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Fruit plants are an important source of vitamins and inorganic salts, and contain a variety of biologically active substances. To date, only persimmons (Diospyros lotus) and kiwifruits (genus Actinidia) have had their sex determinants uncovered with their functions verified experimentally. Some other fruit crops, including grape (Vitis vinifera), papaya (Carica papaya), date palm (Phoenix dactylifera), and strawberry (Fragaria virginiana), are being investigated in order to identify their sex-determination regions and candidate sex-determination genes.
Persimmon
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Persimmons originated in China and have been cultivated for > 1,000 years. In 2014, Japanese scholars identified the genes determining the sex of persimmon plants using diploid D. lotus[5]. Diploid persimmons are dioecious and either androecious (chromosomes XX) or gynoecious (chromosomes XY)[6]. Through de novo whole-genome sequencing and transcriptome approaches, the researchers identified a Y-specific non-coding gene OGI expressed in male flowers[5]. OGI produces male-specific 21-nt small RNAs that specifically target its homologous gene MeGI, a class I homeodomain transcription factor (HD-ZIP) (Fig. 1a)[5]. MeGI is expressed at high levels in the buds and flowers of female plants, sterilizing the androecia and promoting the formation of female flowers (Fig. 1a)[5]. In male flowers, small RNA produced by OGI on the Y chromosome triggers transitive and persistent small RNA production from MeGI, which in turn represses mRNA production of MeGI and allows for the normal development of stamens and the formation of male flowers (Fig. 1a)[5]. Two years later, Akagi et al.[7] identified an epigenetic mechanism regulating sex determination in hexaploid persimmons (D. kaki). In hexaploid persimmons, plants containing Y chromosomes have both female and male flowers. OGI expression is undetectable in developing male flowers, potentially due to the presence of a 268 bp short interspersed nuclear element (SINE)-like insertion in the OGI promoter region of the Y chromosome. However, in male flowers, methylation of the MeGI promoter can activate MeGI small RNA (smMeGI) production, which represses MeGI expression and in turns results in male flower development[7]. Occasional sex reversal from male to female may originate from spontaneous demethylation of the MeGI promoter[7]. Since methylation can be accumulated and reset, the sex of the flowers on the offspring of hexaploid persimmons can change[7]. Hexaploid persimmons, then, have more flexible flower sex determination than do diploid persimmons because of the involvement of DNA methylation. In 2020, Akagi et al.[8] found that MeGI (Chr.13) was derived from its homologous gene Sister of MeGI (SiMeGI) (Chr.4) through whole-genome duplication, suggesting that MeGI acquires a new function as a repressor of stamen development, while SiMeGI still maintains the original function. After that, segmental duplication events produced OGI, the regulator of the MeGI gene, on the Y chromosome, thus completing the path to dioecy[8]. The discovery of the OGI-MeGI system in persimmons provides a good reference for studying other dioecious plants.
Figure 1.
Horticultural crops with known sex-determining genes. (a) The sex-determining genes of persimmon. (b) The sex-determining genes of kiwifruit. (c) The sex-determining genes of asparagus.
Kiwifruit
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Kiwifruit, a fruit consumed worldwide, is native to China and belongs to the genus Actinidia of Actinidiaceae. Most Actinidia species are dioecious with XY males and XX females[9]. Akagi et al.[9,10] identified a female sterility factor, Shy Girl (SyGI), and a male promoter gene, Friendly Boy (FrBy), as sex determinants of kiwifruit. Both genes are encoded in the male-specific region of the Y chromosome (MSY). SyGI is a type-C cytokinin response regulator that is specifically expressed in the rudimentary carpel in developing male flowers (Fig. 1b)[9]. By inhibiting the development of the carpel in male flowers, SyGI acts as a dominant suppressor in female fertility[9]. FrBy exhibits strong expression in tapetal cells during the early stages of anther development[10]. Gene-editing and complementation analyses in Arabidopsis thaliana and Nicotiana tabacum indicated that FrBy acts on male maintenance independently of the SyGI gene[10]. Hermaphrodite kiwifruit can be formed through FrBy gene expression in female kiwifruit[10]. This clearly shows that SyGI and FrBy on the Y chromosome act independently as the female suppressor SuF and the male promotor M in kiwifruit (Fig. 1b)[10].
Grapevine
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Grapevine is a long-lived perennial plant and is one of the most important fruit crops in the world. Wild grapevine (V. vinifera ssp. sylvestris) is dioecious with XY chromosomes, while cultivated grapevine (V. vinifera ssp. vinifera) has reverted to hermaphroditism[11]. The evolution of hermaphroditism was key in the domestication of cultivated grapevine, reflecting the influence of artificial selection pressure on plant sex differentiation[12]. Sex determination in grapevine is proposed to be controlled by a major locus containing three alleles, including male gene M, female gene F, and hermaphroditic gene H, and the dominant epistasis between alleles is M > H > F[13]. Various genetic studies have shown that a region across ~150 kb on chromosome 2 is the sex determination region of grapevine[12−16]. By analyzing 20 Vitis SDR haplotypes and the associated gene expression data, Massonnet et al.[16] identified a recessive allele of VviINP1 as the candidate gene for male sterility, and the M allele of VviYABBY3 as the gene for female sterility. In contrast, Badouin et al.[14] suggested that the upregulation of VviAPT3 on the Y chromosome causes female sterility, and the downregulation of VviAPT3 on the Yh chromosome may trigger reversal to hermaphroditism, thus VviAPT3 is the candidate gene for female sterility. Zou et al.[12] reported results consistent with VviINP1 and VviYABBY3 being the genes determining male sterility and female sterility, respectively, after investigating the pattern of sex-linked SNPs and chromosome painting of these SNPs coupled with allele-specific transcriptome analysis (Table 1). The above results are based primarily on comparative genomic analyses, and should be verified further using biological experiments.
Table 1. The sex-related gene with known function in horticultural crops.
Horticulture
plantsGene name Organs with high expression level Gene function Persimmon OGI Male flowers Promoting the development of stamens MeGI Buds and flowers of female Promoting the formation of female flowers Kiwifruit SyGI The rudimentary carpel of developing male flowers Inhibiting the development of carpel in male flowers FrBy Tapetal cells during early stage of anther development Male maintenance Grapevine VviINP1 Female flowers The candidate gene for male sterility VviYABBY3 Male flowers The candidate gene for female sterility Cucumber CsACS1G (F) Early stages of flower buds development Promoting the formation of female flowers CsACS11 (A) Phloem cells connected to flowers programmed to become female Promoting carpel development CsWIP1 Male flowers Inhibiting carpel development and promoting male flower development CsACS2 (M) Flower buds at different stages of development Inhibiting stamen development CsACO2 Carpel primordia Promoting carpel development Melon CmASC11 Vascular bundles of female flowers Promoting carpel development in female flower CmWIP1(g) The carpel primordia of male flowers Leading to carpel abortion and promoting stamen development CmACS-7(a) The carpel primordia of female and hermaphrodite flowers at the early stages Inhibiting stamens development Watermelon ClWIP1(gy) Carpel primordia Leading to carpel primordia abortion ClACS7 Carpel primordia of flower buds that are determined to develop into full carpels Promoting the development of carpels CitACS4(a) Carpel primordia in pistillate flowers Inhibiting of stamen development Pumpkin CpWEI Undefined Inducing female flower development in undifferentiated asexual buds Asparagus SOFF Flower buds in supermale (weakly) Suppressing female organogenesis aspTDF1 Anther tapetum Promoting anther development Papaya
