Correlative Inhibition of Lateral Bud Growth in Phaseolus vulgaris L. Effect of Application of Indol-3yl-Acetic Acid to Decapitated Plants

In view of the variation in the ability of indol-3yl-aceticacid (IAA) to prevent lateral bud growth on decapitated plantsvarious factors which might influence the response have beeninvestigated in Phaseolus vulgaris L. The effectiveness of IAAapplied in lanolin varied from experiment to experiment. Factorsthat altered the response included the time of year, the concentrationand quantity of IAA, the age of the plant, the type of lanolinand the region of application. In many instances IAA, at a concentrationbelieved to mimic the auxin relations of the intact plant, cancompletely replace the main shoot with respect to the correlativeinhibition of lateral bud growth. The evidence for the involvementof IAA as the primary determinant in apical dominance in P.vulgaris is summarized.     

Levels of Indol-3yl-acetic Acid and Acid Inhibitors in Green and Etiolated Bean Seedlings (Phaseolus vulgaris)

Indol-3yl-acetic acid (IAA) was identified in Phaseolus vulgaris L. Shoot tissue of seedlings, exposed to light for 5 days, had a higher level of IAA than etiolated seedlings of the same age. The content of IAA increased in green seedlings during light treatment for 5–12 days. No increase could be measured in dark-grown seedlings. Inhibitory substances appeared at different R_f-values. The main part was identical to the inhibitor-β complex and occurred in a higher amount in light-grown seedlings than in etiolated taller ones. One part of the inhibitor-complex appeared to be abscisic acid (ABA). It is suggested that both IAA and acid inhibitors may play an important role in the control of stem growth and differentiation, although light effects on other hormones and regulatory systems cannot be ignored.     

The Pathway of Radial Transfer of Photosynthate in Decapitated Stems of Phaseolus vulgaris L.

[¹⁴C]Sucrose was found to be the predominant component of the¹⁴C-photosynthates that accumulated in the free space of decapitatedstems of P. vulgaris plants. The ¹⁴C-photosynthates appearedto occupy the entire free-space volume of the stems at totalsugar concentrations in the range of 3–12 mM. The free-spacesugar levels were found to rapidly decline once photosynthatetransfer to the stems was halted. Moreover, it was found thatestimates of the rate of in vitro sucrose uptake by the stemscould account fully for the decline in free-space sugar levels.Overall, the evidence indicated that at least part of the radialpathway of photosynthate transfer in bean stems involved thestem apoplast. It is tentatively proposed that, based on celland tissue distribution of ¹⁴C-photosynthates, the apoplasticpathway extends from the membrane boundary of the sieve element/companion-cellcomplex to all other cells of the stem. Apoplast, Phaseolus vulgaris L., bean, phloem unloading, photosynthates, symplast     

Identity of Products of Indol-3yl-acetic Acid Oxidation

The product of IAA decomposition which gives the pink colourin the Salkowski reaction is not N-hydroxy-IAA and its identityis still unknown, as is also that of other substances foundduring metabolism of IAA by pea seedlings.     

Hormone-directed Transport of Assimilates in Decapitated Internodes of Phaseolus vulgaris L.

Application of ethylene, indole-3yl-acetic acid (IAA), 6-(benzylamino)-9-(2tetrahydropyranyl)-9-Hpurine (SD8339), or mixtures of IAA, gibberellic acid (GA),and cytokinins, increased the accumulation of ¹⁴C-activity indecapitated internodes of Phaseolus vulgaris seedlings. Differencesbetween treated and untreated tissues with respect to importof labelled assimilate were detected 3 h after application ofa mixture of IAA, GA, and SD8₃₃₉. In longer-term experimentseffects of the growth-regulator mixture on translocation oflabel were greater than those of IAA alone. Inhibitory effectsof abscisic acid on import of assimilate were counteracted bySD8₃₃₉. The ability of internode tissues to import ¹⁴C-photosynthatedeclines with time from decapitation, and a decrease in incorporationof ¹⁴C-leucine into protein was detected after 24 h. There wasan increase in protein and RNA synthesis in internodal tissuesfollowing a 2.5-h pre-treatment of decapitated internodes withIAA, GA, and SD8₃₃₉. Concentrations of 2, 3, 5-triiodobenzoicacid which inhibit ¹⁴C-IAA translocation stimulate protein synthesisin decapitated internodes, and augment the IAA-effect on importof ¹⁴C-photosynthate. ‘Hormone-directed’ assimilatetransport is discussed in relation to confounding effects ofgrowth responses and differential senescence of treated anduntreated tissues. It is suggested that accumulation of labelledassimilate in treated tissues results from effects of growthregulators on synthetic activities at the point of application.     

