Free IAA Levels Research Articles

Changes in peroxidase activity, auxin level and ethylene production during root formation by poplar shoots raised in vitro

Shoots of poplar (Populus tremula × P. tremuloides) were multiplied in vitro and rooted on a rooting medium in the presence of NAA. No rooting occurred in the absence of exogenous auxin. A peak of soluble peroxidase activity, which corresponded to a decrease in the free IAA level in the shoots, preceded rooting These events were considered as corresponding to the initiative phase of rooting. They are preceded by a peak in free IAA activity which might initiate the inductive phase of the rooting process. A burst of ethylene production was measured in both rooting and non-rooting shoots, but the ethylene peak from rooting shoots appeared earlier and was higher. The use of ACC indicated that the exogenous auxin might have enhanced ACC-synthetase activity.

Compartmentation of indole-3-acetic acid metabolism in protoplasts isolated from leaves of wild-type and IAA-overproducing transgenic tobacco plants

Cellular compartmentation of indole-3-acetamide (IAM), indole-3-acetic acid (IAA), and [15N1]IAA synthesised from [15N1]tryptophan was monitored in protoplasts isolated from sterile wild-type tobacco SRI plants, and in IAA-overproducing plants expressing the Agrobacterium tumefaciens T-DNA IAA genes iaaM and iaaH. Indole-3-acetamide was located exclusively in the cytosol of both iaaM and iaaM/iaaH protoplasts, being 75% lower than in iaaM protoplasts, presumably because of conversion into IAA by action of the iaaH-encoded hydrolase. The free-IAA level, however, was raised only 8% in iaaM/iaaH compared to iaaM protoplasts, whereas the level of IAA-conjugates was increased more than fivefold. For both genotypes, the location of IAA conjugates was restricted to the cytosol, while one-third of the free-IAA pool was present in chloroplasts. Transcription of the iaaM gene was increased by fusion to the strong cauliflower mosaic virus (CaMV) 35S promoter. Compared with the wildtype, this led to an 18-fold higher conversion of [15N1]tryptophan to [15N1]IAA, a three- to fourfold increase in free IAA, and a tenfold higher level of IAA conjugates in 35S-iaaM/iaaH protoplasts. Also in these genotypes, IAA conjugates were exclusively cytosolic. There was no major difference between transgenic and wildtype protoplasts in the proportion of chloroplastic to total cellular IAA, although the chloroplastic IAA and [15N1IAA pools in the transformant were threefold and eightfold higher, respectively. Since the IAM pool in transgenic plants is exclusively cytosolic, these findings suggest that the increased chloroplastic [15N1IAA pool in 35S-iaaM/iaaH protoplasts is synthesised in the cytosol but rapidly transported into the chloroplast. Furthermore, the presence of IAA in the chloroplast together with the exclusively cytosolic location of IAA conjugates, suggests the presence of two differentially subcellular pools of IAA. The first is located in the cytosol and mainly regulated by non-decarboxylative catabolism and conjugation (Sandberg et al. 1990, Planta 180, 562–568), whereas the second is located in the chloroplast and is seemingly not directly regulated inside the organelle by either of these two processes. The cytosolic IAA control mechanisms, however, also affect the chloroplastic pool size due to the rapid transport of IAA between the two compartments.

Localization of target cells and improvement ofAgrobacterium-mediated transformation efficiency by direct acetosyringone pretreatment of carrot root discs

Localization of target cells forAgrobacterium-mediated transformation in the carrot root disc model has been achieved after inoculation with a disarmedA. tumefaciens strain harbouring a GUS-intron construct. The first GUS positive cells could be detected on both sides of the discs 48 h after inoculation. The transformed cells were always more numerous on the apical side, mainly localized in the intrafascicular cambium and in the immature phloem strands. The kinetics of free endogenous IAA levels on both sides after wounding have been determined, indicating that rapid IAA accumulation on the apical side was not simply due to polar migration from the basal side. Attempts to optimize transformation efficiency were made by pretreating the discs with various concentrations of acetosyringone (AS) and/or naphthalene acetic acid (NAA). Surprisingly, while 25 μM AS applied to bacteria prior to the inoculations was ineffective, the same AS concentration applied as a pretreatment to the discs strongly increased the number of transformed cells in the target tissues and decreased the lag time for the appearance of the first GUS positive cells. NAA pretreatment on the basal side enhanced the AS effect. AS pretreatment was found both to advance the reentry of competent cells with a potential for cell division into the S phase of the cell cycle and to stimulate bacterial attachment to the cell walls. The relationship between transformation efficiency and DNA synthesis in the host cells is discussed. AS treatment of plant tissues prior to inoculation is proposed as a means of increasing the transformation rates.

