C‐terminal conformational changes in <scp>SCF‐D3</scp> / <scp>MAX2</scp> ubiquitin ligase are required for <scp>KAI2</scp> ‐mediated signaling

Abstract

Karrikins (KARs) are smoke-derived compounds that stimulate seed germination in various plant species, which are perceived by the α/β hydrolase receptor KARRIKIN INSENSITIVE2 (KAI2) (Flematti et al., 2004, 2011; Guo et al., 2013; Kagiyama et al., 2013). The Arabidopsis kai2 mutant shows elongated hypocotyls, increased seed dormancy, altered root system architecture, and decreased root hair length and density, suggesting that KAI2 perceives an endogenous ligand, termed KAI2-ligand (KL) (Waters et al., 2012; Conn & Nelson, 2016; Villaécija-Aguilar et al., 2019). The perception and signal activation of KAR/KL is thought to be similar to that of the phytohormone strigolactone (SL) that plays roles in the regulation of plant growth and development as well as symbiotic interactions with fungi (Cook et al., 1966; Akiyama et al., 2005; Gomez-Roldan et al., 2008; Umehara et al., 2008; Arite et al., 2009). The plant α/β hydrolase DWARF14 (D14) serves as an SL receptor and broadly perceives a variety of SLs that can be classified into two structurally distinct groups: canonical and noncanonical SLs (Arite et al., 2009; Hamiaux et al., 2012; Waters et al., 2012; Kagiyama et al., 2013; Nakamura et al., 2013; Abe et al., 2014; Yao et al., 2016, 2017). Canonical SLs contain a tricyclic lactone-ring connected to a methylbutenolide ring via an enol-ether bridge. Noncanonical SLs may lack the precise tricyclic lactone-ring composition but have the enol ether–D-ring moiety (Yoneyama et al., 2018). Interestingly, D14 and KAI2 enzymes are structurally similar yet exhibit distinct ligand selectivity for particular stereochemistry (Flematti et al., 2016). KAI2 does not recognize natural plant-produced SLs but shows hydrolytic activity toward the enantiomer of 5-deoxystrigol (ent-5DS) (Scaffidi et al., 2014; Flematti et al., 2016). This suggests that KL is a naturally produced butenolide-based chemical. In both D14 and KAI2 pathways, the E3 ligase-associated F-box protein MORE AXILLARY GROWTH 2 (MAX2) or DWARF3 (D3, in rice) plays a key role in mediating signal transduction. MAX2 is an F-box protein and part of the ASK1/SKP1-CULLIN1-F-box (SCF) complex. MAX2 has been shown to directly interact with D14/KAI2 upon ligand perception and to subsequently recruit specific targets for ubiquitylation and proteasomal degradation. While in D14 signaling, the target substrates are SUPPRESSOR OF MAX2 LIKE 6, 7, and 8 (SMXL6/7/8) or DWARF53 (D53 in rice), in KAI2 signaling the target substrates of MAX2 are SMAX1 and SMXL2 (Nelson et al., 2011; Jiang et al., 2013; Zhou et al., 2013; Soundappan et al., 2015). D53/SMXL proteins share a similar secondary structure to the class I Clp ATPase family, which is characterized by N-terminal domain, D1 ATPase domain, M domain, and D2 ATPase domain (Zhou et al., 2013). The D2 domain of D53 was found to be important for SL-dependent D3/MAX2-D14 interaction (Shabek et al., 2018). Similarly, the SMAX1D2 domain was shown to be degraded following treatment with karrikin and the synthetic SL analog rac-GR24 (Khosla et al., 2020). Phylogenetic analyses showed that proteins resembling KAI2 are found throughout land plants and in charophyte algae and that KAI2 signaling is ancestral to D14 signaling (Delaux et al., 2012; Waters et al., 2012, 2015b). Both MAX2 and the D14-MAX2-interaction interface are conserved throughout land plant evolution, but several DDK (D14/DLK2/KAI2) protein groups lack the conserved MAX2 interaction interface and are suggested to function independently of MAX2 (Bythell-Douglas et al., 2017), and the interaction interface between MAX2 and KAI2 is yet to be determined. Recently, we found that the D3/MAX2 C-terminal helix (CTH) plays a significant role in SL perception and metabolism as well as in D3/MAX2-D14-D53/SMXL complex formation (Shabek et al., 2018). Furthermore, we demonstrated that the D3/MAX2 CTH undergoes a conformational switch to potentiate SL signaling (Tal et al., 2022). This was indicated by several key findings: First, a truncated D3 protein