title: Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant.
abstract: Plants' distribution and productivity are adversely affected by low temperature (LT) stress. LT induced proteins were analyzed by 2-DE-nano-LC-MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub. Seedlings were subjected to direct freezing stress (-5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. LT induced secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide an insight in probable LT induced signaling in the secretome.
authors.affiliation: Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi-110007, India.
authors.forename: Ravi
authors.initials: R
authors.lastname: Gupta
authors.forename: Renu
authors.initials: R
authors.lastname: Deswal
mesh_terms: D000064:Acclimatization; D021301:Antifreeze Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification*; D003080:Cold Temperature; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000302:isolation & purification; D031281:Hippophae / Q000737:chemistry* / Q000201:enzymology; D007791:Lactoylglutathione Lyase / Q000737:chemistry / Q000302:isolation & purification; D008858:Microscopy, Phase-Contrast; D061286:Osmometry; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D055571:Secretory Pathway; D015398:Signal Transduction; D013312:Stress, Physiological; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D021301:Antifreeze Proteins; D016326:Extracellular Matrix Proteins; D010940:Plant Proteins; C003427:thaumatin protein, plant; D013482:Superoxide Dismutase; D002688:Chitinases; D007791:Lactoylglutathione Lyase