QUANTITATIVE CORRELATION BETWEEN SPECTROPHOTOMETRIC PHYTOCHROME ASSAY AND PHYSIOLOGICAL RESPONSE

Abstract— Apparent synthesis* of the enzyme lipoxygenase in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_{fr ground state})? through a threshold (all-or-none) mechanism. This response was used to determine physiologically the photostationary states, Λ that is, the [P_fr]/[P_tot] ratios established by different wavelengths in the red and far-red range of the spectrum, including the standard red and far-red sources used in this laboratory (Mohr, 1966). Under the premises (for which justification has been given on previous occasions) that the [P_fr]/[P_tot] ratio for standard red light is 0.8, and that the decay of P_fr is a first-order process with a half-life of 45 min, the [P_fr]/[P_tot] ratios determined physiologically by means of the lipoxygenase response agree with the [P_fr]/[P_tot] ratios determined spectrophotometrically by Hartmann and Spruit (cf. Fig. 9 in Hanke et al., 1969) in hypocotyl hooks of mustard seedlings. In the hook the _fr, that is, the [P_fr]/[P_tot] ratio for standard far-red, is found to be 0.023. In the cotyledons, this ratio is several times higher (Schafer et al., 1972). The conclusion that apparent lipoxygenase synthesis in the cotyledons is controlled by phytochrome located in the hook has been substantiated by further spectrophotometric (Schäfer et al., 1973) and physiological experiments (H. Oelze-Karow and H. Mohr, in preparation). The minimum steepness of the threshold was determined. An increase of the P_fr level from 118 (relative units) to 130 leads to an instantaneous and total suppression of apparent lipoxygenase synthesis; a corresponding decrease from 130 (relative units) to 118 leads to an immediate resumption of apparent LOG synthesis at full speed. It is concluded that an explanation of the experimental facts requires a cooperative effect on the level of P_fr, a high degree of synchrony on the cellular and organismic level and rapid communication between the hypocotyl hook and the cotyledons. *The term ‘apparent synthesis’ is used operationally in the present paper to denote any increase of enzyme activity, although de novo synthesis of lipoxygenase has not so far been rigorously demonstrated. The usual inhibitor experiments (cf. Oelze-Karow et al., 1970) have led to the conclusion that intact RNA and protein synthesis is required for an increase of lipoxygenase activity.     

DIRECT MEASUREMENT OF PHYTOCHROME PHOTOCONVERSION INTERMEDIATES AT HIGH PHOTON FLUENCE RATES

A custom-built modulated split-beam spectrophotometer has been used to measure the absorbance of tissue samples and purified phytochrome whilst exposing the sample to actinic 633 nm laser radiation at fluence rates approaching those of daylight. This approach has allowed the direct observation of the accumulation of phytochrome photoconversion intermediates at high fluence rates. At ca 1250 μmol m^?2 s^?1 upwards of 35% of the total phytochrome was present in the form of photoconversion intermediates in tissues of maize, sunflower and tomato. In other tissues tested (wheat, bean and Amaranthus) and in purified oat phytochrome, rather smaller levels of intermediates accumulated. Upon “lights-off” only a proportion of the accumulated intermediates decayed to far-red absorbing phytochrome (Pfr), the remainder appearing as the red-absorbing form (Pr). Difference spectra suggested that, at high light levels, Pr may be reformed via a photochemical back-conversion of an intermediate in the Pr—Pfr pathway, although the involvement of intermediates in the Pfr—Pr pathway cannot be excluded. The implications of the results for the ecological function of phytochrome are discussed.     

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 PHOTOCONVERSION in vivo. EFFECT OF THE INITIAL Pfr/Ptot RATIO

The equation for phytochrome photoconversion, derived from photoconversion kinetics of purified phytochrome, predicts that the rates of photoconversions starting from low and high Pfr/Ptot₍₀₎, (initial Pfr/Ptot) should be the same for light of the same quality and fluence rate. The situation might be different in vivo. Phytochrome photoconversion rates were measured in excised cotyledons of Cucurbita pepo L. exposed to BL (blue; ?_BL= 0.39; ?, Pfr/Ptot at photoequilibrium) and RI (mixture of red and far red; ?_RI= 0.46) after saturating preirradiations with red and far-red to establish high (0.78) and low (0.02) Pfr/Ptot₍₀₎, respectively. Under BL, the rate of photoconversion is faster when starting from a high than a low Pfr/Ptot₍₀₎; under RI, the rate of photoconversion is faster when starting from a low than a high Pfr/Ptot₍₀₎., No effects of Pfr/Ptot₍₀₎, on photoconversion rates were found in phytochrome solutions exposed to BL and RI. These data provide another indication of the discrepancies between phytochrome photconversion kinetics in vivo and in vitro.     

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.     

PHYTOCHROME MACRO-DISTRIBUTION, LOCAL PHOTOCONVERSION AND INTERNAL PHOTON FLUENCE RATE FOR Cucurbita pepo L. COTYLEDONS

Abstract— Using 7-day-oId cotyledons of Cucurbita pepo L., local phytochrome photoconversions could be measured for blue, red and far-red light. For this purpose, after nonsaturating irradiation, cotyledons were sliced into discs 0.3 to 0.5 mm thick and signals measured. This method also yielded the internal phytochrome distribution of the cotyledons with maximal concentration near the adaxial surface, dropping to about 50% in the center and reaching again about 90% at the abaxial surface. Local phytochrome conversion rates were used to calculate internal fluence rates across the cotyledons. Relative internal fluence rates were also derived from measured reflectances and transmittance according to the Kubelka-Munk theory. The general shape of the internal fluence distribution calculated on the basis of these two methods coincided well. It was observed that the internal local photoconversion is proportional to the penetration depth over a wide range of incident fluences and for all wavelengths tested, showing in addition that reciprocity holds. A method to calculate internal fluence rates by a simplified procedure assuming either linear or exponential functions is described.     

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

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

THE PHOTOCONVERSION OF CHENOPODIUM CHLOROPHYLL PROTEIN

Abstract— Changes in the UV absorption spectrum with the photoconversion of Chenopodium chlorophyll protein, CP668 ⇆ CP743, and with the pH change of the CP668 and CP743 solutions, were measured. The change in the absorption spectrum of the apoprotein caused by the pH change was reversible, whereas that caused by the photoconversion was irreversible. The apoprotein may undergo a proton dissociation or association of the phenolic group in tyrosine residues upon pH change. A photooxidation in CP668 (loss of electron) caused by irradiation of CP668 solution may induce a change in the ionization state of some amino acid residues. The isoelectric points of CP668 and CP743 were determined to be 9.3 and 7.2, respectively.     

PHYTOCHROME and PROTEIN PHOSPHORYLATION

The molecular mode of signal transduction triggered by phytochrome is unknown. One characteristic structural/topographic feature of the physiologically active form (Pfr) of phytochrome is that its tetrapyrrole chromophore becomes preferentially exposed in the Pfr form (compared to the Pr form). Phytochrome in its Pfr form appears to affect phosphorylation of cellular proteins. The literature on the phytochrome-mediated protein phosphorylation has been reviewed in an attempt to search for the role of the chromophore topography of phytochrome in the signal transduction process. In order to initiate a dephosphorylation-phosphorylation cascade as a possible step for the signal transduction, it may interact with a cellular protein kinase to inhibit its activity. This hypothesis has been reviewed with results from phosphorylation inhibition assays by the Pfr form of phytochrome and in light of the inhibition of protein kinase activity by tetrapyrroles in general.     

PHYSIOLOGICAL RELATIONSHIPS BETWEEN PHYTOCHROME EFFECTS ON INTERNODE EXTENSION GROWTH AND DRY MATTER ACCUMULATION INLIGHT-GROWN MUSTARD

