Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase

The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.This work was supported by grants from the Spanish Ministerio de Ciencia y Tecnología and FEDER to MRC (BIO2002-1653 and BIO2005-00367) and AB (BMC2003-06833). AC, PB-P, and OB received doctoral fellowships from the Spanish Ministerio de Educación y Ciencia.Peer reviewe

PLGA cationic nanoparticles, obtained from nano-emulsion templating, as potential DNA vaccines

Polymeric nanoparticles offer advantageous characteristics as gene-delivery vectors such as biocompatibility and biodegradability. With this aim, a smart and innovative strategy was followed here: Cationic PLGA nano-emulsions, prepared by a low energy method, were used as templates to obtain cationic nanoparticles (NPs) able to easily complex with nucleic acids (i.e. plasmid DNA) by electrostatic interactions. The strategy employed to produce stable positively-charged nanoparticles was the use of non-ionic/cationic surfactant mixtures to stabilize template nano-emulsions. This methodology allowed obtaining nanoparticles with reproducible nanometric sizes and positive zeta potential values, appropriate to successfully complex with nucleic acids, resulting in nanometric spherical polyplexes. Nanoparticles, plasmids and polyplexes proved to be biocompatible at the optimal concentration. Therefore, we can conclude that we have designed a novel strategy to efficiently obtain cationic polymeric nanoparticles that can be a promising approach to act as novel non-viral gene-delivery vectors, useful for many applications in gene therapy, such as gene vaccines. © 2019 Elsevier LtdFinancial support from MINECO (grants CTQ2016-80645-R and CTQ2017-84998-P); Generalitat de Catalunya (grants 2014SGR1655 and 2017SGR1778), and CIBER-BBN are acknowledged. CIBER-BBN is an initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation 2013–2016, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Dr. Cristina Fornaguera is grateful to AGAUR for their Predoctoral Fellowship (grant FI-DGR 2012) and to MINECO for their Torres Quevedo Postdoctoral Fellowship(grant PTQ 2015). Authors thank the access to the facilities of TEM-SEM Casanova unit of the Centres Científics i Tecnològics (CCiTUB) of the University of Barcelona (UB) and its staff members for their technical advice.Peer reviewe

PLGA cationic nanoparticles, obtained from nano-emulsion templating, as potential DNA vaccines

Polymeric nanoparticles offer advantageous characteristics as gene-delivery vectors such as biocompatibility and biodegradability. With this aim, a smart and innovative strategy was followed here: Cationic PLGA nano-emulsions, prepared by a low energy method, were used as templates to obtain cationic nanoparticles (NPs) able to easily complex with nucleic acids (i.e. plasmid DNA) by electrostatic interactions. The strategy employed to produce stable positively-charged nanoparticles was the use of non-ionic/cationic surfactant mixtures to stabilize template nano-emulsions. This methodology allowed obtaining nanoparticles with reproducible nanometric sizes and positive zeta potential values, appropriate to successfully complex with nucleic acids, resulting in nanometric spherical polyplexes. Nanoparticles, plasmids and polyplexes proved to be biocompatible at the optimal concentration. Therefore, we can conclude that we have designed a novel strategy to efficiently obtain cationic polymeric nanoparticles that can be a promising approach to act as novel non-viral gene-delivery vectors, useful for many applications in gene therapy, such as gene vaccines.MINECOGrants CTQ2016-80645-R and CTQ2017-84998-PGeneralitat de CatalunyaGrants 2014SGR1655 and 2017SGR1778Spanish National Plan for Scientific and Technical Research and Innovation 2013–2016CIBER-BBNIniciativa Ingenio 2010Consolider ProgramInstituto de Salud Carlos IIIEuropean Regional Development FundAGAURgrant FI-DGR 2012grant PTQ 2015Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

The monoterpene limonene in orange peels attracts pests and microorganisms

Plant volatiles include terpenoids, which are generally involved in plant defense, repelling pests and pathogens and attracting insects for herbivore control, pollination and seed dispersal. Orange fruits accumulate the monoterpene limonene at high levels in the oil glands of their fruit peels. When limonene production was downregulated in orange fruits by the transgenic expression of a limonene synthase (CitMTSE1) in the antisense configuration, these fruits were resistant to the fungus Penicillium digitatum (Pers.) Sacc. and the bacterium Xanthomonas citri subsp. citri and were less attractive to the medfly pest Ceratitis capitata. These responses were reversed when the antisense transgenic orange fruits were treated with limonene. To gain more insight into the role of the limonene concentration in fruit responses to pests and pathogens, we attempted to overexpress CitMTSE1 in the sense configuration in transgenic orange fruits. Only slight increases in the amount of limonene were found in sense transgenic fruits, maybe due to the detrimental effect that excessive limonene accumulation would have on plant development. Collectively, these results suggest that when limonene reaches peak levels as the fruit develops, it becomes a signal for pest and pathogen attraction, which facilitate access to the fruit for pulp consumers and seed dispersers