Protein and gene expression patterns of endo-β-mannanase following germination of rice

Abstract

AbstractThe rice (Oryza sativa L.) cv. Taichung 65, a japonica subspecies, was used to characterize the isoform, protein and gene expression patterns of endo-β-mannanase during and after seed germination. Activity assays and isoform analyses of whole grains or seed parts (scutellum, aleurone layer and starchy endosperm) revealed that seeds began to express endo-β-mannanase activity at 48 h from the start of imbibition at 25°C, after the completion of germination of most seeds. Three isoforms of endo-β-mannanase (pI 8.86, pI 8.92 and pI 8.98) were detected in the aleurone layer and starchy endosperm, but only two (pI 8.86 and pI 8.92) were present in the scutellum. The endo-β-mannanase in the starchy endosperm was mainly from the aleurone layer. Western blot analysis, using a tomato anti-endo-β-mannanase antibody, indicated that an endo-β-mannanase protein was present in an inactive form in dry grains. The amount of this protein decreased in the scutellum, but increased in the aleurone layer during and after germination. Thus, the increase in endo-β-mannanase activity in rice grains may be due to the activation of extant proteins and/or the de novo synthesis of the enzyme. Northern blot analysis showed that four putative rice endo-β-mannanase genes (OsMAN1, OsMAN2, OsMAN6 and OsMANP) were expressed in germinating and germinated rice grains. However, OsMANP was not expressed in the scutellum. The amount of OsMAN6 mRNA decreased after the completion of germination and paralleled the decline in endo-β-mannanase protein. In the aleurone layer, the increase of OsMAN2, OsMAN6 and OsMANP mRNA was prior to the increase of endo-β-mannanase protein.