Comparative proteomics analysis provides new insights into the haustorium development of Taxillus chinensis (DC.) Danser

Comparative proteomics analysis provides new insights into the haustorium development of Taxillus chinensis (DC.) 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Danser Limei Pan, Lingyun Wan, Lisha Song, Lili He, Ni Jiang, Hairong Long, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1033805/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Loranthus ( Taxillus chinensis ) is an important medicinal and parasitic plant that attacks other plants for living. To reveal the mechanisms of haustorium development, we employed an iTRAQ proteomics-based approach to identify differentially abundant proteins (DAPs) of fresh seeds (CK), baby (FB), and adult haustoria (FD). Results A total of 563 and 785 DAPs were successfully quantified in the early/later developmental stage, respectively. Pathway enrichment analysis indicated that the DAPs mainly associated with metabolic pathways, ribosome, phenylpropanoid biosynthesis and photosynthesis. In the meantime, DAPs associated with phytohormone signaling pathway changed markedly. Furthermore, we evaluated the contents change of phytohormone during the haustoria development. These results indicated that phytohormone is very important for haustorium development. qRT-PCR validation showed that the mRNA expression levels were consistent with the protein variation, suggesting that our result were reliable. Conclusions To the best of our knowledge, this is the first haustoria proteomes of loranthus, and our findings will improve our understanding of the molecular mechanism of haustoria development. Plant Physiology and Morphology Plant Molecular Biology and Genetics proteomics T. chinensis haustorium phytohormone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The Taxillus chinensis (DC.) Danser is a member of the family Loranthaceae, which consists of approximately 73 genera and 900 species, which generally consists of aerial hemiparasitic plants ( 1 ). T. chinense is widespread distributed in the southern and southwestern areas of China. T. chinense is also used in Chinese traditional for many years, mainly because its stems and leaves can be used for the treatment of stroke, rheumatoid arthralgia, threatened abortion, angina pectoris, and hypertension ( 2 ). Mulberry, camellia, plum, peach, litchi, carambola, longan, and other economically important plant species can were parasitized by T. chinensis , which is semi-parasitic botanical medicinal materials ( 3 ). T. chinensis absorb nutrient compounds and water from the host plants via a unique multicellular organ termed the haustorium that aids to host attachment and invasion, although they provide their own carbohydrates through photosynthesis ( 1 , 4 ). In general, the initiation of haustorium development in most parasitic plants is triggered by host derived chemical signals terms haustorium-inducing factors (HIFs) after seed germination. Phenolic acids, quinones, flavonoids, and 2,6-dimethoxy-p-benzoquinone (DMBQ) that can be act as HIFs have been well documented ( 5 ). However, the exhaustive mechanisms about the how these compounds are released and then triggered haustorium development are still not well studied and need further research. Besides, some genes have been identified that may be involved in these processes. For example, the TvQR1 gene encoding quinone oxidoreductase is needed to trigger the development of the haustorium of the facultative parasite Triphysaria versicolor ( 6 , 7 ). One possible reason for inducing the development of the haustor is that this enzyme converts quinone to semiquinone, which acts as an intermediate product during the redox cycle. Semiquinone itself or the redox cycle may be involved in haustorium induction signal transduction pathway ( 6 , 8 ). The current model proposes that the phenolic acid derived from the degradation of the host cell wall is oxidized by ROS and oxidase to generate the haustor-inducing factor quinone ( 9 , 10 ). In addition, it has been recently demonstrated that the accumulation of auxin and ethylene can act as early events in haustorium development of the hemiparasitic plant T. versicolor ( 11 ). Some research have observed that zeatin (Z), zeatin nucleotide (ZN), zeatin riboside (ZR), abscisic acid (ABA), and cytokinins (CKs) were significantly deposited in the haustoria of the Rhinanthus–Hordeum vulgare association ( 12 , 13 ). Santalum album is a kind of invasive root hemiparasite. After being pretreated with 2~8mM GA3 for 12 h, the seeds of Santalum album can germinate in vitro on Murashige and Skoog medium or in sand without HIFs trigger within one month ( 14 , 15 , 16 ). All these results suggest that the plant hormones may play a crucial role in controlling haustorium development. With the rapid development in mass spectrometry (MS), next-generation sequencing, and microarray technologies, the researchers has enabled relatively high-throughput analyses of transcriptomes, proteomes, and metabolomes and obtained large-scale snapshot information on transcripts, proteins, and metabolites. The changes in the transcriptome of haustorial development in T. chinense has been investigated and has shown the possible mechanism of T. chinense haustorial development ( 2 ). But study of biological processes at the protein level provides more realistic information compared to the transcript level. This is because proteins have biological functions, and the post-translational process will lead to the production of different protein subtypes ( 17 ). However, few studies have performed proteomic analysis of haustorial development in T. chinense . Methods Plant Material The fresh seeds were collected from Taxillus chinensis (DC.) Danser which parasitized in the mulberry trees that were planted in the experimental field of Guangxi Botanical Garden of Medicinal Plants in China. Then, the seeds were peeled, washed with sterile water, placed on a germination dish, and incubated under the controlled environment (25°C, 80% relative humidity, 10 h:14 h light: dark, 2000 Lx), as previously described (2). The fresh seeds were collected as control (CK). The seeds with protruding seed-type radicle and tiny suction device were collected after ten days of incubation (FB). And twenty days later, seeds with the loranthus haustoria and true leaves were used for the experiments (FD). Then, the samples were immediately frozen in liquid nitrogen, and stored at -80°C until use. Protein extraction Protein was extracted as described in a previous study (18). Briefly, the seeds samples were frozen in liquid nitrogen and pulverized. The samples were mixed with 5 volumes of chilled acetone containing 10% (v/v) trichloroacetic acid (TCA) and incubated at -20℃ overnight. After centrifugation at 6,000 g and 40 min, the supernatant was discarded. The precipitant was washed three times with pre-cooling acetone and was air-dried. The pellet was dissolved in Lysis buffer (8 M urea,120 mM NaCl, 10 mM EDTA, 1% Triton X100 and 1% PMSF dissolved in 50 Mm Tris-HCl PH 8.0). Finally, the supernatant was filtered through 0.22-mm filters after centrifugation at 13,000 g for 10 min at 4 ℃. The concentration of the protein was quantified with the BCA Protein Assay Kit (Bio-Rad, USA) and the quality of the protein sample was measured by SDS-PAGE, respectively. Protein digestion and iTRAQ labeling For each sample, 100 µg protein was used for digestion and iTRAQ labeling. Protein were reduced with 10 mM DTT at 37 ℃ for 