Comparative Biochemistry and Physiology, Part A 146 (2007) S243–S253www.elsevier.com/locate/cbpa

Society for Experimental Biology Annual Main Meeting31st March – 4th April 2007, Glasgow, Scotland

P4–NUTRITIONAL GENOMICS

P4.1PhytonutrientsG. Tucker, (University of Nottingham)

There has been an increasing public awareness of the potentiallink between diet and health over the past 5–10 years. This hasbeen partly fuelled by government initiatives such as the 5-a-day campaign. This campaign has recognised the benefits ofincreased consumption of fruit and vegetables (cereals are alsoimportant) in the prevention of chronic diseases such as cancer,stroke and diabetes. The benefits presumably derive from therange of nutrients that plants can deliver. These include recog-nised micronutrients such as vitamins A, B9 (folate), C and E aswell as less well recognised nutrients such as carotenoids andpolyphenols. However, whilst there is increasing epidemiolo-gical evidence for the benefits of these plant foods themechanism of action of individual nutrients remains unclear.The antioxidant activity associated with many phytonutrientsmay be one explanation but it is becoming increasingly evidentthat more specific nutrient–gene interactions may also beresponsible.There are several strategies being employed to enhance thephytonutrient content of the diet. Diversification of food isperhaps the best approach but supplementation and for-tification are also used to varying effectiveness. One morerecent approach has been the biofortification of cropsutilising either natural variation or genetic modification.There are now several good examples of how thephytonutrient levels in crops can be enhanced in this way,including vitamins A, E, folate, flavonoids and long chainfatty acids.Whilst biofortification may be beneficial in enhancing the dietthere are many factors that impact on nutrient delivery andthese must not be ignored. These include the nature of thecrop (staple crops are better targets than more exotic ones),cultivar, pre-harvest conditions, post-harvest losses (storageand processing), cooking and finally bioavailability. Theseother factors can often have a very significant impact on the

doi:10.1016/j.cbpa.2007.01.562

delivery of phytonutrients in the diet and may represent abetter target for modification.

doi:10.1016/j.cbpa.2007.01.563

P4.2The application of nutritional genomics for determining

optimal micronutrient requirements

R. Elliot, (Institute of Food Research); S. Fairweather-Tait,(University of East Anglia)

The basis for estimating recommended dietary allowances formicronutrients is generally the prevention of deficiency but thisis not necessarily the level at which health-promoting functionaleffects are maximised. Defining optimal health is not easy, andthe problem is compounded by the lack of robust biomarkers ofstatus for a number of micronutrients e.g. zinc, selenium andcopper.In order to calculate optimal intakes, detailed information onmicronutrient bioaccessibility, homeostatic control mechanisms,and the full spectrum of functional effects is required.Mechanistic studies employing in vitro and animal modelshave proven extremely valuable in elucidating aspects ofmicronutrient homeostatic control mechanisms. This kind ofwork also provides one possible route for developing newmarkers of status. Another promising approach is to employ“omic” technologies (i.e. transcriptomics, proteomics andmetabolomics). Indeed, nutritional genomic studies havealready revealed a number of previously undefined functionaleffects of micronutrients. These methods also hold promise forthe development of new robust biomarkers based on diagnosticprofiles of mRNA, protein and/or metabolites. However, to datethe majority of such studies have been performed in modelsystems. This reflects the major challenge of undertaking humannutrition studies where inter-individual variation, volunteercompliance, access to relevant tissues and other ethical

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restrictions pose particular problems. Nevertheless, there is agrowing body of evidence that while the inter-individual varia-tion observed using functional genomic approaches is sub-stantial, intra-individual variation is much less and thatappropriate experimental design may help to address thisproblem. This, in combination with the developments ofmethods such as gene set enrichment analysis that improve thesensitivity of “omic” methods to detect subtle changes, plusresults from a limited number of human studies suggests thatnutritional genomic studies are feasible and hold considerablepromise.

doi:10.1016/j.cbpa.2007.01.564

P4.3Flavonoid biosynthesis

L. Jaakola, (University of Oulu, Finland)

Flavonoids are a class of phenolic metabolites that are widelydistributed in the plant kingdom. These compounds frequentlyserve as pigments in flowers and fruits, but are also involved inmany biological interactions. Flavonoids participate in devel-opmental processes and respond to the environment. They canserve as signal molecules in sexual reproduction and protect theplant against UV radiation, as well as participate in plant–microbe interactions and defence responses. Since flavonoidsare present in a wide range of fruits and vegetables, they form anintegral part of human daily diet. Health benefits of flavonoidshave been under active research during the last decade. Flavo-noids are offered as health-promoting dietary supplements withstrong antioxidant capacity and they can be effective againstcancer, cardiovascular and ageing related diseases. Thesepotential nutritional effects of flavonoids make them anattractive target for genetic engineering strategies aimed atproducing plants with increasing nutritional value. Transcrip-tional control of the structural genes of the flavonoidbiosynthetic pathway has been most intensively studied inrelation to biosynthesis of anthocyanins. Groundbreakingresearch on revealing the expression of the structural andregulatory genes of the flavonoid pathway has been done withornamental plants like snapdragon, petunia and gerbera, inaddition to maize and Arabidopsis. Today, molecular levelinformation on flavonoid biosynthesis is available also fromvarious fruit and vegetable tissues. Our research has focused onbiosynthesis of flavonoids in bilberries and blueberries, whichare among the best sources of anthocyanins.

doi:10.1016/j.cbpa.2007.01.565

P4.4Developmental regulation of ripening in fleshy fruits

G. Seymour, K. Manning, (University of Nottingham)

Fruits are reproductive organs unique to the angiosperms that haveevolved to promote seed dispersal. They are also one of the mostimportant components of the human diet being rich in phytonu-trients such as carotenoids and polyphenols. A diet poor in fruitsand vegetables is considered one of the top ten factors contributingto human mortality. We want to understand the processes thatcontrol fruit development and ripening and to develop morerational approaches to improve the nutritional value and quality ofthese import crop products by harnessing natural variation. It hasbeen known for many years that ethylene is important in initiatingand controlling ripening in climacteric fruits. Recent discoveriesindicate that genes normally associated with floral developmentare responsible for controlling fruit ripening; linking genesinvolved in the evolution of flowers and fruits. In the presentationI will describe our discovery of a gene that plays a central role incontrolling the ripening process. I will then discuss how thisinformation will be used to help build a molecular framework thatconnects the regulatory network controlling ripening to downstream processes affecting key quality attributes.

