Nodule-specific regulation of phosphatidylinositol transfer protein expression in Lotus japonicus.

Phosphatidylinositol transfer proteins (PITPs) modulate signal transduction pathways and membrane-trafficking functions in eukaryotes. Here, we describe the characterization of a gene family from Lotus japonicus that encodes a novel class of plant PITP-like proteins (LjPLPs) and that is regulated in an unusual nodule-specific manner. Members of this gene family were identified based on their nucleotide sequence homology with a previously described cDNA, LjNOD16, which encodes the L. japonicus late nodulin Nlj16. Nlj16 or highly related amino acid sequences are shown to constitute C-terminal domains of LjPLPs and are suggested to function as specific plasma membrane targeting modules. The expression patterns of one member of this gene family (LjPLP-IV) revealed that LjNOD16 mRNA synthesis in nodules is the result of the transcriptional activity of a nodule-specific promoter located in an intron of the LjPLP-IV gene. This intron-borne bidirectional promoter also generates nodule-specific antisense transcripts derived from the N-terminal PITP domain coding region of the LjPLP-IV gene. We propose that Nlj16 protein synthesis and LjPLP-IV antisense transcript generation are components of an elaborate mechanism designed to control LjPLP synthesis and/or functioning in nodules.

Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype.

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1-1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root. No significant differences between har1-1 mutant and wild-type plants were detected with respect to root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.

A protein phosphatase 2C gene, LjNPP2C1, from Lotus japonicus induced during root nodule development.

Symbiotic interactions between legumes and compatible strains of rhizobia result in root nodule formation. This new plant organ provides the unique physiological environment required for symbiotic nitrogen fixation by the bacterial endosymbiont and assimilation of this nitrogen by the plant partner. We have isolated two related genes (LjNPP2C1 and LjPP2C2) from the model legume Lotus japonicus that encode protein phosphatase type 2C (PP2C). Expression of the LjNPP2C1 gene was found to be enhanced specifically in L. japonicus nodules, whereas the LjPP2C2 gene was expressed at a similar level in nodules and roots. A glutathione S-transferase-LjNPP2C1 fusion protein was shown to have Mg2+- or Mn2+-dependent and okadaic acid-insensitive PP2C activity in vitro. A chimeric construct containing the full-length LjNPP2C1 cDNA, under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter, was found to be able to complement a yeast PP2C-deficient mutant (pct1Delta). The transcript level of the LjNPP2C1 gene was found to increase significantly in mature nodules, and its highest expression level occurred after leghemoglobin (lb) gene induction, a molecular marker for late developmental events in nodule organogenesis. Expression of the LjNPP2C1 gene was found to be drastically altered in specific L. japonicus lines carrying monogenic-recessive mutations in symbiosis-related loci, suggesting that the product of the LjNPP2C1 gene may function at both early and late stages of nodule development.

The Lotus japonicus LjNOD70 nodulin gene encodes a protein with similarities to transporters.

A novel nodule-specific gene, LjNOD70, associated with late stages in Lotus japonicus nodule development and/or functioning was characterized. The LjNOD70 gene is a member of a small family of closely related L. japonicus genes. Two major mRNA species corresponding to the LjNOD70 gene were identified in nodules and shown to be the result of a mechanism resembling alternative splicing. The longer, presumably unspliced, mRNA species was shown to contain a single open reading frame (ORF), encoding a polytopic hydrophobic protein, LjN70, with a predicted molecular mass of 70 kDa. The second, presumably spliced, mRNA species was shown to be less abundant in nodules. The absence of the presumptive 'intron' was found to divide the reading frame into an upstream and a downstream ORF encoding the partial N- and C-terminal regions of the LjN70 protein, respectively. The predicted amino acid sequence of nodulin LjN70 revealed structural features characteristic of transport proteins, and was found to share similarity with the oxalate/formate exchange protein of Oxalobacter formigenes. Therefore, we postulate that the L. japonicus LjNOD70 gene family encodes nodule-specific transport proteins, which may have evolved as a result of exon-intron shuffling.

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation.

Novel, highly expressed late nodulin gene (LjNOD16) from Lotus japonicus.

We have isolated a Lotus japonicus cDNA corresponding to a highly abundant, late nodule-specific RNA species that encodes a polypeptide with a predicted molecular mass of 15.6 kD. The protein and its corresponding gene were designated Nlj16 and LjNOD16, respectively. LjNOD16 was found to be expressed only in the infected cells of L. japonicus nodules. Related DNA sequences could be identified in the genomes of both Glycine max and Medicago sativa. In the latter, a homologous mRNA species was detected in the nodules. Unlike LjNOD16, its alfalfa homologs appear to represent low-abundance mRNA species. However, the proteins corresponding to the LjNOD16 and its alfalfa homolog could be detected at similar levels in nodules but not in roots of both legume species. The predicted amino acid sequence analysis of nodulin Nlj16 revealed the presence of a long alpha-helical region and a positively charged C terminus. The former domain has a very high propensity to form a coiled-coil type structure, indicating that nodulin Nlj16 may interact with an as-yet-unidentified protein target(s) in the nodule-infected cells. Homology searches revealed no significant similarities to any known sequences in the databases, with the exception of two related, anonymous Arabidopsis expressed sequence tags.

