A brassinosteroid-cytokinin interaction on ethylene production by etiolated mung bean segments

Three cytokinins and their ribosides were tested in conjunction with brassinosteroid (BR) for their effects on ethylene production in etiolated mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. When varying concentrations of BR were tried in combination with a fixed amount of kinetin (10 μ M ), there was an increase in ethylene production with increasing concentrations of BR up to 3 μ M , and a decline beyond. A stimulation in ethylene production was observed with increasing concentrations of each of the cytokinins used. However, when applied in conjunction with 3 μ M BR, all cytokinin concentrations produced similar stimulatory patterns. When 3 μ M BR or 10 μ M 6-benzylaminopurine (BAP) was applied in conjunction with increasing concentrations of IAA, there was a stimulation in ethylene production with increasing concentrations of IAA. However, 3 μ M BR and 10 μ M BAP together with varying concentrations of IAA failed to alter the level of ethylene production.     

Evidence that brassinosteroid stimulates auxin-induced ethylene synthesis in mung bean hypocotyls between S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid

Brassinosteroid (BR) stimulation of auxin-induced ethylene production and the particular step at which BR acts to promote such synthesis were studied in mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Increasing concentrations of methionine alone and in combination with 3 μ M BR and 10 μ M IAA had a minimal effect on ethylene production. With increasing concentrations of 1-aminocyclopro-pane-1-carboxylic acid (ACC), however, ethylene production increased. BR or IAA further enhanced ethylene production with maximum rates occurring when these compounds were added together with ACC. The addition of 10 μ M CoCl₂ in conjunction with BR and/or IAA resulted in 85–97% inhibition of ethylene production. When 20 μ M cycloheximide was used in conjunction with BR and/or IAA there was a complete inhibition of ethylene production. Total inhibition also resulted when 1.0 μ M aminoethoxy-vinylglycine (AVG) was used in combination with BR and/or IAA. AVG alone had no effect on ACC conversion to ethylene.     

Fusicoccin, an inhibitor of brassinosteroid-induced ethylene production

Fusicoccin was evaluated for its effects on brassinosteroid (BR), indole-3-acetic acid (IAA) and BR + IAA-induced ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC-synthase production by etiolated mung bean ( Vigna radiata L. Rwilez cv. Berken) hypocotyl segments. Fusicoccin inhibition of ethylene and ACC production induced by 2 μ M BR started at concentrations as low as 0.05 μ M . Maximum inhibition occurred at a 1 μ M concentration with no further inhibition at higher concentrations tested. Fusicoccin (1 μ M ) was effective in the inhibition of BR-induced ethylene, ACC and ACC-synthase production at low and high concentrations of BR.
Fusicoccin at concentrations as high as 2 μ M had no effect on ethylene and ACC production promoted by low concentrations of IAA (1 to 10 μ M ). When higher concentrations (100–1000 μ M ) of IAA were used, fusicoccin (1 μ M ) had an inhibitory effect on ethylene and ACC production. Interestingly, fusicoccin (1 μ M ) had little or no effect on ACC-synthase promoted by high concentrations of IAA (1000 μ M ).
When BR and IAA were used in combination, fusicoccin inhibited ethylene and ACC production at concentrations as low as 0.05 μ M with maximum inhibition occurring at 0.5 μ M . At a 1 μ M concentration, fusicoccin was effective in inhibiting the synergistic stimulation of ACC-synthase promoted by BR and IAA.     

Effect of Auxin on Autolysis of Cell Walls in Azuki Bean Epicotyls

Native cell walls of azuki bean epicotyls incubated in bufferautolytically released neutral sugars, abundant in galactose,and uronic acids. Treatment with 10^–5 M IAA of subapicalor basal epicotyl segments for 3 h did not influence the amountof total neutral sugars released from the cell walls duringautolysis. However, the amount of glucose and xylose releasedfrom subapical cell walls was increased by IAA. Pretreatmentwith IAA of subapical epicotyl segments enhanced the solubilizationof neutral sugars from pectinase-treated cell walls during incubationin buffer at pH 5 to 6. The amount of fucose, xylose, and glucosereleased was specifically increased by IAA. Of the sugar fractionsreleased from pectinase-treated cell walls during autolysisand subsequently separated by gel filtration on a ToyopearlHW-40S column, IAA promoted the release of oligosaccharides,consisting mainly of glucose and xylose. These results suggestthat autolytic degradation of xyloglucans is closely relatedto IAA-induced growth of azuki bean epicotyls. (Received May 19, 1989; Accepted January 5, 1990)     

Influence of Gibberellic Acid on Growth and Endogenous Auxin Level in Epicotyl and Hypocotyl Tissues of Normal and Dwarf Bean Plants

Application of gibberellic acid to the apex of dwarf bean plants (cv. Alabaster) stimulated the elongation growth of epicotyl and hypocotyl but showed no significant effect on elongation growth in a normal cultivar (‘Blue Lake’). Gibberellin-treatment of dwarf plants was characterized by about twofold increase in the level of endogenous auxin. Maximum increase in IAA level was observed after 48 h of GA treatment. There was less increase in IAA content in normal bean plants. — Gibberellin treatment to excised epicotyl and hypocotyl sections of either dwarf or normal cultivar showed no effect on elongation growth. However, a considerable increase in the auxin level was observed in the sections of the dwarf cultivar. The maximum effect occurred with only 1 h incubation in basal medium containing gibberellin. — The indolo-α-pyrone spectro-fluoremetric method for IAA determination was used.     

Effects of Brassin-Complex on Auxin and Gibberellin Mediated Events in the Morphogenesis of the Etiolated Bean Hypocotyl

The excised, hooked bean hypocotyl was the system used to determine wheiher the ‘auxin- and gibberellin like’ effect of the lipoidal pollen extract, Brass in-complex (Br), were mediated through, or independent of, auxin and gibberellin. The morphogenetic events of hook opening and hypocotyl elongation in this system are regulated by auxin and gibberellin, respectively. Brassin complex, like IAA, elicited a book closure in (he dark and retarded its opening in red light. This effect was synergized by T1BA, IAA and the presence of the auxin-producing organs, the epicotyl and cotyledons. Br-elicited hook closure was inhibited by the antiauxin. PCIB. Both GA₃ and Br totally reversed the light inhibition of hypocotyl elongation. The GA₃-effect, but nol the Br elicited elongation, was overcome by Ancymidol. Hypocotyl elongation was partially inhibited by TIBA and PCIB. suggesting a possible auxin involvement also in this effect of Br. Br may elicit its growth responses through an effect on endogenous auxin levels, In this way it is different from other lipoidat growth regulators, such as the oleanimins which require the presence of exogenous growth regulators for activity.     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. 2,6-Dicholorobenzonitrile as a new cellulose-synthesis inhibitor

In azuki bean epicotyl sections, 2,6-dichlorobenzonitrile inhibitedcellulose synthesis but showed no effect on the synthesis ofnon-cellulose wall materials and trichloroacetic-acid-insolublecytoplasmic substances. Dichlorobenzonitrile reversed gibberellinpromotion and kinetin inhibition of auxin-induced epicotyl elongationwithout affecting auxin-induced elongation, itself. (Received August 17, 1973; )     

Auxin and Ethylene Control of Growth in Seedlings of Zea mays L. and Avena sativa L

The effects of applied ethylene on the growth of coleoptilesand mesocotyls of etiolated monocot seedlings (oat and maize)have been compared with those on the epicotyl of a dicot seedling(the etiolated pea). Significant inhibition of elongation by ethylene (10 µll^–1for 24 h) was found in intact seedlings of all three species,but lateral expansion growth was observed only in the pea internodeand oat mesocotyl tissue. The sensitivity of the growth of seedlingparts to ethylene is in the decreasing order pea internode,oat coleoptile and oat mesocotyl, with maize exhibiting theleast growth response. Although excised segments of mesocotyland coleoptile or pea internode all exhibit enhanced elongationgrowth in IAA solutions (10^–6–2 ? 10^–5 moll^–1), no consistent effects were found in ethylene. Ethyleneproduction in segments was significantly enhanced by applicationof auxin (IAA, 10^–5 mol l^–6 or less) in all tissuesexcept those of the eat mesocotyl. Segments of maize show a slow rate of metabolism of applied[2-¹⁴C]IAA (30 per cent converted to other metabolites within9 h) and a high capacity for polar auxin transport. Ethylene(10 µl l^–1 for 24 h) has little effect on eitherof these processes. The oat has a smaller capacity for polartransport than maize and the rate ef metabolism of auxin isas fast as in the pea (90 per cent metabolized in 6 h). Althoughethylene pretreatment does not change the rate of auxin metabolismin oat, there is a marked reduction in auxin transport. It is proposed that the insensitivity of maize seedlings toethylene is related to the supply and persistence of auxin whichcould protect the seedling against the effects of applied orendogenously produced ethylene. Although the mesocotyl of oatis sensitive to applied ethylene it may be in part protectedagainst ethylene in vivo by the absence of an auxin-enhancedethylene production system. The results are discussed in relationto a model for the auxin and ethylene control of cell growthin the pea.     

Roles of auxin and gibberellic acid in growth and maturation of epicotyls of Vigna angularis: Cell wall changes

The effects of auxin and gibberellic acid on cell wall composition in various regions of epicotyls of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) were investigated with the following results. (1) Young segments excised from apical regions of the epicotyl elongated in response to added 10⁻⁴ M indole-3-acetic acid (IAA). When the segments were supplied with 50 m M sucrose, the IAA-induced segment growth was accompanied by enhanced overall synthesis of cell wall polysaccharides, such as xyloglucans, polyuronides and cellulose. This IAA effect on the cell wall synthesis is a consequence of extension growth induced by IAA. Gibberellic acid (GA) at 10⁻⁴ M synergistically enhanced the IAA-induced cell wall synthesis as well as IAA-induced extension growth, although GA by itself neither stimulated the cell wall synthesis nor extension growth. In the absence of sucrose, cell wall synthesis was not induced by IAA or GA. (2) In mature segments excised from basal regions of the epicotyl, no extension growth was induced by IAA or GA. GA enhanced the synthesis of xylans and cellulose when the segments were supplied with 50 m M sucrose. IAA had no effect on the cell wall synthesis. These findings indicate that synthesis of polyuronides, xyloglucans and cellulose, which occurs during extension growth of the apical region of the epicotyl, is regulated chiefly by auxin whereas synthesis of xylans and cellulose during cell maturation in the basal region of the epicotyl is regulated by GA.     

Comparison of the effects of epibrassinolide and steroidal estrogens on adventitious root growth and early shoot development in mung bean cuttings

Concentrations of 24-epibrassinolide as low as 0.1 μ M consistently inhibited adventitious root formation and elongation in both hypocotyl and epicotyl cuttings from mung bean ( Phaseolus aureus L.). Similar, but less pronounced, inhibitory effects on root elongation were also observed with estrone sulphate and estradiol sulphate. With regards to root number, estrone sulphate enhanced this in both types of cutting, whereas estradiol sulphate was stimulatory in hypocotyl cuttings but inhibitory in epicotyl cuttings. Brassinolide caused a marked stimulation of epicotyl (but not hypocotyl) elongation and a swelling and splitting of the epicotyl in both types of cutting, whereas estrogens varied in their effect from inhibition of epicotyl growth to no effect. Root-applied brassinolide and estrogen sulphates brought about similar morphological abnormalities in shoots viz. epinasty and inrolling of primary leaves and delayed expansion of the first trifoliate leaf.     

Growth and cell wall changes in azuki bean epicotyls II. Changes in wall polysaccharides during auxininduced growth of excised segments

Changes in cell wall polysaccharides and mechanical propertiesof the cell wall were examined during IAA-induced elongationgrowth of excised azuki bean epicotyl segments under differentgrowth conditions. Sucrose promoted IAA-induced cell elongation,but had very little effect on IAA-induced cell wall loosening.In the absence of sucrose, the amount of galactose in the cellwall decreased during the incubation period. IAA enhanced thedecrease in the galactose level. In the presence of sucrose,on the other hand, IAA induced increases in the amounts of cellulose,galactose and xylose in noncellulosic polysaccharides. TheseIAA-induced increases were not observed in the presence of mannitolat concentrations higher than 0.1 M, although cell wall looseningwas induced by IAA even in the presence of 0.2 M mannitol. (Received November 21, 1978; )     

Reversal of auxin induced inhibition by ethrel

The interaction between exogenous 2-chloroethylphosphonic acid (Ethrel, CEPA) and auxin (both native and synthetic—IAA) was studied on pea and bean seedlings, potato tubers, and processed flax plants. After the addition of ethrel the inhibiting effect of IAA was decreased in all objects and it was found that the concentration of the growth of the regulators played an important role. The growth response of a part of flax hypocotyl, as induced by exogenous auxin produced in the cotyledon, was reversed by ethrel, too. The application of ethrel on the epicotyl apex in beans resulted in the lost of apical dominance of epicotyl and in the growth of lateral buds together with the epicotyl. When stimulating the growth, ethrel reverses the inhibitions through the decrease in the auxin content (from an inhibiting, supraoptimum level to an optimum one which already stimulates growth). In objects with a low content of endogenous auxin the ethrel induced the decrease in the auxin content and shows an inhibiting effect on growth.     

Indoleacetic Acid and Molecular Differentiation Evidence for Interaction

Proteins of hypocotyls of bean were studied by electrophoresis. Proteins were extracted from hypocotyl segments of various stages of development starting with the relatively undifferentiated hook regions and proceeding by 2 cm segments down the hypocotyl. The proteins were the soluble (pH 7.4), the basic nuclear (histones), acidic ribonuclear and acidic chromosomal. Soluble proteins reflected differentiation of the hypocotyl in that lower hypocotyl segments had more different protein types than did the hook region. Indoleacetic acid (IAA) at 10^?6M when applied to the lower hypocotyl appeared to induce still more different proteins. However, at 10^?3M, IAA appeared to induce molecular dedifferentiation in that hypocotyl protein patterns began to resemble those of the hook. Histones also reflected differentiation, the hook having more histone types than the lower hypocotyl. IAA had no effect on histones. The hook region had two types of acidic chromosomal proteins, the lower hypocotyl one. When lower hypocotyl segments were incubated in 10^?3M IAA, the protein pattern resembled that of the hook in that the second protein normally present in the hook and not in the hypocotyl was in fact induced in the hypocotyl. The hook had two acidic ribonuclear proteins, the lower hypocotyl one. IAA did not affect this protein. These experiments suggest that IAA in some manner regulates molecular (protein) differentiation. It is further suggested that IAA accomplishes this control through the acidic nuclear proteins which are closely associated with genetic material and which reflect differentiation and are also affected by IAA.     

A scanning electron microscopic study of IAA-induced tumors in bean (Phaseolus vulgaris L.) embryos

A scanning electron microscopic study of indole-3-acetic acid (IAA) induced tumour in the hypocotyl region of bean embryos shows a distinct morphological, structural and topographical change from the non-treated bean embryos. The IAA-induced tumour surface, in the hypocotyl region, shows distinct cell enlargement, some cellular proliferation in the parenchymatous tissue, total destruction of the epidermis and stomata and some variation in trichome structures. In dole-3-acetic acid inhibits the normal growth of the epicotyl, and, as age progresses, adventitious roots appear all over the surface. When IAA-depletion occurs, epicotyl growth resumes, which indicates that this tumour formation in bean embryos is an IAA-dependent tumour.     

Characteristics of hook formation by bean seedlings

Explants were isolated from 6-day-old etiolated bean seedlings (Phaseolus vulgaris L. cv. Black Valentine) containing the cotyledons with 4 mm of hypocotyl just below the node and/or the epicotyl. During incubation on distilled water, uneven growth of the hypocotyl or epicotyl occurred resulting in the formation of a hook. The more rapid growth of the side which became convex was not dependent upon the presence of the slower growing concave side. It was concluded that the main axis has an intrinsic capacity for asymmetric growth. The growth leading to hook formation was inhibited by α-naphthaleneacetic acid at concentrations above 0.2 milligram per liter.     

A Possible Double Role for Brassinolide in the Reorientation of Cortical Microtubules in the Epidermal Cells of Azuki Bean Epicotyls

Brassinolide, at 10^–8M or higher, enhanced the elongationof epicotyl segments from azuki bean seedlings that was inducedby IAA, but it did not enhance the increase in fresh weightof the segments, an indication that brassinolide suppressedthe lateral expansion of the segments. The additional elongationcaused by brassinolide was completely prevented in the presenceof 10^–5 M cremart, which disrupted the cortical microtubules(MTs) in epidermal cells in the segments, and in the presenceof 10^–6M 2,6-dichlorobenzonitrile, an inhibitor of thesynthesis of cellulose. Brassinolide at 10^–7M, appliedtogether with IAA, increased the percentage of epidermal cellswith transversely oriented cortical MTs. Brassinolide appearsto enhance the longitudinal expansion and suppress the lateralexpansion of epicotyl cells by organizing cortical MTs transverselyto the cell axis and, thereby, causing the deposition of cellulosemicrofibrils in the same orientation. Brassinolide by itself, at 10^–8M or higher, induced theelongation of epicotyl segments and the elongation caused bybrassinolide was partially prevented by 10^–5M cremart,results that suggest that brassinolide regulates cell expansionvia at least two processes, an MT-dependent process and an MT-independentprocess. Brassinolide by itself increased the percentage ofepidermal cells with transversely oriented cortical MTs. Since,in azuki bean epicotyls, the percentage of cells with transverseMTs is increased only by the combination of auxin and gibberellinbut not by either alone, brassinolide applied alone seems toplay a double role, similar to that of auxin and of gibberellin,in organizing cortical MTs. (Received September 2, 1994; Accepted November 16, 1994)     

Epidermis integrity and epicotyl growth in azuki bean

In order to verify if epidermis integrity played a determinant role in epicotyl elongation induced by fusicoccin (FC), buffers at different pH's, and indoleacetic acid (IAA), we studied the short-term kinetics of elongation growth, the increase of fresh weight in long-term treatment, and the H⁺ excretion in intact, abraded, and peeled azuki bean epicotyl sections. We demonstrated that the epidermis is more sensitive to IAA, whereas the cortex is highly responsive to protons. Our data are consistent with the acid growth theory. In addition, our studies support the idea that the epidermis may be the tissue target for auxin, but its integrity is necessary for IAA-induced elongation.     

Inhibition of RNA Synthesis and Auxin-Induced Cell Wall Extensibility and Growth by Actinomycin D

A linear stress strain analyzer was used to determine the effects of inhibitors of RNA and protein synthesis on auxin-induced increases in cell wall extensibility. With etiolated soybean hypocotyl, maize mesocotyl and Avena coleoptile sections and light-grown pea internode sections, inhibition of RNA synthesis resulted in inhibition of auxin-induced extensibility changes and cell expansion. The results with both actinomycin D and cycloheximide support an earlier conclusion that unstable cell constituents, presumably enzymes, are essential for cell wall loosening induced by auxin as well as for cell elongation.     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.     

Untersuchungen zum Wirkungsmechanismus von Indolyl-3-Essigsäure mit Hilfe von schwerem Wasser

Summary The auxin-induced cell elongation and the formation of indoleacetyl-aspartic acid (IAAsp) of pea epicotyl sections and Agrostemma hypocotyl sections are inhibited by heavy water. The formation of IAAsp requires a specific enzyme. The lack of IAAsp in D₂O-treated plant tissues may be due to an influence of D₂O on the induction or on the synthesis of that enzyme. Treatment of plant sections with synthetic IAAsp has no effect on the growth of the sections in D₂O. Indole-3-acetic acid (IAA) increases the incorporation of ³²P-orthophosphate into ribosomal and soluble RNA of pea epicotyl sections in H₂O but not in D₂O. The synthesis of ribosomal RNA is decreased by heavy water.The effects of IAA and D₂O on the soluble proteins of pea sections have been studied by PAA-gel electrophoresis. D₂O does not change the pattern of protein bands in comparison with the H₂O-control, but prevents the probably IAA-induced alteration of the Rf-value of one protein band on the pherogram. It is assumed that the inhibition of auxin-induced reactions in the D₂O-medium is due to the stabilizing effect of heavy water on allosteric proteins. The results of this work support the hypothesis that IAA acts as allosteric effector.