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Proteomic analysis of Zanthoxylum nitidum seeds dormancy release: Influence of stratification and gibberellin
Industrial Crops & Products 122 (2018) 7–15
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Proteomic analysis of Zanthoxylum nitidum seeds dormancy release: Influence of stratification and gibberellin Qiang Lu, Zhen Shan Zhang1, Ruo Ting Zhan, Rui He
T
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Key Laboratory of Chinese Medicinal Resource from Lingnan, Ministry of Education and Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Seed dormancy Zanthoxylum nitidum iTRAQ Cold stratification GA
Zanthoxylum nitidum (Roxb.) DC. is one of the most important economic crops on account of its value as a Chinese herbal medicine (Zanthoxyli radix), health product and daily chemical raw materials. Zanthoxylum nitidum seeds are characterized to be an intermediate physiological dormancy that requires three months of cold stratification (4 °C) or gibberellin (GA) treatment (300 mg L−1, soaking for two days) to relieve seed dormancy and promote germination. Exploring the changes of proteins and related biosynthetic and metabolic pathways during seed dormancy release will contribute to expand our knowledge of this process. In this study, mature Zanthoxylum nitidum seeds from Pingyuan County, Guangdong province were treated with cold stratification and GA solution. The differently expressed proteins after treatments were characterized by iTRAQ coupled LC–MS/ MS. In total, 484 proteins showed that at least 1.3-fold differences in abundance were detected after treatments; a majority of the differently abundant proteins are involved in the respiration-related metabolism processes, biosynthesis of amino acids and translation during the dormancy release of Zanthoxylum nitidum seeds. This study is the first to use the iTRAQ-based proteomics approach to analyse the proteome of Zanthoxylum nitidum seeds, pointing out the direction for our further research on the pathways and even the key genes and proteins associated with the dormancy release of Zanthoxylum nitidum seeds.
1. Introduction
attention of many researchers. At present, the propagation of Zanthoxylum nitidum is mainly carried out by cutting propagation and seed reproduction (Sun et al., 2008). As a way of asexual reproduction, cutting propagation has many merits, including preferably maintaining the desirable characteristics, reproducing a large number of plants in a short period, etc (Jia et al., 2015). However, this approach is lacking the competence of developing tap roots of plant, which does not meet the medicinal purpose of using the roots that mainly accumulates effective components. Seed propagation can retain the integrity of the plant to the best as a primitive breeding method, and is considered an ideal propagation method for Zanthoxylum nitidum in herbal farming. However, long germination process and low emergence rate of Zanthoxylum nitidum seeds were found in field. These phenomena were also found in indoor germination tests and seed dormancy was determined. It is important and necessary to clarify the types of dormancy in Zanthoxylum nitidum seeds and find the methods of breaking dormancy, to improve breeding efficiency and promote sustainable utilization of Zanthoxylum nitidum resource. Seed dormancy is one of the least understood phenomena in the field of seed biology and could be considered simply as a block to the completion of germination of an
Zanthoxylum nitidum (Roxb.) DC. is a woody climber of the rue family (Rutaceae). The dried root of Zanthoxylum nitidum is a frequentlyused traditional Chinese medicine (namely Radix zanthoxyli), which has been included into every edition of Chinese Pharmacopoeia since 1977 Edition (Commission, 2015). Radix zanthoxyli has a comprehensive range of pharmacological effects, and is mainly used for traumatic injury, stomachache, toothache, rheumatic arthralgia, snakebite and burns (Chen et al., 2011; Hu et al., 2013; Li and Wang, 2013). The root (Radix zanthoxyli) is generally harvested after 5–6 years of growth, and the plants of Zanthoxylum nitidum die after losing their roots. Therefore, the availability of Radix zanthoxyli is very limited. Besides its medicinal value, Zanthoxylum nitidum is a significant raw material of some health products and household chemicals (Liu et al., 2005). However, wild Zanthoxylum nitidum has been excessively collected and the growth environment for the plants was severely damaged, resulted in a sharp drop of the resource quantity and rise of adulterants in market. Therefore, establishing the system of domestic cultivation and rapid proliferation for Zanthoxylum nitidum has drawn ⁎
1
Corresponding author. E-mail address: [email protected] (R. He). Current address: Analysis & Test Center, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China.
https://doi.org/10.1016/j.indcrop.2018.05.044 Received 2 January 2018; Received in revised form 4 April 2018; Accepted 20 May 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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The iTRAQ labelling procedure was performed following the instructions provided in the iTRAQ labelling kit (AB Sciex U.K. Limited). For each protein sample, 200 μg of protein was denatured, and the cysteine residues were blocked as described in the iTRAQ protocol. The protein samples were digested with sequencing-grade modified trypsin (Sigma) overnight at 37° C. The resultant peptide mixture was labeled with iTRAQ tags 113–119 and 121, respectively. The labeled samples were combined and vacuum dried. The labelled peptides were reconstructed in elution buffer before being subjected to strong cation exchange (SCX) fractionation and reverse-phase nanoliquid chromatography/tandem MS (LC–MS/MS) analysis.
intact viable seed under favourable conditions (Finch-Savage and Leubner-Metzger, 2006). Due to the significance of seed dormancy in the plant life cycle, particularly in agriculture and vegetative restoration, the studies concerning the regulatory mechanisms underlying seed dormancy are necessary (Graeber et al., 2012). The molecular regulation of seed dormancy has been studied in model species such as Arabidopsis thaliana and found that the main regulators included hormones, dormancy-specific genes and chromatin modifiers (Nee et al., 2017). Will results of studies on this species allow us to make broad generalizations about the basic mechanisms of seed dormancy? The answer is still uncertain. At present, more seed biologists have begun to adopt genetic approaches and omics technologies to study the seed dormancy mechanism of non-model organisms, such as European beech (Pawlowski, 2007), Norway maple (Pawlowski, 2009), sycamore (Pawlowski and Staszak, 2016), Cunninghamia lanceolata (Cao et al., 2016), Ornamental peach (Kanjana et al., 2016), American ginseng (Qi et al., 2015) and so on. In the present study, GA treatment and cold stratification were found that could release dormancy and improve the germination rate of Zanthoxylum nitidum seeds. We hypothesized that these two treatments can alter the genes expression and further influence the protein synthesis related to dormancy release and then enhance the germination rate in Zanthoxylum nitidum seeds. The objective of this study was to conduct proteome analysis among seeds treated by dormancy release methods stated above and obtain an understanding of the molecular mechanism during dormancy-release of Zanthoxylum nitidum seeds and to prove the hypothesis. Being applicable to a variety of samples and successfully identified great quantity and dynamic range of proteins, isobaric tags for relative and absolute quantification (iTRAQ) has become one of the most popular and powerful tools for proteome analysis (Wiese et al., 2007). Therefore, we adopted iTRAQ technology to identify and characterize the composition of the large number of proteins, especially those differentially abundant during dormancy progression.
2.3. Bioinformatics analysis Protein identification was carried out by processing the tandem mass spectra with ProteinPilot™ Software 5.0 (AB Sciex U.K. Limited), and then the obtained tandem mass spectra were searched against the Viridiplantae subset of Uniprot database, which contains a total of 3, 671, 389 sequences. Only proteins with at least two unique peptides and unused value of ≥1.3 were considered for further analysis. Identified proteins were quantified according to the total intensity of the assigned peptides. To screen for differentially expressed proteins in the largest range and prevent missing meaningful proteins, ratio changes in expression > 1.3 or < 0.77 and with a coefficient of variation (CV) value ≤0.5 were set as cutoff values (Gan et al., 2007; Hollander et al., 2012). The screened differentially expressed proteins (DEPs) were used for GO (gene ontology) annotation and classification analysis by QuickGO (Binns et al., 2009). GO database is an internationally standardized gene functional classification system, provides a set of dynamically updated standard glossaries to fully describe the attributes of genes and gene products in organisms. The GO project mainly depicts the differentially abundant proteins from three significant aspects including biological process, cellular component and molecular function. Furthermore, in KEGG (Kyoto Encyclopedia of Genes and Genomes) database, the selected differentially abundant proteins were annotated and classified into various biosynthetic and metabolic pathways and signal transduction pathways (Kanehisa et al., 2012). Some of the most crucial pathways the DEPs involved were selected after analyzing these pathways. The interaction networks among the DEPs were established by STRING (the Search Tool for the Retrieval of Interacting Genes/Proteins) database (Jensen et al., 2009).
2. Materials and methods 2.1. Seed materials and treatment conditions Freshly matured fruits were collected from of Zanthoxylum nitidum plants in Pinyuan county (N 24°30′, E 115°48′, a.s.l. 160 m), Guangdong Province, China on September 29, 2014. The fruits were shelled after naturally dried in the shade. The obtained seeds were then stored in silver sands containing approximately 15% water at 4 °C before use. Before germination test and extracting proteins, the seeds were divided into three groups: ZN, seeds were soaked in water under 25 °C for 2 days; ZNGA, seeds were soaked in 300 mg L−1 GA solution under 25 °C for 2 days; ZNS, seeds were treated with cold stratification at 4 °C for 3 months followed by soaking in water under 25 °C for 2 days. Approximately 10 g treated seeds were taken from each group, instantly frozen in liquid nitrogen and stored at −80 °C before protein extraction. Meanwhile, a germination test was carried out for each group. Based on the recommendations of the International Seed Testing Association (ISTA, 2016) and previous optimization tests (lab data not shown), the optimal condition of germination test was confirmed: the seeds (three replicates of 50 seeds each) were cultivated at 15 °C in semienclosed plastic trays with moist fine sand in the light (500 lx/0 lx (14 h/10 h)). Seed dormancy release is assessed by significant difference in seed germination rate.
