Direct regeneration in vitro and transient GUS expression in Mentha×piperita

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient -glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l^–1) and 6-benzylaminopurine (2 mg l^–1), followed by selection of regenerated shoots with 200 mg 1^–1 kanamycin.Abbreviations BA 6-benzylaminopurine - GUS -glucuronidase - MS Murashige and Skoog (1962) - NAA -naphthalenacetic acid - PIG particle inflow gun - SEM scanning electron microscope     

In situ detection of expression of thegus reporter gene in transgenic plants: ten years of blue genes

Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus (uidA) gene ofEscherichia coli, which encodes a -glucuronidase (E.C. 3.2.1.31; GUS), have been the most widely used during the last ten years. The apparent simplicity of the histochemical GUS assay has been a major factor in the increase in articles usinggus genes. However, over the last four years, there have been occasional reports expressing doubts concerning the specificity of the observed localizations based on discrepancies between results obtained with GUS histochemistry and immunocytochemistry and/orin situ hybridization. This brief review compares the results obtained with immunocytochemistry with those obtained with various GUS substrates for histochemical studies. Certain sources of artefact are discussed, as are the limits that should be imposed on interpretation of GUS histochemistry results at the organ, tissue and cell levels.     

Spurious localizations of diX-indigo microcrystals generated by the histochemical GUS assay

For several models expressing theuidA orgus reporter gene with or without a presequence for mitochondrial targeting, we have demonstrated that the compartmentation of -glucuronidase (E.C. 3.2.1.31) activity was not in agreement within situ localization of the diX-indigo microcrystals generated by the cytoenzymological GUS assay. These crystals were generally associated with the various cytomembranes and lipid inclusions. Experiments with purified -glucuronidase or withgus-expressing bacteria incubated with 5-bromo-4-chloro-3-indolyl--d-glucuronide and maize oil-phosphate buffer emulsion indicated that the intermediate products resulting from the GUS assay actively diffused and crystallized preferentially in association with lipids, sometimes far from the site of enzyme activity. This phenomenon could not be suppressed by the addition of potassium ferricyanide in the incubation medium. These findings are discussed with regard to previously reported biochemical and histochemical data on animal tissues, and focus on the necessity for caution in studies of tissue-specific gene expression using the GUS assay, particularly for lipid-rich plant models.     

Electron microscopy and X-ray microanalysis as tools for fine localization of the β-glucuronidase activity in transgenic plants harbouring the GUS reporter gene

Summary The GUS reporter gene encoding -glucuronidase is very useful in various domains of plant genetic engineering. A method for ultrastructural detection of its activity was developed using 35S-GUS transgenic tobacco root tips. Short glutaraldehyde prefixation at 4°C preserved up to 70% enzyme activity and was followed by brief incubation in X-Glu, strong postfixations, then quick dehydration at low temperature before resin embedding. In these conditions, transgenic cells were well preserved and displayed electron dense indigo precipitates with a crystalline structure as shown by electron diffraction. Due to other dense structures in the tissues, controls of the nature of the reaction product (diX-indigo) were necessary. A first control was carried out by means of X-ray microanalysis in order to check the presence of bromine. Other controls, including incubated non-transformed tissues, non-incubated or boiled transgenic roots as well as transgenic samples incubated with the specific -glucuronidase inhibitor, D-saccharic acid-1,4-lactone, were also carried out. The discussion points out the potential uses but also the limits of the method, non-specific localizations of the diX-indigo microcrystals being possible.Abbreviations X-Glu 5-bromo-4-chloro-3-indolyl--D-glucuronic acid - diX-indigo 5,5-dibromo-4,4-dichloro-indigo - MUG methyl umbelliferyl glucuronide - 4-MU 4-methyl umbelliferone - EDTA ethylene diamine tetraacetic acid disodium salt - PB phosphate buffer - CaMV cauliflower mosaic virus     

Chemical and histochemical analysis of 'Quatre Saisons Blanc Mousseux', a Moss Rose of the Rosa x damascena group

BACKGROUND AND AIMS: Moss roses are old garden roses covered with a mossy growth on flower pedicel and calyx. This moss releases a pine-scented oleoresin that is very sticky and odoriferous. Rosa x centifolia 'muscosa' was the first moss rose to be obtained by bud-mutation but, interestingly, R. x damascena 'Quatre Saisons Blanc Mousseux' was the first repeat-blooming cultivar, thus interesting breeders. In the present study, the anatomy of these sports (i.e. bud-mutations) is characterized and the volatile organic compounds (VOCs) produced by the moss versus the petals are identified. They are compared between the two lines and their respective parents. METHODS: Anatomy of the moss is studied by environmental scanning electron microscopy and histochemical light microscopy. Sudan Red IV and Fluorol Yellow 088 are used to detect lipids, and 1-naphthol reaction with N,N-dimethyl-p-phenylenediamine to detect terpenes (Nadi reaction). Head-space or solid/liquid extraction followed by gas chromatography and mass spectrometry are used to identify VOCs in moss, trichomes and petals. KEY RESULTS: Moss of the two cultivars has the same structure with trichomes on other trichomes but not exactly the same VOCs. These VOCs are specific to the moss, with lots of terpenes. An identical VOC composition is found in leaves but not in petals. They are nearly the same in the moss mutants and in the respective wild types. CONCLUSIONS: Sepals of moss roses and their parents have a specific VOC pattern, different from that of the petals. The moss corresponds to a heterochronic mutation with trichomes developing on other trichomes. Such a mutation has probably appeared twice and independently in the two lines.     

