A novel calcium-binding protein is expressed in Brassica pistils and anthers late in flower development

We describe the cloning and characterization of PCP, a novel calcium-binding protein that is expressed predominantly in the pistils and anthers of Brassica flowers late in flower development. A PCP cDNA - isolated from a subtracted cDNA library enriched in transcripts present in the pistil late in flower development - potentially encodes a 175 amino acid protein with a calculated molecular weight of 19.1 kDa. Other than limited homology to a repetitive C-terminal polyacidic region of PCP, none of the sequences in the GenBank database shares identity to PCP. This unique protein was purified from an Escherichia coli expression system and shown to bind calcium in a specific manner, both in a protein blot assay and by equilibrium dialysis. PCP binds 29 mol of calcium per mol of PCP protein with an apparent affinity constant of 3.2 x 10(2)/M, values consistent with the presence of a high capacity/low-affinity calcium-binding domain. PCP-specific mRNAs are detected predominantly in the stigma and style of pistils excised from open flowers; much lower levels of expression are seen in anthers of open flowers and in root and leaf tissue. Expression in the pistil steadily increases during flower development and peaks at flower opening. A PCP-specific antibody first detects the protein in pistils at one day prior to flowering, with higher levels of the protein seen in the pistils of open flowers. A low level of the protein is present in anthers of open flowers; however, PCP is not detected in either root or leaf extracts. The pattern of PCP expression is consistent with a possible role for PCP in pollen-pistil interactions or in pistil development. The results are also discussed in light of the central role calcium maintains in pollen tube growth and fertilization.