No abstract

Previous studies have indicated that phytochrome regulates Ca2" fluxes across the plasma membrane of plant cells. In this study we investigated whether phytochrome can also regulate such fluxes across mitochondrial membranes, using the Ca2"-sensitive dye murexide to monitor the uptake and release of Ca2" by mitochondria. The results showed that Ca2" fluxes in these organelles could be photoreversibly altered, red light diminishing the net uptake rate and far-red light restoring this rate to its dark control level. Treatment ofthe mitochondria with ruthenium red blocked their Ca2+ uptake. In the presence of this inhibitor, red light induced a net efflux of Ca2`from the mitochondria, and subsequent far-red light reduced this efflux to nearly zero, the dark control level. Light-induced rate changes in Ca2`flux, both with and without the inhibitor, persisted for several minutes in the dark and remained photoreversible through several irradiations for as long as 30 min. The purity of the mitochondrial preparation wasjudged to be about 80% by electron microscopic morphometry; most of the phytochrome present was localized on the mitochondria in the preparation by using immunocytochemical methods. Taken together with previous findings, the results suggest that red light activation of phytochrome would initiate an increase in the cytosolic Ca2`concentration. The results are integrated with the fact that calmodulin is a component ofplant cell cytoplasms to construct a model postulating that phytochrome directs photomorphogenesis in part through its regulation of Ca2+ and calmodulin-controlled enzyme activities.

Previously published extinction coefficients for phytochrome were based on indirect colorimetric estimates of protein weight, mostly by the Lowry method, using bovine serum albumin as a standard. This paper reports revised values based on quantitative amino acid analyses of highly purified phytochrome. The molar extinction coefficient of the red-absorbing form of phytochrome with 120000mol. wt subunits was found to be approximately 102000cm-' per 120000mol. wt at 667nm. The molar extinction coefficients at 667 nm for phytochrome with 120000 mol. wt subunits and for the 60000 mol. wt chromopeptide of P, were found to be approximately the same for samples of equivalent purity. Included in the amino acid analyses of phytochrome with 120000 mol. wt subunits are values for cysteine (1 1) and cystine (I), measured by two different techniques that gave the same results and agreed well with the estimate of total half-cystine (14) obtained by an independent method. Tryptophan was quantitated by analysis of a p-toluene sulfonic acid hydrolysate.

After electrophoresis on sodium dodecyl sulfate polyacrylamide gels, bleached chromopeptides of phytochrome can have their blue color restored by soaking the gels in 20% trichloroacetic acid. The blue bands have a broad absorption maximum between 630 and 655 nm, characteristic of denatured phytochrome. The restored color retains most of its intensity for up to 2 h, but gradually bleaches again until it disappears completely within 24 h. This visualization method is used to identify the number and sizes of phytochrome chromopeptides produced by limited tryptic digestion. The results reveal some structural requirements for photoreversibility and are consistent with a model of phytochrome structure that predicts a high degree of symmetry within the native subunit.