Production and Emission of Volatile Compounds by Petal Cells

We localized the tissues and cells that contribute to scent biosynthesis in scented and non-scented Rosa × hybrida cultivars as part of a detailed cytological analysis of the rose petal. Adaxial petal epidermal cells have a typical conical, papillate shape whereas abaxial petal epidermal cells are flat. Using two different techniques, solid/liquid phase extraction and headspace collection of volatiles, we showed that, in roses, both epidermal layers are capable of producing and emitting scent volatiles, despite the different morphologies of the cells of these two tissues. Moreover, OOMT, an enzyme involved in scent molecule biosynthesis, was localized in both epidermal layers. These results are discussed in view of results found in others species such as Antirrhinum majus, where it has been shown that the adaxial epidermis is the preferential site of scent production and emission.Key Words: floral scent, petal epidermis, Rosa, terpenes, volatilesMany plant species produce volatile compounds and these molecules serve a range of purposes. For example, compounds that are emitted from leaves are generally required for the defence of the plant against insect predators. On the other hand, floral compounds attract beneficial insects, leading to pollination of the flower. In leaves, scent compounds are very often synthesised in specialized cells grouped in structures termed trichomes or secretory glands. In many flowers, it is well documented that floral fragrance is produced by the corolla,¹ although other flower parts, such as the stamens in Ranunculus acris,² sometimes play an important role in fragrance emission. In some flowers, in particular those belonging to the Orchidaceae family, scent is emitted by specialized areas of the petal, which have been termed osmophores by Vogel.³ However, in most flowers, when petals produce scent, it is thought to be emitted by all the cells of the petal in a diffusive manner.⁴ In many flowers, such as roses, the adaxial petal epidermal cells have a conical-papillate shape whereas the cells of the abaxial epidermis are flat (Fig. 1).⁵ The shape of these conical cells is controlled by a Myb-factor named MIXTA in Antirrhinum majus⁶ and their shape has been shown to play a role in the diffusion of light, thereby enhancing the attractiveness of the flower.⁷ Flowers of the mixta mutant have flat adaxial petal epidermal cells that reflect less light⁸ and as a consequence attract less insects.⁹ Along the same lines, Kolosova et al.¹⁰ demonstrated that S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT), an enzyme involved in scent biosynthesis, was localized in the conical cells of the inner epidermal layer and to a much lesser extent, in the cells of the outer epidermis of the lobes of snapdragon petals. On the basis of these latter observations, some authors have proposed that the papillate cell shape could enhance the diffusion of scent molecules or influence its directionality and be of adaptive significance not only by enhancing light reflection but also by enhancing scent production.^11,12Open in a separate windowFigure 1Hand-made cross-section of Rosa × hybrida petal; Ad, adaxial epidermis; Ab, abaxial epidermis; P, spongy parenchyma. Bar = 20 µm.To test the hypothesis that the adaxial epidermis is a privileged site for the production and emission of scent, we chose a highly scented flower, the rose. Contrary to what was expected, we found that both the adaxial and abaxial epidermal layers of the petal were sites of scent production and emission. We were able to show that NaDi reagent stained purple droplets in both epidermal layers of the rose petal, indicating that they both contain terpenes. Several different techniques, including the analysis of epidermal peels and epidermal layer-specific headspace analysis failed to detect a strong difference between the production and emission of scent in the two epidermal layers. Moreover, the detection of OOMT protein, an enzyme involved in 3,5-dimethoxytoluene production, in both the abaxial and adaxial epidermis, indicated that biosynthesis of at least some phenolic scent compounds occurs in both tissues. It will be interesting to extend this approach using in situ hybridization or immunolocalization to determine whether other pathways such as terpene metabolism are also active in the abaxial epidermis.It is striking to note that in Clarkia breweri, which has actinomorphic flowers like the rose, expression of the S-adenosyl-L-methionine:(iso) eugenol O-methyltransferase (IEMT) gene seems to occur in both epidermal layers.¹³ A. majus flowers have a different structure, they are highly zygomorphic with a flower shape that is adapted for bee pollination and includes specialized cell types in different parts of the flower (the lobes and the tube). To determine whether emission of scent in highly specialized flowers such as A. majus is linked to cell shape, it would be very useful to know whether mixta mutant flowers which have flat epidermal cells are impaired in their capacity to emit scent. However, the explanation may not be as simple. A recent study of the synthesis and emission of methyl benzoate showed that in Nicotiana suaveolens, as in the rose, both epidermal layers of the petal lobes are involved in scent production, whereas in Stephanotis floribunda, SAMT, another enzyme involved scent biosynthesis, is localized only in the adaxial epidermis and subepidermal regions of the flower petal lobes.¹⁴ It is intriguing to note that N. suaveolens has bullate to rugose epidermal cell layers on both sides of the petal whereas S. floribunda has tight flat to bullate epidermal cells.The reasons for the differences in the potential for scent emission of the two petal epidermal layers in the rose and other species are not known. However, our results and a survey of the literature clearly indicate that, in petals, epidermal cells may have diverse shapes and that the shape of the cells is not necessarily a reliable indicator of the secretory potential of those cells. It will be interesting to see whether common structural features and/or molecular factors are responsible for the differences between these various cell types.     

Functional diversification in the Nudix hydrolase gene family drives sesquiterpene biosynthesis in Rosa × wichurana

Roses use a non‐canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1‐2. In order to study the function of the RwNUDX1‐2 protein, we analyzed the volatile profiles of an F₁ progeny generated by crossing R. chinensis cv. ‘Old Blush’ with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1‐2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E‐farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1‐2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E‐farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1‐2 gene co‐localized with the QTL for E,E‐farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed.