1. Introduction

Wheat is adapted to temperate regions of the world and was one of the first crops to be domesticated. Spread over all continents, it is today one of the most important food source for human beings. Bread wheat (Triticum aestivum) belongs to the tribe Triticeae, and has three genomes A, B and D, each organised in seven homoeologous chromosome groups. The diploid progenitors of the three genomes A, B, and D have been identified in Triticum uratu, Aegilops speltoides and Aegilops tauschii, although the progenitor of B genome is still matter of debate ^[1]. Throughout their evolutionary history, multiple polyploidization events occurred between species of the Triticum and Aegilops genera and human manipulation of wild species led to the domestication of different cultivated lineages ^{[2, 3, 4, 5]}.

Wheat grains are consumed under many different forms, such as flour for leavened, flat and steamed breads, biscuits, cookies, cakes, breakfast cereal, pasta, noodles, couscous and for fermentation to make beer, alcohol, vodka and biofuel. It is composed by starch, proteins and other compounds, which accumulate in significant quantities during its development, in particular grain storage proteins, are responsible for the quality of the end product. The majority of them are prolamins, which account for > 90 % of the total protein content in the wheat grain. Different quality of grain storage proteins produced by the same genotype under different environmental condition has stimulated research on protein folding and assembling.

Secretory protein folding and disulphide bond formation takes place within the Endoplasmic reticulum (ER lumen), but the precise mechanisms involved, and the role of other proteins such as molecular chaperones, are not fully understood ^{[6, 7]}. PDI plays an important role in assisting protein folding and assembly, catalyzing thiol-disulfide oxidation, reduction and isomeration, this latter occurring directly by intramolecular disulfide rearrangement or through cycles of reduction and oxidation ^{[8, 9, 10]}. During the maturation of the secretory proteins, disulfide bonds cross-linking specific cysteines are added to stabilize a protein or to join covalently different polypeptides. These bonds are crucial for the stability of the final protein structure, thus mispairing of cysteine residues can prevent proteins from attaining their native conformation and lead to misfolding ^[11].

Typical PDI/Classical PDI is the most prominent member of a family of related proteins (PDI-like) characterised by one, two or three thioredoxin-like active domains ^[12]. It has been cloned and sequenced in many plant species, such as alfalfa ^[13], barley ^[14], maize ^[15], castor bean ^[16] and soybean ^{[17, 18, 19, 20]}, common and durum wheat ^{[21, 22]}. A detailed knowledge of the complexity and diversity of genes encoding PDI and PDI-like proteins in A. thaliana, wheat and other plant species was described by ^{[23, 24, 25]}.

Wheat genes coding for typical PDI in bread wheat have been located in homoeologous chromosome group four ^[26]. Analyses performed on 23 species of Triticum and Aegilops ^[27], indicated that PDI restriction fragments were highly conserved within each species and confirmed

The expression analysis of the typical PDI homoeologous genes located on chromosomes 4A, 4B and 4D of bread wheat cv Chinese Spring (CS) ^[28] showed that the PDI transcripts, although constitutively present at a low-level in all the analyzed tissues, are equally abundant in the developing caryopses, but are differentially expressed in spikelets, roots and leaves ^{[21, 28]}. The PDI-4A transcription was higher in spikelets that of PDI-4B were higher in roots while the PDI-4 D transcripts were more abundant in leaves. The transcription levels of the three genes were higher in the early stage of seed development (6-14 DAA) and decreased during middle to late stage of (18-34 DAA) grain filling ^[28]. Within the upstream putative promoter region of the three homoeologous genes cloned from bread wheat cv CS, respectively 1352 bp for PromPDI-4A, 1370 bp for PromPDI-4B and 1292 bp for PromPDI-4D long, several cis-acting elements involved in endosperm specific expression were detected, consistently with the higher PDI expression detected in the kernels.

The variability and evolutionary relationship in a ~700 bp region, comprising ~600 bp of the 5’ upstream putative promoter region and 88 bp of the first exon of the typical PDI gene from the diploid species Triticum uratu (AA) Aegilops speltoides (BB) and Aegilops tauschii (DD) has been reported ^{[30, 31, 32]}. This paper reports the cloning and characterization of ~1350 bp of the 5’ upstream putative promoter region and 88 bp of the first exon of the typical PDI gene from the diploid species Triticum uratu (AA) Aegilops speltoides (BB) and Aegilops tauschii (DD).

