Manipulation of flavour and aroma compound sequestration and release using a glycosyltransferase with specificity for terpene alcohols.

Glycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP-glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)-hex-3-enol, contribute strongly to the 'grassy-green' aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over-expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan-3-ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over-expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over-expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3-methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening. Yauk YK1, Ged C, Wang MY, Matich AJ, Tessarotto L, Cooney JM, Chervin C, Atkinson RG

Apple skin patterning is associated with differential expression of MYB10

Background: Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. ‘Royal Gala’) produce fruit with a defined fruit pigment pattern, but in the case of ‘Honeycrisp’ apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown. Results: Here we show that striped areas of ‘Honeycrisp’ and ‘Royal Gala’ are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in ‘Honeycrisp’ and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5’ of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in ‘Honeycrisp’ red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. Conclusions: Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in ‘Honeycrisp’. We suggest that methylation in the MYB10 promoter is more variable in ‘Honeycrisp’ than in ‘Royal Gala’, leading to more variable color patterns in the peel of this cultivar.https://doi.org/10.1186/1471-2229-11-9

Consumption of an Anthocyanin-Rich Extract Made From New Zealand Blackcurrants Prior to Exercise May Assist Recovery From Oxidative Stress and Maintains Circulating Neutrophil Function: A Pilot Study

Aim: To evaluate blackcurrant anthocyanin-rich extract (BAE) consumption on time- and dose-dependent plasma anthocyanin bioavailability and conduct a pilot study to explore the potential effect of BAE in promoting recovery from exercise-induced oxidative stress, and maintenance of circulating neutrophil function.Methods: Time- and dose-dependent blackcurrant anthocyanin bioavailability was assessed using LC-MS in 12 participants over 6 h after the ingestion of a placebo or BAE containing 0.8, 1.6, or 3.2 mg/kg total anthocyanins. In a separate pilot intervention exercise trial, 32 participants consumed either a placebo or 0.8, 1.6, or 3.2 mg/kg BAE (8 individuals per group), and then 1 h later performed a 30 min row at 70% VO2max. Blood was collected during the trial for oxidative, antioxidant, inflammatory, and circulating neutrophil status.Results: Consumption of BAE caused a time- and dose-dependent increase in plasma anthocyanins, peaking at 2 h after ingestion of 3.2 mg/kg BAE (217 ± 69 nM). BAE consumed 1 h prior to a 30 min row had no effect on plasma antioxidant status but hastened the recovery from exercise-induced oxidative stress: By 2 h recovery, consumption of 1.6 mg/kg BAE prior to exercise caused a significant (P < 0.05) 34 and 32% decrease in post-exercise plasma oxidative capacity and protein carbonyl levels, respectively, compared to placebo. BAE consumption prior to exercise dose-dependently attenuated a small, yet significant (P < 0.01) transient 13 ± 2% decline in circulating neutrophils observed in the placebo group immediately post-exercise. Furthermore, the timed consumption of either 1.6 or 3.2 mg/kg BAE attenuated a 17 ± 2.4% (P < 0.05) decline in neutrophil phagocytic capability of opsonised FITC-Escherichia coli observed 6 h post-exercise in the placebo group. Similarly, a dose-dependent increase in neutrophil surface expression of complement receptor-3 complex (CR3, critical for effective phagocytosis of opsonised microbes), was observed 6 h post-exercise in both 1.6 and 3.2 mg/kg BAE intervention groups.Conclusions: Consumption of BAE (>1.6 mg/kg) 1 h prior to exercise facilitated recovery from exercise-induced oxidative stress and preserved circulating neutrophil function. This study provides data to underpin a larger study designed to evaluate the efficacy of timed BAE consumption on post-exercise recovery and innate immunity

Preparation and reactions of some organic derivatives of manganese carbonyl compounds

New orthomanganated aryl ketones of type 1 (Figure 1) have been prepared, including some in which the donor ketone carbonyl group is incorporated into a fused ring. Analogous derivatives of benzoates and heteroaromatic carboxylic acid esters are also described, together with several orthorheniated compounds. Single crystal X-ray structure determinations of η²-5-(1,4-benzopyronyl)tetracarbonylmanganese (13) and η²-(4-acetyl-2,5-dimethylthien-3 yl) tetracarbonylrhenium (32) are reported. Reactions of orthomanganated aryl ketones with SO₂ and with the electronically and structurally related cumulenes, N-sulfinylaniline (PhN=S=O), N-sulfinylbenzenesulfonamide (PhS(O)₂N=S=O) and the disulfonylsulfodiimide [PhS(O)₂N]₂S have been investigated. SO₂ inserts efficiently into the Mn-Cₐᵣᵧₗ bond of a range of orthomanganated ketones, to give the corresponding S-sulfinato complex e.g. (43) (Figure 1). The X-ray crystal structure of (43) was determined and it is the first reported example of a six-membered metallocycle incorporating an O, Mn and S atom. In contrast, reaction of PhN=S=O with a number of orthomanganated acetophenones led to the corresponding orthomanganated imines e.g. (38) (Figure 1). The structure of η²-3-chloro-2-[1-(N-phenylimino)ethyl]-phenyl-tetracarbonylmanganese (44) was determined by single crystal X-ray diffraction. PhS(O)₂NSO and [PhS(O)₂N]₂S did not react with orthomanganated p-methoxyacetophenone (39) under similar conditions. The palladium- and thermally-promoted coupling reactions of orthomanganated aryl ketones and esters with alkenes have been explored in detail. In the presence of Pd(II), η²-(o-acetylaryl)tetracarbonylmanganese compounds undergo coupling reactions with methyl acrylate, methyl vinyl ketone, acrylonitrile, acrolein, vinyl acetate and allyl alcohol. The coupling reactions normally proceed in excellent yield and produce three main products, arylalkene, arylalkane and indene, the ratios of which vary with reaction conditions and substrate (Scheme 1; Path (I)). Orthomercurated aryl ketones obtained via transmetalation of orthomanganated ketones with mercuric chloride allowed a comparison of the product distribution for Pd(II)-promoted methyl acrylate coupling of an orthomercurated precursor 4-acetyl-2,5-dimethylthien-3-ylmercury(II) chloride (112) and the corresponding orthomanganated precursor η²-(4-acetyl-2,5-dimethylthien-3-yl)tetracarbonylmanganese (41) under identical conditions. Cyclisation to give the indene-type product methyl 1,3,6- trimethyl-4H-cyclopenta[c]thiophene-5- carboxylate (53) (whose crystal structure is reported) was found to be much more strongly favoured in the Mn than the Hg case precluding the possibility that the only role of both replaced metals is in transmetalation with palladium prior to alkene coupling. This conclusion is supported by similar reactions with phosphite or phosphine derivatives of cyclomanganated aryl ketones e.g. (145), (146) (Figure 2), and by reactions of orthorheniated substrates e.g. (32) (Figure 2). In the absence of Pd(II), alkenes were also found to insert efficiently into the Mn-Cₐᵣᵧₗ bond of orthomanganated ketones at ca 80 °C. In this case, the major products were found to be arylalkane, indene and a mixture of the diastereoisomeric indanols (Scheme 1; Path (II)). Arylalkene products were formed in very small or indetectable amounts. Possible reaction pathways for both the Pd(II) and the thermally initiated reactions are presented. Several sterically crowded 3-substituted orthomanganated esters and ketones were reacted with ICl to give the corresponding 2-iodo-3-substituted compounds (e.g. Equation 1). 3-Substituted 2-iodo esters formed via this route are of interest as potential precursors for the formation of 5-substituted 9-oxo-9H-xanthene-4 acetic acid (XAA) compounds, which exhibit antitumour activity

Regioisomeric preferences in the orthomanganation of meta-substituted acetophenones and isopropyl benzoates, and application of iodo-demanganation with iodine chloride to the synthesis of 2-iodo-3-O-substituted and other ortho-iodo arylcarbonyl compounds

Synthesis is reported of regioisomeric ortho-[Mn(CO)4] derivatives of aryl ketones (2-chlorothioxanthen-9-one and 3′,4′-methylenedioxyacetophenone) and esters (isopropyl 3-methoxy- and 3-acetoxy-benzoate), as well as of the single ortho-[Mn(CO)4] products from methyl 4-methoxy- and 3,5-dimethoxy-benzoate. Factors influencing the preference for manganation at the crowded positions ortho to the C=O in meta-substituted aryl ketones and esters are considered. The frequency of the lowest energy metal carbonyl stretching mode is useful in the structural assignment of the regioisomers. Isopropyl 2-iodo-3-methoxy- and 2-iodo-3-acetoxy-benzoates are obtained by reaction of iodine chloride to replace the Mn(CO)4 group at the crowded 2-position of the corresponding orthomanganated 3-O-substituted benzoate esters. ortho-Iodoacetophenones were prepared likewise, without α-iodination, some with iodine in crowded positions (2′-iodo-3′-methoxy-, -3′,4′-methylenedioxy- and -3′,4′,5′-trimethoxy-acetophenone), others with O-protected 2′-hydroxy groups (2′-benzyloxy- and 2′-t-butyldimethylsilyloxy-3′,4′-dimethoxy-6′-iodobenzene and 2′-t-butyldimethylsiloxy-6′-iodobenzene). Potential general synthetic routes to derivatives of 2-iodo-3-hydroxy-, 2-iodo-3,4-dihydroxy-, and 2-iodo-6-hydroxy-aryl carbonyl compounds are indicated. The corresponding routes to 3-iodo-4-acetyl-2,5-dimethylthiophene and 3-iodo-2-acetylthiophene are also reported