doi:10.1016/j.cbpa.2007.01.566

P4.5Rational metabolic engineering of transgenic plants for the

synthesis of long chain polyunsaturated fatty acids

J. Napier, R. Haslam, M. Venegas-Caleron, O. Sayanova,(Rothamsted Research)

There is now considerable evidence as to the importance of longchain polyunsaturated fatty acids (LC-PUFAs) in humannutrition. Unfortunately, current sources are either in severedecline (fish oils) or expensive (via microbial fermentation),leading to the search for an alternative source. We have beenevaluating the possibility of producing LC-PUFAs in transgenicplants to provide a sustainable source of these importantnutrients, although no native higher plant species synthesisethese fatty acids. We have transgenically assembled the primarybiosynthetic pathway for LC-PUFAs in both model and cropspecies. Our data indicate that whilst the transgenic synthesis ofLC-PUFAs such as arachidonic acid and eicosapentaenoic acidis clearly feasible, a number of factors limit the efficientreconstitution of this pathway. We have attempted to addressthis problem in a systematic manner by firstly identifyingdifferent metabolic “bottlenecks” and then seeking geneticinterventions to overcome them. For example, it seems likelythat a generic bottleneck resides within the primary LC-PUFAbiosynthetic pathway as a result of the “substrate dichotomy”between the desaturases and elongases which catalyze thereactions. Desaturases require phosphoglycerolipid-linked sub-strates, whereas elongases require acyl-CoA substrates: lack ofacyl-exchange between these two metabolic pools (phospho-glycerolipids, acyl-CoAs) reduces the flux through the pathway.Thus, enhanced transgenic synthesis of LC-PUFAs mightrequire the additional expression of acyltransferase genes(from LC-PUFA-synthesising organisms) to maximise the

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accumulation of these non-native products. I will review ourprogress in the production of LC-PUFAs in transgenic plants, aswell as our approaches to overcoming various bottlenecks.

doi:10.1016/j.cbpa.2007.01.567

P4.6Food allergens

M. Alcocer, (University of Nottingham)

The prevalence of food allergy and other allergic diseases hasincreased during recent decades. Food allergy is nowrecognised as a worldwide problem in Westernised nationsand food-induced anaphylaxis has become the leading singlecause of anaphylactic reaction treated in hospitals in the USAand UK. Despite advances in diagnosis and the characterisationof many food allergens at the molecular level, our basicunderstanding of what makes an allergen remains limited.Although counter-intuitive, most known food allergens, parti-cularly plant food allergens, can be classified within a smallnumber of families suggesting that some common mechanism(s) of recognition might be taking place.In order to study food allergens at the molecular level somemodel protein systems have been proposed and will be brieflydiscussed. Recent findings from these systems question manyaspects of the current methodology used in the evaluation ofallergenicity of proteins employed by official governmental ins-titutions, normally based on stability and sequence comparison.Surprisingly our experiments are showing that other non-proteiccomponents present in the food matrix might play an essentialrole during the early sensitization phase in vivo, a long-standingclinical observation. Further, data will be presented that suggestthat allergenicity in this plant system is the result of an intrinsiccharacteristic of the allergen and reflects early decisions (pola-rization) taken by the immune system at the dendritic cell level.

doi:10.1016/j.cbpa.2007.01.568

P4.7Acrylamide in food is a plant and agronomic science issue

N. Halford, N. Muttucumaru, M. Parry, P. Shewry, (RothamstedResearch); A. Dodson, S. Elmore, D. Mottram, (University ofReading)

Acrylamide is considered to be a probable carcinogen and hasneurological and reproductive effects. In 2002 it was found inmany cooked foods, causing considerable concern within thefood industry. It is formed from asparagine and sugars throughthe Maillard reaction at the high temperatures achieved duringfrying, baking and roasting.Foods with the highest levels of acrylamide (commonly severalhundred parts per billion) include those derived from potato,coffee, wheat and other cereals. Proposals for lowering

acrylamide levels include reducing cooking times and tempera-tures and lowering the pH. However, these approaches mayreduce colour and flavour development. An alternative is toreduce the levels of asparagine and/or sugars in cereal seeds,potato tubers and other plant-derived raw materials.We have discovered that sulphate deprivation during thecultivation of wheat causes a huge increase in grain asparaginelevels, shown effects of temperature and water availability ongrain asparagine accumulation and demonstrated a clearrelationship between levels of free asparagine in the grain andacrylamide formation. Experiments with potato have revealed amore complex relationship between the levels of asparagine andsugars in tubers and the amount of acrylamide that is formed, butlike those with wheat they have identified clear targets forimprovement and confirm that genetic and agronomicapproaches could play a significant part in mitigating theacrylamide problem. Acrylamide in food is therefore as much aplant and agronomic science issue as a food processing one.

1. Muttucumaru et al. (2006) J. Food Agric. Chem. 54,8951–8955.

doi:10.1016/j.cbpa.2007.01.569

P4.8Progress in mapping the Arabidopsis ionome

D. Salt, I. Baxter, B. Lahner, L. Yakubov, (Purdue University)

Altering the ability of plants to take up and sequester mineralscould have a dramatic impact on both plant and human health.Furthermore, understanding the pathways by which metalsaccumulate in plants will enable the engineering of plants toeither exclude toxic metals or extract them from the soil. We haveemployed mineral nutrient and trace element profiling, usinginductively coupled plasma–mass spectrometry (ICP-MS), as atool to determine the biological significance of connectionsbetween a plant's genome and its elemental profile or “ionome”.Our focus is on genes that control uptake and accumulation ofsolutes, including Ca, K, Mg, P (macronutrients in fertilizer), Co,Cu, Fe, Li, Mn, Mo, Ni, Se, Zn, (micronutrients of significance toplant and human health) andAs, Cd, Na and Pb (elements causingagricultural or environmental problems). To date we haveanalyzed the shoot ionome of over 60,000 Arabidopsis plants.This includes the completion of several Arabidopsis forwardgenetic screen including 6000 fast neutron mutagenized lines(Lahner et al., 2003 Nat. Biotechnol. 21:1215), 4000 EMSmutagenized lines grown under Fe or P deficiency conditions, anda screen of over 100 Arabidopsis accession and severalrecombinant inbred populations including Cvi-1×Ler-2, Bay-0×Sha, Col-4×Ler-0 and Col-0×Van-0 for variation in the shootionome. We have successfully used PCR-based positionalcloning, DNA microarray based approaches, and QTL mappingto locate multiple loci that cosegregate with the ionomic traits ofinterest, and such approaches are allowing the identification ofgenes involved in regulating the ionome (for example Rus et al.,

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2006 Plos Genetics 2(12): e210.). By varying the concentrationsof multiple nutrients in the soil, we have observed severalunexpected alterations in the ionome, including significantdifferences in the accumulation of macro- and micronutrients inresponse to changing soil iron levels. In a complementary reversegenetic approach we have also characterized over 1000 uniquesequenced T-DNA insertional alleles for genes that affect theshoot ionome. To maximize the value of this ionomics approach,we have developed a publicly searchable online databasecontaining ionomic information on over 60,000 plants (www.purdue.edu/dp/ionomics; Baxter et al., 2007 Plant Physiol 143(2)in press), and the database is being updated regularly.

doi:10.1016/j.cbpa.2007.01.570

P4.9Selenium concentrations in UK wheat and biofortificationstrategies

F. Zhao, S. McGrath, C. Gray, J. Lopez-Bellido, (Rothamsted)

μμSelenium is essential for humans and animals but has noknown function in plants. Dietary intake of selenium is low in alarge number of people worldwide. In some European countries,dietary Se intakes have decreased significantly in recentdecades. For example, average Se intake of UK adults hasdecreased by more than 40% from the mid 1970s to 1995. Themain reason for this trend is the decreased importation of bread-making wheat from North America, which generally containsmuch more Se than European wheat. Our survey of 452 grainsamples of bread-making wheat produced in the UK shows arange of 6–858 μg Se/kg dry weight with a mean and median of32 and 22 μg Se/kg, respectively. Furthermore, 91% of thesamples contained <50 μg Se/kg, which is considered to beinsufficient for the requirements of humans and animals. Themain reason for the generally low Se status in UK wheat is a lowSe supply from soil. Other soil factors such as pH, organicmatter content and the availability of sulphate also have a majorinfluence on selenium uptake by plants. Because cereals are animportant source of Se to humans, Se biofortification of wheatwould have a desirable effect on increasing human Se intake.Results from a field trial show that grain Se concentration wasincreased by 3–27 fold by additions of 10–20 g Se/ha as sodiumselenate at the stem extension stage. Other strategies of Sebiofortification, such as genetic improvement to enhance Seaccumulation potential, should also be explored.

doi:10.1016/j.cbpa.2007.01.571

P4.10Genetic aspects of mineral biofortification

P. White, (SCRI); M. Broadley, (University of Nottingham)

Humans require at least 22 mineral elements. These can all besupplied by an appropriate diet. Nevertheless, over 60% of theworld's 6 billion people are Fe deficient, over 30% are Zndeficient, 30% are I deficient and about 15% are Se deficient(White and Broadley, TiPS 10: 586–593, 2005). Deficiencies ofCa, Mg and Cu are also common. Mineral malnutrition can beaddressed through supplementation, food fortification or dietarydiversification, but these interventions have had limited success.‘Biofortification’ is a complimentary strategy that aims toincrease bioavailable concentrations of essential elements inedible portions of crop plants by applyingmineral fertilizers and/or growing crops that accumulate minerals more effectively.Since fertilisers impose a financial and environmental burden,and many infertile soils contain sufficient minerals to supportmineral-dense crops if they became phytoavailable, there isconsiderable interest in breeding for mineral-dense crops thatproduce high yields on infertile soils. This presentation will firstdescribe the contrasting mineralogies of angiosperm orders andquantify the phylogenetic impacts on species' mineral composi-tion. These data support the hypothesis that a change from bean-rich to cereal-rich diets can increase the incidence of Ca, Zn andFe deficiencies in rural populations. It will then considervariation in mineral composition within plant orders and species.The survey supports the hypothesis that sufficient geneticvariation exists in most crop species to breed mineral-densecrops for infertile soils. Such studies provide the platform toidentify genes that underpin agriculturally-useful mineralogicaltraits and develop molecular markers to assist plant breeding.

doi:10.1016/j.cbpa.2007.01.572

P4.11Natural genetic variation in the mineral nutrient

composition of Brassica oleracea

M. Broadley, M. Meacham, (University of Nottingham); H.Bowen, J. Hammond, R. Hayden, A. Mead, G. Teakle,(University of Warwick); G. King, (Rothamsted Research); P.White, (Scottish Crop Research Institute)

Plants require at least 14 mineral elements to complete their lifecycles, which are acquired primarily from the soil. The mineralcomposition of plant tissues varies widely due to environmentalfactors, such as soil mineral availability, and due to plantdevelopmental and genetic factors. Substantial natural geneticvariation in the mineral composition of plant tissues occursbetween populations and varieties of the same species. Thiswithin-species variation is being used in genetic biofortificationprogrammes, i.e. to breed staple crops with higher mineralcontents in their edible portions to alleviate human dietarymineraldeficiencies. A significant proportion of the variation in the leafcomposition of some minerals (e.g. Ca, K, Mg, Si, Zn) has alsobeen attributed to phylogenetic effects occurring above thespecies level, for example, at the family level and above. Here, weconducted a thorough, species-wide, genetic dissection ofmineralcomposition using Brassica oleracea L. (Brassicaceae) as a

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genetic model. Lines from a B. oleracea ‘C genome’ diversityfoundation set (DFS) and commercial (F1) accessionswere grownwith different external phosphorus (P) supplies under bothglasshouse and field conditions. Tissue mineral composition wasmeasured using inductively-coupled plasma emission spectro-metry. Among 455 accessions selected to capture the majority ofspecies-level genetic diversity, substantial species-wide geneticvariation in mineral composition was observed. For example,leaf-Ca, Fe, Mg and Zn varied by 2, 14, 2 and >20-foldrespectively. Subsequently, quantitative trait loci (QTL) formineral composition in B. oleracea were established, underglasshouse and field conditions, using a population of doubled-haploid (DH) lines derived from a B. oleracea var. alboglabra Xvar. italica cross (the ‘AGDH’mapping population). These can beused to inform genetic biofortification programmes.

doi:10.1016/j.cbpa.2007.01.573

P4.12Rice biofortification

R. Abilgos-Ramos, E. Murchie, N. Anukul, M. Bennett,(University of Nottingham); E. Corpuz-Arocena, G.D.G.Garcia, R. Manaois, M. Julaton, (PhilRice); J. Stangoulis, R.Graham (University of Adelaide)

