Metabolomic characterization of strawberry cultivars during postharvest

The cultivated strawberry (Fragaria x ananassa) is the berry most consumed worldwide and is well-known for its delicate flavour and nutritional characteristics. However, strawberries possess a very short postharvest shelf-life due to their high respiration rate and their susceptibility to water loss, mechanical damage and fungi deterioration. Extension of fruit shelf-life is a major economic goal, and measures are commercially taken to delay senescence. These procedures include low temperature, controlled atmosphere and/or chemical treatments, being the first one the most commonly applied. To improve our understanding of the molecular and biochemical mechanisms underlying the deterioration of fruit quality attributes during senescence, we monitored the metabolomic profiles of five commercial strawberry cultivars under different postharvest treatments. Ripe fruits of F x ananassa cv. ‘Amiga’, ‘Camarosa’, ‘Candonga’, ‘Fortuna’ and ‘Santa Clara’ were harvested and kept at 4ºC during three, six and ten days in normal, CO2-enriched and O3-enriched atmospheres. We used a combination of GC-TOF-MS, LC-MS and GC-SPME-MS to identify and semi-quantify 49 primary metabolites (sugars, amino and organic acids), 132 polar secondary metabolites and 70 volatile compounds in all different treatments along postharvest stages. Multivariate statistical approaches, including hierarchical cluster analysis, partial least squares discriminant analyses and k-means clustering, were used to characterize the variation in metabolite content during the strawberry fruit postharvest life and to identify the biochemical pathways which are most affected in the senescence processes. Here, we present the main changes in volatile compounds, primary and secondary metabolites as a consequence of postharvest storage, highlighting the differences between cultivars and treatments. Network-based methods will allow us to point out the regulatory factors and molecular mechanisms underlying fruit senescence.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The balance between fumarate and malate plays an important role in plant development and postharvest quality in tomato fruit

Organic acids, produced as intermediates of the tricarboxylic cycle, play a crucial role in the plant primary metabolism and are considered as being ones of the most important quality traits in edible fruits. Even if they are key metabolites in a multitude of cellular functions, little is known about their physiological relevance and regulation. Transgenic tomato (Solanum lycopersicum) plants expressing constitutively a bacterial maleate isomerase, which converts reversibly maleate to fumarate, were generated in order to improve our knowledge about the role of organic acids in the crop and fruit metabolism. Growth and reproduction were affected by the unbalance of tricarboxylic cycle intermediates, as a dwarf phenotype and a flowering delay were observed in the transgenic plants. In addition, a delay in chlorophyll synthesis, a decrease in the numbers of stomata and significant changes in some photosynthetic parameters indicated alterations in central primary metabolism. Postharvest was also impaired, as transgenic fruits showed increased water lost and deterioration, indicating a possible role of the organic acids in cell wall metabolism. Finally, preliminary metabolomics analysis pointed out important changes during fruit ripening in flavor-related metabolites, such as acids and sugars, revealing the importance of organic acids in fruit metabolism. Taken together, these data indicate a pivotal role of tricarboxylic cycle intermediates, such as malate or fumarate, as regulatory metabolites. Besides their role in quality fruit characteristics, they are involved in a multitude of functions including growth and photosynthesis.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Metabolomics profiling of strawberry (Fragaria x ananassa) F1 population to characterize flavour and nutritional traits

The cultivated strawberry (Fragaria x ananassa) is a highly consumed fruit known for its delicate flavour and nutritional characteristics. However, as fruit quality attributes have been lost after years of traditional breeding, new technological tools, such as high throughput metabolomics, are necessary for the identification of factors responsible of these traits. Here we present the metabolomics profiling for the content of primary and secondary metabolites of a 95 F1 individuals strawberry population derived from genotype “1392”, selected for its superior flavour, and “232” (Zorrilla-Fontanesi et al., 2011; Zorrilla-Fontanesi et al., 2012). Metabolite profiling was performed on mature fruits of the strawberry population using gas chromatography hyphenated to time-of-flight mass spectrometry for primary metabolites and ultra performance liquid chromatography Exactive Orbitrap tandem mass spectrometry for secondary metabolites.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Sugar deficiency causes changes in cuticle permeability and cell wall composition that influence fruit postharvest shelf-life

