Indole-3-acetic Acid Biosynthesis Pathway Research Articles

Endogenous cAMP elevation in Brassica napus causes changes in phytohormone levels

ABSTRACT In higher plants, the regulatory roles of cAMP (cyclic adenosine 3′,5′-monophosphate) signaling remain elusive until now. Cellular cAMP levels are generally much lower in higher plants than in animals and transiently elevated for triggering downstream signaling events. Moreover, plant adenylate cyclase (AC) activities are found in different moonlighting multifunctional proteins, which may pose additional complications in distinguishing a specific signaling role for cAMP. Here, we have developed rapeseed (Brassica napus L.) transgenic plants that overexpress an inducible plant-origin AC activity for generating high AC levels much like that in animal cells, which served the genetic model disturbing native cAMP signaling as a whole in plants. We found that overexpression of the soluble AC activity had significant impacts on the contents of indole-3-acetic acid (IAA) and stress phytohormones, i.e. jasmonic acid (JA), abscisic acid (ABA), and salicylic acid (SA) in the transgenic plants. Acute induction of the AC activity caused IAA overaccumulation, and upregulation of TAA1 and CYP83B1 in the IAA biosynthesis pathways, but also simultaneously the hyper-induction of PR4 and KIN2 expression indicating activation of JA and ABA signaling pathways. We observed typical overgrowth phenotypes related to IAA excess in the transgenic plants, including significant increases in plant height, internode length, width of leaf blade, petiole length, root length, and fresh shoot biomass, as well as the precocious seed development, as compared to wild-type plants. In addition, we identified a set of 1465 cAMP-responsive genes (CRGs), which are most significantly enriched in plant hormone signal transduction pathway, and function mainly in relevance to hormonal, abiotic and biotic stress responses, as well as growth and development. Collectively, our results support that cAMP elevation impacts phytohormone homeostasis and signaling, and modulates plant growth and development. We proposed that cAMP signaling may be critical in configuring the coordinated regulation of growth and development in higher plants.

NIT24 and NIT29-mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice.

Auxin plays a pivotal role in the co-evolution of plants and microorganisms. Xanthomonas oryzae pv. oryzicola (Xoc) stands as a significant factor that affects rice yield and quality. However, the current understanding of Xoc's capability for indole 3-acetic acid (IAA) synthesis and its mechanistic implications remains elusive. In this study, we performed a comprehensive genomic analysis of Xoc strain RS105, leading to the identification of two nitrilase enzyme family (NIT) genes, designated as AKO15524.1 and AKO15829.1, subsequently named NIT24 and NIT29, respectively. Our investigation unveiled that the deletion of NIT24 and NIT29 resulted in a notable reduction in IAA synthesis capacity within RS105, thereby impacting extracellular polysaccharide production. This deficiency was partially ameliorated through exogenous IAA supplementation. The study further substantiated that NIT24 and NIT29 have nitrilase activity and the ability to catalyse IAA production invitro. The lesion length and bacterial population statistics experiments confirmed that NIT24 and NIT29 positively regulated the pathogenicity of RS105, suggesting that NIT24 and NIT29 may regulate Xoc invasion by affecting IAA synthesis. Furthermore, our analysis corroborated mutant strains, RS105_ΔNIT24 and RS105_ΔNIT29, which elicited the outbreak of reactive oxygen species, the deposition of callose and the upregulation of defence-related gene expression in rice. IAA exerted a significant dampening effect on the immune responses incited by these mutant strains in rice. In addition, the absence of NIT24 and NIT29 affected the growth-promoting effect of Xoc on rice. This implies that Xoc may promote rice growth by secreting IAA, thus providing a more suitable microenvironment for its own colonization. In summary, our study provides compelling evidence for the existence of a nitrilase-dependent IAA biosynthesis pathway in Xoc. IAA synthesis-related genes promote Xoc colonization by inhibiting rice immune defence response and affecting rice growth by increasing IAA content in Xoc.

Colletotrichum truncatum, an endophytic fungus derived from Musa acuminata (AAA group): antifungal activity against Aspergillus isolated from COVID-19 patients and indole-3-acetic acid (IAA) production

Fungal endophyte is a fungal that lives in plant organism as mutualism association. The role of fungal endophyte is a growth promoter or/and microbial pathogen inhibitor. This study investigated antifungal activity of Colletotrichum truncatum E10, an endophytic fungus derived from Musa acuminata (AAA group), against 7 isolates of Aspergillus obtained from lower respiratory samples of COVID-19 patients. In addition, IAA production of this strain was also observed. All isolates of Aspergillus were identified using MALDI-TOF MS. The fungal endophyte, C. truncatum E10, was screened for IAA induction with and without 0.1, 2 and 8 mg/mL of L-tryptophan based on colorimetric method using Salkowski reagent which produced pinkish to reddish solution indicating the presence of IAA. Antagonist activity was based on dual culture assay measured in colony growth inhibition (CGI). C. truncatum E10 produced the highest IAA concentration of 112.81±0.12 µg/mL when 8 mg/mL of L-tryptophan added. The strong antagonist activities were shown by C. truncatum E10 against 5 Aspergillus isolates including 2 A. fumigatus: sp442/6 (CGI=57.83±5.11%) and sp269/11 (CGI=53.01±8.52%), 1 A. niger sp26/7 (CGI=57.83±15.33%) and 2 A. flavus: sp26/7 (CGI=56.63±13.63%) and sp36/7 (CGI=57.23±0.85%), whereas the colony growth inhibition (CGI) of other 2 isolates including A. fumigatus sp567/6 and A. flavus sp269/11 were less than 50%. In this study, C. truncatum E10 produced substances that inhibited human fungal pathogen including A. fumigatus, A. flavus and A. niger. Moreover, it can produce IAA activity. Further investigations are being conducted to expand the plant growth promotion effects and determine IAA biosynthesis pathway. For antifungal activity, the bioactive metabolites produced by this endophytic fungal isolate should be characterized to specify the effective compounds. Asian Australas. J. Biosci. Biotechnol. 2023, 8 (2), 23-29

Growth Phase Influence the Gene Expression and Metabolite Production Related to Indole-3-Acetic Acid (IAA) Biosynthesis by Serratia plymuthica UBCF_13.

<b>Background and Objective:</b> The optimization of the indole-3-acetic acid (IAA) producing capability of <i>Serratia plymuthica</i> UBCF_13 has been intensively studied. This work tried to reveal the effect of growth phases on IAA production, gene expression and metabolite synthesis related to the IAA biosynthesis pathway. <b>Materials and Methods:</b> The growth curve and IAA production were measured every 3 hrs. The putative IAA biosynthesis pathway was investigated based on the UBCF_13 genome. To identify the possible pathway of IAA biosynthesis in UBCF_13, we applied the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and High-Performance Liquid Chromatography (HPLC) analysis to measure the transcript levels of each gene and indole metabolite production based on tryptophan treatment at different times of incubation. <b>Results:</b> The optimal IAA production on colorimetric assay was at 9 hrs of incubation (initial stationary phase). The level expression of <i>puuC</i>, <i>DDC</i>, <i>oxdA</i>, <i>amiE</i>, <i>nthA</i> and <i>nthB</i> have been upregulated maximum in 3 hrs of culture time (lag phase), except <i>tyrB</i> and <i>ipdC</i>. The highest transcript level of the genes was found in nitrile hydratase genes (<i>nthA</i> and <i>nthB</i>) and indole-3- acetamide (IAM) has been detected as the only intermediate in the crude extract of UBCF_13 thus the IAM pathway may be used to produce IAA. The maximum IAA production on HPLC analysis was found at 21 hrs of incubation (late stationary phase). <b>Conclusion:</b> This study gives a new insight that the best time to measure gene expression and intermediates related to the IAA biosynthetic pathway in bacteria was found at a specific growth phase.

Auxin-Producing Bacteria from Duckweeds Have Different Colonization Patterns and Effects on Plant Morphology.

The role of auxin in plant–microbe interaction has primarily been studied using indole-3-acetic acid (IAA)-producing pathogenic or plant-growth-promoting bacteria. However, the IAA biosynthesis pathway in bacteria involves indole-related compounds (IRCs) and intermediates with less known functions. Here, we seek to understand changes in plant response to multiple plant-associated bacteria taxa and strains that differ in their ability to produce IRCs. We had previously studied 47 bacterial strains isolated from several duckweed species and determined that 79% of these strains produced IRCs in culture, such as IAA, indole lactic acid (ILA), and indole. Using Arabidopsis thaliana as our model plant with excellent genetic tools, we performed binary association assays on a subset of these strains to evaluate morphological responses in the plant host and the mode of bacterial colonization. Of the 21 tested strains, only four high-quantity IAA-producing Microbacterium strains caused an auxin root phenotype. Compared to the commonly used colorimetric Salkowski assay, auxin concentration determined by LC–MS was a superior indicator of a bacteria’s ability to cause an auxin root phenotype. Studies with the auxin response mutant axr1-3 provided further genetic support for the role of auxin signaling in mediating the root morphology response to IAA-producing bacteria strains. Interestingly, our microscopy results also revealed new evidence for the role of the conserved AXR1 gene in endophytic colonization of IAA-producing Azospirillum baldaniorum Sp245 via the guard cells.

Participating mechanism of a major contributing gene ysnE for auxin biosynthesis in Bacillus amyloliquefaciens SQR9.

The phytohormone indole-3-acetic acid (IAA) has been demonstrated to contribute to the plant growth-promoting effect of rhizobacteria, but the IAA biosynthesis pathway in rhizobacteria remains unclear. The ysnE gene, encoding a putative tryptophan acetyltransferase, has been demonstrated to be involved in and strongly contribute to IAA production in Bacillus, but the mechanism is unknown. In this study, to investigate how ysnE participates in IAA biosynthesis in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens SQR9, differences in the produced IAA biosynthesis intermediates between wild-type SQR9 and ΔysnE were analyzed and compared, and the effects of different intermediate compounds on the production of IAA and the accumulation of other intermediates were also investigated. The results showed that the mutant ΔysnE produced more indole-3-lactic acid (ILA) and tryptamine (TAM) than the SQR9 wild-type strain (nearly 1.6- and 2.1-fold), while the production of tryptophol (TOL) was significantly decreased by 46%. When indole-3-pyruvic acid (IPA) served as the substrate, the concentration of ILA in the ΔysnE fermentation broth was much higher than that of the wild type, while IAA and TOL were significantly lower, and ΔysnE was lower than SQR9 in IAA and TOL with the addition of TAM. The TOL content in the ΔysnE fermentation broth was much lower than that in the wild-type SQR9 with the addition of ILA. We suggest that ysnE may be involved in the IPA and TAM pathways and play roles in indole acetaldehyde (IAAld) synthesis from IPA and TAM and in the conversion of ILA to TOL.

High-Level Production of Indole-3-acetic Acid in the Metabolically Engineered Escherichia coli.

Indole-3-acetic acid (IAA) is a critical plant hormone that regulates cell division, development, and metabolism. IAA synthesis in plants and plant-associated microorganisms cannot fulfill the requirement for large-scale agricultural production. Here, two novel IAA biosynthesis pathways, tryptamine (TAM) and indole-3-acetamide (IAM), were developed for IAA production by whole-cell catalysis and de novo biosynthesis in an engineered Escherichia coli MG1655. When 10 g/L l-tryptophan was used as a substrate, an MIA-6 strain containing a heterologous IAM pathway had the highest IAA titer of 7.10 g/L (1.34 × 103 mg/g DCW), which was 98.4 times more than MTAI-5 containing the TAM pathway by whole-cell catalysis. De novo IAA biosynthesis was optimized by improving NAD(P)H availability, resulting in an increased IAA titer of 906 mg/L obtained by the MGΔadhE::icd strain, which is 29.7% higher than the control. These strategies exhibit the potential for IAA production in engineered E. coli and possible industrial applications.

Auxin-producing fungal endophytes promote growth of sunchoke

Endophytic fungi were able to protect their host plants against pathogens and promote plant growth. No previous studies have been conducted on the growth promotion of sunchoke by endophytic fungi. This research was the first to characterize plant growth promoting properties of endophytic fungi including, Macrophomina phaseolina BUP2/3 and Diaporthe phaseolorum BUP3/1 isolated from sunchoke and Daldinia eschscholtzii 2NTYL11, Trichoderma koningii ST-KKU1, Trichoderma erinaceum ST-KKU2, Macrophomina phaseolina SS1L10 and Macrophomina phaseolina SS1R10 from medicinal plants. Also, their plant growth promoting efficiency in artichoke plants under greenhouse condition was evaluated. The highest phosphate solubility and production of extracellular enzymes including amylase, protease, cellulase and xylanase were found after 5 and 21 days of incubation, respectively. Moreover, Indole-3-Acetic Acid (IAA) in the crude extracts of fungi isolated from Jerusalem artichoke was higher than that from medicinal plants (p ≤ 0.05). HPLC analysis suggested the putative IAA biosynthesis pathways in our fungi were via Indole-3-lactic acid (ILA) and Indole-3-acetamide (IAM). In the pot experiment, the values of height, diameter, chlorophyll content and leaf dry weight in sunchoke plants inoculated with fungal endophytes were significantly higher than those of un-inoculated plants. Therefore, this study suggested that fungal endophytes could be used as a biofertilizer for promoting growth of sunchoke.

Biosynthetic pathway of indole-3-acetic acid in ectomycorrhizal fungi collected from northern Thailand.

Indole-3-acetic acid (IAA) is an imperative phytohormone for plant growth and development. Ectomycorrhizal fungi (ECM) are able to produce IAA. However, only a few studies on IAA biosynthesis pathways in ECM fungi have been reported. This study aimed to investigate the IAA biosynthesis pathway of six ECM cultures including Astraeus odoratus, Gyrodon suthepensis, Phlebopus portentosus, Pisolithus albus, Pisolithus orientalis and Scleroderma suthepense. The results showed that all ECM fungi produced IAA in liquid medium that had been supplemented with L-tryptophan. Notably, fungal IAA levels vary for different fungal species. The detection of indole-3-lactic acid and indole-3-ethanol in the crude culture extracts of all ECM fungi indicated an enzymatic reduction of indole-3-pyruvic acid and indole-3-acetaldehyde, respectively in the IAA biosynthesis via the indole-3-pyruvic acid pathway. Moreover, the tryptophan aminotransferase activity confirmed that all ECM fungi synthesize IAA through the indole-3-pyruvic acid pathway. Additionally, the elongation of rice and oat coleoptiles was stimulated by crude culture extract. This is the first report of the biosynthesis pathway of IAA in the tested ECM fungi.

Genome-based identification and analysis of the genes involved in auxin biosynthesis and signal transduction during tea plant leaf development

Auxin plays crucial roles in the process of plant growth and development. Among all the organs of the tea plant, the leaves are the main edible part for humans. The appearance and endoplasm of tea leaves directly affects taste, quality, and economic value. The metabolism of Indole-3-acetic acid (IAA) in tea plants changes the growth and development of leaves in certain aspects. However, the molecular mechanism of IAA metabolism during the growth and development in tea leaves remains unclear. To explore the molecular mechanism of IAA metabolism in tea leaves, the samples were collected from the three different development stages of tea leaves. Cell morphology and IAA contents were also investigated during tea leaves development. A total of 16 genes involved in IAA biosynthesis and signal transduction pathways were identified based on the genome sequence of Camellia sinensis. CsTSA, CsIAA27, CsTIR1 and CsCH3-6 expression patterns exhibited positive correlation with IAA concentration in tea leaves. In contrast, CsYUC1, CsCYP83B1 and CsLAX1 transcriptions were negatively correlated with IAA contents during tea leaves ripening. The interaction of IAA metabolism related proteins was also discussed. The results indicated that the growth and development of tea leaves could be regulated by IAA metabolism in the form of related-genes differentially expression and protein-protein interaction. This study provided a further glimpse to IAA metabolic pathways in leaf during tea plant growth and development.

An Historical Review of Phenylacetic Acid.

Plant hormone biology is an ever-evolving field and as such, novel avenues of research must always be sought. Technological and theoretical advancement can also allow for previously dismissed research to yield equally interesting insights into processes now that they are better understood. The auxin phenylacetic acid (PAA) is an excellent example of this. PAA is a plant auxin that also possesses substantial antimicrobial activity. It has a broad distribution and has been studied in bacteria, fungi, algae and land plants. Research on this compound in plants was prominent in the 1980s, where its bioactivity and broad distribution were frequently examined. Unfortunately, due to the strong interest in the quintessential auxin, indole-3-acetic acid (IAA), research on PAA quickly petered out. Recently, several groups have resumed investigations on this hormone in plants, yet, little is known about PAA biology and its physiological role is unclear. PAA biosynthesis from the amino acid Phe invites direct comparisons with previously studied IAA biosynthesis pathways, and recent work has shown that PAA metabolism and signaling appears to be similar to that of IAA. However, given the large gap between previous work and recent investigations, a historical review of this auxin is required to renew our understanding of PAA. Here, previous work on PAA is reassessed in light of recent research in plants and serves as a synthesis of current knowledge on PAA biology.

Characterization of indole-3-pyruvic acid pathway-mediated biosynthesis of auxin in Neurospora crassa.

Plants, bacteria and some fungi are known to produce indole-3-acetic acid (IAA) by employing various pathways. Among these pathways, the indole-3-pyruvic acid (IPA) pathway is the best studied in green plants and plant-associated beneficial microbes. While IAA production circuitry in plants has been studied for decades, little is known regarding the IAA biosynthesis pathway in fungal species. Here, we present the first data for IAA-producing genes and the associated biosynthesis pathway in a non-pathogenic fungus, Neurospora crassa. For this purpose, we used a computational approach to determine the genes and outlined the IAA production circuitry in N. crassa. We then validated these data with experimental evidence. Here, we describe the homologous genes that are present in the IPA pathway of IAA production in N. crassa. High-performance liquid chromatography and thin-layer chromatography unambiguously identified IAA, indole-3-lactic acid (ILA) and tryptophol (TOL) from cultures supplemented with tryptophan. Deletion of the gene (cfp) that encodes the enzyme indole-3-pyruvate decarboxylase, which converts IPA to indole-3-acetaldehyde (IAAld), results in an accumulation of higher levels of ILA in the N. crassa culture medium. A double knock-out strain (Δcbs-3;Δahd-2) for the enzyme IAAld dehydrogenase, which converts IAAld to IAA, shows a many fold decrease in IAA production compared with the wild type strain. The Δcbs-3;Δahd-2 strain also displays slower conidiation and produces many fewer conidiospores than the wild type strain.

Indole-3-acetic acid production via the indole-3-pyruvate pathway by plant growth promoter Rhizobium tropici CIAT 899 is strongly inhibited by ammonium

Like many rhizobia, Rhizobium tropici produces indole-3-acetic acid (IAA), an important signal molecule required for root hair infection in rhizobia-legume symbioses. However, the IAA biosynthesis pathway and its regulation by R. tropici are still poorly understood. In this study, IAA synthesis and the effects of mineral N in IAA production by R. tropici CIAT 899 were verified by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). Furthermore, expression of genes related to IAA biosynthesis and metabolism were evaluated by RT-qPCR. Results indicated that IAA production by CIAT 899 was 12 times lower in the presence of NH4+. Moreover, it was found that indole-3-pyruvate (IPyA) is the major IAA biosynthesis intermediate. Genes y4wE, lao and iorA were identified by analysis of R. tropici genome in silico and were upregulated by tryptophan, indicating a possible role of these genes in IAA biosynthesis by CIAT 899. In conclusion, we show that IPyA is the major pathway for IAA biosynthesis in CIAT 899 and that its production is strongly inhibited by NH4+. Although present results arose from in vitro experiments, they provide new insight into the role of nitrogen in early events related to legume nodulation.

Low nitrogen stress stimulating the indole-3-acetic acid biosynthesis of Serratia sp. ZM is vital for the survival of the bacterium and its plant growth-promoting characteristic.

Serratia sp. ZM is a plant growth-promoting (PGP) bacterial strain isolated from the rhizospheric soil of Populus euphratica in northwestern China. In this study, low nitrogen supply significantly stimulated the production of indole-3-acetic acid (IAA) in Serratia sp.ZM. The inoculation of the bacterium to wheat seedlings improved plant growth compared with the uninoculated group, and the stimulating effect was more prominent under low nitrogen stress. Inactivation of the predicted key gene in the IAA biosynthesis pathway impaired IAA production and significantly hampered mutant growth in poor medium. Furthermore, the IAA-deficient mutant lost the PGP effect under either normal or low nitrogen conditions in plant experiments. This study revealed the significant impact of environmental nitrogen levels on IAA production in the PGP strain and the vital effect of IAA on resistance physiology of both the bacterium and host plant. The characteristics of Serratia sp. ZM also indicated its application potential as a biofertilizer for plants, especially those suffering from poor nitrogen soil.

The auxins, IAA and PAA, are synthesized by similar steps catalyzed by different enzymes

ABSTRACTOne of the fundamental plant growth substances, indole-3-acetic acid (IAA), belongs to a class of phytohormones known as auxins. The main IAA biosynthesis pathway involves the conversion of tryptophan to indole-3-pyruvic acid, which is in turn converted to IAA. The two enzymes responsible for these conversions, members of the TAA1 and YUCCA gene families, respectively, have recently been implicated in the synthesis of another auxin, phenylacetic acid (PAA). While there is some evidence to support this theory, there are also some concerns. Here we address the question: to what extent does the TAA1/YUCCA system contribute to the biosynthesis of PAA? In addition, we highlight the importance of measuring auxin metabolites and conjugates in addressing such questions.

Comparison of phytohormone biosynthesis and signal transduction pathways in developing and abortive hazelnut ovules

In hazelnuts, ovule abortion is associated with the formation of empty nuts, resulting in serious yield losses. Phytohormones play important roles in plant growth and development; however, the mechanisms through which they regulate ovule abortion in hazelnut are largely unknown. RNA-seq identified 1209 differentially expressed genes with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation between developing and abortive ovules in hazelnut. Hormone signal transduction (ko04075), and several phytohormone biosynthesis pathways, were found to be significantly enriched after pathway enrichment analysis. Compared with developing ovules, the levels of indole-3-acetic acid (IAA), gibberellins (GA), cytokinin (CTK), and salicylic acid (SA) were found to be significantly lower in abortive ovules, while levels of abscisic acid (ABA), ethylene (ETH), and jasmonic acid (JA) were found to be significantly higher. Consistent with these results, some important rate-limiting enzymes involved in the biosynthesis of phytohormones, including 9-cis-epoxycarotenoid dioxygenase, ACC synthase, and phospholipase A1 were significantly up-regulated in abortive ovules. Some key genes in the ETH, JA, and ABA signal transduction pathways were highly upregulated, including ethylene-responsive transcription factor 1 (ERF1/2) and the serine/threonine-protein kinase CTR1 in the ETH signal pathway, jasmonate ZIM domain-containing protein (JAZ) and the transcription factor MYC2 in the JA signal pathway, and ABA responsive element binding factor (ABF) in the JA signal pathway. Thus, ABA, ETH, and JA signaling were more active in abortive ovules than in developing ovules. Expression levels of NCED, ABF, ACS, ERF1/2, PLA1, and MYC2 revealed by q-RT-PCR were consistent with the results obtained by differentially expressed genes (DEG) analysis. Our data suggest that multiple phytohormone signaling pathways are involved in the development of hazelnut ovules. Inhibition of IAA, GA, CTK, and SA biosynthesis and signaling pathways, and activation of ABA, ETH, and JA biosynthesis and signaling pathways may contribute to the formation of abortive ovules in hazelnut.

Indole-3-Acetic Acid Is Produced by Emiliania huxleyi Coccolith-Bearing Cells and Triggers a Physiological Response in Bald Cells.

Indole-3-acetic acid (IAA) is an auxin produced by terrestrial plants which influences development through a variety of cellular mechanisms, such as altering cell orientation, organ development, fertility, and cell elongation. IAA is also produced by bacterial pathogens and symbionts of plants and algae, allowing them to manipulate growth and development of their host. They do so by either producing excess exogenous IAA or hijacking the IAA biosynthesis pathway of their host. The endogenous production of IAA by algae remains contentious. Using Emiliania huxleyi, a globally abundant marine haptophyte, we investigated the presence and potential role of IAA in algae. Homologs of genes involved in several tryptophan-dependent IAA biosynthesis pathways were identified in E. huxleyi. This suggests that this haptophyte can synthesize IAA using various precursors derived from tryptophan. Addition of L-tryptophan to E. huxleyi stimulated IAA production, which could be detected using Salkowski's reagent and GC × GC-TOFMS in the C cell type (coccolith bearing), but not in the N cell type (bald). Various concentrations of IAA were exogenously added to these two cell types to identify a physiological response in E. huxleyi. The N cell type, which did not produce IAA, was more sensitive to it, showing an increased variation in cell size, membrane permeability, and a corresponding increase in the photosynthetic potential quantum yield of Photosystem II (PSII). A roseobacter (bacteria commonly associated with E. huxleyi) Ruegeria sp. R11, previously shown to produce IAA, was co-cultured with E. huxleyi C and N cells. IAA could not be detected from these co-cultures, and even when stimulated by addition of L-tryptophan, they produced less IAA than axenic C type culture similarly induced. This suggests that IAA plays a novel role signaling between different E. huxleyi cell types, rather than between a bacteria and its algal host.

Indole-3-acetic acid biosynthetic pathways in the basidiomycetous yeast Rhodosporidium paludigenum.

Microorganisms produce plant growth regulators, such as auxins, cytokinins and gibberellins, to promote plant growth. Auxins are a group of compounds with an indole ring that have a positive effect on plant growth. Indole-3-acetic acid (IAA) is a plant growth hormone classified as an indole derivative of the auxin family. IAA biosynthesis pathways have been reported and widely studied in several groups of bacteria. Only a few studies on IAA biosynthesis pathways have been conducted in yeast. This study aimed to investigate IAA biosynthesis pathways in a basidiomycetous yeast (Rhodosporidium paludigenum DMKU-RP301). Investigations were performed both with and without a tryptophan supplement. Indole compound intermediates were detected by gas chromatography-mass spectrometry. Indole-3-lactic acid and indole-3-ethanol were found as a result of the enzymatic reduction of indole-3-pyruvic acid and indole-3-acetaldehyde, in IAA biosynthesis via an indole-3-pyruvic acid pathway. In addition, we also found indole-3-pyruvic acid in culture supernatants determined by high-performance liquid chromatography. Identification of tryptophan aminotransferase activity supports indole-3-pyruvic acid-routed IAA biosynthesis in R. paludigenum DMKU-RP301. We hence concluded that R. paludigenum DMKU-RP301 produces IAA through an indole-3-pyruvic acid pathway.

Effect of IAA on in vitro growth and colonization of Nostoc in plant roots.

Nostoc is widely known for its ability to fix atmospheric nitrogen and the establishment of symbiotic relationship with a wide range of plants from various taxonomic groups. Several strains of Nostoc produce phytohormones that promote growth of its plant partners. Nostoc OS-1 was therefore selected for study because of the presence of putative ipdC gene that encodes a key enzyme to produce Indole-3-acetic acid (IAA). The results indicated that both cellular and released IAA was found high with increasing incubation time and reached to a peak value (i.e., 21 pmol mg-1ch-a) on the third week as determined by UPLC-ESI-MS/MS. Also the Nostoc OS-1 strain efficiently colonized the roots and promoted the growth of rice as well as wheat under axenic conditions and induced ipdC gene that suggested the possible involvement of IAA in these phenotypes. To confirm the impact of IAA on root colonization efficiency and plant promoting phenotypes of Nostoc OS-1, an ipdC knockout mutant was generated by homologous recombinant method. The amount of releasing IAA, in vitro growth, root colonization, and plant promoting efficiency of the ipdC knockout mutant was observed significantly lower than wild type strain under axenic conditions. Importantly, these phenotypes were restored to wild-type levels when the ipdC knockout mutant was complemented with wild type ipdC gene. These results together suggested that ipdC and/or synthesized IAA of Nostoc OS-1 is required for its efficient root colonization and plant promoting activity.

Comparison of indole derivatives as potential intermediates of auxin biosynthesis in Arabidopsis

Biosynthesis of indole-3-acetic acid (IAA) is crucial for the regulation of plant growth and morphological changes. However, the pathways of IAA biosynthesis have not been completely understood. Although indole derivatives have been proposed to be intermediates of the pathway, it has not been thoroughly elucidated whether the proposed intermediates are indeed converted into an active auxin, IAA, or intermediate metabolites other than IAA. In the present work, we examined 11 indole derivatives whether they are indeed IAA intermediates. The indole derivatives were examined by measuring their effects on recovery from auxin-deficiency conditions caused by l-aminooxyphenylpropionic acid (AOPP), which we previously identified as an IAA biosynthesis inhibitor. We estimated auxin activity of each indole derivative by analyzing auxin-inducible marker gene expression and root morphology in Arabidopsis. We found that indole-3-pyruvic acid most effectively recovered seedlings from auxin deficiency among the indole derivatives examined, followed by Nhydroxyl tryptamine and indole-3-acetonitrile, with effective recovery from the auxin deficiency caused by AOPP. We also found that tryptophol recovered the root growth defects. Among the indole derivatives examined, only indole-3-acrylic acid did not show effective recovery from auxin deficiency. These results provide information on which compounds are reliable intermediates of IAA biosynthesis pathways in Arabidopsis.