Sexual reproduction in angiosperms encompasses a remarkable diversity of breeding systems. Most flowering plant species produce bisexual (hermaphroditic) flowers bearing both male and female organs, a condition that facilitates self-pollination and reproductive assurance (Barrett, 2002; Renner, 2014). However, a minority of angiosperms have evolved unisexual flowers, giving rise to monoecious species, in which separate male and female flowers develop on the same individual, and dioecious species, in which male and female flowers are borne on distinct individuals (Charlesworth, 2002). Dioecy has evolved independently hundreds of times across the angiosperm phylogeny and occurs in approximately 5–6% of flowering plant species (Renner, 2014). In dioecious lineages, sex is often determined by sex chromosomes, which may range from homomorphic pairs with limited differentiation to highly heteromorphic systems resembling those of animals (Ming et al., 2011). The evolution of sex chromosomes in plants is thought to proceed through the progressive suppression of recombination around a sex-determining locus, leading to the accumulation of sex-linked genes and, eventually, morphological differentiation of the sex chromosome pair (Bergero and Charlesworth, 2009).

Despite the presence of genetic sex-determining mechanisms, sexual expression in many plant species is not rigidly fixed. In both dioecious and monoecious plants, the phenotypic sex of flowers can be modulated by environmental conditions, developmental stage, and phytohormone signaling, a phenomenon broadly referred to as sexual lability or sexual plasticity (Cossard and Pannell, 2021; Käfer et al., 2022). Sexual plasticity has been documented across diverse taxa, including Carica papaya (papaya), Spinacia oleracea (spinach), Mercurialis annua, and Amborella trichopoda (Anger et al., 2017; Cossard and Pannell, 2021; Lin et al., 2016). The triggers that shift phenotypic sex or floral sex ratios vary widely and include natural population variability, reproductive pressure, abiotic or biotic stress, and targeted molecular interventions with plant growth regulators (Dennis Thomas, 2004; J. Zhang et al., 2017). Among the phytohormones implicated in sex determination, ethylene and gibberellins (GAs) have emerged as central players, with evidence from multiple plant families linking their biosynthesis and signaling to the control of male versus female flower development (Chailakhyan and Timiriazev, 1979; Chandler, 2011; Diggle et al., 2011).

Cannabis sativa L. is a predominantly dioecious species with dimorphic male and female flowers on separate individuals (Bonini et al., 2018). The species has 10 chromosome pairs (2n = 20), comprising nine autosomal pairs and one sex-chromosome pair: males are typically XY with heteromorphic sex chromosomes, whereas females are XX with a homomorphic pair (Carey et al., 2026; Prentout et al., 2020). Pistillate flowers (female flowers; FF) comprise an ovary enclosed by two bracts bearing glandular trichomes and terminate in two elongated stigmas (Leme et al., 2020; Spitzer-Rimon et al., 2019). Staminate flowers (male flowers; MF) consist of a simple perianth and typically five stamens positioned opposite the sepals (Schilling et al., 2020). The glandular trichomes found in female inflorescences produce a diversity of cannabinoids with substantial pharmacological interest (Andre et al., 2016). In recent years, the cannabis genome has been sequenced and assembled (Grassa et al., 2021; Van Bakel et al., 2011), and interest in the genetic and molecular basis of sex determination has grown considerably (Adal et al., 2021; Chen et al., 2025; Monthony et al., 2026; Orozco et al., 2026; Prentout et al., 2020; Shi et al., 2025).

As observed in other dioecious plants, sex expression in C. sativa is not determined solely by sex chromosomes. Under specific environmental or experimental conditions, cannabis plants can display flowers that do not align with their chromosomal sex (XX/XY karyotype), a phenomenon recognized for decades (Chailakhyan and Timiriazev, 1979; Mohan Ram and Sett, 1982). Among the hormonal regulators implicated in this plasticity, ethylene emerged as a key modulator of feminization: inhibition of ethylene signaling using silver thiosulfate (STS) or silver nitrate reliably induces male flowers on XX plants (induced male flowers; IMF), whereas exogenous applications of ethylene or the ethylene-releasing compound ethephon induce feminization of XY plants (induced female flowers; IFF) (Flajšman et al., 2021; Garcia-de Heer et al., 2025; Monthony et al., 2026; Moon et al., 2020). Conversely, GAs have been recognized as modulators of sex expression since the pioneering work demonstrating that exogenous GA₃ applications can induce male flowers on XX plants (Galoch, 1978; Ram and Jaiswal, 1972). These chemical interventions now form the basis of feminized seed production in the cannabis industry, yet the underlying molecular mechanisms governing induced sex change have remained poorly understood until recently.

Recent transcriptomic and multi-omics investigations have begun to uncover the molecular architecture of ethylene-mediated sexual plasticity in cannabis. A comprehensive study by Monthony et al. (2026) integrated over 130 RNA-seq libraries with ethylene pathway metabolite profiling and whole-genome sequencing across three XX and XY genotypes treated with STS and ethephon, respectively. This work demonstrated that ethylene-mediated sexual plasticity involves both systemic and local signaling components, where early transcriptional activation of ethylene biosynthesis and signaling genes occurred within 18 hours of treatment in leaves, prior to the emergence of flowers, while the new phenotypic sexual identity in developing flowers involved distinct sets of genes differentially regulated in each chromosomal sex. Other transcriptomic studies have similarly identified ethylene-related genes as candidates for sex determination (Adal et al., 2021), and network ontology analyses have further highlighted hormone-related gene expression changes associated with sexual plasticity (Orozco et al., 2026). Together, these findings have established ethylene as a primary hormonal driver of sexual plasticity in cannabis but also raised the question of whether other phytohormone pathways, particularly GAs, participate in this regulatory network.

GAs comprise a large family of tetracyclic diterpenoid phytohormones that regulate diverse aspects of plant growth and development, including stem elongation, flowering, fertility, and reproductive organ development (Bao et al., 2020; Pimenta Lange and Lange, 2006; Plackett et al., 2011; Wilson et al., 1992; Yamaguchi, 2008; Yu et al., 2004). Although more than one hundred gibberellins have been identified, only a small subset, primarily GA₁ and GA₄ in plants, are bioactive (Shani et al., 2024), with the remaining acting as precursors or inactive forms (Hedden and Thomas, 2012). In planta GA activity largely depends on the balance between GA production and deactivation (Yamaguchi, 2008). GA biosynthesis is a complex, multi-step process beginning in the plastid with a 20-carbon precursor, geranylgeranyl diphosphate (GGPP), which undergoes enzymatic transformation to form GA₁₂, the common precursor for all GAs in plants (He et al., 2020; Shani et al., 2024). From GA₁₂, two parallel branches are commonly described: a non-13-hydroxylated route yielding GA₄ and a 13-hydroxylated route in which GA₁₂ is hydroxylated by GA13-oxidase (GA13ox) to GA₅₃ and ultimately converted to GA₁. Following synthesis in the endoplasmic reticulum, both GA₁₂ and GA₅₃ are converted in the cytosol via GA20-oxidases (GA20ox), which generate C19 precursors (e.g., GA9/GA20), and GA3-oxidases (GA3ox), which convert these into the bioactive GAs GA₄/GA₁ (Shani et al., 2024). GA deactivation is classically mediated by GA2-oxidases (GA2ox), which can also remove precursors from the biosynthetic pool.

Perception and signaling of bioactive GAs occur primarily in the nucleus, where GA binds to the soluble receptor GA INSENSITIVE DWARF1 (GID1) (Griffiths et al., 2007; Hirano et al., 2008) and promotes formation of a complex that triggers DELLA protein degradation via SLEEPY1 (SLY1) or its homolog SNEEZY (SNE/SLY2), both F-box subunits of SCF E3 ubiquitin ligase complexes (Ariizumi et al., 2011; McGinnis et al., 2003). DELLA proteins, belonging to the GRAS family, function as master growth repressors that integrate multiple phytohormone signals; their GA-dependent degradation de-represses downstream developmental programs (Davière and Achard, 2013). DELLA activity is further modulated by SPINDLY (SPY) and SCARECROW-LIKE (SCL). When GA activity is low, DELLAs accumulate and activate feedback mechanisms that modulate transcriptional regulation of GA metabolic genes, including GA20ox, GA3ox, and GA2ox, in several systems (Fukazawa et al., 2014; Hedden and Thomas, 2012). Downstream of these core signaling events, GA-responsive genes are induced, including members of the GA-stimulated transcripts (GASA/GAST1-like) family, which encode small, secreted, cysteine-rich peptides that serve as integration nodes for multiple hormonal pathways (Aubert et al., 1998; Qu et al., 2016; Roxrud et al., 2007).

An additional feature of GA biology is its spatial organization. GA biosynthesis, deactivation, perception, and response need not occur in the same cells or tissues: expression of early and late biosynthetic enzymes can be spatially separated, requiring movement of pathway intermediates for local production of bioactive GA (Binenbaum et al., 2018; Olszewski et al., 2002). GAs and their precursors can also move between cells and organs, while localized biosynthesis, catabolism, and transport together generate tissue- and developmental-stage-specific GA distributions (Dayan, 2016; Rizza and Jones, 2019).

