Dataset Information

Genetic variation of ?-tocopherol methyltransferase gene contributes to elevated ?-tocopherol content in soybean seeds.

ABSTRACT: BACKGROUND: Improvement of ?-tocopherol content is an important breeding aim to increase the nutritional value of crops. Several efforts have been conducted to improve the ?-tocopherol content in soybean [Glycine max (L.) Merr.] through transgenic technology by overexpressing genes related to ?-tocopherol biosynthesis or through changes to crop management practices. Varieties with high ?-tocopherol content have been identified in soybean germplasms. The heritability of this trait has been characterized in a cross between high ?-tocopherol variety Keszthelyi Aproszemu Sarga (KAS) and low ?-tocopherol variety Ichihime. In this study, the genetic mechanism of the high ?-tocopherol content trait of KAS was elucidated. RESULTS: Through QTL analysis and fine mapping in populations from a cross between KAS and a Japanese variety Ichihime, we identified ?-TMT3, which encodes ?-tocopherol methyltransferase, as a candidate gene responsible for high ?-tocopherol concentration in KAS. Several nucleotide polymorphisms including two nonsynonymous mutations were found in the coding region of ?-TMT3 between Ichihime and KAS, but none of which was responsible for the difference in ?-tocopherol concentration. Therefore, we focused on transcriptional regulation of ?-TMT3 in developing seeds and leaves. An F5 line that was heterozygous for the region containing ?-TMT3 was self-pollinated. From among the progeny, plants that were homozygous at the ?-TMT3 locus were chosen for further evaluation. The expression level of ?-TMT3 was higher both in developing seeds and leaves of plants homozygous for the ?-TMT3 allele from KAS. The higher expression level was closely correlated with high ?-tocopherol content in developing seeds. We generated transgenic Arabidopsis plants harboring GUS gene under the control of ?-TMT3 promoter from KAS or Ichihime. The GUS activity assay showed that the activity of ?-TMT3 promoter from KAS was higher than that of Ichihime. CONCLUSIONS: The genetic variation in ?-TMT3, which plays a major role in determining ?-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 ?-tocopherol content.

SUBMITTER: Dwiyanti MS

PROVIDER: S-EPMC3220646 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Genetic variation of γ-tocopherol methyltransferase gene contributes to elevated α-tocopherol content in soybean seeds.

Dwiyanti Maria S MS Yamada Tetsuya T Sato Masako M Abe Jun J Kitamura Keisuke K

BMC plant biology 20111107

<h4>Background</h4>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 ...[more]

PMID: 22053941

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Project description:BackgroundTocopherols are natural antioxidants with both in vivo (vitamin E) and in vitro activity. Sunflower seeds contain predominantly alpha-tocopherol (>90% of total tocopherols), with maximum vitamin E effect but lower in vitro antioxidant action than other tocopherol forms such as gamma-tocopherol. Sunflower germplasm with stable high levels of gamma-tocopherol (>85%) has been developed. The trait is controlled by recessive alleles at a single locus Tph2 underlying a gamma-tocopherol methyltransferase (gamma-TMT). Additionally, unstable expression of increased gamma-tocopherol content in the range from 5 to 85% has been reported. The objective of this research was to determine the genetic basis of unstable expression of high gamma-tocopherol content in sunflower seeds.ResultsMale sterile plants of nuclear male sterile line nmsT2100, with stable high gamma-tocopherol content, were crossed with plants of line IAST-1, with stable high gamma-tocopherol content but derived from a population that exhibited unstable expression of the trait. F2 seeds showed continuous segregation for gamma-tocopherol content from 1.0 to 99.7%. Gamma-tocopherol content in F2 plants (average of 24 individual F3 seeds) segregated from 59.4 to 99.4%. A genetic linkage map comprising 17 linkage groups (LGs) was constructed from this population using 109 SSR and 20 INDEL marker loci, including INDEL markers for tocopherol biosynthesis genes. QTL analysis revealed a major QTL on LG 8 that corresponded to the gamma-TMT Tph2 locus, which suggested that high gamma-tocopherol lines nmsT2100 and IAST-1 possess different alleles at this locus. Modifying genes were identified at LGs 1, 9, 14 and 16, corresponding in most cases with gamma-TMT duplicated loci.ConclusionsUnstable expression of high gamma-tocopherol content is produced by the effect of modifying genes on tph2a allele at the gamma-TMT Tph2 gene. This allele is present in line IAST-1 and is different to allele tph2 present in line nmsT2100, which is not affected by modifying genes. No sequence differences at the gamma-TMT gene were found associated to allelic unstability. Our results suggested that modifying genes are mostly epistatically interacting gamma-TMT duplicated loci.

Project description:BackgroundThe essential sulfur-containing amino acid methionine plays a vital role in plant metabolism and human nutrition. In this study, we aimed to elucidate the regulatory role of the first committed enzyme in the methionine biosynthesis pathway, cystathionine γ-synthase (CGS), on methionine accumulation in tobacco seeds. We also studied the effect of this manipulation on the seed's metabolism.ResultsTwo forms of Arabidopsis CGS (AtCGS) were expressed under the control of the seeds-specific promoter of legumin B4: feedback-sensitive F-AtCGS (LF seeds), and feedback-insensitive T-AtCGS (LT seeds). Unexpectedly, the soluble content of methionine was reduced significantly in both sets of transgenic seeds. Amino acids analysis and feeding experiments indicated that although the level of methionine was reduced, the flux through its synthesis had increased. As a result, the level of protein-incorporated methionine had increased significantly in LT seeds by up to 60%, but this was not observed in LF seeds, whose methionine content is tightly regulated. This increase was accompanied by a higher content of other protein-incorporated amino acids, which led to 27% protein content in the seeds although this was statistically insignificantly. In addition, the levels of reducing sugars (representing starch) were slightly but significantly reduced, while that of oil was insignificantly reduced. To assess the impact of the high expression level of T-AtCGS in seeds on other primary metabolites, metabolic profiling using GC-MS was performed. This revealed significant alterations to the primary seed metabolism manifested by a significant increase in eight annotated metabolites (mostly sugars and their oxidized derivatives), while the levels of 12 other metabolites were reduced significantly in LT compared to wild-type seeds.ConclusionExpression of T-AtCGS leads to an increase in the level of total Met, higher contents of total amino acids, and significant changes in the levels of 20 annotated metabolites. The high level of oxidized metabolites, the two stress-associated amino acids, proline and serine, and low level of glutathione suggest oxidative stress that occurs during LT seed development. This study provides information on the metabolic consequence of increased CGS activity in seeds and how it affects the seed's nutritional quality.

Project description:Soybean seeds are primary sources of natural tocopherols used by the food and pharmaceutical industries, owing to their beneficial impacts on human health. Selection for higher tocopherol contents in seeds along with other desirable traits is an important goal in soybean breeding. In order to identify the genomic loci and candidate genes controlling tocopherol content in soybean seeds, the bulked-segregant analysis technique was performed using a natural population of soybean consisting of 1525 accessions. We constructed the bulked-segregant analysis based on 98 soybean accessions that showed extreme phenotypic variation for the target trait, consisting of 49 accessions with extremely-high and 49 accessions with extremely-low tocopherol content. A total of 144 variant sites and 109 predicted genes related to tocopherol content were identified, in which a total of 83 genes were annotated by the gene ontology functions. Furthermore, 13 enriched terms (p < 0.05) were detected, with four of them found to be highly enriched: response to lipid, response to abscisic acid, transition metal ion transmembrane transporter activity, and double-stranded DNA binding. Especially, six candidate genes were detected at 41.8−41.9 Mb genomic hotspots on chromosome 5 based on ANNOtate VARiation analysis. Among the genes, only Glyma.05G243400 carried a non-synonymous mutation that encodes a “translation elongation factor EF1A or initiation factor IF2gamma family protein” was identified. The haplotype analysis confirmed that Glyma.05G243400 exhibited highly significant variations in terms of tocopherol content across multiple experimental locations, suggesting that it can be the key candidate gene regulating soybean seed tocopherols. The present findings provide novel gene resources related to seed tocopherols for further validation by genome editing, functional characterization, and genetic improvement targeting enhanced tocopherol composition in soybean molecular breeding.

Dataset Information

Genetic variation of ?-tocopherol methyltransferase gene contributes to elevated ?-tocopherol content in soybean seeds.

Publications

Genetic variation of γ-tocopherol methyltransferase gene contributes to elevated α-tocopherol content in soybean seeds.

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