Phosphoproteomics Reveals the Biosynthesis of Secondary

Subscriber access provided by BUFFALO STATE

Article

Phosphoproteomics reveals the biosynthesis of secondary metabolites in Catharanthus roseus under ultraviolet-B radiation Zhuoheng Zhong, Shengzhi Liu, Wei Zhu, Yuting Ou, Hisateru Yamaguchi, Keisuke Hitachi, Kunihiro Tsuchida, Jingkui Tian, and Setsuko Komatsu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00267 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 30, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Phosphoproteomics reveals the biosynthesis of secondary metabolites in Catharanthus roseus under ultraviolet-B radiation

Zhuoheng Zhong 1, 2), Shengzhi Liu 1), Wei Zhu 1), Yuting Ou 1), Hisateru Yamaguchi 3), Keisuke Hitachi 3), Kunihiro Tsuchida 3), Jingkui Tian 1, *), Setsuko Komatsu 2, *)

1

College of Biomedical Engineering & Instrument Science, Zhejiang University,

Hangzhou 310027, P. R. China 2

Faculty of Life and Environmental and Information Sciences, Fukui University of

Technology, Fukui 910-8505, Japan 3

Institute for Comprehensive Medical Science, Fujita Health University, Toyoake 470-

1192, Japan

*To whom correspondence should be addressed: Jingkui Tian, College of Biomedical Engineering & Instrument Science, Zhejiang University, Tianmushan Road 148, Hangzhou 310027, P. R. China. Tel: 86-57188273823, E-mail address: [email protected] Setsuko Komatsu, Faculty of Life and Environmental and Information Sciences, Fukui University of Technology, Gakuen 3-6-1, Fukui 910-8505, Japan. Tel: 81-766-29-2466. E-mail address: [email protected]

1 ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abbreviations: UV, ultraviolet; TDC, tryptophan decarboxylase; DAHP, 3-deoxy-Darabino-heptulosonate-7-phosphate; MAPK, mitogen-activated protein kinase; PolyMAC, polymer-based metal-ion affinity capture; LC, liquid chromatography; MS, mass spectrometry; PD, protein discoverer; FDR, false discovery rate; GC, gas chromatography; TOF, time of flight; FBPA, fructose-bisphosphate aldolase; TPI, triose phosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RuBisCO, ribulose biphosphate carboxylase/oxygenase; LSU, large subunit; SSU, small subunit; APX, ascorbate peroxidase; GR, glutathione reductase, PRX, peroxiredoxin; PR proteins, pathogenesis related proteins; CBB, Coomassie brilliant blue; ROS, reactive oxygen species


Page 2 of 48

Page 3 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT Ultraviolet (UV)-B radiation acts as an elicitor to enhance the production of secondary metabolites in medicinal plants. To investigate the mechanisms, which lead to secondary metabolites in C. roseus under UVB radiation, a phosphoproteomic technique was used. ATP content increased in the leaves of C. roseus under UVB radiation. Phosphoproteins related to calcium such as calmodulin, calcium-dependent kinase, and heat shock proteins increased. Phosphoproteins related to protein synthesis/ modification/ degradation and signaling intensively changed. Metabolomic analysis indicated that the metabolites classified with pentoses, aromatic amino acids, and phenylpropanoids, accumulated under UVB radiation. Phosphoproteomic and immunoblot analyses indicated that proteins related to glycolysis and the reactiveoxygen species scavenging system was changed under UVB radiation. These results suggest that UVB radiation activates calcium related pathway and reactive-oxygen species scavenging system in C. roseus. These changes lead to the upregulation of proteins which are responsible for the redox reactions in secondary metabolism and are important for the accumulation of secondary metabolites in C. roseus under UVB radiation.

Keywords: phosphoproteomics, Catharanthus roseus, ultraviolet B, photosynthesis, secondary metabolites



Introduction Catharanthus roseus, which produces secondary metabolites such as indole alkaloids, is the most extensively investigated medicinal plant. The alkaloids isolated from C. roseus are comprised of a group of 130 indole alkaloids, some of which exhibit great pharmacological activities, such as serpentine and ajmalicine.1 C. roseus remains as the only natural source for anti-cancer drugs such as vinblastine and vincristine.2 Their formation requires the coupling of monomeric alkaloid precursors, which are vindoline and catharanthine, to α-3’,4’-anhydrovinblastine.3 Several alkaloids can absorb ultraviolet (UV) B light and serve putatively to protect plant from harmful radiation.4 UVB acts as an elicitor to enhance the biosynthesis of alkaloids such as catharanthine and vindoline in suspension cultures of C. roseus.5 Understanding of UVB response in C. roseus provides important insights for improving the alkaloid content in medicinal plants. In order to clarify the mechanism of UVB response in C. roseus, the effect on transcripts levels was intensively analyzed. Ouwerkerk et al.6 reported that UVB induced the mRNA expression of strictosidine synthase and tryptophan decarboxylase (TDC). Ramani et al.7 revealed that the transcript of 3-deoxy-D-arabino-heptulosonate7-phosphate (DAHP) synthase increased in suspension-cultured cells under UVB radiation. In addition, Nishanth et al.8 indicated that the gene expression levels of TDC, a senescence-associated gene, strictosidine synthase, peroxidase 1, and superoxide dismutase were upregulated, while those of geraniol-10-hydroxylase, deacetylvindoline4-O-acetyltransferase, and secologanin synthase genes were downregulated under UV treatment. These studies suggested that indole-alkaloid metabolism is affected by UV radiation, especially UVB. Pretreatment of suspension-cultured cells of C. roseus with inhibitors of calcium fluxes/kinases blocked the production of catharanthine and inhibited the gene 4 ACS Paragon Plus Environment

Page 4 of 48

Page 5 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


expression of strictosidine synthase, TDC, and DAHP synthase under UVB radiation.7,9 Raina et al.10 isolated a mitogen-activated protein kinase (MAPK) gene from C. roseus and proved that MAPK induced auto-phosphorylation/ the phosphorylation of the myelin basic protein through UV treatment. García-Gómez et al.11 confirmed that the degree of phosphorylation of c-Jun N-terminal kinase and p38-like MAPK was higher in suspension-cultured cells of Dunaliella tertiolecta exposed to UV treatment. These studies indicated the involvement of signaling components in regulating responses in plants under UV stress. Kellner et al.12 generated a draft genome sequence of C. roseus using the shotgun sequence approach. Transcriptomic and proteomic analyses are now available for C. roseus, which enable the establishment of a database that includes the whole genome. Improved genome assembly and annotation methods were applied to yield a wellannotated genome database.13 The whole genome sequence database contains information about the genes in plant-specialized metabolic pathways14, which facilitates the identification of transcripts/ proteins related to secondary metabolism in omics analysis.15 Plant response to UVB radiation is an important step for the subsequent accumulation of metabolites. The molecular basis of signaling events during UVB radiation that leads to the metabolism changes of the indole alkaloids is largely unknown. To identify the phosphorylated proteins, which mediate responses under UVB radiation and lead to the enhanced biosynthesis of secondary metabolites, a quantitative phosphoproteomic approach was used. In this study, a polymer-based metal-ion affinity capture (PolyMAC) technique was applied for phosphopeptide enrichment. Phosphopeptides were identified using a gel-free/label-free proteomic technique. Furthermore, phosphoproteomic results were confirmed by metabolomic and immunoblot analyses. 5 ACS Paragon Plus Environment


Materials and methods Plant material and treatment Seeds of C. roseus (provided by College of Pharmacy, Zhejiang University, Hangzhou, China) were sown and cultivated in a greenhouse for 45 days with temperature maintained at 25-28°C and 80% relative humidity. For control group samples, leaves were directly collected from the seedlings. For UVB group samples, leaves were collected from the seedlings, which were exposed to UVB for 1 h. The radiation intensity for UVB was 1345 µW cm-2. As three biological replicates, seeds were sown on three different days (Supplemental Figure 1). The fresh samples were immediately ground into fine powder in liquid nitrogen with a mortar and pestle, lyophilized, and stored at −80°C for further analysis (Supplemental Figure 2).

ATP-content measurement ATP-content measurement was performed using ATP Content Assay kit (Solarbio, Beijing, China) according to manufacturer’s protocol. A portion (15 mg) of leaves was mixed with 1 mL of extraction buffer provided by kit and ground on ice. The homogenates were centrifuged at 8,000 x g at 4°C for 10 min. The resulting supernatant was collected, mixed with 500 µL of chloroform, and centrifuged with 10,000 x g at 4°C for 3 min. The supernatant (100 µL) was collected and mixed with reaction solution provided by kit. ATP content was determined from the increase in absorbance at 340 nm before and after reaction by comparison with absorbance increase of the ATP standards. ATP content = (ODtest-ODcontrol)/(ODstandard-ODzero) ×standard concentration×sample volume/sample weight. ATP content was calculated as µmol per dry weight (g) according to previous study.16


Page 6 of 48

Page 7 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Protein extraction A portion (12 mg) of leaves was homogenized with a mortar and pestle in 500 µL of lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, and protease/phosphatase inhibitor cocktail (Abcam, Buelingame, CA, USA). The homogenates were centrifuged with 15,000 x g at 4°C for 10 min. The supernatant was collected and centrifuged with 15,000 x g at 4°C for 10 min. The method of Bradford17 was used to determine the protein concentration with bovine serum albumin used as the standard.

