THE IN VIVO SPECTROPHOTOMETRIC ASSAY OF PHYTOCHROME IN TWO MATURE DICOTYLEDONOUS PLANTS*

Abstract— Mature Sinapis alba L. and Impatiens parviflora DC. were treated with the herbicide norfiuorazon prior to development of the third or second leaf, respectively. This treatment yielded a partially bleached plant capable of normal growth and development. The bleached leaves were used for spectrophotometric phytochrome assay. In mature plants an almost constant level of phytochrome is maintained under continuous white light. The dark kinetics and the response of the phytochrome system to light of various qualities provide further evidence of the stable character of the phytochrome system.     

CHANGES IN THE RATES OF PHOTOCONVERSION OF PHYTOCHROME DURING ETIOLATION IN MUSTARD SEEDLINGS

Abstract— The relative phytochrome photoconversion rates in cotyledons and hypocotylar hook of etiolating mustard ( Sinapis alba L.) seedlings were measured between 16 and 96 h after sowing. It was found that at constant fluence rates photoconversion rate in red light increases in both organs with time whereas the photoconversion rate in far-red (756 nm) light decreases with time of development. Since the isosbestic point remains constant, it was concluded that the observed changes cannot be attributed to changes of extinction coefficients. It was not possible, however, to decide whether the observed changes are due to changes of light attenuation or quantum yields.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.     

PHOTORECEPTOR INTERACTION IN PLANT PHOTOMORPHOGENESIS: THE LIMITS OF EXPERIMENTAL TECHNIQUES AND THEIR INTERPRETATIONS

Abstract— Problems concerning the interpretation of interactions of higher plant photomorphogenetic receptors are discussed. The theory that action of a blue light photoreceptor serves only to maintain responsiveness to phytochrome (Responsiveness Theory) is demonstrated to be unable to be properly tested with present techniques. This theory is also unable to explain experimental results any better than an alternative theory that a blue light photoreceptor may require the presence of the active form of phytochrome to express its activity (Presence Theory). This tatter theory is also incapable of being fully tested. There does not appear to be an adequate current theory to explain photoreceptor interactions. Other issues discussed include the use of displacement transducers in growth studies, the induction of phytochrome-type responses by blue light, and the relative importance of the photoreceptors. New data are introduced on the effect of blue light in the end-of-day growth response to phytochrome of the light-grown Cucumis sativus L. hypocotyl, and on the light equivalence principle in the same species.     

PHOTOTRANSFORMATION KINETICS OF UNDEGRADED OAT AND PEA PHYTOCHROME INITIATED BY LASER FLASH EXCITATION OF THE RED-ABSORBING FORM

Abstract— Phototransformation at 2°C of the red-absorbing form of phytochrome (Pr) to the far-redabsorbing form (Pfr) was studied with both undegraded oat ( Avena sativa L., cv. Garry) and undergraded pea Pisum sativum L., cv. Alaska) phytochrome. Phototransformation was initiated by a 15-ns laser pulse with maximum emission near 600 nm and output power of 30 mJ. The first resolvable transformation intermediate exhibited relative to Pr a maximum absorbance increase near 700 nm and was fully present at the earliest time measured, which was 60 ns after the flash. This intermediate absorbance decayed by two reactions for oat phytochrome (half-lives of 11 and 140 μs assuming parallel reactions) and by three for pea phytochrome (half-lives of 14, 280 and 1600 μs assuming parallel reactions). The kinetics of the slowest reaction for pea phytochrome, however, might be somewhat distorted by an instrument artifact. The appearance of the far-red-absorbing phytochrome, as monitored by absorbance increase at 720 nm, occurred by at least two reactions for both oat (half-lives of 47 and 250 ms assuming parallel reactions) and pea (half-lives of 170 and 770 ms assuming parallel reactions) phytochrome. The possibility of slower reactions was not tested. Assays for possible proteolysis of the phytochrome samples studied here indicated that the presence of degraded phytochrome could not account for the observed multiphasic kinetics except possibly for one phase of the triphasic intermediate decay seen with pea phytochrome.     

STUDIES ON THE PROTEIN CONFORMATION OF PHYTOCHROME

Abstract— The extinction coefficients for large rye phytochrome were found to be: Fluorescence and circular dichroism spectra of large- and small-molecular-weight rye phytochrome give no evidence for a protein conformational change on phototransformation of phytochrome. The large molecule has a fluorescence emission peak at 331 nm when excited at 290 nm, and an excitation peak for this emission at 288 nm. The circular dichroism spectra indicate that large rye phytochrome has about 17–20% a-helix content, 30%β-structure and 50% random coil, and that the small rye phytochrome has about 10–13%α-helix content. The ultraviolet difference spectra for large and small rye phytochrome are similar and differ from the difference spectrum of the small oat phytochrome in the relative size of the 296–298 nm peak. The difference spectra may reflect changes in chromophore absorbance and in the environment of amino acid residues near the chromophore, particularly of tyrosine, and perhaps of tryptophan and cysteine.     

PHYTOCHROME and THE DEVELOPMENT OF PHOTOPHOSPHORYLATION CAPACITY—AN APPRAISAL OF THE EXPERIMENTAL APPROACH*

Abstract— Development of the capacity for photophosphorylation (= total capacity for light-driven ATP formation) in the mustard ( Sinapis alba L.) cotyledons is strongly influenced by a red light pulse pretreatment which operates through phytochrome. The present report deals with several objections raised against the in situ assay of the rate of photophosphorylation. Experimental evidence is given in support of the assumption that the linear increase of the ATP content of the cotyledons as measured over 1.5 min after the onset of saturating white light (370 Wm^-2) in fact represents the maximum rate of photophosphorylation ('capacity'). Moreover, it is confirmed that control by phytochrome of the development of the photophosphorylation capacity and of the capacity for chlorophyll synthesis are unrelated phenomena. The failure of development of the capacity for photophosphorylation in isolated cotyledons from dark-grown seedlings cannot be attributed to deficiencies of chlorophyll synthesis.
It is concluded that the photophosphorylation response is particularly useful to study the mechanism of phytochrome (P_fr) action in case of a response which involves a threshold reaction and an interorgan (hook→cotyledon) cooperation.     

RESPONSE OF LIGHT-GROWN WILD-TYPE and LONG HYPOCOTYL MUTANT CUCUMBER PLANTS TO END-OF-DAY FAR-RED LIGHT

Abstract— A long-hypocotyl mutant ( lh ) of cucumber ( Cucumis sativus L.) has been studied which has previously been shown to lack phytochrome control of growth in de-etiolated seedlings and thought to be modified with respect to the light-stable type of phytochrome. We have analyzed the response of lh mutant and isogenic wild-type (WT) plants to daily treatment with end-of-day far-red light (EODFR). Only the WT responded to this treatment resulting in a large increase in internode length; an increase in petiole length; changes in leaf development (increased area, decreased thickness and reduction in indentation); redistribution of dry matter from leaf blades to stem; increased apical dominance and promotion of tendril formation. There were only small or no significant effects on chlorophyll and total carotenoid content, chlorophyll alb ratio, soluble protein levels and net photosyn-thetic rates. The lh mutant failed to respond to EODFR treatment, and had the appearance of a shade-avoiding plant growing in extreme shade. The lh mutant appears to completely lack the phytochrome responses attributable to the type of phytochrome that is active in shade detection. A discussion of the possible roles of the stable and labile types of phytochrome in light grown plants follows.     

THE CONTROL BY PHYTOCHROME OF NITRATE REDUCTASE IN THE CURD OF LIGHT-GROWN CAULIFLOWER

Abstract— The activity of nitrate reductase from the curd of light-grown cauliflower ( Brassica oleracea (L) var botrytis (DC) 'St. Hilary') is modulated by nitrate and by light. Using broad-band sources of equal photosynthetically active radiation but with different proportions of red and far-red light, a linear relationship between nitrate reductase activity and ψ(Estimated phytochrome photoequilibrium) was obtained. This relationship, apparent after 8 h incubation, was maintained and little altered after 48 h incubation. The linearity was apparent between ψE 0.26 and ψE 0.69; ψE 0.26 being no more effective than a dark control. Far-red reversibility confirmed the involvement of phytochrome. Brief pulses of red light were also used to establish a range of phytochrome photoequilibria within the tissue. Again a linear relationship between ψ and nitrate reductase activity was obtained with a threshold for the response at ψ 0.3. With both monochromatic and broad-band sources it was seen that neither photon fluence rate nor duration of exposure affected the final activity of the enzyme and that phytochrome was acting solely through ψ (or [Pfr] since phytochrome is stable in this tissue) to bring about these responses.     

IN SlTU ESTIMATION OF SPATIAL DISTRIBUTION OF PHYTOCHROME ALONG THE AXIS OF THE ACTINIC BEAM

Non-stationary photoconversions of the two forms of the photochromic sensory plant pigment phytochrome are theoretically analyzed. It is shown that from actual in situ measurements of changes of absorbances caused by these photoconversions in a sequence of exposures to actinic light, one can calculate the spatial distribution of phytochrome along the path of the actinic beam. The necessary condition of such a calculation is the existence of a screen with a known spatial distribution, which must be obtained in an independent experiment. Thus, the possibility is proved to measure, instead of phytochrome spatial distribution, that of the screen which is a much larger chemical and optical body inside the object. Expressions to calculate the spatial distribution of phytochrome and to estimate the errors involved are presented in the present paper.     

PHYTOCHROME REGULATION OF STEM GROWTH AND INDOLE-3-ACETIC ACID LEVELS IN THE lv AND Lv GENOTYPES OF Pisum

Phytochrome influences stem elongation and the mechanism for this is not understood. The levels of indole-3-acetic acid (IAA) were analyzed in an leLv genotype of Pisum sativum L. which responded to end-of-day far-red light by doubling growth rate. The IAA levels in epidermal peels increased 40% after far-red light whereas IAA levels of the entire stem tissue changed insignificantly. This increase was reversible by red light. Under light-grown conditions, the lv mutation increases stem elongation rates by 2–3-fold and is thought to block the transduction of a phytochrome signal. Analysis of the short-term stem elongation kinetics of dark- and light-grown Lv and lv seedlings suggests that lv blocks the action of the light-stable form of phytochrome. The higher growth rate of lv plants was found to be associated with abnormally high epidermal IAA levels typical of far-red treated Lv plants. End-of-day far-red treatments did not substantially increase epidermal IAA levels in lv plants. These observations support the view that phytochrome regulation of stem elongation may occur in part through modulation of epidermal IAA levels. The lv mutation may result in increased internode growth in part by blocking the ability of phytochrome to decrease epidermal IAA levels.     

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.     

PHOTOCONTROL OF PHYTOCHROME DESTRUCTION AND BINDING IN DICOTYLEDONOUS vs MONOCOTYLE-DONOUS SEEDLINGS. THE INFLUENCE OF WAVELENGTH AND IRRADIANCE

Abstract— The irradiance and wavelength dependence of phytochrome destruction in vivo was analysed in etiolated cotyledons of Cucurbita pepo L. and etiolated seedlings of Amaranthus caudatus L. In contrast to grass seedlings, the rate of P _tot destruction could only be saturated by light sources that establish relatively high P _fr levels (about 50% of total phytochrome, corresponding to the photostationary state established by 693 nm light). To explain the irradiance dependence of P _tot destruction in dicots at irradiances above 0.1 Wm^-2, where the light reaction is at least one order of magnitude faster than P _fr destruction, we suggest there is a fast intercalary dark reaction between photoreaction and destruction. This dark reaction is probably—as in grass seedlings—the binding of P _fr to a receptor site. We conclude that the differences between dicots and grass seedlings with respect to the phytochrome system are of a quantitative rather than a qualitative nature.     

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.     

APPARENT INCREASE IN PHOTODETECTABLE PHYTOCHROME IN THE PRESENCE OF CALCIUM CARBONATE*

Abstract— –Using squash seedling extracts ( Cucurbita pepo L.), we describe an apparent increase in photodetectable phytochrome content that is depended upon the use of either CaCO₃ or starch as a scattering agent and is also a function of preirradiation with red light of either intact tissue or crude extracts prior to sample preparation for spectrophotometric assay. This apparent increase in photodetectable phytochrome content requires about 1 h for full expression after addition of the scattering agent. If irradiations are given in vitro , the increase is partially reversed by far red light when using squash extracts, and is fully reversed with Aoena and Zea extracts. For squash extracts the magnitude of this increase. which is typically between 20 and 40%. is quantitatively correlated with both the amount of Pfr produced by a brief red irradiation and with the proportion of the total phytochrome pool that is pelletable upon centrifugation at 20,000 g . The correlation with pelletable phytochrome does not hold for Auena and Zea , at least when irradiations are gwen in uitro . The increase in photodetectable phytochrome may result from changed phytochrome extinction, changed phototransformation quantum yields, and/or specific trapping of phytochrome by the scattering agent as it settles. An important consequence of these data is that they indicate a need for caution when using a scattering agent during spectrophotometric assay of phytochrome.     

PHYTOCHROME ACTION AT HIGH PHOTON FLUENCE RATES: RAPID EXTENSION RATE RESPONSES OF LIGHT-GROWN MUSTARD TO VARIATIONS INFLUENCE RATE and RED: FAR-RED RATIO*

Abstract— Extension growth rate of light-grown mustard (Sinapis alba L.) seedlings was monitored continuously using a sensitive linear displacement transducer system. When high fluence rates (ca 2 mmol m^?2 s^_1) of mixed red and far-red light were presented to the growing internodes from fibre optic probes, fluctuations in extension rate occurred during the first 30 min. High red: far-red ratios (R: FR) caused growth deceleration, whilst low R: FR caused transitory growth acceleration. These changes in extension rate were not exactly as predicted from the proportions of Pr (the red-absorbing form of phytochrome) and Pfr (the far-red absorbing form of phytochrome) calculated to be established by the light sources. Nevertheless, the data demonstrate that phytochrome is able to control extension growth at fiuence rates approaching those of summer sunlight, thereby providing the capacity to sense the presence of neighbouring vegetation before shading seriously compromises photosynthesis. Varying fiuence rate over two orders of magnitude whilst maintaining R: FR constant evoked transient fluctuations in extension rate. At high R: FR, a 100-fold step down in fiuence rate led, after a lag of ca 10 min, to a transient (i.e. 20 min) deceleration of extension that was followed by a marked transient (i.e. 20 min) acceleration. After a 100-fold step up in fiuence rate, a transient (i.e. 20 min) acceleration only was observed, beginning after a lag of ca 10 min. When R: FR was low, neither a step-down nor a step-up in fluence rate resulted in appreciable fluctuations in extension rate. The data are discussed in relation to the possible role played by the accumulation of photoconversion intermediates using a simple computer model for simulating active phytochrome concentrations at high fluence rates. The possibility that the mechanism for the photoperception of light quality by phytochrome may be capable of rapid adaptation to fluence rate fluctuations is proposed.     

THE TEMPERATURE DEPENDENCE OF Pfr ACTION GOVERNS THE UPPER TEMPERATURE LIMIT FOR GERMINATION IN LETTUCE

In most cultivars of lettuce (Lactuca saliva L,), red light acting through the red/far-red reversible phytochrome system promotes full germination within the20–30°C range, but at progressively higher temperatures germination declines sharply. The relationship between this upper ternperature limit for germination and the temperature dependence of phytochrome action was investigated in Grand Rapids lettuce. In fresh seeds the GT₅₀ (temperature giving half maximal germination) was ca 29–30°C. In these seeds, escape from far-red reversibility did not occur at 35°C, a temperature above the GT₅₀, but occurred rapidly at 27°C, a temperature below the upper limit. Increasing periods of dark pretreatment at high temperature (35°C) or increasing concentrations of the germination inhibitor coumarin caused a progressive decline in the GT₅₀, Escape from photoreversibility did not occur at 27°C in seeds in which the GT₅₀ had been reduced to less than 25°C by coumarin or by prolonged high temperature pretreatment. These results indicate that there is a close correlation between the position of the upper temperature limit for germination, and the temperature dependence of phytochrome action. We conclude that factors that alter the upper temperature limit for germination do so by changing the temperature dependence of phytochrome action.     

PHYTOCHROME CONTROL OF EXTRACELLULAR PEROXIDASE ACTIVITY IN MUSTARD INTERNODES: CORRELATION WITH GROWTH, and COMPARISON WITH THE EFFECT OF WOUNDING

