The White Clover enod40 Gene Family. Expression
Patterns of Two Types of Genes Indicate a Role
in Vascular Function1
Erika Varkonyi-Gasic2 and Derek William Richard White*
Plant Breeding and Genomics, AgResearch, Private Bag 11008, Palmerston North, New Zealand
Enod40 is one of the genes associated with legume nodule development and has a putative role in general plant organogenesis. We have isolated a small enod40 gene family from white clover (Trifolium repens), with three genes designated
Trenod40-1, Trenod40-2, and Trenod40-3, all containing the conserved enod40 regions I and II. Trenod40-1 and Trenod40-2 share
over 90% homology in the transcribed regions and high levels of similarity in their upstream regulatory sequences.
Trenod40-1 and Trenod40-2 are similar to the enod40 genes of legumes forming indeterminate nodules (group II) and are
predominantly expressed in nodules. Trenod40-3 shares only 32.8% identity with Trenod40-1 and Trenod40-2 within the
transcribed region. Trenod40-3 is similar to the enod40 genes of legumes with determinate nodules (group I) and is not
predominantly expressed in nodules. To our knowledge, this is the first report of both group I- and group II-type enod40
genes being expressed in a single legume species. In situ hybridization studies revealed that Trenod40 genes were highly
expressed in non-symbiotic tissues, particularly in stolon nodes during nodal root and lateral shoot development. High
levels of Trenod40 transcripts were also present in the vascular bundles of mature plant organs, mainly at sites of intensive
lateral transport, suggesting a role in vascular tissue function. The expression pattern of Trenod40 genes was analyzed further
using Trenod40 promoter-gus fusions in transgenic white clover and tobacco (Nicotiana tabacum), indicating that white clover
and tobacco share the regulatory mechanisms for Trenod40-1/2 promoters and some aspects of Trenod40-3 regulation.
It has been proposed that genes with nodulespecific expression and their products, nodulins,
have a role in the development and function of
nitrogen-fixing nodules on the roots of legumes
(Verma et al., 1986; Nap and Bisseling, 1990). Based
on the time of expression during nodule development, nodulins are divided into early nodulins, with
a putative role in the rhizobial infection process, and
late nodulins, with a role in nitrogen fixation (Govers
et al., 1987). One of the earliest nodulin genes is
enod40, a gene implicated in the early stages of development of both determinate and indeterminate
nodules (Yang et al., 1993). The two types of nodules
can be distinguished by their growth pattern and the
origin of their primordia. In tropical legumes such as
soybean (Glycine max) or bean (Phaseolus vulgaris),
nodules originate from the root outer cortex. These
nodules are determinate, because the nodule meristem ceases to divide at an early stage of development and the cells in the nodule central tissue are at
1
This work was supported by the New Zealand Foundation for
Research, Science, and Technology (grant no. C10X0021). This
paper was written in partial fulfillment of the PhD thesis of E.V.-G.
to the Faculty of Biology, University of Belgrade.
2
Present address: Genesis Research and Development, P.O. Box
50, Auckland, New Zealand.
* Corresponding author; e-mail [email protected];
fax 64 – 6 –351– 8042.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010916.
a similar stage of development (Newcomb et al., 1979).
Indeterminate nodules arise from the inner cortex on
the roots of temperate legumes, such as alfalfa (Medicago sativa), white clover (Trifolium repens), and pea
(Pisum sativum). They are characterized by a persistent
meristem that continuously differentiates into specific
zones, representing successive stages of nodule development (Newcomb et al., 1979; Vasse et al., 1990).
In both types of nodules, expression of enod40 is
induced in the root pericycle before the division of
the cortical cells that give rise to a nodule primordium, and subsequently in the dividing cortical cells
and the nodule primordium (Kouchi and Hata, 1993;
Yang et al., 1993). In later stages of indeterminate
nodule development, enod40 is expressed in the meristem and the infection zone adjacent to the meristem
(Asad et al., 1994; Fang and Hirsch, 1998). The intensity of expression decreases across the older parts of
the nodule, and this decline in expression coincides
with the start of amyloplast accumulation in the
Rhizobium-infected cells of the interzone between the
infection and fixation zones (Vijn et al., 1995). This
expression before and during the early stages of nodule development indicated a possible function for the
enod40 genes in nodule organogenesis (Mylona et al.,
1995). A role in the initiation of cortical cell divisions
was proposed, because over expression of enod40 in
transgenic Medicago truncatula plants resulted in extensive cortical cell division in the absence and an
increased rate of nodulation in the presence of Rhizobium (Charon et al., 1997, 1999). However, enod40
expression in the root pericycle and nodule progen-
Plant Physiology, July 2002, Vol. 129,
pp. 1107–1118,
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Varkonyi-Gasic and White
Figure 1.
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(Legend appears on facing page.)
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Plant Physiol. Vol. 129, 2002
White Clover enod40 Gene Family
itor cells is not necessarily accompanied by divisions
of cortical cells (Minami et al., 1996; Mathesius et al.,
2000). Furthermore, enod40 expression persists in the
pericycle of the vascular bundles of mature nodules,
indicating an additional role in nodule function
(Kouchi and Hata, 1993; Yang et al., 1993).
Expression of enod40 genes is not confined to nodules, because transcripts have been detected in nonsymbiotic meristematic tissues, such as developing
lateral roots (Asad et al., 1994; Papadopoulou et al.,
1996; Fang and Hirsch, 1998), young leaf and stipule
primordia (Asad et al., 1994; Corich et al., 1998), stem
and root procambial cells (Asad et al., 1994; Corich et
al., 1998), and embryonic tissues, namely ovules and
embryos (Flemetakis et al., 2000). Enod40 gene homologs have also been identified in the non-legume
species, tobacco (Nicotiana tabacum; Matvienko et al.,
1996) and rice (Oryza sativa; Kouchi et al., 1999),
indicating a more general role in plants. A role in
plant development has been suggested, because introduction of an enod40 antisense construct arrested
the callus growth of alfalfa explants, whereas embryos over expressing enod40 developed into teratomas (Crespi et al., 1994).
In both legumes and non-legumes, enod40 genes
encode transcripts of about 0.7 kb that are characterized by the absence of a long open reading frame
(ORF). Computer analysis of the full-length enod40
nucleotide sequences indicated a stable RNA structure, a property characteristic for biologically active
RNAs, i.e. riboregulators (Crespi et al., 1994). In addition, enod40 mRNA did not copurify with polysomes, and only a fraction copurified with monosomes (Asad et al., 1994). However, reporter gene
fusions with M. truncatula enod40 cDNA demonstrated translation of both region I and region II
ORFs (Sousa et al., 2001), and in vitro transcription of
soybean enod40 demonstrated translation of two peptides from the short overlapping ORFs in region I,
suggesting a polycistronic nature for enod40 mRNA
(Röhrig et al., 2002). Therefore, the mechanism of
enod40 action is still to be elucidated. To date, it is
unclear whether it is the enod40 RNA, a small peptide
of region I, or a number of peptides directly translated from enod40 short ORFs that are biologically
active.
Analysis of all legume enod40 genes identified to
date revealed the clustering of these genes into two
groups, based on the percentage of nucleotide sequence similarity and the length of the putative
ENOD40 peptide (Kouchi et al., 1999; Flemetakis et
al., 2000). Enod40 genes of legumes with determinate
nodules are clustered in group I, and all encode a
putative peptide of 12 amino acids; whereas enod40
genes of legumes with indeterminate nodules cluster
in group II, and their region I ORF corresponds to a
peptide of 13 amino acids. In plants where two different enod40 genes were detected, such as soybean
(Minami et al., 1996), alfalfa (Fang and Hirsch, 1998),
and Lotus japonicus (Flemetakis et al., 2000), both
genes were clustered within the same enod40 gene
group.
In this paper, we describe the isolation and characterization of three distinct white clover enod40
genes, two of which are similar in sequence and
nodule expression to those commonly characterized
from legumes forming indeterminate nodules, and a
single unusual enod40 gene that is not predominantly
expressed in nodules, with a higher sequence similarity to the enod40 genes of legumes with determinate nodules. Expression analysis of these enod40
genes in symbiotic and non-symbiotic tissues of
white clover and expression of their promoters in
transgenic white clover and tobacco suggests a role at
sites of intensive lateral transport of solutes.
RESULTS
At Least Three Different enod40 Genes Are
Expressed in White Clover Stolon Nodes
Partial enod40 cDNA was obtained by PCR from
cDNA prepared from stolon nodes of white clover,
using degenerate oligonucleotide primers with homology to the conserved domains of legume enod40
genes. The nucleotide sequence of several clones revealed the presence of two distinct classes, designated Trenod40-1/2 and Trenod40-3 (for T. repens
enod40), both with homology to known enod40 genes.
To isolate and characterize the full-length Trenod401/2 and Trenod40-3 cDNA, 5⬘- and 3⬘-RACE was
performed with sequence-specific oligonucleotide
primers. The nucleotide sequence of the resulting
clones indicated that three different Trenod40 cDNAs,
designated Trenod40-1, Trenod40-2 (corresponding to
Trenod40-1/2 cDNA class), and Trenod40-3, were
present in white clover nodes.
To identify the number of Trenod40 genes present
in white clover, genomic DNA digested with BamHI
and EcoRI was subjected to Southern-blot analysis.
The presence of two hybridizing bands when
Trenod40-1 cDNA was used as a probe suggests a
small gene family, comprising the Trenod40-1 and
Trenod40-2 genes. Trenod40-3 cDNA probe identified
only one gene (Fig. 1A). Inverse PCR and genomic
walking were used to obtain the upstream and down-
Figure 1. Structure and number of white clover enod40 genes. A, Detection of Trenod40 genes by Southern analysis. White
clover genomic DNA was digested with BamHI (B) or EcoRI (E) and hybridized with the Trenod40-1 (left) or and Trenod40-3
(right) cDNA probes. B, Nucleotide sequences of the Trenod40 transcripts. Identical nucleotides in all of the transcripts are in
shaded boxes. Conserved region I and region II are underlined. C, Comparison of the Trenod40-1 and Trenod40-2 promoters.
Homologous regions are presented as shaded boxes.
Plant Physiol. Vol. 129, 2002
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Varkonyi-Gasic and White
stream regulatory regions of each Trenod40 gene, and
genomic DNA fragments containing the transcribed
region and regulatory sequences of each Trenod40
gene were obtained by PCR using primers designed
from the distal ends of upstream and downstream
regions. The sequence of transcribed regions of
genomic clones Trenod40-1, Trenod40-2, and Trenod40-3
was identical to the sequence of corresponding cDNA
clones, indicating that the Trenod40 genes contain no
introns.
