1. Introduction
Sex determination and expression in plants is a complex biological process influenced by a combination of genetic, environmental, physiological, biological, and anthropogenic factors. Male plants of dioecious spinach varieties exhibit rapid senescence and a short flowering period, whereas monoecious plants exhibit slower senescence and longer flowering periods and are capable of self-fertilization for seed production. Therefore, the study of sex-related genes in monoecious spinach is critical for breeding. Sex differentiation in plants is governed by both intrinsic factors, such as endogenous hormones, and extrinsic factors, such as environmental conditions. These factors collectively govern and regulate plant sex determination [
1]. Studies have shown that plant sex is determined by sex chromosomes (XY system), with the sex-determining gene on the Y chromosome playing a pivotal role [
2,
3]. Most species in the plant kingdom are monoecious (with bisexual flowers, bearing bisexual flowers, where both pistils and stamens are located in the same flower, or unisexual flowers, where a flower contains only pistils or stamens), and approximately 6% of angiosperms are dioecious, which is characterized by separate male and female flowers located on different plants [
4]. The sex-determination system varies significantly across breeding systems. In dioecious species, sex determination is typically controlled by nuclear genes located on sex chromosomes. Only a subset of species exhibits heteromorphic sex chromosomes, similar to most animals. Dioecy in plants may have evolved from monoecy, with sex chromosomes arising from a pair of autosomes [
5].
Spinach (
Spinacia oleracea L.) is an annual and biennial herbaceous plant belonging to the Amaranthaceae family. It is widely cultivated and highly valued worldwide as a nutritious leafy vegetable rich in vitamins (such as vitamin C and vitamin K) and minerals (including iron and calcium). Spinach can be cultivated year-round and serves as an important edible green vegetable during the spring, autumn, and winter seasons [
6]. Although most spinach plants are dioecious, monoecious individuals are occasionally observed [
7]. Sex determination in dioecious spinach is governed by a pair of
X/Y alleles located on the largest chromosome [
8]. Spinach exhibits an
XY sex-determination system, with no significant size difference between the
X and
Y chromosomes [
7]. Spinach exhibits three types of sex expression: male, female, and monoecious. Spinach flowers are unisexual. Most spinach varieties are dioecious, with distinct male and female plants typically showing a 1:1 segregation ratio. In spinach, sex determination is controlled by sex-determining genes on sex chromosomes. Previous studies have shown that
X/Y genes control dioecy in spinach and the
Xm gene governs monoecy. The
Y chromosome is dominant over both
X and
Xm, and
Xm is dominant over
X [
9]. Therefore, the spinach genotypes are as follows: male plants are
XY or
XmY, monoecious plants are
XmXm or
XmX, and female plants are
XX. Sex determination and differentiation are crucial biological processes in unisexual flower development. Spinach serves as a model plant for studying plant sex determination and differentiation mechanisms [
10]. Despite these advances, research on the
Xm gene responsible for monoecy in spinach is still limited, and positional cloning has not yet been achieved. Furthermore, the molecular mechanisms underlying the sex determination network in monoecious spinach remain poorly understood, significantly hindering the breeding efficiency of spinach.
In higher plants, plant hormones, such as auxin (IAA), ethylene, gibberellin (GA), and abscisic acid, play multiple roles in plant growth and development [
1,
11,
12], with many exhibiting pleiotropic effects. IAA induces female flower development in cucumber, lemon, and hemp. GA promotes male flower development in cucumber, melon, asparagus, and hemp but induces female flowers in maize. These hormones also influence sex differentiation in monoecious and dioecious species. Collectively, these findings show that no single hormone has a universal effect on sex determination in monoecious and dioecious plants, indicating that each species has its own hormonally regulated mechanism for sex expression. GA is a major plant hormone that participates in normal plant growth and development and influences sex expression in many plant species [
13,
14,
15,
16,
17]. Among plant hormones, GA plays a key role in regulating flowering in the model plant
Arabidopsis thaliana. Although over 100 GAs have been identified in plants, only a few are considered biologically active, including GA1, GA3, GA4, and GA7 [
18]. GAs were initially recognized for their effects on stem elongation, with exogenous GA3 application reversing the dwarf phenotypes of
Pisum sativum [
19] and
Zea mays mutants [
20], enabling them to reach heights comparable to mature plants. GAs are essential at multiple stages of plant growth and development, including seed germination, floral induction, leaf elongation, and fruit growth. GAs have also been shown to promote male flower formation in several species, including spinach [
21], hemp [
22,
23], and cucumber. Although GA typically promotes male floral development, it is not universal across all species. For example, exogenous GA3 application in
Z. mays results in feminization of the terminal inflorescence [
24]. Exogenous GA treatment of spinach leads to the transformation of 78% of pistils into stamens [
21]. GA3 has been shown to enhance the masculinizing effect on individual female spinach plants, resulting in complete conversion and functional stamens [
25].
