Richard Craig

RNA isolation from recalcitrant plant tissue

The isolation of high-quality RNA from various tissues (leaves, pedicels, glandular trichomes) of garden geranium (Pelargonium xhortorum) using various published methods is difficult due to numerous oxidizing compounds. A new RNA extraction method was developed through the combination and modification of two separate procedures (Rochester et al., 1986; Manning 1991). In addition to geranium tissues, this method is successful when used with other recalcitrant tissues such as mature needles of white pine (Pinus strobus) and mature leaves of poinsettia (Euphorbia pulcherrima). RNA quality was judged by spectrophotometric readings, denaturing agarose gels, and successful reverse transcription.

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50s of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.

Analysis of trichome exudate from mite-resistant geraniums.

Trichome exudate from mite-resistant geraniums (Pelargonium horlorum) was analyzed, principally by mass spectrometry and NMR spectroscopy. The exudate was found to consist of two anacardic acid derivatives,o-pentadecenylsalicylic acid ando-heptadecenylsalicylic acid. Bioassays established a moderate toxicity of these compounds to the two-spotted spider mite,Tetranychus urticae. The production of these compounds in geraniums was correlated with the two complementary dominant genes previously reported for host resistance to spider mites.

Geranium defensive agents. IV. Chemical and morphological bases of resistance

Insect-resistant and -susceptible geranium plants (Pelargonium xhortorum) were chemically and morphologically compared to assess the relative importance of resistance components. Glandular and nonglandular trichome density and quantification of trichome exudate (anacardic acids) from inbred resistant and susceptible lines were determined. Three different extraction procedures were employed to quantify the anacardic acids: (1) capillary solvent extract, (2) leaf wipe, and (3) whole leaf extract. The results support the conclusion that tall glandular trichomes and the exudate they produce are the most important factors in small pest resistance in the geranium. In addition, it was shown that tall glandular trichome densities are less and the ability of the plant to express the anacardic acids as exudate on the trichome head exterior is lacking in the susceptible lines analyzed, even though all lines possessed the capability to synthesize the anacardic acids. A plant line of intermediate resistance character was shown to possess high densities of tall glandular trichomes but resembled susceptible plants in lacking the ability to express the anacardic acids as an exudate in appreciable quantities.

Fatty acid incorporation in the biosynthesis of anacardic acids of geraniums

Abstract Geraniums, Pelargonium xhortorum, have been shown to possess tall glandular trichomes which secrete anacardic acids, a viscous, sticky exudate which provides defence against small pest species. Pest-resistant geraniums possess primarily C 22 - and C 24 -unsaturated (ω-5) anacardic acids with lesser amounts of the C 22 - and C 24 -saturated side chain structures. Previous workers have suggested that anacardic acids are biosynthesized from fatty acids by addition of two carbon units. To investigate this hypothesis, [ 14 C]lauric, -myristic, -palmitic, -stearic and -oleic acids were administered to leaves and floral buds of pest-resistant geraniums. Palmitate and stearate were shown to be the respective precursors for C 22 and C 24 anacardic acids with saturated side chains. A major portion of the applied [ 14 C]stearate was degraded and subsequently incorporated into the C 22 - and C 24 -saturated and unsaturated (ω-5) anacardic acids indicating an indirect pathway of biosynthesis also exists. Therefore, the ω-5 anacardic acids must be the result of ω-5 fatty acid precursors rather than the desaturation of saturated anacardic acids. Data supports the hypothesis that anacardic acids are biosynthesized from saturated and ω-5 unsaturated C 16 and C 18 fatty acids by chain elongation through the addition of three acetate groups, which then undergo partial reduction and dehydration, followed by intramolecular aldol condensation, to give the aromatic ring in a manner similar to that proposed for the biosynthesis of salicylic acid.

Effects of mite resistance mechanism of geraniums on mortality and behavior of foxglove aphid (Acyrthosiphon solani Kaltenbach)

Geraniums (Pelargonium xhortorum Bailey) possess a pest-resistance mechanism, based on glandular trichomes and the exudate they produce, that has been shown to be effective against the two-spotted spider mite (Tetranychus urticae Koch). Using an intact plant bioassay, the effectiveness of the resistance mechanism was determined for another potential pest, the foxglove aphid (Acyrthosiphon solani Kaltenbach). Comparisons were made between plant lines previously analyzed for their degree of resistance to mites, as well as their glandular trichome density and trichome exudate production. Over 100 aphid adults were bioassayed on each of the five plant lines used in the experiment. In addition to adult aphid survival, the production and survival of nymphs was determined in this bioassay. The results indicate that plant lines that are resistant to the two-spotted spider mite are also resistant to the foxglove aphid, while lines susceptible to mites are susceptible to the aphids. To evaluate the physical impediment features of the trichome exudate, the behavior of foxglove aphid nymphs was compared on two geranium lines, one a resistant line with high trichome densities and large quantities of exudate and the second a susceptible line with few trichomes and reduced exudate. A third leaf surface type was produced by washing the exudate from resistant leaves using a mildly basic buffer solution prior to the bioassay. Aphid behavior was divided into five categories: feeding or probing, resting, wandering, struggling, and immobilized. On both susceptible leaves and resistant leaves from which the exudate had been removed by washing, the aphids settled quickly and were observed with inserted stylets during most of the observation intervals. In contrast, aphids on the unwashed resistant leaf surfaces often became ensnared in the sticky trichome exudate and had difficulty in settling to probe the leaf. Physical entrapment by glandular trichome exudate appears to be an important aspect of aphid resistance in geraniums.

