Tomato Seed Germination. Osmotic Pretreatment and Far Red Inhibition

At 25 °C germination of tomato (Lycopersicon lycopersicum)seeds is inhibited by continuous and intermittent far red illumination.It is also inhibited by a single 30 min far red irradiationgiven 8 h from the start of imbibition. The incubation of seedsin a mannitol solution inhibitory for germination has no effecton the final germination percentage after seeds are subsequentlytransferred to water. A 30 min far red irradiation at the timeof transfer results in partial inhibition of germination. Thisinhibition can be released by the continuation of osmotic incubationfor several days before the transfer to water. At the end ofa 7 d dark period of osmotic incubation, inhibition of subsequentgermination in water can be realized only by continuous farred illumination. Seeds osmotically pretreated for 7 d and afterwardsdried-back show a mean time to 50% germination significantlylower than that of untreated seeds. Moreover, besides singleand intermittent, even continuous far red light has no inhibitoryeffect on the germination of these seeds. It is concluded that,in addition to the already known germination advantages, osmoticpresowing treatment also induces the ability of seeds to germinateunder unfavourable light conditi.     

Separation of Light-Induced [C]ent-Kaurene Metabolism and Light-Induced Germination in Grand Rapids Lettuce Seeds

The effect of light on the metabolism of [¹⁴C]kaurene in light-requiring lettuce seeds (Lactuca sativa L. cv Grand Rapids) was investigated. Seeds were soaked in a solution of [¹⁴C]ent-kaurene in methylene chloride with 0.01% Tween-20, dried, and incubated in 20% polyethylene glycol (PEG) to prevent seedling development. Labeled metabolites were extracted and analyzed by high performance liquid chromatography and gas chromatography-radio counting. [¹⁴C]ent-Kaurenol and [¹⁴C]ent-kaurenal were identified in seeds incubated in constant white light, while no ethyl acetate-soluble metabolites were found in seeds incubated in the dark. In time course experiments using acid scarified seeds, metabolism began after 18 hours of incubation and greatly increased after 24 hours of incubation in 20% PEG. By 48 hours, several unidentified, more polar metabolites were found. Germination was induced in seeds imbibed in 20% PEG by 4 hours of red or 4 hours of white light following 20 hours in the dark, and was fully reversed by 2 hours of far red light. However, in metabolism experiments, [¹⁴C]ent-kaurene oxidation was observed only with constant white light. These results indicate that although ent-kaurene oxidation is a light sensitive step in the biosynthesis of gibberellins in Grand Rapids lettuce seeds, ent-kaurene metabolism is not required for light-induced germination.     

Eco-physiological studies on desert plants

Summary The O₂ uptake and RQ of germinating negatively photoblastic Zygophyllum coccineum seeds were studied during the first fourty hours after soaking in water under dark or light conditions. Five phases could be distinguished in the course of O₂ uptake in water in dark. The respiration course in light did not differ from that in dark till the 12th hour after soaking, then it deviated showing a low level, The RQ values in dark and light are nearly identical; both had sharp decline between the 8th and the 16th hour after soaking. Light, by blocking germination, affected the O₂ uptake.Moisture stress simulated by mannitol solutions caused a decrease in the O₂ uptake of seeds and seedlings. Na₂SO₄ caused a reduction and a lag in the time of maximum O₂ uptake. NaCl showed a particular effect on respiration of germinating seeds causing earlier rise and decrease in O₂ uptake than in water. NaCl was effective in rising the O₂ uptake of seedlings specially those supplied with glucose. Its effect was not pronounced on starved seedlings. O₂ uptake of seedlings germinated in NaCl was considerably high when compared with that of seeds germinating in the same medium 40 hours after soaking.The decreasing effect of moisture stress caused by mannitol on O₂ uptake was reversed by diluting the medium or replacing it with water. Values of O₂ uptake on number of seedlings basis are different from those calculated on fresh weight basis due to the difference in water content of seedlings germinated under various conditions.     

