Genistein-Specific G6DT Gene for the Inducible Production of Wighteone in Lotus japonicus.

Prenylated isoflavonoids have been found in several legume plants, and they possess various biological activities that play important roles in both plant defense and human health. However, it is still unknown whether prenylated isoflavonoids are present in the model legume plant Lotus japonicus. In the present study, we found that the prenylated isoflavonoid wighteone was produced in L. japonicus when leaf was supplemented with genistein. Furthermore, a novel prenyltransferase gene, LjG6DT, was identified, which shared high similarity with and was closely related to several known prenyltransferase genes involved in isoflavonoid biosynthesis. The recombinant LjG6DT protein expressed in yeast exhibited prenylation activity toward genistein as an exclusive substrate, which produced wighteone, a prenylated genistein at the C-6 position that occurs normally in legume plants. The LjG6DT-green fluorescent protein (GFP) fusion protein is targeted to plastids. The transcript level of LjG6DT is induced by glutathione, methyl jasmonate and salicylic acid, implying that LjG6DT is involved in stress response. Overexpression of LjG6DT in L. japonicus hairy roots led to increased accumulation of wighteone when genistein was supplied, indicating that LjG6DT is functional in vivo. Feeding assays with the upstream intermediate naringenin revealed that accumulation of wighteone in L. japonicus was dependent on genistein supplementation, and accumulation of wighteone is competed by genistein methylation. This study demonstrated that phytoalexin wighteone is inducibly produced in L. japonicus, and it provides new insight into the biosynthesis and accumulation of prenylated isoflavonoids in legume plants.

Phytochemical profile and antimicrobial properties of Lotus spp. (Fabaceae).

The phytochemical profile and antimicrobial activity of cultivar (cv.) extracts of Lotus uliginosus (cvs. Trojan and Serrano), L. tenuis (cv. Larrañaga) and L. corniculatus (cv. São Gabriel) were investigated. The phytochemical analysis revealed tannins, coumarins and flavonoids in all extracts, with variations among cultivars, showing genotypic variability. By High Performance Liquid Chromatographic method, the cvs. Larrañaga and São Gabriel showed the highest percentage of catechin and epicatechin, respectively, and presented rutin, which was not detected in the other ones. These genotypes showed antifungal activity but not antibacterial one. The cv. Larrañaga inhibited the mycelia growth of Alternaria sp. and Fusarium graminearum while the cv. São Gabriel was active only against Alternaria sp. The cultivars showed the greatest amounts of secondary metabolites and demonstrated significant activity against filamentous fungi. The results provide a direction for further research about pharmacological use of Lotus spp.

The cis-acting CTTC-P1BS module is indicative for gene function of LjVTI12, a Qb-SNARE protein gene that is required for arbuscule formation in Lotus japonicus.

The majority of land plants live in symbiosis with arbuscular mycorrhizal fungi from the phylum Glomeromycota. This symbiosis improves acquisition of phosphorus (P) by the host plant in exchange for carbohydrates, especially under low-P availability. The symbiosome, constituted by root cortex cells accommodating arbuscular mycorrhizal fungal hyphae, is the site at which bi-directional exchange of nutrients and metabolites takes place. Uptake of orthophosphate (Pi) in the symbiosome is facilitated by mycorrhiza-specific plant Pi transporters. Modifications of the potato Pi transporter 3 (StPT3) promoter were analysed in transgenic mycorrhizal roots, and it was found that the CTTC cis-regulatory element is necessary and sufficient for a transcriptional response to fungal colonization under low-Pi conditions. Phylogenetic footprinting also revealed binary combination of the CTTC element with the Pi starvation response-associated PHR1-binding site (P1BS) in the promoters of several mycorrhiza-specific Pi transporter genes. Scanning of the Lotus japonicus genome for gene promoters containing both cis-regulatory elements revealed a strong over-representation of genes involved in transport processes. One of these, LjVTI12, encoding a member of the SNARE family of proteins involved in membrane transport, exhibited enhanced transcript levels in Lotus roots colonized with the arbuscular mycorrhizal fungus Glomus intraradices. Down-regulation of LjVTI12 by RNA interference resulted in a mycorrhiza-specific phenotype characterized by distorted arbuscule morphology. The results highlight cooperative cis-regulation which integrates mycorrhiza and Pi starvation signaling with vesicle trafficking in symbiosome development.

Structural analysis of K+ dependence in L-asparaginases from Lotus japonicus.

The molecular features responsible for the existence in plants of K+-dependent asparaginases have been investigated. For this purpose, two different cDNAs were isolated in Lotus japonicus, encoding for K+-dependent (LjNSE1) or K+-independent (LjNSE2) asparaginases. Recombinant proteins encoded by these cDNAs have been purified and characterized. Both types of asparaginases are composed by two different subunits, α (20 kDa) and ß (17 kDa), disposed as (αß)2 quaternary structure. Major differences were found in the catalytic efficiency of both enzymes, due to the fact that K+ is able to increase by tenfold the enzyme activity and lowers the K(m) for asparagine specifically in LjNSE1 but not in LjNSE2 isoform. Optimum LjNSE1 activity was found at 5-50 mM K+, with a K(m) for K+ of 0.25 mM. Na+ and Rb+ can, to some extent, substitute for K+ on the activating effect of LjNSE1 more efficiently than Cs+ and Li+ does. In addition, K+ is able to stabilize LjNSE1 against thermal inactivation. Protein homology modelling and molecular dynamics studies, complemented with site-directed mutagenesis, revealed the key importance of E248, D285 and E286 residues for the catalytic activity and K+ dependence of LjNSE1, as well as the crucial relevance of K+ for the proper orientation of asparagine substrate within the enzyme molecule. On the other hand, LjNSE2 but not LjNSE1 showed ß-aspartyl-hydrolase activity (K(m) = 0.54 mM for ß-Asp-His). These results are discussed in terms of the different physiological significance of these isoenzymes in plants.

Cloning and functional characterization of LjPLT4, a plasma membrane xylitol H(+)- symporter from Lotus japonicus.

Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation.

Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

Functional characterization of an unusual phytochelatin synthase, LjPCS3, of Lotus japonicus.

In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.

Polyubiquitin promoter-based binary vectors for overexpression and gene silencing in Lotus japonicus.

In this study, we compared the transcriptional activities between Cauliflower mosaic virus (CaMV)35S promoter and polyubiquitin (Ljubq1) promoter from Lotus japonicus using beta-glucuronidase (gus) reporter gene in transgenic plants of L. japonicus. The promoter analysis demonstrated that the Ljubq1 promoter possessed higher activity than the CaMV35S promoter in leaves, stems, roots, nodules, and pollen. Finally, we created GATEWAY conversion technology-compatible binary vectors for over-expression and RNA interference under the Ljubq1 promoter. These materials could provide alternative choice for studies in L. japonicus.

Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes.

Two photorespiratory mutants from Lotus japonicus, namely Ljgln2-1 and Ljgln2-2, deficient in plastidic glutamine synthetase (GLN2), were analysed at the molecular level. Both mutants showed normal levels of Gln2 mRNA, indicating that they were affected post-transcriptionally. Complete sequencing of full-length Gln2 cDNAs revealed the presence of a single point mutation on each mutant, leading to G85R and L278H amino acid replacements, respectively. Different types of experimental approaches, including heterologous expression and complementation tests in Escherichia coli, showed that both GLN2 mutant proteins completely lacked of biosynthetic and transferase enzyme activities. Moreover, it was also shown that while GLN2-1 mutant protein was assembled into a less stable inactive octamer, GLN2-2 mutant protein was unable to acquire a proper quaternary structure and was rapidly degraded. Therefore, the mutations analysed are the first of their type affecting the stability and/or the quaternary structure of the GLN2 enzyme. The kinetic parameters of purified recombinant GLN2 were determined. The enzyme showed positive cooperativity towards ammonium and Mg(2+). Thiol compounds stimulated by twofold the biosynthetic activity but not the transferase activity of recombinant GLN2 and were able to alter the kinetics towards glutamate of the enzyme. Moreover, the biosynthetic activity of recombinant GLN2 was stimulated by more than tenfold by the presence of free Mg(2+).

Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus.

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.

Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots.

The roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary younger nitrogen-fixing root-nodule symbiosis with bacteria. Plant symbioses with bacteria and fungi require a set of common signal transduction components that redirect root cell development. Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. Surprisingly, both twin proteins are localized in the plastids of root cells, indicating a previously unrecognized role of this ancient endosymbiont in controlling intracellular symbioses that evolved more recently.

Cloning and expression pattern of a novel microspore-specific gene encoding hypersensitive-induced response protein (LjHIR1) from the model legume, Lotus japonicus.

In order to understand the microspore and pollen development, recently, we have isolated a number of anther-specific genes in the model legume, Lotus japonicus. From these anther-specific genes, we identified one novel microspore-specific gene, LjImfb-c82. In order to determine the molecular characterization of LjImfb-c82, full-length cDNA clone was first isolated and sequenced. It encoded a protein of 286 amino acids (LjHIR1), which had sequence similarity to Hypersensitive-Induced Response like protein. LjHIR1 was specifically expressed in microspore on the in situ hybridization experiment. From the sequence similarity to prohibitin-domain protein, the LjHIR1 might be related to ion channel regulation in microspore development.

Lotus japonicus LjKUP is induced late during nodule development and encodes a potassium transporter of the plasma membrane.

The KUP family of potassium transporters in plants is large but poorly characterized. We isolated and characterized the first KUP transporter from a legume, LjKUP of Lotus japonicus. Although expressed throughout plants, LjKUP transcript levels were highest in nodules. Induction of LjKUP expression occurred late during nodule development, at a time of rapid organ expansion. A high level of LjKUP expression was maintained in mature, full-sized nodules. However, induction of LjKUP expression was independent of symbiotic nitrogen fixation (SNF), and occurred in ineffective nodules resulting from mutations in either the plant or its microsymbiont, Mesorhizobium loti. Heterologous expression of LjKUP in Escherichia coli showed that the protein is able to transport potassium. Transient expression of a GFP-LjKUP fusion protein in Arabidopsis cells indicated a plasma membrane location for the transporter. Taken together, the results indicate that LjKUP is a potassium transporter of the plasma membrane, which may play roles in cell expansion during nodule development and in ion homeostasis during SNF.

Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle.

The symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to the arrested infection thread growth from the epidermis into the cortex. Here, we describe that crk is also affected in male fertility that causes the production of small pods with few seeds. Under in vitro conditions, pollen germination and tube growth were markedly reduced in the crk mutant. A swollen tip phenotype with disorganized filamentous actin (F-actin) was observed in the mutant pollen tubes after prolonged in vitro culture. During pollen development, the striking difference noted in the mutant was the small size of the microspores that remained spherical. Histological examination of ovule development, as well as outcrosses of the mutant as female to wild type as male, showed no evidence of abnormality in the female gametophyte development. Based on these findings, the Crk gene, aside from its role in the infection process during nodulation, is also involved in male gametophyte development and function. Therefore, this gene represents a connection between nodule symbiosis, polar tip growth, and other plant developmental processes.

Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers.

Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.

cDNA cloning, mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus.

