Regulatory effects of gibberellin and cytokinin on citrus peel cell wall metabolism

Regulatory effects of gibberellin and cytokinin on citrus peel cell wall metabolism | 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 Regulatory effects of gibberellin and cytokinin on citrus peel cell wall metabolism Xun Wang, Yuping Wang, Defa Cao, Panpan Gao, Mingfei Zhang, Jiaxian He, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6059599/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jun, 2025 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version Abstract During citrus fruit development, exogenous gibberellin (GA) and 6-benzylaminopurine (6-BA, a synthetic cytokinin (CTK)) are both known to promote citrus peel thickness; however, the differences in their regulatory mechanisms on cell wall metabolism in citrus peels remain unclear. In this study, we found that GA treatment significantly increased cell wall polysaccharides in citrus peels, such as pectin and cellulose, whereas 6-BA treatment led to a notable accumulation of lignin. RNA-sequencing data revealed that several fruit ripening-related cell wall degradation genes, such as PME3, PL18, and EXPA1/8, exhibited decreased expression levels in both GA and 6-BA treatments. Additionally, a set of cell wall polysaccharide synthesis genes was upregulated in response to GA treatment but was largely downregulated in 6-BA-treated peels. Conversely, a group of lignin biosynthesis genes was upregulated in 6-BA-treated peels. GA treatment inhibited DELLA proteins (encoded by RGA and GAI) in the GA signaling pathway, whereas 6-BA treatment increased the expression of B-ARRs (ARR1 and ARR2) in the CTK signaling pathway. Furthermore, GA treatment elevated endogenous CTK levels, while 6-BA treatment also enhanced endogenous GA content, suggesting a reciprocal interaction between these two hormonal pathways. citrus peels cell wall gibberellin cytokinin DELLAs B-ARRs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction As the citrus fruit develops, the fruit gradually expands and the peel becomes thinner. Some modern citrus cultivars have been bred to develop extremely thin peels for easy peeling. However, excessively thin peels may lead to fruit cracking during growth [1], resulting in production loss. Therefore, maintaining appropriate peel thickness is crucial for alleviating fruit cracking in citrus [2]. Peel thickening will result from increased cell wall material deposition. Fruit cell wall is composed of polysaccharides such as pectin, hemicellulose, and cellulose, and often contains glycoproteins and lignin [3]. Pectins play a central role in regulating the rheological properties of the cell wall, which are crucial for plant growth [4]. Pectin is composed of unbranched homogalacturonan (HG) and branched rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) [5]. Hemicellulose is a diverse group of polysaccharides, including xyloglucan (XyG) and xylan in dicot plants. XyG forms cross-links between cellulose microfibrils, creating a strong yet extensible XyG-cellulose network, which serves as the primary load-bearing structure of the cell wall [6]. Cellulose, a fibrous, tough, and water-insoluble substance, consists of unbranched β -(1,4)-linked glucan chains [7]. Lignin is an unordered polymer derived from phenylalanine, composed of aromatic monomers [8]. Lignin is a key component of both primary and secondary cell walls [9]. Expansins, which non-enzymatically relax the cell wall, play a critical role in cell expansion by loosening and softening the wall [10]. Fruit development is regulated by a variety of expansin genes, which show distinct expression patterns [11]. Phytohormones are essential to the regulation of fruit development and maturation. Ethylene and abscisic acid are generally associated with fruit ripening, while fruit growth (including in fruit cell division and expansion) involves other phytohormones, such as gibberellin (GA) and cytokinin (CTK) [12]. GA is extensively used in fruit production to induce parthenocarpic fruits, resulting in fruit sizes equal to or larger than those of pollinated fruits [13]. GA primarily promotes cell expansion during fruit development [13], and it also enhances cell division [14]. GA-induced thickening of fruit peels is associated with an increase in cellulose content in the cell walls [15]. Furthermore, studies in non-fruit plant organs have demonstrated that GA can enhance lignin deposition [16, 17] and increase the synthesis of cell wall polysaccharides, such as hemicellulose [18]. CTKs are a diverse group of plant growth regulators with wide-ranging effects on plant growth and development [19]. CTKs promote fruit enlargement through either cell division or expansion, thereby facilitating fruit development [19]. GA mediates its effects by overcoming the inhibition imposed by DELLA proteins in the GA signaling pathway [20]. DELLA proteins act as transcriptional regulators for numerous aspects of plant growth and development, including seed germination, flowering, hypocotyl hook formation, fruit development, and defense responses [20]. In contrast, B-type ARABIDOPSIS RESPONSE REGULATORS (Type-B RRs) are essential for the initial transcriptional response to CTK [21]. Type-B ARRs function as positive transcription factors in the CTK signaling pathway, regulating the expression of target genes involved in plant growth, development, and responses to abiotic stress [22]. The Arabidopsis genome contains 22 ARR genes, with ARR1, 2, and 10–14, as well as 18–21, classified as Type-B ARRs [23]. Antagonistic crosstalk between GA and CTK is a common feature in plant physiological processes [24]. In fruits, both GA and CTK promote growth, albeit through distinct mechanisms. GA primarily promotes cell expansion, whereas CTK enhances cell division [13, 25]. Brenner et al. found that CTK suppresses the expression of GA biosynthesis genes ( GA3ox and GA20ox ) while promoting the expression of DELLA genes ( GAI and RGA ) [26]. However, the interaction between GA and CTK is species- and tissue-dependent. For instance, [27] showed that in Brassica napus seedling development, GA promotes CTK accumulation by modulating RGA-related CKX gene expression. In prior experiments, we confirmed the role of GA and 6-benzylaminopurine (6-BA, a synthetic CTK) in thickening citrus peels. However, the molecular mechanisms through which GA and 6-BA induce peel thickening, as well as the differences in their effects, remain unclear. In this study, we investigated the effects of GA and 6-BA on peel thickening in Citrus Mandarin ( Citrus reticulata Blanco) cultivar Asumi, a variety known for its relatively thin peels, making it an ideal model for studying peel thickening. Our objective was to elucidate the molecular mechanisms underlying GA- and 6-BA-induced peel thickening. 2. Materials and Methods 2.1. Plant materials The study was conducted using four-year-old Citrus Mandarin ( Citrus reticulata Blanco) cultivar Asumi trees, which were grafted in Citrus junos rootstock. The experiment was carried out in Sichuan, China. The experimental trees exhibited uniform height, fruit-setting rates, and growth potential. All trees were healthy and free from pests and diseases. Three treatments were applied: gibberellin (GA), 6-benzylaminopurine (6-BA), and water (as the control check, CK). Each treatment included five trees, with each tree serving as a biological replication. The concentration of GA was 20 mg/L and 6-BA was 4 mg/L, which were determined in preliminary experiment (data not shown). Spraying treatments were stated at the beginning of the fruit enlargement period. The first spraying was on Jul 20 th , 2023. Three applications were performed at approximately 10-day intervals. All spraying was conducted in the morning on sunny days. The spray solution was applied evenly to the foliage and the abaxial surfaces of the leaves until visible water droplets formed without dripping. The first sampling was conducted on the day of the first spraying treatment (July 20 th ), when was at the beginning of the fruit expansion stage, designated as DAT0 (0 day after treatment). The second sampling was conducted 30 days after the first spraying treatment (August 20 th ), when was at the mid-stage of fruit expansion, designated as DAT30. The third sampling was conducted 60 days after the first spraying treatment (September 20 th ), when was at the end of the fruit expansion period, designated as DAT60. Fruits were harvested from the periphery of the tree canopy in four directions: southeast, southwest, northwest, and north. Approximately 10 fruits were collected per tree. 2.2. Measurement of citrus peel thickness The pericarp thickness was measured using a vernier caliper. Each fruit was longitudinally cut into two halves along the equatorial plane using a sharp knife. The thickness of the pericarp at the equatorial region, specifically at the point where the vesicle lobes contact the pericarp, was measured. Measurement was taken three times for each of the ten fruits to ensure accuracy and consistency. 