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BackgroundSclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species.ResultsBased on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants.ConclusionsSsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

BackgroundSclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species.ResultsBased on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants.ConclusionsSsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

We established conditions for the regeneration of natural maize zygotes isolated from pollinated plants with the goal of investigating the molecular control of early embryogenesis in higher plants. Viable zygotes were excised from embryo sacs by minimal enzymatic digestion and microdissection. Viable zygotes transferred to coculture with androgenic microspores from barley developed into embryo-like structures in 61% of the cases. No development was observed when zygotes were cultured in the presence of maize anthers undergoing androgenetic embryogenesis. Zygote-derived embryo-like structures regenerated into fertile plants through secondary embryogenesis when transferred to solid medium. The first zygotic division was asymmetrical and bipolar structures similar to pretransitional embryos observed in planta were later produced as observed using light and electron microscopy. Conditions for efficient microinjection of DNA into zygotes were established. Calcofluor and PATAG staining of zygotes showed that cell wall regeneration occurred as early as 20 min after enzymatic isolation and that after 2 hr, each zygote was bordered with cell wall material. Through quantitative microphotometry, DNA synthesis during the first cell cycle of the zygote was shown to occur between isolation and 12 hr of culture. Microinjection of two types of reporter genes (GUS gene and anthocyanin regulatory genes) demonstrates transient expression in plant zygotes. On average, 3.5% of microinjected zygotes showed transgenic expression. Reporter gene expression was observed in zygotes at different time points of their first cell cycle.

Lavandula pedunculata (Mill.) Cav. subsp. lusitanica, Lavandula stoechas L. subsp. stoechas and Lavandula viridis l'Hér. are three lavender taxa that belong to the botanical section Stoechas and are widely used as aromatherapy, culinary herb or folk medicine in many Mediterranean regions. The analysis of their bioactive volatile constituents revealed the presence of 124 substances, the most abundant being the bicyclic monoterpenes fenchone, camphor and 1,8-cineole that give these three species their respective chemotypes. Most noteworthy was fenchone which, with its reduced form fenchol, made 48% of the total volatile constituents of L. pedunculata while present at 2.9% in L. stoechas and undetectable in L. viridis. In order to provide a molecular explanation to the differences in volatile compounds of these three species, two monoterpene synthases (monoTPS) and one sesquiterpene synthase (sesquiTPS) were cloned in L. pedunculata and functionally characterized as fenchol synthase (LpFENS), α-pinene synthase (LpPINS) and germacrene A synthase (LpGEAS). The two other lavender species contained a single orthologous gene for each of these three classes of TPS with similar enzyme product specificities. Expression profiles of FENS and PINS genes matched the accumulation profile of the enzyme products unlike GEAS. This study provides one of the rare documented cases of chemotype modification during plant speciation via changes in the level of plant TPS gene expression, and not functionality. [ABSTRACT FROM AUTHOR]

In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a ( E)-β-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and ( E)-β-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender. [ABSTRACT FROM AUTHOR]

Meiosis is often described as a special case of cell division since it differs from mitosis in having two nuclear divisions without an intervening S-phase. It will be of great interest to uncover what molecular mechanisms underlie these special features of meiosis. We previously reported that the tardy asynchronous meiosis (tam) mutant of Arabidopsis (Arabidopsis thaliana) is slower in cell cycle progression in male meiosis. Here we report that TAM encodes the A-type cyclin, CYCA1;2. The point mutation in tam replaced a conserved threonine with an isoleucine in the linker region between the α4 and α5 helices of the first cyclin fold. By studying the dynamics of a CYCA1;2-green fluorescent protein fusion protein under the control of the CYCA1;2 promoter, we found that the fusion protein was most abundant at pachytene, but was undetectable from late prophase I until telophase II. Nonetheless, cell cycle progression in tam was delayed in both pachytene and meiosis II. We conclude either that the CYCA1;2 produced in prophase I indirectly regulates meiosis II progression, or that a very low level of CYCA1;2 directly regulates meiosis II progression. Either of these scenarios is a deviation from the typical mode of action of mitotic cyclins in mitosis and meiosis I, in which each nuclear division is coupled with a peak of expression of mitotic cyclins. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) is a favorable method to study gene function by posttranscriptional gene silencing in plants. Here we describe a methodology of graft-accelerated VIGS in rose aimed at obtaining posttranscriptional gene silencing in the flower. The resulting phenotype can be observed within 5-6 weeks post infiltration. By using this method, we successfully silenced the expression of several genes involved in processes such as scent production, petal coloration, or flower architecture. We showed that graft-accelerated VIGS was faster, more efficient, and more convenient than conventional methods previously developed in rose such as agroinfiltration of young plantlets and in vitro cultured tissues or seeds.