Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi Network(TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the interactome of the Transport Protein Particle II (TRAPPII) complex, required for TGN structure and function. Together with yeast-two-hybrid screens, this identified shaggy-like kinases (GSK3/AtSKs) as TRAPPII interactors. Kinase assays and pharmacological inhibition provided in vitro and in vivo evidence that AtSKs target the TRAPPII-specific subunit AtTRS120. We identified three GSK3/AtSK phosphorylation sites in AtTRS120. These sites were mutated, and the resulting AtTRS120 phosphovariants subjected to a variety of single and multiple stress conditions. The non-phosphorylatable TRS120 mutant exhibited enhanced adaptation to multiple stress conditions and to osmotic stress whereas the phosphomimetic version was less resilient. This suggests that the TRAPPII phosphostatus mediates adaptive responses to abiotic stress factors. AtSKs are multitaskers that integrate a broad range of signals. Similarly, the TRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

Co-Immunoprecipitation: Co-immunoprecipitation experiments were carried out as described previously (Rybak et al, 2014). Briefly, seedling lysates were incubated with GFP-trap beads (Chromotek). After washing away all non-binding proteins, 70 μl 2x NuPAGE LDS + 25 mM dithiothreitol (DTT) buffer (ThermoFisher, US) was added and boiled at 70°C for 10 min to denature the bait and all interaction partners. In-gel trypsin digestion was performed according to standard procedures (Shevchenko et al, 2006). Briefly, the samples were run on a NuPAGE 4-12% Bis-Tris Protein Gel (Thermofisher Scientific, US) for 3 min. Subsequently, the still not size-separated single protein band per sample was cut out of the gel, reduced (50 mM DTT), alkylated (55 mM chloroacetamide) and digested overnight with trypsin (trypsin-gold, Promega). The resulting peptides were analysed by mass spectrometry. In vitro kinase assays: In vitro kinase assays with mass-spectrometry readout were performed to determine specific phosphorylation sites. For each reaction, 10 μg of substrate (TRS120-T2) and different dilutions of the kinase (1:5, 1:10, 1:100) were incubated for 15, 30, and 120 min in a kinase buffer (20 mM Tris HCl, 100 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1 mM ATP). For the negative controls, one sample with the highest kinase concentration and the longest incubation time was incubated in a kinase buffer without ATP. For the kinase dead control, the kinase was heat-inactivated prior to the incubation with its substrate. To stop the reaction, samples were heated at 95°C for 5 min. After reduction (10 mM DTT), alkylation (55 mM CAA) and protein digestion (trypsin-gold, Promega) the resulting peptides were purified using self-packed StageTips (C18 material, 3M Empore) and analyzed by mass spectrometry. More detailed information can be found in the paper's supporting methods. LC-MS/MS data acquisition and analysis Generated peptides were analyzed on a Dionex Ultimate 3000 RSLCnano system coupled to a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific) in data-dependent acquisition mode. Peptide identification and quantification was performed using the software MaxQuant(version 1.6.1.0) (Cox & Mann, 2008; Tyanova et al, 2016a) with its built-in search engine Andromeda (Cox et al, 2011) and an Arabidopsis thaliana reference database (Araport). For a targeted analysis of the kinase assay data set, MS1 chromatograms from selected phosphopeptides were extracted and analyzed using the Skyline software (MacLean et al, 2010). More detailed information can be found in the paper’s supporting information.

Seed were surface sterilized, stratified at 4◦C for two days and plated on 1/2 MS medium supplemented with 1% sucrose and B5 Vitamins (Sigma; https://www.sigmaaldrich.com). Plates were incubated at 22°C in constant light (80 μmol m-2s-1). Seven-day-old plate-grown seedlings were used for co-immunoprecipitation. Co-Immunoprecipitation: For CLUB/AtTRS130:GFP we used 3 g of inflorescences per IP experiment. For TRS120-GFP, 3 g of light-grown seedlings were harvested at day 7. For bikinin treatment, seedlings were treated for 30 min with 25 μM bikinin at day 7; mock treatments were conducted in parallel. In the case of PPZ, seedlings were treated in 10 ml of a PPZ solution (end concentration on plate: 2 µM). Mock treatments were conducted in parallel. In vitro kinase assay: Glutathione S-transferase (GST)-BIN2 (Li & Nam, 2002) and GST-T2-TRS120 phosphovariants were expressed in Escherichia coli (Rosetta-gamiTM strain) under constant shaking for 20 h at 25°C or 20 h at 18°C, respectively. The expressed proteins were affinity purified with GST-tags. After sonication of the samples in 1x PBS, 1 mM PMSF, 1 mg/ml lysozyme and 1% Triton X-100, the bacterial cell rests were centrifuged for 30 min at 16,000 g. Supernatants were incubated for 2 h with Glutathione SepharoseR 4 Fast Flow Beads (GE Healthcare) while rotating. After washing the samples five times, the GST tagged proteins were eluted with 10 mM glutathione and concentrated by ultra-filtration.