Normalizing Development of Cultured Triticum aestivum L. Embryos: I. LOW OXYGEN TENSIONS AND EXOGENOUS ABA

Wheat (Triticum aestivum L.) embryos form in dynamically-regulatedovular environments. Our objectives were to improve developmentof cultured immature wheat embryos by simulating, in vitro,abscisic acid (ABA) levels and O₂ tensions as found in wheatovules during zygotic embryogenesis. We characterized from intactwheat kernels embryo respiration, embryo morphology and embryoand endosperm + ABA levels at 13, 19 and 25 d post-anthesis(DPA). Young (13 DPA) embryos were then excised and culturedin vitro, where they were exposed to 0·2 or 2·Ommol m^–3 ±ABA and 2.·1, 2·5 or 7·4mol m^–3 (6, 7 and 21%, respectively) gaseous O₂. At 6and 12 d in culture, + ABA levels, embryo respiration and embryomorphology were characterized by treatment. Thirteen-day-oldembryos from two different plant populations differed by 17-foldin initial ABA content. However, this difference did not affectprecocious germination in vitro, nor did it affect the amountof exogenous ABA required to reduce precocious germination by40%. In this respect, embryos from both populations were equallysensitive to exogenous ABA. Cavity sap O₂ levels (2·1to 2·5 mol m^–3) were much more effective in preventingprecocious germination of cultured embryos than were cavitysap levels of ABA (0·2 to 2·0 mmol m^–3).The combination of physiological levels of both ABA and O₂ largelynormalized DW accumulation and embryo morphology without alteringendogenous + ABA levels. Residual respiration of cultured embryoswas higher than that of embryos grown in situ, and was not influencedby the exogenous O₂ and ABA treatments Key words: Abscisic acid, embryo development, oxygen tensions, respiration, wheat     

Contrasted Effects of CO2 on the Regulation of Dormancy and Germination in Xanthium pennsylvanicum and Setaria faberi Seeds

The effects of CO₂ on dormancy and germination were examinedusing seeds of cocklebur (Xanthium pennsylvanicum Wallr.) andgiant foxtail (Setaria faberi Herrm.). The rate of germinationof the giant foxtail seeds as well as cocklebur was promotedby exogenously applied CO₂ at a concentration of 30 mmol mol^-1regardless of the sowing conditions. However, seeds which failedto germinate in the presence of CO₂, entered a secondary phaseof dormancy under unfavourable germination conditions. If CO₂was applied to seeds under conditions such as water stress imposedwith a 200 mol m^-3 mannitol solution, a hypoxic atmosphere of100 mmol mol^-1 O₂ or a treatment of 0·1 mol m^-3 ABA,development of secondary dormancy was accelerated. These contrastedeffects of CO₂ were observed in ecological studies. Under naturalfield conditions germination of buried giant foxtail seeds respondedpositively to CO₂ during a period of release from primary dormancyfrom Feb. to May, but CO₂ accelerated secondary dormancy commencingin early Jun. In other words, in the presence of CO₂, both theenvironmental conditions and the germination states of the seedsclearly showed secondary dormancy-inducing effects. Thus, itseems that CO₂ has contrasted effects on regulation of dormancyand germination of seeds depending on the germination conditions.Copyright1995, 1999 Academic Press Xanthium pennsylvanicum, cocklebur, Setaria faberi, giant foxtail, CO₂, water stress, hypoxia, ABA, germination, secondary dormancy     

Quantifying the sensitivity of barley seed germination to oxygen, abscisic acid, and gibberellin using a population-based threshold model

Barley (Hordeum vulgare L.) seeds (grains) exhibit dormancyat maturity that is largely due to the presence of the glumellae(hulls) that reduce the availability of oxygen (O₂) to the embryo.In addition, abscisic acid (ABA) and gibberellins (GAS) interactwith O₂ to regulate barley seed dormancy. A population-basedthreshold model was applied to quantify the sensitivities ofseeds and excised embryos to O₂, ABA, and GA, and to their interactiveeffects. The median O₂ requirement for germination of dormantintact barley seeds was 400-fold greater than for excised embryos,indicating that the tissues enclosing the embryo markedly limitO₂ penetration. However, embryo O₂ thresholds decreased by anotherorder of magnitude following after-ripening. Thus, increasesin both permeability of the hull to O₂ and embryo sensitivityto O₂ contribute to the improvement in germination capacityduring after-ripening. Both ABA and GA had relatively smalleffects on the sensitivity of germination to O₂, but ABA andGA thresholds varied over several orders of magnitude in responseto O₂ availability, with sensitivity to ABA increasing and sensitivityto GA decreasing with hypoxia. Simple additive models of O₂–ABAand O₂–GA interactions required consideration of theseO₂ effects on hormone sensitivity to account for actual germinationpatterns. These quantitative and interactive relationships amongO₂, ABA, and GA sensitivities provide insight into how dormancyand germination are regulated by a combination of physical (O₂diffusion through the hull) and physiological (ABA and GA sensitivities)factors. Key words: Abscisic acid, barley, germination, gibberellin, Hordeum vulgare L., model, oxygen, sensitivity Received 2 August 2007; Revised 14 November 2007 Accepted 19 November 2007     

The action of cyclic-AMP on GA3 controlled responses IV. Characteristics of the promotion of seed germination in Lactuca sative variety 'Spartan Lake' by gibberellic acid and cyclic 3,5'-adenosine monophosphate

GA₃ (1 mM) promotes the germination of dark grown seeds of Lactucasativa variety "Spartan Lake" above levels obtained when theseseeds are germinated in the light for 18 hr. Cyclic-AMP alsopromotes the germination of these seeds in the dark but to amuch smaller extent than that obtainable with 1 mM GA₃ and equivalentto levels obtained with 1 µM GA₃. Combinations of lowconcentrations of GA₃ and cyclic-AMP significantly increasedgermination over levels obtained with either compound alone,promoting germination at times to near optimum levels. GA₃ isrequired for less than half the incubation period to bring aboutmaximum germination. ABA was found to inhibit the GA₃ and cyclic-AMP induced responseas well as the response obtained with combinations of the nucleotideand hormone. Theophylline and caffeine in combination with lowGA₃ concentrations promoted germination over seeds treated withthe hormone alone. Of a variety of adenine containing compoundstested only ADP and ATP gave responses similar to those obtainedwith cyclic-AMP. Some of the implications of these results arediscussed. (Received January 16, 1973; )     

ABA and Low Temperature Induce Freezing Tolerance via Distinct Regulatory Pathways in Wheat

The role of ABA in the induction of freezing tolerance was investigatedin two wheat (T. aestivum L.) cultivars, Glenlea (spring var)and Fredrick (winter var). Exogenous application of ABA (5x10^–5M for 5 days at 24°C) increased the freezing tolerance ofintact plants by only 3°C (LT₅₀) in both cultivars. Maximalfreezing tolerance (LT₅₀ of –9°C for Glenlea and –17°Cfor Fredrick) could only be obtained with a low temperaturetreatment (6/2°C; day/night) for 40 days. These resultsshow that exogenously applied ABA cannot substitute for lowtemperature requirementto induce freezing tolerance in intactwheat plants. Furthermore, there was no increase in the endogenousABA level of wheat plants during low temperature acclimation,suggesting the absence of an essential role for ABA in the developmentof freezing tolerance in intact plants. On the other hand, ABAapplication (5x10^–5 M for 5 days at 24°C) to embryogenicwheat calli resulted in an increase of freezing tolerance similarto that achieved by low temperature. However, as in intact plants,there was no increase in the endogenous ABA level during lowtemperature acclimation of calli. These results indicate thatthe induction of freezing tolerance by low temperature is notassociated with an increase in ABA content. Using an antibodyspecific to a protein family associated with the developmentof freezing tolerance, we demonstrated that the induction offreezing tolerance by ABA in embryogenic wheat calli was correlatedwith the accumulation of a new 32 kDa protein. This proteinis specifically induced by ABA but shares a common antigenicitywith those induced by low temperature. These results suggestthat ABA induces freezing tolerance in wheat calli via a regulatorymechanism different from that of low temperature. (Received June 15, 1993; Accepted September 16, 1993)     

Involvement of Cyanogens in the Promotion of Germination of Cocklebur Seeds in Response to Various Nitrogenous Compounds, Inhibitors of Respiratory and Ethylene

Nitrogenous inhibitors of respiration, namely, KCN, NaN₃ andNH₂OH, which promote the germination of cocklebur (Xanthiumpennsylvanücum Wallr.) seeds, enhanced the accumulationof cyanogenic compounds. Urea and cyanamide, which were ineffective,did not. Part of the exogenously applied KCN was converted tocyanogenic glycosides and lipids, but these compounds were onlyproduced at low levels in NaN₃-treated seeds. Exposure to NH₂OHcaused a slight accumulation of both types of cyanogenic compounds.Thiourea, effective in stimulating germination, did not increasethe cyanogenic compounds, which suggests that the mechanismof its effect on germination is different from that of othernitrogenous compounds. Thiocyanate had a slightly promotiveeffect and caused minor increases in the levels of cyanogeniccompounds. Ethylene increased the metabolic utilization of the accumulatedcyanogens in response to KCN or NaN₃. The response to ethyleneof seeds in secondary dormancy was restored only after pre-treatmentwith KCN or NaN₃. The response occurred once the level of cyanogenshad increased. By contrast, enhancement of the effects of KCNand NaN₃ on germination by propyl gallate or benzohydroxamate,inhibitors of CN-resistant respiration, was accompanied by theincreased accumulation of cyanogens within the seeds that hadbeen exposed to KCN or NaN₃ in combination with the other typesof respiratory inhibitors. Thus, it is suggested that endogenouscyanogens might be involved in the germination of cockleburseeds. (Received December 20, 1995; Accepted April 9, 1996)     

Hormonal Regulation of Cormel Dormancy in Gladiolus grandiflorus

The germination of dormant gladiolus corrnels is promoted bycold storage and by treatment with 6-benzyladenine (BA). Itis inhibited by abscisic acid (ABA) and by gibberellin A₃. Ethrelpromotes the germination of dormant cormels but inhibits thegermination of non-dormant ones. ABA was identified by ORD,GLC, TLC, and wheat coleoptile bioassay as the major endogenousgrowth inhibitor controlling cormel germination. The ABA levelin dormant cormels (summer crop stored at 25 °C) is fiveto 10 times that in non-dormant ones (winter crop, or cold-storedsummer crop). The ABA content of the deeply dormant varietyTexas is higher than that of the less-dormant variety Friendship.     

The Role of ABA in the Control of Apple Seed Dormancy Re-appraised by Combined Gas Chromatography-Mass Spectrometry

Measured by GC—MS²—SIR³, endogenous ABA⁴ in embryonicaxes of seeds of Malus pumila L. cv. Golden Delicious decreased8-fold and cotyledon ABA by only 60%, during 10–50 d ofstratification at 5 ?C, after ABA leaching during an initial24 h soaking. During stratification, the percentage germinationof embryos transferred to 17?C showed a significant linear dependenceon log_e of ABA levels in the axes at transfer. Between 50 and70 d, ABA levels increased markedly in axes and testa both ofstratified seeds and seeds allowed to re-dry at 17 ?C afterinitial soaking. The ability of fully stratified axes with elevatedendogenous ABA to germinate indicated that stratification haddecreased their ABA sensitivity. Changes in cotyledon ABA couldnot account for the promotory effect of cotyledons on germinationduring the first 30 d of stratification. Loss of testa inhibitionof germination during stratification was not linked with changesin testa ABA. Stratification markedly increased the sequestrationin the axes of exogenous ABA supplied via the cotyledons. Changesboth in axis ABA levels and sensitivity were thus correlatedwith dormancy release, but subject to modifying control by thecotyledons and testa not involving ABA. Rehydration of driedseeds affected axis ABA later during storage via mechanismsunconnected with dormancy. Key words: ABA, seed dormancy, stratification     

