Dynamic root exudation of sorgoleone and its in planta mechanism of action.

The oily droplets exuded from the root hairs of sorghum are composed of a 1:1 ratio of sorgoleone and its lipid resorcinol analogue. The production of these droplets appears to be suppressed when c. 20 microg of exudate mg(-1) root dry weight accumulates at the tip of the root hairs. However, more exudate is produced following gentle washing of the roots with water, suggesting that the biosynthesis of lipid benzoquinones and resorcinols is a dynamic process. Sorgoleone interferes with several molecular target sites, including photosynthetic electron transport, in in vitro assays. However, the in planta mechanism of action of sorgoleone remains controversial because it is not clear whether this lipid benzoquinone exuding from the roots of sorghum is taken up by roots of the receiving plants and translocated to their foliage where it must enter the chloroplast and inhibit PSII in the thylakoid membrane. Experiments designed to test the in planta mode of action of sorgoleone demonstrated that it has no effect on the photosynthesis of older plants, but inhibits photosynthesis in germinating seedlings. Sorgoleone is not translocated acropetally in older plants, but can be absorbed through the hypocotyl and cotyledonary tissues. Therefore, the mode of action of sorgoleone may be the result of inhibition of photosynthesis in young seedlings in concert with inhibition of its other molecular target sites in older plants.

The majority of in vitro macrophage activation exhibited by extracts of some immune enhancing botanicals is due to bacterial lipoproteins and lipopolysaccharides.

We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacterially derived components when tested in in vitro monocyte/macrophage activation systems. In experiments using RAW 264.7 and mouse peritoneal macrophages the majority (85-98%) of the activity within extracts from eight immune enhancing botanicals was eradicated by treatment with agents (lipoprotein lipase and polymyxin B) known to target these two bacterial components. Alfalfa sprouts exhibited the highest activity of those botanicals tested but the appearance of this activity during the germination of surface sterilized seeds was abolished by the presence of antibiotics. These studies indicate that the majority of the in vitro macrophage activating properties in extracts from these botanicals can be attributed to the presence of lipoproteins and lipopolysaccharides derived from bacteria and that bacterial endophytes may be a significant source of these components.

Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway.

Salvinorin A, a neoclerodane diterpenoid, isolated from the Mexican hallucinogenic plant Salvia divinorum, is a potent kappa-opioid receptor agonist. Its biosynthetic route was studied by NMR and HR-ESI-MS analysis of the products of the incorporation of [1-(13)C]-glucose, [Me-(13)C]-methionine, and [1-(13)C;3,4-(2)H2]-1-deoxy-D-xylulose into its structure. While the use of cuttings and direct-stem injection were unsuccessful, incorporation of (13)C into salvinorin A was achieved using in vitro sterile culture of microshoots. NMR spectroscopic analysis of salvinorin A (2.7 mg) isolated from 200 microshoots grown in the presence of [1-(13)C]-glucose established that this pharmacologically important diterpene is biosynthesized via the 1-deoxy-D-xylulose-5-phosphate pathway, instead of the classic mevalonic acid pathway. This was confirmed further in plants grown in the presence of [1-(13)C;3,4-(2)H2]-1-deoxy-D-xylulose. In addition, analysis of salvinorin A produced by plants grown in the presence of [Me-(13)C]-methionine indicates that methylation of the C-4 carboxyl group is catalyzed by a type III S-adenosyl-L-methionine-dependent O-methyltransferase.

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker.

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata (L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone. In in vitro assays, the threonine (Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of cross-resistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R/S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.