Open Access Research article

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

Eleonora Egidi1, Francesco Sestili1, Michela Janni14, Renato D’Ovidio1, Domenico Lafiandra1, Aldo Ceriotti2, William H Vensel3, Donald D Kasarda3 and Stefania Masci1*

Author Affiliations

1 DAFNE, Tuscia University, Viterbo, Italy

2 IBBA, CNR, Milan, Italy

3 USDA, ARS, WRRC, Albany, CA, USA

4 Present address: Institute of Plant Genetics (IGV), CNR, Via Amendola 165/A, 70126 Bari, Italy

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BMC Plant Biology 2014, 14:64  doi:10.1186/1471-2229-14-64

Published: 14 March 2014

Abstract

Background

Wheat 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.

Results

We 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.

Conclusions

Our 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.

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