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.

ABSTRACT

The alga Mougeotia has a large central chloroplast whose positioning is regulated by photoactivation of phytochrome, possibly via modulation of cytosolic calcium (Serlin B, Roux SJ [1984] Proc NatI Acad Sci USA 81: 6368-6372). We used the patch clamp technique to examine the effects of red and far-red light on ion channel activity in the plasma membrane of Mougeotia protoplasts to determine if ion channels play a role in chloroplast movement. Patch clamping in the cell-attached mode reveals two channels of about 2 and 4 picoamperes amplitude at 0 millivolt (inside pipette) and estimated conductances of 30 and 65 picosiemens. They are activated by red light irradiation after a lag period of about 2 to 5 minutes. Far-red light, when applied immediately after red light irradiation, reverses this activation; otherwise it has no effect. This result implicates phytochrome.

The activity of a Mg2"-dependent ATPase present in highly purified preparations of Arena mitochondria was photoreversibly modulated by red/far-red light treatments. These results were obtained either with mitochondria isolated from plants irradiated with white light prior to the extraction or with mitochondria isolated from unirradiated plants only when purified phytochrome was exogenously added to the reaction mixture. Red light, which converts phytochrome to the far red-absorbing form (Pfr) depressed the ATPase activity, and far-red light reversed this effect. Addition of exogenous CaRC2 also depressed the ATPase activity, and the kinetics of inhibition were similar to the kinetics of the Pfr effects on the ATPase. The calcium chelator, ethyleneglycol-bis4fl-amino-ethyl ether)-N,N'-tetracetic acid, blocked the effects of both CaC12 and Pfr on the ATPase. These results are consistent with the interpretation that Pfr promotes a release of Ca2" from the mitochondrial matrix, thereby inducing an increase in the concentration of intermembranal and extramitochondrial Ca2+.

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