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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OBJECTIVE: The 'standard' postoperative follow-up of patients with differentiated thyroid cancer (DTC) has been based upon serum thyroglobulin (Tg) measurement and (131)I whole body scan ((131)I-WBS) after thyroid hormone (T(4)) treatment withdrawal. However, (131)I-WBS sensitivity has been reported to be low. Thyroid hormone withdrawal, often associated with hypothyroidism-related side effects, may now be replaced by recombinant human thyroid stimulating hormone (rhTSH). The aim of our study was to evaluate the diagnostic accuracy of (131)I-WBS and serum Tg measurement obtained after rhTSH stimulation and of neck ultrasonography in the first follow-up of DTC patients. DESIGN: Ninety-nine consecutive patients previously treated with total thyroidectomy and (131)I ablation, with no uptake outside the thyroid bed on the post-ablative (131)I-WBS (low-risk patients) were enrolled. METHODS: Measurement of serum Tg and (131)I-WBS after rhTSH stimulation, and ultrasound examination (US) of the neck. RESULTS: rhTSH-stimulated Tg was 1 ng/ml (Tg+) in 21 patients, including 6 patients with Tg levels >5 ng/ml. (131)I-WBS was negative for persistent or recurrent disease in all patients (i.e. sensitivity = 0%). US identified lymph-node metastases (confirmed at surgery) in 4/6 (67%) patients with stimulated Tg levels >5 ng/ml, in 2/15 (13%) with Tg>1<5 ng/ml, and in 2/78 (3%) who were Tg-negative. CONCLUSIONS: (i) diagnostic (131)I-WBS performed after rhTSH stimulation is useless in the first follow-up of DTC patients; (ii) US may identify lymph node metastases even in patients with low or undetectable serum Tg levels

Euclid:Validation of the MontePython forecasting tools

The Euclid mission of the European Space Agency will perform a survey of weak lensing cosmic shear and galaxy clustering in order to constrain cosmological models and fundamental physics. We expand and adjust the mock Euclid likelihoods of the MontePython software in order to match the exact recipes used in previous Euclid Fisher matrix forecasts for several probes: weak lensing cosmic shear, photometric galaxy clustering, the cross-correlation between the latter observables, and spectroscopic galaxy clustering. We also establish which precision settings are required when running the Einstein-Boltzmann solvers CLASS and CAMB in the context of Euclid. For the minimal cosmological model, extended to include dynamical dark energy, we perform Fisher matrix forecasts based directly on a numerical evaluation of second derivatives of the likelihood with respect to model parameters. We compare our results with those of other forecasting methods and tools. We show that such MontePython forecasts agree very well with previous Fisher forecasts published by the Euclid Collaboration, and also, with new forecasts produced by the CosmicFish code, now interfaced directly with the two Einstein-Boltzmann solvers CAMB and CLASS. Moreover, to establish the validity of the Gaussian approximation, we show that the Fisher matrix marginal error contours coincide with the credible regions obtained when running Monte Carlo Markov Chains with MontePython while using the exact same mock likelihoods. The new Euclid forecast pipelines presented here are ready for use with additional cosmological parameters, in order to explore extended cosmological models

Euclid:Validation of the MontePython forecasting tools

The Euclid mission of the European Space Agency will perform a survey of weak lensing cosmic shear and galaxy clustering in order to constrain cosmological models and fundamental physics. We expand and adjust the mock Euclid likelihoods of the MontePython software in order to match the exact recipes used in previous Euclid Fisher matrix forecasts for several probes: weak lensing cosmic shear, photometric galaxy clustering, the cross-correlation between the latter observables, and spectroscopic galaxy clustering. We also establish which precision settings are required when running the Einstein-Boltzmann solvers CLASS and CAMB in the context of Euclid. For the minimal cosmological model, extended to include dynamical dark energy, we perform Fisher matrix forecasts based directly on a numerical evaluation of second derivatives of the likelihood with respect to model parameters. We compare our results with those of other forecasting methods and tools. We show that such MontePython forecasts agree very well with previous Fisher forecasts published by the Euclid Collaboration, and also, with new forecasts produced by the CosmicFish code, now interfaced directly with the two Einstein-Boltzmann solvers CAMB and CLASS. Moreover, to establish the validity of the Gaussian approximation, we show that the Fisher matrix marginal error contours coincide with the credible regions obtained when running Monte Carlo Markov Chains with MontePython while using the exact same mock likelihoods. The new Euclid forecast pipelines presented here are ready for use with additional cosmological parameters, in order to explore extended cosmological models

Improvements in cosmological constraints from breaking growth degeneracy

The key probes of the growth of a large-scale structure are its rate f and amplitude σ8. Redshift space distortions in the galaxy power spectrum allow us to measure only the combination fσ8, which can be used to constrain the standard cosmological model or alternatives. By using measurements of the galaxy-galaxy lensing cross-correlation spectrum or of the galaxy bispectrum, it is possible to break the fσ8 degeneracy and obtain separate estimates of f and σ8 from the same galaxy sample. Currently there are very few such separate measurements, but even this allows for improved constraints on cosmological models

Euclid:Constraining linearly scale-independent modifications of gravity with the spectroscopic and photometric primary probes

The future Euclid space satellite mission will offer an invaluable opportunity to constrain modifications to general relativity at cosmic scales. We focus on modified gravity models characterised, at linear scales, by a scale-independent growth of perturbations while featuring different testable types of derivative screening mechanisms at smaller nonlinear scales. We consider 3 specific models, namely Jordan-Brans-Dicke (JBD), the normal branch of Dvali-Gabadadze-Porrati (nDGP) gravity and $k$-mouflage (KM) gravity. We provide forecasts from spectroscopic and photometric primary probes by Euclid on the cosmological parameters and the extra parameters of the models, respectively, $\omega_{\rm BD}$, $\Omega_{\rm rc}$ and $\epsilon_{2,0}$, which quantify the deviations from general relativity. This analysis will improve our knowledge of the cosmology of these modified gravity models. The forecasts analysis employs the Fisher matrix method applied to weak lensing (WL); photometric galaxy clustering (GC$_{ph}$); spectroscopic galaxy clustering (GC$_{sp}$) and the cross-correlation (XC) between GC$_{ph}$ and WL. For the Euclid survey specifications we define three scenarios, characterised by different cuts in $\ell$ and $k$, to assess the constraining power of nonlinear scales. For each model we consider two fiducial values for the corresponding model parameter. In an optimistic setting at 68.3\% confidence interval, with Euclid alone we find the following percentage relative errors: for $\log_{10}{\omega_{\rm BD}}$, with a fiducial value of $\omega_{\rm BD}=800$, 35% using GC$_{sp}$ alone, 3.6% using GC$_{ph}$+WL+XC and 3.3% using GC$_{ph}$+WL+XC+GC$_{sp}$; for $\log_{10}{\Omega_{\rm rc}}$, with a fiducial value of $\Omega_{\rm rc}=0.25$, we find respectively 90%, 20% and 17%; finally, for $\epsilon_{2,0}=-0.04$ respectively 5%, 0.15% and 0.14%. (abridged

Use of SSR and retrotransposon-based markers to interpret the population structure of native grapevines from Southern Italy

Native grapevines are the quintessential ele- ments of Southern Italy winemaking, and genomic char- acterization plays a role of primary importance for preservation and sustainable use of these unexploited genetic resources. Among the various molecular techniques available, SSR and retrotransposons-based markers result to be the most valuable for cultivars and biotypes distinc- tiveness. A total of 62 accessions including 38 local grape cultivars were analyzed with 30 SSR, four REMAP and one IRAP markers to assess their genetic diversity and obtain a complete genomic profiling. The use of VrZAG79, VrZAG112, VVS2, VVMD25 and VVMD5 combined with retrotransposon-based markers proved to be the most dis- criminating and polymorphic markers for the rapid and unambiguous identification of minority grapevines from Campania region, which is considered one of the most appreciated Italian districts for wine production. Results revealed 58 SSR marker-specific alleles, 22 genotype- specific SSR alleles, and four REMAP and IRAP private bands. Cases of synonymy and homonymy were discov- ered. In conclusion, we provided evidences that the inte- grating SSR and retrotransposon-based markers is an effective strategy to assess the genetic diversity of autochthonous grapes, allowing their easy identification