PHYTOCHROME CONTROL OF ITS OWN SYNTHESIS IN Sorghum vulgare AND Avena sativa

The accumulation of phytochrome in the dark was measured for Avena sativa seedlings after a white light pretreatment and for Sorghum vulgare seedlings after continuous red or far-red light treatments, using the herbicide Norflurazon to prevent greening under continuous irradiation. In both cases the accumulation of phytochrome depends on the state of the phytochrome at the light-dark transition: high P_fr levels (red light pulse) led to a slower rate of phytochrome accumulation than lower P_fr levels (long wavelength far-red (RG 9) light pulse). Poly-(A⁺)-RNA was isolated fromA. sativa seedlings grown for 48 h in darkness + 24 h WL + light pulse (5 min) (red, RG 9 light, red followed by RG 9 light or RG 9 followed by red light pulse) + 19 h darkness. The poly-(A⁺)-RNA was translated in a rabbit reticulocyte lysate system and the translation products were immunoprecipitated by specific anti-phytochrome antibodies. It was demonstrated that the activity of mRNA coding for phytochrome was under phytochrome control.     

APPEARANCE OF PHOTOPHOSPHORYLATION CAPACITY: THRESHOLD vs GRADED CONTROL BY PHYTOCHROME

Abstract— It is shown that in attached mustard cotyledons graded control of chlorophyll synthesis by physiologically active phytochrome (P_fr) and threshold control by P_fr of the 'potential capacity' to photophosphorylate are totally different phytochrome actions even though both controls are essential for the build-up of the same functional complex, the machinery for photophosphorylation. The essential findings are as follows: The action of P_fr (made by a 1 min red light pulse) on the capacity and efficiency of photophosphorylation is rapid—detectable after 15 min and completed after 30 min—whereas the action of P_fr on chlorophyll formation is slower—only detectable 45 min after the original red light pulse (R). Detailed escape studies (loss of full reversibility of the inductive effect of a R pulse by far-red) show that the effect of a R pulse on chlorophyll synthesis remains fully reversible for 45 min whereas the action of P_fr on the capacity for photophosphorylation is very fast (occurring within 2 min). Control of capacity for photophosphorylation is a threshold response (whereby the threshold value is approximately 1.25% P_fr based on total phytochrome at 36 h = 100%) whereas control by P_fr of chlorophyll synthesis is graded. Control of capacity for photophosphorylation by P_fr only operates if the hypocotyl hook is connected to the cotyledons for at least 2 min after the inductive R pulse, i.e. until full escape from reversibility has occurred, whereas chlorophyll formation in the cotyledons is not affected by the separation of hook and cotyledons.     

A NOVEL EFFECT OF UV-B IN A HIGHER PLANT (SORGHUM VULGARE)

Abstract— Two non-photosynthetic photoreceptors (phytochrome and the usual blue/UV light photoreceptor) were previously found to be involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings (Drumm and Mohr, 1978). The decisive point is that phytochrome can act only after a blue/UV light effect has occurred. On the other hand, the expression of the blue/UV light effect is controlled by phytochrome ('obligatory sequential action'). A strong positive interaction between the blue/UV-A and the UV-B part of the spectrum was found, in addition to the above sequential action: an inductive effect of blue or UV-A light can only express itself fully if short wavelength UV (approximately 300–320nm. UV-B range) is also given, either after the blue/UV-A light or simultaneously. Since even small amounts of the UV-B are strongly effective it is probable that this effect plays a role under natural conditions and may not be considered as a mere laboratory artifact.     

CONTROL OF CHLOROPHYLL b BIOSYNTHESIS BY PHYTOCHROME

In the mustard seedling cotyledons, chlorophyll b appears from the very beginning in white light provided that a red light pulse pretreatment was given 12 h prior to the onset of white light. The red light pulses act through phytochrome. Without pretreatment no chlorophyll b is detectable at least during the first 60 min after the onset of white light (25°C). Biogenesis of chlorophyll b specifically depends on the action of phytochrome during the pre-steady state period as well as during the steady state period of chlorophyll accumulation. In light pulse experiments, it was found that formation of chlorophyll b takes place stoichiometrically at the cost of chlorophyll(ide) a.     

