An RNA-Seq based gene expression atlas of the common bean
© O’Rourke et al.; licensee BioMed Central Ltd. 2014
Received: 8 May 2014
Accepted: 24 September 2014
Published: 6 October 2014
Common bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species.
Combining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form.
These data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.
KeywordsPhaseolus vulgaris cv Negro jamapa Common bean RNA-Seq Symbiotic nitrogen fixation Expression atlas SRP046307
Common bean (Phaseolus vulgaris L.), Pv, is an important source of proteins, micronutrients and calories for over three hundred million people worldwide, mostly throughout Latin America and Africa where beans are an important component of traditional diets. The high levels of dietary protein (between 20 and 25%) and micronutrients in beans complement the high carbohydrates found in maize and cassava . In addition to their important contribution to human health, legumes are also important contributors to biological nitrogen (N). N is a primary nutrient limiting plant production , with the acquisition and assimilation of N second only to photosynthesis for plant growth and development . Despite the international importance of Pv, both in terms of economics and consumption, it has lagged behind Medicago truncatula, Lotus japonicus, soybean, and other legumes in terms of genetic resources. cDNA libraries have been used to investigate phosphate stress, resistance to bean rust, and leaf development [3–7]. Sequence information for Pv was greatly enhanced by using Roche 454 technology coupled with mRNA sequences to assemble 59,295 unigene sequences, , though these data are not yet publicly available. Most recently, the genome sequence and predicted gene calls for Pv G 19833 has been made publicly available (http://www.phytozome.net). This resource provides a platform for Pv genomic and comparative genomic analyses . Sequence conservation and genetic colinearity between Pv and soybean (Glycine max L. merr) [10, 11] which diverged from a common ancestor approximately 19 million years ago [12, 13], allows genomic information to be leveraged from one species to the other.
In this study we utilized RNA-seq to characterize expression profiles for the transcriptome of common bean (Pv cv. Negro Jamapa). Gene expression profiles were analyzed from 24 unique samples from seven distinct tissues; roots, nodules, stems, flowers, leaves, pods, and seeds throughout development. Our data was used as the foundation for The Pv Gene Expression Atlas (Pv GEA) database, available at http://plantgrn.noble.org/PvGEA/. We utilized the expression profiles of all predicted genes in Pv to examine the biological processes related to seed and pod development, nodulation and symbiosis, and changes in gene expression due to nitrogen availability.
Results and discussion
Phaseolus vulgaris gene expression atlas (PvGEA)
To facilitate additional use of the RNA-Seq data generated for these analyses, we have developed a web-accessible database, The Pv Gene Expression Atlas (Pv GEA), available at http://plantgrn.noble.org/PvGEA/. This database was built using a similar database structure, web application, architecture and tools as the LegumeIP platform  to retrieve and visualize the gene expression patterns using RNA-seq data. To facilitate the mining of the data included in Pv GEA, we have provided the capability to: (i) visualize expression profiles of genes of interest, (ii) identify genes exhibiting certain expression patterns in specific tissues, (iii) identify genes and gene expression patterns based on http://www.phytozome.net annotation terms; and (iv) download the entire data set, either raw or normalized, in tabular form to facilitate the analysis of more complicated biological questions. Using the predicted gene calls of the G 19833 Pv genome to build the Pv GEA database means it can be easily expanded to integrate RNA-Seq data from future experiments. Currently, Pv GEA includes gene expression profiles from 24 samples isolated from roots, root nodules, stems, leaves, flowers, pods, and seeds at various developmental stages under ideal growth conditions. Included in this dataset are transcripts from eight samples including nodule, root, and leaf tissues for plants having either fix + or fix- root nodules; providing preliminary data on the impact of nodulation and N fixation on gene expression, an important biological process for legumes.
