β-Mannosidase (EC 3.2.1.25) activity during and following germination of tomato (Lycopersicon esculentum Mill.) seeds. Purification, cloning and characterization

Abstract.

β-Mannosidase, a high-salt-soluble enzyme, increases in activity in seeds of tomato prior to the completion of germination. This increase occurs in both the lateral and micropylar endosperm and becomes more evident during post-germinative seedling growth. The β-mannosidase activity profile is similar to that of endo β-mannanase although it is the first to increase in the lateral endosperm. Tomato seed β-mannosidase was purified to homogeneity and its cDNA (LeMside1) obtained by 3′-RACE PCR using oligonucleotide sequences based on four peptide sequences obtained from the purified enzyme. The derived amino acid sequence of the tomato β-mannosidase shows the enzyme is a member of the Glycosyl Hydrolases Family 1 (GHF1) but has a very low sequence identity with that of β-mannosidases from non-plant sources; no other plant sequence for the enzyme is known. There appears to be only one gene encoding β-mannosidase in tomato, the sequence of which has been determined (LeMSide2). Its expression occurs first in the micropylar endosperm, and then declines after germination. This is followed by an increase in its expression in the lateral endosperm, which precedes that of the gene for endo β-mannanase. Expression of the β-mannosidase gene increases appreciably in the growing seedling embryo. With this report, the cloning of all three of the enzymes involved in galactomannan mobilization (endo β-mannanase, α-galactosidase and β-mannosidase) in tomato seeds has now been achieved.