Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicumstrain CPAC 15
© Barros de Carvalho et al; licensee BioMed Central Ltd. 2013
Received: 14 January 2013
Accepted: 28 February 2013
Published: 6 March 2013
Biological nitrogen fixation in root nodules is a process of great importance to crops of soybean [Glycine max (L.) Merr.], as it may provide the bulk of the plant’s needs for nitrogen. Legume nodulation involves several complex steps and, although studied for many decades, much remains to be understood.
This research aimed at analyzing the global expression of genes in soybean roots of a Brazilian cultivar (Conquista) inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. To achieve this, we used the suppressive subtractive hybridization (SSH) technique combined with Illumina sequencing. The subtractive library (non-inoculated x inoculated) of soybean roots resulted in 3,210 differentially expressed transcripts at 10 days after inoculation were studied. The data were grouped according to the ontologies of the molecular functions and biological processes. Several classes of genes were confirmed as related to N2 fixation and others were reported for the first time.
During nodule formation, a higher percentage of genes were related to primary metabolism, cell-wall modifications and the antioxidant defense system. Putative symbiotic functions were attributed to some of these genes for the first time.
KeywordsSubtractive library Differential expression of genes Nodulation
Soybean (Glycine max (L.) Merr.) has significant agronomic and nutritional relevance because of the high concentrations of protein and oil in its grains. Concomitant with the high protein content, the legume shows a strong demand for nitrogen (N) for optimal development and grain productivity . Although atmospheric N2 is abundant, no eukaryotic organism is able to directly assimilate it, due to the strong triple bond linking the atoms [2, 3]. However, when growing in N-depleted soils, much of soybean’s need for N can be obtained via biological nitrogen fixation (BNF) in root nodules, through the symbiotic association with bacteria, collectively called rhizobia, belonging mainly to the species Bradyrhizobium japonicum and Bradyrhizobium elkanii[4, 5].
In Brazil, estimates indicate that the value of BNF by soybean root nodules is equivalent to U$7 billion/year; furthermore, BNF is an environmentally beneficial process, in comparison to the use of synthetic N fertilizers [4, 5].
The establishment of the symbiosis starts with molecular interactions between the rhizobia and the host plant, involving a succession of complex processes that lead to profound changes in both symbionts [3, 6]. The plant releases molecular signals, in particular flavonoid compounds, that are primary inducers of rhizobial nodulation genes. The induction of this class of genes leads to the biosynthesis of Nod factors, rhizobial signals that trigger specific responses in the root hairs of the host plant; plant cells perceive the presence of Nod factors—and of the rhizobia—through cell-surface receptors on the roots [7–10]. Many molecular events are triggered in a coordinated manner, leading to morphological and physiological changes in the host plant, necessary for a successful symbiosis .
Given the complexity of the symbiosis, studies of transcriptional profiles during nodulation are important to gain greater understanding of the nodulation process. Studies conducted with soybean, e.g. Brechenmacher et al. , evaluated gene-expression profiles in roots inoculated with B. japonicum (USDA 110), and elucidated reduced plant defenses. Furthermore, a complex regulatory mechanism in the plant was detected, enabling it to adapt to changes in its nutritional status. Another study of the transcriptome of soybean roots inoculated with strain USDA 110 showed rapid changes in gene expression in response to inoculation, with the pattern modified in accordance with the various stages of nodule development and function .
Brazil is the second most prolific producer of soybean worldwide, and probably the country where BNF has been exploited most successfully [4, 5]. However, no investigation of transcriptomics with Brazilian strains and cultivars have been reported. This study aimed at analyzing the global expression of genes in soybean roots of cultivar Conquista inoculated with B. japonicum strain CPAC 15 (=SEMIA 5079), both broadly used in Brazil, through the suppressive subtractive hybridization (SSH) technique  combined with sequencing Illumina analysis.
Results and discussion
The SSH library of the Brazilian cultivar Conquista, at 10 days after inoculation (DAI) with B. japonicum strain CPAC 15, which is broadly used in commercial inoculants in Brazil, resulted in 4,621,072 reads.
Among the 3,776 sequences identified as up-regulated, 3,210 were automatically recorded by the AutoFACT tool and grouped into functional categories (GO), according to the ontologies of molecular function (1,764 sequences) and biological process (1,728 sequences), using Blast2GO software . The remaining 566 sequences presented no similarity with a known protein of the database.
Classification of biological processes triggered in soybean roots (cv. Conquista) in response to inoculation with strain CPAC 15 of B. japonicum
I- Metabolic process
Cellular metabolic process
Primary metabolic process
Secondary metabolic process
Nitrogen compound metabolic process
Small molecule metabolic process
I.a- Macromolecule metabolic process
Protein metabolic process
II- Response to stimulus
Response to chemical stimulus
Response to abiotic stimulus
Response to biotic stimulus
II.a- Response to stress
II.b- Response to endogenous stimulus
Response to hormone stimulus
III- Developmental process
Multicellular organismal development
III.a- Anatomical structure development
IV.a- Establishment of localization
V- Biological regulation
Regulation of biological process
Regulation of biological quality
VI- Cellular component organization or biogenesis
Cellular component organization or biogenesis at cellular level
Cellular component organization
VII- Cellular component biogenesis
IX- Multi-organism process
Response to other organism
Furthermore, studies with Lotus japonicus indicate that genes involved in across-membrane transport, hormone metabolism, cell-wall modification and signal transduction were also induced by the presence of the bacteria [17, 18].
