The role of acidification in gibberellic acid- and fusicoccin-induced elongation growth of lettuce hypocotyl sections

The roles of gibberellic acid (GA₃) and fusicoccin (FC) in the elongation growth and acidification of the medium by excised hypocotyl sections of lettuce (Lactuca sativa L.) were investigated. Hypocotyl sections incubated in buffer without GA₃ elongate optimally at pH 4.0–4.25 while sections incubated with GA₃ show the same growth between pH 4.25 and 6.0. Preincubation of sections at pH 6.0 for 6 h does not affect the subsequent elongation response to acidic medium (pH 4.25); however, the sections become refractory to further acid treatment after their initial burst of growth in response to pH 4.25. Sections made refractory to acid are responsive to GA₃ application, however, and the rate of growth in response to GA₃ of sections pretreated for 6 h at pH 4.25 is 85% of that of sections pretreated at pH 6.0. Although preincubation of sections for 48 h in medium at pH 6.0 abolishes the GA₃ response, it does not affect the response to buffer at pH 4.25. FC stimulates elongation growth in letuce hypocotyls at an optimal concentration of 1 M, and pretreatment of sections at pH 4.25 does not affect this elongation response. Although both GA₃ and FC increase elongation of the section, neither causes appreciable acidification of the medium. Addition of KCl or NaCl to FC-treated sections causes rapid medium acidification but addition of salts to GA₃-treated tissue does not cause acidification. Abrasion of the hypocotyl to remove the cuticle does not enhance acidification of the medium by the sections nor deos it affect elongation of the sections in response to GA₃ or FC. Medium acidification by the sections is not a passive process since it is abolished both by low temperature (2° C) and metabolic inhibitors (carbonyl cyanide-m-chlorophenyl-hydrazone, azide). The acidification of the medium by barley (Hordeum vulgare L.) roots in response to FC is also dependent on the presence of KCl. We conclude that the acid-growth hypothesis does not explain GA₃- or FC-induced elongation in lettuce hypocotyls.Abbreviations FC tusicoccin - GA₃ gibberellic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - MES 2-(N-morpholino)ethanesulphonic acid - Tris tris-(hydroxymethyl)aminomethane     

Calcium and gibberellin-induced elongation of lettuce hypocotyl sections

The relationship between calcium ions and gibberellic acid (GA₃)-induced growth in the excised hypocotyl of lettuce (Lactuca sativa L.) was investigated. The short-term kinetics of growth responses were measured using a linear displacement transducer. Test solutions were added either as drops to the filter paper on which the hypocotyl stood (non-flow-past) or by switching solution flowing past the base of hypocotyl (flow-past), resulting in differences in growth behavior. Drops of CaCl₂ added at a high concentration (10 mM) inhibited growth within a few minutes. This inhibition was reversed by ethylenediaminetetraacetic acid (EDTA). Drops of EDTA or ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid caused a rapid increase in growth rate. Growth induced by EDTA was not further promoted by GA₃. A continuous H₂O flow resulted in growth rates comparable to those in response to GA₃. Addition of CaCl₂ to the flow-past medium inhibited growth and this inhibition was reversed by a decrease in CaCl₂ concentration. The growth rate was found to be a function of CaCl₂ concentration. When a constant CaCl₂ concentration was maintained by the flow-past medium, a shift in pH from 5.5 to 4.25 had no obvious effect on hypocotyl elongation. Gibberellic acid was found to reverse the inhibitory effect of CaCl₂, causing an increase in growth rate similar to that found previously when GA₃ was added to hypocotyls grown in H₂O under non-flow-past conditions. We propose that gibberellin controls extension growth in lettuce hypocotyl sections by regulating the uptake of Ca²⁺ by the hypocotyl cells.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid - GA gibberellin - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

Short-term kinetics of elongation growth of gibberellin-responsive lettuce hypocotyl sections

