Patterns of inheritance of acylsugar acyl groups in selected interspecific hybrids of genus Nicotiana.

Glandular trichomes on the surface of Solanaceae species produce acyl sugars that are species-, and cultivar-specific. Acyl sugars are known to possess insecticidal, antibiotic, and hormone-like properties, and as such have great potential as a class of naturally occurring pesticides and antibiotics. The objective of this work was to analyze the acyl composition of acyl sugars in the leaf trichome exudate from selected Nicotiana species and to follow the inheritance of acyl content in their hybrids. Trichome exudates were collected, and the acyl profiles of acyl sugars were identified via GC-MS. The variations in acyl group inheritance in the hybrids (a single parent resemblance, missing, complementary, and novel groups) matched the patterns described in the literature for a variety of secondary metabolites. However, we did not find a complementation of major parental acyl groups. Instead, in some hybrids we observed a dynamic change in the proportions of acyl groups, distinguishing the acyl group profiles as novel. We observed paternal (i.e. N. tabacum cv. Turkish Samsun × N. benthamiana hybrids) and maternal (i.e. N. tabacum cv. Samsun-nn × N. otophora) inheritance patterns, novel acyl profiles (N. excelsior hybrids), and missing acyl groups (N. excelsiana). Selective inheritance of some acyl groups in the hybrids of N. benthamiana (4- and 5-methylheptanoic isomers) or N. alata (octanoate) was found. Suggestions are given to explain certain patterns of inheritance. The data presented here contribute to the body of knowledge about the effect of interspecific hybridization on the secondary metabolites by including acylsugar acyl groups that have not been studied previously.

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

KEY MESSAGE: Phylloplanins 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. ABSTRACT: Tobaccos 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.

Root-selective expression of AtCAX4 and AtCAX2 results in reduced lamina cadmium in field-grown Nicotiana tabacum L.

To assess the impact of enhanced root vacuole cadmium (Cd) sequestration on leaf Cd accumulation under a low Cd dose, as generally occurs in agriculture, leaf Cd accumulation was examined in field-grown tobacco plants expressing genes encoding the high-capacity-Cd, tonoplast-localized, divalent cation/H antiporters AtCAX4 and AtCAX2 (AtCAX, Arabidopsis cation exchanger). It has been shown previously that root tonoplast vesicles isolated from plants expressing these genes, directed by root-selective promoters, show enhanced Cd transport activity, and young plants show enhanced root Cd accumulation when grown in solution culture containing 0.02 microM Cd, a moderate Cd dose. In this article, we present results which show that the lower leaves of mature plants expressing AtCAX2 or AtCAX4, under the control of two different root-selective promoters, accumulate 15%-25% less lamina Cd than control plants when grown in the field (3 years, three different collection methods). Reciprocal grafting experiments of AtCAX2 shoots onto control roots (and vice versa), grown in solution culture with 0.005 microM Cd, indicated that the root controls Cd translocation and accumulation in the shoot in control and AtCAX2 and AtCAX4 tobacco plants exposed to low Cd concentration. The results are consistent with a model in which supplementation of Cd/H antiporter activity in root cell tonoplasts enhances root Cd sequestration, resulting in decreased translocation of Cd to the shoot of field-grown plants. These results suggest that human Cd intake from food and tobacco use could be reduced via the enhancement of root vacuolar sequestration of this pollutant.

Phylloplane proteins: emerging defenses at the aerial frontline?

The phylloplane, or leaf surface, is an interkingdom crossroads between plants and microorganisms, and secretion of antimicrobial biochemicals to aerial surfaces is thought to be one defensive strategy by which plants deter potential pathogens. Secondary metabolites on leaf surfaces are well documented but antimicrobial phylloplane proteins have only recently been identified. In this review, we describe the physical structures and biochemicals of the phylloplane and briefly discuss protein-based surface defenses of animals. We also review the emerging evidence pertaining to antimicrobial phylloplane proteins and mechanisms by which proteins can be released to the phylloplane, including biosynthesis (e.g. phylloplanins) by specific trichomes and delivery in guttation fluid from hydathodes. Future research should lead to exciting advances in our understanding of the phylloplane and to useful biotechnological interventions.

Natural variability in acyl moieties of sugar esters produced by certain tobacco and other Solanaceae species.

A unique feature of glandular trichomes of plants in the botanical family Solanaceae is that they produce sugar esters (SE), chemicals that have been shown to possess insecticidal, antifungal, and antibacterial properties. Sugar esters of tobacco (Nicotiana tabacum) provide pest resistance, and are important flavor precursors in oriental tobacco cultivars. Acyl moieties of SEs in Nicotiana spp., petunia, and tomato are shown to vary with respect to carbon length and isomer structure (2-12 carbon chain length; anteiso-, iso-, and straight-chain). Sugar esters and their acyl groups could serve as a model to explore the basis of phenotypic diversity and adaptation to natural and agricultural environments. However, information on the diversity of acyl composition among species, cultivars, and accessions is lacking. Herein, described is the analysis of SE acyl groups found in 21 accessions of Nicotiana obtusifolia (desert tobacco), six of Nicotiana occidentalis subsp. hesperis, three of Nicotiana alata, two of N. occidentalis, four modern tobacco cultivars, five petunia hybrids, and one accession each of a primitive potato (Solanum berthaultii) and tomato (Solanum pennellii). A total of 20 different acyl groups was observed that were represented differently among cultivars, species, and accessions. In Nicotiana species, acetate and iso- and anteiso-branched acids prevailed. Straight-chain groups (2-8 carbons) were prominent in petunias, while octanoic acid was prominent in N. alata and N. × sanderae. Two unexpected acyl groups, 8-methyl nonanoate and decanoate were found in N. occidentalis subsp. hesperis. Longer chain groups were found in the petunia, tomato, and potato species studied.

Enhancing tonoplast Cd/H antiport activity increases Cd, Zn, and Mn tolerance, and impacts root/shoot Cd partitioning in Nicotiana tabacum L.

Sequestration mechanisms that prevent high concentrations of free metal ions from persisting in metabolically active compartments of cells are thought to be central in tolerance of plants to high levels of divalent cation metals. Expression of AtCAX2 or AtCAX4, which encode divalent cation/proton antiporters, in Nicotiana tabacum cv. KY14 results in enhanced Cd- and Zn-selective transport into root tonoplast vesicles, and enhanced Cd accumulation in roots of plants exposed to moderate, 0.02 muM Cd in solution culture (Korenkov et al. in Planta 225:403-411, 2007). Here we investigated effects of expressing AtCAX2 and AtCAX4 in the same lines on tolerance to growth with near-incipient toxicity levels of Cd, Zn and Mn. Less growth inhibition (higher tolerance) to all three metals was observed in 35S::AtCAX2 and FS3::AtCAX4 expressing plants. Consistent with the tolerance observed for Cd was the finding that while root tonoplast vesicle proton pump activities of control and FS3AtCAX4 expressing plants grown in 3 muM Cd were similarly reduced, and vesicle proton leak was enhanced, root tonoplast vesicle antiporter activity of these plants remained elevated above that in controls. We suggest that CAX antiporters, unlike tonoplast proton pump and membrane integrity, are not negatively impacted by high Cd, and that supplementation of tonoplast with AtCAX compensates somewhat for reduced tonoplast proton pump and proton leak, and thereby results in sufficient vacuolar Cd sequestration to provide higher tolerance. Results are consistent with the view that CAX2 and CAX4 antiporters of tonoplast play a role in tolerance to high, toxic levels of Cd, Zn, and Mn in tobacco.

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.

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 beta-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.

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

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 regions of both genes in AS and S orientations were

Different elongation pathways in the biosynthesis of acyl groups of trichome exudate sugar esters from various solanaceous plants.

Two common pathways are known for elongation of aliphatic acids via acetate in biological organisms: the fatty acid synthase (FAS) and the alpha-ketoacid elongation (alphaKAE) pathways. The alphaKAE route is utilized in many biosynthetic pathways, including the tricarboxylic acid cycle, leucine biosynthesis, and in formation of coenzyme B, glucosinolates, alpha-ketoadipate, sugar-ester acyl acids, short-chain alcohols of yeast and Clostridium species, 2-amino-4-methylhex-4-enoic acid, and l-gamma-phenyl butyrine. In the FAS route, both carbons from acetyl-acyl carrier protein are retained per elongation cycle, while in the alphaKAE route only one carbon from acetyl-coenzyme A is retained. Available evidence indicates that different members of the family Solanaceae may use one or the other of these elongation mechanisms in the synthesis of acyl groups of trichome-exuded sugar esters. In both, precursors for elongation are derived from branched-chain amino acid metabolism. Here we compared radiolabeling patterns in sugar-ester acyl groups from trichomes (the specific tissue in which sugar esters are synthesized) of the tobaccos, Nicotiana benthamiana, N. gossei, N. glutinosa, of Petunia x hybrida cv. Falcon Red & White, and Datura metel, and epidermal peels of Lycopsersicon pennellii after their synthesis from [2-(14)C]-, [1-(14)C]- and [1,2-(14)C]acetate. Recovered acyl acids were purified and then degraded to determine label distribution between the carboxyl termini and the remainder of the molecules. Six- and 20-h incubations were studied, and membrane fatty acids were monitored as internal controls for FAS-mediated elongation. Results are consistent with participation of alphaKAE in synthesis of sugar-ester acyl groups of tobaccos and petunia, but apparently FAS is utilized in the formation of these groups in L. pennellii and D. metel.

Pathway for the biosynthesis of 4-methyl-1-hexanol volatilized from petal tissue of Nicotiana sylvestris.

Compounds volatilized from plant tissues play important roles in plant-insect and plant-herbivore interactions and are important to food quality/preference, and to the perfume and flavorant industries. While the chemistry of plant volatiles is well understood, less is known about the biosynthesis of this diverse group of compounds. This is particularly the case for non-terpenoid components such as volatile acyclic alcohols and their esters. Here we have studied metabolic pathways leading to formation of the anteiso-branched alcohol 4-methyl-1-hexanol volatilized by petal tissue of Nicotiana sylvestris. Evidence presented supports the involvement of steps in the pathways of both biosynthesis and degradation of isoleucine to form 2-oxo-3-methylvaleric acid then 2-methylbutyryl CoA. Results indicate that 2-methylbutyryl CoA is then elongated by addition of one acetate molecule via fatty acid synthase, followed by reduction to yield 4-methyl-1-hexanol. This pathway is in contrast to elongation of 2-oxo-3-methylvaleric acid via alpha-keto acid elongation leading to the formation of 4-methylhexanoyl acyl groups of tobacco leaf-trichome-secreted sugar esters.

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.

Isolation and characterization of the CYP71D16 trichome-specific promoter from Nicotiana tabacum L.

Trichomes are specialized epidermal cells that produce secretions that are thought to provide a first line of defence against pests and pathogens. Many trichome-secreted compounds are used commercially as flavourings, medicines, etc. Described here is the cloning and characterization of the promoter of a tobacco trichome-specific P450 gene, CYP71D16. This promoter is shown to direct the specific expression of the reporter gene, beta-glucuronidase (GUS), in glandular trichomes of Nicotiana tabacum cv. T.I. 1068 at all developmental stages. With the full promoter, GUS activity was predominantly in the gland cell, with less in the stalk cell adjacent to the gland, and in lower stalk cells. GUS staining was also observed in the most distal trichome stalk cells of non-glandular trichomes found on variety T.I. 1112. Promoter deletion analysis revealed that the region from -223 to +111 bp is sufficient to direct trichome-specific expression, but not strong gland expression. Examination of the literature suggests that this is the first characterized trichome-specific-promoter shown to function at all stages of plant development. This promoter may provide efficient bioengineering to enhance pest and pathogen resistance, and for molecular farming based on the trichome gland system.