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Review

Deciphering the Molecular Mechanisms That Control Ovule Development in Pomegranate

by
Yujie Zhao
1,2,3,
Hidenori Sassa
4,
Ming Li
1,2,3,
Yifei Miao
1,2,3,
Xiaoyan Zhu
1,2,3,
Pengbo Hao
1,2,3,
Ran Wan
1,2,3,
Kunxi Zhang
1,2,3,
Liu Cong
1,2,3,
Yawen Shen
1,2,3,
Yu Liu
1,2,3,
Miaomiao Wang
1,2,3,
Jiangli Shi
1,2,3,
Shangwei Song
1,2,3,
Tuanhui Bai
1,2,3,
Jian Jiao
1,2,3,
Zhaohe Yuan
5 and
Xianbo Zheng
1,2,3,*
1
College of Horticulture, Henan Agricultural University, Zhengzhou 450046, China
2
International Joint Laboratory of Henan Horticultural Crop Biology, Henan Agricultural University, Zhengzhou 450046, China
3
Henan Apple Germplasm Innovation and Utilization Engineering Research Center, Henan Agricultural University, Zhengzhou 450046, China
4
Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan
5
College of Forestry, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Horticulturae 2026, 12(1), 26; https://doi.org/10.3390/horticulturae12010026 (registering DOI)
Submission received: 19 November 2025 / Revised: 24 December 2025 / Accepted: 25 December 2025 / Published: 26 December 2025
(This article belongs to the Special Issue From Structure to Function: Anatomical and Reproductive Studies in Native and Cultivated Horticultural Plants)
Browse Figure
Versions Notes

Abstract

Plant seed number depends on ovule number initiated within the carpels, and it serves as a primary factor shaping fruit yield. Pomegranate trees exhibit bisexual flowers and functional male flowers. Pomegranate have anatropous ovules which are bitegmic and crassinucellate. Bisexual flowers possess the fertile pistil, while functional male flowers have abnormally developed ovules, a small ovary with few chambers, and a short style. The formation of functional male flowers is due to abnormal and stagnant development of ovule integument. Ovule number directly determines the yield of pomegranate seeds. Recent studies have highlighted the molecular mechanisms through which ovule-related genes regulate pomegranate ovule development. Pomegranate PgCRC and PgINO genes positively regulate the increase in the number of ovules, and PgBEL1 to synergistically regulate seed development. PgAGL11 (the SEEDSTICK orthologous gene) promotes ovule development in transgenic Arabidopsis. PgSEP protein can bridge interactions among PgBEL1, PgSTK and PgAG, which regulate ovule development. At the level of post-transcriptional regulation, PgmiRNA167, PgmiRNA164 and PgmiRNA160 are differentially expressed during pomegranate flower development, and PgmiR166a interacts with its target genes to affect ovule development. This review summarizes the key regulators of ovule development and their molecular pathways, integrating these interactions into a model that describes pomegranate ovule development.

1. Introduction

In flowering plants, the ovules upon fertilization develop into seeds. Understanding the mechanisms regulating ovule number and development is essential, as these factors ultimately determine seed number and, thus, yield. Pomegranate trees produce bisexual and functional male flowers [1]. Bisexual flowers have fully developed gynoecia with numerous anatropous ovules. But ovules in functional male flowers are rudimentary, shriveled and exhibit various stages of degeneration [1,2,3]. Previous research has shown that ovule primordia, along with the outer and inner integument primordia, are formed in bisexual flowers. The integuments initiate as circular ridges at the nucellus base and grow upward to encase it, and the outer integument rapidly grows to completely enclose the inner integument [3,4]. However, integument primordia are not observed in functional male flowers [3,4,5]. The number of ovules in flowers can influence the aril number and fruit size in pomegranate. The aril is the edible tissue of pomegranate, which is derived from the outer integument [5]. Therefore, investigating the mechanisms regulating ovule development has become a significant research area in plant science. The pomegranate genome has provided the data foundation for the ovule development research [6,7]. Based on previous findings, we constructed a regulatory network involving CRC (CRABS CLAW), INO (INNER NO OUTER), BEL1 (BELL1), and MADS-box genes that govern pomegranate ovule development [4]. Furthermore, microRNAs (miRNAs) have been implicated in pomegranate ovule sterility at the level of post-transcriptional regulation [8]. The functions of transcription factors and miRNAs in pomegranate flower and seed development and their regulatory relationships have been studied in previous research [3,4,6,7,8]. Ovule development determines ovule number and seed characteristics, and this research lays the foundation for breeding large-fruited pomegranate varieties with plump seeds and high juiciness. In this review, we summarize the current understanding of both molecular and hormonal mechanisms that regulate pomegranate ovule formation and development.

