Abstract: Brief red illumination (10 min/8 hr) of Lemna gibba L. G-3 growing heterotrophically in the dark increases the growth of the plants and results in a substantial increase in the levels of mRNA for two major chloroplast polypeptides. These two nuclear-coded polypeptides are the light-harvesting chlorophyll a/b-protein, an intrinsic thylakoid membrane protein, and the small subunit of the stromal enzyme ribulose 1, 5-bisphosphate carboxylase [RuP2; 3-phospho-D-glycerate carboxylyase (dimerizing), E.C.4.1.1.39]. The effect of 10 min red illumination on the dark growth of the plants is reversed by immediate far-red illumination, but the effect on the mRNA levels is not. However, this latter response can be reversed by far-red light if the time between the beginnings of the red and far-red illumination is reduced to one minute. Thus phytochrome is the photoreceptor mediating both responses, and the effect on amounts of the translatable mRNAs has a remarkably short escape time.

Abstract: Germination of Rumex obtusifolius L. seeds (nutlets) is low in darkness at 25° C. Germination is stimulated by exposure to 10 min red light (R) and also by a 10-min elevation of temperature to 35° C. A 10-min exposure to far-red light (FR) can reverse the effect of both R (indicating phytochrome control) and 35° C treatment. Fluence-response curves for this reversal of the effect of R and 35° C treatments are quantitatively identical. Treatment for 10 min with light of wavelenght 680, 700, 710 and 730 nm, after R and 35° C treatment, demonstrates that germination induced by 35° C treatment results from increased sensitivity to a pre-existing, active, far-red-absorbing form of phytochrome (Pfr) in the seeds.

Abstract: Detailed action spectra are presented for the inhibition of hypocotyl extension in dark-grown Sinapis alba L. seedlings by continuous (24 h) narrow waveband monochromatic light between 336 nm and 783 nm. The results show four distinct wavebands of major inhibitory action; these are centred in the ultra-violet (λmax=367 nm), blue (λmax=446 nm), red (λmax=653 nm) and far-red (λmax=712 nm) wavebands. Previous irradiation of the plants with red light (which also decreases Ptot) causes decreased inhibitory action by all wavelengths except those responsible for the red light inhibitory response. Pre-irradiation did not alter the wavelength of the action maxima. It is concluded that ultra-violet and blue light act mainly on a photoreceptor which is different from phytochrome.

Abstract: Previous studies have indicated that phytochrome regulates Ca(2+) fluxes across the plasma membrane of plant cells. In this study we investigated whether phytochrome can also regulate such fluxes across mitochondrial membranes, using the Ca(2+)-sensitive dye murexide to monitor the uptake and release of Ca(2+) by mitochondria. The results showed that Ca(2+) fluxes in these organelles could be photoreversibly altered, red light diminishing the net uptake rate and far-red light restoring this rate to its dark control level. Treatment of the mitochondria with ruthenium red blocked their Ca(2+) uptake. In the presence of this inhibitor, red light induced a net efflux of Ca(2+) from the mitochondria, and subsequent far-red light reduced this efflux to nearly zero, the dark control level. Light-induced rate changes in Ca(2+) flux, both with and without the inhibitor, persisted for several minutes in the dark and remained photoreversible through several irradiations for as long as 30 min. The purity of the mitochondrial preparation was judged to be about 80% by electron microscopic morphometry; most of the phytochrome present was localized on the mitochondria in the preparation by using immunocytochemical methods. Taken together with previous findings, the results suggest that red light activation of phytochrome would initiate an increase in the cytosolic Ca(2+) concentration. The results are integrated with the fact that calmodulin is a component of plant cell cytoplasms to construct a model postulating that phytochrome directs photomorphogenesis in part through its regulation of Ca(2+) and calmodulin-controlled enzyme activities.

Abstract: — Slow destruction of the far-red-absorbing form of phytochrome (Pfr), which has been observed in light-grown oat and maize, occurs in light- and dark-grown Amaranthus, Pharbitis, and Brassica seedlings as well. Destruction of Pfr in these seedlings shows two phases: if a high level of Pfr is produced in dark-grown seedlings, the destruction is fast in the beginning and then slows after a low Pfr level has been reached. Slow Pfr destruction is predominant in light-grown tissue.

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.

Abstract: . It is well established that seedlings of mustard (Sinapis alba L.) synthesize juvenile anthocyanin only if treated with light pulses or continuous light. The light effects are considered to be due to the operation of phytochrome. Here we show that the responsiveness of anthocyanin synthesis to a saturating red light pulse or to continuous far-red light varies as a function of time and is strongly influenced by a light pretreatment prior to competence. Competence appears approximately 25 h after sowing. The starting point of anthocyanin synthesis, which is 27 h after sowing, and the lag- phase of this response, which is 2 h, are not affected by light pretreatments prior to competence. It is concluded that quantitative interpretations of phytochrome responses based entirely on properties of phytochrome can no longer be considered adequate.

Abstract: The specific activity of arginine decarboxylase (ADC; l-arginine carboxylase; EC 4.1.1.19) rises steadily over an 8 hour experimental period in the growing buds and subapical epicotyl internodes of 6-day-old totally etiolated pea seedlings. Treatment with red light (R) completely annuls this rise in epicotyls but increases it in buds, thus paralleling the opposite effects of R on the growth of these two organs. Far red light (FR) reverses both effects of R on ADC and is, in turn, reversed by R, indicating phytochrome control. Effects in both organs are clearly seen within 2 hours. By 6 hours after R, the post-irradiation rise in ADC specific activity in buds is 3 times greater than that of the dark controls. Over the same period, ADC specific activity in epicotyls is inhibited by 56% relative to dark controls, reflecting zero net change after R and a continued rise in the dark. Cycloheximide inhibits the rise in ADC activity in both rapidly growing organs (epicotyls in dark and buds after R) but is without effect in both slower growing organs. Actinomycin D inhibits only in dark grown epicotyls, whereas chloramphenicol produces no inhibition in any system tested. ADC is the first enzyme to show a two-way, organ-specific response to phytochrome conversion from Pr to Pfr. This finding is discussed in relation to the growing evidence that polyamines formed from arginine may be important growth regulators in plants, as well as in microbial and animal cells.

Abstract: The single, ribbon-shaped chloroplast in the filamentous green algaMougeotia performs orientational movements with respect to light. The chain of reaction involves phytochrome as the photoreceptor pigment to perceive the light signal differentiated by wavelength and direction, calcium probably to convert the light signal into a chemical message and actomyosin to respond to this message and to move the chloroplast accordingly.Precise reorientation of the chloroplast is proposed to be brought about by a dual function of phytochrome: regulation of the cellular level of calciumand regulation of membrane anchorage sites to actin.

Abstract: Endogenous ethylene production of tobacco leaves was similar in light and in darkness. However, the rate of conversion of exogenously applied l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene was reversibly inhibited by light. Virus-stimulated ethylene production, during the hypersensitive reaction of tobacco leaves to tobacco mosaic virus, was likewise inhibited by light. Under such circumstances ethylene production is limited at the level of the conversion of ACC to ethylene. Inhibition of the increase in ACC-stimulated ethylene production by cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide after shifting leaf discs from light to darkness indicated that de novo protein synthsis was involved. Regulation of ACC-dependent ethylene production by reversible oxidation/reduction of essential SH groups, as suggested by Gepstein and Thimann (1980, Planta 149, 196–199) could be excluded. Instead, regulation of the ACC-converting enzyme at the level of both synthesis/degradation and activation/inactivation is suggested. Phytochrome was not involved in light inhibition, but low intensities of either red or blue light decreased the rate of ACC conversion. Dichlorophenyldimethylurea counteracted the inhibitory effect of light, indicating that (part of) the photosynthetic system is involved in the light inhibition. The ethylene production of Pharbitis cotyledons grown in darkness or light, either in the presence of absence of the inhibitor of carotenoid synthesis, SAN 9789 (norflurazon), supported this view.

Abstract: The biological activity of the plant photochromic photoreceptor, phytochrome, is generally thought to reside in Pfr the far-red-absorbing form, whilst the red-absorbing Pr is inactive. This view is based solely on incomplete correlations between spectrophotometrically detectable Pfr and responses in etiolated tissues given brief light treatments1,2. Studies of light-grown seedlings have recently revealed an inverse, linear relationship between phytochrome-regulated extension growth and phytochrome photoequilibrium (defined as Pfr/(Pr+Pfr) and estimated from the spectral photon distribution of the actinic radiation)3–10. Such a relationship could only fit the ‘Pfr as active form’ theory if Pr+Pfr (that is, Ptotal) remained constant and independent of the light sources used. Here I report data from herbicide-bleached, light-grown maize seedlings which show that Ptotal is strongly dependent on the wavelength distribution of the radiation and that the amount of Pfr does not correlate with growth response.

Abstract: Relative quantum responsivity curves for inhibition of hypocotyl elongation in Sinapis alba L. seedlings previously grown in white light confirm that a marked “end of day” inhibition response can be induced by a monochromatic light treatment (30 min) at the end of the light period. In dark grown seedlings, however, no growth inhibition can be induced by a 30 min monochromatic light treatment. A prerequisite for an induction response appears to be a pretreatment with continuous light. Far red light is most effective with blue and red light showing a lesser effectiveness. The light pretreatment also shows a marked fluence rate dependency with respect to its ability to allow an induction response to manifest itself. The pretreatment required shows all the characteristics of a classical “HIR” response. The appearance of the effect in plants treated with the herbicide SAN 9789 seems to exclude chlorophyll as being the photoreceptor.

Abstract: The photostationary equilibrium between the Pr and Pfr forms of phytochrome shows a strong solvent deuterium isotope effect. Phytochrome transformation from the Pr to the Pfr form exhibits a small deuterium isotope effect, in Tris-D2O upon irradiation with red light, only after a photocycling of the phytochrome. In contrast, both the photoreversion and dark reversion of Pfr show an enhanced rate in D2O. In addition to the shift in the photostationary equilibrium in D2O, another pronounced effect of D2O on phytochrome is reflected in a significant enhancement of the fluorescence quantum yield of phytochrome (Pr). This result is interpreted in terms of the primary reaction involving an intramolecular proton transfer and its consequence in the phototransformation of phytochrome. It is further proposed that a tyrosyl residue acts as a general acid catalyst in the Pr to Pfr phototransformation, which is slower in D2O than in H2O. The D2O solvent isotope effect on the photoreversion and dark reversion of Pfr is explained on the basis of acid catalysis, probably a specific acid catalysis by deuteronium ion.

Abstract: Blue and red light induce stomatal opening in Commelina comrnunis L. 3-(3, 4-di-chlorophenyl)-l, l-dimethylurea (DCMU) slightly inhibits stomatal opening in blue light, while being strongly inhibitory in the presence of red light. Similar inhibition of red-light-induced opening is obtained by far red. Far red added to red light causes closure of open stomata almost to the degree obtained in darkness. Parallel to its effect on stomatal movement, far red increases 86Rb leakage from guard cells. The antagonism of red and far red possibly indicates the involvement of phytochrome. It is proposed that chlorophyll fluorescence in the far red region, increased by DCMU, is responsible for the inhibitory effect of red light. Similarly, the stimulatory effect of blue light may be due to its fluorescence in the red region.

Abstract: — Phototransformation at 2°C of the red-absorbing form of phytochrome (Pr) to the far-redabsorbing form (Pfr) was studied with both undegraded oat (Avena sativa L., cv. Garry) and undergraded pea Pisum sativum L., cv. Alaska) phytochrome. Phototransformation was initiated by a 15-ns laser pulse with maximum emission near 600 nm and output power of 30 mJ. The first resolvable transformation intermediate exhibited relative to Pr a maximum absorbance increase near 700 nm and was fully present at the earliest time measured, which was 60 ns after the flash. This intermediate absorbance decayed by two reactions for oat phytochrome (half-lives of 11 and 140 μs assuming parallel reactions) and by three for pea phytochrome (half-lives of 14, 280 and 1600 μs assuming parallel reactions). The kinetics of the slowest reaction for pea phytochrome, however, might be somewhat distorted by an instrument artifact. The appearance of the far-red-absorbing phytochrome, as monitored by absorbance increase at 720 nm, occurred by at least two reactions for both oat (half-lives of 47 and 250 ms assuming parallel reactions) and pea (half-lives of 170 and 770 ms assuming parallel reactions) phytochrome. The possibility of slower reactions was not tested. Assays for possible proteolysis of the phytochrome samples studied here indicated that the presence of degraded phytochrome could not account for the observed multiphasic kinetics except possibly for one phase of the triphasic intermediate decay seen with pea phytochrome.

Abstract: The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.

Abstract: The binding of phytochrome to Cibacron Blue 3GA was utilized to develop a new affinity purification procedure for phytochrome. Brushite-purified phytochrome from rye (Secale cereale c.v. Cougar) was bound to agarose-immobilized blue dye in 0.1 molar potassium phosphate (pH 7.8), contaminating proteins washed out with 0.5 molar KCl, and homogeneous phytochrome eluted with 10 millimolar flavin mononucleate. Ninety-five per cent of the phytochrome applied bound, and 60 to 65% was eluted, giving a 25 to 30% yield for the complete one-day procedure. Affinity-purified rye phytochrome was identical to conventionally purified phytochrome in its behavior on sodium dodecyl sulfate gels, in gel exclusion chromatography, in sedimentation in sucrose density gradients and in its spectral properties.

Abstract: Nitrite reductase in the excised etiolated leaves of maize showed the photoreversibility by red and far-red light. Five minutes of red light illumination lead to a 130% increase in the enzyme activity which was reversed by far-red light. The kinetics of nitrite reductase activity under continuous far-red light showed a lag phase of 1 hr.

Abstract: The peroxisomal enzyme, urate oxidase (EC 1.7.3.3), and the next enzyme of the urate pathway, allantoinase (EC 3.5.2.5), demonstrate a lightmediated rise of activity in the cotyledons of mustard (Sinapis alba L.). The capacity of the peroxisomes for urate breakdown, marked by the time course of urate oxidase, develops distinctly later than the two other peroxisome functions (fatty acid breakdown, “glyoxysomal” function; glycolate breakdown, “leaf peroxisomal” function). The light effect on urate oxidase and allantoinase is mediated through the phytochrome system in all three seedling organs (cotyledons, hypocotyl, radicle), as revealed by induction/reversion experiments with red/far-red light pulses and continuous irradiation with far-red light (high irradiance reaction of phytochrome). Both enzyme activities can be induced by phytochrome in the seedling cotyledons only during a sensitive period of about 48 h prior to the actual light-mediated rise of activity, making it necessary to assume the existence of a long-lived intermediate (“transmitter”) in the signal response chain connecting enzyme formation to the phytochrome system. Detailed kinetic investigation, designed to test whether urate oxidase and allantoinase are controlled by phytochrome via the same signal response chain (coordinate induction), revealed large differences between the two enzymes: (i) a different onset of the loss of reversibility of a red light induction by a far-red light pulse (=onset of transmitter formation=coupling point; 48 h/24 h after sowing for urate oxidase/allantoinase); (ii) a different onset of the response (=onset of competence for transmitter= starting point; 72 h/48 h); (iii) full loss of reversibility (=completion of transmitter formation) is reached at different times (independence point, 90 h/52 h). These differences show that phytochrome controls urate oxidase and allantoinase via separate signal response chains. While urate oxidase can be localized in the peroxisomal fraction isolated from crude organelle extracts of the cotyledons by density gradient centrifugation, most of the allantoinase activity found in the peroxisomal fraction did not appear to be an integral part of the peroxisome but originated presumably from adhering membrane fragments.

Abstract: In background white light, supplementary far-red (λmax 700 nm) is an order of magnitude less effective than supplementary far-red (λmax 739 nm) in the stimulation of stem extension in Sinapis alba. The relationship between phytochrome photoequilibrium and extension rate increase for the two supplementary far-red treatments is, however, very similar. This evidence indicates that phytochrome cycling is not involved in the phytochrome control of stem extension in light-grown Sinapis alba and that the response to supplementary far-red light is not fluence rate (irradiance) dependent.

Abstract: The electric potential difference changes observed on etiolated oat coleoptiles in response to phytochrome transformation have been further studied using contacts on the coleoptile surface. Results are given, at 0.4 second resolution, for the first 1.5 minutes after saturating flashes of light each lasting 1 second. Responses to initial red (662 nanometers), to far red (about 700 nanometers and above) 10 minutes later, and to second red 10 minutes later still, all have time courses that are approximately Gaussian sigmoid in shape. The response to far red is of opposite sign to the response to red. Approximate magnitudes of the three changes, 1 minute after the light flash, are +6 millivolts for red, −10 millivolts for far red, +3.5 millivolts for the second red. It is argued that the observations reflect a hyperpolarization of the plasmalemma of coleoptile cells following red light and depolarization following far red. The response to red is not produced by a change in membrane permeability to K + . The mechanism could include a change to Na + or Cl − permeability or a modulation of an electrogenic pump: enhanced H + extrusion, Ca 2+ extrusion, or Cl − uptake. The response to far red could be produced by the reverse of one of those changes. The Gaussian curve is fitted to the data to determine the time at which the responses begin. Each response begins 4.5 seconds after the start of the flash of light. These delays are not related to the time course of phytochrome pelletability or redistribution in the cell. The delays may be due to some interaction of the transformed phytochrome with the plasma-lemma. Alternatively, the transformed phytochrome may interact quickly with some other structure which initiates a signal that takes 4.5 seconds to reach the plasmalemma.

Abstract: Phytochrome was shown to bind to agarose-immobilized Cibacron blue 3GA. A higher affinity of the dye for the putative biologically active form (Pfr) than the inactive form (Pr) of phytochrome was observed. Effective general eluants of Pr included 40% (vol/vol) ethylene glycol, 1% Triton X-100, or 0.5 M potassium iodide. Increasing ionic strength (1 M KCl) did not effectively elute phytochrome. Of the natural cofactors that have been reported to be analogues of the dye, NAD+, NADH, NADP+, cyclic AMP, AMP, ADP, ATP, and coenzyme A at a concentration of 10 mM would not elute phytochrome. At 10 mM, FMN eluted at least 65% of the bound Pr, whereas FAD eluted 40%. Blue dextran/agarose was found to bind Pfr but exhibited essentially no affinity for Pr. Phytochrome that was bound as Pfr could be subsequently released by photoconversion to Pr. Because of the high degree of selectivity that the blue dye and its dextran conjugate exhibit for the Pfr form of phytochrome and the known property of the dye as an analogue of natural ligands of proteins, it is proposed that the dye and its conjugate may be used as probes of a binding domain on the phytochrome protein that is important to its biochemical action.

Abstract: Using light-grown plants of Sinapis alba an analysis has been made of the effect on extension growth of adding far red light to a background photosynthetic source. It has been possible to distinguish between the increase in fluence rate and the reduction of the amount of phytochrome present as Pfr, which are both consequences of the addition of supplementary far red light, and to determine that the response of increased extension growth is due only to the latter. It is shown that the degree of fluence rate dependency varies with photoequilibrium and the significance of this interaction is discussed in terms of the mode of action of phytochrome and of its role in the natural light environment.

Abstract: Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (Pfr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.

Abstract: In germinating seedlings of Sinapis alba nitrate reductase activity as assayed in vivo becomes accessible to phytochrome control between 15 and 17 h after sowing. Phytochrome operates via the high irradiance reaction to control nitrate reductase activity in the period 15 to 20 h after sowing. Both continuous red light and far-red light elicit this response with a strong fluence rate dependency being apparent in each case. The induction of nitrate reductase activity by light pulses at 20 h after sowing is greatly influenced by red light pre-treatments (operating through phytochrome) given between 0 and 15 h after sowing. Low fluence rate pre-treatments reduce the effectiveness of a subsequent pulse to below the level of a dark control whilst high fluence rate pre-treatments greatly increase the effectiveness of a subsequent pulse.

Abstract: Accumulation of betalain (amaranthin) in the seedling of Amaranthus caudatus, var. viridis, is inducible by light. Since the apparent lag-phase of amaranthin accumulation after the onset of light is of the order of 3 h, light induction experiments could be performed up to 3 h after the onset of light without interference with actual synthesis. The intricate induction phenomena can be explained as follows: The inductive light operates through phytochrome and through a blue/UV photoreceptor (‘cryptochrome’). A phytochrome-dependent ‘High Irradiance Reaction’ is of minor importance. However, there is a strong, specific interaction between the light effects mediated through phytochrome and cryptochrome in the sense that the ‘extent of the reversible response’ — (response obtained with a particular light treatment terminated with a saturating red light pulse) minus (response obtained with the same light treatment when terminated with a saturating 756 nm light pulse) —increases with increasing Pfr level and total fluence rate during the induction period. It is concluded that light induced amaranthin synthesis is, in fact, a convenient biochemical model system of photomorphogenesis in the case when phytochrome and cryptochrome operate simultaneously in mediating photomorphogenesis.