Plant growth conditions and treatments
The seeds of s17235 and s17033 were germinated in mannitol (7.5% concentration) and deionized water for 24 h, as described by Zhu et al13. The samples were collected in 24 h, immediately frozen and stored in liquid nitrogen for protein and RNA extraction. Three biological replicates were conducted for each treatment.
Protein extraction, digestion and iTRAQ labelling
Total proteins were extracted using the cold acetone method. Samples were ground to power in liquid nitrogen, then dissolved in 2mL lysis buffer (8 M urea, 2% SDS, 1x Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland), followed by sonication on ice for 30 min and centrifugation at 13 000 rpm for 30min at 4℃. The supernatant was transferred to a fresh tube. For each sample, proteins were precipitated with ice-cold acetone at -20℃ overnight. The precipitations were cleaned with acetone three times and re-dissolved in 8M Urea by sonication on ice. Protein quality was examined with SDS-PAGE.
BCA protein assay was used to determine the protein concentration of the supernatant. 100 µg protein per condition was transferred into a new tube and adjusted to a final volume of 100µL with 8M Urea. 11µL of 1M DTT (DL-Dithiothreitol) was added and samples were incubated at 37°C for 1hour. Then 120µL of the 55mM iodoacetamide was added to the sample and incubated for 20 minutes protected from light at room temperature.
For each sample, proteins were precipitated with ice-cold acetone, then re-dissolved in 100µL TEAB. Proteins were then tryptic digested with sequence-grade modified trypsin (Promega, Madison, WI) at 37°C overnight. The resultant peptide mixture was labeled with iTRAQ tags 113 through 118. The labeled samples were combined and dried in vacuum.
Strong cation exchange (SCX) fractionation and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis
The combined labeled samples were subjected to the SCX fractionation column connected with a high performance liquid chromatography (HPLC) system. The peptide mixture was re-dissolved in the buffer A (buffer A: 20mM ammonium formate in water, pH10.0, adjusted with ammonium hydroxide), and then fractionated by high pH separation using Ultimate 3000 system (Thermo Fisher scientific, MA, USA) connected to a reverse phase column (XBridge C18 column, 4.6mm x 250 mm, 5µm, (Waters Corporation, MA, USA). High pH separation was performed using a linear gradient starting from 5% B to 45% B in 40 min (B: 20mM ammonium formate in 80% ACN, pH 10.0, adjusted with ammonium hydroxide). The column was re-equilibrated at initial conditions for 15 min. The column flow rate was maintained at 1mL/min and column temperature was maintained at 30℃. Twelve fractions were collected; each fraction was dried in a vacuum concentrator for the next step.
Peptide fractions were resuspended with 30µl solvent C respectively (C: water with 0.1% formic acid; D: ACN with 0.1% formic acid), separated by nanoLC and analyzed by on-line electrospray tandem mass spectrometry. The experiments were performed on an Easy-nLC 1000 system (Thermo Fisher Scientific, MA, USA) connected to a Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, MA, USA) equipped with an online nano-electrospray ion source. 10µl peptide sample was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100µm x 2cm), with a flow of 10µl/min for 3 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75µm x 15cm) with a linear gradient, from 3% D to 32% D in 120 min. The column was re-equilibrated at initial conditions for 10 min. The column flow rate was maintained at 300nL/min. The electrospray voltage of 2kV versus the inlet of the mass spectrometer was used.
The fusion mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 350–1550) were acquired with a mass resolution of 120K, followed by sequential high energy collisional dissociation (HCD) MS/MS scans with a resolution of 30K. The isolation window was set as 1.6 Da. The AGC target was set as 400000. MS/MS fixed first mass was set at 110. In all cases, one microscan was recorded using dynamic exclusion of 45 seconds.
Database search
The mass spectrometry data were transformed into MGF files with Proteome Discovery 1.2 (Thermo, Pittsburgh, PA, USA) and analyzed using Mascot search engine (Matrix Science, London, UK; version 2.3.2). Mascot database was set up for protein identification using Vigna angularis L reference transcriptome or Vigna angularis L database in NCBInr/SwissProt/Uniprot/IPI, and the PacBio SMRT and Illumina Sequencing data published by Zhu et al18. Mascot was searched with a fragment ion mass tolerance of 0.050 Da and a parent ion tolerance of 10.0 PPM.
Protein identification and quantification
The Mascot search results were averaged using medians and quantified. Proteins with fold change in a comparison >1.2 or <0.83 and unadjusted significance level p < 0.05 were considered differentially expressed.
GO Enrichment analysis
Gene Ontology (GO) is an international standardized gene functional classification system which offers a dynamic-updated controlled vocabulary and a strictly defined concept to comprehensively describe properties of genes and their products in any organism. GO has three ontologies: molecular function, cellular component and biological process. The basic unit of GO is GO-term. Each GO-term belongs to a type of ontology.
GO enrichment analysis provides all GO terms that significantly enriched in DEPs comparing to the genome background, and filter the DEGs that correspond to biological functions. Firstly, all DEPs were mapped to GO terms in the Gene Ontology database (http://www.geneontology.org/), gene numbers were calculated for every term, significantly enriched GO terms in DEGs comparing to the genome background were defined by hypergeometric test. The calculated p-value was gone through FDR Correction, taking FDR ≤ 0.05 as a threshold. GO terms meeting this condition were defined as significantly enriched GO terms in DEPs. This analysis was able to recognize the main biological functions that DEPs exercise.
Pathway enrichment analysis
Pathway-based analysis was conducted by blasting against for KEGG database (https://www.kegg.jp/kegg/pathway.html). Pathway enrichment analysis identified significantly enriched metabolic pathways or signal transduction pathways in DEPs comparing with the whole genome background. The calculated p-value was gone through FDR Correction, taking FDR ≤ 0.05 as a threshold. Pathways meeting this condition were defined as significantly enriched pathways in DEPs.
RNA extraction and qRT-PCR
Total RNA was extracted using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), and then treated with RNasefree DNase (Invitrogen, Gaithersburg, MD, USA). The purified RNA was reverse transcribed using the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The qRT-PCR reactions were performed in CFX96™ Real-Time PCR Detection System (Bio-Rad, USA). The gene specific primers were listed in additional Table S5 Each reaction was conducted in 10 µl mixture containing 5 µl of SYBR green (SYBR@ Premix Ex Taq™ (TliRNaseH Plus), TAKARA, Japan), 0.3 µl forward and reverse primers (10 µM), respectively, 2 µl cDNA template, and 2.4 µl ddH2O. The reactions for each gene were conducted in triplicate with the thermal cycling conditions as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 57°C for 30s. The primer specificity was confirmed by melting curve analysis. Relative expression levels of the genes were calculated using the 2−ΔΔCT method43.
Ectopic expression of VaVHA-c and RT-PCR
Four weeks old N. benthamiana plants grown in a growth room (24°C, 16 h/8 h light/dark, 100 µM m− 2 s− 1 white light) were used for VaVHA-c ectopic expression. The coding sequence (CDS) region of VaVHA-c was amplified with gene specific primers and was inserted into PVX-LIC vector as described by Zhao et al44, and confirmed by sequencing. The resultant construct VaVHA-c- PVX-LIC was introduced into Agrobacterium tumefaciens GV3101 via the freeze-thaw method, and then introduced into tobacco (Nicotiana benthimiana) by infiltration method44 (Zhao et al. 2016). The empty vector PVX-LIC was introduced into tobacco as negative control. The experiment was performed three times with at least 5 plants for each construct. After 7 d of infiltration, the leaves were harvested for RNA extraction and RT-PCR analysis, and the plants were treated by withholding water. The phenotype of plants was photographed at 15 d after water withholding. Gene-specific primers were used for RT-PCR and actin was used to normalize the reaction as described by Sha et al43.
Physiological Parameter Measurements and Statistical Analysis
The activity of superoxide dismutase (SOD), peroxidase (POD), content of proline and malondialdehyde (MDA), and water Loss were analyzed as described by Zhou et al45. Data analysis were conducted by Microsoft Excel 2016 and the software of SPSS 16.0. The significance was analyzed by One-way ANOVA test. Tukey multiple comparison test was used to compared differences at 0.05 significance level.