Study of Transcriptional Regulatory Network Controlling Strawberry Fruit Ripening and Quality

Ponencia invitadaRipening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project, we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. Also, we are using a collection of around two hundred wild strawberry (Fragaria vesca) accessions to identify loci involved in important traits such as aroma, size or resistance to pathogens. Finally, we are implementing the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect it might open opportunities for engineering this species to improve traits of economic importance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Identification and characterization of RIPENINGLESS, a key transcription factor in the regulation of fruit development and ripening in the cultivated strawberry Fragaria × ananassa

Despite the advances that have allowed to elucidate transcription factors (TFs) involved in the development and ripening of non-climacteric strawberry fruits, key regulators of these processes remain to be identified. Among the plant TFs, the BEL class of Homeobox genes has been shown to control meristem formation and/or maintenance, organ morphogenesis, organ position, and several aspects of the reproductive phase in Arabidopsis thaliana (Bhatt et al., 2004; Byrne et al., 2003; Roeder et al., 2003; Smith and Hake, 2003). In this work, we have identified RIPENINGLESS (FaRPL), a BEL-like Homeodomain TF of the cultivated strawberry (Fragaria × ananassa), that shows an increase in its expression during ripening, peaking at the turning stage. To functionally characterize the role of FaRPL, we have established stable silencing (RNAi) and over-expression lines under the 35S promoter. FaRPL-RNAi lines showed a high rate of abortions. However, some lines achieved to develop full fruits, being firmer and with a lower water content than those of the control. Furthermore, the ripening progress was significantly delayed in these RNAi lines, in which a high percentage of fruits displayed a complete blockage at a stage similar to the turning stage in wt. Interestingly, fruits overexpressing FaRPL showed aberrant shapes, being enlarged in the base of the receptacles. All these results support an important role of this TF in fruit development and ripening. Finally, gene expression and hormone analyses point to an essential role of FaRPL in the regulation of ABA biosynthesis, being this phytohormone the main promoter of strawberry fruit ripening.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in arabidopsis

Background: The wall-associated kinases (WAKs) serve as pectin receptors. Results: A pectin methyl esterase and two transcription factor mutants suppress a dominant WAK allele. Conclusion: De-esterification of pectin is required for WAK activation though EDS1 and PAD4. Significance: The results provide a mechanism for the state of pectins to activate two different pathways. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc

Aroma map in European woodland strawberry

Woodland strawberry (Fragaria vesca, 2x) is the diploid closest ancestor of the cultivated strawberry (Fragaria ´ annassa, 8x) and the model species for genetic studies in the Fragaria genus. It is naturally distributed all across Europe and it is appreciated for their delicate aroma and flavor. Aiming to describe the genetic and organoleptic diversity of European woodland strawberry and decipher the genetic control of its characteristic volatile compounds, we have sequenced and metabolically-phenotyped a diverse collection of 199 geographically distant European accessions. The metabolic profiling of the lines includes a set of 100 unambiguosly identified volatiles. This study has revealed genetic and metabolic differences between subpopulations with different geographical origin. In addition, Genome Wide Association Analysis points to several candidate genetic regions controlling the accumulation of volatiles compounds sharing common biosynthetic pathways. Specifically, we have detected SNPs associated to the accumulation of methyl ketones and their corresponding alcohols mapping to a small region of chromosome 4 with a reduced set of candidate genes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Aroma Map in European Woodland Strawberry

Woodland strawberry (Fragaria vesca, 2x) is a wild, diploid ancestor of the cultivated strawberry (Fragaria ananassa, 8x), the most economically important berry crop. F. vesca is very appreciated for its intense fruity aroma, characterized by a unique combination of volatile compounds, which are absent, or accumulated at lower rates, in the commercial strawberry varieties. In addition, F. vesca presents a wide genetic diversity and it is naturally distributed across Europe. Our aim is to describe the genetic and organoleptic diversity of European woodland strawberry and decipher the genetic control of its characteristic volatile compounds. A collection of 199 accessions representing the European genetic diversity of F. vesca has been re-sequenced obtaining a set of 1.8M SNPs. In addition, the volatilome of ripe fruits was quantified in two independent harvests by GCMS providing a set of 100 unambiguously identified compounds. This study has revealed genetic and metabolic differences between subpopulations with different geographical origin. In addition, Genome-Wide Association Analysis has revealed genetic regions significantly associated to the accumulation of several metabolites that contribute to strawberry aroma, such as terpenes (alpha-farnesene, alpha-pinene, alpha-terpineol, linalool, myrtenol), lactones (g-decalactone), eugenol and mesifurane among others.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

