Into Laakso

A functional genomics approach toward the understanding of secondary metabolism in plant cells

Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonate-mediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general.

Analysis of fatty acids by gas chromatography, and its relevance to research on health and nutrition

Abstract Gas chromatography (GC) has been an indispensable analytical technique ever since the first exciting steps in the application of fatty acid determinations in oilseed plant breeding, biosynthesis and human metabolism. Present-day GC methods with high-quality capillary columns allow sensitive and reproducible fatty acid analyses, as well as the characterization of complex mixtures of geometric isomers when combined with other chromatographic separations and spectroscopic identification. Ordinary GC analysis is well suited for a detailed follow-up of the changes in human tissue fatty acids derived from dietary fats providing, however, that all the steps in the methodology are carefully optimized. Plasma fatty acids act as excellent indicators, and the use of substitute fats can be found as dose-dependent correlations. Analysis of phospholipid (PL) fatty acid composition is especially useful for recognizing the competitive capability of essential fatty acids present in a particular dietary fat. A clear response is observed even at the level of minor fatty acids, i.e. both increased and decreased use of trans fatty acids are indicated by changes in the most abundant octadecenoic trans isomers. In addition to the expected associations with serum lipids, plasma fatty acid data are also useful in monitoring relationships with lipid oxidation parameters. GC analysis of fatty acids still has its traditional uses, but it is now faced with new challenges. Since the effects that arise from even moderate amounts of dietary essential fatty acids cannot be neglected, continuing research aimed at their requirements is of prime importance. Modification of fatty acid compositions by metabolic engineering offers good possibilities for producing new oilseed crops with a more balanced α-linolenic/linoleic acid (LA) ratio, preferably combined with a high oleic acid (OA) content. In the future, particular attention has to be paid to the proportions of polyunsaturated fatty acids (PUFAs) in the diet, which are the factors that finally determine the apparently unique balance of tissue n −3 and n −6 fatty acids and eicosanoids decisive for human health.

High-performance liquid chromatographic determination of oligomeric procyanidins from dimers up to the hexamer in hawthorn

An HPLC method using UV diode array detection was developed for analysing procyanidins qualitatively and quantitatively up to the hexameric level in hawthorn samples. The analysed compounds included procyanidin dimers B-2, B-4 and B-5, procyanidin trimers C-1, epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin and epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin, a tetramer D-1 and a pentamer E-1 both consisting of (-)-epicatechin units linked through C-4beta/C-8 bonds. The concentrations of two unknown tetramers and a hexamer F were also quantified. The oligomeric procyanidins (OPs) were specifically determined due to the development of a method for isolating them from hawthorn during sample preparation. The pattern of oligomeric procyanidins in the leaves, flowers and fruits was similar, but the concentrations varied depending on the part of the plant. The concentration in leaves was 1.6%, in flowers 1.2% and in fruits 0.2% of the dry mass. The method was validated with respect to repeatability, recovery, linearity, and sensitivity. The repeatability for the quantitative analytical method of all the OPs in leaves was 7.7%, in flowers 8.8%, and in fruits 12.3%. The recovery of the main OPs ranged from 91 to 97%. The correlation coefficients of calibration curves were between 0.997 and 1.000. The limits of quantitation for different procyanidin standards were 0.05-0.12 mg/ml, when 10 microl of each standard solution was injected into the HPLC.

Variation in the essential oil composition of Artemisia annua L. of different origin cultivated in Finland

Seven batches of seeds of Artemisia annua L. (Asteraceae) of different origin were grown in Finland. The leaf essential oils were obtained by hydrodistillation in yields of 0.4–0.9% (v/w). About 30 compounds were identified and 19 key compounds, representing 87–96% of the total oil, are listed. There was a large variability in the essential oil composition in plants of different seed origin. Camphor, artemisia ketone, germacrene-D and β-caryophyllene were detected as the major compounds. One oil had an exceptionally high content of (+)-α-pinene. Cluster analysis grouped the oils according to their origin, which shows that the essential oil composition is under strict genetic control. During the growing period the essential oil content and the amounts of artemisia ketone and 1,8-cineole reached a maximum about 2 weeks before budding. Camphor reached its maximum 2–3 weeks earlier. The enantiomeric ratios of (−)-camphene/(+)-camphene and (−)-α-pinene/(+)-α-pinene were determined for the first time in an A. annua oil and studied during a growing period.

Secretion of Secondary Metabolites by ATP-Binding Cassette Transporters in Plant Cell Suspension Cultures

The substrate specificity of the yeast ( Saccharomyces cerevisiae ) pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporters is extended to plant secondary metabolites of the tropane alkaloid family. Functional analysis of yeast PDR5 genes in transgenic tobacco ( Nicotiana

Major human plasma lipid classes determined by quantitative high-performance liquid chromatography, their variation and associations with phospholipid fatty acids

An HPLC method with evaporative light-scattering detection (ELSD) was optimized and validated for the simultaneous quantitation of cholesteryl esters (CEs), triacylglycerols (TGs), free cholesterol (FC) and phosphatidylcholine (PC) in human plasma. The separation of CEs from TGs, the most variable plasma lipid class, was improved by speeding up the gradient steps and by increasing the re-equilibration time between runs. The calibrations were made at levels of 0.14-14 microg lipid/injection. The intra- and inter-day precision values of the method ranged between 1.9 and 4.5 and 2.3-7.2% (RSD, n=6), respectively, including determinations at two concentration levels. In comparison to other lipid classes, quantitation of PC proved to be equally repeatable despite its lowest detector response. The relative recoveries varied from 97.0 to 110.3%, showing good accuracy of the method. The methodological variation of the lipid classes covered 0.6-3.1% of their total variation in the study population (n=48). The CE/FC ratio showed an even closer relationship with phospholipid linoleic acid (18:2n-6; r=0.65, P<0.001) than with serum cholesterol levels, while eicosapentaenoic acid (20:5n-3) was significantly associated with PC (r=0.41, P<0.01). The CE/FC ratio increased (P<0.01) during soyabean oil substitution and the level of PC increased (P<0.01) during cold-pressed rapeseed oil substitution.

