Polarotropic Response Research Articles

Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism

Using gene targeting by homologous recombination in Ceratodon purpureus, we were able to knock out four phytochrome photoreceptor genes independently and to analyze their function with respect to red light dependent phototropism, polarotropism, and chlorophyll content. The strongest phenotype was found in knock-out lines of a newly described phytochrome gene termed CpPHY4 lacking photo- and polarotropic responses at moderate fluence rates. Eliminating the atypical phytochrome gene CpPHY1, which is the only known phytochrome-like gene containing a putative C-terminal tyrosine kinase-like domain, affects red light-induced chlorophyll accumulation. This result was surprising, since no light dependent function was ever allocated to this unusual gene.

The Mobility of Phytochrome within Protonemal Tip Cells of the Moss Ceratodon purpureus, Monitored by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a versatile tool for investigating the mobilities of fluorescent molecules in cells. In this article, we show that it is possible to distinguish between freely diffusing and membrane-bound forms of biomolecules involved in signal transduction in living cells. Fluorescence correlation spectroscopy was used to measure the mobility of phytochrome, which plays a role in phototropism and polarotropism in protonemal tip cells of the moss Ceratodon purpureus. The phytochrome was loaded with phycoerythrobilin, which is fluorescent only in the phytochrome-bound state. Confocal laser scanning microscopy was used for imaging and selecting the xy measuring position in the apical zone of the tip cell. Fluorescence correlation was measured at ancient z-positions in the cell. Analysis of the diffusion coefficients by nonlinear least-square fits showed a subcellular fraction of phytochrome at the cell periphery with a sixfold higher diffusion coefficient than in the core fraction. This phytochrome is apparently bound to the membrane and probably controls the phototropic and polarotropic response.

Molecular structure and light-signal perception mechanism of phytochrome: A visual pigment in plants.

Molecular structure and molecular mechanism of photoreception of phytochrome, a photoreceptor for morphogenetic responses in green plants, are reviewed. In the first half, domain structures are discussed based on their proteolytic sites, phosphorylation sites, dimerization sites, e. t.c. And recent topics related to their possible functions, e.g. transmitter module of bacteria sensor protein, ATP-binding motif analogue, are summerized. In the second half, the quartenary molecular and the chromophore structures are discussed based on small-angle Xray scattering and resonance Raman scattering, respectively, and the molecular mechanism of polarotropic responses analyzed based on linear dichroism of oriented phytochrome preparations is discussed.

A MODEL FOR THE MOLECULAR STRUCTURE AND ORIENTATION OF THE CHROMOPHORE IN A DIMERIC PHYTOCHROME MOLECULE*

Abstract—A model for the molecular structure and orientation of red‐light absorbing form of phytochrome (P,) chromophores in a dimeric molecular model of Pr is proposed. A chromophore model with probable molecular structures was generated to reproduce the absorption spectrum produced by its π‐electron conjugating system. The model has C5‐Z, syn, C10‐E, anti and C15‐Z, syn configurations and a protonation at a C‐ring nitrogen. Orientation of the chromophore model in the dimeric phytochrome molecular was analyzed by displaying the atoms of the chromophore, the coordinates of which were converted into those with respect to the molecular axes to the dimeric molecule, on a 3‐D graphic workstation. The conversions were performed by using the azimuthal angles between the Z axis of the dimeric molecule (axis of 2‐fold rotational symmetry) and the dipole moments of the electronic transition at the blue‐ (384 nm) and red‐ (667 nm) absorbing bands of the chromophore, which were calculated as 55.5° and 59.3°, respectively, based on linear dichroism of the oriented phytochrome molecules. The result demonstrates that the long axis of the P, chromophore lies almost parallel to the Y axis of the molecular model, and that the tetrapyrrolic chromophore is well contained within the flat chromophoric domain without protruding from it, a configuration that assures that the chromophore is protected against aqueous environments. The model may explain the rotation angle of the transition moment of the red‐absorbing band, induced by the phototransformation from Pr to Prr which we measured as smaller than that measured in nonoriented preparations by a photoselection technique. The model also suggests a molecular basis for the polarotropic response of phytochrome.

