This is a preprint version of our artical published in Journal of Proteomics Title: Arabidopsis proteome responses to the smoke-derived growth regulator karrikin by Jana Baldrianová, Martin Černý, Jan Novák, Petr L. Jedelský, Eva Divíšková and Břetislav Brzobohatý See Journal of Proteomics for the final version: doi:10.1016/j.jprot.2015.02.011 A preprint is an author's own write-up of research results and analysis that has not been peer-reviewed, nor had any other value added to it by a publisher (such as formatting, copy editing, technical enhancement etc.). 1 2 TITLE PAGE 3 4 Title: Arabidopsis proteome responses to the smoke-derived growth regulator 5 karrikin 6 7 Running head: 8 Karrikin-response proteome in Arabidopsis 9 10 Authors: 11 Jana Baldrianová1*, Martin Černý1*, Jan Novák1, Petr L. Jedelský2'1', Eva Divíšková1 and 12 Břetislav Brzobohatý1 13 (1) Laboratory of Plant Molecular Biology, Institute of Biophysics AS CR, v.v.i. and CEITEC 14 - Central European Institute of Technology, Mendel University in Brno, Zemědělská 1, CZ- 15 613 00 Brno, Czech Republic 16 (2) Department of Cell Biology, Faculty of Science, Charles University in Prague, Viničná 7, 17 CZ-128 43 Prague, Czech Republic 18 (t) Present address: Research Department, Faculty of Arts, Charles University in Prague, 19 Náměstí Jana Palacha 2, CZ-116 38 Prague, Czech Republic 20 21 *These authors contributed equally to this work. 22 Jana Baldrianová 51197@mail.muni.cz 23 Martin Černý martincernv83 @ gmail.com 24 Jan Novák novakhonza@ atlas.cz 25 Petr L. Jedelský petr.jedelský @natur.cuni.cz, petr.jedelský @ seznam.cz 1 1 Eva Divíšková diviskova.e@seznam.cz 2 Břetislav Brzobohatý brzoboha@ibp.cz 3 4 Corresponding author: 5 Name: Břetislav Brzobohatý 6 Address: Mendel University in Brno, Zemědělská 1, CZ-61300 Brno, Czech Republic 7 Phone: +420-545133359 8 Fax: +420-545133295 9 E-mail: brzoboha@ibp.cz 10 11 Number of figures: 6 12 Number of tables: 6 13 Supplementary tables: 4 14 Supplementary figures: 5 15 2 1 ABSTRACT 2 Karrikins are butenolide plant growth regulators in smoke from burning plant material that 3 have proven ability to promote germination and seedling photomorphogenesis. However, the 4 molecular mechanisms underlying these processes are unclear. Here we provide the first 5 proteome-wide analysis of early responses to karrikin in plants (Arabidopsis seedlings). 6 Image analysis of two-dimensionally separated proteins, Rubisco-depleted proteomes and 7 phosphoproteomes, together with LC-MS profiling, detected >1900 proteins, 113 of which 8 responded to karrikin treatment. All the differentially regulated proteins (except HSP70-3) are 9 novel karrikin-responders, and most are involved in photosynthesis, carbohydrate metabolism, 10 redox homeostasis, transcription control, proteosynthesis, protein transport and processing, or 11 protein degradation. Our data provide functionally complementary information to previous 12 identifications of karrikin-responsive genes and evidence for a novel karrikin signalling 13 pathway originating in chloroplasts. We present an updated model of karrikin signalling that 14 integrates proteomic data and is supported by growth response observations. 15 16 Keywords 17 smoke, proteome, growth regulators, karrikin, butenolide 3 1 1. Introduction 2 Plant growth and development processes are controlled by numerous signalling molecules, 3 both endogenous and exogenous. Some, including auxins, cytokinins, abscisic acid, ethylene 4 and gibberellins, have been known for decades, while others have only been discovered 5 recently and both their functions and signalling mechanisms are poorly understood. The latter 6 include karrikins, butenolides identified in smoke from burning plant material [1, 2]. These 7 heterocyclic molecules, containing a five-membered butenolide ring fused to a six-membered 8 pyran ring, promote germination of fire-following plant species. To date, six compounds with 9 similar action have been identified in smoke, KARi being the most effective [3-5]. Karrikins 10 also induce responses in the model plant Arabidopsis, notably stronger promotion of seed 11 germination than gibberelins, 1-aminocyclopropane-l-carboxylic acid and epi-brassinolide 12 [6], and seedling photomorphogenesis, including inhibition of hypocotyl elongation and 13 cotyledon expansion [7, 8]. Relatively little is known about karrikin signalling, but it has 14 apparent similarity to signalling mediated by the structurally related plant hormones 15 strigolactones. A proposed model summarized by Waters and co-workers [9] postulates that 16 karrikin binding may induce conformational changes in a/|3-fold hydrolase KAI2 and its 17 association with MAX2, an F-box component of E3 ubiquitin-protein ligase, thereby targeting 18 signalling repressor(s) for degradation. SMAX1 and SMAXl-like proteins act downstream of 19 MAX2 and could be the hypothetical repressors. Further, karrikin signalling requires HY5 for 20 full photomorphological responses, induces auxin-response genes and recruits gibberellins by 21 increasing transcription of gibberellin oxidases (GA3oxl, 2). The involvement of proteasome- 22 mediated signalling and similarity to strigolactone signalling indicates that proteome 23 dynamics could mediate at least some karrikin responses. However, no proteome-wide 24 analysis of karrikin signalling has been previously reported. We recently showed that state-of- 25 the-art proteomic analyses can elucidate early links between plant hormone cytokinin 4 1 signalling and temperature perception [10-12]. We have now applied this approach to identify 2 early karrikin-response proteins and refine the emerging model of karrikin action in 3 Arabidopsis. 5 1 2. Materials and methods 2 2.1. Plant material, growth conditions and KARi treatment for proteomic experiments 3 Unless specified otherwise, seeds of Arabidopsis thaliana ecotype Columbia (Col-0) were 4 surface-sterilized and sown on Uhelon 120T (Silk & Progress, Brnenec, Czech Republic). 5 Meshes were placed on Murashige and Skoog medium (pH 5.7) solidified with 1% (w/v) 6 agar, stratified at 4 °C for 48 h and cultivated at 21°C/19°C day/night temperatures, with a 16 -2 -1 7 h photoperiod (90 umol m" s" light intensity) for 7 d in an AR-36L growth chamber 8 (Percival, Perry, IA, USA). For proteome profiling, Uhelon mesh supporting 7-day-old 9 seedlings was transferred onto liquid MS medium supplemented with mock (ddHiO) or 1 uM 10 KARi (kindly provided by Dr. Martin Vlk, Czech Technical University, Prague, Czech 11 Republic) and incubated for 15 min. Seedlings were then rapidly harvested, dried, flash - 12 frozen and ground in liquid nitrogen. For morphological and chlorophyll fluorescence 13 analysis, a Uhelon mesh supporting 6-day-old seedlings was transferred onto liquid MS 14 medium supplemented with mock (ddHiO) or 1 uM KARi and cultivation was continued for 15 an additional 24 h under the environmental conditions specified above. Seedlings were 16 divided into two sets for determining chlorophyll fluorescence parameters and morphological 17 variables, root length and cotyledon area (using ImageJ software; http://rsbweb.nih.gov/ij/). 18 The whole experiment was performed in three biological replicates. 19 20 2.2. Protein extraction, 2-DE analysis and protein identification 21 Arabidopsis proteome and sub-proteomes were extracted, prepared and analysed essentially 22 as previously described. Briefly, total protein was extracted by acetone/trichloroacetic acid 23 extraction [10, 13, 14], a Rubisco-depleted proteome was prepared [11, 15], and 24 phosphoroteins were enriched [10]. Portions of each extract, 500 or 150 ug, were loaded onto 25 18 or 7 cm IPG strips (Bio-Rad, http://www.bio-rad.com/), respectively, isoelectrically 6 1 focused (linear pH gradient, 4-7), and subsequently resolved by SDS-PAGE. Gels were 2 stained with colloidal Bio-Safe Coomassie G-250 (Bio-Rad), digitally imaged and analysed 3 using Decodon Delta 2D software (http://www.decodon.com). Responses to KARi treatment 4 of proteins corresponding to detected spots were deemed significant if there was an absolute 5 KARi/mock spot volume ratio >1.4, with t-test p value<0.05, and similar profiles in two 6 biological replicates of the total protein extract and Rubisco-depleted proteome (three 7 technical replicates per sample), or three biological replicates for phosphoproteome 8 comparison (two technical replicates per sample). Selected protein spots were digested with 9 trypsin. The dried tryptic peptides were each dissolved in 10 ul of 0.1% trifluoroacetic acid 10 and purified using ZipTip C18 tips and eluted directly on sample plate with 10 mg ml"1 11 CHCA in 50% v/v acetonitrile and 0.1% trifluoroacetic acid. Spectra were acquired using 12 4800 Plus MALDI TOF/TOF analyzer (AB Sciex) equipped with a Nd:YAG laser (355 nm) 13 with firing rate 200 Hz. All spots were measured in MS mode and then up to ten strongest 14 precursors were selected for MS/MS which was performed with 1 kV collision energy and 15 operating pressure of collision cell set to 10"6 Torr. MS and MS/MS spectra were searched by 16 local Mascot v. 2.1 (Matrix Science) against TAIR10 database of Arabidopsis protein 17 sequences. Database search criteria were as follows: enzyme - trypsin; taxonomy - 18 Arabidopsis thaliana; fixed modification - carbamidomethylation; variable modification - 19 methionine oxidation; peptide tolerance - 80 ppm, allowed one missed cleavage; MS/MS 20 tolerance - 0.2 Da. 21 22 2.3. LC-MS proteome profiling 23 Quantitative proteomic analyses were also performed using a gel-free shotgun protocol based 24 on nano-HPLC and MS/MS. Briefly, two independently grown replicates, each consisting of 25 approximately 300 Arabidopsis seedlings cultivated as described above, were extracted by 7 1 acetone/TCA and phenol extraction then digested in solution with immobilized trypsin beads 2 (Promega, http://www.promega.com/). The resulting peptides were desalted, dried and 3 dissolved in 0.5% (v/v) formic acid in 5% (v/v) acetonitrile, then analysed by nanoflow C18 4 reverse-phase liquid chromatography using a 15 cm Ascentis Express Column (0.1 mm inner 5 diameter; Sigma-Aldrich) and a Dionex Ultimate 3000 RSLC nano UPLC system (Thermo, 6 www.thermoscientific.com) directly coupled to a CaptiveSpray nanoESI source (Bruker) and 7 an UHR maXis impact q-TOF mass spectrometer (Bruker, www.bruker.com). Peptides were 8 eluted with up to a 180-min, 4% to 40% acetonitrile gradient. Raw files obtained from the MS 9 analysis were analysed by Profile Analysis 2.1 (Bruker) and MS precursors with significant 10 differences (absolute ratio >1.5, with t-test p-values <0.05) were targeted and identified in 11 consecutive MS/MS analyses. MS/MS spectra were acquired in an intensity dependent mode 12 at a rate of 2-20 Hz with a maximum of 20 precursor ions and a MS spectra rate of 2 Hz. Data 13 from MS/MS data-dependent measurements were processed by DataAnalysis 4.1 (Bruker) 14 and searched against the TAIR10 Arabidopsis database using the Mascot 2.4 (Database search 15 criteria: trypsin; variable modifications - methionine oxidation, NQ deamidation, ST 16 phosphorylation; peptide tolerance - 10 ppm; allowed one missed cleavage; MS/MS tolerance 17 - 0.06 Da) and Bruker's ProteinScape percolator algorithms (target FDR<1%) to identify 18 source proteins (using high-confidence peptides, p <0.05 with at least one distinct proteotypic 19 peptide per protein). Quantitative differences were further manually validated by comparing 20 corresponding peptide ion signal peak areas in Skyline 1.4 (MacCossLab Software; 21 https://skvline.gs.washington.edu). 22 23 2.4. Proteomic data analysis 24 Information about functions of identified proteins was collected from available literature and 25 the UniProt, UniGene (http://www.ncbi.nlm.nih.gov/unigene), TAIR (http://www. 8 1 Arabidopsis.org), KEGG (http://www.genome.jp/kegg/pathway.html), conserved domains 2 (http://www.ncbi.nlm.nih.gov/Structure/index.shtml), and homology 3 (http://blast.ncbi.nlm.nih.gov/Blast.cgi) databases. Protein-protein interactions were evaluated 4 by String 9.1 (http ://string-db .org/, [16]). Overrepresentation of Gene Ontology categories 5 was analysed by the BioMaps module of VirtualPlant 1.3 (http://virtualplant.bio.nvu.edu/cgi- 6 bin/vpweb/, [17]), using the Arabidopsis thaliana TAIR 10 genome, TAIR/TIGR GO 7 biological process assignments, and p-values of over-representation < 0.05. OriginPro 9.0 was 8 then used for PC A analysis. In the presentation of results, numbers following named proteins 9 are spot numbers, while the alphanumerical designations L and P with numbers (e.g. L23 and 10 P58) are codes assigned to differentially expressed proteins detected in the LC-MS and 11 phosphoproteome analyses, respectively. 12 9 1 3. Results 2 3.1. Proteomic responses to KARi-treatment 3 To obtain insights into early proteomic responses to karrikin, 7-day-old Arabidopsis seedlings 4 were treated with 1 uM KARi for 15 minutes. The experimental design is outlined in Figure 5 1. First, whole proteome changes were monitored by 2-DE. To increase coverage of 6 seedlings' proteomes, we combined standard denaturative acetone/TCA whole protein 7 extraction with native extraction followed by immunodepletion of Rubisco or affinity-based 8 isolation of phosphoproteins. The isolated proteins were subjected to 2-DE (Fig. 2). Image 9 analysis of the resulting proteome maps yielded 114 spots with at least 1.4-fold absolute 10 variation between control and karrikin-treated samples in all biological replicates (P<0.05; 11 Fig. 2), which were subjected to protein identification. Altogether, 74 proteins were identified 12 in the 85 spots, including three protein mixtures, by MALDI TOF/TOF MS analysis followed 13 by Mascot database searches against the TAIR 10 database (Additional file 1: Table SI). Of 14 these proteins, 63 and 17 were detected in comparisons of the total protein extract with the 15 Rubisco depleted sub-proteome and phosphoproteome, respectively. 16 To complement results from the protein-based 2-DE analysis (which provides 17 excellent qualitative data) we applied a more sensitive peptide-based quantitative approach: 18 LC-MS shotgun proteomic analysis of total soluble proteins. In total, we quantified relative 19 peptide abundances in 1890 proteins, detected in at least nine repeated LC-MS experiments, 20 represented by more than 5600 peptides with 3.3 peptides per protein on average and average 21 sequence coverage of 12.3%. Comparisons after Skyline validation of MS spectra and 22 elimination of low-confidence peptides (e.g. peptides susceptible to non-enzymatic 23 modifications [18]) revealed 49 differentially regulated proteins quantified by 109 unique 24 peptides (for details, see Additional files 2, 3). 10 1 Combining the 2-DE and LC-MS datasets revealed limited overlaps of the 2 differentially regulated proteins/proteoforms in the sub-proteomes. In total, we detected 113 3 karrikin-responsive proteins, 15 present in several proteoforms. Only 11 of these differentially 4 regulated proteins were detected in multiple subproteome fractions, most of which probably 5 represent different proteoforms of the same protein (Additional file 4: Table S4). Analysis of 6 the Rubisco-depleted proteome further validated results from total protein extract 2-DE and 7 detected 9 additional karrikin-responsive proteins (Supplementary Fig. SI). 8 Determination of subcellular locations of the identified differentially regulated 9 proteins, using the SUBA database (http://suba.plantenergy.uwa.edu.au/; [19]), indicated that 10 62 (50%) and 32 (26%) are chloroplastic and cytosolic, respectively. Corresponding 11 proportions for all proteins identified by LC-MS were both ca. 30% (Table S3). Thus, 12 chloroplastic proteins are significantly overrepresented in the karrikin-responsive proteome. 13 14 3.2. Novel links in karrikin responses 15 Our proteomic analyses detected only one Arabidopsis gene identified as being karrikin- 16 responsive in previous transcriptomic analyses (Heat shock 70kDa protein 3; L23, decreased 17 after KARi-treatment). This protein is induced during germination [20] and is involved in 18 protein-protein interactions with several partners, including the deubiquitinating enzyme 19 AMSH3, which is involved in intracellular trafficking [21], and a ubiquitination target [22]. 20 Further, its close human homolog HSPA8 (>75% identity) participates in transcription control 21 [23]. The remaining 112 differentially regulated proteins we detected are novel KARi- 22 responsive factors. However, our protein-protein interaction network and functional 23 enrichment of metabolic pathways analyses showed that significant proportions of these 24 proteins have functional matches or complement previously identified karrikin-responsive 25 genes. These are proteins involved in carbon fixation and photosynthesis, RNA metabolism, 11 1 lipid metabolism and glutathione metabolism. Categories 'Citric acid cycle', 'Protein 2 transport', proteasome mediated processes, and ribosome components were highlighted only 3 in karrikin-response proteome (Fig. 3). All identified karrikin-responsive proteins were 4 divided according to their predominant function into the following categories: Photosynthesis 5 and carbohydrate metabolism; Redox homeostasis; Transcription; Proteosynthesis, protein 6 transport and processing; and Protein degradation (Tables 1-5, respectively). The remaining 7 categories (each represented by less than five proteins) are grouped in Table 6. 8 9 3.3. Proteins involved in photosynthesis and carbohydrate metabolism are rapidly 10 regulated in karrikin responses 11 The most significant overlap between known karrikin-responsive genes and the karrikin- 12 responsive proteins we identified was among those involved in carbohydrate metabolism (7 13 genes and 17 proteins; Fig. 3). The significantly regulated proteins included 23 enzymes and 14 seven structural components of the photosynthetic apparatus, some present in multiple 15 proteoforms (Table 1). KARi-treatment increased levels of enzymes involved in chlorophyll 16 biosynthesis (protoporphyrinogen oxidase 1; porphobilinogen deaminase). However, it 17 appeared to have generally negative effects on photoassimilatory proteins, including: Calvin 18 cycle enzymes (Rubisco - 18IL44IP18IP21IP31; chaperonin 60, required for Rubisco folding - 19 46; Rubisco activase - P22IP30; glyceraldehyde-3-phosphate dehydrogenase - L6IP25IP26; 20 fructose-bisphosphate aldolase - L13IL34; transketolase - 72; phosphorybulokinase - P20) 21 and enzymes involved in both downstream and upstream metabolic pathways of carbon 22 fixation (carbonic anhydrase) and carbohydrate metabolism (aldolase 1-epimerase, 23 fructokinase, and nucleotide-sugar biosynthesis enzymes). Similarly, it reduced levels of 24 constituents of PS I (light harvesting complex - 36; chlorophyll binding protein - 33; PS I 12 1 reaction centre - L11IL33), PSII (Oxygen-evolving enhancer proteins - L26IP14IL29) and 2 ATP synthase (L43). 3 4 3.4. Proteasome-mediated signalling components in the KARi responsive proteome 5 KAI2, MAX2 and ubiquitin-proteasome signalling is the only reported molecular mechanism 6 of karrikin perception. Thus, proteins involved in ubiquitin-dependent processes in the 7 karrikin-responsive proteome should be particularly interesting. We were not able to detect 8 KAI2 (At4g37470), nor MAX2 on protein level. However, KARi treatment increased levels 9 of a proteasome subunit (76) and two deubiquitinating enzymes (9, 82) (Table 2). One, 10 ubiquitin carboxyl-terminal hydrolase 14, is important for development and its mutation 11 results in enlarged endosperm nuclei and defective embryos [24]. The treatment also 12 upregulated the ubiquitin receptor protein RAD23, which reportedly interacts with a 13 proteasome docking subunit and participates in recognition of ubiquitinated substrates [25]. 14 Products of proteasome activity are predominantly peptides up to 12 amino acids long that are 15 subsequently processed by peptidases. Accordingly, we detected significant changes in 16 abundance of two dipeptidases (L38, L49) and one tripeptidase (L30). Our data also indicate 17 more potential links with the ubiquitin-dependent pathway, e.g. the changes in levels of Heat 18 shock 70kDa (ubiquitinated, interacts with deubiquitinating enzyme) described above, but the 19 evidence is less conclusive. An alpha/beta domain-containing hydrolase with an uncertain 20 function and expected mitochondrial location (L31) was also upregulated. However, sequence 21 comparison does not indicate that the hydrolase is a mitochondrial counterpart of KAI2 22 (identity <12%). 23 24 3.5. Transcription regulators involved in KARi responses 13 1 Previous transcriptomic analyses have detected five KAR-responsive Arabidopsis genes 2 involved in transcription control, but the proportion of genes covered by the analyses (and 3 proteins covered by our analyses) is too low to pinpoint any significant over-representation in 4 this category. However, our supervised analyses detected upregulation of several relevant 5 proteins (Table 3), including two (DNA topoisomerase-like protein, 48, and Nucleosome 6 assembly protein, 10) indicating that KAR-treatment induces DNA uncoiling. Another three 7 proteins are classified as mediators (19, 53) or a probable mediator (L35) of RNA polymerase 8 II, which directly participates in transcription regulation. Further, all these mediators share 9 sequence similarity with Heat shock 70kDa protein 3 (> 60% for the first two and >92% for 10 L35) which is also apparently a transcription mediator (Supplementary Fig. S2). Furthermore, 11 KARi-treatment affected RNA processing, or at least expression of two RNA helicases — 14 12 (expected to be involved in nonsense-mediated mRNA decay and ribosome biogenesis) and 13 56 (pre-mRNA splicing, mRNA export from nucleus) — and ribonucleoprotein At2g37220. 14 At2g37220 was significantly affected by KARi-treatment in all analysed proteome fractions 15 (38IL17IP19), contains an RRM domain and plays an expected role in chloroplastic RNA 16 processing. We also detected posttranslational modification(s) in Cold shock protein 2 (59 - 17 increased, 77 - decreased), an RNA chaperone that is regulated by cold and developmental 18 signals [26]. 19 20 3.6. The early karrikin-response proteome regulates proteosynthesis 21 Translation regulates all physiological processes, directly or indirectly. Accordingly, we 22 detected 28 karrikin-responsive proteins (35% of all differentially expressed proteins) 23 involved in ribosome assembly (26IL4IL14IL15IL18IL22IL24IL27), translation initiation 24 (75IL39IP1IP12), aminoacyl t-RNA synthesis (54), elongation (5I13I67IP8IP28), and protein 25 processing and transport (7I8I12I46I57IL25IL32IL41IL45IL46). Overall, KARi-treatment 14 1 seemed to inhibit proteosynthesis (at least cytosolic proteosynthesis) and related processes 2 (Table 4). However, the responses are probably complex. For example, changes in ribosome 3 composition may have severe effects on plant growth and development [27]. Mutant 4 phenotypes are known for two KAR-responsive ribosomal proteins identified here: 50S 5 ribosomal protein Lll (slow growth, pale green; [28]) and 60S ribosomal protein L23 6 (embryo defective; [29]). Since ribosomal proteins have been implicated in auxin and 7 cytokinin signalling [11, 30, 31], some KAR-effects are probably results of stimulation or 8 interference with ribosome-mediated signalling of endogenous hormones. 9 10 3.7. Similarities in response to plant hormones and KAR treatment 11 Karrikin is not recognized as a plant hormone because it may not be endogenously 12 synthesised, but since it has similar effects to known plant hormones we compared GO- 13 category KARi responses with those elicited by abscisic acid, auxin (IAA), brassinosteroids 14 (BR), cytokinin (CK), ethylene, jasmonic acid (JA), gibberellins (GA) and salicylic acid (SA) 15 (Fig. 4). Our analysis covered 128 karrikin-responsive genes and 113 proteins, and 3453, 16 1245, 861, 672, 973, 348, 778 and 541 TAIR-annotated genes linked to the respective 17 hormones [32, 33]. Altogether, over 900 GO categories are significantly overrepresented in 18 sets of genes or proteins responding to at least one of the listed substances. The diversity of 19 processes apparently up- or down-regulated by them (number of overrepresented GO 20 categories) decreased in the order JA>SA>IAA>ethylene>ABA>GA>KAR>CK>BL. The 21 GO category 'Response to karrikin' (which does not yet include proteins identified here) is 22 not significantly enriched in sets that reportedly respond to plant hormones, unsurprisingly as 23 no responses to any of the listed phytohormones have been detected for >50% of karrikin- 24 responsive genes. Similarly, only 28 of the proteins identified here reportedly respond to 25 phytohormones: 15, 10, 9, 2 and 1 to ABA, JA, SA, IAA and BR, respectively. Nevertheless, 15 1 our PCA indicates that the differences between hormonal and KARi responses is not 2 dramatic. The first dimension, covering nearly 67% of total variability, clusters KAR- and 3 BR-responsive proteins in the same group. Unfortunately, we could not include strigolactone 4 in the analysis, as there are too few annotated strigolactone-responsive genes. 5 6 3.8. Fluorescence measurements suggest photosynthesis is modulated in karrikin 7 responses 8 To obtain further insights into karrikin effects on photosynthetic processes we measured 9 chlorophyll fluorescence parameters of Arabidopsis seedlings cultivated on medium 10 supplemented with 1 |jM KARi for the last 24 h of a 7d cultivation period (Supplementary 11 Fig. S3, S4). Then we measured the seedling's cotyledon surface area, root length and 12 maximum PSII quantum yield in a dark-adapted state (Fv/Fm) to acquire information about 13 photosynthetic processes following light adaptation (Supplementary Fig. S4). Calculated 14 Fv/Fm ratios did not differ between karrikin- and mock-treated seedlings, indicating that 15 properties of PSII and associated processes were not significantly affected. However, as 16 already described, the proteomic analyses indicate that KARi treatment reduced levels of PSI 17 constituents and enzymes involved in carbon photoassimilation. This is likely to repress 18 processes required for efficient light adaptation and hence the quantum efficiency of PSII 19 (OPSII). Accordingly, 24 hours after transfer to KARi-supplemented medium we detected a 20 slight decrease in this parameter. We also observed an increase in NPQ (non-photochemical 21 quenching), which corresponds to thermal energy dissipation and may correlate with a 22 decrease in energy transfer to PSI. However, the changes in the photosynthetic parameters 23 were not statistically significant at P<0.05. 24 16 1 4. Discussion 2 4.1. Profiling early proteomic responses to karrikin in Arabidopsis 3 Karrikin responses have been previously examined in bioassays, selected mutants and 4 transcriptomic analyses [3, 6-8, 34-39], but not (to our knowledge) in proteome-wide 5 analyses. However, protein abundance is not dependent only on transcription, and 6 posttranslational modifications can rapidly activate or inactivate enzymes without degradation 7 or de novo synthesis (e.g. [40, 41]). Thus, proteome analysis can complement transcriptomic 8 analysis and extend insights into molecular processes. Karrikins were discovered as 9 germination-promoting substances, but their effect is not limited to this developmental stage. 10 They also promote cotyledon expansion and inhibit hypocotyl elongation of Arabidopsis 11 seedlings [8], eliciting responses at nanomolar to micromolar concentrations [4], like plant 12 hormones. This prompted us to employ the approach we established for identifying cytokinin- 13 responsive proteins [10, 12], combining affinity depletion of Rubisco and phosphoproteome 14 isolation with 2-DE - MALDI TOF/TOF and LC-MS identification of differentially regulated 15 proteins. Preliminary experiments confirmed that micromolar KARi elicits reported growth 16 responses [8] under experimental conditions similar to those applied in our proteomic 17 experiments, promoting both root and cotyledon growth when applied for the last 24 h of a 7- 18 day cultivation period (Fig. 5). 19 20 4.2. Down-regulation prevails in proteomic responses to karrikin 21 Karrikin treatment reportedly up-regulates transcription of most (>80%) differentially 22 expressed genes [8], but most (69%) early karrikin-response proteins we detected were down- 23 regulated: 97% in the phosphoprotein-enriched fraction and -62% in sets detected in both the 24 LC-MS and total protein 2-DE (including Rubisco-depleted fraction) analyses. Interestingly, 25 we observed similar proportions in analyses of early cytokinin (-67%) and temperature-shock 26 (-62%) response proteins [10, 12]. We hypothesise that processes that reduce 17 1 protein/proteoform abundance may be preferentially employed in rapid responses to stimuli 2 because they are faster than protein synthesis. 3 4 4.3. Plastids - the site of missing links in karrikin signalling? 5 Early karrikin-response proteins we detected in seedlings are localized mainly to the 6 chloroplast (50% of the set). The plastidic genome of vascular plants has lost most of its 7 original content and encodes fewer than 100 open reading frames [42, 43]. Thus, plastid 8 development and function are highly dependent on the nucleus and largely under its control. 9 However, plastids also generate signals that modulate nuclear gene expression via several 10 pathways, including redox signalling [42]. Accordingly, we identified 11 differentially 11 expressed proteins involved in redox homeostasis (Table 2). Of these, six have a chloroplast 12 location, including monothiol glutaredoxin-S12 (L12), thioredoxin-like protein CDSP32 (25) 13 and thioredoxin Ml (LI). Thioredoxins (Trx) participate in day/night metabolism switches, 14 including light-dependent regulation of the Calvin-Benson cycle, but they also apparently 15 participate in protein targeting, cell-to-cell trafficking and gene regulation [44] and CK 16 responses [11, 45]. The m-type of Trx is also essential for chloroplast development [46]. 17 Thus, Trx could be the missing link between light signalling and transcription factor HY5. 18 19 4.4. Posttranslational control in KARi responses 20 Our 2-DE data provide several indications that targeted proteasome-mediated protein 21 degradation is not the only posttranslational mechanism involved in karrikin signalling. First, 22 differences between theoretical and observed pI/MW values indicate that >50% of 23 significantly regulated proteins detected in our 2-DE analysis are probably posttranslationally 24 modified proteoforms (Additional file 1: Table SI). Identification and characterization of 25 these modifications detected in the total protein extract/Rubisco depleted proteome 18 1 comparison is beyond the scope of this work. However, our targeted phosphoproteome 2 analysis indicates that changes in phosphorylation status are involved, at least indirectly. We 3 have previously demonstrated that the phosphoprotein enrichment protocol we employed is 4 fairly specific by validation with phospho-specific staining and MS/MS sequencing [10, 12]. 5 Here, we supplemented our previous validation by Western blot analysis (Supplementary Fig. 6 S5). We were able to follow dynamics in phosphoproteome, but the biological relevance of 7 detected phosphorylations mostly remains to be elucidated. For some of them, even the 8 function of a protein itself is not clear. Phosphorylation could be involved in a direct karrikin 9 signalling mechanism, but with the combined sensitivity of 2-DE analysis and MALDI-MS 10 we detected regulatory phosphorylation for only 17 proteins, all of which seem to be 11 influenced indirectly. Literature and a homology search indicate that one detected 12 dephosphorylation (of Rubisco; P18, P21, P31) would be inhibitory [47] while another (of the 13 cytosolic enzyme triosephosphate isomerase; P29) would be activating [48], which correlates 14 with observed regulations in our dataset. Rubisco activase (P22, P30) has multiple 15 phosphorylation sites, some also with a potential on/off function [49]. Elongation factor Tu 16 (P8) is one of four detected phosphoproteins involved in proteosynthesis. Phosphorylation of 17 this chloroplastic protein prevents ternary complex formation [50] and its regulation in 18 response to karrikin likely represents an increased proteosynthesis in chloroplasts. The last 19 phosphorylation which should be discussed is that of chloroplastic protein glyceraldehyde-3- 20 phosphate dehydrogenase (GAPDH; P25, P26). Although the prominent role of GAPDH is 21 commonly known to be in carbohydrate metabolism, its homolog also participates in 22 intracellular trafficking. Tisdale (2002) reported that phosphorylation of GAPDH influences 23 microtubule dynamics in the early secretory pathway [51]. In this respect, we can note that 24 chloroplastic proteins like Trigger factor-like protein TIG (L41, protein exporter) or 19 1 Chaperone protein ClpCl (57) indicate that there are changes in chloroplast protein 2 trafficking after karrikin treatment. 3 4 4.5. Karrikin signalling - an updated model 5 Considerable time may be required for manifestation of transcriptional control at the protein 6 level. For example, heat stress induces significant increases in Hsp70 transcript levels, 7 peaking within minutes, but significant changes (gradual rises) in HSP70 protein levels only 8 occur after an hour [52]. Thus, we believe that the protein dynamics revealed by our data 9 cannot correlate with transcript levels and originate in posttranscriptional and/or 10 posttranslational control. This is consistent with recent indications that posttranscriptional, 11 translational, posttranslational and degradation govern protein concentrations at least as 12 strongly as transcription (see e.g. [53]). Proteasome-dependent degradation could explain 13 some observed karrikin responses, and even increases in protein abundance could result from 14 degradation of repressors. However, the sheer number of differentially expressed 15 chloroplastic proteins we detected indicates involvement of an alternative proteasome- 16 independent and chloroplast-based signalling pathway. We did not pinpoint a novel karrikin 17 receptor per se, but our data provide evidence for several novel signal transduction pathways. 18 We have already discussed karrikin-responsive proteins involved in ribosome biogenesis 19 (posttranscriptional control) and redox signalling that could participate in retrograde 20 chloroplast-to-nucleus signalling, and the whole concept is illustrated in Fig. 6. Further, we 21 observed a decrease in levels of phospholipase D (42), representing a link to calcium 22 signalling and crosstalk with endogenous hormones like ABA or CK [54, 55]. 23 24 4.6. Could inhibition of photosynthesis stimulate growth? 20 1 Karrikin treatment for 24 h increased root length and cotyledon area in Arabidopsis seedlings 2 (Fig. 5), in accordance with reported effects, but seemingly conflicting with indications from 3 our proteome analysis that it reduces photoassimilation. Furthermore, our fluorescence 4 measurements suggest that karrikin reduces photoassimilatory energy transfer and increases 5 thermal energy dissipation (although it does not affect PSII properties), which should reduce 6 photosynthetic efficiency. We hypothesize that for a limited period compensatory 7 mechanisms may be induced by such changes, including cotyledon expansion, which 8 increases plants' photosynthetic area. As karrikin signalling reportedly requires HY5 for full 9 photomorphological responses [8], we examined the abovementioned responses in a hy5 10 mutant, which responded largely as wild-type Col-0. Thus, HY5 function is likely not critical 11 for these responses (Supplementary Fig. S3). 12 13 5. Conclusions 14 We have pioneered a proteomic analysis of karrikin responses in the model plant Arabidopsis 15 thaliana. Combination of a traditional 2-DE approach with robust LC-MS analysis resulted in 16 identification of numerous novel karrikin-response proteins that provide novel targets for 17 detailed mechanistic studies using, e.g., mutants and transgenic plants. The data obtained 18 fundamentally deepen our understanding of karrikin roles in chloroplast functions. Further, 19 the comparative analysis provided novel indications of a chloroplast-based signalling pathway 20 that operates in parallel to KAI2-mediated karrikin signalling. 21 21 1 Author contributions 2 JB, MC, JN, PLJ and ED performed the experiments and analysed the data. BB and MC 3 conceived the study and wrote the manuscript. All the authors approved the final manuscript. 4 5 Competing interests 6 The authors declare that they have no competing interests. 7 8 Acknowledgement 9 We thank Dr. Martin Vlk for supplying KARi and Prof. Johannes van Staden for discussions 10 during preparatory phases of this work, which was supported by grants P305/12/2144 (CSF), 11 TE02000177 (TACR), 1M06030 (MEYS CR), MSM0021620858 (P.L.J.; MEYS CR), and 12 funds from the ERDF for 'CEITEC-Central European Institute of Technology' 13 (CZ. 1.05/1.1.00/02.0068). Access to the MetaCentrum computing facilities provided under 14 the Projects of Large Infrastructure for Research, Development, and Innovations program 15 (LM2010005), funded by the Ministry of Education, Youth, and Sports of the Czech Republic 16 is highly appreciated. MC. was supported by the Operational Program Education for 17 Competitiveness - European Social Fund (Project CZ.l.07/2.3.00/30.0017, for Postdocs in 18 Biological Sciences at MENDELU). 19 20 Appendix A. Supplementary data 21 The data sets supporting the results of this article are included within the article and its 22 additional files. 23 22 1 2 References 3 [1] Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. A compound from smoke that 4 promotes seed germination. Science 2004;305(5686):977. 5 [2] Van Staden J, Jager A, Light M, Burger B. Isolation of the major germination cue from 6 plant-derived smoke. South African J Bot 2004;70:654-659. 7 [3] Light M, Daws M, Van Staden J. Smoke-derived butenolide: towards understanding its 8 biological effects. South African J Bot 2009;75(1): 1-7. 9 [4] Chiwocha SD, Dixon KW, Flematti GR, Ghisalberti EL, Merritt DJ, Nelson DC, 10 Riseborough JM, Smith SM, Stevens JC. Karrikins: A new family of plant growth regulators 11 in smoke. Plant Science 2009; 177(4):252-256. 12 [5] Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. Identification of alkyl substituted 13 2 H-furo [2, 3-c] pyran-2-ones as germination stimulants present in smoke. J Agric Food 14 Chem 2009;57(20):9475-9480. 15 [6] Nelson DC, Riseborough J, Flematti GR, Stevens J, Ghisalberti EL, Dixon KW, Smith 16 SM. Karrikins discovered in smoke trigger Arabidopsis seed germination by a mechanism 17 requiring gibberellic acid synthesis and light. Plant Physiol 2009;149(2):863-873. 18 [7] Waters MT, Smith SM. KAI2- and MAX2-mediated responses to karrikins and 19 strigolactones are largely independent of HY5 in Arabidopsis seedlings. Mol Plant 20 2013;6(l):63-75. 21 [8] Nelson DC, Flematti GR, Riseborough J, Ghisalberti EL, Dixon KW, Smith SM. 22 Karrikins enhance light responses during germination and seedling development in 23 Arabidopsis thaliana. Proc Natl Acad Sci U S A 2010;107(15):7095-7100. 24 [9] Waters MT, Scaffidi A, Sun YK, Flematti GR, Smith SM. The karrikin response system of 25 Arabidopsis. Plant J 2014;79(4):623-631. 23 1 [10] Černý M, Dyčka F, Bobal'ova J, Brzobohatý B. Early cytokinin response proteins and 2 phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome 3 profiling. J Exp Bot 2011;62(3):921-937. 4 [11] Černý M, Kuklová A, Hoehenwarter W, Fragner L, Novák O, Rotková G, Jedelsky PL, 5 Žákova K, Smehilová M, Strnad M, Weckwerth W, Brzobohatý B. Proteome and metabolome 6 profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to 7 cytokinin down- and up-regulation. J Exp Bot 2013;64(14):4193-4206. 8 [12] Černý M, Jedelský PL, Novák J, Schlosser A, Brzobohatý B. Cytokinin modulates 9 proteomic, transcriptomic and growth responses to temperature shocks in Arabidopsis. Plant 10 Cell Environ 2014;37(7):1641-1655. 11 [13] Hradilová J, Rehulka P, Rehulková H, Vrbová M, Griga M, Brzobohatý B. Comparative 12 analysis of proteomic changes in contrasting flax cultivars upon cadmium exposure. 13 Electrophoresis 2010;31(2):421-431. 14 [14] Lochmanová G, Zdráhal Z, Konečná H, Koukalová S, Malbeck J, Souček P, Válková M, 15 Kiran NS, Brzobohatý B. Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: 16 a proteomic analysis. J Exp Bot 2008;59(13):3705-3719. 17 [15] Černý M, Skalák J, Kůrková B, Babuliaková E, Brzobohatý B. Using a commercial 18 method for Rubisco immunodepletion in analysis of plant proteome. Chemické listy 19 2011;105:640-642. 20 [16] Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, Lin J, 21 Minguez P, Bork P, von Mering C, Jensen LJ. STRING v9.1: protein-protein interaction 22 networks, with increased coverage and integration. Nucleic Acids Res 2013 ;41 (Database 23 issue):D808-15. 24 1 [17] Katari MS, Nowicki SD, Aceituno FF, Nero D, Keifer J, Thompson LP, Cabello JM, 2 Davidson RS, Goldberg AP, Shasha DE, Coruzzi GM, Gutierrez RA. VirtualPlant: a software 3 platform to support systems biology research. Plant Physiol 2010;152(2):500-515. 4 [18] Černý M, Skalák J, Cerna H, Brzobohatý B. Advances in purification and separation of 5 posttranslationally modified proteins. J Proteomics 2013;92:2-27. 6 [19] Heazlewood JL, Verboom RE, Tonti-Filippini J, Small I, Millar AH. SUBA: the 7 Arabidopsis Subcellular Database. Nucleic Acids Res 2007;35(Database issue):D213-8. 8 [20] Sung DY, Vierling E, Guy CL. Comprehensive expression profile analysis of the 9 Arabidopsis Hsp70 gene family. Plant Physiol 2001;126(2):789-800. 10 [21] Isono E, Katsiarimpa A, Müller IK, Anzenberger F, Stierhof Y, Geldner N, Chory J, 11 Schwechheimer C. The deubiquitinating enzyme AMSH3 is required for intracellular 12 trafficking and vacuole biogenesis in Arabidopsis thaliana. Plant Cell 2010;22(6):1826-1837. 13 [22] Saracco SA, Hansson M, Scalf M, Walker JM, Smith LM, Vierstra RD. Tandem affinity 14 purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis. Plant J 15 2009;59(2):344-358. 16 [23] Yahata T, de Caestecker MP, Lechleider RJ, Andriole S, Roberts AB, Isselbacher KJ, 17 Shioda T. The MSG1 non-DNA-binding transactivator binds to the p300/CBP coactivators, 18 enhancing their functional link to the Smad transcription factors. J Biol Chem 19 2000;275(12):8825-8834. 20 [24] Doelling JH, Yan N, Kurepa J, Walker J, Vierstra RD. The ubiquitin-specific protease 21 UBP14 is essential for early embryo development in Arabidopsis thaliana. Plant J 22 2001;27(5):393-405. 23 [25] Lin Y, Sung S, Tsai H, Yu T, Radjacommare R, Usharani R, Fatimababy AS, Lin H, 24 Wang Y, Fu H. The defective proteasome but not substrate recognition function is responsible 25 1 for the null phenotypes of the Arabidopsis proteasome subunit RPN10. Plant Cell 2 2011;23(7):2754-2773. 3 [26] Sasaki K, Kim M, Imai R. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 is a RNA 4 chaperone that is regulated by cold and developmental signals. Biochem Biophys Res 5 Commun 2007;364(3):633-638. 6 [27] Rosado A, Sohn EJ, Drakakaki G, Pan S, Swidergal A, Xiong Y, Kang B, Bressan RA, 7 Raikhel NV. Auxin-mediated ribosomal biogenesis regulates vacuolar trafficking in 8 Arabidopsis. Plant Cell 2010;22(1):143-158. 9 [28] Pesaresi P, Varotto C, Meurer J, Jahns P, Salamini F, Leister D. Knock-out of the plastid 10 ribosomal protein Lll in Arabidopsis: effects on mRNA translation and photosynthesis. Plant 11 J2001;27(3):179-189. 12 [29] Meinke DW, Meinke LK, Showalter TC, Schissel AM, Mueller LA, Tzafrir I. A 13 sequence-based map of Arabidopsis genes with mutant phenotypes. Plant Physiol 14 2003;131(2):409-418. 15 [30] Brenner WG, Schmulling T. Transcript profiling of cytokinin action in Arabidopsis roots 16 and shoots discovers largely similar but also organ-specific responses. BMC Plant Biol 17 2012;12:112. 18 [31] Rosado A, Li R, van de Ven W, Hsu E, Raikhel NV. Arabidopsis ribosomal proteins 19 control developmental programs through translational regulation of auxin response factors. 20 Proc Natl Acad Sci U S A 2012;109(48):19537-19544. 21 [32] Nemhauser JL, Hong F, Chory J. Different plant hormones regulate similar processes 22 through largely nonoverlapping transcriptional responses. Cell 2006;126(3):467-475. 23 [33] Goda H, Sasaki E, Akiyama K, Maruyama-Nakashita A, Nakabayashi K, Li W, Ogawa 24 M, Yamauchi Y, Preston J, Aoki K, Kiba T, Takatsuto S, Fujioka S, Asami T, Nakano T, 25 Kato H, Mizuno T, Sakakibara H, Yamaguchi S, Nambara E, Kamiya Y, Takahashi H, Hirai 26 1 MY, Sakurai T, Shinozaki K, Saito K, Yoshida S, Shimada Y. The AtGenExpress hormone 2 and chemical treatment data set: experimental design, data evaluation, model data analysis 3 and data access. Plant J 2008;55(3):526-542. 4 [34] Waters MT, Nelson DC, Scaffidi A, Flematti GR, Sun YK, Dixon KW, Smith SM. 5 Specialisation within the DWARF 14 protein family confers distinct responses to karrikins and 6 strigolactones in Arabidopsis. Development 2012; 139(7): 1285-1295. 7 [35] Soös V, Sebestyen E, Juhäsz A, Light ME, Kohout L, Szalai G, Tandori J, Van Staden J, 8 Baläzs E. Transcriptome analysis of germinating maize kernels exposed to smoke-water and 9 the active compound KAR1. BMC Plant Biol 2010; 10:236. 10 [36] Soös V, Sebestyen E, Juhäsz A, Pinter J, Light ME, Van Staden J, Baläzs E. Stress- 11 related genes define essential steps in the response of maize seedlings to smoke-water. Funct 12 Integr Genomics 2009;9(2):231-242. 13 [37] Nelson DC, Flematti GR, Ghisalberti EL, Dixon KW, Smith SM. Regulation of seed 14 germination and seedling growth by chemical signals from burning vegetation. Annu Rev 15 Plant Biol 2012;63:107-130. 16 [38] Nelson DC, Scaffidi A, Dun EA, Waters MT, Flematti GR, Dixon KW, Beveridge CA, 17 Ghisalberti EL, Smith SM. F-box protein MAX2 has dual roles in karrikin and strigolactone 18 signaling in Arabidopsis thaliana. Proceedings of the National Academy of Sciences 19 2011;108(21):8897-8902. 20 [39] Light ME, Burger BV, Staerk D, Kohout L, Van Staden J. Butenolides from plant- 21 derived smoke: natural plant-growth regulators with antagonistic actions on seed germination. 22 J Nat Prod 2010;73(2):267-269. 23 [40] Nelson CJ, Li L, Jacoby RP, Millar AH. Degradation rate of mitochondrial proteins in 24 Arabidopsis thaliana cells. J Proteome Res 2013;12(7):3449-3459. 27 1 [41] Černý M, Doubnerová V, Müller K, Ryšlavá H. Characterization of 2 phosphoenolpyruvate carboxylase from mature maize seeds: properties of phosphorylated and 3 dephosphorylated forms. Biochimie 2010;92(10): 1362-1370. 4 [42] Chi W, Sun X, Zhang L. Intracellular signaling from plastid to nucleus. Annual review of 5 plant biology 2013;64:559-582. 6 [43] Heidt HW, Piechulla B. Plant Biochemistry, 4th Edition. London: Academic Press; 2010. 7 [44] Meyer Y, Belin C, Delorme-Hinoux V, Reichheld J, Riondet C. Thioredoxin and 8 glutaredoxin systems in plants: molecular mechanisms, crosstalks, and functional 9 significance. Antioxid Redox Signal 2012; 17(8): 1124-1160. 10 [45] Bhargava A, Clabaugh I, To JP, Maxwell BB, Chiang Y, Schaller GE, Loraine A, Kieber 11 JJ. Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq 12 in Arabidopsis. Plant Physiol 2013;162(l):272-294. 13 [46] Wang P, Liu J, Liu B, Feng D, Da Q, Wang P, Shu S, Su J, Zhang Y, Wang J, Wang H. 14 Evidence for a role of chloroplastic m-type thioredoxins in the biogenesis of photosystem II in 15 Arabidopsis. Plant Physiol 2013;163(4):1710-1728. 16 [47] Kaul R, Saluja D, Sachar R. Phosphorylation of small subunit plays a crucial role in the 17 regulation of RuBPCase in moss and spinach. FEBS letters 1986;209(l):63-70. 18 [48] Lee D, Pan Y, Kanner S, Sung P, Borowiec JA, Chowdhury D. A PP4 phosphatase 19 complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination. Nat 20 Struct Mol Biol 2010;17(3):365-372. 21 [49] Boex-Fontvieille E, Daventure M, Jossier M, Hodges M, Zivy M, Tcherkez G. 22 Phosphorylation pattern of Rubisco activase in Arabidopsis leaves. Plant Biol (Stuttg) 23 2014;16(3):550-557. 28 1 [50] Alexander C, Bilgin N, Lindschau C, Mesters JR, Kraal B, Hilgenfeld R, Erdmann VA, 2 Lippmann C. Phosphorylation of elongation factor Tu prevents ternary complex formation. J 3 BiolChem 1995;270(24):14541-14547. 4 [51] Tisdale EJ. Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein 5 kinase CtA, and plays a role in microtubule dynamics in the early secretory pathway. Journal 6 of Biological Chemistry 2002;277(5):3334-3341. 7 [52] Palmblad M, Mills DJ, Bindschedler LV. Heat-shock response in Arabidopsis thaliana 8 explored by multiplexed quantitative proteomics using differential metabolic labeling. J 9 Proteome Res 2008;7(2):780-785. 10 [53] Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic 11 and transcriptomic analyses. Nature Reviews Genettics 2012;13: 227-232. 12 [54] Romanov GA, Kieber JJ, Schmülling T. A rapid cytokinin response assay in Arabidopsis 13 indicates a role for phospholipase D in cytokinin signalling. FEBS Lett 2002;515(l-3):39-43. 14 [55] Guo L, Mishra G, Markham JE, Li M, Tawfall A, Welti R, Wang X. Connections 15 between sphingosine kinase and phospholipase D in the abscisic acid signaling pathway in 16 Arabidopsis. J Biol Chem 2012;287(11):8286-8296. 17 18 19 29 1 Figure Legends 2 Fig. 1 - Experimental design. 3 4 Fig. 2 - Effects of KARi on the proteome and phosphoproteome of Arabidopsis seedlings. 5 Average two-dimensional gel electrophoresis maps of total protein extracts and RuBisCO- 6 depleted proteomes (A) and phosphoproteomes (B) of 7-day-old Arabidopsis seedlings treated 7 with karrikin/mock buffer for 15 min. Differentially regulated protein spots are indicated. See 8 Tables 1-6 and Additional files for detailed information on the corresponding identified 9 proteins. Proteins were separated in the first and second dimensions by IPG — 7 cm or 18 cm 10 strips, pH 4-7 - followed by 8-20% (7 cm) or 11% (18 cm) SDS-PAGE then visualized by 11 Bio-Safe Coomassie G250 staining. Isoelectric points (pi) and migrating positions of 12 molecular mass (kDa) markers are marked. For details see Materials and methods. Decodon 13 Delta 2D was used to compensate for the PAGE background and thus optimize the visibility 14 of all differentially regulated protein spots. 15 16 Fig. 3 - Protein-protein interaction network constructed using STRING (http://string-db.org; 17 [16]). Karrikin-responsive genes (TAIR, Nelson and co-workers [8]; blue) and proteins 18 identified here (brown) were analysed for protein-protein interaction networks. String analysis 19 indicates that two sets overlap in several categories (purple). Categories 'Flavonoid 20 biosynthesis' and 'Light induced processes' are extensively regulated according to the 21 transcriptomic data, while significant effects of karrikin on the TCA cycle, protein transport 22 and proteasome-mediated processes are apparent only in our proteomics data. 23 24 Fig. 4 - Functional classification of genes and proteins responsive to phytohormones and 25 karrikin. PCA analysis indicates that karrikin has distinct effects, with some similarities to 30 1 those of recognised plant hormones (ABA, abscisic acid; BR, brassinosteroids; CK, cytokinin; 2 ethylene; IAA, auxin; JA, jasmonic acid; GA, gibberellins; SA, salicylic acid). Although the 3 overlap between previously found karrikin-responsive genes (Ktair) and novel karrikin- 4 responsive proteins (Kprot) identified here is minimal (protein L23, Heat shock 70kDa protein 5 3), the similarity in over-represented GO categories cluster them in the same group. Data for 6 principal component analysis were mined from the TAIR database (9/2014) and large-scale 7 hormonal transcriptomic datasets [32, 33], followed by functional classification using 8 BioMaps [17]. GO categories of interest are marked. For details, see Materials and Methods. 9 10 Fig. 5 - Effects of prolonged KARi treatment on Arabidopsis seedlings. 11 Selected morphological parameters of 7-day-old Col-0 seedlings exposed for the last 24 h to 1 12 \xM KARi. Photographs of representative seedlings for each treatment are shown. Data shown 13 are means of two biological replicates, each with >40 seedlings per replicate, and standard 14 errors (SE; error bars). Asterisks indicate statistically significant (Student's t-test p<0.05) 15 differences. 16 17 18 Fig. 6 - Integration of karrikin-response proteins into a karrikin signalling model. 19 Proteasome-dependent signalling: Karrikin binding to KAI2 initiates a proteasome-dependent 20 cascade and regulates proteins involved in ubiquitination and a di/tripeptide cleavage (Table 21 5), accounting for observed reductions in abundance of proteins involved in transcription 22 control (Table 3). Rapid changes in the chloroplastic proteome indicate the presence of an 23 alternative signalling pathway originating in chloroplasts. Karrikin negatively affects proteins 24 involved in photosynthesis and subsequent carbon assimilation (Table 1). Consequently, 25 reductions in ATP fluxes from photosynthesis would likely be compensated (at least 31 1 temporarily) by increases in pyruvate dehydrogenase activity, TCA cycle fluxes and 2 respiration rates, accompanied by reductions in lipid biosynthesis (Table 6). Alterations in 3 photoassimilation modulate redox homeostasis (Table 2) and through retrograde signalling 4 may influence gene expression. Signalling from both pathways thus converges on 5 proteosynthesis and protein transport (Table 4). 6 7 8 9 32 1 2 Suppementary data 3 4 Table SI - Differentially regulated proteins detected by 2-DE-based analysis. 5 Table S2 - Differentially regulated proteins detected by LC-MS-based analysis. 6 Table S3 - Proteins identified in LC-MS based analysis. 7 Table S4 - Overlaps of differentially regulated proteins in sets analysed by 2-DE and LC-MS. 8 Figure SI. Differentially regulated protein spots revealed after Rubisco depletion. 9 Figure S2. Sequence analysis of HSP70 proteins. 10 Figure S3. Effects of prolonged KARi treatment on Arabidopsis seedlings. 11 Figure S5. Western blot detection of phosphorylated proteins. 12 33 Tables Table 1 - Proteins involved in photosynthesis and carbohydrate metabolism Spot/protein AGI Name Abundance no- KARi:mock 51 AT5G66530 Aldose 1-epimerase family protein 1 L43 ATCG00120 ATP synthase subunit alpha, chloroplastic II 22|P5 AT3G01500 Carbonic anhydrase 1 III! 33 AT1G29910 Chlorophyll a-b binding protein 2 1 23 AT5G61410 D-ribulose-5-phosphate-3-epimerase T 16 AT1G20020 Ferredoxin-NADP reductase T 30|L9 AT1G66430 Fructokinase •!■ >L1 •!■ ~L L6 AT1G42970 Glyceraldehyde-3-phosphate dehydrogenase i P25 AT1G42970 Glyceraldehyde-3-phosphate dehydrogenase i P26 AT1G42970 Glyceraldehyde-3-phosphate dehydrogenase i L26 AT5G66570 Oxygen-evolving enhancer protein 1-1 i AT3G50820 Oxygen-evolving enhancer protein 1-2 P14 AT5G66570 Oxygen-evolving enhancer protein 1-1 ii 44 AT1G06680 Oxygen-evolving enhancer protein 2-1 TT L29 AT4G05180 Oxygen-evolving enhancer protein 3-2 II L7 AT1G56190 Phosphoglycerate kinase 2 II 35 AT2G45790 Phosphomannomutase T P20 AT1G32060 Phosphoribulokinase 1 36 AT3G61470 Photosystem I light harvesting complex protein 1 L33 AT4G28750 Photosystem I reaction center subunit IV A II L11 AT2G20260 Photosystem I reaction center subunit IV B 1 L36 AT5G08280 Porphobilinogen deaminase TT L13 AT2G21330 Probable fructose-bisphosphate aldolase 1 II 28|L34 AT4G38970 Probable fructose-bisphosphate aldolase 2 Till L28 AT4G01690 Protoporphyrinogen oxidase 1 T L3 AT1G23740 Quinone oxidoreductase-like protein TT 18|L44 ATCG00490 Ribulose bisphosphate carboxylase large chain III P18 ATCG00490 Ribulose bisphosphate carboxylase large chain 1 P21 ATCG00490 Ribulose bisphosphate carboxylase large chain II P31 ATCG00490 Ribulose bisphosphate carboxylase large chain II P22|P30 AT2G39730 Rubisco activase ■!■ •!■1 •!■ •!■ 72 AT3G60750 Transketolase-1, chloroplastic i L20 AT2G45290 Transketolase-2, chloroplastic TT P29 AT3G55440 Triosephosphate isomerase, cytosolic 1 P27 AT3G29360 UDP-glucose 6-dehydrogenase 2 1 Spot/protein AG I Name Abundance no. KARi:mock 21 AT1G07890 L-ascorbate peroxidase II 64 AT4G08390 L-ascorbate peroxidase 1 L2 AT1G11840 Lactoylglutathione lyase-like protein II L8 AT1G63940 Monodehydroascorbate reductase TT L12 AT2G20270 Monothiol glutaredoxin-S12 T 49 AT1G65980 Peroxiredoxin-2B T 32 AT1G67280 Probable lactoylglutathione lyase/glyoxalase T 24 AT3G10920 Superoxide dismutase [Mn] T 25 AT1G76080 Thioredoxin-like protein CDSP32 TT L1 AT1G03680 Thioredoxin M1 II 43 AT2G17420 Thioredoxin reductase 2 1 Table 2 - Proteins involved in redox homeostasis Table 3 - Proteins involved in transcription Spot/protein AGI no. Name Abundance KARi:mock 59|77 L21 P9 48 L23 53 19 P2|P3 10 L35 AT4G38680 AT3G01540 AT5G11200 AT3G15950 AT3G 09440 AT5G28540 AT5G42020 AT1G73230 AT4G26110 AT5G02500 38|L17|P19 AT2G37220 Cold shock protein 2 tílii DEAD-box ATP-dependent RNA helicase 14 tt DEAD-box ATP-dependent RNA helicase 56 || DNA topoisomerase-like protein f Heat shock 70 kDa protein 3 | Mediator of RNA polymerase II transcription subunit 37a ü Mediator of RNA polymerase II transcription subunit 37f tt Nascent polypeptide-associated complex NAC ü\l Nucleosome assembly protein 1 -like 1 tt Probable mediator of RNA polymerase II transcription a subunit 37e Ribonucleoprotein At2g37220 tlili Table 4 - Proteins involved in proteosynthesis, protein transport and processing Spot/protein AGI Name Abundance KAR i: mock 57 AT5G50920 Chaperone protein ClpC1 T 46 AT1G55490 Chaperonin 60 subunit beta 1 1 L27 AT3G63140 Chloroplast stem-loop binding protein of 41 kDa a II 5 AT5G13650 Elongation factor family protein II 67 AT4G29060 Elongation factor Ts 1 13|P8 AT4G20360 Elongation factor Tu llll P12 AT1G10840 Eukaryotic translation initiation factor 3 subunit H II P1 AT1G26630 Eukaryotic translation initiation factor 5A-2 1 L32 AT4G24280 Heat shock 70 kDa protein 6 II L45 AT5G49910 Heat shock 70 kDa protein 7 II L46 AT4G37910 Heat shock 70 kDa protein 9 1 8 AT1G79920 Heat shock 70 kDa protein 15 1 L25 AT3G15520 Peptidyl-prolyl cis-trans isomerase CYP37 II 7 AT3G25230 Rotamase FKBP 1 II P28 AT1G57720 Probable elongation factor 1-gamma 2 1 54 AT5G52520 prolyl-tRNA synthetase 1 12 AT2G47470 Protein disulfide-isomerase like 2-1 1 26 AT5G24490 Putative 30S ribosomal protein 1 P15|P16 AT1G07140 Ran-binding protein 1 homolog a •!■ •!■1 •!■ •!■ L39 AT5G38640 Translation initiation factor elF-2B delta subunit-like protein rr 75 AT3G13920 Translational initiation factor 4A-1 T L41 AT5G55220 Trigger factor-like protein TIG TT L18 AT2G37270 40S ribosomal protein S5-1 II L24 AT3G11510 40S ribosomal protein S14-2 II L14 AT2G21580 40S ribosomal protein S25-2 TT L4 AT1G32990 50S ribosomal protein L11 TT L15 AT2G27720 60S acidic ribosomal protein P2-1 II L22 AT3G04400 60S ribosomal protein L23 1 Table 5 - Proteins involved in protein degradation Spot/protein AGI Name Abundance no- KARi:mock L49 AT4G30920 Leucyl aminopeptidase 3 T 11 AT4G38220 Peptidase M20/M25/M40 family protein T 39 AT5G38470 Ubiquitin receptor RAD23d II L38 AT5G36210 Putative S9 Tyrosyl aminopeptidase II 76 AT5G35590 Proteasome subunit alpha type-6-A T L30 AT4G20850 Tripeptidyl peptidase 2 II 82 AT1G51710 Ubiquitin carboxyl-terminal hydrolase 6 T 9 AT3G20630 Ubiquitin carboxyl-terminal hydrolase 14 1 Table 6 - Proteins involved in diverse cellular processes Spot/protein AGI Name Abundance no- KAR i: mock 71 AT2G38040 Acetyl-coenzyme A carboxylase carboxyl transferase [[ subunit alpha 55 AT5G59880 Actin depolymerizing factor 3 | 50 AT3G23810 Adenosylhomocysteinase 2 | L5 AT1G34430 At1g34430/F7P12_2 t L10 AT2G07698 ATP synthase subunit alpha, mitochondrial t 2 AT1G48030 Dihydrolipoyl dehydrogenase 1 | 3 AT3G48990 4-coumarate-CoA ligase-like 10 | L47 AT5G14910 || Heavy metal transport/detoxification superfamily protein L42 AT5G63890 Histidinol dehydrogenase t 15 AT1G53240 Malate dehydrogenase 1, mitochondrial t L40 AT5G41970 Metal-dependent protein hydrolase TT L37 AT5G26000 Myrosinase 1 || 4 AT5G11670 NADP-dependent malic enzyme 2 U 62 AT1G79690 Nudix hydrolase 3 I L16 AT2G28900 Outer envelope pore protein 16-1, chloroplastic || 42 AT3G15730 Phospholipase D alpha 1 || 56 AT2G35040 Phosphoribosylaminoimidazolecarboxamide | formyltransferase L19 AT2G38410 VHS and GAT domain-containing protein TÍ L31 AT4G24160 Hydrolase, alpha/beta fold family protein TT P24 AT2G38230 Pyridoxal biosynthesis protein PDX1.1 || 65 AT5G09650 Soluble inorganic pyrophosphatase 1 1 47 AT3G14540 Terpenoid synthase 19 1 68 AT1G13930 Uncharacterized protein T 70 AT2G37660 NAD(P)-binding Rossmann-fold-containing protein 1 L48 AT3G61260 Remorin-like protein T 66 AT1G20260 V-type proton ATPase subunit B3 1 Figurel KARi / mock treated Arabtdopsis seedlings Acetone/TCA precipitation Native extraction Total protein extract Rutxsco immunodepletion Phenol extraction Trypsin digest i IC-MS label-free profiling t 2 DE 2 DE \ - iJlÉl Image analysis LC-MS/MS identification Trypsin digest -1— 1 MAlDI-TOF/TOf MS analysis 1 Phosphoprotein enrichment 2-DE Identified KAR, responsive proteins Figure2 Figure3 Legend Protein transport Karr=km response Figure4 00 0 1 0.2 0 3 0,4 0.5 0.6 0 20 40 PC1 (667%) RESPIRAT [Fig. W JtoN [Table 1] [Table 2] N "4 [Table 4] ■ KAO- [Table 3]