Current
strategies
for
vitamin
E
biofortification
of
crops
Laurent
Me`ne-Saffrane´ and
Se´bastien
Pellaud
VitaminEreferstofourtocopherolsandfourtocotrienolsthat
areexclusivelysynthesizedbyphotosyntheticorganisms.
Whilea-tocopherolisthemostpotentvitaminEcompound,itis
notthemainformconsumedsincethecompositionofmost
majorcropsisdominatedbyg-tocopherol.Nutritionalstudies
showthatpopulationsofdevelopedcountriesdonotconsume
enoughvitaminEandthatalargeproportionofindividuals
exhibitplasmaa-tocopheroldeficiency.Followingthe
identificationofvitaminEbiosyntheticgenes,severalstrategies
includingmetabolicengineering,classicbreedingandmutation
breeding,havebeenundertakentoimprovethevitaminE
contentofcrops.Inadditiontoprovidingcropsinwhichvitamin
Econtentisenhanced,thesestudiesarerevealingthe
bottleneckslimitingitsbiosynthesis.
Address
UniversityofFribourg,DepartmentofBiology,Chemindumuse´e,10,
CH-1700Fribourg,Switzerland
Correspondingauthor:Me`ne-Saffrane´,Laurent(
laurent.mene-saffrane@unifr.ch)
Introduction
VitaminEencompasseseightorganiccompoundswitha chromanol ring substituted with one to three methyl groupsthatarecollectivelycalledtocochromanols. Vita-min E isoforms with a saturated prenyl side chain are called tocopherols, while the ones with an unsaturated prenylsidechainarenamedtocotrienols(Figure1a&b). Tocopherolandtocotrienolforms(a-,b-,g-,andd-)differ bythenumberandpositionofmethylsubstituentsonthe chromanol ring. The vitamin E activity of each form differs greatly due to the different affinities between specifictocochromanolsandthelivera-tocopherol trans-fer protein, which preferentially binds a-tocopherol [1]
(Table 1).Other tocochromanols such as
plastochroma-nol-8(PC-8; Figure 1c) andtocomonoenols(Figure 1d) havebeenidentifiedinedibleplantsregularlyconsumed
by humans[2,3].The vitaminE activityof theselatter formsisunknownyet.
VitaminEwasoriginallyidentifiedasanutritionalfactor essential foranimal reproduction [4].Since,ithasbeen shownthatvitaminEisapowerfullipidantioxidantthat protects cell membranes from the damaging effects induced byfreeradicals[5,6].Inaddition, several toco-chromanol isoforms show beneficial roles in delaying brain aging [7], reducing the risk of developing Alzheimer’s disease [7], or inhibiting lung cancer [8]. Tocopherols are found in virtually all photosynthetic plants, notably in vegetableoiland nuts,whereas toco-trienols are mainly encountered in monocot seeds. Despite its wideavailability, nutritional surveys clearly demonstratethatalargeportionofWesternpopulations donotmeetthevitaminErecommendeddietary allow-ance[9,10,11].Whilethelong-termeffectsonhealthof chronic vitamin Edeficiencyare not definitively estab-lished for humans, the oxidative stress associated with vitaminEdeficiencystronglyadvocatesforimprovingits quality andquantityinthehumandiet.
Updateonthetocochromanolbiosyntheticpathway
StrategiesimplementedtoenhancevitaminEcontentin cropsderivefromourcurrentknowledgeof tocochroma-nol biosynthesis, which has been recently reviewed
[12,13].Thissectionwillbrieflysummarize
tocochroma-nol metabolism and introduce vitamin E biosynthetic genes recentlyidentified (Figure2).
Tocochromanolsareamphipathicmoleculescomposedof a polar chromanol ring built around the phenolic com-pound homogentisic acid (HGA), and a hydrophobic isoprenoid side chain. HGA is produced from 4-hydro-xyphenylpyruvate(HPP)byap-hydroxyphenylpyruvate dioxygenase(HPPD;Figure2a).Inplants,HPPderives fromtyrosinedegradationthatnotablyinvolvesaspecific tyrosineaminotransferase[14].Incontrast,cyanobacteria produceHPPdirectlyfromchorismateand/orprephenate withabifunctionalchorismatemutase/prephenate dehy-drogenasenamed TyrA[15,16].
