Plant materials, growth conditions and light sources
The Cabinent Sauvignon plants grown for eight years in Yunnan were chosen as experimental materials. In order to ensure consistent test condition, eighteen plants with consistent growth were selected as experimental materials. Each plant maintains 5 to 7 branches, and main stems of each plant were kept at 1250mm high. Selected plants were transplanted into pots which were 260mm in height and 370mm in diameter. Each pot contains vermiculite, red soil, and humus, of which the proportion of soil was 1:1:3. We placed Eighteen pots in a glasshouse where temperature and humidity were 0-dayled (day and night temperature were 25/18℃，relatively humidity was 40%). In consideration of meeting the environmental conditions of the local wine grape regions as much as possible, we carried out meteorological monitoring on three major wine grape regions in Yunnan province and took the meteorological data of recent three years as a reference (2015-2017), found that the annual average UV-B radiation intensity was in between10-15w/m2. The strongest UV-B intensity in the daytime was 22w/ m2 (data was collected from the local meteorological database). The Lights were supplied by Photoelectric Instrument Factory of Beijing Normal University, Beijing, China (Philips. 20w/m2, 6lamp tubes).
We set up three experimental groups with total 18plants, one 0-day group (CK) and two treatment groups. Each group contains 2lamps, 3 pots as three replicates (6plants of Cabinent Sauvignon). According to preliminary work, the phenotype appeared after 3-day irradiation; thereby in later experiment the duration of UV-B treatment was justified to 3 days and 7 days. The experiment was conducted from 10:00 am to 14:00 pm each day after dormant plants began to sprout and each branch has 5-6 leaves. In order to ensure the consistency of measurement time, the 7-day exposure group was carried out 4 days in advance. One-thirds of the new leaves and developing leaves including the meristem were directly exposed to light (as described in Fig.8). The radiation intensity was measured by Hand-hold UV irradiation meter (Mode type: LS126. Manufacture: Linshang Technology. Co, Shenzhen, China. Measuring range of wavelength is 380nm-760nm). When plants get perpendicular to the tube, and were 10-15cm away from the lamp, the instant exposure intensity was around 3.8w/m2. When the distance between the tube and the plants increased to 30-40cm, the exposure intensity decreased to 2.7-2.5w/m2. The longer the distance was, the weaker the radiation would be. Every two pots of plants under each lamp were adjusted continuously during the test to ensure the evenness of light exposure.
Anatomical observation and photosynthetic index determination
The leaves completely exposure to UV-B but capable of function were selected for anatomic and transcriptomic analysis. Among which, 3-5 leaves from each plant were cut into small pieces in the size of 5mmx5mm then put into the pre-configured glutaraldehyde fixer for one week. All samples were cleaned, embedded with resin for slicing. Each embedded section was cut into 5μm thick slice by the Leica EM UC7 ultra-thin slicer (manufacture：Germany) for observation under 20-fold microscope. We performed photosynthetic measurement on three groups from 10:00 am every hour until 2:00 pm on the third and seventh day of exposure43.
Proteomic materials and methods
The TMT (Tandem Mass Tags) technology innovated by Thermo SCIENTIFIC. Co. USA, was used to measure the expression level of proteins. Samples were gathered right after lights off from treated plant treated for 3 days（exposure time was 12 hours）, so did the 7-day treatment group. For the untreated samples, we collected samples for three times: 0day, 3 days and 7 days; collecting time was consistent with the treatment groups. A total of leaves of the same size were selected, stored in liquid nitrogen then delivered to Novogene technology Co. (Beijing, China) for proteomic and transcriptomic analysis.
Total protein extract
The samples were individually milled to a power in a mortar with liquid nitrogen then mixed 150 mg of the powder from each sample with 1 ml of lysis buffer containing Tris-base (pH 8), 8M Urea, 1% SDS, complete protease inhibitor cocktail (Sigma) in a glass homogenizer. The homogenate was incubated on ice for 20 min and then centrifuged at 12000 g for 15 min at 4°C and protein concentration was determined with a Bradford assay. Then 4 volumes 10 mM DTT were added in cold acetone to a sample extract, Vortexed well, placed sample at -20°Cfor 2 h to overnight. Centrifuged and collected pellet to wash twice with cold acetone. Finally dissolve the pellet by dissolution buffer containing Tris-base (pH=8), 8M Urea.
The supernatant from each sample, containing precisely 0.1mg of protein, DTT reduction,
iodoacetamide alkylation, and was digested with Trypsin Gold (Promega, Madison, WI) at 37°C for 16h. After trypsin digestion, peptide was desalted with C18 cartridge to remove the high urea, and desalted peptides were dried by vacuum centrifugation.
TMT labeling of peptides
Desalted peptides were labeled with TMT10-plex reagents (TMT10plex™ Isobaric Label Reagent
Set, Thermofisher), following the manufacturer’s instructions. For 0.1mg of peptide, 1 unit of labeling reagent was used. Peptides were dissolved in 30µl of 0.1 M triethylammonium bicarbonate solution
(TEAB, pH 8.5), and the labeling reagent was added to 20µl of acetonitrile. After incubation for 1 h, the reaction was stopped with 50 mM Tris/HCl (pH 7.5). Differently labeled peptides were mixed equally and then desalted in 100 mg SCX columns (strata-x-c, Phenomenex: 8B-S029-EBJ).
A ~600 microgram TMT-labeled peptide mix was fractionated using a C18 column (waters BEH
C18 4.6 × 250 mm, 5 µm) on a Rigol L3000 HPLC operating at 1ml/min. The column oven was set
as 50 °C. Mobile phases A (2% acetonitrile, 20mM NH4FA, adjusted pH to 10.0 using NH3·H2O) and
B (98% acetonitrile, 20mM NH4FA, adjusted pH to 10.0 using NH3ÀH2O) were used to develop a
gradient elution. The solvent gradient was set as follows: 3–8% B, 5min; 8–18% B, 12 min; 18–32% B,
11 min; 32–45% B, 7 min; 45–80% B, 3 min; 80% B, 5 min; 80–5%，0.1min，5% B, 7 min The tryptic peptides were monitored at UV 214 nm. Eluent was collected every minute and then merged to 15 fractions. The samples were dried under vacuum and reconstituted in 20µl of 0.1% (v/v) FA, 3% (v/v) acetonitrile in water for subsequent analyses.
Fractions from the first dimension RPLC were dissolved with loading buffer and then separated by a C18 column (150μm inner-diameter, 360μm outer-diameter×15cm, 1.9μm C18, Reprosil-AQ Pur, Dr. Maisch). Mobile phase A consisted of 0.1% formic acid in water solution, and mobile phase B consisted of 0.1% formic acid in acetonitrile solution; a series of adjusted 60mingradients according to the hydrophobicity of fractions eluted in 1D LC with a flow rate of 300 nL/min was applied. Q-Exactive HF-X mass spectrometer was operated in positive polarity mode with capillary temperature of 320°C.Full MS scan resolution was set to 60000 with AGC target value of 3e6 for a scan range of 350-1500m/z. A data-dependent top 40 method was operated during which HCD spectra was obtained at 15000 MS2 resolution with AGC target of 1e5 and maximum IT of 45ms, 1.6 m/z isolation window, and NCE of 30, dynamically excluded of 60s.
The identification and quantitation of protein
The resulting spectra from each fraction were searched by the search engines: Proteome Discoverer 2.2 (PD 2.2, Thermo). The searched parameters as follows: A mass tolerance of 10 ppm for precursor ion scans and a mass tolerance of 0.02 Da for the product ion scans were used. Carbamidomethyl was specified in PD 2.2 as fixed modifications. Oxidation of methionine, acetylation of the N-terminus and TMT 10-plex of lysine were specified in PD 2.2 as variable modifications. A maximum of 2 miscleavage sites were allowed. For protein identification, protein with at least 1 unique peptide was identified at FDR less than 1.0% on peptide and protein level, respectively. Proteins containing similar peptides and could not be distinguished based on MS/MS analysis were grouped separately as protein groups. Reporter Quantification (TMT 10-plex) was used for TMT quantification. The protein quantitation results were statistically analyzed by Mann-Whitney Test.
Sample collection and preparation
RNA quantification and qualification
RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
Library preparation for Transcriptome sequencing
A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
Clustering and sequencing
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Quality 0-day Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reads mapping to the reference genome
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.4 and paired- end clean reads were aligned to the reference genome using Hisat2 v2.0.4. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
Quantification of gene expression level
HTSeq v0.9.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels (Trapnell, Cole, et al., 2010).
Differential expression analysis
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for 0-dayling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed44,45.
GO and KEGG enrichment analysis of differentially expressed genes
The GO enrichment analysis was performed of differentially expressed genes by the GOseq R package. The gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. For KEGG analysis, we used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways.