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Saponins are a diverse family of naturally occurring plant triterpene or steroid glycosides that have a wide range of biological activities. They have been shown to permeabilize membranes and in some cases membrane disruption has been hypothesized to involve saponin/cholesterol complexes. We have examined the interaction of steroidal saponin 1688-1 with lipid membranes that contain cholesterol and have a mixture of liquid-ordered (Lo) and liquid-disordered (Ld) phases as a model for lipid rafts in cellular membranes. A combination of atomic force microscopy (AFM) and fluorescence was used to probe the effect of saponin on the bilayer. The results demonstrate that saponin forms defects in the membrane and also leads to formation of small aggregates on the membrane surface. Although most of the membrane damage occurs in the liquid-disordered phase, fluorescence results demonstrate that saponin localizes in both ordered and disordered membrane phases, with a modest preference for the disordered regions. Similar effects are observed for both direct incorporation of saponin in the lipid mixture used to make vesicles/bilayers and for incubation of saponin with preformed bilayers. The results suggest that the initial sites of interaction are at the interface between the domains and surrounding disordered phase. The preference for saponin localization in the disordered phase may reflect the ease of penetration of saponin into a less ordered membrane, rather than the actual cholesterol concentration in the membrane. Dye leakage assays indicate that a high concentration of saponin is required for membrane permeabilization consistent with the supported lipid bilayer experiments.

*SUMMARY Cyclic peptides (CPs) are produced in a very wide range of taxa. Their biosynthesis generally involves either non-ribosomal peptide synthases or ribosome-dependent production of precursor peptides. Plants within the Caryophyllaceae and certain other families produce CPs which generally consist of 5–9 proteinogenic amino acids. The biological roles for these CPs in the plant are not very clear, but many of them have activity in mammalian systems. There is currently very little known about the biosynthesis of CPs in the Caryophyllaceae. A collection of expressed sequence tags from developing seeds of Saponaria vaccaria was investigated for information about CP biosynthesis. This revealed genes that appeared to encode CP precursors which are subsequently cyclized to mature CPs. This was tested and confirmed by the expression of a cDNA encoding a putative precursor of the CP segetalin A in transformed S. vaccaria roots. Similarly, extracts of developing S. vaccaria seeds were shown to catalyze the production of segetalin A from the same putative (synthetic) precursor. Moreover, the presence in S. vaccaria seeds of two segetalins, J [cyclo(FGTHGLPAP)] and K [cyclo(GRVKA)], which was predicted by sequence analysis, was confirmed by liquid chromatography/mass spectrometry. Sequence analysis also predicts the presence of similar CP precursor genes in Dianthus caryophyllus and Citrus spp. The data support the ribosome-dependent biosynthesis of Caryophyllaceae-like CPs in the Caryophyllaceae and Rutaceae.

Introduction Saponins are widely distributed complex plant glycosides possessing a variety of structure-dependent bioactivities. Quantitation of individual saponins is difficult due to lack of available standards, mainly as a consequence of purification difficulties. Determination of total saponin content can be problematic, often relying on non-specific methods based on butanol solubility, haemolytic activity or formation of coloured derivatives. Objective To develop a general quantitative method based on the use of the readily available cardenolides, digitoxin (1) and digoxin (2), as internal standards in an HPLC-PAD-based analysis. Methodology The cardenolides were run at a variety of concentrations to establish linearity and reproducibility of detector response and then evaluated as internal standards for quantitation of triterpene saponins in several plant-derived extracts by HPLC-PAD. Mixtures of saponins, largely freed from other extractables, were obtained by fractionation of total extracts on solid phase extraction columns (SPE) employing a water–methanol gradient and used for construction of calibration curves. Saponin identification and structural information was obtained via a single quadrupole mass detector using electrospray ionisation in negative ion mode (ESI−). Results Saponin contents in six samples from five species were determined and compared with literature results and a gravimetric method based on butanol–water partitioning. Results were generally consistent with literature reports and superior to gravimetric butanol–water partitioning. Conclusion Digitoxin and digoxin are useful as internal standards in HPLC estimation of saponin content. Saponins from different species having similar structures and molecular weights afford similar calibration curves. Copyright © 2008 John Wiley & Sons, Ltd.

Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a β-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria.

A high-performance liquid chromatographic method using photodiode array and single quadrupole electrospray mass detection for analysis and profiling of bisdesmosidic saponins in Saponaria vaccaria seed was developed. Profiles of seed extract from three different plant sources were obtained and found to contain the same saponins, albeit in different proportions. Several known saponins were identified by selected ion extraction of quasi-molecular ions from the total ion chromatogram and confirmed by their mass spectra. Application of high cone voltages afforded mass spectra containing key diagnostic fragments and relatively strong singly charged quasi-molecular ions. In addition to previously identified saponins, several new quillaic acid and gypsogenin bisdesmosides could be detected via mass spectral analysis. Five of these were tentatively identified as pentose homologues of known saponins, having an added xylosyl residue linked to the 3-O-glucuronyl group (1 --> 3). The stereochemistry and identity of the xylosyl linkage in the new saponins was determined by chemical means. Previously reported vaccaric or segetalic acid-type bisdesmosides could not be detected in any of the extracts.

Nine quillaic acid and five gypsogenin bisdesmosides were isolated from roots of Saponaria officinalis L. (Caryophyllaceae). Seven of the quillaic acid saponins possessed a 3-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl unit, but differed from each other in oligosaccharide units linked to the C-28 ester. The five gypsogenin saponins isolated from the roots all possessed the 3-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl unit, with their oligosaccharide units linked to the C-28 ester differing. Structures were elucidated by extensive 1D and 2D NMR spectroscopy and mass spectrometry. The saponins were evaluated for growth inhibitory activity in two human cancer cell lines and hemolytic activity in sheep red blood cells.

The structural and conformational requirements of a carrier responsible for the saturable component of the uptake of the plant hormone (+)-abscisic acid in barley suspension culture cells have been probed through the use of a defined series of optically pure ABA analogues. Two analogues showed a tenfold increase over (+)-ABA in inhibiting the uptake of radiolabelled (+)-ABA. Results from different studies (molecular modelling, low temperature NMR spectroscopy and assays with sterically rigid analogues) have shown the likely conformation of ABA in the binding site of the carrier is that with the sidechain in an equatorial-like orientation. Structure/activity studies show that the C-1 carboxylic acid is essential for binding, the C-4 carbonyl is moderately important while the C-1′ hydroxyl group is not important for binding to the carrier.

In vivo measurements of the enzymatic hydroxylation of the phytohormone (+)-abscisic acid to hormonally inactive ( -)-phaseic acid in corn cell cultures revealed that (+)-abscisic acid 8' hydroxylase activity was induced by (+)-abscisic acid treatment. This induction was blocked by the protein synthesis inhibitor cycloheximide and by the transcription inhibitor cordycepin. Following an induction treatment with abscisic acid, the amount of induced enzyme was measured by addition of both cycloheximide, to prevent further induction, and fresh abscisic acid as substrate for the induced enzyme. Phaseic acid production was related to the amount of enzyme induced. The experimental system was optimized for studying enzyme induction and the substrate specificity of the induced enzyme. The induced 8' hydroxylase was specific for (+)-abscisic acid, whereas the low basal activity also hydroxylated (-)-abscisic acid at the 7' position to form 7'-hydroxy abscisic acid. After induction, the 8' hydroxylase was rapidly degraded with a half-life of approximately 2 h. Phaseic and (-)-abscisic acid were weak inducers; however, the unstable oxidation product 8'-hydroxy abscisic acid exhibited 42% of the activity of (+)-abscisic acid. When corn cells were placed under water stress by increasing concentrations of mannitol in the culture medium, 8' hydroxylase induction was suppressed. The experimental system described will be useful for further studies of the physiological and hormonal factors that modulate abscisic acid metabolism and for testing potential enzyme inhibitors and hormone analogues.

Abscisic acid (ABA) was functionalized at the 3′-position by reaction of its α-2′,3′-epoxide with 1,4-butanedithiol to afford the corresponding derivative having a six-atom linker arm containing a terminal aulphhydryl group. This derivative was coupled with epoxy activated Sepharose 6B affording a gel containing ca 1.4 mg of (+)-ABA ml−1. This gel was shown to bind specifically an anti-ABA antibody from hybridoma culture broth, and its binding capacity was determined. The (+)-ABA analogue possessing the thiol linker arm was also derivatized by reaction with a fluorescent derivatizing agent and these compounds were shown to inhibit cress seed germination.

Unnatural phaseic acid (U-PA) was identified by chiral GC as a minor component of the metabolite mixture obtained from the medium of a maize cell suspension culture treated with unnatural (−)( R )-abscisic acid (U-ABA). When U-[3′,5′,5′,7′,7′,7′]-hexadeutero-ABA was fed to the cell suspension, d 6 -PA was obtained, establishing its origin from the exogenously-added U-ABA. The major metabolise produced was identified as U-7′-hydroxyabscisic acid (U-7′-HOABA [ d 5 -7′-HOABA from d 6 -U-ABA]. Confirmation of the assignments was obtained by HPLC/LC/CFSIMS analysis of the metabolise mixtures. This result corroborates a previous report of the formation of U-PA from racemic ABA in avocado mesocarp tissue.

