Expression of multi-domain type III antifreeze proteins from the Antarctic eelpout (Lycodichths dearborni) in transgenic tobacco plants improves cold resistance

Abstract Type III antifreeze proteins (AFPIIIs) are a group of small globular proteins found in some polar fishes to protect them against freezing damage. Transgenic expression of AFPs has been shown to confer cold tolerance to commercially important plants and animals. We have previously isolated multiple AFPIII genes in the Antarctic eelpout (Lycodichthys dearborni) that encode larger AFPIII isoforms with up to 12 of the conventional domains. Here we have introduced the fish AFPIII genes that encode for the monomer (ld1), dimer (ld2), trimer (ld3) and tetramer (ld4) AFPIII isoforms in tobacco plants. Pot-grown 4-week-old transgenic tobacco plants were exposed to cold stress at 4 °C for 30 days and the results show that ld1, ld2, ld3 and ld4 transgenic plants present relatively lower electrolyte leakage and lower content of malondialdehyde (MDA), but accumulated higher content of proline when compared to control plants. This indicates considerable improved membrane integrity under low temperature stress and improvement of the plant cold resistance. The plants transformed with the AFPIII tetramer- and trimer-domains demonstrated a higher cold-tolerant levels when compared with plants transformed with the dimer- and monomer AFPIII domains. Our study further supports that fish AFPIIIs, especially the multidomain proteins, protect cells from non-freezing hypothermic stresses, apart from there well-known function as ice inhibitors molecules at freezing temperature.