Regulatory mechanism of GA₃ application on grape (Vitis vinifera L.) berry size

Gibberellin A₃ (GA₃) is often used as a principal growth regulator to increase plant size. Here, we applied Tween-20 (2%)-formulated GA₃ (T1:40 mg/L; T2:70 mg/L) by dipping the clusters at the initial expansion phase of 'Red Globe' grape (Vitis vinifera L.) in 2018 and 2019. Tween-20 (2%) was used as a control. The results showed that GA₃ significantly increased fruit cell length, cell size, diameter, and volume. The hormone levels of auxin (IAA) and zeatin (ZT) were significantly increased at 2 h (0 d) -1 d after application (DAA0-1) and remained significantly higher at DAA1 until maturity. Conversely, ABA exhibited an opposite trend. The mRNA and non-coding sequencing results yielded 436 differentially expressed mRNA (DE_mRNAs), 79 DE_lncRNAs and 17 DE_miRNAs. These genes are linked to hormone pathways like cysteine and methionine metabolism (ko00270), glutathione metabolism (ko00480) and plant hormone signal transduction (ko04075). GA₃ application reduced expression of insensitive dwarf 2 (GID2, VIT_07s0129g01000), small auxin-upregulated RNA (SAUR, VIT_08s0007g03120) and 1-aminocyclopropane-1-carboxylate synthase (ACS, VIT_18s0001g08520), but increased SAUR (VIT_04s0023g00560) expression. These four genes were predicted to be negatively regulated by vvi-miR156, vvi-miR172, vvi-miR396, and vvi-miR159, corresponding to specific lncRNAs. Therefore, miRNAs could affect grape size by regulating key genes GID2, ACS and SAUR. The R2R3 MYB family member VvRAX2 (VIT_08s0007g05030) was upregulated in response to GA₃ application. Overexpression of VvRAX2 in tomato transgenic lines increased fruit size in contrast to the wild type. This study provides a basis and genetic resources for elucidating the novel role of ncRNAs in fruit development.