The biosynthesis of the anti-microbial diterpenoid leubethanol in Leucophyllum frutescens proceeds via an all-cis prenyl intermediate.

Serrulatane diterpenoids are natural products found in plants from a subset of genera within the figwort family (Scrophulariaceae). Many of these compounds have been characterized as having anti-microbial properties and share a common diterpene backbone. One example, leubethanol from Texas sage (Leucophyllum frutescens) has demonstrated activity against multi-drug-resistant tuberculosis. Leubethanol is the only serrulatane diterpenoid identified from this genus; however, a range of such compounds have been found throughout the closely related Eremophila genus. Despite their potential therapeutic relevance, the biosynthesis of serrulatane diterpenoids has not been previously reported. Here we leverage the simple product profile and high accumulation of leubethanol in the roots of L. frutescens and compare tissue-specific transcriptomes with existing data from Eremophila serrulata to decipher the biosynthesis of leubethanol. A short-chain cis-prenyl transferase (LfCPT1) first produces the rare diterpene precursor nerylneryl diphosphate, which is cyclized by an unusual plastidial terpene synthase (LfTPS1) into the characteristic serrulatane diterpene backbone. Final conversion to leubethanol is catalyzed by a cytochrome P450 (CYP71D616) of the CYP71 clan. This pathway documents the presence of a short-chain cis-prenyl diphosphate synthase, previously only found in Solanaceae, which is likely involved in the biosynthesis of other known diterpene backbones in Eremophila. LfTPS1 represents neofunctionalization of a compartment-switching terpene synthase accepting a novel substrate in the plastid. Biosynthetic access to leubethanol will enable pathway discovery to more complex serrulatane diterpenoids which share this common starting structure and provide a platform for the production and diversification of this class of promising anti-microbial therapeutics in heterologous systems.

Stemarane Diterpenes and Diterpenoids.

: In this article the scientific activity carried out on stemarane diterpenes and diterpenoids, isolated over the world from various natural sources, was reviewed. The structure elucidation of stemarane diterpenes and diterpenoids was reported, in addition to their biogenesis and biosynthesis. Stemarane diterpenes and diterpenoids biotransformations and biological activity was also taken into account. Finally the work leading to the synthesis and enantiosynthesis of stemarane diterpenes and diterpenoids was described.

Phytofiltration of arsenic and cadmium by using an aquatic plant, Micranthemum umbrosum: phytotoxicity, uptake kinetics, and mechanism.

Arsenic (As) and cadmium (Cd) are noxious and carcinogenic pollutants that can be removed from water by using emerging, ecofriendly, phytofiltration technology that employs Micranthemum umbrosum. After culturing M. umbrosum for 7 days in a hydroponic experiment, accumulation of 1219±44.11 µg As g(-1) and 799.40±30.95 µg Cd g(-1) were observed in the leaves, from 1000 µg As L(-1) and 1000 µg Cd L(-1) of water, respectively. Plant and water samples were analyzed for assessing the As and Cd accumulations, translocations, phytotoxic effects, uptake mechanisms and kinetics, and for evaluating the potential of M. umbrosum in As and Cd phytofiltration. The uptake pattern was leaf>stem>root for both pollutants. The plant showed higher resistance to As than to that to Cd. Uptake of inorganic As species was much greater than that of organic As and was found at above the substrate concentration. However, Cd showed similar uptake pattern to that of inorganic As species, and the data was better fit to a non-linear than a linear model. Low molecular weight substances that have thiol group(s) may be responsible for the binding of As in plants whereas Cd showed a different mechanism to that of As. M. umbrosum showed good As phytofiltration capabilities without any phytotoxic effects, but it was found to be a moderate accumulator of Cd with some phytotoxic effect compare to some other previously studied plant.

The dynamics of recovery and growth: how defoliation affects stored resources.

Growth rate varies widely among species and the trade-off between growth rate and storage or maintenance traits is a principal axis of variation between species. Many plant species have substantial root stores, but very little is known about how growth rate modifies responses of these stores to defoliation and other stresses. Species with different growth rates are predicted to respond in distinct ways, because of variation in the pre-defoliation allocation to storage. Here, we quantified the dynamics of stored carbohydrates in seven species with varying growth rate, following defoliation in a pot experiment. For faster growing species, there was significant reduction in carbohydrate concentration following defoliation, followed by relatively fast recovery, whereas for slower growing species, carbohydrate concentration levels remained relatively invariant across treatments. Results for total carbohydrates mirrored those for concentration, but were not as significant. Our findings were consistent with the idea that faster growing species respond more rapidly than slower growers to defoliation, through changes in carbohydrate pool concentrations. Growth rate as an indicator of life-history and ecological strategy may therefore be key to understanding post-defoliation recovery and storage strategies.

