Functional characterization os satrawberry (Fragaria x Ananassa) fruit-specific and ripening-related genes involved in aroma and anthochyanins biosynthesis

Along the development of this thesis, we have studied the transcriptomic changes that occur in the receptacle of the strawberry fruit (Fragaria x ananassa cv Camarosa) during ripening using an oligo microarray platform of strawberry fruit. This analysis allowed us to select several target genes with biotechnological importance potentially involved in the process of fruit ripening, since they are determinants of some relevant organoleptic properties that directly influence the final quality of strawberry fruit. One of the selected genes (FaAAT2) showed significant homology of sequence with genes of higher plants encoding alcohol acyltransferases (AATs), proteins involved in generating the characteristic aroma of ripe strawberry fruit. These enzymes are involved in the final step in the biosynthesis of volatile esters, catalyzing the esterification of acyl moiety of acyl-CoA with an alcohol. In this work, we have observed a clear correlation between the increase in FaAAT2 gene expression and the increase of volatile esters biosynthesis along the strawberry maturation. Furthermore, it has been found that their expression is negatively regulated by auxin synthesized in the achenes. On the other hand, we evaluated the enzymatic activity of FaAAT2 protein recombinant derived from the full-length FaAAT2 cDNA using a wide variety of acyl-CoA and alcohols as substrates. The recombinant enzyme showed activity in the presence of straight chain alcohols and aromatic alcohols in combination with acetyl-CoA, although it had preference for the cinnamyl alcohol as acceptor of acyl groups. The analysis of potential substrates for this enzyme present in the strawberry fruit indicated that the FaAAT2 protein always has preference to C6-C10 alcohols, being more active with hexanol and heptanol followed by octanol. Therefore, our results suggest that the FaAAT2 protein can produce esters as hexyl acetate and octyl acetate present in the strawberry fruit. After the transient silencing of FaAAT2 gene expression, we observed a significant reduction of volatiles esters in the transgenic strawberry fruit, suggesting that the FaAAT2 enzyme could be related with the synthesis of volatile compounds involved in the final aroma of the strawberry fruit. The second gene selected for our study was a transcription factor belonging to the MYB family (FaMYB10), which is involved in regulating the metabolism of flavonoids/ phenylpropanoids during the strawberry fruit ripening. The expression of this gene is fruit receptacle specific, inducible along its maturation and regulated by auxin and abscisic acid. Furthermore, transcriptome analysis of transgenic fruits with the FaMYB10 expression transiently silenced indicated that many of the genes involved in the metabolism of flavonoids/phenylpropanoids [Early-regulated Biosynthesis Genes (EBGs): CHS, CHI, F3H, FLS; Late-regulated Biosynthesis Genes (LBGs): DFR, UFGT; and genes of the general pathway: PAL, C4H, 4CL] may be regulated by this transcription factor which also seems to repress genes involved in the biosynthesis of proanthocyanidins (PAs) (Glycosyltransferases, F3'5'H, LDOX/ANS, LAR) in immature fruit receptacle. We have also found that the FaMYB10 gene induces the expression of GST and MATE transporters and appears to regulate other transcription factors involved in the strawberry maturation. Thus, our results indicate that the FaMYB10 transcription factor plays a key role in the strawberry fruit ripening acting as an important part of the signal transduction cascade during this process.Durante el desarrollo de esta tesis doctoral, se ha procedido al estudio de los cambios transcriptómicos que se producen en el receptáculo de fruto de fresa (Fragaria x ananassa cv Camarosa) durante su maduración empleando una plataforma de microarrays de oligos de fruto de fresa. Este análisis nos permitió seleccionar varios genes diana potencialmente implicados en el proceso de maduración del fruto y con gran importancia biotecnológica, ya que son determinantes de algunas de las propiedades organolépticas que influyen directamente en la calidad final del fruto de fresa. Uno de los genes seleccionados (FaAAT2) presentó homología significativa de secuencia con genes de plantas superiores que codifican alcohol aciltransferasas (AATs), proteínas implicadas en la generación del aroma característico del fruto de fresa maduro. Estas enzimas participan en el último paso de la biosíntesis de ésteres volátiles, catalizando la esterificación de un resto acilo de acil-CoA con un alcohol. En este trabajo hemos determinado que existe una clara correlación entre el incremento de expresión del gen FaAAT2 y el aumento de la biosíntesis de ésteres volátiles a lo largo de la maduración de la fresa. Además, se ha comprobado que su expresión está regulada negativamente por las auxinas sintetizadas en los aquenios. Por otra parte, se analizó la actividad enzimática de la proteína recombínante FaAAT2 obtenida a partir del ADNc completo del gen FaAAT2 empleando una amplia variedad de acil-CoA y alcoholes. La enzima recombinante obtenida mostró actividad en presencia de alcoholes de cadena lineal y alcoholes aromáticos combinados con acetil CoA, aunque mostró preferencia por el cinnamil alcohol como aceptor de grupos acilos. El análisis de los posibles sustratos presentes en el fruto de fresa para esta enzima indicó que la proteína FaAAT2 mostró siempre preferencia por alcoholes C6-C10, siendo más activa con hexanol seguida de octanol y heptanol. Por tanto, nuestros resultados sugieren que la proteína FaAAT2 puede producir ésteres como hexil acetato y octil acetato presentes en el fruto de fresa. Paralelamente y mediante el silenciamiento transitorio de la expresión del gen FaAAT2, se observó una reducción significativa de la producción de volátiles en el fruto de fresa, lo que sugiere que la enzima FaAAT2 estaría implicada en la síntesis de compuestos volátiles y contribuiría de forma importante al aroma final del fruto de fresa. El segundo gen seleccionado para su estudio fue un factor de transcripción perteneciente a la familia MYB (FaMYB10) implicado en la regulación del metabolismo de los flavonoides/fenilpropanoides durante la maduración del fruto de fresa. La expresión de dicho gen resultó ser específica de receptáculo de fruto, inducible a lo largo de la maduración de éste y regulada por auxinas y ácido abscísico. Por otra parte, el análisis transcriptómico de frutos transgénicos con la expresión del gen FaMYB10 silenciada de forma transitoria indicó que muchos de los genes implicados en el metabolismo de flavonoides/fenilpropanoides [Early-regulated Biosynthesis Genes (EBGs): CHS, CHI, F3H, FLS; Late-regulated Biosynthesis Genes (LBGs): DFR , UFGT; y genes de la ruta general: PAL, C4H, 4CL] podrían estar regulados por este factor de transcripción, mientras que reprimiría genes involucrados en la biosíntesis de las proantocianidinas (PAs) (Glicosiltransferasas, F3´5´H, LDOX /ANS, LAR) en receptáculo de frutos inmaduros. Hemos comprobado también que el gen FaMYB10 activa la expresión de los transportadores GST y MATE y parece regular muchos factores de transcripción implicados en la maduración de la fresa. Por tanto, nuestros resultados indican que el factor de transcripción FaMYB10 juega un papel importante en el proceso de maduración del fruto de fresa actuando como parte importante de la cascada de transducción de señales durante este proceso

Title: p38δ Regulates IL6 Expression Modulating ERK Phosphorylation in Preadipocytes.

IL6 is an essential cytokine in metabolism regulation and for intercommunication among different organs and tissues. IL6 produced by different tissues has different functions and therefore it is very important to understand the mechanism of its expression in adipose tissue. In this work we demonstrated that IL6 expression in mouse preadipocytes, like in human, is partially dependent on Wnt5a and JNK. Using mouse preadipocytes lacking each one of the p38 SAPK family members, we have shown that IL6 expression is also p38γ and p38δ dependent. In fact, the lack of some of these two kinases increases IL6 expression without altering that of Wnt5a. Moreover, we show that the absence of p38δ promotes greater ERK1/2 phosphorylation in a MEK1/2 independent manner, and that this increased ERK1/2 phosphorylation state is contributing to the higher IL6 expression in p38δ-/- preadipocytes. These results suggest a new crosstalk between two MAPK signaling pathway, p38δ and ERK1/2, where p38δ modulates the phosphorylation state of ERK1/2.JMC-G was recipient of a Ramón y Cajal contract (RYC2015–17867). SD-C and CMM-Q were recipients of Fellowships from the Junta de Extremadura. SG-J was a recipient of a Fellowship from the Universidad de Extremadura. This work was supported by BFU 2017–85547-P grant from the Spanish Ministry of Economy and IB18014 grant from Junta de Extremadura to JMC-G and GR15164 grant from Junta de Extremadura to FC All Spanish and Junta de Extremadura funding are co-sponsored by the European Union FEDER program.S