Properties Of Phytochrome Research Articles

Protein purification with C-terminal fusion of maltose binding protein.

For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography. After overexpression in yeast a 125-fold enrichment could be achieved. The spectral properties of phytochrome B were not impaired by the fusion and purification. These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification.

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH‐PIGMENT MUTANT OF TOMATO

Abstract— A high‐pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light‐labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence‐response relationships for anthocyanin synthesis after a blue‐light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far‐red light (FR)‐absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6‐fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild‐type and hp‐mutant seedlings, despite the latter accumulating 9‐fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence‐rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24‐h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.

Phytochrome in Norflurazon‐treated seedlings of Pharbitis nil

The properties of phytochrome have been measured by dual‐wavelength spectropho‐tometry in the cotyledons of the short‐day plant Pharbitis nil Choisy cv. Violet, where it is known to play a role in flower induction. In plants de‐etiolated by a single white light period (4 h or longer), destruction of the far‐red absorbing form of phytochrome (Pfr) was twice as rapid as after 10 min red light. A small fraction of Pfr was stable. After de‐etiolation by a period of white light (6 h or longer) the rapid decrease of Pfr during the first 30 min was accompanied by a rapid increase of the red absorbing form of phytochrome (Pfr). This rapid increase of Pfr is probably due to dark reversion. Long term synthesis of phytochrome was inhibited by the presence of Pfr. Phytochrome synthesised in darkness showed the etiolated‐plant type characteristics and underwent rapid destruction upon photoconversion to Pfr. The stable Pfr identified here is possibly that pool of phytochrome associated with the long term promotive process in flower induction, and the rapidly reverting Pfr is that pool associated with the night break inhibition of flowering.

Comparison of the spectral properties of phytochrome associated with plant membranes and phospholipid liposomes

Comparison of the spectral properties of phytochrome associated with plant membranes and phospholipid liposomes

Time‐dependent changes in the responsiveness to light of phytochrome‐mediated anthocyanin synthesis

Abstract. It is well established that seedlings of mustard (Sinapis alba L.) synthesize juvenile anthocyanin only if treated with light pulses or continuous light. The light effects are considered to be due to the operation of phytochrome. Here we show that the responsiveness of anthocyanin synthesis to a saturating red light pulse or to continuous far‐red light varies as a function of time and is strongly influenced by a light pretreatment prior to competence. Competence appears approximately 25 h after sowing. The starting point of anthocyanin synthesis, which is 27 h after sowing, and the lag‐ phase of this response, which is 2 h, are not affected by light pretreatments prior to competence. It is concluded that quantitative interpretations of phytochrome responses based entirely on properties of phytochrome can no longer be considered adequate.

Purification of phytochrome by Affi-Gel Blue chromatography; an effect of lumichrome on purified phytochrome

Lumichrome, a common contaminant from the thermal and photochemical degradation of flavin mononucleotide (FMN), tightly binds to phytochrome purified by the Affi-Gel Blue chromatographic method. FMN is used as an eluant. Lumichrome bound to phytochrome negates the dithionite acceleration of phytochrome's dark-reversion. Thus, FMN, for the elution of phytochrome from an Affi-Gel Blue column, must be free of lumichrome. Some modification of the Affi-Gel Blue method is also described. The modified procedure yields a large-molecular-weight phytochrome (from oat) in an electrophoretically pure form. The use of lumichrome as a probe for the structure and properties of phytochrome has been discussed briefly.

STUDIES ON PLANT BILE PIGMENTS—9. PHOTOOXIDATION OF A-DIHYDROBILINDIONE AS PHYTOCHROME MODEL IN ACID MEDIUM

— The photooxidation of octaethyl-2, 3-dihydrobilindione (2) as a model for the Pr-chromophore has been studied in acidic methanol. When 2 is titrated with HCl to the cation and irradiated under N2 with an excess of I2, violin and rhodin type pigments are formed bearing methoxy-substituents at either C-4,5 or C-15,16, respectively. The reaction is slowed down as compared to neutral conditions, it is no longer regioselective at the C-5 methine bridge, and the formation of dimers is suppressed. From the data of this and earlier chemical model studies on the photooxidation of 2, and the known properties of phytochrome, a reaction for the Pr Pfr phototransformation is suggested, in which the chromophore is attacked by the protein moiety in a sequence of oxidation and nucleophilic substitution, which may lead to a primary redox signal for the physiological response.

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

Properties of phytochrome in gymnosperms

Addition of water to dry seeds of Pinus spp. increased the detectable phytochrome immediately and the level reached after 2 h in darkness was retained for at least 20 h at 20° C. The in-vivo difference spectra of phytochrome in Pinus seeds showed absorption maxima at approximately 656 nm and at 710 nm to 715 nm. An isosbestic point was observed at about 680 nm. Shifts towards longer wavebands were obtained especially with tissue containing substantial amounts of chlorophyll and are, therefore, not due to diverse types of phytochrome. Embryo tissue of Ginkgo biloba showed also a maximum in R at 655 nm but the peak in FR occurred at a longer wavelength, 725 nm. This was confirmed by determining action spectra for the phototransformations Pr⇌Pfr.The dark reactions of phytochrome in Pinus differed from those in Ginkgo. Following a short exposure to R light, the total quantity of photoreversible pigment in Pinus seeds remained constant for several hours in darkness at room temperature. Dark reversion of Pfr occurred extremely rapidly and tPfr 50 was only 0.3 h. In Ginkgo embryos total phytochrome in darkness following a brief exposure to R light was not completely stable. Reversion of Pfr was much slower and tPfr 50 was slightly less than 2 h.It is concluded that, at least as regards the spectral qualities, the phytochrome in Gymnospermae differs from that of Angiospermae and is apparently also not identical in Coniferae and Ginkgoinae.

Properties of Phytochrome

Properties of Phytochrome

Photoperiodism: An Effect of Darkness during the Light Period on Critical Night Length

The critical night length of the short-day plant Lemna perpusilla, grown with sucrose, increases roughly 3 hours under cycles with "light periods" composed of darkness preceded and ended by brief exposures to light. Although plants so grown are white, the effect is due neither to the absence of photosynthesis nor to insufficient total energy. It is inconsistent with current ideas on photoperiodic timing but may be explained by a hypothesis based on reported properties of phytochrome.