Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog
ORIGINAL ARTICLE
Tyamagondlu V.Venkatesh ?Balasulojini Karunanandaa Daniel L.Free ?Jeannie M.Rottnek ?Susan R.Baszis Henry E.Valentin
Identi?cation and characterization of an Arabidopsis homogentisate phytyltransferase paralog
Received:14July 2005/Accepted:7November 2005/Published online:12January 2006óSpringer-Verlag 2006
Abstract Tocochromanols (tocopherols and tocotrie-nols)are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition.There is considerable interest in identifying the genes involved in tocochromanol biosynthesis to allow trans-genic modi?cation of both tocochromanol levels and tocochromanol composition in agricultural crops.The ?rst committed reaction in tocopherol biosynthesis is the condensation of homogentisic acid (HGA)with phy-tyldiphosphate or geranylgeranyldiphosphate,catalyzed by the homogentisate phytyltransferase (VTE2)or by the homogentisate geranylgeranyl transferase (HGGT).In this study,we describe the identi?cation of conserved amino acid sequences within VTE2and HGGT and the application of these conserved sequences for a motif analysis resulting in the discovery of a VTE2-paralog in the Arabidopsis genome.We designated this new gene VTE2-2and renamed the old VTE2to VTE2-1.Seed-speci?c expression of VTE2-2in Arabidopsis resulted in increased seed-tocopherol levels,similar to the trans-genic expression of VTE2-1.Bioinformatics analysis revealed that VTE2-2is conserved in both monocotyle-donous and dicotyledonous plants and is distinct from VTE2-1and HGGT.
Keywords Arabidopsis ?Homogentisate
phytyltransferase ?Homogentisate prenyltransferase ?Motif analysis ?Tocopherol ?Vitamin E
Abbreviations CTP:Chloroplast target peptide ?GGDP:Geranylgeranyldiphosphate ?HGA:Homogentisic acid ?HGGT:Homogentisate
geranylgeranyltransferase ?HMM:Hidden Markov Model ?MPSS:Massively Parallel Signature
Sequencing ?TNF:Tumor necrosis factor ?PDP:Phytyldiphosphate ?PUFA:Polyunsaturated fatty
acids ?VTE2:Homogentisate phytyltransferase ?VTE3:2-methyl-6-phytylbenzoquinol methyltransferase
Introduction
Tocopherols and tocotrienols function as the principle lipid-soluble oxidation chain-breaking antioxidants in biological membranes and lipoproteins (Liebler 1998;
Brigelius-Flohe
et al.2002).In contrast to cytoplasmatic systems,such as the glutathione redox cycle,or the superoxide dismutase,which depend on enzymatic inactivation of oxygen radicals,the antioxidant reac-tions of tocochromanols do not require involvement of enzymes.The physiological role of tocopherols and tocotrienols is thought to be the protection of polyun-saturated fatty acids (PUFA)from lipid oxidation by quenching free radicals in cell membranes and other lipophilic environments (Kamal-Eldin and Appelqvist 1996).Tocopherols and tocotrienols occur in four major isoforms each:a -,b -,c -,and d -tocopherol,and a -,b -,c -,and d -tocotrienol (Pongracz et al.1995;Fig.1).Tocotrienols are distinguished from tocopherols by the presence of three double bonds in the isoprenoid side chain,and greek letters refer to the grade of methylation on the aromatic head group,with a -tocopherol or a -tocotrienol being the highest methylated isoforms
Electronic Supplementary Material Supplementary material is available for this article at https://www.wendangku.net/doc/679302715.html,/10.1007/s00425-005-0180-1and is accessible for authorized users.
Tyamagondlu V.Venkatesh,and Balasulojini Karunanandaa have equally contributed.
T.V.Venkatesh ?B.Karunanandaa
Monsanto Company,800N.Lindbergh Boulevard,St.Louis,MO 63167,USA
D.L.Free ?J.M.Rottnek ?S.R.Baszis
Monsanto Company,700Chester?eld Parkway West,Chester?eld,MO 63017,USA
H.E.Valentin (&)
Monsanto Company,Calgene Campus,1920Fifth Street,Davis,CA 95616,USA
E-mail:henry.e.valentin@https://www.wendangku.net/doc/679302715.html, Tel.:+1-530-7922136Fax:+1-530-7922454
Planta (2006)223:1134–1144DOI 10.1007/s00425-005-0180-1
(Fig.1).Of these,a -tocopherol has the highest vitamin E activity in animals and humans (Sheppard et al.1993;Bramley et al.2000),presumably due to its preferred retention and distribution throughout the mammalian body (Traber and Sies 1996).
