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.     

INTRACELLULAR DICHROIC ORIENTATION OF THE BLUE LIGHT-ABSORBING PIGMENT AND THE BLUE-ABSORPTION BAND OF THE RED-ABSORBING FORM OF PHYTOCHROME RESPONSIBLE FOR PHOTOTROPISM OF THE FERN Adiantum PROTONEMATA

The intracellular localization and orientation of the receptors for the blue light-induced phototropism in the fern Adianrum protonemata, phytochrome and the blue light-absorbing pigment, were investigated by combining the techniques of cell centrifugation and of microbeam irradiation with linearly polarized light. The phototropic response was induced in the cells even after they had been centrifuged basipetally to spin down the endoplasm from the apical region. When a polarized blue microbeam was given to a flank of the apical region of the protonema, the phototropic response after compensation of phytochrome effect by far-red light was most effectively induced when the polarization plane was parallel to the long axis of the cell. If protonemata were pre-irradiated with blue and far-red light, the phototropic response was mediated through phytochrome alone. If such pre-irradiated protonemata were similarly irradiated with a polarized blue microbeam, polarized light vibrating parallel to the cell axis was again most effective in inducing the response. These results indicate that both the blue light-absorbing pigment and the phytochrome responsible for the blue light-induced phototropism in Adiantum are confined to the plasma membrane and/or the ectoplasm and that the transition moments of their blue-absorption bands are nearly parallel to the cell surface.     

A POSSIBLE CONTROL BY A PHYTOCHROME-LIKE PHOTORECEPTOR OF CHLOROPHYLL SYNTHESIS IN THE GREEN ALGA Ulva rigida

Chlorophyll synthesis is stimulated by red light pulses in the green alga Ulva rigida C. Aghard. Chlorophyll synthesis in darkness is greater after longer red light pulses (30 min) than after shorter red light pulses (5 min). Chlorophyll synthesis was higher after red light pulses of 14 Wm_-2 fluence rate than after those of 7 Wm_-2. The effect of red light showed some far-red reversibility. The reversion by far-red light was higher after red light pulses of 4 min than after those of 30 min. These results indicate the existence of a rapid induction of chlorophyll synthesis during the red light pulses and a fast escape from photoreversibility. The percentage of reversion is also affected by the fluence rate of the light pulses. The reversion was reduced by about 15% when the photon fluence rate was increased from 7 to 14 Wm_-2. Reversion was also observed when red and far-red light pulses were applied successively. Thus, phytochrome or a phytochrome-like photoreceptor could be involved in the induction of chlorophyll synthesis in Ulva rigida.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

LIGHT-INDUCED LINEAR ACTION DICHROISM IN PHOTOREVERSIBLY PHOTOCHROMIC SENSOR PIGMENTS—I. THEORY

Abstract— With a photoreversibly photochromic regulator pigment such as phytochrome, linear action dichroism could theoretically be obtained after photoselection even if the molecules are initially randomly oriented: If randomly oriented P_fr (fed-absorbing phytochrome) molecules are partially converted to P_fr (far-red absorbing phytochrome) molecules by plane-polarized red light, those molecules will preferentially be converted which have their 'red' transition moments nearly parallel to the electric vector of the red light. The effect of subsequent plane-polarized far-red light will depend on the plane of polarization. A general theory is developed for how this can be used to determine whether or not the transition moment changes direction during conversion. The pigment need not be isolated, since only physiological reactions (such as germination or chromatic adaptation) are measured.     

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.     

COMPARATIVE PHOTOCHEMICAL ANALYSIS OF HIGHLY PURIFIED 124 KILODALTON OAT and RYE PHYTOCHROMES in vitro

Abstract A direct comparison of the photochemical interconversions between red (P_r-) and far-red (P_fr-) absorbing forms of highly-purified 124 kDa oat and rye phytochromes under identical experimental conditions was performed. In two different buffer systems at 5°C, the quantum yields for the P_r to P_tr and P_fr to P_r phototransformations under constant red and far-red illumination, φ _r and φ_fr respectively, were determined to be 0.152-0.154 and 0.060-0.065 for oat preparations and 0.172-0.174 and 0.074-0.078 for rye preparations. These values as well as the wavelength dependence of the photoequilibrium produced under continuous illumination throughout the visible and near-ultraviolet spectrum were based on the absorption spectra of the two phytochrome preparations and revised molar absorption coefficients. The molar absorption coefficients were estimated by quantitative amino acid analysis and shown to be identical for the two monocot phytochromes (i.e. 132 mM ⁻¹ cm⁻¹ at the red absorption maximum for the P_r form). Because these measurements were performed under identical experimental conditions, including buffer, temperature, light fluence rate, and instrumentation, the differences observed must reflect structural features inherent to the two different monocotyledonous phytochromes.     

Phototropism: a "simple" physiological response modulated by multiple interacting photosensory-response pathways

Phototropism is the process by which plants reorient growth of various organs, most notably stems, in response to lateral differences in light quantity and/or quality. The ubiquitous nature of the phototropic response in the plant kingdom implies that it provides some adaptive evolutionary advantage. Upon visual inspection it is tempting to surmise that phototropic curvatures result from a relatively simple growth response to a directional stimulus. However, detailed photophysiological, and more recently genetic and molecular, studies have demonstrated that phototropism is in fact regulated by complex interactions among several photosensory systems. At least two receptors, phototropin and a presently unidentified receptor, appear to mediate the primary photoreception of directional blue light cues in dark-grown plants. PhyB may also function as a primary receptor to detect lateral increases in far-red light in neighbor-avoidance responses of light-grown plants. Phytochromes (phyA and phyB at a minimum) also appear to function as secondary receptors to regulate adaptation processes that ultimately modulate the magnitude of curvature induced by primary photoperception. As a result of the interactions of these multiple photosensory systems plants are able to maximize the adaptive advantage of the phototropic response in ever changing light environments.     

MODE OF COACTION OF PHYTOCHROME AND BLUE LIGHT PHOTORECEPTOR IN CONTROL OF HYPOCOTYL ELONGATION

Abstract The rate of hypocotyl longitudinal growth in seedlings of Sesamum indicum L. is strongly inhibited by continuous blue light (cBL)† and slightly by continuous far-red light while continuous red light (cRL) or red light pulses are hardly effective from 60 h after sowing onwards. Between 36 and 60 h after sowing the growth rate responds to red light pulses the effect of which is fully reversible by long wavelength far-red light. When seedlings are kept in cBL for 3 days and then treated with red light hypocotyl growth rate responds strongly. However, RL effectiveness decreases with time after transfer from BL to RL. BL → darkness transfer experiments with different levels of P_fr established at the beginning of darkness show that after a BL pretreatment phytochrome (P_fr) alone is capable of fully controlling growth rate. When white light (WL) is given no BL effect is detectable in weak WL. Only high light fluxes maintain a typical BL growth rate. At medium WL fluxes elongation rate returns gradually to the dark rate. The simplest explanation of the data is that light absorbed by a separate BL photoreceptor is necessary to maintain responsivity to P_fr. With increasing age of the seedlings the requirement for BL increases strongly. On the other hand, brief light pulses—given to demonstrate photoreversibility of phytochrome—remain equally effective provided that responsivity to P_fr exists.     

PHYTOCHROME CONVERSION BY ULTRAVIOLET LIGHT

Abstract— Light absorbed primarily by the protein of phytochrome is active in transforming both the red and far-red absorbing forms. P _r and P _fr. The ratio of quantum yields for the conversions of P _r and P _fr by u.v. radiation (φ_r/φ_fr)_u.v.= 1.5 and does not differ significantly from the ratio obtained with red and far-red light absorbed directly by the chromophores (φ_r/φ_fr)v_vis. Thus, the efficiency of energy transfer from protein to chromophore is essentially the same for both forms of the chromoprotein. The ratio of the relative quantum yields for u.v. and visible light (φ^r)^u.v./(φ^r)^vis was 0.32 indicating that 30–35 per cent of the light energy absorbed by the protein was transferred to the chromophore.     

Pfr PHYTOCHROME AND SUCROSE REQUIREMENT FOR RHYTHMIC LEAFLET MOVEMENT IN ALBIZZIA

Abstract— Albizzia leaflets open and close with a circadian periodicity without damping during extended dark periods if supplied with sucrose and irradiated with red each rhythmic cycle. In the absence of a carbohydrate source, the rhythm damps irrespective of phytochrome status and the differential effects of red and far-red light treatments tend to disappear after 2 or 3 cycles. The rhythm also damps if leaflets are exposed to red only once, or if they are exposed to far-red once or cyclically. Sucrose does not prevent such damping but affects the stable angle assumed; the rhythm damps in the closed position in the presence of sucrose, and in the partially open position in its absence. Sucrose appears to have two distinct effects; it interacts with P _fr to promote manifestation of the rhythm and it reduces the average angle, whether the P _fr level is low or high.     

PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

MULTIPLE ACTION OF FAR-RED LIGHT IN PHOTOPERIODIC INDUCTION and CIRCADIAN RHYTHMICITY

Abstract— It is generally accepted that phytochrome influences the photoperiodic induction of flowering through its interaction with the circadian clock mechanism. We have attempted to separate the effects of phytochrome on the clock mechanism from those that mediate flowering directly by examining a number of responses that are unrelated to flowering but are also regulated by the circadian clock. Gas exchange measurements of both CO₂ and H₂0 vapor were monitored under light conditions (200 μmol m ² s⁻¹) where the addition of far-red energy is required for the maximal promotion of flowering. In addition, photosynthetic capacity and maximal transpiration rates were measured in plants grown under continuous dim (20 μmol m⁻² S⁻') light, with or without supplemental far-red, by exposing them briefly to saturating fluxes (1000 μmol m⁻² s^-l) of light. Net CO₂ fixation was very weakly rhythmic in plants grown under both high and low light and this weak oscillation was completely suppressed by far-red light. Far-red also suppressed the rhythm in transpiration under high light, but the rhythm was immediately reinstated when the far-red light was removed. The phase of this rhythm was also reset with the next peak always occurring15–18 h after the far-red was turned off. When grown under dim light, the transpiration rhythm was not suppressed and the amplitude of the oscillation was more than doubled. Far-red light appears to interact with the rhythm in transpiration in a manner suggesting that the stomatal rhythm may be coupled to the same clock oscillator that regulates the flowering rhythm.     

Photoreception and Phototropism in Phycomyces: Antagonistic Interactions between Far-UV, Blue, and Red Light

Abstract— Phototropism of the sporangiophore of the fungus Phycomyces is mediated by UV and blue light. Classical phototropism action spectra with maxima near 280, 370 and 450 nm indicate a flavin-like photoreceptor. Blue light mediates positive phototropism while far-UV light mediates negative phototropism. To better understand the mode of interaction of far-UV with blue light we performed phototropism experiments in which sporangio-phores were placed for 4 h between sources of 280 and 454 nm light coming from opposite directions. The fluence rates of the far-UV were chosen such that unilateral light alone elicited 90° of negative bending. For blue light, moderate fluence rates were applied that elicited about 40° bending. Under conditions of bilateral irradiation the blue light substantially reduced the far-UV elicited phototropism. In the presence of tonic red light the antagonism between far-UV and blue light was greatly reduced. Red light, which by itself is phototropically ineffective, also reduced phototropic bending elicited by either far-UV or blue light. These observations are taken as indications for the existence of a red light-absorbing intermediate of the blue-light receptor. Because the far-UV/ blue-light antagonism disappeared almost completely in the presence of tonic red light, the antagonism may occur at the level of this receptor intermediate.     

Light Regulation of Phytochrome Content in Wild-type and Aphototropic Mutants of the Moss Ceratodon purpureus

In filaments of the moss Ceratodon purpureus , phototropism is controlled by the photoreceptor phytochrome. Thirty-three aphototropic mutants with a proposed defect in phytochrome-chromophore biosynthesis were isolated and analyzed. The phototropic response of those mutants was rescued with the precursor of the phytochrome chromophore, biliverdin. Phytochrome spectral activity was measured in 19 arbitrarily chosen mutants. All contained low but still measurable quantities of photoactive phytochrome; the highest level was around 15% of the wild-type. The level of total phytochrome (apophytochrome and holophytochrome) as assayed by immunoblotting was indistinguishable from wild-type. The content of photoactive phytochrome in Ceratodon is light-regulated. Phytochrome of wild-type kept for 24 h in red light was reduced to 50% as compared to dark controls but was unaffected by blue. The red-light-induced decrease was partially reversible by far-red light, indicating that phytochrome itself is the photoreceptor for this response. This regulation was further analyzed with the mutant ptr114 , which contains 15% photoactive phytochrome as compared to the wild-type. In this mutant, continuous red light given for 6 days decreased the level of spectrally active phytochrome down to 25% of dark controls, whereas the amount of phytochrome found on immunoblots was hardly reduced. This indicates that the loss of phytochrome affects only the holoprotein and implies that Ceratodon phytochrome is specifically degraded as a far-red-absorbing phytochrome.     

INDEPENDENT EFFECTS OF PHYTOCHROME AND NITRATE ON NITRATE REDUCTASE AND NITRITE REDUCTASE ACTIVITIES IN MAIZE

Abstract— Involvement of phytochrome in the regulation of nitrate reductase (NR) and nitrite reductase (NIR) activities in excised, etiolated leaves of Zea mays (L.) variety 'Ganga-5' is demonstrated using low energy and high irradiance responses of phytochrome action. Photoreversibility by far-red light of red light stimulated increases in NR and NIR activities was lost by 2 h. Red light given to the leaves, when induction by NO^-₃, was saturated, further increased both enzyme activities. Even if red light was given 4–8 h before NO^-₃, it still increased both NR and NIR activities.     

ANALYSIS OF LIGHT-CONTROLLED ANTHOCYANIN FORMATION IN COLEOPTILES OF Zea mays L.: THE ROLE OF UV-B, BLUE, RED AND FAR-RED LIGHT

Abstract— Light-induced anthocyanin formation in Zea mays L. coleoptiles was investigated in seven different varieties of this species. Under the test conditions, four varieties showed practically no response to any waveband used (UV, continuous red and continuous far-red), two responded strongly to both UV and far-red, and one showed a strong response only to far-red. The radiation-sensitive varieties showed, however, only a very weak response to continuous red light. In those varieties sensitive to far-red light, a pretreatment with continuous red light led to a greatly enhanced response to UV or in one case the manifestation of a response to UV that was previously lacking. Further investigations in one radiation-sensitive variety (INRA) showed that the UV response was to UV-B radiation below 350 nm. The UV response, as well as the far-red and blue responses in this variety, showed fluence-rate dependency. Red light was almost ineffective and showed only a very weak fluence-rate dependency.     

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.     

APPEARANCE OF PHOTOPHOSPHORYLATION CAPACITY: THRESHOLD vs GRADED CONTROL BY PHYTOCHROME

Abstract— It is shown that in attached mustard cotyledons graded control of chlorophyll synthesis by physiologically active phytochrome (P_fr) and threshold control by P_fr of the 'potential capacity' to photophosphorylate are totally different phytochrome actions even though both controls are essential for the build-up of the same functional complex, the machinery for photophosphorylation. The essential findings are as follows: The action of P_fr (made by a 1 min red light pulse) on the capacity and efficiency of photophosphorylation is rapid—detectable after 15 min and completed after 30 min—whereas the action of P_fr on chlorophyll formation is slower—only detectable 45 min after the original red light pulse (R). Detailed escape studies (loss of full reversibility of the inductive effect of a R pulse by far-red) show that the effect of a R pulse on chlorophyll synthesis remains fully reversible for 45 min whereas the action of P_fr on the capacity for photophosphorylation is very fast (occurring within 2 min). Control of capacity for photophosphorylation is a threshold response (whereby the threshold value is approximately 1.25% P_fr based on total phytochrome at 36 h = 100%) whereas control by P_fr of chlorophyll synthesis is graded. Control of capacity for photophosphorylation by P_fr only operates if the hypocotyl hook is connected to the cotyledons for at least 2 min after the inductive R pulse, i.e. until full escape from reversibility has occurred, whereas chlorophyll formation in the cotyledons is not affected by the separation of hook and cotyledons.     

INTERPRETATION OF THE 'DICHROIC ORIENTATION' OF PHYTOCHROME

The dichroic orientation of phytochrome observed both in the phytochrome-mediated phototropism in Adiantum protonemata and in the phytochrome-mediated chloroplast movement in Mougeotia were analyzed in terms of the orientation of the transition moment associated with the long-wavelength absorption band, assuming that phytochrome, associated with the plasma membrane, rotates around the normal to the membrane. The orientation of the long-wavelength transition moment of the phytochrome chromophore was calculated using the zero-differential overlap approximation of the molecular orbital theory for ir-electrons. The results indicate that the orientation of the long-wavelength transition moment mainly changes later than 2 ms after red light excitation of P_r, and that the different dichroic orientations of P_r and P_fr can be attributed to the change in the angle of the long-wavelength transition moment of phytochrome with the plasma membrane from 18^o to 72^o during phototransformation.