Correlation of Zn2+ content with aflatoxin content of corn.

Forty-nine samples from the 1983 Virginia corn harvest were analyzed for aflatoxin, zinc, copper, iron, and manganese content. Values (mean +/- standard deviation) were as follows: aflatoxin, 117 +/- 360 micrograms/kg; zinc, 22.5 +/- 3.4 mg/kg; copper, 2.27 +/- 0.56 mg/kg; iron, 40.8 +/- 18.7 mg/kg; and manganese, 5.1 +/- 1.1 mg/kg. Aflatoxin levels positively correlated with zinc (Spearman correlation coefficient, 0.385; P less than 0.006) and copper levels (Spearman correlation coefficient, 0.573; P less than 0.0001). Based on biochemical data in the literature, we believe that the correlation with zinc is important and that there may be a cause-and-effect relationship between zinc levels in corn and aflatoxin levels which are produced upon infection with Aspergillus flavus or A. parasiticus. Control of aflatoxin contamination in field corn by decreasing the zinc levels may be feasible, but no methods to decrease zinc levels are currently available.     

Spatio-Temporal Metabolite Profiling of the Barley Germination Process by MALDI MS Imaging

MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals’ germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest.     

The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfalfa

Summary We have shown by physical and genetic means that there are two megaplasmids in all strains of Rhizobium meliloti that we have studied. Megaplasmids from several strains of R. meliloti were mobilized to Agrobacterium tumefaciens and to other Rhizobium strains using the Tn5-Mob system. We were also able to resolve these two megaplasmids in agarose gels for most strains, and to show that only one of them hybridized to nif and nod genes. Transfer of this plasmid, the pSym, to Agrobacterium, R. leguminosarum, and R. trifolii strains conferred on these recipients the ability to nodulate alfalfa ineffectively. The second megaplasmid did not appear to have a direct role in nodule initiation. However, we were able to complement extracellular polysaccharide (EPS^-) mutants of R. meliloti by transferring this second megaplasmid into them. Furthermore, Tn5-induced EPS^- mutants of R. meliloti 2011, which produced ineffective (Fix^-) nodules of abnormal morphology, were shown by hybridization and complementation to carry mutations in this second megaplasmid. This demonstrates that both megaplasmids of R. meliloti are necessary for the effective nodulation of alfalfa.     

Structural relationship of an apolipoprotein (a) phenotype (570 kDa) to plasminogen: homologous kringle domains are linked by carbohydrate-rich regions

At least six allelic forms of apolipoprotein(a), differing in molecular mass, could be detected by immunoblot analysis. One of these phenotypes with a molecular mass of 570 kDa has been investigated. After reduction and carboxymethylation it was digested with trypsin and the resulting peptides were separated by gel filtration and reverse phase HPLC. The tryptic fragments sequenced comprised a total of 356 amino acids. The N-terminus of apo(a) was highly homologous to the start of the kringle 4 domain from human plasminogen and the majority of the tryptic peptides isolated was also homologous to sequences from this kringle. At least five homologous "kringle 4" domains are present in apolipoprotein(a) whereby one domain occurs more frequently than the others. A carbohydrate-rich peptide was also obtained in high yield. This glycopeptide connects two "kringle 4" domains and contains one N-glycoside within the kringle and six potential O-glycosides in the linking region. From the recovery it can be estimated that this peptide occurs several times within the whole apolipoprotein (a) sequence. The high carbohydrate content is in sharp contrast to that of human plasminogen. Other peptides sequenced indicate that apo (a) also contains domains homologous to the kringle 5 and protease regions of plasminogen. No unique peptides were found. These studies suggest that apolipoprotein (a) could have arisen through duplication of specific regions from the human plasminogen gene. The size heterogeneity of apo (a) might then be explained by differences in the numbers of gene duplications.     

Identification of a Cryptococcus neoformans gene that directs expression of the cryptic Saccharomyces cerevisiae mannitol dehydrogenase gene.

The Mtl gene from Cryptococcus neoformans, which confers the ability of Saccharomyces cerevisiae Sc4l YJO to grow on mannitol with substantial NAD-dependent mannitol dehydrogenase activity, was identified. Purifications and characterizations of this enzyme show that it is found in polyploid strain BB1, and the peptide sequence of the enzyme helped identify the saccharomyces gene encoding this mannitol dehydrogenase activity. On the other hand, the Mtl gene of C. neoformans encodes a 346-amino-acid protein which is not mannitol dehydrogenase but a regulatory element which is active in a heterologous fungus.     

Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L.) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst. Received: 23 November 1998 / Accepted: 23 June 1999     

Lipid microdomain polarization is required for NADPH oxidase-dependent ROS signaling in Picea meyeri pollen tube tip growth

The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca²⁺ gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.