Gibberellin A3 and Abscisic Acid Cause the Reorientation of Cortical Microtubules in Epicotyl Cells of the Decapitated Dwarf Pea

To determine whether or not the changes in the orientation ofmicrotubules (MTs) that are induced by GA₃ and ABA result fromchanges in the rate of epicotyl elongation caused by these hormones,we examined the effects of GA₃ and ABA on the orientation ofMTs in epidermal cells of decapitated epicotyls of the dwarfpea (Pisum sativum cv. Little Marvel), in which neither GA₃nor ABA causes changes in the rate of epicotyl elongation. Cuttings taken from GA₃-pretreated seedlings were decapitatedand treated with ABA. ABA eliminated the GA₃-induced predominanceof transverse MTs and treatment with ABA resulted in a predominanceof longitudinal MTs in the decapitated cuttings. However, ABAdid not reduce the rate of epicotyl elongation in these samples.Cuttings taken from ABA-pretreated seedlings were decapitatedand treated with GA₃. GA₃ caused the orientation of MTs to changefrom longitudinal to transverse in the decapitated cuttings.However, GA₃ had no promotive effect on elongation of theseepicotyls. The results indicate that both ABA and GA₃ have the abilityto change the orientation of MTs by mechanisms that do not involvechanges in the rate of cell elongation. (Received August 18, 1992; Accepted January 18, 1993)     

The action of cyclic AMP on GA3 controlled responses VI. Characteristics of the promotion of light-inhibited seed germination in Phacelia tanacetifolia by GA3 and cyclic 3',5'-adenosine monophosphate

GA₃ and cyclic AMP were found to promote the germination (inlight) of light inhibited Phacelia tanacetifolia seed to thelevels obtained with dark controls. The GA₃ and cyclic AMP promotedgermination was inhibited by ABA and cycloheximide but not by6-methyl purine. The hormone and the nucleotide were not requiredfor the entire incubation period to obtain maximum germination. Theophylline and caffeine also promoted germination of theseseeds and 8-bromoadenosine cyclic AMP was found to be a betterpromoter of germination than cyclic AMP. Of a variety of nucleotidestested, only cyclic AMP promoted germination. This study supportsthe hypothesis that cyclic AMP is involved in some manner inGA₃-induced seed germination. (Received January 22, 1974; )     

Role of ABA in Maturation of Rapeseed Embryos

Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investigate the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development.     

Characterization pf Spring Wheat Genotypes Differing in Drought-Induced Abscisic Acid Accumulation: I. DROUGHT-STRESSED ABSCISIC ACID PRODUCTION

Abscisic acid (ABA) accumulation in response to drought stresswas studied in F₅ and F₆ progeny of a cross between two springwheat (Triticum aestivum L.) genotypes contrasting in ABA accumulation:TW269/9 (high ABA) and Highbury (low ABA). Selection in earliergenerations had been made on the basis of ABA accumulation indetached and partially dehydrated samples of the fourth leafof the main stem (MSL4). Detached-leaf tests with MSL4 from several low and high ABAselections at F₅ confirmed differences found in ABA accumulationin the F₄ generation. Approximately two-fold differences weremaintained at F₅ in both cabinet-grown and field-grown plants. Measurements of the ABA concentrations present in flag leavestaken from drought-stressed plants in the field gave variableresults. Several selections at F₅ which differed in ABA contentby about 70% in detached-leaf tests with MSL4, contained, overall,the same concentration of ABA in both low and high ABA selectionsas plants became water-stressed. However, low and high ABA F₆selections which were sampled on five occasions over severalweeks differed consistently in the level of ABA present fora particular degree of water stress. The high ABA selectionscontained c. 50% more ABA than low ABA selections.Possible explanationsare given for the inconsistencies between ABA levels from lowand high ABA selections in detached-leaf and attached-leaf tests. Key words: Wheat Triticum aestivum, Abscisic acid, ABA, Drought stress     

