Research:
Auxin.Transp.
Vascular.Dev.
Nutritional Genomics
Key areas of research:
Identification of Key Loci that Regulate Ascorbate Pool Size
The laboratory currently has EC funding to study the gene(s) that regulate ascorbate levels. Several quantitative trait loci (QTL) have been identified in the Arabidopsis Cvi x Ler recombinant population. At least one of the QTL regulating ascorbate levels will be characterised at the molecular level as part of the EC NATURAL programme (Swarup et al, 2002, Garratt et al 2003).
Background
Hydrogen peroxide (H2O2) detoxification is mediated by peroxidases, specifically ascorbate peroxidase (APx) by coupling the reduction of H2 to the monovalent oxidation of ascorbate, yielding monodehydroascorbate (MDA). As well as its primary role in H2O2 detoxification, ascorbate also serves as an antioxidant in many other detoxification reactions, such as the scavenging of hydroxyl radicals and the reduction of tocopheryl radicals produced by a-tocopherol, by quenching autocatalytic lipid peroxidation.
Ascorbate is regenerated from MDA in several parallel reactions. The MDA produced by the membrane-bound APx is reduced by ferredoxin. In the stroma, MDA reductase (MDAR) catalyses the reduction of MDA by NADPH. Alternatively, MDA can dissociate into ascorbate and dehydroascorbate (DHA); the latter is re-reduced by stromal dehydroascorbate reductase (DHAR), with glutathione (GSH) as electron donor; glutathione reductase (GR) then re-reduces the oxidised glutathione (GSSG) back to its reduced form (Halliwell and Gutteridge, 1999). There is evidence that peroxidase activity and concentrations of reactive oxygen species (ROS), such as H2O2, increase during senescence and fruit ripening (Lester, 2000; Eskin and Robinson, 2001). In respiring cells, up to 5% of the total oxygen may be reduced to form ROS (Eskin and Robinson, 2001). During post-harvest storage, particularly of processed material, the percentage of oxygen reduced to form ROS increases, leading to cellular damage, in particular, the peroxidation of membrane lipids. The latter process has been recognised as a key factor in the loss of membrane selective permeability and fluidity during senescence, leading eventually to loss of cellular integrity (Hong et al., 2000). By increasing the accumulation of acscorbate and thereby enhancing the oxyradical scavenging and antioxidant capacities of crop plants, the pre- and post-harvest performance of the crop plants and their products will be improved, reducing and/or delaying lipid peroxidation, delaying rates of senescence and improving shelf-life, appearance and nutritional content.
Plants also provide the major source of vitamin C in the human nutrition. The accumulation of ROS have been associated with over 100 disorders (Halliwell and Gutteridge, 1999), ranging from rheumatoid arthritus and haemorrhagic shock through cardiomyopathy and cystic fibrosis to gastrointestinal ischaemia and AIDS, and have been shown to make a significant contribution to the disease pathology. Disease-associated oxidative stress could also result from diminished antioxidants and it has been shown that intensive care patients frequently exhibit low levels of ascorbate in body fluids. Therefore any enhancement in the antioxidant status of human nutrition would be of direct benefit to the consumer.
Aims of Research
As the levels of antioxidants, in particular ascorbate, are variable from crop to crop and can decline seriously post harvest and during processing, the identification of genes that regulate ascorbate levels would provide useful molecular tools for plant breeders. The laboratory has identified several quantitative trait loci (QTL) that regulate ascorbate (ASC) accumulation in vegetative tissue. The molecular genetic factors determining and influencing the ascorbate pool size are largely unknown, therefore the primary aim of our research is the cloning of the QTL regulating ascorbate levels and its characterisation at a molecular, biochemical and physiological level. Transcription factors are likely to play a key regulatory function and behave as quantitative trait loci (QTL) since changes in their levels of expression can have major effects on the traits they control. Quantitative genetics therefore represents a powerful approach to identify loci that have large effects on a target trait. This work is being conducted as part of the EC framework IV funded NATURAL programme (LINK).
Nutritional Enhancement of Folic Acid and Folates
Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-MS/MS) is being utilised to identify and quantify polyglutamated folates in plant tissue, human serum and tissue. This work is being conducted in collaboration with the Centre for Analytical Bioscience, Pharmacy.
 
|
|
Fig. 1. The electrospray ionization tandem mass spectra of 5-methyltetrahydrofolate with collision-induced dissociation (CID) in mass profile mode, allowing identification of fragment ions for multiple reaction monitoring (MRM) analysis. |
Background
Folates play a pivotal role in the methylation cycle and DNA synthesis by acting as cofactors for carbon one transfer (Scott et al 2000). Plant photorespiration also requires two tetrahydrofolate-dependent reactions, specifically for the conversion of glycine into serine. Reduced dietary intake of folates has been associated with bone marrow defects resulting in anaemia, increased risk of cardiovascular disease (CVD) and stroke, in addition to increased risk of spina bifida and other neural tube defects (NTDs). Any reduction in folate status is reflected by a rise in plasma homocysteine levels, which is directly associated with increased occurrence of CVD. Food consists of a complex mixture of polyglutamated tetrahydrofolates (typically 3 to 11), characterised according two their carbon one attachments, such as formyl (-CHO), methylene (-CH2-) and methyl (-CH3). These polyglutamated folates are converted to a monoglutamated form of 5-methyltetrahydrofolate at the brush border membrane of the jejunal mucosa by pteroylpolygutamate hydrolase (conjugase). The average intake of folates, irrespective of background, location and affluence is rarely greater than 200mg a day, which is less than optimal. It has been estimated that to reduce the risk of CVD and NTDs, dietary intake of natural folates needs to be increased to 600mg a day.
Despite the importance of folates, genes coding for many of the biosynthetic and redox enzymes in addition to regulatory sequences have yet to be characterised.
Aims of Research
The development of this tool for the quantitative metabolic profiling of Arabidopsis and other plant species, targeting all the tetrahydrofolates and their respective polyglutamated forms, in addition to key metabolites in their biosynthesis. One of the many applications will be the identification of key QTLs for regulating the biosynthesis of tetrahydrofolates.
References
Swarup K, Garratt L C, Tucker G A and Bennett M J (2002), Nutritional Genomics: Identification of key loci that regulate ascorbate pool size. Free Radical Research, 36, S1, 106.
Garratt L C, Swarup K, Tucker G A and Bennett M J(2003), Using nutritional genomics to identify key loci that regulate ascorbate pool size. J. Exp. Bot, in press.
Eskin N A M and Robinson D S (2001), Food shelf life stability: chemical, biochemical, and microbiological changes, Boca Raton, CRC Press.
Hong Y, Wang T, Hudak K A, Schade F, Froese C D and Thompson J E (2000), An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence, Proc Natl Acad Sci USA, 97, 8717-8722.
Lester G E (2000), Polyamines and their cellular anti-senescence properties in honey dew muskmelon fruit, Plant Sci, 160, 105-112.
Halliwell B and Gutteridge J M C (1999), Free radicals in biology and medicine, Oxford, Oxford University Press.
Scott J, Rébeille F and Fletcher J (2000) Folic acid and folates: the feasibility for nutritional enhancement in plant foods. Journal of the Science of Food and Agriculture, 80, 795-824.
- last updated on: 9/11/2004 -