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International audience; Consumption of cereals contaminated by mycotoxins poses health risks. For instance, Fumonisins B, mainly produced by Fusarium verticillioides and Fusarium proliferatum, and the type B trichothecene deoxynivalenol, typically produced by Fusarium graminearum, are highly prevalent on cereal grains that are staples of many cultural diets and known to represent a toxic risk hazard. In Peru, corn and other cereals are frequently consumed on a daily basis under various forms, the majority of food grains being sold through traditional markets for direct consumption. Here, we surveyed mycotoxin contents of market-bought grain samples in order to assess the threat these mycotoxins might represent to Peruvian population, with a focus on corn. We found that nearly one sample of Peruvian corn out of six was contaminated with very high levels of Fumonisins, levels mostly ascribed to the presence of F. verticillioides. Extensive profiling of Peruvian corn kernels for fungal contaminants could provide elements to refine the potential risk associated with Fusarium toxins and help define adapted food safety standards.

Consumption of cereals contaminated by mycotoxins poses health risks. For instance, Fumonisins B, mainly produced by Fusarium verticillioides and Fusarium proliferatum, and the type B trichothecene deoxynivalenol, typically produced by Fusarium graminearum, are highly prevalent on cereal grains that are staples of many cultural diets and known to represent a toxic risk hazard. In Peru, corn and other cereals are frequently consumed on a daily basis under various forms, the majority of food grains being sold through traditional markets for direct consumption. Here, we surveyed mycotoxin contents of market-bought grain samples in order to assess the threat these mycotoxins might represent to Peruvian population, with a focus on corn. We found that nearly one sample of Peruvian corn out of six was contaminated with very high levels of Fumonisins, levels mostly ascribed to the presence of F. verticillioides. Extensive profiling of Peruvian corn kernels for fungal contaminants could provide elements to refine the potential risk associated with Fusarium toxins and help define adapted food safety standards. This research was funded by the French National Alliance for Life Sciences and Health, grant number ENV201408. S.B. has received funding from the European Union's Horizon 2020 Framework program under the Marie Sklodowska-Curie Actions, agreement number 823935. P.V.O. was supported by a postdoctoral fellowship from the French National Research Institute for Sustainable Development (IRD) (04077858). JPC was a recipient of a doctoral fellowship from the Peruvian National Council for Science and Technology (212-2015-FONDECYT).

International audience; Doubled haploid (DH) technology produces strictly homozygous fertile plant thanks to doubling the chromosomes of a haploid embryo/seedling. Haploid embryos are derived from either male or female germ line cells and hold only half the number of chromosomes found in somatic plant tissues, albeit in a recombinant form due to meiotic genetic shuffling. DH production allows to rapidly fix these recombinant haploid genomes in the form of perfectly homozygous plants (inbred lines), which are produced in two rather than six or more generations. Thus, DH breeding enables fast evaluation of phenotypic traits on homogenous progeny. While for most crops haploid embryos are produced by costly and often genotype-dependent in vitro methods, for maize, two unique in planta systems are available to induce haploid embryos directly in the seed. Two "haploid inducer lines", identified from spontaneous maize mutants, are able to induce embryos of paternal or maternal origin. Although effortless crosses with lines of interest are sufficient to trigger haploid embryos, substantial improvements were necessary to bring DH technology to large scale production. They include the development of modern haploid inducer lines with high induction rates (8-12%), and methods to sort kernels with haploid embryos from the normal ones. Chromosome doubling represents also a crucial step in the DH process. Recent identification of genomic loci involved in spontaneous doubling opens up perspectives for a fully in planta DH pipeline in maize. Although discovered more than 60 years ago, maize haploid inducer lines still make headlines thanks to novel applications and findings. Indeed, maternal haploid induction was elegantly diverted to deliver genome editing machinery in germplasm recalcitrant to transformation techniques. The recent discovery of two molecular players controlling haploid induction allowed to revisit the mechanistic basis of maize maternal haploid induction and to successfully translate haploid induction ability to other crops.

Apomictic plants reproduce asexually through seeds by avoiding both meiosis and fertilization. Although apomixis is genetically regulated, its core genetic component(s) has not been determined yet. Using profiling experiments comparing sexual development in maize (Zea mays) to apomixis in maize-Tripsacum hybrids, we identified six loci that are specifically downregulated in ovules of apomictic plants. Four of them share strong homology with members of the RNA-directed DNA methylation pathway, which in Arabidopsis thaliana is involved in silencing via DNA methylation. Analyzing loss-of-function alleles for two maize DNA methyltransferase genes belonging to that subset, dmt102 and dmt103, which are downregulated in the ovules of apomictic plants and are homologous to the Arabidopsis CHROMOMETHYLASEs and DOMAINS REARRANGED METHYLTRANSFERASE families, revealed phenotypes reminiscent of apomictic development, including the production of unreduced gametes and formation of multiple embryo sacs in the ovule. Loss of DMT102 activity in ovules resulted in the establishment of a transcriptionally competent chromatin state in the archesporial tissue and in the egg cell that mimics the chromatin state found in apomicts. Interestingly, dmt102 and dmt103 expression in the ovule is found in a restricted domain in and around the germ cells, indicating that a DNA methylation pathway active during reproduction is essential for gametophyte development in maize and likely plays a critical role in the differentiation between apomictic and sexual reproduction.

Transposable elements have been widely used as mutagens in many organisms. Among them, the maize transposable element En/Spm has been shown to transpose efficiently in several plant species including the model plant Arabidopsis, where it has been used for large-scale mutagenesis. To determine whether we could use this transposon as a mutagen in the model legume plant Medicago truncatula, we tested the activity of the autonomous element, as well as two defective elements, in this plant, and in Arabidopsis as a positive control. In agreement with previous reports, we observed efficient excision of the autonomous En/Spm element in A. thaliana. This element was also active in M. truncatula, but the transposition activity was low and was apparently restricted to the tissue culture step necessary for the production of transgenic plants. The activity of one of the defective transposable elements, dSpm, was very low in A. thaliana and even lower in M. truncatula. The use of different sources of transposases suggested that this defect in transposition was associated with the dSpm element itself. Transposition of the other defective element, I6078, was also detected in M. truncatula, but, as observed with the autonomous element, transposition events were very rare and occurred during tissue culture. These results suggest that the En/Spm element is rapidly inactivated in the regenerated plants and their progeny, and therefore is not suitable for routine insertion mutagenesis in M. truncatula.

International audience; Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.

Culture and genetics rely on two distinct but not isolated transmission systems. Cultural processes may change the human selective environment and thereby affect which individuals survive and reproduce. Here, we evaluated whether the modes of subsistence in Native American populations and the frequencies of the ABCA1*Arg230Cys polymorphism were correlated. Further, we examined whether the evolutionary consequences of the agriculturally constructed niche in Mesoamerica could be considered as a gene-culture coevolution model. For this purpose, we genotyped 229 individuals affiliated with 19 Native American populations and added data for 41 other Native American groups (n = 1905) to the analysis. In combination with the SNP cluster of a neutral region, this dataset was then used to unravel the scenario involved in 230Cys evolutionary history. The estimated age of 230Cys is compatible with its origin occurring in the American continent. The correlation of its frequencies with the archeological data on Zea pollen in Mesoamerica/Central America, the neutral coalescent simulations, and the FST-based natural selection analysis suggest that maize domestication was the driving force in the increase in the frequencies of 230Cys in this region. These results may represent the first example of a gene-culture coevolution involving an autochthonous American allele. Fil: Hünemeier, Tábita. Universidade Federal do Rio Grande do Sul; Brasil Fil: Amorim, Carlos Eduardo Guerra. Universidade Federal do Rio Grande do Sul; Brasil Fil: de Azevedo, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentina Fil: Contini, Veronica. Universidade Federal do Rio Grande do Sul; Brasil Fil: Acuña Alonzo, Víctor. Instituto Nacional de Antropología e Historia. Escuela Nacional de Antropología e Historia; México Fil: Rothhammer, Francisco. Universidad de Chile. Facultad de Medicina; Chile Fil: Dugoujon, Jean Michel. Centre National de la Recherche Scientifique; Francia. Université Paul Sabatier; Francia Fil: Mazières, Stephane. Centre National de la Recherche Scientifique; Francia. Aix-Marseille Université; Francia. Anthropologie Bio-Culturelle, Droit, Éthique et Santé; Francia Fil: Barrantes, Ramiro. Universidad de Costa Rica; Costa Rica Fil: Villarreal Molina, María Teresa. Instituto Nacional de Medicina Genómica. Laboratorio de Genómica de Enfermedades Cardiovasculares; México Fil: Paixão Côrtes, Vanessa Rodrigues. Universidade Federal do Rio Grande do Sul; Brasil Fil: Salzano, Francisco M.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Canizales Quinteros, Samuel. Universidad Nacional Autónoma de México; México Fil: Ruiz-Linares, Andres. University College London; Reino Unido Fil: Bortolini, Maria Cátira. Universidade Federal do Rio Grande do Sul; Brasil

Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.

Apomixis is a form of asexual reproduction through seeds in angiosperms. Apomictic plants bypass meiosis and fertilization, developing offspring that are genetically identical to their mother. In a genetic screen for maize (Zea mays) mutants mimicking aspects of apomixis, we identified a dominant mutation resulting in the formation of functional unreduced gametes. The mutant shows defects in chromatin condensation during meiosis and subsequent failure to segregate chromosomes. The mutated locus codes for AGO104, a member of the ARGONAUTE family of proteins. AGO104 accumulates specifically in somatic cells surrounding the female meiocyte, suggesting a mobile signal rather than cell-autonomous control. AGO104 is necessary for non-CG methylation of centromeric and knob-repeat DNA. Digital gene expression tag profiling experiments using high-throughput sequencing show that AGO104 influences the transcription of many targets in the ovaries, with a strong effect on centromeric repeats. AGO104 is related to Arabidopsis thaliana AGO9, but while AGO9 acts to repress germ cell fate in somatic tissues, AGO104 acts to repress somatic fate in germ cells. Our findings show that female germ cell development in maize is dependent upon conserved small RNA pathways acting non-cell-autonomously in the ovule. Interfering with this repression leads to apomixis-like phenotypes in maize.

C4 grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C4 photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C4 differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C4 specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.

The comparison of the chromosome numbers of today's species with common reconstructed paleo-ancestors has led to intense speculation of how chromosomes have been rearranged over time in mammals. However, similar studies in plants with respect to genome evolution as well as molecular mechanisms leading to mosaic synteny blocks have been lacking due to relevant examples of evolutionary zooms from genomic sequences. Such studies require genomes of species that belong to the same family but are diverged to fall into different subfamilies. Our most important crops belong to the family of the grasses, where a number of genomes have now been sequenced. Based on detailed paleogenomics, using inference from n = 5–12 grass ancestral karyotypes (AGKs) in terms of gene content and order, we delineated sequence intervals comprising a complete set of junction break points of orthologous regions from rice, maize, sorghum, and Brachypodium genomes, representing three different subfamilies and different polyploidization events. By focusing on these sequence intervals, we could show that the chromosome number variation/reduction from the n = 12 common paleo-ancestor was driven by nonrandom centric double-strand break repair events. It appeared that the centromeric/telomeric illegitimate recombination between nonhomologous chromosomes led to nested chromosome fusions (NCFs) and synteny break points (SBPs). When intervals comprising NCFs were compared in their structure, we concluded that SBPs (1) were meiotic recombination hotspots, (2) corresponded to high sequence turnover loci through repeat invasion, and (3) might be considered as hotspots of evolutionary novelty that could act as a reservoir for producing adaptive phenotypes.

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C4 photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.

We examined the justifications invoked by the German government in April 2009 to suspend the cultivation of the genetically modified maize varieties containing the Bt insect-resistance trait MON810. We have carried out a critical examination of the alleged new data on a potential environmental impact of these varieties, namely two scientific papers describing laboratory force-feeding trials on ladybirds and daphnia, and previous data on Lepidoptera, aquatic and soil organisms. We demonstrate that the suspension is based on an incomplete list of references, ignores the widely admitted case-by-case approach, and confuses potential hazard and proven risk in the scientific procedure of risk assessment. Furthermore, we did not find any justification for this suspension in our extensive survey of the scientific literature regarding possible effects under natural field conditions on non-target animals. The vast majority of the 41 articles published in 2008 and 2009 indicate no impact on these organisms and only two articles indicate a minor effect, which is either inconsistent during the planting season or represents an indirect effect. Publications from 1996 to 2008 (376 publications) and recent meta-analyses do not allow to conclude on consistent effects either. The lower abundance of some insects concerns mainly specialized enemies of the target pest (an expected consequence of its control by Bt maize). On the contrary, Bt maize have generally a lower impact than insecticide treatment. The present review demonstrates that the available meta-knowledge on Cry1Ab expressing maize was ignored by the German government which instead used selected individual studies. Electronic supplementary material The online version of this article (doi:10.1007/s11248-009-9297-5) contains supplementary material, which is available to authorized users.

A-Maize-ing Maize is one of our oldest and most important crops, having been domesticated approximately 9000 years ago in central Mexico. Schnable et al. (p. 1112 ; see the cover) present the results of sequencing the B73 inbred maize line. The findings elucidate how maize became diploid after an ancestral doubling of its chromosomes and reveals transposable element movement and activity and recombination. Vielle-Calzada et al. (p. 1078 ) have sequenced the Palomero Toluqueño ( Palomero ) landrace, a highland popcorn from Mexico, which, when compared to the B73 line, reveals multiple loci impacted by domestication. Swanson-Wagner et al. (p. 1118 ) exploit possession of the genome to analyze expression differences occurring between lines. The identification of single nucleotide polymorphisms and copy number variations among lines was used by Gore et al. (p. 1115 ) to generate a Haplotype map of maize. While chromosomal diversity in maize is high, it is likely that recombination is the major force affecting the levels of heterozygosity in maize. The availability of the maize genome will help to guide future agricultural and biofuel applications (see the Perspective by Feuillet and Eversole ).

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on ∼1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses., Author Summary Maize is a major cereal crop and key experimental system for eukaryotic biology. Previous investigations of the maize genome at the sequence level have primarily focused on analyses of genome survey sequences and BAC contigs. Here we used a comprehensive set of resources to construct an ordered and oriented 22-Mb sequence from chromosome 4 that represents 1% of the maize genome. Genome annotation revealed the presence of 544 genes that are interspersed with transposable elements (TEs), which occupy 83.8% of the sequence. Fifty-one genes were involved in 14 tandem gene clusters and most appear to have arisen after lineage divergence. TEs, especially helitrons, were found to contain gene fragments and were widely distributed in gene-rich regions. Large inversions and unequal gene deletion between the two homoeologous maize regions were the main contributors to synteny disruption among maize, sorghum, and rice. We also show that small RNAs are primarily associated with TEs across the region. Comparison of this ordered and oriented sequence with the corresponding uncurated region in the whole genome sequence of maize resulted in improvements in TE annotation that will ultimately enhance detection sensitivity and characterization of TEs. Doing so is likely to improve the specificity of gene annotations.

This work was supported by the Institut National de la Recherche Agronomique (doctoral fellowship to C.E. and grant no. AIP300), the French Ministry of Research (grant no. ACI2003 [Biologie du Développement et Physiologie Intégrative] to C.M. and T.S.), and Agence Nationale de la Recherche Génoplante (award to F.T.).; International audience; Root hydraulic conductivity in plants (Lp(r)) exhibits large variations in response to abiotic stimuli. In this study, we investigated the impact of dynamic, aquaporin-mediated changes of Lp(r) on leaf growth, water potential, and water flux throughout the plant. For this, we manipulated Lp(r) by subjecting roots to four independent treatments, with aquaporin inhibitors applied either to transpiring maize (Zea mays) plants grown in hydroponics or to detopped root systems for estimation of Lp(r). The treatments were acid load at pH 6.0 and 5.0 and hydrogen peroxide and anoxia applied for 1 to 2 h and subsequently reversed. First, we established that acid load affected cell hydraulic conductivity in maize root cortex. Lp(r) was reduced by all treatments by 31% to 63%, with half-times of about 15 min, and partly recovered when treatments were reversed. Cell turgor measured in the elongating zone of leaves decreased synchronously with Lp(r), and leaf elongation rate closely followed these changes across all treatments in a dose-dependent manner. Leaf and xylem water potentials also followed changes in Lp(r). Stomatal conductance and rates of transpiration and water uptake were not affected by Lp(r) reduction under low evaporative demand. Increased evaporative demand, when combined with acid load at pH 6.0, induced stomatal closure and amplified all other responses without altering their synchrony. Root pressurization reversed the impact of acid load or anoxia on leaf elongation rate and water potential, further indicating that changes in turgor mediated the response of leaf growth to reductions in Lp(r).

