Bioinformatic analyses and identification of PDI8 homologs
To identify homologs of PDI8, BLAST (Basic Local Alignment Search Tool) searches were performed against both the National Center for Biotechnology Information (NCBI) non-redundant (nr) protein sequence database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the Phytozome v10 (http://phytozome.jgi.doe.gov), using the bb’ region of PDI8 as the search query sequence due to its absence of homology to the other 13 PDIs from Arabidopsis. Whenever possible, incomplete or incorrectly annotated protein sequences were corrected based on available expressed sequence tag (EST) sequences. All sequences and their corresponding accession numbers are provided in Additional file 4, with alterations to the original source sequences highlighted in yellow.
Signal peptide cleavage site prediction for PDI8 was performed using the program SignalP (v. 4.1) (http://www.cbs.dtu.dk/services/SignalP/; ). The predicted locations of TMDs were obtained using the hidden Markov model-based membrane protein topology prediction program, TMHMM (v. 2.0) (http://www.cbs.dtu.dk/services/TMHMM/; ). Protein secondary structure predictions for α-helices and β-strands were obtained using the program SPIDER2 (http://sparks-lab.org/yueyang/server/SPIDER2/; ).
Generation of Arabidopsis transgenic plants
A fragment containing the PDI8 coding sequence was inserted between the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences of binary vector pCAMBIA1302 to create the PDI8 over-expression construct, pC1302[35s
:PDI8]. The PDI8 coding sequence was amplified from first-strand cDNA prepared from 7-day-old Arabidopsis ecotype Columbia-0 (Col-0) seedlings using a forward primer containing an engineered SphI restriction site (5’-TCG GCA TGC GTT CGT TAA AGT TAC TCC TTT G-3’), and a reverse primer containing a BstEII site (5’-AAG GGT CAC CAA ACT AGT CCT CTT TTT TGT CAC-3’). The incorporated restriction sites in these primer sequences (and all subsequent primers described in this report) are underlined. The PDI8 cDNA fragment was ligated between the NcoI and BstEII restriction sites of pCAMBIA1302. For antisense expression of PDI8, a PDI8 cDNA fragment was amplified from cDNA (as above) using a forward primer containing a BstEII restriction site (5’-AAA TGG TGA CCT CAT GAG ATC GTT AAA GTT ACT CCT TTG TTG-3’), and a reverse primer with an SpeI site (5’-AAG TGG GTC AAC ACT AGT CCT CTT TTT TGT CAC T-3’). The cDNA fragment was ligated in the antisense orientation between the SpeI and BstEII cloning sites of pCAMBIA1302 to generate the construct pC1302[35S
Promoter expression studies were performed using stably transformed transgenic lines harboring the construct PDI8
:GUS, which contains a 2.3-kb PDI8 promoter fragment transcriptionally fused to the β-glucuronidase (GUS) reporter gene, gusA. The PDI8 promoter fragment was amplified from Arabidopsis (Col-0) genomic DNA, using a forward primer with a SacI site (5’- TTT GAG CTC GTA GAA GTT TGC TTG AAT ATT CA-3’) and a reverse primer with an NcoI site (5’-AAC CCA TGG CGA TCT GAT TTT CAG ACC AAA C-3’). The gusA gene was amplified from pCAMBIA1304 using a forward primer with an NcoI site (5’-TGA CCA TGG TAG ATC TGA CTA GTT TAC GTC-3’) and a reverse primer with a BstEII site (5′-CTC CGG TCA CCT ATT GTT TGC CTC CCT GCT GCG-3′). The PDI8
:GUS fusion was assembled in pCAMBIA1302 by cloning the gusA PCR fragment between the NcoI and BstEII sites of the vector to create the intermediate construct pC1302[35S
:GUS]. The PDI8 promoter fragment was then cloned between the SacI and NcoI sites of pC1302[35s
:GUS] to produce the final construct, pC1302[PDI8
:PDI8] and pC1302[PDI8
:GUS] constructs were introduced into wild-type Arabidopsis (Col-0) plants by Agrobacterium-mediated transformation, using the floral dip method . Initial transformants were obtained by selecting for hygromycin resistance in the T1 generation, and the presence of an intact transgene determined by PCR. Homozygous transgenic lines were subsequently identified by screening for the occurrence of 100 % hygromycin-resistance in the T3 generation.
