Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins
© Barkla et al. 2016
Received: 8 December 2015
Accepted: 2 May 2016
Published: 10 May 2016
Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant.
In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance.
This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.
KeywordsProteomics Trichome Salinity Salt tolerance Crassulacean acid metabolism (CAM) Ionomics Chloride Sodium V-ATPase Single cell-type
Single-cell-type analysis is a powerful experimental approach, allowing for the capture of information from specific cell types that would normally be lost due to the heterogeneity of cells in a tissue, giving us greater insight into the role of specialized cells. In plants, successful single cell type analysis has been undertaken for only a handful of cell types, including pollen grains, but also, due to ease of isolation, cells of the epidermis, such as root hairs, guard cells and trichomes . Trichomes are highly differentiated cell types found on the aerial epidermis of most plants. These specialized cells vary morphologically and functionally, with roles in plant defence, stress tolerance, water collection, seed dispersal and leaf structure. They can range from simple unicellular hair-like extensions to multicellular complex appendages [2, 3]. They are classified as non-secreting or glandular-secreting trichomes; the latter can secrete a vast array of substances including lipophilic compounds, proteins, ions, sugars and secondary plant products [4, 5]. Halophyte plant species have evolved several different types of trichomes ranging from bi- or multi-cellular glands of the Poaceae, which actively excrete salt , to non-glandular (non-secreting) trichomes called epidermal bladder cells (EBC). These EBC are attached to either the epidermis via stalk cells, as in the Chenopodiaceae , or stalk-less as in the Mesembryanthemaceae . In the halophyte Mesembryanthemum crystallinum these single celled EBC are present on leaves, stems and flower buds. Cell morphology changes with plant age and metabolic/stress state of the plant. In young plants the EBC are small and flattened to the leaf surface and stem, whereas in adult plants that are undergoing Crassulacean acid metabolism (CAM), and particularly those exposed to salt, the cells swell up and appear as liquid filled balloons. On average, the diameter of EBC can be 1 mm with an average cell volume of 500 nl; although volumes in excess of 5 μl have been reported [8, 9].
Early work on the physiology of EBC in M. crystallinum concluded that these cells were predominantly involved in water storage during times of reduced water availability . However, we now know that they are also substantial stores for sodium ions. EBC have been shown to accumulate as much as 1.2 M Na+ which is thought to be sequestered into the large central vacuole [8, 11]. Evidence that EBC are essential for salt tolerance of M. crystallinum, comes from studying a mutant that had reduced numbers of these specialized cells . Mutant plants showed diminished Na+ accumulation capacity, reduced leaf and stem water content and a significant reduction in seed number, however the gene involved was not identified.
Initial proteomic profiling of the EBC extract isolated from salt-treated adult M. crystallinum plants by single cell sampling techniques and shot-gun LC-MS/MS was only able to identify 84 proteins at high confidence. These belonged to diverse functional classes, including proteins involved in ion and water homeostasis, but also photosynthesis related proteins and proteins associated with CAM . Understanding which proteins are critical and central for bladder cell function and adaptation to salt stress can only be obtained by direct comparisons between EBC from untreated plants and plants that have been salt-treated. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label and label-free approach to identify salt-responsive proteins in the EBC extract. Using these approaches, we were able to identify 438 proteins at high confidence and show significant changes between treatment conditions in 54 of these. In order to confirm these results a number of the proteins were then validated by western blot analysis. In parallel, an ionomics analysis was carried out to determine the ion profile of the bladder cells and how this may change under salinity stress with the accumulation of sodium in these cells. These results, combined with our previous transcriptomics and metabolomics profiling data, allow for an integrated view of the adaptive responses occurring in the bladder cells to salt-treatment.
Quantitative proteomic analysis
To identify salt-responsive proteins in EBC extracts, a study combining complementary 2D-DIGE and 1D-PAGE with label-free LC-MS/MS (GeLC-MS/MS) based quantification methods was performed to compare proteins in extract from salt-treated plants to EBC extract from untreated control plants with the aim to maximize the coverage of the proteome. The use of distinctive approaches, which exploit alternative technologies requiring unique sample handling procedures, helped to obtain a greater coverage of the salt-responsive proteome.