Background Horizontal transfers (HTs) refer to the transmission of genetic material between phylogenetically distant species. Although most of the cases of HTs described so far concern genes, there is increasing evidence that some involve transposable elements (TEs) in Eukaryotes. The availability of the full genome sequence of two cereal species, (i.e. rice and Sorghum), as well as the partial genome sequence of maize, provides the opportunity to carry out genome-wide searches for TE-HTs in Poaceae. Results We have identified an LTR-retrotransposon, that we named Route66, with more than 95% sequence identity between rice and Sorghum. Using a combination of in silico and molecular approaches, we are able to present a substantial phylogenetic evidence that Route66 has been transferred horizontally between Panicoideae and several species of the genus Oryza. In addition, we show that it has remained active after these transfers. Conclusion This study constitutes a new case of HTs for an LTR-retrotransposon and we strongly believe that this mechanism could play a major role in the life cycle of transposable elements. We therefore propose to integrate classe I elements into the previous model of transposable element evolution through horizontal transfers.

International audience; Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N(6)-(buta-2,3-dienyl)adenine (HA-8) and N(6)-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 A resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.

Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1–3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant.

International audience; To determine the effect of nematode infection on the response of snails to selected toxins, we infected Helix aspersa with 0-, 0.25-, 1-, or 4-fold the recommended field dose of a commercial nematode application for agricultural use. In the first experiment, the snails also were exposed to cadmium via food and soil at concentrations of 0, 30, 60, 120, or 240 mg/kg in a full-factorial design. In the second experiment, snails were infected with nematodes and also fed either Bt (expressing Bacillus thuringiensis toxin) maize or non-Bt maize. The snails were weighed at the beginning and end (after four weeks) of the experiments, and mortality was checked daily. Neither exposure of snails to nematodes nor exposure of snails to cadmium or Bt toxin affected the survival rates of snails. The number of dead snails was highest for combinations of nematode treatments with cadmium concentrations of 120 and 240 mg/kg. In both experiments (Bt and cadmium), the growth rate decreased with increasing nematode dose. The Bt maize was not harmful to the snails in the absence of nematodes, but infected snails grew faster when fed non-Bt maize. The growth rate of snails exposed to cadmium decreased with exposure to increasing Cd concentrations and differed significantly between the no-nematode treatment and the treatments with nematode doses of one- and fourfold the recommended field dose. Snails treated with the highest dose of nematodes accumulated the highest cadmium concentrations.

International audience; Soil isolates of Aspergillus section Flavi from Mazandaran and Semnan provinces with totally different climatic conditions in Iran were examined for aflatoxins (AFs; B and G types), cyclopiazonic acid (CPA) and sclerotia production. A total of 66 Aspergillus flavus group strains were identified from three species viz. Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius in both locations. A. flavus (87.9%) was found to be the prominent species followed by A. nomius (9.1%) and A. parasiticus (3.0%). Only 27.5% of A. flavus isolates were aflatoxigenic (B(1) or B(1) and B(2)), out of which approximately 75% were capable to producing CPA. All the A. parasiticus and A. nomius isolates produced AFs of both B (B(1) and B(2)) and G (G(1) and G(2)) types, but did not produce CPA. Sclerotia production was observed in only 4 isolates of A. flavus among all 66 isolates from three identified species. A. flavus isolates were classified into various chemotypes based on the ability to produce aflatoxins and CPA. In this study, a new naturally occurring toxigenic A. flavus chemotype comprising of two strains capable of producing more AFB(2) than AFB(1) has been identified. A relatively larger proportion of aflatoxigenic A. flavus strains were isolated from corn field soils of Mazandaran province which indicate a possible relationship between high levels of relative humidity and the incidence of aflatoxin-producing fungi. The importance of incidence of Aspergillus section Flavi in corn field soils regard to their mycotoxin production profiles and crop contamination with special reference to climatic conditions is discussed.