Moreover, we also identified in our library nodulin-21 , nodulin-22 , nodulin-26 , nodulin-36 , among others, which are recognized as induced genes in the tested condition, giving further confidence in SSH data.
For a more focused discussion of our results, categories were selected based on the high level of gene expression (RPKM, reads per kilobase of exon per million mapped reads), in comparison to the RPKM values of genes validated by RT-qPCR (Additional file 1: Table S1). Therefore, we assume that other genes with greater values of RPKM are also up-regulated during soybean nodulation.
Previous studies of the effects of inoculation of soybean with B. japonicum, at various stages of nodulation, ranging from hours (12, 24 and 48), days (4, 8 and 16) and weeks (2, 5 and 10) [2, 11, 23] after inoculation have reported profound changes in plant metabolism, that varied with the growth stage analyzed . Thus, our study is in accordance with Hayashi et al.  because the Nod factors perception cause metabolic changes, and here we can observed that the metabolic pathways most active in the presence of rhizobia were glycolysis and the Krebs cycle (Additional file 2: Figures S1 and S2). Indeed, for the development and functioning of nodules, it is necessary to allocate plant sources of C to the new organs [26, 27]. Similarly, genes involved in the breakdown of sucrose, glycolysis and synthesis of amino acids were found to be differentially expressed in L. japonicus, consistent with these processes being accelerated during nodulation . The glycolytic pathway and Krebs cycle are closely linked, representing the main ways of acquiring energy. Consistent with the N2-fixing symbiosis being an energy-demanding process [26, 29, 30], the expression of genes related to both pathways increased. Additional file 1: Table S2 presents the identified genes related to energetic pathways; of these, Glyma14g36850.1 (fructose-biphosphate aldolase) had the highest level of expression based on its RPKM value. This enzyme participates in the production of dicarboxylic acids for rhizobial and carbon skeletons for the assimilation of N by the plant . According to Brechenmacher et al. , this induction effect occurs from the eighth day after inoculation.
Genes that encode the soybean enzymes that participate in the metabolism of glutathione in the presence of B. japonicum strain CPAC 15
As shown by Chang et al. , changes in redox state are observed at various stages of nodulation. They occurred during early symbiotic interactions in Medicago sativa and Phaseolus vulgaris. Transcripts present in the subtractive library, which participate of antioxidant defense system, are shown in Table 2. In Medicago truncatula, inhibition of the synthesis of glutathione resulted in fewer nodules on inoculated roots, showing the critical role of glutathione in nodule morphogenesis [31, 35].
Signal-transduction genes are important at the various stages of the symbiotic interaction, as they lead to the coordinated development of epidermal and cortical cells needed to permit rhizobial penetration and nodule initiation [8, 36]. It is known that the processes of recognition and signaling triggered by the rhizobia activate genes related to nodulation; some of them have been characterized in soybean, such a receptor kinases NARK (nodule auto regulation receptor kinase)  and NORK (nodulation receptor kinase) , protein kinase type LRR (leucine rich repeat) encoded, respectively, by genes Glyma12g04390 and Glyma09g33510. Libault et al.  identified other genes related to the LRR proteins that are differentially regulated during nodulation.
Genes selected, based on RPKM, from some biological processes induced in soybean roots inoculated with strain CPAC 15 of B. japonicum
LRR (Leucine rich repeat)
LRR (Leucine rich repeat)
Response to stimulus
PDR (Pleiotropic drug resistance)
Glycoside hydrolase 19
Glycoside hydrolase 19
Amino acid transporter
Amino acid transporter
Amino acid transporter
Also noteworthy are genes encoding calmodulins (Table 3), important proteins that participate in the transduction of signals triggered by the interaction of Nod factors to specific receptors on the surface of the root and, thus, allowing the expression of nodulins . The most common calmodulin genes identified in the nodulation of soybean are Glyma15g35070.1 and Glyma08g24360.1 , and here three other genes were detected (encoding Glyma05g13900.1, Glyma13g03910.1, Glyma20g10820.1), whose participation in nodulation had not been emphasized.
Response to stimulus
The category that combined the second largest number of transcripts was that of response to stimulus, which includes genes related to abiotic and biotic factors, including stress and defense responses and reactions to endogenous stimuli. These genes are involved in morphological, biochemical and physiological changes stimulated by inoculation with B. japonicum. Among the identified genes in this category we emphasize the calreticulin, protein PDR and glycoside hydrolases 19 (Table 3).
The calreticulins have great functional diversity; they participate in protein folding (chaperones), in the homeostasis of intracellular Ca2+ and signaling, and are also crucial for the growth and development of plants. Moreover, they are potent regulators of the plant in response to various stresses, including the activation of mechanisms of resistance to infections by pathogens [43–45]. These proteins have been broadly studied in animals, but their characterization in plant models remains limited . Among the identified genes corresponding to calreticulins, Glyma20g23080.1 showed a high level of expression. Interestingly, no studies of the activities of these proteins during the nodulation have been reported.
Another gene detected in our study was related to a PDR-like-ABC transporter-family protein, involved in the responses to biotic and abiotic stresses in plants and fungi . These proteins have been identified as transporters involved in the secretion of genistein in soybean roots in response to B. japonicum, thus with an important role in nodulation.
Genes encoding glycoside hydrolases-19 were also detected. They are also known as chitinases and, among other functions in the symbiosis, it is believed that they participate in the perception and degradation of Nod factors [48, 49]. Xie et al.  showed an increase in activity of chitinase in the presence of Nod factors of rhizobia in soybean roots, suggesting that these enzymes can regulate the perception of Nod factors during nodulation.
Genes related to transport accounted for about 10% of differentially expressed genes; Table 3 displays the genes with the highest expression levels. This category was also expressed in the nodulation of M. truncatula and includes, among others, genes encoding membrane-transport proteins. Aquaporins, membrane proteins responsible for transport, especially of water, are expressed [52–54]. The soybean genes encoding aquaporins most commonly found on nodulation are Nodulin-26 (Glyma08g12650, Glyma-19g22210) found in the symbiosome [11, 42]. Other genes encoding aquaporins were identified in our study (Table 3). During nodulation in Medicago truncatula, the aquaporins were the most expressed membrane proteins from early to relatively late stages of nodulation , emphasizing their importance during nodule organogenesis.
Other transporters are important for the acquisition of nutrients by root cells and the symbiosome . Expression was detected of genes related to ABC transport, amino acid transport, and oligopeptide, potassium and sulfate transport (Table 3).
Genes encoding soybean enzymes active involved in carbohydrate metabolism
A study by Kaewsuralikhit et al. , of soybean at 12 DAI, showed elevated expression of pectinesterase, one of the enzymes responsible for cell-wall degradation during the formation of nodules, which also occurs in Sesbania rostrata. In Medicago truncatula, a pectinesterase was up-regulated and cellulase was induced on the third and fourth days post-inoculation . And in the present study, we also identified the gene that encode pectinesterase 10 DAI, confirming that this gene is induced only some days post-inoculation, because in the early hours, the pectinesterase gene showed as down-regulated .
Another important enzyme with a high level of expression in this study was sucrose synthase (Nodulin-100), which, among other known functions in nodulation, contributes significantly to development of cell wall .
SSH validation by RT-qPCR and proteomics
Two genes, represented by Glyma16g06940 and Glyma18g05710, were chosen for SSH validation by RT-qPCR. We took the genes that showed RPKM values of 460.98 and 397.18 respectively, in order to verify the sensitivity and quality of the subtraction. Expression levels of both genes were up-regulated at the same time, 10 DAI (Additional file 1: Table S1).
Proteomics was used as a supplemental functional analysis, in view to validate the transcriptional data. In parallel with the RNA extraction, we also made the protein extraction of both conditions (inoculated and non-inoculated). Two-dimensional gel electrophoresis profiles of the two conditions were compared with each other. Representative spots, which showed a significant higher volume in the inoculated condition, were selected and identified by mass spectrometry.
Two spots were successfully identified and one of the selected spots didn’t fit into the statistical parameters of identification. These two proteins identified were also found in the subtractive library data, which represents a functional confirmation of the transcriptomic analysis. Two of them were only detected in the inoculated condition: a putative glutathione-S-transferase (Glyma12g28670.2). The other identified protein was a sucrose synthase (Glyma15g20180.3), which had a 1.47 fold change in relation to the non-inoculated condition (Additional file 2: Figure S5). It is also worth mentioning that both spots identified correspond to the same Glyma IDs identified by the RNAseq analysis.
By using the SSH technique, it was possible to identify the main biological processes triggered in the Brazilian soybean cultivar Conquista after inoculation with the commercial strain CPAC 15 of B. japonicum. Among the main processes, we may highlight the metabolic pathways of primary metabolism, cell-wall modification and antioxidant-defense systems. Putative functions for some of these genes were assigned for the first time in the Bradyrhizobium-soybean symbiosis.
The analysis of transcriptional profiles of soybean in the presence of B. japonicum is essential to understand the symbiosis. Some transcripts have been previously described in nodulated soybean; however, novel genes were firstly described and could be related to the Brazilian germoplasm (plant genotype and bacterium), both studied by this standpoint for the first time.
Soybean seeds of cultivar Conquista MG/BR46 were surface sterilized  and germinated on absorbent paper moistened with sterile distilled water, at 22 ± 2°C (in the dark) for three days, and seedlings were transferred to sterile plastic bags containing 200 mL of N-free nutrient solution .
Inoculum preparation and plant inoculation
The B. japonicum strain used for inoculation was CPAC 15 (=SEMIA 5079), which has been used in commercial inoculants in Brazil, since 1992, for its outstanding symbiotic efficiency and competitiveness [4, 5]. The bacterium was grown until the exponential phase of growth in yeast-mannitol broth (YMB) . The cells were centrifuged and washed with saline solution (NaCl 0.85%). Aliquots of washed cell suspension were counted in YMB medium, indicating a concentration of 2.27 × 107 cells mL–1.
The experiment had a fully randomized design with three replicates, each consisting of 20 plants per treatment. Treatments consisted of: soybean roots inoculated with strain CPAC 15 and non-inoculated soybean. For the inoculated treatment, 1 mL of the inoculum was applied at the base of each radicle. The plants were grown under greenhouse condition with a 12-h day/night period and mean temperature of 25–28°C/15–18°C (day/night) for ten days. Subsequently, the roots were separated from shoots, immediately frozen in liquid nitrogen and stored at −80°C.
RNA extraction and isolation of mRNA
Total RNA was isolated from the roots of each treatment using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. After extraction, total RNA was analyzed for quality using the Thermo Scientific NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The mRNA was obtained from 2 μg of total RNA using the FastTrack MAG mRNA Isolation Kit (Invitrogen), according to the manufacturer’s specifications.
Construction of the suppressive subtractive hybridization (SSH) cDNA library
This study is a component of a consortium named Genosoja (Brazilian Soybean Genome Consortium). Before the development of all experiments of the Genosoja project, the techniques of RNA extraction, SSH, RNAseq and qPCR were validated in several laboratories, and a standard protocol was applied, including the bioinformatics analyses. The experimental validations were published in a special edition of the Journal “Genetics and Molecular Biology” (v.35, n.1, 2012, http://www.scielo.br/scielo.php?script=sci_issuetoc&pid=1415-475720120002&lng=en&nrm=iso). A procedure included in the validation was the comparison of SSH and superSAGE strategies, and resulted in congruent results. Due to the high costs we have decided to follow the SSH strategy.
The SSH library was constructed using a PCR-Select cDNA Subtration Kit (Clontech Laboratories Inc, Mountain View, CA, USA), according to the manufacturer’s instructions. A cDNA library was built, containing clones from the subtraction of inoculated plants versus non-inoculated plants. This subtractive hybridization was performed using sample cDNAs (tester) from inoculated plants, which was subtracted with cDNAs (driver) from non-inoculated plants. This kind of hybridization is called forward subtraction, to identify the induced genes (up-regulated).
Sequencing and bioinformatics
The pool of cDNA resulting from the subtractive library was transferred directly to the sequencing reaction with the Genome Analyzer GAII technology Illumina, carried out by Fasteris S.A., in Switzerland.
The reads from sequencing were aligned against the GENOSOJA database (http://www.lge.ibi.unicamp.br/soybean) . The generated sequences were assembled and preliminarily analyzed at the LGE (Laboratory of Genomic and Expression at UNICAMP, Campinas, São Paulo, Brazil). First, the reads from sequencing were aligned in the reference genome of soybean (Phytozome database)  using SOAP software , allowing a maximum of two mismatches.
In addition, a level of inference of gene expression by reads per kilobase of exon per million mapped reads (RPKM) was assigned, making it possible to infer the expression level of genes in the subtractive library .
AutoFACT software  was used for automatic annotation of the sequences, through several BLASTx searches (e-value cutoff of 1e-5) against protein databases including NR (non-redundant from NCBI), Swissprot  and KEGG . Subsequently, the transcripts were subjected to functional categorization, which was performed using the Gene Ontology database (http://www.geneontology.org), with Blast2GO , enabling clustering of genes according to the biological processes ontology (level 2) and molecular function (level 3). The most relevant metabolic pathways (based on the KEGG database) were also identified.
SSH validation by real-time qPCR analysis
Quantitative real-time PCR experiments were performed to validate the expression of two genes, whose RPKM values were relatively low, in order to compare other genes in the library. Therefore, a new plant-inoculation experiment was performed, under the conditions previously described. After extraction of total RNA, the samples were treated with deoxyribonuclease I, amplification grade (Invitrogen), according to the manufacturer’s instructions. The cDNA synthesis was carried out using a SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen), according to the manufacturer’s instructions.
Primers were designed using PrimerExpress 3.0 (Applied Biosystems, Foster City, CA, USA), and the sequences are available on Additional file 1: Table S3, along with the sequences of the primers for the reference genes for β-actin  and F-box , used as endogenous controls.
For the RT-PCR reactions, Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) was used. Each sample was run in triplicate along with the corresponding non-template controls containing water instead of cDNA. Amplification reactions were performed using a 7300 Real-Time PCR System thermal cycler (Applied Biosystems). The amplification cycles were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. For each sample, a threshold cycle (Ct) value was calculated based on the amplification curves by selecting the optimal ΔRn (emission of reporter dye over starting background fluorescence) in the exponential part of the amplification plot. The specificity of the amplified products was evaluated by dissociation-curve analyses. The relative linear amount of target molecules relative to the calibrator was calculated according to Pfaffl , with significant differences determined with the REST 2009 software (p < 0.05) (Relative Expression Software Tool ).
Whole-cell proteins of soybean roots were extracted, from both the inoculated and non-inoculated treatments, following the simplified method described by Rodrigues et al. . IPG strips (pH 4–7, 13 cm, GE Healthcare, Uppsala, Sweden) were rehydrated overnight with aliquots of 350 μg of solubilized proteins. Next, the strips were submitted to isoelectric focalization and SDS-PAGE as described by Batista and Hungria . Gels were stained overnight with Comassie Brilliant Blue (CBB) R-350 (GE Healthcare), destained in a solution of 40% ethanol and 10% acetic acid and scanned (ImageScanner LabScan v. 5.0).
Spots were strictly identified in the high-resolution digitalized gel images and analyzed by Image Master 2D Platinum v 5.0 software (GE Healthcare). Ratios of mean normalized spot volumes were calculated. All selected spots were manually confirmed and statistically evaluated (p < 0.05) upon Student’s t-test, using XLSTAT (Addinsoft, France, add-in to Microsoft Excel).
Spots which showed a significantly higher volume in the inoculated condition were excised and processed as described before , with trypsin (Gold, mass spectrometry grade, Promega, Madison, WI) at 37°C overnight. Tryptic peptides (0.5 μL) were mixed with saturated solution of HCCA (α-cyano-4-hydroxy-cinamic acid) in 50% acetonitrile, 0.1% TFA (trifluoroacetic acid). The mixture was spotted onto a MALDI sample plate and allowed to crystallize at room temperature. The same procedure was used for the standard peptide calibration mix (Bruker Daltonics).
For mass spectra acquisition, a MALDI-TOF-MS Autoflex spectrometer (Bruker Daltonics) was operated manually in the LIFT mode for MALDI-TOF/TOF, using the FlexControl 3.0 (Bruker Daltonics) software.
PMFs and MS/MS ions generated were searched against the public database NCBInr (National Center for Biotechnology Information non-redundant)/Viridiplantae, using the Mascot software v. 2.3 (Matrix Science). For protein searches, monoisotopic masses were used, considering a peptide tolerance of 150 ppm and allowance of one missed cleavage. When MS/MS was carried out, a tolerance of 0.3 Da was acceptable. Carbamidomethylation of cysteine and oxidation of methionine were considered fixed and variable modifications, respectively.
Identifications were only validated when the Mowse (molecular weight search) score was significant, above the recommended cutoff score. The spectrometry datasets are available at PRIDE (http://ebi.ac.uk/pride/) with the experiment accession number 14846 (Username: review03737, Password: +6Nx + E^Y).
Author-recommended internet resource
Site for the data http://www.lge.ibi.unicamp.br/soybean. In Browse, subtractive libraries.
To Dr. Elisete R.P. Rodrigues and Aline Schiavon for helping in the conduction of the experiments. To Marcelo F. Carazzolle and Gonçalo A.G. Pereira for helping in the bioinformatics analysis and Dr. Allan R.J. Eaglesham for suggstions on the manuscript. G.A.B. Carvalho acknowledges an MSc fellowship from CAPES (Coordenação de Aperfeiçoamento do Pessoal de Nível Superior), J.S.S. Batista a postdoc fellowship from CNPq (National Council for Scientific and Technological Development) and M Hungria a research fellowship from CNPq (300547/2010-2). Project funded by CNPq projects Genosoja, MAPA (577933/2008-6) and Repensa (562008/2010-1). Approved for publication by the Editorial Board of Embrapa Soja as manuscript 10/2012.
- Graham PH, Vance CP: Legumes: importance and constraints to greater use. Plant Physiol. 2003, 131 (3): 872-877. 10.1104/pp.017004.PubMed CentralView ArticlePubMed
- Brechenmacher L, Kim MY, Benitez M, Li M, Joshi T, Calla B, Lee MP, Libault M, Vodkin LO, Xu D, Lee SH, Clough SJ, Stacey G: Transcription profiling of soybean nodulation by Bradyrhizobium japonicum. Mol Plant Microbe Interact. 2008, 21 (5): 631-645. 10.1094/MPMI-21-5-0631.View ArticlePubMed
- Oldroyd GE, Murray JD, Poole PS, Downie JA: The rules of engagement in the legume- rhizobial symbiosis. Annu Rev Genet. 2011, 45: 119-144. 10.1146/annurev-genet-110410-132549.View ArticlePubMed
- Hungria M, Campo RJ, Mendes IC, Graham PH: Contribution of biological nitrogen fixation to the N nutrition of grain crops in the tropics: the success of soybean (Glycine max L. Merr.) in South America. Nitrogen nutrition and sustainable plant productivity. Edited by: Singh RP, Shankar N, Jaiwal PK. 2006, Houston, Texas: Studium Press, LLC, 43-93.
- Hungria M, Franchini JC, Campo RJ, Crispino CC, Moraes JZ, Sibaldelli RNR, Mendes IC, Arihara J: Nitrogen nutrition of soybean in brazil: contributions of biological N2 fixation and N fertilizer to grain yield. Can J Plant Sci. 2006, 86 (4): 927-939. 10.4141/P05-098.View Article
- Stougaard J: Regulators and regulation of legumes root nodule development. Plant Physiol. 2000, 124 (2): 531-540. 10.1104/pp.124.2.531.PubMed CentralView ArticlePubMed
- Ferguson BJ, Mathesius U: Signaling interactions during nodule development. J Plant Growth Regul. 2003, 22: 47-72. 10.1007/s00344-003-0032-9.View Article
- Limpens E, Bisseling T: Signaling in symbiosis. Curr Opin Plant Biol. 2003, 6 (4): 343-350. 10.1016/S1369-5266(03)00068-2.View ArticlePubMed
- Stacey G, Libault M, Brechenmacher L, Wan J, May GD: Genetics and functional genomics of legume nodulation. Curr Opin Plant Biol. 2006, 9 (2): 110-121. 10.1016/j.pbi.2006.01.005.View ArticlePubMed
- Ferguson BJ, Indrasumunar A, Hayashi S, Lin MH, Lin YH, Reid DE, Gresshoff PM: Molecular analysis of legume nodule development and autoregulation. J Integr Plant Biol. 2010, 52 (1): 61-76. 10.1111/j.1744-7909.2010.00899.x.View ArticlePubMed
