INTERORGAN CORRELATION IN A PHYTOCHROME-MEDIATED RESPONSE IN THE MUSTARD SEEDLING

Abstract —Phytochrome ( P_fr )-mediated threshold control of apparent lipoxygenase (LOG) synthesis in the cotyledons of the mustard seedling depends on the presence of the hypocotylar hook. With isolated cotyledons, no suppression by light of apparent LOG synthesis can be detected. As soon as the cotyledons are separated from the hook, the control of apparent LOG synthesis by P_fr is completely lost. The data are consistent with the hypothesis advanced in a previous paper [Oelze-Karow, H. and H. Mohr (1973) Photochem. Photobiol. 18, 319–330] that apparent LOG synthesis in the cotyledons is controlled by phytochrome located in the hypocotylar hook. The data support the concept [De Greef, J. A. and R. Caubergs (1972) Arch. Int. Physiol. Biochim. 80, 959–960] that a very precise and highly ordered biophysical recognition and transmission system of light signals exists in plants.     

AN ATTEMPT TO LOCALIZE THE THRESHOLD REACTION IN PHYTOCHROME-MEDIATED CONTROL OF LIPOXYGENASE SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —Synthesis* of the enzyme lipoxygenase (LOG)? in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_fr) through a threshold (all-or-none) mechanism. The data of the present paper confirm the previous assumption (Oelze-Karow and Mohr, 1973) that the primary reaction of P_fr (P_fr+ X → P_frX ? P_frX‘) is the site of the highly cooperative threshold reaction. Suppression of LOG synthesis depends on the presence of P_frX’. However, P_frX‘ is only stable above the threshold level of P_fr. If the level of P_fr is below the threshold, P_frX is stable, and no suppression of LOG synthesis occurs. As long as the level of P_fr remains below the threshold, no destruction of P_fr takes place. Destruction of P_fr occurs only as long as [P_fr]?is above the threshold level. Thus the simplest formulation of the actual threshold reaction in the LOG response is P_frX?_frX’ state at [P_fr] below threshold no P_fr destruction LOG synthesis suppressed state at [P_fr] above threshold P_fr destruction(¹k_d LOG synthesis unimpaired The reversible threshold reaction is thus an integral part of the “primary reaction” of P_fr occurring at the “matrix” specific for the LOG response. The data and conclusions on the LOG response are consistent with an “open phytochrome-receptor model” recently advanced by E. Schäfer (1975). The data are not consistent with the concept that a rapid dark reversion (P_fr→P_r) exists in dicotyledonous seedlings and that the degree of P_fr dark reversion strongly depends on the initial photostationary state, φ?, established by a saturating light pulse.     

THE PHOTORECEPTORS INVOLVED IN ANTHOCYANIN SYNTHESIS

Abstract— Experiments with irradiation sequences where red precedes far-red lead to the conclusion that, in turnip, phytochrome is the only pigment mediating anthocyanin synthesis in red and far-red. Results from experiments where far-red precedes red, however, suggest that more than one reaction is involved. A possible interpretation is that the 'high-energy' reaction in far-red and the low energy red/far-red reversible reaction are mediated by two different forms of phytochrome.
The 'high-energy' reaction in blue light does not appear to depend on phytochrome.     

PHYTOCHROME-MEDIATED LOSS OF POLYRIBOSOMES IN ETIOLATED MUSTARD HYPOCOTYLS

Abstract. Continuous far-red light operating through phytochrome reduces the rate of hypocotyl lengthening by a factor of 8. Consonant with this was a reduction of up to 12% in the proportion of total cytoplasmic ribosomes present as polysomes. A 2% reduction was elicited by brief red light. Evidence is presented which argues against these changes arising from a change in extractability or nucleolytic activity. The speed of the response (1.5 h) is much slower than that for inhibition of lengthening (15 min). The data therefore indicate that phytochrome is unlikely to act by controlling the general rate of translation in this instance.     