2. Overview of Ovule Development in Plants

Seed number depends on the number of ovules initiated within the carpels. Ovule number is determined by ovule spacing and the length of the placenta [9]. Flower fertility and ovule number are the crucial factors determining yield. Plant ovule development has been extensively studied in detail. Arabidopsis thaliana flower development is divided into 20 stages, with ovule development primarily occurring during stages 9–12 [10,11]. Plant ovule development includes ovule cell fate determination, the ovule primordium initiation and ovule morphogenesis [9,12,13]. Periclinal cell divisions within the placenta sub-epidermal tissue initiate ovule primordium formation at stage 9, megaspore mother cells (MMCs) are identified at stage 10, integument initiation and megasporogenesis are observed at stage 11, and the embryo sac develops at stage 12 [14,15]. In A. thaliana, ovule primordia are formed in carpel medial meristem (CMM) as lateral organs from the placenta [16,17]. The critical step in ovule development is to determine the number and position of ovule primordia in the CMM [9]. Ovule primordia arise from the placental tissue at stage 10. Ovules are finger shaped, and their number and shape at each placenta show only minor differences [11]. The outer and inner integuments develop from the chalaza. The outer integument presents asymmetrical growth by heightened cell divisions on the side facing the central septum. The outer and inner integuments grow upward, gradually enclosing the nucellus. By the stage of maturity, the outer integument has fully overgrown the inner integument. The faster growth of the abaxial integuments induces curvature in the developing ovules [18]. The integuments, of sporophytic origin, ultimately form the seed coat. While the ovules in petunia and tomato possess a single integument, Arabidopsis forms two integuments [19,20].
In recent decades, several research studies have identified genes involved in ovule initiation, ovule identity determination and development in some species, such as A. thaliana, petunia, rice, and tomato (Table 1) [11,21,22,23,24]. ANT (AINTEGUMENTA) has been verified to promote ovules and floral organ initiation [25,26]. CUC (CUP SHAPED COTYLEDON) transcription factors, CUC1, CUC2 and CUC3, are expressed in boundaries of aboveground organ primordia, and CUC2 has been verified to regulate ovule initiation [13,27]. OsPID (OsPINOID) plays a crucial role in the regulation of rice stigma and ovule initiation [28]. In Arabidopsis, EPFL1 (Epidermal Patterning Factor-Like1) and EPFL2 promote ovule initiation and nascent ovule elongation. EPFL1 and EPFL2 synergy with CUC boundary genes regulates ovule number and shape [9]. Key genes such as BELL1 (BEL1), SEEDSTICK (STK), SHATTERPROOF (SHP), and AGAMOUS (AG) have been identified as being involved in determining ovule identity [21,29]. Mutations in AG homologues, rice osmads13 mutant, petunia fbp7/fbp11 double mutant and stk/shp1/shp2 triple mutant in Arabidopsis, lead to homeotic transformation of ovules into carpels [21,22,29,30,31]. VvAGL11 (AGAMOUS-LIKE11, an ortholog of Arabidopsis STK), is negatively regulated by VvBPC1 (BASIC PENTACYSTEINE), and VvBPC1 protein interacts with VvBELL1, regulating grape seed development [32]. BEL1 interacts with the ovule identity MADS-box factors (STK and SHP) when they dimerize with SEPALLATA (SEP) proteins [33]. Ovule integument development is regulated by intricate genetic interactions, such as INO and ANT, and establishing their expression regions in early ovule primordia is crucial for integument formation [34,35,36]. CRC has been verified to play a vital role in ovule primordia initiation, differentiation and development [17,24,37,38]. Additionally, TCP4 protein interacts with AG, SEPALLATA3 (SEP3) and BEL1 to strengthen the connection of BEL1 with AG-SEP3 [39]. This research in model plants can be used as reference for ovule development of woody plants.

3. Key Genes Involved in Pomegranate Ovule Development

Pomegranate fruit bears many ovules, and can therefore serve as a representative system for ovule development studies in fruit trees. Previous studies have confirmed that INO, CRC, BEL1, NST, and MADS-box genes (such as STK, SEP and AG) are involved in regulating ovule development in pomegranate (Table 2) [4,83,84].

3.1. BEL1—A Regulator of Pomegranate Ovule Development

BELL is a critical regulator of ovule development. BEL1 is expressed throughout the ovule primordia in the initial stages, and BEL1 is essential for INO expression [85]. Ovules of the bel1 mutant lack the inner integument and exhibit abnormal development of the outer integument. In ovule development, BEL1 negatively regulates AG gene expression [40], and the loss of AG regulation in the bel1 mutant results in the conversion of ovule into carpels [86,87]. In outer integument development, BEL1 forms the protein complex with AG and SEP to regulate INO expression [33]. BEL1 is required for proper morphogenesis of the ovule integuments.
Our previous studies have demonstrated that PgBEL1 was highly expressed in the inner and outer seed coat [88]. Specifically, when the bud vertical diameter was 5.1–12.0 mm, PgBEL1 expression levels were significantly higher in bisexual flowers than those in functional male flowers. The expression level of PgBEL1 in bisexual flowers was 16.6 times higher than that in functional male flowers at a bud vertical diameter of 5.1–8.0 mm, 2.4 times at 8.1–10.0 mm, and 1.8 times at 10.1–12.0 mm [89]. To investigate the function of PgBEL1, transgenic lines of overexpression PgBEL1 was constructed [90]. The siliques of overexpression PgBEL1 A. thaliana lines normally developed, and seed numbers were substantially lower than in the wild-type (WT). This result contradicts other findings reports [85,86,87], which may be due to the heterologous expression, species-specific interactions or altered meristem identity. So pomegranate transgenic is necessary to verify the gene’s function.
To explore the relationships between INO, CRC, BEL1, and SEP, their expression patterns in flowers, siliques, stems, and leaves of WT and overexpression PgBEL1 transgenic lines were investigated (Supplementary Figure S1). In flowers, AtCRC expression level in overexpression PgBEL1 lines were significantly higher than that in WT. During young leaf development, AtINO and AtCRC transcriptional levels in overexpression PgBEL1 were significantly higher than that in WT. In young siliques of overexpression PgBEL1 transgenic lines, the expression levels of AtINO and AtSEP3 were higher than those in WT. These results suggested that PgBEL1 enhances the expression of AtINO and AtSEP3 at the transcript level during early silique development.

3.2. MADS-Box Genes—The Regulators of Ovule Identity and Development in Pomegranate

In Arabidopsis, STK, SHP1, SHP2, and AG have been identified as key regulators of ovule identity, with AG, STK and SHP playing redundant roles in promoting ovule development [21]. Our previous studies demonstrated that PgSEP1, PgSEP3a, PgSEP3b, PgSTK, and PgSHP were highly expressed in both the functional male flower and bisexual flower [91]. AG showed differential expression pattern between functional male and bisexual flowers of pomegranate during key stages of ovule development [3]. Overexpression of PgAGL11 leads to phenotypes such as shortened stamens, a thicker style, and elongated mastoid cells on the stigma surface. PgAGL11 (STK orthologous gene) expression level is notably higher in bisexual flowers compared to that in functional male flowers, which suggested that PgAGL11 regulates ovule development in pomegranate [83]. RT-qPCR analysis further revealed that PgSTK expression levels in bisexual flowers were higher than those in functional male flowers (Supplementary Figure S2). At pomegranate ovule sterility stages (P3–P5, 8.1–14.0 mm), the expression level of PgSEPs in bisexual flowers is higher compared to that in functional male flowers. These results indicate that PgSTK and PgSEPs are involved in pomegranate ovule development.
The regulatory relationships between BEL1, AG and STK have been elucidated [33]. In addition, the interaction between BEL1 protein and the AG-SEP dimer is necessary to suppress the carpel identity function of AG during ovule integument development [33]. In pomegranate, PgBEL1 could not interact with PgAG and PgSTK at the protein level [4]. SEP is crucial for the ovule development; it is actively involved in the formation of MADS-box protein higher-order transcription factor complexes [92]. Without SEP proteins, the ovule identity complex fails to form, consequently preventing the initiation of the ovule identity pathway [93]. Our results confirmed that PgSEPs protein interacts with PgAG, PgSTK and PgBEL1 [4]. In pomegranate, there was no protein correlation between PgBEL1, PgSTK and PgAG. PgSEP functioned as the molecular interaction that bridged PgBEL1 and MADS-box genes to regulate pomegranate ovule development.
The stem cell maintenance gene WUS (WUSCHEL) plays a vital role in specifying nucellus identity and indirectly regulating integument formation [33,34,94,95]. The suppression of WUS by AG is primary for terminating floral meristem activity, and WUS can induce AG expression in developing flowers [96,97]. WUS is ectopically expressed in the chalaza of bel1 mutant, indicating that the AG-SEP-BEL1 complex restricts WUS expression domain [33]. In a previous study, we demonstrated that PgWUS expression was higher in bisexual flowers than in functional male flowers (bud vertical diameter: 5.1–10.0 mm), and PgWUS expression in pistils was 1.5 times that in the stamens of pomegranate flowers [89]. WUS expression serves as a marker for nucellus determination in the ovule, and the WUS protein acts non-cell autonomously to regulate integument growth [17,34]. In pomegranate, PgWUS protein could interact with PgBEL1 and PgAG [4]. These results suggest that PgWUS is involved in the regulation pathway of pomegranate flower and ovule development.

3.3. INO and CRC Genes—The Positive Regulator of Pomegranate Ovule Development

The Arabidopsis YABBY family member INO is essential for formation of the outer integument [44,98,99], and the ovules of ino mutants are without outer integument [85]. INO is specifically expressed in the abaxial layer of the outer integument [17]. During ovule development in Arabidopsis, the growth of the outer integument occurs primarily to the side of the ovule facing the gynoecium basal region. This asymmetry is regulated by SUP, which restricts INO expression to the basal side of the gynoecium [100]. CRC can substitute for INO in promoting integument development, but CRC does not respond to SUP regulation [101]. Recent research has confirmed that brassinosteroids (BRs) regulate the outer integument development partially by BZR1-mediated INO transcriptional regulation [102]. VviINO, a gene homologous to INO in Vitis vinifera, partially complements the asymmetric growth of outer integument in ino mutants [45]. In pomegranate, PgINO was expressed in the vital developmental stages (5.1–25.0 mm) of bisexual flowers [103]. INO showed down-regulation in functional male flowers at the key stage of ovule development cessation [3]. Functional studies have confirmed that PgINO is involved in the development of flower organs and seeds [4]. PgINO protein interacted with the ovule identity factors PgBEL1, whereas PgINO could not interacte with MADS-box proteins [4]. These results showed that PgINO plays a critical role in regulating the differentiation of ovule and seeds.
CRC functions as the transcriptional activator in carpel closure and nectary development, and is also involved in floral meristem termination by transcriptional repression [104]. It plays a critical role in proper gynoecium growth [105]. The crc mutation consistently reduces stylar growth and results in the carpels’ incomplete medial fusion [105,106]. Arabidopsis carpels of crc mutants are broader, shorter, and contain fewer ovules, indicating that CRC is involved in ovule primordia initiation [105,106,107]. In tomato, SlCRCa and SlCRCb operate as positive regulators of floral meristem determinacy [108]. CRC and INO, combined with KAN (KANADI) and ETT (ETTIN), regulate the adaxial–abaxial patterning of carpels [109]. PeDL1 (the gene homologous to CRC in orchids) overexpression causes ovules abnormal development [37]. The complex of CRC with members of the chromatin remodelling epigenetically represses WUS expression through histone deacetylation, to ensure the termination of floral stem cell activity [108]. Recent research has indicated that CRC is required for plant ovule differentiation and development [37,38]. In pomegranate, PgCRC are expressed in bisexual and functional male flowers [103]. PgCRC expression promotes seed and seed coat development, and PgCRC protein directly interacts with PgBEL1 [4]. These results reveal a role for PgCRC in ovule and seed development, as PgCRC participates in the BEL1-INO ovule regulatory pathway in the plant. Our findings confirmed that PgCRC protein interacts with PgBEL1 and they participate in the traditional INO-BEL1-MADS ovule development regulatory network in woody fruit trees [4].

