Lotus Corniculatus Plants Research Articles

A system for tissue-specific copper-controllable gene expression in transgenic plants: nodule-specific antisense of aspartate aminotransferase-P2.

A vector system, based on copper controllable gene expression, has been developed to give control over place as well as time of expression of an introduced gene. This system consists of two elements: (1) the yeast ace1 gene encoding a metallo-regulatory transcription factor, ACE1, under control of either an organ-specific or a constitutive promoter; and (2) a gene of interest under control of a chimaeric promoter consisting of the 46 bp TATA fragment of the CaMV 35S RNA promoter linked to four repeats of the ACE1 binding site. The functioning of the system in an organ-specific manner was tested in nodulated Lotus corniculatus plants which consisted of non-transformed shoots plus transformed hairy root tissue 'wild-type tops/transgenic roots'. After addition of copper ions to the plant nutrient solution, beta-glucuronidase (GUS) expression was visualized either specifically in nodules or in both roots and nodules when the ace1 gene was placed under control of the nod45 promoter or the CaMV 35S RNA promoter, respectively. The nodule-specific system was used to express antisense constructs of aspartate aminotransferase-P2 in transgenic Lotus corniculatus plants. When expression was induced by the addition of copper ions to the plant nutrient solution aspartate aminotransferase-P2 activity declined dramatically, and a decrease of up to 90% was observed in nodule asparagine concentration.

Minimal enhancer elements of the leghemoglobinlba andlbc3 gene promoters fromGlycine max L. have different properties

The characteristics of the soybean leghemoglobin lba gene promoter were analyzed and important promoter elements from the lba and lbc3 promoters were compared using transgenic Lotus corniculatus plants. A 5' deletion analysis of the lba promoter delimited two cis-acting elements controlling expression: a distal positive element (-1254, -884) required for expression and a proximal element (-285, -60) essential for full-level activity. In contrast to the corresponding region of the lbc3 promoter, the lba proximal element is unable to control expression from the heterologous CaMV 35S enhancer. The upstream positive element of the lba gene contains a position- and orientation-independent enhancer between positions (-1091, -788). The sequence of this enhancer region is conserved in the lbc3 gene upstream (-1333, -1132) of the previously assigned strong positive element (SPE; -1090, -947). The present analysis revealed some of the properties of this extended lbc3 SPE element. The extended element (-1364, -947) functions in both orientations from 5' locations whereas the SPE2 subcomponent (-1364, -1154) containing the conserved sequence is only active in the correct orientation. Removal of the SPE2 by internal deletion demonstrates that the SPE2 subcomponent is indispensable for the activity of the lbc3 upstream positive element. These results indicate that the upstream positive elements of the lba and lbc3 genes possess different properties although their conserved minimal enhancer sequence has similar function. This may reflect the differential expression of the two lb genes of Glycine max L.

Interaction of a Rhizobial DNA-Binding Protein with the Promoter Region of a Plant Leghemoglobin Gene

A nucleotide sequence was identified approximately 650 bp upstream of the Sesbania rostrata leghemoglobin gene Srglb3 start codon, which interacts specifically with a proteinaceous DNA-binding factor found in nodule extracts but not in extracts from leaves or roots. The binding site for this factor was delimited using footprinting techniques. The DNA-binding activity of this factor was found to be heat stable, dependent on divalent cations, and derived from the (infecting) Azorhizobium caulinodans bacteria or bacteroids (A. caulinodans bacterial binding factor 1, AcBBF1). A 9- to 10-kD protein was isolated from a free-living culture of A. caulinodans that co-purifies with the DNA-binding activity (A. caulinodans bacterial binding protein 1, AcBBP1) and interacts specifically with its target (S. rostrata bacterial binding site 1, SrBBS1). The amino acid sequence of the N-terminal 27 residues of AcBBP1 was determined and was found to share significant similarity (46% identity; 68% similarity) with a domain of the herpes simplex virus major DNA-binding protein infected cell protein 8 (ICP8). An insertion mutation in the SrBBS1 was found to result in a substantial reduction of the expression of a Srglb3-gus reporter gene fusion in nodules of transgenic Lotus corniculatus plants, suggesting a role for this element in Srglb3 promoter activity. Based on these results, we propose that (a) bacterial transacting factor(s) may play a role in infected cell-specific expression of the symbiotically induced plant lb genes.

Phage sensitivity and host range of Rhizobium strains isolated from root nodules of temperate legumes

Twenty-five Rhizobium strains were isolated from root nodules of Astragalus spp. (10), Hedysarum alpinum (7), Glycyrrhiza pallidiflora (3) and Ononis arvensis (5). The sensitivity of these strains to bacteriophages of Rhizobium loti, R. meliloti, R. galegae and R. leguminosarum was studied. Phages specific to R. loti strains were shown to induce the phage lysis of several Astragalus, Hedysarum and Ononis rhizobia. Ten R. loti strains tested for nodulation abilities on the plant hosts under investigation were able to develop nitrogen-fixing nodules on the Ononis arvensis roots. On the other hand, rhizobia from Ononis and Glycyrrhiza could form an effective symbiosis with Lotus corniculatus plants, so these bacteria are considered to belong to the Rhizobium loti taxon. Bacterial strains isolated from Astragalus and Hedysarum were observed to cross-nodulate their plant hosts as well as Oxytropis campestris, Glycyrrhiza uralensis and Ononis arvensis plants, whereas they could not nodulate Lotus plants. It is concluded that these Rhizobium strains comprise a cross-inoculation group related to Rhizobium loti. ei]{gnR O D}{fnDixon}

A two-component nodule-specific enhancer in the soybean N23 gene promoter.

The two positive cis elements in the soybean nodulin N23 gene promoter were investigated in transgenic Lotus corniculatus plants and shown to constitute a two-component nodule-specific enhancer. Equal quantitative contributions from the two components were suggested by the similar expression level of chimeric N23-chloramphenicol acetyltransferase genes after deletion of either the distal positive element (PE-A, -320 to -298) or the proximal positive element (PE-B, -257 to -165). A combined effect of the two elements was indicated by orientation-dependent effects in the N23 promoter, and by the observation that neither PE-A nor PE-B separately was able to confer any activity to the cauliflower mosaic virus 35S minimal promoter. Reactivation of the minimal N23 and the minimal cauliflower mosaic virus 35S promoters by the inverted complete element (PE-AB) further suggested that PE-AB is a nodule-specific enhancer containing two equally strong enhancer components. Two 12-bp sequence motifs, InvA and InvB, constituting an inverted repeat, were identified as the core of the enhancer components PE-A and PE-B, respectively. Point mutations in InvA or InvB resulted in lower expression levels and mutations in both abolished enhancer activity. Point mutations in two nodulin consensus sequences, 5'-CTCTT and 5'-AAAGAT located downstream of PE-AB, resulted in a decreased level of expression, confirming the involvement of these two motifs in nodulin gene expression. The binding site for the nodule-specific trans-acting factor, NAT2, present in the PE-A segment, was removed without affecting expression significantly. This interaction is, therefore, dispensable for enhancer activity.

Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc 3 and N23 gene promoters

The qualitative and quantitative contributions of four separate cis-acting DNA elements controlling the root nodule-specific soybean leghemoglobin lbc3 gene were analyzed in transgenic Lotus corniculatus plants. Expression from internal deletions in the 5' region between positions -49 and -1956 was monitored from a CAT reporter gene. The strong positive element (SPE; -1090, -947) responsible for high-level expression was demonstrated to be an organ-specific element by deleting proximal nodule-specific control elements. Deletion of the downstream qualitative organ-specific element (OSE; -139, -102) containing the putative nodulin consensus sequences 5'AAAGAT and 5'CTCTT resulted in a low expression level. Efficient SPE enhancement is therefore dependent on the organ-specific element, which by itself does not enhance expression. This quantitative effect of the immediate upstream region carrying the consensus sequences was also found in hybrid promoter studies using the soybean nodulin N23 gene promoter, suggesting the involvement of these motifs in a regulatory mechanism for nodulin genes. Deletion of the lbc3 negative element (NE, -102, -49) linking the SPE and OSE onto the TATA box did not lead to unregulated expression. These results indicate that interaction between positive, negative and neutral qualitative elements controls lbc3 expression. Binding of the nuclear protein NAT2 at the lbc3 weak positive element (WPE; -230, -170) is probably not directly required for this mechanism.

Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants.

The gln-gamma gene, which specifies the gamma subunit of glutamine synthetase in Phaseolus vulgaris L., has been isolated and the regulatory properties of its promoter region analyzed in transgenic Lotus corniculatus plants. A 2-kilobase fragment from the 5'-flanking region of gln-gamma conferred a strongly nodule-enhanced pattern of expression on the beta-glucuronidase reporter gene. Parallel studies on the promoter of another glutamine synthetase gene (gln-beta) showed that a 1.7-kilobase fragment directed 20-fold to 140-fold higher levels of beta-glucuronidase expression in roots than in shoots. Histochemical localization of beta-glucuronidase activity in nodules of the transgenic plants indicated that the chimeric gln-gamma gene was expressed specifically in the rhizobially infected cells; expression of the gln-beta construct was detected in both cortical and infected regions of young nodules, and became restricted to the vascular tissue as the nodule matured. We conclude that gln-beta and gln-gamma genes are differentially expressed both temporally and spatially in plant development and that the cis-acting regulatory elements responsible for conferring these contrasting expression patterns are located within a 2-kilobase region upstream of their coding sequences.

5′ Analysis of the soybean leghaemoglobin lbc 3 gene: regulatory elements required for promoter activity and organ specificity

The soybean leghaemoglobin lbc(3) gene promoter was analysed in transgenic Lotus corniculatus plants. Hybrid-promoter constructions and 5' deletions were studied using chimeric genes composed of the various promoters, the chloramphenicol acetyltransferase (CAT) coding sequence and the lbc(3) 3' flanking region. A 5' Bal31 deletion series mapped a strong positive regulatory element between -1100 and -950. A weaker element located between -230 and -170 defined the minimum 5' region required for detectable promoter activity. Reactivation of inactive promoters with deletion endpoints between -230 and the transcription initiation site was obtained employing the constitutive cauliflower mosaic virus (CaMV) 35S enhancer. The position of cis regulatory element(s) required for nodule-specific expression was defined to 37 bp between -139 and -102. This region contains sequences conserved in other leghaemoglobin and nodulin genes. No indispensable control elements were found on the lbc(3) 3' flanking region.

Selective Eating of the Acyanogenic Form of the Plant Lotus corniculatus L. by Various Animals

WILD populations of the tetraploid plant Lotus corniculatus L. (birds' foot trefoil) are polymorphic for the presence or absence of a cyanogenic glucoside. This character is genetically controlled1, the gene responsible for producing the glucoside being dominant over its allelomorph. Hydrocyanic acid is evolved on hydrolysis of the glucoside when the stems or leaves are injured. Corkill2 has shown that with Trifolium repens L. the change in the colour of sodium picrate paper from yellow to brick red is proportional to the amount of hydrocyanic acid produced and hence to the glucoside content of the plant. The plants are scored from 0 (no glucoside) to 6 by matching the colour-change in the picrate paper against a series of standards.