Lotus Corniculatus Plants Research Articles

Analysis of the contribution of Lotus corniculatus to soil carbon content in a rice-pasture rotation system

Productivity in continuous rice cropping agroecosystems (RR) is lower than in rice-pasture rotation agroecosystems with pastures based on legumes (RP). This may be attributed to changes in soil carbon (C) and nitrogen (N) levels. However, there is a lack of quantitative information about the contribution of the legume crop in RP agroecosystems in terms of soil quality indicators. The current study analyzed an RR and an RP in order to contribute to the understanding of the underlying processes involved in C and N dynamics in the soil-plant environment, finding better soil quality indexes in RP than in RR, and an interaction between N and C parameters. The percentage of total N explained 90 % of the C pool index (CPI) variability, and 72 % of the percentage of particulate organic C (%POC) variability. Furthermore, a biological N fixation (BNF) study performed in RP showed that almost 80 % of the N content in the aerial biomass of Lotus corniculatus plants originated from the legume–rhizobia symbiosis. Taken together, our results suggest that the soil N derived from the BNF associated with L. corniculatus plants could be a key factor for increasing soil C content, thereby improving soil structure.

Biotechnology for remediation and restoring the ecological status of soil in the places of permanent disposition of the troops by oil-destructor preparation on the basis of the microbial-plant association

The object of the study is a preparation of ecotoxicant destructor based on bacterial strains of Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas délhiensis and associations of nodule bacteria Rhizobium loti with Lotus corniculatus. It is established, that during the simultaneous use of the bacteria and the legume Lotus corniculatus the rate of oxidation of petroleum hydrocarbons increases three times in comparison with the degradation rate of the pollutant after the separate use of each bacterial strain. We have received the preparation of ecotoxicant destructor – dry heterogeneous mass consisting of viable microbial cells of strains of P. delhiensis and R. lotus, seeds of leguminous plant Lotus corniculatus and delignified sawdust. Oil destructive activity of the preparation is proved in the course of microfield experiments. The degradation of ecotoxicants is proved by capillary gas chromatography-mass spectrometry with mass-selective detection. Bacteria of the strains, included in the preparation, are non-pathogenic for humans and animals, biocompatible, environmentally safe, stable, unpretentious to nutritional needs, technological, do not persist in environmental objects in the absence of a substrate for destruction. The technology of the production of the preparation is developed. The results of these studies demonstrate the possibility of practical use of the degradative potential of the preparation in the course of the implementation of the measures for soil reclamation, cleaning up ecotoxicants and restoring its ecological status.

First report of Epicoccum nigrum causing disease in Lotus corniculatus in Argentina

Lotus corniculatus is a species adapted to field conditions in the most important cattle production region in Argentina and constitutes a very valuable forage species. In January 2018, black leaf spots were observed on L. corniculatus (Fig. 1) in field plots in Chascomús, Buenos Aires. These spots were amphigenous, circular to irregular, 2-8 mm diameter, close to the leaf margin and reddish brown. Single lesions often coalesced to form larger lesions and became dark brown (Fig. 1). Fungal isolates were obtained from the shoot lesions and were incubated on oatmeal agar, malt extract and potato dextrose agar (Crous et al., 2) at 25°C in darkness. Macroscopic features of colonies on these media and mycelia characteristic were characterised under a microscope (Fig. 2) and conformed morphologically most closely to the genus Epicoccum (Chen et al., 1). Total genomic DNA was extracted from fresh mycelia and PCR amplification was done using the ITS4 and ITS5 primers (White et al., 5) designed to amplify the internal transcribed spacer (ITS) region. The amplified PCR products were purified and sequenced (GenBank Accession No. MH042300). A BLAST search revealed 99% identity to E. nigrum (KX815296 and MF510615). An inoculation trial was done as follows. Lotus corniculatus plants were grown in a growth chamber at 25°C at 30-40% relative humidity with a 12 hr photoperiod. Thirty-day-old leaves were placed on Petri plates containing water agar medium. Leaves were sprayed with a conidia solution prepared by agitating the fungal lawn with distilled water in a petri dish culture, then suspending in 0.05% Tween 20 to a final concentration of 105 conidia/ml. Control leaves were sprayed with water containing 0.05% Tween 20. Petri plates were maintained at 25°C, 100% relative humidity for 16 days. The inoculated L. corniculatus plants were observed to be wilted with a yellow appearance and black spots (Fig. 3). The causal fungus was isolated and the macro- and microscopic characteristics were consistent with the inoculated fungus. The genus Epicoccum was reported in Argentina as a seed pathogen in Lotus spp. (Sisterna & Lori, 4). The isolate in the current study had 98% identity with E. nigrum (KC568289 and KY303832) that caused leaf spot in Lablab purpureus and brown leaf spot in loquat, respectively (Mahadevakumar et al., 3; Wu et al., 6). To our knowledge, this is the first report of E. nigrum causing leaf spot in L. corniculatus in Argentina.

Differential phenotypic and genetic expression of defence compounds in a plant-herbivore interaction along elevation.

Elevation gradients impose large differences in abiotic and biotic conditions over short distances, in turn, likely driving differences in gene expression more than would genetic variation per se, as natural selection and drift are less likely to fix alleles at such a narrow spatial scale. As elevation increases, the pressure exerted on plants by herbivores and on arthropod herbivores by predators decreases, and organisms spanning the elevation gradient are thus expected to show lower levels of defence at high elevation. The alternative hypothesis, based on the optimal defence theory, is that defence allocation should be higher in low-resource habitats such as those at high elevation, due to higher costs associated with tissue replacement. In this study, we analyse variation with elevation in (i) defence compound content in the plant Lotus corniculatus and (ii) gene expression associated with defence against predators in the specific phytophagous moth, Zygaena filipendulae. Both species produce cyanogenic glycosides (CNglcs) such as lotaustralin and linamarin as defence mechanisms, with the moth, in addition, being able to sequester CNglcs from its host plant. Specifically, we tested the assumption that the defence-associated phenotype in plants and the gene expression in the insect herbivore should covary between low- and high-elevation environments. We found that L. corniculatus accumulated more CNglcs at high elevation, a result in agreement with the optimal defence theory. By contrast, we found that the levels of expression in the defence genes of Z. filipendulae larvae were not related to the CNglc content of their host plant. Overall, expression levels were not correlated with elevation either, with the exception of the UGT33A1 gene, which showed a marginally significant trend towards higher expression at high elevation when using a simple statistical framework. These results suggest that the defence phenotype of plants against herbivores, and subsequent herbivore sequestration machineries and de novo production, are based on a complex network of interactions.

Comparative ionomics and growth factors alteration in Lotus corniculatus under salt stress.

In order to understand the response of Lotus corniculatus to salt stress, ions content and some growth factors were analyzed in three varieties of this plant, namely, Ardebil, Karaj, and Jolfa. Lotus corniculatus plants were exposed to 0, 50, 100, and 150 mM NaCl in hydroponic condition. The amounts of anions (Cl-and NO3-) and cations (K+ and Na+) in leaves as well as shoot and root dry weights, stem and root length, leaf area, and number of leaves were analyzed. The amount of Na+ and Cl- significantly increased in all varieties. However, with an increase in NaCl concentration, K+ and No3- content decreased significantly in all varieties (p<0.05). Growth factors (root and shoot length and weight, leaf area, and leaf number) declined significantly with increasing NaCl concentration. Considering alterations in growth factors and ions content, it seems that Jolfa and karaj have a higher capacity to tolerate salt stress particularly in100 and 150 mM NaCl compared to Ardebil.

