Article pubs.acs.org/jpr
S‑Nitrosylation Analysis in Brassica juncea Apoplast Highlights the Importance of Nitric Oxide in Cold-Stress Signaling Ankita Sehrawat and Renu Deswal* Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi 110007, India S Supporting Information *
ABSTRACT: Reactive nitrogen species (RNS) including nitric oxide (NO) are important components of stress signaling. However, RNS-mediated signaling in the apoplast remains largely unknown. NO production measured in the shoot apoplast of Brassica juncea seedlings showed nonenzymatic nitrite reduction to NO. Thiol pool quantification showed cold-induced increase in the protein (including Snitrosothiols) as well as non protein thiols. Proteins from the apoplast were resolved as 109 spots on the 2-D gel, while Snitrosoglutathione-treated (a NO donor), neutravidin-agarose affinity chromatography-purified S-nitrosylated proteins were resolved as 52 spots. Functional categorization after MALDITOF/TOF identification showed 41 and 38% targets to be metabolic/cell-wall-modifying and stress-related, respectively, suggesting the potential role(s) of S-nitrosylation in regulating these responses. Additionally, identification of cold-stress-modulated putative S-nitrosylated proteins by nLC−MS/MS showed that only 38.4% targets with increased S-nitrosylation were secreted by classical pathway, while the majority (61.6%) of these were secreted by unknown/nonclassical pathways. Cold-stress-increased dehydroascorbate reductase and glutathione Stransferase activity via S-nitrosylation and promoted ROS detoxification by ascorbate regeneration and hydrogen peroxide detoxification. Taken together, cold-mediated NO production, thiol pool enrichment, and identification of the 48 putative Snitrosylated proteins, including 25 novel targets, provided the preview of RNS-mediated cold-stress signaling in the apoplast. KEYWORDS: apoplast, nitric oxide, thiol pool, S-nitrosylation, cold stress (45%) or metabolic (25%).8 For the identification of low abundant targets, the most commonly used approaches are the selective depletion of RuBisCO (the most abundant plant protein) and the subcellular proteome analysis. In our previous study, RuBisCO depletion was performed using immunoaffinity chromatography, which showed improved S-nitrosoproteome coverage of the cold-modulated targets.9 Here subcellular Snitrosoproteome analysis is attempted to get low abundant targets. Subcellular analysis provides deeper insight into the role of Snitrosylation in different subcellular compartments and also gives hints regarding S-nitrosylation-mediated protein translocation in the sub-organelles. For example, S-nitrosylation of NPR1 (a master regulator of the defense genes) at Cys 156 prevents its translocation to the nucleus from the cytosol.10 Subcellular S-nitrosylation has been analyzed in the mitochondria11,12 and peroxisomes.13 In pea mitochondrial extracts, differential S-nitrosylation of respiration- and photorespirationrelated targets in salinity stress was shown.12 Similarly, in peroxisomes, four photorespiration-related enzymes were
1. INTRODUCTION Cold stress influences the plant growth, development, and crop productivity.1 In response to the cold stress, vast changes in the gene expression take place, which modulate the metabolome and manifest different physio-biochemical responses, culminating in membrane stability, increased accumulation of osmolytes (proline), soluble sugars, and amino acids.2 Furthermore, increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is also observed in cold stress.3,4 RNS refers to nitric oxide (NO) and NO-related molecules such as peroxynitrite (ONOO−), dinitrogen trioxide (N2O3), and S-nitrosothiols (SNOs). S-Nitrosoglutathione (GSNO), a low-molecular-mass SNO, is produced by the reaction of NO with glutathione (GSH, a non-protein-based thiol), while high-molecular-mass SNOs are produced by binding of NO to the protein based thiol.5 These SNOs mediate S-nitrosylation, a NO-based PTM, which regulates genes, phytohormone signaling, and cell death.6,7 Although, recently more than 700 S-nitrosylated proteins have been identified in plants, the list of regulatory/low abundant S-nitrosylated targets is still scanty. The reason for this is the masking of these by the abundant targets; for example, in a recent study in Arabidopsis, 70% of the endogenous S-nitrosylated proteins were either photosynthetic © 2014 American Chemical Society
Received: January 22, 2014 Published: March 19, 2014 2599
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Control (RT) seedlings were kept at 25 °C. Seedlings were treated with sodium nitroprusside (SNP, a NO donor, 50−250 μM) or 2-phenyl-4,4,5,5-tetramethylimidazolone-1-oxyl-3oxide (cPTIO, a NO scavenger, 250 μM) under control and cold conditions. After treatment, seedlings were rinsed with doubledistilled water, blotted onto a filter paper, and used for apoplastic protein extraction.
identified as S-nitrosylated targets13 and highlighted the role of S-nitrosylation in regulating the photorespiration. Plant extracellular space or apoplast is an important site for stress signal perception or initial signaling in response to the environmental stimuli.14,15 Apoplastic proteome analysis of Arabidopsis thaliana,16 Oryza sativa,17 Glycine max,18 Populus deltoides,19 Zea mays,20 and Hippophae rhamnoides21 identified targets involved in carbohydrate metabolism, proteolytic events, defense responses, signaling networks, and redox reactions. Interestingly, changes in the leaf apoplast of Medicago truncatula in wounding stress showed differential expression of 28 extracellular proteins involved in ROS-dependent and -independent signaling pathways.22 These studies without a doubt proved apoplast to be a communication channel between plant and environment during stress. However, whether these targets are regulated by NO via S-nitrosylation has not yet been analyzed. Brassica juncea is an oil-yielding winter crop, and India is the second largest cultivator of Brassica spp. after China.23 B. juncea frequently faces yield loss due to extreme variations in the temperature. In 2013 (October to December), minimum temperature varied from 3 to 16 °C (http://www.iari.res.in) in northern India. Additionally, cold spells, fog, and intermittent rains add to the yield loss. In India, 40% of annual edible oil requirements is met by import, and this demand is likely to increase in future. Therefore, it is important to analyze the mechanism of cold-tolerance to bridge this gap between demand and supply by developing cold-tolerant Brassica. Our previous work on the analysis of the effect of cold stress on S-nitrosylation in the crude24 and RuBisCOdepleted fractions9 was on the B. juncea seedlings (7 days old); therefore, to further enrich the cold-stress-modulated B. juncea S-nitrosoproteome, for this study also, seedlings were chosen. Different components of NO signaling including NO, nitrite, and SNOs were measured in cold stress (2 to 96 h, 4 °C). Furthermore, total thiol status, including protein and nonprotein thiols, was also quantified to investigate the interaction of NO with these and their role in maintaining redox homeostasis. We have recently proposed NO and cold-stress cross-talk,4 but the details of NO signaling in apoplast in response to any stress is currently unknown. Therefore, an attempt was made to identify apoplastic S-nitrosylated targets. A total of 48 (24 GSNO-treated and 24 cold-responsive) putative S-nitrosylated proteins were identified by MALDITOF/TOF and nLC−MS/MS. The results for the first time indicated the role of S-nitrosylation in regulating apoplastic redox and stress-related proteins (including the antioxidant enzymes).
