IRRADIATION‐ENHANCED PHYTOCHROME PELLETABILITY IN AVENA: PIGMENT RELEASE BY Mg2+‐GRADIENT ELUTION

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Abstract
Abstract— Gradient elution is used to facilitate controlled withdrawal of Mg2+ from phytochrome‐rich particulate fractions from irradiated Avena sativa L. shoots. The bound pigment from red‐irradiated tissue is released in a discrete band when the Mg2+ falls to just below 1 mM. This phytochrome has an apparent molecular weight of ˜300 kilodaltons upon gel filtration, indistinguishable from that of the unbound pigment in the same extract and from that in the 50,000 × g supernatant from non‐irradiated Avena. This indicates that the bound phytochrome is released as a soluble molecule at a Mg2+ concentration above that which permits release of the particulate binding partner from other particulate components. These findings appear to preclude the possibility that the phytochrome‐binding partner association can be selectively preserved at a Mg2+ level that would permit separation and analysis of phytochrome‐bearing particles without the complication of Mg2+‐induced membrane and RNP aggregation. “Cycled” Pfr (that from tissue irradiated with a red‐far red sequence prior to homogenization) is released at 0.1 to 0.2 mM Mg2+. This indicates that “cycled Pr is more tenaciously bound by the particulate fraction than is Pfr. This effect is photoreversible both by further in vivo and subsequent in vitro irradiations, suggesting that the state of the pigment, rather than of the binding partner, directly controls the tenacity of the interaction. Increasing concentrations of KCl release the pigment from the particulate fraction in the presence of 10 mM Mg2+; increasing Triton X100 concentrations do not. This confirms the ionic nature of the pigment‐particulate fraction interaction and indicates strongly that the phytochrome is located external to any membrane vesicles present (although not necessarily that it is bound directly to such vesicles). The data further suggest that phospholipid polar head groups are not primarily responsible for the binding.