Photoregulation of hypocotyl growth: geotropic evidence for the operation of two photosystems

Abstract. The rate of curvature of etiolated cress ( Lepi-dium sativum L. ) hypocotyls in response to gravity (negative geotropism) was retarded by red or blue light; far-red irradiation was without effect. The timing of the irradiation period in relation to the presentation for geostimulus markedly affected the response. When seedlings were irradiated during the 1–2 h period of geostimulus, blue light was more effective than red at retarding curvature; when seedlings were irradiated prior to geostimulus, only red light affected geocurvature. These results are interpreted as a further example of the kinetically distinct effects of red and blue light on hypocotyl development. Blue light elicited a rapid, immediate response effective only during the period of irradiation; red light induced a response characterized by a lag period and persistence in subsequent darkness. Etiolated mustard seedlings showed similar responses to light and gravity. The results are discussed in relation to the possibility that two photosystems operate in hypocotyl growth.     

Reorientation of microtubules at the outer epidermal wall of maize coleoptiles by phytochrome,blue-light photoreceptor,and auxin

Summary The effects of red and blue light on the orientation of cortical microtubules (MTs) underneath the outer epidermal wall of maize (Zea mays L.) coleoptiles were investigated with immunofluorescent techniques. The epidermal cells of dark-grown coleoptiles demonstrated an irregular pattern of regions of parallel MTs with a random distribution of orientations. This pattern could be changed into a uniformly transverse MT alignment with respect to the long cell axis by 1 h of irradiation with red light. This response was transient as the MTs spontaneously shifted into a longitudinal orientation after 1–2 h of continued irradiation. Induction/reversion experiments with short red and far-red light pulses demonstrated the involvement of phytochrome in this response. In contrast to red light, irradiation with blue light induced a stable longitudinal MT alignment which was established within 10 min. The blue-light response could not be affected by subsequent irradiations with red or far-red light indicating the involvement of a separate blue-light photoreceptor which antagonizes the effect of phytochrome. In mixed light treatments with red and blue light, the blue-light photoreceptor always dominated over phytochrome which exhibited an apparently less stable influence on MT orientation. Long-term irradiations with red or blue light up to 6 h did not reveal any rhythmic changes of MT orientation that could be related to the rhythmicity of helicoidal cell-wall structure. Subapical segments isolated from dark-grown coleoptiles maintained a longitudinal MT arrangement even in red light indicating that the responsiveness to phytochrome was lost upon isolation. Conversely auxin induced a transverse MT arrangement in isolated segments even in blue light, indicating that the responsiveness to blue-light photoreceptor was eliminated by the hormone. These complex interactions are discussed in the context of current hypotheses on the functional significance of MT reorientations for cell development.Abbreviations MT cortical microtubule - P_r, P_fr red and far-red absorbing form of phytochrome     

THE PHOTOINHIBITION OF GROWTH IN ETIOLATED STEM SEGMENTS. III. FAR-RED REVERSIBILITY OF BLUE LIGHT EFFECTS IN PISUM

Bertsch , Walter F. (Yale U., New Haven, Conn.) The photoinhibition of growth in etiolated stem segments. III. Far-red reversibility of blue light effects in Pisum. Amer. Jour. Bot. 50(8):754–760. Illus. 1963.—Etiolated pea stem segments were used to study the relationship between blue light effects and the red, far-red reversible photoinhibition of growth. The irradiation periods were too short to give an appreciable effect of high-energy, nonreversible photoreactions. Filters which were used to isolate blue light are specified, contaminations by other wavelengths being extremely small. It was found that photoinhibitions due to uncontaminated blue light were reversible by subsequent far-red irradiation, regardless of whether a broad band of blue wavelengths, or the 4358-A emission line of mercury, was used. Blue light (4800 A) was about 0.3-1% as effective as red (6600 A) light. Changes in the incubation medium caused the same changes in photosensitivity to blue as to red irradiations. These observations are presented as evidence supporting the hypothesis that the pigment moiety of phytochrome absorbs blue light.     

Photocontrol of spore germination in the fern Thelypteris kunthii

The light requirement for germination in spores of the fern Thelypteris kunthii (Desv.) Morton was fully satisfied by a long period of continuous red light or partially by intermittent, short periods of red light. Red light-potentiated spore germination was inhibited by brief far-red light irradiation, indicating phytochrome involvement. Repeated exposure of spores to prolonged red and short far-red irradiations, or exposure of red-potentiated spores to far-red light after an extended period in darkness, led to their escape from inhibition of germination by far-red light. Prolonged irradiation of spores with blue light before or after red light treatment partially antagonized the effect of red light.     

