel electrophoresis and column chromatography using DEAE-cellulose. The two isoenzymes which ere possibly both glyco proteins, appear in barley endosperm soon after anthesis. One appears capable of unprimed activity, and may be associated with the initiation of a-1,2 glucans, which then serve as primers for starch synthetase. This disappears by 13–15 days after anthesis. The other isoenzyme is capable of some unprimed activity but undergoes modification between 15 and 20 days after anthesis, resulting in the loss of unprimed activity. The relevance of the results to initiation of starch synthesis and to starch synthetase in amyloplasts is discussed.     

Enzymes of starch metabolism in leaves and berries of Vitis vinifera

Leaves of Vitis vinifera L., cv. Cabernet Sauvignon contained 2.0 mg of starch per g fresh weight, whereas young green berries and maturing grape berries contained less than 0.03 mg of starch, despite the presence of abundant substrates (reducing sugars and sucrose) in berries for starch synthesis. the activities of several enzymes likely to be involved in starch synthesis were determined in extracts of berries and leaves. Fractionation procedures resulted in final recoverable ADPglucose-starch glucosyltransferase activity which was 2–3 times the activity measured in crude extracts of leaves. Compared to leaves, berries contained low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase. These enzymes increased only 2- to 3-fold from young to maturing berries. ADPglucose-starch glucosyltransferase activity in the absence of added primer was found in leaf extracts but not in berry extracts. The activities of UDP-glucose pyrophosphorylase, phosphorylase and amylase were comparable in both leaves and berries and increased 6- to 7-fold during berry development. The low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase probably account for the paucity of starch in grape berries.     

Characteristics of α-glucan phosphorylase from Chlorella vulgaris

α-Glucan phosphorylase from Chlorella vulgaris has been partially purified. In the direction of glucan phosphorolysis the apparent K_m for Pi was ca 2.4 mM at pH 7.1. In the direction of glucan synthesis the K_m for G1P was ca 0.12 mM at pH 6.2. The enzymic activity was inhibited by physiological concentrations of ADP, ATP, ADPG and UDPG. In the direction of starch degradation in the presence of 2.4 mM Pi the I_0.5 values for ADP and ATP were ca 1.6 and 2.9 mM, respectively, while in the direction of synthesis in the presence of 0.12 mM G1P the values were ca 0.23 and 1.4 mM, respectively. The Hill plots for starch degradation showed n values of 2.2 for ADP and 2.2 for ATP and values of 1.5 and 1.2, respectively, for starch synthesis. Both ADPG and UDPG were linear competitive inhibitors either with respect to Pi or with respect to GIP. The K_i values for ADPG and UDPG in the direction of phosphorolysis were shown to be ca 0.11 and 0.51 mM, respectively, and those in the direction of synthesis 0.033 and 0.15 mM, respectively.     

Occurrence of more than one important source of ADPglucose linked to glycogen biosynthesis in Escherichia coli and Salmonella

To explore the possible occurrence of sources, other than GlgC, of ADPglucose linked to bacterial glycogen biosynthesis we characterized Escherichia coli and Salmonella DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery. These mutants displayed the expected glycogen-less phenotype but accumulated ADPglucose. Importantly, DeltaglgCAP cells expressing the glycogen synthase encoding glgA gene accumulated glycogen. Protein chromatographic separation of crude extracts of DeltaglgCAP mutants and subsequent activity measurement analyses revealed that these cells possess various proteins catalyzing the conversion of glucose-1-phosphate into ADPglucose. Collectively these findings show that enterobacteria possess more than one important source of ADPglucose linked to glycogen biosynthesis.     

Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity

ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, and is a highly regulated enzyme in the pathway of starch synthesis. In Arabidopsis thaliana, the enzyme is a heterotetramer, containing two small subunits encoded by the APS1 gene and two large subunits encoded by the APL1-4 genes. TILLING (Targeting Induced Local Lesions IN Genomes) of a chemically mutagenised population of A. thaliana plants identified 33 novel mutations in the APS1 gene, including 21 missense mutations in the protein coding region. High throughput measurements using a robotised cycling assay showed that maximal AGPase activity in the aps1 mutants varied from <15 to 117% of wild type (WT), and that the kinetic properties of the enzyme were altered in several lines, indicating a role for the substituted amino acid residues in catalysis or substrate binding. These results validate the concept of using such a platform for efficient high-throughput screening of very large populations of mutants, natural accessions or introgression lines. AGPase was estimated to have a flux control coefficient of 0.20, indicating that the enzyme exerted only modest control over the rate of starch synthesis in plants grown under short day conditions (8 h light/16 h dark) with an irradiance of 150 μmol quanta m⁻² s⁻¹. Redox activation of the enzyme, via reduction of the intermolecular disulphide bridge between the two small subunits, was increased in several lines. This was sometimes, but not always, associated with a decrease in the abundance of the APS1 protein. In conclusion, the TILLING technique was used to generate an allelic series of aps1 mutants in A. thaliana that revealed new insights into the multi-layered regulation of AGPase. These mutants offer some advantages over the available loss-of-function mutants, e.g. adg1, for investigating the effects of subtle changes in the enzyme's activity on the rate of starch synthesis.     

Leaf photosynthesis and carbohydrates of CO₂-enriched maize and grain sorghum exposed to a short period of soil water deficit during vegetative development

Among C₄ species, sorghum is known to be more drought tolerant than maize. The objective was to evaluate differences in leaf gas exchanges, carbohydrates, and two enzyme activities of these nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) C₄ subtype monocots in response to water deficit and CO₂ concentration ([CO₂]). Maize and sorghum were grown in pots in sunlit environmental-controlled chambers. Treatments included well watered (WW) and water stressed (WS) (water withheld at 26 days) and daytime [CO₂] of 360 (ambient) and 720 (elevated) μmol mol⁻¹. Midday gas exchange rates, concentrations of nonstructural carbohydrates, and activities of sucrose-phosphate synthase (SPS) and adenosine 5′-diphosphoglucose pyrophosphorylase (ADGP) were determined for fully expanded leaf sections. There was no difference in leaf CO₂ exchange rates (CER) between ambient and elevated [CO₂] control plants for both maize and sorghum. After withholding water, leaf CER declined to zero after 8 days in maize and 10 days for sorghum. Sorghum had lower stomatal conductance and transpiration rates than maize, which resulted in a longer period of CER under drought. Nonstructural carbohydrates of both control maize and sorghum were hardly affected by elevated [CO₂]. Under drought, however, increases in soluble sugars and decreases in starch were generally observed for maize and sorghum at both [CO₂] levels. For stressed maize and sorghum, decreases in starch occurred earlier and were greater at ambient [CO₂] than at elevated [CO₂]. For maize, drought did not meaningfully affect SPS activity. However, a decline in SPS activity was observed for drought-stressed sorghum under both [CO₂] treatments. There was an increase in ADGP activity in maize under drought for both [CO₂] treatments. Such a response in ADGP to drought, however, did not occur for sorghum. The generally more rapid response of maize than sorghum to drought might be related to the more rapid growth of leaf area of maize.