The Effect of EGTA, Calcium Channel Blockers (Lanthanum Chloride and Nifedipine) and their Interaction with Abscisic Acid on Seed Germination of Brassica juncea cv. RLM-198

Recent investigations have shown that abscisic acid (ABA) dependsupon the availability of Ca²⁺ for its action in certain systems.In order to check whether ABA requires Ca²⁺ to inhibit seedgermination, the effects of ABA, CaCl₂, EGTA (a Ca²⁺ chelator)and lanthanum chloride and nifedipine (Ca²⁺ channel blockers),individually and in combination, on seed germination of Brassicajuncea L. cv. RLM-198 were studied. ABA inhibited germinationin a concentration dependent manner. ABA-induced inhibitionwas abolished with the lapse of time after application. Calciumneither affected germination nor altered the ABA-induced inhibition.EGTA. La³⁺ and nifedipine suppressed seed germination when testedindividually and further elevated the ABA effect, in a synergisticway, when applied simultaneously. They also prevented the time-dependentreduction in ABA-induced inhibition. The findings indicate thatCa²⁺ is not essential for ABA to cause germination inhibition. EGTA, Ca²⁺ channel blockers, abscisic acid, seed germination, Brassica juncea     

Abscisic Acid levels and seed dormancy

Dormant seeds from Fraxinus species require cold-temperature after-ripening prior to germination. Earlier, we found that abscisic acid (ABA) will inhibit germination of excised nondormant embryos and that this can be reversed with a combination of gibberellic acid and kinetin. Using Milborrow's quantitative “racemate dilution” method the ABA concentration in 3 types of Fraxinus seed and pericarp were determined. While ABA was present in all tissues, the highest concentration was found in the seed and pericarp of dormant F. americana. During the chilling treatment of F. americana the ABA levels decreased 37% in the pericarp and 68% in the seed. The ABA concentration of the seed of the nondormant species, F. ornus, is as low as that found in F. americana seeds after cold treatment. Experiments with exogenously added ABA solutions indicate that it is unlikely that the ABA in the pericarp functions in the regulation of seed dormancy. However, the ABA in the seed does seem to have a regulatory role in germination.     

Respiration of Grand Rapids lettuce (Lactuca sativa L.) seeds in relation to chemical and photocontrol of germination

A 10-min irradiation with red light (R) of ‘Grand Rapids’lettuce (Lactuca sativa L.) seeds increased respiratory rates(QO₂ and QCO₂) over those of far red (FR)-irradiated seeds within2 to 3 hr. Differences in respiration between R- and FR-irradiatedseeds became more pronounced with time. The respiration, indarkness at 25?C, of seeds air dried after germination promotingimpregnation treatments with 1.0 mM GA₃+0.5 mM kinetin in acetonewas stimulated slightly by the 6th hour after planting and markedlyby the 12th and 24th hour. Results with seeds in GA₃+kinetindissolved in water were similar. ABA, which inhibits germinationand seedling growth, prevented stimulation in respiration oflight-induced seeds. ABA also repressed respiration when usedwith GA₃+kinetin. The germination and growth retardant, isopropylN-(3-chlorophenyl) carbamate (CIPC), did not affect the respirationof either R- or FR-treated seeds. A comparison of the effectsof chemical and light treatments on respiration indicate thatchemical and photocontrol mechanisms are not identical. (Received May 17, 1976; )     

Cyclic AMP Promotion and Abscisic Acid Inhibition of {alpha}-Amylase Activity in the Seeds of Rice (Oryza sativa L.)

