Epigenetic regulation of fruit shape determination by JAGGED gene in Capsella rubella

Epigenetic regulation of fruit shape determination by JAGGED gene in Capsella rubella | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Epigenetic regulation of fruit shape determination by JAGGED gene in Capsella rubella Yang Dong, Tian-Feng Lü, Xiao-Yu Chen, Nicola Trozzi, Wen He, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8265099/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Fruits that protect the seeds and aid their dispersal are a key feature distinguishing angiosperms from other land plants, and fruit shape varies widely between species. However, it remains largely unknown how local growth behaviors are coordinated during development to generate fruits with characteristic sizes and shapes. Members of the Capsella genus in the mustard family produce unique fruits with an evolutionarily derived heart shape, making it an exceptional system suited for addressing this question. In this study, we demonstrate that the Capsella rubella C2H2-zinc finger transcription factor JAGGED (CrJAG) controls fruit shape. Whole-mount live imaging analysis indicates that CrJAG regulates valve development by promoting both cell division and anisotropic growth. Ectopic expansion of the valve epidermal cells in Crjag mutants is accompanied by mis-patterned endoreduplication events. CrJAG physically interacts with the histone chaperones Capsella rubella MULTICOPY SUPPRESSOR OF IRA1 members, increasing the chromatin accessibility and thereby sustaining the expression of genes involved in fruit morphogenesis. The resulting closed chromatin state in Crjag fruits is characterized by a reduction of the active histone marker (H3K18ac) and an increase of the repressive marker (H3K27me3). Expression analyses and pharmacological treatments indicate that the developmental defects in Crjag fruits are largely attributable to down-regulation of the key cell-cycle regulator CrAUR2 . Collectively, our findings suggest that fine-tuning the cell cycle via epigenetic modification represents an additional, essential layer of regulation critical to organ development. Biological sciences/Plant sciences Biological sciences/Plant sciences/Plant development Fruit Morphogenesis Capsella rubella Development Epigenetic Regulation Histone Modification Cell Division Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The evolutionary success of angiosperms is largely attributable to their ability to produce fruits 1 , 2 . The fruit is developed from the female reproductive organs, gynoecium, after pollination 3 . The fruits enclose, nourish and protect the developing seeds from harsh environments, and upon maturation, they facilitate seed dispersal and colonization of new habitats through diverse mechanisms 3 . Variations in fruit shape are associated with specific dispersal strategies 3 . Accordingly, the developmental program underlying fruit shape determination must be robustly regulated, as variations in this process would result in adaptive penalties due to limited dispersal 4 , 5 . Recently, the heart-shaped fruit of Capsella has been established as a model for understanding the developmental genetics of fruit shape determination 5 . We previously showed that development of the heart shape is promoted by localized auxin biosynthesis at the apex of the valve tips 6 , 7 . This auxin maximum in the apical region reshapes the ovate spheroid gynoecium into a heart-shaped fruit through dynamic changes in cell growth and cell division 8 . However, how cell growth and division are coordinated during the heart-shape fruit development remain largely unknown. Results and Discussion The Crjag mutant produces deformed fruits with reduced cell divisions and cell growth To identify the genetic components controlling the development of heart-shaped fruits in Capsella , we carried out a forward genetic screen of an EMS-induced Capsella rubella (Cr22.5) mutant population. One mutant, EMS1196B , was identified based on its strong defects in fruit development with compromised growth in the fruit valves (Fig. 1 a, b; Ref. 9). Bulked segregation analysis (BSA) revealed that the causal gene of the EMS1196B encodes a C2H2 zinc finger transcription factor (Carub.0002s1499), which is orthologous to the Arabidopsis JAGGED ( JAG ) gene 9 – 11 . We henceforth refer to the EMS1196B as Capsella rubella JAGGED ( CrJAG ) in the following sections. Development of the heart-shaped fruit in Wild type Capsella (CrWT ) starts from an ovate spheroid gynoecium soon after pollination at stage 12 (Extended Data Fig. 1 a). From stage 13 onwards, reshaping of fruit growth initiates from the apical parts of the valves and gradually leads to the formation of the heart shape by stage 14 (Extended Data Fig. 1 a and Ref. 6). In the Crjag mutant, however, this fruit reshaping process is abolished because valve outgrowth is significantly compromised, resulting in a fruit with an inverted triangular shape at stage 14 (Extended Data Fig. 1 b). To uncover the cellular basis of the Crjag phenotype, we performed a large-scale comparative live-imaging analysis between CrWT and Crjag fruits from developmental stage 12 (fertilization) to stage 14, when the most significant defects of the Crjag mutant are observed (Extended Data Fig. 1 a, b). Cell growth parameters, including growth rate (cell area expansion), cell proliferation, and anisotropy (direction of cell expansion), were visualized using lineage-tracking maps (Fig. 1 c, d). For each genotype, we conducted two biological replicates (see Methods). Analysis of the cell size heatmaps demonstrated a consistent distribution pattern between replicates (Fig. 1 c, d and Supplementary Fig. 1). Fruit morphogenesis in CrWT is characterized by the acropetal differentiation of epidermal cells in the valves in which cell expansion initiated from the base at stage 13 (Fig. 1 c). At stage 14, large cells are predominantly found at the base while smaller cells are distributed at the tip (Fig. 1 c and Extended Data Fig. 1 c and Ref. 8). In Crjag fruits, however, cell size increase occurs as early as stage 12 and ectopic large cells are observed in the apical and middle parts of the valves at stage 14 (Fig. 1 d and Extended Data Fig. 1 d). These data suggest that valve differentiation process is accelerated in Crjag fruits. During organ morphogenesis, cell growth contributes substantially to organ size once cells exit mitosis 12 . Analysis of areal growth rates reveals a pronounced acceleration of growth in CrWT valves from stage 13 to 14, consistent with the rapid increase in fruit size during this period (Fig. 1 e, f). In contrast, Crjag valves exhibit the opposite trend: growth rate is higher at stage 13 and decreases at stage 14 (Fig. 1 e, f). Comparative mapping and quantification of cell proliferation rates reveal a substantial reduction in cell division events (approximately 20-fold reduction in Crjag compared with CrWT) in the valve epidermis (Fig. 1 g, h and Extended Data Fig. 1 e). In addition to cell growth and division, a region of high anisotropic cell growth at the apical part of the fruit near the replum is critical for pushing the upper part of the valve outward to create the heart shape (Fig. 1 i, j). This pattern of anisotropic growth is completely lost in the upper part of Crjag fruits, with only residual anisotropic growth detected in the lower part of stage-13 Crjag fruits (Fig. 1 i, j). In summary, this comparative organ-wide cellular analysis suggests that CrJAG controls fruit shape determination by coordinating the differentiation process in the valves. Ectopic cell expansion coincides with increases in endoreduplication in the Crjag fruits In eukaryotic cells, the enlargement of cell volume is often associated with endoreduplication, a process in which chromosomal DNA is duplicated without undergoing mitosis 13 . In plant organs, cell size is strongly correlated with DNA ploidy levels 14 , 15 . In Crjag fruits, valve epidermal cells are ectopically expanded throughout the valves compared with WT (Fig. 1 c, d; mostly evident in stage-14 fruits). To assess the contribution of endoreduplication to cell size changes and fruit shape development, we performed comparative flow cytometry of nuclei from CrWT and Crjag valves of stage-17 fruits. Compared to CrWT fruits, where most nuclei had a 2C DNA content, Crjag fruits exhibited a striking increase in the population of 4C and 8C nuclei (Extended Data Fig. 2 a, b). Moreover, quantification of the average number of endocycles per nucleus using the Endoreduplication Index (EI, Ref. 16) revealed a significant increase in EI in Crjag compared with CrWT (Extended Data Fig. 2 c). Altogether, these data indicate that the ectopic expansion of epidermal cells in the Crjag valves is accompanied by increase in endoreduplication events. Genetic validation of CrJAG The mutation responsible for the Crjag phenotype disrupts the donor site of the third intron that integrates this 244-bp intron with the fourth exon (Fig. 2 a; Ref.9). The resulting frameshift introduces a premature stop codon within the intron sequence, producing a truncated 115 amino acid (aa) protein instead of the 252-aa WT CrJAG protein (Fig. 2 a). To confirm the role of CrJAG in fruit shape development, we generated an independent mutant allele of Carub.0002s1499 ( Crjag ge , “ ge ” indicating “genome editing”) using the CRISPR/Cas9 system (Fig. 2 a and Supplementary Fig. 2a). A single base-pair insertion in the second exon caused a frameshift that generated a 40-aa peptide (Fig. 2 a and Supplementary Fig. 2a). The fruits of the Crjag ge mutant perfectly phenocopied the Crjag fruit phenotype, and F 1 plants from a cross between Crjag and Crjag ge showed no genetic complementation (Fig. 2 b). Moreover, a GFP-tagged CrJAG protein driven by the native CrJAG promoter ( pCrJAG :CrJAG:GFP) effectively complements the Crjag fruits defects (Fig. 2 b and Supplementary Fig. 2b). Collectively, these results provide compelling genetic evidence that the developmental defects observed in Crjag are caused by a loss-of-function mutation in Carub.0002s1499, which encodes a C2H2 transcription factor orthologous to Arabidopsis JAG . CrJAG is dynamically expressed during fruit development To explore the expression pattern of the CrJAG gene during fruit development, we generated a pCrJAG :GUS reporter line by fusing a 4.5 kb promoter fragment to the β-glucuronidase (GUS) reporter gene. In agreement with the strong developmental defects in Crjag fruits, GUS expression was specifically localized to the lateral apical region of the ovary and to the valves during gynoecium patterning and fruit development (Fig. 2 c). Its expression in the ovary was initially detected at stage 8 and subsequently became stronger, reaching a maximum in the lateral valve regions at stages 12 and 13 (Fig. 2 c). After stage 14, when the heart shape is established, expression decreased gradually (Fig. 2 c). In addition, strong CrJAG expression was also detected in the valve margins from stage 12 to 14 (Fig. 2 c). Notably, the continuous and dynamic expression of CrJAG in Capsella valves contrasts with the expression of JAG in Arabidopsis gynoecium, where JAG displays transient valve-specific expression before stage 9, after which expression becomes restricted to the valve margins 11 . This divergence in expression patterns may reflect promoter differences between these orthologous genes, as seen for INDEHISCENT 6 . Indeed, the Arabidopsis jag mutant produces fruits similar to WT without obvious morphological alterations except that apical region of the fruit valves is slightly downturned (Extended Data Fig. 3 a, b; Ref.10). CrJAG interacts with histone chaperones to regulate fruit development To identify proteins that interact with CrJAG in fruit shape determination, we resorted to two independent approaches, i.e. immunoprecipitation followed by mass spectrometry (IP-MS) with a GFP antibody against CrJAG:GFP fusion proteins from Crjag;pCrJAG:CrJAG:GFP complemented fruits, and yeast two-hybrid (Y2H) library screening using CrJAG-BD as a bait on the library prepared from stage-10 to stage-13 fruits, when CrJAG are highly expressed (see Methods). Because the full-length CrJAG-BD exhibited strong auto-activation activity in the Y2H system, we performed a systematic truncation analysis to exclude the region responsible for auto-activation and used it for the library screening (Supplementary Fig. 3). An overlap analysis of the proteins identified from these two independent approaches yielded a list of four high-confidence candidate interactors (Fig. 3 a, b and Supplementary Data 1). Among these, the histone chaperone MULTICOPY SUPPRESSOR OF IRA1 3 (CrMSI3) was of particular interest, as its homologs have prominent roles in plant development 17 – 19 . The other three candidates are orthologous to Arabidopsis LWD2 ( LIGHT-REGULATED WD2 ), ISI1 ( IMPAIRED SUCROSE INDUCTION1 ), and a tetratricopeptide repeat (TPR)-containing protein, which have been implicated in light responses and physiological processes 20 – 22 , respectively. These candidates were excluded for further analysis in this study. MSI proteins belong to a family of WD40 histone chaperones conserved in yeast, mammals, and plants 23 . The Capsella genome encodes five closely related homologs, CrMSI1 to CrMSI5 (Fig. 3 c). While no interaction and only weak interaction were detected with CrMSI5 and CrMSI1, respectively (Fig. 3 d), CrJAG exhibits strong interaction with CrMSI2, CrMSI3, and CrMSI4 in the Y2H assays (Fig. 3 d). These interactions were further substantiated in vivo by BiFC and CoIP experiments (Fig. 3 e, f and Supplementary Fig. 4). Consistent with these interactions, CrJAG, CrMSI2, CrMSI3, and CrMSI4 were co-localized in the nucleus when transiently expressed in protoplasts (Extended Data Fig. 4 ). Furthermore, promoter reporter analysis revealed that pCrMSI2:GUS and pCrMSI3:GUS showed strong expression in developing valves, overlapping with the CrJAG expression domain, whereas CrMSI4 promoter activity is weakly detected in the valves (Fig. 4 a-c). To test whether CrMSI2 and CrMSI3 are required for Capsella fruit shape determination, we generated knock-out mutants by CRISPR/Cas9 (Supplementary Fig. 5). Unexpectedly, neither single loss-of-function mutants in Crmsi2 , Crmsi3 , nor the Crmsi2 ; Crmsi3 double mutant shows any detectable fruit defects (Fig. 4 d). Homozygous high-order Crmsi mutants could not be recovered, suggesting that further loss of CrMSI function may have led to embryo lethal 23 , 24 . Given the weak but detectable expression of CrMSI4 in fruits, it may function redundantly with CrMSI2 and CrMSI3 proteins in regulating fruit shape development. MSI proteins are histone chaperones without DNA binding domains, their target specificity is determined by the transcription factors they interact with 23 – 25 . To further unveil the functional importance of the CrJAG-CrMSI2/3 protein complex in fruit development, we fused the DNA-binding domain (DBD) of CrJAG to either CrMSI2 or CrMSI3 with the idea that amino acid sequence outside the DNA-binding domain of CrJAG may function primarily to recruit MSI1-like proteins. We examined their ability to complement the Crjag fruit defects (Fig. 4 e). Whereas the control construct ( pCrJAG :DBD:GFP) failed to complement the fruit defects, either pCrJAG :DBD-CrMSI2:GFP or pCrJAG :DBD-CrMSI3:GFP construct effectively rescued the Crjag fruit defects (Fig. 4 f). In summary, these expression and genetic analyses suggest that CrJAG plays a crucial role in fruit development by directly interacting with MSI1-like proteins in Capsella . The Crjag phenotype is attributed to the down-regulation of its target genes To dissect the molecular mechanism underlying CrJAG-regulated fruit development in Capsella , we first performed a comparative transcriptomic analysis of stage-13 fruits (when shape changes begin) between CrWT and Crjag (Extended Data Fig. 1 a, b). This analysis identified 3,574 differentially expressed genes (DEGs, |logFC|>1, FDR < 0.05), of which 1,913 were up-regulated and 1,661 were down-regulated (Extended Data Fig. 5 a, b). Next, we performed a CUT&Tag experiment using a GFP antibody against CrJAG:GFP from Crjag;pCrJAG :CrJAG:GFP stage-13 fruits. This experiment identified 18,054 CrJAG:GFP binding peaks associated with 7,445 genes (see Methods). Integration of the RNA-seq and CUT&Tag datasets revealed 454 up-regulated DEGs and 373 down-regulated DEGs as CrJAG targeted genes (Extended Data Fig. 5 a, b and Supplementary Data 2). Gene Ontology (GO) enrichment analysis of the 373 down-regulated direct targets revealed significant enrichment of genes in GO terms related to the cellular developmental process (GO: 0048869), cell cycle (GO:0007049), and microtubule-based process (GO: 0007017) (Extended Data Fig. 5 d). In contrast, the 454 up-regulated direct targets were enriched for response to stimulus (GO: 0050896) and transmembrane transport (GO: 0055085) (Extended Data Fig. 5 c). These results corroborate the live-imaging data and suggest that the Crjag fruit defects are primarily due to the down-regulation of genes involved in cell cycle and differentiation, whereas the up-regulated genes appear to appear to have minimal phenotypic contribution. The CrJAG–CrMSI2/3 complex governs