Promotion and inhibition of {alpha}-amylase production in barley endosperm by cyclic 3',5'-adenosine monophosphate and adenosine diphosphate

The possibility that gibberellin-induced -amylase synthesisin barley endosperm might be mediated by cyclic-3',5'-adenosinemonophosphate (3',5'-AMP) was examined. Promotion of -amylasesynthesis by 3',5'-AMP (5 mM) was observed in the absence ofgibberellic acid (GA₃) and in combination with GA₃ at concentrationsbelow 2 mµM. When combined with gibberellin at concentrationsabove 2 mµM, however, 3',5'-AMP reduced the amount of-amylase obtained. The cyclic nucleotide showed slight activityat concentrations as low as 0.05 mM. These promotions were shownto be due to increased synthesis of -amylase rather than toan increased secretion of the enzyme. Of a variety of adeninecompounds and nucleoside diphosphates tested only 3',5'-AMPand adenosine diphosphate (ADP) induced -amylase synthesis.Longer incubation times were necessary to obtain maximal -amylaseinduction with the nucleotides than with GA₃. ADP and 3',5'-AMPwere about one third and one fifth as active, respectively,as GA₃ in promoting -amylase synthesis, although GA₃ was morethan 10⁷ times more effective. AMO-1618 did not inhibit theaction of the nucleotides and methanolic extracts of the nucleotidesshowed no gibberellin-like activity. Both nucleotides were synergisticwith GA₃ in overcoming the inhibitory effects of acetate andcitrate buffers on -amylase synthesis. (Received February 24, 1969; )     

The Effects of Gibberellic Acid on Organelle Biogenesis in the Endosperm of Germinating Castor Bean Seeds

Mature seeds of castor bean (Ricinus communis L.) were imbibedin tap water or 0.3 mM GA₃, planted in vermiculite moistenedwith tap water or 0.3 mM GA₃, and incubated at 32 ?C. Duringthe course of germination, GA₃ promoted marked increases inthe activities of the glyoxysomal marker enzyme isocitrate lyaseand certain mitochondrial marker enzymes, but did not affectthe ER marker enzyme choline phosphotransferase. Glyoxysomaland ER protein and phospholipid were not increased in amountby GA₃, whereas mitochondrial protein and phospholipid were.SDS-polyacrylamide gels of the glyoxysomal matrix polypeptidesfrom GA₃-treated beans exhibited two polypeptides additionalto those found to be common to both GA₃-treated and controltissue. Incorporation of CDP-(methyl ¹⁴C)-choline into intactendosperm tissue and the distribution amongst the glyoxysomes,mitochondria, and ER of newly synthesized phosphatidyl-(methyl¹⁴C)-choline was unchanged by GA₃.     

The action of cyclic-AMP on GA3 controlled responses V. Similarities in the induction of isocitrate lyase in hazel seeds by gibberellic acid and cyclic 3',5'-adenosine monophosphate

Cyclic-AMP was found to promote the induction of isocitratelyase in hazel seeds. Similarities in the induction of thisenzyme by GA₃ and cyclic-AMP were found in the (1) total amountof enzyme produced, (2) the time course for the production ofthis enzyme, (3) the amount of enzyme secreted into the medium,and (4) the inhibition of enzyme production induced by ABA,cycloheximide and 6-methyl purine. Of a variety of nucleotidestested only cyclic-AMP consistently promoted high levels ofisocitrate lyase. Cyclic-AMP also promoted a similar increase in the fresh weightof the embryo to that obtained with GA₃ treated seeds. Thisinitial study is suggestive of the possibility that cyclic-AMPmay be involved in GA₃ mediated responses in hazel seeds. (Received May 4, 1973; )     

NaCl-and Gibberellic Acid-induced Changes in the Content of Auxin and the Activities of Cellulase and Pectin Lyase During Leaf Growth in Rice (Oryza sativa)

