Role of IAA-oxidase in the formation of ethylene from 1-aminocyclopropane-1-carboxylic acid

The IAA-oxidase system of olive tree (Olea europea) in the presence of its substrate, IAA, and cofactors, DCP and Mn², forms ethylene from 1-aminocyclopropane-l-carboxylic acid (ACC) bound as a Schiffs base to pyridoxal phosphate. Similarly, olive leaf discs upon incubation with ACC liberate considerable amounts of ethylene. The results suggest that this IAA-oxidase system may be the one active in the last step in the biosynthesis of ethylene from methionine.     

Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants

Inflorescence stalks produced the highest amount of ethylene in response to IAA as compared with other plant parts tested. Leaf age had an effect on IAA-induced ethylene with the youngest leaves showing the greatest stimulation. The highest amount of IAA-induced ethylene was produced in the root or inflorescence tip with regions below this producing less. Inflorescence stalks treated with IAA, 2,4-D, or NAA over a range of concentrations exhibited an increase in ethylene production starting at 1 microM with increasingly greater responses up to 100 microM, followed by a plateau at 500 microM and a significant decline at 1000 microM. Both 2,4-D and NAA elicited a greater response than IAA at all concentrations tested in inflorescence stalks. Inflorescence leaves treated with IAA, 2,4-D, or NAA exhibited the same trend as inflorescence stalks. However, they produced significantly less ethylene. Inflorescence stalks and leaves treated with 100 microM IAA exhibited a dramatic increase in ethylene production 2 h following treatment initiation. Inflorescence stalks showed a further increase 4 h following treatment initiation and no further increase at 6 h. However, there was a slight decline between 6 h and 24 h. Inflorescence leaves exhibited similar rates of IAA-induced ethylene between 2 h and 24 h. Light and high temperature caused a decrease in IAA-induced ethylene in both inflorescence stalks and leaves. Three auxin-insensitive mutants were evaluated for their inflorescence's responsiveness to IAA. aux2 did not produce ethylene in response to 100 microM IAA, while axr1-3 and axr1-12 showed reduced levels of IAA-induced ethylene as compared with Columbia wild type. Inflorescences treated with brassinolide alone had no effect on ethylene production. However, when brassinolide was used in combination with IAA there was a dramatic increase in ethylene production above the induction promoted by IAA alone.     

Ethylene-induced leaf abscission in cotton seedlings : the physiological bases for age-dependent differences in sensitivity

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°) leaves but had no effect on the fully expanded first true (1°) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3° leaves occurred while <50% abscission of the 1° leaves was observed. On a leaf basis, endogenous levels of free IAA in 1° leaves were approximately twice those of 3° 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 [¹⁴C]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° leaves abscised faster than 1° leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3° explants was not appreciably inhibited by exogenous IAA while 1° 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° and 3° 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.     

Light quality regulation of endogenous levels of auxin,abscisic acid and ethylene production in petioles and leaves of wild type and ACC deaminase transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> seedlings

Brassica napus L. seedlings responded to low red to far-red (R/FR) ratio by elongating petioles and decreasing leaf expansion. These typical shade avoidance traits were correlated with significantly decreased endogenous indole-3-acetic acid (IAA) levels and significantly increased endogenous abscisic acid (ABA) levels and ethylene production. The transgenic (T) B. napus line bearing the bacterial ACC deaminase gene, did not respond to low R/FR ratio with altered petiole and leaf growth and less ethylene (especially by petioles) was produced. As with WT seedlings, T seedlings had significantly lower IAA levels in both petioles and leaves under low R/FR ratio. However, ABA levels of low R/FR ratio-grown T seedlings either increased (petioles) or were unaltered (leaves). Our results further suggest that low R/FR ratio regulates endogenous IAA levels independently of ethylene, but there may be an interaction between ABA and ethylene in leaf development.     

The effect of plant growth regulator treatments on the levels of ethylene emanating from excised turgid and wilted wheat leaves

Abscisic acid (ABA) inhibits the production of ethylene induced by water stress in excised wheat leaves and counteracts the stimulatory effect of 6-benzyladenine (BA) on this process. The stimulatory effect of BA and the inhibitory effect of ABA were equally pronounced whether external or endogenous ethylene levels were determined. When leaves were sprayed or floated on solutions of BA, indole-3-acetic acid (IAA), gibberellic acid (GA₃), or ABA, the relative activities of these growth regulators on stress-induced ethylene at 10^-4 mol l^-1 were BA>IAA >GA₃>controls>ABA. In non-stressed leaves, however, where the levels of ethylene produced were 2–20 times smaller, the relative activities were IAA >BA>GA₃>controls>ABA. The effects of BA and ABA spray treatment on water stress induced ethylene were closely similar whether the solutions were applied 2 or 18 h prior to the initiation of water stress. The relationships between the levels of endogenous growth regulators in the plant and ethylene release induced by water stress are discussed.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - ABA abscisic acid - GLC gas-liquid chromatography - _leaf leaf water potential     

Ethylene Promotes Elongation Growth and Auxin Promotes Radial Growth in Ranunculus sceleratus Petioles

Submergence induces elongation in the petioles of Ranunculus sceleratus L., after a rise in endogenous ethylene levels in the tissue. Petioles of isolated leaves also elongate 100% in 24 hours when treated with ethylene gas, without a change in the radius. Application of silver thiosulfate, aminoethoxyvinylglycine (AVG), abscisic acid (ABA), or methyl jasmonate inhibits this elongation response. Gibberellic acid treatment promotes ethylene-induced elongation, without an effect on the radius. Indoelastic acid (IAA) induces radial growth in the petioles, irrespective of the presence or absence of added ethylene. High concentrations of IAA will also induce elongation growth, but this is largely due to auxin-induced ethylene synthesis; treatment with silver thiosulfate, AVG, ABA, or methyl jasmonate inhibit this auxin-promoted elongation growth. However, the radial growth induced by IAA is not affected by gibberellic acid, and not specifically inhibited by ABA, methyl jasmonate, silver thiosulfate, or AVG. These results support the idea that petiole cell elongation during “accommodation growth” can be separated from radial expansion. The radial expansion may well be regulated by IAA. However, effects of high levels of IAA are probably anomalous, since they do not mimic normal developmental patterns.     

Carbohydrates stimulate ethylene production in tobacco leaf discs : I. Interaction with auxin and the relation to auxin metabolism

Various naturally occurring carbohydrates, applied at a concentration range of 1 to 100 mm, stimulated ethylene production for several days in indoleacetic acid (IAA)-treated or untreated tobacco (Nicotiana tabacum L. cv `Xanthi') leaf discs. The lag period for this sugar-stimulated ethylene production was 8 to 12 hours after excision in the untreated leaf discs, but less than 2 hours in the IAA-treated ones. Among the tested carbohydrates, 12 were found to increase synergistically ethylene production, with d-galactose, sucrose, and lactose being the most active; mannitol and l-glucose had no effect. The extent and duration of the increased ethylene production was dependent upon the type of sugar applied, the tissue's age, and the existence of both exogenous IAA and sugar in the medium. Sucrose appeared to elicit a continuous IAA effect for 48 hours, as expressed by increased ethylene production, even when IAA was removed from the medium after a 4-hour pulse. Sucrose stimulated both the uptake and decarboxylation of [1-¹⁴C]IAA, as well as the hydrolysis of the esteric and amide IAA conjugates formed in the tissue after application of free IAA. This gradual hydrolysis was accompanied by a further accumulation of a third IAA metabolite. Moreover, synthetic indole-3-acetyl-l-alanine increased ethylene production mainly with sucrose, and this effect was accompanied by its increased decarboxylation and turnover pattern suggesting that release of free IAA was involved. An esteric IAA conjugate, tentatively identified by GC retention time was found to be the major component (84%) of the naturally occurring IAA conjugates in tobacco leaves. Accordingly the sucrose-stimulated ethylene production in tobacco leaves can be ascribed mainly to the sucrose-stimulated hydrolysis of the esteric IAA conjugate.     

Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants,and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 [plus or minus] 7%, SD) than in old leaves (36 [plus or minus] 8%), whereas there was no difference between young (73 [plus or minus] 8%) and old internodes (70 [plus or minus] 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 [plus or minus] 10%) for both leaves and internodes, independent of the total IAA level or tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.     

Involvement of Abscisic Acid in Ethylene-Induced Cotyledon Abscission in Cotton Seedlings

Cotton (Gossypium hirsutum L. cv LG102) seedlings raised from seeds exposed to 100 [mu]M norflurazon (NFZ) during imbibition contained reduced levels of free abscisic acid (ABA) and were visibly achlorophyllous. Exposure of untreated cotton seedlings to ethylene concentrations >1 [mu]L/L for 24 h resulted in cotyledon abscission. In contrast, exposure of NFZ-treated seedlings to concentrations of ethylene [less than or equal to]50 [mu]L/L elicited no cotyledon abscission. Application of ABA, an ABA analog, or jasmonic acid to NFZ-treated seedlings restored ethylene-induced abscission. Isolated cotyledonary node explants prepared from NFZ-treated seedlings exhibited an altered dose-response pattern of ethylene-induced petiole abscission. Endogenous levels of free IAA were unaltered in NFZ-treated seedlings. Ethylene treatment (50 [mu]L/L, 24 h) had no effect on free indoleacetic acid (IAA) levels in either control or NFZ-treated seedlings. Levels of conjugated (ester plus amide) IAA were substantially increased in NFZ-treated seedlings regardless of ethylene treatment. These results indicate that endogenous ABA plays an essential, but physiologically undefined, role in ethylene-induced cotyledon abscission in cotton.     