Tissue samples isolated from Phaseolus vulgaris cv. Negro jamapa for RNA-Seq analysis
Fully expanded 2nd trifoliate leaf tissue from plants provided with fertilizer
Leaf tissue collected 5 DAI with effective rhizobium
Leaf tissue from fertilized plants collected at the same time of LE and LI
Leaf tissue collected 21 DAI with effective rhizobium
Leaf tissue collected 21 DAI with ineffective rhizobium
All stem internodes above the cotyledon collected at the 2nd trifoliate stage
Shoot tip, including the apical meristem, collected at the 2nd trifoliate stage
Young flowers, collected prior to floral emergence
Young pods, collected 1 to 4 days after floral senescence. Samples contain developing embryos at globular stage
Pods approximately 9 cm long, associated with seeds at heart stage (pod only)
Pods between 10 and 11 cm long, associated with stage 1 seeds (pod only)
Pods between 12 and 13 cm long associated with stage 2 seeds (pod only)
Heart stage seeds, between 3 and 4 mm across and ~7 mg
Stage 1 seeds, between 6 and 7 mm across and ~50 mg
Stage 2 seeds, between 8 and 10 mm across and 140–150 mg
Root tips, 0.5 cm of tissue, collected from fertilized plants at 2nd trifoliate stage of development
Whole roots, including root tips, collected at the 2nd trifoliate stage of development
Whole roots separated from 5 day old pre-fixing nodules
Whole roots from fertilized plants collected at the same time as RE and RI
Whole roots separated from fix + nodules collected 21 DAI
Whole roots separated from fix- nodules collected 21 DAI
Pre-fixing (fix+) nodules collected 5 DAI
Fix + nodules collected 21 DAI
Fix- nodules collected 21 DAI
Differentially expressed genes between tissue types
To ensure our transcriptome analysis was reliable we visualized the expression profile of the purine and ureide biosynthesis pathways (Additional file 5) and conducted qPCR on 85 additional genes (for details see experimental procedures and Additional file 6). In warm season legumes, such as soybean and Pv, purine biosynthesis is known to be highly up regulated in nodules . Our data is consistent with this as the genes in this pathway are highly up regulated in nitrogen fixing nodules compared to all other tissues (Additional file 5). The enzyme uricase (Additional file 3c) degrades ureate to allantoin, which is the main supply of N for plant nutrition. In our Pv data allantoinase, the enzyme responsible for allantoin degradation, is highly expressed early in seed and pod development, likely providing N to developing seeds (Additional file 3d). Expression of uricase and allantoinase in aerial tissues suggests ureides are degraded after being transported from the nodules. Leaves, seeds, and pods can then utilize the released NH3 and CO2 in a variety of cellular processes. These results are consistent with reports of high ureide levels observed in developing Pv seeds  and high allantoinase enzyme activity throughout pod development and seed filling as measured by Thomas et al. . Additionally, Pellissier et al.  reported allantoin transporters highly expressed in developing pods and seed coats of Pv. The results of these three studies combined with the gene expression patterns observed in our data highlight the importance of ureide metabolism in aerial tissues to provide N for developing tissues.
Gene expression analysis
Genes exhibiting tissue specific expression (Additional file 2 and Additional file 3a) are involved in a variety of gene ontology biological processes. Genes uniquely expressed in leaves are involved in amino acid phosphorylation, DNA and protein binding. Genes uniquely expressed in seed tissues include processes such as carbohydrate metabolism, as exemplified by a starch branching enzyme (SBE), which is important in amylopectin synthesis, a carbohydrate precursor . In our data, Pv SBE expression (Phvul.005G040300.1) is highest in developing seeds (Additional file 3e). These results are consistent with the high carbohydrate composition of Pv seeds reported by Broughton et al. . Nodule specific transcripts annotate as involved in oxidoreductase activity, amino acid phosphorylation and membrane transport/signaling are highly and uniquely expressed; reflecting the importance of nutrient transport to the nodule and the high energy cost of N fixation. In root tissues, genes involved in pectinecterase, carbohydrate metabolism, iron ion binding, oligopeptide membrane transport, and lipid metabolisms are uniquely expressed. Expression of these genes illustrates the role of the root in nutrient acquisition and the importance of root growth for plant health.
Seed development and metabolism
Abscisic acid (ABA) is a key hormone in seed development, important in developing desiccation tolerance and entrance into dormancy . ABA accumulation in seeds is both temporally and spatially regulated . We found high expression of ABA biosynthesis genes (Phvul.002G018700.1 and Phvul.005G031500.1) in developing seeds, with expression decreasing as the seeds matured (Additional file 3g). ABA biosynthesis is regulated by 9-cis-EPOXYCAROTENOID DIOXYGENASE (NCED9) . The expression pattern of NCED9 (Additional file 3 h) and ABA biosynthesis genes (Additional file 3 g) in Pv developing seeds is consistent with those from developing seeds in Arabidopsis [33–35].
Trehalose biosynthesis is important in regulating both seed composition and nodule metabolism [36, 37]. In seeds, TREHALOSE 6 PHOSPHATE SYNTHASE 1 (TPS1), the enzyme responsible for converting glucose-6-phosphate to trehalose-6-phosphate is thought to regulate sugar utilization . In Arabidopsis thaliana tps1 null mutants, both sucrose and starch content of seeds dramatically increased . In Pv, TPS1 expression drops dramatically as the seed develops (Additional file 3i), corresponding with increased SS expression. We hypothesize that, as in Arabidopsis, the reduced TPS1 expression promotes increased carbohydrate biosynthesis in Pv seeds.
Comparing 5 DAI pre-fixing nodules (N5) and 21 DAI fix + nodules (NE) revealed 2,932 differentially expressed genes (Additional file 14). Comparing 21 DAI fix + and fix- nodules (NE vs NI) identified 2,953 differentially expressed genes (Additional file 15). Additionally, we found 245 nodule specific genes; genes expressed in any and/or all the nodule tissues sampled, but not expressed in any other tissues (Additional file 4). Comparing these nodule specific genes to those identified in two soybean gene atlases [26, 41] identified 21 nodule specific homologs common to both species (Additional file 16) including seven TFs and four transporters. The conserved expression of these genes highlights the importance of regulating gene expression, but also the exchange of nutrients between nodules and the plant roots. Five of these sequences have no known annotation, though nodule specific expression in both species suggests these are important candidates for characterization in future nodulation and nitrogen fixation research.
Cognate genes involved in nodule development and the establishment of N fixation have been identified and cloned from multiple species [42, 43]. Using BLASTN, the homologous sequences in common bean were identified and gene expression patterns were visualized as a heat-map (Figure 5). Upon further analysis of these nodule cognate genes, we detected three expression profiles: those highly up regulated early in nodule development (N5), those highly up regulated in 21DAI fix + nodules (NE), and those highly up regulated in 21 DAI fix- nodules (NI).
The autoregulation of nodulation (AON) pathway mediates nodule formation . ASTRAY and UFD1a proteins, both expressed in leaves, function in the AON pathway . ASTRAY, encodes a bZIP TF that interacts with a nodulation autoregulation receptor kinase (NARK) [44, 45]. UFD1a expression indicates the presence of Q, a root derived signal induced upon compatible rhizobial infection . In soybean, three candidates for Q have been identified, all of which are CLE peptides . Pv encodes a single CLE homolog (Phvul.005G097000.1), which is highly up regulated in N5 and NE, but noticeably absent in NI (Figure 5). Surprisingly, we observed aerial AON genes (ASTRAY and UFD1a) expressed higher in leaves of plants inoculated with fix- rhizobia than in leaves of plants inoculated with fix + rhizobia (Figure 5). We hypothesize the fix- inoculated plant may up regulate the AON pathway to minimize resources allocated to nodules as part of a survival strategy.
Early in nodule development (N5) nod factor receptors (NFR) and nodulation signaling pathway (NSP) TFs are highly expressed (Figure 5). The early calcium spiking response induces both a calmodulin dependent protein kinase (CCaMK/DMI3), which is required and sufficient for nodule organogenesis [47–49], and nuclear porin proteins (NUPs)  (Figure 5). Additionally, genes involved in infection thread formation and elongation including ERN1, FLOT, VPY, PUB1 and RPG[50–58] are highly up regulated in N5 (Figure 5). VPY and PUB1, which are up regulated in N5 and NE, are involved in rhizobial recognition, attachment, entry, and initiation of the infection thread [53, 58]. Nodule organogenesis involves the altered differentiation and division of root cortical cells prior to the formation of the nodule primordia. In Medicago truncatula, these processes are dependent on ENOD40, which is highly expressed in N5 (Figure 5). HAP2, which promotes nodule development and the release of bacteria from the infection thread  is expressed highest in N5, but remains elevated in NE (Figure 5).
Gene Ontology (GO) categories statistically over-represented in NE enhanced genes
Number of genesa
Chorismate Mutase Activity
IMP Biosynthetic Process
Response to Stress
Cysteamine Dioxygenase Activity
Aromatic Amino Acid Biosynthesis
Purine Nucleotide Biosynthesis
Purine Base Biosynthesis
Among the nodule cognate genes, those most highly expressed in NE are: NIN transcription factors, CYCLOPS, and CERBERUS with expression profiles increasing from 5 DAI to 21 DAI in fix + nodules (2X, 2.4X, and 4X respectively, Figure 5). CYCLOPS expression is required for rhizobia infection. Nodule organogenesis is dependent on CERBERUS gene expression [49, 67–69].
The primary function of nodules is to fix N2 to NH3. Nitrogenase, the enzyme responsible for nitrogen fixation, requires sulfur and a near anaerobic environment to function. The gene encoding SYMBIOSOME SULFATE TRANSPORTER 1 (SST1), which transports sulfur into bacteroids , is expressed 15 fold higher in NE than in NI (Figure 5). The genes encoding leghemoglobin, which sequesters oxygen , are only expressed in NE (Figure 5). Once N2 is reduced to usable ammonia it must be assimilated for use and distribution throughout the plant. NADH-dependent glutamate synthase (NADH-GOGAT) is a key enzyme in ammonia assimilation . Two NADH-GOGAT genes (Phvul.009G053900.1 and Phvul.001G076400.1) are expressed 5 and 10-fold higher in NE than in NI (Figure 5), reflecting the difference in effectiveness. Transcripts encoding glutamine synthase, uricase, and allantoinase (Additional file 3 j, c, and d), each involved in primary ammonia assimilation, exhibit similar expression patterns. Nod41 was identified in Pv by Olivares et al.  as important in controlling defense responses during symbiotic interactions and maintaining the integrity of the uninfected root nodule cells. Our data is consistent with this hypothesis as Nod41 is expressed 7-fold higher in 21 DAI fix + nodules than in 21 DAI fix- nodules (Figure 5).
Genes up regulated in NI include those involved in the GO processes of autophagy and early senescence including ubiquitination, proteolysis, peptidyolysis and apoptosis. Respiratory burst oxidase homolog (RBOH) genes, which generate reactive oxygen species (ROS) , are up regulated four-fold in NI. Increased ROS production is a common defense response to pathogen attack (ie: ineffective rhizobia) and in response to abiotic stress, including nitrogen deficiency. Additionally, we observed high expression of leucine rich repeat (LRR) genes in NI (Additional file 19), likely reflecting a defense response as the plant reacts to invading bacteria . Also highly expressed in NI are genes involved in oxidation-reduction processes, membrane transport, protein binding, and amino acid phosphorylation. Among the nodule cognate genes are those encoding a second group of NIN and NSP TFs (Figure 5). This result suggests a group of alternative TFs may be induced in NI versus NE.
Type A-response regulators (RRs) negatively regulate cytokinin signaling . In lotus, RRs are rapidly induced following rhizobial inoculation in root hairs and dividing cortical cells , repressing the cytokinin signaling pathway . Inhibition of the cytokinin-signaling pathway may contribute to plant and bacterial cell differentiation. In Pv, the gene encoding RR5 is more highly expressed in NI than in NE (Figure 5). Genes encoding both CASTOR and POLLUX, both of which are required for the activation of voltage gated calcium (Ca2+) channels , are highly expressed in NI (Figure 5). The high expression of CASTOR and POLLUX genes in NI may suggest that at 21 DAI the plant is still attempting to establish SNF or may reflect the induction of Ca2+ channels by ROS as described in Pisum sativum[78, 79].
Roots and nitrogen
Gene expression profiles of Pv roots were examined from plants grown under three conditions 1) those from NO3− fertilized plants (RF), 2) those from plants with fix + effective nodules (RE), and 3) those derived from plants having fix- ineffective nodules (RI). RF and RE roots had adequate N for growth, while RI roots were N deficient. We identified 1,714 genes differentially expressed between the three 21 DAI root samples (RI, RE, and RF) (Additional file 20). The majority of these genes (1,668 genes) are differentially expressed between fertilized roots (RF) and nodulated roots (either RE or RI).
In the presence of abundant NO3−, plants will preferentially take up and utilize NO3− rather than develop SNF. NO3− transporters exhibit either low (NRT1) or high (NRT2 and NRT3) NO3− affinity [80–84]. Examination of the expression patterns of NO3− transporters in our root samples revealed plants provided with NO3− as a fertilizer induce NRT1 and NRT3 gene expression, reflecting the abundance of available NO3− (Figure 6d). In –N roots, only members of the high affinity NRT2 gene family are up regulated (Figure 6d). N-deficient plants may up regulate N transporters in an attempt to increase the N content of the plant to mitigate –N stress. Members of the low affinity NRT1 gene family are also up regulated in the roots of N2 fixing plants (Figure 6d). The constitutive expression of NRT1.1 is consistent with the recent evidence suggesting that it serves as both an N sensor and transporter [85–87]. Expression of NRT1.1 in fix + plants may be involved in N sensing.
Once N is within the plant it must be assimilated. Glutamine synthetase (GS) functions as a primary enzyme for NH4 assimilation produced from N2 fixation or NO3− nutrition [88, 89]; synthesizing glutamine from NH3 and glutamate (Additional file 3j). In SNF plants, the majority of glutamine is committed to the de-novo purine biosynthesis pathway. Alternatively, glutamine may be reduced by GOGAT. Consistent with previous studies , NADH-GOGAT expression is highest in roots (particularly YR) and NE while Fd-GOGAT is expressed highest in leaf tissues (Additional file 3 k). Plants provided with NO3− fertilization utilize glutamine in the synthesis of asparagine via asparagine synthetase (AS) , which is most highly expressed in fertilized root (RF) tissues (Additional file 3 l).
Our data shows increased expression of auxin response factors unique to –N leaves (Figure 6b). This gene expression pattern indicates increased auxin levels in –N leaves, supporting auxin as the N signal. The availability of N for proper growth and development is likely monitored throughout the plant. Auxin has been proposed as an N status mediator, signaling from root to shoot . Under low N and other nutrient stress conditions in the shoot, increased auxin is transported to the roots to enhance lateral root development, a hallmark response of –N plants [46, 80, 87].
This study provides a resource for global analysis of gene expression patterns in Pv of 24 samples from seven unique tissues across important developmental time points. The publicly available gene atlas, Pv GEA, will facilitate the use of this data for researchers querying gene expression patterns within various biological processes, as evidenced by Additional file 3. Additionally, by comparing gene expression patterns in developing seeds of Pv to those in Glycine max, we were able to identify differences potentially responsible for altered seed composition between the two closely related species. Finally, our analysis of N uptake and utilization revealed the N source is an important component of the N pathway and has a long-term effect on gene expression patterns.
Plant materials and growth conditions
Phaseolus vulgaris cv. Negro jamapa seeds were grown as described by O’Rourke et al. . At the emergence of the unifoliate, pots were assigned to one of three nitrogen (N) treatments; inoculated with Rhizobium tropici CIAT899 (fix+), Rhizobium giardini 6917 (fix-), or fertilized with a full nutrient solution. Pots assigned to the fertilization treatment were watered daily with a nutrient solution of 9 mM KNO3, 2.5 mM Ca(NO3)2, 1.0 mM Ca(H2PO4)2, 1.0 mM MgSO4, 12 μM Fe (as FeEDTA), 4.0 μM MnCl2, 22.0 μM H3BO3, 0.4 μM NaMoO4, and 1.6 μM CuSO4. Twenty-four tissue samples were collected throughout development and across all N treatments (for details see Table 1). For each nitrogen treatment, a representative plant was chosen and leaf, root, and nodule tissue samples were collected. Two plants were maintained in full nutrient solution fertilized pots. From these plants, root, leaf, flower, stem, seed and pod tissues were collected (see Table 1 for details). All tissue collected for RNA -Seq analysis was immediately flash frozen in liquid nitrogen.
RNA extraction and expression analysis
Total RNA was purified from 24 tissue samples using RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). For RNA-Seq analysis, RNA samples were shipped on dry ice overnight to National Center for Genome Resources (NCGR, Santa Fe, NM) for sequencing as described by Severin et al. . Illumina reads generated from all 24 samples are available at the NCBI SRA browser, accession number SRP046307. Illumina reads passing quality control standards, approximately 25 million sequences per sample or 596 million 36 bp sequences total, were mapped to the Phaseolus vulgaris v1.0 genome available at http://www.phytozome.net using the program Bowtie . Reads were also mapped to the predicted transcripts to account for splicing events. Reads that mapped to more than one location were counted at each mapping location. Of the 596 million reads generated, 406 million (89%) mapped to the genome with 14% of those mapping to non-coding regions. Raw gene expression counts were normalized using the RPKM (reads/Kb/Million) method [92, 93] using custom R scripts. To ensure expression profiles were not statistical artifacts as described by earlier studies [94, 95], we determined an RPKM of 3 represents a 2X coverage across the coding region, assuming equal distribution, and would be the minimum level at which a gene would be considered expressed; genes with an RPKM < 3 were considered silent. Transcripts differentially expressed between libraries were identified using NOIseq . Differentially expressed transcripts were required to have > 2-fold change in expression between samples and a probability of differential expression > 0.9. Additionally, one of the two sequences was required to have an RPKM > 3. Heatmaps illustrating expression patterns of various subgroups of transcripts were generated in R as described by Severin et al..
To identify genes exhibiting enhanced expression in NE, we determined the Euclidian distance between Z-scores for each gene. A threshold of two Euclidian distances was established as significant; genes within the threshold were identified as co-expressed.
Real time quantitative RT-PCR (qPCR) and housekeeping genes
RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) from three biological replicates of tissues grown in growth chambers under the same conditions described above. Transcript specific primers were designed using Primer3 (Frodo.wi.mit.edu). The qPCR analysis was run as described by O’Rourke et al.  for 85 genes identified as differentially expressed by NOIseq (Additional file 6). 92% of the qPCR experiments confirmed the differential expression measured by NOIseq analysis in at least two of the three biological replicates.
Genes exhibiting stable expression profiles between tissues and across growth conditions were identified as described by Severin et al.  (Additional file 22). The 10% of transcripts with the lowest CV were selected as potential housekeeping genes. This suite of stably expressed transcripts may be useful in future experiments for normalizing gene expression patterns across a variety of experimental conditions, or tissues . One of these genes (Phvul.006G165300.1) was successfully utilized as a housekeeping sequence in the qPCR analysis. Comparing the housekeeping genes proposed in this study to the eleven potential housekeeping genes identified for Pv under biotic and abiotic stress by Borges et al.  found seven genes common to both lists, illustrating the utility of this list for multiple experimental conditions.
Acetylene reductase assay
Acetylene reduction assays were performed as described by Vance et al.  with the following modifications. Plant roots from six biological replicates of each sample (roots inoculated with fix + R. tropici CIAT899 at 5 and 21 DAI and roots inoculated with fix – R. giardini 6917 21 DAI) were placed in 500 ml airtight glass containers equipped with serum stoppers. 50 ml of air was removed from each container and replaced with 50 ml of ethylene; samples were incubated at room temperature for one hour, at which time 10 ml of gas was withdrawn from the container for analyses as previously described by Vance et al. .
Nodule In Situs
A partial coding sequence for Pv leghemoglobin (645 bp) was PCR amplified and cloned into pBSSK+. Nodules inoculated with fix + R. tropici CIAT899 and fix – R. giardini 6917 were collected 21 DAI and analyzed as described by Sbabou et al. .
Availability of supporting data
The expression data used in this study is publicly available at the NCBI short read archive; accession SRP046307. Additionally, the raw and normalized datasets can be downloaded and explored at the Phaseolus vulgaris Gene Expression Atlas (Pv GEA) website, http://plantgrn.noble.org/PvGEA/.
This work was facilitated, in part, using resources at the University of Minnesota Supercomputing Institute.
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