International audience; Grass cell wall degradability is conventionally related to the lignin content and to the ferulic-mediated cross-linking of lignins to polysaccharides. To better understand the variations in degradability, 22 maize inbred lines were subjected to image analyses of Fasga- and Mäule-stained stem sections and to chemical analyses of lignins and p-hydroxycinnamic acids. For the first time, the nearness of biochemical and histological estimates of lignin levels was established. Combination of histological and biochemical traits could explain 89% of the variations for cell wall degradability and define a maize ideotype for cell wall degradability. In addition to a reduced lignin level, such an ideotype would contain lignins richer in syringyl than in guaiacyl units and preferentially localized in the cortical region rather than in the pith. Such enrichment in syringyl units would favor wall degradability in grasses, contrary to dicots, and could be related to the fact that grass syringyl units are noticeably p-coumaroylated. This might affect the interaction capabilities of lignins and polysaccharides.

In animals, early embryonic development is largely dependent on maternal transcripts synthesized during gametogenesis. Recent data in plants also suggest maternal control over early seed development, but the actual timing of zygotic genome activation is unclear. Here, we analyzed the timing of the maternal-to-zygotic transition during early Zea mays seed development. We show that for 16 genes expressed during early seed development, only maternally inherited alleles are detected during 3 d after fertilization in both the embryo and the endosperm. Microarray analyses of precocious embryonic development in apomictic hybrids between maize and its wild relative, Tripsacum, demonstrate that early embryo development occurs without significant quantitative changes to the transcript population in the ovule before fertilization. Precocious embryo development is also correlated with a higher proportion of polyadenylated mRNA in the ovules. Our data suggest that the maternal-to-zygotic transition occurs several days after fertilization. By contrast, novel transcription accompanies early endosperm development, indicating that different mechanisms are involved in the initiation of endosperm and embryo development.

A unigene set of 1411 contigs was constructed from 2629 redundant maize expressed sequence tags (ESTs) mapped on the maizeDB genetic map. Rice orthologous sequences were identified by blast alignment against the rice genomic sequence. A total of 1046 (74%) maize contigs were associated with their corresponding homologues in the rice genome and 656 (47%) defined as potential orthologous relationships. One hundred and seventeen (8%) maize EST contigs mapped to two distinct loci on the maize genetic map, reflecting the tetraploid nature of the maize genome. Among 492 mono-locus contigs, 344 (484 redundant ESTs) identify collinear blocks between maize chromosomes 2 and 4 and a single rice chromosome, defining six new collinear regions. Fine-scale analysis of collinearity between rice chromosomes 1 and 5 with maize chromosomes 3, 6 and 8 shows the presence of internal rearrangements within collinear regions. Mapping of maize contigs to two distinct loci on the rice sequence identifies five new duplication events in rice. Detailed analysis of a duplication between rice chromosomes 1 and 5 shows that 11% of the annotated genes from the chromosome 1 locus are found duplicated on the chromosome 5 paralogous counterpart, indicating a high degree of re-organisations. The implications of these findings for map-based cloning in collinear regions are discussed.

Effects of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA) on endomembrane structures and lipid synthesis were compared in maize root cells and tobacco Bright Yellow-2 cells. Immunofluorescence and electron microscopy studies showed that NDGA altered the structure and distribution of the endoplasmic reticulum (ER) within 1 h but not of the Golgi apparatus whereas, as shown previously, BFA altered that organization of the Golgi apparatus and, only subsequently, of the ER. Biochemical studies revealed that both drugs and especially BFA led to a strong inhibition of the phytosterol biosynthetic pathway: BFA led to accumulation of sterol precursors. The importance of phytosterols in membrane architecture and membrane trafficking is discussed.

