ABSTRACT Arbuscular mycorrhiza fungi (AMF) can colonize the roots of _Amorpha fruticosa_, a perennial leguminous woody shrub and form arbuscular mycorrhiza (AM). AMF have significant

promoting effects on _A. fruticosa_ growth as the intensity of fungal colonization increases. Taking AMF-_A. fruticosa_ symbionts as the experimental material, gel-free isobaric tags for

relative and absolute quantification (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to investigate the expression of _A. fruticosa_

mycorrhizal proteins at the maturation stage. A total of 3,473 proteins were identified, of which 77 showed dramatic changes in their root expression levels; 33 increased and 44 decreased.

membrane and transport-related (4%), cellular structure-related (2.5%), signaling transduction-related (11%) and unknown proteins (5%). The results of the study provide a foundation for

further investigation of the metabolic characteristics and molecular mechanisms of AM. SIMILAR CONTENT BEING VIEWED BY OTHERS METABOLOMICS AND TRANSCRIPTOMICS TO DECIPHER MOLECULAR

MECHANISMS UNDERLYING ECTOMYCORRHIZAL ROOT COLONIZATION OF AN OAK TREE Article Open access 21 April 2021 TISSUE-SPECIFIC SIGNATURES OF METABOLITES AND PROTEINS IN ASPARAGUS ROOTS AND

EXUDATES Article Open access 01 April 2021 COMPARATIVE SINGLE-NUCLEUS RNA-SEQ ANALYSIS REVEALED LOCALIZED AND CELL TYPE-SPECIFIC PATHWAYS GOVERNING ROOT-MICROBIOME INTERACTIONS Article Open

access 03 April 2025 INTRODUCTION Arbuscular mycorrhiza (AM), representing the most widely distributed mutualistic root symbiosis in nature, are the result of long-term evolution between

plant and soil fungi. AM fungi (AMF) are obligate symbionts and are recalcitrant to pure culture on synthetic media; they grow only in living plants. Their hereditary variability and

heterogeneous characteristics are essential for colonizing a large number of potential host plants. However, different host plants may simultaneously induce the expression of different

symbiosis-related genes. AMF are some of the most widespread microorganisms and they can form symbionts with more than two-thirds of the vascular plants in natural or artificial ecosystems.

These plants include important agricultural species, such as wheat, rice and the model plant _Populus trichocarpa_1,2. The foundation of mycorrhizal symbiosis is the ability of AMF, using

their multicore hyphae, to provide nutrients (especially phosphorous) to host plants that have long-distance illiquidity3. New physiological and molecular evidence has shown that, for

phosphorus, the mycorrhizal pathway (MP) is operational regardless of plant growth responses. Meanwhile, the contribution of the direct pathway (DP) is decreased, which results in a greater

dependence of host plants on the nutrients that AMF provide4. AMF can utilize only simple carbon and nitrogen sources from their hosts to complete their life cycles. This may be due to the

loss of some enzyme-encoding genes and to macromolecular synthesis defects that have arisen during the long-term evolution of symbiosis with plants5,6. AM play a significant role in

promoting the growth of host plants and researchers have increased their efforts to study the interactions between AMF and host plants. Recorbet and colleagues have compared the root

proteome responses of _Medicago truncatula_ upon colonization with two AM fungi, i.e., _Glomus mosseae_ (GM) and _G. intraradices_, using two-dimensional electrophoresis (2-DE)7. They found

42 symbiosis proteins; of these, 32 could be confidently identified and retrieved following MS/MS and matching with a database encompassing 21 fungal proteins. To test the mechanisms by

which shoots of Cd-treated mycorrhizal plants avoid metal toxicity, Aloui has performed a 2-DE/MALDI-TOF-based comparative proteomic analysis of the _M. truncatula_ shoot responses upon

mycorrhization and Cd exposure8. finding that Cd triggers an opposite response than mycorrhization, which is coupled with an increase in molecular chaperones in the shoots of mycorrhizal

plants relative to those that are metal-free. Wang has studied the dynamic changes in maize leaf protein expression profiles under AMF colonization9. In that study, the differentially

expressed proteins in maize leaves were separated by 2-DE and the results reveal 21 differentially expressed gel spots in maize leaves. Among them, 8 proteins were successfully identified.

With the development of molecular biology techniques, quantitative analysis of the differences in protein expression profiles during the colonization process of pathogenic or symbiotic

microorganisms has become possible; these techniques have played a critical role in analyzing pathogenic mechanisms. Isobaric tags for relative and absolute quantification (iTRAQ) represents

one of the new and powerful techniques for simultaneous analysis of multiple samples and provide relative quantification of hundreds of proteins. iTRAQ reagents produce high-quality,

reproducible results from complex samples and iTRAQ has thus become widely used. However, iTRAQ-based studies on the symbiotic mechanisms of AMF and host plants have rarely been reported.

