Phytochrome is the photoreceptor that controls red light-mediated morphogenesis in higher plants. It exists in two photointerconvertible forms, a red light-absorbing form, Pr, and a far-red light-absorbing form, Pfr. Because photoconversion of Pr to Pfr by a brief light pulse decreases the in vivo half-life of this chromoprotein by a factor of =100, this system offers a unique way to modulate the turnover rate of a specific protein and hence study the mechanisms responsible for selective protein degradation. In etiolated oat [Avena sativa (L.)] seedlings, degradation of phytochrome as Pfr follows zero-order kinetics as measured both spectrally and by ELISA, with 50% of Pfr lost in 130 min at 270C. Immunoblot analysis of the destruction process with anti-oat phytochrome immunoglobulins reveals that degradation involves the loss of the 124-kDa phytochrome monomer and that proteolytic intermediates of apparent molecular mass lower than 124 kDa do not accumulate to detectable levels in vivo (<0.015% of total phytochrome). The latter observation suggests that proteolytic breakdown of the protein is extremely rapid. However, a series of polypeptides with higher apparent molecular mass and recognized by anti-phytochrome immunoglobulins (principally 129 and 134 kDa) appears after photoconversion to Pfr. These polypeptides represent no more than a few percent of the total immunologically detectable phytochrome pool and have incremental differences in apparent molecular mass of 5 kDa. They appear within 5 min after Pfr formation, reach maximal levels between 90 and 180 min, and decline thereafter. These polypeptides and others of apparent molecular mass up to 160 kDa are also detectable with immunoglobulins directed against either oat or human ubiquitin, indicating that they are ubiquitin-phytochrome conjugates. Since ubiquitin conjugation is involved in intracellular protein turnover and since formation and degradation of Pfr-ubiquitin conjugates coincide with the turnover of Pfr, these data suggest that the Pfr form of phytochrome is degraded via a ubiquitin-dependent proteolytic pathway.Although intracellular protein degradation is essential to cellular physiology and development, little is known about the molecular mechanisms involved in this process. In eukaryotes, several different ATP-dependent pathways for protein degradation have been identified. The best characterized is a cytoplasmic pathway involving the small (76 amino acids), highly conserved polypeptide ubiquitin (1, 2). Ubiquitin and various components ofthe proteolytic pathway have been detected in all eukaryotes thus far investigated, including mammals, yeast, and higher plants (1-3). In this pathway, ubiquitin is enzymatically ligated to proteins destined for catabolism and then is released to a free, functional form during the degradation of the target proteins. Ligation is accomplished via an unusual peptide linkage between the carboxyl-terminal glycine of ubiquitin and free a-and Eamino groups on proteins by a multi-enzyme system that require...

Action spectra for the inhibition by continuous (24-hour) irradiation of hypocotyl growth in 54-hour-old Sinapis alba L. seedlings were measured using seedlings which had had four different pretreatments. These seedlings were either (a) dark-grown with a high total phytochrome level, (b) darkgrown with a low total phytochrome level, (c) light-grown with chlorophyll, or (d) light-grown with no chlorophyll Itreated with 4-chloro-5-(methylamino)-2-(a,a,a-trifluoro-m-tolyl)-3(2H)-pyridazinone (San 9789)1.The resulting action spectra show that the blue, red, and far-red (716 to action spectrum, although this may be partially due to differences in irradiation time and mode (14,15). A loss of the response to FR light with increasing age or after a light pretreatment has also been reported (2,5,27). The identity of the photoreceptor(s) is not entirely clear. Candidates which have been considered are Chl/photosynthesis, phytochrome, and, for the B part of the spectrum, the "blue light receptor." Evidence suggests no direct role for photosynthesis (14), although it is possible that it may affect the final expression of responses in green plants. Hartmann (7-9) has shown that it is possible to explain the FR action peak on the basis ofphytochrome alone. The receptor(s) responsible for the B action maximum has not been identified.To clarify some of these points and also to determine the form of the action spectrum in green plants where screening by Chl will occur and total phytochrome will be lower than in dark-grown plants, action spectra for the inhibition of hypocotyl elongation in Sinapis alba L. seedlings have been measured. The measurements were carried out on (a) dark-grown plants with a high level of Ptot, (b) dark-grown plants with a low level of P,ot, (c) MATERIALS AND METHODSSinapis alba L. seeds (harvest 1975) were obtained from Asgrow Co., Freiburg-Ebnet, W. Germany, selected and sown on filter paper in plastic boxes as described by Mohr (19)

Treatment of the mustard (Sinapis alba L.) seedling with the herbicide SAN 9789 inhibits synthesis of colored carotenoids and interferes with the formation of plastid membrane lipids without affecting growth and morphogenesis significantly. In farred light, which is hardly absorbed by chlorophyll, development of plastid ultrastructure, synthesis of ribulosebisphosphate carboxylase and synthesis of chlorophyll are not affected by SAN 9789. It is concluded that normal phytochrome actions on plastid structural development, protein and chlorophyll syntheses are not affected by the absence of carotenoids provided that there is no significant light absorption in chlorophyll. The findings show that the inhibition of synthesis of one set of plastid membrane components (the carotenoids) does not stop synthesis of other components such as chlorophyll and does not halt membrane assembly. Supplementary experiments with the closely related compound SAN 9785, which affects the amount and composition of plastid lipids but not carotenoid and chlorophyll syntheses, suggest that the effect of the herbicide SAN 9789 is due exclusively to its inhibition of synthesis of colored carotenoids. In the presence of SAN 9789 white or red light at high fluence rate causes photodestruction of chlorophyll and ribulosebisphosphate carboxylase and photodecomposition of thylakoids. These effects are interpreted as resulting exclusively from the self-photooxidation and photosensitizing action of chlorophyll once the protection by carotenoids of chlorophyll against self- and sensitized photooxidation is lost.

The herbicide, 4-chloro-5-(methybano)-2-(aaa-trifluoro-m-tolyl)- In many plant species sublethal doses of 4-chloro-5-(methylamino)-2-(a,a,a-trifluoro-m-tolyl)-3(2H)-pyridazinone (Norflurazon; hereafter referred to as San 9789, manufacturer's code no.) inhibit carotenoid synthesis (2). When exposed to white light these plants do not accumulate Chl pigments, but appear to grow and develop as well as untreated plants for several days.Our interest in this herbicide evolved from the possibility of using it as a tool to study phytochrome spectrophotometrically in light-grown plants. Normally ChM fluorescence makes spectrophotometric measurements in light-grown plants impossible (18). Furthermore, R3/FR-reversible-fluorescence yield changes, unrelated to phytochrome, produce apparent A changes which could be confused with phytochrome (6

(14).Subsequent experiments with daylength extensions, rather than dark interruptions, found that a mixture of R and FR was more effective for promotion than R alone (7, 21, 31). The action spectrum for this response (27) shows a single peak in the R-FR region between 710 and 720 nm and some action in the blue at high irradiances. Such responses have been termed "high irradiance responses" (HIR) (28), to distinguish them from the low energy R/FR reversible responses, and are assumed to be a complex function of the photostationary state between Pr and Pfr (13).Although Hartmann (13)