Trace metal and metalloid contamination levels in soils and in two native plant species of a former industrial site: evaluation of the phytostabilization potential.

This study aimed at identifying the extent and type of contamination of a former lead smelting site in the area of Marseille, France, dating from the industrial revolution, and to evaluate environmental hazards and opportunities for phytoremediation, a promising sustainable technology. Amongst the native plants growing in this semiarid shrub ecosystem, two perennials Globularia alypum L. and Rosmarinus officinalis L. were selected. Twenty-one soil/plant couples were collected and seventeen additional soil samples were added to better characterize the soil pollution of the area. A multi-contamination by Pb, As, Sb, Zn, Cu was demonstrated, with huge variations within the contamination levels. The soils highest concentrations were encountered along the horizontal chimney and on the slag heaps area. However, both sites differed from each other. The former was characterized by the highest Pb, As and Sb concentrations that could reach 130, 7.0 and 9.0gkg(-1) respectively, the latter, by high Cu, Fe, Mn, S concentrations, even if it was also heavily contaminated by Pb and Zn. G. alypum and R. officinalis were shown to be metal-tolerant and to accumulate trace metals and As. Due to the low bioconcentration and translocation factors determined, both species may not be used for phytoextraction, but seem to be good candidates for phytostabilization.

Nemesia root hair response to paper pulp substrate for micropropagation.

Agar substrates for in vitro culture are well adapted to plant micropropagation, but not to plant rooting and acclimatization. Conversely, paper-pulp-based substrates appear as potentially well adapted for in vitro culture and functional root production. To reinforce this hypothesis, this study compares in vitro development of nemesia on several substrates. Strong differences between nemesia roots growing in agar or in paper-pulp substrates were evidenced through scanning electron microscopy. Roots developed in agar have shorter hairs, larger rhizodermal cells, and less organized root caps than those growing on paper pulp. In conclusion, it should be noted that in this study, in vitro microporous substrates such as paper pulp lead to the production of similar root hairs to those found in greenhouse peat substrates. Consequently, if agar could be used for micropropagation, rooting, and plant acclimatization, enhancement could be achieved if rooting stage was performed on micro-porous substrates such as paper pulp.

Evolution of petaloid sepals independent of shifts in B-class MADS box gene expression.

Attractive petals are an integral component of animal-pollinated flowers and in many flowering plant species are restricted to the second floral whorl. Interestingly, multiple times during angiosperm evolution, petaloid characteristics have expanded to adjacent floral whorls or to extra-floral organs. Here, we investigate developmental characteristics of petaloid sepals in Rhodochiton atrosanguineum, a close relative of the model species Antirrhinum majus (snapdragon). We undertook this in two ways, first using scanning electron microscopy we investigate the micromorphology of petals and sepals, followed by expression studies of genes usually responsible for the formation of petaloid structures. From our data, we conclude that R. atrosanguineum petaloid sepals lack micromorphological characteristics of petals and that petaloid sepals did not evolve through regulatory evolution of B-class MADS box genes, which have been shown to specify second whorl petal identity in a number of model flowering plant species including snapdragon. These data, in conjunction with other studies, suggests multiple convergent pathways for the evolution of showy sepals.

Physiological response of Cu and Cu mine tailing remediation of Paulownia fortunei (Seem) Hemsl.

The physiological responses and Cu accumulation of Paulownia fortunei (Seem) Hemsl. were studied under 15.7-157 µmol L(-1) Cu treatments in liquid culture for 14 days; the impacts of Cu concentration in the seedlings were evaluated under Cu mine tailing culture with acetic acid and EDTA treatment for 60 days. Results showed that the concentrations of Chl-a, Chl-b and Carotenoids significantly increased (p < 0.05) at 15.7-78.7 µmol L(-1)Cu treatment and significantly decreased at 157 µmol L(-1) treatment after 14 days of Cu exposure. The activities of superoxide dismutase (SOD) and catalase (CAT) significantly increased as Cu levels were enhanced and the activities of both SOD and CAT under 157 µmol L(-1) Cu stress were 2.9 and 1.9 times higher than that of control, respectively. The concentrations of proline and soluble sugars in the leaves of P. fortunei significantly increased as the Cu concentrations were elevated. Cu concentrations in roots, stems and leaves of P. fortunei increased significantly as Cu levels increased and reached 1911, 101 and 93 µg g(-1) dry weights (DW) at 157 µmol L(-1) Cu treatment, respectively. The seedlings of P. fortunei cultivated in Cu tailing experienced unsuccessful growth and loss of leaves in all treatments due to poor nutrition of the Cu tailing. The dry weight of P. fortunei increased under all the treatments of acetic acid after 60 days exposure. However, dry weight significantly decreased under both levels of EDTA. The Cu concentrations increased significantly in roots and decreased in leaves when each was treated with both concentrations of acetic acid. The Cu concentrations in the roots, stems and leaves increased significantly, and the concentrations of Cu in the stems and leaves under the treatment of 2 µmol L(-1) EDTA reached 189.5 and 763.1 µg g(-1) DW, respectively. The result indicated that SOD, CAT, proline and soluble sugars played an important role in coping with the oxidative stress of copper. Acetic acid could promote growth and EDTA at the experimental levels, which could also enhance Cu absorption and translocation into the stems and leaves of P. fortune. Furthermore, acetic acid and EDTA could be rationally utilized in Cu-contaminated soil.