Our group has studied the symbiotic relationship between plants and fungi at the mRNA level, Zhang xingxing identified 30 symbiosis-related genes expressed in _Amorpha fruticosa_ roots

colonized by GM at different stages by using mRNA differential-display PCR (DDRT-PCR). The expressed genes were confirmed by reverse Northern blotting. Eleven fragments were sequenced and

putatively identified by homologous alignment and these genes were found to relate to defense and signal transduction10. Kong xiangshi also has found 47 symbiosis-related unigenes during AMF

treatment by using suppression subtractive hybridization (SSH) and subsequent Gene Ontology (GO) database, BLAST annotation and literature searches to categorize each of the identified

genes. Among the expressed genes, those related to plant metabolism and stress and defense show important roles during the symbiotic process of AMF-_A. fruticosa_11. Based on our previous

work, we have continued to use _A. fruticosa_, a perennial leguminous woody shrub plant, as a host. Using AMF-_A. fruticosa_ symbionts as the experimental materials, we used iTRAQ combined

with 2-D LC-MS/MS to investigate the expression of _A. fruticosa_ mycorrhizal proteins at the maturation stage. The results of the study provide a theoretical basis for the further analysis

of the metabolic characteristics and molecular mechanisms of symbiosis between AMF and _A. fruticosa_. RESULTS AMF COLONIZATION The colonization percentage of _A. fruticosa_ roots is shown

in Fig. 1. At day 5, the roots were relatively small and only a few hyphae were detected. Most of the spores were not in contact with the host roots and were mainly in their vegetative

growth stage12,13. The infection rate began to increase at day 10 and the growth status of GM-inoculated seedlings was significantly better than that of non-inoculated plants. The

colonization percentage increased rapidly at day 15. At that time, there was a large quantity of mycelium-infected roots that increased over time. The colonization percentage reached its

peak at the vigorous phase (i.e., 30 days) and a large number of vesicles and arbuscules were observed within the roots. At 40 days, we observed that the hyphae had extended from the plant

root surface and had infected neighboring roots. No colonization by AM was observed in the non-inoculated plants, because the mixed soil used for culture had been autoclaved thoroughly.

IDENTIFICATION OF SYMBIOSIS-RELATED PROTEINS USING ITRAQ LC-MS/MS The roots of _A. fruticosa_ changed their protein levels when colonized by GM and mutualistic symbionts formed. Using an

iTRAQ approach, 86 differentially expressed symbiosis-related proteins were successfully identified. Among them, 77 were plant proteins, with 33 proteins showing increases and 44 showing

decreases (Table 1) and 9 were fungal proteins (Table 2). More detailed information in supplementary information. AMF proteins play an import role in symbiotic systems, but they show high

expression in only the AMF themselves. The low overall concentration of AMF proteins and the limitations of the technology, resulted in few AMF proteins being detected14. CLASSIFICATION OF

SYMBIOSIS-RELATED PROTEINS The GO database, BLAST annotations and information reported in the literature were used to categorize each of the identified proteins11. The 77 differentially

expressed proteins in _A. fruticosa_ were categorized into different functional classes and assigned to 11 categories. The functional categories are shown in Fig. 2; they include

metabolism-related (32%), protein folding and degradation-related (22%), energy-related (10%), protein synthesis-related (8%), stress and defense-related (24%), transcription-related (6%),

membrane and transport-related (4%), cellular structure-related (2.5%), signaling transduction-related (11%) and unknown (5%). Among these classes, proteins related to plant metabolism,

protein folding and degradation and energy (totaling 64% of the identified proteins) play important roles during the symbiotic process of AMF-_A. fruticosa._ DISCUSSION Previous studies have

demonstrated that colonization is a multi-step, genetically regulated process under the control of specific loci15,16. AMF interact with host plants as cell walls, cell membranes and

cellular components undergo dramatic changes17. During the colonization process, functional proteins are induced to express and regulate this process, ultimately forming stable mutualistic

symbionts. SIGNALING-RELATED PROTEINS Mutualistic symbionts are the result of a mutual recognition and interaction process between AMF and plant signaling molecules. During the colonization

process, signal transduction occurs so that the symbiotic partners recognize each other and the host plants decrease their defense responses. At the same time, AMF are prepared to colonize

and to form appressoria and, subsequently, to form arbuscules, vesicles and spores. Because of their mutual nutritional relationship, a real-time dynamic signal dialogue between fungi and

host plants is continually present. In this study, we found that the protein levels of Rho GDP-dissociation inhibitor 1 and somatic embryogenesis receptor-like kinase were significantly

increased in the symbiotic roots. Rho GDP-dissociation inhibitor 1, a regulator of Rho GTPase, regulates the balance of Rho GTPase bound to GTP or GDP. There are 2 conformational states of

Rho GTPase: the GTP-bound ‘active’ state and the GDP-bound ‘inactive’ state, in which GTP has been hydrolyzed to GDP18. As a member of the subfamily of small G proteins, Rho GTPase regulates

a number of important signal-transduction pathways in eukaryotic cells. Rho GTPase, called Rop (Rho-related GTPase) in plants, has different isomers in animals and fungi19. Rho GTPases are

widely distributed in plants and the corresponding genes in _Arabidopsis_, maize, barley, rice, peas and alfalfa have been cloned20,21,22. Rho GTPases participate in the regulation of a

variety of cellular processes, e.g., gene expression, cell wall synthesis, H2O2 production, actin rearrangement processes, signal transduction pathways of MAP kinase23,24 and cytoskeletal

assembly and reassembly, to produce a variety of cellular responses. As a regulatory factor, RhoGDI1 was significantly increased in _A. fruticosa_ AM. Clearly, this protein is closely

related to signal transduction between _A. fruticosa_ and GM. Multiple somatic embryogenesis receptor-like kinases (SERKs) have been defined, including the leucine-rich repeat receptor-like

kinase (LRR-RLK) subfamily members and a family of transmembrane signal-transduction proteins25. They are characterized by a predicted signal sequence, a single transmembrane region and a

cytoplasmic kinase domain. These features suggest that some SERK family protein kinases may play pivotal roles in communication between cells and the environment or in cell-cell

interactions. Currently, SERK genes have been cloned from various plant species. The AtSERK3 gene participates in the brassinolide (brassinosteroid, BR) signal-transduction pathway. BR is an

important hormone that regulates plant growth and development. Functional analysis has shown that the _Arabidopsis thaliana_ mutant became a dwarf when the AtSERK3 gene is knocked out26.