PHYTOCHROME-STIMULATED REGENERATION OF PROTOCHLOROPHYLL IN COTYLEDONS OF THE MUSTARD SEEDLING

Abstract —It has been shown [Kasemir, H., U. Oberdorfer and H. Mohr, Photochem. Photobiol. (1973) 18 , 481–486] that elimination of the lag phase of chlorophyll a (Chl) accumulation in continuous white light is due exclusively to the action of phytochrome (P_fr). In the present paper we show that the action of P_fr on the lag phase of Chl accumulation can be understood quantitatively as a consequence of the action of P_fr on the initial rate of protochlorophyll (PChl) regeneration. Disappearance of PChl (or formation of Chl) can be excluded as a control signal for the light-mediated changes in rate of PChl regeneration. The P_fr control of PChl regeneration does not discriminate between PChl 650 and PChl 637. The action of P_fr on the PChl regeneration is a relatively fast process (time lag < 3 h). On the other hand, the effect remains stable over long periods (at least 24 h) in darkness.     

ON THE PHYSIOLOGICAL SIGNIFICANCE OF DARK REVERSION OF PHYTOCHROME IN THE MUSTARD SEEDLING

Abstract —We present physiological evidence using the threshold control of lipoxygenase synthesis† by P_fr in the mustard seedling (LOG response) that there is no dark reversion of phytochrome
which would be relevant for this response. Such Ptr which can be detected with the lipoxygenase response disappears exclusively through degradation with a half-life of 45 min at 25°C. De novo synthesis of P*_r in the hypocotylar hook takes place at a constant rate (zero order rate constant) irrespective of the level of P_r or P*_tot, i.e. there is no detectable feedback control of P_r synthesis during the period of experimentation. The data of the present paper are consistent with a quantitative phytochrome model (Scheme 1) which has been advanced and treated quantitatively in the companion paper (Oelze-Karow and Mohr, 1976).     

THE ACTION SPECTRUM AND DOSE RESPONSE STUDIES OF UNSCHEDULED DNA SYNTHESIS IN NORMAL HUMAN FIBROBLASTS

Abstract— Excision repair of DNA damage by UV has been assessed in normal human fibroblasts in culture by measuring unscheduled DNA synthesis. Dose response experiments indicated that the same chromophore was involved in UV-induced damage and excision repair at three different wavelengths between 260 and 300 nm. Action spectra for unscheduled DNA synthesis were determined at wavelengths between 260 and 320 nm 30 min after irradiation using 2 doses of UV, 100 J m^-2and 10Jm^-2. Experiments at the lower dose were carried out because it appeared that repair was saturated with the higher dose at 260 and 280 nm. To explore this part of the spectrum further, experiments were performed with different doses at 260 and 280 nm and unscheduled DNA synthesis assessed 30 min and 24 h after irradiation. At 24 hr after irradiation a significantly greater amount of unscheduled DNA synthesis occurred at 280 nm. It is suggested, therefore, that both DNA and protein are concerned in the absorption of UV which leads to DNA damage and excision repair.     

PHYTOCHROME-MEDIATED CONTROL OF PROLAMELLAR BODY REORGANIZATION AND PLASTID SIZE IN MUSTARD COTYLEDONS

Abstract— The development of plastids in the palisade parenchyma cells of the cotyledons of mustard seedlings ( Sinapis alba L.) was studied by electron microscopy. In darkness the etioplasts undergo a sequence of morphogenic changes previously recognized in principle in bean and barley leaves, as summarized by Rosinski, J. and W. G. Rosen (1972) Quart. Rev. Biol. 47 , 160–190. From 12 to 36 h after sowing, an increase in the percentage of etioplast profiles with paracrystalline prolamellar bodies can be observed. Thereafter, the degree of organization and size of the prolamellar bodies decrease. 60 h after sowing, the etioplasts show only remnants of prolamellar bodies with irregularly spaced tubules. Continuous far-red light, which is considered to operate via phytochrome, counteracts the decay of organization of the prolamellar body and strongly increases the size of the plastids. The effect of continuous far-red light (onset of light 36 h after sowing) can be substituted by 12 h of far-red light given between 36 and 48 h after sowing. It is shown with red and far-red light pulses that the morphogenic effect of long-term far-red light on plastid size and appearance of the prolamellar body is exclusively due to phytochrome (P_fr). Changes by light in the amounts of protochlorophyll(ide) or chlorophyll(ide) do not affect these results. The action of P_fr on the structure of the prolamellar body is a relatively fast process, occurring within 3 h. Formation of thylakoids does not seem to be under phytochrome control. Rather, this response seems to be related to the protochlorophyll(ide)→ chlorophyll(ide) a transformation.     

STIMULATION OF THE SHIBATA SHIFT BY PHOTOCHROME IN THE COTYLEDONS OF THE MUSTARD SEEDLING SINAPIS ALBA L.

Abstract— In the cotyledons of the mustard seedling Sinapis alba L. the duration of the Shibata shift can be greatly shortened by a pretreatment with light pulses prior to the protochlorophyllide– chloro-phyllide a photoconversion. It was shown that the light pulses act through photochrome (P _fr ). Since reversibility of a red light pulse induction by a far-red light pulse is rapidly lost (within 2 min) it is concluded that at least the initial action of P_fr occurs rapidly in this response. On the other hand, the effect of a red light pulse on the rate of protochlorophyll regeneration in the mustard seedling cotyledons is fully reversible by a far-red light pulse for more than 5 min. It is concluded that control of protochlorophyll regeneration and control of the Shibata shift by phytochrome cannot be consequences of the same initial action of P_fr Apparently P_fr controls both phenomena independently.     

LIGHT-INDUCED SYNTHESIS OF ANTHOCYANIN IN CARROT CELLS IN SUSPENSION—IV. THE ACTION SPECTRUM

Abstract— Using carrot cell suspension in 2,4-dichlorophenoxyacetic acid (2,4-D)-depleted culture medium, fluence-response curves for the formation of anthocyanin were determined at various wavelengths from 250 to 800 nm. In the fluence-response curves at wavelengths between 260 and 330 nm, the response showed a sharp fluence-dependent increase after the fluence exceeded threshold level at the respective wavelength. Such a sharp increase in response was not observed by light at 450 nm or longer wavelengths, although the response obtained by higher fluence of such light was always higher than that in the dark control. Action spectra determined at the sharp increasing phase of the response showed the single peak at 280 nm which equals the absorption maximum of UV-B photoreceptor.
Although red (R)-light alone had a minor effect on anthocyanin accumulation, it modulated the action of UV-B light. That is, when carrot cells were irradiated with R-light either before or after UV-B irradiation, anthocyanin formation was greatly enhanced above the level enhanced by UV-B light alone. The most effective wavelength for this enhancement was 660 nm. The effect of R-light on the anthocyanin formation of the UV-B irradiated cells was reversed by immediately following it with far-red light, suggesting the involvement of phytochrome in the R-effect.     

PHYTOCHROME-MEDIATED THRESHOLD CONTROL OF HYPOCOTYL GROWTH IN PARTLY DE-ETIOLATED MUSTARD (Sinapis alba L.) SEEDLINGS

Abstract— Hypocotyl elongation in etiolated mustard ( Sinapis alba L.) seedlings is known to be controlled by phytochrome (Pfr) through a threshold mechanism. The Pfr threshold value required to suppress hypocotyl growth was low (3 times 10⁻²% Pfr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%). In the present study the question was addressed whether the threshold control by Pfr of hypocotyl elongation also operates in light-pretreated, partly de-etiolated seedlings after transfer to darkness. The experimental results show that this is the case. Calculation of the threshold level in far-red light pretreated seedlings led to a very low value (3 times 10⁻⁷%) compared to etiolated seedlings (3 times 10⁻²%). In red light pretreated seedlings the threshold level was calculated to be 9 times 10⁻⁷%. Since the light pretreatment affected the rate of degradation of phytochrome strongly (half-life of Ptot in continuous red light was found to be 35 min in far-red pretreated instead of 47 min in etiolated material), the difference in threshold level between far-red and red pretreated material cannot be interpreted unambiguously. However, the conclusion can be drawn that light nretreatment strongly increases the degradation rate of Pfr and decreases the threshold level.     

THE EFFECT OF PROLONGED LIGHT EXPOSURE ON THE EFFECTIVENESS OF PHYTOCHROME IN ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS*

Abstract— The hypocotyl of the tomato ( Lycopersicon esculentum ) seedling synthesizes large amounts of anthocyanin if exposed to prolonged light. Single light pulses are totally ineffective. The involvement of phytochrome can be shown by light pulse treatments following a prolonged light exposure. It is predominantly the action of blue/UV light which leads to a high responsiveness of anthocyanin synthesis towards phytochrome. Moreover, the data suggest a phytochrome-independent action of blue/UV light, in particular of UV-B, on anthocyanin synthesis.     

THE MODE OF INTERACTION BETWEEN BLUE (UV) LIGHT PHOTORECEPTOR AND PHYTOCHROME IN ANTHOCYANIN FORMATION OF THE SORGHUM SEEDLING

Two non-photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (P_fr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without P_fr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.     

MULTIPLICATIVE ACTION OF A UV-B PHOTORECEPTOR and PHYTOCHROME IN ANTHOCYANIN SYNTHESIS

Using 290-nm light, which excites only a UV-B photoreceptor, and 385- and 660-nm light, which activate only phytochrome, the fluence rate-response curves of monochromatic irradiations for anthocyanin synthesis in the first internodes of broom sorghum (Sorghum bicolor Moench, cv. Acme Broomcorn) were analyzed. Although the two photoreceptors absorbed light independently, they multiplicatively increased the action of each other. Accordingly, when the fluence rates of both wavelengths were changed together, the resulting slopes of the fluence rate-response curves of double-log plots were steep compared with the slopes obtained with the respective monochromatic irradiations. The slopes of fluence rate-response curves for monochromatic irradiations at 325 to 345 nm were steeper than those at other wavelengths. This difference was shown to be due to the multiplicative actions of both photoreceptors.     

AN ANALYSIS OF PHYTOCHROME-MEDIATED THRESHOLD CONTROL OF HYPOCOTYL GROWTH IN MUSTARD (Sinapis alba L.) SEEDLINGS†

Hypocotyl elongation in mustard (Sinapis alba L.) seedlings is known to be controlled by phytochrome (P_fr) through a threshold response. This phytochrome-mediated threshold response was studied in detail with the following results: (i) The P_fr threshold value required to suppress hypocotyl growth is much lower (0.03% P_rr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%) than those threshold valued observed previously in threshold control by hook phytochrome of appearance of 'potential capacity for photophosphorylation' and lipoxygenase appearance in the mustard cotyledons (1.25% P_tr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%). This probably explains why hypocotyl elongation is so extremely sensitive to light, (ii) The P_fr threshold value controlling hypocotyl growth is a system constant, independent of total phytochrome content, developmental age and actual growth rate, (iii) Threshold control of hypocotyl elongation is unaffected by the removal of the cotyledons and half of the hook. However, removal of the whole hook totally eliminates any light control over the residual hypocotyl growth, (iv) After termination of the threshold control, the hypocotyl growth rate immediately returns to precisely that found in untreated dark control even though the partial growth rates of the different parts of the hypocotyl are quite different, relative to their dark controls. Obviously, the organ grows as an integrated unit.
It is concluded that the all-or-none threshold control over hypocotyl growth is exerted from the plumular hook. It appears that the hook can send off phytochrome all-or-none signals in both directions, to the cotyledons and to the hypocotyl.     

EFFECT OF BLUE/UV LIGHT ON ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS IN THE ABSENCE OF BULK CAROTENOIDS

Abstract Anthocyanin synthesis in the hypocotyl of tomato ( Lycopersicon esculentum ) seedlings responds strongly and specifically to blue/UV light while the response to red and far-red light, operating through phytochrome, is weak. The herbicide Norflurazon (SAN 9789) was used to inhibit synthesis of colored carotenoids almost completely without affecting growth and development measurably. Even though carotenoid content was reduced to less than 2% of normal and the fluence rate response function for blue and UV light was linear within the experimental range, Norflurazon treatment did not reduce seedling sensitivity toward blue/UV light. It was concluded that at least'bulk'carotenoids are not the photoreceptor chromophore of the blue/UV photoreceptor pigment.     

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.     

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-MEDIATED RAPID CHANGES IN THE LEVEL OF PHOSPHOINOSITIDES IN ETIOLATED LEAVES OF Zea mays

The role of the active form of phytochrome in Zea mays on the polyphosphoinositide cycle was studied. As little as 15 s of red irradiation of etiolated leaves immediately increased the level of phosphatidylinositol bisphosphate (PIP₂) 3–6-fold compared to unirradiated leaves. The elevated level of PIP₂ decreased with longer red irradiations up to 5 min, but remained higher than in unirradiated leaves. The level of PIP₂ decreased if red irradiation was followed by far-red irradiation. Far-red alone had no effect. Levels of phosphatidylinositol phosphate (PIP) and phosphatidylinositol did not change significantly. Since red irradiation significantly changed PIP, but not PIP, photocontrol appears to be at the PIP kinase and phospholipase level. In related studies of the effect of light on phospholipids, 5 min of red irradiation induced significant decreases in phosphatidylcholine and phosphatidylethanola-mine.