The Andromonoecious Sex Determination Gene Predates the Separation of Cucumis and Citrullus Genera

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 A locus. We previously showed, in two Cucumis species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, ACS-7/ACS2. To test whether the ACS-7/ACS2 function is conserved in cucurbits, we cloned and characterized ClACS7 in watermelon. We demonstrated co-segregation of ClACS7, the homolog of CmACS-7/CsACS2, with the A locus. Sequence analysis of ClACS7 in watermelon accessions identified three ClACS7 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. ClACS7, like CmACS-7/CsACS2 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, ACS7, likely predated the separation of the Cucumis and Citrullus genera.


Introduction
The majority of angiosperms are hermaphroditic producing exclusively bisexual flowers.Sex determination is a developmental evolutionary process that leads to unisexual flowers in 10% of the species [1].Monoecious species (5-6%) exhibit male and female flowers on the same plant.Dioecious species (5-6%) have separate male and female individuals.There has been concerted effort in the last few decades to pinpoint the master genes controlling sex determination and to understand the evolutionary forces that drive transitions between sexual morphs [2,3].In these investigations, several plant families have emerged as model systems [4][5][6][7].The choice of the Cucurbitaceae is justified by the wide spread of sex among the species of this plant family.Out of 800 investigated species, 460 are monoecious and 340 are dioecious.Throughout evolution of Cucurbitaceae, there have been also numerous shifts between monoecy and other sexual morphs, which makes this plant family a practical model to investigate the molecular mechanisms controlling sex determination.
In monoecious Cucurbitaceae species, such as Cucumis melo, floral primordia are initially bisexual with sex determination occurring by the selective developmental arrest of either the stamen or the carpel, resulting in unisexual flowers [8].Cloning and characterization of natural mutations leading to transition from monoecy to different sexual morphs in melon and cucumber has brought new insight to the monoecy sex determination pathway.Three master sex genes, Monoecious, Androecious and Gynoecious genes, interact to explain the gender and the position of the flower on the plant [9][10][11][12].In this model the expression of the carpel inhibitor, CmWIP1, encoded by the gynoecious gene, is dependent on non-expression of CmACS11, encoded by the androecious gene.The expression of the stamina inhibitor, CmACS-7, encoded by the monoecious gene is dependent on non-expression of CmWIP1.In monoecious and andromonoecious plants, male flowers develop because of non-expression of CmACS11 that permits the expression of CmWIP1.Although sex determination occurred many times independently during evolution [1,13,14], one would expect that related species use common sexual mechanisms.We previously showed that in melon and cucumber, two species that diverged more than 10 million years ago (Mya) share the same pathway leading to monoecy [10].For instance the melon and the cucumber monoecious gene are orthologs and encode for the rate-limiting enzyme in ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylic acid synthase (ACS).In both species, the ACS genes, referred to as CmACS-7 in melon and CsACS2 in cucumber, are expressed in carpel primordia, and loss of enzymatic activity leads to stamen development [10].
Similarly, the melon and the cucumber androecious genes that encode for an ACS are also orthologs [11].In both species, the ACS genes, referred to as CmACS11 in melon and CsACS11 in cucumber, are expressed in carpel primordia, and loss of enzymatic activity leads to stamen development [11].
In melon and cucumber, two species of the Cucumis genus, our previous studies [9,10] have shown that the sexual transition from monoecy to andromonoecy results from loss-of-function mutations in CmACS-7 and CsACS2, respectively.In watermelon, a monoecious cucurbit, the recessive a locus controls the andromonoecious sexual phenotype and acts as CmACS-7 and CsACS2 [15].Recently, a QTL analysis has shown that the major QTL controlling the watermelon andromonoecy is located on chromosome 3 close to the Cla011230 gene encoding an ACC synthase enzyme [16].
In the present study, we investigated whether components of the monoecy pathway are conserved between the Cucumis and Citrullus genus that diverged 20 Mya [17].To test this hypothesis, we cloned the andromonoecious (a) gene from Citrullus lanatus (watermelon) and characterized natural mutations leading to sexual transition between monoecy and andromonoecy.We showed that, like in melon and cucumber, an ACS gene, ClACS7, inhibits stamina development in female flowers and loss of enzymatic activity leads to stamen development.ClACS7 is syntenic to CmACS-7 in melon and CsACS2 in cucumber.Based on this finding, we concluded that the ACS7 function is ancestral to the divergence of the Cucumis and the Citrullus genus.

Plant material and segregating population
The genetic linkage analysis of the A locus and ClACS7 was carried out using a segregating population obtained from a cross between a monoecious accession, Citrullus lanatus var.citroides, PI 296341, and an andromonoecious Citrullus lanatus var.lanatus variety Halep Karasi.The resultant F1 was backcrossed with Halep Karasi.
Plants were grown in the greenhouse in summer at Montfavet, France, under standard agronomic conditions and evaluated for the sex of their flowers at each node along the main stem and side branches.

Plant genotyping
To identify plants carrying recombination events, plant DNA was extracted from each individual of the backcross segregating population.Determination of the sexual phenotype was performed for all plants.For genotyping, a 556 bp DNA fragment of the ClACS7 gene was amplified with primers listed in S1 Table and sequenced.Polymorphisms within this fragment were used to determine recombinant alleles.

Sequence analysis
Multiple sequence alignment of full-length protein sequences of ACS was performed using the ClustalW (http://www.ebi.ac.uk/Tools/clustalw2). Phylogenetic trees were constructed by MEGA6 (http://www.megasoftware.net/index.html)based on the Neighbor-Joining method.The three-dimensional structure predictions were generated using the Geno3D server (http:// geno3d-pbil.ibcp.fr)and the visualization was carried out using the Chimera server (http:// www.cgl.ucsf.edu/chimera).

Quantitative RT-PCR experiments
Total RNA was extracted from frozen leaves, stems and flowers with the Trizol reagent (Invitrogen, France).First strand cDNA synthesis and primer design were performed as decribed in [11].Primers sequences are listed in S1 Table .To check specificity of the designed primers, all amplicons were sequenced and blasted against NCBI database.Quantitative RT-PCR reactions were performed as described in [11].A negative control without cDNA, technical replicates on three independent synthesis of cDNA (derived from the same RNA sample), and three independent biological experiments were performed in all cases.To compare data from different PCR runs and cDNA samples, CT values for ClACS7 were normalized to the CT value of ClActin2 (primers shown in S1 Table ).ClACS7 relative expressions were determined as described in [9].

In situ hybridization
ClACS7 in situ hybridisation was performed as described in [18].Primers used for this experiment are listed in S1 Table.
The ClACS7 enzymes expression and purification were performed as described in [9], using BL21(DE3)pLYS cells expressing the pClACS7 constructs.

Enzyme activity assays
Enzyme activity was determined by monitoring the MTA formation by differential spectroscopy recorded on an Uvikon 940 spectrophotometer (Biotek-Kontron Instruments, France) as according to [19].Namely, specific activity measurement were performed on 3 different enzyme preparations by incubating 60 μM SAM in 100 mM KPhos buffer pH 8.5 (0.2 ml) and deaminase (8 μg for 0.2 ml) in the presence of 5 μM PLP in each quartz cuvette for 3 min at 25°C after addition of the purified enzyme (1 to 2 μg).The conversion of the MTA produced by ACC into an inosine derivative was monitored at 265 nm (Δε = -7740 M -1 .cm - ) and the specific activity was expressed as nmol of MTA formed per min per mg of protein.

Cla011230/ClACS7 is the watermelon orthologue of CmACS-7/CsACS2
To test whether the genetic determinant controlling monoecy to andromonoecy sexual transition is conserved between the Citrullus and Cucumis genus and whether the watermelon ortholog of CmACS-7/CsACS2 could be encoded by the watermelon A gene, we isolated all the ACS genes from melon, cucumber and watermelon.Both cucurbit genomes contain 8 ACS genes and phylogenetic analysis determined that Cla011230, hereafter ClACS7, CmACS-7 and CsACS2 define a monophyletic clade (Fig 1A).ClACS7 encodes an ACC synthase of 444 amino acids that share 92% amino acid sequence identity with CmACS-7 and CsACS2 and all the polymorphic residues are located in non-conserved positions (S1 Fig) .Sequence analysis shows that ClACS7, CmACS-7 and CsACS2 are highly synthenic and share conserved gene structure with 3 exons and 2 introns (Fig 1B and 1C).Based on this, we concluded that ClACS7 is most likely the watermelon ortholog of CmACS-7 and CsACS2.versus andromonoecy in watermelon, melon and cucumber, the high sequence identity between ClACS7, CmACS-7 and CsACS2 and the co-segregation of the A gene with ClACS7, we concluded that it is likely that the A gene encodes for ClACS7.

