INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

It has been proposed that genes with nodule-specific 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 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 progenitor 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 non-symbiotic 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

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

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 Trenod40-1/2 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 downstream 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.

View larger version (87K): [in this window] [in a new window]   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.

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).

View larger version (31K): [in this window] [in a new window]   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).

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 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.

View larger version (46K): [in this window] [in a new window]   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.

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 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).

View larger version (124K): [in this window] [in a new window]   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 µm in A and 100 µm in B through D.

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 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).

View larger version (163K): [in this window] [in a new window]   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 µm (A-H and J), 25 µm (I and L), or 200 µm (K).

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. 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).

View larger version (131K): [in this window] [in a new window]   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.

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.

View larger version (66K): [in this window] [in a new window]   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.

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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

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, 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., 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 induction 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 xylem-associated 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 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 short-distance 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 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

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

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 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 gene-specific 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 phosphate-buffered 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 µm) 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 µg mL¹ 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 µm) were mounted onto slides for observation and photographed.