Glutathione synthesis is regulated by nitric oxide in <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

Glutathione (GSH) is one of the main antioxidants in plants. Legumes have the specificity to produce a GSH homolog, homoglutathione (hGSH). We have investigated the regulation of GSH and hGSH synthesis by nitric oxide (NO) in Medicago truncatula roots. Analysis of the expression level of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) after treatment with sodium nitroprusside (SNP) and nitrosoglutathione (GSNO), two NO-donors, showed that γ-ecs and gshs genes are up regulated by NO treatment whereas hgshs expression is not. Differential accumulation of GSH was correlated to gene expression in SNP-treated roots. Our results provide the first evidence that GSH synthesis pathway is regulated by NO in plants and that there is a differential regulation between gshs and hgshs in M. truncatula. G. Innocenti and C. Pucciariello have contributed equally to the work.     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

The distributional changes and role of microtubules in Nod factor-challenged<Emphasis Type="Italic"> Medicago sativa</Emphasis> root hairs

The normal tip-growing pattern exhibited by root hairs of legumes is disrupted when the hair is exposed to Nod factors generated by compatible bacteria capable of inducing nodule formation. Since microtubules (MTs) play an important role in regulating directionality and stability of apical growth in root hairs [T.N. Bibikova et al. (1999) Plant J 17:657–665], we examined the possibility that Nod factors might affect the MT distribution patterns in root hairs of Medicago sativa L. We observed that Nod factor application caused rapid changes in the pattern of MTs starting as early as 3 min after perfusion. Within 3 to 10 min after Nod factor application, first endoplasmic and then cortical MTs depolymerised, initially at the proximal ends of cells. Twenty minutes after exposure to Nod factors, a transverse band of microtubules was seen behind the tip, while almost all other MTs had depolymerised. By 30 min, very few MTs remained in the root hair and yet by 1 h the MT cytoskeleton re-formed. When Nod factors were applied in the presence of 10 M oryzalin or 5 M taxol, the MTs appeared disintegrated while the morphological effects, such as bulging and branching, became enhanced. Compared to the treatments with oryzalin or taxol alone, the combinatory treatments exhibited higher growth rates. Since microtubule reorganization is one of the earliest measurable events following Nod factor application we conclude that microtubules have an important role in the early phases of the signalling cascade. Microtubule involvement could be direct or a consequence of Nod factor-induced changes in ion levels.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-003-1097-1Abbreviations BNM buffered nodulation medium - CLSM confocal laser scanning microscopy - MT microtubule     

Structural and transcriptional analysis of<Emphasis Type="Italic"> S</Emphasis>-locus F-box genes in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S₂, very similar to S₂-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S₂ also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S₁S₅ and S₂S₄, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S₂-specific primers, TAC clones containing both AhSLF-S₂ and its homologs were subsequently identified (S₂TAC, S₅TACa, S₄TAC, and S₁TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S₂, -S₁, -S₄ and -S₅) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S₂ were identified. In total, three F-box genes (AhSLF-S₂, -S₂A and -S₂C) in S₂TAC (51 kb), three (AhSLF-S₄, -S₄A and -S₄D) in S₄TAC (75 kb), two (AhSLF-S₅ and -S₅A) in S₅TACa (55 kb), and two (AhSLF-S₁ and -S₁E) in S₁TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S₂C, -S₄D and -S₁E) were specifically expressed in pollen, similar to AhSLF-S₂, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535     

<Emphasis Type="Italic">Medicago truncatula</Emphasis> syntaxin SYP132 defines the symbiosome membrane and infection droplet membrane in root nodules

Symbiotic association of legume plants with rhizobia bacteria culminates in organogenesis of nitrogen-fixing root nodules. In indeterminate nodules, plant cells accommodate rhizobial infection by enclosing each bacterium in a membrane-bound, organelle-like compartment called the symbiosome. Numerous symbiosomes occupy each nodule cell; therefore an enormous amount of membrane material must be delivered to the symbiosome membrane for its development and maintenance. Protein delivery to the symbiosome is thought to rely on the plant secretory system; however, the targeting mechanisms are not well understood. In this study, we report the first in-depth analysis of a syntaxin localized on symbiosome membranes. Syntaxins help define a biochemical identity to each compartment in the plant secretory system and facilitate vesicle docking and fusion. Here, we present biochemical and cytological evidence that the SNARE MtSYP132, a Medicago truncatula homologue of Arabidopsis thaliana Syntaxin of Plants 132, localizes to the symbiosome membrane. Using a specific anti-MtSYP132 peptide antibody, we also show that MtSYP132 localizes to the plasma membrane surrounding infection threads and is most abundant on the infection droplet membrane. These results indicate that MtSYP132 may function in infection thread development or growth and the early stages of symbiosome formation. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Proteomic Profiling Unravels Insights into the Molecular Background Underlying Increased <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>-Tolerance of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity. Requests concerning seeds from the Medicago truncatula lines F83.005-5 and F83.005-9 should be addressed to Jean-Marie Prospéri, INRA-SGAP Laboratory, Laboratoire de Ressources Génétiques et d’Amélioration des Luzernes méditerranéennes, Mauguio, France, jean-marie.prosperi@ensam.inra.fr.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

The<Emphasis Type="Italic"> GAOLAOZHUANGREN2</Emphasis> gene is required for normal glucose response and development of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Recent studies of glucose (Glc) sensing and signaling have revealed that Glc acts as a critical signaling molecule in higher plants. Several Glc sensing-defective Arabidopsis mutants have been characterized in detail, and the corresponding genes encoding Glc-signaling proteins have been isolated. However, the full complexity of Glc signaling in higher plants is not yet fully understood. Here, we report the identification and characterization of a new Glc-insensitive mutant, gaolaozhuangren2 (glz2), which was isolated from transposon mutagenesis experiments in Arabidopsis. In addition to its insensitivity to Glc, the glz2 plant exhibits several developmental defects such as short stature with reduced apical dominance, short roots, small and dark-green leaves, late flowering and female sterility. Treatment with 4% Glc blocked expression of the OE33 gene in wild-type plants, whereas expression of this gene was unchanged in the glz2 mutant plants. Taken together, our results suggest that the GLZ2 gene is required for normal glucose response and development of Arabidopsis.Mingjie Chen and Xiaoxiang Xia contributed equally to this work.     

Efficient transformation of<Emphasis Type="Italic"> Medicago truncatula</Emphasis> cv. Jemalong using the hypervirulent<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain AGL1

The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter-gus/gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4–5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T₁ descendents.Communicated by R.J. Rose     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

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     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Thermostable malate synthase of<Emphasis Type="Italic"> Streptomyces thermovulgaris</Emphasis>

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40–60 °C) than those of scMS (28–37 °C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9–91.4% amino acid identities, with stMS possessing slightly more charged residues (~31%) than its mesophilic counterparts (~28–29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.