3. Results 3.1. Seed germination rate in three different groups As shown in Fig. 1, the germination rate of Zanthoxylum nitidum seeds in the control group is only 2.7% and the lowest among the three groups, the germination rate of seeds treated with GA (300 mg L−1) is 22% and significantly higher than the control group (P < 0.01), the germination rate of seeds after cold stratification is 30.7% and significantly higher than the control group (P < 0.01) and the GA treatment group (P < 0.01). 3.2. Protein identification and quantification To uncover the significant metabolic proteins involved in the dormancy alleviation mechanism of Zanthoxylum nitidum seeds, iTRAQbased quantitative proteomic characterization of differently treated seeds was conducted. A global profiling of quantitative proteome was acquired on seeds at different dormant stage after water, cold stratification and GA treatment with two biological replicates. A toatl of 7540 unique peptides and 1207 corresponding proteins were identified. Among them, 1047 and 1022 proteins were identified in group ZNS vs ZN and ZNGA vs ZN respectively. Protein abundances that changed less than 1.3-fold were discarded when statistical significance is detected by
2.2. Protein extraction, iTRAQ labelling and liquid chromatographytandem mass spectrometry (LC–MS/MS) separation Total proteins were extracted using the cold acetone method (Wu et al., 2014). Standard BCA assay was used to detect protein concentration (Sigma Sigma-Aldrich (Shanghai) Trading Co. Ltd. (China)). 8
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Fig. 1. Germination rate of Zanthoxylum nitidum seeds under a suitable condition after three different treatments. ZN, seeds were soaked in water under 25 °C for 2 days; ZNGA, seeds were soaked in 300 mg L−1 Gibberellin solution under 25 °C for 2 days; ZNS, seeds were treated with cold stratification at 4 °C for 3 months followed by soaking in water under 25 °C for 2 days. Germination conditions: temperature, 15 °C; illumination, 500 lx/0 lx (14 h/10 h); germinating bed, sand bed. Each group contains three biological replicates and each replicate contains 50 seeds.
Fig. 2. Venn diagram showing the number of proteins differentially expressed between upregulated proteins of ZNS vs ZN, downregulated proteins of ZNS vs ZN, upregulated proteins of ZNGA vs ZN and downregulated proteins of ZNGA vs ZN.
the ANOVA test (P < 0.05). Finally, 420 DEPs including 208 upregulated and 212 downregulated were screened out in ZNS vs ZN, and 198 proteins including 109 upregulated and 89 downregulated in ZNGA vs ZN. The results showed that, consistent with the differences of the germination rate of Zanthoxylum nitidum seeds, the number of differentially expressed and upregulated proteins in ZNS vs ZN is higher than that in ZNGA vs ZN.
metabolic process and biosynthetic process. In the upregulation profile of ZNGA vs ZN, DEPs mainly experienced small molecule metabolic process, carbohydrate metabolic process, translation, transport and catabolic process. In the downregulation profile of ZNGA vs ZN, DEPs mainly experienced small molecule metabolic process, cellular nitrogen compound metabolic process, carbohydrate metabolic process, biosynthetic process and catabolic process. In molecular function, the differentially abundant proteins in upregulated profile of ZNS vs ZN were mainly related to oxidoreductase activity, ion binding, lyase activity, peptidase activity and structural constituent of ribosome. While in downregulated profile of ZNS vs ZN, they were mainly related to oxidoreductase activity, ion binding, peptidase activity, lyase activity and structural constituent of ribosome. In upregulated profile of ZNGA vs ZN, DEPs were mainly related to oxidoreductase activity, ion binding, peptidase activity, lyase activity and GTPase activity. In downregulated profile of ZNGA vs ZN, DEPs were mainly related to ion binding, oxidoreductase activity, DNA binding, isomerase activity and structural constituent of ribosome. In cellular component, the differentially abundant proteins in upregulated profile of ZNS vs ZN mainly distributed in intracellular, cytoplasm, ribosome, extracellular region and mitochondrion. While in downregulated profile of ZNS vs ZN, they mainly distributed in intracellular, cytoplasm, protein complex, ribosome and cytosol. In upregulated profile of ZNGA vs ZN, DEPs were mainly distributed in intracellular, mitochondrion, cytoplasm, ribosome and protein complex. In downregulated profile of ZNGA vs ZN, DEPs were mainly distributed in intracellular, cytoplasm, nucleus, cytosol and ribosome. According to KEGG analysis (Fig. 5), in the ZNS vs ZN profile, the proteins mainly experienced carbon metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, biosynthesis of amino acids and ribosome. In ZNGA vs ZN, the proteins mainly experienced carbon metabolism, biosynthesis of amino acids, glycolysis/gluconeogenesis, ribosome and glyoxylate and dicarboxylate metabolism. Following annotation, amino acid codes were sequentially mapped into biosynthetic pathways. As Fig. 6 shows, we find that the DEPs were evenly mapped into all the parts of amino acid biosynthetic pathway in KEGG; proteins involved in amino acid biosynthetic pathway were very active during the seed stratification process (ZNS vs ZN), and most of DEPs were upregulated, such as enolase [EC:4.2.1.11], aspartatesemialdehyde dehydrogenase [EC:1.2.1.11] and ketol-acid reductoisomerase [EC:1.1.1.86], these were about 2 fold changed, and
3.3. Protein expression profiles between different treatments A total of 484 proteins were identified over 1.3-fold significant changes during the dormancy release of Zanthoxylum nitidum seeds. Then four protein expression profiles were established: compared with control group (ZN), 126 proteins were just upregulated in the ZNS group; 26 proteins were only upregulated in the ZNGA group; 160 of proteins were only downregulated in the ZNS group; 38 of proteins were only downregulated in the ZNGA group; 76 of proteins were upregulated both in the ZNS group and ZNGA group; 45 of proteins were downregulated both in the ZNS group and ZNGA group; 6 of proteins were upregulated in the ZNS group and downregulated in the ZNGA group; 7 of proteins were upregulated in the ZNGA group and downregulated in the ZNS group (Fig. 2). As shown in Fig. 2, most of the common differentially expressed proteins in ZNS vs ZN and ZNGA vs ZN are upregulated or downregulated proteins. This shows that the molecular changes in ZNS and ZNGA group may overlap. 3.4. Functions and pathways analysis of differentially expressed proteins To show the global features of the specific functional categories enriched in Zanthoxylum nitidum seeds, gene ontology (GO) category enrichment analysis was carried to categorize all 484 proteins with 1.3fold difference based on GO Slim classification for vegetation. As Figs. 3 and 4 shows, proteins with differential abundances are annotated into various biological process, cellular component and molecular function after GO analysis. In biological process category, the differentially abundant proteins in the upregulation profile of ZNS vs ZN mainly experienced small molecule metabolic process, carbohydrate metabolic process, cellular nitrogen compound metabolic process, transport, catabolic process and biosynthetic process; while in the downregulation profile of ZNS vs ZN, they mainly experienced small molecule metabolic process, carbohydrate metabolic process, catabolic process, cellular nitrogen compound 9
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Fig. 3. Gene ontology (GO) of the differentially expressed proteins identified between ZNS and ZN, red representing up-regulated proteins and green representing down-regulated proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
biosynthesis of amino acids and translation. And both ZNS vs ZN and ZNGA vs ZN showed fewer changes in their proteomes of plant hormone signal transduction.
glyceraldehyde 3-phosphate dehydrogenase [EC:1.2.1.12], aconitate hydratase [EC:4.2.1.3] and glycine hydroxymethyltransferase [EC:2.1.2.1] were even more than 5 fold upregulated. Whereas, D-3phosphoglycerate dehydrogenase [EC:1.1.1.95], phosphoglycerate kinase [EC:2.7.2.3], pyruvate kinase [EC:2.7.1.40] and glycine hydroxymethyltransferase [EC:2.1.2.1] were over 2 fold downregulated in this pathway (Fig. 6, Table S1). According to results mentioned above, we found similar functional proteome changes focusing on respiratory related pathways,
3.5. Protein–protein interaction analysis of differentially expressed proteins In order to illuminate the interactions of the proteins taking part in the dormancy release, the differentially abundant proteins in the research was used to build the protein–protein interaction (PPI) network
Fig. 4. Gene ontology (GO) of the differentially expressed proteins identified between ZNGA and ZN, red representing up-regulated proteins and green representing down-regulated proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 10
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Fig. 5. Histogram of the distribution of differentially abundant proteins according to their predicted KEGG signaling pathways of the proteins identified as involved in Zanthoxylum nitidum seed dormancy release of (A) ZNS vs ZN and (B) ZNGA vs ZN.
alleviate seed dormancy or act through distinct signaling pathways remains unclear. In order to observe some characteristics of these two treatments at the molecular level, we conducted a proteomics study on Zanthoxylum nitidum seeds experienced these two treatments along with freshly harvested seeds using iTRAQ technique. In germination test, the germination rate of GA-treated seeds was lower than that of the seeds after cold stratification. Correspondingly, the number of DEPs in the GA-treated group was lower than that in the stratification group. Based on the KEGG results and GO results, a majority of functional proteome changes were found similar, though the numbers of DEPs of the two groups differred greatly. Here, we will analyze the characteristics of differentially expressed proteome mainly from the following three aspects.
