Plant Science 152 2000 67 – 77
A small gene family of broad bean codes for late nodulins containing conserved cysteine clusters
Martin Fru¨hling, Ulrike Albus, Natalija Hohnjec, Gerhard Geise, Alfred Pu¨hler, Andreas M. Perlick
Uni6ersita¨t Bielefeld, Lehrstuhl fu¨r Genetik, Postfach
100131
, D-
33501
Bielefeld, Germany Received 5 August 1999; received in revised form 25 October 1999; accepted 25 October 1999
Abstract
Five transcripts encoding different members of a nodulin family with conserved cysteine clusters Cys-X
4
-Asp-Cys and Cys-X
4
-Cys were identified in broad bean root nodules. They displayed homologies to the early nodulins PsENOD3 and PsENOD14 and the late nodulin PsNOD6 from pea. In addition to the occurence of putative secretory signal peptides, the spatial
distribution of the cysteine residues was comparable in both the broad bean and the pea nodulins. Based on tissue print hybridizations, we found that the corresponding broad bean genes VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 were
expressed in the interzone II – III and the nitrogen fixing zone III of mature nodules whereas the gene VfNOD-CCP4 was first induced in the prefixing zone II. A strong expression of the VfNOD-CCP2 gene only could be detected the interzone II – III region.
Sequence analysis of a genomic VfNOD-CCP1 clone isolated revealed the presence of one intron seperating a first exon encoding the signal peptide from a second exon encoding the cysteine cluster domain of this nodulin. Apart from the multiple presence of
the common nodulin motifs AAAGAT and CTCTT on both DNA strands of the putative VfNOD-CCP1 promoter region a sequence element resembling the organ specific element of the soybean lbc3 gene promoter was identified. © 2000 Published by
Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Symbiotic nitrogen fixation; Tissue print hybridization; Metal binding; Vicia faba L. www.elsevier.comlocateplantsci
1. Introduction
The interaction of soil bacteria belonging to the genera Rhizobium, Bradyrhizobium and Azorhizo-
bium with leguminous plants leads to the forma- tion of a novel plant organ — the root nodule
[2,23,43]. In mature nodules, nitrogen-fixation is carried out by the microsymbiont.
The elicitation and development of an effective symbiosis involves a mutual exchange of specific
signals between both partners [37] and is accompa- nied by the expression of specific genes in both
rhizobia and their host plants [23,45]. Initially, nodules develop from primordia by differentiation
of specialized tissues. The nodules are infected by the symbiotic bacteria via infection threads [18].
After their release from these tubular structures, the bacteria are enclosed by a modified plant-
derived membrane peribacteriod membrane and differentiate into bacteriods capable of reducing
atmospheric nitrogen. The central tissues of fully developed indeterminate nodules formed on tem-
perate legumes display a characteristic zonation. An apical meristem is followed by the prefixing
zone II, the interzone IIIII rich in amyloplasts, the nitrogen fixing zone III, and the senescence
zone IV, according to the nomenclature of Vasse et al. [44].
The sequence data reported will appear in the EMBL database under the accession numbers AJ243461 and AJ243462 VfNOD-
CCP1, AJ243463
VfNOD-CCP2, AJ243464
VfNOD-CCP3, AJ243465 VfNOD-CCP4, and AJ243466 VfNOD-CCP5, respec-
tively. Corresponding author. Tel.: + 49-521-106-5631; fax: + 49-521-
106-5626. E-mail
address
:
andreas.perlickgenetik.uni-bielefeld.de A.M. Perlick
0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 1 9 - 8
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
