초록
Freezing-resistant plants can survive subzero temperatures by withstanding extracellular ice formation. During cold acclimation, their leaves accumulate antifreeze proteins (AFPs) that are secreted into the apoplast and have the ability to modify the normal growth of ice crystals. Three barley, two wheat and two rye cultivars were grown under two different temperature regimes (20/16$^{\circ}C$ and 5/2$^{\circ}C$, day/night). Apoplastic proteins from winter cereals were separated by SDS-PAGE and detected with antisera to AFPs from winter rye. Apoplastic proteins accumulated to much higher levels in cold-acclimated (CA) leaves compared with nonacclimated (NA) ones in winter cereals. After cold acclimation, the protein concentration of apoplastic extracts increased significantly from 0.088 $mgmL^{-1}$ to 0.448 $mgmL^{-1}$, with about 5-fold increment. Also, the apoplastic protein content per gram leaf fresh weight in CA leaves ranged from 31 $\mu\textrm{g}$ $(gFW)^{-1}$ to 120 $\mu\textrm{g}$ $(gFW)^{-1}$ with an averaged value of 77 $\mu\textrm{g}$ $(gFW)^{-1}$, and coefficients of variation of 54.9%. The CA leaves in Musketeer (a Canadian winter rye cultivar) showed the greatest AFPs and antifreeze activity followed by 'Geurumil' (a Korean winter wheat cultivar), and 'Dongbori l' (Korean facultative barley cultivar). The proteins secreted into the wheat leaf apoplast at CA condition were more numerous than those observed in winter rye, where two $\beta$-1,3-glucanase-like proteins (GLPs), two chitinase-like proteins (CLPs) and two thaumatin-like proteins (TLPs) accumulated during cold acclimation. The proteins in barley leaf apoplast at CA conditions were a little different from those in wheat leaves. The AFPs were various among and within species. More freezing-resistant cultivars had more clear and numerous bands than less freezing-resistant ones. The high determination coefficient ($R^2$ =91 %) between freezing resistance and AFPs per gram leaf fresh weight indicated that the amount of AFPs was highly related to freezing resistance in winter cereal crops.