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.     

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.     

BLUE LIGHT INDUCED PHOTOTRANSFORMATION OF PHYTOCHROME IN THE PRESENCE OF FLAVIN*

Abstract— Irradiation of the P_r form of phytochrome in the presence of flavin mononucleotide (FMN) which absorbs the actinic blue light yields P_fr at a rate greater than that in the absence of FMN. The actinic blue light absorbed by FMN enhances the phototransformation of P_r via the energy transfer from the former to the latter. On the other hand, the photoreversion of P_fr was inhibited by the presence of FMN when illuminated with blue light. The lack of photo-enhancement of the reversion of P_r, by blue light suggests that the P_fr chromophore (acceptor) transition dipole is virtually perpendicular to the FMN transition dipole, as the result of a chromophore reorientation in the P_r→P_fr phototransformation. The fact that blue light absorbed by flavin preferentially enhances the forward phototransformation of phytochrome while inhibiting the reversion may have an important implication in the high irradiance responses in plants in terms of a preferential accumulation of P_fr by blue light excitation.     

THE DOSE RESPONSE CURVE IN PHYTOCHROME-MEDIATED ANTHOCYANIN SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —The dose response curve for light (phytochrome)-induced anthocyanin synthesis was determined in the mustard seedling. The curve gives the amount of anthocyanin (A) synthesized within 24 h as a function of the amount of P_fr* produced by a brief light pulse. The [P_fr] response curve is composed of two linear parts with very different slopes ( a _1,2) connected by a relatively narrow transient range (curved segment). The [P_fr] response curve extrapolates precisely through zero [P_fr]. The reciprocity law is valid over the whole range investigated (up to 320 s of irradiation). It is concluded that the initial (or primary) reaction of P_fr (P_fr+ X → P_frX) does not involve any significant cooperativity in the case of phytochrome-mediated anthocyanin synthesis. It is speculated that the linear parts of the [P_fr] response curve truly reflect the mode of phytochrome action ( A = a _1,2 [P_fr]; X does not come into play since it is not rate limiting) whereas the curved segment represents a transition of the reaction matrix of P_fr. The large difference between a₁ and a₂ seems to indicate that the physiological effectiveness of a given amount of P_fr (or P_frX) is determined by [P_fr] through a P_fr-induced change in the reaction matrix.     

DEPENDENCE OF Pfr/Ptot-RATIOS ON LIGHT QUALITY and LIGHT QUANTITY

Abstract— Not only the spectral distribution of the light source determines the relative proportion of phytochrome in the P_fr(P_r) form, the P_fr/P_tot-ratio also depends strongly on the fluence rate of the irradiation. This dependence has been observed in the cotyledons of etiolated mustard seedlings for blue light of fluence rates below 20 Wm_-2. It has also been observed for white light and seems to be a characteristic of the phytochrome system resulting from the involvement of phytochrome thermal reactions as well as P_r P_fr photoconversions. The fluence rate dependence of P_fr/P_tot-ratios can be used to analyze the characteristic transformations of the phytochrome system. Phototransformations together with a fast thermal transformation (τ_½⋍ 3min) are consistent with the results obtained for blue and white light.     

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.     

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.     

VARIATION IN THE RATES OF SYNTHESIS AND DEGRADATION OF PHYTOCHROME IN COTYLEDONS OF CUCURBITA PEPO L. DURING SEEDLING DEVELOPMENT

Abstract— The time courses for P_r appearance, P_r disappearance and P_fr destruction have been analysed in cotyledons of Cucurbita pepo L. after different preirradiation programs. In etiolated seedlings the rate of P_r appearance is low in young seedlings reaching a maximum in 3.5–5 day old seedlings then decreasing rapidly with increasing age. The rate of P_fr destruction is very low in young seedlings, increases rapidly up to the 4th day and then remains almost constant. The disappearance of P_r becomes significant for seedlings older than 45 days. These reactions seem not to be influenced by short preirradiations. However, after prolonged preirradiation, a degree of control of P, appearance and/or disappearance by the "internal clock appears to be operative.     

PHYTOCHROME INTERMEDIATES IN VIVO–II. CHARACTERISATION OF INTERMEDIATES BY DIFFERENCE SPECTROPHOTOMETRY

Abstract— In epicotyl tissue of Pisum , irradiation of P_r at – 196°C forms a stable product P₆₉₈, whereas P_fr forms a stable product P₆₅₀. On warming P₆₉₈, dark transformation to P_r predominates. On warming P₆₅₀ to – 70°C an intermediate P₆₉₀ is formed which bleaches on further warming to –10°C. When tissue is cooled to –196°C during actinic irradiation, difference spectra for subsequent warming to –10°C indicate that P_r, P_fr and an intermediate P₇₁₀ are formed from weakly absorbing intermediates. Complete photoconversion of P_r to P_fr is not possible at temperatures below –5°C. As the temperature is reduced, the amount of P_fr produced from P_r decreases, while P₇₁₀ increases. P₇₁₀ can be photoconverted at –20°C and above, ultimately forming P_r, but in contrast to P_fr it is not photoconvertible at –196°C.     

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.     

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).     

EVIDENCE FOR A SEQUENTIAL PATHWAY FROM Pr TO Pfr OF THE PHOTOTRANSFORMATION OF 124-kDa OAT PHYTOCHROME

Abstract. Phototransformation kinetics of 124-kDa oat phytochrome at 298 K after a red (660-nm) laser flash excitation were recorded at different wavelengths. The kinetics of the dark relaxation processes for lumi-R to P_fr can be satisfactorily described by only 3 rate constants: k = 28000 s^-1 370 s^-1 and 20 s^-1. The first rate constant is due to the decay of lumi-R to meta -Ra. The latter two rate constants correspond to processes establishing the far-red (>700 nm) absorption band. No meta -Rb could be detected. From the wavelength dependency of the amplitudes of these two rates, parallel pathways in the formation of P_fr could be excluded. A unique sequential pathway for the dark relaxation leading to P_fr seems to be an intrinsic property of 124-kDa phytochrome, however. Assuming a sequential pathway, molar extinction coefficients for intermediates have been calculated. These values agree with molar extinction coefficients obtained from low-temperature spectra. The process with a rate constant of 370 s^-1 corresponds to absorbance changes for the formation of meta -Rc from meta -Ra and the rate constant of 20 s^-1 describes the absorbance changes due to the transformation of meta -Rc to P_fr.     

RELATIONSHIP BETWEEN PHYTOCHROME PHOTOCONVERSION and RESPONSE

Abstract— The action of phytochrome on appearance of NADH-dependent glutamate synthase (NADH-GOGAT) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) was studied in the cotyledons of the mustard ( Sinapis alba L.) seedling. It was found that the [P_fr]-response curves are composed of two branches with two very different slopes (a₁ a₂). This explains the biphasic fiuence response curves reported previously. While a, is the same with both enzymes, a, is much higher in NADH-GOGAT than in Fd-GOGAT. However, the transition from the high (a₁) to the low (a₁) slope occurs at the same P_fr/P_tot ratio irrespective of the steepness of a₁ While the appearance of NADH-GOGAT is very sensitive to small amounts of P_rr (high a₁), the response is not sensitive to the "High Irradiance Reaction" (HIR) of phytochrome. On the other hand, appearance of Fd-GOGAT (relatively low a₁) exhibits the usual HIR. It is concluded that the presently available models of phytochrome action, including the dimeric model, are not adequate to account for the actual data. At present it appears that a multiplicity of primary actions of phytochrome exist, and that it is the availability of the primary reactant (X, _1,2,3) which determines transduction or non-transduction of the phytochrome signal.     

SIGNIFICANCE OF FLUENCE-RESPONSE DATA IN PHYTOCHROME-INITIATED SEED GERMINATION

Abstract. A model was developed to describe changes in fluence-response kinetics in terms of total phytochrome level (P_tot), level of the factor X interacting with active phytochrome (P_fr), P_frX equilibrium constant, seed sensitivity to P_fr-X interaction, and variation in phytochrome sensitivity within the seed population. Under conditions of stable X levels and stable population variation, the model predicted that a change in any of the other components will result in a parallel fluence-response curve on a probit-logarithmic plot. The linearity of the subsaturation plot is dependent on the ratio of P_tot to X concentrations. The model showed that changes in threshold response fluences can result from many causes other than changes in total phytochrome [P_tot>]. Changes in response-saturating fluences when maximal germination is less than 100% are predicted to be due to limiting levels of X. Changes in slope of fluence-response curves can be explained by changes in seed population variation by this model. Rumex crispus L. fluence-response data for germination is best explained by this model in terms of neither changes in P_tot nor X levels altering kinetics.     

