Inactivation of GH3.5 by COP1-mediated K63-linked ubiquitination promotes seedling hypocotyl elongation

Liu, Yongting; Xie, Yinpeng; Xu, Dongqing; Deng, Xing Wang; Li, Jian

doi:10.1038/s41467-025-58767-6

Download PDF

Article
Open access
Published: 14 April 2025

Inactivation of GH3.5 by COP1-mediated K63-linked ubiquitination promotes seedling hypocotyl elongation

Nature Communications volume 16, Article number: 3541 (2025) Cite this article

9383 Accesses
2 Citations
9 Altmetric
Metrics details

Subjects

Abstract

CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), which was first discovered as a central repressor of photomorphogenesis in Arabidopsis, destabilizes proteins by ubiquitination in both plants and animals. However, it is unclear whether and how Arabidopsis COP1 mediates non-proteolytic ubiquitination to regulate photomorphogenesis. Here, we show that COP1-mediated lysine 63 (K63)-linked polyubiquitination inhibits the enzyme activity of GRETCHEN HAGEN 3.5 (GH3.5), a synthetase that conjugates amino acids to indole-3-acetic acid (IAA), thereby promoting hypocotyl elongation in the dark. We show that COP1 physically interacts with and genetically acts through GH3.5 to promote hypocotyl elongation. COP1 does not affect GH3.5 protein stability; however, it suppresses GH3.5 activity through K63-linked ubiquitination in the dark, inhibiting the endogenous conversion of IAA to IAA-amino acid conjugates. Further, light regulates IAA metabolism by suppressing the inhibitory effect of COP1 on the function of GH3.5 and its homologs. Our results shed light on the non-proteolytic role of COP1-mediated ubiquitination and the mechanism by which light regulates auxin metabolism to modulate hypocotyl elongation.

Role of myo-inositol during skotomorphogenesis in Arabidopsis

Article Open access 15 October 2020

A distinct giant coat protein complex II vesicle population in Arabidopsis thaliana

Article 07 October 2021

Arabidopsis COP1 guides stomatal response in guard cells through pH regulation

Article Open access 05 February 2024

Introduction

One of the most important environmental cues, light plays a crucial role in seedling morphogenesis. Hypocotyl elongation is among the most remarkable seedling morphogenic events. Rapid hypocotyl elongation in the dark (e.g., under soil) favors seedling exposure to light, and light inhibits excessive hypocotyl elongation¹. In Arabidopsis, different photoreceptors have evolved to perceive specific wavelengths of sunlight. Light-activated photoreceptors suppress the accumulation and activity of central repressors to release the function of light-responsive transcription factors, thereby suppressing hypocotyl elongation and promoting photomorphogenesis².

The ubiquitin-proteasome system for protein degradation is conserved in eukaryotes and modulates plant growth and development processes, including seedling photomorphogenesis³. Ubiquitin ligase (E3) attaches ubiquitin moieties to substrates and targets them to the 26S proteasome for degradation. During light-controlled morphogenesis in Arabidopsis seedlings, COP1 functions as an E3 ligase and plays a critical role in destabilizing key signaling factors to repress photomorphogenesis in the dark². In Arabidopsis, COP1 is a Really Interesting New Gene (RING)-type E3 that contains three distinct domains from its N-terminus to the C-terminus: RING, coiled-coil, and WD40 repeat domains. The RING domain normally mediates the interaction of COP1 with the ubiquitin-conjugating enzyme (E2). The coiled-coil domain mediates the interaction with accessory proteins, and the WD40 domain mediates the interaction with substrate proteins⁴. Light signaling tightly controls the E3 activity of COP1. Light-activated photoreceptors, such as phytochromes and cryptochromes, modulate the physical interaction of COP1 and its accessory proteins SUPPRESSOR OF phyA-105 (SPAs), thereby inhibiting the E3 activity of COP1 in vivo^5,6. Additionally, photoactivated cryptochromes prevent the interaction of COP1 with its substrate, thereby inhibiting COP1-mediated ubiquitination⁷.

Ubiquitination involves successive enzymatic reactions mediated by three enzymes, namely, the ubiquitin-activating enzyme (E1), E2, and E3. E1 catalyzes the formation of a high-energy thioester bond between itself and ubiquitin, thereby activating ubiquitin. The activated ubiquitin is then transferred to E2, and E3 interacts with both E2 and the substrate and serves as a scaffold to transfer ubiquitin from E2 to the substrate. An isopeptide bond is formed between the C-terminal glycine residue of the ubiquitin and the ε-amino group of the lysine residue of the substrate⁸. A ubiquitin protein contains seven lysine (K) residues, namely, K6, K11, K27, K29, K33, K48, and K63. Each lysine on ubiquitin serves as an acceptor site for the next ubiquitin, thereby forming polyubiquitin chains with different linkages⁸. K48-linked ubiquitination is the most common ubiquitin modification type in animals and plants^{9,10,11,12,13}. Proteins labeled with K48-linked ubiquitin chains are usually targeted to the 26S proteasome for degradation¹⁴. K63 ubiquitination is the second most common type. Rather than triggering protein degradation, K63 ubiquitination in animals plays a non-proteolytic role in many cases by regulating protein interaction, subcellular localization, and biochemical activity¹⁵. In plants, the function of K63 ubiquitination is involved in processes such as regulating cargo endocytosis and stabilizing proteins^16,17. However, only a few exact E3-substrate pairs of K63 ubiquitination have been reported in plants^18,19,20,21. Although there are more than 1400 E3s in Arabidopsis, only two, PLANT U-BOX 25/26 (PUB25/PUB26)¹⁹ and ARABIDOPSIS TOXICOS EN LEVADURA 31 (ATL31)²⁰, have been identified as attaching K63 chains to known substrates in vivo. More than 50 substrates of Arabidopsis COP1 have been identified to date, all of which are degraded by COP1-mediated ubiquitination²². However, whether COP1 also has a non-proteolytic ubiquitination role is currently unknown.

As the first discovered phytohormone, auxin plays a fundamental role in cell expansion²³. Inhibition of auxin biosynthesis impairs hypocotyl elongation in the dark, suggesting that auxin promotes this process²⁴. Moderate auxin levels can promote cell elongation, but excessive auxin levels inhibit it²⁵. Therefore, auxin homeostasis, which is regulated by auxin biosynthesis and metabolism, is crucial for auxin-driven plant growth. IAA is the predominant natural auxin in plants²⁶, and its inactivation of IAA mainly involves GRETCHEN HAGEN 3 (GH3) and DIOXYGENASE FOR AUXIN OXIDATION (DAO)²⁷. GH3 genes encode acyl amido synthetases that conjugate different substrates with amino acids. In Arabidopsis, there are 19 members of the GH3 gene family, which are divided into three groups according to sequence homology and substrate preference^28,29. Group II GH3 proteins, which consist of eight members (GH3.1–GH3.6, GH3.9, and GH3.17), inactivate IAA mainly redundantly by IAA conjugation with aspartic acid (Asp) or glutamic acid (Glu)³⁰. The IAA-amino acid conjugates can then be irreversibly oxidized to oxIAA-amino acid conjugates by DAO proteins³¹. Genetic evidence has shown that the group II GH3 family is redundantly involved in inhibiting hypocotyl elongation. Transgenic plants overexpressing any single group II GH3 family member, such as GH3.2, GH3.5, and GH3.6, show short hypocotyls and a dwarf phenotype in the light^32,33,34,35. In contrast, the absence of the entire group II GH3 family in Arabidopsis dramatically promotes hypocotyl elongation in light^36,37. Interestingly, auxin induces the expression of several group II GH3s, including GH3.1–GH3.6, although group II GH3s play a role in auxin inactivation, suggesting that they act as negative feedback regulators to prevent active auxin overaccumulation³⁸.

Although numerous studies have uncovered the mechanisms underlying light-regulated auxin biosynthesis and signal transduction³⁹, it is unclear whether and how light regulates auxin metabolism. Here, we showed that COP1 attached K63 ubiquitin chains to GH3.5 in the dark and inhibited the enzymatic activity of GH3.5, rather than regulating its protein stability, thereby regulating auxin metabolism in vivo. Our results shed light on the non-proteolytic role of COP1-mediated K63 ubiquitination and provide insight into light-regulated auxin metabolism.

Results

Screening of potential substrates for COP1-mediated K63 ubiquitination

We used linkage-nonspecific and -specific ubiquitin antibodies to determine the global ubiquitination level in cop1 mutants grown in the dark in order to determine whether COP1 contributes to the formation of K63 polyubiquitin chains in Arabidopsis. The total ubiquitination level, which represented a mixture of various ubiquitin chains, decreased significantly in the cop1-4 and cop1-6 mutants (Supplementary Fig. 1a). Further, similar to the double mutant of UBC35 and UBC36^40,41, the E2s that specifically catalyze the formation of K63-linked ubiquitin chains in Arabidopsis, K63-linked ubiquitin chain accumulation was also significantly impaired in cop1-4 and cop1-6 (Supplementary Fig. 1b). These results suggest that COP1 is involved in the formation of K63 ubiquitin chains in the dark. Thus, we performed two types of immunoprecipitation-mass spectrometry (IP-MS) to screen potential substrates for COP1-mediated K63 ubiquitination (Supplementary Fig. 2). We used the K63 tandem ubiquitin binding entity (K63 TUBE)⁴² to capture proteins with K63 ubiquitin chains from Col and cop1-4, and then identified proteins whose K63 ubiquitination was dependent on COP1. We used GFP-Trap® beads (ChromoTek) to co-immunoprecipitate COP1-interacting proteins from YFP-COP1 transgenic plants⁴³. The proteins that were simultaneously detected by these two strategies were suspected to be the candidates of interest (Supplementary Fig. 2). Thus, we found that GH3.6, a member of the GH3 synthetases family, was a substrate candidate for COP1-mediated K63 ubiquitination.

