iTRAQ and RNA-Seq Analyses Provide New Insights into Regulation

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iTRAQ and RNA-Seq analyses provide new insights into regulation mechanism of symbiotic germination of Dendrobium officinale seeds (Orchidaceae) Juan Chen, Sisi Liu, Annegret Kohler, Bo Yan, Hong Mei Luo, Xiaomei Chen, and Shun Xing Guo J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 10, 2017

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Journal of Proteome Research

iTRAQ and RNA-Seq analyses provide new insights into regulation mechanism of symbiotic germination of Dendrobium officinale seeds (Orchidaceae) Juan Chen1, Si Si Liu1, Annegret Kohler2, Bo Yan1, Hong Mei Luo1, Xiao Mei Chen1, Shun Xing Guo1* 1

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine,

Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, P. R. China 2

UMR 1136 INRA/Université de Lorraine, Interactions Arbres/Micro-organismes, INRA, Institut

National de la Recherche Agronomique, Centre INRA de Nancy, Champenoux, 54280, France.

ABSTRACT: Mycorrhizal fungi colonize orchid seeds and induce germination. This so-called symbiotic germination is a critical developmental process in the lifecycle of all orchid species. However, the molecular changes that occur during orchid seed symbiotic germination remains largely unknown. To better understand the molecular mechanism of orchid seed germination, we performed a comparative transcriptomic and proteomic analysis of the Chinese traditional medicinal orchid Dendrobium officinale for exploring the change in protein expression at the different developmental stages during asymbiotic and symbiotic germination and identifying the

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key proteins that regulate the symbiotic germination of orchid seeds. Among 2256 identified plant proteins, 308 were differentially expressed across three developmental stages during asymbiotic and symbiotic germination and 229 were differentially expressed during symbiotic germination compared to asymbiotic development. Of these, 32 proteins were co-up-regulated at both the proteomic and transcriptomic levels during symbiotic germination compared to asymbiotic germination. Our results suggest that symbiotic germination of D. officinale seeds shares a common signaling pathway with asymbiotic germination during the early germination stage. However, compared to asymbiotic germination, fungal colonization of orchid seeds appears to induce higher and earlier expression of some key proteins involved in lipid and carbohydrate metabolism and thus improves the efficiency of utilization of stored substances present in the embryo. This study provides new insight into the molecular basis of orchid seed germination.

KEYWORDS: Orchidaceae, mycorrhizal interaction, seed germination, proteome, transcriptome, carbohydrate metabolism, defense reaction INTRODUCTION The family Orchidaceae, which contain more than 25,000 species, is renowned for its extraordinary morphological diversity.1 As the most highly evolved family in the plant kingdom, Orchidaceae displays a unique mycorrhizal symbiosis with particular fungal lineages (Rhizocotina-like fungi) and specific interactions between flowers and pollinators.2, 3 Therefore, Orchidaceae are generally viewed as having evolved close biological interactions in many of their features and are also regarded as an excellent model for investigating the ecology and evolution of biological interactions.4, 5


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The seeds of all orchid species are minute with a simple embryo and minimal nutrient reserves.6 In these species, seed germination and development in nature necessarily depend on colonization by appropriate mycorrhizal fungi for carbohydrates and nutrient supplies.7 Since Bernard’s pioneering works in 1899, symbiotic relationship between orchid seeds and fungi has been the subject of many further investigations,8 and progress has been made in understanding the diversity and specificity of orchid mycorrhizal fungi 2, 9 and the morphological changes that occur in host cells and mycobionts during their interaction.10-12 However, the molecular process of symbiotic germination induced by mycorrhizal fungi remains unclear. Orchid mycorrhizal association has three basic characteristics: fungal penetration, formation of pelotons (hyphae coils) and degradation of pelotons.2 However, pelotons formation and degradation usually occurred simultaneously over a short time period so that it is not easily to temporally resolve these two processes during the symbiotic germination of orchid seeds.13 Fungi are usually thought to provide a source of carbohydrate during symbiotic germination.14 Although previous studies suggested that the translocation of nutrients from fungi to plant cells occurs via the degradation of intercellular pelotons, subsequent experiments showed that embryo and protocorm growth occurs prior to hyphae degradation, indicating that the nutrient supply is likely obtained from living fungal pelotons. 13 Recently, isotope-labeling experiments were used to demonstrate that orchid plants acquire the carbon, nitrogen and phosphorous from their fungal partners and that carbon and nitrogen are transferred from symbiotic fungi to host plants by living pelotons or digested hyphae.12, 15, 16 These remarkable findings provided important information that formed a basis for further exploration of the role of fungi in orchid mycorrhizal symbiosis.


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Seed germination is considered to be a determining factor in the propagation of plant species.17 At present, symbiotic germination (seeds inoculated in a barren medium containing mycorrhizal fungi) and asymbiotic germination (seeds grown on sugar-containing medium in the absence of fungi) are two choices for orchid propagation. Experiments have shown that symbiotic germination improves the seed germination rate and the rate of seedling growth of some orchid species.18, 19 A prime example of this is the commercial cultivation of Gastrodia elata by seed germination with Armillaria sp. in China. 20 Several recent publications on orchid symbiosis have opened the door to molecular studies of the mechanism of symbiotic germination of orchid seeds. Valadares et al.21 analyzed differences in protein expression between the mycoheterotrophic and the photosynthetic stages of Oncidium sphacelatum inoculated with mycorrhizal fungi using iTRAQ and showed that proteins related to the homeostasis of reactive oxygen species, the defense reaction and carotenoid biosynthesis are involved in the seed development. Perotto et al. reported that few genes related to plant defense were significantly up-regulated other than nodulin-like genes expressed in the plant protocorm containing the fungal hyphae, supporting the existence of mutualism between orchid and their fungal partner. 22 By comparing gene expression in infected protocorms and ungerminated seeds of D. officinale, our previous studies identified several up-regulated genes encoding calmodulin during symbiotic protocorm development using suppression subtractive hybridization.23 Mostly recently, comparative transcriptome analysis of gene expression in Gastrodia elata in response to fungus symbiosis provided evidence that monooxygenases and glycosyltransferases may participate in the biosynthetic pathway that produces the medicinal compounds gastrodin.24 To better understand the molecular process of symbiotic germination of orchid seed, we performed a comparative proteomic analysis using isobaric tags for relative and absolute


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quantification (iTRAQ) coupled with RNA-Seq transcriptomic analysis across three different developmental stages of asymbiotic and symbiotic germination. The aims of this study were i) to compare protein expression differences at adjacent developmental stage after symbiotic or asymbiotic germination; ii) to obtain a profile of the protein expression patterns associated with asymbiotic and symbiotic germination at individual developmental stages of D. officinale; iii) to identify proteins that are significantly differentially expressed due to fungal stimulation during seed germination in D. officinale; and iv) to attempt to elucidate how the proteome responds to fungus-induced selection. Our data represent an important step toward the elucidation of gene and protein regulatory networks that are active during the symbiotic germination and it will contribute to a better understanding of the orchid seed development and biology. MATERIALS AND METHODS Seed samples collection Seeds of D. officinale were collected from an artificial cultivation greenhouse in Xishuangbanna, Yunnan province, in November 2012. Mature capsules were surface sterilized for 15–60 s in a solution containing 75% ethanol, followed by incubation in sodium hypochlorite solution containing 2.5% available chlorine for 10 min and three rinsed in distilled water. Axenic seeds were stored at 4°C in wax paper packets inside 1.5 ml sterilized tubes, containing sterilized silica gel. Seed germination experiments Prior to sowing, the seeds were immersed in sterile distilled water and subjected to ultrasonic treatment 1-2 min; seeds that sank to the bottom of the tube were selected for experiments. For symbiotic germination (SG), the seeds were suspended in sterile water and a single drop of the seed suspension was dispensed onto 1.5 cm ! 1.5 cm autoclaved nylon cloths on the oatmeal


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agar plates (OMA, 0.25% oat meal and 1% agar) in a 9 cm petri dish. Then, four 5 mm colonies of Tulasnella sp. S6 on PDA medium were placed in each OMA petri dish (containing orchid seeds) for symbiotic germination. For asymbiotic germination (AG), the seeds suspension was dropped onto the 1/2 MS culture medium without fungi (Figure 1). The plates were sealed with plastic film and incubated under a 12/12 h L/D cycle at 25°C. Seed germination and protocorm development were assessed by examining the cultures under a dissecting stereomicroscope every three days. Samples at three different developmental stages of AG and SG were both analyzed using the RNA-Seq and iTRAQ methods. The developmental stages analyzed included stage 2 (when the embryo swells, enlarges and emerges from the coat; germination stage), stage 3 (when the embryo differentiates into a protomeristem; the protocorm stage) and stage 4 (when the first leaf emerges; seedling stage). The developmental stages were defined according to Stewart et al.25 An ungerminated seed sample and a free-living fungus (Tulasnella sp.) were also analyzed as control (Table 1). All collected seed material were immediately frozen in liquid nitrogen and stored at !80 °C prior to RNA and protein extraction. Protein extraction Protein was extracted from eight samples using a plant total protein extraction kit (PE0230, Sigma) following the protocol supplied by the manufacturer. For extraction, 200 mg of seeds was ground in liquid nitrogen to a fine powder and the powder was re-suspended in 1.5 ml methanol solution with 1% cocktail and incubated for 30 minutes at !20 °C. After centrifugation, the precipitate was washed three times with 1.5 ml acetone for 30 min at !20 °C, then vacuum-dried and stored at !80 °C.


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Protein powder was re-solubilized in 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and 65 mM DTT. The protein concentration was determined by the Bradford method with bovine serum albumin as the standard26 and protein purity was examined by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). iTRAQ labeling and mass spectrometry analysis iTRAQ labeling and mass spectrometry analysis were performed at the Beijing Proteome Research Center (BPRC) following the methods described by Unwin et al.27 Each 75 µg protein sample was reduced using 2 µL of reducing agent supplied by the manufacturer (iTRAQ Reagent 8-plex kit, AB Sciex) and then incubated at 60 °C for 1 h. Cysteine residues were blocked using methyle methanethiosulfonate (MMTS) at room temperature for 10 min followed by trypsin digestion (modified trypsin, Promega) at 37°C for 16 h. Prior to labeling, the reagents were dissolved in 50 µL of isopropanol, the contents of one vial were transferred to individual sample tubes, and the pH of the solution was adjusted to 7.5. A total of eight samples were labeled with iTRAQ tags 113-121 using iTRAQ 8-plex kits (Applied Biosystems, USA) according to the manufacturer’s protocol (Table S1, Supporting Information). After labeling and quenching, the samples were combined and further fractionated using SCX. For SCX chromatography using the HPLC (Rigol L-3000, Beijing, China), the iTRAQ-labelled peptide mixture was reconstituted with 1 mL buffer A (25mM NaH2PO4 in 25% ACN, pH 3.0) and loaded onto an SCX column (4.6 " 250 mm Ultremex SCX column, Phenomenex, USA). The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer A for 10 min, followed by a gradient of 8-35% buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 3.0) for 11 min and another gradient of 35-80% buffer B for 1 min. The system was then maintained in 80% buffer B for 3 min before equilibration with buffer A for 10 min prior to the next injection. The


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absorbance of the eluate at 214 nm was monitored, and fractions were collected every 1 min. The eluted peptides were pooled as 10 fractions, desalted on a Strata X C18 column (Phenomenex) and vacuum-dried. The fractionated samples were analyzed on a NanoLC Eksigent 425 system coupled to a TripleTOFTM 5600 plus Systerm (AB Sciex, Concord, ON). Each fraction was resuspended in buffer A (5% ACN, 0.1% FA) and centrifuged at 20,000 g for 10 min. The supernatant was loaded on a C18 trap column (5 µm, ID100 µm, 20 mm length), and the peptides were eluted onto an analytical C18 column (1.9 µm, ID75 µm, 100 mm length) packed in-house. Then, a 45min gradient of 2 to 35% buffer B (95% ACN, 0.1% FA) was run at a flow rate of 300 nL/min, followed by 5-min linear gradient to 80% buffer B, and maintenance at 80% buffer B for 4 min, and a final return to 5% in Buffer B over 1 min. The parameters used for spectrometry on the TripleTOF 5600 mass spectrometer were as follows: ionspray voltage floating (ISVF) = 2300 V, curtain gas (CUR) = 30, ion source gas 1 (GS1) = 15, interface heater temperature (IHT) = 150, and declustering potential (DP) 80 V. The TOF scan parameters were set as follows: 0.25 s TOF MS accumulation time in the mass range of 350-1250 Da, followed by product ion scan of 0.1 s accumulation time in the mass range of 100-1500 Da. The switching criteria were set to ions greater than m/z = 350 and smaller than m/z = 1250 with charge state of 2 to 5 and an abundance threshold of > 120 cps. Former target ions were excluded for 15 s, and former ions were excluded after one repeat. The maximum number of candidate ions per cycle was 30 spectra. IDA Advanced “rolling collision energy (CE)” and “Adjust CE when using iTRAQ Reagent” were required. 28 Generation of peak lists was performed with the PeakView 2.1(AB Sciex). The data were processed with Proteome Discoverer 1.3 software.


