Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns.

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Exogenous application of the apocarotenoid retinaldehyde negatively regulates auxin-mediated root growth.

Root development is essential for plant survival. The lack of carotenoid biosynthesis in the phytoene desaturase 3 (pds3) mutant results in short primary roots (PRs) and reduced lateral root formation. In this study, we showed that short-term inhibition of PDS by fluridone suppresses PR growth in wild type, but to a lesser extent in auxin mutants of Arabidopsis (Arabidopsis thaliana). Such an inhibition of PDS activity increased endogenous indole-3-acetic acid levels, promoted auxin signaling, and partially complemented the PR growth of an auxin-deficient mutant, the YUCCA 3 5 7 8 9 quadruple mutant (yucQ). The exogenous application of retinaldehyde (retinal), an apocarotenoid derived from ß-carotene, complemented the fluridone-induced suppression of root growth, as well as the short roots of the pds3 mutant. Retinal also partially complemented the auxin-induced suppression of root growth. These results suggest that retinal may play a role in regulating root growth by modulating endogenous auxin levels.

The benzoylphenylurea derivative BPU17 acts as an inhibitor of prohibitin and exhibits antifibrotic activity.

Inflammation-induced choroidal neovascularization followed by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) is a cause of neovascular age-related macular degeneration (nAMD). RPE-derived myofibroblasts overproduce extracellular matrix, leading to subretinal fibrosis. We already have demonstrated that benzylphenylurea (BPU) derivatives inhibit the function of cancer-associated fibroblasts. Here, we investigated the anti-myofibroblast effects of BPU derivatives and examined such BPU activity on subretinal fibrosis. A BPU derivative, BPU17, exhibits the most potent anti-myofibroblast activity among dozens of BPU derivatives and inhibits subretinal fibrosis in a mouse model of retinal degeneration. Investigations with primary cultured RPEs reveal that BPU17 suppresses cell motility and collagen synthesis in RPE-derived myofibroblasts. These effects depend on repressing the serum response factor (SRF)/CArG-box-dependent transcription. BPU17 inhibits the expression of SRF cofactor, cysteine and glycine-rich protein 2 (CRP2), which activates the SRF function. Proteomics analysis reveals that BPU17 binds to prohibitin 1 (PHB1) and inhibits the PHB1-PHB2 interaction, resulting in mild defects in mitochondrial function. This impairment causes a decrease in the expression of CRP2 and suppresses collagen synthesis. Our findings suggest that BPU17 is a promising agent against nAMD and the close relationship between PHB function and EMT.

Chemical inhibition of the auxin inactivation pathway uncovers the roles of metabolic turnover in auxin homeostasis.

The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

Radicle Growth Regulation of Root Parasitic Plants by Auxin-related Compounds.

Root parasitic plants in the Orobanchaceae, such as Striga and Orobanche, cause significant damage to crop production. The germination step of these root parasitic plants is induced by host-root-derived strigolactones. After germination, the radicles elongate toward the host and invade the host root. We have previously discovered that a simple amino acid, tryptophan (Trp), as well as its metabolite, the plant hormone indole-3-acetic acid (IAA), can inhibit radicle elongation of Orobanche minor. These results suggest that auxin plays a crucial role in the radicle elongation step in root parasitic plants. In this report, we used various auxin chemical probes to dissect the auxin function in the radicle growth of O. minor and Striga hermonthica. We found that synthetic auxins inhibited radicle elongation. In addition, auxin receptor antagonist, auxinole, rescued the inhibition of radicle growth by exogenous IAA. Moreover, a polar transport inhibitor of auxin, N-1-naphthylphthalamic acid, affected radicle bending. We also proved that exogenously applied Trp is converted into IAA in O. minor seeds, and auxinole partly rescued this radicle elongation. Taken together, our data demonstrate a pivotal role for auxin in radicle growth. Thus, manipulation of auxin function in root parasitic plants should offer a useful approach to combat these parasites.

Arinole, a novel auxin-stimulating benzoxazole, affects root growth and promotes adventitious root formation.

The triple response phenotype is characteristic for seedlings treated with the phytohormone ethylene or its direct precursor 1-aminocyclopropane-carboxylic acid, and is often employed to find novel chemical tools to probe ethylene responses. We identified a benzoxazole-urea derivative (B2) partially mimicking ethylene effects in a triple response bioassay. A phenotypic analysis demonstrated that B2 and its closest analogue arinole (ARI) induced phenotypic responses reminiscent of seedlings with elevated levels of auxin, including impaired hook development and inhibition of seedling growth. Specifically, ARI reduced longitudinal cell elongation in roots, while promoting cell division. In contrast to other natural or synthetic auxins, ARI mostly acts as an inducer of adventitious root development, with only limited effects on lateral root development. Quantification of free auxins and auxin biosynthetic precursors as well as auxin-related gene expression demonstrated that ARI boosts global auxin levels. In addition, analyses of auxin reporter lines and mutants, together with pharmacological assays with auxin-related inhibitors, confirmed that ARI effects are facilitated by TRYPTOPHAN AMINOTRANSFERASE1 (TAA1)-mediated auxin synthesis. ARI treatment in an array of species, including Arabidopsis, pea, tomato, poplar, and lavender, resulted in adventitious root formation, which is a desirable trait in both agriculture and horticulture.

Investigation of physiological roles of UDP-glycosyltransferase UGT76F2 in auxin homeostasis through the TAA-YUCCA auxin biosynthesis pathway.

Cellular auxin (indole-3-acetic acid, IAA) levels are coordinately regulated by IAA biosynthesis and inactivation. IAA is synthesized through sequential reactions by two enzymes, TAA1 and YUCCA, in a linear indole-3-pyruvic acid (IPA) pathway. TAA1 converts tryptophan to IPA, and YUCCA catalyzes the oxidative decarboxylation of IPA into IAA. Arabidopsis UDP-glycosyltransferase UGT76F2 (At3g55710) was previously reported to catalyze the glycosylation of IPA and consequently modulate IAA levels. We carefully analyzed the physiological roles of UGT76F2 and its close homolog UGT76F1 (At3g55700) in IAA homeostasis. We generated two independent ugt76f1 ugt76f2 double null Arabidopsis mutants (ugt76f1f2) with a 2.7 kb deletion, along with two independent ugt76f2 single null mutants by CRISPR/Cas9 gene editing technology. Surprisingly, these null mutants exhibited indistinguishable phenotypes from the wild-type seedlings under our laboratory conditions. Our results indicate that UGT76F1 and UGT76F2 do not play important roles in regulating IAA biosynthesis via IPA glycosylation.

Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition.

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Role of CRP2-MRTF interaction in functions of myofibroblasts.

Inflammatory response induces phenotypic modulation of fibroblasts into myofibroblasts. Although transforming growth factor-ßs (TGF-ßs) evoke such transition, the details of the mechanism are still unknown. Here, we report that a LIM domain protein, cysteine-and glycine-rich protein 2 (CSRP2 [CRP2]) plays a vital role in the functional expression profile in myofibroblasts and cancer-associated fibroblasts (CAFs). Knock-down of CRP2 severely inhibits the expression of smooth muscle cell (SMC) genes, cell motility, and CAF-mediated collective invasion of epidermoid carcinoma. We elucidate the following molecular bases: CRP2 directly binds to myocardin-related transcription factors (MRTF-A/B [MRTFs]) and serum response factor (SRF) and stabilizes the MRTF/SRF/CArG-box complex to activate SMC gene expression. Furthermore, a three-dimensional structural analysis of CRP2 identifies the amino acids required for the CRP2-MRTF-A interaction. Polar amino acids in the C-terminal half (serine-152, glutamate-154, serine-155, threonine-156, threonine-157, and threonine-159 in human CRP2) are responsible for direct binding to MRTF-A. On the other hand, hydrophobic amino acids outside the consensus sequence of the LIM domain (tryptophan-139, phenylalanine-144, leucine-153, and leucine-158 in human CRP2) play a role in stabilizing the unique structure of the LIM domain.Key words: CRP2, 3D structure, myocardin-related transcription factor, myofibroblast, cancer-associated fibroblasts.

Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay.

Actin-related protein 5 (ARP5) inhibits the differentiation of skeletal, smooth, and cardiac muscle tissues, and ARP5 expression increases or decreases according to physiological and pathological changes in the muscle differentiation status. However, the regulatory mechanisms of ARP5 expression are largely unknown. Here, we identified a novel Arp5 mRNA isoform that contains premature termination codons in alternative exon 7b and is thus targeted by nonsense-mediated mRNA decay (NMD). In mouse skeletal muscle cells, switching from the canonical Arp5 isoform, i.e., Arp5(7a), to the NMD-targeted isoform Arp5(7b) occurred during differentiation, suggesting that Arp5 expression is regulated by alternative splicing coupled to NMD (AS-NMD). We developed an original method to accurately quantify the proportion of both Arp5 isoforms and measured higher levels of Arp5(7b) in muscle and brain tissues, where ARP5 is less expressed. The 3' splice site in Arp5 exon 7 has an unusual acceptor sequence that often leads to the skip of the authentic splice site and the use of the cryptic splice site localized 16 bases downstream. When the unusual acceptor sequence was mutated to the usual one, the Arp5(7b) isoform was barely detectable. The expression of several splicing factors involved in 3' splice site recognition was reduced after muscle differentiation. Additionally, knockdown of splicing factors increased the levels of Arp5(7b) and decreased the expression of Arp5(7a). Furthermore, strong positive correlations were found between Arp5 expression and the levels of these splicing factors in human skeletal and cardiac muscle tissues. Thus, Arp5 expression in muscle tissues is most likely regulated by the AS-NMD pathway.

The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism.

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues.

Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants.

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the "dehydrogenase momilactone A synthase" gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

Local regulation of auxin transport in root-apex transition zone mediates aluminium-induced Arabidopsis root-growth inhibition.

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.

A luciferase reporter assay for ecdysone agonists using HEK293T cells.

Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators.

Fluorescent Auxin Analogs Report Two Auxin Binding Sites with Different Subcellular Distribution and Affinities: A Cue for Non-Transcriptional Auxin Signaling.

The complexity of auxin signaling is partially due to multiple auxin receptors that trigger differential signaling. To obtain insight into the subcellular localization of auxin-binding sites, we used fluorescent auxin analogs that can undergo transport but do not deploy auxin signaling. Using fluorescent probes for different subcellular compartments, we can show that the fluorescent analog of 1-naphthaleneacetic acid (NAA) associates with the endoplasmic reticulum (ER) and tonoplast, while the fluorescent analog of indole acetic acid (IAA) binds to the ER. The binding of the fluorescent NAA analog to the ER can be outcompeted by unlabeled NAA, which allows us to estimate the affinity of NAA for this binding site to be around 1 µM. The non-transportable auxin 2,4-dichlorophenoxyacetic acid (2,4-D) interferes with the binding site for the fluorescent NAA analog at the tonoplast but not with the binding site for the fluorescent IAA analog at the ER. We integrate these data into a working model, where the tonoplast hosts a binding site with a high affinity for 2,4-D, while the ER hosts a binding site with high affinity for NAA. Thus, the differential subcellular localization of binding sites reflects the differential signaling in response to these artificial auxins.

GH3 Auxin-Amido Synthetases Alter the Ratio of Indole-3-Acetic Acid and Phenylacetic Acid in Arabidopsis.

Auxin is the first discovered plant hormone and is essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the main auxin and plays pivotal roles in intercellular communication through polar auxin transport. Phenylacetic acid (PAA) is another natural auxin that does not show polar movement. Although a wide range of species have been shown to produce PAA, its biosynthesis, inactivation and physiological significance in plants are largely unknown. In this study, we demonstrate that overexpression of the CYP79A2 gene, which is involved in benzylglucosinolate synthesis, remarkably increased the levels of PAA and enhanced lateral root formation in Arabidopsis. This coincided with a significant reduction in the levels of IAA. The results from auxin metabolite quantification suggest that the PAA-dependent induction of GRETCHEN HAGEN 3 (GH3) genes, which encode auxin-amido synthetases, promote the inactivation of IAA. Similarly, an increase in IAA synthesis, via the indole-3-acetaldoxime pathway, significantly reduced the levels of PAA. The same adjustment of IAA and PAA levels was also observed by applying each auxin to wild-type plants. These results show that GH3 auxin-amido synthetases can alter the ratio of IAA and PAA in plant growth and development.

