Soluble and Cell Wall-Associated β-Galactosidases from Cold-Grown Winter Rape ( Brassica napus L., var. oleifera L.) Leaves

Abstract

Summary Soluble and cell wall associated (solubilized from cell wall preparations by 0.5 M NaCl) β-galactosidases were found in non-acclimated or cold-acclimated leaves of winter rape (Brassica napus L., var. L. [itoleifera} L.). Gel filtration of soluble proteins on a Sephadex G-200 column revealed one fraction with β-galactosidase activity with a molecular weight close to 65 kDa. The temperature optimum for soluble and cell wall-associated β-galactosidases was 50 °C and 55 °C, respectively. The cell wall associated form showed higher thermostability at 60 °C. Both fractions had a similar pH optimum of 4.0. The Km against p-nitrophenyl β-D-galactopyranoside was 0.25 mM and 50 mM and the Vmax values 2.80 and 1.42 μmol (mg protein)-1 min-1 for the cell wall-associated and soluble form, respectively. The 3-week growth of plants in the cold (2 °C) brought about a 22 % and 38 % decrease in the total and specific activities of the soluble form, respectively. The activity of the cell wall-associated form decreased by ca. 70 % during the treatment, independently of the calculation basis. The cold-induced decrease in the enzyme activities was associated with the increase of the Km value for the soluble form and with the decrease of Vmax values for both soluble and wall-associated enzymes. The transient frost (-5 °C for 18 h followed by 6 h at 2 °C), applied in darkness, markedly stimulated the activity of the cell wall-associated enzyme in the cold-acclimated leaves. It resulted in decreased Km and increased Vmax values for both soluble and cell wall-associated forms. The possible meaning of cold- and frost-induced changes in the β-galactosidase activities for cold acclimation of winter rape leaves is discussed.