Studying the role of the strawberry Fra protein family in the flavonoid metabolism during fruit ripening

Strawberry fruits are highly appreciated worldwide due to their pleasant flavor and aroma and to the health benefits associated to their consumption. An important part of these properties is due to their content in secondary metabolites, especially phenolic compounds, of which flavonoids are the most abundant in the strawberry fruit. Although the flavonoid biosynthesis pathway is uncovered, little is known about its regulation. The strawberry Fra a (Fra) genes constitute a large family of homologs of the major birch pollen allergen Bet v 1 and for which no equivalents exist in Arabidopsis. Our group has shown that Fra proteins are involved in the formation of colored compounds in strawberries (Muñoz et al., 2010), which mainly depends on the production of certain flavonoids; that they are structurally homologs to the PYR/PYL/RCAR Arabidopsis ABA receptor, and that they are able to bind flavonoids (Casañal et al., 2013). With these previous results, our working hypothesis is that the Fra proteins are involved in the regulation of the flavonoids pathway. They would mechanistically act as the ABA receptor, binding a protein interactor and a ligand to regulate a signaling cascade and/or act as molecular carriers. The main objective of this research is to characterize the Fra family in strawberry and gain insight into their role in the flavonoid metabolism. By RNAseq expression analysis in ripening fruits we have identified transcripts for 10 members of the Fra family. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each Fra protein. Then, our next approach was to identify the proteins that interact with Fras and their ligands to gain knowledge on the role that these proteins play in the flavonoids pathway. To identify the interacting partners of Fras we have performed a yeast two hybrid (Y2H) screening against cDNA libraries of strawberry fruits at the green and red stages. A protein that shares a 95% homology to the Heat stress transcription factor A-4-C like of Fragaria vesca (HSA4C) interacts specifically with Fra1 and not with other family members, which suggests functional diversification of Fra proteins in specific signaling pathways. The Y2H screening is not yet saturated, so characterization of other interacting proteins with other members of the Fra family will shed light on the functional diversity within this gene family. This research will contribute to gain knowledge on how the flavonoid pathway, and hence, the fruit ripening, is regulated in strawberry; an economically important crop but for which basic research is still very limited. References: Muñoz, C, et al. (2010). The Strawberry Fruit Fra a Allergen Functions in Flavonoid Biosynthesis. Molecular Plant, 3(1): 113–124. Casañal, A, et al (2013). The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding Metabolic Intermediates. Journal of Biological Chemistry, 288(49): 35322–35332.

RGS14 (414)-mediated prevention of an episodic memory loss: a study of molecular mechanism

A large proportion of human populations suffer memory impairments either caused by normal aging or afflicted by diverse neurological and neurodegenerative diseases. Memory enhancers and other drugs tested so far against memory loss have failed to produce therapeutic efficacy in clinical trials and thus, there is a need to find remedy for this mental disorder. In search for cure of memory loss, our laboratory discovered a robust memory enhancer called RGS14(414). A treatment in brain with its gene produces an enduring effect on memory that lasts for lifetime of rats. Therefore, current thesis work was designed to investigate whether RGS14(414) treatment can prevent memory loss and furthermore, explore through biological processes responsible for RGS-mediated memory enhancement. We found that RGS14(414) gene treatment prevented episodic memory loss in rodent models of normal aging and Alzheimer´s disease. A memory loss was observed in normal rats at 18 months of age; however, when they were treated with RGS14(414) gene at 3 months of age, they abrogated this deficit and their memory remained intact till the age of 22 months. In addition to normal aging rats, effect of memory enhancer treatment in mice model of Alzheimer´s disease (AD-mice) produced a similar effect. AD-mice subjected to treatment with RGS14(414) gene at the age of 2 months, a period when memory was intact, showed not only a prevention in memory loss observed at 4 months of age but also they were able to maintain normal memory after 6 months of the treatment. We posit that long-lasting effect on memory enhancement and prevention of memory loss mediated through RGS14(414) might be due to a permanent structural change caused by a surge in neuronal connections and enhanced neuronal remodeling, key processes for long-term memory formation. A neuronal arborization analysis of both pyramidal and non-pyramidal neurons in brain of RGS14(414)-treated rats exhibited robust rise in neurites outgrowth of both kind of cells, and an increment in number of branching from the apical dendrite of pyramidal neurons, reaching to almost three times of the control animals. To further understand of underlying mechanism by which RGS14(414) induces neuronal arborization, we investigated into neurotrophic factors. We observed that RGS14 treatment induces a selective increase in BDNF. Role of BDNF in neuronal arborization, as well as its implication in learning and memory processes is well described. In addition, our results showing a dynamic expression pattern of BDNF during ORM processing that overlapped with memory consolidation further support the idea of the implication of this neurotrophin in formation of long-term memory in RGS-animals. On the other hand, in studies of expression profiling of RGS-treated animals, we have demonstrated that 14-3-3ζ protein displays a coherent relationship to RGS-mediated ORM enhancement. Recent studies have demonstrated that the interaction of receptor for activated protein kinase 1 (RACK1) with 14-3-3ζ is essential for its nuclear translocation, where RACK1-14-3-3ζ complex binds at promotor IV region of BDNF and promotes an increase in BDNF gene transcription. These observations suggest that 14-3-3ζ might regulate the elevated level of BDNF seen in RGS14(414) gene treated animals. Therefore, it seems that RGS-mediated surge in 14-3-3ζ causes elevated BDNF synthesis needed for neuronal arborization and enhanced ORM. The prevention of memory loss might be mediated through a restoration in BDNF and 14-3-3ζ protein levels, which are significantly decreased in aging and Alzheimer’s disease. Additionally, our results demonstrate that RGS14(414) treatment could be a viable strategy against episodic memory loss.