Identification of Leaf Proteins Differentially Accumulated between Wheat Cultivars Distinct in Their Levels of Drought Tolerance

<div><p>The drought-tolerant ‘Ningchun 47’ (NC47) and drought-sensitive ‘Chinese Spring’ (CS) wheat (<i>Triticum aestivum</i> L.) cultivars were treated with different PEG6000 concentrations at the three-leaf stage. An analysis on the physiological and proteomic changes of wheat seedling in response to drought stress was performed. In total, 146 differentially accumulated protein (DAP) spots were separated and recognised using two-dimensional gel electrophoresis. In total, 101 DAP spots representing 77 unique proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These proteins were allocated to 10 groups according to putative functions, which were mainly involved in carbon metabolism (23.4%), photosynthesis/respiration (22.1%) and stress/defence/detoxification (18.2%). Some drought stress-related proteins in NC47, such as enolase, 6-phosphogluconate dehydrogenase, Oxygen-evolving enhancer protein 2, fibrillin-like protein, 2-Cys peroxiredoxin BAS1 and 70-kDa heat shock protein, were more upregulated than those in CS. Multivariate principal components analysis revealed obvious differences between the control and treatments in both NC47 and CS, while cluster analysis showed that the DAPs displayed five and six accumulation patterns in NC47 and CS, respectively. Protein–protein interaction network analysis showed that some key DAPs, such as 2-Cys peroxiredoxin BAS1, RuBisCO large subunit-binding protein, 50S ribosomal protein L1, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase isoenzyme and 70-kDa heat shock protein, with upregulated accumulation in NC47, had complex interactions with other proteins related to amino acid metabolism, carbon metabolism, energy pathway, signal transduction, stress/defence/detoxification, protein folding and nucleotide metabolism. These proteins could play important roles in drought-stress tolerance and contribute to the relatively stronger drought tolerance of NC47.</p></div

Principal components analysis (PCA) of the set of 101 DAP spots in NC47 and CS.

<p>(A) PCA of individual experimental samples in NC47. (B) PCA of 69 differentially accumulated protein spots in NC47. (C) PCA of individual experimental samples in CS. (D) PCA of 77 differentially accumulated protein spots in CS. Red spots indicate an upregulated protein; green spots indicate a downregulated protein. CS-0, CS-15, CS-20, CS-25 and CS-30 represent the 0, 15%, 20%, 25% and 30%, respectively, of the PEG6000 concentration treatment in CS. NC47-0, NC47-15, NC47-20, NC47-25 and NC47-30 represent the 0, 15%, 20%, 25% and 30%, respectively, of the PEG6000 concentration treatment in NC47.</p

Morphological and physiological changes in seedlings of two wheat (<i>Triticum aestivum</i> L.) cultivars, ‘Chinese Spring’ (CS) and ‘Ningchun 47’ (NC47), during 48 h of PEG-mediated drought stress.

<p>(A) Morphological changes of wheat seedlings in response to different PEG6000 concentration treatments. (B) Leaf relative water content (RWC) analysis. (C) Proline content analysis. (D) Soluble sugar content analysis.</p

Functional distribution of identified differentially accumulated proteins in CS and NC47.

<p>Functional distribution of identified differentially accumulated proteins in CS and NC47.</p

Comparison of some important DAPs in seedling leaves of NC47 and CS.

<p>^aCyto, cytoplasm; P: plastid; Mito: mitochondria; Nucl: nuclear; PM: Plasma membrane.</p><p>^b"N" represent the DAP spot was no obvious difference (< 2.0-fold).</p><p>Comparison of some important DAPs in seedling leaves of NC47 and CS.</p

Hierarchical cluster analysis of DAP spots in NC47 and CS.

<p>Red colour indicates a positive abundance in protein spots; green colour denotes a negative abundance in protein spots. (A) Cluster analysis of 69 differentially accumulated proteins in NC47. (B) Cluster analysis of 77 differentially accumulated proteins in CS. 0, 15%, 20%, 25% and 30% represent the PEG6000 concentration treatment.</p

Network of key DAPs in NC47 involved in drought adaptation and tolerance.

<p>Interactions of the DAPs are extracted by searching the STRING database with a confidence cutoff of 0.700. The interaction network is reconstructed using the Cytoscape software. Red colour represents the upregulated proteins and green colour shows the downregulated proteins in NC47 under high PEG6000 concentration stress. Blue colour indicates the signal transduction-related protein.</p

Expression of CD133 promoted autophagosome formation.