The Trenod40-1 and Trenod40-2 transcribed regions
were largely identical; however, an 18-bp insertion
was present in the 3⬘ end of the Trenod40-2 cDNA,
resulting in 91% overall identity between the two
transcripts. Trenod40-3 cDNA was found to share
only 32.8% identity with Trenod40-1 and Trenod40-2,
the identity being highest in the conserved domains
and the 5⬘ terminus (Fig. 1B). Homologous regions
were also identified in both proximal and distal parts
of Trenod40-1 and Trenod40-2 upstream regulatory
regions (Fig. 1C). The Trenod40-3 upstream regulatory region had low levels of homology to Trenod40-1
and Trenod40-2 only in the proximal 100 nucleotides.
Trenod40-3 Is an Unusual Legume enod40
A comparison of the nucleotide sequences of the
enod40 genes reported so far indicates that two major
groups of enod40 genes have evolved within the legume family (Kouchi et al., 1999; Flemetakis et al.,
2000). White clover Trenod40-1 and Trenod40-2 cDNAs
both have a high level of homology with group II,
clustering with enod40 genes of other legumes that
form indeterminate nodules. Surprisingly, Trenod40-3
clusters with enod40 genes of legumes with determinate nodules (Fig. 2A). Trenod40-1 and Trenod40-2
share the putative peptide of conserved region I that
is 13 amino acids long. The Trenod40-3 conserved
region I encodes for a putative peptide of 12 amino
acids (Fig. 2B). However, region I of all of the
Trenod40 genes contains the conserved nucleotides
present in all other enod40 genes. Region II of all of
the Trenod40 genes contains the majority of the conserved nucleotides previously identified in legume
and non-legume enod40 genes as the region II consensus sequence (Kouchi et al., 1999).
To compare the expression of Trenod40 genes in
symbiotic and non-symbiotic tissues of white clover,
a northern gel-blot analysis was performed using
Trenod40-1 and Trenod40-3 cDNA probes (Fig. 3). The
Trenod40-1 probe detects the combined expression
pattern of Trenod40-1 and Trenod40-2 attributable to
the high sequence homology between these two
genes. Both Trenod40-1/2 and Trenod40-3 transcripts
were found in stolon nodes, with increased levels
detected in more mature nodes. Both transcripts were
also detected in internodes and non-nodulated roots,
whereas they were almost undetectable in stolon tips,
leaves, and immature inflorescence. As expected, the
1110
Figure 2. Phylogenetic analysis of legume enod40 genes and putative ENOD40 peptides. A, Distance tree of legume enod40 cDNA
sequences deduced from the alignment of cDNA sequences starting
20 nucleotides 5⬘ to region I. The alignment was performed using
CLUSTAL V. The scale represents the distance between sequences. B,
Multiple alignment of region I encoded putative oligopeptides from
legumes and nonlegumes. The conserved amino acids are boxed.
The conserved nucleotides present in all legume and nonlegume
enod40 transcripts are presented below. Plant species and GenBank
database accession numbers used in alignments are as follows: Tr,
white clover (1, AF426838; 2, AF426831; and 3, AF426840, this
work), Lj, L. japonicus (1, AJ271787; 2, AJ271788); Sr, Sesbania
rostrata (Y12714); Gm, soybean (1, D13503; 2, D13504); Pv, bean
(X86441); Ms, alfalfa (X80263); Mt, Medicago truncatula (X80264);
Vs, vetch (Vicia sativa; X83683); Ps, pea (X81064); Nt, tobacco
(X98716); and Os, rice (ABO24054).
highest level of Trenod40-1/2 transcript was detected
in nodules. Surprisingly, the Trenod40-3 transcript,
although not entirely absent from nodule tissue,
showed a significantly lower hybridization signal
than that detected in non-symbiotic tissues.
The pattern of expression obtained by in situ hybridization of nodule sections with a Trenod40-1 antisense RNA probe was identical to that shown previously for indeterminate nodules. The transcripts
were detectable in all central tissues of developing
nodules and in the apex, namely the meristematic
and invasion zone of mature nodules (Fig. 4, A and
B). Throughout the process of nodule maturation and
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Plant Physiol. Vol. 129, 2002
White Clover enod40 Gene Family
Figure 3. Northern-blot analysis of poly(A) RNA prepared from stolon tips (ST), stolon internodes, and nodes at different
maturation stages (first visible internode IN5 to mature internode IN⬎8; first visible node N5 to mature node N⬎8), leaf (L),
immature inflorescence (F), non-nodulating root (R), and nodules (NOD). The blotted RNAs were hybridized with the
Trenod40-1 and Trenod40-3 cDNA probes. Trenod40-1 cDNA probe detected the combined expression of Trenod40-1 and
Trenod40-2 (Trenod40-1/2). Integrity of each RNA sample was determined by reprobing with the white clover ubiquitin cDNA.
in the fully mature nodules, the transcripts were
detectable in the nodule vascular bundles. A strong
hybridization signal was also detected in the emerging lateral root (data not shown). No hybridization
signal was detected with the Trenod40-1 sense probe,
which was used as a negative control (Fig. 4C);
whereas the Trenod40-3 probe gave only a weak hybridization signal in the apical meristems and invasion zone of root nodules (Fig. 4D).
Trenod40s Are Expressed in Developing Lateral
Organs and in the Vascular Tissue of Mature Organs
More detailed spatial localization of Trenod40 transcripts in non-symbiotic tissues was obtained using
in situ hybridization. White clover stolon sections
taken from nodes and internodes of different maturity were hybridized with Trenod40-1 and Trenod40-3
antisense RNA probes. High expression of both
Figure 4. Localization of Trenod40 transcripts in white clover nodules. The blue-purple precipitate corresponds to
hybridization signals. A, Longitudinal section of a mid-mature nodule, hybridized with the Trenod40-1 antisense probe.
Transcripts were detected in all central tissues and vascular bundles. Transcripts were also detected in the lateral root stele.
B, Longitudinal section of a mature nodule. Transcripts were detected in the meristem, infection zone, and the vascular
bundle. C, Longitudinal section of a mature nodule. No hybridization signal was detected with the sense Trenod40-1 probe.
D, Longitudinal section of a mid-mature nodule, hybridized with the Trenod40-3 antisense probe. A very weak signal was
detected in the meristem and infection zone. lr, Lateral root; m, meristem; iz, infection zone; and vb, vascular bundle. Bar
represents 200 ␮m in A and 100 ␮m in B through D.
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Varkonyi-Gasic and White
Figure 5. Localization of Trenod40 transcripts in the shoot. The blue-purple precipitate corresponds to hybridization signals.
A through C, Sections of nodal roots at different stages of development, hybridized with a Trenod40-1 antisense probe. A,
Nodal root primordium of node N5. B, Nodal root of node N6, before emergence. C, Advanced stage of nodal root
emergence on node N6. Expression levels remained high in the zone that corresponded to vascular initials. D, Nodal root
of node N6, hybridized with the Trenod40-3 antisense probe. Markedly high expression was detected in the root cap and
the developing vascular cylinder. E, Transverse section of the axillary shoot on node N7, hybridized with the Trenod40-1
antisense probe. A strong hybridization signal was detected in vascular tissue of the axillary shoot base. F and G, Sections
of lateral shoots on nodes N6 and N8, respectively, hybridized with the Trenod40-3 antisense probe. H, Transverse section
of node N6 hybridized with the sense Trenod40-3 probe. In this case, no significant hybridization signal is visible. I, Leaf
trace of node N6, hybridized with the Trenod40-1 antisense probe. Expression was detected in parenchyma surrounding
xylem vessels and the phloem or phloem-cambium region. J, Immature inflorescence, transverse section. No hybridization
was detected with the Trenod40-1 antisense probe. K, Mature inflorescence, transverse section. Elevated expression of
Trenod40-1/2 was detected in the vascular tissue. L, Higher magnification of K. nrp, Nodal root primordium; lt, leaf trace;
vi, vascular initials; rc, root cap; ls, lateral shoot; ph, phloem; x, xylem; ia, inflorescence axis; and p, pedicel. Bar represents
100 ␮m (A–H and J), 25 ␮m (I and L), or 200 ␮m (K).
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Plant Physiol. Vol. 129, 2002
White Clover enod40 Gene Family
Trenod40-1/2 and Trenod40-3 was observed in a developing nodal root. A strong hybridization signal
detected with the antisense Trenod40-1 probe was
localized in the dividing cells of the primordium of
the adventitious root that is formed in the cortex of
the first visible node, designated N5 (Fig. 5A). Expression is continued in later stages, in the second
visible node (N6), as the developing nodal root
grows through the cortex of the stolon (Fig. 5B). At a
slightly advanced stage of nodal root emergence, the
signal is weakening (Fig. 5C) but remains strong in
the zone that corresponds to the vascular initials. A
similar expression pattern in nodal roots was observed with the Trenod40-3 antisense RNA probe,
where transcripts were localized mainly in the developing root cap, meristematic cells, and vascular initials of the nodal root (Fig. 5D).
The developing lateral shoot of node N7 also has
up-regulation of Trenod40-1/2 expression, which is
confined to the vascular tissue at the base of the
lateral shoot (Fig. 5E). Trenod40-3 was also expressed
in developing lateral shoots, with high levels of expression localized mainly in the vascular tissue but
also in other tissues of both the axillary bud of node
N6 (Fig. 5F) and more mature lateral shoot of node
N8 (Fig. 5G). No significant hybridization signal was
detected when stolon sections with developing nodal
roots and the axillary shoot were hybridized to sense
Trenod40-3 RNA (Fig. 5H).
Expression of Trenod40 genes was also detected in
the stolon vascular bundles. The Trenod40-1/2 transcript was localized in the phloem-cambium region
of all vascular bundles and the parenchyma surrounding xylem vessels in the leaf vascular traces
(Fig. 5I). The hybridization signal in the xylem parenchyma was detectable in the internode, then increased significantly in the node, and was not detectable in the petiole vascular bundles. In addition,
Trenod40-1/2 transcripts were found to accumulate
in mature flowers. Although no hybridization signal
above background was detected in the young inflorescence (Fig. 5J), a strong signal was observed in the
vascular tissue of mature inflorescence, after the onset of senescence in the lower rows of florets. The
hybridization signal was localized in the vascular
bundles of the inflorescence axes and in the vascular
bundles of the pedicels that connect individual florets to the inflorescence axes (Fig. 5, K and L). The
expression pattern of Trenod40-3 in stolon vascular
bundles and mature inflorescence axes was similar to
the pattern described for Trenod40-1/2 (data not
shown).