The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, and postharvest shelf-life. Tomato fruits with reduced expression of the tomato gene LIN5 encoding cell wall invertase exhibits decreases transpirational water loss. Transcriptomic, biochemical, histological, and biomechanical analysis identified several unsual features of RNAi-LIN5 cuticles and the data indicate that, perturbation of endogenous fruit sugar levels affects the composition of the tomato cuticle and cell wall architecture which are an integral and regulated part of the ripening program affecting the postharvest shelf-life. A model is proposed in which sugar levels affects the cuticle formation which has a direct effect in softening of intact tomato fruit both directly, by providing a physical support, and indirectly, by regulating water status.University of Málaga, Campus de Excelencia Internacional de Andalucia Tech. Spanish Ministry of Science and Innovation (Ramón and Cajal contract, RYC2011-09170

Candidate gene for branched-chain amino acid content in strawberry

Cultivated strawberry (Fragaria × ananassa) is a berry crop widely appreciated worldwide due to its nutritional and organoleptic properties. However, the main breeding targets in these crops have been the control of flowering (for fruit yield), runnering (for vegetative propagation) and the trade-off between the two. For this reason, nowadays, breeders are mainly focused on recovering fruit quality attributes in order to gain consumers acceptance. Those characteristics are determined by the metabolic composition of the fruit, thus the modulation of its metabolism can help to reach this aim. Regarding aroma, esters are the main responsible of this trait in strawberry fruit. Some of them are derived from the catabolism of branched-chain amino acids (valine, leucine and isoleucine). Although the synthesis of these amino acids has been well stablished in strawberry fruit, its degradation remains unclear. Using a F1 F. × ananassa mapping population we identified a stable QTL (Quantitative Trait Loci) for these amino acids. Based on its differential gene expression in contrasting lines for these metabolites and its annotation, we identified an amino acid transporter as a candidate gene for the regulation of branched-chain amino acid content in strawberry. To elucidate the involvement of this gene in branched-chain amino acid metabolism, we have done functional analysis based on transient overexpression lines on Nicotiana benthamiana showing changes in amino acids content. Moreover, the subcellular localization of the candidate gene in N. benthamiana suggested its location in the plasmatic reticulum. Currently, we are performing further experiments in order to elucidate the role of this gene on strawberry volatile metabolism.This work was supported by grants RTI 2018-099797-B-100 (Ministerio de Ciencia, Innovación y Universidades, Spain) and UMA18-FEDERJA-179 (FEDER-Junta Andalucía). In addition, we acknowledge partial funding by PY20_00408 (PAIDI 2020-Junta de Andalucía). The attendance to this meeting was supported by Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The molecular control of tomato fruit quality traits: the trade off between visual attributes, shelf life and nutritional value

Tomato (Solanum lycopersicum) is an established model to study fleshy fruit development and ripening and is an important crop in terms of its economic and nutritional value. Tomato fruit quality is a function of metabolite content which is prone to physiological changes related to fruit development and ripening. It has been described some ripening tomato mutants, delayed fruit deterioration (DFD), non-ripening (NOR) and ripening-inhibitor (RIN) which substantially extend “shelf life” in tomato for up to several months when defined in terms of softening, water loss and resistance to postharvest biotic infection. However, it is not known whether this extension in “shelf life” is in fact a desirable objective from the perspective of nutritional quality of the fruits. The aim of this work was to use a metabolomics approach join to genomic tools to characterize compositional changes (sugars, amino acids, organic acids and carotenoids) of non-softening tomato mutants reported (DFD, NOR and RIN) in comparison with the normally softening fruits (Ailsa Craig and M82) during ripening and postharvest shelf-life. Important results related with ripening gene expression and metabolic evolutions are shown

