Phosphate mobilization in grains of Hordeum distichon

When decorticated grains were germinated at 14·5°, inorganic substances moved from the endosperm, mainly the aleurone layer, to the embryo. The level of Pi rose in the embryo and endosperm, and the embryo appeared to accumulate Pi against a concentration gradient. The level of organic-P declined in the endosperm, particularly in the alcurone layer. Separated aleurone layers, incubated at 25°, released only small amounts of organic or inorganic phosphate. However, when incubated with gibberellic acid (GA₃), a massive release of Pi occurred at the expense of organic phosphates within the tissue. This release followed a sigmoid pattern with time following a lag and was virtually complete in 6 days. Allowing the aleurone layer to dry before incubating with GA₃ reduced or abolished the lag period of Pi release and only marginally depressed the total amount ultimately freed. In contrast, α-amylase production was depressed by the longer periods of drying. The major phosphate of the aleurone was phytate (meso-inositol hexaphosphate, IP₆), but traces of 1P₄, IP₃, IP₂, IP₁, Pi and unidentified phosphates were detected. During incubation with GA₃ the IP₆ content fell, and the lower esters of inositol rose slightly and then fell in a pattern indicating that the phosphate groups of each IP₆ molecule were being sequentially hydrolysed. After 6 days incubation, the tissue phosphates were reduced to a very low level. Attempts to isolate aleurone grains containing phytate were unsuccessful.     

Influence of the Seed Envelope and Growth Regulators Upon Seed Dormancy in Witchweed (Striga lutea Lour.)

Seed of the angiospermous parasite, witchweed (Striga luteaLour.), normally germinate poorly or not at all unless adequatelypretreated and exposed to a germination stimulant which is obtainedfrom plant root exudates. Under certain conditions, scarificationof the seed envelope promoted germination in the absence ofthe stimulant. With adequately (2-week) pretreated seed, a cutor puncture through the aleurone at the radicular end inducedtypical germination. A puncture through the aleurone elsewhereon the seed induced little or no germination. A cut throughthe aleurone at the cotyledon end or centre of the seed inducedatypical germination. A puncture through the aleurone elsewhereon the seed induced little or no germination. A cut throughthe aleurone at the cotyledon end or centre of the seed inducedatypical germination in which the radicle elongated but didnot penetrate the intact envelope over the radicle. Incubationin oxygen did not promote germination regardless of the siteof seed scarification. Ten per cent carbon dioxide reduced thegermination of punctured seed. Seed germinated equally wellwhen scarified over the radicle and incubated in air, nitrogen,darkness, or light. Brief treatments with sulphuric acid alsoinduced germination. The softening effect of stimulatory acidtreatments upon the aleurone was first evident 6 h later andonly occurred at the radicular end of the seed. Indol-3yl-aceticacid inhibited and ethylene stimulated germination of scarifiedand non-sacrified seed. Gibberellic acid(GA₃) had no apparenteffect upon nonscarified seed but promoted germination of scarifiedseed. Inadequately (I-day) or excessively (12- to 16-week) pretreatedseed germinated poorly or not at all when treated with the germinationstimulant, ethylene, GA₃, or sulphuric acid. Some seed germinatedslowly when scarified over the radicle, but the germinationrates and totals were less than those of scarified seed pretreatedfor 2 weeks. Additions of stimulant, ethylene or GA₃, aceleratedthe germination rates of scarified but inadequately pretreatedseed. Of the treatments tested, only GA₃, increased the slowgermination of the excessively pretreated scarified seed. Results indicated that the aleurone restrained radicle elongation.Scarification over the radicle removed the restraint and permittedradicle emergence. However, the ability of the radicle to elongate,as influenced by time of seed pretreatment and exogenous stimulant,GA₃ or ethylene, determined whether or not the scarified seedgerminated.     

Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

Influence of Gibberellic Acid and the Rht3 Gene on Choline and Phospholipid Metabolism in Wheat Aleurone Tissue

The effect of gibberellic acid (GA3) on phospholipid metabolismand -amylase production was studied in aleurone tissue of twonear-isogenic lines of wheat (Triticum aesuvum L.). Incubationof embryoectomized seeds from a GA-responsive line (rht3, tall)with GA₃ caused the induction of -amylase activity after a lagphase of 30 h. In the case of embryoectomized seeds from a ‘GA-insensitive’line (Rh13, dwarf), however, the lag phase was extended up to50 h. During the first 14 h following imbibition, GA₃ inhibitedcholine uptake and its subsequent incorporation into phosphatidylcholine in the Rhr3 line but not in the rht3 line. GA₃ promotedphospholipid breakdown in both the lines during this period,however. GA₃ also terminated independent turnover of the cholineN-methyl groups in phosphatidyl choline and promoted turnoverof the whole choline headgroup. These results are discussedin relation to the possibility that phosphatidyl choline turnoveris an integral part of the GA₃ signal-transduction mechanismin aleurone tissue. Key words: GA3, Rht3 gene, choline, phospholipid     

The Effect of Calmodulin Antagonists on Gibberellic Acid-induced Enzyme Secretion in Barley Aleurone Layers

Calmodulin activity was detected and assayed in barley aleuronecells. The effect of calmodulin antagonists on GA₃-induced enzymesynthesis and secretion in barley aleurone layers was also investigated.These calmodulin antagonists (chlorpromazine, haloperidol) inhibitedonly GA₂-induced -amylase secretion. This inhibitory effectwas intensified after 6 h of GA₃-incubation. This leads us tosuggest that some calmodulin-controlled mechanism is involvedin GA₂-induced -amylase secretion. Hordeum vulgare L., barley aleurone cells, gibberellic acid, -amylase secretion, calmodulin, calmodulin antagonist     

Gibberellic acid-induced secretion of hydrolases in barley aleurone layers

Activities of phosphatases in the aleurone layers of a husklessbarley, Ehime-hadaka No. 1, were enhanced in the absence ofgibberellic acid (GA₃), while the enzyme secretion was absolutelydependent upon its presence. GA₃ was required for both inductionand secretion of a-amylase. The longer the preincubation ofthe tissue without GA₃, the longer was the lag period beforesecretion of both a group of phosphatases and a-amylase. Changesin the fine structure of aleurone cells were also investigated.Characteristics of the phase transition from enzyme accumulationto enzyme secretion seemed to be a development of a bundledtype of endoplasmic reticulum. ¹Present address: Institute of Biological Sciences, The Universityof Tsukuba, Ibaraki 300-31, Japan. (Received August 25, 1975; )     

Effects of Gibberellins on Seed Germination of Phytochrome-Deficient Mutants of Arabidopsis thaliana

Experiments were carried out to explore the involvement of gibberellins(GAs) in the light-induced germination of Arabidopsis thaliana(L.) Heynh, using wild type (WT) and phytochrome-deficient mutants(phyA, phyB and phyAphyB deficient in phytochrome A, B and Aplus B, respectively). Seed germination of WT and phytochrome-deficientmutants was inhibited by uniconazole (an inhibitor of an earlystep in biosynthesis of GA, the oxidation of ent-kaurene) andprohexadione (an inhibitor of late steps, namely, 2rß-and 3rß-hydroxylation). This inhibition was overcomeby simultaneous application of 10^-5 M GA₄. The relative activityof GAs for promoting germination of uniconazole-treated seedswas GA₄>GA₁=GA₉>GA₂₀. The wild type and the phyA and phyBmutants had an increased response to a red light pulse in thepresence of GA₁, GA₄, GA₉, GA₂₀ and GA₂₄ but there were no significantdifferences in activity of each GA between the mutants. Therefore,neither phytochrome A nor hytochrome B appears to regulate GAbiosynthesis from GA₁₂ to GA₄ during seed germination, sincethe conversion of GA₁₂ to GA₉ is regulated by one enzyme (GA20-oxidase). However, GA responsiveness appears to be regulatedby phytochromes other than phytochromes A and B, since the phyAphyBdouble mutant retains the photoreversible increased responseto GAs after a red light pulse. (Received February 13, 1995; Accepted July 11, 1995)     

Effects of Gibberellins and Their Methyl Esters on Dark Germination and Antheridium Formation in Lygodium japonicum and Anemia phyllitidis

The effects of GA₃, GA₄ and GA₉ and their methyl esters on darkspore germination and antheridium formation of the ferns Lygodiumjaponicum and Anemia phyllitidis were investigated. Althoughall induced both germination and antheridium formation in Lygodium,only the gibberellins induced these effects inAnemia. (Received August 28, 1986; Revision received November 14, 1986. )     

Specific binding of gibberellin A1 to aleurone grain fractions from wheat endosperm

A subcellular fraction enriched in aleurone grains isolatedin glycerol from aleurone layers of wheat endosperm specificallyand reversibly bound GA₁-(³H). Specific binding of GA₁ to otherfractions including spherosomes, nuclei, mitochondria, and plasmamembranes was negligible. The K_d of binding to aleurone grainswas 1.5 µM and the number of specific binding sites 0.45pmoles per mg protein. The presence of Ca⁺⁺ ions was absolutelyrequired for binding. Abscisic acid which inhibits giberellinaction in vivo prevented specific GA₁-binding in vitro. GA₁-bindingto aleurone grains is important to the primary action of thehormone which may involve mobilization of reserves from thealeurone grain-spherosome complex for utilization in membranebiogenesis. ¹ Present address: Section of Cytology, Yale University Schoolof Medicine, New Haven, CT 06510, U.S.A. ³ Present address: Laboratoire de Biologie V?g?tale, Ecole NormaleSup?rieure, 24 rue Lhomond, 75231 Paris, France. (Received March 28, 1977; )     

Phosphoinositides in barley aleurone layers and gibberellic Acid-induced changes in metabolism

Phospholipids of barley (Hordeum vulgare L. cv Himalaya) aleurone layers were labeled with myo-[2-³H]inositol or [³²Pi], extracted, and analyzed by physical (chromatography) and chemical (deacylation) techniques. Three phospholipids were found to incorporate both myo-[2-³H]inositol and [³²Pi]—phosphatidylinositol, phosphatidylinositol-monophosphate, and phosphatidylinositol-bisphosphate. Stimulation of [³H]inositol prelabeled aleurone layers with GA₃ showed enhanced incorporation of label into phosphatidylinositol within 30 seconds and subsequent rapid breakdown. Stimulation of phosphatidylinositol labeling observed in these studies is the earliest response of aleurone cells to gibberellic acid reported.     

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; )     

INTERACTION OF GIBBERELLIN A3, AND INORGANIC PHOSPHATE IN TOBACCO SEED GERMINATION

1. Investigation was made on the influence of inorganic phosphateupon the germination of positively photoblastic tobacco seed(Nicotiana tabacum L. var. uirginica (AGDH.) COM. "Bright Yellow")induced by GA₃, GA₃M, kinetin, red light, and ammonium saltsof various organic acids.
2. Inorganic phosphate increases theGAs-induced germination, andinhibits the germination causedby ammonium citrate, while itdoes not influence the germinationbrought about by GA₃M, kinetin,and red light.
3. The optimumpH for the GA₃-induced germination lies in the acidicpH range,indicating that the undissociated form of GA₃ is operative.The stimulatory effect of phosphate is, however, not ascribedmerely to the pH control in the mediurr. Phosphate exerts somespecific influence for which the presence of the free carboxylgroup of GAs is required.
4. The observed contrasting effectsof phosphate on the GA₃-inducedgermination (i.e., acceleration),on the one hand, and on theammonium citrate-induced germination(i.e., inhibition), onthe other, were explained by assumingthat the phosphate effectsultimately consist in acceleratingthe uptake of the carboxylicacid into the seeds.
5. GA₃M alsohas an activity of inducing the germination of tobaccoseedwithout light.

¹Present address: Department of Vegetable Crops, Universityof California, Davis, California, U.S.A. (Received March 12, 1962; )     

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; )     

Effects of Repetitive Acid Immersion, Red Light, and Gibberellin A3 Treatments on Phytochrome-mediated Germination Control in Skotodormant Lettuce Seeds