Changes in microtubule and microfibril arrangement during polarotropism inAdiantum protonemata

Polarotropism was induced inAdiantum (fern) protonemata grown under polarized red light by turning the electrical vector 45 or 70 degrees. One hour after the light treatment, tropic responses became apparent in many cells as a slight distortion of the apical dome. Changes in the position of the circumferentially-arranged cortical microtubule band (Mt-band) (Murataet al., 1987) and the arrangement of microfibrils around the subapical part of protonemata were investigated in relation to the polarotropic responses. Twenty minutes after turning the electrical vector, preceding the morphological change of cell shape, the Mt-band began to change its orientation from perpendicular to oblique to the initial growing axis. After 30 min, the Mt-band changed its orientation further under 45 degrees polarized light, but under light rotated 70 degrees, it began to disappear. In phototropic responses induced by local irradiation of a side of the subapical part of a protonema with a non-polarized red microbeam, the Mt-band on the irradiated side disappeared or became faint within 20 min, but neither disappearance nor a change of orientation of Mts occurred on the non-irradiated side. One hour after turning the electrical vector 45 degrees, in half of the cells tested, the innermost layer of microfibrils in the subapical part of the protonema changed its orientation from perpendicular to oblique to the growing axis, corresponding to the changes in the orientation of the Mt-band. After 2 hr, those changes were obvious in all cells examined. The same basic results on the orientation of microfibrils were obtained with protonemata cultured for 2 hr under 70 degrees polarized light. The role of the Mt-band in tropic responses is discussed.

Orientation of the chromophore transition moment in the 4-leaved shape model for pea phytochrome molecule in the red-light absorbing form and its rotation induced by the phototransformation to the far-red-light absorbing form

Orientation of the chromophore transition moment in pea phytochrome (subunit molecular mass, 114 kDa) in the red-light-absorbing form (P r) and its rotation induced by the phototransformation to the far-red-light-absorbing (P fr) form was studied by measuring linear dichroism of oriented phytochrome prepared by a ‘gel squeezing method’. The phytochrome in P r showed a negative linear dichroism. On the contrary, the P fr form showed positive linear dichroism. Assuming that the molecular axis for the two-fold rotational symmetry in the ‘4-leaved shape model’ proposed recently for the dimeric molecular structure of the phytochrome [(1989) FEBS Lett. 247, 139–142], is oriented in parallel with the direction of the external constraint, the average azimuthal angle between the molecular axis and the transition moment of the chromophore was calculated as 59° or 121° for P r and 52° or 128° for P fr. These results explain well the polarotropic responses by phytochrome observed in plant organisms.

Dichroic Orientation of Phytochrome Intermediates in the Pathway from PR to PFR as Analyzed by Double Laser Flash Irradiations in Polarotropism of Adiantum Protonemata

The polarotropic response in protonemata of the fern Adiantum is regulated by phytochrome (Kadota et al. 1984); PR and PFR have been shown to be dichroically oriented parallel and normal to the cell surface, respectively (Kadota et al. 1982). This change in the dichroic orientation of phytochrome during photoconversion was analyzed by a newly-built, polarization plane-rotatable double laser flash irradiator. A polarotropic response was effectively induced with a flash of polarized red (640 nm) light (6×l0−7 s) having the vibration plane of the electrical vector parallel to the protonemal cell axis. When a flash of polarized far-red (710 nm) light (6×l0−7s) was given 30 sec after the red flash, the red flash-induced response was reversed by a far-red flash vibrating normal to the cell axis but not by one vibrating parallel. However, when given 2 μs or 2 ms after the red flash, the polarotropic response was not reversed by a polarized far-red flash vibrating normal to the cell axis but was reversed by a parallel-vibrating flash. These results suggest that the orientation of phototransformation intermediates existing 2 μs or 2 ms after a red flash is still parallel to the cell surface, and that the change in the orientation of phytochrome molecules occurs between 2 ms and 30 s after the red flash.

Phytochrome-mediated polarotropism of Adiantum capillus-veneris L. protonemata as analyzed by microbeam irradiation with polarized light

Perception of polarized light inducing phytochrome-mediated polarotropism in protonemata of the fern Adiantum capillus-veneris L. was analyzed using brief microbeam irradiation with polarized red (R) or far-red light (FR). The polarotropic response inducible by irradiation of the subapical 10-30-μm part with polarized R vibrating parallel to the cell axis was nullified by subsequently giving R at the apical 0-2.5-μm region. This inhibitory effect of R showed an action dichroism, that is, polarized R vibrating normal to the cell axis was effective but the parallel-vibrating R was not. On the other hand, FR irradiation of the extreme tip after irradiation of the whole cell with depolarized R effectively induced a tropic response. This FR effect also showed action dichroism, with parallel-vibrating polarized FR being more effective than FR vibrating normal to the cell axis. When the apical-dome region and the adjacent subapical 10-20-μm region were sequentially irradiated with polarized R vibrating obliquely in different directions, polarotropism took place depending on the vibrating direction of the light given to the apical-dome region. Obliquely vibrating polarized FR given to the apical dome after irradiation of the whole cell with depolarized R also induced polarotropism. Thus, the difference in amount (or percent) of the far-redabsorbing form of phytochrome (Pfr) between the extreme tip and the subapical region appears to be crucial in regulating the direction of apical growth; the difference in Pfr level between the two sides of the protonemal apex may occur mainly at the apical dome. Furthermore, the transition moments of the red-absorbing form of phytochrome (Pr) and Pfr seem to be aligned parallel and normal, respectively, to the cell surface at the periphery of the apical hemisphere.

Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of mutant strains

The phototropic and polarotropic responses of primary chloronemata grown from germinated minated spores of three mutant strains of the moss, Physcomitrella patens, have been studied and compared with those of the wild-type. The mutants and wild-type show the same qualitative tropic responses but differ with respect to the light conditions under which they are expressed. In both the wild-type and mutants the responses are controlled by phytochrome. In monochromatic red light, at low fluence rates, wild-type primary chloronemata grow positively phototropically in unidirectional light or perpendicular to the electrical vector (E) in polarised light; at high fluence rates growth in unidirectional light is lateral to the incident light or, in polarised light, parallel to E. The mutants, however, show only the lateral phototropic or parallel polarotropic responses at all fluence rates of red light tested. In far-red light, the wild-type primary chloronemata adopt a positive phototropic or a perpendicular polarotropic response; the mutants show the same responses but in a lower percentage of filaments. These results and those at other wavelengths indicate either that the mutants are impaired in their ability to adopt the positive phototropic and perpendicular polarotropic responses or that in the mutants the transition between the "low light" (positive phototropic-perpendicular polarotropic) and the "high light" (lateral phototropic-parallel polarotropic) responses is shifted to a lower photon fluence rate. Possible explanations of this phenotypic difference are discussed.

Particle-bound phytochrome from maize and pumpkin.

PHYTOCHROME-induced changes in membrane properties have been inferred from physiological studies for some time1–7. Hendricks and Borthwick6 have proposed that the primary action of phytochrome is indeed the induction of such membrane changes and Smith7 has proposed a mechanism for phytochrome-membrane interaction. The interest in a search for evidence of a direct association between phytochrome and cellular membranes has therefore been considerable. The polarotropic response of Dryopteris8, chloroplast movement in Mougeotia9 and the photoconversion of phytochrome in corn coleoptiles in polarized light10 have all been taken to indicate that at least some phytochrome is located and orientated in the periphery of the cytoplasm or the plasmalemma itself. An immunological approach has been attempted11 and Ptr-induced changes in black lipid membrane permeability have recently been reported12 (Pr and Ptr are the two photo-reversible forms of phytochrome). Rubinstein et al.13 reported that about 4% of the total extractable phytochrome from Avena could be pelleted at 40,000g from a 1,500g supernatant, pH 7.4. Here we report the presence of particle-bound phytochrome in extracts from maize coleoptiles and pumpkin hooks and suggest a mechanism for the interaction of the pigment with its binding site(s).

Red light interactions with blue and ultraviolet light in polarotropism of germlings of a fern and a liverwort.