The pattern of Trenod40-2 expression in white clover mature vascular tissues was confirmed using a
Trenod40-2 promoter-gusA fusion. Strong ␤-glucuronidase (GUS) activity was observed in the apex
and vascular bundles of mature nodules (Fig. 6A),
root vascular tissue, mostly at branching points (Fig.
Plant Physiol. Vol. 129, 2002
6B), mature inflorescence in pedicels that diverge
from the inflorescence axes (Fig. 6C), and the stem
vascular bundles. In young nodes, GUS activity was
mainly confined to leaf traces at the point of petiole
attachment (Fig. 6D); whereas in more mature nodes,
it was also detectable at the base of the axillary shoot
(Fig. 6E). In mature nodes, the GUS activity was also
detected in other stolon vascular bundles and
throughout the vascular tissue of the developing lateral shoot (Fig. 6F). The intensity of histochemical
staining in xylem parenchyma of all leaf traces increased in nodes, as presented schematically in Figure 6G. Cross-sections indicate GUS activity localization in the xylem parenchyma of a leaf trace and the
phloem-cambium region of all vascular bundles, irrespective of the size of the vascular bundle and
number of xylem vessels present (Fig. 6H).
The Trenod40 Promoters Are Active in Tobacco and
Regulated in a Manner Similar to White Clover
In white clover, the Trenod40 genes are expressed in
the xylem parenchyma of the leaf vascular traces and
in the phloem-cambial region of all vascular bundles.
This xylem-associated expression is confined mainly
to nodes rather than internodes. White clover has
three-trace nodes, with two leaf traces traversing two
internodes and one traversing one internode, extending into the petiole. The vascular bundles are collateral, with phloem positioned abaxial of the xylem
and the two separated by cambium. Because tobacco
has one-trace nodes with bicolateral vasculature,
with the external and internal phloem on either side
of the xylem, it was of interest to study the expression of Trenod40 promoters in tobacco stems. All
three Trenod40 promoter-gusA fusions were introduced into tobacco using the Agrobacterium tumefaciens leaf disc transformation technique, and the
stems were examined histochemically for GUS expression. In plants carrying the Trenod40-1 and
Trenod40-2 promoter-driven gusA gene, histochemical staining was observed in the stem vascular tissue,
in the internode on the side of the petiole attachment,
and in the node, at the point of the petiole attachment
(Fig. 7A). Some GUS activity was detected at the base
of the axillary shoot bud. Cross-sections indicated the
localization of GUS activity in the parenchyma surrounding xylem vessels, the internal phloem, and the
cells between xylem and internal phloem. Although
the intensity of staining increased in the node, no
staining was detected in the petiole, as schematically
presented in Figure 7C.
In transgenic tobacco stems carrying the Trenod40-3
promoter-gusA fusion, strong GUS activity was detected in the base of the axillary shoot bud. No histochemical staining was observed in the stem vascular tissue (Fig. 7D).
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Varkonyi-Gasic and White
Figure 6. Localization of GUS activity in transgenic white clover plants containing the pRD40-2 construct. A, Mature
nodules. B, Root. C, Mature inflorescence. D through F, Longitudinal sections of white clover stolon nodes at different
maturity stages. D, Node N5. GUS activity was detected in the leaf trace at the point of petiole attachment (arrow). E, Node
N6. GUS activity was detected in the leaf trace at the point of petiole attachment (arrow) and in the base of the auxiliary
bud (arrowhead). F, Node N9. GUS activity was detected in the node vascular bundles and lateral shoot vascular bundles.
G, Diagram showing the intensity of GUS activity in the xylem of leaf vascular traces in internodes and nodes. H, Cross
section of stolon node N6. GUS is localized in the phloem-cambial region of all bundles and the xylem parenchyma of the
leaf trace bundle on the left. ph, Phloem; c, cambium; and x, xylem.
DISCUSSION
Here, we describe the isolation and characterization of a white clover enod40 gene family, composed
of at least three distinct enod40 genes expressed in
stolon node tissues. Two of the white clover enod40
genes, designated Trenod40-1 and Trenod40-2, share a
significant level of identity. However, the sequence
of cDNA clones and genomic clones containing the
transcribed region and over 2 kb of the upstream
regulatory sequences of each gene, combined with
Southern analysis, indicate that Trenod40-1 and
Trenod40-2 are distinct enod40 genes and not variants
of the same gene. The cDNA sequence divergence is
mainly attributed to an insertion sequence at the 3⬘
end of the molecule, a feature found in other enod40
cDNA clones (Crespi et al., 1994; Flemetakis et al.,
2000), suggesting that the evolution of these genes
proceeded by means of irregular duplication. Both
genes share homology to the group II enod40 genes,
1114
isolated from legumes with indeterminate nodules,
and furthermore, they are expressed in nodules in the
manner described for indeterminate nodules (Yang et
al., 1993; Asad et al., 1994; Vijn et al., 1995).
The Trenod40-3 gene is significantly different and is
more similar to the group I enod40 genes isolated
from legumes with determinate nodules. Region I
potentially encodes an oligopeptide of 12 amino acids, a feature found in both legumes with determinate nodules and a monocot, rice (Kouchi et al.,
1999). This gene is the first legume enod40 gene that is
not predominantly expressed in nodules.
To our knowledge, white clover is the first example
of a legume species where the enod40 gene family has
both group I and group II type genes. When multiple
enod40 genes have previously been isolated from legume species, such as soybean (Kouchi and Hata,
1993), French bean (Papadopoulou et al., 1996), alfalfa (Fang and Hirsch, 1998), S. rostrata (Corich et al.,
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Plant Physiol. Vol. 129, 2002
White Clover enod40 Gene Family
Figure 7. GUS localization in the transgenic tobacco stems. A, Longitudinal stem section of a transgenic tobacco plant
containing the pRD40-2 construct. B, Transverse stem section of a transgenic tobacco plant containing the pRD40-2
construct. C, Diagram showing the intensity of GUS activity in a transgenic tobacco stem. D, Longitudinal stem section of
a transgenic tobacco plant containing the pRD40-3 construct. x, Xylem; ip, internal phloem.
1998), and L. japonicus (Flemetakis et al., 2000), the
sequence of all of the genes identified in each of these
species has been similar to either group I or group II
type. White clover, an allotetraploid species, forms
indeterminant root nodules. Therefore, white clover
is an exception to the previous rule that the type of
root nodule development correlates with the type of
enod40 genes present. The group II and group I type
Trenod40 genes may have been derived separately
from the, as yet unknown, parental species that hybridized to form white clover. As an alternative, both
groups of enod40 genes may not have been detected
previously in a single legume species, because the
type of nodule formed determines the predominant
form of enod40 transcript present in nodules. The
unusual Trenod40-3 transcript is only present at relatively low levels in nodules, but is abundant in some
non-symbiotic tissues. Hence, a more extensive examination of enod40 gene expression, particularly in
non-nodule tissues, may identify the presence of both
group II and group I type genes in many legume
species.
The Trenod40 genes of white clover showed strong
expression during lateral organ initiation and development. Two new sites of expression were detected
in the developing lateral organs of the shoot: adventitious nodal roots, with a pattern of expression similar to that described for lateral roots (Papadopoulou
et al., 1996), and developing lateral shoots, with the
expression of Trenod40-3 being more prominent and
induced both at early and later stages of lateral shoot
development. Thus, Trenod40 genes are potentially
useful molecular markers in studies of nodal root and
lateral shoot development.
The putative role of the enod40 genes has mostly
been argued in favor of organogenesis, such as inPlant Physiol. Vol. 129, 2002
duction of the cortical cell divisions that lead to initiation of nodule primordia (Mylona et al., 1995; Charon et al., 1997). The presence of enod40 transcripts in
developing lateral roots (Papadopoulou et al., 1996)
and embryonic tissues (Flemetakis et al., 2000), together with our findings of Trenod40 expression during early stages of nodal root and lateral shoot development support the hypothesis for a role of enod40
in lateral organ development.
However, expression of Trenod40 genes was not
confined to developing lateral organs, indicating that
expression during lateral organ initiation and development may be derived from a different primary
role. Expression of Trenod40 genes in mature vascular
tissue may provide a better clue as to the primary
role of these genes. A primary role in the differentiation or function of vascular bundles has been suggested for Osenod40, a gene that exhibits a xylemassociated pattern of expression in stem vascular
bundles (Kouchi et al., 1999). Transcripts of the Osenod40 gene were detected only in the parenchyma
cells of developing lateral vascular bundles in rice
stems that conjoin the newly emerging leaf. Such
expression at a stage of differentiation of protoxylem
and metaxylem poles in the vascular bundle of a leaf
that is a strong sink for photosynthates and nutrients
led to the hypothesis that Osenod40 has a role in the
differentiation or function of vascular bundles. Our
findings that Trenod40 expression occurs in the xylem
parenchyma of vascular bundles in white clover
nodes that conjoin both young and fully developed
leaves provide support for a role in vascular tissue
function. Leaf traces are larger than other stolon vascular bundles, and they usually have a larger amount
of xylem (Devadas and Beck, 1971), whereas the xylem parenchyma cells differentiate into transfer cells
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1115
Varkonyi-Gasic and White
with a profusion of wall ingrowths, suggesting a
specialization for the intensive lateral transport of
solutes (Gunning et al., 1970). This transport is most
intensive in the node, where the flow rate in the
vascular bundles is diminished and transfer of solutes into surrounding parenchyma cells or exchange
between xylem and phloem is enhanced (Haeder and
Beringer, 1984). Moreover, Trenod40 expression in the
xylem parenchyma was restricted to leaf traces of a
young node, but was detected in all vascular bundles
of older nodes. The vascular traces of older nodes are
involved in the intensive transport of solutes between established nodal roots, developing lateral
shoots, and apical and basal parts of the stem. A role
in intensive lateral transport has also been proposed
for the pericycle of nodule vascular bundles (Pate et
al., 1969), where enod40 expression is detectable
throughout the onset of nodule maturation, as well as
in the mature nodules. Developing lateral organs are
strong sinks that require very intensive shortdistance transport due to the lack of established vasculature. Finally, expression in mature white clover
inflorescence in the pedicels that connect florets with
the inflorescence axes, after the onset of senescence in
the lower floret whorls, when florets become a strong
sink for metabolites, supports the notion of enod40
involvement in vascular tissue function.
Röhrig et al. (2002) recently reported the in vitro
translation of two peptides of 12 and 24 amino acids
from the short, overlapping ORFs of soybean
ENOD40 mRNA. Both peptides specifically bind to
soybean Nodulin 100, which is a subunit of Suc synthase. Suc synthase catalyzes the conversion of Suc
into UDP-Glc and Fru, thus providing nutrients for
sink tissues, energy for metabolic processes, and precursors for cellulose synthesis. Different isoforms of
Suc synthase (Chourey et al., 1998; Barratt et al.,
2001), phosphorylation level (Subbaiah and Sachs,
2001), and localization in multiple cellular compartments (Haigler et al., 2001; Salnikov et al., 2001)
enable Suc synthase to channel carbon from Suc toward different metabolic fates within the cells. Therefore, Suc synthase acts as a molecular switch between
survival metabolism and growth and differentiation
processes (Haigler et al., 2001). Röhrig et al. (2002)
postulate that enod40 peptides either regulate the Suc
synthase activity or direct the enzyme to specific
intracellular sites, suggesting that enod40 peptides
may contribute to the control of photosynthate use in
plants. This is consistent with the expression of
Trenod40 in developing root and shoot lateral organs,
vascular tissue of mature nodules, inflorescence, and
phloem-cambium region of stem vascular bundles. In
these tissues, Trenod40 may have a role in regulation
of Suc unloading and channeling into distinct metabolic pathways within the cells.
In M. truncatula, only one Suc synthase isoform was
detected in root nodules, but an additional isoform
was present in uninfected roots, stem, and flower
1116
tissue (Hohnjec et al., 1999). It is possible that
Trenod40-1/2 modulate the white clover equivalents
of both isoforms, whereas Trenod40-3 specifically regulates only the non-nodule Suc synthase. However,
expression of Trenod40 in the xylem of mature stem
vascular bundles, suggests that there is for an additional role for Trenod40 transcripts or putative peptides, possibly via a different ligand. Enod40 may
have a more general role in regulation of unloading
of other nutrients, amino acids, and minerals, acting
as a regulator of lateral transport in a fine-tuned
network of auxin, cytokinin, and flavonoid signaling
(Mathesius et al., 2000). The pattern of Trenod40-1
and Trenod40-2 promoter-driven GUS activity in
transgenic tobacco indicate that the mechanisms involved in the activation of the regulatory elements in
these promoters are common for white clover and
tobacco.
Other enod40 promoters have previously been
shown to be active in heterologous plants and controlled in the same manner as the homologous enod40
genes, e.g. the rice Osenod40 promoter provides temporal and spatial expression in soybean nodules
identical to Gmenod40 (Kouchi et al., 1999), and the
Gmenod40 promoter is active in Arabidopsis (Mirabella et al., 1999). However, we found that the
Trenod40-3 promoter was active only in the axillary
bud of tobacco plants, and other aspects of its regulation in white clover stolons were not present in the
heterologous system. One possibility is that there are
tissue specific signals activating the Trenod40-3 promoter in white clover that are absent in tobacco. As
an alternative, the expression found by northern and
in situ hybridization in white clover stolons was due
to sequences located upstream from the 2.1-kb promoter fragment used in the experiment. This promoter fragment provided only transient GUS activity
in immature transgenic white clover plants, and further work is needed to identify and analyze the regulatory regions necessary for controlling all aspect of
expression for this gene.
MATERIALS AND METHODS
Isolation of Trenod40 cDNA Clones
A pair of degenerate primers designated enod40F [5⬘-GGC (A/T)(A/
C)(A/G) (A/C) A(A/T) C(A/C) A TCC ATG GTT CTT-3⬘] and enod40R
[5⬘-GGA (A/G) TC CAT TGC CTT TT-3⬘] were designed based on the two
highly conserved regions of legume enod40 cDNA sequences. PCR products
amplified from white clover (Trifolium repens) node cDNA were cloned into
pGEM-T plasmid vector (Promega, Madison, WI), and the nucleotide sequences of the inserts were determined by the dideoxy chain termination
method using an automated sequencer (ABI310; PE-Applied Biosystems,
Foster City, CA). Two different classes of enod40 cDNAs were identified,
designated Trenod40-1/2 and Trenod40-3.
Full-length clones were isolated by 5⬘- and 3⬘-RACE with the Marathon
cDNA amplification kit (CLONTECH Laboratories, Palo Alto, CA) using
gene-specific oligonucleotides as primers: Trenod40-1/2 5⬘-RACE (5⬘-GTG
ACT TGC CGG TTT GCC ATG CTA-3⬘) and Trenod40-3 5⬘-RACE (5⬘-CTC
CAT ATT CTC ACT GTG ATT ACT-3⬘) were used for the amplification of
specific 5⬘-end sequences. After cloning into pGEM-T and sequencing, the
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Plant Physiol. Vol. 129, 2002
White Clover enod40 Gene Family
gene-specific 3⬘-RACE oligonucleotide primers were designed from the very
5⬘-end sequences of each cDNA class. Two Trenod40-1/2 3⬘-RACE primers
(5⬘-GAT CAG AGA TAC CAA CTT CCC CAC-3⬘ and 5⬘-CTT CCC CAC
TAC CTT CTT TTG T-3⬘) and two Trenod40-3⬘-RACE primers (5⬘-GAT CAG
AAA CTA ACT TCC CCA CTA GCA-3⬘ and 5⬘-CAA ATC TGA AAT CTT
GTA GTT GCT-3⬘) were used for the amplification of full-length cDNAs.
The resulting amplification products were cloned into pGEM-T, and five
clones of each cDNA class were sequenced.
Isolation of Trenod40 Regulatory Regions and
Genomic Clones
Genomic sequences flanking the Trenod40-1/2 transcribed region were
isolated by inverse PCR (Ochman et al., 1988) from EcoRI-digested white
clover genomic DNA, using gene-specific antisense (5⬘-GTG GGG AAG TTG
GTA TCT CTG ATC-3⬘) and sense (5⬘-GGT GTT GTC TTC CTT TGA GAA
GTT GCC-3⬘) primers. Two resulting PCR fragments, corresponding to
flanking regions of Trenod40-1 and Trenod40-2, were cloned into pGEM-T
and sequenced. The flanking regions of the Trenod40-3 transcribed region
were isolated by genomic walking (Siebert et al., 1995), using two genespecific antisense primers (5⬘-CGG ACG ATC AAA ATC AAT GAC TGC
GTC ACG-3⬘ and 5⬘-CTC CAT ATT CTC ACT GTG ATT ACT-3⬘) for the
amplification of upstream and two gene-specific sense primers (5⬘-AAG
TTG TGT GAA AGG GTC CTC A-3⬘ and 5⬘-CTT TGG CTA TAG CTT GGT
AAA CCG-3⬘) for the amplification of downstream regions.
Primers from the distal end of the upstream regulatory sequences and the
downstream regions were designed to amplify, clone, and sequence all three
Trenod40 genes.
Construction of Trenod40 Promoter-gusA Fusions
A BclI site was identified in the junction of the promoters and transcribed
regions of all of the Trenod40 genes. The EcoRI-BclI fragments of Trenod40-1
and Trenod40-2 promoters (2.2 and 3.1 kb, respectively) were individually
ligated into the HindIII-BamHI site of binary vector pRD410 (Datla et al.,
1992) using a HindIII-EcoRI linker to create pRD40-1 and pRD40-2, respectively. The HindIII-BclI fragment of the Trenod40-3 promoter (2.1 kb) was
cloned into pRD410 to create pRD40-3. All pRD40 plasmids were electroporated into Agrobacterium tumefaciens strain LBA4404 and transformed into
Nicotiana tabacum W38 by the leaf disc transformation method (Horsch et al.,
1985). Four of 12 primary transformants for each construct were assayed for
GUS activity. In addition, the pRD40-2 plasmid was introduced in white
clover cv Huia by A. tumefaciens-mediated transformation (Voisey et al.,
1994). Six of 12 primary transformants showed GUS activity in nodules and
stolon nodes. Two of those were chosen for detailed histochemical staining.
Southern and Northern Analysis
Southern blots of white clover genomic DNA were prepared and hybridized by standard protocols (Church and Gilbert, 1984; Chomcszynski, 1992).
Total RNA was extracted from white clover tissues according to Verwoerd
et al. (1989) and used for subsequent poly(A) RNA extraction with the
PolyATtract mRNA isolation system (Promega). One microgram of poly(A)
RNA was subjected to denaturing gel electrophoresis and blotted onto the
membrane using standard methods (Sambrook et al., 1989). The hybridization and washing were performed according to Church and Gilbert (1984).
[␣-32P]dCTP-Labeled probes were prepared from full-length cDNA fragments by random primed labeling.
In Situ Hybridization
Fresh plant material was fixed in 4% (v/v) formaldehyde in phosphatebuffered saline containing 0.1% (v/v) Tween 20, pH 7.4, overnight at 4°C.
Fixed tissues were dehydrated through an ethanol series and histoclear and
embedded in paraffin according to Cox et al. (1984). Sections (10 ␮m) were
cut and mounted on poly-l-Lys-coated slides. Antisense and sense RNA
probes labeled with (DIG)-11-rUTP were transcribed from the full-length
cDNA clones with the T7 and SP6 polymerase (Roche). Sections were
rehydrated, prepared for hybridization according to Steel et al. (1998), and
hybridized overnight at 48°C in 50% (v/v) formamide, 5⫻ SSC and 50 ␮g
Plant Physiol. Vol. 129, 2002
mL⫺1 heparin. After hybridization, the slides were washed, and the probes
were visualized as described by Steel et al. (1998). Finally, slides were
treated with 50% (v/v) 2-mercaptoethanol to prevent oxidation.
GUS Histochemical Assay
GUS activity was assayed as described by Jefferson (1987). After staining,
the tissues were rinsed in 50 mm phosphate buffer, pH 7.0, and cleared in
50% and subsequently absolute ethanol. The stained tissues were used
directly for observation or dehydrated with histoclear, embedded in paraffin, and sectioned. Sections (10–25 ␮m) were mounted onto slides for
observation and photographed.
ACKNOWLEDGMENTS
We acknowledge Nick Roberts, Igor Kardailsky, and Bruce Campbell for
critical discussions.
Received October 9, 2001; returned for revision January 2, 2002; accepted
March 22, 2002.
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