Juan Segura

Up-Regulation of 1-Deoxy-d-Xylulose-5-Phosphate Synthase Enhances Production of Essential Oils in Transgenic Spike Lavender

Spike lavender (Lavandula latifolia) is an aromatic shrub cultivated worldwide for the production of essential oils. The major constituents of these oils are monoterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-Deoxy-d-xylulose-5-P synthase (DXS) catalyzes the first step of the MEP pathway. A cDNA coding for the Arabidopsis (Arabidopsis thaliana) DXS was constitutively expressed in spike lavender. Gas chromatography/mass spectrometry analyses revealed that transgenic plants accumulated significantly more essential oils compared to controls (from 101.5% to 359.0% and from 12.2% to 74.1% yield increase compared to controls in leaves and flowers, respectively). T0 transgenic plants were grown for 2 years, self-pollinated, and the T1 seeds obtained. The inheritance of the DXS transgene was studied in the T1 generation. The increased essential oil phenotype observed in the transgenic T0 plants was maintained in the progeny that inherited the DXS transgene. Total chlorophyll and carotenoid content in DXS progenies that inherited the transgene depended on the analyzed plant, showing either no variation or a significant decrease in respect to their counterparts without the transgene. Transgenic plants had a visual phenotype similar to untransformed plants (controls) in terms of morphology, growth habit, flowering, and seed germination. Our results demonstrate that the MEP pathway contributes to essential oil production in spike lavender. They also demonstrate that the DXS enzyme plays a crucial role in monoterpene precursor biosynthesis and, thus, in essential oil production in spike lavender. In addition, our results provide a strategy to increase the essential oil production in spike lavender by metabolic engineering of the MEP pathway without apparent detrimental effects on plant development and fitness.

Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Deficiency Leads to Altered Root Development and Affects the Sugar and Amino Acid Balance in Arabidopsis

Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD+ to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

RAPD variation within and among natural populations of outcrossing willow-leaved foxglove ( Digitalis obscura L.)

Abstract Random amplified polymorphic DNA (RAPD) markers were used to assess levels and patterns of genetic diversity in Digitalis obscura L. (Scrophulariaceae), an outcrossing cardenolide-producing medicinal plant species. A total of 50 plants from six natural populations on the Iberian Peninsula were analysed by six arbitrarily chosen decamer primers resulting in 96 highly reproducible RAPD bands. To avoid bias in parameter estimation, analyses of population genetic structure were restricted to bands (35 of 96) whose observed frequencies were less than 1–3/n in each population. The analysis of molecular variance (AMOVA) with distances among individuals corrected for the dominant nature of RAPDs (genotypic analysis) showed that most of the variation (84.8%) occurred among individuals within populations, which is expected for an outcrossing organism. Of the remaining variance, 9.7% was attributed to differences between regions, and 5.5% for differences among populations within regions. Estimates of the Wright, Weir and Cockerham and Lynch and Milligan FST from null-allele frequencies corroborated AMOVA partitioning and provided significant evidence for population differentiation in D. obscura. A non-parametric test for the homogeneity of molecular variance (HOMOVA) also showed significant differences in the amount of genetic variability present in the six populations. UPGMA cluster analyses, based on Apostol genetic distance, revealed grouping of some geographically proximate populations. Nevertheless, a Mantel test did not give a significant correlation between geographic and genetic distances. This is the first report of the partitioning of genetic variability within and between populations of D. obscura and provides important baseline data for optimising sampling strategies and for conserving the genetic resources of this medicinal species.

Population genetic study in the Balearic endemic plant species Digitalis minor (Scrophulariaceae) using RAPD markers.

Digitalis minor (Scrophulariaceae) is a cardenolide-producing plant endemic to the eastern Balearic Islands (Mallorca, Menorca, and Cabrera) that occurs in two morphologically distinct varieties: D. minor var. minor (pubescent) and D. minor var. palaui (glabrous). Levels and patterns of genetic diversity in 162 individuals from 17 D. minor populations across the entire geographic range were assessed using random amplified polymorphic DNA (RAPD) markers. Comigrating RAPD fragments tested were found to be homologous by Southern hybridization in both var. minor and var. palaui. To avoid bias in parameter estimation, analyses of population genetic structure were restricted to those RAPD bands that fulfilled the 3/N criterion (observed frequencies were less than 1 - [3/N] in each population) either among or within each island. Analyses of molecular variance (AMOVAs) with distances among individuals corrected for the dominant nature of RAPD (genotypic analysis) showed low values (1.57-17.55%) of between-population variability, indicating a relatively restricted population differentiation as expected for an outcrossing species such as D. minor. Nested AMOVAs demonstrated, however, a not significant partitioning of genetic diversity among Mallorca, Menorca, and Cabrera islands. Estimates of the Wright, Weir, and Cockerham and the Lynch and Milligan F(ST) from null allele frequencies corroborated AMOVA partitioning and provided evidence for population differentiation in D. minor. Our RAPD data did not show significant differences between pubescent and glabrous populations of D. minor, suggesting a failure to find a correlation between the RAPD loci and this morphological trait.

