Rat feeding trials: A comprehensive assessment of contaminants in both genetically modified maize and resulting pellets.

We analyzed a comprehensive set of contaminants in MON810 and NK603 genetically modified (GM) maize, and their non-GM counterparts, used in a rat feeding study (the GMO90 + project). Both the maize grains and the manufactured pellets were characterized. Only minor differences in contaminant levels between GM and corresponding non-GM harvests were evidenced. Fumonisin and deoxynivalenol mycotoxins were the pollutants present in the highest amounts, with concentrations that were however largely below acceptance reference values. Our data reporting slightly lower levels of fumonisin in MON810 compared to its non-GM counterpart corroborate the lower susceptibility of insect resistant Bt maize to fumonisin-producing fungi. Traces of glyphosate (0.016 mg/kg) were evidenced in grains from NK603 treated crops. Regarding the pellets, analysis of more than 650 potentially toxic substances revealed low amounts of various mycotoxins, pesticides and heavy metals. Concentrations of contaminants quantified in the pellets were however far below the maximum level of residues values set by regulatory agencies, and no substantial differences in contaminants between GM and non-GM pellets were observed. Moreover, when comparing the contamination status of grains and pellets, we demonstrate yet again that characterizing the grains is actually not sufficient to foresee the quality of the produced pellets.

Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family.

Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (somatic embryogenesis receptor-like kinase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

Expression patterns of genes encoding HD-ZipIV homeo domain proteins define specific domains in maize embryos and meristems.

A family of homeo box genes with cell layer-specific expression patterns defining subdomains of the embryo and certain meristems has been isolated from maize. These genes encode proteins from the class of plant specific homeo domain-leucine zipper (HD-Zip) transcription factors containing the previously described ZmOCL1 protein, and have been designated ZmOCL2, ZmOCL3, ZmOCL4 and ZmOCL5. ZmOCL3, ZmOCL4 and ZmOCL5, like ZmOCL1, showed essentially L1 or epidermis-specific expression. However, each gene was expressed in a distinct region of the embryonic protoderm during early development, with ZmOCL3 showing suspensor-specific expression, ZmOCL4 transcripts being localized to the adaxial face of the embryo proper and ZmOCL5 showing a more abaxial expression pattern. All three genes were also expressed in vegetative, inflorescence and floral apices, although ZmOCL3 transcripts were excluded from meristems and very young organ primordia. In contrast, ZmOCL2 expression was entirely meristem-specific and was excluded from the L1 layer, appearing instead to be largely restricted to a cell layer directly beneath the L1, especially in floral meristems. This expression pattern is unprecedented and may indicate that cell-layer organization in maize meristems is more complex than that suggested by the classical L1/L2 (outer cell layer/inner cell mass) model. These differing expression patterns indicate that the members of the HD-ZipIV family of maize may not only play roles in defining different regions of the epidermis during embryonic development, but could also be responsible for maintaining cell-layer identity in meristematic regions.

Esr genes show different levels of expression in the same region of maize endosperm.

Esr genes share high homology among each other, code for small hydrophilic proteins, and are expressed in a restricted region of maize endosperm surrounding the embryo. We show here that not only Esr2 but also Esr1 and Esr3 are expressed in maize, and that the relative contribution of Esr1, Esr2 and Esr3 to total Esr mRNA is 17%, 55% and 28%, respectively. DNA sequence analysis of putative promoter fragments ranging from 0.53 kb to 3.54 kb revealed the presence of retrotransposons related to the Zeon and Cinful families in the distal parts of the promoters. The proximal parts show high homology that extended over 504bp between Esr2 and Esr3, and 265bp between Esr1 and the other two genes. The most conspicuous potential cis element is a fully conserved tandem repeat of the sequence CTACACCA close to the respective open reading frames (ORFs). By the analysis of transgenic maize plants carrying promoter-Gus fusions, it was shown that all three cloned upstream fragments contain functional promoters, that the spatial activity of all three Esr promoters is identical, and that the cis element(s) responsible for the expression in the embryo surrounding region reside in the 265 bp upstream of the respective ORFs.

