Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol160.Issue1.2000:

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The promoter of the

Vicia faba

L. gene VfEnod12 encoding an

early nodulin is active in cortical cells and nodule primordia of

transgenic hairy roots of

Vicia hirsuta

as well as in the prefixing

zone II of mature transgenic

V

.

hirsuta

root nodules

Martin Fru¨hling, Gerald Schro¨der, Natalija Hohnjec, Alfred Pu¨hler,

Andreas M. Perlick, Helge Ku¨ster *

Biologie VI(Genetik),Uni6ersita¨t Bielefeld,Fakulta¨t fu¨r Biologie,Postfach100131,D-33501Bielefeld, Germany

Received 6 April 2000; received in revised form 7 July 2000; accepted 9 August 2000

Abstract

A full-length cDNA encoding theVicia fabaL. early nodulin VfEnod12 was isolated. The deduced protein sequence specified a 90 amino acid protein with a MW of 10 206 and contained a putative signal peptide sequence followed by PPX3 repeats

characteristic of Enod12 proteins. The VfEnod12 gene was found to be expressed specifically in root nodules as early as 3 days post inoculation withRhizobium leguminosarumbv.6_iciae_{. In mature nodules, VfEnod12 transcripts were confined to the prefixing}

zone II. A 3.3 kb genomic fragment carrying the complete VfEnod12 coding region was isolated. No intervening sequences were identified in the coding region. A promoter fragment carrying the -692/-41 region mediated reporter gene expression in root cortical cells, nodule primordia and the prefixing zone II of transgenic Vicia hirsuta root nodules. This fragment contained a putative binding site for the transcription factor ENBP1. In contrast to the highly conserved terminal AATAA motif of the ENBP1 binding site of known Enod12 promoters, the VfEnod12 promoter was characterized by an altered terminal AATAT sequence. This alteration did not interfere with VfEnod12 promoter activity in transgenic roots and nodules ofV.hirsuta. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Early nodulin; ENBP1 binding site; Hairy roots; Promoter activity;Vicia faba;Vicia hirsuta

www.elsevier.com/locate/plantsci

1. Introduction

During the symbiotic interaction of rhizobia with legume plants the bacterial microsymbionts induce a developmental programme that leads to the formation of a completely new symbiotic or-gan, the root nodule. The root nodule is infected and the central tissue is subsequently colonized by the microsymbionts [4,9]. In the nodule tissue, the plant provides the environment necessary for the microsymbiont to convert atmospheric dinitrogen to ammonia. During all stages of recognition,

organogenesis and function, a complex network of molecular communication accompanied by the specific activation of genes in both partners is involved [8,22,12]. The crucial signalling factors

that are synthesized by Rhizobium and that are

perceived by the plants are substituted

lipochi-tooligosaccharides, the so-called nodulation

(NOD) factors. These bacterial signal molecules are responsible for the specificity of the legume-Rhizobium interaction [15,34]. Up to now, a num-ber of plant genes expressed exclusively in root nodules in response to rhizobial infection were identified [7,40,23]. The encoded gene products were designated nodulins and were divided into early and late nodulins according to their timing of synthesis [39]. In contrast to late nodulins

com-* Corresponding author. Tel.: +49-521-1065620; fax: + 49-521-1065626.

E-mail address:[email protected] (H. Ku¨ster).

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prising leghemoglobins and enzymes of nodule carbon and nitrogen metabolism, early nodulins are mainly structural proteins involved in infection or nodule organogenesis [32]. One of the first early nodulin genes specifically activated after infection of legume roots by rhizobia are the Enod12 genes. Up to now, Enod12 sequences were isolated from Pisum sati6_um _[13,31], _{Medicago sati}6_a _[1],

Medicago truncatula [26] and Vicia sati6_a _[41].

Enod12 genes code for proline-rich proteins

char-acterized by different numbers of PPX3

pentapep-tide repeats preceded by a signal peppentapep-tide. The two

similar Enod12 genes encoded in P. sati6_um

(PsEnod12a and-b) and M. sati6_a _(MsEnod12a

and-b) differ in the number of PPX3 repeats. The

occurence of PPX3 pentapeptide repeats defines a

category of structural cell-wall proteins designated hydroxyproline rich glycoprotein (HRGPs, [35]). Hence, Enod12 proteins are assumed to be struc-tural components of plant cell walls in root nod-ules involved in the reaction of the plant to an infection by rhizobia [31]. In addition to the

Enod12 proteins, several proline-rich early

nodulins were identified [32], e.g. the well-known Enod5 and Enod2 nodulins as well as the Enod10 [21] and MtPRP4 proteins [44]. Csanadi et al. [6] identified an alfalfa line that does not contain any Enod12 gene. In such plants nodule formation was not impaired, nor was nitrogen fixation reduced.

Obviously, at least in M. sati6_a _{root nodule}

organogenesis and function is not dependent on Enod12 proteins. This might be explained by the existence of similar PRPs that substitute for Enod12 proteins.

In infected roots, PsEnod12 transcripts were localized in cells containing an infection thread and cells placed in front of the growing infection thread leading to the suggestion that Enod12 might be involved in the infection process [31]. Later it was demonstrated that the Enod12 gene from M. truncatula is activated in root hairs as early as 3 h after infection [26]. In mature root nodules, Enod12 transcripts were localized in the prefixing zone II [2,31,41]. In contrast to the PsEnod12a and PsEnod12b genes, which are ex-pressed comparably, for the two Enod12 genes

fromM.sati6_a_{markedly different expression}

char-acteristics were found. MsEnod12a was activated exclusively in the proximal part of the prefixing zone II of root nodules dependent on the presence of an active meristem, whereas MsEnod12b was

detected in root hairs within few hours after appli-cation of NOD factors [2] as it was already demonstrated for PsEnod12a [17] and MtEnod12 [26]. Enod12 genes are also activated in spontanu-ous nodules [27] or by phytohormones [3] indicat-ing that Enod12 gene expression is part of the preexisting plant programme underlying nodule formation.

Using fusions of Enod12 promoters from M.

sati6_a _and _M_. _truncatula _{to the} _gus_{A reporter}

gene, regions mediating activity were identified [3,26]. In case of the PsEnod12 promoters, essen-tial regions were localized within 200 bp upstream of the transcription start sites [42]. Recently, a promoter element was identified in the PsEnod12b promoter that was able to specifically interact with

the transcription factor ENBP1 from Vicia sati6_a

[5]. Interestingly, mutations in this element com-pletely abolished PsEnod12b promoter activity in

transgenic root nodules of Vicia hirsuta [14].

The characteristic expression properties of

Enod12 genes made them valuable tools to analyse

early aspects of legume-Rhizobiuminteractions. To

investigate organ-specific gene expression in broad

bean (Vicia faba L.) nodules, we constructed a

nodule-specific cDNA library by differential hy-bridization [24]. Sequence analysis of a cDNA

from clone group VfNDS-X7 (V6 icia f6aba n6odule

d6ifferential s6creening, group X7) of this library

revealed that this incomplete cDNA encoded a broad bean Enod12 protein. We here report on

the expression properties of a Vicia faba Enod12

gene and analyze the promoter in transgenic hairy

roots and nodules of Vicia hirsuta. We show that

the VfEnod12 promoter fragment isolated is active although it contains a binding site for the tran-scription factor ENBP1 that is altered in a subele-ment that is exactly conserved in all other Enod12 genes identified so far.

2. Methods

2.1. Plant material

Vicia faba L. cv. Kleine Thu¨ringer plants were grown in the greenhouse in sand or Seramis (clay granules). Two days after sowing, the seedlings

were inoculated withRhizobium leguminosarumbv.

6_iciae _{VF39 [28] to obtain infected plants. To}

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sterile soil as described [24]. Vicia hirsuta (hairy tare) seeds were obtained from John Chambers Wild Flower Seeds, Kettering, UK.

2.2. cDNA libraries, genomic libraries and cloning of reporter gene fusions

A nodule cDNA library and a nodule-specific

cDNA library were constructed in lgt11 [18]

from polyA+ _{mRNA isolated from root nodules}

of V. faba L. cv. Kleine Thu¨ringer [24]. A

ge-nomic library of V. faba L. cv. Kleine Thu¨ringer

was prepared in lEMBL3 [10] according to

Sambrook et al. [30]. The complete -1955/-41

VfEnod12 promoter region was PCR-amplified using the M13 reverse primer in conjunction

with an oligonucleotide spanning the -36/-60

re-gion of the VfEnod12 promoter where positions

-36/-41 were modified to form an EcoRI site. As

a template, plasmid pUC18:16-69 was used that contained the 3.3 kb genomic VfEnod12

frag-ment cloned in the EcoRI site of plasmid

pUC18 [45]. The -692/-41 region was

subse-quently released as EcoRV/EcoRI fragment and

was cloned into the SmaI/EcoRI sites in front of

the gusAint gene in plasmid pGUS-INT [19].

From the resulting plasmid, the pVfEnod12-gu

-sAint fusion was subcloned as SphI/SalI

frag-ment into the TL DNA integration vector pIV2 [19] and the resulting plasmid was transferred to A. rhizogenes ARqua1 using E. coli S17-1 medi-ated conjugation [36]. Correct integration in the TL DNA was verified by Southern hybridiza-tions.

2.3. DNA sequencing and sequence analysis

Sequencing reactions were carried out accord-ing to Zimmermann et al. [46] usaccord-ing the ‘Au-toRead Sequencing Kit’ (Pharmacia). Sequencing gels were run on the ‘A.L.F. DNA Sequencer’ (Pharmacia) using sequencing gel mixes of stan-dard composition. All sequences reported here were determined from both strands. Nucleic acid sequences were read using the ‘A.L.F. MAN-AGER V3.0’ software (Pharmacia). Deduced amino acid sequences were analysed according to von Heijne [43] and Straden [37] and by using

the PC/Gene software package (Intelligenetics,

release 6.80).

2.4. Isolation of RNA, northern, cDNA-cDNA and tissue-print hybridizations

Total RNA was isolated from nodules (32 days after sowing), uninfected roots (32 days af-ter sowing), leaves (32 days afaf-ter sowing), seeds (90 days after sowing), epicotyls (8 days after sowing), stems (12 days after sowing) and flow-ers (60 days after sowing) of broad bean accord-ing to Perlick and Pu¨hler [24]. For time-course hybridizations, total RNA was prepared accord-ingly from infected roots harvested in 2 day

in-tervals from day 3 after inoculation with R.

leguminosarum bv. 6_iciae VF39. For Northern

blotting, the amount of 30 mg of total RNA

from different tissues was separated

elec-trophoretically and blotted onto Hybond – N

ny-lon membranes (Amersham) according to

standard protocols [30]. Fifty nanograms of

VfEnod12 probe DNA were labelled with 50 mCi

of [a32_{P]dATP and hybridizations were carried}

out as described [24]. Stringent washes were

car-ried out at room temperature using 2×SSC,

0.1% (w/v) SDS (5 min) and at 68°C using

0.2×SSC, 0.1% (w/v) SDS (twice for 30 min).

cDNA – cDNA hybridizations were carried out according to Fru¨hling et al. [11]. Tissue print hybridizations were carried out as described by Schro¨der et al. [33]. To relate hybridizing regions of the print to distinct nodule zones, nodule sec-tions corresponding to the print were stained for

starch in a solution containing 1% (w/v) KI and

1% (w/v) I2 in distilled water. Subsequently,

stained sections were rinsed in distilled water and photographed at the same magnification as the tissue print filter.

2.5. Induction and analysis of transgenic hairy roots and root nodules of V. hirsuta

V. hirsuta seeds were surface-sterilized and plants were grown in petri dishes in a growth chamber as described [29]. Transgenic hairy

roots of V. hirsuta were generated using A. rhi

-zogenes Arqua1 derivatives and nodulated using R. leguminosarum bv. 6iciae Vh5eSm as reported previously [29]. After 2 – 4 weeks, roots and root nodules were analysed histochemically as re-ported by Ku¨ster et al. [19].

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3. Results and discussion

3.1. Isolation of a full-length cDNA encoding an Enod12 protein from V. faba

During a differential screening for nodulin genes

of V. faba, the cDNA 66-1 from the

cross-hy-bridizing clone group X7 was isolated that con-tained an incomplete Enod12 transcript sequence [24]. A full-length VfEnod12 cDNA was isolated by rescreening the nodule cDNA library with the 66-1 probe. Clone 114 – 96 was sequenced and contained a 533 bp cDNA covering the complete

Enod12 coding region (EMBL accession

AJ277289). The deduced protein sequence con-sisted of 90 amino acids and had a MW of 10 206. According to von Heine [43], the VfEnod12 protein was predicted to contain an N-terminal secretory signal peptide with a cleavage site be-tween amino acids 24 and 25 (Fig. 1). The C-ter-minal region was composed of the proline-rich

PPX3 pentapeptide repeats typical of Enod12

proteins [31]. Comparing VfEnod12 with other Enod12 sequences identified so far, 10 such repeats were identified. In four cases one of the proline residues (NPAYE, TPVHK, PSYGK, HPTES) and in one case both prolines were changed (SHLHV, Fig. 1). In the homologous region, VfEnod12 was almost identical to PsEnod12a [31]. Within 90 amino acids only four changes occured, three in the signal peptide region and one in the

first PPX3 repeat. Nevertheless, four PPX3 repeats

corresponding to PsEnod12a repeats were not present in the broad bean sequence (Fig. 1).

Inter-Fig. 2. Expression of the VfEnod12 gene in broad bean tissues. A Northern blot containing total RNA isolated from root nodules, uninfected roots, leaves, seeds, epicotyls, stems, and flowers was probed with the full-length VfEnod12 cDNA. The size of the hybridizing transcript was determined using molecular weight standards. Abbreviation: kb, kilo bases.

estingly, these four PPX3repeats were also missing

in the PsEnod12b sequence which on the other hand showed a reduced sequence homology with

VfEnod12. Hence, VfEnod12 resembled

PsEnod12a on the sequence level but PsEnod12b on the structural level. Comparable differences

existed for the twoM. sati6a Enod12 nodulins [1].

Here, MsEnod12a is a 93 amino acid protein whereas MsEnod12b is a 113 amino acid protein

containing four additional PPX3 repeat as does

PsEnod12a. Due to the presence of PPX3 repeats,

the structure of VfEnod12 and other Enod12 proteins is very similar to proline-rich proteins (PRPs) found in a variety of plants independently of the presence of any symbiotic interaction. The highest homologies existed with SbPRP3, a 90

amino acid protein with 11 PPX3 repeats present

in the aerial parts of soybean plants ([16], data not

shown). Due to the presence of PPX3 repeats

characteristic of plant cell wall proteins of the hydroxyproline rich glycoprotein (HGRP) type [35], Enod12 proteins are thought to be integral cell wall proteins [31]. Considering this and the homologies identified, we propose that VfEnod12 constitutes a cell wall protein from broad bean root nodules.

3.2. VfEnod12 transcripts accumulate early during root nodule organogenesis

The cDNA 114 – 96 was used as a probe to analyse VfEnod12 gene expression in different tis-sues. An appr. 600 nt transcript corresponding in size to the full-length VfEnod12 cDNA was present exclusively in nodules, whereas no tran-scripts were detected in uninfected roots, seeds,

Fig. 1. Comparison of Enod12 protein sequences from differ-ent legumes. Sequences of Enod12 proteins fromVicia sati6_a

(VsEnod12; [41]), Medicago sati6_a _{(MsEnod12a and b; [1]),} Pisum sati6_um _{(PsEnod12a and b; [13,31]) and} _Medicago truncatula (MtEnod12, [26]) were compared with VfEnod12. The potential cleavage site between the signal peptides and the mature proteins containing characteristic PPX3repeats is

indicated by an arrowhead. The pentapeptide repeats are seperated by gaps. Gaps introduced during sequence align-ment are indicated by dashes. Amino acids identical in at least four Enod12 sequences are inverted.

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epicotyls, shoots, and flowers (Fig. 2). This corre-sponds to the expression pattern of MsEnod12a [2] and MtEnod12 [26], but is in contrast to the PsEnod12a, PsEnod12b and MsEnod12b gene pression [2,13,31]. The latter genes are also ex-pressed at lower levels in other tissues, in particular in flowers. In a time-course experiment, VfEnod12 transcripts accumulated earlier after

in-fection by R. leguminosarum bv. 6_iciae _{than all}

other 19 broad bean nodulin genes identified so far. In detail, VfEnod12 transcripts were present three post inoculation, whereas transcripts from the early nodulin gene VfEnod2 were detected 2 days later and most other nodulin genes including the leghemoglobin genes were detected 6 days later [20,25]. To localize the VfEnod12 site of expres-sion, tissue-print hybridizations were carried out. Fig. 3 shows that VfEnod12 transcripts were de-tected in the prefixing zone II of mature root nodules with the highest amount in the distal part of this region. Apparently, no VfEnod12 tran-scripts were present in the nodule meristem. This type of expression is more similar to the expres-sion pattern observed for the Enod12 gene from

V.sati6_a_{[41] than to the expression patterns of the}

PsEnod12 genes that are expressed in the most distal parts of the prefixing zone II [13,31]. Hence, VfEnod12 constitutes a proline-rich early nodulin that might function as a cell wall protein in the prefixing zone II of broad bean root nodules,

possibly by specifically modifying plant cell walls.

3.3. The VfEnod12 gene contains no introns and is preceded by a putati6_{e ENBP}₁ _{binding site}

In contrast to the two different P. sati6_um

Enod12 genes that are expressed similarly [13,31], the expression properties of the two alfalfa Enod12 genes are different [2]. To be able to

investigate the expression properties of the

VfEnod12 gene using fusions of its promoter to reporter genes, we isolated a broad bean genomic clone containing VfEnod12 sequences. A 3332 bp

EcoRI fragment of this clone contained not only

the complete coding region without any

interven-ing sequences, but also 1955 bp of the 5%_{and 1104}

bp of the 3% region (EMBL accession AJ277288).

The fact that the VfEnod12 gene contains no introns is a common feature of Enod12 genes [1,13,26,41]. Sequence comparisons between the VfEnod12 cDNA 114-96 and the VfEnod12 ge-nomic sequence revealed two nucleotide changes in the coding region and 3 nucleotide changes in

the 3% _{UTR, whereas insertions or deletions were}

not detected (data not shown). Considering the limited allelic variation that exists in our broad bean cultivar [20], the genomic fragment isolated corresponds to the full-length VfEnod12 cDNA.

Further upstream in the 5% region, homologies to

gag proteases were identifed from position -856 onwards indicating the presence of a

retrotranspo-son in the -856/-1955 upstream region of

VfEnod12. The putative gag coding region is ori-ented in the opposite direction of VfEnod12 tran-scription (data not shown).

At position -47/-52 relative to the start codon a

putative TATA-box was localized, whose position was similar to the positions of the TATA-boxes identified in the promoters of the two MsEnod12

genes [2]. Further upstream at position -99/-118 a

sequence element was identified that is ho-mologous to the binding site for the transcription factor ENBP1 that was identified in the promoter of the PsEnod12b gene [5]. The palindromic motif

characteristic of this binding site (TTATT-N7--12

-AATAA, [14]) is present at a similar position appr. 120 bp upstream from the start codon in different Enod12 promoters (Fig. 4). Interestingly, the terminal base of the AATAA subelement that is conserved in all Enod12 promoters isolated so far is changed to AATAT in the VfEnod12

pro-Fig. 3. Localization of VfEnod12 transcripts in root nodules. In A, total RNA from a broad bean root nodule section was transferred to a nylon filter by tissue printing and hybridized against a VfEnod12 antisense riboprobe. In B, the nodule section used for printing was subsequently stained for starch to determine the accumulation of amyloplasts indicating the nodule interzone II/III. VfEnod12 transcripts accumulate in the prefixing zone II and are absent from the nodule meris-tem.

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Fig. 4. Comparison of the binding site for the transcription factor ENBP1 found in the promoters of different Enod12 genes. The sequences of the binding sites for the transcription factor ENBP1 found in the promoters of different Enod12 genes are compared with the conserved termini being under-lined. The positions of these elements relative to the transla-tional start are given on the right.

not observed in non-infected parts of the trans-genic root system including the epidermis and the meristem (Fig. 5B, E). The activity of the VfEnod12 promoter in outer cortical cells that probably contain infection threads conforms to the localization of PsEnod12a transcripts [31]. Re-porter gene expression in root inner cortical cells conforms to results for Enod12 promoters from M. sati6a [2], M. truncatula [26] and P. sati6um [31]. In contrast to the MsENOD12a promoter [3],

Fig. 5.

moter (Fig. 4). Apart from the homologies to the transcription factor binding site, the sequence of

the VfEnod12 promoter is significantly

ho-mologous to the sequences of other Enod12 gene

promoters fromM. sati6a and P. sati6um [1,13,26]

in the region of appr. 200 bp upstream of the start codon with the homologies decreasing in the up-stream regions (data not shown). We concluded that the Enod12 promoter from broad bean con-tained an altered binding site for the transcription factor ENBP1.

3.4. The -692/-41 VfEnod12 promoter containing an altered ENBP1 binding site is sufficient to acti6ate gene expression in early stages of nodule de6_{elopment and in mature root nodules}

The -692/-41 promoter region of the VfEnod12

gene containing the ENBP1 binding site altered in the terminal position and the putative TATA-box

was cloned in front of the gusAint reporter gene

[38] and the resulting fusion was expressed in

transgenic roots and nodules ofV.hirsutausing an

A. rhizogenes based transformation system gener-ating transgenic hairy roots [29]. Resulting hairy roots were assayed histochemically for GUS activ-ity to monitor the expression properties of the cloned region of the VfEnod12 promoter. In inoc-ulated root systems, GUS activity was first de-tected in inner cortical cells and in cells of the outer cortex that probably contain infection threads (Fig. 5A). In subsequent stages of early nodule organogenesis, GUS activity was detected in root nodule primordia (Fig. 5B – D), but not in emerging lateral roots (Fig. 5D). Young emerging nodules expressed the reporter gene throughout the central region (Fig. 5E), whereas in mature root nodules, GUS activity was confined to the prefixing zone II (Fig. 5E – F). GUS staining was

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no expression of the VfENOD12 promoter was seen in emerging lateral roots.

VfEnod12 promoter activity in the prefixing zone II of mature nodules corresponds to the

results obtained in V. faba by tissue-printing (Fig.

3) and resembles the expression of Enod12 pro-moters from other legumes [2,14]. Vijn et al. [42] demonstrated that a 200 bp region of the PsEnod12 promoters is sufficient for an expression in nodules. Christiansen et al. [5] identified the transcription factor ENBP1 that interacted with a specific region of the PsEnod12b promoter. Re-cently, Hansen et al. [14] showed by mutational analysis that the terminal AATAA subelement of

the TTATT-N9-AATAA binding site is essential

for expression of the PsEnod12b promoter in

transgenic hairy roots and nodules of V. hirsuta.

In these experiments, the AATAA element was changed to CCGCC. The expression properties of the PsEnod12b [14] and the VfEnod12 (Fig. 5) promoters in transgenic systems are very similar and therefore should allow a comparison of the results obtained. Our results on the expression of

the VfEnod12 promoter in transgenic V. hirsuta

hairy roots show that the AATAA element can tolerate a limited amount of variation and that such variation exists in a native Enod12 promoter. These data might provide a starting point for additional mutational analyses to elucidate the requirements for a functional ENBP1 binding site.

Acknowledgements

This work was supported by a grant from the

‘Studienstiftung des Deutschen Volkes’ to Gerald Schro¨der.

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(1)

3. Results and discussion

3.1. Isolation of a full

-

length cDNA encoding an

Enod

12 protein from V

.

faba

During a differential screening for nodulin genes

of

V

.

faba

, the cDNA 66-1 from the

cross-hy-bridizing clone group X7 was isolated that

con-tained an incomplete Enod12 transcript sequence

[24]. A full-length VfEnod12 cDNA was isolated

by rescreening the nodule cDNA library with the

66-1 probe. Clone 114 – 96 was sequenced and

contained a 533 bp cDNA covering the complete

Enod12

coding

region

(EMBL

accession

AJ277289). The deduced protein sequence

con-sisted of 90 amino acids and had a MW of 10 206.

According to von Heine [43], the VfEnod12

protein was predicted to contain an N-terminal

secretory signal peptide with a cleavage site

be-tween amino acids 24 and 25 (Fig. 1). The

C-ter-minal region was composed of the proline-rich

PPX

pentapeptide repeats typical of Enod12

proteins [31]. Comparing VfEnod12 with other

Enod12 sequences identified so far, 10 such repeats

were identified. In four cases one of the proline

residues (NPAYE, TPVHK, PSYGK, HPTES)

and in one case both prolines were changed

(SHLHV, Fig. 1). In the homologous region,

VfEnod12 was almost identical to PsEnod12a [31].

Within 90 amino acids only four changes occured,

three in the signal peptide region and one in the

first PPX

repeat. Nevertheless, four PPX

repeats

corresponding to PsEnod12a repeats were not

present in the broad bean sequence (Fig. 1).

estingly, these four PPX

repeats were also missing

in the PsEnod12b sequence which on the other

hand showed a reduced sequence homology with

VfEnod12.

Hence,

VfEnod12

resembled

PsEnod12a on the sequence level but PsEnod12b

on the structural level. Comparable differences

existed for the two

M

.

sati

6 a

Enod12 nodulins [1].

Here, MsEnod12a is a 93 amino acid protein

whereas MsEnod12b is a 113 amino acid protein

containing four additional PPX

repeat as does

PsEnod12a. Due to the presence of PPX

repeats,

the structure of VfEnod12 and other Enod12

proteins is very similar to proline-rich proteins

(PRPs) found in a variety of plants independently

of the presence of any symbiotic interaction. The

highest homologies existed with SbPRP3, a 90

amino acid protein with 11 PPX

repeats present

in the aerial parts of soybean plants ([16], data not

shown). Due to the presence of PPX

repeats

characteristic of plant cell wall proteins of the

hydroxyproline rich glycoprotein (HGRP) type

[35], Enod12 proteins are thought to be integral

cell wall proteins [31]. Considering this and the

homologies identified, we propose that VfEnod12

constitutes a cell wall protein from broad bean

root nodules.

3.2. VfEnod

12 transcripts accumulate early during

root nodule organogenesis

The cDNA 114 – 96 was used as a probe to

analyse VfEnod12 gene expression in different

tis-sues. An appr. 600 nt transcript corresponding in

size to the full-length VfEnod12 cDNA was

present exclusively in nodules, whereas no

tran-scripts were detected in uninfected roots, seeds,

Fig. 1. Comparison of Enod12 protein sequences from differ-ent legumes. Sequences of Enod12 proteins fromVicia sati6_a (VsEnod12; [41]), Medicago sati6_a _{(MsEnod12a and b; [1]),}

Pisum sati6_um _{(PsEnod12a and b; [13,31]) and} _Medicago

truncatula (MtEnod12, [26]) were compared with VfEnod12.

The potential cleavage site between the signal peptides and the mature proteins containing characteristic PPX3repeats is

(2)

epicotyls, shoots, and flowers (Fig. 2). This

corre-sponds to the expression pattern of MsEnod12a

[2] and MtEnod12 [26], but is in contrast to the

PsEnod12a, PsEnod12b and MsEnod12b gene

pression [2,13,31]. The latter genes are also

ex-pressed at lower levels in other tissues, in

particular in flowers. In a time-course experiment,

VfEnod12 transcripts accumulated earlier after

in-fection by

R

.

leguminosarum

bv.

6 _iciae

_{than all}

other 19 broad bean nodulin genes identified so

far. In detail, VfEnod12 transcripts were present

three post inoculation, whereas transcripts from

the early nodulin gene VfEnod2 were detected 2

days later and most other nodulin genes including

the leghemoglobin genes were detected 6 days later

[20,25]. To localize the VfEnod12 site of

expres-sion, tissue-print hybridizations were carried out.

Fig. 3 shows that VfEnod12 transcripts were

de-tected in the prefixing zone II of mature root

nodules with the highest amount in the distal part

of this region. Apparently, no VfEnod12

tran-scripts were present in the nodule meristem. This

type of expression is more similar to the

expres-sion pattern observed for the Enod12 gene from

V

.

sati

6 _a

_{[41] than to the expression patterns of the}

PsEnod12 genes that are expressed in the most

distal parts of the prefixing zone II [13,31]. Hence,

VfEnod12 constitutes a proline-rich early nodulin

that might function as a cell wall protein in the

prefixing zone II of broad bean root nodules,

possibly by specifically modifying plant cell walls.

3.3. The VfEnod

12 gene contains no introns and

is preceded by a putati

6 _{e ENBP}

₁

_{binding site}

In contrast to the two different

P

.

sati

6 _um

Enod12 genes that are expressed similarly [13,31],

the expression properties of the two alfalfa

Enod12 genes are different [2]. To be able to

investigate

the

expression

properties

of

the

VfEnod12 gene using fusions of its promoter to

reporter genes, we isolated a broad bean genomic

clone containing VfEnod12 sequences. A 3332 bp

Eco

RI fragment of this clone contained not only

the complete coding region without any

interven-ing sequences, but also 1955 bp of the 5

%

_{and 1104}

bp of the 3

%

region (EMBL accession AJ277288).

The fact that the VfEnod12 gene contains no

introns is a common feature of Enod12 genes

[1,13,26,41]. Sequence comparisons between the

VfEnod12 cDNA 114-96 and the VfEnod12

ge-nomic sequence revealed two nucleotide changes

in the coding region and 3 nucleotide changes in

the 3

%

_{UTR, whereas insertions or deletions were}

not detected (data not shown). Considering the

limited allelic variation that exists in our broad

bean cultivar [20], the genomic fragment isolated

corresponds to the full-length VfEnod12 cDNA.

Further upstream in the 5

%

region, homologies to

gag proteases were identifed from position -856

onwards indicating the presence of a

retrotranspo-son

in

the

-856/-1955

upstream

region

of

VfEnod12. The putative gag coding region is

ori-ented in the opposite direction of VfEnod12

tran-scription (data not shown).

At position -47/-52 relative to the start codon a

putative TATA-box was localized, whose position

was similar to the positions of the TATA-boxes

identified in the promoters of the two MsEnod12

genes [2]. Further upstream at position -99/-118 a

sequence element was identified that is

ho-mologous to the binding site for the transcription

factor ENBP1 that was identified in the promoter

of the PsEnod12b gene [5]. The palindromic motif

characteristic of this binding site (TTATT-N

7--12

-AATAA, [14]) is present at a similar position

appr. 120 bp upstream from the start codon in

different Enod12 promoters (Fig. 4). Interestingly,

the terminal base of the AATAA subelement that

is conserved in all Enod12 promoters isolated so

far is changed to AATAT in the VfEnod12

(3)

not observed in non-infected parts of the

trans-genic root system including the epidermis and the

meristem (Fig. 5B, E). The activity of the

VfEnod12 promoter in outer cortical cells that

probably contain infection threads conforms to

the localization of PsEnod12a transcripts [31].

Re-porter gene expression in root inner cortical cells

conforms to results for Enod12 promoters from

M

.

sati

6 a

[2],

M

.

truncatula

[26] and

P

.

sati

6 um

[31]. In contrast to the MsENOD12a promoter [3],

Fig. 5.

moter (Fig. 4). Apart from the homologies to the

transcription factor binding site, the sequence of

the

VfEnod12

promoter

is

significantly

ho-mologous to the sequences of other Enod12 gene

promoters from

M

.

sati

6 a

and

P

.

sati

6 um

[1,13,26]

in the region of appr. 200 bp upstream of the start

codon with the homologies decreasing in the

up-stream regions (data not shown). We concluded

that the Enod12 promoter from broad bean

con-tained an altered binding site for the transcription

factor ENBP1.

3.4. The

-692/-41

VfEnod

12 promoter containing

an altered ENBP

1 binding site is sufficient to

acti

6 ate gene expression in early stages of nodule

de

6 _{elopment and in mature root nodules}

The -692/-41 promoter region of the VfEnod12

gene containing the ENBP1 binding site altered in

the terminal position and the putative TATA-box

was cloned in front of the

gus

Aint reporter gene

[38] and the resulting fusion was expressed in

transgenic roots and nodules of

V

.

hirsuta

using an

A

.

rhizogenes

based transformation system

gener-ating transgenic hairy roots [29]. Resulting hairy

roots were assayed histochemically for GUS

activ-ity to monitor the expression properties of the

cloned region of the VfEnod12 promoter. In

inoc-ulated root systems, GUS activity was first

de-tected in inner cortical cells and in cells of the

outer cortex that probably contain infection

threads (Fig. 5A). In subsequent stages of early

nodule organogenesis, GUS activity was detected

in root nodule primordia (Fig. 5B – D), but not in

emerging lateral roots (Fig. 5D). Young emerging

nodules expressed the reporter gene throughout

the central region (Fig. 5E), whereas in mature

root nodules, GUS activity was confined to the

prefixing zone II (Fig. 5E – F). GUS staining was

(4)

no expression of the VfENOD12 promoter was

seen in emerging lateral roots.

VfEnod12 promoter activity in the prefixing

zone II of mature nodules corresponds to the

results obtained in

V

.

faba

by tissue-printing (Fig.

3) and resembles the expression of Enod12

pro-moters from other legumes [2,14]. Vijn et al. [42]

demonstrated that a 200 bp region of the

PsEnod12 promoters is sufficient for an expression

in nodules. Christiansen et al. [5] identified the

transcription factor ENBP1 that interacted with a

specific region of the PsEnod12b promoter.

Re-cently, Hansen et al. [14] showed by mutational

analysis that the terminal AATAA subelement of

the TTATT-N

-AATAA binding site is essential

for expression of the PsEnod12b promoter in

transgenic hairy roots and nodules of

V

.

hirsuta

.

In these experiments, the AATAA element was

changed to CCGCC. The expression properties of

the PsEnod12b [14] and the VfEnod12 (Fig. 5)

promoters in transgenic systems are very similar

and therefore should allow a comparison of the

results obtained. Our results on the expression of

the VfEnod12 promoter in transgenic

V

.

hirsuta

hairy roots show that the AATAA element can

tolerate a limited amount of variation and that

such variation exists in a native Enod12 promoter.

These data might provide a starting point for

additional mutational analyses to elucidate the

requirements for a functional ENBP1 binding site.

Acknowledgements

This work was supported by a grant from the

‘Studienstiftung des Deutschen Volkes’ to Gerald

Schro¨der.

References

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