iTRAQ based protein profile analysis revealed key proteins involved in regulation of drought-tolerance during seed germination in Adzuki bean

Adzuki bean is an important legume crop due to its high-quality protein, fiber, vitamins, minerals as well as rich bioactive substances. However, it is vulnerable to drought at the germination stage. However, little information is available about the genetic control of drought tolerance during seed germination in adzuki bean. In this study, some differential expression proteins (DEPs) were identified during seed germination between the drought-tolerant variety 17235 and drought-sensitive variety 17033 in adzuki bean using iTRAQ method. A total of 2834 proteins were identified in the germinating seeds of these two adzuki beans. Compared with the variety 17033, 87 and 80 DEPs were increased and decreased accumulation in variety 17235 under drought, respectively. Meanwhile, in the control group, a few DEPs, including 9 up-regulated and 21 down-regulated proteins, were detected in variety 17235, respectively. GO, KEGG, and PPI analysis revealed that the DEPs related to carbohydrate metabolism and energy production were significantly increased in response to drought stresses. To validate the proteomic function, the ectopic overexpression of V-ATPase in tobacco was performed and the result showed that V-ATPase upregulation could enhance the drought tolerance of tobacco. The results provide valuable insights into genetic response to drought stress in adzuki bean, and the DEPs could be applied to develop biomarkers related to drought tolerant in adzuki bean breeding projects.

Drought is one of most devastating environmental stress that decreases crop productivity 1 . The occurrence of drought will be more frequent as the global temperature is increasing and the fresh water is lacking 2 . Drought stress impact more seriously on germinating seeds and seedling development phases in most crops, which results in the delaying of seed germination and reduction of the germination rate at a very early developmental stage 3 . The establishment, growth, and productivity of crop is required for a high rate and uniformity of germination under drought. Therefore, understanding the genetic basis involved in seed germination under drought is helpful for further increasing yield potential 3 .
Seed germination is accomplished by a well-orchestrated series of events such as phytohormones and other small molecules mediated interactions with the environment, which signal a suitable environment for germination to ensure plant survival 4 . Specifically, ABA and GAs are considered to be indispensable for seed germination, and their dynamic equilibrium is central to the control of seed dormancy and germination 5 . Drought stress can delay or prevent the seed germination through reducing water availability, changing the mobilization of stored reserves, and affecting the structural organization of proteins 6 . To adapt to drought stress, plants developed the concerted mechanisms such as mechano-receptors, ion transport channels, and secondary signal molecules to Identification of differentially expressed proteins. Drought-responsive proteins were identified by the pairwise comparison of protein abundance between control and MA-treated samples using the iTRAQ data. The criterion of fold change ratio > 1.20 or < 0.83 and p < 0.05 was used to identify differentially expressed proteins (DEPs) between the treatments and control groups.
To investigate the candidate proteins that play roles on the different tolerance of the two varieties, we firstly focus on the DEPs identified with 17235T and 17033T. A total of 167 DEPs were identified, in which 87 were upregulated and 80 down-regulated, respectively (Fig. 1A, Table S3). In the control groups, 30 DEPs were identified between 17235 and 17033CK (Fig. 1A, Table S3). With drought stress, the different protein expression levels in these two varieties could reflect their different abilities of drought tolerance. Notably, 5 DEPs were identified in the two comparisons of 17235T versus 17033T and 17235CK versus 17033CK (Fig. 1B, Table S3), implying these DEPs might be variety-specific proteins. To investigate drought-responsive proteins in different varieties, we analyzed the DEPs in the comparisons of 17235T versus 17235CK and 17033T versus 17033CK. A total of 337 and 267 DEPs were identified in the comparisons of 17235T versus 17235CK and 17033T versus 17033CK, respectively. Among them, 132 up-regulated DEPs and 205 down-regulated DEPs were detected in comparisons of 17235T versus 17235CK. Likewise, 144 DEPs were up-regulated and 123 DEPs were down-regulated in comparisons of 17033T versus 17033CK (Fig. 1A, Table S3). In addition, 62 DEPs were detected in the two comparisons of 17235T versus 17235CK and 17033T versus 17033CK, suggesting that these DEPs might be drought responding proteins.
Functional annotation of drought-responsive proteins. The DEPs in the comparison of 17235T versus 17033T belonged to 111 biological processes, 101 cellular compartments, and 125 molecular functions, respectively (Table S4). In terms of biological process, metabolic process and cellular process were the major groups. Catalytic activity and binding were the top two major molecular functional groups. Cell and cell part were the top two cellular compartments. "structural molecule activity", "structural constituent of ribosome" were enriched by DEPs in the category of molecular function, indicating that structural molecule activity play a major www.nature.com/scientificreports/ role in regulating drought tolerance between different varieties; In the category of cellular component, the GO terms related with several cytosolic proteins were detected, such as "cytosolic large ribosomal subunit", "cytosolic part", "cytosol", "cytosolic ribosome" (Fig. 2). These cytosolic proteins may be involved in the regulation of seed permeation to regulate drought tolerance in germinating seeds. In addition, GO enrichment analysis was performed with the DEPs from the comparisons of 17235T versus 17235CK and 17033T versus 17033CK. In Cellular components, cytoplasm, intracellular part and intracellular were significantly enriched. Peptide metabolic process, cellular amide metabolic process and translation are significantly enriched in biological process. In molecular function. structural molecule activity, guanyl nucleotide binding and GTP binding are significantly enriched (Table s4).
KEGG annotation indicated that the DEPs in comparison of 17235T versus 17033T were assigned to 110 KEGG pathways. Among them, 4 pathway categories, including Ribosome, Phagosome, Fructose and mannose metabolism, and Insulin signaling pathway, were significantly enriched (Table S5). In the comparison of 17235T versus 17235CK, 15 pathways were significantly enriched. Among them, 5 pathways are related to regulation of drought stress, including Ribosome, Peroxisome, Glycolysis / Gluconeogenesis, and Starch and sucrose metabolism as well as Endocytosis (Table S5).

Protein-protein interaction among DEPs.
To predict the relationship among all these DEPs identified in adzuki bean, a protein-protein interaction (PPI) network was generated using the web-tool STRING 9.1. A total of 167 DEPs represented by 99 unique proteins from adzuki bean were shown in the PPI network ( Fig. 3, Table S6). Four functional modules forming tightly-connected clusters were illuminated in the network. Nodes in different colors belong to the major metabolic pathways. In module RNA-binding Proteins, a large number of ribosomal proteins are closely linked to the RNA-binding proteins and transport RNA. It indicated that the genes in ribosome pathway may play key roles in the early germination of adzuki bean seeds under drought stress. Plant cytoskeleton related proteins connected together in module cellular structure, indicating that these genes related to cell structure may mediate seed germination under drought stress. The module of energy metabolism included the proteins related to multiple enzymes involved in the TCA cycle, glycolysis, amino acid metabolism and nitrogen metabolism. These linked proteins also indicated that a synergistic system for carbon and nitrogen metabolism may play important roles in drought response. The distribution of ROS regulation module is relatively scattered, indicating that ROS is produced in different metabolic processes, but it is related to energy metabolism, which also shows that the process of energy metabolism is closely related to the production and regulation of ROS.
In addition, PPI analysis was performed on the differential proteins in the comparison of 17235CK versus 17033CK. A total of 29 DEPs represented by 14 unique proteins from adzuki bean were shown in the PPI network ( Fig. S1, Table S6). These proteins are mainly related to RNA binding and protein translation modification, which is also the difference in the germination period between the two varieties under normal conditions. qRT-PCR assay. To investigate transcription patterns, we used the samples of 17235T and 17033T as ITRAQ proteome to extract RNA, and reversely transcribed for q-PCR. A total of 11 proteins identified by iTRAQ were used for qRT-PCR analyses. Some of these DEPs have been reported to be involved in stress response. The fold changes of protein are shown in Table S3, and the primers for qRT-PCR are listed in Table S7. The internal reference gene actin was used to normalize the transcriptional level of target genes in the calculation of 2 − ΔΔCT . The qRT-PCR results indicated that the gene expression levels of 11 DEPs (A0A0L9VAK7, A0A0L9TF45, A0A0L9TJ92, A0A0L9TQX2, A0A0L9U7B9, A0A0L9UWB5, A0A0L9V113, A0A0L9V7V3, A0A0L9VCX3, A0A0L9VM15, A0A0L9VS04) were consistent with the iTRAQ results (Fig. 4). such as mechano-receptors, ion transport channels, and secondary signal molecules to maintain ion homeostasis as well as cascades of gene activations for hormonal metabolism, signal transduction pathways, and stress responses 7 . PVX based vector can be used to mediate high expression of foreign genes, and the overexpressed protein can move away from the initially infected cells to the uninfected sites 19,20 . Virus-based vectors were used to investigate gene function involved in drought stress 21,22 . In this study, V-ATPase (A0A0L9TJ92) was upregulated in 17235T compared to 17033T, and its expression levels at transcriptomic and proteomic level were consistent. To verify whether this protein can improve the drought tolerant in plant, the full-length cDNA of VaVHA-c was amplified from adzuki bean seeds and was ectopic overexpressed in Nicotiana benthamiana by the PVX virus vector. The ectopic overexpression of VaVHA-c obviously enhanced the drought tolerance in tobacco.