Quantification of indole-3-acetic acid in untransformed and Agrobacterium rhizogenes-transformed pea roots using gas chromatography mass spectrometry

Root segments of Pisum sativum L. were transformed by several strains of Agrobacterium rhizogenes. The resulting hairy roots, as well as apical segments from untransformed pea roots, were used to initiate root lines cultured in vitro. Levels of free IAA were quantified in the sub-cultured lines by gas-chromatography coupled to mass spectrometry, using selected ion monitoring. For most of the cultured untransformed and transformed root lines the IAA content was very small, compared with levels in untransformed intact primary roots. However, an agropine-type hairy root line (incited by strain 15834) contained significantly higher amounts of IAA. The peculiar phenotype of this root line (abundant production of calli) appears to be associated with an increased IAA level, as opposed to most of the hairy root lines, where the extensive secondary root proliferation associated with the hairy-root disease cannot be merely attributed to a markedly enhanced IAA content.

Effects of Ri plasmid rol gene expression on the IAA content of transformed roots of Solanum dulcamara L.

The effects have been investigated of various combinations of rol genes, together with Ri plasmid TR-DNA transcripts 1 and 2, upon free IAA levels in transformed roots of Solanum dulcamara L. Differences between the IAA levels were observed, including a highly significant (p=0.01) difference between the IAA levels of rol− TR+ and rol ABC+ TR+ transformed roots. Possible mechanisms are discussed for the regulation of free IAA levels by rol gene products.

ENDOGENOUS IAA AND WATER UPTAKE IN DEVELOPING COFFEE FLOWER BUDS

Dormant coffee (Coffea arabica L.) flower buds require water stress to stimulate regrowth. A xylem specific watersoluble dye, azosulfamide, was used to quantify the uptake of water by buds after their release from dormancy by withholding water. In non-stressed flower buds, the rate of water uptake was generally slower and variable. In stressed flower buds, the rate of uptake tripled from one day to 3 days after rewatering and preceded the doubling of fresh and dry weight of buds. Free, ester, and amide IAA levels of developing flower buds were measured by GCMS-SIM using an isotope dilution technique with [13C6] IAA as an internal standard. Throughout development, the majority of IAA was present in a conjugated form and the dominant form was amide IAA. The proportions of amide and free IAA changed rapidly after plants were water stressed until day 3, and preceded the doubling of fresh and dry weight. Correlation coefficients of 0.9, 0.7, and 0.7 (p<0.l) were found between auxin content and fresh weight, dry weight, and rate of water uptake, respectively.

In Vivo Modulation of T-DNA Encoded Amidohydrolase Activity in Transformed Tobacco Cells

Auxin autonomous growth of most crown gall tumor cells requires the expression of two auxin biosynthesizing genes (tms 1 and tms 2) from the T-DNA of Agrobacterium tumefaciens. The potential role of the tms 2 locus to affect auxin accumulation was studied by measuring the activity of its gene product, indoleacetamide hydrolase (AH), in cloned cells of tobacco (Nicotiana tabacum) transformed by the A6 strain of Agrobacterium tumefaciens. AH activity followed a consistent pattern over a 30 day culture cycle with a peak at 10 to 14 days. This same pattern was observed in a number of independently isolated clones as well as in uncioned tumor tissue, suggesting that AH activation is a regular process in wounded, transformed cells. Transfer of unwounded tissue to fresh media resulted in a similar pattern of AH activation, but with the peak activity only about 50 percent of the cut tissues. These results show that the tms 2 encoded AH activity is modulated over the culture cycle, and that the modulation is affected by wounding and supplying fresh nutrients in the medium. AH activity correlated closely with free indoleacetic acid levels which suggests that it can be an important determinant in controlling free IAA levels in transformed cells.

The role of indol-3-ylacetic acid in regulation of juvenility inXanthium strumarium L.

Cotyledons ofXanthium strumarium, organs with low sensitivity to photoperiodic treatment show a higher free indol-3-ylacetic acid level (by about 35 %) than the first pair leaves, organs with high sensitivity to photoperiodic treatment. This was seen in plants of three different age groups : A. with the first pair of leaves of 15-20 mm in length; B. with the first pair of leaves having finished their growth and C. with the third leaf of 30-40 mm in length.Changes in free IAA level during the inductive dark period were similar in both cotyledons and leaves of the first pair. The level of IAA rose in the first half of the dark period, began to decrease in the latter half, reaching nearly initial level at its end.Application of IAA (10-4 - 10-2M) to the cotyledons reduced their already low photoperiodic sensitivity resulting in inhibition of flowering (almost 70 % using 10-4M IAA).Elevated free IAA level is assumed to be one of the causal factors of low photoperiodic sensitivity of cotyledons.

Ethylene-Induced Leaf Abscission in Cotton Seedlings

The speed of ethylene-induced leaf abscission in cotton (Gossypium hirsutum L. cv LG-102) seedlings is dependent on leaf position (i.e. physiological age). Fumigation of intact seedlings for 18 hours with 10 microliters per liter of ethylene resulted in 40% abscission of the still-expanding third true (3 degrees ) leaves but had no effect on the fully expanded first true (1 degrees ) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3 degrees leaves occurred while <50% abscission of the 1 degrees leaves was observed. On a leaf basis, endogenous levels of free IAA in 1 degrees leaves were approximately twice those of 3 degrees leaves. Free IAA levels were reduced equally (approximately 55%) in both leaf types after 18 hours of ethylene (10 microliters per liter) treatment. Ethylene treatment of intact seedlings inhibited the basipetal movement of [(14)C]IAA in petiole segments isolated from both leaf types in a dose-dependent manner. The auxin transport inhibitor N-1-naphthylphthalamic acid increased the rate and extent of ethylene-induced leaf abscission at both leaf positions but did not alter the relative pattern of abscission. Abscission-zone explants prepared from 3 degrees leaves abscised faster than 1 degrees leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3 degrees explants was not appreciably inhibited by exogenous IAA while 1 degrees explants exhibited a pronounced and protracted inhibition. The synthetic auxins 2,4-D and 1-naphthaleneacetic acid completely inhibited ethylene-induced abscission of both 1 degrees and 3 degrees explants for 40 hours. It is proposed that the differential abscission response of cotton seedling leaves is primarily a result of the limited abscission-inhibiting effects of IAA in the abscission zone of the younger leaves.

Changes in free IAA level in the leaves of short-and long-day tobacco during flowering and the effect of applied IAA on the transition to flowering

Changes in free IAA level were studied in the leaves of the central stem zone of short-day tobacco (Mcotianatabacum, cv. Maryland Mammoth) and long-day tobacco(Nicotiana silvestris) in inductive photoperiodic regime after 10, 20, 30 and 40 d, respectively. The leaves of SD tobacco Mammoth showed a high free IAA level in vegetative plants kept under long days but it significantly decreased (by ca. 50 %) after 10, 30 and 40 short days, respectively. After 20 short days the IAA level was as high as in the leaves of plants at the beginning of inductive treatment. The changes of freeIAA level in the leaves of LD tobacco N.silvestris were similar to those of SD Mammoth, but the IAA level in this species was significantly lower than that of Mammoth throughout the investigated period. Consequently, the changes observed in N. silvestris were much less pronounced. Plants of both tobacco species were fully induced to flowering by 30 inductive days and this was associated with differentiation of the flower organs.Application of 10 -4 M IAA during the last 10 d of the inductive treatment of 30 d significantly reduced flowering in SD tobacco Mammoth without changing the stem length and apex width. Apex length was slightly reduced. IAA application elicited almost no effect inN. silvestris. The results are discussed with respect to the possible role of IAA in flower induction in SD and LD plants.

Higher flower bud formation in haploid tobacco is connected with higher peroxidase/IAA-oxidase activity, lower IAA content and ethylene production

Haploid tobacco plants (cv. Samsun) form inflorescences with a larger number of flowers than diploid plants. Leaves of haploid plants were shown to have lower free IAA level (by 40 %), higher peroxidase (by 160 %) and IAA-oxidase (by 70 %) activities and produce less ethylene (by 25 %) than leaves of corresponding diploid plants. The increase of peroxidase activity in haploids was due to the increase in the activity of the cathodic isozyme which is known to have high IAA-oxidase activity. It is proposed that higher peroxidase/IAA-oxidase activity in haploid plants may take part in IAA catabolism, at least duringin vitro culture of haploid explants. Lowered IAA level and ethylene production may then be directly correlated with a larger number of flower buds; as a higher IAA level is generally considered to act as a background inhibitor of flowering.

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by &lt;italic&gt;Agrobacterium tumefaciens&lt;/italic&gt; III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissues transformed with Agrobacterium tumefaciens that harbored wild-type, aux− or cyt− Ti plasmids. IAA oxidase activities were similar among the three types of transformed tissue. Metabolites of IAA were examined by following the fate of 3-indolyl-[l-l4C]IAA. IAA conjugates were detected in all transformed tissues as well as in hypocotyl segments of non-transformed carrot seedlings. The rate of formation of IAA conjugates was ten times higher in the tissues transformed with wild-type or cyt− Ti plasmids than in the tissues transformed with aux− Ti plasmids. When the tissues transformed with aux− Ti plasmids were cultured on medium that contained IAA for 6 h, the rate of formation of IAA conjugates in these tissues became as high as that in tissues transformed with wild-type or cyt− Ti plasmids. The tissues transformed with wild-type or cyt− Ti plasmids not only synthesize larger amounts of IAA but also convert a larger amount of free IAA to conjugated IAA than do non-transformed and aux− transformed tissues. Presumably, in carrot, the formation of IAA conjugates decreases the amount of free IAA in the transformed tissues that synthesize large amounts of IAA and, consequently, the level of free IAA can be maintained fairly constant.

Measurement of Indole-3-Acetic Acid in Peach Fruits (Prunus persica L. Batsch cv Redhaven) during Development

The amount of indole-3-acetic acid (IAA) was measured in peach fruits by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution assay with [(13)C(6)]IAA as an internal standard throughout the growing season. Ethylene evolution of the fruit was also measured. IAA levels were 25 nanograms per gram fresh weight, 18 days after anthesis. Both IAA levels and rates of ethylene evolution declined to their lowest levels (7 nanograms IAA per gram fresh weight and 0.01 nanoliter ethylene per gram per hour) in the second stage of fruit growth. Endogenous levels of free-IAA and ethylene evolution increased in the last stage of peach fruit development to 32 nanograms per gram fresh weight and 0.27 nanoliter per gram per hour, respectively. IAA amounts peaked in the ovules 67 days after anthesis.

Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development

Levels of activity of the sucrose catabolizing enzymes, acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), were measured during development of new leaves of Citrus sinensis (L.) Osbeck cv. Shamouti. Soluble acid invertase showed a peak activity of 32 nkat (g fresh weight)−1 at ca 60% of full leaf expansion and rapidly declined toward and after full expansion. There was no concomitant increase in an insoluble form of the enzyme. Sucrose synthase activity, measured in the synthesis direction, declined from 33% of full leaf expansion [10 nkat (g fresh weight)−1] 10, and following, full expansion. Highest sucrose synthase activity, measured in the cleavage direction, was 6 nkat (g fresh weight)−1 and showed little change during development. Acid invertase has a Km of 5 mMfor sucrose, while sucrose synthase had a Km of 118 mM for sucrose. Changes in acid invertase activity correlated with changes in the reducing sugar:sucrose ratio. These results suggest that soluble acid invertase activity is the primary enzyme responsible for sucrose catabolism in the expanding Citrus leaf. Changes in leaf expansion rate and invertase activity did not correlate positively with changes in endogenous free IAA level, as determined by enzyme linked immunoassay.

Auxin Distribution in Spruce Needles

Summary Auxin was extracted from needles of Picea abies as free and as total IAA. It was quantified with the aid of a solid-phase enzyme immunoassay. The dependence of auxin levels on the position of the needle on the tree, shading, season, and age of the tree is described for representative examples with ages between 8 and 25 years. Marked differences were found especially for free IAA levels. The consideration of absolute auxin levels for an evaluation of the health status of conifers is questioned.

Auxin in flowering of short-pay and long-dayChenopodium species

The fluctuation of free IAA under 16 h dark period in shoots (receptor organs of photoperiodic induction) and roots of the short-day plant (SDP)Chenopodium rubrum and in shoots of the long-day plant (LDP)Chenopodium murale is very similar. The data reflect the general adjustment of auxin level to day-length rather than changes due to floral induction. However, the shift in phasing of the circadian rhythm of flowering was accompanied by a change in the position of the’ troughs’ of free IAA levels indicating a possible relationship between the two processes. Periods of higher sensitivity to application of IAA (3. 10-4M) inhibitory to flowering have been observed both during the endogenous rhythm of flowering in the SDPC. rubrum and during induction by days of continuous illumination in the LDPC. murale. There exist common traits in the response of LDP and of SDPChenopodium to auxin treatment. Aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, counteracted some flowering inhibitory effects of IAA when applied simultaneously with it. This suggests that auxin effects in modifying flowering might in fact be due to ethylene.

Effect of Ethephon on IAA Transport, IAA Conjugation, and Antidotal Action of NAA in Relation to Leaf Abscission of Pecan

Abstract Ethephon at 3, 6, and 12 mm induced abscission of mature foliage on pecan [Carya illinoensis (Wangenh) K. Koch] seedlings. This effect was offset to varying degrees using NAA at 3, 6, or 12 mm in which NAA acted as an effective antidote to ethephon and presumably ethylene. Abscission varied with leaf age and with the relative timing of leaf treatment with ethephon and NAA. Leaves treated with NAA 6 days prior to ethephon treatment were much more sensitive to ethephon induced abscission than those treated one day prior to, at time of, or one day after ethephon sprays. There were no abscission response differences among the 3 latter NAA treatment times. Ethephon greatly reduced 14C-IAA transport in leaf midrib tissue and increased the amount of IAA conjugated in leaf tissue without changing free IAA levels. It is suggested that ethephon treatment reduces the amount of IAA transported from the leaf blade to the abscission zone by inhibiting auxin transport in vascular tissue. IAA inactivation by conjugation appears to have no influence on the IAA level reaching the cells of the abscission zone. Chemical names used: (2-chloroethyl)phosphonic acid (ethephon); 1-naphthaleneacetic acid (NAA).

Fluctuation of free IAA under inductive and non-inductive photoperiods in Chenopodium rubrum

Fluctuation in levels of endogenous free IAA has been followed in the SD plant Chenopodium rubrum under photoperiodic conditions inductive or not inductive of flowering. Endogenous IAA was measured fluorimetrically as α-pyrone. The level of IAA shows little fluctuation under continuous illumination. An endogenous rhythm of IAA fluctuation was found in plants transferred from light to continuous darkness, with a natural period of 30 hrs. The ‘troughs’ of minimum IAA level within the endogenous rhythm coincided with the peaks in the endogenous rhythm of flowering response, which possessed the same period length. The concentration of IAA in the shoot always decreased at the end of cycles of dark period that induce flowering. The results are discussed in relation to the role of IAA in flowering of SD plants.

The effect of red light on the level of free IAA

Red light (660 nm) break which cancels the floral inductive effect of the dark period brought about a transitional rise in the level of free IAA in the shoot ofChenopodium rubrum. The higher content of IAA was then found in treated plants at the beginning of the following photoperiod, too. The red light treatment did not change the phase of endogenous rhythm fluctuations of free IAA.

Action of red light on indole-3-acetic-acid status and growth in coleoptiles of etiolated maize seedlings

Brief irradiation of intact etiolated seedlings of maize (Zea mays L.) with red light (R; 30 μW cm(-2), 10 min) reduces the amounts of diffusible and free (solvent-extractable) indole-3-acetic acid (IAA) obtainable from excised coleoptile tips. The effect is transient, the lowest level (30% of the dark control) occurring at about 3 h after irradiation. The free-IAA content of the whole coleoptile and the diffusible-IAA yield from the base of the same organ are similarly reduced, whereas the conjugated-IAA content of the coleoptile is not affected. These results support the view that R inhibits the production of IAA at the coleoptile tip. It is further shown that R inhibits biosynthesis of [(3)H]IAA from [(3)H]tryptophan supplied to the coleoptile tip. The shapes of the fluence-response curves obtained for the reduction of the diffusible-IAA yield by R and far-red light (FR) indicate the participation of two photoreactive systems. One has thresholds at 10(-3) μW s cm(2) of R, five orders of magnitude less than the minimum required for the appearance of spectrophotometrically measurable far-red-absorbing form of phytochrome (Pfr) in vivo, and 10(-1) μW s cm(-2) of FR; its response is linear to the logarithm of fluence exceeding five orders of magnitude. The other system is seen above 10(2) μW s cm(-2) as an increase in the slope of the fluenceresponse curve; its response is FR reversible and related to the Pfr level of total photoreversible phytochrome. Both systems inhibit biosynthesis of IAA from tryptophan. Elongation of the coleoptile is stimulated by R; the stimulation is most apparent in the apical region, and is saturated with a fluence at which bo detectable pfr is formed. Farred light can also saturate this response. Since the endogenous IAA concentration in the coleoptile appears not to be in the inhibitory range, it is concluded that the stimulation of coleoptile elongation is not the result of changes in free-IAA levels.