Diseño y validación de herramientas biotecnológicas para la mejora del valor nutricional de la alfalfa (Medicago sativa L.)

[EN] Alfalfa (Medicago sativa L.), is a forage legume with a significant content of protein, being the most widely cultivated forage around the world. In this species, the protein content decreased during growth processes as well as other resources used by the plant during the flowering process. Alfalfa also contains a lower concentration of condensed tannins or proanthocyanidins (PAs), less than required to remedy the digestive disorder of ruminant livestock causing pasture bloat by production of greenhouse gases as result of the microbial fermentation and the excessive desamination of proteins in the rumen (by-pass effect). These defects on nutrition are reflected in the yield and production of ruminants. Both problematics are difficult to improve by conventional methods. The overall objective of this thesis is to develop molecular tools useful for genetic manipulation of certain traits of agronomic interest in alfalfa, to enhance their nutritional value. At a first step, we have developed an Agrobacterium tumefaciens-mediated transformation protocol via somatic embryogenesis applicable to various genotypes of alfalfa to produce viable embryos in the 50% of the inoculated explants. Chimaeric plants or scapes were almost not detected because most of the transgenic plants contained the T-DNA with all the transgenes incorporated, having thus the indispensable tool to perform the other two objectives. We have isolated two orthologs of the TERMINAL FLOWER 1 (TFL1) gene from alfalfa (MsTFL1c and MsTFL1a). Both genes were constitutively expressed in A. thaliana, and later on in alfalfa, to evaluate their possible use as biotechnological tool for the improvement of the nutritional value of this forage legume, producing a delay in the flowering time and thereby an increase of vegetative development. MsTFL1a performs the same functions as TFL1 in Arabidopsis y its overexpression in this plant produces a remarkable delay of flowering time. In alfalfa, its constitutive expression also produces a delay in flowering. However, this phenotype was not associated to an increase of vegetative growth, being plants of reduced size that appears tied to a limitation in cell proliferation. Moreover, the limitation in cell proliferation was associated with a reduction in the expression levels of certain cell division effector genes, as it is the case of cyclins. Our results support the hypothesis that TFL1-like genes could also participate in signaling pathways that regulate cell differentiation in plants. M. sativa could be a good model to study the possible range of biochemical diversification of these proteins. On the other hand, we have made a multigenic construct in the modular cloning system GoldenBraid 2.0 (35S::Del::Ros1::MtANR::MtLAR). In a first step, it was functionally validated by transient expression in N. benthamiana. In addition, this construct was introduced by stable transformation in two experimental models (A. thaliana and N. tabacum) and later on in M. sativa, where it has been studied the integration capacity and expression levels of the different transgenes. In N. tabacum it has been achieved the activation of both the anthocyanin and PAs biosynthetic pathways. However, it was not able to activate both routes in alfalfa. The TFs Delila and Rosea1 were not able to activate the genes involved in the regulation of these metabolic pathways in alfalfa, or were not sufficient levels of expression of both TFs for this purpose. Therefore, this multigenic construct does not resulted a good biotechnological tool for the activation of PAs biosynthesis in alfalfa. Our results suggest that the mechanisms of transcriptional control of both biosynthetic pathways could be different between plant species, and that additional specific genetic information on the anthocyanin and PAs biosynthesis in legumes is needed to efficiently activate both pathways in this species.[ES] La alfalfa (Medicago sativa L.), es una leguminosa forrajera con un importante contenido en proteína, siendo la más cultivada a nivel mundial. En esta especie, los contenidos protéicos se ven mermados por los procesos de crecimiento y los recursos utilizados por la planta en el proceso de floración. Además, la alfalfa contiene una concentración de taninos condensados o proantocianidinas (PAs) muy inferior a la requerida para subsanar el desorden digestivo del ganado rumiante que genera hinchamiento por gases de efecto invernadero o meteorismo y la excesiva desaminación de las proteínas en el rumen producida por las fermentaciones de la flora microbiana ruminal. Ambas problemáticas son difíciles de abordar mediante métodos convencionales de mejora. El objetivo general de esta Tesis es desarrollar herramientas moleculares de utilidad para la manipulación genética de determinados caracteres de interés agronómico en la alfalfa, con objeto de incrementar su valor nutricional. Para ello, hemos puesto a punto un protocolo de transformación genética mediada por Agrobacterium tumefaciens vía embriogénes somática aplicable a varios genotipos de alfalfa, que permite la producción de embriones viables capaces de germinar y producir plantas completas en el 50% de los explantes inoculados. Prácticamente no se detectó la presencia de quimeras o escapes ya que la mayoría de las plantas obtenidas contenían el T-DNA con todos los transgenes incorporados, disponiéndose por tanto de la herramienta imprescindible para la realización de los otros dos objetivos. Hemos aislado dos ortólogos del gen TERMINAL FLOWER 1 (TFL1) de alfalfa (MsTFL1c y MsTFL1a). Ambos genes se sobreexpresaron constitutivamente en A. thaliana y después en alfalfa para su posible uso como herramienta biotecnológica para producir un retraso en el tiempo de floración y con ello un posible aumento del desarrollo vegetativo. MsTFL1a extiende la fase vegetativa e inflorescente y retrasa la floración en Arabidopsis. En alfalfa su expresión constitutiva también produce retraso de la floración. Sin embargo, a este fenotipo no se le asocia un incremento del desarrollo vegetativo, presentando las plantas un tamaño más reducido que parece vinculado a una limitación en la proliferación celular. Hemos comprobado que este hecho se asocia a una reducción en los niveles de expresión de determinados genes efectores de la división celular, como es el caso de las ciclinas. Nuestros resultados apoyan la hipótesis de que los genes TFL1-like podrían participar adicionalmente en vías de señalización que regulan la diferenciación celular en plantas. También, hemos realizado una construcción multigénica mediante el sistema de clonaje por módulos GoldenBraid 2.0 (35S::Del::Ros1::MtANR::MtLAR). Se ha validado su funcionalidad mediante expresión transitoria en N. benthamiana y se ha introducido de manera estable en dos modelos experimentales: A. thaliana y N. tabacum, y en M. sativa, donde se ha estudiado su capacidad de integración, así como la de expresión de los transgenes. Hemos comprobado que esta construcción multigénica es capaz de activar la ruta de biosíntesis de antocianinas y de PAs en N. tabacum, pero no fue capaz de activar ambas rutas en alfalfa. Los factores transcripcionales Delila y Rosea1 no fueron capaces de activar los genes implicados en la regulación de ambas vías metabólicas en esta especie, o no fueron suficientes sus niveles de expresión para este fin. Por tanto, esta construcción no es una buena herramienta biotecnológica que procure la activación de la ruta de biosíntesis de PAs en la alfalfa. Nuestros resultados sugieren que los mecanismos de control transcripcional de la biosíntesis de antocianinas y PAs son diferentes entre las distintas especies, y que se necesita información genética adicional específica de la biosíntesis de antocianinas y PAs en leguminosas para poder diseñar eficientemente herrami[CA] L'alfals (Medicago sativa L.), és una lleguminosa farratgera amb un important contingut en proteïna, sent la més cultivada a nivell mundial. En aquesta espècie, els continguts proteics es veuen desfavorits amb els processos de creixement i els requeriments utilitzats per la planta en el procés de floració. A més, l'alfals conté una concentració de tanins condensats o proantocianidines (PAs) molt inferior a la requerida per a esmenar el desorde digestiu del bestiar remugant que genera unflament per gasos o meteorisme per la producció de gasos d'efecte hivernacle, i l'excessiva desaminació de les proteïnes en el rumen produïda per les fermentacions de la flora microbiana. Ambdós problemàtiques són difícils d'abordar per mitjà de mètodes convencionals de millora. L'objectiu general d'aquesta Tesi és desenvolupar ferramentes moleculars d'utilitat per a la manipulació genètica de determinats caràcters d'interés agronòmic en l'alfals, a fi d'incrementar el seu valor nutricional. Per a això, hem posat a punt un protocol de transformació mediada per Agrobacterium tumefaciens via embriogènesi somàtica aplicable a diversos genotips d'alfals que permet la producció d'embrions viables capaços de germinar i produir plantes completes en el 50% dels explants inoculats. Pràcticament no es va detectar la presència de fugues, ja que la majoria de les plantes obtingudes contenien el T-DNA amb tots el transgens introduïts, tenint per tant la ferramenta imprescindible per a la realització dels altres dos objectius. Es van aïllar i sobreexpressar constitutivament dos ortòlegs del gen TERMINAL FLOWER 1 (TFL1) d'alfals (MsTFL1c i MsTFL1a) en A. thaliana i en alfals per al seu possible ús com a ferramenta biotecnològica, procurant un retard en el temps de floració i amb això un augment del desenvolupament vegetatiu. MsTFL1a estén la fase vegetativa i inflorescent i retarda la floració en Arabidopsis. En alfals la seua expressió constitutiva també produïx retard de la floració. No obstant això, a aquest fenotip no se li associa un increment del desenvolupament vegetatiu, sent les plantes d'una grandària més reduït que pareix vinculat a una limitació en la proliferació cel·lular. Hem comprovat que la limitació en la proliferació cel·lular s'associa a una reducció en els nivells d'expressió de determinats gens efectors de la divisió cel·lular, com és el cas de les ciclines. Els nostres resultats recolzen la hipòtesi que els gens TFL1-like podrien participar adicionalment en vies de senyalització que regulen la diferenciació cel·lular en plantes. Hem realitzat una construcció multigénica per mitjà del sistema de clonatge per mòduls GoldenBraid 2.0 (35S::Del::Ros1::MtANR::MtLAR). S'ha validat funcionalment per mitjà d'expressió transitòria en N. benthamiana. A més, s'ha introduït de manera estable en dos models experimentals (A. thaliana amb N. tabacum), i finalment en M. sativa, en els que s'ha estudiat la seua capacitat d'integració, així com la d'expressió dels transgens. Hem mostrat que aquesta construcció multigénica és capaç d'activar la ruta de biosíntesi d'antocianines i de PAs en N. tabacum. No obstant això, no van ser capaços d'activar la ruta de biosíntesi d'antocianines i per tant, la de PAs, en alfals. Els factors transcripcionals Delila i Rosea1 no són capaços d'activar els gens implicats en la regulació d'ambdues vies metabòliques en aquesta espècie, o alternativament no van ser prou els seus nivells d'expressió per a este fi. Per tant, aquesta construcció multigénica no és una bona ferramenta biotecnològica per activar la ruta de biosíntesi de PAs en l'alfals. Els nostres resultats suggerixen que els mecanismes de control transcripcional de la biosíntesi d'antocianines i PAs són diferents entre les distintes espècies, i que es necessita informació genètica addicional específica de la biosíntesi d'antocianines i PAs en lleguminoses per aFresquet Corrales, S. (2015). Diseño y validación de herramientas biotecnológicas para la mejora del valor nutricional de la alfalfa (Medicago sativa L.) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/59240TESI

Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis

[EN] Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl-prolyl cis-trans isomerase of the FK506-binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over-expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H+-ATPase) is indirect: over-expression of ROF2 activates K+ uptake, causing depolarization of the plasma membrane, which activates the electrogenic H+ pump. The depolarization of ROF2 over-expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.This work was supported by grants BFU2008-00604 from the Ministerio de Ciencia e Innovacion (Madrid, Spain) and PROMETEO/2010/ 038 of the 'Conselleria de Educacion' (Valencia, Spain). We thank Dr Eugenio Grau (Sequencing Service, Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for sequencing of the various genes, and Dr Vicente Fornes (Instituto de Tecnologia Quimica, Valencia, Spain) for assistance with atomic absorption spectrophotometry. None of the authors has a conflict of interest to declare.Bissoli, G.; Niñoles Rodenes, R.; Fresquet Corrales, S.; Palombieri, S.; Bueso Ródenas, E.; Rubio, L.; Garcia-Sanchez, MJ.... (2012). Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis. Plant Journal. 70(4):704-716. https://doi.org/10.1111/j.1365-313X.2012.04921.xS70471670

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp

[EN] Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloatsafe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.This work was supported by grants BIO2012-39849-C02-01 and BIO2016-75485-R from the Spanish Ministry of Economy and Competitiveness (MINECO) (http://www.idi.mineco.gob.es/portal/site/MICINN) to LAC and a fellowship of the JAE-CSIC program to SF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fresquet-Corrales, S.; Roque Mesa, EM.; Sarrión-Perdigones, A.; Rochina, M.; López-Gresa, MP.; Díaz-Mula, HM.; Belles Albert, JM.... (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLoS ONE. 12(9). https://doi.org/10.1371/journal.pone.0184839Se018483912

Caracterización fenotípica de mutantes de Arabidopsis thaliana en los genes de las prolil isomerasas ROF1 y ROF2

En trabajos previos en levadura se ha demostrado que la sobreexpresión de FPR1, una inmunofilina de tipo FKBP (FK506 binding proteins), confiere tolerancia a los ácidos orgánicos débiles, como el ácido acético y el sórbico. Los FKBPs son receptores de las drogas inmunosupresoras FK506 y rapamicina. Los FKBPs contienen un dominio que posee actividad peptidil-prolil cis/trans isomerasa. Para evaluar la implicación de los FKBPs en la tolerancia a estrés ácido en plantas, hemos generado líneas de Arabidopsis thaliana sobreexpresando un tipo de FKBP, el FKBP65 (ROF2), mediante el promotor constitutivo 35S. Además se han seleccionado mutantes de pérdida de función (knock-out) de ROF1 (FKBP62) y ROF2 (FKBP65). La sobreexpresión de ROF2 confiere tolerancia a estrés por ácido débil. Por otro lado, el mutante de pérdida de función de rof1 presenta mayor resistencia que el control en todos los estreses químicos utilizados, mientras que el mutante rof2 no tenía fenotipo significativo. Además, en ausencia de estrés, los mutantes de pérdida de función realizan la transición floral antes, mientras que las líneas de sobreexpresión de ROF2 son más tardías que la línea silvestre. Por último, se observó que el mutante de pérdida de función rof2 presentaba una pérdida de las ramificaciones en sus raíces. La aplicación exógena de auxinas (IAA) revierte este fenotipo. Este fenotipo se observa en el mutante rof1 de forma menos acusada. Lo que sugiere un papel de ROF1 y ROF2 en el mecanismo de señalización dependiente de auxinas.Fresquet Corrales, S. (2010). Caracterización fenotípica de mutantes de Arabidopsis thaliana en los genes de las prolil isomerasas ROF1 y ROF2. http://hdl.handle.net/10251/1148

Expression analyses of transgenes and key genes involved in the anthocyanin biosynthetic pathway in transgenic <i>Nicotiana tabacum</i> leaves.

<p><b>(A)</b> qRT–PCR analysis of <i>AmRosea1</i>, <i>AmDelila</i>, <i>MtANR</i> and <i>MtLAR</i> transgenes in transformed leaves of <i>N</i>. <i>tabacum</i>. Error bars correspond to the standard deviation of three replicates. The expression value of <i>AmRosea1</i> in plant Nt#5 was set to 1.00 and the expression levels of the rest of transgenes were plotted relative to this value. To normalize the samples the constitutive <i>NtACT8</i> gene was used. <b>(B)</b> RT-PCR expression analysis of key genes involved in the anthocyanin pathway in <i>N</i>. <i>tabacum</i> Nt#6 and Nt#7 transgenic plants. PCR results were obtained after 30 amplification cycles for all genes and 25 cycles for the housekeeping <i>NtACT8</i> gene.</p

Phenotypes of the <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> transgenic tobacco plants.

<p><b>(A)</b> Some of the <i>in vitro</i> regenerated transgenic calli showed an intense purple pigmentation due to accumulation of anthocyanins. <b>(B)</b> <i>In vitro</i> regenerated control plantlet. <b>(C)</b> <i>In vitro</i> regenerated transgenic tobacco plant showing intense purple color in all tissues. <b>(D)</b> Control plant after acclimation in the greenhouse. <b>(E)</b> Transgenic tobacco plant Nt#7 with intense purple pigmentation after acclimation in the greenhouse. <b>(F)</b> Detail of a leaf from the transgenic plant Nt#7 showing intense purple pigmentation in the abaxial side and vascular bundles. <b>(G)</b> Detail of a leaf from the trasngenic plant Nt#5 showing only small patches of purple pigmentation. <b>(H)</b> Entire and disected flower from a control (left) and transgenic plant Nt#7 (right). <b>(I)</b> Carpel and stamens from a disected flower of a control (left) and the Nt#7 transgenic plant (right).</p

Relationship between purple phenotype, transgene expression and PAs production in three T1 <i>Nicotiana tabacum</i> plants.

<p><b>A-D.</b> Different leaf coloured phenotypes in the T1 of <i>N</i>. <i>tabacum</i> transgenic plants Nt#6.1 (green), Nt#6.8 (purple spots), Nt#6.11 (purple patches) and Nt#7.6 (full purple). <b>E.</b> In the lineage of plants Nt#6 and Nt#7 we analyzed by semi-qRT-PCR three plants with the complete set of transgenes showing a weak (Nt#6.8), a middle (Nt#6.11) and a strong purple phenotype (Nt#7.6). In the three plants the four transgenes were properly expressed. To normalize the samples the constituve <i>NtACT8</i> gene was used. <b>F.</b> The Nt#6.11, Nt#6.8 and Nt#7.6 transgenic plants produced PAs as demonstrated by HPLC-MS analysis of leaf extracts when compared with the WT.</p

Expression analyses of transgenes and key genes involved in the anthocyanin biosynthetic pathway in transgenic <i>Nicotiana tabacum</i> leaves.

<p><b>(A)</b> qRT–PCR analysis of <i>AmRosea1</i>, <i>AmDelila</i>, <i>MtANR</i> and <i>MtLAR</i> transgenes in transformed leaves of <i>N</i>. <i>tabacum</i>. Error bars correspond to the standard deviation of three replicates. The expression value of <i>AmRosea1</i> in plant Nt#5 was set to 1.00 and the expression levels of the rest of transgenes were plotted relative to this value. To normalize the samples the constitutive <i>NtACT8</i> gene was used. <b>(B)</b> RT-PCR expression analysis of key genes involved in the anthocyanin pathway in <i>N</i>. <i>tabacum</i> Nt#6 and Nt#7 transgenic plants. PCR results were obtained after 30 amplification cycles for all genes and 25 cycles for the housekeeping <i>NtACT8</i> gene.</p

Anthocyanin and proanthocyanidin biosynthetic pathways and multigenic construct assembly strategy.

<p><b>(A)</b> Schematic representation of the biosynthetic pathways for anthocyanins and proanthocyanidins. Abbreviations: chalcone synthase (CHS); chalcone isomerase (CHI); dihydroflavonol reductase (DFR); flavanone 3-hydroxylase (F3H); flavonol synthase (FLS); leucoanthocyanidin reductase (LAR); anthocyanidin synthase (ANS); anthocyanidin reductase (ANR); uridine diphosphate glucose-flavonoid 3-<i>O</i>-glucosyl transferase (UFGT). Purple colored arrows represent the catalytic steps that are upregulated by the overexpression of the <i>A</i>. <i>majus</i> transcription factors <i>Rosea1 and Delila</i>. The yellow highlighted area indicates the catalytic steps that are overexpressed in this work by the <i>M</i>. <i>truncatula</i> genes introduced in our multigenic construct, whose catalytic steps are highlighted in darker yellow and blue. <b>(B)</b> Premade GBParts, Modules and vectors used in this work. This includes the parts pCaMV35S promoter (GB0030), pTNos (GB0035), three vectors of the pGreenII-based pDGB1 series (Alfa1, Alfa2 and Omega2), one vector of the pCAMBIA pDGB2 series (Omega1) and two preassembled modules that were previously tested by the GB2.0 developers. The first module (GB0129) expresses the two <i>A</i>. <i>majus</i> transcriptional factors <i>Rosea1</i> and <i>Delila</i> that under the control of the CaMV35S promoter. The second module (GB0235) is the hygromycin resistant cassette that is used to select the transformed plants in the stable transformation process. CaMV35S is the Cauliflower Mosaic Virus 35S Promoter; TNos is the Nopaline synthase terminator; PNos is the Nopaline synthase promoter; K^R and S^R stand for bacterial kanamycin and spectinomycin resistance cassettes; LB and RB represent the Left and Right Borders of the T-DNA. <b>(C)</b> GoldenBraid 2.0 multigenic construct <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> generated in this work. The multigenic construct was generated in five steps that include the assembly of the <i>MtANR</i> and <i>MtLAR</i> transcriptional units from its basic parts (Assemblies 1 and 2), the combination of these transcriptional units in a single vector (Assembly 3), the later addition of the <i>A</i>. <i>majus</i> transcriptional factors to the <i>M</i>. <i>truncatula</i> genes (Assembly 4) and finally the incorporation of the hygromycin resistance cassette to generate the multigenic construct that is used in all the experiments of this work. <i>MtANR</i> is the <i>M</i>. <i>truncatula</i> anthocyanidin reductase gene; <i>MtLAR</i> is the <i>M</i>. <i>truncatula</i> leucoanthocyanidin reductase gene.</p

Validation of the multigenic construct by transient expression in <i>Nicotiana benthamiana</i> leaves.

<p><b>(A)</b> Agroinfiltrated <i>N</i>. <i>benthamiana</i> leaf with the <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> multigenic construct showing purple pigmentation. <b>(B)</b> Infiltrated leaf with <i>DsRed</i> (control). <b>(C)</b> Non-infiltrated wild-type leaf. <b>(D)</b> Expression levels of the <i>AmRosea1</i>, <i>AmDelila</i>, <i>MtANR</i> and <i>MtLAR</i> transgenes in agroinfiltrated leaves of <i>N</i>. <i>benthamiana</i>. <i>NbACT8</i> is used as positive control. PCR results were obteined after 30 amplification cycles for all analized genes. <b>(E)</b> RT-PCR analysis of the anthocyanin biosynthetic pathway genes in agroinfiltrated leaves of <i>N</i>. <i>benthamiana</i>. The PCR results were obteined after 30 amplification cycles for all genes and 25 cycles for the housekeeping <i>NbACT8</i> gene. <b>(F)</b> PA levels in agroinfiltrated leaf extracts of <i>N</i>. <i>benthamiana</i> obteined by the dimethylaminocinnamaldehyde (DMACA) colorimetric reaction. The DMACA reaction showed an increase of the PA’s content in the agroinfiltrated leaf area compared with the non-infiltrated WT leaves. The maximum PA levels were found in the plants infiltrated with the multigenic construct. Statistical T-Test values for (-) catechin (n = 3, t = 11.46, df = 4, <i>p</i> = 0.0003) and (-) epicatechin (n = 3, t = 11.46, df = 4, <i>p</i> = 0.0003).</p