Plant materials and treatment
Leafy lettuce (Lactuca sativa L.) GB-30 (bolting resistant, numbered and conserved in our laboratory) was considered the experimental material. The seeds were sown in a sand/soil/peat (1:1:1 v/v) mixture and grown in the Beijing University of Agriculture Experimental Station in Beijing under standard greenhouse conditions (14 h light; 300–1300/mol/(m2 s); 20 ± 2 °C during the day; 13 ± 2 °C at night; 10 h dark; and 50–70% relative humidity). Pest control and water management were performed according to standard practices. When the sixth real leaves were formed, move the lettuce plants under the growing chamber in the following conditions: The temperature was 20 / 13 °C (day/night), the photoperiod was 14/10 h, and the relative humidity was 60% for 2 days of acclimation. Then, divide the plants into two groups. Keep the control group under standard greenhouse conditions where described above. The other group moved to another growth chamber, respectively for 33 / 25 °C high temperature treatment (day/night). Other environmental conditions remain unchanged.
The blossom buds were observed every two days by stereomicroscopy and paraffin-based methods [50] to monitor the progress of flower bud differentiation. A ruler was used to measure the stem length (in cm) of the control and treatment groups every 4 days. Simultaneously, collect the stem samples from control and treatment plant, freeze samples in liquid nitrogen, and store them at − 80 °Crefrigerator for further physiological analysis. On day 8, collect stem samples from the control and treatment plants, freeze samples in liquid nitrogen, and store them at − 80 °C refrigerator for further proteome analysis.
Measurement of sugar components
Approximately 0.2 g samples were ground in 10 mL 80% (v/v) ethanol in a tube, and then the tube was placed in a boiling water bath for 1 h, cooled, and centrifuged at 1000 g for 10 mins. The pellet was extracted two additional times with 10 mL 80% (v/v) ethanol. The supernatants from each extraction were combined and evaporated to dryness in a boiling water bath. The samples were resolubilized in 0.5 mL distilled water and filtered through an acetate filter (0.45-μm pore size, Nalgene, Thermo Fisher Scientific, Waltham, MA).
The contents of galactose, glucose, fructose, and sucrose were determined using high-performance liquid chromatography (HPLC) [51]. The system included a Waters 6000A pump (Millipore, Waters Chromatography Division, Milford, MA), an Inertsil NH2 column (250 mm × 4.6 mm, 5 μm, Dikma Company, Forest Lake, CA) and a Waters 2410 refractive index detector connected to a strip chart recorder. Distilled water, at a flow rate of 10 mL/min, was used as the solvent of the 70% (v/v) acetonitrile. The column temperature was maintained at 35 °C and was preceded by a Waters Bondapak C18/Corasil guard and a set of anion and cation cartridges (deashing guards, Bio-Rad Laboratories, Richmond, CA). All guards were operated at an ambient temperature of 25 °C, and 20 μL samples were injected. Galactose, glucose, fructose, and sucrose were identified and quantified from the retention times and the peak heights of stachyose and raffinose standards. All chemicals were of chromatographic grade in purity. Standards of stachyose and raffinose were purchased from Sigma (St. Louis City MO).
Protein extraction
Extract the samples using the trichloroacetic acid (TCA)/acetone method as previously described with some modifications. Ground each sample into a fine powder in liquid nitrogen at about 2.5 g. Resuspend the powder in 30 mL of 10% (w/v) TCA/acetone (65 mM dithiothreitol (DTT) in a 50-mL tube. For precipitation, store the mixture overnight at least in − 20 °C refrigerator. Centrifuge the mixture at 4 °C at 10,000 rpm for 30 mins, and discard the supernatant. Then add 40 mL precooled acetone and centrifuge at 7000 rpm for 15 mins. Discard supernatant and wash the pellets with acetone for 3 times. Add 200 μL of lysis buffer (SDT buffer (4% (v/v) SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6)) to the precipitate and ultrasonic treat for 30 mins, then place the mixture on ice for 20 mins. Centrifuge at 12,000 rpm at 4 °C for 10 mins and extract the supernatant. Afterwards, vacuum drying the precipitation. Quantitative the supernatant of total protein with BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA).