lacking the CTH does not form a complex with D14–D53 in the presence of SL (Shabek et al., 2018) and an Arabidopsis CRISPR/Cas9 genome edited line of MAX2 with a deletion of the CTH (designated MAX2∆CTH) fully mimics the loss of function phenotypes of max2 (Tal et al., 2022). Second, increasing concentrations of the CTH peptide inhibits the hydrolysis of the ligand Yoshimulactone Green (YLG) by D14 in vitro (Shabek et al., 2018). Moreover, a change in the terminal residue (D720) of D3 results in a conformational change of a dislodged CTH. In the presence of this mutant protein (designated D3D720K), YLG hydrolysis by D14 in vitro is also inhibited, even more than the inhibition by wild-type D3. Lastly, Arabidopsis plants overexpressing the corresponding dislodged CTH mutant (pUBQ:MAX2D693K) exhibit SL loss-of-function phenotypes in a dominant negative manner (Tal et al., 2022). We further showed that the D3/MAX2 CTH conformational switch can be triggered by the presence of a small carboxylate molecule (Tal et al., 2022). Given the dual role of D3/MAX2 in SL and KAR/KL signaling pathways, the function of MAX2 CTH in KAR/KL signaling regulation remains to be addressed. Here, we investigate the effects of the CTH dynamics both in vitro and in Arabidopsis and demonstrate a conserved central role for the CTH dynamics between SL and the KAR/KL signaling pathways. Mutant seeds of Arabidopsis thaliana (Col-0 background) were obtained from the Arabidopsis Biological Research Center (ABRC) including WT seeds and the CS956 for max2-2 (AT2G42620) mutant line. The htl-3 mutant line for kai2 (AT4G37470) was a gift (Toh et al., 2014). For the root hair development assays, max2-1 seeds were used (Stirnberg et al., 2002; Villaécija-Aguilar et al., 2019). All plants were grown in the growth chamber at 22°C with a 16-h : 8-h, light : dark photoperiod. Sunshine Mix #1 Fafard 1P (Sungro Horticulture, Agawam, MA, USA) was used to grow the plants. Germination was assessed on solid medium supplied with ½ Murashige Skoog salt mixture without MES (Caisson labs MSP01). Harvested seeds were collected immediately and stored at −80°C to preserve primary dormancy. Following 70% EtOH seed sterilization and plating, plates were moved to 24 h of white light for 3 d and germinated seeds were counted. KAR-induced germination was performed on water-agar plates supplemented with 10 μM final KAR1, KAR2, (www.olchemim.cz) or the equivalent amount of 70% methanol as control. Seeds were imbibed in 10 μM final KAR1, KAR2, or the equivalent amount of 70% methanol as control for 2 h before plating. Root hair development was assessed according to Villaécija-Aguilar et al. (2021) with some modifications. Images for 20 roots per genotype were taken with a Zeiss Discovery V8 microscope equipped with a Zeiss Axiocam 503 camera. For the quantification of root hair length, the length of all the root hairs visible in the correct plane was measured between 1.5 to 2.5 mm distance from the root tip using Fiji as described previously (Villaécija-Aguilar et al., 2021). The values obtained for one root were averaged to give the root hair length in that region for that root. For the quantification of root hair density, the number of visible root hairs between 1.5 mm and 2.5 mm distance away from the root tip was counted. This provided the density as number per mm in that region per individual root. The full-length rice D3 or D3D720K (O. sativa) and A. thaliana ASK1 were co-expressed as a 6 × His–2 × Msb (msyB) fusion protein and an untagged protein, respectively, in Hi5 suspension insect cells (as described in Shabek et al., 2018). The ASK1-D3/D3D720K (D3 or D3 with D720K mutation) complex was isolated from the soluble cell lysate by Q Sepharose High Performance resin (GE Healthcare, Chicago, IL, USA). 600 mM NaCl eluates were further subjected to Nickel Sepharose Fast Flow (GE Healthcare) and was eluted with 200 mM imidazole. In experiments where 6 × His–2 × Msb-fusion tag was removed, the clarified ASK1-D3/D3D720K complex was cleaved at 4°C for 16 h by TEV (tobacco etch virus) protease and was purified by anion exchange and size exclusion chromatography. For biochemical analysis, both D3-expressing constructs were designed to eliminate a nonconserved 40 residue disordered loop between amino acid 476–514 after affinity purification (as described in Shabek et al., 2018). The resulting D3 HisMsb-fusion protein contains three TEV cleavage sites: between the Msb tag and D3, after T476, and before L514, resulting in a purified split stable form of D3 with D3-NTD (1–476) and CTD (514–720). GST-SMAX1D2 was cloned and expressed as fusion protein in BL21 (DE3) cells. Plasmid construction and protein purifications are detailed in Shabek et al. (2018). BL21 (DE3) cells transformed with the expression plasmid were grown in LB broth at 16°C to an OD600 of c. 0.8 and induced with 0.25 mM IPTG for 16 h. Cells were harvested, resuspended, and lysed in extract buffer (20 or 50 mM Tris, pH 8.0, 200 mM NaCl). For GST-fused proteins, glutathione sepharose (GE Healthcare) was used to isolate the proteins supplement with buffer containing 50 mM Tris–HCl, pH 8.0, 200 mM NaCl, 5 mM DTT. Proteins were purified by elution with 5–8 mM glutathione (Fisher BioReagents, Pittsburgh, PA, USA), followed by anion exchange and size exclusion chromatography. Arabidopsis KAI2 protein was expressed as a 6× His-SUMO fusion protein using the expression vector pSUMO (LifeSensors, Malvern, PA, USA). His-SUMO-KAI2 was isolated from by Ni-NTA resin, and the eluted His-SUMO KAI2 was further separated by anion-exchange. His-SUMO KAI2 was further incubated overnight with TEV protease at a protease/protein ratio of 1 : 1000 at 4°C, and the tag was removed by passing through a Nickel Sepharose. All proteins were further purified by chromatography through a Superdex-200 gel filtration and concentrated by ultrafiltration to 3–10 mg ml−1. YLG (Yoshimulactone Green; Fisher Scientific Company, Pittsburgh, PA, USA) hydrolysis assays were conducted in reaction buffer (50 mM MES pH 6.0, 150 mM NaCl, and 1 mM DTT) in a 50-μl volume on a 96-well, F-bottom, black plate (Greiner Bio-One, Monroe, NC, USA). The final concentration of dimethyl sulfoxide (DMSO) was equilibrated for all samples to final concentration of 0.4%. The intensity of the fluorescence was measured by a Synergy|H1 Microplate Reader (Agilent Technologies, Santa Clara, CA, USA) with excitation by 480 nm and detection by 520 nm. Readings were collected using 13-s intervals over 60 min. Background auto-YLG hydrolysis correction was performed for all samples. Raw fluorescence data were converted directly to fluorescein concentration using a standard curve. Data that were generated in Excel were transferred to GraphPad Prism 9 for graphical analysis. One-way ANOVA was performed to compare each condition with a post hoc Tukey multiple comparison test. All experiments were run with technical triplicates, and independent experiments were performed three times. GST-tagged proteins were expressed in E. coli BL21 cells. Following cell lysis, clarification, and centrifugation, the cell lysate was incubated with GST beads for 2 h at 4°C. Beads were then washed twice with wash buffer containing 50 mM Tris, 150 mM NaCl, 1% Glycerol and 1 mM TCEP. Beads were then washed once with 0.025% BSA blocking solution and twice with wash buffer. For control, GST beads were incubated with 0.05% BSA. Protein-bound GST beads were incubated with purified His-tagged and/or nontagged proteins and 100 μM GR24ent5DS (or Acetone as control) for 30 min on ice. Following two washes with wash buffer, elution was achieved with 50 mM Tris, 10 mM Reduced glutathione pH 7.2, and 5 mM DTT. After addition of fourfold concentrated sample buffer, boiled samples were resolved via SDS-PAGE, and proteins were visualized using Ponceau stain and Western blot with monoclonal anti-His (Invitrogen MA1-21315), and polyclonal anti-GST (Thermo Fisher Scientific, Waltham, MA, USA, CAB4169) antibodies. All uncropped gels are presented in Figs S1 and S2. 