George J. Wagner

Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance

Trichome glands on the surface of many higher plants produce and secrete exudates affecting insects, microbes, and herbivores. Metabolic engineering of gland exudation has potential for improving pest/disease resistance, and for facilitating molecular farming. We identified a cytochrome P450 hydroxylase gene specific to the trichome gland and used both antisense and sense co-suppression strategies to investigate its function. P450-suppressed transgenic tobacco plants showed a ≥41% decrease in the predominant exudate component, cembratriene-diol (CBT-diol), and a ≥19-fold increase in its precursor, cembratriene-ol (CBT-ol). Thus, the level of CBT-ol was raised from 0.2 to ≥4.3% of leaf dry weight. Exudate from antisense-expressing plants had higher aphidicidal activity, and transgenic plants with exudate containing high concentrations of CBT-ol showed greatly diminished aphid colonization responses. Our results demonstrate the feasibility of significantly modifying the natural-product chemical composition and aphid-interactive properties of gland exudates using metabolic engineering. The results also have implications for molecular farming.

Metabolic pathways as enzyme complexes: Evidence for the synthesis of phenylpropanoids and flavonoids on membrane associated enzyme complexes

In earlier studies [G. Hrazdina, G. J. Wagner, and H. W. Siegelman (1978) Phytochemistry 17, 53-56; G. J. Wagner and G. Hrazdina (1984) Plant Physiol. 74, 901-906], evidence was obtained suggesting that the endoplasmic reticulum was a site for phenylpropanoid and flavonoid metabolism in petal tissue, and that (a) multienzyme complex(es) might be involved in this metabolism. Now, the possible role of membrane-bound multienzyme complexes in phenylpropanoid and flavonoid metabolism in three tissues has been investigated by (1) correlating enzyme induction kinetics and rates, (2) examining the molecular weight of putative complexes, (3) channeling of substrates, (4) determining the susceptibility of bound activities to trypsin digestion, and (5) investigating the structurally linked latency of bound activities. Results suggest that at least a part--and possibly the entire pathway--from phenylalanine to flavonoids is membrane (endoplasmic reticulum) associated, and that this metabolism is facilitated by a multienzyme complex. Phenylalanine ammonia lyase, the first enzyme of the biosynthetic sequence, and a flavonoid glucosyltransferase, the last, appear to be located in the lumen of the membranes. Cinnamate 4-hydroxylase is membrane embedded, while other enzyme activities appear to be weakly associated with the cytoplasmic face of endoplasmic reticulum membranes.

Relationship between cadmium, glutathione and cadmium-binding peptides (phytochelatins) in leaves of intact tobacco seedlings

Abstract Most studies of Cd2+ effects on plants have focused on formation of thiol-rich peptides, often called phytochelatins, in cultured cells or roots exposed to moderate-to-high levels of this metal. Less attention has been paid to effects on leaf tissue, lower level exposure, and on glutathione, the most prominent cellular thiol. Here, glutathione and phytochelatin levels were monitored during a 7 day time-course in leaves of intact tobacco seedlings exposed to non-growth-inhibiting, 5, 10, 20, or 40 μM Cd2+. Increased Cd2+ levels in the growth medium resulted in increased accumulation of Cd2+ in leaves. Glutathione levels decreased only slightly during the first hours of Cd2+-exposure and, by day 2, were completely recovered to control levels. Data presented support the contention that in leaves of tobacco seedlings exposed to low levels of Cd2+, complexation with cytosolic glutathione and antiport-mediated transport of Cd2+ ion and its subsequent sequestration into the vacuole may be sufficient mechanisms for accomodation of intracellularly accumulated cadmium. Phytochelatins were synthesized in a sequential order with smaller peptides being synthesized prior to larger peptides. Furthermore, above 5 μM Cd2+ exposure there was a positive correlation between the number of γ-glutamylcysteine repeat units and the endogenous Cd2+ concentration. The stoichiometry of SH in phytochelatins to Cd2+ was not constant, but varied with Cd2+ concentration and duration of Cd2+ exposure. This is in contrast to certain observations made with putative γ-glutamylcysteine dipeptidyl transferase and suggests that the regulation of phytochelatin synthesis in vivo may differ from that in vitro.