by the STRING database. Thirty-six DEPs took part in the respirationrelated metabolism processes were used, “Arabidopsis thaliana” was selected in the item of Organism, and 15 DEPs were annotated in the STRING database and the PPI network was established (Fig. 7, Table S2). Phosphofructokinase family protein (AT1G12000), which is the catalytic subunit of pyrophosphate–fructose 6-phosphate 1-phosphotransferase, catalyzes the phosphorylation of D-fructose 6-phosphate, the first committing step of glycolysis. It also uses inorganic phosphate (PPi) as phosphoryl donor instead of ATP like common ATP-dependent phosphofructokinases (ATP-PFKs), which renders the reaction reversible, and can thus function both in glycolysis and gluconeogenesis. It has the interaction with Aldolase superfamily protein (FBA6), Phosphoglucomutase 3 (PGM3), Pyruvate kinase (AT5G08570), Bifunctional enolase 2/transcriptional activator (LOS2), Glucose-6-phosphate isomerase (AT5G42740), Malate dehydrogenase (c-NAD-MDH1), Phosphoglycerate kinase (PGK), Dihydrolipoyl dehydrogenase 1 (mtLPD1) and Succinyl-CoA ligase, alpha subunit (AT5G23250). PGM3 participates in both the breakdown and synthesis of glucose. LOS2 is a multifunctional enzyme, which acts as an enolase involved in the metabolism and, as a positive regulator of cold-responsive gene transcription. LOS2 exhibited interactions with (PGK, FBA6, Phosphofructokinase family protein (AT1G12000), PGM3, AT5G08570, AT5G42740, Aldehyde dehydrogenase 11A3 (ALDH11A3), c-NADMDH1, mtLPD1, AT5G23250, Aldehyde dehydrogenase (ALDH7B4), Aldehyde dehydrogenase 2B4 (ALDH2B4); bound to the cis-element of the gene promoter of STZ/ZAT10, a zinc finger transcriptional repressor. ALDH2B4 possesses activity on acetaldehyde and glycolaldehyde in vitro. ALDH11A3 is an important means of generating NADPH for biosynthetic reactions. Glyoxalase I homolog (GLX1) catalyzes the conversion of hemimercaptal, formed from methylglyoxal and glutathione, to S-lactoylglutathione. MtLPD1 is a component of the glycine decarboxylase (GDC) or glycine cleavage system as well as part of the alpha-ketoacid dehydrogenase complexes. LPD1 is probably the protein most often associated with the glycine decarboxylase complex while LPD2 is probably incorporated into alpha-ketoacid dehydrogenase complexes.
4.1. Respiratory metabolism pathways are activated during dormancy release From the classification of DEPs associated with dormancy reduction, we found that the metabolic categories are the most representative. Regardless of treatments, a large portion of DEPs were annotated to four main respiratory metabolism pathways: Glycolysis/ Gluconeogenesis, Oxidative phosphorylation, Citrate cycle and Pentose phosphate pathway. Studies have shown that these pathways are not isolated, but are interrelated and interacted at several levels by exchanging metabolic intermediates. Different respiratory substrates enter these pathways at various locations for respiratory metabolism. The respiratory activities of treated seeds increased, indicating that the seeds has completely or partially walked away from the quiescent state (namely seed dormancy) and be ready for germination. Nucleoside diphosphate kinase (ndk) was upregulated in both treatment groups, and its expression was affected by stratification and GA. It takes part in nucleotide metabolism and keeps cellulate ATP with other nucleoside triphosphates in a dynamic equilibrium. It participates in the synthesis of purines and pyrimidines. Nevertheless, it also plays a role in many regulatory functions apart from the housekeeping effect. Lots of factors would trigger the expression of ndk, such as vulnus, heat shock, oxidant stress, pathogenic organism, abscisic acid, and so on (Cho et al., 2004). The content of ndk were altered at seed germination and early seedling growth (Yano et al., 1995). In plants, it may also function in the signal transduction of mitogen-activated protein kinase (MAPK), which is regulated by GA (Choi et al., 1999; Yano et al., 1995). Compared with control group (ZN), the up-regulated level of Adenosylhomocysteinase (ahcY) in the stratification group (11.9 fold)
4. Discussion As is well known, cold stratification or exogenous application of GA could break the seed dormancy in many plants at different degrees (Rascio et al., 1998; Song et al., 2017; Wang et al., 2016). Whereas, whether these two treatments arouse the same potential mechanisms to 11
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Fig. 6. Proteins with differential abundances involved in biosynthesis of amino acids were mapped to the biosynthetic pathway in KEGG, red representing upregulated proteins and blue representing down-regulated proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in amino acid and folate metabolism, and is also one of the main sources of carbon units for certain indispensable biosynthesis processes (Florio et al., 2011). glyA also catalyzes the biosynthesis of 5-formyltetrahydropteroylglutamate, which was indicated to act as a storage form of reduced folates and one-carbon groups in dormant stage cells (Kruschwitz et al., 1994). In our research, cold stratification and GA treatment both improved the content of glyA, showing that one-carbon units are necessary for the embryo formation during the transition from dormancy release to germination. glyA acts also on behalf of a vital crosstalk among amino acid metabolism, one-carbon metabolism, and photorespiration. Serine, a metabolite generated by glyA, serves as a metabolic signal in the transcription control of genes associated with photorespiration (Timm et al., 2013). Therefore, glyA may be involved in regulating the photorespiration, which is essential for the growth and development of seedlings.
was more notable than that in the GA-treated group (5.6 fold). Adenosylhomocysteine competitively inhibits the methyl transferase reactions, which depends on Sadenosylmethionine. Therefore, ahcY may play an important role in controlling DNA or other substrates’ methylation through regulating the level of adenosylhomocysteine in cells. The activity of ahcY decreased in the hog1 mutant of Arabidopsis thaliana, which resulted in the maintenance of methylation being disturbed and the expression of many genes affected. These mutant plants have the characteristics of slow growth, poor fertility and decreased seed germination. When a T-DNA including the gene coding for ahcY complemented the hog1 mutation, DNA methylation, plant growth and seed viability were brought normal (Rocha et al., 2005). Hence, ahcY seems to play a role in the way that regulating DNA methylation, which influences the expression of genes taking charge of seed dormancy release. The level of Glycine hydroxymethyltransferase (glyA) in both treated groups exceeded that in the control group, and the up-regulation in the stratification group particularly outstanding. glyA takes part 12
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molecular activities involved in these genes are important for seed dormancy. At present, we have not yet identified the specific genes associated with the dormancy of Zanthoxylum nitidum seeds. However, we found some changes of the pathways associated with genetic information processing both in ZNS vs ZN and ZNGA vs ZN, especially the pathway of translation. Pawlowski et al. found that at least 22 of the proteins identified as being associated with the dormancy release of sycamore seeds were classified in the genetic information processing category and identified six HSPs that responded to various treatments (Pawlowski and Staszak, 2016). In this study, we found that 60S ribosomal proteins responded to various treatments. 4.4. Plant hormone signal pathways showed fewer changes during dormancy release With more and more studies about the effects of various hormones on dormancy in Arabidopsis thaliana, almost all plant hormones were found to be involved in the regulation of dormancy and germination (Finkelstein et al., 2008; Shu et al., 2016). In this research, KEGG pathway analysis showed some changes of the proteins in the plant hormone signal pathways. In ZNS vs. ZN, the DEPs in the plant hormone signal pathways are mainly associated with auxin, ethylene and jasmonic acid (JA). In ZNGA vs. ZN, the DEPs in the plant hormone signal pathways are mainly associated with auxin and ethylene. The plant hormone auxin participates in almost all aspects of plant growth and development, and responds to a variety of environmental factors (Vanneste and Friml, 2009; Zhao, 2010). Previously, auxin had not been considered as an important player in seed dormancy and germination, in spite of its participation in the cross-linking with abscisic acid (ABA). New genetic and biochemical evidence showed that auxin influences ABA signaling to exert its physiological effect on the control of seed dormancy and germination (Liu et al., 2013; Ye et al., 2016). In ZNS vs. ZN, three DEPs including aldehyde dehydrogenase family 7 member A1, aldehyde dehydrogenase and catalase, were associated with auxin, took part in Tryptophan metabolism, which has an indirect effect on auxin in plant hormone signal pathway. In ZNGA vs. ZN, only one protein catalase was differentially expressed in Tryptophan metabolism. Some researchers have proved that ethylene may has a negative influence on ABA biosynthesis and signaling (Ghassemian et al., 2000). Further studies found that ethylene could offset the impact of ABA to reduce seed dormancy and improve seed germination (Arc et al., 2013; Corbineau et al., 2014). Nevertheless, whether and how ethylene impacts GA biosynthesis and signaling to change seed dormancy and germination is still unknown in a large part. In this study, four proteins including aspartate-semialdehyde dehydrogenase, malate dehydrogenase, malate dehydrogenase and adenosylhomocysteinase differentially expressed after stratification treatment, and these proteins participated in Cysteine and methionine metabolism, which located in the upstream of the plant hormone signal pathway. Therefore, changes of these proteins may produce an important impact. Only one protein, adenosylhomocysteinase, in Cysteine and methionine metabolism appeared significantly upregulated after GA treatment. Seed germination was put off after JA application, showing that JA can inhibit the seed germination process (Nambara et al., 2010). Nevertheless, another study found that JA respectively restrain the transcription of ABA biogenesis genes and motivates ABA-inactivating genes, indicating that JA and ABA has antagonistic effect (Jacobsen et al., 2013). However, the reason why the effect of JA in seed germination is frequently paradoxical remains unknown. In ZNS vs. ZN, four DEPs, alcohol dehydrogenase class-P, acetyl-CoA acyltransferase 1, allene oxide cyclase, and acetyl-CoA acyltransferase are involved in αLinolenic acid metabolism, in which the biosynthesis of JA is located. No DEP relevant to JA was found in Zanthoxylum nitidum seeds after GA treatment. It should be noted that the effects of plant hormones on seed
Fig. 7. The interaction networks were built by 15 DEPs which participate in the respiration-related metabolism processes during the alleviation of seed dormancy. Protein-protein interaction network was analyzed by STRING database. The confidence score was set to medium level ≥0.4. The blue lines represent database evidence; the purple lines represent experimental evidence; yellow lines represent text mining evidence; the black lines represent coexpression evidence; and green lines represent neighborhood evidence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4.2. Dormancy releasing treatments activate the biosynthesis of amino acids In the stratification group and GA-treated group, a large amount of proteins were annotated into Biosynthesis of amino acids, which is a feasible fate for the carbon skeletons from seed storage compound mobilization. β-Aspartyl-semialdehyde (ASA) is a common precursor for the biosynthesis of threonine, methionine and lysine. ASA dehydrogenase (ASADH) locates at the first branch point in the biogenesis pathway of aspartic acid in plants, ASA formation is catalyzed by ASADH via the reductive dephosphorylation of β-aspartyl phosphate utilizing NADPH (Paris et al., 2002). Ketol-acid reductoisomerase is an enzyme in the biosynthesis pathway of branched-chain amino acid, in which it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate (Leung and Guddat, 2009). D-3-phosphoglycerate dehydrogenase takes part in the biosynthesis of L-serine. Aspartate-semialdehyde dehydrogenase (ASADH family protein), Ketol-acid reductoisomerase, and D-3-phosphoglycerate dehydrogenase differently expressed in the treating groups, showing that Biosynthesis of amino acids play a crucial role in seed dormancy alleviation. Our results resembled those of Erwann Arc and Xu Hengheng’s, who put forward that dormancy-relieving treatments can stimulate amino acid biosynthesis and/or recycling pathways (Arc et al., 2012; Xu et al., 2016).
4.3. Dormancy releasing treatments motivate translation activities Seed dormancy is an important and complex adaptive trait that is determined by genetic factors with a substantial environmental influence (Graeber et al., 2012). Some genes have been identified that specially regulate seed dormancy (Nee et al., 2017). These genes have a seed-specific expression pattern and show strong dormancy mutant phenotypes. The main representative genes among this group are DELAY OF GERMINATION 1 (DOG1) and REDUCED DORMANCY 5 (RDO5) (Bentsink et al., 2006; Xiang et al., 2014). Therefore, the 13
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dormancy and germination are cross-linked and the individual analysis about each hormone-related proteins may be insufficient or inadequate. Although plant hormone signal pathways showed fewer changes during the dormancy release of Zanthoxylum nitidum seeds, discussion on this part is still very necessary.
arabidopsis. Plant Cell 12, 1117–1126. Graeber, K., Nakabayashi, K., Miatton, E., Leubner-Metzger, G., Soppe, W.J.J., 2012. Molecular mechanisms of seed dormancy. Plant Cell Environ. 35, 1769–1786. Hollander, A., D'Onofrio, P.M., Magharious, M.M., Lysko, M.D., Koeberle, P.D., 2012. Quantitative retinal protein analysis after optic nerve transection reveals a neuroprotective role for hepatoma-derived growth factor on injured retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 53, 3973–3989. Hu, J., Shi, X.D., Mao, X., Chen, J.G., Zhu, L., Zhao, Q.J., 2013. Antinociceptive activity of Rhoifoline A from the ethanol extract of Zanthoxylum nitidum in mice. J. Ethnopharmacol. 150, 828–834. ISTA, 2016. International Rules for Seed Testing, 2016 ed. International Seed Testing Association, Bassersdorf, Switzerland. Jacobsen, J.V., Barrero, J.M., Hughes, T., Julkowska, M., Taylor, J.M., Xu, Q., Gubler, F., 2013. 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Nambara, E., Okamoto, M., Tatematsu, K., Yano, R., Seo, M., Kamiya, Y., 2010. Abscisic acid and the control of seed dormancy and germination. Seed Sci. Res. 20, 55–67. Nee, G., Xiang, Y., Soppe, W.J.J., 2017. The release of dormancy, a wake-up call for seeds to germinate. Curr. Opin. Plant Biol. 35, 8–14. Paris, S., Wessel, P.M., Dumas, R., 2002. Overproduction, purification, and characterization of recombinant aspartate semialdehyde dehydrogenase from Arabidopsis thaliana. Protein Expr. Purif. 24, 99–104. Pawlowski, T.A., Staszak, A.M., 2016. Analysis of the embryo proteome of sycamore (Acer pseudoplatanus L.) seeds reveals a distinct class of proteins regulating dormancy release. J. Plant Physiol. 195, 9–22. Pawlowski, T.A., 2007. Proteomics of European beech (Fagus sylvatica L.) seed dormancy breaking: influence of abscisic and gibberellic acids. Proteomics 7, 2246–2257. Pawlowski, T.A., 2009. Proteome analysis of Norway maple (Acer platanoides L.) seeds dormancy breaking and germination: influence of abscisic and gibberellic acids. BMC Plant Biol. 9. Qi, J.J., Sun, P., Liao, D.Q., Sun, T.Y., Zhu, J., Li, X.N., 2015. Transcriptomic analysis of American ginseng seeds during the dormancy release process by RNA-Seq. PLoS One 10. Rascio, N., Mariani, P., Dalla Vecchia, F., La Rocca, N., Profumo, P., Gastaldo, P., 1998. Effects of seed chilling or GA(3) supply on dormancy breaking and plantlet growth in Cercis siliquastrum L. Plant Growth Regul. 25, 53–61. Rocha, P., Sheikh, M., Melchiorre, R., Fagard, M., Boutet, S., Loach, R., Moffatt, B., Wagner, C., Vaucheret, H., Furner, I., 2005. The Arabidopsis homology-dependent gene silencingi gene codes for an S-adenosyl-L-homocysteine hydrolase required for DNA methylation-dependent gene silencing. Plant Cell 17, 404–417. Shu, K., Liu, X.D., Xie, Q., He, Z.H., 2016. 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Role of short-term cold stratification on seed dormancy break and germination of alien species in southeastern China. Plant Ecol. 217, 383–392. Wiese, S., Reidegeld, K.A., Meyer, H.E., Warscheid, B., 2007. Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research. Proteomics 7, 340–350. Wu, X.L., Xiong, E.H., Wang, W., Scali, M., Cresti, M., 2014. Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction
5. Conclusion Along with the dormancy release of Zanthoxylum nitidum seeds, the proteomes have also undergone some significant changes both in stratification and GA-treated groups. Our results showed that stratification and GA treatment caused similar proteome changes associated with the dormancy release of Zanthoxylum nitidum seeds. These changes are mainly enriched in respiratory-related metabolic pathways, biosynthesis of amino acids and translation. However, unlike the results of the model organism Arabidopsis thaliana, we found that plant hormone signal pathways showed fewer changes during the dormancy release of Zanthoxylum nitidum seeds. Conflicts of interest The authors declare no conflict of interest. Acknowledgement This research was supported by grant from General university innovation team project of Guangdong province-Innovative research team of Chinese Medicine resources (2016KYTD02). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.05.044. References Arc, E., Chiban, K., Grappin, P., Jullien, M., Godin, B., Cueff, G., Valot, B., Balliau, T., Job, D., Rajjou, L., 2012. Cold stratification and exogenous nitrates entail similar functional proteome adjustments during arabidopsis seed dormancy release. J. Proteome Res. 11, 5418–5432. Arc, E., Sechet, J., Corbineau, F., Rajjou, L., Marion-Poll, A., 2013. ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination. Front. Plant Sci. 4. Bentsink, L., Jowett, J., Hanhart, C.J., Koornneef, M., 2006. Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 103, 17042–17047. Binns, D., Dimmer, E., Huntley, R., Barrell, D., O'Donovan, C., Apweiler, R., 2009. QuickGO: a web-based tool for gene ontology searching. Bioinformatics 25, 3045–3046. Cao, D.C., Xu, H.M., Zhao, Y.Y., Deng, X., Liu, Y.X., Soppe, W.J.J., Lin, J.X., 2016. Transcriptome and degradome sequencing reveals dormancy mechanisms of cunninghamia lanceolata seeds. Plant Physiol. 172, 2347–2362. Chen, J.J., Lin, Y.H., Day, S.H., Hwang, T.L., Chen, I.S., 2011. New benzenoids and antiinflammatory constituents from Zanthoxylum nitidum. Food Chem. 125, 282–287. Cho, S.M., Shin, S.H., Kim, K.S., Kim, Y.C., Eun, M.Y., Cho, B.H., 2004. Enhanced expression of a gene encoding a nucleoside diphosphate kinase 1 (OsNDPK1) in rice plants upon infection with bacterial pathogens. Mol. Cells 18, 390–395. Choi, G., Yi, H., Lee, J., Kwon, Y.K., Soh, M.S., Shin, B.C., Luka, Z., Hahn, T.R., Song, P.S., 1999. Phytochrome signalling is mediated through nucleoside diphosphate kinase 2. Nature 401, 610–613. Commission, C.P., 2015. Chinese Pharmacopoeia, 2015 ed. China Medical Science Press, Beijing. Corbineau, F., Xia, Q., Bailly, C., El-Maarouf-Bouteau, H., 2014. Ethylene, a key factor in the regulation of seed dormancy. Front. Plant Sci. 5. Finch-Savage, W.E., Leubner-Metzger, G., 2006. Seed dormancy and the control of germination. New Phytol. 171, 501–523. Finkelstein, R., Reeves, W., Ariizumi, T., Steber, C., 2008. Molecular aspects of seed dormancy. Annu. Rev. Plant Biol. 387–415. Florio, R., di Salvo, M.L., Vivoli, M., Contestabile, R., 2011. Serine hydroxymethyltransferase: a model enzyme for mechanistic, structural, and evolutionary studies. Biochim. Biophys. Acta-Proteins Proteomics 1814, 1489–1496. Gan, C.S., Chong, P.K., Pham, T.K., Wright, P.C., 2007. Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). J. Proteome Res. 6, 821–827. Ghassemian, M., Nambara, E., Cutler, S., Kawaide, H., Kamiya, Y., McCourt, P., 2000. Regulation of abscisic acid signaling by the ethylene response pathway in
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encoding rice nucleoside diphosphate kinase (ndk) in escherichia-coli and organ-related alteration of ndk activities during rice seed-germination (ORYZA-SATIVA L). Plant Mol. Biol. 27, 1053–1058. Ye, Y.J., Gong, Z.Y., Lu, X., Miao, D.Y., Shi, J.M., Lu, J., Zhao, Y., 2016. Germostatin resistance locus 1 encodes a PHD finger protein involved in auxin-mediated seed dormancy and germination. Plant J. 85, 3–15. Zhao, Y.D., 2010. Auxin biosynthesis and its role in plant development. In: In: Merchant, S., Briggs, W.R., Ort, D. (Eds.), Annual Review of Plant Biology Vol 61. pp. 49–64.
for crop proteomic analysis. Nat. Protoc. 9, 362–374. Xiang, Y., Nakabayashi, K., Ding, J., He, F., Bentsink, L., Soppe, W.J.J., 2014. Reduced dormancy5 encodes a protein phosphatase 2C that is required for seed dormancy in arabidopsis. Plant Cell 26, 4362–4375. Xu, H.H., Liu, S.J., Song, S.H., Wang, W.Q., Moller, I.M., Song, S.Q., 2016. Proteome changes associated with dormancy release of Dongxiang wild rice seeds. J. Plant Physiol. 206, 68–86. Yano, A., Umeda, M., Uchimiya, H., 1995. Expression of functional proteins of cdna-
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Proteomic analysis of Zanthoxylum nitidum seeds dormancy release: Influence of stratification and gibberellin Qiang Lu, Zhen Shan Zhang1, Ruo Ting Zhan, Rui He
T
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Key Laboratory of Chinese Medicinal Resource from Lingnan, Ministry of Education and Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Seed dormancy Zanthoxylum nitidum iTRAQ Cold stratification GA
Zanthoxylum nitidum (Roxb.) DC. is one of the most important economic crops on account of its value as a Chinese herbal medicine (Zanthoxyli radix), health product and daily chemical raw materials. Zanthoxylum nitidum seeds are characterized to be an intermediate physiological dormancy that requires three months of cold stratification (4 °C) or gibberellin (GA) treatment (300 mg L−1, soaking for two days) to relieve seed dormancy and promote germination. Exploring the changes of proteins and related biosynthetic and metabolic pathways during seed dormancy release will contribute to expand our knowledge of this process. In this study, mature Zanthoxylum nitidum seeds from Pingyuan County, Guangdong province were treated with cold stratification and GA solution. The differently expressed proteins after treatments were characterized by iTRAQ coupled LC–MS/ MS. In total, 484 proteins showed that at least 1.3-fold differences in abundance were detected after treatments; a majority of the differently abundant proteins are involved in the respiration-related metabolism processes, biosynthesis of amino acids and translation during the dormancy release of Zanthoxylum nitidum seeds. This study is the first to use the iTRAQ-based proteomics approach to analyse the proteome of Zanthoxylum nitidum seeds, pointing out the direction for our further research on the pathways and even the key genes and proteins associated with the dormancy release of Zanthoxylum nitidum seeds.
1. Introduction
attention of many researchers. At present, the propagation of Zanthoxylum nitidum is mainly carried out by cutting propagation and seed reproduction (Sun et al., 2008). As a way of asexual reproduction, cutting propagation has many merits, including preferably maintaining the desirable characteristics, reproducing a large number of plants in a short period, etc (Jia et al., 2015). However, this approach is lacking the competence of developing tap roots of plant, which does not meet the medicinal purpose of using the roots that mainly accumulates effective components. Seed propagation can retain the integrity of the plant to the best as a primitive breeding method, and is considered an ideal propagation method for Zanthoxylum nitidum in herbal farming. However, long germination process and low emergence rate of Zanthoxylum nitidum seeds were found in field. These phenomena were also found in indoor germination tests and seed dormancy was determined. It is important and necessary to clarify the types of dormancy in Zanthoxylum nitidum seeds and find the methods of breaking dormancy, to improve breeding efficiency and promote sustainable utilization of Zanthoxylum nitidum resource. Seed dormancy is one of the least understood phenomena in the field of seed biology and could be considered simply as a block to the completion of germination of an
Zanthoxylum nitidum (Roxb.) DC. is a woody climber of the rue family (Rutaceae). The dried root of Zanthoxylum nitidum is a frequentlyused traditional Chinese medicine (namely Radix zanthoxyli), which has been included into every edition of Chinese Pharmacopoeia since 1977 Edition (Commission, 2015). Radix zanthoxyli has a comprehensive range of pharmacological effects, and is mainly used for traumatic injury, stomachache, toothache, rheumatic arthralgia, snakebite and burns (Chen et al., 2011; Hu et al., 2013; Li and Wang, 2013). The root (Radix zanthoxyli) is generally harvested after 5–6 years of growth, and the plants of Zanthoxylum nitidum die after losing their roots. Therefore, the availability of Radix zanthoxyli is very limited. Besides its medicinal value, Zanthoxylum nitidum is a significant raw material of some health products and household chemicals (Liu et al., 2005). However, wild Zanthoxylum nitidum has been excessively collected and the growth environment for the plants was severely damaged, resulted in a sharp drop of the resource quantity and rise of adulterants in market. Therefore, establishing the system of domestic cultivation and rapid proliferation for Zanthoxylum nitidum has drawn ⁎
1
Corresponding author. E-mail address: [email protected] (R. He). Current address: Analysis & Test Center, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China.
https://doi.org/10.1016/j.indcrop.2018.05.044 Received 2 January 2018; Received in revised form 4 April 2018; Accepted 20 May 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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The iTRAQ labelling procedure was performed following the instructions provided in the iTRAQ labelling kit (AB Sciex U.K. Limited). For each protein sample, 200 μg of protein was denatured, and the cysteine residues were blocked as described in the iTRAQ protocol. The protein samples were digested with sequencing-grade modified trypsin (Sigma) overnight at 37° C. The resultant peptide mixture was labeled with iTRAQ tags 113–119 and 121, respectively. The labeled samples were combined and vacuum dried. The labelled peptides were reconstructed in elution buffer before being subjected to strong cation exchange (SCX) fractionation and reverse-phase nanoliquid chromatography/tandem MS (LC–MS/MS) analysis.
intact viable seed under favourable conditions (Finch-Savage and Leubner-Metzger, 2006). Due to the significance of seed dormancy in the plant life cycle, particularly in agriculture and vegetative restoration, the studies concerning the regulatory mechanisms underlying seed dormancy are necessary (Graeber et al., 2012). The molecular regulation of seed dormancy has been studied in model species such as Arabidopsis thaliana and found that the main regulators included hormones, dormancy-specific genes and chromatin modifiers (Nee et al., 2017). Will results of studies on this species allow us to make broad generalizations about the basic mechanisms of seed dormancy? The answer is still uncertain. At present, more seed biologists have begun to adopt genetic approaches and omics technologies to study the seed dormancy mechanism of non-model organisms, such as European beech (Pawlowski, 2007), Norway maple (Pawlowski, 2009), sycamore (Pawlowski and Staszak, 2016), Cunninghamia lanceolata (Cao et al., 2016), Ornamental peach (Kanjana et al., 2016), American ginseng (Qi et al., 2015) and so on. In the present study, GA treatment and cold stratification were found that could release dormancy and improve the germination rate of Zanthoxylum nitidum seeds. We hypothesized that these two treatments can alter the genes expression and further influence the protein synthesis related to dormancy release and then enhance the germination rate in Zanthoxylum nitidum seeds. The objective of this study was to conduct proteome analysis among seeds treated by dormancy release methods stated above and obtain an understanding of the molecular mechanism during dormancy-release of Zanthoxylum nitidum seeds and to prove the hypothesis. Being applicable to a variety of samples and successfully identified great quantity and dynamic range of proteins, isobaric tags for relative and absolute quantification (iTRAQ) has become one of the most popular and powerful tools for proteome analysis (Wiese et al., 2007). Therefore, we adopted iTRAQ technology to identify and characterize the composition of the large number of proteins, especially those differentially abundant during dormancy progression.