150 mg of inflorescences of Arabidopsis ecotype Columbia-0 (Col-0) WT and max2-2 mutant and pUBQ:MAX2D693K plants were collected and frozen. Total proteins were extracted to stock concentration of 8–10 mg ml−1 using Minute™ (Invent Biotechnologies Inc., Plymouth, MN, USA, SD-008/SN-009), supplemented with protease inhibitor cocktail (Roche). To monitor protein degradation in the cell-free system, 0.5 μg of purified tagged proteins was incubated at 22°C in a reaction mixture that contained, at a final volume of 12.5 μl, 3 μl of plant extract supplemented with 10 μM GR24ent-5DS, 25 mM Tris–HCl, pH 7.4, 0.625 mM ATP, 5 mM MgCl2, 0.25 μg μl−1 Ub and 0.5 mM DTT. To test the effect of organic acids, samples were incubated with 50 mM of citrate or succinate. Where indicated, the proteasome inhibitor MG132 (Thermo Fisher Scientific, 47-479-01MG) as described previously (Shabek et al., 2018). Reactions were terminated at the indicated times by the addition of fourfold concentrated sample buffer. Boiled samples were resolved via SDS-PAGE, and proteins were visualized using Western blot and polyclonal anti-GST antibodies. For the cell-free ubiquitination assay, a similar protocol was performed using GST-tagged protein with addition of 1 μM MG132 to all samples to inhibit degradation. max2 plant protein extract was supplemented with 0.5 μg purified His-D3 or His-D3D720K and both were copurified with ASK1. Following 1 h of incubation in 27°C, pull down using GST beads was completed. Beads were washed twice with 50 mM Tris, 150 mM NaCl, 1% Glycerol, and 1 mM TCEP. Elution was achieved with 50 mM Tris, 10 mM reduced glutathione pH 7.2 and 5 mM DTT. After addition of fourfold concentrated sample buffer, boiled samples were resolved via SDS-PAGE, and proteins were visualized using Western blot and monoclonal anti-ubiquitin antibody (Fisher Scientific Company, eBioP4D1 (p4D1)). High-molecular-weight-conjugated Ub species were quantified and normalized by their intensity vs the nonconjugated GST-SMAX1D2 species. Quantification of bands was performed via Image lab 6.0.1, Bio Rad. Sequences for AtKAI2 and AtMAX2 were threaded through existing D14–D3 complex structures using SWISS-MODEL with PDB ID: 6BRT and 5HZG as templates. The 3D structure illustration and analysis were generated using PyMOL Molecular Graphics System, Schrödinger, LLC. Sequence conservation was analyzed using 200 KAI2 and D14 sequences from Bythell-Douglas et al. (2017). Alignment was performed in Mega X (Kumar et al., 2018) using the multiple was analyzed via Sequence was generated with the as the in the et al., KAI2 signaling plays a role seed germination and (Waters et al., Furthermore, KAI2 and MAX2 through the to root growth et al., 2019). We MAX2 with dislodged CTH and CRISPR/Cas9 edited Arabidopsis for seed germination white both max2 and kai2 inhibition of seed germination to wild-type seeds of a similar inhibition of germination of MAX2 effect on germination but pUBQ:MAX2D693K seeds germinated with a than In the and pUBQ:MAX2D693K not to for seed germination Furthermore, is a of root hair and root development (Villaécija-Aguilar et al., 2019). in kai2 and max2 to a in root hair length and density Similarly, we that and reduced and with WT or the MAX2 line data on seed germination and root hair development that plays a role in KAI2 To the by which dynamics KAI2 signaling, we the interaction between KAI2 and SMAX1D2 with the dislodged CTH mutant D3D720K from rice by GST pull GST-tagged Arabidopsis SMAX1D2 was generated on to the previously domain of D53 (Zhou et al., 2013; Shabek et al., 2018) In the presence of nontagged KAI2 and GR24ent-5DS, Arabidopsis SMAX1D2 was to pull down both rice D3 and D3D720K Given the hydrolytic activity of KAI2 et al., et al., the ligand Yoshimulactone Green (YLG) et al., was to monitor KAI2 activity in the presence of D3 or D3D720K Interestingly, YLG hydrolysis by KAI2 was decreased in the presence of D3 and to a KAI2 activity was in the presence of results with the effects of D3 and the dislodged form on hydrolysis by D14 (Shabek et al., et al., 2022). Moreover, we showed previously that a specific carboxylate molecule as citrate can the dislodged CTH form of D3 (Tal et al., we the effect of D3 on KAI2 with citrate and as a control. KAI2 hydrolysis was further in the presence of both D3 and citrate with effect in the presence of as control results that the CTH dislodged form of D3 plays a role in the interaction with KAI2 in a similar manner as shown with D14 (Shabek et al., et al., 2022). data that KAI2 and SMAX1 can be by in a dislodged CTH We the ubiquitination and degradation of SMAX1 after by D3/MAX2 in a dislodged CTH To this we subjected SMAX1D2 to a cell-free ubiquitination in the of max2 cell extract and in the presence of KAI2 and D3 or Interestingly, in the presence of D3 or SMAX1D2 undergoes significant suggesting that the dislodged CTH does not the of SMAX1 and D3 Given the loss-of-function phenotypes of dislodged we SMAX1D2 proteasomal degradation. We the protein of SMAX1D2 in the line pUBQ:MAX2D693K and max2 cell extract degradation of SMAX1D2 was in the presence of pUBQ:MAX2D693K of proteins after with WT cell extract of proteins after and was in the max2 We previously showed that citrate is to potentiate the dislodged conformational (Tal et al., we the effect of citrate on SMAX1D2 degradation and found that SMAX1D2 were in the presence of citrate to We further KAI2 by D3/MAX2 using in analyses To that and were analyzed using two distinct of 200 sequences of KAI2 and D14 from species the from algae to (Bythell-Douglas et al., 2017), we found and between D14 at the with D3/MAX2 in both the dislodged and the conformational results further that the CTH conformational of to KAI2 and of SMAX1 in the KAR/KL signaling D3/MAX2 plays a central role in both SL and KAR/KL pathways. The that demonstrated a function for a D3/MAX2 conformational switch and dislodged in the SL signaling the similar dynamics in KAR/KL Here, we the function of in the KAR/KL in plants using and Arabidopsis as well as in to the kai2 loss-of-function pUBQ:MAX2D693K and (Tal et al., plants exhibit white light growth as well as in and root hair development with the Furthermore, reduced germination that is not by in vitro data that KAI2 can be by a dislodged and this interaction KAI2 the complex can recruit and target SMAX1 for ubiquitination and degradation in a similar manner as targets D53 in SL Interestingly, the D2 domain of SMAX1 is but not degraded in the presence of dislodged the phenotypes of and Arabidopsis this suggests that the of SMAX1 to the proteasome yet conformational change in and of the conformational switch may SMAX1 degradation and the of the degradation of D14 is by the conformational switch (Tal et al., but in the of this degradation is independent of MAX2 presence (Waters et al., we showed that are in the presence of D3D720K than in the presence of D3 (Tal et al., 2022). Here, the of SMAX1D2 and found that increased in the presence of both D3 and their roles as E3 the for the in ubiquitination between the two targets remains to be further and may be to as the of of the ligand, or of proteins from species. in analyses of KAI2 by conservation between 200 KAI2 and D14 sequences both in the and the of at the interface with MAX2 than all 200 sequences on and the conserved were conserved by biochemical than on Arabidopsis This suggests that the conserved are for and complex formation and as targets for in the KAR/KL and SL signaling Moreover, in we the ligand that can be perceived by KAI2 and has been to KAR/KL signaling (Scaffidi et al., 2014; Flematti et al., 2016; et al., yet the of the natural KAI2 ligand remains with the KL and yet to be and/or in this to down the of KAR/KL perception and signal transduction. the D3/MAX2 conformational switch does not to as the between SL and we demonstrate that dynamics a conserved and central role in both pathways. is by the and and by the of of Biological and Research for this in the was by the of the to and a to has an in Bio and serves on the Scientific The and data have or that be perceived to the results and/or in this and and designed the and conducted the protein seed and biochemical conducted and and analyzed root hair and the with from and The data that the of this are in the of this S1 uncropped gels and uncropped gels and KAR-induced germination and SMAX1D2 and KAI2 purification and hydrolysis function by CTH dislodged D3. of and their is not for the or of supplied by the than be to the The is not for the or of supplied by the than be to the corresponding for the

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