Late enzymes of vindoline biosynthesis

From differentiated plants of Catharanthus roseus (L.) G. Don we have isolated a specific enzyme of the vindoline biosynthetic pathway catalysing the S-adenosylmethionine-dependent methylation of 11-O-demethyl-17-O-deacetyl-vindoline. The enzyme we named S-adenosyl-L-methionine : 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase. This transferase exhibits a high substrate specificity. Obviously the O-methylation at C-11 precedes the O-acetylation at the C-17 position during the biosynthesis of vindoline.A second enzyme was detected which hydrolyses the acetyl function of vindoline. The distribution of this acetylesterase in C. roseus plants demonstrates that the enzyme is not specifically associated with the vindoline distribution in the plant material. Most probably this enzyme plays no essential role in the biosynthesis of vindoline.     

A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization,purification and kinetic properties

The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, -kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where -kg is the first substrate to bind followed by the binding of O₂ and desacetoxyvindoline. Their K _m values for -kg, O₂ and desacetoxyvindoline are 45 M, 45 M and 0.03 M, respectively. The first product to be released was deacetylvindoline followed by CO₂ and succinate, respectively.Abbreviations -kg -ketoglutarate or 2-oxoglutarate - NMT N-methyltransferase - SAM S-adenosyl-l-methionine - TLC thin layer chromatography - VBL vinblastine - VCR vincristine     

Phytochrome is not involved in the red-light-enhancement of the stomatal blue-light-response in wheat seedlings

The stomatal response to blue light (BL) in wheat seedlings ( Triticum aestivum L. cv. Starke II, Weibull) was enhanced by background red light (R). This enhancement was only slightly affected by the addition of background far-red light (FR). Under similar light treatments, the addition of FR induced a 43% transformation from the far-red-absorbing form towards the red-absorbing form of phytochrome from etiolated oat ( Avena sativa L. cv. Sol II), immobilized on phenyl-sepharose. Furthermore, the enhancement of the stomatal BL-response by 15 min R was not reversed by a subsequent irradiation with 5 min FR. It is concluded that the red-light-enhancement of the stomatal blue-light-response in wheat seedlings does not involve a change in the photostationary state of phytochrome.     

Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus

Various elicitors of hydroxylase, peroxidase, acetyltransferase and inhibitors of oxygenase were added to a Catharanthus roseus cell culture medium to investigate the regulatory effects on tabersonine, vindoline and vinblastine biosynthesis. Hydrogen peroxide was found to be the most effective agent for enhancing the biosynthesis of tabersonine. By adding 20???g/L hydrogen peroxide, the tabersonine concentration reached 9.02?mg/g dry weight (DW) after culturing cell suspensions for 7?days. With the addition of 30???g/L acetyl CoA, the most vindoline (final cell content of 0.33?mg/g DW) was produced. By effective inhibition of lochnericine biosynthesis with the addition of 0.5???mol/L benzotriazole, the cell content of vindoline was increased to 0.42?mg/g DW. An orthogonal experiment consisting of multiple regulation factors was carried out to optimize vinblastine biosynthesis. It was shown that optimal vinblastine biosynthesis was achieved by addition of 5?mg/L acetyl CoA, 20???g/L hydrogen peroxide, 0.5???mol/L benzotriazole, 100?mg/L tryptophan, 100?mg/L loganin and 30?mg/L cerium chloride. Under these conditions, the cell content of vinblastine reached 0.81?mg/g DW. Simultaneous changes in cell content and enzyme activities of Cytochrome P-450 monooxygenases, Deacetylvindoline-O-acetyltransferase and Peroxidase enzyme indicated that these enzymes were closely linked to vinblastine biosynthesis.     

Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2–3 weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S. nodorum.     

CylA is a sequence-specific protease involved in toxin biosynthesis

Journal of Industrial Microbiology & Biotechnology - CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As...     

AtABCG29 is a monolignol transporter involved in lignin biosynthesis

Lignin is the defining constituent of wood and the second most abundant natural polymer on earth. Lignin is produced by the oxidative coupling of three monolignols: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Monolignols are synthesized via the phenylpropanoid pathway and eventually polymerized in the cell wall by peroxidases and laccases. However, the mechanism whereby monolignols are transported from the cytosol to the cell wall has remained elusive. Here we report the discovery that AtABCG29, an ATP-binding cassette transporter, acts as a p-coumaryl alcohol transporter. Expression of AtABCG29 promoter-driven reporter genes and a Citrine-AtABCG29 fusion construct revealed that AtABCG29 is targeted to the plasma membrane of the root endodermis and vascular tissue. Moreover, yeasts expressing AtABCG29 exhibited an increased tolerance to p-coumaryl alcohol by excreting this monolignol. Vesicles isolated from yeasts expressing AtABCG29 exhibited a p-coumaryl alcohol transport activity. Loss-of-function Arabidopsis mutants contained less lignin subunits and were more sensitive to p-coumaryl alcohol. Changes in secondary metabolite profiles in abcg29 underline the importance of regulating p-coumaryl alcohol levels in the cytosol. This is the first identification of a monolignol transporter, closing a crucial gap in our understanding of lignin biosynthesis, which could open new directions for lignin engineering.     

