Characterization of Oat Calmodulin and Radioimmunoassay of Its Subcellular Distribution

Abstract

A protein identifiable as calmodulin has been isolated from oat (Avena sativa, var Garry) tissues. This protein is relatively heat stable, binds to hydrophobic gels, and phenothiazines in a calcium-dependent fashion, and binds to antibody to rat testes calmodulin. Based on its migration on sodium dodecyl sulfate-polyacrylamide gels, ultraviolet absorption spectrum, and amino acid composition, oat calmodulin is essentially identical to calmodulin isolated from other higher plants. Radioimmunoassays indicate that calmodulin is associated with isolated oat protoplasts, mitochondria, etioplasts, and nuclei and also appears to be a component of oat cell wall fractions.Much evidence has accumulated recently to support the hypothesis that Ca 2 plays a major role in mediating the adaptations of plants to certain environmental changes (12,22). As in animals, at least some Ca2-imediated responses in plants are controlled by Ca2-binding regulatory proteins. Among these, the most studied and best characterized is calmodulin (1).We have published reports suggesting a possible role for calmodulin in mediating phytochrome and gravitropic responses in Avena saliva (oats) (2, 21). As part of our ongoing research on this question, we have isolated and characterized calmodulin from oats and have estimated its content, both in intact tissue and in isolated subcellular fractions, by radioimmunoassay. Here we report the results of these experiments.MATERIALS AND METHODS Plant Material. Except where indicated, the starting material for all extractions was taken from the coleoptiles and primary leaves of 3-to 4-d-old dark-grown oat (A vena sativa, var. Garry) seedlings, harvested 5 to 7.5 mm above the seed. The oats were grown on water-saturated vermiculite at 27C.Calmodulin Isolation. Two extraction methods were employed.'Supported by grants from the National Aeronautics and Space Administration (NSG 7480), The National Science Foundation (PCM 81-03429), and The Robert A. Welch Foundation (F 858) to S. J. R.

Cellular Distribution of Calmodulin and Calmodulin-Binding Proteins in Vicia faba L.

“…The second possibility is that much of the CaM in leaves may be loosely associated with the cell wall or may be completely extracellular. The presence of extracellular CaM in both plant (2,30) and animal (19) systems has been documented. The exact role of extracellular CaM in plants is yet to be elucidated, but it has been hypothesized that CaM may be involved in cell wall maintenance (31).…”

The Presence of a Heterotrimeric G Protein and Its Role in Signal Transduction of Extracellular Calmodulin in Pollen Germination and Tube Growth

“…Cauliflower and wheat CaMs were purified by using phenylSepharose 4B affinity chromatography, as described by Biro et al (1984). The final CaM preparation was homogeneous, as determined by electrophoresis in an SDS-polyacryamide gel.…”

“…In plant systems, Biro et al (1984) first reported the presence of CaM in soluble extracts of oat coleoptile cell walls by using radioimmunoassays. In our laboratory, a series of experiments provided evidence for the presence of extracellular CaM.…”

A Signaling Pathway Linking Nitric Oxide Production to Heterotrimeric G Protein and Hydrogen Peroxide Regulates Extracellular Calmodulin Induction of Stomatal Closure in Arabidopsis

“…It has been shown that calmodulin exists not only intracellularly but also extracellularly in many plant species (Biro et al, 1984;Sun et al, 1994Sun et al, , 1995Cui et al, 2005). ExtCaM has been implicated in several important biological functions, such as the promotion of cell proliferation, pollen germination, and tube growth (Sun et al, 1994(Sun et al, , 1995Ma and Sun, 1997;Ma et al, 1999;Cui et al, 2005;Shang et al, 2005).…”