Auxin-Promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: Auxin Dose-Response Curves and Effects of Inhibitors of Polar Auxin Transport

Transport of ¹⁴C-photosynthate in decapitated stems of Phaseolusvulgaris explants was dependent on the concentration of indole-3-aceticacid (IAA) applied to the cut surfaces of the stem stumps. Thephysiological age of the stem influenced the nature of the transportresponse to IAA with stems that had ceased elongation exhibitinga more pronounced response with a distinct optimum. Increasednutrient status of the explants had little influence on theshape of the IAA dose-response curve but increased, by two ordersof magnitude, the IAA concentration that elicited the optimalresponse. Applications of the inhibitor of polar auxin transport,1-(2-carboxyphenyl)-3-phenylpropane-1, 3-dione (CPD), affectedIAA-promoted transport of ¹⁴C-photosynthates. At sub-optimalIAA concentrations, CPD inhibited transport, whereas at supra-optimalIAA concentrations, ¹⁴C-photosynthate transport was marginallystimulated by CPD. Treatment with CPD resulted in a significantreduction in stem levels of [¹⁴C]IAA below the site of inhibitorapplication, while above this point, levels of [¹⁴C]1AA remainedunaltered. The divergent responses of auxin-promoted transportto CPD treatment are most consistent with a remote action ofIAA on photosynthate transport in the decapitated stems. Key words: Auxin, photosynthate, transport     

Degradation of Indol-3yl-acetic Acid in Homogenates and Segments of Cabbage Roots

Plots of reaction rate versus substrate concentration of the enzymatic decarboxylation of IAA yield sigmoid, rather than the usual, hyperbolic curves, suggesting that the IAA oxidase of cabbage roots is an allosteric enzyme. The quantity of this enzyme in roots is so high that the IAA concentration is likely to limit IAA degradation in intact cells. Thus, variations in the level of this enzyme seem not to be essential for the regulation of the endogenous IAA concentration. Cabbage roots contain substances that can inhibit IAA oxidase. These substances are spatially separated from IAA oxidase in intact cells, but the same inhibitors are able to reach the enzyme when added exogenously to tissue segments. The possibility that added IAA is treated by tissue segments in another manner than endogenous IAA is discussed.     

Occurrence of Endogenous Indol-3yI-Aspartic Acid in Light and Dark-Grown Bean Seedlings (Phaseolus vulgaris)

An acid ether-soluble, strongly growth-stimulating substance revealed by the Avena coleoptile straight-growth test in methanol extracts from bean seedlings (Phaseolus vulgaris L.) was identified as indol-3yl-aspartic acid (IAAsp). Points of agreement between synthetic IAAsp and the investigated growth stimulator were indicated by chromatographic behavior, elution volume in gel filtration, mobility in paper electrophoresis, “colour reaction” with DMCA reagent, ability to form indol-3yl-acetic acid (IAA) and aspartic acid after hydrolysis and, finally, biological activity in the Avena test. Furthermore, some experiments demonstrated the occurrence of an inhibitor in extracts from light-grown tissue. This masked the stimulation of IAAsp in the Avena test when the extracts had been chromatographed in isopropanol: NH₃:H₂ O. A comparison of the levels of IAAsp between green and etiolated tissue did not reveal any distinct difference, demonstrating that the IAA conjugate IAAsp does not participate in the regulation of the photomorphogenesis.     

