An RNA-Seq based gene expression atlas of the

common bean
O’Rourke et al.

O’Rourke et al. BMC Genomics 2014, 15:866

http://www.biomedcentral.com/1471-2164/15/866
O’Rourke et al. BMC Genomics 2014, 15:866
http://www.biomedcentral.com/1471-2164/15/866

RESEARCH ARTICLE Open Access

An RNA-Seq based gene expression atlas of the

common bean
Jamie A O’Rourke1,7*, Luis P Iniguez2, Fengli Fu1, Bruna Bucciarelli1,3, Susan S Miller1,3, Scott A Jackson4,
Philip E McClean5, Jun Li6, Xinbin Dai6, Patrick X Zhao6, Georgina Hernandez2 and Carroll P Vance1

Abstract
Background: 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.
Results: 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.
Conclusions: 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.
Keywords: Phaseolus vulgaris cv Negro jamapa, Common bean, RNA-Seq, Symbiotic nitrogen fixation, Expression atlas,
SRP046307

Background in beans complement the high carbohydrates found in

Common bean (Phaseolus vulgaris L.), Pv, is an important maize and cassava [1]. In addition to their important con-
source of proteins, micronutrients and calories for over tribution to human health, legumes are also important
three hundred million people worldwide, mostly through- contributors to biological nitrogen (N). N is a primary nu-
out Latin America and Africa where beans are an import- trient limiting plant production [2], with the acquisition
ant component of traditional diets. The high levels of and assimilation of N second only to photosynthesis for
dietary protein (between 20 and 25%) and micronutrients plant growth and development [1]. Despite the inter-
national importance of Pv, both in terms of economics and
* Correspondence: [email protected]
consumption, it has lagged behind Medicago truncatula,
1
Department of Agronomy and Plant Genetics, University of Minnesota, St. Lotus japonicus, soybean, and other legumes in terms of
Paul, MN 55108, USA genetic resources. cDNA libraries have been used to inves-
7
Current Address: USDA-ARS, Corn Insect Crop Genetics Research Unit, Iowa
State University, Ames, IA 50011, USA
tigate phosphate stress, resistance to bean rust, and leaf de-
Full list of author information is available at the end of the article velopment [3-7]. Sequence information for Pv was greatly
© 2014 O’Rourke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
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enhanced by using Roche 454 technology coupled with on the impact of nodulation and N fixation on gene ex-
mRNA sequences to assemble 59,295 unigene sequences, pression, an important biological process for legumes.
[8], though these data are not yet publicly available. Most The 26,964 transcriptionally active genes identified in
recently, the genome sequence and predicted gene calls our data (RPKM ≥ 3 in at least one tissue) represent 78%
for Pv G 19833 has been made publicly available (www. of the 31,638 predicted genes in Pv; confirming that the
phytozome.net). This resource provides a platform for Pv tissues, time points, and treatments used in this study (as
genomic and comparative genomic analyses [9]. Sequence described in Table 1) affected a majority of the genes in
conservation and genetic colinearity between Pv and soy- the genome and provide an excellent foundation of gene
bean (Glycine max L. merr) [10,11] which diverged from a expression in future experimental comparisons. Pair-wise
common ancestor approximately 19 million years ago analyses identified differentially expressed genes between
[12,13], allows genomic information to be leveraged from both tissues and samples (Table 2 and Additional file 1,
one species to the other. respectively). These comparisons identified 11,010 genes
In this study we utilized RNA-seq to characterize ex- differentially expressed between tissues. Additionally, we
pression profiles for the transcriptome of common bean identified genes differentially expressed within tissues from
(Pv cv. Negro Jamapa). Gene expression profiles were samples collected at developmentally important time-
analyzed from 24 unique samples from seven distinct points (between seeds from three developmental stages
tissues; roots, nodules, stems, flowers, leaves, pods, and (DS): 2,694, four pod DS: 13,125, five leaf DS: 5,401, six
seeds throughout development. Our data was used as root DS: 1,458, and three nodule DS: 1,551). Finally, we
the foundation for The Pv Gene Expression Atlas (Pv identified genes exhibiting either tissue specific expression,
GEA) database, available at http://plantgrn.noble.org/ (Additional file 2 and Additional file 3a), or sample specific
PvGEA/. We utilized the expression profiles of all pre- expression, (Additional file 3b and Additional file 4).
dicted genes in Pv to examine the biological processes To ensure our transcriptome analysis was reliable we vi-
related to seed and pod development, nodulation and sualized the expression profile of the purine and ureide
symbiosis, and changes in gene expression due to nitro- biosynthesis pathways (Additional file 5) and conducted
gen availability. qPCR on 85 additional genes (for details see experimental
procedures and Additional file 6). In warm season le-
gumes, such as soybean and Pv, purine biosynthesis is
Results and discussion known to be highly up regulated in nodules [15]. Our data
Phaseolus vulgaris gene expression atlas (Pv GEA) is consistent with this as the genes in this pathway are
To facilitate additional use of the RNA-Seq data generated highly up regulated in nitrogen fixing nodules compared
for these analyses, we have developed a web-accessible to all other tissues (Additional file 5). The enzyme uricase
database, The Pv Gene Expression Atlas (Pv GEA), avail- (Additional file 3c) degrades ureate to allantoin, which is
able at http://plantgrn.noble.org/PvGEA/. This database the main supply of N for plant nutrition. In our Pv data
was built using a similar database structure, web applica- allantoinase, the enzyme responsible for allantoin degrad-
tion, architecture and tools as the LegumeIP platform [14] ation, is highly expressed early in seed and pod develop-
to retrieve and visualize the gene expression patterns using ment, likely providing N to developing seeds (Additional
RNA-seq data. To facilitate the mining of the data in- file 3d). Expression of uricase and allantoinase in aerial tis-
cluded in Pv GEA, we have provided the capability to: (i) sues suggests ureides are degraded after being transported
visualize expression profiles of genes of interest, (ii) iden- from the nodules. Leaves, seeds, and pods can then utilize
tify genes exhibiting certain expression patterns in specific the released NH3 and CO2 in a variety of cellular pro-
tissues, (iii) identify genes and gene expression patterns cesses. These results are consistent with reports of high
based on www.phytozome.net annotation terms; and (iv) ureide levels observed in developing Pv seeds [16] and
download the entire data set, either raw or normalized, in high allantoinase enzyme activity throughout pod develop-
tabular form to facilitate the analysis of more complicated ment and seed filling as measured by Thomas et al. [17].
biological questions. Using the predicted gene calls of the Additionally, Pellissier et al. [18] reported allantoin trans-
G 19833 Pv genome to build the Pv GEA database means porters highly expressed in developing pods and seed
it can be easily expanded to integrate RNA-Seq data from coats of Pv. The results of these three studies combined
future experiments. Currently, Pv GEA includes gene ex- with the gene expression patterns observed in our data
pression profiles from 24 samples isolated from roots, root highlight the importance of ureide metabolism in aerial
nodules, stems, leaves, flowers, pods, and seeds at various tissues to provide N for developing tissues.
developmental stages under ideal growth conditions. In-
cluded in this dataset are transcripts from eight samples Gene expression analysis
including nodule, root, and leaf tissues for plants having Genes exhibiting tissue specific expression (Additional file 2
either fix + or fix- root nodules; providing preliminary data and Additional file 3a) are involved in a variety of gene
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Table 1 Tissue samples isolated from Phaseolus vulgaris cv. Negro jamapa for RNA-Seq analysis
Organ Sample IDa Sample description Reads Reads Genes
sequencedb mappedc expressedd
Leaves YL Fully expanded 2nd trifoliate leaf tissue from plants provided with fertilizer 24,118,479 21,395,546 19,466
L5 Leaf tissue collected 5 DAI with effective rhizobium 25,297,092 19,762,749 18,966
LF Leaf tissue from fertilized plants collected at the same time of LE and LI 22,692,275 17,581,928 16,103
LE Leaf tissue collected 21 DAI with effective rhizobium 23,366,279 17,714,097 16,081
LI Leaf tissue collected 21 DAI with ineffective rhizobium 23,257,968 18,820,812 18,523
Stem YS All stem internodes above the cotyledon collected at the 2nd trifoliate stage 27,696,970 22,718,724 20,299
ST Shoot tip, including the apical meristem, collected at the 2nd trifoliate stage 25,826,838 22,358,410 21,142
Flower FY Young flowers, collected prior to floral emergence 23,334,037 16,503,930 20,055
Pods PY Young pods, collected 1 to 4 days after floral senescence. Samples contain 26,234,498 17,381,695 12,115
developing embryos at globular stage
PH Pods approximately 9 cm long, associated with seeds at heart stage (pod only) 24,986,174 19,130,051 19,065
P1 Pods between 10 and 11 cm long, associated with stage 1 seeds (pod only) 24,349,622 16,400,591 15,831
P2 Pods between 12 and 13 cm long associated with stage 2 seeds (pod only) 21,647,774 18,018,224 18,311
Seeds SH Heart stage seeds, between 3 and 4 mm across and ~7 mg 28,222,798 22,972,385 18,668
S1 Stage 1 seeds, between 6 and 7 mm across and ~50 mg 21,395,296 17,004,466 16,949
S2 Stage 2 seeds, between 8 and 10 mm across and 140–150 mg 24,696,630 19,949,204 15,363
Roots RT Root tips, 0.5 cm of tissue, collected from fertilized plants at 2nd trifoliate stage of 24,536,948 21,680,391 18,514
development
YR Whole roots, including root tips, collected at the 2nd trifoliate stage of development 25,140,904 20,962,342 19,170
R5 Whole roots separated from 5 day old pre-fixing nodules 27,423,246 23,034,248 19,865
RF Whole roots from fertilized plants collected at the same time as RE and RI 25,121,968 21,858951 20,305
RE Whole roots separated from fix + nodules collected 21 DAI 24,449,104 21,325,105 20,450
RI Whole roots separated from fix- nodules collected 21 DAI 27,834,770 24,188,904 20,697
Nodules N5 Pre-fixing (fix+) nodules collected 5 DAI 23,909,973 20,774,095 19,102
NE Fix + nodules collected 21 DAI 26,350,845 23,035,381 17,011
NI Fix- nodules collected 21 DAI 24,875,317 21,877,973 19,278
a
A two-letter ID assigned to each sample. bNumber of single end 36 bp reads generated for each sample. cNumber of RNA-Seq reads mapping to the genome
using Bowtie. dNumber of predicted genes expressed with an RPKM ≥ 3 in each sample.

ontology biological processes. Genes uniquely expressed (SBE), which is important in amylopectin synthesis, a
in leaves are involved in amino acid phosphorylation, carbohydrate precursor [19]. In our data, Pv SBE expres-
DNA and protein binding. Genes uniquely expressed in sion (Phvul.005G040300.1) is highest in developing seeds
seed tissues include processes such as carbohydrate me- (Additional file 3e). These results are consistent with the
tabolism, as exemplified by a starch branching enzyme high carbohydrate composition of Pv seeds reported by
Broughton et al. [1]. Nodule specific transcripts annotate
Table 2 Differentially expressed genes between tissue as involved in oxidoreductase activity, amino acid phos-
types phorylation and membrane transport/signaling are highly
Seed Pod Stem Leaf Root Nodule and uniquely expressed; reflecting the importance of nu-
Seed 0 909 2,521 446 1,690 840 trient transport to the nodule and the high energy cost of
Pod 665 0 227 844 504 455 N fixation. In root tissues, genes involved in pectinec-
Stem 1,299 946 0 1,847 602 773 terase, carbohydrate metabolism, iron ion binding, oligo-
peptide membrane transport, and lipid metabolisms are
Leaf 1,354 1,111 690 0 1,112 1,084
uniquely expressed. Expression of these genes illustrates
Root 1,986 1,867 731 3,003 0 679
the role of the root in nutrient acquisition and the import-
Nodule 1,554 936 526 1,789 375 0 ance of root growth for plant health.
The number of genes in each cell represents genes up regulated in the The 6,667 known transcription factors (TF) in soybean
column tissue compared to the row tissue. For the comparison Seeds vs Pods
1,574 genes are differentially expressed; 909 up regulated in pods and 665
(downloaded from SoyDB) [20] were compared to Pv genes
up-regulated in seeds. using TBLASTN (e-value 1e-30). This analysis identified
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3,726 putative TFs in the Pv genome, representing 52 of Five other TF families (AS2, AUX, NAC, SBP, and ZD-
the 64 transcription factor families in soybean (Additional HD) showed expression patterns that were statistically sig-
file 7). The 3,726 TFs identified in Pv is almost exactly half nificant in multiple tissues.
the number identified in soybean, as expected since soy-
bean has undergone a whole genome duplication event Seed development and metabolism
not experienced by Pv [12,13]. The average expression of Pv seeds are an integral component of diets around the
TFs in all seed tissues was much lower than that of other world. Unlike soybean seeds, which are valued for high
tissues, including developing pods. Fisher tests confirmed oil and protein content, Pv beans provide high levels of
26 TF families exhibited statistically enhanced or repressed protein and carbohydrates making it a highly nutritious
expression by tissue type (Figure 1). Twenty-one TF fam- food for human consumption. The seed and pod sam-
ilies exhibited altered expression patterns in a single tissue. ples represent an extended time-course collection of the

Figure 1 Transcription factor family expression profile by tissue. Fisher’s test identified 26 transcription factor families with higher or lower
than expected expression in a specific tissue (leaf, blue; pod, red; seed, green; root, purple; nodule, teal). Tissues with statistically significant gene
expression patterns are denoted to the right of the graph; Leaf, L; Pod, P; Seed, S; Root, R; Nodule, N.
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same tissue spanning great developmental changes. Ap- steadily decreasing and 1,236 genes with consistently in-
proximately half (16,292) of the 31,638 predicted genes are creasing expression patterns (Figure 2).
expressed in Pv seeds with 12,182 genes expressed in all Soybean and Pv, although closely related species, have
three stages of seed development examined. In Pv pods distinct seed compositions. While soybean accumulates
17,248 genes are expressed in at least one of the four de- oil and protein, Pv accumulates carbohydrates and pro-
velopmental stages examined. We identified genes differ- tein [1,25]. Comparing the 500 most highly expressed
entially expressed between seeds and pods at the three genes in both Pv and soybean seeds [26] allowed us to
stages of development (8,189 genes; Additional file 8, identify genes important for general seed development
Additional file 9, and Additional file 10), and genes differ- (Figure 3a). Genes involved in carbohydrate biosynthesis
entially expressed within seeds (2,694) and pods (13,125) are highly up regulated in Pv seeds while genes involved
throughout development (Additional file 11 and Additional in fatty acid biosynthesis are highly up regulated in de-
file 12). Additionally, we identified 9,702 genes with con- veloping soybean seeds (Figure 3b and c). The starch
sistently decreasing expression levels as the seed develops, synthase (STS) genes, particularly Phvul.001G082500.1,
including 1,196 TFs. By comparison, 753 genes were iden- are highly expressed in Pv seeds (RPKMs: S1 = 240, S2 =
tified with increasing expression levels as the seed develops 286) but not in soybean seeds (RPKM < 15) (Figure 3b
(Figure 2) including 70 TFs from 25 families, including HB and c). STS is required for amylopectin biosynthesis, a
(Phvul.007G064100.1), MYB (Phvul.001G025200.1), and component of carbohydrates. Similarly, sucrose acts as a
ARF (Phvul.011G080100.1) (Additional file 3f). Members key regulator of seed carbon flux; high sucrose synthase
of three of these TF families (HB, MYB, and ARF) are seed (SS) activity in developing seeds may channel available
specific in Arabidopsis, M. truncatula, and our Pv data carbon towards carbohydrate biosynthesis and away
[21,22]. Additionally, members of these TF families are from fatty acid biosynthesis [27]. In our Pv RNA-Seq
among the TFs identified by Le et al. [23] as differentially data, genes encoding SS are highly expressed in develop-
expressed between structures of developing Phaseolus coc- ing seeds (Figure 3b). In Arabidopsis, SS loss of function
cineus (scarlet runner bean) seeds that are also differen- mutants favor fatty acid and protein biosynthesis over
tially expressed in Pv seeds at different developmental starch biosynthesis in seed development, resulting in a
stages. All these results are consistent with a study by 55% increase of fatty acids and a near 70% reduction in
Hajduch et al. [24], which determined the expression of starch content of mature seeds [28]. The role of SS in
proteins involved in primary and secondary metabolism, regulating carbohydrate synthesis is further supported by
cell growth and division, signal transduction, and protein the low expression in developing soybean seeds (Figure 3c)
synthesis all decrease as the seeds develop. Conversely, in (Severin et al. [26]), which are valued for oil and pro-
the pods we identified 39 genes with expression levels tein. The synthesis of polyunsaturated fatty acids is reg-
ulated by FATTY ACID DESATURASE 2 (FAD2) [29].
In soybeans, FAD2 is highly expressed in developing
seeds while in Pv FAD2 is expressed early in seed and
pod development, but at a much lower level (Figure 3b
and c).
Seed development in multiple species is regulated by
four master TFs: LEAFY COTYLEDON1 (LEC1), LEAFY
COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3
(ABI3), and WRINKLED 1 (WRI1) [21,22,30]. Using
BLASTP, we queried the Arabidopsis protein sequences to
identify homologous sequences in the Pv predicted genes
(Figure 4). The homolog for LEC2 was only weakly
expressed (RPKM = 4) mid-seed development in Pv. Seeds
of Arabidopsis loss of function lec2 mutants accumulated
15% less protein and 30% less oil while the seed starch
content increased five fold as compared to wild type plants
[31]. The altered seed composition of lec2 mutant plants
closely resembles that of Pv, suggesting down regulation
of LEC2 may affect seed composition. LEC2 controls the
gene expression of WRI1, which also exhibits low ex-
Figure 2 Expression trends in seed and pod development. pression patterns in Pv developing seeds (RPKM: SH =
Genes with consistent expression patterns as seeds and pods
5, S1 = 9, S2 = 5). WRI1 expression modulates the ex-
develop, transcription factors denoted in parentheses.
pression of a set of genes controlling late glycolysis and
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Figure 3 (See legend on next page.)

