| Two major types of calcium oxalate crystal idioblasts: raphide and druse, were found in Pistia stratiotes L. Raphides formed very early during organ development, suggesting a function in early tissue calcium regulation and possible aerenchyma creation. Druse idioblasts appear later and are distributed mainly along the vascular system suggesting their function is to sequester calcium directly from the xylem stream.; SDS-PAGE of isolated crystals revealed the presence of proteins of approximately 54, 60 and 62 kD. These “matrix proteins” have a strong affinity for calcium. Immunolabling showed matrix protein is packaged in the Golgi apparatus and transported to the vacuole by Golgi-derived vesicles. These results are the first to demonstrate matrix proteins in plant crystals. A matrix protein was cloned and consists of 522 amino acid residues, giving an apparent molecular weight of 59.2 kD. A high amount of charged residues are distributed unevenly in the peptide chain. Northern blots show matrix protein gene expression is regulated by calcium supply, and in situ hybridization demonstrates that the gene is specifically expressed in the crystal idioblasts. The matrix protein was expressed as a Glutathione S-Transferase (GST) fusion protein in a bacterial system. The expressed matrix protein shows both calcium and oxalic acid binding activities. An antibody raised against the expressed matrix protein can recognize not only expressed protein, but also the native matrix protein in plant tissue sections and partially dissolved crystals.; Dissolution studies further demonstrate matrix protein presence. Upon dissolution of raphide crystals with EDTA, a needle shaped, “ghost” remained. The ghost is flexible, is immunolabeled by matrix protein antibody, and likely plays a role as a framework for crystal deposition. The middle part of the raphide crystal is resistant to dissolution, which may be related to its hypothesized function as the original nucleation site. Partially dissolved crystals can bind more labeled calcium and oxalate than intact crystals indicating the ghost has the ability to bind both ions. The expressed matrix protein was examined for its ability to affect crystallization, and results support what was seen for dissolved crystals where addition of calcium and oxalate result in recrystallization. |