Loss of ClACS7 enzymatic activity leads to andromonoecy
To understand the basis of allelic differences in the A gene, we compared the sequence of ClACS7 in 43 watermelon accessions (Fig 3) selected based on their sexual types.All tested monoecious accessions harbouring the dominant A allele presented the same ClACS7 protein sequence.In contrast, analysis of the sequence obtained from the andromonoecious accessions, harbouring the recessive a allele, revealed three missense mutations, C364W, N264K and L395F leading to two ClACS7 isoforms, C364W and N264K_L395F (Fig 3).When the closest ClACS7 homologous sequences from different plants were aligned, we observed that the residues C 364 and L 395 are conserved across seed plants whereas N 264 is located in a non-conserved region of the protein (Fig 4A) [20].Crystallographic studies have determined that C 364 and L 395 residues are located in close proximity to the active site (Fig 4B -4D) [21].The L 395 residue is located in the conserved box 7 that contains residues involved in the binding of the enzyme substrate SAM (Fig 4B -4D) [21].Surprisingly, the L 395 is located at 3 amino acids of the S 399 and the S399L mutation was previously described as a natural andromonoecious m allele in cucumber [10].
To determine whether hermaphrodite flowers in andromonoecious plants are due to loss of a ClACS7 enzymatic activity, we expressed the different forms of the protein as six-histidine tag (His 6 -ClACS7) fusion proteins in Escherichia coli.The purified proteins were assessed for enzymatic activity in vitro by monitoring 5'-methylthioadenosine (MTA) formation at different PLP concentrations.The K 264 F 395 isoform was found to be totally inactive (Fig 4E and 4F, Table 1).In the presence of high concentrations of PLP (100μM), the W 364 and the ClACS7 isoforms share similar biochemical constants (Table 1).However, at a physiological PLP concentration [9], the W 364 isoform has a reduced enzymatic activity; its V max is approximately 25% of the V max of the ClACS7 isoform (Fig 4E , Table 1).For the K 264 F 395 protein form, to determine whether the amino acid substitution of the conserved residue L 395 is responsible for the inactivation of the enzyme, we expressed and assessed the enzymatic activity of the F 395 isoform.Like the W 364 isoform, at a physiological PLP concentration, the F 395 isoform has a reduced enzymatic activity whereas at 100μM PLP, the F 395 and ClACS7 isoforms have similar biochemical constants (Fig 4E , Table 1).The fact that the L395F amino acid substitution does not lead to a complete ClACS7 enzymatic inactivation suggests that the N264K substitution could also reduce enzymatic activity and thus the complete loss of enzymatic activity observed in the K 264 F 395 isoform is due to a synergic effect of the N264K and L395F substitutions.
Based on this and because the ClACS7 isoform from the monoecious lines was active and isoforms from the andromonoecious lines showed reduced to no enzymatic activity, we concluded that the loss of ClACS7 enzyme activity is likely the cause of the development of hermaphrodite flowers in watermelon and that ethylene production by ClACS7 is necessary for the developmental inhibition of the male organs in the female flowers of monoecious watermelon.This conclusion is reinforced by recent experiments showing that monoecious watermelons treated with an ethylene biosynthesis inhibitor, the 1-aminoethoxyvinylglycine (AVG), develop hermaphrodite flowers [22].Moreover, these data show that, in watermelon, the andromonoecy evolved from monoecy, independently in wild C. lanatus var.citroides accessions and in cultivated C. lanatus var.lanatus.

ClACS7 is expressed in carpel primordia of female and hermaphrodite flowers
Quantitative real-time PCR analysis demonstrates that ClACS7 is highly expressed in female and hermaphrodite flower buds and it is weak or undetectable in buds determined to develop into male flowers.No ClACS7 expression was detected in vegetative tissues (Fig 5A).To investigate in more detail the spatial expression pattern of ClACS7, we carried out in situ hybridizations.ClACS7 mRNA was detected in female and hermaphrodite flower buds at stages where  buds are not morphologically distinguishable.ClACS7 mRNA was strongly localised in the carpel primordia (Fig 5C and 5E).In the male flowers of the monoecious or andromonoecious watermelon lines, no ClACS7 transcript was detected (Fig 5B and 5D).Since ClACS7 is expressed in both female and hermaphrodite flowers (Fig 5 ) and because the loss of ClACS7 activity accounts for the functional variation between the monoecious and andromonoecious watermelon lines (Fig 4 ), we concluded that ClACS7-mediated ethylene production in the carpel primordia prevents the development of the stamina in female flowers and is also not required for carpel development.
Moreover, since the watermelon ClACS7, the melon CmACS-7 and the cucumber CsACS2 have the same expression pattern and since the loss of ClACS7, CmACS-7 and CsACS2 enzymatic activities lead to the sexual transition from monoecy to andromonoecy, we concluded that ClACS7, CmACS-7 and CsACS2 are regulated by mechanisms common to the Citrullus and Cucumis genus and that ethylene signalling and response mechanisms leading to inhibition of stamina development in female flowers of monoecious plants are conserved in the two genera, Citrullus and Cucumis.