COP1 genetically interacts with GH3.5 in the dark

It has already been reported that the GH3 synthetase family in Arabidopsis is divided into three groups^28,29. Group II GH3 genes in Arabidopsis consist of eight members, including GH3.6 and its closest homolog, GH3.5³⁰. Therefore, we investigated the genetic interaction of COP1 and these two GH3 genes by crossing the gh3 mutations into cop1-4. As shown in Fig. 1a, b, the hypocotyls of cop1-4 gh3.6 in the dark were the same length as those of cop1-4. However, in the dark, the hypocotyls of the cop1-4 gh3.5-1 double mutants and cop1-4 gh3.5-1 gh3.6 triple mutants were the same length and significantly longer than those of cop1-4, suggesting that functional GH3.5, but not GH3.6, contributes to the short hypocotyl of cop1-4 in the dark. Knockout of four members, namely, GH3.5, GH3.6, GH3.9, and GH3.17, of group II GH3 genes in cop1-4 further promoted the hypocotyl elongation of cop1-4 in the dark, indicating the obvious contribution of group II GH3 genes to short hypocotyls in cop1-4. We applied nalacin, a chemical inhibitor of group II GH3 enzymes⁴⁴, to Col and cop1-4 seedlings in the dark to further identify the effect of the GH3 family on short hypocotyls in cop1-4 (Fig. 1c, d). The hypocotyls of cop1-4 were significantly elongated when treated with nalacin in the dark. These results confirm that COP1 antagonizes the function of GH3.5 and other group II GH3s to promote hypocotyl elongation in the dark.

**Fig. 1: *COP1* genetically interacts with group II *GH3s* in the dark.**

COP1 physically interacts with GH3.5

As GH3.5, but not GH3.6, played a significant role in COP1-regulated hypocotyl elongation in the dark (Fig. 1a, b), we investigated the physical interaction of COP1 and GH3.5. We purified recombinant GST-GH3.5 and MBP-COP1 using a prokaryotic expression system. The in vitro pull-down assay showed that COP1 interacted directly with GH3.5 (Fig. 2a). The in vivo interaction of COP1 and GH3.5 was confirmed with firefly luciferase complementation imaging (LCI) and coimmunoprecipitation (Co-IP) assays (Fig. 2b, c). Additionally, the bimolecular fluorescence complementation (BiFC) assay in Arabidopsis protoplasts showed a clear interaction between COP1 and GH3.5 in the nucleus (Fig. 2d). To map the subdomain of COP1 that interacted with GH3.5, we first divided full-length COP1 into the N- and C-terminal domains and found that the C-terminal domain of COP1 directly interacted with GH3.5 in vitro (Fig. 2e, f). We then examined the in vivo interaction between the truncated COP1 proteins and GH3.5 using the LCI assay in tobacco leaves. The C-terminal end of COP1 also interacted with GH3.5 in vivo (Fig. 2e, g). However, deletion of the RING, coiled-coil, or WD40 domain in COP1 alone had no effect on the interaction between COP1 and GH3.5 in vivo (Fig. 2e, g). Overall, these results demonstrate that COP1 physically interacts with GH3.5 in the nucleus and that the C-terminus of COP1 is essential for this interaction.

COP1 has no effect on the protein stability of GH3.5 in the dark

Because COP1 and GH3.5 antagonistically regulate hypocotyl elongation in the dark (Fig. 1), it is possible that COP1 regulates the mRNA or protein levels of GH3.5. As shown in Fig. 3a, the mRNA level of GH3.5 was significantly decreased in cop1-4 compared with Col in the dark, suggesting that COP1 maintains GH3.5 expression in the dark. Prior studies have shown that GH3.5 expression is induced by auxin, which may serve as a negative feedback mechanism to balance the auxin pathway with growth in planta³⁸. GH3.5 expression in cop1-4 was partially restored by the application of 2,4-dichlorophenoxyacetic acid (2,4-D), a synthetic auxin analogue (Supplementary Fig. 3), suggesting that the low GH3.5 expression in cop1-4 is partially due to an abnormal auxin pathway in cop1-4.

COP1 functions as a well-known ubiquitin ligase that promotes protein degradation. We generated a GH3.5-GFP/Col overexpressing plant, driven by the UBQ10 promoter and introduced into cop1-4, in order to determine the effect of COP1 on the protein stability of GH3.5. In the dark, the abundance of GH3.5-GFP in Col was the same as that in cop1-4 (Fig. 3b). When etiolated Col and cop1-4 seedlings were treated with cycloheximide (CHX), an inhibitor of protein biosynthesis, the degradation rate of pre-existing GH3.5-GFP in Col was comparable to that in cop1-4 (Fig. 3c, d). These results show that COP1 has no effect on the protein stability of GH3.5 in the dark.

COP1 mediates K63 ubiquitination of GH3.5 in the dark

We first performed an in vitro ubiquitination assay and found that COP1 functioned as a ubiquitin ligase to polyubiquitinate GH3.5 in vitro (Fig. 4a). Transient expression experiments were performed in Arabidopsis protoplasts to further test the in vivo modification of GH3.5 by COP1-mediated ubiquitination, especially K63 ubiquitination. GH3.5-GFP was transiently co-expressed with HA- and FLAG-tagged COP1 (HF-COP1)⁴⁵ in protoplasts in the dark. GH3.5-GFP proteins were immunoprecipitated from protoplasts, and their polyubiquitination level was detected with pan-ubiquitin and K63 linkage-specific ubiquitin antibodies. Compared to the sole expression of GH3.5-GFP in protoplasts, the co-expression of GH3.5-GFP and HF-COP1 in protoplasts resulted in higher levels of pan-ubiquitination and K63 ubiquitination of GH3.5-GFP (Fig. 4b). Further, compared to the expression of wild-type (WT) ubiquitin proteins in protoplasts, K63R mutant ubiquitin expression resulted in impaired ubiquitination of GH3.5 by COP1 (Fig. 4c). These results suggest an important role for COP1 in the K63 ubiquitination of GH3.5 in vivo. Stably overexpressed GH3.5 proteins GH3.5-GFP/Col and GH3.5-GFP/cop1-4 (Fig. 3) were used to determine COP1-mediated K63 ubiquitination of GH3.5 in planta. As shown in Fig. 4d, e, pan-ubiquitination of GH3.5-GFP was significantly decreased in cop1-4 compared to Col. The K48 ubiquitination level of GH3.5-GFP in cop1-4 was comparable to that in Col, which was consistent with the finding that COP1 had no effect on GH3.5 stability (Fig. 3b–d). However, K63 ubiquitination of GH3.5-GFP was significantly reduced in cop1-4 (Fig. 4e). These experiments demonstrate that COP1 mainly attaches K63 ubiquitin chains to GH3.5 in vivo in the dark.

**Fig. 4: COP1 mediates K63-linked ubiquitination of GH3.5 in the dark.**

COP1 inhibits GH3.5 activity by K63 ubiquitination

The genetic interaction of COP1 and GH3.5 showed an inhibitory effect of COP1 on GH3.5 function in the dark (Fig. 1a, b). Because COP1 had no effect on the stability of GH3.5 in the dark (Fig. 3b–d), we determined whether COP1-mediated K63 ubiquitination inhibited the enzyme activity of GH3.5. Prior studies have shown that overexpressing group II GH3 genes promote the conversion of IAA to IAA-amino acid conjugates, thereby increasing seedling resistance to the inhibitory effect of exogenous IAA on primary root growth^30,46. The primary root growth of cop1-4 was significantly resistant to exogenous IAA in the dark (Fig. 5a). Further, the loss of GH3.5 in cop1-4 restored the sensitivity of cop1-4 to IAA treatment (Fig. 5a), indicating that the hyposensitivity of cop1-4 to IAA may be due to the increased function of GH3.5 in cop1-4. A prior study has shown that nalacin inhibits the primary root growth of seedlings by inhibiting group II GH3 activity⁴⁴. We thus examined the primary root growth of cop1-4 when treated with nalacin in the dark. As shown in Fig. 5b, the primary root growth of cop1-4 was hyposensitive to nalacin treatment compared to Col, indicating increased GH3 activity in cop1-4 in the dark.