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Protein identification and quantitation The raw MS/MS data were converted into MGF format by the ProteoWizard tool msConvert, and the exported MGF files were searched using Mascot (v2.3.02) 29 against our RNA-Seq transcriptomic database of D. officinale seeds (PRJNA279934, 119,681 sequences). The Mascot search parameters were listed in the Table S2 in Supporting Information. Quantitatively analysis of the peptides labeled with isobaric tags was performed using IQuant software.30 The main IQuant quantitation parameters are listed in Table S3 in Supporting Information. The results were filtered at 1% false discovery rate (FDR) at peptide level and each protein identified with at least one unique peptide. The tandem mass spectra of five proteins with 1-2 unique peptide as example were showed in Figure S1 in Supporting Information. Proteins with 1.2- fold or greater change and Q-value less than 0.05 were considered to be differentially expressed. Hierarchical clustering of protein expression was conducted using Genesis 1.7.7.27 software.31 RNA extraction and sequencing Total RNA was isolated independently from the before mentioned eight samples. RNA was extracted from 200 mg of seeds using the RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s recommendations and treated with an RNase-free DNase I digestion kit (Aidlab, China) to remove residual genomic DNA. RNA degradation and contamination were monitored using EtBr-stained 1% agarose gels and the purity of the isolated RNA was analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The concentration and integrity of the RNA were assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Each sample had a RIN value above " 6.5, an A260/A280 ratios " 1.8, an A260/A230 ratios " 1.8, and a 28S RNA/18S RNA ratios " 1.0 and at least 20 µg of total RNA (" 250 ng/µl) was used


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subsequently for cDNA library construction. Library construction and sequencing were performed at the Beijing Genomics Institute (BGI) (Shenzhen, China) on an Illumina Hiseq 2000 platform. The general procedure was described as Zhao et al.32 Reassembling, annotation, classification and expression clustering analysis Prior to assembly and mapping, the raw reads were processed by removing reads with adaptors, reads with more than 5% unknown nucleotides, and reads of low quality (base quality # 20) to obtain clean data. All downstream analyses were based on clean, high-quality data. BWA software was used to map the clean reads to the reference genome of D. officinale.33 Gene expression was quantified using the RSEM software package (RNA-Seq by Expectation Maximization). Gene annotation and functional assignments were performed based on the Nr, Nt, COG, Swiss-prot, KEGG and GO databases. The threshold for significantly differentially expressed genes (DEGs) was set at a fold change of $ 2.0 and an FDR value of < 0.001. KEGG pathway enrichment was performed to identify significantly enriched metabolic pathways for comparison with the whole genome background. Protein and RNA correlation analysis To compare the concordance between transcriptome changes and proteome changes during seed germination in D. officinale, a correlation analysis was performed based on quantitative and differentially expressed proteins and genes from the aspects of expression and functional enrichment. Pearson correlation tests were conducted on each compared group, including adjacent developmental stages (stages 0-2, stages 2-3 and stages 3-4) within AG or SG and between AG and SG at the same stage. A flow diagram of the experiment is presented in Figure S2 in Supporting Information. Quantitative RT-PCR


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Six genes that were up-regulated in symbiotic germination of D. officinale were selected for confirmation of their up-regulated expression by quantitative real-time PCR (RT-PCR). All primers were designed using Primer 3.0 software and synthesized by Genewiz Company (China). RT-PCR was performed as the previously described.34 In brief, 1 µg RNA was reversetranscribed to cDNA using a reverse-transcription system (Biorad, USA) and cDNA equivalent to 25 ng of total RNA was used as template for each PCR reaction using SYBR Green supermix (Biorad) with a final concentration of 1.6 mM of each primer. The reaction was run using the lightCycler 480 (Roche Applied, USA) and the thermal-cycling condition parameters were set as 95°C for 3min, 40 cycles of 95°C for15s, 60°C for 30s followed by a melting curve. PCR amplification of three biological replicates was performed and included two distinct technical replicates. Transcript abundance was normalized to that of the EF1-% gene, and the ratios of expression between two conditions were calculated using Pfaffl’s method.35 RESULTS AND DISCUSSION The effects of symbiotic germination compared to asymbiotic germination of D. officinale seeds In the majority of orchid species, seed development can be divided into five stages based on the growth of the embryo25: (i) ungerminated seed (stage 0); (ii) enlarged embryo (stage 1); (iii) embryo emerges from the seed coat (stage 2, germination); (iv) appearance of the protomeristem (stage 3, protocorm); (v) emergence of the first leaf (stage 4, seedling) and (vi) elongation of the first leaf (stage 5) (Figure 1). Germination is usually defined as the emergence of the embryo from the seed coat (stage 2). The protocorm is an important stage of orchid seed development because seeds are fully heterotrophic prior to protocorm formation; after it occurs, they can photosynthesize.


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When D. officinale seeds were inoculated with the mycorrhizal fungus Tulasnella sp. S6, the embryos grew more rapidly and displayed a higher germination rate than seeds that were allowed to undergo asymbiotic germination (Figure S3, Supporting Information). After two weeks, 75.3 % of the seeds had enlarged embryos (stage 1) in SG compared to only 21.7% of the seeds in AG. At three weeks, 52.26% of the seeds had germinated and formed the protocorm structures (stage 3) in SG, but only 26.6% of the AG seeds had germinated (stage 2, grew out from coat). Protocorm formation (stage 3) in AG required at least five weeks. After 12 weeks, 66% of the SG seeds had developed young seedlings (stage 5), whereas only 30.03% of the AG seeds had formed seedlings. Taken together, these results show that seed germination on 1/2 MS medium without fungi is slower than seed germination on the OMA medium with fungi. Overall proteome at various germination stages of D. officinale A total of 242,946 spectra were generated from the eight tested samples, and 7596 peptides and 2256 plant proteins were identified using the cutoff Mascot Percolator Q-value # 0.01. The protein mass, percent coverage, number of peptides matching individual proteins, and accession numbers assigned to each of the identified plant proteins and the functional annotation of the proteins are provided in the Table S4 in Supporting Information. Protein expression changes across adjacent developmental stages during asymbiotic and symbiotic germination of D. officinale Changes in the proteome were first examined between adjacent developmental stages (stage 0-2; stage 2-3 and stage 3-4) in AG and SG. A total of 308 proteins were significantly differentially expressed during at least one more germination stage in AG and SG, and more variation in gene expression during development was observed in SG (277 proteins, 12.28%) than in AG (127 proteins, 5.6%) (Figure 2A, B). When we compared the number of DEPs at adjacent stages in


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seeds grown under asymbiotic or symbiotic conditions, we found that the highest number of DEPs are shared between the two conditions at the early germination stage (A1/C0 and S1/C0, 28.82%), followed by the protocorm stage (A2/A1 and S2/S1, 19.14%) and the seedling stage (A3/A2 and S3/S2, 11.6%) (Figure 2C). The distribution of DEPs at various germination stages of D. officinale is shown in Figure 2D. In AG, 75 DEPs proteins were found at stages 0-2; 58 DEPs were found at stage 2-3 and 36 proteins were differential expressed at stage 3-4. In SG, 144 DEPs, 110 DEPs and 129 DEPs were found at stages 0-2, stages 2-3 and stages 3-4, respectively. An obvious bias toward differential gene expression at the earliest developmental stage (75 DEPs at stages 0-2 versus 36 DEPs at stages 3-4) was observed in AG; a slightly higher number of DEPs was also found at the early stage of development in SG (144 DEPs at stages 0-2 and 129 DEPs at stages 3-4) (Figure 2). To evaluate the expression pattern of DEPs at the different developmental stages in asymbiotic and symbiotic germination assays, we performed a hierarchical clustering analysis of 308 DEPs (Table S5, Supporting Information). As shown in Figure 3A, DEPs present at the early developmental stage (stage 2) were clustered, indicating the occurrence of similar but diverse changes in gene expression in embryos that were asymbiotically and symbiotically germinated. A major difference in the protein expression patterns of plants undergoing symbiotic and asymbiotic germination occurred at the protocorm developmental stage (stage3). Specifically, the samples from SG embryos between stages 2 and 3 were grouped with the samples from AG embryos between stage 3 and 4 instead of with samples obtained from embryos at the same stages, suggesting a general shift of protein regulation toward earlier protocorm development in the symbiotic situation. Taken together, this results indicated that it could AG and SG seeds may share a common metabolism pathway at the early stage (stage 2) of seed germination but that


