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UMR DAP - Equipe DGB; International audience; Plant non-specific lipid transfer proteins (nsLTPs) are encoded by a multigene family and support physiological functions, which remain unclear. We adapted an efficient ligation-mediated polymerase chain reaction (LM-PCR) procedure that enabled isolation of 22 novel Triticum aestivum nsLtp (TaLtp) genes encoding types 1 and 2 nsLTPs. A phylogenetic tree clustered the wheat nsLTPs into ten subfamilies comprising 1-7 members. We also studied the activity of four type 1 and two type 2 TaLtp gene promoters in transgenic rice using the beta-Glucuronidase reporter gene. The activities of the six promoters displayed both overlapping and distinct features in rice. In vegetative organs, these promoters were active in leaves and root vascular tissues while no beta-Glucuronidase (GUS) activity was detected in stems. In flowers, the GUS activity driven by the TaLtp7.2a, TaLtp9.1a, TaLtp9.2d, and TaLtp9.3e gene promoters was associated with vascular tissues in glumes and in the extremities of anther filaments whereas only the TaLtp9.4a gene promoter was active in anther epidermal cells. In developing grains, GUS activity and GUS immunolocalization data evidenced complex patterns of activity of the TaLtp7.1a, TaLtp9.2d, and TaLtp9.4a gene promoters in embryo scutellum and in the grain epicarp cell layer. In contrast, GUS activity driven by TaLtp7.2a, TaLtp9.1a, and TaLtp9.3e promoters was restricted to the vascular bundle of the embryo scutellum. This diversity of TaLtp gene promoter activity supports the hypothesis that the encoded TaLTPs possess distinct functions in planta

Equipe DGB - ex-UMR PIA; International audience; Puroindolines form the molecular basis of wheat grain hardness. However, little is known about puroindoline gene regulation. We previously reported that the Triticum aestivum puroindoline-b gene (PinB) promoter directs beta-glucuronidase gene (uidA) seed-specific expression in transgenic rice. In this study, we isolated a puroindoline-a gene (PinA), analyzed PinA promoter activity by 5' deletions and compared PinA and PinB promoters in transgenic rice. Seeds of PinA-1214 and PinB-1063 transgenic plants strongly expressed uidA in endosperm, in the aleurone layer and in epidermis cells in a developmentally regulated manner. The GUS activity was also observed in PinA-1214 embryos. Whereas the PinB promoter is seed specific, the PinA promoter also directed, but to a lower level, uidA expression in roots of seedlings and in the vascular tissues of palea and pollen grains of dehiscent anthers during flower development. In addition, the PinA promoter was induced by wounding and by Magnaporthe grisea. By deletion analysis, we showed that the "390-bp" PinA promoter drives the same expression pattern as the "1214-bp" promoter. Moreover, the "214-bp" PinA promoter drives uidA expression solely in pollen grains of dehiscent anthers. The presence of putative cis-regulatory elements that may be related to PinA expression is discussed from an evolutionary point of view. By electrophoretic mobility shift assay, we showed that putative cis-elements (WUN-box, TCA motifs and as-1-like binding sites) whose presence in the PinA promoter may be related to wounding and/or the pathogen response form complexes with nuclear extracts isolated from wounded wheat leaves

Common wheat adulteration of durum wheat pasta was quantified using real-time duplex polymerase chain reaction (PCR). The total DNA content of pasta was determined by amplifying part of a wheat gene encoding a lipid transfer protein, and common wheat DNA was quantified by amplifying part of the puroindoline-b gene. Under the conditions defined by this study, for pasta with a theoretical adulteration of 3%, the experimentally determined mean value was 2.6–3.4%, depending on drying temperature. Pure durum wheat pastas were distinguished from adulterated pastas without ambiguity. This study demonstrates the feasibility of using real-time duplex PCR to quantify common wheat adulteration of pasta dried at high temperature, quantification that was impossible with the French official peroxidase-marker method.

