An asparagine residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm

BMC Plant Biology

Research Article

Stefania Masci¹  Eleonora Egidi¹  Renato D’Ovidio¹  Francesco Sestili¹  Domenico Lafiandra¹  Michela Janni²  Aldo Ceriotti³  Donald D Kasarda⁴  William H Vensel⁴ 

[1] DAFNE, Tuscia University, Viterbo, Italy;DAFNE, Tuscia University, Viterbo, Italy;Institute of Plant Genetics (IGV), CNR, Via Amendola 165/A, 70126, Bari, Italy;IBBA, CNR, Milan, Italy;USDA, ARS, WRRC, Albany, CA;

关键词: Asparaginyl endopeptidase;    Gluten protein maturation;    Low molecular weight glutenin subunits;    Proteomic analysis;    Genetic transformation;    Transgenic plants;    Wheat;   

DOI  :  10.1186/1471-2229-14-64

 received in 2014-01-13, accepted in 2014-03-07,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundWheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase.ResultsWe performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing.ConclusionsOur results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.

【 授权许可】

Unknown   
© Egidi et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.