Elucidation of the functions of genes central to diterpene metabolism in tobacco trichomes using posttranscriptional gene silencing.

Abstract. The functions of two key, trichome-expressed genes were assessed using different posttranscriptional gene silencing strategies (PTGS). Efficient RNA interference (RNAi) revealed the function of a cembratriene-ol (CBT-ol) cyclase gene responsible for conversion of geranylgeranyl pyrophosphate to CBT-ols, and verified the function of a P450 gene responsible for conversion of CBT-ols to CBT-diols. CBT-diols are abundant diterpenes that comprise about 60% and 10% of trichome exudate weight and leaf dry weight, respectively, in Nicotiana tabacum, T.I. 1068. The relative efficiencies and levels of suppression using antisense (AS), sense co-suppression (S), and RNAi were compared for these two genes. With a partial cDNA of the P450 gene, the suppression efficiencies (percent of primary transformants with high CBT-ols/CBT-diols) were low, 3.3% for AS and 0% for S plants. In contrast, using RNAi with a partial gene sequence, a knockdown efficiency of about 45% was achieved. For the CBT-ol cyclase gene, no suppression was observed using partial cDNAs in AS or S orientations, while RNAi with a partial gene sequence yielded an efficiency of about 64%. The efficiencies of gene silencing using full-length coding reigons of both genes in AS and S orientations were ≤20%. Our results identify the function of a CBT-ol cyclase gene and demonstrate the efficacy and superiority of RNAi for assessing the functions of two trichome-specific genes that encode enzymes having widely different functions.

Phylloplanins of Tobacco are defensive proteins deployed on aerial surfaces by short glandular trichomes

In plants, defensive proteins secreted to leaf aerial surfaces have not previously been considered to be a strategy of pathogen resistance, and the general occurrence of leaf surface proteins is not generally recognized. We found that leaf water washes (LWW) of the experimental plant Nicotiana tabacum tobacco introduction (TI) 1068 contained highly hydrophobic, basic proteins that inhibited spore germination and leaf infection by the oomycete pathogen Peronospora tabacina. We termed these surface-localized proteins tobacco phylloplanins, and we isolated the novel gene T-Phylloplanin (for Tobacco Phylloplanin) and its promoter from N. tabacum. Escherichia coli–expressed T-phylloplanin inhibited P. tabacina spore germination and greatly reduced leaf infection. The T-phylloplanin promoter, when fused to the reporter genes β-glucuronidase and green fluorescent protein, directed biosynthesis only in apical–tip cell clusters of short, procumbent glandular trichomes. Here, we provide evidence for a protein-based surface defense system in the plant kingdom, wherein protein biosynthesis in short, procumbent glandular trichomes allows surface secretion and deposition of defensive phylloplanins on aerial surfaces as a first-point-of-contact deterrent to pathogen establishment. As yet uncharacterized surface proteins have been detected on most plant species examined.

Critical Review of the Scienceand Options for Reducing Cadmium in Tobacco (Nicotiana Tabacum L. ) and Other Plants

Cadmium (Cd) accumulation in crop plants such as tobacco can lead to human exposure to this carcinogenic metal. Therefore, efforts should be made to minimize the Cd content of soils and crops. We review the options for reducing Cd content of plants, with emphasis on tobacco, a plant that can accumulate relatively high levels of this metal in its leaves. Many studies aimed at understanding Cd biology in plants do not reflect field conditions, because, often of necessity, non-field-like conditions were used. Thus, further study is needed to understand which processes govern Cd uptake, accumulation, etc., under field conditions. Numerous factors, such as soil characteristics, agronomic practices, and environmental conditions, impact the uptake of Cd by plants, including tobacco. Identifying anthropogenic sources of Cd and controlling application may limit Cd accumulation in agricultural fields. Soil remediation strategies may be envisaged to reduce Cd availability to the plant (e.g., soil amendments) or to extract Cd from the soil (e.g., phytoextraction). Another approach for reducing Cd in crops involves genetic modification of the plant to reduce Cd uptake or to change its partitioning in the plant. Indeed, more knowledge has been gained in recent decades regarding the mechanisms governing the transport, accumulation, and compartmentalization of Cd by tobacco and other plants. Several types of genes can be considered for genetic engineering to affect these processes. Although no single remedy appears to exist that might drastically reduce the Cd content of crops, including tobacco, an integrated approach may prove useful.

Studies of the site and mode of biosynthesis of tobacco trichome exudate components.

Detached glandular trichome head preparations and epidermal strips with and without trichome heads were used to identify glandular trichome heads as the site of sucrose ester biosynthesis in tobacco. Carbon dioxide in solution as well as sucrose, glucose, and acetate were shown to serve as precursors to both sucrose esters and duvatrienediol diterpenes in detached trichome heads or epidermal strips, and gaseous CO2 was also efficiently utilized by epidermal strips. Thus, glandular heads can biosynthesize these principal exudate components from a molecule as simple as CO2. While formation of duvatriendiols from all precursors tested and conversion of sucrose and glucose to sucrose esters was light dependent, utilization of acetate to label the 6-O-acetyl group of the glucose moiety of sucrose esters occurred equally well in light and dark. The data suggest that CO2 and/or monosaccharides produced in trichome head cells and perhaps that supplied by other epidermal cells can act as carbon sources for sucrose ester and duvatrienediol biosynthesis which occurs in the glandular trichome head.

Tissue partitioning of cadmium in transgenic tobacco seedlings and field grown plants expressing the mouse metallothionein I gene

Since agricultural crops contribute >70% of human cadmium (Cd) intake, modification of crops to reduce accumulation of this pollutant metal during plant growth is desirable. Here we describe Cd accumulation characteristics of seedlings and field grown tobacco plants expressing the Cd-chelating protein, mouse metallothionein I. The objective of the transformation is to entrap Cd in roots as Cd-metallothionein and thereby reduce its accumulation in the shoot. Transformed and control seedlings were exposed for 15 days in liquid culture at a field soil-solution-like Cd concentration of 0.02 μm. Transformed seedlings ofNicotiana tabacum cultivar KY 14 contained about 24% lower Cd concentration in shoots and about 5% higher Cd concentration in roots than control seedlings. Dry weights of transformed and control tissues did not differ significantly. In the field in 1990, mature transformedN. tabacum cv. KY 14 plants exposed only to endogenous soil Cd contained about 14% lower leaf lamina Cd concentration than did controls. Differences were significant at thep≤0.1 level in 13 of 16 leaf positions. Leaf dry weight did not differ significantly but transformed field plants had 12% fewer leaves and were 9% shorter than the controls. Copper (Cu) concentration was significantly higher (ca10%) in the bottom nine leaf positions of transformed plants suggesting that reduced leaf number and plant height may be due to Cu deficiency or toxicity. Alternatively, somaclonal variation or gene position effects may be involved. No differences were found in zinc levels. WithN. tabacum cv. Petit Havana, transformed seedlings contained no less Cd in shoots but 48% higher Cd concentration in roots. However, dry weights of shoots and roots of transformed seedlings were 25% and 26%, respectively, greater than in controls. In the field, transformed and control plants of this cultivar showed little significant differences in leaf Cd content, plant height or leaf number. Although comparison of additional metallothionein-expressing tobaccos and other plants is needed, results obtained with cultivar KY 14 support the hypothesis that sequestration of Cd in roots as Cd-metallothionein may have potential for reducing Cd content of above root tissues of certain plants.

Biosynthesis of labdenediol and sclareol in cell-free extracts from trichomes of Nicotiana glutinosa

Biosynthesis of the diterpenes labdenediol and sclareol from all trans-geranylgeranyl pyrophosphate was observed in cell-free extracts prepared from leaf midvein epidermal peels of Nicotiana glutinosa 24A. The bioactivities were shown to be exclusively localized in trichomes, to be conferred by a soluble enzyme(s), and to resemble other plant terpene cyclase activities in basic characteristics. Chromatographic methods for protein purification including gel filtration, anion exchange, hydroxyapatite chromatography, and free-flow isoelectric focusing. Thermal inactivation and end-product inhibition experiments did not afford separation of the biosynthetic activities. Results indicate that these labdane diterpenes are direct products of cyclization reactions catalysed by a soluble cyclase(s). To the best of our knowledge, this report represents the first case for direct biosynthesis of diterpene di-alcohols by cyclization of the acyclic precursor. A unified scheme for formation of labdane mono- and di-alcohols in Nicotiana species is presented.

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.