The prenyl side chains of prenylquinols and tocochro-manols all derive from geranylgeranyl pyrophosphate (GGPP) produced by the plastidial methyl erythritol phosphate (MEP)pathway (Figure 2b).Geranylgeranyl pyrophosphatesynthase(GGPPS)11wasrecentlyshown to catalyze the biosynthesis of most plastid GGPP-derived isoprenoids, including tocochromanols [17]. For tocotrienol synthesis, GGPP is directly condensed withHGA.Forsolanesylderivativessuchas
plastoquinol-http://doc.rero.ch
9 (PQ-9) and PC-8, GGPP is converted into solanesyl pyrophosphate (SPP) by solanesyl pyrophosphate synthases[18].Tocopherolsynthesisrequiresthe reduc-tion of GGPP into phytyl pyrophosphate (PPP) by a geranylgeranylreductase(GGR).Invitro,GGRreduces both free GGPP and geranylgeranylated chlorophyll a
(Figure 2c). Recently, thelight-harvesting-like proteins
LIL3:1 and LIL3:2 were shown to be involved in
tocochromanol metabolism through their interaction and stabilization of GGR proteins in the chloroplast membrane[19].InArabidopsisseedsandleaves, tocoph-erolsynthesismostlydependsonthephytolkinaseVTE5 andthephytylphosphatekinaseVTE6thatsequentially phosphorylate phytol into PPP [20,21]. In senescent leaves,thephytolusedfortocopherolsynthesisoriginates mostlyfromchlorophyllhydrolysis[21].InArabidopsis
Figure1 O HO R1 R2 R3 CH3 CH3 CH3 H H H3C H3C O HO R1 R2 R3 CH3 CH3 CH3 H H H3C H3C O HO H3C CH3 CH3 CH3 CH3 CH3 7 (a) (b) (d) (c) O HO R1 R2 R3 CH3 CH3 CH3 CH3 CH3
Current Opinion in Biotechnology
Tocochromanolstructures.Chemicalstructuresoftocopherols(a),tocotrienols(b),plastochromanol-8(c),andtocomonoenols(d).R1,R2,
R3=CH3,a-tocochromanol;R1,R3=CH3,R2=H,b-tocochromanol;R2,R3=CH3,R1=H,g-tocochromanol;R3=CH3,R1,R2=H,
d-tocochromanol.
seeds,chlorophylldegradationandrecyclingcontributes atleast60%totocopherolsynthesis[20].Incontrast,the originofphytolusedfortocopherolsynthesisinhealthy leavesisstillanopenquestion[22].
Thecommittedstepoftocochromanolbiosynthesisisthe condensation of HGA with an isoprenoid side chain mediatedbyvariousprenyltransferases.Tocotrienol syn-thesis is initiated by a homogentisate geranylgeranyl-transferase(HGGT),PC-8synthesisbyahomogentisate solanesyltransferase(HST),andtocopherolsynthesisbya homogentisatephytyltransferase(HPT;Figure2e).The resultingmethylprenylquinolsaremethylatedby methyl-transferase (MT) to form dimethylprenylquinols. Both methyl-anddimethyl-geranylgeranyl-andphytyl-quinols arecyclizedbytocopherol cyclase(TC) toform d-toco-chromanols and g-tocochromanols, respectively. These isoforms are further methylated by the g-tocopherol methyl transferase (g-TMT) to form b-tocochromanols and a-tocochromanols, respectively. For solanesyl deri-vatives,cyclizationofPQ-9byTCproducesPC-8thatis notfurthermethylated inwild-typeplants.
VitaminEbiofortificationthroughimprovingplant tocopherolcomposition
ThefirststrategythatwasassessedtoenhancevitaminE activityin plantsconsistedinimprovingplant tocochro-manolcompositionbyconvertingpreexistingtocopherols into forms exhibiting higher biological potency [23]. Indeed,seedsofthemostabundantlyconsumedoilseed crops(e.g.,soybean,rapeseed,cotton,andoilpalm) accu-mulateprimarilyg-tocopherol,aformthathas10%ofthe vitamin E activity of a-tocopherol (Table 1). Since
conversion into a-tocopherol by overexpression shouldgreatlyenhancethevitaminEactivityinatissue. Thisstrategywasoriginallyattemptedin Arabidopsisin which the g-TMT gene was introduced under a seed-specificpromoter[23].Whileg-tocopheroldominatedthe tocopherol composition of wild-type seeds (>95%), a-tocopherolrepresentedupto95%ofseedtocopherols in the best transgenic event [23]. Subsequently, the successful conversion of g-tocopherol into a-tocopherol via g-TMT overexpression has been reported in many otherplantsincludingsoybean[24–27],shiso[28],lettuce [29],mustard[30],maize[31],andtobacco[32].Because of the higher biological activity of a-tocopherol, most g-TMToverexpressingcropsexhibited5–10timeshigher vitaminEactivitythanuntransformedplants.Inspecies accumulatingd-tocopherol,whichhas3%ofthevitamin E activity of a-tocopherol, g-TMT overexpression also enhanceditsconversionintob-tocopherol,whichat50% of the vitamin E activity of a-tocopherol is 16.6 times morepotent[23–27,30](Figure2andTable1).Because thegainin vitamin Eactivityisvery significantand no adverseeffectsongrowthandfertilityhavebeenreported in g-TMT overexpressing plants, this strategy is today amongthemosteffectiveforvitaminEbiofortificationof crops.
In addition to transgenic approaches, a-tocopherol enrichmentcanalsobeachievedbytraditionalbreeding usingnaturalhigha-tocopherolallelesidentifiedincrop germplasms byQTL studies. Whileseeds of most soy-beanvarietiescontainlowa-tocopherolamounts(<10% ofthetocopherolpool),threevarietieshavingupto53% of a-tocopherol were identified in soybean germplasm [33]. QTLanalysis showedthat higha-tocopherol con-tent correlated with higher expression of g-TMT3, a soybean gene encoding a polypeptide that exhibits 81.8% similarity with the Arabidopsis g-TMT protein [34]. Comparison of g-TMT3 promoter sequences betweenhighandstandarda-tocopherolvarieties identi-fied conserved polymorphisms within the promoter regionsof thehigh a-tocopherolvarieties thatmightbe responsible for the higher g-TMT3 promoter activity. ThishypothesiswasconfirmedintransgenicArabidopsis plantsexpressingtheGUSreportergenefusedtog-TMT3 promotersoriginatingfromstandardorhigha-tocopherol varieties [34]. Thus, soybean germplasm carries mono-genic alleles that increase by at least fivefold the a-tocopherolcontentinseedsandcouldbeintrogressed intostandardvarietiestoimprovetheirvitaminEcontent. Higha-tocopherolcultivarshavebeenidentifiedinother crops including rice [35], maize [36,37], and rapeseed [38]. Collectively, these high a-tocopherol natural var-iantsrepresentpromisingalternativestotransgeniccrops, notably for countries in which the production and/or marketingof plantGMOsare currentlybanned.
VitaminEactivityofnaturalandsynthetictocochromanols.The
biologicalactivityofeachtocochromanolform,giveninIU/mg
andcomparedtotheactivityofa-tocopherol,hasbeen
calcu-lated from the rat fetal resorption assay. Briefly, vitamin
E-depletedvirgin femaleswerematedwithnormalmales.
