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Abstract
ABSTRACT
Fruit development typically follows pollination and fertilisation. However, parthenocarpy, the formation of seedless fruits, represents an alternative pathway. The fruit that grows from the ovary under unpollinated and/or unfertilised conditions is termed parthenocarpic. This trait is not only desirable to consumers but also contributes to yield stability under adverse environmental conditions. In this study, we demonstrate that overexpression of miR156h regulates parthenocarpy under unpollinated conditions via a gibberellic acid (GA)‐dependent pathway. MdSPL13B was found to be involved in MdmiR156h‐modulated GA accumulation and parthenocarpy through directly binding to the promoters of
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Introduction
In most sexually reproducing plants, fruit formation and development typically require the process of pollination and fertilisation. Parthenocarpy refers to the development of fruits without pollination and fertilisation. It ensures fruit yield even under challenging environments (Gorguet et al. 2005; Lukyanenko 1991). Additionally, parthenocarpic fruits are seedless, which enhances their desirability among consumers.
Parthenocarpy can occur naturally or be induced by various stimuli, including hormonal treatments (Qian et al. 2018; Sharif et al. 2022). Endogenous hormone levels in the ovaries of parthenocarpic fruits were observed to be elevated, indicating that plant hormones play a critical role in this process (Li et al. 2014). Naturally occurring parthenocarpy is often associated with specific hormonal pathways. For example, the naturally occurring parthenocarpy in tomato mutants pat (parthenocarpic fruit), pat-2 and pat-3/pat-4 was found to be dependent on the gibberellin pathway (Fos et al. 2000). Both gibberellic acid and auxin are well documented to influence parthenocarpy. During the pollination stage of custard apples, adverse environments can severely impact fruit development and reduce yield. This issue can be mitigated by applying GA3 to induce the formation of seedless fruits (Santos et al. 2016). Conversely, the application of GA biosynthesis inhibitors, such as paclobutrazol, significantly impaired fruit development induced by the auxins indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), whereas the application of GA3 reversed this effect (Serrani et al. 2008). It was also reported that DELLA proteins (in the GRAS family of plant-specific nuclear proteins) inhibited gibberellin signalling, and the silencing of DELLA induced parthenocarpy in tomatoes (Martí et al. 2007).
MicroRNAs (miRNAs) are a major group of small RNAs and influence almost all aspects of agronomic traits through post-transcriptional regulation of gene expression (Wang and Wang 2015). However, the function of miRNAs in parthenocarpy remains ambiguous. Self-incompatibility and male sterility are known to promote fruit set and the production of seedless fruits (Mahajan et al. 2011; Rojas-Gracia et al. 2017; Roque et al. 2019). miRNAs play an important role in regulating floral architecture. Well-studied miRNAs are miR156, miR159 and miR172 (Shi et al. 2018). miR156 regulates vegetative phase change in plants by targeting SQUAMOSA promoter-binding protein-like (SPL) genes and inhibiting the expression and translation of SPL (Wu et al. 2009). In Arabidopsis, the spl8 mutant exhibited abnormal pollen sac formation, reduced pollen counts and reduced fertility (Xing et al. 2010). Constitutive overexpression of miR156 in the spl8 mutant background resulted in complete plant sterility. miR159 targeted GAMYB-like genes (encode a highly conserved family of R2R3 MYB domain transcription factors) and resulted in a series of defects including male sterility (Alonso-Peral et al. 2010). Furthermore, overexpression of SlMIR159 was associated with abnormal ovule development, leading to the formation of seedless tomato fruits (da Silva et al. 2017). The mutation of miR172 in maize led to the development of extra florets in the spikelet without causing pistil abortion in the tassels (Chuck et al. 2007). Similarly, in wheat and rice, overexpression of miR172 altered spike morphogenesis (Debernardi et al. 2017; Lee and An 2012).
Previous studies demonstrated that miRNAs and their target genes were directly or indirectly regulated by histone-modifying Polycomb Group (PcG) proteins in Arabidopsis (Lafos et al. 2011). PcG proteins are crucial epigenetic regulators involved in eukaryotic development. PcG proteins exist as two main multiprotein complexes: Polycomb Repressive Complex1 (PRC1) and PRC2. The primary role of PRC2 is to recruit target genes and catalyse the histone H3 lysine-27 trimethylation (H3K27me3). The core of the PRC2 complex contains Extra Sex Combs (ESC), Enhancer of Zeste [E(Z)], the PcG repressor SU(Z)12 and Nurf55/p55 in Drosophila (Müller et al. 2002). Our previous research identified eight Nurf55/p55 homologues in apple (Wang, Wang, et al. 2022), designated as the multicopy suppressor of IRA (MSI). StMSI1 regulates potato aerial tuber formation by inhibiting StBMI1, a PRC1 member upstream of miR156 (Kumar et al. 2020). It has also been reported that AtMSI1 is epigenetically required for the maintenance of reproductive development (Hennig et al. 2003). Reduced expression of AtMSI1 disrupted ovule development and resulted in complete female sterility (Hennig et al. 2003).