2.3. Extraction of the Apoplastic Proteins
Apoplastic proteins were extracted using vacuum infiltration method following the methodology of Hon et al.25 In brief, cotyledonary leaves and hypocotyls were cut into 2 cm segments. These were rinsed four times with deionized water to remove the cytosolic protein contamination. The sections were vacuum-infiltrated at 10 mmHg using a vacuum pump (Millipore) for 30 min in different buffers including 20 mM sodium acetate (pH 3 and 5.5), 20 mM sodium phosphate (pH 6), and 20 mM HEN [250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, and 0.1 mM neocuproine]. Dried sections were inserted in a 10 mL syringe barrel and placed in a 50 mL falcon tube. The apoplastic fluid was collected by centrifugation at 2000g (Beckman Coulter, Allegra 64R) for 10 min. For the optimization of centrifugal speed, sections infiltrated in 20 mM sodium acetate (pH 3) were centrifuged at different centrifugal speed (1000−4000g) for 10 min. Extracted apoplastic fluid was used immediately after extraction. Proteins were processed using a clean-up step to remove salts and other contaminants. For this, 150 μL of apoplastic fluid was mixed with 600 μL of methanol (4 volumes), 150 μL of chloroform (one volume), and 450 μL (three volumes) of autoclaved MilliQ water. The tube was vortexed and centrifuged at 12 000 rpm (1−15k Sigma) for 5 min at 4 °C. Centrifugation results in the formation of a “protein disc”. The solution above the disc was carefully discarded without damaging the disc. The disc was vortexed in methanol (450 μL, three volumes) and centrifuged at 12 000 rpm for 5 min at 4 °C. The pellet was air-dried, followed by vacuum drying for 5 min on ice. Protein estimation was done by Bradford’s assay using bovine serum albumin (BSA) as a standard.26 2.4. Purity Assessment of the Apoplastic Proteins
Purity was assessed using the glucose-6-phosphate dehydrogenase (G6PDH) assay as described by Noltmann et al.27 To do so, 50 μL of the extract was added in 1 mL reaction mixture [55 mM Tris (pH 8.8), 3.3 mM magnesium chloride, 6 mM NADP and 0.1 M glucose-6-phosphate]. Absorbance was taken at 340 nm for 5 min using a UV-spectrophotometer (Beckman Coulter, DU-730). Additionally, the activity assay was also performed in total protein extract. Total proteins were extracted in 20 mM Tris-HCl (pH 7.0, 1:3 w/v) containing 20% glycerol and 5 mM PMSF. The homogenate was centrifuged at 12 000 rpm for 20 min at 4 °C. The supernatant was used for the activity assay. For the Western blotting, apoplastic and crude proteins (27 μg) were resolved on a 15% SDS-PAGE, transferred by wet transfer (Transblot cell, Biorad), and probed using rabbit antiRuBisCO antibody (1:5000, 2 h) and alkaline phosphatase conjugated antibody (1:2000 for 30 min, Santa Cruz) as primary and secondary antibodies, respectively. Nitroblue tetrazolium and 5-bromo 4-chloro 3-indolyl phosphate were used as substrates.
2. MATERIALS AND METHODS 2.1. Plant Material and Growth Conditions
Brassica juncea var. Pusa Jaikisan seeds were obtained from Indian Agricultural Research Institute, New Delhi, India. Seeds were surface-sterilized with 70% ethanol, soaked overnight in deionized water, and germinated on the wet germination papers. After overnight incubation in the dark, paper rolls were kept in a growth chamber at 25 ± 2 °C under white fluorescent light (270 μmol/m2/s, 16 h light/8 h dark). Seedlings were regularly watered for 7 days. 2.2. Cold-Stress, SNP, and cPTIO Treatments
Cold stress (4 °C) was given to 7 day old seedlings for 2 to 96 h under the same light conditions as previously mentioned. 2600
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2.5. Nitric Oxide and Nitrite Measurements
Bio-Spin 6 columns (Bio-Rad). For the detection of the Snitrosylated proteins by Western blotting, samples after biotinylation were dissolved in 1× sample buffer without any reducing agent and were resolved on a 15% SDS-PAGE. S-nitrosylated proteins were biotinylated using BST and purified by neutravidin-agarose column chromatography.24,33 For 1-DE analysis of putative S-nitrosylated proteins, the eluate after neutravidin affinity chromatography was dissolved in 1× sample buffer. The samples were resolved on a SDS gel (15%). For the 2-DE, purified S-nitrosylated proteins were dissolved in 100 μL of lysis buffer [8 M urea, 2 M thiourea, 2% CHAPS (w/ v), 2% Triton X-100, 50 mM DTT, and 0.75% ampholytes (pH 4−7)]. Cold-stress-induced endogenous S-nitrosylation was analyzed in the apoplastic proteins extracted from cold-treated (6 h) seedlings following the procedure previously mentioned, except that apoplastic fluid was extracted in the dark due to the light-labile nature of SNOs, and GSNO treatment was omitted.