Results from DIGE analysis of control vs salt treated EBC extract
Protein(s) in Spotc
GO biological processe
V-ATPase A subunit
V-ATPase B subunit
NADP-dependent malic enzyme
NADP-dependent malic enzyme
NADP-dependent malic enzyme
NADP-dependent malic enzyme
no proteins identified
Alpha-1,4-glucan protein synthase
cell wall metabolism
no proteins identified
cell wall metabolism
cell wall metabolism
V-ATPase E subunit
no proteins identified
Fructose bisphosphate aldolase,
cell wall metabolism
amino acid metabolism
V-ATPase A subunit
Protein identity following LC-MS/MS (protein threshold 99 %, peptide threshold 95 %, at least two unique peptides) was successfully achieved at high confidence for 19 of the 22 spots (Table 1 and Additional file 2). We were unable to detect protein in three spots (923, 1064, and 1290) and in two of the spots (740 and 1123) we identified more than one protein. These 19 spots corresponded to 14 different proteins. Products expressed from a single gene can migrate to multiple spots on 2D gels for a variety of reasons indicating protein modifications leading to a change in overall protein charge and/or molecular weight (MW) such as splice variants, proteolytic cleavage products, and processed proteins, as well as post-translational modified proteins .
In parallel, a complementary proteomics approach was carried out using GeLC–MS/MS, in which protein, from EBC extracts collected from control and salt-treated plants under the exact same conditions as for the 2D-DIGE analysis, was separated by 1D-GE. Each lane, representing one biological replicate of a total of 3, was then sliced into seven pieces as indicated in Additional file 3. This was followed by in-gel digestion and analysis of the resulting tryptic peptide mixtures by LC-MS/MS. In total, 1731 unique peptides derived from 438 proteins were identified in the six EBC samples. For subsequent analyses, only those proteins that were detected in all three biological replicates of either control or salt-treated samples (or both) by at least two unique peptides were considered (225 proteins). For the analysis of the presence/absence of the proteins in different biological replicates, unweighted spectral counts were used.
Western blot validation of proteomics results
The majority of epidermal cell types do not contain chloroplasts, with the exception of the guard cells , and some trichomes . Although early work raised doubt about the presence or functionality of chloroplasts in M. crystallinum EBC , more recent studies relying on new technologies including proteomics and single cell-type sampling methods have identified proteins associated with photosynthesis [9, 22].
pH and Malate concentration in EBC extract
The development of a high throughput and precise technique to sample the extract of the epidermal bladder cells from Mesembryanthemum crystallinum provides a unique opportunity to understand function and regulation of genes and pathways with concise contextual information from a single cell type. In combination with multi-omics approaches, we can begin to build a comprehensive integrated picture of cellular processes within the bladder cell. In this study, we performed both label and label-free proteomics and carried out ionomics analysis of the EBC extracts from control and salt-treated plants, to complete our omics analysis of these cells [24, 28].
The complementary nature of the quantitative proteomics technologies used in this study (2D-DIGE and 1D-GE label free) is underscored by the lack of overlap in the proteins identified (Tables 1 and 2). This highlights the advantage of combining different approaches and techniques to obtain a greater coverage of the proteome. Differences in sample handling, from the composition of the sample buffers to the gel separation conditions, combined with the physicochemical properties of the proteins in the sample result in unique differences in protein profiling between the two approaches helping to maximize the number of proteins identified. Using GeLC-MS/MS we were able to identify 141 more proteins than had previously been identified employing shotgun LC-MS/MS; an increase of 2.7 fold . These numbers are based on the identification of at least two unique peptides and the protein being present in all three biological replicates of either control or salt-treated samples for GeLC-MS/MS (this study), or two of four biological replicates from our previous LC-MS/MS profiling study . Using this criteria, only 11 proteins were exclusive to the previous LC-MS/MS analysis. Obtaining comprehensive protein profiles from very complex samples is challenging due to the large number of proteins present in the sample over a wide dynamic range in abundance. In the EBC extract a cysteine protease makes up nearly 50 % of the identified spectra in the samples , and is the most abundant protein on SDS-PAGE gels (Additional file 3 - asterisks). The high abundance of this protein would result in an under sampling of the low abundant proteins in the fraction. GeLC-MS/MS helped to overcome this problem by decreasing sample complexity, and when directly compared to LC-MS/MS in this study and others [29, 30], it was shown to perform better in the number of protein identifications, reproducibility of identifications and % coefficient of variance on spectral counts.
