# Source paper archive - Source title: Antagonism between sugar-dependent O-fucosylation and BIN2-mediated phosphorylation regulates metabolism and growth in Arabidopsis - Author(s): Zhang, H., Aizezi, Y., Chaudhary, A., Wang, Z.-Y. - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764934v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764934v1.full.pdf - PDF SHA-256: 2c0c0e18e5f7b5bc65b0a5bf8e8235e80902190ba3486324856d219a67c0b7fe - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 1 Breakthrough Report 2 **Antagonism** **between** **sugar-dependent** _**O**_ **-fucosylation** **and** **BIN2-mediated** 3 **phosphorylation regulates metabolism and growth in Arabidopsis** 4 **Hongliang Zhang** **[1,2*]** **, Yalikunjiang Aizezi** **[1,2,3]** **, Ajeet Chaudhary** **[1,2]** **, Zhi-Yong Wang** **[1,2*]** 5 1 Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, United 6 States. 7 2 Department of Genetics, Stanford University, Stanford, CA 94305, United States. 8 3 Department of Biology, Stanford University, Stanford, CA 94305, United States. 9 10 *Corresponding authors: Zhi-Yong Wang and Hongliang Zhang _._ Department of Plant 11 Biology, Carnegie Institution for Science, Stanford, CA 94305, United States. 12 Email: zywang24@stanford.edu; hzhang10@carnegiescience.edu 13 Short title: _O_ -fucosylation–phosphorylation crosstalk regulates metabolism and growth 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: Zhi-Yong Wang (zywang24@stanford.edu). [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 30 **Abstract** 31 Coordinating sugar sensing, hormone signaling, and carbon metabolism is crucial for plant 32 growth and environmental acclimation. Brassinosteroid (BR)-dependent phosphorylation 33 and sugar-dependent _O_ -glycosylation modify overlapping sets of proteins, but how these 34 modifications co-regulate protein functions remains unknown. Here, we show that SPINDLY 35 (SPY)-catalyzed _O_ -fucosylation competes with BIN2-mediated phosphorylation and 36 antagonistically regulates phosphoenolpyruvate carboxykinase 1 (PCK1), a key 37 gluconeogenic enzyme. SPY associates with and _O_ -fucosylates PCK1 within a peptide 38 containing Ser-62 and Thr-66, two BIN2 phosphorylation sites that regulate PCK1 activity, 39 preventing its phosphorylation by BIN2. PCK1 _O-_ fucosylation and phosphorylation exclude 40 each other and oppositely affect PCK1 activity _in vivo_ . The _spy_ mutants show increased 41 PCK1 phosphorylation, reduced PCK activity, and impaired hypocotyl elongation; BR or 42 BIN2 inhibitor treatments suppressed these phenotypes, indicating that these _spy_ effects 43 result from increased BIN2 phosphorylation. Together, these results reveal functional 44 antagonism between SPY/ _O_ -fucosylation and BIN2/phosphorylation in regulating 45 metabolism and growth. 46 47 **Introduction** 48 Plant growth requires coordination of developmental signals with the availability and use of 49 carbon. Growth-promoting hormones stimulate cell division, expansion, and differentiation, 50 but these processes can proceed only when sufficient sugars provide energy, metabolic 51 intermediates, and substrates for cell wall biosynthesis (Zhang et al., 2016). Conversely, 52 carbon availability must be translated into appropriate developmental outputs rather than 53 merely increasing metabolic flux. In addition, non-growing cells must maintain an 54 appropriate basal level of carbon metabolism to sustain cellular function and viability. Plants 55 therefore require mechanisms that integrate hormonal information about when and where 56 to grow, or to not grow, with metabolic information and metabolic activities. 57 Brassinosteroids (BRs) are central regulators of plant growth and act through the receptor 58 kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) to inhibit BRASSINOSTEROID59 INSENSITIVE 2 (BIN2), a glycogen synthase kinase 3 (GSK3)-like kinase. BIN2 is best 60 known for phosphorylating and inhibiting the BRASSINAZOLE RESISTANT 1 (BZR1)/BRI1 61 EMS SUPPRESSOR 1 (BES1) family transcription factors, thereby coupling BR perception 62 to broad transcriptional programs (He et al., 2002; Li and Nam, 2002; Kim et al., 2009). 63 However, the expanding repertoire of BIN2 substrates indicates that BR signaling also 64 directly regulates diverse cellular processes, including primary metabolism (Kim et al., 2023; 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 65 Zhang et al., 2026). How hormone-regulated phosphorylation is coordinated with sugar 66 status remains poorly understood. 67 One major mechanism by which eukaryotic cells sense nutrient availability is the _O_ 68 glycosylation of nucleocytoplasmic proteins (Hart et al., 2007; Lagerlöf et al., 2016; Bi et al., 69 2023). In Arabidopsis, two related glycosyltransferases catalyze distinct forms of this 70 modification: SECRET AGENT (SEC) transfers _O_ -linked N-acetylglucosamine ( _O_ -GlcNAc) 71 from UDP-GlcNAc, whereas SPINDLY (SPY) transfers _O_ -linked fucose ( _O_ -fucose) from 72 GDP-fucose to serine and threonine residues (Zentella et al., 2016; Zentella et al., 2017; Bi 73 et al., 2023). Because these nucleotide-sugar donors are generated through central 74 carbohydrate metabolism, protein _O_ -glycosylation provides a direct route for cellular sugar 75 status to influence protein function (Hart et al., 2007; Aizezi et al., 2025). Genetic studies 76 show that SEC and SPY have overlapping essential functions, as well as distinct roles in 77 hormone signaling, development, circadian regulation, and stress responses (Hartweck et 78 al., 2002; Xu et al., 2017; Bi et al., 2021; Aizezi et al., 2025). SPY is also required for sugar79 dependent seedling growth, supporting a physiological role for _O_ -fucosylation in coupling 80 carbon availability to growth (Bi et al., 2023). At the molecular level, _O_ -glycosylation can 81 alter protein conformation, stability, activity, localization, or interactions, allowing nutrient 82 information to regulate cellular processes rapidly and independently of transcription (Hart et 83 al., 2007; Aizezi et al., 2025). 84 Modification of serine and threonine residues by either sugars or phosphate creates 85 extensive potential for crosstalk between _O_ -glycosylation and phosphorylation (Hart et al., 86 2011). The two modifications may compete for the same residue, influence modification at 87 nearby residues, or exert opposing effects on protein structure and activity. Antagonistic 88 interactions between _O_ -GlcNAcylation and phosphorylation have been observed in animals 89 (Hart et al., 2011), but their functional importance in plants remains largely unexplored 90 (Aizezi et al., 2025). Recent proteomic studies have identified hundreds of _O_ -GlcNAcylated 91 and _O_ -fucosylated proteins as SEC and SPY substrates (Xu et al., 2017; Bi et al., 2023; 92 Zentella et al., 2023; Shrestha et al., 2024). Independently, proximity labeling combined with 93 quantitative phosphoproteomics identified a broad BIN2 signaling network containing 94 numerous _in vivo_ kinase substrates (Kim et al., 2023). Comparison of these datasets 95 revealed substantial overlap between proteins targeted by _O_ -glycosylation and BIN296 dependent phosphorylation (Kim et al., 2023; Aizezi et al., 2025). Whether sugar-dependent 97 _O_ -glycosylation and BIN2 phosphorylation directly modulate each other on shared 98 substrates, and whether such crosstalk integrates metabolic and BR signals, remain 99 important unanswered questions. 100 A particularly compelling shared target is PHOSPHOENOLPYRUVATE CARBOXYKINASE 101 1 (PCK1, also termed PEPCK1) (Xu et al., 2017; Kim et al., 2023), which catalyzes the ATP102 dependent decarboxylation and phosphorylation of oxaloacetate to produce 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 103 phosphoenolpyruvate, a key step in gluconeogenesis. This reaction enables carbon derived 104 from storage lipids, amino acids, and organic acids to enter carbohydrate biosynthesis 105 (Leegood and Walker, 2003; Walker et al., 2021). PCK1 is especially important during post106 germinative growth, when seedlings must convert stored reserves into soluble sugars before 107 photosynthesis is fully established. Arabidopsis _pck1_ mutants accumulate less soluble sugar 108 and show severe defects in hypocotyl growth and seedling establishment under carbon109 limiting conditions (Rylott et al., 2003; Penfield et al., 2004; Zhang et al., 2026). Beyond early 110 seedling development, PCK functions as a major decarboxylase in PCK-type C 4 111 photosynthesis and in some crassulacean acid metabolism plants (Burnell and Hatch, 1988; 112 Walker and Leegood, 1996; Leegood and Walker, 2003). PCK regulation therefore provides 113 a strategic point for matching sugar production with developmental and environmental 114 demands. 115 A recent study showed that BIN2 directly phosphorylates and inhibits PCK1, whereas BR 116 signaling promotes PCK1 dephosphorylation and activation, establishing PCK1 as a direct 117 link between a growth-hormone pathway and carbon metabolism (Zhang et al., 2026). Its 118 identification as an _O_ -glycosylated protein suggests that sugar status and BR signaling 119 converge on PCK1 through interacting post-translational modifications (Xu et al., 2017). 120 Here, we investigate whether SPY-mediated _O_ -fucosylation modulates BIN2-dependent 121 phosphorylation of PCK1 and how this interplay affects PCK activity and seedling growth. 122 123 **Materials and methods** 124 **Plant materials and growth conditions** 125 _Arabidopsis thaliana_ Columbia-0 (Col-0) was used as the wild type. The _spy-4 allele_ (Swain 126 et al., 2001), _spy-23_ (WiscDsLox241C03) (Woody et al., 2007; Bi et al., 2023), _bzr1-1D_, the 127 semidominant gain-of-function mutant _bin2-1_, and the 35S:PCK1-YFP/Col-0 and PCK1128 YFP/ _bin2-1_ lines (Zhang et al., 2026) were described previously. The _35S:PCK1-YFP/spy-_ 129 _4_ line was generated by crossing 35S:PCK1-YFP/Col-0 with _spy-4_ . A homozygous line was 130 identified in the F2 generation by screening for Basta resistance and the _spy-4_ phenotype 131 and propagated to the F3 generation for experiments. Seeds were surface-sterilized with 132 chlorine gas for 2 h and sown on 120-mm square polystyrene Petri dishes (Greiner Bio-One) 133 containing 0.5× Murashige and Skoog (MS) salts (Duchefa) with the indicated 134 concentrations of sucrose and chemical treatments. Plates were stratified at 4 °C in the dark 135 for 3 d and then grown at 22 °C under a 16-h light/8-h dark photoperiod. For etiolated 136 seedling cultivation, plates were exposed to light for 6 h to synchronize germination before 137 incubation at 22 °C in continuous darkness for the indicated periods. _Nicotiana benthamiana_ 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 138 plants were cultivated in a greenhouse at 20 °C under a 16-h light/8-h dark cycle; 139 approximately 6-week-old plants were used for _Agrobacterium_ -mediated infiltration. 140 **Chemical treatments** 141 Half-strength MS medium was supplemented with the indicated concentrations of 142 propiconazole (PPZ), brassinolide (BL), 30 μM bikinin, or 30 μM SOFTI (Aizezi et al., 2024); 143 corresponding solvent controls were included. 144 **Hypocotyl length measurements** 145 Seedlings were photographed using a digital camera (Nikon D600). Hypocotyl length of 146 seedlings was measured with ImageJ software. 147 **PCK activity assays** 148 Measurement of PCK activity in crude plant extracts was performed as described previously 149 (Zhang et al., 2026). In brief, harvested plant tissues were ground to fine powder in liquid 150 nitrogen. Ground tissue (20 mg) was extracted with 500 μl of 100 mM Bicine-KOH (pH 9.0) 151 containing 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 5 mM β-mercaptoethanol, 1 mM 152 EDTA, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1× protease inhibitor 153 cocktail (Roche, 11873580001), and 1× phosphatase inhibitor (Pierce, A32957). The 154 extracts were vortexed and centrifuged at 14,000 × g for 10 min. PCK decarboxylation 155 activity in the supernatant was determined at 25 °C by monitoring NADH oxidation at 340 156 nm. The reaction mixture contained 65 mM Tris-acetate (pH 7.4), 100 mM KCl, 0.3 mM 157 oxaloacetate, 1 mM ATP, 10 μM MnCl₂, 4 mM MgCl₂, 71.5 mM β-mercaptoethanol, 0.4 mM 158 NADH, 1 U pyruvate kinase, and 2 U lactate dehydrogenase. One unit of PCK activity was 159 defined as the amount of enzyme catalyzing the oxidation of 1 μmol NADH min⁻¹ at 25 °C. 160 **Protein extraction and immunoblotting** 161 Protein extraction and immunoblotting were performed as described previously (Zhang et 162 al., 2025). Briefly, plant material was ground in liquid nitrogen and extracted in Laemmli 163 buffer [125 mM Tris-HCl (pH 6.8), 10% (v/v) 2-mercaptoethanol, 0.01% (w/v) bromophenol 164 blue, 4% (w/v) SDS, and 20% (v/v) glycerol] and incubated at 95 °C for 10 min. Protein 165 samples were separated by standard SDS-PAGE or Phos-tag SDS-PAGE and transferred 166 to polyvinylidene difluoride (PVDF) membranes by semi-dry transfer. Membranes were 167 blocked with 5% (w/v) low-fat milk powder (AppliChem) in TBS-T (0.1% Tween-20) for 1 h 168 at room temperature and probed with anti-PCK1 [1:5,000; (Rojas et al., 2021)], anti-GFP 169 (1:2,000; HT801, Transgen Biotech), anti-His (1:3,000; 27E8, Cell Signaling Technology), 170 anti-mCherry/RFP (1:5,000; ab213511, Abcam), or anti-GST (1:3,000; 26H1, Cell Signaling 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 171 Technology), as indicated. After three 10-min washes in TBS-T, membranes were incubated 172 with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (1:5,000; 173 Fisher Scientific) and washed three times for 10 min in TBS-T. Signals were detected using 174 WesternBright Quantum (Advansta). For the nuclear protein immunoblotting, nuclear protein 175 fractions from WT, _bin2-1_, _spy-4_, and _spy-23_ seedlings were probed with a mixture of anti176 serine- _O_ -fucose and anti-threonine- _O_ -fucose antibodies (1:1,000; GlycoScientific). 177 Coomassie Brilliant Blue staining or the indicated housekeeping protein served as a loading 178 control. 179 **AAL affinity purification and AAL-biotin detection** 180 AAL-agarose affinity purification was used to enrich fucose-containing proteins from agro181 infiltrated _N. benthamiana_ leaves and _Arabidopsis_ seedlings. Around 250 mg infiltrated _N._ 182 _benthamiana_ leaf tissue or _Arabidopsis_ tissue was homogenized in 1.5 mL of extraction 183 buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.5% (v/v) Triton X-100, 2.5 mM 184 2-mercaptoethanol, 20 μM MG132, and 1× cOmplete EDTA-free protease inhibitor cocktail 185 (Sigma-Aldrich). All samples were sonicated for 1 min on ice and centrifuged at 15,000 g for 186 10 min at 4°C to clarify the extracts. An aliquot of each clarified extract was retained for input 187 analysis. 20 μL of extract was mixed with 20 μL of 2× Laemmli sample buffer, and the 188 remaining extract was incubated with 30 μl of AAL-agarose beads (2 mg lectin/ml, AL-1393189 2; Vector Laboratories) for 1.5 h at 4°C with end-over-end rotation. Beads were washed by 190 four washes with TBST-500 and one wash with 20 mM Tris-HCl (pH 7.5). Bound proteins 191 were eluted by heating at 65°C for 10 min in 2× Laemmli sample buffer and analyzed by 192 immunoblotting. 193 For AAL-biotin detection of protein _O_ -fucosylation, protein extracts were resolved by SDS– 194 PAGE, transferred to PVDF membranes, and probed sequentially with biotinylated AAL 195 (1:10,000; Vector Laboratories) for 1 h and horseradish peroxidase-conjugated streptavidin 196 for 1 h (1:5,000; Thermo Scientific). 197 **Transient expression in** _**N. benthamiana**_ **and co-immunoprecipitation assays** 198 _Agrobacterium tumefaciens_ GV3101 strains carrying the indicated constructs were 199 resuspended in infiltration buffer containing 10 mM MES (pH 5.6), 10 mM MgCl₂, 0.01% (v/v) 200 Tween 20, and 150 μM acetosyringone to an OD 600 of 1.0. The suspensions were combined 201 as indicated for each experiment and incubated for 1 h at room temperature before infiltration 202 into the abaxial side of approximately 6-week-old _N. benthamiana_ leaves using a 1 mL 203 needleless syringe. Leaf tissue was harvested 3 d after infiltration for protein extraction. 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 204 Co-immunoprecipitation assays were performed as described previously (Zhang et al., 205 2022). Approximately 200 mg of infiltrated leaf tissue was ground to a fine powder in liquid 206 nitrogen and extracted in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM 207 NaCl, 5 mM EDTA (pH 8.0), 0.1% (v/v) Triton X-100, and 0.2% (v/v) NP-40, supplemented 208 immediately before use with 2 mM phenylmethylsulfonyl fluoride and 1× protease inhibitor 209 cocktail (Roche, 11873580001). After centrifugation for 10 min at 4°C, an aliquot of each 210 clarified extract was retained as the input, and the remaining extract was incubated with anti211 GFP magnetic beads (SM038005, Smart Lifesciences) for 1.5 h at 4°C. The beads were 212 washed three times with lysis buffer, and bound proteins were eluted by boiling in Laemmli 213 sample buffer for 10 min. Input and immunoprecipitated fractions were analyzed by 214 immunoblotting with anti-GFP and anti-mCherry/RFP antibodies, as indicated. 215 **Recombinant protein expression and purification** 216 Recombinant 6×His–SUMO–PCK1, 6×His–SUMO–SPY, 6×His–SUMO–3TPR-SPY [The 217 catalytic domain and three TPR domains of SPY (residues 326–914)], and GST–BIN2 were 218 expressed and purified as described previously (Zhang et al., 2026). Briefly, the proteins 219 were expressed in Rosetta (DE3) _Escherichia coli_ cells by overnight induction with 300 μM 220 IPTG at 16°C for 6×His–SUMO-tagged proteins or 22°C for GST-tagged proteins. Cells were 221 lysed by ultrasonication in 1x PBS supplemented with 1 mM PMSF and 1x protease inhibitor 222 cocktail. After clarification by centrifugation, 6×His–SUMO- and GST-tagged proteins were 223 purified using Ni–NTA agarose (R901-01, Invitrogen) and glutathione agarose (16100, 224 Pierce), respectively, followed by size-exclusion chromatography on a Superdex 200 column 225 (GE Healthcare). Purified proteins were concentrated, aliquoted, and stored at −80°C until 226 use. 227 **Bio-layer interferometry** 228 The interaction between 3TPR-SPY and PCK1 was measured using a GatorPlus biolayer 229 interferometry system (Gator Bio). 3TPR-SPY was biotinylated with EZ-Link NHS-PEG4230 Biotin (Thermo A39259) and immobilized on streptavidin-XT biosensors (Gator Bio 160029) 231 and dipped into PCK1 at 0.125, 0.25, 0.5, and 1 μM for 360 s to record association. The 232 sensor was then dipped into PBS without PCK1 for 360 s to record dissociation. Biosensors 233 loaded with NHS-PEG4-Biotin quenched in 50 mM Tris-Cl (pH = 7.5) was used as a control 234 for nonspecific binding of PCK1 to the sensor surface. The dissociation constant ( _K_ d) was 235 calculated using Gator Bio analysis software based on classical kinetic fitting ( _k_ off/ _k_ on). 236 _**In vitro**_ _**O**_ **-fucosylation assay** 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 237 For _in vitro O_ -fucosylation assays, 5 μg purified recombinant PCK1 protein was incubated 238 with 1 μg recombinant SPY or 3TPR-SPY protein in 1× PBS (pH 7.3) containing 5 mM MgCl₂ 239 and 1 mM GDP-fucose at room temperature for indicated periods or overnight, as described 240 previously (Aizezi et al., 2024). Parallel reactions lacking SPY or GDP-fucose, or containing 241 SPY inhibitor SOFTI, served as controls. 242 **Semi-** _**in vitro**_ **BIN2 kinase assay** 243 Semi- _in vitro_ kinase assays were performed using the reaction conditions described 244 previously with modifications (Zhang et al., 2026). Briefly, PCK1-YFP was 245 immunoprecipitated with anti-GFP magnetic beads from _N. benthamiana_ leaves 246 coexpressing SPY-RFP or the RFP control. Bead-bound PCK1-YFP was incubated with 1.0 247 μg of purified GST-BIN2 in kinase reaction buffer containing 25 mM Tris-HCl (pH 7.4), 12 248 mM MgCl₂, 1 mM DTT, and 1 mM ATP at 25°C for 3 h. PCK1 phosphorylation was analyzed 249 by Phos-tag SDS–PAGE followed by immunoblotting with anti-PCK1 antibody. 250 **Nuclear protein extraction** 251 For isolation of nuclear proteins, approximately 350 mg of frozen Arabidopsis tissue powder 252 was extracted in 1.5 mL of ice-cold buffer 1 (0.4 M sucrose, 10 mM Tris-Cl [pH 8.0], 5 mM 253 dithiothreitol, 2 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail). 254 The homogenate was passed through a 70-μm cell strainer into a 50-mL conical tube and 255 centrifuged at 800 × g for 3 min at 4 °C. The pellet was resuspended in 700 μL of the 256 supernatant and centrifuged at 700 × g for 5 min at 4 °C. The pellet was resuspended in 1 257 mL of buffer 2 (0.25 M sucrose, 10 mM Tris-Cl [pH 8.0], 5 mM dithiothreitol, 10 mM MgCl₂, 258 1% [v/v] Triton X-100, 0.1 mM PMSF, and 1× protease inhibitor cocktail) and centrifuged at 259 700 × g for 5 min at 4 °C. The pellet was resuspended in 500 μL of buffer 3 (1.7 M sucrose, 260 10 mM Tris-Cl [pH 8.0], 5 mM dithiothreitol, 2 mM MgCl₂, 0.15% [v/v] Triton X-100, 0.1 mM 261 PMSF, and 1× protease inhibitor cocktail), layered over a further 500 μL of buffer 3, and 262 centrifuged at 15,000 × g for 10 min at 4 °C. The nuclear pellet was resuspended in 60 μL 263 of 2× SDS sample buffer and heated at 65 °C for 10 min before immunoblot analysis. 264 **Mass spectrometric analysis of PCK1** _**O**_ **-fucosylation** 265 PCK1-YFP was immunoprecipitated from _N. benthamiana_ leaves coexpressing SPY-RFP, 266 resolved by SDS–PAGE, and visualized by Coomassie Brilliant Blue staining. Bands 267 corresponding to PCK1-YFP were excised, reduced, alkylated, and digested in-gel with 268 trypsin. Peptides were analyzed using an Orbitrap Eclipse Tribrid mass spectrometer 269 coupled to an Easy-nLC 1200 system, as described previously (Zhang et al., 2026). MS/MS 270 spectra were searched using Protein Prospector (v6.4.9) against the _Arabidopsis thaliana_ 271 TAIR10 database concatenated with randomized decoy sequences. Carbamidomethylation 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 272 of cysteine was specified as a fixed modification, whereas methionine oxidation, protein N273 terminal acetylation, peptide N-terminal glutamine conversion to pyroglutamate, _O_ 274 fucosylation of serine/threonine, and neutral loss of _O_ -fucose were included as variable 275 modifications. Precursor and fragment ion mass tolerances were set to 10 and 20 ppm, 276 respectively, and the false discovery rate was controlled at 1% for both peptides and proteins. 277 Up to two missed cleavages and three variable modifications per peptide were allowed. 278 **Statistical analysis** 279 Statistical analyses were performed as described previously (Zhang and Kramer, 2018) 280 using IBM SPSS Statistics (version 29.0.2.0). Data are presented as mean ± s.d. or as box 281 plots, as indicated in the figure legends. Comparisons between two groups were performed 282 using two-tailed Student’s _t_ -test. Comparisons among multiple groups were performed using 283 one-way ANOVA followed by Tukey’s or Games–Howell multiple-comparison test. Different 284 letters or asterisks indicate significant differences according to the _P_ values specified in the 285 figure legends. Sample sizes and numbers of biological replicates are provided in the 286 legends. 287 **Accession numbers** 288 Sequence data from this article can be found in the GenBank/EMBL data libraries under the 289 following Arabidopsis thaliana locus identifiers: _PCK1_ (AT4G37870), _SPY_ (AT3G11540), 290 _SEC_ (AT3G04240), _BIN2_ (AT4G18710), _BRI1_ (AT4G39400), _BZR1_ (AT1G75080), and 291 _BES1_ (AT1G19350). 292 293 **Results** 294 **SPY associates with and** _**O**_ **-fucosylates PCK1** 295 To investigate whether PCK1 is modified by SPY, we first analyzed the _O_ -fucosylation of 296 PCK1 in Arabidopsis. We crossed the 35S:PCK1-YFP line with _spy-4_ to generate a 297 homozygous 35S:PCK1-YFP/ _spy-4_ line. PCK1-YFP was enriched by _Aleuria aurantia_ lectin 298 (AAL)-agarose from 35S:PCK1-YFP/Col-0 extracts but not from the _spy-4_ background, 299 despite similar PCK1-YFP abundance in the inputs (Fig. 1a), indicating SPY-dependent _O_ 300 fucosylation of PCK1 _in vivo_ . Mass spectrometry detected _O_ -fucosylation within an N301 terminal PCK1 peptide containing Ser-62, Thr-65, and Thr-66 (Fig. 1b). Ser-62 and Thr-66 302 are known BIN2 phosphorylation sites (Zhang et al., 2026). Co-immunoprecipitation from 303 _Nicotiana benthamiana_ leaves showed that PCK1-YFP associates with SPY-RFP but not 304 the RFP control (Fig. 1c). The S62A/T66A substitutions did not abolish this association. 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 305 Biolayer interferometry (BLI) using purified PCK1 and a truncated SPY protein containing 306 three tetratricopeptide repeat motifs and the catalytic region (3TPR-SPY) further supported 307 direct binding, with an apparent dissociation constant ( _K_ d) of 0.23 nM (Fig. 1d). Incubation 308 of recombinant PCK1 with SPY and GDP-fucose produced a time-dependent increase in 309 AAL-detectable _O_ -fucosylation (Fig. 1e), whereas the SPY inhibitor SOFTI reduced _O_ 310 fucosylation of both PCK1 and SPY (Fig. 1f). In _N. benthamiana_, SPY-RFP increased AAL 311 enrichment of PCK1-YFP more strongly than of PCK1 [AA] -YFP (S62A/T66A), although the 312 mutant retained detectable enrichment (Fig. 1g). Thus, SPY directly associates with and _O_ 313 fucosylates PCK1, and Ser-62/Thr-66 substitutions reduce, but do not eliminate SPY314 dependent PCK1 _O_ -fucosylation. 315 **SPY-mediated** _**O**_ **-fucosylation attenuates BIN2-dependent PCK1 phosphorylation** 316 The proximity of the _O_ -fucosylated PCK1 region to the known BIN2 phosphorylation sites 317 raised the possibility that SPY-dependent _O_ -fucosylation interferes with PCK1 318 phosphorylation. To examine this relationship, we transiently co-expressed PCK1-YFP, 319 BIN2-YFP, and SPY-YFP in _N. benthamiana_ leaves. BIN2-YFP increased PCK1-YFP 320 phosphorylation, whereas co-expression of SPY-YFP reduced the phosphorylated fraction 321 without appreciably changing total PCK1-YFP abundance (Fig. 2a). We then immunopurified 322 PCK1-YFP from leaves co-expressing either SPY-RFP or an RFP control and assayed its 323 subsequent phosphorylation by recombinant GST-BIN2 _in vitro_ . PCK1-YFP isolated from 324 SPY-expressing tissue showed substantially less BIN2-dependent phosphorylation than 325 PCK1-YFP from the RFP control (Fig. 2b). These results indicate that SPY expression, 326 associated with increased PCK1 _O_ -fucosylation, attenuates subsequent BIN2-mediated 327 phosphorylation. 328 We next examined how BR signaling and SPY activity influence PCK1 modifications and 329 enzymatic activity in _Arabidopsis_ . Inhibition of BR biosynthesis with PPZ increased PCK1 330 phosphorylation and decreased the amount of PCK1-YFP recovered by AAL-agarose 331 without markedly changing total PCK1-YFP abundance (Fig. 2c). The phosphorylated PCK1 332 fraction was lower in the AAL-enriched material than in the corresponding input, consistent 333 with an inverse relationship between the two modifications (Fig. 2c). In PCK1-YFP/ _bin2-1_ 334 seedlings, where BIN2 activity is elevated, PCK1 phosphorylation increased while AAL 335 enrichment decreased relative to the WT background (Fig. 2d). Bikinin, a BIN2/GSK3 336 inhibitor _,_ reduced PCK1 phosphorylation, increased AAL enrichment, and partly restored 337 PCK activity, which was approximately 65% of WT in _bin2-1_ without treatment (Fig. 2d,e). 338 We also examined SPY inhibition in PCK1-YFP/Col-0 seedlings. SOFTI decreased AAL 339 enrichment of PCK1-YFP without a marked change in its total abundance, accompanied by 340 greater PCK1 phosphorylation and a reduction of PCK activity to approximately 60% of the 341 control level (Fig. 2f,g). Together, these experiments support reciprocal effects of SPY 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 342 dependent _O_ -fucosylation and BIN2-dependent phosphorylation on PCK1 modification 343 status and activity. 344 **Loss of SPY reduces PCK1 activity by increasing PCK1 phosphorylation** 345 We next asked whether increased phosphorylation contributes to the reduced PCK activity 346 observed in _spy_ seedlings. In 5-d-old seedlings, _spy-4_ and _spy-23_ each showed 347 approximately half the PCK activity of WT, together with a higher proportion of 348 phosphorylated PCK1 (approximately 57% versus 22% in WT _;_ Fig. 3a). Treatment with 1 349 nM BL lowered PCK1 phosphorylation to approximately 17–18% and restored PCK activity 350 in both _spy_ alleles toward the WT level (Fig. 3a). In 3.5-d-old etiolated seedlings grown on 351 sugar-free medium, basal PCK1 phosphorylation was approximately 24–26% in WT and 44– 352 47% in _spy_ mutants, accompanied by lower PCK activity in the mutants (Fig. 3b,c). PPZ 353 increased PCK1 phosphorylation to approximately 51–54% in WT and 67–69% in _spy_ 354 mutants and further lowered PCK activity. Addition of either BL or bikinin reduced the PPZ355 associated phosphorylated fraction and restored activity substantially in both genotypes 356 (Fig. 3b,c). These results indicate that the PCK activity defect in _spy_ seedlings is reversible 357 by inhibiting BIN2-dependent phosphorylation through BR signaling or a kinase inhibitor. 358 _**O**_ **-fucosylation promotes seedling growth and is inhibited by BIN2** 359 To test whether loss of _O_ -fucosylation enhances BIN2-dependent phosphorylation and thus 360 contributes to growth inhibition, we analyzed the phenotypes of _spy_ mutants in the dark. On 361 sugar-free medium, _pck1_ and _spy_ showed short hypocotyls (Fig. 4a,b). On media containing 362 exogenous sucrose, the _pck1_ phenotype was rescued (Fig. 4a,b), as reported previously. 363 However, sucrose did not rescue the short-hypocotyl phenotype of _spy-4_ or _spy_ -23 (Fig. 364 4a,b). Dysfunction in additional SPY substrates likely contributes to the growth defects, as 365 PCK1 is one of hundreds of proteins targeted by SPY. 366 The significant overlap between targets of SPY and BIN2 raises the possibility that SPY 367 antagonizes BIN2 and enhances BR-regulated processes globally. To explore whether BIN2 368 might also influence _O_ -fucosylation of proteins other than PCK1, we examined nuclear 369 protein fractions with antibodies recognizing serine- and threonine-linked _O_ -fucose. Several 370 bands showed lower intensity in the gain-of-function mutant _bin2-1_ than in WT, and were 371 not detected in the _spy_ mutants (Fig. 4c). These observations indicate that BIN2 reduces _O_ 372 fucosylation of multiple proteins in addition to PCK1. 373 **SPY promotes BR response in dark-grown seedlings** 374 To test this possibility, we analyzed the BR responses of _spy_ mutants. The _spy_ mutants 375 displayed shorter hypocotyls than wild type under normal conditions but showed no 376 significant difference from wild type when grown on 2 µM PPZ, or PPZ plus 100 nM 377 brassinolide (BL) or 30 µM bikinin (Fig. 5a,b), indicating that the short-hypocotyl phenotype 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 378 of _spy_ mutants is dependent on BR. We then grew the mutants on various concentrations 379 of PPZ and 2 µM PPZ plus various concentrations of BL. The results show that the _spy-4_ 380 and _spy-23_ mutants were hypersensitive to PPZ treatments, in contrast to _bzr1-1D,_ which is 381 hyposensitive (Fig. 5c,d), but were hyposensitive to exogenous BL: WT reached maximal 382 elongation on 10 nM BL, whereas both _spy_ alleles continued to increase hypocotyl length 383 as BL concentration increased to 100 nM (Fig. 5e). These results suggest that sugar384 dependent _O_ -glycosylation promotes plant growth in part by antagonizing BIN2-dependent 385 phosphorylation. 386 387 **Discussion** 388 Plant growth requires coordination between carbon availability and hormone signaling. 389 Nucleocytoplasmic _O_ -glycosylation links nutrient status to protein regulation, while BIN2390 dependent phosphorylation connects brassinosteroid (BR) signaling to numerous cellular 391 targets (Bi et al., 2023; Kim et al., 2023; Aizezi et al., 2025). To investigate how these 392 modifications interact to regulate protein function, we examined SPY-mediated _O_ 393 fucosylation of PCK1, a key enzyme in carbon metabolism whose activity is directly inhibited 394 by BIN2 phosphorylation (Zhang et al., 2026). Our findings demonstrate reciprocal 395 antagonism between PCK1 _O_ -fucosylation and phosphorylation and show that SPY 396 promotes PCK1 activity by counteracting BIN2-dependent inhibition. PCK1 thus provides a 397 mechanistic connection between sugar-responsive _O_ -fucosylation and BR-regulated 398 phosphorylation in the control of carbon metabolism. 399 The reduced PCK1 activity in the _spy_ mutant supports a positive role for SPY-mediated _O_ 400 fucosylation in PCK1 regulation in vivo. Mass spectrometry detected _O_ -fucosylation within 401 an N-terminal peptide containing Ser-62, Thr-65, and Thr-66. Ser-62 and Thr-66 are 402 established BIN2 phosphorylation sites, and our previous work showed that phosphorylation 403 at these residues inhibits PCK1 by shifting the enzyme from an active hexameric form to an 404 inactive dodecameric form (Zhang et al., 2026). Substitution of Ser-62 and Thr-66 with 405 alanine reduced SPY-dependent _O_ -fucosylation, consistent with overlapping sites of _O_ 406 fucosylation and phosphorylation within this regulatory region. These observations raise two 407 possible mechanisms by which _O_ -fucosylation could promote PCK1 activity: directly 408 influencing enzyme structure or preventing the inhibitory phosphorylation that alters its 409 oligomeric state. 410 Genetic and pharmacological perturbations further support reciprocal antagonism between 411 the two modifications _in vivo_ . SPY overexpression reduced BIN2-mediated PCK1 412 phosphorylation, whereas loss of SPY or its chemical inhibition by SOFTI caused PCK1 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 413 hyperphosphorylation. Conversely, promoting BIN2 activity by inhibiting BR biosynthesis 414 decreased PCK1 _O_ -fucosylation, whereas bikinin treatment increased it. AAL lectin 415 preferentially enriched unphosphorylated PCK1, further supporting an inverse relationship 416 between _O_ -fucosylation and phosphorylation. Together with the mass spectrometry and 417 mutational data, these findings support a model in which the two modifications compete at 418 shared or closely spaced residues within the N-terminal regulatory region. Functional rescue 419 of _spy_ provides further insight into the consequences of this interplay. Treatment with BL or 420 bikinin reversed PCK1 hyperphosphorylation and restored its activity to wild-type levels, 421 demonstrating that relief of inhibitory phosphorylation is sufficient to recover PCK1 activity 422 in the absence of SPY. These results support protection from BIN2-dependent inhibition as 423 a principal mechanism through which SPY-mediated _O_ -fucosylation promotes PCK1 activity. 424 The SPY–BIN2 antagonism demonstrated for PCK1 may extend to other cellular proteins, 425 suggesting a broader regulatory relationship between _O_ -glycosylation and GSK3-mediated 426 phosphorylation. Dark-grown _spy_ seedlings exhibit short hypocotyls, resembling _pck1_ 427 mutants and consistent with reduced PCK1 activity. However, exogenous sugar rescues the 428 short-hypocotyl phenotype of _pck1_ but not that of _spy_, suggesting that additional SPY targets 429 contribute to the growth defects of _spy_ . Previous proteomic studies identified more than 100 430 BIN2 substrates that are also modified by _O_ -GlcNAc and/or _O_ -fucose, highlighting extensive 431 potential for crosstalk between _O_ -glycosylation and phosphorylation. Consistent with this 432 possibility, our anti- _O_ -fucose immunoblot analysis revealed an overall reduction in protein 433 _O_ -fucosylation in the dominant _bin2-1_ mutant, suggesting that BIN2-dependent regulation 434 of _O_ -fucosylation extends beyond PCK1. Broader antagonism between SPY and BIN2 may 435 therefore contribute to the short-hypocotyl and BR-hyposensitive phenotypes of dark-grown 436 _spy_ seedlings. Suppression of these phenotypes by high concentrations of BL or by bikinin 437 further supports involvement of the BR–BIN2 pathway in these growth defects. Together, 438 our findings establish reciprocal antagonism between _O_ -fucosylation and phosphorylation 439 as a mechanism regulating PCK1 and suggest that similar interactions among shared 440 substrates help coordinate carbon metabolism and plant growth. Functional characterization 441 of additional SPY–BIN2 substrates will clarify the scope and physiological significance of 442 this mechanism in integrating nutrient and hormone signaling. 443 444 **Author contributions** 445 H.Z. and Z.W. designed the research. H.Z., Y.A., and A.C. performed experiments. H.Z. and 446 Z.W. analyzed data. H.Z. and Z.W. wrote and edited the manuscript. 447 **Acknowledgement** 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 448 The authors thank Alberto A. Iglesias (Instituto de Agrobiotecnología del Litoral, UNL, 449 CONICET, FBCB, Santa Fe, Argentina) for sharing the anti-PCK1 antibody, and Drs. Cao 450 Son Trinh and Shouling Xu for assistance with mass spectrometry analyses. 451 **Funding** 452 This work was funded by the National Institutes of General Medical Science grants 453 R01GM066258 and S10OD030441. 454 **Conflicts of interest** 455 The authors declare no competing interests. 456 **Data availability statement** 457 All materials and data presented in this study are available from the corresponding author 458 on request. 459 **References** 460 **Aizezi, Y., Yuan, Y., Xu, S.L., and Wang, Z.Y.** (2025). A tale of two sugars: O-GlcNAc and O-fucose 461 orchestrate growth, development, and acclimation in plants. Trends Biochem Sci **50,** 332-343. 462 **Aizezi, Y., Zhao, H., Zhang, Z., Bi, Y., Yang, Q., Guo, G., Zhang, H., Guo, H., Jiang, K., and Wang,** 463 **Z.Y.** (2024). Structure-based virtual screening identifies small-molecule inhibitors of O464 fucosyltransferase SPINDLY in Arabidopsis. 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The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 538 **Zhang, H., Aizezi, Y., Bessho-Uehara, K., Chaudhary, A., Trinh, C.S., Xu, S.L., and Wang, Z.Y.** 539 (2026). Brassinosteroids promote sugar synthesis by inhibiting BIN2 phosphorylation of 540 phosphoenolpyruvate carboxykinase. Nat Plants **12,** 1741-1755. 541 **Zhang, H., Janina, N., Utkur, K., Manivannan, T., Zhang, L., Wang, L., Grefen, C., Schaffrath, R.,** 542 **and Kramer, U.** (2025). Diphthamide formation in Arabidopsis requires DPH1-interacting DPH2 543 for light and oxidative stress resistance. 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Immunoblotting of input (total protein extracts) or AAL556 agarose-enriched samples was probed with an anti-GFP antibody. **b,** MS/MS spectra of 557 fucosylated peptide 62-SAPTTPINQNAAAAFAAVSEEER-84 of PCK1-YFP protein 558 immunoprecipitated from _N. benthamiana_ leaves co-expressing PCK1-YFP with SPY-RFP, 559 containing Ser-62, Thr-65, and Thr-66 residues. **c,** PCK1-YFP was co-immunoprecipitated AA 560 with SPY-RFP in _N. benthamiana_ leaves. PCK1-YFP or PCK1 -YFP was transiently 561 expressed with SPY-RFP or RFP-MH in _N. benthamiana_ leaf. Immunoprecipitation was 562 performed with total protein extracts (input) using anti-GFP beads. Input and 563 immunoprecipitates (IPs) were analyzed by anti-mCherry and anti-GFP antibodies. **d,** 564 Biolayer interferometry (BLI) analysis of the interaction between 3TPR-SPY and PCK1. 565 Biotinylated 3TPR-SPY was immobilized on streptavidin biosensors and exposed to the 566 indicated concentrations of PCK1 for 360 s, followed by dissociation in PBS for 360 s. 567 Biosensors loaded with free biotin and exposed to 1 μM PCK1 served as a negative control. 568 The ( _K_ d ) was 0.23 nM. **e,** Time-course analysis of SPY-mediated _O_ -fucosylation of 569 recombinant PCK1. **f,** In vitro _O_ -fucosylation of PCK1 by the SPY catalytic region (3TPR– 570 SPY) by using 1 mM GDP-fucose with 10 μM (+) or 30 μM (++) of SOFTI. Fucosylation level 571 of PCK1 and 3TPR-SPY was detected by immunoblotting with a biotinylated AAL. PCK1 and 572 3TPR-SPY were detected by immunoblotting with an anti-His antibody. **g,** PCK1-YFP was AA 573 _O_ -fucosylated by SPY in _N. benthamiana_ leaves. PCK1-YFP or PCK1 -YFP was transiently 574 co-expressed with SPY-RFP or RFP-MH in _N. benthamiana_ leaves. _O_ -fucosylated proteins 575 were enriched by AAL-agarose. Immunoblot containing input or AAL-agarose-enriched 576 samples was probed with an anti-GFP or anti-RFP antibody. Values below the blots 577 represent relative band intensities. Coomassie Brilliant Blue (CBB) or Ponceau S staining of 578 the gel blot was used as a loading control. 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 579 **Alt text:** Panel a shows lectin-enriched PCK1 immunoblots, with a band in wild type but not 580 _spy-4_ . Panel b is a fucosylated peptide mass spectrum plotting fragment intensity against 581 mass-to-charge ratio. Panel c shows SPY co-precipitation blots for PCK1 and PCK1 [AA] . 582 Panel d plots binding response against time, with larger responses at higher PCK1 583 concentrations. Panel e shows progressively stronger PCK1 fucosylation bands over time. 584 Panel f shows fucosylation blots with weaker bands at higher SOFTI concentrations. Panel 585 g shows lectin-enriched PCK1 blots, with weaker bands for the PCK1 [AA] under SPY 586 coexpression. 587 588 **Figure 2: Phosphorylation and** _**O**_ **-fucosylation of PCK1 antagonize each other. a,** Co589 expression of SPY with BIN2 inhibits BIN2-mediated PCK1 phosphorylation _in vivo_ . The 590 proteins were transiently co-expressed in _N. benthamiana_ leaves and analyzed by 591 immunoblotting with an anti-GFP antibody to detect protein expression by Phos-tag gel 592 (upper blot) and standard SDS-PAGE (lower blot). **b,** PCK1 immunoprecipitated from _N._ 593 _benthamiana_ leaves co-expressing SPY shows reduced subsequent phosphorylation by 594 recombinant BIN2. PCK1 was immunoprecipitated from leaves co-expressing PCK1 with 595 SPY-RFP or RFP. We incubated the immunoprecipitated PCK1 on beads with GST-BIN2 in 596 kinase buffer, then immunoblotted with an anti-PCK1 antibody. **c,** Immunoblotting detection 597 of total, phosphorylated, and _O_ -fucosylated PCK1 in the 7-day-old _PCK1-YFP/Col-0_ 598 transgenic seedlings grown on 0.5x MS sugar-free medium with or without 2 μM PPZ. Anti599 GFP immunoblotting with input (total protein extract) or AAL-agarose pulled down samples 600 was performed to analyze PCK1 phosphorylation status using SDS-PAGE gel containing 601 Phos-tag (upper panel) or no Phos-tag (lower). **d,** Immunoblotting detection of total, 602 phosphorylated, and _O_ -fucosylated PCK1 in the 16-day-old seedlings of _PCK1-YFP/Col-0_ 603 and _PCK1-YFP/bin2-1_ grown on 0.5x MS with 1% sucrose with or without 30 μM bikinin. **e,** 604 Relative PCK activity of seedlings in ( **d** ). **f,** Immunoblotting detection of total, phosphorylated, 605 and _O_ -fucosylated PCK1 in the 16-day-old seedlings of _PCK1-YFP/Col-0_ on 0.5x MS with 606 1% sucrose with or without 30 μM SOFTI. Anti-GFP immunoblotting with AAL-agarose607 enriched samples to analyze PCK1 fucosylation or input (total protein extract) was 608 performed to analyze PCK1 phosphorylation status using SDS-PAGE gel containing Phos609 tag (lower blot) or no Phos-tag (upper blot). Phosphorylated PCK1 is the shifted band 610 marked with *. CBB: Coomassie Brilliant Blue staining of the gel blot _._ **g,** Relative PCK activity 611 of seedlings in ( **f** ). Data are mean ± s.d., _n_ = 3 ( **e,g** ); ***, _P_ < 0.001, according to two-tailed 612 Student’s _t_ -test ( **g** ), ( **e** ) Significant differences between means are marked by different letters 613 (one-way ANOVA, Tukey’s test, _P_ < 0.05). 614 **Alt text:** Panel a shows PCK1 immunoblots with different combinations of BIN2 and SPY. 615 Panel b shows kinase-assay blots with weaker phosphorylated bands after SPY 616 coexpression. Panel c shows stronger phosphorylated and weaker lectin-enriched PCK1 617 bands with propiconazole. Panel d shows weaker phosphorylated and stronger lectin 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 618 enriched bands with bikinin in _bin2-1_ . Panel e shows higher relative PCK activity with bikinin 619 in a bar graph. Panel f shows stronger phosphorylated and weaker lectin-enriched bands 620 with the SPY inhibitor SOFTI. Panel g shows lower activity with SOFTI in a bar graph. 621 622 **Figure 3: The** _**spy**_ **mutants show increased PCK1 phosphorylation and reduced PCK** 623 **activity. a,** Relative PCK activity and phosphorylation status of WT and _spy_ mutants grown 624 on medium with or without 1 nM brassinolide (BL) for 5 days. **b-c,** Relative PCK 625 decarboxylation activity and PCK1 phosphorylation status of 3.5-day-old WT and _spy_ 626 etiolated seedlings grown on 0.5x MS sugar-free medium containing 2 μM propiconazole 627 (PPZ), 2 μM PPZ plus 100 nM BL, or 2 μM PPZ plus 30 μM bikinin. PCK1 phosphorylation 628 status was analyzed using anti-PCK1 immunoblotting of SDS-PAGE gel containing Phos629 tag (upper panel) or no Phos-tag (lower panel). Phosphorylated PCK1 is the shifted band 630 marked with *. Values below the blots represent the percentage of phosphorylated PCK1 (% 631 p) relative to total PCK1. Data are mean ± s.d., _n_ = 3 biological replicates ( **a-c** ); Significant 632 differences between means are marked by different letters (one-way ANOVA, Tukey’s test, 633 _P_ < 0.05). 634 **Alt text:** Panel a shows relative PCK activity bars above PCK1 immunoblots for wild type 635 and two _spy_ mutants with or without brassinolide. Untreated mutants have lower activity and 636 stronger phosphorylated bands. Brassinolide raises activity and weakens these bands. 