Light-induced degradation of storage starch in turions of Spirodela polyrhiza depends on nitrate

Light induces both the germination of turions of the duckweed Spirodela polyrhiza and the degradation of the reserve starch stored in the turions. The germination photoresponse requires nitrate, and we show here that nitrate is also needed for the light-induced degradation of the turion starch. Ammonium cannot substitute for nitrate in this regard, and nitrate thus acts specifically as signal to promote starch degradation in the turions. Irradiation with continuous red light leads to starch degradation via auto-phosphorylation of starch-associated glucan, water dikinase (GWD), phosphorylation of the turion starch and enhanced binding of alpha-amylase to starch granules. The present study shows that all of these processes require the presence of nitrate, and that nitrate exerts its effect on starch degradation at a point between the absorption of light by phytochrome and the auto-phosphorylation of the GWD. Nitrate acts to coordinate carbon and nitrogen metabolism in germinating turions: starch will only be broken down when sufficient nitrogen is present to ensure appropriate utilization of the released carbohydrate. These data constitute the first report of control over the initiation of reserve starch degradation by nitrate.     

Light induces phosphorylation of glucan water dikinase, which precedes starch degradation in turions of the duckweed Spirodela polyrhiza

Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [betagamma-33P]ATP but not by [gamma-33P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the beta-phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions.     

Association of alpha-amylase and the R1 protein with starch granules precedes the initiation of net starch degradation in turions of Spirodela polyrhiza

In turions of Spirodela polyrhiza (L.) Schleiden, net degradation of storage starch is controlled by a special low fluence response of phytochrome requiring illumination for several days. This light effect has been used to study protein-starch interactions that occur prior to and during net degradation of starch. Following various pretreatments on S. polyrhiza turions, native starch granules were isolated and two fractions of starch-related proteins were distinguished: proteins enclosed within the starch particles (starch-internalized proteins) and those attached to the surface (starch-associated proteins). The pattern of starch-associated proteins as resolved by SDS-PAGE was more complex than that of starch-internalized proteins and varied depending upon the pretreatment of the turions. Two starch associated proteins were identified immunochemically as alpha-amylase (EC 3.2.1.1) and the R1 protein (Lorberth et al. (1998) Nature Biotechnology 16: 473-477). Dark-pretreatment of non-dormant turions does not induce starch net degradation. Under these conditions, alpha-amylase and R1 were bound to the surface of the starch granules. Continuous illumination with red light induces a rapid degradation of starch. Within the first 24 h of illumination the level of starch-associated alpha-amylase transiently increased and subsequently decreased rapidly. Similarly, the amount of the starch-associated R1 also decreased during illumination. The dissociation of both alpha-amylase and R1 from the starch granules preceded the decrease in starch content. However, binding of the two proteins to starch granules remained unchanged when the turions did not perform net starch degradation (as observed during continuous darkness, orthophosphate deficiency, or dormancy of the turions). Thus, during net starch degradation, so far unidentified changes are postulated to occur at the surface of the starch particles that are relevant for protein binding. This conclusion was supported by in vitro studies in which the binding of purified beta-amylase (EC 3.2.1.2) to starch granules isolated from turions following various pretreatments was monitored. The enzyme did bind to starch granules prepared from dark-stored turions (in which starch degradation had not been initiated), but not to those isolated from illuminated (starch degrading) turions.     

A model for starch breakdown in higher plants

An in vitro system for the breakdown of starch granules by mixtures of α- and β-amylase is developed and discussed with reference to information concerning the degradation of starch in vivo. β-Amylase has no action on starch granules and has very little effect on the rate of starch granule digestion by α-amylase. It does, however, affect the product distribution in an α-amylase digest and is considered to attack dextrin intermediates produced by the action of α-amylase on the starch granules.     

Light-induced degradation of starch granules in turions of Spirodela polyrhiza studied by electron microscopy

Spirodela polyrhiza forms turions, starch-storing perennial organs. The light-induced process of starch degradation starts with an erosion of the surface of starch grains. The grain size decreases over a period of red irradiation and the surface becomes rougher. The existence of funnel-shaped erosion structures demonstrates that starch degradation is also possible inside the grains. Neither etioplasts nor clues as to their transition into chloroplasts were found in the storage tissue by transmission electron microscopy. Juvenile chloroplasts always contained the starch grains which remained from amyloplasts. No chloroplasts were found which developed independently of starch grains. Amyloplasts are therefore the only source of chloroplasts in the cells of irradiated turions. The intactness of amyloplast envelope membranes could not be directly proved by electron microscopy. However, the light-induced transition of amyloplasts into chloroplasts provides indirect evidence for the integrity of the envelope membranes throughout the whole process. The starch grains are sequestered from the cytosolic enzymes, and only plastid-localized enzymes, which have access to the starch grains, can carry out starch degradation. In this respect the turion system resembles transitory starch degradation as known from Arabidopsis leaves. On the other hand, with α-amylase playing the dominant role, it resembles the mechanism operating in the endosperm of cereals. Thus, turions appear to possess a unique system of starch degradation in plants combining elements from both known starch-storing systems.     