4. MicroRNA Regulates Ovule Development in Pomegranate

The target genes of conserved microRNAs (miRNAs) are TF-coding (transcription factors, TF) involved in controlling developmental processes [110,111]. The regulation pairs of miRNA-target genes show significant negative correlations at transcriptional levels [111,112,113]. MiRNAs have been implicated in processes as diverse as flowering [114] and ovule development (Table 3) [115,116,117]. miR165 and miR166, which are highly expressed in the small regions of early ovule primordia, restrict the PHB expression domain to promote integument formation [118]. MiRNA-target genes have been identified in pomegranate [119]. PgmiR156, PgmiR157, PgmiR159, PgmiR160, PgmiR172, and PgmiR319 were highly expressed in leaves, functional male and bisexual flowers, and different fruit developmental stages of pomegranate [119,120]. MiR156 negatively regulated the expression of PgSPL5, PgSPL12 and PgSPL13 (SPOROCYTELESS5/12/13), which played roles in pomegranate flower development [121]. Combined analyses of small RNAs and mRNAs indicated that Pg-miR858b affect pomegranate pistil development, and Pg-miR444b.1, Pg-miRN11, Pg-miR166/165a-3p, and Pg-miR952b regulate pomegranate integument and embryo sac development [122]. Pg-miR166a-3p can interact with its targets (Gglean012177.1 (PHB orthologous gene) and Gglean013966.1 (PHAVOLUTA, PHV orthologous gene)), to affect ovule development [122]. Small RNA sequencing analysis also found that known miRNAs were differentially expressed during pomegranate flower development [8], such as miR167, miR164 and miR160, and that the target genes of these known miRNAs were predicted in pomegranate [119].

4.1. MiR166—The Negative Regulator of Pomegranate Ovule Development

MiR166 targets the highly conserved class III homeodomain-leucine zipper (HD-ZIP III) family gene, which includes ATHB8, PHV, PHB, REVOLUTA (REV), and CORONA (CNA) in Arabidopsis thaliana [144,145]. Tight spatial regulation of PHB by miR165/166 is critical for proper ovule morphogenesis [118]. In phb-1d mutants that are insensitive to miR165/166 regulation, the outer integument development is disrupted, underscoring the necessity of miR165/166 activity in restricting PHB expression to specific domains within early ovule primordia to facilitate integument initiation [118,146].
In pomegranate, differential expression analysis between functional male flowers and bisexual flowers revealed 43 known and 14 novel miRNAs with significant expression changes, among which Pg-miR166a-3p was notably upregulated in functional male flowers [122]. This increase in Pg-miR166a-3p expression correlated with the downregulation of its predicted target genes, Gglean012177.1 and Gglean013966.1. Ectopic expression of Pg-miR166a-3p in transgenic lines led to a significant reduction in the number of ovule primordia, while simultaneously increasing the number of floral meristems [122]. These findings confirmed that Pg-miR166a-3p negatively regulates ovule development in pomegranate, likely through post-transcriptional repression of Gglean012177.1 and Gglean013966.1.