2.3. Bioinformatics analysis Protein identification was carried out by processing the tandem mass spectra with ProteinPilot™ Software 5.0 (AB Sciex U.K. Limited), and then the obtained tandem mass spectra were searched against the Viridiplantae subset of Uniprot database, which contains a total of 3, 671, 389 sequences. Only proteins with at least two unique peptides and unused value of ≥1.3 were considered for further analysis. Identified proteins were quantified according to the total intensity of the assigned peptides. To screen for differentially expressed proteins in the largest range and prevent missing meaningful proteins, ratio changes in expression > 1.3 or < 0.77 and with a coefficient of variation (CV) value ≤0.5 were set as cutoff values (Gan et al., 2007; Hollander et al., 2012). The screened differentially expressed proteins (DEPs) were used for GO (gene ontology) annotation and classification analysis by QuickGO (Binns et al., 2009). GO database is an internationally standardized gene functional classification system, provides a set of dynamically updated standard glossaries to fully describe the attributes of genes and gene products in organisms. The GO project mainly depicts the differentially abundant proteins from three significant aspects including biological process, cellular component and molecular function. Furthermore, in KEGG (Kyoto Encyclopedia of Genes and Genomes) database, the selected differentially abundant proteins were annotated and classified into various biosynthetic and metabolic pathways and signal transduction pathways (Kanehisa et al., 2012). Some of the most crucial pathways the DEPs involved were selected after analyzing these pathways. The interaction networks among the DEPs were established by STRING (the Search Tool for the Retrieval of Interacting Genes/Proteins) database (Jensen et al., 2009).
2. Materials and methods 2.1. Seed materials and treatment conditions Freshly matured fruits were collected from of Zanthoxylum nitidum plants in Pinyuan county (N 24°30′, E 115°48′, a.s.l. 160 m), Guangdong Province, China on September 29, 2014. The fruits were shelled after naturally dried in the shade. The obtained seeds were then stored in silver sands containing approximately 15% water at 4 °C before use. Before germination test and extracting proteins, the seeds were divided into three groups: ZN, seeds were soaked in water under 25 °C for 2 days; ZNGA, seeds were soaked in 300 mg L−1 GA solution under 25 °C for 2 days; ZNS, seeds were treated with cold stratification at 4 °C for 3 months followed by soaking in water under 25 °C for 2 days. Approximately 10 g treated seeds were taken from each group, instantly frozen in liquid nitrogen and stored at −80 °C before protein extraction. Meanwhile, a germination test was carried out for each group. Based on the recommendations of the International Seed Testing Association (ISTA, 2016) and previous optimization tests (lab data not shown), the optimal condition of germination test was confirmed: the seeds (three replicates of 50 seeds each) were cultivated at 15 °C in semienclosed plastic trays with moist fine sand in the light (500 lx/0 lx (14 h/10 h)). Seed dormancy release is assessed by significant difference in seed germination rate.
3. Results 3.1. Seed germination rate in three different groups As shown in Fig. 1, the germination rate of Zanthoxylum nitidum seeds in the control group is only 2.7% and the lowest among the three groups, the germination rate of seeds treated with GA (300 mg L−1) is 22% and significantly higher than the control group (P < 0.01), the germination rate of seeds after cold stratification is 30.7% and significantly higher than the control group (P < 0.01) and the GA treatment group (P < 0.01). 3.2. Protein identification and quantification To uncover the significant metabolic proteins involved in the dormancy alleviation mechanism of Zanthoxylum nitidum seeds, iTRAQbased quantitative proteomic characterization of differently treated seeds was conducted. A global profiling of quantitative proteome was acquired on seeds at different dormant stage after water, cold stratification and GA treatment with two biological replicates. A toatl of 7540 unique peptides and 1207 corresponding proteins were identified. Among them, 1047 and 1022 proteins were identified in group ZNS vs ZN and ZNGA vs ZN respectively. Protein abundances that changed less than 1.3-fold were discarded when statistical significance is detected by
2.2. Protein extraction, iTRAQ labelling and liquid chromatographytandem mass spectrometry (LC–MS/MS) separation Total proteins were extracted using the cold acetone method (Wu et al., 2014). Standard BCA assay was used to detect protein concentration (Sigma Sigma-Aldrich (Shanghai) Trading Co. Ltd. (China)). 8
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Fig. 1. Germination rate of Zanthoxylum nitidum seeds under a suitable condition after three different treatments. ZN, seeds were soaked in water under 25 °C for 2 days; ZNGA, seeds were soaked in 300 mg L−1 Gibberellin solution under 25 °C for 2 days; ZNS, seeds were treated with cold stratification at 4 °C for 3 months followed by soaking in water under 25 °C for 2 days. Germination conditions: temperature, 15 °C; illumination, 500 lx/0 lx (14 h/10 h); germinating bed, sand bed. Each group contains three biological replicates and each replicate contains 50 seeds.
Fig. 2. Venn diagram showing the number of proteins differentially expressed between upregulated proteins of ZNS vs ZN, downregulated proteins of ZNS vs ZN, upregulated proteins of ZNGA vs ZN and downregulated proteins of ZNGA vs ZN.
the ANOVA test (P < 0.05). Finally, 420 DEPs including 208 upregulated and 212 downregulated were screened out in ZNS vs ZN, and 198 proteins including 109 upregulated and 89 downregulated in ZNGA vs ZN. The results showed that, consistent with the differences of the germination rate of Zanthoxylum nitidum seeds, the number of differentially expressed and upregulated proteins in ZNS vs ZN is higher than that in ZNGA vs ZN.
metabolic process and biosynthetic process. In the upregulation profile of ZNGA vs ZN, DEPs mainly experienced small molecule metabolic process, carbohydrate metabolic process, translation, transport and catabolic process. In the downregulation profile of ZNGA vs ZN, DEPs mainly experienced small molecule metabolic process, cellular nitrogen compound metabolic process, carbohydrate metabolic process, biosynthetic process and catabolic process. In molecular function, the differentially abundant proteins in upregulated profile of ZNS vs ZN were mainly related to oxidoreductase activity, ion binding, lyase activity, peptidase activity and structural constituent of ribosome. While in downregulated profile of ZNS vs ZN, they were mainly related to oxidoreductase activity, ion binding, peptidase activity, lyase activity and structural constituent of ribosome. In upregulated profile of ZNGA vs ZN, DEPs were mainly related to oxidoreductase activity, ion binding, peptidase activity, lyase activity and GTPase activity. In downregulated profile of ZNGA vs ZN, DEPs were mainly related to ion binding, oxidoreductase activity, DNA binding, isomerase activity and structural constituent of ribosome. In cellular component, the differentially abundant proteins in upregulated profile of ZNS vs ZN mainly distributed in intracellular, cytoplasm, ribosome, extracellular region and mitochondrion. While in downregulated profile of ZNS vs ZN, they mainly distributed in intracellular, cytoplasm, protein complex, ribosome and cytosol. In upregulated profile of ZNGA vs ZN, DEPs were mainly distributed in intracellular, mitochondrion, cytoplasm, ribosome and protein complex. In downregulated profile of ZNGA vs ZN, DEPs were mainly distributed in intracellular, cytoplasm, nucleus, cytosol and ribosome. According to KEGG analysis (Fig. 5), in the ZNS vs ZN profile, the proteins mainly experienced carbon metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, biosynthesis of amino acids and ribosome. In ZNGA vs ZN, the proteins mainly experienced carbon metabolism, biosynthesis of amino acids, glycolysis/gluconeogenesis, ribosome and glyoxylate and dicarboxylate metabolism. Following annotation, amino acid codes were sequentially mapped into biosynthetic pathways. As Fig. 6 shows, we find that the DEPs were evenly mapped into all the parts of amino acid biosynthetic pathway in KEGG; proteins involved in amino acid biosynthetic pathway were very active during the seed stratification process (ZNS vs ZN), and most of DEPs were upregulated, such as enolase [EC:4.2.1.11], aspartatesemialdehyde dehydrogenase [EC:1.2.1.11] and ketol-acid reductoisomerase [EC:1.1.1.86], these were about 2 fold changed, and
3.3. Protein expression profiles between different treatments A total of 484 proteins were identified over 1.3-fold significant changes during the dormancy release of Zanthoxylum nitidum seeds. Then four protein expression profiles were established: compared with control group (ZN), 126 proteins were just upregulated in the ZNS group; 26 proteins were only upregulated in the ZNGA group; 160 of proteins were only downregulated in the ZNS group; 38 of proteins were only downregulated in the ZNGA group; 76 of proteins were upregulated both in the ZNS group and ZNGA group; 45 of proteins were downregulated both in the ZNS group and ZNGA group; 6 of proteins were upregulated in the ZNS group and downregulated in the ZNGA group; 7 of proteins were upregulated in the ZNGA group and downregulated in the ZNS group (Fig. 2). As shown in Fig. 2, most of the common differentially expressed proteins in ZNS vs ZN and ZNGA vs ZN are upregulated or downregulated proteins. This shows that the molecular changes in ZNS and ZNGA group may overlap. 3.4. Functions and pathways analysis of differentially expressed proteins To show the global features of the specific functional categories enriched in Zanthoxylum nitidum seeds, gene ontology (GO) category enrichment analysis was carried to categorize all 484 proteins with 1.3fold difference based on GO Slim classification for vegetation. As Figs. 3 and 4 shows, proteins with differential abundances are annotated into various biological process, cellular component and molecular function after GO analysis. In biological process category, the differentially abundant proteins in the upregulation profile of ZNS vs ZN mainly experienced small molecule metabolic process, carbohydrate metabolic process, cellular nitrogen compound metabolic process, transport, catabolic process and biosynthetic process; while in the downregulation profile of ZNS vs ZN, they mainly experienced small molecule metabolic process, carbohydrate metabolic process, catabolic process, cellular nitrogen compound 9
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Fig. 3. Gene ontology (GO) of the differentially expressed proteins identified between ZNS and ZN, red representing up-regulated proteins and green representing down-regulated proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
biosynthesis of amino acids and translation. And both ZNS vs ZN and ZNGA vs ZN showed fewer changes in their proteomes of plant hormone signal transduction.