In a previous study, overexpression of apple MIR156h in Arabidopsis resulted in a prolonged juvenile stage, shorter siliques and partial seed desiccation without fertility (Sun et al. 2013). Here, we demonstrate that miR156h-SPL13B in apple regulates parthenocarpy by affecting GA content. MdSPL13B is involved in miR156h-mediated parthenocarpy by directly binding to the promoters of ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase 2 (KAO2) and GA 20-oxidase (GA20ox), thereby repressing their transcription and reducing GA accumulation. In addition, MdSPL13B regulates parthenocarpy by binding to the MdMSI1 promoter and regulating the levels of H3K27me3 modifications by or on GA 2-oxidase 8 (GA2ox8) and SPINDLY (SPY). This study elucidates the role of the miR156-SPL module in regulating parthenocarpy, which provides a theoretical framework for identifying candidate genes for parthenocarpy breeding in apple.
Results
Overexpression of
In 2013, we discovered that ectopic expression of MdMIR156h in Arabidopsis resulted in shorter fruit pods and partial seed abortion (Sun et al. 2013). Here, we found that siliques of unpollinated MdMIR156h transgenic Arabidopsis lines were able to expand but had no seeds (Figure S1), which is a characteristic feature of parthenocarpy (Vivian-Smith and Koltunow 1999).
In situ hybridization results revealed that MdmiR156h is expressed at a relatively high level in the receptacle (Figure S2), which serves as the primary edible tissue composing apple fruits. To further investigate the role of miR156h in parthenocarpy, MdMIR156h was transformed into ‘Gala’ apple to generate two overexpression lines (Figure S3A,B). The two overexpression lines were subjected to pollination and unpollination treatments at stage 4 of apple flower development (Liu et al. 2014). After 16 days after flowering (DAF) under unpollination conditions, the stalks of WT ‘Gala’ abscised, while the stalks of MdMIR156h overexpression apple lines remained attached, and fruit development commenced (Figure 1A and Figure S3C). In addition, unpollinated MdMIR156h overexpression apple fruits did not show significant morphological differences compared to the pollinated ones, but the unpollinated MdMIR156h overexpression apple lines exhibited higher fruit set at the fruit ripening stage relative to the unpollinated WT ‘Gala’ (Figure 1B,C and Figure S3D).
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Subsequently, MdMIR156h was ectopically expressed in the tomato line ‘Micro Tom’ (Figure S3E,F). Similar pollination and unpollination treatments were applied to WT ‘Micro Tom’ and MdMIR156h overexpression tomato lines. The results showed that unpollinated MdMIR156h overexpression tomato lines were able to develop fruits without seeds (Figure 1D,E and Figure S3G), similar to the MdMIR156h overexpression apple lines. Meanwhile, to assess the evolutionary conservation of miR156 in regulating parthenocarpy, the orthologous gene SlMIR156a was overexpressed in the tomato line Ailsa Craig ‘AC’ (Cui et al. 2020). Parthenocarpic fruit formation was also observed in SlMIR156a transgenic ‘AC’ (Figure 1F,G and Figure S3H). Taken together, these results indicate that miR156 plays a conserved role in the regulation of parthenocarpy across different plant species.
We found that the mature sequences of MdmiR156h and SlmiR156a are identical (Figure S4C), indicating a high degree of evolutionary conservation of miR156 in these two different plant species, apple and tomato. In previous studies, after the expression of SPLI3 was inhibited by RNAi, most of the phenotypes it exhibited were similar to those of SlMIR156a-OE (Cui et al. 2020). It is speculated that MdmiR156h and SlmiR156a may share similar target genes and regulatory functions. When conducting a comparison of the phylogenetic tree of SlSPL13 with that of the MdSPLs targeted by MdmiR156h in apple, two SPLs were identified to exhibit an extremely close evolutionary relationship with SlSPL13 (Figure S4A). These two SPLs were designated as MdSPL13A (Mdg_14g014950) and MdSPL13B (Mdg_06g013140). We performed RT-qPCR validation and found that MdSPL13B differed most significantly compared to Gala-pol (Figure S4B). RLM-5'RACE analysis verified the presence of a cleavage site on the third exon of MdSPL13B with a cleavage frequency of 8/16 (Figure S4C). Co-expression of MdMIR156h and MdSPL13B demonstrated a significant reduction in β-glucuronidase (GUS) staining and activity, indicating that MdMIR156h suppresses MdSPL13B expression (Figure S4D,E). In addition, the transcription inhibition of MdSPL13B by MdMIR156h was also demonstrated by dual-luciferase activity assay (Figure S4F). Collectively, these results suggest that miR156h targets and downregulates MdSPL13B.
To investigate the role of SPL13B in parthenocarpy, MdSPL13B-overexpressing (MdSPL13B-OE) and SlSPL13B silencing (SlSPL13B-RNAi) transgenic tomato lines were generated (Figure 2A). Phylogenetic analysis of the tomato SPL family revealed that SlSPL13B was most closely related to Solyc01g090730 and Solyc10g018780 (Figure S5A). qRT-PCR assays demonstrated that the expression levels of these two genes in SlSPL13B-RNAi lines showed no significant difference compared with WT ‘Micro Tom’ (Figure S5B), confirming that the RNAi specifically targets SlSPL13B without interfering with other SPL family members. SlSPL13B-RNAi-1 transgenic tomato line produced seedless fruits without pollination, while WT ‘Micro Tom’ and MdSPL13B-OE1 transgenic tomato line did not (Figure 2B–D).
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Abscission is a key indicator of fruit set (Olsson and Butenko 2018). Genetic analysis of MdMIR156h and MdSPL13B in regulating parthenocarpy was verified via a virus-induced gene silencing (VIGS) system (Figure 2E). At stage 4 of flower development, the recombinant plasmid MdSPL13B-IL60 was infiltrated into pollinated or unpollinated WT ‘Gala’ and MdMIR156h-L1 apple ovaries. The IL60 empty vector was included as a negative control. This process generated three transiently transformed plant materials: MdSPL13B-IL60, MdMIR156h-L1 + MdSPL13B-IL60 and IL60 (Figure 2F). After 10 days of treatment, the apple fruit set rate of all pollinated materials remained at about 95%. In contrast, the apple fruit set rate of unpollinated MdMIR156h-L1 was about 55%, while MdSPL13B-IL60 and MdMIR156h-L1 + MdSPL13B-IL60 exhibited an apple fruit set rate of 6% (Figure 2F). These results suggest that MdSPL13B is involved in MdMIR156h-mediated parthenocarpy.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed a significant enrichment of differentially expressed genes (DEGs) within the ‘Plant hormone signal transduction’ pathway (Figure S6A). Thus, it was hypothesized that miR156h may regulate parthenocarpy through phytohormone signalling pathway. A previous study showed that phytohormones, especially auxin and GA, are key regulators of parthenocarpy (Sharif et al. 2022). Therefore, the contents of IAA (indoleacetic acid) and GA in the fruits of WT ‘Gala’ and MdMIR156h overexpression apple lines were measured at 16 DAF. We found no significant difference in IAA content between Gala-pol and MdMIR156h-pol; however, the IAA content in MdMIR156h-par was much lower (Figure 3A, top panel). MdMIR156h-pol fruits accumulated significant higher levels of GA3 and GA4 than Gala-pol, whereas GA levels in MdMIR156h-par were comparable to those in Gala-pol (Figure 3A).
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Among the DEGs, genes involved in GA biosynthesis, such as GA1, KO, KAO1 and GA20ox, and genes involved in GA signalling, such as GIID1C, GASA4 and GASA11, were upregulated in MdMIR156h-pol and MdMIR156h-par fruits compared to Gala-pol (Figure S6B,C). In addition, the GA oxidase gene GA2ox8 and the SPY gene, a negative regulator of GA signalling, were downregulated in MdMIR156h-pol and MdMIR156h-par plants compared to Gala-pol (Figure S6C). The above results suggest that miR156h may regulate parthenocarpy through the GA biosynthesis pathway.
To further validate whether miR156h regulates parthenocarpy through the GA pathway, MdMIR156h overexpression tomato lines were treated with GA and PAC (Paclobutrazol, GA biosynthesis inhibitor). It was found that WT ‘Micro Tom’ and MdMIR156h overexpression tomato lines treated with GA successfully set fruit. In contrast, PAC-treated tomato plants failed to set fruit under unpollinated conditions (Figure 3B,C). Notably, no significant difference in single fruit weight, transverse diameter, and longitudinal diameter was observed between WT ‘Micro Tom’ and MdMIR156h overexpression tomato line fruits following GA treatment. However, MdMIR156h overexpression tomato lines exhibited higher productivity than the other plants (Figure 3D).
Given that miR156h regulates parthenocarpy through GA synthesis and SPL13B is involved in miR156h-mediated parthenocarpy, we further investigated whether SPL13B regulates parthenocarpy via the GA pathway. The MdSPL13B-OE1 and SlSPL13B-RNAi-1 transgenic tomato lines were treated with GA and PAC. All GA-treated tomatoes set fruit successfully, while tomatoes treated with PAC did not (Figure 3E–G).
Transcriptome analysis suggested that MdSPL13B might influence GA synthesis through MdGA20ox2, MdGA1, MdKO, MdKAO1, MdKAO2 and MdGA20ox (Figure S6C). These genes were significantly downregulated in MdSPL13B overexpression apple calli lines and promoter analysis identified GTAC cis-elements in all of them (Figure 4A,B). Electrophoretic mobility shift assay (EMSA) showed that MdSPL13B binds to the promoters of genes involved in GA biosynthesis (MdKO, MdKAO2 and MdGA20ox) (Figure 4C). Binding bands were weakened with the addition of competing probes and disappeared with mutation probes (Figure 4D). Furthermore, chromatin immunoprecipitation qPCR (ChIP-qPCR) and yeast one-hybrid (Y1H) assays confirmed that MdSPL13B directly binds to the promoters of MdKO, MdKAO2 and MdGA20ox (Figure 4E,F). Transcriptional repression of these genes by MdSPL13B was also verified by LUC and GUS staining assays (Figure 4G,H). Taken together, these results indicate that MdSPL13B directly binds to the promoters of GA biosynthesis related genes and participates in MdMIR156h-modulated parthenocarpy.
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Compared with Gala-pol, genes related to GA deactivation and signalling (including MdGA2ox8 and MdSPY) are significantly downregulated in MdMIR156h-pol and MdMIR156h-par (Figure S6C). The transcription of MdGA2ox8 and MdSPY was also significantly altered in MdMIR156h and MdSPL13B overexpression apple lines (Figure S7A–C). However, MdSPL13B did not bind to the promoters of MdGA2ox8 and MdSPY directly (Figure S7D,E), suggesting that MdSPL13B may regulate the transcript of these genes through indirect mechanisms. In Arabidopsis, genes related to GA pathways—for example, GA2ox8, are regulated by H3K27me3 modifications (Zhang et al. 2007). MdMIR156h-OE apple calli lines exhibited a higher level of H3K27me3 than the WT, while MdSPL13B-OE apple calli lines demonstrated the opposite effect (Figure S8). In order to explore this further, nine regions from the promoter, UTR, introns and exons of MdGA2ox8 and MdSPY were selected for analysis (Figure 5A). This result suggests that epigenetic modification may be involved in the transcriptional regulation of MdGA2ox8 and MdSPY by the miR156h-MdSPL13B module. PcG proteins, including MEDEDA (MEA), FERTILISATION-INDEPENDENT SEED (FIS), FERTILISATION INDEPENDENT ENDOSPERM (FIE), MULTICOPY SUPPRESSOR OF IRA1 (MIS1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1) and EMBRYONIC FLOWER2 (EMF2), are known to regulate the target genes of H3K27me3 (Mozgova and Hennig 2015). However, a Y2H assay suggests that MdSPL13B does not interact with the tested proteins directly (Figure S9). Interestingly, the transcription level of MdMSI1 was enhanced in the MdMIR156h-OE apple calli lines, while it showed an opposite trend in the MdSPL13B-OE apple calli lines (Figure 5B). Subsequent Y1H, EMSA and ChIP-qPCR assays showed that MdSPL13B protein interacts with the promoter of MdMSI1 (Figure 5C–E). Furthermore, LUC and GUS assays confirmed that MdSPL13B directly binds to the promoter of MdMSI1 and represses its expression (Figure 5F–H). Taken together, these results suggest that MdSPL13B interacts with the promoter of MdMSI1 and represses its expression.
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To further investigate the role of MdMSI1 in regulating GA metabolism and parthenocarpy, overexpressing MdMSI1 transgenic apple lines were generated (Figure 6A). Fruit stalks were observed attached to the fruit within 16 DAF under unpollinated conditions in MdMSI1 transgenic apple lines, while the fruit stalks abscised in WT ‘Gala’ (Figure 6B). The fresh weight, transverse diameter, longitudinal diameter and fruit set rate of the ovaries in MdMSI1 transgenic apple lines were larger than those in WT ‘Gala’ under unpollinated conditions (Figure 6C). At the fruit ripening stage, unpollinated MdMSI1-OE transgenic apple lines fruits were seedless (Figure 6D and Figure S11). In addition, heterologous overexpression of MdMSI1 in tomato led to seedless fruits under unpollinated conditions (Figure 6E–G and Figure S12). The unpollinated MdMSI1-OE transgenic tomato lines were able to set fruit and had no seeds. These results verified the role of MdMSI1 in regulating parthenocarpy.
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Moreover, the content of H3K27me3 was found significantly higher in MdMSI1-OE apple calli lines than that in the WT, while MdMSI1-RNAi showed the opposite trend (Figure S13A,B). The expression of MdGA2ox8 and MdSPY was significantly downregulated in MdMSI1-OE (Figure S13C). Transcriptome analysis revealed a significant downregulation of MdGA2ox8 and MdSPY in MdMSI1 overexpressed Gala-pollinated (MdMSI1-pol) and MdMSI1 overexpressed Gala-unpollinated (MdMSI1-par) compared to Gala-pol (Figure S14B). The H3K27me3 modification of MdGA2ox8 and MdSPY was significantly enhanced in the MdMSI1-OE transgenic apple calli line and reduced in the MdMSI1-RNAi transgenic apple calli line relative to WT (Figure 7A). Meanwhile, we also examined the H3K27me3 levels of MdKO, MdKAO2 and the results indicated that there was no significant difference between the transgenic calli and WT (Figure S15). The above results indicate that MdMSI1 regulates parthenocarpy through H3K27me3 modification of MdGA2ox8 and MdSPY. KEGG analysis with Gala-pol, MdMSI1-pol and MdMSI1-par identified significant enrichment of DEGs in phytohormone signalling pathways (Figure S14C). No significant difference in IAA content was observed between Gala-pol and MdMSI1-pol, while the IAA content in MdMSI1-par was much lower. GA3 and GA4 accumulated at higher levels in MdMSI1-pol than those in Gala-pol. No significant difference in GA3 content was observed between MdMSI1-par and Gala-pol; however, the content of GA4 was higher in MdMSI1-par than Gala-pol (Figure S14D). GA and PAC treatments of MdMSI1-OE transgenic tomato lines revealed that all GA-treated tomato plants set fruit, and the PAC-treated did not (Figure S16). The phenotypes of MdMSI1-OE transgenic tomato lines under the treatments of GA and PAC mirrored those observed in MdMIR156h-OE transgenic tomato lines. Taken together, these results suggest that MdMSI1 regulates parthenocarpy through the GA signalling pathway.
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To explore the role of MdMSI1 in regulating parthenocarpy alongside MdSPL13B and MdMIR156h, five transiently transformed apple ovaries were generated using the VIGS system: MdSPL13B-TRV, MdMSI1-TRV, MdSPL13B-TRV + MdMSI1-TRV, MdMIR156h-L1 + MdMSI1-TRV and TRV (Figure 7B,D). After 10 days under unpollinated conditions, MdSPL13B-TRV and stably transformed MdMIR156h-L1 exhibited high fruit set compared to the other unpollination treatments (Figure 7C,E). Notably, MdMIR156h-L1 + MdMSI1-TRV exhibited about 20%–40% fruit set after 10 days under unpollinated conditions (Figure 7E). These results suggest that the miR156h-MdSPL13B module may regulate parthenocarpy via an additional pathway involving direct regulation of GA-related genes.
Discussion
Recent studies have identified the miR156/SPL module as a multifunctional toolbox for improving agronomic traits in crops (Wang and Wang 2015), with miR156 being recognised as a key regulator of vegetative phase change (VPC) in plants (Poethig 2013; Teotia and Tang 2015). Reproductive organs of plants are among the most complex organs in terms of structure and function. Multiple miR156/7 targeted SPL genes were required for male and female fertility in Arabidopsis (Xing et al. 2013, 2010). miR156 and miR172 mainly cooperate with each other or play complementary roles in different links when regulating plant functions. In terms of regulating plant flowering, miR156 delays flowering by inhibiting genes of the SPL family (Yu et al. 2012), while miR172 promotes flowering by suppressing genes of the AP2 family (Zhang and Chen 2021). NON-PARTHENOCARPIC FRUIT 1 (NPF1), belonging to the AP2-like transcription factor, plays a crucial role in the formation of parthenocarpic fruits in cucumbers (Nie et al. 2025). However, the role of miR156-SPL in regulating parthenocarpy remains unclear. Our findings revealed that MdMIR156h acts as a positive regulator of parthenocarpy (Figure 1). Transient transformation of MdSPL13B-IL60 in apple by the VIGS system and stable overexpression of MdSPL13B in tomato demonstrated a decreased fruiting rate compared to the overexpressing MdMIR156h, while MdSPL13B-TRV and SlSPL13B-RNAi enhanced parthenocarpy (Figures 2 and 7). The fruit set rate of the MdMIR156h overexpression apple line without pollination was approximately 55%. However, after transiently transforming MdSPL13B-IL60 into the ovary of the MdMIR156h overexpression apple line using the VIGS system, the fruit set rate was significantly reduced to approximately 6% (Figure 2F). These results suggest that the miR156h-MdSPL13B module represents a key regulatory mechanism that controls parthenocarpy.
The development of seedless fruits is regulated by a complex interplay of phytohormones, with IAA and GA playing pivotal roles (He and Yamamuro 2022; Sharif et al. 2022). NPF1 can directly bind to the promoter of the auxin biosynthetic gene YUCCA4 and activate its expression, thereby increasing the auxin content and promoting the formation of parthenocarpy (Nie et al. 2025). There was no significant difference in IAA levels between MdMIR156h-pol and Gala-pol. The bioactive GAs content (including GA3 and GA4) in MdMIR156h-pol was significantly higher than that in Gala-pol (Figure 3A). Interestingly, the GAs content in MdMIR156h-par is lower than that in MdMIR156h-pol. It is possible that pollination stimulates the expression of certain genes related to GA synthesis or metabolism. These genes are independent of the MdMIR156h-SPL regulatory pathway; yet, they have an additive effect on promoting GAs synthesis in conjunction with this pathway. The role of GA in the formation of seedless fruits has been studied in a variety of horticultural crops including tomato, watermelon, apple and pear (Sharif et al. 2022). Consistent with their findings, in this study, we demonstrated that exogenous application of GA promoted the formation of seedless fruits in all tomato lines. In addition, exogenous application of PAC did not induce the formation of seedless fruits (Figure 3). In MdMIR156-OE tomato lines, exogenous application of GA promoted flowering, enhanced fruit set and increased yield by modulating plant structure (Figure 3D).
GA is essential in seed germination, fruit development and ripening. In a previous study, Arabidopsis mir156 mutants demonstrated enhanced seed dormancy and inhibited seedling growth due to the regulation of the GA pathway by IPA1 (Miao et al. 2019). GA regulates the floral transition through SPL targeted by miR156 in Arabidopsis (Yu et al. 2012). During the process of parthenocarpy, there may be a molecular mechanism similar to that of seed dormancy and floral transition regulation in Arabidopsis. The genes related to the GA pathway or metabolism may be affected by the expression regulation of MdSPL13B by miR156, thereby influencing the GAs content and ultimately having an impact on the process of parthenocarpy. In line with these observations, MdSPL13B was shown to directly bind to the genes (MdKO, MdKAO2 and MdGA20ox) in the GA pathway (Figure 4C–H). Collectively, our findings showed that the miR156h-MdSPL13B module regulates parthenocarpy by modulating the GA pathway.
Epigenetic regulation, including DNA methylation, histone modification and chromatin remodelling, plays a crucial role in regulating gene expression without altering DNA sequence. PcG proteins, key epigenetic regulators during development, catalyse H3K27me3 modification and maintain gene silencing in chromatin regions (Schuettengruber et al. 2007). PcG proteins were initially identified in
In this study, we observed that GA content was elevated in MdMSI1 overexpression lines (Figure S14D). Previous studies reported that H3K27me3 and H3K27ac are involved in the photoregulation of gibberellin metabolism (Charron et al. 2009). In
The fruit set rate of silenced MdMSI1 was not different from that of the negative control TRV. However, in the MdMIR156h overexpression apple line with transient silencing of MdMSI1, the fruit set rate of unpollinated ovary was approximately 20%–40% (Figure 7E). Simultaneously, transient silencing both MdSPL13B and MdMSI1 resulted in a higher fruit setting rate compared to silencing MdMSI1 alone (Figure 7C). These results indicate that the miR156h-MdSPL13B module regulates parthenocarpy in a manner that is not fully dependent on MdMSI1. The PRC2 complex further regulates flowering time by epigenetically regulating FLOWERING LOCUS T (FT) and by targeting the transition to the adult stage through the MIR156 (Xu et al. 2016). AtBMI1, a component of the PRC1 complex, has been shown to regulate the expression of miR156 in Arabidopsis (Picó et al. 2015). StBMI1-1 inhibited the expression of miR156; however, overexpression of StMSI1 enhanced the expression of miR156 by downregulating StBMI1-1. Moreover, the transcription of miR156 was elevated in StMSI1-OE lines (Kumar et al. 2020). Additionally, we found that the expression of MdMIR156h was enhanced and the expression of MdSPL13B was suppressed in MdMSI1-OE lines (Figure S17).