Rice is the staple food for more than half of the world'spopulation and the main source of nutrients for most poor inmany Asian countries. The recent report of the World HealthOrganization (WHO) showed that there are more than 3 billioniron-deficient people globally and most of them are the childrenand women in the Asian region. This is because rice containslow level (1–3 mg/kg) of the micronutrient in the polished form(white rice). Breeding efforts for high-iron rice started in 2001under the Asian Development Bank-International Food PolicyResearch Institute (ADB-IFPRI) project (more popularlyknown as the high-iron rice project) with the introduction ofIR68144 (cross between IR72×Zawa Bonday). Crosses weremade between IR68144 with other popular varieties (e.g. IR64)while germplasms were screened for iron and zinc content. Atthe Philippine Rice Research Institute (PhilRice), 538 lines andvarieties were subjected to inductively-coupled plasma-opticalemission spectrometry (ICP-OES) analysis for grain mineralconcentration (7.0–17.8 mg/kg Fe). A 9-month human efficacystudy showed a 20% increase in the body iron stores of thesubjects1 proving that consumption of high-iron rice wouldimprove the nutritional status of iron-deficient populations. Forenhancement to work however, the rice variety must be highyielding, resistant to pests and diseases, and acceptable toconsumers. IR68144 was susceptible to bacterial leaf blight andsheath blight and the grain quality is inferior to IR64, thus,mining of germplasm has continued for other potential parents.Methods (based on Prussian blue staining and colorimetry) weredeveloped for rapid screening of breeding materials (popularand traditional varieties) of the National Agricultural ResearchExperiment Stations (NARES). The use of X-ray fluorescence

spectrometry (XRF) was also employed for non-destructivequantification of grain mineral concentration.

1. Haas J.D., Beard J.L., Murray-Kolb L.E., del Mundo A.M.,Felix A. and Gregorio G.B., Iron-biofortified rice improvesthe iron stores of nonanemic Filipino women. Journal ofNutrition 135:2823–2830 (2005).

doi:10.1016/j.cbpa.2007.01.574

P4.13Wheat nutritional genomics: Remobilisation of nitrogen

and sulfur during grain-filling in wheat

J. Howarth, S. Parmar, P. Barraclough, M. Hawkesford,(Rothamsted Research)

Wheat is the major arable crop grown in the UK with 15 milliontonnes produced annually supplying 85% of the country'srequirement for milling, bread making and animal feeds. Grainquality depends on nutrient availability throughout the devel-opment of the crop and particularly the uptake and internalremobilisation of nutrient resources to the seed during graindevelopment.Rothamsted's Broadbalk experiment has been used for morethan 160 years to study the effects of fertiliser applications onwheat physiology, yield and quality. We have utilised thisresource to study the molecular basis of metabolic processesduring grain filling.Leaf, stem and grain tissues were sampled weekly during thegrain-filling period between anthesis (GS65) grain maturity(GS84). In this period protein, carbohydrates and cell com-ponents in vegetative tissues are remobilised to developinggrain tissues. We hypothesise that processes taking placeaccount for the efficiency of resource remobilisation to thedeveloping grain. N and S-deficiency adversely affect thesystem and grain quality. We have used Affymetrix, metabo-lomics and chemical analyses to study gene expression in leavesand the principal metabolite pools in the leaf and grain tissueson N and S-deficient plots.Field experiments involving a range of wheat varieties grownon low and control levels of N have also been carried out.Genetic and metabolic analyses will be presented highlightingvarietal differences in remobilisation processes.Research funded by BBSRC/DEFRA grant BB/C514066/1.Rothamsted Research receives grant-aided support from theBiotechnology and Biological Sciences Research Council(BBSRC) of the UK.

doi:10.1016/j.cbpa.2007.01.575

P4.14Increasing the iron and zinc contents of plants

M.G.M. Aarts, (Wageningen University)

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To increase the (bioavailable) iron and zinc content in plantsseveral strategies can be followed. The most straightforward is tobreed for enhanced mineral contents in plants. So far, sufficientvariation has been found in most of the important crop species toallow a breeding approach. However, the trait is not easy to breedfor because of strong environmental effects and the absence ofgenetic markers.The alternative is to use a biotechnological approach usinggenetically modified plants, but this requires proper knowledgeon the physiological and genetic aspects of zinc and iron uptakeand translocation. Recently zinc and iron homeostasis isincreasingly studied at the molecular level. We are using themodel species Arabidopsis thaliana and the zinc hyperaccumu-lator Thlaspi caerulescens for better understanding the regula-tion of zinc and iron homeostasis and to identify target genes formodification. These results will be discussed in relation to thegeneral aim to increase iron and zinc content of plants.

doi:10.1016/j.cbpa.2007.01.576

P4.15Probing natural variation and nutrient use efficiency in

Arabidopsis

K. North, S. Kopriva, (John Innes Centre)

One of the challenges for the future is to produce sufficient foodfor the expanding global population. We also have a responsi-bility to minimise the environmental impact of agriculture, forexample by reducing the amount of fertiliser applied to crops.Improvements in nutrient use efficiency in arable crops wouldhelp us to achieve these objectives, as we could increase plantyield per unit of nutrient input. The natural variation found in themodel species Arabidopsis thaliana is a resource we can exploitto help understand the basis for adaptation of plants to differentenvironments. We are using natural variation in different A.thaliana accessions to investigate nutrient use efficiency withessential plant nutrients including nitrate, phosphate andsulphate. We have identified differences in growth with a subsetof accessions, by growing them under nutrient-depletedconditions where some accessions grow better than others ascompared to control conditions. Our aim is to understand thebasis for this — which may be due to improved nutrient useefficiency in particular accessions for example, or a lowerrequirement for the nutrient. Ultimately we hope to establish theunderlying genetic cause of adaptation to survival under nutrientdeprived conditions, with potential agricultural benefits from abetter understanding of nutrient use efficiency in the long term.