The overexpression of the OsSERK1 gene in rice cultivars leads to an increase in host resistance to blast fungus27; in contrast, transcripts of the lettuce LsSERK gene not only are decreased

in _in vitro_ somatic embryonic structures but also easily infect _Sclerotinia_28. Studies have also shown that the SERK gene is closely related to antibiotic stress. Plant root

colonization by AMF results in increased levels of somatic embryogenesis receptor-like kinase, which plays a major role in promoting plant growth and enhancing plant disease resistance.

STRESS AND DEFENSE-RELATED PROTEINS Inoculation with AMF has strong growth-promoting effects on _A. fruticosa_, especially at the mature stage of symbiont formation. These effects are

mediated by increased action of SERK in BR signal-transduction pathways, which have a key role in the regulation of autoimmune responses and of plant root cell elongation and division.

However, such regulation is not determined by a single factor. At an early stage of symbiosis, a weak defense response emerges when roots are stimulated by AMF colonization. Lectin plays a

crucial role in this defense response by recognizing and binding to the sugar molecules of intruders and interfering with their function on plants. Many plant lectins can bind to glucose,

mannitol, galactose or other monosaccharides and they exhibit high affinity to the oligosaccharides of alien plants. Studies have shown that lectins on leguminous tree surfaces can gather

rhizobia around the roots29. As AMF infect the roots of _A. fruticosa_, plant defense responses are initiated, resulting in agglutinin-2 accumulation. Agglutinin-2 is an important factor for

the identification of AMF, similarly to rhizobia. When _A. fruticosa_ is colonized by AMF, the abscisic acid (ABA) content increases rapidly, leading to the closing of plant stomata and

decreased transpiration; this response also activates the genes encoding soluble osmolytes, thus decreasing stress injuries and the impact of stress-induced reactive oxygen and ethylene30.

Therefore, ABA accumulation may stimulate metabolic enzymes to produce a feedback effect31. The major ABA catabolic route is decomposition via ABA 8′-hydroxylase to form phaseic acid.

Therefore, ABA 8′-hydroxylase accumulation in _A. fruticosa_ may represent a mechanism for regulating ABA levels. In multiple rice mapping populations, germin-like protein (GLP) markers have

been associated with quantitative trait loci (QTL) for resistance to rice blast pathogens. At the early stage of rice blast fungus infection or mechanical damage, some OsGLPs are

transiently induced and expressed. Varying 5′ regulatory regions and the differential expression of some protein family members between resistant and susceptible cultivars correspond with

differential hydrogen peroxide (H2O2) accumulation levels after fungal infection32. Wang discovered a new wheat germin-like protein33 that is up-regulated in both resistant and susceptible

plants. It has been speculated to be involved in wheat defense responses. GLP is significantly increased at the early stage of AMF infection in roots of _A. fruticosa_ and it may participate

in biotic stress responses. PROTEIN FOLDING AND DEGRADATION-RELATED PROTEINS During the symbiosis process, the modification and degradation of peptides and proteins are critical for

maintaining cell function. Protein disulfide isomerase, bi-ubiquitin, serine carboxypeptidase, proteasome subunit beta type-6 and subtilisin-like protease SDD1 accumulate in plant roots to

ensure proper cell function. Plants use the proteasome pathway for selective protein degradation and the proteasome plays pivotal roles in removing abnormally modified proteins and

non-targeted proteins. Interactions between bi-ubiquitin and proteasome subunit beta type-6 provide an effective way to degrade proteins. Bi-ubiquitin is highly conserved in eukaryotes and

it is covalently bound to target proteins through post-translational modification to mediate degradation. Serine carboxypeptidase (SCP), an enzyme that catalyzes the hydrolysis of proteins

in eukaryotes, has been found in rice, _Arabidopsis_ and peas. It has been shown that SCP has broad functions in plants, including protein turnover and secondary metabolism synthesis and it

plays an important role in improving plant stress resistance. Liu showed that the expression of _OsBISCPL1_ was induced by rice blast fungi and antiviral signaling molecules (salicylic acid

and jasmonic acid)34 and that overexpression of _OsBISCPL1_ could enhance disease resistance, oxidative stress tolerance and ABA sensitivity in transgenic _Arabidopsis_ plants. _OsBISCPL1_

is expressed ubiquitously and differentially in rice and it is induced by antiviral signaling molecules (BTH, JA, SA and ACC) and is up-regulated by incompatible interactions between rice

and the blast fungus. Liu has shown that the expression of the _ZmSCP_ gene in corn is up-regulated under induction by _Rhizoctonia solani_ and that the ZmSCP protein are associated with

various abiotic stresses35. The subtilisin-like protease SDD1 is a member of the processing-type proteases in eukaryotes. As a preproprotein, it can direct peptides for transport to the

cytoplasm. SDD1 is a crucial gene that regulates stomatal development and encodes a subtilisin-like serine protease. As a processive enzyme, it may activate a protein molecule or a signal

that directs receptors into contact with epidermal cells during stomatal development processes. Liang has shown that the serine protease-encoding gene _SDD1_ is widely expressed acts on the

development of stomata and is also necessary for normal root development36. Protein disulfide isomerase (PDI), a multifunctional protein, is distributed widely in eukaryotic organisms and is

involved in modifying/folding newly synthesized proteins. The catalytic thiol-disulfide exchange reaction to form disulfide is involved in many physiological processes, such as auxiliary

protein folding in the endoplasmic reticulum, reconstruction of misfolded proteins and the repair and refolding of damaged proteins under stress37. Additionally, as a chaperone, PDI can

assemble heterogeneous protein peptides and regulate disulfide bonds in an ATP-dependent manner and it may also be closely related to sugar transport, protein synthesis and other metabolic

processes in eukaryotic organisms. ENERGY-RELATED PROTEINS During the symbiosis process, dihydrolipoyl dehydrogenase, aldehyde dehydrogenase and isocitrate dehydrogenase [NAD] regulatory

subunit 1 accumulated in plant roots. AMF colonization significantly enhances the energy metabolism of plants. The Krebs cycle provides more energy than glycolysis and it is an important

pathway not only an important for sugar metabolism but also for the metabolism of lipids, proteins and nucleic acids, which are eventually oxidized to carbon dioxide and water. Isocitrate

dehydrogenase (IDH) is considered to be the rate-limiting enzyme of the Krebs cycle; it catalyzes decarboxylation to ketoglutarate while reducing NAD+ to NADH38. Therefore, the activity of

NAD-IDH has a significant impact on cellular metabolism. Isocitrate dehydrogenase [NAD] regulatory subunit 1, a regulatory factor, controls the activity of NAD-IDH and thus affects metabolic

activity. Kuhlemeier has explored the energy metabolism of tobacco pollen and has found that, in vegetative tissues39, pyruvate enters the Krebs cycle by pyruvate dehydrogenase (PDH);

however, in reproductive organs, it is converted to acetaldehyde by pyruvate decarboxylase (PDC) and then enters the Krebs cycle via aldehyde dehydrogenase (ALDH) and acetyl coenzyme A

synthetase (ACS). Thus, ALDH plays an important role in the pyruvate metabolism pathway of PDC/ALDH/ACS. Under stress conditions, plant cells quickly accumulate excessive reactive oxygen

species (ROS), which cause oxidative stress and result in the accumulation of large amounts of toxic substance and eventually in plant death40. Aldehydes are an important component of

peroxidation reaction products and they play a crucial role in the oxidation of carboxylic aldehydes, the removal of toxic aldehydes and the reduction of lipid peroxidation, thereby

improving plant tolerance41. As an important member of the pyruvate dehydrogenase family, dihydrolipoyl dehydrogenases ensure the production of oxidatively decarboxylated pyruvate CoA and

CoA then enters the Krebs cycle to produce large amounts of energy for plant growth. CELLULAR STRUCTURE-RELATED PROTEINS Dramatic changes in plant morphology and in the penetrating mycelium,

dynamic reorganization of cytoskeletal elements and organelle transformation occur when arbuscular vesicles develop42. Tubulin is an important component of the cytoskeleton and it plays an

important role in maintaining intracellular structural order and cell morphology. Meanwhile, tubulin is closely related to cellular transport, cell differentiation, cell motility, signal

recognition, cell division and other developmental activities. Mills has revealed dramatic changes in both microtubules and actin arrangement in the host cell and further studies have found

that microtubules and actin rearrangement in the host cell are necessary for expression in non-host plants43. Studies on plant tubulin have primarily been focused on annual plants, such as

_Arabidopsis_, tobacco and rice, but study of the tubulin gene in perennial trees has been rare44. MEMBRANE AND TRANSPORT-RELATED PROTEINS A K+ efflux antiporter and coatomer, which are

membrane and transport-related proteins, respectively, were found in the _A. fruticosa_ mycorrhizea. The K+ efflux antiporter is mainly responsible for maintaining the intracellular ion

balance and regulating the cells’ osmotic pressure. During AMF colonization, AMF invasion affects the ion balance of plant root cells and plants maintain the intracellular ion balance to

stimulate K+ increases. Coatomer, a coat protein, transports vesicles and vesicle-mediated non-selective transport ensures the accurate transport of proteins and lipids. METABOLISM-RELATED

