Rapid Decrease in the Glutamine Synthetase Activity during Imbibition of Thermodormant New York Lettuce Seeds

A high glutamine synthetase (GS) activity was detected in dryseeds of New York lettuce but it decreased rapidly during imbibitionat 35°C. The decrease in GS activity was remarkable at 35°C,but not so at 45°C or at 25°C. The activity of extractedGS was relatively constant even at 35°C. The decrease inGS activity stopped immediately after the addition of cycloheximide(CH). This suggests the presence of a GSinactivating systemin the seeds. The amount of ammonia increased abruptly duringthe early stage of imbibition at 35°C, suggesting the blockageof ammonia-assimilation at high temperature. The GS activitythat decreased to a low level during imbibition at 35°Cfor 12 h increased again during the subsequent imbibition atlow temperature (15CC) before the break of thermodormancy. Ahigher GS activity was found in the embryonic axes than in thecotyledons. Partial purification of GS showed that lettuce seedGS was eluted as a single peak on Biogel A1.5m or DEAE-Sephacel(mol wt: 4.4 x 10⁵). Thus the thermodormancy of New York lettuce seeds may be relatedto inactivation of GS during imbibition at high temperatures,and the activity of GS in the embryonic axes may control thegermination of New York lettuce seeds through the regulationof glutamine formation. (Received May 11, 1983; Accepted September 13, 1983)     

Reversal of induced dormancy in lettuce by ethylene, kinetin, and gibberellic Acid

The germination of lettuce seeds (Lactuca sativa L., cv. Premier Great Lakes) was significantly inhibited by high temperature (32 C), 0.1 mM abscisic acid or 0.4 M mannitol. Ethylene (16 μl/1 of air) partially reversed the dormancy induced by all three inhibitors but only in the presence of 1 mM gibberellic acid (GA) or light. Neither ethylene plus GA nor ethylene plus light were able to promote germination when thermal inhibition was imposed at 36 C. Addition of 0.01 mM kinetin to the ethylene plus GA or light reversed thermodormancy at 36 C. The dormancy imposed by abscisic acid was also reversed by kinetin. Kinetin was unable to reverse the osmotic dormancy imposed by mannitol. The reversal of osmotic dormancy by ethylene or ethylene plus GA was actually inhibited by kinetin but only in the light. Kinetin apparently stimulates cotyledonary growth in the presence of light, and this growth may compete for certain metabolites critical to radicle growth and subsequent germination. Kinetin and ethylene, as demonstrated primarily in the thermodormancy at 36 C and in osmotic dormancy, appear to regulate a common event(s) leading to germination but through mechanisms unique to each respective growth regulator. The regulation of germination by ethylene is absolutely dependent upon an interaction with GA and/or light.     

Inhibition of Photosensitive Lettuce-Seed Germination by Methionine Sulfoximine, a Specific Inhibitor of Glutamine Synthetase

L-Methionine-DL-sulfoximine (MSO), a specific inhibitor of glutaminesynthetase (GS), severely inhibited germination of New York515 Improved and of Grand Rapids lettuce seeds. However, theelongation of excised axes was not affected by MSO. MSO (1 mM)decreased the growth potential of the axes to a level nearlyequal to the restraining force of the seed coats (about 0.4M). When seeds were treated with MSO at 25?C free ammonia accumulatedin the seeds. MSO inhibited germination of several other light-sensitiveor light-hard seeds, but not light-indifferent seeds. (Received June 10, 1983; Accepted October 6, 1983)     

Effects of sodium chloride on prevention of thermodormancy, ethylene and protein synthesis and respiration in Grand Rapids lettuce seeds

Grand Rapids lettuce ( Lactuca saliva L.) seeds entered into a state of secondary dormancy (thermodormancy) when they were imbibed at 40°C for 72 h. The effect of 40°C in inducing thermodormancy was largely reduced by imbibing seeds at 40°C in solutions of polyethylene glycol (PEG), mannitol and NaCl. Despite similar water potentials of solutions, NaCl pretreatment was more effective. Subsequent germination in the dark at 25°C of saline, high-temperature-pretreated seeds required only gibberellic acid (GA₃), as was the case with nonthermodormant seeds. Thermodormancy reduced total respiratory capacity (V_T) and increased the ratio of alternate pathway (V_alt) to cytochrome pathway (V_cyt) respiration. This was prevented by saline pretreatment. Ethylene production and protein synthesis were depressed in thermodormant seeds, and this was partly alleviated by saline pretreatment. The patterns of protein synthesis in saline- and nonsaline-freated seeds at 40°C were similar, differing only in that the saline treated seeds produced in addition a 78 kDa polypeptide. The pattern of protein synthesis at 40°C differed significantly from that at 25°C.     

Rapid disappearance of small fat bodies during the early stage of imbibition of lettuce seeds

It was found that small fat bodies disappeared from New Yorklettuce seeds during the early stage of imbibition at 20?C.The disappearance began about 4 hr after the start of imbibitionand ended before the start of the radicle elongation. It wascompletely suppressed under thermodormancy at 35?C. Red lightand GA treatment promoted the disappearance, and far-red lightand ABA treatment inhibited it. These results suggest that thedisappearance of the small fat bodies may be closely relatedto the control mechanism of lettuce seed germination. (Received April 23, 1977; )     

Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

Induction of secondary dormancy in sunflower seeds by high temperature. Possible involvement of ethylene biosynthesis

High temperature (45°C) inhibits seed germinition and seedling sunflower ( Helianthus annuus L. cv. Mirasol). Treatment of imbibed seeds at 45°C for more than 48 h induces a secondary dormancy, which is associated with progressive decrease of germination ability at optimal temperature (25°C) as well as with abnormal seedling growth. Ethylene (55μl l⁻¹) and 2-chloroethylphosphonic acid (ethephon) (2.5 m M ) improve germination of thermodormant seeds at 25°C. but the abnormal growth of the seedlings remains. O₂-enriched atmosphere and dry storage improve germination and normal seedling growth. The induction of thermodormancy in sunflower seeds seems associated with loss of their ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. Possible effects of high temperature on membranes and ethylene forming enzyme (EFE) are discussed.     

Characterization of ethylene/gibberellic acid control of germination in Lactuca sativa L.

Ethylene production during germination of lettuce seeds (Lactucasativa L., cv. Premier Great Lakes) occurred at two distinctlydifferent rates. A very low rate of ethylene release was observedprior to the 12th hour of incubation at 22?C. The rate of ethyleneproduction, however, increased 100 fold between the 12th and16th hour of incubation. This high rate of ethylene productiononly occurred in the presence of seeds which exhibited a visibleprotrusion of the radicle. The duration of exposure to a supraoptimaltemperature (32?C) was inversely proportional to the percentgermination at 32?C. Ethylene production and growth were notblocked by incubating visibly germinated seeds at 32?C. Exogenous ethylene partially restored germination at 32?C, butonly in the light. Gibberellic acid partially substituted forthe induced light requirement but not for ethylene. It was concludedthat the supraoptimal temperature raised the threshold concentrationof ethylene required for germination. This threshold requirementwas satisfied in the presence of exogenous ethylene. Germinationat 32?C was abo dependent upon the presence of GA. With exogenousethylene present, the GA-mediated system was presumably reinstatedor bypassed by exposing the seeds to either light or GA. Theinitial low rate of ethylene production apparently regulatessubsequent germination but only when present at a minimum thresholdconcentration. Those events initiating germination have obviouslyoccurred prior to the time of radicle emergence. Post-germinationethylene production, therefore, did not break thermodormancy,but occurred simultaneously with radicle emergence. (Received November 29, 1976; )     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )     

The relationships between ethylene production and cell elongation during the initial growth period of chick-pea seeds (Cicer arietinum)

Ethylene production by isolated chick-pea embryonic axes during the initial stages of germination has been studied. Maximum production of ethylene occurs when growth is entirely due to cell elongation and before mitotic activity begins. This peak increases three-fold in the presence of calcium, but it is diminished by osmotic inhibitors, polyamines and abscisic acid (ABA), with a parallel fall in growth rate. Fusicoccin stimulates ethylene production and counteracts the effects of polyethylene glycol; thiourea, which breaks thermodormancy in chick-pea seeds, reduces ethylene production but does not counteract the effects of osmotic inhibitors. Of the polyamines studied, spermine (in low concentrations, 0.1 to 1.0 m M ) is the only one to stimulate ethylene production and cell elongation. It is concluded that there is a close relationship between ethylene production and cell elongation.     

