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

Abstract

Background: The 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.

Results: PDI 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.

Conclusions: High 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.

Figure 1

Wheat PDI family protein domain structures deduced from the cDNA clones. The boxes indicate the domain boundaries predicted by an NCBI conserved domain search. Black boxes in domains a and a’ represent the CXXS/C motif. The N-terminal black box represents a signal peptide that was predicted using SignalP-4.0 euk software.

Figure 2

Splicing sites in TaPDIL1Aα , TaPDIL1Aβ , TaPDIL1Aγ , and TaPDIL1Aδ . Open boxes indicate exons, and solid black lines denote introns. The numbers indicate the size of each exon and intron (bp). The positions of the signal peptide (SP), the two CGHC motifs, and the C-terminal KDEL sequence are indicated.

Figure 3

Expression of recombinant wheat PDI family proteins. Recombinant TaPDIL1Aα (A), TaPDIL1B (B), TaPDIL2 (C), TaPDIL3A (D), TaPDL4D (E), and TaPDIL5A (F) were expressed in E. coli. cells transformed with each expression plasmid and incubated with 0.4 mM IPTG at 30°C for 0 (lane 1) or 20 h (lane 2). Soluble (lane 3) and insoluble (lane 4) fractions were separated from these cells after 20 h by centrifugation after sonication. Each recombinant PDI family protein (arrowheads) was purified by His-tag column chromatography (lane 5) followed by gel filtration chromatography (lane 6). Proteins in each sample were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. (G) The CD spectra of the purified recombinant TaPDIL1Aα (●), TaPDIL1B (○), TaPDIL2 (▲), TaPDIL3A (■), TaPDL4D (♦), and TaPDIL5A (Δ) were determined at a concentration of 0.3 mg/ml. (H) The purified recombinant TaPDIL1Aα (lane 1), TaPDIL1B (lane 2), TaPDIL2 (lane 3), TaPDIL3A (lane 4), TaPDIL4D (lane 5), or TaPDIL5A (lane 6) was detected by western blot analysis with anti-TaPDIL1Aα serum (lanes 1 and 2), anti-TaPDIL2 serum (lane 3), anti-TaPDIL3A serum (lane 4), anti-TaPDIL4D serum (lane 5), or anti-TaPDIL5A serum (lane 6). Asterisks indicate degradation products.

Figure 4

Expression of wheat PDI family proteins in wheat caryopses. (A) Proteins (25 μg) extracted from the caryopses at 25 (lanes 1, 3) or 15 (lanes 2, 4-7) dap were analyzed by western blot with serum against TaPDIL1Aα (lane 1), TaPDIL2 (lane 2), TaPDIL3A (lane 3), TaPDIL4D (lane 4), TaPDIL5A (lane 5), calreticulin (lane 6), or Ero1 (lane 7). (B) TaPDIL1, TaPDIL2, and TaPDIL3 are high mannose-type N-glycosylated proteins in wheat caryopses. The proteins extracted from the caryopses were treated with (+) or without (−) endoglycosidase H (H) or endoglycosidase F (F). The proteins (20 μg for TaPDIL1 and TaPDIL2; 30 μg for TaPDIL3, TaPDIL4, and TaPDIL5) were separated by SDS-PAGE and immunostained with serum against TaPDIL1Aα, TaPDIL2, TaPDIL3A, TaPDIL4D, or TaPDIL5A.

Figure 5

Expression of wheat PDI family proteins in wheat caryopses during maturation. (A) Proteins (25 μg) extracted from caryopses at 5 (lane 1), 10 (lane 2), 15 (lane 3), 25 (lane 4), 30 (lane 5), and 35 dpa (lane 6) as well as mature grain (M) were analyzed by western blot as described in Figure 4A. For detection of gliadins, anti-gliadin serum was used. Total proteins were stained with Coomassie Brilliant Blue (CBB). HMW-GS indicates high molecular glutenin subunit. (B) Levels of TaPDIL1, TaPDIL2, TaPDIL3, TaPDIL4, and TaPDIL5 in caryopses at 5 (1), 10 (2), 15 (3), 25 (4), 30 (5) and 35 dpa (6) and in mature grain (7) were estimated from the band intensities on western blots in A. Values were calculated as a ratio to the value obtained at 5 dpa. Data represent the mean ± standard error of three experiments.

Figure 6

Distribution of wheat PDI family proteins in immature wheat caryopses. Cross sections of caryopses at 10 dpa (A-E) and 20 dpa (F-J) were immunostained with serum against TaPDIL1Aα (A, F), TaPDIL2 (B, G), TaPDIL3A (C, H), TaPDIL4D (D, I), or TaPDIL5A (E, J). Specimens were observed with a stereomicroscope SZX16. Fluorescent images are shown on the right panels in (A-J). Visible light images collected simultaneously are shown on the left in A-J. se, starchy endosperm; a, aleurone layer; pc, pericarp. Scale bar = 1 mm.

Figure 7

Subcellular distribution of wheat PDI family proteins in immature starchy endosperm and aleurone cells. Cross sections of caryopses at 10 dpa were immunostained with a combination of sera against TaPDIL1Aα (A and F, green), TaPDIL2 (B and G, green), TaPDIL3A (C and H, green), TaPDIL4D (D and I, green), or TaPDIL5A (E and J, green) and calreticulin (CRT, red). Specimens were observed with a Confocal Imaging System FV1200. Merged images of red and green are also shown. Endosperm sections and aleurone cells are shown in the left and right panels, respectively. Asterisks indicate A-type starch granules. Arrowheads indicate aleurone grains. White dotted lines show the profile of one aleurone cell. Scale bar = 5 μm.

Figure 8

Distribution of wheat PDI family proteins in the mature aleurone cells and the protein matrix of wheat grains. Cross sections of mature grains were immunostained with sera against TaPDIL1Aα (A, F, K), TaPDIL2 (B, G, L), TaPDIL3A (C, H, M), TaPDIL4D (D, I, N), or TaPDIL5A (E, J, O). Specimens were observed with a fluorescence microscope BZ-9000 (A-E, K-O) and a Confocal Imaging System FV1200 (F-J). Visible light images (gray) collected simultaneously are shown in the middle panels (F-O). Merged fluorescent and visible light images are also shown in the right panels (F-O). a, aleurone layer; pc, pericarp; pm, protein matrix; se, starchy endosperm. Arrowheads indicate aleurone grains. White dotted lines show the profile of one aleurone cell. Asterisks indicate A-type starch granules. Scale bars in A-E = 250 μm. Scale bars in F-J = 5 μm. Scale bars in K-O = 50 μm.