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A cDNA, LeMAN4 , containing an open-reading frame of 1185 bp and putatively encoding an endo -(1,4)-β-mannanase (EC 3.2.1.78) was isolated from red tomato ( Lycopersicon esculentum Mill.) fruit. Native PAGE IEF of fruit protein extracts revealed a single mannanase of p I 9, close to the predicted p I of 8.8 for the mature protein ( M r of 42,400) encoded by LeMAN4 . In mature green fruit LeMAN4 mRNA was detectable, increased markedly at the breaker stage and remained at high levels thereafter. endo -(1,4)-β-Mannanase activity, however, was almost undetectable in the early stages of ripening and did not increase significantly until the pink and red stages of ripeness, possibly reflecting post-transcriptional regulation of the enzyme. Cell wall analysis revealed no net change in non-cellulosic mannosyl residues during ripening, suggesting cell wall mannans may not be the in vivo substrate of this enzyme. Southern analysis indicated that LeMAN4 is a single copy gene although several weakly hybridising bands imply the presence of other related genes. LeMAN4 is not fruit-specific, being expressed in flowers but not in leaves, and is probably not rate-limiting in the ripening-related softening of tomato fruit. |
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