Plasma nicotine and cotinine concentrations in mice after chronic oral nicotine administration and challenge doses

Abstract The relevance of oral nicotine administration to long-term nicotine treatment was investigated. Mice received nicotine in drinking water as the sole fluid for 7 weeks, the nicotine concentration being increased gradually from 50 to 500 μg/ml so that the maximum estimated daily dose of nicotine was 60–65 mg/kg. The fluid intake started to decrease but weight gain did not alter during the first 3 weeks. At 7 weeks the mice drank 47% less and weighed 9% less than the controls. During the 24-h withdrawal, the fluid intake increased above that of the controls and the difference in body weights disappeared. After 7 weeks, the plasma nicotine and cotinine concentrations were about similar to concentrations attained at 1 h after a subcutaneous nicotine dose of 3 mg/kg, and did not differ significantly from those at 2 and 4 weeks. After acute nicotine, the plasma cotinine concentrations rose more slowly and fell more quickly in 12-week-old mice than in 5-week-old-mice. However, the chronic oral administration of nicotine for 7 weeks did not alter the rate of nicotine elimination. Our results indicate that nicotine can be administered to mice chronically and orally through drinking water in doses high enough to induce pharmacologically relevant plasma nicotine concentrations.

Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers.

Oligomeric procyanidins were isolated from the leaves and flowers of hawthorn (Crataegus laevigata). A trimer, epicatechin-(4 beta-->8)-epicatechin-(4 beta-->6)-epicatechin, and a pentamer consisting of (-)-epicatechin units linked through C-4 beta/C-8 bonds have been isolated from hawthorn for the first time, in addition to known procyanidins including dimers B-2, B-4 and B-5, trimers C-1 and epicatechin-(4 beta-->6)-epicatechin-(4 beta-->8)-epicatechin, and tetramer D-1. A fraction containing a hexamer was also found.

Formation of parental-type and novel glycoalkaloids in somatic hybrids between Solanum brevidens and S. tuberosum

Abstract Steroidal glycoalkaloid aglycones (SGAA) in the somatic hybrids between the cultivated potato ( Solanum tuberosum line PDH40) and the non-tuber-bearing wild potato species S. brevidens (accession CPC 2451) were analysed as trimethylsilyl derivatives by gas chromatography-mass spectrometry (GC-MS). The reproducibility of the quantitative results including extraction, hydrolysis, derivatisation of aglycones, and GC-MS ranged 5–7% (C.V.). The leaves of S. tuberosum contained solanidine (396 mg/kg dry weight) and solanthrene (49 mg/kg dry weight), whereas the leaves of S. brevidens contained tomatidine (8173 mg/kg dry weight). In addition to these parental-type SGAAs, all somatic hybrids contained demissidine in the leaves, and the total SGAA contents ranged 290–7774 mg/kg dry weight The pattern of SGAAs produced in the tubers of S. tuberosum and a few tested hybrid lines was similar to the leaves. In the symmetric somatic hybrids which had a known genome composition the proportion of tomatidine of the total SGAA content correlated positively with the genome doses of S. brevidens . Based on the results, a simple hypothesis for the formation of the novel SGAA, demissidine, in the somatic hybrids is proposed. According to this hypothesis, the hydrogenase enzyme of S. brevidens which produces tomatidine from its precursor teinemine by hydrogenating the double bond at position 5 also produces demissidine by hydrogenating the corresponding double bond in solanidine.

Chronic oral nicotine administration affects the circadian rhythm of dopamine and 5-hydroxytryptamine metabolism in the striata of mice

The effect of chronic oral administration of nicotine on the circadian rhythm of striatal dopamine (DA) and 5-hydroxytryptamine (5-HT) was studied in mice. Mice receiving nicotine in their drinking water and control mice drinking tap water were killed at 05:00, 11:00, 15:00 or 21:00 hours on the 50th day of chronic administration. The plasma concentrations of nicotine and cotinine, as well the striatal concentrations of DA, 5-HT and their metabolites 3,4 dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanilic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were estimated. The largest plasma concentrations of nicotine and cotinine were found at 05:00, when they were more than double the concentrations found at the other times studied. This indicates that the mice, typically for nocturnal animals, consumed most of their daily drinking water at night. In the control mice, the striatal DA and 3-MT concentrations showed circadian variation and were lowest at 11:00. The 5-HIAA concentrations also varied, being highest at 11:00. In the nicotine-treated mice the circadian variations in striatal monoamines were altered and more pronounced than in the controls. The concentrations of DA, DOPAC, HVA and 5-HIAA were highest at 11:00 and that of 5-HT at 21:00. The striatal DA, DOPAC, HVA and 5-HIAA concentrations in the nicotine-treated mice were significantly higher at 11:00 and the 5-HT concentrations at 21:00 than in the control mice, and, in contrast to the control mice, in the mice treated with chronic nicotine no circadian rhythm was observed in the 3-MT. No elevation of striatal DA metabolites occurred in the nicotine-treated mice compared with the controls when the plasma nicotine concentration was at its peak at 05:00. This finding suggests development of tolerance to the nicotine-induced changes in striatal DA metabolism. Further, our findings suggest that the chronic administration of nicotine in the drinking water of mice alters the circadian pattern of striatal DA and, to a lesser extent, that of 5-HT, and thus may affect the functions regulated by these transmitters.