glyceraldehyde 3-phosphate dehydrogenase [EC:1.2.1.12], aconitate hydratase [EC:4.2.1.3] and glycine hydroxymethyltransferase [EC:2.1.2.1] were even more than 5 fold upregulated. Whereas, D-3phosphoglycerate dehydrogenase [EC:1.1.1.95], phosphoglycerate kinase [EC:2.7.2.3], pyruvate kinase [EC:2.7.1.40] and glycine hydroxymethyltransferase [EC:2.1.2.1] were over 2 fold downregulated in this pathway (Fig. 6, Table S1). According to results mentioned above, we found similar functional proteome changes focusing on respiratory related pathways,
3.5. Protein–protein interaction analysis of differentially expressed proteins In order to illuminate the interactions of the proteins taking part in the dormancy release, the differentially abundant proteins in the research was used to build the protein–protein interaction (PPI) network
Fig. 4. Gene ontology (GO) of the differentially expressed proteins identified between ZNGA and ZN, red representing up-regulated proteins and green representing down-regulated proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 10
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Fig. 5. Histogram of the distribution of differentially abundant proteins according to their predicted KEGG signaling pathways of the proteins identified as involved in Zanthoxylum nitidum seed dormancy release of (A) ZNS vs ZN and (B) ZNGA vs ZN.
alleviate seed dormancy or act through distinct signaling pathways remains unclear. In order to observe some characteristics of these two treatments at the molecular level, we conducted a proteomics study on Zanthoxylum nitidum seeds experienced these two treatments along with freshly harvested seeds using iTRAQ technique. In germination test, the germination rate of GA-treated seeds was lower than that of the seeds after cold stratification. Correspondingly, the number of DEPs in the GA-treated group was lower than that in the stratification group. Based on the KEGG results and GO results, a majority of functional proteome changes were found similar, though the numbers of DEPs of the two groups differred greatly. Here, we will analyze the characteristics of differentially expressed proteome mainly from the following three aspects.
by the STRING database. Thirty-six DEPs took part in the respirationrelated metabolism processes were used, “Arabidopsis thaliana” was selected in the item of Organism, and 15 DEPs were annotated in the STRING database and the PPI network was established (Fig. 7, Table S2). Phosphofructokinase family protein (AT1G12000), which is the catalytic subunit of pyrophosphate–fructose 6-phosphate 1-phosphotransferase, catalyzes the phosphorylation of D-fructose 6-phosphate, the first committing step of glycolysis. It also uses inorganic phosphate (PPi) as phosphoryl donor instead of ATP like common ATP-dependent phosphofructokinases (ATP-PFKs), which renders the reaction reversible, and can thus function both in glycolysis and gluconeogenesis. It has the interaction with Aldolase superfamily protein (FBA6), Phosphoglucomutase 3 (PGM3), Pyruvate kinase (AT5G08570), Bifunctional enolase 2/transcriptional activator (LOS2), Glucose-6-phosphate isomerase (AT5G42740), Malate dehydrogenase (c-NAD-MDH1), Phosphoglycerate kinase (PGK), Dihydrolipoyl dehydrogenase 1 (mtLPD1) and Succinyl-CoA ligase, alpha subunit (AT5G23250). PGM3 participates in both the breakdown and synthesis of glucose. LOS2 is a multifunctional enzyme, which acts as an enolase involved in the metabolism and, as a positive regulator of cold-responsive gene transcription. LOS2 exhibited interactions with (PGK, FBA6, Phosphofructokinase family protein (AT1G12000), PGM3, AT5G08570, AT5G42740, Aldehyde dehydrogenase 11A3 (ALDH11A3), c-NADMDH1, mtLPD1, AT5G23250, Aldehyde dehydrogenase (ALDH7B4), Aldehyde dehydrogenase 2B4 (ALDH2B4); bound to the cis-element of the gene promoter of STZ/ZAT10, a zinc finger transcriptional repressor. ALDH2B4 possesses activity on acetaldehyde and glycolaldehyde in vitro. ALDH11A3 is an important means of generating NADPH for biosynthetic reactions. Glyoxalase I homolog (GLX1) catalyzes the conversion of hemimercaptal, formed from methylglyoxal and glutathione, to S-lactoylglutathione. MtLPD1 is a component of the glycine decarboxylase (GDC) or glycine cleavage system as well as part of the alpha-ketoacid dehydrogenase complexes. LPD1 is probably the protein most often associated with the glycine decarboxylase complex while LPD2 is probably incorporated into alpha-ketoacid dehydrogenase complexes.
4.1. Respiratory metabolism pathways are activated during dormancy release From the classification of DEPs associated with dormancy reduction, we found that the metabolic categories are the most representative. Regardless of treatments, a large portion of DEPs were annotated to four main respiratory metabolism pathways: Glycolysis/ Gluconeogenesis, Oxidative phosphorylation, Citrate cycle and Pentose phosphate pathway. Studies have shown that these pathways are not isolated, but are interrelated and interacted at several levels by exchanging metabolic intermediates. Different respiratory substrates enter these pathways at various locations for respiratory metabolism. The respiratory activities of treated seeds increased, indicating that the seeds has completely or partially walked away from the quiescent state (namely seed dormancy) and be ready for germination. Nucleoside diphosphate kinase (ndk) was upregulated in both treatment groups, and its expression was affected by stratification and GA. It takes part in nucleotide metabolism and keeps cellulate ATP with other nucleoside triphosphates in a dynamic equilibrium. It participates in the synthesis of purines and pyrimidines. Nevertheless, it also plays a role in many regulatory functions apart from the housekeeping effect. Lots of factors would trigger the expression of ndk, such as vulnus, heat shock, oxidant stress, pathogenic organism, abscisic acid, and so on (Cho et al., 2004). The content of ndk were altered at seed germination and early seedling growth (Yano et al., 1995). In plants, it may also function in the signal transduction of mitogen-activated protein kinase (MAPK), which is regulated by GA (Choi et al., 1999; Yano et al., 1995). Compared with control group (ZN), the up-regulated level of Adenosylhomocysteinase (ahcY) in the stratification group (11.9 fold)
4. Discussion As is well known, cold stratification or exogenous application of GA could break the seed dormancy in many plants at different degrees (Rascio et al., 1998; Song et al., 2017; Wang et al., 2016). Whereas, whether these two treatments arouse the same potential mechanisms to 11
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Fig. 6. Proteins with differential abundances involved in biosynthesis of amino acids were mapped to the biosynthetic pathway in KEGG, red representing upregulated proteins and blue representing down-regulated proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in amino acid and folate metabolism, and is also one of the main sources of carbon units for certain indispensable biosynthesis processes (Florio et al., 2011). glyA also catalyzes the biosynthesis of 5-formyltetrahydropteroylglutamate, which was indicated to act as a storage form of reduced folates and one-carbon groups in dormant stage cells (Kruschwitz et al., 1994). In our research, cold stratification and GA treatment both improved the content of glyA, showing that one-carbon units are necessary for the embryo formation during the transition from dormancy release to germination. glyA acts also on behalf of a vital crosstalk among amino acid metabolism, one-carbon metabolism, and photorespiration. Serine, a metabolite generated by glyA, serves as a metabolic signal in the transcription control of genes associated with photorespiration (Timm et al., 2013). Therefore, glyA may be involved in regulating the photorespiration, which is essential for the growth and development of seedlings.