Use of the plant Bellardia trixago for the enantiospecific synthesis of odorant products.

An easy procedure to obtain extracts enriched in trixagol monomalonylesther (1) from aerial parts of the plant Belladia trixago chemotype Trix was developed. Preparation of (+)-dihydro-gamma-ionone (4) was carried out directly from the extracts with good yields by selective oxidation. Other interesting odorant products as alpha-ambrinol (5), ambraldehyde (6) and the tricyclic compound 7 were synthesized very efficiently using (4) as intermediate.

Salinity effects on protein content, lipid peroxidation, pigments, and proline in Paulownia imperialis (Siebold & Zuccarini) and Paulownia fortunei (Seemann & Hemsley) grown in vitro

We evaluated the effects of saline stress on soluble proteins, lipid peroxidation (TBAR), chlorophyll a, chlorophyll b, beta-carotene, violaxanthin, and proline in Paulownia imperialis and Paulownia fortunei plants grown in vitro. When the propagated plants reached a determined size, they were transferred aseptically to WPM culture medium containing different sodium chloride concentrations (0, 20, 40, 60, 80, and 160 mM) and were sampled at 15 and 30 days. Proline content was determined at 30 days after transfer only. Protein concentration significantly decreased with the highest salt levels in P. imperialis compared to controls in which no sodium chloride was added. In both P. imperialis and P. fortunei, lipid peroxidation significantly increased at 15 days but decreased at 30 days. Chlorophyll a, chlorophyll b, beta-carotene, and violaxanthin significantly decreased with exposure to higher sodium chloride concentrations at 15 and 30 days in both species. Proline content in P. imperialis significantly increased in plants grown in 20 and 40 mM of sodium chloride and decreased in higher sodium chloride concentrations. In P. fortunei, this measure significantly decreased proline content at all salt concentrations in plants exposed to all levels of sodium chloride compared to controls. Our results show that P. imperialis is more tolerant to salt stress at the salinity conditions tested.

[Complex effects of simulated acid rain and Cu on the physiological characteristics of Paulownia fortunei and its detoxification mechanism].

A pot experiment was conducted to study the effects of simulated acid rain (pH 4.0, 5.0) and Cu (0-200 mg x kg(-1)) on the physiological characteristics of Paulownia fortunei and its detoxification mechanism. With no Cu addition, the leaf chlorophyll, carotenoid, O2 division by, H2O2, and MDA contents of P. fortunei had no significant differences between the two acid rain treatments. However, with the addition of 100 and 200 mg Cu x kg(-1), the chlorophyll and carotenoid contents of treatment pH 4.0 were lower, while the O2 divided by, H2O2 and MDA contents were higher thanthose of treatment pH 5.0. The chlorophyll a/b ratio of treatments Cu was higher than that of the control. The leaf Cu content decreased obviously with the increasing acidity of stimulated acid rain, but the root Cu content was in reverse. With increasing Cu addition, both the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) and the total contents of phytochelatins (PCs) and glutathione (GSH) in treatment pH 5.0 increased, while the activities of SOD, POD, CAT and APX in treatment pH 4.0 decreased after an initial increase, and the total contents of glutathione (GSH) decreased greatly in treatment 200 mg Cu x kg(-1). All of these demonstrated that the oxidative stress of high Cu concentration to P. fortunei was aggravated by stimulated acid rain.

Evidence for functional heterogeneity of sieve element-companion cell complexes in minor vein phloem of Alonsoa meridionalis.

Two modes of phloem loading have been proposed, apoplastic and symplastic, depending on the structure of sieve element-companion cell complexes (SE-CCCs) in minor vein phloem. Species are usually classified as either apoplastic or symplastic loaders although the cytology of SE-CCCs in minor veins of the majority of plants indicates that both mechanisms can be simultaneously involved in phloem loading. The functions of structurally different SE-CCCs in minor veins of the stachyose-translocating plant Alonsoa meridionalis were examined. A stachyose synthase gene, AmSTS1, was expressed in intermediary cells but not in the ordinary companion cell of the same vein. In contrast, sucrose transporter AmSUT1 protein was present in ordinary companion cells but not in the neighbouring intermediary cells. These data reveal the principles of phloem sap formation in A. meridionalis and, probably, in many other dicots. The two types of SE-CCCs within one and the same minor vein load different carbohydrates, using contrasting mechanisms for their delivery into the phloem. Lateral sieve pores in the minor vein phloem lead to mixing of the carbohydrates soon after loading. While symplastic and apoplastic pathways can function simultaneously during phloem loading, they are separated at the level of different SE-CCCs combined in phloem endings.

Transfer of selected mineral nutrients and trace elements in the host-hemiparasite association, Cistus-Odontites lutea, growing on and off metal-polluted sites.

The role of a hemiparasitic life-style in plant resistance to toxic trace elements in polluted soils is unclear. Restriction of metal uptake by the host, restriction of metal transfer from host to parasite, or transformation of metals into a less toxic form may play a role. This study analysed the transfer of selected mineral elements from soil to host (Cistus spp.) and from host to hemiparasite (Odontites lutea) at locations with different metal burdens: a Cu-rich serpentine site, Pb-Ba mine spoil and an unpolluted soil. Highest soil-to-host transfer factors for K, Mg, Ca, Zn, Cu and Pb were observed on the unpolluted soil. Statistically significant differences among locations of host-to-parasite transfer factors were only found for Ca and Pb. Restriction of transfer of unfavourable Ca/Mg ratios, characteristic at the serpentine site, and of high Pb and Zn concentrations at the Pb-Ba mine occurred mainly at the soil-host, and not at the host-parasite, level. Odontites lutea was able to withstand enhanced Zn and Pb concentrations and low Fe/Cu ratios in shoot tissue without developing toxicity symptoms. This could be caused by specific metal resistance mechanisms in this hemiparasite and/or the transformation and transfer of these metals into a less toxic form by the metal-tolerant host.

Cardenolide genin pattern in Isoplexis plants and shoot cultures.

An HPLC method for the quantitative analysis of cardenolides was developed and applied. The procedure was optimised for analysing small samples (40 mg dw) of plant and tissue culture material. ISOPLEXIS CANARIENSIS plants obtained from seeds accumulated cardenolides to about 20 - 40 micromol g (-1) dw as calculated from the levels of cardenolide genins released after acidic hydrolysis of methanolic extracts. The relative contents of xysmalogenin, digitoxigenin, uzarigenin and canarigenin were 5 - 15 %, 0 - 5 %, 10 - 15 % and 70 - 90 %, respectively. Shoot cultures were initiated from seeds, established as permanent cultures and cultivated on agar-solidified or in liquid medium. Shoot cultures maintained on solid medium contained an average of about 6 micromol cardenolides g (-1) dw. A relatively high proportion of digitoxigenin was found in two-thirds of the shoot cultures examined. The cardenolide content of amphibian shoot cultures averaged to about 1 micromol g (-1) dw. Plants regenerated from shoot cultures and maintained under hydroponic conditions accumulated the same amount of cardenolides as plants collected in the field. No cardenolides could be detected in callus cultures.

Identification of a dehydration and ABA-responsive promoter regulon and isolation of corresponding DNA binding proteins for the group 4 LEA gene CpC2 from C. plantagineum.

The resurrection plant Craterostigma plantagineum (Scrophulariaceae) is used as a model system to investigate the molecular and biochemical basis of desiccation tolerance. Genes which contribute to desiccation tolerance are expressed during dehydration of this plant. One of the dehydration-induced genes is CpC2, a group 4 LEA gene. The CpC2 promoter was analysed and a core promoter region (CPR) was identified which is critical for the responsiveness of the gene to dehydration and the plant hormone ABA. The CPR motif contains two ABA-response elements (ABRE) and a binding site for HDZIP transcription factors. A yeast one-hybrid screen was performed to isolate CPR binding proteins. This resulted in the isolation of a bZIP transcription factor (CpbZIP1) and three highly conserved CpHistone H3 proteins. Two of these CpHistone H3 proteins are constitutively expressed histone H3 variants which are suggested to be involved in gene regulation via histone modification. The CpbZIP1 belongs to the group S of bZIP genes which possess long 5'-UTRs with a putative regulatory function. A second very similar bZIP clone, CpbZIP2, was isolated which contains a conserved small upstream open reading frame (uORF) within the 5'-leader sequence. A possible regulatory role of the uORF is discussed.