Using a PCR approach, we showed that puroindoline genes are present in diploid and hexaploid Triticum species but absent in tetraploid species, and that T. tauschii is likely to be the donor of the T. aestivum puroindoline genes. Puroindoline-like genes are present in cereals closely related to wheat (barley, oat and rye) and absent in cereals more distantly related to wheat (maize, rice and sorghum). Barley, oat and rye puroindoline-like sequences and primary structure of deduced proteins are highly conserved. In oat, avenoindolines are more closely related to puroindolines than to tryptophanins.

A genomic DNA fragment containing the 5′-upstream sequence and part of the open reading frame corresponding to Triticum aestivum puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to −1068, −388, −210 or −124 upstream of the translation initiation ATG codon and the sequence coding for the first 13 amino acids of the puroindoline-b, were translationally fused to the uidA reporter gene encoding β-glucuronidase and transferred to rice calli via particle bombardment-mediated transformation. The 1068 bp and 124 bp promoters were also transcriptionally fused to the uidA reporter gene. Out of the 196 plants regenerated from transformed rice calli, 118 plants set seeds. No GUS activity was detectable in the stems, roots, leaves or pollen of the transgenic rice which had integrated the puroindoline-b promoter or its deletions; GUS activity was detected only in seeds, except in those having integrated the 124 bp promoter. Within seeds, histological localisation showed GUS activity as being restricted to the endosperm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5′ promoter deletions identified the region between −388 and −210 as essential for endosperm expression, and the region between −210 and −124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the translational and transcriptional fusion genes.

Two cDNA clones, pTaM13.38 and pTd14.13.2, encoding a Triticum aestivum and a Triticum durum thioredoxin h, respectively, were isolated from mid-maturation seed cDNA libraries. The T. aestivum thioredoxin h has a molecular mass of 13.5 kDa and that from T. durum has a molecular mass of 13.8 kDa. These two wheat thioredoxin h are 98.5 % similar and contain the canonical WCGPC active site and the important structural and functional amino acids that are conserved in thioredoxin sequences. The recombinant T. aestivum thioredoxin h (TrxTa) overproduced in BL21(DE3)pLysS was purified to homogeneity by a three-step procedure including heat treatment, anion-exchange chromatography and gel filtration. TrxTa showed a lower stability to high temperature than Escherichia coli thioredoxin or plant thioredoxin m. The molecular mass of TrxTa, determined by mass spectrometry, is 13 391 Da and corresponds to a protein lacking the first methionine residue, as confirmed by its N-terminal end sequence AASAAT. Using the 5,5′-dithiobis(2-nitrobenzoic acid)-reduction assay and monobromobimane revelation we showed that TrxTa is specifically reduced by wheat NADP : thioredoxin reductase (NTR), and not by E. coli NTR. TrxTa is able to reduce identified target proteins i.e. wheat seed α-amylase inhibitors (chloroform/methanol-soluble proteins). The presence of a putative transmembrane domain at the N-terminal end of the two wheat thioredoxins raises the question of whether these proteins are membrane anchored.

UMR DIAPC, UMR DAP équipe DGB; We study here the evolution of genes located in the same physical locus using the recently sequenced Ha locus in seven wheat genomes in diploid, tetraploid, and hexaploid species and compared them with barley and rice orthologous regions. We investigated both the conservation of microcolinearity and the molecular evolution of genes, including coding and noncoding sequences. Microcolinearity is restricted to two groups of genes (Unknown gene-2, VAMP, BGGP, Gsp-1, and Unknown gene-8 surrounded by several copies of ATPase), almost conserved in rice and barley, but in a different relative position. Highly conserved genes between wheat and rice run along with genes harboring different copy numbers and highly variable sequences between close wheat genomes. The coding sequence evolution appeared to be submitted to heterogeneous selective pressure and intronic sequences analysis revealed that the molecular clock hypothesis is violated in most cases

UMR DAP équipe DGB; International audience; Puroindolines are two small, basic cysteine-rich proteins isolated from Triticum aestivum seeds and characterized by a tryptophan-rich domain. They form the molecular basis of wheat grain hardness and display antimicrobial activity that may contribute to plant defence. Their antimicrobial activity is presumed to be due to their hydrophobic tryptophan-rich domain. However, little is known about their mode of action and there is no in vivo evidence that the binding of puroindolines to membranes is mediated by their tryptophan-rich domain. In this study, using a yeast complementation assay, we showed that puroindolines interact with the Saccharomyces cerevisiae plasma membrane. By site-directed mutagenesis of their tryptophan-rich domain, we determined that two tryptophan residues (W41 and W44) are mandatory for interaction of puroindoline-a with the yeast membrane whereas interaction of puroindoline-b depends on lysine residues. These results highlight that other residues than tryptophan play a critical role in the interaction of puroindolines with membranes, and probably their affinity for lipids and antimicrobial activities

A Triticum durum cDNA library prepared from developing endosperm (22 days after flowering (DAF] was screened using synthetic oligonucleotide probes covering part of the CM3 and CM16 N-terminal protein sequences. A full-length cDNA clone (pTd78) encoding the CM16 protein (chloroform/methanol-soluble protein) was isolated and characterized. To our knowledge this is the first characterization of a clone coding for a wheat CM protein. The CM16 protein is synthesized as a preprotein with a signal peptide of 24 residues, the molecular weight of the mature protein being 13,438 Da. As other members of the cereal trypsin/alpha-amylase inhibitor family, the CM16 protein contains 10 cysteine residues, their position being well conserved. In developing endosperm the highest level of CM16 mRNA was detected at mid-maturation.

Equipe DGB, ex-UMR PIA; International audience; Chestnut and chestnut-derived products are quite expensive and thus a possible target for fraudulent labeling. To obtain a French Label of Origin, chestnut producers need to certify the purity of their products. Chestnut-derived products are consumed by people who may suffer from celiac disease or are allergic to cereals. For these reasons, we developed a qualitative PCR-based method to detect cereal and leguminous species in chestnut flour. The presence of common wheat and barley was determined by amplifying part of the puroindoline-a gene, the presence of rye by amplifying part of the secaloindoline-a gene, the presence of durum wheat, rice, maize and chickpea by amplifying part of lipid transfer protein genes, the presence of oat by amplifying part of a thionin gene, the presence of kidney bean by amplifying part of a late embryogenesis- abundant protein, the presence of soybean by amplifying part of a lectin gene and the presence of fava bean by amplifying part of a nodulin gene. A chestnut thaumatine gene was used as control. PCR conditions were optimized to detect 1% of adulteration. Duplex PCR and specially designed sets of primers that allow amplification of several related species limited the number and cost of analyses required. Interpretation of the results is facilitated by using a decision tree

61 ref.; International audience; Gene expression profiles of group 2 (dehydrins) and group 4 Late embryogenesis abundant (Lea) genes in developing seeds of Triticum durum and T. aestivum and in coleoptiles and coleorhizae of T. durum seedlings were monitored by real-time quantitative RT-PCR. The five genes exhibited clear differences in their accumulation pattern in wheat seed and in response to dehydration, low temperature, salinity and ABA. Td29b, Td16 and Td27e gene transcripts accumulate late in embryogenesis as expected for Lea genes, Td11 gene transcripts were present throughout seed development whereas no Td25a gene transcripts were detected in seeds. Drastic changes in the relative levels of Td29b, Td16, Td27e and Td11 transcripts occurred at the shift between the cell expansion and desiccation phases. All genes except the Td11 gene are more highly induced by dehydration in coleorhizae than in coleoptiles. In contrast, response to low temperature, salinity or ABA is higher in coleoptiles than in coleorhizae. Depending on both the gene and on the type of stress, a wide range of induction levels (8- to 100,000-fold) was observed.

