Differential Gene Expression Caused by the F and M Loci Provides Insight Into Ethylene-Mediated Female Flower Differentiation in Cucumber

Abstract

In cucumber (<i>Cucumis sativus</i> L.), the differentiation and development of female flowers are important processes that directly affect the fruit yield and quality. Sex differentiation is mainly controlled by three ethylene synthase genes, <i>F</i> (<i>CsACS1G</i>), <i>M</i> (<i>CsACS2</i>), and <i>A</i> (<i>CsACS11</i>). Thus, ethylene plays a key role in the sex differentiation in cucumber. The "one-hormone hypothesis" posits that <i>F</i> and <i>M</i> regulate the ethylene levels and initiate female flower development in cucumber. Nonetheless, the precise molecular mechanism of this process remains elusive. To investigate the mechanism by which <i>F</i> and <i>M</i> regulate the sex phenotype, three cucumber near-isogenic lines, namely H34 (<i>FFmmAA</i>, hermaphroditic), G12 (<i>FFMMAA</i>, gynoecious), and M12 (<i>ffMMAA</i>, monoecious), with different <i>F</i> and <i>M</i> loci were generated. The transcriptomic analysis of the apical shoots revealed that the expression of the B-class floral homeotic genes, <i>CsPI</i> (<i>Csa4G358770</i>) and <i>CsAP3</i> (<i>Csa3G865440</i>), was immensely suppressed in G12 (100% female flowers) but highly expressed in M12 (∼90% male flowers). In contrast, <i>CAG2</i> (<i>Csa1G467100</i>), which is an AG-like C-class floral homeotic gene, was specifically highly expressed in G12. Thus, the initiation of female flowers is likely to be caused by the downregulation of B-class and upregulation of C-class genes by ethylene production in the floral primordium. Additionally, <i>CsERF31</i>, which was highly expressed in G12, showed temporal and spatial expression patterns similar to those of <i>M</i> and responded to the ethylene-related chemical treatments. The biochemical experiments further demonstrated that <i>CsERF31</i> could directly bind the promoter of <i>M</i> and promote its expression. Thus, <i>CsERF31</i> responded to the ethylene signal derived from <i>F</i> and mediated the positive feedback regulation of ethylene by activating <i>M</i> expression, which offers an extended "one-hormone hypothesis" of sex differentiation in cucumber.