An Evolutionarily Primitive and Distinct Auxin Metabolism in the Lycophyte Selaginella moellendorffii.

Auxin is a key regulator of plant growth and development. Indole-3-acetic acid (IAA), a plant auxin, is mainly produced from tryptophan via indole-3-pyruvate (IPA) in both bryophytes and angiosperms. Angiosperms have multiple, well-documented IAA inactivation pathways, involving conjugation to IAA-aspartate (IAA-Asp)/glutamate by the GH3 auxin-amido synthetases, and oxidation to 2-oxindole-3-acetic acid (oxIAA) by the DAO proteins. However, IAA biosynthesis and inactivation processes remain elusive in lycophytes, an early lineage of spore-producing vascular plants. In this article, we studied IAA biosynthesis and inactivation in the lycophyte Selaginella moellendorffii. We demonstrate that S. moellendorffii mainly produces IAA from the IPA pathway for the regulation of root growth and response to high temperature, similar to the angiosperm Arabidopsis. However, S. moellendorffii exhibits a unique IAA metabolite profile with high IAA-Asp and low oxIAA levels, distinct from Arabidopsis and the bryophyte Marchantia polymorpha, suggesting that the GH3 family is integral for IAA homeostasis in the lycophytes. The DAO homologs in S. moellendorffii share only limited similarity to the well-characterized rice and Arabidopsis DAO proteins. We therefore suggest that these enzymes may have a limited role in IAA homeostasis in S. moellendorffii compared to angiosperms. We provide new insights into the functional diversification of auxin metabolic genes in the evolution of land plants.

Arogenate dehydratases can modulate the levels of phenylacetic acid in Arabidopsis.

Phenylacetic acid (PAA) is one type of natural auxin and widely exists in plants. Previous biochemical studies demonstrate that PAA in plants is synthesized from phenylalanine (Phe) via phenylpyruvate (PPA), but the PAA biosynthetic genes and its regulation remain unknown. In this article, we show that the AROGENATE DEHYDRATASE (ADT) family, which catalyzes the conversion of arogenate to Phe, can modulate the levels of PAA in Arabidopsis. We found that overexpression of ADT4 or ADT5 remarkably increased the amounts of PAA. Due to an increase in PAA levels, ADT4ox and ADT5ox plants can partially restore the auxin-deficient phenotypes caused by treatments with an inhibitor of the biosynthesis of indole-3-acetic acid (IAA), a main auxin in plants. In contrast, the levels of PAA were significantly reduced in adt multiple knockout mutants. Moreover, the levels of PPA are substantially increased in ADT4 or ADT5 overexpression plants but reduced in adt multiple knockout mutants, suggesting that PPA is a key intermediate of PAA biosynthesis. These results provide an evidence that members of the ADT family of Arabidopsis can modulate PAA level via the PPA-dependent pathway.

UDP-glucosyltransferase UGT84B1 regulates the levels of indole-3-acetic acid and phenylacetic acid in Arabidopsis.

Auxin is a key plant growth regulator for diverse developmental processes in plants. Indole-3-acetic acid (IAA) is a primary plant auxin that regulates the formation of various organs. Plants also produce phenylacetic acid (PAA), another natural auxin, which occurs more abundantly than IAA in various plant species. Although it has been demonstrated that the two auxins have distinct transport characteristics, the metabolic pathways and physiological roles of PAA in plants remain unsolved. In this study, we investigated the role of Arabidopsis UDP-glucosyltransferase UGT84B1 in IAA and PAA metabolism. We demonstrated that UGT84B1, which converts IAA to IAA-glucoside (IAA-Glc), can also catalyze the conversion of PAA to PAA-glucoside (PAA-Glc), with a higher catalytic activity in vitro. Furthermore, we showed a significant increase in both the IAA and PAA levels in the ugt84b1 null mutants. However, no obvious developmental phenotypes were observed in the ugt84b1 mutants under laboratory growth conditions. Moreover, the overexpression of UGT84B1 resulted in auxin-deficient root phenotypes and changes in the IAA and PAA levels. Our results indicate that UGT84B1 plays an important role in IAA and PAA homeostasis in Arabidopsis.