The interactive effect of NaCl salinity and gibberellic acidin the activities of cellulase and pectin lyase, and on thecontent of auxin and chlorophyll, has been determined duringleaf growth (fifth from base) in rice. The linear growth, chlorophyllcontent, activity of cellulase, and the auxin level of leaveswere markedly decreased when plants were exposed to salt stress(12 dS m^–1). However pectin lyase activity did not registerany significant alteration in the leaves of salt-stressed plantscompared with the control. Treatment of plants with gibberellicacid (GA₃) (10 ppm) increased the leaf growth and chlorophyllcontent with a concomitant rise in the activity of cellulaseunder stressed as well as non-stressed conditions. A markedincrease in the content of auxin was discernible in the leavesof salt-stressed plants treated with GA₃ compared with non-treatedsalinized ones. An appreciable increment in the activity ofpectin lyase in response to GA₃ administration was detectedonly in the leaves of non-stressed plants. These results indicatethat enhancement of cellulase activity and the augmentationof endogenous auxin content may be involved in the stimulationof rice leaf growth by GA₃ under saline conditions. Oryza sativa, rice, leaf growth, NaCl salinity, gibberellic acid, cellulase, pectin lyase, auxin     

Gibberellin-Induced Changes in the Water Absorption, Osmotic Potential and Starch Content of Cucumber Hypocotyls

The effects of GA₃ on the water absorption, osmotic potentialand starch content of light-grown cucumber (cv. Aonagajibai)hypocotyl sections were examined. GA₃ (100µM.) stimulatedfresh weight increase as well as elongation. It had no effecton the dry weight change in the presence, or absence, of 50mM sucrose although dry weight increased significantly in thepresence of sucrose. In the absence of sucrose the weight wasunchanged. The osmotic potential of the epidermal cells of sectionsincubated with GA₃ was lower than that of the control, and thedifference between the two values was larger in young seedlings.When sections were incubated with sucrose, the osmotic potentialgreatly decreased. This decrease was more marked in the presenceof GA₃. GA₃ reduced the starch content of the sections bothin the presence and absence of sucrose. The total amount ofstarch, however, was markedly increased in its presence. Thedegradation of starch formed in advance from exogenous sucrosein light was not accelerated by GA₃. We discuss a possible rolefor gibberellin in cell elongation, based on our results, interms of cell water relations. (Received January 18, 1983; Accepted July 19, 1983)     

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; )     

The action of cyclic-AMP on GA3 controlled responses III. Characteristics of barley endosperm acid phosphatase induction by gibberellic acid and cyclic 3', 5'-adenosine monophosphate

Studies on the induction of barley endosperm acid phosphataseby GA₃ and cyclic-AMP revealed (1) acid phosphatase inductionby GA₃ and cyclic-AMP is inhibited in a similar manner by ABA,cycloheximide and 6-methyl purine, (2) combinations of GA₃ andphosphodiesterase inhibitors such as caffeine and theophyllinepromoted the level of acid phosphatase induction obtainablewith the hormone alone, and (3) cyclic-AMP and GA₃ may be actingas triggers in the induction of this enzyme. Some of the implicationsof these results are discussed. (Received May 25, 1972; )     

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)     

Effect of GA3, GA4$7, GA5 and GA9 on the sex expression of Luffa acutangula var. H-2

Gibberellins A₃, A_4$7, A₅, and A₉ increase the number of malebuds and flowers, but inhibit the opening of female flowersand shift their position to a higher node in Luffa acutangula.GA₃ is the most effective followed by GA_4$7, GA₅ and GA₉. (Received November 19, 1971; )     

Interaction of Photoperiod, Chilling and Exogenous Gibberellic Acid on Growth of Strawberry Petioles

Gibberellic acid (GA₃) was applied to strawberry plants in varyingdoses. Lengths of successively emergent petioles increased ina manner indicating that they were responsive to exogenous GA₃during both pre- and especially postemergence phases of ontogeny.Exogenous GA₃ interacted with photoperiod and chilling, but,because the responses were complicated by competition from aberrantstem elongation, GA₃ could not be shown to replace the endogenousstimulus completely, although it may do so in part. On the otherhand, a large component of the endogenous stimulus which promotesgrowth in long daylength and after chilling was not replacedby exogenous GA₃. This is interpreted as evidence for non-gibberellinfactors in the endogenous system. It appears that the stimuli generated by long daylength andby chilling are different, but the evidence is against the suggestionthat one mediates the gibberellin system and the other the non-gibberellinone.     

Gibberellin-Induced Suppression of Chloroplast Starch Formation from Exogenous Sucrose in Isolated Epidermis of Light-Grown Cucumber Hypocotyls

The effects of gibberellin A₃(GA₃) on chloroplast starch formationfrom exogenous sucrose in epidermal strips of light-grown cucumber(Cucumis sativus L. cv. Aonagajibai) hypocotyls were studied.Chloroplast starch formation was measured by counting microscopicallythe number of I₂KI stained chloroplasts. In the presence ofsucrose (1–50 mM) and in light, chloroplast starch formationoccurred rapidly for 5 hr and then the rate declined. GA₃ (100µM) simultaneously supplied with sucrose clearly suppressedboth the rate and the degree of starch formation. When the epidermiswas preincubated in the dark with sucrose in the presence orabsence of GA₃ and then post-incubated in light with bufferonly in the presence or absence of GA₃, starch formation wassuppressed to the same degree by GA₃ given either in the pre-or post-incubation period. When the epidermis was preincubatedin the dark with GA₃ alone and then transferred to light forpost-incubation with sucrose alone, GA₃ suppressed the initialrate of starch formation during the post-incubation period.The degradation of chloroplast starch formed in advance fromexogenous sucrose in light was not significantly affected byGA₃ These results are discussed in relation to the mechanismof the GA₃ action and also to the GA₃-induced decrease in theosmotic potential of the cell. (Received February 2, 1982; Accepted May 31, 1982)     

Effects Produced on Tomato Plants, Lycopersicum esculentum, by Seed or Root Treatment with Gibberellic Acid and Indol-3yl-Acetic Acid

Growth in lengths of tomato stems and leaves was acceleratedby 5.0 µg gibberellic acid (GA₂) applied to the seed,or by 5.0, 0.5, and 0.05 µg given to the roots. Treatmentwith 5.0 µg also decreased bud number and lengthened thetime between bud appearance and fruit formation on the firsttruss by 1–8 days. Smaller amounts applied to roots shortenedthis time by 1–4 days. Indol-3yl-acetic acid at 0.5 µghad no effect, nor was simultaneous application of GA₃ and IAAto the roots more effective than GA₃, alone. Single applicationsof very small amounts of GA₃ to seeds or seedling roots thusproved capable of changing growth-rates of stems, leaves, andtrusses.The effects of treating tomatoes with GA₂ and with culturesof Azotobacter chroococcum, which contain small amounts of GA₃,and IAA, are compared.     

The action of cyclic-AMP on GA3 controlled responses II. Similarities in the induction of barley endosperm ATPase activity by gibberellic acid and cyclic 3',5'-adenosine monophosphate

The effect of cyclic-AMP and GA3 on ATPase production in embryolessbarley half-seeds was examined. Similarities in the responseinduced by 5 mM cyclic-AMP and GA₃ were observed in (1) thetotal amount of ATPase produced, (2) the time course for therelease of ATPase, and (3) the effect of abscisic acid on ATPaseinduction by these compounds. Of a variety of adenine nucleotidesexamined only cyclic-AMP was found to promote high levels ofATPase activity. Some of the implications of these results arediscussed. (Received May 11, 1971; )     

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     

Effect of Gibberellic Acid and Ethylene on Peroxidase in Pea Internodes

Ethylene at 5–80 µl l^–1 inhibited elongationand induced swelling in internodes of light-grown normal anddwarf pea plants; GA₃ did not prevent swelling in response toethylene. GA₃ neither inhibited nor enhanced the activity of isoperoxidasesin the internodes, regardless of its effect on their elongation.Ethylene at 80 µl l^–1 enhanced peroxidase in GA₃-untreatedand treated normal and dwarf plants. At 5 µl l^–1,ethylene had only a weak effect on peroxidase activity or none.The enzyme enhancement by ethylene was not related to its effecton cell expansion and seems do be due, at least in part, tochemical injury. Electron microscopy revealed peroxidase activity in the roughER and cell walls, including intercellular spaces. Stainingof walls in ethylene-treated tissues was more pronounced thanin untreated ones. Golgi vesicles did not seem to be involvedin the assembly of the enzyme carbohydrate moiety in ethylene-treatedcells. The peroxidase fraction extracted with 20 mM phosphate buffer,pH 6, and that extracted from wall debris with 1 M NaCl accountedfor 98% of total enzyme activity. Both fractions contained thesame six cathodic isoforms which comprised 85–90% of theiractivity. Electrophoresis did not reveal differences in thequalitative isoenzyme patterns in relation to variety, age,GA₃, or ethylene. The only observed quantitative differenceswere age-dependent. Procedural artefacts during separation of protoplast and wallionically bound peroxidase fractions are discussed.     

Effects of Gibberellic Acid on Floral Development in vivo and in vitro in the Cleistogamous Species, Lamium amplexicaule L.

Using established developmental markers in the corolla and androeciumof Lamium amplexicaule L., we investigated the apparent inductionof open (chasmogamous — CH) from closed (cleistogamous— CL) flowers after application of GA₃ (0.1 mM). In vivotreatment of potentially CL flower primordia caused cell expansionbut not the increased cell division in anthers and corolla necessaryto convert a CL into a CH flower. Floral primordia that appearedto be of undetermined floral type were grown in vitro on a basalmedium supplemented with kinetin (0.1 p.p.m.) and grew to maturityas CL flowers. On media additionally supplemented with GA₃,flowers underwent anthesis, but no true CH flower was produced.Gibberellins appear to be directly responsible for anthesisin the CH flower; but additional, as yet unknown growth factorsare involved in the switch from CL to CH floral form in a developinginflorescence. floral morphogenesis, Lamium amplexicaule L, henbit, cleistogamy, gibberellin, kinetin, anthesis     

Effects of a Plant Growth Regulator, Prohexadione Calcium (BX-112), on the Elongation of Rice Shoots Caused by Exogenously Applied Gibberellins and Helminthosporol, Part II

Prohexadione calcium (BX-112) is a novel plant growth regulatorthat inhibits the late stages of the biosynthesis of gibberellinsin plants. Fourteen kinds of gjbberellin, helminthosporol and'helminthosporic acid were applied simultaneously with BX-112to rice seedlings (Oryza sativa L. ), and their growth-promotingactivities in terms of shoot elongation were examined. The growth-promotingactivities of GA₁, GA₄, GA₁₈, GA₂₂, GA₂₃, GA₃₈, helminthosporoland helminthosporic acid were not inhibited by BX-112, but thoseof GA₅, GA₉, GA₁₅, GA₁₉, GA₂₀, GA₃₁, GA₄₄ and GA₅₃ were inhibited.These results suggest that 3ß-hydroxylation is animportant and necessary step in the biosynthesis of gibberellinsthat promote shoot elongation in rice seedlings. The weak promotionof shoot elongation by GA₂₂ in the presence of BX-112 suggeststhat the effect of a hydroxyl group at C-18 of GA₂₂ might beable to mimic the effect of the 3ß-hydroxyl groupof GA₁. Helminthosporol and helminthosporic acid may promotethe shoot elongation of rice by mimicking physiologically activegibberellins and not by stimulating their biosynthesis. ¹Part I is the previous paper by Nakayama et al. (1990a) ³Present address: Frontier Research Program RIKEN, Wako-shi,Saitama, 351-01 Japan. (Received June 26, 1991; Accepted September 4, 1991)     