Ionomics analysis of elements reveals that the EBC Na/K ratio goes from 0.075 mg/L in the EBC from control plants to 7.133 mg/L in the EBC from salt-treated plants, a 100-fold difference (Additional file 4). Salinity commonly reduces the amount of K in cells from both glycophytes and halophytes ; however, in halophytes Na can substitute for K for turgor generation and cell growth .
The combined accumulation of Na and K usually exceeds Cl by about 35 % in dicotyledonous species and by at least double in halophytic grasses . In this study, while control untreated plants had a combined accumulation of Na and K more than double that of the Cl content (Clav = 3144 mg/L; Naav + Kav = 7985 mg/L), in the salt treated plants the Cl content was 1.4 fold that of Naav + Kav (Additional file 4). Chloride was the most accumulated ion, exceeding Na by 1.4 fold, suggesting an important role of EBC in Cl accumulation and detoxificaiton. Few studies of salt tolerance traits have linked tolerance to chloride homeostasis, and mechanisms of chloride transport into and within cells is poorly understood in comparison to Na transport . Of two possible chloride channels belonging to the CLC family of anion transporters only one, CLC-b, which is thought to be tonoplast localized from studies in Arabidopsis , was found to be significantly upregulated in our RNA-seq analysis of EBC .
Significant increases in the levels of manganese were also detected in the salt-treated plants (Fig. 5). This micronutrient activates decarboxylase, dehydrogenase and oxidase enzymes and is therefore an essential regulator for both glycolysis and CAM enzymes . Additionally, Mn is important for redox systems, as activators of various enzymes including those involved in the detoxification of superoxide radicals  and therefore increases may be linked to stress-induced ROS production.
The view of the EBC as a simple passive storage body for sodium and water is mistaken. Rather, our single-cell-type omics approach, combining proteomics and ionomics in this study, with transcriptomics , and metabolomics data , shows indisputably that these specialized cells are highly metabolically active, with photosynthesis and primary metabolism supporting rapid cell expansion, ion accumulation, compatible solute synthesis and CAM.
Plant materials and growth conditions
Mesembryanthemum crystallinum L. plants were germinated in potting substrate (MetroMix 510; SunGro Horticulture, Bellevue, WA) in a propagation tray. Three weeks following germination, individual seedlings were transplanted to pots containing potting substrate at a density of two plants per 15-cm-diameter pot. The watering regime consisted of daily watering with tap water with a weekly supply of Hoagland’s medium  until plants were 6 weeks old. At that time NaCl treatment (200 mM) was initiated for a period of 14 d with either water (for control plants) or NaCl (for salt-treated plants) supplied daily. Plants were grown in a glasshouse under natural irradiation and photoperiod at, 18.93 latitude and -99.23 longitude and an elevation 1540 m above sea level in the months of March to June. Temperature was maintained at 25 °C ± 3 °C and peak photosynthetic photon flux density was 1300 mmol m−2 s−1 during the middle of the day.
Extraction of bladder cell Sap
Vacuum aspiration was applied to collect bladder cell sap from individual cells on the leaf or stem epidermal surface using a fine gage insulin needle (27G, 13 mm) attached to a collection reservoir maintained on ice. To avoid contamination of cellular contents from underlying cell types the collection needle was oriented horizontally to the leaf or stem axis and the procedure was visualized using a Nikon SMZ645 stereo microscope equipped with a dual arm Nikon MKII fibre optic light source (Nikon, Japan). Extracts for a single plant were pooled to obtain approximately 1 mL of sample (approximately 3000 EBC), representing a single biological replicate and in this way distinct biological replicates as indicated for the individual experiments were collected (2D-DIGE – 4 biological replicates and Label-free Proteomics – 3 biological replicates).
Protein determination in samples
Protein in EBC extracts was measured by a modification of the Bradford method . Triton X-100 [0.5 % (v/v)] was added for 5 min before dilution of the sample and the addition of the dye reagent concentrate (Bio-Rad); the final concentration of Triton X-100 in the assay following dilution was 0.015 % (v/v). Protein in samples prepared for 2D-DIGE analysis was measured by the RCDC Protein Assay Kit (Bio-Rad) according to manufacturer’s instructions. For both methods BSA was employed as the protein standard.
EBC sap was diluted in 2X concentrated TE buffer (final; 10 mM Tris/HCl pH 7.6; 1 mM EDTA pH 8; 0.1 % (w/v) sodium deoxycholate) and samples were precipitated sequentially; first with 72 % (w/v) TCA, followed by 90 % (v/v) acetone. Protein (75 μg) was then desalted/cleaned according to manufacturer’s instructions with the ReadyPrep 2D Cleanup kit (Bio-Rad). The final protein pellet was resuspended in labelling buffer; 30 mM Tris-HCl pH 8.5, 7 M urea, 2 M thiourea, 2 % CHAPS (w/v), 2 % (w/v) amidosulfobetaine-14 (ASB-14). Samples were then labelled with the appropriate CyDye (Cy2, Cy3, or Cy5) according to the strategy outlined in the experimental design (Additional file 5). To each sample, 300 pmol of the appropriate dye was added and samples were incubated for 30 min on ice in the dark. The labelling reaction was stopped by the addition of one μl of 10 mM lysine and incubated on ice for a further 10 min. To avoid CyDye specific artifacts resulting from preferential labelling or variable fluorescence characteristics of the gel matrix or glass plates at the different excitation wavelengths used for acquisition, dye swapping between experimental samples was carried out (Additional file 5). Following labelling, equal volumes of rehydration buffer containing 2X DTT and ampholytes was added to each sample (7 M urea, 2 M thiourea, 2 % (w/v) ASB-14, 2 % (w/v) CHAPS, 100 mM DTT, 1 % (v/v) Bio-Lyte 3–10 ampholytes (Bio-Rad) to give a final concentration of 50 mM DTT and 0.5 % (v/v) Bio-Lyte 3–10 ampholytes. The three different CyDye labelled samples for each gel were then pooled and brought to a final volume of 300 μl with rehydration buffer containing 50 mM DTT and 0.5 % ampholytes.
2D Gel electrophoresis, Gel imaging and image analysis
For rehydration Ready Strip IPG strips (17 cm, linear pH 3–10, Bio-Rad) were layered gel side down onto CyDye labelled samples placed in the Protean IEF tray (Bio-Rad) ensuring bubbles were not trapped under the strip. Strips were carefully covered with 2 ml of mineral oil and active rehydration was carried out overnight in a Protean IEF Cell (Bio-Rad) at 50 V and 20 °C in the dark. Following overnight rehydration of the strips, isoelectric focusing (IEF) was initiated for a total of 40,000 volt hours with a maximum current setting of 50 μA per strip, using a three-step ramping protocol. After IEF the IPG strips were first equilibrated by shaking for 15 min in DTT equilibration buffer (6 M urea, 0.375 M Tris-HCl pH 8.8, 2 % (w/v) SDS, 20 % (w/v) glycerol and 2 % (w/v) DTT), and then for an additional 15 min in iodoacetamide equilibration buffer (6 M urea, 0.375 M Tris-HCl pH 8.8, 2 % (w/v) SDS, 20 % (w/v) glycerol and 2.5 % (w/v) iodoacetamide). Equilibrated gel strips were loaded onto 10 % acrylamide gels cast between low fluorescence glass plates coated on one side with bind silane solution (80 % (v/v) ethanol, 2 % (v/v) glacial acetic acid and 0.001 % (v/v) bind silane). Two fluorescent reference markers were included on opposite sides of the glass plate containing bind silane to facilitate robotic spot picking. Strips were overlaid with 0.5 % (w/v) low melting point agarose in SDS running buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.1 % (w/v) SDS and 0.01 % (w/v) Bromophenol Blue) and SDS-PAGE was carried out using the Ettan Daltsix electrophoresis system (GE Lifesciences) at 10 mA/gel for 1 h followed by 12 mA/gel for a total of 17–20 h, in the dark at 25 °C.
Individual gels were scanned at three different wavelengths using a Typhoon Variable Mode TM 9410 imager (GE Lifesciences) to obtain the images for each of the three CyDyes according to the acquisition conditions outlined in Additional file 6 online. Image analysis was carried out using the DeCyder 2D Software V6.5 following the manufacturer’s instructions (GE Lifesciences) and as described .
Spot picking and protein identification by ESI-LTQ-orbitrap MS/MS
Protein spots of interest were excised from the gels using the Ettan Spot Picker robot (GE Lifesciences) according to the spot pick map generated by the Decyder software for significantly altered spots. Protein spots were sent by overnight courier to the Proteomics Facility at the Institut de Recherches Clinique de Montreal, Canada for processing and MS analysis. Digestion of protein was carried out according to the in-gel method . Protein digests were desalted by solid-phase extraction employing C18-ZipTips from Millipore. Peptides were bound, washed, and then eluted in 15 μl of 1 % (v/v) formic acid in 50 % (v/v) ACN.
The resulting peptide mixtures from each excised spot were analysed by nano LC-MS/MS using a Finnigan MicroAS autosampler and a Surveyor MS pump system coupled to an LTQ-Orbitrap (ThermoFisher Scientific). Forty μL of each peptide mixture was loaded on a C18 precolumn (Symmetry300 C18 5 μm, NanoEase Trap Column, Waters) at 3 μl/min for 15 min in 0.1 % (v/v) formic acid in 5 % (v/v) ACN. Peptides were eluted using a 5–35 % gradient of solvent B (0.1 % (v/v) formic acid in 100 % (v/v) ACN) during 60 min at a flow rate of 300 nl/min with a BioBasic C18 picofrit column (PFC7515/BI/10, NewObjective). Data-dependent acquisition mode was carried out with the Xcalibur software. A Fourier transformed (FT) full scan from 300 to 1800 m/z was acquired by means of the Orbitrap, with resolving power set at 30,000 (400 m/z). The five most intense peaks were sequentially isolated for the MS/MS experiments using collisionally induced dissociation. Dynamic exclusion was set to two and selected ions were placed on the exclusion list for 45 s to prevent duplication of MS/MS data for the same peptide. The MS/MS raw spectra data were converted to DTA files using ThermoElectron Bioworks 3.2 and analyzed by means of Turbo SEQUEST (ThermoFisher Scientific). From a general Viridiplantae_txid33090 database (unknown version, 677107 entries) two decoy databases were generated for M. crystallinum and A. thaliana. Independent searching was carried out using rigorous parameters (Xcorr z = 1: 1.90, z = 2: 2.70, z = 3: 3.50, z = 4: 3.75 and Delta Cn >0.1) and allowing dynamic modifications for cysteine alkylation with iodoacetamide and methionine oxidation.
Label-free quantitative proteomics
EBC extracts (20 μg protein per sample) were precipitated using 1:1 volumes of ethanol/acetone, resuspended in 2.5 % (w/v) SDS Tris/glycine sample buffer, heated at 60 °C for 2 min, and loaded onto a 10 % (w/v) acrylamide mini-gel. Following electrophoresis (200 V for 55 min) gels were stained in Coomassie Blue and each replicate lane was subsequently sliced into seven pieces as indicated in Additional file 3. Gel slices were processed as described above for gel spots. Data from all gel slices representing a single lane or biological replicate were combined for further analysis.
SDS PAGE, staining and western immuno-blotting
Protein samples were precipitated by dilution of the samples 50 fold in 1:1 (v/v) ethanol/acetone and incubated overnight at 30 °C according to the method of Parry et al., . Samples were then centrifuged at 13 000 g for 20 min at 4 °C using an F2402 rotor in a GS15R table top centrifuge (Beckman). Pellets were air dried, re-suspended with sample buffer (2.5 % (w/v) SDS), and heated at 60 °C for 2 min before loading (15 μg of protein per lane) onto 10 % (w/v) linear mini gels (Bio-Rad). After electrophoresis, SDS-PAGE separated proteins were either fixed and stained with Coomassie R250, or electrophoretically transferred onto nitrocellulose membranes (ECL, GE Lifesciences) for western immunoblot analysis as previously described . Digital chemiluminescent images were captured using a C-DiGit Blot scanner (LICOR Biosciences). Primary antibodies used in this study were either commercially available or custom made as indicated. Antibodies purchased from Agrisera (Agrisera, Sweden) included the A. thaliana V-ATPase subunits VHA-A (69 kD; AgriSera Cat# AS09 467 RRID:AB_1832048), VHA-B (55 kD; AgriSera Cat# AS09 503 RRID:AB_1832050), VHA-c (16 kD; AgriSera Cat# AS09 468 RRID:AB_1832051) and VHA-E (29 kD; AgriSera Cat# AS07 213 RRID:AB_1031583); and the general regulatory element 14-3-3 protein GRF (20 kD; AgriSera Cat# AS12 2119). Anti-enolase antibodies were purchased from Santa Cruz Biotechnology (50 kD; cat. #sc7455 AB_640163). Custom and in-house made primary antibodies used included; the M. crystallinum phosphoenol pyruvate carboxylase anti-PEPCase CAM isoform (110 kD) ; the M. crystallinum aquaporin PIP1;4 peptide specific antibody (41 kD) ; M. crystallinum myo-inositol O-methyl transferase anti-IMT antibodies (40 kD) [53, 54]. Dilutions were as follows: VHA-A, VHA-B, VHA-c, were 1/2000; all others were 1/1000.
For analysis of elements, EBC extracts (six biological replicates for each condition) were analysed on a Perkin Elmer NexION 300D ICPMS. The instrument was calibrated for each element using a three-point calibration curve, prepared from certified stock solutions, to provide an R2 coefficient of 0.9999 or greater. The accuracy of the calibration was confirmed by analysing standards that are independent from those of the calibrating solution. Calibration standards were re-analysed every 20 samples to confirm calibration stability and suitable internal standards were used to monitor and correct for instrument drift. Polyatomic interferences were removed using helium gas in Kinetic Energy Discrimination (KED) mode, additionally methane was used in Dynamic Reaction Cell (DRC) to remove interferences on selenium. Ionomics data was checked for outliers (>3 SD from the mean) and one value was removed for each of nickel, lead, zinc, silicon and barium for all subsequent analyses. One-way analyses of variance and principal component analysis (PCA) were done using Genstat software . The PCA was based on the correlation matrix to avoid biasing the results towards trait with high variance. PCA loadings and scores were extracted and the values for components 1 vs 2 plotted.
pH and Malate measurements
The pH of the EBC extract was measured directly in the fluid obtained with a pH micro-electrode PerpHecT Ross microcombination pH electrode (8220BNWP model, Thermo-Fisher Scientific) connected to a pH meter (Accumet, Fisher Scientific).
Malate was quantified enzymatically by a coupled enzyme assay according to Hohorst . The reaction medium contained 50 mM glycylglycine (pH 10), 30 mM L-glutamate, 3 mM NAD+, I U of glutamate oxaloacetate transaminase (GOT, Sigma-Aldrich), 10 U of L-malate dehydrogenase (MDH, Sigma-Aldrich). Malate concentrations were obtained by calculating the difference in the absorbance at 340 nm before and after 20 min incubation at RT. Measurements of pH and malate were made on four independent samples for each time and treatment, and the results for malate were expressed as μmol malate ml−1 of EBC extract.
Fluorescence microscopy was performed using an upright multiphotonic confocal microscope (Olympus FV1000) equipped with an XLPLN 25X W NA1.05 water immersion objective. Stem sections from salt-treated plants were submerged in water for imaging. Laser wavelength 1 = 488 (green) cell wall autofluorescence, Laser wavelength 2 = 635 (red) chloroplast autofluorescence. Chlorophyll autofluorescence was visualized by excitation with a multi-line Argon laser at 635 nm and spectral detector set between 650–750 nm for the emission.
Availability of data and materials section
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD004045.
two dimensional differential in-gel electrophoresis
biological variation analysis
crassulacean acid metabolism
epidermal bladder cell
exponentially modified protein abundance
in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry
inductively coupled plasma - mass spectrometry
inductively coupled plasma - optical emission spectrometry
inositol methyl transferase
myo-inositol 1-phosphate synthase
Liquid chromatography-tandem mass spectrometry
normalized spectral abundance factor
principal component analysis
sodium dodecyl sulfate polyacrylamide gel electrophoresis
total spectra count
weighted spectra count
The authors thank Andrés Saralegui, IBT, for technical support with confocal microscopy, Alicia Hidden, SCPS, for greenhouse support, Dr. Denis Faubert and his team at Institut de Recherches Cliniques de Montréal—Proteomics Discovery Platform for MS analysis and the Environmental Analysis Lab (EAL) at SCU for ICP-MS analysis. BJB acknowledges financial support from Southern Cross Plant Science and DGAPA-UNAM-PAPIIT.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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