Translucent Tissue Defects in Solanum tuberosum L: I. Alterations in Amyloplast Membrane Integrity, Enzyme Activities, Sugars, and Starch Content

Kennebec (cv) potatoes randomly developed translucent areas in their centrally located pith-parenchymal cells during storage. These defective areas were characterized as having reduced starch concentration and increased levels of free sugars (i. e. sucrose and glucose) and inorganic phosphate. Electron micrographs of potato tubers stored at 10° ± 1°C for 8 months indicated that the amyloplast membrane was still intact and continuous around starch granules in both normal and prematurely sweetened tissue. The total activities of phosphorylase and sucrose-6-P synthase were elevated 5.4- and 3.8-fold, respectively, in the defective tissue compared to healthy nonsweetened tubers while there were no significant differences in the levels of sucrose synthase, UDPglucose pyrophosphorylase, invertase, or α-amylase. Total and specific activities of acid phosphatase were only slightly elevated in translucent tissue but their increase was significant (P < 0.05, t test) over that seen in healthy tubers. The premature sweetening in storage may have been indirectly triggered by moisture and heat stress experienced during development. Translucency eventually led to physical deterioration of the tissue.     

Adhesion of Lactobacillus amylovorus to Insoluble and Derivatized Cornstarch Granules

Approximately 70% of the cells in a suspension of the amylolytic bacterium Lactobacillus amylovorus bind to cornstarch granules within 30 min at 25°C. More than 60% of the bound bacteria were removed by formaldehyde (2%) or glycine (1 M) at pH 2.0. More than 90% of the bound bacteria were removed by MgCl₂ (2 M; pH 7.0). Binding of L. amylovorus to cornstarch was inhibited in heat-killed cells and in cells that had been pretreated with glutaraldehyde, formaldehyde, sodium azide, trypsin, or 1% soluble potato starch. Bacterial binding to cornstarch appeared to correlate with both the concentration of cornstarch in the suspension and the amylose content in the granules. The ability of L. amylovorus to adhere to cornstarch granules was reduced for granules that had been extracted with HCl-ethanol, HCl-methanol, HCl-propanol, or HCl-butanol. Chemical derivatization of cornstarch resulted in a wide variety of adhesion responses by these bacteria. For example, 2-O-butyl starch (degree of substitution, 0.09) enhanced adhesion, whereas two palmitate starches (degree of substitution, 0.48 and 0.09) exhibited reduced adhesion activities. 2-O-(2-hydroxybutyl) starch and starch-poly(ethylene-co-acrylic acid) ester showed adhesion activities similar to those of the nonderivatized starch controls.     

Translucent tissue defect in potato (Solanum tuberosum L.) tubers is associated with oxidative stress accompanying an accelerated aging phenotype

Translucent tissue defect (TTD) is an undesirable postharvest disorder of potato tubers characterized by the development of random pockets of semi-transparent tissue containing high concentrations of reducing sugars. Translucent areas turn dark during frying due to the Maillard reaction. The newly released cultivar, Premier Russet, is highly resistant to low temperature sweetening, but susceptible to TTD. Symptoms appeared as early as 170 days after harvest and worsened with time in storage (4–9 °C, 95 % RH). In addition to higher concentrations of glucose, fructose and sucrose, TTD resulted in lower dry matter, higher specific activities of starch phosphorylase and glc-6-phosphate dehydrogenase, higher protease activity, loss of protein, and increased concentrations of free amino acids (esp. asparagine and glutamine). The mechanism of TTD is unknown; however, the disorder has similarities with the irreversible senescent sweetening that occurs in tubers during long-term storage, where much of the decline in quality is a consequence of progressive increases in oxidative stress with advancing age. The respiration rate of non-TTD ‘Premier Russet’ tubers was inherently higher (ca. 40 %) than that of ‘Russet Burbank’ tubers (a non-TTD cultivar). Moreover, translucent tissue from ‘Premier Russet’ tubers had a 1.9-fold higher respiration rate than the average of non-translucent tissue and tissue from non-TTD tubers. Peroxidation of membrane lipids during TTD development resulted in increased levels of malondialdehyde and likely contributed to a measurable increase in membrane permeability. Superoxide dismutase and catalase activities and the ratio of oxidized to total glutathione were substantially higher in translucent tissue. TTD tubers also contained twofold less ascorbate than non-TTD tubers. TTD appears to be a consequence of oxidative stress associated with accelerated aging of ‘Premier Russet’ tubers.     

Pyrophosphorylases in Solanum tuberosum: I. Changes in ADP-Glucose and UDP-Glucose Pyrophosphorylase Activities Associated with Starch Biosynthesis during Tuberization, Maturation, and Storage of Potatoes

Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C. Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. When tubers reached an average diameter of 1 centimeter (0.5 gram average tuber weight) and had already established 58% starch on a dry weight basis, ADP-glucose pyrophosphorylase increased 16- to 24-fold over its activity seen in low starch containing stolon tissue. During this same period UDP-glucose pyrophosphorylase increased approximately 2- to 3-fold. Although participation of UDP-glucose in starch formation can not be neglected, it is suggested that the onset of rapid non-photosynthetic potato tuber starch biosynthesis may be closely related to the simultaneous increase in ADP-glucose pyrophosphorylase activity.     

