A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species

Abstract

Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, 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 CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.

Figure 1. Sequence analysis of CsACS2.

(A) Schematic diagram of CsACS2 gene structure. The numbers indicate the size of the 3 exons (filled boxes) and the 2 introns (black lines) in bps. The grey boxes indicate the 5′ and the 3′ UTR. (B) Alignment of the melon protein CmACS-7 and cucumber CsACS2. The grey boxes indicate the 8 residues that are polymorphic between CsACS2 and CmACS-7. The blue and red boxes indicate the amino acid changes associated with the andromonoecious phenotype in melon and cucumber, respectively. (C) Relationships between CmACS-7, CsACS2 and ACS from Arabidopsis. CsACS1G, BAA33374 and BAA33375 are ACS from cucumber. The distance tree was produced using ClustalW to align the sequences and using a neighbour-joining algorithm to group them. The length of lines connecting the proteins indicates the mean number of estimated substitutions per site (corrected for multiple substitutions). Scale bar, 0.05 substitution per site.

Figure 2. Sequence variation and co-segregation analysis.

(A) CsACS2 sequence variation in the four parental lines used in the co-segragation analysis. Polymorphic sites indicated below the gene model were positioned relative to the first nucleotide of the start codon. The red colored nucleotide indicates the C to T transition leading to the amino acid substitution P209S. Genotypes of the parental lines, Oman, 319H, Elem Female and Erez are ffMMAA, FFmmAA, FFMMAA and ffMMaa, respectively. M and m genotypes on the left indicate whether the accessions harbour the dominant M or the recessive m alleles at the M locus. (B) Linkage analysis of Female (F), androecious (a) and Monoecious (M) loci with CsACS2. SNP positions are indicated relative to the first nucleotide of the start codon of the genomic CsACS2 sequence and are shown in bold in the panel A. At position 1184 and 1267, Oman cultivar carries an A whereas 319H, Elem Female and Erez cultivars carry a G nucleotide. At position 1391, Oman, Erez and Elem Female carry a C, whereas 319H carries a T. “+” and “np” indicate polymorphic and non polymorphic nucleotide position in the plant crosses, respectively. The number of gametes carrying parental combinations of CsACS2 haplotype and the sex genotype, and the number of gametes exhibiting non parental combinations (“recombinant” gametes) are indicated.

Figure 3. Amino acid mutations associated with andromonoecy are located at the active site of CsACS2.

(A) Amino acid alignments of CsACS2, CmACS-7 and homologous proteins from Vitis vinifera (Vv), Arabidopsis thaliana (At), Lycopersicon esculentum (Le), Petunia hybrida (Ph), Antirrhinum majus (Am), Zea mays (Zm), Picea glauca (Pg). Numbers above the alignment indicate the amino acid positions along the CsACS2 protein. Box 1, Box 4 and Box 7 indicate conserved domains in ACS. The conserved residues between ACS and subgroup 1 aminotransferases and the invariant residue in all aminotransferases are indicated by ⋆ and , respectively. Melon A57V and cucumber G33C, P209S and S399L sequence variations are shown above the alignment (B–D) 3D structure model of CsACS2. (B) Superposition of the tomato ACS structure determined by x-ray crystallography , indicated in white and the 3D models of CsACS2, indicated in blue, and of CmACS-7, indicated in pink. The melon and cucumber models were determined using the Geno3D server (http://geno3d-pbil.ibcp.fr). (C and D) Zoom in of the ACS active site. Ball and stick representations show the cofactor PLP in yellow, the competitive inhibitor AVG in green, and the three amino acids G³³ in red, P²⁰⁹ in orange and S³⁹⁹ in magenta associated with the presence of hermaphrodite flowers in cucumber. The amino acid A⁵⁷, previously shown to be associated with andromonoecy in melon, is represented in cyan .

Figure 4. Biochemical characterization of S²⁰⁹, C³³ and L³⁹⁹ CsACS2 isoforms.

(A–C) Initial velocity measured at 50 µM of PLP. Experimental data (circle) were fit into a line following Michaelis-Menten equation. The resulting parameters are indicated in the table (insert). (D) Enzymatic activity of CsACS2 (black bars), S²⁰⁹ (orange bars), C³³ (red bars) and L³⁹⁹ (magenta bars) protein forms. Specific activities were measured on dialyzed enzymes in the presence of 60 µM SAM and various PLP concentrations.

Figure 5. Expression analysis of CsACS2.

Expression of CsACS2 in different organs was determined using quantitative real-time PCR, relative to CsACTIN2 standard. The mean±s.d. of four biological replicate experiments is shown. L, Leaf; S, Stem; Mfl, Male flower; Hfl, Hermaphrodite flower and Ffl, Female flower. Cucumber accessions and their respective genotypes are indicated below the graph.