Studies for the selection of the neutrino events collected in ICARUS T600 at FERMILAB

openThe ICARUS T600 LAr-TPC detector has restarted in 2020 to collect events at Fermilab exposed to the Booster Neutrino Beam (BNB) within the SBN program, to definitively clarify the open questions of the presently-observed neutrino anomalies related to the possible existence of sterile neutrinos. It is also recording neutrino interactions from the NuMI off-axis beam and these events will be studied to obtain in particular neutrino-Argon cross section measurements. The T600 is taking data at shallow-depth, so a large number of cosmic muons are expected to cross the detector randomly in the 1-ms drift time corresponding to the triggered event. The neutrino interactions should be recognized among the events triggered by cosmics and this condition makes it necessary to deploy suitable automatic tools for the identification, selection, and measurement of the neutrino events. These tools should exploit all the available information from the TPC, from the internal PMTs and from the external Cosmic Ray Tagging system surrounding the detector. In the present thesis tools for the selection of the neutrino interactions recorded in the T600 will be developed starting from the available reconstruction tools and applied to the data recorded and MC events to evaluate the performance. The selected events will be also visually studied to improve the selection efficiency of the neutrino interactions. The focus will be in particular on the selection and reconstruction of the quasi elastic muon neutrino interactions fully contained in the detector

Incorporation of natural antioxidants from rice straw into renewable starch films.

Abstract This study showed that rice straw waste is a valuable source for the extraction of water-soluble phenolic compounds that can be successfully incorporated into bioactive starch-based films. The major phenolic compounds in the extract were identified as ferulic, p-coumaric and protocatechuic acid using UHPLC-MS. Homogeneous films with antioxidant properties were produced by melt blending and compression molding and the changes in the physico-chemical properties were evaluated. The produced antioxidant starch films were slightly reddish-colored and exhibited good in-vitro antiradical scavenging activity against DPPH*. The addition of the antioxidant extract improved the oxygen barrier properties without negatively affecting the thermal and the water vapor barrier properties. However, antioxidant starch films turned more brittle with increasing amount of the antioxidant extract, which was probably due to interactions of phenolic compounds with the starch chains. The film forming process induced chain scission of starch molecules in all films, shown in a decrease in molecular weight of native starch from 9.1 × 106 Da to values as low as 1.0–3.5 × 106 Da. This study aids a circular economy by recycling rice straw for the production of bioactive food packaging

A quadruple mutant of Arabidopsis reveals a β-carotene hydroxylation activity for LUT1/CYP97C1 and a regulatory role of xanthophylls on determination of the PSI/PSII ratio

<p>Abstract</p> <p>Background</p> <p>Xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex, to light absorbance and to photoprotection. The first step in xanthophyll biosynthesis from α- and β-carotene is the hydroxylation of ε- and β-rings, performed by both non-heme iron oxygenases (CHY1, CHY2) and P450 cytochromes (LUT1/CYP97C1, LUT5/CYP97A3). The Arabidopsis triple <it>chy1chy2lut5 </it>mutant is almost completely depleted in β-xanthophylls.</p> <p>Results</p> <p>Here we report on the quadruple <it>chy1chy2lut2lut5 </it>mutant, additionally carrying the <it>lut2 </it>mutation (affecting lycopene ε-cyclase). This genotype lacks lutein and yet it shows a compensatory increase in β-xanthophylls with respect to <it>chy1chy2lut5 </it>mutant. Mutant plants show an even stronger photosensitivity than <it>chy1chy2lut5</it>, a complete lack of qE, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. Biochemical analysis reveals that the <it>chy1chy2lut2lut5 </it>mutant is depleted in Lhcb subunits and is specifically affected in Photosystem I function, showing a deficiency in PSI-LHCI supercomplexes. Moreover, by analyzing a series of single, double, triple and quadruple Arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the PSI-PSII ratio. The decrease in the xanthophyll/carotenoid ratio causes a proportional decrease in the LHCII and PSI core levels with respect to PSII.</p> <p>Conclusions</p> <p>The physiological and biochemical phenotype of the <it>chy1chy2lut2lut5 </it>mutant shows that (i) LUT1/CYP97C1 protein reveals a major β-carotene hydroxylase activity <it>in vivo </it>when depleted in its preferred substrate α-carotene; (ii) xanthophylls are needed for normal level of Photosystem I and LHCII accumulation.</p

Color Mutations Alter the Biochemical Composition in the San Marzano Tomato Fruit

San Marzano (SM) is a traditional Italian landrace characterized by red elongated fruits, originating in the province of Naples (Italy) and cultivated worldwide. Three mutations, yellow flesh (r), green flesh (gf) and colorless fruit epidermis (y) were introduced into SM by backcross and the resulting introgression lines (ILs) produced the expected yellow, brown and pink fruit variants. In addition, ILs carrying double combinations of those mutations were obtained. The six ILs plus the SM reference were analyzed for volatile (VOC), non-polar (NP) and polar (P) metabolites. Sixty-eight VOCs were identified, and several differences evidenced in the ILs; overall gf showed epistasis over r and y and r over y. Analysis of the NP component identified 54 metabolites; variation in early carotenoids (up to lycopene) and chlorophylls characterized respectively the ILs containing r and gf. In addition, compounds belonging to the quinone and xanthophyll classes were present in genotypes carrying the r mutation at levels higher than SM. Finally, the analysis of 129 P metabolites evidenced different levels of vitamins, amino acids, lipids and phenylpropanoids in the ILs. A correlation network approach was used to investigate metabolite–metabolite relationships in the mutant lines. Altogether these differences potentially modified the hedonistic and nutritional value of the berry. In summary, single and combined mutations in gf, r and y generated interesting visual and compositional diversity in the SM landrace, while maintaining its original typolog

Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers

BACKGROUND: Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold. RESULTS: In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species. CONCLUSION: Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content

Flavescence dorée-derived leaf yellowing in grapevine (Vitis vinifera l.) is associated to a general repression of isoprenoid biosynthetic pathways

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2020.00896/full#supplementary-materialFlavescence dorée (FD), caused by the phytoplasma Candidatus Phytoplasma vitis, is a major threat to vineyard survival in different European grape-growing areas. It has been recorded in French vineyards since the mid-1950s, and rapidly spread to other countries. In Portugal, the phytoplasma was first detected in the DOC region of Vinhos Verdes in 2006, and reached the central region of the country in 2009. The infection causes strong accumulation of carbohydrates and phenolics in the mesophyll cells and a simultaneous decrease of chlorophylls, events accompanied by a down regulation of genes and proteins involved in the dark and light-dependent reactions and stabilization of the photosystem II (PSII). In the present study, to better elucidate the basis of the leaf chlorosis in infected grapevine cv. Loureiro, we studied the isoprenoid transcriptmetabolite correlation in leaves from healthy and FD-infected vines. Specifically, targeted metabolome revealed that twenty-one compounds (out of thirty-two), including chlorophylls, carotenoids, quinones and tocopherols, were reduced in response to FD-infection. Thereafter, and consistently with the biochemical data, qPCR analysis highlighted a severe FD-mediated repression in key genes involved in isoprenoid biosynthetic pathways. A more diverse set of changes, on the contrary, was observed in the case of ABA metabolism. Principal component analysis (PCA) of all identified metabolites clearly separated healthy from FD-infected vines, therefore confirming that the infection strongly alters the biosynthesis of grapevine isoprenoids; additionally, forty-four genes and metabolites were identified as the components mostly explaining the variance between healthy and infected samples. Finally, transcriptmetabolite network correlation analyses were exploited to display the main hubs of the infection process, which highlighted a strong role of VvCHLG, VvVTE and VvZEP genes and the chlorophylls intermediates aminolevulunic acid and porphobilinogen in response to FD infection. Overall, results indicated that the FD infection impairs the synthesis of isoprenoids, through the repression of key genes involved in the biosynthesis of chlorophylls, carotenoids, quinones and tocopherols.The work was supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the strategic program UIDB/04050/2020. The work was also supported by FCT and European Funds (FEDER/POCI/ COMPETE2020) through the research projects BerryPlastid (PTDC/BIAFBT/28165/2017 and POCI-01-0145-FEDER - 028165), MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01- 0145-FEDER-030341) and GrapeInfectomics (PTDC/ASPHOR/28485/2017). AT was supported by a postdoctoral researcher contract/position within the project “BerryPlastid”. HN was supported by an FCT postdoctoral grant (SFRH/BPD/115518/2016). The work also benefited from the networking activities within the European Cooperation in Science and Technology Action (EUROCAROTEN CA15136).info:eu-repo/semantics/publishedVersio

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer

[EN] Although assisted reproduction technologies (ARTs) are recognised as safe, and most of the offspring seem apparently healthy, there is clear evidence that ARTs are associated with changes in the embryo's developmental trajectory, which incur physiological consequences during the prenatal and postnatal stages of life. The present study aimed to address the influence of early (day-3 embryos) embryo transfer and cryopreservation on embryo survival, size, and metabolome at the preimplantation stage (day-6 embryos). To this end, fresh-transferred (FT) and vitrified-transferred (VT) embryos were compared using naturally-conceived (NC) embryos as a control reference. The results show that as in vitro manipulation was increased (NC < FT < VT), both embryo survival rate (0.91 +/- 0.02, 0.78 +/- 0.05 and 0.63 +/- 0.05, for NC, FT, and VT groups, respectively) and embryo size (3.21 +/- 0.49 mm, 2.15 +/- 0.51 mm, 1.76 +/- 0.46 mm of diameter for NC, FT, and VT groups, respectively) were significantly decreased. Moreover, an unbiased metabolomics analysis showed overall down-accumulation in 40 metabolites among the three experimental groups, with embryo transfer and embryo cryopreservation procedures both exerting a cumulative effect. In this regard, targeted metabolomics findings revealed a significant reduction in some metabolites involved in metabolic pathways, such as the Krebs cycle, amino acids, unsaturated fatty acids, and arachidonic acid metabolisms. Altogether, these findings highlight a synergistic effect between the embryo transfer and vitrification procedures in preimplantation embryos. However, the ex vivo manipulation during embryo transfer seemed to be the major trigger of the embryonic changes, as the deviations added by the vitrification process were relatively smaller.This research was funded by Conselleria d'Educacio, Investigacio, Cultura i Esport, Spain, grant number AICO/2019/272. Ximo Garcia-Dominguez was supported by a research grant from the Ministry of Economy, Industry and Competitiveness of Spain (BES-2015-072429).Garcia-Dominguez, X.; Diretto, G.; Frusciante, S.; Vicente Antón, JS.; Marco-Jiménez, F. (2020). Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer. International Journal of Molecular Sciences. 21(19):1-14. https://doi.org/10.3390/ijms21197116S1142119Rizos, D., Maillo, V., Sánchez-Calabuig, M.-J., & Lonergan, P. (2017). The Consequences of Maternal-Embryonic Cross Talk During the Periconception Period on Subsequent Embryonic Development. Advances in Experimental Medicine and Biology, 69-86. doi:10.1007/978-3-319-62414-3_4Avilés, M., Gutiérrez-Adán, A., & Coy, P. (2010). Oviductal secretions: will they be key factors for the future ARTs? MHR: Basic science of reproductive medicine, 16(12), 896-906. doi:10.1093/molehr/gaq056Li, S., & Winuthayanon, W. (2017). Oviduct: roles in fertilization and early embryo development. Journal of Endocrinology, 232(1), R1-R26. doi:10.1530/joe-16-0302Wale, P. L., & Gardner, D. K. (2015). The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Human Reproduction Update, 22(1), 2-22. doi:10.1093/humupd/dmv034Fleming, T. P., Watkins, A. J., Velazquez, M. A., Mathers, J. C., Prentice, A. M., Stephenson, J., … Godfrey, K. M. (2018). Origins of lifetime health around the time of conception: causes and consequences. The Lancet, 391(10132), 1842-1852. doi:10.1016/s0140-6736(18)30312-xRoseboom, T. J. (2018). Developmental plasticity and its relevance to assisted human reproduction. Human Reproduction, 33(4), 546-552. doi:10.1093/humrep/dey034Vrooman, L. A., & Bartolomei, M. S. (2017). Can assisted reproductive technologies cause adult-onset disease? Evidence from human and mouse. Reproductive Toxicology, 68, 72-84. doi:10.1016/j.reprotox.2016.07.015Ng, K. Y. B., Mingels, R., Morgan, H., Macklon, N., & Cheong, Y. (2017). In vivo oxygen, temperature and pH dynamics in the female reproductive tract and their importance in human conception: a systematic review. Human Reproduction Update, 24(1), 15-34. doi:10.1093/humupd/dmx028Zacchini, F., Sampino, S., Stankiewicz, A. M., Haaf, T., & Ptak, G. E. (2019). Assessing the epigenetic risks of assisted reproductive technologies: a way forward. The International Journal of Developmental Biology, 63(3-4-5), 217-222. doi:10.1387/ijdb.180402gpDuranthon, V., & Chavatte-Palmer, P. (2018). Long term effects of ART: What do animals tell us? Molecular Reproduction and Development, 85(4), 348-368. doi:10.1002/mrd.22970Ramos‐Ibeas, P., Heras, S., Gómez‐Redondo, I., Planells, B., Fernández‐González, R., Pericuesta, E., … Gutiérrez‐Adán, A. (2019). Embryo responses to stress induced by assisted reproductive technologies. Molecular Reproduction and Development, 86(10), 1292-1306. doi:10.1002/mrd.23119Feuer, S., & Rinaudo, P. (2016). From Embryos to Adults: A DOHaD Perspective on In Vitro Fertilization and Other Assisted Reproductive Technologies. Healthcare, 4(3), 51. doi:10.3390/healthcare4030051Feuer, S. K., & Rinaudo, P. F. (2017). Physiological, metabolic and transcriptional postnatal phenotypes ofin vitrofertilization (IVF) in the mouse. Journal of Developmental Origins of Health and Disease, 8(4), 403-410. doi:10.1017/s204017441700023xRomar, R., Funahashi, H., & Coy, P. (2016). In vitro fertilization in pigs: New molecules and protocols to consider in the forthcoming years. Theriogenology, 85(1), 125-134. doi:10.1016/j.theriogenology.2015.07.017Canovas, S., Ivanova, E., Romar, R., García-Martínez, S., Soriano-Úbeda, C., García-Vázquez, F. A., … Coy, P. (2017). DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids. eLife, 6. doi:10.7554/elife.23670Campo, H., García-Domínguez, X., López-Martínez, S., Faus, A., Vicente Antón, J. S., Marco-Jiménez, F., & Cervelló, I. (2019). Tissue-specific decellularized endometrial substratum mimicking different physiological conditions influences in vitro embryo development in a rabbit model. Acta Biomaterialia, 89, 126-138. doi:10.1016/j.actbio.2019.03.004Le Saint, C., Crespo, K., Bourdiec, A., Bissonnette, F., Buzaglo, K., Couturier, B., … Kadoch, I. J. (2019). Autologous endometrial cell co-culture improves human embryo development to high-quality blastocysts: a randomized controlled trial. Reproductive BioMedicine Online, 38(3), 321-329. doi:10.1016/j.rbmo.2018.12.039Sparks, A. (2015). Human Embryo Cryopreservation—Methods, Timing, and other Considerations for Optimizing an Embryo Cryopreservation Program. Seminars in Reproductive Medicine, 33(02), 128-144. doi:10.1055/s-0035-1546826Saenz-de-Juano, M. D., Marco-Jiménez, F., Peñaranda, D. S., Joly, T., & Vicente, J. S. (2012). Effects of Slow Freezing Procedure on Late Blastocyst Gene Expression and Survival Rate in Rabbit1. Biology of Reproduction, 87(4). doi:10.1095/biolreprod.112.100677Saenz-de-Juano, M. D., Vicente, J. S., Hollung, K., & Marco-Jiménez, F. (2015). Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy. PLOS ONE, 10(4), e0125157. doi:10.1371/journal.pone.0125157Saenz-de-Juano, M. D., Marco-Jimenez, F., Schmaltz-Panneau, B., Jimenez-Trigos, E., Viudes-de-Castro, M. P., Peñaranda, D. S., … Vicente, J. S. (2014). Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level. REPRODUCTION, 147(6), 789-801. doi:10.1530/rep-14-0019Vicente, J. S., Saenz-de-Juano, M. D., Jiménez-Trigos, E., Viudes-de-Castro, M. P., Peñaranda, D. S., & Marco-Jiménez, F. (2013). Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation. Cryobiology, 67(3), 321-326. doi:10.1016/j.cryobiol.2013.09.165Marco-Jiménez, F., Lavara, R., Jiménez-Trigos, E., & Vicente, J. S. (2013). In vivo development of vitrified rabbit embryos: Effects of vitrification device, recipient genotype, and asynchrony. Theriogenology, 79(7), 1124-1129. doi:10.1016/j.theriogenology.2013.02.008Lavara, R., Baselga, M., Marco-Jiménez, F., & Vicente, J. S. (2014). Long-term and transgenerational effects of cryopreservation on rabbit embryos. Theriogenology, 81(7), 988-992. doi:10.1016/j.theriogenology.2014.01.030Lavara, R., Baselga, M., Marco-Jiménez, F., & Vicente, J. S. (2015). Embryo vitrification in rabbits: Consequences for progeny growth. Theriogenology, 84(5), 674-680. doi:10.1016/j.theriogenology.2015.04.025Garcia-Dominguez, X., Vicente, J. S., & Marco-Jiménez, F. (2020). Developmental Plasticity in Response to Embryo Cryopreservation: The Importance of the Vitrification Device in Rabbits. Animals, 10(5), 804. doi:10.3390/ani10050804Gupta, A., Singh, J., Dufort, I., Robert, C., Dias, F. C. F., & Anzar, M. (2017). Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage. PLOS ONE, 12(11), e0187268. doi:10.1371/journal.pone.0187268García-Domínguez, X., Marco-Jiménez, F., Puigcerver-Barber, M., Más-Pellicer, A., & Vicente, J. S. (2020). The harmful effect of removing the extracellular vitrification medium during embryo cryopreservation using a nylon mesh device in rabbit. Cryobiology, 93, 44-48. doi:10.1016/j.cryobiol.2020.02.013Marco-Jiménez, F., Jiménez-Trigos, E., Almela-Miralles, V., & Vicente, J. S. (2016). Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method. PLOS ONE, 11(2), e0148661. doi:10.1371/journal.pone.0148661Saenz-de-Juano, M. D., Marco-Jiménez, F., & Vicente, J. S. (2016). Embryo transfer manipulation cause gene expression variation in blastocysts that disrupt implantation and offspring rates at birth in rabbit. European Journal of Obstetrics & Gynecology and Reproductive Biology, 207, 50-55. doi:10.1016/j.ejogrb.2016.10.049Montag, M., Koll, B., Holmes, P., & Ven, H. van der. (2000). Significance of the Number of Embryonic Cells and the State of the Zona Pellucida for Hatching of Mouse Blastocysts In Vitro Versus In Vivo. Biology of Reproduction, 62(6), 1738-1744. doi:10.1095/biolreprod62.6.1738Giritharan, G., Talbi, S., Donjacour, A., Di Sebastiano, F., Dobson, A. T., & Rinaudo, P. F. (2007). Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos. Reproduction, 134(1), 63-72. doi:10.1530/rep-06-0247Van Landuyt, L., Van de Velde, H., De Vos, A., Haentjens, P., Blockeel, C., Tournaye, H., & Verheyen, G. (2013). Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos. Human Reproduction, 28(11), 2943-2949. doi:10.1093/humrep/det356Salilew-Wondim, D., Saeed-Zidane, M., Hoelker, M., Gebremedhn, S., Poirier, M., Pandey, H. O., … Tesfaye, D. (2018). Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation. BMC Genomics, 19(1). doi:10.1186/s12864-018-4826-3Heras, S., De Coninck, D. I. M., Van Poucke, M., Goossens, K., Bogado Pascottini, O., Van Nieuwerburgh, F., … Van Soom, A. (2016). Suboptimal culture conditions induce more deviations in gene expression in male than female bovine blastocysts. BMC Genomics, 17(1). doi:10.1186/s12864-016-2393-zDriver, A. M., Peñagaricano, F., Huang, W., Ahmad, K. R., Hackbart, K. S., Wiltbank, M. C., & Khatib, H. (2012). RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts. BMC Genomics, 13(1). doi:10.1186/1471-2164-13-118Gad, A., Hoelker, M., Besenfelder, U., Havlicek, V., Cinar, U., Rings, F., … Tesfaye, D. (2012). Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions1. Biology of Reproduction, 87(4). doi:10.1095/biolreprod.112.099697Miles, J. R., Blomberg, L. A., Krisher, R. L., Everts, R. E., Sonstegard, T. S., Van Tassell, C. P., & Zuelke, K. A. (2008). Comparative transcriptome analysis of in vivo- and in vitro-produced porcine blastocysts by small amplified RNA-Serial analysis of gene expression (SAR-SAGE). Molecular Reproduction and Development, 75(6), 976-988. doi:10.1002/mrd.20844Bauer, B. K., Isom, S. C., Spate, L. D., Whitworth, K. M., Spollen, W. G., Blake, S. M., … Prather, R. S. (2010). Transcriptional Profiling by Deep Sequencing Identifies Differences in mRNA Transcript Abundance in In Vivo-Derived Versus In Vitro-Cultured Porcine Blastocyst Stage Embryos1. Biology of Reproduction, 83(5), 791-798. doi:10.1095/biolreprod.110.085936Swain, J., Bormann, C., Clark, S., Walters, E., Wheeler, M., & Krisher, R. (2002). Use of energy substrates by various stage preimplantation pig embryos produced in vivo and in vitro. Reproduction, 253-260. doi:10.1530/rep.0.1230253Lee, Y. S. L., Thouas, G. A., & Gardner, D. K. (2015). Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage. Human Reproduction, 30(3), 543-552. doi:10.1093/humrep/deu334Krisher, R. L., Heuberger, A. L., Paczkowski, M., Stevens, J., Pospisil, C., Prather, R. S., … Schoolcraft, W. B. (2015). Applying metabolomic analyses to the practice of embryology: physiology, development and assisted reproductive technology. Reproduction, Fertility and Development, 27(4), 602. doi:10.1071/rd14359Perkel, K. J., & Madan, P. (2017). Spent culture medium analysis from individually cultured bovine embryos demonstrates metabolomic differences. Zygote, 25(6), 662-674. doi:10.1017/s0967199417000417McKeegan, P. J., & Sturmey, R. G. (2012). The role of fatty acids in oocyte and early embryo development. Reproduction, Fertility and Development, 24(1), 59. doi:10.1071/rd11907Sayre, B. L., & Lewis, G. S. (1993). Arachidonic acid metabolism during early development of ovine embryos: A possible relationship to shedding of the zona pellucida. Prostaglandins, 45(6), 557-569. doi:10.1016/0090-6980(93)90019-4Feuer, S. K., Liu, X., Donjacour, A., Simbulan, R., Maltepe, E., & Rinaudo, P. (2017). Transcriptional signatures throughout development: the effects of mouse embryo manipulation in vitro. Reproduction, 153(1), 107-122. doi:10.1530/rep-16-0473Feuer, S. K., Donjacour, A., Simbulan, R. K., Lin, W., Liu, X., Maltepe, E., & Rinaudo, P. F. (2014). Sexually Dimorphic Effect of In Vitro Fertilization (IVF) on Adult Mouse Fat and Liver Metabolomes. Endocrinology, 155(11), 4554-4567. doi:10.1210/en.2014-1465Wang, L.-Y., Le, F., Wang, N., Li, L., Liu, X.-Z., Zheng, Y.-M., … Jin, F. (2013). Alteration of fatty acid metabolism in the liver, adipose tissue, and testis of male mice conceived through assisted reproductive technologies: fatty acid metabolism in ART mice. Lipids in Health and Disease, 12(1). doi:10.1186/1476-511x-12-5Leese, H. J., Guerif, F., Allgar, V., Brison, D. R., Lundin, K., & Sturmey, R. G. (2016). Biological optimization, the Goldilocks principle, and how much islagomin the preimplantation embryo. 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Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase

BACKGROUND: Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. RESULTS: We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. CONCLUSION: The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system

[EN] Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron beta-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites.We thank K. Schreiber and C. Mares (IBMCP, CSIC-UPV, Valencia, Spain) for technical assistance during plant transformation. We thank M. Gasc.on and M.D. G.omez-Jim.enez (IBMCP, CSIC-UPV, Valencia, Spain) for helpful assistance with LSCM analyses. We thank D. Dubbala (IBMCP, CSIC-UPV, Valencia, Spain) for English revision. This work was supported by grants BIO2016-77000-R and BIO2017-83184-R from the Spanish Ministerio de Ciencia e Innovacion (co-financed European Union ERDF), and SBPLY/17/180501/000234 from Junta de Comunidades de Castilla-La Mancha. M.M. was the recipient of a predoctoral fellowship from the Spanish Ministerio de Educacion, Cultura y Deporte (FPU16/05294). G.D. and L.G.G. are participants of the European COST action CA15136 (EUROCAROTEN). L.G.G. is a participant of the CARNET network (BIO2015-71703-REDT and BIO2017-90877-RED).Martí, M.; Diretto, G.; Aragones, V.; Frusciante, S.; Ahrazem, O.; Gómez-Gómez, L.; Daròs, J. (2020). Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system. 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Gene-Metabolite Networks of Volatile Metabolism in Airen and Tempranillo Grape Cultivars Revealed a Distinct Mechanism of Aroma Bouquet Production

Volatile compounds are the major determinants of aroma and flavor in both grapes and wine. In this study, we investigated the emission of volatile and non-volatile compounds during berry maturation in two grape varieties (Airén and Tempranillo) throughout 2010 and 2011. HS-SPME coupled to gas chromatography and mass spectrometry was applied for the identification and relative quantitation of these compounds. Principal component analysis was performed to search for variability between the two cultivars and evolution during 10 developmental stages. Results showed that there are distinct differences in volatile compounds between cultivars throughout fruit development. Early stages were characterized in both cultivars by higher levels of some apocarotenoids such as β-cyclocitral or β-ionone, terpenoids (E)-linalool oxide and (Z)-linalool oxide and several furans, while the final stages were characterized by the highest amounts of ethanol, benzenoid phenylacetaldehyde and 2-phenylethanol, branched-amino acid-derived 3-methylbutanol and 2-methylbutanol, and a large number of lipid derivatives. Additionally, we measured the levels of the different classes of volatile precursors by using liquid chromatography coupled to high resolution mass spectrometry. In both varieties, higher levels of carotenoid compounds were detected in the earlier stages, zeaxanthin and α-carotene were only detected in Airén while neoxanthin was found only in Tempranillo; more variable trends were observed in the case of the other volatile precursors. Furthermore, we monitored the expression of homolog genes of a set of transcripts potentially involved in the biosynthesis of these metabolites, such as some glycosyl hydrolases family 1, lipoxygenases, alcohol dehydrogenases hydroperoxide lyases, O-methyltransferases and carotenoid cleavage dioxygenases during the defined developmental stages. Finally, based on Pearson correlation analyses, we explored the metabolite-metabolite fluctuations within VOCs/precursors during the berry development; as well as tentatively linking the formation of some metabolites detected to the expression of some of these genes. Our data showed that the two varieties displayed a very different pattern of relationships regarding the precursor/volatile metabolite-metabolite fluctuations, being the lipid and the carotenoid metabolism the most distinctive between the two varieties. Correlation analysis showed a higher degree of overall correlation in precursor/volatile metabolite-metabolite levels in Airén, confirming the enriched aroma bouquet characteristic of the white varieties.This work was supported by the “Junta de comunidades de Castilla-La Mancha” (JCCM) [PPII10-0062-7718] and benefited from the networking activities within the European Cooperation in Science and Technology Action CA15136 (EUROCAROTEN). GD was supported by short-term fellowships of the Quality Fruit (FA1106) European Cooperation in Science and Technology actions. OA was funded by FPCYTCLM through the INCRECYT Programme.Peer reviewedPeer Reviewe