Biochemical, biophysical, and physiological aspects of crystal idioblast and calcium oxalate crystal growth in Pistia stratiotes L.

摘要

Calcium (Ca) is essential to the growth and metabolism of higher plants, but at high concentrations is toxic. Crystal idioblasts are specialized cells that serve as high-capacity sinks for excess calcium from surrounding cells and tissues. They sequester excess Ca as insoluable Ca oxalate crystals. This study examines some anatomical, physiological, and biochemical features of crystal idioblasts and Ca oxalate crystals that allow high level Ca sequestration, and specifically examines control and coordination of Ca flux in the cytosol and crystal precipitation within the vacuole during idioblast development.; Calcium oxalate crystals rapidly accumulate within the vacuoles of idioblasts, as large fluxes of calcium are necessary from the apoplast through the cytosol. Idioblasts are shown to have extensive ER, which is implicated in Ca sequestration. An antibody raised to calreticulin (CRT), a high-capacity Ca binding protein, is highly localized to ER subdomains. In situ hybridization demonstrated that the CRT gene is highly expressed in idioblasts. Specialized ER subdomains thus appear to be a critical component controlling Ca flux through the cell prior to vacuolar Ca precipitation.; Little is known about how crystal growth is controlled within the idioblast vacuole. Microauotradiography of 45Ca and 14C-oxalic acid addition to developing crystals demonstrated that raphide crystals develop through bi-directional addition of Ca and oxalate. Raphide crystals stop growing in the middle while still maintaining tip growth, leading to their needle-like shape. Polarizing microscopy and SEM demonstrate a distinct polarity in raphide morphology; one pointed and one blunt end. A crystal nucleation region was also identified, with micro-morphological features consistent with the growth dynamics seen using microautoradiography.; Crystal idioblasts present unique immunocytochemical staining problems. A novel antibody purification method utilizing magnetic beads was used to purify rabbit anti-CRT antibody. The purified antibody was successfully used to clarify earlier immunolocalization results for CRT.