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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 BJ, Vera-Estrella R, Raymond C - BMC Plant Biol. (2016)

Bottom Line: Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum.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.Data are available via ProteomeXchange with identifier PXD004045.

View Article: PubMed Central - PubMed

Affiliation: Southern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, Australia. bronwyn.barkla@scu.edu.au.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Integration of transcript, protein, metabolite and ionome data into EBC metabolic pathways. Data from all studies was obtained from 6-week-old plants treated for 2 weeks with 200 mM NaCl. Bladder cell extract was collected at the end of the dark period. Transcriptomic data from Oh et al., [24] and metabolomics data from Barkla and Vera-Estrella, [28]. T, transcript; P, protein and W, western blot analysis. Red arrows indicate changes in metabolites. Enzyme abbreviations: BAM – ϐ-amylase, HK – hexokinase, PGI – glucose-6P-isomerase, PFK – phosphofructokinase, FBA – aldolase, TPI – triose-P-isomerase, G3PD – glyceraldehyde-3P-dehydrogenase, PGK – phosphoglycerate kinase, PGM – phosphoglycerate mutase, ENO – enolase, PK- pyruvate kinase, FK – fructokinase, CS – citrate synthase, ACO – aconitase, IDH – isocitrate dehydrogenase, α-KGDH - α-ketoglutarate dehydrogenase, SCS – succinyl-CoA synthetase, SDH – succinate dehydrogenase, FUM – fumerase, MDH – malate dehydrogenase, PEPCK – PEP carboxykinase, ME – malic enzyme, PEPC – phosphoenolpyruvate carboxylase, PPDK – pyruvate-Pi-dikinase, CA – carbonic anhydrase, GDH – glutamate dehydrogenase, P5CS - pyrroline-5-carboxylate synthase, P5CR - pyrroline-5-carboxylase reductase, OAT - ornithine aminotransferase, ARG – arginase, INSP - myo-inositol 1-phosphate synthase, IMP – myo-inositol monophosphatase, IMT – inositol methyl transferase, OEP - ononitol epimerase, MAT – methionine adenosyltransferase, SAM - S-adenosyl methionine, SAH - S-adenosylhomocysteine, SAHH – S-adenosylhomocysteine hydrolase, MET – methionine, METS – methionine synthase, ATP-SF - ATP-sulfurylase, APSK - 5’-adenylylsulfate kinase, GOX - glucose oxidase, CAT – catalase
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Fig7: Integration of transcript, protein, metabolite and ionome data into EBC metabolic pathways. Data from all studies was obtained from 6-week-old plants treated for 2 weeks with 200 mM NaCl. Bladder cell extract was collected at the end of the dark period. Transcriptomic data from Oh et al., [24] and metabolomics data from Barkla and Vera-Estrella, [28]. T, transcript; P, protein and W, western blot analysis. Red arrows indicate changes in metabolites. Enzyme abbreviations: BAM – ϐ-amylase, HK – hexokinase, PGI – glucose-6P-isomerase, PFK – phosphofructokinase, FBA – aldolase, TPI – triose-P-isomerase, G3PD – glyceraldehyde-3P-dehydrogenase, PGK – phosphoglycerate kinase, PGM – phosphoglycerate mutase, ENO – enolase, PK- pyruvate kinase, FK – fructokinase, CS – citrate synthase, ACO – aconitase, IDH – isocitrate dehydrogenase, α-KGDH - α-ketoglutarate dehydrogenase, SCS – succinyl-CoA synthetase, SDH – succinate dehydrogenase, FUM – fumerase, MDH – malate dehydrogenase, PEPCK – PEP carboxykinase, ME – malic enzyme, PEPC – phosphoenolpyruvate carboxylase, PPDK – pyruvate-Pi-dikinase, CA – carbonic anhydrase, GDH – glutamate dehydrogenase, P5CS - pyrroline-5-carboxylate synthase, P5CR - pyrroline-5-carboxylase reductase, OAT - ornithine aminotransferase, ARG – arginase, INSP - myo-inositol 1-phosphate synthase, IMP – myo-inositol monophosphatase, IMT – inositol methyl transferase, OEP - ononitol epimerase, MAT – methionine adenosyltransferase, SAM - S-adenosyl methionine, SAH - S-adenosylhomocysteine, SAHH – S-adenosylhomocysteine hydrolase, MET – methionine, METS – methionine synthase, ATP-SF - ATP-sulfurylase, APSK - 5’-adenylylsulfate kinase, GOX - glucose oxidase, CAT – catalase

Mentions: Multi-omics data integration is challenging for plant-derived pathways and particularly for non-model plants, however, better insight into functional networks can be gained if we incorporate data compiled from different technologies. From information from this study and our previous omics analysis of EBC [24, 28], a picture is emerging of a metabolically active cell, photosynthetically active and undertaking CAM, with salinity treatment resulting in decreased abundance of photosynthetic machinery proteins, increases in enzymes involved in photorespiration, glycolysis and proteins specific for CAM. (Fig. 7). High metabolic activity highlights the considerable energy cost to drive compatible solute synthesis and Na accumulation in the cell [31]. From the 54 proteins that showed significant fold changes with salt treatment and were present in all biological replicates, a high proportion of those identified can be classified as transport proteins (Fig. 1a). Of these, four were subunits of the peripheral cytoplasmic V1 sector of the vacuolar H+-ATPase, V-ATPase; VHA-G, VHA-A, VHA-B and VHA-E (Fig. 8). Western blot analysis using subunit specific antibodies confirmed the increase in VHA-B and VHA-E, (Fig. 2) and results were corroborated for VHA-A and VHA-B from our previous EBC transcriptomics study [24]. Transcriptomics data also revealed changes in additional VHA V1 subunits, including VHA-D, VHA-F, VHA-H, but also subunits of the V0 membrane sector, VHA-c and VHA-d (Fig. 8). While these were not detected in the proteomics study, western blot analysis was able to confirm the change in abundance of VHA-c (Fig. 2). These results for EBC extracted total protein are in agreement to results reported for whole leaf microsomal proteomic analysis of M. crystallinum [32]. In that study significant changes in subunits VHA-A, VHA-E, but also VHA-a, were identified; additionally, although not significant, changes in relative abundance were also measured for VHA-B, VHA-G, VHA-H, VHA-c and VHA-d [32]. However, results would comprise a mix of protein originating from up to 15 cell types with diverse functions in the leaf [33]. In plants, the V-ATPase is not only present on the vacuolar membrane (tonoplast) but also endosomal vesicular compartments, where it has a role in luminal pH control, vesicle trafficking and generation of an electrochemical gradient for ion transport. Recent evidence employing V-ATPase mutants showed that the tonoplast localized V-ATPase does not play a role in salt tolerance, as despite lacking a functional tonoplast V-ATPase, mutants were still able to accumulate sodium [34]. Moreover, although capturing multiple full-length transcripts for tonoplast localized Na/H exchangers (NXH), none of them showed significant induction in response to salt [24], and no NHX proteins were identified in our quantitative proteomics analysis (Tables 1 and 2). Therefore, changes in abundance of V-ATPase in EBC observed in this study may be important for energizing the uptake of sodium into endosomal vesicles [35, 36], which are then delivered to and fuse with the tonoplast. Additionally, V-ATPase activity would be essential for turgor generation to facilitate rapid cell expansion of the EBC [37]. The role of the V-ATPase in determining cell shape and size through turgor generations has been demonstrated by studying Arabidopsis VHA-C mutants, which showed reduced cell expansion of specific cell types due to reduced turgor [38].Fig. 7


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 BJ, Vera-Estrella R, Raymond C - BMC Plant Biol. (2016)

Integration of transcript, protein, metabolite and ionome data into EBC metabolic pathways. Data from all studies was obtained from 6-week-old plants treated for 2 weeks with 200 mM NaCl. Bladder cell extract was collected at the end of the dark period. Transcriptomic data from Oh et al., [24] and metabolomics data from Barkla and Vera-Estrella, [28]. T, transcript; P, protein and W, western blot analysis. Red arrows indicate changes in metabolites. Enzyme abbreviations: BAM – ϐ-amylase, HK – hexokinase, PGI – glucose-6P-isomerase, PFK – phosphofructokinase, FBA – aldolase, TPI – triose-P-isomerase, G3PD – glyceraldehyde-3P-dehydrogenase, PGK – phosphoglycerate kinase, PGM – phosphoglycerate mutase, ENO – enolase, PK- pyruvate kinase, FK – fructokinase, CS – citrate synthase, ACO – aconitase, IDH – isocitrate dehydrogenase, α-KGDH - α-ketoglutarate dehydrogenase, SCS – succinyl-CoA synthetase, SDH – succinate dehydrogenase, FUM – fumerase, MDH – malate dehydrogenase, PEPCK – PEP carboxykinase, ME – malic enzyme, PEPC – phosphoenolpyruvate carboxylase, PPDK – pyruvate-Pi-dikinase, CA – carbonic anhydrase, GDH – glutamate dehydrogenase, P5CS - pyrroline-5-carboxylate synthase, P5CR - pyrroline-5-carboxylase reductase, OAT - ornithine aminotransferase, ARG – arginase, INSP - myo-inositol 1-phosphate synthase, IMP – myo-inositol monophosphatase, IMT – inositol methyl transferase, OEP - ononitol epimerase, MAT – methionine adenosyltransferase, SAM - S-adenosyl methionine, SAH - S-adenosylhomocysteine, SAHH – S-adenosylhomocysteine hydrolase, MET – methionine, METS – methionine synthase, ATP-SF - ATP-sulfurylase, APSK - 5’-adenylylsulfate kinase, GOX - glucose oxidase, CAT – catalase
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862212&req=5

Fig7: Integration of transcript, protein, metabolite and ionome data into EBC metabolic pathways. Data from all studies was obtained from 6-week-old plants treated for 2 weeks with 200 mM NaCl. Bladder cell extract was collected at the end of the dark period. Transcriptomic data from Oh et al., [24] and metabolomics data from Barkla and Vera-Estrella, [28]. T, transcript; P, protein and W, western blot analysis. Red arrows indicate changes in metabolites. Enzyme abbreviations: BAM – ϐ-amylase, HK – hexokinase, PGI – glucose-6P-isomerase, PFK – phosphofructokinase, FBA – aldolase, TPI – triose-P-isomerase, G3PD – glyceraldehyde-3P-dehydrogenase, PGK – phosphoglycerate kinase, PGM – phosphoglycerate mutase, ENO – enolase, PK- pyruvate kinase, FK – fructokinase, CS – citrate synthase, ACO – aconitase, IDH – isocitrate dehydrogenase, α-KGDH - α-ketoglutarate dehydrogenase, SCS – succinyl-CoA synthetase, SDH – succinate dehydrogenase, FUM – fumerase, MDH – malate dehydrogenase, PEPCK – PEP carboxykinase, ME – malic enzyme, PEPC – phosphoenolpyruvate carboxylase, PPDK – pyruvate-Pi-dikinase, CA – carbonic anhydrase, GDH – glutamate dehydrogenase, P5CS - pyrroline-5-carboxylate synthase, P5CR - pyrroline-5-carboxylase reductase, OAT - ornithine aminotransferase, ARG – arginase, INSP - myo-inositol 1-phosphate synthase, IMP – myo-inositol monophosphatase, IMT – inositol methyl transferase, OEP - ononitol epimerase, MAT – methionine adenosyltransferase, SAM - S-adenosyl methionine, SAH - S-adenosylhomocysteine, SAHH – S-adenosylhomocysteine hydrolase, MET – methionine, METS – methionine synthase, ATP-SF - ATP-sulfurylase, APSK - 5’-adenylylsulfate kinase, GOX - glucose oxidase, CAT – catalase
Mentions: Multi-omics data integration is challenging for plant-derived pathways and particularly for non-model plants, however, better insight into functional networks can be gained if we incorporate data compiled from different technologies. From information from this study and our previous omics analysis of EBC [24, 28], a picture is emerging of a metabolically active cell, photosynthetically active and undertaking CAM, with salinity treatment resulting in decreased abundance of photosynthetic machinery proteins, increases in enzymes involved in photorespiration, glycolysis and proteins specific for CAM. (Fig. 7). High metabolic activity highlights the considerable energy cost to drive compatible solute synthesis and Na accumulation in the cell [31]. From the 54 proteins that showed significant fold changes with salt treatment and were present in all biological replicates, a high proportion of those identified can be classified as transport proteins (Fig. 1a). Of these, four were subunits of the peripheral cytoplasmic V1 sector of the vacuolar H+-ATPase, V-ATPase; VHA-G, VHA-A, VHA-B and VHA-E (Fig. 8). Western blot analysis using subunit specific antibodies confirmed the increase in VHA-B and VHA-E, (Fig. 2) and results were corroborated for VHA-A and VHA-B from our previous EBC transcriptomics study [24]. Transcriptomics data also revealed changes in additional VHA V1 subunits, including VHA-D, VHA-F, VHA-H, but also subunits of the V0 membrane sector, VHA-c and VHA-d (Fig. 8). While these were not detected in the proteomics study, western blot analysis was able to confirm the change in abundance of VHA-c (Fig. 2). These results for EBC extracted total protein are in agreement to results reported for whole leaf microsomal proteomic analysis of M. crystallinum [32]. In that study significant changes in subunits VHA-A, VHA-E, but also VHA-a, were identified; additionally, although not significant, changes in relative abundance were also measured for VHA-B, VHA-G, VHA-H, VHA-c and VHA-d [32]. However, results would comprise a mix of protein originating from up to 15 cell types with diverse functions in the leaf [33]. In plants, the V-ATPase is not only present on the vacuolar membrane (tonoplast) but also endosomal vesicular compartments, where it has a role in luminal pH control, vesicle trafficking and generation of an electrochemical gradient for ion transport. Recent evidence employing V-ATPase mutants showed that the tonoplast localized V-ATPase does not play a role in salt tolerance, as despite lacking a functional tonoplast V-ATPase, mutants were still able to accumulate sodium [34]. Moreover, although capturing multiple full-length transcripts for tonoplast localized Na/H exchangers (NXH), none of them showed significant induction in response to salt [24], and no NHX proteins were identified in our quantitative proteomics analysis (Tables 1 and 2). Therefore, changes in abundance of V-ATPase in EBC observed in this study may be important for energizing the uptake of sodium into endosomal vesicles [35, 36], which are then delivered to and fuse with the tonoplast. Additionally, V-ATPase activity would be essential for turgor generation to facilitate rapid cell expansion of the EBC [37]. The role of the V-ATPase in determining cell shape and size through turgor generations has been demonstrated by studying Arabidopsis VHA-C mutants, which showed reduced cell expansion of specific cell types due to reduced turgor [38].Fig. 7

Bottom Line: Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum.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.Data are available via ProteomeXchange with identifier PXD004045.

View Article: PubMed Central - PubMed

Affiliation: Southern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, Australia. bronwyn.barkla@scu.edu.au.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.