Endosperm Enzyme Research Articles

Boiling Soluble Proteins Involved in Drought Stress Adaptation of Embryos and Endosperm of Wheat Cultivars

Boiling soluble proteins (BSPs), which constitutes about 0.2% of the total genome, play an key role in stress adaptation. However, drought stress induced regulation of BSPs are still a matter of conjecture. This study primarily focused indicators of oxidative stress like: ROS, lipid peroxidation, injury index and scavenging boiling soluble antioxidant enzymes in embryos and endosperm of different cultivars of wheat under different intensities of drought stress followed by stress recovery. Both in embryos and endosperm of cultivars HD 2967 and PBW 550, MII, MDA and ROS relatively unchanged, minor increased or decreased under severe drought stress conditions. Activities of different boiling soluble antioxidants like BsMDAR, BsTRX.R, BsPAO and BsPDI were substantially higher in the embryos and endosperm of cultivars HD 2967 and PBW 550 under moderate and severe stress conditions, while minor increase or decreased activities were detected in cultivars HD 2851 and HD 2932. Based on present study, it can incidental that cultivars HD2967 and PBW 550 are better equipped to perform biological antioxidant responses to eliminate drought-induced oxidative stress.

Endosperm enzyme activity is responsible for texture and eating quality of cooked rice grains in Japanese cultivars.

Eating quality of cooked rice grains is an important determinant of its market price and consumer acceptance. To comprehensively assess the variation of eating-quality traits in 152 Japanese rice cultivars, we evaluated activities of eight endosperm enzymes related to degradation of starch and cell-wall polysaccharides. Endosperm enzyme activities showed a wide range of variations and were lower in recently developed cultivars than in landraces and old improved cultivars. Activities of most endosperm enzymes correlated significantly with the eating-quality score and surface texture of cooked rice grains. Principal component analysis revealed that rice cultivars with high eating-quality scores had high stickiness of the grain surface and low levels of endosperm enzyme activities. These results suggest that endosperm enzyme activities control texture and eating quality of cooked rice grains in Japanese rice cultivars.

Seed Plumpness of Rice with Inhibition Expression of Starch Branching Enzymes and Starch Properties, Grain Position on Panicle

Cereal crops with inhibition expression of starch branching enzyme (SBE) contain highly resistant starch in the endosperm, and have potential health benefits for human. However, seed plumpness is significantly different, with different inhibition effects of SBE expression, resulting in differently shrunken seeds. In this study, a transgenic resistant starch rice line, which has highly resistant starch and is developed through inhibition expression of SBEs, had non-, slightly, and moderately shrunken seeds with plumpness from high to low. The differently shrunken seeds had significantly different seed weight and starch content. Different morphological starch granules were detected in the endosperm and had similar spatial distribution pattern among the non-, slightly, and moderately shrunken seeds. Starches from differently shrunken seeds had similar amylose content and amylopectin structure. The protein amount of amylose/amylopectin synthesis key enzymes in endosperm was no different between differently shrunken seeds. The primary branch of the panicle had a higher percentage of non-shrunken seeds than did the secondary branch at the same part of the panicle. From the upper part to the base of the panicle, non-shrunken seeds gradually decreased but slightly and moderately shrunken seeds gradually increased. The above results indicated that the differently shrunken seeds in transgenic rice line had the same SBE dosage, and the starch morphology and structure had no relationship with seed plumpness. The grain position on the panicle influenced seed plumpness, but had no effect on starch properties.

On the Roles of Wheat Endosperm ADP-Glucose Pyrophosphorylase Subunits

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.

The Rice Grain Localization of Endosperm Enzyme Activity

The Rice Grain Localization of Endosperm Enzyme Activity

The Rice Endosperm Enzyme Action Effect the Eating Quality of Cooked Rice

The Rice Endosperm Enzyme Action Effect the Eating Quality of Cooked Rice

Comprehensive molecular characterization of the α/β-gliadin multigene family in hexaploid wheat.