2.3. Determination of pectin, hemicellulose, cellulose, and lignin The rind and pulp of the collected samples were separated, and citrus peels were dried in an oven at 60 °C. The dried samples were then ground into a fine powder. To extract the cell wall material, the peels were sequentially treated with ethanol, dimethyl sulfoxide, chloroform-methanol, and acetone. The extracted cell wall material was subsequently used to determine the relative contents of pectin, cellulose, hemicellulose, and lignin. The water-soluble pectin (WSP) and protopectin were quantified by colorimetry using a carbazole-vitriol method [28]. The relative content of cellulose was determined by an anthrone colorimetric method [29]. Hemicellulose content was measured using the phenol-sulfuric acid method [29], and lignin content was determined using the acetyl bromide method [30]. 2.4. Microscopic observation of cellulose and lignin Citrus peels sampled on the third time (September 20, 2023) were used for microscopic observation of cellulose and lignin. Fresh samples from the equatorial region of fruits were cut into pieces measuring 1 cm × 1 cm. The cut samples were immediately fixed in FAA solution (formaldehyde: 5%, glacial acetic acid: 6%, ethanol: 70%) for more than 24 hours. The fixed samples were embedded in paraffin and sectioned perpendicular to the pericarp surface into slices with a thickness of 4 μm. Cellulose and lignin in the tissues were stained using Solid Green Staining and Safranin-O Staining, respectively. The stained sections were observed under an orthogonal fluorescence microscope (Eclipse Ci, Nikon, Japan). The microstructural areas of lignin and cellulose were analyzed using Image-Pro Plus 6.0 software. To quantify cellulose and lignin, the ratio of the Solid Green Staining stained area (cellulose) and the Safranin-O stained area (lignin) of a 200 μm × 180 μm, regarded as an unit area, was calculated. Six unit-areas were randomly selected for analysis, with each serving as a replicate. 2.5. Determination of hormone content The GA and CTK concentrations were determined by double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) kit (CamaiShu Biotech, China). Specimen, standard and HRP-labeled detection antibody were added into the microtiter wells pre-coated with GA and CTK antibodies. After incubation and thorough washing, the wells were color-developed with the substrate Tetramethylbenzidine (TMB), which is converted to blue by peroxidase and to final yellow by acid. The shade of color is positively correlated with GA and CTK in samples. The absorbance (OD) was measured at 450 nm using an enzyme meter (SpectraMax M2, Molecular Devices, US) and the concentration of samples was calculated. The correlation coefficient R value for the linear regression of the standard should be greater than or equal to 0.9900 compared to the expected concentration. The minimum detectable concentration should be less than 1.0 pg/mL for GA determination and 1.0 pmol/mL for CTK determination. Three replicates were done for each sample. 2.6. Analysis of transcriptome sequencing Citrus peels sampled on the second time (Aug 20, 2023) and third time (Sep 20, 2023) were used for transcriptome sequencing analysis, which were designated to be Stage I and Stage II. The citrus peels were rapidly frozen in liquid nitrogen for transcriptome sequencing. Transcriptome sequencing was performed using the Illumina NovaSeq6000 sequencing platform (Biomarker Technologies Co, Ltd.). The Raw data obtained was filtered and aligned with the reference genome Citrus_reticulata.Mangshan.v1.0.genome.fa using a HISAT2 software. A StringTie software was used to assemble the aligned reads [31]. Based on the Count values of genes in each sample, differential expression gene (DEG) screening was carried out using a DESeq2 software [32]. During the detection of differentially expressed genes, a fold change of ≥ 1.5 and a false discovery rate (FDR) < 0.01 were used as the screening criteria. A Hierarchical Clustering Analysis of cell wall metabolic genes and transcription factors was performed using R language. K-means was used for verification. The Factoextra and Cluster packages were used to draw visual scatter plots. 2.7. RNA extraction and Real-time quantitative PCR (RT-qPCR) analysis Another citrus peel sample was ground into fine powder in liquid nitrogen and subjected to total RNA extraction and RT-qPCR. RNA was extracted using total RNA extraction kit (RNAsinple Total RNA Kit, Tiangen Biotech, China). For analysis of mRNA transcription levels, 1 μg total RNA was reverse transcribed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311, TransGen Biotech, China). The RT-qPCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, US) using PerfectStart® Green qPCR SuperMix (Transgen, China). Relative gene expression levels were calculated using the 2 −ΔΔCT method. Three replicates in each were performed. Actin gene was the reference gene. The primers for RT-qPCR were in Supplementary material 1. 3. Results 3.1. Citrus peel thickness in GA and 6-BA treatments During fruit ripening, the peel thickness gradually decreased in both GA, 6-BA, and CK controls (Fig. 1). The peel thickness in both GA and 6-BA treatments was significantly greater than that in the CK control on DAT30, with the difference further increasing by DAT60 (Fig. 1). 3.2. Cell wall material contents in GA and 6-BA treatments The relative content of insoluble pectin in GA and 6-BA treatments was significantly higher compared to the CK control on DAT60 (Fig. 2). The relative content of water-soluble pectin increased during fruit ripening, whereas it was lower in both GA and 6-BA treatments than in the CK control. An increase in hemicellulose content was observed in the GA treatment on DAT30 compared to the CK control. The relative cellulose content in GA treatment was significantly higher than in the CK control on DAT60. Lignin content in the 6-BA treatment increased on both DAT30 and DAT60. Microscopic analysis of paraffin sections revealed the accumulation of cellulose and lignin on DAT60 (Fig. 3). In the GA treatment, cellulose stained with Solid Green Staining (cyan) was more abundant than in the CK control. Image-Pro Plus analysis of the cyan area revealed a significantly larger area in the GA treatment compared to the CK control. In contrast, in the 6-BA treatment, lignin stained with Safranin-O Staining (magenta) was more abundant than in the CK control. The Image-Pro Plus analysis also indicated that the magenta area was significantly larger in the 6-BA treatment than in the CK control. 3.3. Plant hormone contents in GA and 6-BA treatments The contents of GA and CTK were measured. The GA content in citrus peels was elevated in both the GA and 6-BA treatments on DAT30 (Fig. 4). The GA content remained significantly higher in both GA and 6-BA treatments compared to the CK control on DAT60. Additionally, CTK content in citrus peels increased in 6-BA treatment on DAT30. On DAT60, CTK content was also elevated in the GA treatment, whereas it decreased in the 6-BA treatment. 3.4. Differentially expressed genes (DEGs) involved in cell wall metabolism As citrus peels were significantly thickened Aug 20, 2023 and Sep 20, 2023, transcriptome sequencing was performed using the peels sampled on the two stages, designated to be Stage I and Stage II. The RT-qPCR results that validated the results of transcriptome were in Supplementary material 2. The DEGs from transcriptome sequencing in GA(I) vs CK(I) and 6-BA(I) vs CK(I) were 5316 and 4230, respectively, while in GA(II) vs CK(II) and 6-BA(II) vs CK(II), the DEGs were 2231 and 1206, respectively. These results indicate that the majority of differential gene expression in response to GA or 6-BA occurs at stage I, implying a mechanism of phenotype delay to genotype. Therefore, the gene expression at this stage was further analyzed. Genes involved in cell wall metabolism (with average FPKMs > 10) were screened (Supplementary material 3), including those related to pectin HG synthesis, pectin RG-I/II synthesis, hemicellulose (XyG) synthesis, cellulose synthesis, and lignin synthesis. Additionally, genes involved in cell wall degradation were examined, including pectinesterase/pectin methyl‐esterase, polygalacturonase, pectate lyase, β -galactosidase, arabinosyltransferase, endotransglucosylase/hydrolase, and expansin proteins (Supplementary material 3). A total of 43 genes were identified (Fig. 5). In the GA treatment (stage I), compared to the CK control, pectin HG synthesis genes, including galacturonosyltransferases GAUT8 / 4 / 7 , were upregulated, with GAUT8 showing a significantly higher transcription level. The pectin RG synthesis gene rhamnogalacturonan I rhamnosyltransferase ( RRT1 ) was also upregulated. The XyG metabolic genes generally showed increased expression, such as XyG glycosyltransferase ( CSLC12 / 4 ), XyG 6-xylosyltransferase ( XXT2 / 3 ), XyG galactosyltransferase ( MUR3 ), fucosyltransferase ( MUR2 / FUT1 ), and XyG galactosyltransferase ( XLT2 ). All cellulose synthesis genes were upregulated, including cellulose synthase ( CESA1 / 2 / 3 ) and CESA-like D3 ( CSLD3 ). In the 6-BA treatment (stage I), the abovementioned genes involved in cell wall polysaccharide synthesis were mostly downregulated compared to the CK control, while lignin synthesis genes were upregulated, including p-coumaroylshikimate 3′-hydroxylase ( C3’H ), caffeoyl-CoA O-methyltransferase ( CCoAOMT1 ), ferulate 5-hydroxylase ( F5H ), phenylalanine ammonia-lyase ( PAL1 ), cinnamate 4-hydroxylase ( C4H ), caffeic acid O-methyltransferase ( COMT ), cinnamoyl-CoA reductase ( CCR1 ), and cinnamyl alcohol dehydrogenase ( CAD1 ). Several genes involved in fruit cell wall degradation were significantly downregulated in both GA and 6-BA treatments, such as pectin methyl‐esterase ( PME3 ), polygalacturonase ( PG49 ), pectate lyase ( PL18 ), arabinosyltransferase ( ASD1 ), and expansin ( EXPA1 / 8 ). In contrast, other genes, such as PME1 , PG52 , and XyG endotransglucosylase/hydrolase ( XTH23 ), were upregulated. 3.5. Differentially expressed genes involved in plant hormone synthesis and signaling pathways Genes involved in GA and 6-BA synthesis and signaling pathways were analyzed (Fig. 5, Supplementary material 4). In the GA treatment (stage I), the GA synthesis gene GA20ox2 decreased, while the catabolic gene GA2ox1 increased compared to the CK control. Additionally, CKX5 (which downregulates CTK levels) was significantly reduced. In the 6-BA treatment (stage I), the expression of GA synthesis gene GA20ox2 decreased, and GA2ox1 increased compared to the CK control, similar to the GA treatment (stage I). However, in the 6-BA treatment (stage II), the GA synthesis genes KAO and GA20ox2 were upregulated, while GA2ox1 was downregulated compared to the CK control. In the signaling transduction pathway, all identified genes in the GA signaling pathway were downregulated in the GA treatment compared to the CK control, including GID1B / C , GAI , RGA , and GID2 . In the 6-BA treatment, genes in the CTK signaling pathway, such as AHK3 , AHP5 , and B-type ARR ( ARR1 ), were upregulated compared to the CK control. 3.6. Transcription factors associated with cell wall metabolism-related genes A total of 803 transcription factor (TF) genes were identified in the transcriptome data. Of these, 445 and 457 genes (with average FPKMs > 10) were significantly connected with cell wall metabolic genes in the GA and 6-BA treatments, respectively. Cluster analysis using the Hierarchical Clustering method was performed based on correlation analysis. In the GA treatment, the connected TF genes and cell wall metabolic genes were divided into six clusters (Fig. 6). Cell wall degradation genes were mostly located in cluster 4 (Fig. 6). Several TFs that were negatively correlated with multiple cell wall degradation genes were identified in this cluster, including NFYB8 , JAZ10 , APC8 , and NAC017 (Fig. 7; Supplementary material 5). Genes involved in cell wall polysaccharide (pectin, XyG, and cellulose) synthesis were generally placed in cluster 1 of the GA treatment (Fig. 6), where TFs such as EJ2 , RAP2-12 , and ANL2 were positively associated with several cell wall polysaccharide synthesis genes (Fig. 7; Supplementary material 5). In the 6-BA treatment, the connected TF genes and cell wall metabolic genes were similarly divided into six clusters (Fig. 6). Cell wall degradation genes were mostly located in cluster 1 (Fig. 6), where several TFs, including SPL1 , EIN3 , CAMTA2 , and NAC017 , were negatively correlated with multiple cell wall degradation genes (Fig. 7; Supplementary material 6). The genes involved in lignin synthesis were primarily clustered in cluster 4 of the 6-BA treatment (Fig. 6), where TFs such as BLH7 , HAT5 , COL4 , WRKY15 , and ARR1 were positively associated with several lignin synthesis genes (Fig. 7). In contrast, genes involved in cell wall polysaccharide synthesis were generally placed in cluster 2 (Fig. 6), where many TFs, such as NAC047 , DF1 , TCF13 , and ARR2 , were negatively correlated with multiple cell wall polysaccharide synthesis genes (Fig. 7; Supplementary material 6). 4. Discussion 4.1. GA promoted pectin and cellulose accumulation, while 6-BA enhanced lignin accumulation GA and CTK are key phytohormones involved in fruit development, promoting both cell expansion and division [19, 25]. The synthesis of cell wall materials supports cell division and expansion, contributing to fruit enlargement. Previous studies have shown that GA increases cellulose content, thereby strengthening the fruit peel [15]. Although the effects of CTK on the cell wall material of fruit peel are less well understood, it is hypothesized that CTK primarily promotes lignin accumulation, which contributes to plant organ growth, as observed in studies on carrot ( Daucus carota ) taproots [33] and bamboo ( Phyllostachys nigra ) suspension cells [34]. In the present study, as citrus fruits developed, the fruit peel gradually thinned. However, the peel was significantly thicker in the GA and 6-BA (a synthetic CTK) treatments compared to the control. In the GA treatment, the cell wall materials of the peel increased overall, particularly in the relative contents of pectin and cellulose. In contrast, the 6-BA treatment significantly elevated the relative content of lignin. 4.2. GA treatment upregulated cell wall polysaccharide synthesis genes, while 6-BA treatment enhanced lignin synthesis genes In the present study, a set of genes involved in cell wall metabolism was identified (Fig. 5). The GAUT family is responsible for synthesizing pectin homogalacturonan (HG) [35]. Rhamnose transferases (RRTs) facilitate the addition of rhamnose residues to RG-I oligosaccharides [36]. XXT1/2/5 are involved in the first step of side-chain formation in xyloglucan (XyG) [37]. XLT2 and MUR3 add galactose to the second and third xylosyl residues, respectively, of XyG side chains [38]. MUR2/FUT1 adds terminal fucose to the XyG side chain [39]. Cellulose synthase complexes (CSCs), consisting of CESA1, CESA3, and other CESA isoforms, catalyze cellulose glucan chain elongation and are essential for primary cell wall cellulose synthesis [40]. The CSLD gene family, similar to CESA, is implicated in cellulose synthesis [41]. In this study, GA treatment significantly upregulated genes encoding these enzymes, including GAUT8 , RRT1 , XXT2 (homologous to AtXXT1 ), XXT3 (homologous to AtXXT5 ), XLT2 , MUR3 , MUR2 , CESA1 / 2 , and CSLD3 (Fig. 5). Additionally, genes encoding cell wall-modifying enzymes, such as PME1 , PG52 , and XTH23 , which promote the restructuring of pectin and hemicellulose to support new cell wall synthesis [4;6], were also upregulated in GA treatment (Fig. 5). These findings confirm that GA treatment promotes the accumulation of cell wall polysaccharides, including pectin, hemicellulose, and cellulose. In contrast, in the 6-BA treatment, genes involved in the synthesis of pectin and hemicellulose were downregulated (Fig. 5), suggesting that CTK slows the synthesis of these dynamic cell wall components. However, 6-BA treatment significantly upregulated genes involved in lignin synthesis, such as C3'H , CCoAOMT1 , F5H , PAL1 , C4H , COMT , CCR1 , and CAD1 (Fig. 5). These results indicate that the thickening of citrus peel induced by CTK is primarily due to the accumulation of lignin in the cell wall. 4.3. Both GA and 6-BA treatments downregulate fruit ripening-related cell wall degradation genes Several genes associated with cell wall degradation were downregulated in both the GA and 6-BA treatments, including PME3 , PG49 , PL18 , ASD1 , and EXPA1 / 8 (Fig. 5). PME3, a homolog of tomato ( Solanum lycopersicum ) PME ubiquitously 1 ( SLPmeu1 ), is associated with fruit ripening and softening in tomatoes [42]. PG49 is highly expressed in the tomato fruit pericarp and plays a pivotal role in fruit ripening [43]. PL18 , homologous to tomato SlPL , is involved in pectin depolymerization during tomato fruit ripening [28]. ASD1 , an α -L-arabinofuranosidase gene, shows expression patterns coincident with ripening in peach ( Prunus persica (L.) Batsch) and Japanese pear ( Pyrus pyrifolia ) [44, 45]. EXPA1 / 8 encodes expansins, which play a role in cell wall polymer disassembly, leading to fruit softening during ripening [10]. The downregulation of these genes suggests that both GA and 6-BA treatments delayed the ripening process of citrus fruit. This finding aligns with previous reports that both GA and CTK are known to delay fruit ripening [46, 47]. 4.4. Transcription factor involved in cell wall metabolism in the treatments of GA and 6-BA Several transcription factors were detected to be involved in cell wall metabolism. For example, under GA treatment, members of AP2/ERF-ERF family (such as RAP2-12 ) and the NAC transcription factor family were highly associated with the cell wall polysaccharides synthesis. In Arabidopsis research, four AP2/ERF-ERF genes found to directly up-regulate CESA genes, which encode cellulose synthase in primary cell wall [48]. In the 6-BA treatment, MYB transcription factor genes were highly associated with the lignin synthesis. MYB transcript factors belong to one of the largest superfamilies, and their roles in regulating cell wall formation have been widely confirmed. Among them, MYB46 and MYB83, which form the second layer of the main switch of secondary cell wall biosynthesis, coordinate upstream and downstream transcription factors related to secondary wall synthesis [49]. In fruits, such as jujube ( Ziziphus jujuba Mill.), MYBs have been shown to control lignin biosynthesis by regulating CCR and F5H [50]. Additionally, we identified several CAMTA family genes (such as CAMTA2 ) that were associated with fruit ripening-related cell wall degradation genes repressed by GA and 6-BA treatments (Fig. 7, Supplementary material 5 and 6). CAMTA2 is a hyper-DMR-associated gene. Previous research has confirmed that pear CAMTA2 acts as an inhibitor of fruit ripening, as demonstrated by overexpression in transgenic tomato fruits [51]. 