# Source paper archive - Source title: SEC16A/MAG5-mediated ER export shapes ABA receptor homeostasis - Author(s): Jose Antonio Berlanga-Torres, Adela Villasante-Fernandez, Cecilia Costigliolo-Rojas, Jeanette Moulinier-Anzola, Mar Bono, Pedro Luis Rodriguez Egea, Barbara Korbei, Maria Jesus Marcote, Fernando Aniento, Alejandro Ferrando, Borja Belda Palazon - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.06.761007v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.06.761007v1.full.pdf - PDF SHA-256: c06a7e3bc9caab2694b0864374c0c74fbae1b8a1881a03be4c5f2d13214d2aa0 - 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/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. # 1 Title 2 SEC16A/MAG5-mediated ER export shapes ABA receptor homeostasis 3 Authors 4 José Antonio Berlanga-Torres [1], Adela Villasante-Fernández [1], Cecilia Costigliolo-Rojas [1], 5 Jeanette Moulinier-Anzola [2], Mar Bono [1], Pedro L. Rodriguez [1], Barbara Korbei [2], María 6 Jesús Marcote [3], Fernando Aniento [3], Alejandro Ferrando [1], Borja Belda-Palazón [1*] # 7 Author affiliation 8 1 Instituto de Biología Molecular y Celular de Plantas (IBMCP), CSIC, Universitat 9 Politècnica de València, 46022 Valencia, Spain; [2] Department of Biotechnology and Food 10 Sciences, Institute of Molecular Plant Biology, BOKU University, Muthgasse 18, 1190, 11 Vienna, Austria; [3] Departamento de Bioquímica y Biología Molecular, Facultad de 12 Farmacia, Instituto Universitario de Biotecnología y Biomedicina (BIOTECMED), 13 Universitat de València, 46100, Burjassot, Spain. 14 *** Author for correspondence:** Borja Belda-Palazón, bbelda@ibmcp.upv.es # 15 ORCIDs 16 José Antonio Berlanga-Torres, 0009-0002-2295-8087. 17 Adela Villasante-Fernández, 0009-0002-2697-3890. 18 Cecilia Costigliolo-Rojas, 0000-0003-3075-8464. 19 Jeanette Moulinier-Anzola, 0000-0001-8635-7227. 20 Mar Bono, 0000-0002-6495-7069. 21 Pedro L. Rodriguez, 0000-0002-5886-9425. 22 Barbara Korbei, 0000-0002-0439-0435. 23 María Jesús Marcote, 0000-0002-0270-7507. 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Fernando Aniento, 0000-0003-3553-585X. 25 Alejandro Ferrando, 0000-0002-1903-9111. 26 Borja Belda-Palazón, 0000-0003-4926-1917. # 27 Summary 28 - The phytohormone abscisic acid (ABA) controls plant adaptation to environmental 29 stress through PYR/PYL/RCAR receptors. Regulated receptor turnover through 30 ubiquitination and endosomal trafficking, including ESCRT-dependent vacuolar 31 degradation, is a major mechanism controlling ABA signalling. However, whether 32 ABA receptor homeostasis is established during the early secretory pathway remains 33 unknown. 34 - Using complementary cell biological and biochemical approaches, we define a 35 constitutive early secretory pool of the ABA receptors PYR1 and PYL4 associated 36 with the endoplasmic reticulum (ER) and the Golgi apparatus (GA). 37 - Genetic, cell biological and pharmacological analyses reveal that the COPII scaffold 38 protein SEC16A (also known as MAG5) is required for the proper trafficking and 39 homeostasis of this receptor pool. Loss of MAG5 causes intracellular receptor 40 accumulation, impaired trafficking towards late endosomal compartments and 41 enhanced ABA signalling without affecting endogenous ABA accumulation. 42 - Together, our findings establish the early secretory pathway as an integral component 43 of the ABA signalling network and demonstrate that MAG5-mediated ER export 44 shapes ABA receptor homeostasis by coordinating receptor trafficking throughout the 45 endomembrane system, revealing the ER as a key regulatory compartment for ABA 46 receptor homeostasis. # 47 Keywords 48 ABA receptors, ABA signaling pathway, ER, Golgi, COPII, SEC16A/MAG5, secretory 49 pathway. 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. # 50 Introduction 51 The endomembrane system is a highly dynamic network that coordinates intracellular 52 protein trafficking, membrane identity and cellular signalling, thereby enabling plants to 53 continuously adapt their growth and development to changing environmental conditions. 54 Rather than functioning as a constitutive transport route, the secretory pathway is 55 increasingly recognized as a regulatory platform whose organization is remodelled 56 according to developmental stage, secretory demand and environmental stress. Such 57 plasticity not only maintains membrane homeostasis but also determines the abundance, 58 localization and activity of signalling proteins, placing protein trafficking alongside 59 transcriptional and translational regulation as a fundamental mechanism controlling 60 cellular responses (Paez Valencia _et al._, 2016; Wang _et al._, 2020; Aniento _et al._, 2022). 61 Among the signalling pathways that require particularly tight spatial regulation, abscisic 62 acid (ABA) signalling plays a central role in orchestrating plant adaptation to drought, 63 salinity, osmotic stress and other adverse environmental conditions. Perception of ABA 64 by members of the PYRABACTIN RESISTANCE/PYR1-LIKE/REGULATORY 65 COMPONENT OF ABA RECEPTOR (PYR/PYL/RCAR) family initiates a signalling 66 cascade in which clade A PROTEIN PHOSPHATASE 2Cs (PP2Cs) are inhibited, 67 allowing activation of SNF1-RELATED PROTEIN KINASE 2 (SnRK2) kinases and 68 downstream transcriptional and physiological responses (Cutler _et al._, 2010; Yoshida _et_ 69 _al._, 2019). Because ABA signalling controls essential processes including stomatal 70 closure, seed dormancy, germination and stress acclimation, the abundance, subcellular 71 localization and activity of ABA receptors must be tightly coordinated in space and time 72 to ensure signalling outputs that are both robust and rapidly reversible. Consequently, the 73 compartmentalization of ABA receptors has emerged as a central determinant of 74 signalling intensity, duration and sensitivity (Rodriguez _et al._, 2019; Coego _et al._, 2021; 75 Qin _et al._, 2026). 76 Consistent with this concept, ABA receptor homeostasis is regulated through multiple 77 sequential layers acting across the endomembrane system. In addition to transcriptional 78 regulation, receptor abundance is extensively controlled by ubiquitination and selective 79 protein degradation. Several E3 ubiquitin ligases operate at distinct cellular 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 80 compartments, including CRL4 complexes, RBR-type RFA ligases and the plasma 81 membrane-associated RING E3 ligase RSL1, promoting proteasomal or vacuolar 82 degradation depending on receptor localization (Irigoyen _et al._, 2014; Bueso _et al._, 2014; 83 Fernandez _et al._, 2020; Coego _et al._, 2021). However, receptor proteostasis is not solely 84 determined by protein turnover. Members of the C2-domain ABA-related (CAR) protein 85 family recruit specific PYR/PYL receptors to the plasma membrane in a calcium 86 dependent manner, thereby facilitating efficient ABA perception by controlling receptor 87 positioning (Rodriguez _et al._, 2015; Diaz _et al._, 2016). Importantly, ABA signalling also 88 regulates membrane-delimited processes, including the activity of plasma membrane ion 89 channels that control ion and water fluxes during stomatal closure. The spatial 90 organization of ABA signalling components at or near the plasma membrane may 91 therefore directly influence the efficiency and specificity of physiological ABA responses 92 (Geiger _et al._, 2009; Imes _et al._, 2013; Kollist _et al._, 2014). More recently, the 93 endoplasmic reticulum (ER)-associated E3 ubiquitin ligase DOA10A was shown to 94 mono-ubiquitinate ABA receptors, promoting their trafficking through the 95 endomembrane system towards the plasma membrane without affecting receptor stability 96 (Liu _et al._, 2025). This finding fundamentally expanded the role of ubiquitination in ABA 97 signalling, demonstrating that ubiquitin modifications regulate receptor localization 98 independently of protein degradation. Following receptor activation and internalization, 99 components of the ESCRT machinery, including FYVE1/FREE1, FYVE4, VPS23A, 100 ALIX and TOM1-LIKE (TOL) proteins, mediate endosomal sorting and vacuolar 101 degradation of ABA receptors (Belda-Palazon _et al._, 2016; Yu _et al._, 2016; García-León 102 _et al._, 2019; Moulinier-Anzola _et al._, 2024; Chen _et al._, 2025). Together, these studies 103 reveal that receptor abundance and subcellular distribution depend on the coordinated 104 regulation of intracellular trafficking across the endomembrane system. 