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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.


Box plots of the significant changes in ion concentration of EBC from control and salt-treated plants. For each concentration, the box represents the interquartile range (IQR), the bisecting line represents the median, the square symbol represents the mean, the whiskers represent the 95th and 5th percentiles, and the X symbols represent the maximum and minimum values. Elements which are significantly different between treatments with a P < .01 are designated **, while those significantly different with P < 0.05 are designated *
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Fig5: Box plots of the significant changes in ion concentration of EBC from control and salt-treated plants. For each concentration, the box represents the interquartile range (IQR), the bisecting line represents the median, the square symbol represents the mean, the whiskers represent the 95th and 5th percentiles, and the X symbols represent the maximum and minimum values. Elements which are significantly different between treatments with a P < .01 are designated **, while those significantly different with P < 0.05 are designated *

Mentions: The combined use of ICP-MS (Inductively Coupled Plasma - Mass Spectrometry) or ICP-OES (Inductively Coupled Plasma - Optical Emission Spectrometry) enabled the determination of 27 major and trace elements (Al, As, Ba, B, Br, Cd, Ca, Cl, Cr, Co, Cu, Fe, Pb, Hg, Mg, Mn, Mo, Ni, P, K, Se, Si, Ag, Na, S, V, and Zn) in EBC extracts with high accuracy and precision. The elemental concentrations varied by almost seven orders of magnitude (Additional file 4). The least abundant elements measured in the EBC extract from both control and salt-treated plants were Cd, Cr, Pb, Hg, V, Co, all of which were below 0.05 mg/L. The most abundant element in the EBC extract from control plants was K, while the most abundant elements in the EBC extract from salt-treated plants was Cl followed by Na (Additional file 4). Salt-treatment strongly affected the accumulation of numerous elements (Fig. 5). As expected, and has been shown previously [8, 11, 24], Na increased significantly in the EBC (21-fold), from an average of 557 mg/L in control samples to 11,679 mg/L in the samples from salt-treated plants. Cl also showed a large increase of 5.7-fold; from 3144 mg/L in the control extract to 18,000 mg/L in the salt-treated extract. Significant increases were also seen in the abundance of Mn, P, V and Zn (Fig. 5). Elements that showed a significant decrease in abundance relative to control in the samples from salt-treated EBC were K, S, Mg and Co (Fig. 5) with K decreasing 4.5-fold from an average of 7429 mg/L in the EBC from control plants to only 1637 mg/L in salt-treated plants and sulphur decreasing 4-fold from an average of 442 mg/L in control plants to 111 mg/L in salt-treated plants (Additional file 4 and Fig. 5).Fig. 5


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)

Box plots of the significant changes in ion concentration of EBC from control and salt-treated plants. For each concentration, the box represents the interquartile range (IQR), the bisecting line represents the median, the square symbol represents the mean, the whiskers represent the 95th and 5th percentiles, and the X symbols represent the maximum and minimum values. Elements which are significantly different between treatments with a P < .01 are designated **, while those significantly different with P < 0.05 are designated *
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Box plots of the significant changes in ion concentration of EBC from control and salt-treated plants. For each concentration, the box represents the interquartile range (IQR), the bisecting line represents the median, the square symbol represents the mean, the whiskers represent the 95th and 5th percentiles, and the X symbols represent the maximum and minimum values. Elements which are significantly different between treatments with a P < .01 are designated **, while those significantly different with P < 0.05 are designated *
Mentions: The combined use of ICP-MS (Inductively Coupled Plasma - Mass Spectrometry) or ICP-OES (Inductively Coupled Plasma - Optical Emission Spectrometry) enabled the determination of 27 major and trace elements (Al, As, Ba, B, Br, Cd, Ca, Cl, Cr, Co, Cu, Fe, Pb, Hg, Mg, Mn, Mo, Ni, P, K, Se, Si, Ag, Na, S, V, and Zn) in EBC extracts with high accuracy and precision. The elemental concentrations varied by almost seven orders of magnitude (Additional file 4). The least abundant elements measured in the EBC extract from both control and salt-treated plants were Cd, Cr, Pb, Hg, V, Co, all of which were below 0.05 mg/L. The most abundant element in the EBC extract from control plants was K, while the most abundant elements in the EBC extract from salt-treated plants was Cl followed by Na (Additional file 4). Salt-treatment strongly affected the accumulation of numerous elements (Fig. 5). As expected, and has been shown previously [8, 11, 24], Na increased significantly in the EBC (21-fold), from an average of 557 mg/L in control samples to 11,679 mg/L in the samples from salt-treated plants. Cl also showed a large increase of 5.7-fold; from 3144 mg/L in the control extract to 18,000 mg/L in the salt-treated extract. Significant increases were also seen in the abundance of Mn, P, V and Zn (Fig. 5). Elements that showed a significant decrease in abundance relative to control in the samples from salt-treated EBC were K, S, Mg and Co (Fig. 5) with K decreasing 4.5-fold from an average of 7429 mg/L in the EBC from control plants to only 1637 mg/L in salt-treated plants and sulphur decreasing 4-fold from an average of 442 mg/L in control plants to 111 mg/L in salt-treated plants (Additional file 4 and Fig. 5).Fig. 5

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.