JXB Advance Access originally published online on January 24, 2005
Journal of Experimental Botany 2005 56(413):799-806; doi:10.1093/jxb/eri074
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RESEARCH PAPER |
The promoter of the leghaemoglobin gene VfLb29: functional analysis and identification of modules necessary for its activation in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots
1Universität Bielefeld, Fakultät für Biologie, Lehrstuhl für Genetik, PO Box 100131, D-33501 Bielefeld, Germany
2International NRW Graduate School in Bioinformatics and Genome Research, Center for Biotechnology, Universität Bielefeld, D-33594 Bielefeld, Germany
To whom correspondence should be addressed. Fax: +49 (0)521 1065626. E-mail: Helge.Kuester{at}Genetik.Uni-Bielefeld.DE
Received 3 August 2004; Accepted 2 November 2004
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Abstract |
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In this study the further characterization of the Vicia faba leghaemoglobin promoter pVfLb29 is presented that was previously shown to be specifically active in the infected cells of root nodules and in arbuscule-containing cells of mycorrhizal roots. Using promoter studies in transgenic hairy roots of the Pisum sativum mutant RisNod24, disabled in the formation of functional arbuscules, VfLb29 promoter activity is assigned to later stages of arbuscule development. In order to narrow down the regions containing cis-acting elements of pVfLb29, the activity of five VfLb29 promoter deletions (–797/–31 to –175/–31 in relation to the start codon) fused to the gusAint coding region were tested in transgenic V. hirsuta hairy roots. The results specify a promoter region ranging from position –410 to –326 (85 bp) as necessary for gus expression in arbuscule-containing cells, whereas this segment is not involved in the nodule-specific activity. Sequence analysis of the pVfLb29 fragment –410/–326 (85 bp) revealed sequence motifs previously shown to be cis-acting elements of diverse promoters. To investigate the autonomous function of pVfLb29 regions for activation in arbuscule-containing cells, different regions of pVfLb29 from positions –410 to –198 were used to prepare chimeric promoter constructs for trans-activation studies. These fragments alone did not activate the mycorrhiza inactive promoter of the Vicia faba leghaemoglobin gene VfLb3, showing that the activation of pVfLb29 in arbuscule-containing cells is governed by a complex regulatory system that requires at least two modules located between position –410 and –31 of the VfLb29 gene.
Key words: Arbuscules, leghaemoglobin promoter, mycorrhizal roots, root nodules, Vicia faba
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusions and perspectivesReferences |
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The majority of legume species can establish symbiotic associations with bacteria of the genus Rhizobium and arbuscular mycorrhizal fungi of the order Glomeromycota (Schüssler et al., 2001
). The Rhizobium–legume interaction leads to symbiotic nitrogen fixation carried out by differentiated bacteria within a specialized plant organ, the root nodule (Brewin, 1991). The arbuscular mycorrhiza (AM) symbiosis offers several benefits to the host plant, including improved nutrition (mainly phosphorus), enhanced drought resistance, and protection from pathogens (Smith and Read, 1997; Brundrett, 2002). Due to these facts both symbioses have to be looked upon as factors of great importance both for nature ecosystems and for agriculture (Provorov et al., 2002).
For the N2-fixing symbiosis, nutrient exchange occurs in the rhizobia-infected cells of the nitrogen-fixing zone III of root nodules (Provorov et al., 2002). In the case of the AM symbiosis, the major site of exchange between plant and fungus is the arbuscule, a highly branched structure that is formed by the fungus within the inner cortical cells of the root. In addition, the intercellular hyphae are also discussed as sites for the transfer of nutrients (Harrison, 1999). Arbuscules are transient structures since they develop and subsequently degenerate within 6–10 d (Alexander et al., 1989; Dickson and Smith, 2001). Due to the obvious similarities between both symbiotic interactions and the existence of mutations resulting both in nod– and myc– phenotypes, a common genetic basis of the two symbioses was supposed (Duc et al., 1989; Gianinazzi-Pearson, 1997). It was recently shown that both microsymbionts induce a common signalling cascade during initiation of root nodules and AM (Cullimore and Denarie, 2003), and several nodulin genes such as MtEnod11, MtEnod12 (Journet et al., 2001), MsEnod40, MsEnod2 (van Rhijn et al., 1997), PsEnod5 and PsEnod12 (Albrecht et al., 1998), have already been shown to be induced not only during nodulation but also in legume root tissues colonized by arbuscular mycorrhizal fungi.
The most abundant and best-characterized nodule-specific proteins are the leghaemoglobins (Lb), which are expressed in the infected cells just prior to the onset of nitrogen fixation. These oxygen-binding haem proteins are supposed to be responsible for supporting the flux of oxygen to the nitrogen-fixing bacteroids (Appleby, 1984). One member of this family, the Vicia faba Lb gene VfLb29, was shown to be expressed in both symbiotic interactions (Frühling et al., 1997) and, recently, the promoter of this gene was found to be active not only in the infected cells of root nodules but also in arbuscule-containing cells of mycorrhizal roots (Vieweg et al., 2004). A similar highly specific activity in arbuscule-containing cells, as shown for the VfLb29 promoter (Vieweg et al., 2004), has also been demonstrated for the promoters of two phosphate transporter genes, MtPt4 of M. truncatula (Harrison et al., 2002) and Stpt3 of potato (Rausch et al., 2001).
Specific cis-acting elements, which play a role in gene regulation, have traditionally been identified by carrying out deletion analyses of the promoter regions of diverse plant genes. In particular, the structural properties of several nodule-specific promoters were studied that way (Stougaard et al., 1990; Rodriguez-Llorente et al., 2003; Nakawaga et al., 2003). These analyses led to the identification of several regulatory elements, for example, enhancers, organ- or cell-specific elements, and strong positive elements in addition to core promoter motifs. Common with other known leghaemoglobin promoters, pVfLb29 exhibits the two consensus sequence motifs ‘AAAGAT’ and ‘CTCTT’ between positions –193 and –175 (Vieweg et al., 2004). These two motifs were shown to be necessary for promoter activity in the infected cells of root nodules and are part of the organ-specific element (OSE) originally identified in the soybean leghaemoglobin lbc3 promoter reported by Stougaard et al. (1990).
In order to identify regulatory elements in the VfLb29 promoter that are required for its activity in both arbuscule-containing cells of mycorrhizal roots and the infected cells of root nodules, promoter deletion and cross-activation studies in transgenic mycorrhizal roots and nodules of the vetch V. hirsuta were carried out. As far as is known, this is the first approach to identify minimal regulatory regions mediating activity of promoters in arbuscule-containing cells of mycorrhizal roots.
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Materials and methods |
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Plant material and microbial strains
Seeds of the P. sativum mutant RisNod24 (Engvild, 1987
; Sagan et al., 1994) and the corresponding wild-type ‘Finale’ were supplied by Gerard Duc (INRA Dijon, France). V. hirsuta seeds were obtained from John Chambers Ltd., London. A. rhizogenes strain Arqua1 (Quandt et al., 1993) was used for the induction of hairy roots. Nodules were induced on 12–14-d-old transgenic hairy roots of V. hirsuta and P. sativum wild-type by R. leguminosarum strain VF39 (Priefer, 1989). Inoculations with G. intraradices were carried out with a commercially available inoculum based on aseptic liquid medium containing spores (Premier Tech Biotechnologies, Rivière-de-Loup, Québec, Canada).
Construction of promoter–gusAint fusions
The 5' promoter deletions of VfLb29 were amplified by PCR, and their sequences were subsequently confirmed. The amplified regions –534/–31, –410/–31, and –325/–31 were cloned as XhoI/EcoRI fragment, whereas the –797/–31 region was cloned as ClaI/EcoRI and the –175/–31 region as the blunt/EcoRI fragment into plasmid pGUS-INT (Küster et al., 1995). The resulting pVfLb29-gusAint fusions were subsequently cloned as SpeI fragments (filled in using Klenow polymerase) into the SmaI site of the binary vector pRedRoot (Limpens et al., 2004), and the resulting binary vectors were transformed into A. rhizogenes Arqua1 (Quandt et al., 1993).
The pVfLb29 fragments for the construction of the chimeric promoter–gusAint fusions were isolated by PCR and cloned as StuI/BglII in front of the VfLb3 (–356/–1) promoter–gusAint fusion. The resulting chimeric promoter–gusAint fusions VfLb3+(–410/–326), +(–410/–245), and +(–410/–198) were subsequently cloned as SpeI fragments (filled in using Klenow polymerase) into the SmaI site of the binary Vector pRedRoot (Limpens et al., 2003), and the resulting binary vectors were transformed into A. rhizogenes Arqua1 (Quandt et al., 1993).
Induction of transgenic hairy roots and conditions of plant growth
The induction of transgenic roots was performed by a method in open pots as described by Vieweg et al. (2004). Plants were fertilized weekly with half-strength Hoagland's solution (Arnon and Hoagland, 1940) containing 20 µM phosphate and grown in pots with sterilized clay granulate (Seramis, Masterfoods GmbH, Verden, Germany). All plants were grown in growth chambers under a photoperiod of 16 h at 22 °C with a decrease to 18 °C during the dark phase. The relative humidity was set to 70%.
Histochemical analysis of transgenic tissues
The results presented in this study are based on the analysis of a representative number of independent hairy roots for each approach.
Prior to the histochemical analysis of root tissues, transgenic roots were selected by using the binary vector pRedRoot (Limpens et al., 2004) that provides constitutive dsRed gene expression in order to ensure the identification of transgenic hairy roots by dsRed fluorescence. Examination of fluorescence was carried out using a fluorescence stereomicroscope (Leica MZ FL III, Wetzlar, Germany).
Histochemical assays for GUS activity were performed as described by Jefferson et al., (1987). The GUS substrate solution contained 2 mM X-Gluc (5-bromo-4-chloro-3-indolyl-ß-D-glucuronide cyclohexylammonium salt; Biosynth, Switzerland), 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide, 100 mM TRIS pH 7.0, 50 mM sodium chloride pH 7.0, and 0.1% (v/v) Tween-20. Roots were first vacuum-infiltrated prior to incubation in the dark at 37 °C for 1–24 h. GUS assays of hairy roots expressing promoter–gusAint fusions were performed 4 weeks after nodulation and 25 d after inoculation with G. intraradices. For staining of fungal structures, roots were first cleared with 10% KOH (w/v potassium hydroxide dissolved in water) for 10 min at 90 °C and were subsequently assayed using 0.03% (w/v) Chlorazol black E in a lactoglycerol solution (1:1:1 by vol. lactic acid, glycerol, and water) for 4 h at 90 °C as described by Brundrett et al. (1984). Prior to observation, roots were destained with 50% (v/v) glycerol for several days. For GUS staining of root nodules, semi-thin sections of 80–120 µm were prepared with a microtome (Leica VT/000S, Wetzlar, Germany). Examination of tissues was carried out by light microscopy (Olympus BH-2, Hamburg, Germany), and documentation was done using a digital camera (Olympus C-2000Z, Hamburg, Germany).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusions and perspectivesReferences |
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The activity of the VfLb29 promoter is confined to cells containing fully developed arbuscules
The activity of the VfLb29 promoter (Fig. 1) was analysed in transgenic hairy roots induced on the P. sativum mutant RisNod24 (Engvild, 1987
; Sagan et al., 1994). This mutant is not only unable to develop nodules, but is also disabled in the formation of late developmental stages of mycorrhiza symbiosis (Lapopin et al., 1999), since only the formation of deformed and unbranched arbuscules that lack a complete function is admitted by the plant (Fig. 2E). The activity of the VfLb29 promoter (–996/–31 region, GenBank accession number AJ564166) in the mutant background was analysed in order to give information on the start of VfLb29 expression during the development of arbuscules within a cell. The root transformations on the P. sativum RisNod24 mutant and the corresponding wild-type were performed as described in Vieweg et al. (2004).
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In transgenic roots induced on the P. sativum wild-type (cv. Finale), pVfLb29 shows activity in the infected cells of root nodules (Fig. 2A) and in the arbuscule-containing cells of mycorrhizal roots (Fig. 2B). To verify the presence of the mycorrhizal fungus G. intraradices, the GUS-stained roots were subsequently treated with Chlorazole Black E (CBE) which visualizes fungal structures within the root tissue (Fig. 2C). This result is in complete accordance to the VfLb29 promoter activity in other legumes such as V. faba, V. hirsuta, and M. truncatula (Vieweg et al., 2004
).
In the case of the P. sativum RisNod24 mutant, no GUS staining was observed in cells containing the characteristically deformed arbuscules, as verified by subsequent treatment with CBE (Fig. 2E). In the case of no detectable GUS activity concerning the pVfLb29-gusAint transgenic roots of the mutant, it was of importance to verify the transgenic nature of the roots studied. This was ensured using the binary vector pRedRoot (Limpens et al., 2004) that provides constitutive dsRed gene expression in order to enable the identification of transgenic hairy roots by dsRed fluorescence (Fig. 2D). Arbuscules are transient structures which have a life span of 6–10 d including the stages of arbuscule formation, metabolic activity, and subsequent degeneration (Alexander et al., 1989; Dickson and Smith, 2001). It is obvious that these different developmental steps require a sophisticated system of differential gene expression and hence regulation of promoter activity. The results presented here suggest that the factor(s) activating pVfLb29 in arbuscule-containing cells are not related to the induction of arbuscule formation or the pure presence of fungal tissues within the root cell, but rather to a fully developed, probably metabolically active arbuscule. It has previously been speculated that a possible function for VfLb29 concerning the detoxification of nitric oxide to avoid cell death and defence gene induction in symbiotic interactions (Vieweg et al., 2004). It should be noted that there is evidence that processes concerning defence suppression take place specifically in cells containing arbuscules, since an induction of several genes related to defence suppression was already shown to occur exclusively in these cells (Bonanomi et al., 2001; Blee and Anderson, 1996; Salzer et al., 1999). Against this background, it is possible that metabolically active arbuscules produce substances or provoke processes that directly or indirectly induce a cascade of reactions leading to plant defence suppression, and these processes might include factors activating the VfLb29 promoter.
Analysis of VfLb29 promoter deletions in transgenic hairy roots of V. hirsuta
In order to define and characterize regulatory regions of the VfLb29 promoter responsible for the activation in the infected cells of root nodules and the arbuscule-containing cells of mycorrhizal roots, 5' promoter deletions were constructed of different lengths fused to the gusAint coding region (Fig. 3) for their use in a loss-of-function approach. The induction of transgenic roots with these constructs was performed as described by Vieweg et al. (2004) on the legume species V. hirsuta, a close relative to V. faba. As for P. sativum, verification of the transgenic nature of the roots was ensured by the use of the binary vector pRedRoot. After induction of hairy roots, the V. hirsuta plants were inoculated separately with Rhizobium leguminosarum bv. viciae and with the arbuscular mycorrhizal fungus G. intraradices, respectively. After 4 weeks, transgenic roots were identified by dsRed fluorescence and analysed histochemically for ß-glucuronidase (GUS) activity in root nodules and mycorrhizal roots. To verify the presence of arbuscules, GUS-stained roots were subsequently treated with CBE.
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The GUS staining of transgenic roots and nodules transformed with the constructs –797/–31, –534/–31, and –410/–31 showed promoter activity of the same pattern as detected for the entire promoter, both in the infected cells of root nodules shown in Fig. 2G (–410/–31 fusion) and in arbuscule-containg cells shown in Fig. 2F (–410/–31 fusion). The next shorter deletion –325/–31, however, still showed unaltered activity in root nodules (Fig. 2H) but no activity in cells containing arbuscules (data not shown, Fig. 3). This result suggests that cis-acting elements involved in the activity of the VfLb29 promoter in arbuscule-containing cells are located in the sequence between the –410/–31 and the –325/–31 deletion (Figs 1, 3), clearly demonstrating that the activity of the VfLb29 promoter in both symbioses is based on different regulatory regions. This result further implies that although there are obvious analogies between the infected cells of root nodules and the arbuscule-containg cells of mycorrhizal roots, at least one additional trigger is responsible for the activation of pVfLb29 in AM tissues. The shortest deletion –175/–31 shows neither GUS activity in nodules nor in mycorrhizal roots (data not shown, Fig. 3). This construct lacks the consensus motifs of the organ-specific element (OSE) (Figs 1, 3) which was previously shown to be an essential element of leghaemoglobins and other nodulin gene promoters with respect to their expression in the infected cells of root nodules (Stougaard et al., 1990
). Therefore, the lack of promoter activity of the –175/–31 construct in the infected cells of root nodules is probably due to the absence of the OSE consensus sequence.
The VfLb29 promoter contains putative cis-acting elements between position –410 bp and –326 bp
The deletion analysis of the VfLb29 promoter in V. hirsuta suggests the presence of cis-acting elements essential for VfLb29 expression in arbuscule-containing cells between position –410 and –326. At the nucleotide level, it was possible to identify several motifs within these 85 bp (Fig. 1) that represent putative cis-acting elements possibly involved in the activation of pVfLb29. From position –350/–358 a sequence motif AATATTAAA was found (Fig. 1, lower strand) which is also present in the strong positive element (SPE) of the soybean lbc3 promoter (Stougaard et al., 1990) and which was also discovered recently in the soybean phosphoenolpyruvate carboxylase GmPEPC7 promoter as a part of a regulatory element (Nakagawa et al., 2003).
Within the 85 bp fragment (–410/–326) of the VfLb29 promoter, a palindromic sequence motif of 14 bp between position –381 and –351 (Figs 1, 4) that might represent a putative regulatory element was further identified. Interestingly, the left part of this palindromic motif overlaps to some extent with the SPE-like element (Fig. 1). Cross-activation studies of the GmPEPC7 promoter also identified a 14 bp palindromic sequence as part of a switch region involved in its regulatory function (Nakagawa et al., 2003). Alignments with promoters that are also specifically active in arbuscule-containing cells revealed a palindromic sequence motif at position –253 and –226 relative to the start codon in the promoter of the potato phosphate transporter gene StPt3 (Rausch et al., 2001) (Fig. 4). Although the two inverse sequences of the palindrome are exchanged, the position and sequence of this 14 bp palindromic element are similar to that of pVfLb29 (Fig. 4). That finding can be considered as a hint that these palindromic sequences exhibit a cis-acting function for the activation in cells containing arbuscules and that the regulation of these promoters might be based on similar regulatory mechanisms.
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Cross-activation studies indicate a modular structure of the VfLb29 promoter
To investigate the autonomous function of the 85 bp (–410/–326) pVfLb29 region (Fig. 1) in terms of promoter activation in arbuscule-containing cells, chimeric promoter constructs were prepared. In order to show a cross-activation effect of the sequence from –410 to –326 (85 bp) on a promoter that is not active in arbuscule-containing-cells, a –356/–1 promoter fragment of the VfLb3 gene encoding a leghaemoglobin that is expressed in root nodules but not in mycorrhizal roots was used as a target (Vieweg et al., 2004
). The 85 bp pVfLb29 fragment (–410/–326) was amplified by PCR and cloned in front of a VfLb3 (–356/–1) promoter gusAint fusion (Fig. 5). As there was the possibility that regulatory elements interact with promoter regions located downstream from position –326, two additional fragments (–410/–245; –410/–198) were prepared which were also fused to the Lb3 (–356/–1) promoter fragment (Fig. 5). Verification of the transgenic character of the roots was ensured by dsRed fluorescence. After the induction of transgenic roots on V. hirsuta, plants were inoculated with the AM fungus G. intraradices.
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The analysis of roots transformed with the chimeric promoter fusions pLb3+(–410/–326), pLb3+(–410/–245), and pLb3+(–410/–198) revealed no activation effect in arbuscule-containing cells. The verification of the presence of arbuscules was shown by CBE staining, and the general activity of the chimeric promoter-fusions was confirmed by the pVfLb3 specific expression pattern in root nodules (Fig. 2I, construct pLb3+(–410/–198). These results indicate that the activation of pVfLb29 in arbuscule-containing cells probably requires two regulatory modules being located from position –410 to –198 and from position –197 to –31. Within the –410/–198 module, at least one important regulatory element is located in the –410/–326 region. Although this region is not autonomous in mediating expression in arbuscule-containing cells, the loss-of-function approach shows that its presence is nevertheless essential to activate VfLb29 expression in arbuscule-containing cells.
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Conclusions and perspectives |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusions and perspectivesReferences |
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The purpose of this study was the further characterization of the VfLb29 promoter, which was previously shown to be active not only in the infected cells of root nodules but also in arbuscule-containing cells of mycorrhizal roots (Vieweg et al., 2004
). We were able to present functional and structural features of the VfLb29 promoter and narrowed down the minimal regulatory region that mediates activity in arbuscule-containing cells of mycorrhizal roots. As far as is known, this is the first investigation of a promoter that is active in arbuscule-containing cells of mycorrhizal roots using a loss-of-function approach. These results demonstrated that VfLb29 expression in arbuscule-containing cells is mediated by a complex regulatory system. Further characterizations of the structural features and putative cis-acting elements of the VfLb29 promoter presented in this study might contribute to an elucidation of the still unexplored mechanisms that regulate gene expression in cells containing arbuscules. |
Acknowledgements |
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We would like to thank the Deutsche Forschungsgemeinschaft for supporting this work in the frame of the SPP1084 (MolMyk) projects Pe814/1-2 and Ku1478/2-3. We are grateful to Gerard Duc (INRA Dijon, France) for providing seeds of the P. sativum mutant RisNod24 and to Rene Geurts (Wageningen University, Netherlands) for supplying the pRedRoot plasmid prior to publication.
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Footnotes |
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* These two authors contributed equally to this work.
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References |
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