Photolytic Decarboxylation of Carboxyl-14C-labelled Indol-3yl-acetic Acid in Leaves of the Apple Tree

The nature and rate of degradation of carboxyl-¹⁴C-labelledindol-3y1-acetic acid (IAA-[l-¹⁴C]) were studied in apple leaves.The labelled auxin was applied to the cut surface of the growingshoot after the apical part had been removed. The respiratoryCO₂ absorbed by chromatographic paper as Na₂CO₃ then freed byphosphoric acid was quantitatively measured by an internal gascounter. It was found that the concentration of ¹⁴CO₂ evolvedby leaves was 77 times higher in daylight than in darkness.The ratio of ¹⁴CO₂/CO₂ obtained from respiration from the uppersurface of leaf blades was two and seven times higher than thatfrom the lower surface after 15 and 30 h of daylight, respectively.No such differences were noticed in darkness. Similarly, thetotal radioactivity of leaf tissues tripled in daylight, presumablybecause of photosynthetic incorporation of radioactive CO₂ evolvedduring decomposition of LAA. These facts demonstrate the photolyticcharacter of auxin decarboxylation in apple leaves. Prolongeddarkness seemed to provoke a large metabolite withdrawal fromleaves and, to some extent, to protect auxin against decarboxylation.     

Indol-3yl-acetic acid in roots of Zea mays

Indol-3yl-acetic acid was identified in extracts of sterile roots of Zeamays seedlings by means of TLC, chromogenic reactions, GLC and GC-MS.     

Effects of Morphactin on Indol-3yl-acetic Acid Transport, Growth, and Geotropic Response in Cereal Coleoptiles

The effects of the morphactin 2-ehloro-9-hydroxyfluorene-9-carboxylicacid methyl ester [CFM] on growth, geotropic curvature and transportand metabolism of indol-3yl-acetic acid [IAA-5-³H] in the coleoptilesof Zea mays and A vena saliva have been investigated. A strongcorrelation has been found to exist between the inhibition ofthe geotropic response and the inhibition of auxin transport.CFM supplied at concentrations sufficient to abolish auxin transporthas been shown to promote the elongation of Zea, but not ofAvena, coleoptile segments. CFM does not change the patternof metabolism of IAA in Zea coleoptile segments. In these segmentsIAA is metabolized when its concentration is high, but the radioactivitytransported basipetally, or laterally in geotropically stimulatedcoleoptiles, is virtually confined to the IAA molecule. Radioactivityexported into the basal receiver blocks is wholly confined toIAA. It is concluded that CFM inhibits the geotropic responsein coleoptiles by suppression of the longitudinal and lateralauxin transport mechanisms. The growth-promoting propertiesof this substance cannot be linked with its effects on eitherauxin metabolism or transport.     

The Effect of Morphactin (Methyl 2-Chloro-9-hydroxyfluorene-9-carboxylate) on Basipetal Transport of Indol-3-ylacetic Acid in Hypocotyl Sections of Phaseolus vulgaris L.

The effect of morphactin (methyl 2-chloro-9-hydroxyfluorene-9-carboxylate)on basipetal transport of auxin (Indol-3-ylacetic acid-2-¹⁴C)was studied in bean (Phaseolus vulgaris) hypocotyl with thedonor-receiver block method. Morphactin (5 x 10^–6m) reduced IAA (5 x 10^–6m) transportintensity by an average of 83 per cent and auxin transport capacityby 90 per cent, but transport velocity was not affected. Morphactin did not inhibit uptake of IAA into hypocotyl tissue,but it did prevent transfer of IAA from the tissue into receiverblocks. Chromatographic analysis of the tissue after 4 h IAA-2-¹⁴Ctransport showed that 54 per cent of the total activity wasin the form of IAA in the control and 42 per cent in the morphactintreated tissue. No difference was found in the rate of decarboxylationof IAA-1-¹⁴C between control and morphactin treated tissue sections.Nor could any difference between control and morphactin be shownin the radioactivity associated with a TCA ppt fraction. Ina study of the transportable auxin pool, morphactin decreasedthe size of the pool and increased the half-life of decay ofauxin transport from 1•22 h to 3•85 h. In a kineticanalysis of the reversal of morphactin (5 x 10^–6m) inhibitionby increasing concentration of IAA-2-14C (5 x 10^–6m to2 x 10^–5m), it was shown that IAA transport resemblesMichaelis-Menten enzyme reaction kinetics, and that inhibitionby morphactin fitted a ‘mixed type’ model. IAA hada dissociation constant of 8•5 x 10^–6m and morphactinthat of 4•3 x 10^–7m with a K_m for the transport processof 8•5 x 10^–6m.     

Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L

Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed development of Phaseolus vulgaris L. cv. `Taylor's Horticultural.' An internal standard method of gas-liquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in `Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit.     

Tissue Cultures of Phaseolus vulgaris L.

Abstract

Callus production and plant regeneration from different explants of Phaseolus vulgaris L. cv. Giza are reported. Calli cultures were induced from leaf, hypocotyl, embryo and root explants. Rapid growth of callus was achieved by leaf explants cultured on MS salts, B5 vitamins and supplemented with 2,4— dichlorophenoxyacetic acid (2, 4—D)+0.5 mg/l kinetin (kin). Addition of casein hydrolysate at 2 g/l to maintenance medium enhanced callus growth and hindered the early appearance of necrotic parts. This report also provides a detailed method for production of multiple shoots directly from the wounded edges of immature cotyledon explant via organogenesis on 1 mg/l benzyladenine (BA) or indirectly on 0.5 mg/l naphthaleneacetic acid (NAA)+2 mg/l BA. The regeneration of bean plants through the two ways described here (direct or indirect) may be of use in genetic improvement of bean.     

Roles of Ethylene and Indol-3yl-acetic Acid in Petiole Epinasty in Helianthus annuus and the Modifying Influence of Gibberellic Acid

The effect of 12 h exposure to ethylene upon epinastic curvatureand elongation of a 5-cm segment in the attached petiole ofHelianthus annuus has been investigated in either normal orGA₂-treated plants. Curvature of segments occurred rapidly inthe first. 6 h during exposure of normal plants to either 1.0or 40.0 parts/10⁶ ethylene, and continued slowly from 6 to 12h. After the ethylene treatment, recovery from the induced curvaturewas completed in 12 h. In 0.2 parts/10⁶ ethylene, recovery fromthe epinastic curvature began during the second half of thetreatment period. Pre-treatment of plants with 60 µg GA₃,did not change the epinastic response to 40.0 parts/10⁶ ethylene.In 10.0 parts/10⁶ ethylene, recovery commenced towards the endof the treatment period, while in 1.0 parts/10⁶ the onset ofepinasty was delayed by about 6 h. In 0.2 parts/10⁶ ethylenethe epinastic response was slight. Ethylene accelerated elongation in the upper half of the petiolesegment. This response was completed within 12 h in all concentrationsand in both normal and GA₃-treated plants. The mean elongationrate in the lower half was depressed from 4.6 to 1.0 mm 24h^–1in 40.0 parts/10⁶ but immediately afterwards it rose to 14.2mm 24 h^–1. A similar response occurred in 1.0 parts/10⁶.In contrast, the elongation of the lower half of the petiolesegment was stimulated by 0.2 parts/10⁶ ethylene. GA₂-treatedplants showed an initial depression of elongation in the lowerhalf in 10.0 or 40.0 parts/10⁶ ethylene, but in the second partof the treatment period the elongation rate recovered to thatof the control segments. Both 0.2 and 1.0 parts/10⁶ ethylenestimulated elongation growth in the lower half of segments inGA₂-treated plants. Removal of the leaf lamina inhibited segment elongation, butdid not affect the growth response of the upper half to 40.0parts/10⁶ ethylene. In contrast the lower half of the segmentno longer showed the usual growth responses to 40.0 parts/10⁶ethylene, although these were partially retained when 10µgof IAA was applied to the cut end of the petiole.     

Apical Dominance in Phaseolus vulgaris L.

