β-1,3-Glucanase Is Cryoprotective in Vitro and Is Accumulated in Leaves during Cold Acclimation

We have used isolated spinach (Spinacea oleracea L.) thylakoid membranes to investigate the possible cryoprotective properties of class I [beta]-1,3-glucanase (1,3-[beta]-D-glucan 3-glucanohydrolase; EC 3.2.1.39) and chitinase. Class I [beta]-1,3-glucanase that was purified from tobacco (Nicotiana tabacum L.) protected thylakoids against freeze-thaw injury in our in vitro assays, whereas class I chitinase from tobacco had no effect under the same conditions. The [beta]-1,3-glucanase acted by reducing the influx of solutes into the membrane vesicles during freezing and thereby reduced osmotic stress and vesicle rupture during thawing. Western blots probed with antibodies directed against tobacco class I [beta]-1,3-glucanase showed that in spinach and cabbage (Brassica oleracea L.) leaves an isoform of 41 kD was accumulated during frost hardening under natural conditions.