Free Indole-3-acetic Acid Content Research Articles

In vitro rooting of Quercus robur, activated charcoal vs. exogenous auxin: a morphophysiological approach

In plant cultivation, morphophysiological status of the plantlets is crucial. We aimed to assess how activated charcoal (AC) and synthetic auxins, separately and in combination, affect the biochemistry, anatomy, and physiology of Quercus robur plantlets. Two concurrent experiments were conducted using indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as exogenous auxins, with or without AC. Plantlets grown without exogenous auxins and AC displayed weak root development, reduced functionality, lower photosynthetic pigments, and elevated starch and free IAA content. Incorporating IAA or IBA into the medium triggered positive morphophysiological changes, such as aerenchyma formation, increased hydrogen peroxide, and reduced free IAA levels in the in vitro-produced clones. Plantlets cultured with 2 g L−1 AC exhibited higher biomass and more photosynthetic pigments. AC also enhanced the photosynthetic apparatus, as indicated by higher total performance index (PI(TOTAL)) values. Importantly, AC improved rooting success and overall morphophysiological condition, regardless of exogenous auxin presence. These findings provide novel insights into in vitro rooting modulation with AC and exogenous auxins, promising advancements in the growth and performance of Q. robur plantlets.

Allelic variation in the indoleacetic acid-lysine synthase gene of the bacterial pathogen Pseudomonas savastanoi and its role in auxin production.

Indole-3-acetic acid (IAA) production is a pathogenicity/virulence factor in the Pseudomonas syringae complex, including Pseudomonas savastanoi. P. savastanoi pathovars (pvs.) genomes contain the iaaL gene, encoding an enzyme that catalyzes the biosynthesis of the less biologically active compound 3-indole-acetyl-ϵ-L-lysine (IAA-Lys). Previous studies have reported the identification of IAA-Lys in culture filtrates of P. savastanoi strains isolated from oleander (pv. nerii), but the conversion of IAA into a conjugate was not detectable in olive strains (pv. savastanoi). In this paper, we show the distribution of iaaL alleles in all available P. savastanoi genomes of strains isolated from woody hosts. Most strains encode two different paralogs, except for those isolated from broom (pv. retacarpa), which contain a single allele. In addition to the three previously reported iaaL alleles (iaaL Psv, iaaL Psn and iaaL Pto), we identified iaaL Psf, an exclusive allele of strains isolated from ash (pv. fraxini). We also found that the production of IAA-Lys in P. savastanoi pv. savastanoi and pv. nerii depends on a functional iaaL Psn allele, whereas in pv. fraxini depends on iaaL Psf. The production of IAA-Lys was detected in cultures of an olive strain heterologously expressing IaaLPsn-1, IaaLPsf-1 and IaaLPsf-3, but not when expressing IaaLPsv-1. In addition, Arabidopsis seedlings treated with the strains overproducing the conjugate, and thus reducing the free IAA content, alleviated the root elongation inhibitory effect of IAA. IAA-Lys synthase activity assays with purified allozymes confirmed the functionality and specificity of lysine as a substrate of IaaLPsn-1 and IaaLPsf-3, with IaaLPsf-3 showing the highest catalytic efficiency for both substrates. The IAA-Lys synthase activity of IaaLPsn-1 was abolished by the insertion of two additional tyrosine residues encoded in the inactive allozyme IaaLPsv-1. These results highlight the relevance of allelic variation in a phytohormone-related gene for the modulation of auxin production in a bacterial phytopathogen.

Auxin-mediated regulation of arbuscular mycorrhizal symbiosis: A role of SlGH3.4 in tomato.

Most land plants can establish symbiosis with arbuscular mycorrhizal (AM) fungi to increase fitness to environmental challenges. The development of AM symbiosis is controlled by intricate procedures involving all phytohormones. However, the mechanisms underlying the auxin-mediated regulation of AM symbiosis remains largely unknown. Here, we report that AM colonisation promotes auxin response and indole-3-acetic acid (IAA) accumulation, but downregulates IAA biosynthesis genes in tomato(Solanum lycopersicum). External IAA application modulates the AM symbiosis by promoting arbuscule formation at low concentrations but repressing it at high concentrations. An AM-induced GH3 gene, SlGH3.4, encoding a putative IAA-amido synthetase, negatively regulates mycorrhization via maintaining cellular auxin homoeostasis. Loss of SlGH3.4 function increased free IAA content and arbuscule incidence, while constitutively overexpressing SlGH3.4 in either tomato or rice resulted in decreased IAA content, total colonisation level and arbuscule abundance in mycorrhizal roots. Several auxin-inducible expansin genes involved in AM formation or resistance to pathogen infection were upregulated in slgh3.4 mycorrhizal roots but downregulated in the SlGH3.4-overexpressing plants. Taken together, our results highlight a positive correlation between the endogenous IAA content and mycorrhization level, particularly arbuscule incidence, and suggest that the SlGH3.4-mediated auxin homoeostasis and regulation of expansin genes is involved in finely tuning the AM development.

AcERF1B and AcERF073 Positively Regulate Indole-3-acetic Acid Degradation by Activating AcGH3.1 Transcription during Postharvest Kiwifruit Ripening

Ethylene can accelerate the postharvest ripening process of kiwifruit, while indole-3-acetic acid (IAA) delays it. However, the molecular mechanism by which ethylene regulates IAA degradation is unclear. Here, we found that ethephon promotes the degradation of free IAA in kiwifruit. Furthermore, ethylene can promote the expression of AcGH3.1 and enhance its promoter activity. Two ethylene response factors (ERFs), AcERF1B and AcERF073, were obtained using an AcGH3.1 promoter as bait for a yeast one-hybrid screening library. Both AcERF1B and AcERF073 bind to the AcGH3.1 promoter to activate it. Also, AcERF1B/073 enhanced AcGH3.1 expression, decreased the free IAA content, and increased the IAA-Asp content in kiwifruit. In addition, we found that the AcERF1B and AcERF073 proteins directly interact, and this interaction enhanced their binding to the AcGH3.1 promoter. In summary, our results suggest that AcERF1B and AcERF073 positively regulate IAA degradation by activating AcGH3.1 transcription, which accelerated postharvest kiwifruit ripening.

WRKY46 promotes ammonium tolerance in Arabidopsis by repressing NUDX9 and indole-3-acetic acid-conjugating genes and by inhibiting ammonium efflux in the root elongation zone.

Ammonium (NH4+ ) is toxic to root growth in most plants, even at moderate concentrations. Transcriptional regulation is one of the most important mechanisms in the response of plants to NH4+ toxicity, but the nature of the involvement of transcription factors (TFs) in this regulation remains unclear. Here, RNA-seq analysis was performed on Arabidopsis roots to screen for ammonium-responsive TFs. WRKY46, the member of the WRKY transcription factor family most responsive to NH4+ , was selected. We defined the role of WRKY46 using mutation and overexpression assays, and characterized the regulation of NUDX9 and indole-3-acetic acid (IAA)-conjugating genes by WRKY46 via yeast one-hybrid and electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative real-time polymerase chain reaction (ChIP-qPCR). Knockout of WRKY46 increased, while overexpression of WRKY46 decreased, NH4+ -suppression of the primary root. WRKY46 is shown to directly bind to the promoters of the NUDX9 and IAA-conjugating genes (GH3.1, GH3.6, UGT75D1, UGT84B2) and to inhibit their transcription, thus positively regulating free IAA content and stabilizing protein N-glycosylation, leading to an inhibition of NH4+ efflux in the root elongation zone (EZ). We identify TF involvement in the regulation of NH4+ efflux in the EZ, and show that WRKY46 inhibits NH4+ efflux by negative regulation of NUDX9 and IAA-conjugating genes.

Salicylic acid regulates adventitious root formation via competitive inhibition of the auxin conjugation enzyme CsGH3.5 in cucumber hypocotyls.

Exogenous SA treatment at appropriate concentrations promotes adventitious root formation in cucumber hypocotyls, via competitive inhibiting the IAA-Asp synthetase activity of CsGH3.5, and increasing the local free IAA level. Adventitious root formation is critical for the cutting propagation of horticultural plants. Indole-3-acetic acid (IAA) has been shown to play a central role in regulating this process, while for salicylic acid (SA), its exact effects and regulatory mechanism have not been elucidated. In this study, we showed that exogenous SA treatment at the concentrations of both 50 and 100µM promoted adventitious root formation at the base of the hypocotyl of cucumber seedlings. At these concentrations, SA could induce the expression of CYCLIN and Cyclin-dependent Kinase (CDK) genes during adventitious rooting. IAA was shown to be involved in SA-induced adventitious root formation in cucumber hypocotyls. Exposure to exogenous SA led to a slight increase in the free IAA content, and pre-treatment with the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) almost completely abolished the inducible effects of SA on adventitious root number. SA-induced IAA accumulation was also associated with the enhanced expression of Gretchen Hagen3.5 (CsGH3.5). The in vitro enzymatic assay indicated that CsGH3.5 has both IAA- and SA-amido synthetase activity and prefers aspartate (Asp) as the amino acid conjugate. The Asp concentration dictated the functional activity of CsGH3.5 on IAA. Both affinity and catalytic efficiency (Kcat/Km) increased when the Asp concentration increased from 0.3 to 1mM. In contrast, CsGH3.5 showed equal catalytic efficiency for SA at low and high Asp concentrations. Furthermore, SA functioned as a competitive inhibitor of the IAA-Asp synthetase activity of CsGH3.5. During adventitious formation, SA application indeed repressed the IAA-Asp levels in the rooting zone. These data show that SA plays an inducible role in adventitious root formation in cucumber through competitive inhibition of the auxin conjugation enzyme CsGH3.5. SA reduces the IAA conjugate levels, thereby increasing the local free IAA level and ultimately enhancing adventitious root formation.

Overexpression of MsGH3.5 inhibits shoot and root development through the auxin and cytokinin pathways in apple plants.

Phytohormonal interactions are crucial for plant development. Auxin and cytokinin (CK) both play critical roles in regulating plant growth and development; however, the interaction between these two phytohormones is complex and not fully understood. Here, we isolated a wild apple (Malus sieversii Roem) GRETCHEN HAGEN3 (GH3) gene, MsGH3.5, encoding an indole-3-acetic acid (IAA)-amido synthetase. Overexpression of MsGH3.5 significantly reduced the free IAA content and increased the content of some IAA-amino acid conjugates, and MsGH3.5-overexpressing lines were dwarfed and produced fewer adventitious roots (ARs) than the control. This phenotype is consistent with the role of GH3 in conjugating excess free active IAA to amino acids in auxin homeostasis. Surprisingly, overexpression of MsGH3.5 significantly increased CK concentrations in the whole plant, and altered the expression of genes involved in CK biosynthesis, metabolism and signaling. Furthermore, exogenous CK application induced MsGH3.5 expression through the activity of the CK type-B response regulator, MsRR1a, which mediates the CK primary response. MsRR1a activated MsGH3.5 expression by directly binding to its promoter, linking auxin and CK signaling. Plants overexpressing MsRR1a also displayed fewer ARs, in agreement with the regulation of MsGH3.5 expression by MsRR1a. Taken together, we reveal that MsGH3.5 affects apple growth and development by modulating auxin and CK levels and signaling pathways. These findings provide insight into the interaction between the auxin and CK pathways, and might have substantial implications for efforts to improve apple architecture.

Expression of two indole-3-acetic acid (IAA)-amido synthetase (GH3) genes during fruit development of raspberry (Rubus idaeus Heritage)

The conjugation of indole-3-acetic acid (IAA) to amino acids by indole-3-acetic acid (IAA)-amido synthetases (GH3) is an important part of auxin level regulation. However, the auxin conjugation during development of soft fruits such as raspberry is poorly understood. In this study, indole-3-acetic acid (IAA)-amido synthetases in raspberry, designated as RiGH3 (RiGH3.1, RiGH3.5 transcripts) were evaluated during fruit development of raspberry cultivar Rubus idaeus Heritage, and under IAA treatment. The results showed that before to the onset of ripening the fruit size, weight and the expression of IAA-amido synthetase RiGH3.1 transcript levels increased. Then when the fruits attain full development, fruit firmness and titratable acidity decreased, in the contrast to ethylene production and total soluble solids content increasing. However, the RiGH3.5 transcript was found to be expressed primarily in flowers. When compared to untreated control fruit, fruit treated with 1 mM of IAA at white stage, showed an increase of RiGH3.1 transcript during in-vitro assay (10 °C by 18 h). However, no significant change in the levels of RiGH3.5 was observed during IAA treatment. Multiple alignments of the full-length predicted RiGH3.1 protein sequences revealed a high sequence homology with proteins deduced sequences described for other fruit of Rosaceae species. The RiGH3.1 deduced sequence showed the presence of binding motives for IAA and aspartic acid, and indicate that the isolated sequence have the typical motives of GH3.1 protein family. These findings give new insights into the possible role of RiGH3.1 transcripts, and the IAA conjugation (in maintaining the low concentration of free IAA) during raspberry fruit ripening.

Involvement of auxin in the regulation of ammonium tolerance in rice (Oryza sativa L.)

Ammonium (NH4+) is an important nitrogen source and is widely used as a fertilizer in agricultural systems. However, excess NH4+ inhibits root growth, and, subsequently, vegetative shoot growth and yield. This study examines whether auxin is involved in differential NH4+ tolerance in rice (Oryza sativa L.), and how auxin is regulated under high-NH4+ conditions in rice. An NH4+-sensitive (Kasalath, Kas) and an NH4+-insensitive (Koshihikari, Kos) rive cultivar were cultured hydroponically with or without exogenous indole-3-acetic acid (IAA) and auxin biosynthesis inhibitors. Root growth, root area, tissue IAA content, and transcription of genes involved in auxin biosynthesis, conjugation and degradation were determined. pDR5::GUS staining and auxin measurement show that high NH4+ can decrease free IAA content in roots. In addition, quantitative RT-PCR, pharmacology, and genetics analysis suggest that Kos possesses a higher capacity for auxin biosynthesis and a weaker capacity for auxin metabolism compared to Kas under high-NH4+ stress. We conclude that the NH4+-tolerant cultivar possesses a higher capacity to maintain auxin homeostasis under high-NH4+ stress, and that this advantage is incurred by promotion of auxin biosynthesis and a suppression of auxin metabolism.

Influence of Different Yeast Strains on Metabolism of Tryptophan and Indole-3-Acetic Acid During Fermentation

The effect of supplementation of Riesling (Vitis vinifera L.) must with ammonium sulphate (AS) on tryptophan (Trp) and indole-3-acetic acid (IAA) metabolism during alcoholic fermentation with four commercial yeast strains (Uvaferm CEG, Lalvin Cross Evolution, Anchor VIN 13 and Anchor exotic SPH) were examined. AS addition reduced Trp uptake by yeasts from 1% to 15%. Wines produced by Anchor VIN 13 and Uvaferm CEG yeasts with AS addition had the highest concentrations of Trp (0.60 and 0.59 mg/L respectively). Concentrations of total and bound IAA decreased during all treatments. At the end of fermentation the highest concentration of free IAA was measured in the control must inoculated with Anchor VIN 13 yeast (35.83 μg/L) and the lowest in the sample inoculated with Uvaferm CEG yeast (13.08 μg/L). Anchor VIN 13 yeast showed the strongest change in metabolism due to AS addition during fermentation. 2-Aminoacetophenone (2-AAP) formation was not observed in all the studied wines.

Ammonium regulates embryogenic potential in Cucurbita pepo through pH-mediated changes in endogenous auxin and abscisic acid

