Suppression of glucan,water dikinase in the endosperm alters wheat grain properties,germination and coleoptile growth

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down‐regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain‐specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species.     

The distribution of covalently bound phosphate in the starch granule in relation to starch crystallinity

Five selected starches with a 60-fold span in their content of monoesterified starch phosphate were investigated with respect to distribution of glucose 6-phosphate and glucose 3-phosphate residues, amylopectin chain length distributions and gelatinisation properties. The distribution of starch phosphate in the starch granules was determined by preparation of N?geli dextrins followed by quantitative 31P-nuclear magnetic resonance spectroscopy. Total starch phosphate content was positively correlated to the unit chain lengths of the amylopectin as well as to the chain lengths of the corresponding N?geli dextrins. The major part (68-92%) of the total starch phosphate content was partitioned to the hydrolysed (amorphous) parts. Starch-bound glucose 6-phosphate per milligram of starch was 2-fold enriched in the amorphous parts, whereas phosphate groups bound at the 3-position were more evenly distributed. The gelatinisation temperatures of the native starches as determined by differential scanning calorimetry were positively correlated (R(2)=0.75) to starch phosphate content, while crystallinity (gelatinisation enthalpy) and crystal heterogeneity (endotherm peak width) showed no correlations to starch phosphate content. The relations between starch molecular structure, architecture and functional properties are discussed.     

Amylopectin aggregation as a function of starch phosphate content studied by size exclusion chromatography and on-line refractive index and light scattering

Starches with a natural 65-fold span in covalently bound phosphate content were prepared from five different crops including sorghum, cassava, three potato varieties and an exotic ginger plant, Curcuma zedoaria, with extreme starch phosphate content. These starches were subjected to size exclusion chromatography with refractive index detection (SEC/RI). A simple and rapid method for starch solubilisation was used. The conditions during solubilisation (2 M NaOH) and separation (10 mM NaOH, 50°C) were such as enabling >94% recovery of the starch without detectable degradation. The aggregation properties of the starch was investigated using on line refractive index/multi angle laser light scattering (RI/MALLS) detection. Three major regions in the SEC profile were identified, consisting of large amylopectin aggregates, amylopectin particles with radius of gyration (R_g) of approx 200 nm (400 nm blocklets) and amylose. A procedure for correction of light scattering signals spread over the SEC profile as a result of aggregate tailing was developed. The significance of the relative amounts of these three molecular species on standard starch pasting parameters, as measured by a Rapid Visco Analyzer (RVA), was investigated. Starches with a high amount of amylopectin aggregates showed high peak viscosities. Moreover, very high amounts of starch bound phosphate or amylose appears to suppress the content of large aggregates resulting in low viscosity.     

Salinity-dependent regulation of starch and glycerol metabolism in Dunaliella parva

Abstract. The glycerol and starch metabolism of synchronized Dunaliella parva cells as a function of the salinity of the medium has been investigated.
The higher the salinity of the medium the higher is the rate of glycerol synthesis and the endogenous glycerol concentration, whereas starch content and salinity of the medium are inversely related. Upon transfer to a hyperosmotic NaCl-medium cells respond by an immediate increase in glycerol synthesis and an inhibition of starch formation in the light. Under corresponding conditions in darkness, starch degradation is stimulated. In both light and darkness hyperosmotic shocks are followed by a rapid increase in the endogenous pool of inorganic phosphate (P_i). It is suggested that in the light the increase in the endogenous phosphate level inhibits the chloroplast ADPG-pyrophosphorylase (E.G.2.7.7.27), and thereby starch synthesis, and promotes starch phosphorolysis. Photosynthetically produced triosephosphates and triosephosphates derived from starch degradation are converted to glycerol. Also, in the dark the increase in the P_i-level stimulates phosphorolytic starch degradation and thereby synthesis of glycerol. Reasons for the salt stress induced increase in the endogenous P_i-level are discussed.     

Physico-chemical and degradative properties of in-planta re-structured potato starch

Starch re-structured directly in potato tubers by antisense suppression of starch branching enzyme (SBE), granule bound starch synthase (GBSS) or glucan water dikinase (GWD) genes was studied with the aim at disclosing the effects on resulting physico-chemical and enzyme degradative properties. The starches were selected to provide a combined system with specific and extensive alterations in amylose and covalently esterified glucose-6-phosphate (G6P) contents. As an effect of the altered chemical composition of the starches their hydrothermal characteristics varied significantly. Despite of the extreme alterations in phosphate content, the amylose content had a major affect on swelling power, enthalpy for starch gelatinization and pasting parameters as assessed by Rapid Visco Analysis (RVA). However, a combined influence of the starch phosphate and long glucan chains as represented by high amylose or long amylopectin chain length was indicated by their positive correlation to the final viscosity and set back (RVA) demonstrating the formation of a highly hydrated and gel-forming system during re-structuring of the starch pastes. Clear inverse correlations between glucoamylase-catalyzed digestibility and amylopectin chain length and starch phosphate and lack of such correlation with amylose content indicates a combined structuring role of the phosphate groups and amylopectin chains on the starch glucan matrix.     

Action of glucoamylase from Aspergillus niger on phosphorylated substrate

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger was purified to be free from alpha-amylase and phosphatase (glucose 6-phosphate as substrate). The phosphatase was well separated from the glucoamylase by phosphocellulose ion-exchange chromatography. The glucoamylase action was prevented by the esterified phosphate groups of the substrate. Thus, the extensive action of the glucoamylase on potato starch exposed the 6-posphorylglucosyl residue of the starch at the non-reducing terminal and large molecular weight limit dextrins remained. The concomitant action of the phosphatase was necessary for the complete degradation of the starch.     

STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana

Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.     

Enzymatic degradation of and bovine serum albumin release from starch-acetate films

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.     

Phosphorylation of transitory starch is increased during degradation

The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied (32)P orthophosphate into starch. Illuminated cells incorporated (32)P into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with (32)P/(31)P orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch.     

‘Last In–First Out’: seasonal variations of non‐structural carbohydrates,glucose‐6‐phosphate and ATP in tubers of two Arum species

• Knowledge on the metabolism of polysaccharide reserves in wild species is still scarce. In natural sites we collected tubers of Arum italicum Mill. and A. maculatum L. – two geophytes with different apparent phenological timing, ecology and chorology – during five stages of the annual cycle in order to understand patterns of reserve accumulation and degradation.
• Both the entire tuber and its proximal and distal to shoot portion were utilised. Pools of non‐structural carbohydrates (glucose, sucrose and starch), glucose‐6‐phosphate and ATP were analysed as important markers of carbohydrate metabolism.
• In both species, starch and glucose content of the whole tuber significantly increased from sprouting to the maturation/senescence stages, whereas sucrose showed an opposite trend; ATP and glucose‐6‐phosphate were almost stable and dropped only at the end of the annual cycle. Considering the two different portions of the tuber, both ATP and glucose‐6‐phosphate concentrations were higher in proximity to the shoot in all seasonal stages, except the flowering stage.
• Our findings suggest that seasonal carbon partitioning in the underground organ is driven by phenology and occurs independently of seasonal climate conditions. Moreover, our results show that starch degradation, sustained by elevated ATP and glucose‐6‐phosphate pools, starts in the peripheral, proximal‐to‐shoot portion of the tuber, consuming starch accumulated in the previous season, as a ‘Last In–First Out’ mechanism of carbohydrate storage.

     

Physiological rates of starch breakdown in isolated intact spinach chloroplasts

Starch breakdown with rates above 10 μatom carbon per mg chlorophyll per hour has been monitored in spinach chloroplasts and compares favorably with the rates in whole leaves. Intact starch-loaded chloroplasts were prepared from protoplasts to avoid rupture during mechanical homogenization and rapid centrifugation. Particular attention was paid to the identification of all the products of starch degradation and to measuring the actual rates of their accumulation. The products of starch breakdown included triose phosphate, 3-phosphoglycerate, CO₂, glucose, and some maltose. Comparison of the rates of metabolism of added glucose and of the conversion of starch to phosphorylated intermediates showed that starch phosphorolysis was the major pathway leading to phosphorylated endproducts. From the results, the relative contribution of phosphorolysis and hydrolysis to starch breakdown and the contribution of glycolysis and the oxidative pentose phosphate cycle can be estimated. Phosphate has a large influence on the metabolism of the chloroplast in the dark.     

Bioremediation of sago industry effluent and its impact on seed germination (green gram and maize)

In this study, we attempted two investigational systems: one is treatment of sago industry effluent by aerobic bacterial consortium and the other is impact of treated and untreated effluent on seed germination. For the treatment system, the starch degrading bacteria were isolated from sago industry effluent and effluent contaminated soil. The genera, Alcaligenes, Bacillus and Corynebacterium were found efficient in starch degradation. The selected isolates were tested for their efficiency on the degradation of starch both in Mineral Salts Medium (MSM) and in sago industry effluent. About 85% of the starch was degraded in MSM by a bacterial consortium composed of Alcaligenes, Bacillus and Corynebacterium, whereas in effluent the degradation of starch was only 63%. The physico-chemical properties such as electrical conductivity, total solids, suspended solids, dissolved solids, BOD, COD, nitrogen and phosphate were found decreased in effluent after 72 h. The pH of the effluent was relatively increased from 3 to 6.7. The study of seed germination (maize and green gram) was carried out at 25, 50, 75 and 100% concentrations of treated and untreated effluent using soil sowing method. Shoot length, root length, fresh weight, dry weight and chlorophyll content showed an increase when treated effluent was tested whereas a decrease of growth was noticed in untreated effluent tested seedlings. The results revealed that effluent treated by aerobic microorganisms has no negative impact on the seed germination and can be effectively used for irrigation.     

Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.     

Dry weight accumulation and starch-synthesis enzymes in grain of cultured ears of three winter wheat varieties

Detached ears of three winter wheat ( Triticum aestivum L.) varieties were cultured in solution for 12 days with sucrose levels varying from 36.5 to 292 m M. The dry weight and starch content of grains increased asymptotically with the sucrose level in the solution. At 4 days of culture, glucose phosphate isomerase (EC 5.3.1.9) activity grain⁻¹ was lower with 36.5 m M than with higher sucrose levels in the medium; at 8 days, adenosinc diphosphoglucose pyrophosphorylase (EC 2.7.7.27) and (soluble plus bound) starch synthase (EC 2.4.1.21) activities grain⁻¹ were higher with 146 and 292 m M sucrose than with 36.5 and 73 m M sucrose. The multiple regression of starch content over these enzyme activities showed that starch synthase was relatively more important as an independent variable. The dry weight and starch content of grains were higher in the variety Maris Huntsman than in Splendeur and Hobbit. The water content of grains was lower in Splendeur than in the other two varieties. At 4 days the glucose phosphate isomerase, adenosine diphosphoglucose pyrophosphorylase and starch synthase activities grain⁻¹ were smaller in Splendeur than in Hobbit and Maris Huntsman and al 8 days they were higher in Maris Huntsman than in Hobbit and Splendeur. The varietal differences in starch content of grains were related to the activities of glucose phosphate isomerase and especially of starch synthase.     

The effects of diet on the esterification of glycerol phosphate, dihydroxyacetone phosphate and 2-hexadecylglycerol by homogenates of rat adipose tissue.

1. Male rats were fed for 5 weeks after weaning on a diet containing (by weight) 59% of starch or on diets that contained 39% of starch and 20% of either sucrose, beef tallow or corn oil. 2. The rats fed on the beef tallow consumed more energy than did the rats fed on the starch and sucrose diets. The rats fed on the corn oil drank less water than did the other groups of rats. 3. There were no significant differences between the four groups in terms of body-weight gain, epididymal-fat-pad weight and in the size, number and triacylglycerol content of the adipocytes in the fat-pads. 4. There was a significant correlation (P less than 0.001) between the activities of glycerol phosphate acyltransferase and monoacylglycerol acyltransferase in individual rats. Both of these activities were highest in the group fed on the high-starch diet and both correlated with the consumption of glucose by individual rats in the four groups. 5. The percentage of glycerol phosphate converted into diacylglycerol and triacylglycerol was positively correlated with the mean diameters, surface area and triacylglycerol content of the adipocytes for individual rats and was greates in the sucrose-fed rats. 6. The specific activity of dihydroxyacetone phosphate acyltransferase was highest in the rats fed on beef tallow. This activity was positively correlated with the energy intake for all dietary groups over the 5-week feeding period. 7. The results are discussed in terms of the functions of the three routes of glycerolipid synthesis in adipose tissue.     

Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii

We have used phosphate, nitrogen, or carbon limited batch and continuous flow cultures to study how growth and biochemical composition of the dinoflagellate Crypthecodinium cohnii CCMP 316 is affected by nutrient limitation. Specific contents of phosphorous, proteins, and starch were differently affected by nutrient limitation. The specific phosphorous content in C. cohnii varied 10-20 times depending on phosphate availability in the medium. When phosphate was available it was taken up in excess and stored to be re-utilized during phosphate limitation. The specific protein content varied twofold. At most conditions, proteins made up 12-15% of the biomass dry weight but when cells were nitrogen limited, the specific protein content was only half this value. Floridean starch was the major cell constituent of C. cohnii accounting for 40-50% of the biomass dry weight. Only during carbon limitation did the specific starch content decrease. In contrast was the specific lipid content almost unaffected by nutrient availability and lipids accounted for 12-15% of the biomass dry weight irrespectively of which nutrient that was limiting. Lipid production does therefore not depend on nutrient limitation in C. cohnii and lipids are produced even by carbon limited cells. Cultures grown under phosphate limitation resulted in formation of cells with maximal specific contents of all the three major cell constituents; starch, lipid, and protein.     

Entamoeba histolytica zymodemes: exhibition of gamma and delta bands only of glucose phosphate isomerase and phosphoglucomutase may be influenced by starch content in the medium.

Entamoeba histolytica isolated from human can be associated with either symptomatic disease or with asymptomatic carriers. Pathogenic and nonpathogenic strains can be distinguished on the basis of differences in the electrophoretic patterns of four isoenzymes (zymodeme). With glucose phosphate isomerase and phosphoglucomutase, we observed variation of the expression of their gamma bands as a function of starch volume in culture. We cultured E. histolytica strains from different zymodemes in Robinson medium using both a low (2-4 mg/bottle) and a high (12-15 mg/bottle) content of rice starch as supplement. These cultures were monitored by electrophoresis of glucose phosphate isomerase and phosphoglucomutase. Strains having gamma or delta bands exhibited those bands when a high content of starch was used in culture, but did not do so with a low content. Contrarily, strains that never exhibited those bands did not express them when the amount of starch in the culture was increased. However, alpha and beta bands of the same isoenzymes were always present and never showed any variation. The results suggest that expression of gamma and delta bands of glucose phosphate isomerase and phosphoglucomutase are subject to culture conditions and that genes coding for those isoenzymes may be different from those coding for the alpha and beta bands.     

Incorporation of alpha-amylase enzyme and a bioactive filler into hydrophilic, partially degradable, and bioactive cements (HDBCs) as a new approach to tailor simultaneously their degradation and bioactive behavior

Hydrophilic, partially degradable, and bioactive cements (HDBCs) are starch-containing cements intended to degrade partially in the human body and, in so doing, allow for bone ingrowth inside the pores formed during degradation. Therefore, the study of degradation and bioactivity behavior was performed to assess the suitability of the current HDBCs formulations to achieve those aims. The degradation profile of HDBCs was studied under different conditions, including incubation in phosphate-buffered saline (PBS) and PBS supplemented with alpha-amylase at different concentrations. Thermostable alpha-amylase was also added to some formulations to allow control of the degradation rate and its extent. In a second stage the simultaneous phenomena of enzymatic degradation and bioactivity (both in vitro) was studied. We observed that the degradation of starch present in HDBCs can be easily controlled by the amount of alpha-amylase added to the cement and high values of degradation may be achieved if high enough quantities of enzyme are incorporated. However, the maximum degradation extent is much more dependent on the total amount of starch present in the formulation than on the amount of enzyme added to it: for full pore connectivity, the amount of starch should be higher than the percolation threshold for a 3D specimen. Nonetheless, calcium phosphate was able to nucleate and spread in inner pores of the cement, formed due to degradation, if they were interconnected. For a more thorough covering of the pores with calcium phosphates the amount of starch present in HDBCs should be increased to be higher than the percolation threshold.