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.

Antifreeze proteins enable plants to survive in freezing conditions

“…AFGPs have been isolated from five plants including Solanum dulcamara (Duman 1994), Daucus carota (Worrall et al 1998;Smallwood et al 1999), Lolium perenne (Sidebottom et al 200 0;P udn ey e t al. 2003 ), Ammopiptanthus mongolicus (Yong et al 2000;Fei et al 2008) and Hippophae rhamnoides (Gupta and Deswal 2012). The sugar moiety of fish AFGPs interacts with ice.…”

“…Furthermore, treatment of SdAFGP with β-galactosidase or Abrus precatorius lectin also resulted in the loss of activity, suggesting the pivotal roles of galactose in the antifreeze activity of SdAFGP (Duman 1994). In contradiction with these, AFGPs of other plant do not require a sugar moiety for their activity as even deglycosylated AFGPs exhibited similar levels of antifreeze activity (Gupta and Deswal 2012). SdAFGP is homologous to the WRKY transcription factor and is cytoplasmic (Duman 1994), while AFGPs of D. carota and H. rhamnoides are homologous to polygalacturonase inhibitor protein (PGIP) and are apoplastic (Smallwood et al 1999;Gupta and Deswal 2012).…”

Hippophae rhamnoides N-glycoproteome analysis: a small step towards sea buckthorn proteome mining

“…turkestanica) were grown according to the procedure followed by Gupta and Deswal (2012) where seeds were soaked in teepol for 5 min and then washed with tap water followed by sterilization with 70 % ethanol and washing with double distilled water. The seeds were soaked for 5 days in double distilled water followed by incubation in dark for one day and plated on pre-soaked germination paper rolls and kept under white light (270 lmol/m 2 /s, 16 h light/8 h dark) at 24 ± 2°C and were watered regularly.…”

“…Secretome analysis from seedlings showed 61 low temperature responsive targets. Polygalacturonase Inhibitor Protein (PGIP) an antifreeze protein was isolated from the apoplast (Gupta and Deswal 2012). This antifreeze protein has the ability to inhibit growth of ice crystal at a temperature below freezing point and presence of these proteins allows the survival of seedlings at -5°C.…”

Plant proteomics in India and Nepal: current status and challenges ahead

“…AFPs are a class of polypeptides that bind to and inhibit the growth of ice crystals, thereby minimizing the freezing stress-induced damage. A polygalacturonase inhibitor protein showing antifreeze activity was purified by ice adsorption chromatography (Gupta and Deswal 2012). These AFPs are further being analyzed for their biotechnological applications.…”