PHOTOCONTROL OF ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS: A GENETIC APPROACH*

The photocontrol of anthocyanin synthesis in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) has been studied in an aurea (au) mutant which is deficient in the labile type of phytochrome, a high pigment (hp) mutant which has the wild-type level of phytochrome and the double mutant au/hp , as well as the wild type. The hp mutant demonstrates phytochrome control of anthocyanin synthesis in response to a single red light (RL) pulse, whereas there is no measurable response in the wild type and au mutant. After pretreatment with 12 h blue light (BL) the phytochrome regulation of anthocyanin synthesis is 10-fold higher in the hp mutant than in the wild type, whilst no anthocyanin is detectable in the au mutant, thus suggesting that it is the labile pool of phytochrome which regulates anthocyanin synthesis. The au/hp double mutant exhibits a small (3% of that in the hp mutant) RL/far-red light (FR)-reversible regulation of anthocyanin synthesis following a BL pretreatment. It is proposed that the hp mutant is hypersensitive to the FR-absorbing form of phytochrome (P_fr) and that this (hypersensitivity) establishes response to the low level of P_fl. (below detection limits in phytochrome assays) in the au/hp double mutant.     

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH-PIGMENT MUTANT OF TOMATO

Abstract— A high-pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light-labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence-response relationships for anthocyanin synthesis after a blue-light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far-red light (FR)-absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6-fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild-type and hp-mutant seedlings, despite the latter accumulating 9-fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence-rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24-h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.     

PHOTOTRANSFORMATIONS OF PHYTOCHROME

Abstract— –Phytochrome is the photoreversible chromoprotein that controls many aspects of plant growth and development Phototransformations of the red absorbing form (Pr) and the far red absorbing form (Pfr) involve initial photoreactions followed by dark relaxation reactions. Techniques for the study of intermediates of phototransformation and the present picture of intermediates involved in the phototransformations of Pr and Pfr are outlined. The molecular natures of the phototransformations are reviewed in relationship to knowledge of the chemistry of the chromophore and apoprotein. The significance of phytochrome intermediates in understanding the physiology of phytochrome controlled responses is discussed.     

Phytochrome-mediated inhibition of coleoptile growth in rice: age-dependency and action spectra

Phytochrome has been shown to be the major photoreceptor involved in the photo-inhibition of coleoptile growth in Japonica-type rice (Oryza sativa L.). We have characterized this typical photomorphogenetic response of rice using mutants deficient in phytochrome A (phyA) and phytochrome B (phyB) and with respect to age-dependency and action spectra. Seedlings were irradiated with a pulse of light 40 h or 80 h after germination (i.e. at an early or late developmental stage) and the final coleoptile length of these seedlings was determined. A saturating pulse of red light (R) had a stronger effect when it was given in the late stage than in the early stage. It was found that the photoinhibition is mediated by both the phyA and the phyB in the late stage but predominantly by phyB in the early stage. Consistent with many other reported responses, the photo-inhibition in the phyA mutant, which was observed in the early and late developmental stages and is thought to be mediated mainly by phyB, occurred in the low-fluence range (10(1)-10(3) micromol m(-2)) of R and was far-red-light (FR)-reversible; the photo-inhibition in the phyB mutant, which was observed in the late developmental stage and is thought to be mediated mainly by phyA, occurred in the very-low-fluence range (10(-2)-10(0) micromol m(-2)) and was FR-irreversible. The action spectra (350-800 nm at 50 nm intervals) obtained at the two developmental stages using phyA and phyB mutants indicated that both the phyB-mediated low-fluence response and the phyA-mediated very-low-fluence response have a major peak at 650 nm and a minor peak at 400 nm.     

Red Light-Induced Appearance of Phosphotyrosine-like Epitopes on Nuclear Proteins from Pea (Pisum sativum L.) Plumules

Abstract— As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti-phosphotyro-sine antibodies in several pea nuclear proteins. The im-munostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red-light-induced response seen in plumules is reversible by a subsequent far-red-light irradiation, indicating that the likely photoreceptor for this response may be phy-tochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin-matrix preparations. Phytochrome A and the protein bands immuno-stained by anti-phosphotyrosine antibodies can be solu-bilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine-like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far-red light-absorbing form can induce a post-translational epitope change in nuclear proteins in vivo .     