The short-term kinetics of growth of the excised lettuce (Lactuca sativa L.) hypocotyl were characterized with respect to the effects of gibberellic acid (GA₃), indole-3-acetic acid (IAA), KCl and pH. A Hall-device-based, miniaturized, linear displacement transducer was developed to measure the growth of 2-mm hypocotyl sections with 1-m resolution. Following treatment with GA₃, a lag time of less than 10 min was typically followed by an increase in growth rate with two acceleration phases, reaching a final elevated rate within about 1 h. The kinetics of the response to GA₁, a mixture of GA₄ and GA₇, and GA₉ were similar to the response to GA₃. There was no response to IAA treatment either in the presence or absence of GA₃. KCl alone had no effect on the growth rate, but caused an increase in rate when added after GA₃, with a lag time of usually less than 1 h. Responses to pH changes had lag times of a few minutes in all cases. A shift from H₂O to pH 6 buffer inhibited growth, while a shift from H₂O to pH 4 buffer resulted in a transient increase to a rate comparable to that induced by GA₃. A shift from pH 6 to pH 5 caused an increase in growth rate, followed by a gradual decline to an H₂O control rate after more than an hour. The responses to GA₃ at pH 4 and pH 5 were similar to that found for addition of GA₃ to water controls.Abbreviations GA gibberellin - GA₃ gibberellic acid - GA₁, GA₄₊₇, GA₉ gibberellins A₁, A₄₊₇, A₉ - IAA indole-3-acetic acid     

Catecholamine stimulation of the gibberellin action that induces lettuce hypocotyl elongation

Effects of catecholamines and their derivatives on gibberellicacid (GA)-induced lettuce hypocotyl elongation was studied,because catecholamines have a chemical structure similar tothe dihydroconiferyl alcohol that has been isolated from lettucecotyledons as a GA synergist. Epinephrine, norepinephrine, dopamineand 3,4-dihydroxymandelic acid synergistically enhanced thepromoting effect of GA on hypocotyl elongation. In contrast,metanephrine, normetanephrine, DOPA and 3-methoxy-4- hydroxymandelicacid did not enhance the GA effect. The action of catecholamineswas inhibited by trans-cinnamic acid which competitively inhibitedthe action of dihydroconiferyl alcohol; this suggests that thereceptor site for catecholamines is the same as that for dihydroconiferylalcohol. The basic ethyl acetate fraction from lettuce seedlingssynergistically enhanced the GA effect. TLC analyses of thisbasic ethyl acetate fraction revealed that the chromatographicarea corresponding to authentic catecholamines could enhancethe GA effect. From these results, a possible role for catecholamines in theregulation of lettuce hypocotyl elongation caused by GA wasposited, and is discussed here. (Received May 15, 1979; )     

Identification in lettuce seedlings of a catecholamine active in synergistically enhancing the gibberellin effect on lettuce hypocotyl elongation

The occurrence of catecholamines in lettuce seedlings was examined by bioassay and gas chromatography-mass spectrometry (GC-MS), since synthetic catecholamines can synergistically enhance the stimulating effect of gibberellic acid (GA₃) on hypocotyl elongation of decotylized lettuce seedlings. The catecholamine fraction on TLC obtained from lettuce seedlings synergistically enhanced the GA₃ effect on hypocotyl elongation. The analysis of the catecholamine fraction from lettuce seedlings by GC-MS demonstrated the occurrence of dopamine.     

Isolation and chemical identification of a lettuce cotyledon factor, a synergist of the gibberellin action in inducing lettuce hypocotyl elongation

Removal of cotyledons from intact lettuce seedlings retardedhypocotyle elongation caused by gibberellic acid. This inhibitoryeffect of cotyledon excision was alleviated by incubating decotylizedseedlings with a hot water extarct from excised cotyledons orintact seedlings. This active principle was identified as dihydroconiferylalcohol on the basis of its IR, NMR and mass spectra. (Received August 28, 1973; )     

Effects of colchicine and lumicolchicine on hypocotyl elongation,respiration rates and microtubules in gibberellic-acid-treated lettuce seedlings

Gibberellic-acid (GA₃)-induced hypocotyl elongation of intact lettuce (Lactuca sativa L.) seedlings was inhibited by colchicine (4×10^-4 M) but not by lumicolchicine (4×10^-4 M). In excised lettuce hypocotyls, GA₃ (10^-5 M) increased respiration over water controls, while both colchicine and lumicolchicine alone, or in combination with GA₃, reduced respiration. Microtubules were present in the hypocotyls of lumicolchicine-treated seedlings but absent in those treated with colchicine. It is suggested that lumicolchicine is a useful drug to discriminate between the metabolic and microtubule-mediated processes in cell morphogenesis.     

Effects of light on auxin-induced elongation of light-grown cucumber hypocotyl sections

The effect of light on IAA-induced elongation of light-growncucumber hypocotyl sections was examined. There was no differencein the optimal concentration of IAA between light and darknessand the biphasic pattern of IAA-induced elongation also wasobtained in both states. Elongation of the first phase was notaffected by light, but that of the second phase was influencedby the light condition of the first phase; dark incubation forthe first 3 hr resulted in a larger IAA-induced elongation inthe second phase. For the maximum IAA-induced elongation, atleast 3 hr of light exposure in the second phase was necessary.Simultaneously applied photosynthetic inhibitors, DCMU, CMUand o-phenanthroline, synergistically enhanced IAA-induced elongationin light, but not in the dark. They were not effective whenIAA treatment preceded the treatment with them. (Received April 3, 1978; )     

Gibberellin response in lettuce hypocotyl sections

Excised lettuce (Lactuca sativa L.) hypocotyl sections retain the ability to elongate in response to gibberellic acid (GA₃) addition. In 48 hr at 30 C a GA₃-treated segment more than doubles while a control segment elongates less than 50%. Auxin has no detectable effect on this system. Sensitivity to GA₃ is not decreased by apex or root removal. Of the experimental variables tested, temperature, sucrose, and preincubation in water affect growth both with and without GA₃. Blue and far red light inhibit growth without GA₃; this inhibition is reversed by GA₃. Potassium chloride stimulates growth of illuminated sections treated with GA₃ but has no effect on control growth. When sections are incubated in the dark, KCl has a promotive effect on elongation.     

A study of gibberellin homeostasis and cryptochrome-mediated blue light inhibition of hypocotyl elongation

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA(4) in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA(4) content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.     

Gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature interaction

Soybean is an important oilseed crop grown globally. However, two examples of environmental stresses that drastically regulate soybean growth are low light and high-temperature. Emerging evidence suggests a possible interconnection between these two environmental stimuli. Low light and high-temperature as individual factors have been reported to regulate plant hypocotyl elongation. However, their interactive signal effect on soybean growth and development remains largely unclear. Here, we report that gibberellins (GAs) and auxin are required for soybean hypocotyl elongation under low light and high-temperature interaction. Our analysis indicated that low light and high-temperature interaction enhanced the regulation of soybean hypocotyl elongation and that the endogenous GA₃, GA₇, indole-3-acetic acid (IAA), and indole-3-pyruvate (IPA) contents significantly increased. Again, analysis of the effect of exogenous phytohormones and biosynthesis inhibitors treatments showed that exogenous GA, IAA, and paclobutrazol (PAC), 2, 3, 5,-triiodobenzoic acid (TIBA) treatments significantly regulated soybean seedlings growth under low light and high-temperature interaction. Further qRT-PCR analysis showed that the expression level of GA biosynthesis pathway genes (GmGA3ox1, GmGA3ox2 and GmGA3) and auxin biosynthesis pathway genes (GmYUCCA3, GmYUCCA5 and GmYUCCA7) significantly increased under (i) low light and high-temperature interaction and (ii) exogenous GA and IAA treatments. Altogether, these observations support the hypothesis that gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature stress interaction.     

Dihydroconiferyl alcohol as a gibberellin synergist in inducing lettuce hypocotyl elongation. An assessment of structure-activity relationships

Structure-activity relationships of the cotyledon factor wereexamined by testing the effect of various substances structurallyrelated to the cotyledon factor (dihydroconiferyl alcohol) ongibberellin-induced lettuce hypocotyl elongation. The biological activity of the cotyledon factor, 3-(4-hydroxy-3-methoxyphenyl)propan-1-ol, disappeared if the phenolic hydroxy group was maskedwith a methoxy or glucosyl group. Oxidation of the alcoholicgroup in the side chain to a carboxylic group decreased thebiological activity of the cotyledon factor. As to relationshipsbetween the biological activity and length of the alkyl sidechain, the propane type was found to be much more active thanthe methane, ethane or butane type. The presence of a C = Cbond in the alkyl side chain made the cotyledon factor biologicallyinactive. Some antioxidants of indole-3-acetic acid were alsoassayed for cotyledon factor-like activity, since the cotyledonfactor is a polyphenol. However, known antioxidants such asrutin, pyrocatechol, chlorogenic acid, caffeic acid and ferulicacid did not show cotyledon factor-like activity. From these results, structural requirements of the cotyledonfactor as a gibberellin synergist were discussed. (Received June 17, 1975; )     

Effects of cyclic AMP and gibberellic acid on lettuce hypocotyl elongation and mechanical properties of its cell wall

Growth responses of lettuce seedlings to cyclic AMP and gibberellicacid were studied. Both cyclic AMP and sodium dibutyryl cyclicAMP significantly stimulated hypocotyl elongation, althoughelongation by cyclic AMP was smaller than that by gibberellicacid. Substances related to cyclic AMP, i.e. 5'-AMP, 5'-ADPand 5'-ATP, had no growth promoting effects. Examination of mechanical properties of the cell wall by a stress-relaxationtechnique indicated that both cyclic AMP and gibberellic acidcaused cell wall loosening when they stimulated hypocotyl elongation. (Received September 30, 1971; )     

ZEITLUPE positively regulates hypocotyl elongation at warm temperature under light in Arabidopsis thaliana

Hypocotyl cell elongation has been studied as a model to understand how cellular expansion contributes to plant organ growth. Hypocotyl elongation is affected by multiple environmental factors, including light quantity and light quality. Red light inhibits hypocotyl growth via the phytochrome signaling pathways. Proteins of the FLAVIN-BINDING KELCH REPEAT F-BOX 1 / LOV KELCH PROTEIN 2 / ZEITLUPE family are positive regulators of hypocotyl elongation under red light in Arabidopsis. These proteins were suggested to reduce phytochrome-mediated inhibition of hypocotyl elongation. Here, we show that ZEITLUPE also functions as a positive regulator in warmth-induced hypocotyl elongation under light in Arabidopsis.     

Photosensory mechanisms in the lettuce seedling hypocotyl

Summary A number of differences in the responses of Great Lakes lettuce seedlings to blue and far-red light indicate that more than one photo-sensitive pigment is involved in the photo-inhibition of hypocotyl elongation under highenergy conditions. In far-red light the inhibitory effect is restricted to young seedlings and is of limited duration; after 24 hours in far-red a rapid growth rate similar to that of plants maintained in darkness is resumed, despite continued irradiation. The onset of inhibition is relatively slow. Blue light, in contrast, exerts a strongly inhibitory effect on elongation at any age, and a slow rate of growth persists throughout the entire irradiation period. The onset of inhibition is very rapid. Furthermore, even when the inhibition in far-red had already been exhausted after prolonged exposure, transfer to blue light resulted in a prompt reduction in growth rate. Also the effect of far-red is almost completely lost after a pre-irradiation with red light which does not affect the response to blue. It is concluded that the responses to blue and far-red light in Great Lakes lettuce are not mediated by a single pigment system and that a distinct blue-sensitive pigment is present in addition to phytochrome. Red light has a number of different effects depending on conditions: (1) a pretreatment with red light almost completely prevents the inhibitory effect of a subsequent far-red irradiation, (2) a brief terminal treatment with red increases the inhibitory effect of either far-red or blue light; this is reversed by far-red, and (3) prolonged exposure to red light given alone increases the growth rate relative to darkness, because the more rapid elongation rate characteristic of young seedlings continues for longer with red light than in plants grown in darkness throughout.     

Hormone-solute interactions in the lettuce hypocotyl hook

The hypocotyl hook of the lettuce (Lactuca sativa cv. Grand Rapids) seedling is stimulated to a high degree of curvature through a synergistic interaction of ethylene and gibberellic acid in the light. Presentation of various inorganic salts to the seedlings caused extensive alteration of the hormone-induced curvatures, with ammonium and sulfate being the most stimulatory of curvature, and potassium and carbonate being the most inhibitory of curvature. Experiments using organic buffers indicated that the effect was not a pH response. The abilities of various cations and anions to alter the hormonally regulated curvature is suggested as further evidence of solute alteration of hormonal effectiveness. The interpretation is offered that the solutes may be influencing hormonal effectiveness through salting-in and salting-out effects on macro-molecules such as proteins.     

