Characterization of the pea ENOD12B gene and expression analyses of the two ENOD12 genes in nodule, stem and flower tissue

Summary The ENOD12 gene family in pea consists of two different members. The cDNA clone, pPsENOD12, represents the PsENOD12A gene. The second ENOD12 gene, PsENOD12B, was selected from a genomic library using pPsENOD12 as a probe and this gene was sequenced and characterized. The coding regions of the two genes are strikingly similar. Both encode proteins having a signal peptide sequence and a region with pentapeptide units rich in prolines. ENOD12A has a series of rather conserved repeating pentapeptide units, whereas in ENOD12B the number of pentapeptide units is less and these are less conserved. From the amino acid sequence it is obvious that the PsENOD12 genes encode proline-rich proteins which are closely related to proteins that have been identified as components of soybean cell walls (SbPRPs). Previously, Northern blot analyses had shown that ENOD12 genes are expressed in a tissues-pecific manner. A high expression level is found in Rhizobium-infected roots and in nodules, whereas expression in flower and stem is lower. This raised the question of which gene is expressed where and when. The availability of the sequences of both ENOD12 genes allowed us to analyse the expression of the two genes separately. Specific oligonucleotides were used to copy the ENOD12 mRNAs and to amplify the cDNAs in a polymerase chain reaction. It was demonstrated that in all the tissues containing ENOD12 mRNA, both genes PsENOD12A and PsENOD12B are transcribed and that the relative amounts of PsENOD12A and PsENOD12B mRNA within each tissue are more or less equal. Moreover, the expression pattern during infection and nodule development is the same for the two genes. These results show that two closely related genes have the same tissue-specific expression pattern and that the gene that we have isolated is an actively transcribed gene. The 2.7 kb genomic region that contains the PsENODI2B gene has a 41 pb nearly direct repeat in the 5 flanking region of the gene (between -1447 and -1153) and another 14 by direct repeat 3' downstream (between 550 and 626). The region between the AGGA box and the TATA box has a striking homology with the same region in SbPRP genes.     

Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A

We report here that the pea early nodulin genes PsENOD5 and PsENOD12A are induced during the interaction of pea roots and the endomycorrhizal fungus Gigaspora margarita. Using the pea nodulation mutant Sparkle-R25, which is mutated in SYM8, it is shown that SYM8 is essential for the induction of PsENOD5 and PsENOD12Ain pea roots interacting either with Rhizobium or the endomycorrhizal fungus Gigaspora margarita. Our results suggest that mycorrhizal signals activate a signal transduction cascade sharing at least one common step with the Nod factor-activated signal transduction cascade.     

The pea early nodulin gene PsENOD7 maps in the region of linkage group I containing sym2 and leghaemoglobin

The early nodulin gene, PsENOD7, is expressed in pea root nodules induced by Rhizobium leguminosarum bv. viciae, but not in other plant organs. In situ hybridization showed that this gene is transcribed during nodule maturation in the infected cells of the proximal part of the prefixation zone II. At the transition of zone II into interzone II–III, the level of PsENOD7 mRNA drops markedly. PsENOD7 has no significant homology to other genes. RFLP mapping studies have shown that PsENOD7 is located in linkage group I between the leghaemoglobin genes and sym2.     

VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule formation on Vicia sativa roots

We isolated ENOD5, ENOD12 and ENOD40 homologues from Vicia sativa and studied their expression pattern during Rhizobium-induced nodule formation. Comparison of the VsENOD40 nucleotide sequence with the pea, soybean and alfalfa ENOD40 sequences showed that the sequences contain two conserved regions, called region I and region II. Comparison of all the potential open reading frames (ORFs) showed that all the five different ENOD40 clones encode a highly conserved small polypeptide of 12 or 13 amino acids encoded by an ORF located in region I. Furthermore we studied with in situ hybridization the expression pattern of VsENOD5, VsENOD12 and VsENOD40 during Rhizobium-induced nodule formation. Although the expression of these genes is largely similar to that of the pea counterparts, differences where found for the expression of VsENOD12 and VsENOD40 in Vicia. VsENOD12 is expressed in the whole prefixation zone II, whereas in pea ENOD12 is only expressed in the distal part of this zone. VsENOD40 is expressed in the uninfected cells of interzone II–III; while in pea ENOD40 is expressed in both the uninfected and infected cells of this zone.     

Lipo-oligosaccharides of Rhizobium induce infection-related early nodulin gene expression in pea root hairs

This paper shows that lipo-oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection-related early nodulin genes ( PsENOD5 and PsENOD12 ) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12 , but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host-specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.     

Male Gametic Cell-specific Histone <Emphasis Type="Italic">gH2A</Emphasis> Gene of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>: Genomic Structure and Promoter Activity in the Generative Cell

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5′ upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5′ upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.     

cDNA cloning and developmental expression of pea nodulin genes

A cDNA library prepared from pea nodule poly(A)⁺ RNA was screened by differential hybridization with cDNA probes synthesized from root and nodule RNA respectively. From the cDNA clones that hybridized exclusively with the nodule probe five clones, designated pPsNod 6, 10, 11, 13 and 14 and each containing unique sequences, were further characterized together with one leghemoglobin and one root-specific cDNA clone. In vitro translation of RNA selected by the pPsNod clones showed that the corresponding genes encode nodulins with molecular weights ranging from 5 800 to 19 000. During pea root nodule development expression of the five PsNod genes starts more or less concomitantly with the onset of nitrogen fixing activity in the nodules and the time course of appearance and accumulation of the nodulin mRNAs is similar to that of leghemoglobin mRNA. In ineffective pea root nodules expression of the PsNod genes is induced but the final accumulation levels of the mRNAs are markedly reduced to various degrees. The expression of another nodulin gene, designated ENOD2, was followed using a heterologous soybean cDNA clone as probe. In pea root nodules the ENOD2 gene is expressed at least five days before the PsNod and leghemoglobin genes, and in contrast to the PsNod mRNAs the concentration of the ENOD2 mRNA is the same in wild type and fix ^- nodules. The results described suggest that in root nodules several regulatory mechanisms exist which determine the final nodulin mRNA amounts accumulating in the root nodule.     

Expression of<Emphasis Type="Italic"> ENOD40</Emphasis> during tomato plant development

In legumes, ENOD40 expression is increased upon interaction of plants with rhizobia. Little is known of the expression pattern of ENOD40 during other stages of the plant life cycle. Studies of ENOD40 expression in non-legume development may give an indication of the function of the gene. To investigate the ENOD40 expression pattern during plant development, a fusion between the -glucuronidase (GUS) reporter gene and 150 bp of the 5 untranslated region plus 3,000 bp of 5 untranscribed tomato ENOD40 sequence was constructed and introduced into Lycopersicon esculentum Miller. Based on the observed GUS expression patterns in transgenic tomato we speculate that ENOD40 in tomato has a role in counteracting ethylene-provoked responses.Abbreviations GUS -glucuronidase - FISH fluorescence in situ hybridisation - RACE rapid amplification of cDNA ends - RFLC restriction fragment length polymorphism     

Accumulation of extracellular proteins bearing unique proline-rich motifs in intercellular spaces of the legume nodule parenchyma

Summary. Nodulins encoding repetitive proline-rich cell wall proteins (PRPs) are induced during early interactions with rhizobia, suggesting a massive restructuring of the plant extracellular matrix during infection and nodulation. However, the proteins corresponding to these gene products have not been isolated or characterized, nor have cell wall localizations been confirmed. Posttranslational modifications, conformation, and interactions with other wall polymers are difficult to predict on the basis of only the deduced amino acid sequence of PRPs. PsENOD2 is expressed in nodule parenchyma tissue during nodule organogenesis and encodes a protein with distinctive PRP motifs that are rich in glutamate and basic amino acids. A database search for the ENOD2 signature motifs indicates that similar proteins may have a limited phylogenetic distribution, as they are presently only known from legumes. To determine the ultrastructural location of the proteins, antibodies were raised against unique motifs from the predicted ENOD2 sequence. The antibodies recognized nodule-specific proteins in pea (Pisum sativum), with a major band detected at 110 kDa, representing a subset of PRPs from nodules. The protein was detected specifically in organelles of the secretory pathway and intercellular spaces in the nodule parenchyma, but it was not abundant in primary walls. Similar proteins with an analogous distribution were detected in soybean (Glycine max). The use of polyclonal antibodies raised against signature motifs of extracellular matrix proteins thus appears to be an effective strategy to identify and isolate specific structural proteins for functional analysis. Correspondence and reprints: Delaware Biotechnology Institute, Newark, DE 19711, U.S.A.     

Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.     

Identification of two alfalfa early nodulin genes with homology to members of the pea Enod12 gene family

In a search for plant genes expressed during early symbiotic interactions between Medicago sativa and Rhizobium meliloti, we have isolated and characterized two alfalfa genes which have strong sequence similarity to members of the Enod12 gene family of Pisum sativum. The M. sativa genes, MsEnod12A and B, encode putative protein products of 8066 Da and 12849 Da, respectively, each with a signal sequence at the N-terminus followed by a repetitive proline-rich region. Based on their expression during the initial period of nodule development, MsEnod12A and B are alfalfa early nodulin genes.     

Structure,expression, chromosomal location and product of the gene encoding ADH1 inPetunia

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.     

A New Retroposed Gene in <Emphasis Type="Italic">Drosophila</Emphasis> Heterochromatin Detected by Microarray-Based Comparative Genomic Hybridization

A genomic pattern of new gene origination is often dependent on a genomic method that can efficiently identify a statistically adequate number of recently originated genes. The heterochromatic regions have often been viewed as genomic deserts with low coding potential and thus a low flux of new genes. However, increasing reports revealed unexpected roles of heterochromatic regions in the evolution of genes and genomes. We identified recently retroposed genes that originated in heterochromatic regions in Drosophila, by developing microarray-based comparative genomic hybridization (CGH) with multiple species. This new gene family, named Ifc-2h, originated in the common ancestor of the clade of D. simulans, D. mauritiana, and D. sechellia. The sequence features and phylogenetic distribution indicated that Ifc-2h resulted from the retroposition from its parental gene, Infertile crescent (Ifc), and integrated into heterochromatic region of common ancester of the three sibling species 2 million years ago. Expression analysis revealed that Ifc-2h had developed a new expression pattern by recruiting a putative regulatory element from its target sequence. The distribution of indel variation in Ifc-2h of D. simulans and D. mauritiana revealed a significant sequence constraint, suggesting that the Ifc-2h gene may be functional. These analyses cast fresh insight into the evolution of heterochromatin and the origin of its coding regions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.