Maurice E. Snook

Engineering Secondary Metabolism in Maize Cells by Ectopic Expression of Transcription Factors

Manipulation of plant natural product biosynthesis through genetic engineering is an attractive but technically challenging goal. Here, we demonstrate that different secondary metabolites can be produced in cultured maize cells by ectopic expression of the appropriate regulatory genes. Cell lines engineered to express the maize transcriptional activators C1 and R accumulate two cyanidin derivatives, which are similar to the predominant anthocyanin found in differentiated plant tissues. In contrast, cell lines that express P accumulate various 3-deoxy flavonoids. Unexpectedly, P-expressing cells in culture also accumulate phenylpropanoids and green fluorescent compounds that are targeted to different subcellular compartments. Two endogenous biosynthetic genes (c2 and a1, encoding chalcone synthase and flavanone/dihydroflavonol reductase, respectively) are independently activated by ectopic expression of either P or C1/R, and there is a dose–response relationship between the transcript level of P and the degree to which c2 or a1 is expressed. Our results support a simple model showing how the gene encoding P may act as a quantitative trait locus controlling insecticidal C-glycosyl flavone level in maize silks, and they suggest how p1 might confer a selective advantage against insect predation in maize.

Isolation and Characterization of Leu7-Surfactin from the Endophytic Bacterium Bacillus mojavensis RRC 101, a Biocontrol Agent for Fusarium verticillioides

Bacillus mojavensis is an endophytic bacterium patented for control of fungal diseases in maize and other plants. Culture extracts and filtrates from this bacterium were antagonistic to the pathogenic and mycotoxic fungus Fusarium verticillioides. However, the identity of the inhibitory substance from extracts of this bacterium has not been determined. An HPLC-MS analysis of the culture filtrate showed a major ion peak that was identified as a cyclic lipopeptide. Furthermore, collisional ion dissociation (CID) analysis indicated that this lipopeptide was surfactin, a cyclic heptapeptide linked to a β-hydroxy fatty acid. A CID analysis of the peptide moiety was established by deduction and indicated that the peptide sequence consisted of two acidic amino acids and five hydrophobic amino acids with a sequence of Leu-Leu-Asp-Val-Leu-Leu-Glu. These spectra indicated that this bacterium produced Leu(7)-surfactin, which was toxic to F. verticillioides. Production of this cyclic lipopeptide is a characteristic of several species of Bacillus, but this is the first report of this very powerful biosurfactant from this endophytic species.

Reproductive and Sphingolipid Metabolic Effects of Fumonisin B1 and its Alkaline Hydrolysis Product in LM/Bc Mice: Hydrolyzed Fumonisin B1 Did Not Cause Neural Tube Defects

Fumonisins are mycotoxins produced by Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisins are converted to their hydrolyzed analogs by alkaline cooking (nixtamalization). Both fumonisins and hydrolyzed fumonisins are found in nixtamalized foods such as tortillas, and consumption of tortillas has been implicated as a risk factor for neural tube defects (NTD). Fumonisin B(1) (FB(1)) induced NTD when given (ip) to pregnant LM/Bc mice; however, neither the NTD induction potential of hydrolyzed fumonisin B(1) (HFB(1)) nor its affect on sphingolipid metabolism in pregnant mice have been reported. The teratogenic potential of FB(1) and HFB(1) was therefore compared using the LM/Bc mouse model. Dams were dosed (ip) with 2.5, 5.0, 10, or 20 mg/kg (< or = 49 micromol/kg) body weight (bw) HFB(1) on embryonic day (E)7-E8. Negative and positive control groups were given vehicle or 10 mg/kg (14 micromol/kg) bw FB(1), respectively. The high dose of HFB(1) disrupted sphingolipid metabolism, albeit slightly, but did not cause maternal liver lesions or NTD (n = 8-10 litters per group). In contrast, 10 mg/kg bw FB(1) markedly disrupted maternal sphingolipid metabolism, caused hepatic apoptosis in the dams, increased fetal death rates, and decreased fetal weights. Furthermore, NTD were found in all FB(1)-exposed litters (n = 10), and 66 +/- 24% of the fetuses were affected. The findings indicate that HFB(1) does not cause NTD in the sensitive LM/Bc mouse model and only weakly disrupts sphingolipid metabolism at doses up to sevenfold higher (micromole per kilogram body weight basis) than the previously reported lowest observed adverse effect level for FB(1).

A maize QTL for silk maysin levels contains duplicated Myb-homologous genes which jointly regulate flavone biosynthesis.

The maize p1 locus coincides with a major QTL (quantitative trait locus) determining levels of maysin, a C-glycosyl flavone that deters feeding by corn ear-worm. The p1 gene is tightly linked with a second gene, p2, and both genes encode similar Myb-domain proteins. We show here that maize cell cultures transformed with either the p1or p2 genes expressed under a constitutive promoter accumulate transcripts for flavonoid biosynthetic genes, and synthesize phenylpropanoids and C-glycosyl flavones related to maysin. Additionally, maize plants that are deleted for the p1 gene have reduced maysin levels and moderate silk-browning reaction, whereas plants with a deletion of both p1 and p2 have non-detectable silk maysin and non-browning silks. We conclude that both p1 and p2 induce maysin biosynthesis in silk, although the two genes differ in their expression and pigmentation effects in other tissues. These results show that a QTL for flavone biosynthesis actually comprises two tightly linked genes with related functions.

Fumonisin concentrations and in vivo toxicity of nixtamalized Fusarium verticillioides culture material: evidence for fumonisin-matrix interactions.

The toxic potential of nixtamalized foods can be underestimated if, during cooking, reversible fumonisin-food matrix interactions reduce the amount of mycotoxin that is detected but not the amount that is bioavailable. Fusarium verticillioides culture material (CM) was nixtamalized as is (NCM) or after mixing with ground corn (NCMC). Additional portions were sham nixtamalized without (SCM) or with corn (SCMC). Nixtamalization and sham nixtamalization reduced FB(1); CM, NCM, and SCM diets contained 9.08, 2.08, and 1.19 ppm, respectively. FB(1) was further reduced in the NCMC (0.49 ppm) but not the SCMC (1.01 ppm) diets compared to their NCM and SCM counterparts. Equivalent weights of the cooked products, uncooked CM, corn (UC) or nixtamalized UC (NUC) were fed to rats for up to three weeks. Kidney lesions in the NCM-fed group were less severe than in the CM-fed, positive control group and no lesions were found in the NCMC and other groups. Group kidney sphinganine (biomarker of fumonisin exposure) concentrations decreased in the order: CM (absolute concentration (nmol/g)=600-800)>NCM (400-600)>SCM and SCMC (30-90)>NCMC, UC and NUC (<8). Together, these results suggest that mycotoxin-corn matrix interactions during nixtamalization reduce the bioavailability and toxicity of FB(1).

In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

Restriction fragment length polymorphism markers associated with silk maysin, antibiosis to corn earworm (Lepidoptera: Noctuidae) larvae, in a dent and sweet corn cross.

Abstract Maysin, a C-glycosylflavone in maize silk, has insecticidal activity against corn earworm, Helicoverpa zea (Boddie), larvae. Sweet corn, Zea mays L., is a vulnerable crop to ear-feeding insects and requires pesticide protection from ear damage. This study was conducted to identify maize chromosome regions associated with silk maysin concentration and eventually to transfer and develop high silk maysin sweet corn lines with marker-assisted selection (MAS). Using an F2 population derived from SC102 (high maysin dent corn) and B31857 (low maysin sh2 sweet corn), we detected two major quantitative trait loci (QTL). It was estimated that 25.6% of the silk maysin variance was associated with segregation in the genomic region of npi286 (flanking to p1) on chromosome 1S. We also demonstrated that a1 on chromosome 3L had major contribution to silk maysin (accounted for 15.7% of the variance). Locus a1 has a recessive gene action for high maysin with the presence of functional p1 allele. Markers umc66a (near c2) and umc105a on chromosome 9S also were detected in this analysis with minor contribution. A multiple-locus model, which included npi286, a1, csu3 (Bin 1.05), umc245 (Bin 7.05), agrr21 (Bin 8.09), umc105a, and the epistatic interactions npi286 × a1, a1 × agrr21, csu3 × umc245, and umc105a × umc245, accounted for 76.3% of the total silk maysin variance. Tester crosses showed that at the a1 locus, SC102 has functional A1 alleles and B31857 has homozygous recessive a1 alleles. Individuals of (SC102 × B31857) × B31857 were examined with MAS and plants with p1 allele from SC102 and homozygous a1 alleles from B31857 had consistent high silk maysin. Marker-assisted selection seems to be a suitable method to transfer silk maysin to sweet corn lines to reduce pesticide application.

Phenolic constituents in flax bast tissue and inhibition of cellulase and pectinase.

Flax bast tissue was sequentially extracted using hexane, propanol, methanol and water as solvents and extracts were analyzed using reverse phase HPLC and 13C NMR. Results indicated a large variety of aromatic constituents including flavonoids and hydroxy-methoxy cinnamic acids linked to oligosaccharides and hydroxy acids through glycosidic linkages. The extracts inhibited cellulase and pectinase activities and can thus influence retting.

Quantitative gas chromatographic method for the analysis of aliphatic hydrocarbons, terpenes, fatty alcohols, fatty acids and sterols in tobacco

Abstract A rapid and reproducible method is described for the analysis of the major tobacco lipids, including the C 25 C 34 paraffinic hydrocarbons, neophytadiene, phytol, docosanol, squalene, α-tocopherol, β-amyrin, cycloartenol, 24-methylenecycloartanol, palmitic acid, stearic acid, the unsaturated C 18 acids, cholesterol, stigmasterol, campesterol, sitosterol and solanesol. The method is based upon the short-column silicic acid chromatography of saponified lipids from a hexane extract of tobacco. The free lipids are separated into non-polar, polar and terpenoid fractions and quantitated by gas chromatography, with the use of internal standards. The applicability of the method has been demonstrated by the highly reproducigle analyses of a cigarette blend, conventional flue-cured tobaccos and two close-grown tobaccos. The method should also be applicable to analyses of lipids in other natural products, including foodstuffs.

Caffeoyltartronic acid from catnip (Nepeta cataria): A precursor for catechol in lubber grasshopper (Romalea guttata) defensive secretions

Adults of the lubber grasshopper (Romalea guttata) secrete increased amounts of catechol from their defensive glands when fed diets containing only catnip leaves (Nepeta cataria). Model compound bioassays showed that these insects were able to sequester and biomagnify simple phenols, such as catechol and hydroquinone, in their defense gland secretions. Excessive catechol secretions from caffeic acid-fortified diets indicated metabolic pathways exist to perform efficiently more complex biochemical conversions. Reverse-phase HPLC of methanol extracts of catnip revealed only one major caffeoyl-polyphenol as a possible precursor for the observed elevated catechol secretions, when this plant is fed to lubbers. The compound was shown to be caffeoyltartronic acid (CTA). During analysis of CTA by probe-MS or gas chromatography (of its silylated derivative), CTA decomposed by loss of carbon dioxide to form caffeoylglycolic acid (CGA), making identification by these methods ambiguous. Only fast atom bombardment mass spectrometry (FAB-MS, negative mode) gave a true molecular weight. Groundivy (Glecoma hederacea), a relative of catnip, was also shown to contain CTA. The mung bean (Phaseolus radiatus=Vigna radiata), a species totally unrelated to catnip, is the only other reported plant source of CTA. Catnip leaves were found to contain about twice as much CTA as mung bean leaves.