Cyclic AMP and GA₃ stimulated both -amylase activity in riceendosperm and the germination of the seed. In combination theyalso induced germination of ABA-treated seeds but cyclic AMPalone failed to neutralize the inhibitory effect of ABA; withadded kinetin, however, it promoted the -amylase activity ofthe dormant seeds. The enzyme activity decreased as the storageperiod of seeds increased. Cyclic AMP and GA     

Influence of Abscisic Acid on Nitrate Accumulation and Nitrate Reductase Activity in Potato Tuber Slices

Abscisic acid (ABA) at concentrations of 1 to 10 µg.ml^–1suppressed development of nitrate reductase activity in freshtuber slices of Solanum tuberosum L. incubated in KNO₃. Suppressionof activity was evident after 3 hr and continued for 20 hr beforerecovery. This recovery may be due to inactivation of the hormone.Nitrate accumulation was enhanced by ABA. At exogenous NO₃ levelsof 0.1 to 5 mM, the hormone enhanced both NO₃ accumulation andnitrate reductase activity. When applied 24 hr after incubation in NO₃, ABA promoted a markeddecline in enzyme activity in the absence of exogenous NO₃,but was less effective in the presence of NO₃. Slices incubatedin NO₃ and ABA also exhibited increased loss of enzyme activityupon removal of NO₃. Preincubating slices in the hormone for24 hr in a NO₃- free medium resulted in stimulation of nitratereductase activity. Addition of NO₃ resulted in a marked stimulationof enzyme activity over a period of 8–10 hr. The ABA response is not related to tissue levels of free aminoacids and is not affected by different NO₃ sources. These resultssuggest the ABA effect on nitrate reductase activity is influencedby NO₃ status of the cells. Where external NO₃ levels are lowit stimulated NRA while it inhibited activity where NO₃ contentis high. (Received May 12, 1981; Accepted October 12, 1981)     

Seasonal Changes of GA1, GA19 and Abscisic Acid in Three Rice Cultivars

The levels of endogenous gibberellin A₁ and A₁₉ (GA₁ and GA₁₉)and abscisic acid (ABA) in three rice (Oryza sativa L.) cultivars,Nihonbare (normal), Tan-ginbozu (semid-warf) and Tong-il (semi-dwarf),were measured at various stages of internode elongation andear development. GA₁₉ was the main GA in Nihonbare and Tong-ilthroughout the life cycle but was not detected in Tan-ginbozu.The levels of GA₁ in the ears of all three cultivars were lowand reached their maxima after anthesis. Similarly, the earsof all three cultivars contained high ABA levels which peakedafter anthesis. Shoots contained low quantities of ABA throughoutthe life cycle. The roles of GAs and ABA are discussed withrespect to physiological phenomena, such as internode elongation,ear development and dwarfism. (Received May 9, 1981; Accepted July 18, 1981)     

ABA Levels and Sensitivity in Developing Wheat Embryos of Sprouting Resistant and Susceptible Cultivars

A sprouting-resistant and a sprouting-susceptible wheat cultivar were utilized to examine the role of ABA levels and sensitivity responses in wheat embryonic germination. Endogenous embryonic ABA levels were measured in both cultivars throughout grain maturation utilizing a new and sensitive ABA immunoassay. Embryonic ABA levels of each cultivar were similar with the sprouting-susceptible cultivar having about a 25% lower ABA level than that of the sprouting-resistant cultivar. Larger differences between the cultivars were noted in sensitivity to ABA, as measured by capability of ABA to block embryonic germination. ABA inhibited embryonic germination much more effectively in the sprouting-resistant cultivar.     

Induction of alpha-amylase inhibitor synthesis in barley embryos and young seedlings by abscisic Acid and dehydration stress

An endogenous α-amylase inhibitor was found to be synthesized in embryos of developing barley grain (Hordeum vulgare cv Bonanza). Accumulation of this protein occurred late in development (stage IV), at the same time that endogenous abscisic acid (ABA) showed a large increase. The inhibitor could be induced up to 23-fold in isolated immature embryos (stage III) by culture in ABA. Precocious germination was also blocked in stage III embryos by ABA. Dehydration stress on the isolated immature embryos also induced higher levels of the inhibitor and ABA. An even greater response to dehydration stress was observed in young seedlings, where inhibitor content increased 20-fold and ABA increased 80-fold during water stress. The high degree of correlation between ABA and inhibitor contents in in situ embryos, dehydrated embryos and young seedlings, as well as the increase in inhibitor caused by exogenously applied ABA to isolated embryos, suggests that increased α-amylase inhibitor synthesis in response to dehydration stress is mediated by ABA.