Antoaneta B. Kroumova

A pathway for the biosynthesis of straight and branched, odd- and even-length, medium-chain fatty acids in plants.

Pathways and enzymes of fatty acid synthase-mediated, long-even-chain (generally C16-C20) fatty acid synthesis are well studied, and general metabolism involved in short-chain (C4-C7) fatty acid biosynthesis is also understood. In contrast, mechanisms of medium-chain (C8-C14) fatty acid synthesis are unclear. Recent work suggests involvement of chain-elongation-terminating thioesterases in medium-chain fatty acid formation in oilseeds and animals. We have shown that iso- and anteiso-branched and straight, odd- and even-length, short-chain fatty acids esterified in plant-trichome-gland-produced sucrose esters are synthesized by using carbon skeletons provided by modified branched-chain amino acid metabolism/catabolism. The principal enzymes involved are those catalyzing leucine biosynthesis in all organisms and those leading to short-chain alcohols in mutant yeasts and alkyl acids in Clostridium species (products often serving as mammalian pheromones). Here we provide evidence that C10-C12 straight medium-chain and C10-C12 branched medium-chain acyl acids of tomato, C6-C8 straight-chain acyl acids of Petunia, and C6 and C8 branched acyl acids of Nicotiana glutinosa are formed by alpha-ketoacid elongation without participation of fatty acid synthase-mediated reactions or -independent thioesterases. This different metabolism suggests greater integration of amino acid and fatty acid metabolism than previously considered and provides other avenues to study and manipulate not only straight even-length but also odd- and even-length straight and branched medium-chain fatty acid biosynthesis.

Impacts of T-Phylloplanin Gene Knockdown and of Helianthus and Datura Phylloplanins on Peronospora tabacina Spore Germination and Disease Potential

T-phylloplanin proteins secreted to aerial surfaces of tobacco (Nicotiana tabacum) by short procumbent trichomes inhibit spore germination and blue mold disease caused by the oomycete pathogen Peronospora tabacina. Many other plants were found to contain water-washed leaf surface proteins (phylloplanins), but the functions and properties of these are not known. Here we extend earlier evidence for the antifungal activity of T-phylloplanins using a reverse genetics approach. RNA interference of the T-phylloplanin gene in tobacco ‘T.I. 1068’ resulted in loss of T-phylloplanin mRNA and protein, loss of in vitro spore germination inhibition activity, and leaf infection inhibition activity of leaf water washes from RNA interference plants, and young knockdown plants were susceptible to disease. The glycoprotein character, adaxial-leaf-surface enrichment of, and renewability of T-phylloplanins are also described. We also report that leaf water washes of sunflower (Helianthus annuus) and jimson weed (Datura metel), but not soybean (Glycine max), like that of tobacco, possess ProteinaseK- and boiling-sensitive P. tabacina spore germination and tobacco leaf infection inhibition activities. Results establish that T-phylloplaninins of tobacco are active in P. tabacina inhibition, and indicate that leaf surface proteins of certain non-Nicotiana species that are not susceptible to P. tabacina disease can inhibit germination of spores of this oomycete pathogen and inhibit tobacco leaf infection by this pathogen.

Expression of an apoplast-directed, T-phylloplanin-GFP fusion gene confers resistance against Peronospora tabacina disease in a susceptible tobacco

Key messagePhylloplanins are plant-derived, antifungal glycoproteins produced by leaf trichomes. Expression of phylloplanin-GFP fusion gene to the apoplast of a blue mold susceptible tobacco resulted in increased resistance to this pathogen.AbstractTobaccos and certain other plants secrete phylloplanin glycoproteins to aerial surfaces where they appear to provide first-point-of-contact resistance against fungi/fungi-like pathogens. These proteins can be collected by water washing of aerial plant surfaces, and as shown for tobacco and a sunflower phylloplanins, spraying concentrated washes onto, e.g., turf grass aerial surfaces can provide resistance against various fungi/fungi-like pathogens, in the laboratory. These results suggest that natural-product, phylloplanins may be useful as broad-selectivity fungicides. An obvious question now is can a tobacco phylloplanin gene be introduced into a disease-susceptible plant to confer endogenous resistance. Here we demonstrate that introduction of a tobacco phylloplanin gene—as a fusion with the GFP gene—targeted to the apoplasm can increase resistance to blue mold disease in a susceptible host tobacco.

Mechanisms for elongation in the biosynthesis of fatty acid components of epi-cuticular waxes

It is known that branched-chain amino acids can serve as precursors to iso- and anteiso-branched components of epi-cuticular waxes. Keto acid deamination products of Val, Leu and Ile are thought to serve as primers which are elongated by fatty acid synthase. However, the origin of elongation carbons has not been studied directly. Nor has the mechanism for formation of odd-carbon-length, straight- or branched-chain, cuticular ester fatty acids or free odd-carbon-length, straight fatty acid components of waxes been characterized. It is not known that α-oxidation of even-length precursors or elongation of odd-length primers is involved in these cases. Here, we present evidence which substantiates the expectation that elongation of branched as well as straight-chain precursors to wax ester acids occurs by fatty acid synthase catalyzed by addition of two carbon units via acetate. Also, we present evidence which indicates that odd-carbon-length acids can result from elongation of odd-carbon-length primers (at least branched), rather than even-length acids shortened by α-oxidation.

Biochemical observations on medium-chain-length polyhydroxyalkanoate biosynthesis and accumulation in Pseudomonas mendocina.

Certain Pseudomonads are capable of accumulating high levels of medium-chain-length polyhydroxyalkanates (PHAmcl) when grown with carbohydrates as the main carbon source. 3-OH acyl components of PHAmcl are derived from fatty acid synthase (FAS) and these components are accessed by action of 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase (transacylase). However, little is known with regard to the time courses of 3-OH acyl component occurrence and of transacylase activity during PHAmcl induction. Also, little is known with regard to the coupling mechanism between FAS and PHAmcl synthesis or whether the FAS pathway itself is specialized in PHAmcl-producing cells. Our results with regard to the time course of formation of 3-OH acids, 3-OH acyl-ACPs, and PHAmcl are consistent with the view that transacylase provides the key link between FAS and PHAmcl synthase. They also suggest that FAS specialization is not a feature of the mechanism. Further, we observed the formation of a 3-OH 10:0 homopolymer early in the induction phase followed by later formation of a mixed polymer containing 3-OH 8:0 and 3-OH 12:0 in addition to 3-OH 10:0. Early occurrence of 3-OH 10:0-CoA transacylase activity was coincident with homopolymer formation.

The Use of RNAi to Elucidate and Manipulate Secondary Metabolite Synthesis in Plants

RNA interference technology (RNAi, dsRNA-mediated gene silencing) has already had a major impact on the study and manipulation of plant secondary metabolites. To date RNAi has mainly been used as a readily available, rapid, reverse genetics tool to create plants with novel chemical phenotypes, and to determine the phenotypes of genes responsible for the synthesis of many different secondary metabolites. These manipulations have also greatly facilitated the identification and improvement of specific plant-insect and plant-pathogen interactions, and have set the stage for greater exploitation of plants to produce commercially-valuable, plant-derived drugs, flavoring agents, perfumes, etc. RNAi has been used to study and manipulate products that are representatives of all three main groups of plant secondary metabolites, the phenylpropanoids (and allied phenolics), alkaloids, and terpenoids. We predict that because there exists so much diversity in chemical structure among plant secondary metabolites, and RNAi is highly efficient, foreign gene expression together with RNAi will undoubtedly play an increasingly important role in enabling plants to produce renewable chemical feed stocks now obtained from petroleum. Thus, various forms of RNAi will be very important in the post “peak oil” future of agriculture, worldwide. After a brief general introduction to plant secondary metabolites, we will survey recent studies that have used RNAi for phenotyping of secondary metabolite related genes, and to generating novel chemical phenotypes, pointing out the advantages gained. A few examples will be highlighted to exemplify the powerful approach of gene knockdown combined with foreign gene overexpression as a means for creating new secondary-product-based phenotypes (e.g., the blue rose). Where data is available, we will compare knockdown efficiency and stability of antisense, co-suppression and dsRNAi. As a case study, we will describe in more detail how RNAi has been used to chart carbon flow among branch pathways of diterpenoid biosynthesis in trichome glands. Finally we will discuss the potential of artificial miRNAi for S.-Y. Ying (ed.) Current Perspectives in microRNAs (miRNA), 431

Use of Several Natural Products from Selected Nicotiana Species to Prevent Black Shank Disease in Tobacco

SUMMARY Black shank is a major annual disease threat to all types of tobacco worldwide. It is caused by the fungus Phytophthora parasitica var. nicotianae (PPN). The major tobacco growing areas in US - Kentucky, Tennessee and North Carolina can experience devastating losses, reaching in some fields up to 100%. Thus far, the main approaches to control this disease have been creation of resistant varieties, fungicide treatments, and crop rotation. Some fungicides are reported to have negative effects on the environment. The goal of this work was to test the antifungal activity of several natural products that are synthesized by certain Nicotiana species, and secreted to the leaf surface. We hypothesized that phylloplanin, cis-abienol, labdenediol and sclareol can suppress PPN-race 0- and PPN-race 1-caused disease in Burley tobaccos KY 14 and MS KY 14 × L8LC in the greenhouse. We developed methods for leaf surface extraction, spore preparation and soil drench application of the natural compounds tested. Experiments were performed on 5–8 week-old greenhouse grown seedlings. cis-Abienol showed high inhibitory properties toward the disease. Race 0 infection was completely subdued in KY 14 while race 1 infection was reduced by 70–80%, and delayed by 6–10 days in KY 14 and MS KY14 × L8LC. Sclareol was very effective in inhibiting race 0-caused disease in both tobacco cultivars. In MS KY 14 × L8LC race 1 infection was inhibited while in KY 14 it was reduced by 85% and delayed by 6 days. Labdenediol reduced the disease by half in eight week-old KY 14 plants. Tobacco phylloplanin reduced plant infection by both races by 50–60% and delayed the disease by 6–10 days. Phylloplanin was least suppressive in both tobacco cultivars. We consider sclareol to be the best candidate for future studies due to its antifungal properties and availability. cis-Abienol, despite its good antifungal activity, is not feasible for large-scale use due to the production and stability limitations.