**Fig. 5: COP1-mediated K63 ubiquitination inhibits GH3.5 activity.**

We then prepared GST-GH3.5 proteins linked to K63 ubiquitin chains (GST-GH3.5-K63) by COP1-mediated ubiquitination in vitro (Fig. 5c), and we performed an in vitro enzyme reaction to analyze the effect of COP1-mediated K63 ubiquitination on GH3.5 activity. Compared to GST-GH3.5 without ubiquitin chains (GST-GH3.5), the yield of IAA-Asp conjugated by GST-GH3.5-K63 decreased dramatically (Fig. 5d, e). Kinetic analysis of the enzymatic reaction further revealed an almost 6-fold reduction in the catalytic activity of GST-GH3.5-K63 compared to GST-GH3.5 proteins (Fig. 5f). K_m^IAA was only slightly lower for GST-GH3.5-K63 than that for GST-GH3.5 (Fig. 5f). These results suggest that COP1-mediated K63 ubiquitination can strongly inhibit the catalytic activity of GH3.5 synthetase in vitro, possibly through noncompetitive inhibition. We measured the levels of IAA and IAA-amino acid conjugates in the etiolated seedlings to further verify GH3.5 activity inhibition by COP1 in vivo (Fig. 5g). The free IAA content in cop1-4 was comparable to that in Col. However, the contents of IAA-amino acid conjugates, such as IAA-Asp and IAA-Glu, were significantly higher in cop1-4 than in Col. The loss of GH3.5 in cop1-4 significantly reduced the IAA-Asp content in cop1-4. Knockout of more group II GH3 genes (including GH3.5, GH3.6, GH3.9, and GH3.17) in cop1-4 even more significantly reduced the IAA-Asp and IAA-Glu conjugate levels in cop1-4. Altogether, these results demonstrate that COP1-mediated K63 ubiquitination of GH3.5 inhibits its activity in the dark.

Light regulates IAA metabolism by suppressing the function of COP1

Because COP1 is a key repressor of the light signaling pathway and its function is suppressed by light via multiple regulatory mechanisms, we determined whether light also regulated GH3.5 activity in a COP1-associated manner. We first tested the possible role of light in regulating the GH3.5 mRNA and protein levels. RT-qPCR data showed that GH3.5 expression was significantly lower in continuous white light (cWL) than in continuous darkness (cD), and it gradually decreased when Col seedlings switched from dark to light (Supplementary Fig. 4a). Additionally, the differential expression of GH3.5 in dark-grown Col and cop1-4 seedlings disappeared when the seedlings were grown in the light (Supplementary Fig. 4b), suggesting that light partially inhibits GH3.5 expression by repressing the function of COP1. The GH3.5-GFP/Col overexpressing line was used to analyze the protein stability of GH3.5 regulated by light. The protein levels of GH3.5 did not differ when the seedlings were grown under different light conditions (Supplementary Fig. 4c), indicating that light had no effect on the GH3.5 protein stability.

To avoid the interference caused by the light-controlled expression of GH3.5, we used a GH3.5-GFP overexpressing line (Fig. 3) to examine the light-regulated K63 ubiquitination of GH3.5. Both GH3.5-GFP/Col and GH3.5-GFP/cop1-4 seedlings were grown in cD or cWL. GH3.5-GFP proteins were immunoprecipitated from these materials and used to analyze the K63 ubiquitination level. GH3.5-GFP/Col seedlings grown in the light showed a significantly lower K63 ubiquitination level for GH3.5-GFP compared to dark-grown seedlings (Fig. 6a), suggesting that light can suppress K63 ubiquitination of GH3.5. The K63 ubiquitination level of GH3.5-GFP in cop1-4 did not differ in either dark or light, and it was significantly lower than the level in Col grown in the dark (Fig. 6a). This showed that inhibiting the function of COP1 is required for light-suppressed K63 ubiquitination of GH3.5.

**Fig. 6: Light regulates IAA metabolism by suppressing COP1 function.**

To further confirm the in vivo enhancement of GH3.5 activity by light, we measured the IAA and IAA-Asp conjugate levels in seedlings grown in cD, as well as those grown in cWL. In Col, light had no effect on the endogenous IAA content (Fig. 6b) but suppressed IAA-Asp accumulation (Fig. 6c) and promoted oxIAA-Asp accumulation (Fig. 6d), the oxidized product of IAA-Asp (Fig. 6c). A prior study has shown that IAA-Asp can be directly converted to oxIAA-Asp by DAO1³¹. Therefore, the lower content of endogenous IAA-Asp in the light might be due to the faster conversion of IAA-Asp to oxIAA-Asp. As shown in Fig. 6e, the average content of endogenous IAA-Asp and its oxidized form (IAA-Asp + oxIAA-Asp) in light-grown Col was about 62.04 ng g⁻¹ F.W., which was much higher than in dark-grown Col (about 20.90 ng g⁻¹ F.W.). These results suggest that light promotes the production of different IAA-Asp conjugates, consisting of IAA-Asp and oxIAA-Asp. Light-promoted production of IAA-Asp + oxIAA-Asp was significantly attenuated in the gh3(5/6/9/17) quadruple mutant (Fig. 6e), suggesting that light promotes IAA-Asp + oxIAA-Asp production in a GH3-dependent manner. Further, the loss of function of COP1 resulted in constitutive IAA-Asp + oxIAA-Asp overaccumulation in both dark and light (Fig. 6e), suggesting a negative role of COP1 in the light-promoted accumulation of IAA-Asp + oxIAA-Asp. Knockout of GH3.5, GH3.6, GH3.9, and GH3.17 in cop1-4 again inhibited the excessive IAA-Asp + oxIAA-Asp accumulation in cop1-4 in the light (Fig. 6e). To further test whether light-regulated IAA metabolism contributes to the modulation of hypocotyl elongation, we analyzed the hypocotyl lengths of seedlings grown in cD or cWL (Supplementary Fig. 5). Light-inhibited hypocotyl elongation was significantly attenuated in the gh3(5/6/9/17) mutant (Supplementary Fig. 5), which was consistent with the reduced production of IAA-Asp + oxIAA-Asp in gh3(5/6/9/17) when exposed to light (Fig. 6e). Moreover, knockout of the four GH3s in cop1-4 partially restored hypocotyl elongation of cop1-4 when under light (Supplementary Fig. 5), indicating the existence of an inverse correlation between hypocotyl elongation and IAA-Asp + oxIAA-Asp accumulation in cop1-4 (Fig. 6e). These results show that light regulates IAA metabolism by suppressing the inhibitory effect of COP1 on the function of group II GH3s, thereby modulating hypocotyl elongation.

Discussion

Numerous studies have established a comprehensive regulatory network based on COP1-mediated ubiquitination and the degradation of key signaling factors in the regulation of plant growth and development²². However, the non-proteolytic role of ubiquitination in plants is gradually coming into focus^19,47. In this study, we revealed that non-proteolytic ubiquitination of GH3.5 by COP1 promotes hypocotyl elongation in the dark (Fig. 7). In the dark, functional COP1 physically interacts with GH3.5 in the nucleus and mediates the K63 ubiquitination of GH3.5. The enzyme activity of GH3.5 is inhibited by ubiquitination, limiting the conversion of IAA to IAA-Asp conjugates, thereby promoting hypocotyl elongation. In the light, COP1 inactivation impairs K63 ubiquitination of GH3.5, leading to GH3.5 activation. Activated GH3.5 efficiently promotes the conversion of IAA to IAA-Asp conjugates, thereby inhibiting hypocotyl elongation. Thus, the COP1-mediated K63 ubiquitination of GH3.5 plays an important role in light-regulated auxin metabolism and hypocotyl elongation (Fig. 7).

**Fig. 7: A working model illustrating how COP1-mediated K63 ubiquitination regulates hypocotyl elongation.**

Our results showed an interaction between COP1 and GH3.5 in the nucleus (Fig. 2d), indicating that COP1 modulates GH3.5 activity through K63 ubiquitination in the nucleus (Fig. 7). However, the subcellular localization of group II GH3s in Arabidopsis remains unclear³⁸. The GH3s of several other species that produce IAA-amino acid conjugates have been reported to be localized in both the cytoplasm and the nucleus^48,49,50. Moreover, the GFP fluorescence signals from the GH3.5-GFP/Col line indicate that the GH3.5 proteins are localized in both the cytoplasm and nucleus (Fig. 3, Supplementary Fig. 6), supporting the nuclear interaction between COP1 and GH3.5 (Figs. 2d and 7). Classical auxin perception and signaling transduction by TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) receptors occur in the nucleus, and are critical for regulating gene expression and plant growth and development^51,52. It is likely that the nuclear localization of COP1-GH3.5 regulation provides a rapid mechanism to modulate the nuclear auxin pathway to regulate gene expression.