The uptake of [ 3 H]-abscisic acid in barley (Hordeum vulgare L. cv. Heartland) cell cultures was found to be mediated through both non-saturable and saturable components. The kinetic parameters of the saturable component, determined at pH 4.5 and 21° C, showed a K m for natural or (+).ABA of 1.3 ± 0.7 μM and an apparent V max of 7.0 ± 2.8 nmol g −1 cells h −1 . The carrier showed a strong preference for the natural enantiomer of ABA as compared to the unnatural one. Other substances tested, e.g. amino acids, organic acids, and other growth regulators, did not appear to interfere with the carrier-mediated uptake of ABA. At low external concentrations of ABA (below 2.0 μM), the saturable component was greater than the diffusion component

Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the C4-hydroxylation of 2,3-dihydro-3-hydroxy-N(1)-methyltabersonine to the 3,4-dihydroxy derivative. The enzyme showed an absolute requirement for 2-oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). The hydroxylase exhibited specificity for position 4 of various alkaloid substrates. The enzyme exhibited a pH optima between 7 and 8 and an apparent molecular weight of 45,000. The appearance of 4-hydroxylase activity was developmentally regulated and was shown to be inducible by light treatment of seedlings. Substrate specificity studies of this enzyme for indole alkaloid substrate suggested that hydroxylation at position 3 and N-methylation occur prior to hydroxylation at position 4. This is in agreement with previous studies which suggest that C4-hydroxylation is the second to last step in vindoline biosynthesis in Catharanthus roseus.

We report here the synthesis and biological activity of a new persistent abscisic acid (ABA) analog, 8[prime]-methylene ABA. This ABA analog has one additional carbon atom attached through a double bond to the 8[prime]-carbon of the ABA molecule. (+)-8[prime]-Methylene ABA is more active than the natural hormone (+)-ABA in inhibiting germination of cress seed and excised wheat embryos, in reducing growth of suspension-cultured corn cells, and in reducing transpiration in wheat seedlings. The (+)-8[prime]-methylene analog is slightly weaker than (+)-ABA in increasing expression of ABA-inducible genes in transgenic tobacco, but is equally active in stimulating a transient elevation of the pH of the medium of corn cell cultures. In corn cells, both (+)-ABA and (+)-8[prime]-methylene ABA are oxidized at the 8[prime] position. ABA is oxidized to phaseic acid and (+)-8[prime]-methylene ABA is converted more slowly to two isomeric epoxides. The alteration in the ABA structure causes the analog to be metabolized more slowly than ABA, resulting in longer-lasting and more effective biological activity relative to ABA.

Summary Seeds of Catharanthus roseus (L.) G. Don cv. Little Delicata were germinated in the presence of different dark-light regimes. Alkaloid profiles as well as tryptophan decarboxylase and acetyl-CoA: deacetylvindole 0-acetyltransferase were monitored. Growth of seedlings in the dark resulted in an early ubiquitous accumulation of tabersonine as a major alkaloid and in the subsequent day 5–10 cotyledon restricted accumulation of vindoline and its immediate precursors. Transfer of 5-day old seedlings to the light resulted in rapid loss of vindoline precursors followed by a more gradual disappearance of tabersonine and the subsequent enhanced accumulation of vindoline. Although light enhanced vindoline biosynthesis, it was not essential. This sequence of biogenetic events suggested the following biosynthetic pathway: tabersonine → 16-hydroxytabersonine → 16-methoxytabersonine → 16-methoxy-2,3-dihydro-3-hydroxytabersonine → N(1)-methyl-l6-methoxy-2,3-dihydro-3-hydroxytabersonine (i.e. desacetoxyvindoline) → deacetylvindoline → vindoline. The time course of induction indicated that increase of tryptophan decarboxylase activity coincided with tabersonine accumulation, whereas increase of acetyl-CoA: deacetylvindoline 0-acetyltransferase activity coincided with vindoline accumulation. Results suggested that tabersonine biosynthetic pathway enzymes occur in all plant parts whereas the last 5 steps in vindoline biosynthesis are restricted to aerial parts of the plant and that the whole pathway to vindoline biosynthesis is developmentally regulated.

Syntheses of the C 8 and C 10 olefinic units cis - and trans -5-ethyl-1-iodo-hex-4-enes and cis - and trans -7-ethyl-3-iodo-oct-6-enes are described. The Wittig coupling of such units with derivatives of α- and β-thujaketonic acids to give analogs of insect juvenile hormones is discussed.

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615–767 and 916 could not be induced to produce DAT or NMT enzyme activities.

A large body of research has concerned itself with the production of commercially important phytochemicals by using plant cells in culture. These studies have identified several key areas where basic knowledge is generally limiting exploitation [1,2]. One fundamental problem occurs when non-differentiated tissue cultures are unable to produce and to accumulate complex chemicals originally found in specialized tissues or organs in the intact plant [3,4]. It is this need for specialized organs or tissues for metabolite production that has led to the development of rapidly growing and phytochemical producing organized hairy root cultures following genetic transformation of plants with Agrobacterium rhizogenes [5, and references cited therein]. Such cultures offer the possibility of producing root-derived phytochemicals but not those occurring in other plant tissues [5]. These technical innovations, however, fail to address the fundamental regulatory controls that maintain the specific developmental states which induce the production and accumulation of a given phytochemical.