GUS expression analysis
:GUS seedlings were grown vertically on 1/2× LS agar plates [0.8 % (w/v) Gellan Gum (Sigma-Aldrich, St. Louis, MO), 1/2x Linsmaier & Skoog media (Caisson Laboratories, Smithfield, UT) and 1.5 % (w/v) sucrose] for 7 or 14 days at 22 °C under a 16 h-light/8 h-dark cycle. Shoot inflorescences were obtained from 6-week-old PDI8
:GUS plants grown on Farfard Super-Fine Germinating Mix (Sun Gro Horticulture, Agawam, MA) under a 16 h-light/8 h-dark cycle at 25 °C. GUS staining was performed as described . Briefly, the tissue samples were fixed in 90 % ice-cold acetone for 20 min at 25 °C, then washed with staining buffer (50 mM sodium phosphate buffer (pH 7.0), 0.2 % Triton X-100, 2 mM potassium ferrocyanide, and 2 mM potassium ferricyanide) three times on ice, then submerged in staining buffer containing 1 mM 5-bromo-4-chloro-3-indoxyl-β-D-glucuronide cyclohexylammonium salt (X-gluc). The tissues were vacuum infiltrated briefly, then incubated O/N at 37 °C. After staining, the samples were incubated in 70 % ethanol to extract soluble pigments, repeating with fresh 70 % ethanol as necessary. Images of GUS staining in roots and stomata were acquired on an Olympus BX-51 upright microscope, with the samples mounted on glass slides in 50 % glycerol. All other images were taken on an Olympus SZX-12 stereomicroscope, with samples submerged in 70 % ethanol in a petri dish.
Transient expression of spGFP-PDI8 in protoplasts
The creation of the ER marker construct pBL(35S
:ER-mCherry), and the unfused green fluorescent protein (GFP) control construct pBL(35S
:GFP(S65T)), was described previously . The construct pBL(35S
:spGFP-PDI8) was generated by cloning the following arrangement of DNA sequences between the KpnI and BstEII sites of pBL(35S
:GFP(S65T)): a CaMV 35S promoter fragment (KpnI/XhoI), a PDI8 signal peptide coding sequence-GFP(S65T) fragment (XhoI/XmaI), and a PDI8 mature protein cDNA fragment (XmaI/BstEII). The CaMV 35S promoter fragment was amplified from pCAMBIA1302 using a forward primer with a KpnI site (5’-TTC AGG GTA CCT TCA TGG AGT CAA AGA TTC A-3’), and a reverse primer with an XhoI site (5’-ATC TAC TCG AGT CAA GAG TCC CCC GTG-3’). The GFP(S65T) fragment, modified to include the signal peptide sequence at the N-terminus of GFP, was amplified from plasmid HBT95::sGFP(S65T)-NOS using a forward primer with an XhoI site (5’-TTT CTC GAG ATG CGT TCG TTA AAG TTA CTC CTT TGT TGG ATC TCG TTT CTT ACG TTA TCA ATC TCA ATC TCT GCA TCG TCA ATG GTG AGC AAG GGC GAG GAG CTG-3’), and a reverse primer with an XmaI site (5’-AAA CCC GGG CTT GTA CAG CTC GTC CAT GC-3’). The PDI8 mature protein cDNA fragment was amplified from a full-length PDI8 cDNA clone using a forward primer with an XmaI site (5’-ATA CCC GGG TCG TCA GAT GAT CAA TTC ACC CTC-3’) and a reverse primer with a BstEII site (5’-AAG GGT CAC CAA ACT AGT CCT CTT TTT TGT CAC TAG-3’). The construct pBL(35S
:PDI8-GFP-KKED) was generated by replacing the spGFP-PDI8 coding sequence of pBL(35S
:spGFP-PDI8), between restriction sites XhoI and BstEII, with a full-length PDI8 cDNA fragment (XhoI/XmaI) and a GFP(S65T)-KKED fragment (XmaI/BstEII). The PDI8 cDNA fragment was amplified from a PDI8 cDNA clone using a forward primer with an XhoI site (5’-CAG CTC GAG ATG CGT TCG TTA AAG TTA C-3’) and a reverse primer with an XmaI site (5’-ACA CCC GGG GTC CTC TTT TTT GTC ACT AGG CT-3’). The GFP(S65T) fragment, modified to include the KKED putative retention signal of PDI8, was amplified from plasmid HBT95::sGFP(S65T)-NOS using a forward primer with an XmaI site (5’-TAG TCC CGG GAT GGT GAG CAA GGG CGA GGA-3’), and a reverse primer with a BstEII site (5’-AGG ATG GTC ACC TAA TCC TCT TTT TTG CCG TGA GTG ATC-3’).
The procedure for isolating and transfecting protoplasts was adapted from Yoo et al.  and Wu et al. . The abaxial epidermis of rosette leaves from four-week-old Arabidopsis plants was removed using the tape-sandwich method . Mesophyll cells were released by incubating the peeled leaves in 10 mL of enzyme solution (1.5 % cellulase R10, 0.4 % macerozyme R10, 0.4 M mannitol, 20 mM KCl, 20 mM MES, pH 5.7) for 3 h, then mixed gently with 10 mL of W5 solution (154 nM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7). The protoplasts were gently centrifuged at 100 g for 2 min, resuspended in fresh W5 solution to a density of 2 × 105/mL, and incubated on ice for at least 30 min. The W5 solution was then removed, and the protoplasts resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7) to a density of 2 × 105/mL. The protoplasts were transfected by gently mixing 200 μL of protoplasts in MMg solution with 20 μL of plasmid DNA solution (containing ~20 μg of each construct in H2O), and 220 μL of PEG solution (40 % PEG, 0.2 M mannitol, 100 mM CaCl2). After incubating at 25 °C for 5-10 min, transfection was stopped by adding 0.8 mL W5 solution. The protoplasts were centrifuged at 100 g for 2 min, and then resuspended in 1 mL WI solution (0.5 M mannitol, 20 mM KCl, 4 mM MES, pH 5.7). The transfected protoplasts were incubated in the dark at 22 °C for 18 h to allow for transgene expression. Fluorescence was visualized using an Olympus FV-1000 laser scanning confocal microscope at the Biological Electron Microscope Facility (University of Hawaii at Manoa, Honolulu, HI). The excitation/emission filters utilized for fluorescence detection were 488/505–525 nm for GFP(S65T) and 543/585–615 nm for mCherry.
Anti-PDI8 antibody production
Affinity-purified polyclonal rabbit antibodies recognizing PDI8 were generated commercially through YenZym Antibodies, LLC (San Francisco, CA), using a truncated form of recombinant PDI8 as the antigen for both rabbit immunization and affinity purification of the antiserum. For production of the truncated PDI8 protein, a cDNA fragment encoding the central b-b’ region of PDI8 (PDI8bb’, corresponding to residues 138-377 of the PDI8 preprotein sequence) was amplified by RT-PCR using a forward primer with an NdeI site (5’-GCC TAC GCA TAT GGT TGC TCC AGA TGT GCG G-3’) and reverse primer with a BamHI site (5’-CGT GGA TCC CTA TGA GTT GAT AAA TCC CAT GAA-3’). The PDI8
bb’ cDNA fragment was ligated between the NdeI and BamHI sites of the bacterial expression vector pET-15b (EMD Millipore, Billerica, MA), placing the PDI8
bb’ sequence in-frame with the 6xHis-tag of pET-15b. Expression of PDI8bb’ was induced in Escherichia coli strain BL21(DE3) for 5 h at 28 °C by the addition of 0.2 mM IPTG. After induction, the E. coli cells were harvested by centrifugation and lysed using BugBuster Protein Extraction Reagent (EMD Millipore). The His-tagged PDI8bb’ protein was purified from the bacterial lysate by nickel affinity chromatography.
Transmission electron microscopy and immunolabeling
For immunogold labeling analysis, developing roots and apical buds were preserved by high-pressure freezing/freeze-substitution techniques as described in . For immunolabeling, 80 nm thick sections from Lowicryl HM20 resin embedded specimens were placed on formvar-coated gold or nickel slot grids and blocked for 30 min with 2 % (w/v) non-fat dried milk solution in 0.01 M phosphate-buffered saline pH 7.2 containing 0.1 % Tween-20 (PBST). The sections were washed and then incubated with a 10-fold dilution of the primary antibody, anti-PD8, for 2 h at RT. Sections were washed and transferred to a 25-fold dilution of secondary antibody goat anti-rabbit IgG-conjugated to 10 or 15 nm gold particles (Ted Pella, Inc) for 2 h at RT. Sections were washed and then stained with uranyl acetate solution for 2 min and lead citrate for 4 min. All observations were performed using a Hitachi H-7000 transmission electron microscope operated at 80 KV (Hitachi USA, OH).