Anticotyledon factor: Competitive inhibitors of the action of dihydroconiferyl alcohol in stimulating gibberellic acid-induced lettuce hypocotyl elongation

The lettuce cotyledon factor, dihydroconiferyl alcohol, synergisticallyenhanced the stimulating effect of gibberellic acid (GA₃) onhypocotyl elongation of decotylized lettuce seedlings. The actionof dihydroconiferyl alcohol was inhibited by 3-(3,4-dimethoxy-phenyl)propan-l-ol, 3-(3-hydroxy-4-methoxyphenyl) propionic acid, methyl-p-methoxycinnamate,trans-cinnamic acid, p-coumaric acid, ferulic acid, caffeicacid, and synapic acid. Kinetic studies with Lineweaver-Burkplots indicated that these compounds were competitive inhibitorsof dihydroconiferyl alcohol. These inhibitors were termed anticotyledonfactors. The action of dihydroconiferyl alcohol was not influencedby phenylalanine, tyrosine, p-coumaryl alcohol and coniferylalcohol. (Received March 28, 1977; )     

Boron-regulated hypocotyl elongation is affected in Arabidopsis mutants with defects in light signalling pathways

We studied the effect of elevated boron (B) concentrations on the growth and development of Arabidopsis thaliana in vitro with respect to different light conditions. Two basic responses were observed. At high concentrations (above 5 mM) a clear toxicity effect of B on plant growth was apparent. Seedlings were short, stunted and pale. However at concentrations between 1 and 3 mM H₃BO₃, hypocotyl elongation was stimulated in all Arabidopsis ecotypes tested relative to plants grown at 0.1 mM H₃BO₃. The stimulation of hypocotyl elongation by elevated B was proportionally greater with increasing irradiance. We also showed that blue light (BL) and red light (RL) did not alter the sensitivity of Arabidopsis hypocotyls to boron, but, dependent on genotype, BL and RL increased or reduced capacity of boron-induced hypocotyl elongation. Analysis of photomorphogenic mutants indicated the existence of an interaction between boron and light signalling pathways during plant growth and development. This interaction was supported by the observation that the expression of the BOR1 gene in Arabidopsis hypocotyls was stimulated by BL and RL. Our results suggest that in etiolated or light-grown seedlings the stimulation of hypocotyl growth by boron can be mediated by cryptochromes and phytochromes.     

Regulation of lettuce hypocotyl elongation by gibberellic acid. Correlation between cell elongation, stress-relaxation properties of the cell wall and wall polysaccharide content

Lettuce hypocotyl elongation caused by gibberellic acid wasstrongly inhibited by coumarin and dichlobenil, known inhibitorsof cellulose biosyndiesis. Stress-relaxation analysis of thecell wall revealed that gibberellic acid induces a decreasein both minimum relaxation time (To) and relaxation rate (b)and an increase in maximum relaxation time (Tm), when gibberellicacid stimulates hypocotyl elongation. Both coumarin and dichlobenilnullified the effect of gibberellic acid on changes in To, Tmand b values. The content of pectic, hemicellulosic and cellulosic substancesin the cell wall increased per hypocotyl but decreased per unithypocotyl length, in response to gibberellic acid treatment.Particularly, gibberellic acid caused a substantial increasein cellulose content per hypocotyl but a decrease per unit length.A good correlation existed between the decrease in To and thedecrease in hemicellulose content per unit lengdi of the cellwall. The increase in Tm was correlated with the decrease incellulose content per unit length of the cell wall. The decreasein b was correlated with the decrease in the content of bothcellulose and hemicellulose per unit length. Based on these results, we discuss the role of polysaccharidemetabolism of the cell wall in gibberellic acid-induced lettucehypocotyl elongation and the nature of gibberellic acid-inducedbiochemical modifications of the cell wall, which are representedby changes in stress-relaxation properties of the cell wall. ¹Present address: Department of Anatomy, Aichi Medical University,Nagakutecho, Aichigun, Aichi 480-11, Japan. (Received September 22, 1975; )