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize.

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.

ZmOCL1, an HDGL2 family homeobox gene, is expressed in the outer cell layer throughout maize development.

The formation of a morphologically distinct outer cell layer or protoderm is one of the first and probably one of the most important steps in patterning of the plant embryo. Here we report the isolation of ZmOCL1 (OCL for outer cell layer), a member of the HDGL2 (also known as HD-ZIP IV) subclass of plant-specific HD-ZIP homeodomain proteins from maize. ZmOCL1 transcripts are detected very early in embryo development, before a morphologically distinct protoderm is visible, and expression then becomes localised to the protoderm of the embryo as it develops. Subsequently, expression is observed in the L1 cell layer of both the developing primary root and shoot meristems, and is maintained in developing leaves and floral organs. We propose that ZMOCL1 may play a role in the specification of protoderm identity within the embryo, the organisation of the primary root primordium or in the maintenance of the L1 cell layer in the shoot apical meristem. We also show that the expression of ZmOCL1 is different from that of another epidermal marker gene, LTP2 (lipid transfer protein) and, in meristems, is complementary to that of Kn1 (Knotted) which is transcribed only in underlying cell layers.

Activation of hsr203, a plant gene expressed during incompatible plant-pathogen interactions, is correlated with programmed cell death.

hsr203J is a tobacco gene whose activation is rapid, highly localized, and specific for incompatible interactions between tobacco and the bacterial pathogen Ralstonia solanacearum. The effect of other hypersensitive response (HR)-inducing pathogens and elicitors has been tested with transgenic plants containing the hsr203J promoter-GUS reporter gene fusion, and confirms the generality of the preferential inducibility of the hsr203J gene promoter during incompatible interactions: bacterial and viral pathogens inducing an HR in tobacco were able to induce the promoter fusion, as were inducers of HR-like responses such as harpin, elicitins, and PopA1 proteins. A tomato hsr203 homologous cDNA was isolated (Lehsr203) and used to examine the effect of avr gene products on the expression of such genes. Lehsr203 was shown to be rapidly and transiently induced in leaves of the tomato Cf-9 line, following Avr9 product infiltration, but not in those of the Cf-0 line. Among potential effectors of HR or resistance such as H2O2, salicylic acid, methyl jasmonate, and 2,6-dichloro-isonicotinic acid (INA), none is able to induce a significant increase in promoter activation. In contrast, heavy metals that cause leaf necrosis can trigger such an activation. In addition, hsr203-GUS fusion expression is detected in transgenic tobacco lines expressing the bO gene and exhibiting spontaneous HR-like lesions. Taken together, these results demonstrate a strong correlation between hsr203 and genetically controlled cell death in tobacco and tomato. The expression of this gene should be a useful marker for programmed cell death occurring in response not only to diverse pathogens, but also to diverse death-triggering extracellular agents.

ZmEsr, a novel endosperm-specific gene expressed in a restricted region around the maize embryo.

A novel endosperm-specific gene named Esr (embryo surrounding region) has been isolated by differential display between early developmental stages of maize endosperms and embryos. It is expressed in a restricted region of the endosperm, surrounding the entire embryo at early stages (4 to 7 days after pollination, DAP) and ever-decreasing parts of the suspensor at subsequent stages. The expression starts at 4 DAP and is maintained until at least 28 DAP. A minimum of three Esr genes are present in the maize genome and at least two of them map to the short arm of chromosome 1 at position 56. The Esr genes contain no introns and show no significant nucleotide or amino acid sequence homologies to sequences in the databases. The open reading frames encode basic proteins of 14 kDa with presumptive signal peptides at their N-terminal followed by a hypervariable and a conserved region. The gene product may play a role in the nutrition of the developing embryo or in the establishment of a physical barrier between embryo and endosperm.

Characterisation of wheat-rye recombinants with RFLP and PCR probes.

