Phosphoproteomic Analysis of Paper Mulberry Reveals

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Phosphoproteomic Analysis of Paper Mulberry Reveals Phosphorylation Functions in Chilling Tolerance Zhi Pi, Meiling Zhao, Xian-Jun Peng, and Shi-Hua Shen J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b01016 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Journal of Proteome Research

Phosphoproteomic Analysis of Paper Mulberry Reveals Phosphorylation Functions in Chilling Tolerance Zhi Pi,†,‡ Mei-Ling Zhao, †,‡ Xian-Jun Peng† and Shi-Hua Shen†,* †

Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of

Sciences, Beijing 100093, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: chilling tolerance, phosphorylation, paper mulberry, hub protein, CBF/DREB-responsive pathway

1

ACS Paragon Plus Environment


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ABSTRACT: Paper mulberry is a valuable woody species with a good chilling tolerance. In this study, phosphoproteomic analysis, physiological measurement and mRNA quantification were employed to explore the molecular mechanism of chilling (4 °C) tolerance in paper mulberry. After chilling for 6 hours, 427 significantly changed phosphoproteins were detected in paper mulberry seedlings without obvious physiological injury. When obvious physiological injury occurred after chilling for 48 hours, a total of 611 phosphoproteins were found to be significantly changed at the phosphorylation level. Several protein kinases, especially CKII, were possibly responsible for these changes, according to conserved sequence analysis. The results of GO analysis showed that phosphoproteins were mainly responsible for signal transduction, protein modification and translation during chilling. Additionally, transport and cellular component organization were enriched after chilling for 6 and 48 hours, respectively. Based on the protein-protein interaction network analysis, a protein kinases and phosphatases hub protein (P1959) was found to be involved in cross-talk between Ca2+, BR, ABA and ethylene-mediated signaling pathways. We also highlighted the phosphorylation of BpSIZ1 and BpICE1 possibly impacted on the CBF/DREB-responsive pathway. From these results, we developed a schematic for the chilling tolerance mechanism at phosphorylation level. INTRODUCTION Low temperature, as an extreme environment, is responsible for 30-40 % yield reduction in temperate growing areas.1 Along with temperature drop, the plants suffer 2


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both water imbalance and oxidative damage, which are respectively induced by decreasing root hydraulic conductance and reactive oxygen species (ROS) production.2, 3

To adapt to chilling, complex mechanisms such as osmolyte biosynthesis and

transportation,4 membrane lipid unsaturation5 and ROS detoxification6 have evolved. These responses are due to the reprogramming of gene expression, which is mediated by Ca2+ and phytohormones such as ABA,7 auxin,8 BR9 and ethylene.10 Recently, the novel gene COLD1, which interacts with G-proteins leading to the activation of the Ca2+ channel for temperature sensing, has been identified in rice. A SNP of COLD1 caused the adaptation to a cold environment in japonica rice.11 In Arabidopsis thaliana, gene microarray studies reveal that the large majority (approximately 30 %) of the most highly chilling-induced genes belong to the CBF/DREB regulons, suggesting that they play a major role in configuring the low-temperature transcriptome.12, 13 The activity and stabilization of transcription factors such as ICE1 and ABI5 implicated in CBF/DREB

and

phosphorylation14,

ABA-responsive

pathways

are

strictly

dependent

on

15

, ubiquitination16 and SUMOylation.16 Thus, post-translational

modification is an essential strategy for regulation of cellular processes in response to chilling. Phosphorylation is one of most frequent post-translational modifications due to over thousand kinase genes occupying 3-4 % of functional genes in plant.17 Benefiting from the advancement of functional genomic and mass spectrum tools, identification and quantification of phosphorylation is feasible at large scale level. It has been widely 3



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employed for understanding phosphorylation networks in subcellular organelles, such as chloroplast,18 mitochondria19 and plasma membrane;20 downstream substrates of key protein kinases including STN8,21 SnRK22 and OXI1;23 and molecular mechanism of biotic/abiotic stress24,

25

and hormone response.26,

27

To understand how

phosphorylation functions in chilling response and defense, a total of 42 phosphoproteins in leaf sheath of rice have been explored by 2-DE with 32P-labeling visualization and mass spectrometric identification, of which 4 phosphoproteins involved in glycolytic metabolism processes and Ca2+-mediated signaling were demonstrated to be significantly changed after chilling.28 In the following years, 2-DE coupled with Pro-Q Diamond stain was employed to explore chilling-related phosphoproteins in rice roots29 and cell suspensions of A. thaliana.30 Although this information suggested the importance and ubiquity of phosphorylation in response to chilling, it merely focuses on model herbaceous plants and is still far from sufficient to understand the complex mechanism of chilling response owing to the limitation of 2-DE separation ability and staining sensitivity. The development of specific phosphopeptide enrichment and high resolution mass spectrum allows us to explore thousands of phosphoproteins simultaneously.18 Therefore, we applied the TiO2 enrichment and LTQ-Orbitrap mass spectrometry for exploring the phosphoproteome characterization of paper mulberry under chilling. Paper mulberry (Broussonetia papyrifera L.), which belongs Moraceae family, is a dioecious, perennial and woody species.31 The whole plant has high value in paper 4


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making, stock farming and medicine. Thus, most studies are mainly focus on analyzing fiber structure,32 nutrients33 and medicinal ingredients.34 However, its environmental adaptation characterization has been relatively ignored. It natively possesses wide temperature adaption owing to natural distribution in China from Hainan (19°12′N 109°42′E) to Beijing (39°55′N 116°23′E).35 Individuals are tolerant of frost and can overwinter in the place with -25 °C. Unfortunately, there are few studies focus on molecular mechanism of chilling response and defense in paper mulberry, including several previous studies on chilling-related transcriptomics and proteomics.36, 37 Low temperature is still the major limiting factor that significantly restrict the extension of the paper mulberry planting, which presents a challenge to the further production of the paper mulberry. In this study, quantitative phosphoproteomics has been applied to explore the chilling response and defense mechanism in paper mulberry at phosphorylation level. It not only broadens the understanding of how mulberry paper resists to low temperature stress, but also provide the opportunity to discover the candidate phosphorylated proteins and the potential pathway for chilling in paper mulberry.

MATERIALS AND METHODS Plant material and chilling treatments Paper mulberry (Broussonetia kazinoki × Broussonetia papyifera) were cultured on Murashige and Skoog medium in an artificial climatic chamber kept at 26 °C with a 5



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14/10 hours photoperiod (day/night), quantum flux density of 80 µmol/(m 2 s). After 25 days, plantlets were transferred to 4 °C for chilling treatments. Only the last three fully expanded leaves of paper mulberry with the similar growth vigor were harvested in this study, and a mixed sampling strategy was adopted to eliminate differences between individuals. Both short-term (0, 6, 12, 24, 36 and 48 hours) and long-term (0, 2, 4, 6, 8 and 10 days) chilling treatments were set for detecting physiological changes. Samples were rapidly frozen in liquid nitrogen and stored in -80 °C for further protein and RNA extraction. Three treatments (0, 6 and 48 hours treatment) were also prepared for protein extraction. Additionally, the leaves with 0, 2, 6, 12 and 24 hours chilling were sampled for RNA-seq analysis. For quantitative RT-PCR analysis, chilled paper mulberry leaves were respectively sampled at different time points (0, 1, 3, 6, 12, 24, 24 and 48 hours). Three independent biological replicates of control and treated samples were analyzed for physiological and phosphoproteomic analysis. RNA-seq analysis was performed without replicate, while the expression of interesting genes was checked by Q-PCR with three biological replicates. Physiological characteristics measurement Chlorophyll fluorescence was analyzed using a MAXI-IMAGING-PAM chlorophyll fluorometer (Heinz Walz, Effeltrich, Germany). Briefly, the whole plantlets were dark adapted for 30 minutes after treatment, and the maximum quantum yield of PSII (Fv/Fm) was determined for each sample by analyzing fully expanded intact leaves. The method described by Yan et al.38 was employed to measure relative electrolytic 6


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leakage (REL). Four leaves from each treatment were cut off and immersed in tubes with 10 mL deionized water. The tubes were placed on a shaker at 28 °C. Two hours later, the electrical conductivity of bathing solution (Lt) was measured by conductivity meter (INESA instrument, Shanghai, China). Then the tubes were incubated in boiling water for 20 minutes and subsequently cooled down to 28 °C. The electrical conductivity (Lo) was measured again. The relative electrolyte leakage was calculated by the formula: REL= Lt/Lo × 100 Malondialdehyde (MDA) content was determined by the thiobarbituric acid reaction. 39

