Ken-Ichiro Hayashi

Dynamic regulation of auxin (indole-3-acetic acid; IAA) levels is crucial for proper plant growth and development and is finely regulated through biosynthesis, transport, metabolic inactivation, and signal transduction. While O -glucosylation of IAA is well established, the physiological significance of N -glucosylation pathways acting on IAA-amino acid conjugates remains largely unexplored. Here, we identify a UDP-glucosyltransferase in rice ( Oryza sativa ), designated IAAspGT, that catalyzes the N -glucosylation of indole-3-acetyl (IA) amino acid conjugates. Functional analysis revealed that natural allelic variants of IAAspGT differ markedly in enzymatic activity, with the high-activity allele prevalent in indica and the low-activity allele in japonica cultivars. Mutational analysis identified key residues within the glucose-accepting substrate-binding domain that account for this variation. A metabolic perturbation experiment demonstrated that IAAspGT competes with the DAO-mediated oxidation, thereby affecting active auxin levels. Plants harboring the high-activity allele exhibited enhanced root elongation under nutrient-deficient conditions and altered growth allocation between the panicle and other above-ground organs. Haplotype analysis indicated that the high-activity allele is ancestral and widely retained in the wild relatives. These findings establish IA-amino acid N -glucosylation as a previously overlooked metabolic branch that modulates auxin availability and contributes to environmental responses and growth plasticity in rice.

Indole-3-acetic acid (IAA), the predominant natural auxin, plays a crucial role in plant growth and development. However, the molecular mechanism underlying the precise regulation of IAA homeostasis is not fully understood. The GH3 family of IAA–amino acid-conjugating enzymes is crucial for maintaining auxin homeostasis by converting IAA into inactive IAA–amino acid conjugates. Here, we report on Auxurea A (AuR), a diphenylurea compound derived from the structural optimization of a GH3 inhibitor identified through a chemical library screen. AuR specifically inhibited GH3-mediated IAA inactivation both in vivo and in vitro. AuR increased endogenous IAA levels in Arabidopsis (Arabidopsis thaliana) plants, phenocopying the IAA-overaccumulating gh3-septuple mutants. Kinetic analyses revealed that AuR acted as a selective competitive inhibitor of GH3 enzymes. The high-auxin phenotypes induced by AuR were significantly suppressed in the aux1 mutant, which is defective in auxin influx. The AuR-induced phenotypes were also suppressed by AUX1 inhibitors. In contrast, auxin efflux inhibitors enhanced the AuR-induced high-auxin phenotypes. These findings, together with IAA inactivation, underscore the pivotal role of IAA transport between the apoplast and cytosol in maintaining intracellular auxin homeostasis. Notably, AuR exhibited selective activity toward dicotyledonous plants, including tobacco, lettuce, and carrot, but was not effective in monocotyledonous plants, such as rice and barley. These results demonstrate that AuR is a valuable chemical tool for investigating auxin homeostasis and a promising compound for the development of agrochemicals.

Protein knockdown using an improved auxin-inducible degron (AID2) technology has proven to be a powerful tool for studying protein function. The current approach requires the fusion of target proteins with a degron tag, a process typically achieved through CRISPR knock-in. However, knock-in remains challenging in non-model organisms and humans, limiting the broader applicability of AID2. To overcome this limitation, we developed a single-chain antibody AID2 (scAb-AID2) system. This approach employs an adaptor composed of a single-chain antibody fused with a degron, which recognizes a target protein and induces rapid degradation in the presence of the inducer 5-Ph-IAA. We demonstrated that scAb-AID2, in combination with an anti-GFP nanobody, degraded GFP-fused proteins in human cells and Caenorhabditis elegans. Furthermore, we showed that endogenous p53 and H/K-RAS were conditionally degraded in cells expressing an adaptor encoding an anti-p53 nanobody and -RAS monobody, respectively, and led to aphidicolin sensitivity in cell culture and growth inhibition in mouse xenografts. This study paves the way for broader application of AID2-based target depletion in model and non-model organisms and for advancing therapeutic strategies.

Protein knockdown using an improved auxin-inducible degron (AID2) technology has proven to be a powerful tool for studying protein function. The current approach requires the fusion of target proteins with a degron tag, a process typically achieved through CRISPR knock-in. However, knock-in remains challenging in non-model organisms and humans, limiting the broader applicability of AID2. To overcome this limitation, we developed a single-chain antibody AID2 (scAb-AID2) system. This approach employs an adaptor composed of a single-chain antibody fused with a degron, which recognises a target protein and induces rapid degradation in the presence of the inducer 5-Ph-IAA. We demonstrated that scAb-AID2, in combination with an anti-GFP nanobody, degraded GFP-fused proteins in human cells and C. elegans . Furthermore, we showed that endogenous p53 and H/K-RAS were conditionally degraded in cells expressing an adaptor encoding an anti-p53 nanobody and -RAS monobody, respectively, and led to aphidicolin sensitivity in cell culture and growth inhibition in mouse xenografts. This study paves the way for broader application of AID2-based target depletion in model and non-model organisms and for advancing therapeutic strategies.

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 the IPA glycosylation.

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 thorough 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, besides pharmacological assays with auxin-related inhibitors, confirmed that ARI effects are facilitated by TRYPTOPHAN AMINOTRANSFERASE1 (TAA1)-mediated auxin synthesis. ARI treatment resulted in AR formation in an array of species, including Arabidopsis, pea, tomato, poplar, and lavender, a desirable trait in both agriculture and horticulture.

The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.

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 and enhance target depletion. For this purpose, we initially compared PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrated control of cohesin and condensin with AID2 and BromoTag, respectively. We developed a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrated that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we found that co-depletion of ORC1 and CDC6 by the double-degron system completely suppressed DNA replication, and the cells entered mitosis with single-chromatid chromosomes, indicating DNA replication was uncoupled from the cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

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 ofcistotransisomers, consistent with the requirement of thecisconformer 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.

Summary: Lateral root (LR) positioning and development rely on the dynamic interplay between auxin production, transport but also inactivation. Nonetheless, how the latter affects LR organogenesis remains largely uninvestigated. Here, we systematically analyze the impact of the major auxin inactivation pathway defined by GRETCHEN HAGEN3‐type (GH3) auxin conjugating enzymes and DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) in all stages of LR development using reporters, genetics and inhibitors in Arabidopsis thaliana. Our data demonstrate that the gh3.1/2/3/4/5/6 hextuple (gh3hex) mutants display a higher LR density due to increased LR initiation and faster LR developmental progression, acting epistatically over dao1‐1. Grafting and local inhibitor applications reveal that root and shoot GH3 activities control LR formation. The faster LR development in gh3hex is associated with GH3 expression domains in and around developing LRs. The increase in LR initiation is associated with accelerated auxin response oscillations coinciding with increases in apical meristem size and LR cap cell death rates. Our research reveals how GH3‐mediated auxin inactivation attenuates LR development. Local GH3 expression in LR primordia attenuates development and emergence, whereas GH3 effects on pre‐initiation stages are indirect, by modulating meristem activities that in turn coordinate root growth with LR spacing.