Wheat Protein Disulfide Isomerase Research Articles

Functional analysis of TaPDI genes on storage protein accumulation by CRISPR/Cas9 edited wheat mutants

Wheat protein disulfide isomerase (PDI) is involved in the formation of glutenin macropolymers (GMP) and the correct folding and accumulation of storage proteins in endosperm. In present study, seven types of homozygous TaPDI gene edited mutants were obtained by CRISPR/Cas9 technology, which were confirmed by PCR-RE and sequencing. Compared with other mutants and wild type (WT), the grain length and width in mutant PDI-abd-6 which was edited for the three TaPDI homoeologous genes were reduced, and the grain middle parts were slumped. The GMP size in PDI-abd-6 was not significantly different from that in WT, whereas the accumulation of protein bodies (PBs) increased during grain development. The endosperm cells became denser in PDI-abd-6 without sheet-like structure, and the expression level of TaBiP gene was significantly decreased. Particularly, the GMP content in PDI-abd-6 is also decreased significantly. The basic bread and flour rheological parameters in the mutant were negatively changed compared with those in WT. Our results indicated that TaPDI genes affects wheat flour-processing quality by the order of TaPDI-4B, TaPDI-4D, and TaPDI-4A from high to low; the expression of either one TaPDI could be enough to maintain the GMP accumulation and processing properties of wheat dough.

PDI-Regulated Disulfide Bond Formation in Protein Folding and Biomolecular Assembly.

Disulfide bonds play a pivotal role in maintaining the natural structures of proteins to ensure their performance of normal biological functions. Moreover, biological molecular assembly, such as the gluten network, is also largely dependent on the intermolecular crosslinking via disulfide bonds. In eukaryotes, the formation and rearrangement of most intra- and intermolecular disulfide bonds in the endoplasmic reticulum (ER) are mediated by protein disulfide isomerases (PDIs), which consist of multiple thioredoxin-like domains. These domains assist correct folding of proteins, as well as effectively prevent the aggregation of misfolded ones. Protein misfolding often leads to the formation of pathological protein aggregations that cause many diseases. On the other hand, glutenin aggregation and subsequent crosslinking are required for the formation of a rheologically dominating gluten network. Herein, the mechanism of PDI-regulated disulfide bond formation is important for understanding not only protein folding and associated diseases, but also the formation of functional biomolecular assembly. This review systematically illustrated the process of human protein disulfide isomerase (hPDI) mediated disulfide bond formation and complemented this with the current mechanism of wheat protein disulfide isomerase (wPDI) catalyzed formation of gluten networks.

The wPDI Redox Cycle Coupled Conformational Change of the Repetitive Domain of the HMW-GS 1Dx5-A Computational Study.

The repetitive sequence of glutenin plays an important role in dough rheology; however, its interaction with wheat protein disulfide isomerase (wPDI) remains unclear. In this study, the conformations of wild type glutenin repetitive sequence (WRS) from the high molecular weight glutenin subunit (HMW-GS) 1Dx5, an artificially designed glutenin repetitive sequence (DRS) of which the amino acid composition is the same but the primary structure is different, and wPDI under different redox states were simulated. The molecular interactions between the aforementioned repetitive sequences with wPDI under different redox states were further investigated. The results indicated that the repetitive sequences bind to the b and b′ domains of an “open”, oxidized wPDI (wPDIO) which serves as the acceptor state of substrate. The repetitive sequence is partially folded (compressed) in wPDIO, and is further folded in the thermodynamically favored, subsequent conformational transition of wPDIO to reduced wPDI (wPDIR). Compared with the artificially designed one, the naturally designed repetitive sequence is better recognized and more intensively folded by wPDI for its later unfold as the molecular basis of dough extension.

Wheat protein disulfide isomerase improves bread properties via different mechanisms

Gluten network formation by the oxidation of glutenin sulfhydryl group majorly impacts the subsequent dough and bread properties, and an evolutionary list of chemical oxidants has been used as improvers in bread making. A systematic comparison between azodicarbonamide (ADA), Vc, wheat protein disulfide isomerase (wPDI) and disulfide bond formation protein C (DsbC) of their effects on the alveographic characters of dough and texture properties of subsequent bread was performed. Results show that wPDI improves dough alveographic characters and bread texture properties better in most aspects than other reagents. Free sulfhydryl analysis finds that addition of wPDI increased the free sulfhydryl content in both dough and bread. Compare with inorganic reagents and its bacterial homologue, improving the dough and bread properties with less oxidation of sulfhydryl lead to the proposal that wPDI acts by catalyzing the formation of rheologically active disulfide and reduction of inactive ones in a substrate specific manner.

Characterization of wheat endoplasmic reticulum oxidoreductin 1 and its application in Chinese steamed bread

This study investigated characteristics of recombinant wheat Endoplasmic Reticulum Oxidoreductin 1 (wEro1) and its influence on Chinese steamed bread (CSB) qualities. The purified wEro1 monomer, which contained two conserved redox active motif sites, bound to flavin adenine dinucleotide (FAD) cofactor with a molecular weight of ∼47 kDa. wEro1 catalyzed the reduction of both bound and free FAD, and its reduction activity of free FAD reached 7.8 U/mg. Moreover, wEro1 catalyzed the oxidation of dithiothreitol and wheat protein disulfide isomerase (wPDI). Both glutathione and the reduced ribonuclease could work as electron donors for wEro1 in catalyzing the oxidation of wPDI. Additionally, wEro1 supplementation improved the CSB qualities with an increased specific volume of CSB and decreased crumb hardness, which was attributed to water-insoluble wheat proteins increasing and gluten network strengthening. The results give an understanding of the properties and function of wEro1 to facilitate its application especially in the flour-processing industry.