Comparison of anacardic acid biosynthetic capability between insect-resistant and -susceptible geraniums

The garden geranium (Pelargonium xhortorum) has been shown to secrete anacardic acids in the form of a viscous sticky exudate from tall glandular trichomes, and this exudate provides a sticky trap defense against small pest species. The anacardic acids from genetically related pest-resistant and -susceptible plants have been characterized, and resistance has been shown to depend upon the presence ofω5 unsaturated anacardic acids. In this study, the biosynthesis of these anacardic acids was comparatively investigated by incubating [14C]methyl palmìtate, margarate, stearate, oleate and linoleate on floral buds of resistant and susceptible plants. In addition, the incorporation of [14C]valine, -isoleucine, and -leucine into anacardic acids was also studied. Nineteen anacardic acids were quantitated utilizing an improved HPLC technique. Fatty acids and, to a much lesser extent, amino acids were incorporated into anacardic acids. There are at least two pathways of biosynthesis operating: direct elongation, and β-oxidation with reincorporation of the [14C]acetate, the latter being more prevalent in the resistant plant. The amino acids were processed into branched chain anacardic acids, isoleucine being the precursor of the anteiso compounds, and valine the iso branched ones. The major difference between resistant and susceptible plants was the ability of resistant plants, but not the susceptible plants, to synthesizeω5 unsaturated anacardic acids. Both types of plants were capable of directly incorporating14C-labeled fatty acid methy esters into anacardic acids regardless of the plants normal anacardic acid composition, thus bypassing the plants tightly controlled regulation of the chemical structures of anacardic acids. No evidence was found forω5 desaturation of saturated anacardic acids. A revised biosynthesis scheme is presented.

Glandular trichome exudate is the critical factor in geranium resistance to foxglove aphid

Resistance to the foxglove‐aphid (Acyrthosiphon solani Kaltenbach) has been demonstrated in some inbred geranium lines (Pelargonium Xhortorum Bailey). To establish more definitively the cause/effect relationship between tall glandular trichome exudate and resistance, an intact plant bioassay was performed comparing a resistant plant line, a resistant plant line from which the tall glandular trichome exudate had been removed using a basic buffer solution, a susceptible line and a susceptible line treated with the buffer wash. After 5 days of isolation on the respective surfaces, the number of surviving adult aphids as well as the number of nymphs produced and remaining alive were determined. Aphids on the buffer washed, resistant line exhibited mortality and fecundity which was not significantly different from that produced by the susceptible line. In contrast, the untreated resistance line was clearly resistant with lower adult survival and fewer living nymphs. The tall glandular trichome exudate must therefore be a critical factor in geranium resistance to the foxglove aphid.

Geranium defensive agents. III. Structural determination and biosynthetic considerations of anacardic acids of geranium

Ozonolysis, dithioether derivatization, and EI and CI mass spectrometry were used to establish the location of the double bond in the side chain of the two major anacardic acids in geranium (Pelargonium hortorum) trichome exudate. The point of unsaturation was shown to be between C-5 and C-6 counting from the methyl end of the side chain, contradicting the earlier hypothesis that the olefmic bond was probably at the 9–10 position based upon expected biosynthetic considerations. The two major components are thus 6-[(Z)-10′-pentadecenyl]salicylic acid (C22H34O3) and 6-[(Z)-12′-heptadecenyl] salicylic acid (C24H38O3). This may indicate that the precursor of these anacardic acids is a saturated fatty acid since the location of a double bond at the 5–6 position is unusual among the unsaturated fatty acids. Capillary GLC and HPLC of the trichome exudate indicated the presence of small amounts of other anacardic acid analogs possessing such features as odd numbers of carbon atoms and saturated side chains.

Development of a mite bioassay to evaluate plant resistance and its use in determining regeneration of spider mite resistance

The zonal geranium (Pelargonium xhortorum) possesses tall glandular trichomes that secrete anacardic acids, a viscous, sticky exudate which has been suggested as the primary mechanism in two‐spotted spider mite (Tetranychus urticae Koch) resistance. A new bioassay was developed using small Plexiglas® cylinders as chambers for evaluating the resistance of geranium leaves to the two‐spotted spider mite. This bioassay was easy to prepare, required only 24 hours to conduct, exhibited no problems with desiccation, condensation, or mite accountability, and yielded reproducible results. This bioassay was then used to study the regeneration of resistance of attached geranium leaves after they were made mite‐susceptible by removing the excreted anacardic acids with water. Washed leaves regained full resistance after 14 days.