The physiological versus the spectrophotometric status of phytochrome in corn coleoptiles

The influence of red light in altering the phototropic sensitivity of corn coleoptiles (Zea mays L., cultivar Burpee Barbecue Hybrid) is compared with the spectrophotometric status of the phytochrome they contain. The distribution of measurable phytochrome corresponds roughly with the distribution of sensitivity to red light for physiological change. Both phytochrome concentration and red light sensitivity are maximal in the coleoptile tips. Red light pretreatments which reduce total phytochrome by about 50%, however, do not alter subsequent red light sensitivity of the phototropic system. Dosages of red light sufficient to saturate the physiological system are two orders of magnitude too small to induce measurable phytochrome transformation. The log-dosage-response curves for physiological change and for phytochrome transformation do not have the same slopes. The time course for appearance, mainconcentration of the far-red-absorbing form of phytochrome over a broad range of tenance, and decay of the physiological response is independent of the measurable concentrations. The following hypothesis is proposed: the phytochrome mediating the alteration in phototropic sensitivity is only a small proportion of the total present. This small active fraction is physically and kinetically independent of the bulk measurable, and is packaged in some manner which facilitates its transformation in both directions.     

Function of Light in the Light-induced Geotropic Response in Zea Roots

The light doses just above the threshold energy value for inducing the geotropic responsiveness in the roots of Zea mays L., cv. Golden Cross Bantam 70, caused a drastic rise in the NADPH level and a drop in the NADP level in 2-millimeter root tips. Some reducing agents lowered the threshold energy value up to about one-third of the control. From these results, we deduce that light may exert two functions in the geotropic response of Zea primary roots, one being the photochemical transformation of a photoreceptor and the other being the induction of a reduction state in the tissue.     

Spectral dependence of the light-induced geotropic response in Zea roots

The induction by light of geotropic responsiveness in the primary roots of Zea mays L. (cv. Golden Cross Bantam 70) was found to be governed by the all-or-none law. The response was induced by light energies above a threshold value, but the maximal curvature of geo-stimulated roots was constand irrespective of the light energy above that threshold. The action spectrum for this light effect showed a large peak at 650, a small peak at 410, and a shoulder at 663 nm. The effect of red light was not reversed by far-red light. Thus, the geotropic response in Zea roots may not be controlled by phytochrome.     

The Effect of Light on the Geotropic Responses of Phycomyces Sporangiophores

The geotropic responses of Phycomyces sporangiophores were studied under varying intensities of illumination, using a low speed centrifuge and a fixed beam of blue light. This light has a strongly inhibitory effect on the transient geotropic response, reducing it to 36 per cent of its magnitude in darkness. The inhibition does not vary systematically with light intensity over a range of 400-fold. The light sensitivity of the transient geotropic response thus differs from the light-growth response system, which shows the same growth rate in light and darkness. By contrast, the slower long term geotropic response is enhanced by light of moderate intensities, but is strongly inhibited by high intensities. At and above a mean intensity of about 1 µw/cm², the long term response is completely removed. If the intensity is lowered from an inhibitory level, either to darkness or to a low level, the geotropic response appears after a time lag of 20 minutes. Furthermore an increase in intensity from one level to another, both levels normally enhancing, results in a transient reversal in the long term geotropic response, also after a time lag of 20 minutes. Thus it is suggested that light is acting at some intermediate step in the long term geotropic sensory system, a step that normally requires 20 minutes for completion.     

Phytochrome B and the Regulation of Circadian Ethylene Production in Sorghum

The sorghum (Sorghum bicolor L. Moench) cultivar 58M, which contains the null mutant phytochrome B gene, shows reduced photoperiodic sensitivity and exhibits a shade-avoidance phenotype. Ethylene production by seedlings of wild-type and phytochrome B mutant cultivars was monitored every 3 h, and both cultivars were found to produce ethylene in a circadian rhythm, with peak production occurring during the day. The phytochrome B mutant produces rhythmic peaks of ethylene with approximately 10 times the amplitude of the wild-type counterpart with the same period and diurnal timing. The source of the mutant's additional ethylene is the shoot. The diurnal rhythm can be produced with either light or temperature cycles; however, both light and temperature cycles are required for circadian entrainment. The temperature signal overrides the light signal in the production of diurnal rhythms, because seedlings grown under thermoperiods reversed with the photoperiod produced ethylene peaks during the warm nights. To examine the effect of extreme shading on ethylene production, seedlings were grown under dim, far-red-enriched light. This treatment duplicated the phytochrome B mutant's shade-avoidance phenotype in the wild type and caused the wild type to produce ethylene peaks similar to those observed in the mutant. The results confirm that phytochrome B is not required for proper function of circadian timing, but it may be involved in modulating physiological rhythms driven by the biological clock oscillator.     

Light environment in pre- and post-dehiscent fruits affects seed germination in Calotropis procera

Fruits in Calotropis procera can be distinguished into five discrete but contiguous stages on the basis of diameter and seed color. Seeds from dehisced fruits at stage V germinated >80% on moist substratum in darkness. This was rather unexpected because the seeds developed and matured in an FR-enriched microenvironment (R:FR ratio ～0.3) of the chlorophyll-containing maternal tissue and displayed low-fluence response (LFR) mode of phytochrome action. In contrast to >80% dark-germinating seeds from dehisced fruits at stage V, about 50% seeds from undehisced fruits at that stage were dark germinating, whereas another 30% seeds required light for germination. The light-requiring fraction of the seed population did not only respond to a very low-fluence R and to a short FR pulse, but also lacked R–FR reversibility thereby indicating to a very low-fluence response (VLFR) mode of phytochrome action. The present study reporting VLFR to non-dormant seed state transition in C. procera suggested that the state of phytochrome and the subsequent seed germination response in dry-seeded species, besides being determined by the light environment immediately before maturation drying, might also be regulated by a post-dehiscence light signal.     

Relation of Phytochrome-enhanced Geotropic Sensitivity to Ethylene Production

Brief exposure of etiolated pea (Pisum sativum cv. Alaska) seedlings to red light enhances subsequent development of geotropic curvature of the stem. Both this response and inhibition of ethylene production by red light become maximal 8 hours after illumination. Very low concentrations of applied ethylene inhibit development of geotropic curvature, whereas hypobaric treatment enhances geotropic sensitivity by removing endogenous ethylene. Increased geotropic sensitivity after illumination is accompanied by increased lateral migration of ³H-indoleacetic acid in response to gravity, and ethylene inhibits this lateral migration. It is suggested, therefore, that red light-enhanced geotropic sensitivity is caused by increased lateral auxin transport resulting from a reduction in ethylene production after illumination.     

Wirkung von Vorbestrahlung mit Rot- oder Blaulicht auf die geotropische Empfindlichkeit von Maiskoleoptilen

The early geotropic downward bending of corn (Zea mays L.) coleoptiles was found to be influenced by red and blue light. The coleoptiles were illuminated from above and kept in the dark for defined intervals; afterwards they were positioned horizontally and their curvature was monitored for 40 min. After illumination with red light and a 120 min interval and early upward bending instead of an early downward bending was found. This effect was nullified by a far-red illumination administered immediately after exposure to red light. These results indicate that the phytochrome system influences the geotropic reaction. After illumination with blue light and a 30 min interval little downward bending was found. This result corresponds well with the findings of earlier authors who measured the late geotropic reaction, on the basis of the hypothesis that the strength of the early downward bending is a measure of the geotropic sensitivity. The dose-effect curve of the blue light influence on geotropic sensitivity, measured by early downward bending, is very similar to the dose-effect curve of the phototropic curvature of corn coleoptiles. This fact together with the earlier finding of similar adaptation times of about 30 min suggests the existence of some common transducers in the reaction chain of phototropism and geotropism.

---

Abkürzungen HR Hellrot - DR Dunkelrot - D Dunkel - WT Wartezeit - DK Dunkelkontrolle     

Phytochrome-mediated germination of very sensitive oospores

The light receptor and its mode of operation were studied in photosensitive oospores of Nitella furcata subsp. megacarpa (Allen emend. Wood). Brief pulses of light activated maximal germination of post-secondary dormant oospores removed from lake sediments. Fluence response data at 12 wavelengths were used to construct an action spectrum for germination. The shape of the action spectrum with its maximum at 669 nm provides evidence for the involvement of phytochrome. Germination was induced with photon fluences that established as little as 0.01% of the phytochrome in the far red-absorbing form, which suggests that phytochrome was operating in the very low-fluence response mode. The functioning of phytochrome in the very low-fluence response mode in Nitella is similar to that in higher plants.     

Kinetics and time dependence of the effect of far red light on the photoperiodic induction of flowering in wintex barley

Flowering in the long day plant Hordeum vulgare L. var. Wintex barley was enhanced by the addition of far red light to the main light portion of the photoperiod. Far red energy was provided to produce quantum flux ratios (660/730 nm) and phytochrome photoequilibria (Pfr/total phytochrome) equivalent to those reported both beneath a leaf canopy and outside a canopy at twilight. The photoperiodic requirement for long days can be completely eliminated by the addition of far red light. However, both the effect of extending the photoperiod without far red and the addition of far red to 12-hour photoperiods were suboptimal. Maximal stimulation was achieved only when far red was added to continuous light. The duration of the period of maximal apex elongation rate, as well as the reduction of the time required for floral initiation, were saturated by three inductive cycles. When far red energy was provided intermittently during 3 days of continuous light, the ability to respond varied in a circadian manner. This enhancement of flowering by far red appears to be mediated by the “high irradiance response” of phytochrome.     

Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor.     

Modulation of a mitochondrial function by oat phytochrome in vitro

Previous data in the literature have indicated that phytochrome could alter the rate of reduction of exogenously added NADP by a pea mitochondrial preparation in vitro. These results could not be duplicated using a mitochondrial preparation isolated from etiolated oat seedlings. Further experimentation demonstrated that the addition of Pr to the preparation, in combination with a far red light illumination, could significantly reduce the rate of oxidation of NADH by the external dehydrogenases of oat mitochondria. This response was characterized by a 15% decrease in reaction velocity at saturating substrate concentrations and a 2-fold increase in apparent K_m as compared to values obtained after Pfr plus red light treatment. The response was photoreversible, the rate of oxidation of exogenous NADH being determined by the last light illumination given to the mitochondrial preparation. The interaction between phytochrome and the mitochondria was apparently occurring at the level of the inner mitochondrial membrane. A requirement for these results was that the mitochondria be isolated from plants that were illuminated with white or red light before extraction; mitochondria from unirradiated plants showed no dehydrogenase response to treatments with phytochrome plus actinic light.     

Photoinduced Seed Germination of Oenothera biennis L: I. General Characteristics

General characteristics of light-induced germination of Oenothera biennis L. seeds were investigated at 24°C. During dark imbibition, seeds reached maximal respiration in 7 hours and maximal water content and photosensitivity in 24 hours. After dark imbibition of 24 hours, seeds required a long exposure (>36 hours) to red or white light for maximal germination. Two photoperiods (12 and 2 hours) separated by a period of darkness of 10 to 16 hours gave near maximal germination. For the two photoperiod regime, the first light potentiates a reversible phytochrome response by the second light. A 35°C treatment for 2 to 3 hours in the dark immediately prior or subsequent to 8 hours of light caused a higher percentage of germination. A 2 hour treatment at 35°C also potentiates a reversible phytochrome response. Halved seeds germinated at 100% in light or darkness indicating that the light requirement of the seeds is lost in the halving procedure. After-ripened seeds required less light and germinated more rapidly and at higher percentages than seeds tested shortly after maturation.     

The influence of de-etiolation on the sensitivity of Avena coleoptiles to the far-red absorbing form of phytochrome

In photoresponses regulated by phytochrome the effect of a red irradiation is not always reversed by far-red. This applies for instance to the influence of red light on the geotropic reactions of Avena coleoptiles. We could induce red/far-red reversibility by a short de-etiolating exposure to red light about 20 h prior to the experimental irradiations. This, was due to a decrease of the sensitivity to the low level of the far-red absorbing form of phytochrome that is established by far-red. Since etiolated plants react also to a wavelength of 520 nm (green light), it is advisable to expose the coleoptiles to a de-etiolating irradiation prior to manipulations in green safelight in order to prevent the plants from reacting to the green light.     

Regulation of pea epicotyl elongation by blue light : fluence-response relationships and growth distribution

Irradiation with blue light causes a rapid decrease in stem elongation in Pisum sativum. Growing plants under continuous red light allowed us to study the fluence dependence and spatial distribution of blue-induced growth effects without interference from large changes in the ratio of the far-red absorbing form of phytochrome to total phytochrome. The magnitude of the inhibition generated by a 30-second pulse of blue light was linearly related to the log of the fluence applied over two orders of magnitude. Reciprocity held for irradiations with a pulse length shorter than the lag time for the response. The spatial distribution of inhibition was studied by marking the growing zone and photographing the stem at 10-minute intervals before, during, and after a 1-hour exposure to blue light. The region just below the hook does not undergo any perceptible change in growth rate while growth is nearly 100% inhibited in the base of the third internode.     

A photoperiod-insensitive barley line contains a light-labile phytochrome B

Barley (Hordeum vulgare L.) is a long-day plant whose flowering is enhanced when the photoperiod is supplemented with far-red light, and this promotion is mediated by phytochrome. A chemically mutagenized dwarf cultivar of barley was selected for early flowering time (barley maturity daylength response [BMDR]-1) and was made isogenic with the cultivar Shabet (BMDR-8) by backcrossing. BMDR-1 was found to contain higher levels of both phytochrome A and phytochrome B in the dark on immunoblots with monoclonal antibodies from oat (Avena sativa L.) that are specific to different members of the phytochrome gene family. Phytochrome A was light labile in both BMDR-1 and BMDR-8, decreasing to very low levels after 4 d of growth in the light. Phytochrome B was light stable in BMDR-8, being equal in both light and darkness. However, phytochrome B became light labile in BMDR-1 and this destabilization of phytochrome B appeared to make BMDR-1 insensitive to photoperiod. In addition, both the mutant and the wild type lacked any significant promotion of flowering in response to a pulse of far-red light given at the end of day, and the end-of-day, far-red inhibition of tillering is normal in both, suggesting that phytochrome B is not involved with these responses in barley.     

Phytochrome is not involved in the red-light-enhancement of the stomatal blue-light-response in wheat seedlings

The stomatal response to blue light (BL) in wheat seedlings ( Triticum aestivum L. cv. Starke II, Weibull) was enhanced by background red light (R). This enhancement was only slightly affected by the addition of background far-red light (FR). Under similar light treatments, the addition of FR induced a 43% transformation from the far-red-absorbing form towards the red-absorbing form of phytochrome from etiolated oat ( Avena sativa L. cv. Sol II), immobilized on phenyl-sepharose. Furthermore, the enhancement of the stomatal BL-response by 15 min R was not reversed by a subsequent irradiation with 5 min FR. It is concluded that the red-light-enhancement of the stomatal blue-light-response in wheat seedlings does not involve a change in the photostationary state of phytochrome.     

Photophysiology and phytochrome content of long-hypocotyl mutant and wild-type cucumber seedlings

Photomorphogenetic responses have been studied in a cucumber (Cucumis sativus L.) mutant (lh), which has long hypocotyls in white light (WL). While etiolated seedlings of this mutant have a similar phytochrome content and control of hypocotyl elongation as wild type, deetiolation is retarded and WL-grown seedlings show reduced phytochrome control. Spectrophotometric measurements exhibit that WL-grown tissues of the lh mutant (flower petals and Norflurazon-bleached leaves) contain 35 to 50% of the phytochrome level in the wild type. We propose that this is a consequence of a lack of light-stable phytochrome, in agreement with our hypothesis proposed on the basis of physiological experiments. The lh mutant lacks an end-of-day far-red light response of hypocotyl elongation. This enables the end-of-day far-red light response, clearly shown by the wild type, to be ascribed to the phytochrome, deficient in the lh mutant. Growth experiments in continuous blue light (BL) and continuous BL + red light (RL) show that when RL is added to BL, hypocotyl growth remains inhibited in the wild type, whereas the lh mutant exhibits significant growth promotion compared to BL alone. It is proposed that the hypocotyls fail to grow long in low fluence rate BL because photosynthesis is insufficient to sustain growth.     

Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking of Arabidopsis Hypocotyl Protoplasts

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl⁻ and K⁺. The postshrinking volume recovery is achieved by K⁺ and Cl⁻ influxes, with contribution by the proton motive force. External Ca²⁺ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.     

Ethylene and the Responses to Light of Rice Seedlings

The growth of rice seedlings (Oryza satira L.) in the presence of ethylene caused a change in the response to light of coleoptile elongation. In plants grown in air without added ethylene coleoptile elongation was promoted by red, far-red and yellow-green light only in very young seedlings; in older plants irradiation inhibited the growth of the coleoptile. The effect of growing plants in the presence of ethylene was to prolong the period during which light promoted coleoptile growth. Elongation of the first internode was inhibited by light whether or not the seedlings were grown in the presence of ethylene. A correlation existed between the growth effect of an irradiation and the initial decay rate of phytochrome which was established by the treatment. Regardless of wave length, light sources whose intensities were adjusted to produce a decay rate of about 10% per hour or less induced a moderate rate of coleoptile elongation which persisted for a relatively long period. Irradiation with red or yellow-green light of higher intensity which produced a higher rate of phytochrome decay induced a higher rate of coleoptile elongation, but growth stopped after several hours. Other observations, however, showed that one cannot establish a general simple correlation between the rate of elongation of rice coleoptiles under light and the status of measurable phytochrome in the plant.