Abstract— In polarotropism of the chloronema of the fern Dryopteris filix‐mas (L.) Schott and of the germ tube of the liverwort Sphaerocarpos donnellii Aust. a phytochrome action in blue and u.v. was presumed[1, 2]. In the present paper this assumption was tested by simultaneously irradiating with red and blue, and red and near u.v. Red energy is given to shift the phytochrome photoequilibrium in favour of high Pfr/Ptotal concentrations. The data obtained by simultaneous irradiation are consistent with the predictions made under the assumption of a phytochrome involvement in the blue‐ and u.v.‐mediated polarotropic response.

Action spectrum for polarotropism in the chloronema of the fern Dryopteris filix-mas (L.) Schott.

Abstract— A series of dose response curves was worked out under steady state conditions for mediation of the polarotropic response of the chloronema of the fern Dryopreris filix‐mas (L.) Schott. The dose response curves show changes in slope with wavelength. In red and u.v. the slope values are higher than in blue. As a consequence the relative height of the peaks in red, blue, and u.v. and the fine characteristics in blue of the action spectra calculated on the basis of these dose response curves change decisively with different response levels taken for calculation. Therefore, a final decision cannot be made as to what photoreceptor(s) might be involved besides phytochrome. However, at low response levels the action spectrum looks similar to those action spectra described for a variety of other blue‐u.v.‐mediated photoresponses, which generally are believed as being indicative for a flavin. The findings clearly indicate that in cases such as Dryopteris and in cases, where action spectra were done only at one arbitrarily chosen dose, action spectroscopy may not allow the unequivocal identification of the photoreceptor(s) involved.

Dose response behaviour for polarotropism of the germ tube of the liverwort Sphaerocarpos Donnellii aust.

The dose response behaviour for polarotropism of the unicellular germ tube of Sphaerocarpos was studied in blue and near UV. At constant intensities the response is proportional to the logarithm of the exposure duration, and at constant exposure durations the response is proportional to the logarithm of intensity. Neither a polarotropic response to wavelengths > 550 nm, nor a significant influence on response to blue of a pre-, post-, or simultaneous-irradiation with red or far-red could be observed. Also a dark period was without any effect on the polarotropic response to blue. Growth responses under linearly polarized light are described.

Tropic Responses of Funaria Spores to Red Light

The tropic responses of Funaria hygrometrica spores to continuous illumination with red light (610 to 690 mmu) have been studied over the intensity range from 10(-5) through 10(+6) erg/cm(2) second, using both plane polarized light and partial illumination with unpolarized light. From the relative frequency of outgrowth origin in different directions, the following is inferred. (1). The germination direction of chloronemal filaments is directly influenced by red light over this whole intensity range, while that of rhizoids tends to be opposite the chloronema. (2) Three photoreceptor systems direct chloronemal primordia: (a) A low intensity system acting from 10(-5) to 10(-0.5) erg/cm(2) second. It favors their growth from a cell's brightest part(s). Its photoreceptors are disoriented, excited by the electric vector, and probably are dispersed phytochrome molecules. (b) A medium intensity system which acts largely alone only at 10(0.5) erg/cm(2) second but is influential from 10(0) to 10(5) erg/cm(2) second. It likewise favors growth from a cell's brightest part(s); its receptor molecules are also excited electrically, but they are tangentially oriented. (c) A high intensity system which acts alone from 10(5) to 10(6) erg/cm(2) second and is influential down to 10(1) erg/cm(2) second. It favors growth of the chloronemas from a cell's darkest part. Its receptors probably are magnetically excited and tangentially oriented. The polarotropic responses of the chloronemas resemble those directing their origins. One new feature is that under intense (10(6) erg/cm(2) second) plane polarized and vertically directed light, many soon grow to form tight helices.

Der Polarotropismus und Phototropismus der Chloronemen von Dryopteris filix mas (L.) Schott