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(See figure on previous page.)

Figure 3 Comparison of soybean and common bean seeds. (a) Comparing the top 1,500 expressed genes (regardless of seed stage) in
soybean (as reported by Severin et al. [26]) and common bean seeds. (b and c) Expression profiles of genes involved in fatty acid and starch
biosynthesis pathways in developing seed tissues. Glb2 (GLABARA 2), ACoAC (Acetyl CoA Carboxylase), FAD2 and FAD3 (fatty acid desaturase),
AAP1 and 2 (amino acid transporter), PEPC (phosphoenolpyruvate carboxylase), STS (starch synthase), STB (starch branching enzyme), SS (sucrose
synthase). (b) Gene expression profiles in common bean, (c) Gene expression profiles in soybean (as reported by Severin et al. [26]).

fatty acid biosynthesis. The low expression of both (Additional file 3 g) in Pv developing seeds is consistent
LEC2 and WRI1 may relate to the lower oil composition with those from developing seeds in Arabidopsis
of Pv. [33-35].
Abscisic acid (ABA) is a key hormone in seed develop- Trehalose biosynthesis is important in regulating both
ment, important in developing desiccation tolerance and seed composition and nodule metabolism [36,37]. In
entrance into dormancy [30]. ABA accumulation in seeds, TREHALOSE 6 PHOSPHATE SYNTHASE 1
seeds is both temporally and spatially regulated [27]. (TPS1), the enzyme responsible for converting glucose-
We found high expression of ABA biosynthesis genes 6-phosphate to trehalose-6-phosphate is thought to
(Phvul.002G018700.1 and Phvul.005G031500.1) in devel- regulate sugar utilization [32]. In Arabidopsis thaliana
oping seeds, with expression decreasing as the seeds tps1 null mutants, both sucrose and starch content of
matured (Additional file 3g). ABA biosynthesis is seeds dramatically increased [38]. In Pv, TPS1 expres-
regulated by 9-cis-EPOXYCAROTENOID DIOXYGEN- sion drops dramatically as the seed develops (Additional
ASE (NCED9) [32]. The expression pattern of NCED9 file 3i), corresponding with increased SS expression. We
(Additional file 3 h) and ABA biosynthesis genes hypothesize that, as in Arabidopsis, the reduced TPS1

Figure 4 Seed master transcription factor expression. The expression profiles (as Z-scores: red = high, blue = low) of four transcription factors
that regulate seed development in multiple species. Note the low expression of LEC2 (RPKM = 4) and WRI1 (RPKM = 5–9) in developing seeds. See
Table 1 for tissue descriptions.
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expression promotes increased carbohydrate biosyn- [26,41] identified 21 nodule specific homologs common to
thesis in Pv seeds. both species (Additional file 16) including seven TFs and
four transporters. The conserved expression of these genes
Nodule analysis highlights the importance of regulating gene expression,
Legumes have established a unique symbiotic relation- but also the exchange of nutrients between nodules and
ship with Rhizobium, which allows legumes to fix at- the plant roots. Five of these sequences have no known an-
mospheric N2 into biologically useful NH3. For this notation, though nodule specific expression in both species
experiment plants were provided with nutrients con- suggests these are important candidates for characterization
taining NO−3 nitrogen for optimal growth conditions or in future nodulation and nitrogen fixation research.
inoculated with either effective fix + Rhizobium tropici Cognate genes involved in nodule development and
CIAT899 or ineffective fix- Rhizobium giardini 6917 to the establishment of N fixation have been identified and
induce nodulation. Plants inoculated with normal (fix+) cloned from multiple species [42,43]. Using BLASTN,
R. tropici appeared green and healthy, though smaller the homologous sequences in common bean were iden-
than plants provided with nitrate fertilizer (Additional tified and gene expression patterns were visualized as a
file 13a). This phenotype is consistent with previous heat-map (Figure 5). Upon further analysis of these nod-
studies reporting the overall growth of N2 fixing Pv ule cognate genes, we detected three expression profiles:
plants is restricted compared to fertilized plants, likely those highly up regulated early in nodule development
due to altered carbon partitioning [39,40]. Fix + plants (N5), those highly up regulated in 21DAI fix + nodules
inoculated with R. giardini were nitrogen (N) deficient, (NE), and those highly up regulated in 21 DAI fix- nod-
exhibiting severe chlorosis and a stunted phenotype ules (NI).
(Additional file 13a). Small, pre-fixing white nodules The autoregulation of nodulation (AON) pathway medi-
(N5) were isolated from root tissues of plants inoculated ates nodule formation [44]. ASTRAY and UFD1a proteins,
with effective fix + R. tropici five days after inoculation both expressed in leaves, function in the AON pathway
(DAI). At 21 DAI nodules were collected from plants [44]. ASTRAY, encodes a bZIP TF that interacts with a
inoculated with either fix + R. tropici (NE) or fix- R. nodulation autoregulation receptor kinase (NARK) [44,45].
giardini (NI) (Additional file 13b and c respectively). UFD1a expression indicates the presence of Q, a root de-
Microscopic imaging of fix-nodules 21 DAI (NI) re- rived signal induced upon compatible rhizobial infection
vealed early senescing cells with few, if any, infected [44]. In soybean, three candidates for Q have been identi-
cells compared to fix + nodules formed 21 DAI (Additional fied, all of which are CLE peptides [46]. Pv encodes a sin-
file 13d and e). In situ hybridization analysis was used to gle CLE homolog (Phvul.005G097000.1), which is highly
visualize the localization pattern of leghemoglobin tran- up regulated in N5 and NE, but noticeably absent in NI
scripts in these two nodule types. Fix- nodules collected 21 (Figure 5). Surprisingly, we observed aerial AON genes
DAI (NI) exhibited little to no expression of leghemoglo- (ASTRAY and UFD1a) expressed higher in leaves of plants
bin transcripts while fix + nodules collected 21 DAI (NE) inoculated with fix- rhizobia than in leaves of plants ino-
exhibited high expression levels, likely mirroring the bacte- culated with fix + rhizobia (Figure 5). We hypothesize the
roid colonization patterning (Additional file 13f and g) and fix- inoculated plant may up regulate the AON pathway to
directly reflecting the gene expression patterns observed in minimize resources allocated to nodules as part of a sur-
the RNA-Seq data (Figure 5). Nodule acetylene reduction vival strategy.
assays failed to detect nitrogenase activity at 5 DAI in pre- Early in nodule development (N5) nod factor receptors
fixing nodules and at 21 DAI with fix-nodules. Fix + nod- (NFR) and nodulation signaling pathway (NSP) TFs are
ules (and associated roots) from plants 21 DAI reduced highly expressed (Figure 5). The early calcium spiking
320 nm/hr/gfw (roots) of acetylene, indicating high N2 fix- response induces both a calmodulin dependent protein
ation activity. Leaf tissue was also collected at 5 DAI for kinase (CCaMK/DMI3), which is required and sufficient
fix + plants and 21 DAI for both fix + and fix- inoculated for nodule organogenesis [47-49], and nuclear porin pro-
plants (see Table 1). teins (NUPs) [44] (Figure 5). Additionally, genes involved
Comparing 5 DAI pre-fixing nodules (N5) and 21 DAI in infection thread formation and elongation including
fix + nodules (NE) revealed 2,932 differentially expressed ERN1, FLOT, VPY, PUB1 and RPG [50-58] are highly up
genes (Additional file 14). Comparing 21 DAI fix + and fix- regulated in N5 (Figure 5). VPY and PUB1, which are up
nodules (NE vs NI) identified 2,953 differentially expressed regulated in N5 and NE, are involved in rhizobial recogni-
genes (Additional file 15). Additionally, we found 245 nod- tion, attachment, entry, and initiation of the infection
ule specific genes; genes expressed in any and/or all the thread [53,58]. Nodule organogenesis involves the altered
nodule tissues sampled, but not expressed in any other tis- differentiation and division of root cortical cells prior to
sues (Additional file 4). Comparing these nodule specific the formation of the nodule primordia. In Medicago trun-
genes to those identified in two soybean gene atlases catula, these processes are dependent on ENOD40 [59],
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Figure 5 Nodulation gene expression patterns. Expression patterns (as Z-scores) of Pv homologs of genes involved in nodulation and symbiosis
identified in Lotus japonicus, Medicago truncatula, and Glycine max. Red indicates a positive Z-score while blue indicates a negative Z-score. Genes
common to both symbiotic nitrogen fixation and mycorrhizal symbiosis are denoted with an asterisk (*). See Table 1 for tissue descriptions.

which is highly expressed in N5 (Figure 5). HAP2, which Consistent with these results, the two most statistically
promotes nodule development and the release of bacteria significant GO categories among the 402 genes are
from the infection thread [60] is expressed highest in N5, GO:0005215 (P-value = 0.002), associated with transport ac-
but remains elevated in NE (Figure 5). tivity and GO:0006857, associated with oligopeptide trans-
Genes highly expressed in NE are involved in processes port (P-value = 0.007). Increased expression of transporters
such as amino acid biosynthesis, nitrogen metabolism, in nodule tissues is consistent with previous reports [63] of
carbohydrate metabolism, membrane transport, and sulfur nodule organogenesis gene expression. Also important to
assimilation (cysteamine dioxygenase). We identified 402 nodule function is carbohydrate metabolism, which is sta-
genes highly expressed in NE as compared to all other tistically over-represented in genes highly expressed in NE
tissues (Additional file 17 and Additional file 18). These (Table 3, GO:0030246) [63]. Among the 402 genes up regu-
genes are likely involved in the establishment of symbiosis lated in NE are 34 TFs belonging to 18 different families
and symbiotic nitrogen fixation (SNF). Among these 402 and 73 transporters. Four of the TFs belong to the Nodule
genes, 73 encoding a transmembrane domain, 49% of Inception (NIN) family. NIN TFs mediate signals of rhizo-
which relate to transport including Phvul.002G300900.1, bial infection including: root hair curling, infection thread
which encodes a SWEET4 homolog. SWEET genes mediate formation, and the initiation of the nodule primodia
sucrose transport to the phloem [61]. In Arabidopsis, [64-66]. They are also involved in regulating gene expres-
SWEET4 is up regulated by pathogen infection, likely being sion in response to nitrate. Additional highly expressed
co-opted to aid pathogen growth [62]. We hypothesize this genes in NE are members of the shi related sequence (SRS)
function is conserved in Pv upon fix + Rhizobum infection. TF family, with 32% of the familial expression from nodules
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(Figure 1). SRS TFs mediate protein:protein interactions in- Genes up regulated in NI include those involved in the
volved in ubiquitination for targeted proteolysis. Expression GO processes of autophagy and early senescence including
data from both Pv and soybean [26,41] indicates this family ubiquitination, proteolysis, peptidyolysis and apoptosis.
is highly expressed in both roots and nodules of legumes Respiratory burst oxidase homolog (RBOH) genes, which
(Figure 1). generate reactive oxygen species (ROS) [74], are up regu-
Among the nodule cognate genes, those most highly lated four-fold in NI. Increased ROS production is a com-
expressed in NE are: NIN transcription factors, CYC- mon defense response to pathogen attack (ie: ineffective
LOPS, and CERBERUS with expression profiles increas- rhizobia) and in response to abiotic stress, including nitro-
ing from 5 DAI to 21 DAI in fix + nodules (2X, 2.4X, gen deficiency. Additionally, we observed high expression
and 4X respectively, Figure 5). CYCLOPS expression is of leucine rich repeat (LRR) genes in NI (Additional file
required for rhizobia infection. Nodule organogenesis is 19), likely reflecting a defense response as the plant reacts
dependent on CERBERUS gene expression [49,67-69]. to invading bacteria [75]. Also highly expressed in NI are
The primary function of nodules is to fix N2 to NH3. genes involved in oxidation-reduction processes, mem-
Nitrogenase, the enzyme responsible for nitrogen fixation, brane transport, protein binding, and amino acid phos-
requires sulfur and a near anaerobic environment to func- phorylation. Among the nodule cognate genes are those
tion. The gene encoding SYMBIOSOME SULFATE encoding a second group of NIN and NSP TFs (Figure 5).
TRANSPORTER 1 (SST1), which transports sulfur This result suggests a group of alternative TFs may be in-
into bacteroids [70], is expressed 15 fold higher in NE duced in NI versus NE.
than in NI (Figure 5). The genes encoding leghemoglobin, Type A-response regulators (RRs) negatively regulate
which sequesters oxygen [71], are only expressed in NE cytokinin signaling [76]. In lotus, RRs are rapidly in-
(Figure 5). Once N2 is reduced to usable ammonia it must duced following rhizobial inoculation in root hairs and
be assimilated for use and distribution throughout the dividing cortical cells [77], repressing the cytokinin sig-
plant. NADH-dependent glutamate synthase (NADH- naling pathway [48]. Inhibition of the cytokinin-signaling
GOGAT) is a key enzyme in ammonia assimilation [72]. pathway may contribute to plant and bacterial cell differ-
Two NADH-GOGAT genes (Phvul.009G053900.1 and entiation. In Pv, the gene encoding RR5 is more highly
Phvul.001G076400.1) are expressed 5 and 10-fold higher expressed in NI than in NE (Figure 5). Genes encoding
in NE than in NI (Figure 5), reflecting the difference in both CASTOR and POLLUX, both of which are re-
effectiveness. Transcripts encoding glutamine synthase, quired for the activation of voltage gated calcium (Ca2+)
uricase, and allantoinase (Additional file 3 j, c, and d), each channels [74], are highly expressed in NI (Figure 5). The
involved in primary ammonia assimilation, exhibit similar high expression of CASTOR and POLLUX genes in NI
expression patterns. Nod41 was identified in Pv by Olivares may suggest that at 21 DAI the plant is still attempting
et al. [73] as important in controlling defense responses to establish SNF or may reflect the induction of Ca2+
during symbiotic interactions and maintaining the integrity channels by ROS as described in Pisum sativum [78,79].
of the uninfected root nodule cells. Our data is consistent
with this hypothesis as Nod41 is expressed 7-fold higher Roots and nitrogen
in 21 DAI fix + nodules than in 21 DAI fix- nodules Gene expression profiles of Pv roots were examined from
(Figure 5). plants grown under three conditions 1) those from NO−3

