CHARACTERIZATION OF WALL-BOUND INVERTASE ISOFORMS OF PICEA-ABIES CELLS AND REGULATION BY ECTOMYCORRHIZAL FUNGI

In culture, the ectomycorrhiza-forming fungi Amanita muscaria (Pers. e x Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. onl y grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall-bound invertase activity. Therefore, utilization of sucrose by the fungi within a myc orrhizal association is believed to depend on the wall-bound invertase activity of the host. This enzyme activity was studied in the apoplas t of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall-bound isoform of acid invertase were found that fu nction as beta-D-fructofuranoside-fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact c ells with buffer of high ionic strength. In its native form, the ionic ally bound invertase isoform is a monomeric protein with a molecular m ass of 61 kDa, as determined by gel filtration and SDS-PAGE. Glycoprot ein nature of the enzyme was demonstrated with antibodies directed aga inst the digoxigenin-labeled protein. The K(m) values of both enzymes for sucrose, their natural substrate, are relatively high (ionically b ound invertase K(m) = 16 mM, tightly bound invertase K(m) = 8.6 mM). A ctivity of both wall-bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH-optimum and exhibit highest activ ity at pH 4.5, with elevated activity between pH 4.5 and 6.0. Furtherm ore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall-bound invertase of the ho st. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the pl ant invertase by acidification of the cell wall and by fructose uptake . Thus, the fungus itself can regulate its own supply of glucose and f ructose.