Studies of the Mechanism of Enhancement of Phytochrome-dependent Lettuce Seed Germination by Prechilling

Temperature and kinetic studies were performed to examine the mechanism by which prechilling stimulates phytochrome-dependent seed germination in lettuce, Lactuca sativa, L. cv. Grand Rapids. Imbibed seeds were given a short far red irradiation and one day of dark incubation at 20 C to establish very low levels of the far red-absorbing form of phytochrome—(Pfr). Germination was greatly stimulated by subsequent prechilling treatments when they were followed by a second short far red irradiation. Prechilling therefore increased germination sensitivity to the low, normally inhibitory Pfr levels established by far red irradiation. This sensitivity increased with lowered prechilling temperature to a maximum near 4 C. It was linearly dependent upon duration of prechilling at 4 C up to a near maximal response at 10 hours, and it decayed in a converse manner when seeds were returned to 20 C after 10 hours at 4 C. Prechilling also increased germination responses to subsequent periods of high levels of Pfr which were initiated by red and terminated by far red irradiations. High Pfr periods adequate to promote the germination of unchilled seeds produced sharp inflections at 18 C in the dependence of germination on prechilling temperature. Rates of phytochrome potentiation of germination were not affected by prechilling. The response to prechilling fit a mechanism involving homeoviscous adaptation of membrane lipids to temperature.     

Analysis of the Effect of Light and Temperature on the Fluence Response Curves for Germination of Rumex obtusifolius

Both red light (10 minutes) and 35°C treatment (60 minutes) stimulate the germination of seeds of Rumex obtusifolius otherwise maintained in darkness at 25°C. Fluence response curves were determined for the effect of red light to stimulate germination of seeds with and without 35°C treatment. The endogenous far-red absorbing form (Pfr) level in the seeds was determined using short saturating fluences of wavelengths of light which maintain different proportions of phytochrome as Pfr at equilibrium. In the seed batches investigated, the endogenous Pfr level was found to be 4% or less of the total phytochrome. High dark germination after 35°C treatment does not result from an increase in sensitivity of the whole population to Pfr. Calculated fluence response curves for germination which best fit the experimental data suggest that seeds germinate in darkness after 35°C treatment because of a nonphytochrome-related process (overriding factor).     

Tomato seed germination: regulation of different response modes by phytochrome B2 and phytochrome A

Lycopersicon esculentum seeds germinate after rehydration in complete darkness. This response was inhibited by a far-red light (FR) pulse, and the inhibition was reversed by a red light (R) pulse. Comparison of germination in phytochrome-deficient mutants (phyA, phyB1, phyB2, phyAB1, phyB1B2 and phyAB1B2) showed that phytochrome B2 (PhyB2) mediates both responses. The germination was inhibited by strong continuous R (38 micromol m(-2) s(-1)), whereas weak R (28 nmol m(-2) s(-1)) stimulated seed germination. Hourly applied R pulses of the same photon fluence partially replaced the effect of strong continuous R. This response was called 'antagonistic' because it counteracts the low fluence response (LFR) induced by a single R pulse. This antagonistic response might be an adaptation to a situation where the seeds sit on the soil surface in full sunlight (adverse for germination), while weak R might reflect that situation under a layer of soil. Unexpectedly, the effects of continuous R or repeated R pulses were mediated by phytochrome A (PhyA). We therefore suggest that low levels of PhyA in its FR-absorbing form (Pfr) cause inhibition of seed germination produced either by extended R irradiation (by degradation of PhyA-Pfr) or by extended FR irradiation [keeping a low Pfr/R-absorbing form (Pr) ratio].     

Effect of light and temperature on the germination of lettuce seeds

A short period (15–30 min) at 30° C promotes germination of seeds of Lactuca sativa L. cv. Repolhuda in darkness. Far-red light reverses this stimulation, and the escape curves for phytochrome and high-temperature action are quite similar, indicating that the two factors act at a common point in the chain of events leading to germination. It is suggested that high temperature acts by decreasing the threshold of the active, far-red absorbing, form of phytochrome (Pfr) needed to promote germination.Abbreviations FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light     

Inhibition of Prechill-induced Dark Germination in Sorghum halepense (L.) Pers. Seeds by Phytochrome Transformations

A 10 C dark prechilling of johnsongrass [Sorghum halepense (L.) Pers.] seeds, when terminated by a 2-hr, 40 C temperature shift, potentiates about 40% germination at 20 C in darkness. Irradiation of the seeds before, during, and at the end of prechilling with far red light reduces the subsequent germination, although red irradiation after the far red can overcome some of the inhibition. However, either brief red or far red irradiation given immediately after the temperature shift inhibits subsequent germination by one-third to one-half. The results suggest that the far red-absorbing form of phytochrome is a factor in the prechill-induced dark germination and that phytochrome participates in the inhibition of germination by irradiations immediately after the temperature shift.     

