In vitro phytochrome dark reversion process

Thermal reversion of the far-red absorbing form of phytochrome to the red absorbing form in darkness has been investigated in crude and partially purified isolates from a number of etiolated and light grown higher plants. The influence of temperature, aging and urea on the rate of reversion was also determined.

Phytochrome isolated from all higher plants underwent reversion. The reversion proceeded in at least 2 distinct stages; a short rapid initial phase being followed a slow phase which continued for many hours. Reversion rate was highest in phytochrome isolated from green leaves of parsnip (Pastinacea sativa) and lowest in that isolated from etiolated oats (Avena sativa). Although the rate of reversion could be changed by modifying the tertiary structure of the protein component, the large differences in rate appeared to be characteristic of the plant source. Observed in vitro rates of reversion are slower than those occurring in vivo. Removal of other buffer solubilized material during purification had little effect on the rate of reversion of phytochrome isolated from etiolated material.

     

Dependence of phytochrome dark reactions on the initial photostationary state

Summary Under conditions of continuous irradiation, the P _jr destruction rate constants (k _d ) of phytochrome in hooks and cotyledons of squash (Cucurbita pepo L.) seedlings do not depend on the photostationary state and are the same in both organs. On the other hand, the rate constants of the dark reversion and the first destruction step, plotted as a function of ⁰ , show optimum curves with maxima between ⁰ and 0.5. Similar results were obtained for dark reactions of mustard (Sinapis alba L.)-hook phytochrome in vivo. This indicates a cooperative behaviour of these phytochrome dark reactions.Abbreviations P _r red-absorbing form of phytochrome - P _fr far-red-absorbing form of phytochrome - [P _tot] [P _r ]+[P _fr ] - [P _tot] ([P _fr ]/[P _tot]), photostationary state - ⁰ at t=0, immediately after saturating irradiation     

Purification of oat and rye phytochrome

A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A₂₈₀ nm/A₆₆₅ nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.     

Spectral characteristics of phytochrome in vivo and in vitro

The absorption maximum of the far-red absorbing form of phytochrome in the difference spectrum for phototransformation (Pfr max) was investigated in vivo and in in vitro pellets from dark grown Hordeum vulgare L. primary leaves. Exposure of pellets in Honda medium from tissue pre-irradiated with red light to far red light gave a Pfr max of 734 nm, a slightly longer wavelength than was seen in vivo (730 nm). After incubation as the red absorbing form of phytochrome (Pr) for 2 h at 0° C irradiation with red light showed that Pfr max had shifted to shorter wavelength (716 nm) in Honda medium. Further incubation as Pfr for 2 h at 0° C and irradiation with far red light showed that Pfr max had shifted to longer wavelength (726 nm). Similar shifts were also seen in other media, although the peak positions were different. Phytochrome remained pelletable throughout these experiments and Pfr max is compared to that of soluble phytochrome in similar media. The results are interpreted as indicating changes in molecular environment of the putative phytochrome membrane receptor site and that Pfr max can be used to probe the nature of this binding.Abbreviations D Dark - EDTA Ethylene diamine tetra-acetic acid - F far red light - MOPS N-morpholino-3-propane-sulphonic acid - P Phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - Pfr max wavelength maximum of Pfr absorbance in a phototransformation difference spectrum - R red light     

Acceleration of dark reversion of phytochrome in vitro by calcium and magnesium

Rates of dark reversion of the far red-absorbing form of phytochrome, Pfr, to the red-absorbing form, Pr, have been determined in the presence of several salts. Low concentrations of calcium chloride and magnesium chloride (up to 3 mm) accelerated the rate of dark reversion at all stages of purification of phytochrome from etiolated rye (Secale cereale L. cv. Balbo) seedlings. The complex kinetics of the dark reversion could be resolved into two first-order components. The effect of the added divalent cations was on the relative proportion of the fast and slow reacting components, rather than on the rate constants of the two populations. It was possible to reverse the effects of the cations by adding the chelating agents ethylene-bis-(oxyethylene-nitrilo) tetraacetic acid or ethylenediaminetetraacetate. The effect of the divalent cations is not a nonspecific ionic strength effect. The relative proportion of the two populations was also affected by the degree of purity of the phytochrome samples.     

Partial characterization of oat and rye phytochrome

Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (sigma(200)) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a sigma(200) value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein.     

Phototransformation of phytochrome in the dark

Enzymatically generated triplet acetone transfers its energy to the ground state phytochrome and promotes to some extent, in the dark, the conversion of P_r into P_fr and of P_fr into P_r. This is the first report of inverse dark reversion “in vitro”.     

Some spectral properties of pea phytochrome in vivo and in vitro

The transformation difference spectrum for phytochrome (Pr spectrum minus Pfr spectrum) in pea tissue is determined below 560 nanometers and compared with similar data on phytochrome in vitro The difference spectrum in vivo between phytochrome intermediates and Pfr is also shown for comparison with the data on phytochrome solutions. These comparisons show that the peaks in the spectra occurring in the blue wave lengths are shifted to shorter wave lengths and are much enhanced when phytochrome is extracted from the cell and placed in solution. The results indicate that the physicochemical state of phytochrome in the cell may be different from that of the extracted pigment.     