Preg-nant females were subsequently fed with different doses of
specificvitaminEisomersduring21daysafterwhichtheywere
sacrificed.ThevitaminEbiologicalactivitywasdeterminedby
countingthenumberofliving,dead,andresorbedfetuses.One
international unit(IU)correspondstothevitaminE activityof
1mgofthesyntheticall-rac-a-tocopherylacetate
Tocochromanol Activity(IU/mg)Activity(%)
a-Tocopherol 1.49 100 b-Tocopherol 0.75 50 g-Tocopherol 0.15 10 d-Tocopherol 0.05 3 a-Tocotrienol 0.45–0.75 30–50 b-Tocotrienol 0.08 5 g-Tocotrienol Below detection Below detection d-Tocotrienol Below detection Below detection
all-rac-a-Tocopherylacetate(synthetic)1 67
RRR-a-Tocopherylacetate(synthetic) 1.36 91
Figure2 MEP pathway IPP/DMAPP Shikimate pathway Tyr HPP TAT7 HPPD TyrA Pyruvate DXS (a) (b) MPBQ DMPBQ α-tocopherol β-tocopherol γ-tocopherol δ-tocopherol α-tocotrienol β-tocotrienol γ-tocotrienol MGGBQ DMGGBQ TC TC δ-tocotrienol TC HPT HGGT SPP HST PPP (e) Phytyl-P Phytol Chlorophyllgg Chlorophyllphy G4 VTE5 VTE6 (c) 4-maleyl acetoacetate HGO SPS1 SPS2 Chlorophyllide hydrolase MSBQ PQ-9 PC-8 TC GGPP GGPP HGA GGPPS11 GGPP SPP PPP GGR LIL3:1/LIL3:2 tocochromanol synthesis GGR LIL3:1/LIL3:2 G3P HO OH CH3 H3C CH3 CH3 3 H HO OH CH3 H3C CH3 CH3 8 H O CH3 H HO CH3 CH3 CH3 3 H3C O CH3 H HO CH3 CH3 CH3 3 O CH3 H HO CH3 CH3 CH3 3 O CH3 H HO CH3 CH3 CH3 3 H3C O CH3 H HO CH3 CH3 3 O CH3 H HO CH3 CH3 3 H3C O CH3 H HO CH3 CH3 3 H3C O CH3 H HO CH3 CH3 3 O CH3 H HO H3C CH3 CH3 8 HO OH CH3 H3C CH3 CH3 3 H HO OH CH3 CH3 CH3 3 H HO OH CH3 CH3 CH3 8 H HO OH CH3 CH3 CH3 3 H γ-TMT SAM γ-TMT SAM γ-TMT SAM γ-TMT SAM MT SAM MT SAM MT SAM GGPP SPP PPP (d) TC HGA HGA HO OH O HO CH3 H3C CH3 CH3 CH3 O P O P OH OH HO OO CH3 H3C CH3 CH3 CH3 O P O P OH OH HO OO CH3 CH3 CH3 CH3 O P O P OH OH HO O O CH3 CH3 CH3 CH3 CH3 H3C
Current Opinion in Biotechnology
Inordertoidentifythebiosyntheticstepslimiting toco-chromanol accumulation in plants, tocopherol produc-tionwasinvestigatedincellculturesfedwithmetabolic intermediates[15,39,40].Whilethesestudiesconcluded that both HGA and phytol had the greatest effects on vitaminEbiosynthesis,theirconclusionsdifferonwhich compoundisthemostlimiting.Insafflowercellcultures, phytol feeding significantly increased tocopherol pro-ductionafter 3 days ofincubation,while HGA supple-mentation had no effect [39]. After fourteen days of treatment, HGA feeding increased tocopherol content by 3.3 times, while phytol supplementation increased tocopherolcontentby18.4times[39].Thesedata indi-cate that the availability of both tocopherol precursors restrictsvitamin Ebiosynthesis, with phytol being the mostlimiting.Incontrast,insunflowersuspensioncells, HGA feeding stimulated tocopherol accumulation (30%), while phytol supplementation had no effect [40].Finally,insoybeansuspensioncultures,tocopherol contentincreasedtwofoldinresponsetoeitherHGAor phytolfeeding,andfivefoldwhenboth precursorswere added simultaneously [15]. These studies collectively showed that the availability of tocochromanol biosyn-thetic precursor(s) significantly limits vitamin E accu-mulationinplantsandsuggestthatenhancingthe met-abolic pathway(s) producing them in planta might improvevitaminEsynthesis.
VitaminEbiofortificationthroughmetabolicengineering oftocopherolprecursors
ThesecondstrategyaimingatimprovingcropvitaminE activityinvolvesincreasingtheglobalproductionof toco-chromanols. This was attempted by overexpressing tocopherol core biosynthetic genes (VTE), and biosyn-thetic genes producing the aromatic head and/or the isoprenoidsidechainofprenylquinols.
TransgenicapproachestargetingvitaminEcorebiosynthetic genes(VTE)
The committed step of the tocochromanol pathway is catalyzedbyprenyltransferasesthatcondenseHGAwith either PPP (tocopherols) or GGPP (tocotrienols).
depending on the selected gene, the plant organ or species, and the study. Overexpression of HPT genes incanolaandsoybeandidnotsignificantlyincreaseseed tocopherolcontent[15].InArabidopsis,HPT overexpres-sion increased seed tocopherols from 40% to 100% depending onthe study, indicating that this activity is atleastpartiallylimitinginArabidopsisseeds[15,41,42]. In leaves, HPT overexpression induced a 3.6 fold and 4.4 fold increase of tocopherol content in tomato and Arabidopsis, respectively [42,43]. Together, these indi-catethat,whiletheHPTactivityisnotthemajor bottle-neck in seed tocopherol metabolism, it clearly restricts tocopherol synthesisinleaves.
OverexpressionofHGGTgenesincreasedtocochromanol contentupto5timesintobaccoleaves,upto15timesin Arabidopsisleaves,and7–18timesinmaizekernels[44– 46]. In agreement with the preference of HGGT for GGPP, newly synthesized tocochromanols were exclu-sively tocotrienols. In Arabidopsis and tobacco leaves overexpressingHGGT,tocopherolcontentwasnotaltered despitethemassiveaccumulationoftocotrienols[44,46]. Since tocopherol and tocotrienol biosynthetic pathways both share HGA as a common precursor, these data indicatethatArabidopsisandtobaccoleavescansustain tocochromanol synthesisup to 15 and 5 times the WT tocochromanol levels, respectively, withoutHGA being limiting. In transgenic maize kernels the situation is slightly different. While maize HGGT overexpressors containing 7 times higher tocotrienol content showed unchanged tocopherol amounts, lines accumulating 18 times more tocotrienols had an 18% reduction in tocopherols [44,45].Theseindicatethatinmaize, HGA isnotlimitingup toacertainenhancementof tocochro-manol metabolism, but does become limiting athigher levels. In addition, these data also show that HGGT activity is clearly limiting tocotrienol biosynthesis in maize kernels.
TransgenicapproachestargetingHGAavailability
SinceHGAfeedingimprovedtocopherolcontentinsome studies, transgenic approaches aiming at increasing
(Figure2Legend)Biosyntheticpathwaysinvolvedintocochromanolbiosynthesis.Biosyntheticpathwaysformingthetocochromanolprecursors
homogentisicacid(HGA,a),geranylgeranylpyrophosphate(GGPP,b),phytylpyrophosphate(PPP)andsolanesylpyrophosphate(SPP,c),aswell
asprenylquinolsandtocochromanols(e).ThechemicalstructuresofHGA,GGPP,PPPandSPPareshown(d).Compoundabbreviations:
DMAPP,dimethylallylpyrophosphate;DMGGBQ:2,3-dimethyl-6-geranylgeranyl-1,4-benzoquinol;DMPBQ,2,3-dimethyl-6-phytyl-1,4-benzoquinol;
G3P,D-glyceraldehyde-3-phosphate;HPP,4-hydroxyphenylpyruvate;IPP,isopentenylpyrophosphate;MGGBQ,
2-methyl-6-geranylgeranyl-1,4-benzoquinol;MPBQ,2-methyl-6-phytyl-1,4-benzoquinol;MSBQ,2-methyl-6-solanesyl-1,4-benzoquinol;PC-8,plastochromanol-8;PQ-9,
plastoquinol-9;SAM:S-adenosyl-methionine;Tyr,tyrosine.Biosyntheticenzymeabbreviations:DXS,1-deoxy-D-xylulose-5-phosphatesynthase;
HGGT,homogentisicacidgeranylgeranyltransferase;HGO,homogentisicaciddioxygenase;HPPD,p-hydroxyphenylpyruvatedioxygenase;HPT,
homogentisicacidphytyltransferase(VTE2);HST,homogentisicacidsolanesyltransferase;G4,chlorophyllsynthase;GGPPS11,geranylgeranyl
pyrophosphatesynthase11;GGR,geranylgeranylreductase;g-TMT,g-tocopherolmethyltransferase(VTE4);LIL3,lightharvesting-likeprotein3;
MT,methyltransferase(VTE3);SPS,solanesylpyrophosphatesynthase;TAT7,tyrosineaminotransferase7;TC,tocopherolcyclase(VTE1);TyrA,
bifunctionalchorismatemutase/prephenatedehydrogenase;VTE5,phytolkinase;VTE6,phytylphosphatekinase.ThedirectsynthesisofHPPfrom
chorismateand/orprephenateviatheprokaryoticTyrAenzyme(greyarrow)existsincyanobacteriabutnotinhigherplants.
synthesisofthisprecursorinplantahavebeenassessed. Initial attemptsto increase HGA synthesisconsisted in overexpressing HPPD genes (Figure 2a). While HPPD overexpressionslightlyincreasedtocopheroland tocotrie-nolcontentsinseedsoftransgenictobacco[47],itdidnot substantiallyincreasetocochromanolsin tobaccoor Ara-bidopsis leaves, or in Arabidopsis and soybean seeds [15,47–50].Incontrast,inthecyanobacterium Synechocys-tis,HPPDoverexpressionresultedinasevenfoldincrease in tocopherols [15]. The contrasting results of HPPD overexpressionin higherplantsandphotosynthetic bac-terialikelyresultsfromthedifferenceinHPP biosynthe-sisbetweentheseorganisms.While(cyano)bacteriaand yeastproduceHPPdirectlyfromchorismateand/or pre-phenateviaabifunctionalchorismatemutase/prephenate dehydrogenase,itsbiosynthesisinhigherplantsrequires severalenzymaticreactionsinvolvingtyrosineasan inter-mediate [15,16]. Since tyrosine levels are tightly regu-latedin plant cells via feedback inhibition by tyrosine, plant tocochromanol biosynthesis may be restricted by tyrosine availability, whereas photosynthetic bacteria escape this limitation [13,16]. This hypothesis was assessedintransgenicplantsco-expressingHPPDgenes together with bacterial or yeast prephenate dehydro-genases (TyrA and Tyr1, respectively). In Arabidopsis and tobacco leaves, overexpression of HPPD alone did notalter tocopherol contents but itsco-expression with TyrA or Tyr1 induced atwofold and 10-foldincrease in tocochromanols,respectively[48,50].Thisindicatesthat HGAavailabilitylimitstocochromanolsynthesisinleaves ofthesetwospecies.Inseeds,co-expressionofHPPDand TyrA increased tocochromanol amounts in Arabidopsis (+80%),canola(+140%),andsoybean(+160%),indicating thatHGAavailabilityalsopartiallyrestricts tocochroma-nol biosynthesis in seeds [15]. Importantly, newly syn-thesized tocochromanols in plants co-expressing HPPD and TyrA genes were mostly tocotrienols, including in species or tissues that usually do not accumulate them [15,48,50].ThisindicatesthatHGAavailabilityrestricts tocotrienolsynthesisbutisnotsufficientpersetoenhance tocopherolsynthesis.
InArabidopsisandsoybeanseeds,co-expressionofHPPD and TyrAincreased freeHGA content 60-foldand 800-fold,respectively[15].Sincenewly synthesizedHGAis onlypartiallyconvertedintotocochromanols,thesedata collectivelyshowthatHGAavailability isoneofseveral limitations that restrict tocochromanol biosynthesis in plants.Thiswasfurtherdemonstratedbyoverexpressing additionalvitaminEbiosyntheticgene(s)intohigh-HGA transgenicplants[15].Forinstance,HPToverexpression inhigh-HGAArabidopsisandsoybeanfurtherincreased tocochromanol synthesis[15]. The highest tocochroma-nol increase (15-fold) was reported for high-HGA soy-beans in which both HPT and GGR were further over-expressed[15].Intheselatterexamples,newlyproduced tocochromanolsweremostlytocotrienolsconfirmingthat
seed tocopherol biosynthesis is not limited in the first placebyHGAavailability.
AnovelmechanismlimitingHGAavailabilityand toco-chromanolmetabolismhasbeenrecentlyidentifiedinthe soybean MO12 mutant [51]. This deletion mutant notablylacksHGO1,ageneencodinganHGA dioxygen-asethatcatalyzesthedegradationofHGAinto 4-maley-lacetoacetate(Figure2a).SincefreeHGAlevelspartially control tocochromanol biosynthesis, its degradation by HGA dioxygenase might potentially limit vitamin E biosynthesis. Indeed, MO12 mutant seeds contained 30timeshigherHGAamountsandtwiceas much toco-chromanols [51]. As for high-HGA transgenic plants, tocotrienol content was strongly enhanced in MO12 mutant seeds, while tocopherols were unchanged. Col-lectively these data demonstrate that soybean HGA dioxygenase restricts tocochromanol synthesis in seeds bydegradingHGA.Inaddition,itfurtherdemonstrates thatincreasingonly HGAavailability isnotsufficientto boosttocopherolsynthesisin seeds.
