Red Irradiation Research Articles

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca(2+) ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg(2+), Ba(2+) or K(+) could not replace this requirement for Ca(2+). The presence of K(+) did not enhance the Ca(2+)-dependent swelling response. When the Ca(2+)-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca(2+)-channelblocker Verapamil and La(3+) inhibited R-induced swelling. It is proposed that R causes the opening of Ca(2+)-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.

The effect of red and far-red light on proton secretion from mesophyll-cell protoplasts of Vicia faba L.

The influence of red and far-red irradiation on the transport of H(+) and (86)Rb(+) through the plasmalemma was studied using parenchymal protoplasts isolated from Vicia faba leaves. The results indicate that red light stimulates H(+) secretion and the uptake of (86)Rb(+). Moreover, it has been demonstrated that far-red irradiation acts antagonistically with respect to red light in both these processes.

REVERSAL OF CAESIUM INHIBITION OF GROWTH BY POTASSIUM IN HYPOCOTYLS OF TOMATO SEEDLINGS (LYCOPERSICON ESCULENTUM L.)

Stunted, misshapen, necrotic hypocotyls of tomato seedlings (Lycopersicon esculentum L.) germinated on caesium cations in continuous red or white light indicated caesium-associated hypocotyl photo-injury. Continuous, far red irradiation inhibited seedling germination in control and caesium treatments but was reversed by continuous white light. Hypocotyl photo-injury also occurred in far red pretreated seedlings germinated on caesium in continuous white light. Except for suppressed elongation, hypocotyls of caesium-treated seedlings were normal in appearance. Etiolated caesium-treated seedlings placed in continuous white light also exhibited hypocotyl photo-injury. The effects of caesium cations were reversed by potassium cations but not by cations of other alkali metals.

Phytochrome Regulation of the Response to Exogenous Gibberellins by Epicotyls of Vigna sinensis

The elongation rate of cowpea epicotyls from whole cowpea (Vigna sinensis) seedlings and derooted and debladed plants (explants) increased after the main light period (8-hour duration) was extended with either continuous low intensity tungsten light or brief (5 minutes) far-red (FR) irradiation. This end-of-day FR effect was reversed by red (R) irradiation suggesting the involvement of phytochrome. These results confirm and extend those obtained previously with other species. Localization studies indicate the epicotyl to be the site of the photoreceptor. Treatment of cowpea seedlings with paclobutrazol, a gibberellin (GA) biosynthetic inhibitor, abolished the FR promoted epicotyl elongation, indicating a role for GAs in this process. There was no significant difference in epicotyl elongation rates of R plus FR irradiated explants treated with GA(1) or GA(20) and R irradiated explants treated with GA(1). However, R irradiation inhibited subsequent epicotyl elongation of GA(20) treated explants. Moreover, the observation, using GC-MS, that GA(1) and GA(20) are native GAs in cowpea lends support to the concept that phytochrome may control the conversion of endogenous GA(20) to GA(1) in cowpea.

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.

Fluorescence lifetimes and relative quantum yields of 124-kilodalton oat phytochrome in water and deuterium oxide solutions

The lifetimes and relative quantum yields of the three fluorescence components of the P/sub r/ (red-absorbing) form of 124-kDa oat phytochrome in H/sub 2/O and D/sub 2/O solutions have been determined by single-photon-timing techniques. Lifetime and quantum yield of the main component, which is the shortest lived and reflects the photochromic properties of P/sub r/, are not affected by D/sub 2/O. The medium-lifetime component, which is attributable to a photochromic source behaving comparable to that responsible for the main component, is not affected either. Only the least occurring and longest lived component, which is nonphotochromic, i.e., unaffected by red irradiation, markedly increases in lifetime. Thus, its relative contribution to the total fluorescence yield increases in D/sub 2/O. The spectra of the individual fluorescence components, obtained by a global analysis of the decay traces from 124-kDa P/sub r/ in H/sub 2/O solution at different observation wavelengths, differ only slightly in wavelength. The authors conclude that the primary photoreaction in the P/sub r/ transformation to P/sub fr/ (far-red-absorbing form of phytochrome) of the main phytochrome component exhibiting the shortest lived fluorescence does not involve a proton transfer as has been suggested in the literature.