- Libault M, Farmer A, Brechenmacher L, Drnevich J, Langley RJ, Bilgin DD, Radwan O, Neece DJ, Clough SJ, May GD, Stacey G: Complete transcriptome of the soybean root hair cell, a single-cell model, and its alteration in response to Bradyrhizobium japonicum infection. Plant Physiol. 2010, 152 (2): 541-552. 10.1104/pp.109.148379.PubMed CentralView ArticlePubMed
- Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD: Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. PNAS. 1996, 93 (12): 6025-6030. 10.1073/pnas.93.12.6025.PubMed CentralView ArticlePubMed
- Conesa A, Götz S: Blast2GO: a comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics. 2008, 2008: 619832-PubMed CentralView ArticlePubMed
- El Yahyaoui F, Küster H, Ben Amor B, Hohnjec N, Pühler A, Becker A, Gouzy J, Vernié T, Gough C, Niebel A, Godiard L, Gamas P: Expression profiling in Medicago truncatula identifies more than 750 genes differentially expressed during nodulation, including many potential regulators of the symbiotic program. Plant Physiol. 2004, 136 (2): 3159-3176. 10.1104/pp.104.043612.PubMed CentralView ArticlePubMed
- Lohar DP, Sharopova N, Endre G, Peñuela S, Samac D, Town C, Silverstein KA, Vandenbosch KA: Transcript analysis of early nodulation events in Medicago truncatula. Plant Physiol. 2006, 140 (1): 221-234.PubMed CentralView ArticlePubMed
- Starker CG, Parra-Colmenares AL, Smith L, Mitra RM, Long SR: Nitrogen fixation mutants of Medicago truncatula fail to support plant and bacterial symbiotic gene expression. Plant Physiol. 2006, 140 (2): 671-680. 10.1104/pp.105.072132.PubMed CentralView ArticlePubMed
- Kouchi H, Shimomura K, Hata S, Hirota A, Wu GJ, Kumagai H, Tajima S, Suganuma N, Suzuki A, Aoki T, Hayashi M, Yokoyama T, Ohyama T, Asamizu E, Kuwata C, Shibata D, Tabata S: Large-scale analysis of gene expression profiles during early stages of root nodule formation in a model legume, Lotus japonicus. DNA Res. 2004, 11 (4): 263-274. 10.1093/dnares/11.4.263.View ArticlePubMed
- Asamizu E, Nakamura Y, Sato S, Tabata S: Comparison of the transcript profiles from the root and the nodulating root of the model legume Lotus japonicus by serial analysis of gene expression. Mol Plant Microbe Interact. 2005, 18 (5): 487-498. 10.1094/MPMI-18-0487.View ArticlePubMed
- Delauney AJ, Cheon CI, Snyder PJ, Verma DP: A nodule-specific sequence encoding a methionine-rich polypeptide, nodulin-21. Plant Mol Biol. 1990, 14 (3): 449-451. 10.1007/BF00028782.View ArticlePubMed
- Sandal NN, Bojsen K, Marcker KA: A small family of nodule specific genes form soybean. Nucl Acids Res. 1987, 15 (4): 1507-1519. 10.1093/nar/15.4.1507.PubMed CentralView ArticlePubMed
- Miao GH, Verma DP: Soybean nodulin-26 gene encoding a channel protein is expressed only in the infected cells of nodules and is regulated differently in roots of homologous and heterologous plants. Plant Cell. 1993, 5 (7): 781-794.PubMed CentralView ArticlePubMed
- Kouchi H, Hata S: Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Mol Gen Genet. 1993, 238 (1–2): 106-119.PubMed
- Lee H, Hur CG, Oh CJ, Kim HB, Pakr SY, An CS: Analysis of the root nodule-enhanced transcriptome in soybean. Mol Cells. 2004, 18 (1): 53-62.PubMed
- Desbrosses GJ, Stougaard J: Root nodulation: a paradigm for how plant-microbe symbiosis influences host developmental pathways. Cell Host Microbe. 2011, 10 (4): 348-358. 10.1016/j.chom.2011.09.005.View ArticlePubMed
- Hayashi S, Reid DE, Lorenc MT, Stiller J, Edwards D, Gresshoff PM, Ferguson BJ: Transient Nod factor-dependent gene expression in the nodulation-competent zone of soybean (Glycine max [L.] Merr.) roots. Plant Biotechnol J. 2012, 10 (8): 995-1010. 10.1111/j.1467-7652.2012.00729.x.View ArticlePubMed
- Neves MCP, Hungria M: The physiology of nitrogen fixation in tropical grain legumes. Crit Rev Plant Sci. 1987, 6 (3): 267-321. 10.1080/07352688709382252.View Article
- Colebatch G, Desbrosses G, Ott T, Krusell L, Montanari O, Kloska S, Kopka J, Udvardi MK: Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus. Plant J. 2004, 39 (4): 487-512. 10.1111/j.1365-313X.2004.02150.x.View ArticlePubMed
- Colebatch G, Kloska S, Trevaskis B, Freund S, Altmann T, Udvardi MK: Novel aspects of symbiotic nitrogen fixation uncovered by transcript profiling with cDNA arrays. Mol Plant Microbe Interact. 2002, 15 (5): 411-420. 10.1094/MPMI.2002.15.5.411.View ArticlePubMed