To characterize the structure and expression of a large multigene family of α/β-gliadin genes, 90 individual α/β-gliadin genes harboring a promoter region were identified in the wheat cultivar Chinese Spring. These genes were classified into eleven groups by phylogenetic analysis, and the chromosomes they were derived from were determined. Of these genes, 50 had the basic α/β-gliadin domains and six conserved cysteine residues and 16, 16 and 18 of them were, respectively, located on chromosome 6A, 6B and 6D. Six genes had an additional cysteine residue, suggesting that these α/β-gliadins acquired the property of binding other proteins through intermolecular disulphide bands. Expression of α/β-gliadin genes in developing seeds was measured by quantitative RT-PCR using group-specific primers over 3years. Expression patterns of these genes on the basis of accumulated temperature were similar among gene groups, whereas expression levels differed for the 3years. The expression of most α/β-gliadin and other prolamin genes was correlated with the sunshine duration. On the other hand, although all α/β-gliadin genes had a common E-box within the -300 promoter region, some genes showed a particular expression pattern with respect to the sunshine duration, similarly to gene encoding high-molecular weight glutenin subunits and endosperm enzymes. These observations suggested that expression of each α/β-gliadin gene is differentially regulated by multiple regulatory factors.

米飯の食味形成に関与する米胚乳酵素の活性量の品種および栽培地域による変動の解析

米飯の食味形成に関与する米胚乳酵素の活性量の品種および栽培地域による変動の解析

Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis.

The role of the large subunit in redox regulation of the rice endosperm ADP-glucose pyrophosphorylase.

The starch regulatory enzyme ADP-glucose pyrophosphorylase is activated by 3-phosphoglyceric acid (3-PGA) and inhibited by inorganic phosphate (Pi ). The activity of the plastid-localized enzyme is also subject to fine regulation by redox control in response to changing light and sugar levels. The less active oxidized form of the enzyme contains an inter-subunit disulfide bond formed between the pair of small subunit's Cys12 residues of the heterotetrameric enzyme. Although this cysteine residue is not conserved in the small subunits of cereal endosperm cytosolic AGPases, biochemical studies of the major rice endosperm enzyme indicate that the cytosolic isoform, like the plastidial enzymes, is subject to redox control. Kinetic analysis revealed that the reduced forms of the partially purified native and purified recombinant AGPases have 6- and 3.4-fold, respectively, more affinity to 3-PGA, rendering the enzymes more active at lower 3-PGA concentration than the non-reduced enzyme. Truncation of the large subunit by removal of N-terminal peptide resulted in a decrease in 3-PGA affinity and loss of redox response of the enzyme. Site-directed mutagenesis of the conserved cysteine residues at the N-terminal of the large subunit showed that C47 and C58, but not C12, are essential for proper redox response of the enzyme. Overall, our results show that the major rice endosperm AGPase activity is controlled by a combination of allosteric regulation and redox control, the latter through modification of the large subunit instead of the small subunit as evident in the plastid-localized enzyme.

Enhanced heat stability and kinetic parameters of maize endosperm ADPglucose pyrophosphorylase by alteration of phylogenetically identified amino acids

ADP-glucose pyrophosphorylase (AGPase) controls the rate-limiting step in starch biosynthesis and is regulated at various levels. Cereal endosperm enzymes, in contrast to other plant AGPases, are particularly heat labile and transgenic studies highlight the importance of temperature for cereal yield. Previously, a phylogenetic approach identified Type II and positively selected amino acid positions in the large subunit of maize endosperm AGPase. Glycogen content, kinetic parameters and heat stability were measured in AGPases having mutations in these sites and interesting differences were observed. This study expands on our earlier evolutionary work by determining how all Type II and positively selected sites affect kinetic constants, heat stability and catalytic rates at increased temperatures. Variants with enhanced properties were identified and combined into one gene, designated Sh2-E. Enhanced properties include: heat stability, enhanced activity at 37°C, activity at 55°C, reduced Ka and activity in the absence of activator. The resulting enzyme exhibited all improved properties of the various individual changes. Additionally, Sh2-E was expressed with a small subunit variant with enhanced enzyme properties resulting in an enzyme that has exceptional heat stability, a high catalytic rate at increased temperatures and significantly decreased Km values for both substrates in the absence of the activator.

The potato tuber, maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.

Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases

ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis.

The Role of α-Glucosidase in Germinating Barley Grains

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.

Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme

ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses.

Characterization of an Autonomously Activated Plant ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP [Mos(1-198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1-198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1-198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1-198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1-198)/SH2.

Effects of low temperature on grain filling, amylose content, and activity of starch biosynthesis enzymes in endosperm of basmati rice

The effects of low temperature on amylose contents and activities of key enzymes related to starch biosynthesis in basmati rice (Oryza sativa L.) endosperm were investigated. Low temperature treatment prolonged the grain-filling period from 32 days to 53 days, but had no significant effect on the final grain weight. Results showed that low temperature during grain filling had no significant effect on total starch content but increased the amylose content in the mature endosperm of hulled rice by 21%. The measurable activities of sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (AGPase), starch phosphorylase (SPase), starch branching enzyme (SBE), and soluble starch synthase (SS) in endosperms developed at 12°C were lower than those at 22°C when compared at a similar ripening stage on an endosperm basis, but the activity of granule-bound starch synthase (GBSS) was significantly higher than the corresponding activity in endosperms developing at 22°C. These findings suggest that GBSS might play a crucial role in increasing amylose during low-temperature growth conditions.

ADP-glucose pyrophosphorylase from wheat endosperm. Purification and characterization of an enzyme with novel regulatory properties.

ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) was purified and characterized from two wheat (Triticum aestivum L.) tissues: leaf and endosperm. The leaf enzyme, purified over 1,300-fold, was found to be a heterotetramer composed of subunits of 51 and 54 kDa and possessing regulatory properties typical of AGPases from photosynthetic tissues, being mainly regulated by 3-phosphoglycerate (activator; A0.5=0.01 mM) and orthophosphate (inhibitor; I0.5=0.2 mM). Conversely, the enzyme from wheat endosperm was insensitive to activation by 3-phosphoglycerate and other metabolites. It was, however, inhibited by orthophosphate (I0.5=0.7 mM), ADP (I0.5=3.2 mM) and fructose-1,6-bisphosphate (0.5 = 1.5 mM). All of these inhibitory actions were reversed by 3-phosphoglycerate and fructose-6-phosphate. The endosperm enzyme was found to be a heterotetramer composed of subunits of 52 and 53 kDa, which were recognized by antiserum raised to spinach leaf AGPase. The results suggest that wheat endosperm AGPase possesses distinctive regulatory properties that are relevant in vivo.

Subcellular compartmentation and allosteric regulation of the rice endosperm ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (AGPase) catalyzes the first unique step in the starch biosynthetic pathway. Recent studies in barley, wheat, and maize indicate that the major endosperm enzyme exhibits different enzymatic and cellular properties than those found in leaf tissue. To determine how prevalent these properties are in other plants, the AGPase activities of rice endosperm were studied at the enzymatic and cellular levels. Unlike the barley and wheat endosperm enzymes, the rice AGPase was dependent on 3-phosphoglycerate (3-PGA) with more than 40-fold increase in catalytic activity, when assayed under near saturating 3-PGA conditions. The rice enzyme was also inhibited by Pi and this inhibition was reversed by 3-PGA. Subcellular fractionation studies indicated that the bulk (90%) of the AGPase activity was extra-plastidic, located in the cytoplasm. A smaller amount of enzyme activity (10%) was observed associated with the amyloplast fraction. The presence of two AGPase forms was supported by immunoblot analysis using anti-bodies specific for the large or small subunit of the heterotetrameric enzyme. Anti-bodies specific for the maize endosperm AGPase small subunit (BT2) recognized a major 54 kD polypeptide in the cytoplasmic fraction. A second polypeptide at 48 kD was also detected in the cytoplasmic fraction but was more abundant in the amyloplast fraction. Based on results obtained from this study, strategies for increasing starch synthesis by manipulation of AGPase activities in developing seeds and, in turn, seed yields are discussed.

Activation of the Potato Tuber ADP-glucose Pyrophosphorylase by Thioredoxin

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, P(i). It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045-25052) that the enzyme was activated by reduction of the Cys(12) disulfide linkage present in the catalytic subunits. In this study, both reduced thioredoxin f and m from spinach (Spinacia oleracea) leaves reduced and activated the enzyme at low concentrations (10 microM) of activator (3-phosphoglycerate). Fifty percent activation was at 4.5 and 8.7 microM for reduced thioredoxin f and m, respectively, and 2 orders of magnitude lower than for dithiothreitol. The activation was reversed by oxidized thioredoxin. Cys(12) is conserved in the ADP-GlcPPases from plant leaves and other tissues except for the monocot endosperm enzymes. We postulate that in photosynthetic tissues, reduction could play a role in the fine regulation of the ADP-GlcPPase mediated by the ferredoxin-thioredoxin system. This is the first time that a covalent mechanism of regulation is postulated in the synthesis of starch.