Although determinations of the ABA content of lateral buds ofPhaseolus vulgaris revealed no difference between decapitatedand intact control plants in the first 12 h following decapitation,a relative decrease in the ABA content of lateral buds of decapitatedplants was detectable 24 h following decapitation. Shoot decapitationwas also observed to result in a decrease in the ABA contentof stem tissue. The application of IAA to the stem of decapitatedplants prevented these changes and increased the ABA contentof stem tissue relative to that of intact plants. The levelsof IAA and ABA were also determined in the stem tissue fromthe nodes of intact bean plants. The possible interdependenceof these two plant hormones was further investigated by a studyof [2_–14ClABA metabolism. The results are discussed inrelation to the possible role of these hormones in apical dominance. Key words: Apical dominance, Abscisic acid, Indole-3-acetic acid     

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9--D-ribofuranosylpurine (dihydrozeatin riboside), and the 5-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O--D-glucoside and zeatin ribotide-O--D-glucoside. [8-¹⁴C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.Abbreviations TEAB triethyl ammonium bicarbonate - UV ultra-violet - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - TMS trimethyl silyl     

The Cellular Pathway of Radial Transfer of Photosynthates in Stems of Phaseolus vulgaris L.: Effects of Cellular Plasmolysis and p-Chloromercuribenzene Sulphonic Acid

Cellular plasmolysis with l M solutions of mannitol appearedto sever plasmodesmatal interconnections between all cells ofthe stems of Phaseolus vulgaris plants except the sieve element-companioncell (se—cc) complexes. Phloem loading and uptake of [¹⁴C]sucroseby the storage cells of the stems was unimpaired by cellularplasmolysis followed by rehydration of the stem tissues. Accumulationof phloem-transported ¹⁴C-photosynthates of the treated stemswas inhibited in summer-grown plants and unaffected in winter-grownplants indicating that phloem unloading follows a symplasticand a free-space route respectively depending on growth season.At a concentration that did not interfere with cellular metabolism,p-chloromercuribenzene sulphonic acid (PCMBS) applied to thestems blocked [¹⁴C]sucrose loading into the phloem and storagecells of the stem, but had no effect on the pool size of free-spacesugars. This latter response is consistent with a facilitatedmechanism of sugar unloading to the stem free-space. Accumulationof phloem-transported ¹⁴C-photosynthates was stimulated by PCMBSand this effect was most pronounced in winter-grown plants.Cellular plasmolysis followed by rehydration abolished the PCMBSaction on ¹⁴C-photosynthate accumulation. This effect is consistentwith a PCMBS induction of phloem unloading through the stemsymplast. It is proposed that phloem unloading in bean stemsmay follow either a free-space or symplastic route and thatthe latter route is entrained under sink-limited conditions. Phaseolus vulgaris, french bean, stem, phioem unloading, free-space, symplast     

Abscisic Acid Decreases Leaf Na+ Exclusion in Salt-Treated Phaseolus vulgaris L.

Previous results showed that in short-term NaCl-treated beans increased leaf abscisic acid (ABA) concentration was triggered by Na⁺ but not by Cl^-. In this work, the specificity of ABA signaling for Na⁺ homeostasis was studied by comparing the plant’s responses to solutions that modified accumulation of ABA and/or Na⁺ uptake and distribution, such as supplemental Ca²⁺, increased nutrient strength, different isosmotic composition, application of exogenous ABA, fluridone (an ABA inhibitor) and aminooxiacetic acid (AOA, an ethylene inhibitor). After fluridone pretreatment, salt-treated beans had lower Na⁺ uptake and higher leaf Na⁺ exclusion capacity than non-pretreated plants. Moreover, Na⁺ uptake was increased and leaf Na⁺ exclusion was decreased by AOA and ABA. NaCl and KCl similarly increased leaf ABA and decreased transpiration rates, whereas supplemental Ca²⁺ and increased strength nutrient solution decreased leaf ABA and leaf Na⁺. These results show (1) a non-ion-specific increase in ABA that probably signaled the osmotic component of salt, and (2) increased ABA levels that resulted in higher leaf Na⁺ concentrations due to lower Na⁺ exclusion or increased root-shoot Na⁺ translocation.