Differential Proteomic Analysis of Arabidopsis thaliana Genotypes Exhibiting Resistance or Susceptibility to the Insect Herbivore, Plutella xylostella

A proteomic study was conducted to investigate physiological factors affecting feeding behaviour by larvae of the insect, Plutella xylostella, on herbivore-susceptible and herbivore-resistant Arabidopsis thaliana. The leaves of 162 recombinant inbred lines (Rils) were screened to detect genotypes upon which Plutella larvae fed least (P. xylostella-resistant) or most (P. xylostella-susceptible). 2D-PAGE revealed significant differences in the proteomes between the identified resistant and susceptible Rils. The proteomic results, together with detection of increased production of hydrogen peroxide in resistant Rils, suggest a correlation between P. xylostella resistance and the production of increased levels of reactive oxygen species (ROS), in particular H2O2, and that this was expressed prior to herbivory. Many of the proteins that were more abundant in the Plutella-resistant Rils are known in other biological systems to be involved in limiting ROS damage. Such proteins included carbonic anhydrases, malate dehydrogenases, glutathione S-transferases, isocitrate dehydrogenase-like protein (R1), and lipoamide dehydrogenase. In addition, patterns of germin-like protein 3 isoforms could also be indicative of higher levels of reactive oxygen species in the resistant Rils. Consistent with the occurrence of greater oxidative stress in the resistant Rils is the observation of greater abundance in susceptible Rils of polypeptides of the photosynthetic oxygen-evolving complex, which are known to be damaged under oxidative stress. The combined results suggest that enhanced production of ROS may be a major pre-existing mechanism of Plutella resistance in Arabidopsis, but definitive corroboration of this requires much further work

Characterization of the ethanol‐inducible alc gene‐expression system in Arabidopsis thaliana

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to β-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana

Assessment of the flavour of fresh uncooked onions by taste-panels and analysis of flavor precursors, pyruvate and sugars.J Sci Food Agric 85:112–120 CSA

Abstract: Flavour in fresh onions is dominated by volatile sulphenic and thiosulphenic acids that are liberated once alk(en)yl cysteine sulphoxide (ACSO) flavour precursors are cleaved by the enzyme alliinase after tissue disruption. The levels of pyruvate and ACSOs in over 100 samples of onions marketed in the UK were measured, and compared with assessment by taste-panels. There was a linear relationship between the content of ACSOs and pyruvate. Measurements of pyruvate indicated that the marketing classification of some types of onion did not correspond to their pyruvate levels. A significant linear relationship was found between a sensory measure of strength and pyruvate over the range 1.2-9.3 µmol pyruvate g −1 fresh weight. In most cases, when a flavour classification of sweet, mild or strong was applied to a sample of onions based on pyruvate content, the taste-panels agreed with the categorization. The taste-panels were unable to identify a sweet flavour in onions, except at low levels of pyruvate. Taste-panels were able to define a likeability character (attractiveness of flavour) for onions, which correlated with the level of pyruvate. However, for some varieties, the flavour classification or likeability did not correspond to predictions based on pyruvate levels alone. Pyruvate measurements were seen as a suitable method for routine quality control once the characteristics of a variety of onion had been established, but initial evaluations should include well-designed taste-panel assessments

The alc-GR System. A Modified alc Gene Switch Designed for Use in Plant Tissue Culture

The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: β-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants