Citation:
Abstract:
Endo-beta-mannanase is one of the key enzymes involved in the hydrolysis of the mannan-rich cell walls oftomato (Solanum lycopersicon) seeds. Two isoforms of endo-beta-mannanase have been characterized intomato seeds: LeMAN2 is active in the micropylar area prior to germination and LeMAN1 is active aftergermination in all endosperm cells surrounding the cotyledons. To explore whether general mannanase activity in the endosperm cap is sufficient to promote germination, the gene encoding Le.MAN3 was inserted into transgenic tomato plants under the control of a CaMV-35S promoter. Expression of LeMAN3 was evident in the endosperm cap and in the lateral endosperm of the transgenic seeds 10 min after imbibition. An activity test indicated increased activity of endo-beta-mannanase in the transgenic lines relative to thecontrol line in all seed parts, during the first 20 h of imbibition. However, overexpression of LeMAN3 intransgenic seeds inhibited seed germination at both optimal and suboptimal temperatures. Detailed RT-PCR analyses revealed the transcription patterns of the genes encoding the various mannanase isoforms, and indicated a delay in LeMAN2 transcription in the endosperm cap of the transgenic seeds. Interestingly, tissue-print assays indicated similar mannanase activity in the micropylar areas for both transgenic andcontrol seeds. These results indicate that overexpression of active endo-beta-mannanase in the endosperm cap is not sufficient to enable hydrolysis of the cell walls or to promote germination of tomato seeds. Cell-wall hydrolysis in these endosperm cells is under tight control and requires the specific activity of LeMAN2.