Table 3 Gene Ontology (GO) categories statistically over-represented in NE enhanced genes

GO ID P-value Number of genesa Description
GO:0005215 0.00271 14 Transporter Activity
GO:0006857 0.00702 8 Oligopeptide Transport
GO:0004106 0.00871 3 Chorismate Mutase Activity
GO:0006188 0.01178 2 IMP Biosynthetic Process
GO:0006950 0.01773 7 Response to Stress
GO:0047800 0.01929 3 Cysteamine Dioxygenase Activity
GO:0030246 0.01929 3 Carbohydrate Binding
GO:0009073 0.01929 3 Aromatic Amino Acid Biosynthesis
GO:0006164 0.02765 2 Purine Nucleotide Biosynthesis
GO:0009113 0.02765 2 Purine Base Biosynthesis
GO:0030259 0.03735 3 Lipid Glycosylation
a
The number of genes on NE enhanced list with GO ID of interest.
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fertilized plants (RF), 2) those from plants with fix + effect- synthesis of asparagine via asparagine synthetase (AS)
ive nodules (RE), and 3) those derived from plants having [88], which is most highly expressed in fertilized root
fix- ineffective nodules (RI). RF and RE roots had adequate (RF) tissues (Additional file 3 l).
N for growth, while RI roots were N deficient. We identi- Our data shows increased expression of auxin re-
fied 1,714 genes differentially expressed between the three sponse factors unique to –N leaves (Figure 6b). This
21 DAI root samples (RI, RE, and RF) (Additional file 20). gene expression pattern indicates increased auxin levels
The majority of these genes (1,668 genes) are differentially in –N leaves, supporting auxin as the N signal. The
expressed between fertilized roots (RF) and nodulated availability of N for proper growth and development is
roots (either RE or RI). likely monitored throughout the plant. Auxin has been
Comparing gene expression patterns between RI (roots proposed as an N status mediator, signaling from root to
from fix- plants) and both RF and RE identified 426 and
46 genes differentially expressed between root samples re-
spectively (Figure 6c). Additionally, 210 genes were dif-
ferentially expressed between RE and RF. These 210
sequences represent genes differentially expressed due to
the N source (Figure 6c). A similar comparison of leaf tis-
sues collected from each of the plants revealed 116 genes
differentially expressed between + N leaves due to different
N sources (Figure 6a). These 116 genes indicate that the
source of N (either via N2 fixation or NO−3 fertilization)
has a long-term impact on plant gene expression. Among
all 2,641 genes differentially expressed between samples
due to the N source are 340 TFs, the majority of which
are up regulated in –N tissues (Additional file 21 a and b).
In the presence of abundant NO−3 , plants will preferen-
tially take up and utilize NO−3 rather than develop SNF.
NO−3 transporters exhibit either low (NRT1) or high
(NRT2 and NRT3) NO−3 affinity [80-84]. Examination of
the expression patterns of NO−3 transporters in our root
samples revealed plants provided with NO−3 as a fertilizer
induce NRT1 and NRT3 gene expression, reflecting the
abundance of available NO−3 (Figure 6d). In –N roots, only
members of the high affinity NRT2 gene family are up reg-
ulated (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 affin-
ity 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 suggest-
ing that it serves as both an N sensor and transporter
[85-87]. Expression of NRT1.1 in fix + plants may be in-
volved in N sensing.
Once N is within the plant it must be assimilated. Glu-
tamine synthetase (GS) functions as a primary enzyme
for NH4 assimilation produced from N2 fixation or NO−3
nutrition [88,89]; synthesizing glutamine from NH3 and
glutamate (Additional file 3j). In SNF plants, the major- Figure 6 Impact of nitrogen source on gene expression
patterns. Genes differentially expressed between leaf samples (a)
ity of glutamine is committed to the de-novo purine bio-
and root samples (c) due to the nitrogen source. Heatmaps of gene
synthesis pathway. Alternatively, glutamine may be expression profiles, represented by Z-scores; red indicates a positive
reduced by GOGAT. Consistent with previous studies Z-score, blue indicates a negative Z-score. (b) auxin response factor
[72], NADH-GOGAT expression is highest in roots (par- expression in leaves. (d) nitrogen transporter expression; plants
ticularly YR) and NE while Fd-GOGAT is expressed provided with NO−3 up regulate low affinity N transporters 1 and 3,
while N deficient plants up regulate high affinity transporter NRT2.
highest in leaf tissues (Additional file 3 k). Plants pro-
Plants fixing N2, show an increased expression of NRT1.
vided with NO−3 fertilization utilize glutamine in the
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shoot [80]. Under low N and other nutrient stress condi- control standards, approximately 25 million sequences
tions in the shoot, increased auxin is transported to the per sample or 596 million 36 bp sequences total, were
roots to enhance lateral root development, a hallmark mapped to the Phaseolus vulgaris v1.0 genome available
response of –N plants [46,80,87]. at www.phytozome.net using the program Bowtie [91].
Reads were also mapped to the predicted transcripts to
Conclusion account for splicing events. Reads that mapped to more
This study provides a resource for global analysis of gene than one location were counted at each mapping loca-
expression patterns in Pv of 24 samples from seven tion. Of the 596 million reads generated, 406 million
unique tissues across important developmental time (89%) mapped to the genome with 14% of those map-
points. The publicly available gene atlas, Pv GEA, will fa- ping to non-coding regions. Raw gene expression counts
cilitate the use of this data for researchers querying gene were normalized using the RPKM (reads/Kb/Million)
expression patterns within various biological processes, method [92,93] using custom R scripts. To ensure ex-
as evidenced by Additional file 3. Additionally, by com- pression profiles were not statistical artifacts as de-
paring gene expression patterns in developing seeds of scribed by earlier studies [94,95], we determined an
Pv to those in Glycine max, we were able to identify dif- RPKM of 3 represents a 2X coverage across the coding
ferences potentially responsible for altered seed compos- region, assuming equal distribution, and would be the
ition between the two closely related species. Finally, our minimum level at which a gene would be considered
analysis of N uptake and utilization revealed the N expressed; genes with an RPKM < 3 were considered
source is an important component of the N pathway and silent. Transcripts differentially expressed between li-
has a long-term effect on gene expression patterns. braries were identified using NOIseq [96]. Differentially
expressed transcripts were required to have > 2-fold
change in expression between samples and a probability
Methods of differential expression > 0.9. Additionally, one of the
Plant materials and growth conditions
two sequences was required to have an RPKM > 3. Heat-
Phaseolus vulgaris cv. Negro jamapa seeds were grown maps illustrating expression patterns of various sub-
as described by O’Rourke et al. [90]. At the emergence groups of transcripts were generated in R as described
of the unifoliate, pots were assigned to one of three ni- by Severin et al. [26].
trogen (N) treatments; inoculated with Rhizobium tropici To identify genes exhibiting enhanced expression in
CIAT899 (fix+), Rhizobium giardini 6917 (fix-), or fertil- NE, we determined the Euclidian distance between Z-
ized with a full nutrient solution. Pots assigned to the scores for each gene. A threshold of two Euclidian dis-
fertilization treatment were watered daily with a nutrient tances was established as significant; genes within the
solution of 9 mM KNO3, 2.5 mM Ca(NO3)2, 1.0 mM Ca threshold were identified as co-expressed.
(H2PO4)2, 1.0 mM MgSO4, 12 μM Fe (as FeEDTA),
4.0 μM MnCl2, 22.0 μM H3BO3, 0.4 μM NaMoO4, and Real time quantitative RT-PCR (qPCR) and housekeeping
1.6 μM CuSO4. Twenty-four tissue samples were col- genes
lected throughout development and across all N treat- RNA was extracted using the RNeasy Plant Mini Kit
ments (for details see Table 1). For each nitrogen (Qiagen, Valencia, CA, USA) from three biological repli-
treatment, a representative plant was chosen and leaf, cates of tissues grown in growth chambers under the same
root, and nodule tissue samples were collected. Two conditions described above. Transcript specific primers
plants were maintained in full nutrient solution fertilized were designed using Primer3 (Frodo.wi.mit.edu). The
pots. From these plants, root, leaf, flower, stem, seed and qPCR analysis was run as described by O’Rourke et al.
pod tissues were collected (see Table 1 for details). All [90] for 85 genes identified as differentially expressed by
tissue collected for RNA -Seq analysis was immediately NOIseq (Additional file 6). 92% of the qPCR experiments
flash frozen in liquid nitrogen. confirmed the differential expression measured by NOIseq
analysis in at least two of the three biological replicates.
RNA extraction and expression analysis Genes exhibiting stable expression profiles between tis-
Total RNA was purified from 24 tissue samples using sues and across growth conditions were identified as de-
RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). For scribed by Severin et al. [26] (Additional file 22). The 10%
RNA-Seq analysis, RNA samples were shipped on dry of transcripts with the lowest CV were selected as poten-
ice overnight to National Center for Genome Resources tial housekeeping genes. This suite of stably expressed
(NCGR, Santa Fe, NM) for sequencing as described by transcripts may be useful in future experiments for nor-
Severin et al. [26]. Illumina reads generated from all 24 malizing gene expression patterns across a variety of ex-
samples are available at the NCBI SRA browser, acces- perimental conditions, or tissues [97]. One of these genes
sion number SRP046307. Illumina reads passing quality (Phvul.006G165300.1) was successfully utilized as a
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housekeeping sequence in the qPCR analysis. Comparing Additional file 9: Genes differentially expressed between S1 and P1.
the housekeeping genes proposed in this study to the Additional file 10: Genes differentially expressed between S2 and P2.
eleven potential housekeeping genes identified for Pv Additional file 11: Genes differentially expressed as seeds develop.
under biotic and abiotic stress by Borges et al. [98] found Additional file 12: Genes differentially expressed as pods develop.
seven genes common to both lists, illustrating the utility Additional file 13: Nitrogen Deficiency, Plants, Nodules, and In
of this list for multiple experimental conditions. situs. (a) Plant phenotypes, plant on the left provided with nitrate (NO−3 )
fertilizer, middle plant inoculated with fix + rhizobium, plant on right
inoculated with fix- rhizobium (nodules form, but no N2 fixation).
Acetylene reductase assay (b) Effective (fix+) nodules 21 DAI with fix + R. tropici CIAT 899.
Acetylene reduction assays were performed as described by (c) Ineffective (fix-) nodules 21 DAI with fix- R. giardini 6917. (d and e)
Cross section of fix + (d) and fix- (e) nodules stained with toluene blue.
Vance et al. [99] with the following modifications. Plant
Bacteroid structures visible in fix + nodules. Disorganized cellular structure
roots from six biological replicates of each sample (roots evident in (e). (f and g) Transcript abundance of leghemoglobin: high in
inoculated with fix + R. tropici CIAT899 at 5 and 21 DAI fix + nodules (f) but barely detectable in fix- nodules (g), as assessed by in
and roots inoculated with fix – R. giardini 6917 21 DAI) situ hybridizations.
Additional file 14: Genes differentially expressed between NE and N5.
were placed in 500 ml airtight glass containers equipped
Additional file 15: Genes differentially expressed between NE and NI.
with serum stoppers. 50 ml of air was removed from each
Additional file 16: Nodule specific genes common to Pv and
container and replaced with 50 ml of ethylene; samples
Glycine max.
were incubated at room temperature for one hour, at
Additional file 17: Genes highly expressed in NE compared to
which time 10 ml of gas was withdrawn from the container other tissues.
for analyses as previously described by Vance et al. [99]. Additional file 18: Genes highly up regulated in NE.
Additional file 19: LRR gene expression. 151 genes containing an LRR
Nodule In Situs domain expressed in our data. Expression, represented by Z-scores, of
LRR containing sequences is low in developing seeds, but high in
A partial coding sequence for Pv leghemoglobin (645 bp) ineffective nodules. Red indicates a positive Z-score while blue indicates
was PCR amplified and cloned into pBSSK+. Nodules in- a negative Z-score. For tissue description see Table 1.
oculated with fix + R. tropici CIAT899 and fix – R. giardini Additional file 20: Genes differentially expressed between root
6917 were collected 21 DAI and analyzed as described by samples.
Sbabou et al. [100]. Additional file 21: Impact of N on TF expression. Transcription
factors differentially expressed in leaf samples (a) and root samples (b) of
plants provided with NO−3 fertilizer, −N plants (inoculated with
Availability of supporting data fix- rhizobium), and N2 fixing plants (inoculated with fix + rhizobium).
The expression data used in this study is publicly available Heatmaps of gene expression represented by Z-scores; red indicates a
positive Z-score while blue indicates a negative Z-score.
at the NCBI short read archive; accession SRP046307.
Additional file 22: Housekeeping genes.
Additionally, the raw and normalized datasets can be
downloaded and explored at the Phaseolus vulgaris Gene
Expression Atlas (Pv GEA) website, http://plantgrn.noble. Competing interests
org/PvGEA/. The authors declare that they have no competing interests.

