Temperature and other factors affecting chloramphenicol stimulation of the germination of light-sensitive lettuce seeds

Summary D-threo-chloramphenicol at concentrations ranging from 1000 to 3000 g/ml stimulated the germination of the light-sensitive seeds of the lettuce (Lactuca sativa L.) varieties Attractie and Grand Rapids. This stimulatory effect of chloramphenicol was markedly temperature dependent, increasing with decrereasing temperature. Seeds showed little response to chloramphenicol at temperatures of 28°C and above except in the case of light treated Attractie seed. The failure of one batch of Grand Rapids seed to respond to chloramphenicol was associated with the low degree of dormancy in this batch.When the germination of half-seeds or intact excised embryos of Attractie seed was inhibited osmotically with 0.15 M NaCl a stimulatory response to chloramphenicol was obtained suggesting that the site of action was in the embryo itself.Other inhibitors of protein synthesis, cycloheximide, puromycin and p-fluorophenylalanine, did not stimulate germination. Cycloheximide at concentrations of 10 g/ml and above inhibited germination whereas puromycin and p-fluorophenylalanine were relatively ineffective as germination inhibitors.     

Interaction between a plant-derived smoke extract,light and phytohormones on the germination of light-sensitive lettuce seeds

Plant-derived smoke extracts mimics the effect of red light on germination in light-sensitive lettuce seeds and partially overcomes the inhibitory effect of far-red light. Interaction between a smoke extract and gibberellins, cytokinins, abscisic acid and ethephon was investigated. Smoke acted synergistically with GA₃ and increased the sensitivity of the lettuce seeds to ABA. It seems likely that smoke affects membrane permeability or receptor sensitivity rather than influencing the phytochrome system of these seeds.Abbreviations R red light - FR far-red light - SM smoke extract     

The effect of ethylene, octanoic acid and a plant-derived smoke extract on the germination of light-sensitive lettuce seeds

The effects of an aqueous plant-derived smoke extract, octanoic acid and ethylene on germination of light-sensitive Grand Rapids lettuce seeds were investigated. The smoke extract brought about a concentration dependent increase in germination and a complete inhibition of germination at high concentrations. Octanoic acid could not induce germination. Ethylene at concentrations over 5 L L^–1 increased lettuce seed germination, but not to the same degree as smoke. Aqueous smoke in combination with ethylene showed a synergistic effect on germination at suboptimal smoke concentrations. At high smoke concentrations the effect of ethylene was almost completely inhibited. Octanoic acid in combination with ethylene brought about a higher level of germination than with ethylene alone, but only at the highest concentration of octanoic acid tested (1 mM). Standardized hexane and dichloromethane-partitioned smoke extracts and octanoic acid were subjected to TLC separation. The R _f -fractions in the smoke lanes showing activity in the lettuce seed bioassay did not correspond to the R _f -value of octanoic acid. As aqueous smoke can withstand autoclaving and can be separated by TLC and HPLC without loosing activity it is unlikely that the activity of aqueous smoke is linked to ethylene. It thus appears that the active compound in smoke is neither octanoic acid nor ethylene.Abbreviations TLC thin layer chromatography - HPLC High performance liquid chromatography     

The effect of a plant-derived smoke extract on the germination of light-sensitive lettuce seed

A simple and rapid bioassay using seeds of Lactuca sativa L. Grand Rapids has been developed for the detection of germination-enhancing compound(s) in plant-derived smoke extracts. This light-sensitive species germinates within 24 h in the dark at 20 or 25°C and shows responsiveness to smoke-derived extracts over a wide concentration range. For some seed lots where the P _fr level is high and germination in the dark is unacceptably high, a brief (10 min) exposure to far-red light, one hour after the start of imbibition in the dark, is necessary to clearly demonstrate biological activity in the smoke extracts.     

Dependence of gibberellic acid-induced dark germination of lettuce seed on RNA synthesis

Summary Gibberellicacid-induced dark germination of Grand Rapids lettude seed was completely inhibited by 6-azauracil and partly by 2-thiouracil. Other inhibitors of nucleic acid and protein synthesis used were without effect. Inhibition of gibberellic acid-induced dark germination was reversed by uracil but not by thymine, deoxycytidine and orotic acid. The results suggest that gibberellic acid-induced dark germination is dependent on RNA synthesis and not on DNA synthesis.Gibberellic acid-induced lettuce hypocotyl growth was inhibited by all the inhibiters of nucleic acid and protein synthesis used, including actinomycin D, puromycin, chloramphenicol and p-fluorophenylalanine.Approved by the Director of the New York State Agricultural Experiment Station for publication as Journal Paper No. 1507.     