International audience; Nogo recombinant proteins To express Amino-Nogo, the human Nogo-A cDNA for residues 1±1,040 was ligated into pcDNA3.1MycHis (Invitrogen, Burlingame, California) with an in-frame Myc-His tag. We transfected this plasmid into HEK293T cells and Amino-Nogo was puri®ed with a Ni 2+ resin 10. The human Nogo-66 sequence was ligated into pcAP-5 (ref. 10) in frame with the signal sequence, 6´His tag and placental AP coding region. This plasmid was transfected into HEK293 cells, and secreted AP±Nogo was puri®ed by Ni 2+ af®nity chromatography. GST±Nogo-66 has been described 1. Nogo-66 receptor binding assays and expression cloning To detect AP±Nogo binding, cultures were washed with Hanks balanced salt solution containing 20 mM sodium HEPES, pH 7.05, and 1 mg ml-1 bovine serum albumin (BSA) (HBH). The plates were then incubated with AP±Nogo in HBH for 2 h at 23 8C. We detected and quanti®ed bound AP±Nogo as for AP±Sema3A 11. For saturation analysis of AP±Nogo bound to COS-7 cells, bound AP±Nogo protein was eluted with 1% Triton X-100. After heat inactivation of endogenous AP, we measured AP±Nogo using p-nitrophenyl phosphate as substrate. For expression cloning of a Nogo-66 receptor, pools of 5,000 arrayed clones from a mouse adult-brain cDNA library (Origene Technologies, Rockville, Maryland) were transfected into COS-7 cells, and AP±Nogo binding was assessed. We isolated single NgR cDNA clones by sib selection and sequenced them. A Myc±NgR vector was created in pSecTag2-Hygro (Invitrogen) using the signal peptide of pSecTag2 fused to Myc and residues 27±473 of NgR. Human NgR cDNA was predicted from a human genomic cosmid sequence (AC007663). Oligonucleotide primers based on the predicted human cDNA ampli®ed the cDNA from a human adult-brain cDNA library (Origene Technologies). To assess binding of Myc±NgR to the cell membrane, particulate fractions were treated with or without 5 U PI-PLC (Sigma, St. Louis, MO) per mg of HEK293T cell protein for 1 h at 30 8C in HBH. After centrifugation at 100,000g for 1 h, we analysed equal proportions of the soluble and particulate fractions. To assess the physical interaction of NgR with Nogo-66, we incubated the PI-PLC extract (50 mg total protein) with 10 mg GST±Nogo-66 or buffer, or 10 mg GST for 1 h at 23 8C. We added glutathione-coupled agarose to bind GST and associated proteins. We analysed bound proteins by anti-Myc immunoblot. RNA analysis For northern blots, 1 mg poly(A) + RNA from each adult mouse tissue on a nylon membrane (Origene Technologies) was hybridized with a full-length 32 P-labelled probe 1,12. We used digoxegenin-labelled riboprobes (nucleotides 1±1,222) and adult mouse brain sections 1,12 for in situ hybridization. The sense probe produced no signal. Nogo-66 receptor antibodies A GST±NgR (residues 27±447) fusion protein was puri®ed from Escherichia coli and used to immunize rabbits. We diluted immune serum 3,000-fold for immunoblots and 1,000fold for immunohistology on tissue-culture samples that had been ®xed by formalin. Staining of tissue was totally abolished by addition of 5 mg ml-1 GST±NgR. Cell spreading, neurite outgrowth and viral infection To measure spreading rates, subcon¯uent NIH 3T3 ®broblasts or COS-7 cells were plated for 1 h in serum-containing medium before ®xation and staining with rhodaminephalloidin. Glass coverslips were precoated with 100 mg ml-1 poly-L-lysine, washed, and then 3 ml drops of PBS containing 15 pmol Amino-Nogo, 15 pmol GST±Nogo-66, 15 pmol poly-Asp (M r 35 K, Sigma), or no protein were spotted and dried. We added soluble Nogo protein preparations (100 nM) at the time of plating. Amino-Nogo was added alone or after a pre-incubation with a twofold molar excess of anti-Myc 9E10 antibody, or with a twofold excess of anti-Myc plus a twofold excess of puri®ed goat anti-mouse IgG. Chick E5 spinal cord, chick E7±E13 DRG, chick E7 retina and mouse P4 cerebellar neuron culture, growth-cone-collapse assays and neurite-outgrowth assays have been described 1,10±13. Here, outgrowth from dissociated neurons was assessed after 12±24 h. For the substrate-bound experiments, glass chamber slides were coated with 100 mg ml-1 poly-L-lysine, washed, and then 3 ml drops of PBS containing 15 pmol Amino-Nogo, 15 pmol GST±Nogo-66, 15 pmol poly-Asp, or no protein were spotted and dried. After three PBS washes, we coated slides with 10 mg ml-1 laminin. After aspiration of laminin, dissociated neurons were added. For the soluble Nogo experiments, slides were coated with poly-Llysine and laminin in the same fashion, and then 100 nM Amino-Nogo, 100 nM clustered Amino-Nogo, or 100 nM GST±Nogo-66 was added to the culture medium at the time of plating. After 1 d in vitro, some DRG explants were treated for 30 min with 1 unit ml-1 PI-PLC (Sigma) before the growth-cone-collapse assay. An HSV-Myc±NgR stock was prepared as described 10. We infected E7 retinal explants for 24 h. We stained some cultures infected with HSV-Myc±NgR with anti-Myc antibody to verify protein expression. Error bars re¯ect the s.e.m. from 4±8 determinations.