4.2. MiR167—The Putative Positive Regulator of Pomegranate Ovule Development

In Arabidopsis thaliana, miRNAs play a crucial role in modulating auxin signaling pathways, particularly through the post-transcriptional regulation of ARF genes [147]. Among these, miR167 specifically negatively targets ARF6 and ARF8. Transgenic Arabidopsis plants harboring miR167-resistant versions of ARF6 or ARF8 exhibit defects in ovule formation and anther dehiscence, highlighting the essential role of miR167 in reproductive organ development [148]. In this context, miR167a is the principal precursor gene responsible for establishing spatial restriction of ARF6 and ARF8 expression during floral organogenesis. Both ARF6 and ARF8 are necessary for female fertility in Arabidopsis, whereas jasmonic acid (JA) signaling, along with transcription factors MYB21 and MYB24, predominantly influences stamen and petal development [74,149,150]. Similarly, in tomato, ARF6 and ARF8 function in the coordinated development of the gynoecium, stamen, and petals [74]. Notably, in developing Arabidopsis flowers, ARF6 and ARF8 are expressed in stamen filaments, a major site of JA biosynthesis [73,151].
In the ‘Taishanhong’ pomegranate genome, 19 ARF genes have been identified, including three PgARF6 homologs [8]. Both PgmiR167a and PgmiR167d possess binding sites within the coding sequences of PgARF6 genes. Experimental evidence has confirmed a direct regulatory interaction between PgmiR167a and PgARF6a [8]. In bisexual flowers, a negative correlation was observed between PgmiR167d and PgARF6b expression levels, while in functional male flowers, PgmiR167d was inversely correlated with PgARF6a and PgARF6c expression (Supplementary Figure S3A).
Under treatment with exogenous indole-3-butyric acid (IBA) and 6-benzylaminopurine (6-BA), the expression of PgARF6 genes was generally downregulated, with PgARF6c showing significant repression [8]. Moreover, transcriptomic analyses revealed negative correlations between PgARF6 expression and the levels of endogenous zeatin riboside (ZR), BR, gibberellins (GA), and abscisic acid (ABA). Conversely, JA content was positively correlated with PgARF6a and PgARF6c expression under IBA treatment [8]. Collectively, these findings indicate that PgmiR167 and PgARF6 genes are integral to the hormonal regulation of flower and ovule development in pomegranate.

4.3. MiR164—The Putative Positive Regulator of Pomegranate Ovule Development

Plant miR164 plays a multifaceted role in development by mediating the post-transcriptional regulation of transcription factors containing the NAC (NAM/ATAF/CUC) domain. In Arabidopsis thaliana, miR164a non-redundantly regulates leaf margin serration through repression of CUC2 transcripts [70]. Similarly, miR164c restricts supernumerary petal formation during early floral development by downregulating CUC1 and CUC2 in the second floral whorl [131]. Additionally, miR164a and miR164b facilitate auxin-mediated lateral-root emergence by targeting NAC1 for transcript cleavage [132]. In tomato, SlmiR164b is essential for the delineation of shoot and floral boundaries, while SlmiR164a contributes to fruit growth regulation [152]. In maize, five NAC family genes have been identified as putative targets of zma-miR164, with three of these expression patterns inversely correlated with zma-miR164 levels [113]. In Arabidopsis, CUC1 and CUC2 are also implicated in ovule development, in part through their activation of cytokinin biosynthetic pathways [153]. Disruption of miR164-mediated regulation of CUC1 and CUC2 significantly alters the number, positioning, and morphology of carpel margin meristems (CMMs), further emphasizing their importance in reproductive organogenesis [69].
PgNST is a homologous gene of NAC in the pomegranate, PgNST3 protein-binding PgMYB46 promoter, regulating the thickness of the inner seed coat; PgNST3A overexpression results in softer and smaller tomato seed coats [84]. In pomegranate, transcriptomic and small RNA sequencing analyses of bisexual and functional male flowers revealed that the expression levels of PgCUCa and PgCUCb, homologs of CUC genes, gradually declined in both flower types (Supplementary Figure S3B). In contrast, the expression of PgmiR164c-5p exhibited a dynamic pattern, increasing and subsequently decreasing in bisexual flowers, while showing the opposite trend in functional male flowers. These reciprocal expression patterns suggest a regulatory relationship between PgmiR164c-5p and PgCUC genes, consistent with a role for miR164 in promoting ovule development in pomegranate by fine-tuning the expression of CUC genes.

4.4. MiR160—The Predicted Positive Regulator of Pomegranate Ovule Development

Accumulating evidence indicates that miR160 plays a critical role in both vegetative and reproductive development by targeting members of the ARF gene family, particularly ARF10, ARF16 and ARF17 [128,129,154]. In tomato, suppression of sly-miR160 results in the upregulation of SlARF10a, SlARF10b and SlARF17, with SlARF10a exhibiting the most pronounced increase in expression [155,156]. Functional analyses further reveal that sly-miR160a promotes the development of leaves and floral organs through quantitative regulation of its primary target, SlARF10a. In Arabidopsis thaliana, ARF17 is essential for pollen wall formation, and loss-of-function mutants display male sterility, underscoring the role of miR160-targeted ARFs in reproductive organogenesis [157].
In pomegranate, expression profiling during bisexual flower development revealed a gradual decline in PgmiR160a-3p levels, coinciding with a progressive increase in PgARF10 transcript abundance (Supplementary Figure S3C). Similarly, during functional male flower development, the expression of PgARF10 and PgARF16a increased, whereas PgmiR160a-3p expression decreased. These inverse expression trends suggest that PgmiR160a-3p negatively regulates PgARF10 and, potentially, PgARF16a, and that this regulatory axis may be critical for floral and ovule development in pomegranate. Collectively, these findings support the hypothesis that miR160a-3p, through its interaction with ARF transcription factors, contributes to the regulation of ovule development, highlighting the broader role of miRNA-TF networks in coordinating reproductive development in pomegranate.

