Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Ciencias Biológicas, Universidad de Talca, Avenida Lircay s/n, Talca, Chile.
Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Ciencias Biológicas, Universidad de Talca, Avenida Lircay s/n, Talca, Chile.
Plant Physiol Biochem. 2024 Oct;215:108985. doi: 10.1016/j.plaphy.2024.108985. Epub 2024 Jul 29.
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.
智利草莓是智利本土物种，在成熟过程中会强烈软化。其软化与细胞壁降解酶的参与有关，导致细胞壁解体。ABA 处理可以加速 F. chiloensis 果实的软化，伴随着关键细胞壁降解基因表达的增加，然而，直到现在，参与转录调控的分子机制尚未得到研究。因此，研究了属于不同亚家族的两个 MADS 框转录因子 FchAGL9 和 FchSHP 的作用。这两个 TF 都是 II 型 MADS 框家族（MIKC 型）的成员，定位于细胞核内。FchAGL9 和 FchSHP 仅在花和果实组织中表达，随着果实软化而升高，在 C3-C4 阶段表达水平最高。EMSA 实验表明，FchAGL9 结合 RIN 和 SQM 的 CArG 序列，而 FchSHP 仅与 RIN 相互作用。双分子荧光互补和酵母双杂交实验证实了 FchAGL9-FchAGL9 和 FchAGL9-FchSHP 的相互作用。通过同源建模构建异源二聚体结构，得出 FchSHP 单体结合 DNA 的结论。通过 Luciferase-dual 测定的功能验证表明，FchAGL9 可激活 FchRGL 和 FchPG 的启动子，而 FchSHP 可激活 FchEXP2、FchRGL 和 FchPG 的启动子。在 C2 F. chiloensis 果实中过表达 FchAGL9 会增加 FchEXP2 和 FchEXP5 的转录物，而过表达 FchSHP 也会增加 FchXTH1 和 FchPL；在这两种情况下，FchRGL 和 FchPG 的表达都下调。总之，我们提供了证据表明 FchAGL9 和 FchSHP 参与了与 F. chiloensis 软化相关的转录调控。
