Larry G. Butler

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.

Choosing appropriate methods and standards for assaying tannin

Tannins are chemically diverse polyphenolics that have multiple biological activities. Attempts to establish the ecological significance of tannins have been hindered by the complexities of tannin analysis. A multitude of analytical procedures for tannins has been described, but it is difficult for the nonspecialist to select appropriate methods. We have classified the most common procedures for determining tannin as either chemical assays, appropriate for determining the amount and the chemical nature of the tannin in a sample, or as protein-binding assays, suitable for determining the potential biological activity of the tannin in a sample. We have recommended procedures that are particularly reliable and straightforward for general use. We have also considered the problems encountered in selecting appropriate standards for tannin analysis and have recommended standards that are readily available.

Interactions of condensed tannins with selected proteins

Abstract The relative affinities of condensed tannins purified from sorghum, pinto bean, quebracho and wattle for six dissimilar proteins have been determined by a competitive binding assay. The results indicate that tannin/protein interactions may be specific for different tannins as well as for different proteins. The highly specific interactions suggest that the differences in affinity are functionally significant.

Natural astringency in foodstuffs — A molecular interpretation

The structures of plant polyphenols (vegetable tannins) are briefly reviewed. Their interactions with proteins, polysaccharides, and the alkaloid caffeine are discussed at the molecular level, and these fundamental properties are related to the quality of astringency that polyphenols possess. The various ways in which astringency may be modified and ultimately lost are outlined in relation to the aging of red wines, the formation of nonbiological hazes in beers and lagers, and the ripening of fruit.

Phenylalanine ammonia-lyase and hydroxycinnamate: CoA ligase in maize mesocotyls inoculated with Helminthosporium maydis or Helminthosporium carbonum☆

Abstract Phenylalanine ammonia-lyase (E.C. 4.3.1.5) (PAL) and hydroxycinnamate:CoA ligase (E.C. 6.2.1.12) activities were measured in extracts from maize mesocotyls resistant and susceptible to Helminthosporium maydis and resistant to H. carbonum . CoA ligase activity increased in response to infection with H. maydis in both the resistant and susceptible cultivars. Activity began to increase between 6 and 9 h after inoculation and in the resistant cultivar continued to increase throughout a 48-h period. In susceptible cultivars activity ceased to increase at approximately 12 h after inoculation. The results demonstrate that the increase in CoA ligase activity is detectable as early as the onset of penetration by the fungus. No significant change in PAL activity was observed in either resistant or susceptible combinations with H. maydis , suggesting that PAL and CoA ligase are not coordinately regulated in interactions involving this fungus. Neither enzyme was found to change as a result of inoculation of any cultivar with H. carbonum .

Enzymes in non-aqueous solvents

Abstract For a variety of reasons including increased recognition of the large degree of association, by non-polar interaction, of enzymes with other cellular components such as membranes, enzymes are increasingly being investigated in mixed solvents less polar than water. Such solvents may be quite relevant because their polarity more nearly resembles the natural cellular microenvironment than does pure water. The single most important criterion in selecting a non-aqueous solvent is its compatibility with the maintenance of the enzymes catalytic activity, which must be determined experimentally for each enzyme. Non-aqueous solvents have a variety of effects on enzymes: they may bind specifically, compete with substrate binding, dissociate multimers, shift an equilibrium between two enzyme conformations, alter the amount of helix, react with the enzyme, stabilize or destabilize the enzyme, and affect the rate of the catalytic reaction in several different ways. Typically, modest concentrations of hydroxylic solvents have little effect on rates, and may even enhance the rate significantly. Higher concentrations give lower rates, in a solvent-specific and enzyme-specific manner. Hydroxylic solvents may replace water as acceptor of a phosphoryl, glycosyl, or acyl group produced by a hydrolytic enzyme. Non-aqueous solvents also make it possible to run hydrolytic reactions in the reverse direction, forming a condensation product and water as a by-product. Non-aqueous solvents are being extensively used in cryoenzymology as antifreeze agents, in solubilizing and purifying enzymes, and to a lesser degree in two-phase systems in which the non-polar substrate is dissolved in the non-aqueous phase. Liquefied aqueous phenol is an extraordinary solvent for enzymes and other proteins. It is a powerful denaturant which rapidly and irreversibly extracts the enzyme into the phenol-rich phase of a phenol-water system. This property makes phenol useful for removing protein contaminants, and for detecting labile enzyme-substrate intermediates, by extracting substrate covalently bound to enzyme into the phenol-rich phase away from all other substrate, which generally remains in the aqueous phase.

Use of dye-labeled protein as spectrophotometric assay for protein precipitants such as tannin.

Bovine serum albumin has been covalently labeled with Remazol brilliant blue R to provide a substrate for a convenient spectrophotometric assay for protein precipitants. The blue protein is especially useful for measuring protein precipitation by vegetable tannins because its absorption maximum is at a wavelength where plant pigments exhibit minimum absorption. Blue BSA has been used to determine, by competition experiments, the relative affinity of various proteins for tannins. A procedure for purifying condensed tannin from commercially available quebracho extract is described.

Interaction of proteins with sorghum tannin: Mechanism, specificity and significance

The grain of some varieties of sorghum contains 2% or more condensed tannin; many other varieties contain no tannin at all. Agronomic advantages, e.g., resistance to bird depredation, are associated with high-tannin sorghums, which have relatively low nutritional value for nonruminants. The biological effects of tannin are a result of its propensity for binding proteins; both hydrogen bonding and hydrophobic interactions are involved. Sorghum tannins can bind dietary proteins and reduce their digestibility. Purified digestive enzymes are inhibited by tannin, but significant inhibition in vivo is unlikely. Proteins differ greatly in their affinity for tannin. Those with highest affinity are large, have an open structure, contain no bound carbohydrate and are rich in proline. Sorghum proteins of the alcohol-soluble prolamine fraction associate strongly with tannin, are difficult to remove during tannin purification and are found combined with tannin in the indigestible residue after in vitro digestion with pepsin. On germination, the seed may sacrifice a portion of these proteins to bind the tannin that might otherwise interfere with metabolism by inhibiting seed enzymes. During seed development, tannin molecules are relatively short and do not effectively precipitate proteins; as the seed dries, tannins undergo polymerization to an average of ca. 6 flavan-3-ol units/molecule. The antinutritional effects of sorghum tannins can be eliminated by soaking the grain in dilute aqueous alkali, but not by cooking. When rats are put on high-tannin sorghum diets, their parotid glands undergo hypertrophy and produce a group of unique salivary proteins with extremely high affinity for tannin. These proteins contain over 40% proline and are devoid of sulfur-containing and aromatic amino acids. This metabolic adaption may protect rats against tannin by binding and inactivating it immediately when it enters the digestive tract.

Selecting sorghum genotypes expressing a quantitative biosynthetic trait that confers resistance to Striga

Abstract One of the best characterized mechanisms of host resistance to witchweeds ( Striga spp.) is exudation by host plant roots of relatively low amounts of compounds that Striga seeds require as stimulants for germination. We find that all sorghums tested, regardless of whether they are susceptible or resistant to Striga , produce equivalent amounts of sorgoleone, the alkylated hydroquinone we previously identified from sorghum root exudate as the first host-derived Striga germination stimulant to be characterized. In contrast, some highly resistant sorghums produce relatively tiny amounts of another germination stimulant, as yet unidentified but with properties quite different from sorgoleone, whereas all susceptible sorghums produce relatively large amounts. A simple, rapid and nondestructive agar gel assay which detects this second stimulant, but not sorgoleone, reliably distinguishes low ( Striga resistant) and high ( Striga susceptible) stimulating parental genotypes and progenies from crosses among them. Because the lack of a reliable and rapid method for screening germplasm and breeding progenies has hampered the development of crop varieties resistant to Striga , this gel assay is expected to greatly enhance the efficiency of sorghum breeding for Striga resistance.

Structure and evolution of the genomes ofsorghum bicolor andZea mays.

Cloned maize genes and random maize genomic fragments were used to construct a genetic map of sorghum and to compare the structure of the maize and sorghum genomes. Most (266/280) of the maize DNA fragments hybridized to sorghum DNA and 145 of them detected polymorphisms. The segregation of 111 markers was analyzed in 55 F2 progeny. A genetic map was generated with 96 loci arranged in 15 linkage groups spanning 709 map units. Comparative genetic mapping of sorghum and maize is complicated by the fact that many loci are duplicated, often making the identification of orthologous sequences ambiguous. Relative map positions of probes which detect only a single locus in both species indicated that multiple rearrangements have occurred since their divergence, but that many chromosomal segments have conserved synteny. Some sorghum linkage groups were found to be composed of sequences that detect loci on two different maize chromosomes. The two maize chromosomes to which these loci mapped were generally those which commonly share duplicated sequences. Evolutionary models and implications are discussed.