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.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Modification of plant hormone levels and signaling as a tool in plant biotechnology.

Plant hormones are signal molecules, present in trace quantities, that act as major regulators of plant growth and development. They are involved in a wide range of processes such as elongation, flowering, root formation and vascular differentiation. For many years, agriculturists have applied hormones to their crops to either increase the yield, or improve the quality of the commercial product. Nowadays, the knowledge of hormone biosynthesis, degradation and signaling pathways has allowed the utilization of biotechnological tools to further improve the main agricultural crops. Natural or artificial mutants, with impaired functioning of the corresponding genes, have been adopted because of their superior phenotype in specific agricultural traits. In addition, transgenic plants have been generated to regulate internal hormone levels, or their signaling pathways, resulting in some crops that have revolutionized agriculture.The authors are financially supported by the Grant BIO2007-67509-C02-01 (Ministerio de Educación y Ciencia, Spain)

Biosynthesis of L-ascorbic acid in plants: new pathways for an old antioxidant

The biosynthetic pathway of L-ascorbic acid (vitamin C) in plants has been established for several years. However, recent reports describe alternative pathways, revealing a more complex picture of L-ascorbic acid biosynthesis than had been expected. GDP-L-gulose and myo-inositol are proposed as new intermediates in L-ascorbic acid biosynthesis, indicating that part of the animal pathway might also be operating in plants. Enzymatic studies on the GDP-mannose-3 0 ,5 0 -epimerase and L-galactono-1,4-lactone dehydrogenase suggest that they are important regulatory steps for L-ascorbic acid biosynthesis

ttl mutants are impaired in cellulose biosynthesis under osmotic stress

As sessile organisms, plants require mechanisms to sense and respond to the challenging environment, that encompass both biotic and abiotic factors that results in differential development. In these conditions is essential to balance growth and stress responses. As cell walls shape plant growth, this differential growth response cause alterations to the plant cell wall and cellulose is a major component. Therefore, understanding the mechanisms that regulate cellulose biosynthesis is essential to develop strategies to improve plant production. Previous studies have shown that the GSK3 kinase BIN2 modulate cellulose biosynthesis through phosphorylating cellulose synthases and that the expression of cellulose synthases are regulated by Brassinosteroids. Our previous work reveals that the tetratricopeptide-repeat thioreoxin-like (TTL) TTL1, TTL3, and TTL4 genes, in addition to their reported role in abiotic stress tolerance, are positive regulators of BR signaling. We observe association of TTL3 with most core components in traducing BR signalling, such as LRR-RLK BRI1, BIN2 and the transcription factor BES1 that positively regulate cellulose biosynthesis. We show that ttl mutants are affected in cellulose biosynthesis, particularly in osmotic stress conditions. Furthermore, TTL3 associates with LRR-RLKs that have been shown to be important for cellulose biosynthesis such as FEI1 in the FEI1/FEI2/SOS5 pathway. We aim to investigate the mechanisms by which TTL proteins regulate cellulose biosynthesis using a combination of genetics, biochemical, and molecular and cell biology approaches. This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256); (3) Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256); (3) Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Structural basis of the allergenicity to strawberries due to Fra a 1.02

Strawberry fruits are highly valued due to their flavor, aroma, and benefits for human health. Despite this, 30% of the population with food hypersensitivity also shows adverse reactions to strawberry (Franz-Oberdorf et al, 2016). The FaFra a 1 protein family, homologs of the major birch pollen allergen Bet v 1, is involved in this allergenicity to strawberry. By RNAseq we have identified transcripts for 18 members of the FaFra a 1 family (from 1.01 to 1.18) in strawberry fruits. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each FaFra a 1 protein. FaFra a 1.02 (Fra2 from now on) is the most expressed one in red fruits and is also the most allergenic among the family members tested (Muñoz et al. 2010; Franz- Oberdorf et al, 2016). In order to understand the molecular bases of this allergenicity we crystalized Fra2 and obtained its structure by X-ray diffraction. Fra2 showed a very high structural homology to Bet v 1, and we asked whether the two proteins were recognized by the immune system in a similar way. For this, we generated five different mutant versions of Fra2 in sites described as important for allergenicity in Bet v 1 (Fernandes et al, 2016), and studied their potential allergenicity as well as their crystal structures. Three of the mutants had substitutions in loop 4 (E46R, D48R, E46/48A) and the other two facing the cavity (A141F and Q64W). Compared to Fra2, all the mutants showed a significant reduction in their capacity to be recognized by the serum of patients with allergies to Bet v 1, and their crystal structures revealed conformational changes in the Bet v 1- IgG interaction sites. Together, these results support that Fra2 and Bet v 1 have similar allergenic determinants We hope this research will aid in understanding how human IgGs interact with Fra2 and might help in the development of new cultivars with a lesser allergenic potential.Grants BIO2013-44199R and BES-2014-068723 (MINECO). The authors also acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía

The Arabidopsis Synaptotagmin1 is enriched in endoplasmic reticulum-plasma membrane contact sites and confers cellular resistance to mechanical stresses

Eukaryotic endoplasmic reticulum (ER)-plasma membrane (PM) contact sites are evolutionarily conserved microdomains that have important roles in specialized metabolic functions such as ER-PM communication, lipid homeostasis, and Ca2+ influx. Despite recent advances in knowledge about ER-PM contact site components and functions in yeast (Saccharomyces cerevisiae) and mammals, relatively little is known about the functional significance of these structures in plants. In this report, we characterize the Arabidopsis (Arabidopsis thaliana) phospholipid binding Synaptotagmin1 (SYT1) as a plant ortholog of the mammal extended synaptotagmins and yeast tricalbins families of ER-PM anchors. We propose that SYT1 functions at ER-PM contact sites because it displays a dual ER-PM localization, it is enriched in microtubule-depleted regions at the cell cortex, and it colocalizes with Vesicle-Associated Protein27-1, a known ER-PM marker. Furthermore, biochemical and physiological analyses indicate that SYT1 might function as an electrostatic phospholipid anchor conferring mechanical stability in plant cells. Together, the subcellular localization and functional characterization of SYT1 highlights a putative role of plant ER-PM contact site components in the cellular adaptation to environmental stresses

Vitamin C Content in Fruits: Biosynthesis and Regulation

Throughout evolution, a number of animals including humans have lost the ability to synthesize ascorbic acid (ascorbate, vitamin C), an essential molecule in the physiology of animals and plants. In addition to its main role as an antioxidant and cofactor in redox reactions, recent reports have shown an important role of ascorbate in the activation of epigenetic mechanisms controlling cell differentiation, dysregulation of which can lead to the development of certain types of cancer. Although fruits and vegetables constitute the main source of ascorbate in the human diet, rising its content has not been a major breeding goal, despite the large inter- and intraspecific variation in ascorbate content in fruit crops. Nowadays, there is an increasing interest to boost ascorbate content, not only to improve fruit quality but also to generate crops with elevated stress tolerance. Several attempts to increase ascorbate in fruits have achieved fairly good results but, in some cases, detrimental effects in fruit development also occur, likely due to the interaction between the biosynthesis of ascorbate and components of the cell wall. Plants synthesize ascorbate de novo mainly through the Smirnoff-Wheeler pathway, the dominant pathway in photosynthetic tissues. Two intermediates of the Smirnoff-Wheeler pathway, GDP-D-mannose and GDP-L-galactose, are also precursors of the non-cellulosic components of the plant cell wall. Therefore, a better understanding of ascorbate biosynthesis and regulation is essential for generation of improved fruits without developmental side effects. This is likely to involve a yet unknown tight regulation enabling plant growth and development, without impairing the cell redox state modulated by ascorbate pool. In certain fruits and developmental conditions, an alternative pathway from D-galacturonate might be also relevant. We here review the regulation of ascorbate synthesis, its close connection with the cell wall, as well as different strategies to increase its content in plants, with a special focus on fruits

Autophagy is required for strawberry fruit ripening

Autophagy is a catabolic and recycling pathway that maintains cellular homeostasis under normal growth and stress conditions. Two major types of autophagy, microautophagy and macroautophagy, have been described in plants. During macroautophagy, cellular content is engulfed by a double-membrane vesicle called autophagosome. This vesicle fuses its outer membrane with the tonoplast and releases the content into the vacuole for degradation. During certain developmental processes, autophagy is enhanced by induction of several autophagy-related genes (ATG genes). Autophagy in crop development has been studied in relation to leaf senescence, seed and reproductive development, and vascular formation. However, its role in fruit ripening has only been partially addressed. Strawberry is an important berry crop, representative of non-climacteric fruit. We have analyzed the occurrence of autophagy in developing and ripening fruits of the cultivated strawberry. Our data show that most ATG genes are conserved in the genome of the cultivated strawberry Fragaria x ananassa and they are differentially expressed along the ripening of the fruit receptacle. ATG8-lipidation analysis proves the presence of two autophagic waves during ripening. In addition, we have confirmed the presence of autophagy at the cellular level by the identification of autophagy-related structures at different stages of the strawberry ripening. Finally, we show that blocking autophagy either biochemically or genetically dramatically affects strawberry growth and ripening. Our data support that autophagy is an active and essential process with different implications during strawberry fruit ripening.This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie to VS-V grant agreement 844365 project FrATGaria: Identification of the role of autophagy during fruit ripening and stress resistance in Strawberry: Autophagy manipulation as a way to improve Strawberry fitness, by the Ministerio de Economía y Competitividad, co-financed by the European Regional Development Fund (grant BIO2017-82609-R) to MB and by the Plan Propio of the University of Malaga