Tom Shepherd

Effects of environment on the composition of epicuticular wax esters from kale and swede

Abstract The composition of intact leaf epicuticular wax esters of two individual genotypes each of kale and swede grown indoors (I) and outdoors (O) at SCRI, Scotland, and outdoors at Wadenswil in Switzerland (S), were determined by GC-mass spectrometry. For all genotypes (I, O, S) esters were found to consist of unbranched ( n- ) and branched anteiso - ( a- ) and iso - ( i- ) components in the a : a , a : i , i : a , a : n , n : a , n : n and i / n : n / i acid-alcohol combinations. Esterification was non-random, n : n and doubly branched br -/ br - combinations were favoured over mixed n -/ br - combinations. Combinations with extremes of acid and alcohol chain-length were generally uncommon, although longer-chain alcohols were more predominant in some swede esters. There were considerable compositional differences between indoor-grown plants (I) and those grown outdoors (O and S). In general, i : n / n : i , i : a and a : i esters were relatively more abundant in (O and S) and n : n and n : a esters were more abundant in (I), whereas a : n and a : a esters were of similar abundance in all (I, O and S). Generally, (I)-grown plants were found to have proportionally more esters of longer chain-length and (O, S)-grown plants proportionally more esters of shorter chain-length. For kale a : a , n : n , a : n and n : a esters, this was particularly related to variation in alcohol chain-length. There were also major compositional differences between kale and swede esters, long-acid-short-alcohol combinations were more prominent in the former, while short-acid-long-alcohol combinations dominated in the latter.

Transcriptional and metabolic profiles of Lolium perenne L. genotypes in response to a PEG‐induced water stress

Metabolic profiling was carried out in the forage grass Lolium perenne L. (perennial ryegrass) to uncover mechanisms involved in the plants response to water stress. When leaf and root materials from two genotypes, with a contrasting water stress response, were analysed by GC-MS, a clear difference in the metabolic profiles of the leaf tissue under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more susceptible Cashel genotype and an increase in sugars and compatible solutes in the more tolerant PI 462336 genotype. Sugars with a significant increase included: raffinose, trehalose, glucose, fructose and maltose. Increasing the ability of perennial ryegrass to accumulate these sugars in response to a water deficit may lead to more tolerant varieties. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336, which has identified perennial ryegrass genes regulated under water stress.

Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes

Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44 000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene α-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode α-copaene synthase. Other potential ‘flavour genes’, identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5′-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.

Ecological biochemistryEffects of environment on the composition of epicuticular wax from kale and swede

The composition of leaf epicuticular waxes of two genotypes each of kale and swede were determined by gas chromatography-mass spectrometry. Plants were grown indoors (I) and outdoors (O) at SCRI, Scotland, and outdoors at Wadenswil in Switzerland (S). Epicuticular waxes from outdoor-grown plants (O and S) were found to have higher proportions of n-alkanes, octacosanoic acid, primary alcohols and long-chain esters but lower proportions of aldehydes, ketones, ketols and secondary alcohols than waxes from (I)-grown plants. Outdoor-grown plants were also found to have proportionally more shorter chain length compounds and indoor-grown plants proportionally more compounds of longer chain length. Variations in wax composition between genotypes of a species and between species were also observed. Differences in leaf surface wax ultrastructure, between species, and between different growth conditions were detected using scanning electron microscopy. The possible role of leaf wax chemicals in the antixenotic resistance to the turnip root fly, Delia floralis, of certain genotypes was also considered.

Phytochemical Diversity in Tubers of Potato Cultivars and Landraces Using a GC-MS Metabolomics Approach

Phytochemical diversity with respect to a range of polar (including amino acids, organic acids, sugars, and sugar alcohols) and nonpolar (including fatty acids, alkanols, and sterols) metabolites was examined within tubers from a total of 29 genetically diverse potato cultivars and Chilean landraces using a metabolomics approach by gas chromatography-mass spectrometry. From principal component analysis of the polar and nonpolar metabolite data there was insufficient variation to differentiate the majority of cultivars and landraces. Analysis of all polar metabolite profiles revealed separation of two cultivars (Glenna and Morag) from the other cultivars and landraces and a separate cluster of one landrace line, largely due to higher levels of sugars. Pentland Javelin was distinct in containing high levels of many amino acids. The two Solanum tuberosum group phureja cultivars (Inca Sun and Mayan Gold) were not particularly similar and were not separated from the S. tuberosum group tuberosum cultivars. Analysis of the nonpolar metabolite data revealed partial separation of two landrace lines and, on the basis of some minor fatty acids, Mayan Gold was distinct. The differences in metabolite profiles are considered in terms of the taxonomy and breeding history of the cultivars and possible influences from other factors such as developmental stage of the tuber. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed on all metabolites. The significance of high correlations between many amino acids and between several nonpolar metabolites is discussed.

Identification of Fluorinases from Streptomyces sp MA37, Norcardia brasiliensis, and Actinoplanes sp N902‐109 by Genome Mining

The fluorinase is an enzyme that catalyses the combination of S‐adenosyl‐L‐methionine (SAM) and a fluoride ion to generate 5′‐fluorodeoxy adenosine (FDA) and L‐methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80–87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21‐residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X‐ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4‐fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.

Epicuticular wax composition in relation to aphid infestation and resistance in red raspberry (Rubus idaeus L.)

Abstract Epicuticular waxes from the aphid-resistant red raspberry (Rubus idaeus) cultivar Autumn Bliss and the aphid-susceptible cultivar Malling Jewel were collected from the newly emerging crown leaves, and also from the group of four more mature leaves immediately below the crown. Resistance and susceptibility status of the leaves to infestation by the large raspberry aphid, Amphorophora idaei, were determined by bioassay with the insect just prior to collection of the wax. Analysis showed the waxes to consist of a complex mixture of free fatty acids; free primary alcohols and their acetates; secondary alcohols; ketones; terpenoids including squalene, phytosterols, tocopherol and amyrins; alkanes and long chain alkyl and terpenyl esters. Compositional differences which may relate to A. idaei-resistance status were noticeably higher levels of sterols, particularly cycloartenol, together with the presence of branched alkanes, and an absence of C29 ketones and the symmetrical C29 secondary alcohol in wax from the resistant cultivar Bliss. There were also differences between the cultivars in the distribution of individual amyrins and tocopherols and in the chain length distribution for homologues of fatty acids, primary alcohols and alkanes, and these may also be related to resistance to A. idaei. Emerging leaves had lower levels of primary alcohols and terpenes, but higher levels of long-chain alkyl esters, and in general, more compounds of shorter chain-length than the more mature leaves. During bioassay A. idaei displayed a preference to settle on the more mature leaves. This may be due to greater wax coverage and higher levels of the compounds of shorter chain length found in the newly emerged younger leaves at the crown of the plant.

Epicuticular waxes and volatiles from faba bean (Vicia faba) flowers

Abstract The floral bouquet of faba bean flowers was shown to be a complex mixture of some 27 identifiable compounds. In addition to the ubiquitous mono and sesquiterpenes, the porous-polymer entrained volatiles included a diverse range of phenylpropenoids which together accounted for over 7% of the total. Cinnamyl alcohol was also found to be the most abundant free alcohol in the epicuticular wax of faba bean flowers. Two new classes of epicuticular wax esters consisting of saturated C16, C18, C20, C22 and C24 fatty acids esterified with the phenylpropenoid, cinnamyl alcohol and with the diterpene, phytol have been identified.

Raspberry viruses manipulate the behaviour of their insect vectors

Many insect herbivores and plant pathogens influence each other via plant‐mediated mechanisms. Although there is speculation that these interactions may be important in structuring terrestrial food webs, few studies have empirically demonstrated the mechanisms by which pathogens manipulate the behaviour of their insect vectors. We investigated how infection of red raspberry, Rubus idaeus L. (Rosaceae), with two viral pathogens, black raspberry necrosis virus (BRNV) and raspberry leaf mottle virus (RLMV), affected the behaviour of their vector, the large raspberry aphid, Amphorophora idaei Börner (Hemiptera: Aphididae: Macrosiphini). As semi‐persistently transmitted viruses, comparatively little is known about how such viruses affect vector biology. We also examined the effect of infection on plant volatile emissions and amino acid content, which could drive changes in aphid behaviour and performance. Virus‐infected plants were initially more attractive to the aphid and the insects remained on infected plants for 30 min, but were found equally on uninfected plants 12 h after inoculation. Twenty‐seven volatile compounds were identified. Two green leaf volatiles were emitted at higher concentrations by infected plants: (Z)‐3‐hexenyl acetate and 2‐hexenal. In dose‐response assays, (Z)‐3‐hexenyl acetate was attractive to the aphid at concentrations of 50 ng ml−1. When reared on infected plants, aphids took more than 3 days longer to reach adulthood compared with those on uninfected plants, although the number of offspring remained the same. Soluble amino acid (essential and non‐essential) concentrations in raspberry leaves increased more than two‐fold with virus infection. Amino acid composition was dominated by glutamate, accounting for 64 and 77% of the total in uninfected and infected leaves, respectively. Excessive glutamate may have underpinned the negative effects of viral infection on aphid performance. These results demonstrate the capacity of viruses to alter their host plant to manipulate vector behaviour, which may have evolved to be consistent with the transmission requirements of the virus.

A Metabolomics Study of Cultivated Potato (Solanum tuberosum) Groups Andigena, Phureja, Stenotomum, and Tuberosum Using Gas Chromatography−Mass Spectrometry

Phytochemical diversity was examined by gas chromatography-mass spectrometry in tubers of genotypes belonging to groups Andigena, Phureja, Stenotomum, and Tuberosum of the potato, Solanum tuberosum. Polar extracts (mainly amino acids, organic acids, sugars, and sugar alcohols) and nonpolar extracts (mainly fatty acids, fatty alcohols, and sterols) were examined. There was a large range in levels of metabolites, including those such as asparagine, fructose, and glucose, that are important to tuber quality, offering considerable scope for selecting germplasm for breeding programmes. There were significant differences in the levels of many metabolites among the groups. The metabolite profiles of genotypes belonging to Phureja and Stenotomum were similar and different from those of Tuberosum and the majority of Andigena genotypes. There was some agreement with the phylogeny of the groups in that Stenotomum is believed to be the ancestor of Phureja and they are both distinct from Tuberosum. Andigena genotypes could be partially distinguished according to geographical origin, Bolivian genotypes being particularly distinct from those from Ecuador. Biosynthetic links between metabolites were explored by performing pairwise correlations of all metabolites. The significance of some expected and unexpected strong correlations between many amino acids (e.g., between isoleucine, lysine, valine, and other amino acids) and between several nonpolar metabolites (e.g., between many fatty acids) is discussed. For polar metabolites, correlation analysis gave essentially similar results irrespective of whether the whole data set, only Andigena genotypes, or only Phureja genotypes were used. In contrast, for the nonpolar metabolites, Andigena only and Phureja only data sets resulted in weaker and stronger correlations, respectively, compared to the whole data set, and may suggest differences in the biochemistry of the two groups, although the interpretation should be viewed with some caution.