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.

International audience; Correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and NOE spectroscopy (NOESY) experiments have been used to assign sequentially the 1H 500-MHz NMR spectra of a non-specific (ns) lipid-transfer protein extracted from maize seeds. The spin-system identification and sequential assignment were combined with secondary-structure determination to identify most of the proton resonances of this 93-residue protein. From the sequential connectivities it was established that the secondary structure mainly involved four helical fragments: H1, H2, H3 and H4. This secondary structure was compared with that of wheat ns-lipid-transfer protein recently determined. The four helices are located in nearly the same regions, but helix H4 is appreciably longer in the maize protein than in the wheat protein. Comparison of the transfer activities reveals that the maize protein is more efficient than the wheat ns-lipid-transfer protein and that this difference is probably due to the affinity of the lipid for the binding site and not to the interfacial activation, i.e. adsorption of the ns-lipid-transfer protein to the membrane. From these results, it is suggested that helix H4 is a part of the lipid-binding site or contributes to the folding of this site. The present data define the basis for a further modelling of the three-dimensional structure of the maize ns-lipid-transfer protein which will be compared with that of the wheat ns-lipid-transfer protein in order to establish structure/activity relationships for this class of carriers by using natural ns-lipid-transfer protein mutants.

International audience; Consumption of flavonoid-rich foods and beverages is thought to reduce the risk of cardiovascular diseases. Whereas the biological activities of flavonoids have been characterized in vitro, there are no clear experimental data demonstrating that chronic dietary intake and intestinal absorption of flavonoids actually protects the heart against ischemia-reperfusion injury. We tested whether long-term consumption of specific flavonoids (anthocyanins) included in normal food could render the heart of rats more resistant to myocardial infarction. Maize kernels that differed specifically in their accumulation of anthocyanins were used to prepare rodent food in which anthocyanins were either present or absent. Male Wistar rats were fed the anthocyanin-rich (ACN-rich) or the anthocyanin-free (ACN-free) diet for a period of 8 wk. Anthocyanins were significantly absorbed and detected in the blood and urine of only rats fed the ACN-rich diet. In Langendorff preparations, the hearts of rats fed the ACN-rich diet were more resistant to regional ischemia and reperfusion insult. Moreover, on an in vivo model of coronary occlusion and reperfusion, infarct size was reduced in rats that ate the ACN-rich diet than in those that consumed the ACN-free diet (P < 0.01). Cardioprotection was associated with increased myocardial glutathione levels, suggesting that dietary anthocyanins might modulate cardiac antioxidant defenses. Our findings suggest important potential health benefits of foods rich in anthocyanins and emphasize the need to develop anthocyanin-rich functional foods with protective activities for promoting human health.

International audience; The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (phi, psi, chi 1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for alpha-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 +/- 0.6 x 10(-6) M and > 10(-3) M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid-protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid-protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).