Improvements of Modified Wheat Protein Disulfide Isomerases with Chaperone Activity Only on the Processing Quality of Flour

Wheat protein disulfide isomerase (wPDI) with oxidoreductase activity, isomerase activity, and chaperone activity catalyzes the formation of disulfide bonds in gluten in vitro; the addition of wild-type wPDI weakened yet the flour processing quality. To develop the potential flour improvers from wPDI, the modified wPDIs were prepared by the biological or chemical method, and their effects on the processing quality of flour in connection with bread making were investigated by farinograph, texture profile analysis, electrophoresis, size exclusion chromatography, and scanning electron microscopy. A truncated protein of wPDI, fragment AB with oxidoreductase, and isomerase activities only was firstly confirmed to exert deteriorative effects similar to wPDI on the dough and bread quality. Then other two modified wPDIs, mPDI and aPDI, were prepared by site-directed mutagenesis and alkylation, respectively. Both mPDI and aPDI considerably retained the chaperone activity of wPDI but completely lost oxidoreductase and isomerase activities. After adding the appropriate amount of mPDI or aPDI, the stability time of dough was significantly prolonged from 3.60 to 4.15 or 4.13 min, respectively. The enhanced gluten network matrix and decreased hardness and chewiness of bread further suggested that dough was strengthened by the treatment of mPDI or aPDI. Moreover, the formation of gluten network was facilitated by the modified wPDIs with chaperone activity only for the increase amount of gluten macropolymer and the decrease content of SDS-soluble gluten. Consequently, mPDI and aPDI are valid candidates of flour improvers in food industry.

Expression and characterization of protein disulfide isomerase family proteins in bread wheat.

BackgroundThe major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses.ResultsPDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aβ, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm.ConclusionsHigh expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0460-2) contains supplementary material, which is available to authorized users.

Cloning and characterization of wheat PDI (protein disulfide isomerase) homoeologous genes and promoter sequences

The genomic and cDNA sequences of three PDI homoeologous genes located on chromosomes 4A, 4B and 4D of bread wheat and their promoters were cloned and sequenced. The three sequences showed a very high conservation of the coding region and of the exon/intron structure, which consisted of ten exons. The comparison of wheat sequences with those of rice and Arabidopsis showed a significant conservation of the exon/intron structure across the three species. The expression of each gene was analysed by RT-PCR in different plant tissues (roots, coleoptiles, spikelets, leaves and developing caryopses). All the genes showed a higher expression in developing caryopses than in other analysed tissues, wherein some differences were detected. The promoter sequences of the three genes possessed some regulatory motifs typical of endosperm specific expression.

Molecular characterization of gene sequences coding for protein disulfide isomerase (PDI) in durum wheat (Triticum turgidum ssp. durum)

The organisation of the durum wheat genomic sequence (3.5 kb) coding for protein disulfide isomerase (PDI), deduced by comparison between genomic fragments and cDNA sequences (1.5 kb) isolated from immature caryopses, is described. The gene structure consists of ten exons and nine introns. The presence of consensus sequences involved in splicing, such as intron-exon junctions and branchpoint, has been observed and discussed. Although the deduced wheat PDI amino acid sequence exhibited an overall identity of only 31% to that of human PDI, their modular architecture in terms of number, size, location and secondary structure-propensities of the constituent domains are remarkably similar. The comparison of the amino acid sequences with the eight available plant PDI-like sequences showed a high identity with four of them and low with the remaining ones. Analyses of transcription levels showed that the PDI mRNA was present in all analysed tissues, with much higher expression in immature caryopses.

Restriction Fragment Length Polymorphism (RFLP) for protein disulfide isomerase (PDI) gene sequences in Triticum and Aegilops species

RFLP variation revealed by protein disulfide isomerase (PDI) coding gene sequences was assessed in 170 accessions belonging to 23 species of Triticum and Aegilops. PDI restriction fragments were highly conserved within each species and confirmed that plant PDI is encoded either by single-copy sequences or by small gene families. The wheat PDI probe hybridized to single EcoRI or HindIII fragments in different diploid species and to one or two fragments per genome in polyploids. Four Aegilops species in the Sitopsis section showed complex patterns and high levels of intraspecific variation, whereas Ae. searsii possessed single monomorphic fragments. T. urartu and Ae. squarrosa showed fragments with the same mobility as those in the A and D genomes of Triticum polyploid species, respectively, whereas differences were observed between the hybridization patterns of T. monococcum and T. boeoticum and that of the A genome. The single fragment detected in Ae. squarrosa was also conserved in most accessions of polyploid Aegilops species carrying the D genome. The five species of the Sitopsis section showed variation for the PDI hybridization fragments and differed from those of the B and G genomes of emmer and timopheevi groups of wheat, although one of the Ae. speltoides EcoRI fragments was similar to those located on the 4B and 4G chromosomes. The similarity between the EcoRI fragment located on the 1B chromosome of common and emmer wheats and one with a lower hybridization intensity in Ae. longissima, Ae. bicornis and Ae. sharonensis support the hypothesis of a polyphyletic origin of the B genome.

Purification, Characterization, and Intracellular Localization of Glycosylated Protein Disulfide Isomerase from Wheat Grains

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.