Preparation of microsomal membranes and protease protection analysis
For extraction of microsomal membranes, 35S
:PDI8 Arabidopsis seedlings were grown vertically on 1/2× LS agar plates under a 16 h-light/8 h-dark cycle at 22 °C. 7-day-old seedlings were homogenized with a chilled mortar and pestle in ice-cold extraction buffer [40 mM HEPES pH 7.5, 0.4 % polyvinyl polypyrrolidone (PVP), 1 mM MgCl2, 10 mM KCL, and 0.4 M sucrose], at a ratio of 1.5 μL extraction buffer per 1 mg of plant tissue. To remove insoluble debris, the homogenate was centrifuged twice at 1000 g and 4 °C for 3 min, collecting the supernatant after each spin. The total protein homogenate was separated into microsomal and soluble protein fractions by centrifuging as 150 μL aliquots at 21,000 g and 4 °C for 1.5 h . The microsomal pellets were washed once with 150 μL of fresh extraction buffer, recovering the microsomes by spinning at 21,000 g and 4 °C for 45 min and removing the supernatant. Finally, the microsomal pellets were resuspended in a volume of fresh extraction buffer equivalent to the original sample volume (i.e. 150 μL).
For immunoblot detection of PDI8 and BiP, protein samples were separated by SDS-PAGE (10 % polyacrylamide gels) and transferred onto nitrocellulose membranes. An equivalent amount (by volume) of the 35S
:PDI8 total and fractioned protein samples were loaded, equaling ~20 μg protein in the unseparated homogenate, ~14 μg protein in the soluble fraction, and ~7 μg protein in the microsomal fraction. Immunoblot analysis of PDI8 was performed using the anti-PDI8 antiserum at 1:100 dilution, and an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody at 1:2000 dilution supplied in the Amersham ECL Western Blotting Detection Kit (GE Healthcare Bio-Sciences, Pittsburgh, PA). Detection of BiP was performed using the goat anti-BiP primary antibody aC-19 (Santa Cruz Biotechnology, Inc., Dallas, Tx) at 1:1000 dilution, and a donkey anti-goat HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) at 1:3000 dilution.
To determine the membrane topology of PDI8, 35S
:PDI8 resuspended microsomes were incubated at 37 °C for 30 min in extraction buffer alone (negative control), or with 50 μg/mL proteinase K and/or 0.1 % Triton X-100. Each reaction contained ~0.36 μg/μL microsomal protein in a total volume of 60 μL. Proteinase K digestion was stopped by adding 5 mM PMSF to all samples. SDS-PAGE and immunoblot detection of PDI8 was performed as described above, with each lane loaded with 20 μL of sample (~7.2 μg of microsomal protein).
Alkaline phosphatase activity assay
A construct for the heterologous expression of PDI8 in E. coli was generated by cloning the coding sequence for the lumenal portion of PDI8, containing the catalytic a domain and redox-inactive b and b’ domains (PDI8abb’, Fig. 1) between the XmaI and SalI restriction sites of the bacterial expression vector pFLAG-CTS (Sigma-Aldrich, St. Louis, MO). The PDI8
abb’ gene fragment was amplified from a full-length PDI8 cDNA using a forward primer with an XmaI site (5’-TGT CCC GGG AGA TGA TCA ATT CAC CCT CGA C-3’) and a reverse primer with a SalI site (5’-AAT GTC GAC CAT TGA GTT GAT AAA TCC CAT G-3’).
The E. coli strains RI89 (dsbA
+) and RI90 (dsbA::kan1; RI89 genetic background) were obtained from the E. coli Genetic Stock Center (Yale University, New Haven, CT). The pFLAG-PDI8abb’ construct and pFLAG-CTS empty vector were transformed into strain RI90. To measure alkaline phosphatase (PhoA) activity, the cells were grown at 37 °C in M9 minimal media to an OD600 of 0.4–0.6, harvested by centrifugation, washed once with 50 mM Tris-HCl (pH 8.0), and lysed with 0.2 % Triton X-100. PhoA activity was determined using the QuantiChrom Alkaline Phosphatase Assay Kit (BioAssay Systems, Hayward, CA). Briefly, 150 μL of working solution (5 mM magnesium acetate, and 10 mM p-nitrophenyl phosphate in supplied assay buffer, pH 10.5) was added to 50 μL of lysed cells. After quickly mixing, the initial OD405 (t = 0) was measured for each sample, and then re-measured after 4 min (t = 4). PhoA activity (IU/L) was calculated from the OD405 values as described in the kit. The activities reported are averages (±standard deviation) derived from three independent trials.