A simple method for the isolation of intact mesophyll protoplasts from cereal plants

Intact mesophyll protoplasts from cereal plants were easilyprepared by incubating leaves with the abaxial epidermis peeledoff at 20–25?C for 2–3 hr in 0.6 M mannitol containing1% cellulase at pH 5.6. From one gram (fresh weight) of leaves1.5–6?10⁶ protoplasts, more than 90% of which were morphologicallyintact, could be obtained. Protoplasts isolated from wheat,oat, corn and barley were efficiently infected with brome mosaicvirus (BMV), and supported viral multiplication. (Received June 21, 1977; )     

Soya Bean Seed Growth and Maturation In vitro without Pods

Immature Glycine max (L.) Merrill seeds, initially between 50and 450 mg f. wt, were grown and matured successfully in vitro.Excised seeds were floated in a liquid medium containing 5 percent sucrose, minerals and glutamine in flasks incubated at25 °C under 300 to 350 µE m^–2 s^–1 fluorescentlight. During 16 to 21 d in culture, seeds grew to a matured. wt of 100 to 600 mg per seed at an average rate of 5 to 25mg d. wt per seed d^–1 depending on initial size. Growthrates were maximal during the first 8 to 10 d in vitro but declinedwith loss of green colour in the cotyledons. Seed coats rupturedwith rapid cotyledon expansion during the first 2 d in culture.Embryos were tolerant to desiccation and 80 to 90 per cent germinatedif removed from culture before complete loss of green colour.The growth of excised seeds in vitro exceeded the growth ofseeds in detached pods, but when windows were cut in pods topermit direct exposure of seeds to the medium, seed growth wascomparable. Glycine max (L.) Merrill, soya bean, seed culture, seed growth, seed maturation, germination     

The Effect of Changing Temperatures During Imbibition on Ultrastructure in Germinating Pea Embryonic Radicles

Hodson, M. J., Di Nola, L. and Mayer, A. M. 1987. The effectof changing temperatures during imbibition on ultrastructurein germinating pea embryonic radicles.—J. exp. Bot. 38:525–534. Pea seeds were imbibed at 5 °C or at 25 °C for 5–6h and then germinated at the same temperature or transferredto the other temperature for 15 h. After imbibition or germinationthe embryonic axis was removed, fixed and the ultrastructureof the radicle tip examined. Exposure of the seeds to 5 °Cduring imbibition resulted in the cessation of almost all ultrastructuralchanges during subsequent germination at 5 °C. When theseeds were transferred to 25 °C development of ultrastructurewas far slower than expected. In particular, mitochondrial structurefailed to develop, there were few Golgi and little ER, and lipidbodies located near the plasma membrane disappeared or fusedmuch more slowly with the membrane. An attempt is made to relatethe observations on ultrastructure with data previously reportedon the metabolism of phospholipids in the plasma membrane andER of the embryonic axis. A tentative hypothesis to accountfor the effect of temperature of imbibition on subsequent seedgermination is proposed. Key words: Pisum sativum L. cv. Alaska, germination, imbibition, temperature, ultrastructure, lipid bodies, protein bodies, vacuolation, mitochondria     

Effects of Temperature and dl-Strigol on Seed Conditioning and Germination of Witchweed (Striga asiatica)

