Sex determination in plants involves a variety of mechanisms. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling gene M. M was identified as a previously characterized putative 1-aminocyclopropane-1-carboxylic acid synthase gene, while the m allele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.

It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the ‘one-hormone hypothesis’, which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified ‘one-hormone hypothesis’ for sex determination in cucumber is proposed.

In plants, unisexual flowers derived from developmental sex determination form separate stamens and pistils that facilitate cross pollination. In cucumber and melon, ethylene plays a key role in sex determination. Six sex determination-related genes have been identified in ethylene biosynthesis in these Cucumis species. The interactions among these genes are thought to involve ethylene signaling; however, the underlying mechanism of regulation remains unknown. In this study, hormone treatment and qPCR assays were used to confirm expression of these sex determination-related genes in cucumber and melon is ethylene sensitive. RNA-Seq analysis subsequently helped identify the ethylene responsive factor (ERF) gene, CsERF110, related to ethylene signaling and sex determination. CsERF110 and its melon ortholog, CmERF110, shared a conserved AP2/ERF domain and showed ethylene-sensitive expression. Yeast one-hybrid and ChIP-PCR assays further indicated that CsERF110 bound to at least two sites in the promoter fragment of CsACS11, while transient transformation analysis showed that CsERF110 and CmERF110 enhance CsACS11 and CmACS11 promoter activity, respectively. Taken together, these findings suggest that CsERF110 and CmERF110 respond to ethylene signaling, mediating ethylene-regulated transcription of CsACS11 and CmACS11 in cucumber and melon, respectively. Furthermore, the mechanism involved in its regulation is thought to be conserved in these two Cucumis species.

A novel dwarf cucumber mutant, scp-2, displays a typical BR biosynthesis-deficient phenotype, which is due to a mutation in CsDET2 for a steroid 5-alpha-reductase. Brassinosteroids (BRs) are a group of plant hormones that play important roles in the development of plant architecture, and extreme dwarfism is a typical outcome of BR-deficiency. Most cucumber (Cucumis sativus L.) varieties have an indeterminate growth habit, and dwarfism may have its value in manipulation of plant architecture and improve production in certain production systems. In this study, we identified a spontaneous dwarf mutant, super compact-2 (scp-2), that also has dark green, wrinkle leaves. Genetic analyses indicated that scp-2 was different from two previously reported dwarf mutants: compact (cp) and super compact-1 (scp-1). Map-based cloning revealed that the mutant phenotype was due to two single nucleotide polymorphism and a single-base insertion in the CsDET2 gene that resulted in a missense mutation in a conserved amino acid and thus a truncated protein lacking the conserved catalytic domains in the predicted steroid 5α-reductase protein. Measurement of endogenous hormone levels indicated a reduced level of brassinolide (BL, a bioactive BR) in scp-2, and the mutant phenotype could be partially rescued by the application of epibrassinolide (EBR). In addition, scp-2 mutant seedlings exhibited dark-grown de-etiolation, and defects in cell elongation and vascular development. These data support that scp-2 is a BR biosynthesis-deficient mutant, and that the CsDET2 gene plays a key role in BR biosynthesis in cucumber. We also described the systemic BR responses and discussed the specific BR-related phenotypes in cucumber plants.

No abstract

A 14 bp deletion in CsACS2 gene encoding a truncated loss-of-function protein is responsible for elongated fruit shape and perfect flowers in cucumber. In cucumber (Cucumis sativus L.), sex expression and fruit shape are important components of biological and marketable yield. The association of fruit shape and sex expression is a very interesting phenomenon. The sex determination is controlled primarily by the F (female) and M (monoecy) loci. Homozygous recessive mm plants bear bisexual (perfect) flowers, and the fruits are often round shaped. CsACS2 encoding the 1-aminocyclopropane-1-carboxylic acid synthase has been shown to be the candidate gene for the m locus. We recently identified an andromonoecious cucumber line H38 that has bisexual flowers but elongated fruits. To rapidly clone this monoecious gene in H38, we developed a tri-parent mapping strategy, which took advantage of the high-density Gy14 × 9930 cucumber genetic map and the powder of bulk segregant analysis. Microsatellite markers from the Gy14 × 9930 map were used to screen two pairs of unisexual and bisexual bulks constructed from H38 × Gy14 and H38 × 9930 F2 populations. Polymorphic markers were identified and used to quickly develop a framework map and place the monoecious locus of H38 in cucumber chromosome 1. Further fine mapping allowed identification of a novel allele, m-1, at the monoecious locus to control the bisexual flower in H38, which was due to a 14 bp deletion in the third exon of the CsACS2 gene encoding a truncated loss-of-function protein of the cucumber 1-aminocyclopropane-1-carboxylic acid synthase. This new allele provides a valuable tool in understanding the molecular mechanisms of CsACS2 in the relationships of sex determination, fruit shape, and CsACS activities in cucumber.

Owing to its diverse sex types, the cucumber plant has been studied widely as a model for sex determination. In addition to environmental factors and plant hormones, three major genes-F/f, M/m, and A/a-regulate the sex types in the cucumber plant. By combining the bulked segregant analysis (BSA) and the sequence-related amplified polymorphism (SRAP) technology, we identified eight markers linking to the M/m locus. Among them, the two closely linked SRAP markers flanking the M/m locus were the co-dominant marker ME1EM26 and the dominant marker ME1EM23. Further, the co-dominant marker ME8SA7 co-segregated with the M/m locus. With the chromosome walking method using the cucumber genomic bacterial artificial chromosome (BAC) library, we successfully developed a co-dominant SCAR marker S_ME1EM23 from the ME1EM23 sequence. Along with the other two co-dominant SCAR markers S_ME1EM26 and S_ME8SA7 (developed from ME1EM26 and ME8SA7, respectively) in a larger segregating population (900 individuals), the M/m locus was mapped between S_ME1EM26 (5.4 cM) and S_ME1EM23 (0.7 cM), and S_ME8SA7 co-segregated with it.

Sex determination in plants has various mechanism; however, a single gene locus controlling unisexual expression is unique in the Cucurbitaceae plants, particularly cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). In this study, with quantitative RT-PCR method, two sets of near-isogenic lines (NILs) were used to analyze the expression pattern of gene CsACS2 (GenBank accession number FJ529216). Additionally, chemical applications (AgNO3 and AVG) were used to investigate the effect of the plant endogenous ethylene on CsACS2 expression. Expression analysis reveals that endogenous ethylene, which might be derived from F or M itself, could activate the expression of M gene.