The effect of blue light on energy levels in epidermal strips

Red light applied together with blue enhanced stomatal opening in epidermal strips of Commelina communis L. more than red light alone. In red light, stomatal opening was enhanced by exogenously applied ATP and was inhibited by 3-(3,4-dichlorophe-nyl)-l,l-dimethylurea (DCMU), while in the presence of blue light external ATP was almost without effect, and DCMU stimulated stomatal opening. Blue light increased the ATP levels in the epidermal strips. DCMU diminished the amount of ATP in both red light and red + blue light treatments, but did not abolish the stimulatory effect of blue light. Blue light also stimulated the respiration rate of the epidermal strips. Rotenone, which inhibited stomatal opening and respiration rate, abolished the effect of blue light in both processes. These results imply that blue light increases the ATP levels by stimulation of oxidative phosphorylation.     

Phytochrome-membrane interactions as a factor in stomatal opening

Red light enhances stomatal opening in Commelina communis L. This light effect is reversed by far-red irradiation. Pretreatment with filipin, which competitively inhibits phytochrome binding to membranes, also inhibits light-enhanced opening. The pretreatment with filipin is more inhibitory if preceded by red irradiation, than after far-red irradiation. Similar results are obtained with cycloheximide and low temperature, which retard phytochrome synthesis more than its degradation. This result may indicate an enhanced release of phytochrome in the Pfr form from binding sites rather than release of phytochrome in the Pr form. This points towards the possibility that phytochrome degradation and its release from binding sites are coupled.     

The phytochrome-deficient pcd2 mutant of pea is unable to convert biliverdin IXα to 3(Z)-phytochromobilin

During screening of ethylmethane sulphonate-mutagenized pea ( Pisum sativum L.) seedlings under far-red light a mutant line, AF130, was isolated which showed a reduction in both red and far-red light-induced de-etiolation responses. The photomorphogenic phenotype of AF130 results from a single recessive mutation which is not allelic with the previously described phytochrome chromophore biosynthesis mutant pcd1 . This new mutant has been designated pcd2 , for p hytochrome c hromophore d eficient 2. Like pcd1 , etiolated pcd2 seedlings are severely deficient in spectrally active phytochrome and contain wild-type levels of phytochrome A apoprotein which is not substantially depleted by red light treatment. Etioplast preparations from pcd2 seedlings can metabolize heme to biliverdin (BV) IXα, but are unable to convert BV IXα to the phytochrome chromophore, phytochromobilin. The PCD1 and PCD2 genes therefore control consecutive steps in phytochromobilin synthesis. Despite a similarly severe impairment of photomorphogenic responses, pcd2 mutant seedlings do not display the strongly chlorotic phenotype of pcd1 , suggesting that this characteristic of pcd1 does not result from phytochrome deficiency per se , but is a specific effect of the pcd1 mutation. A double mutant between pcd1 and pcd2 was constructed. This mutant is paler than pcd1 and less responsive to red light than either single mutant, but retains a strong response to blue light.     

Light-dependent osmoregulation in pea stem protoplasts. photoreceptors, tissue specificity, ion relationships, and physiological implications

Light-induced changes in the volume of protoplasts bathed in a medium of constant osmolarity are useful indications of light-dependent cellular osmoregulation. With this in mind, we investigated the effect of light on the volume of protoplasts isolated from the elongating stems of pea (Pisum sativum) seedlings raised under red light. The protoplasts were isolated separately from epidermal peels and the remaining peeled stems. Under continuous red light, the protoplasts of peeled stems swelled steadily, but those of epidermal peels maintained a constant volume. Experiments employing far-red light and phytochrome-deficient mutants revealed that the observed swelling is a light-induced response mediated mainly by phytochromes A and B with a little greater contribution by phytochrome A. Protoplasts of epidermal peels and peeled stems shrank transiently in response to a pulse of blue light. The blue light responsiveness in this shrinking response, which itself is probably mediated by cryptochrome, is under the strict control of phytochromes A and B with equal contributions by these phytochromes. We suggest that the swelling response participates in the maintenance of high tissue tension of elongating stems and that the shrinking response is involved in stem growth inhibition. Other findings include the following: The swelling is caused by uptake of K+ and Cl-. The presence of Ca2+ in the bathing medium is required for phytochrome signaling in the swelling response, but not in the response establishing blue light responsiveness. Phytochrome A mediates the two responses in a totally red/far-red light reversible manner, as does phytochrome B.     

The Influence of Light on Geotropism in Cress Roots

Light affects the growth and orientation of roots of cress seedlings(Lepidium sativum L. cv. Curled). The effects are manifest eitheras increased rates of geotropic curvature or, if the roots arehorizontal, as distorted and crinkled forms of growth. Blue,red, and far-red irradiation can bring about these effects,but with differences of detail: at equal fluence rates duringthe period of geostimulus, blue is more effective than red atincreasing the rate of geocurvature; however, with irradiationprior to a geostimulus, only the stimulatory effects of redirradiation persist for 2–4 h of darkness. Short periods(5 min) of radiation, if given at the time of geostimulus, enhancegeocurvature, again with blue most, and far-red least, effective,but there are no clear indications of red/far-red reversibility.The possibility of there being more than one photosystem responsiblefor the effects of white light on the geotropic responsivenessof roots is discussed.     

Effects of red, far-red and blue light on the viscosity of the cytoplasm of wheat leaf cells

The displacement by centrifugation of the cell contents of wheat ( Triticum aestivum L. cy. Weibull's Starke) was studied after various light treatments. In dark-grown leaves the viscosity of the cytoplasm, measured as the time necessary to displace the cell contents, is low, but increases slowly during continuous red irradiation as well as after a short red pulse. The increase after a red light pulse can be nullified by a short far-red irradiation which in itself has no effect. Unlike that found earlier for Elodea densa Casp., and verified in the present study, the cytoplasm of wheat leaves does not show any rapid response to blue light, not even after pretreatment with red light.     

Further support for the involvement of phytoehrome in stomatal movement

The involvement of phytochrome in stomatal movement in Commelina communis L. is indicated by the following observations: 1) Short irradiation with red or blue light causes opening, of isolated stomata and swelling of guard cell protoplasts. This is reversed by subsequent far red irradiation. 2) In a similar way, stomatal response to prolonged irradiation with red or blue light is decreased by concomitant far red irradiation. 3) Pretreatment with filipin, which interferes with phytochrome binding to membranes, decreases stomatal opening in red and blue light. The stomatal responses to blue and red light are modified by DCMU, N₂, CO_2-enriched atmosphere, and CO_2-free air, which are known to affect, among other processes, chlorophyll fluorescence. Increased chlorophyll fluorescence by DCMU, N₂ and CO₂-enriched atmosphere enhanced stomatal opening in blue light and inhibited it in red light. CO₂-free air, which decreases chlorophyll fluorescence, had the opposite effect.     

Amaranthin accumulation in continuous red and blue light by seedlings ofAmaranthus caudatus L.

Four days oldAmaranthus seedlings responded to light treatment with an increase of amaranthin accumulation. With increasing irradiation time, red light caused a saturation effect. Blue light induced a high irradiation response. The blue light effect was reversible to a certain extent by far-red irradiation given at the end of the treatment with blue light. Intermittent red light (3 h red light, 3 h dark, …) caused a higher amaranthin accumulation than 24 h continuous red light. Results obtained with red and blue light are discussed on the basis of the phytochrome system.     

PHOTOMORPHOGENESIS OF THE GAMETOPHYTES OF LYGODIUM JAPONICUM

Protonemata of Lygodium japonicum turn biplanar in both red and blue light regimes and remain filamentous in far-red light. Biplanar gametophytes formed in red light are longer than broad with long, rectangular cells, whereas in blue light they appear broader than long with short, isodiametric cells. Transfer of protonemata of all ages from far-red regime to red or blue light induces a morphological type of growth characteristic of the new light regime. However, only relatively young biplanar forms transferred from red or blue light are able to resume filamentous type of growth in a subsequent regime of far-red light.     

Blue light and phytochrome-mediated stomatal opening in the npq1 and phot1 phot2 mutants of Arabidopsis

Recent studies have shown that blue light-specific stomatal opening is reversed by green light and that far-red light can be used to probe phytochrome-dependent stomatal movements. Here, blue-green reversibility and far-red light were used to probe the stomatal responses of the npq1 mutant and the phot1 phot2 double mutant of Arabidopsis. In plants grown at 50 micromol m-2 s-1, red light (photosynthetic)-mediated opening in isolated stomata from wild type (WT) and both mutants saturated at 100 micromol m-2 s-1. Higher fluence rates caused stomatal closing, most likely due to photo-inhibition. Blue light-specific opening, probed by adding blue light (10 micromol m-2 s-1) to a 100 micromol m-2 s-1 red background, was found in WT, but not in npq1 or phot1 phot2 double mutant stomata. Under 50 micromol m-2 s-1 red light, 10 micromol m-2 s-1 blue light opened stomata in both WT and npq1 mutant stomata but not in the phot1 phot2 double mutant. In npq1, blue light-stimulated opening was reversed by far-red but not green light, indicating that npq1 has a phytochrome-mediated response and lacks a blue light-specific response. Stomata of the phot1 phot2 double mutant opened in response to 20 to 50 micromol m-2 s-1 blue light. This opening was green light reversible and far-red light insensitive, indicating that stomata of the phot1 phot2 double mutant have a detectable blue light-specific response.     

Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores of Adiantum capillus-veneris L.

In the present study, using a newly developed fluorescent differential display technique, we have carried out large-scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern Adiantum capillus-veneris L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo-dT primer. The cDNA was then used as a template for PCR with the oligo-dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 upregulated and four down-regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up-regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser-(Pro) _n repeats and showed homology with cell wall-associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far-red light and photo-irreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet     

Red and blue light-stimulated proton efflux by epidermal leaf cells of the Argenteum mutant of Pisum sativum

Light stimulates leaf expansion in dicotyledons by increasingapoplastic acidification, cell wall loosening and solute accumulationfor turgor maintenance. Red and blue light enhance growth viadifferent photo-systems, but the cellular location and modesof action of these systems is not known. Here, the effect of red and blue light was studied on transportprocesses in epidermal cells of expanding leaves of the Argenteummutant of Pisum satlvum. Both red and blue light caused extraceiiuiaracidification by isolated epidermal tissue, which was stimulatedby extracellular K⁺ and inhibited by DCCD at 0.1 mol m^–3.Acidification induced by red compared with blue light showeddifferent saturating kinetics in fluence rate-response curves.Under near saturating light conditions the effects of red andblue light were additive. The red light-induced acidificationwas inhibited by far-red light while the blue light-inducedacidification was not. Light caused a hyperpoianzation of themembrane potential in epidermal strips, and stimulated ⁸⁶Rb⁺uptake by epidermal protoplasts. These results show that phytochromeand an additional blue light-photoreceptor function in isolatedepidermal cells to promote proton efflux, hyperpolarization,and cation uptake. Key words: Pisum sativum, light-induced acidification, ion transport, epidermis, photoreceptor     

Red-light and blue-light photoreceptors controlling chlorophyll a synthesis in the red alga Porphyra umbilicalis and in the green alga Ulva rigida

Chlorophyll synthesis is stimulated by red light in the green alga Ulva rigida C. Ag. and in the red alga Porphyra umbilicalis (L.) Kützing. Because the effect of red light showed some far-red reversibility in successive red and far-red light treatments, the involvement of phytochrome or a phytochrome-like photoreceptor is suggested. The extent of the response is dependent on exposure and photon fluence rate of red-light pulses. In addition to the effect of red light, a strong stimulation of chlorophyll synthesis by blue light was only observed in Ulva rigida. The effect of blue light shows also some far-red reversibility. In the green alga the accumulated chlorophyll is higher after blue light pulses than after red light pulses. In Porphyra umbilicalis , however, the contrary is observed. In Ulva rigida the involvement of a blue light photoreceptor in addition to phytochrome or a phytochrome-like photoreceptor is proposed. The different responses to red and blue light in both algae are explained in terms of their adaptation to the natural light environment.     

The composition and function of thylakoid membranes from pea plants grown under white or green light with or without far-red light

Pea plants ( Pisum sativum L. ev. Greenfeast) were grown for 2 to 3 weeks in while (˜ 50 μmol photons m⁻² s⁻¹; 400–700 nm) or green (˜ 30 μmol photons m⁻² s ⁻¹ 400–700 nm) light (16 h day/8 h night), with or without far-red light. Supplementary far-red light decreased leaf area and increased internodal length in both white and green light, demonstrating that phytochrome influenced leaf size and plant growth. However, there was no effect of far-red light on chlorophyll a /chlorophyll b ratios, chlorophyll-protein composition, the stoichiometry of electron transport complexes or photosynthetic function of isolated thylakoids. These results suggest that phytochrome is ineffective in modulating the composition and function of thylakoids in pea plants grown at low irradiance. One possible explanation of the ineffectiveness of phytochrome on thylakoids is discussed in terms of the drastic attenuation of red relative to far-red light in green tissue.     

Evidence for a phytochrome-mediated phototropism in etiolated pea seedlings

Entirely etiolated pea seedlings (Pisum sativum, L. cv Alaska) were tested for a phototropic response to short pulses of unilateral blue light. They responded with small curvatures resembling in fluence-dependence and kinetics of development a phytochrome-mediated phototropic response previously described in maize mesocotyls. Irradiations from above with saturating red or far-red light, either immediately before or after the unilateral phototropic stimulus, strongly reduced or eliminated subsequent positive phototropic curvature. Only blue light from above, however, entirely eliminated curvature at all fluences of stimulus. It is concluded that the phototropism is primarily a result of phytochrome action.