RESUMO
Fragaria chiloensis is a Chilean native species that softens intensively during its ripening. Its softening is related to cell wall disassembly due to the participation of cell wall degrading enzymes. Softening of F. chiloensis fruit can be accelerated by ABA treatment which is accompanied by the increment in the expression of key cell wall degrading genes, however the molecular machinery involved in the transcriptional regulation has not been studied until now. Therefore, the participation of two MADS-box transcription factors belonging to different subfamilies, FchAGL9 and FchSHP, was addressed. Both TFs are members of type-II MADS-box family (MIKC-type) and localized in the nucleus. FchAGL9 and FchSHP are expressed only in flower and fruit tissues, rising as the fruit softens with the highest expression level at C3-C4 stages. EMSA assays demonstrated that FchAGL9 binds to CArG sequences of RIN and SQM, meanwhile FchSHP interacts only with RIN. Bimolecular fluorescence complementation and yeast two-hybrid assays confirmed FchAGL9-FchAGL9 and FchAGL9-FchSHP interactions. Hetero-dimer structure was built through homology modeling concluding that FchSHP monomer binds to DNA. Functional validation by Luciferase-dual assays indicated that FchAGL9 transactivates FchRGL and FchPG's promoters, meanwhile FchSHP transactivates those of FchEXP2, FchRGL and FchPG. Over-expression of FchAGL9 in C2 F. chiloensis fruit rises FchEXP2 and FchEXP5 transcripts, meanwhile the over-expression of FchSHP also increments FchXTH1 and FchPL; in both cases there is a down-regulation of FchRGL and FchPG. In summary, we provided evidence of FchAGL9 and FchSHP participating in the transcription regulation associated to F. chiloensis's softening.
Assuntos
Fragaria , Frutas , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS , Proteínas de Plantas , Frutas/genética , Frutas/metabolismo , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fragaria/genética , Fragaria/metabolismoRESUMO
Fragaria chiloensis (Chilean strawberry) is a native species that produces fruit with an exotic pinkish color and a fruity aroma. It has a non-climacteric pattern of fruit ripening, and it is the mother of the commercial Fragaria x ananassa. The ripening of F. chiloensis fruit seems stimulated by ABA, and a complete set of genes participate in its softening, color, and aroma development. In addition, a set of transcription factors regulate the entire process, but few of them have been described. Over the last two decades, RNA-seq was used to identify genes at three fruit development/ripening stages, named C2 (unripe, large green) to C4 (full ripe), in whole fruit and fruit without achenes. A total of 204,754 contigs were assembled considering all samples, obtaining an N50 of 1.125 bp. Differentially expressed genes (DEGs) between two samples were identified, obtaining a total of 77,181 DEGs. Transcripts for genes involved in ABA biosynthesis present high and differential expression during the C2, C3, and C4 stages. Besides, contigs corresponding to ABA receptors, which interact with a regulatory network, are also differentially expressed. Genes associated with cell wall remodeling and those involved in flavonoid synthesis were also differentially expressed. An interaction network was built considering differentially expressed genes for the phenylpropanoid and flavonoid molecular pathways and having FcMYB1 as a transcription factor regulator. Identifying key genes could give an option to control the ripening of this non-climacteric fruit.