Daniel K. Owens

Functional Analysis of a Predicted Flavonol Synthase Gene Family in Arabidopsis

The genome of Arabidopsis (Arabidopsis thaliana) contains five sequences with high similarity to FLAVONOL SYNTHASE1 (AtFLS1), a previously characterized flavonol synthase gene that plays a central role in flavonoid metabolism. This apparent redundancy suggests the possibility that Arabidopsis uses multiple isoforms of FLS with different substrate specificities to mediate the production of the flavonols, quercetin and kaempferol, in a tissue-specific and inducible manner. However, biochemical and genetic analysis of the six AtFLS sequences indicates that, although several of the members are expressed, only AtFLS1 encodes a catalytically competent protein. AtFLS1 also appears to be the only member of this group that influences flavonoid levels and the root gravitropic response in seedlings under nonstressed conditions. This study showed that the other expressed AtFLS sequences have tissue- and cell type-specific promoter activities that overlap with those of AtFLS1 and encode proteins that interact with other flavonoid enzymes in yeast two-hybrid assays. Thus, it is possible that these “pseudogenes” have alternative, noncatalytic functions that have not yet been uncovered.

Biochemical Markers and Enzyme Assays for Herbicide Mode of Action and Resistance Studies

Agricultural Research Service, U.S. Department of Agriculture, Plant Physiologist, Plant Physiologist, Support Chemist, and Biological Science Laboratory Technician, Natural Products Utilization Research Unit

Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (K(m)(app)=67 microM; V(max)=20.45 pKat/microg), kaempferol (K(m)(app)=12 microM; V(max)=11.63 pKat/microg), and myricetin (K(m)(app)=33 microM; V(max)=12.21 pKat/microg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu(2+), Fe(2+), and Zn(2+) as well as UDP (K(i)(app)=69.5 microM), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.

Evolution of resistance to phytoene desaturase and protoporphyrinogen oxidase inhibitors--state of knowledge.

Two major classes of herbicides include inhibitors of protoporphyrinogen oxidase (PPO) and phytoene desaturase (PDS). Plants can evolve resistance to PPO and PDS inhibitors via several mechanisms that include physical changes, resulting in reduced uptake, physiological changes, resulting in compartmentalization or altered translocation, and biochemical changes, resulting in enhanced metabolic degradation or alterations of protein structures, leading to loss of sensitivity to the herbicides. This review discusses the involvement of some of these mechanisms in the various cases of resistance to PDS- and PPO-inhibiting herbicides, and highlights unique aspects of target-site resistance to these herbicides.

Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.

Biosynthesis and Function of Citrus Glycosylated Flavonoids

Citrus is one of the major crops in the world with notable consumption of fresh fruit and processed fruit products. Flavonoids and flavonoid glycosides in Citrus have an impact on consumer acceptance of fruit and fruit products as well as affecting human health. Glycosylation of flavonoids is a key modification process leading to the production of the compounds actually found in plant tissues. Citrus is known for synthesis and accumulation of significant levels of flavanone and flavone glycosides. Biosynthesis of the core flavonoids and modification reactions resulting in the synthesis of flavonoid glycosides in Citrus are reviewed, with emphasis on Citrus flavonoid glycosyltransferases.

Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® cotton

AbstractMain conclusionInsertion of the gene encoding phosphinothricin acetyltransferase (PAT) has resulted in cotton plants resistant to the herbicide glufosinate. However, the lower expression and commensurate reduction in PAT activity is a key factor in the low level of injury observed in theWideStrike®cotton and relatively high level of resistance observed in LibertyLink®cotton. LibertyLink® cotton cultivars are engineered for glufosinate resistance by overexpressing the bar gene that encodes phosphinothricin acetyltransferase (PAT), whereas the insect-resistant WideStrike® cultivars were obtained using the similar pat gene as a selectable marker. The latter cultivars carry some level of resistance to glufosinate which enticed certain farmers to select this herbicide for weed control with WideStrike® cotton. The potency of glufosinate on conventional FM 993, insect-resistant FM 975WS, and glufosinate-resistant IMACD 6001LL cotton cultivars was evaluated and contrasted to the relative levels of PAT expression and activity. Conventional cotton was sensitive to glufosinate. The single copy of the pat gene present in the insect-resistant cultivar resulted in very low RNA expression of the gene and undetectable PAT activity in in vitro assays. Nonetheless, the presence of this gene provided a good level of resistance to glufosinate in terms of visual injury and effect on photosynthetic electron transport. The injury is proportional to the amount of ammonia accumulation. The strong promoter associated with bar expression in the glufosinate-resistant cultivar led to high RNA expression levels and PAT activity which protected this cultivar from glufosinate injury. While the insect-resistant cultivar demonstrated a good level of resistance to glufosinate, its safety margin is lower than that of the glufosinate-resistant cultivar. Therefore, farmers should be extremely careful in using glufosinate on cultivars not expressly designed and commercialized as resistant to this herbicide.

Sarmentine, a natural herbicide from Piper species with multiple herbicide mechanisms of action

Sarmentine, 1-(1-pyrrolidinyl)-(2E,4E)-2,4-decadien-1-one, is a natural amide isolated from the fruits of Piper species. The compound has a number of interesting biological properties, including its broad-spectrum activity on weeds as a contact herbicide. Initial studies highlighted a similarity in response between plants treated with sarmentine and herbicidal soaps such as pelargonic acid (nonanoic acid). However, little was known about the mechanism of action leading to the rapid desiccation of foliage treated by sarmentine. In cucumber cotyledon disc-assays, sarmentine induced rapid light-independent loss of membrane integrity at 100 μM or higher concentration, whereas 3 mM pelargonic acid was required for a similar effect. Sarmentine was between 10 and 30 times more active than pelargonic acid on wild mustard, velvetleaf, redroot pigweed and crabgrass. Additionally, the potency of 30 μM sarmentine was greatly stimulated by light, suggesting that this natural product may also interfere with photosynthetic processes. This was confirmed by observing a complete inhibition of photosynthetic electron transport at that concentration. Sarmentine also acted as an inhibitor of photosystem II (PSII) on isolated thylakoid membranes by competing for the binding site of plastoquinone. This can be attributed in part to structural similarities between herbicides like sarmentine and diuron. While this mechanism of action accounts for the light stimulation of the activity of sarmentine, it does not account for its ability to destabilize membranes in darkness. In this respect, sarmentine has some structural similarity to crotonoyl-CoA, the substrate of enoyl-ACP reductase, a key enzyme in the early steps of fatty acid synthesis. Inhibitors of this enzyme, such as triclosan, cause rapid loss of membrane integrity in the dark. Sarmentine inhibited the activity of enoyl-ACP reductase, with an I50app of 18.3 μM. Therefore, the herbicidal activity of sarmentine appears to be a complex process associated with multiple mechanisms of action.

Secondary product glucosyltransferase and putative glucosyltransferase expression during Citrus paradisi (c.v. Duncan) growth and development

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.

Phytochemicals for Pest Management: Current Advances and Future Opportunities

As with pharmaceuticals, a significant proportion of commercial pesticides are natural molecules or are derived from natural compounds. This review describes some of the past commercial successes of phytochemicals as pesticides by pesticide class as well as current work and future prospects for development of pesticides from plant-derived natural compounds. For example, two compounds isolated by assay-guided fractionation of the essential oil of American beautyberry (Callicarpa americana L.) (Verbenaceae), callicarpenal and intermediol, were found to have very potent insect repellent properties. An analysis of the number of new phytochemicals being discovered yearly and the relatively few bioassays for potential pesticidal activity that most of the known phytochemicals have been subjected to, indicates that this area still has a bright future. Furthermore, chemical modification of these compounds and their use to discover new modes of action greatly expand the scope for future work. In addition, the use of transgene technology holds great promise, not only to protect crops from pests, by imparting production or manipulation of production of pest management phytochemicals, but also for crop/weed allelopathy, as success in this effort would greatly decrease the most used form of synthetic pesticides, herbicides.