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
3
.
1
. Fi6e broad bean cDNAs encode small polypeptides characterized by conser6ed cysteine
clusters Preliminary sequence data indicated that incom-
plete cDNAs from five different clone groups of a broad bean nodule-specific cDNA library [28] en-
coded polypeptides with conserved cysteine clus- ters cysteine cluster proteins: CCPs [12]. The
corresponding genes were designated VfNOD- CCP1, VfNOD-CCP2, VfNOD-CCP3, VfNOD-
CCP4 and VfNOD-CCP5, respectively. To isolate and determine transcript sequences covering the
entire CCP coding regions, we rescreened a nodule cDNA library with appropriate probes. In addi-
tion the RACE-PCR technique was used to com- plete cDNAs which did not extend to the
5-untranslated transcript regions. The sequences of the different cDNAs and RACE fragments
isolated for each of the five individual transcripts showed identity in the regions of overlap. There-
fore it was reasonable to combine these sequences. The open reading frames of the full-length CCP
transcript sequences presented encoded for re- markable small polypeptides. Their calculated
molecular masses ranged from 6.67 kDa for VfNOD-CCP5 to 7.57 kDa for VfNOD-CCP3
Table 1. An alignment of the deduced polypep- tide sequences is shown in Fig. 1. The comparison
on the amino acid level revealed only limited sequence identities between 22.1 VfNOD-CCP3
vs. VfNOD-CCP5 and 43.9 VfNOD-CCP2 vs. VfNOD-CCP4. On the other hand, a bipartite
domain structure of the CCPs was found to be well conserved. Analysis of the hydrophobic N-
terminal polypeptide regions revealed characteris- tics associated with secretory signal peptides.
According to Nielsen et al. [26] each of the five
Table 1 Comparison of five broad bean nodulins with conserved cyteine clusters
a
Primary Amino acid sequence identity to
Transcript Processed
polypeptide polypeptide
VfNOD- kDa
VfNOD- VfNOD-
VfNOD- ENOD3
VfNOD- ENOD14 NOD6
aa kDa
aa CCP2
CCP5 CCP3
CCP1 CCP4
¯ 34.8
31.9 28.6
28.4 27.8
40 23.6
4.45 28.2
7.44 65
VfNOD- CCP1
4.12 62
34.8 ¯
35.7 43.9
24.6 36.2
35.3 34.3
6.98 37
VfNOD- CCP2
31.9 35.7
¯ 43.7
22.1 36.8
40 27.4
7.57 35.2
4.63 VfNOD-
66 CCP3
28.6 43.9
43.7 ¯
30.3 54.3
38.8 VfNOD-
33.8 64
7.42 40
4.67 CCP4
28.4 24.6
22.1 30.3
¯ 29.6
32.4 33
24.2 3.69
VfNOD- 59
6.67 CCP5
a
Abbreviations: aa, amino acids; kDa, kilodaltons.
CCPs meet all the criteria of a functional eukary- otic signal sequence. The cleavage sites were pre-
dicted to
be between
Ser
25
and Thr
26
for VfNOD-CCP1, between Ala
25
and Gln
26
for VfNOD-CCP2, between Gly
26
and Glu
27
for VfNOD-CCP3, between Ala
24
and Asn
25
for VfNOD-CCP4 and between Ala
26
and Ser
27
for VfNOD-CCP5. The most striking feature of the
C-terminal polypeptide domain were four con- served cysteine residues. They were arranged in
two clusters of the form Cys-X
4
-Asp-Cys and Cys- X
4
-Cys, respectively. As is evident from Fig. 1, the distance between the cysteine clusters vary be-
tween 9 amino acids within VfNOD-CCP1 and VfNOD-CCP3
and 14
amino acids
within VfNOD-CCP4. From these results we concluded
that the five CCP transcripts encode members of a small broad bean polypeptide family.
Database searches revealed homologies of the different CCPs to the early nodulins PsENOD3
and PsENOD14
[35] and
the late
nodulin PsNOD6 [16] from pea Fig. 1. We could not
detect any other significant homologies between the broad bean CCPs and protein sequences in the
databases. The homologous pea nodulins dis- played the same characteristics as the CCPs. In
addition to the occurence of putative signal pep- tides, the spatial distribution of the conserved
cysteine residues was comparable. In some cases the amino acid identities between the CCPs and
the pea nodulins exceeded the identities between the members of the broad bean polypeptide fam-
ily. For example VfNOD-CCP4 and PsENOD3 matched in 54.3 of all amino acid residues see
Table 1.
3
.
2
. The CCP transcripts identified are exclusi6ely expressed in root nodules
Our previous analysis showed that the CCP transcripts were expressed strongly in broad bean
root nodules [12]. To analyse the expression pat- terns in greater detail, northern blot hybridizations
were carried out Fig. 2. These experiments re- vealed that the expression of the five CCP genes
under investigation was restricted to nodules. No hybridizing transcripts could be detected in unin-
fected roots, leaves, seeds, epicotyls, stems and flowers see Fig. 2, even after overexposure data
not shown. The mRNAs identified in nodules were about 450 bases long, as judged by RNA
markers. Considering the very short polyA tails in the cDNAs isolated, these transcript lengths
were in accordance to the CCP sequences pre- sented. As determined by cDNA-cDNA hybridiza-
tions data not shown CCP transcripts were first
Fig. 1. Alignment of the deduced amino acid sequences of the broad bean late nodulins VfNOD-CCP1, VfNOD-CCP2, VfNOD-CCP3, VfNOD-CCP4 and VfNOD-CCP5, the late pea nodulin PsNOD6 [16] and the early pea nodulins PsENOD3 and
PsENOD14 [35]. Amino acids identical in at least six of the eight sequences aligned are presented in reverse type. Shadowed amino acids indicate conservative substitutions. Putative signal peptide cleavage sites are marked by vertical arrows. Conserved cysteine
residues are marked by arrowheads. Abbreviation: aa, amino acids.
Fig. 2. Expression of CCP genes in different broad bean tissues. Northern blots containing 30 mg of total RNA from
root nodules, uninfected roots, leaves, seeds, epicotyls, stems and flowers, were hybridized against different CCP cDNA
probes. The probes and the deduced length of hybridizing transcripts are indicated on the right. Abbreviation: kb, kilo-
bases.
3
.
3
. The CCP genes are expressed in central nodule tissues
To elucidate the spatial distribution of the CCP transcripts in broad bean root nodules, tissue print
hybridizations were performed. Longitudinal sec- tions of mature nodules were printed onto nylon
membranes and hybridized to CCP sense and anti- sense riboprobes. Representative results of these
experiments are shown in Fig. 3. To relate hy- bridizing regions to distinct nodule zones we visu-
alized the interzone IIIII in the nodule sections used for printing by staining for starch see Fig.
3B [44]. In general, hybridization signals occured exclusively with the antisense probes data not
shown and were found to be restricted to the central tissues of the nodule Fig. 3A. No hy-
bridization was detected in peripheral tissues or in the nodule meristem. VfNOD-CCP1, VfNOD-
CCP3, VfNOD-CCP4 and VfNOD-CCP5 tran- scripts were located in the interzone IIIII and the
nitrogen fixing zone III. VfNOD-CCP1 transcripts were additionally detectable in the ineffective zone
IV, whereas detection of VfNOD-CCP4 tran- scripts extended to the distal region of the nodule
and comprised large areas of the prefixing zone II. VfNOD-CCP2 transcripts were predominantly
present in the interzone IIIII. In much lower amounts hybridizing VfNOD-CCP2 transcripts
were also found to be dispersed in the distal region of the nitrogen fixing zone III.
3
.
4
. Genomic organsisation of VfNOD-CCP
1
As a step towards the characterization of the genomic organization of CCP nodulin genes, we
isolated a 20 kb VfNOD-CCP1 fragment from a genomic broad bean library. Subcloning resulted
in the identification of a 4.4 kb EcoRISalI frag- ment in clone c 2 – 19, which hybridized to a
VfNOD-CCP1 cDNA. Fig. 4 shows the sequence of a 2424 bp EcoRISphI subfragment of this
clone.
Sequence analysis
revealed that
the VfNOD-CCP1 gene consisted of two exons that
showed sequence identity to the VfNOD-CCP1 transcript sequence determined. The two exons
were interrupted by an 99 bp intron separating the first exon encoding the secretory signal peptide
from the second exon encoding the cysteine cluster domain see Fig. 4. The exonintron boundaries
identified were in good agreement with the consen- detectable in Rhizobium infected broad bean roots
uniformly 7 days after inoculation. The expression of leghemoglobin genes starts earlier at day 6 after
inoculation. Due to the nodule specificity and the onset of CCP expression during nodule formation,
the polypeptides encoded by the CCP genes were classified as late nodulins, according to Nap and
Bisseling [24,25].
sus sequences of splice junctions in genes of di- cotyledonous plants [13]. Upstream of the first
base corresponding to the full length VfNOD- CCP1 transcript sequence, the sequence most simi-
lar to a consensus of sequences surrounding nodulin gene TATA boxes could be identified at
position − 37 to − 25 ACACTATAAATTG, 10 out of 13 bases matched the consensus reported by
Joshi [15]. In addition, an analysis of the com- plete − 1759 + 1 putative promoter region of the
VfNOD-CCP1 gene revealed the multiple presence of the sequence motifs AAAGAT and CTCTT,
characteristic of leghemoglobin and other nodulin gene promoters [34,39], on both strands of the
DNA sequence see Fig. 4. The CTCTT sequence located from position − 173 to − 169 was iden-
tified as part of a short element, which resembled the OSE organ-specific element of the soybean
leghemoglobin lbc3 gene promoter [37] both in sequence and position Fig. 5A. Apart from these
motifs similar to published promoter motifs, a 12 bp inverted repeat and a 17 bp direct repeat were
Fig. 3. Localization of transcripts coding for CCP nodulins in broad bean root nodules. A Representative tissue print hybridizations of longitudinal nodule sections against DIG-labelled antisense ribroprobes are shown for VfNOD-CCP1,
VfNOD-CCP2, VfNOD-CCP3, VfNOD-CCP4 and VfNOD-CCP5. B Microphotographs of the nodule sections used for tissue printing after staining for starch. Cells of the interzone IIIII stained black. In addition, starch was detected in the basal region
of the nodules, which presumably corresponds to the senescence zone IV. C Diagrams of the distribution of hybridizing transcripts in the model nodule were concluded from a comparison of A and B. In the diagrams the nodule zones meristem,
preinfection zone II, interzone IIIII, nitrogen fixing zone III and ineffective zone IV are displayed from the top to the base.
Fig. 4. Sequence of a 2424 bp EcoRISphI fragment from the genomic VfNOD-CCP1 clone c 2 – 19. The deduced amino acid sequence of VfNOD-CCP1 is printed in bold type above the DNA sequence with the extent of a secretory signal peptide being
underlined. The putative signal peptide cleavage site is marked by an vertical arrow. An intron interrupting the VfNOD-CCP1 coding region is displayed in small letters. The first base corresponding to the full length VfNOD-CCP1 transcript sequence is
indicated by an dot and was chosen as position + 1. In the upstream region, the putative TATA box sequence is underlined twice. The sequence element resembling leghemoglobin OSE sequences are shadowed, while nodulin consensus sequences of the type
AAAGAT and CTCTT on both strands of the DNA are underlined. A inverted repeat and a direct repeat are marked by arrows below the sequence.
located on the VfNOD-CCP1 putative promoter Fig. 5B. The significance of these motifs for
theactivity of the putative VfNOD-CCP1 pro- moter has to be demonstrated by the analysis of
promoter-reporter-gene
fusions in
transgenic plants.
4. Discussion
