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.

     

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.     

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.     

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).     

Assembly of synthetic locked phycocyanobilin derivatives with phytochrome in vitro and in vivo in Ceratodon purpureus and Arabidopsis

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.     

In vivo measurement of phytochrome in tomato fruit

Presence of phytochrome in two kinds of tomatoes (Lycopersicon esculentum Mill.), the yellow lutescent strain and cherry tomatoes (L. esculentum Mill. var. cerasiformecv. Red Cherry), was established by measuring the absorption difference spectra of the whole fruit after irradiation with red and with far red light. Phytochrome content was determined in yellow lutescent tomatoes and decreased gradually during the ripening period.