Additional files Authors’ contributions

JAO, LPI, and CPV: conceived the project, analyzed the data, and drafted the
manuscript. FF: performed bioinformatic analyses and initiated database
Additional file 1: Pairwise identification of differentially expressed construction. BB: provided plant tissues and performed RNA extractions.
genes. SS: performed qPCR experiments. SAJ and PEM: conceived the project and
Additional file 2: Tissue specific genes. critically reviewed the manuscript. JL, XD and PXZ: built the gene expression
atlas database. GH: advised on the experimental plan and critically reviewed
Additional file 3: Expression patterns of specific genes of interest.
the manuscript. All authors have read and approved the final manuscript.
Graphs illustrating the expression patterns of genes of interest, individual
tissues on the X-axis, RPKM values, on the Y-axis. (a) tissue specific, (b)
sample specific genes, (c) uricase (d) allantoin degradation, (e) glutamate Acknowledgements
dehydrogenase, (f) transcription factors with increased expression as This work was facilitated, in part, using resources at the University of
seeds develop, (g) ABA1 and ABA2, (h) NCED9, (i) trehalose 6 phosphate, Minnesota Supercomputing Institute.
(j) glutamine synthetase, (k) GOGAT, (l) asparagine synthetase.
Additional file 4: Sample specific genes. Author details
1
Additional file 5: Purine Biosynthesis. Expression patterns of the Pv Department of Agronomy and Plant Genetics, University of Minnesota, St.
homologs in the purine biosynthesis pathway. Genes in this pathway are most Paul, MN 55108, USA. 2Centro de Ciencias Genomicas, Universidad Nacional
highly expressed in effective nodules compared to the rest of the plant. For Autonoma de Mexico, 66210 Cuernavaca, Mor, Mexico. 3USDA-Agricultural
Research Service, Plant Science Research Unit, St. Paul, MN 55108, USA.
tissue descriptions see Table 1. Gene expression is represented by Z-scores; red 4
indicates a positive Z-score while blue indicates a negative Z-score. Center for Applied Genetic Technologies, University of Georgia, Athens, GA
30602, USA. 5Department of Plant Sciences, North Dakota State University,
Additional file 6: qPCR results. Fargo, ND 58105, USA. 6Plant Biology Division, The Samuel Roberts Noble
Additional file 7: Transcription factors. Foundation, Ardmore, OK 73401, USA. 7Current Address: USDA-ARS, Corn
Additional file 8: Genes differentially expressed between SH and PH. Insect Crop Genetics Research Unit, Iowa State University, Ames, IA 50011,
USA.
O’Rourke et al. BMC Genomics 2014, 15:866 Page 14 of 16
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Received: 8 May 2014 Accepted: 24 September 2014 21. Le BH, Cheng C, Bui AQ, Wagmaister JA, Henry KF, Pelletier J, Kwong L,
Published: 6 October 2014 Belmonte M, Kirkbride R, Hovath S, Drews GN, Fischer RL, Okamuro JK,
Harada JJ, Goldberg RB: Global analysis of gene activity during
Arabidopsis seed development and identification of seed-specific
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