PROTEINS During mycorrhizal symbiosis, increased levels of 3-oxoacyl-[acyl-carrier-protein] synthase, neutral ceramidase and caffeic acid 3-O-methyltransferase (COMT), which are

metabolism-related proteins, were observed. Lipid metabolism is one of the basic metabolic pathways in plants. The β-ketoacyl-acyl carrier protein synthase (KASI)-mediated acyl chain

extension is important in the _de novo_ synthesis of fatty acids. Ceramides, which are central molecules in the sphingolipid signaling pathway, play important roles as second messengers in

plants and participate in many significant plant signaling pathways, such as cell growth, proliferation, differentiation, senescence and apoptosis45. Neuraminidase is a key enzyme that

regulates ceramide. Neutral neuraminidase hydrolyzes ceramide to form sphingosine (ref). Liu has found that AtCER is involved in H2O2-induced oxidative stress46. During cell morphogenesis,

lignin plays an important role in the growth and development of vascular tissues and is involved in cell wall lignification, which increases the hardness or compressive strength of the cell

wall. It also promotes the formation of mechanical tissues while also having a major impact on plant lodging, disease and stress resistance47. There are 3 types of monomeric lignin

biosynthesis pathways,: the shikimate pathway, the phenylketonuria pathway and the lignin biosynthesis-specific pathway48. COMT is a key enzyme in the specific lignin pathway and is involved

in the synthesis of S-lignin49. AMF colonization enhanced the synthesis of woody _amorpha_ lignin, thus affecting the growth and development of plants. TRANSCRIPTION AND PROTEIN

SYNTHESIS-RELATED PROTEINS Ribosomal proteins are important components of the ribosome and they have important roles in translation efficiency and ribosome stability. They also participate

in important cellular processes, such as DNA repair, apoptosis and regulation of gene expression; e.g., 40S ribosomal proteins showed significant accumulation in plant roots after AMF

invasion. Nascent polypeptide-associated complex subunit alpha (NAC), which is located at the top of the newly synthesized polypeptide, can reversibly bind to eukaryotic ribosomes and guide

the correct distribution and translocation of newly synthesized polypeptides in the cell. The observed increases in 40S ribosomal protein and NAC levels, combined with folding- and

degradation-associated proteins, ensure the fast and accurate synthesis and distribution of AM symbiosis-related proteins. Transcriptional regulation is an important aspect of the regulation

of gene expression. The results show significant accumulation of histone H3 and histone H4 in the host plant roots. Nucleosomes constitute the basic unit of chromatin in eukaryotes.

Histones, which are structural proteins of chromosomes, play important roles in DNA folding and packing, protecting DNA from digestive enzymes and gene regulation, tumor formation and

apoptosis. The N-terminal amino acids of histones participate in acetylation, methylation, phosphorylation, ubiquitination and other covalent modifications. Studies have shown that histones

may change the structure of chromatin via post-translational modifications, thus modulating gene expression50. UNKNOWN PROTEINS During AMF symbiosis, the expression levels of proteins within

_A. fruticosa_ roots were changed; some proteins disappeared and new symbiosis proteins arose. The functional analysis of symbiotic proteins in _A. fruticosa_, a non-model plant, is not

difficult. Because these proteins were differentially expressed in the symbiotic system, they are targets for future studies. AM-A. FRUTICOSA MOLECULAR REGULATION MODEL By using

bioinformatics analysis, we found that mycorrhizal proteins were involved in several biological processes and cellular activities (Fig. 3) and we verified that the symbiosis formed between

AMF and _A. fruticosa_ is a uniform and harmonious result of symbiotic interactions. METHODS _G. mosseae_ (GM) was harvested from sorghum, which was supplied by the Ecology Laboratory of

Heilongjiang University, by co-culturing for longer than 40 days. Inocula contained a mixture of the rhizosphere that consisted of AM fungal spores, hyphae and mycorrhizal fragments. The

inocula contained approximately 500 spores per 20 g. SEEDLING CULTURE Seeds of _A. fruticosa_ were purchased from the Academy of Agricultural Sciences of Heilongjiang Province. _A.

fruticosa_ seeds were sterilized with 0.4% K2MnO4 for 20 min, rinsed and then covered with a layer of white gauze to keep them moist. Germination was conducted in an incubator at 30 °C for

60 h after soaking for 24 h. The growing medium was 50% peat soil, 30% vermiculite and 20% sand. It was sterilized in an autoclave at 121°C for 2 h and then air dried for 1 week before the

start of the experiments10,11. MYCORRHIZAL COLONIZATION PERCENTAGE DETERMINATION The germinated seeds were then planted in a pre-sterilized mixed matrix and grown under a 16-h photoperiod at

temperatures of 25/18 °C (day/night) with 60% relative humidity. One group was inoculated with GM inoculum and the other was inoculated with sterilized inoculum as a control (CK). Each

treatment was repeated 10 times. A total of 20 pots were arranged randomly and watered every 2 days. The mycorrhizal colonization percentage of the seedlings was determined using the Phillip

and Hayman staining method (KOH bleaching-acid fuchsin stain) with some modifications (Phillips J M, 1970)51. PROTEIN EXTRACTION, PROTEIN QUANTIFICATION AND SDS-PAGE At the maturation

stage, _A. fruticosa_ roots were harvested and total root protein was precipitated with 10% (w/v) trichloroacetic acid (TCA) in acetone at −20 °C overnight. After centrifugation at 40,000 × 

g at 4 °C for 1 h, the pellets were washed 3 times with cold 80% acetone. A 2-D Quant kit (GE Healthcare, USA) was used to determine the protein concentrations. SDS-polyacrylamide gel (12%)

electrophoresis was performed with 30-μg samples at 120-V constant voltage for 2 h. The gel was stained with Coomassie blue and visualized52,53. ITRAQ LABELING The CK group and the GM group

each included 3 biological replicates. After digesting with trypsin, the proteins from the non-infected and infected samples were labeled with iTRAQ reagents 115 (CK1), 116 (CK2), 117 (CK3),

118 (GM1), 119 (GM2) and 121 (GM3) and were then combined following the manufacturer’s protocol at a ratio of 1:1:1:1:1:1 for LC-MS/MS analysis54,55. LC-MS/MS MEASUREMENTS The labeled

samples were pooled and purified using a strong cation-exchange chromatography (SCX) column and were then separated on an analytical column (1.7 μm, 100 μm × 100 mm) at a flow rate of 300 

nL/min using a linear gradient of 5–35% acetonitrile (ACN) over 40 min. The ion spray voltage was 4.5 kV and nitrogen was used as a nebulizing gas (30 psi) and a curtain gas (15 psi). From

each MS scan, the 30 most intense precursor ions were selected for MS/MS fragmentation and were detected at a mass resolution of 30,000 at m/z 40056. Data analysis was performed with a

Triple TOF 5600 System and then the iTRAQ data were compared with the protein sequences of homologous species after genome annotation. PROTEIN IDENTIFICATION Protein Pilot 4.0 (AB Sciex

Inc., USA) was used to simultaneously identify and quantify proteins57,58. Differentially expressed proteins were required to satisfy 3 conditions for identification: (1) each confident

protein identification involved at least 1 unique peptide; (2) the _P_-value was less than 0.05; and (3) changes of greater than 1.2-fold or less than 0.8 fold were considered significant.

All of the identified proteins were classified according to the annotations acquired by using the UniProt knowledge base and the GO database. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE:

Song, F. _et al._ Proteomic analysis of symbiotic proteins of _Glomus mosseae_ and _Amorpha fruticosa_. _Sci. Rep._ 5, 18031; doi: 10.1038/srep18031 (2015). REFERENCES * Fitter, A.,

Heinemeyer, A. & Staddon, P. The impact of elevated CO2 and global climate change on arbuscular mycorrhizas: a mycocentric approach. New Phytologist. 147, 179–187 (2000). Article  CAS 

Google Scholar  * Rosendahl, S. Populations and individuals of arbuscular mycorrhizal fungi. New Phytologist. 178, 253–266 (2008). Article  Google Scholar  * Smith, S. & Read, D.

Mycorrhizal symbiosis from cellular to ecosystem scales. Annual review of plant biology. 62, 227–250 (2008). Article  Google Scholar  * Smith, S. E. & Smith, F. A. Roles of arbuscular

mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales. Annual review of plant biology. 62, 227–250 (2011). Article  CAS  Google Scholar  * Kiers, E. T.

et al. Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis. Science. 333, 880–882 (2011). Article  CAS  ADS  Google Scholar  * Trépanier, M. et al. Dependence of

arbuscular-mycorrhizal fungi on their plant host for palmitic acid synthesis. Applied and environmental microbiology. 71, 5341–5347 (2005). Article  Google Scholar  * Recorbet, G. et al.

Identification of in plant-expressed arbuscular mycorrhizal fungal proteins upon comparison of the root proteomes of _Medicago truncatula_ colonised with two Glomus species. Fungal Genetics

and Biology. 47, 608–618 (2010). Article  CAS  Google Scholar  * Aloui, A. et al. Arbuscular mycorrhizal symbiosis elicits shoot proteome changes that are modified during cadmium stress

alleviation in _Medicago truncatula_. BMC plant biology. 11, 75 (2011). Article  CAS  Google Scholar  * Wang, Z. H., Yuan, K. & Yang, L. F. Identification and Functional Analysis of

Maize Leaf Proteins Responding to the Abuscular Mycorrhizal Fungi (AMF). Chinese Journal of Tropical Agriculture. 33, 40–44 (2013). Google Scholar  * Song, F. Q., Li, J. Z. & Zhang, X.

X. Characterization of expressed genes in the establishment of arbuscular mycorrhiza between _Amorpha fruticosa_ and _Glomus mosseae_. Journal of Forestry Research. 25, 541–548 (2014).

Article  CAS  Google Scholar  * Song, F. Q., Kong, X. S., Li, J. Z. & Chang, W. Screening the related genes in the AM Fungi and _Amorpha fruticosa_ symbiosis with the subtractive

hybridization technique. Scienta Slivae Sinicae. 50, 64–74 (2014). Google Scholar  * Heikham, E., Rupam, K. & Bhoopander, G. Arbuscular mycorrhizal fungi in alleviation of salt stress: a

review. Annals of Botany. 104, 1263–1280 (2009). Article  Google Scholar  * Song, F. Q., Kong, X. S., Dong, A. R. & Liu, X. F. Impact of arbuscular mycorrhizal fungi on the growth and

related physiological indexes of _Amorpha fruticosa_. Journal of Medicinal Plants Research. 6, 3648–3655 (2012). CAS  Google Scholar  * Claudia, H. & Helge, K. A roadmap of cell-type

specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis. BMC Genomics. 14, 306–324 (2013). Article  Google Scholar  * Bonfante, P. et al. The _Lotus

japonicus LjSym4_ gene is required for the successful symbiotic infection of root epidermal cells. Molecular plant-microbe interactions: MPMI. 13, 1109–1120 (2000). Article  CAS  Google

Scholar  * Novero, M. et al. Dual requirement of the _LjSym4_ gene for mycorrhizal development in epidermal and cortical cells of _Lotus japonicus_ roots. New phytologist. 154, 741–749

(2002). Article  CAS  Google Scholar  * Bonfante, P. et al. At the interface between mycorrhizal fungi and plants: the structural organization of cell wall, plasma membrane and cytoskeleton.

Fungal Associations. 9, 45–61 (2001). Article  CAS  Google Scholar  * Etienne Manneville, S. & Hall, A. Rho GTPases in cell biology. Nature. 420, 629–635 (2002). Article  CAS  ADS 

Google Scholar  * Li, H. et al. Arabidopsis Rho-related GTPases: differential gene expression in pollen and polar localization in fission yeast. Plant Physiology. 118, 407–417 (1998).

Article  CAS  Google Scholar  * Winge, P., Brembu, T., Kristensen, R. & Bones, A. M. Genetic structure and evolution of RAC-GTPases in _Arabidopsis thaliana_. Genetics. 156, 1959–1971

(2000). CAS  PubMed  PubMed Central  Google Scholar  * Christensen, T. M. et al. Conserved subgroups and developmental regulation in the monocot rop gene family. Plant physiology. 133,

1791–1808 (2003). Article  CAS  Google Scholar  * Zheng, Z. L. & Yang, Z. The Rop GTPase: an emerging signaling switch in plants. Plant molecular biology. 44, 1–9 (2000). Article  CAS 

Google Scholar  * Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiological reviews. 81, 153–208 (2001). Article  CAS  Google Scholar  * Yang, Z. et al. Small

GTPases versatile signaling switches in plants. The Plant Cell. 14, 375–388 (2002). Article  ADS  Google Scholar  * Shiu, S. H. & Bleecker, A. B. Receptor-like kinases from _Arabidopsis_

form a monophyletic gene family related to animal receptor kinases. Proceedings of the National Academy of Sciences. 98, 10763–10768 (2001). Article  CAS  ADS  Google Scholar  * Noguchi, T.

et al. Brassinosteroid-insensitive dwarf mutants of _Arabidopsis_ accumulate brassinosteroids. Plant Physiology. 121, 743–752 (1999). Article  CAS  Google Scholar  * Hu, H., Xiong, L. &

Yang, Y. Rice SERK1 gene positively regulates somatic embryogenesis of cultured cell and host defense response against fungal infection. Planta. 222, 107–117 (2005). Article  CAS  Google

Scholar  * Santos, M. et al. Suppression of SERK gene expression affects fungus tolerance and somatic embryogenesis in transgenic lettuce. Plant Biology. 11, 83–89 (2009). Article  CAS  ADS

  Google Scholar  * Yin, A. H. & Han, S. F. Relationship of Nodulation with Reactions of Letins of Leguminous Trees with EPS of _Rhizobium_. Journal of Nanjing Forestry University. 29,

88–90 (2005). Google Scholar  * Verslues, P. & Zhu, J. Plant signaling from genes to biochemistry. Biochemical Society Transactions. 33 (2005). * Cutler, A. J. & Krochko, J. E.

Formation and breakdown of ABA. Trends in plant science. 4, 472–478 (1999). Article  CAS  Google Scholar  * Davidson, R. M. et al. Rice germin-like proteins: Allelic diversity and

relationships to early stress responses. Rice. 3, 43–55 (2010). Article  Google Scholar  * Wang, J. M. et al. Cloning and Chromosome Mapping of a Germin-Like Protein Gene in Wheat and Its

Expression in Response to Infection with Wheat Powdery Mildew. Scientia Agricultura Sinica. 42, 3104–3111 (2009). CAS  Google Scholar  * Liu, H. et al. A rice serine carboxypeptidase-like

gene _OsBISCPL1_ is involved in regulation of defense responses against biotic and oxidative stress. Gene. 420, 57–65 (2008). Article  CAS  Google Scholar  * Liu, L. et al. Cloning and

Expression Analysis of Serine Carboxypeptidases in Maize (_Zea mays L_.). Acta Agronomica Sinica. 39, 164–171 (2013). Article  CAS  Google Scholar  * Liang, K. et al. Identification and

Genetic Analysis of Two Putative SDD1 Alleles in _Arabidopsis_. Chin Bull Bot. 3, 318–326 (2010). Google Scholar  * Kim, Y. J. et al. Protein disulfide isomerase-like protein 1-1 controls

endosperm development through regulation of the amount and composition of seed proteins in rice. PlOS one. 7, 44493 (2012). Article  ADS  Google Scholar  * Chen, R. D. & Gadal, P.

Structure, functions and regulation of NAD and NADP dependent isocitrate dehydrogenases in higher plants and in other organisms. Plant Physiology and Biochemistry. 28, 411–427 (1990). CAS 

Google Scholar  * Kuhlemeier, C. Aldehyde dehydrnase in tobacco pollen. Plant Mol Bio. 1, 355–365 (1997). Google Scholar  * Mittler, R. Oxidative stress, antioxidants and stress tolerance.

Trends in plant science. 7, 405–410 (2002). Article  CAS  Google Scholar  * Li, X. L., Yang, C. P. & Xu, X. L. Molecular Cloning and Expression Analysis of Aldehyde Dehydrogenase (ALDH)

Gene Segment in Leymus Chinensis. Chinese Agricultural Science Bulletin. 23, 115–120 (2007). Google Scholar  * Genre, A. & Bonfante, P. Actin versus tubulin configuration in

arbuscule-containing cells from mycorrhizal tobacco roots. New phytologist. 140, 745–752 (1998). Article  CAS  Google Scholar  * Mills, D., Kunoh, H., Keen, N. & Mayama, S. Molecular

aspects of pathogenicity and resistance: requirement for signal transduction. American Phytopathological Society, 257–267 (1996). * Rao, G. D. & Zhang, J. G. Advance of Studies on Plant

Tubulin Gene. World Forestry Research. 26, 11–16 (2013). Google Scholar  * Liang, H. et al. Ceramides modulate programmed cell death in plants. Genes & Development. 17, 2636–2641 (2003).

