Single-step purification and characterization of antifreeze proteins from leaf and berry of a freeze-tolerant shrub seabuckthorn (Hippophae rhamnoides).

Abstract

Seabuckthorn is a freeze-tolerant Himalayan shrub, capable of withstanding temperatures below -40°C. Antifreeze proteins prevent freezing associated damage by restricting ice crystals growth. In the present study, homogenous purification of two antifreeze proteins (41 and 39kDa) from Hippophae rhamnoides leaf and one (41kDa) from berry was performed using ice-affinity chromatography. MS identification and Basic Local Alignment Search Tool search showed homology of berry antifreeze proteins with disease resistance protein while leaf antifreeze proteins showed similarity with transmembrane protein (39kDa) and low temperature induced protein (41kDa) suggesting their role in cold stress signalling. Hexagon shaped ice crystals (Nanoliter osmometer) and ice recrystallization inhibition assay (Splat assay) confirmed higher ice recrystallization inhibition activity of purified leaf (2.5 fold decrease in mean ice crystal size) and berry (2.1 fold decrease) antifreeze proteins. String interactome analysis showed interaction of antifreeze proteins with cold stress modulated targets including pathogenesis related proteins. This probably is the first report of antifreeze proteins purification from naturally growing seabuckthorn. Further validation of these targets may open gates for commercial utilization of this plant growing abundantly in Himalayan regions of India, for crop improvement of freeze susceptible crops or biomedical applications like cryopreservation of tissues and cells.