60 min and then alkylated with 55 mM iodoacetamide (IAM) at room temperature for 30 min in darkness. The urea concentration of protein sample was diluted less than 2 M by adding 100 mM TEAB. Then trypsin was added to the protein pool of each sample with the ratio of protein: trypsin=50:1(mass ratio) at 37℃ overnight and 100: 1 for a second digestion for 4 h. After trypsin digestion, peptide was desalted with the Strata X SPE column and vacuum-dried. Peptide was reconstituted in 20 μL 500 mM TEAB and labeled according to the manufacturer’s protocol. Briefly, dissolutioned the peptide solution in 50 μL isopropanol and added one unit of iTRAQ reagent, incubated for 2 h at room temperature, then pooled and dried by vacuum centrifugation. High Performance Liquid Chromatography (HPLC) Fractionation The peptide was reconstituted with HPLC solution A (2% ACN, PH 10.0) and fractionated into fractions by high PH reverse-phase HPLC using Waters Bridge Peptide BEHC18 (130 Å, 3.5 μm, 4.6*250 mm). Briefly, peptides were first separated with a gradient of 2% to 98% acetonitrile (pH 10) into 72 fractions. The wavelength 250 nm is used for detection of peptides. Then, the peptides were combined into 18 fractions and dried by vacuum centrifugation. The peptide fractions were desalted using Ziptip C18(Millipore, Billerica, MA) according to manufacturer’s instructions. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis Then the experiment was performed by NanoLC 1000 LC-MS/MS using a Proxeon EASY-nLC 1000 coupled to Thermo Fisher Q Exactive. Resuspended the disgestion fractions using 0.1% formic acid and loaded onto a reversed-phase-column (Acclaim PepMap®100 C18, 3μm, 100Å, 75μm× 2cm) at a rate of 5 μL/min in 100% solvent A（0.1M acetic acid in water）. Next, peptides eluted from the trap column were loaded onto a reversed-phase analytical column (Acclaim PepMap®RSLC C18, 2μm, 100Å, 50μm× 15cm). The gradient was comprised of an increase from 15% to 35% solvent B (0.1% FA in 98% ACN) over 45 min, 35% to 98% solvent B during 5 min and keep in 98% in 5 min at a constant flow rate of 300 nl/min on an EASY-nLC1000 system. The eluent was sprayed via NSI source at the 2.0 kv electrospray voltage and then analyzed by tandem mass spectrometry (MS/MS) in Q Exactive. A data-depentent mode in the scan range of 350-2000 m/z was carried out for the mass spectrometry analyses, and the survey scans were captured at a mass resolution of 17500 by the Orbitrap analyzer. In the linear ion trap, 15 of the most intense precursor ions were selected for subsequent decision tree-based ion trap HCD fragmentation at the normalized collision energy of 32% in the MS survey scan with 10.0s dynamic exclusion. Raw data processing The raw data files were searched against the transcriptome database using Swquest software integration in Proteome Discoverer (version 1.3, Thermo Scientific). The search parameters used as follows: Carbamidomethylation (C) was set as fixed modifications; and oxidation (M), acetylation in N-Term were set as variable modification. Trypsin was chosen as enzyme and two missed cleavages were allowed. Mass tolerance of precursor ions was 20 ppm, and a fragment ion tolerance was 0.05 Da, resulting in 1% false discovery rate (FDR). Differentially abundant proteins (DAPs) were identified based on the following criteria: P-values smaller than 0.05 and a mean relative abundance>1.2 or<0.83. Bioinformatics analysis To determine the functional characterization of differentially abundant proteins (DAPs), proteins were mapped with Gene Ontology (GO) annotation base on UniProt-GOA database ( www.http://www.ebi.ac.uk/GOA ) (19). All proteins were grouped into three major categories: biological processes, cellular components, and molecular functions. The metabolic pathway analyses of DAPs were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( http://www.genome.jp/kegg/ ) (20). Phytohormone analysis Phytohormone indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid (ABA) were quantified based on a liquid chromatography and mass spectrometry system (LC-MS, 1120-6460, Agilent, USA). Phytohormone extraction was performed from loranthus seeds based on the previous method (21). The phytohormone were separated by C18 column (Hypersil Gold, 100 mm × 2.1 mm, 1.9 μm, Thermo Fisher Scientific) at flow rate of 0.3 mL/min with a 17 min gradient elution. For Multiple Reaction Monitoring (MRM), the phytohormone were analyzed with negative mode by ESI ion source. Quantitative real-time PCR analysis Total RNA was extracted from each sample using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. FastQuant RT Kit (with gDNase, Tiangen) was used for DNA remove and cDNA synthesis. Specific primer pairs of randomly selected 8 genes for quantitative real-time PCR (qRT-PCR) were designed using Primer Premier 5.0 software and sequences are listed in Table S1. actin was used as reference gene (2). The procedure of qRT-PCR experiment was same as our previous study (22). 2 −ΔΔCt method was used to evaluate the expression levels of transcripts in each sample (23). Each transcript was performed with three biological replicates and three technical replicates. Result Differential abundant proteins (DAPs) analysis To investigate the potential mechanisms involved in the haustorial developmental process in T. chinense , an integrated approach involving LC-MS/MS and iTRAQ labeling was applied to analyze the proteomic changes. A total of 563 proteins were identified as DAPs between FB treatment and control, of which 384 were identified as upregulated and 179 were downregulated proteins, respectively. A total of 785 proteins were identified as DAPs between FD treatment and control, of which 569 and 216 were identified as upregulated and downregulated proteins, respectively (Figure 1). All the DAPs were grouped based on their subcellular localizations. For the FB treatment, 11 subcellular components were identified, including 250 chloroplast-localized proteins (44.4%), 162 cytosol- localized proteins (28.77%), 80 nuclear-localized proteins (14.21%). For the FD treatment, 16 subcellular components were identified, including 325 chloroplast-localized proteins (41.4%), 2433 cytosol- localized proteins (30.96%), 106 nuclear-localized proteins (13.5%) (Figure 1). Functional categorization analysis The biological functions of the DAPs could also be identified by their GO annotations. The DAPs were classified into the three main GO categories (cellular component, biological process, and molecular function). In the early developmental stage (FB), 1546 DAPs (some proteins have more than one GO annotation) were annotated with biological process, 653 DAPs with molecular functions, and 1885 DAPs with cellular component compared to control. In the later developmental stage (FD), 2126 DAPs were annotated with biological process, 945 DAPs with molecular functions, and 2665 DAPs with cellular component compared to control. In the biological process, the most enriched categories were cellular process and metabolic process. In the molecular function, the most enrichment occurred in relation to binding and catalytic activity. In the cellular component, the most enrichment occurred in relation to cell and cell part (Figure 2). Metabolism pathway analysis To further understand molecular mechanisms potentially associated with haustorial developmental in T. chinense , DAPs were subjected to the KEGG pathway database. During the early developmental stage (FB), the proteins with increased abundance were mainly involved in pathways related to metabolic pathways (108 DAPs), photosynthesis (20 DAPs), and carbon fixation in photosynthetic organisms (12 DAPs). the proteins with increased abundance were involved in only three pathways, namely ribosome (52 DAPs), systemic lupus erythematosus (3 DAPs), and flavonoid biosynthesis (3 DAPs). During the later developmental stage (FD), the proteins with increased abundance were mainly involved in pathways related to metabolic pathways (158 DAPs), photosynthesis (23 DAPs), glyoxylate and dicarboxylate metabolism (15 DAPs), and phenylpropanoid biosynthesis (14 DAPs). In addition, the proteins whose abundance decreased were related to also only three pathways, which are ribosome (67 DAPs), systemic lupus erythematosus (4 DAPs), and alcoholism (4 DAPs) (Figure 3). DAPs involved in phytohormone signaling pathway Based on the proteomic analysis, the levels of 19 proteins associated with phytohormone signaling pathway were altered, including auxin-binding protein, mitogen-activated protein kinase, ABC transporter protein, and gibberellin-regulated protein and so on (Table S2). We found that the majority of DAPs related to auxin signaling pathways were significantly increased during the haustorial developmental in T. chinense , with a highest ratio of 2.34 for enhancer of mRNA-decapping protein 4-like (Table S2). Only glutaredoxin was significantly down regulated with a ratio of 0.47 and 0.37, respectively. Furthermore, all four proteins associated with abscisic acid signaling pathway were significantly up regulated with a ratio that ranged from 1.57 to 2.58. In addition to, three DAPs associated with gibberellin signaling pathway abundance was marked down regulated, with a lowest ratio of 0.09 being observed for GAST-like protein after 10 days of haustorial developmental. Phytohormone concentrations To elucidate the correspondence between the level of hormone and abundance of proteins, the IAA, ABA, and GA contents were analyzed by LC–MS. The accumulation of IAA was slightly decreased after 10 days of haustorial developmental. However, content of IAA was significantly increased after 20 days of haustorial developmental compared to the CK and FB. In contrast, the levels of ABA gradually decreased with the haustorial developmental. Interestingly, GA3 contents was initially up regulated, but then down regulated, reaching its highest level at 10 days (Figure 4). Transcriptional expression analysis by qRT-PCR To confirm the correspondence between the transcript level of mRNA and abundance of protein, transcriptional analysis of 8 DAPs was analyzed by qRT-PCR. The results showed that expressions of these gene were similar to those the abundance of their corresponding protein. The results indicate that the method used to confirm DAPs in this experiment is feasible (Figure 5). Discussion Branches and leaves of T. chinensis are widely used in traditional Chinese medicines for treating rheumatism, hypertension, and obesity as well as for preventing miscarriages ( 24 ). The haustorium is an essential parasitic organ used by T. chinensis to penetrate host tissues. However, the molecular mechanism of T. chinensis haustorium developmental is still unclear. To gain insights on protein changes in the developmental reprogramming of haustorial formation in T. chinensis , a comparative proteomic approach based on iTRAQ strategy was undertaken to study detailed proteome changes during its development. A total of 563 and 785 DAPs were successfully identified in in the early/later developmental stage, respectively. These DAPs were functionally classified according to their roles and were enriched in metabolic pathways, ribosome, phenylpropanoid biosynthesis and phytohormone signaling pathway. Our results provide information for the further identification and functional study of proteins in T. chinensis . Some studies have uncovered metabolic pathways have important function in haustorial development. Ichihashi et al proposed that the accumulation of very long chain fatty acid (VLCFAs) involved in the developmental reprogramming of Thesium chinense haustorial formation in natural environment ( 25 ). Besides, some metabolites have been demonstrated response to haustorial development. For example, lignin-related compounds induced haustoria in P. japonicum and S. hermonthica with different specificities. High concentrations of lignin polymers induced haustorium formation. Treatment with laccase, a lignin degradation enzyme, promoted haustorium formation at low concentrations ( 26 ). In our study, 15 up regulated DAPs involved in phenylpropanoid biosynthesis were identified. Phenylpropanoid metabolism is one of the most important metabolisms in plants, contributing to plant development ( 27 ). The biosynthetic pathways leading to lignin is known to be originated from the general phenylpropanoid pathway ( 28 ). Together with these results collectively imply that the accumulation of lignin, leading to the T. chinensis haustorium formation, was regulated key proteins belong to phenylpropanoid metabolism pathway. Phytohormones, such as IAA, GAs, ABA, play crucial roles in regulating plant growth and development and responding to various stresses as previously reported ( 29 , 30 ). Auxin was demonstrated that involved in the formation of cluster-roots (CR) and adventitious roots (AR) ( 31 ). Genes related to auxin singling pathway were significantly enriched in the parasitic plant T. chinense and C. australis. ( 32 ). In the meantime, genes involved in polar auxin transport were also enriched haustorial and prehaustorial stages compared to reference tissues, stems and seedlings of dodder. ( 33 ). Polar auxin transport promotes the formation of local auxin maxima and gradients within tissues and further results in patterns of cell division and differentiation in the root meristem ( 34 ). And the accumulation of auxin level was also observed in the haustoria initiation in sandalwood ( 15 ). Our result demonstrated that content of auxin was significantly increased after 20 days of haustorial developmental, which was consistent with the protein related to auxin abundance. GA3 was also considered as an important regulatory factor in haustorial development of parasitic plants, which may originated from effects of GA3 on xylem formation and elongation ( 35 ). Endogenous GA3 was involved in tracheary element differentiation was reported in Zinnia elegans xylogenic culture ( 36 ). In parasite–host associations, ABA is considered as a root-derived signaling molecule. In general, ABA levels in parasite roots were also higher than in the host root, which to keep stomata closed and the hydraulic conductivity of roots response to higher transpiration ( 12 ). However, our study suggested that the levels of ABA gradually decreased with the haustorial developmental. A more precise mechanism of the ABA function still need further investigation. In the meantime, we observed that the protein abundance related to ABA was significantly up regulated. We speculate these proteins may as negative regulator to control ABA biosynthesis. All these results imply that endogenous auxin, GAs, and ABA may function as an important regulatory factor during the haustorial developmental in T. chinense . Conclusions In conclusion, we studied the protein and phytohormones profiles of the loranthus haustoria development. Some crucial proteins involved in haustorial developmental were successfully identified in this work. More importantly, the findings of this study will improve our understanding of parasitism and contribute to the breeding program of loranthus. Abbreviations DAPs: differentially abundant proteins; CK: fresh seeds; FB: baby haustoria; FD: adult haustoria; qRT-PCR: quantitative real-time PCR; DMBQ: 2,6-dimethoxy-p-benzoquinone; HIF: haustorium-inducing factor; CKs: cytokinins; Z: zeatin; ZR: zeatin riboside; ZN: zeatin nucleotide; ABA: abscisic acid; GA: gibberellin; TCA: trichloroacetic acid; HPLC: high performance liquid chromatography; LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry; FDR: false discovery rate; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; MRM: multiple reaction monitoring; VLCFAs: very long chain fatty acid; CR: cluster-roots; AR: adventitious roots. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material The datasets used and analyzed during the current study could be available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science Foundation of China (81860672, 81960695, and 82173933), the Guangxi Natural Science Foundation, China (2017GXNSFDA198026, 2018GXNSFAA281089, and 2021GXNSFBA075037), the Guangxi Botanical Garden of Medicinal Plants Research and Innovation Team Building Project (GYCH2019008) and the scientific research funding project of Guangxi Botanical Garden of Medicinal Plants (GYJ202012). Authors’ Contributions SW, JF, and LP conceived and designed the experiments. LP, WL, and SJ performed the experiments. LP, HL, JN, LH, and HJ analyzed the data. LP, JX, and HF wrote the manuscript. LP revised the manuscript. All the authors have read and approved the final version of manuscript. Acknowledgments Not applicable. References Moghadamtousi SZ, Kamarudin MNA, Chan CK, Goh BH, Kadir HA. (2014). 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Frontiers in Plant Science . 4: 1–6. Doi: 10.3389/fpls.2013.00042 . Erb M, Meldau S, Howe GA. (2012). Role of phytohormones in insect-specific plant reactions. Trends in Plant Science . 17: 250–259. Doi: 10.1016/j.tplants.2012.01.003 . Pacurar DI, Perrone I, Bellini C. (2014). Auxin is a central player in the hormone cross-talks that control adventitious rooting. Physiologia Plantarum . 151: 83–96. Doi: 10.1111/ppl.12171 . Sun GL, Xu YX, Liu H, Sun T, Zhang JX, Hettenhausen C, Shen GJ, Qi JF, Qin Y, Li J, Wang L, Chang W, Guo ZH, Baldwin IT, Wu JQ. (2018). Large-scale gene losses underlie the genome evolution of parasitic plant Cuscuta australis . Nature Communications . 9: 2683. Doi: 10.1038/s41467-018-04721-8 . Ranjan A, Ichihashi Y, Farhi M, Zumstein K, Townsley B, David-Schwartz R, Sinha NR. (2014). De novo assembly and characterization of the transcriptome of the parasitic weed dodder identifies genes associated with plant parasitism. Plant Physiology. 166: 1186–1199. Doi: 10.1104/pp.113.234864 . Frim J. (2003). Auxin transport — shaping the plant. Current Opinion in Plant Biology . 6:7–12. Doi: 10.1016/S1369526602000031 . Eriksson ME, Israelsson M, Olsson O, Moritz T. (2000). Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length. Nature Biotechnology. 18:784–8. Doi: 10.1038/77355 Tokunaga N, Uchimura N, Sato Y. (2006). Involvement of gibberellin in tracheary element differentiation and lignification in Zinnia elegans xylogenic culture. Protoplasma . 228: 179–187. Doi: 10.1007/s00709-006-0180-4 . Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1033805","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":65271774,"identity":"be2b69c0-8cef-4cb2-adf4-d8e2bc8b935a","order_by":0,"name":"Limei Pan","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Limei","middleName":"","lastName":"Pan","suffix":""},{"id":65271775,"identity":"7516ef02-62ab-4668-a6b9-616c83a9f6cd","order_by":1,"name":"Lingyun Wan","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Wan","suffix":""},{"id":65271776,"identity":"737031a3-b955-4008-9a6f-0a3e18ab80e1","order_by":2,"name":"Lisha Song","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lisha","middleName":"","lastName":"Song","suffix":""},{"id":65271777,"identity":"e2e1e5ad-6aad-4569-b9ee-8034d31e0f2c","order_by":3,"name":"Lili He","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"He","suffix":""},{"id":65271779,"identity":"8a5105c1-1598-4d5b-93c2-2acf322db1cc","order_by":4,"name":"Ni Jiang","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ni","middleName":"","lastName":"Jiang","suffix":""},{"id":65271783,"identity":"8656cd57-770e-45f7-b067-9b99ea62fbdb","order_by":5,"name":"Hairong Long","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hairong","middleName":"","lastName":"Long","suffix":""},{"id":65271784,"identity":"adb7159a-95d0-4c5d-a0bd-fcdb2f9a0f6d","order_by":6,"name":"Juan Huo","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Huo","suffix":""},{"id":65271785,"identity":"296d56f9-68a3-4378-827a-e460d976b7c9","order_by":7,"name":"Xiaowen Ji","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaowen","middleName":"","lastName":"Ji","suffix":""},{"id":65271786,"identity":"bd883491-b086-47a8-9aa8-19cba5d6be26","order_by":8,"name":"Fengyun Hu","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fengyun","middleName":"","lastName":"Hu","suffix":""},{"id":65271787,"identity":"f4102494-8d06-4ecb-a451-e3d166a42ee4","order_by":9,"name":"Jine Fu","email":"","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jine","middleName":"","lastName":"Fu","suffix":""},{"id":65271788,"identity":"4b88aa38-acb3-43eb-8a84-d45fa0b6d6fc","order_by":10,"name":"Shugen Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFACxgZmMM3efODAhx8kaeE5lnhwZg+R9kC0SOQYH+ZgI0K5wfHm5s+FbbVy5gw5Hw4z8DDI84sdIKDlzME26Zltx40tG85uOFxgwWA4c3YCfi1mNxLbmHnbjiVuONi74fAMHoYEg9uEtNx/2PwZqKV+w2GeB4d52IjRcoOxQZq3rSbB4BgPA3Fa7M8ktknznDtguOEMmwEwkCUI+0Wy/fjjzzxldfIG9x8//vDhh408vzQBLVBwGMaQIEo5CNQRrXIUjIJRMApGIAAAHJRLiN3bVnwAAAAASUVORK5CYII=","orcid":"","institution":"Garden of Medicinal Plant","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shugen","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2021-10-30 09:59:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1033805/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1033805/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":16016947,"identity":"2636e680-099e-46e7-bdac-ebbc1e6f1c31","added_by":"auto","created_at":"2021-11-30 15:16:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253378,"visible":true,"origin":"","legend":"Identification of differentially abundant proteins (DAPs) between FB vs CK (a) and FD vs CK (b). Subcellular locations of differentially abundant proteins (DAPs) between FB vs CK (c) and FD vs CK (d).","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/37508c8f7d9afd0dccc7cf9c.jpg"},{"id":16017445,"identity":"73a4075a-6b32-4055-9b24-edb569d53bea","added_by":"auto","created_at":"2021-11-30 15:19:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":317884,"visible":true,"origin":"","legend":"Gene Ontology (GO) analysis of differentially abundant proteins (DAPs) between FB vs CK (a) and FD vs CK (b). And the proteins were annotated by biological process, cellular component, and molecular function.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/4a0b9f7f06281cff35bdbbb1.jpg"},{"id":16015687,"identity":"551cc000-5feb-4c4d-806f-0db00c1083d2","added_by":"auto","created_at":"2021-11-30 15:10:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":345096,"visible":true,"origin":"","legend":"Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially abundant proteins (DAPs) between FB vs CK (a) and FD vs CK (b).","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/9a6a21424c77d875a8635054.jpg"},{"id":16015681,"identity":"11b57efe-07eb-4776-9e32-15a3802111c5","added_by":"auto","created_at":"2021-11-30 15:10:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66286,"visible":true,"origin":"","legend":"Average phytohormone concentrations of IAA, ABA, and GA3 in FB, FD, and control plants. Different letters above the bars indicate significant differences, p \u003c 0.05.