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.     

SIGNIFICANCE OF FLUENCE-RESPONSE DATA IN PHYTOCHROME-INITIATED SEED GERMINATION

Abstract. A model was developed to describe changes in fluence-response kinetics in terms of total phytochrome level (P_tot), level of the factor X interacting with active phytochrome (P_fr), P_frX equilibrium constant, seed sensitivity to P_fr-X interaction, and variation in phytochrome sensitivity within the seed population. Under conditions of stable X levels and stable population variation, the model predicted that a change in any of the other components will result in a parallel fluence-response curve on a probit-logarithmic plot. The linearity of the subsaturation plot is dependent on the ratio of P_tot to X concentrations. The model showed that changes in threshold response fluences can result from many causes other than changes in total phytochrome [P_tot>]. Changes in response-saturating fluences when maximal germination is less than 100% are predicted to be due to limiting levels of X. Changes in slope of fluence-response curves can be explained by changes in seed population variation by this model. Rumex crispus L. fluence-response data for germination is best explained by this model in terms of neither changes in P_tot nor X levels altering kinetics.     

A GENERAL HYPOTHESIS TO INTERPRET 'HIGH ENERGY PHENOMENA' OF PHOTOMORPHOGENESIS ON THE BASIS OF PHYTOCHROME

Abstract— It is demonstrated that the high energy reaction of photomorphogenesis (blue-far-red reaction) in the case of seed germination and hypocotyl lengthening of Lactuca sativa (L.) 'Grand Rapids' is a consequence solely of phytochrome. A hypothesis is presented in which all high energy phenomena can be explained on the basis of phytochrome.     

THE TEMPERATURE DEPENDENCE OF Pfr ACTION GOVERNS THE UPPER TEMPERATURE LIMIT FOR GERMINATION IN LETTUCE

In most cultivars of lettuce (Lactuca saliva L,), red light acting through the red/far-red reversible phytochrome system promotes full germination within the20–30°C range, but at progressively higher temperatures germination declines sharply. The relationship between this upper ternperature limit for germination and the temperature dependence of phytochrome action was investigated in Grand Rapids lettuce. In fresh seeds the GT₅₀ (temperature giving half maximal germination) was ca 29–30°C. In these seeds, escape from far-red reversibility did not occur at 35°C, a temperature above the GT₅₀, but occurred rapidly at 27°C, a temperature below the upper limit. Increasing periods of dark pretreatment at high temperature (35°C) or increasing concentrations of the germination inhibitor coumarin caused a progressive decline in the GT₅₀, Escape from photoreversibility did not occur at 27°C in seeds in which the GT₅₀ had been reduced to less than 25°C by coumarin or by prolonged high temperature pretreatment. These results indicate that there is a close correlation between the position of the upper temperature limit for germination, and the temperature dependence of phytochrome action. We conclude that factors that alter the upper temperature limit for germination do so by changing the temperature dependence of phytochrome action.     

PHOTOBIOLOGICAL CHARACTERIZATION OF A SPORE GERMINATION MUTANT dkgl WITH REVERSED PHOTOREGULATION IN THE FERN Ceratopteris richardii

Abstract— This paper describes the mutant dkgl in the fern Ceratopteris richardii, which shows rapid germination in darkness but is markedly inhibited by white light. Action spectra plotted at 10 nm intervals from 400 to 800 nm are presented for germination responses of wild-type and mutant spores to photon flux densities of 0.004, 0.04 and 0.4 jtmol/mVs. The action spectra for wild-type spores exhibit a sharp phytochrome-mediated peak at 660 nm, a broad peak from 670 to 740 nm resulting from an apparent high irradiance response and no germination below 560 nm. In the corresponding action spectra for mutant spores, the blue region displays rather complex fine structure with prominent minima at 450 and 470 nm, which suggests that cryptochrome is unaltered in these spores. The region from 550 to 640 nm shows the greatest inhibition of spore germination, but this region exhibits no obvious fine structure, which argues rather strongly against the possibility of a unique photoreceptor being active in mutant spores. The mutant spectra resemble the wild-type spectra in the region from 650 to 800 nm, and thus phytochrome seems normal in the mutant spores. The dkgl mutation appears to act late in the phytochrome transduction pathway where a hypothetical coupling protein may regulate the light-sensitive step in spore germination.     

The Heterotrimeric G-protein Complex Modulates Light Sensitivity in Arabidopsis thaliana Seed Germination

Release of dormancy and induction of seed germination are complex traits finely regulated by hormonal signals and environmental cues such as temperature and light. The Red (R):Far-Red (FR) phytochrome photoreceptors mediate light regulation of seed germination. We investigated the possible involvement of heterotrimeric G-protein complex in the phytochrome signaling pathways of Arabidopsis thaliana seed germination. Germination rates of null mutants of the alpha (Gα) and beta (Gβ) subunits of the G-protein (At gpa1-4 and agb1-2 , respectively) and the double mutant ( agb1-2/gpa1-4 ) are lower than the wildtype (WT) under continuous or pulsed light. The Gα and Gβ subunits play a role in seed germination under hourly pulses of R lower than 0.1 μmol m⁻² s⁻¹ whereas the Gβ subunit plays a role in higher R fluences. The germination of double mutants of G-protein subunits with phyA-211 and phyB-9 suggests that AtGPA1 seems to act as a positive regulator of phyA and probably phyB signaling pathways, while the role of AGB1 is ambiguous. The imbibition of seeds at 4°C and 35°C alters the R and FR light responsiveness of WT and G-protein mutants to a similar magnitude. Thus, Gα and Gβ subunits of the heterotrimeric G-protein complex modulate light induction of seed germination by phytochromes and are dispensable for the control of dormancy by low and high temperatures prior to irradiation. We discuss the possible indirect role of the G-protein complex on the phytochrome-regulated germination through hormonal signaling pathways.     

PHYTOCHROME-MEDIATED PHOTOTROPISM IN Adiantum PROTONEMATA-I. PHOTOTROPISM AS A FUNCTION OF THE LATERAL Pfr GRADIENT*

Abstract— Phytochrome-mediated phototropism of the protonema of the fern Adiantum capillus-veneris was studied in view of the hypothesis that phototropism is controlled by the gradient of Pfr (phytochrome in the far-red-absorbing form) across the short axis of the protonema. Fluence-response relationships were investigated using a microbeam irradiation technique that allowed simultaneous stimulation of the two sides of the subapical portion of the protonema with different fluences of red light. Mathematical models describing the tropic response as a function of fluence were derived from the hypothesis in consideration of the minimal phototransformation kinetics of phytochrome. and the fitness of the functions to the experimental data was examined. The analytical results were then evaluated in view of the photochemical properties of phytochrome known from the literature. It is concluded that the extent of the tropic response is determined by the difference in the Pfr concentrations between the two sides of the protonemal cell. It is further suggested that, even if phytochrome exists as a dimer in vivo, the physiological unit of phytochrome is the monomer.     

PHYTOCHROME-MEDIATED GERMINATION: EFFECTS OF BRIEF HIGH TEMPERATURE TREATMENTS

Abstract— Brief high temperature treatments, applied early in the pregerminative period to radiation-requiring seeds of lettuce, inhibited both dark and radiation-induced germination. The inhibition increased with increasing incubation temperature. There was little difference in the germination percentage whether the inductive irradiation was applied before or after the high temperature treatment. When the high temperature treatment preceded induction, there was an increase in the radiation dosage required for maximum germination. These findings are interpreted in terms of lability of physiologically active phytochrome to higher temperatures.     

PHEOMELANIN PHOTOCHEMISTRY. PHOTOLYSIS OF MODEL COMPOUNDS

Abstract— Phytochrome control of nitrate reductase activity has been studied in cotyledons and hypocotyls of light-grown Sinapis alba. Under polychromatic irradiation, an increase in the fluence rate of far-red light added to a constant source of photosynthetically active radiation causes a decrease in the phytochrome photoequilibrium and, in the hypocotyl, this results in decreased nitrate reductase activity. However, in the cotyledons this difference is only observed transiently. In both organs, enzyme activity is correlated with the level of the far-red light absorbing form of phytochrome, P_fr. These correlations are not altered when the fluence rate (with respect to phytochrome) is increased, suggesting that the responses are not fluence rate dependent. The results obtained are consistent with the notion that in fully de-etiolated seedlings, P_ft alone controls nitrate reductase activity.     

DETERMINATION OF EXTINCTION COEFFICIENTS OF OAT PHYTOCHROME BY QUANTITATIVE AMINO ACID ANALYSES

Abstract— Previously published extinction coefficients for phytochrome were based on indirect colori-metric estimates of protein weight, mostly by the Lowry method, using bovine serum albumin as a standard. This paper reports revised values based on quantitative amino acid analyses of highly purified phytochrome. The molar extinction coefficient of the red-absorbing form of phytochrome with 120000mol. wt subunits was found to be approximately 102000cm"₁ per 120000moi. wt at 667nm. The molar extinction coefficients at 667 nm for phytochrome with 120000 mol. wt subunits and for the 60000 mol. wt chromopeptide of P_r were found to be approximately the same for samples of equivalent purity. Included in the amino acid analyses of phytochrome with 120000 mol. wt subunits are values for cysteine (11) and cystine (1), measured by two different techniques that gave the same results and agreed well with the estimate of total half-cystine (14) obtained by an independent method. Tryptophan was quantitated by analysis of a p-toluene sulfonic acid hydrolysate.     