5. Implications for Fruit Production and Quality

Due to the effects of global climate change, the decreasing availability of arable land, and the continuous increase in global population, food security is becoming increasingly challenging [158,159,160]. Consequently, increasing crop yield remains a central objective in agricultural research and breeding programs [161]. Among the key determinants of yield is seed number, which is fundamentally dependent on the number of ovules initiated within the carpels by meristematic tissues [9]. As ovule number directly influences potential seed number, it serves as a primary factor shaping both reproductive success and fruit yield.
Ovules originate from meristematic tissue in the gynoecium, and, following successful fertilization, each ovule gives rise to a seed, while the gynoecium itself matures into the fruit [71,162]. As such, ovule number represents a key heritable trait with significant implications for crop improvement [163]. Ovule number per flower defines the theoretical maximum number of seeds that can be produced, while the final seed number is further constrained by the spatial and developmental limitations imposed by fruit structure, such as the size of the fruit [164]. Thus, coordinated regulation of ovule initiation and fruit development is critical for optimizing seed set and fruit quality.
Importantly, increases in ovule number must also be evaluated in the context of seed viability and quality. Therefore, elucidating the molecular mechanisms that govern ovule development is essential for advancing modern agricultural productivity. Ovule development is tightly regulated by complex gene regulatory networks involving transcription factors that control placenta patterning, ovule primordium initiation, and morphogenesis [12,13,165]. Given the positive correlation between ovule number and gynoecium size, several key genetic determinants of these traits have been identified [163,166]. A comprehensive understanding of these regulatory networks offers new avenues for enhancing crop yield through targeted genetic improvement. As seedlessness is a desirable trait that improves ease of consumption [167], developing new seedless varieties with large, high-quality fruits is a key objective in fruit breeding [79,168]. Seedlessness has been bred into many fruit species, including grape [168], litchi [169], citrus [170], watermelon [171], and pear [79]. Therefore, clarifying the development mechanism of the pomegranate ovule is the basis of exploring the characters of polyovule and functional male flowers in pomegranate.

6. Conclusions

Diverse factors and genetic networks in ovule development indicate that different pathways act cooperatively to ensure the proper establishment and progression of seed through the regulation of hub factors. We propose a model for pomegranate ovule development that explains how the ovule is regulated by the INO, CRC, BEL1, and MADS-box homeodomain protein complex, and miRNAs (Figure 1). PgAGL11 promotes ovule development in transgenic Arabidopsis. PgCRC, PgINO and PgBEL1 regulate ovule development and seed determination. As a protein molecular bridge, the PgBEL1 protein combines with PgCRC, PgINO and PgSEP proteins, which are involved in regulating pomegranate ovule and seed development. The PgARF6s protein function in the cell nucleus and PgmiR167a directly targets PgARF6a and affects its expression. PgmiR166a could interact with its target genes (Gglean013966.1 and Gglean012177.1) to affect ovule development. In addition to these factors regulating ovule development, recent studies have revealed that plant hormones brassinosteroids and cytokinin, as well as the post-translational modifications of proteins (such as acetylation and S-sulfenylation modifications), are also involved in the regulation of ovule development in A. thaliana [44,165,172,173], which provides insights into exploring the mechanisms of pomegranate ovule development from multiple perspectives. This review lays the foundation for elucidating polyovule development and ovule sterility for functional male flowers in pomegranate.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae12010026/s1, Supplementary Figure S1: Expression patterns of ovule identify genes in PgBEL1 transgenic Arabidopsis lines. Several pre-bloom flowers were collected for each transgenic line. Stem, leaf, and young siliques were collected at the same time. Mature siliques were collected when the seed coat turned yellow. Data were means ± SD of three technical replicates. ∗ represents the level of significance difference p < 0.05, ∗∗ represents the level of significance difference p < 0.01 in independent-samples t-test. Supplementary Figure S2: Relative expression of PgAG (A), PgSTK (B), PgSEP1 (C) and PgSEP4 (D) in pomegranate flowers at different development stages. Buds are divided into eight developmental stages, according to Zhao Y.J. (2023) [90]. Data are means ± SD of three technical replicates. Supplementary Figure S3: (A) Relative expression of PgmiR167d and target genes PgARF6s in bisexual and functional male flowers, respectively. (B): Relative expression of PgmiR164c-5p and target genes PgCUCs in bisexual and functional male flowers, respectively. (C): Relative expression of PgmiR160a-3p and target genes PgARF10, PgARF16s, and PgARF17 in bisexual and functional male flowers, respectively. Supplementary Data.

Author Contributions

Writing—original draft preparation, Y.Z.; writing—review and editing, Y.Z., H.S. and J.J.; validation and formal analysis, M.L., Y.M., X.Z. (Xiaoyan Zhu), K.Z., P.H., R.W., L.C., Y.S., Y.L., M.W., J.S., S.S. and T.B.; funding acquisition, Z.Y., J.J. and X.Z. (Xianbo Zheng); the corresponding authors supervised the entire experimental process. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the China Scholarship Council (CSC, 202008320482), the Initiative Project for Talents of Nanjing Forestry University (GXL2014070), the fund for modern agricultural industrial technology systems of Henan province (HARS-22-09-G3), Henan Provincial Science and Technology Research Project (242102110284), and the Basic Research Program of Key Scientific Research Projects of Henan Province Universities (25B210005).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Pomegranate flower transcriptome data (PRJNA754480) and microRNA sequencing data (PRJNA793612) can be downloaded from the NCBI database.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The regulation model of ovule development in pomegranate. The solid lines indicate the confirmed interaction relationships. The dashed lines represent regulatory relationships which are predicted or at the transcriptional level. Arrows indicate positive regulation or activation, and strings represent negative regulation or repression.
Figure 1. The regulation model of ovule development in pomegranate. The solid lines indicate the confirmed interaction relationships. The dashed lines represent regulatory relationships which are predicted or at the transcriptional level. Arrows indicate positive regulation or activation, and strings represent negative regulation or repression.
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Table 1. The list of key genes of ovule development in plants.
Table 1. The list of key genes of ovule development in plants.
SpeciesGene NameGene NumberBiological FunctionReferences
A. thaliana, Populus alba, Oryza sativaAG (AGAMOUS), OsMADS13AT4G18960, Potri.004G064300 (PtAG1), Potri.011G075800 (PtAG2)determine ovule identity, repress carpel development[40,41,42]
A. thaliana, P. albaSTK (SEEDSTICK)AT2G01930determine ovule identity, floral organ determinacy, and ovule differentiation[25,33,41]
A. thalianaBEL1 (BELL1)AT5G41410,determine ovule identity[33,39,40]
A. thalianaSHP (SHATTERPROOF)AT3G58780
AT2G42830		determine ovule identity[21,25]
Petunia hybridaFBP7 (Floral Hinding Protein7)CAA57311determine ovule identity[30,31]
P. hybridaFBP11 (Floral Hinding Protein 11)CAA57445determine ovule identity[30,43]
A. thaliana, Vitis vinifera, Solanum lycopersicum, Nicotiana tabacum, Prunus speciesINO (INNER NO OUTER)AT1G23420, VIT201s0127g00330, Solyc05g005240.1.1determine outer integument formation[44,45,46,47]
A. thalianaSUP (SUPERMAN)AT3G23130determine integument development[48]
A. thaliana, Ginkgo bilobaANT (AINTEGUMENTA)AT4G37750determine ovule primordium initiation and integument development[16,25,26]
A. thalianaLUG (LEUNIG)AT4G32551regulate gynoecium and ovule development[49,50,51]
A. thalianaSEU (SEUSS)AT1G43850regulate ovule primordium[52,53]
A. thalianaPAN (PERIANTHIA)AT1G68640regulate inflorescence, embryo and ovule development[52,54,55]
A. thalianaHLL (HUELLENLOS)AT1G17560regulate integument development[56,57]
A. thalianaSIN 2 (SHORT INTEGUMENTS 2)AT2G41670regulate integument development[58,59]
A. thaliana, V. vinifera, Malus domesticaSEP1 (SEPALLATA1)AT5G15800, VIT_14s0083g01050, Cor0054610.1, MD17G1065400determine the identity of carpels[29,60,61,62]
A. thaliana, Vitis viniferaSEP2 (SEPALLATA2)AT3G02310, VIT_17s0000g05000determine the identity of carpels[29,60]
A. thaliana, V. viniferaSEP3 (SEPALLATA3)AT1G24260, VIT_01s0010g03900, Cor0008190.1determine the identity of carpels, ovules formation[29,60,61,63]
A. thaliana, V. viniferaSEP4 (SEPALLATA4)AT2G03710, VIT_01s0011g00110, Cor0142960.1regulate the development of petals, stamens, carpels, and sepal[60,61,64]
A. thalianaABS (ARABIDOPSIS
BSISTER)		AT5G23260regulate inner seed/coat layer formation[65]
A. thaliana, Cucumis melo,
Phalaenopsis equestris, Physalis floridana, S. lycopersicum		CRC (CRABS CLAW)AT1G69180,
XP_020572967, MN969999, XM_004228801.4, Solyc01g01
0240, Solyc05g012050		carpel identity, nectary development, ovule fate determination[24,37,38,66,67]
A. thalianaZFP11 (ZINC FINGER PROTEIN11)AT2G42410ensure ovule primordia and number development[44]
A. thalianaNZZ (NOZZLE)AT4G27330adaxial–abaxial ovule development[68]
A. thalianaCUC (CUP-SHAPED COTYLEDON)AT3G15170, AT5G53950, At1g76420promote carpel margin meristem formation, regulate ovule number and morphology[9,69,70,71]
A. thalianaKNAT3, KNAT4 (KNOTTED1-LIKE)AT5G25220
AT5G11060		regulate integument and ovule formation[72]
A. thaliana, S. lycopersicumARF6 (AUXIN RESPONSE FACTOR 6)AT1G30330, Solyc00g196060, solyc07g043610/043620regulate ovules, anther, and seed development[73,74]
A. thaliana, S. lycopersicumARF8 (AUXIN RESPONSE FACTOR 8)AT5G37020, EF667342, Solyc03g031970, Solyc02g037530regulate ovules, anther, and seed development[73,74,75]
A. thalianaPIN3 (PINFORMED3)AT1G70940promote the late ovule initiation[76]
A. thalianaPHB (PHABULOSA)AT2G34710determine inner and outer integument development[77]
A. thalianaATS/KAN4 (ABERRANT TESTA SHAPE)AT5G42630regulate integument growth[78]
A. thalianaTCP4 (TEOSINTE BRANCHED1/CYCLOIDEA/PCF)AT3G15030maintain ovule identity[39]
Pyrus communisEIL1 (Ethylene-Insensitive 3-Like)AT5G21120, LOC103928267regulate ovule senescence[79]
A. thaliana, Phalaenopsis aphroditeOAF (Ovule Activating Factor)AT3G10910regulate ovule development[80]
A. thaliana, S. lycopersicumWUS (WUSCHEL)Solyc02g083950regulate the development of ovules[81]
A. thalianaEPFL (EPIDERMAL PATTERNING FACTOR-LIKE)At5g10310, At4g37810regulate ovule number and morphology, promote ovule initiation[9]
A. thalianaERL (ERECTA-LIKE)At5g62230, At5g07180promote seed density[9]
A. thaliana, V. viniferaBPC (BASIC PENTACYSTEINE)AT2G01930 (BPC1),
AT1G14685 (BPC2), AT1G68120 (BPC3), 100233138, 100255909, 100233069, 100233070		regulate ovule development[32,82]
Table 2. The list of key genes of ovule development in pomegranate.
Table 2. The list of key genes of ovule development in pomegranate.
A. thaliana OrthologueGene NumberBiological FunctionReferences
STK (SEEDSTICK)OP828677ovule identity, floral organ determinacy, ovule differentiation[4,83]
AG (AGAMOUS)OP828676regulate reproductive organ identity and meristem determinacy[4]
BEL1 (BELL1)OP828678ovule identity[4]
INO (INNER NO OUTER)OP828670ovule outer integument formation, positive regulation of ovule number[4]
WUS (WUSCHEL)OP828679regulate the development of ovules[4]
SEP1 (SEPALLATA1)OP828672the development of petals, stamens and carpels, and negative regulation of ovule development[4]
SEP3 (SEPALLATA3)OP828673, OP828674determine the identity of carpels, and ovule formation[4]
SEP4 (SEPALLATA4)OP828675regulate the development of stamens, carpels, and sepal, and negative regulation of the number of ovules[4]
CRC (CRABS CLAW)OP828671carpel identity, nectary development, positive regulation of ovule differentiation, and ovule fate determination[4]
NST3/NAC012Pgr01G048970regulate the thickness of the inner seed coat[84]
Table 3. Conserved miRNAs, and their targets and functions in plant flower development.
Table 3. Conserved miRNAs, and their targets and functions in plant flower development.
miRNA NameTarget GenesFunctionReference
miR156/157SPL3/4/5/9vegetative-to-reproductive phase transitions and flowering[123,124]
miR159MYB33/65/101flowering time; floral patterning; anther development[125,126,127]
miR160ARF10/16/17carpel development; floral patterning; predicted positive regulator of ovule development[128,129]
miR164CUC1, CUC2, NAC1petal number control; normal embryonic development; putative positive regulator of ovule development[130,131,132]
miR165/166PHB, PHV, REV, HB15adaxial and abaxial polarity; embryo development; negative regulator of ovule development[133,134,135,136]
miR167ARF6, ARF8floral induction; ovule, embryonic and anther development; putative positive regulator of ovule development[73,127,137]
miR169NF-YA (NUCLEAR TRAN-SCRIPTION FACTOR Y SUBUNIT ALPHA)regulate stamen and carpel development[138]
miR172AP2, TOE1/2/3, SMZ, SNZflowering time; seed and floral development[127,139,140,141]
miR319TCPsregulation of flower development; male and female gametophyte development[142,143]
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Zhao, Y.; Sassa, H.; Li, M.; Miao, Y.; Zhu, X.; Hao, P.; Wan, R.; Zhang, K.; Cong, L.; Shen, Y.; et al. Deciphering the Molecular Mechanisms That Control Ovule Development in Pomegranate. Horticulturae 2026, 12, 26. https://doi.org/10.3390/horticulturae12010026