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     

Differential effects of actinomycin d and cordycepin in lettuce seed germination and RNA synthesis

Intact lettuce seed germination was inhibited by cordycepin but not by actinomycin D; however, when seeds were clipped at the cotyledonary end, actinomycin D partially inhibited germination. Uptake studies with intact seeds using ³H-actinomycin D showed that it was unable to reach the embryo prior to radical protrusion. ³H-Cordycepin uptake studies using intact seeds showed that cordycepin was able to reach the embryo during the first 3 hours of incubation and at subsequent times. The pericarp and endosperm offered resistance to penetration of cordycepin into the embryo. In contrast to actinomycin D, cordycepin markedly inhibited ³H-uridine incorporation into RNA of intact seeds during the first 10 and 12 hours of incubation. About 60% of ³H-adenosine incorporation into poly A-RNA was inhibited by cordycepin during 12 hours of incubation, whereas actinomycin D had little effect. RNA synthesis appears to be essential for seed germination.     

Antagonistic effects of high and low temperature pretreatments on the germination and pregermination ethylene synthesis of lettuce seeds

Red light-induced germination of Grand Rapids lettuce seeds (Lactuca sativa L.) incubated at 20 C was inhibited if the seeds were first imbibed at 30 C for 36 hours. This effect was counteracted by exogenous ethylene and associated with a reduction in the rate at which the seeds produced ethylene throughout the pregermination period. A chilling treatment reversed the effect of a prior imbibition at 30 C on both germination and ethylene production. The possibility that the pretreatments influence germination through their effects on ethylene production is discussed.     

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     

Growth physics and water relations of red-light-induced germination in lettuce seeds

Red light (R) and gibberellins (GA) each induce a water potential decrease in the axes of lettuce (Lactuca sativa L.) embryos resulting in germination of intact seeds (achenes) or an increase in growth of the axes of isolated embryos. The fruit coat and endosperm are a substantial barrier to the penetration of exogeneous GA. Isolated embryos take up 35 times as much [³H]GA₁ as the embryos of intact seeds and respond to less than 1·10^-10 M GA₃ or GA₄₊₇. We calculated that only 1·10^-8 M of either GA₃ or GA₄₊₇ would result in 50% germination if the GA were able freely to penetrate the fruit coat. Exogenous GA₃ or GA₄₊₇, at concentrations insufficient to cause germination, result in an apparent synergistic promotion of germination when suboptimal R is applied. Yet suboptimal concentrations of exogenous GA₃ or GA₄₊₇ and suboptimal R result in only additive increases in the growth response in axes of isolated embryos. Dose-response curves demonstrate quantitative increases in the growth response of the isolated axes after R or GA treatments insufficient to induce germination in intact seeds, indicating that a threshold potential must be achieved by the embryonic axes before germination can occur.Abbreviations FR far=red light - GA gibberellin - PEG poly-ethylene glycol 4000 - P_fr far-red-absorbing phytochrome - R red light III.=Carpita et al. 1979b; IV.=Carpita et al. 1979c     

Effects of long-term storage on phytochrome-mediated germination in lettuce seeds

The effects of long-term seed storage on the physiological properties of phytochrome-mediated germination including water uptake, the temperature and light flunnce dependencies of germination and dark germination were studied. The fluenceresponse relationships of the brief irradiation with monochromatic red (660 nm, 7.5 W m⁻²) and far-red (750 nm, 6.6 W m⁻²) light at various times after sowing were also studied. The samples used consisted of three lots of seeds ofLactuca sativa L. cv. MSU-16, which had been harvested in 1976, 1979 and 1985 and stored dry for 9, 6 and 0 years, respectively, in darkness at 23±2 C until the experiments were carried out in July–August, 1985. Seeds with the longer storage periods showed the higher ability to germinate in both continuous darkness and continuous white fluorescent light at 20–30 C. In the seeds stored for 6 or 9 years, red light irradiation for 20 sec given at 15 min or more after sowing at 25 C induced as high a percent germination (85–95%) as those under continuous white fluorescent light. In the freshly harvested seeds, however, germination under continuous white fluorescent light (46%) was considerably lower than the germination induced by the red pulse (97%). Germination of the seeds decreased when the intervals between sowing and a far-red irradiation for 20 sec increased up to 100 min (or 30 min in the freshly harvested seeds). The far-red pulse given later than 100 min (or 6 hr in the freshly harvested seeds) after sowing resulted in an increased germination up to the dark-germination levels with increasing intervals between sowing and the pulse irradiation. Before or at 3 min after sowing, the seeds stored for 6 or 9 years were responsive to the far-red pulse although they were not or hardly responsive to the red pulse, while the freshly harvested seeds were responsive to both the far-red and the red pulses. These data indicate that normal functions of phytochrome completely survived in the dry seeds during storage at 25 C for as long as 6 or 9 years and that these functions are restored into full operation by means of imbibition. The differences in the dependence of germination on the time and fluence of a single pulse of red or far-red light seems to be related to the smaller water content throughout the imbibition in the seeds with the longer storage periods. The greater ability to germinate in the dark indicates the greater amounts of P_FR or the greater responsivity to P_FR, in the seeds with the longer storage periods.     

The laboratory germination of crisp lettuce seeds under moisture stress

Seeds of the crisp lettuce cultivar Pennlake were germinated using all combinations of six ‘initial’ solutions of polyethylene glycol 6000 (PEG) with osmotic potentials ranging from 0 to -8 bars and seven ‘secondary’ solutions of PEG with osmotic potentials ranging from 0 to -10 bars, to which seeds were moved after 24 or 48 h in the ‘initial’ solution. The number of seeds germinating decreased at more negative osmotic potentials of both ‘initial’ and ‘secondary’ solutions but there was an interaction between germination temperature and the osmotic potential of the ‘initial’ solution. At an ‘initial’ solution osmotic potential of 0 bars germination at 20°C exceeded that at 10°C. As the osmotic potential of the ‘initial’ solution decreased germination at 20°C decreased more than at 10°C so that at the more negative osmotic potentials germination at 10°C exceeded that at 20°C. However seeds ungerminated after 14 days germinated normally when transferred back to water, so that the average final germination was 99.5%. The results suggest that major fluctuations in soil water potential in a seedbed are unlikely to influence seed germination per se provided that a period of 24 to 48 h at 0 bars tension is available at some time. The timing of such a period relative to sowing will have a considerable effect on the time of germination and hence the time of emergence. It is concluded that factors other than the direct effect of soil moisture content on germination are involved in reducing seedling emergence under fluctuating soil moisture conditions in the field.     

Analysis of temperature effect on the germination of New York lettuce seeds

When New York lettuce seeds were imbibed below 20°C in thedark, full germination was observed, whereas none occurred above35°C even under red-light irradiation. Partial treatmentwith high temperature during imbibition revealed the involvementof a thermo-labile process in the germination. This processwas completely inhibited above 30°C in the dark, but re-activatedby the following incubation below 20°C. The longer the seedswere preincubated above 30°C, the longer the period below20°C required for re-activation. The results suggest thatthe thermo-labile process is controlled by a thermo-labile factor.The factor was inactivated slowly at 25°C and rapidly above30°C to a form which could be re-activated below 20°Cbut was irreversibly inactivated when the seeds were imbibedat temperatures above 45°C for more than 40 hr. The escapereaction of the phytochrome system proceeded even at 35°Cwhereas no germination was observed at that temperature. Thus,die germination of lettuce seeds is regulated not only by thephytochrome system but also by the thermolabile factor. (Received August 11, 1975; )     

Interactions between hydrostatic pressure and oxygen concentration on the germination of lettuce seeds

Lettuce seeds were germinated at different hydrostatic pressures and O₂ concentrations. Some combinations of pressure and O₂ concentration stimulated while others retarded germination. The highest germination rates which occurred with pressure application and added O₂ were equalled at atmospheric pressure if the O₂ concentration was increased sufficiently. Increasing pressure generally retarded germination, but the pressure effect was partly offset by added O₂. Sustained pressures affected subsequent germination after pressure was released, but only when O₂ was present. Seeds to which pressure was applied in the absence of O₂ germinated as well after pressure exposure as untreated seeds.     

ATP synthesis in lettuce seeds during ripening

During the ripening of lettuce seeds, ATP, AMP + ADP, and moisture decrease to very low levels, and the ability to produce ATP from AMP + PEP (phosphoenolpyruvate) and the PEP-carboxylase (EC 4.1.1.38) activity is diminished. Malate dehydrogenase (EC 1.1.1.37) and pyruvate kinase (PK) (EC 2.7.1.40) decreased up to 10 days after anthesis, after which a sharp increase occurred.     

Opposing effects of strychnine and brucine on the germination and growth of lettuce seeds

Strychnine and brucine, two related alkaloids that occur in plants belonging to theStrychnos species, were shown to have opposing effects on the elongation of the radicle of lettuce seeds. Strychnine was found to be inhibitory, whereas brucine was found to be stimulating to radicle elongation. Alkaloids, generally, are more commonly known for their inhibitory effects on plant growth rather than on their stimulating effects.Paper No. 2435 of the Journal Series of the South Dakota Agricultural Experiment Station, Brookings, South Dakota, USA.     

Opposing effects of strychnine and brucine on the germination and growth of lettuce seeds

Strychnine and brucine, two related alkaloids that occur in plants belonging to theStrychnos species, were shown to have opposing effects on the elongation of the radicle of lettuce seeds. Strychnine was found to be inhibitory, whereas brucine was found to be stimulating to radicle elongation. Alkaloids, generally, are more commonly known for their inhibitory effects on plant growth rather than on their stimulating effects.     

The growth physics and water relations of red-light-induced germination in lettuce seeds

Using lettuce (Lactuca sativa L., cv. Grand Rapids) embryos in osmotica, we have demonstrated that when the growth rates of the embryonic axes of seeds treated with red (R) or far-red (FR) light are equalized, the axes of R-treated seeds develop a 3.4-bar decrease in water potential (paper No. III).As axial growth begins, reserve protein and phytin decrease rapidly, concomitant with increases in reducing sugars, -amino nitrogen, and inorganic and esterified soluble phosphates. However, no differences between the axes of R-and FR-treated seeds are found with respect to the changes in these compounds, indicating that these changes arise as a result of growth and are not under immediate phytochrome control. Little change in the total lipid content is found in either treatment. The axes of FR-treated seeds hydrolyze endogenous sucrose at a greater rate thant those of R-treated seeds. Axes of R-treated seeds accumulate K⁺ and Na⁺ to a greater extent than those of FR-treated seeds. When potassium salts are added to the incubation medium, R induces increased K⁺ uptake by the axis and greater medium acidification by the axis. Malate and other organic acids and acidic amino acids increase at equal rates in both treatments, indicating that inorganic anions may also be taken up to balance the ionic charges. The results are compatible with the assumption that changes in the osmotic and pressure potentials of the embryonic axes of R-treated seeds are the result of a phytochrome-stimulated proton pump which, in whole dormant seeds, would initiate water-potential changes allowing the embryos to overcome the mechanical restraint of the surrounding seed layers, resulting in germination.Abbreviations FR far-red light - PEG polyethylene glyeol 4000 - Pfr far-red-absorbing form of phytochrome - R red light III=Carpita et al. 1979     

The growth physics and water relations of red-light-induced germination in lettuce seeds

Summary A study of photodormant lettuce embryos germinating in water showed that red light induces an increased rate of water uptake. Determinations of the water potential, carried out by a modified gravimetric technique which eliminates errors introduced by solute penetration into cellular osmotic space, showed that the water potential of embryos germinating in water after dark and red light treatment was equivalent and equal to the osmotic potential of a 0.0 to 0.1 molal mannitol solution. Osmotic potentials of the embryos were determined using two new methods. One of the methods utilizes penetration of deuterated water; the other, penetration of a labeled osmoticum into the tissue. For both light- and dark-treated embryos in water, the osmotic potential was equivalent to that of a 0.34 to 0.41 molal mannitol solution. Lettuce embryos thus require that turgor pressure reach a threshold considerably above zero before growth can occur.     

Actions of gibberellic Acid and phytochrome on the germination of grand rapids lettuce seeds

Red light and gibberellic acid were about equally effective in promoting germination of Grand Rapids lettuce (Lactuca sativa L.) seeds. With initial far red light treatment more than 80% remained dormant in subsequent dark storage. After 2 days of dark storage, red light effectively promoted germination, while gibberellic acid action was weak. With between 2 and 10 days of dark storage, gibberellic acid had little effect, while promotion by red light decreased slowly and finally disappeared. After 10 days of dark storage, both gibberellic acid and red light were required for germination. The dark storage treatment interferes with phytochrome-independent germination processes and cannot be overcome by added gibberellic acid. However, storage may also decrease the effectiveness of endogenous gibberellins. Phytochrome-dependent germination seems to require only low levels of endogenous gibberellin activity or the addition of gibberellic acid. Gibberellins and red light appear to act on germination by regulation of sequential sites of a branched-looped pathway.