NO was measured using in NO measuring system (Innovative instruments) following the manufacturer’s instructions. The system consists of a NO meter, a sensor, and a data acquisition system. NO measurement is based on the principle of diffusion of NO from the sample to the sensor surface through sensor membrane. The experiments were performed following Bethke et al.28 and Modolo et al.29 In brief, 100 μL of apoplastic extract was incubated with L-arginine (125 μM), L-arginine (125 μM) with NADPH (125 μM), NG-nitro-L-arginine methyl ester (LNAME, 125 μM), sodium nitrite (125 μM), potassium nitrate (125 μM) with NADH (125 μM) and tungstate (125 μM) in different sets. To check whether nonenzymatic NO production is pH dependent, we measured NO production in 100 μL of apoplastic protein in 100 μL of 20 mM Tris buffer (pH 7.4) with nitrite (250 μM) or 20 mM sodium acetate buffer (pH 3) with nitrite (250 μM). Nitrite concentration in the apoplastic extract was determined by Griess assay following Wang et al.30 In brief, 0.5 mL of sample was mixed with 0.5 mL of 1% sulphanilamide. To this, 0.5 mL of 0.02% N-(1-naphthyl) ethylene-diamine dihydrochloride (NED) was added. The solution was incubated for 15 min, following centrifugation at 12 000 rpm for 2 min to remove any precipitate. As a control, for each set of sample, a reaction without NED was put and the readings were corrected with it. The absorbance was taken at 540 nm. Standard curve was prepared with sodium nitrite (1−5 μM).
2.8. 2-DE, Image Acquisition, and Data Analysis
Isoelectric focusing was carried out with 200 μg protein in 150 μL of rehydration buffer [8 M urea, 2% CHAPS, 20 mM DTT, 0.002% bromophenol blue, and 0.5% (v/v) IPG buffer (pH 3− 10 or 4−7)], as previously described.24 The proteins were resolved onto 13 cm IPG strip (nonlinear pH 3−10 and 4−7). Electrofocusing was done using an Ettan IPGphor (GE Healthcare, Uppsala, Sweden) at 27 500 V for 8.3 h at 20 °C with a limiting current of 50 μA/strip. Proteins were reduced by incubating the strip with 1% (w/v) DTT and then alkylating with 2.5% (w/v) iodoacetamide in the equilibration buffer [6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl (pH 8.8), and 0.002% bromophenol blue]. The strip was loaded on a 15% SDS-PAGE. The gel was fixed in fixative (50% methanol, 40% water, and 10% acetic acid) for 2 h. Gels (1-D as well as 2-D) were stained by MS-compatible silver staining.34 After removing the fixative, the gel was incubated in 5% methanol for 15 min. It was washed with double-distilled water three times for 5 min each. This is followed by the incubation of the gel in 0.02% sodium thiosulphate solution for 2 min. The gel was washed with double-distilled water three times for 10 s each. Following this, the gel was incubated in 0.2% silver nitrate solution for 25 min in the dark. The gel was washed with double-distilled water three times for 1 min each. After washing, developer (3% sodium carbonate with 0.05% formaldehyde and 2 mL of 0.02% sodium thiosulphate) was added and the gel was shaken until polypeptides/spots develop. The reaction was stopped with 1.4% EDTA solution. Following this, EDTA solution was replaced with double-distilled water. Gels were scanned using AlphaImager (Alpha Innotech), and images were saved in TIFF format. ImageMaster 2D Platinum software (version 6.0, GE Healthcare) was used for the analysis of the 2-D gels. Experimental variations were reduced by performing three independent biological replicate gels for each set. Only those spots detected in all replicates within each set were considered as valid spots. A first level match set was created in which protein spots from three independent biological replicates were compared by the software, and a master gel was created. The gels were normalized in percentage spot volume mode to reduce the differences in protein loading and gel staining. After spot detection, a second level of the match set was created in which master gels were matched and compared. A two-fold increase or decrease in the spot abundance on the 2-D gel of cold-treated S-nitrosylated