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subsequent protein expression at the protocorm and seedling differentiation stages is distinctly different in AG and SG seeds. In addition, proteins with similar or relevant cellular activities in most functional classes were of diverse expression patterns (Figure 3, gray columns). Among these, three apparent clustering were identified, such as “carbohydrate metabolism”, “translation” and “posttranslational modification”. The hierarchical clustering of 308 genes (correlated with 308 proteins) yielded a result similar to that obtained by proteomic analysis (Figure 3B); that is, the genes displayed similar expression patterns at stage 2 in both SG and AG, consistent with the results of the proteomic analysis (Figure 3A). However, from the stage of protocorm development to formation of young seedling, genes expression was not always consistent with proteins expression. For example, in protein expression analysis, the samples obtained at stage 3 of SG clustered with those obtained at stage 4 of AG, but gene expression analysis yielded an opposite result, grouping the samples obtained at stage 4 of SG with those obtained at stage 3 of AG. To explore the differences in metabolic processes at different germination stages in SG and AG in more detail, a KEGG pathway enrichment analysis was conducted, and DEPs present at adjacent stages of AG or SG were compared (Figure 4). At the early germination stage, photosynthetic and antenna proteins, photosynthesis and linoleic acid metabolism were significantly enriched in AG (A1/C0), whereas fatty acid biosynthesis was enriched in SG (S1/C0) (Figure 4). Linoleic acid is well known as one of the two families of essential fatty acids, thus fatty acid biosynthesis is involved into early germination events of D. officinale in both AG and SG. The proteins involved into RNA degradation and in the pentose phosphate pathway are significantly differentially expressed during the protocorm formation stage of AG (A2/A1). In contrast, metabolic pathways (e.g., carbohydrate, amino acid and lipid metabolism) are mainly


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enriched in the protocorm formation stage of SG (Figure 4). Thirty-six DEPs, including 6phosphogluconolactonase, fructose-bisphosphate aldolase, &-D-xylosidase, glyceraldehyde-3phosphate dehydrogenase, glucose dehydrogenase, ribitol dehydrogenase, malate dehydrogenase, oxygen-evolving enhancer protein, 3-oxoacyl-[acyl-carrier-protein] reductase and GDSL lipase/esterase, participated in this pathway. Interestingly, proteins involved into endoplasmic reticulum processing were significantly enriched at the protocorm formation stage of SG (S2/S1) but at the seedling developmental stage of AG (A3/A2). The endoplasmic reticulum (ER) is a site for the production of secretory proteins and has also been reported to be involved in plant defenses against pests and fungi.36 A total of eight DEPs were found to be associated with protein processing in the endoplasmic reticulum at stage 2/stage 1 (protocorm stage/germination stage) of SG, including four heat shock proteins, an endoplasmins, a calreticulin, a luminal-binding protein and a protein of unknown function. This indicates that protein processing in the endoplasmic reticulum may represent an important pathway through which fungal stimulation improves protocorm growth. Taken together, these results suggest that fungal colonization during symbiotic germination induces the higher and/or earlier expression of key proteins involved into lipid and carbohydrate metabolism and in endoplasmic reticulum processing and thereby improves the efficiency of utilization of stored nutrients. Proteomic changes between asymbiotic and symbiotic germination of D. officinale seeds at the same developmental stage A comparison of protein expression levels at various germination stages between AG and SG revealed that a total of 229 proteins underwent significant up- or down-regulation in symbiotic germination compared with asymbiotic germination at one or more developmental stages (Table


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S6, Supporting Information). The highest number of differentially expressed proteins occurred at the seedling stage (stage 4, 154 proteins), following by the protocorm stage (stage 3, 125 proteins) and germination (stage 2, 23 proteins) (Figure 2C). These 229 DEPs were functionally categorized into 11 classes; other than proteins of unknown function (34.9%) proteins, “posttranslational modification” was the most represented functional category (19.2%), followed by “translation” (10.4%), “carbohydrate metabolism” (9.6%), “energy production” (7.9%), “inorganic metabolism”(3.5%), and “lipid metabolism” (3.0%) and “amino acid metabolism” (3.0%). Furthermore, hierarchically cluster analysis of the 229 DEPs identified 9 distinct clusters based on the expression patterns of the proteins during the symbiotic germination of seeds (Den1-Den9 in Figure 5). Interestingly, Cluster 2 (Den2), which contained 14 proteins, showed a distinct trend toward up-regulation during the entire germination process. This group included five cucumisins (a subtilisin-like proteases), mannose-specific lectin, epidermis-specific secreted glycoprotein, a cysteine-rich repeat secretory protein, a basic 7S globulin and acidic endochitinase. These proteins and enzymes are mainly related to “carbohydrate metabolism” and “posttranslational modification” and probably play important roles in the degradation of stored substances and in plant defense mechanisms during the germination of Dendrobium seeds inoculated with fungi. The expression of proteins related to plant photosynthesis (Den3, 42 proteins), such as photosystem II CP43 chlorophyll apoprotein, chlorophyll a-b binding protein, photosystem Q (B) protein, acid phosphatase, linoleate 13S-lipoxygenase, ATP synthase and ribulose bisphosphate carboxylase small chain, is up-regulated at the seedling stage and downregulated at early germination and protocorm stages. This observation is consistent with the morphological differentiation of seeds that occurs at this stage when the germinated seed of D.


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officinale changes its nutritional mode from heterotrophy to photoautotrophy at post-germination stage. Thirty-six DEPs in cluster 9 displayed up-regulated expression at early germination stage and up-regulation or no significant change at post-germination stage. These proteins included ribulose bisphosphate carboxylase, elongation factor 1-alpha, HMG1/2-like protein, 40S and 60S ribosomal protein, acyl-CoA-binding protein transketolase, reticulon-like protein, endoplasmin, plasma membrane ATPase, and others. A few pathways were significantly enriched at the early germination stage (stage 2) in SG compared to AG. At the protocorm formation stage (stage 3), “glyoxylate and dicarboxylate metabolism”, “glutathione metabolism” and “carbon fixation in photosynthetic organisms” were significantly enriched in SG. The glyoxylate cycle is known to be involved in the biosynthesis of carbohydrates from fatty acids. The differential expression of proteins related to this pathway during SG suggests that the presence of fungi may alter the carbon metabolism of host plant after invasion of the seed embryo. A global functional explanation of how DEPs may be involved in symbiotic germination is provided as below. Protein sets associated with carbohydrate metabolism in symbiotic germination of D. officinale During the process of plant seed germination, the starch in the endosperm is gradually degraded to serve as the main source of ATP and to provide precursors for the anabolic reactions that occur in the embryos of most of plants species (e.g., rice, wheat).37 However, orchid seeds are extremely small and have limited nutrient reserves; thus, in most orchid species in nature, germination and the subsequent development of the embryo depend on the presence of mycorrhizal fungi. In the presence of mycorrhizal fungi, starch or cellulose is a suitable carbohydrate for the initiation of germination, but soluble carbohydrates such as sucrose, glucose