Protein enrichment, reduction, alkylation, and digestion Extracted proteins (100 µg) were adjusted to a final volume of 100 µL. Methanol (400 µL) was added to each sample and mixed before addition of 100 µL of chloroform and 300 µL of water. After mixing and centrifugation at 20,000 x g for 10 min to achieve phase separation, the upper phase was discarded and 300 µL of methanol was added to the lower phase, and then centrifuged at 20,000 x g for 10 min. The pellet was collected as the soluble fraction.18 Proteins were resuspended in 50 mM ammonium bicarbonate, reduced with 50 mM dithiothreitol for 30 min at 56C in the dark, and alkylated with 50 mM iodoacetamide for 30 min at 37C in the dark. Alkylated proteins were digested with trypsin and lysyl endopeptidase (Wako, Osaka, Japan) at a 1:100 enzyme/protein ratio for 16 h at 37C. Peptides were desalted with MonoSpin C18 Column (GL Sciences, Tokyo, Japan) and acidified with 1% trifluoroacetic acid.

Phosphopeptide enrichment PolyMAC phosphopeptide enrichment kit (Titanium based, Tymora, West Lafayette, IN, USA) was used to enrich phosphopeptides according to manufacturer’s protocol. Desalted peptides (100 µg) were resuspended in 200 µL of loading buffer 7 ACS Paragon Plus Environment


provided by kit. After short vortexing, 50 µL of PolyMAC beads was added to the sample and shaken vigorously for 20 min. The beads were separated from the solution using a tip with frit. Capture beads were washed once in loading buffer and twice in washing buffer provided by kit. Finally, phosphopeptides were eluted, dried, and used for liquid chromatography (LC)-mass spectrometry (MS) analysis.

Protein identification using nano-liquid chromatography mass spectrometry The LC conditions as well as the MS acquisition conditions are described in the previous study.19 The peptides were loaded onto the LC system (EASY-nLC 1000; Thermo Fisher Scientific, San Jose, CA, USA) equipped with a trap column (Acclaim PepMap 100 C18 LC column, 3 µm, 75 µm ID x 20 mm; Thermo Fisher Scientific), equilibrated with 0.1% formic acid, and eluted with a linear acetonitrile gradient (035%) in 0.1% formic acid at a flow rate of 300 nL min-1. The eluted peptides were loaded and separated on the column (EASY-Spray C18 LC column, 3 µm, 75 µm ID x 150 mm; Thermo Fisher Scientific) with a spray voltage of 2 kV (Ion Transfer Tube temperature: 275oC). The peptide ions were detected using MS (Orbitrap Fusion ETD MS; Thermo Fisher Scientific) in the data-dependent acquisition mode with the installed Xcalibur software (version 4.0; Thermo Fisher Scientific). Full-scan mass spectra were acquired in the FTMS over 375-1,500 m/z with resolution of 120,000. The most intense precursor ions were selected for high-energy collision-induced fragmentation in the ion routing multiple at normalized collision energy of 28%, the MS/MS spectra were acquired in the ITMS. Dynamic exclusion was employed within 60 sec to prevent repetitive selection of peptides.

Mass spectrometry data analysis The MS/MS searches were carried out using MASCOT (Version 2.6.1, Matrix 8 ACS Paragon Plus Environment

Page 8 of 48

Page 9 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Science, London, UK) and SEQUEST HT search algorithms against the UniprotKB Arabidopsis thaliana (2017-10-25) or Catharanthus roseus (version 2)13 (https://datadryad.org/resource/doi:10.5061/dryad.08vv50n) protein database using Proteome Discoverer (PD) 2.2 (Version 2.2.0.388; Thermo Fisher Scientific). The workflow for both algorithms included spectrum files RC, spectrum selector, MASCOT, SEQUEST HT search nodes, percolator, ptmRS, and minor feature detector nodes. PhosphoST, PhosphoY, protein N-terminal acetylation, and oxidation of methionine were set as a variable modification and carbamidomethylation of cysteine was set as a fixed modification. Mass tolerances in MS and MS/MS were set at 10 ppm and 0.6 Da, respectively. Trypsin was specified as protease and a maximum of two missed cleavage was allowed. Target-decoy database searches used for calculation of false discovery rate (FDR) and for peptide identification FDR was set at 1%.

Differential analysis of proteins using mass spectrometry data Label-free quantification was also performed with PD 2.2 using precursor ions quantifiler nodes. For differential analysis of the relative abundance of peptides and proteins between samples, the freely software PERSEUS (version 1.6.5.0)20 was used. Proteins and peptides abundances were transferred into log2 scale. Three biological replicates of each sample were grouped and a minimum of three valid values were required in at least one group. Normalization of the abundances was performed to subtract the median of each sample. Missing values were imputed based on a normal distribution (width = 0.3, down-shift = 1.8). Significance was assessed using Student’s t-test analysis.

Metabolite extraction Metabolites were extracted according to previous method21 with minor 9 ACS Paragon Plus Environment


modifications. A portion (10 mg) of leaves was extracted with 450 μL extraction solution (methanol : H2O = 3:1), added with 10 μL of 0.5 mg mL-1 adonitol as internal standard, and vortexed to mix for 30 sec. Samples were homogenized in ball mill (JXFSTPRP-24; Tianjin Technology, Shanghai, China) for 4 min at 45 Hz, treated with ultra-sonication for 5 min in ice water, and centrifuged with 16,200 x g at 4C for 15 min. The supernatant (300 μL) was transferred to a glass vial and dried in a vacuum concentrator. The residue was resuspended with 80 μL of 20 mg mL−1 methoxyamination hydrochloride in pyridine and incubated for 30 min at 80°C. Subsequently, 100 μL of bis (trimethylsily) trifluoroacetamide containing 1% trimethylchlorosilane (Regis Technologies, Morton Grove, IL, USA) as derivatization reagent was added and incubated for 90 min at 70°C. Finally, derivatized samples were loaded to gas chromatography (GC)- time of flight (TOF)/MS.

Gas chromatography-time of flight/mass spectrometry analysis GC-TOF/MS analysis was performed using an Agilent 7890 GC system (Agilent, Palo Alto, CA, USA) coupled with a Pegasus HT TOF-MS (LECO, St Joseph, MI, USA). The sample (1 μL) was injected to a DB-5MS capillary column coated with 5% diphenyl cross-linked with 95% dimethylpolysiloxane (250 μm ID x 30 m, 0.25 μm film thickness; J &W Scientific, Folsom, CA, USA) in split mode. Helium was used as the carrier gas, the front inlet purge flow was 3 mL min−1, and the gas flow rate through the column was 1 mL min−1. The initial temperature was kept at 80°C for 1 min and raised to 300°C afterwards at a rate of 10°C min−1. The temperature at 300°C was maintained for 10 min. The temperature of injection, transfer line, and ion source were 280, 300, and 230°C, respectively. The energy was -70 eV in the electron impact mode. The MS data were acquired in full-scan mode with 75-650 m/z at a rate of 10 spectra sec−1 after a solvent delay of 3.833 min. 10 ACS Paragon Plus Environment

Page 10 of 48

Page 11 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Functional prediction Protein functions were categorized using MapMan bin codes22 and protein abundance ratio was visualized through MapMan software23 (version 3.6.0RC1; http://mapman.gabipd.org). Pathway mapping of identified proteins was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database24 (http://www.genome.jp/kegg/).

Immunoblot analysis SDS-sample buffer consisting of 60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol was added to protein samples.25 Quantified proteins (10 µg) were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane using a semidry transfer blotter (Nippon Eido, Tokyo, Japan). The blotted membrane was blocked for 5 min in Bullet Blocking One regent (Nacalai Tesque, Kyoto, Japan). After blocking, the membrane was cross-reacted with a 1: 1000 dilution of the primary antibodies for 1 h at room temperature. Anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA, USA) was used as the secondary antibody. After 1 h incubation, signals were detected using TMB Membrane Peroxidase Substrate kit (Seracare, Milford, MA, USA) following the manufacturer’s protocol. Coomassie brilliant blue (CBB) staining was used as loading control. The integrated densities of bands were calculated using Image J software (version 1.8, National Institutes of Health, Bethesda, MD, USA). As primary antibodies, the followings were used: anti-fructose-bisphosphate aldolase (FBPA),26 anti-triose phosphate isomerase (TPI) (provided by Proteomic Laboratory in Fukui University of Technology, Fukui, Japan), anti-glyceraldehyde-3phosphate dehydrogenase (GAPDH) (provided by Proteomic Laboratory in Fukui 11 ACS Paragon Plus Environment


University of Technology, Fukui, Japan), anti-ribulose biphosphate carboxylase/oxygenase (RuBisCO) large subunit (LSU),27 anti-RuBisCO small subunit (SSU),28 anti-RuBisCO activase,28anti-ascorbate peroxidase (APX),29 anti-glutathione reductase (GR) (Agrisera, Vännäs, Sweden), anti-peroxiredoxin (PRX),30 antipathogenesis related (PR) proteins class 1 (PR-1),31 anti-PR proteins class 5 (PR-5),32 and anti-PR proteins class 10 (PR-10)33 antibodies.

Statistical analysis The statistical significance of two groups was evaluated by the Student’s t-test. A pvalue of less than 0.05 was considered as statistically significant.

