Abstract
The TaPR60 gene from bread wheat encodes a small cysteine-rich protein with a hydrophobic signal peptide, predicted to direct the TaPR60 protein to a secretory pathway. It was demonstrated by heterologous expression of recombinant TaPR60 protein that the signal peptide is recognized and cleaved in yeast cells. The full-length gene including promoter sequence of a TaPR60 orthologue was cloned from a BAC library of Triticum durum. A transcriptional promoter-GUS fusion was stably transformed into wheat, barley and rice. The strongest GUS expression in wheat and barley was found in the endosperm transfer cells, while in rice the promoter was active inside the starchy endosperm during the early stages of grain filling. The TaPR60 gene was also used as bait in a yeast two-hybrid screen. Five proteins were identified in the screen, and for some of these prey proteins, the interaction was confirmed by co-immunoprecipitation. The signal peptide binding proteins, TaUbiL1 and TaUbiL2, are homologues of animal proteins, which belong to proteolytic complexes, and therefore may be responsible for TaPR60 processing or degradation of the signal peptide. Other proteins that interact with TaPR60 may have a function in TaPR60 secretion or regulation of this process. Examination of a three dimensional model of TaPR60 suggested that this protein could be involved in binding of lipidic molecules.
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Abbreviations
- ETC:
-
Endosperm transfer cells
- BETL:
-
Basal endosperm transfer layers
- BAP2:
-
Basal layer type antifungal protein
- DAP:
-
Days after pollination
- dbEST:
-
GenBank expressed sequence tag database
- GUS:
-
β-Glucuronidase
- BAC:
-
Bacterial artificial chromosome
- Y2H:
-
Yeast two-hybrid
- Y3H:
-
yeast three-hybrid
- BD:
-
Binding domain
- AD:
-
Activation domain
- UBL:
-
Ubiquitin like domain
- UBA:
-
Ubiquitin associated domain
- APP:
-
Amyloid precursor protein
- BCP:
-
Blue copper protein
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Acknowledgments
We thank Professor U. Grossniklaus for providing us with the collection of pMDC vectors, and Dr. U. Langridge and R. Hosking for assistance with growing plants. This work was supported by the Australian Research Council, the Grains Research and Development Corporation and the Government of South Australia.
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The nucleotide sequences have been deposited in the GenBank database under GenBankAccession Numbers: FJ459807 (TdPR60), FJ459808 (TaUbiL1), FJ459809 (TaUbiL2), FJ459810 (TaBCP), FJ459811 (TaPlp-P), FJ459812 (TaHVA22L).
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11103_2009_9510_MOESM1_ESM.pdf
Supplementary Fig. 1 Alignment of protein sequences of TaUbiL1 (Acc. FJ459808) and TaUbiL2 (Acc. FJ459809) to similar proteins from mammals: mouse Ataxin/Ubiquilin4 (Acc. Q99NB8), human Ubiquilin1 (Acc. EAW62658), rat Ubiquilin1 (Acc. NP_446199). Identical amino acids are in black boxes, similar amino acids are in grey boxes. The UBI domain is underlined with a solid line, the conserved region with a dashed line, and the UBA domain with a dotted line. (PDF 1535 kb)
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Kovalchuk, N., Smith, J., Pallotta, M. et al. Characterization of the wheat endosperm transfer cell-specific protein TaPR60. Plant Mol Biol 71, 81–98 (2009). https://doi.org/10.1007/s11103-009-9510-1
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DOI: https://doi.org/10.1007/s11103-009-9510-1