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or fructose are preferable for asymbiotic in vitro propagation.6, 38, 39 Early experiments showed that orchid seed embryos contain a small amount of starch and that asymbiotic protocorms accumulate high levels of starch and grow slowly in sugar-containing medium compared with the protocorms of embryos infected by fungi.13, 39 It has been suggested that the presence of carbohydrate in the external medium leads to ineffective utilization of internal nutrients in Dactylorhiza purpurella under asymbiotic germination conditions.40 Physiological experiments involving the symbiotic germination of Goodyera repens seeds revealed that the protocorms of embryo infected by fungi grew rapidly without accumulating starch, indicating that symbiotic infection of the orchid protocorm may trigger metabolic changes in the host plant that result in the suppression of starch synthesis.41 In our transcriptomic analysis, we found that 4 genes encoding beta or alfa-amylase (Dendrobium_GLEAN_10069668, 10026401; 10026400; 10050481), key enzymes in related to starch degradation, are significantly up-regulated at the early symbiotic germination stage (S1/A1) with 4.6 to 15.8-fold changes in expression. In addition, 6 genes encoding amylase (Dendrobium_GLEAN_10069669, 10069668, 10026401, 10026400, 10050481, and 10106303) showed increased expression at the symbiotic protocorm stage (S2/A1) with 2- to 60- fold changes in expression compared to asymbiotic protocorms. However, these transcripts were either not identified or did not show significant changes at the proteomic level. Enzymes involved in the pentose phosphate pathway (transketolase) and the glycolysis pathway (pyruvate kinase) also showed increased expression in SG compared to AG. In our study, organic carbon supports by fungi was presumed to induce the expression of host plant enzymes related to the degradation of seed storage substance (starch) at the initial germination stage of D. officinale after fungal invasion. Thus, it is likely that the glycolysis and the Calvin photosynthetic cycle are


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involved in the changes in carbohydrate metabolism that we observed in embryos that received fungal inoculation. Lipid metabolism Orchid seeds have been reported to contain high concentrations of lipid reserves that are converted to starch upon germination.6 Uetake et al. found that lipid reserves were slowly degraded during orchid seed germination.10 During asymbiotic germination of the epiphytic orchid Cattleya, glyoxysomes are absent. However, utilization of lipid may occur when the seeds were incubated with an external source of sucrose.41 In our study, fatty acids metabolism is significantly enriched at the early germination stage during symbiotic germination of D. officinale. Acetyl-CoA carboxylase (ACC) and &-oxidation multifunctional protein are key enzymes in fatty acids biosynthesis and in the catabolism of lipids. We also demonstrated that glyoxysomal fatty acid beta-oxidation multifunctional protein is up-regulated at the early germination stage by more than 1.40-fold change compared to ungerminated seeds and that it is down-regulated during post-germination stage (protocorm and seedling development) of symbiotic germination; however, no significant change in its expression was detected during the process of asymbiotic germination of D. officinale. In addition, we found that acyl-CoA-binding proteins (ACBPs) displayed increasing expression at the early stage (2.0-fold change), the protocorm stage (2.3-fold change) and the seedling developmental stage (1.5-fold change) during the symbiotic germination process in D. officinale. ACBPs are essential proteins in fatty acid metabolism and have divers functions in a diverse manner in developmental and physiological processes such as seed germination, stem cuticle formation, pollen development and leaf senescence.42 Hence, we inferred that the degradation of stored lipid is likely to be a specific process in symbiotic seed germination after fungal inoculation.


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Plant defense mechanisms Seed germination is not only activities a series of metabolic processes such as the degradation of stored substances, the production of energy and the biosynthesis of secondary metabolites but also activates systems that protect the plant against damage due to stress, especially oxidative damage.43 Studies have found the that activities of polyphenol oxidase, ascorbic acid oxidase, peroxidase and catalase are greatly increased in the protocorms of embryos infected with mycorrhizal fungi and that the peak activity of these enzymes often coincides with peak oxygen uptake and digestion of pelotons, indicating that the plant defense reaction occurs during the interaction of orchid plants with their mycobionts.44 Our proteomic analysis showed that a catalase isozyme (Dendrobium_GLEAN_10047013) was present at significantly higher levels during the entire process of SG in D. officinale compared to AG, with 1.95-, 1.69- and 2.05- fold changes at the early germination, protocorm formation and seedling growth stages, respectively. The expression of L-ascorbate peroxidase (Dendrobium_GLEAN_10099098) also increased during the protocorm stage in SG. The gene encoding superoxide dismutase (SOD) (Dendrobium_GLEAN_10000566) was found to be up-regulated at the early germination stage and during proctocorm formation in SG. This finding is in agreement with the results of previous studies showing the up-regulation of SOD in green protocorms.22 Interestingly, increased the expression of acidic endochitinase (Dendrobium_GLEAN_10117734), which functions as a defense against chitin containing fungal pathogens, was identified during the entire germination process in SG relative to AG; its peak expression occurred at the protocorm stage, with a 6.72fold change in SG relative to AG. The results suggest that these proteins regulate the response to fungal stimulation during seed germination of D. officinale after fungal colonization. Signal transduction


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It is generally acknowledged that the plant hormone abscisic acid (ABA) is a positive regulator of dormancy, whereas gibberellins (GAs) cause dormancy and promote the completion of germination, counteracting the effects of ABA.45 Moreover, the abscisic acid receptor PYR1, has been shown to be involved in mycorrhizal establishment during the early symbiosis of AM mycorrhiza.46, 47 In our proteomic database, the regulatory component of the ABA receptor (RCAR)/PYR1/PYL (Dendrobium_GLEAN_10109310) was found to be significantly upregulated in protocorm compared to early-germination seeds in SG (Table S5, Supporting Information). In summary, the data indicate that plant hormone biosynthesis and signal transduction may be involved in the symbiotic germination of D. officinale seeds. Taken together, the results of our comparative proteome analysis show that carbohydrate metabolism, fatty acid metabolism and glutathione metabolism are differentially affected during SG of D. officinale. Fungal invasion appears to trigger metabolic changes in the host plant (nutrient accumulation) and to initiate plant defense reactions during the establishment of symbiosis between D. officinale seeds and mycorrhizal fungi. Integrative analysis of the proteome and transcriptome during seed germination of D. officinale Transcriptomic analysis of the same samples assayed by iTRAQ was performed using the RNASeq method; thus, using our data, it is possible to directly compare transcript and protein expression during seed germination of D. officinale. The concordance analysis of expression changes in transcripts and proteins was conducted at three levels, including the number of identified proteins or genes, the quantitation of proteins and gene transcripts, and their differential expression. The parameters used for correlative analysis and the number of proteins