PHOTOREGULATION OF LOGARITHMIC FLUENCE-RESPONSE CURVES FOR PHYTOCHROME CONTROL OF CHLOROPHYLL FORMATION IN PISUM SATIVUM L*

Abstract— Logarithmic fluence-response curves for red (660 nm) and far red (730 nm) light induction of rapid chlorophyll a (Chi a) accumulation in pea seedlings (Pisum sativum L. cv. Early Alaska) indicate extreme light sensitivity in dark-grown seedlings. The energy requirement for onset of 660 nm light induction is less than 20 μJ m^-2 and for 730 nm is about 1 mJ m^-2. De-etiolation produced by a saturating exposure of red light (3–8 kJ m^-2) 24 h prior to the construction of the logarithmic fluence-response curves resulted in approximately a 3 fold increase in slope for 660 nm light, whereas the energy requirement for onset of induction shifted to about 100 mJ m^-2. In such de-etiolated plant material, far red applied at low incident energies almost completely lost its inductive capacity. The inductive capacity of far red applied as high irradiance over a long period of time (16h) appeared not to be affected by the de-etiolation treatment. Reciprocity failed for both dark-grown and de-etiolated seedlings upon exposures exceeding 1,000s. Nearly identical results were obtained for seedlings de-etiolated by red exposures immediately followed by far red (4.8 kJ m^-2), although this treatment did not lead to any significant decrease in spectrophotometrically measurable phytochrome. Therefore, no simple correlation was observed between the level of phytochrome present and the sensitivity of seedlings for induction of rapid Chl a accumulation. In order to explain this apparent phytochrome paradox the possibility was tested and ruled out for changes in the degree of synchronization of seedlings, or for induction of some sort of circadian rhythmicity in light sensitivity being involved. In addition, no correlation was observed between induction of morphogenic development and changing light sensitivity. These results formed, therefore, additional support for a model for phytochrome action involving its intracellular transport and local concentration during the process of seedling de-etiolation.     

SPECTRAL DEPENDENCE OF A SINGLE AND A SUBSEQUENT SECOND LIGHT PULSE INDUCING BARLEY LEAF UNFOLDING

The unfolding of etiolated barley leaves was induced by two short pulses of light separated by a dark interval. For certain wavelengths of light the effect of the “second light pulse was enhanced when the pulse was given after a 300–2000 s dark interval as compared with its effect when the two light pulses were given simultaneously. We investigated the spectral dependence of the effect of the first, inducing light flash and of the effect of the second Light flash given after a 500 s dark interval. The spectral actinity for the effect of the first flash showed phytochrome involvement. The spectral actinity for the effect of the second light flash, however, was shifted towards shorter wavelengths and the inductive action of red and far red light was attenuated.     

SPECTRAL PROPERTIES,PHOTOTRANSFORMATION KINETICS AND PELLETABILITY OF PHYTOCHROME IN Pharbitis nil Chois*

Abstract— An examination has been made of the involvement of phytochrome in the circadian rhythm of flowering in Pharbitis nil Chois. The peak position of P_fr absorption changes with time after a red light pulse. The shortest absorption wavelength of P_fr occurs at the same time as flowering is inhibited by red light in dark grown, red light pretreated plants. Pelletable and supernatant phytochrome show a similar trend with lowest values found at the time of flower inhibition. Neither phototransformation kinetics nor intermediates of phytochrome which accumulate in white light show such a relationship to the circadian rhythm found in flowering of dark grown P. nil.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.     

