Changes in free and conjugated indole-3-acetic acid during early stage of flower bud differentiation in Polianthes tuberosa

Tuberose (Polianthes tuberosa L. cv. Double) corms at the vegetative, early floral initiation, and flower bud differentiation stages were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. The corms in the vegetative stage contained higher free IAA than those from the early floral initiation stage. Free IAA in corm tissues increased 2.7-fold at flower bud differentiation as compared to the vegetative stage. In the vegetative corms, a marked promotion of leaf differentiation was recorded. In contrast, corms from the early floral initiation stage contained less free IAA, whereas esterified IAA and peptidyl IAA increased dramatically. It is concluded that the level of free IAA in vegetative corms is correlated with leaf differentiation, and that the early floral initiation stage is correlated with a reduction in free IAA and an increase in IAA conjugates in the corms. Moreover, increases in free IAA and decreases in IAA conjugates in the floral differentiation stage, as compared to the early floral initiation stage, indicates that free IAA is correlated with flower development.     

Influence of 5-Methyltryptophan-Resistant Bradyrhizobium japonicum on Soybean Root Nodule Indole-3-Acetic Acid Content

Bradyrhizobium japonicum mutants resistant to 5-methyltryptophan were isolated. Some of these mutants were found to accumulate indole-3-acetic acid (IAA) and tryptophan in culture. In greenhouse studies, nodules from control plants inoculated with wild-type bradyrhizobia contained 0.04, 0.10, and 0.58 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. Nodules from plants inoculated with 5-methyltryptophan-resistant bradyrhizobia contained 0.94, 1.30, and 10.6 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. This manyfold increase in nodule IAA content indicates that the Bradyrhizobium inoculum can have a considerable influence on the endogenous IAA level of the nodule. Further, these data imply that much of the IAA that accumulated in the high-IAA-containing nodules was of bacterial rather than plant origin. These high-IAA-producing 5-methyltryptophan-resistant bacteria were poor symbiotic nitrogen fixers. Plants inoculated with these bacteria had a lower nodule mass and fixed less nitrogen per gram of nodule than did plants inoculated with wild-type bacteria.     

Concentration and Metabolic Turnover of Indoles in Germinating Kernels of Zea mays L

The amounts and rates of metabolic turnover of the indolylic compounds in germinating kernels of sweet corn were determined. Knowledge of pool size and rate of pool turnover has permitted: (a) identification of indole-3-acetyl-myo-inositol as the major chemical form for transport of indole-3-acetic acid (IAA) from endosperm to shoot; (b) demonstration that the free IAA of the endosperm is turning over rapidly with a half-life of 3.2 hours; (c) identification of esters of IAA as the immediate precursors of IAA in the endosperm and shoot; (d) demonstration that neither tryptophan nor tryptamine is a major precursor of IAA for the seed or shoot; (e) identification of IAA-myo-inositol glycosides as precursors of IAA-myo-inositol.     

Movement of Indole-3-acetic Acid and Tryptophan-derived Indole-3-acetic Acid from the Endosperm to the Shoot of Zea mays L

The structures and the concentrations of all of the indolylic compounds that occur in the endosperm of the seeds of corn (Zea mays L.) are known. Thus, it should be possible to determine which, if any, of the indolylic compounds of the endosperm can be transported to the seedling in significant amounts and thus help identify the seed-auxin precursor of Cholodny (1935. Planta 23:289-312) and Skoog (1937. J. Gen. Physiol. 20:311-334). Of interest is the transport of tryptophan, indole-3-acetic acid (IAA), and the esters of IAA, which comprise 95% of the IAA compounds of the seed. We have shown that: (a) IAA can move from the endosperm to the shoot; (b) the rate of movement of IAA from endosperm to shoot is that of simple diffusion; (c) 98% of the transported IAA is converted into compounds other than IAA, or IAA esters, en route; (d) some of the IAA that has moved into the shoot has been esterified; (e) labeled tryptophan applied to the endosperm can be found as labeled IAA in the shoot; and (f) with certain assumptions concerning IAA turnover, the rate of movement of IAA and tryptophan-derived IAA from the endosperm to shoot is inadequate for shoot growth or to maintain IAA levels in the shoot.     