Sugar and starch changes in pea cotyledons during germination

Changes in starch and sugar contents in the cotyledons during germination have been compared in a smooth (cv. Alaska) and a wrinkled (cv. Progress) cultivar of the garden pea ( Pisum sativum L.). In both cultivars there was an initial accumulation of sucrose due to the hydrolysis of sucrosyl oligosaccharides, but galactose did not accumulate in the cotyledons. Starch mobilization in the Progress pea was linear with time and started before the rise in α-amylase (EC 3.2.1.1) activity in the cotyledons; sucrose was synthesized in the cotyledons, and their excision from the axis resulted in an additional accumulation of this sugar. In the Alaska pea, the onset of starch hydrolysis coincided with the rise in α-amylase activity; no accumulation of sucrose was found in excised cotyledons, whilst the sucrose content decreased continuously in attached cotyledons.
The same sugars were found in the cotyledons of both cultivars, suggesting a common pathway for starch breakdown. Maltose, maltotriose and linear malto-dextrins were not present and only trace amounts of glucose were detected, suggesting a degradation of starch by phosphorylase after an initial attack by α-amylase. α-Amylase activity in the cotyledons was higher in the presence of the axis, but was influenced by the water content of the cotyledons. Transient changes in α-amylase activity correlated well with changes in the rate of starch hydrolysis, but after 2–3 days starch mobilization was reduced in excised cotyledons probably due to the resynthesis of starch.     

In-vitro degradation of starch granules isolated from spinach chloroplasts

The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and -amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by -amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.Abbreviations Glc1P Glucose 1-phosphate - Mes 2(N-morpholino)ethanesulfonic acid - Pi Orthophosphate     

Biochemical characterization of a raw starch degrading <Emphasis Type="Italic">α</Emphasis>-amylase from the Indonesian marine bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. ALSHL3

An Indonesian marine bacterial isolate, which belongs to genus of Bacillus sp. based on 16S rDNA analysis and was identified as Bacillus filicolonicus according to its morphology and physiology, produced a raw starch degrading α-amylase. The partially purified α-amylase using a maize starch affinity method exhibited an optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme retained 72% of its activity in the presence of 1.5 M NaCl. Scanning electron micrographs showed that the α-amylase was capable of degrading starch granules of rice and maize. This α-amylase from Bacillus sp. ALSHL3 was classified as a saccharifying enzyme since its major final degradation product was glucose, maltose, and maltotriose.     

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.     

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.     

Activities,Quantitative Changes and Subcellular Localization of |á-Amylase During Development of Apple Fruit

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α-Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α-amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple (Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular-localization studies via immunogold electron-microscopy technique showed that α-amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid-distributed pattern of α-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α-amylase is involved in starch hydrolysis in plastids of the fruit cells.     

New look at starch degradation in Arabidopsis thaliana L. chloroplasts

Transitory starch is accumulated during the day and is the main source of energy for the cell metabolism during the night. The observed periodical starch degradation has become a model often used by scientist in their experiments. Starch granule degradation could be divided into 2 periods: initiation of degradation and digestion of amylopectin and amylose into maltooligosaccharide and their derivative. Key meaning is attributed in this process to beta-amylaze, product of its activity beta-maltose is transported to the cytosole and there it subjects farthest conversions. It has been demonstrated that a number of enzymes take part in the starch degradation process. However, the way of regulating their activity is still not fully explained. There is most important elements effecting rate of starch decomposition: day cycle, starch phosphorylation and regulation of enzyme activity. It proceeds through redox potential, pH changes and phosphorylation of protein involved in starch degradation due specific phosphatases. The purpose of the current work is to systematize the knowledge of the Arabidopsis thaliana L. leaf starch degradation. The results of the recent research cast a new light on the starch degradation process as well as on its control.     

The legwd mutant uncovers the role of starch phosphorylation in pollen development and germination in tomato

Starches extracted from most plant species are phosphorylated. α-Glucan water dikinase (GWD) is a key enzyme that controls the phosphate content of starch. In the absence of its activity starch degradation is impaired, leading to a starch excess phenotype in Arabidopsis and in potato leaves, and to reduced cold sweetening in potato tubers. Here, we characterized a transposon insertion ( legwd::Ds ) in the tomato GWD ( LeGWD ) gene that caused male gametophytic lethality. The mutant pollen had a starch excess phenotype that was associated with a reduction in pollen germination. SEM and TEM analyses indicated mild shrinking of the pollen grains and the accumulation of large starch granules inside the plastids. The level of soluble sugars was reduced by 1.8-fold in mutant pollen grains. Overall, the transmission of the mutant allele was only 0.4% in the male, whereas it was normal in the female. Additional mutant alleles, obtained through transposon excision, showed the same phenotypes as legwd::Ds . Moreover, pollen germination could be restored, and the starch excess phenotype could be abolished in lines expressing the potato GWD homolog ( StGWD ) under a pollen-specific promoter. In these lines, where fertility was restored, homozygous plants for legwd::Ds were isolated, and showed the starch excess phenotype in the leaves. Overall, our results demonstrate the importance of starch phosphorylation and breakdown for pollen germination, and open up the prospect for analyzing the role of starch metabolism in leaves and fruits.     

β-amylase in developing apple fruits: activities, amounts and subcellular localization

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β-amylase is considered one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. The present experiment showed that β-amylase activity was progressively increasing concomitantly with decreasing starch concentrations during apple (Malus domestica Borkh cv. Starkrimson) fruit development. The apparent amount of β-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The subcellular-localization studies via immunogold electron-microscopy technique showed that β-amylase visualized by gold particles was predominantly located in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments. These data proved for the first time that the enzyme is compartmented in its functional sites in plant living cells. The predominantly plastid-distributed pattern of β-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (β-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that β-amylase is involved in starch hydrolysis in plastids of the fruit cells.     

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

A gene encoding the thermostable raw starch digesting α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Activities of enzymes of starch metabolism in developing Norway spruce [Picea abies (L.) Karst.] needles

 Development of spruce needles starts with high levels of starch. These are derived from imported sucrose, and, with some fluctuation, largely vanish during sink/source transition (Hampp et al. 1994, Physiol Plant 90: 299 – 306). In order to get more information about starch metabolism during this period, we collected current year needles of approximately 25-year-old Norway spruce trees [Picea abies (L.) Karst.] for up to 100 days starting from bud break. Levels of extractable activities of α-amylase (EC 3.2.1.1), ADP-glucose pyrophosphorylase (AGP, EC 2.7.7.27), D-enzyme (4-α-D-glucotransferase; EC 2.4.1.25), and of starch phosphorylase (STP, EC 2.4.1.1.) exhibited specific development-related responses. Insoluble starch dissolving α-amylase was close to the limit of detection for up to 70 days after bud break. At this stage, which marked the start of sink/source transition, α-amylase showed a rise in activity which could be related to the activity of sucrose phosphate synthase, a key enzyme of sucrose formation (correlation coefficient r = + 0.93). Similarly, the activity of AGP, a key enzyme of starch synthesis, was low during the initial phase of needle development and started to increase from about 60 days onwards. STP and D-enzyme, both involved in starch cycling, differed from each other. While STP activity changed in parallel to that of AGP, it was only the D-enzyme which showed appreciable rates shortly after bud break. We thus assume that in spruce needles D-enzyme is mainly responsible for starch turnover during the early period of development, whereas needle maturation, i. e. the acquisition of the ability to export photoassimilates, is characterized by an increased turnover of transitory starch – both synthesis (AGP) and degradation (α-amylase, STP) – and this is closely connected to the emergence of activity of the key enzyme of sucrose synthesis, sucrose phosphate synthase. Received: 16 October 1995 / Accepted: 20 February 1996     

Gene Sequence,Bioinformatics and Enzymatic Characterization of α-Amylase from <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis> KZ

A fragment coding for a putative extracellular α-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp coding for a 494 amino acid residues long protein with 99% identity to the α-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ α-amylase (Sfamy KZ) belongs to typical extracellular fungal α-amylases classified in the glycoside hydrolase family 13, subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13 α-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate. The enzyme exhibited pH and temperature optima in the range of 5–6 and 40–50 °C, respectively. Stable adsorption of the enzyme to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed.     

Physiology and enzymology of thermophilic anaerobic bacteria degrading starch

Abstract The capability of secreting thermoactive enzymes exhibiting α-amylase and pullulanase with debraching activity, seems to be widely distributed amongst anaerobic thermophilic bacteria. Interestingly, pullulanase formed by these bacteria displays dual specificity by attacking α-1,6- as well as α-1,4-glycosidic linkages in branched glucose polymers. Unlike the enzyme system of aerobic microorganisms the majority of starch hydrolysing enzymes of anaerobic bacteria is metal indepedent and is extremely thermostable. This enzyme system is controlled by substrate induction and catabolite repression; enzyme expression is accomplished when maltose or maltose-containing carbohydrates are used as substrates. By developing a process in continuous culture we were able to greatly enhance enzyme synthesis and release by anaerobic thermophilic bacteria. An elevation in the specific activities of cell-free amylases and pullulanases could also be achieved by entrapping of bacteria in calcium alginate beads. The unique properties of extracellular enzymes of thermophilic anaerobic bacteria makes this group of organisms suitable candidates for inductrial application.     

Some characteristics of a raw starch digestion inhibitory factor fromAspergillus niger

Summary The effect of an inhibitory factor (IF) fromAspergillus niger 19 on raw starch digestion by pure glucoamylase I of blackAspergillus, pure glucoamylae ofRhizopus niveus, bacterial -amylase, fungal -amylase and various combination was investigated. The IF caused higher inhibition of raw starch hydrolysis by the combined action of glucoamylase and fungal -amylase than of hydrolysis by the individual enzymes. A protein moiety of IF might play an active part in this inhibition phenomenon. The IF was bound to starch granules, preventing hydrolysis by the enzymes, and caused decreased raw starch hydrolysis yields.