Plant Science 160 2000 67 – 75
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, Postfach
100131
, D-
33501
Bielefeld, Germany Received 6 April 2000; received in revised form 7 July 2000; accepted 9 August 2000
Abstract
A full-length cDNA encoding the Vicia faba L. 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 PPX
3
repeats characteristic of Enod12 proteins. The VfEnod12 gene was found to be expressed specifically in root nodules as early as 3 days
post inoculation with Rhizobium leguminosarum bv. 6iciae. 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 of V. 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.comlocateplantsci
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
:
helge.kuestergenetik.uni-bielefeld.de H. Ku¨ster. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 6 2 - 9
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 sati6um [13,31], Medicago sati6a [1],
Medicago truncatula [26] and Vicia sati6a [41]. Enod12 genes code for proline-rich proteins char-
acterized by different numbers of PPX
3
pentapep- tide repeats preceded by a signal peptide. The two
similar Enod12 genes encoded in P. sati6um PsEnod12a and-b and M. sati6a MsEnod12a
and-b differ in the number of PPX
3
repeats. The occurence of PPX
3
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. sati6a 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
from M. sati6a 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. sati6a and M. truncatula to the gusA 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 sati6a [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-Rhizobium interactions. 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 V6icia 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
