Genetic variation of γ-tocopherol methyltransferase gene contributes to elevated α-tocopherol content in soybean seeds
© Dwiyanti et al; licensee BioMed Central Ltd. 2011
Received: 12 April 2011
Accepted: 7 November 2011
Published: 7 November 2011
Improvement of α-tocopherol content is an important breeding aim to increase the nutritional value of crops. Several efforts have been conducted to improve the α-tocopherol content in soybean [Glycine max (L.) Merr.] through transgenic technology by overexpressing genes related to α-tocopherol biosynthesis or through changes to crop management practices. Varieties with high α-tocopherol content have been identified in soybean germplasms. The heritability of this trait has been characterized in a cross between high α-tocopherol variety Keszthelyi Aproszemu Sarga (KAS) and low α-tocopherol variety Ichihime. In this study, the genetic mechanism of the high α-tocopherol content trait of KAS was elucidated.
Through QTL analysis and fine mapping in populations from a cross between KAS and a Japanese variety Ichihime, we identified γ-TMT3, which encodes γ-tocopherol methyltransferase, as a candidate gene responsible for high α-tocopherol concentration in KAS. Several nucleotide polymorphisms including two nonsynonymous mutations were found in the coding region of γ-TMT3 between Ichihime and KAS, but none of which was responsible for the difference in α-tocopherol concentration. Therefore, we focused on transcriptional regulation of γ-TMT3 in developing seeds and leaves. An F5 line that was heterozygous for the region containing γ-TMT3 was self-pollinated. From among the progeny, plants that were homozygous at the γ-TMT3 locus were chosen for further evaluation. The expression level of γ-TMT3 was higher both in developing seeds and leaves of plants homozygous for the γ-TMT3 allele from KAS. The higher expression level was closely correlated with high α-tocopherol content in developing seeds. We generated transgenic Arabidopsis plants harboring GUS gene under the control of γ-TMT3 promoter from KAS or Ichihime. The GUS activity assay showed that the activity of γ-TMT3 promoter from KAS was higher than that of Ichihime.
The genetic variation in γ-TMT3, which plays a major role in determining α-tocopherol concentration, provides significant information about the regulation of tocopherol biosynthesis in soybean seeds. This knowledge will help breeding programs to develop new soybean varieties with high α-tocopherol content.
The vitamin E family comprises tocopherols (α, β, γ, and δ forms) and tocotrienols (α, β, γ, and δ forms). All isoforms possess lipid antioxidant activity, and α-tocopherol possesses the highest vitamin E activity in mammals [1, 2]. Vitamin E is widely used as an antioxidant in foods and oils, as a nutrient additive in poultry and cattle feeds to improve meat quality, and as a supplement in the human diet to help prevent diseases such as cancer and cardiovascular diseases. The market size is expected to grow because of the increasing interest in functional food and increasing demand for meat products. About 85% of commercial vitamin E is synthesized by chemical reaction . This vitamin E usually includes the naturally occurring RRR-α-tocopherol and 7-stereoisomers as secondary products, whose biological activity is only 50%-74% of that of the natural α-tocopherol . Thus, it is very important to increase natural vitamin E production in crops and vegetables .
Tocopherols are present in leaves, stems, flower petals, and seeds of higher plants and green algae [1, 11]. While α-tocopherol is usually the predominant form in leaves, there are diverse variations of tocopherol composition in seeds . For example, in soybean, rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana), most of the tocopherols are γ-tocopherol or δ-tocopherol; in sunflower (Helianthus annuus) and safflower (Carthamus tinctorius) seeds, the content of α-tocopherol is more than 95% of the total tocopherol content [12, 13]. Variations in α-tocopherol content (α-tocopherol weight [μg] per 100 mg seed powder) and concentration (α-tocopherol as a percentage of total tocopherol) have been reported in crops such as maize (from 0.9 to 6.5 μg 100 mg-1), sunflower (>95% in wild type and <10% in mutants), safflower (>85% in wild type and <15% in mutants), rapeseed (α/γ-tocopherol ratio ranged from 0.54 to 1.70) and in the model plant Arabidopsis [12–16]. Previous studies have shown that variation is also present in soybean. Three soybean varieties with α-tocopherol concentration of 20% to 30%, Keszthelyi Aproszemu Sarga (KAS), Dobrogeance, and Dobrudza 14 Pancevo, were identified through analysis of more than 1,000 cultivars and varieties from soybean germplasms collections . These varieties showed higher α-tocopherol content compared to typical cultivars over two planting years, indicating that high α-tocopherol content was a stable trait . QTL analysis using Chinese (Hefeng 25) and Canadian (OAC Bayfield) soybean varieties revealed four QTLs for tocopherol content in linkage groups B2, C2, D1b, and I, which correspond to chromosome 14, 6, 2, and 20, respectively. However, the causal genes involved in these QTLs are yet to be identified .
In our previous study, the genetic characteristics of the high α-tocopherol concentration trait were evaluated in an F2 population derived from a cross between KAS and a typical variety, Ichihime . α-Tocopherol concentration of a typical variety is less than 10% of total tocopherol . Here and in our previous study , α-tocopherol concentration was defined as the ratio of α-tocopherol to total tocopherol, whereas α-tocopherol content was defined as the α-tocopherol weight (μg) per 100 mg soybean seed powder. The broad-sense heritability of the high α-tocopherol concentration trait was estimated to be 0.645 . Two simple sequence repeats (SSR) markers, Sat_167 and Sat_243 on linkage group K (chromosome 9) were strongly correlated with α-tocopherol concentration . The relationships between tocopherol forms were also analyzed; α-tocopherol concentration had no significant correlation with total tocopherol content, whereas γ-tocopherol and α-tocopherol concentrations showed a strong negative correlation .
The strong negative correlation between α-tocopherol concentration and γ-tocopherol concentration suggested that a major gene involved in the biosynthesis pathway of α-tocopherol might be responsible for the trait . Tocopherols are biosynthesized from two precursors, homogentisic acid (HGA) and phytyl diphosphate. The two precursors are condensed by HGA phytyl transferase, generating MPBQ. MPBQ is methylated to become 2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ). MPBQ and DMPBQ are converted by tocopherol cyclase to δ-tocopherol and γ-tocopherol, respectively. The last step of the tocopherol biosynthesis pathway is methylation of δ-tocopherol and γ-tocopherol by γ-tocopherol methyltransferase (γ-TMT), yielding β-tocopherol and α-tocopherol, respectively (Figure 1) .
To elucidate the genetic basis of the high α-tocopherol concentration trait in KAS, we performed QTL analysis and fine mapping for α-tocopherol concentration by using the population derived from a cross between a typical variety Ichihime and the high α-tocopherol variety KAS. The γ-TMT3, which has high similarity to the Arabidopsis VTE4 gene, was located within a QTL region of approximately 75 kb. The expression level of γ-TMT3 was higher in developing seeds of plants with the KAS genotype, and the expression elevation was correlated with an increase in α-tocopherol content. It is also demonstrated that the transient activity of γ-TMT3 promoter from KAS was higher than that of Ichihime.
Mapping the QTL responsible for the high α-tocopherol concentration trait
KAS, a soybean variety with 20% to 30% α-tocopherol concentration, was crossed to the Japanese cultivar Ichihime (α-tocopherol concentration <10%) to obtain a segregating population consisting of 122 F2 plants . These plants were grown in the Hokkaido University greenhouse, where F3 seeds of each F2 plant were obtained and analyzed for their tocopherol composition. A molecular linkage map was constructed using 152 SSR markers that were polymorphic between Ichihime and KAS. The linkage map covered 3401 cM of the soybean genome and consisted of 20 linkage groups that corresponded to the 20 pairs of soybean chromosomes.
Two population groups were used for QTL analysis. The first population (hereafter, "F2 seed population") consisted of F2 seeds from the Ichihime × KAS cross; in this population, tocopherol concentrations were analyzed using the half-seed method (see Materials and Methods). The second population ("F2 plant population") consisted of F2 plants whose tocopherol content and concentration were evaluated by testing the F2:3 seeds. Multiple QTL Mapping (MQM) analysis was performed using MapQTL5, and the QTL threshold values were determined for each trait by using a 1,000-permutation test .
QTL associated with tocopherol concentration or content using F2 seed and F2 plant populations.
For the F2 plant population, QTLs for tocopherol concentrations and contents were analyzed. The same QTL observed in the analysis of the F2 seed population was also detected for α-tocopherol concentration (LOD = 20.2, PVE = 55.0%), γ-tocopherol concentration (LOD = 16.7, PVE = 48.7%), and δ-tocopherol concentration (LOD = 4.8, PVE = 17.0%). Moreover, this QTL was also responsible for α-tocopherol content (LOD = 20.6, PVE = 56.5%) and γ-tocopherol content (LOD = 5.24, PVE = 17.9%). For δ-tocopherol concentration, another QTL was detected in interval Sat_244 and Sat_033 of linkage group M (chromosome 12), with LOD value 5.26 and PVE 22.5%. However, this QTL was not detected in F2 seeds analysis.
It has been reported that four QTLs for tocopherol concentrations and contents were detected from QTL analysis in a segregating population derived from a cross between a Chinese variety (Hefeng 25) and a high α-tocopherol Canadian variety (OAC Bayfield) . However, in this study, no QTL was detected in those regions. This fact suggests that the genetic factor responsible for high α-tocopherol concentration in KAS may be different from that in OAC Bayfield.
Identification of candidate gene in the QTL region
Predicted genes located in QTL region, based on information of Phytozome database.
auxin responsive protein
auxin responsive protein
diphtheria toxin resistance
no function annotation
amidophosphoribosylpyrophosphate transferase domain
amidophosphoribosylpyrophosphate transferase domain
γ-tocopherol methyltransferase (γ-TMT)
no function annotation
no function annotation
DNA topoisomerase type I
Polymorphisms in exon region of γ-TMT3 gene.
3.88 ± 0.32
1.99 ± 0.08
4.84 ± 0.58
19.25 ± 2.22
18.06 ± 2.20
Dobrudza 14 Pancevo
19.38 ± 1.14
Relationship between α-tocopherol concentration and expression levels of γ-TMT genes
The expression level of γ-TMT3 could affect α-tocopherol content and concentration was investigated because the polymorphisms correlated to α-tocopherol concentration were found in the transcriptional regulatory domain of γ-TMT3.
F5-24, an F5 heterogeneous inbred family (HIF)  which was heterozygous for the genomic region surrounding γ-TMT3 and homozygous throughout almost entire genome was used to generate plants homozygous for the γ-TMT3 genomic region from Ichihime and that from KAS; these are referred to as Ichihime lines and KAS lines, respectively. Three lines homozygous for the Ichihime allele (F5-24-10, F5-24-14, and F5-24-15) and three lines homozygous for the KAS allele (F5-24-7, F5-24-18, and F5-24-22) were generated. From each plant, developing seeds were collected at 30, 40, and 50 days after flowering (DAF).
Activity of γ-TMT3 promoter of Ichihime and KAS
γ-TMT3 is the candidate gene for high α-tocopherol concentration in KAS
In the previous study, two SSR markers, Sat_243 and Sat_167 on a linkage group K (chromosome 9) were strongly associated with α-tocopherol concentration. In this study, we confirmed that the QTL in interval Sat_243 and KSC138-17 was associated with α-tocopherol concentration, γ-tocopherol concentration, α-tocopherol content, and γ-tocopherol content. The QTL positively regulated α-tocopherol concentration and α-tocopherol content, and negatively regulated γ-tocopherol concentration and γ-tocopherol content (Table 1), indicating that the candidate gene is directly related to conversion of γ-tocopherol to α-tocopherol. Fine mapping using F5 lines showed that γ-TMT3 was located in a QTL region. This study focused on the molecular characterization of γ-TMT3 gene.
Based on sequencing analysis and gene expression analysis, the nucleotide polymorphisms in γ-TMT3 promoter region might increase the expression level of γ-TMT3 in developing seeds of KAS, and subsequently associated with high α-tocopherol concentration in KAS seeds. Transient GUS assay for the 1.2-kb promoter region of γ-TMT3 from KAS and Ichihime also supported our view that different γ-TMT3 expression between KAS and Ichihime could be, at least partly, attributed to the difference in the promoter sequence, although we cannot exclude the possibility that some cis-elements affecting the γ-TMT3 expression is located outside of 1.2-kb upstream of the transcriptional start site.
Two of the polymorphisms were located in transcription factor binding motifs in the 5'-upstream region of the γ-TMT3 gene in high α-tocopherol soybeans (Figure 7). The first mutation is located in a CAAT box, which acts as an enhancer for gene expression. The canonical sequence of CAAT box is "CCAAT", which is the sequence found in Ichihime. The KAS type is "CAAAT", which is not canonical but is recognized as a functional CAAT box in mouse . At present, we do not know any report that mutation in a CAAT box can enhance gene expression. The second mutation produced a MYB binding site in the KAS promoter; this same sequence ("CTGTTA") is also found in the caffeic acid O-methyltransferase gene promoter of Arabidopsis . In Arabidopsis, the "CTGTTA" motif is recognized by maize MYB transcription factors ZmMYB31 and ZmMYB42 . Further analysis of these cis-elements will provide information of whether these polymorphisms contribute to alteration in the promoter activity.
Regulation of tocopherol content and concentration in soybean
The tocopherol content analysis in this study provides important information about regulation of the tocopherol content and concentration in soybean. In the KAS lines, δ-tocopherol content was lower than in Ichihime lines at 40 DAF. However, the content of γ-tocopherol did not differ between KAS lines and Ichihime lines. Since the peaks from γ-tocopherol and β-tocopherol could not be separated by the analytic method used in this study, it is suggested that increase in β-tocopherol content might mask a decrease in the content of γ-tocopherol. Thus, γ-TMT3 may catalyze both γ-tocopherol and δ-tocopherol conversion to α-tocopherol and β-tocopherol, respectively (Figure 1). The δ-tocopherol decrease and α-tocopherol increase in KAS lines also raises the question of whether γ-TMT3 can also catalyze the methylation of MPBQ to DMPBQ. It is reported that Arabidopsis γ-TMT (VTE4) was not active toward MPBQ in vitro . In soybean, there was little similarity in amino acid sequences between γ-TMTs and MPBQ-MTs, indicating that soybean γ-TMTs might not be active toward MPBQ. Further analysis of the enzymatic activity and substrate specificity of γ-TMT3 will provide more information about the biochemical properties of γ-TMT3.
The possibility of functional differentiation of γ-TMT proteins
γ-TMT1, γ-TMT2, and γ-TMT3 proteins have amino acid similarity more than 90% and two SAM binding domains (Figure 6), suggesting that they all possess the γ-TMT activity that catalyzes the conversion of γ-tocopherol to α-tocopherol. It is elucidated that three γ-TMT genes (γ-TMT1, γ-TMT2, and γ-TMT3) were expressed in leaves and developing seeds where α-tocopherol was synthesized and accumulated (Figure 10, Figure 11). However, it is indicated that alteration in expression level of γ-TMT3 alone could increase both α-tocopherol concentration and α-tocopherol content to up to 2.4 times that of typical soybean (Figure 8A, 9A). If γ-TMT1 or γ-TMT2 mutations are also able to enhance α-tocopherol accumulation, gene pyramiding of these γ-TMT variants will enable us to develop new soybean varieties with higher α-tocopherol concentration or content than KAS. γ-TMT1, γ-TMT2, and γ-TMT3 polypeptides showed differences in their NH2-terminal region (Figure 6), although they shared high amino acid similarity with γ-TMTs found in several other plant species (Figure 6). Interestingly, no plastid signal peptide was predicted in γ-TMT1 and γ-TMT3 based on in silico analysis. α-Tocopherol is known to be localized and be synthesized in plastids , and enzymes involved in its biosynthesis are localized inside the plastid [11, 28]. Further analysis about the subcellular localization of γ-TMT1 and γ-TMT3 might elucidate the functional diversifications in γ-TMT proteins for the regulation of α-tocopherol biosynthesis in soybean.
In this work, we identified a QTL responsible for genetic regulation of the high α-tocopherol concentration in KAS. In addition to regulating α-tocopherol concentration, this QTL also affected γ-tocopherol concentration and δ-tocopherol concentration. Thus it is suggested that a gene underlying this QTL regulates tocopherol concentration. Through fine mapping, γ-TMT3 was identified as a candidate gene for the high α-tocopherol concentration trait. γ-TMT3 encodes γ-tocopherol methyltransferase, which catalyzes the methylation γ-tocopherol to α-tocopherol. The expression of γ-TMT3 in the developing seeds of KAS lines was higher than in the seeds of Ichihime lines. Concomitantly, γ-TMT3 expression was higher in leaves of KAS than in those of Ichihime. Taken these results together, it is concluded that the promoter region polymorphisms caused higher γ-TMT3 expression in KAS, resulting in a higher α-tocopherol concentration. A transient activity analysis of γ-TMT3 promoters showed that the activity of KAS γ-TMT3 promoter was higher than that of Ichihime γ-TMT3 promoter. In this study, it is also demonstrated that genetic variation in the promoter region of γ-TMT3 was associated with both α-tocopherol content and concentration in soybean seeds.
Plant material and growing conditions
A total of 140 F2 seeds derived from crosses between Ichihime and KAS were used for QTL mapping. The distal portion of each seed was cut off and used for tocopherol concentration analysis. The F2 seeds were grown in commercial potting soil (Katakura Chikkarin Co., Ltd., Japan) in the greenhouse of Hokkaido University, Japan (43°0'N, 141°21'E) in 2005. Ten seeds from each plant were collected and bulked for tocopherol concentration analysis. Leaves were harvested from each plant, frozen immediately in liquid nitrogen and stored at -30°C until DNA extraction.
For gene expression and tocopherol quantification analysis in developing seeds, HIF-derived lines were used. An HIF (F5-24) was identified as being heterozygous around the γ-TMT3 locus based on the genotypes of the SSR markers at flanking loci. The plant was selfed to obtain lines that were homozygous for either Ichihime or KAS marker alleles around the γ-TMT3 locus. Three lines homozygous for the Ichihime alleles (24-10, 24-14, 24-15) and three lines homozygous for the KAS alleles (24-7, 24-18, 24-22) were used for analysis; these sets of lines are referred to as Ichihime lines and KAS lines, respectively. All lines were grown at the Hokkaido University experimental farm in June 2008. Seeds of each plant were sampled at 30 days after flowering (DAF), 40 DAF and 50 DAF. The seeds were immediately frozen in liquid nitrogen and stored at -80°C until gene expression and tocopherol content analyses.
Extraction and HPLC analysis of tocopherols
Tocopherols were extracted from mature seeds and analyzed by reverse-phase high performance liquid chromatography (HPLC) following the procedure described by Dwiyanti et al. .
For F2 seeds, a distal portion of the seed was cut off with razor blade and cut into bits. Ten mg of sample was weighed and sonicated in 300 μl of 80% aqueous ethanol for 10 min at room temperature. Hexane (600 μl) was added to the sample for extraction. The sample was let sit at 4°C before being centrifuged at 13,000 rpm for 5 min using a refrigerated centrifuge (Eppendorf centrifuge 5417R, Eppendorf). The upper (hexane) phase was transferred to an HPLC vial (Waters Corp., Japan). Analysis was performed in an HPLC system (Hitachi LaChrom Elite, Hitachi High-Technologies Corp., Japan) with an Inertsil ODS-3 reverse-phase column (3.0 mm × 250 mm, GL Sciences, Japan). Column temperature was maintained at 40°C and separation was performed under isocratic condition for 25 min. Solvent A was acetonitrile, solvent B was methanol, and the ratio of solvent A to solvent B was 75:25 (v/v). Flow rate was 0.5 ml/min. Tocopherols were detected at the wavelength of 295 nm.