- Vance CP, Gantt JS: Control in nitrogen and carbon metabolism in root-nodules. Physiol Plant. 1992, 85 (2): 266-274. 10.1111/j.1399-3054.1992.tb04731.x.View Article
- Curioni PMG, Hartwig UA, Nösberger J, Schuller KA: Glycolytic flux is adjusted to nitrogenase activity in nodules of detopped and argon-treated alfalfa plants. Plant Physiol. 1999, 119 (2): 445-453. 10.1104/pp.119.2.445.PubMed CentralView ArticlePubMed
- Frendo P, Harrison J, Norman C, Jiménez MJH, Van de Sype G, Gilabert A, Puppo A: Glutathione and homoglutathione play a critical role in the nodulation process of Medicago truncatula. Mol Plant Microbe Interact. 2005, 18 (3): 254-259. 10.1094/MPMI-18-0254.View ArticlePubMed
- Chang C, Damiani I, Puppo A, Frendo P: Redox changes during the legume–Rhizobium symbiosis. Mol Plant. 2009, 2 (3): 370-377. 10.1093/mp/ssn090.View ArticlePubMed
- Santos R, Herouart D, Sigaud S, Touati D, Puppo A: Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction. Mol Plant Microbe Interact. 2001, 14 (1): 86-89. 10.1094/MPMI.2001.14.1.86.View ArticlePubMed
- Cárdenas L, Martínez A, Sánchez F, Quinto C: Fast, transient and specific intracellular ROS changes in living root hair cells responding to Nod factors (NFs). Plant J. 2008, 56 (5): 802-813. 10.1111/j.1365-313X.2008.03644.x.View ArticlePubMed
- Pucciariello C, Innocenti G, Van de Velde W, Lambert A, Hopkins J, Clément M, Ponchet M, Pauly N, Goormachtig S, Holsters M, Puppo A, Frendo P: (Homo)glutathione depletion modulates host gene expression during the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti. Plant Physiol. 2009, 151 (3): 1186-1196. 10.1104/pp.109.142034.PubMed CentralView ArticlePubMed
- Ding Y, Oldroyd GE: Positioning the nodule, the hormone dictum. Plant Signal Behav. 2009, 4 (2): 89-93. 10.4161/psb.4.2.7693.PubMed CentralView ArticlePubMed
- Searle IR, Men AE, Laniya TS, Buzas DM, Iturbe-Ormaetxe I, Carroll BJ, Gresshoff PM: Long- distance signaling in nodulation directed by a CLAVATA1-like receptor kinase. Science. 2003, 299 (5603): 109-112. 10.1126/science.1077937.View ArticlePubMed
- Zhu H, Riely BK, Burns NJ, Ané JM: Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics. 2006, 172 (4): 2491-2499.PubMed CentralView ArticlePubMed
- Stracke S, Kistner C, Yoshida S, Mulder L, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J, Szczyglowski K, Parniske M: A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature. 2002, 417 (6892): 959-962. 10.1038/nature00841.View ArticlePubMed
- Capoen W, Goormachtig S, Rycke RD, Schroeyers K, Holsters M: SrSymRK, a plant receptor essential for symbiosome formation. PNAS. 2005, 102 (29): 10369-10374. 10.1073/pnas.0504250102.PubMed CentralView ArticlePubMed
- Oldroyd GE, Downie JA: Calcium, kinases and nodulation signalling in legumes. Nat Rev Mol Cell Biol. 2004, 5 (7): 566-576. 10.1038/nrm1424.View ArticlePubMed
- Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L: Genome sequence of the palaeopolyploid soybean. Nature. 2010, 463: 178-183. 10.1038/nature08670.View ArticlePubMed
- Christensen A, Svensson K, Persson S, Jung J, Michalak M, Widell S, Sommarin M: Functional characterization of Arabidopsis calreticulin1a: a key alleviator of endoplasmic reticulum stress. Plant Cell Physiol. 2008, 49 (6): 912-924. 10.1093/pcp/pcn065.View ArticlePubMed
- Jia XY, He LH, Jing RL, Lia RZ: Calreticulin: conserved protein and diverse functions in plants. Physiol Plant. 2009, 136 (2): 127-138. 10.1111/j.1399-3054.2009.01223.x.View ArticlePubMed
- Gupta D, Tuteja N: Chaperones and foldases in endoplasmic reticulum stress signaling in plants. Plant Signal Behav. 2011, 6 (2): 232-236. 10.4161/psb.6.2.15490.PubMed CentralView ArticlePubMed
- Crouzet J, Trombik T, Fraysse AS, Boutry M: Organization and function of the plant pleiotropic drug resistance ABC transporter family. FEBS Lett. 2006, 580 (4): 1123-1130. 10.1016/j.febslet.2005.12.043.View ArticlePubMed
- Sugiyama A, Shitan N, Yazaki K: Signaling from soybean roots to rhizobium. Plant Signal Behav. 2008, 3 (1): 38-40. 10.4161/psb.3.1.4819.PubMed CentralView ArticlePubMed
- Kasprzewska A: Plant chitinases: regulation and function. Cell Mol Biol Lett. 2003, 8 (3): 809-824.PubMed
- Santos P, Fortunato A, Ribeiro A, Pawlowski K: Chitinases in root nodules. Plant Biotechnol. 2008, 25: 299-307. 10.5511/plantbiotechnology.25.299.View Article
- Xie ZP, Staehelin C, Wiemken A, Broughton WJ, Müller J, Boller T: Symbiosis-stimulated chitinase isoenzymes of soybean (Glycine max (L.) Merr.). J Exp Bot. 1999, 50 (332): 327-333.