4.5. GA treatment promoted endogenous CTK content and 6-BA treatment promoted endogenous GA content GA treatment increased endogenous CTK content (Fig. 4), and the CTK catabolic gene CKX5 was significantly downregulated in GA treatment (Fig. 5). Similar results were observed in a previous study on Brassica napus seedling development [27]. Conversely, 6-BA treatment also promoted endogenous GA content (Fig. 5). GA synthesis genes ( GA20ox and KAO ) were upregulated, while the GA catabolic gene ( GA2ox ) was downregulated in 6-BA treatment. Similar findings were reported in Brassica napus seedlings [27] and tomato fruits [52]. 5. Conclusion GA and 6-BA are growth regulators commonly used in production to promote fruit enlargement. In the present research, we found that citrus peels gradually thinned during fruit development, whereas the thickness of peels treated with GA and 6-BA was significantly greater than that of the CK control. The physiological data, microscopic observations, and RNA-sequencing data indicated that GA treatment promoted the synthesis of cell wall polysaccharides, particularly cellulose, while 6-BA treatment enhanced lignin synthesis in the cell wall. RNA-sequencing data also suggested that both GA and 6-BA may delay fruit ripening. The experimental results provide a theoretical basis for the use of GA and 6-BA to prevent fruit cracking in citrus production. Declarations Supplementary materials Supplementary materials associated with this article can be found, in the online version. Acknowledgements Not applicable. Author contributions XW designed the research and modified the manuscript. YPW analyzed the data and wrote the first draft. DFC performed software analysis and visualization of the data PPG performed the experiments and analyzed the data. MFZ and JXH provide experimental methods. BX, LL, and QFW provide experimental materials. GCS and SYH Assisted in managing the project. ZMX and ZHW supervised and guided the experimental design. Funding This work was supported by The National Key Research and Development Program of China (Grant No. 2023YFD2300600) and Yaan Science and Technology Program, China (Grant No. 23CGZH0004). Data availability The raw sequence data reported in this paper have been deposited in the NationalCenter for Biotechnology Information (NCBI) and the accession number is PRJNA1234498. Ethics approval and consent to participate The experimental research on plants performed in this study complies with institutional, national and international guidelines. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Li J, Chen J. Citrus fruit-cracking: causes and occurrence. Hortic Plant J. 2017;3(6):255–260. Li J. Cell wall metabolism and related gene isolation of pitting fruit peel in citrus. ProQuest Dissertations & Theses. 2009. Zhang B, Gao Y, Zhang L, Zhou Y. 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Differential expression of α- I -arabinofuranosidase and α- I -arabinofuranosidase/β- d -xylosidase genes during peach growth and ripening. Plant Physiol Biochem. 2009;47(7):562–569. Tateishi A, Mori H, Watari J, Nagashima K, Yamaki S, Inoue H. Isolation, characterization, and cloning of α-L-Arabinofuranosidase expressed during fruit ripening of Japanese pear. Plant Physiol. 2005;138(3):1653–1664. Huang H, He W. Application of exogenous cytokinin regulates cytokinin oxidase and antioxidant activity to maintain chlorophyll pigment during ripening of banana fruit. Food Biosci. 2023;55:102998. Reynolds A, Robbins N, Lee HS, Kotsaki E. Impacts and interactions of abscisic acid and gibberellic acid on sovereign coronation and skookum seedless table grapes. Am J Enol Viticult. 2016;67(3):327–338. Saelim L, Akiyoshi N, Tan TT, Ihara A, Yamaguchi M, Hirano K, Matsuoka M, Demura T, Ohtani M. Arabidopsis Group IIId ERF proteins positively regulate primary cell wall-type CESA genes. J Plant Res. 2019;132:117–129. Xiao R, Zhang C, Guo X, Li H, Lu H. MYB transcription factors and its regulation in secondary cell wall formation and lignin biosynthesis during xylem development. Int J Mol Sci. 2021;22(7):3560. Zhang Q, Wang L, Wang Z, Zhang R, Liu P, Liu M, Liu Z, Zhao Z, Wang L, Chen X, Xu H. The regulation of cell wall lignification and lignin biosynthesis during pigmentation of winter jujube. Hortic Res. 2021;8:238. Song B, Yu J, Li X, Li J, Fan J, Liu H, Wei W, Zhang L, Gu K, Liu D et al. Increased DNA methylation contributes to the early ripening of pear fruits during domestication and improvement. Genome Biol. 2024;25(1):87. Ding J, Chen B, Xia X, Mao W, Shi K, Zhou Y, Yu J. Cytokinin-induced parthenocarpic fruit development in tomato is partly dependent on enhanced gibberellin and auxin biosynthesis. PloS one. 2013;8(7):e70080. Additional Declarations No competing interests reported. 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01:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6059599/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6059599/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06705-5","type":"published","date":"2025-06-03T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80321544,"identity":"d79790ce-323d-448d-ab5e-67c0da63d04f","added_by":"auto","created_at":"2025-04-10 13:31:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":336876,"visible":true,"origin":"","legend":"\u003cp\u003eThe thickness of citrus peels. The photo illustrates the peels at DAT60.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/e871f9391e85a3be36d0a10f.png"},{"id":80319904,"identity":"c2663c3f-2bbd-4fa1-b630-3d9fe60f67d5","added_by":"auto","created_at":"2025-04-10 13:15:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31364,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative contents of cell wall materials in citrus peels.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/439341892438f2183a6eb16c.png"},{"id":80319910,"identity":"f1237128-e1b3-4da2-a335-f4d7045602df","added_by":"auto","created_at":"2025-04-10 13:15:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":591561,"visible":true,"origin":"","legend":"\u003cp\u003eThe microscopy observation of citrus peels in GA treatment, 6-BA treatment, and CK treatment. Cellulose was stained in cyan, and lignin was stained in magenta.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/5fac833eb314d977cae9ddbe.png"},{"id":80319903,"identity":"298173bd-3027-4b5a-a204-850934944527","added_by":"auto","created_at":"2025-04-10 13:15:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16244,"visible":true,"origin":"","legend":"\u003cp\u003eGibberellin and cytokinin contents in GA treated and 6-BA treated citrus peels. GA, gibberellin; CTK, cytokinin.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/d1f3d340aacc03b8718bc368.png"},{"id":80320841,"identity":"6915dd3f-8431-4ed6-9423-132cbe13cccd","added_by":"auto","created_at":"2025-04-10 13:23:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":97538,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of genes involved in cell wall metabolism and synthesis and signaling pathway of gibberellin and cytokinin. GA (I) and (II), stage I and II in GA treatment; 6-BA (I) and (II), stage I and II in 6-BA treatment; CK (I) and (II), stage I and II in CK treatment.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/7f9cfcbfec29c61e050a63e9.png"},{"id":80319908,"identity":"0fd6e127-4259-41a8-be9a-9f83b95f6be5","added_by":"auto","created_at":"2025-04-10 13:15:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":205325,"visible":true,"origin":"","legend":"\u003cp\u003eThe PAC scatter plots illustrating the spread of cell wall metabolic genes and transcription factor genes according to Hierarchical ClusteringAnalysis.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/6f68e1c3bb61608ed3033923.png"},{"id":80319907,"identity":"a79828d9-b319-490d-a12e-0f41cc9f9efd","added_by":"auto","created_at":"2025-04-10 13:15:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87285,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation between transcript factor genes and cell wall metabolic genes. Top ten transcript factor genes in Cluster 4 and 1 of GA treatment and in Cluster 1, 4, and 2 of 6-BA treatment are shown. The cell wall metabolic genes in magenta are differentially up-expressed genes comparing to CK, and that in cyan are differentially down-expressed genes.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/e7477bdaf5fb0d2302c6c242.png"},{"id":84242615,"identity":"a8345cd9-bcf4-4ef0-bbac-ec2df676f7b2","added_by":"auto","created_at":"2025-06-09 16:10:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1898729,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/03af98da-5e25-4668-a43a-8441f5aaccb5.pdf"},{"id":80319911,"identity":"491cf40f-6642-4b1c-acf4-83749f0f9e68","added_by":"auto","created_at":"2025-04-10 13:15:39","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":265104,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialsrevisededition.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6059599/v1/a19e445d7a9efb1ca676a63d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulatory effects of gibberellin and cytokinin on citrus peel cell wall metabolism","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs the citrus fruit develops, the fruit gradually expands and the peel becomes thinner. Some modern citrus cultivars have been bred to develop extremely thin peels for easy peeling. However, excessively thin peels may lead to fruit cracking during growth [1], resulting in production loss. Therefore, maintaining appropriate peel thickness is crucial for alleviating fruit cracking in citrus [2].\u003c/p\u003e\n\u003cp\u003ePeel thickening will result from increased cell wall material deposition. Fruit cell wall is composed of polysaccharides such as pectin, hemicellulose, and cellulose, and often contains glycoproteins and lignin [3]. Pectins play a central role in regulating the rheological properties of the cell wall, which are crucial for plant growth [4]. Pectin is composed of unbranched homogalacturonan (HG) and branched rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) [5]. Hemicellulose is a diverse group of polysaccharides, including xyloglucan (XyG) and xylan in dicot plants. XyG forms cross-links between cellulose microfibrils, creating a strong yet extensible XyG-cellulose network, which serves as the primary load-bearing structure of the cell wall [6]. Cellulose, a fibrous, tough, and water-insoluble substance, consists of unbranched \u003cem\u003eβ\u003c/em\u003e-(1,4)-linked glucan chains [7]. Lignin is an unordered polymer derived from phenylalanine, composed of aromatic monomers [8]. Lignin is a key component of both primary and secondary cell walls [9]. Expansins, which non-enzymatically relax the cell wall, play a critical role in cell expansion by loosening and softening the wall [10]. Fruit development is regulated by a variety of expansin genes, which show distinct expression patterns [11].\u003c/p\u003e\n\u003cp\u003ePhytohormones are essential to the regulation of fruit development and maturation. Ethylene and abscisic acid are generally associated with fruit ripening, while fruit growth (including in fruit cell division and expansion) involves other phytohormones, such as gibberellin (GA) and cytokinin (CTK) [12].\u0026nbsp;GA is extensively used in fruit production to induce parthenocarpic fruits, resulting in fruit sizes equal to or larger than those of pollinated fruits [13]. GA primarily promotes cell expansion during fruit development [13], and it also enhances cell division [14]. GA-induced thickening of fruit peels is associated with an increase in cellulose content in the cell walls [15]. Furthermore, studies in non-fruit plant organs have demonstrated that GA can enhance lignin deposition [16, 17] and increase the synthesis of cell wall polysaccharides, such as hemicellulose [18]. CTKs are a diverse group of plant growth regulators with wide-ranging effects on plant growth and development [19]. CTKs promote fruit enlargement through either cell division or expansion, thereby facilitating fruit development [19].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGA mediates its effects by overcoming the inhibition imposed by DELLA proteins in the GA signaling pathway [20]. DELLA proteins act as transcriptional regulators for numerous aspects of plant growth and development, including seed germination, flowering, hypocotyl hook formation, fruit development, and defense responses [20]. In contrast, B-type ARABIDOPSIS RESPONSE REGULATORS (Type-B RRs) are essential for the initial transcriptional response to CTK [21]. Type-B ARRs function as positive transcription factors in the CTK signaling pathway, regulating the expression of target genes involved in plant growth, development, and responses to abiotic stress [22]. The \u003cem\u003eArabidopsis\u003c/em\u003e genome contains 22 ARR genes, with ARR1, 2, and 10–14, as well as 18–21, classified as Type-B ARRs [23].\u003c/p\u003e\n\u003cp\u003eAntagonistic crosstalk between GA and CTK is a common feature in plant physiological processes [24]. In fruits, both GA and CTK promote growth, albeit through distinct mechanisms. GA primarily promotes cell expansion, whereas CTK enhances cell division [13, 25]. Brenner et al. found that CTK suppresses the expression of GA biosynthesis genes (\u003cem\u003eGA3ox\u003c/em\u003e and \u003cem\u003eGA20ox\u003c/em\u003e) while promoting the expression of DELLA genes (\u003cem\u003eGAI\u003c/em\u003e and \u003cem\u003eRGA\u003c/em\u003e) [26]. However, the interaction between GA and CTK is species- and tissue-dependent. For instance, [27] showed that in \u003cem\u003eBrassica napus\u003c/em\u003e seedling development, GA promotes CTK accumulation by modulating RGA-related CKX gene expression.\u003c/p\u003e\n\u003cp\u003eIn prior experiments, we confirmed the role of GA and 6-benzylaminopurine (6-BA, a synthetic CTK) in thickening citrus peels. However, the molecular mechanisms through which GA and 6-BA induce peel thickening, as well as the differences in their effects, remain unclear. In this study, we investigated the effects of GA and 6-BA on peel thickening in\u0026nbsp;Citrus Mandarin (\u003cem\u003eCitrus\u003c/em\u003e \u003cem\u003ereticulata\u003c/em\u003e Blanco) cultivar Asumi, a variety known for its relatively thin peels, making it an ideal model for studying peel thickening. Our objective was to elucidate the molecular mechanisms underlying GA- and 6-BA-induced peel thickening.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e2.1. Plant materials\u003c/p\u003e\n\u003cp\u003eThe study was conducted using four-year-old Citrus Mandarin (\u003cem\u003eCitrus\u003c/em\u003e \u003cem\u003ereticulata\u003c/em\u003e Blanco) cultivar Asumi trees, which were grafted in \u003cem\u003eCitrus\u003c/em\u003e \u003cem\u003ejunos\u003c/em\u003e rootstock. The experiment was carried out in Sichuan, China. The experimental trees exhibited uniform height, fruit-setting rates, and growth potential. All trees were healthy and free from pests and diseases. Three treatments were applied: gibberellin (GA), 6-benzylaminopurine (6-BA), and water\u0026nbsp;(as the control check, CK). Each treatment included five trees, with each tree serving as a biological replication.\u0026nbsp;The concentration of GA was 20 mg/L and 6-BA was 4 mg/L, which were determined in preliminary experiment (data not shown).\u0026nbsp;Spraying treatments were stated at the beginning of the fruit\u0026nbsp;enlargement period. The first spraying was\u0026nbsp;on Jul 20\u003csup\u003eth\u003c/sup\u003e, 2023.\u0026nbsp;Three applications were performed at approximately 10-day intervals.\u0026nbsp;All spraying was conducted in the morning on sunny days. The spray solution was applied evenly to the foliage and the abaxial surfaces of the leaves until visible water droplets formed without dripping.\u0026nbsp;The first sampling was conducted on the day of the first spraying treatment (July 20\u003csup\u003eth\u003c/sup\u003e), when was at the beginning of the fruit expansion stage, designated\u0026nbsp;as DAT0 (0 day after treatment). The second sampling was conducted 30 days after the first\u0026nbsp;spraying treatment (August 20\u003csup\u003eth\u003c/sup\u003e), when was at the mid-stage of fruit expansion, designated\u0026nbsp;as DAT30. The third sampling was conducted 60 days after the first\u0026nbsp;spraying treatment (September 20\u003csup\u003eth\u003c/sup\u003e), when was at the end of the fruit expansion period,\u0026nbsp;designated\u0026nbsp;as DAT60.\u0026nbsp;Fruits were harvested from the periphery of the tree canopy in four directions: southeast, southwest, northwest, and north. Approximately 10 fruits were collected per tree.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.2. Measurement of citrus peel thickness\u003c/p\u003e\n\u003cp\u003eThe pericarp thickness was measured using a vernier caliper. Each fruit was longitudinally cut into two halves along the equatorial plane using a sharp knife. The thickness of the pericarp at the equatorial region, specifically at the point where the vesicle lobes contact the pericarp, was measured. Measurement was taken three times for each of the ten fruits to ensure accuracy and consistency.\u003c/p\u003e\n\u003cp\u003e2.3. Determination of pectin, hemicellulose, cellulose, and lignin\u003c/p\u003e\n\u003cp\u003eThe rind and pulp of the collected samples were separated, and citrus peels were dried in an oven at 60 °C. The dried samples were then ground into a fine powder. To extract the cell wall material, the peels were sequentially treated with ethanol, dimethyl sulfoxide, chloroform-methanol, and acetone. The extracted cell wall material was subsequently used to determine the relative contents of pectin, cellulose, hemicellulose, and lignin. The water-soluble pectin (WSP) and protopectin were quantified by colorimetry using a carbazole-vitriol method\u0026nbsp;[28]. The relative content of cellulose was determined by an anthrone colorimetric method [29]. Hemicellulose content was measured using the phenol-sulfuric acid method\u0026nbsp;[29], and lignin content was determined using the acetyl bromide method\u0026nbsp;[30].\u003c/p\u003e\n\u003cp\u003e2.4. Microscopic observation of cellulose and lignin\u003c/p\u003e\n\u003cp\u003eCitrus peels sampled on the third time (September 20, 2023) were used for microscopic observation of cellulose and lignin. Fresh samples from the equatorial region of fruits were cut into pieces measuring 1 cm × 1 cm. The cut samples were immediately fixed in FAA solution (formaldehyde: 5%, glacial acetic acid: 6%, ethanol: 70%) for more than 24 hours. The fixed samples were embedded in paraffin and sectioned perpendicular to the pericarp surface into slices with a thickness of 4 μm. Cellulose and lignin in the tissues were stained using Solid Green Staining and Safranin-O Staining, respectively. The stained sections were observed under an orthogonal fluorescence microscope (Eclipse Ci, Nikon, Japan). The microstructural areas of lignin and cellulose were analyzed using Image-Pro Plus 6.0 software. To quantify cellulose and lignin, the ratio of the Solid Green Staining stained area (cellulose) and the Safranin-O stained area (lignin) of a 200 μm × 180 μm, regarded as an unit area, was calculated. Six unit-areas were randomly selected for analysis, with each serving as a replicate.\u003c/p\u003e\n\u003cp\u003e2.5. Determination of hormone content\u003c/p\u003e\n\u003cp\u003eThe GA and CTK concentrations were determined by double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) kit (CamaiShu Biotech, China). Specimen, standard and HRP-labeled detection antibody were added into the microtiter wells pre-coated with GA and CTK antibodies. After incubation and thorough washing, the wells were color-developed with the substrate Tetramethylbenzidine (TMB), which is converted to blue by peroxidase and to final yellow by acid. The shade of color is positively correlated with GA and CTK in samples. The absorbance (OD) was measured at 450 nm using an enzyme meter (SpectraMax M2, Molecular Devices, US) and the concentration of samples was calculated. The correlation coefficient R value for the linear regression of the standard should be greater than or equal to 0.9900 compared to the expected concentration. The minimum detectable concentration should be less than 1.0 pg/mL for GA determination and 1.0 pmol/mL for CTK determination. Three replicates were done for each sample.\u003c/p\u003e\n\u003cp\u003e2.6. Analysis of transcriptome\u0026nbsp;sequencing\u003c/p\u003e\n\u003cp\u003eCitrus peels sampled on the second time (Aug 20, 2023) and third time (Sep 20, 2023) were used for transcriptome sequencing analysis, which were designated to be Stage I and Stage II. The citrus peels were rapidly frozen in liquid nitrogen for transcriptome sequencing. Transcriptome sequencing was performed using the Illumina NovaSeq6000 sequencing platform (Biomarker Technologies Co, Ltd.). The Raw data obtained was filtered and aligned with the reference genome Citrus_reticulata.Mangshan.v1.0.genome.fa using a HISAT2 software. A StringTie software was used to assemble the aligned reads [31]. Based on the Count values of genes in each sample, differential expression gene (DEG) screening was carried out using a DESeq2 software [32]. During the detection of differentially expressed genes, a fold change of ≥ 1.5 and a false discovery rate (FDR) \u0026lt; 0.01 were used as the screening criteria. A Hierarchical Clustering Analysis of cell wall metabolic genes and transcription factors was performed using R language. K-means was used for verification. The Factoextra and Cluster packages were used to draw visual scatter plots.\u003c/p\u003e\n\u003cp\u003e2.7. RNA extraction and Real-time quantitative PCR (RT-qPCR) analysis\u003c/p\u003e\n\u003cp\u003eAnother citrus peel sample was ground into fine powder in liquid nitrogen and subjected to total RNA extraction and RT-qPCR. RNA was extracted using total RNA extraction kit (RNAsinple Total RNA Kit, Tiangen Biotech, China). For analysis of mRNA transcription levels, 1 μg total RNA was reverse transcribed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311, TransGen Biotech, China). The RT-qPCR was performed on a\u0026nbsp;CFX96 Touch Real-Time PCR Detection System (Bio-Rad, US) using PerfectStart® Green qPCR SuperMix (Transgen, China). Relative gene expression levels were calculated using the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method. Three replicates in each were performed. Actin gene was the reference gene. The primers for RT-qPCR were in Supplementary material 1.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. Citrus peel thickness in GA and 6-BA treatments\u003c/p\u003e\n\u003cp\u003eDuring fruit ripening, the peel thickness gradually decreased in both GA, 6-BA, and CK controls (Fig. 1). The peel thickness in both GA and 6-BA treatments was significantly greater than that in the CK control on DAT30, with the difference further increasing by DAT60 (Fig. 1).\u003c/p\u003e\n\u003cp\u003e3.2. Cell wall material contents in GA and 6-BA treatments\u003c/p\u003e\n\u003cp\u003eThe relative content of insoluble pectin in GA and 6-BA treatments was significantly higher compared to the CK control on DAT60 (Fig. 2). The relative content of water-soluble pectin increased during fruit ripening, whereas it was lower in both GA and 6-BA treatments than in the CK control. An increase in hemicellulose content was observed in the GA treatment on DAT30 compared to the CK control. The relative cellulose content in GA treatment was significantly higher than in the CK control on DAT60. Lignin content in the 6-BA treatment increased on both DAT30 and DAT60.\u003c/p\u003e\n\u003cp\u003eMicroscopic analysis of paraffin sections revealed the accumulation of cellulose and lignin on DAT60 (Fig. 3). In the GA treatment, cellulose stained with Solid Green Staining (cyan) was more abundant than in the CK control. Image-Pro Plus analysis of the cyan area revealed a significantly larger area in the GA treatment compared to the CK control. In contrast, in the 6-BA treatment, lignin stained with Safranin-O Staining (magenta) was more abundant than in the CK control. The Image-Pro Plus analysis also indicated that the magenta area was significantly larger in the 6-BA treatment than in the CK control.\u003c/p\u003e\n\u003cp\u003e3.3. Plant hormone contents in GA and 6-BA treatments\u003c/p\u003e\n\u003cp\u003eThe contents of GA and CTK were measured. The GA content in citrus peels was elevated in both the GA and 6-BA treatments on DAT30 (Fig. 4). The GA content remained significantly higher in both GA and 6-BA treatments compared to the CK control on DAT60. Additionally, CTK content in citrus peels increased in 6-BA treatment on DAT30. On DAT60, CTK content was also elevated in the GA treatment, whereas it decreased in the 6-BA treatment.\u003c/p\u003e\n\u003cp\u003e3.4. Differentially expressed genes (DEGs) involved in cell wall metabolism\u003c/p\u003e\n\u003cp\u003eAs citrus peels were significantly thickened\u0026nbsp;Aug 20, 2023 and Sep 20, 2023, transcriptome\u0026nbsp;sequencing\u0026nbsp;was performed using the peels sampled on the two stages,\u0026nbsp;designated to be Stage I and Stage II. The RT-qPCR results that validated the results of\u0026nbsp;transcriptome were in\u0026nbsp;Supplementary material\u0026nbsp;2.\u0026nbsp;The DEGs from\u0026nbsp;transcriptome\u0026nbsp;sequencing in GA(I) vs CK(I) and 6-BA(I) vs CK(I) were 5316 and 4230, respectively, while in GA(II) vs CK(II) and 6-BA(II) vs CK(II), the DEGs were 2231 and 1206, respectively. These results indicate that the majority of differential gene expression in response to GA or 6-BA occurs at stage I, implying a mechanism of phenotype delay to genotype. Therefore, the gene expression at this stage was further analyzed.\u003c/p\u003e\n\u003cp\u003eGenes involved in cell wall metabolism (with average FPKMs \u0026gt; 10) were screened (Supplementary material 3), including those related to pectin HG synthesis, pectin RG-I/II synthesis, hemicellulose (XyG) synthesis, cellulose synthesis, and lignin synthesis. Additionally, genes involved in cell wall degradation were examined, including pectinesterase/pectin methyl‐esterase, polygalacturonase, pectate lyase, \u003cem\u003eβ\u003c/em\u003e-galactosidase, arabinosyltransferase, endotransglucosylase/hydrolase, and expansin proteins (Supplementary material 3). A total of 43 genes were identified (Fig. 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the GA treatment (stage I), compared to the CK control, pectin HG synthesis genes, including galacturonosyltransferases \u003cem\u003eGAUT8\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e/\u003cem\u003e7\u003c/em\u003e, were upregulated, with \u003cem\u003eGAUT8\u003c/em\u003e showing a significantly higher transcription level. The pectin RG synthesis gene rhamnogalacturonan I rhamnosyltransferase (\u003cem\u003eRRT1\u003c/em\u003e) was also upregulated. The XyG metabolic genes generally showed increased expression, such as XyG glycosyltransferase (\u003cem\u003eCSLC12\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e), XyG 6-xylosyltransferase (\u003cem\u003eXXT2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e), XyG galactosyltransferase (\u003cem\u003eMUR3\u003c/em\u003e), fucosyltransferase (\u003cem\u003eMUR2\u003c/em\u003e/\u003cem\u003eFUT1\u003c/em\u003e), and XyG galactosyltransferase (\u003cem\u003eXLT2\u003c/em\u003e). All cellulose synthesis genes were upregulated, including cellulose synthase (\u003cem\u003eCESA1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e) and CESA-like D3 (\u003cem\u003eCSLD3\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the 6-BA treatment (stage I), the abovementioned genes involved in cell wall polysaccharide synthesis were mostly downregulated compared to the CK control, while lignin synthesis genes were upregulated, including p-coumaroylshikimate 3′-hydroxylase (\u003cem\u003eC3’H\u003c/em\u003e), caffeoyl-CoA O-methyltransferase (\u003cem\u003eCCoAOMT1\u003c/em\u003e), ferulate 5-hydroxylase (\u003cem\u003eF5H\u003c/em\u003e), phenylalanine ammonia-lyase (\u003cem\u003ePAL1\u003c/em\u003e), cinnamate 4-hydroxylase (\u003cem\u003eC4H\u003c/em\u003e), caffeic acid O-methyltransferase (\u003cem\u003eCOMT\u003c/em\u003e), cinnamoyl-CoA reductase (\u003cem\u003eCCR1\u003c/em\u003e), and cinnamyl alcohol dehydrogenase (\u003cem\u003eCAD1\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral genes involved in fruit cell wall degradation were significantly downregulated in both GA and 6-BA treatments, such as pectin methyl‐esterase (\u003cem\u003ePME3\u003c/em\u003e), polygalacturonase (\u003cem\u003ePG49\u003c/em\u003e), pectate lyase (\u003cem\u003ePL18\u003c/em\u003e), arabinosyltransferase (\u003cem\u003eASD1\u003c/em\u003e), and expansin (\u003cem\u003eEXPA1\u003c/em\u003e/\u003cem\u003e8\u003c/em\u003e). In contrast, other genes, such as \u003cem\u003ePME1\u003c/em\u003e, \u003cem\u003ePG52\u003c/em\u003e, and XyG endotransglucosylase/hydrolase (\u003cem\u003eXTH23\u003c/em\u003e), were upregulated.\u003c/p\u003e\n\u003cp\u003e3.5. Differentially expressed genes involved in plant hormone synthesis and signaling pathways\u003c/p\u003e\n\u003cp\u003eGenes involved in GA and 6-BA synthesis and signaling pathways were analyzed (Fig. 5, Supplementary material 4). In the GA treatment (stage I), the GA synthesis gene \u003cem\u003eGA20ox2\u003c/em\u003e decreased, while the catabolic gene \u003cem\u003eGA2ox1\u003c/em\u003e increased compared to the CK control. Additionally, \u003cem\u003eCKX5\u003c/em\u003e (which downregulates CTK levels) was significantly reduced. In the 6-BA treatment (stage I), the expression of GA synthesis gene \u003cem\u003eGA20ox2\u003c/em\u003e decreased, and \u003cem\u003eGA2ox1\u003c/em\u003e increased compared to the CK control, similar to the GA treatment (stage I). However, in the 6-BA treatment (stage II), the GA synthesis genes \u003cem\u003eKAO\u003c/em\u003e and \u003cem\u003eGA20ox2\u003c/em\u003e were upregulated, while \u003cem\u003eGA2ox1\u003c/em\u003e was downregulated compared to the CK control. In the signaling transduction pathway, all identified genes in the GA signaling pathway were downregulated in the GA treatment compared to the CK control, including \u003cem\u003eGID1B\u003c/em\u003e/\u003cem\u003eC\u003c/em\u003e, \u003cem\u003eGAI\u003c/em\u003e, \u003cem\u003eRGA\u003c/em\u003e, and \u003cem\u003eGID2\u003c/em\u003e. In the 6-BA treatment, genes in the CTK signaling pathway, such as \u003cem\u003eAHK3\u003c/em\u003e, \u003cem\u003eAHP5\u003c/em\u003e, and B-type ARR (\u003cem\u003eARR1\u003c/em\u003e), were upregulated compared to the CK control.\u003c/p\u003e\n\u003cp\u003e3.6. Transcription factors associated with cell wall metabolism-related genes\u003c/p\u003e\n\u003cp\u003eA total of 803 transcription factor (TF) genes were identified in the transcriptome data. Of these, 445 and 457 genes (with average FPKMs \u0026gt; 10) were significantly connected with cell wall metabolic genes in the GA and 6-BA treatments, respectively. Cluster analysis using the Hierarchical Clustering method was performed based on correlation analysis. In the GA treatment, the connected TF genes and cell wall metabolic genes were divided into six clusters (Fig. 6). Cell wall degradation genes were mostly located in cluster 4 (Fig. 6). Several TFs that were negatively correlated with multiple cell wall degradation genes were identified in this cluster, including \u003cem\u003eNFYB8\u003c/em\u003e, \u003cem\u003eJAZ10\u003c/em\u003e, \u003cem\u003eAPC8\u003c/em\u003e, and \u003cem\u003eNAC017\u003c/em\u003e (Fig. 7; Supplementary material 5). Genes involved in cell wall polysaccharide (pectin, XyG, and cellulose) synthesis were generally placed in cluster 1 of the GA treatment (Fig. 6), where TFs such as \u003cem\u003eEJ2\u003c/em\u003e, \u003cem\u003eRAP2-12\u003c/em\u003e, and \u003cem\u003eANL2\u003c/em\u003e were positively associated with several cell wall polysaccharide synthesis genes (Fig. 7; Supplementary material 5).\u003c/p\u003e\n\u003cp\u003eIn the 6-BA treatment, the connected TF genes and cell wall metabolic genes were similarly divided into six clusters (Fig. 6). Cell wall degradation genes were mostly located in cluster 1 (Fig. 6), where several TFs, including \u003cem\u003eSPL1\u003c/em\u003e, \u003cem\u003eEIN3\u003c/em\u003e, \u003cem\u003eCAMTA2\u003c/em\u003e, and \u003cem\u003eNAC017\u003c/em\u003e, were negatively correlated with multiple cell wall degradation genes (Fig. 7; Supplementary material 6). The genes involved in lignin synthesis were primarily clustered in cluster 4 of the 6-BA treatment (Fig. 6), where TFs such as \u003cem\u003eBLH7\u003c/em\u003e, \u003cem\u003eHAT5\u003c/em\u003e, \u003cem\u003eCOL4\u003c/em\u003e, \u003cem\u003eWRKY15\u003c/em\u003e, and \u003cem\u003eARR1\u003c/em\u003e were positively associated with several lignin synthesis genes (Fig. 7). In contrast, genes involved in cell wall polysaccharide synthesis were generally placed in cluster 2 (Fig. 6), where many TFs, such as \u003cem\u003eNAC047\u003c/em\u003e, \u003cem\u003eDF1\u003c/em\u003e, \u003cem\u003eTCF13\u003c/em\u003e, and \u003cem\u003eARR2\u003c/em\u003e, were negatively correlated with multiple cell wall polysaccharide synthesis genes (Fig. 7; Supplementary material 6).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e4.1. GA promoted pectin and cellulose accumulation, while 6-BA enhanced lignin accumulation\u003c/p\u003e\n\u003cp\u003eGA and CTK are key phytohormones involved in fruit development, promoting both cell expansion and division [19, 25]. The synthesis of cell wall materials supports cell division and expansion, contributing to fruit enlargement. Previous studies have shown that GA increases cellulose content, thereby strengthening the fruit peel [15]. Although the effects of CTK on the cell wall material of fruit peel are less well understood, it is hypothesized that CTK primarily promotes lignin accumulation, which contributes to plant organ growth, as observed in studies on carrot (\u003cem\u003eDaucus carota\u003c/em\u003e) taproots [33] and bamboo (\u003cem\u003ePhyllostachys nigra\u003c/em\u003e) suspension cells [34]. In the present study, as citrus fruits developed, the fruit peel gradually thinned. However, the peel was significantly thicker in the GA and 6-BA (a synthetic CTK) treatments compared to the control. In the GA treatment, the cell wall materials of the peel increased overall, particularly in the relative contents of pectin and cellulose. In contrast, the 6-BA treatment significantly elevated the relative content of lignin.\u003c/p\u003e\n\u003cp\u003e4.2. GA treatment upregulated cell wall polysaccharide synthesis genes, while 6-BA treatment enhanced lignin synthesis genes\u003c/p\u003e\n\u003cp\u003eIn the present study, a set of genes involved in cell wall metabolism was identified (Fig. 5). The GAUT family is responsible for synthesizing pectin homogalacturonan (HG) [35]. Rhamnose transferases (RRTs) facilitate the addition of rhamnose residues to RG-I oligosaccharides [36]. XXT1/2/5 are involved in the first step of side-chain formation in xyloglucan (XyG) [37]. XLT2 and MUR3 add galactose to the second and third xylosyl residues, respectively, of XyG side chains [38]. MUR2/FUT1 adds terminal fucose to the XyG side chain [39]. Cellulose synthase complexes (CSCs), consisting of CESA1, CESA3, and other CESA isoforms, catalyze cellulose glucan chain elongation and are essential for primary cell wall cellulose synthesis [40]. The CSLD gene family, similar to CESA, is implicated in cellulose synthesis [41]. In this study, GA treatment significantly upregulated genes encoding these enzymes, including \u003cem\u003eGAUT8\u003c/em\u003e, \u003cem\u003eRRT1\u003c/em\u003e, \u003cem\u003eXXT2\u003c/em\u003e (homologous to \u003cem\u003eAtXXT1\u003c/em\u003e), \u003cem\u003eXXT3\u003c/em\u003e (homologous to \u003cem\u003eAtXXT5\u003c/em\u003e), \u003cem\u003eXLT2\u003c/em\u003e, \u003cem\u003eMUR3\u003c/em\u003e, \u003cem\u003eMUR2\u003c/em\u003e, \u003cem\u003eCESA1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e, and \u003cem\u003eCSLD3\u003c/em\u003e (Fig. 5). Additionally, genes encoding cell wall-modifying enzymes, such as \u003cem\u003ePME1\u003c/em\u003e, \u003cem\u003ePG52\u003c/em\u003e, and \u003cem\u003eXTH23\u003c/em\u003e, which promote the restructuring of pectin and hemicellulose to support new cell wall synthesis [4;6], were also upregulated in GA treatment (Fig. 5). These findings confirm that GA treatment promotes the accumulation of cell wall polysaccharides, including pectin, hemicellulose, and cellulose.\u003c/p\u003e\n\u003cp\u003eIn contrast, in the 6-BA treatment, genes involved in the synthesis of pectin and hemicellulose were downregulated (Fig. 5), suggesting that CTK slows the synthesis of these dynamic cell wall components. However, 6-BA treatment significantly upregulated genes involved in lignin synthesis, such as \u003cem\u003eC3'H\u003c/em\u003e, \u003cem\u003eCCoAOMT1\u003c/em\u003e, \u003cem\u003eF5H\u003c/em\u003e, \u003cem\u003ePAL1\u003c/em\u003e, \u003cem\u003eC4H\u003c/em\u003e, \u003cem\u003eCOMT\u003c/em\u003e, \u003cem\u003eCCR1\u003c/em\u003e, and \u003cem\u003eCAD1\u003c/em\u003e (Fig. 5). These results indicate that the thickening of citrus peel induced by CTK is primarily due to the accumulation of lignin in the cell wall.\u003c/p\u003e\n\u003cp\u003e4.3. Both GA and 6-BA treatments downregulate fruit ripening-related cell wall degradation genes\u003c/p\u003e\n\u003cp\u003eSeveral genes associated with cell wall degradation were downregulated in both the GA and 6-BA treatments, including \u003cem\u003ePME3\u003c/em\u003e, \u003cem\u003ePG49\u003c/em\u003e, \u003cem\u003ePL18\u003c/em\u003e, \u003cem\u003eASD1\u003c/em\u003e, and \u003cem\u003eEXPA1\u003c/em\u003e/\u003cem\u003e8\u003c/em\u003e (Fig. 5). PME3, a homolog of tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) PME ubiquitously 1 (\u003cem\u003eSLPmeu1\u003c/em\u003e), is associated with fruit ripening and softening in tomatoes [42]. \u003cem\u003ePG49\u003c/em\u003e is highly expressed in the tomato fruit pericarp and plays a pivotal role in fruit ripening [43]. \u003cem\u003ePL18\u003c/em\u003e, homologous to tomato \u003cem\u003eSlPL\u003c/em\u003e, is involved in pectin depolymerization during tomato fruit ripening [28]. \u003cem\u003eASD1\u003c/em\u003e, an \u003cem\u003eα\u003c/em\u003e-L-arabinofuranosidase gene, shows expression patterns coincident with ripening in peach (\u003cem\u003ePrunus\u003c/em\u003e \u003cem\u003epersica\u003c/em\u003e (L.) Batsch) and Japanese pear (\u003cem\u003ePyrus\u003c/em\u003e \u003cem\u003epyrifolia\u003c/em\u003e) [44, 45]. \u003cem\u003eEXPA1\u003c/em\u003e/\u003cem\u003e8\u003c/em\u003e encodes expansins, which play a role in cell wall polymer disassembly, leading to fruit softening during ripening [10]. The downregulation of these genes suggests that both GA and 6-BA treatments delayed the ripening process of citrus fruit. This finding aligns with previous reports that both GA and CTK are known to delay fruit ripening [46, 47].\u003c/p\u003e\n\u003cp\u003e4.4. Transcription factor involved in cell wall metabolism in the treatments of GA and 6-BA\u003c/p\u003e\n\u003cp\u003eSeveral transcription factors were detected to be involved in cell wall metabolism. For example, under GA treatment, members of AP2/ERF-ERF family (such as\u0026nbsp;\u003cem\u003eRAP2-12\u003c/em\u003e) and the NAC\u0026nbsp;transcription factor\u0026nbsp;family were highly associated with the cell wall polysaccharides synthesis. In \u003cem\u003eArabidopsis\u003c/em\u003e research, four AP2/ERF-ERF genes found to directly up-regulate CESA genes, which encode cellulose synthase in primary cell wall [48]. In the 6-BA treatment, MYB\u0026nbsp;transcription factor genes were highly associated with the lignin\u0026nbsp;synthesis. MYB transcript factors belong to one of the largest superfamilies, and their roles in regulating cell wall formation have been widely confirmed. Among them, MYB46 and MYB83, which form the second layer of the main switch of secondary cell wall biosynthesis, coordinate upstream and downstream transcription factors related to secondary wall synthesis [49]. In fruits, such as jujube (\u003cem\u003eZiziphus jujuba\u003c/em\u003e Mill.), MYBs have been shown to control lignin biosynthesis by regulating CCR and F5H [50]. Additionally, we identified several CAMTA family genes (such as \u003cem\u003eCAMTA2\u003c/em\u003e) that were associated with fruit ripening-related cell wall degradation genes repressed by GA and 6-BA treatments (Fig. 7,\u0026nbsp;Supplementary material 5 and 6). CAMTA2 is a hyper-DMR-associated gene. Previous research has confirmed that pear \u003cem\u003eCAMTA2\u003c/em\u003e acts as an inhibitor of fruit ripening, as demonstrated by overexpression in transgenic tomato fruits\u0026nbsp;[51].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.5. GA treatment promoted endogenous CTK content and 6-BA treatment promoted endogenous GA content\u003c/p\u003e\n\u003cp\u003eGA treatment increased endogenous CTK content (Fig. 4), and the CTK catabolic gene \u003cem\u003eCKX5\u003c/em\u003e was significantly downregulated in GA treatment (Fig. 5). Similar results were observed in a previous study on \u003cem\u003eBrassica napus\u003c/em\u003e seedling development [27]. Conversely, 6-BA treatment also promoted endogenous GA content (Fig. 5). GA synthesis genes (\u003cem\u003eGA20ox\u003c/em\u003e and \u003cem\u003eKAO\u003c/em\u003e) were upregulated, while the GA catabolic gene (\u003cem\u003eGA2ox\u003c/em\u003e) was downregulated in 6-BA treatment. Similar findings were reported in \u003cem\u003eBrassica napus\u003c/em\u003e seedlings [27] and \u003cem\u003etomato\u003c/em\u003e fruits [52].\u0026nbsp;\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eGA and 6-BA are growth regulators commonly used in production to promote fruit enlargement. In the present research, we found that citrus peels gradually thinned during fruit development, whereas the thickness of peels treated with GA and 6-BA was significantly greater than that of the CK control. The physiological data, microscopic observations, and RNA-sequencing data indicated that GA treatment promoted the synthesis of cell wall polysaccharides, particularly cellulose, while 6-BA treatment enhanced lignin synthesis in the cell wall. RNA-sequencing data also suggested that both GA and 6-BA may delay fruit ripening. The experimental results provide a theoretical basis for the use of GA and 6-BA to prevent fruit cracking in citrus production.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary materials associated with this article can be found, in the online version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXW designed the research and\u0026nbsp;modified the manuscript. YPW analyzed the data and wrote the first draft. DFC performed software analysis and visualization of the data PPG performed the experiments and analyzed the data. MFZ and JXH provide experimental methods. BX, LL, and QFW provide experimental materials. GCS and SYH Assisted in managing the project. ZMX and ZHW supervised and guided the experimental design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by The National Key Research and Development Program of China (Grant No. 2023YFD2300600) and Yaan Science and Technology Program, China (Grant No. 23CGZH0004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the NationalCenter for Biotechnology Information (NCBI) and the accession number is PRJNA1234498.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental research on plants performed in this study complies with institutional, national and international guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi J, Chen J. 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Cytokinin-induced parthenocarpic fruit development in tomato is partly dependent on enhanced gibberellin and auxin biosynthesis. PloS one. 2013;8(7):e70080.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"citrus peels, cell wall, gibberellin, cytokinin, DELLAs, B-ARRs","lastPublishedDoi":"10.21203/rs.3.rs-6059599/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6059599/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"During citrus fruit development, exogenous gibberellin (GA) and 6-benzylaminopurine (6-BA, a synthetic cytokinin (CTK)) are both known to promote citrus peel thickness; however, the differences in their regulatory mechanisms on cell wall metabolism in citrus peels remain unclear. In this study, we found that GA treatment significantly increased cell wall polysaccharides in citrus peels, such as pectin and cellulose, whereas 6-BA treatment led to a notable accumulation of lignin. RNA-sequencing data revealed that several fruit ripening-related cell wall degradation genes, such as PME3, PL18, and EXPA1/8, exhibited decreased expression levels in both GA and 6-BA treatments. Additionally, a set of cell wall polysaccharide synthesis genes was upregulated in response to GA treatment but was largely downregulated in 6-BA-treated peels. Conversely, a group of lignin biosynthesis genes was upregulated in 6-BA-treated peels. GA treatment inhibited DELLA proteins (encoded by RGA and GAI) in the GA signaling pathway, whereas 6-BA treatment increased the expression of B-ARRs (ARR1 and ARR2) in the CTK signaling pathway. Furthermore, GA treatment elevated endogenous CTK levels, while 6-BA treatment also enhanced endogenous GA content, suggesting a reciprocal interaction between these two hormonal pathways.","manuscriptTitle":"Regulatory effects of gibberellin and cytokinin on citrus peel cell wall metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 13:15:34","doi":"10.21203/rs.3.rs-6059599/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-22T20:38:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T09:19:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-10T06:21:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134035494953728386014295780848766436318","date":"2025-04-08T12:26:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T01:10:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51228771307117542362274814296223648562","date":"2025-04-07T01:00:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123886742767976091232569937542233240647","date":"2025-04-06T07:03:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-06T01:42:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-05T04:30:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-04-02T15:06:38+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":"201382e6-cace-47c2-b491-d0eb009605d5","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:03:59+00:00","versionOfRecord":{"articleIdentity":"rs-6059599","link":"https://doi.org/10.1186/s12870-025-06705-5","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-06-03 15:57:46","publishedOnDateReadable":"June 3rd, 2025"},"versionCreatedAt":"2025-04-10 13:15:34","video":"","vorDoi":"10.1186/s12870-025-06705-5","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06705-5","workflowStages":[]},"version":"v1","identity":"rs-6059599","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6059599","identity":"rs-6059599","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}