Beyond their roles in growth regulation, GAs serve as sex determinants across a range of plant lineages: in ferns and most eudicots, GAs generally promote male organ development, while in some monocots such as maize the effect is reversed (Gupta and Chakrabarty, 2013; Vandenbussche et al., 2007). In Arabidopsis thaliana, GA is essential for stamen filament elongation and anther development, and GA-deficient mutants display severe male sterility (Cheng et al., 2004; Plackett et al., 2011). Foundational work in the dioecious species S. oleracea implicated GA signaling in unisexual floral development: GA application promoted male-organ development in female plants, whereas inhibition of GA biosynthesis or proteasome activity induced female organs in males (West and Golenberg, 2018). Although bulk GA concentrations did not differ between the sexes, SpGAI, the only detected DELLA-family gene, showed higher inflorescence expression in females; its silencing promoted male-organ development. These findings identified sex-biased DELLA regulation as a component of the spinach feminizing pathway. Subsequent transcriptomic studies further linked GA-related regulation to spinach floral sex. Li et al. (2020) identified sex-biased co-expression networks in male and female flowers in which auxin- and GA-related genes were prominent. A GAST1/GASA13 ortholog and a GASA6 ortholog were the genes most closely co-expressed with the central hub genes of the female and male networks, respectively, identifying GASA-family genes as candidate components of sex-biased regulatory networks. Similarly, gene regulatory network analysis across female, male, and monoecious spinach flowers implicated GA and ABA signal-transduction pathways as major components of floral regulatory networks (Ma et al., 2024).

Functional evidence was subsequently provided by Zhang et al. (2024). In spinach, GA3 treatment induced functional masculinization of genetically female plants, and silencing GIBBERELLIC ACID INSENSITIVE (SpGAI), which encodes the single DELLA repressor in this system, produced a similar masculinized phenotype with viable pollen. Zhang et al. further showed that SpGAI interacts with the KNOX transcription factor SpSTM to repress the B-class floral identity gene SpPI, regulating female floral development. More recently, Wang et al. (2025) showed that GA treatment can also induce stamen carpelization in male spinach, accompanied by changes in GA, auxin, cytokinin, jasmonate, and ABA profiles. Their functional analyses implicated the anther-associated genes SpAMS and SpPGIP as activators of SpPI. Together, these studies show that GA can influence sex-associated floral development through DELLA-dependent regulation and broader hormone- and anther-developmental networks in spinach.

In C. sativa, a genome-wide association study identified a major sex-determination QTL (QTLSex_det1) containing a gene encoding a GAI-like DELLA protein (Petit et al., 2020), identifying a DELLA-related candidate but not establishing a conserved mechanism (Salentijn et al., 2019). Alter et al. (2024) showed that inflorescence development in female C. sativa is mediated by photoperiod and GA: short-day conditions trigger a decrease in bioactive GA4 at the shoot apex that is required for compact inflorescence formation, whereas exogenous GA3 prevents condensation. Transcriptomic analyses of sex-changed cannabis plants have also identified GA-related genes associated with sexual plasticity. In IMF on XX plants, a putative GA2ox gene is consistently downregulated, suggesting altered transcriptional regulation of GA deactivation in masculinized tissues (Toscani et al., 2026). CsGASA4 was upregulated in both MF and STS-induced IMF relative to untreated FF (Adal et al., 2021), and a cytochrome P450 gene proposed to participate in GA biosynthesis was consistently upregulated in male tissues (Orozco et al., 2026).

Crucially, the GA and ethylene pathways do not operate in isolation. In model species, GA–ethylene crosstalk is mediated largely through DELLA proteins, which serve as integration hubs for both pathways. Ethylene signaling stabilizes DELLA proteins, thereby reducing GA-responsive growth and development (Achard et al., 2003, 2007). Activated ethylene signaling can also reduce levels of bioactive GAs by inhibiting GA biosynthesis enzymes such as GA20ox (Achard et al., 2007), and the ethylene-regulated transcription factor EIN3 can physically interact with the JAZ-family repressors, which themselves interact with DELLAs (Colebrook et al., 2014; Davière and Achard, 2013). In certain developmental contexts, ethylene and GA can act either antagonistically or synergistically: ethylene inhibits root elongation by blocking GA-induced DELLA degradation, while in floral induction GA promotes flowering and ethylene typically delays it (Achard et al., 2007; Sun, 2008). These molecular interactions provide a framework for testing how changes in one pathway may be associated with responses in the other and why ethylene-modulating treatments in cannabis lead to coordinated shifts in both ethylene- and GA-related gene expression.

Despite well-documented phenotypic effects of manipulating GA and ethylene pathways in C. sativa, the transcriptional relationship between the pathways during sex reversal remains poorly characterized. Building on the ethylene-focused experimental framework of Monthony et al. (2026), this study reconstructs the canonical GA pathway in C. sativa and examines GA-related gene expression across treated and untreated plants at multiple time points. We test whether these transcript patterns are associated with ethylene-pathway manipulation, sampling time, and floral phenotype. This descriptive framework identifies candidates for future mechanistic study of GA in cannabis sex reversal.