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.     

Immediate phytochrome action in lettuce seeds and its interaction with gibberellins and other germination promoters

Summary An immediate action of phytochrome (P_FR) was found by a technique of adding subthreshold concentrations of gibberellic acid (GA₃) after irradiation. It was shown that phytochrome (P_FR) is active within 5 mins after its formation. The interaction between P_FR, GA₃ and six other gibberellins (GA₁, GA₄, GA₅, GA₇, GA₉ and GA₁₃) was synergistic. This result is interpreted to mean that P_FR does not produce any of these gibberellins, at least during the first 30 mins. of its action. This conclusion was confirmed by interaction experiments in darkness using GA₃ combined with the other gibberellins. A similar synergism was found between P_FR and kinetin, thiourea and chloramphenicol.     

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.     

Dependence of phytochrome action in seeds on membrane organization

Germination of Amaranthus retroflexus L. seeds imbibed at 40 C is enhanced by establishing the active form of phytochrome before a reduction in temperature to <32 C. The half-time for effectiveness of the lower temperature is about 8 min at 15 C. Isolated membrane fragments of A. retroflexus seeds associated with the fluorescent probe 1,8-anilino-naphthalene sulfonate (ANS) increase in structural order as the temperature is lowered through the 32 C region. The germination response is decreased by the membrane-disruptive substances tris, octonoate, and ethanol. The results show that phytochrome activity is associated with an organized membrane. By using ANS with membrane fragments from Setaria faberi Herrm. seeds, leakage of amino acid was found to be enhanced at temperatures >32 C by a transition in the plasmalemma.     

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.     

Light effects upon dry lettuce seeds

Germination of certain dry seeds (achenes) of Lactuca sativa L. cv. Grand Rapids was increased to ca. 75% after irradiation with 665 nm red light (R; 1x10³ J m^-2); this response was eliminated by far-red light (FR) following the R. The response of dry seeds required an order of magnitude more light than that of wetted seeds, and was not maximal until 48 h after irradiation. Other seeds, which could not be stimulated by R in dry state, showed a partial response after 10 min hydration. Irradiation of dry seeds (or seeds wetted 1 h) with FR (1x10³ J m^-2) reduced dark germination from 26% to 2%. Seeds dehydrated in an oven (60°C, 90 min) showed a decrease in germination if irradiated with R (1x10⁵ J m^-2) before wetting. The results show that phytochrome is present in dry lettuce seeds (and functional in some seed lots) prior to wetting; and that in other seed lots the molecule becomes functional within minutes after wetting the seeds. Transformation of the FR absorbing from of phytochrome (P_FR) to the inactive from (P_R) occurs at lower seed moisture content than the reverse reaction. It appears that dormancy in seeds ripened in sunlight might be assured during seed drying and maturation by the more effective transformation of P_FR to P_R than vice versa as phytochrome is dehydrated.Abbreviations FR far-red - R red - CAL seeds from California - NC seeds from North Carolina (see text)     

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     

Ethylene synthesis in lettuce seeds: its physiological significance

The germination and pregermination ethylene production of Grand Rapids lettuce seeds (Lactuca sativa L.) incubated at 20 C after a red light treatment are inhibited if the seeds are first imbibed at 30 C for 36 hours. In this study, low concentrations of ethylene were found to enhance the germination of seeds pretreated at 30 C more than that of untreated controls. In the presence of high concentrations of ethylene, pretreated seeds and controls germinated at a similar rate. These results are consistent with the view that a prolonged imbibition at 30 C inhibits germination at a lower temperature through its effect on the ethylene production of the seeds. As a further test of the hypothesis, estimates were made of the pregermination ethylene content of untreated seeds and pretreated seeds incubated in the presence of sufficient ethylene to make them germinate as rapidly as untreated seeds. The values obtained were 0.65 and 0.74 nanoliter of ethylene per gram (dry weight) of seeds, respectively.     

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.     