The Relationship between Levels of Endogenous Gibberellins and the Response of Vigna Hypocotyls to Exogenous Indole-3-Acetic Acid

Segments excised from the upper and the lower parts of cowpea(Vigna unguiculata L.) hypocotyls were compared in terms oftheir responses to exogenous indole-3-acetic acid (IAA) in relationto their endogenous levels of gibberellin. Growth of the segmentswas measured continuously during xylem perfusion with a lineardifferential transformer. IAA induced a burst of elongationin the upper segments but only slight promotion of growth inthe lower segments. Treatment with uniconazole, a potent inhibitorof the biosynthesis of gibberellins, reduced the responsivenessof the upper segments to exogenous IAA to about one half ofthe control value. Pre-perfusion with GA₃ of such segments fortwo hours prior to application of IAA, partially restored theresponsiveness to IAA. Analysis by GC/MS identified GA₁, GA₄,GA₉ GA₂₀ and GA₅₁ as native gibberellins in the hypocotyls ofcowpea seedlings. Analysis by GC/SIM also showed that the physiologicallyactive gibberellins (GA₁ and GA₄) were located mainly in theupper part of the hypocotyl and the treatment with uniconazolemarketly reduced the endogenous level of gibberellins thereto less than 11% of the control level. These results suggestthat levels of endogenous gibberellins possibly control theresponse to IAA in these segments. (Received May 12, 1994; Accepted November 15, 1994)     

Unusual Branching in the Seedlings of Lotus japonicus--Gibberellins Reveal the Nitrogen-sensitive Cell Divisions within the Pericycle on Roots

We studied the effects of several plant-growth regulators onthe induction of nodule-like structures on roots of Lotus japonicus,which has been proposed as a candidate for a leguminous plantfor molecular genetic analysis. Contrary to our expectations,the addition of gibberellin A₃ (GA₃) at concentrations of 10^-4M to 10^-4 M resulted in the formation of nodule-like structureson roots when seedlings were plated on nitrogen-free Fahraeusagar medium. GA₄ also induced such outgrowths but was less activethan GA₃. Application of an inhibitor of auxin transport, N-(1-naphthyl)-phthalamicacid (NPA) and of kinetin, which have been reported to inducepseudonodules in other legumes, had no effect on L. japonicus.Microscopic observations showed that GA₃-induced nodule-likestructures were caused by cell divisions within the pericycleon the roots. In addition, the outgrowths elicited by GA₃ couldbe completely suppressed by the addition of 15 mM potassiumnitrate or ammonium nitrate. These results show that the pericyclecells of the roots of L. japonicus are specifically sensitiveto gibberellins and that potential for cell division might bemodulated by nitrogen compounds. We also examined the effectsof ancymidol and uniconazole [S-3307; (E)-1-(4-chIorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol],two synthetic plant-growth retardants. Both compounds at 3 x10^-5 M significantly increased the number of stunted lateralroots. The unusual branching could not be counteracted by theexogenous addition of GA₃ but by 10^-6 M brassinolide. We discussthe physiological role of brassinolide in the initiation oflateral roots. (Received August 4, 1995; Accepted March 11, 1996)     

Gibberellin-Induced Changes in Starch Content in Isolated Chloroplasts of Light-Grown Cucumber Hypocotyls

Gibberellin A₃ (GA₃) was found to reduce the starch contentin chloroplasts isolated from light-grown cucumber (cv. Aonagajibai)hypocotyls. Chloroplasts incubated with 50 mM sucrose showedan increase in starch content. The GA₃-induced reduction occurredboth in the presence and absence of exogenous sucrose. GA₃ hadno effect on the level of starch already formed from exogenoussucrose. (Received April 4, 1984; Accepted June 7, 1984)