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

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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.

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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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Acknowledgements

Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. We are grateful to Cristina Sanches-Gritsch and Raffaella Carzaniga (Rothamsted Research) for discussions and advice on microscopy and to Dr. Marie-Françoise Gautier (INRA, Montpellier) for providing the Pinb::uidA reporter construct and for further advice and discussion.

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Author notes

  1. Paul R. Wiley

    Present address: CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia

  2. Alexandre Evrard

    Present address: Department of Botany and Plant Sciences, University of California, 3220 Batchelor Hall, Riverside, CA, 92521, USA

Authors and Affiliations

  1. Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK

    Paul R. Wiley, Paola Tosi, Alison Lovegrove, Huw D. Jones & Peter R. Shewry

  2. INRA, UMR 1096PIA, 2 place Viala, 34060, Montpellier Cedex 01, France

    Alexandre Evrard

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  1. Paul R. Wiley
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Correspondence to Peter R. Shewry.

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Wiley, P.R., Tosi, P., Evrard, A. et al. Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain. Plant Mol Biol 64, 125–136 (2007). https://doi.org/10.1007/s11103-007-9139-x

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