Action Dichroism Research Articles

Action dichroism in the chloroplasts rearrangements in various plant

Action dichroism in the chloroplasts rearrangements in various plant

Nuclear and cytosolic events of light-induced, phytochrome-regulated signaling in higher plants.

Plants as sessile organisms cannot walk away but have to adapt to changes in their natural environments. Light is obviously the most important external factor, being the energy source. To regulate their fitness, the photosynthetic capacity of plants must be adapted to changes in the ambient light environment. Therefore, plants have evolved a large set of photoreceptors to monitor light quality and quantity, ranging from the UV to the infrared part of the spectrum. At least three different photoreceptor classes have been identified and analyzed: (i) UV‐B receptors, primarily characterized by action spectroscopy (Wellmann, 1983); (ii) the blue UV‐A photoreceptors, cry1 and cry2 and phototropine (Ahmad and Cashmore, 1993; Christie et al ., 1998; Lin et al ., 1998); and (iii) the red/far‐red reversible phytochromes. These photoreceptors control different aspects of plant growth and development, and in this review we focus on the physiological and molecular events regulated by the most characterized plant photoreceptors, the phytochromes. In higher plants, phytochromes are encoded by a small multigene family; five genes ( PHYA–PHYE , Sharrock and Quail, 1989; Clack et al ., 1994) have been identified in Arabidopsis . Phytochromes exist as dimers composed of two 125 kDa polypeptides, each carrying a covalently linked tetrapyrrol chromophore in the N‐terminal domain and dimerization domains in the C‐terminal domain. The photosensory function of the molecule is based on its capacity for reversible interconversion between the red light‐absorbing Pr form and the far‐red light absorbing Pfr form following sequential absorption of red and far‐red light. Photosignal perception by the receptor is followed by conformational changes, and activates, through an as yet poorly understood mechanism, signaling pathways leading to changes in the expression of genes that underlie developmental responses to light (Quail et al ., 1995). In order to finely tune their …

Dual involvement of phytochrome in light-oriented chloroplast movement in Dryopteris sparsa protonemata

The low-fluence-rate response of light-oriented chloroplast movement in Dryopteris sparse protonemata was induced by continuous irradiation with either red (R) or blue light (B). Effects of concomitant irradiation with far-red light (FR) indicated that the photoreceptor of R was phytochrome and that of B, a specific B photoreceptor. Action dichroism of light suggested that the absorption axes of the R-absorbing form of phytochrome and the B photoreceptor were parallel to the cell surface, and that of the FR-absorbing form of phytochrome (P fr), perpendicular. When irradiation was terminated, the B-induced chloroplast arrangement began to decay immediately. In contrast, the R-induced arrangement survived for approximately 3 h, but when R irradiation was terminated with an FR pulse, the decay began after 1 h. When R irradiation was terminated before the response reached the maximum, it stayed at the attained level for 3 h, but did not increase further. These results suggest that the R induction of chloroplast movement required not only the presence of a P fr gradient, but also the continued asorption of R, the former is only effective in determining the direction of the movement and in holding the chloroplasts at the area of higher P fr concentration.

Action dichroism in perception of vectorial photo-excitation in the solar-tracking leaf ofLavatera cretica L.

The leaf lamina ofLavatera cretica L. exhibits a diaphototropic response that discriminates between two opposite, constant vectorial excitations by white light beams whose fluence rates differ by as little as 10% (50 versus 45 μmol·m(-2)·S(-1)). The relationship between the response (angular velocity of laminar reorientation) and the fluence-rate ratio is linear. The lamina similarly discriminates between two such excitations by polarized light, one with the electrical vector transverse to the plane of the two beams (θ) and the opposite one with the vector parallel to that plane ([Symbol: see text]). When two such beams were of equal fluence rate, the lamina reoriented towards the [Symbol: see text] beam. When the fluence rate of the θ beam was maintained at 50 μmol·m(-2)·s(-1) and that of the [Symbol: see text] beam was reduced, the response to the latter (angular velocity of laminar reorientation) was reduced progressively. Further reduction in the fluence rate of the [Symbol: see text] polarized beam eventually resulted in reorientation in the opposite direction (towards the θ beam) and the response to the latter increased progressively with the reduction in fluence rate. The equilibrium was at a [Symbol: see text]/θ ratio of 0.62. Measurements of reflectance of oblique beams of [Symbol: see text] and θ polarized light from the upper laminar surface, and of transmittance of such light ghrough the lamina, eliminated the possibility that optical dichroism of the lamina contributed significantly to these results. The implications of this action dichroism to the postulated mechanism of perception of vectorial excitation by these leaves is discussed.

High-fluence rate responses in the light-oriented chloroplast movement in Adiantum protonemata

Light-oriented chloroplast movement in the protonema of the fern Adiantum capillus-veneris L. was studied using the techniques of 2-h local and whole-cell irradiation. Local irradiation of protonemata with high-fluence rate blue light (BL) or red light (R) moved chloroplasts out of the irradiated area (negative response). Whole-cell irradiation with high-fluence rate BL accumulated chloroplasts at the flanks of cells (parastrophe). In the latter experiment, polarized light having the vibration plane perpendicular to the protonema axis was more effective than unpolarized light, and parallel polarized light was ineffective. All these responses were the reverse of those induced by low-fluence rate R and BL irradiation. A whole-cell irradiation with high-fluence rate R accumulated chloroplasts at the front and the rear of cells (diastrophe) in the same way as that with low-fluence rate R. It was also found that the high-fluence rate BL prevented R actions in chloroplast movement. That the photoceptors involved in these high-fluence rate responses were the same as those for the low-fluence rate responses, a BL photoreceptor for BL and phytochrome for R, was deduced from the effects of local and whole-cell irradiations with R and far-red light as well as from the action dichroism observed with the whole-cell irradiation. Possible mechanisms of the negative responses are discussed.

No Action Dichroism for Light-controlled Fern-spore Germination

Summary Action dichroism for phytochrome-mediated light control of fern-spore germination was searched for in several irradiation programs. The test irradiations consisted of a pair of linearly polarized red pulses with their planes of polarization identical or normal to each other. In no instance did these two combinations give different results, and thus, no action dichroism could be demonstrated. A theoretical calculation clearly shows that an action dichroism should be detected, if phytochrome were structurally fixed, provided some conditions are met. The negative result is discussed as a basis for further improvement of the experiments, for detecting or ruling out an action dichroism in non-directional light responses.

Blue light regulation of stomata in wheat seedlings. II. Action spectrum and search for action dichroism

The stomatal conductance response to low intensity blue light was studied in wheat seedlings (Triticum aestivum L. cv. Starke II, Weibull) under red background illumination. Reciprocity was shown to be valid for illumination times from 10 s up to about 2 min. The action spectrum, constructed from fluence rate response curves, showed a maximum peak at 445–450 nm, another peak at 470 nm, a slight shoulder at 420 nm and a plateau between 370–400 nm. The relationship with action spectra for other blue light responses is discussed. The blue light response of wheat stomata did not exhibit action dichroism (the direction of the electrical vector of polarized blue light did not influence the response of the guard cells).

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.

Light‐induced linear dichroism in photoreversibly photochromic sensor pigments. – V. Reinterpretation of the experiments on in vivo action dichroism of phytochrome

Experiments by several authors on the effects of polarized light on phytochromemediated responses in fern gametophytes and in the green alga Mougeotia have earlier been interpreted as showing that the transition moment of phytochrome in the Pr form is parallel to the plasmalemma, but perpendicular to the plasmalemma for the Pfr form of phytochrome. It is now shown that the experimental results can be interpreted differently, and that they are also consistent with a chromophore rotation of about 30° (instead of 90°), as found for immobilized phytochrome molecules in vitro. Thus there is no evidence for a rotation of the whole phytochrome protein. For the gametophyte of Adiantum it is calculated that the Pr transition moment is inclined 17° to the plasmalemma, and the Pfr transition moment ca 50°, corresponding to an in vivo chromophore rotation of ca 33°; however, these values are very approximate.

