Integrated omics analysis provides preliminary insights into the regulatory network controlling ethephon-promoted calyx abscission in Korla Xiangli (Pyrus × sinkiangensis Yü)

Integrated omics analysis provides preliminary insights into the regulatory network controlling ethephon-promoted calyx abscission in Korla Xiangli (Pyrus × sinkiangensis Yü) | 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 Research Article Integrated omics analysis provides preliminary insights into the regulatory network controlling ethephon-promoted calyx abscission in Korla Xiangli (Pyrus × sinkiangensis Yü) Yue Wen, Lingling Zheng, Yan Lin, Tianyu Sun, Yun Gao, Chen Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8648793/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Calyx abscission is a key horticultural trait affecting fruit quality in Korla Xiangli (Pyrus × sinkiangensis Yü ). Exogenous ethephon treatment has been shown to significantly promote calyx abscission, but the underlying molecular regulatory network and metabolic mechanisms that regulate ethephon-induced abscission remain poorly understood. Elucidation of these processes is essential to optimize calyx management strategies in commercial Korla Xiangli production. Results This study systematically elucidated the molecular regulatory network of ethephon-induced calyx abscission in Korla Xiangli by phenotypic observation, phytohormone profiling, wide-target metabolomics and high-throughput transcriptomic analyses. Treatment with 300 mg·L⁻¹ ethephon significantly accelerated abscission zone formation by two days, with abscission occurring seven days after pollination. The peak daily abscission rate reached 16.90%, which represented a 5.15-fold increase compared with the untreated control. The results demonstrated that the third day after pollination was the critical period for ethephon-mediated calyx abscission regulation. At this stage, ethylene concentration in the abscission zone significantly increased by 33.09% and indole-3-acetic acid content decreased sharply by 49.30%. In contrast, abscisic acid, salicylic acid, gibberellin A₃, and jasmonic acid levels were strongly upregulated, which suggests extensive hormonal regulation associated with abscission initiation. Transcriptome data showed transient induction of ERF1/2 and repression of AUX/IAA and SAUR genes, limiting polar auxin transport and enhancing ethylene sensitivity. Metabolomic profiling revealed 383 differential metabolites, primarily associated with flavonoid, lipid, and terpenoid pathways, collectively promoting ROS regulation, membrane destabilization, and programmed cell death. Network analyses identified MYB, AP2/ERF, and NAC transcription factors and highlighted key hub genes involved in auxin–ethylene crosstalk. Conclusions In this study, we first conducted phenotypic observations of ethephon-induced calyx abscission in Korla Xiangli, and then measured the levels of plant hormones, metabolites and gene expression in the abscission zone. These results provide a theoretical basis and genetic resources for the precise regulation and molecular breeding of calyx abscission in Korla Xiangli. Korla Xiangli (Pyrus × sinkiangensis Yü) calyx abscission ethephon transcriptomics metabolomics hormone signaling Weighted gene co-expression network analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Plant organ abscission is a process that detaches plant tissues or organs from the mother plant and involves coordinated action among multiple factors. The region where separation occurs and its adjacent tissues, is termed the ‘abscission zone’ and plant hormones play pivotal roles in regulating abscission [ 1 ]. For instance, auxin (indole-acetic acid, IAA) maintains abscission zone insensitivity to ethylene (ETH), thereby suppressing abscission [ 2 ]. If IAA levels decline or its polar transport is disrupted in the abscission zone, then ETH signaling is enhanced and the expression of ethylene response factors (ERFs) increases. This activates the transcription and translation of cell wall hydrolases, such as β‑1,4‑glucanase and polygalacturonase, which accelerate cell wall degradation and middle lamella dissolution [ 3 ]. Studies by on tomato and rose plants showed that ETH biosynthesis genes were the key targets of ERFs during abscission and that this regulatory process was finely tuned by abscission signals [ 4 ]. The results highlighted the central role of ERFs in the abscission regulatory network. Abscisic acid (ABA) promotes abscission via two synergistic pathways: first, by directly activating 1‑aminocyclopropane‑1‑carboxylate synthase (ACS) gene expression to enhance ETH biosynthesis and second, by suppressing PIN‑FORMED‑mediated polar auxin transport, thereby further amplifying ETH signaling [ 5 ]. The role played by gibberellin (GA) in abscission zone development and organ abscission is unclear, although most studies have suggested that it has an inhibitory function. Gibberellin probably delays abscission by antagonizing ETH signaling through DELLA‑dependent or DELLA‑independent pathways [ 6 ]. Jasmonic acid (JA), a key hormone involved in plant stress responses, markedly accumulates in abscission zone tissues and accelerates organ abscission by either directly activating programmed cell death (PCD)‑related genes via MYC2/3/4 transcription factors or through synergistic action with 1‑aminocyclopropane‑1‑carboxylic acid [ 7 , 8 ]. Salicylic acid (SA) modulates abscission via crosstalk with immune signaling pathways. Key regulatory components, such as NPR1, EDS1, and PAD4, drive SA biosynthesis and form a positive‑feedback loop with SA. This induces localized reactive oxygen species (ROS) bursts and PCD, which ultimately promote cell separation within the abscission zone [ 9 ]. A continuous series of physiological and biochemical reactions related to cell metabolism and signal transduction occurs during plant organ abscission, and the various metabolites generated in this process regulate abscission progression [ 10 ]. A metabolomic analysis of fruit abscission in blue honeysuckle showed that different metabolites were significantly enriched in various pathways, such as plant hormone signal transduction, starch‑sucrose metabolism, the pentose phosphate pathway, and phenylpropanoid biosynthesis. Among them, m‑hydroxysalicylic acid and 2‑hydroxy‑6‑(8‑tridecenyl)benzoic acid, as core effector molecules of the SA signaling pathway, synergistically regulated the antioxidant defense system, mediated cell wall modification in the abscission zone, and promoted middle lamella degradation. These actions systematically controlled the initiation and progression of fruit abscission [ 11 , 12 ]. Other studies on flower and pod abscission in soybean revealed that there were significant changes to the flavonoid, fatty acid, and amino acid biosynthesis pathways in the abscission zone tissues, and that these changes were closely associated with stress responses and secondary metabolism. Metabolites such as proline and phenylalanine are precursors of osmoregulators and antioxidants and maintain redox homeostasis in abscission zone cells. Also, they indirectly affect lignin and flavonoid accumulation by regulating the metabolic flux of phenylpropanoid biosynthesis, which means that they participate in the remodeling of cell wall structure [ 13 ]. Korla Xiangli (Pyrus × Sinkiangensis Yü) belongs to the Rosaceae family and the Xinjiang system. It is an ancient local cultivar originating from the Korla region of Xinjiang and has a long cultivation history. The fruit can be categorized into decalyx fruit and persistent calyx fruit types. The persistent calyx fruit quality is generally lower than that of decalyx fruit but the proportion of decalyx fruit is high, which limits sustainable commercial production. Therefore, identifying safe and efficient exogenous plant growth regulators to precisely regulate calyx abscission has become a critical technical challenge in Korla Xiangli production. Ethephon is a synthetic, environmentally benign regulator that rapidly decomposes within plants to release ETH, thereby inducing abscission zone formation and organ shedding [ 14 ]. To date, research on ethephon‑mediated organ abscission in horticultural crops has largely focused on flower and fruit drop, particularly tomato pedicels and citrus fruitlets [ 15 , 16 ]. Previous studies have also reported that ethephon treatment of litchi panicles significantly down‑regulated the expression of key auxin‑signaling genes (ARF and Aux/IAA), but up‑regulated ERF genes in the abscission zone [ 17 ]. One study confirmed that spraying 300 mg·L⁻¹ ethephon at the large‑bud stage significantly accelerated calyx abscission in Korla Xiangli, although the molecular mechanisms were unclear [ 18 ]. Further mass‑spectrometry imaging has revealed that ABA and ETH are markedly enriched in the abscission zone of natural de‑calyx fruit, whereas IAA and GA accumulated in the corresponding region of persistent‑calyx fruit [ 19 ]. Nevertheless, the phenotypic dynamics, the hormone and metabolite changes, and the regulatory network constructed by hormone‑related genes during ethephon‑induced calyx abscission require further exploration. Based on this, the present study used the abscission zone of de‑calyx Korla Xiangli fruit to systematically compare the calyx‑abscission phenotypic traits between water‑ and ethephon‑treated samples and combined wide‑target metabolomics to analyze the dynamic responses of key metabolic pathways. High‑throughput transcriptomics and bioinformatics were used to identify ethephon‑induced differentially expressed genes, with a particular focus on core nodes within the plant hormone signaling pathways. This information was then used to identify and validate the key regulatory factors promoting calyx abscission and a molecular regulatory network for ethephon‑induced calyx abscission was constructed. The findings from this study provide a theoretical basis and gene resources for the precise regulation and molecular breeding of calyx abscission in Korla Xiangli and lay a solid foundation for improving its fruit quality. Results Phenotypic observation of ethephon‑induced calyx abscission in Korla Xiangli External morphological changes during calyx abscission The external morphological changes during ethephon‑induced calyx abscission in Korla Xiangli were observed. At 3 DAP, the flowers remained intact with well‑developed stamens and sepals, whereas at 5 DAP, the petals had begun to shed naturally and the anther color was gradually fading. However, but the sepals remained firmly attached to the young fruit. These changes indicated that the plant samples had entered the pre‑abscission‑zone stage (3–5 DAP). Between 7, 9, and 11 DAP, the stamen filaments turned brown and a distinct circular abscission zone became visible at the junction between the sepals and the young fruit. This was the abscission‑zone formation stage. By 13 DAP (post‑abscission‑zone formation), the sepals had fully detached at the abscission zone and had been shed (Fig. 1 ). Calyx abscission followed a similar morphological progression in the CK group and could be divided into the pre‑abscission‑zone, abscission‑zone formation, and post‑formation stages. However, in the CK group, the yellowish ring that indicated abscission zone initiation did not appear until 9 DAP, which was 2 days later than in the ethephon treatment. This demonstrated that exogenous ethephon application significantly accelerated both abscission‑zone formation and calyx abscission in Korla Xiangli. Daily calyx abscission rate The ethephon treatment significantly increased the daily calyx abscission rate (Fig. 2 ). During the first 6 DAP, no calyx abscission was observed in either the ethephon‑treated or CK (water‑treated) groups. Under ethephon induction, calyx abscission began at 7 DAP (consistent with external morphological observations) and peaked at 10 DAP. The daily abscission rate was 16.90% and abscission had ceased by the 13th day. In contrast, calyx abscission started at 9 DAP in the CK group and peaked at 11 DAP. However, the daily abscission rate was only 3.28%. Thus, abscission initiation was delayed by 2 days and the peak was postponed by 1 day in the CK group compared to the ethephon group. The external morphological changes during calyx abscission showed that 3–5 DAP in the ethephon‑treated group corresponded to the pre‑abscission‑zone stage, days 7, 9, and 11 represented the abscission‑zone formation stage, and day 13 was the post‑abscission‑zone stage. Further analyses of the pre‑abscission‑zone stage (3–5 DAP), including an in‑depth investigations of hormones, metabolites, and gene expression, will need to be performed to further elucidate the regulatory mechanism controlling ethephon effects on calyx abscission in Korla Xiangli. Changes in phytohormone content during ethephon‑induced calyx abscission in Korla Xiangli The ETH concentration in the calyx abscission zone of the ethephon treated plants was significantly higher than in the CK plants at 3, 5, and 7 DAP and peaked on day 7 with a 33.09% increase compared to CK (Fig. 3 a); IAA content was markedly lower by 49.30% compared to CK at 3 DAP (Fig. 3 b); and ABA, GA₃, and SA levels had significantly increased by 242.69%, 34.19%, and 31.51%, relative to CK on day 3, respectively (Figs. 3 c, 3 e, 3 f). Conversely, the GA₃ content was significantly lower by 82.88% and 64.64% in the ethephon group at 5 and 7 DAP compared to the control, respectively (Fig. 3 ‑D). The JA content was significantly higher than CK by 42.32% and 126.50% at 3 and 5 DAP, respectively (Fig. 3 e). Note * and ** indicate significant differences at the p < 0.05 and 0.01 levels, respectively. Metabolomic analysis of ethephon‑induced calyx abscission in Korla Xiangli Screening and identification of differential metabolites A total of 1,585 metabolites were identified in this study. Amino acids and their derivatives (145), organic acids (74), and nucleotides and their derivatives (42) were the top three categories among the primary metabolites accounting for 9.15%, 4.67%, and 2.65% of the total, respectively. Flavonoids (248), terpenoids (246), phenolic acids (193), alkaloids (138), and lipids (123) ranked the highest among the secondary metabolites accounting for 17.74%, 15.53%, 12.18%, 8.71%, and 7.77%, respectively, of the secondary metabolites (Fig. 4 a). The PCA results showed that there was a clear separation between the ethephon‑treated and CK samples along the PC1 axis and that they were highly dispersed. This indicated that the two groups had distinct metabolic profiles (Fig. 4 b). A further comparative analysis revealed 383 differential metabolites between the abscission zones of the ethephon and CK treatments, with the highest number and variety observed at 5 DAP (118 metabolites) (Fig. 4 c). Among these, 86 were up‑regulated and 32 were down‑regulated (Fig. 4 d). The lipid levels were significantly lower in the ethephon‑treated group compared to CK at 3 DAP, while terpenoids and alkaloids were notably elevated (Fig. 4 e). However, at 5 DAP the terpenoids, lipids, and flavonoid levels were all significantly higher than in the CK group (Fig. 4 f). After excluding the "others" category, the comparisons showed that there were 11 major classes of differential metabolites. At 3 DAP flavonoids showed the greatest difference, followed by lipids and terpenoids (Fig. 4 e), and at 5 DAP terpenoids showed the greatest difference, followed by lipids