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH-PIGMENT MUTANT OF TOMATO

Abstract— A high-pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light-labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence-response relationships for anthocyanin synthesis after a blue-light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far-red light (FR)-absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6-fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild-type and hp-mutant seedlings, despite the latter accumulating 9-fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence-rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24-h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.     

CONTROL OF HYPOCOTYL GROWTH IN MUSTARD SEEDLINGS AFTER LIGHT-DARK TRANSITIONS

Abstract— Six hours of irradiation with white or far-red light strongly stimulates the 'end of day' inhibition of hypocotyl elongation in dark grown Sinapis alba L. Measurements of both reversion kinetics of the inductive light pulse and 'null point' experiments (Hillman, 1965. 1972) indicate that this response is controlled by stable phytochrome. The extent of the reversible response (i. e. length after far-red light minus length after red light) showed rhythmic oscillations after a 6, 48 and 54 h white light pretreatment. The rhythm is started by the transition from light to dark and neither phase nor amplitude is influenced by the level of P at this transition.     

THE MODE OF INTERACTION BETWEEN BLUE (UV) LIGHT PHOTORECEPTOR AND PHYTOCHROME IN ANTHOCYANIN FORMATION OF THE SORGHUM SEEDLING

Two non-photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (P_fr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without P_fr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.     

ANALYSIS OF Pfr DESTRUCTION IN AMARANTHUS CAUDATUS L EVIDENCE FOR TWO POOLS OF PHYTOCHROME*

Abstract— Kinetics of the destruction of the far red absorbing form of phytochrome (P_fr), measured by in vivo spectroscopy, show two phases: after a saturating red light pulse, rapid first order decay results in the loss of most, but not all, of the detectable P_rr; decay of the rest is much slower. The concentration of the more stable P_fr is positively correlated to the concentration of the total P_fr established at time zero. The linear relationship between total and ‘stable’ P_fr exludes the existence of a threshold level of P_fr for fast destruction. Photoconversion of the P_r (red absorbing form of phytochrome) present during the slow decay, by exposure to a second light pulse, is followed by fast destruction of most of the newly formed P,_r, whereas some P_fr formed by the first pulse still remains. The experiment suggests that not all P_fr molecules are accessible to the same destruction mechanism, i.e. there are two populations of P_fI.     

KINETICS OF Pfr APPEARANCE IN Amaranthus caudatus‡

Abstract— The kinetics of the far-red absorbing form of phytochrome (P_fr) appearance from intermediates in the pathway from the red absorbing form of phytochrome (P_r) to P_fr that accumulate under high fluence rate white light have been investigated in 3-day old dark grown Amaranthus caudatus seedlings. The appearance of P_(r after a 5 s white light pulse was measured over the temperature range -8 to 25^°C in samples flushed with O₂ or N₂. Over the whole temperature range under anaerobic conditions the kinetics of the slowest component of P_fr appearance are faster than in the presence of O₂. Arrhenius plots are linear over this temperature range and indicate the activation energy for the slowest component of P_fr appearance is 44.05 ± 1.97 kJ mol^?1 for O₂ and 53.69 ± 4.86 kJ mol^?1 for N₂.     

RELATIVE IMPORTANCE OF BLUE LIGHT AND LIGHT ABSORBED BY PHYTOCHROME IN GROWTH OF MUSTARD (Sinapis alba L.) SEEDLINGS*

Abstract— Hypocotyl straight growth in mustard (Sinapis alba L.) responds very strongly and in precisely the same way to low fluence rate red (RL) and white light (WL). The effect of weak light can be attributed fully to light absorption by phytochrome. Only with increasing fluence rate an effect of blue light (BL) comes into play which cannot be explained by the action of phytochrome. However, this specific action of BL can be demonstrated in hypocotyl growth of mustard seedlings only up to 5 days after sowing (25°C). With older seedlings control of hypocotyl growth seems to be exerted exclusively via phytochrome. Regarding the far-red light dependent “high irradiance reaction” (FR-HIR) it was found that it plays a dominant role in growth of mustard only during a relatively short period. It tends to disappear in favor of a RL-HIR between 3 and 4 days after sowing. It is concluded that the seedling exhibits a largely endogenous temporal pattern of responsiveness to light. Phototropism of the mustard seedling can be elicited by low fluence rates (< 1 mW m^?2) of unilateral BL. This same light has no effect on straight growth. It is concluded that BL-dependent phototropic growth response of a hypocotyl and the effect of BL on longitudinal growth of the hypocotyl are unrelated phenomena.     