In vivo and in vitro effects of nickel and cadmium on the plasmalemma ATPase from rice (Oryza sativa L.) shoots and roots

Abstract Plasmalemma vesicles isolated from rice (Oryza sativa L.) shoots and roots were used to study the effect of different nickel and cadmium concentrations on the Mg2+-ATPase activity. In vivo application of the metals for 5 or 10 days resulted in a stimulation of the enzyme activity in shoots, while either increases or decreases were observed in roots depending on the concentration and the type of ion tested. In contrast, an inhibitory effect was always observed on shoot and root plasmalemma ATPase activity when these ions were applied in vitro. At the organ level, the content of Cd in plants treated with 0.5 mM metal was higher than that of Ni, especially in roots. No correlation between the metal ion levels in the plasmalemma vesicles and ATPase activties could be established. The possible mechanisms of heavy metal induced changes on ATPase activities are discussed.

The plastidial glyceraldehyde-3-phosphate dehydrogenase is critical for viable pollen development in Arabidopsis

Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work, we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis (Arabidopsis thaliana). Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was down-regulated in gapcp1gapcp2. The fertility of gapcp1gapcp2 was rescued by transforming this mutant with a construct carrying the GAPCp1 cDNA under the control of its native promoter (pGAPCp1::GAPCp1c). However, the GAPCp1 or GAPCp2 cDNA under the control of the 35S promoter (p35S::GAPCp), which is poorly expressed in the tapetum, did not complement the mutant fertility. Mutant GAPCp isoforms deficient in the catalytic activity of the enzyme were unable to complement the sterile phenotype of gapcp1gapcp2, thus confirming that both the expression and catalytic activity of GAPCp in anthers are necessary for mature pollen development. A metabolomic study in flower buds indicated that the most important difference between the sterile (gapcp1gapcp2, gapcp1gapcp2-p35S::GAPCp) and the fertile (wild-type plants, gapcp1gapcp2-pGAPCp1::GAPCp1c) lines was the increase in the signaling molecule trehalose. This work corroborates the importance of plastidial glycolysis in plant metabolism and provides evidence for the crucial role of GAPCps in pollen development. It additionally brings new insights into the complex interactions between metabolism and development.

Genetic diversity and structure of natural and managed populations of Cedrus atlantica (Pinaceae) assessed using random amplified polymorphic DNA.

Cedrus atlantica (Pinaceae) is a large and exceptionally long-lived conifer native to the Rif and Atlas Mountains of North Africa. To assess levels and patterns of genetic diversity of this species, samples were obtained throughout the natural range in Morocco and from a forest plantation in Arbúcies, Girona (Spain) and analyzed using RAPD markers. Within-population genetic diversity was high and comparable to that revealed by isozymes. Managed populations harbored levels of genetic variation similar to those found in their natural counterparts. Genotypic analyses of molecular variance (AMOVA) found that most variation was within populations, but significant differentiation was also found between populations, particularly in Morocco. Bayesian estimates of F(ST) corroborated the AMOVA partitioning and provided evidence for population differentiation in C. atlantica. Both distance- and Bayesian-based clustering methods revealed that Moroccan populations comprise two genetically distinct groups. Within each group, estimates of population differentiation were close to those previously reported in other gymnosperms. These results are interpreted in the context of the postglacial history of the species and human impact. The high degree of among-group differentiation recorded here highlights the need for additional conservation measures for some Moroccan populations of C. atlantica.

Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves

We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene.

An assessment of genetic relationships within the genus Digitalis based on PCR-generated RAPD markers

Abstract RAPD markers were used to study inter-specific variation among six species of the genus Digitalis: D. obscura, D. lanata, D. grandiflora, D. purpurea, D. thapsi and D. dubia, and the hybrid D. excelsior (D. purpurea×D. grandiflora). A total of 91 highly reproducible bands amplified with four arbitrarily chosen decamer primers were obtained. Homology of the co-emigrating RAPD markers was tested by blot hybridisation and sequencing of selected bands. The application of a range of statistical approaches for RAPD data analysis, including distance and parsimony methods, family clustering and the analysis of molecular variance (AMOVA), indicated that these molecular markers were taxonomically informative in Digitalis. The species relationships revealed were fully consistent with those previously obtained using morphological affinities. The hybrid D. excelsior seems to have stronger affinity to the section Digitalis than to Grandiflorae. This is the first known report of the application of RAPD markers for the study of genetic relationships among species of the genus Digitalis.

Random amplified polymorphic DNA assessment of diversity in western Mediterranean populations of the seagrass Posidonia oceanica

Posidonia oceanica is an endemic Mediterranean seagrass species that has often been assumed to contain low levels of genetic diversity. Random amplified polymorfic DNA (RAPD) markers were used to assess genetic diversity among five populations from three geographical regions (north, central, and south) of the western Mediterranean Sea. Stranded germinating seeds from one of the central populations were also included in the analysis. Forty-one putative genets were identified among 76 ramets based on 28 RAPD markers. Genotypic diversity strongly depended on the spatial structure, age, and maturity of the meadows. The lowest clonal diversity was found in the less structured and youngest prairies. Conversely, a high genotypic diversity was found in the highly structured meadows. The genotypic diversity in these meadows was at the same level as in P. australis and higher than previously reported data for P. oceanica populations in the Tyrrhenian Sea near the coast of Italy.