The new developping leaves that was unincoculated in plants with ectopic overexpression of VaVHA-c grew normally, however, the plants in control group inoculated with empty vector wilted after 15 d drought treatment (Fig. 5A). The expression of VaVHA-c could be detected only in the ectopic overexpression of VaVHA-c plants by RT-PCR (Fig. 5B), implying that the enhanced drought tolerance was due to the overexpression of VaVHA-c.
Measurements of physiological parameters indicated that the activity of peroxidase (POD) was increased, and water loss was reduced in the leaves of plants with overexpressed VaVHA-c under drought (Fig. 6).

Discussion
Germination is a complex process involving events associated with the transition of a quiescent dry seed to a metabolically active state 23,24 . The germination efficiency determines the seedling establishment and the proper development of mature plants. In the study, a number of DEPs were detected between the drought-tolerant and www.nature.com/scientificreports/ drought-sensitive varieties (Fig. 1), and 4 functional modules energy metabolism, cellular structure, ROS regulation, RNA-binding proteins were formed tightly-connected clusters in PPI network in this study (Fig. 3), implying that DEPs involved in these processes might played the key roles in regulatory of germination under stress. The cytoskeleton-associated proteins have been determined to be key regulatory molecules in mediating cytoskeleton reorganization in response to multiple environmental signals, such as light, salt, drought and biotic stimuli 25 . Reorganization of cellular structure actin is a central component of the cytoskeleton 3 . Actin and the microtubule network are regulated by many factors such as EF1α, Ca 2+ /CaM, tubulin cofactors (TBCC) [26][27][28][29] . OsADF3-heterologous transgenic Arabidopsis increased drought stress tolerance and up-regulated many downstream drought responsive genes 30 . Overexpression of LreEF1A4 improved seed germination rate under drought stress 31 . In this study, we detected several up-regulated DEPs related to cell structure such as A0A0L9TKB7   Table S8) under drought stress in the drought-tolerant variety.These proteins may provide a vital structural basis for seed germination under drought stress. In addition, the cell wall-related proteins NC_030646.1.428.73, A0A0S3SNK4 (Dirigent protein), NC_030641.1.847.6 (epidermis-specific secreted glycoprotein EP1), A0A0S3SEG8 (expansin) were not consistent with previous studies [32][33][34] , which may be the specific metabolic activity of adzuki bean under drought conditions. Energy materials including carbohydrates, proteins and lipids in seeds are utilized as nutrients and energy sources at the germination stage. Starch and proteins are mobilized via the activation of corresponding amylases and proteases 35 . The carbohydrate can be utilized more easily in metabolism due to its simple molecular structure 36 . The enhanced carbohydrate metabolism would accelerate reserve hydrolysis and impair protein biosynthesis during seed germination 37 . In this study, some DEPs were detected in the pathways related with energy and carbohydrate metabolism, such as alpha-1,4 glucan phosphorylase, pyrophosphate-fructose 6-phosphate 1-phosphotransferase, sucrose synthase. Most of these DEPs were up-regulated, and five seed storage proteins A0A0S3SMN4, A0A0S3SMS9, NC_030638.1.306.23, A4PI99, NC_030642.1.591.12 ( Fig. 7 and Table S8) were significantly down-regulated in the drought-tolerant variety under drought stress, implying that more energy provided might result in the drought-tolerance variety under drought stress.