In conclusion, we propose a model in which the miR156h-MdSPL13B module modulates parthenocarpy in apple. MdMIR156h directly regulates GA pathway-related genes involved in parthenocarpy through MdSPL13B, while MdSPL13B regulates parthenocarpy by binding to the MdMSI1 promoter, influencing the histone modifications of MdGA2ox8 and MdSPY. In addition, MdMSI1 provides feedback regulation on the expression of MdMIR156h and MdSPL13B to control unisexual fruit development in apple. Our findings not only elucidate the role of the miR156-SPL module in regulating parthenocarpy, but also offer new insights for studying unisexual fruit development in different species.
Materials and Methods
Plant Materials and Growth Conditions
WT ‘Gala’ and transgenic apple lines (
WT and transgenic apple, tomato and Arabidopsis flowers were emasculated by removing stamens and stigmas before being subjected to unpollinated conditions. WT and transgenic tomato flowers were subjected to the removal of stamens and stigmas prior to pollination, and the treated flowers were sprayed with 0.1 mM GA3 and 0.1 mM paclobutrazol (PAC), respectively.
Vector Construction and Plant Transformation
Overexpression vectors for MdSPL13B and MdMSI1 were constructed by ligating the full-length coding sequences (CDSs) of MdSPL13B and MdMSI1 into the pRI101-GFP plasmid. The RNAi fragment targeting SlSPL13B and MdMSI1 was amplified from a tomato or apple cDNA library and inserted into the pRI-RNAi plasmid. Stable transformants of apple calli and tomato were transformed into plants by the Agrobacterium-mediated method following an established protocol (An et al. 2021).
Gene Expression Analysis
Total RNA was extracted from plant samples using the RNA Plant Plus Reagent Kit (Tiangen, Beijing, China). cDNA synthesis assay was performed using the PrimeScript RT Reagent Kit (Takara, Dalian, China), followed by RT-qPCR. For miR156h quantitative assays, miR156h-specific stem-loop primers were used. RT-qPCR was performed as described previously (Wang, Yang, et al. 2022). All quantitative primers are listed in Table S1.
The 35S::MdMIR156h and 35S::MdSPL13B-GUS vectors were constructed by inserting the precursor sequence of miR156h into the pRI101-GFP vector and the full-length CDS of MdSPL13B into the pRI101-GUS vector. Promoter sequences of MdKO, MdKAO2, MdGA20ox and MdMSI1 were cloned into the p1300-GN vector. The recombinant plasmids were transferred into Agrobacterium strains GV3101; the transformed Agrobacterium was used to infiltrate tobacco leaves or apple calli, which were incubated for 48 h. GUS staining and activity assay were performed as previously described (Wang, Yang, et al. 2022).
5′
The 5′ RLM-RACE method was used to identify MdSPL13B as a target gene of miR156h. Total RNA was extracted from apple fruits using the RNA Plant Plus Reagent Kit (Tiangen, Beijing, China), followed by treatment with calf intestinal alkaline phosphatase (CIP) and tobacco acid pyrophosphatase. The total RNA was ligated to an RNA adapter by incubating at 37°C for 1 h and then reverse-transcribed to cDNA. The cloned PCR products were inserted into the pEASY Blunt Cloning vector. Primers used in the 5′ RLM-RACE assay are listed in Table S1.
For the yeast one-hybrid (Y1H assay), the full-length MdSPL13B was cloned into the pGADT7 vector to generate MdSPL13B-pGADT7. The promoter segments of MdKO, MdKAO2, MdGA20ox and MdMSI1 were inserted into the pHIS2 vector. The recombinant plasmids were co-transformed into yeast strain Y187 and screened on selective medium SD-Leu/−Trp/−His/ + 30 mM or 80 mM 3-amino-1,2,4-triazole (SD-L/−T/-H+ 30 mM or 80 mM 3-AT) at 30°C for 3 days.
For dual-LUC reporter assay (An et al. 2018), the precursor sequence of miR156h and full-length CDS of MdSPL13B were ligated into the pGreen II 62-SK vector or pGreenII 0800-LUC vector. Promoter sequences of MdKO, MdKAO2, MdGA20ox and MdMSI1 were cloned into the pGreenII 0800-LUC vector. The recombinant plasmids were transformed into Agrobacterium strain GV3101, which was then infiltrated into tobacco leaves and incubated for 48 h. After spraying with 100 mM luciferin on the abaxial surface, tobacco leaves were placed in the dark for 3 min. The luminescence was detected using an in vivo imaging system (Xenogen, Alameda, CA, USA). Primers used in LUC assay are listed in Table S1.