Influence of Na+ on Synthesis of Macromolecules by a Marine Bacterium

Resting cells of Vibrio natriegens acquired the ability to take up (14)C-labeled mannitol in media containing Na(+) and K(+). But, the cells took up a significant quantity of the label as well in the presence of 0.3 m K(+) and no Na(+). The label was distributed throughout the cells in both systems. Cells incubated in mannitol minimal culture medium proliferated and synthesized approximately nine times as much protein in the presence of Na(+) and K(+) as those incubated in the presence of mannitol and 0.3 m K(+). The bacteria did not proliferate in the absence of Na(+). Cells incubated in medium containing mannitol and Na(+) and K(+) synthesized approximately twice the quantity of deoxyribonucleic acid and ribonucleic acid as those incubated in medium containing mannitol and 0.3 m K(+) but no Na(+). A significant amount of mannitolbinding protein was synthesized in the membranes of V. natriegens incubated in the presence of mannitol and Na(+) and K(+), but only a small quantity was produced in medium containing mannitol and 0.3 m K(+) but no Na(+). A binding fraction comprising at least two proteins (both with molecular weight near 34,000) was isolated by gel electrophoresis from other components of a K(2)CO(3)-extract of membrane protein from mannitol-grown cells. This binding fraction mediated phosphorylation of mannitol at the expense of either adenosine triphosphate or phosphoenolpyruvate. It was then found that mannitol-grown, but not broth-grown, cells contained nicotinamide adenine dinucleotide-linked mannitol-1-phosphate dehydrogenase. Neither contained mannitol dehydrogenase.     

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.     

The Relationship between the Effects of Cytokinin on the Expension and Metabolism of Excised Cucumber Cotyledons and Water Stress

Excised cucumber (Cucumis sativus, var. Jin-ian No. 4) cotyledons were incubated with 10 ppm of BA (benzyladenine) or water for 1 h, then thouroughly rinsed with water and grown in darkness on filter paper saturated with different concentrations of mannitol solution. Up to 24 h, the fresh weight, carotenoid, RNA, DNA and lipid contents of cotyledons were determined. Although mannitol solution reduced the effectiveness of BA treatment, in the same condition of osmotic potential, the increases of fresh weight, carotenoid, RNA and DNA contents, as well as the decrease of lipid per cotyledon were always much higher in BA treated tissues. BA enhanced the rate of water uptake by the cotyledons. The fresh weight of BA and 0.2 M mannitol treated cotyledons was equal to that of water control, but the increases of carotenoid and nucleic acid contents and the decrease of lipid were much higher in tho former than the latter. 0.3 M and 0.5 M mannitol solu- tions almost interrupted the water uptake of water and BA treated cotyledons respectively. However, the increases of carotenoid and nucleic acid contents as well as decrease of lipid were still occurred in these conditions. The different osmotic potential did nearly not affect the ratio of the increases of carotenoid and nucleic acid contents between BA treatment and control. It means the effectiveness of BA was almost the same under different osmotic potential It is evident that BA stimulated simultaneously the water uptake and metabolism of the cotyledons. They are probably different processes but closely related to each other.     

Influence of factors affecting germination on respiration of Phacelia tanacetifolia seeds

Summary Germination of the seeds of Phacelia tanacetifolia is inhibited by light. Removal of that part of the covering structures of the seeds which directly covers the radicle allows full germination in light. The rate of O₂ uptake in the seeds increases following imbibition, and reaches the same steady rate in light and in darkness after 3 hr. From the 14 th hour on, dark-imbibed seeds show a linear increase in the rate of respiration. This increase is not observed in dormant seeds incubated in light. In normal dark germination, protrusion of the radicle begins at 12 th hour following soaking, and by the end of 18 th hour approximately 60% of the seeds have germinated. The seeds which have been scarified at the radicle end and germinate readily in light show a steady increase in Q _{O 2}. If scarified seeds are allowed to imbibe 0.3 M mannitol and are then incubated in light, the embryo does not grow and the pattern of O₂ uptake becomes identical with that of intact seeds in light. Mannitol, however, does not inhibit respiration by itself. These observations indicate that the increased O₂ uptake is the result rather than the cause of seed germination, and that light does not cause dormancy by inhibiting O₂ uptake. Measures effective in releasing dormancy (dark incubation, mechanical scarification, gibberellin treatment) do not induce germination by facilitating oxygen entry.This work was supported in part by the Jane Coffin Childs Memorial Fund for Medical Resarch, and by the U. S. Atomic Energy Commission under Contract No. AT (11-1)-1338.     

