The relationship between a large molecular weight (9S) and a small molecular weight (4.5S, 60,000 molecular weight) species of phytochrome was examined to determine if the larger species was an aggregate of the smaller. Alterations of pH, salt concentration, or phytochrome concentration did not cause any observable formation of the large form from the small form. However, in partially purified phytochrome extracts from Secale cereale L. and Avena sativa L., the large form was converted to the small form over time at 4 C in the dark. This breakdown was inhibitable by the protease inhibitor phenylmethanesulfonyl fluoride. When highly purified large molecular weight rye phytochrome was incubated with a neutral protease isolated from etiolated oat shoots, the large phytochrome was converted to the small form without qualitative visible absorbancy changes. The effect of the oat protease could be mimicked by a wide variety of commercial endopeptidases, including trypsin. Examination of the trypsin-induced breakdown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that as the size of the photoreversible unit changes from large to small, the size of its constituent polypeptide chains is reduced from 120,000 to 62,000 molecular weight. These experiments provide evidence that the endogenous breakdown observed in extracts is a result of contaminant protease and, consequently, that the small molecular weight species of phytochrome is an artifact due to proteolysis.Since the early work of Siegelman and Firer (41)

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...

A factor catalyzing the in vitro degradation of oat phytochrome in crude extracts has been shown to be a proteolytic enzyme. The enzyme, an endoprotease, has been purified about 600-fold from dark-grown oat shoots by chromatography on ion exchange and molecular seive gels. The pH-activity curve is broad, with a maximum around pH 6.4. The enzyme is apparently dependent on the presence of reduced sulfhydryl groups for activity: low concentrations of reductants stimulate it, while inhibition has been obtained with a variety of sulfhydryl antagonists. High ionic strength conditions are inhibitory. A molecular weight of 61,500 has been estimated, though autolysis may yield smaller active fragments. An enzyme with similar properties has been isolated from imbibed oat seeds, light-grown oat shoots, and dark-grown rye shoots.

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for transparinaric acid fluorescence was detected at 7'C and -3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20'C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trarns-parinaric acid indicating that significant domains of solid lipid form below 7'C or -3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2'C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29'C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7'C (or -3°C) its completion. Because of the present results, however, this interpretation needs to be modified.Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.The evergreen higher plant Nerium oleander (oleander) acclimates to both hot summer and cool winter conditions and grows actively in these contrasting regimes (2 prevailing temperature is probably an important adaptive mechanism for this and other desert evergreen species, such as Larrea divaricata (9) and A triplex lentiformis (12), which experience large seasonal differences in environmental temperature. The variation in fatty acid composition ofA. lentiformis with growth temperature (I I) suggests that changes in the physical properties of membranes might be the basis for the acclimation.To characterize the oleander membrane lipids, we have utilized ESR4 spectroscopy and measurements of fluorescence intensity and polarization with the probe, trans-parinaric acid (25). Both techniques revealed the same temperature for the separation of solid and fluid phase lipids but indicate a need to reinterpret some previous ESR studies of plant membrane lipids. Furthermore, the results show that the physical properties of N. oleander membrane lipids change during thermal acclimation. MATERIALS AND METHODSRooted cut...

The phase separation temperatures of total leaf phospholipids from warm and cool climate plants were determined in order to explore the relationship of lipid physical properties to a species' thermal habitat. The separation temperatures were determined by measuring the fluorescence intensity and fluorescence polarization of liposomes labeled with the polyene fatty acid probe trans-parinaric acid. To focus on a single climatic region, Mojave Desert dicots (chiefly ephemeral annuals) were examined, with plants grown under identical conditions whenever possible. Winter active species showed lower phase separation temperatures than the summer active species. A group of warm climate annual grasses showed separuition temperatures distinctly higher than those of a group of cool climate grasses, all grown from seed under the same conditions. Growth at low temperature seems correlated with (and may require) a low phase separation temperature. Winter active ephemerals appear genetically programmed to synthesize a mixture of phospholipids which will not phase separate in the usual growth conditions. When the lipids of desert perennials were examined in cool and warm seasons, there was a pronounced seasonal shift in the phase separation temperature, implying environmental influences on lipid physical properties. The relationship of these results to high and low temperature tolerance is discussed.The physical state of membrane lipids may bear a significant relationship to the lower temperature limit for a species' growth or survival. In various crop plants there is a correlation between the occurrence of a lipid phase separation at about 10 C, an abrupt increase in the activation energy of various membrane-bound reactions at this temperature, and the occurrence of metabolic dysfunction at temperatures below this point (7,15,16 The importance of membrane lipids in determining chilling sensitivity has recently been questioned (2,5,6,24 America. Ephemeral species that are known to grow principally during the summer or winter seasons and some perennials that are active throughout the year were examined. For the most part the ephemeral species were grown at a common growth temperature in controlled growth facilities. However, the perennial species were sampled during midwinter and in early summer from natural plants growing in Death Valley, California. The mean daily maximum and minimum temperatures in Death Valley for January and July are 18 C/3 C and 45 C/32 C, respectively; these temperatures indicate the different thermal regimes experienced by plants growing in the winter or summer. We used the fluorescent polyene fatty acid, trans-parinaric acid (22, 23), as a probe for determining the phase boundaries of liposomes of membrane phospholipids extracted from these plants. Unfortunately, it was not possible to use trans-parinaric acid with native membranes of leaf cells or with total lipid preparations because pigments of the leaf quench the probe's fluorescence. In interpreting these studies, we assume that differences...