In addition, storage proteins must be degraded to sustain embryo growth and development until an autotrophic growth is reached. Several protease families are involved in the germination process 38 . Notably, 9 proteases were up-regulated, and 8 protease inhibitors are down-regulated in the drought-tolerance variety under drought ( Fig. 7 and Table S8), suggesting that the seeds of drought-tolerant variety use a large number of proteases to decompose storage proteins to provide energy for their germination and promote seed germination, whereas the sensitive variety seeds decompose more slowly due to protease inhibitors, and inhibit seed germination.
ROS can participate in endosperm weakening during germination through cell wall loosening, but uncontrolled ROS production can lead to oxidative stress and cellular damage, resulting in seed deterioration, preventing germination and early seedling development 39,40 . Some antioxidants such as SOD, POD, glutathione-related enzyme and dehydrogenase was produced to decrease the cellular damage caused by ROS in plants 41 . In this study, several DEPs, including NC_030637.1.1703.10, A0A0L9U8C9, NC_030637.1.740.3, and A0A0S3SSI5, which are involved in regulating ROS homeostasis [42][43][44] , were detected, suggesting maintenance of ROS homeostasis is important for drought tolerance in adzuki bean seed germination.
V-ATPase (Vacuolar H + -ATPases, VHA) regulates the ion balance of cell in plants by pumping H + from the cytosol into the vacuole. V-ATPase was reported to survive plant cell by enhancing its activity under salinity and drought 45,46 . In this study, we found that overexpression of a DEP encoding VaVHA-c could enhance the drought tolerance of plants in Nicotiana benthamiana. Furthermore, the POD activity was increased, and the water loss was reduced in the leaves of overexpressed tobacco plants compared to the control group under drought stress. It is suggested that VaVHA-c might participate in maintenance of ROS homeostasis to regulate the tolerance in plant.

Plant growth conditions and treatments.
This study is complied with relevant institutional, national, and international guidelines and legislation. The landraces s17235 and s17033 were collected from Hubei Province by Institute of Food Crops, Hubei Academy of Agricultural Sciences, China, which is permitted by government of Hubei Province, and Nicotiana benthimiana (N. benthamiana) was kept in our lab of Yangtze University, which is permitted to be used for non-commercial purposes.The seeds of s17235 and s17033 were germinated in mannitol (7.5% concentration) and deionized water for 24 h, as described by Zhu et al. 13 . 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 2 mL lysis buffer (8 M urea, For each sample, proteins were precipitated with ice-cold acetone, then re-dissolved in 100 μL 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 www.nature.com/scientificreports/ re-dissolved in the buffer A (buffer A: 20 mM ammonium formate in water, pH 10.0, 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, 4.6 mm × 250 mm, 5 μm, (Waters Corporation, MA, USA). High pH separation was performed using a linear gradient starting from 5% B to 45% B in 40 min (B: 20 mM 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 1 mL/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 30 μL 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. 10 μL peptide sample was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 μm × 2 cm), with a flow of 10 μL/min for 3 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm × 15 cm) 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 300 nL/ min. The electrospray voltage of 2 kV 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 120 K, followed by sequential high energy collisional dissociation (HCD) MS/MS scans with a resolution of 30 K. The isolation window was set as 1.6 Da. The AGC target was set as 400,000. MS/MS fixed first mass was set at 110. In all cases, one microscan was recorded using dynamic exclusion of 45 s. 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. 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. geneo ntolo gy. 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 adjusted with FDR correction, setting 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/ kegg1. html) 48-50. The significantly enriched metabolic pathways or signal transduction pathways in DEPs were identified by 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.
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 S7. An actin gene (X69885) of adzuki bean was used as internal control. 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 ddH 2 O. 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 30 s. The primer specificity was confirmed by melting curve analysis. Relative expression levels of the genes were calculated using the 2 −ΔΔCT method 51 .
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 al. 52 , 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 N. benthimiana by infiltration method 52  www.nature.com/scientificreports/ re-suspended in infiltration buffer, and infiltrated into leaves of N. benthamiana through needleless syringes. 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 new developping leaves that was uninoculated 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 al 51 .
Physiological parameter measurements and statistical analysis. The new developping leaves that was uninoculated with 10 d water withholding were harvested for analyzing the activity of SOD, POD, content of proline and MDA, and water loss as described by Zhou et al 53 . Data analysis were conducted by Microsoft Excel 2016 and the 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.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.