In many plants, sex determination is controlled by specific genes, and the expression of these genes is often regulated by transcription factors (TFs). For example, in some plants, TFs control the plant’s sex expression by activating or repressing sex-determining gene transcription. The MADS-box gene family plays a pivotal role in numerous stages of the plant developmental cycle, including floral organogenesis, fruit development, and gametophyte formation [
26,
27]. These genes are also involved in biological regulatory processes in plants, such as circadian rhythm regulation, metabolic regulation, and floral transition [
26,
28,
29]. Some MADS-box genes in angiosperms exhibit homologous functions. Although not all MADS-box genes are homologous, they can serve as key meristem identity regulators. CRC genes from the YABBY family influence carpel development and affect male and female flower formation, playing a pivotal role in sex determination in Cucurbitaceae species [
30]. Proposed in 1991 to explain the role of homologous genes in floral organ identity, the ABC model posits that each organ within a floral whorl is determined by the interaction of three distinct organ identity genes [
31,
32,
33,
34]. This model was later expanded to the ABCDE model [
35,
36].
In contemporary plant research, the utilization of transcriptomic data represents a prevalent and popular approach to identify genes associated with sex determination and flowering. Using transcriptomic data from different periods of pepper floral organ development and incorporating the ABCDE model of floral development, 17 ABCDE model candidate genes were identified in pepper by Tang et al. [
37]. The transcriptomes of different sexes were analyzed in papaya, and the expression of 40 plant-conserved miRNAs and 14 papaya-specific miRNAs was detected in 1 or 2 normal flowers and pistils of monoecious, inverted flowers by Lin et al. [
38]. These results suggest that male-to-female sex reversal may be caused by the silencing of androgynous inhibitory sex functions through epigenetic modification in the sex-determination pathway.
AP1 (Apetala1) regulates sepal and petal development and is expressed during early floral development, playing a key role in flower formation [
36]. The PI gene plays a critical role in flower development, particularly in the formation of the second and third whorls of floral organs (i.e., the stamen and pistil). PI expression influences petal and stamen development [
39]. AG (Agamous) is essential for stamen and pistil development. AG is a key gene in floral development, and its absence leads to abnormal reproductive organ development [
40]. SQUA (Squamosa) participates in stamen and pistil formation in some plant species and affects floral morphology [
41]. SEP1 is a member of the SEPALLATA gene family and regulates floral organ development. SEP1 plays an important role in the formation of floral organs, such as petals, stamens, and pistils [
42]. Suppressor of Overexpression of Constans 1 (SOC1) integrates environmental signals and activates the flowering process. SOC1 is typically a part of plants’ biological clock and responds to environmental cues, such as photoperiod and temperature, to regulate flowering [
43].
In this study, RNA sequencing (RNA-seq) was conducted in female and monoecious plants at two flower stages. We conducted a co-expression network analysis and identified many TF genes. These findings provide novel genetic reservoirs for further pinpointing the regulatory mechanism underlying monoecism.
4. Discussion
Spinach is a predominantly dioecious biennial herb; however, monoecious inbred spinach lines, which have a longer flowering period and produce hybrids with high purity and yield, have significant implications for spinach breeding. Therefore, research on the monoecious form of spinach is of considerable importance for breeding programs. In this study, two sampling time points—the first day of flowering and the eighth day of flowering, which show distinct phenotypic differences—were selected for sampling two genotypes. RNA-seq analysis was performed on samples with different genotypes collected at different developmental stages. PCA, GO and KEGG enrichment analyses, co-expression trend analysis, and TF prediction were conducted based on the RNA-seq data. In this study, numerous DEGs involved in pathways related to flower development were identified, influencing processes such as floral organ morphogenesis. In addition, some DEGs were associated with plant hormone signaling pathways and phenylpropanoid biosynthesis.
