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     

Biphasic response of cucumber hypocotyl sections to auxin

The pattern of the response to auxin of cucumber hypocotyl sections,its relation to acid-induced growth and the role of the epidermiswere investigated. In longterm incubation, IAA had practicallyno effect on sections without epidermis (peeled sections). Timecourse measurementsshowed that the response to IAA of nonpeeledsections was biphasic; the first phase started right after IAAtreatment and continued for about 1 hr, then after a periodof 1 hr with a greatly decreased growth rate the second phasestarted with a higher rate. Peeled sections lacked thesecondphase; their growth rate in the first phase depended on theacidity of the incubation medium. These results were confirmedby utilizing one side-peeled sections and measuring the curvatureof the sections which represented the difference in the growthrate between the peeled and nonpeeled sides. The following pointswere suggested. The first phase resembles acid-induced growthand the growth capacity is limited by the epidermis; the secondphase is specific to auxin and requires the presence of theepidermis. The first phase was hardly influenced by temperature,while the second one was greatly affected; its Q,₁₀ being morethan 4.0, suggesting that the first phase is of physical nature. (Received December 26, 1975; )     

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     

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.     

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10⁻⁹ M of the gibberellin GA₄₊₇, whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA₄₊₇ has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA₄₊₇ than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process. Received: 27 February 1999 / Accepted: 21 May 1999     

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.     

The role of light in suppressing hypocotyl elongation in lettuce and Petunia

Summary Hypocotyl elongation in two varieties of Petunia and in Grand Rapids lettuce is shown to be affected by a high-energy reaction and by phytochrome action. These two photoreactions interact in such a way that, on the one hand, shortening of the hypocotyls due to the high-energy reaction can be entirely masked by brief terminal far-red light treatment, while on the other hand, there is no evidence of phytochrome action unless brief exposures to red light are preceded by relatively long exposure of high-intensity.The action spectra for the high-energy reaction show peak effectiveness at wavelengths of 430–450 m, with a minor peak at 660 m in Comanche Petunia, at 700 m in Pink Cascade Petunia, and at 720 m in Grand Rapids lettuce.Prior treatment with DCMU did not reduce the effect of high-intensity light on hypocotyl lengths in lettuce.The nature of the high-energy reaction, and the relation between it and phytochrome action are discussed. Besides these two photoreactions there appears to be a direct effect of light on elongation, blue light preventing, and far-red light accelerating, elongation during actual exposure.With 9 Figures in the Text     

Phytochrome-induced synthesis of ribonuclease de novo in lupin hypocotyl sections

1. Density-labelling with 99 atoms% of (2)H(2)O distinguished pre-existing from newly synthesized ribonuclease molecules in sections of developing hypocotyl tissue. 2. Activity profiles of enzyme extracted from the fraction pelletable at 100000g showed heterogeneity after isopycnic centrifugation in CsCl gradients. 3. Measurement of density shifts of the entire heterogeneous band shows that ribonuclease protein is synthesized de novo in both continuous far-red light and darkness. 4. A twofold increase in enzyme activity after irradiation was accompanied by band-broadening and a significantly faster rate of labelling than in darkness. 5. The conclusion is drawn from the experimental evidence and theoretical arguments presented that phytochrome regulates the synthesis of new enzyme molecules against a background of continuous (dark-rate) synthesis and degradation. 6. Further information has been deposited as Supplementary Publication SUP 50033 (3 pages) at the British Library Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

Regulation of sterol biosynthesis in etiolated mung bean hypocotyl sections

The incorporation of radioactivity into sterols by transmethylation of methionine-[¹⁴C-methyl] was studied in mung bean hypocotyl sections. Young hypocotyl sections (1 cm) synthesized 4 times more radioactive sterols than older sections (5 cm). The transmethylation reactions may be rate limiting in older tissues. Wounding has only a quantitative effect on sterol biosynthesis, as seen by incorporation experiments with MVA-[2-¹⁴C]. Naphthalene acetic acid (NAA) stimulates sterol biosynthesis in both wounded surfaces and intact tissues of mung bean hypocotyl sections.     

Phytochrome and potassium uptake by mung bean hypocotyl sections

Uptake of potassium (K) and ⁸⁶rubidiumlabelled potassium (⁸⁶Rb) by sub-hypocotyl hook sections of Phaseolus aureus L. was inhibited by red light. The effect was reversible with far red light. Using short exposures of high irradiance the effect on ⁸⁶Rb-labelled K uptake was observed after 5 min. The response showed no specificity for a particular anion. Uptake of ⁸⁶Rb-labelled K by sections cut immediately below the cotyledons was enhanced by red light after 10 min incubation and was also far red reversible. These results are interpreted as a rapid phytochrome-induced change in membrane properties resulting in modified K uptake.Abbreviations P Phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - R red light - F far red light     

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; )     

An improved cytokinin bioassay using cultured soybean hypocotyl sections

This paper describes a modified soybean (Glycine max) tissue culture bioassay for cytokinins. Soybean hypocotyls were grown under sterile conditions and sliced into 1-mm sections. Sections were cultured for 5,9,13, or 22 days on a callus medium with zeatin or other cytokinins. The fresh weight of sections increased with the cytokinin concentration from 0.0005 to 1 mum zeatin; 2-fold concentration differences were readily distinguishable at 9 days. The assay should prove to have several advantages over the conventional soybean callus bioassay including convenience, lower variability between tissue samples, and improved resolution. Its specificity is comparable to that of the soybean callus bioassay.     

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; )     

Kinetics of indole acetic acid-induced growth in hypocotyl sections of Vigna mdiata

The kinetics of auxin-induced elongation of segments from Vigna radiata (L.) Wilczek hypocotyls have been investigated using auxanometer measurements. Doseresponse curves were established for several well-defined parameters of the growth response. The experimental data revealed that different kinetic parameters were affected differently by increasing auxin concentrations. The dose-response curves are slightly sigmoid for fresh weight, nearly bell-shaped for total elongation or maximal elongation rate, and nearly linear for maximal growth acceleration. The effects of auxin concentration on regulation of growth orientation are discussed.
A biphasic response is observed mainly with segments taken from the middle of the hypocotyl ('C') which exhibit maximal growth rates. Segments from other levels, with lesser growth potentials, exhibit a very weak response. The two successive phases may then require different maturation states. Kinetics of the acceleration curves are very similar all along the hypocotyl and remain very homogeneous with increasing IAA concentrations.     

A proteinaceous inhibitor of ethylene biosynthesis by etiolated mungbean hypocotyl sections

Summary A protein which reversibly inhibits auxin-induced ethylene synthesis has been isolated and purified from hypocotyls of etiolated mungbean (Phaseolus aureus Roxb.) seedlings. The molecular weight of the inhibitor was estimated to be 112 000 by gel filtration and polyacrylamide gel-electrophoresis. When treated with sodium dodecylsulfate, the inhibitor gave on polyacrylamide gel-electrophoresis a single band corresponding to a molecular weight of 56 000, indicating that it consisted of two subunits with identical molecular weight. The inhibitor does not degrade nor bind indole-3-acetic acid, and has no peroxidase activity.