and flavonoids (Fig. 4 f). KEGG enrichment analysis and key metabolite screening of the differential metabolites The differential metabolites were significantly enriched in a number of pathways at 3 DAP, including linoleic acid metabolism, flavonoid biosynthesis, α‑linolenic acid metabolism, amino acid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, isoflavonoid biosynthesis, lysine biosynthesis, pyrimidine/purine metabolism, and glycine‑serine‑threonine metabolism. Among these, the top five pathways showed significant enrichment ( p < 0.05) (Fig. 5 ). The core pathways comprised of α‑linolenic acid metabolism, linoleic acid metabolism, sesquiterpenoid and triterpenoid biosynthesis, purine metabolism, zeatin biosynthesis, amino acid biosynthesis, C5‑branched dibasic acid metabolism, biotin metabolism, pyrimidine metabolism, and monoterpenoid biosynthesis at 5 DAP. The significantly enriched pathways ( p < 0.05) were the same as those at 3 DAP and still showed increased linoleic acid metabolism, flavonoid biosynthesis, α‑linolenic acid metabolism, amino acid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis (Fig. 5 a). Within these pathways, three primary metabolites were commonly annotated across both comparison groups. These were DL‑ortho‑tyrosine, N‑(1‑deoxy‑1‑fructosyl)phenylalanine, and S‑methylglutathione (Fig. 5 b). The ethephon treatment led to the down‑regulation of 13 secondary metabolites at 3 DAP. These were grouped into six lipids (9‑oxo‑10,12‑octadecadienoic acid, 2‑aminooctadecane‑1,16,18,18‑tetrol, 9,10,13‑trihydroxy‑11‑octadecenoic acid, 9(10)‑epoxyoctadecenoic acid, (9R,10S)‑(12Z)‑9,10‑epoxyoctadecenoic acid, 13S‑hydroperoxy‑9Z,11E‑octadecadienoic acid, and 9,12,13‑trihydroxy‑10,15‑octadecadienoic acid), four flavonoids (kaempferol‑3‑O‑glucoside‑7‑O‑rhamnoside, prunetin, quercetin‑3‑O‑(6″‑O‑malonyl)‑glucoside, and persicoside), two terpenoids (hamamelitannin B and coeloginin), and one alkaloid (pantetheine). In contrast, five metabolites were up‑regulated: two alkaloids (diprophylline and pectolinarin), two flavonoids (limocitrin‑7‑O‑(6″‑acetyl)glucoside and didymin), and one terpenoid (spathulenol). The number of differential metabolites induced by exogenous ethephon sharply decreased at 5 DAP, with only leucocyanidin, 2,3,16,21‑tetrahydroxyolean‑12‑en‑28‑oic acid (platycodin C), lysophosphatidylcholine 20:5, and dihydromarrubin showing significant changes (Fig. 5 C). Transcriptomic analysis of ethephon‑induced calyx abscission in Korla Xiangli Screening of differentially expressed genes (DEGs) A total of 2,141 DEGs were identified out of the 20,456 expressed genes. The majority occurred in the pre‑abscission zone stage at 3 DAP (Fig. 6 a). A total of 679 DEGs were identified at 3 DAP (390 DEGs were significantly up‑regulated and 289 were down‑regulated) in the abscission zone; 581 DEGs were identified at 5 DAP (362 up‑regulated and 219 down‑regulated); 581 DEGs were identified at 7 DAP (292 up‑regulated and 289 down‑regulated); and the number of DEGs was the lowest at 9 DAP, with only 300 genes showing differential expression. Additionally, eight DEGs were commonly annotated across all four time points, which suggested that they were core regulatory factors (Fig. 6 b). KEGG and GO enrichment analysis of differentially expressed genes (DEGs) The DEGs between the ethephon treatment and CK were mapped to obtain their corresponding KEGG pathway information. A total of 8 and 14 significantly enriched KEGG pathways were identified in the 300‑3d‑D_vs_CK‑3d‑D and 300‑5d‑D_vs_CK‑5d‑D comparison groups, respectively (Fig. 7 ). After sorting the KEGG pathways by P‑value, the DEGs in the abscission zone at 3 DAP were mainly enriched in metabolic pathways, sesquiterpenoid and triterpenoid biosynthesis, pentose and glucuronate interconversions, carotenoid biosynthesis, plant hormone signal transduction, α‑linolenic acid metabolism, biosynthesis of secondary metabolites, and photosynthesis‑antenna proteins, among which the first five pathways were significantly enriched ( p < 0.05) (Fig. 7 a). The DEGs at 5 DAP were significantly enriched in sesquiterpenoid and triterpenoid biosynthesis, photosynthesis‑antenna proteins, protein processing in endoplasmic reticulum, biosynthesis of secondary metabolites, motor proteins, and plant hormone signal transduction. The "plant hormone signal transduction" pathway was significantly enriched at both 3 and 5 DAP, which suggested that it played a core role in ethephon‑induced calyx abscission. Therefore, future analyses should further explore the key genes in this pathway that are induced by ethephon. The GO enrichment analysis (Fig. 7 b) showed that the biological processes affected by the DEGs in the ethephon treatment group encompassed reproduction, metabolism, signal transduction, regulation of biological processes and rhythmic processes. The cellular components were dominated by protein complexes and cellular anatomical entities, whereas molecular functions were significantly enriched in categories such as transcription regulator activity, antioxidant activity, molecular transducer activity, and ATP‑dependent activity. Transcription factor analysis The key transcriptional regulators involved in ethephon‑induced calyx abscission in Korla Xiangli were identified by predicting and summarizing the transcription factors (TFs) among the DEGs (Fig. 8 ). A total of 84 differentially expressed TFs belonging to 27 families were identified at 3 and 5 DAP. The top eight TF families most significantly regulated by ethephon were AP2/ERF, MYB, NAC, HSF, AUX/IAA, C2C2‑CO‑like, LOB, and WRKY. A total of 12 TF families were commonly enriched across both time points and the three most abundant families were MYB, AP2/ERF, and NAC. Analysis of DEGs in the plant hormone signal transduction pathways Related genes in the signal transduction pathways of four major phytohormones (IAA, ETH, ABA, JA, and GA) were significantly regulated at both 3 and 5 DAP during ethephon‑induced calyx abscission in Korla Xiangli, (Figs. 9 a, b). Among these, the IAA pathway had the greatest number of enriched DEGs, followed by the ETH pathway. This indicated that these pathways were critical for ethephon‑mediated promotion of calyx abscission. The DEGs in the IAA and JA pathways were predominantly down‑regulated at 3 DAP, which suggested that ethephon had inhibitory effects on these pathways at this stage. In contrast, the DEGs in the JA pathway were mainly up‑regulated at 5 DAP. Conversely, the DEGs in the ETH and ABA pathways were largely up‑regulated, which showed that ethephon strongly activated these pathways. Further analysis of the changes in the genes associated with the four hormone pathways (IAA, ETH, ABA, and JA) that were enriched at both 3 and 5 DAP revealed that genes such as Psin13G007030, Psin17G015890, Psin13G016030, Psin15G013490, Psin16G015870, Psin16G015890, Psin16G010530, Psin09G006830, Psin10G004140, Psin10G004150, Psin10G004340, Psin15G019720, and Psin05G016120 in the IAA pathway were significantly down‑regulated at 3 DAP. Almost all the genes were significantly up‑regulated in the ETH pathway, except for Psin02G006860 and Psin17G019520, and the number of DEGs in these related pathways was notably higher at 3 DAP compared to 5 DAP (Figs. 9 c, d). Additionally, the numbers of genes in the ABA and JA hormone signal transduction pathways were markedly lower than in the IAA and ETH pathways, which further suggested that they played key regulatory roles in IAA and ETH signaling in ethephon‑promoted calyx abscission in Korla Xiangli. The results also suggested that 3 DAP is a critical window for ethephon regulation of calyx abscission. Figure 10 illustrates the hormone signal transduction processes associated with IAA, ETH, ABA, and JA during ethephon‑induced calyx abscission in Korla Xiangli. Specifically, in the ETH signaling pathway, ethephon significantly up‑regulated the expression of ethylene response factor ERF1/2 at 3 DAP. However, ERF1/2 expression had decreased to levels below those of CK by 5 DAP, which meant that it showed a “rise‑then‑fall” pattern. Ethephon had strongly suppressed the expression of AUX/IAA, CH3, and the early auxin‑responsive gene SAUR in the IAA signaling pathway at 3 DAP, which indicated that there was a marked reduction in IAA signaling. Moreover, multiple genes in the ABA signaling pathway showed distinct expression pattern differences between the ethephon treatment and CK. For example, at 3 DAP, ethephon up‑regulated sucrose non‑fermenting‑related protein kinase 2 (SnRK2), which is a positive regulator of the ABA response, and ABA‑responsive element‑binding factor (ABF). However, the core ABA receptor family PYR/PYL was significantly down‑regulated at 5 DAP. Furthermore, MYC2, a central responder in the JA signaling pathway, was significantly down‑regulated by ethephon at both 3 and 5 DAP. These results suggested that these genes probably responded to exogenous ethephon treatment, participated in calyx abscission in Korla Xiangli, and that the IAA and ETH signaling pathways played key roles in this process. Validation of the qRT‑PCR results The number of DEGs was considerably higher at 3 DAP than at 5 DAP; therefore this study focused on the 3‑day time point and 12 DEGs from the plant hormone signal transduction pathways were selected based on their FPKM values for qRT‑PCR validation (Fig. 11 ). The qRT‑PCR results were highly consistent with the FPKM trends, which further confirmed that ethephon significantly suppressed the expression of IAA‑related genes and activated ETH‑related genes in the abscission zone. This result showed that ethephon primarily promoted calyx abscission by modulating the plant hormone signal transduction pathways and that IAA and ETH signaling played central roles in this process. WGCNA analysis of phytohormone‑related differentially expressed genes during ethephon‑induced calyx abscission in Korla Xiangli Previous studies have confirmed that exogenous ethephon significantly affected the levels of hormones such as IAA and ETH. In addition, a large number of DEGs in the IAA and ETH signal transduction pathways were significantly enriched during Korla Xiangli calyx abscission following ethephon treatment. This suggested that IAA and ETH signaling were the key regulatory pathways through which ethephon promotes calyx abscission in Korla Xiangli and that 3 DAP was the critical period for ethephon regulation. Based on this, a dynamic cut‑tree algorithm was used to perform a time‑series association analysis of the phytohormone contents and the differential expression profile at 3 DAP. It identified seven co‑expression modules that were significantly coupled to hormone signal transduction (Fig. 12 a). Excluding the non‑informative MEgrey module, the number of genes in the other modules ranged from 55 (MEturquoise module) to 204 (MEblack module) (Table S2). Among these, the MEyellow module showed a strong negative correlation with IAA levels (r = − 0.93, Fig. 12 b). Within this module, nine core genes that were potentially regulated by ethephon during calyx abscission were further predicted (Fig. 12 c: a). Additionally, the MEblue and MEgreen modules contained 8 and 13 candidate target genes, respectively, whose functions included leucine‑rich repeat extension‑like protein 6, receptor‑like protein kinase (ZmPK1), indole‑3‑acetic acid‑induced protein (ARG13), indole acetic acid‑induced protein 32 (PSK4), protein IDA‑like (IDL 2), transcription factors AP2/ERF and AUX/IAA, and bHLH75 (Figs. 12 c: b, c). This set of genes could be used to elucidate the molecular mechanism by which ethephon promotes calyx abscission in Korla Xiangli through the regulation of the IAA and ETH signal transduction pathways. Discussion Abscission zone formation and identification of the key phases in ethephon‑induced calyx abscission in Korla Xiangli Organ abscission in plants is a complex physiological process that is often accompanied by morphological changes such as wilting and browning [ 22 ]. Previous research has shown that in ‘Korla Fragrant’ pear, a yellow annular abscission zone appears at the junction between the young fruit and the calyx tube around the 8th day after full bloom. This zone is a key marker for calyx abscission [ 18 ]. In this study, ethephon treatment induced the formation of the calyx abscission zone as early as 7 DAP, which was 2 days earlier than in the CK group (9 DAP). This suggested that exogenous ethephon application accelerated the calyx abscission process. Ethephon enhances hydroxyl radical scavenging capacity, the superoxide anion generation rate, and the intracellular Ca²⁺ concentration in abscission zone cells. These changes lead to the rupture of distal cell wall structures and the degradation of cell membranes and organelles into condensed apoptotic bodies, and accelerates PCD to promote abscission [ 23 , 24 ]. Notably, abscission zone cells perceive and respond to abscission signals at an earlier point than when morphological changes become evident. This means that the abscission process is actually initiated before a visible abscission zone can be observed. Therefore, it can be hypothesized that the period before 7 DAP represents the key phase for ethephon‑induced calyx abscission (corresponding to 3 and 5 DAP in this study). Accordingly, the calyx abscission process under ethephon treatment was divided into three consecutive stages: the pre‑abscission zone phase (3–5 DAP), the abscission zone formation phase (7–11 DAP), and the post‑abscission zone phase (13 DAP). This provided a clear temporal framework for the subsequent molecular mechanism analysis. Hormonal regulation in ethephon‑induced calyx abscission in Korla Xiangli Numerous studies have shown that exogenous ethephon promotes abscission by mediating the biosynthesis and signaling of endogenous plant hormones and cell wall modification [ 25 , 26 ]. It is widely recognized that ETH, ABA, JA, and SA promote abscission, while IAA, GA, and CTK generally play inhibitory roles. In this study, following ethephon treatment, the ETH concentration in the calyx abscission zone was significantly higher than that of CK at 3, 5, and 7 DAP and was the most pronounced increase among all the hormones measured. The ABA, GA₃, JA, and SA contents were also significantly elevated compared to CK at 3 DAP, whereas the IAA content showed an opposite trend. The results suggested that ABA and JA indirectly amplified ETH signaling by activating the expression of ETH biosynthesis enzymes (ACS/ACO), which enhanced the abscission response. In addition polar IAA transport in the abscission zone seemed to continuously modulate tissue sensitivity to ETH. When endogenous ETH levels rose, polar IAA transport decreased, which significantly increased the responsiveness of the abscission zone to abscission signals and accelerated the abscission process [ 27 ]. Based on these findings, it is proposed that the biosynthesis, signaling, and interactions between endogenous ETH and IAA constitute one of the core regulatory mechanisms through which exogenous ethephon induces calyx abscission in Korla Xiangli. Therefore, the key genes involved in ETH and IAA synthesis and signaling pathways during the pre‑abscission stage need to be further investigated to clarify the mechanism associated with ethephon‑induced calyx abscission in Korla Xiangli. Regulatory role of metabolites in ethephon‑induced calyx abscission in Korla Xiangli The metabolomic analysis revealed the dynamic changes and potential regulatory roles of metabolites during ethephon‑induced calyx abscission in Korla Xiangli. It detected 1,585 metabolites and demonstrated that ethephon significantly altered the types and contents of metabolites in the calyx abscission zone. The primary metabolites were dominated by amino acids and their derivatives, which were significantly enriched in the amino acid biosynthesis pathways. The secondary metabolites mainly included flavonoids, terpenoids, and lipids, which were primarily annotated to pathways such as flavonoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, linoleic acid metabolism, and α‑linolenic acid metabolism. The dynamic analysis of the metabolites indicated that flavonoid accumulation was the greatest change at 3 DAP, followed by the change in lipid concentration, which showed a downward trend. As potent antioxidants, the early accumulation of flavonoids is probably a stress response to an ethephon‑induced ROS burst. This increase in flavonoids probably scavenges excess free radicals to protect cells from oxidative damage [ 28 ]. In contrast, the significant down‑regulation of lipid metabolites, such as 9‑oxo‑10,12‑octadecadienoic acid, may impair cell membrane stability and actively promote PCD. Furthermore, the significant enrichment of the α‑linolenic acid and linoleic acid metabolism pathways provides a logical link to the observed increase in JA levels as these unsaturated fatty acids are direct precursors for JA biosynthesis. Terpenoids emerged as the most differentially altered metabolite class at 5 DAP and consisted of compounds that are part of the sesquiterpenoid and triterpenoid pathways involved in lignin precursor synthesis and defense responses. These would potentially influence the structural toughness of the abscission zone by modulating cell wall lignification [ 29 ]. Among the primary metabolites, amino acids and their derivatives are directly involved in fundamental physiological activities and defense responses. For instance, in ripening strawberries, ethephon treatment increases the contents of various amino acids, such as alanine, arginine, and glutamine, with the most significant effects observed during the green and white developmental stages, which suggests a close association with plant growth, development, and defense mechanisms [ 30 ]. Organic acids, as core intermediates in the mitochondrial tricarboxylic acid and glyoxylate cycles, regulate photosynthesis and respiration and accumulate under environmental stress to enhance resistance [ 31 ]. In contrast, alkaloids contribute to defense against microbial and viral infections [ 32 ]. Although these metabolites play important roles in plant metabolic networks [ 33 ], their specific mechanisms during organ abscission regulation require further investigation. Ethephon induces calyx abscission in Korla Xiangli by modulating the interaction between ETH and IAA signaling Exogenous plant growth regulators influence organ abscission by regulating hormone biosynthesis, signal transduction, and transport [ 34 ]. Studies on apple have reported that IAA treatment up‑regulated IAA‑responsive genes in the fruit pedicel abscission zone while suppressing key ETH biosynthesis genes and the transcriptional activity of ERFs [ 35 ]. Conversely, ethephon treatment of litchi panicles down‑regulated IAA signaling genes (such as ARF, Aux/IAA, and SAUR) and significantly up‑regulated ERFs [ 36 ]. These findings collectively highlighted the core mechanism by which IAA and ETH signaling antagonistically regulate organ abscission. They also agree with the transcriptomic data for litchi fruit abscission and validate this model for abscission. In the abscission zone, the expression of 47 IAA‑related genes was down‑regulated, while 39 ETH biosynthesis and signaling genes, including the ethylene receptor LcETR2, EIN3‑binding F‑box protein, and multiple ERFs, were significantly up‑regulated [ 37 ]. The functions of hormone‑related genes and pathways in Korla Xiangli have also been reported in other studies [ 38 , 39 ]. This study further revealed that at 3 DAP, exogenous ethephon treatment strongly activated the expression of the key ETH signaling genes Psin13G016570 and Psin16G016440, while simultaneously suppressing core components of IAA signaling (Psin05G016120, Psin13G016030, Psin15G013490, Psin16G015870, Psin16G015890, Psin10G004140, Psin10G004150, Psin10G004340, Psin10G004350, and Psin15G019720) and two ETH‑related genes (Psin13G016570 and Psin16G016440). This pattern aligned with the molecular mechanism reported for litchi fruitlet abscission [ 40 ]. In the ETH signaling pathway, ERF1/2 had a “rise‑then‑fall” expression pattern, whereas key IAA pathway components, such as AUX/IAA, CH3, and the early‑response gene SAUR, were continuously suppressed. This suggested that ethephon rapidly weakened IAA signaling intensity through transcriptional reprogramming. The WGCNA analysis identified the MEyellow module as being strongly negatively correlated with IAA levels (r = − 0.93) and enriched with hub genes, including AUX/IAA and AP2/ERF. This suggested that transcriptional regulatory modules played a integrative role in hormone crosstalk. The mechanism closely resembled the findings for apple pedicel abscission, where either the IAA transport inhibitor NPA or ETH treatment disrupted the IAA‑ETH balance and accelerated abscission by up‑regulating ETH biosynthesis genes, such as MdACS5B and MdACO1 [ 41 ]. Therefore, this study has demonstrated that ethephon drives calyx abscission through a dual pathway that includes “endogenous ETH burst–IAA signal suppression”, which rapidly reshapes the hormonal microenvironment in the abscission zone. Based on the above research findings, a preliminary molecular regulatory model for ethephon‑induced calyx abscission can be proposed for Korla Xiangli (Fig. 13 ), as follows: exogenous ethephon → rapid increase in endogenous ETH concentration in the abscission zone → activation of AP2/ERF transcription factors → suppression of IAA influx carriers AUX/IAA and responsive gene SAUR → disruption of polar IAA transport → decrease in IAA concentration in the abscission zone → attenuation of IAA antagonism toward ethylene → sharp increase in the ETH sensitivity shown by abscission zone cells → initiation of PCD and cell wall degradation programs → calyx abscission. During this process, flavonoids significantly accumulate at the onset of abscission to scavenge ROS, while down‑regulated lipid metabolites compromise membrane stability, which synergistically promotes PCD. This model provides an initial explanation for the mechanism by which ethephon promotes calyx abscission in Korla Xiangli and offers a theoretical reference for studies on organ abscission in other Rosaceae fruit trees. Future research should consider integrating techniques such as DAP‑seq and CUT&Tag to map genome‑wide TF‑binding profiles and could employ virus‑induced gene silencing (VIGS) for functional validation. The results from these types of studies would deepen understanding about the molecular mechanisms underlying ethephon‑induced calyx abscission in Korla Xiangli. Conclusion This study systematically elucidated the molecular mechanism associated with exogenous ethephon‑induced calyx abscission in Korla Xiangli by integrating phenotypic observations, phytohormone profiling, wide‑target metabolomics, and a transcriptome analysis. Foliar spraying with 300 mg·L⁻¹ ethephon at the large‑bud stage significantly accelerated the abscission process and advanced abscission zone formation to 7 DAP (2 days earlier than CK). The peak daily abscission rate was 16.90%, which was a 5.15‑fold increase compared to CK. The third day after pollination was identified as the key regulatory window. At the molecular level, ethephon rapidly increased the endogenous ETH concentration (+ 33.09%) and suppressed IAA levels (− 49.30%) in the abscission zone, which disrupted hormonal homeostasis. The transcriptional analysis revealed that the ETH response factors ERF1/2 followed a “rise‑then‑fall” pattern, while IAA pathway genes, such as AUX/IAA and early‑response gene SAUR, were suppressed, thereby blocking polar IAA transport and enhancing ETH sensitivity in the abscission zone. The metabolomic analysis identified 383 differential metabolites. These were mainly enriched in the flavonoid biosynthesis, α‑linolenic acid metabolism, and terpenoid synthesis pathways. Among these, flavonoids markedly accumulated at the onset of abscission to scavenge ROS, whereas the down‑regulated lipid metabolites compromised membrane stability and jointly promoted PCD. The transcription factor analysis showed that the MYB, AP2/ERF, and NAC families became significantly enriched. The WGCNA identified the MEyellow module as being strongly negatively correlated with IAA levels (r = − 0.93) and identified nine hub genes, including IDA‑like, AP2/ERF, and AUX/IAA, as core regulatory nodes. This study provides a theoretical foundation for the precise chemical regulation of calyx abscission in Korla Xiangli and has identified candidate gene resources, such as ERF1/2 and AUX/IAA, that could be used for molecular breeding. Materials and methods Experimental materials The experiment was conducted at the Korla Xiangli planting base of Xinjiang Qianguoxian Agricultural Planting Co., Ltd., which is located in Xiaolanggan Village, Awati Township, Korla City, Bayingolin Mongol Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China (41°69′N, 86°06′E). The test materials were 20‑year‑old Korla Xiangli (Pyrus × sinkiangensis Yü ) trees pollinated with a mixed pollen blend containing the ‘Yali’ and ‘Dangshan Suli’ varieties. The rootstock was from Pyrus betulifolia (Chinese pear) with a plant spacing of 4 m × 5 m and an open‑center tree training system. All selected plants exhibited uniform and vigorous growth with no signs of pests or diseases. Ethephon spray treatment Selected experimental trees were sprayed with 300 mg·L⁻¹ ethephon solution on April 10, 2024 at the full‑bud stage and an equal amount of clean water was applied to another set of trees as the control (CK). Each treatment was applied to ten trees. Spraying was conducted in the morning on a clear, windless day using a handheld sprayer and the application continued until the flowers were fully wetted and the solution began to drip. The ethephon (85% purity) was purchased from Shanghai Yuanye Bio‑Technology Co., Ltd., Shanghai, China (Product No. S18030, provided for scientific research purposes only). Sample collection The de‑calyx fruits with the different treatments were collected at 3, 5, 7, and 9 days after pollination (DAP). Previous studies had indicated that fruits at the 4th and 5th positions of the inflorescence typically developed as de‑calyx types [ 20 ]; therefore young fruit at these positions were selected before abscission‑zone formation had occurred. During sampling, the junction between the calyx tube and young fruit was transversely excised with a surgical scalpel and then the petals and sepals were manually removed. The retained abscission‑zone tissue included the calyx tube and cells that were proximal to the separation line. After collection, the samples were immediately wrapped in aluminum foil, flash‑frozen in liquid nitrogen, and subsequently stored at − 80°C for further analysis. A total of eight sample groups (4 × 2 treatments) were prepared, each with three biological replicates, for phytohormone quantification, transcriptome sequencing, metabolome sequencing, and qRT‑PCR validation. All sequencing procedures were conducted by Wuhan MetWare Biotechnology Co., Ltd., Wuhan, China. Phenotypic trait observation during calyx abscission External morphological changes associated with calyx abscission The external morphological characteristics of the de‑calyx fruits were observed and photographed using a digital camera at 3, 5, 7, 9, 11, and 13 DAP. Daily calyx abscission rate survey A total of three Korla Xiangli trees were selected from each treatment (six trees in total). In early April, 2024, branches at similar developmental stages were tagged on the east, south, west, and north aspects of the canopy for each treatment. Then 20 branches were randomly selected per treatment, giving a total of 40 branches. Within each treatment, 100 inflorescences were randomly labelled on the selected branches and the number of calyx‑shedding fruits was recorded daily from the third DAP until abscission ceased. The daily calyx abscission rate was calculated as Daily calyx abscission rate (%) = (number of newly de‑calyx fruits per day on labelled branches) / (total number of young fruits on the labelled main branches) × 100%. Phytohormone assay The IAA, ABA, gibberellin A₃ (GA₃), JA, and SA contents in the samples were determined using high‑performance liquid chromatography–tandem mass spectrometry (HPLC‑MS/MS). The sample processing procedure was as follows: abscission‑zone samples that had been stored at − 80°C were rapidly transferred to liquid nitrogen and ground to a fine powder using a grinding mill (30 Hz, 1 min). Approximately 100 mg of the powdered tissue was weighed into a 2 mL centrifuge tube, followed by the sequential addition of 10 µL of mixed internal standard solution (100 ng·mL⁻¹ in methanol) and 1 mL of pre‑cooled extraction solvent (methanol:water:formic acid = 15:4:1, v/v/v). The mixture was vortexed for 10 min and then centrifuged at 4°C and 12,000 rpm for 5 min. The supernatant was transferred to a new 1.5 mL centrifuge tube and completely dried using vacuum centrifugation. Then the residue was reconstituted in 100 µL of 80% methanol aqueous solution (v/v), filtered through a 0.22 µm organic phase membrane filter, transferred to an LC vial, and subjected to LC‑MS/MS analysis. Each treatment contained three biological replicates. Ethylene concentration measurement The ETH concentration was measured according towith modifications [ 21 ]. Approximately 5 g of each sample was weighed and placed into a clean conical flask. The flask was tightly sealed with a rubber stopper and further secured with sealing film to prevent gas leakage. After 24 h, the sealed flask was kept at room temperature for 2 h to equilibrate the internal gas and then gas sampling was performed by slowly withdrawing 1 mL of headspace gas with a syringe for gas chromatography analysis. Nitrogen was used as the carrier gas at a flow rate of 1 mL·min⁻¹ and the injector and column temperatures were both set at 50°C to optimize ETH separation. Separation was achieved using an Agilent HP‑PLOT Q column (30 m × 0.53 mm × 0.40 µm) and detection was carried out using a flame ionization detector set at 200°C. The retention time for ETH was approximately 1.1 min, and quantification was performed using an external standard method. Each treatment contained three independent replicates. Metabolite detection Sample preparation and metabolite extraction The samples were vacuum freeze‑dried for 63 h, ground using a mill (30 Hz, 1 min), and passed through a 40‑mesh sieve. Approximately 50 mg of the powdered sample was weighed into a 2 mL centrifuge tube. Then 1,200 µL of a pre‑cooled (–20°C) 70% methanol aqueous internal standard extraction solution was added to the tube. After thorough mixing by shaking, the mixture was placed in a vortex mixer and vortexed for 30 seconds every 30 minutes, which was repeated six times in total. Following centrifugation, the supernatant was carefully collected, filtered through a microporous membrane, and transferred to a sample vial for subsequent metabolomic analysis. LC‑MS/MS‑based metabolite detection The data acquisition system consisted of an ultra‑performance liquid chromatography (UPLC) unit coupled to a tandem mass spectrometer (MS/MS). The liquid chromatography column temperature was maintained at 40°C and the flow rate was set at 0.35 mL/min during data acquisition. Electrospray ionization was used for the mass spectrometry with the ion source heating temperature adjusted to 500°C. Triple quadrupole scanning was performed in multiple reaction monitoring (MRM) mode and the declustering potential (DP) and collision energy (CE) for each MRM ion pair were optimized using systematic adjustment. A specific set of MRM ion pairs was monitored in each time window and were selected according to the metabolites eluted during that period. Transcriptome sequencing Total RNA extraction and quality assessment Total RNA was extracted from the samples by combining ethanol precipitation with a cetyltrimethylammonium bromide (CTAB)-based method. RNA concentration was determined using a Qubit 4.0 Fluorometer and an MD microplate reader, while RNA integrity was assessed via a Qsep400 bioanalyzer. cDNA library construction and quality control The poly(A) + mRNA was enriched from the total RNA using oligo(dT)-conjugated magnetic beads and then the purified mRNA was fragmented in fragmentation buffer to generate shorter RNA fragments. These fragments served as templates for first-strand cDNA synthesis and were primed with random hexamers during reverse transcription. Second-strand cDNA was then synthesized using buffer, deoxynucleotide triphosphates (dNTPs), and DNA polymerase. The resulting double-stranded cDNA was purified with magnetic beads and subjected to end repair, 3’ adenylation, and sequencing adapter ligation. Finally, fragments of the desired size were selected using magnetic beads and high-quality cDNA libraries were constructed using PCR amplification. Raw data processing CASAVA software was used to perform base calling using image data generated by the Illumina high‑throughput sequencing platform. This process produced the raw sequence reads (raw reads). To ensure data quality, the following quality control steps were applied: (1) removal of reads containing adapter sequences, (2) exclusion of reads with ambiguous bases, and (3) filtering out low‑quality reads (defined as reads where > 50% of bases had an Qphred score ≤ 20). Bioinformatics analysis The clean reads were aligned to the reference genome ( Pyrus sinkiangensis ; available at https://pearomics.njau.edu.cn/sites/default/files/raw/Pyrus_sinkiangensis ) using HISAT2 (v2.0.5). The resulting SAM files were converted to the BAM format using SAMtools and then the genes were quantified using FeatureCounts to generate a count matrix. The gene expression levels were normalized as fragments per kilobase of transcript per million mapped reads (FPKM) and a differential expression analysis was performed using DESeq2 (v1.20.0) with significance thresholds set at |log₂(fold change)| ≥ 1 and p < 0.05. Then GO functional annotation and KEGG pathway enrichment analyses were performed using clusterProfiler. A weighted gene co‑expression network analysis (WGCNA) was performed using the WGCNA package (v1.29), and the co‑expression networks were visualized by Cytoscape (v3.3.0). Quantitative real‑time PCR (qRT‑PCR) validation All the qRT‑PCR primers used in this experiment were designed using the online primer design tool Primer3Plus ( https://www.primer3plus.com/ ) and primer specificity was verified using the DNAMAN design tool. The qRT‑PCR assays were performed using a 2×ChamQ Universal SYBR Master Mix kit from Novizan Biotechnology Co., Ltd., China using the procedure outlined below: (1) Preparation of qRT‑PCR reaction mixtures as described in Table S1; (2) Loading of the prepared reaction system into a real‑time PCR instrument; (3) Use of RhUBI2 as the reference gene for normalization; (4) Each sample was analyzed with three biological replicates and gene expression levels were calculated using the 2⁻ΔΔCT method. Statistical analysis All the data were organized using Microsoft Excel 2019 (Microsoft Corp, Redmond, WA, USA) and the statistical analyses, including a variance analysis combined with t‑testing, were performed using SPSS 26.0 (IBM, Armonk, NY, USA) with the significance level set at p < 0.05. The graphs and charts were generated using Origin 2021 (Originlab, Northampton MA, USA) and GraphPad Prism 9 (San Diego, CA, USA). The figures were assembled and processed using Adobe Photoshop CC 2019 (Adobe Systems, San Jose, CA, USA). Declarations Acknowledgments This study was funded by the postgraduate innovation project of the autonomous region（XJ2024G136）and the second group of tianshan talent training program: youth support talent project (2023TSYCQNTJ0004). Data availability statement All the data generated or analyzed during this study are included in this article and its supplementary information files. The genomic DNA, coding sequences (CDS), and annotation files of Xinjiang pear were downloaded from the Pear Database of Nanjing Agricultural University. The raw transcriptome data have been deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/; BioProject Accession ID PRJNA1404807). CRediT authorship contribution statement Y.W. contributed to writing the original draft, visualization, methodology, data curation, project administration and funding acquisition. L.Z. participated in writing the original draft, visualization, methodology, investigation, formal analysis and data curation. Y.L. and T.S. were responsible for investigation, visualization, formal analysis and data curation. Y.G. contributed to methodology and formal analysis. C.C. and L.W. engaged in formal analysis and data curation. F.Z., C.W., Y.W. and X.Z. participated in investigation and methodology. J.T. was involved in writing-review and conceptualization. All authors reviewed the manuscript. Ethics approval and consent to participate This article does not contain any studies involving human participants or animals performed by the authors. The methods in this study were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Declaration of competing interests The authors declare that they have no financial or personal conflicts of interest that could have influenced the work described in this paper. 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Colleger","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Zhang","suffix":""},{"id":581609766,"identity":"3fcd30fe-b0ac-47a6-8649-3f16d7da1e26","order_by":11,"name":"Jia Tian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACZiCWMGCQY2A4AOMSADxQLcYkaIHSiQ0ISwkAe3bmZw8sCuzSNxw8/EyCocI6sYH97AECDmMzN5AwSM6d2XDMTILhTHpiA09eAgEtDGYSEgbMuf0MB8wkGNsOJzZI8BgQ0ML+DailPp2N4fg3CcZ/RGnhAdlyOIGf4QzQlgZitBzmKQNqOW44s+FMsUXCsXTjNp4c/FrY+49vk5b4Uy1vcOP4xhsfaqxl+9nP4NcCAswSIFLiAANDApBmI6geCBg/gEj+BmLUjoJRMApGwUgEAAu3OySC6kwEAAAAAElFTkSuQmCC","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Jia","middleName":"","lastName":"Tian","suffix":""}],"badges":[],"createdAt":"2026-01-20 11:51:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8648793/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8648793/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101752083,"identity":"993bbacb-e1d6-42d5-a627-18caeb504d9c","added_by":"auto","created_at":"2026-02-03 10:25:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":171597,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the external morphological characteristics of Korla Xiangli during calyx abscission in the water and ethephon treatments. DAP: Days after pollination\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/6f2068635bb13ca07dad45de.png"},{"id":101528906,"identity":"ea37660c-5fa0-4bfc-90df-08fe1da1f541","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262031,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethephon treatment on the daily calyx abscission rate in Korla Xiangli\u003c/p\u003e\n\u003cp\u003eNote: * indicates significant difference at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/df479ecd56d7885c513c9857.png"},{"id":101528908,"identity":"a0fecd3b-bdf3-41fe-b0c3-70e902463dfa","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":286631,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in plant hormone contents during the Korla Xiangli calyx abscission process under ethephon treatment \u003cstrong\u003ea \u003c/strong\u003eETH; \u003cstrong\u003eb \u003c/strong\u003eIAA; \u003cstrong\u003ec \u003c/strong\u003eABA; \u003cstrong\u003ed\u003c/strong\u003e GA\u003csub\u003e3\u003c/sub\u003e; \u003cstrong\u003ee\u003c/strong\u003e JA; \u003cstrong\u003ef\u003c/strong\u003e SA\u003c/p\u003e\n\u003cp\u003eNote: * and ** indicate significant differences at the \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and 0.01 levels, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/7f13315b58a19b06727e4d1b.png"},{"id":102404015,"identity":"e49e853d-5679-482d-9087-433752bb4ba7","added_by":"auto","created_at":"2026-02-11 10:53:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":638539,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolomic data analysis of the abscission zones during calyx abscission in Korla Xiangli. \u003cstrong\u003ea\u003c/strong\u003e: Metabolite category ring chart. \u003cstrong\u003eb\u003c/strong\u003e: PCA score plot of the metabolites. \u003cstrong\u003ec\u003c/strong\u003e: Column statistics for the differential metabolite numbers. \u003cstrong\u003ed\u003c/strong\u003e: Column statistics for the differential metabolite categories. \u003cstrong\u003ee\u003c/strong\u003e: Heat map of the differential metabolites at 3 DAP. \u003cstrong\u003ef\u003c/strong\u003e: Heat map of the differential metabolites at 5 DAP. DAP: Days after pollination\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/abb22097ae0305316e67430d.png"},{"id":101528912,"identity":"50616147-f6a1-46f2-98a0-d81394180627","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":369824,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG enrichment analysis and screening of key metabolites associated with ethephon-induced calyx abscission in Korla Xiangli. \u003cstrong\u003ea\u003c/strong\u003e: KEGG enrichment analysis; \u003cstrong\u003eb\u003c/strong\u003e: Primary metabolite differential expression analysis; and \u003cstrong\u003ec\u003c/strong\u003e: Secondary metabolite differential expression analysis.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/d676e3632728103a4d0cf958.png"},{"id":101752623,"identity":"b2013666-9561-42f0-a25a-4225be7d0b6c","added_by":"auto","created_at":"2026-02-03 10:28:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185129,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ethephon treatment on the transcriptome of the calyx abscission zone in Korla Xiangli. \u003cstrong\u003ea\u003c/strong\u003e: Statistical bar chart for the differentially expressed gene numbers and \u003cstrong\u003eb\u003c/strong\u003e: Venn diagram of the differentially expressed genes\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/de1426a4e143b7d73e855558.png"},{"id":101528909,"identity":"d9c3adee-0329-4138-a80c-280f869bd674","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":337084,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG and GO enrichment analysis of DEGs in the calyx abscission zone of Korla Xiangli under ethephon treatment. \u003cstrong\u003ea\u003c/strong\u003e: KEGG enrichment analysis and \u003cstrong\u003eb\u003c/strong\u003e: GO enrichment analysis\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/9b397cc7bbedd5e3e2b60bb6.png"},{"id":101752624,"identity":"dceccd69-5dc6-4b71-b510-585a20a9db9e","added_by":"auto","created_at":"2026-02-03 10:28:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":401252,"visible":true,"origin":"","legend":"\u003cp\u003eTypes and numbers of differential transcription factor families during ethephon-induced calyx abscission in Korla Xiangli\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/ef07c6937bfcc54bd4769581.png"},{"id":101528916,"identity":"e50cbaae-f8e8-4ffa-ad1b-a3a71cfb1ad0","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":774201,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs in the plant hormone signal transduction pathways during ethephon-induced calyx abscission in Korla Xiangli. \u003cstrong\u003ea \u003c/strong\u003eand \u003cstrong\u003eb\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eDEGs related to the IAA, ETH, ABA, and JA hormone signal transduction pathways. \u003cstrong\u003ec \u003c/strong\u003eand \u003cstrong\u003ed\u003c/strong\u003e: Differential gene expression analysis of the IAA, ETH, ABA and JA hormone signal transduction pathways\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/caf7a024b879432239bb9f16.png"},{"id":101528913,"identity":"c81d2353-096f-4ab9-a437-3bcc5fb88fcd","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":645729,"visible":true,"origin":"","legend":"\u003cp\u003ePathway map for plant hormone signal transduction (IAA、ETH、ABA, and JA) during ethephon-induced calyx abscission in Korla Xiangli at (\u003cstrong\u003ea\u003c/strong\u003e) 3 DAP and (\u003cstrong\u003eb) \u003c/strong\u003e5 DAP.\u003cstrong\u003e \u003c/strong\u003eDAP: Days after pollination\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/3e780c091d6004f0b4a7c470.png"},{"id":101528914,"identity":"31d4535d-3281-4051-9fd5-6114a63cdc38","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":391654,"visible":true,"origin":"","legend":"\u003cp\u003eqRT-PCR validation of 12 differentially expressed genes\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/e73d551151945f3fd97481bb.png"},{"id":101528917,"identity":"8ae45f60-5eee-4474-b45b-3670ebf77b72","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1022447,"visible":true,"origin":"","legend":"\u003cp\u003eCo-expression network for plant hormones during ethephon-induced calyx abscission in Korla Xiangli. \u003cstrong\u003ea\u003c/strong\u003e Identification of seven co-expression modules using the dynamic tree cut method\u003cstrong\u003e;\u003c/strong\u003e \u003cstrong\u003eb\u003c/strong\u003e Correlations between modules and endogenous plant hormones with corresponding P-values; darker colors indicate stronger correlations\u003cstrong\u003e;\u003c/strong\u003e \u003cstrong\u003ec\u003c/strong\u003e Co-expression network diagram of the candidate genes related to plant hormones (a MEyellow; b MEblue; c MEgreen)\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/8d9d81099c8211644182c502.png"},{"id":101528918,"identity":"b6172df4-8fab-49a6-ac9b-1ff5b0fc24b4","added_by":"auto","created_at":"2026-01-30 19:18:03","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":244819,"visible":true,"origin":"","legend":"\u003cp\u003eCoordinated regulatory network for key genes and hormone signals during ethephon-induced calyx abscission in Korla Xiangli\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/833c65bbe2186cbc8dd660b1.png"},{"id":102745805,"identity":"adedd39f-7c8d-4fba-8e75-bd8166c66d36","added_by":"auto","created_at":"2026-02-16 08:54:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7275512,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8648793/v1/83647dfd-02c1-422c-834e-16f05d5b26b0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated omics analysis provides preliminary insights into the regulatory network controlling ethephon-promoted calyx abscission in Korla Xiangli (Pyrus × sinkiangensis Yü)","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant organ abscission is a process that detaches plant tissues or organs from the mother plant and involves coordinated action among multiple factors. The region where separation occurs and its adjacent tissues, is termed the \u0026lsquo;abscission zone\u0026rsquo; and plant hormones play pivotal roles in regulating abscission [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. For instance, auxin (indole-acetic acid, IAA) maintains abscission zone insensitivity to ethylene (ETH), thereby suppressing abscission [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. If IAA levels decline or its polar transport is disrupted in the abscission zone, then ETH signaling is enhanced and the expression of ethylene response factors (ERFs) increases. This activates the transcription and translation of cell wall hydrolases, such as β‑1,4‑glucanase and polygalacturonase, which accelerate cell wall degradation and middle lamella dissolution [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Studies by on tomato and rose plants showed that ETH biosynthesis genes were the key targets of ERFs during abscission and that this regulatory process was finely tuned by abscission signals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The results highlighted the central role of ERFs in the abscission regulatory network. Abscisic acid (ABA) promotes abscission via two synergistic pathways: first, by directly activating 1‑aminocyclopropane‑1‑carboxylate synthase (ACS) gene expression to enhance ETH biosynthesis and second, by suppressing PIN‑FORMED‑mediated polar auxin transport, thereby further amplifying ETH signaling [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The role played by gibberellin (GA) in abscission zone development and organ abscission is unclear, although most studies have suggested that it has an inhibitory function. Gibberellin probably delays abscission by antagonizing ETH signaling through DELLA‑dependent or DELLA‑independent pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Jasmonic acid (JA), a key hormone involved in plant stress responses, markedly accumulates in abscission zone tissues and accelerates organ abscission by either directly activating programmed cell death (PCD)‑related genes via MYC2/3/4 transcription factors or through synergistic action with 1‑aminocyclopropane‑1‑carboxylic acid [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSalicylic acid (SA) modulates abscission via crosstalk with immune signaling pathways. Key regulatory components, such as NPR1, EDS1, and PAD4, drive SA biosynthesis and form a positive‑feedback loop with SA. This induces localized reactive oxygen species (ROS) bursts and PCD, which ultimately promote cell separation within the abscission zone [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A continuous series of physiological and biochemical reactions related to cell metabolism and signal transduction occurs during plant organ abscission, and the various metabolites generated in this process regulate abscission progression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A metabolomic analysis of fruit abscission in blue honeysuckle showed that different metabolites were significantly enriched in various pathways, such as plant hormone signal transduction, starch‑sucrose metabolism, the pentose phosphate pathway, and phenylpropanoid biosynthesis. Among them, m‑hydroxysalicylic acid and 2‑hydroxy‑6‑(8‑tridecenyl)benzoic acid, as core effector molecules of the SA signaling pathway, synergistically regulated the antioxidant defense system, mediated cell wall modification in the abscission zone, and promoted middle lamella degradation. These actions systematically controlled the initiation and progression of fruit abscission [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOther studies on flower and pod abscission in soybean revealed that there were significant changes to the flavonoid, fatty acid, and amino acid biosynthesis pathways in the abscission zone tissues, and that these changes were closely associated with stress responses and secondary metabolism. Metabolites such as proline and phenylalanine are precursors of osmoregulators and antioxidants and maintain redox homeostasis in abscission zone cells. Also, they indirectly affect lignin and flavonoid accumulation by regulating the metabolic flux of phenylpropanoid biosynthesis, which means that they participate in the remodeling of cell wall structure [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKorla Xiangli \u003cem\u003e(Pyrus \u0026times; Sinkiangensis Y\u0026uuml;)\u003c/em\u003e belongs to the Rosaceae family and the Xinjiang system. It is an ancient local cultivar originating from the Korla region of Xinjiang and has a long cultivation history. The fruit can be categorized into decalyx fruit and persistent calyx fruit types. The persistent calyx fruit quality is generally lower than that of decalyx fruit but the proportion of decalyx fruit is high, which limits sustainable commercial production. Therefore, identifying safe and efficient exogenous plant growth regulators to precisely regulate calyx abscission has become a critical technical challenge in Korla Xiangli production. Ethephon is a synthetic, environmentally benign regulator that rapidly decomposes within plants to release ETH, thereby inducing abscission zone formation and organ shedding [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To date, research on ethephon‑mediated organ abscission in horticultural crops has largely focused on flower and fruit drop, particularly tomato pedicels and citrus fruitlets [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previous studies have also reported that ethephon treatment of litchi panicles significantly down‑regulated the expression of key auxin‑signaling genes (ARF and Aux/IAA), but up‑regulated ERF genes in the abscission zone [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. One study confirmed that spraying 300 mg\u0026middot;L⁻\u0026sup1; ethephon at the large‑bud stage significantly accelerated calyx abscission in Korla Xiangli, although the molecular mechanisms were unclear [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Further mass‑spectrometry imaging has revealed that ABA and ETH are markedly enriched in the abscission zone of natural de‑calyx fruit, whereas IAA and GA accumulated in the corresponding region of persistent‑calyx fruit [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nevertheless, the phenotypic dynamics, the hormone and metabolite changes, and the regulatory network constructed by hormone‑related genes during ethephon‑induced calyx abscission require further exploration. Based on this, the present study used the abscission zone of de‑calyx Korla Xiangli fruit to systematically compare the calyx‑abscission phenotypic traits between water‑ and ethephon‑treated samples and combined wide‑target metabolomics to analyze the dynamic responses of key metabolic pathways. High‑throughput transcriptomics and bioinformatics were used to identify ethephon‑induced differentially expressed genes, with a particular focus on core nodes within the plant hormone signaling pathways. This information was then used to identify and validate the key regulatory factors promoting calyx abscission and a molecular regulatory network for ethephon‑induced calyx abscission was constructed.\u003c/p\u003e \u003cp\u003eThe findings from this study provide a theoretical basis and gene resources for the precise regulation and molecular breeding of calyx abscission in Korla Xiangli and lay a solid foundation for improving its fruit quality.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic observation of ethephon‑induced calyx abscission in Korla Xiangli\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eExternal morphological changes during calyx abscission\u003c/h2\u003e \u003cp\u003eThe external morphological changes during ethephon‑induced calyx abscission in Korla Xiangli were observed. At 3 DAP, the flowers remained intact with well‑developed stamens and sepals, whereas at 5 DAP, the petals had begun to shed naturally and the anther color was gradually fading. However, but the sepals remained firmly attached to the young fruit. These changes indicated that the plant samples had entered the pre‑abscission‑zone stage (3\u0026ndash;5 DAP). Between 7, 9, and 11 DAP, the stamen filaments turned brown and a distinct circular abscission zone became visible at the junction between the sepals and the young fruit. This was the abscission‑zone formation stage. By 13 DAP (post‑abscission‑zone formation), the sepals had fully detached at the abscission zone and had been shed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Calyx abscission followed a similar morphological progression in the CK group and could be divided into the pre‑abscission‑zone, abscission‑zone formation, and post‑formation stages. However, in the CK group, the yellowish ring that indicated abscission zone initiation did not appear until 9 DAP, which was 2 days later than in the ethephon treatment. This demonstrated that exogenous ethephon application significantly accelerated both abscission‑zone formation and calyx abscission in Korla Xiangli.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eDaily calyx abscission rate\u003c/h3\u003e\n\u003cp\u003eThe ethephon treatment significantly increased the daily calyx abscission rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). During the first 6 DAP, no calyx abscission was observed in either the ethephon‑treated or CK (water‑treated) groups. Under ethephon induction, calyx abscission began at 7 DAP (consistent with external morphological observations) and peaked at 10 DAP. The daily abscission rate was 16.90% and abscission had ceased by the 13th day. In contrast, calyx abscission started at 9 DAP in the CK group and peaked at 11 DAP. However, the daily abscission rate was only 3.28%. Thus, abscission initiation was delayed by 2 days and the peak was postponed by 1 day in the CK group compared to the ethephon group. The external morphological changes during calyx abscission showed that 3\u0026ndash;5 DAP in the ethephon‑treated group corresponded to the pre‑abscission‑zone stage, days 7, 9, and 11 represented the abscission‑zone formation stage, and day 13 was the post‑abscission‑zone stage. Further analyses of the pre‑abscission‑zone stage (3\u0026ndash;5 DAP), including an in‑depth investigations of hormones, metabolites, and gene expression, will need to be performed to further elucidate the regulatory mechanism controlling ethephon effects on calyx abscission in Korla Xiangli.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eChanges in phytohormone content during ethephon‑induced calyx abscission in Korla Xiangli\u003c/h3\u003e\n\u003cp\u003eThe ETH concentration in the calyx abscission zone of the ethephon treated plants was significantly higher than in the CK plants at 3, 5, and 7 DAP and peaked on day 7 with a 33.09% increase compared to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea); IAA content was markedly lower by 49.30% compared to CK at 3 DAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb); and ABA, GA₃, and SA levels had significantly increased by 242.69%, 34.19%, and 31.51%, relative to CK on day 3, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Conversely, the GA₃ content was significantly lower by 82.88% and 64.64% in the ethephon group at 5 and 7 DAP compared to the control, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e‑D). The JA content was significantly higher than CK by 42.32% and 126.50% at 3 and 5 DAP, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003e* and ** indicate significant differences at the \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 0.01 levels, respectively.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eMetabolomic analysis of ethephon‑induced calyx abscission in Korla Xiangli\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eScreening and identification of differential metabolites\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eA total of 1,585 metabolites were identified in this study. Amino acids and their derivatives (145), organic acids (74), and nucleotides and their derivatives (42) were the top three categories among the primary metabolites accounting for 9.15%, 4.67%, and 2.65% of the total, respectively. Flavonoids (248), terpenoids (246), phenolic acids (193), alkaloids (138), and lipids (123) ranked the highest among the secondary metabolites accounting for 17.74%, 15.53%, 12.18%, 8.71%, and 7.77%, respectively, of the secondary metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The PCA results showed that there was a clear separation between the ethephon‑treated and CK samples along the PC1 axis and that they were highly dispersed. This indicated that the two groups had distinct metabolic profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). A further comparative analysis revealed 383 differential metabolites between the abscission zones of the ethephon and CK treatments, with the highest number and variety observed at 5 DAP (118 metabolites) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Among these, 86 were up‑regulated and 32 were down‑regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The lipid levels were significantly lower in the ethephon‑treated group compared to CK at 3 DAP, while terpenoids and alkaloids were notably elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). However, at 5 DAP the terpenoids, lipids, and flavonoid levels were all significantly higher than in the CK group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). After excluding the \"others\" category, the comparisons showed that there were 11 major classes of differential metabolites. At 3 DAP flavonoids showed the greatest difference, followed by lipids and terpenoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), and at 5 DAP terpenoids showed the greatest difference, followed by lipids and flavonoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKEGG enrichment analysis and key metabolite screening of the differential metabolites\u003c/h3\u003e\n\u003cp\u003eThe differential metabolites were significantly enriched in a number of pathways at 3 DAP, including linoleic acid metabolism, flavonoid biosynthesis, α‑linolenic acid metabolism, amino acid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, isoflavonoid biosynthesis, lysine biosynthesis, pyrimidine/purine metabolism, and glycine‑serine‑threonine metabolism. Among these, the top five pathways showed significant enrichment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The core pathways comprised of α‑linolenic acid metabolism, linoleic acid metabolism, sesquiterpenoid and triterpenoid biosynthesis, purine metabolism, zeatin biosynthesis, amino acid biosynthesis, C5‑branched dibasic acid metabolism, biotin metabolism, pyrimidine metabolism, and monoterpenoid biosynthesis at 5 DAP. The significantly enriched pathways (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were the same as those at 3 DAP and still showed increased linoleic acid metabolism, flavonoid biosynthesis, α‑linolenic acid metabolism, amino acid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Within these pathways, three primary metabolites were commonly annotated across both comparison groups. These were