First, 0.3 g leaves was ground with an ice-cooled mortar and pestle and then extracted

with 3 mL 10 % (w/v) trichloroacetic acid. The homogenate was then centrifuged at 8,000 g for 10 minutes at 4 °C. Then, 3 mL supernatant and 3 mL 0.05 % (w/v) TBA were mixed and heated for 30 min in a boiling water bath and then cooled on ice to stop the reaction. The concentration of MDA was calculated as follows: MDA=6.45(A532 − A600) − 0.56A450 Protein extraction and digestion Protein was extracted using a modified method according to Shen et al.40 Briefly, 0.5-1 g leaves were homogenized in 2 mL of the homogenization buffer containing 20 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM ethylene glycol tetraacetic acid, 5 % 2-mercaptoethanol, 1 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Mixture (Roche, Mannnheim, Germany). Then, the homogenate was centrifuged at 10,000 g for 10 minutes at 4 °C. The supernatant was 7



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mixed with a quarter of the volume of 50 % (w/v) trichloroacetic acid to attain 10 % (w/v) trichloroacetic acid in final mixture. After incubating in an ice bath for 30 minutes, the mixture was centrifuged at 15,000 g for 10 minutes at 4 °C, and the supernatant was discarded. After washing three times with acetone, the pellet was collected by centrifugation and air dried. Then, 3 mg total protein from each sample was resuspended in 1500 µL of 4 % SDS and 0.1 M DTT in 0.1 M Tris-HCl, pH 7.6, at room temperature, and following the filter-aided sample preparation (FASP) protocol described elsewhere.41 Briefly, for every 500 µg protein, 500 µL of 8 M urea in 0.1 M Tris/HCl, pH 8.5 (UA) was added, and samples were centrifuged at 12,000 g at 20 °C for 30 minutes. This step was performed three times. Then, 100 µL of 0.05 M iodoacetamide in 8 M urea was added to the filters, and the samples were incubated in darkness for 20 minutes. Filters were washed twice with 400 µL of 8 M UA, followed by two washes with 400 µL of 50 mM NH4HCO3. Finally, 1 µg/µL trypsin (Promega, Madison, USA) was added in 150 µL of 50 mM NH4HCO3 to each filter. The protein to enzyme ratio was 100:1 (w/w). Samples were incubated overnight at 37 °C, and released peptides were collected by centrifugation. Phosphopeptide enrichment The phosphopeptide enrichment procedure was described elsewhere with some modification.42, 43 In detail, the TiO2 beads (GL Sciences, Tokyo, Japan) were incubated in 800 µL loading buffer containing 65 % ACN and 2 % TFA saturated by glutamic acid. Tryptic peptides (3mg) were resuspended in 800 µL loading buffer. After shaking the 8


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mixture of tryptic peptide and TiO2 beads (1:1, w/w) for 40 minutes and collecting TiO2 beads by centrifugation, 800 µL wash buffer (65 % ACN and 0.1 % TFA) was added and shaken for 40 minutes twice. Subsequently, TiO2 beads were eluted twice with 800 µL elution buffer (500 mM NH4OH and 60 % ACN). The eluates were dried and reconstituted in 0.1 % formic acid for MS analysis. LC-MS/MS Analysis LC was performed on an Easy-nLC System (Thermo Fisher Scientific, Odense, Denmark). Peptides were separated on a 15-cm fused silica emitter packed in-house with the reverse phase material ReproSil-Pur C18AQ, 3 µm resin (Dr. Maisch, Ammerbuch, Germany) with a 55 minutes gradient at 300 nL/min from 5 % to 22 %, and 5 minutes from 22 % to 90 % of 100 % ACN/0.1 % acetic acid (v/v). The quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was operated in the positive ion mode using a data dependent “top 12” method. Survey scans (m/z 300-1500) and MS/MS scans were acquired at a resolution of 70,000 and 17,500 at m/z 200. Up to the top 12 most abundant isotope patterns with charge ≥ 2 from the survey scan were selected with an isolation window of 2 Th and fragmented by HCD with normalized collision energies of 25. For accurate mass measurements, the lock-mass option was employed (445.120025).44 The maximum ion injection times for the survey scan and the MS/MS scans were 50 ms and 100 ms, respectively, and the ion target values were set to 1E6 and 1E5, respectively. Selected sequenced ions were dynamically excluded for 30 seconds. 9



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Protein identification and phosphopeptide quantification Raw mass spectrometric data were analyzed with the MaxQuant software (version 1.5.2.8).45 A time-dependent mass recalibration algorithm was used for recalibration to improve the mass accuracy of precursor ions.46 MS/MS spectra were searched by the Andromeda search engine, which is incorporated into the MaxQuant software suite, against the paper mulberry protein database (28,908 entries), whose protein sequences were predicted based on genome and transcriptome sequencing. For the search, trypsin allowing for cleavage N-terminal to proline (trypsin/P) was chosen as enzyme specificity. Cysteine carbamidomethylation was selected as a fixed modification, while protein N-terminal acetylation, methionine oxidation and phosphorylation on serine/threonine/tyrosine were selected as variable modifications. Maximally, two missed cleavages were allowed. For MS and MS/MS, the tolerances of the main search for peptides were set at 7 ppm and 20 ppm, respectively. A false discovery rate (FDR) of 0.01 for proteins and peptides and a minimum peptide length of 7 amino acids were required. Identified peptides that are all shared between two proteins were combined and reported as one protein group. Proteins matching to the reverse database were filtered out. For high-confident phosphorylation site localization, the following two criteria were defined: their localization probability for the assignment was at least 0.75, and the PTM score difference from the second possible localization assignment was 5 or higher. The median localization probability of such class I sites is almost always higher than 0.99.47 Phosphopeptides were quantified by their extracted ion 10


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chromatograms (‘Intensity’ in MaxQuant),47,

48

enabling the ‘match between runs’

option (time window 2 minutes). Then, normalization followed with imputation as Karpievitch et al.49 described were performed to remove systematic biases and obtain a complete matrix of intensities. Briefly, phosphopeptide intensity distributions were log-transformed to base 2, normalized across biological replicates by linear regression, and standardized by median absolute deviation and mean centering across samples with InfernoRDN.50 Subsequently, missing intensity value was imputed by lowest observed value of each treatment. The number of imputations for each phosphopeptide was restricted to three. Only the phosphopeptides that met the following restrictions were regarded as significantly changed phosphoproteins: (1) phosphorylation site localization probability greater than 0.75 and phosphorylation site score difference not less than 5; (2) the p value of one-way ANOVA analysis < 0.05; and (3) the fold change between each treatments > 2. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD005516.51 Transcriptomics analysis The transcriptomics analyses were prepared according to our former study.52 Briefly, total RNAs were isolated with TRIzol Reagent (Life Technologies, Shanghai, China) from each sample according to the manufacturer’s instructions. RNAs were treated with RNase-free DNase I (Takara, Dalian, China) to remove the DNA residues. The quality and purity of RNAs were assessed with OD260/230 ratio and RNA integrity 11



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number (RIN) using the NanoDrop 2000 (Thermo Fisher, Waltham, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA), respectively. mRNAs were purified from the mixed high-quality total RNA using Oligo (dT) RNA purification. First strand cDNAs were synthesized using Oligo (dT) primer, then second strand cDNAs were synthesized using RNase H and DNA polymerase I. Two different adapters were ligated to the cDNA fragments, which were then purified using second strand AMPure XP beads (Beckman Coulter, California, USA). cDNA templates were enriched by multiplexing PCR with primers 1.0 and 2.0. Barcodes were added to each library during amplification. Raw sequence data were generated by the Illumina pipeline and are available in NCBI’ s Short Read Archive (SRA) database under accession number SRP029966. All of the clean reads were pooled together and assembled to form the global transcriptome of the paper mulberry. To obtain the transcriptome of each sample, raw data were also assembled for each sample. The expression level of every transcript in each sample was calculated by quantifying the number of Illumina reads that mapped to the paper mulberry transcriptome. The raw gene expression counts were normalized using the RPKM (reads per kb per million reads) value. For screening of differentially expressed genes, p values that correspond to differentially expressed genes (DEGs) were obtained via a general

Chi

squared

test

that

was

performed

using

IDEG6

(http://telethon.bio.unipd.it/bioinfo/IDEG6/). The threshold of p values in multiple tests was checked through manipulating the false discovery rate (FDR) value. Among 12


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the five samples, the transcripts with the highest RPKM value more than 5 and the ratio of RPKM between samples of more than 3 (fold change > 3) and an FDR < 0.01 were considered to have significant changes in expression in response to chilling. Quantitative RT-PCR analysis Total RNA was isolated from leaf tissue using TransZol Plant Mini kit (TransGen, Beijing, China), and the DNA was digested using DNase I (Takara, Dalian, China) according to the manufacturer's instructions. RNA integrity was checked on an agarose gel and quantified by using the NanoDrop 2000 (Thermo Fisher, Waltham, USA ). The first-strand cDNA for each sample was prepared from 1 µg of total RNA using SuperScript II reverse transcriptase (Takara, Dalian, China) following the manufacturer’s instructions, and template cDNAs were diluted 5-fold for PCR. Gene-specific primers were designed using the Primer 5 program and are listed in Table S-1. Quantitative RT-PCR analysis was performed by our former study.36 Samples and standards were run in triplicate on each plate using the SYBR® Premix Ex Taq™ II kit (Takara, Dalian, China) on a StepOneTM real-time PCR system (Applied Biosystems, Foster City, USA) following the manufacturer’s recommendations. Real-Time PCR was performed in a 20 µL reaction volume containing 6.8 µL of ddH2O, 10 µL of SYBR® Premix Ex Taq II, 0.4 µL of ROX Reference Dye II, 0.4 µL of forward primer (10 µmol/L), 0.4 µL of reverse primer (10 µmol/L), and 2 µL of template cDNA. The PCR programs were run as follows: 30 seconds of pre-denaturation at 95 °C, 40 cycles of 5 seconds at 95 °C and 30 seconds at 60 °C, followed by steps for dissociation curve 13



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generation. Dissociation curves for each amplicon were carefully examined to confirm the lack of multiple amplicons at different melting temperatures. Relative transcript levels for each sample were obtained using the comparative cycle threshold method using the cycle threshold value of glyceraldehyde-3-phosphate dehydrogenase gene as a control. Bioinformatics Protein

function

was

annotated

through

Blast2GO

software

(https://www.blast2go.com/home). Enrichment analysis was conducted using the Singular

Enrichment

Analysis

(SEA)

tool

in

the

AgriGO

toolkit

(http://bioinfo.cau.edu.cn/agriGO/).53 Customized annotated reference was selected and uploaded based on our genomic sequencing. Under advanced options, the gene ontology type chosen was Plant GO slim. Significantly changed phosphorylation sites were subjected to k-means clustering using MultiExperiment Viewer (MeV). The Euclidean distance of log-ratios normalized to their respective maxima per phosphorylation site was used to measure the distance between regulated phosphorylation sites. The iceLogo program (version 1.2) was used for the analysis of sequence features adjacent to the significantly changed phosphorylation sites.54 The 15 amino acids surrounding each phosphorylation site were extracted and aligned as an experimental set. The reference dataset consisted of non-phosphorylated peptides, which were generated by random sampling from the full sequences of significantly changed 14


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phosphoproteins as Schmidt et al.55 described. In addition, another reference dataset containing all of the non-changed phosphopeptides that were identified in our mass spectrometric analysis was used for calculation. The analysis was visualized using iceLogo figures with percentage difference as scoring system (p < 0.05). The protein-protein interaction of the phosphoproteins was analyzed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (http://string-db.org/) database of physical and functional interactions. The confidence score was set at the high level (> 0.700). The protein-protein interaction network was arranged and displayed using Cytoscape (version3.3.0) software (http://www.cytoscape.org/). Protein domains were searched by Pfam (http://pfam.xfam.org/) and PlantsP (http://plantsp.genomics.purdue.edu/index.html).

RESULTS Physiological changes of paper mulberry after chilling After 25 days growth, paper mulberry plantlets were transferred to 4 °C for 10 days chilling treatment. The growth of paper mulberry was hardly inhibited by chilling (Figure 1A). A slight wilting of leaves occurred after chilling for 2 days and then was gradually aggravated. However, necrotic areas and external discoloration were not caused. According to chlorophyll fluorescence (Figure 1B), leaf age and position were correlated with chilling sensitivity. The top three fully expanded leaves of paper mulberry were highly sensitive to chilling and were selected for further measurement. 15



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In contrast, the older leaves located at the bottom did not show obvious change until 4 days after chilling. The change of Fv/Fm value in the top three fully expanded leaves was calculated and shown in Figure 1C. It was markedly suppressed with prolonged chilling time, and the Fv/Fm nearly reached the lowest point (approximately 0.2) after chilling for 6 days. To understand when PSII was first damaged by chilling, a short-term series of chilling treatments was performed. The results show that no significant change occurred within 1 day, and values of Fv/Fm were decreased from 0.827 to 0.631 after chilling for 2 days. The value of relative electrolytic leakage soared from 8.29 % to 86.99 % during chilling for 10 days (Figure 1D). The significant increase was first observed at 1 day after chilling. It is worth noting that the degree of relative electrolytic leakage increase within the last 4 days after chilling was nearly double times that of the first 6 days. These findings implied that the plasma membrane of paper mulberry was relatively stable within 1 day after chilling, and drastic collapse of plasma membrane occurred after chilling for 6 days. MDA content of leaves presented a gradual rising tendency as chilling time increased (Figure 1E). The significant increase of MDA content was detected within 1 day after chilling, which was earlier than plasma membrane leakage and photoinhibition. At last, MDA content had been increased approximately 2.4-fold comparing to control. Comparative analysis of paper mulberry phosphoproteomics after chilling. 16


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To investigate the phosphorylation involved in chilling tolerance, we evaluated the changes in the phosphoproteome of paper mulberry leaves after chilling for 6 and 48 hours, when non-significant and significant physiological injury was detected. In total, 2,478 phosphopeptides with 3105 phosphorylation sites corresponding to 1,509 phosphoproteins were identified (Table S-2), and the proportions of pS, pT, and pY sites were estimated as 84.7 %, 15.0 %, and 0.3 %, respectively. To evaluate the quality of the complete experiment, correlation analyses for replicates and PCA analysis were performed. Three samples were individually clustered with their replicates with Pearson correlation coefficients between 0.74 and 0.93 (Figure S-1). A total of 933 significantly changed phosphorylation sites corresponding to 719 phosphoproteins were screened out (Figure 2A). Compared to untreated leaves, 509 and 768 phosphorylation sites corresponding to 427 and 611 phosphoproteins were differentially changed after chilling for 6 hours and 48 hours, respectively (Table S-3 and 4). There were 253 and 256 phosphorylation sites up-regulated and down-regulated, respectively, at 6 hours under chilling (Figure 2B). After chilling for 48 hours, a total of 628 and 140 phosphorylation sites were respectively up-regulated and down-regulated at the phosphorylation level (Figure 2B). Between 6 hours treatment and 48 hours treatment, there were 683 phosphorylation sites corresponding to 555 phosphoproteins screened out as significantly changed at the phosphorylation level (Table S-5), of which 568 phosphorylation sites were up-regulated and 115 phosphorylation sites were down-regulated at the phosphorylation level. A total of 16, 17



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22 and 168 phosphorylated sites were uniquely detected after chilling for 0, 6 and 48 hours, respectively (Figure 2B). Then, all of the significantly changed phosphorylation sites were classified into eight groups with four up-regulated and down-regulated patterns (Figure 2C). IceLogo analysis of phosphorylation sites in each cluster. To eliminate any amino acid frequency bias, non-phosphorylated peptides from full-length phosphoproteins were used as a reference dataset (Figure 3A). Two clear consensus motifs ([SP] and [RxxS]) were enriched in the whole clusters and occupied a relatively high percentage. An acidic motif ([SxE]) was also observed in Cluster 4. However, only [SxE] was overrepresented compared to non-changed phosphopeptides (Figure 3B). Additionally, histidine at position -2 presented a preference of Cluster 1, 2, 4 and 7 against both different reference datasets. Glycine residue at position -1 was enriched in Cluster 5 and 8, containing down-regulated phosphopeptides after chilling. Of note, the percentage difference of these residues compared with two reference datasets was always below 10 %, and some did not belong to clear consensus motifs. GO annotation and enrichment analysis of significant changed phosphoproteins Blast2GO and AgriGO were applied to annotate and analyze significantly changed phosphoproteins. From the biological process perspective, protein modification, signal transduction and translation were significantly overrepresented after chilling for 6 and 48 hours (Figure 4A). Amongst protein modification, 77 protein kinases and 15 phosphatases possessed over 70 % of phosphoproteins (Table 1 and Figure 5A). For 18


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signal transduction, the ABA, BR, light, Ca2+ and phosphatidylinositol-mediated signaling accounted for over half of signal transduction terms under 48 hours treatment (Figure 5B). In terms of translation, phosphorylation mainly happened in ribosomal proteins, RNA binding proteins and translational initiation factors after chilling (Figure 5C). Transport was merely enriched after chilling for 6 hours. The majority was protein and ion transport, followed by hormone and water transport (Figure 5D). With chilling duration, cellular component organization was also overrepresented. Cellular component organization was found to contain up to 22 % cytoskeleton organization, when chilling injury was aggravated after 48 hours (Figure 5E). The other phosphoproteins were also involved in the term of cell morphogenesis, chromatin organization, protein complex organization and chloroplast organization, which had 16 %, 15 %, 13 % and 12 %, respectively, of cellular component organization. In addition, protein modification processes were enriched in both Cluster 3 and Cluster 4. Cellular component organization and signal transduction were also overrepresented in Cluster 3. From the molecular function perspective, kinase activity as well as binding for RNA, protein, nucleotide and lipid was overrepresented (Figure 4B). Among these, kinase activity and protein binding were enriched after chilling for 6 hours and 48 hours. Additionally, lipid binding was overrepresented after chilling for 6 hours, and binding for nucleotide and RNA were only enriched after chilling for 48 hours. Regarding cellular component perspective for 6 hour and 48 hour chilling treatments, significantly 19



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changed phosphoproteins were mainly located in the nucleus, plasma membrane, cytosol, thylakoid and ribosome. Transcriptional expression analysis of paper mulberry after chilling After stringent quality checks and data cleaning, we obtained 12, 992, 767, 550 raw reads, totaling 9.164 Gb. The sequencing data output and quality assessment were listed in Table S-6. By applying screening thresholds of 3-fold changes and FDR < 0.01, a total of 5,800 DEGs were detected as being involved in chilling tolerance (Figure 6A). Then, a total of 274 differentially expressed protein kinases and phosphatases were screened out at the transcriptional level in response to chilling, accounting for approximately 5 % of total DEGs (Figure 6B). Relative to the control, 24 protein kinases and phosphatases with 13 up-regulated genes and 11 down-regulated genes were detected after chilling for 2 hours. There were 203 protein kinases and phosphatases detected between control and 6 hours treatment. Among these, 158 genes were up-regulated and 45 genes were down-regulated. In addition, 25 and 40 protein kinases and phosphatases were up-regulated after chilling for 12 and 24 hours, respectively, while 12 and 9 protein kinases and phosphatases were down-regulated. To understand the transcriptional expression pattern corresponding to significantly changed phosphoproteins, RNA-seq of the paper mulberry transcriptome under chilling was performed. It is notable that the expression patterns of 706 corresponding mRNAs contained 554 non-differentially and 152 differentially expressed mRNAs was 20


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determined (Figure 6C and Table S-7). Subsequently, q-PCR was performed to reassess the expression of interesting genes (Figure 6D). The expression of 11 genes, including KEG, SIZ1 and PLC2, was not induced by chilling, while corresponding proteins were significantly changed at the phosphorylation level. The up-regulation of the BSL3 gene occurred after chilling for 24 hours, and changes at the phosphorylation level were detected after chilling for 6 hours. The transcriptional expression of CAT was down-regulated after chilling, while the corresponding protein was significantly phosphorylated. Additionally, the expression pattern of BpDREB1 was analyzed, which increased to the maximum within 6 hours after chilling, and then drastically decreased (Figure S-2). Protein-protein interaction analysis of protein kinases and phosphatases Considering the essential role of protein kinases and phosphatases, 363 protein kinases and phosphatases significantly changed in response to chilling at the transcriptional or phosphorylation levels were used for establishing a protein-protein interaction network (Table 1 and S-8). Finally, a protein-protein interaction network containing 93 nodes and 303 edges was demonstrated through Cytoscape (Figure 7). These proteins showed relationships with MAPK cascades as well as light, ABA, cyclin, ethylene, BR and Ca2+-mediated signaling pathways. A potential hub protein (P1959) was noted in the center of network with 52 first neighbors; these neighbors were spread in a wide distribution of signal transduction, including MAPK cascades as well as ABA, ethylene, BR and Ca2+-mediated signaling pathway. Among these, STN8 21



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with 25 neighbors was associated with cyclin-mediated signaling pathway. For further understanding the features of P1959, functional domain analysis was performed. P1959 contained three domains, namely a protein phosphatase 2C domain, a cyclic nucleotide-binding domain and a protein kinase domain located in order from the N-terminal to C-terminal (Figure S-3). To estimate conservation of phosphorylation sites, sequence alignment was computed with homologous sequence of Morus alba, P. trichocarpa, A. thaliana and Glycine max. The result indicated that three domains and phosphopeptides demonstrate high conservation in all five species.

DISCUSSION Phosphorylation was essential for chilling tolerance in paper mulberry Unlike chilling sensitive crops such as rice, cotton and maize, there was no obvious physiological injury of cellular membrane and chloroplast occurring within 24 hours (Figure 1). The Fv/Fm of chilling sensitive crops such as rice, maize and cotton were generally decreased over 50 % at 7 °C within 20 hours.56 The significant increase of REL and MDA were also reported within several hours after chilling.38, 57 These results suggested that paper mulberry had good chilling resistance, although it failed to resist long-term chilling. Through a combination of phosphoproteomic and transcriptomic approaches, we performed a comprehensive analysis of chilling tolerance in paper mulberry leaves for the first time. Phosphorylation changes widely occurred in chilling tolerance and the 22


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numbers drastically increased with chilling duration, especially after physiological injury occurrence (Figure 2). It is notable that a high proportion of significantly changed phosphoproteins were not induced at the transcriptional level (Figure 6C), indicating the essential role of phosphorylation in chilling tolerance. Subsequently, we employed iceLogo for sequence analysis against non-phosphorylated peptides and non-changed phosphopeptides (Figure 3). The most obvious difference between the two methods was that [SP] and [RxxS] were highly enriched against with non-phosphorylated peptides, but not with non-changed phosphopeptides. A possible reason was that the MAPKs,58 CDKs,58 or CDPKs59 recognizing [SP] or [RxxS] might catalyze not only chilling-responsive proteins but also other proteins. In contrast, [SxE] was constantly overrepresented by different references. Thus, CKIIs might act as an important protein kinase causing increasing protein phosphorylation under chilling.60 Previous phosphoproteomics studies in abiotic stress have showed that phosphorylation changes mainly happened in regulatory proteins involving signal transduction, transcription, translation and transport.61, 62 Similarly, phosphorylation events were implicated in these functional categories against chilling. In addition, chilling-induced phosphorylation contributed to cellular component organization and protein modification (Figure 4). A recent phosphoproteomics study in a chill-tolerant fly showed that the term of cytoskeleton organization was highly enriched, and phosphorylation may play a role in cytoskeleton depolymerization.63 Together with the above phosphoproteomic results, a complex cellular network regulated by 23



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phosphorylation

was concluded,

as

illustrated

in

Page 24 of 67

Figure

8.

Briefly,

the

chilling-responsive signaling was transduced through protein phosphorylation, which was invoked by phytohormones, Ca2+ light and phosphoinositide. The expression of chilling-related proteins was controlled by phosphorylation, mainly involving ribosome and translation initiation factors. During early chilling, phosphorylation also played an important role in the regulation of transport for homeostasis. With physiological injury caused by chilling, the proteins related to cytoskeleton and chloroplast organization were phosphorylated against chilling. A hub protein (P1959) was associated with a number of chilling-responsive signaling pathways. The term of protein modification was highly enriched after chilling for both 6 and 48 hours, containing over 70 % proteins that participated in protein phosphorylation or dephosphorylation (Figure 4A and 5A). Interestingly, a protein kinase and phosphatase hub protein (P1959) interacted with dozens of protein kinases and phosphatases including CIPK23, SRK2E, KEG, BSL1 and CTR1. Its A. thaliana orthologue (AT2G20050) has also been noticed as hub protein among protein kinases in response to oligogalacturonides, which is a class of plant damage-associated molecular patterns.20 The N-terminal and C-terminal part of P1959 exhibited homology with protein phosphatase 2C and cGMP/cAMP-dependent protein kinase, respectively (Figure S-3). In plants, they are considered essential components of Ca2+, ABA, cAMP and cGMP-mediated signaling pathways.64-66 This could account for the interaction of 24


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P1959 with such a large number of protein kinases and phosphatases. However, there were few studies of its molecular functions hitherto, and the impact of P1959 phosphorylation on the protein kinase and phosphatase network is still worthy of future study. The neighbors of P1959 were associated with a number of chilling-responsive signaling and regulated multiple biological processes. When plants are exposed to chilling temperature, Ca2+ is generally accumulated in cytosol67 and regulates several metabolism processes, including ROS scavenging68 and K+ transport.69 The phosphoproteomic analysis in rice has been investigated, and phosphorylation of OsCDPK13 is an important signaling component in the response to cold stress.28 Consistent with this, two BpCDPKs (P315 and P847) were found to be up-regulated at the phosphorylation level after chilling. For the ROS scavenging system, two antioxidant enzymes, namely CAT (P1091) and APX (P1886), were phosphorylated in response to chilling, whose phosphorylation sites belong to [K/RxxS]. This indicated that CDPKs as possible upstream protein kinase play a role in controlling the activity of antioxidant enzymes against chilling. BpCIPK23 (P1406) was also found to exhibit phosphorylation of Thr 190 located within the activation loop. It has been reported to function in phosphorylation of the K+ channel and contributes to K+ uptake.70 It might be responsible for the phosphorylation of two potassium transporters (P1512 and P1669) detected after chilling for 48 hours. In addition to Ca2+-mediated signaling pathway, phosphorylation changes were implicated in phytohormone-mediated 25



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signaling. The BpSRK2E (P2045), BpSRK2I (P1113) and BpKEG (P2676) involved in ABA-mediated signaling pathway were observed to be up-regulated after chilling for 48 hours. The phosphorylation sites of BpSRK2E and BpSRK2I were positioned in the activation loop, where they are possibly responsible for activation of downstream transcription factors.71 BpKEG, as a negative regulator, would be self-degraded after phosphorylation.72 In BR and ethylene-mediated signaling pathway, several protein kinases and phosphatases were identified and shown to be phosphorylated. Although whether these phosphorylation sites were regulatory was unclear, the downstream substrates BpBES1/BZR1 (P675)73 and BpEIN2 (P428)74 exhibited phosphorylation changes at regulatory sites. This finding implied that phosphorylation was implicated and activated these signaling pathways in response to chilling. Phosphorylation was involved in chloroplast movement against chilling In the protein kinase and phosphatase network, a small group contained two phototropins associated with light-mediated signaling (Figure 7). In A. thaliana, phototropins are considered to mediate not only phototropism and stomatal opening, but also chloroplast movement.75, 76 Chloroplast movement is an efficient strategy to alleviate PSII damage and maintain maximal photosynthetic output.77 In plants, many studies have revealed that low temperature can facilitate light-induced chloroplast movement, which is one of the vital mechanisms for chilling tolerance plants.78, 79 In current study, mass spectrometry showed that multiple phosphorylation sites of 26


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BpPHOT1 (P285) and BpPHOT2 (P2400) were up-regulated at the phosphorylation level after chilling for 6 and 48 hours. Autophosphorylation is frequently observed accompanied with activation of phototropins in A. thaliana.80 For AtPHOT1, residues in the hinge region between LOV1 and LOV2 are significantly more phosphorylated under strong light than in darkness.81 A total of five chilling-induced phosphorylation sites in BpPHOT1 were located in the hinge region between the LOV1 and LOV2 domain (Figure S-4). Among these, Ser 386 and Ser 426, which were respectively found to correspond with Ser 350 for AtPHOT1 and Ser 358 for VfPHOT1, underwent autophosphorylation by blue light. The phosphorylation at these sites was also essential for 14-3-3 protein binding.82 The 14-3-3 proteins was thought to bind to the phosphorylated target leading to a change in structure that regulates activity.83 In addition to signaling response, chloroplast movement depends on different numbers of chloroplast-actin filaments on the front and rear regions of the moving chloroplast.84 After chilling, we observed chilling-induced change of CHUP1 (P1546), PMI1 (P319), JAC1 (P2615) and KCA2 (P2751) at the phosphorylation level. They are implicated in multiple steps of chloroplast movement, including generation, maintenance and reorganization of chloroplast-actin filaments.85-87 Although there is relatively little physiological and molecular evidence for explaining chloroplast movement through phosphorylation, chloroplast movement-related proteins were frequently phosphorylated under strong light, suggesting phosphorylation plays an important role.81 AtKCA1 can be phosphorylated at Ser 842 and Ser 846 by CDKA;1, 27



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causing a conformational change in the structure of KCA with implications in folding and dimerization.88 Two significant changed phosphorylation sites of BpKCA2 (Ser 680 and Ser 685) were located in the coiled-coil stalk domain, which contributes to KCA homo- or hetero- dimerization. The phosphorylation site of BpPMI1 was close to the N-terminal side of the cleavage signal, which was only phosphorylated after chilling for 48 hours. It suggested that phosphorylation possibly played a role in PMI1 translocation. Together with these, chloroplast movement could be modulated by phosphorylation and partly account for the gentle decrease of Fv/Fm in the paper mulberry after chilling (Figure 1C). Phosphorylation participated in the regulation of phosphoinositide metabolism. Phosphoinositides, although occupying a small fraction of the plasma membrane, are believed to play key roles in signaling transduction and cytoskeleton organization.89 In the mulberry tree, an increase of phosphoinositides was observed from autumn to winter, suggesting it was closely associated with cold acclimation.90 A total of eight participants in phosphoinositide metabolism were differentially phosphorylated. They were responsible for reactions leading to the generation of different phosphoinositide species. Amongst them, PLC is capable of catalyzing the hydrolysis of phosphatidylinositol

4,5-bisphosphate

into

diacylglycerol

and

inositol

1,4,5-trisphosphate regarded as two important second messenger molecules. The production can not only result in release of Ca2+ into the cytoplasm but also cytoskeleton rearrangement and growth regulation.91, 92 Recent studies have shown that 28


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AtPLC2, as a primary phosphoinositide-specific phospholipase C, contributes to phosphoinositide metabolism, which is not induced by environmental stresses such as dehydration, salinity and low temperature.92, 93 In paper mulberry, no obvious change in the expression of BpPLC2 (P482) was detected during chilling (Figure 6D). Two residues (Ser 270 and Ser 357) were significantly phosphorylated in response to chilling, of which Ser 357 was was located in the catalytic Y domain (Figure S-4). Similarly, corresponding phosphorylation sites have been detected in the catalytic Y domain

of

AtPLC2,

suggesting

regulation

of

enzymatic

activity

though

phosphorylation.93 The present study also showed that phosphoinositide phosphatase SAC2 (P1500) was significantly phosphorylated after chilling for 6 hours. Along with continuous chilling, significant phosphorylation of BpSAC1 (P723) and BpSAC3 (P1569) were detected. The mutant of AtSAC1 is found to cause aberrant organization of the actin cytoskeleton.94 The phosphorylation site of three phosphoinositide phosphatase SACs were located in SAC domain and nearby phosphatase catalytic core, suggesting effect on its activity. Coinciding with phosphoinositide metabolism, the phosphorylation of two phosphatidylinositol transfers (P1963 and P2583) were respectively up-regulated after chilling for 6 and 48 hours, which was required to deliver phosphatidylinositol to plasma membrane.95 It has been reported that phosphorylation of soybean phosphatidylinositol

transfer

alter

interaction

with

membrane

leading

to

phosphatidylinositol transport.96 Thus, phosphorylation was seen as a determinant of 29



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phosphoinositide metabolism and responsible for chilling perception and cytoskeleton organization. Phosphorylation of ICE1 and SIZ1 impacted on BpDREB1 expression. The

phosphorylation

events

in

the

regulatory

components

of

the

CBF/DREB-responsive pathway also caught our attention due to its essential role in chilling tolerance. SIZ1 interacts with ICE1, enhances its stabilization and then facilitates expression of CBF3/DREB1A.97 However, the regulation of SIZ1 was still enigmatic. AtSIZ1 expression was constitutive and unaffected by chilling.97 Consistent with this, the expression of BpSIZ1 in paper mulberry was also not induced by chilling (Figure 6D). Recent study in overexpression and antisense MdSIZ1 mutants has been reported that the SUMO conjugation level does not depend on transcriptional and translational expression.99 These studies implied that the activity of E3 SUMO ligase SIZ1 might be regulated at post-translational level. Phosphoproteomic analysis showed that Ser 495 and Ser 497 of BpSIZ1 (P2340) were significantly phosphorylated in response to chilling. These sites were adjacent to SP-RING domain, where are identified as SUMO attachment and regarded as the essential of multiple self-SUMOyation sites (Figure 9).98 Phosphorylation in the extension of the SUMOylation motif was known to interplay with SUMOylation in both animals and plants.100,

101

Thus, up-regulation of phosphorylation seems to impact on

self-SUMOylation of BpSIZ1 and probably caused the expression changes of BpDREB1 (Figure S-2). 30


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Additionally, the Ser 446 of BpICE1 (P1160) corresponding to Ser 403 of AtICE1 was significantly dephosphorylated after chilling for 6 and 48 hours. The single point mutant of Ser 403 revealed that this residue is necessary for the regulation of transactivation and stabilization.102 It is also speculated to be an O-linked glycosylation site and linked with ubiquitination. Our mass spectrometry analysis provided evidence for the phosphorylation of Ser 446 in ICE1 of paper mulberry (Figure S-4). It was possible that competition between phosphorylation and glycosylation was happened in Ser 446. The dephosphorylation of Ser 446 was enable O-linked glycosylation causing for degradation of ICE1 and further decreasing of BpDREB1 expression (Figure S-2).

CONCLUSION It is important to keep in mind that the changes observed after chilling for 48 hours could also be caused by protein abundance changes. Some details might contribute to distinguish phosphorylation or abundance changes, indirectly. If a protein was significantly expressed or suppressed, this might cause up- or down-regulation of all phosphopeptides. Some phosphoproteins such as P1959, BpEIN2, BpKCA2 with multiple phosphorylation sites contained both unchanged and changed sites, suggesting phosphorylation changes occurred during chilling tolerance. In addition, interesting phosphoproteins such as PLC292 and SIZ197 were reported to be constitutively expressed and not found to be significantly changed at the protein level 31



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in the other species after chilling.103, 104 Despite this limitation, our results showed phosphorylation, an essential component, were responsible for chilling perception and defense in paper mulberry. The phosphorylation changes after chilling for 6 and 48 hours probably depended on CKIIs, MAPKs, CDKs and CDPKs and implicated in Ca2+, phytohormone, phosphoinositide and light-mediated signaling pathway for chilling perception. A hub protein (P1959) was observed to be involved in cross-talk with these signaling pathways. Additionally, transport, cytoskeleton organization and chloroplast movement were regulated against chilling at the phosphorylation level. We also highlighted the phosphorylation of BpSIZ1 and BpICE1, which might interplay with SUMOylation or glycosylation, possibly impacted on the CBF/DREB-responsive pathway and deserved for further study. Finally, we developed a schematic of chilling-related pathways in paper mulberry, which would broaden the insight into chilling response and defense mechanisms at the phosphorylation level. ASSOCIATED CONTENT Supporting Information Figure S-1: The quality control of phosphproteomics analysis. Figure S-2: The expression pattern of BpDREB1 after chilling. Figure S-3: The homology, function domain and phosphorylation sites of P1959. Figure S-4: The phosphorylation sites and function domains of ICE, PLC2 and PHOT1. Table S-1: qRT-PCR primers for the genes corresponding to significantly changed phosphoproteins. Table S-2: All of the 32


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phosphorylation sites identified in paper mulberry leaves. Table S-3: The significantly changed phosphorylation sites between 0 h and 6 h treatment. Table S-4: The significantly changed phosphorylation sites between 0 h and 48 h treatment. Table S-5: The significantly changed phosphorylation sites between 6 h and 48 h treatment. Table S-6: Summary for the sequencing outcomes and the results of transcriptome assembly. Table S-7: The transcriptional expression of genes corresponding to significantly changed phosphoproteins. Table S-8: Differentially expressed protein kinases and phosphatases determined by RNA-seq. Table S-9: The regulatory phosphorylation sites in response to chilling. AUTHOR INFORMATION Corresponding Author * Tel and Fax: +86-10-62836545. E-mail: [email protected]. Author Contributions Z. P. and M. L. Z. performed the experimental work and wrote the manuscript. S. H. S. and X. J. P. jointly directed this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Poverty Relief Project of the Chinese Academy of Sciences (KFJ-FP-24), Huimin Technology Demonstration Project of the National 33



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Modern

Agricultural

Science

and

Technology

Page 34 of 67

Achievements

City

(Z151100001015008), Knowledge Innovation Program through the Chinese Academy of Sciences (KZCX2-YW-359-2) and the National Natural Science Foundation of China (31270653).

ABBREVIATIONS MAPK, Mitogen-activated protein kinase; CDPK, Calcium-dependent protein kinase; CDK, Cyclin-dependent protein kinase; CKII, Casein kinase II; SIZ1, E3 SUMO-protein ligase SIZ1; ICE, Transcription factor ICE1; PLC, Phosphoinositide phospholipase C; PHOT, Phototropin; CAT, Catalase; APX, L-ascorbate peroxidase; CIPK, CBL-interacting serine/threonine-protein kinase; SRK2, SNF1-related protein kinase 2; KEG, E3 ubiquitin-protein ligase KEG; BSL, Serine/threonine-protein phosphatase BSL; CHUP1, Chloroplast unusual positioning 1; KCA, Kinesin-like protein for action based chloroplast movement; PMI, Plastid movement impaired.

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nitric oxide signaling enhance poplar defense against chilling stress. Planta 2015, 242, 1361-1390. (104) Neilson K. A.; Mariani M.; Haynes P. A. Quantitative proteomic analysis of cold-responsive proteins in rice. Proteomics 2011, 11, 1696-706. FIGURES LEGENDS Figure 1. The morphological and physiological changes of paper mulberry after chilling. (A) The morphology changes were photographed under a fluorescent lamp. (B) The chlorophyll fluorescence changes were captured by chlorophyll fluorometer. (C) The changes of Fv/Fm after chilling. (D) The changes of MDA during chilling. (E) The changes of REL during chilling. Error bars represent SD of three replicates at least. Different letters denote significant differences (p < 0.05). Photograph courtesy of Zhi Pi. Copyright 2016. Figure 2. Statistics analysis of phosphoproteomics of paper mulberry after chilling. (A) Venn diagram of significantly changed phosphorylation sites and phosphoproteins distributed in each sample. (B) The up-regulated and down-regulated significantly changed phosphorylation sites in every pairwise comparison. Black columns represent the number of significantly changed phosphorylation sites. Gray columns represent 16, 22 and 168 phosphorylation sites that were uniquely detected after chilling for 0, 6 and 48 hours, respectively corresponding the number shown in brackets. (C) Clustering analysis of significantly changed phosphorylation sites among three treatments. Eight groups were clustered based on k-mean clustering using MultiExperiment Viewer 50


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(MeV). The fold change compared to unchilled treatment was log-transformed to base 2. Figure 3. IceLogo analysis of significantly changed peptides. (A) IceLogo generated after analyzing aligned sequences of each cluster using non-phosphorylated peptides for background correction. (B) IceLogo generated after analyzing aligned sequences of each cluster as compared with non-changed phosphorylation peptides. Figure 4. AgriGO analysis of the significantly changed phosphoproteins after chilling for 6 and 48 hours. (A) Biological process. (B) Molecular function. (C) Cellular component. Significantly overrepresented terms (FDR adjusted p < 0.05) are marked with an asterisk. Figure 5. The pie chart of overrepresented terms. (A) Protein modification. (B) Translation. (C) Transport. (D) Signal transduction. (E) Cellular component organization. Protein modification and translation are significantly enriched after chilling for 6 and 48 hours. Transport, signal transduction and cellular component organization were respectively overrepresented after chilling for 6 and 48 hours. Figure 6. The transcriptional expression of chilling responsive genes in paper mulberry. (A) The up-regulated and down-regulated DEGs in every comparison between control and chilling treatments. (B) The up-regulated and down-regulated protein kinases and phosphatases in every comparison between control and chilling treatments. “Percentage” referred to the percentage of the differentially expressed protein kinases and phosphatases accounting for the total differentially expressed genes of each 51



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pairwise comparison. (C) The number of non-differentially and differentially expressed genes corresponding to significantly changed phosphoproteins. (D) The heat map of selected

phosphoproteins

responding

to

chilling

at

the

transcription

and

phosphorylation level. Black letters mean expression of genes did not significantly change after chilling. Up and down-regulated genes are shown in red and green letters, respectively. The number in brackets represented the code number of phosphorylation sites corresponding to Table S-3 and 4. Figure 7. Protein-protein interaction analysis of protein kinases and phosphatases in response to chilling. The diameters of nodes depend on the number of first neighbors. Red and blue lines present edges of P1959 and STN8, respectively. Blue and yellow nodes mean proteins that change in response to chilling at the transcription or phosphorylation levels corresponding to Table S-8 and Table 1, respectively. Green nodes mean kinases and phosphatases responding at both the transcription and phosphorylation levels. Figure 8. Schematic presentation of systematic chilling response and defense mechanisms in paper mulberry leaves at phosphorylation level. Red and green balls represent up- and down-regulation at the phosphorylation level after chilling. Among these, the phosphorylation regulatory sites were showed in Table S-9. Solid lines represent catalytic reaction, while dotted lines represent material transportation. Red and green arrows indicate phosphorylation and dephosphorylation reactions, respectively. Yellow arrows represent ubiquitination or SUMOylation. Black arrows 52


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mean the other catalytic reaction. The area outlined with red dotted lines represents an unclear phosphorylation cascade containing hub protein P1959. Figure 9. Sequence alignment, structural composition, three-dimensional structure, and tandem mass spectrometry (MS/MS) spectra map for SIZ1 in paper mulberry. (A) BpSIZ1 sequence alignment with Morus alba, Populus trichocarpa, Arabidopsis thaliana and Glycine max. Phosphopeptide detected by MS/MS was underlined. The phosphorylation and SUMOylation sites were labeled by asterisk. (B) Function domain distribution of BpSIZ1. (C) Phosphopeptide MS/MS spectra map showed the phosphorylation sites at Ser 495 and Ser 497.

Table 1. The protein kinases and phosphatases in response to chilling at the phosphorylation level Protein Phosphosite

Description

Peptide

Cluster No.a

ID

ID

P114

77

Probable receptor kinase

_GM(ox)IPLTQIPNVS(ph)R_

3

P206

135

Probable serine/threonine protein kinase

_VVVYS(ph)DR_

3

P206

136


_VVVYSDRLS(ph)SGESR_

3

P285

202

Phototropin-1

_RNS(ph)HAGTR_

1

P285

203

Phototropin-1

_RS(ph)FMGLIR_

1

P285

198

Phototropin-1

_GFPRVS(ph)EDLK_

4

P285

200

Phototropin-1

_RNSES(ph)VAPPR_

4

P285

194

Phototropin-1

_ALS(ph)ESTNRPFIR_

2

P295

211

_VESSSS(ph)AR_

3

Probable LRR receptor-like serine/threonine protein kinase

P315

229

Calcium-dependent protein kinase 13

_FNSLS(ph)VK_

1

P358

280

Probable inactive receptor protein kinase

_AATESES(ph)LSGDYSK_

4

P361

285

Serine/threonine protein kinase dst4 isoform X1

_FSS(ph)LELIGR_

2

P385

303

Phosphatase 2A 55 kDa regulatory subunit B

_GAES(ph)PGVDANGNSFD FTTK_

53


4


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

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P385

304

Phosphatase 2A 55 kDa regulatory subunit B

_SLS(ph)SSITR_

5

P453

377

Probable protein phosphatase 2C 6

_VNS(ph)LLSLPR_

4

P501

429


_AFGDKS(ph)LK_

5

P515

435

Serine/threonine protein kinase PBS1 isoform X1

_LGPVGDNTHVST(ph)R_

2

P515

437

Serine/threonine protein kinase PBS1 isoform X1

_(ac)SCFSCFS(ph)PGRK_

3

P530

450

CDPK-related protein kinase 4

_WPLPPPS(ph)PAK_

1

P530

449

CDPK-related protein kinase 4

_TES(ph)GIFR_

5

P544

459


_APLQS(ph)PTR_

1

P544

461


P544

460


P546

468

Serine/threonine protein kinase BLUS1 isoform X1

P546

465


_NGS(ph)ITEATTSTSEK_

4

P546

469


_RTPS(ph)FSGPLMLPNR_

4

P570

486

Serine/threonine protein kinase dst1

_MSSS(ph)SIPESVIR_

5

Dual specificity tyrosine-phosphorylation-regulated

_VIDLGSSCFETDHLCS(ph)

kinase 4

YVQSR_

_MIEEIRQS(ph)DSENRPSSE ENK_ _AS(ph)AEVLGK_ _QTYSGPLMPGAVLSHSVS (ph)ER_

4 8 3

3

P579

493

P591

2751

Probable receptor protein kinase

_LLDSGESHITT(ph)R_

4

P598

513

Serine/threonine protein kinase EDR1

_AFS(ph)DLNCDR_

3

P628

538

Cyclin-dependent protein kinase G-2 isoform X1

_VVENGTKS(ph)PAER_

5

P664

575

Serine/threonine protein kinase ATM

_EHS(ph)PDEMLSK_

3

P667

580


P682

603

Serine/threonine protein kinase fray2

_GIS(ph)AWNFNLEDLK_

2

P682

602


_FLSGT(ph)LLPDNALSAK_

3

P682

2774


_FLSGT(ph)LLPDNALSAK_

5

P682

601


_FKVTSAELS(ph)PK_

6

P710

642

_VDSGGLS(ph)PDSWR_

3

P710

641

P766

686

P847

748

P893

G-type lectin S-receptor-like serine/threonine protein kinase

_IEELCRS(ph)SLREDPQSDL VK_

G-type lectin S-receptor-like serine/threonine protein

_LLQALVSGDS(ph)FHGVK

kinase

_

Probable LRR receptor-like serine/threonine protein

3

4

_LNEEENTHIS(ph)TR_

5

Calcium-dependent kinase 32

_DGS(ph)LKLDNESR_

3

787

Leucine-rich repeat receptor protein kinase SRF6

_HKS(ph)FDDDDFSK_

3

P893

2808

Leucine-rich repeat receptor protein kinase SRF6

_T(ph)IGVDQGTSR_

5

P920

3129

Mitogen-activated protein kinase 12

P1085

986

Serine/threonine protein kinase GRIK2

_S(ph)EEILHFR_

4

P1113

1008

Serine/threonine protein kinase SRK2I

_SSVLHS(ph)QPK_

3

kinase

_VSFNDAPSAIFWTDY(ph)V ATR_

54


6

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_ISYPETGGGGGS(ph)SHGS

P1186

1075


P1186

1073


_ASHNNPTIPNVS(ph)K_

5

P1192

1081


_DFGS(ph)GVHDAEK_

5

P1192

2862


_MIEDLRPQLT(ph)K_

5

P1204

1095

Mitogen-activated protein kinase kinase kinase 13-A

_S(ph)DDSFGSQFLK_

3

P1204

1096

Mitogen-activated protein kinase kinase kinase 13-A

_NEGLGS(ph)TNQR_

4

P1234

1120

LRR receptor-like serine/threonine protein kinase ERL1 _ITENLS(ph)EK_

8

P1250

1132

CDPK-related protein kinase 5-like

_NS(ph)PALAPASADR_

2

GEAR_

3

P1257

2870

Serine/threonine protein kinase HT1-like

_VWT(ph)MDKNK_

5

P1267

1143

Ethylene response sensor 1

_FTQPLSGS(ph)GR_

6

P1300

1186

SNF1-related kinase regulatory subunit beta-1

_S(ph)NGELGAR_

5

P1326

1205


_S(ph)EHHTVPLSVLLK_

3

P1406

2894

CBL-interacting serine/threonine protein kinase 23

_EDGLLHT(ph)TCGTPNYV APEVINNK_

3

P1413

1278

Protein-tyrosine-phosphatase MKP1

_S(ph)LDEWPK_

3

P1413

1279


_S(ph)LKLPVLAEK_

3

P1429

2901


P1446

1307

Calmodulin-binding receptor-like cytoplasmic kinase 1

P1455

1315

Receptor protein kinase FERONIA

P1455

1316

Receptor protein kinase FERONIA

P1558

2928

Serine/threonine protein kinase STN8

P1568

1422

P1586

1443

P1617

1491

Serine/threonine protein kinase tricorner

_IADFGLASFFDPHQNQPLT (ph)SR_ _RAAEQDIGATHIS(ph)T(ph) QVK_ _GKDPDAS(ph)PGFDGTVT DSR_ _HFS(ph)FSEIK_ _AAPETCAEFLGSFVADKT NT(ph)QFTK_

Serine/threonine protein kinase minibrain-like isoform _VDHHPGGGHWFAAGLS(p X1

h)PNIPGR_

Leucine-rich repeat receptor-like serine/threonine protein kinase

_AS(ph)AETLGR_ _DFSVGNNLS(ph)GALQSD GR_

3

3

3 5 4

6

1

5

P1650

1513


_VNS(ph)LVQLPR_

3

P1674

1532

Serine/threonine protein kinase, isoform 1

_ASCFAGRPS(ph)ER_

3

P1674

1537


P1674

1533


_LSFLPELIAS(ph)AK_

4

P1689

1546

Casein kinase I isoform delta-like

_PVVNPGPS(ph)AER_

7

_VAENTDAENIGS(ph)MTK _

3

P1694

1550

Mitogen-activated protein kinase kinase kinase YODA _AENPTS(ph)PGSR_

3

P1730

1598


_AES(ph)APELR_

3

P1780

1633

Probable LRR receptor-like serine/threonine protein

_LGQSLS(ph)VQK_

3

55



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kinase P1824

1682

Serine/threonine-protein kinase EDR1

P1826

1683

Protein kinase 2B

P1855

1721


_LHKGDMS(ph)PEK_

3

P1855

1720


_DGLGIGFS(ph)GK_

5

P1858

2978


_VNT(ph)LLTLPR_

3

P1858

1723


P1879

1734

Probable inactive leucine-rich repeat receptor kinase

P1921

2983

Serine/threonine protein phosphatase 7

P1922

2984

Casein kinase I isoform alpha

P1926

1782

P1926

1783

P1944

1808

P1959

1822

P1991

Receptor-like serine/threonine protein kinase ALE2 isoform X1 Receptor-like serine/threonine protein kinase ALE2 isoform X1 Cyclin-dependent protein kinase D-3-like

_HSS(ph)PLQR_ _VDTAQSSLVTSAS(ph)GVS K_

_S(ph)PHASSSPLSPNLGVK _ _LANIAGGS(ph)PTLQSSR_ _ET(ph)SVSNENKESVPNEK _ _LAAT(ph)EDEGSTSPLPER _

3 3

4 5 4

3

_GLSGGS(ph)VSEHGFTLR_

2

_GLSGGSVS(ph)EHGFTLR_

7

_NSEFNPHEGPTVLS(ph)PP R_

1

Phosphatase 2C and cyclic nucleotide-binding kinase

_VPVPQVLEVTGSES(ph)PS

domain-containing protein

TFSWISK_

1853

Protein phosphatase 2C 66 isoform 1

_ALLECAS(ph)SR_

5

P2002

1863


_DIS(ph)VVSATR_

7

P2015

3000

Serine/threonine protein kinase STY46 ame

P2045

1905

Serine/threonine-protein kinase SRK2E

_SALLHS(ph)QPK_

4

P2065

1915


_FHSTS(ph)LVVK_

1

P2101

3011


P2101

3012


_HPWPT(ph)FQTSPVYETD QTR_

_APMQT(ph)WSGPLVDPAA VGAPR_ _IADFGLASFFDPNNKQPM T(ph)SR_

3

3

3

3

P2110

1972

Phytochrome A

_VSNS(ph)VSADQQPR_

4

P2128

1987

Receptor-like serine/threonine protein kinase ALE2

_EATEGS(ph)QHISTR_

4

P2167

2029

Serine/threonine protein kinase EDR1 isoform X1

_MLSDDLKRPS(ph)NVEK_

3

P2167

2027

Serine/threonine protein kinase EDR1 isoform X1

_HST(ph)FLSSR_

4

P2178

2049

Serine/threonine protein phosphatase BSL1

_QLS(ph)LDQFENESR_

4

P2221

2082

Serine/threonine protein kinase STY17

_PLMS(ph)PNPMHVR_

7

P2296

2149


P2296

2150


_KEEAGS(ph)PGRDEVATNI GER_ _VHAVPAS(ph)PNSMLR_

56


4 5

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_MIEEIRPSDSESRPS(ph)PE

P2309

2165


P2400

2258

Phototropin-2

_SQS(ph)HDILSK_

4

P2400

2257

Phototropin-2

_NRLS(ph)ENTEQQSAK_

4

P2410

2270

Proline-rich receptor protein kinase PERK1

_FSFDTDTHVST(ph)R_

4

P2424

2284

Calmodulin-binding receptor-like cytoplasmic kinase 3 _YSMS(ph)PQLSR_

3

P2424

2282

Calmodulin-binding receptor-like cytoplasmic kinase 3 _LGS(ph)VHLNMSQVAK_

5

P2424

2283

Calmodulin-binding receptor-like cytoplasmic kinase 3 _VTAS(ph)PFR_

7

P2433

2296

Probable leucine-rich repeat receptor protein kinase

_AAS(ph)RGVGIPQLK_

3

P2443

2300

Serine/threonine protein kinase PBS1

_ILKNEEGGGS(ph)GR_

3

P2443

2301

Serine/threonine protein kinase PBS1

_LGPTGDKS(ph)HVSTR_

3

P2507

2373


P2536

3082

Cyclin-dependent protein kinase C-1

_SFSNDHDANLT(ph)NR_

4

P2536

2394

Cyclin-dependent protein kinase C-1

_EIVTS(ph)PGPEK_

5

P2565

2412

Receptor-like protein kinase TMK3

_LAPEGKAS(ph)FETR_

3

_FTSGLGFRLS(ph)PK_

3

Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase

DNK_

_EHPPS(ph)PNTVTENWPSS R_

3

3

P2605

2448

P2676

2518

E3 ubiquitin-protein ligase KEG

_S(ph)PPASPDNDLTK_

3

P2681

2526

Serine/threonine protein kinase ATG1c

_KVEGFPLSACS(ph)PAR_

5

P2694

2547


_SVVTS(ph)PR_

3

and -tyrosine-phosphatase PTEN2A

P2694

2544


P2694

3105


P2699

2554

Serine/threonine protein kinase CTR1

_LILFGGATALEGNSAASG TPS(ph)SAGSAGIR_ _ITPFGEPPT(ph)PR_ _S(ph)AAGTPEWMAPEVLR _

4 6 4

P2723

3110


_EHLNDPLRT(ph)PLNWR_

7

P2742

2599

Probable phosphatase 2C 4

_SFS(ph)QGGFAFR_

1

P2742

2598

Probable phosphatase 2C 4

_GFMS(ph)GPLDR_

3

a The tendencies of phosphorylation change corresponding to Figure 2C

57



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For TOC only

Photograph courtesy of Zhi Pi. Copyright 2016.

58


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Figure 1. The morphological and physiological changes of paper mulberry after chilling. (A) The morphology changes were photographed under a fluorescent lamp. (B) The chlorophyll fluorescence changes were captured by chlorophyll fluorometer. (C) The changes of Fv/Fm after chilling. (D) The changes of MDA during chilling. (E) The changes of REL during chilling. Error bars represent SD of three replicates at least. Different letters denote significant differences (p < 0.05). Photograph courtesy of Zhi Pi. Copyright 2016. Figure 1 110x115mm (300 x 300 DPI)



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Figure 2. Statistics analysis of phosphoproteomics of paper mulberry after chilling. (A) Venn diagram of significantly changed phosphorylation sites and phosphoproteins distributed in each sample. (B) The upregulated and down-regulated significantly changed phosphorylation sites in every pairwise comparison. Black columns represent the number of significantly changed phosphorylation sites. Gray columns represent 16, 22 and 168 phosphorylation sites that were uniquely detected after chilling for 0, 6 and 48 hours, respectively corresponding the number shown in brackets. (C) Clustering analysis of significantly changed phosphorylation sites among three treatments. Eight groups were clustered based on k-mean clustering using MultiExperiment Viewer (MeV). The fold change compared to unchilled treatment was log-transformed to base 2. Figure 2 168x160mm (300 x 300 DPI)


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Figure 3. IceLogo analysis of significantly changed peptides. (A) IceLogo generated after analyzing aligned sequences of each cluster using non-phosphorylated peptides for background correction. (B) IceLogo generated after analyzing aligned sequences of each cluster as compared with non-changed phosphorylation peptides. Figure 3 80x35mm (300 x 300 DPI)



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Figure 4. AgriGO analysis of the significantly changed phosphoproteins after chilling for 6 and 48 hours. (A) Biological process. (B) Molecular function. (C) Cellular component. Significantly overrepresented terms (FDR adjusted p < 0.05) are marked with an asterisk. Figure 4 91x80mm (300 x 300 DPI)


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Figure 5. The pie chart of overrepresented terms. (A) Protein modification. (B) Translation. (C) Transport. (D) Signal transduction. (E) Cellular component organization. Protein modification and translation are significantly enriched after chilling for 6 and 48 hours. Transport, signal transduction and cellular component organization were respectively overrepresented after chilling for 6 and 48 hours. Figure 5 102x61mm (300 x 300 DPI)



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Figure 6. The transcriptional expression of chilling responsive genes in paper mulberry. (A) The up-regulated and down-regulated DEGs in every comparison between control and chilling treatments. (B) The upregulated and down-regulated protein kinases and phosphatases in every comparison between control and chilling treatments. “Percentage” referred to the percentage of the differentially expressed protein kinases and phosphatases accounting for the total differentially expressed genes of each pairwise comparison. (C) The number of non-differentially and differentially expressed genes corresponding to significantly changed phosphoproteins. (D) The heat map of selected phosphoproteins responding to chilling at the transcription and phosphorylation level. Black letters mean expression of genes did not significantly change after chilling. Up and down-regulated genes are shown in red and green letters, respectively. The number in brackets represented the code number of phosphorylation sites corresponding to Table S-3 and 4. Figure 6 115x74mm (300 x 300 DPI)


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Figure 7. Protein-protein interaction analysis of protein kinases and phosphatases in response to chilling. The diameters of nodes depend on the number of first neighbors. Red and blue lines present edges of P1959 and STN8, respectively. Blue and yellow nodes mean proteins that change in response to chilling at the transcription or phosphorylation levels corresponding to Table S-8 and Table 1, respectively. Green nodes mean kinases and phosphatases responding at both the transcription and phosphorylation levels. Figure 7 150x127mm (300 x 300 DPI)



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Figure 8. Schematic presentation of systematic chilling response and defense mechanisms in paper mulberry leaves at phosphorylation level. Red and green balls represent up- and down-regulation at the phosphorylation level after chilling. Among these, the phosphorylation regulatory sites were showed in Table S-9. Solid lines represent catalytic reaction, while dotted lines represent material transportation. Red and green arrows indicate phosphorylation and dephosphorylation reactions, respectively. Yellow arrows represent ubiquitination or SUMOylation. Black arrows mean the other catalytic reaction. The area outlined with red dotted lines represents an unclear phosphorylation cascade containing hub protein P1959. Figure 8 140x110mm (300 x 300 DPI)


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Figure 9. Sequence alignment, structural composition, three-dimensional structure, and tandem mass spectrometry (MS/MS) spectra map for SIZ1 in paper mulberry. (A) BpSIZ1 sequence alignment with Morus alba, Populus trichocarpa, Arabidopsis thaliana and Glycine max. Phosphopeptide detected by MS/MS was underlined. The phosphorylation and SUMOylation sites were labeled by asterisk. (B) Function domain distribution of BpSIZ1. (C) Phosphopeptide MS/MS spectra map showed the phosphorylation sites at Ser 495 and Ser 497. Figure 9 81x37mm (300 x 300 DPI)


Phosphoproteomic Analysis of Paper Mulberry Reveals

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