Isolation of a heat-stable maize endosperm ADP-glucose pyrophosphorylase variant

Heat stress reduces maize yield and several lines of evidence suggest that the heat lability of maize endosperm ADP-glucose pyrophosphorylase (AGPase) contributes to this yield loss. AGPase catalyzes a rate-limiting step in starch synthesis. Herein, we present a novel maize endosperm AGPase small subunit variant, termed BT2-TI that harbors a single amino acid change of residue 462 from threonine to isoleucine. The mutant was isolated by random mutagenesis and heterologous expression in a bacterial system. BT2-TI exhibits enhanced heat stability compared to wildtype maize endosperm AGPase.The TI mutation was placed into another heat-stable small subunit variant, MP. MP is composed of sequences from the maize endosperm and the potato tuber small subunit. The MP-TI small subunit variant exhibited greater heat stability than did MP. Characterization of heat stability as well as kinetic and allosteric properties suggests that MP-TI may lead to increased starch yield when expressed in monocot endosperms.     

Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.)

Summary An amylose-free potato mutant was isolated after screening 12,000 minitubers. These minitubers had been induced on stem segments of adventitious shoots, which had been regenerated on leaf explants of a monoploid potato clone after Röntgen-irradiation. The mutant character is also expressed in subterranean tubers and in microspores. Starch granules from the mutant showed a strongly reduced activity of the granule bound starch synthase and loss of the major 60 kd protein from the starch granules.     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

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.     

Influence of phloem transport on flower abscission in Hibiscus rosa-sinensis

Summary Flowering cultivars of Hibiscus rosa-sinensis L. were either cross-pollinated or self-pollinated. Fruit set was observed on 52% of the cross-fertilized flowers, while only 4.6% of the self-fertilized flowers were not abscised. Once during fruit and seed growth, the subtending leaf was exposed to ¹⁴CO₂, and translocation of labelled photoassimilate was recorded by macro- and microautoradiography. Phloem transport into the raphe occurred in both fruits with fertilized and fruits with non-fertilized ovules. Since empty ovules showed some sink strength, it is assumed that growth of vegetative seed-tissue signalizes the retardation of completion of the abscission process. During fruit growth a considerable amount of starch is deposited in the distal layer of the abscission zone. Part of this starch is consumed during growth of cross-fertilized fruits.     

Multiplicity of soluble glucan-synthase activity in spinach leaves: Enzyme pattern and intracellular location

Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 2.4.1.21). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO₂-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 2.4.1.1). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.Abbreviations GS glucan synthase - PAGE polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft and from the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors gratefully acknowledge the generous permission to use the laser densitometer of Professor Dr. W. Barz (Biochemie der Pflanzen, Universität Münster, FRG). They are indebted to Dr. H.-J. Witt (Pflanzenphysiologie, Universität Kassel, FRG) for helpful discussions and to Mr. W. Lamkemeyer for skilfull technical assistance.     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

An unusual pattern of carbohydrate utilization in Corallococcus (Myxococcus) coralloides (Myxobacterales)

The myxobacterium, Corallococcus (Myxococcus) coralloides strain Cc c127, could not utilize mono- and disaccharides, but maltotriose and the polysaccharides starch, amylose, amylopectin, and pullulan stimulated growth. Radioactive CO₂ was set free from ¹⁴C-labeled starch. When starch was degraded, small amounts of maltose and glucose accumulated in the culture supernatant. Maltotriose, however, appeared only temporarily. Outside the cells, the trisaccharide could not be split into glucose and maltose. Pullulan was hydrolyzed exclusively into a trisaccharide which during growth was immediately consumed. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphoglucomutase could readily be demonstrated in cell extracts, but fructose-1,6-diphosphate aldolase was present with low activity only. The data suggest that intracellular glucose is metabolized mainly via the pentose phosphate pathway.Prof. Dr. Gerhard Drews gratefully dedicated to his 60th birthday