Nine cDNA clones encoding non-specific lipid transfer proteins (nsLTPs) were isolated from Triticum aestivum and Triticum durum cDNA libraries and characterized. One cDNA is predicted to encode a type 2 nsLTP (7 kDa) while others encode type 1 nsLTPs (9 kDa). All encoded proteins contain an N-terminal signal sequence and possess the characteristic features of nsLTPs. The genomic structures of the wheat nsLtp genes show that type 2 TaLtp7.1a, TaLtp7.2a and type 1 TaLtp9.2b genes lack introns while the other type 1 genes consist of one intron. Construction of a phylogenic tree of Poaceae nsLTPs shows that wheat nsLTPs can be divided into eleven distinct groups and are closely related to barley sequences. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression patterns of nine nsLtp genes were studied during wheat seed development and germination. We identified three different profiles of nsLtp gene transcript accumulation. Whereas TdLtp7.1a, TdLtp9.4a and TdLtp9.7a transcripts were detected during all maturation stages, TdLtp7.2a, TdLtp9.2a, TdLtp9.3a, TdLtp9.5a and TdLtp9.6a transcripts were only present in the first and TdLtp9.1a in the last stages of seed development. Moreover, these nine wheat nsLtp genes are not seed-specific and are also expressed in the coleoptile of young seedlings. The present study revealed the complexity of the wheat nsLtp gene family and showed that the expression of nsLtp genes is developmentally regulated in the seeds, suggesting a specific function for each of the corresponding proteins.

In order to develop QTL applications, eight new loci were mapped on barley chromosome 7 using 124 doubled haploid lines of the North American Barley Genome Mapping Project (NABGMP) progeny ('Steptoe' x 'Morex'). These loci involve six genomic DNA restriction fragment length polymorphisms (RFLPs) and two cDNA-RFLPs including a puroindoline gene. The distribution of these markers on barley chromosome 7 was compared with that of homoeologous wheat counterparts, i.e. wheat group 5. One locus on chromosome 7 was associated with a QTL for β-glucanase activity measured in green and finished barley malt.

In hexaploid bread wheat ( Triticum aestivum L. em. Thell), ten members of the IWMMN ( International Wheat Microsatellites Mapping Network) collaborated in extending the microsatellite (SSR = simple sequence repeat) genetic map. Among a much larger number of microsatellite primer pairs developed as a part of the WMC ( Wheat Microsatellite Consortium), 58 out of 176 primer pairs tested were found to be polymorphic between the parents of the ITMI ( International Triticeae Mapping Initiative) mapping population W7984 x Opata 85 (ITMI pop). This population was used earlier for the construction of RFLP ( Restriction Fragment Length Polymorphism) maps in bread wheat (ITMI map). Using the ITMI pop and a framework map (having 266 anchor markers) prepared for this purpose, a total of 66 microsatellite loci were mapped, which were distributed on 20 of the 21 chromosomes (no marker on chromosome 6D). These 66 mapped microsatellite (SSR) loci add to the existing 384 microsatellite loci earlier mapped in bread wheat.

A type 2 non-specific lipid transfer protein (ns-LTP2) was isolated from wheat ( Triticum durum Desf.) endosperm and the primary structure of the protein was fully determined. The protein is basic with a predicted p I of 8.4 and a molecular mass of 6980 Da. It contains 67 amino acids including eight cysteine residues. Oligonucleotide probes covering part of the protein sequence were designed to isolate the corresponding cDNA that contains an open reading frame of 96 amino acids, including a 29 amino acid signal peptide. The T. durum ns-LTP2 showed 37.3–77.6% identity with other ns-LTP2 and is most closely related to the barley and rice sequences. PCR carried out on wheat genomic DNA indicated that the gene encoding this ns-LTP2 does not contain introns. Within developing seeds, the level of ltp7.1 gene transcripts detected by RT-PCR remained broadly similar from the cellular division phase to dry seed. This contrast with the expression pattern of the ltp9.1 gene, the transcripts of which started to accumulate during the filling phase. During germination, the level of ltp7.1 gene transcripts decreased more rapidly than those of the ltp9.1 gene.

Bread-making quality in wheat (Triticum aestivum) is a complex trait influenced at once by genetic factors and environmental conditions. The development of molecular biology technics together with intervarietal genetic maps provide a useful tool for the understanding of complex agronomic traits such as quality. The aim of our study was to identify QTL for bread-making quality using a doubled-haploid lines population issued from a cross between Courtot and Chinese Spring. Three hundred and eighty loci were mapped using a subset of 106 lines and 187 lines were genotyped for the anchor markers of this map. Between 144 and 172 DH lines were grown under field conditions for three consecutive years (1994 and 1995: one replication; 1996 two replications) and between 106 and 163 were submitted to several technological tests. We were thus able to approach the genetic control of quality traits (strength (W), grain hardness and protein content) that were highly contrasted between the two parents. Despite the fact that the D genome was under represented because of a lack of polymorphism, it appeared that an important part of the variation was explained by the QTL located at different loci than those of the storage proteins (glutenins and gliadins), which seemed to play a minor role compared to other regions of the genome. We are now planning to study some of these regions to identify the genes that could be involved in the baking process.

International audience; Swards of cocksfoot (cvs KM2, Lutetia) and perennial ryegrass (cvs Aurora, Vigor) were grown under full irrigation or severe (80 d) drought in a field experiment in the South of France. Responses of the bases of immature leaves plus enclosed tissues were made during the drought period and after rewatering. By the end of the drought, water content had fallen from 3.0 to 0.8 g(water)g(dm)(-1), and osmotic potential from -1.0 to -4.5 MPa in all cvs. Measured minerals and water-soluble carbohydrates contributed, respectively, c 19 and 44% to osmotic potential in droughted leaf bases. The drought-sensitive cocksfoot cv. Lutetia was characterized by a large proportion of fructans having a low degree of polymerization (DP = 3, 4). As drought progressed, accumulation of dehydrin transcripts and ABA were higher in leaf bases of the sensitive cv. Lutetia than in the resistant cv. KM2. After rewatering, the water status of immature leaf bases returned to control levels in 1-2 d, and then increased further as leaves began to grow and new tissue was produced. High-DP-fructans remained unchanged in leaf bases of 'Lutetia' but were depleted by over 55%, and therefore remobilized, in leaf bases of other cvs after 8 d. It is concluded that enclosed immature leaf bases survive drought by tolerating a low water status and that changes conventionally associated with desiccation tolerance are expressed most strongly in susceptible plants least able to maintain their water supply.

Triticum aestivum thioredoxin h overproduced in Escherichia coli (TrxTa) was purified to homogeneity. TrxTa showed a lower stability than other thioredoxins but was active and exhibited the same reactivity as wheat seed isolated thioredoxin h. Nuclear magnetic resonance and modeling studies revealed a canonical thioredoxin fold.

A molecular-marker linkage map of wheat (Triticum aestivum L. em. Thell) provides a powerful tool for identifying genomic regions influencing breadmaking quality. A variance analysis for kernel hardness was conducted using 114 recombinant inbred lines (F7) from a cross between a synthetic and a cultivated wheat. The major gene involved in kernel hardness, ha (hard), known to be on chromosome arm 5DS, was found to be closely linked with the locus Xmta9 corresponding to the gene of puroindoline-a. This locus explained around 63% of the phenotypic variability but there was no evidence that puroindoline-a is the product of Ha (soft). Four additional regions located on chromosomes 2A, 2D, 5B, and 6D were shown to have single-factor effects on hardness, while three others situated on chromosomes 5A, 6D and 7A had interaction effects. Positive alleles were contributed by both parents. A three-marker model explains about 75% of the variation for this trait.

From a cDNA library prepared from roots of Triticum durum water-stressed seedlings, we have characterized four dehydrin clones. Two clones, pTd27e and pTd16, code for proteins with classical features of dehydrins, i.e. the consensus motif KIKEKLPG that is present beyond the tract of serine residues and at the carboxy terminus. The encoded proteins, Tddhn15 and Tddhn16, show similarities with Triticum aestivum sequences. Two clones, pTd25a and pTd38, code for a dehydrin which lacks the stretch of serine residues and shows sequence similarity to T. aestivum Cor proteins. To correlate T. durum drought tolerance with dehydrin gene expression, we have for four cultivars compared the accumulation of dehydrin transcripts in roots and shoots of seedlings in response to a water-stress, and under application of exogenous ABA. A water-stress time course showed that accumulation of the dehydrin transcripts is delayed in the drought-tolerant cultivars. Also, the level of accumulated transcripts appeared to be greater in the drought-tolerant cultivars than in the drought-sensitive cultivar. A similar result was observed after application of exogenous ABA.