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.     

Phytochrome-mediated Germination Responses in gamma-Irradiated Lettuce Seeds

Sublethal doses of γ-radiation and far red light have some-what analogous, red light reversible, effects on the germination of lettuce seeds (Lactuca sativa L. var. Grand Rapids). However, the mechanism by which γ-radiation retards germination appears to differ from that of far red light. Compared to controls, γ-radiation retarded germination for the first 24 hours; but after 36 or 48 hours of imbibition gemination of treated seeds was higher than that of the controls, whether or not the γ-irradiated seeds received red or far red light. The effects of γ-radiation are more pronounced in seeds containing 15% water at the time of treatment than in those containing only 7% water. The promotive action of red light is operative in the presumed absence of cell division in γ-treated seeds.     

The effect of cell turgor on sugar uptake in strawberry fruit cortex tissue

A reduction in cell turgor has been shown to stimulate sugar uptake in several plant sink tissues and it may regulate the import of assimilate into the sink apoplast, as well as maintain cell turgor. To determine whether cell turgor influences sugar uptake by strawberry (Fragaria x ananassa Duch. cv. Brighton) fruit cortex tissue, disks were cut from greenhouse-grown primary fruit at the green-white stage of development and placed in buffered incubation solutions containing either mannitol or ethylene glycol as an osmoticum. Cell turgor of fruit disks was calculated from the difference between the water potential of bathing solution and tissue solute potential after incubation at various osmolarities. Cell turgor increased when tissue disks were placed into mannitol incubation solutions more dilute than the water potential of fresh tissue (about 415 mOsmol kg^?1). The rate of uptake of [¹⁴C]-sucrose or [¹⁴C]-glucose decreased as osmolarity of the incubation solution increased, i.e. as cell turgor declined. Cell turgor and the rate of [¹⁴C]-sucrose uptake were unaffected when rapidly permeating ethylene glycol was used as an osmoticum. A decrease in cell turgor reduced both the V_max of the saturable (carrier mediated) kinetic component of sucrose uptake, and the slope of the linear (diffusional) component. The sulfhydryl binding reagent p-chloromercuibenzenesulfonic acid, an inhibitor of the plasma membrane sucrose carrier, strongly inhibited only the saturable component of sucrose uptake. Increased uptake of the nonmetabolizable sugar, O-methyl-glucose, at high turgor was similar to that of glucose, indicating that carrier activity was influenced by cell turgor, not cell metabolism. Turgor did not influence efflux of [¹⁴C]-sucrose from disks and had no effect on cell viability. Strawberry fruit cells do not possess a sugar uptake system that is stimulated by a reduction in turgor.     

Precocious Germination during In Vitro Growth of Soybean Seeds

Immature Glycine max (L.) Merrill seeds were grown and matured in liquid medium at 25°C under fluorescent light. In standard medium containing minerals, 146 millimolar sucrose and 62.5 millimolar glutamine (osmolality 0.24), precocious germination seldom occurred with a starting seed size of less than 300 milligrams fresh weight. Frequency of precocious germination increased with increased starting seed size. Sucrose concentration strongly affected precocious germination while glutamine concentration had no effect. Starting with 300 to 350 milligrams fresh weight seeds, treatments which reduced the sucrose concentration or lowered the osmolality of the culture medium stimulated precocious germination, and increased the fresh weight growth but not the dry weight growth of seeds. Increasing the osmolality to 0.38 with sucrose or mannitol prevented precocious germination without reducing dry weight accumulation in seeds. In medium with initially low osmolality, precocious germination was inhibited by addition of 1 to 100 micromolar abscisic acid to the medium without a reduction in seed growth. During growth and maturation of large soybean seeds in vitro, precocious germination and other abnormal tissue growth can be prevented by high sucrose or mannitol concentrations in the medium or by addition of abscisic acid.     

