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Efficient decontamination of zearalenone, the mycotoxin of cereal pathogen, by transgenic yeasts through the expression of a synthetic lactonohydrolase gene.

Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, is converted to a non-estrogenic product by a detoxifying enzyme of Clonostachys rosea. Previously, we investigated whether recombinant Saccharomyces cerevisiae carrying this detoxification gene, zhd101, can remove 2 microg ml(-1) of ZEN in a liquid culture. Although the transgenic yeasts eliminated most of the ZEN, they also converted a significant amount to a poor substrate, beta-zearalenol, which remained in the medium. In this study, we synthesized a codon-optimized zhd101 gene and investigated whether the transgenic yeast strain can overcome the problem of insufficient detoxification of ZEN. Importantly, within 48 h of incubation at 28 degrees C or 8 h of incubation at 37 degrees C, the transgenic yeasts completely eliminated 2 microg ml(-1) of ZEN in the medium without accumulating even a trace amount of beta-zearalenol. The result suggests that incomplete ZEN detoxification attributed to the action of an endogenous yeast beta-reductase can be overcome by simply increasing the expression of the detoxifying gene.

Amino Acid Sequence↗

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw.

The gene ce1A, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the ce1A gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44,803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The ce1A gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 degrees C and pH 4.0. Study of the performance of Ce1A on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. Ce1A treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by Ce1A.

Amino Acid Sequence↗

Two uncoupling protein genes of rice (Oryza sativa L.): molecular study reveals the defects in the pre-mRNA processing for the heat-generating proteins of the subtropical cereal.

The recent finding of uncoupling proteins (UCPs) in plants has demonstrated that plant mitochondria contain at least two distinct types of potential heat-generating protein, i.e. uncoupling proteins (UCPs) and alternative oxidases. In this study, we isolated and characterized two rice genes encoding UCPs with the aim of improving cold tolerance of rice through the modification of UCP expression. The two rice nuclear genes, OsUCP1 and OsUCP2, appeared to encode functional UCPs, as far as the amino acid sequences of the gene products showed. However, our study revealed that the processing of the pre-mRNAs of both genes was defective. The defects in the pre-mRNA processing resulted in multiple abnormal forms of the transcripts, whose translation products seemed not to retain normal UCP activities. No OsUCP cDNA clones corresponding to the normal transcripts were detected in our study. A northern analysis, on the other hand, revealed that neither of the OsUCP genes exhibited cold-enhanced expression, whereas the gene expression for an alternative oxidase was strikingly enhanced in cold-treated leaves of rice. Our present data thus indicate that rice leaves, placed in cold environments, do not express functional UCPs.

Alternative Splicing↗

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus.

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice-maize-wheat synteny.

1,4-alpha-Glucan Branching Enzyme↗

Differential regulation of genes transcribed by nucleus-encoded plastid RNA polymerase, and DNA amplification, within ribosome-deficient plastids in stable phenocopies of cereal albino mutants.

We isolated stable albino plants of barley and maize by inhibiting plastid protein synthesis with streptomycin and propagating bleached seedlings in the absence of antibiotics in vitro. Albino plants are deficient in plastid translation products and plastid ribosomal RNAs, and are stable phenocopies of the barley albostrians and maize iojap mutants, which contain ribosome-free plastids. Once plastid ribosomes are lost they cannot be re-synthesized because about one-third of plastid ribosomal proteins are themselves plastid encoded. The group II/subgroup IIA intron in plastid rpl2 transcripts was not processed in albinos, providing strong evidence for the absence of plastid translation. Photosynthesis-related plastid mRNAs and plastid tRNAs were down-regulated in albino leaves. A differential influence of plastid ribosome deficiency on mRNA levels allowed us to divide genes transcribed by nucleus-encoded plastid RNA polymerase into two groups. Northern analysis revealed increases in the levels of clpP, rpl2, rpl23, rps15 and rpoB mRNAs in total RNA from albino leaves relative to those in green leaves. In contrast, albinism did not increase the band intensities of rps2 and rps4 messages. Plastid ribosome-associated factor(s) or plastid-encoded product(s) play a role in the initiation, termination, processing or stability of transcripts containing trnG(UCC) and rps4. Excision and 100-fold amplification of a 5.2-kb region of plastid DNA encompassing the trnG(UCC) and trnE(UUC) genes was observed in one of four albino barley plants. Gene amplification was correlated with the accumulation of abundant novel transcripts derived from regions flanking the trnG(UCC) gene.

Base Sequence↗

A gene coding for an RPS2 protein is present in the mitochondrial genome of several cereals, but not in dicotyledons.

A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2.

Amino Acid Sequence↗

Soil-borne rye mosaic and European wheat mosaic virus: two names for a furovirus with variable genome properties which is widely distributed in several cereal crops in Europe.

PCR and nucleotide sequence analyses have revealed that Soil-borne rye mosaic furovirus (SBRMV) which we have recently described is widely distributed in Europe. In Northern Germany, Poland and Denmark the virus affects mainly rye and triticale, but in France and Italy it is wheat which becomes infected. The partial RNA 1 and RNA 2 sequences which were determined for the various SBRMV sources form several clusters, but so far no correlation between molecular differences and the type of host which becomes infected under natural conditions was detected. European wheat mosaic furovirus which was recently described by Diao et al. (1999) [Virology 261: 331-339] is closely related to a French source of SBRMV.

Base Sequence↗

Effect of host defense chemicals on clonal distribution and performance of different genotypes of the cereal aphid Sitobion avenae.

Five microsatellite loci were used to study the genetic variability and population structure of Sitobion avenae (Hemiptera: Aphididae) on some of its host plants. Individuals were collected in Chile from different cultivated and wild Poaceae. Forty-four multilocus genotypes were found among the 1052 aphids analyzed, of which four represented nearly 90% of the sample. No specialist genotypes were found, although some preferred hosts endowed with chemical defenses, i.e., hydroxamic acids (Hx), while others preferred comparatively undefended hosts. Performances of some predominant and some rare genotypes were evaluated on plants differing in their Hx levels. Significant differences in performance were found among clones, the two most common genotypes showing no differences in performance among all hosts tested, and the rare genotypes showing enhanced performance on the host with highest Hx level. A hypothesis is proposed whereby the appearance of rarer genotypes is in part related to the presence of Hx.

Animals↗

Changes over time in the allelochemical content of ten cultivars of rye (Secale cereale L.).

Published studies focused on characterizing the allelopathy-based weed suppression by rye cover crop mulch have provided varying and inconsistent estimates of weed suppression. Studies were initiated to examine several factors that could influence the weed suppressiveness of rye: kill date, cultivar, and soil fertility. Ten cultivars of rye were planted with four rates of nitrogen fertilization, and tissue from each of these treatment combinations was harvested three times during the growing season. Concentrations of a known rye allelochemical DIBOA (2,4-dihydroxy-1,4-(2H)benzoxazine-3-one) were quantified from the harvested rye tissue using high performance liquid chromatography (HPLC). Phytotoxicity observed from aqueous extracts of the harvested rye tissue correlated with the levels of DIBOA recovered in harvested tissue. The amount of DIBOA in rye tissue varied depending on harvest date and rye cultivar, but was generally lower with all cultivars when rye was harvested later in the season. However, the late maturing variety 'Wheeler' retained greater concentrations of DIBOA in comparison to other rye cultivars when harvested later in the season. The decline in DIBOA concentrations as rye matures, and the fact that many rye cultivars mature at different rates may help explain why estimates of weed suppression from allelopathic agents in rye have varied so widely in the literature.

Amaranthus↗

Natural occurrence of mycotoxins in staple cereals from Ethiopia.