doi:10.1016/j.cbpa.2007.01.577

P4.16Plant sulfate assimilation—introduction to a smelly world

S. Kopriva, (John Innes Centre)

Sulfur is essential for life as a constituent of amino acidscysteine and methionine, many coenzymes and prostheticgroups, and a variety of secondary metabolites. Secondarysulfur containing compounds are important for defenseagainst biotic and abiotic stress and are often determiningsmell and taste of vegetables. Adequate sulfur supply istherefore necessary to obtain high yield and quality crops.However, sulfur deficiency has become a serious problem foragriculture as the atmospheric sulfur deposits declined lately.The only form of sulfur available to plants is inorganicsulfate. Sulfate is reduced to sulfide and incorporated intocysteine in the sulfate assimilation pathway. In the last decadea great progress in understanding sulfate reduction in plantshas been made. Sulfate assimilation is highly regulated in ademand-driven manner and tightly interconnected withassimilation of carbon and nitrogen. Here, a brief introductionwill be given into the variety of sulfur containing compoundsand their importance for plant metabolism and in the keyaspects of sulfate assimilation.

doi:10.1016/j.cbpa.2007.01.578

P4.17Sulfate assimilation in lower plants and algae: Surprisinglessons from sequenced genomes

S. Kopriva, (John Innes Centre)

Sulfate assimilation is a pathway providing reduced sulfur forthe synthesis of cysteine, methionine, co-enzymes such asiron–sulfur centres, thiamine, lipoic acid, or coenzyme A, andmany secondary metabolites, e.g. glucosinolates or alliins.The pathway is relatively well understood in flowering plants,but very little information exists on sulfate assimilation inlower plants and algae. Since the finding of a putative 3′-phosphoadenosine 5′-phosphosulfate (PAPS) reductase inPhyscomitrella patens, an enigmatic enzyme thought toexist in fungi and some bacteria only, it has been evidentthat sulfur metabolism in lower plants may substantially differfrom seed plant models. The genomic sequencing of twobasal plant species, the Bryophyte Physcomitrella patens, andthe lycophyte Selaginella moellendorffii, and of several algalspecies including Chlamydomonas reinhardtii, Thalassiosirapseudonana or Phaeodactylum tricornutum, opens up thepossibility to search for differences between lower and higherplants and algae at the genomic level. The genomes of thesespecies contain a surprising number of new enzyme variantsand fusion. Also the complexity of several gene familiesinvolved in sulfate assimilation is substantially different in thevarious genomes. The consequences for regulation of thepathway and evolution of sulfate assimilation in plants will bediscussed.

doi:10.1016/j.cbpa.2007.01.579

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P4.18Adenosine 5′-phosphosulphate kinase: The enzyme at the

interface between primary and secondary sulphatemetabolism

S. Garton, S. Kopriva, (JIC)

Sulphur is present in many key metabolites of primary andsecondary metabolism. In primary metabolism sulphate is usedfor the synthesis of cysteine, methionine, and many co-enzymesand in secondary metabolism for the synthesis of metabolitessuch as glucosinolates, phytoallexins and sulphoflavonoids,which play an important role in plant protection against bothstress and pathogen attack. Despite the importance of thesesulphur-containing secondary molecules, virtually nothing isknown about the control of their synthesis. However, under-standing the regulation of sulphur partitioning between primaryand secondary metabolism is key to improving sulphur usageand nutrition in arable crops. Common to both assimilationpathways is adenosine 5′-phosphosulphate (APS). APS reduc-tase (APR) converts APS to sulphite which is further reducedand incorporated into cysteine, while APS kinase (APK) formsPAPS, the sulphate donor for sulphotransferases whichsynthesise sulphated secondary compounds. In higher plantsthis is the point at which irreversible partitioning occurs, sincehigher plants do not possess PAPS reductase. Studying APKtherefore provides an opportunity to investigate not only theregulation of sulphate partitioning between primary andsecondary metabolism, but also the integration of sulphurassimilation with defence mechanisms. The Arabidopsis geno-me encodes four APK sequences; two of which have beendemonstrated to be functional (APK1 and 2) and two of whichwere only predicted (APK3 and 4). Using a combination ofbiochemical, genetic and molecular techniques, we are system-atically characterising and determining the role of eachindividual isoform, and how they influence plant nutrition,performance and protection from stresses.

doi:10.1016/j.cbpa.2007.01.580

P4.19Regulatory function of the cysteine synthase proteincomplex in transgenic tobacco

R. Hell, M. Wirtz, F. Liszewska, (University of Heidelberg)

Cysteine synthesis in plants is a two step process that constitutesthe entry of reduced sulfur from assimilatory sulfate reductioninto metabolism. The catalyzing enzymes, serine acetyltransfer-ase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL),reversibly form a hetero-oligomeric protein complex, calledcysteine synthase complex (CSC) that is present in cytosol,plastids and mitochondria. Dominant-negative mutation of theCSC showed the crucial function for the regulation of cysteinebiosynthesis in vivo. A SAT from Arabidopsis was over-expressed in the cytosol of transgenic tobacco plants in either

enzymatically active or inactive form that were both shown tointeract efficiently with endogenous tobacco OAS-TL proteins.Expression of active SAT resulted in 40-fold increase of SATactivity and strong elevations of the reaction intermediate OAS,cysteine, glutathione, methionine and total sulfur contents intransgenic lines. However, expression of inactive SAT exceededby far all enhancing effects compared to expression of activeSAT. Up to 30-fold increased cysteine levels were observed.This de-regulation of the CSC was based on competition ofinactive transgenic SAT with endogenous tobacco SAT forbinding to OAS-TL in the protein complex. Expression levels oftobacco SAT and OAS-TL and the corresponding enzymeactivities remained unaffected. Flux control coefficients sug-gested that the accumulation of OAS and cysteine in both typesof transgenic plants was accomplished by different mechan-isms. The contributions of plastidic and mitochondrial cysteinesynthesis to regulation are investigated. The data provideevidence that the CSC and its subcellular compartmentationplay a crucial role in the control of cysteine biosynthesis.

doi:10.1016/j.cbpa.2007.01.581

P4.20Plants in the human diet

J. Buttriss, (British Nutrition Foundation)

Archaeological records demonstrate that cultivation and storageof grains and legumes was already established by 10,000 BCand, by 100 AD, rye and oat cultivation was widespread acrossEurope. Today's global food economy means that now in the21st century we have access, all year round, to a diverse rangeof fruits, vegetables, grains, legumes, nuts, herbs, spices andplant derived drinks. Such foods can make a considerablecontribution to nutrient intake and it is now widely recognisedthat fruit, vegetable and wholegrain consumption is positivelyassociated with health and with reduced risk of conditions suchas stroke, coronary heart disease, type 2 diabetes, some cancers,cognitive decline and possibly some lung conditions and eyeproblems such as cataract and age-related macular degeneration.Various mechanisms to explain these associations have beensuggested, some involving nutrients and others non-nutrientssubstances in plants with bioactive properties, and these will besummarised. Despite this body of evidence, ready access tosuch foods and recent government and retailer publicitycampaigns, many people are consuming lower amounts thanrecommended for good health. The challenge to plant breedersis to optimise nutritional contribution and the challenge to thefood chain, in general, is to provide and present the diversity ofplant foods available to us in ways that meet consumer needs interms of taste, appearance and price, and encourage increasedconsumption.

