Root restriction affected anthocyanin composition and up-regulated the transcription of their biosynthetic genes during berry development in ‘Summer Black’ grape

Root restriction was applied to ‘Summer black’ grape (Vitis vinifera L. × Vitis labrusca L.) to investigate its effect on anthocyanin biosynthesis in grape berry during development. Anthocyanin composition and expression patterns of 16 genes in anthocyanin pathway were thus analyzed. The results showed that the anthocyanin levels in berry skin were significantly increased and the anthocyanin profile was enriched. Gene expression pattern revealed that the increased anthocyanins coincide with the up-regulated expression of all 16 genes investigated, including phenylalanine ammonia-lyase, 4-coumarate CoA ligase, chalcone synthase 1, chalcone synthase 2, chalcone synthase 3, chalcone isomerase, flavanone 3-hydroxylase 1, flavanone 3-hydroxylase 2, flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), di-hydroflavonol 4-reductase, leucoanthocyanidin dioxygenase, O-methyltransferases (OMT), UDP-glucose:flavonoid 3-O-glucosyl-transferase (3GT), UDP-glucose:flavonoid 5-O-glucosyl-transferase (5GT) and glutathione S-transferase (GST). The increased total anthocyanins predominantly resulted from the increase of tri-hydroxylated, methoxylated and mono-glycosylated rather than di-hydroxylated, non-methoxylated, and di-glycosylated forms, which might be due to the differential regulation of F3′5′H/F3′H, OMT and 3GT, respectively.     

Inhibition of Abscisic Acid 8′-Hydroxylase Affects Dehydration Tolerance and Root Formation in Cuttings of Grapes (Vitis labrusca L. × Vitis vinifera L. cv. Kyoho) Under Drought Stress Conditions

The effects of an inhibitor (Abz-E3M) of abscisic acid (ABA) 8′-hydroxylase, which is a primary enzyme of ABA catabolism, on dehydration tolerance and root formation in grape cuttings under drought conditions were investigated. Cuttings of ‘Kyoho’ grape (Vitis labrusca L. × Vitis vinifera L.) were sprayed with 100 μM of Abz-E3M and subjected to water deficit conditions at the stage when their first leaves fully expanded. The physiological and morphological changes in the leaves and basal portions of the cuttings were determined. In Abz-E3M-treated leaves, lower ABA metabolite and higher ABA and indole-3-acetic acid (IAA) concentrations were observed. Compared to the untreated control leaves, higher water potential was significantly maintained in Abz-E3M-treated leaves. Abz-E3M applications resulted in lower proline accumulation and 2,2-diphenyl-2-picrylhydrazyl radical scavenging activity in the leaves and led to enhanced dehydration tolerance. In addition, the percentage of rooted cuttings was significantly increased by Abz-E3M application. In the basal portion of Abz-E3M-treated cuttings, endogenous IAA concentrations and the gene expressions of VvARF6 and VvARF8, which are positive regulators of adventitious root formation, were significantly increased. Moreover, the expression levels of the negative regulator, VvARF17, were significantly lower. These results suggested that the inhibition of ABA 8′-hydroxylase enhanced dehydration tolerance and adventitious rooting and may be an effective strategy for achieving drought stress tolerance in grape cuttings.

     

Enlarged analogues of uniconazole,new azole containing inhibitors of ABA 8′-hydroxylase CYP707A

We enlarged the uniconazole (UNI) molecule to find a specific inhibitor of abscisic acid (ABA) 8′-hydroxylase, and synthesized various UNI derivatives that were substituted with hydrophilic and hydrophobic groups at the 4-chlorine of the phenyl group of UNI using click chemistry. Considering its potency in ABA 8′-hydroxylase inhibition, its small effect on seedling growth, and its ease of application, UT4, the UNI derivative containing the C₄ alkyltriazole, was the best candidate for a highly selective inhibitor of ABA 8′-hydroxylase.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Reduction of Plant-Specific Arabinogalactan-Type O-Glycosylation by Treating Tobacco Plants with Ferrous Chelator 2,2′-Dipyridyl

Plant specific O-glycosylation of proteins includes the attachment of arabinogalactan to hydroxyproline (Hyp) residues. These Hyp residues are generated from peptidyl proline residues by the action of prolyl 4-hydroxylase which requires the ferrous ion. We investigated the effect of the ferrous chelator, 2,2′-dipyridyl on tobacco plants, and found that such treatment reduced the arabinogalactosylation of proteins.     

dl-[2-14C]p-CHLOROPHENYLALANINE AS AN INHIBITOR OF TRYPTOPHAN 5-HYDROXYLASE

The distribution in vivo of dl -[2-¹⁴C]p-chlorophenylalanine (p-CP) in regions and subcellular fractions of the rat brain was determined. The half-lives of p-CP and its metabolite p-chlorophenylpyruvic acid (p-CPPA) in plasma and brain were correlated with the development of inhibition of cerebral tryptophan 5-hydroxylase (EC 1.99.1.4). There was active transamination in vivo of p-CP and p-CPPA in the brain. Transport of indolealkylamino acids into brain was impaired by p-CP. Inhibition of tryptophan 5-hydroxylase could not be reversed by administration of large doses of l -tryptophan, l -tyrosine, or l -phenylalanine. After administration of [2-¹⁴C]p-CP in vivo, appreciable radioactivity was bound to cerebral proteins, including those with tryptophan 5-hydroxylase activity, as well as to phenylalanine 4-hydroxylase (EC 1.99.1.2) purified from liver. Amino acid analysis of the acid hydrolysate of purified, radioactive hepatic phenylalanine 4-hydroxylase showed over 80 per cent of the radioactivity to be present as p-CP. Neither the inhibition in vivo nor in vitro of tryptophan 5-hydroxylase could be reversed by dialysis; in controls, dialysis resulted in marked loss of enzyme activity. After incubation for 5 min with p-CP in vitro, enzymic activity was inhibited 60 per cent. In vitro, p-CPPA labelled protein much more extensively than p-CP, yet inhibited the enzyme less. Some of the label from p-CPPA was removable by dialysis.     

De novo biosynthesis of garbanzol and fustin in Streptomyces albus based on a potential flavanone 3-hydroxylase with 2-hydroxylase side activity

Flavonoids are important plant secondary metabolites, which were shown to have antioxidant, anti-inflammatory or antiviral activities. Heterologous production of flavonoids in engineered microbial cell factories is an interesting alternative to their purification from plant material representing the natural source. The use of engineered bacteria allows to produce specific compounds, independent of soil, climatic or other plant-associated production parameters. The initial objective of this study was to achieve an engineered production of two interesting flavanonols, garbanzol and fustin, using Streptomyces albus as the production host. Unexpectedly, the engineered strain produced several flavones and flavonols in the absence of the additional expression of a flavone synthase (FNS) or flavonol synthase (FLS) gene. It turned out that the heterologous flavanone 3-hydroxylase (F3H) has a 2-hydroxylase side activity, which explains the observed production of 7,4′-dihydroxyflavone, resokaempferol, kaempferol and apigenin, as well as the biosynthesis of the extremely rare 2-hydroxylated intermediates 2-hydroxyliquiritigenin, 2-hydroxynaringenin and probably licodione. Other related metabolites, such as quercetin, dihydroquercetin and eriodictyol, have also been detected in culture extracts of this recombinant strain. Hence, the enzymatic versatility of S. albus can be conveniently exploited for the heterologous production of a large diversity of plant metabolites of the flavonoid family.     

Metabolic engineering and mechanical investigation of enhanced plant autoluminescence

The fungal bioluminescence pathway (FBP) was identified from glowing fungi, which releases self-sustained visible green luminescence. However, weak bioluminescence limits the potential application of the bioluminescence system. Here, we screened and characterized a C3′H1 (4-coumaroyl shikimate/quinate 3′-hydroxylase) gene from Brassica napus, which efficiently converts p-coumaroyl shikimate to caffeic acid and hispidin. Simultaneous expression of BnC3′H1 and NPGA (null-pigment mutant in A. nidulans) produces more caffeic acid and hispidin as the natural precursor of luciferin and significantly intensifies the original fungal bioluminescence pathway (oFBP). Thus, we successfully created enhanced FBP (eFBP) plants emitting 3 × 10¹¹ photons/min/cm², sufficient to illuminate its surroundings and visualize words clearly in the dark. The glowing plants provide sustainable and bio-renewable illumination for the naked eyes, and manifest distinct responses to diverse environmental conditions via caffeic acid biosynthesis pathway. Importantly, we revealed that the biosynthesis of caffeic acid and hispidin in eFBP plants derived from the sugar pathway, and the inhibitors of the energy production system significantly reduced the luminescence signal rapidly from eFBP plants, suggesting that the FBP system coupled with the luciferin metabolic flux functions in an energy-driven way. These findings lay the groundwork for genetically creating stronger eFBP plants and developing more powerful biological tools with the FBP system.     

Cooked Odor of Euphausia pacifica Hansen

Quinoxaline and benzimidazole derivatives obtained from L-rhamnose and L-fucose under deoxygenated, weakly acidic, heated conditions were studied using GLC, HPLC, and NMR.

Four quinoxalines and one benzimidazole were obtained from L-rhamnose (RHA-I, II, III, III′, and IV) and L-fucose (FUA-I, II, III, IV, and V) in an acidic solution (MeOH-AcOH-H₂I = 8 : 1 : 2) at 80°C. The total yield of the products as sugar was about 80% from either rhamnose or fucose.

The structure of RHA-I was (2′S)-2-methyl-3-(2′-hydroxypropyl)quinoxaline; RHA-II, (2′R,3′S)-2-(2′,3′-dihydroxybutyl)quinoxaline; RHA-III, (1′S,2′S,3′S)-2-(1′2′3′-trihydroxybutyl)quinoxaline[2-(L-arabino-1′,2′,3′-trihydroxybutyl)quinoxaline]; RHA-III′, 2-(L-ribo-1′,2′,3′-trihydroxybutyl)quinoxaline; and RHA-IV, 2-(L-manno-1′,2′,3′,4′-tetrahydroxypentyl)-benzimidazole, and the structure of FUA-I was the same as RHA-I; FUA-II, (2′S, 3′S)-2-(2′, 3′-dihydroxybutyl)quinoxaline; FUA-III, (1′R, 2′R, 3′S)-2-(1′,2′,3′-trihydroxybutyl)quinoxaline [2-(L-xylo-1′,2′,3′-trihydroxybutyl)quinoxaline; FUA-IV, 2-(L-lyxo-1′,2′,3′-trihydroxybutyl)-quinoxaline; and FUA-V, 2-(L-galacto-1′,2′,3′,4′-tetrahydroxypentyl)benzimidazole. These results suggest no significant difference for the pathways of quinoxaline and benzimidazole formation between L-rhamnose and L-fucose. Possible pathways are proposed for each sugar.     

The B-ring hydroxylation pattern of anthocyanins can be determined through activity of the flavonoid 3′-hydroxylase on leucoanthocyanidins

Main conclusion

In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3′ position by flavonoid 3’-hydroxylase.

Abstract

The cytochrome P450-dependent monooxygenases flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3′ or 3′ and 5′, respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3′H and F3′5′H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3′H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3′-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3′,5′-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3′Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.     

Nucleic Acid Related Compounds. LXXXI. Efficient General Synthesis of Purine (Amino,Azido, and Triflate)-Sugar Nucleosides

Abstract

Treatment of 3′,5′-O-(tetraisopropyldisiloxanyl)adenosine and its arabino epimer with trifluoromethanesulfonyl chloride/DMAP gave the 2′-triflates in high yields. Displacements (LiN₃/DMF) and deprotection gave 2′-azido-2′-deoxyadenosine and its arabino epimer which were reduced with Bu₃SnH/AIBN/DMAC/benzene (or Staudinger reduction) to give 2′-amino-2′-deoxyadenosine and its epimer. Oxidation of 2′,5′-bis-O-(tert-butyldimethylsilyl)adenosine, stereoselective reduction, triflation, azide displacement, deprotection, and reduction gave 3′-amino-3′-deoxyadenosine.     

Identification and functional analysis of anthocyanin biosynthesis genes in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.     

Synthesis,In Vitro Anti-HIV Activity,and Biological Stability of 5′-O-Myristoyl Analogue Derivatives of 3′-Fluoro-2′,3′-Dideoxythymidine (FLT) as Potential Bifunctional Prodrugs of FLT

Abstract

A group of 5′-O-myristoyl analogue derivatives of FLT (2) were evaluated as potential anti-HIV agents that were designed to serve as prodrugs to FLT. 3′-Fluoro-2′,3′-dideoxy-5′-O-(12-methoxydodecanoyl)thymidine (4) (EC₅₀ = 3.8 nM) and 3′-fluoro-2′,3′-dideoxy-5′-O-(12-azidododecanoyl)thymidine (8) (EC₅₀ = 2.8 nM) were the most effective anti-HIV-1 agents. There was a linear correlation between Log P and HPLC Log retention time for the 5 ′-O-FLT esters. The in vitro enzymatic hydrolysis half-life (t½), among the group of esters (3–8) in porcine liver esterase, rat plasma and rat brain homogenate was longer for 3′-fluoro-2′,3′-dideoxy-5 ′-O-(myristoyl)thymidine (7), with t½ values of 20.3, 4.6 and 17.5 min, respectively.     

Synthesis of New Potential Lipophilic Co‐Drugs of 2‐Chloro‐2′‐deoxyadenosine (Cladribine, 2‐CdA,Mavenclad®, Leustatin®) and 6‐Azauridine (z6U) with Valproic Acid

2‐Chloro‐2′‐deoxyadenosine (cladribine, 1 ) was acylated with valproic acid ( 2 ) under various reaction conditions yielding 2‐chloro‐2′‐deoxy‐3′,5′‐O‐divalproyladenosine ( 3 ) as well as the 3′‐O‐ and 5′‐O‐monovalproylated derivatives, 2‐chloro‐2′‐deoxy‐3′‐O‐valproyladenosine ( 4 ) and 2‐chloro‐2′‐deoxy‐5′‐O‐valproyladenosine ( 5 ), as new co‐drugs. In addition, 6‐azauridine‐2′,3′‐O‐(ethyl levulinate) ( 8 ) was valproylated at the 5′‐OH group (→ 9 ). All products were characterized by ¹H‐ and ¹³C‐NMR spectroscopy and ESI mass spectrometry. The structure of the by‐product 6 (N‐cyclohexyl‐N‐(cyclohexylcarbamoyl)‐2‐propylpentanamide), formed upon valproylation of cladribine in the presence of N,N‐dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X‐ray crystallography. Cladribine as well as its valproylated co‐drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS‐3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol‐12‐myristate 13‐acetate (PMA)‐differentiated human THP‐1 macrophages. The most important result of these experiments is the finding that only the 3′‐O‐valproylated derivative 4 exhibits a significant antitumor activity while the 5′‐O‐ as well as the 3′,5′‐O‐divalproylated cladribine derivatives 3 and 5 proved to be inactive.     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.     

Crystal structure of guanylyl-2′,5′-cytidine dihydrate: An analogue of msDNA-RNA junction in stigmatella aurantiaca

Sequence analysis of msDNA from bacterium such as Stigmatella aurantiaca, Myxococcus xanthus and Escherichia coli B revealed that the guanine residue of the single-stranded RNA is linked to the cytosine residue of the msDNA through a 2′–5′ instead of a conventional 3′–5′ phosphodiester bond. We have now obtained the crystal structure of the self-complementary dimer guanylyl-2′,5′-cytidine (G2′p5′C) that occurs at the msDNA-RNA junction. G2′p5′C crystallizes in the orthorhombic space group P2₁2₁2₁ with a = 8.376(2), b = 16.231(5), c = 18.671(4). CuK ∝ intensity data were collected on a diffractometer in the ω ?2θ scan mode. The amount of 1699 out of 2354 reflections having I ≥ 3σ (F) were considered observed. The structure was solved by direct methods and refined by full-matrix least squares to a R factor of 0.054. The conformation of the guanine base about the glycosyl bond is syn (χ₁ = ?54°) and that of cytosine is anti (χ₂ = 156°). The 5′ and 2′ and ribose moieties show C2′-endo and C3-endo mixed puckering just like in A2′p5′A, A2′p5′C, A2′p5U, and dC3′p5′G. Charge neutralization in G2′p5′C is accomplished through protonation of the cytosine base. An important feature of G2′p5′C is the stacking of guanine on ribose 04′ of cytosine similar to that seen in other 2′–5′ dimers. G2′p5′C, unlike its 3′–5′ isomer, does not form a miniature double helix with the Watson-Crick base-pairing pattern. Comparison of G2′p5′C with A2′p5′C reveals that they are isostructural. A branched trinucleotide model for the msDNA-RNA junction has been postulated. © 1994 John Wiley & Sons, Inc.