It has been well established that COP1 targets key signaling factors for ubiquitination and proteasomal degradation in the dark²². In contrast, we demonstrated the non-proteolytic role of COP1-mediated ubiquitination in this study. Prior reports have shown that the same E3 ligase can ubiquitinate different substrates with different ubiquitin chains in mammals and plants. For example, CELLULAR INHIBITOR OF APOPTOSIS-1 (cIAP1), a mammalian RING-type E3 ligase, directs proteasomal degradation of RECEPTOR-INTERACTING PROTEIN KINASE 1 (RIPK1) through K48 ubiquitination, thereby repressing cell death⁵³. K63 ubiquitination of RECEPTOR INTERACTING PROTEIN 1 (RIP1), mediated by cIAPs, alters RIP1-containing signaling complex assembly and thus facilitates cancer cell survival⁵⁴. PUB25/PUB26 target BOTRYTIS-INDUCED KINASE 1 (BIK1) for degradation to suppress the immune response of Arabidopsis⁵⁵. Otherwise, they can restore MYB15 transcription factor accumulation through K63 ubiquitination in response to prolonged cold exposure¹⁹. IPA1 INTERACTING PROTEIN1 (IPI1) is also a RING domain-containing E3 ligase in Oryza sativa. It ubiquitinates the same substrate, IDEAL PLANT ARCHITECTURE1 (IPA1), with different ubiquitin chains in different tissues to modulate the architecture of rice¹⁸. Further efforts are required to decipher the mechanism by which COP1 determines the linkage types of ubiquitin chains. With regard to RING-type E3-mediated ubiquitination, linkage specificity is mainly determined by the interactive E2 enzyme^56,57. UBC13, a member of the E2 family, specifically catalyzes K63 ubiquitin chain formation in yeast, mammals, and plants¹⁶. The RING-type E3 ligase cIAP1 can cooperate with UBC13 to catalyze K63 ubiquitination of RIP1 in mammals⁵⁴. It is reasonable to assume that COP1 can pair up with UBC35/UBC36 (UBC13 homologs in Arabidopsis)⁴¹ to mediate K63 ubiquitination of GH3.5. Additionally, COP1 conditionally serves as a subunit of a multimeric CULLIN4-DNA DAMAGE BINDING PROTEIN 1^COP1–SPA (CUL4–DDB1^COP1–SPA) E3 ligase complex. In this E3 complex, COP1-SPA functions as a substrate receptor to recruit substrate proteins, while RING BOX 1 (RBX1), an evolutionarily conserved RING domain-containing protein, recruits E2 enzymes to the complex^58,59. The mammalian CULLIN1-based E3 ligase complex likely recruits UBC13 via RBX1 to promote K63 ubiquitination of the substrate⁶⁰. Whether CUL4–DDB1^COP1–SPA E3 complex also recruits UBC35/UBC36 via RBX1 to mediate K63 ubiquitination in Arabidopsis remains to be investigated in the future. It also remains a possibility that since COP1 itself has the RING domain for potential E3 catalytic activity^61,62, it is also feasible that E3 activity for K63 ubiquitination is derived only from COP1/SPA complexes (or even free COP1 alone), without CUL4 association. In the case of legume–rhizobium symbiosis, phosphorylation of Lotus japonicus NFR-INTERACTING RING-TYPE E3 LIGASE 1 (NIRE1) in response to rhizobium inoculation induces the switch of NIRE1 from K48 to K63 ubiquitination activity²¹. COP1 has been reported to be modified at the post-translational level, for example by ubiquitination, SUMOylation, and phosphorylation^43,63,64. Whether these modifications are responsible for the selection of chain linkages by COP1 should also be considered.

GH3.5 is a member of the group II GH3 family, which comprises eight members and redundantly catalyzes IAA to IAA-amino acid conjugates³⁸. Our results showed that functional GH3.5 and its three homologous genes contribute to short hypocotyls in cop1-4 (Fig. 1). To further assess the role of group II GH3s in COP1-regulated hypocotyl elongation, we mutated COP1 in the Col and gh3-septuple (knockout of the entire group II GH3 family except GH3.9) lines. The cop1-4 mutant has a C-to-T mutation in COP1 genomic DNA, which changes the Gln²⁸³ codon (CAA) to a stop codon (UAA)⁶⁵. According to the mutational information of cop1-4, we generated independent cop1-4 like (cop1-4l) mutants in Col and gh3-septuple backgrounds using the CRISPR/Cas9 system (Supplementary Fig. 7a). The hypocotyls of the cop1-4l gh3-septuple mutant were the same length as those of the gh3-septuple mutant and much longer than those of the cop1-4l single mutant (Supplementary Fig. 7b, c). This genetic interaction indicates that the entire group II GH3 family plays a very important role in COP1-regulated hypocotyl elongation. It will be worthwhile to analyze the correlation between COP1 and the individual members of group II GH3s in detail in the future.

Our results showed that COP1-mediated K63 ubiquitination suppresses GH3.5 enzyme activity (Figs. 4 and 5). Nonetheless, the molecular mechanism of the inhibition of GH3.5 activity by ubiquitination is still unclear. Our kinetic analysis of the enzymatic reaction showed only a slight decrease in K_m^IAA for GH3.5 when GH3.5 was modified by COP1-mediated K63 ubiquitination (Fig. 5f), suggesting that this ubiquitination does not affect the affinity of GH3.5 for IAA in vitro. GH3.5 catalyzes IAA-Asp formation via a two-step reaction mechanism. In the first step, the intermediate IAA-adenosine monophosphate (IAA-AMP) is formed in the presence of adenosine triphosphate (ATP); in the second step, AMP is displaced by Asp, resulting in the end product IAA-Asp⁶⁶. Similar to the crystal structures of other members of the GH3 family^44,67, GH3.5 has a large N-terminal domain and a small C-terminal domain, and they are connected by a flexible hinge loop⁴⁴. The active catalytic sites are located at the interface of the N- and C-terminal domains. The rotation of the C-terminal domain through the hinge loop allows residues around the active site to interact with substrates, which is necessary for the success of a continuous two-step enzymatic reaction^44,66,67. The binding of K63 ubiquitin chains to GH3.5 by COP1 may disrupt this conformational change, thereby inhibiting the GH3.5-mediated enzymatic reaction. Further efforts to identify the ubiquitination sites on GH3.5 will be helpful in investigating the structural mechanism underlying the inhibition of GH3.5 activity by COP1-mediated ubiquitination.

In summary, our study reveals the non-proteolytic role of COP1-mediated K63 ubiquitination in the regulation of photomorphogenesis and sheds light on the mechanism by which light regulates auxin metabolism to modulate seedling photomorphogenesis.

Methods

Plant materials, growth conditions and phenotypic analysis

The gh3.5-1 (SALK_033434C), gh3.6 (SALK_013458C), gh3(5/6/9/17), gh3(1/2/3/4/5/6/17) septuple mutant, ubc35-1 ubc36-1, cop1-4, cop1-6, and YFP-COP1/cop1-6 have been reported previously^{37,40,41,43,65,68,69}. Seeds were surface sterilized with 15% (v/v) sodium hypochlorite solution for 10 min and then grown on Murashige and Skoog (MS) medium (Sigma-Aldrich, pH 5.7) supplemented with 1% (w/v) sucrose (Sangon Biotech) and 0.6% (w/v) agar (Sigma-Aldrich). After stratification at 4 °C in the dark for 2 days, the plates were transferred to continuous white light (cWL, 50 μmol m⁻² s⁻¹) at 22 °C for 12 h to induce seed germination. Seeds were then grown at 22 °C in continuous darkness or under other light conditions as indicated. Tobacco (Nicotiana benthamiana) plants were grown in a greenhouse under a 16 h light/8 h dark cycle at 28 °C. Four-day-old etiolated Arabidopsis seedlings were photographed with a camera (EOS80D, Canon), and hypocotyl or root lengths were calculated with ImageJ software.

Plasmid construction and generation of transgenic plants

To generate pSY736-YFP^N-COP1 and pSY735-YFP^C-GH3.5 constructs for the BiFC experiment, the coding sequence (CDS) of COP1 was amplified by PCR and cloned into the SalI/SpeI sites of the pSY736 vector⁷⁰, GH3.5 CDS was amplified and inserted into the SpeI/BamHI sites of the pSY 735 vector⁷⁰.

To generate cLUC-COP1 constructs for LCI assays, the full-length CDS and various truncated CDS of COP1 were cloned into the KpnI/SalI sites of the pCAMBIA1300-cLUC vector⁷¹. To generate the GH3.5-nLUC construct, the full-length CDS of GH3.5 was inserted into the KpnI/SalI sites of the pCAMBIA1300-nLUC vector⁷¹.

To generate pSY738-35S:2×MYC-Ub and pSY738-35S:2×MYC-Ub-K63R constructs for transient expression in Arabidopsis protoplasts, 2×MYC and ubiquitin (Ub) DNA fragments were amplified individually, Ub-K63R mutation DNA was obtained by primer-based site-directed mutagenesis using wild-type Ub as template, then 2×MYC together with Ub/Ub-K63R was cloned into the SalI/NotI sites of the pSY 738 vector⁷⁰ using the ClonExpress II One Step Cloning Kit (Vazyme).

The N- and C-terminal DNA fragments of COP1 were cloned into the EcoRI/SalI sites of the pMAL-c2X vector, to generate the pMAL-c2X-MBP-COP1-N and pMAL-c2X-MBP-COP1-C constructs. For purification of GST-GH3.5 and His-UBC10 recombinant proteins, the GH3.5 CDS was inserted into the BamHI/EcoRI sites of the pGEX4T-1 vector, the UBC10 CDS was amplified from the Col cDNA and inserted into the BamHI/EcoRI sites of the pET28a vector. To generate His-Ub and His-Ub-K63 (all lysine residues of ubiquitin were substituted by arginine, except K63) constructs, Ub and Ub-K63 fragments were cloned into the BamHI/EcoRI sites of the pET28a vector.

