Journal of the American Society for Horticultural Science, 1992
A protocol for in vitro propagation in flowering ash (Fraxinus ornus L.) has been developed. Shoo... more A protocol for in vitro propagation in flowering ash (Fraxinus ornus L.) has been developed. Shoot apices or nodal segments from aseptically grown seedings or shoot apices from adult trees were used as initial explants. Highest shoot multiplication rates were obtained when the explants were cultured for 30 days in liquid Rugini induction medium supplemented with BA followed by 30 days on solidified Rugini multiplication medium without growth regulators. Regenerated shoots were rooted on Heller medium containing auxins alone or in combination with BA. Rooting percentages up to 71% (juvenile material) or 50% (adult material) were obtained in the presence of NAA and BA, and were not improved by treating the basal end of the shoots with concentrated NAA solutions. Following conventional procedures, regenerated plants were transferred to soil with more than 80% success. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).
The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pa... more 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)C6]glucose. GC-MS-analysis of 1,8-cineole and camphor indicated that the C5-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.
This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolyti... more This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of g-aminobutyrate, which in turn affect plant development.
... PEG precipitation, a required step for PCR amplification of DNA from wild plants of Digitalis... more ... PEG precipitation, a required step for PCR amplification of DNA from wild plants of Digitalis obscura L. de Castillo Agudo L, Gavidia I, Pérez-Bermúdez P, Segura J. Universidad de Valencia, Burjassot, Spain. PMID: 7619473 [PubMed - indexed for MEDLINE]. Publication Types: ...
Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essent... more 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 product...
Dulce de Leche (caramel jam) is a typical Argentine dairy product similar in composition to sweet... more Dulce de Leche (caramel jam) is a typical Argentine dairy product similar in composition to sweetened condensed milk. Its flow properties were measured for solids content between 55 and 70% and temperatures between 25 and 70°C. Thixotropicity of the product was quantified using Weltmann's model and decreased with solids content. The coefficient of thixotropic breakdown was correlated with temperature for
We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich ex... more We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior characteristics. We also report adventitious bud differentiation from isolated embryos of C. atlantica. Neither auxin treatments nor other methods tested, including infection with Agrobacterium rhizogenes, were effective in inducing root initiation.
... No correlation between the metal ion levels in the plasmalemma vesicles and ATPasc activities... more ... No correlation between the metal ion levels in the plasmalemma vesicles and ATPasc activities could be establish-ed. The possible mechanisms of heavy metal induced changes on ATPase activities are discussed, Key words." cadmium; nickel; ATPase: plasmalemma; rice ...
Plant metabolism is highly coordinated with development. However, an understanding of the whole p... more 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.
Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration ... more Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantly increased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels were observed in the regenerated plants, with approximately 50% being haploid.
Successful propagation of seedlings and mature trees of Sorbus dornestica L. has been achieved by... more Successful propagation of seedlings and mature trees of Sorbus dornestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75-85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.
ABSTRACT Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants (Oryza sa... more ABSTRACT Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants (Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K+, Mg2+-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 mM KCl and 3 mM ATP with apparent Km (ATP) and Vmax of 0.75 mM and 79 μmol (mg protein)−1 h−1, respectively. With 50 mM KCl and 7 mM ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 mM and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate.
We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the... more 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.
Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarki... more 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.
Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation a... more 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.
ABSTRACT Effects of gibberellic acid on morphogenesis and cardenolide accumulation in in vitro cu... more ABSTRACT Effects of gibberellic acid on morphogenesis and cardenolide accumulation in in vitro cultures of Digitalis obscura are reported. Cultures were established from hypocotyls or leaves of axenic seedlings and shoot tips of mature plants. Irrespective of the kind of explant, gibberellic acid by itself did not induce morphogenesis but modified those morphogenic responses promoted by auxins and/or cytokinins, always inhibited organogenesis and favoured both embryo formation and conversion into plants. Gibberellic acid did not significantly affect cardenolide content of plants regenerated from juvenile or mature D. obscura explants.
Cardenolide content in 49 wild-growing Digitalis obscura plarits from six natural populations on ... more Cardenolide content in 49 wild-growing Digitalis obscura plarits from six natural populations on the Iberiari Peninsula was determined by HPLC. Series A arid B glycosides were the predominant cardenolides in all samples, but absolute values varied among and within populations. Hierarchic analysis of variarice showed that the proportion of variation attributable to individuals was significantly higher thari that attributable to population differences. Furthermore, arialyses from greenhouse-grown plarits, generated from seeds collected from selected parental genotypes, attributed nearly all the variability in cardenolide content to single plarits. Some individual plants showed a leaf content above 3500 j..tg/g dry wt of series A and B cardenolides and will be selected for genetic improvement of Digitalis obscura.
ABSTRACT Cardenolide formation was investigated in hypocotyl cultures of Digitalis obscura L. est... more ABSTRACT Cardenolide formation was investigated in hypocotyl cultures of Digitalis obscura L. established on MS medium with growth regulators selected to promote either callus formation, caulogenesis, embryogenesis or rhizogenesis. Examination of the cardenolide composition of leaves from mature plants was also performed. Several compounds of series A and C were identified by HPLC in in vitro and in vivo grown tissues, no marked differences being found between their cardenolide patterns. RIA determinations showed that shoot- or embryo-forming calli accumulated significantly higher amounts of digoxigenin derivatives than undifferentiated or rhizogenic cultures. In any case, field plants presented the highest contents of these metabolites.
