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

Background: Improvement of alpha-tocopherol content is an important breeding aim to increase the nutritional value of crops. Several efforts have been conducted to improve the alpha-tocopherol content in soybean [Glycine max (L.) Merr.] through transgenic technology by overexpressing genes related to alpha-tocopherol biosynthesis or through changes to crop management practices. Varieties with high alpha-tocopherol content have been identified in soybean germplasms. The heritability of this trait has been characterized in a cross between high alpha-tocopherol variety Keszthelyi Aproszemu Sarga (KAS) and low alpha-tocopherol variety Ichihime. In this study, the genetic mechanism of the high alpha-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 gamma-TMT3, which encodes gamma-tocopherol methyltransferase, as a candidate gene responsible for high alpha-tocopherol concentration in KAS. Several nucleotide polymorphisms including two nonsynonymous mutations were found in the coding region of gamma-TMT3 between Ichihime and KAS, but none of which was responsible for the difference in alpha-tocopherol concentration. Therefore, we focused on transcriptional regulation of gamma-TMT3 in developing seeds and leaves. An F-5 line that was heterozygous for the region containing gamma-TMT3 was self-pollinated. From among the progeny, plants that were homozygous at the gamma-TMT3 locus were chosen for further evaluation. The expression level of gamma-TMT3 was higher both in developing seeds and leaves of plants homozygous for the gamma-TMT3 allele from KAS. The higher expression level was closely correlated with high alpha-tocopherol content in developing seeds. We generated transgenic Arabidopsis plants harboring GUS gene under the control of gamma-TMT3 promoter from KAS or Ichihime. The GUS activity assay showed that the activity of gamma-TMT3 promoter from KAS was higher than that of Ichihime. Conclusions: The genetic variation in gamma-TMT3, which plays a major role in determining alpha-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 alpha-tocopherol content.