To generate UBQ10_pro:GH3.5-GFP transgenic plants, Arabidopsis UBQ10 fragment⁷², GH3.5 and GFP CDS fragments were recombined into the SalI/BamHI sites of the pCAMBIA1300 vector using the ClonExpress II One Step Cloning Kit (Vazyme). The Agrobacterium tumefaciens strain GV3101 carrying the construct was used to transform Col plants. Homozygous transgenic plants were screened on MS medium with an addition of 50 mg/L hygromycin. The cop1-4 like (cop1-4l) mutants were generated using the CIRSPR/Cas9 system⁷³ in the background of Col and gh3-septuple. Two target sequences for COP1 were selected using the CRISPR-P web tool (http://crispr.hzau.edu.cn/CRISPR/)⁷⁴. All constructs were confirmed by DNA sequencing. The primer sequences used in this study are listed in Supplementary Table 1.

Western blot analysis

Arabidopsis seedlings were harvested, ground into powder, and lysed in protein extraction buffer containing 8 M urea, 100 mM NaH₂PO₄, 100 mM Tris-HCl (pH 8.0), 1× phenylmethanesulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (Roche). Samples were centrifuged at 13,000 × g for 10 min at 4 °C, then the supernatants were transferred into new tubes. The total protein concentration was quantified using the Bradford assay reagent (Bio-Rad). The same amounts of total proteins were boiled for 10 min with 1× SDS loading buffer at 100 °C and separated in 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels, followed by immunoblotting detection. Immunoblotting results were visualized using chemiluminescence imaging system (Tanon).

All antibodies used in this study are listed below: anti-MBP (#E8032S, New England Biolabs, 1:5000 dilution), anti-GST (AE006, Abclonal, 1:1000 dilution), anti-Flag (F3165, Sigma-Aldrich, 1:2000 dilution), anti-MYC (C3956, Sigma-Aldrich, 1:2000 dilution), anti-GFP (M20004, Abmart, 1:5000 dilution), anti-COP1 (1:1000 dilution, homemade), anti-Actin (AC009, Abclonal, 1:2000 dilution), anti-Histone H3 (H0164, Sigma-Aldrich, 1:5000 dilution), anti-HSP (AbM51099-31-PU, Beijing Protein Innovation, 1:5000 dilution), anti-pan-Ub (sc-8017, Santa Cruz, 1:1000 dilution), anti-K63-Ub (5621S, Cell Signaling Technology, 1:1000 dilution), and anti-K48-Ub (8081S, Cell Signaling Technology, 1:1000 dilution).

Expression and purification of recombinant proteins

The corresponding constructs were transformed into the Escherichia coli strain BL21. A small amount of bacterial cells was inoculated into 5 mL LB liquid medium with the appropriate antibiotic and cultured overnight at 37 °C with shaking at 220 rpm. Then 100 μL bacterial cultures were inoculated into 100 mL fresh LB medium and grown at 37 °C with 220 rpm shaking until the optical density (OD₆₀₀) reached 0.5. 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and the cultures were shaken at 16 °C (120 rpm) for another 16 h to induce the expression of GST-GH3.5, MBP-COP1, MBP-COP1-N, MBP-COP1-C, His-UBC10, His-Ub or His-Ub-K63 proteins. The bacterial cultures were then centrifuged at 3783 × g for 7 min at 4 °C and the pellets were collected.

For purification of His-tagged proteins, the pellets were resuspended with 15 mL pre-chilled lysis buffer I (50 mM NaH₂PO₄.H₂O, 300 mM NaCl, 20 mM imidazole, pH 8.0). After sonication and centrifugation at 16,639 × g for 30 min at 4 °C, the supernatant was incubated with pre-cleaned 80 μL Ni-NTA agarose beads (QIAGEN) for 2 h at 4 °C. The beads were then collected and washed three times with lysis buffer I. The recombinant proteins were eluted with elution buffer I (50 mM NaH₂PO₄.H₂O, 300 mM NaCl, 250 mM imidazole, 10% [v/v] glycerol, 1× protease inhibitor cocktail, pH 8.0). To quantify the protein concentration, the purified proteins were separated in an SDS-polyacrylamide gel with 1 μg bovine serum albumin (BSA) as reference standard. The gel stained with Coomassie blue was imaged and used to quantify protein concentration using ImageJ software.

The purification of GST- and MBP-tagged proteins was also carried out according to the method described above with some modifications. Bacterial cells expressing GST- or MBP-tagged proteins were resuspended in lysis buffer II (20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10% [v/v] glycerol) and sonicated. GST-tagged proteins and MBP-tagged proteins were captured with Glutathione Sepharose^TM 4B beads (GE Healthcare) and amylose resin (New England Biolabs), respectively. Finally, GST-tagged proteins and MBP-tagged proteins were eluted with elution buffer II (40 mM Tris-HCl [pH 9.0], 300 mM NaCl, 10% [v/v] glycerol, 20 mM GSH, 1× protease inhibitor cocktail) and elution buffer III (40 mM Tris-HCl [pH 9.0], 300 mM NaCl, 10% [v/v] glycerol, 50 mM maltose, 1× protease inhibitor cocktail), respectively.

For the ubiquitination assay, the imidazole-containing elution buffer in which the His-UBC10, His-Ub and His-Ub-K63 proteins were stored was replaced by TBS + 10% glycerol using the Amicon® Pro Purification System (Millipore).

In vitro pull-down assay

1 μg GST-GH3.5 and 1 μg MBP-COP1 were incubated in 1 mL binding buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.25% NP40) at 4 °C for 1 h. Then, 15 μL amylose resins (New England Biolabs) pre-washed with TBS buffer were added into the binding buffer and incubated for another 1 h. The resins were then washed three times with wash buffer (25 mM Tris-HCl [pH 7.5], 500 mM NaCl, 0.5% NP40), and the proteins were subsequently eluted in 1× SDS loading buffer at 100 °C for 10 min. The eluted proteins were detected by immunoblotting with anti-MBP and anti-GST antibodies.

Co-IP assay

0.6 g seedlings grown in the dark for 4 d were homogenized in liquid nitrogen and resuspended in Co-IP lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.05% (v/v) Tween-20, 1 mM PMSF and 1× protease inhibitor cocktail. After centrifugation at 13,000 × g for 10 min at 4 °C, protein concentrations were determined using the Bradford method. 2 mg total proteins were incubated with 15 μL anti-GFP nanobody agarose beads (KTSM1301, AlpalifeBio) in 2 mL Co-IP lysis buffer for 2 h at 4 °C. The anti-GFP beads were then washed three times at 4 °C with 500 μL wash buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.05% (v/v) Tween-20 and 1× protease inhibitor cocktail. The immunoprecipitated proteins were eluted in 1× SDS loading buffer at 100 °C and analyzed by immunoblotting with anti-GFP, anti-COP1 and anti-actin antibodies.

LCI assay

The pCAMBIA1300-P19 (suppress RNA silencing)⁷⁵, the GH3.5-nLUC, and cLUC-COP1 constructs⁷¹ were individually transformed into the GV3101 strain. GV3101 colonies were inoculated in 2 mL LB medium with gentamycin and kanamycin and cultured overnight at 28 °C with shaking at 220 rpm. Then 50 μL cultures were inoculated in 5 mL LB medium containing 10 mM MES (pH 5.6) and 40 μM acetosyringone and grown for 16 h at 28 °C. After centrifugation, the pellets were resuspended in buffer containing 10 mM MES (pH 5.6), 10 mM MgCl₂ and 200 μM acetosyringone. The required bacterial solutions were mixed with a final OD₆₀₀ = 0.5 of each bacterium. Subsequently, the bacteria were kept in the dark at room temperature for 3 h and then infiltrated into the fully expanded tobacco leaves using a 1 mL needleless syringe. After 3 d of growth in the dark, the leaves were infiltrated with 1 mM luciferin (E1605, Promega), and the luciferase signal was captured by Night SHADE LB 985 system (Berthold Technologies).

BiFC assay

For protoplast isolation, the healthy and well-developed leaves of 4-week-old Arabidopsis plants were selected. Protoplasts were isolated from 0.5 to 1 mm wide leaf strips in the freshly prepared enzyme solution containing 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, 1.5% (w/v) cellulase R10, 0.4% (w/v) macerozyme R10, 10 mM CaCl₂, 0.1% (w/v) BSA⁷⁶. After enzymolysis in the dark for 3 h at room temperature with slow shaking (60 rpm), the solution was filtered with a nylon mesh to remove undigested leaf tissue. Then the protoplasts were collected by centrifugation at 100 × g for 2 min and washed twice with 5 mL W5 solution containing 2 mM MES (pH 5.7), 154 mM NaCl, 125 mM CaCl₂, and 5 mM KCl. The protoplasts were then resuspended in MMG solution containing 4 mM MES (pH 5.7), 0.4 M mannitol, 15 mM MgCl₂. 200 μL protoplasts were gently mixed with 10 μg of each plasmid. Then 250 μL of PEG-calcium solution containing 0.2 M mannitol, 100 mM CaCl₂, 40% (w/v) PEG4000 was added and mixed gently. After 20 min of transfection at room temperature, 1 mL W5 solution was added to stop the transfection. The protoplasts were collected and resuspended in 1 mL WI solution containing 4 mM MES (pH 5.7), 0.5 M mannitol, 20 mM KCl. After 14 h of culture in the dark at 22 °C, the protoplasts were then mounted on microscope slides and the fluorescence signals of YFP and mCherry were detected using a Zeiss LSM880 confocal microscope (Carl Zeiss). YFP fluorescence was excited with a 514-nm laser, and the emission spectra were recorded from 519 to 603 nm. The mCherry fluorescence was excited with a 561-nm laser, and the emission spectra were recorded from 590 to 628 nm.