The tocochromanol biosynthesis begins with the prenylation of homogentisic acid (HGA)with phy-tyldiphosphate (PDP)catalyzed by the homogentisate phytyltransferase (VTE2;Collakova and DellaPenna 2001;Schledz et al.2001;Savidge et al.2002),or by prenylation of HGA with geranylgeranyldiphosphate (GGDP)catalyzed by the homogentisate geranylgera-nyl transferase (HGGT;Cahoon et al.2003).HGA and PDP plus GGDP are derived from the shikimate pathway (Norris et al.1998)and the methylerythritol-phosphate pathway (Rohmer 2003),respectively (Fig.1).The reaction products of this ?rst committed step,2-methyl-6-phytylbenzoquinol,or 2-methyl-6-ger-anylgeranylbenzoquinol are subsequently cyclized and methylated resulting in the formation of various tocopherol isoforms (Fig.1).Previous evidence suggests that the ?rst committed reaction in tocopherol biosynthesis regulates tocopherol ?ux and to some extent tocopherol composition (Savidge et al.2002;Cahoon et al.2003;Collakova and DellaP-enna 2003a ,b ;Karunanandaa et al.2005).VTE2has for these reasons been identi?ed to be critical for tocopherol biosynthesis pathway engineering.Interestingly,VTE2was discovered via bioinformatics approaches in parallel by two di?erent groups (Collakova and DellaPenna 2001;Savidge et al.2002).In this study,we report on further bioinformatics characterization of homogentisate phytyltransferases leading to the identi?cation of four conserved sequence motifs and on the application of two of these motifs for database searches to speci?cally search homogentisate prenyltransferase sequences.Application of these motifs for additional database searches identi?ed a VTE2paralog in the Arabidopsis genome.This new VTE2paralog was named VTE2-2and the previously known VTE2was renamed VTE2-1.Transgenic overexpression of this new gene supports a function of this new gene in tocopherol
biosynthesis.
Fig.1Homogentisate prenyltransferase reaction in context of the tocopherol biosynthetic pathway.Tocopherols and tocotrienols with major isoforms (a )and schematic drawing of the homogentisate prenyltransferase reaction (b ).MEP methylerythritol phosphate
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Material and methods
Bioinformatics analysis
BLAST2.2algorithms(Altschul et al.1997)and the non-redundant amino acid database were downloaded from NCBI(https://www.wendangku.net/doc/679302715.html,).Pro?le Hidden Markov Models(HMMs)for the motifs de-scribed were built and pro?le HMM searches using these motifs were performed using HMMER2.0software as described in the users manual(https://www.wendangku.net/doc/679302715.html,/). Gene prediction software FGENESH was procured from Softberry Inc.,NY,USA.Dicot model provided with this software was used to predict coding regions from Arabidopsis genomic DNA sequences.Multiple sequence alignments using ClustalX(Thompson et al. 1997)were performed as described in the user’s manual (http://www.igbmc.u-strasbg.fr/BioInfo/ClustalX/Top. html).Multiple sequence alignments were visualized and edited using GeneDoc(Nicholas et al.1997).The resi-dues were shaded using conserved residue shading mode set to level4using default settings.Similar amino acid residues conserved in all columns were shaded dark. Phylogenetic analysis was conducted using MEGA ver-sion2.1(Kumar et al.2001).Separate neighbor-joining trees were generated using both p-distance and gamma distance amino acid substitution models.Both pair wise and complete deletion approaches for handling gaps or missing data were applied separately for each model. The trees were tested by bootstrapping,performing500 replications.Subsequently,the trees were rooted for the Chloro?exus aurantiacus VTE2.
For further functional characterization of VTE2-2 the expression patterns of VTE2-1and VTE2-2genes were analyzed in the Arabidopsis Massively Parallel Signature Sequencing(MPSS)data set available at https://www.wendangku.net/doc/679302715.html,/at/(Meyers et al.2004).MPSS produces short sequence signatures chosen from a de-?ned position within an mRNA,and the relative abun-dance of these signatures in a given library represents a quantitative estimate of expression of that gene.The MPSS signatures are17bp in length,and can uniquely identify>95%of all genes in Arabidopsis. Arabidopsis transformation
Using standard cloning techniques the VTE2-2full-length cDNA was excised from an in house EST clone (pMON69960)to generate binary vectors for sense and antisense expression under the control of the seed-spe-ci?c Napin promoter(Kridl et al.1991)and the con-stitutive e35S promoter(McPherson and Kay1994)as described(Valentin et al.2003).The plant binary vec-tors,pMON69963,and pMON69965were used to transform Arabidopsis thaliana Col-0for seed-speci?c sense and antisense expression of At-VTE2-2,respec-tively.Binary vectors,pMON69964and pMON69966were used for constitutive sense and antisense expression of VTE2-2,respectively.The binary vectors were trans-formed into Agrobacterium tumefaciens strain ABI by electroporation(Bio-Rad Electroprotocol Manual, Dower et al.1988).Transgenic Arabidopsis plants were obtained by Agrobacterium-mediated transformation as described(Valvekens et al.1988;Bechtold et al.1993; Bent et al.1994).Transgenic plants were selected by sprinkling the transformed seed onto selection plates containing MS basal salts(4.3g/l),Gamborg’s B-5, 500·(2.0g/l),sucrose(10g/l),Mes(0.5g/l),phytagar (8g/l),carbenicillin(250mg/l),cefotaxime(100mg/l), plant preservation medium(2ml/l),and kanamycin (60mg/l)and then vernalizing them at4°C in the ab-sence of light for2–4days.Subsequently,the seed were transferred to23°C,and16/8h light/dark cycle for5–10days until seedlings emerged.Once,one set of true leaves were formed on the kanamycin resistant seedlings, plantlets were transferred to soil and grown to maturity. Transgenic lines generated through kanamycin selection were grown under16h light and8h dark. Tocochromanol analysis
The tocochromanol content was analyzed on R2 Arabidopsis seed as described by Savidge et al.(2002). Northern analysis
RNA was isolated from100mg fully matured but not dried,Arabidopsis siliques.Tissue samples were ground with0.25g polyvinylpolypyrrolidone in liquid nitrogen. While the ground tissues were frozen,10ml REC.8+ bu?er containing50mM Tris–HCl(pH9.0),800mM NaCl,10mM EDTA,0.5%CTAB and0.5%b-mer-captoethanol was added to each sample,mixed and centrifuged for5min at18,000g in a Sorvall centrifuge, model Super T21.The supernatant was?ltered through miracloth(Calbiochem)onto3ml chloroform,mixed and centrifuged again at18,000g for5min.The super-natant was separated and extracted twice with an equal volume of phenol:chloroform mix(1:1,v/v)and then ethanol precipitated.The ethanol pellet was re-sus-pended in50mM EDTA.Equal amounts of total RNA (20l g)were fractionated on1.2%agarose gels con-taining1M formaldehyde.Gels were blotted onto nylon membranes(Ambion)according to the manufacturer’s instructions and hybridized in Sigma PerfectHyb TM bu?er,containing10mg/ml of salmon sperm DNA at 65°C.A cDNA fragment(Xho I/Bam HI)of the Ara-bidopsis VTE2-2gene was labeled and used as a gene-speci?c probe.Probe labeling with32P-dCTP (1.85MBq)was performed using Amersham Redi-prime TM labeling kit(Amersham Biosciences Corp.). Blots were washed in2·SSC,0.1%SDS at65°C and exposed to X-ray?lm(BioMax MS?lm,Sigma).After detection of VTE2-2mRNA the blot was stripped with
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0.1·SSC containing0.1%SDS to remove the radiola-belled VTE2-2probe.Subsequently the blot was again pre-hybridized and hybridized with a rDNA radiola-belled probe using the same methodology described above.After2h hybridization with this probe,the blot was washed and exposed to X-ray?lm.
