Novel starch-related enzymes and carbohydrates.

In chloroplasts, both biosynthesis and degradation of starch are strictly regulated but the mechanisms involved are still incompletely understood. Recent studies revealed two novel and regulatory relevant aspects in the biochemistry of starch: the phosphorylation of starch and the starch-related metabolism of cytosolic heteroglycans. Starch phosphorylation occurs by a sequential action of two plastidial enzymes, the glucan, water dikinase (GWD; EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD; EC 2.7.9.5). Both enzymes utilize ATP as dual phosphate donor and transfer the terminal phosphate group to water whereas the beta-phosphate is used for esterification of glucosyl moieties. The metabolism of starch-derived degradation products is closely linked to recently discovered cytosolic heteroglycans that possess, as prominent constituents, arabinose, galactose, glucose and fucose. The pattern of glycosidic linkages is highly complex comprising more than 25 different bonds. During the dark period the size distribution or the amount of the cytosolic heteroglycans increases depending on the plant species. As revealed by in vitro 14C labeling assays, the heteroglycans act as both glucosyl acceptors and donors for two cytosolic glucosyl transferases, the phosphorylase (EC 2.4.1.1) and the transglucosidase (EC 2.4.1.25) and, at least in part, both enzymes utilize the same glucosyl acceptor and donor sites. In mutants of Arabidopsis thaliana L. that are deficient in the cytosolic transglucosidase both the structure and (bio)chemical properties of the heteroglycans are altered.

Purification of chloroplast alpha-1,4-glucan phosphorylase from spinach leaves by chromatography on Sepharose-bound starch.

Chloroplast alpha-1,4-glucan phosphorylase (EC 2.4.1.1) has been purified to homogeneity from spinach leaves as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purification procedure is composed of (NH4)2SO4 precipitation, ion-exchange chromatography, and chromatography on Sepharose-bound starch. In order to achieve binding of the chloroplast phosphorylase, a previously described Sepharose-glucan gel (Steup, M. Schächtele, C. and Latzko, E. (1980) Planta 148, 168-173) was modified by introducing hydrophobic groups in addition to the covalently bound starch. The chloroplast phosphorylase exhibited complete binding to this type of gel and could be eluted by a mixture of soluble glucan and NaCl. For the purified chloroplast phosphorylase, sodium dodecyl-sulfate polyacrylamide gel electrophoresis and pyridoxal phosphate determination resulted in a molecular weight estimation of about 110,000 per monomer. The apparent molecular weight of the native enzyme, as determined by polyacrylamide density gradient electrophoresis and gel filtration on Sephadex G-200, was 200,000 and 220,000, respectively. The data indicate that the chloroplast phosphorylase is a dimer with a molecular weight higher than that of the non-chloroplast phosphorylase.

Polypeptides of the chloroplast envelope membranes as visualized by immunochemical techniques.

The polypeptides of relative molecular masses (Mr) 22,000, 29,000, and 36,000 represent three major constituents of the chloroplast envelope of spinach (Spinacia oleracea L.) leaves. The Mr 22,000 polypeptide has been localized in the outer membrane, whereas the two other peptides have been attributed to the inner envelope membrane (Joyard et al., 1983). The Mr 29,000 polypeptide has been identified as the "phosphate translocator" (Flügge and Heldt, 1979). In this investigation, we studied the three envelope polypeptides by means of immunocytochemistry. Using indirect immunofluorescence, all three polypeptides were visualized in cryostat sections of formaldehyde-fixed leaf tissue. They were found in both palisade and spongy parenchyma cells and in guard cells, as indicated by a strong fluorescence in the chloroplast periphery. In contrast, fluorescein isothiocyanate or protein A-gold labeling of isolated fixed chloroplasts resulted only in visualization of the Mr 22,000 polypeptide, a constituent of the outer membrane. We further studied the morphological distribution and frequency of this peptide by electron microscopic evaluation of platinum-carbon replicas after freeze-etching or label-fracture and of ultra-thin sections. By use of these three methods, the polypeptide was found to be randomly distributed in the outer envelope membrane and easily accessible to the immunomarker. Average marker density, as obtained by freeze-etching and label-fracture, was approximately 130 gold particles per square micron.

Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase.

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.

The role of plastidial glucose-6-phosphate/phosphate translocators in vegetative tissues of Arabidopsis thaliana mutants impaired in starch biosynthesis.

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.

Blue light-dependent regulation of cytoplasmic ribosomal RNA synthesis in Chlorella.

Effect of blue and red light on ribosomal RNA synthesis in autotrophic synchronous cultures of Chlorella pyrenoidosa (strain 211-8b) is studied by pulse labeling experiments with tritiated guanosine. Nucleic acids were separated by electrophoresis on polyacrylamide gels. Compared with darkness and red light (679 nm), blue light (457 nm) of equal quantum flux (0.5-5x10(-10) Einstein cm-2 s-1) stimulates incorporation into ribosomal RNA. This blue light effect is observed in the cytoplasmic ribosomal RNA after 5 min of illumination, whereas the stimulation of chloroplast ribosomal RNA synthesis by blue light appears later. Maturation of chloroplast ribosomal RNA is slower than that of cytoplasmic ribosomal RNA. The blue light effect on the cytoplasmic ribosomal RNA formation does not require chloroplast RNA or protein synthesis as shown by inhibitor studies with rifampicin or lincomycin. The blocking of cytoplasmic protein synthesis by cycloheximide inhibits the blue light effect on ribosomal RNA formation. It is concluded that the cytoplasmic ribosomal RNA transcription is controlled by a blue light sensitive system.

[Effect of blue and red light on the synthesis of ribosomal RNA in Chlorella]. / Die Wirkung von blauem und rotem Licht auf die Synthese ribosomaler RNA bei Chlorella

In autotrophic cultures of Chlorella pyrenoidosa (strain 211-8b) incorporation of tritiated guanosine and uridine into ribosomal RNA is stimulated by light. Blue light of wavelengths around 457 nm is considerably more effective than red light around 679 nm (5-10(-10) Einstein cm-2 sec-1 for both). This effect can be demonstrated for young daughter cells (at the end of the dark period) and for older cells (at the end of the light period). It is shown to depend on a regulation of rRNA-synthesis. The blue light dependent enhancement of incorporation is more pronounced in the cytoplasmic rRNA (25 and 18 s) than in the chloroplast rRNA (23 and 16 s). Blue light of low intensity (1-10(-10) Einstein cm-2 sec-1) has nearly the same effectivity as the fivefold intensity, whereas red light of equal quantum fluxes enhances incorporation only slightly compared with the dark control. The blue light dependent enhancement of rRNA-synthesis continues in the following darkness in contrary to that caused by red light. This enhancement is also found in DCMU-poisened cultures. In contrast to this, is red light in presence of DCMU, incorporation into rRNA is nearly the same as in dark. It is concluded that the regulation of rRNA-synthesis in red light is closely connected to complete photosynthesis, while in blue light an additional regulation takes place independent of photosynthesis.

Intracellular localization of phosphorylases in spinach and pea leaves.

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.

Polysaccharide Fraction from Higher Plants which Strongly Interacts with the Cytosolic Phosphorylase Isozyme : I. Isolation and Characterization.

From leaves of Spinacia oleracea L. or from Pisum sativum L. and from cotyledons of germinating pea seeds a high molecular weight polysaccharide fraction was isolated. The apparent size of the fraction, as determined by gel filtration, was similar to that of dextran blue. Following acid hydrolysis the monomer content of the polysaccharide preparation was studied using high pressure liquid and thin layer chromatography. Glucose, galactose, arabinose, and ribose were the main monosaccharide compounds. The native polysaccharide preparation interacted strongly with the cytosolic isozyme of phosphorylase (EC 2.4.1.1). Interaction with the plastidic phosphorylase isozyme(s) was by far weaker. Interaction with the cytosolic isozyme was demonstrated by affinity electrophoresis, kinetic measurements, and by (14)C-labeling experiments in which the glucosyl transfer from [(14)C]glucose 1-phosphate to the polysaccharide preparation was monitored.

α-1,4-glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.) : II. Peptide patterns and immunological properties. A comparison with other phosphorylase forms.

Peptide patterns and immunological properties of the cytoplasmic and chloroplastic α-1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves have been studied and were compared with those of phosphorylases from other sources. The two spinach leaf phosphorylases were immunologically different; a limited cross-reactivity was observed only at high antigen or antibody concentrations. Peptide mapping of the two enzymes resulted in complex patterns composed of more than 20 fragments; but no peptide was electrophoretically identical in both proteins. Approximately 13 to 15 of the fragments exhibited antigeneity but no cross-reactivity of any peptide was observed. Therefore, the two compartment-specific phosphorylase forms from spinach leaves represent isoenzymes possessing different primary structures. Peptide patterns of potato tuber and rabbit muscle phosphorylase were different from those of the two spinach leaf enzymes. Although the potato tuber phosphorylase resides in the plastidic compartment and is kinetically closely related to the chloroplastic spinach enzyme, it reacted more strongly with the anti-cytoplasmic-phosphorylase immunoglobulin G. Similar results were obtained with rabbit muscle phosphorylase. These observations support the assumption that the chloroplast-specific phosphorylase isoenzyme has a higher structural diversity than does the cytoplasmic counterpart.

α-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.) I. In situ localization by indirect immunofluorescence.

Antisera were raised against two forms of α-1,4-glucan phosphorylase (EC 2.4.1.1) which had been purified from leaves of Spinacia oleracea L. Immunoglobulin G preparations were isolated from the antisera, and their specificity was ensured by immunoplobulin G preparations were used for in situ localization of the two phosphorylase forms in spinach leaf thin sections by indirect immuno-fluorescence. Both enzyme forms were present in the palisade and spongy parenchyma and in the guard cells, but their intracellular distribution was complementary. One phosphorylase form (designated as the chloroplastic form) was restricted to the stromal space of chloroplasts whereas the other (the non-chloroplastic form) was present only in the cytoplasm of chloroplast-containing cells. Thus, the phosphorylases represent two distinct compartment-specific enzyme forms which reside within the same photosynthetically active mesophyll cell.

Mode of glucan degradation by purified phosphorylase forms from spinach leaves.

The glucan specifity of the purified chloroplast and non-chloroplast forms of α-1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves (Steup and E. Latzko (1979), Planta 145, 69-75) was investigated. Phosphorolysis by the two enzymes was studied using a series of linear maltodextrins (degree of polymerization ≦11), amylose, amylopectin, starch, and glycogen as substrates. For all unbranched glucans (amylose and maltodextrins G5-G11), the chloroplast phosphorylase had a 7-10-fold higher apparent affinity (determined by initial velocity measurements) than the non-chloroplast phosphorylase form. For both enzyme forms, the minimum chain length required for a significant rate of phosphorolysis was five glucose units. Likewise, phosphorolysis ceased when the maltodextrin was converted to maltotetraose. With the chloroplast phosphorylase, maltotetraose was a linear competitive inhibitor with respect to amylose or starch (K i-0.1 mmol 1(-1)); the inhibition by maltotetraose was less pronounced with the non-chloroplast enzyme. In contrast to unbranched glucans, the non-chloroplast phosphorylase exhibited a 40-, 50-, and 300-fold higher apparent affinity for amylopectin, starch, and glycogen, respectively, than the chloroplast enzyme. With respect to these kinetic properties the chloroplast phosphorylase resembled the type of "maltodextrin phosphorylase".

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.

Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L.) by exogenously supplied carbohydrates in excised leaf discs.

A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia oleracea L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other plants, animals and prokaryotes. Escherichia coli cells carrying the entire cDNA for Pho1 expressed an active phosphorylase, which resembled the properties of the plastidic isozyme of spinach with respect to its low affinity to glycogen. Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity. Plastidic phosphorylase was transcribed in flowers and leaves, but the highest Pho1 transcript levels were found in mature fruits/seeds. This is in agreement with the enzyme activity levels, as Pho1 activity was detected in all tissues tested, but the highest activity was also present in mature fruits/seeds. Since developing seeds are strong sink organs, which import sucrose and accumulate starch, this observation may indicate that plastidic phosphorylase plays a role in starch formation. The assumption has been tested further by a series of induction experiments in which leaf discs from spinach and potato plants were incubated with various carbohydrates. Following incubation, phosphorylase steady-state transcript levels as well as levels of neutral sugars and starch were determined. A similar induction behaviour was found for Pho1 from spinach and Pho1a from potato, indicating the presence of related sugar signal transduction pathways in these two species. In addition, the expression of Pho1a and Agp4 (the large submit of ADPglucose synthase) from potato seems to be partly coordinately regulated by carbohydrates. These data may suggest that the regulation of Pho1 expression is linked to the carbohydrate status of the respective tissue.

Reversible binding of the starch-related R1 protein to the surface of transitory starch granules.

Intact starch granules were isolated from leaves of Solanum tuberosum L. (and from Pisum sativum L.), and the patterns of starch-associated proteins were determined by SDS-PAGE. Depending on the pretreatment of the leaves the protein patterns varied: a 160 kDa compound was present in the starch-associated protein fraction when the leaves were darkened and performed net starch degradation. However, following illumination (i.e. during net starch biosynthesis) the 160 kDa protein was recovered almost exclusively in a soluble state. The 160 kDa protein was identified to be the recently described starch-related R1 protein. In in vitro assays recombinant R1 did bind to starch granules isolated from either illuminated or darkened leaves. However, binding to the latter was more effective. It is concluded that, depending upon the metabolic state of the cells, the starch granule surface changes and thereby affects binding of the R1 protein.

Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels.

A method for the analysis of multiple forms of glucan-degrading enzymes is described. The procedure consists of the separation of the proteins by electrophoresis or isoelectric focusing in glucan-free polyacrylamide gels followed by the nondenaturing electrophoretic transfer into a second polyacrylamide layer which contains immobilized glucans. The method combines the resolving power of electrophoretic separations in glucan-free media with the sensitivity of amylase activity detection in amylose-containing polyacrylamide gels. The procedure is especially useful when samples containing low amylase activity, but a large number of multiple enzyme forms, are to be analyzed.

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves.

The non-chloroplastic α-glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.

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 α-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 α-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.

Characterization of starch breakdown in the intact spinach chloroplast.

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with (14)C-starch photosynthetically from (14)CO(2). Starch breakdown was dependent upon inorganic phosphate and the (14)C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of (14)C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.

Photoregulation of transfer and 5S ribosomal RNA synthesis in Chlorella.

The effect of blue and red light on the synthesis of transfer and 5S ribosomal RNA in autotrophic cultures of Chlorella pyrenoidosa was studied by pulse labeling experiments with tritiated guanosine. Compared with darkness or red light (679 nm), blue light (457 nm) of low intensities (quantum flux: 0.5-5×10(-10) mol photons cm(-2) s(-1)) stimulated incorporation of guanosine into transfer and 5S ribosomal RNA within the first 5 min of illumination. This blue light effect was abolished by cycloheximide, an inhibitor of protein synthesis on 80S ribosomes, but not by rifampicin, an inhibitor of chloroplastic transcription, nor by lincomycin, an inhibitor of chloroplastic translation. The rifampicin-insensitive synthesis of transfer and 5S ribosomal RNA was nuclear transcription as shown by RNA-DNA hybridization. The blue light effect on nuclear RNA synthesis was not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea, an inhibitor of photosynthesis electron transport. Further evidence for a photosynthesis-independent photocontrol of RNA synthesis was provided by experiments with the colorless mutant 125a of Chlorella vulgaris. Blue light stimulated incorporation of guanosine into cytoplasmic 25S and 18S ribosomal RNA as well as into transfer and 5S ribosomal RNA, whereas incorporation in red light was the same as that of the dark control.