Conversion of indole-3-ethanol to indole-3-acetic Acid in cucumber seedling shoots

Indoleethanol-¹⁴C was applied to intact cucumber seedlings and to hypocotyl segments. The presence of indoleacetic acid-¹⁴C in tissue extracts was demonstrated by thin layer radiochromatography. There was no evidence of conversion of indoleacetic acid to indoleethanol. It is suggested that the growth-promoting activity of indoleethanol is due to its conversion to indoleacetic acid.     

Tracheid production in response to indole-3-acetic acid varies with internode age in Pinus sylvestris stems

Summary Different concentrations of indole-3-acetic acid (IAA) in lanolin were applied to the cambial region of approximately 10- and 34-year-old internodes in the main stem of Pinus sylvestris (L.) trees during the tracheid production period. After 5 weeks of treatment, the radial width of xylem produced in both ages of internode was positively related to exogenous IAA concentration measured at 0, 1 and 3 cm directly below the application site. Tracheid production in response to exogenous IAA in the 34-year-old internode was approximately one-half of that in the 10-year-old internode. The endogenous IAA level in the 7-, 17- and approximately 34-year-old internodes of similar trees was measured by radioimmunoassay, using gas chromatography-selected ion monitoring-mass spectrometry for validation. No consistent relationship was found between xylem radial width and IAA concentration. The data indicate that the cambium's ability to respond to exogenous IAA is qualitatively the same in 1-year-old shoots and older internodes. However, as the internode ages, there is a decrease in the extent of the response and in the optimal IAA level for inducing tracheid production.     

Effect of defoliation on tracheid production and the level of indole-3-acetic acid in Abies balsamea shoots

To simulate feeding by the spruce budworm ( Choristoneura fumiferana Clem.), the apical current-year shoots on 1-year-old branches in the uppermost whorl of 6-year-old balsam fir [ Abies balsamea (L.) Mill.] trees were either removed completely by debudding before the start of the growing season or defoliated 0, 50, 90 or 100% shortly after budbreak. Debudded branches were treated at the apical end with 0, 0.1 or 1.0 mg of indole-3-acetic acid (IAA) (g lanolin)⁻¹. Ninety % of the 1-year-old needles were also removed from some of the experimental branches. After ca 4 weeks of growth, the radial width of new xylem and the level of IAA were determined in the 1-year-old internode. The IAA content was measured by radioimmunoassay.
The removal or defoliation of current-year shoots inhibited tracheid production and decreased the IAA level. Exogenous IAA stimulated tracheid production and increased the IAA level in debudded branches. Current-year shoot defoliation also inhibited current-year shoot elongation. The inhibitory effect of current-year needle removal on all parameters generally increased with increasing intensity of defoliation. The removal of 1-year-old needles did not affect the IAA level or current-year shoot elongation, nor did it influence tracheid production in branches with current-year shoots. However, removal of 1-year-old needles inhibited tracheid production in debudded branches supplied with exogenous IAA. The results indicate that (1) IAA is involved in the control of tracheid production in the 1-year-old internode, (2) IAA is supplied primarily by current-year shoots, and (3) defoliation by the spruce budworm inhibits tracheid production partly by decreasing the supply of IAA.     

Seasonal variation in respiration of 1-year-old shoots of scots pine exposed to elevated carbon dioxide and temperature for 4 years

Sixteen 20-year-old Scots pine (Pinus sylvestris L.) trees growing in the field were enclosed for 4 years in environment-controlled chambers that maintained: (1) ambient conditions (CON); (2) elevated atmospheric CO2 concentration (ambient + 350 micro mol mol-1; EC); (3) elevated temperature (ambient +2-6 degrees C; ET); or (4) elevated CO2 and elevated temperature (ECT). The dark respiration rates of 1-year-old shoots, from which needles had been partly removed, were measured over the growing season in the fourth year. In all treatments, the temperature coefficient of respiration, Q10, changed with season, being smaller during the growing season than at other times. Respiration rate varied diurnally and seasonally with temperature, being highest around mid-summer and declining gradually thereafter. When measurements were made at the temperature of the chamber, respiration rates were reduced by the EC treatment relative to CON, but were increased by ET and ECT treatments. However, respiration rates at a reference temperature of 15 degrees C were reduced by ET and ECT treatments, reflecting a decreased capacity for respiration at warmer temperatures (negative acclimation). The interaction between season and treatment was not significant. Growth respiration did not differ between treatments, but maintenance respiration did, and the differences in mean daily respiration rate between the treatments were attributable to the maintenance component. We conclude that maintenance respiration should be considered when modelling respiratory responses to elevated CO2 and elevated temperature, and that increased atmospheric temperature is more important than increasing CO2 when assessing the carbon budget of pine forests under conditions of climate change.     

Ethylene-enhanced catabolism of [C]indole-3-acetic Acid to indole-3-carboxylic Acid in citrus leaf tissues

Exogenous [¹⁴C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole-3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [¹⁴C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGIu). Increased formation of ICGIu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGIu were identified by combined gas chromatography-mass spectrometry. Formation of ICGIu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene reduces endogenous IAA levels.     

Developmental regulation of indole-3-acetic acid turnover in Scots pine seedlings

Indole-3-acetic acid (IAA) homeostasis was investigated during seed germination and early seedling growth in Scots pine (Pinus sylvestris). IAA-ester conjugates were initially hydrolyzed in the seed to yield a peak of free IAA prior to initiation of root elongation. Developmental regulation of IAA synthesis was observed, with tryptophan-dependent synthesis being initiated around 4 d and tryptophan-independent synthesis occurring around 7 d after imbibition. Induction of catabolism to yield 2-oxindole-3-acetic acid and irreversible conjugation to indole-3-acetyl-N-aspartic acid was noticed at the same time as de novo synthesis was first detected. As a part of the homeostatic regulation IAA was further metabolized to two new conjugates: glucopyranosyl-1-N-indole-3-acetyl-N-aspartic acid and glucopyranosyl-1-N-indole-3-acetic acid. The initial supply of IAA thus originates from stored pools of IAA-ester conjugates, mainly localized in the embryo itself rather than in the general nutrient storage tissue, the megagametophyte. We have found that de novo synthesis is first induced when the stored pool of conjugated IAA is used up and additional hormone is needed for elongation growth. It is interesting that when de novo synthesis is induced, a distinct induction of catabolic events occurs, indicating that the seedling needs mechanisms to balance synthesis rates for the homeostatic regulation of the IAA pool.     

Studies on the oxidation of indole-3-acetic Acid by peroxidase enzymes. I. Colorimetric determination of indole-3-acetic Acid oxidation products

The method described here is based on a brief report by Harley-Mason and Archer. It involves the use of p-dimethylaminocinnamaldehyde (DMACA), a vinylogue of Ehrlich's reagent, as a color reagent for indoles. Colorimetric analyses of indoleacetic acid (IAA) oxidation reaction mixtures were made with the DMACA reagent as a solution rather than a spray. DMACA reagent will yield a wine-red color with IAA oxidation products in solution. Under similar conditions DMACA reacts with authentic IAA to yield only slight coloration at best. In comparison with other indoles, DMACA is more relative with IAA oxidation reaction products than either Salkowski or Ehrlich's reagents. Data discussed support a concept that the color produced with DMACA is due to the presence of tautomeric oxidation product(s) of IAA.     

Esters of indole-3-acetic Acid from Avena seeds

The present studies showed that about 80% of the indole-3-acetic acid extractable from Avena kernels by aqueous acetone was esterified to polymers precipitable by ammonium sulfate and ethanol or acetone. The polymers were positively charged, being adsorbed to cation exchange columns at a pH of 3, or below, and eluted at a pH greater than 4. The polymers were heterogeneous with respect to size, about 5,000 to 20,000 daltons, and charge, exhibiting apparent pK_a values of 4.2 and 4.7. The polymer fractions contained esterified IAA, anthrone-reactive material that liberated glucose upon acid hydrolysis, phenolic compounds, and peptidic material with a high proportion of hydrophobic amino acids. Since the esterified IAA was unstable, establishing polymer purity was not possible, and the designation IAA-glucoprotein fraction was adopted.     

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; )     

Morphogenic responses of debudded tobacco plants to gibberellic Acid and indole-3-acetic Acid

Stem applications of indole-3-acetic acid (IAA) or gibberellic acid (GA) did not prevent or alter tumor or teratoma formation in debudded tobacco plants (Nicotiana tabacum L., var. One Sucker). The materials produced intense (in case of GA) and moderate (in case of IAA) stem proliferations when applied to debudded plants but were without effect on intact plants.

The results suggest that debudding-tumors are probably not related to or a result of an auxin or gibberellin deficit and that total debudding has a marked physiological effect on the plant. The altered physiological condition of the debudded plant, indicated by its responses to IAA and GA, may likely be related to tumor and teratoma formation.

     

Arabidopsis seedlings over-accumulated indole-3-acetic acid in response to aminooxyacetic acid

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.     

Free and conjugated indole-3-acetic Acid in developing bean seeds

The changes in conjugated indole-3-acetic acid (IAA) levels compared to the levels of free IAA have been analyzed during the development of bean (Phaseolus vulgaris L.) seed using quantitative mass spectrometry. Free and ester-linked IAA levels are both relatively high in the early stages of seed development but drop during seed maturation. Concomitantly, the amide-linked IAA becomes the major form of IAA present as the seed matures. In fully mature seed, amide IAA accounts for 80% of the total IAA. The total IAA pool in the seed is maintained at approximately the same level (150-170 nanograms/seed) once the level of free IAA has attained its maximum. Thus, the amount of amide IAA conjugates that accumulate in mature seed is closely related to the amounts of free and ester-linked IAA that disappeared from the rapidly growing seed. Analysis of developing bean pods, from which the seeds were taken for analysis, showed very low levels of both ester and amide-linked IAA conjugates. The pattern of changes seen in the levels of free and conjugated IAA in developing bean seed supports our prior hypothesis suggesting a role of IAA conjugates in the storage of the phytohormone in the seed.     

Stimulation of indole-3-acetic acid production in Rhizobium by flavonoids

Flavonoids activate nod gene expression in Rhizobium resulting in the synthesis of Nod signals which trigger organogenesis in the host plant. This paper shows that nod-inducers also stimulate the production of the phytohormone IAA (indole-3-acetic acid).     

Oxidation of indole-3-acetic Acid-amino Acid conjugates by horseradish peroxidase

The stability of 21 amino acid conjugates of indole-3-acetic acid (IAA) toward horseradish peroxidase (HRP) was studied. The IAA conjugates of Arg, Ile, Leu, Tyr, and Val were oxidized readily by peroxidase. Those of Ala, β-Ala, Asp, Cys, Gln, Glu, Gly, and Lys were not degraded and their recovery was above 92% after 1 hour incubation with HRP. A correlation between the stability of IAA conjugates toward peroxidase-catalyzed oxidation and the hydrophobicity of the amino acid moiety conjugated to IAA was demonstrated. Polar amino acid conjugates of IAA are more resistant to HRP-catalyzed oxidation.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

Regulation of indole-3-acetic Acid biosynthetic pathways in carrot cell cultures

2,4-Dichlorophenoxyacetic acid (2,4-D) promotes the accumulation of tryptophan-derived indole-3-acetic acid (IAA) in carrot cell cultures during callus proliferation by a biosynthetic pathway that is apparently not active during somatic embryo formation. The effects of 2,4-D were examined by measuring the isotopic enrichment of IAA due to the incorporation of stable isotope-labeled precursors (deuterium oxide, [¹⁵N]indole, and ²H₅-l-tryptophan). Enrichment of IAA from deuterium oxide is similar in both cultured hypocotyls and cell suspension cultures in the presence and absence of 2,4-D, despite the large differences in absolute IAA concentrations. The enrichment of IAA due to the incorporation of [¹⁵N]indole is also similar in callus proliferating in the presence of 2,4-D and in embryos developing in the absence of 2,4-D. The incorporation of ²H₅-l-tryptophan into IAA, however, is at least 7-fold higher in carrot callus cultures proliferating in the presence of 2,4-D than in embryos developing in the absence of 2,4-D. Other experiments demonstrated that this differential incorporation of ²H₅-l-tryptophan into IAA does not result from differential tryptophan uptake or its subsequent compartmentation. Thus, it appears that differential pathways for IAA synthesis operate in callus cultures and in developing embryos, which may suggest that a relationship exists between the route of IAA biosynthesis and development.     

Evidence for production of the phytohormone indole-3-acetic acid by cyanobacteria

The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.     

Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris)

The antagonistic effects of ethylene and Ag⁺ on the metabolism of [1-¹⁴C]indole-3-acetic acid (IAA) and on the rates of ethylene production were studied in tobacco leaf discs ( Nicotiana rustica var. Brasilia ). During the first 10 h of incubation, Ag⁺-pretreated leaf discs contained more free [¹⁴C]IAA than untreated ones due to decreased oxidative decarboxylation, and the discs also produced more ethylene. Exogenously supplied ethylene nullified these effects of Ag⁺. However, the most pronounced effect of Ag⁺ in increasing ethylene production, as well as the strongest antagonistic effect of exogenous ethylene, were found between 24 and 48 h of incubation. During this time span no effect on the level of free IAA and on its decarboxylation could be observed. It is suggested that ethylene exerted its autoinhibitory effect by a feedback control on the IAA-induced ethylene biosynthesis. Possible mechanisms for the autoinhibitory effect of ethylene are discussed.