Free radical and free radical scavenger effects on indole-3-acetic acid levels and ethylene production

Stable free radicals, together with horseradish peroxidase, promoted degradation of indole-3-acetic acid (IAA). These reactions were retarded by the free radical scavengers Bromoxynil, Na-benzoate and kinetin. Certain free radicals promoted, but the free radical scavenger Bromoxynil retarded ethylene production in apple slices and mung bean stem tissues. The interdependency of free radicals and free radical scavengers in systems controlling IAA levels and ethylene production is discussed.     

Hormones in Relation to Stress Recovery in Populus robusta Cuttings

The relationships between changes in WSD,² stomatal resistance,and hormone levels during stress and stress recovery have beeninvestigated in a perennial plant, Populus robusta, over anextended stress period, involving a number of stress cyclesof differing duration. There were marked increases in WSD, stomatal resistance, freeABA levels, and ethylene levels in leaves and stems during stressimposition. However, during stress recovery, WSD declined mostrapidly, while decreases in other parameters were related tothe duration of the preceding stress. In general, the declinein stomatal resistance and free ABA levels to control valueswas protracted. Although there were increases in bound ABA levels,above control values, there was no close correlation with changesin free ABA levels. The concentrations of bound ABA observedin the latter phases of these experiments were higher than freelevels. No clear pattern of change in IAA concentration emerged in responseto stress or stress recovery. Although ethylene concentrations rise in response to initialstress imposition and fall during relief, subsequent stressand stress relief periods were without significant effect. The results are discussed in relation to the role of endogenousgrowth regulators in the response of plants to stress and inparticular the fate of stress-induced ABA.     

磷脂酶Dδ对拟南芥叶片衰老过程中内源ROS和激素含量的影响

活性氧（ROS）和植物激素是植物衰老过程中重要的内在或者外在的调控因子。我们发现,相对于离体诱导的衰老过程,在脱落酸（ABA）和乙烯（ethylene）促进的衰老过程中有较多的活性氧积累;在对拟南芥磷脂酶Dδ（PLDδ）缺失型突变体的研究中发现,与野生型相比,突变体在衰老过程中产生较少的活性氧。我们比较了上述两种基因型的离体叶片在离体、ABA和ethylene三种衰老处理下内源的ABA、茉莉酸甲酯（MeJA）、玉米素核苷（Zeatin Riboside, ZR）和吲哚乙酸（IAA）的含量变化,发现每一种激素对上述三种衰老处理的响应模式都很相似。在离体诱导的衰老中,两种基因型拟南芥的内源激素含量没有差异;而在ABA促进的衰老过程中,PLDδ缺失型突变体叶片中的MeJA的含量较低,ZR和IAA含量较高;在乙烯促进的衰老过程中,突变体中的ABA和MeJA的含量较低,ZR和IAA含量较高。上述内源激素的这种变化可能有助于延缓突变体的衰老。     

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.     

不同氮营养下炭疽病菌侵染对菜心叶片内源激素的影响

研究了6种不同氮营养水平下炭疽病菌对菜心叶片乙烯释放量、脱落酸(ABA)和吲哚乙酸(IAA)含量的影响及其与抗病性的关系.结果表明,在感病过程中,所有氮营养处理乙烯释放量呈单峰曲线变化,并在接种后的第4天至第6天达到峰值,适宜氮、低氮营养比高氮或不施肥可抑制乙烯产生和ABA的合成,维持体内乙烯、ABA的稳定.适宜氮、低氮处理感病后IAA含量一直上升,而高氮或不施肥处理的IAA呈单峰曲线变化,并在接种后4～6d达到峰值,随着致病时间的延长,适宜氮、低氮营养比高氮或不施肥可提高炭疽病菌对IAA的诱导.表明氮营养炭疽病内源激素之间存在密切的关系,维持植株体内的激素平衡是提高植株耐病的机理之一.     

Effect of glyphosate on ethylene production in tobacco callus

Glyphosate (N-phosphonomethylglycine) caused a significant decrease or a slight increase in ethylene production in tobacco callus (Nicotiana tabacum L.) depending on the concentration of indole-3-acetic acid (IAA) present in the medium. IAA stimulated ethylene production, but a pretreatment with glyphosate greatly reduced the IAA-induced ethylene production. Inasmuch as glyphosate treatment promoted the metabolism of IAA, the decrease in ethylene production induced by glyphosate is attributed to the rapid loss of free IAA in the treated tissue.     

Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato

In this study we investigated the role of ethylene in the formation of lateral and adventitious roots in tomato ( Solanum lycopersicum ) using mutants isolated for altered ethylene signaling and fruit ripening. Mutations that block ethylene responses and delay ripening – Nr ( Never ripe ), gr ( green ripe ), nor ( non ripening ), and rin ( ripening inhibitor ) – have enhanced lateral root formation. In contrast, the epi ( epinastic ) mutant, which has elevated ethylene and constitutive ethylene signaling in some tissues, or treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC), reduces lateral root formation. Treatment with ACC inhibits the initiation and elongation of lateral roots, except in the Nr genotype. Root basipetal and acropetal indole-3-acetic acid (IAA) transport increase with ACC treatments or in the epi mutant, while in the Nr mutant there is less auxin transport than in the wild type and transport is insensitive to ACC. In contrast, the process of adventitious root formation shows the opposite response to ethylene, with ACC treatment and the epi mutation increasing adventitious root formation and the Nr mutation reducing the number of adventitious roots. In hypocotyls, ACC treatment negatively regulated IAA transport while the Nr mutant showed increased IAA transport in hypocotyls. Ethylene significantly reduces free IAA content in roots, but only subtly changes free IAA content in tomato hypocotyls. These results indicate a negative role for ethylene in lateral root formation and a positive role in adventitious root formation with modulation of auxin transport as a central point of ethylene–auxin crosstalk.     

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.     

Auxins in the development of an arbuscular mycorrhizal symbiosis in maize

While the levels of free auxins in maize (Zea mays L.) roots during arbuscular mycorrhiza formation have been previously described in detail, conjugates of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with amino acids and sugars were neglected. In this study, we have therefore determined free, ester and amide bound auxins in roots of maize inoculated with Glomus intraradices during early stages of the colonization process. Ester conjugates of IAA and IBA were found only in low amounts and they did not increase in AM colonized roots. The Levels of IAA and IBA amide conjugates increased 20 and 30 days past inoculation (dpi). The formation of free and conjugated IBA but not IAA was systemically induced during AM colonization in leaves of maize plants. This implicated a role for auxin conjugate synthesis and hydrolysis during AM. We have therefore investigated the in vivo metabolism of 3H-labeled IBA by TLC but only slight differences between control and AM-inoculated roots were observed. The activity of auxin conjugate hydrolase activity measured with three different putative substrates showed a decrease in infected roots compared to controls. The fluorinated IBA analog TFIBA inhibited IBA formation in leaves after application to the root system, but was not transported from roots to shoots. AM hyphae were also not able to transport TFIBA. Our results indicate complex control mechanisms to regulate the levels of free and conjugated auxins, which are locally and systemically induced during early stages of the formation of an arbuscular mycorrhizal symbiosis.     

Endogenous indole-3-acetic Acid in the stem of tobacco in relation to flower neoformation as measured by mass spectroscopic assay

The contents of free indole-3-acetic acid (IAA) and alkali-labile, conjugated IAA were measured in relation to a `floral gradient' present in epidermis and subepidermis tissues of flowering plants of Nicotiana tabacum by capillary gas-chromatographic spectrometric analysis by selected ion monitoring (GC-SIM-MS) using 2,4,5,6,7-penta deutero IA (²H₅-IAA) as an internal standard. In floral axes, floral branches and stems with floral branches, free IAA levels (dry weight) were 387, 253, and 417 nanograms, and bound IAA levels were 99, 1089, and 268 nanograms. In vegetative tissue of the first plus second internodes (measured from top), and of the 11th to 13th internodes, free IAA levels were 826 and 500 nanograms, and bound IAA levels were 1421 and 286 nanograms, respectively. Since flower-forming ability of excised cells from the epidermis and subepidermis shows a gradient in an in vitro system, but levels of IAA in these tissues do not, there thus appears to be no correlation between flower-forming ability (in vitro) and endogenous IAA levels (at the time of excision) in tobacco stem tissues.     

Effects of a short-day treatment on pool size, synthesis, degradation and transport of 3-indole-acetic acid in Scots pine (Pinus sylvestris L.) seedlings

The effects of a short-day treatment on pool-size, synthesis, degradation, and transport of 3-indole-acetic acid (IAA) in Scots pine ( Pinus sylvestris L.) seedlings were studied. The short-day treatment decreased the pool of free IAA in the needles but did not significantly affect the pool-sizes of free IAA in the shoot-axis, hypocotyls, cotyledons and roots. The bound alkali-hydrolysable IAA-pools were not affected by the short-day treatment. The synthesis and degradation of radioactive IAA were also unaffected by the short-day treatment. The transport of radioactive IAA was greatest from needles to roots, and this transport was significantly decreased by the short-day treatment.