Characterisation and immunolocalisation of a pollen-specific calmodulin-binding protein from rice

A novel calmodulin (CaM)-binding protein of rice (Oryza sativa pollen CaM-binding protein, OsPCBP) was isolated. It contains six tetratrcopeptide repeats (TPRs) with a molecular mass of ~76.7 kDa. Database searches show that OsPCBP is conserved in monocots and dicots. The results from CaM-agarose pull-down assays show that OsPCBP binds to CaM in a Ca²⁺-dependent manner and its CaM-binding domain (CBD) is located in a segment VSKGWRLLALVLSAQQRY. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis have demonstrated that OsPCBP is a pollen-specific and late-expressed gene. Immunolocalisation showed that OsPCBP was localised in the amyloplast and intine during the late stage of pollen development. However, upon pollen hydration, there was some leakage of protein and the distribution of OsPCBP in the intine varied with the duration of hydration. After pollen germination, much OsPCBP was observed in the pollen tube wall. These results indicate the relationship of OsPCBP with starch accumulation and its involvement in pollen germination.