Rapid Bioassay for Auxin

A rapid bioassay is described for the detection of growth substances. The bioassay is simple to perform under ordinary laboratory or classroom conditions and the sensitivity to auxin is comparable to other bioassays, including those using Avena first internode sections. The bioassay is performed on isolated first internode sections from partly etiolated bean plants (Phaseolus vulgaris L. cv. Bush Burpee Stringless Bush Bean). The test material is applied to one side and near the base of the section. Auxin treatments induce curvature and the distance of the lateral displacement of the apical portion of the segment is proportional to the concentration applied. Routinely, the indole 3-acetic acid response is measured 1 to 3 h after treatment. The sections also respond to gibberellic acid (GA₃), which induces an increase in overall length, but unlike auxin, GA₃ does not cause curvature. The test compounds are applied via a small filter paper disk that provides a convenient means for studying interactions of two or more compounds by co-applications. Auxin-induced growth can be monitored with a sensitive motion detector. Data are presented which confirm the usefulness of the assay.     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Indole-3-acetic Acid (IAA) and IAA Conjugates Applied to Bean Stem Sections: IAA Content and the Growth Response

High resolution growth recording techniques and reverse isotope dilution analysis were used to study the relationship between indole-3-acetic acid (IAA) concentration and curvature of excised bean (Phaseolus vulgaris L. cv Bush Burpee Stringless) first internode sections unilaterally treated with hormone. The maximum rate of curvature occurred rapidly (within 25 minutes) and was proportional to the log of the amount of applied IAA recovered in the tissue. The rate of curvature decreased after 30 minutes although little or no lateral migration of applied IAA occurred and tissue levels of IAA increased. The biologic activity of IAA-amino acid conjugates was found to be directly related to the amount of free IAA, resulting from their hydrolysis, which could be recovered from the tissue.     

THE RELATIONSHIP OF GIBBERELLIN AND AUXIN IN PLANT GROWTH

No synergism was found between IAA and gibberellin in the Avenucurvature test and this bioassay thus measures changes in diffusibleauxin resulting from gibberellin treatment and not a synergisticaction of the gibberellin on the curvature response to auxin.Gibberellin treatment causes an increase in diffusible auxinfrom the stem apex of dwarf pea (Pisum sativum L. var. LittleMarvel) 24 to 48 hours before the elongation response in thestem. The increase in diffusible auxin in the stem apex of Centaureacyanus L. var. Blue Boy occurs four to six days before the boltingresponse to gibberellin treatment under short days. The stemtissues of both the dwarf pea and Centaurea show an elongationresponse to IAA when the IAA is applied in a manner simulatingthe stem apex. Thus the growth of the dwarf pea and the boltingof Centaurea brought about by treatment with gibberellin aredependent on an increase in diffusible auxin. ¹Present address: Biological Institute, College of General Education,University of Tokyo, Komaba, Meguro, Tokyo.     

Brassinolide, a growth-promoting steroidal lactone

Brassinolide (BR), a naturally-occurring-steroidal lactone from rape ( Brassica napus L.) pollen, was compared with auxin for activity in a number of bioassay systems. Responses similar to IAA were elicited by BR in bioassays based upon bean hypocotyl hook opening, elongation of maize mesocotyl, pea epicotyl and azuki bean epicotyl sections, and fresh weight increase in Jerusalem artichoke (2,4-D used) and pea epicotyl sections. The azuki bean and dwarf pea epicotyl bioassays were much more responsive to BR than IAA (at 10 μ M ). Responses approximately two-fold greater in magnitude were elicited by IAA in the maize mesocotyl, bean hypocotyl hook and Jerusalem artichoke bioassays. Little or no response was elicited by BR (0.01 to μ M ) in the cress root or decapitated pea-lateral bud bioassays. A powerful synergism between BR and IAA was observed in the azuki bean, pea epicotyl and bean hypocotyl hook bioassays. Although, as previously reported, other steroidal substances are active in some of the bioassay systems tested, none compared with BR in magnitude and diversity of elicited responses.     

Effect of brassinosteroids on growth and proton extrusion in the alga Chlorella vulgaris Beijerinck (Chlorophyceae)

Brassinolide, as a plant hormone, promotes growth of a number of plant species. Similar effects are induced by its epimer 24-epibrassinolide. In this paper we discuss the effects of brassinosteroids on the growth and proton extrusion in the green alga Chlorella vulgaris (Chlorophyceae). At concentrations between 10^–15 and 10^–8 m, brassinolide and 24-epibrassinolide induce a significant stimulation of growth and H⁺ extrusion. The growth was associated with an increase in the capability of algal cells to acidify the medium, where brassinolide is biologically more active than 24-epibrassinolide.Abbreviations BL brassinolide - BR(s) brassinosteroid(s) - epiBL 24-epibrassinolide - DW dry weight - IAA indole-3-acetic acid     

Oxindole-3-acetic Acid: Physical Properties and Lack of Influence on Growth

The auxin activity of OAA was studied. The assays used were the Avena coleoptile curvature and section tests, and the first internode test. OAA was completely inactive to these assays. Physical tests indicated no color reaction with modified Salkowski reagent (Gordon-Weber) and no activity of any of the biochromatograms run with OAA. These data indicate that OAA is inactive as an auxin due to its characteristics of structural difference from IAA and further lends some speculation as to the site of activity of IAA as an auxin.     

Investigations on the Mechanism of the Brassinosteroid Response: VI. Effect of Brassinolide on Gravitropism of Bean Hypocotyls

Brassinosteroids are steroidal lactones of plant origin that promote growth of a number of plant systems, and particularly the growth induced by auxins. Biologically active brassinosteroids (BR) also promote the growth of gravisensitive hypocotyls of 7-day-old light grown Phaseolus vulgaris when gravistimulated. Brassinolide-mediated promotion of curvature of gravistimulated internodes occurs in the absence of exogenously supplied indole-3-acetic acid (IAA). This is in contrast to the BR-promoted bending of vertically positioned bean hypocotyls, which is dependent upon exogenous IAA. Brassinosteroid treatment increased the graviperception of young internode tissues and the bending of the gravistimulated sections as well as the subsequent reversal of bending after the sections were placed vertically. These results indicate that BR sensitizes bean hypocotyls to gravistimulation and potentiates the action of a growth factor that induces gravitropic growth.     

Transport and Degradation of Auxin in Relation to Geotropism in Roots of Phaseolus vulgaris

The movement of auxin in Phaseolus vulgaris roots has been examined after injection of IAA^?3H into the basal root/hypocotyl region of intact, dark-grown seedlings. Only a portion of the applied IAA^?3H was transported unchanged to the root tip. The major part of the chromatographed, labelled compounds translocated to the roots was indole-3-acetylaspartic acid (IAAsp) and an unidentified compound running near the front in isopropanol, ammonia, water. The velocity of the auxin transport (7.2 mm per hour) was calculated from scintillation countings of methanol extracts from serial sections of the root. An accumulation of radioactive compounds in the extreme root tip, was observed 5 h after the injection of IAA. The influence of exogenous IAA on the geotropical behaviour of the bean seedling roots was examined. Pretreated roots were stimulated for 5 min in the horizontal position and then rotated parallel to the horizontal axis of the klinostat for 60 or 90 min. The resulting geotropic curvature of IAA-injected and control roots showed significantly different patterns of development. When the stimulation was started 5 h after application of the auxin, the geotropic curvature became larger in roots of the injected plants than in the controls. If, however, the translocation period was extended to 20 h the geotropic curvature was significantly smaller in the roots of the injected plants. The auxin injection did not significally affect the rate of root elongation. The change in geotropical behaviour of the roots is interpreted as a result of the influence of the conversion products of the applied IAA on the geotropical responsiveness.     

Effects of the steroidal alkaloid tomatine in auxin bioassays and its interaction with indole-3-acetic acid

Summary The steroidal alkaloid tomatine did not enhance elongation of oat coleoptile and first internode sections, or of wheat coleoptile sections. Higher concentrations of the alkaloid inhibited elongation and interacted antagonistically with IAA. Although 10^-4 M tomatine alone did not influence elongation of oat coleoptile sections, it did reduce growth response to exogenous IAA. Tomatine concentrations less than 10^-4 M did not influence response to IAA. The auxin activity of tomatine, reported by Vendrig, was therefore not confirmed.     

The etiolated cucumber hypocotyl weight-growth test: A sensitive,easy and ultrafast bioassay for IAA

A sensitive, easy and fast bioassay is described for the detection of IAA. The bioassay consists of measuring the weight increase of hypocotyl sections from etiolated cucumber seedlings incubated in a simple medium containing 2 mM KCl, 0.1 mM CaCl and 10 mg/l chloramphenicol (pH 7). The sensitivity of the test is comparable to that of the Avena first internode test, exhibiting a significant response at an IAA concentration as low as 1 ng/ml. The bioassay requires only 3 h, and is easy to perform. The slope of the log-linear concentration-response curve is moderately steep giving good accuracy. The assay is insensitive to the pH of the media between 4 to 7 and can be performed in the presence or absence of buffer. The growth of the etiolated hypocotyl sections is insensitive to GA and to kinetin except at high concentrations.     

Interaction between cortical cylinder and epidermis during auxin-mediated growth in pea internodes

The interaction between cortical cylinder (cortex plus vascular tissue) and epidermis during auxin (indole-3-acetic acid, IAA)-induced growth of third internode sections from red light-grown pea seedlings (Pisum sativum L. cv. Alaska) was investigated. A quantitative comparison of the relative effects of IAA on growth of intact and peeled sections showed that intact segments are nearly 20-fold more sensitive to IAA than peeled cortical cylinders. Tissue tension, determined with the ‘split section test’, was constant during IAA-induced growth of intact sections. Peeled sections also displayed a small amount of tissue tension, which was likewise independent of IAA. The incorporation of myo-[2-³H(N)]inositol ([³H]Ins) into non-cellulosic polysaccharides in the cell walls was stimulated by IAA in both the cortical cylinder and the epidermis by + 70% and + 55%, respectively, after 4 h. A mich higher amount of incorporation was detectable in the epidermis than in the cortical cylinder on a unit weight basis. During a 4-h growth period in IAA the cortical cylinder lost about 50 μg of its initial dry weight per section whereas the epidermis increased in dry weight by about + 24 μg. We conclude that during IAA-induced long-term growth the cortical cylinder (1) provides the driving force for organ growth, (2) responds to IAA by an increase in matrix cell wall synthesis and (3) releases material, some of which is transferred to the attached epidermal cells.     

Strongly acidic auxin indole-3-methanesulfonic Acid: synthesis of [C]indole-3-methanesulfonic Acid and studies of its chromatographic, spectral, and biological properties

A radiochemical synthesis is described for [¹⁴C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [¹⁴C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles.     

Juvenility and endogenous rooting substances inCastanea sativa Mill

The rooting response to exogenous auxin of cuttings in a juvenile phase of growth from plants ofCastanea sativa Mill. was determined and simultaneously the rooting potential of the water extracts was evaluated in presence of IAA by a bean rooting test. The level of the extractable rooting promoters was high in the cuttings which exhibited the highest percentage of rooting. An inhibition of the effect of IAA on rooting was detected in the cuttings which showed the lowest rooting response, the histogram differing not much from that of the adult plant. The results indicate that in chestnut the juvenile condition, easy rooting, is associated with high levels of endogenous rooting promoters.     

Is indolyl-3-acetic acid involved in the wave-like pattern of auxin efflux from Pinus sylvestris stem segments?

The coleoptile curvature bioassay of agar diffusates from successive cambial-region sections of Pinus silvestris L. stem tissue reveals the existence of a wave-like pattern, possibly of auxin translocation. Spectrofluorometric determinations of indolyl-3-acetic acid (IAA) in these diffusates gave only a poor correlation. Probably another phytohormone, also an auxin, plays a more important role in the pattern.
The amplification in the amplitude of the pattern, brought about by apically applied IAA, cannot be induced by the synthetic auxin, 2,4-dichlorophenoxyacetic acid.     

THE TIMING OF AND EFFECT OF TEMPERATURE ON AUXIN-INDUCED HYPONASTIC CURVATURE OF THE BEAN PRIMARY LEAF BLADE

Detailed examination of the hyponastic curvature of the primary bean leaf blade in response to indoleacetic acid (IAA) shows that curvature begins within 15 min after application and increases to a maximal rate at 20 to 30 min. A second application of IAA results in a second curvature maximum when applied 1.5 hr or more after the first. Washing experiments indicate IAA uptake is largely complete by about 20 min after application, suggesting the return to planar form is accompanied by the uptake and passage of a wave of IAA through the responding cells. The rate of curvature decreases as the temperature is lowered, particularly below 14 C; at low concentrations (10^–4 m) the rate of response to 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid is slower than that for IAA and naphthaleneacetic acid. These differences are proposed to reflect the involvement of the polar auxin transport system in the response. The leaves of bean seedlings exposed to 4 C develop hyponastic curvatures when returned to normal growth temperature; 5 min treatment is sufficient to induce this response, and with longer treatments, greater curvatures are obtained. This curvature is inhibited by application of 2,3,5-triiodobenzoic acid (TIBA) to the undersurface of the leaf at the beginning of the cold treatment. The results are consistent with a model of planar plageotropic growth regulation in the leaf blade in which auxin produced by cells in the upper portion of the blade is transported by the polar transport system through cells in the lower portion that are growth limited by auxin supply. The hyponastic and epinastic effects caused by exogenous application of auxin or TIBA and of cold treatments are considered to result from changes in this auxin supply.     

Changes in Endogenous Gibberellin and Auxin Activities during First Internode Elongation in Tulip Flower Stalk

Dark treatment during the most active period of tulip shootgrowth induced rapid elongation of the first internode. Endogenousfree-form gibberellin and diffusible auxin in the first internodeincreased while bound-form gibberellin decreased after the darktreatment. Alternating dark and light treatments at 24-h intervalscaused increases in elongation of the first internode and theamounts of free-form gibberellin and diffusible auxin in thedark but their decreases in the light. TIBA treatment at thefirst node inhibited both the elongation and the increase indiffusible auxin, but did not affect the gibberellin amount.Ancymidol application prior to the dark treatment inhibitedthe increase in both free-form gibberellin and diffusible auxin.Application of gibberellin A₃ increased both elongation of thefirst internode and the amount of diffusible auxin. It alsocaused recovery from ancymidol-mediated reduction in elongationand diffusible auxin content. Dark-induced elongation of thefirst internode was inhibited when all organs above the firstinternode were excised, but endogenous free-form gibberellinincreased and bound-form gibberellin decreased. After excision,elongation of the first internode occurred only when both GA₃and IAA were applied exogenously, or when IAA was applied withdark treatment. These results indicate that dark-induced elongationof the first internode of tulip is promoted by auxin, whichis transported from the upper organs into the first internodedue to stimulation from the dark-induced increase in free-formgibberellin. Free- and bound-form gibberellins changed complementarilywith the dark and light treatments. An interconversion systembetween the two forms in the first internode and its dependenceon light conditions are also discussed. (Received June 23, 1984; Accepted March 5, 1985)     

Rhodethrin: a novel indole terpenoid ether produced by Rhodobacter sphaeroides has cytotoxic and phytohormonal activities

A novel metabolite was isolated from the culture supernatants of Rhodobacter sphaeroides OU5 when grown on l-tryptophan as sole source of nitrogen under photoheterotrophic conditions. It was identified by IR, NMR (¹H, ¹³C) and MS as an indole terpenoid ether [3-hydroxy-6-(1H-indol-3-yloxy)-4-methylhexanoic acid] and is named as rhodethrin. Rhodethrin at 0.5 μM gave positive test in auxin bioassay and initiated early rooting in tissue-cultured plants than IAA at 5 μM. Rhodethrin has cytotoxic activity against Sup-T₁ lymphoma and Colo-125 cancer cell lines at 10 nM.     

Auxin activity of substituted benzoic acids and their effect on polar auxin transport

Six dichloro-, 3 trichloro-, 2 triiodo-, and 3 heterosubstituted benzoic acids (amiben, dinoben, dicamba), and N-1-naphthylphthalamic acid have been tested for effects on growth and on polar auxin transport. Growth activity with and without kinetin was measured by effects on fresh and dry weights of 30-day cultures of fresh tobacco pith. Transport inhibition was measured by following uptake and output of IAA-2-¹⁴C through 10 mm bean epicotyl sections. The distribution of callus growth on vascularized tobacco stem segments was also observed. Avena first internode extension assays established the relative activities: dicamba > amiben > dinoben suggested by pith growth results. Growth effects of active compounds were similar with and without kinetin, except that amiben was less active with kinetin, while 2,3,6-trichlorobenzoic acid was more active with kinetin than alone. The weak auxin activity of NPA was confirmed. Transport experiments showed that NPA was the most inhibitory compound tested, followed by TIBA. Other compounds tested were at least 300 times less inhibitory to IAA transport. The best growth promoters were the least inhibitory to transport, and the most effective transport inhibitors were at best poor auxins. It is suggested that the weak auxin and auxin synergistic activity of TIBA (and perhaps 2,3-dichlorobenzoic acid) in extension growth tests arises from its inhibition of transport of endogenous or added auxin out of the sections, rather than from its intrinsic auxin activity. Chemically induced apolar callus growth on vascularized tobacco stem explants can arise from inhibition of native auxin transport, apolar growth stimulation by auxinic action of the test compound, or both.     

Synthesis and biological activity of brassinolide and its 22β,23β-isomer: Novel plant growth-promoting steroids

Brassinolide (2α,3α,22α, 23α-tetrahydroxy-24α-methyl-B-homo-7-oxa-5α-cholestan-6-one), a novel plant growth-promoting steroid isolated from rape pollen, and its hitherto unknown 22β, 23β-isomer were synthesized from a C-24 epimeric 60:40 mixture of 22-dehydrocampesterol (24α-methyl) and brassicasterol (24β-methyl) from oysters. The method of synthesis favored the formation of the 22β, 23β-isomer by better than 4:1. Comparative plant growth-promoting capabilities of brassinolide, both natural and synthetic, and its three side chain $\text{cis}$-glycolic isomers in the bean second internode bioassay showed that the natural and synthetic brassinolides were equally active and caused splitting of the internode at the 0.1 μg level. The least active was the 22β,23β-isomer of brassinolide. The isomers with the 22α, 23α and 24β, and the 22β, 23β and 24β configurations were highly active and were required at about 10 times the concentration of brassinolide to cause the same physiological response. In the bean first internode bioassay, an auxin-induced growth test system which employs isolated bean plant segments, the isomer with 22β, 23β and 24β configuration caused a greater response than brassinolide. Two of the four tetrahydroxy ketones obtained in the synthesis of the isomers were also active in both assays.