DnaJA4 is a SREBP-regulated chaperone involved in the cholesterol biosynthesis pathway

Using subtractive hybridization technique in 3T3-L1 adipocytes overexpressing constitutively active SREBP2, we have identified a DnaJ/Hsp40 chaperone, DnaJA4, as a new SREBP-responsive gene. SREBP2 regulation was demonstrated by changes in DnaJA4 mRNA under conditions of altered sterol status that were strictly parallel to that of well-characterized SREBP targets (LDL receptor and HMG-CoA reductase). The role of SREBP2 was further established using adenoviral overexpression of a dominant negative SREBP2, which abolished cholesterol-regulated changes in DnaJA4 expression. To determine the functional significance of this regulation, DnaJA4 was overexpressed in COS cells, which induced a specific increase in the synthesis of cholesterol from acetate. We also observed that DnaJA4 overexpression increased the activity and the protein content of HMG-CoA reductase, the rate limiting enzyme in this pathway. At the molecular level, DnaJA4 overexpression did not alter HMG-CoA reductase stability or mRNA levels, suggesting a co-translational effect of the chaperone. In the DnaJ/Hsp40 family, DnaJA4 uniquely exhibited SREBP-regulated expression, and also responded to heat shock. Through its responsiveness to SREBP, and its stimulatory effect on cholesterol synthesis, the DnaJA4 chaperone can be viewed as a new player in cholesterol synthesis. These data suggest a link between molecular chaperones, heat stress and cholesterol synthesis.     

The paramutated SULFUREA locus of tomato is involved in auxin biosynthesis

The tomato (Solanum lycopersicum) sulfurea mutation displays trans-inactivation of wild-type alleles in heterozygous plants, a phenomenon referred to as paramutation. Homozygous mutant plants and paramutated leaf tissue of heterozygous plants show a pigment-deficient phenotype. The molecular basis of this phenotype and the function of the SULFUREA gene (SULF) are unknown. Here, a comprehensive physiological analysis of the sulfurea mutant is reported which suggests a molecular function for the SULFUREA locus. It is found that the sulf mutant is auxin-deficient and that the pigment-deficient phenotype is likely to represent only a secondary consequence of the auxin deficiency. This is most strongly supported by the isolation of a suppressor mutant which shows an auxin overaccumulation phenotype and contains elevated levels of indole-3-acetic acid (IAA). Several lines of evidence point to a role of the SULF gene in tryptophan-independent auxin biosynthesis, a pathway whose biochemistry and enzymology is still completely unknown. Thus, the sulfurea mutant may provide a promising entry point into elucidating the tryptophan-independent pathway of IAA synthesis.     

An ankyrin repeat protein is involved in anthocyanin biosynthesis in Arabidopsis

The ankyrin domain is one of the most common protein motifs in eukaryotic proteins. Repeated ankyrin domains are ubiquitous and their mediation of protein-protein interactions is involved in a number of physiological and developmental responses such as the cell cycle, signal transduction and cell differentiation. A novel putative phytochrome-interacting ankyrin repeat protein 2 (PIA2) containing three repeated ankyrin domains was identified in Arabidopsis. An in vitro pull-down and phosphorylation assay revealed that PIA2 is phosphorylated and interacts directly with oat phytochrome A. The N-terminal domain of PIA2 was specifically phosphorylated, whereas interactions between the domains of PIA2 and phytochrome A had no Pr/Pfr preference. PIA2 was ubiquitously expressed in most tissues and was localized in both the nucleus and the cytoplasm independent of treatment with light of specific wavelengths. Anthocyanin accumulation in seedlings grown under far-red light, a typical phenotype of wild-type plants, was reduced in a loss-of-function mutant of PIA2 (pia2), whereas anthocyanin accumulation was increased in an overexpressing plant (PIA2-OX). The gene expression of UDP-flavonoid-3'-glucosyl-transferase (UF3GT), a major enzyme in the anthocyanin biosynthesis processes, was decreased in pia2 knockout plants suggesting that decreased anthocyanin was because of the decreased expression of UF3GT. Our results suggest that PIA2 plays a role in the anthocyanin biosynthesis during seedling development as a novel phytochrome-interacting protein.     

Light activation of vindoline biosynthesis does not require cytomorphogenesis in Catharanthus roseus seedlings

Upon illumination, the cotyledons of Catharanthus roseus seedlings readily synthesise vindoline from late biosynthetic intermediates, which accumulate in etiolated seedlings. The cellular localisation of tryptophan decarboxylase (TDC) and desacetoxyvindoline 4-hydroxylase (D4H), which catalyse the first and penultimate reactions of vindoline biosynthesis, was identified by immunocytochemistry in developing seedlings. The expression of TDC was restricted to the upper epidermis of cotyledons, whereas that of D4H was confined to laticifer cells. Light exposure of etiolated seedlings significantly induced D4H enzyme activity without changing the steady-state levels of D4H immunoreactive protein or modifying the cellular distribution of D4H expression in dark-grown seedlings. These results suggest that the early and late stages of vindoline biosynthesis occupy different cellular compartments, even in the early phases of etiolated seedling development. The role of light in activating the late stages of vindoline biosynthesis does not, therefore, seem to be related to the formation of the laticifer and idioblast cell types. It is concluded that light is not required for formation of these cell types, whereas regulatory factors, restricted to idioblasts and laticifers, may respond to light to activate localised expression of the late stages of vindoline biosynthesis.     

Mechanisms involved in the biosynthesis of polysaccharides

The mechanisms for the biosynthesis of three polysaccharides are presented: (i) starch synthesized by starch synthase and adenosine diphospho glucose; (ii) dextran synthesized by Leuconostoc mesenteroides B-512FMC dextransucrase and sucrose; and (iii) Acetobacter xylinum cellulose synthesized by cellulose synthase, uridine diphospho glucose, and bactoprenol phosphate. All three enzymes were pulsed with substrates, containing ¹⁴C-glucose and chased with the same nonlabeled substrates. When the polysaccharides were isolated, reduced, and hydrolyzed, the pulsed reactions gave ¹⁴C-glucitol, which was significantly decreased in the chase reaction. These experiments definitively show that all three polysaccharides are biosynthesized by the addition of glucose to the reducing-ends of the growing polysaccharides and not by the addition to the nonreducing-ends of primers. Additional evidence indicates that glucose and the polysaccharides are covalently attached to the active-sites of the enzymes. A two catalytic-site insertion mechanism at one active-site is proposed for the biosyntheses. Two of the polysaccharides are α-linked glucans, starch and dextran, and cellulose is a β-linked glucan, known for several years to require a bactoprenol lipid phosphate intermediate. It is shown how this intermediate is involved in determining that β-linkages are synthesized. Other β-linked polysaccharides: bacterial cell wall peptidomurein, Salmonella O-antigen polysaccharide, and Xanthanomonas camprestris xanthan, are heteropolysaccharides, with the later two also being hetero-linked polysaccharides, with the β-linkage at the reducing-end of the repeating unit. All three require bactoprenol lipid phosphate intermediates and are biosynthesized by the addition of the repeating units to the reducing-end of a growing polysaccharide chain, with the formation of a β-linkage.     

Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase

From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.The enzyme shows a high selectivity towards different substrates. The acetyl-CoA-dependent transferase also catalyses the reverse reaction by hydrolysis of the 17-O-acetyl group of vindoline in the presence of free CoA. This enzyme is localized only in vindoline-containing plant parts, but was so far not detectable in cell suspension cultures of C. roseus. The enzyme allows the synthesis of labelled vindoline with high specific activity, applicable for instance as tracer for radioimmunoassays of vindoline.     

The terminal O‐acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A‐dependent acyl transfer

The gene encoding acetyl CoA:deacetylvindoline 4-O-acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross-reacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of ≈ 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O-acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.     

Multicellular compartmentation of catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate

In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed.     

Jasmonate is involved in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana

Coronatine-inducible tyrosine aminotransferase (TAT), which catalyses the transamination from tyrosine to p-hydroxyphenylpyruvate, is the first enzyme of a pathway leading via homogentisic acid to plastoquinone and tocopherols, the latter of which are known to be radical scavengers in plants. TAT can be also induced by the octadecanoids methyl jasmonate (MeJA) and methyl-12-oxophytodienoic acid (MeOPDA), as well as by wounding, high light, UV light and the herbicide oxyfluorfen. In order to elucidate the role of octadecanoids in the process of TAT induction in Arabidopsis thaliana (L.) Heynh., the jasmonate-deficient mutant delayed dehiscence (dde1) was used, in which the gene for 12-oxophytodienoic acid reductase 3 is disrupted. The amount of immunodetectable TAT was low. The enzyme was still fully induced by coronatine as well as by MeJA although induction by the latter was to a lesser extent and later than in the wild type. Treatment with MeOPDA, wounding and UV light, however, had hardly any effects. Tocopherol levels that showed considerable increases in the wild type after some treatments were much less affected in the mutant. However, starting levels of tocopherol were higher in non-induced dde1 than in the wild type. We conclude that jasmonate plays an important role in the signal transduction pathway regulating TAT activity and the biosynthesis of its product tocopherol.