Plant Molecular Biology

, Volume 64, Issue 1–2, pp 125–136

Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain

  • Paul R. Wiley
  • Paola Tosi
  • Alexandre Evrard
  • Alison Lovegrove
  • Huw D. Jones
  • Peter R. Shewry
Article

Abstract

The purolindolines are small cysteine-rich proteins which are present in the grain of wheat. They have a major impact on the utilisation of the grain as they are the major determinants of grain texture, which affects both milling and baking properties. Bread and durum wheats were transformed with constructs comprising the promoter regions of the Puroindoline a (Pina) and Puroindoline b (Pinb) genes fused to the uidA (GUS) reporter gene. Nine lines showing 3:1 segregation for the transgene and comprising all transgene/species combinations were selected for detailed analysis of transgene expression during grain development. This showed that transgene expression occurred only in the starchy endosperm cells and was not observed in any other seed or vegetative tissues. The location of the puroindoline proteins in these cells was confirmed by tissue printing of developing grain, using a highly specific monoclonal antibody for detection and an antibody to the aleurone-localised 8S globulin as a control. This provides clear evidence that puroindolines are only synthesised and accumulated in the starchy endosperm cells of the wheat grain.

Keywords

Grain texture Location Puroindolines Transgenic wheat 

Abbreviations

BSA

Bovine serum albumin

diX-indigo

5,5′-Dibromo-4,4′-dichloro-indigo (diX-indigo)

DTT

Dithiothreitol

ER

Endoplasmic reticulum

ESI-MS

Electrospray ionisation–mass spectrometry

GUS

β-Glucuronidase

MCP

Microchannel plate

NBT-BCIP

Nitro blue tetrazolium 5-bromo-4-chloro-3-indoyl-phosphate

PAT

Phosphinothricin acetyltransferase

PBS

Phosphate buffered saline

PCR

Polymerase chain reaction

Pin

Puroindoline

PPT

Phosphinothricin

Q-Tof

Quadrolpole time-of-flight

SDS

Sodium dodecyl sulphate

X-Gluc

5-Bromo-4-chloro-3-indolyl-β-d-glucuronic acid

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Paul R. Wiley
    • 1
    • 2
  • Paola Tosi
    • 1
  • Alexandre Evrard
    • 3
    • 4
  • Alison Lovegrove
    • 1
  • Huw D. Jones
    • 1
  • Peter R. Shewry
    • 1
  1. 1.Rothamsted ResearchHertfordshireUK
  2. 2.CSIRO Plant IndustryCanberraAustralia
  3. 3.INRA, UMR 1096PIAMontpellier Cedex 01France
  4. 4.Department of Botany and Plant SciencesUniversity of CaliforniaRiversideUSA

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