Sample preparation, 2-D differential in-gel electrophoresis (2D-DIGE), gel staining, image analysis, and protein identification by LC-MS/MS

The fold change in abundance of different proteins was analyzed in the form of ratios for eight different sets of comparisons based on group assignment (Table ​(Table1).1). Four are ratios of LS to ES PCG and SG: (i) the ratio of LS to ES PCG before senescence (G/E), (ii) the ratio of LS to ES PCG after senescence (H/F), (iii) the ratio for LS to ES SG before senescence (C/A), and (iv) the ratio of LS to ES SG after senescence (D/B). Similarly, the other four sets were as follows: (v) the ratio for after to before senescence in ES PCG (F/E), (vi) the ratio for after to before senescence in LS PCG (H/G), (vii) the ratio for after to before senescence in ES SG (B/A), and (viii) the ratio for after to before senescence in LS SG (D/C).

One gram of leaf tissue samples was ground to powder under liquid nitrogen using a mortar and pestle. Three hundred microliters of 2-D cell lysis buffer (30 mM Tris-HCl, pH 8.8, containing 7 M urea, 2 M thiourea, and 4% CHAPS) was added to this ground tissue and subjected to sonication on ice (Hurkman and Tanaka, 1986). Next, the tubes were shaken for 30 min on a shaker at room temperature. Then, samples were centrifuged for 30 min at 4°C at 25,000 × g, and the supernatant was collected. The concentration of protein was measured in the supernatant by Bio-Rad protein assay buffer with BSA as a standard following the standard manufacturer's guidelines (Peterson, 1983). Lysate samples were diluted with 2-D lysis buffer to a concentration of 5 mg/ml.

The 2D-DIGE were performed by Applied Biomics (Hayward, CA) following the protocol described in Robbins et al. (2013) and Das et al. (in press). Briefly, 1.0 μl of diluted Cy Dye was added to 30 μg of protein lysate (1:5 diluted with DMF from 1 nM/μl stock), followed by a short vortexing. The tubes were kept under dark on ice for 30 min followed by the addition of 1.0 μl of 10 mM lysine to each of the samples and vortexing; then, the reaction was kept in the dark on ice for additional 15 min. A pooled protein sample was prepared containing equal amounts of all 24 samples and was labeled with Cy2 using the same protocol. The Cy2 labeled sample was used as an internal control to compare gel-to-gel variations. For one gel, 3 samples consisting of Cy2, Cy3, and Cy5 labeled samples were mixed with 2X 2-D sample buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, 2% pharmalytes, and a trace amount of bromophenol blue). Then, 100 μl of destreak solution and rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/ml DTT, 1% pharmalytes, and a trace amount of bromophenol blue) was also added to make a final volume of 350 μl for the 18 cm IPG strip (pH 3-10). This was mixed well and spun before loading the labeled samples into the strip holder.

After loading the labeled samples into the IPG strip holder, 18 cm strips were put facedown, and 1.5 ml of mineral oil was added on the top of the strips. This was followed by the protocol provided (Amersham BioSciences) and isoelectric focusing (IEF) was carried out in the dark at 20°C. After IEF, the IPG strips were incubated in freshly made equilibration buffer I (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, a trace amount of bromophenol blue, and 10 mg/ml DTT) for 15 min with slow shaking. Then, the strips were rinsed in freshly prepared equilibration buffer II (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, a trace amount of bromophenol blue, and 45 mg/ml iodacetamide) for 10 min with slow shaking. The strips were then rinsed in the SDS gel running buffer once, followed by their transfer into the SDS gel (12.5% acrylamide SDS gel prepared using low florescent glass plates). They were then sealed with 0.5% (w/v) agarose solution (in SDS gel running buffer). Running of the SDS gels was carried out at 15°C and stopped when the dye front ran out of the gels.

After the SDS-PAGE images were scanned using Typhoon TRIO (GE Healthcare Bioscience, Pittsburgh, PA, USA), and analysis of the scanned images was conducted using Image Quant software (version 6.0, GE Healthcare). An in-gel analysis of the images was conducted using DeCyder software, version 6.5 (GE Healthcare Bioscience). For this step, a difference in-gel analysis (DIA) tool made by DeCyder software was used. The value of the estimated number of spots was 3000. For the sake of low experimental variation, an automated tool made by DeCyder software was chosen for background subtraction and normalization of visible protein spots. The DIA datasets, along with the images, were put into the biological variation analysis (BVA) module made by DeCyder software. The spot intensity data were normalized with the internal standard sample that was labeled with Cy2 dye. The protein spots ratios that had increased or decreased abundance by 1.5-fold and had a Student's t-test value of p ≤ 0.05 were considered to be differentially abundant, along with the extra condition that the spot should be present and analyzed in all 2D gels. These ratios were calculated by the DeCyder image analysis software from spot volumes. As per the manufacturer's instructions the spot ratios were calculated as follows: (volume of secondary image spot/volume of primary image spot). This ratio indicated the change in spot volume between the two images. These ratio values were normalized, so that the modal peak of volume ratios was zero (since the majority of proteins are not up or down regulated). This ratio parameter is referred to as the volume ratio. In all DeCyder 2D Software DIA tables (Supplementary Tables 1, 2) the volume ratio is expressed in the range of 1–1,000,000 for increases in spot volumes and −1 to −1,000,000 for decreases in spot volumes. Values between −1 and 1 are not represented, hence a two-fold increase and decrease is represented by 2 and −2, respectively (and not 2 and 0.5 as might have been expected) (GE Healthcare Bio-Sciences, Pittsburgh, USA).

Protein spots that were statistically significant (see Section IEF, SDS-PAGE, Image Scan and Data Analysis) with a p ≤ 0.1 and cut off value of 1.5-fold were picked up using the Ettan Spot Picker (GE Healthcare). The picked gel spots were washed a few times, digested with modified porcine trypsin protease (Trypsin Gold, Promega), and then desalted using Zip-tip C18 (Millipore, Billerica, MA, USA) (Robbins et al., 2013; Gupta et al., 2014; Hayashi et al., 2015; Das et al., in press). Peptides were eluted from the Zip-tip with 0.5 μl of matrix solution (α-cyano-4-hydroxycinnamic acid, 5 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid, and 25 mM ammonium bicarbonate) and spotted onto a MALDI plate (Robbins et al., 2013; Hayashi et al., 2015; Das et al., in press).

MALDI-TOF (MS) and TOF/TOF (tandem MS/MS) were performed on a 5800 mass spectrometer (AB Sciex). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 2000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 2000 laser shots per fragmentation spectrum on each of the 10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions) (Gupta et al., 2014; Hayashi et al., 2015; Das et al., in press).

Analysis of the MS/MS results were performed using GPS Explorer, version 3.5, which was equipped with a MASCOT search engine (Matrix science). A search in the database of the National Center for Biotechnology Information non-redundant (NCBInr) and in Phytozome v.10.3 (http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvirgatum) was performed without constraining the protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine, oxidation of methionine residues, and one missed cleavage allowed in the search parameters (Table ​(Table22 along with Supplementary Tables 5, 6). Candidates with either protein score C.I.% (Confidence Interval) or Ion C.I.% greater than 95 were considered significant.

Proteins identified by 2D-DIGE followed by Mass Spectrometry.