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Planta

, Volume 236, Issue 1, pp 79–89 | Cite as

The soybean F3′H protein is localized to the tonoplast in the seed coat hilum

  • Kyoko TodaEmail author
  • Haruko Kuroiwa
  • Kalaiselvi Senthil
  • Norimoto Shimada
  • Toshio Aoki
  • Shin-ichi Ayabe
  • Setsuko Shimada
  • Masaaki Sakuta
  • Yasumasa Miyazaki
  • Ryoji Takahashi
Original Article

Abstract

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3′-hydroxylase (F3′H) gene (sf3′h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3′h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3′h1 was not detected in To7G. A truncated sf3′h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3′H was detected using microsomal fractions from yeast transformed with sf3′h1 from To7B, but not from To7G. These results indicate that sf3′h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3′h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3′h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3′H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

Keywords

Antioxidant activity Cytochrome P450 Flavonoid 3′-hydroxylase Soybean Tonoplast 

Abbreviations

CYP

Cytochrome P450

DAPI

4′-6-Diamidino-2-phenylindole

DPBA

Diphenylboric acid 2-aminoethyl ester

DPPH

1,1-Diphenyl-2-picrylhydrazyl

F3′H

Flavonoid 3′-hydroxylase

HPLC

High-performance liquid chromatography

NILs

Near-isogenic lines

ROS

Reactive oxygen species

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Kyoko Toda
    • 1
    Email author
  • Haruko Kuroiwa
    • 2
  • Kalaiselvi Senthil
    • 1
    • 6
  • Norimoto Shimada
    • 3
    • 7
  • Toshio Aoki
    • 3
  • Shin-ichi Ayabe
    • 3
  • Setsuko Shimada
    • 4
  • Masaaki Sakuta
    • 4
  • Yasumasa Miyazaki
    • 5
  • Ryoji Takahashi
    • 1
  1. 1.National Institute of Crop ScienceTsukubaJapan
  2. 2.Department of Life Science, College of ScienceRikkyo UniversityTokyoJapan
  3. 3.Department of Applied Biological ScienceNihon UniversityFujisawaJapan
  4. 4.Department of BiologyOchanomizu UniversityTokyoJapan
  5. 5.Forestry and Forest Products Research InstituteTsukubaJapan
  6. 6.Department of BiochemistryAvinashilingam UniversityCoimbatoreIndia
  7. 7.Tokiwa Phytochemical Co., LtdSakuraJapan

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