The effect of osmotics on plant cells: a study using 1H NMR relaxation and self-diffusion of water.

The osmotic changes in root cells of Zea mays L. under the effect of mannitol (concentration range 1-15%; range of osmotic pressures: -0.13 to -2.01 MPa) were studied by measuring time and concentration dependence of water self-diffusion constant (Deff) and proton spin-lattice relaxation time (T1) using proton NMR relaxation spectroscopy. In addition, in vivo uptake of Mn2+ by roots after their incubation in mannitol solutions were studied. At low (less than or equal to 5%) and medium (5-10%) concentrations of the osmoticum dehydration takes place proportional to the concentration, whereas membrane destruction occurs at higher concentrations (10-15%). It seems that there is a distribution of cells within root tissue regarding their sensitivity to osmotic stress.     

Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments

Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [⁸⁶Rb⁺] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10⁻⁷ molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [⁸⁶Rb⁺] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [⁸⁶Rb⁺] flux is not a general feature of phytochrome action.     

Seed Water Relations and the Regulation of the Duration of Seed Growth in Soybean

Soybean [Glycine max (L.) Merrill] seeds and cotyledons weregrown in an in vitro culture system to investigate the relationshipsbetween cell expansion (net water uptake by the seed) and drymatter accumulation. Seeds or cotyledons grown in a completenutrient medium containing 200 mol m^–3 sucrose continueddry matter accumulation for up to 16 d after in planta seedsreached physiological maturity (maximum seed dry weight). Seedor cotyledon water content increased throughout the cultureperiod and the water concentration remained above 600 g kg^–1fresh weight. These data indicate that the cessation of seeddry matter accumulation is controlled by the physiological environmentof the seed and is not a pre-determined seed characteristic.Adding 600 mol m^–3 mannitol to the medium caused a decreasein seed water content and concentration. Seeds in this mediumstopped accumulating dry matter at a water concentration ofapproximately 550 g kg^–1. The data suggest that dry matteraccumulation by soybean seeds can continue only as long as thereis a net uptake of water to drive cell expansion. In the absenceof a net water uptake, continued dry matter accumulation causesdesiccation which triggers maturation. Key words: Glycine max (L.) Merrill, solution culture, duration of seed growth, water content, dry matter accumulation     

Turnover of the Intracellular Mannitol Pool of Fucus spiralis L. (Fucales, Phaeophyta) during Osmotic Shock

13C {¹H} nuclear magnetic resonance spectroscopy has been usedto study the turnover of the low-molecular weight carbohydratemannitol in the marine brown alga Fucus spiralis L. During incubationof plants in seawater medium with ¹³C enriched bicarbonate,all of the NMR-visible ¹³C appeared in mannitol; no significant¹³C-labelling of laminaran was observed in seawater, in a hypo-salineor in a hyper-saline medium. Pulse-chase experiments showedthat intracellular mannitol was subject to turnover (half-time20 h), the rate of carbon assimilation into mannitol being slightlygreater than the dissimilation rate, possibly due to net mannitolsynthesis during growth. Fucus spiralis was conservative in the use of mannitol as anintracellular osmoticum. In a hypo-saline medium, mannitol showedno substantial change while potassium, the major cellular cation,was reduced. In contrast, mannitol increased substantially overa 12 h incubation period in a hyper-saline medium, whereas potassiumcontent remained constant. Mannitol assimilation and dissimilation rates were not affectedsignificantly by transfer to a hypo-saline medium. The increasedmannitol content of plants incubated in a hyper-saline mediumappeared to be due to a significant increase in the mannitolassimilation rate. Key words: Mannitol, Fucus, Phaeophyta, ¹³C-NMR, osmotic adjustment     

Phytochrome, nitrate movement, and induction of nitrate reductase in etiolated pea terminal buds