A Quantitative Estimation of Alkali-labile Indole-3-Acetic Acid Compounds in Dormant and Germinating Maize Kernels

An estimate has been made of the quantities of alkali-labile esters of indoleacetic acid (IAA) in kernels of sweet corn (Zea mays). The amount is between 70 to 90 mg of IAA per kilogram of dry kernels. About one-half of the IAA is present as high molecular weight esters and the remaining one-half as esters of myo-inositol. Free IAA, which may have existed in the kernels, or may have resulted from ester hydrolysis during isolation or storage, amounts to between 1 to 10% of the esterified IAA. Five newly observed low molecular weight indoleacetyl compounds are described and their chromatographic behavior reported. The total IAA content of corn kernels and intact seedlings decreases during germination, declining to about 10% of the original content during 96 hr of germination. Difficulties in obtaining quantitative results and the possible physiological significance of these results is discussed.     

Free and Conjugated Indoleacetic Acid (IAA) Contents in Transgenic Tobacco Plants Expressing the iaaM and iaaH IAA Biosynthesis Genes from Agrobacterium tumefaciens

The Agrobacterium tumefaciens T-DNA gene iaaM was introduced by leaf-disc transformation into transgenic tobacco (Nicotiana tabacum) plants expressing the iaaH gene. Regenerated calli were screened for the presence of indole-3-acetamide (IAM), by gas chromatography-multiple ion monitoring-mass spectrometry, and IAM-containing calli were further analyzed for free and conjugated indoleacetic acid (IAA). It was found that transgenic calli on average contained twice as much free IAA and three times more conjugated IAA than calli from wild-type plants. About 40% of the transformed calli could be regenerated to plants. The distribution of free and conjugated IAA was measured in transformed plants with a normal phenotype and compared with equivalent wild-type plants. The IAA content of transgenic plants was only slightly increased, whereas IAA-conjugate levels were enhanced significantly. These data suggest that conjugation of IAA may serve as a regulatory mechanism, contributing to maintenance of steady-state IAA pool sizes during tobacco growth and development.     

Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-¹⁴C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.     

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.     

Alkyl esters from pinus radiata foliage epicuticular wax

Wax esters from the epicuticular wax of juvenile and mature-tree Pinus radiata foliage have been shown by capillary column GC-MS to consist mainly of short chain (C₆–C₁₂)alkanols esterified with long chain acids (C₂₄–C₃₂) and long chain alkanols (C₂₄–C₃₂) esterified with short chain acids (C₆-C₁₄) in a non-random manner. Mature-tree foliage wax esters also contained nonacosan-10-ol esterified with dodecanoic and tetradecanoic acids.     

Effect of Deseeding on the Indole-3-acetic Acid Content of Shoots and Roots of Zea mays Seedlings

We wished to determine the effect of endosperm removal on the amounts of free and esterified indole-3-acetic acid (IAA) in young Zea mays seedlings. The increases of IAA derived from endosperm and from biosynthesis, but without correction for catabolic losses, were 0.9 picomole of free IAA per shoot per hour, and 1.1 picomoles per shoot per hour of ester IAA. After deseeding, free IAA in the shoot declines by 40% following kernel removal and total (free + ester) IAA declines at a rate of about 1 picomole per shoot per hour. A slight, but insignificant increase of ester IAA occurs following endosperm removal. In the primary roots, the decreases of free IAA and total (free + ester) IAA are accelerated by seed removal. Thus, the endosperm appears to be a major source of IAA for the shoot and root.     

Response to Gravity by Zea mays Seedlings : I. Time Course of the Response

Gravistimulation induces an asymmetric distribution of free indole-3-acetic acid (IAA) in the cortex-epidermis of the Zea mays L. cv `Stowells Evergreen' mesocotyl within 15 minutes, the shortest time tested. IAA was measured by an isotope dilution method as the pentaflurobenzyl ester. The per cent IAA in the lower half of the mesocotyl cortex was 56 to 57% at 15, 30, and 90 minutes after stimulus initiation. Curvature is detectable in the mesocotyl within 3 minutes after beginning gravitropic stimulation. The rate of curvature of the mesocotyl increases during the first 60 minutes to a maximum of about 30° per hour. Thus, the growth asymmetry continues to increase for 45 minutes after hormone asymmetry is established.

Free IAA occurs predominantly in the stele of the mesocotyl whereas esterified IAA is mainly in the mesocotyl cortex-epidermis. This compartmentation may permit determining in which tissue the hormone asymmetry arises. Current data suggest the asymmetry originated in the stele.

     

Relationship between Indole-3-Acetic Acid Levels in Apple (Malus pumila Mill) Rootstocks Cultured in Vitro and Adventitious Root Formation in the Presence of Indole-3-Butyric Acid

In vitro rooting response and indole-3-acetic acid (IAA) levels were examined in two genetically related dwarfing apple (Malus pumila Mill) rootstocks. M.26 and M.9 were cultured in vitro using Linsmaier-Skoog medium supplemented with benzyladenine (BA), indole-3-butyric acid (IBA), and 1,3,5-trihydroxybenzoic acid (PG). Rooting response was tested in Lepoivre medium supplemented with IBA and PG. IBA concentrations of 12.0 and 4.0 micromolar induced the maximum rooting percentages for M.9 and M.26, respectively. At these concentrations rooting response was 100% for M.26 and 80% for M.9. Free and conjugated IAA levels were determined in M.26 and M.9 shoots prior to root inducing treatment by high performance liquid chromatography with fluorescence detection and validated by gas chromatography-mass spectrometry using ¹³[C₆]IAA as internal standard. Basal sections of M.26 shoots contained 2.8 times more free IAA than similar tissue in M.9 (477.1 ± 6.5 versus 166.6 ± 6.7 nanograms per gram fresh weight), while free IAA levels in apical sections of M.26 and M.9 shoots were comparable (298.0 ± 4.4 versus 263.7 ± 9.3 nanograms per gram fresh weight). Conjugated IAA levels were significantly higher in M.9 than in M.26 indicating that a greater proportion of total IAA was present as a conjugate in M.9. These data suggest that differences between M.26 and M.9 rooting responses may be related to differences in free IAA levels in the shoot base.     

Sterols and steryl esters in some Brassica and Sinapis seeds

The qualitative and quantitative composition of sterols in the free form and esterified to fatty acids was studied in seed oils from Brassica napus, B. campestris, B..iuncea, B. nigra, Sinapis alba and S. aruefisis (Brassica kaber). Sitosterol, followed by campesterol, predominated in both the free and the esterified sterols. The free sterols were richer in brassicasterol (ca 10–20%) than the steryl esters (3–10%). Small amounts of δ⁵-avenasterol and δ⁷-stigmastenol were also found in the Brassica oils, often more in the esterified than in the free form. The quantity of sterols was studied only in Brassica campestris, which had ca 0.3 % in the free as well as in the esterified form. In Sinapis alba, ca 10% of the sterols in the free form and 20 % in the esterified sterols were δ⁵-avenasterol. This compared to only a few per cent in both the free and esterified sterols in the Brassica oils. Similarly, ca 2 % of cholesterol was found among the sterols of Sinapis alba but only traces in the Brassica oils. The similarity of sterol compositions among the cultivated brassicas and wild mustard (Sinapis arvensis), and the specific characteristics of the sterols in white mustard (Sinapis alba) adds further weight to the suggestion that wild mustard should be treated as Brassica kaber and strengthens the generic separation of Sinapis alba.     

Translocation and Metabolism of Endosperm-Applied [2-C] Indoleacetic Acid in Etiolated Avena sativa L. Seedlings

The role of free indole-3-acetic acid (IAA) in the endosperm of Avena sativa L. seedlings was investigated to determine its contribution to free IAA in the shoot. [2-¹⁴C]IAA was injected into the endosperm of darkgrown seedlings and the transport and metabolism of the [¹⁴C]-labeled compounds determined. It was concluded that translocation of free IAA directly from the endosperm is probably not a significant source of free IAA in the shoot, mainly because even small amounts of [¹⁴C]IAA introduced into the endosperm were rapidly metabolized. This suggested that, in Avena, free IAA does not normally exist in the liquid endosperm.     

Influence du milieu de culture sur la composition en sterols de Leptosphaeria typhae

C₂₇, C₂₈ and C₂₉ sterols have been isolated from a Leptosphaeria typhae culture grown in vitro in light on a synthetic medium. These products were characterized by GLC and MS. Saturated and mono-, di- and tri-unsaturated sterols are present, both free and esterified. There are significant differences between these sterols and those in the same fungus grown on “oat water”. Unexpectedly, cholesterol was detected in the latter case.     

Tiller formation in rice is altered by overexpression of OsIAGLU gene encoding an IAA-conjugating enzyme or exogenous treatment of free IAA

Optimization of plant architecture is important for cultivation and yield of cereal crops in the field. Tillering is an essential factor used to determine the overall architecture of cereal crops. It has long been recognized that the development of branching patterns is controlled by the level and distribution of auxin within a plant. To better understand the relationship between auxin levels and tillering in rice, we examined rice plants with increased or decreased levels of free IAA. To decrease IAA levels, we selected the rice IAA-glucose synthase gene (OsIAGLU) from the rice genome database based on high sequence homology with IAA-glucose synthase from maize (ZmIAGLU), which is known to generate IAAglucose conjugate from free IAA. The OsIAGLU gene driven by the Cauliflower Mosaic Virus 35S promoter was transformed into a rice cultivar to generate transgenic rice plants constitutively over-expressing this gene. The number of tillers and panicles significantly increased in the transgenic lines compared to the wild-type plants, while plant height and panicle length decreased. These results indicate that decreased levels of free IAA likely enhance tiller formation in rice. To increase levels of free IAA, we treated rice plants with three different concentrations of exogenous IAA (1 μM, 10 μM and 100 μM) twice a week by spraying. Exogenous IAA treatment at concentrations of 10 μM and 100 μM significantly reduced tiller number in three different rice cultivars. These results indicate that exogenously applied IAA inhibits shoot branching in rice. Overall, auxin tightly controls tiller formation in rice in a negative way.     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.     

Euphorbia escula L. Root and Root Bud Indole-3-Acetic Acid Levels at Three Phenologic Stages

Endogenous indoleacetic acid (IAA) levels of Euphorbia esula L. primary root and root buds were examined at three phenologic stages. High performance liquid chromatography coupled with fluorescence detection and gas chromatography-mass spectrometry, using ¹³C₆[benzene ring]-indole-3-acetic acid as internal standard, were used to measure root bud free and bound IAA levels in vegetative, full flower, and post-flower plants. Highest levels of free IAA (103 nanograms per gram fresh weight) were found in root buds during full flower. Esterified and amide IAA increased significantly in root buds of full flower and post-flower plants, but were not detectable in root buds of vegetative plants. Primary rootfree IAA was highest in vegetative and full flower plants (34.5 nanograms per gram fresh weight) and decreased by 50% in post-flower plants.     

Red Light-Regulated Growth : I. Changes in the Abundance of Indoleacetic Acid and a 22-Kilodalton Auxin-Binding Protein in the Maize Mesocotyl

We examined the changes in the levels of indoleacetic acid (IAA), IAA esters, and a 22-kilodalton subunit auxin-binding protein (ABP1) in apical mesocotyl tissue of maize (Zea mays L.) during continuous red light (R) irradiation. These changes were compared with the kinetics of R-induced growth inhibition in the same tissue. Upon the onset of continuous irradiation, growth decreased in a continuous manner following a brief lag period. The decrease in growth continued for 5 hours, then remained constant at 25% of the dark rate. The abundance of ABP1 and the level of free IAA both decreased in the mesocotyl. Only the kinetics of the decrease in IAA within the apical mesocotyl correlated with the initial change in growth, although growth continued to decrease even after IAA content reached its final level, 50% of the dark control. This decrease in IAA within the mesocotyl probably occurs primarily by a change in its transport within the shoot since auxin applied as a pulse moved basipetally in R-irradiated tissue at the same rate but with half the area as dark control tissue. In situ localization of auxin in etiolated maize shoots revealed that R-irradiated shoots contained less auxin in the epidermis than the dark controls. Irradiated mesocotyl grew 50% less than the dark controls even when incubated in an optimal level of auxin. However, irradiated and dark tissue contained essentially the same amount of radioactivity after incubation in [¹⁴C]IAA indicating that the light treatment does not affect the uptake into the tissue through the cut end, although it is possible that a small subset of cells within the mesocotyl is affected. These observations support the hypothesis that R causes a decrease in the level of auxin in epidermal cells of the mesocotyl, consequently constraining the growth of the entire mesocotyl.