fruit shape determination by sustaining the expression of CrAUR2 MSI1-like proteins bind to histones and recruit protein complexes that regulate chromatin accessibility, thereby modulating gene expression 19 , 24 , 25 . To pinpoint the target genes of the CrJAG–CrMSI2/3 complex in fruit shape determination, we performed comparative Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) on stage 12–14 fruits from CrWT and Crjag . We focused on chromatin regions that were closed in Crjag compared with WT and integrated these data with the 373 directly down-regulated genes (Supplementary Data 2). This analysis revealed that 138 of the 373 down-regulated direct targets were associated with loss of chromatin accessibility in the Crjag fruits (Fig. 5 a, b and Supplementary Data 2). GO analysis of the 138 target genes identified genes related to the cell cycle (GO:0007049) that are significantly enriched (Extended Data Fig. 6 a). Among the 10 genes associated with cell cycle (Extended Data Fig. 6 b), CrAURORA2 ( CrAUR2 ) caught our attention, as mutations in AUR orthologs in Arabidopsis result in defects in cell divisions, endoreduplication and deformed organs 26 . Using GUS reporter lines, we observed strong pCrARU2:GUS expression in developing valves, consistent with a role in valve morphogenesis (Extended Data Fig. 7). Strikingly, this valve expression is almost abolished in Crjag fruits (Extended Data Fig. 7), indicating that CrJAG is required for the transcriptional maintenance of CrARU2 in valves. Indeed, chromatin immunoprecipitation (ChIP) analysis further confirmed that CrJAG, CrMSI2 and CrMSI3 are co-associated with the identical regulatory regions within the CrAUR2 locus (Fig. 5 e). Moreover, the DNA binding of CrMSI2 and CrMSI3 is dependent on CrJAG, as their ChIP enrichment is abolished in a Crjag mutant (Fig. 5 e). These data indicate that CrJAG is essential for recruiting CrMSI proteins to the CrAUR2 locus, thereby sustaining its expression. In agreement, expression of CrAUR2 was perfectly restored in pCrJAG :DBD-CrMSI2:GFP and pCrJAG :DBD-CrMSI3:GFP fruits, but not in pCrJAG :DBD:GFP fruits (Fig. 5 f). AUR kinase activity can be selectively blocked by ATP-competitive inhibitor, Aurora Kinase Inhibitor II 27 . In agreement, Aurora Kinase Inhibitor II treatment on WT stage-12 fruits predominantly suppressed heart-shaped fruit development (Extended Data Fig. 8a). Subsequent flow cytometric analysis indicates that the effect of Aurora Kinase Inhibitor II on fruit shape determination is associated with a significant increase in 4C nuclei and EI in the valve tissues and a decrease in 2C nuclei, in a pattern similar to that observed in the Crjag valves (Compare Extended Data Fig. 8b-d with Extended Data Fig. 2 a-c). In contrast, Crjag mutant fruits failed to respond to Aurora Kinase Inhibitor II treatment (Extended Data Fig. 8e-h), implying that Aurora Kinase activity is severely compromised in the mutant. Notably, the strong phenotypic effect of Aurora Kinase Inhibitor II on CrWT fruit shape development appears inconsistent with the limited cell division activity observed in the epidermis by live imaging analysis (Fig. 1 g, h). This discrepancy suggests that the key cell divisions driving heart-shaped fruit development may occur either prior to stage 12 or within the subepidermal layers, which were not accessible in time-lapse live-imaging experiment. Collectively, these expression and pharmaceutical results provide strong evidence that CrJAG-CrMSI complexes exert their function by directly regulating cell-cycle related genes, such as CrAUR2, in determining fruit shape. In Arabidopsis , JAG directly represses the expression of the cell cycle inhibitors KIP RELATED PROTEIN 4 ( KRP4 ) and KRP2 , thereby promoting the growth of the distal region of petals 28 . However, these genes were not identified in our analysis, indicating that CrJAG may regulates its target genes in an organ specific manner and further suggest a conserved role of JAG in plant organ development by regulating divergent components involved in the cell cycle machinery. Changes in chromatin accessibility on CrJAG target genes are correlated with alterations in H3K18ac and H3K27me3 levels In the nucleus, open chromatin increases genome accessibility to transcription factors and the transcription machinery, thereby activating transcription, whereas closed chromatin represses transcription by limiting access 29 . During development, chromatin accessibility and gene expression are dynamically regulated by covalent histone modifications 30 , 31 . In Arabidopsis , MSI family proteins interact with HISTONE DEACETYLASE19 (HDA19) in a histone deacetylase complex and with CUL4–DDB1 in the Polycomb repressive complex 2 (PRC2), thereby dynamically modifying histone marks and regulating gene expression 19 , 32 – 35 . We next investigated whether the impact of the CrJAG–CrMSI2/3 complex on chromatin accessibility and target genes’ expression is linked to alterations in histone modification patterns. To test this, we performed comparative CUT&Tag profiling of eight histone marks in a fruit sample between WT and Crjag , including the repressive mark H3K27me3 and seven active marks (H3K4me1, H3K4me3, H3K9ac, H3K14ac, H3K18ac, H3K23ac, and H3K27ac) 30 . In agreement with the reduced gene expression in the Crjag fruits, changes in chromatin accessibility are predominantly found in the regulatory regions of the 138 direct target genes (Fig. 5 b). Among the eight histone modifications profiled, six (H3K4me1, H3K4me3, H3K9ac, H3K14ac, H3K23ac, and H3K27ac) showed no consistent change in the Crjag mutant compared to CrWT across the 138 target genes (Fig. 5 c and Supplementary Data 3). In contrast, H3K18ac and H3K27me3 exhibited a pronounced, reciprocal pattern of enrichment (reduced enrichment of H3K18ac, increased enrichment of H3K27me3) that was significantly altered in Crjag (Fig. 5 c and Supplementary Data 3). This shift in the epigenetic landscape is particularly evident around the proximal promoter region, where it coincides with altered chromatin accessibility (Fig. 5 b, d and Supplementary Fig. 6). Notably, this coordinated change in histone modification and accessibility is observed in 3 (including CrAUR2) of the 10 genes involved in cell-cycle regulation (Fig. 5 d; Supplementary Fig. 6; Supplementary Data 3). Thus, the shutdown of chromatin accessibility and gene expression in these target genes can be largely explained by reciprocal changes in covalent modification levels of H3K18ac and H3K27me3. However, how H3K18ac and H3K27me3 are modified on the target genes by the CrJAG-CrMSI2/3 complex is an intriguing question that warrants investigation in future studies. Concluding remarks In this study, we dissected the mechanism by which the C2H2 zinc finger transcription factor CrJAG regulates fruit morphogenesis through coordinated cell division and expansion. During heart-shaped fruit development, CrJAG recruits histone chaperones CrMSI2/3 to sustain the expression of target genes associated with cell-cycle and differentiation process, such as CrAUR2 , to promote fruit morphogenesis (Fig. 6 ). The CrJAG-CrMSI2/3 complex modulates chromatin accessibility, likely by altering the deposition of H3K18ac and H3K27me3 histone markers, to facilitate their proper expression of the target genes during fruit shape development (Fig. 6 ). While rewiring of developmental genes in new regulatory networks is known to be a primary mechanism for organ development, our work demonstrates that direct epigenetic modulation by a core developmental regulator represents an essential, additional layer of regulation for precise organ morphogenesis. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Y.D. initiated, conceived and designed the research with great support from L.Ø. Y.D. secured the funding and supervised this study. T.L. performed the research and collected the data with assistance from the other authors. X.-Y.C., Y.H. and Y.-J.Z. did the bioinformatic analysis on the RNA-seq, CUT&Tag-seq and ATAC-seq data. N.T., Y.Z. and M.M. conducted the live cell imaging data analysis. W.H. and Q.Y. did the SEM analysis and GUS staining. L.-M.L., C.-B.L., Y.-N.S. and C.J. were involved in large-scale genotyping analysis. A.S. and M.L. characterized the original Crjag mutant. H.-Z.K. was involved in routine discussion and provided valuable input to this research. Y.D. and T.L. outlined, drafted the manuscript and prepared the figure. All authors participated in the discussion of the data and contributed to the production of the final version of the manuscript. Acknowledgements We are grateful to Prof. Guo-Zheng Qin (IB-CAS) and Dr. Guang-Tong Gao (IB-CAS) for providing the pCambia1300-cHA, pCambia1300-cFLAG, pBI-cYFP, pBI-nYFP plasmids and for their gorgeous assistance in the Co-IP analysis. We thank Prof. Jun Xiao (IGDB-CAS) and Dr. Xue-Lei Lin (IGDB-CAS) for the technical assistance in the CUT&Tag experiment. We are also indebted to Prof. Adrienne Roeder (Cornell University), Prof. Hong-Yan Shan (IB-CAS) and Dr. Jeonghwan Ahn (IB-CAS) for their critically reading the manuscript and providing comments before submission. This work was supported by grants from the National Natural Science Foundation of China (32221001, 32170227, 32470227 to Y.D.), grants from the National Key Research and Development Program of China (2022YFF1301704 to Y.D.) and the SNSF Starting Grant (PS00-3_234905 to M.M.). Data availability RNA-seq, CUT &Tag and ATAC-seq data generated in this study are deposited in the NCBI database under Bioproject PRJNA1420675 (accession codes, SAMN55237242-SAMN55237278. Live-cell imaging data analyzed in this study are deposited in Figshare platform ( https://figshare.com/s/c3538ceaf40bf75da753 ). Materials and Methods Plant materials, transformation and growth conditions All Capsella materials used in the study were in the Cr22.5 ecotype background. The Arabidopsis materials are in Col-0 ecotype. Seeds were sterilized and germinated following the described protocol 36 . The 10-day-old seedlings were transplanted into soil in the controlled environment room (CER) at 22°C under long day conditions (16 h light / 8 h dark). The tobacco ( Nicotiana benthamiana ) plants used in this study were grown at 22°C under long day (16 h light / 8 h dark) conditions. 3-week-old seedlings were used for agroinfiltration. Scanning electron microscopy (SEM) and phenotypic analysis For SEM analysis, inflorescence samples were rapidly fixed with formalin-acetic acid-alcohol (FAA) for 48 hours at room temperature. Gynoecia or fruits at different developmental stages were dissected from the inflorescence under a microscope in 70% alcohol. Dehydration was performed with a series of graded ethanols (80%, 85%, 90%, 95%, 100%, each for 30 min) followed by critical point drying with CO 2 . The samples were subsequently sputter-coated with gold and analysed on an S-4800 FESEM system (Hitachi, Japan). For whole-mount fruit images, stage-17 fruits of each genotype were collected and photographed using a Nikon D850 camera with a 105 mm prime lens. Live-cell imaging and cell behavior analysis To conduct live-cell imaging analysis, the pUBQ10:acyl-YFP reporter was introgressed into the Crjag background by crossing. The plants were cultivated on soil in the CER to the bolting stage. For both CrWT and Crjag , the stage-12 fruits were dissected and imaged, capturing YFP signals at 24-hour intervals using a Zeiss inverted laser confocal microscope (Zeiss LSM 980) equipped with a water immersion objective (×25/0.95). Confocal images were acquired at a resolution of 1024×1024, with 0.5 µm in the Z-stacks. Between each time point, the samples were grown on Petri dishes containing 1/2 MS medium supplemented with vitamins (PM1011, Coolaber) and 1% sucrose in the CER under long-day conditions (22°C, 16 hours light/8 hours dark) to allow development. The images were stitched and analyzed using MorphoGraphX software 37 . Heatmaps showing the differences between two consecutive time points were generated, with the heatmap being shown on the fruit stage at the later time point. To quantify growth anisotropy, cell area ratio and proliferation, the fluorescence was projected into a mesh, cell outlines were segmented, and relationships between cells were tracked across successive time points. For each genotype, two biological replicates were conducted, and representative growth or proliferation maps were obtained from a single experiment. To quantify cell division events, parameters were extracted using MorphoGraphX software and the Wilcoxon test was used to test significance. Plasmid construction and plant transformation To generate the promoter-GUS reporter plasmids, the promoter regions of CrJAG (-4549 bp upstream of ATG), CrMSI2 (-560 bp), CrMSI3 (-1043 bp), CrMSI4 (-4267 bp), CrAUR2 (-1354 bp) 8 were isolated using PrimeSTAR GXL DNA Polymerase (R050A, Takara) from genomic DNA. The DNA fragments were gel-purified and then inserted upstream of the GUS gene in the pCambia 1301 vector. To generate the pCrJAG : CrJAG : GFP complementary plasmid, the promoter and coding sequences of CrJAG were recombined by PCR, and then the PCR products were purified and fused in-frame with the GFP sequence in the pCambia 1302 backbone. To construct the chimeric complementary plasmids, the DNA-binding domain of CrJAG (DBD, 117 bp, plus start codon, 40 aa) was amplified from pCrJAG : CrJAG : GFP plasmids, then recombined with the coding sequences of CrMSI2 or CrMSI3 by PCR. The PCR products were gel-purified and inserted into the pCambia 1302 backbone. To construct the CaMV 35S overexpression plasmids for subcellular localization, the coding sequence of the genes were amplified and inserted in-frame with GFP, driven by the 35S promoter, in the pCambia 1302 vector. To generate the CRISPR/Cas9 gene editing plasmids, the gRNAs targeting the first or second exon of the target genes were designed using the CRISPR-P 2.0 software 38 . The gRNA sequences were synthesized and then integrated into the binary vector by the Golden Gate system as previously described 6 . All vectors were verified by sequencing and introduced into Agrobacterium tumefaciens strain GV3101. Details of the primers are listed in Supplementary Table S1 . The transformation of Capsella followed the floral dipping method described 36 . Positive transformants were selected using 40 mg/L Hygromycin on MS medium. For each construct, at least 10 independent transgenic lines were produced for further analysis. Gene expression and GUS staining For gene expression analysis, stage-13 fruits were collected and frozen immediately in liquid nitrogen, then stored at -80°C before RNA isolation. Total RNA was isolated with the SV Total RNA Isolation System (Z3100, Promega) according to the manufacturer’s instructions. The RNAs were subsequently subjected to library construction and RNA-seq analysis. The GUS histochemical analysis was performed following the described protocol 6 . For confocal microscopy, fruit samples were dissected under a microscope, and GFP signals were imaged using a laser scanning confocal microscope (Zeiss LSM 980) with excitation and emission wavelengths of 488 nm and 509 nm, respectively. Yeast-two hybrid library construction and protein-protein interaction analysis To construct the yeast-two-hybrid library for CrJAG interactor screening, fruits from stage-10 to stage-13 were collected and frozen immediately in liquid nitrogen for RNA isolation. Total RNA was isolated, and mRNA was purified using the Oligotex mRNA Midi Kit (70042, Thermo Fisher) according to the manufacturer’s instructions. cDNAs were subsequently synthesized using the Revert Aid First Strand cDNA Synthesis Kit. A high-quality cDNA library was constructed using the CloneMiner II cDNA Library Construction Kit (A11180, Thermo Fisher). The cDNAs were ligated with a three-frame attB1 adapter by T4 ligase and then purified using the cDNA size fractionation columns. The purified cDNAs were inserted into the pDONR222 vector using BP Clonase (11789020, Thermo Fisher) and recombined into the pGADT7-DEST vector using LR Clonase (11791020, Thermo Fisher) to generate the yeast-two-hybrid cDNA library. To avoid the autoactivating activity of CrJAG , a series of truncated versions of CrJAG were amplified from the pCrJAG : CrJAG : GFP plasmid and then inserted into pGBK-T7 (Clontech) to generate the bait plasmids. The respective plasmids were verified by sequencing and then transformed into the yeast strain Y2H gold with pGAD-T7 (Clontech). Positive transformants were selected on synthetic dropout (SD) medium without tryptophan and leucine (-WL). The autoactivating activity was tested by serial dilutions on SD medium without tryptophan, leucine, histidine, and adenine (-WLHA) according to the manufacturer’s protocols. The yeast-two hybrid library screening was performed according to the manufacturer’s instructions (630439, Clontech) For verifying the CrJAG-CrMSIs interactions, the coding sequences of CrMSI1 , CrMSI2 , CrMSI3 , CrMSI4 , and CrMSI5 were amplified from the Capsella inflorescences cDNAs and then inserted into pGAD-T7 to generate prey plasmids. The interaction was tested as described above. For the Biomolecular Fluorescence Complementation (BiFC) experiment, the full-length CDS without stop codon of CrJAG and CrMSI2 , CrMSI3 , and CrMSI4 were amplified and inserted into pBI-cYFP and pBI-nYFP backbones, respectively. The constructs were verified by sequencing and then transformed into the Agrobacterium strain GV3101. Agrobacteria were inoculated in YEB medium overnight to OD600 of 1.0-1.2 and then resuspended with infiltration buffer (50 mM MES, pH 5.6, 0.5% glucose, 2 mM NaPO 4 , and 15 mg/L acetosyringone) to OD600 of 0.4–0.5. A mixed Agrobacterium suspension containing nYFP and cYFP plasmids at a 1:1 ratio was infiltrated into 3-week-old Nicotiana benthamiana leaves. The plants were incubated under weak light for 36 hours before signal checking. Protein interaction was judged by the presence of YFP signal in the nucleus using a laser scanning confocal microscope (Zeiss LSM 980) with excitation at 488 nm and emission at 509 nm, respectively. For the Co-IP experiment, the full-length coding sequences without stop codon of CrJAG , CrMSI2 , CrMSI3 and CrMSI4 were inserted into pCambia 1300-cHA or pCambia 1300-cFLAG backbones, respectively. The positive plasmids were verified by sequencing and then transformed into the Agrobacterium strain GV3101. They were co-infiltrated into Nicotiana benthamiana leaves as described above. The infiltrated leaves were collected 36 h after infiltration and ground into fine powder in liquid nitrogen. About 0.5 g sample was used for total protein isolation with 1 mL protein extraction buffer [50 mM Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM NaF, 2 mM Na 3 VO 4 , 0.5% NP-40, 1 mM PMSF, 1 mM DTT and 1×Protease Inhibitor Cocktail]. 