Phytochrome-induced Germination, Endosperm Softening and Embryo Growth Potential in Datura ferox Seeds: Sensitivity to Low Water Potential and Time to Escape to FR Reversal

In Datura ferox seeds, the far-red absorbing form of phytochrome(Pfr) induces endosperm softening, larger embryo growthpotential,and germination. We investigated the effect of exposing theseeds to a range of water potentials in the presence of Pfronits induction of these responses. In addition, the escape timeto far-red-light (FR) reversal of the three responses wasdetermined. Low water potential inhibited Pfr action on endosperm softeningand germination in a similar way. In both cases, a 50% reductionin the response to a saturating red-light (R) irradiation wasobserved at a water potential of c. —0·5 MPa andtherewas very good correlation between the percentage numberof seeds with softened endosperm at 45 h after R and germinationat 72 h after R (R2=0·95). In contrast, the effect ofdecreasing the external water potential on Pfr induction ofa larger embryogrowth potential was more complex. Moderate decreasesin water potential (—0·3 to —0·5 MPa)enhanced Pfr action and thegrowth potential of the embryos waslarger (20—25%) than the water controls; water potentialsbelow —0·7 MPa inhibited the Pfr stimulus. The escape time to FR reversal of the R effect was shorter forthe increase in embryo growth potential than for endospermsoftening.Twenty-four h after R, the embryo response had escaped in morethan 80% of the population whereas endospermsoftening and germinationwere susceptible to FR inhibition in 100% of the seeds. These results indicate that in D. ferox seeds the increase inembryo growth potential is not sufficient for germination andthatendosperm softening is a necessary condition. Key words: Germination, dormancy, phytochrome, endosperm softening, water potential     

Interaction of light and temperature on seed germination of Rumex obtusifolius L.

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.Abbreviations FR far-red light - P phytochrome - Pr red-absorbing form of P - Pfr far-red-absorbing form of P - R red light     

Interaction of light and temperature on the germination of Rumex obtusifolius L.

Seeds (nutlets) of Rumex obtusifolius L. fail to germinate in darkness at 25° C, but are stimulated by short exposure to red light (R) the effectiveness of which can be negated by a subsequent short exposure to far red light (F) indicating phytochrome control. Short periods of elevated temperature treatment (e.g. 5 min at 35° C) can induce complete germination in darkness. Although short F cannot revert the effect of 35° C treatment, cycling the phytochrome pool by exposure to short R before short F results in reversion of at least 50% of the population. Prolonged or intermittent F can also revert the germination induced by 35° C treatment. The effect of elevated temperature treatment is interpreted on the basis of two possible models; (i) that it increases the sensitivity of the seeds to a low level of pre-existing active form of phytochrome (Pfr) (ii) that it induces the appearance of Pfr in the dark. In both cases it is envisaged that elevated temperature treatment and Pfr control germination at a common point in the series of reactions that lead to germination.Abbreviations D Dark - F far red light - P phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - R red light     

Light Actions in the Germination of Cocklebur Seeds: I. DIFFERENCES IN THE LIGHT RESPONSES OF THE UPPER AND LOWER SEEDS

Germination responses to light were studied in the upper andlower seeds of cocklebur (Xanthium pennsylvanicum Wallr.). Thelower seed was dark-germinating and negatively photoblastic;the upper one had a red-light (R) requirement and was positivelyphotoblastic. Germination of the lower seeds was inhibited bya prolonged single irradiation with R, blue (B) or far-red (FR)light applied during imbibition. The maximal inhibitory effectof a single irradiation occurred 9 h and 13 h after the startof soaking at 33 °C and 23 °C, respectively. However,the inhibitory effect of R differed from that of B and FR, byonly delaying germination. A single exposure to B or FR lightcould be replaced by intermittent B or FR irradiation, and theireffects were repeatedly reversible by the following R irradiation.If the upper seeds were not exposed to R during imbibition,they failed to germinate even at 33 °C which was optimalfor germination, and the promotive effect of R increased withdelay of its application time. The photoperceptive locus incocklebur seeds was the axial tissue for all B, R and FR. Lightreceived by the cotyledonary tissue had little effect. Germinationdimorphism in response to light is discussed with respect tothe phytochrome content and the ageing of axial tissues. Key words: Blue light, Dimorphism, Far red light, Germination, Red light, Xanthium seed     