105 Despite these advances, the spatial distribution of ABA receptors within the early 106 secretory pathway remains poorly understood. Export of newly synthesized membrane 107 and secretory proteins from the ER occurs at specialized ER exit sites (ERES) through 108 the assembly of COPII-coated carriers (Brandizzi & Barlowe, 2013). This process is 109 initiated by activation of the small GTPase SAR1 by the ER-localized guanine nucleotide 110 exchange factor SEC12, promoting SAR1 membrane insertion and recruitment of the 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 111 inner coat complex SEC23/SEC24, which mediates cargo selection. Assembly of the 112 outer coat complex SEC13/SEC31 subsequently drives COPII carrier formation and ER 113 to-Golgi transport, whereas cycles of SAR1 GTP binding and hydrolysis coordinate coat 114 assembly, carrier scission and coat disassembly. Consequently, modulation of COPII 115 dynamics enables cells to rapidly adjust secretory flux and protein distribution in 116 response to developmental and environmental cues (Brandizzi, 2018; Aniento _et al._, 117 2022) 118 Supporting this view, increasing evidence indicates that the architecture and activity of 119 the early secretory pathway is actively remodelled during environmental adaptation 120 (Brandizzi, 2018; Aniento _et al._, 2022). A recent study demonstrated that ABA remodels 121 COPII dynamics and early secretory trafficking, revealing that hormone signalling 122 directly targets the machinery responsible for ER export (Li _et al._, 2021). These 123 observations suggest that modulation of ER export constitutes an additional regulatory 124 layer controlling signalling specificity by regulating the availability of selected signalling 125 proteins before they enter downstream trafficking pathways. 126 A central organizer of ER export is SEC16, a large peripheral membrane protein that 127 plays a unique role in COPII assembly because it is not a structural component of the coat 128 itself but instead acts as a master scaffold that spatially organizes ER exit sites. SEC16 129 orchestrates the spatial organization and stability of ER exit sites by coordinating COPII 130 assembly and turnover, and this role is broadly conserved across eukaryotes (Connerly _et_ 131 _al._, 2005; Watson _et al._, 2006; Brandizzi & Barlowe, 2013). In _Arabidopsis thaliana_, two 132 closely related SEC16 homologues, SEC16A (also known as MAG5) and SEC16B, have 133 been identified. Although both proteins share high sequence similarity, MAG5 is the 134 predominant isoform required for ER exit site organization and efficient ER-to-Golgi 135 trafficking, whereas SEC16B appears to play a more limited or partially redundant role in 136 early secretory trafficking (Takagi _et al._, 2013). Given that ABA signalling remodels the 137 early secretory pathway through the regulation of COPII-dependent trafficking (Li _et al._, 138 2021), MAG5 represents an attractive candidate for coupling hormone signalling with 139 early secretory trafficking. 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 140 Here, we demonstrate that the early secretory pathway constitutes a previously 141 unrecognized regulatory layer controlling ABA receptor homeostasis. By combining cell 142 biology, genetic and biochemical approaches, we show that MAG5 regulates the 143 homeostasis of a distinct ER-Golgi-associated receptor pool, whose abundance and 144 subcellular organization depend on functional ER export. Disruption of MAG5 alters 145 receptor distribution and abundance and enhances ABA sensitivity, revealing that early 146 secretory trafficking actively shapes hormone perception rather than merely supporting 147 constitutive protein secretion. Together, our findings establish the ER–Golgi interface as 148 an active signalling hub that integrates membrane trafficking with hormone perception, 149 expanding current models of ABA signalling during plant stress adaptation. # 150 Materials and Methods 151 Plant materials and growth conditions 152 _Arabidopsis thaliana_ ecotype Columbia-0 (Col-0) was used as the wild-type control. All 153 mutant and transgenic lines used in this study are listed in Table **S1** . Unless otherwise 154 indicated, plants were grown under long-day conditions (16 h light/8 h dark; 22°C/18°C) 155 with a light intensity of 100-120 μmol m⁻² s⁻¹. Surface-sterilized seeds were sown on 156 half-strength Murashige and Skoog (½ MS) medium with vitamins and MES (Duchefa 157 M0255) supplemented with 1.03 g/L of MES and 0.8% (w/v) phytoagar (pH 5.7). Plates 158 were sealed with Micropore™ tape, stratified in darkness at 4°C for 2 d, and then 159 transferred to the growth chamber. # 160 Molecular cloning and plasmid construction 161 Gateway® cloning technology (Invitrogen) was used for the generation of all constructs 162 unless otherwise indicated. PCR-amplified coding sequences were first cloned into the 163 Gateway entry vector pDONR™/Zeo by BP recombination and subsequently transferred 164 into the appropriate Gateway destination vectors by LR recombination according to the 165 manufacturer's instructions. All constructs were verified by sequencing. A complete list 166 of primers, plasmids and constructs used in this study is provided in Tables **S2** and **S3** . # 167 Arabidopsis transformation 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 168 Stable transgenic _A. thaliana_ lines were generated by _Agrobacterium tumefaciens_ 169 mediated transformation of flowering Col-0 plants using the floral dip method (Clough & 170 Bent, 1998). T2 progeny were screened to identify lines carrying a single transgene 171 insertion, and homozygous T3 lines were used for subsequent analyses. All transgenic 172 lines used in this study are listed in Table **S1** . # 173 ABA and salt sensitivity assays during germination and early seedling 174 development 175 Surface-sterilized seeds were sown on ½ MS medium supplemented with 0.3 or 0.5 µM 176 ABA, and 100 or 150 mM NaCl. Following stratification, plates were transferred to long 177 day growth conditions and seed germination, and early seedling development were 178 monitored over time. Germination was scored as radicle emergence 48-72 h after transfer 179 to the growth chamber, whereas early seedling establishment was quantified 72-96 h after 180 transfer as the percentage of seedlings developing fully expanded green cotyledons. For 181 each genotype and treatment, at least 50 seeds were analysed per biological replicate. # 182 Primary root growth assays 183 For primary root growth assays, surface-sterilized seeds were germinated on ½ MS 184 medium and grown for 5 d under long-day conditions. Seedlings were then transferred to 185 fresh ½ MS plates supplemented with 5 µM of ABA, and allowed to grow for an 186 additional 8 d. At the end of the treatment, seedlings were subsequently transferred to 187 fresh agar plates, carefully straightened and photographed. Primary root growth was 188 quantified from the scanned images using Fiji/ImageJ (Schindelin _et al._, 2012). # 189 ABA quantification 190 Endogenous ABA was quantified in 6-day-old seedlings. Approximately 100 mg of fresh 191 tissue was extracted with methanol/water/acetic acid (80:19:1, v/v/v) containing 192 deuterium-labelled ABA as an internal standard. Following centrifugation, the 193 supernatant was dried under vacuum, resuspended in 1% (v/v) acetic acid and purified by 194 reverse-phase solid-phase extraction. The eluate was dried, resuspended in 5% (v/v) 195 acetonitrile containing 1% (v/v) acetic acid, and analysed by ultra-high-performance 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 196 liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). 197 ABA was quantified using a Q Exactive Orbitrap mass spectrometer (Thermo Fisher 198 Scientific, Waltham, MA, USA) operated in selected ion monitoring (SIM) mode at the 199 Hormone Quantification Service of the IBMCP (Valencia, Spain). # 200 Infrared thermography and stomatal aperture measurements 201 Infrared thermography was performed on 6-week-old _A. thaliana_ plants grown under 202 short-day conditions (8 h light/16 h dark, 21°C/19°C, 100 μmol m⁻² s⁻¹). Plants were 203 sprayed with 20 mM MES (pH 5.7) containing 0.02% (v/v) Silwet L-77 supplemented 204 with the indicated concentration of ABA or an equivalent volume of DMSO as a mock 205 control. Thermal images were acquired 3 h after treatment using an FLIR E95 infrared 206 camera (FLIR Systems) equipped with a 42° lens. Images were analysed using FLIR 207 Tools software. Mean leaf temperature was determined from 15 independent regions of 208 interest distributed across 4-6 rosette leaves per plant. At least six plants were analysed 209 per genotype in each experiment, and experiments were performed independently at least 210 twice. 211 Stomatal aperture was measured in cotyledons of 8-day-old seedlings. Seedlings were 212 harvested directly into 95% ethanol after 2–3 h of light exposure and fixed for 3–4 h. 213 Samples were subsequently incubated for 10 min each in 70%, 50% and 20% ethanol and 214 cleared overnight in a 1:3:8 glycerol:water:chloral hydrate solution. Cotyledons were 215 mounted with the abaxial surface facing upwards and imaged using a 20× objective. At 216 least one image from a comparable region of the cotyledon was acquired for each of 9 217 individual seedlings, with at least 20 stomata quantified per seedling. Stomatal 218 parameters were quantified using ImageJ. Stomatal aperture was expressed as the ratio of 219 stomatal pore width to length. # 220 Transient expression in Nicotiana benthamiana 221 Transient expression assays were performed in fully expanded leaves of 4-5-week-old 222 _Nicotiana benthamiana_ plants by _A. tumefaciens_ -mediated infiltration as described 223 previously (Belda-Palazón & Rodriguez, 2020, 2022). Briefly, _A. tumefaciens_ strains 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 224 carrying the indicated binary constructs were grown overnight, collected by 225 centrifugation and resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES, pH 226 5.6, and 150 μM acetosyringone) to an OD 600 of 2.0. Bacterial suspensions were 227 incubated at room temperature for 3-5 h before infiltration. Immediately before 228 infiltration, cultures carrying the desired constructs were mixed and diluted in infiltration 229 buffer to the final OD 600 required for each experiment, resulting in a final OD 600 of 0.25 230 0.5 for each individual construct. 231 For co-expression and colocalization experiments, the indicated fluorescent protein 232 fusions were co-expressed with the corresponding organelle markers. For bimolecular 233 fluorescence complementation (BiFC) assays, _A. tumefaciens_ cultures carrying the 234 indicated YFP [N] and YFP [C] fusion constructs were co-infiltrated together with fluorescent 235 subcellular markers (Belda-Palazón _et al._, 2012). Leaves were analysed by confocal laser 236 scanning microscopy 72-96 h after infiltration. A complete list of constructs used is 237 provided in Table **S2** . # 238 Pharmacological treatments and fluorescent probes 239 For pharmacological treatments, 5-day-old _A. thaliana_ seedlings expressing the indicated 240 fluorescent reporters in the corresponding genetic backgrounds were incubated in liquid 241 ½ MS medium supplemented with the indicated compounds or an equivalent volume of 242 DMSO (mock) for 1 h. Brefeldin A (BFA; TargetMol) was used at 50 μM and 243 wortmannin (WM; TargetMol) at 33 μM. 244 For endocytic labelling, seedlings were incubated with FM4-64 (2 μM; Sigma-Aldrich) 245 for 10 min following BFA treatment. To visualize late acidic endosomal/vacuolar 246 compartments, following WM treatment seedlings were incubated with LysoTracker™ 247 Deep Red (1 μM; Thermo Fisher Scientific) for an additional 10 min before confocal 248 imaging. All treatments were performed immediately prior to confocal microscopy. # 249 Confocal laser scanning microscopy 250 Confocal imaging was performed using a Stellaris 8 laser scanning confocal microscope 251 (Leica Microsystems) equipped with a HC PL APO 40×/1.30 OIL objective and HyD 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 252 detectors. GFP/YFP fluorescence was excited at 488 nm and detected between 500 and 253 530 nm, whereas RFP/mCherry fluorescence was excited at 561 nm and detected 254 between 600 and 630 nm. FM4-64 was excited at 488 nm and detected between 610 and 255 630 nm, whereas LysoTracker™ Deep Red was excited at 647 nm and detected between 256 660 and 680 nm. For multicolour imaging, fluorophores were acquired sequentially to 257 avoid spectral overlap. Pinhole size was adjusted to 1 Airy unit for each channel. Images 258 were acquired using Leica LAS X software and processed using Fiji/ImageJ. # 259 Subcellular fractionation and sucrose density gradient centrifugation 260 Microsomal membrane fractions were prepared from two-week-old wild-type (Col-0) _A._ 261 _thaliana_ seedlings. Approximately 1 g of frozen seedling tissue was ground to a fine 262 powder in liquid nitrogen and homogenized in three volumes of ice-cold homogenization 263 buffer (20 mM Tris-HCl, pH 8.0, 250 mM sucrose, 5% glycerol, 5 mM MgCl₂, 5 mM 264 KCl, 5 mM EDTA and 14 mM β-mercaptoethanol) supplemented with cOmplete™, 265 Mini, EDTA-free Protease Inhibitor Cocktail (Roche). Homogenates were incubated on 266 ice for 30 min with gentle inversion every 5 min, filtered through one layer of Miracloth 267 and centrifuged at 8,000 × g for 10 min at 4°C to remove cell debris and large organelles. 268 The resulting supernatant (S8) was ultracentrifuged at 100,000 × g for 30 min at 4°C to 269 obtain the soluble (S100) and microsomal membrane (P100) fractions. The P8 and P100 270 pellets were resuspended in homogenization buffer. An aliquot of each fraction was 271 subsequently mixed with 4× Laemmli sample buffer, heated at 70°C for 10 min, cooled 272 on ice, and analysed by SDS–PAGE and immunoblotting. 273 For density gradient fractionation, the P100 microsomal fraction was layered onto 274 continuous 10-50% (w/v) sucrose gradients prepared in gradient buffer containing 40 275 mM Tris-HCl (pH 8.5), 20 mM KCl and 10 mM MgCl₂. Gradients were centrifuged 276 overnight at 35,000 rpm in a SW40 Ti rotor (Beckman Coulter) at 4°C without brake. 277 Following centrifugation, gradients were collected manually from the top into sixteen 278 fractions of equal volume (F1-F16). Proteins were precipitated overnight at -20°C by 279 addition of one volume of 20% trichloroacetic acid (TCA), centrifuged at 12,000 × g for 280 15 min at 4°C, washed twice with cold acetone, air-dried, and resuspended in 2× 281 Laemmli sample buffer. Samples were subsequently heated at 70°C for 10 min, cooled on 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 282 ice and equal volumes from each fraction were analysed by SDS-PAGE and 283 immunoblotting using antibodies against PYR1 and organelle-specific marker proteins 284 (Table **S4** ). # 285 Protein extraction and immunoblot analysis 286 For immunoblot analyses, approximately 100 mg of frozen _A. thaliana_ tissue was ground 287 to a fine powder in liquid nitrogen and homogenized in two volumes of 2× Laemmli 288 extraction buffer (126 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.05% 289 bromophenol blue and 4% β-mercaptoethanol). Homogenates were incubated on ice for 290 20-30 min with intermittent vortexing, boiled at 95°C for 10-15 min, cooled on ice and 291 centrifuged at 12,000 × g for 15 min. The resulting supernatants were used as total 292 protein extracts. Equal volumes of protein extracts were separated by SDS-PAGE and 293 transferred onto PVDF membranes. 294 Membranes were blocked in 5% (w/v) non-fat dry milk in TBS-T and incubated 295 overnight at 4°C with the indicated primary antibodies. Following incubation with 296 horseradish peroxidase-conjugated secondary antibodies, immunoreactive bands were 297 detected using enhanced chemiluminescence (ECL). Band intensities were quantified 298 using Fiji/ImageJ and normalized to the corresponding loading controls. Antibodies used 299 in this study are listed in Table **S4** . # 300 RNA extraction and RT-qPCR analysis 301 For gene expression analyses, total RNA was extracted from whole _A. thaliana_ seedlings 302 using NZY Total RNA Isolation kit (NZYtech) according to the manufacturer's 303 instructions. RNA samples were treated with the TURBO DNA-free™ Kit (Invitrogen, 304 Thermo Fisher Scientific). RNA concentration and purity were determined using a 305 NanoDrop™ One spectrophotometer (Thermo Fisher Scientific). First-strand cDNA was 306 synthesized from 1 μg of total RNA using the PrimeScript™ RT reagent Kit (Takara Bio 307 Inc.) following the manufacturer's instructions. 308 Quantitative real-time PCR (RT-qPCR) was performed using HOT FIREPol® 309 EvaGreen® qPCR Mix Plus (Solis Biodyne) in a final reaction volume of 10 μL 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 310 containing 0.4 μL of cDNA and 500 nM of each primer. Reactions were run on a 311 QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). Relative transcript 312 abundance was calculated using the 2 [−ΔCt], with _PDF2_ used as the internal reference gene. 313 Each biological replicate was analysed in three technical replicates. Primer sequences are 314 listed in Table **S3** . # 315 Statistical analyses 316 Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software). Data 317 are presented as mean ± SEM. Statistical significance was assessed by one-way or two 318 way ANOVA followed by Tukey's or Dunnett's test multiple comparison test, or by 319 Student's t-test when appropriate. Details of the statistical tests, sample sizes and 320 significance levels are provided in the corresponding figure legends. # 321 Results 322 PYR1 and PYL4 constitutively occupy a previously unrecognized ER- 323 Golgi-associated subpopulation 324 A recent study demonstrated that mono-ubiquitination promotes ABA receptor 325 trafficking towards the plasma membrane, suggesting that receptor localization may 326 already be regulated during early secretory trafficking (Liu _et al._, 2025). Whether ABA 327 receptors constitutively occupy a distinct ER-Golgi-associated subpopulation before 328 undergoing this regulated trafficking, however, has remained unknown. To address this 329 question, we examined the subcellular distribution of functional PYR1 and PYL4 330 reporters using complementary imaging and biochemical approaches. 331 To investigate whether ABA receptors are associated with the early secretory pathway, 332 we first analysed the subcellular distribution of GFP-PYR1 and GFP-PYL4 translational 333 fusions by confocal microscopy in _N. benthamiana_ using markers of distinct 334 endomembrane compartments (Nelson _et al._, 2007; Serra-Soriano _et al._, 2014; Bernat 335 Silvestre _et al._, 2022). In agreement with previous reports, both receptors displayed 336 predominant nucleocytoplasmic localization together with fluorescence at the cell 337 periphery (Belda-Palazon _et al._, 2016). In addition, both receptors consistently 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 338 accumulated in a network-like reticular pattern and in discrete intracellular puncta (Figs 339 **1a,b** ). The reticular signal overlapped with the ER luminal marker ChFP-KDEL, whereas 340 a subset of the intracellular puncta colocalized with the cis-Golgi marker ManI-RFP, 341 indicating that both PYR1 and PYL4 are present within the early secretory pathway. 342 Time-lapse confocal imaging further showed that the ER-associated PYR1 signal 343 remained dynamically associated with the ER network during its continuous remodelling 344 (Video/Movie **S1** ). 345 Since PYR1 and PYL4 are soluble proteins lacking transmembrane domains, their 346 association with the ER-Golgi endomembrane system prompted us to investigate whether 347 receptor interactions with membrane-associated proteins occur at these compartments. 348 CAR proteins are peripheral membrane proteins and well-established interactors of ABA 349 receptors (Rodriguez _et al._, 2015; Diaz _et al._, 2016; Cui _et al._, 2023). We therefore 350 analysed the subcellular localization of PYR1-CAR1 and PYL4-CAR1 complexes by 351 BiFC. BiFC fluorescence was detected at the cell periphery, as previously reported 352 (Rodriguez _et al._, 2015; Diaz _et al._, 2016), but also in a reticular network and in discrete 353 intracellular puncta (Fig. **1c,d** ). Co-expression with the ER marker KDEL revealed 354 extensive overlap of the reticular BiFC signal with the ER network, whereas co 355 expression with the cis-Golgi marker ManI showed that the punctate BiFC structures 356 corresponded to Golgi-associated compartments. Line-scan intensity profiles further 357 supported the spatial coincidence of the BiFC signal with both ER and cis-Golgi markers 358 (Fig. **1c,d** ). Moreover, disruption of the membrane-binding capacity of CAR1 by the 359 DADA mutations largely abolished the ER- and Golgi-associated BiFC pattern (Fig. **S1** ) 360 (Diaz _et al._, 2016), indicating that membrane association of CAR1 is required for 361 localization of the receptor-CAR complexes within the early secretory pathway. 362 To validate these observations under native conditions, we next analysed the subcellular 363 localization of PYR1 and PYL4 in transgenic _A. thaliana_ lines. Importantly, PYR1 364 localization was examined using a functional genomic _pPYR1::PYR1-GFP_ reporter 365 expressed under the control of its native promoter and endogenous regulatory sequences 366 (Moulinier-Anzola _et al._, 2024), thereby minimizing potential artefacts associated with 367 transient heterologous expression or constitutive overexpression, and providing an 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 368 independent validation of the observations obtained by transient expression in _N._ 369 _benthamiana_ . Consistent with the observations in _N. benthamiana_, PYR1-GFP displayed 370 nucleocytoplasmic fluorescence together with a reticular network and discrete 371 intracellular puncta. Co-localization with the ER membrane marker RFP-p24δ5 (Bernat 372 Silvestre et al., 2020) revealed that the reticular PYR1 signal was associated with the ER 373 network, whereas co-expression with the cis-Golgi marker ManI-RFP showed that a 374 subset of PYR1-positive puncta corresponded to Golgi-associated compartments. GFP 375 PYL4 exhibited a similar subcellular distribution, indicating that association with the 376 early secretory pathway is a common feature of distinct ABA receptors (Fig. **1e,f**, **S2** ). 377 To independently validate these observations, total protein extracts from wild-type (Col 378 0) seedlings were subjected to differential centrifugation to isolate a microsomal 379 membrane fraction (P100), which was subsequently resolved by continuous sucrose 380 density gradient centrifugation (Fig. **1g,h** ). Differential centrifugation efficiently 381 separated soluble and membrane fractions, as indicated by the enrichment of the ER 382 marker CNX1/2 and the plasma membrane marker H+-ATPase in the P100 fraction, 383 whereas β-tubulin was predominantly recovered in the soluble S100 fraction. Consistent 384 with its partial membrane association, PYR1 was detected in both soluble and 385 microsomal fractions, indicating that a substantial fraction of the protein is membrane 386 associated (Fig. **1g** ). Within the sucrose gradient, PYR1 was broadly distributed across 387 membrane fractions, with its abundance progressively increasing from fraction F11 388 onwards. Notably, PYR1 showed substantial co-fractionation with the ER marker 389 CNX1/2 across the ER-enriched region (F12-F16), exhibiting a first accumulation peak in 390 fraction F14 that closely overlapped with the maximum abundance of CNX1/2, followed 391 by a second and more pronounced peak in the densest fraction (F16) (Fig. **1h** ). In 392 contrast, the plasma membrane marker H+-ATPase displayed a distinct distribution, 393 reaching maximal abundance in lighter fractions, whereas residual β-tubulin was detected 394 only in fraction F3, confirming the efficient enrichment of membrane compartments. 395 Together, these biochemical analyses corroborate microscopy observations and provide 396 independent evidence for the existence of a constitutive ER-associated membrane pool of 397 PYR1 consistent with its localization within the early secretory pathway. 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 398 Collectively, these results identify a previously unrecognized constitutive ER–Golgi 399 associated subpopulation of PYR1 and PYL4, establishing the early secretory pathway as 400 an active regulatory compartment controlling ABA receptor homeostasis. 401 402 **Figure 1. PYR1 and PYL4 define a constitutive receptor pool associated with the early secretory** 403 **pathway.** (a, b) Representative confocal images of _N. benthamiana_ epidermal cells transiently expressing 404 GFP-PYR1 or GFP-PYL4 together with the ER marker ChFP-KDEL (a) or the cis-Golgi marker ManI-RFP 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 405 (b). (c, d) Representative BiFC confocal images following transient co-expression in _N. benthamiana_ 406 epidermal cells of CAR1-YFP [N] and YFP [C] -PYR1 or YFP [C] -PYL4 together with ChFP-KDEL(c) and ManI 407 RFP (d). (e, f) Confocal images of root epidermal cells from transgenic _A. thaliana_ seedlings expressing 408 _pPYR1::PYR1-GFP_ or _35S::GFP-PYL4_ together with the ER marker RFP-p24δ5 (e), or the cis-Golgi 409 marker ManI-RFP (f). Insets show magnified regions indicated by the yellow boxes. Fluorescence intensity 410 profiles along the yellow lines are also shown on the right. Scale bars: 10 μm. (g) Immunoblot analyses of 411 protein extracts from wild-type (Col-0) seedlings after subcellar fractionation, using the indicated 412 antibodies. Total protein extract (Total), soluble fraction after centrifugation at 8,000 × g (S8), pellet after 413 centrifugation at 8,000 × g (P8), soluble supernatant after ultracentrifugation at 100,000 × g (S100) and 414 microsomal membrane pellet after ultracentrifugation at 100,000 × g (P100). CNX1/2, H [+] -ATPase and β 415 tubulin were used as markers for the ER, plasma membrane and cytosolic fractions, respectively. (h) 416 Immunoblot analyses of protein extracts after continuous sucrose density gradient fractionation of the P100 417 microsomal fraction, using the indicated antibodies. The corresponding densitometric analysis is shown on 418 the right. # 419 MAG5 is required for the normal endomembrane distribution and trafficking 420 of ABA receptors 421 The identification of a constitutive ER-Golgi-associated subpopulation of ABA receptors 422 prompted us to investigate its relationship with the early secretory trafficking machinery. 