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AMF symbiosis in sand dunes is the key for maintenance of stable vegetation. The main goal of this work was to determine the effects of environmental and temporal factors on AMF living in sand dunes (Gulf of Valencia, Spain). Soil samples were collected seasonally at 6 sites, during 2 yrs, from three habitats and four plant species and the frequency and relative abundance of AMF was examined. AMF were more frequent in mobile than in embryonic dunes, in spring and in sites with old vegetation. Ten AMF species were identified, their distribution depending mainly on the anthropogenic disturbance of the site. Gigasporaceae Cetraspora sp. and Dentiscutata sp. preferred undisturbed soil whereas Diversisporaceae, Glomeraceae and other Gigasporaceae were associated with recently restored soils. All AMF species were found in all plant species although Corymbiglomus corymbiforme was mainly associated with Echinophora spinosa. Our results might be of help for Mediterranean sand dune restoration.

The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in developing spike lavender ( Lavandula latifolia Med) was analyzed using specific inhibitors and on the basis of 13 C-labeling experiments. The presence of mevinolin (MEV), an inhibitor of the MVA pathway, at concentrations higher than 0.5 μM significantly reduced plant development, but not the synthesis of chlorophylls and carotenoids. On the other hand, fosmidomycin (FSM), an inhibitor of the MEP pathway, at concentrations higher than 20 μM blocked the synthesis of chlorophyll, carotenoids and essential oils, and significantly reduced stem development. Notably, 1.2 mM MVA could recover the phenotype of MEV-treated plants, including the normal growth and development of roots, and could partially restore the biosynthesis of photosynthetic pigments and, to a lesser extent, of the essential oils in plantlets treated with FSM. Spike lavender shoot apices were also used in 13 C-labeling experiments, where the plantlets were grown in the presence of [U– 13 C 6 ]glucose. GC-MS-analysis of 1,8-cineole and camphor indicated that the C 5 -precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the methylerythritol phosphate (MEP) pathway. However, on the basis of the isotopologue profiles, a minor contribution of the MVA pathway was evident that was increased in transgenic spike lavender plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first enzyme of the MVA pathway. Together, these findings provide evidence for a transport of MVA-derived precursors from the cytosol to the plastids in leaves of spike lavender.

[EN] Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme, This work has been funded by the Regional Government of Valencia (PROMETEO/2009/075, Generalitat Valenciana); the Spanish Ministerio de Educacion y Cultura, FPU fellowship to I. M-P (AP2007/04606); and Fundacion Ramon Areces, fellowship to I. M-P.

Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used.

The increasing interest in natural products from a renewable source has encouraged growers to reintroduce indigo-producing crops into the European agriculture. We studied agronomic conditions (sowing date, plant density, nitrogen fertilization, irrigation rate, seedling transplanting) influencing production of the blue pigment indigo, from Isatis tinctoria and I. indigotica crops in a Mediterranean region of Spain (Valencia). I. tinctoria was more suitable for cultivation in our climate conditions than I. indigotica . Indigo yield from Spanish I. tinctoria trials was greater than in Northern and Central Europe. Furthermore, indigo production was maintained when water and nitrogen supplies were significantly restricted, showing that I. tinctoria is not a high-demanding crop.

Summary Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation and adventitious bud differentiation, by varying the amount and source of plant hormones. Shoot regeneration was also obtained after infection of D. minor with the wild-type Agrobacterium tumefaciens strain 82.139, which induced shooty tumours. These shoots were not transgenic, as revealed by nopaline assays and the use of a C58pMP90/T139GUS-INT strain harbouring the intron inactivated gusA gene. Plants were easily rooted and transplanted into the greenhouse. Shoot cultures of D. minor accumulated up to 226 μg cardenolides per g dry mass when cultured on agar-solidified medium. Cardenolide content was significantly lower in submerged shoot cultures, the effect being related to a high incidence of hyperhydricity. Greenhouse-grown plants presented the highest amounts of these metabolites. This is the first report on micropropagation of the Balearic endemic medicinal plant species D. minor . The induction of caulogenic galls by A. tumefaciens wild strain 82.139 opens new possibilities for the biotechnological improvement of Digitalis spp.

Agrobacterium -mediated transformation has been used to introduce the yeast halotolerant HAL2 as well as the npt II and uid A marker genes into tomato ( Lycopersicon esculentum ) cv. UC82B. Five to six percent of the explants produced transgenic plants. HAL2 expressing transformants were allowed to self-pollinate and salt tolerance assays were performed in vitro on progenies from two independent transgenic plants with different levels of expression of the transgene. In vitro salt tolerance was evaluated according to the level of growth of hypocotyl-derived calli as well as the rooting capability of isolated shootlets on MS-modified medium supplemented with NaCl. Under salt stress, callus formation from hypocotyl explants was higher on both transgenic-derived progenies than in the control. In addition, progenies from the plant with the highest expression of the transgene (2H20b), also showed a higher level of root production on NaCl-supplemented medium. These results suggested a positive effect of the yeast HAL2 gene on the level of salt tolerance in progenies derived from transgenic plants.

Summary Hypocotyl sections from 20-day-old seedlings of Digitalis obscura L. were used to examine the effect of IAA on somatic embryogenesis. Although embryogenesis was obtained with all IAA concentrations (0.1, 0.5 and 1 ppm) and under both illumination conditions tested (16 h photoperiod and darkness), incubation in photoperiod increased yield of embryos and shortened the embryo induction time. Optimum results were obtained with 1 ppm IAA. About 30 % of the embryos obtained developed into complete plantlets when transferred to basal medium without growth regulators.