The role of phytochrome in the induction of nitrate reductase of etiolated field peas (Pisum arvense L.) was examined. Terminal bud nitrate concentration increased in darkness, and the increase correlated with induction of nitrate reductase following brief exposure of intact plants to red, blue, far red, and white lights. Brief light exposure of intact plants stimulated nitrate uptake and induction of nitrate reductase by terminal buds subsequently excised and incubated on nitrate solution in darkness; exposure of excised buds in contact with nitrate led to less uptake but more induction. Nitrate and nitrate reductase activity both declined during incubation with water, irrespective of light treatment. Nitrate enrichment of intact terminal buds and uptake into excised buds and increases in nitrate reductase activity were all red/far red reversible. Dimethyl sulfoxide (1%, v/v) and sugars (sucrose 0.5%, glucose 1, w/v), although stimulating nitrate uptake into excised tissue in darkness, failed to enhance nitrate reductase activity over dark controls. Phytochrome may regulate nitrate reductase via both nitrate movement and a general mechanism such as enhancement of protein synthesis.     

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.     

Influence of osmotic stress on the respiration of potato tuber callus

Potato tuber ( Solanum tuberosum L. cv. Bintje) callus shows a decrease in fresh weight and an increase in dry weight upon transfer to nutrient medium supplemented with 0.3 or 0.5 M mannitol. The osmolarity of the intracellular fluid increases simultaneously. Probably mannitol is taken up from the medium till the osmolarity of the tissue is in equilibrium with that of the medium. After osmotic adaptation, on a medium with 0.5 M mannitol, growth is negligible, although the tissue retains its viability.
Respiration increases upon transfer to medium with extra mannitol, especially when expressed on a fresh weight basis. On this basis cytochrome and alternative pathway capacities do not change appreciably. The respiratory increase is exclusively caused by an increased engagement of the alternative pathway. The participation of this pathway in uninhibited respiration increases from about 10 to 90% upon transfer to medium with extra mannitol. The increase in respiration is partly correlated with the decrease in fresh weight upon transfer. Per disc, the capacities of the cytochrome and alternative pathway decline. Yet, total respiration per disc significantly increases due to the increased participation of the alternative pathway. This results in an almost equal ATP-production per disc before and after transfer. We suggest, that the alternative pathway functions as a reserve capacity in potato callus, which is switched on when ATP-production coupled to the cytochrome pathway is impaired.     

Abscisic acid and osmoticum prevent germination of developing alfalfa embryos,but only osmoticum maintains the synthesis of developmental proteins

Developing seeds of alfalfa (Medicago sativa L.) acquire the ability to germinate during the latter stages of development, the maturation drying phase. Isolated embryos placed on Murashige and Skoog medium germinate well during early and late development, but poorly during mid-development; however, when placed on water they germinate well only during the latter stage of development. Germination of isolated embryos is very slow and poor when they are incubated in the presence of surrounding seed structures (the endosperm or seed coat) taken from the mid-development stages. This inhibitory effect is also achieved by incubating embryos in 10^?5 M abscisic acid (ABA). Endogenous ABA attains a high level during mid-development, especially in the endosperm. Seeds developing in pods treated with fluridone (1-methyl-3-phenyl-5[3-(trifluoromethyl)-phenyl]-4(1H)-pyridinone) contain low levels of ABA during mid-development, and the endosperm and seed coat only weakly inhibit the germination of isolated embryos. However, intact seeds from fluridone-treated pods do not germinate viviparously, which is indicative that ABA alone is not responsible for maintaining seeds in a developing state. Application of osmoticum (e.g. 0.35 M sucrose) to isolated developing embryos prevents their germination. Also, in the developing seed in situ the osmotic potential is high. Thus internal levels of osmoticum may play a role in preventing germination of the embryo and maintaining development. Abscisic acid and osmoticum impart distinctly different metabolic responses on developing embryos, as demonstrated by their protein-synthetic capacity. Only in the presence of osmoticum do embryos synthesize proteins which are distinctly recognizable as those synthesized by developing embryos in situ, i.e. when inside the pod. Abscisic acid induces the synthesis of a few unique proteins, but these arise even in mature embryos treated with ABA. Thus while both osmoticum and ABA prevent precocious germination, their effects on the synthetic capacity of the developing embryo are quite distinct. Since seeds with low endogenous ABA do not germinate, osmotic regulation may be the more important of these two factors in controlling seed development.