423 MAG5 is a core organizer of ER exit sites that defines specialized ER domains where 424 COPII-mediated transport is initiated. To examine their spatial relationship, we analysed 425 _A. thaliana_ plants co-expressing the functional ER exit site marker _pMAG5::MAG5-GFP_ 426 together with RFP-PYR1 (Takagi _et al._, 2013; Moulinier-Anzola _et al._, 2024). 427 Confocal microscopy revealed that MAG5 accumulated in discrete punctate structures 428 corresponding to ER exit sites, whereas PYR1 exhibited a broader intracellular 429 distribution. Although a substantial proportion was detected outside MAG5-positive 430 puncta, a subset of receptor-containing puncta closely associated with MAG5 (Fig. **2a** ), 431 consistent with the transient recruitment of a fraction of the receptor population to ER 432 exit sites during secretory trafficking. A similar spatial association between MAG5 and 433 PYR1 was also observed following transient co-expression in _N. benthamiana_ leaf 434 epidermal cells (Fig. **S3a** ), indicating that this spatial relationship is reproducible in an 435 independent heterologous expression system. Moreover, BiFC complexes formed by 436 CAR1 and PYR1 remained closely associated with RFP–MAG5-positive compartments 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 437 (Fig. **S3b** ), and time-lapse confocal imaging showed that this spatial association was 438 maintained during the dynamic movement of these structures (Video/Movie **S2** ). 439 Together, these observations further support the close association of ABA receptors with 440 MAG5-positive early secretory compartments. 441 To determine whether the association between ABA receptors and MAG5 was 442 maintained following perturbation of membrane trafficking, seedlings were treated with 443 BFA, which induces the formation of BFA compartments by disrupting Golgi-TGN/EE 444 (trans-Golgi network/early endosome) trafficking, or WM, which causes the swelling of 445 multivesicular bodies/ prevacuolar compartments (MVBs/PVCs) (Ritzenthaler _et al._, 446 2002; Tse _et al._, 2004). Following BFA treatment, MAG5 and PYR1 accumulated within 447 the same BFA-induced compartments, which were typically stained by FM4-64 (Fig. **2b** ). 448 Likewise, following WM treatment MAG5 remained closely associated with PYR1 in 449 enlarged LysoTracker-positive acidic compartments, consistent with the WM-induced 450 swelling of MVBs/PVCs (Fig. **2b** ) (Tse _et al._, 2004; Xu _et al._, 2014). These observations 451 indicate that the spatial association between ABA receptors and MAG5 is preserved 452 following pharmacological perturbation of membrane trafficking. Interestingly, although 453 MAG5 is primarily regarded as an organizer of ER exit sites, it was consistently detected 454 in BFA-induced compartments and WM-induced late endosomal structures together with 455 PYR1, suggesting that its association with PYR1 is maintained beyond steady-state ER 456 exit sites during pharmacological perturbation of membrane trafficking. 457 To further assess the in _vivo_ association between ABA receptors and MAG5, BiFC assays 458 were performed in _N. benthamiana_ leaves. Reconstituted YFP fluorescence was detected 459 for both the MAG5-PYR1 and MAG5-PYL4 combinations, whereas no signal was 460 observed in the corresponding negative controls (Figs **2c, S4** ). The BiFC signal exhibited 461 a reticular pattern together with discrete punctate structures that largely overlapped with 462 the ER marker ChFP-KDEL, indicating that the association occurs at ER-associated 463 membrane domains. Although BiFC does not distinguish between direct physical 464 interaction and indirect association within protein complexes or membrane domains, 465 these observations are consistent with a close _in vivo_ association between MAG5 and 466 ABA receptors. 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 467 468 **Figure 2. MAG5 associates with ABA receptors within the early secretory pathway.** (a) Confocal 469 images of root epidermal cells from transgenic _A. thaliana_ seedlings co-expressing _pMAG5::MAG5-GFP_ 470 and _35S::RFP-PYR1_ . Insets show magnified regions indicated by the yellow boxes. Fluorescence intensity 471 profiles along the yellow line are shown on the right. (b) Subcellular distribution of MAG5-GFP and RFP 18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 472 PYR1 in root epidermal cells following pharmacological treatments. Five-day-old seedlings were treated 473 with 33 μM WM for 1 h and stained with LysoTracker™ Deep Red, or with 50 μM BFA for 1 h followed 474 by FM4-64 staining. Arrows indicate representative compartments containing both MAG5-GFP and RFP 475 PYR1 within WM-induced late endosomal structures or BFA bodies. (c) Representative BiFC images 476 following transient co-expression of YFP [N] -MAG5 and YFP [C] -PYR1 or YFP [C] -PYL4 together with ChFP 477 KDEL in _N. benthamiana_ epidermal cells. Scale bars: 10 μm. 478 The close association between ABA receptors and MAG5 suggested that MAG5 is 479 functionally required for receptor trafficking. To test this hypothesis, we compared the 480 subcellular distribution and trafficking of PYR1 and PYL4 in wild-type and _mag5-3_ 481 mutant plants (Takagi _et al._, 2013). Comparison of wild-type and _mag5-3_ seedlings 482 revealed an apparent alteration in the steady-state distribution of PYR1 and PYL4, 483 characterized by a more prominent reticular pattern (Fig. **3a** ), indicating that disruption of 484 MAG5-dependent ER export alters the normal subcellular organization of ABA 485 receptors. 486 Given the established role of MAG5 in organizing ER exit sites and maintaining ER– 487 Golgi interface integrity (Takagi _et al._, 2013), we next examined whether disruption of 488 MAG5 altered the organization of downstream endomembrane compartments by 489 analysing BFA-induced membrane reorganization. FM4-64 staining revealed the 490 formation of characteristic, well-rounded BFA bodies in wild-type root cells. In contrast, 491 BFA body formation was markedly altered in the _mag5-3_ mutant, resulting in fewer, 492 smaller and morphologically aberrant BFA bodies (Fig. **3b** ), indicating that disruption of 493 MAG5 alters the organization of BFA-induced endomembrane compartments. In 494 agreement with the altered morphology and reduced formation of BFA bodies, 495 redistribution of GFP–PYR1 and GFP–PYL4 into FM4-64-labelled BFA bodies appeared 496 less pronounced in the _mag5-3_ mutant, where receptor fluorescence remained more 497 diffusely distributed throughout the cell (Fig. **3c** ). 498 To determine whether MAG5-dependent ER export influences receptor trafficking 499 towards late endosomal compartments, seedlings were treated with WM. In wild-type 500 plants, PYR1 and PYL4 accumulated within enlarged LysoTracker-positive acidic 501 structures induced by WM (Fig. **3d** ), consistent with their delivery to late endosomal 502 MVBs/PVCs (Takáč _et al._, 2012). In contrast, accumulation of PYR1 within these 19 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 503 structures was markedly reduced in the _mag5-3_ mutant, indicating that disruption of 504 MAG5 impairs the normal trafficking of ABA receptors towards late endosomal 505 compartments, a pathway previously implicated in the vacuolar degradation of ABA 506 receptors (Belda-Palazon _et al._, 2016; Yu _et al._, 2016; García-León _et al._, 2019; 507 Moulinier-Anzola _et al._, 2024; Chen _et al._, 2025). Together, these observations 508 demonstrate that MAG5 is required for the normal intracellular trafficking of ABA 509 receptors through the endomembrane system, leading to their delivery to late endosomal 510 compartments. 20 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 511 512 **Figure 3. MAG5 is required for the normal trafficking of ABA receptors through the endomembrane** 513 **system.** a) Representative confocal images of root epidermal cells from wild-type (Col-0) and _mag5-3_ 514 seedlings expressing _35S::GFP-PYR1_ or _35S::GFP–PYL4_ . (b) Representative confocal images showing 515 FM4-64-labelled BFA bodies in wild-type (Col-0) and _mag5-3_ root epidermal cells following treatment 516 with 50 μM BFA for 1 h and subsequent staining with FM4-64. The graph on the right shows the 21 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 517 percentage of cells containing characteristic BFA bodies, defined as well-rounded FM4-64-positive 518 structures larger than 2.5 μm in diameter. Data represent the mean ± SEM (Col-0, n=32 images containing 519 853 cells from 10 plants; _mag5-3_, n=26 images containing 961 cells from 10 plants). P-values ( _P_ ) denote 520 statistically significant differences (Student's t-test). (c) Representative confocal images of root epidermal 521 cells expressing _35S::GFP–PYR1_ or _35S::GFP–PYL4_ in Col-0 or _mag5-3_ background following BFA 522 treatment and FM4-64 staining. (d) Representative confocal images of root epidermal cells expressing 523 _35S::GFP–PYR1_ or _35S::GFP–PYL4_ in Col-0 or _mag5-3_ background, following treatment with 33 μM 524 WM for 1 h and staining with LysoTracker™ Deep Red. Arrows indicate representative WM-induced 525 LysoTracker-positive late endosomal compartments. Scale bars: 10 μm # 526 MAG5-dependent trafficking regulates ABA receptor abundance 527 The altered intracellular trafficking of ABA receptors observed in the _mag5-3_ mutant 528 prompted us to examine whether disruption of MAG5 also affects receptor abundance. 529 Immunoblot analysis revealed a significant increase in both PYR1 and PYL4 protein 530 levels in the _mag5-3_ mutant compared with wild-type plants under control conditions 531 (Fig. **4a,b** ). 532 To determine whether the increased receptor abundance reflected changes in gene 533 expression, transcript levels of PYR1 and PYL4 were analysed by RT-qPCR. In contrast 534 to the marked increase in protein accumulation, transcript abundance did not differ 535 significantly between wild-type and _mag5-3_ plants (Figs **4c** ). 536 Together, these results indicate that MAG5-dependent trafficking regulates ABA receptor 537 abundance through a post-transcriptional mechanism. 22 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 538 539 **Figure 4. MAG5-dependent trafficking regulates ABA receptor abundance through a post-** 540 **transcriptional mechanism.** (a) Immunoblot analysis of PYR1 and PYL4 protein accumulation in wild 541 type (Col-0) and _mag5-3_ seedlings using the indicated antibodies. ACTIN was used as a loading control. 542 (b) Densitometric quantification of PYR1 and PYL4 protein levels shown in (a). Protein abundance was 543 normalized to ACTIN and referenced to wild-type (Col-0). Data represent the mean ± SEM (n = 4 544 independent biological replicates). P-values ( _P_ ) denote statistically significant differences (Student's t-test). 545 (c) Relative transcript levels of PYR1 and PYL4 in wild-type (Col-0) and _mag5-3_ seedlings determined by 546 RT-qPCR. Gene expression was normalized to PDF2. Data represent the mean ± SEM (n = 3 independent 547 biological replicates). # 548 Loss of MAG5 enhances ABA signalling and ABA responses 549 To investigate the functional consequences of MAG5 loss of function on ABA signalling, 550 we analysed ABA-dependent developmental and physiological responses using well 23 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 551 characterized ABA-hypersensitive ( _hab1-1 abi1-2_ ) or ABA-insensitive ( _snrk2.2 snrk2.3_ ) 552 mutants as controls (Saez _et al._, 2006; Fujii _et al._, 2007). Compared with wild-type 553 plants, the _mag5-3_ mutant displayed enhanced sensitivity to exogenous ABA, as 554 evidenced by reduced radicle emergence, cotyledon greening and slightly stronger 555 inhibition of primary root growth (Fig. **5a,b** ). Similar ABA hypersensitivity was also 556 observed in two additional independent _mag5_ alleles ( _mag5-1_ and _mag5-2_ ). By contrast, 557 the closely related _sec16b-1_ mutant exhibited a substantially weaker phenotype than 558 _mag5_ alleles, consistent with the lower expression of SEC16B relative to MAG5 reported 559 previously (Fig. **S5** ) (Takagi _et al._, 2013). Together, these results indicate that enhanced 560 ABA responses are predominantly associated with disruption of MAG5 function. 561 To determine whether this enhanced ABA responsiveness also affected stress adaptation, 562 we examined early growth under salt stress. In agreement with their ABA-hypersensitive 563 phenotype, _mag5-3_ seedlings displayed a stronger inhibition of radicle emergence and 564 cotyledon greening in the presence of 100 and 150 mM NaCl, closely resembling the 565 ABA-hypersensitive _hab1-1 abi1-2_ mutant, whereas wild-type seedlings were 566 considerably less affected (Fig. **5c** ). These results indicate that disruption of MAG5 567 enhances physiological responses to salt stress, in line with increased ABA signalling. 568 To determine whether the enhanced ABA sensitivity of _mag5-3_ was accompanied by 569 increased activation of ABA signalling, we analysed the expression of the ABA 570 responsive marker genes _RD29A_, _RD29B_, _ABI5_ and _RAB18_ (Fujii _et al._, 2007). Under 571 mock conditions, transcript levels were similarly low in wild-type, _mag5-3_ and the ABA 572 insensitive _snrk2.2 snrk2.3_ mutant (Fig. **5d** ). Following ABA treatment, _RD29A_, _RD29B_, 573 _ABI5_ and _RAB18_ were strongly induced in wild-type seedlings, whereas induction was 574 almost completely abolished in _snrk2.2 snrk2.3_, confirming the effectiveness of the 575 treatment. By contrast, ABA-induced expression of both marker genes was significantly 576 higher in _mag5-3_ than in wild-type plants (Fig. **5d)**, demonstrating that disruption of 577 MAG5 enhances ABA signalling at the transcriptional level. 578 To exclude the possibility that the enhanced ABA sensitivity of _mag5-3_ resulted from 579 altered endogenous ABA accumulation, we quantified ABA levels under control, 580 exogenous ABA treatment and salt stress conditions. Endogenous ABA concentrations 24 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 581 were comparable between wild-type and _mag5-3_ seedlings under control conditions and 582 following salt treatment. Upon treatment with 0.3 µM ABA, however, _mag5-3_ seedlings 583 accumulated significantly less ABA than wild-type seedlings (Fig. **5e** ). Thus, the 584 enhanced ABA sensitivity of _mag5-3_ cannot be explained by increased ABA 585 accumulation. Instead, these results support the conclusion that disruption of MAG5 586 enhances ABA responsiveness at the level of signal perception or transduction rather than 587 hormone accumulation. 588 Because ABA promotes stomatal closure, thereby reducing transpirational cooling and 589 increasing leaf temperature, we next examined whether enhanced ABA signalling in 590 _mag5-3_ was associated with altered stomatal responses using infrared thermography 591 (Jones & Leinonen, 2003; Wang _et al._, 2004; Munemasa _et al._, 2015; Vaidya _et al._, 592 2019). Infrared thermography revealed that _mag5-3_ leaves exhibited significantly higher 593 leaf temperatures than wild-type plants under both mock and ABA-treated conditions 594 (Fig. **5f** ). Notably, _mag5-3_ displayed elevated leaf temperatures even in the absence of 595 exogenous ABA, suggesting enhanced responsiveness to endogenous ABA. Following 596 ABA treatment, leaf temperature increased in all genotypes but was significantly higher 597 in _mag5-3_ than in wild-type plants, approaching the response observed in the ABA 598 hypersensitive control _hab1-1 abi1-2_ . To directly assess whether this thermographic 599 phenotype was associated with altered stomatal behaviour, we measured stomatal 600 aperture in seedling cotyledons. Consistent with the thermographic phenotype, _mag5-3_ 601 seedlings displayed a significantly reduced stomatal aperture compared with Col-0 (Fig. 602 **S6** ). Together, these results support enhanced stomatal closure in the absence of MAG5 603 and provide physiological evidence that altered early secretory trafficking affects not 604 only growth-related ABA responses but also stomatal regulation. 25 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 605 606 **Figure 5. Loss of MAG5 enhances ABA signalling without altering endogenous ABA accumulation.** 607 (a) ABA sensitivity during seed germination. Representative images of wild-type (Col-0), the ABA 608 hypersensitive mutant _hab1-1 abi1-2_, and _mag5-3_ seedlings grown on control conditions or media 609 supplemented with 0.3 or 0.5 µM ABA. Quantification of radicle emergence (left) and cotyledon greening 610 (right) of the genotypes is shown. Data represent the mean ± SEM (n = 3 independent biological replicates). 611 P-values ( _P_ ) denote statistically significant differences from wild-type (Col-0) within the same treatment 26 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 612 (two-way ANOVA followed by Dunnett's multiple comparisons test). (b) Primary root growth of wild-type 613 (Col-0), _mag5-3_, and _hab1-1 abi1-2_ seedlings grown on control medium or medium supplemented with 5 614 µM ABA. Data are presented as mean ± SEM (n=29-40 seedlings). P-values ( _P_ ) denote statistically 615 significant differences from wild-type (Col-0) within the same treatment (Student's t-test). (c) Salt 616 sensitivity during early seedling establishment. Representative images of wild-type (Col-0), _hab1-1 abi1-2_, 617 and _mag5-3_ seedlings grown on control medium or supplemented with 100 and 150 mM NaCl. 618 Quantification of radicle emergence (left) and cotyledon greening (right) is shown below. Data represent 619 the mean ± SEM (n = 3 independent biological replicates). P-values ( _P_ ) denote statistically significant 620 differences from wild-type (Col-0) within the same treatment (two-way ANOVA followed by Dunnett's 621 multiple comparisons test). (d) ABA-responsive gene expression. Relative transcript levels of _RD29B_, 622 _RD29A_, _RAB18_ and _ABI5_ determined by RT-qPCR in wild-type (Col-0), the ABA-insensitive mutant 623 _snrk2.2 snrk2.3_, and _mag5-3_ seedlings grown for 6 d on control medium or supplemented with 0.3 μM 624 ABA. Data represent the mean ± SEM (n = 4 independent biological replicates). P-values ( _P_ ) denote 625 statistically significant differences from wild-type (Col-0) within the same treatment (two-way ANOVA 626 followed by Dunnett's multiple comparisons test). (e) Endogenous ABA quantification in wild-type (Col-0) 627 and _mag5-3_ seedlings under the same treatments indicated in (a) and (c). P-values ( _P_ ) denote statistically 628 significant differences from wild-type (Col-0) within the same treatment (Student's t-test). (f) Infrared 629 thermography analysis of rosette leaf temperature in wild-type (Col-0), _hab1-1 abi1-2_, and _mag5-3_ plants 630 following control or ABA treatment (20 µM). Representative thermal images are shown together with the 631 corresponding quantification of leaf temperature. Same infrared thermography image was cropped for 632 showing wild-type (Col-0) and _mag5-3_ contiguously. Violin box plots representing the leaf measurements 633 of 18 plants. P-values ( _P_ ) denote statistically significant differences from wild-type (Col-0) within the same 634 treatment (two-way ANOVA followed by Dunnett's multiple comparisons test). # 635 Discussion 636 ABA receptor homeostasis depends on the coordinated regulation of intracellular 637 trafficking and protein turnover. Over the past decade, considerable progress has 638 identified multiple ubiquitin ligases and endosomal sorting components that control 639 receptor abundance through proteasomal or vacuolar degradation (Coego _et al._, 2021; 640 Qin _et al._, 2026). In contrast, whether early secretory trafficking contributes to receptor 641 homeostasis has remained largely unexplored. Our findings identify MAG5-dependent 642 ER export as a regulatory checkpoint contributing to ABA receptor homeostasis by 643 controlling receptor trafficking at the earliest stages of the secretory pathway (Fig. **6** ). 644 Based on our findings and previous studies (Coego _et al._, 2021; Li _et al._, 2021; Cui _et al._, 645 2023; Liu _et al._, 2025), we propose a model in which a constitutive pool of ABA 27 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 646 receptors is maintained at the ER, where receptor abundance and trafficking competence 647 may already be regulated through interactions with quality-control and trafficking factors. 648 Receptors that successfully exit the ER through MAG5- and COPII-dependent ER exit 649 sites are subsequently delivered to the Golgi apparatus (GA) and the TGN. Along this 650 secretory itinerary, receptors may acquire additional regulatory partners, including CAR 651 proteins, before being sorted either to the plasma membrane or into the endosomal 652 pathway, ultimately reaching MVBs/PVCs and the vacuole for degradation through 653 ESCRT-dependent trafficking. Disruption of MAG5 is predicted to alter the normal 654 distribution of ABA receptors across the endomembrane system by reducing the efficient 655 delivery of ABA receptors to downstream endomembrane compartments while 656 simultaneously limiting their progression towards endosomal sorting and vacuolar 657 degradation. Such redistribution would alter the balance between receptor pools available 658 for ABA perception and signalling but also, those committed to degradation, providing a 659 plausible explanation for the increased accumulation of PYR1 and PYL4 observed in 660 _mag5-3_ mutants despite impaired trafficking through the secretory pathway. Together, 661 this model provides a conceptual framework linking early secretory trafficking with the 662 establishment of ABA receptor homeostasis across the endomembrane system (Fig. **6** ). 663 28 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 664 **Figure 6. Proposed model for the role of MAG5-dependent ER export in the establishment of ABA** 665 **receptor homeostasis.** ABA receptors are proposed to maintain a constitutive pool associated with the ER, 666 where receptor abundance and trafficking competence may already be regulated through interactions with 667 quality-control and trafficking factors, including the ER-associated E3 ubiquitin ligase DOA10A and CAR 668 proteins. Receptors that successfully exit the ER through MAG5- and COPII-dependent ER exit sites 669 (ERES) are subsequently delivered to the Golgi apparatus (GA) and the trans-Golgi network/early 670 endosome (TGN/EE). During their progression through the secretory pathway, receptors may associate 671 with additional regulatory factors before being sorted either to the plasma membrane, where RSL1 672 promotes receptor ubiquitination and endocytosis, or into the endosomal pathway, leading to multivesicular 673 bodies/prevacuolar compartments (MVBs/PVCs) and ESCRT-dependent vacuolar degradation. Disruption 674 of MAG5-dependent ER export is proposed to alter receptor distribution throughout the endomembrane 675 system while limiting progression towards endosomal sorting and vacuolar degradation. Such redistribution 676 increases the proportion of receptors available for ABA perception and signalling, thereby enhancing ABA 677 responsiveness without affecting endogenous ABA accumulation. 678 Consistent with this model, disruption of another core COPII component, SAR1A, also 679 results in enhanced ABA sensitivity, suggesting that COPII-dependent ER export 680 represents a broader regulatory layer controlling ABA signalling rather than a mechanism 681 unique to MAG5. Our findings are particularly intriguing in light of this work, 682 demonstrating that ABA actively remodels the early secretory pathway by inducing 683 SAR1A expression and promoting the formation of specialized COPII vesicles required 684 for the trafficking of stress-associated membrane proteins (Li _et al._, 2021). Together with 685 these observations, our data support a reciprocal regulatory relationship between ABA 686 signalling and the early secretory pathway. Whereas ABA signalling enhances COPII 687 function through SAR1A-dependent remodelling of ER export, our results indicate that 688 efficient COPII-mediated trafficking is required to establish the abundance and 689 intracellular distribution of ABA receptors. Moreover, our pharmacological analyses 690 indicate that the consequences of MAG5 disruption extend well beyond ER exit sites. In 691 addition to the reduced accumulation of PYR1 and PYL4 in WM-induced late endosomal 692 compartments, the altered morphology and reduced formation of BFA bodies in _mag5-3_ 693 are in line with the established role of MAG5 in maintaining the organization of ER exit 694 sites and efficient ER export (Takagi et al., 2013). Rather than reflecting a direct defect in 695 endosomal trafficking, these observations are more consistent with a global perturbation 696 of membrane flux across the endomembrane system. Previous studies of the ER–Golgi 29 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 697 tethering proteins MAG2 and MAG4, together with the ER exit site organizer MAG5, 698 demonstrated that disruption of distinct trafficking steps at the ER–Golgi interface leads 699 to a common phenotype characterized by defective cargo export, accumulation of 700 secretory cargo within ER-derived MAG bodies, and impaired delivery of vacuolar 701 proteins to protein storage vacuoles (Li _et al._, 2006; Takahashi _et al._, 2010; Takagi _et al._, 702 2013). These studies further suggested that defects in early secretory trafficking disrupt 703 the dynamic balance between anterograde and retrograde membrane transport. Within 704 this framework, impaired COPII-dependent ER export would reduce the continuous 705 supply of membrane and cargo to downstream compartments, thereby progressively 706 altering the organization and trafficking capacity of the Golgi and TGN/EE system. 