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants.

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5'-Srglb3-uidA-3'nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.

Use of differential display to identify novel Sesbania rostrata genes enhanced by Azorhizobium caulinodans infection.

Upon infection of the tropical legume Sesbania rostrata with Azorhizobium caulinodans ORS571, nodules are formed on the roots as well as on the stems. Stem nodules appear at multiple predetermined sites consisting of dormant root primordia, which are positioned in vertical rows along the stem of the plant. We used the differential display method to isolate and characterize three cDNA clones (differential display; didi-2, didi-13, and didi-20), corresponding to genes whose expression is enhanced in the dormant root primordia after inoculation. Database searches revealed that the deduced (partial) didi-2 gene product shares significant similarity with hydroxyproline-rich cell wall proteins. The (partial) didi-13 and didi-20 products are similar to chitinases and chalcone reductases, respectively. Transcripts corresponding to the cDNA clones didi-2 and didi-13 were first detectable 1 day after inoculation. In contrast, didi-20 transcripts were found at low levels in uninfected root primordia and were enhanced significantly around 3 days after inoculation. In addition, a cDNA was isolated (didi-42) that corresponds to the previously identified leghemoglobin gene Srlb6. These studies show that differential display is a useful method for the isolation of infection-related genes.

iaglu, a gene from Zea mays involved in conjugation of growth hormone indole-3-acetic acid.

Plants contain most of the growth hormone indole-3-acetic acid (IAA) in conjugated forms believed to be inactive in promoting growth. The iaglu gene, which controls the first step in the biosynthesis of the IAA conjugates of Zea mays, encodes (uridine 5'-diphosphate-glucose:indol-3-ylacetyl)-beta-D-glucosyl transferase. Protein synthesized by Escherichia coli that contained cloned 1-O-beta-D-indol-3-ylacetyl-glucose complementary DNA (cDNA) was catalytically active. The predicted amino acid sequence of the cDNA was confirmed by amino-terminal sequencing of the purified enzyme. Homologous nucleotide sequences were found in all plants tested. The blockage or enhancement of iaglu expression may permit regulation of plant growth.

Site-specific mutagenesis of the nodule-infected cell expression (NICE) element and the AT-rich element ATRE-BS2* of the Sesbania rostrata leghemoglobin glb3 promoter.

Sesbania rostrata leghemoglobin glb3 (Srglb3) promoter sequences responsible for expression in infected cells of transgenic Lotus corniculatus nodules were delimited to a 78-bp Dral-Hinfl fragment. This region, which is located between coordinates -194 to -116 relative to the start codon of the Srglb3 gene, was named the nodule-infected cell expression (NICE) element. Insertion of the NICE element into the truncated nopaline synthase promoter was found to confer a nodule-specific expression pattern on this normally root-enhanced promoter. Within the NICE element, three distinct motifs ([A]AAAGAT, TTGTCTCTT, and CACCC[T]) were identified; they are highly conserved in the promoter regions of a variety of plant (leg)hemoglobin genes. The NICE element and the adjacent AT-rich element (ATRE-BS2*) were subjected to site-directed mutagenesis. The expression patterns of nine selected Srglb3 promoter fragments carrying mutations in ATRE-BS2* and 19 with mutations in the NICE element were examined. Mutations in ATRE-BS2* had varying effects on Srglb3 promoter activity, ranging from a two- to threefold reduction to a slight stimulation of activity. Mutations in the highly conserved (A)AAAGAT motif of the NICE element reduced Srglb3 promoter activity two- to fourfold, whereas mutations in the TCTT portion of the TTGTCTCTT motif virtually abolished promoter activity, demonstrating the essential nature of these motifs for Srglb3 gene expression. An A-to-T substitution in the CACCC(T) motif of the NICE element also abolished Srglb3 promoter activity, while a C-to-T mutation at position 4 resulted in a threefold reduction of promoter strength. The latter phenotypes resemble the effect of similar mutations in the conserved CACCC motif located in the promoter region of mammalian beta-globin genes. The possible analogies between these two systems will be discussed.

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.

Glutamine synthetase in Lupinus luteus. Identification and preliminary characterization of nodule-specific cDNA clone.

Two glutamine synthetase (GS) cDNA clones from L. luteus were identified and characterized. The nucleotide sequence analysis proved that they represent highly homologous but distinct mRNA species. Northern blot hybridization revealed that pc LINGS encodes the nodule-specific subunit of the GS while pcLIGS1 represents the nonspecific one present in nodule tissue as well as in uninfected roots.

Cloning and identification of lupin nodule specific cDNA sequences.

Yellow lupin nodule specific sequences were selected by screening of cDNA library prepared from lupin nodule poly(A)+RNA. From about 3,000 clones containing fragments of lupin DNA 150-1,500 base pair long, 7% of clones carrying nodule specific sequences were identified. Among them the most abundant sequence species, represented by 32% clones, encodes leghemoglobin. Another abundant species designated pLN13 is represented by 13% clones. The Northern blot analysis of lupin mRNA confirmed nodule specificity of the cloned sequences. The nucleotide sequence of one clone, pLN281 of 225 bp, is presented.