In recent years, the bacterial genetics of nodule formation and nitrogen fixation have been exten-
sively studied [9,10]. An increasing number of plant genes specifically expressed in nodules,
termed nodulin genes [42], have been identified and subdivided into early and late nodulin genes
according to the time point of their expression [23,45]. Early nodulin genes ENODs are associ-
ated with organogenesis and bacterial invasion of the root nodule. The sequences of many early
nodulins ENOD2 and ENOD12, for example suggest that they represent proline-rich proteins
most probably involved in cell wall biosynthesis. In general, late nodulin gene NOD products are
thought to be involved in nodule function and include the oxygen transporter leghemoglobin, en-
zymes
of carbon
and nitrogen
metabolism, proteins located in the peribacteroid interface as
well as a number of proteins, the functions of which remain to be identified [6].
To investigate the organ-specific gene expression in broad bean Vicia faba L. root nodules, we
have constructed a nodule-specific cDNA library of approximately 700 independent cDNAs [28]. In
addition to different leghemoglobins [12], tran- scripts homologous to the early nodulin genes
PsENOD
2
[41], PsENOD
5
and PsENOD
12
[35] were isolated [28]. We also detected a family of at
least five nodule-specifically expressed broad bean genes
encoding glycine-rich
proteins [20,36],
VfNOD
28 32
the homologue of MsNOD
25
[19,21] and VfNOD
32
encoding a narbonin-like nodulin with homologies to chitinases [29].
In this paper, we report on the characterization of five broad bean transcripts encoding a family of
late nodulins with conserved cysteine-clusters. We describe the spatial and temporal expression of the
transcripts in broad bean nodules and finally we report on the isolation and analysis of a corre-
sponding genomic sequence.
2. Methods
2
.
1
. Biological material, cDNA and genomic libraries
Broad bean plants Vicia faba L. cv. Kleine Thu¨ringer were grown in the greenhouse in sterile
clay granules Seramis using surface-sterilized seeds saturated Ca-hypochlorite, 30 min. Nodu-
lated plants were obtained by inoculation of 2 day-old seedlings with Rhizobium leguminosarum
bv. 6iciae VF39 [30]. Flowers and seeds were analysed from field grown plants inoculated 3 days
after sowing. The nodule cDNA library was con- structed in lgt11 from polyA
+
RNA isolated from root nodules of V. faba L. cv. Kleine
Thu¨ringer [28]. A broad bean genomic library was prepared in lEMBL3 [11] according to Sambrook
et al. [33].
2
.
2
. Library screening, isolation of nucleic acids and recombinant DNA techniques
Recombinant lgt11 and lEMBL3 phages were plated and screened for positive clones as de-
scribed previously [20]. Three rounds of screening were performed to obtain single positive plaques.
Isolation of phage DNA was carried out using standard protocols [33]. Plasmid DNA was iso-
lated from E. coli XL1-Blue using the ‘Plasmid Mini Kit’ Qiagen according to the manufactur-
er’s instructions. Probe DNA was extracted from agarose gels using the ‘QIAEX Gel Extraction Kit’
Qiagen. For Northern blotting, RNA was iso- lated from nodules 32 days after sowing, unin-
fected roots 32 days after sowing, leaves 32 days after sowing, seeds 90 days after sowing, epi-
cotyls 8 days after sowing, stems 12 days after sowing and flowers 60 days after sowing of
broad
beans using
standard protocols
[5]. PolyA
+
RNA fractions were isolated by one cycle of oligodT-cellulose chromatography.
The ‘5-AmpliFINDER RACE KIT’ Clontech was used to perform 5 RACE reactions. The
cDNA synthesis was carried out according to the manufacturer’s instructions using gene-specific
primers. For subsequent PCR amplification of 5 cDNA fragments gene-specific nested primers were
used. To generate overlapping sequencing clones, exonuclease III digestions were carried out using
the ‘Double Stranded Nested Deletion Kit’ Phar- macia according to the manufacturer’s instruc-
tions. All other in vitro DNA manipulations were carried out using standard protocols [33].
2
.
3
. DNA sequencing and analysis Sequencing reactions have been carried out ac-
cording to Zimmermann et al. [46] using 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. DNA se-
quence data were read using the ‘A.L.F. MAN- AGER V3.0’ software Pharmacia and analysed
using the programme ‘ANALYSEQ’ [38] and ‘LFASTA’ based on [27]. Multiple sequence
alignments were carried out using the program ‘CLUSTAL’ [14] from the PCGene software
package IntelliGenetics, release 6.8. Predictions of signal peptides and their cleavage sites were
carried out using the ‘SignalP world wide web server’ release V1.1 [26].
2
.
4
. Northern and cDNA-cDNA hybridizations Northern blotting and hybridizations were car-
ried out as described previously [28]. About 50 ng of probe DNA isolated from agarose gels were
labelled with 50 mCi of a
32
P-dATP according to Feinberg and Vogelstein [8]. Stringent washes were
carried out at room temperature using 2 × SSC, 0.1 wv SDS 5 min and at 68°C using 0.2 ×
SSC, 0.1 wv SDS twice for 30 min each.
For cDNA-cDNA hybridizations, 0.2 mg EcoRI digested DNA from different cDNA clones was
separated electrophoretically and blotted onto Hy- bond-N nylon membranes Amersham using stan-
dard protocols
[33]. Radioactively
labeled first-strand cDNA probes were synthesized from 1
mg polyA
+
RNA according to Fru¨hling et al. [12] and used immediately for hybridization. Fil-
ters were hybridized in a solution containing 50 mM Na phosphate pH 7.0, 5 × SSC, 0.1 wv
lauroylsarcosin, 2
wv blocking
reagent Boehringer, 7 wv SDS and 50 vv for-
mamide for 48 h at 42°C. Stringent washes were carried
out as
described for
Northern hybridizations.
2
.
5
. Tissue print hybridizations Longitudinal sections of mature broad bean
nodules harvested 32 days after sowing were printed on Hybond-N nylon membranes Amer-
sham as described [36]. Hybridizations against digoxigenin-labeled antisense riboprobes were car-
ried out as reported by Ku¨ster et al. [20]. Stringent washes and detection of hybridizing transcripts
were carried out according to Kessler [17]. As a control, prints were hybridized against the corre-
sponding sense probes. In none of the cases ex- pression above background was observed.
To relate hybridizing regions to nodule zones, sections used for tissue-printing were stained for
starch in Lugol’s solution containing 1 wv KI and 1 wv I
2
in distilled water. Stained sections were photographed at the same magnification as
the tissue-print filters.
3. Results