2. Materials and Methods

A total of three accessions Triticum uratu (IG 44831, AA), Aegilops speltoides (IG 46812, BB) and Aegilops tauschii (AE 1068, DD) were used in this study. Single plant per accessions was grown and used for DNA extraction. Flag leaves were collected at heading stage from plants grown in greenhouse (January- June 2008), immediately frozen in liquid nitrogen and kept at −80 ^◦C until use. About 200 mg of leaf tissue was ground in liquid nitrogen and genomic DNA was extracted using Sigma Gen Elute Plant Genomic DNA Kit (G2N-350, Sigma Aldrich, St. Louis, Mo.).

Primers were designed on the basis of the known homoeologous PDI promoter and gene sequences isolated from bread wheat cv Chinese Spring ^[28], using DNAMAN 4.15 program. Putative promoters of typical PDI were amplified by using the following primer pairs PDIAPDF1-PDIAPDR1 and PDIAPDF2-PDIAPDR2 for A Genome, PDIAPDF3- PDIAPDR3 and PDIAPDF2- PDIAPDR4 for B genome and PDIAPDF3- PDIAPDR1 and PDIAPDF2- PDIAPDR6 for D genome (Table 1), cloned and sequenced in each accession.

PCR reaction mixture included 10 ng/μl genomic DNA, 0.20 mM dNTPs 5ul, 0.05 units/µl of Taq 0.50 µl (go-taq, Promega), 0.40μM primer 2.0 µl (0.20µM per each), 5X buffer 10 µl and 25.5 µl of ddH2O used per reaction (final volume of 50 μl). PCR condition included initial denaturation step at 95°C for three min, then 32 cycles of (95°C for one min, 59°C for 35 sec, 72°C for one min) and final elongation at 72°C for amplification of the sequences from the A , B and D Genome.

Genome specific primer pair (Table 1) was used to amplify, clone and sequence the DNA extracted from one plant each of the three wild relatives. PCR products of expected size were excised from the gel, purified using the High Pure Purification kit (Roche) according to manufacturer’s instructions, and cloned into the pGEM-T easy plasmid vector (Promega). Plasmids were transformed by heat shock into Escherichia coli strain DH5_α. Bacteria were plated onto LB medium containing ampicillin, X-Gal and IPTG, and recombinant plasmids were identified by blue/white screening. For each primer combination two independent PCR reactions were performed and a total of six clones were sequenced. Plasmid DNA for sequencing reaction was prepared from three ml overnight cultures using a plasmid miniprep kit (Qiagen). Sequencing was performed on both strands by the ABI PRISM 377 DNA sequencer (PE Applied Biosystem) using an ABI Prism Dye Terminator sequencing kit (PE Applied Biosystem) and sequenced both with vector and sequence specific primers.

Sequences were analysed in Chromas version 2.3 (https://technelysium.com.au/chromas.html) to identify any unresolved bases and subjected to visual inspection. Analysis of three new sequences were done along with the three homoeologous gene and promoter sequences from CS (GPDI-4A, AJ868102; GPDIA-4B, AJ868103; GPDI-4D, AJ868104; PromPDI4A, AJ868108; PromPDI4B, AJ868109; PromPDI4D, AJ868110; ^[28]). Sequences were then searched for regulatory elements in PlantCARE (https://sphinx.rug.ac.be:8080/PlantCARE/), a database of plant promoters and (https://www.dna.affrc.go.jp/htdocs/PLACE/; ^[33]) PLACE database. The six sequences were multiple aligned with Clustal X software version 2.011 ^[34], IUB as DNA weight matrix with default parameters. A phylogenetic tree was constructed on obtained data by using the neighbour-joining (NJ) method ^[35], using MEGA version4 with neighbour joining option and 1000 bootstrap ^[36]. All three cloned sequences have been submitted in EMBL Nucleotide Sequence Database with sequence ID HE588130, HE588131 and HE588132 for Triticum uratu (IG 44831), Aegilops speltoides (IG 46812) and Aegilops tauschii (AE 1068) respectively.

3. Results

The PDI promoter sequences from one plant each of Triticum uratu (IG 44831), Aegilops speltoides (IG 46812) and Aegilops tauschii (AE 1068) were amplified using the following primer pairs (Table 1) corresponding respectively to sequences comprising putative PDI promoter and part of the first exons of the PDI gene belonging to the A, B and D genome was visualized by gel electrophoresis. Two independent PCR reactions were performed for each of the two primer pair/DNA combinations used and in all cases a specific amplification product of the expected electrophoretic mobility was obtained and cloned. For every each cloned amplification product three clones were randomly chosen and sequenced, for a total of 6 clones for each primer pair/DNA combination. The six DNA sequences deriving from the same primer pair/DNA combination resulted 100% identical.