Article  CAS  Google Scholar  * Liu, R. H., Jiang, W. B. & Yu, D. Q. The Response of _AtCER_ to Oxidative Stress in _Arabidopsis thaliana_. Acta Botanica Yunnaica. 31, 326–334 (2009).

Article  CAS  Google Scholar  * Li, B. et al. Advance on Key Enzyme Gene (COMT) Involved in Lignin Biosynthesis. Molecular Plant Breeding. 1, 117–124 (2010). ADS  Google Scholar  * Hamada,

K. et al. 4-Coumarate: coenzyme A ligase in black locust (_Robinia pseudoacacia_) catalyses the conversion of sinapate to sinapoyl-CoA. Journal of plant research. 117, 303–310 (2004).

Article  CAS  Google Scholar  * Kang, C. et al. Carbon Emission Flow in Networks. Sci. Rep. 2, 479, 10.1038/srep00479 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  *

O'Malley, D. M. et al. The role of laccase in lignification. The Plant Journa. 4, 751–757 (1993). Article  CAS  Google Scholar  * Wei, Q. D. et al. Advancement of Histone and Its

Relationship with Regulation of Gene Expression. Journal of Tropical Medicine. 6, 596–598 (2006). CAS  Google Scholar  * Phillips, J. & Hayman, D. Improved procedures for clearing roots

and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British mycological Society. 55, 158–161 (1970). Article  Google

Scholar  * Sophie, A., Leslie, M. H. & Sona, P. ABA-Dependent and Independent G-Protein Signaling in _Arabidopsis_ Roots Revealed through an iTRAQ Proteomics Approach. Journal of

proteome research. 10, 3107–3122 (2011). Article  Google Scholar  * Zhang, K. R., Carolyn, M., Charles, H. H. & Michael, A. D. The _Medicago truncatula_ Small Protein Proteome and

Peptidome. Journal of Proteome Research. 5, 3355–3367 (2006). Article  CAS  Google Scholar  * Liu, J. Y. et al. Comparative analysis of proteomic profile at different development stages of

_Volvariella volvacea_ by iTRAQ-coupled 2D LC-MSMS. Microbiology China. 39, 853–864 (2012). ADS  Google Scholar  * Pan, X. Q. et al. iTRAQ Protein Profile Analysis of Tomato _Green-ripe_

Mutant Reveals New Aspects Critical for Fruit Ripening. Journal of Proteome Research. 13, 1979–1993 (2014). Article  CAS  Google Scholar  * Alexey, C., Consuelo, M. V., Neus, V. & Roman,

A. Z. Functional Identification of Target by Expression Proteomics (FITExP) reveals protein targets and highlights mechanisms of action of small molecule drugs. Scientific Reports. 5,

11176, 10.1038/srep11176 (2015). Article  Google Scholar  * Jose, A. M. et al. Quantitative proteomic analysis of host—pathogen interactions: a study of _Acinetobacter baumannii_ responses

to host airways. BMC Genomics. 16, 422–443 (2015). Article  Google Scholar  Download references ACKNOWLEDGEMENTS We sincerely thank Prof. sixue Chen for greatly improved the manuscript. This

study was supported by National Natural Science Foundation of China (31070576 and 31270535), Natural Science Foundation of Heilongjiang Province of China (No. ZD201206), Excellent Youth

Foundation of Heilongjiang Province of China (No. JC201306) and High-level Talents Support Program of Heilongjiang University (Ecological Restoration Team). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Heilongjiang University, Harbin, Heilongjiang, China Fuqiang Song, Dandan Qi, Xuan Liu, Xiangshi Kong, Yang Gao, Zixin Zhou & Qi Wu Authors * Fuqiang Song View author

publications You can also search for this author inPubMed Google Scholar * Dandan Qi View author publications You can also search for this author inPubMed Google Scholar * Xuan Liu View

author publications You can also search for this author inPubMed Google Scholar * Xiangshi Kong View author publications You can also search for this author inPubMed Google Scholar * Yang

Gao View author publications You can also search for this author inPubMed Google Scholar * Zixin Zhou View author publications You can also search for this author inPubMed Google Scholar *

Qi Wu View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS F.S. designed the research, D.Q. wrote the main manuscript text of chinese and

interpreted the proteomics data, X.K. and X.L. wrote the main manuscript text of english and edited language, Y.G., Z.Z. and Q.W. extract proteins. All authors reviewed the manuscript.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION RIGHTS AND PERMISSIONS This work is

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the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Song, F., Qi, D., Liu, X. _et

al._ Proteomic analysis of symbiotic proteins of _Glomus mosseae_ and _Amorpha fruticosa_. _Sci Rep_ 5, 18031 (2016). https://doi.org/10.1038/srep18031 Download citation * Received: 30 June

2015 * Accepted: 10 November 2015 * Published: 10 December 2015 * DOI: https://doi.org/10.1038/srep18031 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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