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Papaya is major fruit crop growing in tropical and subtropical regions, and is a trioecious species with male, female, and hermaphrodite flowers on separate plants[17]. The sex determination system in papaya is particularly intriguing, not only because it has three sex types, but because it shows frequent sex reversal caused by environmental factors[18]. Genetically, papaya sex determination is controlled by three chromosomes; in addition to the X and Y chromosomes, papaya has a unique sex chromosome Yh, such that the genotype of males is XY, females is XX, and hermaphrodites is XYh[18]. In 2008, scientists found that DNA methylation and heterochromatinization played an important role in the early stage of evolution of the papaya Y chromosome, and proposed that Yh chromosomes were likely present at the initial stage of sex differentiation[19]. The male-specific region of the Y chromosome (MSY), which determines male flower development, was estimated to account for only 10% of the Y chromosome[20]. The hermaphrodite-specific region of Yh (HSY) determines hermaphrodite flower development[21]. In 2015, the HSY and MSY sequences were aligned and their sequence similarity was estimated to be 99.60%[22]. Further analysis showed that the Yh chromosome originated from the Y chromosome and arose approximately 4,000 years ago, well after plant domestication in Mesoamerica (> 6,200 years ago), but coinciding with the rise of Maya civilization[22]. Therefore, it was speculated that the evolution of the Yh chromosome resulted from the domestication of papaya[22]. Both MSY and HSY are approximately 8.1 Mb from the LG1 (Linkage Group 1) centromere, and recombination with the X chromosome is suppressed[20,21]. Urasaki et al.[17] found a MADS-box transcription factor specific to the Y and Yh chromosomes which is expressed only in male and bisexual plants. The MADS-box gene encodes a protein with 85% similarity to the Short Vegetative Phase (SVP) protein in Arabidopsis, a well-known transcriptional regulator of flowering time. By comparing the sequences of MSY and HSY, it was found that the SVP-like gene (CpSVPL) on the Y chromosome encodes a complete protein, but on the Yh chromosome encodes an incomplete protein because of transposon insertion[23]. Furthermore, CpSVPL had one SNP associated with the three sex genotypes, and was highly expressed in the male and female sterile flowers (abnormal hermaphrodite flowers), which lacked the fourth whorl structure[24]. These results suggest that the SVP-like gene is a candidate gene for sex determination in papaya, but further analysis, including gene knockout in the male or overexpression in the female plants, is required to definitively determine its role.
Other fruit plants
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Sex determination has been investigated in many other fruit plants, but specific sex-determination genes have not been fully identified. In 2019, researchers decoded the red bayberry (Morella rubra) genome by combining genome sequencing and transcriptome sequencing, and confirmed that its sex determination followed the ZW model[25]. In this system, the sex is determined by the genotype of the egg cell, and males are homogametic (ZZ) and females are heterogametic (ZW)[25]. A 59 kb female-specific region (FSR) located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, was identified and cloned. Further transcriptome data revealed that four female-specific genes (MrCPS2, MrASP2, MrSAUR2, and MrFT2) in the FSR region were expressed only in female flower buds. MrCPS2 and MrASP2 were most highly expressed during the female flower initiating stage, whereas MrSAUR2 and MrFT2 were most highly expressed during the flower primordium formation period. This demonstrated that MrCPS2 and MrASP2 were key initiating factors[25].
The date palm (Phoenix dactylifera L.) is one of the most economically important crops in North Africa, the Middle East, and South Asia[26]. The date palm is dioecious, with separate male and female trees. The sex determination region was found in the approximately 5–13 Mb region of the long arm of LG12[26,27]. Torres et al.[28] sequenced the genomes of 15 female and 13 male Phoenix trees representing all 14 species, and found that only four genes contained sequences conserved in all Phoenix males. Among them, CYP703 and GPAT3 are male-specific genes and are critical for male flower development, while a LOG-like gene appears translocated into the Y-linked region and may play a role in suppressing female flowers. The remaining gene encodes a cytidine deaminase and was not expressed in male or female flowers. The role of these genes in date palm and other plants of Phoenix requires further investigation.
