Citronellol biosynthesis in pelargonium is a multistep pathway involving progesterone 5ß-reductase and/or iridoid synthase-like enzymes.

Citronellol is a pleasant-smelling compound produced in rose (Rosa spp.) flowers and in the leaves of many aromatic plants, including pelargoniums (Pelargonium spp.). Although geraniol production has been well studied in several plants, citronellol biosynthesis has been documented only in crab-lipped spider orchid (Caladenia plicata) and its mechanism remains open to question in other species. We therefore profiled 10 pelargonium accessions using RNA sequencing and gas chromatography-MS analysis. Three enzymes from the progesterone 5ß-reductase and/or iridoid synthase-like enzymes (PRISE) family were characterized in vitroand subsequently identified as citral reductases (named PhCIRs). Transgenic RNAi lines supported a role for PhCIRs in the biosynthesis of citronellol as well as in the production of mint-scented terpenes. Despite their high amino acid sequence identity, the 3 enzymes showed contrasting stereoselectivity, either producing mainly (S)-citronellal or a racemate of both (R)- and (S)-citronellal. Using site-directed mutagenesis, we identified a single amino acid substitution as being primarily responsible for the enzyme's enantioselectivity. Phylogenetic analysis of pelargonium PRISEs revealed 3 clades and 7 groups of orthologs. PRISEs from different groups exhibited differential affinities toward substrates (citral and progesterone) and cofactors (NADH/NADPH), but most were able to reduce both substrates, prompting hypotheses regarding the evolutionary history of PhCIRs. Our results demonstrate that pelargoniums evolved citronellol biosynthesis independently through a 3-step pathway involving PRISE homologs and both citral and citronellal as intermediates. In addition, these enzymes control the enantiomeric ratio of citronellol thanks to small alterations of the catalytic site.

UV-A and UV-B combined with photosynthetically active radiation change plant growth, antioxidant capacity and essential oil composition of Pelargonium graveolens.

BACKGROUND: The different wavelengths of solar radiation incident on earth [herein: Photosynthetically Active Radiation (PAR) , Ultra Violet-A (UV-A) and Ultra Violet-B (UV-B)] and their spectral balance not only have an impact on plants' growth, morphology and physiology, but also are important for the quality and quantity of plant secondary metabolites. MATERIAL AND METHODS: In an outdoor study we addressed the effects of PAR intensity and UV-A and UV-B on the growth, yield, phenolic and flavonoid content, antioxidant activity and essential oil composition of Pelargonium graveolens L'Hér. The experiment was performed with split plots in a randomized complete block design with three replications. During the growth, two PAR intensities (ambient PAR and reduced PAR) and four UV treatments (ambient UV, enhanced UV-A, enhanced UV-B and enhanced UVA + B) were applied. RESULTS: High PAR intensity decreased the length and width of leaf, the height of plant and fresh weight of aerial parts, and increased the dry weight of aerial parts. Enhanced UV-B irradiation was associated with reduced plant height, leaf expansion and fresh and dry weight of aerial parts. Interestingly, the negative effect of UV-B radiation on morphology and growth of plant was largely alleviated by high PAR intensity. The amount of total phenols and flavonoids, antioxidant activity and essential oil production of P. graveolens strongly increased with the increase of UV-B irradiation and PAR. On the other hand, UV-A radiation did not significantly influence total phenol and flavonoid content, antioxidant activity and essential oil composition. Moreover, the combination of high PAR intensity and UV-B led to further increases in total flavonoid content and antioxidant capacity. Both high PAR intensity and enhanced UV-B increased the percentage of geraniol in essential oil, leading to a slight reduction of citronellol/geraniol ratio which is a marker of quality for rose geranium essential oil. CONCLUSIONS: Overall, we conclude that UV-B irradiation was associated to reduction of plant growth and yield, while, the adverse effect of UV-B irradiation on the plant was mitigated by high PAR intensity. On the other hand, both high PAR and enhanced UV-B boosted the production of phenols, flavonoids and essential oil. Considering that the lower citronellol/geraniol ratio is the most important indicator for the economic value of rose geranium essential oil, reducing citronellol/geraniol ratio under enhanced UV-B radiation and/or high PAR is likely to be favorable.