Results
Bioinformatic analysis
Motif analysis I:Arabidopsis thaliana and Synechocystis sp.PCC6803VTE2genes(At-VTE2,and Sy-VTE2, respectively)have been described previously(Collakova and DellaPenna2001;Schledz et al.2001;Savidge et al. 2002).Using the deduced amino acid sequence of Sy-VTE2,the Nostoc and Anabaena VTE-genes were iden-ti?ed from GenBank(gi|17230940,and gi|46135490, respectively)based on sequence homology.In addition, sequences homolog to the At-VTE2and Sy-VTE2de-duced amino acid sequences were obtained from soy-bean(Glycine max),leek(Allium porrum)and Cuphea pulcherrima cDNA libraries(GenBank accession no. DQ231059,DQ231057,and DQ231058,respectively). The deduced VTE2amino acid sequences from Arabid-opsis,leek,soybean,Cuphea pulcherrima,Synechocystis, Nostoc,and Anabaena were aligned and four motifs conserved in all VTE2sequences were identi?ed(Fig.S1 in supplemental material).These motifs have been numbered1.1,2.1,3.1and4.1,respectively.They cor-respond to amino acids99–112,167–174,285–298,and 371–390of At-VTE2-1respectively.
We used Hidden Markov Model(HMM)pro?le techniques(Eddy1998)to test the speci?city and sensi-tivity of these motifs.Pro?le HMMs are statistical models of multiple sequence alignments.They capture position-speci?c information about how conserved each column of the alignment is,and which residues are likely to be sensitive in identifying proteins sharing homology to a conserved domain of interest.Pro?le HMM models built from multiple alignments of these motifs were used to search all sequences of the NCBI non-redundant amino acid database available on March2003.Motifs 1.1through4.1identi?ed all previously known deduced VTE2amino acid sequences present in the database at high speci?city.Surprisingly,this search identi?ed two new genomic Arabidopsis VTE2variants representing contradicting predicted protein sequences from the same area of genomic Arabidopsis DNA.When both of these sequences(gi15229898and gi10998133)were used to search the non-redundant amino acid database,the BLAST search results indicated that these sequences were most related to VTE2sequences from cyanobac-teria and Arabidopsis VTE2-1(from here on refered to as VTE2-1).However,gi15229898appeared to encode a much larger protein of970amino acids,which was homologous to VTE2-1over its carboxy terminal half.A blast search using the561amino-terminal deduced amino acids of gi15229898that were not homologues to VTE2-1,identi?ed homology to deduced amino acid sequences from genes of unknown function from Musa acuminate(gi|40850572,53%identity over310amino acids),Oryza sativa(gi|50930768,44%identity over335 amino acids),Zea mays(gi|54652075,41%identity over 224amino acids),and TNF receptor-associated factor5 from Gallus gallus(gi5|4020685,27%identity over179 amino acids).PFAM(https://www.wendangku.net/doc/679302715.html,/Soft-ware/Pfam/index.shtml)analysis of this region identi?ed a conserved TRAF-type zinc?nger(Zf-TRAF)domain. Mammalian signal transducers associated with the cytoplasmic domain of the75kDa tumor necrosis factor (TNF)receptor are known to contain this domain.The presence of a putative Zf-TRAF domain may therefore indicate a regulatory function of the gi|15229898amino terminal sequence.Analysis of plant EST databases indicated that the deduced amino terminal sequence of gi|15229898that was not homologous to VTE2-1may code for a gene of functional signi?cance which is con-served in plants.EST sequences of this gene were iden-ti?ed in multiple plant species including Arabidopsis thaliana,Brassica napus,Zea mays and Triticum aes-tivum(data not shown).However,all cDNA sequences with homology to gi|15229898lacked the carboxyter-minal region that contained sequence similarities to VTE2,suggesting that the N-terminal region of gi|15229898is transcribed independent from the se-quence that was homolog to VTE2-1.
