Expression and location of endo-β-mannanase during the ripening of tomato fruit, and the relationship between its activity and softening

Abstract

Endo-beta-mannanase is thought to play a role in tomato fruit ripening by participating in the degradation of cell walls. Its spatial and temporal expression during ripening was examined, as was the relationship between its activity and softening of the fruit using a large number of tomato lines, and by suppression of transcription of the endo-beta-mannanase (LeMan4a) gene. Immunolocalization studies showed that the enzyme is expressed in the fruit cell wall at all ripening stages, but it is not active during the initial green stage; this is not due to the presence of inhibitors of its activity, nor due to changes in its mRNA sequence. Transient expression in onion epidermal cells of endo-beta-mannanase transcripts fused to green fluorescent protein resulted in the expressed enzyme being localized to the cell walls. Transgenic tomato plants expressing a GUS gene attached to the LeMan4a promoter showed that this occurs initially during ripening in the skin and outer pericarp of the fruit, and later in the skin and throughout the pericarp. Fruit firmness and activity of endo-beta-mannanase were not strongly correlated during ripening of many lines of tomato. Several plants of cv. Micro-Tom were transformed using RNA interference (mRNAi) and antisense RNA strategies to suppress transcription of the LeMan4a gene. When endo-beta-mannanase activity was much reduced in the transgenic fruits, their firmness was higher compared to those of control fruits at the turning and orange-color stages, but at the red-ripe stage firmness was similar between the two fruit types. We suggest that while the enzyme does participate in fruit ripening it alone is not sufficient to cause hydrolysis of the cell walls which results in their weakening; it likely plays a cooperative role with other known wall-modifying enzymes, and/or is involved in cell wall rearrangement.