Two photobiological pathways of phytochrome A activity, only one of which shows dominant negative suppression by phytochrome B

The plant receptor phytochrome A (phyA) mediates responses like hypocotyl growth inhibition and cotyledon unfolding that require continuous far-red (FR) light for maximum expression (high-irradiance responses, HIR), and responses like seed germination that can be induced by a single pulse of FR (very-low-fluence responses, VLFR). It is not known whether this duality results from either phyA interaction with different end-point processes or from the intrinsic properties of phyA activity. Etiolated seedlings of Arabidopsis thaliana were exposed to pulses of FR (3 min) separated by dark intervals of different duration. Hypocotyl-growth inhibition and cotyledon unfolding showed two phases. The first phase (VLFR) between 0.17 and 0.5 pulses.h-1, a plateau between 0.5 and 2 pulses.h-1 and a second phase (HIR) at higher frequencies. Reciprocity between fluence rate and duration of FR was observed within phases, not between phases. The fluence rate for half the maximum effect was 0.1 and 3 mumol.m-2.s-1 for hourly pulses of FR (VLFR) and continuous FR (HIR), respectively. Overexpression of phytochrome B caused dominant negative suppression under continuous but not under hourly FR. We conclude that phyA is intrinsically able to initiate two discrete photoresponses even when a single end-point process is considered.     

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris-I DEMONSTRATION AND QUANTITATIVE CHARACTERIZATION OF THE PHOTOCHROMIC SYSTEM Pr⇔ Ii700USING NANOSECOND-LASER PULSES*,†

Abstract— Spores of Dryopteris paleacea and D. filix-mas are positively photoblastic with an optimum in the action spectrum around 665 nm. Light is perceived by phytochrome and the relationship between germination and mole fraction of the far-red-absorbing form of this pigment, P_fr, was investigated with saturating irradiations between 662 and 747 nm under low-fluence-rate conditions. These control irradiations establish a proportion of the total phytochrome, P,_tot, as P_fr with P_fr/P_tot–φ at equilibrium. These φ -values were calculated according to data for native oat phytochrome (Kelly and Lagarias, 1985, Biochemistry 24, 6003) and the spectral characteristics of the interference filters. With this method a linear relationship could be found between φ and germination from 2 to 70% for D. paleacea and from 2 to 90% for D. filix-mas, if probit germination was plotted vs probit φ This correlation formed the basis of investigating the phytochrome photoconversion by dye-laser pulses of 380 ± 30 ns under high-fluence-rate conditions, and thus to test quantitatively the impact of the photoreversibility of intermediate reactions of the photoconversion and the red-absorbing form of phytochrome, P_fr on the final P_fr-level. Spore germination was initiated by a single-laser pulse in the range from 592 to 700 nm. The most effective wavelengths were 649 and 660 nm in both species, and at saturation maximal germination (ca. 50%) was obtained from 592 to 665 nm for D. paleacea or ca. 60% germination from 592 to 670 nm for D. filix-mas. Both saturation levels correspond to a ø-value between 0.40 and 0.45. This significantly diminished photoconversion is a consequence of the high-fluence-rate conditions during the laser pulse which establishes the photochromic system between P_r and a set of very early intermediates, Iⁱ_700, (= P_r? Iⁱ₇₀₀). This system can be described by the extinction coefficients of P_r and the intermediates Iⁱ₇₀₀, and by the quantum yields, 4,φ for the forward and reverse reactions as φ If φ is calculated, assuming a quantum yield of 1:1 for both reactions and with the extinction coefficients of P_r and Iⁱ_7(l() (= lumi-R) given by Eilfeld and Riidiger (1985, Z. Naturforsch. 40c , 109), significantly higher values are calculated for / as compared to φ found in the control experiments. These results can be explained either: (i) with a quantum yield ratio φ_pr-φ1700: φ_1700φpr=1:1 and an assumed additional dark reaction leading from Iⁱ₇₀₀ or later intermediates back to P_r: or (ii) with a quantum yield ratio φ_{pr φ 1700}: φ_{1700 φpr}=1:2. In this case all Iⁱ₇₀₀ have to relax to P_fr. In this case all Iⁱ₇₀₀ have to relax to P_fr.     

Biogenic Nanoparticle-Mediated Augmentation of Seed Germination,Growth, and Antioxidant Level of Eruca sativa Mill. Varieties

A study was undertaken to examine the influence of biogenic nanoparticles synthesized from Tridax procumbens on different parameters of seed germination, seedling growth, and various biochemical parameters in four Eruca sativa varieties having low percentage of germination. Seeds were treated with different concentrations (30 and 40 ppm) of biogenic nanoparticles, of which 30 ppm was found to be the most effective and was therefore used for subsequent studies. Initially, the effect of biogenic nanoparticles on germination percentage, speed of germination, coefficient of germination, mean germination time, shoot and root length, fresh and dry matter, and vigor index was studied. From the experiments performed and the results obtained, it was evident that the treatment with biogenic nanoparticles decreased the electrolyte leakage and level of malondialdehyde as compared to control. The treatment with biogenic nanoparticles enhanced the levels of proline and ascorbic acid and stimulated the antioxidant enzyme activities resulting in the reduced level of reactive oxygen species. These activities were found to be variety-dependent. The possible involvement of biogenic nanoparticles in the production of new pores in seed coat during their penetration, resulting in the influx of the nutrients inside the seed, is suggested. This accelerated seed germination is followed by rapid seedling growth. The present findings indicated that biogenic nanoparticles promote seed germination in E. sativa by overcoming the detrimental effects of reactive oxygen species (ROS) and improving the antioxidative defense system which finally result in increased seedling growth.     

Light quality influences indigo precursors production and seed germination in Isatis tinctoria L. and Isatis indigotica Fort

Isatis tinctoria L. and Isatis indigotica Fort. are biennial herbaceous plants belonging to the family of Cruciferae that are used as a source of natural indigo and show several morphological and genetic differences. Production of indigo (indigotin) precursors, indican (indoxyl beta-D glucoside) and isatan B (indoxyl ketogluconate), together with seed germination ability were compared in Isatis tinctoria and Isatis indigotica grown under six different light conditions (darkness, white, red, far red, blue, yellow light) at 25 degrees C. Light quality influenced both germination and production of indigo precursors in the two Isatis species. Different responsiveness to far red and blue light was observed. Indeed, a detrimental effect on germination by blue and far red light was found in I. tinctoria only. Different amounts of isatan B were produced under red and far red light in the two Isatis species. In I. tinctoria, the level of main indigo precursor isatan B was maximal under red light and minimal under far red light. Whereas in I. indigotica far red light promoted a large accumulation of isatan B. The photon fluence rate dependency for white and yellow light responses showed that the accumulation of indigo precursors was differently influenced in the two Isatis species. In particular, both white and yellow light enhanced above 40 micromol m(-2) s(-1) the production of isatan B in I. indigotica while only white light showed a photon fluence dependency in I. tinctoria. These results suggest a different role played by the labile and stable phytochrome species (phyA and phyB) in the isatan B production in I. tinctoria and I. indigotica. I. indigotica, whose germination percentage was not influenced by light quality, demonstrated higher germination capability compared with I. tinctoria. In fact, I. tinctoria showed high frequency of germination in darkness and under light sources that establish high phytochrome photoequilibrium (red, white and yellow light). Germination in I. tinctoria was negatively affected by far red and blue light. I. indigotica seeds appear to be indifferent to canopy-like light (far red). Our results provide further insights on the distinct behaviour of I. tinctoria and I. indigotica that belong to two different genetic clusters and different original environments.     

ACTION SPECTRUM FOR PHOTOREVERSION OF THE ACTIVE Pfr-FRACTION OF Mougeotia in vivo

Abstract— The dichroic oriented fraction of the far-red light absorbing form of phytochrome (Pfr) in the green alga Mougeotia was characterized by action spectroscopy. Microbeam irradiations had to be used for the induction of chloroplast movement in Pfr-containing cells, because of the special dichroic absorption characteristics of the red light absorbing form of phytochrome (Pr) and Pfr in the alga. Fluence-response curves were elaborated especially in the far-red spectral region by reverting Pfr to Pr at the flanks of the cells and thus generating Pfr-gradients. Linearly polarized light vibrating perpendicularly to the cell axis was used, thus corresponding to the S,-transition moments of Pfr at the flanks of the cells. The action spectrum is characterized by a peak at approximately 715 nm and a very pronounced decrease towards 728 and 734 nm. The data indicate that the spectral absorption of the active Pfr-fraction in green Mougeotia is shifted towards shorter wavelengths as compared to extracted phytochrome from etiolated or even green higher plants. This "blue shift" seems to be typical for Pfr from green lower plants.     

PHYTOCHROME AND EFFECTS OF SHADING ON GROWTH OF WOODLAND PLANTS

qrowth of Circaea lutetiaim plants was studied in various locations in or near a mixed deciduous woodland. Morphological changes resulting from increased shading included increases in leaf area ratio, specific leaf area and specific water content. Parallel measurements with a spectroradi-ometer confirmed that shading involved a reduction in both light fluence rate and light quality (e.g. red/far-red ratio). Phytochrome P_fr/P status was also studied by spectrophotometric measurements on Avena seedling test material and by biological (Lactuca seed germination) assay. Attempts were made to demonstrate phytochrome controlled changes in plant morphology under controlled environment, using both end-of-day far-red treatment and far-red enrichment of the main light period. Effects of natural shading were most clearly simulated by varying light fluence rate while maintaining a constant but high red/far-red ratio     

PHYTOCHROME-CONTROLLED FERN-SPORE GERMINATION: PHASE-SPECIFIC MODULATION BY ELEVATED TEMPERATURES*

The phytochrome-mediated spore germination in Dryopteris is inhibited by raising the temperature from 22 to 27 or 32°C. As reported earlier (W. Haupt (1990) Photochem. Photobiol. 52,57–59), the elevated temperature is effective during the “coupling phase”, i.e. when P_fr, the active form of phytochrome, starts the processes leading to germination. In addition, a strong effect has now been found in the “preinduction phase”, i.e. when the spore develops its competence to react to P_fr. This latter effect is overcome, if in the subsequent coupling phase a high level of P_fr is allowed to act for a long time. Moreover, the inhibiting effect in a2–3 day preinduction phase is most obvious in or even restricted to its terminal 24 h. It is proposed that the temperature acts by reversibly abolishing the cell's competence to react to P_fr, and that this effect can extend into the coupling phase. Not yet understood is an “inversion” effect in D. paleacea: if the temperature is raised in the earlier parts of the preinduction phase, the effect of P_fr in the subsequent coupling phase is enhanced rather than inhibited.     

KINETIC ANALYSIS OF A VERY RAPIDLY REVERTING POPULATION OF HIGH-MOLECULAR-WEIGHT PHYTOCHROME*

Abstract— Purified high-mol-wt phytochrome preparations in the absence of ethylenediaminetetraacetic acid or 2-mercaptoethanol may exhibit lower far-red extinction in the far-red-absorbing form (Pfr) than has previously been reported. Kinetic analyses suggest that such preparations of phytochrome in the Pfr form consist either of two populations of Pfr, one with normal extinction and one with greatly reduced far-red extinction, or of one population of Pfr in which each molecule possesses both one or more high far-red extinction chromophores and one or more low far-red extinction chromophores. The low-extinction form of Pfr undergoes reversion to the red-absorbing form of phytochrome with a half-life of approximately 2 min at 3°C and may represent as much as 35 per cent of the total Pfr present.     

THE INFLUENCE OF LIGHT UPON SEED GERMINATION. POSSIBLE INTERPRETATIONS OF DATA

Abstract— The response of seeds to irradiation by white light varies with the species: induction of germination, inhibition of germination or both according to the time-irradiation. The low energy reaction (LER) which acts via phytochrome can explain many cases of influence of light upon germination. It seems possible that a second reaction, the high energy reaction (HER), acts through another photoreceptor.     

THE CONTROL BY PHYTOCHROME OF NITRATE REDUCTASE IN THE CURD OF LIGHT-GROWN CAULIFLOWER

Abstract— The activity of nitrate reductase from the curd of light-grown cauliflower ( Brassica oleracea (L) var botrytis (DC) 'St. Hilary') is modulated by nitrate and by light. Using broad-band sources of equal photosynthetically active radiation but with different proportions of red and far-red light, a linear relationship between nitrate reductase activity and ψ(Estimated phytochrome photoequilibrium) was obtained. This relationship, apparent after 8 h incubation, was maintained and little altered after 48 h incubation. The linearity was apparent between ψE 0.26 and ψE 0.69; ψE 0.26 being no more effective than a dark control. Far-red reversibility confirmed the involvement of phytochrome. Brief pulses of red light were also used to establish a range of phytochrome photoequilibria within the tissue. Again a linear relationship between ψ and nitrate reductase activity was obtained with a threshold for the response at ψ 0.3. With both monochromatic and broad-band sources it was seen that neither photon fluence rate nor duration of exposure affected the final activity of the enzyme and that phytochrome was acting solely through ψ (or [Pfr] since phytochrome is stable in this tissue) to bring about these responses.