In vivo PHYTOCHROME-MEDIATED PERCEPTION OF REFLECTED LIGHT SIGNALS

The spectrophotometric assay of phytochrome in vivo in etiolated plant material was used to determine the effects of changes in reflected light on the state of the photoreceptor in etiolated seedlings exposed simultaneously to direct and reflected light. Changes in reflected light that were small in terms of the total (direct + reflected) radiation incident on the seedlings produced detectable changes in the state of phytochrome in vivo. The contribution of reflected light to the state of phytochrome in vertical organs was greater than expected from its low contribution to total incident light. These data from laboratory studies complement and are consistent with results of field studies on the effects of light reflected from neighboring vegetation on plant growth under natural radiation conditions.     

KINETICS OF PHYTOCHROME PELLETABILITY*

Abstract— The pelletability of P_r from maize coleoptiles was studied as a function of the delay time between a red and a far-red light pulse given in vivo. The obtained curve can be resolved into three parts. The two slowest reactions have half lives of 40 s and 3.6 min at 0°C. Furthermore, a break in the Arrhenius plot from the slowest reaction of the curve indicates that either the phytochrome “receptor” or the phytochrome molecule itself undergoes a jump in the Arrhenius activation energy at 20°C. These data are in good agreement with kinetic studies of phytochrome pelletability also discussed in this paper.     

INTRACELLULAR DICHROIC ORIENTATION OF THE BLUE LIGHT-ABSORBING PIGMENT AND THE BLUE-ABSORPTION BAND OF THE RED-ABSORBING FORM OF PHYTOCHROME RESPONSIBLE FOR PHOTOTROPISM OF THE FERN Adiantum PROTONEMATA

The intracellular localization and orientation of the receptors for the blue light-induced phototropism in the fern Adianrum protonemata, phytochrome and the blue light-absorbing pigment, were investigated by combining the techniques of cell centrifugation and of microbeam irradiation with linearly polarized light. The phototropic response was induced in the cells even after they had been centrifuged basipetally to spin down the endoplasm from the apical region. When a polarized blue microbeam was given to a flank of the apical region of the protonema, the phototropic response after compensation of phytochrome effect by far-red light was most effectively induced when the polarization plane was parallel to the long axis of the cell. If protonemata were pre-irradiated with blue and far-red light, the phototropic response was mediated through phytochrome alone. If such pre-irradiated protonemata were similarly irradiated with a polarized blue microbeam, polarized light vibrating parallel to the cell axis was again most effective in inducing the response. These results indicate that both the blue light-absorbing pigment and the phytochrome responsible for the blue light-induced phototropism in Adiantum are confined to the plasma membrane and/or the ectoplasm and that the transition moments of their blue-absorption bands are nearly parallel to the cell surface.     

RAPID TRANSMISSION OF A PHYTOCHROME SIGNAL FROM HYPOCOTYL HOOK TO COTYLEDONS IN MUSTARD (Sinapis alba L.)

Abstract— In mustard ( Sinapis alba L.) cotyledons appearance of 'potential capacity' to phosphorylate is controlled by phytochrome (P_fr) through a threshold, all-or-none reaction. Phytochrome operates only if the hypocotyl hook is connected to the cotyledons for at least 2.5 min after the end of a 1 min inductive light pulse. Partial irradiations of hook and cotyledons show that in the case of induction of 'potential capacity' rapid transmission of the P_fr signal occurs from the hypocotyl hook to the cotyledons. Phytochrome within the cotyledons is totally ineffective in this regard. A 3 min red light pulse suffices for full transmission of the signal. It seems that an explanation of the facts we have observed requires a revision of the present views about communication between the organs of a plant.     

PHYTOCHROME CONTROL OF EXTRACELLULAR PEROXIDASE ACTIVITY IN MUSTARD INTERNODES: CORRELATION WITH GROWTH, and COMPARISON WITH THE EFFECT OF WOUNDING

Abstract— The effects of phytochrome status on extracellular peroxidase activity were investigated in Sinapis alba L. seedlings grown for 12 days under continuous white light and transferred to darkness after a red light or a far-red light pulse. The rates of extension growth and dry matter accumulation in the first internode were increased by the far-red light pulse. Extracellular proteins, obtained by low speed centrifugation of intact internodes infiltrated with CaCl₂, were separated by isoelectrofocusing, and four extracellular acidic peroxidases were resolved, the most active being A3 and A4 (both ˜60 kD). The activity of A4 was reduced by the far-red light pulse perceived by phytochrome, while the activity of A3 was unaffected. The promotion of internode extension growth caused by far-red light is biphasic [Casal and Smith (1989) Plant, Cell Environ. 12 ,511–50]. Changes in peroxidase activity were detected prior to the second, but not to the first phase of the internode growth promotion. The effects on both growth and peroxidase activity were virtually restricted to the upper half of the internode and, once established, did not subsequently increase in magnitude.
In contrast to the effects mediated by phytochrome, blue light pretreatments affected growth but not extracellular peroxidase activity. Wounding the internode reduced extension growth, increased the activity of A3, but caused no significant effects on A4.
Other extracellular proteins, separated in sodium dodecyl sulphate polyacrylamide gels and stained with Coomassie blue, showed no significant differences. The concentration of extracellular proteins was higher in the upper than the lower half of the internode.
Results are discussed in terms of phytochrome effects in light grown plants, peroxidase activitv-arowth relationships, and extracellular peroxidase isoform functions.     

LIGHT PROMOTION OF SEED GERMINATION IN Datura ferox IS MEDIATED BY A HIGHLY STABLE POOL OF PHYTOCHROME

Abstract— The duration of the far-red light-absorbing form of phytochrome (Pfr) of the photoreceptor pool involved in the control of seed germination was investigated for Datura ferox seeds. These seeds require both Pfr and alternating temperatures (20/30°C) to germinate. After 24 h imbibition (25°C), the seeds received pretreatment-light pulses providing different phytochrome photoequilibria (Pfr/P), followed by a 24 h dark incubation (25°C), and test-light pulses providing different Pfr/P immediately prior to transfer to alternating temperatures. Germination increased with increasing Pfr/P provided by the test-light pulses, but was unaffected by the pretreatment-light pulses. This suggests that phytochrome synthesis, phytochrome degradation and phytochrome-mediated changes in response to phytochrome were negligible. In other experiments, red light-pretreatment pulses were followed by dark incubations (25°C) of different duration before transfer to alternating temperatures. The proportion of Pfr remaining after the 25°C incubation period was estimated by comparing germination rates with those of seeds that received test-light pulses of known calculated Pfr/P immediately prior to the start of the cycles of alternating temperatures. More than 80% of the Pfr established by a Pfr/P= 0.87 light pulse was present and active even after 48 h dark incubation at 25°C. Surprisingly, when a pretreatmentlight pulse providing a Pfr/P= 0.70 was given, the reduction in [Pfr] was significantly faster.
Germination of Datura ferox seeds is under the control of a highly stable (type II like) phytochrome pool. Apparently, this pool follows Pfr dark reversion to the red light-absorbing form, the times to reach half the original Pfr pool being > 96 h or <14 h after light pulses providing Pfr/P= 0.87 or 0.70, respectively.