Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.Abbreviations 4Cl-IAA 4-chloro-indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA Indole-3-acetic acid - IAA-C5-BSA, IAA-C1-BSA, IAA-NI-BSA hapten-carrier-conjugates with IAA linked to bovine serum albumin through the C5-position, the carboxyl group, and the indole nitrogen, respectively - IAA-Me the methylester of IAA This study was supported by the Danish Research Council (SJVF 13-4148 and 13-4547 to P.U.) and by The Research Center for Plant Biotechnology.     

Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments

Branca, C, De Lorenzo, G. and Cervone, F. 1988. Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments. - Physiol. Plant. 72: 499–504.
α-D-galacturonide oligomers (OG) were prepared by partial hydrolysis of sodium polypectate with an homogeneous Aspergillus niger endopolygalacturonase (EC 3.2.1.15). OG, obtained after digestion for 10, 20, 30, 60, 120 min and 24 h, were assayed for their ability to interfere with the IAA-induced elongation of pea ( Pisum sativum L. cv. Alaska) stems. Maximum inhibiting activity was exhibited by oligomers with an approximate degree of polymerization higher than 8. Inhibition by longer OG was much lower, and the products of the 24 h digestion and the unhydrolysed polypectate were ineffective. The addition of OG to pea stems caused a parallel shift to the right of the IAA dose-effect curve. The shift depended on the amount of OG used, showing that oligogalacturonides behave as competitive antagonists of IAA. The presence of OG caused the disappearance of the second maximum of the elongation rate and reduced the first maximum. OG were also tested for their ability to inhibit IAA-induced ethylene evolution of pea stem segments. Maximal inhibition was obtained with OG of the same size as those that interfered with IAA-induced elongation. Inhibition of the auxin action seemed to be specific as OG did not interfere with the activity of gibberellic acid (GA₃) or kinetin. It was concluded that oligogalacturonides strongly interfere with the activity of IAA, although they are by themselves incapable to influence the elongation of pea stem segments directly.     

Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: II. Metabolic Requirements

The 2- to 4-fold rise in particle-bound β-glucan synthetase (uridine diphosphate-glucose: β-1, 4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca²⁺ at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by α, α′-dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor.     

INTERACTION OF SUGARS AND AUXINS IN PEA EPICOTYL SECTION GROWTH

Purves , W. K., and A. W. Galston . (Yale U., New Haven, Conn.) Interaction of sugars and auxins in pea epicotyl section growth. Amer. Jour. Bot. 47 (8): 665–669. Illus. 1963.—The nature and magnitude of the response of “S₁” etiolated pea-epicotyl sections to auxin are determined by the concentration of sugar in the growth medium. For example, the concentration of IAA inducing maximum elongation shifts through at least 3 orders of magnitude in response to varying sucrose concentrations, from ca. 10^–4 M, with no sucrose, to 10^–7 M, with 2% sucrose. Similarly, the inhibitory action of high levels of IAA on elongation occurs only in the presence of sucrose. By contrast, although sucrose also promotes water uptake, it affects the IAA optimum for this process only slightly. The action of IAA on growth can be detected immediately, but the growth response to sucrose occurs only after a 6–8 hr. lag. If tissues are supplied with sucrose, then 1-hr. exposures to IAA can be as effective on growth as continuous 20-hr. exposures, depending on the time at which such exposures are given. Thus, 10^—4 M IAA applied in the presence of 2% sucrose is markedly inhibitory to elongation in hours 1–3, relatively inactive in hours 4–6, and strongly promotive after hour 7. The change from inhibitory to promotive action thus coincides in time with the length of the lag period for sucrose action.     

Effects of benzisoxazole and benzisothiazole on tomato plant regeneration in vitro

The effects of two synthetic auxins, BOA and BIA, on plant regeneration in vitro have been studied on explants of tomato cotyledons. The activity of these substances on cell elongation has also been tested on pea stem segments. It has been found that BOA is particularly effective in inducing the formation of shoots but has a weak activity on cell elongation, while BIA, which is more effective in inducing cell elongation, is less active in morphogenesis. It is concluded that (1) the two activities are not related to each other, (2) the receptors involved in the two processes are probably different, (3) thechemical structure of the auxin may be an important factor in organogenetic processes.Abbreviations BIA 1,2-benzisothiazole-3-acetic acid - BOA 1,2-benzisoxazole-3-acetic acid - IAA indoleacetic acid - MS Murashige & Skoog medium     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Auxin structure and activity on tomato morphogenesis in vitro and pea stem elongation

In order to understand better the relationship between auxin structure and activity on morphogenesis and cell elongation, six different auxins were tested on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. The auxins were: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1, 2-benzisoxazole-3-acetic acid (BOA), 1,2-benzisothiazole-3-acetic acid (BIA), 1-naphthalenacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). All these compounds obey the minimum requirement rules for auxin activity and all were effective on cell elongation. At the dose of 10 M and in the absence of cytokinin, they all, except 2,4-D, induced roots, while in the presence of cytokinin they induced shoots, roots, hairy root-like filaments (HRLF) or callus depending on their concentration. The morphogenetic pattern did not change by varying cytokinin concentration. We conclude that auxin structure plays a minor role in morphogenesis or cell elongation, because it is only responsible for variations in the level of auxin activity.     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.     