GO enrichment analysis revealed that the biological processes related to corolla petal development and carbohydrate metabolic processes were the most significantly enriched pathways. Carbohydrates, a crucial organic compound in plants, play pivotal roles in plant growth, development, and metabolism. They not only serve as a primary energy source but also participate in critical physiological processes, such as cell wall biosynthesis, signal transduction, and stress responses [
47]. In the context of cellular components, the chloroplast and plastid nucleoids are closely associated with chloroplast structure and function, potentially influencing plant growth and development through the regulation of photosynthesis [
48]. At the molecular function level, several pathways are linked to DNA-binding activity, which is essential for cellular processes. DNA-binding activity is involved in plant hormone signaling, developmental processes, stress responses, and cell division and differentiation.
KEGG enrichment analysis revealed the most significantly enriched DEGs. Plant hormones are crucial signaling molecules regulating plant growth, development, and stress responses. Hormones, such as GAs, IAA, and BRs, are indispensable for plant growth and development [
49]. These plant hormones modulate gene expression and cellular activities through intricate signal transduction pathways, thereby influencing physiological processes. In addition, the “MAPK signaling pathway—plant” is involved in a wide range of cellular processes, including growth, development, and responses to environmental stimuli.
Co-expression trend analysis identified three gene clusters whose enrichment patterns closely aligned with those observed in previous enrichment analyses. It is hypothesized that these three clusters reflect the expression trends of sex-related genes. GO and KEGG enrichment analysis of these clusters revealed that they are strongly associated with pathways such as GA signaling, nuclear development, DNA-binding, chloroplast association, sexual reproduction, and IAA response, which is consistent with the DEG enrichment results.
TFs play a crucial role in processes such as floral development, fruit ripening, and root development in plants. By predicting the TFs for all DEGs and comparing them with genes representative of the ABCDE model, 27 genes were identified. These genes regulate floral development by influencing the formation of the calyx, corolla, stamens, and carpels.
Based on functional enrichment analysis, co-expression trend analysis, and TF prediction, the
SOV3g004950 gene, encoding a MADS-box protein and showing high similarity to representative genes in the ABCDE model of floral organ development, including PI and SQUA, was enriched in the flower development pathway. The
SOV1g020280,
SOV2g002560,
SOV2g030600, and
SOV4g052720 genes were significantly enriched in the DNA-binding molecular function category and exhibited high similarity to key genes in the ABCDE model, such as AP1, PI, SQUA, SEP1, and SOC1. In addition, SOV1g000430 was involved in the flower, corolla, and petal development pathways, showing high homology to genes regulating pollen development in
Arabidopsis. The
SOV1g002920 and
SOV2g009980 genes were involved in plant hormone signaling pathways and encoded receptor-like kinases, which are highly similar to the EMS1 receptor-like kinase. These kinases are essential for plant reproductive development, particularly in microsporocyte formation, with EMS1 playing a key role in ensuring proper meiosis and anther development. The
SOV4g045350 gene belongs to the molecular function category of DNA binding and encodes a SANT domain-containing protein. This protein regulates the expression of specific genes and modulates the chromatin status, contributing to plant sex determination and floral organ development. It is a member of the RADIALIS (RAD) gene family, which influences dorsal petal identity and regulates floral symmetry. Masuda et al. [
50] demonstrated that DkRAD, a gene from hexaploid persimmon, shared high homology with RADIALIS, and expression analysis showed that DkRAD likely promoted pistil development by activating MYB73 and modulating the IAA signaling pathway. Furthermore, DkRAD overexpression in
Arabidopsis and
Nicotiana models has been shown to induce excessive pistil growth.
We identified nine genes associated with sex determination in monoecious spinach plants. Among them, the SOV3g004950, SOV1g020280, SOV2g002560, SOV2g030600, and SOV4g052720 genes exhibited high homology with genes involved in the floral development model. SOV1g000430 was involved in the pathways of flower development, corolla development, and petal development, with significant expression differences across different developmental stages and genotypes. SOV1g002920 and SOV2g009980 influenced floral development through plant hormone signaling pathways. SOV4g045350 was identified as a key gene involved in pistil growth.