Patterns of food utilization by the germinating lettuce seeds

The embryo excised from seed of Grand Rapids lettuce (Lactuca sativa L.) can be cultured in distilled water. Complete digestion of the endosperm and transfer of nutrients from the endosperm to the embryo occur in the germinating seed with fat as the source of food. The fat is utilized for respiration, synthesis of amino acids, and to a degree, converted to sucrose. ¹⁴C-Glucose administered to the seed is quickly converted to sucrose in the endosperm and translocated to the embryo. Radioactivity associated with the glucose remains predominantly in the carbohydrate fraction, and much of it is incorporated into what is believed to be cell wall polysaccharides. Relatively little isotope is distributed in the amino or organic acids.     

RNA synthesis and the germination of light-sensitive lettuce seeds

Summary The germination of lettuce seeds is inhibited by the nucleotide base analogue 6-methylpurine. RNA synthesis has been measured during imbibition and germination as ³²P-phosphate incorporation into RNA species as fractionated by polyacrylamide gel electrophoresis. Seeds were surface sterilized and imbibed in the presence of various antibiotics. RNA preparations from lettuce seeds were coelectrophoresed with ³H-RNA prepared from bacteria to check for bacterial contamination of the seeds. There is a much higher rate of RNA synthesis in illuminated, germinating seeds as compared to dark, non-germinating seeds. This difference does not develop until after 12 hours of imbibition at 27°, which is the time of onset of germination and radicle growth.This investigation was supported by a contract from the United States Department of Agriculture (No. 616-15-3). Journal paper of the Purdue Agriculture Experiment Station.     

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.     

Endosperm and pericarp involvement in the supercooling of imbibed lettuce seeds

Fully hydrated lettuce (Lactuca sativa L.) seeds showed dual freezing exotherms (−9 and −18°C), even after 10 hours imbibition. Only the −9°C exotherm was observed in seeds imbibed for 20 hours, but without external nucleation, all water in the embryo supercooled. Results indicate that the endosperm acts as a barrier to ice propagation. Other experiments suggest that the pericarp may also protect the embryo under certain freezing conditions.     

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     

Light effects on endogenous levels of gibberellins in photoblastic lettuce seeds

Gibberellin A₁ (GA₁), 3-epi-GA₁ GA₁₇, GA₁₉, GA₂₀, and GA₇₇ were identified by Kovats retention indices and full-scan mass spectra from gas chromatography-mass spectrometry analysis of a purified extract of mature seeds of photoblastic lettuce (Lactuca sativa L. cv. Grand Rapids). Non-13-hydroxylated GAs such as GA₄ and GA₉ were not detected even by highly sensitive radioimmunoassay. These results show that the major biosynthetic pathway of GAs in lettuce seeds is the early-13-hydroxylation pathway leading to GA₁, which is suggested to be physiologically active in lettuce seed germination. Quantification of endogenous GAs in the lettuce seeds by gas chromatography-selected ion monitoring using deuterated GAs as internal standards indicated that the endogenous level of GA₁ increased to a level about three times that of dark control 6 h after a brief red light irradiation, and that far-red light given after red light suppressed the effect of red light. The contents of GA₂₀ and GA₁₉ were not affected by the red light irradiation. Evidence is also presented that 3-epi-GA₁ is a native GA in the lettuce seeds.     

Influence of dimethyl sulfoxide on freezing resistance of lettuce seeds

DTA of lettuce seeds was used to study the cryoprotective mode of action of DMSO. Lettuce seeds were imbibed for 16 hr in 5, 10, 15 or 20% aqueous solutions of DMSO either with or without a 6 hr preimbibition in water. Seeds were frozen to postsecondary exotherm temperatures; exotherms were recorded; and germination was tested after freezing. DMSO had at least a twofold effect on the lettuce seeds: it increased the degree of supercooling, and it imparted a capacity to survive following freezing beyond secondary exotherm levels.