was more notable than that in the GA-treated group (5.6 fold). Adenosylhomocysteine competitively inhibits the methyl transferase reactions, which depends on Sadenosylmethionine. Therefore, ahcY may play an important role in controlling DNA or other substrates’ methylation through regulating the level of adenosylhomocysteine in cells. The activity of ahcY decreased in the hog1 mutant of Arabidopsis thaliana, which resulted in the maintenance of methylation being disturbed and the expression of many genes affected. These mutant plants have the characteristics of slow growth, poor fertility and decreased seed germination. When a T-DNA including the gene coding for ahcY complemented the hog1 mutation, DNA methylation, plant growth and seed viability were brought normal (Rocha et al., 2005). Hence, ahcY seems to play a role in the way that regulating DNA methylation, which influences the expression of genes taking charge of seed dormancy release. The level of Glycine hydroxymethyltransferase (glyA) in both treated groups exceeded that in the control group, and the up-regulation in the stratification group particularly outstanding. glyA takes part 12
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molecular activities involved in these genes are important for seed dormancy. At present, we have not yet identified the specific genes associated with the dormancy of Zanthoxylum nitidum seeds. However, we found some changes of the pathways associated with genetic information processing both in ZNS vs ZN and ZNGA vs ZN, especially the pathway of translation. Pawlowski et al. found that at least 22 of the proteins identified as being associated with the dormancy release of sycamore seeds were classified in the genetic information processing category and identified six HSPs that responded to various treatments (Pawlowski and Staszak, 2016). In this study, we found that 60S ribosomal proteins responded to various treatments. 4.4. Plant hormone signal pathways showed fewer changes during dormancy release With more and more studies about the effects of various hormones on dormancy in Arabidopsis thaliana, almost all plant hormones were found to be involved in the regulation of dormancy and germination (Finkelstein et al., 2008; Shu et al., 2016). In this research, KEGG pathway analysis showed some changes of the proteins in the plant hormone signal pathways. In ZNS vs. ZN, the DEPs in the plant hormone signal pathways are mainly associated with auxin, ethylene and jasmonic acid (JA). In ZNGA vs. ZN, the DEPs in the plant hormone signal pathways are mainly associated with auxin and ethylene. The plant hormone auxin participates in almost all aspects of plant growth and development, and responds to a variety of environmental factors (Vanneste and Friml, 2009; Zhao, 2010). Previously, auxin had not been considered as an important player in seed dormancy and germination, in spite of its participation in the cross-linking with abscisic acid (ABA). New genetic and biochemical evidence showed that auxin influences ABA signaling to exert its physiological effect on the control of seed dormancy and germination (Liu et al., 2013; Ye et al., 2016). In ZNS vs. ZN, three DEPs including aldehyde dehydrogenase family 7 member A1, aldehyde dehydrogenase and catalase, were associated with auxin, took part in Tryptophan metabolism, which has an indirect effect on auxin in plant hormone signal pathway. In ZNGA vs. ZN, only one protein catalase was differentially expressed in Tryptophan metabolism. Some researchers have proved that ethylene may has a negative influence on ABA biosynthesis and signaling (Ghassemian et al., 2000). Further studies found that ethylene could offset the impact of ABA to reduce seed dormancy and improve seed germination (Arc et al., 2013; Corbineau et al., 2014). Nevertheless, whether and how ethylene impacts GA biosynthesis and signaling to change seed dormancy and germination is still unknown in a large part. In this study, four proteins including aspartate-semialdehyde dehydrogenase, malate dehydrogenase, malate dehydrogenase and adenosylhomocysteinase differentially expressed after stratification treatment, and these proteins participated in Cysteine and methionine metabolism, which located in the upstream of the plant hormone signal pathway. Therefore, changes of these proteins may produce an important impact. Only one protein, adenosylhomocysteinase, in Cysteine and methionine metabolism appeared significantly upregulated after GA treatment. Seed germination was put off after JA application, showing that JA can inhibit the seed germination process (Nambara et al., 2010). Nevertheless, another study found that JA respectively restrain the transcription of ABA biogenesis genes and motivates ABA-inactivating genes, indicating that JA and ABA has antagonistic effect (Jacobsen et al., 2013). However, the reason why the effect of JA in seed germination is frequently paradoxical remains unknown. In ZNS vs. ZN, four DEPs, alcohol dehydrogenase class-P, acetyl-CoA acyltransferase 1, allene oxide cyclase, and acetyl-CoA acyltransferase are involved in αLinolenic acid metabolism, in which the biosynthesis of JA is located. No DEP relevant to JA was found in Zanthoxylum nitidum seeds after GA treatment. It should be noted that the effects of plant hormones on seed
Fig. 7. The interaction networks were built by 15 DEPs which participate in the respiration-related metabolism processes during the alleviation of seed dormancy. Protein-protein interaction network was analyzed by STRING database. The confidence score was set to medium level ≥0.4. The blue lines represent database evidence; the purple lines represent experimental evidence; yellow lines represent text mining evidence; the black lines represent coexpression evidence; and green lines represent neighborhood evidence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4.2. Dormancy releasing treatments activate the biosynthesis of amino acids In the stratification group and GA-treated group, a large amount of proteins were annotated into Biosynthesis of amino acids, which is a feasible fate for the carbon skeletons from seed storage compound mobilization. β-Aspartyl-semialdehyde (ASA) is a common precursor for the biosynthesis of threonine, methionine and lysine. ASA dehydrogenase (ASADH) locates at the first branch point in the biogenesis pathway of aspartic acid in plants, ASA formation is catalyzed by ASADH via the reductive dephosphorylation of β-aspartyl phosphate utilizing NADPH (Paris et al., 2002). Ketol-acid reductoisomerase is an enzyme in the biosynthesis pathway of branched-chain amino acid, in which it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate (Leung and Guddat, 2009). D-3-phosphoglycerate dehydrogenase takes part in the biosynthesis of L-serine. Aspartate-semialdehyde dehydrogenase (ASADH family protein), Ketol-acid reductoisomerase, and D-3-phosphoglycerate dehydrogenase differently expressed in the treating groups, showing that Biosynthesis of amino acids play a crucial role in seed dormancy alleviation. Our results resembled those of Erwann Arc and Xu Hengheng’s, who put forward that dormancy-relieving treatments can stimulate amino acid biosynthesis and/or recycling pathways (Arc et al., 2012; Xu et al., 2016).
4.3. Dormancy releasing treatments motivate translation activities Seed dormancy is an important and complex adaptive trait that is determined by genetic factors with a substantial environmental influence (Graeber et al., 2012). Some genes have been identified that specially regulate seed dormancy (Nee et al., 2017). These genes have a seed-specific expression pattern and show strong dormancy mutant phenotypes. The main representative genes among this group are DELAY OF GERMINATION 1 (DOG1) and REDUCED DORMANCY 5 (RDO5) (Bentsink et al., 2006; Xiang et al., 2014). Therefore, the 13
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dormancy and germination are cross-linked and the individual analysis about each hormone-related proteins may be insufficient or inadequate. Although plant hormone signal pathways showed fewer changes during the dormancy release of Zanthoxylum nitidum seeds, discussion on this part is still very necessary.
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5. Conclusion Along with the dormancy release of Zanthoxylum nitidum seeds, the proteomes have also undergone some significant changes both in stratification and GA-treated groups. Our results showed that stratification and GA treatment caused similar proteome changes associated with the dormancy release of Zanthoxylum nitidum seeds. These changes are mainly enriched in respiratory-related metabolic pathways, biosynthesis of amino acids and translation. However, unlike the results of the model organism Arabidopsis thaliana, we found that plant hormone signal pathways showed fewer changes during the dormancy release of Zanthoxylum nitidum seeds. Conflicts of interest The authors declare no conflict of interest. Acknowledgement This research was supported by grant from General university innovation team project of Guangdong province-Innovative research team of Chinese Medicine resources (2016KYTD02). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.05.044. References Arc, E., Chiban, K., Grappin, P., Jullien, M., Godin, B., Cueff, G., Valot, B., Balliau, T., Job, D., Rajjou, L., 2012. Cold stratification and exogenous nitrates entail similar functional proteome adjustments during arabidopsis seed dormancy release. J. Proteome Res. 11, 5418–5432. Arc, E., Sechet, J., Corbineau, F., Rajjou, L., Marion-Poll, A., 2013. ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination. Front. Plant Sci. 4. Bentsink, L., Jowett, J., Hanhart, C.J., Koornneef, M., 2006. Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 103, 17042–17047. Binns, D., Dimmer, E., Huntley, R., Barrell, D., O'Donovan, C., Apweiler, R., 2009. QuickGO: a web-based tool for gene ontology searching. Bioinformatics 25, 3045–3046. Cao, D.C., Xu, H.M., Zhao, Y.Y., Deng, X., Liu, Y.X., Soppe, W.J.J., Lin, J.X., 2016. Transcriptome and degradome sequencing reveals dormancy mechanisms of cunninghamia lanceolata seeds. Plant Physiol. 172, 2347–2362. Chen, J.J., Lin, Y.H., Day, S.H., Hwang, T.L., Chen, I.S., 2011. New benzenoids and antiinflammatory constituents from Zanthoxylum nitidum. Food Chem. 125, 282–287. Cho, S.M., Shin, S.H., Kim, K.S., Kim, Y.C., Eun, M.Y., Cho, B.H., 2004. Enhanced expression of a gene encoding a nucleoside diphosphate kinase 1 (OsNDPK1) in rice plants upon infection with bacterial pathogens. Mol. Cells 18, 390–395. Choi, G., Yi, H., Lee, J., Kwon, Y.K., Soh, M.S., Shin, B.C., Luka, Z., Hahn, T.R., Song, P.S., 1999. Phytochrome signalling is mediated through nucleoside diphosphate kinase 2. Nature 401, 610–613. Commission, C.P., 2015. Chinese Pharmacopoeia, 2015 ed. China Medical Science Press, Beijing. Corbineau, F., Xia, Q., Bailly, C., El-Maarouf-Bouteau, H., 2014. Ethylene, a key factor in the regulation of seed dormancy. Front. Plant Sci. 5. Finch-Savage, W.E., Leubner-Metzger, G., 2006. Seed dormancy and the control of germination. New Phytol. 171, 501–523. Finkelstein, R., Reeves, W., Ariizumi, T., Steber, C., 2008. Molecular aspects of seed dormancy. Annu. Rev. Plant Biol. 387–415. Florio, R., di Salvo, M.L., Vivoli, M., Contestabile, R., 2011. Serine hydroxymethyltransferase: a model enzyme for mechanistic, structural, and evolutionary studies. Biochim. Biophys. Acta-Proteins Proteomics 1814, 1489–1496. Gan, C.S., Chong, P.K., Pham, T.K., Wright, P.C., 2007. Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). J. Proteome Res. 6, 821–827. Ghassemian, M., Nambara, E., Cutler, S., Kawaide, H., Kamiya, Y., McCourt, P., 2000. Regulation of abscisic acid signaling by the ethylene response pathway in
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