Greg Tucker

Phenolic compounds and their role in oxidative processes in fruits

Phenolic compounds occur in all fruits as a diverse group of secondary metabolites. Hence, they are a component of the human diet although data for dietary intakes and metabolic fate are limited. Their role in oxidation processes, as either antioxidants or substrates in browning reactions, is examined. They are characterised by high chemical reactivity and this complicates their analysis.

Tomato exo-(1-->4)-beta-D-galactanase. Isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related cDNA clone.

An exo-(1->4)-[beta]-D-galactanase was isolated from ripe tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig and cv Better Boy) using anion-exchange, gel filtration, and cation-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most active fraction revealed a predominant protein band at 75 kD and several minor bands. A 30-amino acid N-terminal sequence from this 75-kD protein showed a high degree of homology with other recently identified [beta]-galactosidase/galactanase proteins from persimmon and apple fruits (I.-K. Kang, S.-G. Suh, K.C. Gross, J.-K. Byun [1994] Plant Physiol 105: 975–979; G.S. Ross, T. Wegrzyn, E.A. MacRae, R.J. Redgwell [1994] Plant Physiol 106: 521–528) and with the predicted polypeptide sequence encoded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldsbrough, W.R. Woodson [1991] Plant Mol Biol 17: 61–71). The enzyme focused to a single band of [beta]-galactosidase activity on an isoelectrofocusing gel at pH 9.8. The enzyme was specific for (1->4)-[beta]-D-galactan substrates with a pH optimum of 4.5. The only reaction product detected was monomeric galactose, indicating that the enzyme was an exo-(1->4)-[beta]-D-galactanase. [beta]-Galactanase activity increased at the onset of ripening in normal fruit, but no similar increase was detected in the nonripening mutants nor and rin. A tomato homolog (pTom[beta]gal 1) was isolated using the SR12 cDNA clone from carnation as a probe. This clone showed 73% identity at the amino acid level with [beta]-galactosidase-related sequences from apple and asparagus and 66% identity with SR12. pTom[beta]gal 1 is a member of a gene family. Northern analysis demonstrated that pTom[beta]gal 1 expression was ripening related in normal fruits, with lower levels apparent in the nonsoftening mutants.

The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening.

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.

Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct

Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5′ end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectinesterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit.This paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression.

Isolation and identification of phenolic compounds in Citrus sinensis

Various methods were compared for the recovery of phenolic compounds from sweet orange. The need for careful consideration of sample preparation was demonstrated. In the case of cinnamic acids, hydroxide-treatment was essential to liberate the bound acids. Ferulic acid was the predominant phenol in alkali-treated extracts. Positive and negative ion electrospray ionisation mass spectra were systematically investigated for a range of phenols. Detection limits were significantly lower in negative ion than in positive ion mode. However, positive ion mode provided additional structural information for many of the compounds.

The mechanisms of hydrothermal deconstruction of lignocellulose: New insights from thermal–analytical and complementary studies

Graphical abstract Highlights ► Thermal analysis provides real-time data on hydrothermal reactions under realistic conditions. ► From DSC, hemicellulose hydrolysis has low enthalpy change but xylose degradation is exothermic. ► Hydrothermal exothermic degradation reactions may be similar to early stage biomass pyrolysis. ► DMTA shows that the polymeric structure of lignin in biomass is degraded at high temperatures. ► Hydrothermal reactions are effective at greater than 50% solids content.

Compositional changes in cell wall polymers during mango fruit ripening

Abstract Softening of mango fruit has been investigated by analysis of ripening related changes in the composition of the fruit cell walls. There is an apparent overall loss of galactosyl and deoxyhexosyl residues during ripening, the latter indicating degradation of the pectin component of the wall. The loss of galactose appears to be restricted to the chelator soluble fraction of the wall pectin, whilst loss of deoxyhexose seems to be more evenly distributed amongst the pectin. The chelator soluble pectin fraction is progressively depolymerised and becomes more polydisperse during ripening. These changes are similar to those occurring in other fruit and are related to the action of wall hydrolases during ripening.

Soluble lipoxygenase isoforms from tomato fruit

Extraction of maximum lipoxygenase activity from tomato fruits was shown to require the presence of 0.1% Triton X-100. The detergent appeared to have a dual role improving recovery and preventing enzyme inactivation, especially during mechanical homogenization. Ultracentrifugation of total lipoxygenase activity revealed that the majority (96%) was of a soluble form, with very little associated with the membrane fraction. By ammonium sulphate fractionation and anion-exchange chromatography, soluble lipoxygenase was purified 46-fold. Separation of lipoxygenase activity by isoelectric focusing and detection by in-gel activity staining resulted in the identification of two predominant isoforms with pI values of 4.8 and 5.0, one of which appears to be ripening-specific. On examination of reaction products by HPLC, both isoforms appeared to exclusively produce 9-hydroperoxides.

Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borage Δ6-desaturase

The improvement of nutritional quality is one potential application for the genetic modification of plants. One possible target for such manipulation is the modification of fatty acid metabolism. In this work, expression of a borage Δ6-desaturase cDNA in tomato (Lycopersicon esculentum L.) has been shown to produce γ-linolenic acid (GLA; 18:3 Δ6,9,12) and octadecatetraenoic acid (OTA; 18:4 Δ6,9,12,15) in transgenic leaf and fruit tissue. This genetic modification has also, unexpectedly, resulted in a reduction in the percentage of linoleic acid (LA 18:2 Δ9,12) and a concomitant increase in the percentage of α-linolenic acid (ALA; 18:3 Δ9,12,15) in fruit tissue. These changes in fatty acid composition are thought to be beneficial for human health.

Effects of cultivar and harvest maturity on ripening of mangoes during storage

Low-temperature storage trials were undertaken with mango cultivars grown in Senegal. Three cultivars were studied in detail: Amelie, Kent and Sensation. The fruit were harvested at various physiological maturities and stored at 12°C for periods up to 21 days. The response to storage at 12°C was dependent on cultivar, fruit maturity at harvest and also on the date during the season on which a particular harvest was taken. The most apparent effect of fruit maturity on subsequent storage behaviour was observed in ‘Amelie’. Ripening was retarded more effectively in immature than in mature fruit. ‘Kent’ showed similar effects of fruit maturity on storage behaviour and when mature fruit harvested on different dates during the mango season were compared it became apparent that the extent of ripening in storage increased as the harvesting season progressed. ‘Sensation’ mangoes ripened rapidly in store irrespective of fruit maturity at harvest.