The introgression of genetic material from alien species into wheat has become an important tool in modern wheat breeding. Ideally, only the trait of interest and no flanking material should be transferred. Random recombination between the genetic material is therefore of paramount importance. In a model system, we examined 17 recombinants putatively between chromosome 1D of wheat and 1R of rye with 60 random RFLP and three PCR markers. The recombinants had been generated by removing the normal effect of the Ph1 gene in the wheat background. Amongst the nine short-arm recombinants, three breakpoints were identified but no differentiation could be made between the five proximal recombinants. For the eight long-arm recombinants analysed only two breakpoints were identified with 36 markers. However, only a single RFLP marker was able to differentiate between the recombinants. Indeed the long-arm results are consistent with the possibility that only the rye telomeric region had been transferred. These results indicate either a strong clustering of the RFLP markers near the centromere or else imply that recombination induced between wheat and rye in the absence of the normal effect of the Ph1 gene occurs at only restricted sites. The results allow new primary recombinants to be selected for intercrossing to generate secondary recombinants which are expected to have a smaller interstitial rye segment than that present in DR-A1.

Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences.

The rye-specific R173 family of repeated DNA sequences consists of ca. 15,000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins greater than 20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheat Glu-D1 gene.

Polymerase chain reaction based mapping of rye involving repeated DNA sequences.

A novel type of polymerase chain reaction (PCR) marker was developed for the mapping of cereal rye (Secale cereale). Primer pairs were synthesized targeting the insertion sites of three individual copies of the R173 family of rye specific repeated DNA sequences. While one primer was derived from a sequence within the respective R173 element, the second primer corresponded to a flanking region. The complex banding patterns obtained in rye allowed not only the mapping of the three R173 elements to certain chromosome regions of 1RS (the short arm of rye chromosome 1) but also the mapping of an additional 3-10 easily identifiable bands per primer pair to other rye chromosomes. Linkage mapping of a polymorphic 1R band derived from three rye cultivars demonstrated the presence of nonallelic, dominant markers in two independent crosses. Because of the high copy number of the R173 family (15,000 copies per diploid rye genome), its dispersion over the entire length of all chromosomes and the high number of markers obtained per primer pair, PCR markers based on the R173 family provide an almost unlimited source for well-spaced markers in rye mapping.

Isolation and characterization of wheat-rye recombinants involving chromosome arm 1DS of wheat.

The introgression of genetic material from alien species is assuming increased importance in wheat breeding programs. One example is the translocation of the short arm of rye chromosome 1 (1RS) onto homoeologous wheat chromosomes, which confers disease resistance and increased yield on wheat. However, this translocation is also associated with dough quality defects. To break the linkage between the desirable agronomic traits and poor dough quality, recombination has been induced between 1RS and the homoeologous wheat arm IDS. Seven new recombinants were isolated, with five being similar to those reported earlier and two havina new type of structure. All available recombinantsw ere characterized with DNA probes for the loci Nor-R1, 5SDna-R1, and Tel-R1. Also, the amount of rye chromatin present was quantified with a dispersed rye-specific repetitive DNA sequence in quantitative dot blots. Furthermore, the wheat-rye recombinants were used as a mapping tool to assign two RFLP markers to specific regions on chromosome arms 1DS and 1RS of wheat and rye, respectively.

The calcium messenger system and the kinetics of elastase release from human neutrophils in patients with abdominal aortic aneurysms.

Aortic elastase and neutrophil elastase is higher in patients with abdominal aortic aneurysms. The purpose of this study was to determine if these proteolytic elevations occur after abdominal aortic aneurysms have been repaired. Specifically, we studied the stimulation and inhibition of elastase degranulation from neutrophils in postoperative abdominal aortic aneurysm patients compared to aortic occlusive disease patients. Neutrophil elastase was determined in postoperative abdominal aortic aneurysm and aortic occlusive disease patients in response to calcium and the ionophore A23187. Inhibition of elastase release was determined with the calcium channel blocking agent Verapamil. Neutrophil elastase secretion was significantly higher in the abdominal aortic aneurysm patients (47%) versus aortic occlusive disease (20%) (p less than .05), while the effect of Verapamil in blocking this response was significantly lower in the abdominal aortic aneurysm patients (14%) compared to aortic occlusive disease patients (27%) (p less than .02). The time for degranulation to occur was longer in the abdominal aortic aneurysm patients (14.7 minutes) versus aortic occlusive disease patients (3.5 minutes), but the rate of secretion was not different between the two groups. These data indicate that, (1) neutrophils secrete more elastase in response to a calcium stimulus in abdominal aortic aneurysm patients; (2) it takes longer to secrete the increased amount of elastase in abdominal aortic aneurysm patients since the rate of secretion is similar between the two groups; and (3) Verapamil blocks elastase secretion ineffectively in abdominal aortic aneurysm patients. We conclude that the proteolytic alterations in abdominal aortic aneurysm patients are more likely a primary event and not a response to the abdominal aortic aneurysm and that Verapamil is a poor drug to use to medically manipulate the protease system in abdominal aortic aneurysm patients.

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit.

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.

Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens.

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.

DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions.

DNA transfer from Agrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyle-donous plant Zea mays, was analysed using the recently developed technique of agroinfection. Agroinfection of Z. mays with maize streak virus using strains of A. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterial virC genes. In contrast, agroinfection of the control host Brassica rapa with cauliflower mosaic virus was less dependent on the virC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intact virA, B, D and G functions were absolutely necessary, mutants in virE were attenuated. Agroinfection of maize was effective in the absence of an exogenously supplied vir gene inducer, and indeed wounded Z. mays tissues were found to produce substance(s) which induced the expression of A. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function of Agrobacterium vir genes.

virG of Agrobacterium tumefaciens plasmid pTiC58 encodes a DNA-binding protein.

Virulence genes of the Agrobacterium tumefaciens Ti plasmid are positively regulated by the products of virA and virG. To study the DNA-binding properties of the VirG protein, a translational fusion between virG and the trpE gene of Escherichia coli was constructed, and antiserum was raised against the encoded fusion protein. Using this antiserum, a protein of Mr congruent to 29,000, a size similar to that calculated from the virG nucleotide sequence, was detected in an E. coli strain harbouring a virG expression vector. Both the virG protein and the fusion protein were found, by filter-binding and gel retardation analyses, to bind DNA nonspecifically. These data support an existing model for the two-component regulatory systems of bacteria.

Two chromosomal loci involved in production of exopolysaccharide in Agrobacterium tumefaciens.

The chromosomal locus pscA (exoC) of Agrobacterium tumefaciens LBA4301 has been cloned by complementation of the avirulent and exopolysaccharide (EPS)-deficient mutant LBA4301 pscA. We have also identified a new locus, termed psdA (polysaccharide depression) and located 16 kilobases from pscA in the A. tumefaciens chromosome, that negatively affects EPS production when it is present in more than one copy in A. tumefaciens LBA4301. Subcloning, transposon mutagenesis, and transcriptional analysis have been conducted for both loci and indicate that pscA and psdA are transcribed in the same orientation. Acidic-EPS assays showed that psdA depresses succinoglycan production and that its negative effect increases with the copy number of the gene. Virulence tests of psdA transconjugants on Datura stramonium showed no visible alteration in virulence, while LBA4301 pscA was totally avirulent.

Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58.

The virulence (vir) region of pTiC58 was screened for promoter activities by using gene fusions to a promoterless lux operon in the broad-host-range vector pUCD615. Active vir fragments contained the strongly acetosyringone-inducible promoters of virB, virC, virD, and virE and the weakly inducible promoters of virA and virG. Identical induction patterns were obtained with freshly sliced carrot disks, suggesting that an inducer is released after plant tissue is wounded. Optimal conditions for vir gene induction were pH 5.7 for 50 microM acetosyringone or sinapic acid. The induction of virB and virE by acetosyringone was strictly dependent on intact virA and virG loci. An increase in the copy number of virG resulted in a proportional, acetosyringone-independent increase in vir gene expression, and a further increase occurred only if an inducing compound and virA were present.