2.6. Thiol and S-Nitrosothiol (SNO) Pool Measurement in the Apoplast
Thiol pool was measured following Ivanov and Kerchev31 with some modifications. In brief, the apoplastic extract considered as supernatant (S1) was processed by adding freshly prepared prechilled trichloroacetic acid [TCA, 60% (w/v)] to make the final saturation of 1% (v/v). The solution was incubated at 4 °C for 5 min and centrifuged at 15 000g for 30 min at 4 °C, and both the supernatant (S2) and the pellet (P1) were used for the thiol measurement. P1 was resuspended in a detergent solution [6 M urea, 0.5% w/v SDS, 20 mM EDTA in 0.3 M Tris−HCl (pH 7.8)]. To prevent metal ion contamination, we used a separate set of glassware. Additionally, to prevent the oxidation of thiols, we performed all steps at low temperature. A blank was prepared for each fraction (S1, S2, and P1) without 5-5′dithiobis,2-nitrobenzoic acid (DTNB). As a control, a separate set of reactions was put without protein to correct the reading due to DTNB. Protein estimation was performed by Bradford assay using S1. Different fractions for the analysis were: available thiol groups (ATGs), S1−S2; total protein thiols (TPTs), pellet obtained after trichloroacetic acid (TCA) precipitation (P1); buried thiols, TPT − ATG; lowmolecular-weight thiols (LMTs), S2; and total thiols, TPT + LMT. The results were expressed as μmol -SH/mg protein (for protein-based thiols) and μmol -SH/g FW (nonprotein thiols and total thiols). SNOs were measured following Saville method,32 as previously described in Abat and Deswal.24 Extraction as well as the assay was performed in the dark to protect lightdependent decomposition of SNOs. 2.7. Detection and Purification of the Putative S-Nitrosylated Proteins
S-Nitrosylated proteins were detected using Biotin switch technique (BST) following Jaffrey and Snyder33 and Abat and Deswal.24 GSNO, GSH, and DTT were removed using Micro 2601
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samples in comparison with the control was considered as upor down-accumulated spots, respectively.
using the Shimadzu Prominence nano HPLC system (Shimadzu) coupled to a 5600 TripleTOF mass spectrometer (AB Sciex). Peptides after trypsin digestion were loaded onto an Agilent Zorbax 300SB-C18 apparatus (3.5 μm, Agilent Technologies) and separated with a linear gradient of water/ acetonitrile/0.1% formic acid (v/v/v). The peak list obtained was submitted to MASCOT (ver 2.1) search engine. The MS files (from MALDI-TOF/TOF and nLC−MS/MS) were searched using the “MS/MS Ions Search” on the Matrix Science public server (http://matrixscience.com), while the PMF (peptide mass fingerprinting) files were searched using “Peptide Mass Fingerprinting Server”. These were searched against the NCBInr 20131113 (34 201 960 sequences; 12 001 222 213 residues) with Viridiplantae (Green Plants, 1 620 576 sequences) in taxonomy. Other search parameters were the same, as described in Abat and Deswal,24 with mass values, monoisotopic; protein mass, unrestricted; fixed modification, carbamidomethylation; variable modification, methionine oxidation; peptide mass tolerance, 100 ppm; fragment mass tolerance, ± 0.6 Da; maximum trypsin missed cleavage, 1; and instrument type, MALDI-TOF-TOF for MALDI-TOF/TOF analysis and ESI-QUAD-TOF for nLC−MS/MS analysis. Only the significant top hit identified by MASCOT probability analysis (p < 0.05) was selected. For PMF (MALDI-TOF), the number of peptides matched is only for assignments made with high confidence (≥95%). For the MS/MS (MALDI-TOF-TOF and nLC−MS/MS), the peptides with an individual ion score above the threshold value are considered to be of high confidence (≥95%).
2.9. Protein Identification by MALDI-TOF/TOF and nLC−MS/MS
S-Nitrosylated polypeptides from the 1-D gel (for nLC−MS/ MS) and spots from the 2-D gel (for MALDI-TOF/TOF and nLC−MS/MS) were manually excised and destained.35 In brief, the excised gel slice was kept in an acetonitrile-rinsed eppendorf. To this, 500 μL of Milli-Q water was added and the tube was vortexed for a min. This washing step was repeated five times. Next, the gel slice was incubated with 50 μL of freshly prepared destain solution (30 mM potassium ferricyanide and 100 mM sodium thiosulfate) for 5 min with vortexing in between. When the brown color disappears, the destain solution was removed. The gel slice was washed with 500 μL of Milli-Q water thrice. The destained gel slice was used for the trypsin digestion. For trypsin digestion, gel slice was dehydrated with 50 μL of solution A [2:1, mixture of acetonitrile (ACN)/50 mM ammonium bicarbonate (ABC)] for 5 min. The supernatant was removed, 50 μL of 25 mM ABC was added, and the gel slice was rehydrated for 2 min. The gel slice was again dehydrated with 50 μL of solution A for 5 min and incubated in 25 mM ABC for 2 min. The dehydration and rehydration step was repeated twice, as mentioned. The gel slice was incubated with 20 μL trypsin (20 ng/μL, gold mass spectroscopy grade, Promega, Madison, WI) in 50 mM ammonium bicarbonate, pH 7.8 on ice for 30 min. After the gel slice has completely absorbed trypsin, 25 mM ABC is added in the tube to cover the gel slice. Gel slice was incubated at 37 °C for 16 h, and the tube was periodically checked to make sure that it is completely submerged in 25 mM ABC. After digestion, the supernatant was stored and gel slice was vortexed with 100 μL 20% ACN and 1% formic acid. These extractions were added to the supernatant, and it was reduced to powder form using a CentriVap Benchtop Vacuum Concentrators (Labconco). MALDI-TOF/TOF was performed to identify the putative S-nitrosylated targets from GSNO-treated apoplastic fluid, and nLC-ESI-MS/MS was used for the identification of the coldresponsive putative S-nitrosylated proteins. For the MALDI experiments, precalibrated ABI 4800 plus MALDI-TOF-TOF analyzer (Applied Biosystem) was used, and experiments were done in the Department of Biochemistry, South Campus, University of Delhi, New Delhi, India, as previously described.36 In brief, trypsin-digested samples were mixed with 50 mM ammonium acetate and desalted using zip-tip C18 (Millipore, USA). C18 zip-tip was washed with 100% acetonitrile and equilibrated with 0.1% trifluoroacetic acid (TFA). The samples were passed five times through the zip-tip for proper binding with the C18 column. The column was washed with 0.1% TFA to remove the salts, and the samples were eluted from the tip using 50% ACN in 5 mL. The desalted samples were mixed with α-cyano-4-hydroxycinnamic acid in equal ratio. The reconstituted peptides were spotted on a 384well LC MALDI steel plate. Spots were illuminated with laser intensity of 3500. A total of 1200 spectra were recorded. For the MS/MS analysis, 25 precursors/peptides were selected from each spot. Each precursor was fragmented in the collision cell. Peptide fragmented with 4300 laser intensity and 2500 shots were used for data processing. Identification by nLC−MS/MS was done at Proteomics International by electrospray ionization mass spectrometry
2.10. Location Prediction
The subcellular location of the identified targets was predicted by the TargetP program (www.cbs.dtu.dk/services/TargetP).37 The presence of a signal peptide was predicted by SignalP-4.0 server (www.cbs.dtu.dk/services/SignalP/). For those proteins without typical signal peptide sequences, SecretomeP program (http://www.cbs.dtu.dk/services/SecretomeP-1.0) was used to analyze whether a nonclassical secretion mechanism is involved.38 2.11. Prediction of S-Nitrosylation Sites in the Identified Targets
For the identification of possible S-nitrosylation sites in the apoplastic proteins, two computational programs were used, namely, dbSNO (http://csb.cse.yzu.edu.tw/SNOSite/index. html)39 and GPS-SNO (Group-Based Prediction System, http://sno.biocuckoo.org).40 2.12. Dehydroascorbate Reductase and Glutathione S-Transferase Activity Assay