Results ATP-content change in leaves of C. roseus under UVB radiation ATP provides phosphate group for protein phosphorylation. Leaves were treated without or with UVB radiation, ATP was extracted, and ATP content was determined (Supplemental Figures 1 and 2). ATP contents were 1.96 µmol g-1 and 3.07 µmol g-1 in untreated and UVB-treated groups, respectively (Figure 1). These results indicated that ATP content significantly increased in leaves of C. roseus under UVB radiation.

Identification and functional analysis of phosphoproteins in leaves of C. roseus under UVB radiation To understand the phosphorylation status of proteins in C. roseus under UVB radiation, phosphoproteomic analysis was performed. Proteins were extracted using leaves without or with UVB radiation as material. Phosphopeptides were enriched using PolyMAC beads and analyzed by LC-MS/MS (Supplemental Figure 3). Using Catharanthus roseus protein database, 242 phosphoproteins were changed in leaves of 12 ACS Paragon Plus Environment

Page 12 of 48

Page 13 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


C. roseus under UVB radiation compared to control (Table 1). Using Arabidopsis thaliana protein database, 22 phosphoproteins were identified (Supplemental Table 1). Functional category of phosphoproteins was determined using MapMan bin codes. A total of 242 phosphoproteins were mapped and classified into 28 categories (Figure 2). Apart from 54 proteins which were without ontology, the other phosphoproteins were mainly related to protein synthesis/ post-translational modification/ degradation, signaling, RNA transcriptional regulation, transport, and cell organization/ vesicle transport (Figure 2). In redox related pathway, thioredoxin family protein, Fe superoxide dismutase, and cytochrome B5 isoform increased. In glycolysis related pathway, phosphoenolpyruvate carboxylase, aldehyde dehydrogenase, GAPDH, and phosphoglucose isomerase increased (Table 1). To visualize the effect of UVB radiation to photosynthesis, phosphoproteins related to photosynthesis were submitted to MapMan software and mapped (Figure 3). The abundance of phosphoproteins related to light reaction and Calvin cycle intensively changed by UVB radiation. In addition, phosphorylated RuBisCO increased with a fold change of 8.4812. (Figure 3).

Classification of changed metabolites in leaves of C. roseus under UVB radiation To explore the change of metabolites in C. roseus under UVB radiation, GCTOF/MS based metabolomic analysis was performed. Metabolites were extracted from leaves without or with UVB radiation. In total, 187 metabolites were identified in the leaves of C. roseus. Out of them, 110 metabolites were significantly changed (Supplemental Table 2) and searched against KEGG database (Figure 4). Identified metabolites were mainly classified into sugars, organic acids, amino acids, alcohols, and phosphates. The number of increased metabolites related to sugars, organic acids, and amino acids was 11, 18, and 19, respectively, which accounts for 46%, 75%, and 95% 13 ACS Paragon Plus Environment


of all significantly changed metabolites in each categorized group (Figure 4). To visualize the effect of UVB radiation, metabolites related to sugars, organic acids, and amino acids were mapped using KEGG database (Figures 5 and 6). Metabolites related to the upstream of glycolysis such as glucose-1-phosphate, glucose6-phosphate, fructose-6-phosphate decreased. In addition, glucose, isomaltose, and cellobiose also decreased. On the other hand, metabolites related to another upstream of glycolysis such as xylose, xylulose, lyxose, ribitol, and arabitol increased. Furthermore, citrate, fumarate, and malate in organic acids group, which are involved in tricarboxylic acid cycle, increased (Figure 5). Metabolites related to lipid synthesis (valine, leucine, isoleucine, glycine, and alanine), beta-alanine metabolism (spermidine, 4aminobutanoate, malonate, and aspartate), arginine biosynthesis (glutamine, glutamate, and N-acetylglutamate), and lysine degradation (lysine and glutarate) increased. Out of metabolites related to aromatic-compound biosynthesis, indole-alkaloid related metabolites such as tryptophan and shikimate increased. Phosphorylation related amino acids such as serine, threonine, and tyrosine increased (Figure 6).

Immunoblot analysis of proteins involved in glycolysis, photosynthesis, redox, and pathogenesis in leaves of C. roseus under UVB radiation To further investigate the presence of proteins in the control group and UVB group, immunoblot technique was used. Proteins were extracted using leaves without or with UVB radiation as material. Proteins involved in glycolysis (FBPA, TPI, and GAPDH), photosynthesis (RuBisCO LSU, RuBisCO SSU, and RuBisCO activase), redox (APX, GR, and PRX), and pathogenesis (PR-1, PR-5, and PR-10) were analyzed by immunoblot (Supplemental Table 3). CBB staining pattern was used as loading control (Supplemental Figure 4). In glycolysis, the abundance of FBPA decreased, while the abundance of TPI and GAPDH increased under UVB radiation (Figure 7, Supplemental 14 ACS Paragon Plus Environment

Page 14 of 48

Page 15 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5). In photosynthesis, the abundance of RuBisCO LSU and RuBisCO SSU increased while RuBisCO activase did not change under UVB radiation (Figure 7, Supplemental Figure 6). In redox pathway, the abundance of APX and PRX significantly increased under UVB radiation (Figure 8). In pathogenesis related pathway, the abundance of PR-10 decreased under UVB radiation (Figure 8). These results indicated that UVB radiation significantly affected glycolysis, photosynthesis, and the redox pathway in C. roseus.

Discussion Calcium-dependent signaling pathway is activated and may be involved in secondary metabolism in C. roseus under UVB radiation UVB radiation triggers accumulation of secondary metabolites, such as phenolic compounds34 and carotenoids.35 Understanding of plant response to this effective abiotic elicitor is necessary for the development of natural drug manipulation. In the present study, ATP content in leaves of C. roseus increased under UVB radiation (Figure 1). Phosphoproteomic analysis indicated that, altered phosphoproteins related to signaling composed the second biggest category. Among them, proteins related to calcium were the most abundant (Figure 2; Table 1). Previous proteomic analysis indicated that the abundance of several kinases including pyruvate kinase, phosphoribulokinase, and hexokinase-1, increased after UVB radiation.36 Pretreatment of C. roseus with kinase/ calcium-channel inhibitors blocked the accumulation of catharanthine under UVB radiation.9 The present result with previous reports suggest that calcium-dependent signaling pathway may play a vital role in response of C. roseus to UVB radiation. Calcium is a divalent cation which plays important roles in not only plant cell metabolism but also signal transduction.37 By UVB radiation, phosphorylated proteins in C. roseus related to Ca2+ transportation, such as cation exchanger and Ca2+-ATPase, 15 ACS Paragon Plus Environment


increased; In the meanwhile, calmodulin-binding family protein, calcium-binding family protein, and calcium-dependent protein kinase increased as well (Table 1). Calcium regulated the activities of target proteins directly or via calmodulin.38 After binding to Ca2+, calmodulin was capable of activating down-steam protein kinases and proteins in plant cells.38 These results together suggest that calcium-related pathway is activated in C. roseus by UVB radiation. As a powerful secondary messenger in many signaling pathways, including those driven by abscisic acid and jasmonates, calcium was interfered with secondary metabolism.39 Calcium enhanced accumulation of secondary metabolites such as phenolics and flavonoids.40 Heterologous expression of a calcium-dependent protein kinase induced the biosynthesis of phytoalexins in Rubia cordifolia.41 Similar regulatory mechanisms may occur in C. roseus, which may contribute to enhanced production of alkaloids.

Dynamic changes in glycolysis promote ATP production in C. roseus under UVB radiation Glycolysis followed with tricarboxylic acid cycle is fundamental pathway to produce energy for plant survival. Phosphoproteomic, metabolomic, and immunoblot analyses indicated dynamic changes in this pathway (Figures 2, 5, and 7). Glycolysis is divided into the preparatory and the pay-off phases. In the preparatory phase, the phosphorylation of glucose and fructose-6-phosphate consumes 2 ATP molecules. In the pay-off phase, the dephosphorylation of glycerate-1,3-diphosphate and phosphoenolpyruvate produce 4 ATP molecules.42 In the present study, phosphorylated proteins involved in the payoff phase such as GAPDH and aldehyde dehydrogenase increased (Table 1); whereas metabolites involved in the preparatory phase decreased (Figure 5). FBPA, which is at the turning point between two phases, decreased, suggesting that the energy-consuming step is inhibited in glycolysis. GAPDH was 16 ACS Paragon Plus Environment

Page 16 of 48

Page 17 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reported as a responsive protein to abiotic stress such as flooding43 and salt.44 Upregulation of GAPDH may activates the pentose-related upstream of glyceraldehyde3-phosphate (Figure 5), which promotes the upregulation of preparatory phase of glycolysis in C. roseus under UVB radiation. Tricarboxylic acid cycle, which utilizes acetyl-CoA derived from pyruvate can provide not only ATP for fundamental physiological activities45, but also intermediates for many metabolic pathways46. In the present study, proteins such as pyruvate dehydrogenase and phosphoenolpyruvate carboxylase, increased (Table 1). Pyruvate dehydrogenase catalyzes the conversion of pyruvate to acetyl-CoA47. Phosphoenolpyruvate carboxylase catalyzes the carboxylation of phosphoenolpyruvate to oxaloacetate48. Present results indicate that proteins responsible for the conversions which allow metabolites enter to tricarboxylic acid cycle, increased. Taken together, the preparatory phase in glycolysis is inhibited, which saves ATP; furthermore, an alternative pathway to produce substrates for tricarboxylic acid cycle is activated and leads to ATP production in C. roseus under UVB radiation.

Oxidative stress induces secondary-metabolites biosynthesis in C. roseus under UVB radiation UVB radiation represents as one kind of the oxidative stress which induced the production of reactive oxygen species (ROS) and threated plant survival. 49 After production of ROS, ROS scavenging system was activated in response to environmental stress in plants.50 The expression levels of peroxidase and superoxide dismutase were upregulated under UV treatment.8 In the present study, phosphorylated proteins involved in ROS scavenging such as thioredoxin family protein, superoxide dismutase, and cytochrome B5 increased under UVB radiation (Table 1). Immunoblot analysis indicated that the abundance of peroxiredoxin significantly increased (Figure 8). The 17 ACS Paragon Plus Environment


thioredoxin family is a big protein category which includes thioredoxins, glutaredoxins, peroxiredoxins, protein disulfide isomerases and so on.51 While phosphorylation of thioredoxin enhanced its activity in cancer cells52, the effect of phosphorylation to thioredoxin in plants remained poorly investigated. Taken together, UVB is likely to activate ROS scavenging system especially thioredoxin-related pathway in C. roseus. Phosphorylation may regulate the enzymatic activity of proteins involved ROS scavenging, which may induce the change of specialized redox reactions in plants and protect plant from stress. Zandalina et al.53 reported that oxidative stress induced accumulation of secondary metabolites with antioxidant activities. In C. roseus, aromatic amino acids/ phenylpropanoids significantly accumulated under UVB radiation (Figure 6), which is consistent with the previous finding. In addition, phosphorylated proteins related to secondary metabolism such as oxidoreductase, elicitor-activated gene 3-2 (cinnamyl alcohol dehydrogenase), and 2-oxoglutarate and Fe(II)-dependent oxygenase increased in C. roseus under UVB radiation (Table 1). Cytochrome B5 was an obligate electron shuttle protein for syringyl lignin biosynthesis in Arabidopsis.54 Cinnamyl alcohol dehydrogenase, which belongs to the family of oxidoreductase, was involved in the conversions of various phenylpropenyl aldehyde derivatives into the corresponding monolignols via electron transport.55 Present results with previous knowledge suggest that cytochrome B5 is essential for plant secondary metabolism and may associate with the activity of oxidoreductase through electron transport. Furthermore, oxygenase was responsible for the catalyzation of oxidative transformations such as hydroxylation, ring closure, and epoxidation reactions56. The biosynthesis of alkaloid in C. roseus such as vindoline required hydroxylation.57 Taken together, the change of oxygenase may be involved in the biosynthesis of alkaloid in C. roseus under UVB radiation.


Page 18 of 48

Page 19 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conclusion C. roseus is the main source for secondary metabolites, especially indole alkaloids.1 Indole alkaloids such as catharanthine and vindoline accumulated in C. roseus under UVB radiation.5 Calcium fluxes and kinase activities were affected as well.9 This study explored the regulatory mechanisms of the biosynthesis of secondary metabolites in leaves of C. roseus under UVB radiation. The main findings are as follows: (i) calcium-dependent protein phosphorylation was activated; (ii) proteins and metabolites related to glycolysis underwent dynamic changes; (iii) the tricarboxylic acid cycle was upregulated; (iv) the ROS scavenging system was activated; (v) phosphoproteins responsible for redox reactions in secondary metabolism increased; and (vi) metabolites related to secondary metabolism such as aromatic amino acids and phenylpropanoids accumulated in C. roseus under UVB radiation. These results suggest that UVB radiation causes oxidative stress and activates calcium-dependent protein phosphorylation/dephosphorylation in C. roseus. ATP production is promoted by regulation of glycolysis. Phosphorylation of proteins which are involved in redox reactions in secondary metabolism are affected.

Supporting information Supplemental Table 1. List of phosphoproteins altered under UVB radiation in leaves of C. roseus identified using Catharanthus roseus protein database. Supplemental Table 2. List of metabolites altered under UVB radiation in leaves of C. roseus. Supplemental Table 3. Estimated size of proteins involved in immunoblot analysis Supplemental Figure 1. Schematic map of sample preparation for three independent replications. Supplemental Figure 2. Experimental design for phosphoproteomic investigation of C. 19 ACS Paragon Plus Environment


roseus under UVB radiation. Supplemental Figure 3. The example spectrum of phosphorylated peptide. Supplemental Figure 4. The Coomassie brilliant blue staining pattern of proteins used for immunoblot analysis. Supplemental Figure 5. Blots of the entire membrane which used in Figure 7. Supplemental Figure 6. Blots of the entire membrane which used in Figure 8.

Accession codes For MS data, RAW data, peak lists and result files have been deposited in the ProteomeXchange Consortium58 via the jPOST59 partner repository under data-set identifiers PXD013575.

Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding sources This work was supported by the National Science Foundation of China (81872973) and Zhejiang Provincial Science and Technology Planning Project (2016C04005).

References (1) Heijden, R.; Jacobs, D.; Snoeijer, W.; Hallard, D.; Verpoorte, R. The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr. Med. Chem. 2004, 11, 607-628. (2) Qu, Y.; Safonova, O.; De Luca, V. Completion of the canonical pathway for 20 ACS Paragon Plus Environment

Page 20 of 48

Page 21 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


assembly of anticancer drugs vincristine/vinblastine in Catharanthus roseus. Plant J. 2019, 97, 257-266. (3) Costa, M. M.; Hilliou, F.; Duarte, P.; Pereira, L. G.; Almeida, I.; Leech, M.; Memelink, J.; Barcelo, A. R.; Sottomayor, M. Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant Physiol. 2008, 146, 403-417. (4) Binder, B. Y.; Peebles, C. A.; Shanks, J. V.; San, K.Y. The effects of UV-B stress on the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots. Biotechnol. Prog. 2009, 25, 861-865. (5) Ramani, S.; Jayabaskaran, C. Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by Ultraviolet-B light. J. Mol. Signal. 2008, 3, 9. (6) Ouwerkerk, P. B.; Hallard, D.; Verpoorte, R.; Memelink, J. Identification of UV-B light-responsive regions in the promoter of the tryptophan decarboxylase gene from Catharanthus roseus. Plant Mol. Biol. 1999, 41, 491-503. (7) Ramani, S.; Patil, N.; Jayabaskaran, C. UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells. J. Mol. Signal. 2010, 5, 13. (8) Nishanth, M. J.; Sheshadri, S. A.; Rathore, S. S.; Srinidhi, S.; Simon, B.; Expression analysis of cell wall invertase under abiotic stress conditions influencing specialized metabolism in Catharanthus roseus. Sci. Rep. 2018, 8, 15059. (9) Ramani, S.; Chelliah, J. UV-B induced signaling events leading to enhanced production of catharanthine in Catharanthus roseus cell suspension cultures. BMC Plant Biol. 2007, 7, 61. (10) Raina, S. K.; Wankhede, D. P.; Jaggi, M.; Singh, P.; Jalmi, S. K.; Raghuram, B.; Sheikh, A. H.; Sinha, A. K. CrMPK3, a mitogen activated protein kinase from 21 ACS Paragon Plus Environment


Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids. BMC Plant Biol. 2012, 12, 134. (11) García-Gómez, C.; Parages, M. L.; Jiménez, C.; Palma, A.; Mata, M. T.; Segovia, M. Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation. J. Exp. Bot. 2012, 63, 5259-5274. (12) Kellner, F.; Kim, J.; Clavijo, B. J.; Hamilton, J. P.; Childs, K. L.; Vaillancourt, B.; Cepela, J.; Habermann, M.; Steuernagel, B.; Clissold, L.; Mclay, K.; Buell, C. R.; O’Connor, S.E. Genome-guided investigation of plant natural product biosynthesis. Plant J. 2015, 82, 680-692. (13) Franke, J.; Kim, J.; Hamilton, J. P.; Zhao, D.; Pham, G. M.; Wiegert‐Rininger, K.; Crisovan, E.; Newton, L.; Vaillancourt, B.; Tatsis, E.; Buell, C. R.; O’Connor, S. E. Gene discovery in Gelsemium highlights conserved gene clusters in monoterpene indole alkaloid biosynthesis. ChemBioChem. 2019, 20, 83-87. (14) Hoopes, G. M.; Hamilton, J. P.; Kim, J.; Zhao, D.; Wiegert-Rininger, K.; Crisovan, E.; Buell, C. R. Genome assembly and annotation of the medicinal plant Calotropis gigantea, a producer of anticancer and antimalarial Cardenolides. G3 (Bethesda) 2018, 8, 385-391. (15) Chakraborty, P. Herbal genomics as tools for dissecting new metabolic pathways of unexplored medicinal plants and drug discovery. Biochim. Open 2018, 6, 9-16. (16) Pan, D.; Wang, L.; Tan, F.; Lu, S.; Lv, X.; Zaynab, M.; Chen, C. L.; Abubakar, Y. S.; Chen, S.; Chen, W. Phosphoproteomics unveils stable energy supply as key to flooding tolerance in Kandelia candel. J. Proteomics 2018, 176, 1-12. (17) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. 22 ACS Paragon Plus Environment

Page 22 of 48

Page 23 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Komatsu, S.; Han, C.; Nanjo, Y.; Altaf-Un-Nahar, M.; Wang, K.; He, D.; Yang, P. Label-free quantitative proteomic analysis of abscisic acid effect in early-stage soybean under flooding. J. Proteome Res. 2013, 12, 4769-4784. (19) Li, X.; Rehman, S. U.; Yamaguchi, H.; Hitachi, K.; Tsuchida, K.; Yamaguchi, T.; Sunohara, Y.; Matsumoto, H.; Komatsu, S. Proteomic analysis of the effect of plantderived smoke on soybean during recovery from flooding stress. J. Proteomics 2018, 181, 238–248. (20) Tyanova, S.; Temu, T.; Sinitcyn, P.; Carlson, A.; Hein, M. Y.; Geiger, T.; Mann, M.; Cox, J. The Perseus computational platform for comprehensive analysis of (prote) omics data. Nat. Methods 2016, 13, 731-740. (21) Shingaki-Wells, R. N.; Huang, S.; Taylor, N. L.; Carroll, A. J.; Zhou, W.; Millar, A. H. Differential molecular responses of rice and wheat coleoptiles to anoxia reveal novel metabolic adaptations in amino acid metabolism for tissue tolerance. Plant Physiol. 2011, 156, 1706-1724. (22) Usadel, B.; Nagel, A.; Thimm, O.; Redestig, H.; Blaesing, O. E.; Rofas, N. P.; Selbig, J.; Hannemann, J.; Piques, M. C.; Steinhauser, D.; Scheible, W. R.; Gibon, Y.; Morcuende, R.; Weicht, D.; Meyer, S.; Stitt, M. Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes and comparison with known responses. Plant Physiol. 2005, 138, 1195-1204. (23) Usadel, B.; Poree, F.; Nagel, A.; Loshe, M.; Czedik-Eysenberg, A.; Sitt, M. A guide to using MapMan to visualize and compare omics in plants: a case study in the crop species, maize. Plant Cell Environ. 2009, 32, 1211-1229. (24) Kanehisa, M.; Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28, 27-30. (25) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680-685. 23 ACS Paragon Plus Environment


(26) Konishi, H.; Yamane, H.; Maeshima, M.; Komatsu, S. Characterization of fructosebisphosphate aldolase regulated by gibberellin in roots of rice seedling. Plant Mol. Biol. 2004, 56, 839-848. (27) Hashimoto, M.; Komatsu, S. Proteomic analysis of rice seedlings during cold stress. Proteomics 2007, 7, 1293-1302. (28) Zhang, Z.; Komatsu, S. Molecular cloning and characterization of cDNAs encoding two isoforms of ribulose-1, 5-bisphosphate carboxylase/oxygenase activase in rice (Oryza sativa L.). J. Biochem. 2000, 128, 383-389. (29) Komatsu, S.; Yamamoto, A.; Nakamura, T.; Nouri, M. Z.; Nanjo, Y.; Nishizawa, K.; Furukawa, K. Comprehensive analysis of mitochondria in roots and hypocotyls of soybean under flooding stress using proteomics and metabolomics techniques. J. Proteome Res. 2011, 10, 3993-4004. (30) Nishizawa, K.; Komatsu, S. Characteristics of soybean 1-Cys peroxiredoxin and its behavior in seedlings under flooding stress. Plant Biotech. 2011, 28, 83-88. (31) Rakwal, R.; Komatsu, S. Role of jasmonate in the rice (Oryza sativa L.) selfdefense mechanism using proteome analysis. Electrophoresis 2000, 21, 2492-2500. (32) Konish, H.; Ishiguro, K.; Komatsu, S. A proteomic approach towards understanding blast fungus infection of rice grown under different levels of nitrogen fertilization. Proteomics 2001, 1, 1162-1171. (33) Hashimoto, M.; Kisseleva, L.; Sawa, S.; Furukawa, T.; Komatsu, S.; Koshiba, T. A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant Cell Physiol. 2004, 45, 550-559. (34) Surjadinata, B. B.; Jacobo-Velázquez, D. A.; Cisneros-Zevallos, L. UVA, UVB and UVC light enhances the biosynthesis of phenolic antioxidants in fresh-cut carrot through a synergistic effect with wounding. Molecules 2017, 22, 668. 24 ACS Paragon Plus Environment

Page 24 of 48

Page 25 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(35) Moreira-Rodríguez, M.; Nair, V.; Benavides, J.; Cisneros-Zevallos, L.; JacoboVelázquez, D. A. UVA, UVB light, and methyl jasmonate, alone or combined, redirect the biosynthesis of glucosinolates, phenolics, carotenoids, and chlorophylls in broccoli sprouts. Int. J. Mol. Sci. 2017, 18, 2330. (36) Zhu, W.; Yang, B.; Komatsu, S.; Lu, X.; Li, X.; Tian, J. Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus. Front. Plant Sci. 2015, 16, 582. (37) Ahmad, P.; Abdel Latef, A. A.; Abd Allah, E. F.; Hashem, A.; Sarwat, M.; Anjum, N. A.; Gucel, S. Calcium and potassium supplementation enhanced growth, osmolyte secondary metabolite production, and enzymatic antioxidant machinery in cadmiumexposed chickpea (Cicer arietinum L.). Front. Plant Sci. 2016, 7, 513. (38) Vitrac, X.; Larronde, F.; Krisa, S.; Decendit, A.; Deffieux, G.; Mérillon, J. M. Sugar sensing and Ca2+-calmodulin requirement in Vitis vinifera cells producing anthocyanins. Phytochemistry 2000, 53, 659-665. (39) Martins, V.; Garcia, A.; Costa, C.; Sottomayor, M.; Gerós, H. Calcium-and hormone-driven regulation of secondary metabolism and cell wall enzymes in grape berry cells. J. Plant Physiol. 2018, 231, 57-67. (40) Xu, W.; Peng, H.; Yang, T.; Whitaker, B.; Huang, L.; Sun, J.; Chen, P. Effect of calcium on strawberry fruit flavonoid pathway gene expression and anthocyanin accumulation. Plant Physiol. Biochem. 2014, 82, 289-298. (41) Shkryl, Y. N.; Veremeichik, G. N.; Bulgakov, V. P.; Zhuravlev, Y. N. Induction of anthraquinone biosynthesis in Rubia cordifolia cells by heterologous expression of a calcium-dependent protein kinase gene. Biotechnol. Bioeng. 2011, 108, 1734-1738. (42) Dashty, M. A quick look at biochemistry: carbohydrate metabolism. Clin. Biochem. 2013, 46, 1339-1352. (43) Wang, X.; Sakata, K.; Komatsu, S. An integrated approach of proteomics and 25 ACS Paragon Plus Environment


computational genetic modification effectiveness analysis to uncover the mechanisms of flood tolerance in soybeans. Int. J. Mol. Sci. 2018, 19, 1301. (44) Cho, J. I.; Lim, H. M.; Siddiqui, Z. S.; Park, S. H.; Kim, A. R.; Kwon, T. R.; Lee, S. K.; Park, S.C.; Jeong, M. J.; Lee, G. S. Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants. Biotechnol. Lett. 2014, 36, 1641-1648. (45) Nazaret, C.; Heiske, M.; Thurley, K.; Mazat, J. P. Mitochondrial energetic metabolism: a simplified model of TCA cycle with ATP production. J. Theor. Biol. 2009, 258, 455-464. (46) Nunes-Nesi, A.; Araújo, W. L.; Obata, T.; Fernie, A. R. Regulation of the mitochondrial tricarboxylic acid cycle. Curr. Opin. Plant. Biol. 2013, 16, 335-343. (47) Patel, M. S.; Roche, T. E. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J. 1990, 4, 3224-3233. (48) O’Leary, B.; Park, J.; Plaxton, W. C. The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem. J. 2011, 436, 15-34. (49) He, Y. Y.; Häder, D. P. UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine. J. Photochem. Photobiol. B 2002, 66, 115-124. (50) Das, K.; Roychoudhury, A. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front. Environ. Sci. 2014, 2, 53. (51) Lee, S.; Kim, S. M.; Lee, R. T. Thioredoxin and thioredoxin target proteins: from molecular mechanisms to functional significance. Antioxid. Redox Signal. 2013, 18, 1165-1207. 26 ACS Paragon Plus Environment

Page 26 of 48

Page 27 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(52) Chen, X.; Tang, W.; Liu, S.; Yu, L.; Chen, Z. Thioredoxin-1 phosphorylated at T100 is needed for its anti-apoptotic activity in HepG2 cancer cells. Life Sci. 2010, 87, 254-260. (53) Zandalinas, S. I.; Sales, C.; Beltrán, J.; Gómez-Cadenas, A.; Arbona, V. Activation of secondary metabolism in citrus plants is associated to sensitivity to combined drought and high temperatures. Front. Plant Sci. 2017, 7, 1954. (54) Gou, M.; Yang, X.; Zhao, Y.; Ran, X.; Song, Y.; Liu, C. J. Cytochrome b5 is an obligate electron shuttle protein for syringyl lignin biosynthesis in Arabidopsis. Plant Cell. 2019, 31, 1344-1366. (55) Kim, S. J.; Kim, K. W.; Cho, M. H.; Franceschi, V. R.; Davin, L. B.; Lewis, N. G. Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations. Phytochemistry, 2007, 68, 1957-1974. (56) Martinez, S.; Hausinger, R. P. Catalytic mechanisms of Fe (II)-and 2-oxoglutaratedependent oxygenases. J. Biol. Chem. 2015, 290, 20702-20711. (57) Besseau, S.; Kellner, F.; Lanoue, A.; Thamm, A. M. K.; Salim, V.; Schneider, B.; Geu-Flores, F., Hofer, R,; Guirimand, G.; Guihur, A.; Oudin, A.; St-Pierre, B.; Wercjk-Reichhart, D.; Burlat, V.; Luca, V. D.; O’Connor, S. E.; Courdavault, V. A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organdependent manner in Catharanthus roseus. Plant Physiol. 2013, 163, 1792-1803. (58) Vizcaíno, J. A.; Côté, R. G.; Csordas, A.; Dianes, J. A.; Fabregat, A.; Foster, J. M.; Griss, J.; Alpi, E.; Birim, M.; Contell, J.; O'Kelly, G.; Schoenegger, A.; Ovelleiro, D.; Pérez-Riverol, Y.; Reisinger, F.; Ríos, D.; Wang, R.; Hermjakob, H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 2013, 41, D1063-D1069. (59) Okuda, S.; Watanabe, Y.; Moriya, Y.; Kawano, S.; Yamamoto, T.; Matsumoto, M.; 27 ACS Paragon Plus Environment


Takami, T.; Kobayashi, D.; Araki, N.; Yoshizawa, A. C.; Tabata, T.; Sugiyama, N.; Goto, S.; Ishihama, Y. jPOSTrepo: an international standard data repository for proteomes. Nucleic Acids Res. 2017, 45, D1107-D1111.


Page 28 of 48

Page 29 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure legends

Figure 1. ATP content in leaves of C. roseus under UVB radiation. Leaves were treated without (white column) or with UVB radiation (grey column) and ATP was extracted. After addition of reaction solution, ATP content was determined from the increase in absorbance at 340 nm before and after reaction. ATP content was calculated as µmol per dry weight (g). Data are shown as means ± SD from three independent biological replicates. Student’s t-test was used to compare values between control and UVB radiation. Asterisk indicates a significant change (*p < 0.05).

Figure 2. Functional categories of changed phosphoproteins in leaves of C. roseus under UVB radiation. Leaves were treated with UVB radiation. Untreated plant was used as control. Proteins were extracted, reduced, alkylated, and digested. Phosphopeptides were enriched and analyzed using LC-MS/MS (Table 1). White and black columns indicate increased and decreased phosphoproteins under UVB radiation, respectively, compared to control. Functional category of phosphoproteins was determined using MapMan bin codes. A total of 242 phosphoproteins were mapped and classified into 28 categories. Abbreviations are as follows: RNA, RNA regulation of transcription/ processing/ binding; cell, cell organization/ vesicle transport/ division and PTM, post-translational modifications. “not assigned” indicates protein without ontology. “others” indicates protein with other functions.

Figure 3. Mapping on photosynthesis metabolism of changed phosphoproteins in leaves of C. roseus under UVB radiation. Changed phosphoproteins were submitted to the MapMan software and mapped to photosynthesis pathway. Each square indicates one mapped protein. Color indicates the fold change value of a differentially changed 29 ACS Paragon Plus Environment


protein. Red and blue colors indicate an increase and decrease in fold change values under UVB radiation, respectively, compared to control.

Figure 4. Classification of changed metabolites in leaves of C. roseus under UVB radiation. Metabolites were extracted from leaves treated with UVB radiation and analyzed using GC-TOF/MS. Untreated plant was used as control. Student’s t-test was used to compare values between control group and UVB group. Functional category of significantly changed metabolites was searched against KEGG database. A total of 110 metabolites were classified into 16 categories. White and black columns indicate increased and decreased metabolites under UVB radiation, respectively, compared to control. “not assigned” indicates metabolites without ontology.

Figure 5. Mapping of changed metabolites under UVB radiation to pathway involved in sugar metabolism, glycolysis, and tricarboxylic acid cycle. Pathway map was determined based on KEGG database. Each circle indicates one metabolite. Red and blue circles indicate increased and decreased metabolites under UVB radiation, respectively, compared to control. Metabolites neither detected nor changed are marked in white. Dashed lines indicate omission of complex reactions.

Figure 6. Mapping of changed metabolites under UVB radiation to pathway involved in organic acid and amino acid metabolism. Pathway map was determined based on KEGG database. Each circle indicates one metabolite. Different color indicates different ratio range of quantities of metabolites, which is calculated using UVB divided by control. Metabolites neither detected nor changed are marked in white. Dashed lines indicate omission of complex reactions.


Page 30 of 48

Page 31 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Immunoblot analysis of proteins involved in glycolysis and photosynthesis pathways. Proteins were extracted from leaves of C. roseus without (white column) or with UVB radiation (grey column). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto membranes. The membranes were cross-reacted with anti-FBPA, anti-TPI, anti-GAPDH, anti-RuBisCO LSU, anti-RuBisCO SSU, and anti-RuBisCO activase antibodies. CBB staining pattern was used as loading control (Supplemental Figure 3). Picture shows three independent biological replicates. The integrated densities of bands were calculated using ImageJ software. Data are shown as the means ± SD from three independent biological replicates. Student’s t-test was used to compare values between control and UVB radiation. Asterisk indicates a significant change (*p < 0.05).

Figure 8. Immunoblot analysis of proteins involved in redox and pathogenesis pathways. Proteins were extracted from leaves of C. roseus without (white column) or with UVB radiation (grey column). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto membranes. The membranes were cross-reacted with anti-APX, anti-GR, anti-PRX, anti-PR-1, anti-PR-5, and anti-PR-10 antibodies. CBB staining pattern was used as loading control (Supplemental Figure 3). Picture shows three independent biological replicates. The integrated densities of bands were calculated using ImageJ software. Data are shown as the means ± SD from three independent biological replicates. Student’s t-test was used to compare values between control and UVB radiation. Asterisks indicate a significant change (**p < 0.01, *p < 0.05).



Page 32 of 48

Table 1. List of phosphoproteins altered in leaves of C. roseus under UVB radiation. no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

protein ID a description CRO_T128764 eukaryotic elongation factor 5A-1 CRO_T119334 leucine-rich repeat protein kinase family protein CRO_T134832 ribulose-bisphosphate carboxylases CRO_T137967 putative WEB family protein CRO_T128592 pyruvate dehydrogenase complex E1 alpha subunit CRO_T135610 pyruvate orthophosphate dikinase CRO_T107361 autoinhibited Ca(2+)-ATPase CRO_T127037 eukaryotic translation initiation factor 4A isoform 2 CRO_T118301 2Fe-2S ferredoxin-like superfamily protein CRO_T101504 SET domain group CRO_T114609 P-glycoprotein CRO_T121911 phototropin CRO_T102115 SNF1-related protein kinase regulatory subunit CRO_T124199 ubiquitin-specific protease family C19-related protein CRO_T121086 calcium-binding EF hand family protein CRO_T119412 tobamovirus multiplication 2A CRO_T125672 dynamin-like protein CRO_T132447 glutamate decarboxylase CRO_T132061 ubiquitin-like superfamily protein CRO_T138166 tetratricopeptide repeat-like superfamily protein CRO_T124532 ARF-GAP domain CRO_T138569 transducin/WD40 repeat-like superfamily protein CRO_T133299 MIF4G domain-containing protein CRO_T110410 eukaryotic translation initiation factor 4G CRO_T136477 heat repeat protein ILITYHIA CRO_T114831 urease accessory protein G CRO_T102879 translation elongation factor EF1A CRO_T129565 sorting nexin CRO_T116372 villin CRO_T131761 ribosomal protein L10 family protein CRO_T126954 plasma membrane intrinsic protein 2;5

M.P.P. b fold change c -log(p-value) 3 10.1400 2.6724 2 8.7365 4.0989 1 14 1

8.4812 7.7604 7.5523

4.2719 3.1404 1.8478

3 2 1

7.5120 7.3444 7.2404

4.5051 2.9948 3.4267

2

7.2196

3.7409

1 4 5 4

7.0522 7.0367 6.9693 6.9664

3.5543 4.5968 1.4431 2.1544

7

6.7966

1.9291

5

6.7617

3.1672

3 4 1 3 6

6.5415 6.5382 6.4888 6.4757 6.4240

2.5712 1.7847 3.8418 2.7067 1.9286

1 5

6.3338 6.3162

3.5188 1.4369

1 3

6.2839 6.2377

2.6064 3.5070

1 1 1 1 1 3

6.2293 6.1436 6.0538 6.0022 5.9967 5.9867

3.0617 4.3153 2.6848 2.9083 1.9487 2.8400

2

5.8134

2.9227


Page 33 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

CRO_T109260 ubiquitin-specific protease family 4 C19-related protein CRO_T131097 H(+)-ATPase 3 CRO_T124414 early-responsive to dehydration 1 stress family protein CRO_T122628 sister chromatid cohesion 1 protein 1 CRO_T120156 polygalacturonase 10 CRO_T103362 thioredoxin family protein 2 CRO_T127722 autoinhibited H(+)-ATPase isoform 3 CRO_T127798 major facilitator superfamily protein 2 CRO_T128078 plastid-lipid associated protein PAP 1 CRO_T107978 pentatricopeptide repeat superfamily 2 protein CRO_T124369 ubiquitin-specific protease family 2 C19-related protein CRO_T136801 photosystem II reaction center 1 protein A CRO_T135049 alpha/beta-Hydrolases superfamily 2 protein CRO_T114984 protein of unknown function 1 (DUF1022) CRO_T110696 calcium-binding EF hand family 8 protein CRO_T119023 BSD domain-containing protein 3 CRO_T140949 60S acidic ribosomal protein family 2 CRO_T138602 COP9 signalosome subunit 6A 1 CRO_T114282 MAR-binding filament-like protein 1 CRO_T109992 calcium-dependent lipid-binding 1 family protein CRO_T119122 HSP20-like chaperones superfamily 2 protein CRO_T110778 phosphatidylinositol 3- and 4-kinase 2 family protein CRO_T131971 stress-inducible protein 1 CRO_T123693 arginine/serine-rich splicing factor 1 CRO_T111384 ATP binding cassette subfamily B1 1 CRO_T110214 MAP kinase 2 CRO_T113357 Octicosapeptide/Phox/Bem1p 1 family protein CRO_T137977 protein phosphatase 2A, regulatory 1 subunit PR55 CRO_T103993 phosphoenolpyruvate carboxylase 8 CRO_T121150 winged-helix DNA-binding 2 transcription factor family protein CRO_T102271 BAH domain; TFIIS helical bundle- 3 like domain CRO_T133624 CBS/octicosapeptide/Phox/Bemp1 1 33 ACS Paragon Plus Environment

5.8023

2.8294

5.7230 5.6635

2.8311 2.3832

5.6455 5.6282 5.6216 5.5749 5.5245 5.4522 5.4419

3.0980 2.5253 2.2652 3.2458 3.5837 2.6224 1.9259

5.3746

2.3851

5.2928

3.4270

5.2791

2.8491

5.2247

2.2744

5.2076

1.4054

5.1371 5.1359 5.1145 5.0877 5.0836

3.6220 1.7156 2.9072 3.0696 1.7996

5.0135

1.8003

4.9385

2.8487

4.9360 4.9320 4.9316 4.9178 4.8722

2.1909 6.6640 2.9830 2.4535 1.8117

4.8235

2.9558

4.8137 4.7899

2.9794 3.2092

4.7628

1.4496

4.7557

1.9412


64 65 66 67 68

CRO_T128982 CRO_T118369 CRO_T107578 CRO_T102291 CRO_T113891

69 70 71

CRO_T112624 CRO_T122727 CRO_T103020

72 73 74 75 76 77

CRO_T128060 CRO_T101186 CRO_T126499 CRO_T137286 CRO_T111333 CRO_T126626

78

CRO_T119138

79 80 81

CRO_T128873 CRO_T138848 CRO_T117808

82

CRO_T102960

83

CRO_T111083

84 85

CRO_T140837 CRO_T112617

86 87

CRO_T100566 CRO_T134028

88 89 90

CRO_T129975 CRO_T113191 CRO_T112447

91 92

CRO_T140929 CRO_T138307

93

CRO_T119283

94 95 96

CRO_T100074 CRO_T115327 CRO_T140625

domains-containing protein hyccin domain containing protein 1 calcium-dependent protein kinase 1 acyl-CoA binding protein 2 splicing factor, putative 3 calmodulin-binding motif family 3 protein K+ efflux antiporter 3 methyl-CPG-binding domain 3 Phosphoribulokinase / Uridine 3 kinase family trehalose phosphatase/synthase 2 target of Myb protein 4 ENTH/VHS/GAT family protein 2 RNA-binding protein-related 2 MA3 domain-containing protein 4 RNA-binding KH domain1 containing protein alpha/beta-Hydrolases superfamily 1 protein ankyrin repeat-containing 2B 1 Fe superoxide dismutase 4 ABC-2 type transporter family 4 protein stomatal cytokinesis defective 1 protein SIT4 phosphatase-associated family 2 protein cation efflux family protein 1 NAD(P)-binding Rossmann-fold 1 superfamily protein ARF-GAP domain 4 pentatricopeptide repeat superfamily 1 protein conserved hypothetical protein 1 high mobility group A 2 transducin/WD40 repeat-like 2 superfamily protein pleiotropic drug resistance 2 DNA glycosylase superfamily 1 protein NSP (nuclear shuttle protein)3 interacting GTPase sucrose phosphate synthase 1F 5 BRI1 suppressor 1-like protein 4 cation exchanger 1


Page 34 of 48

4.7496 4.7471 4.6673 4.6668 4.6571

1.3618 1.8627 2.7994 2.0627 1.8458

4.6522 4.6519 4.6026

1.3216 2.0476 1.4837

4.4740 4.4665 4.4518 4.4458 4.4396 4.4350

2.9154 1.5875 1.6924 1.4835 1.5560 2.5722

4.4077

2.6670

4.3985 4.3648 4.3562

3.7781 2.3930 1.4078

4.3490

2.6664

4.3471

2.6145

4.3301 4.3049

1.6275 1.4232

4.2935 4.2798

1.3152 2.3982

4.2773 4.2669 4.2378

1.6260 2.0469 1.8852

4.2309 4.2232

2.0976 2.0230

4.2169

1.5198

4.2036 4.1764 4.1544

1.4274 1.7517 3.1193

Page 35 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128

CRO_T129457 germin 1 CRO_T136447 malectin/receptor-like protein kinase 2 family protein CRO_T139458 protein kinase family protein 1 CRO_T125752 PB1 domain-containing protein 1 tyrosine kinase CRO_T118655 transducin family protein 2 CRO_T110763 cyclase associated protein 2 CRO_T118031 conserved hypothetical protein 2 CRO_T113034 DNA mismatch repair protein 3 MSH6 CRO_T105570 TCP family transcription factor 1 CRO_T124492 ARF GAP-like zinc finger3 containing protein ZIGA4 CRO_T130288 plasma membrane intrinsic protein 2 1C CRO_T117202 small ubiquitin-like modifier 2 CRO_T109998 Beige/BEACH domain containing 3 protein CRO_T128033 pyrophosphorylase 2 CRO_T129002 calmodulin-binding transcription 1 activator protein CRO_T125959 RING/U-box superfamily protein 2 with ARM repeat domain CRO_T141167 DHHC-type zinc finger family 4 protein CRO_T123404 calcium-dependent protein kinase 1 family protein CRO_T123068 C2H2 zinc-finger protein 1 SERRATE (SE) CRO_T132939 auxin transport protein (BIG) 1 CRO_T130058 WPP domain protein 2 CRO_T109116 multidrug resistance-associated 2 protein CRO_T118246 ribosomal L5P family protein 2 CRO_T120249 exocyst complex component sec5 2 CRO_T108225 leucine-rich repeat protein kinase 1 family protein CRO_T127805 oxidoreductases 4 CRO_T113477 Early-responsive to dehydration 4 stress protein CRO_T112228 Vps51/Vps67 family protein 1 CRO_T122780 WUS-interacting protein 1 CRO_T134154 DHHC-type zinc finger family 2 protein CRO_T135998 elicitor-activated gene 3-2 2 CRO_T119749 beta-amylase 1 35 ACS Paragon Plus Environment

4.1378 4.1360

1.5454 3.2849

4.0959 4.0778

1.7916 1.7307

4.0745 4.0623 4.0205 4.0084

2.3864 3.2958 1.7504 1.9796

3.9876 3.9653

1.9375 1.3654

3.9643

1.9010

3.9500 3.9445

2.0143 2.6550

3.9305 3.9288

1.8305 1.3216

3.8673

1.3638

3.8206

2.0201

3.7713

1.4390

3.7647

2.1873

3.7414 3.7370 3.7308

2.6533 3.7625 1.6524

3.7117 3.7075 3.7043

1.9730 1.4526 2.4850

3.7024 3.6742

2.4226 1.3580

3.6589 3.6354 3.6140

1.3897 1.9686 1.7552

3.6139 3.6002

2.9581 1.4773


129 130 131 132 133

CRO_T113629 CRO_T118252 CRO_T101502 CRO_T136550 CRO_T137138

134 135 136 137 138 139 140

CRO_T133953 CRO_T124586 CRO_T104334 CRO_T127304 CRO_T121603 CRO_T103840 CRO_T137419

141

CRO_T133136

142

CRO_T130421

143 144

CRO_T120876 CRO_T130723

145

CRO_T120918

146

CRO_T129877

147 148 149

CRO_T140077 CRO_T137277 CRO_T117115

150

CRO_T125990

151

CRO_T132355

152 153

CRO_T119641 CRO_T132919

154 155 156 157 158 159 160

CRO_T125894 CRO_T114863 CRO_T124156 CRO_T121358 CRO_T117309 CRO_T127983 CRO_T133359

161

CRO_T111354

SAP domain-containing protein 4 exocyst complex component sec10 2 CCT motif family protein 2 nuclear factor Y, subunit B1 1 trafficking protein complex II1 specific subunit protein kinase superfamily protein 1 CP12 domain-containing protein 3 ENTH/VHS family protein 1 COP1-interacting protein-related 1 pectin lyase-like superfamily protein1 integrin-linked protein kinase family1 AMP deaminase, 4 putative/myoadenylate deaminase, splicing factor PWI domain2 containing protein alpha/beta-Hydrolases superfamily 2 protein auxin efflux carrier family protein 1 protein of unknown function, 2 DUF584 calcium-dependent lipid-binding 1 family protein Octicosapeptide/Phox/Bem1p 4 family protein neurofilament protein-related 10 elongation factor Ts family protein 10 Tetratricopeptide repeat like 2 superfamily protein L-fucokinase/GDP-L-fucose 1 pyrophosphorylase Ypt/Rab-GAP domain of gyp1p 2 superfamily protein BSD domain-containing protein 2 Malectin/receptor-like protein 1 kinase family protein HOPM interactor 1 lipid phosphate phosphatase 1 MUTL-homologue 2 conserved hypothetical protein 1 ribosomal protein L5 2 protein kinase superfamily protein 3 leucine-rich repeat transmembrane 1 protein kinase NRAMP metal ion transporter 3 family protein


Page 36 of 48

3.5843 3.5563 3.5492 3.5252 3.5131

2.2332 2.1056 2.3106 1.3055 2.2203

3.5032 3.5000 3.4811 3.4708 3.4505 3.4375 3.4374

2.8897 1.5572 1.3059 2.0099 1.3249 1.3392 1.9026

3.4246

2.4050

3.4053

2.2184

3.3979 3.3957

1.9820 2.4669

3.3849

4.1839

3.3672

1.7656

3.3449 3.3285 3.3226

1.7942 2.8680 1.8812

3.3221

2.4468

3.2949

2.1814

3.2668 3.2489

1.5402 2.0304

3.2433 3.2394 3.2386 3.2284 3.2118 3.1807 3.1673

2.4972 1.5645 2.6612 2.0946 2.7489 2.2406 1.7586

3.1622

1.5847

Page 37 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


162 163 164 165

CRO_T127336 CRO_T105738 CRO_T106048 CRO_T112511

166 167 168

CRO_T126886 CRO_T120604 CRO_T112939

169

CRO_T137837

170 171

CRO_T135029 CRO_T110259

172

CRO_T102890

173 174 175 176 177 178

CRO_T115911 CRO_T125922 CRO_T125982 CRO_T114149 CRO_T121711 CRO_T127405

179

CRO_T133407

180 181 182 183

CRO_T120464 CRO_T138590 CRO_T136681 CRO_T128906

184 185

CRO_T138508 CRO_T115358

186

CRO_T127980

187 188

CRO_T129680 CRO_T127327

189

CRO_T138605

190 191 192

CRO_T122908 CRO_T139715 CRO_T117701

193

CRO_T131505

early nodulin-like protein RING/U-box superfamily protein CTC-interacting domain leucine-rich repeat protein kinase family protein mRNA-decapping enzyme subunit WEB family protein tonoplast monosaccharide transporter SPFH/Band 7/PHB domaincontaining membrane-associated protein family Protein kinase superfamily protein calcium-dependent phospholipidbinding protein protein phosphatase 2C family protein haemoglobin cytochrome B5 isoform E DUF632 domain containing protein ARM repeat superfamily protein WEB family protein phosphoprotein phosphatase inhibitors curculin-like (mannose-binding) lectin family protein nuclear factor Y, subunit B3 WPP domain interacting protein H(+)-ATPase protein-protein interaction regulator family protein ubiquitin-protein ligase ribosomal protein S19e family protein phosphoenolpyruvate carboxylase family protein conserved hypothetical protein SMAD/FHA domain-containing protein protein phosphatase 2C family protein calcium dependent protein kinase time for coffee homeodomain-like superfamily protein DNA-binding storekeeper proteinrelated transcriptional regulator

1 2 1 1

3.1605 3.1529 3.1350 3.1228

3.3567 2.3633 2.0663 2.7114

1 1 1

3.0752 3.0623 3.0613

1.5977 3.1055 2.1424

3

3.0243

2.1108

1 1

3.0211 3.0169

1.7385 1.5708

1

3.0001

2.3294

1 2 2 2 2 2

2.9977 2.9801 2.9755 2.9648 2.9546 2.9398

2.4440 1.7788 1.8671 2.2698 1.4107 1.9155

1

2.9380

1.4322

1 3 1 1

2.8897 2.8625 2.8287 2.7981

1.4159 1.6334 1.4290 1.9611

1 2

2.7740 2.7700

1.8403 3.0096

1

2.7472

4.2924

1 1

2.7217 2.7107

2.1625 1.8223

2

2.6830

1.7431

1 1 1

2.6769 2.6506 2.6451

1.9641 1.5661 2.3233

1

2.6357

1.8838



194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224

CRO_T118816 DEK domain-containing chromatin 1 associated protein CRO_T105024 photosynthetic electron transfer A 2 CRO_T122919 Presenilin-2 1 CRO_T133593 PDI-like 1-2 2 CRO_T104316 ribosomal protein S11 2 CRO_T124111 major facilitator superfamily protein 1 CRO_T137925 protein kinase superfamily protein 2 CRO_T129534 casein kinase 1-like protein 1 CRO_T117780 syntaxin of plants 2 CRO_T121805 bZIP transcription factor family 1 protein CRO_T123851 HXXXD-type acyl-transferase 1 family protein CRO_T107522 subunits of heterodimeric actin 1 filament capping protein Capz superfamily CRO_T126348 aldehyde dehydrogenase 11A3 2 CRO_T120400 Adenine nucleotide alpha 1 hydrolases-like superfamily protein CRO_T118752 Foie gras liver health family 1 1 domain containing protein CRO_T111000 RING/U-box superfamily protein 2 CRO_T115439 glycosyl hydrolase family protein 1 CRO_T116703 signal responsive 1 CRO_T114943 copper ion binding 1 CRO_T124267 RAB GTPase homolog G3A 1 CRO_T127792 CBS domain-containing protein 1 with a domain of unknown function (DUF21) CRO_T108517 pleiotropic drug resistance 2 CRO_T115988 white-brown complex homolog 1 protein CRO_T108435 protein of unknown function 1 (DUF761) CRO_T124162 translation elongation factor EF1B, 6 gamma chain CRO_T123063 protein of unknown function 1 (DUF630 and DUF632) CRO_T125662 Rieske (2Fe-2S) domain-containing 2 protein CRO_T132315 villin 6 CRO_T126030 plant VAP homolog 1 CRO_T122567 protein of unknown function 1 (DUF668) CRO_T119886 RNA-binding family protein 1


Page 38 of 48

2.6355

1.4022

2.6171 2.6132 2.6112 2.5662 2.5656 2.5487 2.5396 2.5197 2.4962

1.7036 3.2139 4.2225 1.3410 1.6697 1.7366 1.8244 1.3063 1.6425

2.4714

1.3240

2.4643

1.4047

2.4389 2.4296

2.3061 1.9000

2.4236

2.0537

2.3904 2.3430 2.3277 2.2787 2.2702 2.2367

2.1724 1.9973 1.7204 1.6402 1.5078 2.3878

2.2243 2.2059

2.6128 1.4073

2.1444

2.0348

2.1313

1.4738

2.1120

1.5714

2.0520

1.8735

2.0470 2.0260 2.0119

1.4824 1.4668 1.8846

1.9748

1.3869

Page 39 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242

CRO_T122570 tubulin beta-1 chain 1 CRO_T122877 tetratricopeptide repeat like 2 superfamily protein CRO_T115981 ribosomal protein S6 2 CRO_T120072 glyceraldehyde-3-phosphate 7 dehydrogenase C2 CRO_T109159 ATPase F0 complex 7 CRO_T129788 phosphoglucose isomerase 3 CRO_T106860 annexin 4 CRO_T103197 2-oxoglutarate and Fe(II)-dependent 1 oxygenase superfamily protein CRO_T117189 cytochrome B5 isoform B 2 CRO_T110797 plastid division 2 1 CRO_T109948 general regulatory factor 2 CRO_T106461 rhamnose biosynthesis 1 CRO_T126078 rubredoxin-like superfamily protein 3 CRO_T124261 copper ion binding;;zinc ion binding 1 protein CRO_T135012 HXXXD-type acyl-transferase 1 family protein CRO_T113737 -ketoacyl-acyl carrier protein 1 synthase I CRO_T133060 methyl esterase 3 CRO_T138466 SHV3-like 4

a “protein

1.9598 1.9400

2.2411 1.9200

1.9341 1.9213

1.7501 1.3971

1.9139 1.8838 1.8477 1.8217

1.5330 2.1655 2.8948 1.9154

1.8179 1.6452 1.6178 1.5191 1.4389 1.3653

2.8000 1.4223 1.3847 1.8914 1.6700 1.3723

1.3456

1.6238

1.2887

1.5291

-2.1828 -2.5778

1.3974 1.9375

ID” is determined according to Catharanthus roseus protein database. bM.P.P.

means the number of matched phosphopeptides. c “fold change” indicates log2 fold change of identified phosphoproteins under UVB radiation compared to control.



Figure 1 190x275mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 40 of 48

Page 41 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60







Page 42 of 48

Page 43 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60







Page 44 of 48

Page 45 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60







Page 46 of 48

Page 47 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60





For TOC Only 190x275mm (96 x 96 DPI)


Page 48 of 48

Phosphoproteomics Reveals the Biosynthesis of Secondary

Recommend Documents