Dynamics of indole-3-acetic acid and indole-3-ethanol during development and germination of Pinus sylvestris seeds

Indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified in immature seeds of Pinus sylvestris L. by combined gas chromatography-mass spectrometry. Indole-3-methanol was tentatively identified using multiple ion monitoring. Anatomical investigations of seeds, as well as measurements of free and alkali-hydrolysable IAA and IEt, were made during seed development and germination. Levels of free IAA and IEt decreased during seed development. In the later stages of seed maturation most IAA and IEt were present in alkali-hydrolysable forms. Bound IAA and bound IEt rapidly decreased during germination, while levels of free IAA and IEt increased dramatically for a short period.     

Changes in abscisic acid and indole-3-acetic acid in axillary buds of Elytrigia repens released from apical dominance

Growth of axillary buds on the rhizomes of Elytrigia repens (L) Nevski is strongly dominated by the rhizome apex, by mechanisms which may involve endogenous hormones. We determined the distribution of indole-3-acetic acid (IAA) and abscisic acid (ABA) in rhizomes and measured (by gas-chromatography-mass spectrometry) their content in axillary buds after rhizomes were decapitated. The same measurements were also made in buds induced to sprout by removing their subtending scale leaves. The ABA content tended to be higher in the apical bud and in the axillary buds than in the adjacent internodes, and tended to decline basipetally in the internodes and scale leaves. IAA was similary distributed, except that there was less difference between the buds and other rhizome parts. After rhizomes were decapitated, the ABA content of the first axillary bud declined to 20% of that of control values within 24 h, while the IAA content showed no marked tendency to change. The ABA content also declined within 12 h in the first axillary bud after rhizomes were denuded, while the content of IAA tended to increase after 6 h. These changes occurred before the length of the first axillary bud increased 24–48 h after rhizomes were decapitated or denuded. We conclude that the release of axillary buds from apical dominance in E. repens does not require IAA content to be reduced, but is associated with reduced ABA content.     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

Level of endogenous indole-3-acetic acid in the stem of Pinus sylvestris in relation to the seasonal variation of cambial activity

Abstract. Gas chromatography – selected ion monitoring – mass spectrometry was used to measure the level of indole-3-acetic acid (IAA) in the cambial region at the top and bottom of the branchless portion of the main stem of three large Scots pine trees, at weekly intervals from 28 April to 13 July. During this period, the cambium reactivated from the dormant state and entered its 'grand' period of xylem and phloem production, which was monitored by microscopy. The total amount of IAA (ng cm⁻²) increased steadily from 28 April until late June, and thereafter remained constant. In contrast, the concentration of IAA (ng g⁻¹ fresh weight) was high at the start of cambial reactivation, declined when the number of differentiating tracheids began to increase, and then rose as the number of cells decreased. The timing and magnitude of the changes in xylem and phloem production and in IAA level were similar at the two sampling positions. It is concluded that the seasonal changes in cambial activity in the conifer stem cannot be ascribed simply to a fluctuation in the level of endogenous IAA in the cambial region.     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

Population variation and diurnal changes in the content of indole-3-acetic acid of pine seedlings (Pinus sylvestris L.) grown in a controlled environment

Pine seedlings ( Pinus sylvestris L.) were grown in a growth chamber under simulated summer conditions to an age of eight weeks after the beginning of seed germination. Single seedlings were analyzed for fresh weight, shoot and root lengths, and content of indole-3-acetic acid (IAA). The first three variables were normally distributed with standard deviations of 29%, 17% and 18%, respectively. The IAA content had a standard deviation of 39%, and this variable was not normally distributed. If this finding is of general significance, population variation must be considered when experiments involving IAA analyses are planned, and statistical methods based on a normally distributed population cannot be used to evaluate the result of such analyses unless samples of at least 20–30 individuals are analyzed. There were no correlations between the content of IAA and any of the three other variables. The content of IAA showed pronounced diurnal changes, rising from 15 ng g⁻¹ (fresh weight) in the morning to 42 ng g⁻¹ in the late evening. The initial rate of change was about 10% h⁻¹. Obviously, short-term fluctuations must be checked if long-term changes in IAA content are to be studied. IAA could also be released from the acidic buffer fraction by means of alkaline hydrolysis. This "bound alkali-hydrolysable" IAA did not show short-term fluctuations.     

Regulation of enzymic oxidation of indole-3-acetic acid by phenols: Structure-activity relationships

Mono- and diphenols were tested for their effects on the decarboxylation of [1-¹⁴C]IAA catalysed by purified horseradish peroxidase (EC 1.11.1.7) in the presence or absence of 2,4-dichlorophenol (DCP). The number of hydroxyl groups and their position relative to each other and the nature and position of other substituents on the aromatic ring were found to affect the activity. Although the effects were complex, the following generalizations may be made. (1) Monophenols produce activation when no other cofactor is present. p-Substituted monophenols are more active than o- or m-compounds. In the presence of DCP, the activity varies from slight activation to strong inhibition. (2) m-Diphenols also produce activation in the absence of other cofactors while o- and p-diphenols, with the exception of 3,4-dihydroxyacetophenone and 3,4-dihydroxypropiophenone, produce strong inhibition in the presence or absence of DCP. The o-diphenolsare degraded in the IAA-oxidizing enzyme system and thus produce only a temporary inhibition. (3) m-Diphenols and 3,4-dihydroxyacetophenone produce a sustained inhibition in the presence of DCP. (4) Substitution at position 2 significantly alters the activity of m-diphenols. (5) O-Methylation alters the activity of most o-diphenols. In the absence of DCP, o-methoxyphenols and certain other phenols such as 3,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone either promote or inhibit IAA oxidation depending on concentration.     

Definitive identification of indole-3-acetic acid and abscisic acid in shoots of Coleus blumei by gas chromatography-mass spectrometry

Mass spectra provide definitive identification of indole-3-acetic acid and abscisic acid in shoots of Coleus blumei, a species used for studying the hormone control of plant development since the early 1930s.     

Circadian changes in endogenous concentrations of indole-3-acetic acid,melatonin, serotonin,abscisic acid and jasmonic acid in Characeae (Chara australis Brown)

Giant-celled Characeae (Chara australis Brown), grown for 4 months on 12/12 hr day/night cycle and summer/autumn temperatures, exhibited distinct concentration maxima in auxin (indole-3-acetic acid; IAA), melatonin and serotonin about 4 hr after subjective daybreak. These concentration peaks persisted after 3 day pretreatment in continuous darkness: confirming a circadian rhythm, rather than a response to “light on.” The plants pretreated for 3 d in continuous light exhibited several large IAA concentration maxima throughout the 24 hr. The melatonin and serotonin concentrations decreased and were less synchronized with IAA. Chara plants grown on 9/15 hr day/night cycle for 4 months and winter/spring temperatures contained much smaller concentrations of IAA, melatonin and serotonin. The IAA concentration maxima were observed in subjective dark phase. Serotonin concentration peaks were weakly correlated with those of IAA. Melatonin concentration was low and mostly independent of circadian cycle. The “dark” IAA concentration peaks persisted in plants treated for 3 d in the dark. The plants pretreated for 3 d in the light again developed more IAA concentration peaks. In this case the concentration maxima in melatonin and serotonin became more synchronous with those in IAA. The abscisic acid (ABA) and jasmonic acid (JA) concentrations were also measured in plants on winter regime. The ABA concentration did not exhibit circadian pattern, while JA concentration peaks were out of phase with those of IAA. The data are discussed in terms of crosstalk between metabolic pathways.     

New phenolic inhibitors of the peroxidse-catalysed oxidation of indole-3-acetic acid

Previously we reported two metabolites of the insecticide carbofuran as persistent inhibitors of the peroxidase-catalysed oxidtion ofindole-3-acetic acid. In searching for more active inhibitors of this type, we have found that 5-hydroxy-2,2-dimethylchromene (β-tubanol), 2′,6′-dihydroxycetophenone oxime, 5-hydroxy-2,2-dimethylchroman, 2′,6′-dihydroxyacetophenone and 2,6-dihydroxybenzoic acid methyl ester were more active than the carbofuran metabolite 7-hydroxy-2,2-dimethyl-3-oxo-2,3-dihydrobenzofuran. Resorcinol, 5-hydroxy-2,2-dimethylchroman-4-one, 3-hydroxy-5-methoxy-2,2-dimethylchroman-4-one and 5-hydroxy-2-methylchrom-4-one were also inhibitory but with less activity. The new inhibitors differed from the well-known phenolic inhibitors such as caffeic acid in inhibition kinetics as demonstrated by the rate of disappearance of indole-3-acetic acid, the rate of formation of the oxidation products, and the transient spectral change in the enzyme.     

Effects of water stress on cellular ultrastructure and on concentrations of endogenous abscisic acid and indole-3-acetic acid in Fatsia japonica leaves

Ultrastructural alterations in mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in water-stressed field-grown plants of Fatsia japonica. Under water deficit cellular membranes were modified and an increase in vesicles was observed. The main damage to the chloroplasts included thylakoid swelling and disruption of the chloroplast envelope. Concomitant variations in abscisic acid and indole-3-acetic acid were observed. Despite the expected increased in endogenous ABA concentration in relation to water stress, after the highest concentration of ABA, observed at predawn in severely stressed plants (29-1), there was a sharp decline from 2768 pmol g fw^–1 to 145 pmol g fw^–1; thus in severely stressed plants ABA levels were not related to changes in bulk leaf ABA contents. Water stress did not influence the concentrations of indole-3-acetic acid, although the increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - leaf water potential     

Mediation of tropisms by lateral translocation of endogenous indole-3-acetic acid in maize coleoptiles

Abstract. The hypothesis that tropic responses result from lateral auxin gradients was examined in coleoptiles of red-light-grown maize ( Zea mays L.) by measuring endogenous IAA (indole-3-acetic acid) using a physicochemical method. Phototropic stimulation (unilateral blue light; 8s at 0.33 μmol m⁻²s⁻¹) was found to induce a lateral gradient of solvent-extractable IAA in a subapical zone (2-7mm from the tip). The gradient occurred in advance of the bending response, with a decrease of IAA in the irradiated half and a compensatory increase in the shaded half. The maximal gradient measured was about 1:2 (irradiated: shaded). Diffusible IAA, obtained from the cut end of an excised coleoptile tip (3mm long, with its base split by 1mm), was similarly redistributed between the two sides, indicating that IAA is laterally translocated in the tip and that the resulting IAA gradient migrates to the subapical zone. A smaller gradient was induced in a basal zone (12-17mm from the tip). This gradient was initiated about 20 min later than that at the subapical zone, in agreement with a similar delay of bending observed in this zone. Gravitropic stimulation (60° from the vertical) also resulted in a lateral gradient of extractable IAA in the subapical zone, the gradient preceding the bending response. It is concluded that the tropisms of maize coleoptiles are mediated by IAA gradients, which are most likely caused by lateral IAA transport as the Cholodny-Went theory of tropisms describes. From IAA measurement data, the mean velocity of basipetally-polar transport of endogenous IAA was estimated to be 12 mm h⁻¹.     

The influence of the extraction procedure on yield of indole-3-acetic acid in plant extracts

Different types of plant material, including both dry and swollen maize kernels, swollen bean seeds, bean seedlings and dry rose seeds, were extracted by different methods and the yield of IAA was determined with the indolo-α-pyrone method. Extraction of dry maize kernels during short time experiments, varying from 3 to 24 h, gave the highest IAA yield when methanol was the extractant and a significant lower yield when diethyl ether or dichloromethane were used. The duration of the extraction period increased the yield with all the extractants. Progressive extractions for several days or weeks had little effect on the yield when 100% acetone was used in contrast to methanol and ether as extractants, which increased the yield during prolonged extraction. Extractions of tissue treated to 100°C for 1 h contradicted the hypothesis that IAA is enzymatically liberated during ether extraction. Water in the extractant solvents increased the yields. This was most pronounced when aqueous acetone was used instead of 100% acetone. Increased extraction temperature augmented the IAA yields. The yield of IAA from other types of tissue extracted with methanol for periods of 3 or 24 h was, however, independent of the duration of the extraction time. This indicates that some tissues contain less not easily extractable IAA than dry maize kernels. The terms “free” and “bound” IAA are discussed; they should be replaced by “easily extractable” and “not easily extractable” IAA. The results also show that IPyA in vitro can partly be converted to IAA during extraction and fractionation.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Effect of laterally applied gibberellin A4/7 on cambial growth and the level of indole-3-acetic acid in Pinus sylvestris shoots

Gibberellin A_4/7 (GA_4/7) was applied in lanolin or ethanol around the circumference at the midpoint of the previous-year terminal of dormant Pinus sylvestris seedlings. After cultivating the seedlings under environmental conditions favorable for growth for up to 10 weeks, cambial growth was measured as the radial widths of xylem and phloem, and the level of indole-3-acetic acid (IAA) was determined by combined gas chromatography-mass spectrometry using [¹³₆](IAA) as the internal standard. In intact seedlings, both 1 mg GA_4/7 g^?1 lanolin and 50 mg GA_4/7 I^?1 ethanol increased phloem production and the cambial region IAA level in the current-year terminal, without significantly altering its longitudinal growth. In the previous-year terminal, 1 mg GA_4/7 g^?1 lanolin promoted phloem production at the application point and increased the cambial region IAA level above this point, whereas 50 mg GA_4/7 I^?1 ethanol stimulated the production of both xylem and phloem at the treatment site and elevated the cambial region IAA level beneath it. Laterally applied GA_4/7 at 50 mg I^?1 ethanol stimulated xylem and phloem production in debudded previous-year terminals treated at the apical cut surface with 1 mg IAA g^?1 lanolin, but not in those treated with plain lanolin. However, the promotion of cambial growth in debudded terminals treated apically with 1 mg IAA g^?1 lanolin and laterally with 50 mg GA_4/7 I^?1 ethanol was not associated with an elevated IAA content in the cambial region. The results indicate that exogenous GA_4/7 can promote xylem and phloem production provided an IAA source is present, and that it or a metabolic product acts directly, rather than indirectly by stimulating longitudinal growth and/or raising the cambial region IAA level.     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

Interaction of ethylene with indole-3-acetic acid in regulation of rooting in pea cuttings

Cuttings of pea (Pisum sativum L. cv Marma) were treated with 1-aminocyclopropane-l-carboxylic acid (ACC). This treatment caused increased ethylene production and reduction of root formation. The effect of 0.1 mM ACC on the level of endogenous indole-3-acetic acid (IAA) in the rooting zone and in the shoot apex was analyzed by gas chromatography-single ion monitoring mass spectrometry or by high pressure liquid chromatography with fluorimetric detection (HPLC). Concentrations of indole-3-acetylaspartic acid (IAAsp) in the stem bases were also determined using HPLC. The ACC treatment had little effect on the IAA level in the base measured after 24 h, but caused a considerable decrease during the 3 following days. IAAsp increased in the base on days 1, 2 and 3 and then declined. The build up of IAAsp in the base was not affected by ACC during the first two days of the treatment, but later this conjugate decreased more rapidly than in controls. No effect of the ACC treatment was found on the level of IAA in the apex. IAA (1 µM) applied to the cuttings during 24 h reduced the number of roots formed. The possibility that IAA-induced ethylene is involved in this response was investigated.Our results support earlier evidence that the inhibitory effect of ethylene on rooting in pea cuttings is due to decreased IAA levels in the rooting zone. The inhibitory effect of applied IAA is obtained if the internal IAA level is maintained high during the first 24 h, whereas stimulation of rooting occurs if the internal IAA level remains high during an extended period of time. Our results do not support the suggestion that ethylene mediates the inhibitory effect of applied IAA.     

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.     

Immunocytochemical localization of indole-3-acetic acid in primary roots of Zea mays

Colloidal gold-labelled antibody was used to localize indole-3-acetic acid in caps of primary roots of Z. mays cv. Kys. Gold particles accumulated on the nucleus, vacuoles, mitochondria, and some dictyosomes and dictyosome-derived vesicles. This is the first localization of indole-3-acetic acid in dictyosomes and dictyosome-derived vesicles and indicates that dictyosomes and vesicles constitute a pathway for indole-3-acetic acid movement in and secretion from root cap cells. Our findings provide cytochemical evidence to support the hypothesis that indole-3-acetic acid plays an important role in root gravitropism.     

The onset of starch degradation during banana ripening is concomitant to changes in the content of free and conjugated forms of indole-3-acetic acid

Banana (Musa acuminata AAA cv. Nanicão) slices were infiltrated with mannitol (control) and mannitol plus indole-3-acetic acid (IAA); then, some important ripening parameters like starch degradation, synthesis of ethylene and respiration were monitored. The contents of free-IAA and conjugated forms of IAA (ester and amide) were analyzed, by GC-MS-SIM, throughout the ripening in both banana slices and whole bananas. The starch degradation of IAA-treated slices was delayed for several days, but there was no difference between control and IAA-treated slices in the ethylene and respiration profiles. On day zero after infiltration, free-IAA levels were 500-fold higher in IAA-treated slices than in the control slices, but within 72 hours they declined to values 15-fold higher than those in the control group, with concomitant increase in IAA-ester. Similar to the banana slices, the onset of starch degradation occurred in whole bananas only when the free-IAA concentration was about 4 ng/g FW. The results herein suggest that IAA levels play a role during banana ripening in events like starch degradation with the consequence of banana sweetening.