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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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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DOI: https://doi.org/10.1007/s11103-007-9139-x