DL‑ortho‑tyrosine, N‑(1‑deoxy‑1‑fructosyl)phenylalanine, and S‑methylglutathione (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The ethephon treatment led to the down‑regulation of 13 secondary metabolites at 3 DAP. These were grouped into six lipids (9‑oxo‑10,12‑octadecadienoic acid, 2‑aminooctadecane‑1,16,18,18‑tetrol, 9,10,13‑trihydroxy‑11‑octadecenoic acid, 9(10)‑epoxyoctadecenoic acid, (9R,10S)‑(12Z)‑9,10‑epoxyoctadecenoic acid, 13S‑hydroperoxy‑9Z,11E‑octadecadienoic acid, and 9,12,13‑trihydroxy‑10,15‑octadecadienoic acid), four flavonoids (kaempferol‑3‑O‑glucoside‑7‑O‑rhamnoside, prunetin, quercetin‑3‑O‑(6\u0026Prime;‑O‑malonyl)‑glucoside, and persicoside), two terpenoids (hamamelitannin B and coeloginin), and one alkaloid (pantetheine). In contrast, five metabolites were up‑regulated: two alkaloids (diprophylline and pectolinarin), two flavonoids (limocitrin‑7‑O‑(6\u0026Prime;‑acetyl)glucoside and didymin), and one terpenoid (spathulenol). The number of differential metabolites induced by exogenous ethephon sharply decreased at 5 DAP, with only leucocyanidin, 2,3,16,21‑tetrahydroxyolean‑12‑en‑28‑oic acid (platycodin C), lysophosphatidylcholine 20:5, and dihydromarrubin showing significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eTranscriptomic analysis of ethephon‑induced calyx abscission in Korla Xiangli\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScreening of differentially expressed genes (DEGs)\u003c/h2\u003e \u003cp\u003eA total of 2,141 DEGs were identified out of the 20,456 expressed genes. The majority occurred in the pre‑abscission zone stage at 3 DAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). A total of 679 DEGs were identified at 3 DAP (390 DEGs were significantly up‑regulated and 289 were down‑regulated) in the abscission zone; 581 DEGs were identified at 5 DAP (362 up‑regulated and 219 down‑regulated); 581 DEGs were identified at 7 DAP (292 up‑regulated and 289 down‑regulated); and the number of DEGs was the lowest at 9 DAP, with only 300 genes showing differential expression. Additionally, eight DEGs were commonly annotated across all four time points, which suggested that they were core regulatory factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eKEGG and GO enrichment analysis of differentially expressed genes (DEGs)\u003c/h2\u003e \u003cp\u003eThe DEGs between the ethephon treatment and CK were mapped to obtain their corresponding KEGG pathway information. A total of 8 and 14 significantly enriched KEGG pathways were identified in the 300‑3d‑D_vs_CK‑3d‑D and 300‑5d‑D_vs_CK‑5d‑D comparison groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). After sorting the KEGG pathways by P‑value, the DEGs in the abscission zone at 3 DAP were mainly enriched in metabolic pathways, sesquiterpenoid and triterpenoid biosynthesis, pentose and glucuronate interconversions, carotenoid biosynthesis, plant hormone signal transduction, α‑linolenic acid metabolism, biosynthesis of secondary metabolites, and photosynthesis‑antenna proteins, among which the first five pathways were significantly enriched (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The DEGs at 5 DAP were significantly enriched in sesquiterpenoid and triterpenoid biosynthesis, photosynthesis‑antenna proteins, protein processing in endoplasmic reticulum, biosynthesis of secondary metabolites, motor proteins, and plant hormone signal transduction. The \"plant hormone signal transduction\" pathway was significantly enriched at both 3 and 5 DAP, which suggested that it played a core role in ethephon‑induced calyx abscission. Therefore, future analyses should further explore the key genes in this pathway that are induced by ethephon.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe GO enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) showed that the biological processes affected by the DEGs in the ethephon treatment group encompassed reproduction, metabolism, signal transduction, regulation of biological processes and rhythmic processes. The cellular components were dominated by protein complexes and cellular anatomical entities, whereas molecular functions were significantly enriched in categories such as transcription regulator activity, antioxidant activity, molecular transducer activity, and ATP‑dependent activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranscription factor analysis\u003c/h2\u003e \u003cp\u003eThe key transcriptional regulators involved in ethephon‑induced calyx abscission in Korla Xiangli were identified by predicting and summarizing the transcription factors (TFs) among the DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). A total of 84 differentially expressed TFs belonging to 27 families were identified at 3 and 5 DAP. The top eight TF families most significantly regulated by ethephon were AP2/ERF, MYB, NAC, HSF, AUX/IAA, C2C2‑CO‑like, LOB, and WRKY. A total of 12 TF families were commonly enriched across both time points and the three most abundant families were MYB, AP2/ERF, and NAC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of DEGs in the plant hormone signal transduction pathways\u003c/h2\u003e \u003cp\u003eRelated genes in the signal transduction pathways of four major phytohormones (IAA, ETH, ABA, JA, and GA) were significantly regulated at both 3 and 5 DAP during ethephon‑induced calyx abscission in Korla Xiangli, (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, b). Among these, the IAA pathway had the greatest number of enriched DEGs, followed by the ETH pathway. This indicated that these pathways were critical for ethephon‑mediated promotion of calyx abscission. The DEGs in the IAA and JA pathways were predominantly down‑regulated at 3 DAP, which suggested that ethephon had inhibitory effects on these pathways at this stage. In contrast, the DEGs in the JA pathway were mainly up‑regulated at 5 DAP. Conversely, the DEGs in the ETH and ABA pathways were largely up‑regulated, which showed that ethephon strongly activated these pathways. Further analysis of the changes in the genes associated with the four hormone pathways (IAA, ETH, ABA, and JA) that were enriched at both 3 and 5 DAP revealed that genes such as Psin13G007030, Psin17G015890, Psin13G016030, Psin15G013490, Psin16G015870, Psin16G015890, Psin16G010530, Psin09G006830, Psin10G004140, Psin10G004150, Psin10G004340, Psin15G019720, and Psin05G016120 in the IAA pathway were significantly down‑regulated at 3 DAP. Almost all the genes were significantly up‑regulated in the ETH pathway, except for Psin02G006860 and Psin17G019520, and the number of DEGs in these related pathways was notably higher at 3 DAP compared to 5 DAP (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec, d). Additionally, the numbers of genes in the ABA and JA hormone signal transduction pathways were markedly lower than in the IAA and ETH pathways, which further suggested that they played key regulatory roles in IAA and ETH signaling in ethephon‑promoted calyx abscission in Korla Xiangli. The results also suggested that 3 DAP is a critical window for ethephon regulation of calyx abscission.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e illustrates the hormone signal transduction processes associated with IAA, ETH, ABA, and JA during ethephon‑induced calyx abscission in Korla Xiangli. Specifically, in the ETH signaling pathway, ethephon significantly up‑regulated the expression of ethylene response factor ERF1/2 at 3 DAP. However, ERF1/2 expression had decreased to levels below those of CK by 5 DAP, which meant that it showed a \u0026ldquo;rise‑then‑fall\u0026rdquo; pattern. Ethephon had strongly suppressed the expression of AUX/IAA, CH3, and the early auxin‑responsive gene SAUR in the IAA signaling pathway at 3 DAP, which indicated that there was a marked reduction in IAA signaling. Moreover, multiple genes in the ABA signaling pathway showed distinct expression pattern differences between the ethephon treatment and CK. For example, at 3 DAP, ethephon up‑regulated sucrose non‑fermenting‑related protein kinase 2 (SnRK2), which is a positive regulator of the ABA response, and ABA‑responsive element‑binding factor (ABF). However, the core ABA receptor family PYR/PYL was significantly down‑regulated at 5 DAP. Furthermore, MYC2, a central responder in the JA signaling pathway, was significantly down‑regulated by ethephon at both 3 and 5 DAP. These results suggested that these genes probably responded to exogenous ethephon treatment, participated in calyx abscission in Korla Xiangli, and that the IAA and ETH signaling pathways played key roles in this process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eValidation of the qRT‑PCR results\u003c/h2\u003e \u003cp\u003eThe number of DEGs was considerably higher at 3 DAP than at 5 DAP; therefore this study focused on the 3‑day time point and 12 DEGs from the plant hormone signal transduction pathways were selected based on their FPKM values for qRT‑PCR validation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The qRT‑PCR results were highly consistent with the FPKM trends, which further confirmed that ethephon significantly suppressed the expression of IAA‑related genes and activated ETH‑related genes in the abscission zone. This result showed that ethephon primarily promoted calyx abscission by modulating the plant hormone signal transduction pathways and that IAA and ETH signaling played central roles in this process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWGCNA analysis of phytohormone‑related differentially expressed genes during ethephon‑induced calyx abscission in Korla Xiangli\u003c/h2\u003e \u003cp\u003ePrevious studies have confirmed that exogenous ethephon significantly affected the levels of hormones such as IAA and ETH. In addition, a large number of DEGs in the IAA and ETH signal transduction pathways were significantly enriched during Korla Xiangli calyx abscission following ethephon treatment. This suggested that IAA and ETH signaling were the key regulatory pathways through which ethephon promotes calyx abscission in Korla Xiangli and that 3 DAP was the critical period for ethephon regulation. Based on this, a dynamic cut‑tree algorithm was used to perform a time‑series association analysis of the phytohormone contents and the differential expression profile at 3 DAP. It identified seven co‑expression modules that were significantly coupled to hormone signal transduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ea). Excluding the non‑informative MEgrey module, the number of genes in the other modules ranged from 55 (MEturquoise module) to 204 (MEblack module) (Table S2). Among these, the MEyellow module showed a strong negative correlation with IAA levels (r = \u0026minus;\u0026thinsp;0.93, Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eb). Within this module, nine core genes that were potentially regulated by ethephon during calyx abscission were further predicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ec: a). Additionally, the MEblue and MEgreen modules contained 8 and 13 candidate target genes, respectively, whose functions included leucine‑rich repeat extension‑like protein 6, receptor‑like protein kinase (ZmPK1), indole‑3‑acetic acid‑induced protein (ARG13), indole acetic acid‑induced protein 32 (PSK4), protein IDA‑like (IDL 2), transcription factors AP2/ERF and AUX/IAA, and bHLH75 (Figs.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ec: b, c). This set of genes could be used to elucidate the molecular mechanism by which ethephon promotes calyx abscission in Korla Xiangli through the regulation of the IAA and ETH signal transduction pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eAbscission zone formation and identification of the key phases in ethephon‑induced calyx abscission in Korla Xiangli\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOrgan abscission in plants is a complex physiological process that is often accompanied by morphological changes such as wilting and browning [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Previous research has shown that in \u0026lsquo;Korla Fragrant\u0026rsquo; pear, a yellow annular abscission zone appears at the junction between the young fruit and the calyx tube around the 8th day after full bloom. This zone is a key marker for calyx abscission [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, ethephon treatment induced the formation of the calyx abscission zone as early as 7 DAP, which was 2 days earlier than in the CK group (9 DAP). This suggested that exogenous ethephon application accelerated the calyx abscission process. Ethephon enhances hydroxyl radical scavenging capacity, the superoxide anion generation rate, and the intracellular Ca\u0026sup2;⁺ concentration in abscission zone cells. These changes lead to the rupture of distal cell wall structures and the degradation of cell membranes and organelles into condensed apoptotic bodies, and accelerates PCD to promote abscission [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Notably, abscission zone cells perceive and respond to abscission signals at an earlier point than when morphological changes become evident. This means that the abscission process is actually initiated before a visible abscission zone can be observed. Therefore, it can be hypothesized that the period before 7 DAP represents the key phase for ethephon‑induced calyx abscission (corresponding to 3 and 5 DAP in this study). Accordingly, the calyx abscission process under ethephon treatment was divided into three consecutive stages: the pre‑abscission zone phase (3\u0026ndash;5 DAP), the abscission zone formation phase (7\u0026ndash;11 DAP), and the post‑abscission zone phase (13 DAP). This provided a clear temporal framework for the subsequent molecular mechanism analysis.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHormonal regulation in ethephon‑induced calyx abscission in Korla Xiangli\u003c/h2\u003e \u003cp\u003eNumerous studies have shown that exogenous ethephon promotes abscission by mediating the biosynthesis and signaling of endogenous plant hormones and cell wall modification [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It is widely recognized that ETH, ABA, JA, and SA promote abscission, while IAA, GA, and CTK generally play inhibitory roles. In this study, following ethephon treatment, the ETH concentration in the calyx abscission zone was significantly higher than that of CK at 3, 5, and 7 DAP and was the most pronounced increase among all the hormones measured. The ABA, GA₃, JA, and SA contents were also significantly elevated compared to CK at 3 DAP, whereas the IAA content showed an opposite trend. The results suggested that ABA and JA indirectly amplified ETH signaling by activating the expression of ETH biosynthesis enzymes (ACS/ACO), which enhanced the abscission response. In addition polar IAA transport in the abscission zone seemed to continuously modulate tissue sensitivity to ETH. When endogenous ETH levels rose, polar IAA transport decreased, which significantly increased the responsiveness of the abscission zone to abscission signals and accelerated the abscission process [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Based on these findings, it is proposed that the biosynthesis, signaling, and interactions between endogenous ETH and IAA constitute one of the core regulatory mechanisms through which exogenous ethephon induces calyx abscission in Korla Xiangli. Therefore, the key genes involved in ETH and IAA synthesis and signaling pathways during the pre‑abscission stage need to be further investigated to clarify the mechanism associated with ethephon‑induced calyx abscission in Korla Xiangli.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRegulatory role of metabolites in ethephon‑induced calyx abscission in Korla Xiangli\u003c/h2\u003e \u003cp\u003eThe metabolomic analysis revealed the dynamic changes and potential regulatory roles of metabolites during ethephon‑induced calyx abscission in Korla Xiangli. It detected 1,585 metabolites and demonstrated that ethephon significantly altered the types and contents of metabolites in the calyx abscission zone. The primary metabolites were dominated by amino acids and their derivatives, which were significantly enriched in the amino acid biosynthesis pathways. The secondary metabolites mainly included flavonoids, terpenoids, and lipids, which were primarily annotated to pathways such as flavonoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, linoleic acid metabolism, and α‑linolenic acid metabolism. The dynamic analysis of the metabolites indicated that flavonoid accumulation was the greatest change at 3 DAP, followed by the change in lipid concentration, which showed a downward trend. As potent antioxidants, the early accumulation of flavonoids is probably a stress response to an ethephon‑induced ROS burst. This increase in flavonoids probably scavenges excess free radicals to protect cells from oxidative damage [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In contrast, the significant down‑regulation of lipid metabolites, such as 9‑oxo‑10,12‑octadecadienoic acid, may impair cell membrane stability and actively promote PCD. Furthermore, the significant enrichment of the α‑linolenic acid and linoleic acid metabolism pathways provides a logical link to the observed increase in JA levels as these unsaturated fatty acids are direct precursors for JA biosynthesis. Terpenoids emerged as the most differentially altered metabolite class at 5 DAP and consisted of compounds that are part of the sesquiterpenoid and triterpenoid pathways involved in lignin precursor synthesis and defense responses. These would potentially influence the structural toughness of the abscission zone by modulating cell wall lignification [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the primary metabolites, amino acids and their derivatives are directly involved in fundamental physiological activities and defense responses. For instance, in ripening strawberries, ethephon treatment increases the contents of various amino acids, such as alanine, arginine, and glutamine, with the most significant effects observed during the green and white developmental stages, which suggests a close association with plant growth, development, and defense mechanisms [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Organic acids, as core intermediates in the mitochondrial tricarboxylic acid and glyoxylate cycles, regulate photosynthesis and respiration and accumulate under environmental stress to enhance resistance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In contrast, alkaloids contribute to defense against microbial and viral infections [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although these metabolites play important roles in plant metabolic networks [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], their specific mechanisms during organ abscission regulation require further investigation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthephon induces calyx abscission in Korla Xiangli by modulating the interaction between ETH and IAA signaling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExogenous plant growth regulators influence organ abscission by regulating hormone biosynthesis, signal transduction, and transport [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Studies on apple have reported that IAA treatment up‑regulated IAA‑responsive genes in the fruit pedicel abscission zone while suppressing key ETH biosynthesis genes and the transcriptional activity of ERFs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Conversely, ethephon treatment of litchi panicles down‑regulated IAA signaling genes (such as ARF, Aux/IAA, and SAUR) and significantly up‑regulated ERFs [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These findings collectively highlighted the core mechanism by which IAA and ETH signaling antagonistically regulate organ abscission. They also agree with the transcriptomic data for litchi fruit abscission and validate this model for abscission. In the abscission zone, the expression of 47 IAA‑related genes was down‑regulated, while 39 ETH biosynthesis and signaling genes, including the ethylene receptor LcETR2, EIN3‑binding F‑box protein, and multiple ERFs, were significantly up‑regulated [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The functions of hormone‑related genes and pathways in Korla Xiangli have also been reported in other studies [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This study further revealed that at 3 DAP, exogenous ethephon treatment strongly activated the expression of the key ETH signaling genes Psin13G016570 and Psin16G016440, while simultaneously suppressing core components of IAA signaling (Psin05G016120, Psin13G016030, Psin15G013490, Psin16G015870, Psin16G015890, Psin10G004140, Psin10G004150, Psin10G004340, Psin10G004350, and Psin15G019720) and two ETH‑related genes (Psin13G016570 and Psin16G016440). This pattern aligned with the molecular mechanism reported for litchi fruitlet abscission [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In the ETH signaling pathway, ERF1/2 had a \u0026ldquo;rise‑then‑fall\u0026rdquo; expression pattern, whereas key IAA pathway components, such as AUX/IAA, CH3, and the early‑response gene SAUR, were continuously suppressed. This suggested that ethephon rapidly weakened IAA signaling intensity through transcriptional reprogramming.\u003c/p\u003e \u003cp\u003eThe WGCNA analysis identified the MEyellow module as being strongly negatively correlated with IAA levels (r = \u0026minus;\u0026thinsp;0.93) and enriched with hub genes, including AUX/IAA and AP2/ERF. This suggested that transcriptional regulatory modules played a integrative role in hormone crosstalk. The mechanism closely resembled the findings for apple pedicel abscission, where either the IAA transport inhibitor NPA or ETH treatment disrupted the IAA‑ETH balance and accelerated abscission by up‑regulating ETH biosynthesis genes, such as MdACS5B and MdACO1 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, this study has demonstrated that ethephon drives calyx abscission through a dual pathway that includes \u0026ldquo;endogenous ETH burst\u0026ndash;IAA signal suppression\u0026rdquo;, which rapidly reshapes the hormonal microenvironment in the abscission zone.\u003c/p\u003e \u003cp\u003eBased on the above research findings, a preliminary molecular regulatory model for ethephon‑induced calyx abscission can be proposed for Korla Xiangli (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e), as follows:\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eexogenous ethephon \u0026rarr; rapid increase in endogenous ETH concentration in the abscission zone \u0026rarr; activation of AP2/ERF transcription factors \u0026rarr; suppression of IAA influx carriers AUX/IAA and responsive gene SAUR \u0026rarr; disruption of polar IAA transport \u0026rarr; decrease in IAA concentration in the abscission zone \u0026rarr; attenuation of IAA antagonism toward ethylene \u0026rarr; sharp increase in the ETH sensitivity shown by abscission zone cells \u0026rarr; initiation of PCD and cell wall degradation programs \u0026rarr; calyx abscission.\u003c/p\u003e \u003cp\u003eDuring this process, flavonoids significantly accumulate at the onset of abscission to scavenge ROS, while down‑regulated lipid metabolites compromise membrane stability, which synergistically promotes PCD. This model provides an initial explanation for the mechanism by which ethephon promotes calyx abscission in Korla Xiangli and offers a theoretical reference for studies on organ abscission in other Rosaceae fruit trees. Future research should consider integrating techniques such as DAP‑seq and CUT\u0026amp;Tag to map genome‑wide TF‑binding profiles and could employ virus‑induced gene silencing (VIGS) for functional validation. The results from these types of studies would deepen understanding about the molecular mechanisms underlying ethephon‑induced calyx abscission in Korla Xiangli.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study systematically elucidated the molecular mechanism associated with exogenous ethephon‑induced calyx abscission in Korla Xiangli by integrating phenotypic observations, phytohormone profiling, wide‑target metabolomics, and a transcriptome analysis. Foliar spraying with 300 mg\u0026middot;L⁻\u0026sup1; ethephon at the large‑bud stage significantly accelerated the abscission process and advanced abscission zone formation to 7 DAP (2 days earlier than CK). The peak daily abscission rate was 16.90%, which was a 5.15‑fold increase compared to CK. The third day after pollination was identified as the key regulatory window. At the molecular level, ethephon rapidly increased the endogenous ETH concentration (+\u0026thinsp;33.09%) and suppressed IAA levels (\u0026minus;\u0026thinsp;49.30%) in the abscission zone, which disrupted hormonal homeostasis. The transcriptional analysis revealed that the ETH response factors ERF1/2 followed a \u0026ldquo;rise‑then‑fall\u0026rdquo; pattern, while IAA pathway genes, such as AUX/IAA and early‑response gene SAUR, were suppressed, thereby blocking polar IAA transport and enhancing ETH sensitivity in the abscission zone. The metabolomic analysis identified 383 differential metabolites. These were mainly enriched in the flavonoid biosynthesis, α‑linolenic acid metabolism, and terpenoid synthesis pathways. Among these, flavonoids markedly accumulated at the onset of abscission to scavenge ROS, whereas the down‑regulated lipid metabolites compromised membrane stability and jointly promoted PCD. The transcription factor analysis showed that the MYB, AP2/ERF, and NAC families became significantly enriched. The WGCNA identified the MEyellow module as being strongly negatively correlated with IAA levels (r = \u0026minus;\u0026thinsp;0.93) and identified nine hub genes, including IDA‑like, AP2/ERF, and AUX/IAA, as core regulatory nodes.\u003c/p\u003e \u003cp\u003eThis study provides a theoretical foundation for the precise chemical regulation of calyx abscission in Korla Xiangli and has identified candidate gene resources, such as ERF1/2 and AUX/IAA, that could be used for molecular breeding.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eExperimental materials\u003c/h2\u003e \u003cp\u003eThe experiment was conducted at the Korla Xiangli planting base of Xinjiang Qianguoxian Agricultural Planting Co., Ltd., which is located in Xiaolanggan Village, Awati Township, Korla City, Bayingolin Mongol Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China (41\u0026deg;69\u0026prime;N, 86\u0026deg;06\u0026prime;E). The test materials were 20‑year‑old Korla Xiangli \u003cem\u003e(Pyrus\u003c/em\u003e \u0026times; \u003cem\u003esinkiangensis\u003c/em\u003e Y\u0026uuml;\u003cem\u003e)\u003c/em\u003e trees pollinated with a mixed pollen blend containing the \u0026lsquo;Yali\u0026rsquo; and \u0026lsquo;Dangshan Suli\u0026rsquo; varieties. The rootstock was from \u003cem\u003ePyrus betulifolia\u003c/em\u003e (Chinese pear) with a plant spacing of 4 m \u0026times; 5 m and an open‑center tree training system. All selected plants exhibited uniform and vigorous growth with no signs of pests or diseases.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEthephon spray treatment\u003c/h2\u003e \u003cp\u003eSelected experimental trees were sprayed with 300 mg\u0026middot;L⁻\u0026sup1; ethephon solution on April 10, 2024 at the full‑bud stage and an equal amount of clean water was applied to another set of trees as the control (CK). Each treatment was applied to ten trees. Spraying was conducted in the morning on a clear, windless day using a handheld sprayer and the application continued until the flowers were fully wetted and the solution began to drip. The ethephon (85% purity) was purchased from Shanghai Yuanye Bio‑Technology Co., Ltd., Shanghai, China (Product No. S18030, provided for scientific research purposes only).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eThe de‑calyx fruits with the different treatments were collected at 3, 5, 7, and 9 days after pollination (DAP). Previous studies had indicated that fruits at the 4th and 5th positions of the inflorescence typically developed as de‑calyx types [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]; therefore young fruit at these positions were selected before abscission‑zone formation had occurred. During sampling, the junction between the calyx tube and young fruit was transversely excised with a surgical scalpel and then the petals and sepals were manually removed. The retained abscission‑zone tissue included the calyx tube and cells that were proximal to the separation line. After collection, the samples were immediately wrapped in aluminum foil, flash‑frozen in liquid nitrogen, and subsequently stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further analysis. A total of eight sample groups (4 \u0026times; 2 treatments) were prepared, each with three biological replicates, for phytohormone quantification, transcriptome sequencing, metabolome sequencing, and qRT‑PCR validation. All sequencing procedures were conducted by Wuhan MetWare Biotechnology Co., Ltd., Wuhan, China.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePhenotypic trait observation during calyx abscission\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003eExternal morphological changes associated with calyx abscission\u003c/h2\u003e \u003cp\u003eThe external morphological characteristics of the de‑calyx fruits were observed and photographed using a digital camera at 3, 5, 7, 9, 11, and 13 DAP.