Photoinduced Seed Germination of Oenothera biennis L: I. General Characteristics

General characteristics of light-induced germination of Oenothera biennis L. seeds were investigated at 24°C. During dark imbibition, seeds reached maximal respiration in 7 hours and maximal water content and photosensitivity in 24 hours. After dark imbibition of 24 hours, seeds required a long exposure (>36 hours) to red or white light for maximal germination. Two photoperiods (12 and 2 hours) separated by a period of darkness of 10 to 16 hours gave near maximal germination. For the two photoperiod regime, the first light potentiates a reversible phytochrome response by the second light. A 35°C treatment for 2 to 3 hours in the dark immediately prior or subsequent to 8 hours of light caused a higher percentage of germination. A 2 hour treatment at 35°C also potentiates a reversible phytochrome response. Halved seeds germinated at 100% in light or darkness indicating that the light requirement of the seeds is lost in the halving procedure. After-ripened seeds required less light and germinated more rapidly and at higher percentages than seeds tested shortly after maturation.     

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.     

Photoinduced Seed Germination of Oenothera biennis L: II. Analysis of the Photoinduction Period

The photoinduction period of Oenothera biennis L. seed germination was analyzed by varying the photoinduction temperature and by substituting red light pulses for continuous red light. At 24°C, seeds require 36 hours of continuous red light for maximal percent germination. The optimal photoinduction temperature is 32°C, with higher and lower temperatures being strongly inhibitory. A 30 minute exposure to far-red light, given immediately after a red light period of 1 to 36 hours, reduces germination by about 25%. Seeds escape from far-red inhibition with a half-time of 5 to 10 hours, depending on the length of the red exposure that precedes the far-red light. Periodic 15 minute pulses of red light can substitute for continuous red light in stimulating germination. Ted red light pulses, with 6 hours of darkness between successive pulses, cause maximal germination. The response to periodic red light is fully reversible by far-red light. Probit analysis of the periodic light response shows that as the length of the dark periods between successive pulses increases, less incident light is needed to induce germination but the population variance in light sensitivity remains constant. Probit analysis of the temperature response shows that as the photoinduction temperature increases from 16 to 32°C, less incident light is needed to induce germination and the population variance in light sensitivity also increases.     

Effects of Helminthosporium victoriae Toxin on Germination and Aleurone Secretion by Resistant and Susceptible Seeds

Oat seeds of cultivars susceptible and resistant to Helminthosporium victoriae were held for various times in pathogen-produced, host-specific toxin solutions; control seeds were in water. Seeds were then washed thoroughly and incubated on moist paper, or dried and stored for 2-3 weeks before germination was attempted. In both cases, germination of susceptible seeds was prevented by previous exposure to toxin for 1 hour or more. Control seeds and treated resistant seeds grew normally. Toxin did not affect O₂ uptake or loss of carbohydrates from seeds for the first 12 hours of imbibition. After 12 hours, toxin-treated susceptible seeds had higher respiration and lost more carbohydrates than did control seeds. Experiments with embryoless seeds showed that toxin blocked synthesis and secretion of α-amylase by susceptible but not by resistant aleurone cells. Resting aleurone cells were exposed briefly to toxin, then dried and stored until all toxin was gone. Susceptible aleurone cells treated in this way failed to produce α-amylase following exposure to gibberellic acid, while controls and resistant treated aleurone tissues produced amylase. Susceptibility or resistance to toxin appears to be expressed in resting and metabolically active tissues.     

Temperature and photocontrol of onoclea spore germination

Germination of Onoclea sensibilis L. spores is controlled by light and temperature. Temperatures of 30 C can induce maximal germination in the dark to a level of 60 to 95% of that induced by a saturating dose of red light (0.38 joules/square meter) providing the spores are placed at the elevated temperature immediately after being sown. Maximum dark germination occurs with a minimum exposure of 16 to 24 hours at 30 C, suggesting that the temperature treatment is required for the induction of germination rather than for the germination process per se. Interaction of temperature and light for induction of germination shows nonadditive behavior. Germination induced by light and temperature applied consecutively never exceeded that which could be induced by a saturating dose of red light alone. Imbibition of the spores at 25 C in the dark for 12 or more hours prior to incubation at 30 C results in a loss of thermosensitivity. Dose response curves for red light induction of germination after varying times of imbibition at 25 C show no concomitant loss of sensitivity of the spores to red irradiation. This suggests that the mechanism and/or pathway of thermoinduction of germination differs from that of photoinduction. The loss of thermosensitivity as a result of presoaking at 25 C can be prevented if the spores are imbibed at 25 C in osmotic agents such as 0.3 molar mannitol or 0.1 gram per liter of polyethylene glycol 400 or in 0.08% dimethylsulfoxide or 10 micrograms per milliliter of herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3-(2H)pyridazinone). The latter two substances are hypothesized to act upon membranes. These results suggest that the degree of hydration and possibly changes in membrane properties play a role in the change in sensitivity of Onoclea spores to temperature.     

Water Content and Phytochrome-induced Potential Germination Responses in Lettuce Seeds

Grand Rapids lettuce seeds (Lactuca sativa L.) with 6 to 8% water content show no light-induced germination responses, whereas in seeds with 15% or more water content, germination is promoted or retarded by red and far red light respectively. By adjusting seed water content, persistent potentiated responses to light are induced in the seeds at seed water levels much below that required for germination itself. Alternate moistening and drying of seeds in conjunction with red and far red irradiations show that potentiated responses may be phototransformed only with sufficient seed water. However, the process of drying and remoistening has no effect on potentiated responses.     

Changes in Phytochrome Expressed by Germination of Amaranthus retroflexus L. Seeds

Effects of red (600 to 680 nanometers) and far red (700 to 760 nanometers) irradiances on Amaranthus retroflexus L. seeds indicate that synthesis of phytochrome in the red-absorbing form takes place in water-imbibed nongerminating seeds at 35 C. After 96 hours in darkness, conversion of about 0.10% phytochrome to the far red-absorbing form induces 50% germination. Continuous far red radiation at 35 C with an irradiance of 0.4 × 10⁻¹⁰ Einsteins per square centimeter per second caused photoinactivation of phytochrome about equal to the rate of synthesis. Germination of seeds at 35 C, following far red irradiation adequate to establish the photostationary state, is enhanced by holding at 26 C for 16 minutes. Germination is unaffected relative to controls at constant temperature, if the period at 26 C precedes irradiation. The results indicate a quick response to action of phytochrome in a germination process.     

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.     

Phytochrome Control of Cell Wall-bound Hydroxyproline Content in Etiolated Pea Epicotyls

The red light inhibition of growth of the intact pea (Pisum sativum L. cv. Alaska) third internode was correlated with an increase in the content of cell wall-bound hydroxyproline. These changes were detected 3 hours after irradiation, and possibly at 1 hour. Far red light reversed the effects of red light. The iron chelator α,α′-dipyridyl reversed the red light effects on both growth and hydroxyproline content. Using segments incubated in vitro, no phytochrome-mediated change in hydroxyproline content could be observed, perhaps because of an overwhelming wounding response. If plants were irradiated in situ and grown for 8 hours before excision and incubation of segments, some enhancement of hydroxylation by red light was detectable both colorimetrically and radioisotopically. The red light inhibition of segment growth was reversed by α,α′-dipyridyl. These results are examined in reference to the role of extensin in normal and induced growth cessation.     

Seed Dormancy in Red Rice : III. Response to Nitrite, Nitrate, and Ammonium Ions

Sodium nitrite at 10 millimolar breaks dormancy of dehulled red rice (Oryza sativa). While germination is light independent, low pH conditions (pH 3) are required for maximum response. Water and buffer controls at pH 3 remain dormant. The response to nitrite occurs at 25 and 30°C but is reduced at 20°C, although nondormant seeds germinate readily at this temperature. The contact time for response to nitrite is less than 2 h at the start of imbibition. Seeds imbibed first in water show reduced germination when subsequently transferred to nitrite. Dehulled seeds show little or no response to nitrate and ammonium ions.

Intact seeds remain dormant in the presence of nitrite or nitrate unless partially dry-afterripened. The pH dependence of nitrite sensitivity is reduced in intact, afterripening seeds. In highly dormant seeds, vacuum infiltration experiments suggest that the hull restricts uptake of nitrite.

     

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).     

Photoregulation of beta-Tubulin mRNA Abundance in Etiolated Oat and Barley Seedlings

The effect of light on the abundance of β-tubulin mRNA was measured in etiolated Avena sativa L. and Hordeum vulgare L. seedlings. Slot blot analysis employing an oat β-tubulin cDNA clone was used to measure β-tubulin mRNA levels. White light induced a 45% decrease in oat β-tubulin mRNA abundance by 2 hours after transfer. A saturating red light pulse induced 40 and 55% decreases in β-tubulin mRNA levels in oats and barley, respectively. Recovery of β-tubulin mRNA levels was observed after a red light pulse but not after transfer to continuous white light. The red light induced decrease in oat β-tubulin mRNA abundance was not reversible by a subsequent far-red light treatment. The mesocotyl portion of etiolated oat seedlings exhibited a more dramatic decrease in β-tubulin mRNA abundance in response to red light than did the coleoptile portion. The results indicate that the well-documented effects of red light on the growth of etiolated seedlings are accompanied by changes in the expression of the β-tubulin genes.     

Amylase synthesis and stability in crested wheatgrass seeds at low water potentials

Drying of seeds of Agropyron desertorum (Fisch. ex Link) Schult. did not result in breakdown of α-amylase nor impair the ability of seeds to resume its synthesis when moistened again. β-Amylase activity did not change during 5 days of germination at a water potential of 0 atmosphere nor during 40 days of incubation at −40 atmospheres. Seeds synthesized α-amylase at 0, −20, and −40 atmospheres, but not at −60 atmospheres. At 0 and −20 atmospheres, the log of α-amylase activity was linearly related to hastening of germination. But at −40 atmospheres, seeds synthesized α-amylase during a time when there was little hastening of germination. Thus, it appears that other biochemical reactions are less drought-tolerant than synthesis of α-amylase. It is concluded that inhibition of α-amylase synthesis is not a controlling factor in the germination of these seeds at low water potentials.     

The effects of SAN 9789 and light on phytochrome and the germination of lettuce seeds

Photoblastic seeds (akenes) of lettuce (Lactuca sativa (L.) cv. Grand Rapids) were treated with SAN 9789 [4-chloro-5-(methylamine)-2-a, a, a,-trifluoro-m-tolyl-3-(2H)-pyridasinone]. The seeds weere placed in Petri dishes on filter paper soaked with water or SAN solution. The treatment increased the germination in darkness from 17% for water to 78% for SAN treated seeds. An irradiation with 5 min red light gave a germination of 98% both in water and in SAN. In water the effect of red irradiation could be reversed with a short irradiation (8 min) of far red light (17% germination), while in SAN solution the far red reversibility was poor (92% germination). If the far red light was given repeatedly (5 min per h) it had a slightly larger effect. If given continuously for 24 hours, the germination in water was decreased to 0.3% and in SAN solution to 9%. Possible mechanisms for the SAN effect are discussed.     

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.     

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.     

Long-Lasting Light Effects in Imbibed Kalanchoë blossfeldiana Seeds in the Presence of Gibberellic Acid

Two brief red (R) irradiations, separated by 24 hours, given to Kalanchoë blossfeldiana Poelln. cv Feuerblüte seeds, made secondarily dormant by a prolonged dark incubation period on water and transferred to GA₃, induce very low germination. Some effect of these irradiations is preserved, however, during a long dark interval in fully imbibed seeds and greatly increases the germination induced by another brief R exposure. This long-lasting light effect is, at 20°C, only lost after a dark interval of about 1 month. It can also be induced by two brief far-red (FR) exposures. Its preservation is temperature-dependent, low temperatures being favorable. Light-induced changes in the ATP-content were demonstrated during preservation and expression of the long-lasting light effect, indicating a long-lasting metabolic change. In seeds with primary dormancy sown on GA₃, an analogous long-lasting light effect is induced by one or two brief R or FR irradiations, even when they are given before germination can take place. The presence of GA₃, which was shown to induce a very low fluence germination response in Kalanchoë seeds, is required for the occurrence of the long-lasting light effect. The data suggest long-term preservation of some effect(s) of Pfr rather than persistent presence of Pfr itself.     

Photoinduced Seed Germination of Oenothera biennis L: III. Analysis of the Postinduction Period by Means of Temperature

The postinduction period of Oenothera biennis L. seed germination was examined by temperature treatments. For all experiments, seeds received a standard 24 hour/24°C preinduction period and 12 hour/32°C photoinduction period. Germination is inhibited by postinduction temperatures above 32°C. When seeds are briefly incubated at 44°C and then transferred to 28°C, they germinate at a much lower percentage than 28°C controls. When thermally inhibited seeds are placed in the dark at 28°C for 20 hours, they can be promoted to germinate by a single pulse of red light. Seeds incubated at 12°C or below immediately after photoinduction enter a lag period in which they germinate slowly or not at all for a long time and then resume germination. The length of the lag period is exponentially related to the postinduction temperature. When seeds are incubated at a low temperature and then transferred to a warm temperature, they germinate much more rapidly than seeds not incubated at a low temperature. A model is proposed which is consistent with these and additional results. In the model, a germination promoter is irreversibly formed from a precursor and the synthesis of the precursor is favored at low temperatures and its degradation is favored at high temperatures.     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : III. Sensitivity of Germination to Water Potential and Abscisic Acid during Development

Muskmelon (Cucumis melo L.) seeds are germinable 15 to 20 days before fruit maturity and are held at relatively high water content within the fruit, yet little precocious germination is observed. To investigate two possible factors preventing precocious germination, the inhibitory effects of abscisic acid and osmoticum on muskmelon seed germination were determined throughout development. Seeds were harvested at 5-day intervals from 30 to 65 days after anthesis (DAA) and incubated either fresh or after drying on factorial combinations of 0, 1, 3.3, 10, or 33 micromolar abscisic acid (ABA) and 0, −0.2, −0.4, −0.6, or −0.8 megapascals polyethylene glycol 8000 solutions at 30°C. Radicle emergence was scored at 12-hour intervals for 10 days. In the absence of ABA, the water potential (Ψ) required to inhibit fresh seed germination by 50% decreased from −0.3 to −0.8 megapascals between 30 and 60 DAA. The Ψ inside developing fruits was from 0.4 to 1.4 megapascals lower than that required for germination at all stages of development, indicating that the fruit Ψ is sufficiently low to prevent precocious germination. At 0 megapascal, the ABA concentration required to inhibit germination by 50% was approximately 10 micromolar up to 50 DAA and increased to >33 micromolar thereafter. Dehydration improved subsequent germination of immature seeds in ABA or low Ψ. There was a linear additive interaction between ABA and Ψ such that 10 micromolar ABA or −0.5 megapascal osmotic potential resulted in equivalent, and additive, reductions in germination rate and percentage of mature seeds. Abscisic acid had no effect on embryo solute potential or water content, but increased the apparent minimum turgor required for germination. ABA and osmoticum appear to influence germination rates and percentages by reducing the embryo growth potential (turgor in excess of a minimum threshold turgor) but via different mechanisms. Abscisic acid apparently increases the minimum turgor threshold, while low Ψ reduces turgor by reducing seed water content.     

Persistence of red light induction in lettuce seeds of varying hydration

Incubation of lettuce seeds (Lactuca sativa L. cv. Grand Rapids) in 0.3 m mannitol allows sufficient water uptake to make seeds fully sensitive to red light. But germination is possible only after lowering the osmotic potential of the incubation medium. The red light induction of these incompletely hydrated seeds can be reversed by far red light. Their reversibility declines with time at a slower rate than seeds incubated in water. About half the seeds in 0.3 m mannitol respond to far red light when all seeds in water have escaped control by far red light. Close to 100% of the seeds remain sensitive to far red exposure if 0.6 m mannitol is used as osmoticum. The retention of the original red light stimulus is inversely related to the concentration of the incubation medium.The fresh weight of viable seeds incubated with water or with an osmoticum increases rapidly during the first 5 hours, then remains stationary for about 12 hours. After that only germinating seeds experience a second increase in fresh weight. Heat-killed seeds do not show such a discontinuity in water uptake.