Abstract— The effects of phytochrome status on extracellular peroxidase activity were investigated in Sinapis alba L. seedlings grown for 12 days under continuous white light and transferred to darkness after a red light or a far-red light pulse. The rates of extension growth and dry matter accumulation in the first internode were increased by the far-red light pulse. Extracellular proteins, obtained by low speed centrifugation of intact internodes infiltrated with CaCl₂, were separated by isoelectrofocusing, and four extracellular acidic peroxidases were resolved, the most active being A3 and A4 (both ˜60 kD). The activity of A4 was reduced by the far-red light pulse perceived by phytochrome, while the activity of A3 was unaffected. The promotion of internode extension growth caused by far-red light is biphasic [Casal and Smith (1989) Plant, Cell Environ. 12 ,511–50]. Changes in peroxidase activity were detected prior to the second, but not to the first phase of the internode growth promotion. The effects on both growth and peroxidase activity were virtually restricted to the upper half of the internode and, once established, did not subsequently increase in magnitude.
In contrast to the effects mediated by phytochrome, blue light pretreatments affected growth but not extracellular peroxidase activity. Wounding the internode reduced extension growth, increased the activity of A3, but caused no significant effects on A4.
Other extracellular proteins, separated in sodium dodecyl sulphate polyacrylamide gels and stained with Coomassie blue, showed no significant differences. The concentration of extracellular proteins was higher in the upper than the lower half of the internode.
Results are discussed in terms of phytochrome effects in light grown plants, peroxidase activitv-arowth relationships, and extracellular peroxidase isoform functions.     

ANALYSIS OF PHOTOTRANSFORMATION INTERMEDIATES IN THE PATHWAY FROM THE RED-ABSORBING TO THE FAR-RED-ABSORBING FORM OF AVENA PHYTOCHROME BY A MULTICHANNEL TRANSIENT SPECTRUM ANALYZER

Phototransformation of the red-absorbing form of phytochrome (Pr) to the far-red-absorbing form (Pfr) was followed with a custom-built transient spectrum analyzer. Large phytochrome, which consisted of approximately 120000-dalton monomers, was immunopurified or conventionally purified from etiolated oat (Avena sativa L., cv. Garry) shoots. Phototransformation was initiated by exciting Pr with a 115-mJ, 600-ns half-width, 655-nm laser pulse. Absorption spectra were recorded on a microsecond time scale at predetermined times after the flash. It has been reported earlier that flash excitation of large oat Pr produces a transformation intermediate with maximum absorbance near 700 nm in a difference spectrum and that this intermediate decays by two kinetically distinct reactions. Difference spectra for these two reactions are indistinguishable. Both show bleaching centered at 690 nm with no detectable associated absorbance increase between 570 and 830 nm. Subsequent appearance of absorbance at 724 nm, which presumably but not necessarily represents the appearance of Pfr, had earlier been shown to occur by two kinetically distinct reactions for large oat phytochrome. Data presented here indicate in addition the occurrence of a third, slower reaction. Difference spectra for the two faster reactions are indistinguishable, both with maxima near 728 nm and minima near 650 nm. The difference spectrum for the slowest component, however, was qualitatively different exhibiting a maximum near 722 nm with no corresponding minimum. About 15-20% of the absorbance increase at 724 nm occurred by this slowest reaction, which exhibited a half-life of 3 s at 25°C and a Q₁₀ of 1.2 for immunopurified and 1.5 for conventionally purified phytochrome. The percentage occurring by this reaction was independent of temperature over the range studied (1-25dEC). For immunopurified phytochrome the enthalpy of activation, Gibbs free energy of activation, and entropy of activation of this slowest reaction were found to be about lOkJ-mol^-1, 75kJ.mol^-1, and -220 J.mol^-1 K^-1, respectively, and for conventionally purified phytochrome 25kJ.mol^-1, 75kJ.mol^-1and —170 J.mol^-1 K^-1, respectively. The thermodynamic characteristics of this reaction indicate that it may involve a significant ordering of the protein moiety as it transforms to Pfr.     

THE ''HIGH-IRRADIANCE RESPONSE'' IN ANTHOCYANIN FORMATION AS RELATED TO THE PHYTOCHROME LEVEL

Abstract— The involvement of phytochrome in light-mediated anthocyanin synthesis in the mustard seedling ( Sinapis alba L.) under inductive conditions (law of reciprocity valid) was shown previously (Drumm and Mohr, 1974). In the present paper the hypothesis (Hartmann, 1966) is checked that light-mediated anthocyanin synthesis in continuous high-irradiance far-red light ('high-irradiance response') is also due exclusively to phytochrome. The data indicate that the effectiveness of the far-red light is indeed a function of total phytochrome [ P_total ]* and therewith [ P_fr ]*. The data are not consistent with the suggestion (Schneider and Stimson, 1972) that photosynthesis (in particular, photosystem I) is involved in the 'high-irradiance response' of photomorphogenesis.