The aggregation States of phytochrome from etiolated rye and oat seedings

Initial extracts from etiolated plants contained two aggregates of phytochrome. A major fraction was almost excluded by Sephadex G-200 and was within the fractionation range of Sepharose 4 B. A minor fraction was within the fractionation range of Sephadex G-200. Modifications of a previous isolation procedure which allowed retention of this aggregation state are reported. With respect to gel filtration, the major fraction of phytochrome from oat and rye seedlings was identical. The aggregates of rye and oat phytochrome were also separated by diethylaminoethyl cellulose chromatography.     

The in vivo properties of Amaranthus phytochrome

Summary Phytochrome has been measured in etiolated seedling of Amaranthus caudatus. The phytochrome content increases from the time of germination until 72 hr from sowing, after which it remains constant at 27.5x10^-3 (OD) units per 200 seedlings. After a saturating dose of red light P _fr decays in the dark to a form not detectable photometrically. There is no evidence for the process of dark reversion of P _fr to P _fr found in other dicotyledons. Even in the presence of azide, a selective inhibitor of decay, the process of dark reversion is not observed. The decay of P _fr has been investigated at different temperatures and follows first order decay kinetics throughout. Over the temperature range 15–30° the Q ₁₀ of decay remained constant at 4.3.The photostationary states of phytochrome (P _fr /P _total )maintained by mixed red/far-red light have been measured in both seedlings and partially purified protein extracts, with good agreement. The rate of phytochrome decay can be manipulated by changing the P _fr /P _total ratio. The lag period before a decay curve becomes exponential is characteristic of a particular P _fr /P _total ratio and represents the time for attainment of the photostationary state. The effect of energy on decay has been investigated under red and blue light. The rate of phytochrome decay is dependent on the P _fr /P _total ratio and only becomes energy dependent when the light intensity is so low that the photostationary state is never attained.The process of apparent phytochrome synthesis has been found in Amaranthus. After reducing the phytochrome to a low level by red light treatment a rate of apparent synthesis of 1.35×10^-4 (OD) units per hr per 200 seedlings was observed, levelling off at 29% of the original phytochrome level.Under white tungsten lights of high intensity there is a deviation from the expected first order decay kinetics. The nature of this low rate of decay cannot be explained at the present time.     

The loss of phytochrome photoreversibility in vitro

Killer, a substance extracted from stem tissue of etiolated pea seedlings (Pisum sativum L. v. Alaska), interacts specifically with the far-red-absorbing form of phytochrome (Pfr) in vitro in a temperature-independent, rapid, stoichiometric fashion to cause a loss of phytochrome photoreversibility. The chromatographic, solubility, and spectral properties of partially purified fractions indicate that Killer is a cyclic, unsaturated molecule containing ionizible hydroxyl groups; its molecular weight is unknown, although probably low. Possible mechanisms by which the Killer-phytochrome interaction results in the loss of photoreversibility are discussed.I=Fox, 1975     

Short-term reactions of phytochrome in Mougeotia?

Summary Phytochrome controlled chloroplast movement in Mougeotia is induced by flashes of polarized red light. Two subsequent flashes, separated by a dark interval of a few seconds, are much more effective than two simultaneous flashes; a maximal cumulative effect is reached if the duration of the dark interval is 30 ms or longer. We propose two light reactions in series, separated by a very fast dark reaction. Preliminary evidence is given that the energy requirement for these light reactions is different. It is suggested that the two reactions are related in some way to free and bound phytochrome.Dedicated to Prof. Dr. E. Bünning on the occasion of his seventieth birthday.     

The effect of gibberellic acid on phytochrome measurement in vivo

Phytochrome changes in hypocotyls of light grown mustard seedlings (Sinapis alba L.) and the phytochrome destruction in cotyledons of etiolated plants have been analysed as influenced by exogenous gibberellic acid (GA₃). The rate of phytochrome appearance in hypocotyls was decreased after GA₃ treatment. Destruction of phytochrome in cotyledons of etiolated seedlings was significantly accelerated by the same hormone treatment. This effect was also observed in the hypocotyls of light grown seedlings.     

A detailed analysis of phytochrome decay and dark reversion in mustard cotyledons

Summary During the first 10 min after a saturating dose of red light, 72 h dark-grown mustard cotyledons show no phytochrome decay. Within the same time interval there exists a transient form of P _fr (=P _fr ^T ) which is no longer photoconvertible at 0°C, but is at 25°C. This P _fr ^T converts in the dark to P _fr and P _r . These dark reversions take about 10 min. After a lag phase of 10 min the P _fr decay can be described by a single, first order kinetic curve. The time courses of these reactions are functions of the time of etiolation.Research supported by DAAD and by Deutsche Forschungsgemeinschaft (SFB 46).