637 Panel b compares wild type and _spy-4_ under propiconazole alone or with brassinolide or 638 bikinin; combined treatments raise activity and weaken phosphorylated bands. Panel c 639 shows activity bars and blots for wild type and _spy-23_ ; combined treatments likewise raise 640 activity and weaken phosphorylated bands. 641 642 **Figure 4:** _**O**_ **-fucosylation promotes seedling growth and is inhibited by BIN2. a,** 643 Photograph of 5-day-old WT, _pck1_, _spy-4_, and _spy-23_ seedlings grown on 0.5x MS medium 644 with or without 1% sucrose. Scale bars, 10 mm. **b,** Hypocotyl length of seedlings in ( **a** ). _n_ = 645 20 seedlings. The central lines within the box plot represent the medians, the box represents 646 the interquartile range (IQR), and the whiskers extend to the minima and maxima. Significant 647 differences between means are marked by different letters (one-way ANOVA, Games– 648 Howell test, _P_ < 0.05). **c,** Detection of _O_ -fucosylated proteins in WT, _bin2-1_, and _spy_ mutants 649 by immunoblotting. Total nuclear proteins were extracted and immunoblotted with a mixture 650 of anti-Serine- _O-_ fucose and anti-Threonine- _O-_ fucose antibodies. CBB: Coomassie Brilliant 651 Blue staining of the gel blot. 652 **Alt text:** Panel a shows photographs of wild-type, _pck1_, _spy-4_ and _spy-23_ seedlings with or 653 without sucrose. Panel b shows box plots and individual data points for hypocotyl length; 654 sucrose-treated _pck1_ seedlings have lengths similar to wild type, while both _spy_ mutants 18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764934; this version posted November 12, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764934) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 655 remain shorter. Panel c shows an immunoblot of _O_ -fucosylated nuclear proteins; several 656 _bin2-1_ bands are fainter than wild type, and _spy_ bands are barely visible. 657 658 **Figure 5: SPY promotes BR response in dark-grown seedlings. a,** Photograph of 3.5659 day-old WT and _spy_ seedlings grown in the dark on 0.5x MS sugar-free medium containing 660 2 μM propiconazole (PPZ), 2 μM PPZ plus 100 nM brassinolide (BL), or 2 μM PPZ plus 30 661 μM bikinin. Scale bar, 10 mm. **b,** Hypocotyl length of seedlings shown in ( **a** ). _n_ = 25 662 seedlings. **c,** Photograph of 5-day-old WT, _spy_ mutants, and _bzr1-1D_ seedlings grown in the 663 dark on 0.5x MS medium with 1% sucrose containing different concentrations of PPZ in the 664 dark. Scale bars, 10 mm. **d,** Hypocotyl length of seedlings in ( **c** ). _n_ ≥ 12 seedlings. The 665 central lines within the box plots ( **b,d** ) represent the medians, the box represents the 666 interquartile range (IQR), and the whiskers extend to the minima and maxima. Percentage 667 values indicate the relative reduction in hypocotyl length at each PPZ concentration 668 compared with the untreated control of the same genotype. Significant differences between 669 means ( **b** ) or between means within each genotype ( **d** ) are marked by different letters (one670 way ANOVA, Tukey’s test, **b** ; Games–Howell test, **d** ; _P_ < 0.05). **e,** Hypocotyl length of 5-day671 old seedlings of WT, _spy_, and _bzr1-1D_ mutants grown on 0.5x MS medium or 0.5x MS 672 medium containing 2 μM PPZ plus various concentrations of BL. Data are mean ± s.d., _n_ = 673 12. 674 **Alt text:** Panel a shows photographs of wild-type and _spy_ seedlings. Panel b shows box 675 plots of hypocotyl length with individual points; adding brassinolide or bikinin to 676 propiconazole increases length. Panel c shows photographs of wild-type, _spy_ and _bzr1-1D_ 677 seedlings across propiconazole doses. Panel d shows corresponding box plots; low doses 678 shorten _spy_ proportionally more than wild type, while _bzr1-1D_ remains longest. Panel e plots 679 hypocotyl length across brassinolide concentrations, including untreated controls. Under 680 propiconazole, increasing brassinolide produces a rise then fall in wild type, a smaller rise 681 in _spy_ and a decline in _bzr1-1D_ ; lengths converge at higher doses. 682 19 bo pep t do [ttps//doog/06898/06](https://doi.org/10.64898/2026.11.06.764934) 06 693 ; tseso posted oe be, 06 ecopygt ode otspe (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) # available under a CC-BY 4.0 International license P . **a** _PCK1-YFP_ **b** **62-** **-84** _PCK1-YFP_ Col-0 _spy-4_ 130 PCK1-YFP AAL-pull down 100 130 PCK1-YFP Input CBB **c** PCK1-YFP - + + PCK1 [AA] -YFP - - - + PCK1-YFP - + + **d** Association Dissociation SPY-RFP RFP-MH 180 130 100 SPY-RFP PCK1-YFP - - + - + + _K_ d = 0.23 nM Bait: 3TPR-SPY biotin Analyte: PCK1 (µM) Bait: biotin Analyte: PCK1 (µM) 1.6 1.2 0.8 0.4 0 **f** 1 0.5 0.25 0.125 1 360 720 Time (s) PCK1 Anti-mCherry Anti-GFP Anti-mCherry Anti-GFP **e** 70 55 40 130 100 180 130 100 70 55 40 130 100 CBB Incubation PCK1 with SPY IP **-** **-** **+** **++** **-** **+** **+** **+** SOFTI GDP-fucose PCK1 3TPR-SPY PCK1 3TPR-SPY **-** **+** SPY-RFP RFP-MH PCK1-YFP 130 PCK1-YFP AAL-pull down anti-GFP 1.00 2.72 1.48 anti-His AAL-biotin 100 70 100 70 0.16 1.00 0.72 0.30 Input **g** PCK1-YFP PCK1 [AA] -YFP PCK1-YFP - + + [AA] -YFP - - - + SPY-RFP - - + + RFP-MH - - + + - + - 130 PCK1-YFP 100 180 130 100 70 55 40 SPY-RFP RFP-MH Input 0 2 4 8 24 (h) 100 AAL-biotin 70 anti-GFP anti-RFP Ponceau **Figure 1: SPY associates with and** _**O**_ **-fucosylates PCK1.** **a,** Detection of _O_ -fucosylated PCK1-YFP in PCK1-YFP/Col-0 and PCK1-YFP/ _spy-4_ seedlings. _O_ -fucosylated proteins were pulled down by AAL-agarose. Immunoblotting of input (total protein extracts) or AAL-agarose-enriched samples was probed with an anti-GFP antibody. **b,** MS/MS spectra of fucosylated peptide 62-SAPTTPINQNAAAAFAAVSEEER-84 of PCK1-YFP protein immunoprecipitated from _N. benthamiana_ leaves co-expressing PCK1-YFP with SPY-RFP, containing Ser-62, Thr-65, and Thr-66 residues. **c,** PCK1-YFP was co-immunoprecipitated with SPY-RFP in _N. benthamiana_ leaves. PCK1-YFP or PCK1 [AA] -YFP was transiently expressed with SPY-RFP or RFP-MH in _N. benthamiana_ leaf. Immunoprecipitation was performed with total protein extracts (input) using anti-GFP beads. Input and immunoprecipitates (IPs) were analyzed by anti-mCherry and anti-GFP antibodies. **d,** Biolayer interferometry (BLI) analysis of the interaction between 3TPR-SPY and PCK1. Biotinylated 3TPR-SPY was immobilized on streptavidin biosensors and exposed to the indicated concentrations of PCK1 for 360 s, followed by dissociation in PBS for 360 s. Biosensors loaded with free biotin and exposed to 1 μM PCK1 served as a negative control. The ( _K_ d ) was 0.23 nM. **e,** Time-course analysis of SPY-mediated _O_ -fucosylation of recombinant PCK1. **f,** In vitro _O_ -fucosylation of PCK1 by the SPY catalytic region (3TPR–SPY) by using 1 mM GDP-fucose with 10 μM (+) or 30 μM (++) of SOFTI. Fucosylation level of PCK1 and 3TPR-SPY was detected by immunoblotting with a biotinylated AAL. PCK1 and 3TPR-SPY were detected by immunoblotting with an anti-His antibody. **g,** PCK1-YFP was _O_ -fucosylated by SPY in _N. benthamiana_ leaves. PCK1-YFP or PCK1 [AA] -YFP was transiently co-expressed with SPYRFP or RFP-MH in _N. benthamiana_ leaves. _O_ -fucosylated proteins were enriched by AAL-agarose. Immunoblot containing input or AAL-agaroseenriched samples was probed with an anti-GFP or anti-RFP antibody. Values below the blots represent relative band intensities. Coomassie Brilliant Blue (CBB) or Ponceau S staining of the gel blot was used as a loading control. bo pep t do [ttps//doog/06898/06](https://doi.org/10.64898/2026.11.06.764934) 06 693 ; tseso posted oe be, 06 ecopygt ode otspe (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) **a** **b** **c** AAL-pull down _35S:PCK1-YFP_ + _35S:BIN2-YFP_ + + + PCK1-YFP/Col-0 _35S:SPY-YFP_ _35S:YFP_ 130 Phos-tag 100 180 130 100 PCK1-YFP SPY-YFP PCK1-YFP BIN2-YFP YFP CBB p-PCK1 PCK1 + + + p-PCK1-YFP ATP **+** **-** _PCK1-YFP_ + + + + + + _RFP_ + + - - + _SPY-RFP_ - - + + - + GST-BIN2 - + - + - 130 Phos-tag 130 SDS-PAGE 70 55 35 55 ***** 100 130 100 100 130 100 1.5 1.0 0.5 0.0 phostag phostag Input SDS-PAGE PCK1 **e** **f** **d** **e** **f** **g** PCK1-YFP/Col-0 1.5 130 100 130 100 130 100 AAL-pull down ***** CBB 130 100 130 100 130 100 PCK1-YFP/ _bin2-1_ AAL-pull down ***** Phos-tag CBB Input Input 1.0 0.5 0.0 PCK1-YFP/Col-0 PCK1-YFP/ _bin2-1_ **Figure 2: Phosphorylation and** _**O**_ **-fucosylation of PCK1 antagonize each other. a,** Co-expression of SPY with BIN2 inhibits BIN2-mediated PCK1 phosphorylation _in vivo_ . The proteins were transiently co-expressed in _N. benthamiana_ leaves and analyzed by immunoblotting with an anti-GFP antibody to detect protein expression by Phos-tag gel (upper blot) and standard SDS-PAGE (lower blot). **b,** PCK1 immunoprecipitated from _N. benthamiana_ leaves co-expressing SPY shows reduced subsequent phosphorylation by recombinant BIN2. PCK1 was immunoprecipitated from leaves co-expressing PCK1 with SPY-RFP or RFP. We incubated the immunoprecipitated PCK1 on beads with GSTBIN2 in kinase buffer, then immunoblotted with an anti-PCK1 antibody. **c,** Immunoblotting detection of total, phosphorylated, and _O_ -fucosylated PCK1 in the 7-day-old _PCK1-YFP/Col-0_ transgenic seedlings grown on 0.5x MS sugar-free medium with or without 2 μM PPZ. Anti-GFP immunoblotting with input (total protein extract) or AAL-agarose pulled down samples was performed to analyze PCK1 phosphorylation status using SDS-PAGE gel containing Phos-tag (upper panel) or no Phos-tag (lower). **d,** Immunoblotting detection of total, phosphorylated, and _O_ fucosylated PCK1 in the 16-day-old seedlings of _PCK1-YFP/Col-0_ and _PCK1-YFP/bin2-1_ grown on 0.5x MS with 1% sucrose with or without 30 μM bikinin. **e,** Relative PCK activity of seedlings in ( **d** ). **f,** Immunoblotting detection of total, phosphorylated, and _O_ -fucosylated PCK1 in the 16day-old seedlings of _PCK1-YFP/Col-0_ on 0.5x MS with 1% sucrose with or without 30 μM SOFTI. Anti-GFP immunoblotting with AAL-agaroseenriched samples to analyze PCK1 fucosylation or input (total protein extract) was performed to analyze PCK1 phosphorylation status using SDSPAGE gel containing Phos-tag (lower blot) or no Phos-tag (upper blot). Phosphorylated PCK1 is the shifted band marked with *. CBB: Coomassie Brilliant Blue staining of the gel blot _._ **g,** Relative PCK activity of seedlings in ( **f** ). Data are mean ± s.d., _n_ = 3 ( **e,g** ); ***, _P_ < 0.001, according to two-tailed Student’s _t_ -test ( **g** ), ( **e** ) Significant differences between means are marked by different letters (one-way ANOVA, Tukey’s test, _P_ < 0.05). bo pep t do [ttps//doog/06898/06](https://doi.org/10.64898/2026.11.06.764934) 06 693 ; tseso posted oe be, 06 ecopygt ode otspe (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 1.5 1.0 0.5 0.0 100 **a** **b** 1.5 1.0 0.5 0.0 100 ***** **c** 1. ~~5~~ 1. ~~0~~ 0. ~~5~~ 0. ~~0~~ 100 ***** 70 70 70 70 22 14 57 57 18 17 100 70 BL - + - - + + WT % p 26 51 27 26 47 67 29 30 % p 24 54 27 24 44 69 27 27 % p PPZ - + + + - + + + BL - - + - - - + Bikinin - - - + - - - + WT _spy-4_ BL [-] - + - - - + - - - + - - - + 100 70 100 70 PPZ - + + + - + + + Bikinin WT _spy-23_ **Figure 3: The** _**spy**_ **mutants show increased PCK1 phosphorylation and reduced PCK activity. a,** Relative PCK activity and phosphorylation status of WT and _spy_ mutants grown on medium with or without 1 nM brassinolide (BL) for 5 days. **b-c,** Relative PCK decarboxylation activity and PCK1 phosphorylation status of 3.5-day-old WT and _spy_ etiolated seedlings grown on 0.5x MS sugar-free medium containing 2 μM propiconazole (PPZ), 2 μM PPZ plus 100 nM BL, or 2 μM PPZ plus 30 μM bikinin. PCK1 phosphorylation status was analyzed using anti-PCK1 immunoblotting of SDS-PAGE gel containing Phos-tag (upper panel) or no Phos-tag (lower panel). Phosphorylated PCK1 is the shifted band marked with *. Values below the blots represent the percentage of phosphorylated PCK1 (% p) relative to total PCK1. Data are mean ± s.d., _n_ = 3 biological replicates ( **a-c** ); Significant differences between means are marked by different letters (one-way ANOVA, Tukey’s test, _P_ < 0.05). bo pep t do [ttps//doog/06898/06](https://doi.org/10.64898/2026.11.06.764934) 06 693 ; tseso posted oe be, 06 ecopygt ode otspe (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) **a** **b** - S + S 2 ~~5~~ **c** 180 130 100 70 55 40 2 ~~0~~ anti- _O-_ fucose 1 ~~5~~ 1 ~~0~~ ~~5~~ ~~0~~ CBB **Figure 4:** _**O**_ **-fucosylation promotes seedling growth and is inhibited by BIN2. a,** Photograph of 5-day-old WT, _pck1_, _spy-4_, and _spy-23_ seedlings grown on 0.5x MS medium with or without 1% sucrose. Scale bars, 10 mm. **b,** Hypocotyl length of seedlings in ( **a** ). _n_ = 20 seedlings. The central lines within the box plot represent the medians, the box represents the interquartile range (IQR), and the whiskers extend to the minima and maxima. Significant differences between means are marked by different letters (one-way ANOVA, Games–Howell test, _P_ < 0.05). **c,** Detection of _O_ fucosylated proteins in WT, _bin2-1_, and _spy_ mutants by immunoblotting. Total nuclear proteins were extracted and immunoblotted with a mixture of anti-Serine- _O-_ fucose and anti-Threonine- _O-_ fucose antibodies. CBB: Coomassie Brilliant Blue staining of the gel blot. bo pep t do [ttps//doog/06898/06](https://doi.org/10.64898/2026.11.06.764934) 06 693 ; tseso posted oe be, 06 ecopygt ode otspe (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) **a** **b** WT _spy-4_ _spy-23_ PPZ - + + + - + + + - + + + _spy-23_ - + + + - - + - + + + - - + BL Bikinin **c** - + + + - - + - - - + - - - + - - - + 10 5 0 PPZ BL Bikinin - + + + - - + - - - + - + + + - - + - - - + - + + + - - + - - - + PPZ (μM) 0 0.1 0.25 0.5 1 2 **d** 25 **e** 25 15 10 5 0 BL (nM) 0 0 10 50 100 200 20 20 15 10 5 1 PPZ (μM) PPZ (μM) 0 2 2 2 2 2 Inhibition **-** 23 55 70 82 87 **-** 41 70 77 81 81 **-** 48 70 80 82 82 **-** 8 17 20 18 21 23 55 70 82 87 (%) **Figure 5: SPY promotes BR response in dark-grown seedlings. a,** Photograph of 3.5-day-old WT and _spy_ seedlings grown in the dark on 0.5x MS sugar-free medium containing 2 μM propiconazole (PPZ), 2 μM PPZ plus 100 nM brassinolide (BL), or 2 μM PPZ plus 30 μM bikinin. Scale bar, 10 mm. **b,** Hypocotyl length of seedlings shown in ( **a** ). _n_ = 25 seedlings. **c,** Photograph of 5-day-old WT, _spy_ mutants, and _bzr1-1D_ seedlings grown in the dark on 0.5x MS medium with 1% sucrose containing different concentrations of PPZ in the dark. Scale bars, 10 mm. **d,** Hypocotyl length of seedlings in ( **c** ). _n_ ≥ 12 seedlings. The central lines within the box plots ( **b,d** ) represent the medians, the box represents the interquartile range (IQR), and the whiskers extend to the minima and maxima. Percentage values indicate the relative reduction in hypocotyl length at each PPZ concentration compared with the untreated control of the same genotype. Significant differences between means ( **b** ) or between means within each genotype ( **d** ) are marked by different letters (one-way ANOVA, Tukey’s test, **b** ; Games–Howell test, **d** ; _P_ < 0.05). **e,** Hypocotyl length of 5-dayold seedlings of WT, _spy_, and _bzr1-1D_ mutants grown on 0.5x MS medium or 0.5x MS medium containing 2 μM PPZ plus various concentrations of BL. Data are mean ± s.d., _n_ = 12.