Protein digestion and iTRAQ labeling
According to the Wi’sniewski and colleagues [52] described FASP application for protein digestion, and according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA) using the 8-plex iTRAQ reagent are labeled by the peptide mixture. In brief, incorporate each 200-μg protein sample into 30 μL of SDT buffer (4% (v/v) SDS, 100 mM DTT, 150 mM Tris-HCl, pH 8.0). Use UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) and repeated ultrafiltration (Microcon units, 30 kDa) to remove the detergent, DTT, and other low-molecular-mass components. Then, add 100 μL of 0.05 M iodoacetamide (C2H4INO) in UA buffer to block reduced L-Cysteine residues, and incubate the samples in darkness for 20 mins. Flush the filter with 100 μL UA buffer solution 3 times and 100 μL DS buffer solution (50 mM triethylammonium bicarbonate at pH 8.5) 2 times. Finally, add 2 μg of trypsin (Promega, Madison, WI, USA) into 40 μL of DS buffer to digest the protein suspensions overnight at 37 °C, and collect the resulting peptides as filtrate. In the solution of 0.1% (g/L) concentration, the UV spectral density at 280 nm was determined by extinction coefficient of 1.1, and the peptide content was calculated according to the frequencies of tryptophan and tyrosine in vertebrate proteins. Dissolve each iTRAQ reagent into 70 μL of ethanol and add them to the relevant peptide mixture for labelling. The experiment was conducted in three separate biological duplications, each containing three plant pools. Mark the three separate biological duplications of the control as (CK1)-113, (CK2)-114, and (CK3)-115; mark the three separate biological duplications of the treatment as (H1)-116, (H2)-117, and (H3)-118.
Separation of peptides by strong cation exchange (SCX) chromatography
The iTRAQ-labeled peptides by strong cation exchange (SCX) chromatography using AKTA filter system (GE Healthcare, Chicago, IL, USA) for separation. Reconstitute and acidify the dried peptide mixture with 2 mL of buffer A (10 mM KH2PO4 in 25% (v/v) acetonitrile (ACN), pH 2.7) and loaded into a PolySULFOETHYL 4.6 × 100 mm column (5 μm, 200 Å, PolyLC Inc., Columbia, MD, USA). Use of buffer B (500 mM KCl, 10 mM KH2PO4 in 25% (v/v) ACN, pH 2.7) to 1 mL/min flow elution peptide, gradient is as follows: 0–8% buffer B (500 mM KCl, 10 mM KH2PO4 in 25% ACN, pH 3.0) for 22 mins, 8–52% buffer B for 22–47 min, 52–100% buffer B for 47–50 min, 100% buffer B for 50–58 min. Then, after 58 mins, buffer B was reset to 0%. Absorbance was measured at 214 nm, elution was monitored, and fractions were collected per minute. Combine the collected moieties into 15 fractions and desalt them on C18 cartridges (Empore™ SPE C18 cartridges (standard density), bed inner diameter (I. D.) 7 mm, volume 3 mL; Sigma, St. Louis, MO, USA). Each component by vacuum centrifugal concentration, restructuring in the 40 mu L 0.1% (v/v) acetic acid. Store all samples at − 80 °C refrigerator until LC-MS/MS analysis.
Analysis of liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS)
Performing Experiments on a Q-Exactive mass spectrometer with the addition of an Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific). Nano-LC-MS /MS analysis was performed by injecting 10 μl of each component. Load the peptide mixture (1–2 μg) onto a C18 reversed-phase column (Thermo Scientific Easy Column, 10 cm length, 75 μm I.D., 3 μm resin) in buffer A (0.1% (v/v) formic acid) and separate the mixture with a linear-gradient of buffer B (80% (v/v) acetonitrile (C2H3N) and 0.1% (v/v) methanoic acid) at a flow rate of 250 nL / min, control it by IntelliFlow technology, for 140 mins. MS data were obtained using a data-dependent top 10 method, which dynamically select the most richest precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. Determine the target value of automatic gain control (pAGC) is based on prediction, and the dynamic elimination time is 60 s. The resolution of the survey scans was set to 70,000 at m/z 200, and the resolution of the HCD spectra was set to 17,500 at m/z 200. The normalized collision energy was 30 eV, and the underfill ratio was defined as 0.1%, which specifies the minimum percentage of the target value might be reached at the maximum fill time. The instrument operates in peptide recognition mode.
Database search and protein quantification
MS/MS mass spectra were searched using the MASCOT engine (Matrix Science, London, UK; version 2.2) flushbonading into Proteome Discoverer 1.3 (Thermo Electron, San Jose, CA, USA) against Lactuca.Unigene.pep.fasta (a lettuce protein database which has 53,584 entries in total, was translated from the transcriptome and created by our research group). The material here was G-B30, which is Consistent with the experimental study of varieties). Use the following settings for protein identification: peptide mass tolerance = 20 ppm; MS/MS tolerance = 0.1 Da; enzyme = trypsin; max missed cleavage = 2; fixed modification: carbamidomethyl (C), iTRAQ 8-plex (K), iTRAQ 8-plex (N-term); variable modification: oxidation (M), iTRAQ 8-plex (Y). The false discovery rate (FDR) of protein identification was ≤0.01. At least one kind of unique peptides involved every highly credible protein identification.
Protein relative quantification was based on the reporting ion signal intensity, it reflected the relative abundance of the peptide. As described above, the protein ratios (fold change) of different test groups (high temperature treatment/control) were obtained by using the ratios of reported ions labelled with different isotopes. For differentially expressed proteins (DEPs), one can use a protein containing at least two unique spectra, and only these unique spectra, for quantification. Only fold changes ≥1.20 or ≤ 0.83 (the ratios with p-values < 0.05 and expected cutoff values < 0.05 with 95% confidence) were considered significant. Median intensities were used for normalization, and outliers were removed automatically (the quantitative protein ratio was normalized by the median ratio in MASCOT).
Bioinformatic analysis of proteins
Function classification analysis was carried out by using Blast2GO software (http://www.geneontology.org) [53]. The KEGG Orthology (KO) data was retrieved using an online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) and then mapped to pathways in the KEGG database [54]. The corresponding KEGG pathways were extracted. Perform enrichment analysis for further explore the effects of different abundance proteins on the physiological process of cells and to determine the internal associations between different abundance proteins. Gene Ontology (GO) enrichment analysis was performed for three ontologies (biological process (BP), molecular function (MF), and cellular component (CC)). The derived p-values were further adjusted using the multi-test Benjamini-Hochberg correction, and only functional categories and pathways with p-values < 0.05 were considered significant.
Total RNA extraction and RT-qPCR analysis
Transcript levels of genes associated with DEPs were determined using real-time quantitative polymerase chain reaction (RT-qPCR). For total RNA extraction, extract stems using an RNA Rapid Extraction Kit (Aidlab Biotech, Beijing, China) according to the operation manual. Use the Reverse Aid First Strand cDNA Synthesis Kit (TaKaRa Biotech, Beijing, China) to reverse-transcribe RNA to cDNA. The process was as follows: Mix RNA (2 μg) with 1 μL Oligo d (T) 18 (0.5 μg/μL), 2 × TS Reaction Mix (10 μL) and TransScript RT/RI Enzyme Mix (1 μL) with an extra 20 μL of RNase-free Water. Mix the mixture gently and incubate at 42 °C for 15 mins. Terminate the reaction by incubation at 85 °C for 5 s, and store the cDNAs of the product at − 20 °C. Use the cDNA samples as a template, and then mix them with 200 nmol primer and SYBR Green PCR Real Master Mix (TakaRa, Kusatsu, Japan) for real-time PCR analysis using Bio-Rad CFX 96 real-time PCR instruments and CFX manager software ver 3.0 (Bio-Rad Laboratories, California, USA). The PCR temperature procedure is as follows: 95 °C of predegeneration for 3 mins, 40 cycles of denaturation at 95 °C for 20 s, annealing at 59 °C for 20 s, and extension at 72 °C for 20 s. Use the 18S sequence as an internal standard for standardization.
Statistical analysis
All tests were performed in triplicate. For stem length measurements, each biological replicate came from 6 samples of 6 plants. 5 samples per biological replicate from 6 plants were observed during flower bud differentiation. For physiological and proteomic analysis, 3 different stems were combined into a single biological sample and performed 3 times to produce 3 independent biological replicates (of three pooled stems). The data provided means ± SDs of three replications and were statistically analyzed using analysis of variance (ANOVA) with SPSS 10.0 (International Business Machines, Corporation (IBM), Chicago, IL, USA). To identify significant differences among groups (p < 0.05, p < 0.01) Tukey’s test was used. Figures representing the physiological parameters were generated automatically with both Origin Pro 8.0 SR4 (Origin Lab, Northampton, MA, USA) and Microsoft Office PowerPoint 2007.