<p><b>A.</b> LM3 cells were transfected with either empty vector or p3xFlag-CD133 and cultured in the LGM for 6 h and analyzed by transmission electron microscope. Arrows indicated autophagosomes. The number of autophagosomes per cell per cross-sectioned cells were counted (right graph) (mean±SD (n = 15). <b>B.</b> Huh-7 cells were placed on the coverslips overnight and cultured with fresh CM and LGM media for 3 h and subjected to confocal microscopic analysis. Magnified images (400X) are shown beside. Representative CD133-LC3 double-positive foci are indicated by arrows. CD133(red) and LC3(green). The scale bars represent 10 µm. <b>C.</b> CD133-Cherry and LC3-GFP were transfected into LM3 cells. Cells were observed with Leica confocal analytic system in EBSS. Images showed here were selected from a series of records (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056878#pone.0056878.s002" target="_blank">movie S1</a>). The scale bar represents 10 µm. D. Western blotting showed CD133 levels in fragmentation of membrane or cytoplasm of cells after culturing in the LGM for 8 h. E-cadherin was as membrane control; GADPH as cytosol control.</p

CD133/Prominin-1-Mediated Autophagy and Glucose Uptake Beneficial for Hepatoma Cell Survival

<div><p>CD133/Prominin-1 is a pentaspan transmembrane protein that has been frequently used as a biomarker for cancer stem cells, although its biological function is unclear. The aim of our study was to explore the intrinsic functions of CD133 membrane protein in hepatoma cells during autophagy, apoptosis, tumorigenesis and cell survival through expression or downregulation of CD133. In this study, CD133 was found to be dynamically released from plasma membrane into cytoplasm in both of complete medium(CM) and low glucose medium (LGM), and LGM promoted this translocation. Expression of CD133 enhanced autophagic activity in LGM, while silencing CD133 attenuated this activity in HCC LM3 and Huh-7 cells, suggesting that CD133 is associated with autophagy. Immunofluorescence and time-lapsed confocal techniques confirmed that CD133 was associated with autophagy marker, microtubule-associated protein light chain3 (LC3) and lysosome marker during the glucose starvation. We further found that Huh-7 cells with stable expression of shCD133 (Huh-7sh133) impaired the ability of cell proliferation and formation of xenograft tumors in the NOD/SCID mice. Although loss of CD133 did not affect the rates of glucose uptake in Huh-7con and Huh-7sh133 cells under the CM, Huh-7sh133 cells obviously died fast than Huh-7con cells in the LGM and decreased the rate of glucose uptake and ATP production. Furthermore, targeting CD133 by CD133mAb resulted in cell death in HepG2 cells, especially in the LGM, via inhibition of autophagic activity and increase of apoptosis. The results demonstrated that CD133 is involved in cell survival through regulation of autophagy and glucose uptake, which may be necessary for cancer stem cells to survive in tumor microenvironment.</p></div

CD133 was released into cytoplasm and participated in autophagy.

<p><b>A.</b> LGM condition promoted translocation of CD133 from membrane to cytoplasm. Huh-7 cells were stained by antibody for CD133 after culturing in CM and LGM for 3 h and counterstained by Hoechst. IF: Immunofluoresence. <b>B.</b> Autophagy in LM3 cells. Transient expression of plasmids as indicated together with GFP-LC3 in LM3 cells. Representative pictures were selected from observation at 1 h. <b>C.</b> Percentages of autophagic cells. Autohagic cells were measured by an individual cell with more than six puncta and expressed as mean±SD (n = 5). <b>D.</b> Levels of CD133 and LC3-II proteins in LM3 cells. p3xFlag-CD133 and pSuper-shCD133 were transfected into LM3 cells as indicated. Cells were harvested at 6 h after culturing in the LGM with or without 50 uM Chloroquine. <b>E.</b> CD133 levels were associated with LC3 levels. Under low glucose starvation for 3 h, silencing CD133 in Huh-7 cells reduced the level of LC3 (left). Isolated CD133⁺ huh-7 cells produced higher level of LC3-II than that in CD133⁻ Huh-7 cells (right). Densitometry analysis was done by normalization of CD133 and LC3-II levels with their own α-tubulin or β-actin and shown in graphs under immunoblots. <b>F.</b> Autophagy inhibitor abolished CD133-induced autophagy in LM3 cells. 3-MA (5 mM) inhibited CD133-induced increase of LC3-II in the LGM. Densitometry analysis was carried out by normalization of LC-II levels with GAPDH and shown under Western blotting panels.</p