EMSA
MdSPL13B-HIS was generated by inserting MdSPL13B into the pET-32a vector with His-tag. The recombinant plasmid was transformed into
CHIP assay was conducted according to the method described by Cui et al. Transgenic apple calli expressing 35S::MdMIR156h-GFP, 35S::MdSPL13B-GFP, 35S::MdMSI1-GFP, MdSPL13B-RNAi and MdMSI1-RNAi were analysed using a ChIP test kit (Beyotime, Shanghai, China) with GFP antibody or anti-H3K27Me3 antibody. Precipitated DNA fragments were recovered, and specific primers were used for RT-qPCR analysis. Primers used in the ChIP assay are listed in Table S1.
Y2H assays were performed as previously described (Jiang et al. 2024). The interaction between MdSPL13B and PcG proteins was studied by Y2H assays. The recombinant plasmids were transformed into the Y2H Gold yeast strain and grown on SD-Leu/−Trp. After 2–3 days of incubation, the single clones were transferred onto SD-Ade/-His/−Trp/−Leu. Primers used in Y2H assays are listed in Table S1.
Transcriptome Analyses
RNA sequencing (RNA-seq) assay was performed as previously described (Liu et al. 2023). 16-day-old apple fruits from WT ‘Gala’ and transgenic apple lines were used for transcriptome analysis. Total RNA was extracted from apple fruits, and RNA-seq libraries were constructed and analysed. Differentially expressed genes (DEGs) were identified by the following criteria: Log2[fold change] ≥ 1 and q-value ≤ 0.05.
Phytohormone Measurement
The contents of IAA, GA3 and GA4 were measured using liquid chromatography tandem mass spectrometry (LC–MS/MS). The plant materials were ground to a powder in liquid nitrogen and extracted with methanol/water at 4°C. The supernatant, collected after centrifugation at 4°C and 12 000 × g, was evaporated to dryness under a stream of nitrogen and reconstituted in methanol/water. The supernatant was analysed by LC–MS (Sciex 4500; Sciex).
Virus-Induced Gene Silencing Assays
The full-length CDS of MdSPL13B was inserted into the IL60-2 vector. A 200–300 bp fragment of the non-conserved regions of the CDSs of MdSPL13B and MdMSI1 was reverse-ligated into TRV2. The resulting constructs were MdSPL13B-IL60, MdSPL13B-TRV and MdMSI1-TRV, which were introduced into Agrobacterium LBA4404. Transformed Agrobacteria were infiltrated into apple ovaries using a vacuum plug as described previously (Jiang et al. 2024). Transformed apple ovaries were placed at room temperature for 10 days.
In Situ Hybridization
Apple flowers at the third stage of floral development were fixed in FAA (containing 10% formaldehyde, 5% acetic acid and 50% ethanol). After fixation, the tissues were subjected to sequential ethanol dehydration and xylene clearing, followed by embedding and sectioning into 8-μm-thick slices. The miR156h probe was an oligonucleotide modified with locked nucleic acid (LNA) and labelled using the DIG oligonucleotide 3′-end labelling method.
Statistical Analysis
Statistical analyses were performed using DPS v. 9.01 and graphed with GraphPad Prism 8. One-way analysis of variance (ANOVA), followed by Tukey's test was used to determine the statistical significance. Different letters indicate significant differences (p < 0.05).
Acknowledgements
We thank Dr. Jun-Hong Zhang (Huazhong Agricultural University) for providing the transgenic tomato material SlmiR156a; Dr. Zhen Gao and Zong-Bao Fan (Shandong Agricultural University) for their technical support in RLM-RACE; Dr. Jiu-Cheng Zhang (Shandong Agricultural University) for support in tomato cultivation. We are also deeply grateful to Dr. Shu-Nong Bai (Peking University) for critical discussion. This work was supported by the National Key Research and Development Program of China ( 2023YFD2301000, 2022YFD1201700), National Natural Science Foundation of China (32172538, 32272683) and China Agriculture Research System of MOF and MARA (CARS-27).
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Accession Numbers
All data that support the findings of this study are available within the article and its Supporting Information or from the corresponding author upon reasonable request. The accession codes are listed as follows: MdMIR156h: EB146843; MdSPL13B: Mdg_06g013140; SlSPL13B: Solyc05g015840; MdKO: Mdg_08g003390; MdKAO2: Mdg_06g003050; MdGA20ox: Mdg_01g006410; MdGA2ox8: Mdg_16g000690; MdSPY: Mdg_14g002790; MdMSI1: Mdg_02g019090.
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