When supplied as the sole nitrogen source, ammonium can induce somatic embryogenesis in some plant species, including pumpkin. To get an insight into the involvement of phytohormones in this adaptive developmental response to nitrogen availability, the status of endogenous abscisic acid (ABA), indole-3-acetic acid (IAA), and their conjugates was analyzed in an ammonium-induced embryogenic pumpkin culture. Results were compared with endogenous hormone status in an embryogenic culture induced by exogenously supplied 2,4-dichlorophenoxyacetic acid (2,4-D). In the NH4 +-induced somatic embryogenic culture, the maximum concentration of ABA was measured after 21 days of cultivation, while in the 2,4-D-induced culture ABA peaked after 7 days of cultivation, which suggested a possible activation of different ABA-regulated stress responses. The NH4 +-induced embryogenic culture was also characterized by increased accumulation of endogenous IAA to the level similar or higher than the one induced by 2,4-D. A considerable decrease (threefold) in endogenous IAA content was observed after buffering the NH4 +-medium with MES or after nitrate re-supply. In contrast, buffering the NH4 +-medium increased endogenous ABA by five times. Significant ammonium- and pH-related changes were also detected in ascorbate and glutathione redox status, and discussed regarding the corresponding changes in cell size and mitotic activity in the cultures. Furthermore, a negative correlation between free IAA content and the activity of ascorbate peroxidase (EC 1.11.1.11) and auxin-sensitive peroxidase CpAPRX (EC 1.11.1.7) was observed. The results strongly suggest a mediating role of external pH in the regulation of endogenous phytohormones and the ascorbate–glutathione cycle during ammonium-induced acquisition of embryogenic competence in pumpkin.

Auxin production by the plant trypanosomatid Phytomonas serpens and auxin homoeostasis in infected tomato fruits.

Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of γ-proteobacteria. Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic compounds, quantified by high performance liquid chromatography, increased in P. serpens media in response to amounts of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with P. serpens the concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates, keeping unaltered the concentration of free IAA. Ethanol also accumulated in P. serpens-conditioned media, as the result of a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a three-dimensional model of the enzyme.

SEPALLATA1/2-suppressed mature apples have low ethylene, high auxin and reduced transcription of ripening-related genes

Fruit ripening is an important developmental trait in fleshy fruits, making the fruit palatable for seed dispersers. In some fruit species, there is a strong association between auxin concentrations and fruit ripening. We investigated the relationship between auxin concentrations and the onset of ethylene-related ripening in Malus × domestica (apples) at both the hormone and transcriptome levels. Transgenic apples suppressed for the SEPALLATA1/2 (SEP1/2) class of gene (MADS8/9) that showed severely reduced ripening were compared with untransformed control apples. In each apple type, free indole-3-acetic acid (IAA) concentrations were measured during early ripening. The changes observed in auxin were assessed in light of global changes in gene expression. It was found that mature MADS8/9-suppressed apples had a higher concentration of free IAA. This was associated with increased expression of the auxin biosynthetic genes in the indole-3-acetamide pathway. Additionally, in the MADS8/9-suppressed apples, there was less expression of the GH3 auxin-conjugating enzymes. A number of genes involved in the auxin-regulated transcription (AUX/IAA and ARF classes of genes) were also observed to change in expression, suggesting a mechanism for signal transduction at the start of ripening. The delay in ripening observed in MADS8/9-suppressed apples may be partly due to high auxin concentrations. We propose that, to achieve low auxin associated with fruit maturation, the auxin homeostasis is controlled in a two-pronged manner: (i) by the reduction in biosynthesis and (ii) by an increase in auxin conjugation. This is associated with the change in expression of auxin-signalling genes and the up-regulation of ripening-related genes.

Root‐derived auxin contributes to the phosphorus‐deficiency‐induced cluster‐root formation in white lupin (Lupinus albus)

Formation of cluster roots is a typical morphological response to phosphorus (P) deficiency in white lupin (Lupinus albus), but its physiological and molecular mechanisms are still unclear. We investigated the role of auxin in the initiation of cluster roots by distinguishing the sources of auxin, measuring the longitudinal distribution patterns of free indole-3-acetic acid (IAA) along the root and the related gene expressions responsible for polar auxin transport (PAT) in different developmental stages of cluster roots. We found that removal of shoot apex or primary root apex and application of auxin-influx or -efflux transport inhibitors, 3-chloro-4-hydroxyphenylacetic acid, N-1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid, to the stem did not affect the number of cluster roots and the free-IAA concentration in the roots of P-deficient plants, but when these inhibitors were applied directly to the growth media, the cluster-root formation was greatly suppressed, suggesting the fundamental role of root-derived IAA in cluster-root formation. The concentration of free IAA in the roots was higher in P-deficient plants than in P-adequate ones, and the highest in the lateral-root apex and the lowest in the mature cluster roots. Meanwhile the expression patterns of LaAUX1, LaPIN1 and LaPIN3 transcripts related to PAT was consistent with concentrations of free IAA along the lateral root, indicating the contribution of IAA redistribution in the cluster-root development. We proposed that root-derived IAA plays a direct and important role in the P-deficiency-induced formation of cluster roots.

A GH3 family member, OsGH3-2, modulates auxin and abscisic acid levels and differentially affects drought and cold tolerance in rice.

Plant responses to abiotic stresses are coordinated by arrays of growth and developmental processes. Indole-3-acetic acid (IAA) and abscisic acid (ABA) play critical roles in developmental programmes and environmental responses, respectively, through complex signalling and metabolism networks. However, crosstalk between the two phytohormones in the stress responses remains largely unknown. Here, it is reported that a GH3 family gene, OsGH3-2, encoding an enzyme catalysing IAA conjugation to amino acids, is involved in the modulation of ABA level and stress tolerance. Expression of OsGH3-2 was induced by drought but was suppressed by cold. Overexpression of OsGH3-2 in rice caused significant morphological aberrations related to IAA deficiency, such as dwarfism, smaller leaves, and fewer crown roots and root hairs. The overexpressing line showed significantly reduced carotene, ABA, and free IAA levels, greater stomata aperture, and faster water loss, and was hypersensitive to drought stress. However, the overexpressing line showed increased cold tolerance, which was due to the combined effects of reduced free IAA content, alleviated oxidative damage, and decreased membrane penetrability. Furthermore, expression levels of some ABA synthesis- and stress-related genes were significantly changed in the overexpression line. It was conclude that OsGH3-2 modulates both endogenous free IAA and ABA homeostasis and differentially affects drought and cold tolerance in rice.

Time-Course Analysis of Levels of Indole-3-Acetic Acid and Expression of Auxin-Responsive GH3 Genes in Betula platyphylla

Indole-3-acetic acid (IAA) is a natural hormone that plays an important role in the essential physiological processes of plant growth, development, and environmental response. To maintain IAA homeostasis in plants, some members of the GH3 gene family may mediate the conjugation of amino acids to indole-3-acetic acid. In this study, nine birch BpGH3 genes were cloned, including three BpGH3.5 genes, three BpGH3.6 genes, two BpGH3.9 genes, and a single BpGH3.3 gene, that were likely to be auxin-responsive genes. Time-course expression analysis of these nine BpGH3 genes using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was conducted using birch plant tissues collected during a 1-year growth cycle. Further, we determined the endogenous free IAA content in birch during this period by using liquid chromatography tandem mass spectrometry. The results indicated that the IAA levels changed remarkably throughout the study period; the level remained relatively stable during the developmental stage from June 1 to July 1, and IAA was present in the birch leaves throughout the study period. The correlation between the expression of BpGH3 genes and IAA levels was analyzed using Pearson correlation analysis. The results revealed a significant negative correlation between the expressions of four BpGH3 genes, namely, BpGH3.3 (p < 0.05), BpGH3.5a (p < 0.01), BpGH3.5b (p < 0.01), and BpGH3.9a (p < 0.01) and the free IAA level, thereby suggesting that these genes might play roles in IAA regulation.

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.

Quantitation of endogenous levels of IAA, IAAsp and IBA in micro-propagated shoots of hybrid chestnut pre-treated with IBA

Endogenous levels of indole-3-acetic acid (IAA), indole-3-acetylaspartic acid (IAAsp) and indole-3-butyric acid (IBA) were measured during the first 8 d of in vitro rooting of rootstock from the chestnut ‘M3’ hybrid by high performance liquid chromatography (HPLC). Rooting was induced either by dipping the basal ends of the shoots into a 4.92-mM IBA solution for 1 min or by sub-culturing the shoots on solid rooting medium supplemented with 14.8-μM IBA for 5 d. For root development, the induced shoots were transferred to auxin-free solid medium. Auxins were measured in the apical and basal parts of the shoots by means of HPLC. Endogenous levels of IAA and IAAsp were found to be greater in IBA-treated shoots than in control shoots. In extracts of the basal parts of the shoots, the concentration of free IAA showed a significant peak 2 d after either root inductive method and a subsequent gradual decrease for the remainder of the time course. The concentration of IAAsp peaked at day 6 in extracts of the basal parts of shoots induced with 14.8-μM IBA for 5 d, whereas shoots induced by dipping showed an initial increase until day 2 and then remained stable. In extracts from basal shoot portions induced by dipping, IBA concentration showed a transient peak at day 1 and a plateau between day 2 and 4, in contrast to the profile of shoots induced on auxin-containing medium, which showed a significant reduction between 4 and 6 d after transferred to auxin-free medium. All quantified auxins remained at a relatively low level, virtually constant, in extracts from apical shoot portions, as well as in extracts from control non-rooting shoots. In conclusion, the natural auxin IAA is the signal responsible for root induction, although it is driven by exogenous IBA independently of the adding conditions.

The involvement of indole-3-acetic acid in the control of stem elongation in dark- and light-grown pea ( Pisum sativum) seedlings

We investigated the role of auxin on stem elongation in pea (Pisum sativum L.) grown for 10d in continuous darkness or under low-irradiance blue, red, far red and white light. The third internode of treated seedlings was peeled and the tissues (epidermis and cortex+central cylinder) were separately analyzed for the concentration of free and conjugated indole-3-acetic acid (IAA). Under red, far red and white light internode elongation was linearly related with the free IAA content of all internode tissues, suggesting that phytochrome-dependent inhibition of stem growth may be mediated by a decrease of free IAA levels in pea seedlings. The correlation between IAA and internode elongation, however, did not hold for blue light-grown seedlings. The hypothesis that the growth response under low-irradiance blue light might be correlated with the lack of phytochrome B signalling and changes in gibberellin metabolism is discussed in view of current knowledge on hormonal control of stem growth.

Physiological analysis of transgenic Arabidopsis thaliana plants expressing one nitrilase isoform in sense or antisense direction

The biosynthesis of IAA in Arabidopsis thaliana can proceed via different pathways, which involve indole-3-acetonitrile (IAN) as an intermediate. The enzyme nitrilase, which converts IAN to the auxin indole-3-acetic acid (IAA), might be a key enzyme in this biosynthetic pathway. To elucidate the role of nitrilase during the development of the plant, we have investigated transgenic Arabidopsis plants, carrying one of the four nitrilase isoforms so far identified in Arabidopsis in either sense or antisense direction. The plants have been analyzed for their phenotype, auxin content, nitrilase transcript and protein amounts, and nitrilase activity. Analysis of free IAA content in seedlings did not reveal any prominent differences between the different lines, whereas the total IAA content was slightly increased in the line overexpressing NIT2 (sNIT2). In two of the antisense lines (aNIT1 and aNIT2) the total auxin content was about 75% lower than in the wild type, while IAN was increased in these two antisense lines by about 2-fold. In sNIT2 plants the IAN content was slightly decreased compared with the wild type. When IAN was added to the medium, a strong increase in IAA content was found in sNIT2 seedlings (ca. 22-fold), which was much lower in wild type and antisense plants. In mature plants levels of free IAA were decreased by about 25% and 50% in the antisense lines aNIT2 and aNIT1, respectively, whereas the IAA content in the other lines did not differ much. sNIT2/aNIT2 plants were further characterized because it was shown by Northern and Western analysis as well as enzyme activity measurements that sNIT2 plants overexpress nitrilase constitutively. The results with these plants have shown that i) both nitrilase 1 and nitrilase 2 accept the same substrates, and ii) overexpression of nitrilase does not lead to a visible phenotype because in vivo IAN might be the limiting factor in these plants. Application of IAN leads to an increase in total nitrilase protein as shown by Western blotting. Using transgenic lines with promoter-GUS fusions for each NIT gene, it was shown that i) NIT1 is highly expressed in seedlings and ii) NIT2 is strongly induced (21-fold) after IAN application.