The length of the cloned region upstream of the translation start codon was 1334 bp for Triticum uratu (AA, accession IG 44831), 1332 bp for Aegilops speltoides tauschii (DD, accession AE 1068); the three sequences showed 92.38 % identity in the promoter region (Figure 2). The specificity and uniqueness of their respective amplification products was confirmed through cloning all fragments and sequencing.

A high degree of conservation was detected in the putative promoter sequences of the entire three wild genomes cloned. Differences were due to both nucleotide substitutions and short insertions/deletions. The promoter sequence of Triticum uratu (AA, accession IG 44831) showed 99.40 % identity with one deletion and nucleotide substitution. Aegilops tauschii (DD, accession AE 1068) promoter sequence showed 99.04 % identity with few insertion and nucleotide substitution. Whereas promoter sequence of Aegilops speltoides (BB, accession IG 46812) showed only 89.59 % identity with large number of deletions and nucleotide substitution.

The evolutionary relationship between six sequences, of the putative promoter was studied by phylogeny reconstruction. These included one cloned sequence from Triticum uratu, Aegilops speltoides, Aegilops tauschii for a total of 3 new sequences and the three homoeologous sequences from CS previously reported ^[28]. The phylogenetic tree was constructed using three different methods namely the neighbour-joining (NJ) method, Minimum Evolution (ME) and Maximum Parsimony (MP) methods. As the results of the three methods were similar, only NJ tree is presented here (Figure 1).

Figure 1. Phylogenetic tree of PDI gene promoter sequence of Triticum urartu (TU AA), Aegilops speltoides (AS BB) and Aegilops taushcii (TT DD) with three homoeologous promoter sequences from CS

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Table 1. List of primers used for the isolation of PDI promoter analysis

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Figure 2. Multiple sequence alignment of PDI promoter sequences from Triticum uratu (TU_AA), Aegilops speltoides (AS_BB) and Aegilops tauschii (TT_DD)

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Table 2. Main regulatory motifs found in the 5’ Upstream of typical PDI gene sequence in Triticum aestivum, Triticum urartu, Aegilops speltoides and Aegilops taushcii in both positive and negative strands from ATG

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The search for regulatory motifs in the promoter sequences of Triticum uratu (IG 44831), Aegilops speltoides (IG 46812) and Aegilops tauschii (AE 1068) upstream the coding region in the database of plant promoters (PlantCARE) and PLACE database [https://www.dna.affrc.go.jp/htdocs/PLACE/; ^[33]] detected, a TATA box located at −79 nt from the start codon, and a number of different cis-acting regulatory elements (Table 2) including several motifs (AACA, prolamin box, GCN4, Skn-1) involved in the regulation of endosperm specific genes ^{[Plant Cell 9(2): 171-184.">41, 42, Plant Journal, 23(3): 415-421.">43, Plant Journal, 14(6): 673-683.">44]}.

Several CAAT-boxes were present in both (+) strand and (-) strands of A, B and D genome sequences controlling endosperm and tissue specific expression. AACA motif was present in (+) strand of all genomes, eight in A genome, eleven in B genome and 10 in D genome, but in (-) strand, only five in A, seven in B and six in D genome were identified. A prolamine-box was discovered on (+) strands at position -226 bp in Triticum uratu and at position -227 bp in Aegilops speltoides. No prolamine-box was detected in the target region of Aegilops tauschii. The prolamine-box was present in an identical position in the corresponding promoter sequences of the PDI gene in CS located on homoeologous chromosomes 4A and 4B of CS as described in previous study ^[28]. Skn-1 like element were found at position -480 bp, -481bp and -480 bp on (+) strand respectively in the A, B and D chromosomes. In the homoeologous sequences of CS, ^[28] identified them at position -480bp, -481bp and -479bp respectively in the sequence of the putative PDI promoter from genome 4A, 4B and 4D. In addition to the above-mentioned regulatory elements (AACA, GCN-4, prolamin box, Skn-1,) involved in the regulation of endosperm specific genes, other regulatory motifs identified in the promoter region was CAAT-box responsible for tissue specific gene expression. Search for regulatory motifs have identified several cis-acting regulatory elements and other motifs conferring endosperm specific and tissue specific expression. All promoter elements identified in partial promoter region of three wild species reported previously was reconfirmed in our present study ^[32].

4. Discussion

The promoter sequence of the three homoeologous genes in hexaploid wheat Triticum aestivum cv Chinese Spring showed overall identity of 89% in our previous study; high degree of conservation was found in the 700 nt proximal sequence, with identity exceeding 93%, when compared to distal region with 80% identity ^[28]. In our present study overall identity was 92 % among three sequences from wild realtives; high degree of conservation was found in 650 nt proximal sequence, with identity exceeding 94 %, when compared to distal region with 89 % identity.

