Auxin Does Not Alter the Permeability of Pea Segments to Tritium-labeled Water

The possibility of an auxin effect on the permeability of pea (Pisum sativum L. ev. Alaska) segments to tritium-labeled water has been investigated by three separate laboratories, and the combined results are presented. We were unable to obtain any indication of a rapid effect of indoleacetic acid on the efflux of ³HHO when pea segments previously “loaded” for 90 minutes with ³HHO were transferred to unlabeled aqueous medium with indoleacetic acid. We were able to confirm that segments pretreated with ³HHO plus indoleacetic acid for 60 to 90 minutes can show an enhanced ³HHO release as compared with minus indoleacetic acid controls. However, this phenomenon appears to be due to an increased uptake of ³HHO during the prolonged indoleacetic acid pretreatment, and therefore we conclude that auxin does not alter the permeability of pea segments to ³HHO in either short term or long term tests. We confirm previous reports that the uptake of ³HHO in pea segments proceeds largely through the cut surfaces, and that the cuticle is a potent barrier to ³HHO flux.     

Relazioni ormonali nello sviluppo dell'ovario delle orchidee II. Azione delle auxine in segmenti isolati dell'asse fiorale

Abstract

HORMONAL RELATIONS IN THE DEVELOPMENT OF ORCHID OVARY. II. — The effects of auxins on isolated parts of the flower axis. — The changes in fresh weight of segments of column, ovary and peduncle of Cymb[icaron]dium sp., treated in vitro with auxins (IAA, EtIA and NAA) indicate that these parts of the flower react promptly and independently to the three auxins tested. Expansion is very small in the ovary, and is more and more large moving towards the apex of the flower (fig. 1). The apical part of the column reacts somewhat more rapidly to auxin and tolerates higher concentration than the proximal part.

Naphtalenacetic acid proved to be the most active of the three auxins tested. This finding is in agreement with the higher efficiency of this substances as an inducer of parthe-nocarpy and its ability to induce large necroses of the column, when applied to the flower on the intact plant.

The plots of auxin activity against concentration of the auxins suggest thai the lower activity of IAA might be due to its destruction by indoleacetic acid oxidase.     

Elongation of Stem Internodes in the Japanese Morning Glory (Pharbitis nil) in Relation to Auxin Destruction

The relationship between auxin destruction and stem internode elongation was investigated in the vines of the Japanese morning glory (Pharbitis nil Choisy). In young plants an age-dependent gradient was demonstrated in which the decreasing rate of elongation of older internodes correlated with an increasing ability of such tissue to destroy indoleacetic acid. Fragments of tissue from old internodes when incubated with indoleacetic acid (IAA), destroyed the hormone immediately and rapidly; in contrast, young, rapidly elongating internode tissue destroyed IAA only after a lag of several hours. In older plants the gradient was more erratic towards the middle of the plant but old and young tissue behaved as in young plants, i.e., old internodes destroyed IAA rapidly whereas young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing an internal environment in which auxin is rapidly destroyed.     

Regulation of Auxin Levels in Coleus blumei by Ethylene

An investigation of the effects of ethylene pretreatment on several facets of auxin metabolism in Coleus blumei Benth “Scarlet Rainbow” revealed a number of changes presumably induced by the gas. Transport of indoleacetic acid-1-¹⁴C in excised segments of the uppermost internode was inhibited by about 50%. Decarboxylation of indoleacetic acid-1-¹⁴C by enzyme breis was not affected by the pretreatment. Levels of extractable native auxin in upper leaf and apical bud tissue of the pretreated plants were approximately one-half of those present in untreated plants. The rate of formation of auxin from tryptophan by enzyme breis from pretreated plants was approximately one-half that occurring in incubation mixtures containing the enzyme system from untreated plants. The conjugation of indoleacetic acid-1-¹⁴C in a form characterized chromatographically as indoleacetylaspartic acid was increased 2-fold in the upper stem region of plants pretreated with ethylene.     

Mechanism of Auxin-induced Ethylene Production

Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.     

Osservazioni Sull'effetto Inibente di Elevate Pressioni Osmotiche su Diversi Tipi di Crescita

Abstract

On the inhibition of seeds germination and of growth by cell enlargement by the osmotic pressure of the medium. — The mechanism of inhibition by osmotic pressure (O.P.) of the medium on growth and respiration of germinating wheat, castor bean and lettuce sèeds and of etiolated pea internode segments was investigated.

The following results were obtained:

1 - External osmotic pressure (up to 0.3 M) of various substances such as mannitol, urea, glucose, NaCl, was shown to inhibit the germination and growth of lettuce, wheat and castor bean seeds.

2 - a) A remarkable decrease of the development of respiration during the first 48 h of germination was demonstrated in embryos of wheat seeds germinated and maintained in mannitol solutions at concentration from 0,2 to 0,3 M.

b) A slight but reproducible inhibition of óxygen uptake by O.P. was also observed in embryos isolated from wheat seeds germinated in water for 24 and 34 h and transported respectively in water or into 0,2 M mannitol solutions.

This is interpreted as indicating that high external O.P. inhibits both the respiratory metabolism and the development with time of enzyme systems supporting respiration.

3 - Mannitol solutions (0,2–0,3 M) inhibited completely growth by cell enlargement in pea internode sections, while they did not at all affect oxygen uptake and protein synthesis ( ¹⁴ C - leucine incorporation). The stimulatory effect of auxin on pea elongation was almost completely suppressed by mannitol, whereas the hormone stimulation of respiration remained unchanged.

These data are interpreted as indicating that in tissues, presenting an advanced differentiation, high external O.P. inhibits growth by a direct physico-chemical mechanism; while the inhibitory effect in embrional tissues seems to comprehend, besides this direct effect, a complicated metabolic component, apparently influencing protein synthesis.     

Auxin Enhancement of mRNAs in Epidermis and Internal Tissues of the Pea Stem and Its Significance for Control of Elongation

The epidermis has been considered the site of auxin action on elongation of stems and coleoptiles. To try to identify mRNAs that might mediate auxin stimulation of cell enlargement, we compared, using in vitro translation assays, mRNA enhancement by indoleacetic acid (IAA) in the epidermis, with that in the internal tissues, of pea (Pisum sativum L., cv Alaska) third internode segments. We used seedlings that had been grown under red light, which enables the epidermis to be peeled efficiently from the internode. Most of the `early' IAA enhancements previously reported using etiolated peas, plus several hitherto undescribed enhancements, occur in both the epidermis and the internal tissue of the light-grown plants after 4 hours of IAA treatment. These enhancements, therefore, do not fulfill the expectation of elongation-specific mRNAs localized to the epidermis. One epidermis-specific IAA enhancement does occur, but begins only subsequent to 1 hour (but before 4 hours) of auxin treatment. Similarly, the previously mentioned IAA enhancements common to epidermis and internal tissue do not begin, in the light-grown plants, within 1 hour of IAA treatment. Since IAA stimulates elongation in light-grown internodes within 15 minutes, it appears that none of these mRNAs can be responsible for auxin induction of elongation. We confirmed, with our methods, the previous reports that some of these mRNAs are enhanced by IAA within 0.5 hour in etiolated internodes. This indicates that we could have detected an early enhancement in light-grown tissue had it occurred.     

Effect of gibberellic Acid on elongation and longevity of coleus petioles

The effects of gibberellic acid on the longevity and elongation of variously aged, debladed petioles of Coleus blumei were studied, with particular reference to the hypotheses 1) that auxin increases longevity by increasing growth, and 2) that gibberellic acid acts by increasing the endogenous levels of auxin.

Gibberellic acid, substituted for the leaf blades, significantly decreased longevity of younger petioles, as measured by days or hours to abscission. Gibberellic acid also decreased the longevity resulting from 0.1% indoleacetic acid. This is the opposite of the effect expected if it is increasing auxin levels in the petiole.

In its effect on elongation of younger petioles, however, gibberellic acid did act in the direction expected if it were increasing effective levels of auxin in the petiole. The elongation rate from 0.1% gibberellic acid plus 0.1% indoleacetic acid in lanolin was as large or larger than that for 1.0% indoleacetic acid.

Petioles which were 10 or more weeks old (i.e., at positions 5+ below the apical bud were not affected by 0.1% gibberellic acid in either longevity or rate of elongation, with or without 0.1% indoleacetic acid. Since 1.0% indoleacetic acid increases both longevity and elongation rate of these petioles over 0.1% indoleacetic acid, gibberellic acid is clearly not acting on older petioles as if it were increasing effective auxin levels).

     

Promotion of Xyloglucan Metabolism by Acid pH

Like indoleacetic acid, buffers of acidic pH, which stimulate elongation of pea (Pisum sativum var. Alaska) stem tissue, induce the appearance within the tissue of a watersoluble xyloglucan polymer that probably arises from previously deposited wall material. Neutral pH buffers, which inhibit the elongation response to indoleacetic acid in this tissue, inhibit indoleacetic acid-induced increase in soluble xyloglucan. The findings provide further evidence that release of soluble xyloglucan from the cell walls of pea results from the biochemical action on the cell wall that is responsible for wall extension. The data also indicate that treatment of tissue with either auxin or acidic pH has a similar biochemical effect on the cell wall. This is consistent with the H⁺ secretion theory of auxin action.     

Turnover of cell wall polysaccharides in elongating pea stem segments

Turnover of cell wall polysaccharides and effects of auxin thereon were examined after prelabeling polysaccharides by feeding pea (Pisum sativum var. Alaska) stem segments ¹⁴C-glucose, then keeping the tissue 7 hours in unlabeled glucose with or without indoleacetic acid. There followed an extraction, hydrolysis, and chromatography procedure by which labeled monosaccharides and uronic acids were released and separated with consistently high recovery. Most wall polymers, including galacturonan and cellulose, did not undergo appreciable turnover. About 20% turnover of starch, which normally contaminates cell wall preparations but which was removed by a preliminary step in this procedure, occurred in 7 hours. Quantitatively, the principal wall polymer turnover process observed was a 50% decrease in galactose in the pectinase-extractable fraction, including galactose attached to a pectinase-resistant rhamnogalacturonan. Other pectinase-resistant galactan(s) did not undergo turnover. No turnover was observed in arabinans, but a doubling of radioactivity in arabinose of the pectinase-resistant, hot-acid-degradable fraction occurred in 7 hours, possibly indicating conversion of galactan into arabinan. None of the above changes was affected by indoleacetic acid, but a quantitatively minor turnover of a pectinase-degradable xyloglucan was found to be consistently promoted by indole-acetic acid. This was accompanied by a reciprocal increase in water-soluble xyloglucan, suggesting that indoleacetic acid induces conversion of wall xyloglucan from insoluble to water-soluble form. The results indicate a highly selective pattern of wall turnover processes with an even more specific influence of auxin.     

THE EFFECTS OF AUXINS AND KINETIN ON XYLEM DIFFERENTIATION IN THE PEA EPICOTYL

Sorokin , Helen P., S. N. Mathur , and Kenneth V. Thimann . (Harvard U., Cambridge, Mass.) The effects of auxins and kinetin on xylem differentiation in the pea epicotyl. Amer. Jour. Bot. 49(5): 444–454. Illus. 1962.—Treatment of isolated segments from the second internode of etiolated ‘Alaska’ pea epicotyls with indoleacetic acid or 2,4-D results in: (1) activation of fascicular cambium, and initiation of some interfascicular cambium, resulting in abundant production of secondary xylem, and in formation of hyperplastic tissue; (2) partial or even total occlusion of proto- and metaxylem. The secondary xylem formed consists of short vessel members with scalariformly reticulate or pitted walls, which often lack vertical connection with each other, being interrupted by unlignified cells. When IAA is used, the hyperplastic growth mainly takes the form of root primordia, whereas 2,4-D initiates the formation of callus, but not of root primordia. The growth of this callus causes a characteristic split at the base of the internode. Treatment with kinetin, alone or in combination with the auxin, changes the above structure markedly. It leads to the initiation, over the entire circumference of the core of the internode, of a still more active cambium, which forms several layers of secondary xylem; this consists mainly of long vessel members with pitted walls. Hyperplastic growth is completely absent, and the xylem does not become occluded. Thus the effect of kinetin is to make the xylem more normal and to alter the epicotyl structure from herbaceous to more-or-less woody.     

Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-triiodobenzoic acid

Summary The application of 2,3,5-triiodobenzoic acid (TIBA, 10 mg·g^-1 in lanolin) to the stem of intact pea seedlings (Pisum sativum L.) inhibited the basipetal transport of ¹⁴C from indoleacetic acid-1-¹⁴C (IAA-1-¹⁴C) applied to the apical bud, but not the transport of ¹⁴C in the phloem following the application of IAA-1-¹⁴C or sucrose-¹⁴C to mature foliage leaves. It was concluded that fundamentally different mechanisms of auxin transport operate in these two pathways.When TIBA was applied at the same time as, or 3.0 h after, the application of IAA-1-¹⁴C to the apical bud, ¹⁴C accumulated in the TIBA-treated and higher internodes; when TIBA was applied 24.0 h before the IAA-1-¹⁴C, transport in the stem above the TIBA-treated internode was considerably reduced. TIBA treatments did not consistently influence the total recovery of ¹⁴C, or the conversion of free IAA to indoleaspartic acid (IAAsp). These results are discussed in relation to the possible mechanism by which TIBA inhibits auxin transport,.Attention is drawn to the need for more detailed studies of the role of the phloem in the transport of endogenous auxin in the intact plant.Abbreviations TIBA 2,3,5-triiodobenzoic acid - IAAsp indoleaspartic acid     

Effects of the auxin-inhibiting substances raphanusanin and benzoxazolinone on apical dominance of pea seedlings

The effects of the auxin-inhibiting substances raphanusanin ((3R*,6S*)-3-[methoxy (methylthio) methyl]-2-pyrrolidinethione, raphanusanin B)and benzoxazolinone (6-methoxy-2-bezoxazolinone, MBOA) on apical dominance of pea(Pisum sativum L. cv. Alaska) seedlings were studied.Application of raphanusanin B or MBOA to the apical bud, internode, or lateralbud of pea seedlings released apical dominance in either intact orindole-3-acetic acid (IAA )-treated, decapitated plants. These results suggestthat the auxin-inhibiting substances raphanusanin B and MBOA have activity inreleasing apical dominance. Conversely, the auxin transport inhibitors2,3,4-triiodobenzoic acid (TIBA) and 1-naphthylphthalamic acid (NPA) did notstimulate lateral bud growth when they were applied directly to the lateralbud,although application to the apical bud or internode released apical dominance.Therefore, the mode of action of raphanusanin B and MBOA in apical dominance isclearly different from that of auxin transport inhibitors. Raphanusanin B andMBOA may suppress the synthesis of growth-inhibiting factor(s) of the lateralbud induced by endogenous auxin transported from the apical bud or exogenouslyapplied auxin, and/or the action of the factor(s).     

Ethylene and auxin-induced cell growth in relation to auxin transport and metabolism and ethylene production in the semi-aquatic plant,Regnellidium diphyllum

Cell elongation in the rachis of the semiaquatic fern Regnellidium diphyllum is induced by the addition of ethylene or indoleacetic acid (IAA). Experiments with whole plants or rachis segments have shown that ethylene-induced growth requires the presence of auxin. Ethylene does not cause a modification in either endogenous auxin levels or in the extent of auxin metabolism but auxin transport is reduced. Rates of ethylene production in Regnellidium are not altered by either mechanical excitation or by the addition of auxin. A two-hormone control of cell expansion is proposed in which an initial, auxin-dependent growth event pre-conditions the cells to a further subsequent (or synchronous) ethylene-dependent growth event.Abbreviation IAA indole-3yl-acetic acid     

Auxin-induced longitudinal-to-transverse reorientation of cortical microtubules in nonelongating epidermal cells of azuki bean epicotyls

Summary In epidermal cells of azuki bean (Vigna angularis) epicotyl segments, that were sequentially treated with an auxin-free solution and an auxin solution, cortical microtubules changed their orientation from longitudinal to transverse. Auxin caused the reorientation of microtubules from longitudinal to transverse in segments that were kept under anaerobic conditions and, therefore, showed no elongation, indicating that auxin can regulate the orientation of microtubules by a mechanism that does not involve auxin-induced change in the rate of cell elongation.Abbreviations DMSO dimethylsulfoxide - GA₃ gibberellic acid - IAA indoleacetic acid - MT microtubule - PBS phosphate-buffered saline     

Localization and partial characterization of the extracellular proteins centrifuged from pea internodes

The proteins removed from the extracellular space of dark-grown pea ( Pisum sativum L. cv. Alaska) internode sections by centrifugation were studied. A large number of proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These proteins ranged in size from 10 to 150 kdalton and their removal from the cell wall was greatly facilitated by the presence of salts of divalent and trivalent cations in the infiltration medium. Pulse-labelling experiments with [³⁵S)-methionine showed that many of the proteins extracted from the cell wall incorporated radioactivity and that treatment with indoleacetic acid (IAA) altered the pattern of radiolabel incorporation. One of the proteins centrifuged from pea internode sections possessed per-oxidase (EC 1.11.1.7) activity. The activity of this peroxidase increased less in auxin-treated internode segments than in untreated controls. Antibodies were raised to the total protein fraction extracted by centrifugation and used to localize antigens on protein blots. Most of the proteins centrifuged from pea internode sections were stained by a dye coupled to the cell wall antiserum. Light microscopic immunohistochemical studies showed that the proteins centrifuged from dark-grown pea internodes were localized almost exclusively in the cell wall and intercellular spaces of pea internode tissue. Light microscopic immunohistochemistry also showed that antibodies to extracted proteins penetrate into the apoplast of abraded pea internode segments and split pea stems. These antibodies did not influence growth of IAA-treated or control tissue.     

Effects of calcium and kinetin on growth and cell wall composition of pea epicotyls

Kinetin and CaCl₂, in the presence of indoleacetic acid, promoted lateral expansion of epicotyls of decapitated and derooted Alaska pea seedlings (Pisum sativum L.) and inhibited their elongation. This growth response was correlated with the development of cell walls unusually rich in pectic uronic acids. Epicotyls in calcium-auxin solutions continued to enlarge and to add new wall material long after tissues in auxin only had stopped. Longitudinal enlargement, associated with the development of walls poor in pectic uronic acids, was favored by KCl, MgCl₂, and ethylenediaminetetraacetate. The last of these agents promoted the loss of ⁴⁵Ca from the epicotyls. Seedings grown in vermiculite moistened with CaCl₂, KCl, or MgCl₂ solutions did not differ in appearance or in the composition of their walls. They responded similarly to experimental treatment except that the decapitated epicotyls of the MgCl₂-grown plants suffered an absolute loss of pectic uronate when incubated in that salt.     

Chromatographic degradation of phloridzin

Phloridzin, the main phenolic glucoside in apple leaves, has been found to undergo transformation during chromatography. When chromatographed repeatedly in ammoniacal solvents, at least 2 new derivatives appeared. One of these was identified as phloretic acid. When bioassayed in the presence of indole-3-acetic acid this substance behaved as though it promoted the destruction of the auxin. Comparative bioassay with naphthaleneacetic acid suggested that phloretic acid acts on indoleacetic acid destruction via stimulation of indoleacetic acid oxidase. However, at low concentration and in presence of a small amount of phloridzin it also showed a synergistic effect with indoleacetic acid.

A substance with the same characteristics was obtained directly from apple leaves, which are known to contain phloridzin when the extracts were chromatographed only once in the same (alkaline) solvent. While not completely confirmed, this suggests that phloretic acid is normally present in apple leaves, where it may affect growth there by promoting indoleacetic acid oxidation.

     

Auxin-induced synthesis of TB-RNA in pea stems

The effect of 2,4-dichlorophenoxyacetic acid on RNA synthesisin etiolated pea internode segments was studied. Auxin stimulatedsynthesis of TB-RNA only in short period incubation, suggestingits primary importance in auxin action. The nucleotide compositionof newly synthesized TB-RNA seems to be modified under auxininfluence. (Received February 8, 1969; )