The CM (chloroform-methanol-soluble) proteins are low-molecular-weight cysteine-rich proteins that are found in wheat and barley endosperms. A cDNA clone encoding a Triticum durum (T. durum) CM3 protein has been isolated from a mid-maturation seed cDNA library. The T. durum CM3 protein is synthesized as a precursor including a signal peptide (SP) of 25 residues. Northern blot analysis showes that in developing seed the highest level of CM3 protein mRNA is detected at mid-maturation. The hybridization patterns obtained by Southern blot analysis indicated that T. durum CM proteins are encoded by a small multigene family. The similarity between the wheat and barley CM proteins encoded by homologous chromosomes is much higher than that between each of the three members of the T. durum family. All CM proteins contain ten cysteine residues organized in a conserved cysteine motif.

From a mid-maturation seed cDNA library we have isolated cDNA clones encoding two Triticum aestivum puroindolines. Puroindoline-a and puroindoline-b, which are 55% similar, are basic, cystine-rich and tryptophan-rich proteins. Puroindolines are synthezised as preproproteins which include N- and C-terminal propeptides which could be involved in their vacuolar localization. The mature proteins have a molecular mass of 13 kDa and a calculated isoelectric point greater than 10. A notable feature of the primary structure of puroindolines is the presence of a tryptophan-rich domain which also contains basic residues. A similar tryptophan-rich domain was found within an oat seed protein and a mammalian antimicrobial peptide. The ten cysteine residues of puroindolines are organized in a cysteine skeleton which shows similarity to the cysteine skeleton of other wheat seed cystine-rich proteins. Northern blot analysis showed that puroindoline genes are specifically expressed in T. aestivum developing seeds. No puroindoline transcripts as well as no related genes were detected in Triticum durum. The identity of puroindolines to wheat starch-granule associated proteins is discussed as well as the potential role of puroindolines in the plant defence mechanism.

A new basic protein has been isolated from wheat endosperm by Triton X-114 phase partitioning. It contains five disulfide bridges and is composed of equal amounts of a polypeptide chain of 115 amino acid residues and of the same chain with a C-terminus dipeptide extension. The most striking sequence feature is the presence of a unique tryptophan-rich domain so that this protein isolated from wheat seeds has been named puroindoline. The similar phase partitioning behavior in Triton X-114 of this basic eystine-rich protein and of purothionins suggests that puroindoline may also be a membranotoxin that might play a role in the defense mechanism of plants against microbial pathogens.

Polyacrylamide-slab gel electrophoresis showed that the nature of peroxidases in wheat is a genetic character; different genomes control the synthesis of specific peroxidases. The growing conditions have no influence on isozyme patterns. Within a single species the electrophoretic patterns of peroxidases may differ from one variety to another. Thus, two classes of Triticum aestivum can be distinguished. With Triticum durum the dissimilarity of enzymograms is particularly important.

The cDNA encoding a wheat ( Triticum durum ) lipid transfer protein of 9 kDa was inserted into an Escherichia coli expression vector, pIH902, and expressed in the bacteria as a fusion with the maltose binding protein. The fusion protein was then purified to homogeneity and subjected to factor Xa cleavage, Although complete cleavage of the fusion protein was obtained, the expected lipid transfer protein was not recovered; it appears to be degraded during protease digestion, However, a fluorescent lipid transfer assay demonstrated that the fusion protein has an activity identical to that of the wheat-purified lipid transfer protein. Thus, this expression system should allow further understanding of the structure/function relationships of wheat lipid transfer proteins.