Plant materials and experimental design
Wild Rhododendron chrysanthum tissue seedlings were exposed to 4 ℃ for 12 h were used as the experimental group (EG). Wild Rhododendron chrysanthum tissue seedlings were exposed to normal atmospheric temperature for 12 h were used as the control group (CG). Both EG and CG The leaves excised from six-month-old plants of the EG and the CG were immediately used for protein extraction. To ensure adequate coverage, three biological replicates of each group (i.e., six plants) were collected.
Chlorophyll fluorescence measurement
Chlorophyll fluorescence induction parameters of EG and CG leaves were carried out with the Maxi-version of the Imaging-PAM (Walz, Germany). Before measurement, the plants were kept in darkness for 30 min to allow all reaction centers to open. Then, the fourth leaf from the top of each plant was detached and clamped onto the holder. The minimal fluorescence (Fo) of dark-adapted leaves was recorded during the weak measuring pulses of 0.5 μmol m-2 s-1 and the maximal quantum yield of PSII photochemistry (Fm) was obtained upon application of a 0.5 s saturation light pulse of 2,800 μmol m-2 s-1. The intensity of actinic light setting used in all trials was 230 μmol m-2 s-1. The maximal quantum yield of Photosystem II photochemistry (Fv/Fm), effective quantum yield of PSII photochemistry (Fv’/Fm’), nonphotochemical quenching (NPQ), photochemical quenching (qP), and electron transport rate (ETR) were calculated using ImagingWin version 2.39 software (Walz). Statistical analysis was performed by using SAS 9.4. All data are represented as the means ± SD with three biological independent replications.
H2O2 Content and antioxidant enzyme activity assays
The activity of catalase (CAT) and the content of H2O2 within leaves were determined by Plant CAT ELISA kit and Plant H2O2 ELISA kit (Shanghai Enzyme Biotechnology Co., Ltd., China) according to the manufacturers instructions.
Two grams of leaves were ground in liquid nitrogen and mixed with lysis buffer (8 M urea, 2 mM EDTA, 10 mM DTT and 1% Protease Inhibitor Cocktail). The mixture was sonicated three times on ice using a high-intensity ultrasonic processor (Scientz, China). The remaining debris were removed by centrifugation at 20,000×g at 4℃ for 10 min. The protein in the supernatant was precipitated with cold 15% TCA at -20℃ for 4 h. After centrifugation at 4℃ for 3 min, the remaining precipitates were washed with cold acetone three times. Finally, the protein was dissolved in buffer (8 M urea, 100 mM TEAB, pH 8.0), and the protein concentration in the supernatant was estimated with a 2-D Quant kit (GE Healthcare, USA) according to the manufacturer’s instructions. To ensure adequate coverage, three biological replicates of each group were collected.
Proteomics and bioinformatics analysis
After extraction, proteins were digested into peptides. TMT labeling, HPLC fractionation and LC-MS/MS were then used to analyze and quantify the dynamic changes of the proteome. To ensure adequate coverage, three biological replicates (i.e., six samples) were collected. The MS/MS data were processed using the Mascot search engine (v.2.3.0). Tandem mass spectra were searched against the SwissProt Green Plant database. For protein quantification, the MASCOT software package in NCBI were used in the present work. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate the protein pathway. The protein-protein interaction network was obtained from the String database and the interactions between proteins were performed using Cytoscape software (3.4.0).
TMT/iTRAQ Labeling (optional)
After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit/iTRAQ kit. Briefly, one unit of TMT/iTRAQ reagent were thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted and dried by vacuum centrifugation.
LC-MS/MS Analysis and Database Search
The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75 μm i.d.). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.
The resulting MS/MS data were processed using Maxquant search engine (v.184.108.40.206). Tandem mass spectra were searched against human uniprot database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification and Acetylation modification and oxidation on Met were specified as variable modifications. FDR was adjusted to < 1% and minimum score for modified peptides was set > 40.
Protein Functional Enrichment
Proteins without detailed annotation were annotated by searching against the NCBI non-redundant protein database2 using PSI and PHI-BLAST programs3. Protein functional classification was performed on the basis of combination of information from KEGG pathway database4, UniProt database5, and the Gene Ontology protein database6, as well as literature.
Enrichment of Gene Ontology analysis
Proteins were classified by GO annotation into three categories: biological process, cellular compartment and molecular function. For each category, a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially modified protein against all identified proteins. The GO with a corrected p-value < 0.05 is considered significant.
Enrichment of pathway analysis
Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially modified protein against all identified proteins. The pathway with a corrected p-value < 0.05 was considered significant. These pathways were classified into hierarchical categories according to the KEGG website.
Enrichment of protein domain analysis
For each category proteins, InterPro (a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites) database was researched and a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially modified protein against all identified proteins. Protein domains with a corrected p-value < 0.05 were considered significant.
Phosphoprotein Homology Modeling
Three-dimensional structural models for phosphoproteins were generated using SWISS-MODEL comparative protein modeling server8(Biasini et al., 2014). Structures were visualized and analyzed using the Swiss-PdbViewer software (version 3.7). Functional domains were predicted by InterPro: the integrative protein signature database9.