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of Pfr to Pr, in pelletable pea phytochrome.Low temperature spectrophotometry was used to study the phototransformation of Pfr to Pr in 1000–7000 g pelletable fractions extracted from dark grown pea (Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far‐red light. At ‐170°C, far‐red irradiation of the pelletable phytochrome which had been pre‐irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I660), the difference spectrum of which showed a marked ab‐sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I660 was converted to another intermediate (I660) when it was warmed above ‐80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to Pfr by red irradiation and also underwent dark reversion to Pfr at ‐60°C. I660 formed Pr if the temperature was above ‐10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.

The aurea mutant of tomato is deficient in spectrophotometrically and immunochemically detectable phytochrome

The aurea locus mutant (au (w)) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158-165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.

The effect of deuterium oxide on light-sensitive Paulownia tomentosa seed germination

The effect of deuterium oxide on the germination of light-sensitive Empress tree ( Paulownia tomentosa) seeds was studied. Kinetics of germination in both light- and gibberellic acid (GA 3)-induced seeds was decelerated by increasing concentrations of deuterium oxide (D 2O) in the medium. Deuterium oxide in increasing concentrations also changed the duration of the light necessary for germination, from 5 min in H 2O to more than 3 days in 60% D 2O. A far-red (FR) pulse reversed the effect of inductive, prolonged red (R) irradiation; maximum germination could subsequently be restored by only 5 min of red light. The escape from FR reversibility was delayed from 1 day in water to 2 or 3.5 days in D 2O. The light reactions of D 2O-treated seeds resemble those of skotodormant seeds of the same species.

Soluble-starch-synthesizing and sucrose-degrading enzymes in Onoclea spores

Sucrose degradation and starch accumulation in imbibed spores of Onoclea sensibilis occur slowly in the dark and are accelerated two- to four-fold when irradiated with low fluence of red light. The accelerated rates of starch accumulation and sucrose degradation begin 4–6 hr after irradiation and precede the completion of germination. The effect of 5 min of red irradiation on the extractable activities of the enzymes involved in starch and sucrose metabolism was investigated as a possible means by which light accelerates the flux of metabolites through these pathways. The accumulation of starch from exogenous sucrose in unirradiated spores and the specific radioactivity of newly synthesized starch in irradiated and unirradiated spores were determined to see if the lack of sucrose degradation and starch synthesis in unirradiated spores is due to limited availability of substrates. Irradiation did not increase the maximum catalytic activities of the soluble starch synthesizing or soluble sucrose degrading enzymes during the 24 hr period following irradiation when compared with those in the unirradiated spores. Incubation of unirradiated spores on exogenous sucrose increased starch accumulation to a level comparable to that in irradiated spores incubated on distilled water suggesting that enzymes for sucrose catabolism and starch anabolism are active in situ in unirradiated spores. Irradiation caused a decrease in the specific radioactivity of starch without decreasing the incorporation of label. Therefore, light regulation of starch synthesis and sucrose degradation in Onoclea spores is not a result of enhanced activity of the soluble enzymes investigated. With respect to starch synthesis, irradiation causes an increase in availability of substrates that can be utilized for starch synthesis.

Evidence for Phytochrome Regulation of Gibberellin A20 3β-Hydroxylation in Shoots of Dwarf (lele) Pisum sativum L.

The effect of light on the dwarfing allele, le, in Pisum sativum L. was tested as the growth response to gibberellins prior to or beyond the presumed block in the gibberellin biosynthetic pathway. The response to the substrate (GA(20)), the product (GA(1)), and a nonendogenous early precursor (steviol) was compared in plants bearing the normal Le and the deficient lele genotypes in plants made low in gibberellin content genetically (nana lines) or by paclobutrazol treatment to tall (cv Alaska) and dwarf (cv Progress) peas. Both genotypes responded to GA(1) under red irradiation and in darkness. The lele plants grew in response to GA(20) and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA(20) and steviol in both light and darkness. The red effects on lele plants were largely reversible by far-red irradiation. It is concluded that the deficiency in 3beta-hydroxylation of GA(20) to GA(1) in genotype lele is due to a Pfr-induced blockage in the expression of that activity.

