Mol. Cells, Vol. 18, No. 1, pp. 53-62
M olecules
and
Cells
/
KSMCB 2004
Analysis of the Root Nodule-enhanced Transcriptome in Soybean
Hyoungseok Lee, Cheol-Goo Hur1, Chang Jae Oh, Ho Bang Kim, Sun-Yong Park1, and Chung Sun An*
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea;
1
Korea Research Institute Bioscience and Biotechnology, Genome Research Center, Daejeon 305-333, Korea.
(Received March 5, 2004; Accepted May 25, 2004)
For high throughput screening of root nodule-enhanced
genes, cDNA libraries specific for three different developmental stages of soybean root nodules were constructed after inoculation with Bradyrhizobium japonicum USDA110. 5,469 cDNA clones were sequenced and
grouped into 2,511 non-redundant (nr) ESTs consisting
of 769 contigs and 1,742 singletons. Using similarity
searches against several public databases we constructed a functional classification of the ESTs into root
nodule-specific nodulin genes, stress-responsive genes
and genes related to carbon and nitrogen metabolism.
We also constructed a cDNA microarray with 382 selected clones that appeared to be up-regulated in the
root nodule. Using the microarray we compared the
transcript levels of uninfected roots and root nodules
from four developmental stages. We identified 81 genes
that were differentially expressed, and grouped them
into seven clusters according to the similarity of their
expression profiles, using a hierarchical clustering algorithm. Clusters 1, 2, 3, and 6, comprised of 58 genes,
showed root nodule-enhanced expression. The information from this study will be used to analyze the roles of
root nodule-specific genes and signaling pathways during root nodule development.
Keywords: EST; Microarray; Root Nodule; Soybean.
Introduction
Nitrogen-fixing nodules are formed on the roots of legumes as a result of infection by the genus Rhizobium. The
establishment of a root nodule and its subsequent development involve complex interactions between the two
organisms resulting in structural and biochemical changes
* To whom correspondence should be addressed.
Tel: 82-2-880-6678; Fax: 82-2-872-6881
E-mail: [email protected]
in both partners (Beringer et al., 1979). Several events are
involved in this plant-bacterium interaction, including invasion of the root hair followed by development of the infection thread, release of bacteria into cortical cells, development of the peribacteroid membrane, and differentiation
of the bacteria into bacteroids. A group of host gene products, termed nodulins, play essential roles in nodule formation and functional symbiosis (Legocki and Verma, 1980).
The nodulin genes characterized so far are divided into
early and late genes according to the time of appearance of
their mRNAs (Nap and Bisseling, 1990). It has been suggested that the early nodulins are involved in the infection
process and/or nodule morphogenesis (Gloudemans and
Bisseling, 1989), and the late ones in nodule function and
maintenance (Richter et al., 1991).
In the last two decades random sequencing of expressed sequence tags (ESTs) has become a valuable
method for gene discovery (Hillier et al., 1996; Marra et
al., 1999; Sterky et al., 1998). There have been multitissue EST projects in several plant species, as well as
more specialized, tissue-specific projects on root hairenriched Medicago truncatula tissue and root nodules
(Covitz et al., 1998; Fedorova et al., 2002), woodforming tissues of poplar (Sterky et al., 1998), and hairy
root of ginseng (Chung et al., 2003). cDNA microarray
technology is also widely used to identify global gene
expression patterns during organ development. Its sensitivity and reliability have been demonstrated in the analysis of a variety of phenomena including fruit ripening
(Aharoni et al., 2002), the hypersensitive response to
pathogens (Schenk et al., 2000), and the wound response
(Cheong et al., 2002).
To date, more than 330,000 expressed sequence tags
(ESTs) from various organs of soybean have been registered in dbEST soybean (Shoemaker et al., 2002), and
three studies on root nodule-specific ESTs have been reported in the model legumes, M. truncatula and Lotus
japonicus (Fedorova et al., 2002; Journet et al., 2002;
Szczyglowski et al., 1997). Although several groups have
54
The Root Nodule-enhanced Transcriptome in Soybean
/
identified novel nodulins in early and late stage soybean
nodules by gene cloning (Jacobs et al., 1987; Kouchi and
Hata, 1993; Richter et al., 1991; Sandal et al., 1987; Sengupta-Gopalan et al., 1986), and by proteomic approaches
(Legocki and Verma, 1980; Panter et al., 2000), there are
no reports of large-scale nodule-specific EST or microarray experiments in soybean. We have used EST and
cDNA microarray technologies to identify large numbers
of genes specifically involved in this symbiosis in soybean. We describe here the isolation of numerous genes
that are up-regulated during root nodule development
many of which encode functions that have not previously
been implicated in the symbiosis.
Materials and Methods
Bacterial strain and plant material To nodulate soybean [Glycine max (L.)] cultivar Backtae, seeds were imbibed on paper
towels moistened with distilled water. After three days, the
sprouted seeds were inoculated with 100 ml of approximately 5
× 107 colony-forming units/ml (cfu/ml) of Bradyrhizobium japonicum strain USDA 110, which had been cultured in YEM
(Vincent, 1970), and suspended in buffered nodulation medium
(BNM, Ehrhardt et al., 1992). The seedlings were grown in pots
filled with vermiculite at 27°C in a 16 h/8 h light/dark cycle in a
growth chamber, and watered with 0.5× BNM during growth. To
isolate root nodules, plants were removed from the vermiculite,
washed in tap water and the nodules removed with forceps. As a
control, total RNA was isolated from the roots of uninoculated
plants. The harvested tissues were frozen in liquid nitrogen and
stored at –80°C.
RNA Isolation and cDNA library construction Nodule-specific
soybean cDNA libraries were created from RNA prepared from
root nodules 10 days after inoculation (DAI), 5 weeks after inoculation (WAI), and at 10 WAI. Total RNA was isolated as
previously described (Uhde-Stone et al., 2003), and poly(A)enriched RNA was prepared using an Oligotex mRNA mini kit
(Qiagen, USA). RNase-free DNase (Promega, USA) was used to
remove any genomic DNA contamination in the RNA samples
during RNA purification. cDNA libraries were constructed as
previously described (Kwon and An, 2003). Briefly, cDNA was
prepared using a ZAP-cDNA synthesis kit, directionally ligated to
ZAP II, and packaged with Gigapack III Gold packaging extracts
(Stratagene, USA). Plasmids containing cDNA inserts were excised with Ex-Assist helper phage and propagated in SOLR cells
according to the manufacturer’s instructions (Stratagene, USA).
The libraries were designated AN01, AN02, and AN03.
EST analysis ESTs were generated by 5′ end sequencing and
the resulting sequences were automatically edited to remove
vector and bacterial sequences, as well as ambiguous regions.
Individual ESTs were assembled into groups of contigs representing unique transcripts using the CAP3 program. Consensus
sequences of all the contigs were generated based on 75% homology over a minimum of 30 bp. The individual ESTs were
searched against the GenBank nr database using the BLASTX
algorithm. EST hits on the same target gene were manually assembled into single contigs. On the basis of the BLASTX results,
we classified ESTs into three categories: known, unknown, and
no hit. A known EST was declared when the score was greater
than 80 and the E-value less than 10−14. Unknown was defined
as a score greater than 40 and less than 80, and an E-value of
less than 10−14 but greater than 10−2. No hit was defined as a
score of less than 40 with an E-value greater than 10−2. The
soybean EST database is available at http://kropbase.snu.ac.kr/
cgi-bin/soybean_db_idx.cgi. The functional assignment of ESTs
was based on comparisons with the Arabidopsis thaliana annotation database in the Munich Information Center for Protein
Sequences (MIPS) using the BLASTX algorithm.
Preparation of the cDNA microarray To prepare a 382-cDNA
microarray, plasmid DNA was isolated using a Millipore plasmid kit (Millipore, USA) and inserts were PCR-amplified using
T3 and T7 promoter primers. The PCR cycles were 94°C for 2
min of initial denaturation, followed by 94°C for 45 s, 55°C for
45 s, and 72°C for 2 min (for a total of 30 cycles), ending with a
10 min extension at 72°C. The PCR products were examined by
1% agarose gel electrophoresis, purified on Sephadex G-50
columns, dried and resuspended in 50% DMSO. The DNAs
were spotted with an OmniGrid Microarrayer (GeneMachines,
USA) onto silanized glass slides (CMT-GAPS, Corning,
USA) which were crosslinked with 300 mJ/cm2 of short wave
ultraviolet irradiation (Stratalinker, Stratagene, USA) and stored
in a desiccator until use.
Preparation of probes for microarray hybridization cDNA
probes were prepared from total RNA extracted from triplicate
sets of uninoculated soybean roots, roots harvested at 4 DAI,
and root nodules at 10 DAI, 5 WAI, and 10 WAI. The cDNA
from 4 DAI roots and 10 DAI, 5 WAI, and 10 WAI root nodules
was labeled with Cy5, and the cDNA from the uninoculated
roots with Cy3 (to be used as a control in all the hybridizations).
In each case 80 µg of total RNA was reverse transcribed in the
presence of Cy3- or Cy5-dUTP (Hegde et al., 2000). In brief, 24
µl of the RNA and 4.5 µg of oligo(dT) (Bionics, Korea) were
warmed at 65°C for 10 min and cooled on ice. Thereafter we set
up the following reaction in a total of 40 µl: 24 µl of the RNA
oligo(dT) mixture, 1× first strand reaction buffer, 10 mM dithiothreitol, 0.5 mM dATP, dCTP, and dGTP, 0.3 mM dUTP, 50 µm
Cy3- or Cy5-dUTP (Perkin Elmer, USA), and 400 units of SuperscriptIII (Invitrogen, USA) and incubated it for 2 h at 50°C.