All sequences were grouped into two major clusters, one containing the sequences derived from A genome, and one containing two subclusters each formed respectively by the B and D genome sequences. The sequence from Aegilops speltoides (BB) closely clustered together with the sequence located on the chromosome 4B of CS. The homoeologous B genome of hexaploid wheat is closely related to the genome of Aegilops speltoides that is proposed to be its wild progenitor ^{[37, 38]}. The sequence from the Aegilops tauschii (DD) accessions closely clustered with sequence located on the 4D chromosome of CS. Several studies underline that Aegilops tauschii, also known as wild goat grass from the Middle East, crossed with modern versions of emmer wheat to produce bread wheat ^{[38, 39, 40]}. As expected also sequence of Triticum uratu (AA) closely clustered with the sequence from CS located on chromosome 4A.

Our present study had shown strong evolutionary relationship from three new sequences of Triticum uratu, Aegilops speltoides and Aegilops tauschii cloned in this study with previously identified three homoeologous sequences from CS. Similar result was also obtained from sequences of partial promoter region from Triticum uratu, Aegilops speltoides and Aegilops tauschii reported previously ^{[32, 49, 50]}.

Former studies in wheat had been restricted to the characterization of genes encoding the typical PDI and their promoter, and the cloning and characterization of complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species, which is of special interest for its involvement in determining bread making qualities and technological properties of flour. The interest of extending the study to promoters of wild species is due to high conservation of gene, due to their relevant metabolic functions, as well as to the interesting expression pattern found in previous work. Present study confirmed the conservation of important motifs conferring the endosperm specific expression with evolutionary relationship between wild relatives and bread wheat (Triticum aestivum cv Chinese Spring). An intensive analysis carried out on the transcription levels of nine PDI and PDI-like genes in a set of 29 samples of wheat, including tissues, developmental stages and temperature stresses, showed their constitutive although very variable expression. Highly diversified expression rates and expression patterns were evidenced not only by genes belonging to different phylogenetic groups, but also in close paralog genes. This variable expression pattern may be due to differing regulatory elements and their numbers. The very high expression of the gene TaPDIL1-1, encoding the typical PDI, in the developing caryopses, which is consistent with its hypothesised role in the folding, aggregation and deposition of seed storage proteins was confirmed. This function of the typical PDI has been demonstrated experimentally in maize, rice and soybean ^{[23, 24, 45, 46, 47, 48, 51, 52]}.

Future studies should involve characterization of proximal and distal end of the Typical PDI promoter in diploid and tetraploid progenitor from diverse geographic origin of world to determine to determine the similarity and identity. Functional analysis of Chinese spring PDI promoter (full and partial sequence) driving the GUS gene and selective silencing of the PDI and PDI-like genes in wheat plants. The characterization of the regulatory motifs through the expression studies of the progressive deletions of their promoters, as well as the expression analysis of the PDI gene from accession of wild and cultivated wheat is currently under way. This analysis will help in elucidating the function of some regulatory elements controlling the spatial and temporal specific expression of the PDI and PDI –like genes.

5. Conclusions

The very high expression of the gene TaPDIL1-1, encoding the typical PDI, in the developing caryopses, which is consistent with its hypothesised role in the folding, aggregation and deposition of seed storage proteins was confirmed. This function of the typical PDI has been demonstrated experimentally in maize, rice and soybean. Future studies should involve characterization of proximal and distal end of the Typical PDI promoter in diploid and tetraploid progenitor from diverse geographic origin of world to determine to determine the similarity and identity. Functional analysis of Chinese spring PDI promoter (full and partial sequence) driving the GUS gene and selective silencing of the PDI and PDI-like genes in wheat plants. The characterization of the regulatory motifs through the expression studies of the progressive deletions of their promoters, as well as the expression analysis of the PDI gene from accession of wild and cultivated wheat is currently under way. This analysis will help in elucidating the function of some regulatory elements controlling the spatial and temporal specific expression of the PDI and PDI –like genes.

Acknowledgement

I thank Shalini Narasimhan for helping me in editing grammatical errors and finalizing the manuscript. My Ph.D programme scholarship entitled “International Doctoral Scholarship for Agrobiodiversity” from Scuola Superiore Sant’ Anna Piazza Martiri della Libertà, 33 - 56127 Pisa, Italy from February 2007 to February 2010 is highly acknowledged.

Conflict of Interest

The authors declare that they have no conflict of interest.

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