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/ff18fbfcc900fd42925944c5.jpg"},{"id":16016337,"identity":"ab59c852-29ff-4b9e-a1d8-7ed41ff8802e","added_by":"auto","created_at":"2021-11-30 15:13:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":119084,"visible":true,"origin":"","legend":"Analysis of the corresponding transcript levels of proteins by qRT-PCR.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/d73bc0eeff68251cc49cf675.jpg"},{"id":17150087,"identity":"3227bd0b-5da0-42ad-9748-829451219aea","added_by":"auto","created_at":"2022-01-10 07:14:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":793866,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/5e8dc1f7-c354-43b2-877f-bc62ca59e774.pdf"},{"id":16015682,"identity":"e7e6fe40-3c92-4539-ba81-b3efa916f1ba","added_by":"auto","created_at":"2021-11-30 15:10:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10521,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/9e61c052fb685fb55bca6229.xlsx"},{"id":16016339,"identity":"5f44d840-4f99-40c1-80d8-0531ff9aca33","added_by":"auto","created_at":"2021-11-30 15:13:43","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14824,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1033805/v1/912b063a4e11facaf8b66df9.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eComparative proteomics analysis provides new insights into the haustorium development of \u003cem\u003eTaxillus chinensis\u003c/em\u003e (DC.) Danser\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe \u003cem\u003eTaxillus chinensis\u003c/em\u003e (DC.) Danser is a member of the family Loranthaceae, which consists of approximately 73 genera and 900 species, which generally consists of aerial hemiparasitic plants (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). \u003cem\u003eT. chinense\u003c/em\u003e is widespread distributed in the southern and southwestern areas of China. \u003cem\u003eT. chinense\u003c/em\u003e is also used in Chinese traditional for many years, mainly because its stems and leaves can be used for the treatment of stroke, rheumatoid arthralgia, threatened abortion, angina pectoris, and hypertension (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Mulberry, camellia, plum, peach, litchi, carambola, longan, and other economically important plant species can were parasitized by \u003cem\u003eT. chinensis\u003c/em\u003e, which is semi-parasitic botanical medicinal materials (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eT. chinensis\u003c/em\u003e absorb nutrient compounds and water from the host plants via a unique multicellular organ termed the haustorium that aids to host attachment and invasion, although they provide their own carbohydrates through photosynthesis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn general, the initiation of haustorium development in most parasitic plants is triggered by host derived chemical signals terms haustorium-inducing factors (HIFs) after seed germination. Phenolic acids, quinones, flavonoids, and 2,6-dimethoxy-p-benzoquinone (DMBQ) that can be act as HIFs have been well documented (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, the exhaustive mechanisms about the how these compounds are released and then triggered haustorium development are still not well studied and need further research. Besides, some genes have been identified that may be involved in these processes. For example, the TvQR1 gene encoding quinone oxidoreductase is needed to trigger the development of the haustorium of the facultative parasite \u003cem\u003eTriphysaria versicolor\u003c/em\u003e (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). One possible reason for inducing the development of the haustor is that this enzyme converts quinone to semiquinone, which acts as an intermediate product during the redox cycle. Semiquinone itself or the redox cycle may be involved in haustorium induction signal transduction pathway (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The current model proposes that the phenolic acid derived from the degradation of the host cell wall is oxidized by ROS and oxidase to generate the haustor-inducing factor quinone (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In addition, it has been recently demonstrated that the accumulation of auxin and ethylene can act as early events in haustorium development of the hemiparasitic plant \u003cem\u003eT. versicolor\u003c/em\u003e (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Some research have observed that zeatin (Z), zeatin nucleotide (ZN), zeatin riboside (ZR), abscisic acid (ABA), and cytokinins (CKs) were significantly deposited in the haustoria of the \u003cem\u003eRhinanthus\u0026ndash;Hordeum vulgare\u003c/em\u003e association (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). \u003cem\u003eSantalum album\u003c/em\u003e is a kind of invasive root hemiparasite. After being pretreated with 2~8mM GA3 for 12 h, the seeds of \u003cem\u003eSantalum album\u003c/em\u003e can germinate in vitro on Murashige and Skoog medium or in sand without HIFs trigger within one month (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). All these results suggest that the plant hormones may play a crucial role in controlling haustorium development.\u003c/p\u003e \u003cp\u003eWith the rapid development in mass spectrometry (MS), next-generation sequencing, and microarray technologies, the researchers has enabled relatively high-throughput analyses of transcriptomes, proteomes, and metabolomes and obtained large-scale snapshot information on transcripts, proteins, and metabolites. The changes in the transcriptome of haustorial development in \u003cem\u003eT. chinense\u003c/em\u003e has been investigated and has shown the possible mechanism of \u003cem\u003eT. chinense\u003c/em\u003e haustorial development (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). But study of biological processes at the protein level provides more realistic information compared to the transcript level. This is because proteins have biological functions, and the post-translational process will lead to the production of different protein subtypes (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, few studies have performed proteomic analysis of haustorial development in \u003cem\u003eT. chinense\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fresh seeds were collected from \u003cem\u003eTaxillus chinensis\u0026nbsp;\u003c/em\u003e(DC.) Danser which parasitized in the mulberry trees that were planted in the experimental field of Guangxi Botanical Garden of Medicinal Plants in China. Then, the seeds were peeled, washed with sterile water, placed on a germination dish, and incubated under the controlled environment (25\u0026deg;C, 80% relative humidity, 10 h:14 h light: dark, 2000 Lx), as previously described (2). The fresh seeds were collected as control (CK). The seeds with protruding seed-type radicle and tiny suction device were collected after ten days of incubation (FB). And twenty days later, seeds with the loranthus haustoria and true leaves were used for the experiments (FD). Then, the samples were immediately frozen in liquid nitrogen, and stored at -80\u0026deg;C until use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein was extracted as described in a previous study (18). Briefly, the seeds samples were frozen in liquid nitrogen and pulverized. The samples were mixed with 5 volumes of chilled acetone containing 10% (v/v) trichloroacetic acid (TCA) and incubated at -20℃ overnight. After centrifugation at 6,000 g and 40 min, the supernatant was discarded. The precipitant was washed three times with pre-cooling acetone and was air-dried. The pellet was dissolved in Lysis buffer (8 M urea,120 mM NaCl, 10 mM EDTA, 1% Triton X100 and 1% PMSF dissolved in 50 Mm Tris-HCl PH 8.0). Finally, the supernatant was filtered through 0.22-mm filters after centrifugation at 13,000 g for 10 min at 4 ℃. The concentration of the protein was quantified with the BCA Protein Assay Kit (Bio-Rad, USA) and the quality of the protein sample was measured by SDS-PAGE, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein digestion and iTRAQ labeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each sample, 100 \u0026micro;g protein was used for digestion and iTRAQ labeling. Protein were reduced with 10 mM DTT at 37 ℃ for 60 min and then alkylated with 55 mM iodoacetamide (IAM) at room temperature for 30 min in darkness. The urea concentration of protein sample was diluted less than 2 M by adding 100 mM TEAB. Then trypsin was added to the protein pool of each sample with the ratio of protein: trypsin=50:1(mass ratio) at 37℃ overnight and 100: 1 for a second digestion for 4 h. After trypsin digestion, peptide was desalted with the Strata X SPE column and vacuum-dried. Peptide was reconstituted in 20 \u0026mu;L 500 mM TEAB and labeled according to the manufacturer\u0026rsquo;s protocol. Briefly, dissolutioned the peptide solution in 50 \u0026mu;L isopropanol and added one unit of iTRAQ reagent, incubated for 2 h at room temperature, then pooled and dried by vacuum centrifugation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh Performance Liquid Chromatography (HPLC)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Fractionation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe peptide was reconstituted with HPLC solution A (2% ACN, PH 10.0) and fractionated into fractions by high PH reverse-phase HPLC using Waters Bridge Peptide BEHC18 (130 \u0026Aring;, 3.5 \u0026mu;m, 4.6*250 mm). Briefly, peptides were first separated with a gradient of 2% to 98% acetonitrile (pH 10) into 72 fractions. The wavelength 250 nm is used for detection of peptides. Then, the peptides were combined into 18 fractions and dried by vacuum centrifugation. The peptide fractions were desalted using Ziptip C18(Millipore, Billerica, MA) according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThen the experiment was performed by NanoLC 1000 LC-MS/MS using a Proxeon EASY-nLC 1000 coupled to Thermo Fisher Q Exactive. Resuspended the disgestion fractions using 0.1% formic acid and loaded onto a reversed-phase-column (Acclaim PepMap\u0026reg;100 C18, 3\u0026mu;m, 100\u0026Aring;, 75\u0026mu;m\u0026times; 2cm) at a rate of 5 \u0026mu;L/min in 100% solvent A（0.1M acetic acid in water）. Next, peptides eluted from the trap column were loaded onto a reversed-phase analytical column (Acclaim PepMap\u0026reg;RSLC C18, 2\u0026mu;m, 100\u0026Aring;, 50\u0026mu;m\u0026times; 15cm). The gradient was comprised of an increase from 15% to 35% solvent B (0.1% FA in 98% ACN) over 45 min, 35% to 98% solvent B during 5 min and keep in 98% in 5 min at a constant flow rate of 300 nl/min on an EASY-nLC1000 system. The eluent was sprayed via NSI source at the 2.0 kv electrospray voltage and then analyzed by tandem mass spectrometry (MS/MS) in Q Exactive. A data-depentent mode in the scan range of 350-2000 m/z was carried out for the mass spectrometry analyses, and the survey scans were captured at a mass resolution of 17500 by the Orbitrap analyzer. In the linear ion trap, 15 of the most intense precursor ions were selected for subsequent decision tree-based ion trap HCD fragmentation at the normalized collision energy of 32% in the MS survey scan with 10.0s dynamic exclusion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRaw data processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data files were searched against the transcriptome database using Swquest software integration in Proteome Discoverer (version 1.3, Thermo Scientific). The search parameters used as follows: Carbamidomethylation (C) was set as fixed modifications; and oxidation (M), acetylation in N-Term were set as variable modification. Trypsin was chosen as enzyme and two missed cleavages were allowed. Mass tolerance of precursor ions was 20 ppm, and a fragment ion tolerance was 0.05 Da, resulting in 1% false discovery rate (FDR). Differentially abundant proteins (DAPs) were identified based on the following criteria: P-values smaller than 0.05 and a mean relative abundance\u0026gt;1.2 or\u0026lt;0.83.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the functional characterization of differentially abundant proteins (DAPs), proteins were mapped with Gene Ontology (GO) annotation base on UniProt-GOA database (\u003ca href=\"http://www.http:/www.ebi.ac.uk/GOA\"\u003ewww.http://www.ebi.ac.uk/GOA\u003c/a\u003e) (19). All proteins were grouped into three major categories: biological processes, cellular components, and molecular functions.\u0026nbsp;The metabolic pathway analyses of DAPs were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (\u003ca href=\"http://www.genome.jp/kegg/\"\u003ehttp://www.genome.jp/kegg/\u003c/a\u003e) (20).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhytohormone analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhytohormone indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid (ABA) were quantified based on a liquid chromatography and mass spectrometry system (LC-MS, 1120-6460, Agilent, USA). Phytohormone extraction was performed from loranthus seeds based on the previous method (21). The phytohormone were separated by C18 column (Hypersil Gold, 100 mm\u0026thinsp;\u0026times;\u0026thinsp;2.1 mm, 1.9 \u0026mu;m, Thermo Fisher Scientific) at flow rate of 0.3 mL/min with a 17 min gradient elution. For Multiple Reaction Monitoring (MRM), the phytohormone were analyzed with negative mode by ESI ion source.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from each sample using TRIzol reagent (Invitrogen) according to the manufacturer\u0026apos;s protocol. FastQuant RT Kit (with gDNase, Tiangen) was used for DNA remove and cDNA synthesis. Specific primer pairs of\u0026nbsp;randomly selected 8 genes\u0026nbsp;for quantitative real-time PCR (qRT-PCR) were designed using Primer Premier 5.0 software and sequences are listed in Table S1. actin was used as reference gene (2). The procedure of qRT-PCR experiment was same as our previous study (22). 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was used to evaluate the expression levels of transcripts in each sample (23). Each transcript was performed with three biological replicates and three technical replicates.