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Benzoic acid inhibits peach root growth and lateral root emergence by disrupting auxin homeostasis through salicylic acid accumulation.

We established a non-sterile root transformation system in peach seedlings. Using this system, we demonstrated that BA treatment inhibits plant growth and lateral root emergence by SA-mediated disruption of auxin distribution. Allelopathic autotoxins, particularly benzoic acid (BA), are recognized as primary contributors to peach (Prunus persica) replant disease; however, the molecular mechanisms by which BA disrupts root development remain poorly understood. BA treatment significantly reduced stem and root length and inhibited lateral root emergence without affecting lateral root initiation. To investigate the underlying mechanism at cellular resolution, we established a non-sterile Agrobacterium rhizogenes-based root transformation system achieving 27.11% transformation efficiency. Auxin biosynthesis (PpYUC10), influx transport (PpAUX1), and response (PpARF19) genes were markedly downregulated following BA treatment. Transgenic roots expressing the DR5::GUS auxin reporter exhibited reduced DR5 activity in root tips and suppressed expression in tissues surrounding lateral root primordia, indicating impaired auxin signaling at both developmental sites. Hormone profiling revealed a non-significant trend toward reduced auxin metabolites alongside significant accumulation of salicylic acid (SA), an auxin-antagonistic hormone, and its storage conjugate SA 2-O-β-glucoside. Supporting a causal role for SA, exogenous SA phenocopied BA-induced root growth inhibition, whereas co-treatment with IAA or the SA-biosynthesis inhibitor aminoindan-1-phosphonic acid (AIP) significantly rescued lateral root number and root fresh weight. Multi-treatment RNA-seq identified "response to auxin" and "response to salicylic acid" as the most enriched GO terms in BA-treated roots, and AIP treatment restored the expression of key auxin-related genes while reversing BA-induced SA-pathway changes. Together, these findings suggest that BA-induced SA accumulation suppresses auxin biosynthesis, transport, and signaling, thereby inhibiting peach root growth and lateral root emergence. This study elucidates the molecular basis of BA autotoxicity and establishes a transformation platform for functional genomic studies in Prunus.

Indoleacetic Acids

[Model for auxin receptor].

Literary data on correlation between the structure and auxin activity of aryl-, arylalkan- and aryloxyalkancarboxylic acids and those of 3-indolylacetic acid are reviewed. The structure of auxins is compared to that of their inactive structural analogs in terms of a literary hypothesis on the bifunctional binding of auxins to the receptor, which allowed to construct a model for the structure of the auxin receptor (site responsible for intrinsic binding of auxins). The geometric parameters of this receptor site and specific arrangement of specific auxin binding sites have been proposed. It is demonstrated that during auxin absorption on the receptor the conformation of the latter is changed, which may be accounted for as a primary functional effect of auxins on the receptor.

Carboxylic Acids

Long-day photoperiod promotes growth of pea (Pisum sativum L.) via auxin biosynthesis and polar transport.

Photoperiodic sensitivity is an essential factor that may affect agricultural practices under current climate scenarios. This study used pea (Pisum sativum) to examine effects of varying photoperiods on growth and photosynthetic parameters and then reveal the mechanistic basis of this process by linking them with tissue-specific distribution of auxin and regulation of related genes. This was achieved by transcriptome sequencing, genome-wide gene family identification, and expression pattern analysis. Best results in terms of growth and yield were obtained with a 20 h/4 h light/dark photoperiod and these plants had the highest content of endogenous indole-3-acetic acid (IAA) in both the shoot apex and the root. Genes consistently upregulated with prolonged light exposure were significantly enriched in pathways related to light signal transduction, photosynthetic carbon metabolism, and phytohormone signal transduction. Through genome-wide identification, we characterized the TAA/TAR and YUCCA families (key gene families involved in auxin biosynthesis) as well as the PIN family (responsible for auxin polar transport) in pea. Extending the light duration positively affected expression of several genes related to auxin biosynthesis and transport, among them members of the Elongated Hypocotyl (HY) and Phytochrome-Interacting Factor (PIF) families being key light-induced transcription factors, PsTAR2, the principal gene regulating auxin biosynthesis, as well as PsPIN4, PsPIN5, PsPIN11, and PsPIN13 which mediate polar auxin transport. By elucidating mechanisms underlying the coordinated regulation of pea growth by light and auxin, this work provides a significant reference for photoperiod research on long-day crops for both protected- and field-based horticulture.

Auxin

Separation of two responses to auxin by means of cytokinin inhibition.

A continuous growth apparatus was used to measure the effect of cytokinin on auxin-induced elongation. The soybean hypocotyl segments elicited a bi-phasic response to auxin that appeared to be two overlapping responses. The first response, which began 12 min after auxin addition, was not inhibited by cytokinin, even after long preincubation in cytokinin, but the second response to auxin, which began about 30 min after auxin addition, was completely inhibited by cytokinin. Such overlapping reactions are shown, depending on the amount of overlap, to yield a variety of summation reactions, many of which resemble rate-time curves that have been previously reported. We have shown that the transient first phase of auxin-induced elongation is very similar to acid-activated growth, while the second phase is long lasting and very likely identical to the long-term response to auxin, as extensively studied in Avena, soybean, and other elongating cells.

Cell Division

Auxin-induced ARF transcription factor degradation defines tissue boundaries.

How organs partition themselves into discrete domains with distinct functions is a fundamental question in biology. The gynoecium of flowering plants provides an excellent system to address this question. Here, we show that the boundary between the stigma and style at the gynoecium apex is established by the complementary distribution of the phytohormone auxin and the Auxin Response Factor (ARF), ETTIN (ETT). Mechanistically, auxin induces ETT protein destabilization via the ubiquitin-proteasome pathway. A short sequence motif within an intrinsically disordered region is required for this auxin-triggered degradation. Disruption of this motif leads to ectopic ETT accumulation at the gynoecium apex and consequently abolishes stigma-style boundary development. We further demonstrate that this previously unrecognized mode of auxin-induced ARF instability is evolutionarily conserved among ETT orthologs across angiosperms. In summary, this study reveals how graded auxin distribution affects ARF transcription factor activity, contributing to the establishment of the stigma-style boundary, ensuring correct gynoecium formation and reproductive success in flowering plants.

Indoleacetic Acids

A FERONIA-MPK3/6-WRKY3/4 module links auxin signaling to lateral root development in Arabidopsis.

The phytohormone auxin orchestrates root development through intricate signaling networks. In the non-canonical auxin pathway, both the transmembrane kinase (TMK)-mediated signaling and the mitogen-activated protein kinase (MAPK) cascade are shown to be involved in the auxin-regulated lateral root (LR) formation. However, the role and mechanism of the receptor-like kinase FERONIA (FER) in this process remain unclear. Here, quantitative proteomic and phosphoproteomic analyses of Arabidopsis roots identify FER, MPK3/6, and WRKY3/4 as auxin-responsive components. Further analyses reveal that FER functions as a negative regulator of LR development by modulating cell division patterns within LR primordia. FER interacts with and phosphorylates MPK3/6, which then phosphorylate the transcription factors WRKY3 and WRKY4 to form a repressive module that ultimately suppresses LR organogenesis. Collectively, our findings define a FER-MPK3/6-WRKY3/4 signaling module that negatively regulates LR formation, demonstrating a previously unknown integration of FER-mediated signaling into the MAPK cascade in auxin-triggered organogenesis.

Arabidopsis

Quantitative trait locus qLDC5 regulates primary root branching in an auxin-dependent manner.

L-type lateral root (LLR) density determines root system architecture, affecting nutrient acquisition in rice (Oryza sativa L.), particularly under low-phosphorus conditions. Previous studies identified genotypic differences in LLR density and a quantitative trait locus (QTL) enhancing LLR density on crown roots (qLDC5). We showed that LLR densities on crown and primary roots were closely correlated and confirmed higher LLR density on primary roots in qLDC5 donor DJ123 compared with the African variety NERICA4 using X-ray micro-computed tomography. We confirmed the qLDC5 effect in a field experiment for LLR density on primary roots. LLR densities on primary and crown roots, therefore, appear under similar genetic control. Developmental analyses revealed that DJ123 and NDJ188-a derivative line harboring qLDC5-initiate more lateral root primordia than NERICA4, with a higher proportion progressing to elongation, but that exogenous auxin application reversed this ranking. Within qLDC5, auxin biosynthesis gene OsYUCCA2 and auxin response factor OsARF15 were up-regulated in DJ123. Transcriptome analysis revealed an indirect auxin-mediated regulatory network underlying LLR variation. Differentially expressed genes in DJ123 and NDJ188 were enriched for ent-kaurene and gibberellin metabolism, including the robust induction of OsGA2ox5. These findings suggest qLDC5 increases lateral root density by coordinating gibberellin, auxin, and terpene pathways.

Oryza

Synthesis of auxins from tryptophan and tryptophan-precursors by fungi isolated from mycorrhizae of pine (Pinus silvestris L.).

Fungi isolated from mycorrhizae of pine required tryptophan for auxin synthesis. More auxins were found in culture grown with pyrogallol than in those without this compound. The fungi studied produced also auxins from other than tryptophan compounds. Indole employed with serine was more suitable for the production of auxins than indole or anthranilic acid used separately. The active compounds showing auxin activity were located on the chromatograms at Rf 0.2--0.4 and 0.3--0.5 with the solvent system isopropanol, ammonia, water (10:1:1 v/v).

Culture Media

Deciphering acquired resistance mechanisms to sustained auxin-inducible protein degradation in cells and mice.

Targeted protein degradation is a favorable strategy for studying the immediate downstream effects of protein loss-of-function. An appealing platform among these technologies is the auxin-inducible degron (AID) system. Although this system has been applied extensively to cell and animal models, degradation resistance to long-term auxin treatment has not been studied. With the advent of the new AID2 system, cellular toxicity due to the high concentrations of auxin required in the original AID1 system is no longer a concern, making it possible to study protein degradation over extended periods. In this study, we derived multiple miniAID-tagged knock-in human cell lines and a Ctcf-miniAID knock-in mouse strain to investigate mechanisms of degradation resistance. We revealed four independent resistance mechanisms, including a nonsense mutation in the CTCF coding sequence that removed the miniAID peptide, a missense point mutation in the miniAID coding region that disrupted ubiquitin complex targeting, and silencing of the OsTIR1 adaptor protein. Resistance to auxin degradation was also acquired in mouse primary CtcfminiAID/miniAID knock-in B-ALL cells through missense mutations of the OsTIR1(F74G) protein in vivo and ex vivo. In summary, our innovative study expands our understanding of the AID system and cautions careful consideration of design for future applications in mammalian system.

CTCF

Enhancement of soybean RNA polymerase I by auxin.

When etiolated soybean seedlings are treated with the synthetic auxin, 2,4-dichlorophenoxy-acetic acid, cells of the mature hypocotyl become swollen and proliferate abnormally. This abnormal growth induced by auxin coincides with a 5- to 8-fold increase in the alpha-amanitin-insensitive RNA polymerase associated with isolated chromatin or nuclei. The alpha-amanitin-sensitive RNA polymerase activity of the auxin-treated hypocotyl was similar to that of control tissue. The increase in RNA polymerase I activity of chromatin and nuclei was maintained after solubilization and fractionation on DEAE-cellulose. Auxin thus appears to enhance RNA synthetic activity (i.e., ribosomal RNA) in mature soybean tissue by altering RNA polymerase I directly rather than by altering RNA polymerase I directly rather than by altering the chromatin template.

2,4-Dichlorophenoxyacetic Acid

OsMYB8-OsARF12/25 module fine-tunes tiller angle via auxin signaling pathway in rice.

