Control processes in the induction and relief of thermoinhibition of lettuce seed germination : actions of phytochrome and endogenous ethylene

Germination of lettuce seeds (Lactuca sativa L. cv Grand Rapids) in the dark was nearly 100% at 20°C but was inhibited at 27°C and higher temperatures (thermoinhibition). A single 5-minute exposure to red light completely overcame the inhibition at temperatures up to 28°C, above which the effectiveness of single light exposures gradually declined to reach a negligible level at 32°C. However, the promotive effect of light could be extended to 34°C by repeated irradiations. At any one temperature, increased frequency of irradiations increased germination percentage, and with each degree increase in temperature, increasingly frequent irradiations were necessary to elicit maximal germination. Loss of the effectiveness of single irradiations with increase in temperature may result either from acceleration of the thermal reversion of the far red-absorbing form of phytochrome or decrease in seed sensitivity toward a given percentage of the far red-absorbing form of phytochrome. Using continuous red light to induce germination, the role of endogenous C₂H₄ in germination at 32°C was studied. Ethylene evolution from irradiated seeds began to increase 2 hours prior to radicle protrusion, whereas the dark-incubated (nongerminating) seeds produced a low, constant amount of C₂H₄ throughout the 24 hour incubation period. Inhibition of C₂H₄ synthesis with 2-aminoethoxyvinyl glycine and/or inhibition of C₂H₄ action with 2,5-norbornadiene blocked the promotive effect of light. Exogenous C₂H₄ overcame these blockages. The results showed that participation by endogenous C₂H₄ was essential for the light-induced relief of thermoinhibition of lettuce seed germination. However, light did not act exclusively via C₂H₄ since exogenous C₂H₄ alone in darkness did not promote germination.     

Photomanipulation of phytochrome in lettuce seeds

Seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) were imbibed and given either short irradiation with red or far red light prior to drying or dried under continuous red or far red light. Seeds treated with either short or continuous red germinate in darkness, whereas seeds treated with either short or continuous far red require a short exposure to red light, after a period of imbibition, to stimulate germination. Irradiation of dry red seeds with far red light immediately before sowing results in a marked inhibition of germination. This result was predicted since far red-absorbing form phytochrome can be photoconverted to the intermediate P650 (absorbance maximum 650 nm) in freeze-dried tissue. A similar far red treatment to continuous red seeds is less effective and it is concluded that in these seeds a proportion of total phytochrome is blocked as intermediates between red-absorbing and far red-absorbing form phytochrome, which only form the far red-absorbing form of phytochrome on imbibition. The inhibition of dry short red seeds by far red light can be reversed by an irradiation with short red light given immediately before sowing, confirming that P650 can be photoconverted back to the far red-absorbing form of phytochrome. The results are discussed in relation to seed maturation (dehydration) on the parent plant.     

The problem of reduced nicotinamide adenine dinucleotide oxidation in glyoxysomes

Phototransformation of phytochrome in lettuce seeds (Lactuca sativa L. var. Grand Rapids) was examined by testing germination responses of seeds irradiated at various temperatures. Temperature variations from 0 to 50 C had no influence on the germination of partially hydrated seeds (about 15% water content) irradiated with either red or far red light prior to imbibition. At −15 C far red light more effectively retarded germination than red light promoted it. No effective phototransformation was detected at −79 C or −196 C.     

Phytochrome and Temperature Relations in <Emphasis Type="Italic">Lactuca sativa</Emphasis> L. Grand Rapids Seed Germination after Thermo-dormancy

THE phytochrome reaction involved in the response of seeds of the lettuce cultivar Grand Rapids to light is such that exposure to short periods of illumination with red light (660 nm) promotes its germination, but far red light (730 nm) inhibits it at all temperatures from 5° to 25° C^1–8. There is, however, considerable variation between different seed stocks, which depends largely on the history of the parent plant⁹ and of the seeds after harvest¹⁰.     

