The Arabidopsis PARKIN-like E3 ligase ARIADNE5 regulates plant development by targeting TCP4 for degradation

The Arabidopsis PARKIN-like E3 ligase ARIADNE5 regulates plant development by targeting TCP4 for degradation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Arabidopsis PARKIN-like E3 ligase ARIADNE5 regulates plant development by targeting TCP4 for degradation Genji Qin, Ning Wang, Yu Cao, Yongqi Wu, Jingqiu Lan, Yutao Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7027676/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Plant organ size and morphology are crucial agronomic traits that influence both plant fitness and crop productivity. The TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors play pivotal roles in shaping plant morphology, yet the precise regulatory mechanisms governing their activity remain incompletely understood. Here, we identify the PARKIN-like E3 ubiquitin ligase ARIADNE5 (ARI5) as a key regulator of TCP4 stability, mediating its ubiquitination and subsequent degradation. We identified ARI5 interacted with TCP4 using immunoprecipitation-mass spectrometry (IP-MS). The ari5 ari7 double mutant displays flatter leaves, shorter gynoecia, accelerated cotyledon opening in darkness, and earlier flowering. These phenotypes are strikingly opposite to those observed in tcp -deficient mutants. ARI5 and its close homolog ARI7 exhibit overlapping expression patterns with TCP4 , and their encoded proteins colocalize with TCP4 in the nucleus. ARI5 possesses E3 ubiquitin ligase activity, and promotes the ubiquitin-dependent degradation of TCP4. Our findings not only establish ARI5 as a critical regulator of plant organ morphology but also uncover a post-translational regulatory mechanism that fine-tunes TCP4 activity and thus cell division during organ morphogenesis through proteasomal degradation, highlighting the evolutionary conservation of PARKIN-like E3 ligases in modulating cell division across plants and humans. Biological sciences/Plant sciences/Plant development/Plant morphogenesis Biological sciences/Plant sciences/Plant molecular biology Biological sciences/Plant sciences/Plant development/Leaf development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Plant organ size and morphology represent agronomical traits critical for plant fitness and agricultural productivity. The final size and shape of plant organs are established through the precise spatiotemporal control of cellular processes, particularly the coordination of cell division and differentiation programs 1 . The TEOSINTE BRANCHED1/ CYCLOIDEA/PCF (TCP) family transcription factors plays crucial roles in regulating leaf, flower and other organ development by coordinating cell proliferation and differentiation 2 , 3 . The TCP family exhibits high evolutionary conservation among plant species. All TCP proteins possess a conserved TCP domain that facilitates dual functions in DNA binding and protein-protein interactions 4 , 5 . Phylogenetic analysis based on sequence variations divides TCP proteins into Class I and Class II 4 . Class II TCPs can be further categorized into CINCINNATA (CIN)-like TCPs and CYC/TB1-like TCPs. In Arabidopsis thaliana , the CIN-like TCP subgroup comprises eight members: TCP2, TCP3, TCP4, TCP5, TCP10, TCP13, TCP17, and TCP24 3,6 . Functionally, Class II TCPs typically regulate organ morphogenesis by suppressing cell proliferation while promoting cellular differentiation, whereas Class I TCPs generally exert opposing effects on these cellular processes 3 . For example, in Antirrhinum majus , the loss of TCP function in the cin mutant leads to leaf curvature and wavy margins with delayed cell differentiation 7 . Similarly, in Arabidopsis , mutations in multiple CIN -like TCP genes result in curled leaves due to prolonged cell division 8 – 10 . Beyond simple leaves, TCP transcription factors also regulate compound leaf development. In Solanum lycopersicum , overexpression of the CIN -like TCP homolog LACEOLATE ( LA ) accelerates cell differentiation, yielding simplified leaves, whereas the deficiency of LA extends the cell division phase, generating super-compound leaves 11 , 12 . Genetic perturbation of TCP activity alters final leaf morphology by promoting leaf cell differentiation in a threshold activity manner 8 , 12 . Beyond their well-characterized functions in leaf morphogenesis, CIN-like TCP transcription factors play multifaceted roles throughout plant development. During early seedling establishment, the higher-order tcp3/4/10 mutant displays delayed cotyledon opening during photomorphogenesis, whereas overexpression of TCP4 causes precocious cotyledon expansion in etiolated seedlings 13 . At the reproductive phase, CIN-like TCPs regulate flowering time by modulating key floral factors, including APETALA1 ( AP1 ), FRUITFULL ( FUL ), and LEAFY ( LFY ) 14 . After flowering, TCP4 exerts pleiotropic effects on floral organogenesis, influencing both petal size and pigmentation, and the ovule development 15 , 16 . Furthermore, TCPs govern apical gynoecium development and the ovule development through precise control of cell division, as evidenced by the elongated style phenotype observed in septuple tcp2/3/4/5/10/13/17 mutants and the excessive growth of ovule integuments in the duodecuple tcp2/3/4/5/10/13/17/24/1/12/18/16 mutant 16 , 17 . Collectively, these findings highlight that precise spatiotemporal control of TCP activity is crucial for balancing cell proliferation and differentiation, thereby shaping organ size and morphology. The activity of CIN-like TCP transcription factors is precisely regulated at transcriptional, post-transcriptional, and post-translational levels to ensure spatiotemporal control of their function, enabling plants to adapt to environmental conditions through developmental plasticity 6 . For example, at the transcriptional level, RABBIT EARS (RBE) directly suppresses TCP4 and TCP5 expression to regulate petal morphogenesis 18 . Post-transcriptionally, microRNA319 (miR319) targets TCP2 , TCP3 , TCP4 , TCP10 , and TCP24 , as revealed by the jagged and wavy-Dominant ( jaw-D ) mutant in which overexpression of miR319 causes the curled leaves similar to tcp multiple mutants 19 , 20 . At the protein level, ARMADILLO BTB ARABIDOPSIS PROTEIN1 (ABAP1) controls cell proliferation through interaction with TCP24 21 . The chromatin remodeler BRAHMA (BRM) associates with TCP4 to activate the expression of ARABIDOPSIS RESPONSE REGULATOR16 ( ARR16 ), thereby repressing cytokinin signaling 22 . During photomorphogenesis, TCP2 is stabilized by CRYPTOCHROME1 (CRY1) under blue light, but undergoes rapid degradation in darkness 23 . The phytoplasma effector SAP11 also destabilizes CIN-like TCPs, producing the characteristic jaw-D curly leaf phenotype 24 . The stability of TCP5 and TCP17 are elevated under high temperatures 25 , 26 . More recently, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)/SUPPRESSOR OF PHYA-105 (SPA1) E3 ubiquitin ligases has been reported to target TCP3 protein for degradation in darkness and in short day 27 . In rice, The F-box protein RCN127 facilitates ubiquitin-mediated degradation of OsTB1 and OsTCP19 to enhance rice tillering and grain yields 28 . However, the molecular mechanisms underlying the degradation of CIN-like TCP proteins remain largely unknown. The ubiquitin (Ub)-proteasome system (UPS) serves as a crucial protein degradation pathway that dynamically modulates the activity of transcriptional regulators, playing pivotal roles in hormone signaling, stress responses, and various plant development processes 29 – 32 . In plants, the UPS enables morphological plasticity and rapid physiological adaptation to environmental and internal cues through proteome remodeling 29 . The UPS consists of the 26S proteasome and three enzymatic components responsible for substrate ubiquitination: the Ub-activating enzyme (E1), Ub-conjugating enzymes (E2), and Ub ligases (E3). Ubiquitination initiates with ATP-dependent Ub activation by E1, forming a thioester bond. Ub is subsequently transferred to E2, while E3 ligases confer substrate specificity by catalyzing Ub transfer from E2-Ub to target proteins 29 . The Arabidopsis genome encodes over 1,400 E3 ligases, which can be categorized into three major classes based on their characteristic domains: REALLY INTERESTING NEW GENE (RING)/U-box, HOMOLOGOUS TO THE E6AP CARBOXYL TERMINUS (HECT), and RING-between-RING (RBR) 29 , 33 , 34 . The RBR family represents a unique class of E3 ligases distinguished by two RING domains (RING1 and RING2) separated by an in-between-RING (IBR) domain, are evolutionarily conserved across eukaryotes. For example, PARKIN is an extensively studied RBR-type E3 ligase associated with Parkinson's disease in humans 35 – 37 . Mutations in the PARKIN gene lead to juvenile parkinsonism 35 . In animals, the PARKIN-like E3 ligase ARIADNE1 (ARI1) exhibits ubiquitous expression and is essential for proper differentiation of multiple cell types in Drosophila melanogaster 38 . The Arabidopsis genome harbors sixteen PARKIN-like ARI proteins 39 . However, their enzymatic activities and substrates are still unknown. We previously demonstrated that TIE1 interacts with Class II TCP transcription factors to regulate leaf development and shoot branching 40 , 41 . We further identified TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1), a RING-type E3 ubiquitin ligase that targets TIE1 for degradation, thereby releasing TCP activity during leaf morphogenesis 42 . In this study, we reveal that the Arabidopsis PARKIN-like protein ARI5 physically interacts with TCP4 and exhibits E3 ubiquitin ligase activity. Genetic analysis showed that disruption of ARI5 and its homolog ARI7 resulted in several developmental phenotypes that were opposite to those observed in the jaw-5D mutant. We further demonstrate that TCP4 is an unstable protein whose degradation is mediated by ARI5. Our findings establish ARI5 as a key regulator of plant development and uncover a molecular mechanism by which plants precisely modulate developmental processes through the spatiotemporal ARI5-mediated control of CIN-like TCP protein stability. Results ARI5 is a PARKIN-like protein interacting with TCP4 To elucidate the regulatory mechanisms of TCPs, we generated a 35S-MYC-mTCP4 transgenic line, in which the MYC tag was fused with a microRNA319 (miR319)-resistant form of TCP4 ( mTCP4 ) under the control of the CaMV 35S promoter. Using inflorescence tissues from stable 35S-MYC-mTCP4 transgenic plants, we performed Immunoprecipitation-Mass Spectrometry (IP-MS) to identify potential TCP4 interactors. The identified proteins included previously reported TCP4-associated factors, such as TOPLESS/TOPLESS-RELATED proteins (TPL/TPRs), HISTONE DEACETYLASE 15 (HDA15), and other known interacting transcription factors such as TCP5 and KNAT3 (Supplementary Table 1) 5 , 40 , 43 – 46 , validating the reliability of the results. Additionally, novel interacting candidates including ARI5, 26S PROTEASOME REGULATORY SUBUNIT S2 1A (RPN1A), RPN1B, RPN2A and RPN2B were identified among the results (Supplementary Table 1). Given the reported instability of TCP proteins and the limited understanding of their degradation mechanisms, we focused on ARI5 due to its homology to the well-characterized human PARKIN E3 ligase (Fig. 1 a), whose function in plants remains unexplored. To characterize the ARI5 protein, we first conducted a comprehensive bioinformatic analysis. ARI5 encodes a 552-amino acid protein that features an N-terminal Leucine-rich1 domain, a canonical RBR domain comprising a C3HC4 RING1 region, a C5HC IBR region, and a shorter C3HC4 RING2 region, as well as a C-terminal Leucine-rich2 domain containing a nuclear localization signal (NLS) motif (Supplementary Fig. 1a, b). Protein sequence alignment and phylogenetic analysis revealed that ARI5 exhibits sequence and structure similarity to the human PARKIN protein within the RBR domain (Fig. 1 a, Supplementary Fig. 1c). The Arabidopsis genome encodes 16 ARI proteins, among which ARI5 clusters with ARI6 and ARI7 in subgroup B (Supplementary Fig. 2). ARI6 has been previously characterized as a pseudogene 39 , while the function of ARI5 and ARI7 remains uncharacterized. We then conducted a series of experimental assays to confirm the interaction between ARI5 and TCP4. First, yeast two-hybrid (Y2H) assays were performed by generating bait vectors with ARI5 or ARI7 fused to the DNA-binding domain (DBD) and prey vectors with each of the 11 Class II TCPs fused to the activation domain (AD). The results demonstrated that ARI5 interacted with TCP1, TCP3, TCP4, TCP10, TCP13, TCP17, and TCP18, while ARI7 exhibited interactions with all Class II TCPs (Fig. 1 b,c). Second, firefly luciferase complementation imaging (LCI) assays were carried out to further validate the interaction between ARI5 and TCP4. The construct 35S-cLUC-ARI5 was generated by fusing ARI5 to the sequence encoding the C-termimal part of luciferase (LUC) under the control of the CaMV 35S promoter, while 35S-mTCP4-nLUC was generated by fusing a miR319-resistant mTCP4 to the sequence encoding the N-terminal part of LUC, also under the control of the CaMV 35S promoter. The constructs 35S-cLUC-ARI5 and 35S-mTCP4-nLUC, along with control combinations, were co-expressed in distinct regions of one tobacco leaf. In three independent replicates, strong fluorescence signals were consistently detected in regions co-transformed with 35S-cLUC-ARI5 and 35S-mTCP4-nLUC, whereas no fluorescence was observed in regions expressing the control combinations (Fig. 1 d), indicating a specific interaction between ARI5 and TCP4 in planta . Third, co-immunoprecipitation (Co-IP) assays were performed using GFP-tagged ARI5 and Myc-tagged TCP4 transiently expressed in tobacco leaves. The results confirmed that TCP4 co-immunoprecipitated with ARI5 (Fig. 1 e), further supporting their interaction in planta . Disruption of ARI5 and ARI7 results in antagonistic phenotypes to tcp -deficient mutants To investigate the roles of ARI5 and ARI7 , we created an ari5 ari7 double mutant in which the function of ARI5 and ARI7 was knocked-out using CRISPR/Cas9 technology. Sequencing analysis indicated that the ari5 ari7 double mutant possessed a 2-bp frameshift deletion in the first exon of ARI5 and a large 858-bp deletion in the ARI7 coding sequence, both of which resulted in premature termination and expression of short truncated products (Supplementary Fig. 3). To generate single mutants, the ari5 ari7 double mutant was backcrossed to wild-type (WT) Arabidopsis, and subsequent progeny genotyping identified both ari5 and ari7 single mutants. The ari5 single mutant showed no discernible phenotypic differences compared to WT control. However, the ari7 single mutant exhibited significantly flatter leaves, and the ari5 ari7 double mutant displayed more severe flatter leaves than ari7 , indicating that ARI5 and ARI7 had functional redundancy (Fig. 2 a,c). This phenotype was opposite to curled leaves of tcp null mutantsand jaw-5D in which miR319b is overexpressed to reduce TCP2 , TCP3 , TCP4 , TCP10 , and TCP24 transcript levels (Fig. 2 a,c) 10 , 40 . To further elucidate the cellular basis of the observed phenotypes, we conducted a comparative analysis of abaxial epidermal cells in leaves from ari5 ari7 , jaw-5D , and WT controls. Intriguingly, while ari5 ari7 mutants produced smaller leaves (Fig. 2 a, Supplementary Fig. 4a,b), their epidermal cells exhibited a significant increase in size compared to that of WT (Fig. 2 b,d). Conversely, jaw-5D mutants displayed a reduction in epidermal cell dimensions relative to ari5 ari7 and WT controls (Fig. 2 b,d), in consistent with the previous reports that Class II TCPs usually inhibit cell proliferation and promote cell differentiation 3 . These results indicate that ARI5 and ARI7 enhance cell proliferation to drive leaf curvature, functioning antagonistically to TCP4, which suppresses cell proliferation to inhibit leaf curvature 3 . We then explored additional phenotypes related to TCP transcription factors. Firstly, our previous work has demonstrated that TCP4 acts as an antagonist of PIF3 and promotes light-induced cotyledon opening 13 . To investigate this phenotype, we examined cotyledon opening in 4-d-old seedlings of ari5 ari7 , 35S-MYC-mTCP4 overexpression lines, and WT plants grown in the dark. The results showed that cotyledons of WT seedlings remained largely closed under the dark condition (Fig. 2 e,h). In contrast, ari5 ari7 and 35S-MYC-mTCP4 seedlings exhibited significantly more opening cotyledons than WT seedlings after 4 d of dark growth (Fig. 2 f-h), supporting the previously identified roles of TCP4 as a positive regulator of cotyledon opening 13 . Secondly, CIN-like TCP transcription factors are known to promote flowering 14 . We assessed the flowering time of ari5 ari7 , jaw-5D , and WT control plants. Our results indicated that ari5 ari7 flowered significantly earlier than WT plants, whereas jaw-5D exhibited delayed flowering compared to WT (Fig. 2 i,j), consistent with previous reports about the positive regulation of flowering by CIN-like TCPs 14 . Lastly, we have previously reported that Class II TCPs inhibit the elongation of the style in the apical gynoecium 17 . We measured the style length in ari5 ari7 , jaw-5D , and WT plants. The results showed that ari5 ari7 produced significantly shorter and wider styles than WT, while jaw-5D exhibited longer and thinner styles than WT (Fig. 2 k-o). Scanning electron microscopy (SEM) analysis revealed that the cell number along the longitudinal axis of the style was reduced in ari5 ari7 mutants but increased in jaw-5D . Conversely, in the transverse direction, the cell number was elevated in ari5 ari7 but diminished in jaw-5D (Fig. 2 p-t). These findings further support the antagonistic relationship between ARI5/ARI7 and Class II TCPs. To verify whether the observed phenotypes in ari5 ari7 mutants were specifically caused by the disruption of ARIs, we generated a 35S-GFP-ARI5g construct, in which the genomic sequence of ARI5 was fused to GFP under the control of the CaMV 35S promoter. We transformed 35S-GFP-ARI5g into ari5 ari7 , and found that the expression of ARI5 in ari5 ari7 complemented the defective phenotypes including the smaller rosette size and impaired leaf flatness phenotypes (Fig. 2 u,v, Supplementary Fig. 4a,b), providing compelling evidence that ARI5 plays a critical role in regulating plant development. The observation of multiple antagonistic phenotypes between ari5 ari7 mutants and tcp -deficient mutants strongly indicates that ARI5 and ARI7 likely modulate plant development by negatively regulating the activity of TCP transcription factors. ARI5 and ARI7 exhibit overlapping expression patterns with TCP4 To provide more evidences supporting that ARI5 and ARI7 could inhibit the activity TCP4 through physical interaction, we first generated ARI5pro-GUS or ARI7pro-GUS constructs in which a 1077-bp ARI5 promoter or a 769-bp ARI7 promoter were used to drive the β-glucuronidase ( GUS ) reporter gene. Following transformation into Arabidopsis, histochemical GUS staining revealed consistent expression patterns in eighteen independent ARI5pro-GUS or in fifteen independent ARI7pro-GUS transgenic lines. We selected the stable ARI5pro-GUS-5 and ARI7pro-GUS-2 for detailed spatial and temporal expression analysis. The results revealed that both ARI5 and ARI7 were prominently expressed in the vasculature of all leaves in 8-d-old light- or dark-grown seedlings (Fig. 3 a,d,g,h,j-l). As leaf development progressed, a distinct spatiotemporal expression pattern emerged, with GUS staining gradually diminishing from distal to proximal regions and from medial to marginal regions in both ARI5pro-GUS and ARI7pro-GUS transgenic lines (Fig. 3 a-f). Both ARI5 and ARI7 were detected in the shoot apical meristem (SAM) (Fig. 3 a-f). Under dark-grown conditions, ARI7 exhibited strong expression in the apical hook and closed cotyledons (Fig. 3 j-l), whereas relatively weaker GUS staining was observed in these tissues in ARI5pro-GUS seedlings (Fig. 3 g-i). During reproductive development, both ARI5 and ARI7 had a ubiquitous expression in the pistils and pollen (Fig. 3 m,n). The spatial and temporal expression patterns of ARI5 and ARI7 significantly overlap with those of TCP genes, including TCP4 47 , and correlate well with the developmental phenotypes observed in ari5 ari7 double mutants, further supporting their functional relevance with TCP genes. ARI5 is co-localized with TCP4 in the nuclei To determine the subcellular localization of ARI5 and ARI7, we generated 35Spro-GFP-ARI5g and 35Spro-GFP-ARI7g constructs, utilizing the CaMV 35S promoter to drive GFP fusion with ARI5 or ARI7 genomic sequences. 35Spro-NLS-RFP was generated as a nuclear marker in which RFP fusion with a nuclear localization signal (NLS) sequence was driven by the CaMV 35S promoter. Initially, we transiently co-introduced the two constructs alongside 35Spro-NLS-RFP into tobacco leaves. The observation revealed a clear overlap of green and red fluorescence, indicating both ARI5 and ARI7 could be localized to the nuclei (Fig. 4 a-f). Subsequently, we established the stable Arabidopsis transgenic lines that expressed GFP-tagged ARI5 or ARI7. Confocal microscopy showed that the GFP fluorescence coincided with DAPI-stained nuclei, thereby corroborating the nuclear presence of ARI5 and ARI7 (Fig. 4 g-n). To further investigate the co-localization of ARI5 and TCP4, we generated 35S-RFP-mTCP4, in which miR319-resistant TCP4 was fused to RFP and driven by the CaMV 35S promoter. Transient expression of ARI5-GFP and TCP4-RFP in tobacco leaves revealed a significant co-localization of the two proteins within the nucleus (Fig. 3 o-q). These results provide the spatial evidence that supports the interaction between ARI5 and TCP4. ARI5 functions as an active E3 ubiquitin ligase ARI5 harbors a canonical RBR domain exhibiting conserved structural homology to the human PARKIN E3 ligase (Fig. 1 a, Supplementary Fig. 1), a well-established regulator of ubiquitin-dependent proteolysis in neurodegenerative pathways 36 . To investigate whether ARI5 could possess E3 ligase activity, we employed a bacterial ubiquitination reconstitution assay 48 , using Escherichia coli BL21 as an expression host. We co-expressed an Arabidopsis E1 (UBA1), an Arabidopsis E2 (UBC8), the MYC-tagged ARI5 and a His-FLAG-tagged Ub (ubiquitin protein) in BL21 cells. Immunoblot analysis indicated that MYC-ARI5, Ub, UBA1 and UBC8 were indeed expressed in the cells with anti-MYC, anti-FLAG and anti-S, respectively. Strikingly, reactions containing all the UBA1, UBC8, MYC-ARI5 and Ub exhibited distinct polyubiquitin laddering patterns, while the polyubiquitin ladder in control reactions lacking any single component (E1, E2, E3, or Ub) was completely absent. These data indicate that ARI5 is an active E3 ligase (Fig. 5 ). To determine the functional contribution of the two conserved RING motifs (RING1 and RING2) within the RBR domain, we generated domain-specific truncations including ARI5ΔR1-MYC with intact RING2, ARI5ΔR2-MYC with intact RING1, and ARI5ΔR12-MYC without both RING1 and RING2. Ubiquitination assays indicated that deletion of RING1 or RING2 significantly attenuated polyubiquitin chain formation, though residual mono-ubiquitination signals persisted (Fig. 5 ). ARI5-ΔR2 exhibited stronger mono-ubiquitination intensity than ARI5-ΔR1, suggesting that RING1 plays more important roles than RING2. Complete deletion of both RING domains (ARI5-ΔR12) abolished all ubiquitination activity, indicating the requirement of both RINGs for the full E3 activity of ARI5 (Fig. 5 ). ARI5 targets TCP4 for degradation through ubiquitination Based on the findings that ARI5 functions as a PARKIN-like E3 ligase and interacts with TCP4, we hypothesized that ARI5 mediates TCP4 ubiquitination to facilitate its proteasomal degradation. To test this hypothesis, we first investigated whether TCP4 undergoes ubiquitination in plants. We performed immunoprecipitation (IP) to enrich MYC-tagged TCP4 from 7-d-old light-grown seedlings, leaves or flowers of 35S-MYC-mTCP4 transgenic plants. Subsequent immunoblot analysis using anti-Ub or anti-MYC antibodies demonstrated that TCP4 is subject to ubiquitination modification (Fig. 6 a). To investigate whether ARI5 mediates TCP4 ubiquitination, we used bacterial ubiquitination reconstitution assay by co-expressing UBA1, UBC8, MYC-tagged ARI5, His-FLAG-tagged Ub, and MBP-HA-tagged TCP4 in E. coli cells. Immunoblot analysis using anti-MYC antibody confirmed the self-ubiquitination activity of ARI5, while anti-FLAG detection revealed characteristic ubiquitinated protein ladders (Fig. 6 b). Notably, anti-HA immunoblot analysis demonstrated the presence of polyubiquitin ladders only in reactions containing the complete ubiquitination system (UBA1, UBC8, ARI5, and Ub) with TCP4, while ubiquitination ladders were absent in the control reactions lacking any one of these components (Fig. 6 b). These results suggest that ARI5 mediates TCP4 ubiquitination. To further investigate the role of ARI5 in regulating TCP4 stability in planta , we generated a 35S-MYC-mTCP4 ari5 ari7 line through genetic crosses between a 35S-MYC-mTCP4 transgenic line and an ari5 ari7 mutant. To compare TCP4 degradation kinetics, we treated 35S-MYC-mTCP4 and 35S-MYC-mTCP4 ari5 ari7 plants with the protein synthesis inhibitor cycloheximide (CHX) to inhibit de novo protein synthesis. Quantitative analysis of three independent biological replicates revealed that TCP4 undergoes time-dependent degradation in both genetic backgrounds (Fig. 6 c, d). However, the degradation rate was significantly slower in 35S-MYC-mTCP4 ari5 ari7 compared to 35S-MYC-mTCP4, with statistically significant differences observed at both 12 h and 24 h time points ( P < 0.05) (Fig. 6 c, d). These results suggest that ARI5 promotes TCP4 protein degradation through ubiquitination. The genetic interactions between ARI and CIN -like TCP genes To further investigate the negative regulation of TCP4 stability by the PARKIN-like E3 ligase ARI5, we analyzed genetic interactions between ARI mutants and CIN -like TCP mutants. We generated an ari5 ari7 jaw-5D triple mutant by crossing the ari5 ari7 double mutant with jaw-5D . Phenotypic analysis revealed that the ari5 ari7 double mutant exhibited significantly flatter leaves compared to WT, while jaw-5D mutants showed more pronounced leaf curling, consistent with above observations (Fig. 2 a,c, Fig. 7 a,b, Supplementary Fig. 5a). The introduction of the jaw-5D mutation into the ari5 ari7 background resulted in a phenotypic shift from flat to curled leaves (Fig. 7 a,b, Supplementary Fig. 5a). Specifically, the leaf curvature and wavy margin phenotypes observed in the jaw-5D ari5 ari7 triple mutant were comparable to those of the jaw-5D single mutant (Fig. 7 b, Supplementary Fig. 5a), indicating that jaw-5D is epistatic to ari5 ari7 . Furthermore, additional phenotypic traits of ari5 ari7 , such as early cotyledon opening in etiolated seedlings (Fig. 7 c-f, Supplementary Fig. 5b), early flowering (Fig. 7 k, Supplementary Fig. 5c), and shorter gynoecium styles (Fig. 7 g-j, Supplementary Fig. 5d,e), were also suppressed by the jaw-5D mutation. These genetic interactions strongly suggested that the phenotypes of ari5 ari7 are dependent on the presence of functional CIN -like TCPs , in consistent with the above data that ARI5 promotes the degradation of TCP4. We then generated overexpression lines by transforming the 35S-GFP-ARI5g and 35S-GFP-ARI7g constructs into WT Arabidopsis. Phenotypic analysis revealed that 15 out of 127 35S-GFP-ARI5g independent transgenic lines and 27 out of 144 35S-GFP-ARI7g ones exhibited curly leaves. From these lines, we selected stable overexpression lines, namely 35S-GFP-ARI5g-2 and 35S-GFP-ARI5g-8 for ARI5 , and 35S-GFP-ARI7g-4 and 35S-GFP-ARI7g-8 for ARI7 , as representative lines for further analysis. Reverse transcription quantitative PCR (RT-qPCR) confirmed the successful overexpression of ARI5 and ARI7 in these transgenic lines (Supplementary Fig. 5f,g). Quantitative measurements of leaf curvature demonstrated that the 35S-GFP-ARI5g-2, 35S-GFP-ARI5g-8, 35S-GFP-ARI7g-4, and 35S-GFP-ARI7g-8 lines exhibited significantly more severe leaf curvature compared to WT (Fig. 7 l,m, Supplementary Fig. 5h-k). This phenotype contrasts with the flatter leaf morphology observed in ari5 ari7 , providing further evidence that ARI5 and ARI7 play critical roles in regulating leaf morphogenesis. To determine whether increased TCP4 levels could rescue the leaf curvature phenotype observed in ARI overexpression lines, we crossed the TCP4 overexpression line 35S-MYC-mTCP4 with the ARI overexpression line 35S-GFP-ARI5g-8. Introduction of 35S-MYC-mTCP4 significantly suppressed the leaf curvature phenotype of 35S-GFP-ARI5g-8, supporting the hypothesis that the excessive degradation of TCPs by the overexpression ARI5 leads to more curled leaves (Fig. 7 n). We next compared the abundance of TCP4 protein in 35S-MYC-mTCP4, 35S-MYC-mTCP4 35S-GFP-ARI5g-8, and control lines. The results revealed that ARI5 overexpression significantly reduced TCP4 protein levels in the 35S-MYC-mTCP4 background (Fig. 7 o). We further used the 35S-MYC-mTCP4 35S-GFP-ARI5g-8 line to perform co-immunoprecipitation (Co-IP) assays. MYC-tagged TCP4 successfully co-immunoprecipitated with GFP-tagged ARI5 (Fig. 7 p), again confirming a physical interaction between the two proteins in vivo . Immunoblot analysis using an anti-Ub antibody demonstrated that ubiquitinated TCP4 was more abundant in the 35S-MYC-mTCP4 35S-GFP-ARI5g-8 line compared to 35S-MYC-mTCP4 (Fig. 7 p). These findings strongly suggest that ARI5 regulates plant development by precisely modulating the abundance of TCP4 through ubiquitin-mediated degradation. Discussion In this study, we demonstrate that the PARKIN-like E3 ligases ARI5 and its homolog ARI7 mediate the degradation of CIN-like TCP proteins, thereby regulating diverse plant developmental processes. Disruption of ARIs leads to phenotypes that are antagonistic to those observed in tcp -deficient mutants, including flatter leaves, enhanced cotyledon opening in darkness, earlier flowering, and shorter gynoecium styles in the ari5 ari7 double mutant. We show that ARI5 is developmentally regulated and exhibits spatiotemporal expression patterns overlapping with TCP4 . Biochemical characterization reveals that ARI5 possesses E3 ligase activity and physically interacts with TCP4. We further show that TCP4 protein is regulated by degradation, and ARI5 mediates its ubiquitination and degradation. Based on these findings, we propose a mechanistic model for ARI5 function. ARI5 functions as an active PARKIN-like E3 ligase. ARI5 controls the stability and degradation of TCP4, and the spatially restricted TCP4 accumulation governs cell proliferation and differentiation, thereby determining organ morphology (Fig. 8 ). Our data demonstrate that ARI5 acts as an important organ developmental regulator that shapes organs by spatiotemporally targeting TCP4 for degradation. As central integrators of environmental cues and endogenous signals, TCP transcription factors precisely orchestrate cell division and differentiation to regulate plant developmental plasticity 3 . Among external stimuli, light and temperature emerge as pivotal regulators of TCP activity 6 . Photoperiodic conditions directly influence both TCP protein stability and DNA-binding affinity. For examples, TCP17 undergoes 26S proteasome-mediated degradation under light conditions but accumulates under shade despite transcriptional downregulation 26 . Similarly, TCP2 forms complexes with CRYs under blue light to stabilize the protein and promote photomorphogenesis, whereas it is rapidly degraded in darkness 23 . Temperature fluctuations also exert significant regulatory effects, with elevated temperatures inducing the accumulation of TCP5, TCP13, and TCP17 25 . Beyond environmental modulation, intrinsic regulatory networks govern TCP activity through multiple mechanisms, including microRNA-mediated suppression, hormonal signaling cascades, and controlled protein turnover 6 . The miR319 family directly cleaves TCP transcripts, and its ectopic expression leads to leaf curling and delayed flowering by the coordinated suppression of multiple TCP genes 19 , 20 . Phytohormones including auxin, gibberellins, and brassinosteroids fine-tune TCP activity to maintain the balance between growth and differentiation 6 . Additionally, diverse proteolytic pathways converge to regulate TCP protein stability. For instance, the F-box protein KISS ME DEADLY (KMD) targets TCP14 for degradation to modulate cytokinin responses, a pathway exploited by pathogens to manipulate host development 49 . A striking illustration of pathogen-mediated TCP subversion involves the phytoplasma effector SAP11 from the Aster Yellows Witches' Broom (AY-WB) strain. Upon secretion into host cells, SAP11 induces the degradation of multiple TCPs, including TCP4, resulting in leaf curling and developmental defects phenocopying tcp mutants 24 . Intriguingly, SAP bypass canonical ubiquitination by directly binding their substrates and recruiting the 26S proteasome subunit RPN, thereby circumventing the need for E3 ligase-mediated ubiquitination 50 . Our prior research revealed the complexity of ubiquitin-dependent TCP regulation, demonstrating that TEAR1 indirectly modulates TCP activity by ubiquitinating their interactor TIE1, which recruits TPL/TPR corepressors to suppress TCP target gene expression 40 . In this study, we identify the PARKIN-like E3 ligase ARI5 as a direct regulator of TCP4 through ubiquitin-mediated degradation. As an RBR-type E3 ligase, ARI5 ubiquitinates TCP4 to promote its proteasomal turnover. Unlike TEAR1, which enhances TCP activity by degrading the TCP repressor TIE1, ARI5 directly suppresses TCP4 activity. Given the observed environmental modulation of TCP degradation, we propose that ARI5 and its homologs may not only regulate TCP stability in response to developmental cues but also fine-tune TCP activity under some environmental conditions, thereby shaping organ growth and morphogenesis in adapting to environmental fluctuations. The PARKIN-like E3 ligase ARI family exhibits remarkable evolutionary conservation across both plants and other organisms 39 . However, very few ARIs in plants has been characterized. The medicinal plant Hypericum perforatum (St. John's wort) employs an intriguing reproductive strategy called apospory, where gametophytes develop directly from sporophytic somatic cells without undergoing meiosis 51 . This mechanism enables heterozygosity fixation in crops. Genetic studies have identified HpARI7 as a candidate gene associated with the apospory locus, yet its precise molecular function and physiological substrates remain to be elucidated 51 . Recently, OsRBRL1 , a rice ARI homolog, functions as a viral sensor that recognizes coat proteins of Rice stripe virus (RSV) and Rice dwarf virus (RDV). During viral infection, OsRBRL1 is transcriptionally upregulated and mediates the ubiquitination and subsequent degradation of NOVEL INTERACTOR OF JAZ 3 (NINJA3), a negative regulator of the jasmonic acid (JA) signaling pathway, thereby activating plant antiviral defenses 52 . In this study, we demonstrate that Arabidopsis ARI5 and ARI7 regulate multiple developmental processes. We establish ARI5 as an active E3 ubiquitin ligase that targets the key developmental regulator TCP4 for degradation, thereby precisely controlling organ morphogenesis. These findings suggest that plant ARI proteins act as crucial modulators that may orchestrate diverse biological processes in plants through the degradation of selective proteins. The evolutionary conservation and functional diversification of PARKIN-like E3 ligases extends to functional parallels between plant ARI5 and its human counterpart. While human PARKIN is best characterized for its cytoplasmic role in mitochondrial quality control through mitophagy, emerging evidence reveals its involvement in cell cycle regulation 53 , 54 . Notably, PARKIN mediates the ubiquitination and degradation of Cyclin E, a key regulator of G1/S phase transition, thereby controlling neuronal progenitor cell division during brain development 54 . Intriguingly, our findings demonstrate that plant ARIs regulate TCP transcription factors, which directly control the expression of cell cycle regulators such as CYCLIND3b and CYCB1;1 during leaf development 7 , 55 . This functional convergence where PARKIN-like proteins in different organisms modulate cell proliferation through distinct cyclin-related pathways highlights their conserved role in growth regulation across eukaryotes. Our study reveals that ARI5 mediates nuclear degradation of TCP4, despite its dual localization in both cytoplasmic and nuclear compartments. This observation suggests that, similar to PARKIN, ARI5 might have additional cytoplasmic substrates. In human, the cytoplasmic functions of PARKIN are well-established, including mitochondrial clearance and proteasomal regulation 36 . Future studies should investigate whether ARI5 regulates cytoplasmic targets analogous to the mitochondrial substrates of PARKIN. Conversely, our discovery that plant ARIs target transcription factors raises the intriguing possibility that PARKIN might similarly regulate transcriptional regulators involved in neurodevelopment under specific conditions. Our work establishes ARI5 as an active E3 ligase that shapes plant development through targeted degradation of TCP4, a master regulator of developmental plasticity. This regulatory mechanism enables plants to flexibly adjust developmental programs, thereby shaping organ morphology to adapt to fluctuating environmental conditions. Given the evolutionary conservation of PARKIN-like E3 ligases, and the plant-specific conservation of TCP proteins, our findings may offer molecular breeding strategies for crop improvement through the manipulation of ARI-TCP regulatory modules to optimize developmental and stress-responsive traits, and also provide insights into PARKIN function relevant to Parkinson's disease research. Methods Plant materials and growth conditions The Arabidopsis Columbia-0 (Col-0) ecotype type was used. Seeds of WT, ari5 , ari7 , ari5 ari7 , jaw-5D and other plant materials or mutants were surface-sterilized with 75% ethanol for 10 min, rinsed twice with 100% ethanol, and air-dried in a clean bench. After cold stratification at 4°C for 2 d, the dried seeds were germinated on half-strength Murashige and Skoog (MS) medium at 22°C under long-day conditions (16-h light and 8-h dark). Seedlings of Arabidopsis thaliana or Nicotiana benthamiana were grown in soil in a greenhouse under the same conditions. Generation of binary constructs and transformation The genomic sequences of ARI5 and ARI7 were amplified and cloned into pENTR/D-TOPO (Invitrogen) to generate pENTR-ARI5g and pENTR-ARI7g. The entry vectors were recombined with pK7WGF2 (Invitrogen) via LR reaction to generate 35S-GFP-ARI5g and 35S-GFP-ARI7g constructs. Similarly, promoter regions (1077-bp ARI5pro and 769-bp ARI7pro) were cloned into pENTR/D-TOPO, then recombined with pB7GUSWG0 (Invitrogen) to generate ARI5pro-GUS and ARI7pro-GUS. All constructs were transformed into Agrobacterium tumefaciens GV3101 for Arabidopsis transformation by floral dip 56 . Yeast two-hybrid assays To test the interaction between ARI5/7 and TCPs, we used previously described AD-TCP2/3/4/5/10/13/17/18/24 prey constructs 57 , and generated AD-TCP1/17 by LR recombination of pENTR-TCP1/17 with pDEST22 (Invitrogen). For bait constructs, we cloned 1659-bp ARI5 and 1990-bp ARI7 cDNA into pENTR/D-TOPO, then recombined them with pDEST32 (Invitrogen) to create DBD-ARI5/7. Bait and prey constructs (or pDEST22 control) were co-transformed into yeast strain AH109. The yeasts were grown in a 28°C incubator for 72 h. Selection was performed using SD-Leu-Trp-His medium (Coolaber). Firefly luciferase complementation assays For luciferase complementation assays, mTCP4 and ARI5 were cloned into pCambia1300-nLUC and pCambia1300-cLUC via LR recombination, respectively. Agrobacterium strains carrying these constructs were co-infiltrated into tobacco leaves. The tobacco plants were grown under a weak light for 12 h and were transferred to long-day conditions for 32 h. The infiltrated leaves were harvested, injected with 200 µM luciferin, and dark-adapted for 10 min. Luminescence was captured using a NightOWL II LB983 CCD system (180 sec exposure, 10% intensity, high gain mode) with indiGO software. Immunoprecipitation and Western blot Co-IP assays were performed first in tobacco leaves through transient co-expression of 35S-mTCP4-MYC with either 35S-GFP-ARI5 or 35S-GFP, followed by validation in 7-d-old Arabidopsis seedlings expressing 35S-mTCP4-MYC alone or in combination with 35S-GFP-ARI5. For TCP4 immunoprecipitation, tissues at different developmental stages (7-d seedlings, 21-d leaves, and 21-d flowers) were harvested from 35Spro-MYC-mTCP4 transgenic lines. Total proteins were extracted using buffer (0.1 M HEPES at pH 7.5, 5 mM EGTA, 5 mM EDTA, 5 mM NaF, 50 mM phosphoglycerol, 1% Triton X-100, 10% glycerin, and protease inhibitor cocktail, PMSF) and incubated with anti-GFP (MBL, D153-10) or anti-MYC (MBL, M047-11) beads for 3 h. After five PBS washes, bound proteins were eluted by boiling in 5×SDS buffer (7 min, 100°C), separated by 10% SDS-PAGE, and transferred to PVDF membranes (Millipore). Immunodetection used anti-MYC (ABclonal, AE070), anti-GFP (Sangon, D110008), and anti-Ub (Abcam, ab7254) antibodies, with visualization on a Tanon 5200 Multi system. CRISPR/Cas9-induced mutants The ari5 ari7 double mutant was generated using a UBQ10 promoter-driven CRISPR/Cas9 system 58 . We cloned the sgRNA construct pAtU6-ARI5target1-ARI5target2-ARI7target1-ARI7target2 into pUBQ10-Cas9-P2A-GFP-rbcS-E9t ( Kpn I/ Spe I sites) and transformed it into WT plants. Target locus mutations were verified by sequencing PCR products. The Cas9-free double mutant ari5 ari7 was obtained by genetic segregation. The primers are listed in Supplementary Table 2. Staining and microscopy ARI5pro-GUS and ARI7pro-GUS transgenic lines were fixed in 90% acetone (20 min, ice), then washed with phosphate buffer (0.34 M Na₂HPO₄·12H₂O, 0.01 M NaH₂PO₄·2H₂O, 0.4 mM K₄[Fe(CN)₆]·3H₂O, 0.5 mM K₃[Fe(CN)₆]) and stained overnight at 37°C in GUS solution (0.5 mg/mL X-Gluc). After destaining in 75% ethanol, patterns were documented using a Leica M205 FCA stereoscope. The 35S-GFP-ARI5g or 35S-GFP-ARI7g and 35S-NLS-RFP or 35S-RFP-mTCP4 constructs were used to express fusion proteins in tobacco leaves. Infiltrated leaves were incubated in a growth chamber for 48 h before observing using a Zeiss Axio Imager M2 