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and genes in the correlative analysis are listed in Table S7 and Table S8 in Supporting Information, respectively. In the present study, more than 95% of proteins identified in iTRAQ proteome analysis were also covered by the transcriptomic analysis, while less than 5% of protein were identified only by iTRAQ, suggesting that iTRAQ and transcriptomic analysis are complementary in profiling candidate proteins involving in specific physiology progress, such as seed germination of Orchidaceae. Many studies have demonstrated the preference of iTRAQ in assessing differences in proteins of low-abundance and discovery of unknown or only suspected members of core protein complexes.48, 49 When changes in expression in the transcriptome and proteome in different samples were compared, a poor correlation between the levels of quantitative proteins and those of their gene transcripts was observed using Pearson correlation test (R =-0.05–0.24, Figure 6). However, when the analysis included the DEG and DEP datasets only, the transcriptome and proteome during seed germination of D. officinale showed a better correlation (Figure S4, Supporting Information) (rPearson = -0.5253–0.8611). Spearman correlation analysis also showed the higher correlation coefficient, mainly distributed in type of DEPs and DEGs with the same or opposite expression trends (Figure S5, Supporting Information). Previously published results have also demonstrated that the degree of correlation between the transcriptome and the proteome is generally low (27%–40%).50 The study of Hu et al. also revealed poor correlations (rPearson = 0.06–0.24) between mRNA and protein ratios when they conducted comparative proteomic and transcriptomic profiling of developing fibers from an elite cultivar and a wild varity.51 A possible explanation for this result is that the proteins underwent transcriptional ; translational and posttranslation modification, resulting in protein changes that were not always consistent with


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RNA expression. In addition, comparative analysis of the differentially expressed genes and proteins revealed only a small amount of overlap in transcriptomic level and proteomic level. Further analysis of DEGs and DEPs with the same expression trends (indicated by the red dot in Figure 6) assayed by iTRAQ and RNA-Seq revealed that 66 proteins are significantly upregulated in at least one adjacent germination stage in AG and SG (Table S9, Supporting Information). At the early germination stage, 20 and 37 proteins are differentially co-upregulated in AG and SG, respectively. Of the 20 proteins that are co-up-regulated at the early germination stage in AG, 9 proteins are also up-regulated at the same stage in symbiotic germination, suggesting that these proteins play a core role in the regulation of seed germination in D. officinale. An abscisic acid receptor protein, PYR1, displayed significantly increased expression at the transcriptomic and proteomic levels at the early germination stage of symbiotic growth relative to ungerminated seeds. Thirty-two proteins were found to be differentially up-regulated at both the transcriptomic and proteomic level during symbiotic germination compared to the asymbiotic situation (Table S10, Supporting Information). These proteins are involved into nitrogen metabolism (glutamine synthetase nodule isozyme), symbiotic signals transduction (calreticulin), lipid metabolism (acylCoA-binding protein), the defenses reaction (epidermis-specific secreted glycoprotein EP1), the utilization of storage proteins (subtilisin-like protease, basic 7S globulin), and other functions. Genes encoding mannose-binding lectins were also detected significantly up-regulated in the green protocorm in the presence of the mycorrhizal fungus.21 The expression profiles of six coexpressed genes, including encoding calreticulin, superoxide dismutase, cucumisin, late embryogenesis abundant protein, epidermis-specific secreted glycoprotein and subtilisin-like protease were validated by quantitative real-time PCR (RT-PCR) at three germination stages


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during AG and SG (Figure 7). The RT-PCR results displayed a similar expression trend with our bioinformatics analysis based on transcriptome and proteome of D. officinale seed germination. The present study has two limitations. The absence of biological replicates may lead to the false positive results. In order to avoid the false positive results, we firstly performed the integrative analysis of RNA-Seq and iTRAQ. Secondly, we did the KEGG enrichment analysis to descrease the false positive expression of individual genes. Thirdly, we selected six candidate genes to validate their expression using the RT-PCR method. In addition, protein and RNA were extracted from developmental stages seeds. However, The same stage could stay for a relatively long time and thus, there can be differences between e.g. the young protocorms and older ones. The age of plant material also should therefore be taken into account in future work. CONCLUSION In this study, a comprehensive dataset generated by combining technologies for transcriptomic and proteomic analyses provided a global proteomics perspective on the complex metabolic processes that occur during seed development in D. officinale. Based on a comparison of changes in the proteomes of germinating seeds after inoculation with fungi, a key finding of our study is that protein regulation at the beginning of seed germination is very similar under asymbiotic and symbiotic germination conditions, but that protein expression at the protocorm formation stage (S2/S1) in SG closely resembles a later developmental stage (seedling stage, A3/A2) of AG. The temporal expression patterns of genes and proteins indicated that a common pathway occurs in asymbiotic and symbiotic germination at the early germination stage of D. officinale. The similar expression profiles of genes (proteins) at the early germination stage (stage2) in symbiotic and asymbiotic growth indicates that the substance stored in embryonic cells may be sufficient for the initiation of germination (allowing the embryo to emerge from the seed coat) but insufficient


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for subsequent protocorm growth and seedling development. Fungal colonization may alter host metabolism (especially carbohydrate and lipid metabolism) and improve the utilization of nutrients. At the same time, fungal invasion of the embryonic cells triggers the plant defense reaction and induces the expression of genes related to signal transduction pathways. Multiple cross-interaction of these molecular signals (ROS, Ca2+) with signals generated by plant hormones or sugars are probably required for the germination of orchid seeds. Further investigation of changes in metabolites and other components of germinating seeds will contribute to full comprehension of the molecular mechanism of symbiotic germination induced by fungi. These data will serve as an invaluable resource for research in orchid seed biology. ACKNOWLEDGMENTS We thank Prof. Francis MARTIN for helpful discussions and the technician Wen-Jie WANG for preparation of plant samples. This project was supported by the Natural Science Foundation of China (81573527, 81573526), the Basic scientific research operation cost of state-leveled public welfare scientific research courtyard (YZ-12–14) and the Program for Innovative Research Team in IMPLAD (PIRTI-IT1302). AUTHOR INFORMATION Corresponding Author * E-mail for SXG: [email protected]; Tel/Fax: +86-10-57833231 Notes: The authors declare no competing financial interest. The original transcriptomic data has been deposited in public NCBI and SRA database (accession No. PRJNA279934) (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA279934). The mass spectrometry proteomics data by iTRAQ have been deposited to the ProteomeXchange


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Consortium (http:// proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD006102 (Username [email protected], password: gv4NNm75). SUPPORTING INFORMATION Table S-1. Labeling samples information of Dendrobium officinale seeds in our iTRAQ experiments. Table S-2. Mascot search parameters used in protein identification by iTRAQ analysis of D. officinale seeds. Table S-3. Quantification parameters used in our relative quantification of protein by iTRAQ analysis of D. officinale seeds. Table S-4. Detailed information of 2256 plant proteins identified by iTRAQ during seed germination of D. officinale. Table S-5. 308 differentially expressed proteins (DEPs) in at least one more germination stage during asymbiotic and symbiotic germination of D. officinale. A protein was considered to be DEP if it met fold change $ 1.2 or # 0.83 and Q value < 0.05. A1, A2, A3 represent developmental stage 2, stage3, stage4 in asymbiotic germination and S1, S2, S3 represent stage2, stage 3, and stage 4 in symbiotic germination, C0 represents ungerminated seed. AG: asymbiotic germination; SG: symbiotic germination. Red color: A protein was up-regulated expression in the given compared group; green color, a protein was down-regulated and gray color: protein no significant expression change at the given compared group. Table S-6. 229 differentially expressed proteins (DEPs) in at one more development stage during symbiotic germination compared to asymbiotic germination of D. officinale. A


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protein was considered to be DEP if it met fold change $ 1.2 or # 0.83 and Q value < 0.05. Red color means up-regulated proteins, green color means down-regulated protein and gray color means no significant change in the given compared group, respectively. Table S-7. Key parameters used for correlation analysis between transcriptome and proteome of seed germination of D. officinale. Table S-8. Correlation analysis of the transcriptome and proteome reveals the number relationship of proteins and genes on identification, quantitative and differentially expressed aspects. Table S-9. 66 co-up-regulated proteins at transcriptomic and proteomic level across at least one more adjunct developmental stage during asymbiotic or symbiotic germination of D. officinale. A protein was identified as co-up-regulated protein if it met: i) fold change $ 1.2 (iTRAQ) and 2.0 (RNA-Seq); ii) Q value < 0.05(iTRAQ) and FDR < 0.001(RNA-Seq). Yellow color: co-up-regulated proteins; red color: up-regulated proteins only in iTRAQ; orange color: up-regulated genes only in RNA-Seq; gray color: proteins and genes without significant expression change. ** The tandem mass spectra were showed in Figure S1 in Supporting Information. Table S-10. 32 Co-up-regulated proteins at transcriptomic and proteomic level during symbiotic germination compared to asymbiotic germination of D. officinale. A protein was identified as co-up-regulated protein if it met: i) fold change $ 1.2 (iTRAQ) and 2.0 (RNA-Seq); ii) Q value < 0.05(iTRAQ) and FDR < 0.001(RNA-Seq). Yellow color: co-up-regulated proteins; red color: up-regulated proteins only in iTRAQ; orange color: up-regulated genes only in RNASeq; gray color: proteins and genes without significant expression change. * gene expression was


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validated by qPCRgenes for RT-PCR. **The tandem mass spectra were showed in Figure S1 in Supporting Information Figure S-1. Examples of tandem mass spectra of iTRAQ for the differentially expressed proteins (DEPs) with low number of peptide. Five DEPs during seed germination of D. officinale were selected from Table S9 and Table S10 in Supporting Information (marked **), including Dendrobium_GLEAN_10057503, Dendrobium_GLEAN_10089327, Dendrobium_GLEAN_10117734, Dendrobium_GLEAN_10122630 and Dendrobium_GLEAN_10052315. Left column is the tandem mass spectra of the proteins and right column is the region containing the reporter ions of spectra (m/z:113-121) associated with the spectrum of proteins. Figure S-2. RNA-Seq and iTRAQ 8-plex workflow of asymbiotic and symbiotic germination of D. officinale seeds. Stage2: early germination; Stage3: protocorm stage; Stage4: seedling stage; A1, A2, A3 represent developmental stage 2, stage3, stage4 in asymbiotic germination and S1, S2, S3 represent stage2, stage 3, and stage 4 in symbiotic germination, C0 represent ungermination seed. Figure S-3. Comparative analysis of seed germination at all stages over 12 weeks time of D. officinale. A. Seed development in symbiotic condition. B. Seed development in asymbiotic conditions. C. A quantitive proportion of seeds at all stage compared total germination seed in 12th week. AG: asymbiotic germination; SG: symbiotic germination. Figure S-4. Comparison of expression ratios from transcriptomic (y-axis) and protomic (xaxis) profiling based on differentially expressed proteins and correlated genes at each compared group. Log2 expression ratios were calculated in adjacent development stage during


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asymbiotic (left columns) and symbiotic germination (middle columns) and at the same stage between symbiotic and asymbiotic germination (right columns). Red point represents both proteins and genes are differentially expressed. AG: asymbiotic germination; SG: symbiotic germination. Figure S-5. Summary of correlation coefficient of quantitative correlation results (6 types) in each compared group. 6 types: correlation result of all quantitative protein and the mRNA (blue); the differentially expressed proteins and genes with the same trend (green); the differentially expressed proteins and genes with the opposite trend (yellow); both proteins and RNA no change (orange); proteins no change but genes differentially expressed (red); proteins differentially expressed but genes no change (pale red). A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. REFERENCES 1. Dressler, R.L. How many orchid species? !Selbyana 2004, 26, 155–158. 2. Smith, S. E.; Read, D. J. Mycorrhizal symbiosis, 3rd edn. Academic press: San Diego, 2008× pp 419–506. 3. Cozzolino, S.; Widmer, A. Orchid diversity: an evolutionary consequence of deception? Trends Ecol. Evol. 2005, 20, 487–494. 4. Selosse, M.A. The latest news from biological interactions in orchids: in love, head to toe. New Phytol. 2014, 202, 337-340. 5. Bronstein, J. L.; Armbruster, W. S.; Thompson, J. N. Understanding evolution and complexity of species interactions using orchids as a model system. New Phytol. 2014, 202, 373–375. 6. Arditti, J. Factors affecting the germination of orchid seeds. Botanical Review 1967, 33, 1–97.


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Figure legends Figure 1. Morphological characters and the seed developmental stages of Dendrobium officinale (A) plants, (B) flowers, (C) fruit, (D) seeds, (E) seed symbiotic germination for 7 days, (F) seed asymbiotic germination for 21 days. G-J: light microscope observation for developmental stages of seeds. (G) stage 1-2, (H) stage 3, (I) stage 4, (J) stage 5. (Images A and B taken by Juan Chen; C-F taken by Bo Yan; G-J taken by Si Si Liu. Copyright 2017). Figure 2. Venn diagram showing the numbers of differentially expressed proteins (DEPs) in various comparative groups of D. officinale seeds. A. DEPs at adjacent stage of asymbiotic germination; B. DEPs at adjacent stage of symbiotic germination; C. the number of common expressed proteins at adjacent stage between asymbiotic and symbiotic condition; D. the number of DEPs at each development stage; E. DEPs at the same stage between asymbiotic and symbiotic germination. A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. Figure 3. The expression patterns of 308 differentially expressed proteins (DEPs) and 308 differentially expressed genes (DEGs) at adjacent development stage during asymbiotic and symbiotic germination of D. officinale. A. Expression pattern of 308 DEPs (iTRAQ); B. Expression pattern of correlated 308 DEGs. Heat map was conducted on a log2 fold change. Upor down-regulated proteins or genes are shown in red and green, respectively; black corresponds to no significant change and gray represents missing data. The KOG functional category (gray columns) was assigned to each protein: Energy production (C); Amino acid metabolism (E); F, Nucleotide transport (F); Carbohydrate metabolism (G); Coenzyme metabolism (H); Lipid