Tiller angle is a critical determinant of rice plant architecture and significantly impacts grain yield by influencing planting density and photosynthetic efficiency. Although auxin signaling is known to affect tiller angle in rice, the detailed regulatory networks remain largely unknown. In this study, we identify OsMYB8, an R2R3-MYB transcription factor, as a positive regulator of rice tiller angle. Functional analyses revealed that loss-of-function mutants of OsMYB8 exhibited reduced tiller angles and a more compact architecture, while overexpression of OsMYB8 resulted in more expanded tiller angles. Further investigations found that OsMYB8 might negatively regulate the shoot gravitropic response by disrupting asymmetric auxin distribution. At the molecular level, OsMYB8 directly binds to the promoters of 2 auxin response factors, OsARF12 and OsARF25, and represses their transcription. Genetic analyses confirmed that OsMYB8 acts upstream of OsARF12 and OsARF25 in regulating rice tiller angle. Our finding elucidates a previously uncharacterized OsMYB8-OsARF12/25 transcriptional module that fine-tunes auxin signaling to regulate tiller angle in rice, and offers valuable genetic targets for the optimization of rice architecture and yield potential.

Oryza

Production of auxins by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.).

Qualitative and quantitative studies were carried out on the production of auxins by Coryneform bacteria, the only bacterial types isolated from roots of pine seedlings. Almost all isolates were capable of producing auxins in tryptophan containing media. In media without this amino acid only trace or no auxins were produced. Most of the bacteria studied synthesized auxins located on the chromatograms run with isopropanol, ammonia, water (10:1:1 v/v) at Rf 0.3--0.5. Moreover substances with Rf values 0.05--0.2 and 0.8--1.0 were produced by some strains. No plant growth inhibitors detected with the Avena coleoptiles biotest were produced by the bacteria studied.

Biological Assay

Effect of the culture medium and incubation time on auxins production by bacteria isolated from the roots of pine seedlings (Pinus silvestris l.).

Studies were performed on the effect of culture medium and incubation time on the production of auxins by bacteria. The bacteria studied produced more auxins in the mineral medium containing glucose and tryptophan than in that enriched with casamino acids and yeast extract. The amount of auxins elaborated depended both upon the strain and the age of the culture. Some strains produced the largest amounts of these substances after 7 days of incubation while others required a longer period. Most of the substances showing auxin activity were located on the chromatograms at Rf 0.3-0.4 and 0.8-1.0.

Actinomycetales

Stereoselectivity and affinity in molecular pharmacology. III. Structural aspects in the mode of action of natural and synthetic auxins.

Analysis of available potency estimates for 35 pairs of enantiomeric arylcarboxylic acids with auxin activity (flax-root-growth inhibition test) revealed extensive correlations between the activity of the more potent and less potent isomers, as well as between the log of the ratio of potencies and the log potency of the more active isomer when structurally similar analogs are compared. 5 structural subgroups were discernible (n, eudismic-affinity quotient (EAQ), r2); (1) arylpropionic acids (6, -0.36, 0.66); (2) 2-naphthoxy-carboxylic acids (6, +1.07, 0.99); (3) 1-naphthoxycarboxylic acids (3, +1.56, 0.96); (4) ortho-substituted phenoxycarboxylic acids (10, +0.97, 0.96) and (5) ortho-unsubstituted phenoxycarboxylic acids (10, +0.56, 0.70). For achiral lower homologs such as auxin itself 3-indolyl-acetic acid (IAA), phenoxyacetic acid and 1-naphthoxyacetic acid, extrapolated potencies were found to agree well with experimental values. On the basis of these observations an auxin receptor is postulated and binding arrangements are described which explain most of the experimental data available. A 3-point attachment when allowed is the only binding mode compatible with the reported data.

Carboxylic Acids

Synthesis of auxins by fungi isolated from the roots of pine seedings (Pinus silvestris L.) and from soil.

Synthesis of auxins by fungi grown with and without tryptophan has been studied. 26 out of 30 fungal strains produced detectable amounts of auxins in tryptophan contain media. 18 strains produced but very small amounts of auxins in media without this amino acid. By means of paper chromatography, chromogenic reagents and biotest three active substances could be distinguished. They were found on the chromatograms run with isopropanol, ammonia, water (10:1:1 v/v) at Rf 0.05--0.2, 0.3--0.5 and 0.8--1.0. Most strains produced active substances with Rf 0.3--0.5.

Ascomycota

Analysis of Cytoplasmic RNA Decay Targets Using the Auxin Degron System.

RNA degradation in mammalian cells is performed by multiple enzymes and cofactors making it difficult to identify the specific impact of each of them separately. The auxin-inducible degron system enables direct depletion of a protein of interest limiting the time of depletion and thus reducing secondary effects due to cell adaptation. In this chapter, using XRN1 as an example of cytoplasmic RNA decay enzyme, we describe a combination of methods to introduce the auxin-inducible degron by CRISPR-Cas9, together with downstream analyses of RNA levels after protein depletion.

Indoleacetic Acids

The CsTBH-CsROP2 Module Regulates Waterlogging Tolerance via Auxin-Mediated Adventitious Root Formation in Cucumber.

Cucumber (Cucumis sativus L.) requires frequent irrigation due to its shallow root system and high transpiration rate of the aboveground parts. However, it is also prone to waterlogging damage. Therefore, understanding its response to waterlogging is crucial for breeding waterlogging-tolerant varieties. Although Rho of Plants GTPases play well-established roles in regulating development and stress signalling, their functions in plant adaptation to waterlogging stress has yet to be fully elucidated. Here, we identified nine CsROP genes in the cucumber genome, which exhibit evolutionary diversification but retain conserved functional domains. Functional analysis revealed that CsROP2 acts as a negative regulator of adventitious root formation. It modulates auxin accumulation in hypocotyl vascular bundles, thereby suppressing adventitious root development and enhancing waterlogging sensitivity. The HD-Zip I transcription factor CsTBH directly binds the CsROP2 promoter and activates its expression. Our study uncovers a CsTBH-CsROP2 module that governs adventitious rooting and waterlogging tolerance by modulating auxin homeostasis. These findings provide new insights into the crosstalk between developmental programmes and stress signalling pathways and offer potential genetic targets for improving stress resilience in cucumber and other crops.

CsROP2

Functional analysis of a GWAS pleiotropic hotspot suggests an auxin biosynthesis gene (AhPDS1), regulating pod development in peanut (Arachis hypogaea L.).

Peanut productivity and quality improvement rely on understanding the genetic factors influencing pod and seed size. This study aims to identify genetic factors and regulatory mechanisms influencing pod and seed size in peanuts. Herein, a genome-wide association study (GWAS) was conducted using 390 accessions from 15 peanut growing regions to analyze pod and seed traits across multiple planting seasons. A significant phenotypic variation was observed, with broad-sense heritability ranging from 53.6 to 85.4%. Strong correlations between pod and seed traits further suggest potential for co-selection in breeding efforts. A pleiotropic hotspot on chromosome B06 was strongly associated with six pod and seed traits. A peanut pod size regulator AhPDS1 (PODSIZE-1, Ahy_B06g085516) homolog of Arabidopsis thaliana YUCCA4 (AtYUC4, AT5G11320), involved in auxin biosynthesis, was selected as a candidate regulating pod and seed size. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmed higher AhPDS1 expression in large pod as compared with the small pod genotypes. Subcellular localization showed AhPDS1 to be predominantly cytoplasmic, and GUS reporter assays indicated widespread expression in roots, stems, leaves, flowers, and pods, suggesting a broad functional role. Further overexpression of AhPDS1 in Arabidopsis and rice enhanced pod, seed, and grain sizes via the indole-3-pyruvic acid pathway in transgene lines. These findings highlight AhPDS1 as a potential target for peanut molecular breeding, offering opportunities to enhance pod size via auxin biosynthesis and support sustainable crop improvement.

Arachis