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

Characterization of ripe fruit epidermis-specific transcription factors in strawberry

The epidermis is the external cell layer in direct contact with the environment, and it plays essential biological roles. Transcriptome analysis (RNA-seq) of Fragaria vesca fruit receptacles at four ripening stages (green, white, turning and red) and of different tissue types of receptacles (pith, vascular bundles, cortex and epidermis) at two ripening stages (green and red) allowed us to infer tissue- and stage-specific Gene Regulatory Networks (GRN). Due to the potential role of the epidermis in defense and in the differential anthocyanin accumulation pattern that shows at the ripe stage of F. vesca fruits (the skin is red, while the inner part is white), we have focused on the GRN of the ripe epidermis. In this study, we aim at the functional characterization of two transcription factors (TFs) that constituted the main hubs of this GRN: a MYB-like gene, and a member of the NAC family of TFs. A MapMan analysis of the genes constituting the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions in this tissue at the ripe stage. Using the Luciferase/Renilla (Luc/Ren) system, the interaction of the MYB and NAC TFs with their wax-related putative targets was validated. To gain insight into the target genes of these two TFs, we mapped the genome-wide binding sites using DAP-seq analyses. Consistently, MYB bound to a set of genes involved in cuticle formation and flavonoid biosynthesis, while a number of genes involved in solute transport were enriched among the NAC targets. Currently, we are generating CRISPR/Cas9 mutant lines to functionally characterize these two TFs. Furthermore, we are performing protein interaction assays to decipher whether the MYB and NAC TFs interact with each other and with other TFs from the red epidermis GRN.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Role of ripe fruit epidermis-specific FvMYB29-FvbHLH transcription factor complexes in strawberry

Transcriptome changes during strawberry fruit ripening have been previously reported. However, we have identified genes with tissue- and stage-specific patterns in the receptacles of Fragaria vesca coupling LCM with RNA-seq analysis. In the study, we have focused on the Gene Regulatory Network at the epidermis in ripe fruits, since it is the external cell layer in direct contact with the environment and it plays an important role in defense, and, in contrast to receptacles of the commercial species, it is the only part of the fruit that accumulates anthocyanins. Consistently, a GO functional analysis of this GRN showed enrichment in genes involved in flavonoid and wax biosynthesis. Three out of the several ripe epidermis-specific TFs were selected to study their biological role, one of them belonging to the MYB family (FvMYB29), and two bHLH-like proteins (FvbHLH22 and FvbHLH67). Protein interaction assays revealed that the FvMYB29 protein physically interacts with the two FvbHLHs. Genome-wide binding sites of these TFs were identified by DAP-seq, revealing that genes involved in flavonoid biosynthesis and cuticle composition are among the FvMYB29 targets, which was validated by transactivation assays. However, transactivation assays with different combinations of FvMYB29 and the two FvbHLH showed that the latter modulates the activation of transcription of the targets. Consistently with the role of FvMYB29 in the cuticle formation, stable FvMYB29-overexpressing lines showed a misregulation of genes related to cutin and wax biosynthesis in ripe fruits and leaves. Furthermore, FvMYB29-overexpressing fruits presented cuticular nanoridges. On the other hand, young leaves of FvMYB29-overexpression lines showed denser epicuticular waxes in the abaxial surface and an alteration in wax composition but compared to the control. All these results support the role of the FvMYB29-FvbHLH TF complex as an important regulator of cuticle structure in F. vesca.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech