Role of aluminum in red-to-blue color changes in <Emphasis Type="Italic">Hydrangea macrophylla</Emphasis> sepals

Red, purple, and blue sepals on selected cultivars of Hydrangea macrophylla were analyzed for their aluminum content. This content was determined to be a function of the sepal color with red sepals possessing 0–10 μg Al/g fresh sepal, purple sepals having 10–40 μg Al/g fresh sepal, and blue sepals containing greater than 40 μg Al/g fresh sepal. Accordingly, the threshold aluminum content needed to change H. macrophylla sepals from red to blue was about 40 μg Al/g fresh sepal. Higher aluminum concentrations were incorporated into the sepals, but this additional aluminum did not affect the intensity or hue of the blue color. These observations agreed with a chemical model proposing that the concentration of the blue Al³⁺-anthocyanin complex reached a maximum when a sufficient excess of aluminum was present. In addition, the visible absorbance spectra of harvested red, purple, and blue sepals were duplicated by Al³⁺ and anthocyanin (delphinidin-3-glucoside) mixtures in this model chemical system.     

Copigments in the blueing of sepal colour of Hydrangea macrophylla

From blue sepals of Hydrangea macrophylla, copigments which show a blueing effect on the hydrangea anthocyanin were isolated and identified as 3-p-coumaroylquinic acid and 3-caffeoylquinic acid. 5-Caffeoylquinic acid (chlorogenic acid) which was also found in the blue sepals, however, did not show such a blueing effect though it acted as a copigment. Likewise, the 4-esters of p-coumaroyl- and caffeoylquinic acids (not found in sepals) produced purple rather than blue colours. The facts suggest that the stereostructures of 3-p-coumaroyl- and 3-caffeoylquinic acids are effective for molecular interaction between the p-coumaroyl or caffeoyl residue in the compounds and the anthocyanin. The anthocyanin in red and blue sepals of hydrangea was confirmed to be delphinidin 3-monoglucoside.     

Blueing of sepal colour of Hydrangea macrophylla

Blue and red sepals of Hydrangea macrophylla were quantitatively analyzed for aluminium, anthocyanin (delphinidin 3-glucoside) and copigments (caffeoyl- and p-coumaroylquinic acids). All the blue sepals examined contained both Al and copigments (especially 3-caffeoylquinic acid) in considerable amounts. In in vitro experiments using 3- and 5-caffeoylquinic acids, Al and delphinidin 3-glucoside, it was shown that 3-caffeoylquinic acid and Al formed a blue complex with the anthocyanin. Absorption spectra of the blue complex were practically identical with those of the blue solutions obtained from blue hydrangea sepals by extraction with 4 M NACl. In contrast, 5-caffeoylquinic acid (chlorogenic acid) which was also present in hydrangea sepals gave only a red-purple colour with Al and the anthocyanin. Neither 3-caffeoylquinic acid nor Al independently produced blue colour when mixed with the anthocyanin in the mole ratios of 1–30, this being the range that the compounds were found in blue sepals. These results suggest that blue colour of hydrangea sepals is due mainly to the blue complex of delphinidin 3-glucoside-aluminium-3-caffeoylquinic acid. The role of aluminium may be to stabilize an interaction between the quinic ester and the anthocyanin.     

Flower Colour Modification of Chrysanthemum by Suppression of F3'H and Overexpression of the Exogenous Senecio cruentus F3'5'H Gene

Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium ‘LPi’ and C × morifolium ‘LPu’, that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3’H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3′H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3’H using RNAi and overexpressed the Senecio cruentus F3′5′H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3′H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3′5′H only exhibited F3′H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum.     

Change of color and components in sepals of chameleon hydrangea during maturation and senescence

The sepal color of a chameleon hydrangea, Hydrangea macrophylla cv. Hovaria™ ‘Homigo’ changes in four stages, from colorless to blue, then to green, and finally to red, during maturation and the senescence periods. To clarify the chemical mechanism of the color change, we analyzed the components of the sepals at each stage. Blue-colored sepals contained 3-O-sambubiosyl- and 3-O-glucosyldelphinidin along with three co-pigments, 5-O-p-coumaroyl-, 5-O-caffeoyl- and 3-O-caffeoylquinic acids. The contents of glycosyldelphinidins decreased toward the green-colored stage, with a coincident increase in the number of chloroplasts. During the last red colored stage, the two species of 3-O-glycosyldelphinidin almost disappeared, and another two anthocyanins, 3-O-sambubiosyl- and 3-O-glucosylcyanidin, increased in amounts. Mixing of 3-O-glycosylcyanidins, co-pigments, and Al³⁺ in a buffered solution at pH 3.0-3.5 gave not a blue, but a red, colored solution that was the same as that of the sepal color of the 4th stage. Sepals of hydrangea grown in an highland area also turned red in autumn, and contained the same cyanidin glycosides. The red coloration of the hydrangea during senescence was due to a change in anthocyanin biosynthesis.     

Flower colour and cytochromes P450

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.     

Biosynthesis of the Phytoalexin Pisatin : Isoflavone Reduction and Further Metabolism of the Product Sophorol by Extracts of Pisum sativum

NADPH-dependent reduction of 2′,7-dihydroxy-4′,5′-methylenedioxyisoflavone to the isoflavanone sophorol, a proposed intermediate step in pisatin biosynthesis, was detected in extracts of Pisum sativum. This isoflavone reductase activity was inducible by treatment of pea seedlings with CuCl₂. The timing of induction coincided with that of the 6a-hydroxymaackiain 3-O-methyltransferase, which catalyzes the terminal biosynthetic step. Neither enzyme was light inducible. Further NADPH-dependent metabolism of sophorol by extracts of Cucl₂-treated seedlings was also observed; three products were radiolabeled when [³H]sophorol was the substrate, one of which is tentatively identified as maackiain.     

Genes for the Biosynthesis of the Fungal Polyketides Hypothemycin from Hypomyces subiculosus and Radicicol from Pochonia chlamydosporia

Gene clusters for biosynthesis of the fungal polyketides hypothemycin and radicicol from Hypomyces subiculosus and Pochonia chlamydosporia, respectively, were sequenced. Both clusters encode a reducing polyketide synthase (PKS) and a nonreducing PKS like those in the zearalenone cluster of Gibberella zeae, plus enzymes with putative post-PKS functions. Introduction of an O-methyltransferase (OMT) knockout construct into H. subiculosus resulted in a strain with increased production of 4-O-desmethylhypothemycin, but because transformation of H. subiculosus was very difficult, we opted to characterize hypothemycin biosynthesis using heterologous gene expression. In vitro, the OMT could methylate various substrates lacking a 4-O-methyl group, and the flavin-dependent monooxygenase (FMO) could epoxidate substrates with a 1′,2′ double bond. The glutathione S-transferase catalyzed cis-trans isomerization of the 7′,8′ double bond of hypothemycin. Expression of both hypothemycin PKS genes (but neither gene alone) in yeast resulted in production of trans-7′,8′-dehydrozearalenol (DHZ). Adding expression of OMT, expression of FMO, and expression of cytochrome P450 to the strain resulted in methylation, 1′,2′-epoxidation, and hydroxylation of DHZ, respectively. The radicicol gene cluster encodes halogenase and cytochrome P450 homologues that are presumed to catalyze chlorination and epoxidation, respectively. Schemes for biosynthesis of hypothemycin and radicicol are proposed. The PKSs encoded by the two clusters described above and those encoded by the zearalenone cluster all synthesize different products, yet they have significant sequence identity. These PKSs may provide a useful system for probing the mechanisms of fungal PKS programming.     

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental ¹³C/¹⁵N/²H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2′ of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively ¹³C₉/¹⁵N₃/²H_{(1′,2″,3′,4′,5′,5″)}-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively ¹³C₉/¹⁵N₃/²H_{(1′,2″,3′,4′,5′,5″)}-dC and ¹³C₉/¹⁵N₂/²H_{(1′,2″,3′,4′,5′,5″)}-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective ²H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1′–H2′ and C2′–H2′ resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments.     

Functional characterization of key structural genes in rice flavonoid biosynthesis

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl ^w locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T⁶⁴ M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3′H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3′H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pasteurella Bacteriophage Sex Specific in Escherichia coli

Phage H, thought to be specific for Pasteurella pestis, was shown to plate efficiently on F⁻ strains of Escherichia coli but not on F⁺, F′, or Hfr strains. The phage was adsorbed rapidly to F⁻ strains but was not adsorbed to strains carrying F. Comparison with seven other reported female-specific phages showed that, although phage H was similar to the other phages in some characteristics, the exceptionally low efficiency of plating (<10⁻⁹) on F-containing cells makes phage H a particularly useful female-specific phage.