Characterization of a c-tocopherol methyltransferasemutant gene in wild (Carthamus oxyacanthus M. Bieb.)and cultivated safflower (C. tinctorius L.)

Marıa J. Garcıa-Moreno •

Jose M. Fernandez-Martınez •

Leonardo Velasco • Begona Perez-Vich

Received: 11 March 2014 / Accepted: 12 May 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Safflower (Carthamus tinctorius L.) seeds

contain a high proportion of tocopherols ([90 %) in the

a-tocopherol form. A mutant with a high concentration

of c-tocopherol ([85 %) was identified in germplasm

of wild safflower (Carthamus oxyacanthus M. Bieb.)

that showed strong introgression of C. tinctorius, which

allowed selection of individuals of both species with

high concentrations of either a- or c-tocopherol. The

trait is controlled by a c-tocopherol methyltransferase

(c-TMT) locus. The objective of this research was to

identify c-TMT sequence mutations associated with the

high c-tocopherol trait. Full length genomic and cDNA

sequences of the c-TMT gene were obtained from

plants of C. tinctorius and C. oxyacanthus with both

tocopherol profiles. Sequences from high c-tocopherol

plants showed an 11 bp deletion in exon 6 of the c-TMT

gene that disrupted the reading frame and created a

premature stop codon, resulting in a predicted protein

with a drastically altered amino acid sequence down-

stream the frameshift site. The data suggested that the

frameshift mutation was underlying the c-TMT loss

of function mutant allele that determines the high

c-tocopherol phenotype. The characterized sequence

change of 11 bp deletion could be used directly as a

functional marker for introgression of the high

c-tocopherol trait into elite safflower cultivars.

Keywords Carthamus tinctorius � Carthamus

oxyacanthus � Frameshift mutation � c-tocopherol

methyltransferase � Safflower �Wild safflower

Introduction

Tocopherols (vitamin E) are the main antioxidants in

oil seeds. They consist of a hydrophilic head group, the

chromanol ring, and an isoprenoid-derived lipophilic

side-chain (DellaPenna and Mene-Saffrane 2011).

Tocopherols occur in four forms, named as a-, b-, c-

and d-tocopherol, that differ in the number and position

of methyl substituents on the chromanol ring, which

determines their different in vivo and in vitro antiox-

idant activities. a-tocopherol exerts maximum in vivo

antioxidant activity (Chow 2000), whereas c- and d-

tocopherol have more antioxidant activity in vitro

(Yanishlieva et al. 2002; Isnardy et al. 2003; Marmesat

et al. 2008). a-tocopherol is the predominant tocoph-

erol derivative in most plant tissues except in seeds,

where c-tocopherol is the predominant form (DellaP-

enna and Pogson 2006). Sunflower and safflower are

notable exceptions in which a-tocopherol is also

predominant in the seeds (Padley et al. 2004).

Electronic supplementary material The online version ofthis article (doi:10.1007/s10681-014-1149-6) contains supple-mentary material, which is available to authorized users.

M. J. Garcıa-Moreno � J. M. Fernandez-Martınez �L. Velasco (&) � B. Perez-Vich

Instituto de Agricultura Sostenible (IAS-CSIC),

Alameda del Obispo s/n, 14080 Cordoba, Spain

e-mail: lvelasco@ias.csic.es

123

Euphytica

DOI 10.1007/s10681-014-1149-6

Tocopherols are synthesized exclusively in photo-

synthetic microorganisms and plants. The synthesis of

tocopherols utilizes cytosolic aromatic amino acid

metabolism for head group synthesis and the plastidic

deoxyxylulose 5-phosphate pathway for the synthesis

of the isoprenoid-derived tail group (DellaPenna and

Mene-Saffrane 2011). a-tocopherol is synthesized

from methylation of c-tocopherol, catalyzed by a

c-tocopherol methyltransferase (c-TMT). In sun-

flower seeds, with naturally occurring high a-tocoph-

erol levels, the causative gene for increased

c-tocopherol accumulation has been demonstrated to

be a c-TMT (Hass et al. 2006; Garcıa-Moreno et al.

2012), whereas in soybean seeds, with naturally

occurring high c-tocopherol content, c-TMT muta-

tions resulted in increased a-tocopherol accumulation

(Dwiyanti et al. 2011). In maize, genome-wide

association studies allowed the identification of

c-TMT specific haplotypes associated with high levels

of a-tocopherol (Li et al. 2012; Lipka et al. 2013).

c-TMT genes have been isolated from different

species, including the model species Synechocystis

sp. PCC6803 and Arabidopsis (Shintani and DellaP-

enna 1998; Bergmuller et al. 2003), and the oilseed

crops sunflower (Hass et al. 2006), canola (Endrigkeit

et al. 2009), and soybean (Dwiyanti et al. 2011). In

sunflower and soybean, no mutations in the c-TMT

coding sequence could be associated with the mutant

phenotype (Hass et al. 2006; Dwiyanti et al. 2011).

A natural mutant with high concentration of c-

tocopherol ([85 %) in the seeds was identified in

germplasm of wild safflower (Carthamus oxyacan-

thus) that showed strong introgression of Carthamus

tinctorius L., which allowed selection of individuals of

both species with high seed concentrations of either a-

([90 %) or c-tocopherol ([85 %) (Velasco et al.