\u0026nbsp;\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eDifferential abundant proteins (DAPs) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential mechanisms involved in the haustorial developmental process in \u003cem\u003eT. chinense\u003c/em\u003e, an integrated approach involving LC-MS/MS and\u0026nbsp;iTRAQ\u0026nbsp;labeling was applied to analyze the proteomic changes. A total of 563 proteins were identified as DAPs between FB treatment and control, of which 384 were identified as upregulated and 179 were downregulated proteins, respectively. A total of 785 proteins were identified as DAPs between FD treatment and control, of which 569 and 216 were identified as upregulated and downregulated proteins, respectively (Figure 1). All the DAPs were grouped based on their subcellular localizations. For the FB treatment, 11 subcellular components were identified, including 250 chloroplast-localized proteins (44.4%), 162 cytosol- localized proteins (28.77%), 80 nuclear-localized proteins (14.21%). For the FD treatment, 16 subcellular components were identified, including 325 chloroplast-localized proteins (41.4%), 2433 cytosol- localized proteins (30.96%), 106 nuclear-localized proteins (13.5%) (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional categorization analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biological functions of the DAPs could also be identified by their GO annotations. The DAPs were classified into the three main GO categories (cellular component, biological process, and molecular function). In the early developmental stage (FB), 1546 DAPs (some proteins have more than one GO annotation) were annotated with biological process, 653 DAPs with molecular functions, and 1885 DAPs with cellular component compared to control. In the later developmental stage (FD), 2126 DAPs were annotated with biological process, 945 DAPs with molecular functions, and 2665 DAPs with cellular component compared to control. In the biological process, the most enriched categories were cellular process and metabolic process. In the molecular function, the most enrichment occurred in relation to binding and catalytic activity. In the cellular component, the most enrichment occurred in relation to cell and cell part (Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolism pathway analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further understand molecular mechanisms potentially associated with haustorial developmental in \u003cem\u003eT. chinense\u003c/em\u003e, DAPs were subjected to the KEGG pathway database. During the early developmental stage (FB), the proteins with increased abundance were mainly involved in pathways related to metabolic pathways (108 DAPs), photosynthesis (20 DAPs), and carbon fixation in photosynthetic organisms (12 DAPs). the proteins with increased abundance were involved in only three pathways, namely ribosome (52 DAPs), systemic lupus erythematosus (3 DAPs), and flavonoid biosynthesis (3 DAPs). During the later developmental stage (FD), the proteins with increased abundance were mainly involved in pathways related to metabolic pathways (158 DAPs), photosynthesis (23 DAPs), glyoxylate and dicarboxylate metabolism (15 DAPs), and phenylpropanoid biosynthesis (14 DAPs). In addition, the proteins whose abundance decreased were related to also only three pathways, which are ribosome (67 DAPs), systemic lupus erythematosus (4 DAPs), and alcoholism (4 DAPs) (Figure 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDAPs involved in phytohormone signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the proteomic analysis, the levels of 19 proteins associated with phytohormone signaling pathway were altered, including auxin-binding protein, mitogen-activated protein kinase, ABC transporter protein, and gibberellin-regulated protein and so on (Table S2). We found that the majority of DAPs related to auxin signaling pathways were significantly increased during the haustorial developmental in \u003cem\u003eT. chinense\u003c/em\u003e, with a highest ratio of 2.34 for enhancer of mRNA-decapping protein 4-like (Table S2). Only glutaredoxin was significantly down regulated with a ratio of 0.47 and 0.37, respectively. Furthermore, all four proteins associated with abscisic acid signaling pathway were significantly up regulated with a ratio that ranged from 1.57 to 2.58. In addition to, three DAPs associated with gibberellin signaling pathway abundance was marked down regulated, with a lowest ratio of 0.09 being observed for GAST-like protein after 10 days of haustorial developmental.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhytohormone\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the correspondence between the level of hormone and abundance of proteins, the IAA, ABA, and GA contents were analyzed by LC\u0026ndash;MS. The\u0026nbsp;accumulation\u0026nbsp;of IAA was slightly decreased after 10 days of haustorial developmental. However, content of IAA was significantly increased after 20 days of haustorial developmental compared to the CK and FB. In contrast, the levels of ABA gradually decreased with the haustorial developmental. Interestingly, GA3 contents was initially up regulated, but then down regulated, reaching its highest level at 10 days (Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptional expression analysis by qRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the correspondence between the transcript level of mRNA and abundance of protein, transcriptional analysis of 8 DAPs was analyzed by qRT-PCR. The results showed that expressions of these gene were similar to those the abundance of their corresponding protein. The results indicate that the method used to confirm DAPs in this experiment is feasible (Figure 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBranches and leaves of \u003cem\u003eT. chinensis\u003c/em\u003e are widely used in traditional Chinese medicines for treating rheumatism, hypertension, and obesity as well as for preventing miscarriages (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The haustorium is an essential parasitic organ used by \u003cem\u003eT. chinensis\u003c/em\u003e to penetrate host tissues. However, the molecular mechanism of \u003cem\u003eT. chinensis\u003c/em\u003e haustorium developmental is still unclear. To gain insights on protein changes in the developmental reprogramming of haustorial formation in \u003cem\u003eT. chinensis\u003c/em\u003e, a comparative proteomic approach based on iTRAQ strategy was undertaken to study detailed proteome changes during its development. A total of 563 and 785 DAPs were successfully identified in in the early/later developmental stage, respectively. These DAPs were functionally classified according to their roles and were enriched in metabolic pathways, ribosome, phenylpropanoid biosynthesis and phytohormone signaling pathway. Our results provide information for the further identification and functional study of proteins in \u003cem\u003eT. chinensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSome studies have uncovered metabolic pathways have important function in haustorial development. Ichihashi et al proposed that the accumulation of very long chain fatty acid (VLCFAs) involved in the developmental reprogramming of \u003cem\u003eThesium chinense\u003c/em\u003e haustorial formation in natural environment (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Besides, some metabolites have been demonstrated response to haustorial development. For example, lignin-related compounds induced haustoria in \u003cem\u003eP. japonicum\u003c/em\u003e and \u003cem\u003eS. hermonthica\u003c/em\u003e with different specificities. High concentrations of lignin polymers induced haustorium formation. Treatment with laccase, a lignin degradation enzyme, promoted haustorium formation at low concentrations (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In our study, 15 up regulated DAPs involved in phenylpropanoid biosynthesis were identified. Phenylpropanoid metabolism is one of the most important metabolisms in plants, contributing to plant development (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The biosynthetic pathways leading to lignin is known to be originated from the general phenylpropanoid pathway (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Together with these results collectively imply that the accumulation of lignin, leading to the \u003cem\u003eT. chinensis\u003c/em\u003e haustorium formation, was regulated key proteins belong to phenylpropanoid metabolism pathway.