AMA Style

Zhao Y, Sassa H, Li M, Miao Y, Zhu X, Hao P, Wan R, Zhang K, Cong L, Shen Y, et al. Deciphering the Molecular Mechanisms That Control Ovule Development in Pomegranate. Horticulturae. 2026; 12(1):26. https://doi.org/10.3390/horticulturae12010026

Chicago/Turabian Style

Zhao, Yujie, Hidenori Sassa, Ming Li, Yifei Miao, Xiaoyan Zhu, Pengbo Hao, Ran Wan, Kunxi Zhang, Liu Cong, Yawen Shen, and et al. 2026. "Deciphering the Molecular Mechanisms That Control Ovule Development in Pomegranate" Horticulturae 12, no. 1: 26. https://doi.org/10.3390/horticulturae12010026

APA Style

Zhao, Y., Sassa, H., Li, M., Miao, Y., Zhu, X., Hao, P., Wan, R., Zhang, K., Cong, L., Shen, Y., Liu, Y., Wang, M., Shi, J., Song, S., Bai, T., Jiao, J., Yuan, Z., & Zheng, X. (2026). Deciphering the Molecular Mechanisms That Control Ovule Development in Pomegranate. Horticulturae, 12(1), 26. https://doi.org/10.3390/horticulturae12010026

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