Transgenicapproachestargetingtheisoprenoidsidechain synthesis
Several transgenic approaches targeting PPP synthesis have been assessed in plants. It has been shown that Arabidopsislinesoverexpressingthephytolkinasegene (VTE5) accumulated wild-type tocopherol amounts in transgenicseeds[20].Inaddition,Arabidopsistransgenic lines overexpressing the phytyl phosphate kinase gene (VTE6) accumulatedat best 15% more g-tocopherol in seeds[21].Together,thesedatashowthatthe conver-sionofphytolintoPPPisnotasignificantbottleneckin vitaminEsynthesis,atleastin Arabidopsisseeds. Becausephytolhasbeenshowntobelimiting,thesedata suggestthatpathway(s)involvedinGGPPsynthesis,and/ orchlorophylldegradation,and/orthereductionofGGPP intoPPPmightregulateisoprenoidavailabilityinplants, andthereforetocochromanolsynthesis.Thefirst hypoth-esishasbeenpartiallydemonstratedintransgenic Arabi-dopsisinwhichoverexpressionofthe1-deoxy-D-xylulose
5-phosphate synthase (DXS), the first enzyme of the MEP pathway (Figure 2b), doubled tocopherol content in transgenicArabidopsisseedlings[52].Itwaslatershown thatDXSoverexpressioninmatureArabidopsisleavesdid notsignificantlyalterisoprenoidcontents,indicatingthat DXSactivitymightbelimitingonlyinyoungtissues[53]. A second mechanism controlling PPP availability has been identified in tobacco in which the constitutive expressionof CHLP,ageneencodingaGGR,induced asixfoldincreaseoftocopherolsinleavesandathreefold increase in seeds (Patent US 6,624,342 B1). To date, theseamountsarethehighesttocopherolincreases ever reportedinbothseedsandleaves,indicatingthat reduc-tionofGGPPintoPPPislikelythemostlimitingstepof
mentwiththeresultsofthehigh-HGAtransgenicplants overexpressingbothHPPDand TyrA orTyr1 that accu-mulated mainly unsaturated tocochromanols (tocotrie-nols) rather than saturated ones (tocopherols)
[15,48,50]. However, these data still do not explain
why high-HGA transgenic soybeans expressing GGR overaccumulatedtocotrienolsinsteadoftocopherols[15].
Conclusions
&
future
challenges
Duringthelasttwodecades,verysignificantprogresshas beenmadeonourunderstandingofvitaminE biosynthe-sisinphotosyntheticorganismsandatotalof19different genes that impact or are required for tocochromanol synthesishavebeenidentifiedinplants(Figure2). Sev-eralstrategieshavebeenundertakentoimprovevitamin Econtent in crops.To date, theoverexpression of the g-TMTgeneisbyfarthemostpotent methodavailable. Regardless of the host or the transgene origin, g-TMT overexpressorsandnaturallyelevatedexpressioninhigh a-tocopherol accessions exhibit the highest vitamin E increasesthusfarreportedinplants.
Thenumerousmetabolicengineeringstudiespublished sofargreatlyimprovedourunderstandingof both toco-trienolandtocopherolmetabolismsinplants.Inspecies naturally producing tocotrienols such as monocot seed, tocotrienolcontentisstronglyrestrictedbyHGGT expres-sion. In contrast, tocopherol content is not strongly affected by HPT overexpression in seeds. In addition, the massive accumulation of tocotrienols rather than tocopherols in transgenic plants overaccumulating free HGAoroverexpressingHGGTdemonstratedthat tocoph-erolsynthesisisnotprimarilyregulatedbytheavailability offreeHGA.Sincephytylpyrophosphatemostlycomes from the degradation and recycling of chlorophylls, at leastin senescent leavesand seedsaccumulating chlor-ophyllssuchas Arabidopsis,it suggests thatidentifying thegenesandregulatorymechanism(s)involvedin chlo-rophyll turnover will likely open up new horizons for vitamin E biofortification of crops. This new frontier might be challenging since chlorophylls are essential forplantsurvivalandmetabolismandtheirbiosynthesis andturnoverare bothtightlyregulatedin plant tissues. Thisalsoquestionstheoriginofphytol/phytyl pyrophos-phateandtocopherolsynthesisinspecieswhoseseedsdo notproduce chlorophylls.
Acknowledgements
ThisworkwassupportedbytheDepartmentofBiologyoftheUniversityof
Fribourg,Switzerland.
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