Journal of the American Society for Horticultural Science, 1992
A protocol for in vitro propagation in flowering ash (Fraxinus ornus L.) has been developed. Shoo... more A protocol for in vitro propagation in flowering ash (Fraxinus ornus L.) has been developed. Shoot apices or nodal segments from aseptically grown seedings or shoot apices from adult trees were used as initial explants. Highest shoot multiplication rates were obtained when the explants were cultured for 30 days in liquid Rugini induction medium supplemented with BA followed by 30 days on solidified Rugini multiplication medium without growth regulators. Regenerated shoots were rooted on Heller medium containing auxins alone or in combination with BA. Rooting percentages up to 71% (juvenile material) or 50% (adult material) were obtained in the presence of NAA and BA, and were not improved by treating the basal end of the shoots with concentrated NAA solutions. Following conventional procedures, regenerated plants were transferred to soil with more than 80% success. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).
The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pa... more 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)C6]glucose. GC-MS-analysis of 1,8-cineole and camphor indicated that the C5-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.
This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolyti... more This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of g-aminobutyrate, which in turn affect plant development.
... PEG precipitation, a required step for PCR amplification of DNA from wild plants of Digitalis... more ... PEG precipitation, a required step for PCR amplification of DNA from wild plants of Digitalis obscura L. de Castillo Agudo L, Gavidia I, Pérez-Bermúdez P, Segura J. Universidad de Valencia, Burjassot, Spain. PMID: 7619473 [PubMed - indexed for MEDLINE]. Publication Types: ...
Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essent... more 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 product...
Dulce de Leche (caramel jam) is a typical Argentine dairy product similar in composition to sweet... more Dulce de Leche (caramel jam) is a typical Argentine dairy product similar in composition to sweetened condensed milk. Its flow properties were measured for solids content between 55 and 70% and temperatures between 25 and 70°C. Thixotropicity of the product was quantified using Weltmann's model and decreased with solids content. The coefficient of thixotropic breakdown was correlated with temperature for
We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich ex... more We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior characteristics. We also report adventitious bud differentiation from isolated embryos of C. atlantica. Neither auxin treatments nor other methods tested, including infection with Agrobacterium rhizogenes, were effective in inducing root initiation.
... No correlation between the metal ion levels in the plasmalemma vesicles and ATPasc activities... more ... No correlation between the metal ion levels in the plasmalemma vesicles and ATPasc activities could be establish-ed. The possible mechanisms of heavy metal induced changes on ATPase activities are discussed, Key words." cadmium; nickel; ATPase: plasmalemma; rice ...
Plant metabolism is highly coordinated with development. However, an understanding of the whole p... more 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.
Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration ... more Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantly increased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels were observed in the regenerated plants, with approximately 50% being haploid.
Successful propagation of seedlings and mature trees of Sorbus dornestica L. has been achieved by... more Successful propagation of seedlings and mature trees of Sorbus dornestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75-85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.
ABSTRACT Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants (Oryza sa... more ABSTRACT Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants (Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K+, Mg2+-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 mM KCl and 3 mM ATP with apparent Km (ATP) and Vmax of 0.75 mM and 79 μmol (mg protein)−1 h−1, respectively. With 50 mM KCl and 7 mM ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 mM and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate.
We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the... more 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.
Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarki... more 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.
Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation a... more 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.
ABSTRACT Effects of gibberellic acid on morphogenesis and cardenolide accumulation in in vitro cu... more ABSTRACT Effects of gibberellic acid on morphogenesis and cardenolide accumulation in in vitro cultures of Digitalis obscura are reported. Cultures were established from hypocotyls or leaves of axenic seedlings and shoot tips of mature plants. Irrespective of the kind of explant, gibberellic acid by itself did not induce morphogenesis but modified those morphogenic responses promoted by auxins and/or cytokinins, always inhibited organogenesis and favoured both embryo formation and conversion into plants. Gibberellic acid did not significantly affect cardenolide content of plants regenerated from juvenile or mature D. obscura explants.
Cardenolide content in 49 wild-growing Digitalis obscura plarits from six natural populations on ... more Cardenolide content in 49 wild-growing Digitalis obscura plarits from six natural populations on the Iberiari Peninsula was determined by HPLC. Series A arid B glycosides were the predominant cardenolides in all samples, but absolute values varied among and within populations. Hierarchic analysis of variarice showed that the proportion of variation attributable to individuals was significantly higher thari that attributable to population differences. Furthermore, arialyses from greenhouse-grown plarits, generated from seeds collected from selected parental genotypes, attributed nearly all the variability in cardenolide content to single plarits. Some individual plants showed a leaf content above 3500 j..tg/g dry wt of series A and B cardenolides and will be selected for genetic improvement of Digitalis obscura.
ABSTRACT Cardenolide formation was investigated in hypocotyl cultures of Digitalis obscura L. est... more ABSTRACT Cardenolide formation was investigated in hypocotyl cultures of Digitalis obscura L. established on MS medium with growth regulators selected to promote either callus formation, caulogenesis, embryogenesis or rhizogenesis. Examination of the cardenolide composition of leaves from mature plants was also performed. Several compounds of series A and C were identified by HPLC in in vitro and in vivo grown tissues, no marked differences being found between their cardenolide patterns. RIA determinations showed that shoot- or embryo-forming calli accumulated significantly higher amounts of digoxigenin derivatives than undifferentiated or rhizogenic cultures. In any case, field plants presented the highest contents of these metabolites.
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Papers by Juan Segura