The resulting Cy3 and Cy5 probes were paired according need,
and unincorporated nucleotides were removed with a PCR
cleaning kit (Qiagen, USA), and the cleaned probes precipitated
with absolute EtOH.
Microarray hybridization and analysis The concentrated
cDNA probes were suspended in 10 µl of nuclease free water
Hyoungseok Lee et al.
55
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Table 1. Primers used in the semi-quantitative RT-PCR.
PCR primers (5′ → 3′)
Genes
1-deoxy-D-xylulose 5-phosphate reductoisomerase
Polyamine oxidase
Tyrosine aminotransferase
Succinoaminoimidazole carboximide
ribonucleotide synthetase
Polygalacturonase
Nicotianamine aminotransferase B
Thiamine biosynthetic enzyme
UDP-glucose:salicylic acid glucosyl transferase
Integral membrane protein
Forward
CAACCTCCTAGGCTCCTATT
TGTAACCGTGTACACCAAAA
ACACCAGATCCGTTGTTCGT
Reverse
CAAGACTCTTCCAGCTTCAC
CACCATCTGGGAACAAGTAT
GTTTGCTCGCAATCTCAGCA
GATATATACGACGCTGGGGA
CAATATGAGGCCATGCTCCA
ATTATGGTGCCACGGGCAA
AAGCTACTGATTCTGTTTC
CTTGACATGAACAAGGCAGA
CATGCATCAGTTCTCCAAGCT
TCTCTATGTTCTCACCAGCA
TGCTGATGAAGGTGGATCCAT
ATGGATTGCTAGGATTGATG
GAGACAAAGGTGTGGATGGT
GAGGAGAGGAATGAGGGCAT
ACAAGTGGAAGTGCCTGGAC
/
Table 2. Soybean nodule cDNA libraries from this study.
CDNA
library
AN01
AN02
AN03
Total
Developmental
stage of nodule
2 WAI
5 WAI
10 WAI
Total No. of
ESTs
No. of
Singletons
No. of
contigs
No. of
NR sequence
2,021
1,824
1,624
5,469
1,690
1,777
1,745
1,993
205
516
209
815
1,895
1,293
1,954
2,748
BLAST search result
Known
1,331
1,048
1,744
1,904
Unknown
319
184
159
607
No Hit
245
061
051
0237
/
and 10 µl of preheated (55°C) formamide-based hybridization
buffer (50% formamide, 8× SSC, 1% SDS, 4× Denhardt’s solution). They were layered on a cDNA microarray slide, covered
with 22 × 22 cover slip and incubated in a water bath at 42°C in
a hybridization chamber (GenomicTree, Korea) for 16 h. The
slides were washed once in 2× SSC for 5 min, twice in 0.2×
SSC for 3 min, then scanned in a Genepix 4000B scanner (Axon
Instruments, USA). We performed twelve hybridizations, consisting of three sets of four experiments.
To identify genes differentially expressed in the nodules and
roots, we performed background intensity subtraction and printtip normalization. Using image analysis software, signal intensities were determined for each dye at each element of the array,
and the ratio of Cy5 to Cy3 intensity was calculated. Signal
intensities were averaged from three independent experiments
and the Cy5/Cy3 fluorescence ratios were log transformed (base
2 for simplicity) in order to treat inductions and repressions of
identical magnitude as numerically equal but of opposite sign.
For clustering analysis of the microarray data, we used Quintet
(KRIBB, Korea) to generate hierarchical clusters and selforganizing maps (SOM) for these clusters.
Reverse transcription-PCR Semi-quantitative RT-PCR was
performed to analyze the expression of selected genes during
nodule development using appropriate PCR primers (Table 1).
Primers for amplifying actin were used as a quantitative control
(Lee et al., 2004). Total RNA (1 µg) was used as template for
reverse transcription after treatment with RNase-free DNase
(Promega, USA). The PCR cycles were 95°C for 5 min of initial
denaturation, followed by 94°C for 15 s, 52°C for 50 s, and
72°C for 50 s (for a total of 25 cycles), ending with a 5 min
extension at 72°C. The amplified products were separated on a
1% agarose gel.
Results
Generation of ESTs from soybean nodules at different
developmental stages Three cDNA libraries were constructed from the soybean nodules at 2 weeks after inoculation (WAI) with B. japonicum, at 5 WAI, and at 10 WAI,
and named AN01, AN02, and AN03, respectively. Singlepass 5′ sequencing of these yielded 5,469 sequences. The
non-redundant sequences among these were clustered into
a total of 815 contigs and 1,993 singletons, giving a total
of 2,748 non-redundant (nr) ESTs (Table 2). Using the
BLASTX algorithm (Altschul et al., 1990), these nr ESTs
were translated into their corresponding amino acid sequences and searched against the nr protein database of
GenBank. 1,904 ESTs were matched to known or predicted genes and gene products. Each contig sequence is
clustered with overlapping sequences and therefore presumably represents a unique transcript. The variability in
the number of ESTs comprising each contig therefore
probably reflects differences in the abundance of the transcripts of the corresponding genes. As seen in Table 3, 13
of the 20 most abundant contigs in terms of numbers of
ESTs, are known nodulins, as expected.
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The Root Nodule-enhanced Transcriptome in Soybean
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Table 3. The 20 most abundant contigs from the root nodule ESTs.
No. of ESTs
Contig ID
cl6ct776cn825
a
In this
work
Nodulea
Roota
637
59
0
E-Value
Score
e-110
399
Strongest BLASTX Hit
Nodulin 44 (G. max, P04672)
cl12ct292cn313
131
44
4
3e-74
281
Leghemoglobin A (G. max, P02238)
cl12ct291cn312
102
22
0
5e-75
282
Leghemoglobin C1 (G. max, P02235)
cl6ct775cn824
59
28
0
5e-92
339
Nodulin C51 (G. max, P04671)
cl12ct290cn310
48
22
0
7e-76
285
Leghemoglobin C3 (G. max, P02237)
cl12ct289cn309
46
39
0
9e-76
285
Leghemoglobin C2 (G. max, P02236)
cl15ct30cn31
41
6
0
4e-94
346
Nodulin 20 (G. max, P08960)
cl15ct29cn30
37
10
0
e-101
370
Nodulin 22 (G. max, P08961)
cl113ct137cn145
35
4
0
4e-96
353
Nodulin 26B (G. max, P08863)
cl55ct72cn75
35
2
2
e-127
457
Hypersentive-induced protein (Z. mays, AF236375)
cl77ct98cn103
23
21
22
e-148
525
Phosphatase, putative (G. max, CAD57680)
cl112ct133cn141
17
38
9
0
1170
Nodulin 100, sucrose synthase (G. max, AAC39323)
cl66ct79cn82
16
18
4
5e-49
197
Nodulin 36B (G. max, Q02919)
cl77ct97cn101
15
21
21
e-132
472
Hydrolase, putative (G. max, AAM94615)
cl122ct146cn154
13
5
1
7e-62
239
Nodulin 16 (G. max, P23233)
cl12ct288cn306
13
8
1
e-41
171
Nodulin 93 (G. max, Q02921)
cl566ct632cn674
13
81
102
e-161
571
Sali3-2 protein, aluminium-induced (G. max,T08896)
cl127ct159cn167
12
1
1
0
679
ABC transporter family (A. thaliana, NP190357)
cl27ct32cn34
12
0
0
e-103
378
Unknown protein (A. thaliana, BAB10214)
cl349ct406cn437
12
0
0
0
1151
Cytochrome P450 83D1p (G. max, T05940)
Frequencies Values represent frequencies of ESTs transcribed in the nodule and the root from the soybean gene index (GmGI).
In addition to BLASTX using the nr protein database of
GenBank, we identified the protein in the Arabidopsis
database (MATDB) in the Munich Institute of Protein
Sequences (MIPS) using the highest BLASTX score to
identify the functional category of each nr EST. A total of
402 nr ESTs could be automatically assigned to 13 functional classes in the MIPS classification system, while
known nodulins were manually assigned (Fig. 1). 15% of
the classified ESTs were assigned to the category of metabolism, 10% each to cell rescue, defense, cell death, and
aging, and 8% to known nodulins.
Construction of cDNA microarrays To select candidate
genes that might be primarily regulated in the root nodule,
we investigated the numbers of sequences homologous
with our nr ESTs represented in public soybean EST database. For this, we made two sub-databases, one the nodule EST sub-database comprising 7,733 ESTs, the other
the root EST sub-database comprising 13,750 ESTs. Afterward, homology searches were done using the 2,748 nr
ESTs from our root nodule ESTs as queries, and both
nodule and root EST sub-databases as subjects using the
BLASTN algorithm. By this process, the frequency of the
corresponding ESTs in root and nodule sub-databases was
Fig. 1. Functional classification according to the MIPS classification scheme. 402 ESTs were grouped into 14 groups.
counted for each nr EST in our EST database; some of the
results are shown in Table 3. 264 ESTs were represented
only in the nodule and 72 in both organs, but with significantly enhanced expression in the nodule. In addition to
these 336 ESTs, 46 ESTs with annotations involving nodule functions were selected manually. In the end a total of
Hyoungseok Lee et al.
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compasses many of the nodulin genes. In general the first
significant increases in transcript levels were detected at
10 DAI and were maintained or augmented up to 10 WAI.
We identified 34 known nodulin genes in our EST analysis; 25 genes in soybean, six in other legumes, and three
in non-legume species. Of the 25 soybean genes 18 were
in cluster 2. Beside the nodulin genes, nine genes were
assigned to metabolism. In addition, we identified three
cluster 2 genes, remorin, peroxidase, and a RING-H2 zinc
finger protein, whose homologues showed noduleenhanced expression in the nodules of M. truncatula (Fedorova et al., 2002). Cluster 3 contained only one gene, a
WRKY transcription factor, expressed specifically at 5
WAI, and cluster 6 also has only one gene, a nodulin 3like gene, expressed at 10 WAI.
Fig. 2. Cluster analysis of 58 cDNA clones shown to be upregulated during root nodule development. Classification of the
clones was based on the similarity of their expression profiles
using the hierarchical clustering technique. The following experiments are on the x axis. 1, root versus 4 DAI (days after
inoculation) root; 2, root versus 10 DAI nodule; 3, root versus 5
WAI (weeks after inoculation) nodule; 4, root versus 10 WAI
nodule.
382 cDNA inserts from the selected ESTs were arrayed at
low density on microarray slides to identify genes whose
expression is differentially regulated during root nodule
development.
Differential gene expression during root nodule development From the cDNA microarray analysis, 81 of the
382 genes showed significant changes in expression during nodule development. Their expression patterns were
grouped into seven clusters using the hierarchical clustering method. Two large clusters and five small clusters,
each with distinct expression patterns, were apparent
(data not shown). Among them, the genes in clusters 1, 2,
3, and 6 were up-regulated in the nodule (Fig. 2). The
expression ratios of the 58 genes in these four clusters are
shown in Table 4.
Cluster 1 consists of seven genes whose transcript levels were not changed in 4 DAI and 10 DAI nodules, but
increased in 5 and 10 WAI nodules, hence specifically in
mature and senescent nodules. Of four genes whose functions were assigned to metabolism, two cysteine synthases are known to be involved in metabolism related to
nitrogen. Also one transcription factor, ethylene-responsive
element binding protein 1, and one cysteine proteinase in
this cluster, are expressed abundantly in late stage soybean nodules. Cluster 2, the largest, has 49 genes and en-
Expression of selected genes assessed by semi-quantitative RT-PCR To confirm the microarray data, we performed some semi-quantitative RT-PCR analyses with
gene-specific primer sets (Table 1). We analyzed two
genes in cluster 1 and seven in cluster 2, and used actin as
a quantitative control (Fig. 3). The results of the RT-PCR
analysis agreed in most cases with the profiles derived
from the microarray data.
/
Discussion
We used three successive procedures to select noduleenhanced transcripts. In the first step we constructed nodule-specific cDNA libraries and EST databases. Second,
by in silico analysis comparing our database with public
soybean EST database, we selected candidate genes that
seemed to be more highly expressed in nodules than in
control roots. Third, by cDNA microarray analysis, we
examined the expression patterns of selected genes during
nodule development. As a result, 58 genes were identified
as nodule-enhanced genes; among them, 20 are known
nodulin genes, nine are of unknown function, and six including a cysteine proteinase, coproporphyrinogen oxidase, and remorin, were identified as nodule-enhanced
genes by EST and expression analysis in nodules of M.
truncatula and L. japonicus (Fedorova et al., 2002;
Szczyglowski et al., 1997). On the other hand, many
genes had different expression pattern in the different
legume species; for example, the transcripts of phytochelatin synthetase, bark agglutinin precursor, and B12D protein were up-regulated exclusively in M. truncatula, while
those encoding polyamine oxidase, ethylene-responsive
element binding protein, and RpoB transcripts were upregulated only in soybean. These differences may result
from natural variation between species or from differences in the techniques used in the two studies. For example we analyzed expression patterns by both computational approaches and cDNA microarrays, while in the
58
The Root Nodule-enhanced Transcriptome in Soybean
/
Fig. 3. Semi-quantitative RT-PCR of selected genes that showed enhanced expression during root nodule development. Gene identities are indicated on the right side of each panel. Lane R, root as quantitative control; lane 1, root 4 DAI; lane 2, nodule 10 DAI; lane
3, nodule 5 WAI; lane 4, nodule 10 WAI.
study of M. truncatula only computational methods were
employed (Fedorova et al., 2002). Finally, we have identified 23 nodule-enhanced genes that are new at least to the
field of large-scaled transcriptome analysis.
Although ESTs and cDNA microarrays have been used
on a limited scale to identify nodule-enhanced soybean
genes (Jacobs et al., 1987; Kouchi and Hata, 1993; Richter et al., 1991; Sandal et al., 1987; Sengupta-Gopalan et
al., 1986), this is the first report to employ these approaches on a large scale to identify genes related to soybean-Rhizobium symbiosis. Recently, four publications on
the proteome of the peribacteroid membrane of symbiosomes from various legume species, have reported the
presence of a number of proteins functioning as transporters, molecular chaperones, signaling proteins, and various
metabolic enzymes (Catalano et al., 2004; Panter et al.,
2000; Saalbach et al., 2002; Wienkoop and Saalbach,
2003). We found several of these proteins including the
F1-ATPase delta subunit, sugar transporter, integral membrane protein, protein disulfide isomerase, chaperonin 60
alpha chain, and malate dehydrogenase in our soybean
root nodule EST database and used them in the 382cDNA microarray. Although their expression levels in
nodules were not as high as some nodulins, they were
enhanced as much as two to five fold (data not shown).
Although we did not identify any novel ESTs from the
soybean root nodules, these results along with our data for
the frequencies of the ESTs (Table 3) and the RT-PCR
analysis of selected genes (Fig. 3) demonstrate that our
EST and microarray approach to identifying noduleenhanced genes was reasonably successful.
The following is a brief discussion of the noduleenhanced genes in the three major functional categories.
Genes involved in metabolism The bacterium produces
the nitrogenase complex to reduce nitrogen to ammonia,
while the host plants provide energy from photosynthesis
and various other metabolites. It follows that soybean
genes related to various metabolic processes are expressed during symbiosis (Beringer et al., 1979). The
functions of 13 of the 58 up-regulated genes were assigned to metabolism. Among them, five, i.e., two cysteine synthases, tyrosine aminotransferase, nicotianamine
aminotransferase B, and succinoaminoimidazole carboximide ribonucleotide synthetase, are classified in nitrogen
metabolism. In particular two of them, the cysteine synthases, are the terminal enzymes of cysteine synthesis.
Cysteine cluster proteins are known to be present in nodules of pea (Scheres et al., 1990), broad bean (Kato et al.,
2002), and M. truncatula (Fedorova et al., 2002). It is
possible that the function of the cysteine synthases of
soybean root nodules may be to supply the cysteine for
various cysteine cluster proteins. In addition to the genes
involved in nitrogen metabolism, two genes encoding
polygalacturonase and glycosyl transferase, which are
assigned to carbon metabolism, two encoding coproporphyrinogen oxidase and a thiamin biosynthetic enzyme,
assigned to the metabolism of vitamins and cofactors, and
three encoding cytochrome P450 monooxygenase, UDPglucose:salicylic acid glucosyltransferase, and polyamine
oxidase functioning in secondary metabolism, were also
up-regulated in the soybean root nodule. These results
Hyoungseok Lee et al.
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Table 4. Functional classification of transcripts up-regulated in the soybean root nodule (Cluster 1, 2, 3, and 6).
EST ID
Cluster 1
Metabolism
AN02016F07
AN02018G07
AN02002D03
AN03001F03
Transcription
cl157ct199cn218
Strongest BLASTX Hit
Nodule developmental stage
4 DAI
10 DAI
5 WAI
10 WAI
Cysteine synthase (Spinacia oleracea, BAA01279)
Cysteine synthase (G. max, AF452451)
1-deoxy-D-xylulose 5-phosphate reductoisomerase
(Z. mays, CAC03581)
Polyamine oxidase (A. thaliana, BAC43225)
-0.17527
-0.43315
0.819503
0.278987
-0.1591
-1.17178
3.653666
2.386178
2.688039
3.736222
1.97382
1.227413
0.073529
0.057025
2.450013
1.271627
Ethylene-responsive element binding protein 1
(G. max, AAM45475)
-0.11394
-0.18533
2.969707
0.89363
Protein destination
AN02006E07
Cysteine proteinase (G. max, BAA06030)
Unclassified, unknown proteins
AN03008D06
At5g16360.1
1.76716
0.276625
3.908089
3.094974
-0.45339
0.032898
2.61737
3.283059
Cluster 2
Metabolism
cl649ct724cn773
cl604ct677cn723
0.410262
-0.48492
4.069916
2.87653
2.455869
2.426556
5.534338
2.632074
Tyrosine aminotransferase (A. thaliana, NP_200208)
Succinoaminoimidazolecarboximide ribonucleotide
synthetase (Vigna unguiculata, AAL48317)
Beta-1,4-N-acetylglucosaminyltransferase
(A. thaliana, NP_172759)
Polygalacturonase (A. thaliana, NP_195292)
Thiamin biosynthetic enzyme (G. max, BAA88226)
Coproporphyrinogen oxidase (G. max, CAA50400)
Cytochrome P450 monooxygenase
(Cicer arietinum, CAD31843)
Nicotianamine aminotransferase B
(Hordeum vulgare, BAA87053)
UDP-glucose:salicylic acid glucosyltransferase
(Nicotiana tabacum, AF190634)
0.727592
2.065705
2.423111
1.092563
-2.47727
-0.22257
0.461948
0.197091
7.782655
5.471685
4.387613
4.204318
5.217395
1.745241
4.300804
3.989846
5.504981
3.966187
4.141745
5.31058
-0.16523
2.274022
0.696865
3.134205
-0.21944
1.553638
2.190294
5.313119
Glycolate oxidase (Lens culinaris, 1803516A)
0.251307
2.539481
0.608502
2.718349
0.770395
5.529406
4.895271
5.023331
0.36232
0.125743
-0.17425
3.91691
5.763927
1.177731
2.093165
4.807397
2.370692
3.433199
4.94928
1.296266
Cellular rescue, defense, cell death and ageing
AN01006C12
Peroxidase (G. max, AF145350)
0.528484
2.873938
3.12138
2.957388
Plant hormonal regulation
cl632ct706cn754
Gibberellin 20-oxidase (Phaseolus vulgaris, T11848)
1.855501
2.315797
2.501222
2.874458
0.061328
2.65016
2.902327
3.393847
AN02011G10
cl309ct363cn389
cl1ct1cn1
cl552ct623cn665
AN01017F01
AN02009G09
cl299ct353cn379
Energy
cl287ct341cn366
Transcription
AN03013A02
RpoB (G. max, AAL07334)
Cellular communication/signal transduction
cl253ct305cn330
Remorin 1 (L. esculentum, AF123265)
AN02002A11
14-3-3-like protein (Pisum sativum, CAB42547)
AN03006G06
Inositol 1,3,4-trisphosphate 5/6-kinase-like protein
(A. thaliana, NP_567334)
Unclassified, unknown protein
AN01010D06
(Continued)
Auxin-induced protein (A. thaliana, NP_180016)
60
The Root Nodule-enhanced Transcriptome in Soybean
/
Nodule developmental stage
EST ID
Strongest BLASTX Hit
4 DAI
10 DAI
5 WAI
10 WAI
AN03008F09
cl198ct245cn267
cl547ct618cn660
cl247ct299cn323
AN03007B05
AN02019H11
cl230ct281cn303
cl591ct664cn709
AN01005E05
cl336ct391cn420
AN02002C07
AN02005B02
AN03015F04
Resistant specific protein-1(4) (Vigna radiata, BAC22499)
F-box protein family (A. thaliana, NP_189030)
RING-H2 zinc finger protein (A. thaliana, AAM66032)
At1g19180 (A. thaliana, AAL87391)
At1g21460.1
At4g39235.1
Hypothetical protein (A. thaliana, NP_176128)
Unknown protein (Saccharomyces cerevisiae, AAL79278)
Unknown protein (A. thaliana, AAL60024)
Unknown protein (A. thaliana, AAN13116)
Unknown protein (O. sativa, AC098566)
Prolyl 4-hydroxylase, alpha subunit (O. sativa, AC068923)
Integral membrane protein (A. thaliana, AAD17424)
2.447672
0.707786
0.787688
1.675975
0.089254
-0.32142
0.090157
-0.36468
0.552937
-0.45606
-1.10649
-0.88452
1.837438
2.559374
5.123451
2.567572
1.564894
4.925796
3.047452
5.16215
1.411966
6.79795
2.965997
3.205291
4.255725
2.660995
1.519024
6.142547
5.656819
4.806285
2.353765
5.107818
3.453145
2.081727
4.661285
4.035498
2.41315
2.889657
1.175476
2.303462
7.322282
5.484843
1.502134
2.956531
4.408153
3.375886
3.386288
4.919842
3.996326
4.102879
3.315073
2.736692
Leghemoglobin C1 (G. max, P02235)
Leghemoglobin C2 (G. max, P02236)
Leghemoglobin C3 (G. max, P02237)
Hemoglobin, 2-on-2 (A. thaliana)
Nodulin 20 (G. max, P08960)
Nodulin 21 (G. max, P16313)
Nodulin 22 (G. max, P08961)
Nodulin 22 (G. max, P08961)
Nodulin 24 (G. max, P04145)
Nodulin 26B (G. max, P08863)
Nodulin 35, uricase (G. max, BAA13184)
Nodulin 36B, early (G. max, Q02919)
Nodulin 44 (Nodulin E27) (G. max, P04672)
Nddulin 55-2, early (G. max, Q02917)
Nodulin 6l (G. max, AF434718)
Nodulin 93 Early (G. max, Q02921)
Nodulin C51 (G. max, P04671)
Nodulin Nlj16, late (Lotus japonicus, AAC49692)
Nodulin-16 (G. max, CAA38204)
-0.21868
-0.07827
0.064438
0.786141
-0.06405
0.848704
-0.56838
-0.77968
-0.06684
-0.14529
-0.24232
-0.05228
1.204219
0.03798
-0.55615
-1.14944
-0.10639
-0.58216
0.557936
6.072787
6.314265
6.724354
2.327818
8.108586
4.867635
9.400088
5.41842
6.566009
5.817806
4.222555
5.7062
6.421188
3.680623
4.855964
4.354611
10.23526
2.359448
7.613594
5.810749
5.882737
6.33176
2.99174
6.228092
5.343719
7.025001
4.455443
5.929285
5.891865
4.176018
0.893629
5.96872
1.724324
4.445715
5.97144
7.170326
3.819925
7.177962
5.82704
6.169947
6.74856
2.831268
6.145062
4.901672
6.603087
4.463273
5.38838
6.222952
4.869505
2.823086
5.91081
2.910271
6.051122
6.000292
7.574951
4.40942
8.919058
1.07141
-1.29268
2.7729
-1.31867
0.353109
2.818226
-0.30378
-2.91668
Known nodulins
cl12ct109cn119
cl12ct109cn115
cl12ct110cn116
AN03007H02
cl15ct18cn18
cl584ct656cn700
cl63ct74cn77
cl15ct17cn17
cl23ct25cn25
cl113ct143cn157
cl34ct39cn41
cl66ct78cn81
cl6ct484cn521
cl302ct356cn382
cl172ct215cn235
AN02016F12
cl6ct483cn520
AN02021A09
cl102ct130cn143
Cluster 3
Transcription
AN02002D08
Transcription factor WRKY4
(Petroselinum crispum, AF204925)
Cluster 6
Known nodulin
cl446ct507cn544
Nodulin 3 like protein (M. truncatula, CAA69976)
reflect the nature of the active metabolism in developing
nodules.
Genes involved in protein degradation Protein degradation has an important role in the process of senescence
and in the regulation of various plant hormone responses.
We found that the expression of a cysteine proteinase was
dramatically increased at 5 WAI and at 10 WAI during
root nodule senescence, whereas it was down-regulated in
the developing root nodules. Cysteine proteinases are particularly abundant among proteinases and well-known
senescence-associated proteins (Noh and Amasino, 1999).
However, the microarray data showed that only AN03008D06 of the seven cysteine proteinase genes was signifi-
Hyoungseok Lee et al.
/
cantly enriched in late stage nodules. Thus, AN03008D06
encoding a vacuolar-located protease appears to play a
role during root nodule senescence. Also, transcripts of an
F-box family protein and a RING-H2 zinc finger protein
accumulated during root nodule development, particularly
at 5 WAI and 10 WAI, late stages of root nodule development. These proteins are components of an SCF (for
SKP1, Cullin, and F box protein) E3 ubiquitin ligase
complex, and the F box is important for recognition of the
cognate SCF complex. The subunit containing the RINGH2 motif (Rbx1/Hrt1/Roc1) has also been identified as an
essential SCF component. The F-box subunit interacts
directly with targets for ubiquitination via a C-terminal
protein–protein interaction domain. Formation of a polyubiquitin chain on the substrate targets it for destruction
by the 26S proteasome, which regulates responses to six
major plant hormones, i.e., auxin, jasmonic acid, GA, cytokinin, brassinosteroid, ABA, and ethylene (Itoh et al.,
2003; Potuschak et al., 2003). During root nodule development, protein degradation and various effects of plant
hormones may well regulate the development of the root
nodule and associated metabolic pathways. The F-box and
zinc finger proteins may be essential components of a 26S
proteasome complex functioning specifically in the soybean root nodule.
Known nodulin genes As originally defined, nodulin
genes are those expressed exclusively in nodules (Legocki
and Verma, 1980). However that definition has been
modified recently because a number of nodulin genes are
expressed to a limited extent in other plant organs (Coba
de la Pena et al., 1997; Fedorova et al., 2002; Kapranov et
al., 1997; Mathesius et al., 2001). Also in our in silico
analysis (data not shown) soybean nodulin 26 was detected in flower and hypocotyl, and soybean early nodulin
N93 in root, shoot, and seedlings.
Although Nlj16 was up-regulated with other soybean
nodulin genes, expression of another five of the nine
nodulin genes with homologues in other legume species
did not increased significantly in our microarray analysis.
Furthermore, homologues of ENOD8.3 and MtN3 of M.
truncatula and of a nodulin-like protein in Arabidopsis
were actually down-regulated during soybean nodule
development (data not shown). These results suggest that
the genes needed for nodule development and the network
of interactions between them may differ depending on the
legume species.
In conclusion, we have extended our understanding of
the soybean gene products involved in symbiotic nitrogen
fixation by identifying 58 genes that appear to be upregulated in root nodules, including 20 nodulin genes.
Our analyses also revealed 23 genes with enhanced expression in nodules that had been previously overlooked,
such as UDP-glucose:salicylic acid glucosyltransferase,
polygalacturonase, and polyamine oxidase related to me-
61
tabolism, and an F-box protein and RING-H2 zinc finger
protein related to the SCF complex. Additional experiments, such as larger cDNA microarray analyses, in situ
hybridization, genomic clones and promoter elements
analysis, will certainly further our understanding of the
coordinated gene expression in the soybean root nodule.
The information from this study will be useful in further
analysis of the roles of nodule specific genes and their
signaling network during root nodule development.
Acknowledgments This work was supported by a grant from the
Crop Functional Genomics Center (Project No. M101-KG01000103K0701-01810, Korea) to C. S. An. The Brain Korea 21 Project
of the Ministry of Education supported H. Lee and C. J. Oh.
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