100 µL total protein mixture was kept as input and the remaining mixture was subjected to immunoprecipitation using DYKDDDDK (FLAG)-Nanoab-Magnetic beads (FNM-25-1000, Lablead) for 1 h at 4°C. The beads were then washed four times with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1 mM DTT. The input and IP samples were boiled for 10 mins in SDS loading buffer, and the eluates were analyzed by immunoblotting with anti-HA (MBL, Japan, 1:10000) or anti‐FLAG (MBL, Japan, 1:10000) antibody. Immunoprecipitation-Mass Spectrometry (IP-MS) analysis About 3 g of fruits containing stage 10–13 from Crjag; pCrJAG : CrJAG : GFP and wild type were collected and crosslinked with 1% formaldehyde in PBS buffer (150 mM NaCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO4, 2.7 mM KCl, pH 7.4). The fruits samples were ground into fine powder in liquid nitrogen, and nuclei were isolated with Honda buffer [0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM HEPES (KOH, pH 7.4), 0.5% Triton X-100, 10 mM MgCl 2 and 1×Protease Inhibitor Cocktail]. The nuclei were then resuspended in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM DTT, and 1× Protease Inhibitor Cocktail) and sonicated for 24 cycles with 30 seconds on and 30 seconds off. The DNA was digested with Benzonase (E1014-25KU, Millipore) for 1 h at 4°C by gentle rotation. The protein extracts were then subjected to immunoprecipitation using Pierce Protein A/G Magnetic Beads (88847; Thermo Fisher), incubated with monoclonal anti-GFP antibody (ab290, abcam) at 4°C for 4 h. After immunoprecipitation, the beads were washed twice with PBS buffer plus 0.5% NP-40, and once with PBS buffer and water, respectively. The beads were digested with 2 µg of trypsin and in 50 mM NH 4 HCO 3 solution overnight at 37°C. The peptides were purified with C18 stage tip and then processed for mass spectrometry on an Orbitrap Exploris 480 machine (Thermo Fisher). Cleavage under targets & tagmentation (CUT&Tag) experiment The CUT&Tag experiment was performed following described methods with minor modifications 39 . Briefly, stage-10 to stage-13 fruits collected from either CrWT, Crjag or Crjag;pCrJAG:CrJAG:GFP rescue lines were chopped with a razor blade in HBM buffer (25 mM Tris-HCl pH 7.6, 0.44 M sucrose, 10 mM MgCl 2 , 0.1% Triton-X-100, 0.2 M spermidine, 1× Protease Inhibitor Cocktail, 1 mM PMSF, 10 mM β-Mercaptoethanol). The nuclei suspension was filtered with a 40 µm cell strainer. The extracted nuclei were washed twice with HBB buffer (25 mM Tris-HCl pH 7.6, 0.44 M sucrose, 10 mM MgCl 2 , 0.1% Triton-X-100, 10 mM β-Mercaptoethanol) and then incubated in 50 µL primary antibody buffer [2 mL wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and 1× Cocktail inhibitor) supplemented with 8 µL 0.5 M EDTA and 6.7 µL 30% BSA] at 4°C overnight. 1 µL of primary antibody (1:50, anti-GFP, ab290, abcam; anti-H3K18ac, 07-354, Millipore; anti-H3K27me3, 07-449, Millipore) was used for each sample. The primary antibody was removed after centrifugation at 1200 g for 2 min. Subsequently, the nuclei were incubated with secondary antibody [1:100, Guinea pig anti-rabbit IgG (Heavy & Light Chain) antibody, ABIN101961, Easybio] in 50 µL wash buffer at 4°C for 2 h. The nuclei were resuspended with 100 µL CT-300 buffer (20 mM HEPES pH 7.5, 450 mM NaCl, 0.5 mM spermidine and 1×Protease Inhibitor Cocktail) supplied with pA-Tn5 (1:150) and incubated at 4°C for 3 h. After incubation, the nuclei were washed twice with 600 µL CT-300 buffer, followed by incubation at 37°C for 1 h with 300 µL tagmentation buffer (1 mL CT-300 buffer plus 10 µL 1 M MgCl 2 ). To stop the tagmentation reaction, 10 µL 0.5 M EDTA, 3 µL 10% SDS and 2.5 µL 20 mg/mL Protinease K were added r and incubated at 50°C for 1 h. The DNA was extracted with phenol: chloroform: isoamyl alcohol (25:24:1), precipitated with ethanol, and then dissolved in ddH 2 O. RNA-seq, ATAC-seq, and CUT&Tag-seq data analysis For RNA-sequencing analysis, total RNA was extracted from stage-13 fruits of the respective genotypes. The libraries were constructed using the MGIEasy RNA Library Prep Kit and then sequenced on the DNBSEQ-T7 platform to generate 150 bp paired-end reads. Low-quality reads and adapter sequences were trimmed to generate clean reads using fastp (version 0.24.0) 40 . The sequences were then mapped and annotated using the Capsella rubella v1.1 genome sequence 41 with STAR (version 2.7.11b) 42 . Gene expression levels were calculated as fragments per kilobase of transcript per million fragments (FPKM) using an R script. Differentially Expressed Genes (DEGs) were identified as those with a fold change ≥ 2 and a False Detection Rate (FDR) < 0.05 using the R package DESeq2 (version 1.42.1) 43 . GO enrichment analysis of Biological Process (BP) on the DEGs was performed using the R packages enrichplot (version 1.22.0) and clusterProfiler (version 4.10.1) 44 . Boxplots were drawn using the R package ggplot2 (version 3.5.2) 45 . For the RNA-seq experiment, at least three independent biological replicates were conducted for each genotype. The ATAC-seq experiment was performed following the protocol previously described 8 . Briefly, ~ 0.5 g of stage-12 to stage-14 Crjag fruits were harvested and ground into fine powder in liquid nitrogen. Approximately 50,000 nuclei were collected for DNA library construction. The library was sequenced on the Illumina Novaseq 6000 platform to generate 150 bp paired-end reads. The raw reads were filtered (reads shorter than 35 bp and bases with a quality value less than Q10) using fastp (version 0.24.0) 40 to generate clean FASTQ files. The sequences were then mapped back to the Capsella rubella v1.1 genome 41 using Bowtie2 (version 2.5.4) 46 . Duplicate reads were removed by sambamba (version 1.0.1) 47 and bedtools (version 2.31.1) 48 . Peaks were called using MACS2 (version 2.1.4) 49 with a screening criterion of FDR < 0.05. The CrWT stage 12–14 fruit ATAC data were published previously 8 . Differentially enriched peaks between CrWT and Crjag were identified using the R package DiffBind (version 3.12.0). Significant closed chromatin accessibility in Crjag was defined as fold change < 0.8 and FDR < 0.05. DeepTools (version 3.5.6) 50 was used to map the density distribution of sequencing reads in the upstream and downstream regions of the Transcription Start Site (TSS) and the Transcription End Sits (TES) of each gene. The profile plot was generated using the plotProfile function. For visualization, datasets were converted to bigwig format using bamCoverage in DeepTools (version 3.5.6) with a bin size of 1 bp and normalized by the RPKM method, then visualized using Integrative Genomics Viewer (IGV, version 2.4.14) 51 . Gene annotation was performed by ChIPseeker (version 1.38.0) 52 using a 2 kb promoter-proximal window. The ATAC-seq experiments were performed with two independent biological replicates. For CUT&Tag-seq data analysis, the library was constructed by amplifying 10–12 cycles using Q5 High-Fidelity DNA Polymerase (M0491S, NEB) and purified by AMPure XP beads (A63881, Beckman) according to the manufacturer’s instructions. Primers used for library amplification are listed in Supplementary Table S1 . The library was sequenced on the Illumina Nova X Plus platform to generate 150 bp paired-end reads. The raw reads were filtered using fastp (version 0.24.0) to generate clean FASTQ files. After filtering low-quality reads, the sequences were mapped back to the Capsella rubella v1.1 genome using Bowtie2 (version 2.5.4). Duplicate reads were removed by sambamba (version 1.0.1) and bedtools (version 2.31.1). Peaks were called using MACS2 (version 2.1.4) with a p value of 0.01. The bigwig files were generated by DeepTools (version 3.5.6) for visualization using IGV (version 2.16.2). Gene annotation was performed with a 2 kb promoter-proximal window using ChIPseeker (version 1.38.0) 52 . For each antibody and genotype, two biological replicates were conducted. For data integration, Venn diagrams were generated with the R package VennDiagram (version 1.7.3) 53 . Protoplast transient expression and ChIP-qPCR analysis Protoplast isolation and transient protein expression were performed with the published protocols 54 . Briefly, leaves from 10-day-old young seedlings were sliced and digested with Enzyme buffer I (1.0% cellulase R10, 0.5% macerozyme R10, 0.5% pectinase, 20 mM KCl, 20 mM MES, 0.6 M mannitol, 10 mM CaCl 2 , 0.1% BSA) rotating at 60 rpm and 25°C for 2.5 h. The protoplasts were collected by filtering with a 40 µm cell strainer. 35S:CrJAG/CrMSI2/CrMSI3/CrMSI4:GFP and AtUBQ10:NLS-mCherry plasmids were prepared using a QIAGEN Plasmid Maxi Kit according to the manufacturer’s instructions. A total of 1000 ng plasmids (35S:CrJAG : AtUBQ10:NLS-mCherry = 1:1) were co-transformed into protoplasts with a PEG-mediated heat-shock method (42°C, 3mins). The protoplasts were incubated under weak light at 26°C for 18 h. The subcellular localization of CrJAG:GFP, CrMSI2:GFP, CrMSI3:GFP, and CrMSI4:GFP proteins was detected on a Zeiss 980 confocal microscope with AtUBQ10:NLS-mCherry as a nuclear-localized marker. The ChIP-qPCR analysis was performed using modified protocols described 6 . Briefly, protoplasts transformed with pCambia 1300-cFLAG/CrMSI2/CrMSI3:GFP and CrJAG-cFLAG/CrMSI2/CrMSI3:GFP were collected and crosslinked with 1% formaldehyde for 10 mins at room temperature and stopped with 125 mM glycine for 5 min. The protoplasts were resuspended in RIPA buffer and sonicated for 24 cycles with 30 s on and 30 s off. The samples were then subjected to immunoprecipitation using an anti-GFP antibody (ab290, Abcam) or an anti-FLAG antibody (F3165, Sigma-Aldrich) pre-incubated with Pierce Protein A/G magnetic beads (88847, Thermo Fisher) at 4°C for 4.5 h. The beads were washed, then reverse-crosslinked by adding 10% sodium dodecyl sulfate (SDS) and incubating at 65°C for 12 h. The proteins in the DNA complex were digested with Proteinase K at 45°C for 1 h. The DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol, then resuspended in water (W4502, Sigma-Aldrich). qPCR was performed on a qTOWER PCR System (Analytik Jena AG) using SYBR Premix Ex Taq. Chemical treatment and cell flow cytometry For chemical treatment, stage-12 fruits were collected and transferred onto 0.4% agar half-strength Murashige and Skoog (1/2 MS) medium plates containing 100 µM Aurora kinase inhibitor II (17541, Cayman) or mock solution (DMSO) for 5 days. The phenotypes were recorded using a Nikon D850 camera with a 105 mm prime lens. For the cell-flow cytometry analysis, nuclei were isolated from fruits in 250 µL PVPK12-mGB 2 buffer [30 mM sodium citrate, 45 mM MgCl 2 , 20 mM MOPS, 20 mM NaCl, 20 mM EDTA Na 2 ·2H 2 O, 0.1% (v/v) Triton X-100, 0.5% (v/v) Tween-20, 10 µL/mL 1–2% PVPK12, pH 7.0] and filtered through a 40 µm nylon mesh. The nuclei were stained with 4 µg/mL DAPI. For each sample, approximately 20,000 nuclei were analyzed using a BD LSRFortessa flow cytometer (BD, USA). Quantitative analysis of ploidy level was conducted using FlowJo software (v10.10). EI was calculated from the ploidy histograms using the formula: EI = 4C% + 2 × 8C%. 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Supplementary Files LvExtendedDataFigures2026223.docx Extended Data Figure Legends Extended Data Fig. 1 Comparative phenotypic analysis of the CrWT and Crjag fruits a,b, SEM images of CrWT (a) and Crjag (b) fruits from developmental stages 8 to 14. b,c, Scatterplots of cell size distribution along the vertical axis of the CrWT (b) and Crjag (c) fruits at the time point 0, 48 h and 96 h. The y axis measures distance from the fruit base (0 μm) to the fruit apex. Each point represents an individual cell. The purple line and shading indicate the average cell size and the 95 percent confidence interval, respectively. d, Bar plot showing the proportion of dividing vs. non-dividing cells between CrWT and Crjag fruits during 0-48 h and 48-96 h, with 8.3% of cells dividing during 0-48 h and 4.2% during 48-96h in CrWT fruits, and 0.4% of cells dividing during 0-48 h and 0.2% during 48-96 h in Crjag fruits. Scale bars, 100 μm (a,b). Extended Data Fig. 2 Reduced valve growth in Crjag is coincided with mis-patterned endoreduplication events a, Representative ploidy profiles of valve tissues from stage-17 fruits of CrWT, Crjag and Crjag;pCrJAG:CrJAG:GFP complemented line. Approximately 20,000 nuclei per sample were analyzed. b, Proportion of cells with different ploidy levels in CrWT, Crjag , and Crjag;pCrJAG:CrJAG:GFP fruits. c, Quantification of Endoreduplication Index (EI) between CrWT, Crjag and Crjag;pCrJAG:CrJAG:GFP fruits. In b,c, the values represent the means of six biological replicates and the error bars represent s.d. *p < 0.05, **p < 0.01, ***p <0.001, ****p <0.0001 (Student’s t test). Extended Data Fig. 3 Mutation of JAG in Arabidopsis results in little effect on fruit development a, Genotyping of the jag ge mutant generated by CRISPR/Cas9 in Arabidopsis . The jag ge mutant is a loss-of-function allele with a single-base pair insertion in exon 4, resulting in a frameshift that gives rise to a 167 aa protein. The guide RNA and PAM sequences are indicated by red and blue characters, respectively. The red triangles indicate the gRNA target site. b, Comparison of the developmental defects in the reproductive organs between WT and jag ge . Scale bars, 1 mm (b). Extended Data Fig. 4 Subcellular localization of CrJAG and CrMSIs proteins Subcellular localization of CrJAG:GFP, CrMSI2:GFP, CrMSI3:GFP and CrMSI4:GFP proteins in CrWT leaf protoplast cells transiently expressing the 35S promoter-driven plasmids. The nuclear localized mCherry protein was used as a marker to visualize the nucleus. Scale bars represent 20 μm. Extended Data Fig. 5 Identification of CrJAG target genes by RNA-seq and CUT&Tag-seq a, Venn diagram showing the overlap of the up-regulated DEGs in Crjag fruits and genes identified from CrJAG:GFP CUT&Tag. b, Venn diagram showing the overlap of the down-regulated DEGs in Crjag fruits and genes identified from CrJAG : GFP CUT&Tag. c, Gene Ontology (GO) terms enriched in the 454 CrJAG : GFP targeted up-regulated DEGs. d, Gene Ontology (GO) terms enriched in the 373 CrJAG : GFP targeted down-regulated DEGs. Extended Data Fig. 6 Identification of CrJAG-CrMSI complex target genes a, Gene Ontology (GO) terms enriched in the 138 targeted down-regulated DEGs by CrJAG-CrMSI complex. b, Molecular identities of the 10 candidate genes involved in cell cycle process with CrAUR2 (Carub.0004s0570) shown in bold. Extended Data Fig. 7 Expression analysis of pCrAUR2:GUS reporters GUS staining analysis of the pCrAUR2:GUS reporter line showing the activity expression of CrAUR2 during fruit development. Strong CrAUR2 expression is detected in the CrWT valves from stages 12 to 14. This expression is strongly compromised in the Crjag background. Scale bars, 500 μm. Extended Data Fig. 8 Aurora Kinase Inhibitor II treatment suppresses fruit valve development a, Morphology of mock and AUR Inhibitor II treated CrWT fruits. b, Representative ploidy profiles of valve tissues from mock- and AUR inhibitor II-treated CrWT fruits. Approximately 20,000 nuclei per sample were analyzed. c, Proportion of cells with different ploidy level from mock- and AUR inhibitor II-treated CrWT fruits. d, Quantification of the Endoreduplication Index (EI) of mock and AUR inhibitor II-treated CrWT fruits. e, Morphology of mock and AUR inhibitor II treatment of Crjag fruits. f, Representative ploidy profiles of valve tissues from mock- and AUR inhibitor II-treated Crjag fruits. Approximately 20,000 nuclei per sample were analyzed. g, Proportion of cells with different ploidy levels from mock- and AUR inhibitor II-treated Crjag fruits. h, Quantification of Endoreduplication Index (EI) in mock- and AUR inhibitor II-treated Crjag fruits. In c, d, g and h, the values represent the means of six biological replicates and the error bars represent s.d. ns, not significant, *p < 0.05, **p < 0.01 (Student’s t test). Scale bars, 5 mm (a,e). LvSupplementaryData1CandidategenelistofCrJAGinteractor.xlsx Supplementary Data 1 Candidate gene list of CrJAG interactor LvSupplementaryData2CrJAGtargetgenelist.xlsx Supplementary Data 2 CrJAG target gene list LvSupplementaryData3Hitonemidificationpatternofthe138CrJAGtargetgenes.xlsx Supplementary Data 3 Hitone midification pattern of the 138 CrJAG target genes SupplementaryFig.1620260223.docx Supplementary Fig. 1-6 LvSupplemantaryTable1Primersusedinthisstudy.docx Supplemantary Table 1 Primers used in this study rs.pdf Reporting Summary Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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University","correspondingAuthor":false,"prefix":"","firstName":"Yan-Jun","middleName":"","lastName":"Zan","suffix":""},{"id":595304025,"identity":"1b8c4686-fbb3-443e-8200-b5a2144eb136","order_by":14,"name":"Michael Lenhard","email":"","orcid":"https://orcid.org/0000-0001-8661-6911","institution":"University of Potsdam","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Lenhard","suffix":""},{"id":595304026,"identity":"46722128-7a83-4887-9a8c-1b220b93e5c3","order_by":15,"name":"Hongzhi Kong","email":"","orcid":"https://orcid.org/0000-0002-0034-0510","institution":"Institute of Botany, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hongzhi","middleName":"","lastName":"Kong","suffix":""},{"id":595304027,"identity":"bd372efc-98d4-44d3-a990-e379b15611d9","order_by":16,"name":"Mateusz Majda","email":"","orcid":"https://orcid.org/0000-0003-3405-2901","institution":"University of Lausanne","correspondingAuthor":false,"prefix":"","firstName":"Mateusz","middleName":"","lastName":"Majda","suffix":""},{"id":595304028,"identity":"b47d56df-f10d-4dce-a0bf-42315ca2031a","order_by":17,"name":"Lars Østergaard","email":"","orcid":"https://orcid.org/0000-0002-8497-7657","institution":"John Innes Centre","correspondingAuthor":false,"prefix":"","firstName":"Lars","middleName":"","lastName":"Østergaard","suffix":""}],"badges":[],"createdAt":"2025-12-03 02:31:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8265099/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8265099/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104398665,"identity":"b94ea385-bbff-40e2-98d2-c830fdd286ce","added_by":"auto","created_at":"2026-03-11 12:03:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2851157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative live-cell imaging analysis reveals the cellular basis of the shape defects in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrjag\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e fruits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e, Fruit morphology of CrWT (\u003cstrong\u003ea\u003c/strong\u003e) and \u003cem\u003eCrjag\u003c/em\u003e (\u003cstrong\u003eb\u003c/strong\u003e) at stage 17. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e, Quantitative depiction of valve development using heatmaps of cell sizes in the CrWT (\u003cstrong\u003ec\u003c/strong\u003e) and \u003cem\u003eCrjag\u003c/em\u003e (\u003cstrong\u003ed\u003c/strong\u003e) fruit during 96 h of time-lapse live-cell imaging analysis, corresponding to developmental stages 12, 13 and 14 (0 h, 48 h and 96 h, respectively). Cell size is quantified by cell area (μm\u003csup\u003e2\u003c/sup\u003e). In total, 10,481 and 6,738 cells were segmented for CrWT and \u003cem\u003eCrjag\u003c/em\u003e fruits, respectively. \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e, Heatmaps of cell growth (the rate of cell area increase) of the CrWT (\u003cstrong\u003ee\u003c/strong\u003e) and \u003cem\u003eCrjag\u003c/em\u003e (\u003cstrong\u003ef\u003c/strong\u003e) fruit at 48 h and 96 h time points. \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e, Heatmaps showing cell division status of CrWT(\u003cstrong\u003eg\u003c/strong\u003e) and \u003cem\u003eCrjag\u003c/em\u003e (\u003cstrong\u003eh\u003c/strong\u003e) fruit at 48 h and 96 h time points. \u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e, Heatmaps of cell anisotropy (the ratio of cell expansion in the maximum and minimum principal directions) of the CrWT (\u003cstrong\u003ei\u003c/strong\u003e) and \u003cem\u003eCrjag\u003c/em\u003e (\u003cstrong\u003ej\u003c/strong\u003e) fruit at 48 h and 96 h time points. Scale bars, 5 mm\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e) and, 200 μm\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ec\u003c/strong\u003e–\u003cstrong\u003ej\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"LvFigure120251203.png","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/14ce3dca5c40a3feb5f9ecf7.png"},{"id":104398286,"identity":"bd408a34-f9d9-410d-9e1c-7d715c612214","added_by":"auto","created_at":"2026-03-11 12:01:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":662891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic validation and expression analysis of CrJAG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Molecular characterization of \u003cem\u003eCrjag\u003c/em\u003e and \u003cem\u003eCrjag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e alleles. The \u003cem\u003eCrjag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e was generated by CRISPR/Cas9 with a single-base pair insertion in exon 2, resulting in a frameshift that gave rise to a 40-amino-acid (aa) protein. The guide RNA (gRNA) and PAM sequences are indicated by red and blue characters, respectively. The red triangles indicate the target site of the gRNA. \u003cstrong\u003eb\u003c/strong\u003e, Fruit morphology of CrWT, \u003cem\u003eCrjag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e,\u003cem\u003e Crjag/Crjag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eCrjag;pCrJAG:CrJAG:GFP \u003c/em\u003ecomplementary line at stage 17. \u003cstrong\u003ec\u003c/strong\u003e, GUS staining analysis of the \u003cem\u003epCrJAG:GUS\u003c/em\u003e reporter line showing the dynamic expression pattern of \u003cem\u003eCrJAG\u003c/em\u003e during fruit development. Strong \u003cem\u003eCrJAG\u003c/em\u003e expression is detected in the valves from stages 12 to 14. Scale bars, 5 mm\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) and 500 μm (\u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"LvFigure220251202.png","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/496d86297bc8077614bebcbd.png"},{"id":104398588,"identity":"30928213-d0a2-49bb-886b-0070f9644566","added_by":"auto","created_at":"2026-03-11 12:03:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1205295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrJAG interacts with histone chaperones CrMIS2 proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Venn diagram showing the overlap of protein interactors identified from IP-MS and Y2H library screening. \u003cstrong\u003eb\u003c/strong\u003e, Molecular identities of the four candidate proteins with the MSI1 protein shown in bold. \u003cstrong\u003ec\u003c/strong\u003e, Neighbor-joining tree of proteins encoded by the \u003cem\u003eMSI \u003c/em\u003egenes from the \u003cem\u003eCapsella \u003c/em\u003eand\u003cem\u003eArabidopsis \u003c/em\u003egenome, bootstrap values over 50% (1,000 replicates) are indicated\u003cem\u003e \u003c/em\u003efor each branch. The \u003cem\u003eDamaged DNA Binding 1 (DDB1)-binding WD40\u003c/em\u003e (\u003cem\u003eDWD1\u003c/em\u003e) gene was used as an outgroup. \u003cstrong\u003ed\u003c/strong\u003e, Yeast-two-hybrid analysis of the interaction of CrJAG protein with CrMSI1-5 proteins. \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e, Verification of the protein interaction of CrJAG-CrMSI2, CrJAG-CrMSI3 and CrJAG-CrMSI4 using BiFC analysis (\u003cstrong\u003ee\u003c/strong\u003e) and Co-IP experiment after transient expression in tobacco leaves (\u003cstrong\u003ef\u003c/strong\u003e). Scale bars, 50 μm (\u003cstrong\u003ee\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"LvFigure320260130.png","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/250198330a6b1e267386fd55.png"},{"id":103567152,"identity":"0dafa48a-7171-4dde-a362-4e3a4ad75c71","added_by":"auto","created_at":"2026-02-27 07:29:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2061174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CrJAG-CrMSIs complex is required for fruit shape determination in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCapsella\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e, GUS staining analysis of \u003cem\u003epCrMSI2:GUS\u003c/em\u003e (\u003cstrong\u003ea\u003c/strong\u003e), \u003cem\u003epCrMSI3:GUS\u003c/em\u003e (\u003cstrong\u003eb\u003c/strong\u003e), and \u003cem\u003epCrMSI4:GUS\u003c/em\u003e (\u003cstrong\u003ec\u003c/strong\u003e) reporter lines showing the expression pattern of these genes during fruit development. \u003cstrong\u003ed\u003c/strong\u003e, Fruit morphology of \u003cem\u003eCrmsi2\u003c/em\u003eand \u003cem\u003eCrmsi3\u003c/em\u003e single, and \u003cem\u003eCrmsi2\u003c/em\u003e;\u003cem\u003e Crmsi3\u003c/em\u003e double mutants at stage 17. \u003cstrong\u003ee\u003c/strong\u003e, Schematic illustration of the chimeric constructs of CrJAG(DBD) and CrMSI proteins used for the complementation test. The total protein size is shown. \u003cstrong\u003ef\u003c/strong\u003e, Fruit morphology of \u003cem\u003eCrjag\u003c/em\u003e, \u003cem\u003eCrjag; pCrJAG:DBD:GFP\u003c/em\u003e, \u003cem\u003eCrjag; pCrJAG:DBD-CrMSI2:GFP \u003c/em\u003eand \u003cem\u003eCrjag; pCrJAG:DBD-CrMSI3:GFP \u003c/em\u003eat stage 17, showing that the DBD-CrMSI2 and DBD-CrMSI3 chimeric proteins successfully complement the \u003cem\u003eCrjag\u003c/em\u003e fruit shape defects. Scale bars, 500 μm (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e) and 5 mm (\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"LvFigure420251202.png","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/017cd9f99aad25d5e98f2d78.png"},{"id":103567147,"identity":"007492f1-e445-4f5e-abd4-7821aa5e8820","added_by":"auto","created_at":"2026-02-27 07:29:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CrJAG-CrMSIs complex regulates fruit shape development via epigenetic mechanism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Venn diagram showing the 138 genes identified from \u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e CUT\u0026amp;Tag, down-regulated DEGs in the \u003cem\u003eCrjag\u003c/em\u003e fruits, and closed chromatin accessibility in \u003cem\u003eCrjag\u003c/em\u003e fruits by ATAC-seq. \u003cstrong\u003eb\u003c/strong\u003e, Metaplots displaying the ATAC-seq signals of the 138 \u003cem\u003eCrJAG \u003c/em\u003etargeted genes, showing decreased ATAC-seq signals in the proximal promoter region and 3′regulatory region in the \u003cem\u003eCrjag\u003c/em\u003e compared with CrWT. TSS, Transcription Starting Site; TES, Transcription Ending Site. \u003cstrong\u003ec\u003c/strong\u003e, Venn diagram showing the overlap of down-regulated targeted genes and changes in H3K27me3 and H3K18ac modification levels. \u003cstrong\u003ed\u003c/strong\u003e, Snapshots showing the chromatin binding peaks of \u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e, ATAC-seq signals, H3K27me3 and H3K18ac modification signals in the genomic regions of \u003cem\u003eCrAUR2\u003c/em\u003e between CrWT and \u003cem\u003eCrjag\u003c/em\u003e fruit samples. The double arrowheads (A-C) indicate the regions used for ChIP analysis. The full epigenetic landscape is provided in Supplementary Fig. 6. \u003cstrong\u003ee\u003c/strong\u003e, ChIP-qPCR analysis in the CrWT protoplasts showing CrMSI2:GFP and CrMSI3:GFP association with the same DNA region as\u003cem\u003e \u003c/em\u003eCrJAG:GFP on the \u003cem\u003eCrAUR2\u003c/em\u003e locus, and that these associations depend on the presence of CrJAG:GFP protein. \u003cstrong\u003ef\u003c/strong\u003e, Gene expression analysis of \u003cem\u003eCrAUR2\u003c/em\u003e in different genetic backgrounds. The expression level was quantified as FPKM from RNA seq data. In \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e, the values represent the means of three biological replicates and the error bars represent s.d. ns, not significant, *p \u0026lt; 0.05, **p \u0026lt; 0.01 (Student’s t test).\u003c/p\u003e","description":"","filename":"LvFigure520251202.png","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/a7fbb7f14849d373a5a9def0.png"},{"id":103567157,"identity":"4d6478e6-4ad7-400b-a099-5258a859ee12","added_by":"auto","created_at":"2026-02-27 07:29:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":195031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CrJAG–CrMSIs complex orchestrates fruit shape determination via epigenetic regulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring fruit morphogenesis, CrJAG recruits CrMSI2/3 to downstream target genes. This complex locally reduces H3K27me3 and increases H3K18ac via an as‑yet‑unidentified mechanism (dashed line), thereby promoting chromatin accessibility and gene expression. A key target of CrJAG-CrMSIs in fruit shape determination, \u003cem\u003eCrAUR2\u003c/em\u003e, exemplifies this regulatory logic. In the \u003cem\u003eCrjag\u003c/em\u003e mutant, loss of CrJAG function abolishes CrMSI recruitment. Consequently, H3K27me3 enrichment rises while H3K18ac declines, leading to reduced chromatin accessibility, decreased target gene expression, and impaired fruit morphogenesis.\u003c/p\u003e","description":"","filename":"LvFigure620260129.png","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/e5f9a34dad1d2549d6f69030.png"},{"id":105032589,"identity":"1caef72e-763e-487c-912c-be1e9aef5269","added_by":"auto","created_at":"2026-03-20 07:02:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8555469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/8f5fa2c7-efee-430b-b4fc-dc24517da7c5.pdf"},{"id":104398700,"identity":"b833bb9e-d8aa-418b-b8da-eee2e1932fd0","added_by":"auto","created_at":"2026-03-11 12:03:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2582969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Figure Legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 1 Comparative phenotypic analysis of the CrWT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrjag\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e fruits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e, SEM images of CrWT (\u003cstrong\u003ea\u003c/strong\u003e) and \u003cem\u003eCrjag\u003c/em\u003e (\u003cstrong\u003eb\u003c/strong\u003e) fruits from developmental stages 8 to 14.\u003cstrong\u003e b\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e, Scatterplots of cell size distribution along the vertical axis of the CrWT (\u003cstrong\u003eb\u003c/strong\u003e) and \u003cem\u003eCrjag\u003c/em\u003e (\u003cstrong\u003ec\u003c/strong\u003e) fruits at the time point 0, 48 h and 96 h. The \u003cem\u003ey\u003c/em\u003e axis measures distance from the fruit base (0 μm) to the fruit apex. Each point represents an individual cell. The purple line and shading indicate the average cell size and the 95 percent confidence interval, respectively. \u003cstrong\u003ed\u003c/strong\u003e, Bar plot showing the proportion of dividing vs. non-dividing cells between CrWT and\u003cem\u003e Crjag\u003c/em\u003e fruits during 0-48 h and 48-96 h, with 8.3% of cells dividing during 0-48 h and 4.2% during 48-96h in CrWT fruits, and 0.4% of cells dividing during 0-48 h and 0.2% during 48-96 h in \u003cem\u003eCrjag\u003c/em\u003e fruits. Scale bars, 100 μm (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 2 Reduced valve growth in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrjag\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is coincided with mis-patterned endoreduplication events\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Representative ploidy profiles of valve tissues from stage-17 fruits of CrWT, \u003cem\u003eCrjag\u003c/em\u003e and \u003cem\u003eCrjag;pCrJAG:CrJAG:GFP\u003c/em\u003e complemented line. Approximately 20,000 nuclei per sample were analyzed. \u003cstrong\u003eb\u003c/strong\u003e, Proportion of cells with different ploidy levels in CrWT, \u003cem\u003eCrjag\u003c/em\u003e, and \u003cem\u003eCrjag;pCrJAG:CrJAG:GFP \u003c/em\u003efruits. \u003cstrong\u003ec\u003c/strong\u003e, Quantification of Endoreduplication Index (EI) between CrWT, \u003cem\u003eCrjag\u003c/em\u003e and \u003cem\u003eCrjag;pCrJAG:CrJAG:GFP \u003c/em\u003efruits. In \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e, the values represent the means of six biological replicates and the error bars represent s.d. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt;0.001, ****p \u0026lt;0.0001 (Student’s t test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 3 Mutation of JAG in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e results in little effect on fruit development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Genotyping of the \u003cem\u003ejag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e mutant generated by CRISPR/Cas9 in \u003cem\u003eArabidopsis\u003c/em\u003e. The \u003cem\u003ejag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e mutant is a loss-of-function allele with a single-base pair insertion in exon 4, resulting in a frameshift that gives rise to a 167 aa protein. The guide RNA and PAM sequences are indicated by red and blue characters, respectively. The red triangles indicate the gRNA target site. \u003cstrong\u003eb\u003c/strong\u003e, Comparison of the developmental defects in the reproductive organs between WT and\u003cem\u003e jag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e. Scale bars, 1 mm (\u003cstrong\u003eb\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 4 Subcellular localization of CrJAG and CrMSIs proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubcellular localization of CrJAG:GFP, CrMSI2:GFP, CrMSI3:GFP and CrMSI4:GFP proteins in CrWT leaf protoplast cells transiently expressing the 35S promoter-driven plasmids. The nuclear localized mCherry protein was used as a marker to visualize the nucleus. Scale bars represent 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 5 Identification of CrJAG target genes by RNA-seq and CUT\u0026amp;Tag-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Venn diagram showing the overlap of the up-regulated DEGs in \u003cem\u003eCrjag\u003c/em\u003e fruits and genes identified from \u003cem\u003eCrJAG:GFP\u003c/em\u003e CUT\u0026amp;Tag. \u003cstrong\u003eb\u003c/strong\u003e, Venn diagram showing the overlap of the down-regulated DEGs in \u003cem\u003eCrjag\u003c/em\u003e fruits and genes identified from \u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e CUT\u0026amp;Tag. \u003cstrong\u003ec\u003c/strong\u003e, Gene Ontology (GO) terms enriched in the 454 \u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e targeted up-regulated DEGs. \u003cstrong\u003ed\u003c/strong\u003e, Gene Ontology (GO) terms enriched in the 373 \u003cu\u003e\u003cem\u003eCrJAG\u003c/em\u003e\u003c/u\u003e:\u003cem\u003eGFP\u003c/em\u003e targeted down-regulated DEGs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 6\u003c/strong\u003e \u003cstrong\u003eIdentification of CrJAG-CrMSI complex target genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Gene Ontology (GO) terms enriched in the 138 targeted down-regulated DEGs by CrJAG-CrMSI complex. \u003cstrong\u003eb\u003c/strong\u003e, Molecular identities of the 10 candidate genes involved in cell cycle process with \u003cem\u003eCrAUR2\u003c/em\u003e (Carub.0004s0570) shown in bold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 7\u003c/strong\u003e \u003cstrong\u003eExpression analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epCrAUR2:GUS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reporters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGUS staining analysis of the \u003cem\u003epCrAUR2:GUS\u003c/em\u003e reporter line showing the activity expression of \u003cem\u003eCrAUR2\u003c/em\u003e during fruit development. Strong \u003cem\u003eCrAUR2\u003c/em\u003e expression is detected in the CrWT valves from stages 12 to 14. This expression is strongly compromised in the \u003cem\u003eCrjag\u003c/em\u003e background. Scale bars, 500 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 8\u003c/strong\u003e \u003cstrong\u003eAurora Kinase Inhibitor II treatment suppresses fruit valve development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Morphology of mock and AUR Inhibitor II treated CrWT fruits. \u003cstrong\u003eb\u003c/strong\u003e, Representative ploidy profiles of valve tissues from mock- and AUR inhibitor II-treated CrWT fruits. Approximately 20,000 nuclei per sample were analyzed. \u003cstrong\u003ec\u003c/strong\u003e, Proportion of cells with different ploidy level from mock- and\u003cem\u003e \u003c/em\u003eAUR inhibitor II-treated CrWT fruits. \u003cstrong\u003ed\u003c/strong\u003e, Quantification of the Endoreduplication Index (EI) of mock and AUR inhibitor II-treated CrWT fruits. \u003cstrong\u003ee\u003c/strong\u003e, Morphology of mock and AUR inhibitor II treatment of \u003cem\u003eCrjag\u003c/em\u003e fruits. \u003cstrong\u003ef\u003c/strong\u003e, Representative ploidy profiles of valve tissues from mock- and AUR inhibitor II-treated \u003cem\u003eCrjag\u003c/em\u003e fruits. Approximately 20,000 nuclei per sample were analyzed. \u003cstrong\u003eg\u003c/strong\u003e, Proportion of cells with different ploidy levels from mock- and\u003cem\u003e \u003c/em\u003eAUR inhibitor II-treated \u003cem\u003eCrjag\u003c/em\u003e fruits. \u003cstrong\u003eh\u003c/strong\u003e, Quantification of Endoreduplication Index (EI) in mock- and AUR inhibitor II-treated \u003cem\u003eCrjag\u003c/em\u003e fruits. In \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e, the values represent the means of six biological replicates and the error bars represent s.d. ns, not significant, *p \u0026lt; 0.05, **p \u0026lt; 0.01 (Student’s t test). Scale bars, 5 mm (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"LvExtendedDataFigures2026223.docx","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/1220d1f0fb70547d5cea2096.docx"},{"id":104398021,"identity":"cf85d94c-91e5-419c-b0cf-77f36e021f40","added_by":"auto","created_at":"2026-03-11 11:59:24","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27334,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 1 Candidate gene list of CrJAG interactor\u003c/p\u003e","description":"","filename":"LvSupplementaryData1CandidategenelistofCrJAGinteractor.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/6caa2468d34a657c3c3e6f61.xlsx"},{"id":104397885,"identity":"2c00ce7f-40d8-4244-a9ae-baf0cf1352c4","added_by":"auto","created_at":"2026-03-11 11:58:37","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":93223,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 2 CrJAG target gene list\u003c/p\u003e","description":"","filename":"LvSupplementaryData2CrJAGtargetgenelist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/449806c58c9703440d965f77.xlsx"},{"id":103567145,"identity":"cd759e3c-7cba-4b2c-b8fa-427b6212cbdf","added_by":"auto","created_at":"2026-02-27 07:29:05","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21568,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 3 Hitone midification pattern of the 138 CrJAG target genes\u003c/p\u003e","description":"","filename":"LvSupplementaryData3Hitonemidificationpatternofthe138CrJAGtargetgenes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/50046e1437e0c8d073143ef5.xlsx"},{"id":104398287,"identity":"2ad90e12-33c2-42a0-ab41-f19092b3c12a","added_by":"auto","created_at":"2026-03-11 12:01:19","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3233762,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig. 1-6\u003c/p\u003e","description":"","filename":"SupplementaryFig.1620260223.docx","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/dd54a336ae067a59ab9f5865.docx"},{"id":103567150,"identity":"bed216e0-0ef9-41d7-be3f-21265e2b7ca0","added_by":"auto","created_at":"2026-02-27 07:29:05","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":26511,"visible":true,"origin":"","legend":"Supplemantary Table 1 Primers used in this study","description":"","filename":"LvSupplemantaryTable1Primersusedinthisstudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/8f6cb06a9167e8d9249c466b.docx"},{"id":103567154,"identity":"4e149892-1725-4700-9627-99f9f84c8bdd","added_by":"auto","created_at":"2026-02-27 07:29:05","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":99960,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"rs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8265099/v1/7a6f7838e50af5d32d90de20.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Epigenetic regulation of fruit shape determination by JAGGED gene in Capsella rubella","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe evolutionary success of angiosperms is largely attributable to their ability to produce fruits\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The fruit is developed from the female reproductive organs, gynoecium, after pollination\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The fruits enclose, nourish and protect the developing seeds from harsh environments, and upon maturation, they facilitate seed dispersal and colonization of new habitats through diverse mechanisms\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Variations in fruit shape are associated with specific dispersal strategies\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Accordingly, the developmental program underlying fruit shape determination must be robustly regulated, as variations in this process would result in adaptive penalties due to limited dispersal\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Recently, the heart-shaped fruit of \u003cem\u003eCapsella\u003c/em\u003e has been established as a model for understanding the developmental genetics of fruit shape determination\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. We previously showed that development of the heart shape is promoted by localized auxin biosynthesis at the apex of the valve tips\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This auxin maximum in the apical region reshapes the ovate spheroid gynoecium into a heart-shaped fruit through dynamic changes in cell growth and cell division\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, how cell growth and division are coordinated during the heart-shape fruit development remain largely unknown.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eCrjag\u003c/b\u003e \u003cb\u003emutant produces deformed fruits with reduced cell divisions and cell growth\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify the genetic components controlling the development of heart-shaped fruits in \u003cem\u003eCapsella\u003c/em\u003e, we carried out a forward genetic screen of an EMS-induced \u003cem\u003eCapsella rubella\u003c/em\u003e (Cr22.5) mutant population. One mutant, \u003cem\u003eEMS1196B\u003c/em\u003e, was identified based on its strong defects in fruit development with compromised growth in the fruit valves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b; Ref. 9). Bulked segregation analysis (BSA) revealed that the causal gene of the \u003cem\u003eEMS1196B\u003c/em\u003e encodes a C2H2 zinc finger transcription factor (Carub.0002s1499), which is orthologous to the \u003cem\u003eArabidopsis JAGGED\u003c/em\u003e (\u003cem\u003eJAG\u003c/em\u003e) gene\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We henceforth refer to the \u003cem\u003eEMS1196B\u003c/em\u003e as \u003cem\u003eCapsella rubella JAGGED\u003c/em\u003e (\u003cem\u003eCrJAG\u003c/em\u003e) in the following sections.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDevelopment of the heart-shaped fruit in Wild type \u003cem\u003eCapsella\u003c/em\u003e (CrWT ) starts from an ovate spheroid gynoecium soon after pollination at stage 12 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). From stage 13 onwards, reshaping of fruit growth initiates from the apical parts of the valves and gradually leads to the formation of the heart shape by stage 14 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Ref. 6). In the \u003cem\u003eCrjag\u003c/em\u003e mutant, however, this fruit reshaping process is abolished because valve outgrowth is significantly compromised, resulting in a fruit with an inverted triangular shape at stage 14 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). To uncover the cellular basis of the \u003cem\u003eCrjag\u003c/em\u003e phenotype, we performed a large-scale comparative live-imaging analysis between CrWT and \u003cem\u003eCrjag\u003c/em\u003e fruits from developmental stage 12 (fertilization) to stage 14, when the most significant defects of the \u003cem\u003eCrjag\u003c/em\u003e mutant are observed (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Cell growth parameters, including growth rate (cell area expansion), cell proliferation, and anisotropy (direction of cell expansion), were visualized using lineage-tracking maps (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). For each genotype, we conducted two biological replicates (see Methods). Analysis of the cell size heatmaps demonstrated a consistent distribution pattern between replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d and Supplementary Fig.\u0026nbsp;1). Fruit morphogenesis in CrWT is characterized by the acropetal differentiation of epidermal cells in the valves in which cell expansion initiated from the base at stage 13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). At stage 14, large cells are predominantly found at the base while smaller cells are distributed at the tip (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Ref. 8). In \u003cem\u003eCrjag\u003c/em\u003e fruits, however, cell size increase occurs as early as stage 12 and ectopic large cells are observed in the apical and middle parts of the valves at stage 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). These data suggest that valve differentiation process is accelerated in \u003cem\u003eCrjag\u003c/em\u003e fruits. During organ morphogenesis, cell growth contributes substantially to organ size once cells exit mitosis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Analysis of areal growth rates reveals a pronounced acceleration of growth in CrWT valves from stage 13 to 14, consistent with the rapid increase in fruit size during this period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). In contrast, \u003cem\u003eCrjag\u003c/em\u003e valves exhibit the opposite trend: growth rate is higher at stage 13 and decreases at stage 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). Comparative mapping and quantification of cell proliferation rates reveal a substantial reduction in cell division events (approximately 20-fold reduction in \u003cem\u003eCrjag\u003c/em\u003e compared with CrWT) in the valve epidermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). In addition to cell growth and division, a region of high anisotropic cell growth at the apical part of the fruit near the replum is critical for pushing the upper part of the valve outward to create the heart shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j). This pattern of anisotropic growth is completely lost in the upper part of \u003cem\u003eCrjag\u003c/em\u003e fruits, with only residual anisotropic growth detected in the lower part of stage-13 \u003cem\u003eCrjag\u003c/em\u003e fruits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j). In summary, this comparative organ-wide cellular analysis suggests that \u003cem\u003eCrJAG\u003c/em\u003e controls fruit shape determination by coordinating the differentiation process in the valves.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEctopic cell expansion coincides with increases in endoreduplication in the\u003c/b\u003e \u003cb\u003eCrjag\u003c/b\u003e \u003cb\u003efruits\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn eukaryotic cells, the enlargement of cell volume is often associated with endoreduplication, a process in which chromosomal DNA is duplicated without undergoing mitosis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In plant organs, cell size is strongly correlated with DNA ploidy levels\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eCrjag\u003c/em\u003e fruits, valve epidermal cells are ectopically expanded throughout the valves compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d; mostly evident in stage-14 fruits). To assess the contribution of endoreduplication to cell size changes and fruit shape development, we performed comparative flow cytometry of nuclei from CrWT and \u003cem\u003eCrjag\u003c/em\u003e valves of stage-17 fruits. Compared to CrWT fruits, where most nuclei had a 2C DNA content, \u003cem\u003eCrjag\u003c/em\u003e fruits exhibited a striking increase in the population of 4C and 8C nuclei (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Moreover, quantification of the average number of endocycles per nucleus using the Endoreduplication Index (EI, Ref. 16) revealed a significant increase in EI in \u003cem\u003eCrjag\u003c/em\u003e compared with CrWT (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Altogether, these data indicate that the ectopic expansion of epidermal cells in the \u003cem\u003eCrjag\u003c/em\u003e valves is accompanied by increase in endoreduplication events.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic validation of\u003c/b\u003e \u003cb\u003eCrJAG\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe mutation responsible for the \u003cem\u003eCrjag\u003c/em\u003e phenotype disrupts the donor site of the third intron that integrates this 244-bp intron with the fourth exon (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; Ref.9). The resulting frameshift introduces a premature stop codon within the intron sequence, producing a truncated 115 amino acid (aa) protein instead of the 252-aa WT CrJAG protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To confirm the role of \u003cem\u003eCrJAG\u003c/em\u003e in fruit shape development, we generated an independent mutant allele of Carub.0002s1499 (\u003cem\u003eCrjag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e, \u0026ldquo;\u003cem\u003ege\u003c/em\u003e\u0026rdquo; indicating \u0026ldquo;genome editing\u0026rdquo;) using the CRISPR/Cas9 system (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;2a). A single base-pair insertion in the second exon caused a frameshift that generated a 40-aa peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;2a). The fruits of the \u003cem\u003eCrjag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e mutant perfectly phenocopied the \u003cem\u003eCrjag\u003c/em\u003e fruit phenotype, and F\u003csub\u003e1\u003c/sub\u003e plants from a cross between \u003cem\u003eCrjag\u003c/em\u003e and \u003cem\u003eCrjag\u003c/em\u003e\u003csup\u003e\u003cem\u003ege\u003c/em\u003e\u003c/sup\u003e showed no genetic complementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Moreover, a GFP-tagged CrJAG protein driven by the native CrJAG promoter (\u003cem\u003epCrJAG\u003c/em\u003e:CrJAG:GFP) effectively complements the \u003cem\u003eCrjag\u003c/em\u003e fruits defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;2b). Collectively, these results provide compelling genetic evidence that the developmental defects observed in \u003cem\u003eCrjag\u003c/em\u003e are caused by a loss-of-function mutation in Carub.0002s1499, which encodes a C2H2 transcription factor orthologous to \u003cem\u003eArabidopsis JAG\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCrJAG\u003c/b\u003e \u003cb\u003eis dynamically expressed during fruit development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the expression pattern of the \u003cem\u003eCrJAG\u003c/em\u003e gene during fruit development, we generated a \u003cem\u003epCrJAG\u003c/em\u003e:GUS reporter line by fusing a 4.5 kb promoter fragment to the β-glucuronidase (GUS) reporter gene. In agreement with the strong developmental defects in \u003cem\u003eCrjag\u003c/em\u003e fruits, GUS expression was specifically localized to the lateral apical region of the ovary and to the valves during gynoecium patterning and fruit development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Its expression in the ovary was initially detected at stage 8 and subsequently became stronger, reaching a maximum in the lateral valve regions at stages 12 and 13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). After stage 14, when the heart shape is established, expression decreased gradually (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In addition, strong \u003cem\u003eCrJAG\u003c/em\u003e expression was also detected in the valve margins from stage 12 to 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Notably, the continuous and dynamic expression of \u003cem\u003eCrJAG\u003c/em\u003e in \u003cem\u003eCapsella\u003c/em\u003e valves contrasts with the expression of \u003cem\u003eJAG\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e gynoecium, where \u003cem\u003eJAG\u003c/em\u003e displays transient valve-specific expression before stage 9, after which expression becomes restricted to the valve margins\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This divergence in expression patterns may reflect promoter differences between these orthologous genes, as seen for \u003cem\u003eINDEHISCENT\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Indeed, the \u003cem\u003eArabidopsis jag\u003c/em\u003e mutant produces fruits similar to WT without obvious morphological alterations except that apical region of the fruit valves is slightly downturned (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b; Ref.10).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCrJAG interacts with histone chaperones to regulate fruit development\u003c/h2\u003e \u003cp\u003eTo identify proteins that interact with CrJAG in fruit shape determination, we resorted to two independent approaches, i.e. immunoprecipitation followed by mass spectrometry (IP-MS) with a GFP antibody against CrJAG:GFP fusion proteins from \u003cem\u003eCrjag;pCrJAG:CrJAG:GFP\u003c/em\u003e complemented fruits, and yeast two-hybrid (Y2H) library screening using CrJAG-BD as a bait on the library prepared from stage-10 to stage-13 fruits, when CrJAG are highly expressed (see Methods). Because the full-length CrJAG-BD exhibited strong auto-activation activity in the Y2H system, we performed a systematic truncation analysis to exclude the region responsible for auto-activation and used it for the library screening (Supplementary Fig.\u0026nbsp;3). An overlap analysis of the proteins identified from these two independent approaches yielded a list of four high-confidence candidate interactors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b and Supplementary Data 1). Among these, the histone chaperone MULTICOPY SUPPRESSOR OF IRA1 3 (CrMSI3) was of particular interest, as its homologs have prominent roles in plant development\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The other three candidates are orthologous to \u003cem\u003eArabidopsis LWD2\u003c/em\u003e (\u003cem\u003eLIGHT-REGULATED WD2\u003c/em\u003e), \u003cem\u003eISI1\u003c/em\u003e (\u003cem\u003eIMPAIRED SUCROSE INDUCTION1\u003c/em\u003e), and a tetratricopeptide repeat (TPR)-containing protein, which have been implicated in light responses and physiological processes\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, respectively. These candidates were excluded for further analysis in this study.\u003c/p\u003e \u003cp\u003eMSI proteins belong to a family of WD40 histone chaperones conserved in yeast, mammals, and plants\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eCapsella\u003c/em\u003e genome encodes five closely related homologs, CrMSI1 to CrMSI5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). While no interaction and only weak interaction were detected with CrMSI5 and CrMSI1, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), CrJAG exhibits strong interaction with CrMSI2, CrMSI3, and CrMSI4 in the Y2H assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These interactions were further substantiated \u003cem\u003ein vivo\u003c/em\u003e by BiFC and CoIP experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f and Supplementary Fig.\u0026nbsp;4). Consistent with these interactions, CrJAG, CrMSI2, CrMSI3, and CrMSI4 were co-localized in the nucleus when transiently expressed in protoplasts (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, promoter reporter analysis revealed that \u003cem\u003epCrMSI2:GUS\u003c/em\u003e and \u003cem\u003epCrMSI3:GUS\u003c/em\u003e showed strong expression in developing valves, overlapping with the \u003cem\u003eCrJAG\u003c/em\u003e expression domain, whereas \u003cem\u003eCrMSI4\u003c/em\u003e promoter activity is weakly detected in the valves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). To test whether CrMSI2 and CrMSI3 are required for \u003cem\u003eCapsella\u003c/em\u003e fruit shape determination, we generated knock-out mutants by CRISPR/Cas9 (Supplementary Fig.\u0026nbsp;5). Unexpectedly, neither single loss-of-function mutants in \u003cem\u003eCrmsi2\u003c/em\u003e, \u003cem\u003eCrmsi3\u003c/em\u003e, nor the \u003cem\u003eCrmsi2\u003c/em\u003e;\u003cem\u003eCrmsi3\u003c/em\u003e double mutant shows any detectable fruit defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Homozygous high-order \u003cem\u003eCrmsi\u003c/em\u003e mutants could not be recovered, suggesting that further loss of \u003cem\u003eCrMSI\u003c/em\u003e function may have led to embryo lethal\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Given the weak but detectable expression of \u003cem\u003eCrMSI4\u003c/em\u003e in fruits, it may function redundantly with CrMSI2 and CrMSI3 proteins in regulating fruit shape development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMSI proteins are histone chaperones without DNA binding domains, their target specificity is determined by the transcription factors they interact with\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To further unveil the functional importance of the CrJAG-CrMSI2/3 protein complex in fruit development, we fused the DNA-binding domain (DBD) of CrJAG to either CrMSI2 or CrMSI3 with the idea that amino acid sequence outside the DNA-binding domain of CrJAG may function primarily to recruit MSI1-like proteins. We examined their ability to complement the \u003cem\u003eCrjag\u003c/em\u003e fruit defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Whereas the control construct (\u003cem\u003epCrJAG\u003c/em\u003e:DBD:GFP) failed to complement the fruit defects, either \u003cem\u003epCrJAG\u003c/em\u003e:DBD-CrMSI2:GFP or \u003cem\u003epCrJAG\u003c/em\u003e:DBD-CrMSI3:GFP construct effectively rescued the \u003cem\u003eCrjag\u003c/em\u003e fruit defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). In summary, these expression and genetic analyses suggest that \u003cem\u003eCrJAG\u003c/em\u003e plays a crucial role in fruit development by directly interacting with MSI1-like proteins in \u003cem\u003eCapsella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eCrjag\u003c/b\u003e \u003cb\u003ephenotype is attributed to the down-regulation of its target genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo dissect the molecular mechanism underlying CrJAG-regulated fruit development in \u003cem\u003eCapsella\u003c/em\u003e, we first performed a comparative transcriptomic analysis of stage-13 fruits (when shape changes begin) between CrWT and \u003cem\u003eCrjag\u003c/em\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). This analysis identified 3,574 differentially expressed genes (DEGs, |logFC|\u0026gt;1, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05), of which 1,913 were up-regulated and 1,661 were down-regulated (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Next, we performed a CUT\u0026amp;Tag experiment using a GFP antibody against CrJAG:GFP from \u003cem\u003eCrjag;pCrJAG\u003c/em\u003e:CrJAG:GFP stage-13 fruits. This experiment identified 18,054 CrJAG:GFP binding peaks associated with 7,445 genes (see Methods). Integration of the RNA-seq and CUT\u0026amp;Tag datasets revealed 454 up-regulated DEGs and 373 down-regulated DEGs as CrJAG targeted genes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b and Supplementary Data 2). Gene Ontology (GO) enrichment analysis of the 373 down-regulated direct targets revealed significant enrichment of genes in GO terms related to the cellular developmental process (GO: 0048869), cell cycle (GO:0007049), and microtubule-based process (GO: 0007017) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). In contrast, the 454 up-regulated direct targets were enriched for response to stimulus (GO: 0050896) and transmembrane transport (GO: 0055085) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). These results corroborate the live-imaging data and suggest that the \u003cem\u003eCrjag\u003c/em\u003e fruit defects are primarily due to the down-regulation of genes involved in cell cycle and differentiation, whereas the up-regulated genes appear to appear to have minimal phenotypic contribution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe CrJAG\u0026ndash;CrMSI2/3 complex governs fruit shape determination by sustaining the expression of\u003c/b\u003e \u003cb\u003eCrAUR2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMSI1-like proteins bind to histones and recruit protein complexes that regulate chromatin accessibility, thereby modulating gene expression\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To pinpoint the target genes of the CrJAG\u0026ndash;CrMSI2/3 complex in fruit shape determination, we performed comparative Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) on stage 12\u0026ndash;14 fruits from CrWT and \u003cem\u003eCrjag\u003c/em\u003e. We focused on chromatin regions that were closed in \u003cem\u003eCrjag\u003c/em\u003e compared with WT and integrated these data with the 373 directly down-regulated genes (Supplementary Data 2). This analysis revealed that 138 of the 373 down-regulated direct targets were associated with loss of chromatin accessibility in the \u003cem\u003eCrjag\u003c/em\u003e fruits (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b and Supplementary Data 2). GO analysis of the 138 target genes identified genes related to the cell cycle (GO:0007049) that are significantly enriched (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the 10 genes associated with cell cycle (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), \u003cem\u003eCrAURORA2\u003c/em\u003e (\u003cem\u003eCrAUR2\u003c/em\u003e) caught our attention, as mutations in \u003cem\u003eAUR\u003c/em\u003e orthologs in Arabidopsis result in defects in cell divisions, endoreduplication and deformed organs\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Using GUS reporter lines, we observed strong \u003cem\u003epCrARU2:GUS\u003c/em\u003e expression in developing valves, consistent with a role in valve morphogenesis (Extended Data Fig.\u0026nbsp;7). Strikingly, this valve expression is almost abolished in \u003cem\u003eCrjag\u003c/em\u003e fruits (Extended Data Fig.\u0026nbsp;7), indicating that \u003cem\u003eCrJAG\u003c/em\u003e is required for the transcriptional maintenance of \u003cem\u003eCrARU2\u003c/em\u003e in valves. Indeed, chromatin immunoprecipitation (ChIP) analysis further confirmed that CrJAG, CrMSI2 and CrMSI3 are co-associated with the identical regulatory regions within the \u003cem\u003eCrAUR2\u003c/em\u003e locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Moreover, the DNA binding of CrMSI2 and CrMSI3 is dependent on CrJAG, as their ChIP enrichment is abolished in a \u003cem\u003eCrjag\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). These data indicate that CrJAG is essential for recruiting CrMSI proteins to the \u003cem\u003eCrAUR2\u003c/em\u003e locus, thereby sustaining its expression. In agreement, expression of \u003cem\u003eCrAUR2\u003c/em\u003e was perfectly restored in \u003cem\u003epCrJAG\u003c/em\u003e:DBD-CrMSI2:GFP and \u003cem\u003epCrJAG\u003c/em\u003e:DBD-CrMSI3:GFP fruits, but not in \u003cem\u003epCrJAG\u003c/em\u003e:DBD:GFP fruits (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eAUR kinase activity can be selectively blocked by ATP-competitive inhibitor, Aurora Kinase Inhibitor II\u003csup\u003e27\u003c/sup\u003e. In agreement, Aurora Kinase Inhibitor II treatment on WT stage-12 fruits predominantly suppressed heart-shaped fruit development (Extended Data Fig.\u0026nbsp;8a). Subsequent flow cytometric analysis indicates that the effect of Aurora Kinase Inhibitor II on fruit shape determination is associated with a significant increase in 4C nuclei and EI in the valve tissues and a decrease in 2C nuclei, in a pattern similar to that observed in the \u003cem\u003eCrjag\u003c/em\u003e valves (Compare Extended Data Fig.\u0026nbsp;8b-d with Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). In contrast, \u003cem\u003eCrjag\u003c/em\u003e mutant fruits failed to respond to Aurora Kinase Inhibitor II treatment (Extended Data Fig.\u0026nbsp;8e-h), implying that Aurora Kinase activity is severely compromised in the mutant. Notably, the strong phenotypic effect of Aurora Kinase Inhibitor II on CrWT fruit shape development appears inconsistent with the limited cell division activity observed in the epidermis by live imaging analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). This discrepancy suggests that the key cell divisions driving heart-shaped fruit development may occur either prior to stage 12 or within the subepidermal layers, which were not accessible in time-lapse live-imaging experiment. Collectively, these expression and pharmaceutical results provide strong evidence that CrJAG-CrMSI complexes exert their function by directly regulating cell-cycle related genes, such as CrAUR2, in determining fruit shape.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, JAG directly represses the expression of the cell cycle inhibitors \u003cem\u003eKIP RELATED PROTEIN 4\u003c/em\u003e (\u003cem\u003eKRP4\u003c/em\u003e) and \u003cem\u003eKRP2\u003c/em\u003e, thereby promoting the growth of the distal region of petals\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, these genes were not identified in our analysis, indicating that CrJAG may regulates its target genes in an organ specific manner and further suggest a conserved role of JAG in plant organ development by regulating divergent components involved in the cell cycle machinery.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in chromatin accessibility on CrJAG target genes are correlated with alterations in H3K18ac and H3K27me3 levels\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the nucleus, open chromatin increases genome accessibility to transcription factors and the transcription machinery, thereby activating transcription, whereas closed chromatin represses transcription by limiting access\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. During development, chromatin accessibility and gene expression are dynamically regulated by covalent histone modifications\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eArabidopsis\u003c/em\u003e, MSI family proteins interact with HISTONE DEACETYLASE19 (HDA19) in a histone deacetylase complex and with CUL4\u0026ndash;DDB1 in the Polycomb repressive complex 2 (PRC2), thereby dynamically modifying histone marks and regulating gene expression\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. We next investigated whether the impact of the CrJAG\u0026ndash;CrMSI2/3 complex on chromatin accessibility and target genes\u0026rsquo; expression is linked to alterations in histone modification patterns. To test this, we performed comparative CUT\u0026amp;Tag profiling of eight histone marks in a fruit sample between WT and \u003cem\u003eCrjag\u003c/em\u003e, including the repressive mark H3K27me3 and seven active marks (H3K4me1, H3K4me3, H3K9ac, H3K14ac, H3K18ac, H3K23ac, and H3K27ac) \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In agreement with the reduced gene expression in the \u003cem\u003eCrjag\u003c/em\u003e fruits, changes in chromatin accessibility are predominantly found in the regulatory regions of the 138 direct target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Among the eight histone modifications profiled, six (H3K4me1, H3K4me3, H3K9ac, H3K14ac, H3K23ac, and H3K27ac) showed no consistent change in the \u003cem\u003eCrjag\u003c/em\u003e mutant compared to CrWT across the 138 target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and Supplementary Data 3). In contrast, H3K18ac and H3K27me3 exhibited a pronounced, reciprocal pattern of enrichment (reduced enrichment of H3K18ac, increased enrichment of H3K27me3) that was significantly altered in \u003cem\u003eCrjag\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and Supplementary Data 3). This shift in the epigenetic landscape is particularly evident around the proximal promoter region, where it coincides with altered chromatin accessibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, d and Supplementary Fig.\u0026nbsp;6). Notably, this coordinated change in histone modification and accessibility is observed in 3 (including CrAUR2) of the 10 genes involved in cell-cycle regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed; Supplementary Fig.\u0026nbsp;6; Supplementary Data 3). Thus, the shutdown of chromatin accessibility and gene expression in these target genes can be largely explained by reciprocal changes in covalent modification levels of H3K18ac and H3K27me3. However, how H3K18ac and H3K27me3 are modified on the target genes by the CrJAG-CrMSI2/3 complex is an intriguing question that warrants investigation in future studies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Concluding remarks","content":"\u003cp\u003eIn this study, we dissected the mechanism by which the C2H2 zinc finger transcription factor CrJAG regulates fruit morphogenesis through coordinated cell division and expansion. During heart-shaped fruit development, CrJAG recruits histone chaperones CrMSI2/3 to sustain the expression of target genes associated with cell-cycle and differentiation process, such as \u003cem\u003eCrAUR2\u003c/em\u003e, to promote fruit morphogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The CrJAG-CrMSI2/3 complex modulates chromatin accessibility, likely by altering the deposition of H3K18ac and H3K27me3 histone markers, to facilitate their proper expression of the target genes during fruit shape development (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). While rewiring of developmental genes in new regulatory networks is known to be a primary mechanism for organ development, our work demonstrates that direct epigenetic modulation by a core developmental regulator represents an essential, additional layer of regulation for precise organ morphogenesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003c/p\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eY.D. initiated, conceived and designed the research with great support from L.Ø. Y.D. secured the funding and supervised this study. T.L. performed the research and collected the data with assistance from the other authors. X.-Y.C., Y.H. and Y.-J.Z. did the bioinformatic analysis on the RNA-seq, CUT\u0026amp;Tag-seq and ATAC-seq data. N.T., Y.Z. and M.M. conducted the live cell imaging data analysis. W.H. and Q.Y. did the SEM analysis and GUS staining. L.-M.L., C.-B.L., Y.-N.S. and C.J. were involved in large-scale genotyping analysis. A.S. and M.L. characterized the original \u003cem\u003eCrjag\u003c/em\u003e mutant. H.-Z.K. was involved in routine discussion and provided valuable input to this research. Y.D. and T.L. outlined, drafted the manuscript and prepared the figure. All authors participated in the discussion of the data and contributed to the production of the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful to Prof. Guo-Zheng Qin (IB-CAS) and Dr. Guang-Tong Gao (IB-CAS) for providing the pCambia1300-cHA, pCambia1300-cFLAG, pBI-cYFP, pBI-nYFP plasmids and for their gorgeous assistance in the Co-IP analysis. We thank Prof. Jun Xiao (IGDB-CAS) and Dr. Xue-Lei Lin (IGDB-CAS) for the technical assistance in the CUT\u0026amp;Tag experiment. We are also indebted to Prof. Adrienne Roeder (Cornell University), Prof. Hong-Yan Shan (IB-CAS) and Dr. Jeonghwan Ahn (IB-CAS) for their critically reading the manuscript and providing comments before submission. This work was supported by grants from the National Natural Science Foundation of China (32221001, 32170227, 32470227 to Y.D.), grants from the National Key Research and Development Program of China (2022YFF1301704 to Y.D.) and the SNSF Starting Grant (PS00-3_234905 to M.M.).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eRNA-seq, CUT \u0026amp;Tag and ATAC-seq data generated in this study are deposited in the NCBI database under Bioproject PRJNA1420675 (accession codes, SAMN55237242-SAMN55237278. Live-cell imaging data analyzed in this study are deposited in Figshare platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/s/c3538ceaf40bf75da753\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003ePlant materials, transformation and growth conditions\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eAll \u003cem\u003eCapsella\u003c/em\u003e materials used in the study were in the \u003cem\u003eCr22.5\u003c/em\u003e ecotype background. The \u003cem\u003eArabidopsis\u003c/em\u003e materials are in Col-0 ecotype. Seeds were sterilized and germinated following the described protocol\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The 10-day-old seedlings were transplanted into soil in the controlled environment room (CER) at 22°C under long day conditions (16 h light / 8 h dark). The tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) plants used in this study were grown at 22°C under long day (16 h light / 8 h dark) conditions. 3-week-old seedlings were used for agroinfiltration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eScanning electron microscopy (SEM) and phenotypic analysis\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eFor SEM analysis, inflorescence samples were rapidly fixed with formalin-acetic acid-alcohol (FAA) for 48 hours at room temperature. Gynoecia or fruits at different developmental stages were dissected from the inflorescence under a microscope in 70% alcohol. Dehydration was performed with a series of graded ethanols (80%, 85%, 90%, 95%, 100%, each for 30 min) followed by critical point drying with CO\u003csub\u003e2\u003c/sub\u003e. The samples were subsequently sputter-coated with gold and analysed on an S-4800 FESEM system (Hitachi, Japan). For whole-mount fruit images, stage-17 fruits of each genotype were collected and photographed using a Nikon D850 camera with a 105 mm prime lens.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eLive-cell imaging and cell behavior analysis\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eTo conduct live-cell imaging analysis, the \u003cem\u003epUBQ10:acyl-YFP\u003c/em\u003e reporter was introgressed into the \u003cem\u003eCrjag\u003c/em\u003e background by crossing. The plants were cultivated on soil in the CER to the bolting stage. For both CrWT and \u003cem\u003eCrjag\u003c/em\u003e, the stage-12 fruits were dissected and imaged, capturing YFP signals at 24-hour intervals using a Zeiss inverted laser confocal microscope (Zeiss LSM 980) equipped with a water immersion objective (×25/0.95). Confocal images were acquired at a resolution of 1024×1024, with 0.5 µm in the Z-stacks. Between each time point, the samples were grown on Petri dishes containing 1/2 MS medium supplemented with vitamins (PM1011, Coolaber) and 1% sucrose in the CER under long-day conditions (22°C, 16 hours light/8 hours dark) to allow development. The images were stitched and analyzed using MorphoGraphX software \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Heatmaps showing the differences between two consecutive time points were generated, with the heatmap being shown on the fruit stage at the later time point. To quantify growth anisotropy, cell area ratio and proliferation, the fluorescence was projected into a mesh, cell outlines were segmented, and relationships between cells were tracked across successive time points. For each genotype, two biological replicates were conducted, and representative growth or proliferation maps were obtained from a single experiment. To quantify cell division events, parameters were extracted using MorphoGraphX software and the Wilcoxon test was used to test significance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003ePlasmid construction and plant transformation\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eTo generate the promoter-GUS reporter plasmids, the promoter regions of \u003cem\u003eCrJAG\u003c/em\u003e (-4549 bp upstream of ATG), \u003cem\u003eCrMSI2\u003c/em\u003e (-560 bp), \u003cem\u003eCrMSI3\u003c/em\u003e (-1043 bp), \u003cem\u003eCrMSI4\u003c/em\u003e (-4267 bp), \u003cem\u003eCrAUR2\u003c/em\u003e (-1354 bp)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e were isolated using PrimeSTAR GXL DNA Polymerase (R050A, Takara) from genomic DNA. The DNA fragments were gel-purified and then inserted upstream of the \u003cem\u003eGUS\u003c/em\u003e gene in the \u003cem\u003epCambia\u003c/em\u003e1301 vector. To generate the \u003cem\u003epCrJAG\u003c/em\u003e:\u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e complementary plasmid, the promoter and coding sequences of \u003cem\u003eCrJAG\u003c/em\u003e were recombined by PCR, and then the PCR products were purified and fused in-frame with the GFP sequence in the \u003cem\u003epCambia\u003c/em\u003e1302 backbone. To construct the chimeric complementary plasmids, the DNA-binding domain of \u003cem\u003eCrJAG\u003c/em\u003e (DBD, 117 bp, plus start codon, 40 aa) was amplified from \u003cem\u003epCrJAG\u003c/em\u003e:\u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e plasmids, then recombined with the coding sequences of \u003cem\u003eCrMSI2\u003c/em\u003e or \u003cem\u003eCrMSI3\u003c/em\u003e by PCR. The PCR products were gel-purified and inserted into the \u003cem\u003epCambia\u003c/em\u003e1302 backbone. To construct the CaMV 35S overexpression plasmids for subcellular localization, the coding sequence of the genes were amplified and inserted in-frame with GFP, driven by the 35S promoter, in the \u003cem\u003epCambia\u003c/em\u003e1302 vector. To generate the CRISPR/Cas9 gene editing plasmids, the gRNAs targeting the first or second exon of the target genes were designed using the CRISPR-P 2.0 software\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The gRNA sequences were synthesized and then integrated into the binary vector by the Golden Gate system as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. All vectors were verified by sequencing and introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. Details of the primers are listed in Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe transformation of \u003cem\u003eCapsella\u003c/em\u003e followed the floral dipping method described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Positive transformants were selected using 40 mg/L Hygromycin on MS medium. For each construct, at least 10 independent transgenic lines were produced for further analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eGene expression and GUS staining\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eFor gene expression analysis, stage-13 fruits were collected and frozen immediately in liquid nitrogen, then stored at -80°C before RNA isolation. Total RNA was isolated with the SV Total RNA Isolation System (Z3100, Promega) according to the manufacturer’s instructions. The RNAs were subsequently subjected to library construction and RNA-seq analysis.\u003c/p\u003e\u003cp\u003eThe GUS histochemical analysis was performed following the described protocol \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. For confocal microscopy, fruit samples were dissected under a microscope, and GFP signals were imaged using a laser scanning confocal microscope (Zeiss LSM 980) with excitation and emission wavelengths of 488 nm and 509 nm, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eYeast-two hybrid library construction and protein-protein interaction analysis\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eTo construct the yeast-two-hybrid library for CrJAG interactor screening, fruits from stage-10 to stage-13 were collected and frozen immediately in liquid nitrogen for RNA isolation. Total RNA was isolated, and mRNA was purified using the Oligotex mRNA Midi Kit (70042, Thermo Fisher) according to the manufacturer’s instructions. cDNAs were subsequently synthesized using the Revert Aid First Strand cDNA Synthesis Kit. A high-quality cDNA library was constructed using the CloneMiner II cDNA Library Construction Kit (A11180, Thermo Fisher). The cDNAs were ligated with a three-frame attB1 adapter by T4 ligase and then purified using the cDNA size fractionation columns. The purified cDNAs were inserted into the pDONR222 vector using BP Clonase (11789020, Thermo Fisher) and recombined into the pGADT7-DEST vector using LR Clonase (11791020, Thermo Fisher) to generate the yeast-two-hybrid cDNA library.\u003c/p\u003e\u003cp\u003eTo avoid the autoactivating activity of \u003cem\u003eCrJAG\u003c/em\u003e, a series of truncated versions of \u003cem\u003eCrJAG\u003c/em\u003e were amplified from the \u003cem\u003epCrJAG\u003c/em\u003e:\u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e plasmid and then inserted into pGBK-T7 (Clontech) to generate the bait plasmids. The respective plasmids were verified by sequencing and then transformed into the yeast strain Y2H gold with pGAD-T7 (Clontech). Positive transformants were selected on synthetic dropout (SD) medium without tryptophan and leucine (-WL). The autoactivating activity was tested by serial dilutions on SD medium without tryptophan, leucine, histidine, and adenine (-WLHA) according to the manufacturer’s protocols. The yeast-two hybrid library screening was performed according to the manufacturer’s instructions (630439, Clontech)\u003c/p\u003e\u003cp\u003eFor verifying the CrJAG-CrMSIs interactions, the coding sequences of \u003cem\u003eCrMSI1\u003c/em\u003e, \u003cem\u003eCrMSI2\u003c/em\u003e, \u003cem\u003eCrMSI3\u003c/em\u003e, \u003cem\u003eCrMSI4\u003c/em\u003e, and \u003cem\u003eCrMSI5\u003c/em\u003e were amplified from the \u003cem\u003eCapsella\u003c/em\u003e inflorescences cDNAs and then inserted into pGAD-T7 to generate prey plasmids. The interaction was tested as described above.\u003c/p\u003e\u003cp\u003eFor the Biomolecular Fluorescence Complementation (BiFC) experiment, the full-length CDS without stop codon of \u003cem\u003eCrJAG\u003c/em\u003e and \u003cem\u003eCrMSI2\u003c/em\u003e, \u003cem\u003eCrMSI3\u003c/em\u003e, and \u003cem\u003eCrMSI4\u003c/em\u003e were amplified and inserted into pBI-cYFP and pBI-nYFP backbones, respectively. The constructs were verified by sequencing and then transformed into the \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101. Agrobacteria were inoculated in YEB medium overnight to OD600 of 1.0-1.2 and then resuspended with infiltration buffer (50 mM MES, pH 5.6, 0.5% glucose, 2 mM NaPO\u003csub\u003e4\u003c/sub\u003e, and 15 mg/L acetosyringone) to OD600 of 0.4–0.5. A mixed \u003cem\u003eAgrobacterium\u003c/em\u003e suspension containing nYFP and cYFP plasmids at a 1:1 ratio was infiltrated into 3-week-old \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. The plants were incubated under weak light for 36 hours before signal checking. Protein interaction was judged by the presence of YFP signal in the nucleus using a laser scanning confocal microscope (Zeiss LSM 980) with excitation at 488 nm and emission at 509 nm, respectively.\u003c/p\u003e\u003cp\u003eFor the Co-IP experiment, the full-length coding sequences without stop codon of \u003cem\u003eCrJAG\u003c/em\u003e, \u003cem\u003eCrMSI2\u003c/em\u003e, \u003cem\u003eCrMSI3\u003c/em\u003e and \u003cem\u003eCrMSI4\u003c/em\u003e were inserted into \u003cem\u003epCambia\u003c/em\u003e1300-cHA or \u003cem\u003epCambia\u003c/em\u003e1300-cFLAG backbones, respectively. The positive plasmids were verified by sequencing and then transformed into the \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101. They were co-infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves as described above. The infiltrated leaves were collected 36 h after infiltration and ground into fine powder in liquid nitrogen. About 0.5 g sample was used for total protein isolation with 1 mL protein extraction buffer [50 mM Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM NaF, 2 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 0.5% NP-40, 1 mM PMSF, 1 mM DTT and 1×Protease Inhibitor Cocktail]. 100 µL total protein mixture was kept as input and the remaining mixture was subjected to immunoprecipitation using DYKDDDDK (FLAG)-Nanoab-Magnetic beads (FNM-25-1000, Lablead) for 1 h at 4°C. The beads were then washed four times with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1 mM DTT. The input and IP samples were boiled for 10 mins in SDS loading buffer, and the eluates were analyzed by immunoblotting with anti-HA (MBL, Japan, 1:10000) or anti‐FLAG (MBL, Japan, 1:10000) antibody.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eImmunoprecipitation-Mass Spectrometry (IP-MS) analysis\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eAbout 3 g of fruits containing stage 10–13 from \u003cem\u003eCrjag; pCrJAG\u003c/em\u003e:\u003cem\u003eCrJAG\u003c/em\u003e:\u003cem\u003eGFP\u003c/em\u003e and wild type were collected and crosslinked with 1% formaldehyde in PBS buffer (150 mM NaCl, 10 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO4, 2.7 mM KCl, pH 7.4). The fruits samples were ground into fine powder in liquid nitrogen, and nuclei were isolated with Honda buffer [0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM HEPES (KOH, pH 7.4), 0.5% Triton X-100, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 1×Protease Inhibitor Cocktail]. The nuclei were then resuspended in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM DTT, and 1× Protease Inhibitor Cocktail) and sonicated for 24 cycles with 30 seconds on and 30 seconds off. The DNA was digested with Benzonase (E1014-25KU, Millipore) for 1 h at 4°C by gentle rotation. The protein extracts were then subjected to immunoprecipitation using Pierce Protein A/G Magnetic Beads (88847; Thermo Fisher), incubated with monoclonal anti-GFP antibody (ab290, abcam) at 4°C for 4 h. After immunoprecipitation, the beads were washed twice with PBS buffer plus 0.5% NP-40, and once with PBS buffer and water, respectively. The beads were digested with 2 µg of trypsin and in 50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e solution overnight at 37°C. The peptides were purified with C18 stage tip and then processed for mass spectrometry on an Orbitrap Exploris 480 machine (Thermo Fisher).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eCleavage under targets \u0026amp; tagmentation (CUT\u0026amp;Tag) experiment\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eThe CUT\u0026amp;Tag experiment was performed following described methods with minor modifications\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Briefly, stage-10 to stage-13 fruits collected from either CrWT, \u003cem\u003eCrjag\u003c/em\u003e or \u003cem\u003eCrjag;pCrJAG:CrJAG:GFP\u003c/em\u003e rescue lines were chopped with a razor blade in HBM buffer (25 mM Tris-HCl pH 7.6, 0.44 M sucrose, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1% Triton-X-100, 0.2 M spermidine, 1× Protease Inhibitor Cocktail, 1 mM PMSF, 10 mM β-Mercaptoethanol). The nuclei suspension was filtered with a 40 µm cell strainer. The extracted nuclei were washed twice with HBB buffer (25 mM Tris-HCl pH 7.6, 0.44 M sucrose, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1% Triton-X-100, 10 mM β-Mercaptoethanol) and then incubated in 50 µL primary antibody buffer [2 mL wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and 1× Cocktail inhibitor) supplemented with 8 µL 0.5 M EDTA and 6.7 µL 30% BSA] at 4°C overnight. 1 µL of primary antibody (1:50, anti-GFP, ab290, abcam; anti-H3K18ac, 07-354, Millipore; anti-H3K27me3, 07-449, Millipore) was used for each sample. The primary antibody was removed after centrifugation at 1200 g for 2 min. Subsequently, the nuclei were incubated with secondary antibody [1:100, Guinea pig anti-rabbit IgG (Heavy \u0026amp; Light Chain) antibody, ABIN101961, Easybio] in 50 µL wash buffer at 4°C for 2 h. The nuclei were resuspended with 100 µL CT-300 buffer (20 mM HEPES pH 7.5, 450 mM NaCl, 0.5 mM spermidine and 1×Protease Inhibitor Cocktail) supplied with pA-Tn5 (1:150) and incubated at 4°C for 3 h. After incubation, the nuclei were washed twice with 600 µL CT-300 buffer, followed by incubation at 37°C for 1 h with 300 µL tagmentation buffer (1 mL CT-300 buffer plus 10 µL 1 M MgCl\u003csub\u003e2\u003c/sub\u003e). To stop the tagmentation reaction, 10 µL 0.5 M EDTA, 3 µL 10% SDS and 2.5 µL 20 mg/mL Protinease K were added r and incubated at 50°C for 1 h. The DNA was extracted with phenol: chloroform: isoamyl alcohol (25:24:1), precipitated with ethanol, and then dissolved in ddH\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eRNA-seq, ATAC-seq, and CUT\u0026amp;Tag-seq data analysis\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eFor RNA-sequencing analysis, total RNA was extracted from stage-13 fruits of the respective genotypes. The libraries were constructed using the MGIEasy RNA Library Prep Kit and then sequenced on the DNBSEQ-T7 platform to generate 150 bp paired-end reads. Low-quality reads and adapter sequences were trimmed to generate clean reads using fastp (version 0.24.0)\u003csup\u003e40\u003c/sup\u003e. The sequences were then mapped and annotated using the \u003cem\u003eCapsella rubella\u003c/em\u003e v1.1 genome sequence\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e with STAR (version 2.7.11b)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Gene expression levels were calculated as fragments per kilobase of transcript per million fragments (FPKM) using an R script. Differentially Expressed Genes (DEGs) were identified as those with a fold change ≥ 2 and a False Detection Rate (FDR) \u0026lt; 0.05 using the R package DESeq2 (version 1.42.1)\u003csup\u003e43\u003c/sup\u003e. GO enrichment analysis of Biological Process (BP) on the DEGs was performed using the R packages enrichplot (version 1.22.0) and clusterProfiler (version 4.10.1)\u003csup\u003e44\u003c/sup\u003e. Boxplots were drawn using the R package ggplot2 (version 3.5.2)\u003csup\u003e45\u003c/sup\u003e. For the RNA-seq experiment, at least three independent biological replicates were conducted for each genotype.\u003c/p\u003e\u003cp\u003eThe ATAC-seq experiment was performed following the protocol previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Briefly, ~ 0.5 g of stage-12 to stage-14 \u003cem\u003eCrjag\u003c/em\u003e fruits were harvested and ground into fine powder in liquid nitrogen. Approximately 50,000 nuclei were collected for DNA library construction. The library was sequenced on the Illumina Novaseq 6000 platform to generate 150 bp paired-end reads. The raw reads were filtered (reads shorter than 35 bp and bases with a quality value less than Q10) using fastp (version 0.24.0)\u003csup\u003e40\u003c/sup\u003e to generate clean FASTQ files. The sequences were then mapped back to the \u003cem\u003eCapsella rubella\u003c/em\u003e v1.1 genome\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e using Bowtie2 (version 2.5.4)\u003csup\u003e46\u003c/sup\u003e. Duplicate reads were removed by sambamba (version 1.0.1)\u003csup\u003e47\u003c/sup\u003e and bedtools (version 2.31.1)\u003csup\u003e48\u003c/sup\u003e. Peaks were called using MACS2 (version 2.1.4)\u003csup\u003e49\u003c/sup\u003e with a screening criterion of FDR \u0026lt; 0.05. The CrWT stage 12–14 fruit ATAC data were published previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Differentially enriched peaks between CrWT and \u003cem\u003eCrjag\u003c/em\u003e were identified using the R package DiffBind (version 3.12.0). Significant closed chromatin accessibility in \u003cem\u003eCrjag\u003c/em\u003e was defined as fold change \u0026lt; 0.8 and FDR \u0026lt; 0.05. DeepTools (version 3.5.6)\u003csup\u003e50\u003c/sup\u003e was used to map the density distribution of sequencing reads in the upstream and downstream regions of the Transcription Start Site (TSS) and the Transcription End Sits (TES) of each gene. The profile plot was generated using the plotProfile function. For visualization, datasets were converted to bigwig format using bamCoverage in DeepTools (version 3.5.6) with a bin size of 1 bp and normalized by the RPKM method, then visualized using Integrative Genomics Viewer (IGV, version 2.4.14)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Gene annotation was performed by ChIPseeker (version 1.38.0)\u003csup\u003e52\u003c/sup\u003e using a 2 kb promoter-proximal window. The ATAC-seq experiments were performed with two independent biological replicates.\u003c/p\u003e\u003cp\u003eFor CUT\u0026amp;Tag-seq data analysis, the library was constructed by amplifying 10–12 cycles using Q5 High-Fidelity DNA Polymerase (M0491S, NEB) and purified by AMPure XP beads (A63881, Beckman) according to the manufacturer’s instructions. Primers used for library amplification are listed in Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. The library was sequenced on the Illumina Nova X Plus platform to generate 150 bp paired-end reads. The raw reads were filtered using fastp (version 0.24.0) to generate clean FASTQ files. After filtering low-quality reads, the sequences were mapped back to the \u003cem\u003eCapsella rubella\u003c/em\u003e v1.1 genome using Bowtie2 (version 2.5.4). Duplicate reads were removed by sambamba (version 1.0.1) and bedtools (version 2.31.1). Peaks were called using MACS2 (version 2.1.4) with a \u003cem\u003ep\u003c/em\u003e value of 0.01. The bigwig files were generated by DeepTools (version 3.5.6) for visualization using IGV (version 2.16.2). Gene annotation was performed with a 2 kb promoter-proximal window using ChIPseeker (version 1.38.0)\u003csup\u003e52\u003c/sup\u003e. For each antibody and genotype, two biological replicates were conducted.\u003c/p\u003e\u003cp\u003eFor data integration, Venn diagrams were generated with the R package VennDiagram (version 1.7.3)\u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eProtoplast transient expression and ChIP-qPCR analysis\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eProtoplast isolation and transient protein expression were performed with the published protocols\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Briefly, leaves from 10-day-old young seedlings were sliced and digested with Enzyme buffer I (1.0% cellulase R10, 0.5% macerozyme R10, 0.5% pectinase, 20 mM KCl, 20 mM MES, 0.6 M mannitol, 10 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.1% BSA) rotating at 60 rpm and 25°C for 2.5 h. The protoplasts were collected by filtering with a 40 µm cell strainer. 35S:CrJAG/CrMSI2/CrMSI3/CrMSI4:GFP and AtUBQ10:NLS-mCherry plasmids were prepared using a QIAGEN Plasmid Maxi Kit according to the manufacturer’s instructions. A total of 1000 ng plasmids (35S:CrJAG : AtUBQ10:NLS-mCherry = 1:1) were co-transformed into protoplasts with a PEG-mediated heat-shock method (42°C, 3mins). The protoplasts were incubated under weak light at 26°C for 18 h. The subcellular localization of CrJAG:GFP, CrMSI2:GFP, CrMSI3:GFP, and CrMSI4:GFP proteins was detected on a Zeiss 980 confocal microscope with AtUBQ10:NLS-mCherry as a nuclear-localized marker.\u003c/p\u003e\u003cp\u003eThe ChIP-qPCR analysis was performed using modified protocols described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Briefly, protoplasts transformed with \u003cem\u003epCambia\u003c/em\u003e1300-cFLAG/CrMSI2/CrMSI3:GFP and CrJAG-cFLAG/CrMSI2/CrMSI3:GFP were collected and crosslinked with 1% formaldehyde for 10 mins at room temperature and stopped with 125 mM glycine for 5 min. The protoplasts were resuspended in RIPA buffer and sonicated for 24 cycles with 30 s on and 30 s off. The samples were then subjected to immunoprecipitation using an anti-GFP antibody (ab290, Abcam) or an anti-FLAG antibody (F3165, Sigma-Aldrich) pre-incubated with Pierce Protein A/G magnetic beads (88847, Thermo Fisher) at 4°C for 4.5 h. The beads were washed, then reverse-crosslinked by adding 10% sodium dodecyl sulfate (SDS) and incubating at 65°C for 12 h. The proteins in the DNA complex were digested with Proteinase K at 45°C for 1 h. The DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol, then resuspended in water (W4502, Sigma-Aldrich). qPCR was performed on a qTOWER PCR System (Analytik Jena AG) using SYBR Premix Ex Taq.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eChemical treatment and cell flow cytometry\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eFor chemical treatment, stage-12 fruits were collected and transferred onto 0.4% agar half-strength Murashige and Skoog (1/2 MS) medium plates containing 100 µM Aurora kinase inhibitor II (17541, Cayman) or mock solution (DMSO) for 5 days. The phenotypes were recorded using a Nikon D850 camera with a 105 mm prime lens.\u003c/p\u003e\u003cp\u003eFor the cell-flow cytometry analysis, nuclei were isolated from fruits in 250 µL PVPK12-mGB\u003csub\u003e2\u003c/sub\u003e buffer [30 mM sodium citrate, 45 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM MOPS, 20 mM NaCl, 20 mM EDTA Na\u003csub\u003e2\u003c/sub\u003e·2H\u003csub\u003e2\u003c/sub\u003eO, 0.1% (v/v) Triton X-100, 0.5% (v/v) Tween-20, 10 µL/mL 1–2% PVPK12, pH 7.0] and filtered through a 40 µm nylon mesh. The nuclei were stained with 4 µg/mL DAPI. For each sample, approximately 20,000 nuclei were analyzed using a BD LSRFortessa flow cytometer (BD, USA). Quantitative analysis of ploidy level was conducted using FlowJo software (v10.10). EI was calculated from the ploidy histograms using the formula: EI = 4C% + 2 × 8C%. Statistical analysis was performed in GraphPad Prism (v10.1.2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eQuantification and statistical analysis\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eAll statistics were collected and analyzed using Microsoft Excel and GraphPad Prism (v10.1.2). All data are presented as means ± SD with sample sizes marked by solid dots and described in the figure legends. Comparisons between groups were performed with Microsoft Excel and GraphPad Prism (v10.1.2) using Student’s t-test, and significance levels are marked as: * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSoltis PS, Folk RA, Soltis DE (2019) Darwin review: angiosperm phylogeny and evolutionary radiations. Proc. R. Soc. 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Int J Mol Sci 23:3419\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fruit Morphogenesis, Capsella rubella, Development, Epigenetic Regulation, Histone Modification, Cell Division","lastPublishedDoi":"10.21203/rs.3.rs-8265099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8265099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFruits that protect the seeds and aid their dispersal are a key feature distinguishing angiosperms from other land plants, and fruit shape varies widely between species. However, it remains largely unknown how local growth behaviors are coordinated during development to generate fruits with characteristic sizes and shapes. Members of the \u003cem\u003eCapsella\u003c/em\u003e genus in the mustard family produce unique fruits with an evolutionarily derived heart shape, making it an exceptional system suited for addressing this question. In this study, we demonstrate that the \u003cem\u003eCapsella rubella\u003c/em\u003e C2H2-zinc finger transcription factor JAGGED (CrJAG) controls fruit shape. Whole-mount live imaging analysis indicates that CrJAG regulates valve development by promoting both cell division and anisotropic growth. Ectopic expansion of the valve epidermal cells in \u003cem\u003eCrjag\u003c/em\u003e mutants is accompanied by mis-patterned endoreduplication events. CrJAG physically interacts with the histone chaperones \u003cem\u003eCapsella rubella\u003c/em\u003e MULTICOPY SUPPRESSOR OF IRA1 members, increasing the chromatin accessibility and thereby sustaining the expression of genes involved in fruit morphogenesis. The resulting closed chromatin state in \u003cem\u003eCrjag\u003c/em\u003e fruits is characterized by a reduction of the active histone marker (H3K18ac) and an increase of the repressive marker (H3K27me3). Expression analyses and pharmacological treatments indicate that the developmental defects in \u003cem\u003eCrjag\u003c/em\u003e fruits are largely attributable to down-regulation of the key cell-cycle regulator \u003cem\u003eCrAUR2\u003c/em\u003e. Collectively, our findings suggest that fine-tuning the cell cycle via epigenetic modification represents an additional, essential layer of regulation critical to organ development.\u003c/p\u003e","manuscriptTitle":"Epigenetic regulation of fruit shape determination by JAGGED gene in Capsella rubella","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 07:29:00","doi":"10.21203/rs.3.rs-8265099/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"62ad873e-6475-472a-8ee5-e8029644a9ce","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63343644,"name":"Biological sciences/Plant sciences"},{"id":63343645,"name":"Biological sciences/Plant sciences/Plant development"}],"tags":[],"updatedAt":"2026-04-30T13:06:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 07:29:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8265099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8265099","identity":"rs-8265099","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}