WAVELENGTH‐DEPENDENT ACTION DICHROISM: A THEORETICAL CONSIDERATION

Abstract— For a model structure the wavelength dependence of action dichroism is computed as a function of the angle(△) between the transition moments at two different wavelengths. With as small an angle as △α= 20. inversion of action dichroism for parallel vs perpendicularly vibrating radiation is expected for different absorption bands if the sensory pigment molecules are in proper orientations. Thus published data on opposite action dichroism in the blue vs near‐UV range are compatible with flavins as sensory pigments, although the calculations must not be considered as proof for the flavin hypothesis.

LIGHT‐INDUCED LINEAR DICHROISM IN PHOTOREVERSIBLY PHOTOCHROMIC SENSOR PIGMENTS–II. CHROMOPHORE ROTATION IN IMMOBILIZED PHYTOCHROME

Abstract‐Large phytochrome immobilized via anti‐phytochrome immunoglobulin bound to Sepharose beads was irradiated to saturation with unpolarized far‐red light. The apparent absorbance level was recorded in a dual wavelength spectrophotometer with both measuring beams set to either 660 or 730 nm and polarized perpendicular to each other. The sample was then irradiated with red polarized light. The apparent change in absorbance obtained after this irradiation indicated that purified phytochrome could show linear dichroism. From the absorbance values obtained it was computed that the direction of the long‐wavelength transition moment changes by either 32 or 148o, when phytochrome is transformed from Pr to Pfr. Considering the model of Hahn and Song (1981) the latter value appears more likely. In light of these results, the conclusions drawn from in vivo experiments on action dichroism in Dryopteris (Etzold, 1965), Adiantum (Kadota et al., 1982) and Mougeoutia (Haupt. 1970), which point to a 90o rotation. should be reconsidered.

LIGHT‐INDUCED LINEAR ACTION DICHROISM IN PHOTOREVERSIBLY PHOTOCHROMIC SENSOR PIGMENTS—I. THEORY

Abstract— With a photoreversibly photochromic regulator pigment such as phytochrome, linear action dichroism could theoretically be obtained after photoselection even if the molecules are initially randomly oriented: If randomly oriented Pfr (fed‐absorbing phytochrome) molecules are partially converted to Pfr (far‐red absorbing phytochrome) molecules by plane‐polarized red light, those molecules will preferentially be converted which have their ‘red’ transition moments nearly parallel to the electric vector of the red light. The effect of subsequent plane‐polarized far‐red light will depend on the plane of polarization. A general theory is developed for how this can be used to determine whether or not the transition moment changes direction during conversion. The pigment need not be isolated, since only physiological reactions (such as germination or chromatic adaptation) are measured.

Phytochrome-controlled chloroplast orientation in mougeotia: Action dichroism in double-flash experiments

The phytochrome-mediated chloroplast orientation of Mougeotia has been induced by flashes of red light of a duration of 3 msec each. Two flashes are more than additive if separated by a proper dark interval. As a result of this paper, in the ascending part of the fluence-response curves there is no difference in the action dichroism of the two flashes: whenever one flash is given with the electrical vector normal to the cell axis, for the other flash parallel vibrating light is at least as effective as normal vibrating light. This does not support the earlier hypothesis that phytochrome in the red absorbing form (P r) changes its dichroic property as an effect of the first flash [1]. Instead, the same interpretation is favored as in lettuce and oat [2,3], according to which the second flash attains its full effect not until dark relaxations of phytochrome intermediates back to P r are completed, thus photoconverting more P r to P fr than without the dark interval. It cannot be excluded, however, that in Mougeotia additional effects have to be taken into consideration. The fluence-response curves of the second flash exhibit a particular shape, viz. dropping with higher fluences after having passed an optimum value. This levelling of the P fr gradient had to be expected if the flash duration were short compared with the sequence of reactions from P r to P fr and hence no photostationary state could be established during the flash. The drop of the fluence-response curve shows an interesting action dichroism.

Blitzlichtinduktion der Chloroplastenbewegung bei Mougeotia: Wirkung unterschiedlicher Spektralbereiche and Polarisationsrichtungen: Induction of Chloroplast Movement in Mougeotia with Light Flashes: Spectral Sensitivity and Action Dichroism

Summary Phytochrome-mediated chloroplast movement is induced by flashes of red light with a duration of about 3 ms each. If two flashes are given (-double-flash- inductions), they are separated by a dark interval of 20 s. The spectral distribution of the applied flashes has its maximum near 640 nm and 670 nm, respectively. The following results have been obtained: 1) The effect of a single flash is strongly reduced by preirradiation with far-red (FR; Fig. 1). Without FR, 670 nm is double as effective as 640 nm (Fig. 2). 2) In double-flash experiments after saturating FR with the 2nd flash kept constant, 640 and 670 nm are about equally effective when given as a first flash (Fig. 3). The -difference in the spectral sensitivity has been expected: in the first condition (1), we start with part of the phytochrome in the Pfr form. Thus longer wavelength light transforms Pfr back to Pr at the cell flanks, enhancing the intracellular Pfr gradient which is responsible for the chloroplast movement. In the second condition (2), however, the flash can act only on Pr. We conclude that the spectral sensitivity can be used as an indicator to decide whether the flash acts mainly on Pr or on Pfr. 3) In double-flash experiments after saturating FR with the 1st flash kept constant, 640 and 670 nm are equally effective as a second flash (Fig. 5). We conclude: although the first red flash is expected to have transformed some Pr to Pfr, the effect of the second flash is still mainly its action on Pr rather than on Pfr. 4) Polarized red light as a first flash is about equally effective with the electrical vector oriented parallel or perpendicularly to the cell axis (Fig. 7). Only the second flash exhibits the well-known action dichroism in its typical form. We conclude that after saturating FR the first flash acts mainly on unoriented Pr, i. e. -free- phytochrome. The main effect of the first flash is assumed to be -binding- of Pr to Verwendete Abkurzungen: HR (R) = Hellrot (ca. 640 nm); MR = Mittelrot (ca. 670 nm) ; DR (FR) = Dunkelrot (> 700 nm) ; Pr, Pfr = Phytochrom in der Hellrot- bzw. Dunk elrot-absorbierenden Form.

Phytochrome-mediated response in Mougeotia to very short laser flashes

Chloroplast orientation to the light direction in the green alga Mougeotia could be induced with high efficiency by flashes of 100 ns duration and a wavelength of 694 nm if we started with phytochrome mainly in the P fr form. With increasing fluences, however, an optimum was surpassed, and no response was found in the highest fluences. In addition, the well-known action dichroism disappeared near the optimum fluence and even may have been reversed. In contrast, after saturating FR, i.e. with phytochrome in the P r form, no response could be induced, neither by single nor by repetitive laser flashes. These preliminary results do not as yet allow far-reaching conclusions.

Lichtorientierte Chloroplastenbewegung bei Hormidium flaccidum: Verschiedene Methoden der Lichtrichtungsperception und die wirksamen Pigmente

Summary Over a broad range of intensities of white light, the chloroplast orients itself so as to expose its face to the light by approaching either the front or the rear cell wall in relation to the light direction. At medium intensities, the lens effect of the cell determines whether the chloroplast goes to the rear (in air as surrounding medium) or to the front (in oil). At higher as well as at low intensities, however, the orientation is always to the front regardless of any lens effect; hence it is assumed that an absorption gradient due to intracellular shading is formed. This points to photoreceptor systems localized in different cell compartments whose physiological separation will be discussed in this paper. Both these systems are characterized by an action dichroism: the «lens system» (system A) operates if polarized light vibrates perpendicular to the cell axis, whereas the «shadow system» (system B) is mainly observed in light vibrating parallel to the cell axis (fig. 5). System A can be partly inhibited by potassium iodide, which does not influence system B (fig. 2). It is suggested that a flavin at or near the plasmalemma is the photoreceptor pigment of system A. This is favoured by the following experiments and observations: After 5 days in darkness, system A becomes less effective for the orientation in relation to system B (fig. 4); at the same time, the level of flavin mononucleotide (FMN) decreases by about 40 %. If then FMN is added, the efficiency of system A is quickly regained. Hence this system may be compared with the photoreceptor system of chloroplast orientation in many other plants, which is attributed to flavin compounds associated with the plasmalemma. In system B, in addition to blue light, red light is also effective to some degree (fig. 1). Whereas polarized blue light must vibrate parallel to the cell axis in order to be effective, polarized red light must vibrate perpendicularly (fig. 8). The red effect can be selectively abolished by inhibitors of photosynthesis, e.g. by DCMU. From this, it is concluded that dichroic oriented chlorophyll is the photoreceptor pigment of system B (fig. 9). Since, however, the blue light effect of system B is not inhibited by DCMU and since this blue light is much more effective than red light, an additional photoreceptor pigment also localized in the chloroplast is suggested, this may be a carotenoid. Thus the observed chloroplast orientation is always an interaction of different photoreceptor systems establishing different absorption gradients according to the surrounding medium (which influences the pathways of the light rays), the light intensity, the wavelength and the polarization plane. The contribution of each of these systems can be furthermore modified by experimental manipulation (cf. fig. 3).

Untersuchungen zum Photoreceptorproblem beim tonischen Blaulicht-Effekt der Starklichtbewegung des Mougeotia-Chloroplasten

Summary If irradiated simultaneously from different directions with blue light of high intensity and with red light of low intensity, the chloroplasts of Mougeotia are oriented by red rather than by short wave length light. The main effect of blue light seems to be, in this case, to change the chloropla sts from a positive to a negative phototact ic response, oriented by phytochrome. Thus, the additional given blue light has a tonic effect, i. e. independent of its direction (SCHONBOHM, 1966 b, 1967 a). It seems to be possible, that the difference between the action spectra of the high intensity movement of Mougeotia-chloroplasts (SCHONBOHM, 1963) and the action spectra of chloroplast movements of other objects (HAUPT, 1963; HAUPT and SCHONBOHM, 1970) is caused by the fact that both photoperception in phytochrome and photoperception in a yellow pigment arc represented by the action spectra of chloroplast movement of Mougeotia obtained when irradiating only with a single radiation source. In the following experiments we will record action spectra for the tonic blue light effect only; we have to take care of the action-dichroism, which we have found in former investigations (SCHCNBOHM, 1968). Polarized red light vibrating perpendicularly (r ⊥ or parallel (r ∥) to the cell axis is given simultaneously with blue light (bl). The following results have been obtained in the present investigations. 1. a) The action spectrum for the tonic effect of unpolarized blue light (bl *. with r ⊥ does not differ remarkably from the action spectra for the high intensity movement induced by blue light only (ef. fig . 2 with fig. 1). b) If we compare the action spectra for the effect of unpolarized blue light (bl *. with or without r ⊥ with the action spectra for the tonic effect of polarized blue light (bl ∥. with r ⊥) in the latter we can see a pl ateau reaching from ca. 450 nm to 470 nm instead of one peak near 465 nm (fig. 4). In the latter action spectra we do not find a relativel y high peak in the near ultraviolet as we did in the former action spectra (bl *, with or without r ⊥). 2. a) It has been demonstrated that polarized blue light (λ : ca. 400-500 nm) is tonically much more effective, if vibrating parallel to the cell axis (bi ∥, with r ⊥ than if vibrating perpendicularly (bl ⊥, wirh r ⊥) (fig. 3). b) This action dichroism is inverse, if Mougeotia is irradiated with ultraviolet light (λ = 363 nm) rather than with polarized visible blue light (λ > 400 nm) (fig. 7). c) If the wavelength of the blue light is longer than 400 nm the action dichroism for the tonic blue light effect remains constant during the whole day, whereas it is changing in a diurnal rhythm in near ultraviolet light (fig. 8). d) Action dichroism for the tonic blue light effect of the high intensity movement of Mougeotia-chloroplasts cannot be caused by an action dichroism of a countercurrent «low intensity movement» which is induced simultaneously by the same intensities of polarized blue light as are needed for high intensity movement (ef. fig. 9 and 10).