FaRIF Transcription Factor Plays a Key Role in the Regulation of Fruit Ripening in the Cultivated Strawberry Fragaria x ananassa

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 (35S::RIF-RNAi) and overexpressing (35S::RIF-GFP) 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, by RNA-seq, of the silenced lines showed a significantly altered expression of genes involved in the flavonoids pathway, as well as genes of the metabolism of the main sugars of the fruit. Metabolomics analysis confirmed these changes in the transgenic fruits. Both, transcriptomic and metabolomics data, were in agreement with the general phenotype observed in the fruits of the FaRIF-silenced lines. All together, our results support a main role of FaRIF in the control of relevant ripening-associated processes in strawberry fruit.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

Characterization of ripe fruit epidermis-specific transcription factors in strawberry

Transcriptome changes during strawberry fruit ripening have been previously reported using either complete fruits or achenes (actual fruits) and receptacles (fleshy part) separately. In order to perform a more detailed study, we have performed a tissue- and stage-specific transcriptome analysis in receptacles of Fragaria vesca fruits, allowing us to infer Gene Regulatory Networks (GRN) in each tissue and stage. In the study, we have focused on the epidermis at the ripe stage, since it plays an important role in defense, as it is the external cell layer in direct contact with the environment, and, in contrast to receptacles of the commercial species, it is the only part of the fruit that accumulates anthocyanins. MapMan analysis of the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions. Three out of the several TFs found among the main hubs in this GRN were selected to study their biological role, one of them belonging to the MYB family, and two bHLH genes. Protein interaction assays revealed that the MYB protein physically interacts with the two bHLHs, leading to the subcellular relocalization from the cytoplasm to the nucleus in one of them. DAP-seq analyses showed that the bHLH TFs do not bind DNA by themselves, but that genes involved in cuticle formation and flavonoid biosynthesis are among the MYB targets, which were validated by a transactivation assay using the Luciferase/Renilla system. Consistently, MYB-overexpressing stable lines exhibited an upregulation of genes related to cuticle and wax biosynthesis in ripe fruits, and an accumulation of higher amounts of epicuticular waxes in young leaves compared to the WT. We are currently establishing RNAi and CRISPR lines for these three ripe-epidermis specific TFs to further investigate their biological role and performing analyses to understand the effect on gene expression of the interaction between them.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Characterization of ripe fruit epidermis-specific transcription factors in strawberry

Transcriptome changes during strawberry fruit ripening have been previously reported using either complete fruits or achenes (actual fruits) and receptacles (fleshy part) separately. In order to perform a more detailed study, we have performed a tissue- and stage-specific transcriptome analysis in receptacles of Fragaria vesca fruits, allowing us to infer Gene Regulatory Networks (GRN) in each tissue and stage. In the study, we have focused on the epidermis at the ripe stage, since it plays an important role in defense, as it is the external cell layer in direct contact with the environment, and, in contrast to receptacles of the commercial species, it is the only part of the fruit that accumulates anthocyanins. MapMan analysis of the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions. Three out of the several TFs found among the main hubs in this GRN were selected to study their biological role, one of them belonging to the MYB family, and two bHLH genes. Protein interaction assays revealed that the MYB protein physically interacts with the two bHLHs, leading to the subcellular relocalization from the cytoplasm to the nucleus in one of them. DAP-seq analyses showed that the bHLH TFs do not bind DNA by themselves, but that genes involved in cuticle formation and flavonoid biosynthesis are among the MYB targets, which were validated by a transactivation assay using the Luciferase/Renilla system. Consistently, MYB-overexpressing stable lines exhibited an upregulation of genes related to cuticle and wax biosynthesis in ripe fruits, and an accumulation of higher amounts of epicuticular waxes in young leaves compared to the WT. We are currently establishing RNAi and CRISPR lines for these three ripe-epidermis specific TFs to further investigate their biological role and performing analyses to understand the effect on gene expression of the interaction between them.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec