Anthocyanin production in callus cultures of Cleome rosea: Modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 μM 2,4-D. Reddish-pink regions were observed on callus surface after 6–7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH₄⁺/NO₃⁻, 70 g L⁻¹ sucrose and supplementation with 0.90 μM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Cellobiose catabolism in the haloalkaliphilic hydrolytic bacterium <Emphasis Type="Italic">Alkaliflexus imshenetskii</Emphasis>

Cellobiose metabolism was studied in Alkaliflexus imshenetskii, a haloalkaliphilic hydrolytic bacterium capable of utilizing certain polymers of plant origin, as well as mono- and disaccharides. The major products of cellobiose fermentation by the bacterium were succinate and acetate, and formate was a minor product. Cellobiose could be split into glucose molecules by both β-glucosidase (hydrolytic pathway) and phosphorylase (phosphorolytic pathway); the activity of the former enzyme was two orders of magnitude higher (3600 nmol/(min mg) versus 36 nmol/(min mg)). In cell extracts of the bacterium, high activities of the Embden-Meyerhof-Parnas pathway enzymes—hexokinase, glucose-phosphate isomerase, and phosphofructokinase—were revealed, as well as the activities of glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and key enzymes of the Entner-Doudoroff pathway—6-phospho-gluconate dehydratase and 2-keto-3-deoxy-6-phospho-gluconate aldolase. Neither the activity of the key enzyme of the hexose-mono-phosphate pathway, 6-phospho-gluconate dehydrogenase, nor the activities of the key enzymes of the modified Entner-Doudoroff pathway, glucose dehydrogenase and 2-keto-3-deoxy-gluconate kinase, were revealed.     

Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells

In plants several ‘starch-related’ enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several β‐glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site‐saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl β‐glucosides, methyl α‐glucoside and cellobiose. In comparison with the wild‐type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413–420. © 2010 Wiley Periodicals, Inc.     

Activation of guanine-β-d-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

The deoxyguanosine (GdR) analog guanine-ß-d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.     

Rice endosperm-specific plastidial α-glucan phosphorylase is important for synthesis of short-chain malto-oligosaccharides

Previous genetic studies have indicated that the type L α-glucan phosphorylase (Pho1) has an essential role during the initiation process of starch biosynthesis during rice seed development. To gain insight into its role in starch metabolism, we characterized the enzymatic properties of the Pho1 recombinant form. Pho1 has significantly higher catalytic efficiency toward both linear and branched α-glucans in the synthesis direction than in the degradation direction with equilibrium constants for the various substrates ranging from 13 to 45. Pho1 activity is strongly inhibited by its own reaction product (Pi) in the synthesis reaction (K_i = 0.69 mM) when amylopectin is the primer substrate, but this inhibition is less pronounced (K_i = 14.2 mM) when short α-glucan chains are used as primers. Interestingly, even in the presence of Pi alone, Pho1 not only degrades maltohexaose but also extends them to synthesize longer MOSs. Production of a broad spectrum of MOSs (G4-G19) was stimulated by both Pi and Glc1P in an additive fashion. Thus, even under physiological conditions of high Pi/Glc1P, Pho1 extends the chain length of short MOSs which can then be used as subsequent primer by starch synthase activities. As ADP-glucose strongly inhibits Pho1activity, Pho1 likely operates only during the initial stage and not during maturation phase of starch synthesis.     

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.