Changes in macromolecular movement accompany organogenesis in thin cell layers of Torenia fournieri.

A range of fluorescently labelled probes of increasing molecular weight was used to monitor diffusion via the symplast in regenerating thin cell layer (TCL) explants of Torenia fournieri. An increase in intercellular movement of these molecules was associated with the earliest stages of vegetative shoot regeneration, with the movement of a 10 kDa dextran (FD 10000) observed between epidermal cells prior to the appearance of the first cell divisions. A low frequency of dextran movement in thin cell layers maintained under non-regenerating conditions was also observed, indicating a possible wound induced increase in intercellular movement. Dextran movement between epidermal cells reached a peak by day 4 of culture and then declined as cell division centres (CDCs) formed, became meristematic regions and finally emerged as adventitious shoots. Within CDCs, testing with small fluorescent probes (CF: carboxyfluorescein, mw 376 Da and F(Glu)3: fluorescein-triglutamic acid, mw 799 Da) revealed a mosaic of cell isolation and regions of maintained symplastic linkage. Within shoots, surface cells of the presumptive apical meristem permitted the intercellular movement of 10 kDa dextrans but epidermal cells of the surrounding leaf primordia did not permit dextran movement. In some cases, intercellular movement of CF was maintained within leaf primordia. Symplastic movement of labelled dextrans during regeneration in Torenia thin cell layers represents a significant increase in the basal size exclusion limit (SEL) of this tissue and reveals the potential for intercellular trafficking of developmentally related endogenous macromolecules.

AmSUT1, a sucrose transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis.

A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80-91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.

Utilization of glycine and serine as nitrogen sources in the roots of Zea mays and Chamaegigas intrepidus.

Glycine and serine are potential sources of nitrogen for the aquatic resurrection plant Chamaegigas intrepidus Dinter in the rock pools that provide its natural habitat. The pathways by which these amino acids might be utilized were investigated by incubating C. intrepidus roots and maize (Zea mays) root tips with [(15)N]glycine, [(15)N]serine and [2-(13)C]glycine. The metabolic fate of the label was followed using in vivo NMR spectroscopy, and the results were consistent with the involvement of the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) in the utilization of glycine. In contrast, the labelling patterns provided no evidence for the involvement of serine:glyoxylate aminotransferase in the metabolism of glycine by the root tissues. The key observations were: (i) the release of [(15)N]ammonium during [(15)N]-labelling experiments; and (ii) the detection of a characteristic set of serine isotopomers in the [2-(13)C]glycine experiments. The effects of aminoacetonitrile, amino-oxyacetate, and isonicotinic acid hydrazide, all of which inhibit GDC and SHMT to some extent, and of methionine sulphoximine, which inhibited the reassimilation of the ammonium, supported the conclusion that GDC and SHMT were essential for the metabolism of glycine. C. intrepidus was observed to metabolize serine more readily than the maize root tips and this may be an adaptation to its nitrogen-deficient habitat. Overall, the results support the emerging view that GDC is an essential component of glycine catabolism in non-photosynthetic tissues.

PkMADS1 is a novel MADS box gene regulating adventitious shoot induction and vegetative shoot development in Paulownia kawakamii.

Direct regeneration of shoot buds in vitro is an important technique in plant genetic manipulation. We describe the isolation and functional characterization of a novel MADS box cDNA (PkMADS1) from Paulownia kawakamii leaf explants undergoing adventitious shoot regeneration. mRNA gel blot analysis confirmed the expression of PkMADS1 in the shoot-forming cultures, but no signal was observed in the callus-forming cultures. PkMADS1 transcripts were also detected in shoot apices, but not in root apices, initial leaf explants or the flower. In situ hybridization revealed that its expression was restricted to developing shoot primordia in the excised leaf cultures, suggesting a role for this gene in adventitious shoot formation. Transgenic Paulownia plants over-expressing the PkMADS1 gene showed some changes in phenotype, such as axillary shoot formation. In the antisense transformants, shoots were stunted and had altered phyllotaxy, and, in some lines, the shoot apical meristem appeared to have been used up early during shoot development. Leaf explants from the antisense transgenic plants showed a tenfold decrease in shoot regeneration compared with explants from sense transformants or wild-type. Our results show that PkMADS1 is a regulator of shoot morphogenesis.