Proteins were incubated without or with S-nitroso-L-cysteine (CysNO, 100−500 μM) in the dark for 20 min at 25 °C. CysNO was prepared in the laboratory following Zhang and Hogg, 2004.41 For the DTT treatment, after incubation with CysNO (250 μM), the samples were incubated with DTT (10 mM) in the dark for 40 min. CysNO and DTT were removed using Micro Bio-Spin 6 columns (Bio-Rad). Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was assayed following the method of Dalton et al.42 by measuring the increase in absorbance at 265 (for 2 min) due to ascorbate formation. The reaction mixture (1 mL) contained 12 μg of protein extract, 50 mM potassium phosphate buffer (pH 7.0), 2 mM GSH, and 1 mM dehydroascorbate (DHA). The reaction rate was corrected for the nonenzymatic reduction of 2602
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Figure 1. Contamination-free apoplastic proteins extraction and their resolution on 1-D and 2-D gels. (A) SDS-PAGE (15%) profile of the apoplastic proteins extracted in buffers (SA, sodium acetate; SP, sodium phosphate; HEN; Hepes, EDTA, and neucoproine) with different pH. Lower panel: immunoblot probed with anti-RuBisCO antibody. (B) Glyceraldehyde 6 phosphate dehydrogenase (G6PDH) activity in the apoplastic and crude fractions to check the purity of the apoplastic fluid extracted at 2000g. (C,D) 2-D gels of the apoplastic proteins resolved on 15% SDSPAGE following first dimension on nonlinear IPG strip (pH 3−10 and 4−7). Gels were stained by MS-compatible silver staining. The data in panel B represent mean ± SD from three independent experiments. Asterisk (*) indicates significant differences in the activity in crude and apoplast with p ≤ 0.05.
DHA by GSH. A factor of 0.98 was considered to account for the small contribution to the absorbance by the oxidized GSSG. The activity was calculated using an extinction coefficient of 14 mm−1 cm−1 and was expressed in μmol/min/mg protein. Glutathione S-transferase (GST) activity (EC 2.5.1.18) was measured spectrophotometrically following Arasimowicz et al.43 The reaction mixture (1 mL) contained 12 μg of protein extract, 0.1 M sodium phosphate buffer (pH 7.6), 20 mM 1chloro-2,4-dinitrobenzene (CDNB), and 20 mM GSH. The absorbance was measured at 340 nm, and the activity was calculated using extinction coefficient of 9.6 mm−1 cm−1.
quantitative change between spot abundance on the 2-D gel of control and cold-treated S-nitrosylated samples.
3. RESULTS AND DISCUSSION 3.1. Extraction of Contamination-Free Apoplastic Proteins and Their Resolution by 2-DE
Because apoplast is the first line of defense against stress, understanding the role of NO in regulating the stress signaling is crucial. The information about NO signaling in the apoplast is scanty. Therefore, to analyze this, apoplastic proteins were extracted by vacuum infiltration in different buffers [20 mM sodium acetate (pH 3 and 5.5), 20 mM sodium phosphate (pH 6), and 20 mM HEN (pH 7.7)]. Out of these, sodium acetate (pH 3 and 5.5) provided RuBisCO (the most abundant plant protein) free apoplastic fluid, while proteins extracted with sodium phosphate and HEN buffer showed RuBisCO contamination (Figure 1A). As a quality check, the activity of G6PDH (a cytosolic marker enzyme) was analyzed. Purified G6PDH from Leuconostoc mesenteroides, used as a positive control, showed 32 units/mg activity, indicating that the activity assay is working optimally. G6PDH activity was undetectable in the apoplastic fractions extracted in sodium acetate (pH 3), while 10, 12, and 12.5% of the crude activity (0.077 unit/mg ± 0.004) was observed in sodium acetate (pH 5.5), sodium phosphate (pH 6), and HEN (pH 7.7), respectively (Figure
2.13. Statistical Analysis
For each experiment, the tissue to be extracted was divided in five sets (with 10 g tissue in each set) for thorough washing and removal of the cytoplasmic proteins. The extracted apoplastic fluid was pooled and was considered as one biological set. All experiments were performed in three biological and three technical replicates. Experimental data shown in the NO measurement, thiol pool, SNO pool analysis, and the enzymatic activity assays were analyzed with SPSS 16.0 statistical program (SPSS, Chicago, IL). Two-way ANOVA was performed considering cold stress and time interval as two independent factors. The differences between individual means were compared by Duncan’s multiple comparison test. Student’s t test (p < 0.05) was used to determine any significant 2603
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1B). Because