Effects of red and far red lights on nodulation and nitrogen fixation in soybean (Glycine max L.Merr)

Rooted leaves and plants were irradiated at the end of the photoperiod with red light for 15 minutes, while another group was also irradiated with red light and immediately afterwards with far- red light. The reduction in Pfr/Ptotal established by far red irradiation promoted nodulation probably by affecting an hormonal balance.

Photobiology of Phytochrome-Mediated Growth Responses in Sections of Stem Tissue from Etiolated Oats and Corn

The far-red reversibility of the phytochrome-controlled stimulation of elongation of coleoptile sections by low fluence red light has been characterized in subapical coleoptile sections from dark-grown Avena sativa L., cv Lodi seedlings. The fluence dependence of the far-red reversal was the same whether or not the very low fluence response is also expressed. The capacity of far-red light to reverse the red light-induced response began to decline if the far-red light was given more than 90 minutes after the red irradiation. Escape was complete if the far red irradiation was given more than 240 minutes after the red irradiation. Sections consisting of both mesocotyl and coleoptile tissue from dark-grown Avena seedlings were found to have physiological regulation of the very low fluence response by indole 3-acetic acid and low external pH similar to that seen for sections consisting entirely of coleoptile tissue. The fluence-dependence of the red light-induced inhibition of mesocotyl elongation was studied in mesocotyl sections from dark grown Zea mays L. hybrid T-929 seedlings. Ten micromolar indole 3-acetic acid stimulates the control elongation of the sections, while at the same time increasing the sensitivity of the tissue for the light-induced inhibition of growth by a factor of 100.

The joint action of phytochrome and alternating temperatures in the control of seed germination in Dactylis glomerata

The promoting effect of light and alternating temperatures on the germination of seeds of three contrasting populations of Dactylis glomerata L. was studied. Irradiation treatments using broad band low irradiance light sources resulted in red/far‐red reversible effects, demonstrating the involvement of phytochrome in germination control. Reduction of germination by far‐red below the level of a dark control indicated the presence of high pre‐existing levels of the active form of phytochrome (Pfr) in some individuals. The capacity for dark germination at 21/11°C (12 h/12 h) was shown to be dependent on Pfr. Although some individuals were capable of germination at constant temperatures following red irradiation, in most seeds germination was dependent on the presence of Pfr and alternating temperatures. Some individuals responded to a single red irradiation, although a large proportion of seeds required high levels of Pfr to be maintained for long periods. Previously published dose response curves for alternating temperatures and a measured dark reversion time of 48 h for Pfr established by a single 60 min red irradiation, provided firm evidence of a direct correlation between the requirements for repeated irradiation and number of alternating temperature cycles.

Characterization of a Light-modulated ATPase Activity in a Microsomal Fraction from Cucumber Hypocotyls

The properties of ATPase activity in a microsomal fraction from etiolated hypocotyls of Cucumis sativus L. were investigated. The ATPase required Mg 2+ , was stimulated by K + and was partially inhibited by vanadate. The effect of K + was unspecific and suggested that, at the tested concentration (50 mM), K + stimulated ATPase activity by facilitating H + extrusion by the ATPase and in turn its further activity. This assumption was supported by the effect of the K + and pH gradients on K + stimulation. The stimulation by K + was increased by red light treatments, while red irradiation in vivo followed by far red irradiation in vitro was less stimulatory. This implies that phytochrome modulates the K + stimulation. Ionophores such as gramicidin and nigericin also stimulated the ATPase activity in the presence of K + . This effect was prominent only after far red irradiation in vitro .

Photoregulation of Proton Extrusion by Cucumber Hypocotyls and the Role of ATPase Activity

Summary The photoregulation of H + extrusion by hypocotyl segments of Cucumis sativus L. was studied. Irradiation with red light (to give Pfr) enhanced H + efflux in the presence of Mg 2+ and K + . This effect of red light was decreased by far red irradiation (to give Pr). The acidification was inhibited by sodium orthovanadate. These results imply that H + extrusion is coupled to plasma membrane ATPase activity. In vivo pretreatment with filipin, which interferes with phytochrome association with membranes, reduced both photoregulation of ATPase activity and H + extrusion. A similar effect was found after preirradiation with red light before a prolonged dark period at 4 °C, a treatment which is assumed to lower phytochrome levels.