Gi|10998133encoded a441amino acid protein that was highly similar to VTE2-1but it appeared to miss approximately40–50amino acids from its carboxy ter-minus.Gene prediction algorithms are often mistaken in predicting terminal exons(Zhang2002).Such predicted proteins are often found to have misannotated N-ter-minal and/or C-terminal sequences and require further veri?cation.In order to verify the GenBank sequence prediction,the nucleotide sequence of a BAC clone that corresponded to the Arabidopsis genomic sequence that contained VTE2-motifs(gi|12408742|gb|A-C016795.6|ATAC016795,10,0835bp)was analyzed, and its coding sequences were predicted using the FGENESH gene prediction program(Solovyev2001). FGENESH identi?ed28coding regions on this BAC clone(data not shown).To identify new homogentisate phytyltransferase proteins among these28sequences,all 28predicted amino acid sequences were searched against the non-redundant amino acid database using BLAST tool(Altschul et al.1997).Predicted protein no.25(base 80,571to83,410on the BAC clone),with a length of402 amino acids was most similar to gi10998133(441amino acids),the carboxyl terminal half of gi15229898(970aa) and other deduced VTE2amino acid sequences.The deduced amino acid sequence of protein no.25aligned well with other VTE2s over the entire length of the protein(data not shown).To provide functional and transcriptional evidence and to con?rm the coding se-quence for this gene,plant EST sequence databases were searched.Several ESTs from Arabidopsis matched the
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amino terminal and carboxy terminal portions of this gene(data not shown).ESTs with signi?cant similarity were also identi?ed from other plant species(data not shown).The new gene was named VTE2-2(GenBank accession no.DQ231060)and previously known VTE2s from plant sources were renamed VTE2-1.The deduced At-VTE2-2amino acid sequence is quite distinct from At-VTE2-1and is about32%identical(Figs.2and3). Similar as all plant derived VTE2-1sequences repre-sented in Fig.2,the deduced amino acid sequences of all VTE2-2represented in Fig.2contain a predicted chlo-roplast target peptide(CTP)according to ChloroP (Emanuelsson et al.1998).
Motif analysis II:Additional EST database searches using the previously identi?ed motifs revealed that At-VTE2-2orthologs were present in soybean and rice as well.These newly identi?ed VTE2-2sequences were cloned and their sequences included in the multiple se-quence alignment used in the previous motif analysis,for further motif optimization.The resulting new motifs were designated 1.2, 2.2, 3.2,and 4.2(Fig.4)corre-sponding to motifs1.1,1.2,3.1and4.1from previous motif analysis(Fig.S1).Pro?le HMM models were built and HMM searches were performed using the optimized motifs on the NCBI non-redundant amino acid data-base,containing more than2.1million sequences(Nov. 2004).The results of this search are shown in Table1. All four motifs identi?ed all previously known hom-gentisate phytyltransferase genes from plants and cy-anobacteria found in the database.In addition to VTE2-1and VTE2-2sequences,these motifs also identi?ed the recently identi?ed HGGT sequences from monocotyle-don plants(Cahoon et al.2003).Motifs3.2and4.2were found to be highly speci?c to VTE2type amino acid sequences as evident by lower E values.Motif4.2also identi?ed a VTE2related sequences from the photo-synthetic bacterium Chloro?exus aurantiacus.Motif1.2 is also speci?c to VTE2type genes;however,its sensi-tivity for HGGT sequences is low as indicated by higher E values.Motif2.2identi?ed?ve bacterial ubiA pre-nyltransferase sequences in addition to VTE2s and HGGT s as it spans the conserved catalytic domain
of Fig.2Neighbour-joining tree of homogentisate prenyltransferas-
es.The tree has been rooted for Chloro?exus aurantiacus(Ca)
VTE2.Branch length calculated based on amino acid substitu-
tions per site is shown below(0.05=5%di?erence between
sequences).Blank squares VTE2-1sequences from plants,blank
diamonds,VTE2-2sequences from plants,?lled squares HGGT
sequences from plants,?lled triangles VTE2sequences from
cyanobacteria,blank circle VTE2from photobacterium.Abbre-
viations:An Anabaena,Ap Allium porrum,At Arabidopsis
thaliana,Cp Cuphea pulcherrima,Cw,Crocosphaera watsonii,
Gm Glycine max,Gv Gloeobacter violaceus,Ms Medicago sativa,
No Nostoc,Os Oryza sativa,Sy Synechocystis,Ta Triticum
aestivum,Te Trichodesmium erythraeum,Zm Zea mays.Refer-
ence numbers:An-VTE2,gi|46135490;Ap-VTE2-1,accession no.
DQ231057;At-VTE2-1,gi|21281072;At-VTE2-2,accession no.
DQ231060;Ca-VTE2,gi|53795310;Cp-VTE2-1,accession
no.DQ231058;Cw-VTE2,gi|45525087;Gm-VTE2-1,accession
no.DQ231059;Gm-VTE2-2,accession no.DQ231061;Gv-VTE2,
gi|37519852;Hv-HGGT,gi|33391138;Ms-VTE2-1,gi|51949754;
No-VTE2,gi|17230940;Os-HGGT,gi|33391144;Os-VTE2-1,
gi|51536170;Os-VTE2-2,gi|50938601;Sy-VTE2,gi|16330366;
Ta-HGGT,gi|33391142;Ta-VTE2-1,accession no.DQ231056;
Te-VTE2,gi|48893591;Zm-VTE2-1,accession no.DQ231055 1138
prenyltransferases (Collakova and DellaPenna 2001).However,the E -values for ubiA sequences were higher by several orders of magnitude,indicating a lower se-quence homology and greater sequence divergence of tocochromanol related prenyltransferases from ubiA type
prenyltransferases.
Fig.3Multiple alignment of selected homogentisate prenyltransferases.Motif 1,2,3,and 4are labeled with a bold line.Species abbreviations At Arabidopsis thaliana,Hv Hordeum vulgare,Sy Synechocystis,Ca Chloro?exus aurantiacus
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Fig.4Optimized VTE-2sequence motifs identi?ed based on multiple alignment of VTE2sequences.Numbers on the right border represent the number of amino acid residues in each motif. Consensus sequences are shown for each motif.Abbreviations:An Anabaena,Ap Allium porrum,At Arabidopsis thaliana,Cp Cuphea pulcherrima,Gm Glycine max,No Nostoc,Sy Synechocystis,Ta Triticum aestivum,Zm Zea mays
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Phylogenetic analysis
Four neighbor-joining trees were generated using two di?erent amino acid substitution models(see Materials and methods)and two di?erent approaches for handling gaps or missing data.All four neighbor-joining trees clustered the homogentisate phytyltransferases in four clades.One representing plant VTE2-1sequences,a separate clade representing plant VTE2-2sequences,a third representing HGGT sequences,and a fourth clade representing bacterial homogentisate phytyltransferases.
A tree generated using p distance and complete deletion model is shown in Fig.2.The topology of the tree suggests that the bacteria and cyanobacteria harbor only one homogentisate phytyl transferase gene.Dicotyle-donous and monocotyledonous plants harbor two and three homogentisate prenyltransferase genes,respec-tively.
Expression pattern of VTE2-1and VTE2-2
in Arabidopsis
For characterization of the functional signi?cance of VTE2-2the Arabidopsis expression patterns of the two VTE2-paralogs were characterized in silico.While the frequency of VTE2-1and VTE2-2hits in public and proprietary cDNA-libraries from?ower buds,seed,si-lique,roots and leaves suggested a similar expression pattern of these two genes(data not shown),a search in the more quantitative Arabidopsis MPSS data set (https://www.wendangku.net/doc/679302715.html,/at/)revealed a preferred
Table1HMM search of non-redundant NCBI amino acid databases
Gi No.Species Gene Length Motif1.2Motif2.2Motif3.2Motif4.2Comments 30681973Arabidopsis thaliana VTE2-29540.072 2.9E-110.0119.2E-09N-terminal sequence
misannotated 50938601Oryza sativa(japonica
cultivar-group)
VTE2-23790.072 1.4E-110.0113E-08
51963498Oryza sativa(japonica
cultivar-group)VTE2-21040.072Incomplete sequence,not
detected by blast search
10998133Arabidopsis thaliana VTE2-24410.072 2.9E-110.011C-terminal sequence
misannotated 21281072Arabidopsis thaliana VTE2-13930.031 1.1E-120.00547.9E-09
3004556Arabidopsis thaliana VTE2-1210 1.1E-12Incomplete sequence 51536164Oryza sativa(japonica
cultivar-group)
VTE2-1188 6.20E-07Incomplete sequence
51536170Oryza sativa(japonica
cultivar-group)VTE2-1402 1.3E-120.0026 3.6E-09N-terminal sequence
misannotated
51949754Medicago sativa VTE2-14110.031 1.6E-120.0054 1.1E-07
16330366Synechocystis sp.
PCC6803
VTE23080.011 4.4E-120.00268E-08
17230940Nostoc sp.PCC7120VTE23180.0137.2E-130.0017 2.5E-08
23125851Nostoc punctiforme
PCC73102
VTE23220.013 1.9E-120.0021 5.4E-08
37519852Gloeobacter violaceus
PCC7421
VTE22980.56 1.3E-110.00178.80E-06
45525087Crocosphaera
watsonii WH8501
VTE23150.17 2.5E-110.0299.2E-08
46135490Anabaena variabilis
ATCC29413
VTE23180.0137.2E-130.0017 2.5E-08
48893591Trichodesmium
erythraeum IMS101
VTE23490.046 3.5E-120.0021 1.5E-07
53795310Chloro?exus aurantiacus VTE2300 5.8E-097.80.024
33391138Hordeum vulgare HGGT408 3.5 5.2E-110.0093 1.70E-06
33391142Triticum aestivum HGGT4082 5.2E-110.0093 1.70E-06
33391144Oryza sativa(japonica
cultivar-group)
HGGT4040.62 1.3E-110.0095 1.50E-06
51535642Oryza sativa(japonica
cultivar-group)
HGGT270 1.3E-110.0095 1.50E-06Short Sequence
15921742Sulfolobus tokodaii str.7ubiA0.06
15897501Sulfolobus solfataricus P2ubiA0.12
41719117Methanococcoides
burtonii DSM6242
ubiA0.17
15668454Methanocaldococcus
jannaschii DSM2661
ubiA0.17
45359240Methanococcus
maripaludis S2
ubiA 2.1
Numbers in motif columns indicate E values
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expression of VTE2-1in in?orescence tissue,and a preferred expression of VTE2-2in leaf (Fig.5).Expression levels in callus,root and silique tissues were found to be similar.
Overexpression and antisense of VTE2-2in Arabidopsis Expression of At-VTE2-2under napin promoter control resulted in 38%increased seed tocopherol levels in the R2seed population of all events (Table 2).This increase is slightly less than the increase obtained with VTE2-1using the same promoter and 3￠-UTR (Savidge et al.2002),but it represents a signi?cant change in total seed tocopherol content.Constitutive expression of VTE2-2under e35S promoter control resulted in a signi?cant tocopherol increase of 13%compared to the vector control (Table 2).b -Tocopherol was not detected in any of the VTE2-2over expressing lines,and c -tocotrienol was found only in trace amounts (￡7ppm)of some samples harboring the napin driven expression construct for VTE2-2(Table 2).Mean tocopherol levels of all events in Arabidopsis seed harboring seed-speci?c and constitutive VTE2-2antisense constructs were not sig-ni?cantly di?erent from tocopherol levels found in control seed populations.However,these constructs represented simple antisense constructs,which are fre-quently found to provide only a low percentage of events that exhibit the antisense e?ect.
Expression analysis of transgenic VTE2-2expressers Siliques from seven selected R3,kanamycin resistant,transgenic lines originating from transformation with pMON69963and an empty vector control line were used for Northern analysis to detect the expression level of the VTE2-2transgene.All transgenic lines exhibited a signal migrating at a molecular mass of 1.2kb
corresponding with the expected molecular mass of the VTE2-2transgene mRNA that was not present in wild-type control preparations.Transgenic lines with low,medium and high tocopherol increase were used for this experiment.The transgene expression level in the lines analyzed correlated with the level of tocopherol increase in R2seed (Fig.6).
Discussion
In this study,we describe a motif analysis approach which identi?ed a set of conserved motifs in VTE2-1and HGGT amino acid sequences.Application of these motifs in sequence database searches resulted in the discovery of a VTE2-paralog in the Arabidopsis gen-ome.A function of this new gene in tocopherol bio-synthesis was con?rmed through over expression in Arabidopsis resulting in increased seed tocopherol lev-els.Interestingly,this new gene maps to the same loca-tion as a so far not characterized mutation on the top of chromosome 3(pds2)that caused a tocopherol de?cient phenotype (Norris et al.1995).
The conserved motifs were identi?ed through align-ment of homologous sequences.Pro?le HMMs con-structed from multiple alignments of these motifs were used in sequence database searches.In comparison to PFAM domains (https://www.wendangku.net/doc/679302715.html,)the motifs used here are small,vary in size from 12to 30amino acids,and are speci?c to a subset of proteins of a large protein family.In contrast,the corresponding PFAM motif is approximately 260amino acids long and identi?es a large group of prenyltransferases including ubiA and VTE2orthologs found in multiple taxonomic groups.Motifs 3.2and 4.2described in this paper are speci?c to VTE2and HGGT sequences found in bacteria and higher plants.While motif 2covers the catalytic domain found in other ubiA family prenyltransfrases,the func-tional signi?cance of the other conserved domains identi?ed in this study is not known.Additional studies using VTE2or HGGT genes mutagenized in the con-served regions identi?ed here may help to decipher the structural and biochemical functions of these amino acid sequences.We found this approach sensitive in identi-fying short sequences that can be easily missed by se-quence similarity search methods like BLAST.
It is interesting to note that all other tocopherol pathway genes appear to occur as single copy genes in Arabidopsis.The 2-methyl-6-phytylbenzoquinolmethyl-transferase (VTE3)even appears to carry out reactions in two di?erent metabolic pathways,the methylation of 2-methyl-6-phytylbenzoquinol for tocopherol biosyn-thesis,and the methylation of the plastoquinol precursor 2-methyl-6-solanylbenzoquinol (Cheng et al.2003;Motohashi et al.2003).In this context it may appear surprising to ?nd two VTE2paralogs in the Arabidopsis genome.However,VTE2catalyzes the ?rst committed reaction in tocopherol biosynthesis.These reactions frequently coincide with enzymes regulating the
pathway
Fig.5Expression pattern of VTE2paralogs in Arabidopsis.The expression pattern are based on analysis of the Arabidopsis MPSS data set (https://www.wendangku.net/doc/679302715.html,/at/)
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?ux.Indeed several recent publications suggest a ?ux limitation for tocopherol biosynthesis by VTE2under certain growth conditions (Savidge et al.2002;Cahoon et al.2003;Collakova and DellaPenna 2003a ,b ;Kar-unanandaa et al.2005).The occurrence of two VTE2paralogs may provide a broader ?exibility in tocopherol ?ux regulation,or it may help to separate tocopherol ?ux regulation from plastoquinol biosynthesis.This would however require di?erences in substrate speci?city for the two VTE2paralogs.A slight di?erence in the substrate spectrum of VTE2-1and VTE2-2may be suggested by the observation that some Arabidopsis seed samples expressing VTE2-2under napin promoter con-trol contained traces of c -tocotrienol (Table 2),while a similar construct expressing VTE2-1did not result in tocotrienols accumulation (Karunanandaa et al.2005).VTE2-2might therefore also help to explain the accu-mulation of tocotrienols in transgenic Arabidopsis in the presence of high HGA-levels.
Previous studies (Rippert et al.2004;Karunanandaa et al.2005)have demonstrated the accumulation of
substantial tocotrienol levels in plants co-expressing a prephenate dehydrogenase and a p-hydroxypenylpyru-vate dioxygenase.The accumulation of tocotrienols could not be explained on the basis of currently pub-lished data,as previous studies did not detect prenyl-transferase activity of the Arabidopsis VTE2-1with the tocotrienol precursor GGDP (Collakova and DellaP-enna 2001).The identi?cation of VTE2-2and the data presented in Table 2might suggest that this enzyme can utilize GGDP as substrate,and thereby solve the pre-vious dilemma on how tocotrienols were formed.Whe-ther VTE2-2has a function in plastoquinol biosynthesis or ful?lls other as yet to be identi?ed physiological functions will require additional experimentation,such as enzyme assays using a variety of di?erent substrates,or parallel complementation experiments to elucidate if the pds2mutant as described by Norris et al.(1995)can be complemented by VTE2-1or VTE2-2or both.
Analysis of the Arabidopsis MPSS data set suggested that compared to VTE2-2the VTE2-1gene is expressed at substantially higher levels in in?orescence and at lower expression levels in leaf.Further enzymatic char-acterization of both VTE2expression products with an emphasis on substrate preferences will help to decipher the physiological function of these two genes.
Acknowledgements Tyamagondlu V.Venkatesh and Balasulojini Karunanandaa have equally contributed to this work.We would like to thank Jon Schumeke and team with Taqman analysis and Jing Dong Liu for critical review of the manuscript.
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Table 2Sense and antisense expression of VTE2-2in Arabidopsis thaliana Plasmid
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