The physiological relationships between the effects of phytochrome photoequilibrium (Pfr/P) on internode extension growth and dry matter accumulation were investigated in white light (WL)-grown Sinapis alba L. seedlings. After 11 days under continuous WL, the seedlings were exposed: (a) to pulses of light providing different Pfr/P, followed by 24 h darkness (D); (b) to pulses of light providing different Pfr/P, followed by 3 h D and 24 h continuous WL; (c) to continuous WL with or without supplementary far-red light (to reduce Pfr/P); or (d) to pulses of light providing different Pfr/P followed by D, in factorial combination with either water or a saturating (0.2 M) sucrose solution applied to one of the leaves. In D (“a” and “d”) low, compared to high Pfr/P increased both internode extension growth and dry weight to the same extent. Under WL (“b” and “c”) low PfrlP promoted internode extension growth but had no proportional effects on internode dry weight. Sucrose promoted internode extension growth with a lag of at least 8 h (compared to the rapid effect of low Pfr/P) and did not reduce the effect of low Pfr/P. These results indicate that Pfr/P effects on internode extension growth are not the consequence of changes in photoassimilate translocation from the leaves. Under WL, PfdP effects on internode length occur partially at the expense of internode dry matter per unit length.     

PHOTOTRANSFORMATIONS OF PHYTOCHROME

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

有机化合物结构与性能的关系——激发能的同系线性规律

有机化合物的光谱及激发能存在着良好的同系线性规律。前文曾用HMO法对多联苯体系的同系线性规律进行了讨论,说明了对联多苯的共轭作用比间联多苯强,而后者共轭作用很弱,这可从光谱得到证实。近来,徐光宪等探讨了同系线性规律的量子化学基础,根据多烯中键长交替现象提出了MHMO法。     

PHOTOCONTROL OF HYPOCOTYL ELONGATION IN LIGHT-GROWN Cucumis sativus L. PHOTOSYNTHETIC REQUIREMENT FOR A FLUENCE RATE DEPENDENT PHYTOCHROME RESPONSE

We investigated the role of photosynthesis in the photocontrol of extension growth of the hypocotyl of light-grown Cucumis sativus L. Previous work [Gaba and Black (1985b) Plant Physiol. 79 , 1011] demonstrated that the inhibition of cucumber hypocotyl elongation is a fluence rate dependent response in red light. However, the relative contributions of phytochrome and photosynthesis to the photon flux dependent inhibition response were not clear. Here we have shown that photoperception by the foliar cotyledons as well as the hypocotyl itself are responsible for fluence rate dependence in red light. The inhibitor of photosynthesis diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] reduced both the magnitude of inhibition and the fluence rate dependency in red light, indicating an involvement of photosynthesis. Furthermore, the growth of non-pigmented seedlings (treated with the herbicide norflurazon) was less inhibited by red light, with no fluence rate dependency. In particular, inhibition due to cotyledon photoperception was completely lost in non-pigmented (norflurazon-treated) plants, and much reduced by diuron treatment. Hypocotyl-perceived red light inhibition was only slightly reduced by treatment with norflurazon and diuron. Photosynthesis was compared directly to photo-inhibition of growth: the photon flux response curve of oxygen evolution of green Cucumis cotyledons was distinctly different from that of hypocotyl inhibition. In conclusion, photosynthesis is an essential requirement for the cotyledon-perceived inhibition, but the response itself is not due to photosynthesis.     

聚丁二烯的链节结构与其氧化性能的关系

利用不同催化剂体系的定向聚合,可使丁二烯聚合成双键在主链和侧基中分布不同的聚合物。对于这些聚合物的氧化性能,尽管早期有人利用不同单体单元的均聚物和共聚物如天然胶、古塔坡胶以及丁钠、丁锂、丁苯、丁基胶等作过比较,认为丁二烯橡胶分子主链和乙烯侧基上所含双键的比例可以极大地影响其反应能力,主链上双键在氧化时较活泼,而乙烯侧基上的双键则在热交联时较活泼。     

RELATIONSHIP BETWEEN SURVIVAL AND THYMINE-DIMER EXCISION IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. light and excision of thymine dimers of bacterial strains E. coli B/r hcr ⁺ thy- trp ^-, E. coli B/r hcr ^-thy^- trp ^-, and E, coli 15 T^- 555–7 thy ^- meth ^- trp ^- arg ^- has been studied.
The prestarvation increased the resistance of all the strains but reasonably inhibited excision of thymine dimers. Thus the enhancement of u.v. resistance after amino acid prestarvation was not due to more complete excision of thymine dimers.