doi:10.1016/j.cbpa.2007.01.582

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P4.21Ascorbic acid homeostasis and its effects on plant growth

and defence

C. Foyer, (University of Newcastle)

Ascorbic acid (AsA) is an abundant antioxidant metabolite inplants. It is synthesized in mitochondria and is transported to allsub-cellular compartments including the apoplast, where it isthe major redox buffer modulating physiological and stressresponses. AsA is a signal-transducing molecule relayinginformation on cellular redox state. The amount of AsAaccumulated in leaves is modulated by environmental andmetabolic cues. The roles of AsA in the cytoplasmic andapoplastic compartments of the plant cell, will be consideredwith regard to the control of gene expression, growth anddefence. Micro-array data will be used to illustrate how AsAlevels might be involved in pathways that control geneexpression in planta, with particular emphasis on signallingthrough plant hormones. Evidence from studies using Arabi-dopsismutants that are deficient in AsA (vtc; vtc1 and vtc2) willbe used to show that low AsA abundance leads to micro lesions,expression of pathogenesis-related proteins and enhanced basalresistance against infections caused by Pseudomonas syringae.In these mutants low AsA limits cell expansion and triggersprogrammed cell death in a coordinated manner activatingsystemic enhanced resistance and enhancing basal resistancewhen cell growth has ceased. Finally, the importance ofcompartment-specific changes in AsA will be considered,with particular focus on the apoplastic AsA pool in controllingsignal transduction processes. The AsA pool in the apoplast isspecifically affected by manipulating AO activity and thismodulates different signalling pathways that effect pathogenresistance, photorespiration, calcium signalling and sensitivityto hormones. High AO activity alters responses to auxin andgibberellic acid. The responses of seedlings to the application ofthese hormones were damped when the apoplastic AsA poolwas oxidized.

doi:10.1016/j.cbpa.2007.01.583

P4.22The role of VTC2 in vitamin C biosynthesis in Arabidopsisthaliana

N. Smirnoff, J. Dowdle, (Exeter University); T. Ishikawa,(Shimane University, Japan)

Current genetic and biochemical evidence suggests thatascorbate (vitamin C) is synthesised by a two step oxidationof L-galactose (L-Gal). L-Gal is produced from GDP-mannosevia GDP-L-Gal and L-Gal 1-P. Other pathways via uronic acidintermediates have also been proposed to contribute toascorbate synthesis. The enzyme responsible for convertingGDP-L-Gal to L-Gal 1-P has not been identified to date. Wehave now shown that the A. thaliana VTC2 gene encodes a

novel enzyme, GDP-L-Gal phosphorylase that produces L-Gal1-P from GDP-L-Gal. A. thaliana also expresses a second geneencoding GDP-L-Gal phosphorylase. We made double mutantswhich do not express VTC2 and its homologue. Growth ofdouble mutant seedlings was arrested after initial cotyledonexpansion but normal growth could be restored by ascorbatesupplementation. The results show that ascorbate is essential forplant growth. Also, it is evident that the proposed uronic acidpathways of ascorbate synthesis cannot provide sufficientascorbate to sustain the survival of A. thaliana. The expressionof both genes is light-regulated and increased by jasmonates.We propose that VTC2/GDP-L-Gal phosphorylase plays amajor role in controlling ascorbate biosynthesis. The implica-tions for engineering ascorbate-enriched plants and forunravelling the functions of ascorbate in plants will bediscussed.

doi:10.1016/j.cbpa.2007.01.584

P4.23Enhancing seed phytosterol accumulation by modifying the

regulation of HMG-CoA reductase by SnRK1

S. Hey, N. Halford, (Rothamsted Research)

Dietary phytosterols have been shown to be effective at reducingblood cholesterol levels in humans. The regulation of phytos-terol biosynthesis in seeds is therefore a target for biotechnol-ogists as it offers potential for increasing phytosterol levels inseed oils. HMG-CoA reductase is a key enzyme involved inmevalonate derived phytosterol biosynthesis and it is inactivatedby phosphorylation by Snf1-related protein kinase 1 (SnRK1).With the aim of increasing seed phytosterol levels, transgenictobacco plants were produced expressing modified ArabidopsisHMG-CoA reductase sequences in which the serine residuephosphorylated by SnRK1 was removed. These plants werethen compared with both wildtype tobacco plants and with thoseexpressing an unmodified Arabidopsis HMG-CoA reductasegene. Removal of HMG-CoA reductase from SnRK1 regulationled to significant increases in seed phytosterol accumulation.Our results suggest that modification of the regulatory control ofHMG-CoA reductase by phosphorylation represents a potentialroute for increasing phytosterols in seed oils of crop plants.

1. Hey et al. Plant Biotechnology Journal 2006 4: 219–229.

doi:10.1016/j.cbpa.2007.01.585

P4.24Nutritional genomics of broccoli–human interactions

R. Mithen, (Institute of Food Research)

Epidemiological studies provide evidence that diets rich inbroccoli reduce the risk of cancer of the colon, lung, breast and

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prostate, particularly in the 50% of the populations that have atleast one functional GSTM1 allele. Experimental studies withcell and animal models have demonstrated the complexbiological activity of sulforaphane, the isothiocyanate derivedfrom 4-methysulphinylbutyl glucosinolate that accumulates inbroccoli, which is consistent with this compound being themajor anticarcinogenic component of broccoli. In this presenta-tion, I described, firstly, the genetic basis to glucosinolateaccumulation in broccoli, and the development of broccolicultivates that deliver enhanced levels of sulforaphane uponconsumption. Secondly, I discuss the effects of consuming highglucosinolate broccoli on gene expression in gastric andprostate tissue following both short and long term dietaryintervention studies.