Arabidopsis GH3.5-GFP seedlings grown in continuous darkness for 4 d were soaked in 50 μM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, D9542, Sigma-Aldrich) solution. After staining for 5 min, the finished samples were mounted on glass slides. Images were captured using a confocal microscope. The GFP signal was excited at 488 nm and the emission spectra were recorded in a range from 500 to 550 nm. The DAPI signal was excited at 405 nm and the emission spectra were recorded in a range from 425 to 475 nm.

Quantitative real-time PCR

Total RNA was isolated using the Eastep® Super Total RNA Extraction Kit (LS1040, Promega). For reverse transcription, 1 μg total RNA was used as a template to synthesize first-strand cDNAs using the Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (11141ES60, Yeasen). The RT-qPCR assay was performed using the Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (11184ES08, Yeasen) with the ABI QuantStudio^TM 6 Flex Real-Time PCR System (Thermo Fisher Scientific). The relative expression levels of different genes were analyzed using the 2^−ΔΔCt method and normalized to those of PP2A. All primers for RT-qPCR assay are listed in Supplementary Table 1.

In vitro ubiquitination assay

60 μL ubiquitination reaction mixtures contained 50 nM His-UBE1 (#E-304-050, Boston Biochem), 100 ng His-UBC10, 1 μg COP1-SPA4 complex⁷⁷, 4 μg His-Ub and 330 ng GST-GH3.5 in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM ATP, and 10 μM ZnCl₂. After incubation for 2 h at 30 °C, the reaction was terminated by addition of 5× SDS loading buffer. The polyubiquitination of GH3.5 was detected with anti-GST and anti-pan-Ub antibodies.

In vivo ubiquitination assays

For the transient expression experiment, Arabidopsis protoplasts containing different vector combinations were incubated in the dark for 12 h. Total proteins were extracted with Co-IP lysis buffer with the addition of various deubiquitinating enzyme (DUB) inhibitors, including 5 mM N-ethylmaleimide (NEM, Sigma-Aldrich), 5 mM 1,10-phenanthroline monohydrate (Sigma-Aldrich), and 50 μM 2,6-diaminopyridine-3,5-bis(thiocyanate) (PR-619, Sigma-Aldrich). GH3.5-GFP proteins were immunoprecipitated with anti-GFP agarose beads (KTSM1301, AlpalifeBio) at 4 °C. Then the anti-GFP beads were washed three times with lysis buffer containing DUB inhibitors. Ubiquitination of GH3.5-GFP was detected with the corresponding antibodies.

For the ubiquitination assay in planta, 4-day-old dark-grown Col, GH3.5-GFP, and GH3.5-GFP/cop1-4 seedlings were used to immunoprecipitate GH3.5-GFP proteins with GFP-trap magnetic agarose beads. Polyubiquitination of GH3.5-GFP was detected with anti-pan-Ub, anti-K63 Ub, anti-K48 Ub and anti-GFP antibodies.

GH3.5 enzymatic activity experiment and kinetic analysis

To test the enzymatic activity of GH3.5, GST-GH3.5 proteins linked to K63 ubiquitin chains (GST-GH3.5-K63) were generated by a COP1-mediated in vitro ubiquitination assay. In parallel, an in vitro ubiquitination reaction was performed without GST-GH3.5. After completion of the reaction, GST-GH3.5 proteins were added into the mixture, which was considered as GST-GH3.5 without ubiquitin chains (GST-GH3.5). To enrich GST-GH3.5 and GST-GH3.5-K63 proteins, the ubiquitination mixtures were incubated with GST beads in 1 mL TBS buffer at 4 °C for 1 h. Subsequently, the beads were washed three times with TBS buffer at 4 °C and used to catalyze IAA to IAA-Asp. The beads were mixed with 200 μL reaction buffer containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl₂, 0.15 mM ATP, 1 mM DTT, 10 mM Asp, and 2 mM IAA to start the reaction. The reactions were carried out at 25 °C with gentle rotation on a vertical mixer. After incubation for 30 min, each reaction was quenched by adding 7 μL concentrated hydrochloric acid. An equal volume of ethyl acetate was mixed with the reaction solution on a vortex. The mixture was then centrifuged at 13,000 × g for 10 min at 4 °C, and the supernatant was transferred into a new tube. The liquid-liquid extraction was repeated three times. The supernatants were combined and evaporated to dryness at room temperature. Then 100 μL methanol was added to redissolve the solutes containing IAA-Asp. The production of IAA-Asp was quantified by ultra-performance liquid chromatography tandem mass-spectrometry (UPLC–MS/MS) system (Thermo Vanquish UHPLC system; Q Exactive mass spectrometer; Thermo Scientific, USA)⁴⁴. Gradient elution was performed with solvent A (water with 0.1% formic acid) and solvent B (methanol). The mobile phase was delivered at a flow rate of 0.3 mL/min under the following gradient conditions: 0-0.3 min: 0–5% B, 0.3-1 min: 5–41% B, 1–4 min: 41–45% B, 4–4.2 min: 45–80% B, 4.2–4.5 min: 80-100% B, holding at 100% B for 2 min, then back to 5% B in 0.2 min and holding at 5% B for 1.3 min. The effluent was introduced into the mass spectrometer with the following optimal settings: positive ionization mode; spray voltage, 3800 V; capillary temp, 320 °C; sheath gas flow, 40 Arb; auxiliary gas flow, 11 Arb; aux gas heater temp, 370 °C; scan range, 100–350 m/z. Three independent replicates were used for each treatment. Raw data were analyzed using Xcalibur 2.1 (Thermo Scientific, USA) with default settings. The peak of IAA-Asp was detected in the mass range (m/z 291.09495–291.09787).

The kinetic parameters were determined through initial velocity experiments⁷⁸. Before analyzing the steady-state kinetics, the conditions under which GST-GH3.5-catalyzed production of IAA-Asp was linear over time were determined. Subsequently, the enzymatic reaction, which was catalyzed by approximately 125 ng GST-GH3.5, was carried out at various IAA concentrations (0.004, 0.008, 0.02, 0.05, 0.2, and 0.5 mM) for 5 min to calculate the initial velocities. The production of IAA-Asp was quantified using UPLC–MS/MS⁴⁴. Initial velocities as a function of IAA concentrations were fitted to the Michaelis–Menten equation using GraphPad Prism software (version 8.0). The parameters for the UPLC-MS/MS analyzes are listed in Supplementary Table 2.

Measurement of IAA and IAA metabolites

The contents of endogenous IAA, IAA-Asp, IAA-Glu, and oxIAA-Asp were determined by the Wuhan Greensword Creation Technology Company (http://www.greenswordcreation.com) with UHPLC-MS/MS. 0.1 g of 4-day-old Arabidopsis seedlings grown in continuous darkness or continuous white light (35 μmol m⁻² s⁻¹) were harvested, frozen in liquid nitrogen and ground into fine powder. Three to five biological replicates were prepared. Each sample was extracted with 1.0 mL 80% methanol (v/v) at 4 °C for 12 h and then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was collected and evaporated under mild nitrogen stream at 35 °C followed by re-dissolving in 100 µL H₂O/ACN (90/10, v/v) for UHPLC-MS/MS analysis (Thermo Scientific Ultimate 3000 UHPLC coupled with TSQ Quantiva). Formic acid in water (0.1%, v/v, solvent A) and ACN (solvent B) were employed as mobile phases at a flow rate of 0.4 mL/min. A gradient of 0–8 min 5%–40% B, 8–9 min 40–90% B, 9–12 min 90% B, 12–12.5 min 90–5% B, and 12.5–15 min 5% B was employed. For MS analysis, ion source conditions were as follows: Sheath gas flow, 35 Arb; auxiliary gas flow, 10 Arb; sweep gas flow, 8 Arb; collision gas, 1.5 mTorr; ion transfer tube temperature, 350 °C; vaporizer temperature, 300 °C; spray voltage, 3.5 kV. Data acquisition and analysis were performed using a Thermo Scientific Xcalibur 2.1 data system. The parameters for the UPLC-MS/MS analyzes are listed in Supplementary Table 2.

Primer sequences

All primer sequences used in this study are listed in Supplementary Table 1.

Statistics and reproducibility

The statistical analyzes in this study were performed with GraphPad Prism (version 8.0) and provided in the Source Data file. P values and different lowercase letters above the histogram represent statistically significant differences between groups. For hypocotyl length measurements, n indicates the number of seedlings used in the experiment. BiFC, LCI and western blot experiments were performed at least twice with similar results.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

References

Krahmer, J. & Fankhauser, C. Environmental control of hypocotyl elongation. Annu. Rev. Plant Biol. 75, 489–519 (2024).
Article CAS PubMed Google Scholar
Han, X., Huang, X. & Deng, X. W. The photomorphogenic central repressor COP1: conservation and functional diversification during evolution. Plant Commun. 1, 100044 (2020).
Article PubMed PubMed Central Google Scholar
Vierstra, R. D. The ubiquitin–26S proteasome system at the nexus of plant biology. Nat. Rev. Mol. Cell Biol. 10, 385–397 (2009).
Article CAS PubMed Google Scholar
Yi, C. & Deng, X. W. COP1 – from plant photomorphogenesis to mammalian tumorigenesis. Trends Cell Biol. 15, 618–625 (2005).
Article CAS PubMed Google Scholar
Sheerin, D. J. et al. Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex. Plant Cell 27, 189–201 (2015).
Article CAS PubMed PubMed Central Google Scholar
Wang, H., Ma, L.-G., Li, J.-M., Zhao, H.-Y. & Deng, X. W. Direct interaction of Arabidopsis cryptochromes with COP1 in light control development. Science 294, 154–158 (2001).
Article ADS CAS PubMed Google Scholar
Ponnu, J., Riedel, T., Penner, E., Schrader, A. & Hoecker, U. Cryptochrome 2 competes with COP1 substrates to repress COP1 ubiquitin ligase activity during Arabidopsis photomorphogenesis. Proc. Natl Acad. Sci. USA 116, 27133–27141 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Damgaard, R. B. The ubiquitin system: from cell signalling to disease biology and new therapeutic opportunities. Cell Death Differ. 28, 423–426 (2021).
Article PubMed PubMed Central Google Scholar
Kim, D. Y., Scalf, M., Smith, L. M. & Vierstra, R. D. Advanced proteomic analyses yield a deep catalog of ubiquitylation targets in Arabidopsis. Plant Cell 25, 1523–1540 (2013).
Article CAS PubMed PubMed Central Google Scholar
Saracco, S. A. et al. Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis. Plant J. 59, 344–358 (2009).
Article CAS PubMed PubMed Central Google Scholar
Maor, R. et al. Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants. Mol. Cell Proteom. 6, 601–610 (2007).
Article CAS Google Scholar
Ohtake, F., Saeki, Y., Ishido, S., Kanno, J. & Tanaka, K. The K48-K63 branched ubiquitin chain regulates NF-κB signaling. Mol. Cell 64, 251–266 (2016).
Article CAS PubMed Google Scholar
Jacobson, A. D. et al. The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 S proteasome. J. Biol. Chem. 284, 35485–35494 (2009).
Article CAS PubMed PubMed Central Google Scholar
Smalle, J. & Vierstra, R. D. The ubiquitin 26S proteasome proteolytic pathway. Annu. Rev. Plant Biol. 55, 555–590 (2004).
Article CAS PubMed Google Scholar
Deng, L., Meng, T., Chen, L., Wei, W. & Wang, P. The role of ubiquitination in tumorigenesis and targeted drug discovery. Sig. Transduct. Target Ther. 5, 11 (2020).
Article CAS Google Scholar
Romero-Barrios, N. & Vert, G. Proteasome-independent functions of lysine-63 polyubiquitination in plants. N. Phytol. 217, 995–1011 (2018).
Article Google Scholar
Saeed, B. et al. K63-linked ubiquitin chains are a global signal for endocytosis and contribute to selective autophagy in plants. Curr. Biol. 33, 1337–1345.e1335 (2023).
Article CAS PubMed Google Scholar
Wang, J. et al. Tissue-specific ubiquitination by IPA1 INTERACTING PROTEIN 1 modulates IPA1 protein levels to regulate plant architecture in rice. Plant Cell, 29, 697–707 (2017).
Wang, X. et al. PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis. Plant Cell 35, 3585–3603 (2023).
Article PubMed PubMed Central Google Scholar
Hasegawa, Y. et al. The TGN/EE SNARE protein SYP61 and the ubiquitin ligase ATL31 cooperatively regulate plant responses to carbon/nitrogen conditions in Arabidopsis. Plant Cell 34, 1354–1374 (2022).
Article PubMed PubMed Central Google Scholar
Li, H. et al. Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation. Mol. Plant 17, 1090–1109 (2024).
Article CAS PubMed Google Scholar
Zhou H., Deng X. W. The molecular basis of CONSTITUTIVE PHOTOMORPHOGENIC1 action during photomorphogenesis. J. Exp. Bot., 76, 664–676 (2024).
Du, M., Spalding, E. P. & Gray, W. M. Rapid auxin-mediated cell expansion. Annu. Rev. Plant Biol. 71, 379–402 (2020).
Article CAS PubMed PubMed Central Google Scholar
Nishimura, T. et al. Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis. Plant J. 77, 352–366 (2014).
Article CAS PubMed Google Scholar
Cao, M. et al. TMK1-mediated auxin signalling regulates differential growth of the apical hook. Nature 568, 240–243 (2019).
Article ADS CAS PubMed Google Scholar
Ludwig-Mueller, J. Auxin conjugates: their role for plant development and in the evolution of land plants. J. Exp. Bot. 62, 1757–1773 (2011).
Article CAS Google Scholar
Casanova-Saez, R., Mateo-Bonmati, E. & Ljung, K. Auxin metabolism in plants. Cold Spring Harb. Perspect. Biol. 13, a039867 (2021).
Article CAS PubMed PubMed Central Google Scholar
Staswick, P. E., Tiryaki, I. & Rowe, M. L. Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation. Plant Cell 14, 1405–1415 (2002).
Article CAS PubMed PubMed Central Google Scholar
Terol, J., Domingo, C. & Talón, M. The GH3 family in plants: Genome wide analysis in rice and evolutionary history based on EST analysis. Gene 371, 279–290 (2006).
Article CAS PubMed Google Scholar
Staswick, P. E. et al. Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell 17, 616–627 (2005).
Article CAS PubMed PubMed Central Google Scholar
Hayashi, K.-i. et al. The main oxidative inactivation pathway of the plant hormone auxin. Nat. Commun. 12, 6752 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Nakazawa, M. et al. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length. Plant J. 25, 213–221 (2001).
CAS PubMed Google Scholar
Takase, T. et al. ydk1-D, an auxin-responsive GH3 mutant that is involved in hypocotyl and root elongation. Plant J. 37, 471–483 (2004).
Article CAS PubMed Google Scholar
Park, J.-E. et al. An Arabidopsis GH3 gene, encoding an auxin-conjugating enzyme, mediates phytochrome B-regulated light signals in hypocotyl growth. Plant Cell Physiol. 48, 1236–1241 (2007).
Article CAS PubMed Google Scholar
Zhang, Z. et al. Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction. Plant Physiol. 145, 450–464 (2007).
Article CAS PubMed PubMed Central Google Scholar
Casanova-Saez, R. et al. Inactivation of the entire Arabidopsis group II GH3s confers tolerance to salinity and water deficit. N. Phytol. 235, 263–275 (2022).
Article CAS Google Scholar
Guo, R. et al. Local conjugation of auxin by the GH3 amido synthetases is required for normal development of roots and flowers in Arabidopsis. Biochem. Biophys. Res. Commun. 589, 16–22 (2022).
Article CAS PubMed Google Scholar
Wojtaczka, P., Ciarkowska, A., Starzynska, E. & Ostrowski, M. The GH3 amidosynthetases family and their role in metabolic crosstalk modulation of plant signaling compounds. Phytochemistry 194, 113039 (2022).
Lv, B., Zhu, J., Kong, X. & Ding, Z. Light participates in the auxin-dependent regulation of plant growth. J. Integr. Plant Biol. 63, 819–822 (2021).
Article CAS PubMed Google Scholar
Wen, R. et al. UBC13, an E2 enzyme for Lys63-linked ubiquitination, functions in root development by affecting auxin signaling and Aux/IAA protein stability. Plant J. 80, 424–436 (2014).
Article CAS PubMed Google Scholar
Romero-Barrios, N. et al. Advanced cataloging of lysine-63 polyubiquitin networks by genomic, interactome, and sensor-based proteomic analyses. Plant Cell 32, 123–138 (2020).
Article CAS PubMed Google Scholar
Sims, J. J. et al. Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nat. Methods 9, 303–309 (2012).
Article CAS PubMed PubMed Central Google Scholar
Lin, F. et al. Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis. Proc. Natl Acad. Sci. USA 114, 6617–6622 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Xie, Y. et al. Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation. Proc. Natl Acad. Sci. USA 119, e2209256119 (2022).
Article CAS PubMed PubMed Central Google Scholar
Peng J. et al. COP1 positively regulates ABA signaling during Arabidopsis seedling growth in darkness by mediating ABA-induced ABI5 accumulation. Plant Cell, 34, 2286–2308 (2022).
Park, J.-E. et al. GH3-mediated auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis. J. Biol. Chem. 282, 10036–10046 (2007).
Article CAS PubMed Google Scholar
Yang, K. & Xiao, W. Functions and mechanisms of the Ubc13-UEV complex and lysine 63-linked polyubiquitination in plants. J. Exp. Bot. 73, 5372–5387 (2022).
Article PubMed Google Scholar
Zhang, S. et al. The auxin response factor, OsARF19, controls rice leaf angles through positively regulating 3-5 and 1. Plant Cell Environ. 38, 638–654 (2015).
Article PubMed Google Scholar
Jiang, L. et al. MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance. Plant J. 109, 1271–1289 (2022).
Article CAS PubMed Google Scholar
Ai, G. et al. SlGH3.15, a member of the GH3 gene family, regulates lateral root development and gravitropism response by modulating auxin homeostasis in tomato. Plant Sci. 330, 111638 (2023).
Article CAS PubMed Google Scholar
Middleton, A. M. et al. Data-driven modeling of intracellular auxin fluxes indicates a dominant role of the er in controlling nuclear auxin uptake. Cell Rep. 22, 3044–3057 (2018).
Article CAS PubMed Google Scholar
Tang, W., Yu, Y. & Xu, T. The interplay between extracellular and intracellular auxin signaling in plants. J. Genet. Genomics, 52, 14–23 (2024).
Annibaldi, A. et al. Ubiquitin-mediated regulation of RIPK1 kinase activity independent of IKK and MK2. Mol. Cell 69, 566–580.e565 (2018).
Article CAS PubMed PubMed Central Google Scholar
Bertrand, M. J. M. et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol. Cell 30, 689–700 (2008).
Article CAS PubMed Google Scholar
Wang, J. et al. A regulatory module controlling homeostasis of a plant immune kinase. Mol. Cell 69, 493–504.e496 (2018).
Article CAS PubMed Google Scholar
Ye, Y. H. & Rape, M. Building ubiquitin chains: E2 enzymes at work. Nat. Rev. Mol. Cell Biol. 10, 755–764 (2009).
Article CAS PubMed PubMed Central Google Scholar
Brillada, C. & Trujillo, M. E2 ubiquitin-conjugating enzymes (UBCs): drivers of ubiquitin signalling in plants. Essays Biochem. 66, 99–110 (2022).
Article CAS PubMed Google Scholar
Gray, W. M., Hellmann, H., Dharmasiri, S. & Estelle, M. Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function. Plant Cell 14, 2137–2144 (2002).
Article CAS PubMed PubMed Central Google Scholar
Hotton, S. K. & Callis, J. Regulation of cullin RING ligases. Annu. Rev. Plant Biol. 59, 467–489 (2008).
Article CAS PubMed Google Scholar
Zhang, Q. et al. FBXW7 facilitates nonhomologous end-joining via k63-linked polyubiquitylation of XRCC4. Mol. Cell 61, 419–433 (2016).
Article CAS PubMed PubMed Central Google Scholar
Stoop-Myer, C., Torii, K. U., McNellis, T. W., Coleman, J. E. & Deng, X.-W. The N-terminal fragment of Arabidopsis photomorphogenic repressor COP1 maintains partial function and acts in a concentration-dependent manner. Plant J. 20, 713–717 (1999).
Article CAS PubMed Google Scholar
Osterlund, M. T., Hardtke, C. S., Wei, N. & Deng, X. W. Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405, 462–466 (2000).
Article ADS CAS PubMed Google Scholar
Xu, D. et al. The RING-finger E3 ubiquitin ligase COP1 SUPPRESSOR1 negatively regulates COP1 abundance in maintaining COP1 homeostasis in dark-grown Arabidopsis seedlings. Plant Cell 26, 1981–1991 (2014).
Article CAS PubMed PubMed Central Google Scholar
Lin, X.-L. et al. An Arabidopsis SUMO E3 ligase, SIZ1, negatively regulates photomorphogenesis by promoting COP1 activity. PLoS Genet. 12, e1006016 (2016).
Article PubMed PubMed Central Google Scholar
McNellis, T. W. et al. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains. Plant Cell 6, 487–500 (1994).
CAS PubMed PubMed Central Google Scholar
Westfall, C. S. et al. Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis. Proc. Natl Acad. Sci. USA 113, 13917–13922 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Westfall, C. S. et al. Structural basis for prereceptor modulation of plant hormones by GH3 proteins. Science 336, 1708–1711 (2012).
Article ADS CAS PubMed Google Scholar
Pierdonati, E. et al. Cytokinin-dependent control of GH3 group II family genes in the Arabidopsis root. Plants Basel 8, 94 (2019).
Article CAS PubMed PubMed Central Google Scholar
Gonzalez-Lamothe, R., El Oirdi, M., Brisson, N. & Bouarab, K. The conjugated auxin indole-3-acetic acid-aspartic acid promotes plant disease development. Plant Cell 24, 762–777 (2012).
Article CAS PubMed PubMed Central Google Scholar
Bracha-Drori, K. et al. Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J. 40, 419–427 (2004).
Article CAS PubMed Google Scholar
Chen, H. M. et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 146, 368–376 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Grefen, C. et al. A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J. 64, 355–365 (2010).
Article CAS PubMed Google Scholar
Wang, Z.-P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).
Article PubMed PubMed Central Google Scholar
Lei, Y. et al. CRISPR-P: A web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol. Plant 7, 1494–1496 (2014).
Article CAS PubMed Google Scholar
Yin, Y. et al. Two interacting basic helix-loop-helix transcription factors control flowering time in rice. Plant Physiol. 192, 205–221 (2023).
Article CAS PubMed PubMed Central Google Scholar
Yoo, S.-D., Cho, Y.-H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).
Article CAS PubMed Google Scholar
Wang, Y. et al. Structural insight into UV-B-activated UVR8 bound to COP1. Sci. Adv. 8, eabn3337 (2022).
Article CAS PubMed PubMed Central Google Scholar
Chen, Q., Zhang, B., Hicks, L. M., Wang, S. & Jez, J. M. A liquid chromatography–tandem mass spectrometry-based assay for indole-3-acetic acid–amido synthetase. Anal. Biochem. 390, 149–154 (2009).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

We are grateful to Prof. Wei Xiao (Capital Normal University) for providing the ubc35-1 ubc36-1 mutant, Prof. Yunde Zhao (University of California, San Diego) for providing the gh3(5/6/9/17) and gh3-septuple mutants, Prof. Hongwei Guo (Southern University of Science and Technology) and Prof. Kai Jiang (Yunnan University) for providing the chemical inhibitor nalacin, Prof. Ping Yin (Huazhong Agricultural University) for providing the recombinant COP1-SPA4 complex, and Prof. Jigang Li (China Agricultural University) for providing the pCAMBIA1307-35S:HF-COP1 construct. We thank Ms. Suying Gao (Southern University of Science and Technology) for her help with UPLC-MS experiments. We thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript. This work was supported by grants from the National Key R&D Program of China (Grant No. 2024YFA1306701 to X.W.D.), the National Natural Science Foundation of China (Grant No. 32200198 to J.L.), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Shandong Provincial Natural Science Foundation (Grant No. SYS202206 and ZR2021ZD30 to X.W.D.), Southern University of Science and Technology (Y01226026 to X.W.D.), and the Shenzhen Science and Technology Program (Grant No. ZDSYS20230626091659010).

Author information

Authors and Affiliations

State Key Laboratory of Wheat Improvement, Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences in Weifang, Weifang, Shandong, China
Yongting Liu & Xing Wang Deng
Shenzhen Key Laboratory of Plant Genetic Engineering and Molecular Design, Institute of Plant and Food Science, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China
Yongting Liu, Yinpeng Xie & Xing Wang Deng
State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China
Yinpeng Xie
State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, National Center for Soybean Improvement, College of Agriculture, Nanjing Agricultural University, Nanjing, China
Dongqing Xu
Peking-Tsinghua Center for Life Sciences, School of Advanced Agricultural Sciences, Peking University, Beijing, China
Xing Wang Deng
College of Life Sciences, Nanjing Normal University, Nanjing, China
Jian Li

Authors

Yongting Liu
View author publications
Search author on:PubMed Google Scholar
Yinpeng Xie
View author publications
Search author on:PubMed Google Scholar
Dongqing Xu
View author publications
Search author on:PubMed Google Scholar
Xing Wang Deng
View author publications
Search author on:PubMed Google Scholar
Jian Li
View author publications
Search author on:PubMed Google Scholar

Contributions

X.W.D., and J.L. conceived the project and designed experiments; Y.L., Y.X., D.X., and J.L. performed experiments; Y.L., Y.X., X.W.D., and J.L. analyzed data; Y.L., X.W.D., and J.L. wrote the paper.

Corresponding authors

Correspondence to Xing Wang Deng or Jian Li.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Enamul Huq, Jing Bo Jin, Meng Chen and the other, anonymous, reviewer for their contribution to the peer review of this work. A peer review file is available

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Reporting Summary

Transparent Peer Review file

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Liu, Y., Xie, Y., Xu, D. et al. Inactivation of GH3.5 by COP1-mediated K63-linked ubiquitination promotes seedling hypocotyl elongation. Nat Commun 16, 3541 (2025). https://doi.org/10.1038/s41467-025-58767-6

Download citation

Received: 23 August 2024
Accepted: 28 March 2025
Published: 14 April 2025
DOI: https://doi.org/10.1038/s41467-025-58767-6