Daniel P. O'Keefe

Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent.

Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke

The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites.

Gland Development on Leaf Surfaces of Nepeta racemosa

Trichomes present on the leaf surface of catmint (Nepeta racemosa L.) were examined using both scanning and transmission electron microscopy. Three types of trichomes were observed: (a) multicellular, uniseriate, nonglandular hairs, (b) small capitate glands with two cells in the head, and (c) large, peltate glands with four secretory/head cells. Secretory cells of capitate glands exhibited typical transfer cell morphology, with an abundance of RER and Golgi bodies, indicative of secretion of mainly hydrophilic substances. Mature peltate glands exhibited a distended cuticle and large subcuticular cavity, presumably containing the essential oils. Morphologically well-developed peltate glands were observed on young leaf primordia, with development occurring earlier at the leaf apex and along the midvein than at the leaf margins or base. Four major stages in the development of the peltate gland secretory cells were identified. The first stage was marked by highly vacuolated cytoplasm and normal distribution of subcellular organelles. The second stage, during which there was substantial accumulation of material in the subcuticular cavity, was marked by an abundance of tubular endomembrane elements in proximity to plastids with little internal differentiation. Retraction from the cell wall and an unusual scalloped appearance to the plasmalemma characterized a third stage. Presence of a large subcuticular cavity, an exaggerated retraction of the plasmalemma, and a band of peripheral mitochondria were typical of a fourth, presumably mature stage.

Identification of constitutive and herbicide inducible cytochromes P-450 in Streptomyces griseolus

The elevated soluble cytochrome P-450 content of Streptomyces griseolus cells found after growth in the presence of sulfonylurea herbicides has been shown to be the result of the appearance of one predominant cytochrome P-450 form. This cytochrome P-450 is the major soluble protein found to increase in amount following herbicide treatment, and functions as part of a sulfometuron methyl hydroxylase system. A second minor inducible cytochrome P-450 has been observed only in cells grown in the presence of chlorimuron ethyl, and a third cytochrome P-450 has been found to be present in all cells independent of the presence of sulfonylurea inducers. The three cytochrome P-450 isozymes are distinguishable primarily by their anion exchange properties; however, spectral properties, substrate inducibility, and enzymatic activity provide several further distinguishing features. The recognition of these inducible, xenobiotic metabolizing cytochromes P-450 in S. griseolus provides the only known description of monooxygenase proteins related to herbicide metabolism in bacteria.

Induction of cytochrome P-450-dependent sulfonylurea metabolism in Streptomyces griseolus.

Inducible cometabolism of several sulfonylurea herbicides by Streptomyces griseolus has been shown to occur by hydroxylation, O-dealkylation, or deesterification reactions. Only after growth of the bacterium in the presence of sulfonylurea did cell-free extracts exhibit NAD(P)H-dependent sulfonylurea metabolism. These extracts were shown to contain elevated levels of soluble cytochrome P-450 and exhibit sulfonylurea induced difference spectra consistent with binding of substrate to cytochrome(s) P-450. These results establish the presence of an inducible cytochrome P-450-dependent sulfonylurea metabolizing system in S. griseolus.

Cytochrome P-450-catalysed monoterpenoid oxidation in catmint (Nepeta racemosa) and avocado (Persea americana); evidence for related enzymes with different activities

A cytochrome P-450 present in ripening avocado (Persea americana) fruit mesocarp (CYP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.

Structure and function of the chloroplast cytochrome bf complex

The chloroplast cytochrome bf complex is an intrinsic multisubunit protein from the thylakoid membrane consisting of four polypeptides: cytochrome f, a two heme containing cytochrome b6, the Rieske iron-sulfur protein, and a 17 kD polypeptide of undefined function. The complex functions in electron transfer between PSII and PSI, where most mechanisms suggest that the transfer of a single reducing equivalent from plastoquinol to plastocyanin results in the translocation of two protons across the membrane. Primary sequence analyses, dichroism studies, and functional considerations allow the construction of an approximate structural model of a monomeric complex, although some evidence exists for a dimeric structure. Resolution of the properties of the two cytochrome b6 hemes has relied upon the availability of purified solubilized complex, while evidence in the thylakoid suggests the difference between the two hemes are not as great in situ. Such variability in the spectroscopic and electrochemical properties of the cytochrome b6 is a major concern during the experimental use of the purified complex. There is a general consensus that the complex contains a plastoquinol oxidizing (Qz) site, although the evidence for a plastoquinone reduction (Qc) site, called for in most mechanistic hypotheses, is less substantive. Probably the most severe challenge to the so called Q-cycle mechanism comes from experimental observations made with cytochrome b6 initially reduced, where proposed interpretations more closely resemble a b-cycle than a Q-cycle. Although functional during cyclic electron transfer, the role of the complex and its possible interaction with other proteins, has not been completely resolved.

Sites of cytochrome b-563 reduction, and the mode of action of DNP-INT and DBMIB in the chloroplast cytochrome b-563/f complex

When ferredoxin is the reductant of cytochrome b‐563 in the chloroplast cytochrome b‐563/f complex, the reaction rate varies with pH in a manner which permits identification of a plastoquinone/plastosemiquinone couple as an intermediate in the reaction. The requirement for the inhibitor, DNP‐INT, in this reaction, suggests that there are two sites in the complex where plastosemiquinone can react with cytochrome(s) b‐563. The ineffectiveness of DBMIB in promotion of ferredoxin‐mediated cytochrome b‐563 reduction has led to a model where DBMIB and DNP‐INT inhibit different electron transfer steps at the same, or overlapping, sites in the complex.

Isolation and characterization of Streptomyces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism

SummaryMetabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450SU1 and P-450SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450SU2 in the absence of P-450SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.