Spectral properties and chromophore rotation of phytochrome bound to substituted Sepharose

Brushite purified phytochrome from Avena sativa L. cv. Sol II was bound to phenyl Sepharose, octyl Sepharose, CNBr‐activated Sepharose and to anti‐phytochrome immunoglobulins immobilized on Sepharose. The spectral properties of phytochrome bound to anti‐phytochrome immunoglobulins and to phenyl Sepharose were similar to phytochrome in solution. Phytochrome bound to CNBr‐activated Sepharose or to octyl Sepharose showed reduced Pfr formation after red irradiation. The reversal to Pr with far‐red light was only partial but a further increase at 667 nm took place slowly in the dark. A peak at 657 nm was seen in the difference spectrum between CNBr‐activated Sepharose‐bound phytochrome kept in darkness and the identical sample immediately after a far‐red irradiation.The change in linear dichroism at 660 nm and 730 nm, induced by plane polarized red or far‐red light, was measured. It was computed that the long‐wavelength transition moment of phytochrome had an average rotation angle of 31.5° or 180°–31.5°. The substrate used for immobilization had a limited effect on the rotation angle. Phytochrome immobilized on CNBr‐activated Sepharose gave an angle of 27.8° and phytochrome immobilized on phenyl Sepharose gave an angle of 32.6°.

Phytochrome control of gene expression in radish seedlings. III. Evidence for a rapid control of the ribulose 1,5 bisphosphate carboxylase small subunit gene expression by red light

The effect of red and far-red light on the level of the mRNA encoding the small subunit (SSU) of ribulose 1,5 bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) from radish cotyledons was investigated. Northern blot analysis of RNA with a cDNA probe showed that both long (12–36 h) far-red irradiation and short (1–5 min) red irradiation brings about an increase in SSU mRNA concentration which was prevented by a subsequent far-red light exposure. Far-red light was effective in reversing the red light effect provided that it was given soon after (<10 min) the red light pulse. The red light mediated increase in SSU mRNA level did not occur in presence of α-amanitin. Our results suggest that phytochrome control of SSU gene expression is exerted at the transcriptional level.

Anaesthetic stimulation of Amaranthus albus seed germination: Interaction with phytochrome

Exposure to solutions of acetaldehyde, ethanol and n‐propanol ranging from 0.06 to 0.6 M for about 5 h induced photosensitivity in imbibed Amaranthus albus L. seeds which normally would be insensitive. When the seeds were made dormant by pre‐treattnent with continuous incandescent light for 24 h, n‐propanol promoted germination np to 80% provided the active form of phytochrome was present. Pre‐treatment of seeds with continuous incandescent light terminated by far‐red reduced the n‐propanol‐stimulated germination to about 33% which could again be increased by a red irradiation. Red light and n‐propanol also interacted in seeds made insensitive to red treatment by 20°C in darkness. Loss of the n‐propanol stimulation resulted from treatment in continuous far‐red for 2 or more days, even when a red irradiance and n‐propanol treatment were combined. It is suggested that n‐propanol may act at a membrane level.

PROBIT ANALYSIS OF LOW AND VERY‐LOW FLUENCE‐RESPONSES OF PHYTOCHROME‐CONTROLLED Kalanchoe blossfeldiana SEED GERMINATION*

Abstract— Kalanchoë blossfeldiana seeds are light‐requiring for seed germination. On water or KNO3 solution and irradiated with several daily red (R) irradiations, the seeds show a low‐fluence (LF) response which is far‐red (FR) reversible. Incubated on gibberellic acid (GA3) the seeds show a very‐low‐fluence (VLF) response which can be saturated with red as well as with far red light. As germination is a quantal response, the sub‐optimal segments of the dose‐response curves are analysed by means of probit analysis in order to calculate the seed population parameters. There is a linear relation between the probit of the germination response and the logarithm of the fluence. Moreover, the slope for the VLF as well as for the LF response is the same. The VLF requires about 8 × 104 times less fluence than the LF. VLF saturation with FR requires about 200 times more fluence than with R.Although, GA3 and KNO3 modulate VLF and LF, respectively, there is no direct influence on the phytochrome‐phototransformations. Once Pfr is formed (in VLF or LF, or preserved in dry seeds) germination is proportional to the GAS concentration (for VLF and dark germination) or proportional to the KNO, concentration (for LF). The non‐photochemical events leading to germination seem to be triggered by a similar action mechanism for both GA, and KNO3.