Previous results had shown that vertically directed polarized red light orients the growth of horizontally growing chloronemas of Dryopteris filix mas (L.) Schott in either direction perpendicular to the plane of vibration, i. e. the electric vector. This polarotropic response has been investigated more closely. 1. The effect was measured in terms of angle formed during bending of preoriented chloronemas in response to a turn of the vibration plane by 50°. 2. The orientation perpendicular to the vibration plane occurs throughout the visible spectrum. The action spectrum in the visible region has two peaks: one around 665 nm and another one, about hundred times higher, in the blue. 3. From the action spectrum and other results it must be assumed that polarotropism in the blue and the red far-red region is mediated by two different pigment systems. 4. As had been previously shown, far-red reduces the width of the chloronemas as compared to red and dark. This effect is clearly visible in the region between 707 and 747 nm. Its intensity dependence parallels that of polarotropism by the same wavelengths, and on the basis of this observation and other data it may be assumed that both effects are mediated by the same pigment. 5. The morphogenetic response, that is the development of a prothallium from the chloronema in blue light requires much higher doses than the blue light polarotropic response and starts at intensities at which saturation of polarotropism has almost been reached. From this it is assumed that these effects are mediated by two different pigment systems. 6. Partially illuminated chloronema tips react by bending toward the side of the illuminated flank. This is the case in both red and blue light. 7. From the results with polarized light and partial illumination it is inferred that both blue and red light absorbing pigment molecules are dichroic and located close to or in the cell wall with their axes of maximum absorption parallel to the cell surface. 8. These results allow a detailed description of the optics which are responsible for the absorption gradient in phototropism by unpolarized red and blue light. The gradient is caused by the screening effect, the lens effect creating a by-passed subequatorial zone, and possibly by the orientation of the dichroic pigment molecules, all of them leading to a gradient with more absorption on the side facing the light. 9. By dropping rice starch grains on chloronema tips and following their position during positive phototropic bending it was determined that the tip curvature is caused by bulging of the inner rather than by increased growth of the outer side. Thus in polarotropism and positive phototropism the side that absorbs more light has the higher growth rate. 10. The effect of polarized red light is not reversed by subsequent polarized farred but is instead slightly increased. 11. Adaptation by red light reduces the sensitivity to polarized red, but enhances the effect of polarized far red light as compared to dark adaptation. 12. Adaptation by red light also reduces the sensitivity to polarized blue light. 13. Unpolarized far-red partially reverses the effect of red light adaptation on red light sensitivity. 14. Unpolarized far-red given after polarized red light reduces the effect of the latter. 15. Assuming that phytochrome controls the effect in the red and far-red, all results can be satisfactorily interpreted only if one assumes that the axis of maximum absorption of phytochrome turns by 90° during the transition of the red absorbing form (Pr) to the far red absorbing form (Pfr), leading to an orientation of the Pfr molecules perpendicular to the cell surface. 16. From the interaction between red and far-red and red and blue it is assumed that adaptation by red light consists of a saturation with Pfr as well as with intermediate products formed subsequently to Pfr. Only saturation with the latter would interfere with the blue light response. 17. Growth measurements together with other results indicate that although the average growth rate of the chloronemas does not change over several days, the growth rate of individual chloronemas shows considerable fluctuations, perhaps representing regular oscillations with periods of several hours duration.

THE CONTROL OF PLANT GROWTH AND DEVELOPMENT BY LIGHT

THE CONTROL OF PLANT GROWTH AND DEVELOPMENT BY LIGHT

Tropistic responses of zygotes of the Fucaceae to polarized light

The following picture is drawn from experiments on two members of the Fucaceae, Fucus furcatus and Pelvetia fastigiata: 1. 1. In their “polarotropic” response to unidirectional illumination with plane polarized visible light, the zygotes tend to germinate in the plane of vibration, and “subequatorially” (from 90 ° to 135 ° away from the source). (Fig. 1.) Up to half the embryos so produced may be bipolar forms. 2. 2. The tropistic response to similar unpolarized light is dual. Under some partially defined conditions it is subequatorial; under others, directly away from the source. 3. 3. The polarotropic response and the subequatorial response to unpolarized light fade out over the same intensity range (Fig. 4). 4. 4. The polarotropic action spectra (Fig. 3) belong to the phototropic group of spectra as found with other organisms (Table VI). 5. 5. It is concluded that in all three tropistic responses to light, growth tends to occur where certain photoreceptor molecules absorb the least light. 6. 6. It is suggested that under unpolarized illumination the subequatorial and peripheral photoreceptors absorb least light primarily because they are by-passed by the cell's focusing action (Fig. 5), while under polarized illumination the subequatorial photoreceptors in the vibration plane absorb least light because they are periclinally oriented (Fig. 6).