Action spectra for light-induced germination in dormant lettuce seeds

Fluence response curves for red light-induced germination of thermodormant (TD) seeds of Lactuca sativa L. show two regions that differ in their light sensitivity. In the region of high sensitivity, the germination responses differ between seed batches and can be altered by dark storage or far red irradiation. Induction of germination in far red dormant (FRD) seeds requires far higher fluences. Action spectra for induction to 60% germination were determined for these various response types. Spectra for the regions of low sensitivity response are similar for TD and FRD seeds. In comparison, the action spectrum for the highly sensitive response in TD seeds is significantly shifted to longer wavelengths. Analogous differences exist in the action spectra for far red reversal of the red induced germination responses. Germination induction in the low sensitivity region shows repeated red-far red reversibility. Far red reversal of red induction in the high sensitivity region does not saturate even at the highest far red fluences available and requires increased red fluences for subsequent reinduction. A model quantitatively accounting for these observations is presented. It is pointed out that action spectra of processes involving photoreversible pigments with partly overlapping absorption spectra in general are not identical with the absorption spectra of the partners. They should depend upon the degree of phototransformation required to elicit a given physiological response. In the case of induction of lettuce seed germination the observed action spectra can be interpreted as reflecting different requirements for P _fr of the various response types. Our results do not necessitate the assumption of spectroscopically different forms of phytochrome in these seeds.Abbreviations TD thermodormant - FRD far red dormant - P phytochrome - P _r red absorbing form of P - P _fr far red absorbing form of P     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

Light Actions in the Germination of Cocklebur Seeds: V. EFFECTS OF ETHYLENE, CARBON DIOXIDE AND OXYGEN ON GERMINATION IN RELATION TO LIGHT

Esashi, Y., Hase, S. and Kojima, K. 1987. Light actions in thegermination of cocklebur seeds. V. Effects of ethylene, carbondioxide and oxygen on germination in relation to light.–J.exp. Bot. 38: 702–710. Effects of ethylene, CO² and O² on the germination of after-ripenedupper cocklebur (Xanthium pennsylvanicum Wallr.) seeds wereexamined in relation to pre-irradiation by red (R) or far-red(FR) light In order to remove the pre-existing Pfr, seeds weresoaked in the dark for various periods prior to light irradiationand gas treatments. Regardless of light, 0.3 Pa C₂H₄ promotedgermination at 23 ?C, but it strongly inhibited germinationwhen applied at 33 ?C, the optimal temperature for the germinationof this seed. However, delayed application of C₂H₄ during 33?C incubation stimulated germination independently of lightin a similar manner to that seen at 23 ?C. It is, therefore,suggested that the germination-regulating action of C2H4 iscompletely independent of phytochrome. In contrast, the germination-promoting effect of 3–0 kPaCO₂ was pronounced only when the seeds were previously irradiatedby R, regardless of temperature, suggesting that CO₂ actionto promote germination depends upon Pfr. A synergism betweenCO₂ and C₂H₄ at 23 ?C was observed only in the germination ofseeds pre-irradiated by R, while at 33 ?C an antagonism occurredindependently of light. The stimulation of C₂H₄ production byCO₂ was most striking in the cotyledonary tissue pre-irradiatedby R. However, the R-dependent enhancement of CO₂-stimulatedC₂H₄ production was negated by the subsequent FR and it wasnot found in the presence of 1-aminocyclopropane-1-carboxylicacid (ACC). Moreover, the R dependency of the germination-promotingCO₂ effect disappeared in the presence of C₂H₄. The R-dependentC₂H₄ production enhanced by CO₂ may thus be involved, at leastpartially, in some step of conversion from methionine to ACC. The germination-promoting effect of C₂H₄, but not CO₂, was enhancedby O₂ enrichment regardless of light. However, the germination-promotingeffect of pure O₂ itself appeared to depend upon pre-irradiationwith R Key words: Carbon dioxide, cocklebur seed, ethylene, far-red light, germination, oxygen, red light, Xanthium pennsyloanicum     

Photoregulation of Phytochrome Synthesis in Germinating Embryos of Avena sativa L.

Hilton, J. R. and Thomas, B. 1987. Photoregulation of phytochromesynthesis in germinating embryos of Avena sativa L.—J.exp. Bot. 38: 1704–1712. The effect of light on the accumulation of phytochrome in germinatingAvena embryos was determined. A quantitative ELISA using monoclonalantibody AFRC MAC 56 was used to measure specifically type 1(or dark) phytochrome. A pulse of red light given after 14 himbibition but prior to the onset of type 1 phytochrome synthesis,strongly inhibited subsequent type 1 phytochrome accumulation.This effect of red light at 14 h was reversible by far-red lightindicating the involvement of phytochrome. Red light also inhibitedphytochrome synthesis after 18 h and 24 h imbibition but after24 h, far-red light did not reverse the effect. The effect ofred light treatment at 18 h was reversed by giving a pulse offar-red light at any time up to 30 h. Seed germination was notinfluenced by light under the conditions of these experiments.It is proposed that type 2 (or light) phytochrome may be responsiblefor photoregulation of type 1 phytochrome synthesis in germinatingAvena embryos. Key words: Photoregulation, phytochrome, seed.     

An action spectrum in the blue for inhibition of germination of lettuce seed

Summary Using a 2-h irradiation period at constant quantum irradiance, a complete action spectrum for inhibition of germination of lettuce seed has been obtained. Action maxima were near 470 and 720 nm, the latter being the most active wavelength. It was also shown, under conditions where light inhibition cannot occur, that phytochrome potentiation of germination is maximal at all wavelengths below 700 nm, including the highly active blue region. Evidence was presented for promotion of germination by a 2-h irradiation in the red which cannot be explained on the basis of conversion of phytochrome to the active form.Abbreviations Bl blue - FR far-red, P_FR far-red-absorbing form of phytochrome - R red Supported in part by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171 and the Graduate School Research Funds.     

Phytochrome in seeds of Amaranthus caudatus

Summary Dry seeds of Amaranthus caudatus show little or no photoreversible absorption changes, attributable to phytochrome. During imbibition phytochrome appears in two phases, one immediately after sowing and the second after about 8 hr. Experiments at different temperatures and under continuous illumination with red, far-red and blue light suggest that there are two pools of phytochrome. The first phase in the appearance of phytochrome could be due to the change in optical properties of the sample on hydration or to rehydration of inactive phytochrome, or both. The second phase probably represents phytochrome synthesis. It is absent at 0° and precedes the water uptake associated with germination by some 10 hr. This second pool of phytochrome does not accumulate in red and blue illuminated seeds indicating that the rate of P _fr decay is more rapid than the rate of phytochrome synthesis. The difference spectra of phytochrome in both 2 hr imbibed seeds and 72 hr old seedlings show peaks of absorption at 663 and 735 nm. The presence of P _fr in dark imbibed seeds and the process of inverse reversion of P _r to P _fr in darkness have been demonstrated. The results are discussed in relation to previous hypotheses for the mechanism of photocontrol of Amaranthus seed germination.     

Red light-induced phytochrome relocation into the nucleus in Adiantum capillus-veneris

Phytochromes in seed plants are known to move into nuclei in a red light-dependent manner with or without interacting factors. Here, we show phytochrome relocation to the nuclear region in phytochrome-dependent Adiantum capillus-veneris spore germination by partial spore-irradiation experiments. The nuclear or non-nuclear region of imbibed spores was irradiated with a microbeam of red and/or far-red light and the localization of phytochrome involved in spore germination was estimated from the germination rate. The phytochrome for spore germination existed throughout whole spore under darkness after imbibition, but gradually migrated to the nuclear region following red light irradiation. Intracellular distribution of PHY-GUS fusion proteins expressed in germinated spores by particle bombardment showed the migration of Acphy2, but not Acphy1, into nucleus in a red light-dependent manner, suggesting that Acphy2 is the photoreceptor for fern spore germination.     

Effects of ethylene and carbon dioxide on the germination of osmotically inhibited lettuce seed

Lettuce seeds (Lactuca sativa L.) used in this study germinated 98% at 25 C in light or dark. Their germination was completely inhibited by 0.20 m NaCl, 0.35 m mannitol, or polyethylene glycol 6000 (−7 bars) under continuous light when germination tests were made in Petri dishes. Approximately 50% germination occurred in sealed flasks due to endogenously produced C₂H₄ and CO₂. Removal of either or both gases prevented germination. In the presence of endogenous CO₂, addition of C₂H₄ (0.5 to 16 microliters/liter) stimulated 95 to 100% germination (after 5 days) only in the light, but the rate of germination was dependent on C₂H₄ concentration. At 16 microliters/liter C₂H₄, full germination occurred within 72 hours. Addition of up to 3.2% CO₂ had no adverse effect on the C₂H₄ action. Higher concentrations or the complete absence of CO₂ reduced both rate and total germination. CO₂ alone was ineffective.     

Spectrophotometric characterization of phytochromes in dimorphic cocklebur seeds in relation to capacity for germination

Phytochrome was measured spectrophotometrically in different tissues of the upper (positively photoblastic) and lower (negatively photoblastic) seeds of the cocklebur (Xanthium pennsylvanicum Wallr.). Axial parts of the seeds, in particular parts of the radicle, contained high levels of phytochrome, while cotyledonary parts contained only low levels. These results were consistent with the distribution of the light-sensitive areas of the seeds that were associated with germination. Phytochrome levels in both types of dimorphic seeds increased gradually with increasing duration of dark imbibition for 4–8 h, then the rates of increase in levels of phytochrome accelerated. In both types of seed, some phytochrome was measurable even before imbibition. In the lower seeds, up to 20% of the phytochrome was occasionally observed as P_fr in samples imbibed in darkness for a short time (up to 12 h). A slight blue shift of the peak of P_T in the difference spectrum of phytochrome was observed in the case of lower seeds imbibed for 0–2 h. These results suggest that, to some extent, the lower axes contain dehydrated P_fr or intermediate(s) in the photoconversion of phytochrome. The dark reactions of P_fr were also examined in excised axes of both types of dimorphic seed after they had been pre-imbibed for 16 h in darkness. Dark destruction of P_fr was observed in both types of seed. In addition, net increases in levels of P_r were observed in the dark controls and in the samples irradiated with red light after the level of P_fr diminished. No ‘inverse’ dark reversion from P_r to P_fr was detected. Thus, after 16 h of imbibition, there were no differences in terms of properties of phytochrome between the two types of seed, and the different responses to light of upper and lower seeds might depend mainly on a difference in the physiological state of the two types of seed rather than the properties of phytochrome.     

Dark Reversion of Phytochrome in Lettuce Seeds Stored in a Water-saturated Atmosphere

Dark reversion of the far red-absorbing form of phytochrome, which does not occur in dry lettuce (Lactuca sativa var. Grand Rapids) seeds, appears to take place in seeds stored in a water-saturated atmosphere. The water content (approximately 70% after 10 days) of such seeds is insufficient to support germination; however the treatment enhances germination in seeds stored for 1 to 5 days, but this enhancement subsequently disappears, and the effect of extended storage (up to 28 days) is inhibiting. The half-time for dark far red-absorbing phytochrome reversion is 7 to 8 days, and at this time it can be completely reversed by exposing the seeds to a flash of red light. Storage of more than 7 to 8 days decreases red light enhancement of germination.     

Spectral quality influence of light during development of Arabidopsis thaliana plants in regulating seed germination

During development of Arabidopsis thaliana plants, the spectralquality of light can influence the phytochrome controlled germinationof the progeny. Plants grown under light rich in far red energiesproduce light requiring seed that contain phytochrome in thered absorbing or inactive form. Plants grown under light deficientin far red energies produce dark germinating seed that containphytochrome converted mainly to the far red absorbing or activeform. These responses are determined locally within the developingseed without influence from the vegetative portion of the plant.The developing seed is sensitive to spectral changes throughoutembryogenesis until the seed begins to dehydrate just beforefull maturation. At that point the phytochrome is stabilizedin the form photoinduced before dehydration. In relation tothe induction of phytochrome controlled germination responses,the developing seed appears to act independently of the parentplant. (Received April 16, 1974; )     

Phytochrome Control of Germination of Rumex crispus L. Seeds Induced by Temperature Shifts

High germination of curly dock (Rumex crispus L.) seeds is evident after suitable imbibition and temperature shift treatment, but germination at constant temperatures fails without an input of far red-absorbing form of phytochrome. Preliminary imbibitions at high temperatures (30 C) sharply reduce germination induced by temperature shifts. High germination may be restored by low energies of red radiation, or by brief far red adequate for the photosteady state. Prolonged far red during imbibition also nullifies temperature shift-induced germination. After prolonged far red, high germination may be restored by red radiation of an energy dependent upon the duration of the far red treatment. The evidence supports the conclusion that dark germination induced by temperature shifts arises from the interaction of pre-existent far red-absorbing form of phytochrome in the mature seeds with the temperature shift.     

Phytochrome Transformation and Action in Seeds of Rumex crispus L. during Secondary Dormancy

Promotion of germination by red light fails after prolonged dark imbibition of Rumex crispus L. seeds, indicative of a secondary dormancy. The degree and rate of inception of the dormancy increases with increasing temperature. Following establishment of the dormancy, germination response to red light can be restored by either prolonged cold treatment or brief high temperature shifts. Loss of phytochrome was not a factor in the initial establishment of the dormancy. When the seeds are in secondary dormancy, the chromophore of phytochrome can be transformed to the far red-absorbing form, but the far red-absorbing form cannot induce germination. The responses to changes in temperature suggested dependence of germination on order disorder transitions in components of the seeds.     

Mediation of phytochrome in the inductive action of low temperature on dark germination of lettuce seed at supra-optimal temperature

The induction of dark germination in light-requiring lettuce (Lactuca sativa) seed at supraoptimal temperatures by cold treatment (in darkness) was partly reversed by a brief far-red irradiation made at time of transfer, and even more so when the irradiation was made at the beginning of the cold pretreatment. When the inhibitory far-red irradiation was followed by additional cold treatment, the promotion was greatly restored. The promotive effects of brief irradiations with red light were further enhanced by a following cold period, before transfer to the supraoptimal temperature. These results are interpreted as indicating that the active (far-red absorbing) form of phytochrome is pre-existing in the dry seed, and interacts with a co-factor which is built-up during imbibition. The rate of build-up of this co-factor, as well as of the dark inactivation of active phytochrome increase with temperature. The products of the interaction pass through a photo-labile thermo-stable phase, before becoming photo-stable as well.     

Interaction of carbon dioxide and ethylene in overcoming thermodormancy of lettuce seeds

The combination of ethylene with CO₂ will completely overcome the thermodormancy of lettuce (Lactuca sativa L.) seeds at 35 C. This combination is effective if it is added to seeds either at the start or after several days of imbibition. The action of ethylene is dependent upon the CO₂ level present in the atmosphere surrounding the seeds. When CO₂ is trapped by KOH the ethylene effect is essentially nil.     

A seed respiration-based index of cold-sensitivity during imbibition in four macrothermal species

A research was carried out to evaluate the influence of temperature on seed respiration response of maize, cotton, grain sorghum and sunflower during imbibition, and to define reliable indices for a fast evaluation of cold-sensitivity at germination level in plants. The seed respiration activity was measured during seed imbibition at 25 °C (optimal) and 15 °C (suboptimal) constant temperatures, using a homemade respiration chamber adapted to an infrared gas analyzer. At 15 °C, sunflower and sorghum maintained high levels of seed germination (≥90 %), whilst this last dropped in cotton (36.7 %) and maize (27.8 %). With respect to this, cotton and maize seem to be cold sensitive during germination. Instantaneous seed respiration during imbibition versus temperature or thermal time could not be used as a good indicator for cold tolerance, since the levels of CO₂ recorded at 15 °C in cotton (higher than the other species) and maize (similar to that of sorghum and sunflower) did not correspond to adequate seed germination. Differently, the rates (b coefficient of linear regressions) of accumulation of CO₂ respired at optimal and suboptimal temperatures during the first hours of imbibition (up to approximately 24 h from the start of experiment), were significantly different in maize and cotton, whilst they did not differ in sorghum and sunflower. Therefore, the shift between slopes may represent a reliable index for seed cold-sensitivity assessment during early germination.