Action of Phytochrome During Prechilling of Amaranthus retroflexus L. Seeds

Dark germination of Amaranthus retroflexus L. seeds at 35° increased after several days of prechilling at 20° or lower. Irradiation with far-red light for short periods during the early hours of a prechilling period at 10° inhibited subsequent dark germination at 35°. The inhibition was completely reversible with red light. Far-red irradiation in the latter part of the prechilling period was less effective. Increased dark germination of A. retroflexus seeds following a prechilling period at 20° or less is attributed to action of preexistent P_FR, the far-red absorbing form of phytochrome, within the seeds. Inactivation of P_FR was found to proceed ca. 4 times more rapidly at 25° than at 20°. Failure of imbibition temperatures above 20° to increase dark germination of A. retroflexus seeds is attributed to the rapid thermal reversion of pre-existent P_FR. We suggest that the action of prechilling (layering) on many other seed kinds arises in a similar way.     

Effects of Temperature and Various Red or Far-red Irradiations on Phytochrome- and Gibberellin A3-mediated Germination Control in Partially Hydrated Lettuce Seeds

Dark reversion of phytochrome in partially hydrated lettuceseeds (Lactuca sativa cv. Grand Rapids) is temperature dependent.After initial red irradiation (R) the higher the storage temperature,the higher the dark reversion rate. Following dark moist storage(DMS) at 30 ?C for 15 d none of the seeds receiving initialR germinated, whereas seeds stored at 0 ?C germinated nearlyas well (about 80%) as unstored controls. The half-time fordark reversion at 20 ?C and 30 ?C is 9 d and 3 d respectively.Repeated R treatments given at 5 d intervals during DMS at 20?C and 30 ?C maintained a high germination capacity. With threeor more R treatments the effect of high temperature largelydisappeared. Dark reversion of phytochrome was not observed in partiallyhydrated lettuce seeds receiving continuous red irradiation(cont R) for two or more days. The promotive effect of contR could be reversed at any time with a brief far-red irradiation(FR), indicating that the phytochrome system remained fullyphototransformable. With continuous far-red light (cont FR)the ability of gibberellin A₃ (GA₃) to stimulate germinationdisappeared and response to GA₃ also diminished in cont R followedby FR but at a slower rate indicating the induction of secondarydormancy in these partially hydrated seeds. This induction ofdormancy was retarded by repetitive or cont R but was enhancedby cont FR. The results of this study suggest a role for theaccumulated stable intermediates of phytochrome transformationin partially hydrated seeds with repeated or continuous R treatmentsand different effects of GA₃ and R in the regulation of germination. Key words: Phytochrome, Lactuca sativa, Seed germination, Temperature, Dark reversion of phytochrome, Seed water content     

Phytochrome A Mediates the Promotion of Seed Germination by Very Low Fluences of Light and Canopy Shade Light in Arabidopsis

Seeds of the wild type (WT) and of the phyA and phyB mutants of Arabidopsis thaliana were exposed to single red light (R)/far-red light (FR) pulses predicted to establish a series of calculated phytochrome photoequilibria (Pfr/P). WT and phyB seeds showed biphasic responses to Pfr/P. The first phase, i.e. the very-low-fluence response (VLFR), occurred below Pfr/P = 10-1%. The second phase, i.e. the low-fluence response, occurred above Pfr/P = 3%. The VLFR was similarly induced by either a FR pulse saturating photoconversion or a subsaturating R pulse predicted to establish the same Pfr/P. The VLFR was absent in phyA seeds, which showed a strong low-fluence response. In the field, even brief exposures to the very low fluences of canopy shade light (R/FR ratio < 0.05) promoted germination above dark controls in WT and phyB seeds but not in the phyA mutant. Seeds of the phyA mutant germinated normally under canopies providing higher R/FR ratios or under deep canopy shade light supplemented with R from light-emitting diodes. We propose that phytochrome A mediates VLFR of A. thaliana seeds.     

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.     

Phytochrome Control of Cellulase Activity in Datura ferox L. Seeds and its Relationship with Germination

The influence of phytochrome on endosperm softening and cellulaseactivity was studied on light-stimulated Datura ferox seeds.Endosperm softening preceded the earliest signs of radicle protrusion,and there was good correlation between the % of seeds with softendosperm at 48 h after R and germination at 96 h after R. Cellulaseactivity was stimulated by R and the increase in activity preceded,by more than 24h, radicle protrusion and endosperm softening.The effect of R was reversed by FR, but, by delaying the irradiationwith FR until cellulase activity had increased significantly,it was observed that removing Pfr did more than just stop anyfurther increase, the level of cellulase activity decreasedin about 24 h close to the dark controls. Cellulase activitywas decreased by a FR irradiation even when more than 60% germinationhad escaped from reversion. These results indicate that phytochromeinfluence on cellulase is not an indirect consequence of thestimulus of germination and that the continuous presence ofPfr is required for the cellulase activity to remain high. Thepossibility that cellulase and other degrading enzymes may bepart of the mechanism of light-induced germination is discussed. Key words: Phytochrome, germination, cellulase     

Elementary processes of photoperception by phytochrome A for high-irradiance response of hypocotyl elongation in Arabidopsis

Elementary processes of photoperception by phytochrome A (PhyA) for the high-irradiance response (HIR) of hypocotyl elongation in Arabidopsis were examined using a newly designed irradiator with LED. The effect of continuous irradiation with far-red (FR) light could be replaced by intermittent irradiation with FR light pulses if given at intervals of 3 min or less for 24 h. In this response, the Bunsen-Roscoe law of reciprocity held in each FR light pulse. Therefore, we determined the action spectrum for the response by intermittent irradiation using phyB and phyAphyB double mutants. The resultant action spectrum correlated well with the absorption spectrum of PhyA in far-red-absorbing phytochrome (Pfr). Intermittent irradiation with 550 to 667 nm of light alone had no significant effect on the response. In contrast, intermittent irradiation with red light immediately after each FR light pulse completely reversed the effect of FR light in each cycle. The results indicate that neither red-absorbing phytochrome synthesized in darkness nor photoconverted Pfr are physiologically active, and that a short-lived signal is induced during photoconversion from Pfr to red-absorbing phytochrome. The mode of photoperception by PhyA for HIR is essentially different from that by PhyA for very-low-fluence responses and phytochrome B for low-fluence responses.     

Light and nitrate interaction in phytochrome-controlled germination ofPaulownia tomentosa seeds

Pulsed light and nitrate exhibit an interactive effect on the germination ofPaulownia tomentosa Steud. seeds that require long periods of light irradiation. Two pulses of red light (R), separated by an adequately long dark interval, substitute for continuous prolonged irradiation. A far-red (FR) pulse given at the beginning of the dark interval inhibits germination, while it has no effect if given at the end. The requirement for certain ratios of the far-red-absorbing form of phytochrome/total phytochrome (P_fr/P_tot) differs when a FR+R-pulse is given as the first or second of two pulses (FR+R or R) separated by a dark interval. An equal decrease of the P_fr/P_tot ratio leads to a more pronounced decrease in germination when the pulse of the same FR+R ratio is given as the second pulse at the end of the dark interval. The length of dark interval between light pulses needed for maximal germination, differed in (i) seeds with a natural requirement for long periods of light irradiation from that in (ii) seeds with their long light requirement imposed by two weeks of imbibition in darkness or by (iii) imbibition in 40% heavy water. However, a single R pulse was sufficient to induce a high percentage of germination if the seeds were supplied with KNO₃ (10 mM) from the onset of imbibition up to the onset of light. This effect decreased with a delayed time of application, and was prevented if FR preceded the KNO₃ application. We dedicate this paper to Professor Hans Mohr on the occasion of his 60th birthday     

The Interactive Effects of Phytochrome, Nitrate and Thiourea on the Germination Response to Alternating Temperatures in Seeds of Ranunculus sceleratus L.: A Quantal Approach

Probert, R. J., Gajjar, K. H. and Haslam, I. K. 1987. The interactiveeffects of phytochrome, nitrate and thiourea on the germinationresponse to alternating temperatures in seeds of Ranunculussceleratus L.: A quantal approach.—J. exp. Bot. 38: 1012–1025. The interactive effects of phytochrome, potassium nitrate andthiourea on the germination response to alternating temperaturesin achenes (seeds) of Ranunculus sceleratus L. were studied.Using thermogradient bars, high levels of germination were recordedover a broad range of alternating temperatures providing seedsreceived daily irradiations. Reduced germination in temperaturecycles with a relatively long warm phase was related to thelevel of the active form of phytochrome (Pfr). Dose-responseexperiments to red light (R) and temperature shifts showed thatthe actions of Pfr and alternating temperatures were interdependent.Maximum germination was recorded when intermittent pulses ofR were combined with daily 4 h temperature shifts from 16°Cto 26°C. Whilst probit analysis showed that potassium nitrateand thiourea both increased population sensitivity to temperatureshifts, thiourea was a more potent stimulant. Although the effectof both chemicals was dependent on phytochrome photo-equilibriumthe threshold level of Pfr required for thiourea action wasclearly much lower than that required for nitrate action. Thioureapotentiated a response to daily temperature shifts even whenPfr was at a low, normally inhibitory level. These results indicatedifferent mechanisms of action for potassium nitrate and thioureain relation to phytochrome controlled seed germination. Key words: Phytochrome, nitrate, thiourea, alternating temperatures, germination     

Prevention of Action of Far-Red-Absorbing Phytochrome in Rumex crispus L. Seeds by Ethanol

Phytochrome-enhanced germination of curled dock (Rumex crispus L.) seeds is further stimulated by pretreatments in solutions of 0.5 to 2 molar methanol and 0.03 to ≥ 0.3 molar 2-propanol during a 2-day 20°C imbibition. Similar pretreatments in 0.1 molar ethanol, acetaldehyde, and n-propanol inhibit phytochrome-enhanced germination. If exposure to ethanol is delayed until 16 hours after a red irradiation, seeds escape the ethanol inhibition indicating a mechanism other than toxicity. The rate of escape from ethanol inhibition roughly parallels the escape from phytochrome control in seeds held in water only, indicating possible ethanol effects on phytochrome. It was found that ethanol pretreatment prevents the far-red absorbing form of phytochrome (Pfr) from acting but does not accelerate dark decay or prevent transformation. Ethanol inhibition may be prevented if ethanol pretreatment is at 10°C instead of 20°C, or may be overcome by transferring ethanol-pretreated seeds to 10°C in water. Similarly, ethanol inhibition can be overcome by a 2-hour 40°C temperature shift concluding the pretreatment. It is proposed that the ethanol causes perturbations at a membrane which prevent Pfr from acting.     

The very-low-fluence and high-irradiance responses of the phytochromes have antagonistic effects on germination, mannan-degrading activities, and DfGA3ox transcript levels in Datura ferox seeds

Seed germination can be promoted by the modes of action of two of the phytochromes: the low-fluence response (LFR), which is the classical red (R)-far-red (FR) reversible response and the very-low-fluence response (VLFR) that can be saturated by extremely low levels of Pfr, which can be elicited by a saturating FR pulse. The Datura ferox seed population used in this work had acquired the capacity to germinate through a VLFR after pretreatment in a water-saturated atmosphere (WSA) at constant 25 degrees C. After 12 d in WSA germination after a FR pulse was 82%, while it was less than 10% in darkness. It was found that the VLFR of germination is associated with increments in the embryo growth potential (EGP) and in the activity of two enzymes related to the weakening of the micropylar region of the endosperm (ME); endo-beta-mannanase and beta-mannosidase. The FR pulse also significantly stimulated the expression of DfGA3ox, a GA 3beta-hydroxylase, suggesting that the promotion of germination by the VLFR is associated with an increase in the synthesis of active gibberellins. The promotive action of the VLFR on germination is reduced when the FR pulse is immediately followed by a continuous FR treatment for 24 h (FRc). The effect of FRc cannot be reproduced by hourly FR pulses during the same period, showing that the antagonistic effect of FRc is a high-irradiance response (HIR). The action of the HIR in germination is associated with a decrease of both the mannan-degrading enzyme activity and the expression of DfMan in the ME, whereas no changes in the EGP were observed. The HIR also inhibits the accumulation of DfGA3ox in embryos, indicating that its action on germination is mediated, at least in part, through the modulation of active GA contents in seeds. This is the first report of a gene that participates in the VLFR-HIR antagonism in seeds.     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction