Cell type-specific protoberberine alkaloid accumulation inThalictrum flavum

Berberine is a common benzylisoquinoline alkaloid with potent antimicrobial properties, which suggest it functions to protect some plants from pathogen challenge. Berberine was identified as the major alkaloid in meadow rue (Thalictrum flavum ssp. glaucum), a medicinal member of the Ranunculaceae, and was detected in seeds and all organs of the plant. The high level of berberine in roots, rhizomes, and older petioles is mainly responsible for the intense yellow color of these organs. In rhizomes, protoberberine alkaloids were detected throughout the pith and, to a lesser extent, the cortex, but were absent from the vascular tissues. Similarly, protoberberine alkaloids were detected in the rib parenchyma of older petioles. In roots, alkaloid accumulation was detected only in the endodermis at the onset of secondary growth. Rather than being sloughed off, the endodermis was found to undergo extensive anticlinal division leading to an expanding cellular cylinder that ultimately displaced all external tissues. Endodermal-specific protoberberine alkaloid accumulation continued throughout root development, but was extended to include 3 to 4 layers of smaller pericycle cells in the oldest roots near the base of the stem. The cell type-specific accumulation of antimicrobial alkaloids and the unusual development of the endodermis and pericycle in T. flavum roots support the putative role of berberine in plant defense.     

Immunocytological localization of two enzymes involved in berberine biosynthesis

Using post-embedding immunogold techniques the cytological localization of the two branchpoint enzymes of isoquinoline biosynthesis, berberine bridge enzyme (BBE) and (S)-tetrahydroprotoberberine oxidase (STOX), was demonstrated. Electron-microscopic examination revealed their exclusive compartmentation within vesicles. After these vesicles have fused with the central vacuole, they release their contents, resulting in a characteristic electron-dense precipitate at the tonoplast. Vesicles of similar structure could be identified in young meristematic tissues of roots or shoots from different Berberis species and Papaver somniferum L. The appearance of electron-dense osmiophilic material is strictly correlated with the alkaloid content of the tissue. Immunocytological staining of P. somniferum tissue with antibodies directed against BBE led to a characteristic labeling of electron-dense aggregates in idioblasts that are not connected to the laticifer system. This localization demonstrates the strictly cytological separation of benzophenanthridine and morphine biosyntheses within this plant.     

The localization within plant cells of enzymes involved in arginine biosynthesis

Studies were carried out to determine the distribution of the following: (1) carbamoyl phosphate synthetase (EC 2.7.2.9), (2) ornithine carbamoyltransferase (EC 2.1.3.3), (3) argininosuccinate synthetase (EC 6.3.4.5), and (4) argininosuccinate lyase (EC 4.3.2.1) in soybean cells grown in suspension culture. Protoplasts were produced from the soybean cells by treatment with cellulase (EC 3.2.1.4) and pectinase (EC 3.2.1.15); the protoplasts were then ruptured by osmotic shock with distilled water. This treatment was followed by differential centrifugation and sucrose density gradient centrifugation to isolate various organelle fractions including mitochondria and plastids. Examination of these fractions using specific enzyme assays showed that carbamoylphosphate synthetase and ornithine carbamoyltransferase were localized in a fraction found to be composed primarily of plastids. Argininosuccinate synthetase and argininosuccinate lyase appeared to be associated with either the cytosol or a membrane fraction in close association with the cytosol such as the endoplasmic reticulum or protoplast membrane.     

靛蓝生物合成酶的研究进展

靛蓝色素是人类所知最古老的色素之一,广泛用于食品、医药和印染工业。靛蓝最初由植物中提取获得,作为一种色泽艳丽、无毒无害、能够生物降解的环保型染料,深受青睐。生物合成靛蓝因其绿色、高效、节约土地、安全稳定等优点,作为取代目前植物提取及化学合成的主流途径,受到广泛关注。在过去的几十年中,许多天然酶和工程酶已筛选用于合成靛蓝。本文概述了能够催化靛蓝生物合成的酶及相关应用,并探讨了当前存在的问题和未来研究方向,为推进基于生物合成的靛蓝工业化生产奠定基础。     

Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation

Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 on the B. subtilis chromosome, it encodes five poly-peptides with extensive similarity to enzymes involved in glycogen and starch metabolism in both prokaryotes and eukaryotes. The operon is presumably expressed by an Eσ^E-controlled promoter, which was previously identified downstream from trnB. We have observed glycogen biosynthesis in B. subtilis exclusively on media containing carbon sources that allow efficient sporulation. Sporulation-independent synthesis of glycogen occurred after integration of an Eσ^A controlled promoter upstream of the operon.     

Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts

Flavanone synthase, chalcone-flavanone isomerase and UDP-glucose; anthocyanidin-3-O-glucosyltransferase activities of protoplasts and subcellular fractions of protoplasts of Hippeastrum and Tulipa were investigated. Subcellular fractions studied were intact vacuoles, cytosol and particulate components of protoplasts less the vacuole. The cytosol fraction had the highest activity of the three enzymes studied. Results similar to those found for Hippeastrum were obtained with fractions from leaves and petals of Tulipa. The increase in flavanone synthase activity in the cytosol fraction from petals of Hippeastrum during development paralleled the increase in anthocyanin content of the petals.     

Activation of chloroplast-localized enzymes of quinolizidine alkaloid biosynthesis by reduced thioredoxin

Previous studies from the laboratory of the authors have shown that the tetracyclic quinolizidine alkaloids are synthesized in leaf chloroplasts of Lupinus polyphyllus. Additionally, alkaloid formation reveals a light dependent diurnal rhythm in vivo. The present study shows that the principal biosynthetic enzymes, lysine decarboxylase and 17-oxosparteine synthase, assayed in acetone powder extracts and isolated chloroplasts of L. polyphyllus, were activated by reduced E. coli thioredoxin. Since both enzymes display optimal activity at pH 8 and were rather inactive at pH 7, both thioredoxin and the light mediated shift in the hydrogen ion concentration of the chloroplast stroma from pH 7 to pH 8 may be involved in the light controlled alkaloid formation.Abbreviations DTE dithioerythritol     

Genetic locus encoding functions involved in biosynthesis and outer membrane localization of xanthomonadin in Xanthomonas oryzae pv. oryzae

Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus Xanthomonas. We have characterized a genetic locus (pig) from Xanthomonas oryzae pv. oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production. Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis. The fourth ORF has no homologue in the database. For the first time, we report that a putative cytoplasmic membrane protein encoded in the pig locus is required for outer membrane localization of xanthomonadin in X. oryzae pv. oryzae. We also report the identification of a novel 145-bp palindromic Xanthomonas repetitive intergenic consensus element that is present in two places in the pig locus. We estimate that more than 100 copies of this element might be present in the genome of X. oryzae pv. oryzae and other xanthomonads.     

Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation

The COOH-terminal alpha-amidation of bioactive peptides is a 2-step process catalyzed by two separable enzymatic activities both derived from the peptidylglycine alpha-amidating monooxygenase (PAM) precursor. Two forms of PAM mRNA (rPAM-1 and -2), differing by the presence or absence of optional Exon A, were previously characterized; both encode precursors predicted to have an NH2-terminal signal sequence, an intragranular domain containing both enzymatic activities, and a single transmembrane domain followed by a short, cytoplasmic COOH-terminal domain. In this report, two novel types of PAM mRNA were identified in adult rat atrium. A cDNA of each type was sequenced, and the results indicate that rPAM-3 and -4 could be related to each other and to the previously characterized rat PAM cDNAs by alternative mRNA splicing. Deletion of a 258-nucleotide segment (optional Exon B) encoding the transmembrane domain from rPAM-3 and the presence of a novel 3'-exon in rPAM-4 mean that both rPAM-3 and -4 mRNAs encode precursor proteins that have an NH2-terminal signal peptide but lack a transmembrane domain. The rPAM-4 precursor protein lacks the region of the PAM precursor catalyzing the second step in the alpha-amidation reaction. Low levels of rPAM-3 and -4 type mRNA were detected in atrium. Utilizing the polymerase chain reaction, two major patterns of distribution of forms of PAM mRNA were found. In the heart and central nervous system, PAM mRNAs both containing and lacking optional Exon A were prevalent and almost all of the PAM mRNAs detected contained optional Exon B. In the pituitary and submaxillary glands, PAM mRNAs lacking optimal Exon A were prevalent, as were PAM mRNAs lacking all or part of optional Exon B. Since the distribution of PAM activity between soluble and membrane fractions is tissue-specific and developmentally regulated and since rPAM-4 lacks an enzymatic portion of the PAM precursor, the tissue-specific expression of these forms of rat PAM mRNA is expected to be of functional significance.     

Cell type specific localization of sphingomyelin biosynthesis

We have studied the incorporation of [(14)C]serine and of [(3)H]sphingosine into sphingomyelin in the presence or absence of brefeldin A (BFA) in three different cell types. Administration of BFA (1 microgram/ml) to fibroblasts for 24 h increased the incorporation of label into sphingomyelin 1.5-3 fold compared with untreated controls. In contrast, BFA strongly decreased sphingomyelin biosynthesis (4-5 fold) in cerebellar neurons as well as in neuroblastoma cells. The effect of BFA on glycosphingolipid formation, however, was similar in all three cell types studied: an increased labeling of the precursor glycolipids GlcCer, LacCer, GM3 and GD3 was paralleled by a decreased formation of complex gangliosides, GM1, GD1a, GT1b and GQ1b. Our data therefore suggest that in neuronal cells sphingomyelin synthesis, like the formation of complex gangliosides, is localized primarily distal to the BFA block, in a post-Golgi compartment, most probably the trans-Golgi network, whereas in fibroblasts sphingomyelin biosynthesis is mainly localized prior to the BFA block, in the Golgi apparatus, as has been shown for LacCer, GlcCer, GM3 and GD3 synthases.     

Isolation and properties of enzymes involved in prostaglandin biosynthesis.

Prostaglandin (PG) endoperoxide synthetase was purified until homogeneity had been attained. The pure enzyme displays both cyclooxygenase and peroxidase activity, in accordance with the work of MIYAMOTO et al. (J. biol. Chem. 252, 2629--2636 (1976)). This enzyme therefore converts arachidonic acid into PGH2. Glutathione S-transferases, in the presence of glutathione, convert PGH2 into a mixture of PGF2alpha, PGE2 and PGD2. A new transferase in sheep lung gives mainly PGF2alpha and PGD2. Isolation and properties of these enzymes will be discussed. Finally, progress will be reported on the isolation of a soluble enzyme from various rat organs such as lung and spleen, which forms almost exclusively prostaglandin D.