The occurrence of mycotoxins in barley, sorghum, teff (Eragrostis tef) and wheat from Ethiopia has been studied. Samples were analyzed for aflatoxin B(1) (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) using high performance liquid chromatography (HPLC) and for fumonisins (FUM) using enzyme linked immunosorbent assay (ELISA). AFB1 and OTA were detected in samples of all the four crops. AFB1 was detected in 8.8% of the 352 samples analyzed at concentrations ranging from trace to 26 microg kg(-1). OTA occurred in 24.3% of 321 samples at a mean concentration of 54.1 microg kg(-1) and a maximum of 2106 microg kg(-1). DON occurred in barley, sorghum and wheat at 40-2340 microg kg(-1) with an overall incidence of 48.8% among the 84 mainly 'suspect' samples analyzed; NIV was co-analyzed with DON and was detected at 40 microg kg(-1) in a wheat sample and at 50, 380, and 490 microg kg(-1) in three sorghum samples. FUM and ZEN occurred only in sorghum samples with low frequencies at concentrations reaching 2117 and 32 microg kg(-1), respectively. The analytical results indicate higher mycotoxin contamination in sorghum, which could be related to the widespread storage of sorghum grain in underground pits leading to elevated seed moisture contents. This is the first report on the occurrence of OTA in teff.

Aflatoxin B1↗

Molecular evidence for asymmetric evolution of sister duplicated blocks after cereal polyploidy.

Polyploidy (genome duplication) is thought to have contributed to the evolution of the eukaryotic genome, but complex genome structures and massive gene loss during evolution has complicated detection of these ancestral duplication events. The major factors determining the fate of duplicated genes are currently unclear, as are the processes by which duplicated genes evolve after polyploidy. Fine-scale analysis between homologous regions may allow us to better understand post-polyploidy evolution. Here, using gene-by-gene and gene-by-genome strategies, we identified the S5 region and four homologous regions within the japonica genome. Additional phylogenomic analyses of the comparable duplicated blocks indicate that four successive duplication events gave rise to these five regions, allowing us to propose a model for this local chromosomal evolution. According to this model, gene loss may play a major role in post-duplication genetic evolution at the segmental level. Moreover, we found molecular evidence that one of the sister duplicated blocks experienced more gene loss and a more rapid evolution subsequent to two recent duplication events. Given that these two recent duplication events were likely involved in polyploidy, this asymmetric evolution (gene loss and gene divergence) may be one possible mechanism accounting for the diploidization at the segmental level.

Chromosomes, Artificial, Bacterial↗

Effect of a barley breakfast cereal on blood glucose and insulin response in normal and diabetic patients.

Prowashonupana (Prowash) is a shrunken-endosperm, short awn, waxy starch, hulless barley with low starch, high fiber, high protein, and a relatively high concentration of free sugars. The study was designed to compare equivalent breakfast meals (w/w) of Prowash and oatmeal for glycemic response in diabetic and non-diabetic subjects. A commercial liquid meal replacer (LMR) was included as a reference standard. A substantial reduction of the post-prandial glycemic peak following ingestion of Prowash was observed as compared to LMR or oatmeal. In the non-diabetic subjects, the maximal rise in glucose from baseline was 26.3 +/- 3.9 mg/dL after LMR, 41.3 +/- 3.9 mg/dL after oatmeal and 6.4 +/- 2.7 mg/dL after Prowash (p < 0.01). The maximal increase in glucose in the diabetic patients was 69.9 +/- 4.5 mg/dL after LMR, 80.8 +/- 8.8 mg/dL after oatmeal and 28.4 +/- 3.5 mg/dL after Prowash (p < 0.01). The maximal increase in insulin post-LMR was 33.9 +/- 3.6 mIU/ml in the diabetic patients and 54.0 +/- 9.8 mIU/ml in the non-diabetic controls. Oatmeal elicited a maximal insulin increase of 29.9 +/- 4.2 mIU/ml in the control subjects and 21.4 +/- 2.5 mIU/ml in the diabetic patients. In contrast, the maximal insulin increase after Prowash was 8.6 +/- 1.5 mIU/ml in the non-diabetic controls and 6.8 +/- 1.2 mIU/ml in the diabetic patients (p < 0.01).

Avena↗