Transcriptional regulation of de novo biosynthesis of cyanogenic glucosides throughout the life-cycle of the burnet moth Zygaena filipendulae (Lepidoptera)

The six-spotted burnet moth Zygaena filipendulae (Lepidoptera) utilize the two cyanogenic glucosides (CNglcs) linamarin and lotaustralin as deterrents against predators throughout the entire life cycle. CNglcs can be hydrolyzed and bioactivated by β-glucosidases, resulting in the release of toxic hydrogen cyanide. CNglcs are retained through metamorphosis, probably involved in mating communication, and transferred during mating from the male to the female as a nuptial gift. CNglcs can be biosynthesized de novo by Z. filipendulae larvae, but may also be sequestered from their food plant Lotus corniculatus (Fabaceae). These two strategies are tightly linked and adjusted according to the CNglc content and composition of the food plant in order to balance CNglc homeostasis in the larva. In this study, the amounts of CNglcs and transcript levels of the biosynthetic genes were monitored in all life-stages and tissues of Z. filipendulae. During pupation, transcription of the biosynthetic genes is turned off and the CNglc content slowly declines. In females but not males, transcription of the biosynthetic genes is re-activated at the end of pupation. Eggs and embryos do not biosynthesize CNglcs de novo, but are endowed with CNglcs following eclosion of the female. Similarly to larvae, de novo biosynthesis in female adults takes place in the integument from which CNglcs are then transported to other organs. This study demonstrates that Z. filipendulae has evolved the ability to adjust the production of CNglcs throughout its life-cycle for optimal utilization in defense and possibly other metabolic functions, while at the same time avoiding intoxication.

Sequestration, tissue distribution and developmental transmission of cyanogenic glucosides in a specialist insect herbivore

Considering the staggering diversity of bioactive natural products present in plants, insects are only able to sequester a small number of phytochemicals from their food plants. The mechanisms of how only some phytochemicals are sequestered and how the sequestration process takes place remains largely unknown. In this study the model system of Zygaena filipendulae (Lepidoptera) and their food plant Lotus corniculatus is used to advance the knowledge of insect sequestration. Z. filipendulae larvae are dependent on sequestration of the cyanogenic glucosides linamarin and lotaustralin from their food plant, and have a much lower fitness if reared on plants without these compounds. This study investigates the fate of the cyanogenic glucosides during ingestion, sequestration in the larvae, and in the course of insect ontogeny. To this purpose, double-labeled linamarin and lotaustralin were chemically synthesized carrying two stable isotopes, a 2H labeled aglucone and a 13C labeled glucose moiety. In addition, a small amount of 14C was incorporated into the glucose residue. The isotope-labeled compounds were applied onto cyanogenic L. corniculatus leaves that were subsequently presented to the Z. filipendulae larvae. Following ingestion by the larvae, the destiny of the isotope labeled cyanogenic glucosides was monitored in different tissues of larvae and adults at selected time points, using radio-TLC and LC-MS analyses. It was shown that sequestered compounds are taken up intact, contrary to earlier hypotheses where it was suggested that the compounds would have to be hydrolyzed before transport across the gut. The uptake from the larval gut was highly stereo selective as the β-glucosides were retained while the α-glucosides were excreted and recovered in the frass. Sequestered compounds were rapidly distributed into all analyzed tissues of the larval body, partly retained throughout metamorphosis and transferred into the adult insect where they were distributed to all tissues. During subsequent mating, isotope labeled cyanogenic glucosides were transferred from the male to the female in the nuptial gift.

The glutathione peroxidase gene family of Lotus japonicus: characterization of genomic clones, expression analyses and immunolocalization in legumes

Despite the multiple roles played by antioxidants in rhizobia-legume symbioses, little is known about glutathione peroxidases (GPXs) in legumes. Here the characterization of six GPX genes of Lotus japonicus is reported. Expression of GPX genes was analysed by quantitative reverse transcription-polymerase chain reaction in L. japonicus and Lotus corniculatus plants exposed to various treatments known to generate reactive oxygen and/or nitrogen species. LjGPX1 and LjGPX3 were the most abundantly expressed genes in leaves, roots and nodules. Compared with roots, LjGPX1 and LjGPX6 were highly expressed in leaves and LjGPX3 and LjGPX6 in nodules. In roots, salinity decreased GPX4 expression, aluminium decreased expression of the six genes, and cadmium caused up-regulation of GPX3, GPX4 and GPX5 after 1 h and down-regulation of GPX1, GPX2, GPX4 and GPX6 after 3-24 h. Exposure of roots to sodium nitroprusside (a nitric oxide donor) for 1 h increased the mRNA levels of GPX4 and GPX6 by 3.3- and 30-fold, respectively. Thereafter, the GPX6 mRNA level remained consistently higher than that of the control. Immunogold labelling revealed the presence of GPX proteins in root and nodule amyloplasts and in leaf chloroplasts of L. japonicus and other legumes. Labelling was associated with starch grains. These results underscore the differential regulation of GPX expression in response to cadmium, aluminium and nitric oxide, and strongly support a role for GPX6 and possibly other GPX genes in stress and/or metabolic signalling.

Alteration of O-specific polysaccharide structure of symbiotically defective Mesorhizobium loti mutant 2213.1 derived from strain NZP2213.

Mesorhizobium loti mutant 2213.1 derived from the wild-type strain NZP2213 by Tn5 mutagenesis showed impaired effectiveness of symbiosis with the host plant Lotus corniculatus (Turska-Szewczuk et al., 2007 Microbiol Res, in press). The inability of lipopolysaccharide (LPS) isolated from the mutant 2213.1 strain or de-O-acetylated LPS of the parental cells to inactivate phage A1 particles implicated alterations in the LPS structure. The O-specific polysaccharide of the mutant was studied by chemical analyses along with (1)H and (13)C NMR spectroscopy, which clearly confirmed alterations in the O-chain structure. 2D NMR data showed that the mutant O-polysaccharide consists of a tetrasaccharide repeating unit containing non-substituted as well as O-acetylated or O-methylated 6-deoxytalopyranose residues. Additionally, an immunogold assay revealed a reduced number of gold particles on the mutant bacteroid cell surface, which could result from both a diminished amount of an O-antigenic determinant in mutant LPS and modifications of structural epitopes caused by alterations in O-acetylation or O-methylation of sugar residues. Western immunoblot assay of alkaline de-O-acetylated lipophilic M. loti NZP2213 LPS showed no reactivity with homologous serum indicating a role of O-acetyl groups in its O-specificity.

Niche separation in space and time between two sympatric sister species—a case of ecological pleiotropy

We investigate the niche separation in space and time between the Palearctic sister species Leptidea sinapis and L. reali (Lepidoptera, Pieridae) in central Sweden. Using field sampling, we show that L. reali is a habitat specialist confined to meadows, whereas L. sinapis is a habitat generalist also inhabiting forests. This difference in habitat utilization was corroborated by experimental release of laboratory-reared L. sinapis and L. reali in two adjacent forest and meadow habitats during their natural flight period; virtually all recaptured L. reali that were released in the forest were later caught in the meadow, whereas L. sinapis shifted equally often from meadow to forest as in the opposite direction. In the field, both species fly in May–June, but L. reali appears on average a week earlier in spring and has a substantial second generation in July, whereas L. sinapis is practically univoltine. When overwintered pupae were incubated under identical conditions in the laboratory, females did, however, not differ in phenology, and L. sinapis males actually emerged earlier than L. reali males. When larvae were reared at 23°C on the host plant Lotus corniculatus at a range of daylengths, both species produced a substantial proportion of directly developing individuals at an 18.5 h daylength or longer. When reared at 23°C and a 22 h daylength, L. reali showed an overall higher propensity to develop directly than L. sinapis on plant species originating from both the meadow and the forest habitat. Both Leptidea species showed a lower propensity to enter direct development on forest associated plants than on meadow associated plants. Hence, we suggest that the difference in phenology and voltinism between L. sinapis and L. reali is largely the result of environmentally implemented ecological pleiotropic effects caused by the between-species difference in habitat preference.

Studies on the overexpression of the soybean GmNHX1 in Lotus corniculatus: The reduced Na+ level is the basis of the increased salt tolerance

The full length cDNA coding for a novel vacuolar Na+/H+ antiporter (GmNHX1) was cloned from soybean and determined to consist of 2591 bp with a 5′-untranslated region of 464 bp, an open reading frame (ORF) of 1641 bp, and a 3′-untranslated region of 486 bp. The deduced protein sequence contains 546 aa with the typical characters of the vacuolar Na+/H+ antiporters, and shares high similarity with that of AtNHX1, OsNHX1 and AgNHX1. The soybean genome showed a single copy of the GmNHX1 gene. Northern blot analysis demonstrated that the expression of the GmNHX1 was tissue-specific, increased by ABA treatment, NaCl, KCl, LiCl and dehydration stress, and lower in leaves but higher in roots and hypocotyls of salt-tolerant than salt-sensitive cultivars. The GmNHX1 was overexpressed under the control of a tandem cauliflower mosaic virus (CaMV) 35S promoter in the model leguminous plant Lotus corniculatus L. and conferred salt-tolerance of the transgenic plants. Measurements of Na+ and K+ contents in both roots and shoots demonstrated that the plantlets of GmNHX1-overexpressing lines had lower Na+ and K+ content, and higher K+/Na+ ratio than the controlled lines that were transformed with the empty vector, which indicates that the salt-tolerance conferred by GmNHX1 is closely related with decreased accumulation of Na+ in the transgenic plants.

The role of nectar sources for oviposition decisions of the common blue butterfly Polyommatus icarus

Neural limitations on information processing have been shown to play an important role for host plant specialization in herbivorous insects. The necessity of fast and accurate decisions favors the adoption of a few high‐contrast signals, which selects against the use of multiple resources. Many species face a similar problem when searching for adult food sources and the simultaneous need to fulfill both search tasks can lead to a potential conflict. Some insects use the same host plant species for both adult and larval nutrition, which makes it possible to decrease the number of search images and thus potentially increase efficiency of the choices. The aim of this study was to investigate if there is a connection between choice of nectar sources and choice of oviposition host plant. In a laboratory experiment, females of Polyommatus icarus preferred to oviposit on Lotus corniculatus plants with flowers over those without flowers. Observations of behavioral sequences also revealed that oviposition often followed immediately after nectaring. The results suggest that nectar availability could play an important role in oviposition decisions of P. icarus and can provide one explanation to why some phytophagous insects not always choose the host plant that gives the best offspring performance.

Transformation of Lotus corniculatus Plants with Escherichia coli Asparagine Synthetase A: Effect on Nitrogen Assimilation and Plant Development

Asparagine and glutamine are major forms of nitrogen in the phloem sap of many higher plants. In vascular plants, glutamine-dependent asparagine synthetase (AS) is the primary source of asparagine. In Escherichia coli, asparagine is synthesized by the action of two distinct enzymes, AS-A which utilizes ammonia as a nitrogen donor, and AS-B which utilizes both glutamine and ammonia as substrates, but with a preference for glutamine. In this study, the possibility to endow plants with ammonia-dependent AS activity was investigated by heterologous expression of E. coli asnA gene with the aim to introduce a new ammonium assimilation pathway in plants. The bacterial gene is placed under the control of light-dependent promoters, and introduced by transformation into Lotus corniculatus plants. Analysis of transgenic plants has revealed a phenomenon of transgene silencing which has prevented asnA expression in several transgenics. The asnA-expressing plants are characterized by premature flowering and reduced growth. A significant reduction of the total free amino acid accumulation in transgenic plants is observed. Surprisingly, the content of asparagine in wild-type plants is about 2.5-fold higher than that of transgenic plants. While glutamine levels in transgenic plants are about 3–4-fold higher than those in wild-type plants, aspartate levels are significantly lower. Transformation with asnA also induced a significant reduction of photosynthesis when measured under saturated light and ambient CO2 conditions.

Arbuscular mycorrhizal fungi influence life history traits of a lepidopteran herbivore.

Results from pot and microcosm studies in the greenhouse have shown that plant growth and foliar chemistry is altered by the presence and species composition of arbuscular mycorrhizal fungi (AMF). The growth and survival of herbivores which feed on plants could, as a consequence, also be affected by these mutualistic soil fungi. Consequently, interactions between AMF, plants and herbivores could occur. To test this, larvae of the common blue butterfly, Polyommatus icarus (Lycaenidae), were fed with sprigs of Lotus corniculatus (Fabaceae) plants which were inoculated with one of two different AMF species, with a mixture of these AMF species or with sprigs of plants which were not inoculated with AMF. Survival and larval weight of third instar larvae fed with plants colonised by AMF were greater than those of larvae fed with non-mycorrhizal plants. Survival of larvae feeding on non-mycorrhizal plants was 1.6times lower than that of larvae feeding on plants inoculated with a mixture of AMF species and 3.8times lower than that of larvae feeding on plants inoculated with single AMF species. Furthermore, larvae fed with non-mycorrhizal plants attained only about half the weight of larvae fed with mycorrhizal plants after 11days of growth. These differences in larval performance might be explained by differences in leaf chemistry, since mycorrhizal plants had a 3times higher leaf P concentration and a higher C/N-ratio. Our results, thus, show that the presence of belowground mutualistic soil fungi influences the performance of aboveground herbivores by altering their food quality. Larval consumption, larval food use and adult lipid concentrations of the common blue butterfly differed between larvae which were fed with plants inoculated with different AMF species. This suggests that the performance of herbivores is not only influenced by the presence of AMF but also depends on the identity of the AMF species colonising the host plants. Moreover, a significant interaction term between AMF species and maternal identity of the larvae occurred for adult dry weight, indicating that the performance of offspring from different females was differently influenced by AMF species composition. To our knowledge, these results show for the first time that the species composition of AMF communities can influence life-history traits of butterfly larvae and possibly herbivores in general.

Effect of crop rotation and soil cover on alteration of the soil microflora generated by the culture of transgenic plants producing opines.

The culture of transgenic Lotus corniculatus plants producing opines, which are bacterial growth substrates, leads to the selection of rhizospheric bacteria able to utilize these substrates. We have investigated the fate of the opine-utilizing community over time under different experimental conditions following elimination of selective pressure exerted by the transgenic plants. These plants were removed from the soil, which was either left unplanted or replanted with wild-type L. corniculatus or wheat plants. The density of opine-utilizing bacteria in the fallow soils remained essentially unchanged throughout the experiment, regardless of the soil of origin (soil planted with wild-type or transgenic plants). When wild-type Lotus plants were used to replace their transgenic counterparts, only the bacterial populations able to utilize the opines were affected. Long-term changes affecting the opine-utilizing bacterial community on Lotus roots was dependent upon the opine studied. The concentration of nopaline utilizers decreased, upon replacement of the transgenic plants, to a level similar to that of normal plants, while the concentration of mannopine utilizers decreased to levels intermediate between transgenic and normal plants. These data indicate that: (i) the opine-utilizing bacterial populations can be controlled in the rhizosphere via plant-exudate engineering; (ii) the interaction between the engineered plants and their root-associated micro-organisms is transgene specific; and (iii) alterations induced by the cultivation of transgenic plants may sometimes be persistent. Furthermore, opine-utilizing bacterial populations can be controlled by crop rotation. Therefore, favouring the growth of a rhizobacterium of agronomic interest via an opine-based strategy appears feasible.

Detecting decline in a formerly widespread species: how common is the common blue butterfly Polyommatus icarus?

Grid square distribution maps have been used widely to measure rates of decline and target conservation resources. However, common species that may have many populations per grid square can decline substantially from within squares without being lost from entire squares. In order to quantify this process, fine scale population and habitat data have been collected for the common blue butterfly Polyommatus icarus in a 35 km2 area of fragmented landscape in north Wales. Present day habitat associations, determined from over 2000 transect walks, combined with data on historical and present day habitat distributions reveal that the species has declined by about 74% since 1901. Similar data concerning the species major host plant Lotus corniculatus indicate a decline of 46%. Based on 1 km2 grid maps, neither species have been assessed as declining at all. These results suggest that apparently ‘common’ species may have declined just as much as many of Britain's rare species: using present methods of assessment these declines are undetected.

Transcription of a maize cDNA in Lotus corniculatus is regulated by T-DNA methylation and transgene copy number

Lotus corniculatus plants transformed with a maize cDNA (G1L) encoding a sulphur-rich γ-zein were obtained by using two fusion genes: one with the CaMV 35S promoter, the other with the ribulose bisphosphate carboxylase small subunit (rbcS) promoter. The highest expression of G1L mRNA was found in plants transformed with G1L under the rbcS promoter. The steady state level of G1L mRNA in the leaves was generally directly correlated with the G1L copy number. However, due to a transcriptional block, no G1L mRNA was detected in some of the 35S-G1L multi-copy transformants. Analyses with methylation-sensitive restriction enzymes revealed that the T-DNA of the silenced 35S-G1L transformants was methylated. T-DNA copy number, G1L silencing activity, and the state of methylation were strictly correlated in primary transformants. A cross between two 35S-G1L transformed plants, one (S) with the T-DNA methylated and the other (NS) without, showed that: (1) the methylated state of T-DNA passed through meiosis; and (2) when T-DNA copies from the two parents were combined in the progeny, the unmethylated T-DNA copies of parent NS became methylated at different levels and G1L mRNA production was dependent on the degree of methylation.

A strong constitutive positive element is essential for the ammonium-regulated expression of a soybean gene encoding cytosolic glutamine synthetase.

In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5' promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3' deletions were fused to a -90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium-regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non-legume background to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility.

Nodule parenchyma-specific expression of the sesbania rostrata early nodulin gene SrEnod2 is mediated by its 3' untranslated region

The early nodulin Enod2 gene encodes a putative hydroxyproline-rich cell wall protein and is expressed exclusively in the nodule parenchyma cell layer. The latter finding suggests that the Enod2 protein may contribute to the special morphological features of the nodule parenchyma and to the creation of an oxygen diffusion barrier. The Enod2 gene of the stem-nodulating legume Sesbania rostrata (SrEnod2) is induced specifically in roots by the plant hormone cytokinin, and this induction occurs at a post-transcriptional level. Here, we characterize the cis determinant(s) in the SrEnod2 locus responsible for nodule parenchyma-specific expression and show that the 3' untranslated region (UTR) of the SrEnod2 gene is both required and sufficient for directing chimeric reporter gene expression in the nodule parenchyma of transgenic Lotus corniculatus plants. Moreover, we show that the SrEnod2 3' UTR does not act as a tissue-specific enhancer element. By conducting a detailed deletion analysis of the 5' and 3' SrEnod2 regions, we delimited the minimal promoter of the SrEnod2 gene, and it appears that the 5' flanking sequences are not essential for nodule parenchyma-specific expression. This finding is in contrast with the report that the 5' upstream region of the soybean Enod2 gene directs nodule parenchyma-specific expression, indicating that different mechanisms may be involved in regulating the expression of these two genes. We definitively demonstrate that the cis element(s) for tissue-specific expression is located within the 3' UTR of a plant nuclear gene.

MsEnod12A Expression Is Linked to Meristematic Activity During Development of Indeterminate and Determinate Nodules and Roots

During development of nodules and roots, the alfalfa early nodulin gene MsEnod12A is expressed adjacent to the meristem. Using transgenic alfalfa carrying a MsEnod12A promoter-gusA fusion, we investigated the regulation of MsEnod12A expression in mature nodules and roots by GUS assays and reverse transcription-PCR. We found that in alfalfa indeterminate nodules induced by various Fix- Rhizobium meliloti mutants and in spontaneous nodules devoid of rhizobia, MsEnod12A was expressed at the distal end when a persistent meristem was present. However, this gene was not expressed when the meristem was lacking in nodules arrested in development. The MsEnod12A-gusA fusion was introduced into Lotus corniculatus plants that form determinate nodules devoid of a persistent meristem. Using these plants we found MsEnod12A-gusA expression only in young nodules and a disappearance in mature nodules. Moreover, when alfalfa roots were treated with auxins a lateral band of MsEnod12A expression was observed surrounding the club-shaped root apex and coinciding with induced lateral meristematic activities. Thus, in all cases MsEnod12A expression was associated with meristematic activities, suggesting that MsEnod12A plays a role in the differentiation processes of nodule and root cells and that it may serve as molecular tool for analyzing meristem establishment during nodule development.