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metabolism (I); Translation (J); Transcription (K); Replication (L); Cell wall biogenesis (M); Posttranslational modification (O); Inorganic ion metabolism (P); Secondary metabolites (Q); General function prediction only (R); Signal transduction mechanisms (T); Cytoskeleton (Z). A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. Figure 4. KEGG pathway enrichment analysis of differentially expressed proteins (DEPs) during asymbiotic and symbiotic germination of D. officinale seeds. Left columns: pathway enrichment at adjacent development stages during asymbiotic germination; Middle columns: pathway enrichment at adjacent development stages during symbiotic germination; Right columns: pathway enrichment at the same stage between asymbiotic and symbiotic germination. stage 2: early germination; stage 3: protocorm formation, stage 4: seedling development; A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. Abbreviation: AG, asymbiotic germination; SG, symbiotic germination. Figure 5. Hierarchical clustering analysis of 229 DEPs between symbiotic and asymbiotic germination of D. officinale. Expression ratios were plotted in a heatmap on a log2 scale. The red and green colors indicate up- and down-regulation in symbiotic germination compared to asymbiotic germination. Black represents no significant expression change. A1, A2, A3 represent developmental stage 2, stage3, stage4 in asymbiotic germination and S1, S2, S3 represent stage2, stage 3, and stage 4 in symbiotic germination, C0 represent ungermination seed.


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Figure 6. Comparison of expression ratios from transcriptomic (y-axis) and proteomic (xaxis) profiling based on quantitative proteins and correlated genes at each compared group. Log2 expression ratios were calculated in adjacent development stage during asymbiotic (left columns) and symbiotic germination (middle columns) and at the same stage between symbiotic and asymbiotic germination (right columns). Significant expression changes were labeled in colors: blue point, proteins only; green point, transcripts only; red point, both. A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. AG: asymbiotic germination; SG: symbiotic germination. Figure 7. Six co-expressed proteins were selected for quantitative RT-PCR analysis. PCR amplification was performed at three development stages during asymbiotic and symbiotic germination of D. officinale and three biological replicates and two distinct technical replicates for each sample were included. Expression levels were calculated by the E!''Ct method normalized against EF1-% expression.


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Table1. Seed development stages of D.officinale and the samples analyzed in our study Germination Germination description stage

Stage0 Stage1 Stage2 Stage3 Stage4 Stage5

Asymbiotic germination(AG) Symbiotic germination(SG) experiement experiement day after sowing day after sowing ID ID C0 0 C0 0

No germination, visuable embryo Enlarged embryo, production of rhizoid(s) Continued embryo enlargement, rupture of testa A1 (germination) Appearance of protomeristem (protocorm) A2 Emergence of first leaf (Seedling) A3 Elongation of first leaf

18 day 36 day 50 day

S1 S2 S3

12 day 20 day 30 day


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Figure 1. KEGG pathway enrichment analysis of differentially expressed proteins (DEPs) during asymbiotic and symbiotic germination of D. officinale seeds. Left columns: pathway enrichment at adjacent development stages during asymbiotic germination; Middle columns: pathway enrichment at adjacent development stages during symbiotic germination; Right columns: pathway enrichment at the same stage between asymbiotic and symbiotic germination. stage 2: early germination; stage 3: protocorm formation, stage 4: seedling development; A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. Abbreviation: AG, asymbiotic germination; SG, symbiotic germination. 151x92mm (300 x 300 DPI)



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Figure 2. Venn diagram showing the numbers of differentially expressed proteins (DEPs) in various comparative groups of D. officinale seeds. A. DEPs at adjacent stage of asymbiotic germination; B. DEPs at adjacent stage of symbiotic germination; C. the number of common expressed proteins at adjacent stage between asymbiotic and symbiotic condition; D. the number of DEPs at each development stage; E. DEPs at the same stage between asymbiotic and symbiotic germination. A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. 54x42mm (300 x 300 DPI)


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Figure 3. The expression patterns of 308 differentially expressed proteins (DEPs) and 308 differentially expressed genes (DEGs) at adjacent development stage during asymbiotic and symbiotic germination of D. officinale. A. Expression pattern of 308 DEPs (iTRAQ); B. Expression pattern of correlated 308 DEGs. Heat map was conducted on a log2 fold change. Up-or down-regulated proteins or genes are shown in red and green, respectively; black corresponds to no significant change and gray represents missing data. The KOG functional category (gray columns) was assigned to each protein: Energy production (C); Amino acid metabolism (E); F, Nucleotide transport (F); Carbohydrate metabolism (G); Coenzyme metabolism (H); Lipid metabolism (I); Translation (J); Transcription (K); Replication (L); Cell wall biogenesis (M); Posttranslational modification (O); Inorganic ion metabolism (P); Secondary metabolites (Q); General function prediction only (R); Signal transduction mechanisms (T); Cytoskeleton (Z). A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. 104x83mm (300 x 300 DPI)



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Figure 4. KEGG pathway enrichment analysis of differentially expressed proteins (DEPs) during asymbiotic and symbiotic germination of D. officinale seeds. Left columns: pathway enrichment at adjacent development stages during asymbiotic germination; Middle columns: pathway enrichment at adjacent development stages during symbiotic germination; Right columns: pathway enrichment at the same stage between asymbiotic and symbiotic germination. stage 2: early germination; stage 3: protocorm formation, stage 4: seedling development; A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. Abbreviation: AG, asymbiotic germination; SG, symbiotic germination. 150x96mm (300 x 300 DPI)


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Figure 5. Hierarchical clustering analysis of 229 DEPs between symbiotic and asymbiotic germination of D. officinale. Expression ratios were plotted in a heatmap on a log2 scale. The red and green colors indicate upand down-regulation in symbiotic germination compared to asymbiotic germination. Black represents no significant expression change. A1, A2, A3 represent developmental stage 2, stage3, stage4 in asymbiotic germination and S1, S2, S3 represent stage2, stage 3, and stage 4 in symbiotic germination, C0 represent ungermination seed. 105x74mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Comparison of expression ratios from transcriptomic (y-axis) and proteomic (x-axis) profiling based on quantitative proteins and correlated genes at each compared group. Log2 expression ratios were calculated in adjacent development stage during asymbiotic (left columns) and symbiotic germination (middle columns) and at the same stage between symbiotic and asymbiotic germination (right columns). Significant expression changes were labeled in colors: blue point, proteins only; green point, transcripts only; red point, both. A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. AG: asymbiotic germination; SG: symbiotic germination. 151x150mm (300 x 300 DPI)


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Figure 7. Six co-expressed proteins were selected for quantitative RT-PCR analysis. PCR amplification was performed at three development stages during asymbiotic and symbiotic germination of D. officinale and three biological replicates and two distinct technical replicates for each sample were included. Expression levels were calculated by the E−∆∆Ct method normalized against EF1-α expression 150x144mm (300 x 300 DPI)



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For TOC only 109x106mm (300 x 300 DPI)


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iTRAQ and RNA-Seq Analyses Provide New Insights into Regulation

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