Plasma membrane lipid remodeling during cold acclimation is mediated by the ER-PM contact sites-localized synaptotagmins 1 and 3

Cold acclimation is the capacity of certain plants to increase their freezing tolerance in response to a period of low non-freezing temperatures. Cold acclimation involves a series of biochemical and physiological adaptations, including a deep transcriptional reprogramming and drastic changes in the lipid composition of cellular membranes in order to prevent the freeze-induced damage (1). While a profound knowledge has been acquired on the regulation of gene expression triggered by cold-acclimation, very little is known about the mechanisms governing the cold-induced changes in membranes’ lipid composition. In this study we report that in Arabidopsis, the constitutively expressed Synaptotagmin 1 (SYT1) and the cold-induced homolog Synaptotagmin 3 (SYT3) are essential for cold- acclimated freezing tolerance and for the lipid remodelling of the plasma membrane during cold-acclimation. SYT1 and SYT3 are phospholipid-binding proteins located in Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PMcs), conserved structures defined as regions of the cortical ER in close apposition to the PM (2). ER-PMcs facilitate the non-vesicular lipid transport between ER and PM in yeast and mammals, and are essential for lipid homeostasis (3). In contrast to the high and ubiquitous SYT1 expression, SYT3 expression is low and mainly restricted to meristemoids, young stomata, and old primary root. TIRF microscopy analyses show that during cold acclimation there is an increase of SYT1::SYT1:GFP and SYT3::SYT3:GFP signals as spots at the PM. High-resolution lipidome analyses show the over-accumulation of phosphatidylinositols phosphate (PIPs) and glycerolipids in vivo in syt1 and specially syt1/syt3 mutant plants compared to WT in one-week cold-acclimated plants. Interestingly, protein-lipid overlay assays (membrane-strips and PIP-strips) reveal PIPs and glycerolipids as major interactors for both, SYT1 and SYT3. Here we show that 1) Arabidopsis SYT1 and SYT3 are induced by cold, 2) SYT1 and SYT3 localize to ER-PMcs, 3) the specific lipids that directly interact with SYT1 and SYT3 accumulate in syt1/syt3 mutant after cold acclimation, and 4) syt1/syt3 show reduced cold acclimated freezing tolerance. We propose that SYT1 and SYT3 have essential roles in ER-PMcs mediated lipid remodelling during cold acclimation, which in turn leads to freezing tolerance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

QTLs mapping for primary metabolites responsible of the organoleptic and nutritional characteristics of strawberry (Fragaria x ananassa)

The cultivated strawberry (Fragaria x ananassa) is the berry fruit most consumed worldwide and is well-known for its delicate flavour and nutritional properties. However, fruit quality attributes have been lost or reduced after years of traditional breeding focusing mainly on agronomical traits. To face the obstacles encountered in the improvement of cultivated crops, new technological tools, such as genomics and high throughput metabolomics, are becoming essential for the identification of genetic factors responsible of organoleptic and nutritive traits. Integration of “omics” data will allow a better understanding of the molecular and genetic mechanisms underlying the accumulation of metabolites involved in the flavour and nutritional value of the fruit. To identify genetic components affecting/controlling? fruit metabolic composition, here we present a quantitative trait loci (QTL) analysis using a 95 F1 segregating population derived from genotypes ‘1392’, selected for its superior flavour, and ‘232’ selected based in high yield (Zorrilla-Fontanesi et al., 2011; Zorrilla-Fontanesi et al., 2012). Metabolite profiling was performed on red stage strawberry fruits using gas chromatography hyphenated to time-of-flight mass spectrometry, which is a rapid and highly sensitive approach, allowing a good coverage of the central pathways of primary metabolism. Around 50 primary metabolites, including sugars, sugars derivatives, amino and organic acids, were detected and quantified after analysis in each individual of the population. QTL mapping was performed on the ‘232’ x ‘1392’ population separately over two successive years, based on the integrated linkage map (Sánchez-Sevilla et al., 2015). First, significant associations between metabolite content and molecular markers were identified by the non-parametric test of Kruskal-Wallis. Then, interval mapping (IM), as well as the multiple QTL method (MQM) allowed the identification of QTLs in octoploid strawberry. A permutation test established LOD thresholds for each metabolite and year. A total of 132 QTLs were detected in all the linkage groups over the two years for 42 metabolites out of 50. Among them, 4 (9.8%) QTLs for sugars, 9 (25%) for acids and 7 (12.7%) for amino acids were stable and detected in the two successive years. We are now studying the QTLs regions in order to find candidate genes to explain differences in metabolite content in the different individuals of the population, and we expect to identify associations between genes and metabolites which will help us to understand their role in quality traits of strawberry fruit.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. The work was supported by the MINECO (grant AGL2012-40066-C02-02 ; Spain). SO aknowledges the support by Spanish Ministry of Science and Innovation (Ramón and Cajal contract, RYC2011-09170). DP has received a predoctoral grant from MINECO (grant BES-2013-062856). JJM and IA has been supported by the grant AGL2012-40066-C02-01). The authors also aknowlegde the support by the University de Málaga, Campus de Excelencia Internacional de Andalucía