Adhesion of Bifidobacteria to Granular Starch and Its Implications in Probiotic Technologies

Adhesion of 19 Bifidobacterium strains to native maize, potato, oat, and barley starch granules was examined to investigate links between adhesion and substrate utilization and to determine if adhesion to starch could be exploited in probiotic food technologies. Starch adhesion was not characteristic of all the bifidobacteria tested. Adherent bacteria bound similarly to the different types of starch, and the binding capacity of the starch (number of bacteria per gram) correlated to the surface area of the granules. Highly adherent strains were able to hydrolyze the granular starches, but not all amylolytic strains were adherent, indicating that starch adhesion is not a prerequisite for efficient substrate utilization for all bifidobacteria. Adhesion was mediated by a cell surface protein(s). For the model organisms tested (Bifidobacterium adolescentis VTT E-001561 and Bifidobacterium pseudolongum ATCC 25526), adhesion appeared to be specific for α-1,4-linked glucose sugars, since adhesion was inhibited by maltose, maltodextrin, amylose, and soluble starch but not by trehalose, cellobiose, or lactose. In an in vitro gastric model, adhesion was inhibited both by the action of protease and at pH values of ≤3. Adhesion was not affected by bile, but the binding capacity of the starch was reduced by exposure to pancreatin. It may be possible to exploit adhesion of probiotic bifidobacteria to starch granules in microencapsulation technology and for synbiotic food applications.     

Thermostable, Raw-Starch-Digesting Amylase from Bacillus stearothermophilus

An endospore-forming thermophilic bacterium, which produced amylase and was identified as Bacillus stearothermophilus, was isolated from soil. The amylase had an optimum temperature of 70°C and strongly degraded wheat starch granules (93%) and potato starch granules (80%) at 60°C.     

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.     

Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 °C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both α-1,4 and α-1,6 linkages of starch molecule in the potato starch.     

Expression of an amylosucrase gene in potato results in larger starch granules with novel properties

Main conclusion

Expression of amylosucrase in potato resulted in larger starch granules with rough surfaces and novel physico-chemical properties, including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. Starch is a very important carbohydrate in many food and non-food applications. In planta modification of starch by genetic engineering has significant economic and environmental benefits as it makes the chemical or physical post-harvest modification obsolete. An amylosucrase from Neisseria polysaccharea fused to a starch-binding domain (SBD) was introduced in two potato genetic backgrounds to synthesize starch granules with altered composition, and thereby to broaden starch applications. Expression of SBD–amylosucrase fusion protein in the amylose-containing potato resulted in starch granules with a rough surface, a twofold increase in median granule size, and altered physico-chemical properties including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. These effects are possibly a result of the physical interaction between amylosucrase and starch granules. The modified larger starches not only have great benefit to the potato starch industry by reducing losses during starch isolation, but also have an advantage in many food applications such as frozen food due to its extremely high freeze–thaw stability.     

Use of polyethylene glycol in isolation and assay of stable, enzymically active starch granules from developing wheat endosperms

A procedure using polyethylene glycol (PEG), molecular weight 1000, was developed for the isolation of starch granules from wheat endosperm. Immature endosperm tissue was cut repeatedly in 300 millimolar PEG 1000 and filtered through Miracloth. Centrifugation separated a pellet from a supernatant with inhibitory activity. The pellet contained several enzyme activities, including soluble and bound components of starch synthase, starch phosphorylase, and sucrose synthase activities. The starch phosphorylase activity was unaffected by several washings with 300 millimolar PEG 1000 but was lost when the granules were washed once without PEG or washed with sucrose, glycerol, or sorbitol (up to 30%, w/v). The fraction of starch synthase, remaining on the granules after a wash without PEG (the `bound' activity) was not affected by the addition of 30% sorbitol to the wash buffer. This fraction became larger with grain development (0.2-0.7).

To obtain high activity, PEG was required not only during isolation of granules but also in the assay of both starch phosphorylase and starch synthase giving optimum activity at 225 to 255 millimolar. PEG reduced the requirement for glycogen as primer with soluble starch synthase. However, the `bound' starch synthase activity was unaffected by PEG. PEG of different size were compared by their effects in the assay of starch granules: with increase in molecular size, the same effect was obtained at ever lower polymer concentration (w/v) down to a limit.

Treatment of granules with Triton X-100 did not affect their starch synthase activity, but it removed the capacity to incorporate label from UDP [¹⁴C]G into non-starch polymers.

It is concluded that PEG, like some other active compounds (ethanol Na₃-citrate, and Ficoll) could mediate enzyme-primer interaction by exclusion.

     

Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial beta-amylases

Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them beta-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized beta-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.     

Characterization of a Potato Starch-digesting Bacterium and Its Production of Amylase

A bacterium which can utilize potato starch granules as sole carbon source was isolated and identified as Bacillus circulans from its physiological and biochemical properties. Scanning electron microscopic observation of potato starch granules recovered from the culture broth revealed that granules were degraded gradually from their surface resulting in elongated granules with layered structures on their surface. This bacterium produced extracellular amylase which can digest potato starch granules in vitro. The amylase has a unique property in that it produces only maltohexaose from gelatinized starch in the early stage of the reaction. For the production of this amylase potato starch was found to be most effective while soluble sugars including gelatinized starch and maltose had little effect.     

Non‐GMO potato lines,synthesizing increased amylose and resistant starch,are mainly deficient in isoamylase debranching enzyme

Solanum tuberosum potato lines with high amylose content were generated by crossing with the wild potato species Solanum sandemanii followed by repeated backcrossing to Solanum tuberosum lines. The trait, termed increased amylose (IAm), was recessive and present after three generations of backcrossing into S. tuberosum lines (6.25% S. sandemanii genes). The tubers of these lines were small, elongated and irregular with small and misshaped starch granules and high sugar content. Additional backcrossing resulted in less irregular tuber morphology, increased starch content (4.3%–9.5%) and increased amylose content (29%–37.9%) but indifferent sugar content. The amylose in the IAm starch granules was mainly located in peripheral spots, and large cavities were found in the granules. Starch pasting was suppressed, and the digestion‐resistant starch (RS) content was increased. Comprehensive microarray polymer profiling (CoMPP) analysis revealed specific alterations of major pectic and glycoprotein cell wall components. This complex phenotype led us to search for candidate IAm genes exploiting its recessive trait. Hence, we sequenced genomic DNA of a pool of IAm lines, identified SNPs genome wide against the draft genome sequence of potato and searched for regions of decreased heterozygosity. Three regions, located on chromosomes 3, 7 and 10, respectively, displayed markedly less heterozygosity than average. The only credible starch metabolism‐related gene found in these regions encoded the isoamylase‐type debranching enzyme Stisa1. Decreased expression of mRNA (>500 fold) and reduced enzyme activity (virtually absent from IAm lines) supported Stisa1 as a candidate gene for IAm.     

Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping

Background

Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis.

Methods

Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme.

Results

Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5 μm in diameter.

Conclusions

Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before.

General significance

μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Physicochemical and Binder Properties of Starch Obtained from Cyperus esculentus

The purpose of this study was to isolate starch from the tubers of Cyperus esculentus L. and evaluate its physicochemical and binder properties. Extraction of starch using sodium metabisulfite yielded 37 g of starch per 100 g of the tubers. Scanning electron microscopy indicated that Cyperus starch consists of oval to elliptical particles with a smooth surface. Cyperus starch demonstrates a narrow particle size distribution with a mean of 8.25 μm. Cyperus starch conforms well to United States Pharmacopeia standards established for widely used starches like maize and potato. The X-ray powder diffraction pattern and moisture sorption profile of Cyperus starch were comparable to that of maize starch. Cyperus starch had lower swelling power than maize and potato starch, indicative of stronger associative forces within the granules. Carr’s index and Hausner ratio indicate that Cyperus starch should have comparable flow properties with respect to maize and potato starch. Cyperus starch was employed as binder for the formulation of metronidazole tablets. Formulations containing 5%, 7.5%, and 10% Cyperus starch were compared with those containing 10% potato starch. At 10% binder concentration, the tablets containing Cyperus starch exhibited better hardness and negligible friability as compared with those with potato starch. Although the binder concentration had a significant effect on the disintegration time of the tablets, it did not seem to affect the dissolution profile. These results indicate that Cyperus starch provides excellent binding properties without compromising drug release characteristics and should be explored in pharmaceutical formulations.Key words: Cyperus esculentus, Cyperus starch, pharmaceutical excipient, physicochemical evaluation, starch characterization     

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.     

Starch biosynthesis: experiments on how starch granules grow in vivo

Four varieties of starch granules from potato, wheat, maize, and rice were fractionated into homogeneous 10-μm-sized ranges. The size with the largest amount of granules was reacted with ADP-[¹⁴C]Glc, washed, and peeled into 7−9 layers, using a controlled peeling process, involving 90:10 volume proportions of Me₂SO-H₂O at 10 °C. All of the starches showed biosynthesis of starch throughout the granules. Starch synthase activities were determined for each of the layers. Three of the starches had a relatively large amount of synthase activity in the second layer, with only a small amount in the first layer. Potato starch had the largest amount of activity in the first layer. Starch synthase activity was found to alternate between higher and lower activities throughout all of the varieties of granules, showing that the synthesis was not uniform and also was not exclusively occurring at the surface of the starch granules, which had previously been hypothesized. From these results and our previous studies on the mechanism of starch chain elongation by the addition of d-glucose to the reducing end of a growing chain that is covalently attached to the active site of starch synthase, a hypothesis is proposed for how starch granules grow in vivo.