707 Consequently, ABA receptors would be delivered less efficiently to late endosomes and 708 vacuoles, reducing their progression through degradative pathways while simultaneously 709 favoring their accumulation in earlier secretory compartments. 710 This interpretation is in agreement with the emerging view that the endomembrane 711 system operates as an integrated trafficking network, in which perturbations arising at 712 early secretory steps propagate throughout the endomembrane system and ultimately 713 influence downstream trafficking events. Our observations fit well within this conceptual 714 framework, as defects in MAG5-dependent ER export are accompanied by altered 715 organization of BFA-sensitive compartments and reduced accumulation of ABA 716 receptors in WM-induced late endosomal structures. In line with this idea, recent studies 717 indicate that COPII components participate in membrane remodelling events extending 718 beyond conventional ER-to-Golgi transport, revealing unexpected functional connections 719 between ER exit sites and downstream degradative membrane systems (Liao _et al._, 2024; 720 Chung _et al._, 2024). Although our observations do not demonstrate a direct role for 721 MAG5 in late endosomal trafficking, they support the emerging view that early secretory 722 trafficking and endosomal sorting are functionally integrated processes rather than 723 independent transport pathways. This bidirectional interplay reinforces the concept that 724 the early secretory pathway is not merely a constitutive transport route but a dynamic 725 signalling hub that both responds to and shapes hormone signalling (Aniento _et al._, 726 2022). 30 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 727 An emerging theme in ABA signalling is that receptor homeostasis is coordinated in a 728 compartment-specific manner rather than through a single degradation pathway (Coego _et_ 729 _al._, 2021; Qin _et al._, 2026). Different E3 ubiquitin ligases regulate distinct subcellular 730 pools of ABA receptors, collectively establishing spatial control over receptor abundance 731 and turnover. For instance, the plasma membrane-associated RSL1 promotes 732 ubiquitination and endocytosis of PYR1 and PYL4, leading to their subsequent sorting 733 through the ESCRT pathway for vacuolar degradation, whereas the related RFA family 734 members regulate receptor turnover in the cytosol and nucleus through proteasomal 735 degradation. Likewise, the CRL4-DDA1 complex targets nuclear ABA receptors, 736 whereas the ER-associated E3 ligase DOA10A promotes receptor mono-ubiquitination 737 and relocalization towards membrane compartments (Liu _et al._, 2025). Our findings 738 extend this compartmentalized view of receptor regulation by placing the ER–Golgi 739 interface upstream of the previously described compartment-specific regulatory 740 mechanisms. 741 Importantly, the physiological consequences of ABA receptor trafficking extend beyond 742 the control of receptor abundance itself. Previous work on FYVE1/FREE1, FYVE4, 743 VPS23A, ALIX and TOL proteins has shown that perturbation of receptor endocytosis, 744 endosomal sorting and vacuolar degradation alters ABA receptor homeostasis and 745 hormone responsiveness, affecting both developmental and physiological ABA 746 responses, including stomatal regulation (Belda-Palazon _et al._, 2016; Yu _et al._, 2016; 747 García-León _et al._, 2019; Moulinier-Anzola _et al._, 2024; Chen _et al._, 2025). This is 748 particularly relevant because ABA signalling controls not only transcriptional and growth 749 responses but also rapid membrane-delimited processes required for adaptation to water 750 stress. The recruitment of specific ABA receptors to the plasma membrane by CAR 751 proteins and the DOA10A-dependent regulation of receptor trafficking towards 752 membrane compartments further highlight the importance of receptor positioning for 753 ABA signalling (Diaz _et al._, 2016; Liu _et al._, 2025). In this context, our finding that 754 disruption of MAG5 alters ABA receptor distribution and affects both growth-related 755 ABA responses and stomatal regulation extends the functional relevance of receptor 756 trafficking to the early secretory pathway. Although our data do not directly establish 757 how MAG5-dependent ER export affects receptor abundance at the plasma membrane, 31 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 758 they support a model in which perturbations at the ER–Golgi interface alter receptor 759 distribution and ultimately influence distinct ABA signalling outputs. 760 A major implication of our findings is the identification of a constitutive ER-associated 761 pool of ABA receptors, indicating that the ER functions as an active regulatory 762 compartment rather than merely a transient station of the secretory pathway. In this 763 context, receptor export is likely coordinated with additional processes, including post 764 translational modifications such as DOA10A-mediated mono-ubiquitination and the 765 association with CAR proteins, which together may determine receptor trafficking 766 competence and progression through COPII-dependent ER export. Defining how these 767 mechanisms are integrated with COPII-mediated trafficking will be essential for 768 understanding how receptor homeostasis is dynamically regulated. 769 Collectively, our work expands current models of ABA signalling by demonstrating that 770 receptor trafficking is an integral determinant of hormone responsiveness, revealing that 771 the ER–Golgi interface contributes directly to the establishment of ABA receptor 772 homeostasis. # 773 Acknowledgements 774 We thank Ikuko Hara-Nishimura for the _pMAG5::MAG5-GFP_, _mag5-1_, _mag5-2_, _mag5-3_ 775 and _sec16b-1_ _A. thaliana_ transgenic lines. The authors acknowledge the assistance of the 776 IBMCP Scientific and Technical Services, including the Advanced Microscopy Unit, the 777 Plant Growth Facility and the Hormone Quantification Facility, for their support during 778 the course of this work. We gratefully acknowledge the financial support that made this 779 study possible. This research was funded by the Spanish Ministry of Science, Innovation 780 and Universities (MCIU), grant numbers PID2022-142412NB-I00 (awarded to B.B.-P. 781 and A.F.), PID2020-113847GB-I00 (awarded de F.A and M.J.M.), and CNS2024-154566 782 (awarded to B.B.-P.), and by the CIDEGENT program of excellence of GVA, grant 783 number CIDEXG/2022/27 (awarded to B.B.-P.). This work was also supported by grant 784 PID2023-147322OB-I00, funded by MCIU/AEI/10.13039/501100011033 and the ERDF 785 (“A way of making Europe”) (awarded to P.L.R.). B.K. and J.M.-A. acknowledge 786 funding from the Austrian Science Fund (FWF) through grants PAT1121425 and 787 P34748. J.A.B.-T. and A.V.-F. were respectively the beneficiary of PhD contracts 32 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.761007; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.761007) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 788 CIDEXG/2022/27-01 and CIDEXG/2022/27-02, funded by GVA. We also acknowledge 789 financial support from the institutional grant CEX2025-001626-S (Center of Excellence 790 Severo Ochoa) funded by MCIU/AEI/10.13039/501100011033. # 791 Competing interests 792 None declared. # 793 Author contributions 794 J.A.B.-T., A.V.-F., C.C.-R. M.B., R.M. and B.B.-P. conducted experiments. F.A., 795 M.J.M., J.M.-A., B.K., P.L.R. provided tools and expertise on vesicle trafficking and 796 ABA signalling, actively supporting the conceptual work. B.B.-P. and A.F. conceived 797 and designed the project. B.B.-P. directed and supervised all research activities and 798 prepared figures. B.B.-P. wrote the manuscript. F.A., M.J.M., J.M.-A., B.K., P.L.R and 799 A.F. reviewed the manuscript. 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