Seed conditioning and germination in witchweed (Striga asiatica(L.) Kuntze) were temperature-dependent. With higher conditioningtemperatures, shorter conditioning time was required for germinationwith terminal dl-strigol (strigol) treatment at 30 °C. Maximumgermination (80–100%) was obtained by conditioning inwater at 20, 25, 30 and 35 °C for 14, 7, 5 and 3 d, respectively,and terminally treating with 10^–6 M strigol at 30 °C.Seeds conditioned in 10^–8 M strigol instead of water germinatedmuch less with the same terminal strigol treatment. Generally,conditioning was slower when seeds were conditioned in strigolrather than water. The reduction in germination rate by pretreatmentin strigol or pretreatment at low temperatures could be overcomeby increasing the terminal strigol concentration in the germinationtest. Conditioned seeds did not germinate at 10 and 15 °Cwith a terminal 10^–6 M strigol treatment but yielded closeto maximum germination at 25, 30 and 35 °C with the sameterminal strigol treatment. To obtain maximum germination, boththe minimum conditioning temperature and the minimum germinationtemperature for conditioned seeds were 20 °C. Factors suchas conditioning time, and strigol concentration and temperatureduring conditioning and/or germination determine whether seedsremain in the conditioning phase or shift to a germination phase. dl-Strigol, germination stimulation, parasitic plants, seed conditioning, seed germination, Striga asiatica, temperature, weed control     

The protective role of selenium in recalcitrant Acer saccharium L. seeds subjected to desiccation

Freshly harvested silver maple (Acer saccharinum L.) seeds were soaked in either sodium selenite (10 mg/L) or water for 6 h. After washing and air drying, seeds were desiccated at 22 °C at a RH of 45-50% to comparable water levels from 50 to 12%. Germination capacity was significantly higher in seeds treated with selenium and desiccated [from 50 to 40, 35 and 30% of water content (WC)] than in water-soaked seeds. At 20% WC, the seeds from both treatments had low viability (approximately 20%). The electrolyte leakage and the MDA content were significantly lower in the embryonic axes of seeds soaked in selenite than in seeds soaked in water. We also found that the activity of glutathione peroxidase (GPX) of embryonic axes from selenium-treated seeds that were not desiccated, or from seeds that were desiccated to 40 and 35% WC, was significantly higher than that of non-treated axes. No difference in GPX activity was detected in cotyledons. This was confirmed by activity staining of GPX after native PAGE of proteins extracted from embryonic axes and cotyledons. An increase in glutathione reductase (GR) activity was also observed in embryonic axes of seeds treated with selenium and dried to 35 and 30% WC compared to non-treated samples. Selenium appeared to have no such effect on cotyledons.     

Mechanism of seed priming in circumventing thermodormancy in lettuce

Lettuce (Lactuca sativa L. cv Minetto) seeds were primed in aerated solutions of 1% K₃PO₄ or water at 15°C in the dark for various periods of time to determine the manner by which seed priming bypasses thermodormancy. Seeds which were not primed did not germinate at 35°C, whereas those which were primed for 20 h in 1% K₃PO₄ or distilled H₂O had up to 86% germination. The rate of water uptake and respiration during priming were similar regardless of soak solution. Cell elongation occurred in both water and 1% K₃PO₄, 4 to 6 h prior to cell division. Both processes commenced sooner in water than K₃PO₄. Radicle protrusion (germination) occurred in the priming solution at 21 h in water and 27 h in 1% K₃PO₄.

Respiration, radicle protrusion and cell division consistently occurred sooner in primed (redried) seeds compared to nonprimed seeds when they were imbibed at 25°C. Cell division and elongation commenced after 10 h imbibition in primed (redried) seeds imbibed at 35°C. Neither process occurred in nonprimed seeds. Respiratory rates were higher in both primed and nonprimed seeds imbibed at 35°C compared to those imbibed at 25°C, although radicle protrusion did not occur in nonprimed seeds which were imbibed at 35°C. It is apparent that cell elongation and division are inhibited during high temperature imbibition in nonprimed lettuce seeds. Seed priming appears to lead to the irreversible initiation of cell elongation, thus overcoming thermodormancy.

     