Deciphering the dynamics of metabolic pathways influencing by controlled atmosphere during post-harvest physiology of cultivated strawberry fruit

Strawberry (Fragaria × ananassa) fruit is highly appreciated due to its delicate aroma, flavor and nutritional value. However, fruits are highly perishable and deteriorate quickly at ambient temperature. Controlled atmosphere storage is commonly used to prevent fruit decay; however it affects fruit quality causing physiological disorders. In the present work, High-throughput metabolomics technologies allow the quantitation of (relative) metabolite levels and allow determine the metabolic dynamics associated with postharvest in different controlled atmosphere storage. Five varieties of strawberry fruits (F. ananassa cvs. Camarosa, Candonga, Amiga, Santa Clara, and Fortuna) with different aroma, taste, and postharvest behavior were stored at 4ºC in two different atmosphere compositions, i) 10% CO2 and ii) 0.35 ppm O3 at 0, 3, 6, and 10 days after harvesting and compared to fruits stored at 4ºC. Novel methods for analyzing the resulting multiple data tables revealed preserved dynamics of metabolic processes across species. We identify key metabolites, which prime the fruit to cope with different decay situations, which likely greatly accelerate the design and the improvement of plant breeding programs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia e Innovación, Spain (Ramón and Cajal contract). Ministerio de Economía y Competitividad, through the Grants AGL2012-40066-CO2-02)

Effect of environmental factors on wild strawberry primary metabolic profile

Climate factors such as temperature and precipitation vary significantly over continental scales, strongly structuring biomes along latitudinal gradients, and resulting in species differently adapted either genetically or plastically to cope with their local climate. However, climate change will likely alter these biomes. Thus, it is expected that Nordic regions, historically colder and rainier, will tend to have higher temperatures and less rainfall, which might lead to changes in the distribution of plants leading to novel patterns of local adaptation and maladaptation. In this study we aim to study how plant traits vary with latitude and in response to different temperature and drought conditions in order to find genetic determinants of climate adaptation. Our group is focused in determining the role of the metabolic profiling to that adaptation. For that purpose, we use the woodland strawberry (Fragaria vesca) as the model organism. In particular, we have analyzed 16 different genotypes that have been grown in five common gardens located in Belgium, Sweden, Finland, and Spain, in which drought treatments were also performed. Here, we present the chemical analysis (primary metabolism) in leaves of these genotypes in order to better understand how environmental factors can alter the primary metabolic profiles of F. vesca accessions grown in different locations.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. BiodivERsA project PlantCline -(PCI2020-120719-2) from “Programación Conjunta Internacional” program, Ministerio de Ciencia e Innovación