microscope. For transgenic Arabidopsis, hypocotyls were DAPI-stained (1 mg/mL, 10 min) to confirm nuclear localization of GFP-ARI5/7. In vitro ubiquitination assay The constructs pACYCDuet-ARI5-MYC, pACYCDuet-ARI5-MYC-AtUBC8-S, pACYCDuet-ARI5(ΔR1)-MYC-AtUBC8-S, pACYCDuet-ARI5(ΔR2)-MYC-AtUBC8-S, pACYCDuet-ARI5(ΔR12)-MYC-AtUBC8-S, pCDFDuet-MBP-TCP4-HA and pCDFDuet-MBP-TCP4-HA-AtUBA1-S were generated by Gibson assembly (SC612, Genesand) 48 . Escherichia coli strain BL21 (DE3) containing different combinations of the expression vectors were cultured in LB medium at 37°C until OD600 reached 0.5, at which point protein expression was induced with 0.5 mM IPTG followed by overnight incubation at 18°C. For the ARI5 self-ubiquitination assay, 300 µL of bacterial culture was directly lysed in 100 µL of 1×SDS loading buffer. The TCP4 ubiquitination assay required larger-scale processing, where 20 mL of culture was pelleted, resuspended in 10 mL PBS, and subjected to sonication. After centrifugation, the insoluble fraction was solubilized in 500 µL of 1×SDS buffer. All samples were separated by SDS-PAGE and analyzed by Western blotting using anti-MYC (ABclonal, AE009), anti-HA (ABclonal, AE008), anti-FLAG (Sigma, A8592), and anti-S (HUABIO, EM50105) antibodies. In vivo degradation assay To assess TCP4 protein stability, 14-d-old seedlings from 35S-MYC-mTCP4 and 35S-MYC-mTCP4 ari5 ari7 transgenic plants were treated with 100 µM cycloheximide (CHX) in ½ MS solution for a time-course experiment. After treatment, seedlings were flash-frozen in liquid nitrogen and ground to a fine powder. Total proteins were extracted in extraction buffer for 1 h, followed by centrifugation (2 min, 12000× g ). Protein samples were analyzed by western blot using anti-MYC (AE009, ABclonal) and anti-Actin (AC009, ABclonal) antibodies, with ACTIN serving as the loading control. MYC-mTCP4 protein levels were quantified relative to ACTIN using ImageJ software. Analysis of leaf curvature, hook curvature, style lenghth and width For the leaf curvature analysis, a 1 mm transverse section of the sixth leaves were excised at the leaf widest point perpendicular to the proximal axis. The leaf segments were imaged, with the α angle quantified using ImageJ. For hook curvature assessment in etiolated seedlings, plates were foil-wrapped and incubated vertically at 22°C for 4 d before imaging. Style morphology was examined by emasculating flowers at flower developmental stage 11, with apical gynoecium observed under a Leica M205 FCA stereoscope at 48 h post-emasculation. All morphometric parameters (leaf curvature angle, hook curvature, and style dimensions) were measured using ImageJ, with statistical significance determined by one-way ANOVA. Microscopy analysis For scanning electron microscope (SEM) observation of the style, the flowers of WT, ari5 ari7 and jaw-5D were emasculated at flower developmental stage 11. The experiments utilized an integrated system consisting of an FEI Quanta FEG 450 scanning electron microscope (Thermo Fisher Scientific) and a Quorum PP3010T workstation (Quorum Technologies), which features a cryo-preparation chamber directly connected to the microscope. Plant samples were frozen in subcooled liquid nitrogen (− 210°C) and then transferred under vacuum to the cold stage of the chamber for sublimation (− 90°C, 5 min) and platinum sputter coating (10 mA, 30 s). Imaging was conducted using an electron beam at 5 kV and 3.5 nA with a working distance of 9.9 mm. For leaf cell morphology analysis, leaves from 21-d-old plants were fixed with ethanol : acetic acid : H 2 O (8.4 mL : 2 mL : 3.6 mL) for 16 h. Samples underwent sequential dehydration in 100% ethanol for 30 min twice and 70% ethanol for 30 min, followed by clearing in chloral hydrate solution (Leagene, DM0350) for 12 h. Cleared samples were imaged using DIC optics on a Zeiss Axio Imager M2 microscope. RNA extraction and quantitative real-time polymerase chain reaction assay Total RNA was isolated from 7-d-old 35S-GFP-ARI5g, 35S-GFP-ARI7g, and wild-type seedlings using the Plant RNA Extraction Kit (TaKaRa, 9769). For cDNA synthesis, 1.5 µg of total RNA was reverse transcribed using M-MLV Reverse Transcriptase (Promega, M170A). Quantitative PCR was performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific) with UltraSYBR Mixture (CWBIO, CW2601M). Gene expression levels were normalized to ACTIN2 and calculated using the 2 −ΔΔCT method. All primer sequences are provided in Supplemental Table 2. Declarations Material availability All materials needed to replicate the work are available. Reporting Summary Further methodological details are available in the linked Nature Portfolio Reporting Summary. Data availability The sequences of ARI5 (At1G05890), ARI6 (AT1G63760), ARI7 (At2G31510), TCP1 (AT1G67260), TCP2 (AT4G18390), TCP3 (AT1G53230), TCP4 (AT3G15030), TCP5 (AT5G60970), TCP10 (AT2G31070), TCP12 (AT1G68800), TCP13 (AT3G02150), TCP17 (AT5G08070), TCP18 (AT3G18550), TCP24 (AT1G30210) are available at TAIR (https:// www.arabidopsis.org/). Acknowledgments We thank Dongping Lu (Shanghai Jiao Tong University) for kindly providing the plasmids used in the in vitro ubiquitination assay; Jun Hu, Zhen-Yang Kong, and Ying-Chun Hu for assistance in SEM experiments at the Core Facilities of School of Life Science, Peking University; and Dong Liu and Qi Zhang for assistance in MS experiments at the National Center for Protein Sciences, Peking University. This work was supported by the National Natural Science Foundation of China (Grants No. 32370355 and 323B2008) and the Excellent Research Group Project of the National Natural Science Foundation of China (Grant No. 32488102). Author contributions G.Q. conceived and designed the project. N.W conducted the experiments. G.Q., N.W., Y.C., Y.Wu, J.L. and Y.Wang analyzed the data. G.Q. and N.W. wrote the manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at . 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legend\u003c/p\u003e","description":"","filename":"Figure111.png","url":"https://assets-eu.researchsquare.com/files/rs-7027676/v1/4fe3ad50e011f0ca40c1495f.png"},{"id":87184581,"identity":"0a1987b7-845f-414f-a06c-75719fed6270","added_by":"auto","created_at":"2025-07-21 10:12:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1722732,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure112.png","url":"https://assets-eu.researchsquare.com/files/rs-7027676/v1/430eec3e6cb985650dd06e23.png"},{"id":87183248,"identity":"36416cba-4da2-43d2-a8a1-5c29ad9dc3aa","added_by":"auto","created_at":"2025-07-21 10:04:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1281603,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure 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legend\u003c/p\u003e","description":"","filename":"Figure118.png","url":"https://assets-eu.researchsquare.com/files/rs-7027676/v1/5c3a61470193265296244907.png"},{"id":87184579,"identity":"d3552835-acd5-47d1-a5da-73b48ff802f7","added_by":"auto","created_at":"2025-07-21 10:12:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":189423,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1110.png","url":"https://assets-eu.researchsquare.com/files/rs-7027676/v1/4b883ed147008fcb39c572f9.png"},{"id":87186525,"identity":"c5862600-1481-45f3-b291-5db42b31ab2a","added_by":"auto","created_at":"2025-07-21 10:28:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7739914,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7027676/v1/6f6dddf0-d96d-404b-a415-2c72e8696252.pdf"},{"id":87183247,"identity":"24022e81-029a-41ae-92f6-74b9f1139555","added_by":"auto","created_at":"2025-07-21 10:04:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3005673,"visible":true,"origin":"","legend":"Supplemetary Figures and Tables","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7027676/v1/6adfc2b57dfe9400d7682c02.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The Arabidopsis PARKIN-like E3 ligase ARIADNE5 regulates plant development by targeting TCP4 for degradation","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant organ size and morphology represent agronomical traits critical for plant fitness and agricultural productivity. The final size and shape of plant organs are established through the precise spatiotemporal control of cellular processes, particularly the coordination of cell division and differentiation programs\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The TEOSINTE BRANCHED1/ CYCLOIDEA/PCF (TCP) family transcription factors plays crucial roles in regulating leaf, flower and other organ development by coordinating cell proliferation and differentiation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe TCP family exhibits high evolutionary conservation among plant species. All TCP proteins possess a conserved TCP domain that facilitates dual functions in DNA binding and protein-protein interactions\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Phylogenetic analysis based on sequence variations divides TCP proteins into Class I and Class II\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Class II TCPs can be further categorized into CINCINNATA (CIN)-like TCPs and CYC/TB1-like TCPs. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the CIN-like TCP subgroup comprises eight members: TCP2, TCP3, TCP4, TCP5, TCP10, TCP13, TCP17, and TCP24\u003csup\u003e3,6\u003c/sup\u003e. Functionally, Class II TCPs typically regulate organ morphogenesis by suppressing cell proliferation while promoting cellular differentiation, whereas Class I TCPs generally exert opposing effects on these cellular processes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For example, in \u003cem\u003eAntirrhinum majus\u003c/em\u003e, the loss of TCP function in the \u003cem\u003ecin\u003c/em\u003e mutant leads to leaf curvature and wavy margins with delayed cell differentiation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Similarly, in \u003cem\u003eArabidopsis\u003c/em\u003e, mutations in multiple \u003cem\u003eCIN\u003c/em\u003e-like \u003cem\u003eTCP\u003c/em\u003e genes result in curled leaves due to prolonged cell division\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Beyond simple leaves, TCP transcription factors also regulate compound leaf development. In \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, overexpression of the \u003cem\u003eCIN\u003c/em\u003e-like \u003cem\u003eTCP\u003c/em\u003e homolog \u003cem\u003eLACEOLATE\u003c/em\u003e (\u003cem\u003eLA\u003c/em\u003e) accelerates cell differentiation, yielding simplified leaves, whereas the deficiency of \u003cem\u003eLA\u003c/em\u003e extends the cell division phase, generating super-compound leaves\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Genetic perturbation of TCP activity alters final leaf morphology by promoting leaf cell differentiation in a threshold activity manner\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Beyond their well-characterized functions in leaf morphogenesis, CIN-like TCP transcription factors play multifaceted roles throughout plant development. During early seedling establishment, the higher-order \u003cem\u003etcp3/4/10\u003c/em\u003e mutant displays delayed cotyledon opening during photomorphogenesis, whereas overexpression of \u003cem\u003eTCP4\u003c/em\u003e causes precocious cotyledon expansion in etiolated seedlings\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. At the reproductive phase, CIN-like TCPs regulate flowering time by modulating key floral factors, including \u003cem\u003eAPETALA1\u003c/em\u003e (\u003cem\u003eAP1\u003c/em\u003e), \u003cem\u003eFRUITFULL\u003c/em\u003e (\u003cem\u003eFUL\u003c/em\u003e), and \u003cem\u003eLEAFY\u003c/em\u003e (\u003cem\u003eLFY\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. After flowering, TCP4 exerts pleiotropic effects on floral organogenesis, influencing both petal size and pigmentation, and the ovule development\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, TCPs govern apical gynoecium development and the ovule development through precise control of cell division, as evidenced by the elongated style phenotype observed in septuple \u003cem\u003etcp2/3/4/5/10/13/17\u003c/em\u003e mutants and the excessive growth of ovule integuments in the duodecuple \u003cem\u003etcp2/3/4/5/10/13/17/24/1/12/18/16\u003c/em\u003e mutant\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Collectively, these findings highlight that precise spatiotemporal control of TCP activity is crucial for balancing cell proliferation and differentiation, thereby shaping organ size and morphology.\u003c/p\u003e \u003cp\u003eThe activity of CIN-like TCP transcription factors is precisely regulated at transcriptional, post-transcriptional, and post-translational levels to ensure spatiotemporal control of their function, enabling plants to adapt to environmental conditions through developmental plasticity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. For example, at the transcriptional level, RABBIT EARS (RBE) directly suppresses \u003cem\u003eTCP4\u003c/em\u003e and \u003cem\u003eTCP5\u003c/em\u003e expression to regulate petal morphogenesis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Post-transcriptionally, microRNA319 (miR319) targets \u003cem\u003eTCP2\u003c/em\u003e, \u003cem\u003eTCP3\u003c/em\u003e, \u003cem\u003eTCP4\u003c/em\u003e, \u003cem\u003eTCP10\u003c/em\u003e, and \u003cem\u003eTCP24\u003c/em\u003e, as revealed by the \u003cem\u003ejagged and wavy-Dominant\u003c/em\u003e (\u003cem\u003ejaw-D\u003c/em\u003e) mutant in which overexpression of \u003cem\u003emiR319\u003c/em\u003e causes the curled leaves similar to \u003cem\u003etcp\u003c/em\u003e multiple mutants\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. At the protein level, ARMADILLO BTB ARABIDOPSIS PROTEIN1 (ABAP1) controls cell proliferation through interaction with TCP24\u003csup\u003e21\u003c/sup\u003e. The chromatin remodeler BRAHMA (BRM) associates with TCP4 to activate the expression of \u003cem\u003eARABIDOPSIS RESPONSE REGULATOR16\u003c/em\u003e (\u003cem\u003eARR16\u003c/em\u003e), thereby repressing cytokinin signaling\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. During photomorphogenesis, TCP2 is stabilized by CRYPTOCHROME1 (CRY1) under blue light, but undergoes rapid degradation in darkness\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The phytoplasma effector SAP11 also destabilizes CIN-like TCPs, producing the characteristic \u003cem\u003ejaw-D\u003c/em\u003e curly leaf phenotype\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The stability of TCP5 and TCP17 are elevated under high temperatures \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. More recently, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)/SUPPRESSOR OF PHYA-105 (SPA1) E3 ubiquitin ligases has been reported to target TCP3 protein for degradation in darkness and in short day\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In rice, The F-box protein RCN127 facilitates ubiquitin-mediated degradation of OsTB1 and OsTCP19 to enhance rice tillering and grain yields\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanisms underlying the degradation of CIN-like TCP proteins remain largely unknown.\u003c/p\u003e \u003cp\u003eThe ubiquitin (Ub)-proteasome system (UPS) serves as a crucial protein degradation pathway that dynamically modulates the activity of transcriptional regulators, playing pivotal roles in hormone signaling, stress responses, and various plant development processes\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In plants, the UPS enables morphological plasticity and rapid physiological adaptation to environmental and internal cues through proteome remodeling\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The UPS consists of the 26S proteasome and three enzymatic components responsible for substrate ubiquitination: the Ub-activating enzyme (E1), Ub-conjugating enzymes (E2), and Ub ligases (E3). Ubiquitination initiates with ATP-dependent Ub activation by E1, forming a thioester bond. Ub is subsequently transferred to E2, while E3 ligases confer substrate specificity by catalyzing Ub transfer from E2-Ub to target proteins\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The Arabidopsis genome encodes over 1,400 E3 ligases, which can be categorized into three major classes based on their characteristic domains: REALLY INTERESTING NEW GENE (RING)/U-box, HOMOLOGOUS TO THE E6AP CARBOXYL TERMINUS (HECT), and RING-between-RING (RBR)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The RBR family represents a unique class of E3 ligases distinguished by two RING domains (RING1 and RING2) separated by an in-between-RING (IBR) domain, are evolutionarily conserved across eukaryotes. For example, PARKIN is an extensively studied RBR-type E3 ligase associated with Parkinson's disease in humans\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Mutations in the \u003cem\u003ePARKIN\u003c/em\u003e gene lead to juvenile parkinsonism\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In animals, the PARKIN-like E3 ligase ARIADNE1 (ARI1) exhibits ubiquitous expression and is essential for proper differentiation of multiple cell types in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The Arabidopsis genome harbors sixteen PARKIN-like ARI proteins\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, their enzymatic activities and substrates are still unknown.\u003c/p\u003e \u003cp\u003eWe previously demonstrated that TIE1 interacts with Class II TCP transcription factors to regulate leaf development and shoot branching\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. We further identified TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1), a RING-type E3 ubiquitin ligase that targets TIE1 for degradation, thereby releasing TCP activity during leaf morphogenesis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In this study, we reveal that the Arabidopsis PARKIN-like protein ARI5 physically interacts with TCP4 and exhibits E3 ubiquitin ligase activity. Genetic analysis showed that disruption of \u003cem\u003eARI5\u003c/em\u003e and its homolog \u003cem\u003eARI7\u003c/em\u003e resulted in several developmental phenotypes that were opposite to those observed in the \u003cem\u003ejaw-5D\u003c/em\u003e mutant. We further demonstrate that TCP4 is an unstable protein whose degradation is mediated by ARI5. Our findings establish ARI5 as a key regulator of plant development and uncover a molecular mechanism by which plants precisely modulate developmental processes through the spatiotemporal ARI5-mediated control of CIN-like TCP protein stability.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eARI5 is a PARKIN-like protein interacting with TCP4\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the regulatory mechanisms of TCPs, we generated a 35S-MYC-mTCP4 transgenic line, in which the \u003cem\u003eMYC\u003c/em\u003e tag was fused with a microRNA319 (miR319)-resistant form of \u003cem\u003eTCP4\u003c/em\u003e (\u003cem\u003emTCP4\u003c/em\u003e) under the control of the CaMV 35S promoter. Using inflorescence tissues from stable 35S-MYC-mTCP4 transgenic plants, we performed Immunoprecipitation-Mass Spectrometry (IP-MS) to identify potential TCP4 interactors. The identified proteins included previously reported TCP4-associated factors, such as TOPLESS/TOPLESS-RELATED proteins (TPL/TPRs), HISTONE DEACETYLASE 15 (HDA15), and other known interacting transcription factors such as TCP5 and KNAT3 (Supplementary Table\u0026nbsp;1) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, validating the reliability of the results. Additionally, novel interacting candidates including ARI5, 26S PROTEASOME REGULATORY SUBUNIT S2 1A (RPN1A), RPN1B, RPN2A and RPN2B were identified among the results (Supplementary Table\u0026nbsp;1). Given the reported instability of TCP proteins and the limited understanding of their degradation mechanisms, we focused on ARI5 due to its homology to the well-characterized human PARKIN E3 ligase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), whose function in plants remains unexplored.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo characterize the ARI5 protein, we first conducted a comprehensive bioinformatic analysis. ARI5 encodes a 552-amino acid protein that features an N-terminal Leucine-rich1 domain, a canonical RBR domain comprising a C3HC4 RING1 region, a C5HC IBR region, and a shorter C3HC4 RING2 region, as well as a C-terminal Leucine-rich2 domain containing a nuclear localization signal (NLS) motif (Supplementary Fig.\u0026nbsp;1a, b). Protein sequence alignment and phylogenetic analysis revealed that ARI5 exhibits sequence and structure similarity to the human PARKIN protein within the RBR domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;1c). The Arabidopsis genome encodes 16 ARI proteins, among which ARI5 clusters with ARI6 and ARI7 in subgroup B (Supplementary Fig.\u0026nbsp;2). ARI6 has been previously characterized as a pseudogene\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, while the function of ARI5 and ARI7 remains uncharacterized.\u003c/p\u003e \u003cp\u003eWe then conducted a series of experimental assays to confirm the interaction between ARI5 and TCP4. First, yeast two-hybrid (Y2H) assays were performed by generating bait vectors with ARI5 or ARI7 fused to the DNA-binding domain (DBD) and prey vectors with each of the 11 Class II TCPs fused to the activation domain (AD). The results demonstrated that ARI5 interacted with TCP1, TCP3, TCP4, TCP10, TCP13, TCP17, and TCP18, while ARI7 exhibited interactions with all Class II TCPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). Second, firefly luciferase complementation imaging (LCI) assays were carried out to further validate the interaction between ARI5 and TCP4. The construct 35S-cLUC-ARI5 was generated by fusing \u003cem\u003eARI5\u003c/em\u003e to the sequence encoding the C-termimal part of luciferase (LUC) under the control of the CaMV 35S promoter, while 35S-mTCP4-nLUC was generated by fusing a miR319-resistant \u003cem\u003emTCP4\u003c/em\u003e to the sequence encoding the N-terminal part of LUC, also under the control of the CaMV 35S promoter. The constructs 35S-cLUC-ARI5 and 35S-mTCP4-nLUC, along with control combinations, were co-expressed in distinct regions of one tobacco leaf. In three independent replicates, strong fluorescence signals were consistently detected in regions co-transformed with 35S-cLUC-ARI5 and 35S-mTCP4-nLUC, whereas no fluorescence was observed in regions expressing the control combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), indicating a specific interaction between ARI5 and TCP4 \u003cem\u003ein planta\u003c/em\u003e. Third, co-immunoprecipitation (Co-IP) assays were performed using GFP-tagged ARI5 and Myc-tagged TCP4 transiently expressed in tobacco leaves. The results confirmed that TCP4 co-immunoprecipitated with ARI5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), further supporting their interaction \u003cem\u003ein planta\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDisruption of\u003c/b\u003e \u003cb\u003eARI5\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eARI7\u003c/b\u003e \u003cb\u003eresults in antagonistic phenotypes to\u003c/b\u003e \u003cb\u003etcp\u003c/b\u003e\u003cb\u003e-deficient mutants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the roles of \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e, we created an \u003cem\u003eari5 ari7\u003c/em\u003e double mutant in which the function of \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e was knocked-out using CRISPR/Cas9 technology. Sequencing analysis indicated that the \u003cem\u003eari5 ari7\u003c/em\u003e double mutant possessed a 2-bp frameshift deletion in the first exon of \u003cem\u003eARI5\u003c/em\u003e and a large 858-bp deletion in the \u003cem\u003eARI7\u003c/em\u003e coding sequence, both of which resulted in premature termination and expression of short truncated products (Supplementary Fig.\u0026nbsp;3). To generate single mutants, the \u003cem\u003eari5 ari7\u003c/em\u003e double mutant was backcrossed to wild-type (WT) Arabidopsis, and subsequent progeny genotyping identified both \u003cem\u003eari5\u003c/em\u003e and \u003cem\u003eari7\u003c/em\u003e single mutants. The \u003cem\u003eari5\u003c/em\u003e single mutant showed no discernible phenotypic differences compared to WT control. However, the \u003cem\u003eari7\u003c/em\u003e single mutant exhibited significantly flatter leaves, and the \u003cem\u003eari5 ari7\u003c/em\u003e double mutant displayed more severe flatter leaves than \u003cem\u003eari7\u003c/em\u003e, indicating that \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e had functional redundancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c). This phenotype was opposite to curled leaves of \u003cem\u003etcp\u003c/em\u003e null mutantsand \u003cem\u003ejaw-5D\u003c/em\u003e in which \u003cem\u003emiR319b\u003c/em\u003e is overexpressed to reduce \u003cem\u003eTCP2\u003c/em\u003e, \u003cem\u003eTCP3\u003c/em\u003e, \u003cem\u003eTCP4\u003c/em\u003e, \u003cem\u003eTCP10\u003c/em\u003e, and \u003cem\u003eTCP24\u003c/em\u003e transcript levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To further elucidate the cellular basis of the observed phenotypes, we conducted a comparative analysis of abaxial epidermal cells in leaves from \u003cem\u003eari5 ari7\u003c/em\u003e, \u003cem\u003ejaw-5D\u003c/em\u003e, and WT controls. Intriguingly, while \u003cem\u003eari5 ari7\u003c/em\u003e mutants produced smaller leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;4a,b), their epidermal cells exhibited a significant increase in size compared to that of WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,d). Conversely, \u003cem\u003ejaw-5D\u003c/em\u003e mutants displayed a reduction in epidermal cell dimensions relative to \u003cem\u003eari5 ari7\u003c/em\u003e and WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,d), in consistent with the previous reports that Class II TCPs usually inhibit cell proliferation and promote cell differentiation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These results indicate that ARI5 and ARI7 enhance cell proliferation to drive leaf curvature, functioning antagonistically to TCP4, which suppresses cell proliferation to inhibit leaf curvature\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then explored additional phenotypes related to TCP transcription factors. Firstly, our previous work has demonstrated that TCP4 acts as an antagonist of PIF3 and promotes light-induced cotyledon opening\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. To investigate this phenotype, we examined cotyledon opening in 4-d-old seedlings of \u003cem\u003eari5 ari7\u003c/em\u003e, 35S-MYC-mTCP4 overexpression lines, and WT plants grown in the dark. The results showed that cotyledons of WT seedlings remained largely closed under the dark condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee,h). In contrast, \u003cem\u003eari5 ari7\u003c/em\u003e and 35S-MYC-mTCP4 seedlings exhibited significantly more opening cotyledons than WT seedlings after 4 d of dark growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h), supporting the previously identified roles of TCP4 as a positive regulator of cotyledon opening\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Secondly, CIN-like TCP transcription factors are known to promote flowering\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We assessed the flowering time of \u003cem\u003eari5 ari7\u003c/em\u003e, \u003cem\u003ejaw-5D\u003c/em\u003e, and WT control plants. Our results indicated that \u003cem\u003eari5 ari7\u003c/em\u003e flowered significantly earlier than WT plants, whereas \u003cem\u003ejaw-5D\u003c/em\u003e exhibited delayed flowering compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei,j), consistent with previous reports about the positive regulation of flowering by CIN-like TCPs\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Lastly, we have previously reported that Class II TCPs inhibit the elongation of the style in the apical gynoecium\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We measured the style length in \u003cem\u003eari5 ari7\u003c/em\u003e, \u003cem\u003ejaw-5D\u003c/em\u003e, and WT plants. The results showed that \u003cem\u003eari5 ari7\u003c/em\u003e produced significantly shorter and wider styles than WT, while \u003cem\u003ejaw-5D\u003c/em\u003e exhibited longer and thinner styles than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek-o). Scanning electron microscopy (SEM) analysis revealed that the cell number along the longitudinal axis of the style was reduced in \u003cem\u003eari5 ari7\u003c/em\u003e mutants but increased in \u003cem\u003ejaw-5D\u003c/em\u003e. Conversely, in the transverse direction, the cell number was elevated in \u003cem\u003eari5 ari7\u003c/em\u003e but diminished in \u003cem\u003ejaw-5D\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ep-t). These findings further support the antagonistic relationship between ARI5/ARI7 and Class II TCPs.\u003c/p\u003e \u003cp\u003eTo verify whether the observed phenotypes in \u003cem\u003eari5 ari7\u003c/em\u003e mutants were specifically caused by the disruption of ARIs, we generated a 35S-GFP-ARI5g construct, in which the genomic sequence of \u003cem\u003eARI5\u003c/em\u003e was fused to \u003cem\u003eGFP\u003c/em\u003e under the control of the CaMV 35S promoter. We transformed 35S-GFP-ARI5g into \u003cem\u003eari5 ari7\u003c/em\u003e, and found that the expression of \u003cem\u003eARI5\u003c/em\u003e in \u003cem\u003eari5 ari7\u003c/em\u003e complemented the defective phenotypes including the smaller rosette size and impaired leaf flatness phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eu,v, Supplementary Fig.\u0026nbsp;4a,b), providing compelling evidence that \u003cem\u003eARI5\u003c/em\u003e plays a critical role in regulating plant development.\u003c/p\u003e \u003cp\u003eThe observation of multiple antagonistic phenotypes between \u003cem\u003eari5 ari7\u003c/em\u003e mutants and \u003cem\u003etcp\u003c/em\u003e-deficient mutants strongly indicates that ARI5 and ARI7 likely modulate plant development by negatively regulating the activity of TCP transcription factors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eARI5\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eARI7\u003c/b\u003e \u003cb\u003eexhibit overlapping expression patterns with\u003c/b\u003e \u003cb\u003eTCP4\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo provide more evidences supporting that ARI5 and ARI7 could inhibit the activity TCP4 through physical interaction, we first generated ARI5pro-GUS or ARI7pro-GUS constructs in which a 1077-bp \u003cem\u003eARI5\u003c/em\u003e promoter or a 769-bp \u003cem\u003eARI7\u003c/em\u003e promoter were used to drive the \u003cem\u003eβ-glucuronidase\u003c/em\u003e (\u003cem\u003eGUS\u003c/em\u003e) reporter gene. Following transformation into Arabidopsis, histochemical GUS staining revealed consistent expression patterns in eighteen independent ARI5pro-GUS or in fifteen independent ARI7pro-GUS transgenic lines. We selected the stable ARI5pro-GUS-5 and ARI7pro-GUS-2 for detailed spatial and temporal expression analysis. The results revealed that both \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e were prominently expressed in the vasculature of all leaves in 8-d-old light- or dark-grown seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,d,g,h,j-l). As leaf development progressed, a distinct spatiotemporal expression pattern emerged, with GUS staining gradually diminishing from distal to proximal regions and from medial to marginal regions in both ARI5pro-GUS and ARI7pro-GUS transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-f). Both \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e were detected in the shoot apical meristem (SAM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-f). Under dark-grown conditions, \u003cem\u003eARI7\u003c/em\u003e exhibited strong expression in the apical hook and closed cotyledons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej-l), whereas relatively weaker GUS staining was observed in these tissues in ARI5pro-GUS seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i). During reproductive development, both \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e had a ubiquitous expression in the pistils and pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em,n). The spatial and temporal expression patterns of \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e significantly overlap with those of \u003cem\u003eTCP\u003c/em\u003e genes, including \u003cem\u003eTCP4\u003c/em\u003e\u003csup\u003e47\u003c/sup\u003e, and correlate well with the developmental phenotypes observed in \u003cem\u003eari5 ari7\u003c/em\u003e double mutants, further supporting their functional relevance with \u003cem\u003eTCP\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eARI5 is co-localized with TCP4 in the nuclei\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine the subcellular localization of ARI5 and ARI7, we generated 35Spro-GFP-ARI5g and 35Spro-GFP-ARI7g constructs, utilizing the CaMV 35S promoter to drive \u003cem\u003eGFP\u003c/em\u003e fusion with \u003cem\u003eARI5\u003c/em\u003e or \u003cem\u003eARI7\u003c/em\u003e genomic sequences. 35Spro-NLS-RFP was generated as a nuclear marker in which \u003cem\u003eRFP\u003c/em\u003e fusion with a nuclear localization signal (NLS) sequence was driven by the CaMV 35S promoter. Initially, we transiently co-introduced the two constructs alongside 35Spro-NLS-RFP into tobacco leaves. The observation revealed a clear overlap of green and red fluorescence, indicating both ARI5 and ARI7 could be localized to the nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f). Subsequently, we established the stable Arabidopsis transgenic lines that expressed GFP-tagged ARI5 or ARI7. Confocal microscopy showed that the GFP fluorescence coincided with DAPI-stained nuclei, thereby corroborating the nuclear presence of ARI5 and ARI7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-n). To further investigate the co-localization of ARI5 and TCP4, we generated 35S-RFP-mTCP4, in which miR319-resistant \u003cem\u003eTCP4\u003c/em\u003e was fused to \u003cem\u003eRFP\u003c/em\u003e and driven by the CaMV 35S promoter. Transient expression of \u003cem\u003eARI5-GFP\u003c/em\u003e and \u003cem\u003eTCP4-RFP\u003c/em\u003e in tobacco leaves revealed a significant co-localization of the two proteins within the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eo-q). These results provide the spatial evidence that supports the interaction between ARI5 and TCP4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eARI5 functions as an active E3 ubiquitin ligase\u003c/b\u003e \u003c/p\u003e \u003cp\u003eARI5 harbors a canonical RBR domain exhibiting conserved structural homology to the human PARKIN E3 ligase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;1), a well-established regulator of ubiquitin-dependent proteolysis in neurodegenerative pathways\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To investigate whether ARI5 could possess E3 ligase activity, we employed a bacterial ubiquitination reconstitution assay\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, using \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 as an expression host. We co-expressed an Arabidopsis E1 (UBA1), an Arabidopsis E2 (UBC8), the MYC-tagged ARI5 and a His-FLAG-tagged Ub (ubiquitin protein) in BL21 cells. Immunoblot analysis indicated that MYC-ARI5, Ub, UBA1 and UBC8 were indeed expressed in the cells with anti-MYC, anti-FLAG and anti-S, respectively. Strikingly, reactions containing all the UBA1, UBC8, MYC-ARI5 and Ub exhibited distinct polyubiquitin laddering patterns, while the polyubiquitin ladder in control reactions lacking any single component (E1, E2, E3, or Ub) was completely absent. These data indicate that ARI5 is an active E3 ligase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the functional contribution of the two conserved RING motifs (RING1 and RING2) within the RBR domain, we generated domain-specific truncations including ARI5ΔR1-MYC with intact RING2, ARI5ΔR2-MYC with intact RING1, and ARI5ΔR12-MYC without both RING1 and RING2. Ubiquitination assays indicated that deletion of RING1 or RING2 significantly attenuated polyubiquitin chain formation, though residual mono-ubiquitination signals persisted (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). ARI5-ΔR2 exhibited stronger mono-ubiquitination intensity than ARI5-ΔR1, suggesting that RING1 plays more important roles than RING2. Complete deletion of both RING domains (ARI5-ΔR12) abolished all ubiquitination activity, indicating the requirement of both RINGs for the full E3 activity of ARI5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eARI5 targets TCP4 for degradation through ubiquitination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the findings that ARI5 functions as a PARKIN-like E3 ligase and interacts with TCP4, we hypothesized that ARI5 mediates TCP4 ubiquitination to facilitate its proteasomal degradation. To test this hypothesis, we first investigated whether TCP4 undergoes ubiquitination in plants. We performed immunoprecipitation (IP) to enrich MYC-tagged TCP4 from 7-d-old light-grown seedlings, leaves or flowers of 35S-MYC-mTCP4 transgenic plants. Subsequent immunoblot analysis using anti-Ub or anti-MYC antibodies demonstrated that TCP4 is subject to ubiquitination modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). To investigate whether ARI5 mediates TCP4 ubiquitination, we used bacterial ubiquitination reconstitution assay by co-expressing UBA1, UBC8, MYC-tagged ARI5, His-FLAG-tagged Ub, and MBP-HA-tagged TCP4 in \u003cem\u003eE. coli\u003c/em\u003e cells. Immunoblot analysis using anti-MYC antibody confirmed the self-ubiquitination activity of ARI5, while anti-FLAG detection revealed characteristic ubiquitinated protein ladders (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Notably, anti-HA immunoblot analysis demonstrated the presence of polyubiquitin ladders only in reactions containing the complete ubiquitination system (UBA1, UBC8, ARI5, and Ub) with TCP4, while ubiquitination ladders were absent in the control reactions lacking any one of these components (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These results suggest that ARI5 mediates TCP4 ubiquitination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the role of ARI5 in regulating TCP4 stability \u003cem\u003ein planta\u003c/em\u003e, we generated a 35S-MYC-mTCP4 \u003cem\u003eari5 ari7\u003c/em\u003e line through genetic crosses between a 35S-MYC-mTCP4 transgenic line and an \u003cem\u003eari5 ari7\u003c/em\u003e mutant. To compare TCP4 degradation kinetics, we treated 35S-MYC-mTCP4 and 35S-MYC-mTCP4 \u003cem\u003eari5 ari7\u003c/em\u003e plants with the protein synthesis inhibitor cycloheximide (CHX) to inhibit \u003cem\u003ede novo\u003c/em\u003e protein synthesis. Quantitative analysis of three independent biological replicates revealed that TCP4 undergoes time-dependent degradation in both genetic backgrounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). However, the degradation rate was significantly slower in 35S-MYC-mTCP4 \u003cem\u003eari5 ari7\u003c/em\u003e compared to 35S-MYC-mTCP4, with statistically significant differences observed at both 12 h and 24 h time points (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003eThese results suggest that ARI5 promotes TCP4 protein degradation through ubiquitination.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe genetic interactions between\u003c/b\u003e \u003cb\u003eARI\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCIN\u003c/b\u003e\u003cb\u003e-like\u003c/b\u003e \u003cb\u003eTCP\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the negative regulation of TCP4 stability by the PARKIN-like E3 ligase ARI5, we analyzed genetic interactions between \u003cem\u003eARI\u003c/em\u003e mutants and \u003cem\u003eCIN\u003c/em\u003e-like \u003cem\u003eTCP\u003c/em\u003e mutants. We generated an \u003cem\u003eari5 ari7 jaw-5D\u003c/em\u003e triple mutant by crossing the \u003cem\u003eari5 ari7\u003c/em\u003e double mutant with \u003cem\u003ejaw-5D\u003c/em\u003e. Phenotypic analysis revealed that the \u003cem\u003eari5 ari7\u003c/em\u003e double mutant exhibited significantly flatter leaves compared to WT, while \u003cem\u003ejaw-5D\u003c/em\u003e mutants showed more pronounced leaf curling, consistent with above observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b, Supplementary Fig.\u0026nbsp;5a). The introduction of the \u003cem\u003ejaw-5D\u003c/em\u003e mutation into the \u003cem\u003eari5 ari7\u003c/em\u003e background resulted in a phenotypic shift from flat to curled leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b, Supplementary Fig.\u0026nbsp;5a). Specifically, the leaf curvature and wavy margin phenotypes observed in the \u003cem\u003ejaw-5D ari5 ari7\u003c/em\u003e triple mutant were comparable to those of the \u003cem\u003ejaw-5D\u003c/em\u003e single mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, Supplementary Fig.\u0026nbsp;5a), indicating that \u003cem\u003ejaw-5D\u003c/em\u003e is epistatic to \u003cem\u003eari5 ari7\u003c/em\u003e. Furthermore, additional phenotypic traits of \u003cem\u003eari5 ari7\u003c/em\u003e, such as early cotyledon opening in etiolated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-f, Supplementary Fig.\u0026nbsp;5b), early flowering (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ek, Supplementary Fig.\u0026nbsp;5c), and shorter gynoecium styles (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-j, Supplementary Fig.\u0026nbsp;5d,e), were also suppressed by the \u003cem\u003ejaw-5D\u003c/em\u003e mutation. These genetic interactions strongly suggested that the phenotypes of \u003cem\u003eari5 ari7\u003c/em\u003e are dependent on the presence of functional \u003cem\u003eCIN\u003c/em\u003e-like \u003cem\u003eTCPs\u003c/em\u003e, in consistent with the above data that ARI5 promotes the degradation of TCP4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then generated overexpression lines by transforming the 35S-GFP-ARI5g and 35S-GFP-ARI7g constructs into WT Arabidopsis. Phenotypic analysis revealed that 15 out of 127 35S-GFP-ARI5g independent transgenic lines and 27 out of 144 35S-GFP-ARI7g ones exhibited curly leaves. From these lines, we selected stable overexpression lines, namely 35S-GFP-ARI5g-2 and 35S-GFP-ARI5g-8 for \u003cem\u003eARI5\u003c/em\u003e, and 35S-GFP-ARI7g-4 and 35S-GFP-ARI7g-8 for \u003cem\u003eARI7\u003c/em\u003e, as representative lines for further analysis. Reverse transcription quantitative PCR (RT-qPCR) confirmed the successful overexpression of \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e in these transgenic lines (Supplementary Fig.\u0026nbsp;5f,g). Quantitative measurements of leaf curvature demonstrated that the 35S-GFP-ARI5g-2, 35S-GFP-ARI5g-8, 35S-GFP-ARI7g-4, and 35S-GFP-ARI7g-8 lines exhibited significantly more severe leaf curvature compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el,m, Supplementary Fig.\u0026nbsp;5h-k). This phenotype contrasts with the flatter leaf morphology observed in \u003cem\u003eari5 ari7\u003c/em\u003e, providing further evidence that \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e play critical roles in regulating leaf morphogenesis.\u003c/p\u003e \u003cp\u003eTo determine whether increased \u003cem\u003eTCP4\u003c/em\u003e levels could rescue the leaf curvature phenotype observed in \u003cem\u003eARI\u003c/em\u003e overexpression lines, we crossed the \u003cem\u003eTCP4\u003c/em\u003e overexpression line 35S-MYC-mTCP4 with the \u003cem\u003eARI\u003c/em\u003e overexpression line 35S-GFP-ARI5g-8. Introduction of 35S-MYC-mTCP4 significantly suppressed the leaf curvature phenotype of 35S-GFP-ARI5g-8, supporting the hypothesis that the excessive degradation of TCPs by the overexpression \u003cem\u003eARI5\u003c/em\u003e leads to more curled leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003en). We next compared the abundance of TCP4 protein in 35S-MYC-mTCP4, 35S-MYC-mTCP4 35S-GFP-ARI5g-8, and control lines. The results revealed that \u003cem\u003eARI5\u003c/em\u003e overexpression significantly reduced TCP4 protein levels in the 35S-MYC-mTCP4 background (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eo). We further used the 35S-MYC-mTCP4 35S-GFP-ARI5g-8 line to perform co-immunoprecipitation (Co-IP) assays. MYC-tagged TCP4 successfully co-immunoprecipitated with GFP-tagged ARI5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep), again confirming a physical interaction between the two proteins \u003cem\u003ein vivo\u003c/em\u003e. Immunoblot analysis using an anti-Ub antibody demonstrated that ubiquitinated TCP4 was more abundant in the 35S-MYC-mTCP4 35S-GFP-ARI5g-8 line compared to 35S-MYC-mTCP4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep). These findings strongly suggest that ARI5 regulates plant development by precisely modulating the abundance of TCP4 through ubiquitin-mediated degradation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate that the PARKIN-like E3 ligases ARI5 and its homolog ARI7 mediate the degradation of CIN-like TCP proteins, thereby regulating diverse plant developmental processes. Disruption of \u003cem\u003eARIs\u003c/em\u003e leads to phenotypes that are antagonistic to those observed in \u003cem\u003etcp\u003c/em\u003e-deficient mutants, including flatter leaves, enhanced cotyledon opening in darkness, earlier flowering, and shorter gynoecium styles in the \u003cem\u003eari5 ari7\u003c/em\u003e double mutant. We show that \u003cem\u003eARI5\u003c/em\u003e is developmentally regulated and exhibits spatiotemporal expression patterns overlapping with \u003cem\u003eTCP4\u003c/em\u003e. Biochemical characterization reveals that ARI5 possesses E3 ligase activity and physically interacts with TCP4. We further show that TCP4 protein is regulated by degradation, and ARI5 mediates its ubiquitination and degradation. Based on these findings, we propose a mechanistic model for ARI5 function. ARI5 functions as an active PARKIN-like E3 ligase. ARI5 controls the stability and degradation of TCP4, and the spatially restricted TCP4 accumulation governs cell proliferation and differentiation, thereby determining organ morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Our data demonstrate that ARI5 acts as an important organ developmental regulator that shapes organs by spatiotemporally targeting TCP4 for degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs central integrators of environmental cues and endogenous signals, TCP transcription factors precisely orchestrate cell division and differentiation to regulate plant developmental plasticity\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Among external stimuli, light and temperature emerge as pivotal regulators of TCP activity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Photoperiodic conditions directly influence both TCP protein stability and DNA-binding affinity. For examples, TCP17 undergoes 26S proteasome-mediated degradation under light conditions but accumulates under shade despite transcriptional downregulation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Similarly, TCP2 forms complexes with CRYs under blue light to stabilize the protein and promote photomorphogenesis, whereas it is rapidly degraded in darkness\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Temperature fluctuations also exert significant regulatory effects, with elevated temperatures inducing the accumulation of TCP5, TCP13, and TCP17\u003csup\u003e25\u003c/sup\u003e. Beyond environmental modulation, intrinsic regulatory networks govern TCP activity through multiple mechanisms, including microRNA-mediated suppression, hormonal signaling cascades, and controlled protein turnover\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The miR319 family directly cleaves \u003cem\u003eTCP\u003c/em\u003e transcripts, and its ectopic expression leads to leaf curling and delayed flowering by the coordinated suppression of multiple \u003cem\u003eTCP\u003c/em\u003e genes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Phytohormones including auxin, gibberellins, and brassinosteroids fine-tune TCP activity to maintain the balance between growth and differentiation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Additionally, diverse proteolytic pathways converge to regulate TCP protein stability. For instance, the F-box protein KISS ME DEADLY (KMD) targets TCP14 for degradation to modulate cytokinin responses, a pathway exploited by pathogens to manipulate host development\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. A striking illustration of pathogen-mediated TCP subversion involves the phytoplasma effector SAP11 from the Aster Yellows Witches' Broom (AY-WB) strain. Upon secretion into host cells, SAP11 induces the degradation of multiple TCPs, including TCP4, resulting in leaf curling and developmental defects phenocopying \u003cem\u003etcp\u003c/em\u003e mutants\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Intriguingly, SAP bypass canonical ubiquitination by directly binding their substrates and recruiting the 26S proteasome subunit RPN, thereby circumventing the need for E3 ligase-mediated ubiquitination\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Our prior research revealed the complexity of ubiquitin-dependent TCP regulation, demonstrating that TEAR1 indirectly modulates TCP activity by ubiquitinating their interactor TIE1, which recruits TPL/TPR corepressors to suppress \u003cem\u003eTCP\u003c/em\u003e target gene expression\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In this study, we identify the PARKIN-like E3 ligase ARI5 as a direct regulator of TCP4 through ubiquitin-mediated degradation. As an RBR-type E3 ligase, ARI5 ubiquitinates TCP4 to promote its proteasomal turnover. Unlike TEAR1, which enhances TCP activity by degrading the TCP repressor TIE1, ARI5 directly suppresses TCP4 activity. Given the observed environmental modulation of TCP degradation, we propose that ARI5 and its homologs may not only regulate TCP stability in response to developmental cues but also fine-tune TCP activity under some environmental conditions, thereby shaping organ growth and morphogenesis in adapting to environmental fluctuations.\u003c/p\u003e \u003cp\u003eThe PARKIN-like E3 ligase ARI family exhibits remarkable evolutionary conservation across both plants and other organisms\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, very few ARIs in plants has been characterized. The medicinal plant \u003cem\u003eHypericum perforatum\u003c/em\u003e (St. John's wort) employs an intriguing reproductive strategy called apospory, where gametophytes develop directly from sporophytic somatic cells without undergoing meiosis\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. This mechanism enables heterozygosity fixation in crops. Genetic studies have identified \u003cem\u003eHpARI7\u003c/em\u003e as a candidate gene associated with the apospory locus, yet its precise molecular function and physiological substrates remain to be elucidated\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Recently, \u003cem\u003eOsRBRL1\u003c/em\u003e, a rice ARI homolog, functions as a viral sensor that recognizes coat proteins of \u003cem\u003eRice stripe virus\u003c/em\u003e (RSV) and \u003cem\u003eRice dwarf virus\u003c/em\u003e (RDV). During viral infection, \u003cem\u003eOsRBRL1\u003c/em\u003e is transcriptionally upregulated and mediates the ubiquitination and subsequent degradation of \u003cem\u003eNOVEL INTERACTOR OF JAZ 3\u003c/em\u003e (NINJA3), a negative regulator of the jasmonic acid (JA) signaling pathway, thereby activating plant antiviral defenses\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In this study, we demonstrate that \u003cem\u003eArabidopsis\u003c/em\u003e ARI5 and ARI7 regulate multiple developmental processes. We establish ARI5 as an active E3 ubiquitin ligase that targets the key developmental regulator TCP4 for degradation, thereby precisely controlling organ morphogenesis. These findings suggest that plant ARI proteins act as crucial modulators that may orchestrate diverse biological processes in plants through the degradation of selective proteins.\u003c/p\u003e \u003cp\u003eThe evolutionary conservation and functional diversification of PARKIN-like E3 ligases extends to functional parallels between plant ARI5 and its human counterpart. While human PARKIN is best characterized for its cytoplasmic role in mitochondrial quality control through mitophagy, emerging evidence reveals its involvement in cell cycle regulation\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Notably, PARKIN mediates the ubiquitination and degradation of Cyclin E, a key regulator of G1/S phase transition, thereby controlling neuronal progenitor cell division during brain development\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Intriguingly, our findings demonstrate that plant ARIs regulate TCP transcription factors, which directly control the expression of cell cycle regulators such as CYCLIND3b and CYCB1;1 during leaf development\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. This functional convergence where PARKIN-like proteins in different organisms modulate cell proliferation through distinct cyclin-related pathways highlights their conserved role in growth regulation across eukaryotes. Our study reveals that ARI5 mediates nuclear degradation of TCP4, despite its dual localization in both cytoplasmic and nuclear compartments. This observation suggests that, similar to PARKIN, ARI5 might have additional cytoplasmic substrates. In human, the cytoplasmic functions of PARKIN are well-established, including mitochondrial clearance and proteasomal regulation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Future studies should investigate whether ARI5 regulates cytoplasmic targets analogous to the mitochondrial substrates of PARKIN. Conversely, our discovery that plant ARIs target transcription factors raises the intriguing possibility that PARKIN might similarly regulate transcriptional regulators involved in neurodevelopment under specific conditions.\u003c/p\u003e \u003cp\u003eOur work establishes ARI5 as an active E3 ligase that shapes plant development through targeted degradation of TCP4, a master regulator of developmental plasticity. This regulatory mechanism enables plants to flexibly adjust developmental programs, thereby shaping organ morphology to adapt to fluctuating environmental conditions. Given the evolutionary conservation of PARKIN-like E3 ligases, and the plant-specific conservation of TCP proteins, our findings may offer molecular breeding strategies for crop improvement through the manipulation of ARI-TCP regulatory modules to optimize developmental and stress-responsive traits, and also provide insights into PARKIN function relevant to Parkinson's disease research.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePlant materials and growth conditions\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe Arabidopsis Columbia-0 (Col-0) ecotype type was used. Seeds of WT, \u003cem\u003eari5\u003c/em\u003e, \u003cem\u003eari7\u003c/em\u003e, \u003cem\u003eari5 ari7\u003c/em\u003e, \u003cem\u003ejaw-5D\u003c/em\u003e and other plant materials or mutants were surface-sterilized with 75% ethanol for 10 min, rinsed twice with 100% ethanol, and air-dried in a clean bench. After cold stratification at 4°C for 2 d, the dried seeds were germinated on half-strength Murashige and Skoog (MS) medium at 22°C under long-day conditions (16-h light and 8-h dark). Seedlings of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e or \u003cem\u003eNicotiana benthamiana\u003c/em\u003e were grown in soil in a greenhouse under the same conditions.\u003c/p\u003e\u003cp\u003e \u003cb\u003eGeneration of binary constructs and transformation\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe genomic sequences of \u003cem\u003eARI5\u003c/em\u003e and \u003cem\u003eARI7\u003c/em\u003e were amplified and cloned into pENTR/D-TOPO (Invitrogen) to generate pENTR-ARI5g and pENTR-ARI7g. The entry vectors were recombined with pK7WGF2 (Invitrogen) via LR reaction to generate 35S-GFP-ARI5g and 35S-GFP-ARI7g constructs. Similarly, promoter regions (1077-bp ARI5pro and 769-bp ARI7pro) were cloned into pENTR/D-TOPO, then recombined with pB7GUSWG0 (Invitrogen) to generate ARI5pro-GUS and ARI7pro-GUS. All constructs were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 for Arabidopsis transformation by floral dip\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e \u003cb\u003eYeast two-hybrid assays\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTo test the interaction between ARI5/7 and TCPs, we used previously described AD-TCP2/3/4/5/10/13/17/18/24 prey constructs\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, and generated AD-TCP1/17 by LR recombination of pENTR-TCP1/17 with pDEST22 (Invitrogen). For bait constructs, we cloned 1659-bp ARI5 and 1990-bp ARI7 cDNA into pENTR/D-TOPO, then recombined them with pDEST32 (Invitrogen) to create DBD-ARI5/7. Bait and prey constructs (or pDEST22 control) were co-transformed into yeast strain AH109. The yeasts were grown in a 28°C incubator for 72 h. Selection was performed using SD-Leu-Trp-His medium (Coolaber).\u003c/p\u003e\u003cp\u003e \u003cb\u003eFirefly luciferase complementation assays\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor luciferase complementation assays, mTCP4 and ARI5 were cloned into pCambia1300-nLUC and pCambia1300-cLUC via LR recombination, respectively. Agrobacterium strains carrying these constructs were co-infiltrated into tobacco leaves. The tobacco plants were grown under a weak light for 12 h and were transferred to long-day conditions for 32 h. The infiltrated leaves were harvested, injected with 200 µM luciferin, and dark-adapted for 10 min. Luminescence was captured using a NightOWL II LB983 CCD system (180 sec exposure, 10% intensity, high gain mode) with indiGO software.\u003c/p\u003e\u003cp\u003e \u003cb\u003eImmunoprecipitation and Western blot\u003c/b\u003e \u003c/p\u003e\u003cp\u003eCo-IP assays were performed first in tobacco leaves through transient co-expression of 35S-mTCP4-MYC with either 35S-GFP-ARI5 or 35S-GFP, followed by validation in 7-d-old Arabidopsis seedlings expressing 35S-mTCP4-MYC alone or in combination with 35S-GFP-ARI5. For TCP4 immunoprecipitation, tissues at different developmental stages (7-d seedlings, 21-d leaves, and 21-d flowers) were harvested from 35Spro-MYC-mTCP4 transgenic lines.\u003c/p\u003e\u003cp\u003eTotal proteins were extracted using buffer (0.1 M HEPES at pH 7.5, 5 mM EGTA, 5 mM EDTA, 5 mM NaF, 50 mM phosphoglycerol, 1% Triton X-100, 10% glycerin, and protease inhibitor cocktail, PMSF) and incubated with anti-GFP (MBL, D153-10) or anti-MYC (MBL, M047-11) beads for 3 h. After five PBS washes, bound proteins were eluted by boiling in 5×SDS buffer (7 min, 100°C), separated by 10% SDS-PAGE, and transferred to PVDF membranes (Millipore). Immunodetection used anti-MYC (ABclonal, AE070), anti-GFP (Sangon, D110008), and anti-Ub (Abcam, ab7254) antibodies, with visualization on a Tanon 5200 Multi system.\u003c/p\u003e\u003cp\u003e \u003cb\u003eCRISPR/Cas9-induced mutants\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe \u003cem\u003eari5 ari7\u003c/em\u003e double mutant was generated using a UBQ10 promoter-driven CRISPR/Cas9 system\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. We cloned the sgRNA construct pAtU6-ARI5target1-ARI5target2-ARI7target1-ARI7target2 into pUBQ10-Cas9-P2A-GFP-rbcS-E9t (\u003cem\u003eKpn\u003c/em\u003e I/\u003cem\u003eSpe\u003c/em\u003e I sites) and transformed it into WT plants. Target locus mutations were verified by sequencing PCR products. The Cas9-free double mutant \u003cem\u003eari5 ari7\u003c/em\u003e was obtained by genetic segregation. The primers are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e \u003cb\u003eStaining and microscopy\u003c/b\u003e \u003c/p\u003e\u003cp\u003eARI5pro-GUS and ARI7pro-GUS transgenic lines were fixed in 90% acetone (20 min, ice), then washed with phosphate buffer (0.34 M Na₂HPO₄·12H₂O, 0.01 M NaH₂PO₄·2H₂O, 0.4 mM K₄[Fe(CN)₆]·3H₂O, 0.5 mM K₃[Fe(CN)₆]) and stained overnight at 37°C in GUS solution (0.5 mg/mL X-Gluc). After destaining in 75% ethanol, patterns were documented using a Leica M205 FCA stereoscope.\u003c/p\u003e\u003cp\u003eThe 35S-GFP-ARI5g or 35S-GFP-ARI7g and 35S-NLS-RFP or 35S-RFP-mTCP4 constructs were used to express fusion proteins in tobacco leaves. Infiltrated leaves were incubated in a growth chamber for 48 h before observing using a Zeiss Axio Imager M2 microscope. For transgenic Arabidopsis, hypocotyls were DAPI-stained (1 mg/mL, 10 min) to confirm nuclear localization of GFP-ARI5/7.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eubiquitination assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe constructs pACYCDuet-ARI5-MYC, pACYCDuet-ARI5-MYC-AtUBC8-S, pACYCDuet-ARI5(ΔR1)-MYC-AtUBC8-S, pACYCDuet-ARI5(ΔR2)-MYC-AtUBC8-S, pACYCDuet-ARI5(ΔR12)-MYC-AtUBC8-S, pCDFDuet-MBP-TCP4-HA and pCDFDuet-MBP-TCP4-HA-AtUBA1-S were generated by Gibson assembly (SC612, Genesand)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eEscherichia coli\u003c/em\u003e strain BL21 (DE3) containing different combinations of the expression vectors were cultured in LB medium at 37°C until OD600 reached 0.5, at which point protein expression was induced with 0.5 mM IPTG followed by overnight incubation at 18°C.\u003c/p\u003e\u003cp\u003eFor the ARI5 self-ubiquitination assay, 300 µL of bacterial culture was directly lysed in 100 µL of 1×SDS loading buffer. The TCP4 ubiquitination assay required larger-scale processing, where 20 mL of culture was pelleted, resuspended in 10 mL PBS, and subjected to sonication. After centrifugation, the insoluble fraction was solubilized in 500 µL of 1×SDS buffer. All samples were separated by SDS-PAGE and analyzed by Western blotting using anti-MYC (ABclonal, AE009), anti-HA (ABclonal, AE008), anti-FLAG (Sigma, A8592), and anti-S (HUABIO, EM50105) antibodies.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003edegradation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess TCP4 protein stability, 14-d-old seedlings from 35S-MYC-mTCP4 and 35S-MYC-mTCP4 \u003cem\u003eari5 ari7\u003c/em\u003e transgenic plants were treated with 100 µM cycloheximide (CHX) in ½ MS solution for a time-course experiment. After treatment, seedlings were flash-frozen in liquid nitrogen and ground to a fine powder. Total proteins were extracted in extraction buffer for 1 h, followed by centrifugation (2 min, 12000×\u003cem\u003eg\u003c/em\u003e). Protein samples were analyzed by western blot using anti-MYC (AE009, ABclonal) and anti-Actin (AC009, ABclonal) antibodies, with ACTIN serving as the loading control. MYC-mTCP4 protein levels were quantified relative to ACTIN using ImageJ software.\u003c/p\u003e\u003cp\u003e \u003cb\u003eAnalysis of leaf curvature, hook curvature, style lenghth and width\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor the leaf curvature analysis, a 1 mm transverse section of the sixth leaves were excised at the leaf widest point perpendicular to the proximal axis. The leaf segments were imaged, with the α angle quantified using ImageJ. For hook curvature assessment in etiolated seedlings, plates were foil-wrapped and incubated vertically at 22°C for 4 d before imaging. Style morphology was examined by emasculating flowers at flower developmental stage 11, with apical gynoecium observed under a Leica M205 FCA stereoscope at 48 h post-emasculation. All morphometric parameters (leaf curvature angle, hook curvature, and style dimensions) were measured using ImageJ, with statistical significance determined by one-way ANOVA.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMicroscopy analysis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor scanning electron microscope (SEM) observation of the style, the flowers of WT, \u003cem\u003eari5 ari7\u003c/em\u003e and \u003cem\u003ejaw-5D\u003c/em\u003e were emasculated at flower developmental stage 11. The experiments utilized an integrated system consisting of an FEI Quanta FEG 450 scanning electron microscope (Thermo Fisher Scientific) and a Quorum PP3010T workstation (Quorum Technologies), which features a cryo-preparation chamber directly connected to the microscope. Plant samples were frozen in subcooled liquid nitrogen (− 210°C) and then transferred under vacuum to the cold stage of the chamber for sublimation (− 90°C, 5 min) and platinum sputter coating (10 mA, 30 s). Imaging was conducted using an electron beam at 5 kV and 3.5 nA with a working distance of 9.9 mm.\u003c/p\u003e\u003cp\u003eFor leaf cell morphology analysis, leaves from 21-d-old plants were fixed with ethanol : acetic acid : H\u003csub\u003e2\u003c/sub\u003eO (8.4 mL : 2 mL : 3.6 mL) for 16 h. Samples underwent sequential dehydration in 100% ethanol for 30 min twice and 70% ethanol for 30 min, followed by clearing in chloral hydrate solution (Leagene, DM0350) for 12 h. Cleared samples were imaged using DIC optics on a Zeiss Axio Imager M2 microscope.\u003c/p\u003e\u003cp\u003e \u003cb\u003eRNA extraction and quantitative real-time polymerase chain reaction assay\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTotal RNA was isolated from 7-d-old 35S-GFP-ARI5g, 35S-GFP-ARI7g, and wild-type seedlings using the Plant RNA Extraction Kit (TaKaRa, 9769). For cDNA synthesis, 1.5 µg of total RNA was reverse transcribed using M-MLV Reverse Transcriptase (Promega, M170A). Quantitative PCR was performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific) with UltraSYBR Mixture (CWBIO, CW2601M). Gene expression levels were normalized to ACTIN2 and calculated using the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method. All primer sequences are provided in Supplemental Table\u0026nbsp;2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eMaterial availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll materials needed to replicate the work are available.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReporting Summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther methodological details are available in the linked Nature Portfolio Reporting Summary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences of ARI5 (At1G05890), ARI6 (AT1G63760), ARI7 (At2G31510), TCP1 (AT1G67260), TCP2 (AT4G18390), TCP3 (AT1G53230), TCP4 (AT3G15030), TCP5 (AT5G60970), TCP10 (AT2G31070), TCP12 (AT1G68800), TCP13 (AT3G02150), TCP17 (AT5G08070), TCP18 (AT3G18550), TCP24 (AT1G30210) are available at TAIR (https:// www.arabidopsis.org/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dongping Lu (Shanghai Jiao Tong University) for kindly providing the plasmids used in the \u003cem\u003ein vitro\u003c/em\u003e ubiquitination assay; Jun Hu, Zhen-Yang Kong, and Ying-Chun Hu for assistance in SEM experiments at the Core Facilities of School of Life Science, Peking University; and Dong Liu and Qi Zhang for assistance in MS experiments at the National Center for Protein Sciences, Peking University. This work was supported by the National Natural Science Foundation of China (Grants No. 32370355 and 323B2008) and the Excellent Research Group Project of the National Natural Science Foundation of China (Grant No. 32488102). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.Q. conceived and designed the project. N.W conducted the experiments. G.Q., N.W., Y.C., Y.Wu, J.L. and Y.Wang analyzed the data. G.Q. and N.W. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e is available for this paper at .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u003c/strong\u003e should be addressed to G.Q.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeer review information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions information\u003c/strong\u003e is available at .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note:\u003c/strong\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGonzalez N, Vanhaeren H, Inze D (2012) Leaf size control: complex coordination of cell division and expansion. 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Plant Physiol 186:434\u0026ndash;451\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Chen H (2019) A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis. \u003cem\u003eaBIOTECH\u003c/em\u003e 1, 6\u0026ndash;14\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7027676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7027676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant organ size and morphology are crucial agronomic traits that influence both plant fitness and crop productivity. The TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors play pivotal roles in shaping plant morphology, yet the precise regulatory mechanisms governing their activity remain incompletely understood. Here, we identify the PARKIN-like E3 ubiquitin ligase ARIADNE5 (ARI5) as a key regulator of TCP4 stability, mediating its ubiquitination and subsequent degradation. We identified ARI5 interacted with TCP4 using immunoprecipitation-mass spectrometry (IP-MS). The \u003cem\u003eari5 ari7\u003c/em\u003e double mutant displays flatter leaves, shorter gynoecia, accelerated cotyledon opening in darkness, and earlier flowering. These phenotypes are strikingly opposite to those observed in \u003cem\u003etcp\u003c/em\u003e-deficient mutants. \u003cem\u003eARI5\u003c/em\u003e and its close homolog \u003cem\u003eARI7\u003c/em\u003e exhibit overlapping expression patterns with \u003cem\u003eTCP4\u003c/em\u003e, and their encoded proteins colocalize with TCP4 in the nucleus. ARI5 possesses E3 ubiquitin ligase activity, and promotes the ubiquitin-dependent degradation of TCP4. Our findings not only establish ARI5 as a critical regulator of plant organ morphology but also uncover a post-translational regulatory mechanism that fine-tunes TCP4 activity and thus cell division during organ morphogenesis through proteasomal degradation, highlighting the evolutionary conservation of PARKIN-like E3 ligases in modulating cell division across plants and humans.\u003c/p\u003e","manuscriptTitle":"The Arabidopsis PARKIN-like E3 ligase ARIADNE5 regulates plant development by targeting TCP4 for degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 10:04:27","doi":"10.21203/rs.3.rs-7027676/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0638e10d-209a-44a3-88f1-3aaa5eeaf1cf","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51739271,"name":"Biological sciences/Plant sciences/Plant development/Plant morphogenesis"},{"id":51739272,"name":"Biological sciences/Plant sciences/Plant molecular biology"},{"id":51739273,"name":"Biological sciences/Plant sciences/Plant development/Leaf development"}],"tags":[],"updatedAt":"2026-02-02T07:26:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-21 10:04:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7027676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7027676","identity":"rs-7027676","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}