- Benedito VA, Li H, Dai X, Wandrey M, He J, Kaundal R, Torres-Jerez I, Gomez SK, Harrison MJ, Tang Y, Zhao PX, Udvardi MK: Genomic inventory and transcriptional analysis of Medicago truncatula transporters. Plant Physiol. 2010, 152 (3): 1716-1730. 10.1104/pp.109.148684.PubMed CentralView ArticlePubMed
- Kjellbom P, Larsson C, Johansson I, Karlsson M, Johanson U: Aquaporins and water homeostasis in plants. Trends Plant Sci. 1999, 4 (8): 308-314. 10.1016/S1360-1385(99)01438-7.View ArticlePubMed
- Johansson I, Karlsson M, Johanson U, Larsson C, Kjellbom P: The role of aquaporins in cellular and whole plant water balance. Biochim Biophys Acta. 2000, 1465 (1–2): 324-334.View ArticlePubMed
- Uehlein N, Fileschi K, Eckert M, Bienert GP, Bertl A, Kaldenhoff R: Arbuscular mycorrhizal symbiosis and plant aquaporin expression. Phytochemistry. 2007, 68 (1): 122-129. 10.1016/j.phytochem.2006.09.033.View ArticlePubMed
- Benedito VA, Dai X, He J, Zhao PX, Udvardi MK: Functional genomics of plant transporters in legume nodules. Funct Plant Biol. 2006, 33 (8): 731-736. 10.1071/FP06085.View Article
- Kaewsuralikhit S, Yokoyama T, Kouchi H, Arima Y: Comprehensive analysis of plant gene expression in soybean root nodules at different growth stages. Soil Sci Plant Nutrit. 2006, 51 (4): 535-547.View Article
- Lievens S, Goormachtig S, Herman S, Holsters M: Patterns of pectin methylesterase transcripts in developing stem nodules of Sesbania rostrata. Mol Plant Microbe Interact. 2002, 15 (2): 164-168. 10.1094/MPMI.2002.15.2.164. 2002View ArticlePubMed
- Koch K: Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 2004. 2004, 7 (3): 235-246.View Article
- Vincent JM: A manual for the practical study of root nodule bacteria. 1970, Oxford: Science Blackwell publishing, 164-
- Broughton WJ, Dilworth MJ: Methods in legume-rhizobium technology: plant nutrient solutions. Handbook for rhizobia. Edited by: Somasegaran P, Hoben HJ. 1970, Hawaii: NifTAL Project and University of Hawaii, 245-249.
- Nascimento LC, Costa GGL, Binneck E, Pereira GAG, Carazzolle MF: A web-based bioinformatics interface applied to the GENOSOJA Project: Databases and pipelines. Genet Mol Biol. 2012, 35 (1): 203-211. 10.1590/S1415-47572012000200002.PubMed CentralView ArticlePubMed
- Li R, Li Y, Kristiansen K, Wang J: SOAP: short oligonucleotide alignment program. Bioinformatics. 2008, 24 (5): 713-714. 10.1093/bioinformatics/btn025.View ArticlePubMed
- Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008, 5 (7): 621-628. 10.1038/nmeth.1226.View ArticlePubMed
- Koski LB, Gray MW, Lang BF, Burger G: AutoFACT: an automatic functional annotation and classification tool. BMC Bioinforma. 2005, 6: 151-10.1186/1471-2105-6-151.View Article
- Suzek BE, Huang H, McGarvey P, Mazumder R, Wu CH: UniRef: comprehensive and non-redundant UniProt reference clusters. Bioinformatics. 2007, 23 (10): 1282-1288. 10.1093/bioinformatics/btm098.View ArticlePubMed
- Kanehisa M, Goto S: KEGG: Kyoto encyclopedia of genes and genomes. Nucl Acids Res. 2000, 28 (1): 27-30. 10.1093/nar/28.1.27.PubMed CentralView ArticlePubMed
- Stolf-Moreira R, Lemos EGM, Abdelnoor RV, Beneventi MA, Rolla AAP, Pereira SS, Oliveira MCN, Nepomuceno AL, Marcelino-Guimarães FC: Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean. Pesq Agrop Brasil. 2011, 46 (1): 58-65. 10.1590/S0100-204X2011000100008.View Article
- Libault M, Thibivilliers S, Bilgin DD, Radwan O, Benitez M, Clough SJ, Stacey G: Identification of four soybean reference genes for gene expression normalization. Plant Genome. 2008, 1: 444-454.View Article
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res. 2001, 29 (9): e45-10.1093/nar/29.9.e45.PubMed CentralView ArticlePubMed
- Pfaffl MW, Horgan GW, Dempfle L: Relative expression software tool (REST) for group- wise comparison and statistical analysis of relative expression results in real-time PCR. Nucl Acids Res. 2002, 30 (9): e36-10.1093/nar/30.9.e36.PubMed CentralView ArticlePubMed
- Rodrigues EP, Torres AR, Batista JSS, Huergo L, Hungria M: A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots. Genet Mol Biol. 2012, 35 (1): 348-352. 10.1590/S1415-47572012000200016.PubMed CentralView ArticlePubMed
- Batista JSS, Hungria M: Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains. J Proteomics. 2012, 75 (4): 1211-1219. 10.1016/j.jprot.2011.10.032.View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.