INTERACTION BETWEEN PHYTOCHROME AND THE BLUE LIGHT PHOTORECEPTOR SYSTEM IN Mougeotia*

Abstract— Face-to-profile chloroplast movement in Mougeotia was induced by sequences of strong blue and red short irradiations. This type of response occured only when blue light was applied prior to or simultaneously with red light, and far-red irradiation was necessary after the sequence to cancel the remaining gradient of the far-red absorbing form of phytochrome P_fr. The dependence of the response magnitude on blue and red light sequences was studied for a wide range of light durations and dark intervals. The relationship between the response and the dark interval points to the lack of direct coupling between phytochrome and blue-absorbing “cryptochrome”. It was postulated that a photoproduct having a life-time of2–3 min is formed by the blue-light-mediated reaction. This photoproduct interacts with phytochrome during its transformation or with its final P_fr form.     

Phototaxis in the cyanobacterium Synechocystis sp. PCC 6803: role of different photoreceptors

The second cyanobacterial phytochrome Cph2 from Synechocystis sp. PCC 6803 was suggested as a part of a light-stimulated signal transduction chain inhibiting movement toward blue light. Cph2 has the two bilin binding sites, cysteine-129 and cysteine-1022, that might be involved in sensing of red/far-red and blue light, respectively. Here, we present data on wavelength dependence of the phototaxis inhibition under blue light, indicating that Cph2 itself is the photoreceptor for this blue light response. We found that inhibition of blue-light phototaxis in wild-type cells occurred below the transition point of about 470 nm. Substitution of cysteine-1022 with valine led to photomovement of the cells toward blue light (cph2(-) mutant phenotype). Analysis of mutants lacking cysteine-129 in the N-terminal chromophore binding domain indicated that this domain is also important for Cph2 function or folding of the protein. Furthermore, putative blue-light and phytochrome-like photoreceptors encoded by the Synechocystis sp. PCC 6803 genome were inactivated in wild-type and cph2 knockout mutant background. Our results suggest that none of these potential photoreceptors interfere with Cph2 function, although inactivation of taxD1 as well as slr1694 encoding a BLUF protein led to cells that reversed the direction of movement under blue light illumination in mutant strains of cph2.     

PHYTOCHROME-MEDIATED PHOTOTROPISM AND DIFFERENT DICHROIC ORIENTATION OF Pr AND Pfr IN PROTONEMATA OF THE FERN ADIANTUM CAPILLUS-VENERIS L.

Abstract— Single-celled protonemata of Adiantum capillus-veneris were cultured under continuous red light for 6 days and then in the dark for 15 h. Brief local exposure of a flank (5 times 20 /mi) of the subapical region of a protonema to a microbeam of red light effectively induced a phototropic response toward the irradiated side. The degree of the response was dependent upon the fluence of the red light. Red/far-red reversibility was typically observed in this photoreaction, showing that phytochrome was the photo-receptive pigment. When the flank was irradiated with a microbeam of linearly polarized red and far-red light, red light with an electrical vector parallel to the cell surface was most effective. However, the far-red light effect was most prominent when its electrical vector was normal to the cell surface. These polarized light effects indicate the different dichroic orientation of P_r (red-light-absorbing form of phytochrome) and P_r (far-red-light-absorbing form of phytochrome) at the cell flank.     

PHYTOCHROME-MEDIATED PHOTOTROPISM IN PROTONEMATA OF THE MOSS Ceratodon purpureus BRID

Abstract— Protonemata of the moss Ceratodon purpureus cultured in white light were transferred to darkness for 3 days and then used for phototropic experiments. Irradiation of the apical region of vertically position protonemata with small beams (0.2 mm) of red light induced a growth response towards the irradiated side (positive phototropism). The phototropic response showed irradiance dependence. The effect of red light was completely reversed by far-red light following red light irradiations, demonstrating that phytochrome was the photoreceptor pigment. Far-red light or UV-blue light had no influence on either bulging or phototropism. Experiments with linearly polarized red or far-red light showed a different dichroic distribution of phytochrome in its different forms, the red-absorbing form, P_r and the far-red-absorbing form, P_fr. Red light with a vibration plane parallel to the long axis of the filaments was most effective. The effectiveness of far-red light was expressed best when its vibration plane was 90° to the electrical vector of the inductive red light.     

