Proteome analysis reveals a systematic response of cold-acclimated seedlings of an exotic mangrove plant Sonneratia apetala to chilling stress.

Low temperature in winter was the most crucial abiotic stress that limits the mangrove afforestation northward. Previous study demonstrated that Sonneratia apetala initially transplanted to high latitude area exhibited a stronger plasticity of cold tolerance. To clarify the underlying mechanism, the physiological and proteomic responses to chilling stress were investigated in S. apetala leaves. Our results found that cold-acclimated seedlings had lower relative electrolyte leakage and MDA content than non-acclimated seedlings. On the contrary, higher chlorophyll content and photosynthetic capacity were observed in cold-acclimated seedlings. With proteomic analyses, the differentially accumulated proteins (DAPs) involved in ROS scavenging, photosynthesis and energy metabolism, carbohydrate metabolism, cofactor biosynthesis, and protein folding were suggested to play important roles in enhancing the cold tolerance of S. apetala. However, the down-regulation DAPs were suggested as a tradeoff between plant growth and chilling response. By the protein-protein interaction analyses, translation elongation factor G, chlorophyll A-B binding protein and ascorbate peroxidase 1 were suggested as the important regulators in cold-acclimated S. apetala seedlings under chilling stress. Based on the above results, a schematic diagram describing the mechanism of cold tolerance of exotic mangrove species S. apetala that was achieved by cold acclimation was presented in this study. SIGNIFICANCE: The major environmental factor limits the mangrove afforestation northward is the low temperature in winter. Previous study reported that Sonneratia apetala grew in high latitude exhibited a higher cold tolerance than that in low latitude, which was suggested as a result of cold acclimation. To further understand "how cold acclimation enhance the cold tolerance in S. apetala", the response of S. apetala subjected to chilling stress with or without cold acclimation was investigated in this study at the physiological and proteomic aspects. Our physiological results showed that S. apetala seedlings treated with cold acclimation exhibited a higher tolerance under chilling stress than that without cold acclimation. By using the comparative proteomic approaches and bioinformatic analyses, various biological processes were suggested to play an important role in enhancing the cold tolerance of S. apetala under chilling stress, such as ROS scavenging, photosynthesis and energy metabolism, carbohydrate metabolism, cofactor biosynthesis, and protein folding. Among these differentially accumulated proteins, translation elongation factor G (eEF-G), chlorophyll A-B binding protein (CAB) and ascorbate peroxidase 1 (APX1) were identified as the hub proteins function in coordinated regulating ROS scavenging, photosynthesis and protein biosynthesis in chloroplast and subsequently enhanced the cold tolerance of S. apetala under chilling stress. Our results provided a further understanding of cold acclimation in improving the cold tolerance in exotic mangrove species S. apetala.

Proteomic analysis on mangrove plant Avicennia marina leaves reveals nitric oxide enhances the salt tolerance by up-regulating photosynthetic and energy metabolic protein expression.

Avicennia marina (Forsk.) Vierh is one of the most salt-tolerant mangrove species. Our previous study revealed that nitric oxide (NO) enhanced the salt tolerance of A. marina by promoting salt secretion and Na+ sequestration under salt stress. However, little is known about the regulation of NO on proteomic profiling for this mangrove species. In this study, we used sodium nitroprusside (SNP), an NO donor, to investigate the regulatory mechanism of NO on salt tolerance of A. marina according to physiological and proteomic aspects. Photosynthesis data showed that the reduction in photosynthesis caused by high salinity treatment (400 mM NaCl) could be partially recovered by addition of SNP (100 µM). Further analysis revealed that the high salinity treatment could induce not only the stomatal limitation but also non-stomatal limitation on photosynthetic reduction, while SNP addition could restore the non-stomatal limitation, implying that the application of SNP was beneficial to the metabolic process in leaves. Proteomic analysis identified 49 differentially expressed proteins involved in various biological processes such as photosynthesis, energy metabolism, primary metabolism, RNA transcription, protein translation and stress response proteins. Under high salinity treatment, the abundances of proteins related to photosynthesis, such as ribulose-phosphate 3-epimerase (RPE, spot 3), RuBisCO large subunit (RBCL, spot 4, 5, 24), RuBisCO activase A (RCA, spot 17, 18) and quinine oxidoreductase-like protein isoform 1 (QOR1, spot 23), were significantly decreased. However, the abundance of proteins such as RBCL (spot 5, 9) and QOR1 (spot 23) were increased by SNP addition. In addition, exogenous NO supply alleviated salt tolerance by increasing the accumulation of some proteins involved in energy metabolism (spot 15), primary metabolism (spot 25, 45, 46), RNA transcription (spot 36) and stress response proteins (spot 12, 21, 26, 37, 43). The transcriptional levels of nine selected proteins were mostly consistent with their protein abundance except spot 46. Overall, the presented data demonstrated that NO has a positive effect on improving salt tolerance in A. marina by regulating the protein abundance involved in photosynthesis, energy metabolism, primary metabolism and stress response.

Molecular cloning, expression and purification of lactoferrin from Tibetan sheep mammary gland using a yeast expression system.

This paper reports the successful expression of a lactoferrin gene-obtained from the mammary gland tissue of Tibetan sheep-in the yeast Pichia pastoris GS115 using pPICZαA as the recombinant plasmid and α-factor signal sequence for secretion. The recombinant lactoferrin was purified by ammonium sulfate precipitation, ion-exchange column chromatography and gel-filtration chromatography, and it had a molecular mass of 76kDa. We obtained an expression yield of >60mgL(-1) and specific activity of 2533.33Umg(-1). The antimicrobial activities and iron-binding behaviors of recombinant lactoferrin indicated that it was correctly folded and functional. Additionally, recombinant lactoferrin inhibited the growth of Escherichia coli JM109 and Staphylococcus aureus. These findings indicate that recombinant lactoferrin is a potential antibiotic for use on humans. This study also demonstrates the successful expression of recombinant lactoferrin using the eukaryotic host organism P. pastoris, paving the way for protein engineering using this gene.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid).

INTRODUCTION: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). METHODS AND MATERIALS: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells' location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells' VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. RESULTS: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K(+). This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. CONCLUSION: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

[Secretion and expression of vascular endothelial growth factor and interleukin-8 by SH-SY5Y human neuroblastoma cells].

To establish vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide (PLLA) micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay (ELISA) and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 microm and 4 microm, and a nominal pillar spacing of 2 microm and 7 microm. We found that the secretion and mRNA expression of VEGF and/or IL-8 by SH-SY5Y cells on 2-2 microm (pillar diameter-spacing), 4-2 microm and 4-7 microm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 microm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, cells on topographic substrates showed significant changes in morphology (spreading area, perimeter and roundness), and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SY5Y cells.