MODE OF COACTION BETWEEN UV-A AND LIGHT ABSORBED BY PHYTOCHROME IN CONTROL OF APPEARANCE OF RIBULOSE-1.5-BISPHOSPHATE CARBOXYLASE IN THE SHOOT OF MILO (Sorghum vulgare Pers.)

Abstract— In shoots of milo ( Sorghum vulgare Pers.) appearance of ribulosebisphosphate carboxylase (RuBPCase) and of translatable mRNA for its small subunit is stimulated strongly by red light (R, operating through phytochrome) and UV-A light (UV-A). Ultraviolet-A is more effective than R.
The mode of coaction between phytochrome and light absorbed by the blue/UV-A light photoreceptor ('cryptochrome') was analyzed in detail in case of enzyme appearance. Fluence rate dependencies, lagphases and the time course of the response are compatible with the view that UV-A intensifies a process which is occurring in R alone albeit at a lower rate.
With both light qualities the light effect is fully reversible by far-red light up to 1 h. This means that during this period only phytochrome (P_fr) controls the terminal response, i.e. the actual appearance of RuBPCase. During this 1 h period after the onset of light UV-A or R have no effect on the level of translatable mRNA for the small subunit of RuBPCase indicating that it requires more than 1 h for the light signal to affect gene expression.
When R and UV-A are given longer onset of escape from full reversibility is observed at the same time for both light qualities in the case of RuBPCase appearance. The extent of the reversible response is greater after UV-A pretreatment than after a R pretreatment.
It is argued that the data are consistent with the concept that phytochrome (P_fr) controls the terminal photoresponse, in the present case appearance of RuBPCase, while light absorbed via cryptochrome leads to an increase in responsiveness of the RuBPCase producing machinery towards P_fr.     

A PHYTOCHROME STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: I700 IS A MANDATORY INTERMEDIATE IN THE PrPfr PHOTOTRANSFORMATION

Abstract— The phototransformation of native (124 kDa)oat phytochrome, P_r P_fr, Has been studied at 10C by two laser/ two-color flash photolysis. the overall P_rP_fr reaction yield did not vary with temperature within the range4–21C. Foloeing the excitation of P_r with a single 15 ns laser flash at 650nm, the formation of P_fr was quantitavely measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. the second laser flash causes at 1.0 s after the initial laser flash a depletion of the uintermediate I₇₀₀ as welll as a reduction of the P_fr absorption at 730 nm. The depletion of I₇₀₀ correlates quantitavely with the reduction of P_fr formation. The absorpton spectra of I₇₀₀ and of the following intermendiate, I_bi, were calculated assuming that the amount of P_r, which is photoconverted by a single laser, equals the amount of P_fr formed.     

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.     

PHYTOCHROME DESTRUCTION IN DARK - AND LIGHT-GROWN AMARANTHUS CAUDATUS SEEDLINGS

Abstract— Slow destruction of the far-red-absorbing form of phytochrome (P_fr), which has been observed in light-grown oat and maize, occurs in light- and dark-grown Amaranthus, Pharbitis , and Brassica seedlings as well. Destruction of P_fr in these seedlings shows two phases: if a high level of P_fr is produced in dark-grown seedlings, the destruction is fast in the beginning and then slows after a low P_fr level has been reached. Slow P_fr destruction is predominant in light-grown tissue.     

MODELS FOR THE CHROMOPHORE STRUCTURE OF THE PHOTOTRANSFORMATION INTERMEDIATES OF PEA PHYTOCHROME IN VITRO

Abstract— The changes in the chromophore structure of pea phytochrome during phototransformation in vitro from the red-light-absorbing form (P_r) to the far-red-light-absorbing form (P_fr), and from P_fr to P_r, were analysed in terms of wavelength and oscillator strength of absorption, using the zero-differential overlap approximation of the molecular orbital theory for electrons. The effect of a point-charge and a point-dipole on the optical absorption of phytochromobilin intermediates were examined using the stationary perturbation theory for degenerate states. The results indicate that the cis-trans photoisomerization of the pyrrole ring D, if any, occurred within 10 μs after a laser-flash excitation of the phytochrome, and that the conformations of phytochromobilin and the protein moiety of phytochrome were not significantly changed during the period of examination of phototransformation in either direction.     

THE TRANSFORMATION OF PHYTOCHROME TO ITS PHYSIOLOGICALLY ACTIVE FORM

Abstract— The phototransformation of phytochrome ( P_r ) to its physiologically active form (P_fr) can be best described by a tautomeric proton shift in ring A without a substantial con formational change of the chromophore. The electronic aspect of this transformation is discussed.