Free Indole-3-acetic Acid Levels Research Articles

The auxin-responsive CsSPL9-CsGH3.4 module finely regulates auxin levels to suppress the development of adventitious roots in tea (Camellia sinensis).

The cutting technique is extensively used in tea breeding, with key emphasis on promoting the growth of adventitious roots (ARs). Despite its importance in tea cultivation, the mechanisms underlying AR development in tea remain unclear. In this study, we demonstrated the essential role of auxins in the initiation and progression of AR and established that the application of exogenous 1-naphthaleneacetic acid-enhanced AR formation in tissue-cultured seedlings and cuttings. Then, we found that the auxin-responsive transcription factor CsSPL9 acted as a negative regulator of AR development by reducing the levels of free indole-3-acetic acid (IAA) in tea plants. Furthermore, we identified CsGH3.4 as a downstream target of CsSPL9, which was activated by direct binding to its promoter. CsGH3.4 also inhibited AR development and maintained low levels of free IAA. Thus, these results revealed the inhibitory effect of the auxin-responsive CsSPL9-CsGH3.4 module on AR development by reducing free IAA levels in tea. These findings have significant theoretical and practical value for enhancing tea breeding practices.

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.

Metabolic profiles of 2-oxindole-3-acetyl-amino acid conjugates differ in various plant species.

Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß-d-glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography-tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana, pea (Pisum sativum L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.

Auxin plays a role in the adaptation of rice to anaerobic germination and seedling establishment.

Auxin is well known to stimulate coleoptile elongation and rapid seedling growth in the air. However, its role in regulating rice germination and seedling establishment under submergence is largely unknown. Previous studies revealed that excessive levels of indole-3-acetic acid(IAA) frequently cause the inhibition of plant growth and development. In this study, the high-level accumulation of endogenous IAA is observed under dark submergence, stimulating rice coleoptile elongation but limiting the root and primary leaf growth during anaerobic germination (AG). We found that oxygen and light can reduce IAA levels, promote the seedling establishment and enhance rice AG tolerance. miRNA microarray profiling and RNA gel blot analysis results show that the expression of miR167 is negatively regulated by submergence; it subsequently modulates the accumulation of free IAA through the miR167-ARF-GH3 pathway. The OsGH3-8 encodes an IAA-amido synthetase that functions to prevent free IAA accumulation. Reduced miR167 levels or overexpressing OsGH3-8 increase auxin metabolism, reduce endogenous levels of free IAA and enhance rice AG tolerance. Our studies reveal that poor seed germination and seedling growth inhibition resulting from excessive IAA accumulation would cause intolerance to submergence in rice, suggesting that a certain threshold level of auxin is essential for rice AG tolerance.

Quantitative analysis of auxin metabolites in lychee flowers.

To investigate the modulation of endogenous indole-3-acetic acid (IAA) level by biosynthesis and inactivation during floral development, IAA and its metabolites were analyzed by LC-ESI/MS/MS in Lychee (Litchi chinensis Sonn.) flowers. In the bloomed flowers, the level of free IAA was higher in males than in females. In contrast, the total sum level of IAA metabolites was higher in females than in males, suggesting a higher biosynthetic activity of IAA in the females before the bloom. A detailed time-course analysis from the bud stage to the developing flower stage showed higher levels of IAA in females than males. The major metabolites were oxidized IAA in both sexes. The results suggest that IAA is involved in the maturation of female floral tissues in lychee, and oxidative metabolism plays an essential role in controlling the free IAA levels therein.

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.

Indole-3-acetate beta-glucosyltransferase OsIAGLU regulates seed vigour through mediating crosstalk between auxin and abscisic acid in rice.

SummarySeed vigour is an important trait for direct seeding in rice. In this study, indole‐3‐acetate beta‐glucosyltransferase OsIAGLU was cloned in rice, and its roles on seed vigour were mainly investigated. Disruption of OsIAGLU resulted in low seed vigour in rice. Quantitative RT‐PCR analysis showed that the expressions of OsIAGLU were relatively higher in the late developing and the early germinating seeds and were significantly induced by indole‐3‐acetic acid (IAA) and abscisic acid (ABA). Transcriptome analysis revealed that the IAA‐ and ABA‐related genes were involved in the OsIAGLU regulation of seed vigour in rice. The higher levels of free IAA and ABA were identified in germinating seeds of osiaglu mutants compared to wild‐type (WT) plants. When treated with exogenous IAA and ABA, the osiaglu mutants and WT plants showed sensitivity to ABA while not IAA, but the exogenous IAA amplified ABA‐induced reduction of seed vigour in rice. The continuously higher expressions of ABA‐INSENSITIVE 3 (OsABI3) and OsABI5 occurred in germinating seeds of osiaglu mutants compared to WT plants. The regulation of seed vigour by OsIAGLU might be through modulating IAA and ABA levels to alert OsABIs expression in germinating seeds in rice. Based on analysis of single‐nucleotide polymorphism data of rice accessions, two haplotypes of OsIAGLU that positively correlated with seed vigour were identified in indica accessions. This study provides important insights into the roles of OsIAGLU on seed vigour and facilitates the practical use of OsIAGLU in rice breeding.

A bacterial assay for rapid screening of IAA catabolic enzymes

BackgroundPlants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.ResultsIn this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.ConclusionsThis new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

Overexpression of OsIAAGLU reveals a role for IAA-glucose conjugation in modulating rice plant architecture.

OsIAAGLU could catalyze the reaction of IAA with glucose to generate IAA-glucose. Overexpression of OsIAAGLU in rice resulted in altered rice shoot architecture and root gravitropism. The distribution and levels of indole-3-acetic acid (IAA) within plant tissues are well known to play vital roles in plant growth and development. An important mechanism of regulating free IAA levels in monocots is formation of IAA ester conjugates. In this study, a cytosol-localized protein encoded by the rice gene of indole-3-acetic acid glucosyltransferase (OsIAAGLU) was found to catalyze the reaction of free IAA with glucose to generate IAA-glucose. Expression of OsIAAGLU could be induced by IAA and NAA. The number of tillers and leaf angle was significantly increased with a concomitant decrease in plant height and panicle length in the transgenic rice lines overexpressing OsIAAGLU compared to the wild-type (WT) plants. Phenotypes of iaaglu mutants constructed using the CRISPR/Cas9 system had no obvious differences with WT plants. Furthermore, overexpression of OsIAAGLU resulted in reduced sensitivity to IAA/NAA and altered gravitropic response of the roots in the transgenic plants. Free IAA contents in the leaves, root tips, and lamina joint of OsIAAGLU-overexpressing transgenic lines were lower than those of WT plants. These results support that OsIAAGLU could play a regulatory role in IAA homeostasis and rice architecture.

Deciphering the growth pattern and phytohormonal content in Saskatoon berry (Amelanchier alnifolia) in response to in vitro cytokinin application

Clonal propagation plays a critical integral role in the growth and success of a global multi-billion dollar horticulture industry through a constant supply of healthy stock plants. The supply chain depends on continuously improving the micropropagation process, thus, understanding the physiology of in vitro plants remains a core component. We evaluated the influence of exogenously applied cytokinins (CKs, N6-benzyladenine = BA, isopentenyladenine = iP, meta-topolin = mT, 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine = mTTHP) in Murashige and Skoog (MS)-supplemented media on organogenic response and accumulation of endogenous CK and indole-3-acetic acid (IAA) metabolites. The highest shoot proliferation (30 shoots/explant) was obtained with 20 μM mT treatment. However, the best quality regenerants were produced in 10 μM mT treatment. Rooting of Amelanchier alnifolia in vitro plantlets was observed at the lowest CK concentrations, with the highest root proliferation (3 roots/explant) in 1 μM mTTHP regenerants. Similar to the organogenic response, high levels of endogenous bioactive CK metabolites (free bases, ribosides, and nucleotides) were detected in mT and mTTHP-derived regenerants. The level of O-glucosides was also comparatively high in these cultures. All CK-treated plants had high levels of endogenous free IAA compared to the control. This may suggest an influence of CKs on biosynthesis of IAA.

Arabidopsis NITRILASE 1 Contributes to the Regulation of Root Growth and Development through Modulation of Auxin Biosynthesis in Seedlings.

Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant Arabidopsis thaliana a second group of Brassicaceae-specific nitrilases (NIT1-3) has been found. This so-called NIT1-subfamily has been associated with the conversion of indole-3-acetonitrile (IAN) into the major plant growth hormone, indole-3-acetic acid (IAA). However, apart of reported functions in defense responses to pathogens and in responses to sulfur depletion, conclusive insight into the general physiological function of the NIT-subfamily nitrilases remains elusive. In this report, we test both the contribution of the indole-3-acetaldoxime (IAOx) pathway to general auxin biosynthesis and the influence of altered nitrilase expression on plant development. Apart of a comprehensive transcriptomics approach to explore the role of the IAOx route in auxin formation, we took a genetic approach to disclose the function of NITRILASE 1 (NIT1) of A. thaliana. We show that NIT1 over-expression (NIT1ox) results in seedlings with shorter primary roots, and an increased number of lateral roots. In addition, NIT1ox plants exhibit drastic changes of both free IAA and IAN levels, which are suggested to be the reason for the observed phenotype. On the other hand, NIT2RNAi knockdown lines, capable of suppressing the expression of all members of the NIT1-subfamily, were generated and characterized to substantiate the above-mentioned findings. Our results demonstrate for the first time that Arabidopsis NIT1 has profound effects on root morphogenesis in early seedling development.

Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants

In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses.

Tracking biochemical changes during adventitious root formation in olive (Olea europaea L.)

The activity of oxidative enzymes and the levels of free auxins were determined during adventitious root formation in olive explants. Rooting trials were performed both with in vitro-cultured microshoots of the cultivar ‘Galega Vulgar’, treated with indole-3-butyric acid (IBA) and with salicylhydroxamic acid (SHAM)+IBA, as well as with semi-hardwood cuttings of the cultivars ‘Galega Vulgar’ (difficult-to-root) and ‘Cobrançosa’ (easy-to-root), treated with IBA. The auxin (IBA) was used in all experiments as a rooting promoter, while SHAM was used in micropropagation trials as rooting inhibitor, providing a negative control. Free indole-3-acetic acid (IAA) and IBA concentrations were determined in microshoots, as well as in semi-hardwood cuttings, throughout the rooting period at pre-established time-points. At the same time-points, the enzymatic activity of polyphenol oxidases (PPO), peroxidases (POX), and IAA oxidase (IAAox) was evaluated in the microshoots. Microshoots treated with SHAM+IBA revealed higher POX and IAAox activity, as well as lower PPO activity, than those treated only with IBA. IAA levels were higher in IBA-treated microshoots during induction phase, but lower during early initiation phase. In contrast, free IBA levels were higher in microshoots treated with SHAM+IBA during induction, but lower during initiation. A similar pattern of free auxin levels was observed in semi-hardwood cuttings of the two contrasting cultivars under evaluation. The similarities found on the auxin patterns of microshoots treated with SHAM and those of semi-hardwood cuttings of the difficult-to-root olive cultivar allow considering SHAM a reliable control for when simulation of a difficult-to-root behavior is necessary. The inhibitory effect of SHAM in root formation could be related with 1) the inhibition of alternative oxidase (AOX), leading to a downregulation of phenylpropanoid biosynthetic pathways, which would decrease the concentration of phenolic substrates for PPO; 2) an increase in IAAox activity resulting in lower free IAA levels or; 3) a defective conversion of IBA into IAA.

Conversion of Indole-3-Butyric Acid to Indole-3-Acetic Acid in Shoot Tissue of Hazelnut (Corylus) and Elm (Ulmus)

Indole-3-butyric acid (IBA) is an endogenous compound that appears to regulate both lateral and adventitious root formation in many plant species and is also the auxin most available commercially for application to promote rooting. IBA is converted to indole-3-acetic acid (IAA) by β-oxidation in the peroxisomes. This process has been observed in a number of plant species and has been shown to be critical for normal root development in response to treatment with IBA. In this study, we investigated this process in hybrid hazelnut (Corylus americana × C. avellana), American elm (Ulmus americana), and Cathedral hybrid elm (U. pumila × U. davidiana var. japonica ‘Cathedral’), in which adventitious rooting is a major bottleneck for vegetative propagation, and the efficacy of IBA treatment is highly variable across different cultivars and at different collection times. Using differentially stable isotope-labeled IBA and IAA tracer and internal standard, respectively, and using gas chromatography coupled with selected reaction monitoring mass spectrometry, IBA-derived IAA was measured in shoot tissue treated with stable isotope-labeled IBA. In elm, higher levels of IBA-to-IAA conversion were generally observed in cultivars which formed adventitious roots most easily in softwood stem cutting trials. IBA-to-IAA conversion was observed in hazelnut genotypes with different rooting abilities and suggested a complex relationship exists between IBA conversion and root organogenesis. In both hazelnut and elm, endogenous free IAA levels were not significantly different across the genotypes examined. High rates of root formation is a key trait for establishment of large-scale production systems. Screening for optimal rates of IBA-to-IAA conversion may facilitate selection against genotypes which respond poorly to exogenous IBA and are thus difficult to propagate using hormone treatment.

Putative role of IAA during the early response of Brassica napus L. to Plasmodiophora brassicae

This study examined the role of indole-3-acetic acid (IAA) during the early stage of infection of rapeseed (Brassica napus L.) by the obligate biotrophic protist Plasmodiophora brassicae Woron., which causes clubroot disease. Treatment of infected B. napus seedlings with exogenous IAA promoted the development of clubroot disease, including an increase in the number and size of galls, whereas treatment with N-1-naphthylphthalamic acid (NPA) attenuated the disease and altered the location and size of the root nodules, resulting in a reduced disease index. An increased level of free IAA was detected in the roots at 3 and 7 days after inoculation (DAI) compared with the control, suggesting the activation of IAA signaling in the early stage of P. brassicae infection. Moreover, IAA treatment caused no significant change in the germination rate of the resting spores but resulted in enhanced root hair infection. RT–PCR and quantitative real-time PCR (qPCR) were used to analyze the expression of IAA homeostasis-related genes (3, 7, and 10 DAI) to identify gene expression patterns in the roots and leaves of B. napus after inoculation. Five IAA biosynthesis-related genes examined in this study were induced in the roots after inoculation with P. brassicae but not in the leaves. The rapid induction of BnAAO4 expression at 3 DAI may be responsible for the overproduction of IAA during the early stages of infection. This study suggests that IAA acts as a signaling molecule that putatively stimulates root hair infection in the early response of B. napus to P. brassicae infection.

Knockout of GH3 genes in the moss Physcomitrella patens leads to increased IAA levels at elevated temperature and in darkness

Two proteins of the GRETCHEN HAGEN3 (GH3) family of acyl acid amido synthetases from the moss Physcomitrella patens conjugate indole-3-acetic acid (IAA) to a series of amino acids. The possible function of altered auxin levels in the moss in response to two different growth perturbations, elevated temperatures and darkness, was analyzed using a) the recently described double knockout lines in both P. patens GH3 genes (GH3-doKO) and b) a previously characterized line harboring an auxin-inducible soybean GH3 promoter::reporter fused to β-glucuronidase (G1-GUS). The GUS activity as marker of the auxin response increased at higher temperatures and after cultivation in the darkness for a period of up to four weeks. Generally, the double knockout plants grew more slowly than the wild type (WT). The altered growth conditions influenced the phenotypes of the double knockout lines differently from that of WT moss. Higher temperatures negatively affected GH3-doKO plants compared to WT which was shown by stronger loss of chlorophyll. On the other hand, a positive effect was found on the concentrations of free IAA which increased at 28 °C in the GH3-doKO lines compared to WT plants. A different factor, namely darkness vs. a light/dark cycle caused the adverse phenotype concerning chlorophyll concentrations. Mutant moss plants showed higher chlorophyll concentrations than WT and these correlated with higher free IAA in the plant population that was classified as green. Our data show that growth perturbations result in higher free IAA levels in the GH3-doKO mutants, but in one case – growth in darkness – the mutants could cope better with the condition, whereas at elevated temperatures the mutants were more sensitive than WT. Thus, GH3 function in P. patens WT could lie in the regulation of IAA concentrations under unfavorable environmental conditions.

Transcriptome analysis reveals the role of BpGH3.5 in root elongation of Betula platyphylla × B. pendula

The GH3 family comprises a set of early auxin-responsive genes. Several previous studies have proven that GH3 family proteins play important roles in various aspects of plant growth and development. Our previous studies have found that the overexpression or suppression of BpGH3.5 in birch tree can result in shorter primary and lateral root compared to non-transgenic control, however, the molecular mechanism underlying is not clear. To further reveal the roles of BpGH3.5 in root elongation of birch (Betula platyphylla × B. pendula), we carried out transcriptome analysis on BpGH3.5-overexpressed (G1 and G5), BpGH3.5-suppressed (AG1), and non-transgenic (NT) birch. Compared with the NT line, 3,320 and 5,212 unigenes were found to be differentially expressed in the BpGH3.5 overexpressed and suppressed lines, respectively. Many differentially expressed unigenes related to cell proliferation and growth were downregulated in both the BpGH3.5-overexpressed and suppressed plants, which is consistent with the short-root phenotypes observed in these lines. We found that indole-3-acetic acid (IAA) biosynthesis and signaling pathways were affected in the BpGH3.5-overexpressed and suppressed plants. Interestingly, Free IAA levels were significantly lower in BpGH3.5-overexpressed and BpGH3.5-suppressed birch than in NT plants. This suggested that the expression levels of BpGH3.5 may play a role in maintaining the balance of free IAA in birch and the decreased IAA levels may be responsible for the short-root phenotype of the transgenic birch.

IAA–amido synthetase activity and GH3 expression during development of pea seedlings

Indole-3-acetic acid (IAA) conjugation is an integral component of a sensitive mechanism for the regulation of free auxin level in the cell, in addition to hormone synthesis, degradation and transport. One mechanism of regulating both the level and biological activity of hormone involves the conjugation of IAA to amino acids through IAA–amido synthetases. These enzymes belong to the family of GH3 proteins encoded by early-auxin responsive genes, which are conserved in monocots and dicots. In this investigation, we identified pea IAA–Asp synthetase as a member of the GH3 family proteins based on amino acid sequence determined by tandem mass spectrometry (LC–MS/MS). IAA–Asp synthetase was highly homologous (54–84 % amino acid identity) to the GH3 proteins from Populus trichocarpa, Glycine max, Riccinus communis, Nicotiana tabacum, and Arabidopsis thaliana. Moreover, we studied the subcellular distribution and activity of IAA amido synthetase in developing pea seedlings. The cytoplasmic localization of GH3 IAA amido synthetase was indicated in 12-day-old pea seedlings by immunofluorescence method using anti-AtGH3.5 antibodies. This finding was confirmed by the measurement of the enzyme activity in the subcellular fractions collected by differential centrifugation. The most of IAA–Asp synthetase activity (88 % of the total activity) was detected in the 100,000×g supernatant fluid fraction containing soluble cytoplasmic proteins. 3- and 6-day-old pea seedlings showed a weak IAA–Asp synthetase activity and GH3 mRNA level. Twelve-day-old pea stems exhibited 19-fold, and 4-fold higher levels of transcript and the enzyme activity, respectively than 3-day-old plants. We hypothesized that IAA–amido synthetase expression and activity depend on developmental stage of vegetative tissues and this is associated with changes in free IAA level.

A Role for a Dioxygenase in Auxin Metabolism and Reproductive Development in Rice

Indole-3-acetic acid (IAA), the natural auxin in plants, regulates many aspects of plant growth and development. Extensive analyses have elucidated the components of auxin biosynthesis, transport, and signaling, but the physiological roles and molecular mechanisms of auxin degradation remain elusive. Here, we demonstrate that the dioxygenase for auxin oxidation (DAO) gene, encoding a putative 2-oxoglutarate-dependent-Fe (II) dioxygenase, is essential for anther dehiscence, pollen fertility, and seed initiation in rice. Rice mutant lines lacking a functional DAO display increased levels of free IAA in anthers andovaries. Furthermore, exogenous application of IAA or overexpression of the auxin biosynthesis gene OsYUCCA1 phenocopies the dao mutants. We show that recombinant DAO converts the active IAA into biologically inactive 2-oxoindole-3-acetic acid (OxIAA) invitro. Collectively, these data support a key role of DAO in auxin catabolism and maintenance of auxin homeostasis central to plant reproductive development.

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.