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.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 11. Ultrastructural changes in scutellar epithelium

The ultrastructural changes occurring in the scutellar epithelium cells of rice seeds have been studied during germination and early seedling growth. During this time, several prominent structural changes occur, including (a) formation, development, and proliferation of organelles such as mitochondria, rough endoplasmic reticulum, free ribosomes, and Golgi apparatus; (b) folded structural modification of plasmamembranes in later stages; and (c) conspicuous decrease in lipid-storing spherosomes. Glyoxysome-like electron dense particles are detectable but their formation is much less prominent. It is conceivable that all these structural changes are related to the enhancement of the metabolic activities of the epithelial cells including the synthesis of hydrolytic enzymes such as α-amylase and their secretion into the endosperm tissues. Some enzyme activities characteristic of mitochondria and glyoxysomes have been determined using the crude scutellar extracts, and the results dealing with the low activities of the glyoxylate cycle enzymes and palmitoyl-coenzyme A oxidase appear to indicate that fatty acid breakdown is possibly via mitochondrial β-oxidation, although we reserve a definitive conclusion on the glyoxysomes being nonfunctional in fatty acid oxidation in rice seedlings.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 15. Immunochemical study on multiple forms of amylase

The formation of multiple forms of amylases in germinating rice (Oryza sativa L. cv Kimmaze) grains was examined by means of isoelectric focusing, cross-immunoelectrophoresis, and rocket-line immunoelectrophoresis followed by a reaction of enzymic characterization by using β-limit dextrin or starch as substrate. The constituents detected by isoelectric focusing were identified as three electrophoretically heterogeneous antigens. The major α-amylase bands A and B corresponded to a same antigen, the main portion of which was produced within 2 days' germination. The bulk of α-amylase D appeared between 2 and 4 days' germination. Component E, a debranching enzyme according to its action on the β-limit dextrin, already exists in the ungerminated seeds; its amount decreases within the first 2 days of germination and increases again thereafter.

Evidence showing that β-amylase (band C) is produced by the scutellum at an early stage of germination was provided. The enzyme appeared in a suspension of the scutellum after a prolonged incubation.

     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation

The biosynthetic mechanism of α-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmazé) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of α-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.

In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of α-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of α-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of α-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.

The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of α-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of α-amylase were found to consist of three isozymes. The in vitro translated precursor forms of α-amylase consisted of four multiple components. These results indicate that heterogeneity of α-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice α-amylase isozymes are encoded by multiple genes.

     

Enzymic mechanism of starch synthesis in ripening rice grains VI. Isozymes of starch synthetase

The DEAE-cellulose column chromatography has shown two differentforms of starch synthetase, which are referred to as fractionsI and II in extracts of rice seeds (non-waxy and waxy varieties)harvested at the milky stage. Similarly treated leaf extractsof rice (non-waxy) and maize (non-waxy) also demonstrate dieexistence of two major isozyme fractions. In all enzyme preparationstested, ADP-glucose was the sole glucosyl donor and UDP-glucosewas totally inactive. Rechromatography, on a DEAE-cellulosecolumn, of two enzyme fractions (I and II) separated from non-waxyrice seed extracts did not alter their elution patterns. Someof their enzymic properties were compared, in particular, theirglucosyl-acceptor (primer) specificities. Regardless of potentamylase activities in the two fractions, notable differenceswere observed in that fraction I utilized the long-chain oligosaccharides[maltododecaose] and various types of high molecular -glucansmore readily than fraction II. However, short-chain oligosaccharides[maltose, maltotriose and maltotetraose] were utilized morereadily by fraction II than by fraction I. A possible role forthe two starch synthetase isozymes in starch synthesis in plantcells is discussed. (Received January 5, 1971; )     

Enzymic mechanism of starch stynthesis in ripening rice grains. VII. Purification and enzymic properties of sucrose synthetase

Sucrose synthetase (UDP-glucose:d-fructose-2-glucosyltransferase, EC 2.4.1.13) from ripening rice seeds was purified by ammonium sulfate fractionation and column chromatography of microgranular DEAE-cellulose (DE-32) and Neusilin (MgO· Al₂O₃·2SiO₂). An enzyme preparation obtained was homogeneous as examined by polyacrylamide gel electrophoresis. The enzyme, having a molecular weight, 4.0 × 10⁵, consists of 4 identical subunits, each having a molecular weight, 1.0 × 10⁵.Examination of reaction kinetics of both sucrose synthesis and cleavage catalyzed by sucrose synthetase revealed that the rate of synthesis follows a Michaelis-Menten equation having the following parameters: K_m(fructose)_UDP-glucose, 6.9 mm; K_m(fructose)_ADP-glucose, 40 mm; K_m(UDP-glucose), 5.3 mm; and K_m(ADP-glucose), 3.8 mm. The cleavage reaction yielded the following values: K_m(UDP), 0.8 mm; K_m(ADP), 3.3 mm; and K_m(sucrose)_UDP, 290 mm. In the latter reaction the rate deviated from the Michaelis equation when ADP was used as the glucose acceptor, the n value being 1.6 by the Hill plot analysis and S_0.5(sucrose)_ADP, 400 mm. At high concentration of ADP the cleavage reaction was inhibited, while the synthesis reaction was inhibited with high concentrations of fructose.     

Carbamoyl phosphate synthetase activity from the cotyledons of developing and germinating pea seeds

Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Pisum sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but decreased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and germination.     

Glutamine synthetase isozymes in germinating barley seeds

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of ammonia assimilation in higher plants. In the present study the subunit composition and localization of GS in germinating barley ( Hordeum vulgare ) seed have been clarified. Analysis of the GS polypeptide composition by immunoblotting revealed two different polypeptides. A and B, with a molecular mass of 42 and 40 kDa, respectively. In the scutellum subunit A was already present in the ungerminated seed and remained unchanged, whereas subunit B appeared on day 2 and increased about 5-fold during germination. Polypeptide B also appeared later during germination in the aleurone layer, roots and weakly in the etiolated shoots. By immunogold microscopy, GS was detected in the scutellum and the aleurone layer of barley seeds during germination. Subcellular localization of GS on ultrathin cryosections showed that a cytosolic isozyme was present in the scutellum. Our study confirms that only a cytosolic GS is expressed in barley seed, and its subunit composition changes during germination.     

Auxin-dependent breakdown of xyloglucan in cotyledons of germinating nasturtium seeds

Rapid mobilisation of storage products, including xyloglucan, in cotyledons of germinating nasturtium (Tropaeolum majus L.) normally starts about 7–8 d after imbibition and growth of the seedling at 20–25° C. Levels of activity of endo-1,4--glucanase (EC 3.2.1.4) in cotyledons, as assayed viscometrically with xyloglucan as substrate, varied in parallel with the rate of breakdown of xyloglucan. When cotyledons were excised from the seedling axis and incubated on moist filter paper at any point before 7 d, the catabolic reactions which normally occurred in the intact seedling were suspended. If, however, cotyledons excised at 8 d were incubated in 10^–6 M 2,4-dichlorophenoxyacetic acid, a rise in endo-1,4--glucanase (xyloglucanase) activity was observed and a sharp decrease in fresh and dry weight as well as xyloglucan levels ensued at rates comparable to those observed in cotyledons attached to the seedling. Neither gibberellin nor kinetin treatments promoted xyloglucan breakdown or enhanced xyloglucanase activity. Addition of auxin to excised cotyledons before 7 d did not evoke premature breakdown, indicating that the tissue became receptive to auxin only at this time. The triggering process took place in darkness and was unaffected by various light-dark cycles. It is concluded that the sudden degradation of xyloglucan which occurs in nasturtium seeds about a week after germination begins is the result of enhanced activity of a depolymerizing xyloglucanase, this activity being evoked by auxin originating in the emerging seedling axis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - GA₃ gibberellic acid - kDa kilodalton The authors are pleased to acknowledge the technical assistance of Alexander Marcus and valuable discussions with Dr. Vladimir Farkas. This study was supported by a scholarship to A.H. from the Deutsche Forschungsgemeinschaft (FRG) and a grant to G.M. from the Natural Sciences and Engineering Research Council of Canada.     

Sucrose controls storage lipid breakdown on gene expression level in germinating yellow lupine (Lupinus luteus L.) seeds

This study revealed that cytosolic aconitase (ACO, EC 4.2.1.3) and isocitrate lyase (ICL, EC 4.1.3.1, marker of the glyoxylate cycle) are active in germinating protein seeds of yellow lupine. The glyoxylate cycle seems to function not only in the storage tissues of food-storage organs, but also in embryonic tissue of growing embryo axes. Sucrose (60 mM) added to the medium of in vitro culture of embryo axes and cotyledons decreased activity of lipase (LIP, EC 3.1.1.3) and activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). The opposite effect was caused by sucrose on activity of cytosolic ACO, ICL as well as NADP⁺-dependent (EC 1.1.1.42) and NAD⁺-dependent (EC 1.1.1.41) isocitrate dehydrogenase (NADP-IDH and NAD-IDH, respectively); activity of these enzymes was clearly stimulated by sucrose. Changes in the activity of LIP, ACO, NADP-IDH, and NAD-IDH caused by sucrose were based on modifications in gene expression because corresponding changes in the enzyme activities and in the mRNA levels were observed. The significance of cytosolic ACO and NADP-IDH in carbon flow from storage lipid to amino acids, as well as the peculiar features of storage lipid breakdown during germination of lupine seeds are discussed.     

Starch grain breakdown in cotyledon cells of germinating mung bean seeds

Summary Ultrastructural aspects of the breakdown of starch grains during the mobilisation of reserves in Phaseolus aureus Roxb. seed germination are described. The starch grains show erosion from within leading to the formation of a hollow shell. The erosion is accompanied by intrusion of cytoplasm into the shell. No evidence of a vesicular transport system to or from the eroding face was found.     

Sucrose metabolism in green algae. I. The presence of sucrose synthetase and sucrose phosphate synthetase.

The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae: Chlorella vulgaris and Scenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.     

Sucrose metabolism in green algae I. The presence of sucrose synthetase and sucrose phosphate synthetase

Summary The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae:Chlorella vulgaris andScenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.Dedicated toLuis F. Leloir on his seventieth birthday.     

Sucrose transport from source to sink seeds in rice

In many higher plants, sucrose is loaded as a major carbon photoassimiliate into the phloem apoplastically by sucrose transporters (SUTs) and unloaded in sink tissues, where it serves as a storage material, carbohydrate backbone, and energy source. In sink tissues, a proportion of sucrose molecules are converted by cell wall invertases (CINs) into hexose that is imported into cells by monosaccharide transporters (MSTs). Thus, in developing seeds, co-ordinated regulation of SUTs, CINs, and MSTs is crucial in carbon distribution. Here, we summarize current efforts on the identification of SUTs, CINs, and MSTs in rice.