Cloning And Functional Analyses Of GA Signal Transduction Factors CsGAIP And CsGAMYB1in Cucumber

Cucumber (Cucumis sativus L.) is a typical monoecious vegetable with individual male and female flowers, and the sex determination directly affects the product yield. Extensive studies have been performed previously on the mechanism of cucumber female flower development, while the molecular regulation of male flower development has been neglected, which greatly hinders the understanding of sex expression in cucumber. Gibberellin (GA) is an important plant hormone that can promote male flower formation in cucumber, but the regulatory mechanism remains elusive. In this study, two GA signaling factors, CsGAIP (a repressor of GA action) and CsGAMYB1(a positive regulator for GA signaling pathway), were identified from the monoecious cucumber. Through expression, subcellular localization and functional analyses, we researched the regulator mechanism of CsGAIP and CsGAMYB1in male flower formation of cucumber. The main results are as follows:CsGAIP belongs to DELLA family in cucumber, and it contains DELLA and VHYNP domains in the N-terminal region. CsGAIP is predominantly expressed in stems and male flower buds. During male flower development, CsGAIP RNA was found in the inflorescence meristem, floral meristem, vascular strands of the early stage, developing sepals, petals, stamens, carpels of the hermaphrodite stage and microsporocytes, anther wall, pollen grains of the later stages, while the expression in the hermaphrodite stage is the highest. Further, CsGAIP protein is localized in nucleus. Ectopic expression of CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6mutant, as well as inhibit stamen development of wide-type Arabidopsis through transcriptional repression of floral homeotic genes APETALA3(AP3) and PISTILLATA (PI). CsGAMYB1belongs to GAMYB family in cucumber, and it contains an R2R3repeat domain and three conserved motifs Box1, Box2and Box3, which are typical structures in GAMYB family. CsGAMYB1is predominantly expressed in male flower buds of cucumber, where its expression is upregulated by GA3treatment. During flower development, CsGAMYB1RNA was found in the inflorescence meristem, floral meristem of the early stage and developing sepals, petals, stamens, carpels of the hermaphrodite stage. For male flowers, CsGAMYB1is predominately expressed in the microsporocytes, anther wall and pollen grains. For female flowers, the expression of CsGAMYB1is detected in the developing ovary, but the signal is weaker. Further, CsGAMYB1protein is localized in nucleus. CsGAMYB1can partially rescue stamen development and fertility phenotypes of Arabidopsis myb33myb65double mutant. However, overexpression of CsGAMYB1in wild-type Arabidopsis resulted in male sterility with dose-dependent manner. Besides, we generated the CsGAMYB1-RNAi transgenic plants in cucumber and found that knockdown of CsGAMYB1decreases the ratio of nodes with male and female flowers, but no effect on expression of F and M genes and ethylene production, suggesting that CsGAMYB1can regulate sex expression of cucumber, and ethylene is not involved in this process.