The Andromonoecious Sex Determination Gene Predates the Separation of <i>Cucumis</i> and <i>Citrullus</i> Genera

<div><p>Understanding the evolution of sex determination in plants requires the cloning and the characterization of sex determination genes. Monoecy is characterized by the presence of both male and female flowers on the same plant. Andromonoecy is characterized by plants carrying both male and bisexual flowers. In watermelon, the transition between these two sexual forms is controlled by the identity of the alleles at the <i>A</i> locus. We previously showed, in two <i>Cucumis</i> species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, <i>ACS-7</i>/<i>ACS2</i>. To test whether the <i>ACS-7/ACS2</i> function is conserved in cucurbits, we cloned and characterized <i>ClACS7</i> in watermelon. We demonstrated co-segregation of <i>ClACS7</i>, the homolog of <i>CmACS-7/CsACS2</i>, with the <i>A</i> locus. Sequence analysis of <i>ClACS7</i> in watermelon accessions identified three <i>ClACS7</i> isoforms, two in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed and assayed the activity of the three protein isoforms. Like in melon and cucumber, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. <i>ClACS7</i>, like <i>CmACS-7/CsACS2</i> in melon and cucumber, is highly expressed in carpel primordia of buds determined to develop carpels and not in male flowers. Based on this finding and previous investigations, we concluded that the monoecy gene, <i>ACS7</i>, likely predated the separation of the <i>Cucumis</i> and <i>Citrullus</i> genera.</p>