Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 111, Issue 1, pp 1–13

Ectopic overexpression of peach GDP-d-mannose pyrophosphorylase and GDP-d-mannose-3′,5′-epimerase in transgenic tobacco

  • Tsuyoshi Imai
  • Yusuke Ban
  • Toshiya Yamamoto
  • Takaya Moriguchi
Original Paper

Abstract

We created transgenic tobacco overexpressing peach GDP-d-mannose pyrophosphorylase (PpGMPH) and GDP-d-mannose-3′,5′-epimerase (PpGME), which are involved in the main l-ascorbate (Asc) biosynthetic pathway in plants. Despite an apparent increase in enzymatic activity in the PpGMPH-overexpressing primary transgenic (T0) lines, no significant increase in Asc pool size was observed in leaves. For PpGME-overexpressing T0 lines, Asc pool size was also unchanged, although transcriptional overexpression was confirmed. To reinforce the biosynthetic pathway from d-mannose-1-phosphate to GDP-l-galactose, we further created PpGMPH- and PpGME-double overexpressing crossed transgenic (dOx T1) lines. Transcriptional overexpression of PpGMPH and PpGME transgenes was confirmed, and the Asc content in the dOx T1 lines was slightly elevated in young leaves, albeit not significantly. Western blot analyses revealed protein amounts of GMPH and GME were similar among dOx T1, parental T0 and non-transgenic (SR1) lines in three tissue types tested: leaves, flower buds and immature fruits. Moreover, a similar amount of GMPH and GME proteins were detected in young leaves (YL) and old leaves (OL), although Asc content in YL is roughly double as that in OL. In vitro assay using recombinant PpGME suggested the equilibrium of GME reaction is unfavorable to forward Asc biosynthesis at this step. Finally, to investigate substrate availability in relation to Asc pool size, we added exogenous GDP-d-mannose (5 mM) or l-galactose (5 mM) to tobacco leaf discs; the former resulted in no increase in Asc content, whereas a significant increase was observed in the latter, not only in the dOx T1 lines, but also in the original line SR1. Similar treatments were also conducted under high light, which resulted in a further increase in the increment of Asc content in the l-galactose-fed discs irrespective of transgene expression level. Taken together, both l-galactose availability and light rather than upper biosynthetic gene expression levels of GMPH/GME are critical determinant of Asc content in tobacco leaves, indicating complex modulation of this compound.

Keywords

l-ascorbate GDP-d-mannose-3′,5′-epimerase GDP-d-mannose pyrophosphorylase Overexpression Transgenic tobacco 

Abbreviations

Asc

l-Ascorbate

CaMV

Cauliflower mosaic virus

DHA

Dehydroascorbate

dOx T1

Double overexpressing crossed transgenic

DTT

Dithiothreitol

FB

Flower buds

GalUAR

d-Galacturonate reductase

GDH

l-Galactose dehydrogenase

GGP

GDP-l-galactose phophorylase

GLDH

l-Galactono-1,4-lactone dehydrogenase

GME

GDP-d-mannose-3′,5′-epimerase

GMPH

GDP-d-mannose pyrophosphorylase

GPP

l-Galactose-1-phosphate phosphatase

IF

Immature fruits

Km

Kanamycin

MS

Murashige and Skoog’s

OL

Old leaves

ROS

Reactive oxygen species

SDS

Sodium dodecyl sulfate

TLC

Thin layer chromatography

YL

Young leaves

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Tsuyoshi Imai
    • 1
    • 2
  • Yusuke Ban
    • 2
    • 3
  • Toshiya Yamamoto
    • 1
    • 2
  • Takaya Moriguchi
    • 1
    • 2
  1. 1.National Institute of Fruit Tree Science (NIFTS)National Agriculture and Food Research Organization (NARO)TsukubaJapan
  2. 2.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan
  3. 3.Grape and Persimmon Research Division, NIFTSNAROHigashi-hiroshimaJapan

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