François Bernier

Regulation by biotic and abiotic stress of a wheat germin gene encoding oxalate oxidase, a H2O2-producing enzyme.

Germins and germin-like proteins (GLPs) constitute a ubiquitous family of plant proteins that seem to be involved in many developmental and stress-related processes. Wheat germin has been extensively studied at the biochemical level: it is found in the apoplast and the cytoplasm of germinating embryo cells and it has oxalate oxidase activity (EC 1.2.3.4). Germin synthesis can also be induced in adult wheat leaves by auxins and by a fungal pathogen but it remains to be determined whether the same gene is involved in developmental, hormonal and stress response. In this work, we have studied the expression of one of the wheat germin genes, named gf-2.8, in wheat as well as in transgenic tobacco plants transformed with either this intact gene or constructs with GUS driven by its promoter. This has allowed us to demonstrate that expression of this single gene is both developmentally and pathogen- regulated. In addition, we show that expression of the wheat gf-2.8 germin gene is also stimulated by some abiotic stresses, especially the heavy metal ions Cd2+, Cu2+ and Co2+. Several chemicals involved in stress signal transduction pathways were also tested: only polyamines were shown to stimulate expression of this gene. Because regulation of the wheat gf-2.8 germin gene is complex and because its product results in developmental and stress-related release of hydrogen peroxide in the apoplast, it is likely that it plays an important role in several aspects of plant growth and defence mechanisms.

REGULATED EXPRESSION OF A WHEAT GERMIN GENE IN TOBACCO : OXALATE OXIDASE ACTIVITY AND APOPLASTIC LOCALIZATION OF THE HETEROLOGOUS PROTEIN

Wheat (Triticum aestivum) germin is a homopentameric glycoprotein whose synthesis is allied with seed germination. Germin pentamers show an unusual resistance to dissociation and possess an oxalate oxidase (OxO) activity. In order to increase our knowledge of germin gene expression, the function(s) of germin during development and possible uses in plant genetic engineering, an in vivo expression system is required. To this end, a gene for germin, named gf-2.8, was studied by expressing either promoter-GUS fusions or the intact gene in transgenic tobacco (Nicotiana tabacum) plants. Heterologous gene transcription was monitored in vitro and in vivo by GUS or OxO activity and was found to occur in developing seeds and in seedlings. This transcription was stimulated by auxins, as would be expected because of the presence of putative auxin-responsive elements in the promoter of the gf-2.8 gene. Auxin stimulation also extended to young leaves since OxO activity could be detected in treated but not in untreated leaves. The biochemical characteristics of wheat germin were also conserved in a transgenic host: the OxO activity was present under the form of a doublet co-migrating with germin G and G′ isoforms. Also, germin distributed between a soluble and an apoplastic fractions despite the fact that wheat cell wall substantially differs from tobacco cell wall. Therefore, tobacco constitutes a suitable host for in vivo studies of this monocotyledon gene.

Arabidopsis thaliana germin-like proteins: common and specific features point to a variety of functions.

Abstract. Germin-like proteins (GLPs) are ubiquitous plant proteins encoded by diverse multigene families. It is not known whether they share germins unusual biochemical properties and oxalate oxidase activity. Using specific antibodies, we have studied three GLPs (AtGER1, AtGER2 and AtGER3) in Arabidopsis thaliana (L.) Heynh. as well as in transgenic tobacco (Nicotiana tabacum L.) plants overexpressing these proteins. Like wheat (Triticum aestivum L.) germin, these Arabidopsis GLPs are associated with the extracellular matrix (ECM) and they also seem to exist as two glycosylated isoforms. However, none of them is an oxalate oxidase. Although GLPs display several conserved features, each has its specific characteristics. Both AtGER2 and AtGER3 are oligomeric proteins that share germins resistance to pepsin and to dissociation by heat and SDS. In contrast, AtGER1 seems to exist as a monomer. The GLPs may interact with the ECM in a variety of ways, since each is efficiently extracted by different conditions. In addition, germins and GLPs all bind Cibacron Blue, a dye often but not exclusively used for the purification of enzymes having nucleotide cofactors. In the case of AtGER2, binding to the dye is so tight that it almost allows a one-step purification of this protein. The variety of sequences, expression patterns and biochemical features indicates that GLPs could be a class of receptors localized in the ECM and involved in physiological and developmental processes as well as stress response.

The legumin boxes and the 3' part of a soybean β-conglycinin promoter are involved in seed gene expression in transgenic tobacco plants

Abstractβ-conglycinin is one of the major seed storage proteins in soybean. It is composed of three subunits, namely α, α′ and β. The expression of β-conglycinin is highly regulated, being restricted to the embryo during the mid-maturation phase of embryogeny. Two series of constructs were made with the α′ subunit promoter and the GUS reporter gene to investigate the cis-acting elements involved in the regulated expression of this promoter. The activity of each construct was tested in transgenic tobacco plants.In the first series of constructs, we checked if the ‘legumin box’, a sequence found in most legume seed storage protein genes as well as in other seed-specific genes, is involved in the regulated expression of the α′ subunit of the β-conglycinin gene in tobacco. To this end, both copies of the α′ subunit promoter legumin boxes were mutagenized in vitro. The transcriptional activity of the single mutants and the double mutant were compared with that of the wild-type promoter. Our results show that the legumin boxes act together to increase transcription of the β-conglycinin α′ subunit gene by about a factor of ten. This is the first demonstration of a function for the legumin box in transcriptional regulation.In the second series of experiments, we wished to determine if the 3′ part of the promoter (the CCAAT and TATAA region) contains important regulatory elements. We found that this small fragment (−82 to +13 bp) can confer by itself a low level of seed-specific gene expression. Chimaeric promoters constructed from parts of the α′ subunit promoter and of the constitutive CaMV 35S promoter were also analysed. These constructs also revealed the importance of the CCAAT and TATAA region of the α′ subunit promoter in seed-specific gene expression.

cDNA sequence, genomic organization and differential expression of three Arabidopsis genes for germin/oxalate oxidase-like proteins

Wheat germin is a protein expressed during germination which possesses an oxalate oxidase activity. Germin-type oxalate oxidases have been extensively studied in monocotyledons (wheat and barley) where they are thought to have important functions for development, stress response and defence against pathogens. In contrast, almost nothing is known about the germin-like proteins found in dicotyledons, gymnosperms and myxomycetes. In this work, cDNA clones for three genes (ATGER1, ATGER2 and ATGER3) encoding germin-like proteins, initially characterized as expressed sequence tags (ESTs), from Arabidopsis thaliana cDNA libraries were further characterized. In addition, we isolated and sequenced a Brassica napus cDNA which was strongly homologous to the cDNA for ATGER1. Sequence analysis and secondary structure predictions of the proteins encoded by these cDNAs showed that they possess all the characteristic features of members of the germin family and of the germin/seed globulins/sucrose binding protein superfamily. Sequence comparisons and mapping demonstrated the existence of at least two different gene families in the A. thaliana genome encoding a minimum of three genes for germins. These three genes have been mapped in three different location on the Arabidopsis genome. By northern blot hybridizations we found that these genes are differentially regulated. ATGER1 was expressed during germination, like wheat germin, but also in leaves whereas ATGER2 transcripts were exclusively found in developing embryos, like wheat pseudo-germin. ATGER3 mRNAs were found in leaves and flowers and their abundance was shown to vary during the circadian cycle.

The DING family of proteins: ubiquitous in eukaryotes, but where are the genes?

PstS and DING proteins are members of a superfamily of secreted, high‐affinity phosphate‐binding proteins. Whereas microbial PstS have a well‐defined role in phosphate ABC transporters, the physiological function of DING proteins, named after their DINGGG N termini, still needs to be determined. PstS and DING proteins co‐exist in some Pseudomonas strains, to which they confer a highly adhesive and virulent phenotype. More than 30 DING proteins have now been purified, mostly from eukaryotes. They are often associated with infections or with dysregulation of cell proliferation. Consequently, eukaryotic DING proteins could also be involved in cell–cell communication or adherence. The ubiquitous presence in eukaryotes of proteins structurally and functionally related to bacterial virulence factors is intriguing, as is the absence of eukaryotic genes encoding DING proteins in databases. DING proteins in eukaryotes could originate from unidentified commensal or symbiotic bacteria and could contribute to essential functions. Alternatively, DING proteins could be encoded by eukaryotic genes sharing special features that prevent their cloning. Both hypotheses are discussed.

Digital 3D imaging system for rapid response on remote sites

A compact digital 3D imaging system based on laser triangulation was designed for applications requiring a rapid response on remote sites. Heritage, forensic and industrial applications are among the best fields to benefit from this new technology. This paper focuses on such aspects as the acquisition, calibration, verification, and model creation. These aspects were all optimized to create a versatile system that is compact, i.e. hand portable to a remote site. Emphasis is placed on accuracy verification and monitoring which are critical factors for obtaining high-quality reconstruction of 3D models from multiple range images. A summary of the experimental results acquired in 1997 and 1998 at a number of sites in Italy is presented in this paper.

Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum

A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.

Interaction-centric modelling for interactive virtual worlds: the APIA approach

Conceptual modelling studies the different abstraction methods of the real world. The conception and the execution of virtual worlds depend strongly on the type of conceptual models. Existing modelling methods such as object-oriented modelling are not appropriate when the main concern is the dynamic reusability and interoperability. Such a reusability and interoperability must be free of any human intervention. This paper presents a new paradigm named interaction-centric modelling (ICM) that increases reusability and interoperability of virtual entities and behaviours.

cDNA Cloning Of Physarum polycephalum Stage-Specific mRNAs

A useful approach to the study of differentiation consists of isolating and characterizing the genes specifically expressed in the various stages and then studying their regulation. This chapter describes the methodology used in our laboratory to clone sequences from stage-specific mRNAs of Physarum polycephalum. It involved the preparation of intact poly (A)+ RNA and the construction of cDNA libraries for four developmental stages (amoebae, plasmodia, spores, and spherules). These libraries were then screened by differential hybridization, and the specificity of the positive clones was confirmed by Northern blot hybridization. The quality of the results obtained at each step was controlled, and the methodology was found to be of general use in the identification of Physarum stage-specific mRNAs.