Suppression of a ripening-related endo-1,4-beta-glucanase in transgenic pepper fruit does not prevent...

The function of the ripening-related endo-1,4-beta-D-glucanase (EGase) CaCel1 in fruit softening was investigated by suppression of CaCel1 gene expression in transgenic pepper (Capsicum annuum L.) plants using constitutive expression of a truncated sense CaCel1 transgene. In suppressed lines, immunodetectable CaCel1 protein and extractable CMCase activity were reduced to at or below the limit of detection in ripe mature red fruit, suggesting that in pepper ripening-related CMCase activity is the product of a single gene. However, the abundances of two mRNAs derived from the CaCel1 gene by differential transcription initiation were affected differently in suppressed lines. Accumulation of a 1.7 kb CaCel1 transcript was strongly suppressed, whereas the abundance of a 2.1 kb CaCel1 transcript was only partially reduced. This implies that the 1.7 kb mRNA is responsible for producing CaCel1 protein, while the 2.1 kb mRNA is translationally inactive, and as such is recalcitrant to co-suppression. Chelator-soluble polyuronides exhibited little or no depolymerization during ripening, but matrix glycans including xyloglucan were extensively depolymerized. Depolymerization of non-xyloglucan matrix glycans was the prominant cell wall change observed during pepper ripening. However, the lack of CaCel1 activity in suppressed fruit had no detectable effect on ripening-related matrix glycan depolymerization, which occurred at wild-type levels. Recombinant CaCel1 protein purified from a transgenic pepper line over-expressing functional CaCel1 was active against pepper matrix glycans in vitro, and showed greater activity against non-xyloglucan polysaccharides than against xyloglucan. Transgenic suppression of CaCel1 EGase activity has not identified the natural cell wall substrate for this enzyme, and shows that activities other than CaCel1 are responsible for the depolymerization of matrix glycans occurring during ripening in pepper.