The Relation of Germination of Wheat to Water Potential

The relation of moisture potential to the germination of wheatwas studied by supplying water to the seeds in the vapour phase,at controlled relative humidities. An apparatus for this purposeand a method of attaining the necessary precise control of temperatureare described. The critical level of moisture potential at which germinationis completely inhibited was not reached. Even when, as a resultof infection, the duration of the experiments was limited to20 days, up to 20 per cent, of the seeds germinated at a potentialof –320 metres of water or a pF of 4.5. This is considerablydrier than Permanent Wilting Point, which is represented bya potential of –160 metres of water, or pF 4.2. Thereis a marked effect of moisture potential on the time taken togerminate, as times of emergence varied from 2 to over 20 daysover a range of potentials of from zero to –320 metresof water, but it is probable that very little further germinationwould have occurred after 15 days had not fungal infection enabledthe seeds to obtain liquid water by bridging the air-gap betweenseed and water source. This infection is probably responsiblefor the reduction in viability of the seeds at the lower potentials,as there is an indication that under conditions where germinationis postponed considerably, seeds exhibit a reduced germinatingcapacity probably due to an increased susceptibility to infection. The failure of some individual seeds to germinate at potentialswhich allow other seeds to germinate is not due to failure toattain a critical moisture content. In soil, the percentage germination at low potentials is similarto that observed without soil, but the reduction in the totalgerminating capacity, or viability, after 20 days is not asgreat, and this is probably due to the reduced incidence ofvisible infection observed under these conditions.     

Effect of Temperature, Light, Nutrients and Dehardening on Abscisic Acid Induced Cold Hardiness in Bromus inermis Leyss Suspension Cultured Cells

The effects of culture conditions on abscisic acid (ABA)-inducedfreezing tolerance were determined in smooth bromegrass Bromusinermis Leyss cv. Manchar) cell suspension cultures. Bromegrasscultures initiated with 2 g fr wt of cells achieved maximumfreezing tolerances (greater than –32?C) at 25 to 30?Cin the presence of 75 to 100 µM ABA. High levels of freezingtolerance induced by ABA were correlated with high growth ratesat 25 and 30?C. In control cells, incubation at 10?C inducedoptimum levels of hardiness with minimal growth. Prolonged exposure(6 weeks) of cells to 3?C, with or without ABA, increased freezingtolerance only by several degrees. Exogenous ABA concentrationsgreater than 100 µM were not inhibitory to growth. Repeatedexposure to ABA, however, retarded growth and made the cellstolerant to temperatures below –40?C. Removal of ABA fromthe medium resulted in dehardening of the cells both at 25 and3?C. Nitrogen had a marginal effect on ABA-induced hardeningat 25?C, but inhibited age-dependent hardening of control cellcultures. Light had no effect on the freezing tolerance of culturedcells. Addition of 10% sucrose, 30 min prior to freezing, tobromegrass cells treated with ABA for 4 days increased freezingtolerance more than 15?C. These observations are discussed inrelation to the contrasting behaviour of the low temperatureand photoperiod dependent cold acclimation of plants (Received July 14, 1989; Accepted October 23, 1989)     

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     

The Effect of Stratification and Thidiazuron Treatment on Germination and Protein Synthesis of Pyrus serotina Rehd cv. Niauli

Breakage of seed dormancy in Pyrus serotina Rhed cv. Niauliis achieved by cold treatment. The chilling time necessary forgermination of Pyrus serotina Rhed cv. Niauli seeds was determined,and synthesis of polypeptides during stratification and thidiazuron(TDZ) treatments was investigated. The chilling time requiredwas about 21 d. The longer the chilling time, the higher thegermination percentage. The effect of 200 µM TDZ on seedgermination was equivalent to 14-21 d of chilling, and TDZ couldbe used as a substitute for cold treatment. Use of in vivo ³⁵S-methionine label and two-dimensional gel electrophoretic analysisof polypeptides revealed that the patterns of untreated (dormant)and chilled embryonic axes differed significantly in their polypeptideprofiles. The difference became conspicuous after prolongedchilling exposure. During 28 d of stratification followed by5 d of incubation at 25 °C, changes were noted in 35 solublepolypeptides (ESPs) and 38 pellet polypeptides (EPPs) from theembryonic axis. The changes included increase, decrease or newlyinduced synthesis. Common patterns of changes in soluble andpellet polypeptides of embryonic axes, can be broken down intofour categories. The possible functions and roles of these fourcategories of polypeptides in breaking seed dormancy and germinationare discussed. Although the magnitude of polypeptide changesare not as extensive in seeds receiving TDZ treatment as afterstratification, similar polypeptide changes were co-inducedby the two treatments.Copyright 1994, 1999 Academic Press Stratification, protein synthesis, pear, Pyrus serotina, Thidiazuron, TDZ