The Dark Reactions of Rye Phytochrome in Vivo and in Vitro

Abstract

The dark reactions of Secale cereale L. cv. Balbo phytochrome have been investigated in coleoptile tips and in extensively purified extracts of large molecular weight phytochrome. Destruction, but not reversion, was detected in vivo.The effects of various inhibitors of an in vitro phytochromedegrading protease did not support a view of proteolytic attack as the basis of in vivo destruction. In vitro, rye phytochrome (about 240,000 molecular weight) reverted extremely rapidly, even at 5 C. The reversion curves were resolved into two first order components. The previously studied 60,000 nmolecular weight species, obtained by controlled proteolysis of large rye phytochrome, showed a similar two-component pattern, but a much slower over-all reversion rate. This reduction in rate was caused mainly by the reversion of a greater percentage of the small phytochrome as the slow component. Sodium dithionite markedly accelerated the reversion rate of both large and small forms, but oxidants, at concentrations low enough to avoid chromophore destruction, had no effect. Both large and small crude Avena sativa L. phytochrome showed two-component reversion kinetics.Though the destruction and reversion reactions of phytochrome have long been known in vivo (7,17), their precise role in the physiological action of the pigment system has not been established. Reversion had been assigned a major role in photoperiodic timing (3, 16), but more recent work has questioned this view (12). Many of the "paradoxes" of phytochrome physiology involve these reactions (17). For example, though reversion can easily be detected in partially purified phytochrome from various grasses (8,10,27), no in vivo reversion of coleoptile tip phytochrome has ever been seen (6, 14. 31). Phytochrome from many dicots shows reversion and destruction in vivo (17, 18), though there are at lease two plants, Amnaranthus (19) and pumpkin (2), which show only Pfr destruction. Partially purified extracts often show mainly reversion (10, 27), especially at temperatures near 0 C, though there is a recent report of in vitro destruction (24).Work reported elsewhere (29,30) rye phytochrome was also degraded by the protease, yet the phytochrome alone was quite unstable to incubation at 25 C, so evidently other factors are involved in loss of photoreversibility in vitro. With a knowledge of the properties of the protease (29, 30), experiments reported here were undertaken for assessment of the involvement of the enzyme in in vivo destruction by the use of suitable inhibitors and activators.Recent work in our laboratory (15) has shown that the previously studied 60,000 mol wt phytochrome unit (27, 33) is a stable fragment produced by limited proteolysis. Indeed, a much larger molecule has now been isolated and studied. We feel that this material (about 9 S) is probably native phytochrome. Its absorption spectrum is identical (in the region above 500 nm) to that of the fragment.The previous work on in vitro reversion of highly purified grass phytochrome can be summa...

Partial Characterization of Oat and Rye Phytochrome

“…The ability of the oat protease fraction isolated from darkgrown oats (43) (24) showing that the inhibitor blocks the spontaneous breakdown of 8S oat and rye species into 4S species corroborates the view that oat phytochrome, as it has been described, is an artifact produced during isolation.…”

“…Figure 9 shows the electrophoretic pattern of oat phytochrome (A) and rye photochrome (B) 43,000: mol wt of the dimer, 86,000). Densitometric scans of gel B. using a Gilford gel scanner with the Gilford 2000 spectrophotometer, showed 57% of the stained material in the light chain and 43% in the heavy chain (25).…”

“…Further evidence favoring this thesis is provided in the present work by additional comparison of oat phytochrome with rye phytochrome subjected to mild proteolysis, either with commercial proteases or with an oat shoot protease described by Pike and Briggs (43). A brief account of portions of this work has appeared elsewhere (12).…”

“…The oat protease preparation had 500 units/mg as measured by a dye-bound collagen (Azocoll) assay (36,43 (5). The column reservoir was fitted as a Marriot flask, and the liquid level of the hydrostatic head was 25 cm above the column outlet.…”

Partial Purification and Characterization of a Phytochrome-degrading Neutral Protease from Etiolated Oat Shoots

“…Experiments to determine differential lability in this system were performed with the pigment initially in the desired form. Since the red-irradiated sample would be expected to revert (41,42), Pfr would only be present during the early stages of the reaction. Crude phytochrome (without added factor) is less stable when left as Pfr than as Pr.…”