Phytochrome-regulated Starch Degradation in Germinating Turions of Spirodela polyrhiza

Abstract— Turions of the duckweed Spirodela polyrhiza contain about 70% starch on the dry weight basis. The rate of starch degradation in nondormant turions was highest in continuous red light (cR) followed by continuous blue light (cB), whereas continuous far red light (cFR) is almost ineffective. Continuous R could be substituted by repeated R pulses; this effect was not photoreversible by FR pulses applied after hourly R pulses. This suggested that R-dependent high irradiance response is the mode of phytochrome action in mediating starch degradation. Comparing the mode of phytochrome action with that of phytochrome mutants of other plants it can be assumed that phytochrome B is the photoreceptor involved. Application of the translation inhibitor cycloheximide immediately stopped the phytochrome-dependent starch degradation demonstrating the involvement of de novo synthesis of proteins.     

PHYTOCHROME SYNTHESIS DURING IMBIBITION IN EMBRYONIC AXES OF Pisum sativum L.*

Abstract— The exact quantity of phytochrome in crude homogenates (2kS) prepared from embryonic axes of Pisum sativum during imbibition at 25°C on 0,2% agar was estimated optically. The problem of the scattering factor was solved by using highly purified phytochrome as an internal standard. The content of phytochrome protein moiety in diluted samples of the crude homogenates of the axes was also determined by an enzyme-linked immunosorbent assay (ELISA). Phytochrome was not detectable either spectropho-tometrically or immunochemically in 2kS of dormant dry axes. Embryonic axes quickly absorbed water during the first1–2 h after the start of imbibition, after which the fresh weight stayed at a constant level for a further 10 h. The content of spectrophotometrically detectable phytochrome increased during imbibition in the dark, reaching about 0.2 μ.g/axis after 12 h. The amount of phytochrome in 2kS of axes in the light was so small that only about 0.05 μg/axis was detected after 12 h. The content of immunochemically detectable phytochrome greatly increased up to ca. 0.5 μg/axis after 12 h of dark incubation. In 2kS of the light-grown axes the content of the phytochrome protein was ca. one fourth lower than in dark-grown axes. We conclude that the appearance and increase of phytochrome in fragments of imbibed embryonic axes were caused by de novo synthesis and that the contents of both photometrically detectable phytochrome and its protein moiety in the light-grown samples were lower than those in the dark throughout the early germination process.     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

ACTION SPECTRA FOR INHIBITION OF HYPOCOTYL GROWTH OF WILD-TYPE PLANTS AND OF THE hy2 LONG-HYPOCOTYL MUTANT OF Arabidopsis thaliana L.

Abstract— An analysis was made by action spectroscopy, using the Okazaki Large Spectrograph, of the inhibition of hypocotyl elongation of wild-type plants and the hy2 mutant of Arabidopsis thaliana. Two day old etiolated seedlings were irradiated for 8 h with monochromatic light and left in the dark for 16 h before measurement of hypocotyl length. Spectrophotometric measurement showed that levels of phytochrome in the etiolated tissue of the hy2 mutant were less than 9% of those in the wild type. The action spectra of the wild type looked like those of high irradiance response and showed peaks at 375, 450, 625 and 725 nm, whereas the action spectra of hy2 showed only the peaks at 375 and 450 nm. Monochromatic light of wavelengths longer than 500 nm had no significant inhibitory effects on hy2 plants. Blue and UV-A light were about five times more effective in the wild type than in hy2 plants. Severe inhibitory effects were observed with UV-B light. It is concluded that inhibition of the growth of the hypocotyl involves combined actions of phytochrome and a putative blue/UV-A photoreceptor(s).     

AN OSCILLATING SYSTEM REGULATING DEVELOPMENT OF PLANTS

Phytochrome conversion shifts the developmental pattern of dark-grown bean (Phaseolus vulgaris) seedlings. Red light was found to initiate rapid oscillations in a system that links illumination with subsequent growth. A single 8 s flash of red light increased the average leaf weight measured 24 h later by 8%. When total illumination was kept constant but the interval between two 4 s flashes was varied, the resulting leaf weight increase was not uniform but depended on the length of the interval between flashes and showed a series of deep minima followed by sharp maxima. Weight increase at the maximum was 60% greater than at the minimum. The minimum-maximum transitions recurred every 45 s for at least 22 min. Four s of far red light interposed between the two red flashes abolished the oscillation. Temperature between 15 and 30°C had no significant influence on the period of the oscillation but the period varied directly with the duration of the initial red flash. A 2 s initial flash resulted in a 35 s period, while 40 s of red light caused a 2 min period. Oat (Avena sativa) and radish (Raphanus sativus) seedlings were found to possess similar oscillating mechanisms of growth regulation. The lack of pronounced temperature effects as well as our other findings, suggest that this oscillating system may participate in the time measuring as well as growth regulating mechanisms by which phytochrome controls circadian periodicity and development.     

EXTENSION GROWTH AND ANTHOCYANIN RESPONSES OF PHOTOMORPHOGENIC TOMATO MUTANTS TO CHANGES IN THE PHYTOCHROME: PHOTOEQUILIBRIUM DURING THE DAILY PHOTOPERIOD

Four genotypes of tomato (Lycopersicon esculentum Mill.) in the genetic background ‘Aha Craig’ were used: an aurea (au) mutant, deficient in the bulk light-labile phytochrome pool; a high pigment (hp) mutant, showing exaggerated phytochrome responses at the time of de-etiolation; the au,hp double mutant and the isogenic wild type (WT). A dramatic increase in plant height resulting from an increase in the length of all internodes for each of the genotypes studied was observed upon reduction of the red light: far-red light photon ratio (R:FR) from 6.90 to 0.13 by addition of FR for the whole photoperiod. A concomitant increase in leaf length was also observed. Since au and au,hp mutants, deficient in the bulk light-labile phytochrome pool, respond to this reduction in the R:FR these data demonstrate that the phytochrome pool that mediates this response is present and fully functional. Anthocyanin was detectable in the comparably developed young growing leaves of the WT and hp mutant under the high R:FR, but not in the au and au,hp mutants, suggesting that the potential for anthocyanin synthesis is correlated with the presence of the bulk light-labile phytochrome pool. The kinetics of anthocyanin decrease in the young growing leaves were investigated in the hp mutant and the results suggest a very rapid cessation of flavonoid biosynthesis upon reduction of the R:FR. The functions of different phytochrome types are discussed.     

CONTROL OF HYPOCOTYL GROWTH IN MUSTARD SEEDLINGS AFTER LIGHT-DARK TRANSITIONS

Abstract— Six hours of irradiation with white or far-red light strongly stimulates the 'end of day' inhibition of hypocotyl elongation in dark grown Sinapis alba L. Measurements of both reversion kinetics of the inductive light pulse and 'null point' experiments (Hillman, 1965. 1972) indicate that this response is controlled by stable phytochrome. The extent of the reversible response (i. e. length after far-red light minus length after red light) showed rhythmic oscillations after a 6, 48 and 54 h white light pretreatment. The rhythm is started by the transition from light to dark and neither phase nor amplitude is influenced by the level of P at this transition.     

Phytochrome Control of Anthocyanin Biosynthesis in Tomato Seedlings: Analysis Using Photomorphogenic Mutants

Anthocyanin biosynthesis has been studied in hypocotyls and whole seedlings of tomato (Lycoperskon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. In white light (WL)/dark (D) cycles the fri¹ mutant, deficient in phytochrome A (phyA), shows an enhancement of anthocyanin accumulation, whereas the tri¹ mutant, deficient in phytochrome Bl (phyBl) has a WT level of anthocyanin. Under pulses of red light (R) or R followed by far-red light (FR) given every 4 h, phyA is responsible for the non-R/FR reversible response, whereas phyBl is partially responsible for the R/FR reversible response. From R and blue light (B) pretreatment studies, B is most effective in increasing phytochrome responsiveness, whereas under R itself it appears to be dependent on the presence of phyBl. Anthocyanin biosynthesis during a 24 h period of monochromatic irradiation at different flu-ence rates of 4 day-old D-grown seedlings has been studied. At 660 nm the fluence rate-response relationships for induction of anthocyanin in the WT are similar, yet complex, showing a low fluence rate response (LFRR) and a fluence rate-dependent high irradiance response (HIR). The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both the LFRR and HIR. The fri¹ mutant lacks the LFRR while retaining a normal HIR. In contrast, a transgenic tomato line overexpressing the oat PHYA3 gene shows a dramatic amplification of the LFRR. The tri¹ mutant, retains the LFRR but lacks the HIR, whereas the fri¹, tri¹ double mutant lacks both components. Only an LFRR is seen at 729 nm in WT; however, an appreciable HIR is observed at 704 nm, which is retained in the tri¹ mutant and is absent in the fri¹ mutant, indicating the labile phyA pool regulates this response component.     

Fluorescence and photochemical characterization of phytochromes A and B in transgenic potato expressing Arabidopsis phytochrome B

Phytochrome in etiolated sprouts of wild type (WT) potato and its transgenic strains (DARA5 and DARA12) expressing Arabidopsis thaliana phytochrome B (phyB) was investigated using low-temperature (85 K) fluorescence spectroscopy and photochemistry. Phytochrome content, [Ptot], position of the Pr emission and excitation spectra, lambda(max), and extent of the Pr-->lumi-R, gamma1, and Pr-->Pfr, gamma2, phototransformations (at 85 and 273 K, respectively) were shown to vary in the transgenic lines and WT depending on tissue used (upper vs. lower parts of etiolated sprouts) and light-induced phytochrome depletion. Differences in the parameters between the transgenic lines and WT were detected which were interpreted in terms of the two phenomenological Pr types: a labile Pr' with gamma1 approximately 0.5 consisting of a major phytochrome A (phyA) fraction (phyA') and a relatively conserved Pr" with gamma1 = 0 comprising a minor phyA fraction (phyA") and phyB. Both DARA lines had higher [Pr"] as compared with WT in the lower parts of etiolated stems, especially after light-induced phytochrome depletion (residual phytochrome in DARA5 and DARA12 made up to one-third of its initial level vs. <5% in WT). These differences were associated with the expression of Arabidopsis phyB in the DARA lines and its higher light stability than that of phyA. Arabidopsis phyB expressed in potato was characterised by lambda(max) = 683/669 nm in the emission/excitation (absorption) spectra and gamma1 = 0. PhyB also revealed a relatively low gamma2 (approx. 0.5) and its early red drop as compared with the gamma2 wavelength dependence for phyA. This is believed to contribute to the lower signalling ability of phyB and to confine the region (red) of its physiological activity.     

LIGHT PROMOTION OF SEED GERMINATION IN Datura ferox IS MEDIATED BY A HIGHLY STABLE POOL OF PHYTOCHROME

Abstract— The duration of the far-red light-absorbing form of phytochrome (Pfr) of the photoreceptor pool involved in the control of seed germination was investigated for Datura ferox seeds. These seeds require both Pfr and alternating temperatures (20/30°C) to germinate. After 24 h imbibition (25°C), the seeds received pretreatment-light pulses providing different phytochrome photoequilibria (Pfr/P), followed by a 24 h dark incubation (25°C), and test-light pulses providing different Pfr/P immediately prior to transfer to alternating temperatures. Germination increased with increasing Pfr/P provided by the test-light pulses, but was unaffected by the pretreatment-light pulses. This suggests that phytochrome synthesis, phytochrome degradation and phytochrome-mediated changes in response to phytochrome were negligible. In other experiments, red light-pretreatment pulses were followed by dark incubations (25°C) of different duration before transfer to alternating temperatures. The proportion of Pfr remaining after the 25°C incubation period was estimated by comparing germination rates with those of seeds that received test-light pulses of known calculated Pfr/P immediately prior to the start of the cycles of alternating temperatures. More than 80% of the Pfr established by a Pfr/P= 0.87 light pulse was present and active even after 48 h dark incubation at 25°C. Surprisingly, when a pretreatmentlight pulse providing a Pfr/P= 0.70 was given, the reduction in [Pfr] was significantly faster.
Germination of Datura ferox seeds is under the control of a highly stable (type II like) phytochrome pool. Apparently, this pool follows Pfr dark reversion to the red light-absorbing form, the times to reach half the original Pfr pool being > 96 h or <14 h after light pulses providing Pfr/P= 0.87 or 0.70, respectively.     

SPECTRAL PROPERTIES,PHOTOTRANSFORMATION KINETICS AND PELLETABILITY OF PHYTOCHROME IN Pharbitis nil Chois*

Abstract— An examination has been made of the involvement of phytochrome in the circadian rhythm of flowering in Pharbitis nil Chois. The peak position of P_fr absorption changes with time after a red light pulse. The shortest absorption wavelength of P_fr occurs at the same time as flowering is inhibited by red light in dark grown, red light pretreated plants. Pelletable and supernatant phytochrome show a similar trend with lowest values found at the time of flower inhibition. Neither phototransformation kinetics nor intermediates of phytochrome which accumulate in white light show such a relationship to the circadian rhythm found in flowering of dark grown P. nil.