\u003c/p\u003e \u003cp\u003ePhytohormones, such as IAA, GAs, ABA, play crucial roles in regulating plant growth and development and responding to various stresses as previously reported (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Auxin was demonstrated that involved in the formation of cluster-roots (CR) and adventitious roots (AR) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Genes related to auxin singling pathway were significantly enriched in the parasitic plant \u003cem\u003eT. chinense\u003c/em\u003e and \u003cem\u003eC. australis.\u003c/em\u003e (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In the meantime, genes involved in polar auxin transport were also enriched haustorial and prehaustorial stages compared to reference tissues, stems and seedlings of dodder. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Polar auxin transport promotes the formation of local auxin maxima and gradients within tissues and further results in patterns of cell division and differentiation in the root meristem (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). And the accumulation of auxin level was also observed in the haustoria initiation in sandalwood (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Our result demonstrated that content of auxin was significantly increased after 20 days of haustorial developmental, which was consistent with the protein related to auxin abundance. GA3 was also considered as an important regulatory factor in haustorial development of parasitic plants, which may originated from effects of GA3 on xylem formation and elongation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Endogenous GA3 was involved in tracheary element differentiation was reported in Zinnia elegans xylogenic culture (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In parasite\u0026ndash;host associations, ABA is considered as a root-derived signaling molecule. In general, ABA levels in parasite roots were also higher than in the host root, which to keep stomata closed and the hydraulic conductivity of roots response to higher transpiration (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). However, our study suggested that the levels of ABA gradually decreased with the haustorial developmental. A more precise mechanism of the ABA function still need further investigation. In the meantime, we observed that the protein abundance related to ABA was significantly up regulated. We speculate these proteins may as negative regulator to control ABA biosynthesis. All these results imply that endogenous auxin, GAs, and ABA may function as an important regulatory factor during the haustorial developmental in \u003cem\u003eT. chinense\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we studied the protein and phytohormones profiles of the loranthus haustoria development. Some crucial proteins involved in haustorial developmental were successfully identified in this work. More importantly, the findings of this study will improve our understanding of parasitism and contribute to the breeding program of loranthus.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDAPs: differentially abundant proteins; CK: fresh seeds; FB: baby haustoria; FD: adult haustoria; qRT-PCR: quantitative real-time PCR; DMBQ: 2,6-dimethoxy-p-benzoquinone; HIF: haustorium-inducing factor; CKs: cytokinins; Z: zeatin; ZR: zeatin riboside; ZN: zeatin nucleotide; ABA: abscisic acid; GA: gibberellin; TCA: trichloroacetic acid; HPLC: high performance liquid chromatography; LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry; FDR: false discovery rate; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; MRM: multiple reaction monitoring; VLCFAs: very long chain fatty acid; CR: cluster-roots; AR: adventitious roots. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study could be available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81860672, 81960695, and 82173933), the Guangxi Natural Science Foundation, China (2017GXNSFDA198026, 2018GXNSFAA281089, and 2021GXNSFBA075037), the Guangxi Botanical Garden of Medicinal Plants Research and Innovation Team Building Project (GYCH2019008) and the scientific research funding project of Guangxi Botanical Garden of Medicinal Plants (GYJ202012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSW, JF, and LP conceived and designed the experiments. LP, WL, and SJ performed the experiments. LP, HL, JN, LH, and HJ analyzed the data. LP, JX, and HF wrote the manuscript. LP revised the manuscript. All the authors have read and approved the final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoghadamtousi SZ, Kamarudin MNA, Chan CK, Goh BH, Kadir HA. (2014). Phytochemistry and biology of \u003cem\u003eLoranthus parasiticus\u003c/em\u003e Merr, a commonly used herbal medicine. \u003cem\u003eAmerican Journal of Chinese Medicine.\u003c/em\u003e 42:23\u0026ndash;35. Doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1142/S0192415X14500025\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei SG, Wan LY, He LL, Wei Y, Long HR, Ji XW, Fu JE, Pan LM. (2020). 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Doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00709-006-0180-4\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"proteomics, T. chinensis, haustorium, phytohormone","lastPublishedDoi":"10.21203/rs.3.rs-1033805/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1033805/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLoranthus (\u003cem\u003eTaxillus chinensis\u003c/em\u003e) is an important medicinal and parasitic plant that attacks other plants for living. To reveal the mechanisms of haustorium development, we employed an iTRAQ proteomics-based approach to identify differentially abundant proteins (DAPs) of fresh seeds (CK), baby (FB), and adult haustoria (FD).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 563 and 785 DAPs were successfully quantified in the early/later developmental stage, respectively. Pathway enrichment analysis indicated that the DAPs mainly associated with metabolic pathways, ribosome, phenylpropanoid biosynthesis and photosynthesis. In the meantime, DAPs associated with phytohormone signaling pathway changed markedly. Furthermore, we evaluated the contents change of phytohormone during the haustoria development. These results indicated that phytohormone is very important for haustorium development. qRT-PCR validation showed that the mRNA expression levels were consistent with the protein variation, suggesting that our result were reliable.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTo the best of our knowledge, this is the first haustoria proteomes of loranthus, and our findings will improve our understanding of the molecular mechanism of haustoria development.\u003c/p\u003e","manuscriptTitle":"Comparative proteomics analysis provides new insights into the haustorium development of Taxillus chinensis (DC.) 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