Phytochrome Photoconversion Research Articles

Cryo-Electron Microscopy of Arabidopsis thaliana Phytochrome A in Its Pr State Reveals Head-to-Head Homodimeric Architecture.

Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered Z/E isomerization of a covalently bound bilin chromophore underlies phytochrome photoconversion between the red-absorbing Pr and far-red-absorbing Pfr states. Compared to bacterial phytochromes, the molecular mechanisms of signal propagation to the C-terminal module and its regulation are little understood in plant phytochromes, not least owing to a dearth of structural information. To address this deficit, we studied the Arabidopsis thaliana phytochrome A (AtphyA) at full length by cryo-electron microscopy (cryo-EM). Following heterologous expression in Escherichia coli, we optimized the solvent conditions to overcome protein aggregation and thus obtained photochemically active, near-homogenous AtphyA. We prepared grids for cryo-EM analysis of AtphyA in its Pr state and conducted single-particle analysis. The resulting two-dimensional class averages and the three-dimensional electron density map at 17 Å showed a homodimeric head-to-head assembly of AtphyA. Docking of domain structures into the electron density revealed a separation of the AtphyA homodimer at the junction of its photosensor and effector modules, as reflected in a large void in the middle of map. The overall architecture of AtphyA resembled that of bacterial phytochromes, thus hinting at commonalities in signal transduction and mechanism between these receptors. Our work paves the way toward future studies of the structure, light response, and interactions of full-length phytochromes by cryo-EM.

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.

Crystal Structure of Deinococcus Phytochrome in the Photoactivated State Reveals a Cascade of Structural Rearrangements during Photoconversion

Phytochromes are photochromic photoreceptors responsible for a myriad of red/far-red light-dependent processes in plants and microorganisms. Interconversion is initially driven by photoreversible isomerization of bilin, but how this alteration directs the photostate-dependent changes within the protein to actuate signaling is poorly understood. Here, we describe the structure of the Deinococcus phytochrome photosensory module in its near complete far-red light-absorbing Pfr state. In addition to confirming the 180° rotation of the D-pyrrole ring, the dimeric structure clearly identifies downstream rearrangements that trigger large-scale conformational differences between the dark-adapted and photoactivated states. Mutational analyses verified the importance of residues surrounding the bilin in Pfr stabilization, and protease sensitivity assays corroborated photostate alterations that propagate along the dimeric interface. Collectively, these data support a cooperative "toggle" model for phytochrome photoconversion and advance our understanding of the allosteric connection between the photosensory and output modules.

Assembly of Synthetic Locked Chromophores with Agrobacterium Phytochromes Agp1 and Agp2

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14-C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5-C6 syn C15=C16 Z C14-C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14-C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.

Effect of end-of-day irradiations on polyamine accumulation in petal cultures of Araujia sericifera.

We have studied photoperiodic control and the effect of phytochrome photoconversion at the end-of-day (EOD) on polyamine (PA) accumulation in petal explants of Araujia sericifera. Petals from immature flowers were cultured under long (LD) and short (SD) days. Light was provided by Gro-lux fluorescent lamps (90-100 &mgr;mol m-2 s-1). Red (R), far red (FR), red followed by far-red (R-FR) and far-red followed by red (FR-R) light treatments were applied daily at the end of the photoperiod. The free and bound putrescine (Put), spermidine (Spd) and spermine (Spm) fractions in petal explants were determined 40 days after the beginning of the culture. We also aimed to clarify the involvement of PA changes by using two inhibitors of PA biosynthesis: D-l-alpha-difluoromethylarginine (DFMA) and methylglyoxal bis(guanylhydrazone) (MGBG). We found PA accumulation to be under photoperiodic control, and the inhibitory effect of DFMA on this accumulation suggests that arginine decarboxylase (ADC) is the major pathway for Put biosynthesis. Polyamine levels were higher under LD, mainly as a result of the accumulation of free and bound Put. FR-EOD treatment, which dramatically reduced the R : FR ratio after LD, increased the accumulation of PA, mainly as free Put and free and bound Spd. Sequential R-FR and FR-R-EOD treatments strongly increased bound Spd. The concentration of MGBG used increased total PA accumulation, mainly as Put. However, all EOD light treatments dramatically reduced Put accumulation in the presence of MGBG. This may be due to a dual role of FR light in PA accumulation: (1) FR per se stimulates PA production, probably via ADC, and (2) in the presence of MGBG, FR inhibits Put accumulation, probably via ethylene production.

Effects of light quality on somatic embryogenesis in Araujia sericifera.

The effects of photoperiod, light quality and end-of-day (EOD) phytochrome photoconversion on somatic embryogenesis (SE) of Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 8- and 16-h photoperiods using Gro-lux fluorescent lamps. The photon fluence rate was 90-100 µmol m-2 s-1 and the red (R):far-red (FR) ratio was 98. R, FR, R followed by FR (R-FR) and FR followed by R (FR-R) light treatments were applied for 3 weeks at the end of the photoperiods. In a set of experiments, DL-alpha-difluoromethylarginine (DFMA) or methylglyoxal bis(guanylhydrazone) (MGBG), both inhibitors of polyamine biosynthesis, were added to the culture medium in order to study the involvement of polyamine metabolism. The level of SE was the same in long (LD) and short (SD) days. Thus, the light effect was accomplished after 8 h. All EOD treatments that decreased the Pfr level inhibited SE when applied after SD, but not after LD. The FR-R treatment after LD caused an additional stimulatory effect on SE, even in the presence of polyamine inhibitors. DFMA inhibited SE in both SD and LD, but MGBG did not modify SE in either SD or LD. The R, FR and R-FR treatments did not alter the level of SE when applied after LD in the presence of DFMA or MGBG. However, these treatments decreased SE after SD when the medium contained polyamine inhibitors. Our results suggest that Gro-lux lamps, which produce an extremely high R:FR ratio, promote SE in A. sericifera and a timing response to phytochrome photoconversion during photoperiodic induction. Thus, our data corroborate the involvement of phytochromes and polyamines in SE in A. sericifera, which responded as a light-dominant long-day plant.

A computer simulation of aggregated phytochrome photoconversion

In order to check the relevance of possible phytochrome aggregation forms for the diversity of action spectra and dose-response curves for light sensing by plants, a model for phytochrome photoconversion was produced for different aggregates. Under saturating light treatments, a computer simulation of dimer and aggregated dimer (tetramers and hexamers) photoconversion provided similar compositions of various aggregates in red and green light, which differed from blue and far-red light. These aggregates could account for the variable action spectra only if the nature of the primary transducing components is taken into account. The initial kinetics of aggregated phytochrome appear to be slower in comparison to the monomeric form, and there were transient peaks of certain aggregates in red and green light. A single peak occurred under blue light, but not in far-red, only if dimers are aggregated into tetramers and hexamers. This finding suggests that the initial kinetics of aggregated phytochrome are not sufficient to account for the variable dose-response curves in light-mediated responses by plants, so again the feature of signal transduction components may play a decisive role.

Phytochrome-controlled phototropism of protonemata of the moss ceratodon purpureus: physiology of the wild type and class 2 ptr-mutants

Phototropism and polarotropism in protonemata of the moss Ceratodon purpureus are controlled by the photoreceptor phytochrome. One class of phototropism mutants is characterised by growing randomly when kept for a prolonged time (5 d or longer) in unilateral red light. It was found that a subclass of these mutants grows faster than the wild type, the rate of cell division and the length of the cells being increased. This difference is found for light-grown and dark-grown filaments. It is therefore suggested that the mutant phenotype neither results from a defect in phytochrome photoconversion nor from a defect in phytochrome-gradient formation. Instead, it is possible that a factor which is involved in both signal transduction of phototropism and regulation of cell size and cell division is deregulated. If dark-grown mutant filaments are phototropically stimulated for 24 h, they show a weak phototropic response. Phototropism and polarotropism fluence-rate effect curves for mutants were flattened and shifted to higher fluence rates compared with those for the wild type. With wild-type filaments, a previously unreported response was observed. At a low fluence rate, half of the filaments grew positively phototropically, while the other half grew negatively phototropically. It seems that under these conditions, a phytochrome gradient with two maxima for the far-red-absorbing form of phytochrome (Pfr) within the cross-section of the cell is displayed by the response of the filaments. At higher fluence rates, all filaments of the wild type grew towards the light. These data and results from microbeam irradiation experiments and from phototropism studies with filaments growing within agar, indicate that light refraction plays an important role in the formation of the Pfr gradient in phototropism of Ceratodon.