Developmental changes in the gibberellin-induced growth response in stem segments of light-grown pea genotypes

The effects of GA on stem elongation were studied using segments from one tall and three dwarf light-grown pea genotypes varying in endogenous hormone content. Stem segments were cut at two distinct ages: when the fourth internode was at about 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Light microscopy and flow cytometry were used to demonstrate that GA does not induce cell division in excised pea stem segments. The growth studied here was strictly elongation. Measurement of final segment length after 48 hours and high resolution measurement of growth kinetics over 20 hours using an angular position transducer were done on segments treated with hormone solutions. Our data indicate that the action of GA on stem elongation can be classified into two distinct modes. The first, apparent in early-expansion stem segments, shows distinct growth kinetics and is independent of the endogenous IAA concentration of the segments. Quantitation of IAA by GC/MS in early-expansion segments of wild type pea incubated with gibberellin shows that an increase in IAA concentration is part of the GA response in such segments. The second mode of GA action is evinced in mid-expansion segments. Whereas there is no short term (<20 h) response to GA alone (as determined by growth kinetics), there is a long term (48 h) response whose magnitude decreases across the genotypes with decreasing endogenous hormone content. Growth responses indicate that in mid-expansion segments exogenous GA acts by enhancing IAA action but appears to be unable to augment endogenous IAA content. Contradictory reports of the response of excised stem segments to GA can be reconciled when tissue genotype and developmental stage are considered.     

Short-term response of Pisum stem segments to indole-3-acetic acid

The short-term response of green pea stem segments to indole-3-aceticacid (IAA) was investigated by continuously recording stem elongationwith a differential transformer. Stem segment elongation promotedby IAA began following a latent period after application. Thelatent period was more effectively shortened by raising thetemperature rather than the concentration of IAA; it was reducednearly to 0 min by treatment at 40?C. The length of the latentperiod was not affected by turgor pressures of stem cells, thoughthe rate of stem growth was diminished at lower turgor pressures.Stems pretreated with actinomycin D for 60 min, cycloheximidefor 30 min or colchicin for 6 hr were similar to untreated stemsin their short term response to IAA. This implies that the initiallypromoted elongation does not result from the activity of enzymessynthesized during the latent period by the action of IAA. (Received April 5, 1973; )     

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg²⁺ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K _m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k _cat/K _m = 682,608.7 and 5080 s⁻¹ M⁻¹, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.     

The involvement of indole-3-acetic acid in the control of stem elongation in dark- and light-grown pea (Pisum sativum) seedlings

We investigated the role of auxin on stem elongation in pea (Pisum sativum L.) grown for 10d in continuous darkness or under low-irradiance blue, red, far red and white light. The third internode of treated seedlings was peeled and the tissues (epidermis and cortex+central cylinder) were separately analyzed for the concentration of free and conjugated indole-3-acetic acid (IAA). Under red, far red and white light internode elongation was linearly related with the free IAA content of all internode tissues, suggesting that phytochrome-dependent inhibition of stem growth may be mediated by a decrease of free IAA levels in pea seedlings. The correlation between IAA and internode elongation, however, did not hold for blue light-grown seedlings. The hypothesis that the growth response under low-irradiance blue light might be correlated with the lack of phytochrome B signalling and changes in gibberellin metabolism is discussed in view of current knowledge on hormonal control of stem growth.     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

Effect of Gibberellic Acid and Cycocel on Tryptophan Metabolism and Auxin Destruction in the Sunflower Seedling

A comparative study of tryptophan conversion in different regions of the sunflower seedling indicates that the regions most active in converting tryptophan on a pathway to auxin are the root apical segments and young leaves; next highest in activity is the cotyledonary tissue. The stem apex proper with leaf primordia is less active than the above regions in converting the auxin precursor. Hypocotyl tissue was observed to be least active. Pre-treatment of the apical bud region of the stem with gibberellic acid (GA) gives rise to tryptophan conversion rates which are 2.1 times those in untreated seedlings. The enhanced tryptophan conversion in the apical bud is followed by an increased elongation rate of the 1st internode which is 2.2 times that in the 1st internode of untreated seedlings. Treatment of the seedlings with Cycocel [(2-chloroethyl)trimethylamnionium chloride] does not reduce tryptophan conversion in the apical bud region of the seedling although elongation of the stem is greatly retarded. Indoleacetic acid (IAA) destruction in cell free preparations as well as in whole sections of the elongating region of the seedling stem was studied. IAA-1-¹⁴C destruction rates with the release of ¹⁴CO₂ in whole sections of 1st internode tissue were approximately 3 times those in cell free preparations of the same region. No significant changes in IAA destruction rates in seedlings pre-treated with GA or Cycocel were observed.     

Molecular properties of 4-substituted indole-3-acetic acids affecting pea pericarp elongation

Pea (Pisum sativum L.) fruit naturally contain the auxins, indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA). However, only 4-Cl-IAA can substitute for the seeds in maintaining pea fruit growth in planta. The importance of the substituent at the 4-position of the indole ring was tested by comparing the molecular properties of 4-X-IAA (X = H, Me, Et, F, or Cl) and their effect on the elongation of pea pericarps in planta. Structure-activity is discussed in terms of structural data derived from X-ray analysis, computed conformations in solution, semiempirical shape and bulk parameters, and experimentally determined lipophilicities and NH-acidities. The size of the 4-substituent, and its lipophilicity are associated with growth promoting activity of pea pericarp, while there was no obvious relationship with electromeric effects.     

Auxin movement in corn coleoptiles

Summary Movement of radioactive auxins was analysed in corn coleoptile sections. The results support the idea that processes involved in the transport of indoleacetic acid (IAA) are specific for growth-promoting auxins.Inhibition of IAA transport by triiodobenzoic acid is caused by a reversible block of the exit; the auxin held back remains in the transport pool. The observed increase in immobilization may be a secondary effect caused by the increased concentration of free IAA in the tissue.Auxin molecules are most likely transported by anon-covalent mechanism. IAA and naphthaleneacetic acid (NAA) move through the cell and exit as free molecules. A search for a transient auxin complex, chaseable as required for any transport carrier intermediate, yielded negative results. No¹⁸O was lost from NAA labeled with¹⁸O in the carboxyl group during transport of the auxin through coleoptile tissue.After application of IAA to auxin-depleted tissue, the transport rate undergoes oscillations with a period length of ca. 25 min.The movement of the auxin 2.4-dichlorophenoxyacetic acid which is usually sluggish, increased several times if some IAA was added. Auxin, thus, stimulates its own transport.A model is discussed in which auxin-binding to the plasma membrane and reversible changes of membrane conformation may provide a basis for active secretion and for the observed cooperativity. Leo Brauner zum 70. Geburtstag gewidmet.     

Stimulatory effects of 2,4-dichlorophenoxyacetic acid and of 1-naphthylacetic acid on sucrose level,invertase activity and sucrose utilization in the latex ofHevea brasiliensis

Summary Treatment of the bark ofHevea brasiliensis with 2,4-dichlorophenoxyacetic acid (2,4-D) or l-naphthylacetic acid (NAA) greatly increases sucrose level, invertase activity and sucrose utilization in the latex; the efficacy of 2,4-D is considerably greater than that of NAA. The greater sucrose utilization is the consequence of increased invertase activity. The changes occur as soon as the first tapping following bark treatment. It is suggested that the rise in both sucrose level and utilization in the latex serum mediate the effect of auxins on latex production. This is most likely related to a faciliation of latex outflow resulting from an increase in the osmotic and turgor pressure in the laticiferous tissue, as well as to enhanced regeneration of latex.The latex invertase has been found to be of a weakly alkaline type, with a sharp pH optimum at 7.15–7.20 in citrate-phosphate buffer. Its activity falls of rapidly on the acid side, being almost zero at pH 6.4. Since the natural pH of latex generally varies between pH 6.5 and 7.0, it is suggested that pH is an important factor in the regulation of invertase activity in the latex, and that the limiting nature of invertase-mediated sucrose hydrolysis in latex serum is caused by unfavourable conditions for invertase activity rather than by a scarcity of this enzyme.Expert of the International Atomic Energy Agency.     

Invertase and auxin-induced elongation in internodal segments of Phaseolus vulgaris

A close positive correlation was observed between segment elongation and the specific activity of soluble acid invertase in stem segments of P. vulgaris incubated for 21 hr in the presence of IAA or of several synthetic auxins and auxin analogues. Optimum concentrations for the stimulation of growth and invertase activity were similar and varied from 10^?6 M (2,4-D) through 10^?5 M (IAA, IBA, α-NAA, β-NAA) to greater than 10^?4 (IPA, PoAA, trans-cinnamic acid). The weak activity of trans-cinnamic acid, a competitive inhibitor of auxin action, may have resulted from cis-trans isomerization during incubation. The concentration of hexose sugars in the segments fell during incubation in the presence of auxin, the greatest decline in hexose concentration occurring in the presence of compounds exhibiting the greatest stimulation of growth.     

Indoleacetic Acid-Induced Decrease of the Ribomiclease Activity in vivo

A study has been made on the influence of indole-3-acetic acid (IAA) on the ribonuclease (RNase) activity in wheat coleoptile sections and green pea stem sections. The hormonal effects on the enzyme activity, ribonncleic acid (RNA) metabolism and growth have been compared. Addition of 10^?5M IAA to the plant sections causes their RNase activity to decrease and their elongation to increase. Removal of the added IAA results in increasing enzyme activity and decreasing growth. The altered enzyme activities are paralleled by opposite changes in the RNA net synthesis. Administration of crystalline RNase to the plant tissue depresses growth. There is thus evidence that the in vivo effect of IAA on the RNase activity is of importance for the hormonal regulation of RNA metabolism and growth. The IAA-induced reduction in the enzyme activity involves cellular metabolism. The effect can be suspended by means of p-chloromercuribenzoate. A possible mechanism for the reduction is discussed.