Contrasting Metabolisms in Green and White Leaf Sectors of Variegated Pelargonium zonale-An Integrative Transcriptomic and Metabolomic Study.

The photosynthetically active green leaf (GL) and non-active white leaf (WL) tissues of variegated Pelargonium zonale provide an excellent model system for studying processes associated with photosynthesis and sink-source interactions, enabling the same microenvironmental conditions. By combining differential transcriptomics and metabolomics, we identified the main differences between these two metabolically contrasting tissues. Genes related to photosynthesis and associated pigments, the Calvin-Benson cycle, fermentation, and glycolysis were strongly repressed in WL. On the other hand, genes related to nitrogen and protein metabolism, defence, cytoskeletal components (motor proteins), cell division, DNA replication, repair and recombination, chromatin remodelling, and histone modifications were upregulated in WL. A content of soluble sugars, TCA intermediates, ascorbate, and hydroxybenzoic acids was lower, while the concentration of free amino acids (AAs), hydroxycinnamic acids, and several quercetin and kaempferol glycosides was higher in WL than in GL. Therefore, WL presents a carbon sink and depends on photosynthetic and energy-generating processes in GL. Furthermore, the upregulated nitrogen metabolism in WL compensates for the insufficient energy from carbon metabolism by providing alternative respiratory substrates. At the same time, WL serves as nitrogen storage. Overall, our study provides a new genetic data resource for the use of this excellent model system and for ornamental pelargonium breeding and contributes to uncovering molecular mechanisms underlying variegation and its adaptive ecological value.

Cytosolic geraniol and citronellol biosynthesis require a Nudix hydrolase in rose-scented geranium (Pelargonium graveolens).

Geraniol, citronellol and their esters are high-value acyclic monoterpenes used in food technology, perfumery and cosmetics. A major source of these compounds is the essential oil of rose-scented geraniums of the genus Pelargonium. We provide evidence that their biosynthesis mainly takes place in the cytosol of glandular trichomes via geranyl monophosphate (GP) through the action of a Nudix hydrolase. Protein preparations could convert geranyl diphosphate (GDP) to geraniol in in vitro assays, a process which could be blocked by inorganic phosphatase inhibitors, suggesting a two-step conversion of GDP to geraniol. Pelargonium graveolens chemotypes enriched in either geraniol or (-)-citronellol accumulate GP or citronellyl monophosphate (CP), respectively, the presumed precursors to their monoterpenoid end products. Geranyl monophosphate was highly enriched in isolated glandular trichomes of lines producing high amounts of geraniol. In contrast, (-)-isomenthone-rich lines are depleted in these prenyl monophosphates and monoterpene alcohols and instead feature high levels of GDP, the precursor to plastidic p-menthane biosynthesis. A Nudix hydrolase cDNA from Pelargonium glandular trichomes, dubbed PgNdx1, encoded a cytosolic protein capable of hydrolyzing GDP to GP with a KM of about 750 nm but is only weakly active towards farnesyl diphosphate. In citronellol-rich lines, GDP, GP and CP were detected in nearly equimolar amounts, while citronellyl diphosphate was absent, suggesting that citronellol biosynthesis may proceed by reduction of GP to CP in this species. These findings highlight the cytosol as a compartment that supports monoterpene biosynthesis and expands the roles of Nudix hydrolases in the biosynthesis of plant volatiles.

GC-MS Profiling and Antineoplastic Activity of Pelargonium Inquinans Ait Leaves on Acute Leukaemia Cell Lines U937 and Jurkat.

We investigated the antineoplastic activities of extracts of Pelargonium inquinans leaves, a plant native to South Africa on acute leukemia cell lines, U937 and Jurkat and the inflammatory effect (nitric oxide and cyclo-oxygenase-2) on RAW 264.7 cells. The extracts of Pelargonium inquinans have significant cytotoxicity especially on U937 cells and pro-inflammatory release of nitric oxide on RAW 264.7 macrophages. The GC-MS study of the essential oil showed it had more than a hundred compounds. This study showed that Pelargonium inquinans have antineoplastic and anti-inflammatory activities which can be further explored in In Vivo studies.

[Rose-scented geranium essential oil from Algeria (Pelargonium graveolens L'Hérit.): Assessment of antioxidant, anti-inflammatory and anticancer properties against different metastatic cancer cell lines]. / Essence aromatique du Géranium Odorant (Pelargonium graveolens L'Hérit.) d'Algérie : exploration des propriétés antioxydante, anti-inflammatoire et anticancéreuse (anti-angiogénique et cytotoxique), in vitro et in ovo, vis-à-vis de différentes lignées cellulaires cancéreuses métastasiques.

INTRODUCTION: The study of natural products is one of the strategies implemented for the discovery of new compounds that can be used in cancer therapy. Aromatic herbs and medicinal plants found in Algeria and their anti-angiogenesis and cytotoxic potentials against cancer have not been much explored. OBJECTIVES: Our work aimed to evaluate the antioxidant, anti-inflammatory and anticancer properties of the essential oil (EO) extracted from rose-scented geranium (Pelargonium graveolens) and its major (citronellol) and characteristic (linalool) constituents. RESULTS: The chemical composition of EO was determined with chromatographic analysis and revealed the presence of citronellol as the major compound (25.84%). A strong chelating power of terpene alcohols (IC50=1.58±0.23mg/mL for citronellol) was found, with a significant difference (P<0.05) compared with the standard antioxidants used (L-ascorbic acid and butylated hydroxyanisole). The EO is distinguished by an interesting anti-inflammatory effect with the lowest IC50 (4.63±0.3mg/mL), and it constitutes a good stabilizer of the erythrocyte membrane. Citronellol also exhibited the best anti-inflammatory effect (IC50=0.74±0.09mg/mL). We also assessed the anticancer effect of EO on two main pathways involved in cancer development, angiogenesis and cell proliferation, using in ovo bioassays with a chorio-allantoic membrane (CAM) of chicken eggs and in vitro assays of its cytotoxicity on different metastatic breast cancer (MDA-MB-231), gastric (AGS) and melanoma (MV3) cell lines. In the CAM model, the density of micro-vessels is 75±10 in the group supplemented with EO compared to 140±9 for the control group (b-FGF). In addition, the EO significantly reduced the number of newly formed vessels. The cytotoxicity was evaluated using the cell proliferation inhibition method and cell viability was measured using the MTT test. Results revealed that the treatment of cancer lines with different concentrations of EO reduces the rate of cell viability in a dose-dependent manner. EO showed the greatest cytotoxicity on the AGS line with an inhibition rate of 92.87±0.13% at the highest dose (4µL/mL), followed by the MV3 line (88.76±0.96%). CONCLUSION AND PROSPECTS: Data demonstrated that rose-scented geranium EO has an antitumor potential on metastatic cancer cell lines. It is distinguished by its antiproliferative, anti-angiogenic, and anti-inflammatory activities. Medicinal plants might contain new molecules, with new structures, which could become lead candidate among future anticancer drugs.

Distinct metabolic pathways drive monoterpenoid biosynthesis in a natural population of Pelargonium graveolens.

Pelargonium graveolens is a wild predecessor to rose-scented geranium hybrids prized for their essential oils used as fragrances and flavorings. However, little is known about their biosynthesis. Here we present metabolic evidence that at least two distinct monoterpene biosynthetic pathways contribute to their volatile profiles, namely, cyclic p-menthanes such as (-)-isomenthone and acyclic monoterpene alcohols such as geraniol and (-)-citronellol and their derivatives (referred to here as citronelloid monoterpenes). We established their common origin via the 2C-methyl-d-erythritol-4-phosphate pathway but found no indication these pathways share common intermediates beyond geranyl diphosphate. Untargeted volatile profiling of 22 seed-grown P. graveolens lines demonstrated distinct chemotypes that preferentially accumulate (-)-isomenthone, geraniol, or (-)-citronellol along with approximately 85 minor volatile products. Whole plant 13CO2 isotopic labeling performed under physiological conditions permitted us to measure the in vivo rates of monoterpenoid accumulation in these lines and quantify differences in metabolic modes between chemotypes. We further determined that p-menthane monoterpenoids in Pelargonium are likely synthesized from (+)-limonene via (+)-piperitone rather than (+)-pulegone. Exploitation of this natural population enabled a detailed dissection of the relative rates of competing p-menthane and citronelloid pathways in this species, providing real time rates of monoterpene accumulation in glandular trichomes.

Performance of rose scented geranium (Pelargonium graveolens) in heavy metal polluted soil vis-à-vis phytoaccumulation of metals.

An investigation was carried out to evaluate the effect of heavy metal toxicity on growth, herb, oil yield and quality and metal accumulation in rose scented geranium (Pelargonium graveolens) grown in heavy metal enriched soils. Four heavy metals (Cd, Ni, Cr, and Pb) each at two levels (10 and 20 mg kg-1 soil) were tested on geranium. Results indicated that Cr concentration in soil at 20 mg kg-1 reduced leaves, stem and root yield by 70, 83, and 45%, respectively, over control. Root growth was significantly affected in Cr stressed soil. Nickel, Cr, and Cd concentration and accumulation in plant increased with higher application of these metals. Chromium, nickel and cadmium uptake was observed to be higher in leaves than in stem and roots. Essential oil constituents were generally not significantly affected by heavy metals except Pb at 10 and 20 ppm, which significantly increased the content of citronellol and Ni at 20 ppm increased the content of geraniol. Looking in to the higher accumulation of toxic metals by geranium and the minimal impact of heavy metals on quality of essential oil, geranium can be commercially cultivated in heavy metal polluted soil for production of high value essential oil.

Microbe-based technology ameliorates glandular trichomes, secondary metabolites and antioxidants in Pelargonium graveolens L'Hér.

BACKGROUND: Despite the vast exploration of microbes for plant health, there is a lack of knowledge about the synergistic effects of specific microorganisms in sustainable agriculture, especially in medicinal plants such as Pelargonium graveolens L'Hér. The aim of this study was to evaluate how synergistic microbes Trichoderma harzianum ThU, Glomus intraradices and Bacillus subtilis CIM affected crop productivity, secondary metabolites and glandular trichome number in P. graveolens. RESULTS: The results demonstrated a significant (P < 0.05) increase in plant growth, secondary metabolites, total chlorophyll, carotenoids, carbohydrates, total phenolics, total flavonoids, free radical-scavenging activity and total antioxidant capacity of P. graveolens treated with synergistic bioinoculants as compared with the control. Most interestingly, an increase in essential oil by 32% in the treatment with all three microbes was observed. Furthermore, the principal aroma compounds citronellol and geraniol also increased in the same treatment. A positive and direct correlation was observed between essential oil content and number of glandular trichomes in all treatments. CONCLUSION: The present study highlights an explicit amalgamation of prospective microbes showing potential for synergism that act as biostimulants in enhancing plant production and improving the antioxidant and aroma profile of P. graveolens. © 2016 Society of Chemical Industry.

Cellulase activity screening using pure carboxymethylcellulose: application to soluble cellulolytic samples and to plant tissue prints.

Reliable, rapid and inexpensive detection of cellulolytic enzymes that can be used for a wide variety of biological and environmental samples are currently in high demand. Here, a new cellulase detection protocol is described that circumvents problems observed with popular agar-based methods by exploiting the ability of carboxymethylcellulose (CMC) to form gel-like surfaces on its own. These pure CMC-layers are sensitive to cellulolytic degradation and stainable by Gram's iodine without showing unwelcome reactions with other enzymes. The staining intensity negatively correlates with the enzyme activity and can be used for quantification. Cellulase activities are not obstructed by high sugar contents (e.g., in plant material) which limit the applicability of other quantification methods, making our new method particularly attractive for screening of plant extracts. A useful variant of this new method is its applicability to plant tissue prints for spatial mapping of the cellulolytic activity in a zymogram-like fashion.

Changes in growth, essential oil yield and composition of geranium (Pelargonium graveolens L.) as affected by growing media.

BACKGROUND: Using proper growing medium is known to be an effective way to improve crop growth and yield. However, the effects of growing media on geranium essential oil have scarcely ever been examined in detail. In this research, the effects of different growing media (soil, sand, pumice, perlite and perlite + cocopeat) on growth, oil yield and composition of geranium were studied. RESULTS: Growth was significantly improved in soilless-grown plants compared with soil-grown plants. Oil yield of soilless-grown plants (except for pumice) was about threefold higher than that of soil-grown plants. The increase in oil yield was correlated with higher leaf dry weight (r² = 0.96), as oil content was not affected. The citronellol/geranium ratio of oil was clearly affected by growing media, ranging from 5:1 in soil culture to 3:1 in soilless culture. The latter is acceptable for perfumery. CONCLUSION: Compared with soil, soilless media could produce higher yields of high-quality geranium oil that fits market requirements. Growth, oil yield and composition of plants grown in sand (a cheap and abundant growing medium) were not significantly different from those of plants grown in perlite and perlite + cocopeat.

A comparative UPLC-orbitrap-MS-based metabolite profiling of three Pelargonium species cultivated in Egypt.

Several Pelargonium species are cultivated mainly to produce essential oils used in perfume industry and for ornamental purposes. Although the chemical composition and biological activities of their essential oils were extensively investigated, there is limited information about the chemical composition of their non-volatile constituents. In this study, we report an Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)-based metabolomics approach for the annotation and analysis of various metabolites in three species; P. graveolens, P. denticulatum, and P. fragrans utilizing The Global Natural Product Social Molecular Networking (GNPS) and multivariate data analyses for clustering of the metabolites. A total of 154 metabolites belonging to different classes were annotated. The three species are good sources of coumarins, benzoic acid derivatives, organic acids, fatty acids, and phospholipids. However, the highest level of flavonols (mono- and di-O-glycosides) and cinnamic acid derivatives was found in P. graveolens and P. denticulatum, whereas tannins and flavone C-glycosides were abundant in P. fragrans. The metabolic profiles clarified here provide comprehensive information on the non-volatile constituents of the three Pelargonium species and can be employed for their authentication and possible therapeutic applications.

The fitness of pelargonium cuttings affects the relationship between the photochemical activity of the photosynthetic apparatus and rooting ability.

Pelargoniums cultivated for ornamental purposes rely on efficient vegetative propagation. This study researched applicability of chlorophyll fluorescence for validating the physiological conditions of pelargonium cuttings. Results indicated a correlation between the chlorophyll fluorescence and rooting potential. The ET0/RC values were negatively correlated with the rooting efficiency between the varieties and the duration of cold storage. A negative correlation was observed between OJIP parameters, representing energy flow in thylakoids, and chlorophyll content in cuttings with lower nutritional status. The phenomenological energy fluxes for leaf cross-sections and the number of active PSII reaction centers in the not-excited state (RC/CS0) increase with raised chlorophyll concentration. This imply the influence of rooting ability on the demand for photoassimilates in pelargonium cuttings, which can be detected early on through chlorophyll fluorescence analysis but not chlorophyll content measurements. Chlorophyll fluorescence evaluation, along with specific OJIP test parameters such as the performance indices PIABS and PItotal, prove useful for predicting rooting efficiency in relation to the nutritional status of cuttings, suggesting the effects of cuttings cold storage and discerning varietal differences in rooting. This study establishes the pragmatic application of chlorophyll fluorescence assessment for elucidating the physiological intricacies of pelargonium cuttings and factors influencing rooting efficiency.

Isoprenyl diphosphate synthases of terpenoid biosynthesis in rose-scented geranium (Pelargonium graveolens).

The essential oil of Pelargonium graveolens (rose-scented geranium), an important aromatic plant, comprising mainly mono- and sesqui-terpenes, has applications in food and cosmetic industries. This study reports the characterization of isoprenyl disphosphate synthases (IDSs) involved in P. graveolens terpene biosynthesis. The six identified PgIDSs belonged to different classes of IDSs, comprising homomeric geranyl diphosphate synthases (GPPSs; PgGPPS1 and PgGPPS2), the large subunit of heteromeric GPPS or geranylgeranyl diphosphate synthases (GGPPSs; PgGGPPS), the small subunit of heteromeric GPPS (PgGPPS.SSUI and PgGPPS.SSUII), and farnesyl diphosphate synthases (FPPS; PgFPPS).All IDSs exhibited maximal expression in glandular trichomes (GTs), the site of aroma formation, and their expression except PgGPPS.SSUII was induced upon treatment with MeJA. Functional characterization of recombinant proteins revealed that PgGPPS1, PgGGPPS and PgFPPS were active enzymes producing GPP, GGPP/GPP, and FPP respectively, whereas both PgGPPS.SSUs and PgGPPS2 were inactive. Co-expression of PgGGPPS (that exhibited bifunctional G(G)PPS activity) with PgGPPS.SSUs in bacterial expression system showed lack of interaction between the two proteins, however, PgGGPPS interacted with a phylogenetically distant Antirrhinum majus GPPS.SSU. Further, transient expression of AmGPPS.SSU in P. graveolens leaf led to a significant increase in monoterpene levels. These findings provide insight into the types of IDSs and their role in providing precursors for different terpenoid components of P. graveolens essential oil.

Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA.

While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

In situ molecular analysis of plant tissues by live single-cell mass spectrometry.

We report the development of a rapid, direct molecular analysis of live, single plant cells viewed under a video microscope in their natural environment. A nanoelectrospray tip was used to extract the contents of a single leaf, stem, or petal cell from Pelargonium zonale, and the samples were analyzed on an Orbitrap mass spectrometer by nanoelectrospray ionization. Around a thousand m/z peaks belonging to metabolites and other compounds in each sample were obtained and processed by using statistical tools to find the cell specific molecular peaks. Hybrid high-resolution mass spectrometry analysis was performed to confirm the structure of specific metabolites from the analyzed samples. This method is useful for identifying specific molecules in live single cells from plant tissue and will allow different cell types and stages from different sites in the plant to be compared with morphological observations.

Analysis of the photosynthetic response induced by variation potential in geranium.

Electrical signals (action and variation potentials) caused by environmental stimuli induce a number of physiological responses in plants including changes in photosynthesis; however, mechanisms of these changes remain unclear. We investigated the influence of the variation potential on photosynthesis in geranium (Pelargonium zonale) under different conditions (control, low external CO2 concentration, and actinic light absence). The variation potential caused by lamina burning induced a reduction in photosynthesis (decreases in effective quantum yields of photosystem I and II, CO2 assimilation rate, and stomatal conductance) in unstimulated leaves under control conditions. Changes in the majority of light-stage parameters (photosystem I and II quantum yields, coefficients of photochemical and non-photochemical quenching, quantum yield of non-photochemical energy dissipation in photosystem I due to donor-side limitation) were correlated with a decrease in CO2 assimilation rate. The changes were similar to those caused by lowering [CO2]; their magnitudes decreased both under low external CO2 concentration and without actinic light. These results support the hypothesis that Calvin cycle inactivation plays a key role in photosynthetic response induced by electrical signals. However, a decrease in electron transport through the PSI acceptor side also induced by variation potential was not correlated with a decrease in the CO2 assimilation rate and did not depend on the external CO2 concentration or actinic light intensity. Thus, we suggest that there are two different mechanisms of light-stage inactivation induced by the variation potential in geranium: one strongly dependent on dark-stage inactivation and one weakly dependent on dark-stage inactivation.

The 13C/12C isotopic signal of day-respired CO2 in variegated leaves of Pelargonium × hortorum.

In leaves, although it is accepted that CO(2) evolved by dark respiration after illumination is naturally (13) C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces (13) C-depleted or (13) C-enriched CO(2). Here, we applied equations described previously for mesocosm CO(2) exchange to investigate the carbon isotope composition of CO(2) respired by autotrophic and heterotrophic tissues of Pelargonium × hortorum leaves, taking advantage of leaf variegation. Day-respired CO(2) was slightly (13) C-depleted compared to organic matter both under 21% O(2) and 2% O(2). Furthermore, most, if not all CO(2) molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly (13) C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO(2) refixation and the specific nitrogen metabolism in white leaf areas.

Genetic alterations for increased coumarin production lead to metabolic changes in the medicinally important Pelargonium sidoides DC (Geraniaceae).

The medicinal plant Pelargonium sidoides is fast becoming threatened due to the overharvest of its tubers from the wild to produce a phytopharmaceutical for treating respiratory infections. The action of the coumarins is implicated in the efficacy of the commercial herbal extract with the highly oxygenated coumarins exhibiting the best anti-bacterial and anti-viral activity. Through this work we aimed at exploring the metabolic effects of Agrobacterium rhizogenes transformation. After confirmation of transgenesis using PCR amplification of the rol A (320 bp), rol B (400 bp) and rol C (600 bp) genes, metabolite profiles indicated a high level of variability between the different transgenic clones but these had more compounds compared to non-transgenic control cultures. This was represented by a two- to four-fold increase in detected metabolites in transgenic clones. We quantified several commercially important coumarins, flavonoids and phenolic acids. One of the clones had six out of nine of these metabolites. Overall, the concentration of these metabolites of interest were significantly changed in transgenic root cultures, for instance shikimic acid was recorded at the highest level in clone A4T-A. Production of key metabolites at significantly higher concentrations due to transgenesis and positive anti-bacterial activity exhibited by transgenic roots lends support to the idea of developing these clones as an alternative source that will allow for sustainable access to economically valuable secondary compounds of P. sidoides.

Light absorption by isolated chloroplasts and leaves: effects of scattering and 'packing'.

Light absorption was quantified in the following systems: isolated chloroplasts and leaves of spinach (Spinacea oleracea L.), a mutant of geranium (Pelargonium zonale L.) widely differing in pigment content, and coleus (Coleus blumei Benth.) at different stages of leaf ontogenesis. For these species and pea (Pisum sativum L.), scattering-compensated absorption spectra of chloroplast suspensions are presented. Comparison of leaf and chloroplast spectra showed considerable changes in the extent of the 'package' effect and the lengthening of the effective optical path in a leaf. The difference between leaf and isolated chloroplast absorption could be quantitatively described by adapting Duysens's treatment of flattening. It was found that the accumulation of chlorophyll in leaves is accompanied by a monotonous enhancement of the package effect. The results are discussed with special reference to the role of light scattering in leaf optics, light utilization in photosynthesis and wavelength-dependent light gradients in a leaf.