Phytochrome-mediated photocontrol of the germination of the scentless mayweed, Matricaria inodora L., and its sensitization by nitrate and temperature

The emergence of Matricaria inodora ( M. perforata) may poorly respond to modified light exposure during tillage. Therefore, the influence of light, nitrate and temperature on the germination of M. inodora has been studied under laboratory conditions. Germination of a native seed batch levels off around 77% after 7 to 9 days at 20°C, if sown in 0.01 M KNO 3 or NaNO 3 with a daily photoperiod of 14 h weak white light. In 0.01 M NaCl or in bidistilled water germination reaches 48 ± 5%. One saturating red-light pulse, applied 12 h after imbibition in 0.01 M KNO 3, gives only 12 ± 3% germination after 7 days, but 5 daily repeated red-pulses produce 75% germination. Ten twofold daily repeated red-light pulses are equivalently efficient, and if all immediately followed by a saturating far-red pulse, perfect reversibility down to the far-red level of 7 ± 3% germination is found. The dark germination was 1.7 ± 0.7%. A fluence-response curve, elaborated for 10 red-pulses, gives an exponential pattern with half-saturating pulse fluence around 35 J m −2, as indicative of phytochrome photoconversion. The influence of additional temperature pretreatment in darkness was tested, followed by half-saturating red-light pulses every 12 or 24 h. Chilling at 2 °C was ineffective up to 6 days, but slightly sensitizing after 21 days, giving around 30% and 46% germination, respectively. Preincubation for 6 days between 20 and 36 °C was more efficient and led to 58–68% germination at 20 °C. A thermoperiodic pretreatment with daily 12 h at 10 °C and 12 h at 30 °C was most efficient, giving 73 ± 4% germination. For all these pretreatments dark germination never exceeded 3%. Elevated dark germination occurred when water-imbibed achenes, being exposed and dried in red-light of 1 W m −2, have been reimbibed in darkness. If drying in red-light was for 6 h to a water content of 20% and reimbibition was in water or 0.01 M KNO 3, dark germination increased to 22 ± 5% or 40 ± 6%, respectively. This dark germination was far-red reversible down to 5 ± 2% with half-escape time around 5 h. If drying in red-light was for 24 h to reach the storing content of water around 11%, the dark germination after reimbibition in water or 0.01 M KNO 3 was only 4 ± 2% and 13 ± 2%, respectively. All corresponding controls for drying and reimbibition in darkness, for both water and nitrate, never exceeded 1% germination. This means that drying of seed in light — but not in darkness — increases emergence after the next rainfall and that the nitrate ion acts as a sensitizer for preformed P fr and facilitates phytochrome-mediated germination. Thus, for maximum germination of surface exposed M. inodora a thermoperiodic pretreatment, daily repeated light exposures and nitrate are needed. This is typical of the shallow germinating ancestor occurring in coastal ranges, the Sea Mayweed, Tripleurospermum maritimum, and may explain why the emergence of M. inodora is scarcely reduced after lightless tillage.

Phytochrome photoconversion

ABSTRACTThe spectral properties of native and modified phytochromes and the molecular events during phytochrome photoconversion, , are reviewed. Steady‐state and time‐resolved absorption spectra of native phytochrome A, as well as recombinant phytochromes (oat and potato phytochrome A and potato phytochrome B) reconstituted with phycocyanobilin and phytochromobilin as chromophores, are analysed. The vinyl double bond, present at position 18 in phytochromobilin and substituted by an ethyl group in phycocyanobilin, has a considerable influence on the photo‐transformation kinetics of phytochromes A and B, evidently due to a strong interaction of this region of the chromophore with the protein surrounding. The kinetics of the phototransformation of potato phytochrome B differs from that of oat phytochrome A (wild‐type and recombinant), indicating that the chromophore‐protein interaction in phytochrome B is different from that in phytochrome A. It remains to be seen whether this difference is due to the di‐ versus monocotyledon origin of the phytochromes. Optoacoustic spectroscopy, applied to native oat phytochrome A, afforded thermo‐dynamic, structural and kinetic parameters of the Pr→I700 and the I700→Pr phototransformations. Raman and infrared spectroscopic data for wild‐type phytochrome A suggest that the protonated chromophore in Pr undergoes torsions around two single bonds in addition to the Z→E isomerization of the 15,16 double bond, and that all transients, possibly with the exception of IbI, are protonated at the central pyrrole ring.

遠赤色光の付加による光質変化の評価と蔓性および蔓なしインゲンマメの生長変化

The growth changes caused by the increase of far-red light were investigated in pole-type and bush-type kidney beans (Pheseolus vulgaris L. cv Kentucky Wonder and Celina) . Irradiation with a wavelength over 700 nm was added to the fluorescent lamps to increase far-red light. The light quality variation with supplementary far-red light was presented by phytochrome photoequilibria (Pfr/P) . To estimate the Pfr/P, the SPD method was used, which was calculated by the spectral photon flux distribution of irradiation and the phytochrome photoconversion. The calculated Pfr/P was compared with the measured Pfr/P derived from the method of Holmes and Smith (1975) . The calculated Pfr/P was expressed in a rectangular hyperbolic equation of the R/FR ratio, and this Pfr/P and the measured Pfr/P showed low level difference (<8%) . Therefore, by using the SPD method, estimation of Pfr/P could be possible without the analysis of phytochrome. As the far-red light increased and Pfr/P decreased, the length and dry weight of the stem in both the pole-type and the bush-type kidney beans increased and the proportion of the stem dry weight relative to the whole dry weight increased. The increase of stem length was great when the Pfr/P was above 0.77, and small when below 0.77. The stem length increased through increases of both the number of nodes and the internode length in the case of pole-type, while in the case of bush-type beans, only through the internode length.

Absence of phyB inhibits hypocotyl elongation in dark‐grown Ih cucumber seedlings: an active role for PrB

ABSTRACTCucumber (Cucumis sativus L.) seedlings carrying the long hypocotyl (Ih) mutation, which confers a lack of B‐type phytochrome (phyB), were significantly shorter than their near‐isogenic wild‐type counterparts when grown in complete darkness. Relative growth rates determined for 5 mm hypocotyl regions were lower in Ih seedlings in all growing regions, and the zone of elongation was less extensive in Ih hypocotyls. Digital imaging microscopy revealed that the pattern of epidermal cell lengths along the stem axis differed between the Ih mutant and the iso‐genic wild‐type. These findings (and the fact that experiments were conducted under conditions where phytochrome photoconversion to the far‐red‐absorbing form does not occur) suggest that the red‐absorbing form of phyB (PrB) is an active positive regulator of development in etiolated plants.

PHYTOCHROME PHOTOCONVERSIONin vivo. EFFECT OF THE INITIAL Pfr/Ptot RATIO

Abstract—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 differentin vivo. Phytochrome photoconversion rates were measured in excised cotyledons ofCucurbita pepoL. exposed to BL (blue; ϕBL= 0.39; ϕ, Pfr/Ptot at photoequilibrium) and RI (mixture of red and far red; ϕRI= 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 kineticsin vivoandin vitro.

DIRECT MEASUREMENT OF PHYTOCHROME PHOTOCONVERSION INTERMEDIATES AT HIGH PHOTON FLUENCE RATES

Abstract—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.

Photocontrol of biological processes in monocotyledonous plants after relaxation of phytochrome system

Effects of a short-term pre-irradiation of etiolated wheat seedlings by radiation of various wavelengths and various irradiances on the rates of photophospho-rylation, Hill reaction, and chlorophyll accumulation were studied. The curves, constructed for the dependence of inhibitory effect in different processes on pre-irradiance doses, have a saturation level corresponding to photostationary state of the phytochrome system and a pre-threshold site where the inhibition is absent. For an interpretation of the experimental data we suggest a general model that includes both the threshold concept of Mohr and the presence of an additional control mechanism not sensitive to far-red radiation. According to the model, the phytochrome-controlled biological effect is pro-portional to the time during which the level of active phytochrome is higher than the threshold one and may be analysed according to the inhibition degree. The difference in control by red (RR) and green radiation (GR) pretreatments is attributed to the difference of the phytochrome photoconversion rate under RR and GR.

LIGHT PERCEPTION BY PHYTOCHROME in vivo*

Abstract—The rate of phytochrome photoconversion in cotyledons and hypocotyls of dark‐grown seedlings of Cucurbita pepo varies significantly depending on the characteristics of the light field and the position of the sample in the light field. The overall pattern of the effects of light reflectance conditions and angle of incidence of light on phytochrome photoconversion in vivo is predictable and consistent with the results of physiological studies; however, the extent of the effects is much more difficult to predict with a satisfactory degree of approximation. The differences between phytochrome photoconversion kinetics measured in vivo and those predicted from photochemical parameters of purified phytochrome are rather large and suggest that the use of predictions as estimates of the state of phytochrome in vivo may be subject to significant limitations.

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris‐I DEMONSTRATION AND QUANTITATIVE CHARACTERIZATION OF THE PHOTOCHROMIC SYSTEM Pr⇔ Ii700USING NANOSECOND‐LASER PULSES*,†

Abstract— Spores of Dryopteris paleacea and D. filix‐mas are positively photoblastic with an optimum in the action spectrum around 665 nm. Light is perceived by phytochrome and the relationship between germination and mole fraction of the far‐red‐absorbing form of this pigment, Pfr, was investigated with saturating irradiations between 662 and 747 nm under low‐fluence‐rate conditions. These control irradiations establish a proportion of the total phytochrome, P,tot, as Pfr with Pfr/Ptot–φ at equilibrium. These φ ‐values were calculated according to data for native oat phytochrome (Kelly and Lagarias, 1985, Biochemistry 24, 6003) and the spectral characteristics of the interference filters. With this method a linear relationship could be found between φ and germination from 2 to 70% for D. paleacea and from 2 to 90% for D. filix‐mas, if probit germination was plotted vs probit φThis correlation formed the basis of investigating the phytochrome photoconversion by dye‐laser pulses of 380 ± 30 ns under high‐fluence‐rate conditions, and thus to test quantitatively the impact of the photoreversibility of intermediate reactions of the photoconversion and the red‐absorbing form of phytochrome, Pfr on the final Pfr‐level. Spore germination was initiated by a single‐laser pulse in the range from 592 to 700 nm. The most effective wavelengths were 649 and 660 nm in both species, and at saturation maximal germination (ca. 50%) was obtained from 592 to 665 nm for D. paleacea or ca. 60% germination from 592 to 670 nm for D. filix‐mas. Both saturation levels correspond to a ø‐value between 0.40 and 0.45. This significantly diminished photoconversion is a consequence of the high‐fluence‐rate conditions during the laser pulse which establishes the photochromic system between Pr and a set of very early intermediates, Ii700, (= Pr⇔ Ii700). This system can be described by the extinction coefficients of Pr and the intermediates Ii700, and by the quantum yields, 4,φ for the forward and reverse reactions as φ If φ is calculated, assuming a quantum yield of 1:1 for both reactions and with the extinction coefficients of Pr and Ii7(l() (= lumi‐R) given by Eilfeld and Riidiger (1985, Z. Naturforsch.40c, 109), significantly higher values are calculated for / as compared to φ found in the control experiments.These results can be explained either: (i) with a quantum yield ratio φpr‐φ1700: φ1700φpr=1:1 and an assumed additional dark reaction leading from Ii700 or later intermediates back to Pr: or (ii) with a quantum yield ratio φpr φ 1700: φ1700 φpr=1:2. In this case all Ii700 have to relax to Pfr. In this case all Ii700 have to relax to Pfr.

Phytochrome Photoconversion in Vivo

The measured rates of phytochrome photoconversion in vivo, in etiolated cabbage (Brassica oleracea L.) seedlings and cucumber (Cucumis sativus L.) cotyledons, under blue, red, and far red irradiation, are significantly different from those predicted on the basis of the spectral photon flux distributions of the light sources and optical parameters of purified phytochrome. The geometrical relationships between the light source and the irradiated sample affect the rate of phytochrome photoconversion, which is significantly faster in cabbage seedling laying flat on white, wet filter paper than in seedlings in a vertical position. Light reflected from the white filter paper on the bottom of the dish contributes significantly to phytochrome photoconversion. Substituting the white filter paper with a less reflective black one results in a significant decrease of the rate of phytochrome photoconversion in cucumber cotyledons.

On the Estimation of the Partial Quantum Yields for phytochrome photoconversion by a nonlinear least‐squares method

AbstractA nonlinear least‐squares method is presented which enables the estimation of the partial quantum yields of a photoreversible two component system by fitting the corresponding kinetic equation directly to experimentally measured data.The principles of this method are discussed with reference to the photochromic transformation of phytochrome as an adequate model. It is demonstrated that this method yields sufficiently accurate and highly reliable results.In addition, the advantage of the nonlinear least‐squares method when compared with the so‐called formal integration method and with the initial rate approach is emphasized.

Cryptochrome, Phytochrome, and the Photoregulation of Anthocyanin Production under Blue Light

The principle of equivalent light action predicts that two light treatments (wavelengths ;lambda(1) and lambda(2)) producing the same Pfr/P ratio (phi(lambda1) = phi(lambda2)) and the same rate of phytochrome photoconversion (k(lambda1) = k(lambda2)) are perceived by phytochrome as being the same and should produce the same effect. The results of experiments based on the principle of equivalent light action indicate that cryptochrome is involved in the photoregulation of anthocyanin production elicited by blue light in tomato seedlings. This was also the case for one strain of cabbage seedlings. For another strain of cabbage seedlings, the results suggest that cryptochrome is either not involved or that the state of phytochrome is the principal limiting factor.