Discussion
Most of the species of the Cucurbitales order have unisexual flowers and are mostly monoecious (>75% of the species) bearing both male and female flowers on the same plant.Andromonoecy, a major sexual system in which plants carry both male and perfect bisexual flowers, has evolved independently numerous times [23] and is found in ~4,000 species in 33 angiosperm families [24].Investigations on the genetic inheritance of the different sexual morphs in the Cucurbitaceae plant family have described that the andromonoecious trait is controlled by a recessive monogenic locus [5,18,25,26].In our previous studies, we have demonstrated that the sexual transition from monoecy to andromonoecy, in melon and cucumber, is controlled by the common gene CmACS-7/CsACS2 [9,10].In this context, the genetic determinant controlling the monoecy is likely to have been established before the speciation of melon and cucumber, two species of the genus Cucumis that have diverged over 10 My ago [17].
In this work, we described the isolation and characterization of the watermelon, Citrullus lanatus, andromonoecious (a) locus and showed that the (a) locus encodes for a rate-limiting enzyme in ethylene biosynthesis, ClACS7, the orthologous gene of the melon CmACS-7 and cucumber CsACS2.Sequence analysis of monoecious and andromonoecious watermelon accessions revealed two ClACS7 isoforms in the andromonoecious lines, the C364W and the N264K_L395F.Based on the kinetic parameters of the ClACS7 protein isoforms, 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 in andromonoecious lines.Expression pattern analysis showed that ClACS7 is specifically expressed in carpel primordia of female and hermaphrodite flowers and no expression was detected in male flowers and the vegetative tissues, leaves and stems.On the basis of enzymatic activity and gene expression studies and as previously described in melon and cucumber [9,10], we concluded that ClACS7-mediated ethylene production in the carpel primordia prevents the development of the stamina in female flowers and is also not required for carpel development.
Altogether, the conserved gene structure, the genomic synteny, the conserved expression patterns and the fact that ClACS7, CmACS-7 and CsACS2 loss-of-function activities lead to hermaphrodite flower development suggest that ClACS7, CmACS-7 and CsACS2 derive from a common ancestral gene that controls monoecy before the divergence of the Citrullus and Cucumis genera, 20 My ago [17].
Recently, a study on sex determination in squash, Cucurbita pepo, has linked the partial andromonoecy phenotype, i.e. an incomplete conversion of female into hermaphrodite flowers, to the CpACS27A gene, an ACS gene homologous to CmACS-7 and CsACS2 [27].The similar expression pattern in the carpels of carpel-bearing flowers (female and hermaphrodite) suggest that ClACS7, CmACS-7, CsACS2 and CpACS27A share common regulatory mechanisms along evolution and that monoecy is controlled by an ancestral gene that precedes the Cucumis, Citrullus and Cucurbita genus divergence 30 My ago [17].
Female flowers develop on the branches because of expression of CmACS11 that represses the expression of CmWIP1, thus releasing the expression of CmACS-7 that inhibits the stamina development.If nonfunctional CmACS-7 is expressed, hermaphrodite instead of female flowers develop.Androecious plants are obtained by loss of function of CmACS11 that leads to expression of CmWIP1 in all the flowers of the plants.Gynoecious plants are obtained by inactivation of CmWIP1 function.Hermaphrodite plants are obtained by inactivation of CmWIP1 and CmACS-7 [9-12].
To map ClACS7 relative to the andromonoecious (a) locus, we first sequenced the ClACS7 gene in four watermelon lines, Sugar Baby, Charleston Grey, PI 296341 which are monoecious (A) harboring male and female flowers(Fig 2A and 2B), and Halep Karasi, an andromonoecious (a) line harboring male and hermaphrodite flowers (Fig 2A and 2C).PI 296341 and Halep Karasi were used to generate the mapping population.Among the four accessions, we observed 19 DNA polymorphisms in the introns and 7 in the exons (Fig 2D).Out of the 7 polymorphisms in

Fig 1 .
Fig 1.  Phylogeny relationship of cucurbit ACS protein family and syntenic and gene structure analysis of watermelon, ClACS7, melon, CmACS-7, and cucumber, CsACS2, genes.(A) Using the ClACS7 protein sequence as the query in tBLASTn searches, ACS protein family members were identified from watermelon, cucumber and melon.Protein sequences were aligned and used to generate the neighborjoining phylogenetic tree with 1,000 bootstrap replicates.The percentage of replicate trees in which the

Fig 2 .
Fig 2. ClACS7 sequence variation and co-segregation analysis.(A-C) watermelon flower types from monoecious and andromonoecious plants.Monoecious watermelon bears male (A) and female (B) flowers whereas andromonoecious bears male (A) and hermaphrodite (C) flowers.sg, stigma; st, stamen.(D) ClACS7 sequence variation in monoecious and andromonoecious watermelon lines used in the cosegregation 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 G transition leading to the amino acid substitution C364W.Genotypes of the parental lines, PI 296341 and Halep Karasi are AAGyGy and aaGyGy, respectively.A and a genotypes on the left indicate whether the accessions harbour the dominant A or the recessive a alleles at the A locus.(E) Linkage analysis of andromonoecious (A) locus with ClACS7.The observed sexual phenotype, the number of plants, the genotype at the SNP 1477 and the genotypephenotype linkage frequency are indicated.doi:10.1371/journal.pone.0155444.g002

Fig 3 .
Fig 3. Analysis of ClACS7 DNA sequence variation in 43 watermelon accessions representing all the sexual morphs of the species.Polymorphic sites indicated below the gene model were positioned relative to the first nucleotide of the start codon.The red boxes point out the nucleotide polymorphisms leading to the missense mutations, N264K, C364W and L395F leading to andromonoecious sexual morph.Presence of male, female or hermaphrodite flowers in the accessions is indicated by (x).doi:10.1371/journal.pone.0155444.g003

Fig 4 .
Fig 4. Molecular characterization of ClACS7 alleles.(A) Amino acid alignments of ClACS7 isoform with homologous proteins from Cm (Cucumis melo), Cs (Cucumis sativus), Vv (Vitis vinifera), At (Arabidopsis thaliana), Le (Lycopersicum esculentum), Ph (Petunia hybrida), Zm (Zea mays) and Pg (Picea glauca).Numbers above the alignment indicate the amino acid positions along the ClACS7 protein.Box 7 indicates a conserved domain in ACS proteins.N264K, C364W and L395F amino acid polymorphisms are shown above the alignment.(B-D) Model of the three-dimensional structure of ClACS7.(B) Superposition of the tomato ACS 3D crystal structure (1IAY.pdb)indicated in blue and the 3D model of ClACS7 indicated in pink.The threedimensional structure were generated using the Geno3D server (http://geno3d-pbil.ibcp.fr)and the visualization was carried out using the Chimera server (http://www.cgl.ucsf.edu/chimera).(C and D) Zoom in on the active site of ClACS7.The residues that are polymorphic in the different ClACS7 isoforms are depicted in red.The cofactor PLP is indicated by purple ball and stick.The competitive inhibitor AVG (aminoethoxyvinylglycine) is indicated by yellow ball and stick.(E and F) Biochemical characterization of ClACS7, W 364 , F 395 and K 264 F 395 enzyme isoforms.(E) Enzymatic activity of ClACS7 (black bars), W 364 (pink bars), F 395 (orange bars) and K 264 F 395 (blue bars) protein forms.Specific activities were measured on dialyzed enzymes in the presence of 60 μM SAM and various PLP concentrations.(F) Initial velocity of the ClACS7 isoforms measured at 50 mM of PLP.Experimental data were fit into a line following Michaelis-Menten equation.doi:10.1371/journal.pone.0155444.g004

Fig 5 .
Fig 5. Expression patterns of ClACS7 in watermelon flowers.(A) Quantitative RT-PCR of ClACS7 in reproductive organs, male or female flower buds from monoecious watermelon and male or hermaphrodite flower buds from andromonoecious watermelon, collected at stage 4, and in the vegetative tissues, leaves (L) and stems (S); shown are the mean ± standard deviation (s.d.) of three biological replicates.(B-E) ClACS7 in situ expression in flower buds at stages 4 in male (B) and female (C) flowers of monoecious watermelon and male (D) and hermaphrodite (E) flowers of andromonoecious watermelon.C, Carpel; St, Stamen; P, Petal; S, Sepal.Bars, 100 μm.doi:10.1371/journal.pone.0155444.g005

Table 1 .
The kinetic parameters, Km and Vm, measured at 5μM and 100μM PLP for ClACS7, W 364 , K 264 F 395 and F 395 protein isoforms.