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

Is a member of udp- glycosyltransferase regulating ellagitannins metabolism in strawberry?

Fresh strawberries are a popular and important component of the human diet. The demand for highquality fruits is increasing globally, challenging breeders to develop modern strawberry cultivarsthat fulfill all desired characteristics. Fruit flavor and nutritional characteristics are key quality traitsand ones of the main factors influencing consumer preference. Shikimate and phenylpropanoidpathways produce many chemical compounds, like phenolic compounds, polyphenols and tannins,thatarehighlyvaluable in human nutrition offering antioxidant protection and contribute to the prevention of some diseases. Among them, the hydrolysable tannins, like ellagitanins and ellagic acid, have an effect on health in some human diseases such as breast and prostate cancers or neurodegenerative diseases (Basu et al., 2014). In a previous study, Pott et al., (2020) found 110 stable QTL for secondary metabolism by studying the F1 population derived from the crossing of ‘232’ and ‘1392’ (Zorrilla Fontanesi et al., 2011). Among these QTLs, we highlighted one that was responsible for 50-70% of the variation of ellagic acid hexose. A RNAseq with contrasting lines in ellagic acid hexose content was performed to reveal 11 differentially expressed genes (DEGs) with a false discovery ratio under 0.05. Candidate genes were functionally annotated using MapMan software. One gene raised as a candidate gene, annotated as UDP-glycosyltransferase suggesting to participate in the glycosylation of ellagic acid. In addition, we found that the gene expression of this candidate was negatively correlated with proanthocyanidin and flavan-3-ols content, providing an evidence of possible metabolic flux redirection through the synthesis of ellagitannins. However, further experiments are needed to confirm the role of this gene in the synthesis of ellagitannins.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). JGV acknowledges the EMERGIA Programme (EMERGIA20_00309-Junta de Andalucía). J.M. thanks to Ministerio de Ciencia, Innovación y Universidades (PRE2019-091188). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

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

Independent mutations in a single locus, the transcriptional factor MYB10, control natural variation in fruit color among Fragaria species

External and internal fruit color are important traits in strawberry (Fragaria spp.) breeding programs, where different preferences are sought depending on whether the fruits are produced for fresh consumption or processing. Therefore, there is a great interest in the development of predictive markers that effectively speed the development of new cultivars with increased consumer acceptance and/or which address processed fruit industry´s preferences. In order to identify loci controlling fruit color variation, two mapping populations were generated: one crossing diploid F. vesca parentals and another interspecific population between two octoploid species: the cultivated and the Chilean strawberry, F. x ananassa and F. chiloensis. Both populations allowed the detection of a QTL spanning a region of the F. vesca linkage group 1 (LG I) that includes the MYB10 gene, a known key regulator of anthocyanin biosynthesis. Mapping by sequencing in the F. vesca population revealed an LTR retrotransposon inserted in the third exon of FvMYB10, which produces a premature stop codon, and co-segregates with white fruits in the entire population. Genotyping by Sanger sequencing of additional white-fruited F. vesca accessions resulted in the identification of another three independent mutations in MYB10, two of them not previously described1. In octoploid strawberry, a mayor QTL on LG I-3 controls about 55% variation in internal flesh color and is associated with an insertion in the promoter region of FcMYB10. Similar insertions have been detected in other F. chiloensis accessions bearing white fruits. In all cases, transient over-expression of FvMYB10 restored anthocyanin biosynthesis and red color in fruit flesh and skin, indicating that lack of function of MYB10 was the underlying cause of white fruits in all analyzed cases

Genomics tools available for unravelling mechanisms underlying agronomical traits in strawberry with more to come

In the last few years, high-throughput genomics promised to bridge the gap between plant physiology and plant sciences. In addition, high-throughput genotyping technologies facilitate marker-based selection for better performing genotypes. In strawberry, Fragaria vesca was the first reference sequence obtained in the Rosoideae sub-family. This genome has a high level of synteny with genomes of other species of diploid and polyploid Fragaria, but it also provides a reference for species like Rubus and Rosa for functional genomics. Many tools for genetic, genomic and functional analyses were introduced over the last 10 years and these tools are still evolving. For genotyping, many studies have used simple sequence repeats (SSRs) but whole genome sequencing is now a mature technology and facilitates the development of genotyping chips and other genetic approaches such as genome wide association studies (GWAS). Furthermore, microarray-based technologies have been eclipsed by RNA-seq, the high-throughput sequencing of RNA. These new approaches have led to advances in our understanding of the genetically complex octoploid species, and have revolutionized functional genomics. For all genetic and genomic studies, novel material such as complex crosses such as NILs and EMS have appeared in addition to the classical segregating population. With all these tools, strawberry now emerges as a plant model, not only for studying fruit quality but also for deciphering the mechanisms controlling various aspects of plant biology. Selective examples will be described to illustrate the latest research on strawberry and what is coming from other model species.Peer reviewe

The NAC transcription factor FaRIF is a key regulator of fruit ripening in strawberry

In contrast to climacteric fruits such as tomato, the knowledge on key regulatory genes controlling the ripening of strawberry, a non-climacteric fruit, is still limited. NAC transcription factors are proteins that mediate different developmental processes in plants. In this work, we have identified and characterized FaRIF (Ripening Inducing Factor), a novel NAC transcription factor which is highly expressed and induced in strawberry receptacles during ripening. Functional analysis establishing stable transgenic lines with RNAi, driven by either the constitutive 35S or the ripe receptacle-specific EXP2 promoters, and overexpression constructs showed that FaRIF controls critical ripening-related processes such as fruit softening and pigment and sugars accumulation. Physiological, metabolomic and transcriptomic analyses of receptacles of FaRIFsilenced and overexpression lines point to FaRIF as a key regulator of strawberry fruit ripening from early developmental stages, controlling ABA biosynthesis and signaling, cell wall degradation and modification, the phenylpropanoid pathway, and the balance of the aerobic/anaerobic metabolism, being therefore a target to be modified/edited to control the quality of strawberry fruits.ERC Starting Grant ERC-2014-StG 63813

Identification and characterization of the NAC transcription factor FaRIF, a key regulator of strawberry fruit ripening

Strawberry is becoming a model for studying the molecular mechanism of ripening in non-climacteric fruits. However, a limited number of transcriptional regulators of this process have been identified so far. In this study, we have identified and characterized a gene encoding for a NAC transcription factor (TF), named as FaRIF (Ripening Inducing Factor). FaRIF expression presents a fruit-specific pattern, which is upregulated during ripening. In order to functionally characterize this TF, we have generated silencing and overexpressing stable transgenic lines. While the RNAi lines showed an apparent delay of fruit ripening, the overexpressing lines displayed an acceleration of this process. Transcriptomic analysis of the silenced lines showed a significantly altered expression of genes involved in development, hormone metabolism, flavonoid pathway, and cell-wall disassembly, being many of these confirmed by phenotypical and metabolomics analysis. Our results support a main role of FaRIF in the control of relevant ripening-associated processes in strawberry fruit

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development

FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid-1,4-lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9-specific CArG box was identified in the promoters of these genes