2005). The nearly complete replacement of a- by c-

tocopherol confers enhanced thermoxidative stability

to the oil and opens up new uses for safflower oil

(Fernandez-Cuesta et al. 2014). High c-tocopherol

content was controlled by partially recessive alleles at

one single locus (Velasco et al. 2005). The locus was

initially named Tph1 (Velasco et al. 2005) but later

renamed as Tph2 to be consistent with sunflower

nomenclature (Garcıa-Moreno et al. 2011). Through a

candidate gene approach, Garcıa-Moreno et al. (2011)

found co-segregation between Tph2 and a c-TMT

locus, which was partially sequenced from C. tincto-

rius plants with either high a-tocopherol content or

high c-tocopherol content. Even though that study

provided strong support on the involvement of a

c-TMT locus in the altered tocopherol phenotype, no

sequence polymorphisms associated with the trait

were identified. The objective of this research was to

obtain the full-length genomic and cDNA sequences

of the c-TMT locus co-segregating with Tph2 from

C. tinctorius and C. oxyacanthus lines with either high

a- or high c-tocopherol content in order to identify

mutations associated with the high c-tocopherol trait.

Materials and methods

Plant materials

IASC-1 is a safflower line with c-tocopherol content

([85 %) developed from C. oxyacanthus accession PI

426472, which showed variability for tocopherol

profile as well as strong introgression of cultivated

safflower plant traits (Velasco et al. 2005). Three

plants from accession PI 426472 derived from seeds

having either high a-tocopherol ([90 %; PI426472-

A5 and PI426472-A12) or high c-tocopherol content

([85 %; PI426472-A10) were also used in the study.

CL-1 is a nuclear male sterile C. tinctorius line

characterized by high a-tocopherol content ([95 %)

isolated from germplasm accession PI 560161, which

in turn is derived from the germplasm line UC-148

(Heaton and Knowles 1980). Rancho is a high oil

Spanish safflower cultivar with high a-tocopherol

content in seeds ([95 %; Fernandez-Martınez et al.

1986).

Twelve seeds of IASC-1, CL-1, Rancho, PI426472-

A5, PI426472-A10, and PI426472-A12 were analysed

for tocopherol profile by the half-seed technique as

described below. All the seeds of CL-1, Rancho,

PI426472-A5 and PI426472-A12 showed high

a-tocopherol content ([95 %). All the seeds of

IASC-1 and PI426472-A10 showed high c-tocopherol

content ([92 %). The seeds were germinated and the

plants grown in the field in Cordoba, Spain in 2011.

Analysis of tocopherol profile

Individual half seeds, i.e. small seed pieces excised

from the seed part distal to the embryo, were placed

into a 10 ml tube for tocopherol analyses, conducted

following the procedure reported by Goffman et al.

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(1999). After 2 ml of isooctane were added, the half

seeds were crushed as finely as possible with a

stainless-steel rod. The samples were stirred and

extracted overnight at room temperature in darkness

(extraction time about 16 h). After extraction, the

samples were stirred again, centrifuged and filtered.

25 lL of the extract were analysed by high perfor-

mance liquid chromatography (HPLC) using a fluo-

rescence detector (Waters 474, Waters Corporation,

Milford, MA, USA) at 295 nm excitation and 330 nm

emission and isooctane/tert-butylmethylether (94:6)

as eluent at an isocratic flow rate of 0.8 ml min-1.

Chromatographic separation of tocopherols was per-

formed on a LiChrospher 100 diol column

(250 9 3 mm I.D.) with 5 lm spherical particles,

connected to a silica guard column (LiChrospher Si

60, 5 9 4 mm I.D.). The peak areas of the individual

tocopherols were corrected according to their previ-

ously calculated response factors: a-T = 1.0; b-

T = 1.80; c = 1.85; d-T = 2.30.

DNA and RNA extraction and cDNA synthesis

For DNA extraction, ten fully expanded leaves were

cut from two individual plants of IASC-1, CL1,

Rancho, PI426472-A5, PI426472-A10, and PI426472-

A12. Leaf tissue was lyophilised and ground to a fine

powder in a laboratory mill. DNA was isolated from

ground leaf tissue using a modified version of the

protocol described by Rogers and Bendich (1985). For

RNA extraction, developing seeds were collected

from each line at 20 days after flowering and imme-

diately frozen in liquid nitrogen. Total RNA was

extracted using a Plant RNA Isolation Kit (Qiagen

GmbH, Hilden, Germany), and provided the template

for subsequent cDNA synthesis (QuantiTect

Rev. Transcription Kit, Qiagen GmbH, Hilden,

Germany). Both RNA extraction and cDNA synthesis

were carried out according to the manufacturer’s

instructions.

Cloning of the full-length genomic DNA

and cDNA of the c-TMT gene and sequence

analysis

To obtain the full-length genomic c-TMT sequence,

safflower c-TMT cDNA ends were first isolated using

50 and 30 rapid amplification of cDNA ends (RACE)

on RNA isolated from developing seeds of cultivar

Rancho. RACE-PCR analyses were performed using

the SMARTer RACE cDNA amplification kit (Clon-

tech Laboratories, Takara Bio Company, Mountain

View, CA, USA) using c-TMT specific primers

designed according to the partial nucleotide sequence

of safflower c-TMT (Garcıa-Moreno et al. 2011). The

primers were R54_ESP: CGGGAAGATAAAA

GCTGGAACAAATT for the 50 end and F9_ESP:

ACTCTCCGATCACCGTTCTGCTCAG for the 30

end. RACE fragments were separated on a 1.5 %

agarose gel before being excised and purified by

means of the QIAquick gel extraction kit (Qiagen

GmbH, Hilden, Germany). The purified products were

ligated in a T/A vector (pCR2.1) and cloned with the

TOPO-TA cloning kit (Invitrogen Life Technology,

Carlsbad, CA, USA) as described by the manufacturer.

Transformed E. coli were plated on selective media

containing ampicillin and X-gal. Ten white colonies

were picked from the plate and cultured overnight.

The QIAprep Spin Miniprep Kit (Qiagen GmbH,

Hilden, Germany) was used for plasmid DNA extrac-

tion. Restriction enzyme digestion was performed to

confirm the presence of the insert. Sequencing in both

forward and reverse orientations of the cloned frag-

ments (two clones per fragment) was performed at

GATC Biotechnology (Konstanz, Germany) using the

M13 forward and reverse sequencing primers.

Sequence analysis was conducted with the aid of

Vector NTI Advance 10.3.0 software (Invitrogen, San

Diego, CA, USA). 50 and 30 sequences overlapped and

were grouped in one contig.

Full-length c-TMT genomic sequences were iso-

lated from genomic DNA from plants of high c-

tocopherol lines IASC-1 and PI426472-A10 and high

a-tocopherol lines CL-1, Rancho, PI426472-A5 and

PI426472-A12 using primers designed from the 50 and

30 UTR sequences (RACE-CT-F5: CCATTATC

GATACAAAATACGAC and RACE-CT-R7: ACA-

CAAAAACAGTAGTAGCATAATTTA) and long-

distance PCR using AccuPrime High Fidelity Taq

DNA polymerase (Invitrogen Life Technology, Carls-

bad, CA, USA) according to the manufacturer’s

instructions. Amplification products were isolated,

cloned and sequenced (three clones per line) as

described above, with the exception that sequencing

was carried out using the M13 forward and reverse

sequencing primers and c-TMT internal primers.

The full-length cDNA of the c-TMT gene was also

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amplified using the same primers RACE-CT-F5 and

RACE-CT-R7, and isolated, cloned and sequenced

from IASC-1, CL-1 and Rancho, as described above.

Sequence analyses, cDNA, genomic DNA and protein

sequence alignments, and primer design were per-

formed using Vector NTI Advance 10.3.0 software.

Amino acid sequences were analysed for the presence

of functional domains by using the Conserved Domain

Database (Marchler-Bauer et al. 2005).

Amino acid sequences of c-TMT homologs from

cyanobacteria (Synechococcus sp. PCC 7002 [ACA

99779.1]), green algae (Chlamydomonas reinhardtii

[XP_001694470.1]), and plants (Lotus japonicus

[AAY52459.1], Medicago truncatula [ACJ84366.1],

Glycine max (NP_001240883.1), Arabidopsis thali-

ana [NP_176677.1], Brassica napus [ACD03285.1],

Zea mays [NP_001105914.1], Oryza sativa

[EAZ24304.1], Triticum aestivum [CAI77219.2],

Perilla frutescens [AEP68180.1], Solanum tuberosum

[ADV36922.1], Helianthus annuus [ABB52801.1],

Lactuca sativa [ADC91915.1], and Artemisia sph-

aerocephala [ACS34775.1]) were obtained from

NCBI GenBank (http://www.ncbi.nlm.nih.gov). The

sequences were aligned with those obtained in this

study using the AlignX program in the Vector NTI

software suite. A phylogenetic tree of the proteins was

constructed by using the neighbor-joining method in

MEGA 5.0 software (Tamura et al. 2011). A bootstrap

test was performed 1000 times to assess the reliability

of the phylogenetic tree.

Results

c-TMT genomic sequences from high a-tocopherol

C. tinctorius lines Rancho and CL-1 were 3374 bp

and 3393 bp long, respectively (Supplementary

Fig. S1; GenBank accession numbers JX035782

for Rancho and HM028671.2 for CL-1). Genomic

sequences from high a-tocopherol C. oxyacanthus

selections PI426472-A5 and PI426472-A12 were

3371 bp long and showed no differences between

them (Supplementary Fig. S1; GenBank accession

number JX035783). Genomic sequences from high

c-tocopherol C. tinctorius line IASC-1 and high c-

tocopherol C. oxyacanthus selection PI426472-A10

were 3407 bp long and showed no differences

between them (Supplementary Fig. S1; GenBank

accession number HM028672.2). BLAST search

for all the c-TMT genomic sequences described

above revealed a strong significant homology to

other plant c-TMT, especially to those of other

members of the Asteraceae such as sunflower

(2e-67–2e-62), lettuce (4e-65), and A. sphaerocep-

hala (1e-51).

c-TMT genomic DNA sequences from IASC-1 and

CL-1 were compared to the partial sequence (around

one half of the gene) of the safflower c-TMT locus

(Tph2) previously obtained from these lines (Garcıa-

Moreno et al. 2011). The complete genomic DNA

sequence from CL-1 contained a fragment showing no

differences with the 1875 bp CL-1 c-TMT_F9/

Heli_TMT_R54 fragment reported by Garcıa-Moreno

et al. (2011), corresponding to the wild-type Tph2

allele in CL-1 (high a-tocopherol). Similarly, the

genomic DNA sequence from IASC-1 contained a

fragment identical to the 1593 bp Cart-F6/

Heli_TMT_R54 fragment reported by Garcıa-Moreno

et al. (2011), corresponding to the mutant-type tph2

allele in IASC-1 (high c-tocopherol).

cDNAs isolated from Rancho and CL-1 were

1033 bp long, and that from IASC-1 was 1016 bp

long (Supplementary Fig. S2). Alignment of genomic

and cDNA sequences revealed the presence of five

introns and six exons in the c-TMT gene, as reported in

sunflower (Hass et al. 2006). Nucleotide identity of

full-length genomic sequences between high a-

tocopherol lines CL-1, Rancho, PI426472-A5, and

PI426472-12 was 98 %. Nucleotide identity between

sequences of these high a-tocopherol lines and those

from the high c-tocopherol lines IASC-1 and

PI426472-A10 was slightly lower, 97 % for

PI426472-A5 and PI426472-12, and 96 % for CL-1

and Rancho. Alignment of sequences from these lines

allowed the identification of 179 SNP and 21 INDEL,

both in coding and non-coding regions (Supplementary

Fig. 1 Comparison of the amino acid sequences of c-

tocopherol methyltransferase (c-TMT) from high a-tocopherol

Carthamus tinctorius lines CL-1 and Rancho, high c-tocopherol

C. tinctorius line IASC-1, high a-tocopherol C. oxyacanthus

selections PI426472-A5 and PI426472-A12, and high c-tocoph-

erol C. oxyacanthus selection PI426472-A10 with those of other

other plants, green algae and cyanobacteria. Blocks surrounded

by black solid boxes show the location of the conserved residues

in SAM-binding sites. A block surrounded by black discontin-

uous box is the Rubisco large subunit methyltransferase

consensus motif, as reported by Liscombe et al. (2010). The

residue color scheme is as follows: yellow, identical; blue, highly

similar; green, weakly similar; black, different

c

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1 85IASC-1 and PI426472-A10 (1) ------------------------------------------------------------------MSTAVVVTADEQQQ--LKK

PI426472-A5 and PI426472-A12 (1) ------------------------------------------------------------------MSTAVVVTADEQQQ--LKKCL-1 (1) ------------------------------------------------------------------MSTAVVVTTDEQQQQQLKK

Rancho (1) ------------------------------------------------------------------MSTAVVVTAEEHQQQQLKKLactuca sativa (ADC91915.1) (1) ----------------------------------------------------------------------MATAADEQQQQQLKK

Helianthus annuus (ABB52801.1) (1) ------------------------------------------------------MATTAVGVSATPMTEKLTAADDDQQQQKLKKArtemisia sphaerocephala (ACS34775.1) (1) -------------------------------------------------------------------------------------

Solanum tuberosum (ADV36922.1) (1) MGSPCYSACTIQSLNPTCPSSSSSSVIFALHKPQIHSNIIQNYTRRRIITCSSNSRRRMASVTALNAVSSSSVEVGIQNQQELKKPerilla frutescens (AEP68180.1) (1) ------------------------------------------------------------------------MAEMETEMETLRK

Glycine max (NP_001240883.1) (1) -----------------------------------------------------------------MSVEQKAAGKEEEG--KLQKMedicago truncatula (ACJ84366.1) (1) ------------------MVVTTTRISSLLHCTHTFPQHHRDTIITTTTTTLNSRRRKGSLRVSMAAVKEVMVVMEEEEKKKLQL

Lotus japonicus (DQ013360.1) (1) -------------MATMMMSIFPPPPSVASLFILSHCTHTIRVQSTTQFTGFSIRTRTRDCSRILLTEEREMAVMEEKK--LLQTBrassica napus (ACD03285.1) (1) ----------------------MKATLAPSSLISLPRHKVSSLRSPSLLLQSQRPSSALMTTTTASRGSVAVTAAATSSFEALRE

Arabidopsis thaliana (NP_176677.1) (1) ---------------------MKATLAAPSSLTSLPYRTNSSFGSKSSLLFRS-PSSSSSVSMTTTRGNVAVAAAATS-TEALRKOryza sativa Japonica Group (EAZ24304.1) (1) ------------------------------------------------MAPTLSSSS---------------T--AAAAPPGLKE

Zea mays (NP_001105914.1) (1) -----------------MAHAALLHCSQSSRSLAACRRGS--HYRAPSHVPRHSRRLRRAVVSLRPMASS-TAQAPATAPPGLKETriticum aestivum (CAI77219.2) (1) -----------------MANSAALLHSLLSTAWTPRRRLDRASATRLAPSPGLSCRSSRPTRSVRPMASSTTAARADAAPPGLKE

Chlamydomonas reinhardtii (XP_001694470.1) (1) ------------------------MPSTALQGHTLPSSSACLGRATRHVCRVSTRSRRAVTVRAGPLETLVKPLTTLGKVSDLKVSynechococcus sp. PCC 7002 (ACA99779.1) (1) ------------------------------------------------------------------------------MGAQLYQ

86 170IASC-1 and PI426472-A10 (18) GIAEFYDESSGIWENIWGEHMHHGFYDTNAVVELSD--HRSAQIRMVEEALRFASVS--DDPAKKPRSIVDVGCGIGGSSRYLAR

PI426472-A5 and PI426472-A12 (18) GIAEFYDESSGIWENIWGEHMHHGFYDTNAVVELSD--HRSAQIRMVEEALRFASVS--DDPAKKPRSIVDVGCGIGGSSRYLARCL-1 (20) GIAEFYDESSGIWENIWGEHMHHGFYDTNAVVELSD--HRSAQIRMVEEALRFASVS--DDPAKKPRSIVDVGCGIGGSSRYLAR

Rancho (20) GIAEFYDESSGIWENIWGEHMHHGFYDTNAVVELSD--HRSAQIRMVEEALRFASVS--DDPAKKPRSIVDVGCGIGGSSRYLSRLactuca sativa (ADC91915.1) (16) GIAEFYDESSGMWENIWGEHMHHGFYDTDAVVELSD--HRAAQIRMIEQSLLFASVP--DDPVKKPKTIVDVGCGIGGSSRYLAR

Helianthus annuus (ABB52801.1) (32) GIAEFYDESSGMWENIWGEHMHHGYYNSDDVVELSD--HRSAQIRMIEQALTFASVS--DDLEKKPKTIVDVGCGIGGSSRYLARArtemisia sphaerocephala (ACS34775.1) (1) -------ESSGFWENIWGEHMHHGFYDPGAVVEISD--HRSAQIRMIEQSLVFASVP--EDSLEKPKSIVDVGCGIGGSARYLSR

Solanum tuberosum (ADV36922.1) (86) GIADLYDESSGIWEDIWGDHMHHGYYEPQSSVELSD--HRAAQIRMIEKALSFAAIS--EDPAKKPTSIVDVGCGIGGSSRYLAKPerilla frutescens (AEP68180.1) (14) GIAEFYDESSGVWENIWGDHMHHGFYEPAADVSISD--HRAAQIRMIEESLRFASLS--DNTTEKPKNIVDVGCGIGGSSRYLAR

Glycine max (NP_001240883.1) (19) GIAEFYDESSGIWENIWGDHMHHGFYDPDSTVSVSD--HRAAQIRMIQESLRFASLLS-ENPSKWPKSIVDVGCGIGGSSRYLAKMedicago truncatula (ACJ84366.1) (68) GIAEFYDESSGIWENIWGDHMHHGFYDPDSTVSVSD--HRAAQIRMIENSLTFASLS--EDQSKWPKSVVDVGCGIGGSSRYLAK

Lotus japonicus (DQ013360.1) (71) GIAEFYDESSGLWEDMWGDHMHHGFYEQDVTVSVSD--HRVAQIRMIEESLRFAALS--EDPAKKPESIVDVGCGIGGSSRYLAKBrassica napus (ACD03285.1) (64) GIAEFYNETSGLWEEIWGDHMHHGFYDPDSSVQLSDSGHREAQIRMIEESLRFAGVTE-EE--KKIKRVVDVGCGIGGSSRYIAS

Arabidopsis thaliana (NP_176677.1) (63) GIAEFYNETSGLWEEIWGDHMHHGFYDPDSSVQLSDSGHKEAQIRMIEESLRFAGVTD-EEEEKKIKKVVDVGCGIGGSSRYLASOryza sativa Japonica Group (EAZ24304.1) (21) GIAGLYDESSGVWESIWGEHMHHGFYDAGEAASMSD--HRRAQIRMIEESLAFAAVP--DDAEKKPKSVVDVGCGIGGSSRYLAN

Zea mays (NP_001105914.1) (66) GIAGLYDESSGLWENIWGDHMHHGFYDSSEAASMAD--HRRAQIRMIEEALAFAGVPASDDPEKTPKTIVDVGCGIGGSSRYLAKTriticum aestivum (CAI77219.2) (69) GIAGLYDESSGLWESIWGEHMHHGFYDSGEAASMSD--HRRAQIRMIEEALAFAAVP--DDPTNKPKTIVDVGCGIGGSSRYLAN

Chlamydomonas reinhardtii (XP_001694470.1) (62) GIANFYDESSELWENMWGEHMHHGYYPKGAPVKSNQ----QAQIDMIEETLKVAGVT---Q----AKKMVDVGCGIGGSSRYISRSynechococcus sp. PCC 7002 (ACA99779.1) (8) QIREFYDASSPLWESIWGEHMHHGFYGLGGTERLNR---RQAQIELIEEFLAWGKVEQ--VG-----NFVDVGCGIGGSTLYLAD

171 255IASC-1 and PI426472-A10 (99) KYGAECHGITLSPVQAERAQALAAAQGLADKVSFQVADALNQPFPDGKFDLVWSMESGEHMPDKLKFVSELARVAAPGATIIIVT

PI426472-A5 and PI426472-A12 (99) KYGAECHGITLSPVQAERAQALAAAQGLADKASFQVADALNQPFPDGKFDLVWSMESGEHMPDKLKFVSELARVAAPGATIIIVTCL-1 (101) KYGAECHGITLSPVQAERAQALAAAQGLADKASFQVADALNQPFPDGKFDLVWSMESGEHMPDKLKFVSELARVAAPGATIIIVT

Rancho (101) KYGAECNGITLSPVQAERAQALAAAQGLADKASFQVADALNQPFPDGKFDLVWSMESGEHMPDKLKFVSELARVAAPGATIIIVTLactuca sativa (ADC91915.1) (97) KYGAECHGITLSPVQAERAQALAATQGLADKVSFQVADALNQPFPDGKFDLVWSMESGEHMPDKLKFVSELARVAAPGATIIIVT

Helianthus annuus (ABB52801.1) (113) KYGAECHGITLSPVQAERANALAAAQGLADKVSFQVADALNQPFPDGKFDLVWSMESGEHMPDKLKFVSELTRVAAPGATIIIVTArtemisia sphaerocephala (ACS34775.1) (75) KYGAECCGITLSPVQAERAQALAAAQGLASKVSFQVADALNQPFPDGKFDLVWSMESGEHMPDKLKFVSELARVAAPGATIIIVT

Solanum tuberosum (ADV36922.1) (167) KYGATAKGITLSPVQAERAQALADAQGLGDKVSFQVADALNQPFPDGQFDLVWSMESGEHMPNKEKFVGELARVAAPGGTIILVTPerilla frutescens (AEP68180.1) (95) KYGANCQGITLSPVQAQRAQQLADAQGLNGKVSFEVADALNQPFPEGKFDLVWSMESGERMPDKKKFVNELVRVAAPGGRIIIVT

Glycine max (NP_001240883.1) (101) KFGATSVGITLSPVQAQRANSLAAAQGLADKVSFEVADALKQPFPDGKFDLVWSMESGEHMPDKAKFVGELARVAAPGGTIIIVTMedicago truncatula (ACJ84366.1) (149) KFGANCVGITLSPVQAERANALAAAQGLADKVSFQVADALQQPFPDGQFDLVWSMESGEHMPNKPKFVGELARVAAPGGTIIIVT

Lotus japonicus (DQ013360.1) (152) KFQAKSVGITLSPVQAQRANALAASQGLADKVSFQVADALEQPFPDGQFDLVWSMESGEHMPDKPKFVGELARVAAPGGTIIIVTBrassica napus (ACD03285.1) (146) KFGAECIGITLSPVQAKRANDLAAAQSLSHKVSFQVADALEQPFEDGIFDLVWSMESGEHMPDKAKFVKELVRVAAPGGRIIIVT

Arabidopsis thaliana (NP_176677.1) (147) KFGAECIGITLSPVQAKRANDLAAAQSLAHKASFQVADALDQPFEDGKFDLVWSMESGEHMPDKAKFVKELVRVAAPGGRIIIVTOryza sativa Japonica Group (EAZ24304.1) (102) KYGAQCYGITLSPVQAERGNALAAEQGLSDKVSFQVGDALEQPFPDGQFDLVWSMESGEHMPDKRQFVSELARVAAPGARIIIVT

Zea mays (NP_001105914.1) (149) KYGAQCTGITLSPVQAERGNALAAAQGLSDQVTLQVADALEQPFPDGQFDLVWSMESGEHMPDKRKFVSELARVAAPGGTIIIVTTriticum aestivum (CAI77219.2) (150) KYGAQCSGITLSPVQAERGNALAAAQGLSDKASFQVADALEQPFPDGQFDLVWSMESGEHMPNKQKFVSELARVAAPGATIIIVT

Chlamydomonas reinhardtii (XP_001694470.1)(136) KFGCTSNGITLSPKQAARANALSKEQGFGDKLQFQVGDALAQPFEAGAFDLVWSMESGEHMPDKKKFVSELARVCAPGGTVIVVTSynechococcus sp. PCC 7002 (ACA99779.1) (83) KFNAQGVGITLSPVQANRAIARATEQNLQDQVEFKVADALNMPFRDGEFDLVWTLESGEHMPNKRQFLQECTRVLKPGGKLLMAT

256 340IASC-1 and PI426472-A10 (184) WCHRDLSPTEESLRPEEEKILNKICSSFYLPAWCSTADYIKLLESLSLQDIKAADWSPNVAPFWPAVIKTALSWTGITSLLRSGW

PI426472-A5 and PI426472-A12 (184) WCHRDLSPTEESLRPEEEKILNKICSSFYLPAWCSTADYIKLLESLSLQDIKAADWSPNVAPFWPAVIKTALSWTGITSLLRSGWCL-1 (186) WCHRDLSPTEESLRPEEEKILNKICSSFYLPAWCSTADYIKLLESLSLQDIKAADWSPNVAPFWPAVIKTALSWTGITSLLRSGW

Rancho (186) WCHRDLSPTEESLRPEEEKILNKICSSFYLPAWCSTADYIKLLESLSLQDIKAADWSPNVAPFWPAVIKTALSWTGITSLLRSGWLactuca sativa (ADC91915.1) (182) WCHRDLLPPEKSLRPEEEKILNKICSGFFLPAWCSTADYVKLLESISLQDIKAEDWSGNVAPFWPAVIKTALSWKGITSLLRSGW

Helianthus annuus (ABB52801.1) (198) WCHRDLNPGEKSLRPEEEKILNKICSSFYLPAWCSTADYVKLLESLSLQDIKSADWSGNVAPFWPAVIKTALSWKGITSLLRSGWArtemisia sphaerocephala (ACS34775.1) (160) WCHRDLSPNEKSLRPEEKRILEKICDGFYLPAWCSTADYIKLLESLS---LKAADWSDNVAPFWPAVVKSALTWNGLTSLLQSGW

Solanum tuberosum (ADV36922.1) (252) WCHRDLSPSEESLTPEEKELLNKICKAFYLPAWCSTADYVKLLQSNSLQDIKAQDWSENVAPFWPAVIKSALTWKGLTSVLRSGWPerilla frutescens (AEP68180.1) (180) WCHRDLSPSEESLRQEEKDLLNKICSAYYLPAWCSTADYVKLLDSLSMEDIKSADWSDHVAPFWPAVIKSALTWKGITSLLRSGW

Glycine max (NP_001240883.1) (186) WCHRDLGPDEQSLLPWEQDLLKKICDSYYLPAWCSTSDYVKLLESLSLQDIKSADWSPFVAPFWPAVIRTALTWNGLTSLLRSGLMedicago truncatula (ACJ84366.1) (234) WCHRDLRPDEESLQQWEKDLLKKICDSFYLPEWCSTADYVKLLETMSLQDIKSADWSPFVAPFWPAVIRSALTWKGFTSILRSGL

Lotus japonicus (DQ013360.1) (237) WCHRDLGPAEESLQPWEQNLLKRICDAFYLPAWCSTADYVKLLESHSLQDIKSADWSPFVAPFWPAVIRSAFTWKGLTSLLRSGMBrassica napus (ACD03285.1) (231) WCHRNLSPGEEALQPWEQNLLDRICKTFYLPAWCSTSDYVDLLQSLSLQDIKCADWSENVAPFWPAVIRTALTWKGLVSLLRSGM

Arabidopsis thaliana (NP_176677.1) (232) WCHRNLSAGEEALQPWEQNILDKICKTFYLPAWCSTDDYVNLLQSHSLQDIKCADWSENVAPFWPAVIRTALTWKGLVSLLRSGMOryza sativa Japonica Group (EAZ24304.1) (187) WCHRNLEPSEESLKPDELNLLKRICDAYYLPDWCSPSDYVKIAESLSLEDIRTADWSENVAPFWPAVIKSALTWKGLTSLLRSGW

Zea mays (NP_001105914.1) (234) WCHRNLDPSETSLKPDELSLLRRICDAYYLPDWCSPSDYVNIAKSLSLEDIKTADWSENVAPFWPAVIKSALTWKGFTSLLTTGWTriticum aestivum (CAI77219.2) (235) WCHRNLAPSEDSLKPDELNLLKKICDAYYLPDWCSPSDYVKIAESLSLEDIKTADWSENVAPFWPAVIQSALTWKGLTSLLRSGW

Chlamydomonas reinhardtii (XP_001694470.1)(221) WCHRVLGPGEAGLREDEKALLDRINEAYYLPDWCSVADYQKLFEAQGLTDIQTRDWSQEVSPFWGAVIATALTSEGLAGLAKAGWSynechococcus sp. PCC 7002 (ACA99779.1) (168) WCHRPTDSVAGTLTPAEQKHLEDLYRIYCLPYVISLPDYQAIATECGLENIETADWSTAVAPFWDQVIDSALTPEAVFGILKAGW

341 386IASC-1 and PI426472-A10 (269) KTIRGAMAMPLMIEGFNQILHHYM----------------------

PI426472-A5 and PI426472-A12 (269) KTIRGAMAMPLMIEGFKKDLIKFSIITCRKPE--------------CL-1 (271) KTIRGAMAMPLMIEGFKKDLIKFSIITCRKPE--------------

Rancho (271) KTIRGAMAMPLMIEGFKKDLIKFSIITCRKPK--------------Lactuca sativa (ADC91915.1) (267) KTIRGAMVMPSMIEGFKKDVIKFSIITCKKPE--------------

Helianthus annuus (ABB52801.1) (283) KSIRGAMVMPLMIEGFKKDVIKFSIITCKKPE--------------Artemisia sphaerocephala (ACS34775.1) (242) KTIRGALVMPLMIEGFQKGVIKFSIITCKKPE--------------

Solanum tuberosum (ADV36922.1) (337) KTIKAALAMPLMIEGYKKGLIKFAIITCRKPE--------------Perilla frutescens (AEP68180.1) (265) KTIRGAMVMPLMIEGYKKGVIKFAIITCRKPAS-------------

Glycine max (NP_001240883.1) (271) KTIKGALAMPLMIKGYKKDLIKFSIITCRKPE--------------Medicago truncatula (ACJ84366.1) (319) KTIKGALAMPLMIEGFRKGVIKFAIITCRKPENADGQ---------

Lotus japonicus (DQ013360.1) (322) KTIKGALAMPLMIEGFKKGVIKFAIVTCRKPENVEIE---------Brassica napus (ACD03285.1) (316) KSIKGALTMPLMIEGYKKGVIKFGIITCQKPL--------------

Arabidopsis thaliana (NP_176677.1) (317) KSIKGALTMPLMIEGYKKGVIKFGIITCQKPL--------------Orza sativa Japonica Group (EAZ24304.1) (272) KTIRGAMVMPLMIEGYKKGLIKFTIITCRKPETTQ-----------

Zea mays (NP_001105914.1) (319) KTIRGAMVMPLMIQGYKKGLIKFTIITCRKPGAA------------Triticum aestivum (CAI77219.2) (320) KTIKGALVMPLMIQGYKKGLIKFSIITCHKPQAAIEGEPEAASPRV

Chlamydomonas reinhardtii (XP_001694470.1)(306) TTIKGALVMPLMAEGFRRGLIKFNLISGRKLQQ-------------Synechococcus sp. PCC 7002 (ACA99779.1) (253) QTLQGALALDLMKSGFRRGLIRYGLLQATKPKA-------------

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Fig. S1). c-TMT coding sequences were translated.

High a-tocopherol C. tinctorius Rancho and CL-1

encoded 302 amino acids. High a-tocopherol C.

oxyacanthus selections PI426472-A5 and PI426472-

A12 encoded 300 amino acids. High c-tocopherol C.

tinctorius line IASC-1 and high c-tocopherol C.

oxyacanthus selection PI426472-A10 encoded 292

amino acids (Fig. 1). All the amino acid sequences

showed conserved S-adenosylmethionine (SAM)-

binding domains (Fig. 1).

c-TMT sequences from high a-tocopherol

(PI426472-A5 and PI426472-12) and high c-tocoph-

erol lines (IASC-1 and PI426472-A10) derived from

C. oxyacanthus differed for six synonymous and one

non-synonymous SNP and for an 11 bp long INDEL

in coding regions (Supplementary Fig. S1 and S2).

The non-synonymous SNP (C–T) implied a change of

alanine in PI426472-A5 and PI426472-12 to valine in

IASC-1 and PI426472-A10 that was not associated

with the mutant phenotype, since in that position

valine was also found in sunflower c-TMT amino acid

sequences (Fig. 1). However, the 11 bp long deletion

found in exon 6 in high c-tocopherol lines IASC-1 and

PI426472-A10 disrupted the reading frame, which

drastically altered the protein sequence downstream of

the frameshift site and created a stop codon that led to

a premature termination of the peptide chain (Fig. 1).

The predicted truncated protein in high c-tocopherol

lines IASC-1 and PI426472-A10 had 292 amino acids

instead of 300–302 in high a-tocopherol lines as well

as strong changes in the C-terminal amino acid

sequence (Fig. 1).

Amino acid sequences were aligned with other

c-TMT amino acid sequences from several plant

species as well as from green algae and cyanobacteria

(Fig. 1). Except for the N-terminal region, C. tincto-

rius and C. oxyacanthus c-TMT amino acid sequences

shared high similarity with c-TMT from other plant

species. Phylogenetic analysis classified C. tinctorius

and C. oxyacanthus c-TMT amino acid sequences

close to those from the Asteraceae species L. sativa,

H. annuus, and Artemisia sphaerocephala, within the

dicots group (Fig. 2). Amino acid similarity between

c-TMT from Carthamus and other species of the

Asteraceae was 90 % for L. sativa, 89 % for

H. annuus, and 86 % for A. sphaerocephala. The last

16 amino acids were highly conserved across plant

species (Fig. 1), with the exception of high c-tocoph-

erol lines IASC-1 and PI426472-A10, in which the

11 bp INDEL mutation in exon 6 resulted in a

completely different amino acid sequence in this

region, not found in the c-TMT amino acid sequence

of any other plant, green algae or cyanobacteria

(Fig. 1).

Fig. 2 Neighbour-joining phylogenetic tree of c-tocopherol

methyltransferase (c-TMT) proteins obtained after aligning c-

TMT amino acid sequences from Carthamus tinctorius and C.

oxyacanthus with c-TMT of other plants, green algae and

cyanobacteria. GenBank accession numbers are indicated after

the species name. The bootstrap values (percent) are indicated

above the supported branches. The scale bar indicates the

distance corresponding to five changes per 100 amino acid

positions

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123

Discussion

In a previous study, Garcıa-Moreno et al. (2011) isolated

and sequenced a c-TMT locus co-segregating with the

Tph2 gene underlying high c-tocopherol content in

safflower. The Tph2-c-TMT sequenced fragment was

predicted to contain around one half of the gene. The

complete c-TMT sequences isolated in this research

from IASC-1 and CL-1 harboured a region 100 %

identical to the Tph2-c-TMT alleles from these lines

characterized by Garcıa-Moreno et al. (2011), thus

confirming that the sequences obtained in this study

correspond to the Tph2-c-TMT locus associated with

the high c-tocopherol trait in safflower. This confirma-

tion was relevant, since Garcıa-Moreno et al. (2011)

found evidences of duplicated c-TMT loci in safflower.

In soybean and sunflower, c-TMT genes have been

found to underlie mutant phenotypes showing altered

seed tocopherol profiles. In soybean, with naturally

occurring high levels of c- and d-tocopherol and low

levels of a-tocopherol (\10 %) in seeds, plant material

with increased a-tocopherol content (20–30 %) was

identified in germplasm evaluations (Ujiie et al. 2005).

The increased a-tocopherol content was associated with

a c-TMT gene (Dwiyanti et al. 2011). The authors found

no nucleotide polymorphisms involved in the increased

a-tocopherol content within the coding region of the

c-TMT gene. However, nucleotide polymorphisms in

the c-TMT promoter region and a higher activity of the

c-TMT promoter in lines with increased a-tocopherol

content led the authors to conclude that sequence

changes in the promoter might underlie the increased

seed a-tocopherol levels. In sunflower, a c-TMT gene is

also responsible for high levels of c-tocopherol ([85 %

in mutant lines compared to\1 % in conventional lines)

in the seeds. As in soybean, no polymorphisms within

the coding region of the c-TMT gene were related to the

mutant phenotype (Hass et al. 2006; Garcıa-Moreno

et al. 2012). In the model plant Arabidopsis, a c-TMT

gene was also identified underlying high c-tocopherol

content in leaves of mutant lines. The mutant alleles

vte4-1 and vte4-2 carried null mutations in the VTE4

gene encoding the c-TMT, which consisted in a point

mutation at the highly conserved 50-splicing site of

intron 3, and in a T-DNA insertion into the coding

sequence of exon 3, respectively (Bergmuller et al.

2003). In the present research, c-TMT nucleotide

sequences from high a-tocopherol and high c-tocoph-

erol lines differed for six synonymous SNP, one non-

synonymous SNP, and an 11 bp long INDEL in coding

regions. The non-synonymous SNP (C–T) implied a

change of alanine (in high a-tocopherol lines) to valine

(in high c-tocopherol lines) that was not associated with

the mutant phenotype, as valine has been found at that

position in c-TMT amino acid sequences from sun-

flower lines with either high a-tocopherol content or

high c-tocopherol content (Hass et al. 2006; Garcıa-

Moreno et al. 2012). However, the 11 bp long deletion

found in exon 6 in high c-tocopherol lines disrupted the

reading frame and created a premature stop codon,

which resulted in a predicted truncated c-TMT protein

showing a drastically altered amino acid sequence at the

C-terminal region, not found in any other plant, green

algae or cyanobacteria c-TMT amino acid sequence.

This mutation was expected to disrupt the Tph2-c-TMT

locus resulting in a nearly complete null allele. This was

supported by the fact that this c-TMT C-terminal region

was highly conserved in plants. In fact, it has been

proposed to be an active site for c-TMT post-transla-

tional modification (Magnani et al. 2007; Liscombe

et al. 2010). Frameshift mutations generally result in

loss of function changes since they drastically alter the

protein sequence downstream of the frameshift site,

besides creating premature stop codons (Cupples 2003).

The data suggested that the c-TMT frameshift mutation,

only found in plants with high seed c-tocopherol

content, is the causal DNA sequence difference between

alleles of the c-TMT gene underlying the high

c-tocopherol phenotype in plants of C. tinctorius and

C. oxyacanthus. The identification of sequence changes

associated with the c-tocopherol phenotype will allow

the development of functional markers for marker

assisted introgression of the high c-tocopherol trait into

elite safflower cultivars.

Acknowledgments The research was funded by the Spanish

Ministry of Science and Innovation and the European Union

FEDER funds (research project AGL2007-62834). The authors

are grateful to Dr. Marıa J. Gimenez and Dr. Fernando Piston

(Instituto de Agricultura Sostenible, CSIC) for their advice on

experimental procedures.

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