The female sex chromosome in strawberry is ZW, and the male sex chromosome is ZZ. In 2013, Tennessen et al.[29] identified the sex-determination region of the gynodioecious diploid wild strawberry (Fragaria vesca ssp. bracteate), and found that male-sterile genes were located in a gene-dense 338 kb region of chromosome 4. Then, in 2015, Ashman et al.[30] found a new sex-determination region in a 1.769 Mb region on chromosome 6. In the absence of the MS allele at the LG4 locus (identified by Tennessen et al.[29]), a dominant R allele located at the novel locus fine-mapped on LG6 can restore fertility[30]. In 2018, a 13 kb female-specific fragment which is a conserved, mobile W-specific SDR was identified in wild North American octoploid strawberries (Fragaria)[31]. The SDR could be translocated repeatedly, increasing the size of the female-specific hemizygous sequence on the W sex chromosome and revealing a new potential mechanism for expansion and diversification of incipient sex chromosomes[31].
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In recent years great progress has been made in our understanding of flower sex determination. With the rapid development of genetic and genomic technologies, key sex-determination genes have been discovered in a few economically important horticultural plants. In view of this progress, research on the following topics will be of significance in the future:
Development of model plant systems for the study of flower sex differentiation. The mechanism of sex differentiation in plants is complex. Although the community has made great progress in understanding this mechanism, plants from different families or lineages seem to have evolved different strategies and different genes associated with distinct reproductive pathways. For instance, the kiwifruit, persimmon, and asparagus all have different sex determination systems. It is difficult to dissect the underlying codes, especially for perennial fruit trees, which take years to flower for the first time. Identifying model plants to study flower sex formation is a good way to circumvent this obstacle. These model plants should be closely related (from the same lineage), have similar flower sex types and a relatively short life cycle, be easy to genetically manipulate, etc.
The application of deep sequencing technologies and comparative genomics. With the rapid development of these techniques, it has become easier to execute comparative studies among close species. Lower cost of deep sequencing and higher efficiency of bioinformatics analysis will accelerate genome sequencing and make it feasible to sequence a large number of genomes of closely related species or plants and explore their similarities and differences at the genome level. This will benefit our understanding of the evolution and diversification of flower sex determination systems in plants.
Phytohormone networks. Hormone signaling is one of the key factors in the regulation of sex differentiation in horticultural plants (e.g., ethylene and its crosstalk with gibberellins in Cucurbitaceae). It is generally believed that ethylene promotes female flower development and GA stimulates male flower development. Investigating various hormone signals and their crosstalk in flower sex determination is an exploratory area of great significance.
Once sex determining genes and associated regulatory networks are uncovered, design and development of plants with unisexual or bisexual flowers will become feasible and of practical value. In this way, cross pollination of self-fertilizing plants can be used to avoid inbreeding depression, and bisexual flowers can ensure sufficient fertility and yield for many monoecious and diecious plants.
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About this article
Cite this article
Zheng J, Xia R. 2022. Flower development and sex determination in horticultural crops. Fruit Research 2:9 doi: 10.48130/FruRes-2022-0009
Flower development and sex determination in horticultural crops
- Received: 21 November 2021
- Accepted: 16 June 2022
- Published online: 29 June 2022
Abstract: Horticultural crops are extremely valuable due to their high nutritional value, and fruits, in particular, provide indispensable vitamins and minerals. Fruit yield of edible crops is closely related to the number of flowers, which are often unisexual. The mechanism of sex differentiation in plants with unisexuality is complex, and research investigating this mechanism is in great demand. Sex determinants were first discovered in Cucurbitaceae (e.g., cucumber, melon, watermelon), and in recent years, with the rapid development of deep sequencing technologies and genomics, they have also been deciphered in some dioecious plants (e.g., persimmon, kiwifruit, asparagus). This has deepened our understanding of the evolution and diversification of sexual reproductive systems. This review summarizes recent research investigating flower sex-determination genes and their working networks, focusing on horticultural crops. Perspectives on future research in flower sex differentiation are also discussed.