doi:10.1016/j.cbpa.2007.01.586

P4.25Novel methods for folate detection

D. Barrett, A. Santoyo Castelazo, C. Ortori, M. Bennett,(Nottingham University)

Folates in nature exist in diverse chemical forms depending onthe substitution of one-carbon units, the oxidation state of thepterin ring and the length of the glutamyl side chain. Thismetabolic complexity complicates the task of measuring folatesin biological matrices. Until recently, it was not possible todetermine all forms of folate simultaneously. We report the useof reversed-phase ion-pair chromatography coupled to electro-spray ionisation on a quadrupole linear ion trap mass spectro-meter for the simultaneous profiling of all folate-basedmetabolites including natural folates, their polyglutamatylderivatives and their biosynthetic precursors in plant and animaltissue. Full, quantitative analysis was obtained for 16 folates anda ‘semi-quantitative’ analysis was possible for all other folateswith up to eight conjugated glutamate residues by reference tostructurally-related calibration standards. The precision, accu-racy and recovery of the method were generally within theaccepted guidelines for a quantitative bioanalytical method andthe method was linear over the range 0.2 to 10 ng of individualfolate per sample. The method has been applied to profile mono-and polyglutamated tetrahydrofolates (including subcellularanalysis) in a range of plant species, including Arabidopsis,spinach, Brassica and wheat; and has also been successfullyapplied to the profiling of folates in mammalian tissue.

doi:10.1016/j.cbpa.2007.01.587

P4.26Characterising the functional importance of folatepolyglutamylation in plants

P. Mehrshahi, G. Tucker, M. J. Bennett, (University ofNottingham)

Tetrahydrofolate (THF) functions as substrates and coenzymesin single carbon transfer reactions necessary to synthesisecomponents of DNA, RNA, and proteins. Humans lack thecomplete folate biosynthetic pathway and thus rely on dietaryintake to meet their requirements. A significant proportion ofTHF is polyglutamylated, a process catalyzed by folylpolyglu-tamate synthetase (FPGS), which adds glutamate residues to thepara-aminobenzoate moiety. The polyglutamate chain has to beremoved prior to absorption of THF, in the small intestine.In plants, the polyglutamate chain is the principal means bywhich THF is retained and is compartmentalised within cells.Furthermore, polyglutamated THF is the preferred substrates forenzymes such as methionine synthase, serine hydroxymethyl-transferase and thymidylate synthase (Ravanel et al., 2004;Huang et al., 1998; Kamb et al., 1992).The objectives of this study have been to systematically explorethe role of THF polyglutamylation on one-carbon metabolismand plant development. In Arabidopsis, FPGS is encoded by 3genes which have been reported to encode cytosolic, plastidicand mitochondrial localised isoforms (Ravanel et al., 2001).Knockout mutants have been isolated in all 3 FPGS genes. Wewill report our latest results characterising single and multipleFPGS mutant phenotypes.

1. Kamb A., Finer-Moore J., Calvert A.H., Stroud R.M. (1992)Structural basis for recognition of polyglutamyl folates bythymidylate synthase. Biochemistry 31: 9883–9890.

2. Ravanel S., Cherest H., Jabrin S., Grunwald D., Surdin-KerjanY., Douce R., Rebeille F. (2001) Tetrahydrofolate biosynthesisin plants: zolecular and functional characterization of dihy-drofolate synthetase and three isoforms of folylpolyglutamatesynthetase in Arabidopsis thaliana. Proc. Nat. Ac. Sci. 98:15360–15365.

3. Ravanel S., Block M.A., Rippert P., Jabrin S., Curien G.,Rebeille F., Douce R. (2004) Methionine Metabolism inPlants. J. Biol. Chem. 279: 22548–22557.

doi:10.1016/j.cbpa.2007.01.588

P4.27Dissecting the genetic control of folate homeostasis in plants

S. Gonzalez-Jorge, P. Mehrshahi, M.J. Bennett, (University ofNottingham)

Folates act as essential cofactors in the one-carbon metabolismin cells, which are important for DNA replication and division,de novo synthesis of pyrimidines, purines, methionine, histidineand serine. Unlike plants, mammals and humans cannotsynthesise their own folate and therefore require a dietarysource of folate, enhanced by fortification or supplementation.As folate deficiency is endemic to both developing anddeveloped countries, a growing international interest andresearch has been directed towards enhancing folate abundance.We have probed the mechanisms controlling folate homeostasisin plants by selecting Arabidopsis lines with altered sensitivity

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towards the antifolate, methotrexate (MTX). We demonstratethat resistance results from reduced polyglutamation which isknown to enhance MTX efflux from cells.MTX resistance could also result from reduced cellular uptake.Methotrexate resistance screens were carried out on a muta-genised Arabidopsis population and the Ler/Cvi near-isogenicline (NIL) population. We will report our latest results.

doi:10.1016/j.cbpa.2007.01.589

P4.28Metabolite regulation of gene expression during the

heterotrophic to autotrophic transition in developingseedling of Arabidopsis

E. Allen, M. Hooks, (University of Wales Bangor), A. Moing,M. Marcourt, D. Tomos, D. Rolin, T. Ebbels (Imperial CollegeLondon)

Seed germination and development represents a unique stage ofplant development where metabolic programs that are gearedinitially towards catabolism of stored carbon reserves (hetero-trophy) undergo a transition to permit fixation of inorganiccarbon into organic compounds by photosynthesis (autotrophy).This transition involves a transcriptional reprogramming todismantle catabolic and produce the photosynthetic machinery.Because this transcriptional programming revolves aroundmetabolic changes, a key question is the role of metabolicsignals in regulating this process. Expanding on our work onacetate regulation of gene expression during seedling develop-ment, we are taking a holistic approach to identify other potentialsignallingmetabolites. Using 1H-NMR,we have profiled a groupof metabolites in samples from imbibed seeds to seedlings 8 daysafter imbibition. We have also profiled expressed genes atcorresponding time points usingmicroarrays.We have calculatedcorrelations among differentially expressed genes andmetabolitelevels in order to determine potential metabolic signals for futurestudy. Interestingly, Spring Embedding models of metabolite andgene expression networks have shown that metabolite profilesfrom day 2 samples group with profiles from day 0 (imbibedseeds) and day 1, but that gene expression profiles from day 2samples group with those from day 3 to day 8. This suggests thatmetabolic programming of embryos within seeds establish apattern that may precede reprogramming of gene expression.

doi:10.1016/j.cbpa.2007.01.590

P4.29Natural genetic variation in zinc (Zn) accumulation inBrassicaceae species

S.Ó. Lochlainn, R. Fray, V. Mills, M. Broadley, (University ofNottingham); H. Bowen, J. Hammond, (Warwick HRI); G.King, (Rothamsted Research); P. White, (The Scottish CropResearch Institute)

Zinc (Zn) is an essential plant nutrient. Most plant species havea leaf Zn concentration ([Zn]leaf) <0.1 mg Zn g−1 dry weight(DW), although substantial natural genetic variation occurs. Forexample, within the Brassicaceae family, several species ofThlaspi and Arabidopsis accumulate >10 mg Zn g−1 DW.Within a diversity foundation set of Brassica oleracea L.(BoDFS), representing most of the allelic variation within thisspecies, there is >20-fold variation in [Zn]leaf. Quantitative traitloci (QTL) impacting on the Zn composition of B. oleraceaunder glasshouse and field conditions have also beenestablished using a population of doubled-haploid (DH) linesderived from a B. oleracea var. alboglabra X var. italica cross(the ‘AG’ mapping population). Since over two billion peopleworldwide are likely to suffer from dietary zinc (Zn) deficiency,exploiting this natural genetic variation in [Zn]leaf throughbreeding could help to alleviate this situation. Here, we aretesting the hypothesis that specific Zn-transporters can be usedas potential markers for breeding crops with altered [Zn]leaf.First, the physiological function of selected Zn-transporters,which are highly expressed in Thlaspi caerulescens J. & C.Presl (a Zn hyperaccumulator), are being determined in plantausing transformation-based approaches. These genes include P-type HMA (heavy-metal-associated domain-containing)ATPases from T. caerulescens. Second, associations betweenQTLs impacting on [Zn]leaf in B. oleracea and Brassicaorthologues of candidate Zn-transporter genes are beingdetermined through comparative sequence analysis within theBrassicaceae.

doi:10.1016/j.cbpa.2007.01.591

P4.30Genetic variation associated with glucosinolate hydrolysis

and postharvest performance in rocket (Arugula) species

C. Wagstaff, (University of Reading)

Rocket (Arugula) is a cruciferous crop used extensively in babyleaf salads. Its peppery taste is indicative of high glucosinolatecontent. Hydrolysis of glucosinolates by the enzyme myrosi-nase results in the formation of epithionitriles or isothiocya-nates, and in Arabidopsis the specificity of this conversion isconferred by the locus ESM1. Both isothiocyanates andepithionitriles are produced by cruciferous plants as a defenceagainst herbivores, but isothiocyanates are of interest in humannutrition for their anticancer properties, therefore identificationof ESM1 orthologues in edible crucifers is of potential value tobreeding programmes in these species.Several cultivars are grown commercially from wild rocket(Diplotaxis) and salad rocket (Eruca) species and these havevarying properties of shelf life and taste. The postharvestlongevity of a number of cultivars was compared by visualinspection and determination of membrane leakage by con-ductivity measurements following different preharvest growingconditions. Homologues of ESM1 were obtained from rocketcultivars and comparisons made of the genomic sequence.

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Expression during shelf life was also quantified and theimplications of these findings will be discussed.

doi:10.1016/j.cbpa.2007.01.592

P4.31A QTL for grain yield on 7AL of wheat is activated by

ABA and low nutrient treatments during flag leaf ontogeny

S. Quarrie, A. Kaminska, J. Barnes, A. Gennaro, (University ofNewcastle), D. Dodig, (ARIS)

Studies with wheat doubled haploids from the cross ChineseSpring (CS)×SQ1 trialled over 24 year× treatment× locationsidentified a frequent yield QTL on 7AL associated with a locusfor SSR psp3094, expressed mainly under stressed conditions.SQ1 alleles increased yield as well as biomass and flag leaf (FL)width. Near-isogenic lines (NILs) for the 7AL yield QTL weremade with CS or SQ1 alleles in an SQ1 background, selectingwith psp3094. In a preliminary field trial, the SQ1 allele wasassociated with 21% higher yield/ear, significantly higher FLchlorophyll content and wider FLs due to more cell files acrossthe leaf. In a controlled environment experiment, eight CS-alleleand eight SQ1-allele NILs, with two replicate plants/NIL grownin high nutrient compost, were given either no treatment, low-light, low-nutrient or abscisic acid (ABA) spray treatments for 2weeks during flag leaf emergence on the main stem, tillers 1 and2. No significant differences between CS-NILs and SQ1-NILs inFL width were found in any treatment on shoots with FL celldivision already complete. However, for FLs still undergoingcell division, final leaf width significantly differed between CS-NILs and SQ1-NILs, but only in ABA- and low-nutrienttreatments (by 11.0% and 13.9%, respectively). Therefore, the7AL yield QTL probably results from allele differences in anutrient-regulated gene which determines termination of FLlamina lateral meristematic activity during leaf ontogeny, per-haps via an ABA-signalling pathway. A rice AINTEGUMENTAcandidate gene homologue has an ABRE −607 bp upstream.

doi:10.1016/j.cbpa.2007.01.593

P4.33Role of sulphate transporters in selenium uptake

E. Cabannes, P. Buchner, M. Hawkesford, (RothamstedResearch), (Rothamsted Research); M. Broadley, (Universityof Nottingham); P. White, (The Scottish Crop ResearchInstitute)

Selenium (Se) is an essential micronutrient for animals.Selenium deficiency in the human diet is associated with healthdisorders including cancer, thyroid dysfunction, and reducedimmune functions. In the UK, wheat is an important source ofbioavailable selenium, although suboptimal levels are oftenpresent. The ability of some plants to hyperaccumulate seleniumcan be used to better understand selenium uptake and then todevelop wheat varieties with increased level of seleniumcompounds.Plants take up selenium as selenate from the soil. Selenate andsulphate are thought to be transported through the same proteinsand to compete in the uptake process. To investigate the role ofsulphate transporters in selenate uptake, sulphate transporterswere cloned from both selenium hyperaccumulating species(Astragalus racemosus) and non accumulating (Astragalussinicus, Astragalus glycyphyllos) species. The nucleotide andderived amino acid sequences indicate that they belong to thehigh affinity Group 1 type of sulphate transporters that isbelieved to contain the main transporters involved in the uptakeof sulphate and selenate by roots. The sequences obtained showsubstitutions and deletions between the Astragalus species thatmight be associated with differences in selenium transportcharacteristics. A functional analysis of the cloned transportersusing a yeast expression system is ongoing in order to comparesulphate and selenate transport and specifically to look atcompetition between the two ions.

doi:10.1016/j.cbpa.2007.01.595