Dry lettuce seeds (Lactuca sativa L. cv. Grand Rapids), whichreceived 5 min far-red light (FR) 0.5 h after the onset of waterimbibition, showed 17% and 50% germination without and withacid immersion treatment (pH 0.1) for 1 h and rinsing with water,respectively. The acid treatment caused only 6% germinationor less in FR-treated seeds held for 10 to 30 d in dark storage.The 10 to 30 d skotodormant seeds did not respond to red light(R) or gibberellin A₃ (GA₃) singly, but showed 84% or higherpercentage germination if 1 h acid immersion was given beforeR or GA₃. The 20 d skotodormant seeds, which received R treatmentat day 10 but remained dormant showed 89% germination with onlyacid treatment. Similar values were obtained with 30 d skotodormantseeds which received one or two R treatments at day 10 or 20,i.e. the only requirement for these R-treated dormant seedswas an acid immersion. This releases the skotodormancy and rendersthe seeds more sensitive to R or GA₃, but the skotodormancywas initiated again if no light or hormone treatments were givenimmediately. The repetitive R or GA₃ treatments, which did notcause skotodormant seeds to germinate, lessened the degree ofskotodormancy. The germination of these skotodormant seeds canonly be induced by the synergistic action of R and GA₃. In thisstudy, GA₃ caused higher germination percentages in R-treatedskotodormant seeds than R stimulated in GA3-treated seeds. Itis suggested that (i) repetitive R or Ga₃ treatments maintaina high endogenous level of the far-red-absorbing form of phytochrome(P_fr) and GA activity, respectively, (ii) the accumulated stableintermediates of phytochrome persist in fully-imbibed skotodormantseeds for up to 20 d, without phytochrome expressing its functionuntil the seeds are acidified and (iii) a model is formulatedto interpret the results of acidification, growth promotersand R effects on germination of light-sensitive lettuce seeds. Key words: Phytochrome, Latuca saliva, seed germination, dark reversion of phytochrome, gibberellin A₃, acidification, skotodormancy     

Effects of Gibberellic Acid on the Activation, Synthesis, and Release of Acid Phosphatase in Barley Seed

Acid phosphatase activity was present in unimbibed barley seed,but rose during incubation of embryoless half-seeds and isolatedaleurone layers, and was further increased by 10^–6 M gibberellicacid (GA₃). Release of total acid phosphatase activity fromhalf-seeds and aleurone layers was markedly enhanced by GA₃.Inhibitor studies with cycloheximide and actinomycin D suggestedthat de novo synthesis of acid phosphatase occurred followingimbibition. Gel nitration, electrophoresis, and [¹⁴C]leucineincorporation studies revealed that a single molecular formof acid phosphatase was present in dry seed, whereas on incubationtwo further forms arose. A proportion of the three molecularforms of the enzyme was synthesized de novo. Gibberellic acidstimulated activation, but not de novo synthesis, of all threemolecular forms of acid phosphatase. Although a small amountof one of the molecular forms was secreted in the absence ofGA₃, the presence of gibberellin greatly increased secretionof the same form of acid phosphatase.     

Uptake of Gibberellin Methyl Esters by Spores and Induction of Dark Germination in Lygodium japonicum

In the fern Lygodium japonicum, the effect of the exogenousapplication of two gibberellin methyl esters, gibberellin A₄methyl ester (GA₄Me) and gibberellin A₂₀ methyl ester (GA₂₀Me)on spore germination in the dark and uptake of GA₄Me and GA₂₀Meby spores was investigated. Tritiated GA₄Me and GA₂₀Me wereprepared and used as radioactive tracers. The activity of GA₄Mewas more than 100-fold that of GA₂₀Me for the induction of sporegermination. When treated for 24 h, the activity for inducingspore germination remained after removal of the gibberellinmethyl esters from the medium. The amount of GA₄Me taken upby spores was more than three times that of GA₂₀Me throughoutthe 24 h time course of treatment. The uptake of both gibberellinmethyl esters was proportional to the external concentrationfor the range of concentrations between 10^–9 M and 10^–6M. When treated with the tritiated gibberellin methyl estersat 10^–6 M and 10^–7 M for 24 h, most of the gibberellinmethyl esters taken up by the spores were not metabolized. Althoughthe uptake of the two gibberellin methyl esters differed by3- to 5- fold, their abilities to induce spore germination differedby more than 100-fold. Therefore, the difference in the activityof the two gibberellin methyl esters regarding the inductionof spore germination could not be explained solely by the differencein their uptake. (Received January 11, 1988; Accepted May 26, 1988)     

Effects of Chemicals Often Regarded as Germination Stimulants on Seed Conditioning and Germination of Witchweed (Striga asiatica)

Gibberellin A₃ (GA₃), kinetin and zeatin promoted germinationof conditioned witchweed (Striga asiatica [L.] Kuntze) seedssimilar to dl-strigol and a natural stimulant from corn rootexudates. GA₃ promoted germination only with scarified seeds.However, these regulators, as well as dl-strigol and naturalstimulants, inhibited the conditioning of seeds and their subsequentgermination. On the other hand, abscisic acid induced 74% germinationof conditioned seeds but had no effect on seed conditioning.Other chemicals such as sucrose, NaNO₃, vitamin B₁, L-methionine,nicotinamide, indoleacetic acid and colchicine had no apparenteffect on seed conditioning, while sucrose, L-methionine andinositol induced about 30 % germination of conditioned seeds.Seeds generally required either a longer conditioning time ora higher terminal dl-strigol concentration to eliminate anyinhibition of seed conditioning induced by a compound. dl-Strigol, germination stimulation, parasitic plants, seed conditioning, seed germination, Striga asiatica (L.) Kuntze, weed control, growth regulating substances     

Effect of Gibberellin on Mature Euonymus europaeus L. Seeds

The release from dormancy of Euonvmus europaeus L embryos bya brief treatment with GA₃ has been studied During 48 h incubationof dormant embryos in GA-free medium, phospholipid levels increasedat first, then declined sharply over the last 6 h When the embryoswere placed in GA₃ medium during this 6 h period levels of totalphospholipids as well as of phosphatidylethanolamine increasedwhilst phosphatidylinositol and phosphatidylcholine declinedslightly Fine structural changes stimulated by a brief GA₃ treatmentwere of different character depending on tissue region (1) ‘destructive’changes occurred in the superficial procortical parenchyma onthe hypocotyl/radicle boundary, involving autolysis and decompartmentationof organelles, (2) ‘positive’ changes occurred inregions close to root and shoot apical meristems, involvingdegradation of protein bodies and their conversion into vacuoles,and the proliferation of various organelles A number of differenceswere noted when the changes in GA₃-treated embryos were comparedwith those induced by low temperature, which also overcomesdormancy The results suggest that germination is accompaniedby different cytological events depending on whether it is inducedby cold or GA₃ The growth of embryos in which dormancy was overcomeby GA3 was due to the activation of the apical root meristemclose to the quiescent centre, whilst in embryos in which germinationwas induced by low temperature, the periphery of hypotocotyl/radicleboundary was the site of activation Euonymus europaeus L, dormant embryo, fine structure, phospholipids, GA₃ and cold treatments     

Effect of Gibberellic Acid on the Ultrastructure and {alpha}-Amylase Activity of Aleurone Cells of Avena sativa L.

The -amylase activity and ultrastructure of aleurone cells inseeds of Avena sativa L. were studied using seed halves withembryo (embryo seeds) which had imbibed water and seed halveswithout embryo (embryo-less seeds) which had imbibed water withor without GA₃. -Amylase activity was detected in the aleurone layers of embryoseeds that had imbibed water and embryo-less seeds that hadimbibed GA₃-water. The ultrastructure of aleurone cells withdetectable -amylase activity showed marked changes in the roughsurfaced endoplasmic reticulum (rER), in the flattened sacculesforming stacks and in the aleurone grains. The progressive changesin the rER were as follows: first, the number of slender rERincreased; then, the inner space became wider and showed roundor oval profile; and finally, the rER became slender again witha reduced number of adhering ribosomes. The flattened sacculesforming stacks were appressed to the surface of aleurone grains.With time, they decreased in number and finally disappeared.In parallel with the decrease of flattened saccules, digestionof proteinaceous material inside the aleurone grains proceeded. (Received February 24, 1987; Accepted September 3, 1987)     

Biological Activities of the Methyl Ester of Gibberellin A73, a Novel and Principal Antheridiogen in Lygodium japonicum

The synthetic methyl ester of GA₇₃ (GA₇₃-Me) and the naturalantheridiogen of Lygodium japonicum showed almost the same activityto induce the formation of antheridia in dark-grown protonemataof L. japonicum at concentrations of 10^-14 M and higher. Thus,it appears that the principal antheridiogen in L. japonicumis GA₇₃-Me. GA₇₃-Me inhibited formation of ar-chegonia in light-grownprothallia of L. japonicum at concentrations of 10^-11 M andhigher and induced germination of spores in the dark in thisspecies at the same range of concentrations. GA₇₃(free acidform) promoted growth of seedlings of dwarf rice and hypocotylsof cucumber seedlings at dosages of and above 1 and 100 ng/plant,respectively. Eight compounds related to GA₇₃-Me, includingantheridiogens of Anemia phyllitidis and Anemia mexicana, wereactive in inducing an-theridial formation in L. japonicum, althoughtheir activities were considerably lower than that of GA₇₃-Me. (Received August 24, 1988; Accepted November 28, 1988)