Abstract
Western blotting, ELISA and 1H-NMR spectroscopy showed that RNAi down-regulation of the wheat Gsp-1 gene resulted in reduced contents of both arabinogalactan peptide (AGP) and grain softness protein (GSP-1) in mature wheat grains confirming that these components are encoded by the same gene. A small increase in grain hardness and decrease in the viscosity of aqueous extracts of the transgenic lines also indicated small effects on functional properties. Immunolocalisation using a novel wheat AGP monoclonal antibody in conjunction with confocal microscopy showed that the major form of AGP which was eliminated in knockout lines is located within the cell, probably in the vacuole, and not in the plasma membrane or cell wall. However, clear localisation of the AGP epitope to the plasma membrane was observed in both control and transgenic lines and probably resulted from the presence of one or more separate forms of arabinogalactan protein. The existence of such additional form(s) was also indicated by 1H-NMR spectroscopy which showed that the ratio of arabinose to galactose differed between the control and transgenic lines.
Highlights
The Gsp-1 genes of wheat are proposed to encode precursor proteins that are post-translationally processed to give two components: the arabinogalactan peptide and the grain softness protein (GSP-1) (Elmorjani et al, 2013)
ERÀ2ctR2.ERÀ3ctR3); where ET, ER1, ER2, ER3 are the efficiencies of the target (Pina, Pinb or Gsp-1) and the three reference genes used (Prosm, TaSDH and TaGAPDH); ctT, ctR1, ctR2, ctR3 are the corresponding numbers of cycles at threshold fluorescence set for the reactions; and the denominator of the expression is the geometric average of the relative expression of the three reference genes
It was concluded that phenotypic differences observed between pairs of transgenic were not secondary effect resulting from the suppression of Pin gene expression
Summary
The Gsp-1 genes of wheat are proposed to encode precursor proteins that are post-translationally processed to give two components: the arabinogalactan peptide (comprising 15 amino acids) and the grain softness protein (GSP-1) (the major form comprising 113 amino acids with 10 cysteine residues) (Elmorjani et al, 2013). It can be hypothesised that the AGP/GSP precursor protein is synthesised on the endoplasmic reticulum (ER), transferred into the lumen (a signal sequence of 19 amino acids being predicted) and post-translationally processed (proline hydroxylation, O-glycosylation) in the ER and Golgi before being proteolytically cleaved in the vacuole (Wilkinson et al, 2013) In support of this hypothesis, Elmorjani et al (2013) showed that the GSP precursor protein is further proteolytically cleaved at the N- and Ctermini to give four forms comprising between 110 and 113 residues. Immunolabelling shows that the major form of AGP encoded by Gsp-1 has an intracellular location, probably within the vacuoles that give rise to protein bodies, while analysis of mature grain shows small but statistically significant effects on both grain texture and on the viscosity of aqueous extracts of endosperm flour
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