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eDaily calyx abscission rate survey\u003c/h2\u003e \u003cp\u003eA total of three Korla Xiangli trees were selected from each treatment (six trees in total). In early April, 2024, branches at similar developmental stages were tagged on the east, south, west, and north aspects of the canopy for each treatment. Then 20 branches were randomly selected per treatment, giving a total of 40 branches. Within each treatment, 100 inflorescences were randomly labelled on the selected branches and the number of calyx‑shedding fruits was recorded daily from the third DAP until abscission ceased. The daily calyx abscission rate was calculated as\u003c/p\u003e \u003cp\u003eDaily calyx abscission rate (%) = (number of newly de‑calyx fruits per day on labelled branches) / (total number of young fruits on the labelled main branches) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003ePhytohormone assay\u003c/h2\u003e \u003cp\u003eThe IAA, ABA, gibberellin A₃ (GA₃), JA, and SA contents in the samples were determined using high‑performance liquid chromatography\u0026ndash;tandem mass spectrometry (HPLC‑MS/MS). The sample processing procedure was as follows: abscission‑zone samples that had been stored at \u0026minus;\u0026thinsp;80\u0026deg;C were rapidly transferred to liquid nitrogen and ground to a fine powder using a grinding mill (30 Hz, 1 min). Approximately 100 mg of the powdered tissue was weighed into a 2 mL centrifuge tube, followed by the sequential addition of 10 \u0026micro;L of mixed internal standard solution (100 ng\u0026middot;mL⁻\u0026sup1; in methanol) and 1 mL of pre‑cooled extraction solvent (methanol:water:formic acid\u0026thinsp;=\u0026thinsp;15:4:1, v/v/v). The mixture was vortexed for 10 min and then centrifuged at 4\u0026deg;C and 12,000 rpm for 5 min. The supernatant was transferred to a new 1.5 mL centrifuge tube and completely dried using vacuum centrifugation. Then the residue was reconstituted in 100 \u0026micro;L of 80% methanol aqueous solution (v/v), filtered through a 0.22 \u0026micro;m organic phase membrane filter, transferred to an LC vial, and subjected to LC‑MS/MS analysis. Each treatment contained three biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eEthylene concentration measurement\u003c/h2\u003e \u003cp\u003eThe ETH concentration was measured according towith modifications [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Approximately 5 g of each sample was weighed and placed into a clean conical flask. The flask was tightly sealed with a rubber stopper and further secured with sealing film to prevent gas leakage. After 24 h, the sealed flask was kept at room temperature for 2 h to equilibrate the internal gas and then gas sampling was performed by slowly withdrawing 1 mL of headspace gas with a syringe for gas chromatography analysis. Nitrogen was used as the carrier gas at a flow rate of 1 mL\u0026middot;min⁻\u0026sup1; and the injector and column temperatures were both set at 50\u0026deg;C to optimize ETH separation. Separation was achieved using an Agilent HP‑PLOT Q column (30 m \u0026times; 0.53 mm \u0026times; 0.40 \u0026micro;m) and detection was carried out using a flame ionization detector set at 200\u0026deg;C. The retention time for ETH was approximately 1.1 min, and quantification was performed using an external standard method. Each treatment contained three independent replicates.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMetabolite detection\u003c/h3\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation and metabolite extraction\u003c/h2\u003e \u003cp\u003eThe samples were vacuum freeze‑dried for 63 h, ground using a mill (30 Hz, 1 min), and passed through a 40‑mesh sieve. Approximately 50 mg of the powdered sample was weighed into a 2 mL centrifuge tube. Then 1,200 \u0026micro;L of a pre‑cooled (\u0026ndash;20\u0026deg;C) 70% methanol aqueous internal standard extraction solution was added to the tube. After thorough mixing by shaking, the mixture was placed in a vortex mixer and vortexed for 30 seconds every 30 minutes, which was repeated six times in total. Following centrifugation, the supernatant was carefully collected, filtered through a microporous membrane, and transferred to a sample vial for subsequent metabolomic analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eLC‑MS/MS‑based metabolite detection\u003c/h2\u003e \u003cp\u003eThe data acquisition system consisted of an ultra‑performance liquid chromatography (UPLC) unit coupled to a tandem mass spectrometer (MS/MS). The liquid chromatography column temperature was maintained at 40\u0026deg;C and the flow rate was set at 0.35 mL/min during data acquisition. Electrospray ionization was used for the mass spectrometry with the ion source heating temperature adjusted to 500\u0026deg;C. Triple quadrupole scanning was performed in multiple reaction monitoring (MRM) mode and the declustering potential (DP) and collision energy (CE) for each MRM ion pair were optimized using systematic adjustment. A specific set of MRM ion pairs was monitored in each time window and were selected according to the metabolites eluted during that period.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eTranscriptome sequencing\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eTotal RNA extraction and quality assessment\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the samples by combining ethanol precipitation with a cetyltrimethylammonium bromide (CTAB)-based method. RNA concentration was determined using a Qubit 4.0 Fluorometer and an MD microplate reader, while RNA integrity was assessed via a Qsep400 bioanalyzer.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003ecDNA library construction and quality control\u003c/h3\u003e\n\u003cp\u003eThe poly(A)\u0026thinsp;+\u0026thinsp;mRNA was enriched from the total RNA using oligo(dT)-conjugated magnetic beads and then the purified mRNA was fragmented in fragmentation buffer to generate shorter RNA fragments. These fragments served as templates for first-strand cDNA synthesis and were primed with random hexamers during reverse transcription. Second-strand cDNA was then synthesized using buffer, deoxynucleotide triphosphates (dNTPs), and DNA polymerase. The resulting double-stranded cDNA was purified with magnetic beads and subjected to end repair, 3\u0026rsquo; adenylation, and sequencing adapter ligation. Finally, fragments of the desired size were selected using magnetic beads and high-quality cDNA libraries were constructed using PCR amplification.\u003c/p\u003e\n\u003ch3\u003eRaw data processing\u003c/h3\u003e\n\u003cp\u003eCASAVA software was used to perform base calling using image data generated by the Illumina high‑throughput sequencing platform. This process produced the raw sequence reads (raw reads). To ensure data quality, the following quality control steps were applied: (1) removal of reads containing adapter sequences, (2) exclusion of reads with ambiguous bases, and (3) filtering out low‑quality reads (defined as reads where \u0026gt;\u0026thinsp;50% of bases had an Qphred score\u0026thinsp;\u0026le;\u0026thinsp;20).\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eThe clean reads were aligned to the reference genome (\u003cem\u003ePyrus sinkiangensis\u003c/em\u003e; available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pearomics.njau.edu.cn/sites/default/files/raw/Pyrus_sinkiangensis\u003c/span\u003e\u003cspan address=\"https://pearomics.njau.edu.cn/sites/default/files/raw/Pyrus_sinkiangensis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using HISAT2 (v2.0.5). The resulting SAM files were converted to the BAM format using SAMtools and then the genes were quantified using FeatureCounts to generate a count matrix. The gene expression levels were normalized as fragments per kilobase of transcript per million mapped reads (FPKM) and a differential expression analysis was performed using DESeq2 (v1.20.0) with significance thresholds set at |log₂(fold change)| \u0026ge; 1 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Then GO functional annotation and KEGG pathway enrichment analyses were performed using clusterProfiler. A weighted gene co‑expression network analysis (WGCNA) was performed using the WGCNA package (v1.29), and the co‑expression networks were visualized by Cytoscape (v3.3.0).\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eQuantitative real‑time PCR (qRT‑PCR) validation\u003c/h2\u003e \u003cp\u003eAll the qRT‑PCR primers used in this experiment were designed using the online primer design tool Primer3Plus (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.primer3plus.com/\u003c/span\u003e\u003cspan address=\"https://www.primer3plus.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and primer specificity was verified using the DNAMAN design tool. The qRT‑PCR assays were performed using a 2\u0026times;ChamQ Universal SYBR Master Mix kit from Novizan Biotechnology Co., Ltd., China using the procedure outlined below:\u003c/p\u003e \u003cp\u003e(1) Preparation of qRT‑PCR reaction mixtures as described in Table S1;\u003c/p\u003e \u003cp\u003e(2) Loading of the prepared reaction system into a real‑time PCR instrument;\u003c/p\u003e \u003cp\u003e(3) Use of RhUBI2 as the reference gene for normalization;\u003c/p\u003e \u003cp\u003e(4) Each sample was analyzed with three biological replicates and gene expression levels were calculated using the 2⁻ΔΔCT method.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the data were organized using Microsoft Excel 2019 (Microsoft Corp, Redmond, WA, USA) and the statistical analyses, including a variance analysis combined with t‑testing, were performed using SPSS 26.0 (IBM, Armonk, NY, USA) with the significance level set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The graphs and charts were generated using Origin 2021 (Originlab, Northampton MA, USA) and GraphPad Prism 9 (San Diego, CA, USA). The figures were assembled and processed using Adobe Photoshop CC 2019 (Adobe Systems, San Jose, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the postgraduate innovation project of the autonomous region（XJ2024G136）and the second group of tianshan talent training program: youth support talent project (2023TSYCQNTJ0004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated or analyzed during this study are included in this article and its supplementary information files. The genomic DNA, coding sequences (CDS), and annotation files of Xinjiang pear were downloaded from the Pear Database of Nanjing Agricultural University. The raw transcriptome data have been deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/; BioProject Accession ID PRJNA1404807).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.W. contributed to writing the original draft, visualization, methodology, data curation, project administration and funding acquisition. L.Z. participated in writing the original draft, visualization, methodology, investigation, formal analysis and data curation. Y.L. and T.S. were responsible for investigation, visualization, formal analysis and data curation. Y.G. contributed to methodology and formal analysis. C.C. and L.W. engaged in formal analysis and data curation. F.Z., C.W., Y.W. and X.Z. participated in investigation and methodology. J.T. was involved in writing-review and conceptualization. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies involving human participants or animals performed by\u003c/p\u003e\n\u003cp\u003ethe authors. The methods in this study were carried out in accordance with relevant guidelines and\u003c/p\u003e\n\u003cp\u003eregulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or personal conflicts of interest that could have\u003c/p\u003e\n\u003cp\u003einfluenced the work described in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXu P, Chen H, Cai W. Transcription factor CDF4 promotes leaf senescence and floral organ abscission by regulating abscisic acid and reactive oxygen species pathways in Arabidopsis. EMBO Rep. 2020;21(7):e48967.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Li J. Molecular events involved in fruitlet abscission in litchi. 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Mol Biotechnol. 2007;36(1):9\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Korla Xiangli (Pyrus × sinkiangensis Yü), calyx abscission, ethephon, transcriptomics, metabolomics, hormone signaling, Weighted gene co-expression network analysis","lastPublishedDoi":"10.21203/rs.3.rs-8648793/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8648793/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCalyx abscission is a key horticultural trait affecting fruit quality in Korla Xiangli \u003cem\u003e(Pyrus\u003c/em\u003e \u0026times; \u003cem\u003esinkiangensis\u003c/em\u003e Y\u0026uuml;\u003cem\u003e).\u003c/em\u003e Exogenous ethephon treatment has been shown to significantly promote calyx abscission, but the underlying molecular regulatory network and metabolic mechanisms that regulate ethephon-induced abscission remain poorly understood. Elucidation of these processes is essential to optimize calyx management strategies in commercial Korla Xiangli production.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThis study systematically elucidated the molecular regulatory network of ethephon-induced calyx abscission in Korla Xiangli by phenotypic observation, phytohormone profiling, wide-target metabolomics and high-throughput transcriptomic analyses. Treatment with 300 mg\u0026middot;L⁻\u0026sup1; ethephon significantly accelerated abscission zone formation by two days, with abscission occurring seven days after pollination. The peak daily abscission rate reached 16.90%, which represented a 5.15-fold increase compared with the untreated control. The results demonstrated that the third day after pollination was the critical period for ethephon-mediated calyx abscission regulation. At this stage, ethylene concentration in the abscission zone significantly increased by 33.09% and indole-3-acetic acid content decreased sharply by 49.30%. In contrast, abscisic acid, salicylic acid, gibberellin A₃, and jasmonic acid levels were strongly upregulated, which suggests extensive hormonal regulation associated with abscission initiation. Transcriptome data showed transient induction of \u003cem\u003eERF1/2\u003c/em\u003e and repression of AUX/IAA and SAUR genes, limiting polar auxin transport and enhancing ethylene sensitivity. Metabolomic profiling revealed 383 differential metabolites, primarily associated with flavonoid, lipid, and terpenoid pathways, collectively promoting ROS regulation, membrane destabilization, and programmed cell death. Network analyses identified MYB, AP2/ERF, and NAC transcription factors and highlighted key hub genes involved in auxin\u0026ndash;ethylene crosstalk.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn this study, we first conducted phenotypic observations of ethephon-induced calyx abscission in Korla Xiangli, and then measured the levels of plant hormones, metabolites and gene expression in the abscission zone. These results provide a theoretical basis and genetic resources for the precise regulation and molecular breeding of calyx abscission in Korla Xiangli.\u003c/p\u003e","manuscriptTitle":"Integrated omics analysis provides preliminary insights into the regulatory network controlling ethephon-promoted calyx abscission in Korla Xiangli (Pyrus × sinkiangensis Yü)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 19:17:58","doi":"10.21203/rs.3.rs-8648793/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-14T05:31:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T08:52:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-16T12:15:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203321849800399224345267175882438402899","date":"2026-02-13T09:47:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224462668357710913188923362494035767309","date":"2026-02-13T07:54:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210505927729460442248816783058574265250","date":"2026-01-30T08:35:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-28T01:02:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-27T16:36:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T17:44:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T17:42:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-01-20T11:08:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"58f8f638-984a-46f3-81be-aebf4ab28b28","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T08:39:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 19:17:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8648793","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8648793","identity":"rs-8648793","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}