For F3 seeds derived from F2 plants, five seeds from each plant were bulked and ground into fine powder. Seed powder (50 mg) was weighed into a 15-ml test tube. The powder was sonicated in 1 ml of 80% aqueous ethanol for 15 minutes at room temperature. After incubation at 4°C for 30 min, the sample was centrifuged for 10 min at 2,500 rpm in a Tomy RL-100 centrifuge (Tomy Seiko Co., Japan). The upper phase was transferred to an HPLC vial. Analysis was performed in an HPLC system (Hitachi LaChrom Elite, Hitachi High-Technologies Corp., Japan) with same column as used for the F2 seed analysis. Column temperature was maintained at 40°C and separation was performed under isocratic condition for 25 min. The mobile phase was acetonitrile:methanol at a ratio 90:10 (v/v) ratio. Flow rate was 0.5 ml/min. Tocopherols were detected at the wavelength of 295 nm. Each analysis was performed twice.
Tocopherol extraction and quantification of developing seeds were performed based on a procedure developed previously  with several modifications. Twenty mg of freeze-dried seed powder was stirred in 1 ml cold acetone. The sample was sonicated at room temperature for 20 min. After the sonication, the sample was incubated at 4°C for 30 min. Centrifugation was performed twice, at 13,000 rpm for 10 min each time using a refrigerated centrifuge (Eppendorf centrifuge 5417R, Eppendorf). The upper solution was transferred into an HPLC vial. The analysis was performed using a Hitachi LaChrom Elite with a reverse-phase column (Inertsil ODS-3, 4.6 mm × 250 mm). The column temperature was maintained at 40°C. The analysis was performed under isocratic condition, with a mobile phase of ethyl acetate:75% methanol at a ratio of 50:50 (v/v). Tocopherols were detected by UV light with the detection wavelength set at 295 nm. Each analysis was performed three times.
Tocopherol content in the sample was calculated against the peak area of dl-tocol (Tama Biochemical Co. Ltd. Japan). dl-Tocol was added into the 80% ethanol or acetone used in the extraction at a concentration of 3 μg/ml.
Leaves from each F2 plants were sampled and stored at -30°C until DNA extraction. Genomic DNA isolation was performed according to the CTAB method as described by Dwiyanti . About 0.2 g of leaf tissue ground in liquid nitrogen was added to 700 μl of cetyl trimethyl ammonium bromide (CTAB) extraction buffer. After 30 min incubation at 60°C, the extract was mixed with 700 μl of chloroform:isoamyl alcohol (24:1 v/v), and centrifuged at 10,000 rpm for 5 min in a refrigerated centrifuge Tomy MR150 (Tomy Seiko Co., Japan). The aqueous solution was transferred to a 1.5-ml tube, and mixed with 500 μl of cold isopropanol for nucleic acid precipitation. Crude nucleic acids were collected by centrifugation at 10,000 rpm for 5 min in a refrigerated centrifuge Tomy MR150 (Tomy Seiko Co., Japan). The nucleic acid pellet was washed with 150 μl of 70% ethanol and the remaining liquid was evaporated. The pellet was then dissolved in TE buffer. RNA was precipitated by lithium chloride as described in . About 20 ng of total DNA was used as the template for PCR analysis.
SSR markers were selected from the soybean consensus linkage map  to cover all soybean linkage groups and tested for polymorphism between Ichihime and KAS. Additional SSR markers were developed based on the soybean genomic database Phytozome  and soybean SSR database BARCSOYSSR_1.0 . Genotypes of 148 selected SSR markers were determined in F2 plants. The DNA band for each marker was amplified by using the PCR procedure described previously . Amplified products were separated on either 3% Agarose S (Wako Pure Chemical Industries, Ltd), 4% NuSieve Agarose S (Cambrex Bio Science Rockland, Inc.), or 10% polyacrylamide gel. The gel was stained with ethidium bromide, and DNA bands were photographed under UV light.
Genetic mapping and QTL analysis
A linkage map based on the genotypes of 152 SSR markers in 122 F2 plants was constructed using MapManager QTX . Map distances were calculated in centiMorgans (cM) by using the Kosambi function.
QTL analyses for α-tocopherol concentration, γ-tocopherol concentration and δ-tocopherol concentration were carried out in both F2 seeds and F2 plants. For F2 plants, QTL analyses for α-tocopherol content and γ-tocopherol content were also performed. Permutation analysis (1,000 times) was performed to determine the genome-wide minimum significant LOD threshold score. Based on the analysis result, QTLs with LOD score exceeding 2.8 were regarded as effective loci. Initial QTL mapping was performed by using the interval mapping (IM) method provided in MapQTL 5.0 . Markers flanking the QTLs were used as cofactors in QTL mapping by using the MQM method in the same program.
F5 plants were generated from F2 plants by using the single-seed-descent method. These F5 plants were planted at the Hokkaido University experimental farm, Japan (43°0'N, 141°21'E) in June 2007. Ten seeds from each plant were bulked for tocopherol concentration analysis, and the leaves of each plant were used for DNA genotyping. Tocopherol quantification was performed with the same method used for F3 seeds. DNA was extracted from leaves with the CTAB method.
Primers used for fine mapping.
Nucleotide sequence (5' to 3')
Phylogenetic analysis and plastid transit peptide prediction
Amino acid sequences of γ-TMT1, γ-TMT2, and γ-TMT3 were obtained from the Phytozome database . Amino acid sequences of γ-TMT homologs from cyanobacteria (Synechococcus sp. PCC 7002 [ACA99779.1]), green algae (Chlamydomonas reinhardtii [CA159122.1]), plants (Lotus japonicus [DQ013360.1], Medicago truncatula [AY962639.1], Arabidopsis [AT1G64970], sunflower [DQ229832.1 and DQ229834.1], rapeseed [EU637012.1, EU637013.1, EU637014.1. EU637015.1], maize [AJ634706.1], rice (Oryza sativa L.) [BAD07529.1], wheat (Triticum aestivum L.) [CA177219.2], and Perilla frutescens [AF213481.1]) were obtained from TAIR  and NCBI GenBank . The sequences were aligned by the ClustalW function in MEGA 4.0 software . A phylogenetic tree of the proteins was constructed by using the neighbor-joining method in MEGA 4.0 software . A bootstrap (resampling) test was performed 1,000 times to determine the distances between proteins. Plastid transit peptide prediction was performed using ChloroP 1.1 .
Gene cloning and sequencing
Genomic DNA samples from high α-tocopherol soybean varieties (KAS, Dobrogeance, and Dobrudza 14 Pancevo) and typical soybean varieties (Ichihime, Toyokomachi, and Williams 82) were isolated by the CTAB method described in the genotyping section. Primer pairs were designed based on γ-TMT3 (Glyma09g35680.1) genomic information . γ-TMT3 fragments were amplified by using the following PCR conditions: initial denaturation step at 95°C for 5 min; followed by 35 cycles of 95°C for 30 s, annealing temperature for 30 s, 72°C for 1 min; followed by a final extension step at 72°C for 7 min. PCR products were separated in 1% Agarose S gel (Wako Pure Chemical Industries, Ltd). Expected amplification products were excised from the gel, precipitated with ethanol and ligated into the pGEM-T Easy vector (Promega Corp.). Vectors containing DNA fragments were transformed into Escherichia coli strain JM109. After overnight culture, plasmids were isolated by using Wizard SV Plus Minipreps (Promega Corp.). DNA fragments were treated with a Big Dye Terminator Cycle Sequencing ver.3.1 kit (Applied Biosystems) with the following reaction conditions: 30 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 2 min. DNA fragments were sequenced by using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) and the sequences were aligned using the BioEdit Sequence Alignment Editor .
Total RNA was extracted from developing seeds or leaves following the lithium chloride precipitation procedure  with several modifications. After frozen tissue (about 200 mg) was ground to a fine powder in liquid nitrogen, 150 μl of Tris-saturated phenol (pH 8.0) and 500 μl of extraction buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 100 mM NaCl, 1% SDS) were added to the frozen powder. The mixture was ground thoroughly. Three-hundred μl of chloroform:isoamyl alcohol (24:1 v/v) was added to the sample, the solution was vortexed, and the aqueous and organic layers were separated by centrifugation (15,000 rpm, 10 min, 4°C) in a refrigerated centrifuge (HITACHI Himac CF15RX II, Tokyo, Japan). The aqueous phase was transferred into a 1.5-ml tube. The chloroform:isoamyl alcohol treatment was performed twice. The RNA was precipitated by the addition of 0.3 volumes of 10 M lithium chloride. After being stored at 4°C overnight, the solution was centrifuged (15,000 rpm, 15 min, 4°C). The RNA pellet was dried by leaving the tube opened on ice. The RNA pellet was resuspended in RNase free water.
DNA was removed from the resuspended pellet by DNase I treatment. Ten units of DNase I (TaKaRa) and DNase I buffer was added into the RNA solution. The mixture was incubated at 37°C for 30 min. RNA was precipitated again in the presence of 0.3 M sodium acetate and 2.5 volumes of ethanol. The RNA pellet was dried, and again resuspended in RNase free water.
Quantitative RT-PCR analysis
Each cDNA was synthesized from 1 μg of total RNA by using the M-MLV reverse transcriptase system (Invitrogen) with random hexamer primers according to the manufacturer's instructions. After synthesis, one volume of cDNA was diluted with four volumes of nuclease-free water.
Primers used for gene expression analysis.
Nucleotide sequence (5' to 3')
Bioinformatic analysis of the promoter sequences
The upstream 1.3 kb regions of γ-TMT3 from Ichihime, Toyokomachi, Williams 82, KAS, Dobrogeance, and Dobrudza 14 Pancevo were analyzed. Regulatory elements in these regions were analyzed using program PLACE  and PLANTCARE .
Generation of transgenic Arabidopsis harboring GUS gene under the control of γ-TMT3 promoter
The 1.2 kb region upstream the transcriptional start site in the γ-TMT3 promoter was amplified from Ichihime and KAS, cloned into PCR®8/GW/TOPO® vector (Invitrogen). The plasmids were sequenced. The promoter fragments were inserted into a plant expression vector pMDC100  containing a β-glucuronidase (GUS) reporter gene . The construct was introduced into Agrobacterium tumefaciens strain EHA105. Arabidopsis thaliana ecotype Columbia plants were transformed with A. tumefaciens harboring the expression vector using a floral-dip method .
GUS histochemical and activity analyses
For GUS histochemical assay of transgenic Arabidopsis, leaves from T2 plants were soaked with staining solution containing 1 mg ml-1 of 5-bromo-4-chloro-3-indoyl-β-d-glucuronide (X-Gluc) based on protocol described by . The soaked leaves were vacuumed for 10 minutes and incubated overnight at 37°C. The chlorophylls were removed by a rinse with 99.5% ethanol after staining treatment.
For GUS activity assay, crude protein was extracted from leaves of T2 plants with 200 μl of extraction buffer containing 50 mM of sodium phosphate (pH 7.0), 10 mM of EDTA (pH 8.0), 0.1% of SDS, and 0.1% of Triton X-100. Sixteen μl of the extract was mixed with 50 μl of 1 mM 4-methylumbelliferyl-β-D-glucuronide (4-MUG) and 34 μl of extraction buffer, and incubated at 37°C for 0 min, 30 min, and 60 min. The reactions were stopped by adding 200 μl of 0.2 M sodium carbonate. The fluorescence of 4-methylumbelliferone (4-MU) derived from the reaction was measured using Wallac ArvoTM 1420 Multilabel Counter (Perkin Elmer). Protein content in the extracts was determined using Quick Start™ Bradford Protein Assay Kit (Bio-Rad Laboratories). GUS activity was expressed as pmol 4-MU·min-1·mg protein.
Sequence data from this article can be found in the GenBank/EMBL/DDBJ data libraries under the following accession number: Ichihime γ-TMT3 promoter (AB617792), KAS γ-TMT3 promoter (AB617793), Toyokomachi γ-TMT3 promoter (AB617794), Williams 82 γ-TMT3 promoter (AB617799), KAS γ-TMT3 coding sequence (AB617795), Ichihime γ-TMT3 coding sequence (AB617796), KAS γ-TMT3 genome (AB617797), and Ichihime γ-TMT3 genome (AB617798).
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants-in-Aid for Scientific Research, 19380001. Project for Utilizing Advanced Technologies in Agriculture, Forestry and Fisheries, 18063. We would like to thank Dr. Satoshi Watanabe for helpful advice in developing SSR markers for fine mapping. We would like to thank Ms. A. Shibayama, Ms. K. Ishimoto, and Ms. K. Asano for their technical assistance.
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