A full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situ hybridization revealed that the mRNA level is high in expanding root cells but low in dividing root tip ones. The transcript is also abundant in vascular bundles and the basal portions of mature nodules. L. japonicus squalene synthase has an unusual Asp residue near the active site, where mammalian enzymes have Gln, and replacement of the Gln by Glu has been reported to cause severe inactivation. Site-directed mutagenesis of the L. japonicus enzyme and assaying in vitro showed that this Asp residue can be substituted by not only Gln but also Glu, suggesting that the local structure of plant squalene synthases is different from that of mammalian enzymes.

cDNA cloning and biochemical characterization of S-adenosyl-L-methionine: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase, a critical enzyme of the legume isoflavonoid phytoalexin pathway.

Formononetin (7-hydroxy-4'-methoxyisoflavone, also known as 4'-O-methyldaidzein) is an essential intermediate of ecophysiologically active leguminous isoflavonoids. The biosynthetic pathway to produce 4'-methoxyl of formononetin has been unknown because the methyl transfer from S-adenosyl-L-methionine (SAM) to 4'-hydroxyl of daidzein has never been detected in any plants. A hypothesis that SAM: daidzein 7-O-methyltransferase (D7OMT), an enzyme with a different regiospecificity, is involved in formononetin biosynthesis through its intracellular compartmentation with other enzymes recently prevails, but no direct evidence has been presented. We proposed a new scheme of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor and subsequent dehydration. We now cloned a cDNA encoding SAM: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) through the screening of functionally expressed Glycyrrhiza echinata (Fabaceae) cDNAs. The reaction product, 2,7-dihydroxy-4'-methoxyisoflavanone, was unambiguously identified. Recombinant G. echinata D7OMT did not show HI4'OMT activity, and G. echinata HI4'OMT protein free from D7OMT was partially purified. HI4'OMT is thus concluded to be distinct from D7OMT, and their distant phylogenetic relationship was further presented. HI4'OMT may be functionally identical to (+)-6a-hydroxymaackiain 3-OMT of pea. Homologous cDNAs were found in several legumes, and the catalytic function of the Lotus japonicus HI4'OMT was verified, indicating that HI4'OMT is the enzyme of formononetin biosynthesis in general legumes.

A novel HMG A-like protein binds differentially to the AT-rich regions located in the far distal and proximal parts of a soybean glutamine synthetase gene (GS15) promoter.

In soybean (Glycine max L.) ammonium provided externally or as the result of symbiotic nitrogen fixation stimulates the transcription of GS15, a gene encoding cytosolic glutamine synthetase. Strong constitutive positive expression (SCPE), silencer-like and organ-specific elements, located respectively in the distal, the central and the proximal region of the promoter are required to control the ammonium responsiveness of the gene expression [Tercé-Laforgue et al. (1999) Plant Mol. Biol. 39: 551]. It was hypothesized that the correct spatial conformation of the promoter permitted the cooperative action of these three cis-acting elements. Further investigations were therefore required to ascertain this hypothesis. A nodule nuclear protein, binding to a 66 bp AT-rich DNA fragment containing a 13 bp AT-rich repeated sequence (AT-1) and located just downstream of the SCPE element, was identified using a gel retardation assay. A cDNA clone likely to code for this protein was isolated using the yeast one-hybrid system. It encodes a novel DNA binding protein (AT-1SNBP) similar to HMG A proteins but exhibiting a higher molecular weight. AT-1SNBP appears to be encoded by a single gene that is expressed in roots, root nodules and leaves of soybean. Since two other 13 bp AT-rich repeated sequences (AT-2 and AT-3) were localized in the organ-specific element, we have quantified the binding affinity of AT-1SNBP to these sequences. We demonstrate that AT-1SNBP binds differentially to DNA fragments containing AT-1, AT-2 and AT-3 and that its binding affinity depends on the presence of adjacent sequences. This result suggests that AT-1SNBP may be an architectural protein involved in maintaining the spatial conformation of the GS15 promoter, thus facilitating the interaction between the distal and proximal regulatory elements.

Lotus japonicus gene Ljsbp is highly conserved among plants and animals and encodes a homologue to the mammalian selenium-binding proteins.

We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a polypeptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both.