{"paper_id":"1d4d73f9-7030-4efc-aa51-bb98b8a9fa7e","body_text":"A multilevel investigation to reveal the regulatory mechanism of lignin accumulation in juice sac granulation of pomelo | 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 A multilevel investigation to reveal the regulatory mechanism of lignin accumulation in juice sac granulation of pomelo Luning LIU, YiRan Chen, Weilin Wu, Qiuyou Chen, ZhiJiao Tian, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3958230/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 May, 2024 Read the published version in BMC Plant Biology → Version 1 posted 8 You are reading this latest preprint version Abstract Granulation of juice sacs is a physiological disorder, which affects pomelo fruit quality. Here, the transcriptome and ubiquitinome of the granulated juice sacs were analyzed in Guanxi pomelo. We found that lignin accumulation in the granulated juice sacs was regulated at transcription and protein modification levels. In transcriptome data, we found that the genes in lignin biosynthesis pathway and antioxidant enzyme system of the granulated juice sacs were significantly up-regulated. However, in ubiquitinome data, we found that ubiquitinated antioxidant enzymes increased in abundance but the enzyme activities decreased after the modification, which gave rise to reactive oxygen species (ROS) contents in granulated juice sacs. This finding suggests that ubiquitination level of the antioxidant enzymes is negatively correlated with the enzyme activities. Increased H 2 O 2 is considered to be a signalling molecule to activate the key gene expressions in lignin biosynthesis pathway, which leads to the lignification in granulated juice sacs of pomelo. This regulatory mechanism in juice sac granulation of pomelo was further confirmed through the verification experiment using tissue culture by adding H 2 O 2 or dimethylthiourea (DMTU). Our findings suggest that scavenging H 2 O 2 and other ROS are important for reducing lignin accumulation, alleviating juice sac granulation and improving pomelo fruit quality. Antioxidant enzyme H2O2 juice sac granulation lignin pomelo ROS transcriptome ubiquitinome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Pomelo is a citrus fruit, native to China and now widely grown around the world, with high nutritional value [1]. Juice sac granulation, one of the serious physiological disorders in citrus fruits, occurs during harvest and post-harvest storage of the fruits. The occurrence of pomelo juice sac granulation degrades fruit quality [2]. Recently, there have been several transcriptome researches regard to the juice sac granulation of citrus. Yao et al. [3] reported that the changes in gene expressions based on the transcriptome datasets in the metabolism of sugar and organic acid might be related to juice sac granulation by modulating cell wall components in post-harvest Ponkan ( Citrus reticulata Blanco cv. Ponkan). In addition, Yao et al. [4] revealed the molecular basis of the granulation of juice sacs in navel orange ( Citrus sinensis L. Osbeck) through the analyses of metabolome, transcriptome and methylome profilings. Kang et al. [5] indicated that the accumulated cell wall components such as lignin, cellulose and protopectins were closely related to the juice sac granulation by comparing the transcriptome profiles and physiological properties in different juice sacs of Huyou fruit ( Citrus changshanensis ). Ubiquitination is a crucial post-translational modification (PTM) of protein, which is important for regulating protein localization, functions and interactions in biological cells [6]. Previous proteomic investigations indicate that tens-of-thousands of ubiquitination sites on thousands of proteins are identified. It appears that most proteins will be ubiquitinated at some point in their cellular lifetime. The degradation of large number proteins in cells are controlled by ubiquitin-proteasome system [7]. The increasing evidence shows that protein ubiquitination involves virtually all cellular processes and almost all events in the entire life cycle of plants [8]. Lu et al. [9] reported the ubiquitinome of senescing rose petals were changed, suggesting that protein degradation and autophagy, transport of substances and brassinosteroid signaling played important roles in petal senescence. He et al. [10] found that protein ubiquitination played a widely regulating role in rice seed germination by analyzing the ubiquitinome profiles. Cheng et al. [11] highlighted that protein ubiquitination, especially the proteins related to primary metabolism, involved in responding to post-harvest pathogen infection of sweet orange fruit. Furthermore, several investigations reported that protein ubiquitination played crucial roles in fruit ripening and quality. The degradation of ubiquitinated MADS-box proteins was involved in modulating fruit development and ripening of bananas [12]. Ubiquitinated MdbHLH3 regulated apple fruit ripening and quality by promoting ethylene biosynthesis [13]. Auxin receptor TIR1, as positive regulator of auxin signaling, coordinated tomato fruit development and ripening by depredating auxin repressors via the ubiquitin/26S proteasome pathway [14]. Previous studies indicated that ROS and cell wall compositions are important intracellular factors affecting juice sac granulation of citrus fruit [15,16]. ROS play a role in plant growth and development, acting both as crucial signal transduction molecules and on the other as toxic by-products that accumulate in cells under various stresses [17]. Superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and glutathione peroxidase (GPX) are antioxidant enzymes acting as ROS scavengers. It has been reported that excessive ROS is scavenged via increasing the activities of SOD and CAT in post-harvest Majia pomelo fruits coated with chitosan, which delays the juice sac granulation [18]. Moreover, the increased cell wall components such as lignin, pectin, and cellulose are closely associated with juice sac granulation of citrus [19]. Among them, the lignin accumulation is considered to be the most important factor causing juice sac granulation [20]. However, PTM such as protein ubiquitination related to the juice sac granulation of pomelo has not been reported. Although many studies of transcriptome and proteome have been carried out in granulated juice sacs of citrus fruits, we are not very clear about the molecular regulatory ways regarding to pomelo juice sac granulation. Therefore, it is necessary to screen genes and ubiquitinated proteins on a large scale by transcriptome and ubiquitinome profiles analyses in order to reveal the molecular basis of the juice sac granulation in pomelo. Results Phenotypic characteristics and lignin content in granulated juice sacs of pomelo Pomelo juice sac granulation obviously influences fruit quality. The results of the observation showed that the phenotypic characteristics of pomelo granulated juice sacs were markedly different from those of normal juice sacs in pomelo (Fig.1a). The normal juice sacs were transparent and it was possible for the juice sacs to spill out after being cut. However, the granulated juice sacs became rough, hard, cloudy white or yellow. In addition, the lignin content was significantly increased by 2.48 times in the granulated juice sacs compared with normal ones (Fig. 1b). These results indicate that the phenotype characteristics and quality of juice sacs has been changed in the granulated pomelo. Identification and functional annotation of differentially expressed genes (DEGs) in granulated juice sacs of pomelo The transcriptome data showed that at least 23,247 genes were detected in one sample, of which 5,990 DEGs were identified (Supplementary Table S1). Gene Ontology (GO) enrichment showed that the entries related to antioxidant enzymes such as oxidoreductase activity, hydrogen peroxide catabolic process and oxidoreductase activity were significantly enriched (Fig. 2A and Supplementary Table S2). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that DEGs was enriched to 22 pathways ( P < 0.05), among which phenylpropanoid biosynthesis pathway is the most significant one (Fig. 2b and Supplementary Table S3). Furthermore, the genes encoding antioxidant enzymes including SOD[Fe] , SOD[Mn] , CAT , GSTs and GPX were also identified (Supplementary Table S5), which were notably upregulated in granulated juice sacs (Fig. 4A). In addition, the lignin biosynthesis genes PALs , 4CL , HCT , CCoAMTs , CCR , CADs and PODs in phenylpropanoid pathway were identified (Supplementary Table S4), among which PALs , 4CL , CADs and PODs were significantly upregulated in granulated juice sacs (Supplementary Fig. S1) that was consistent with the data of lignin contents (Fig. 1B). Above results indicate that antioxidant enzymes and lignin biosynthesis genes are closely related to the physiological changes in granulated juice sacs of pomelo. To verify the validation of transcriptome data, we selected 10 genes in antioxidant enzyme genes and lignin biosynthesis genes as candidate genes for quantitative real-time PCR (qRT-PCR) validation. The results confirmed the gene expression trends were consistent with DEGs data (Supplementary Fig. S2), showing that our transcriptome result is true and reliable. Identification and functional annotation of deferentially ubiquitinated proteins (DUPs) in granulated juice sacs of pomelo Supplementary Table S5 showed that 505 ubiquitination sites in 314 DUPs were identified in granulated juice sacs. The DUPs were annotated by GO enrichment analysis and divided into three categories including biological processes, cellular components and molecular functions (Fig. 3 and Supplementary Table S6). In the biological processes, the DUPs were found to be involved in a large number of processes, such as purine ribonucleoside metabolic, purine nucleoside biosynthetic, adenosine metabolic, tricarboxylic acid metabolic and citrate metabolic processes (Fig. 3a). In the cellular components, most DUPs were enriched in the cytosol, symplast, plasmodesma, and cell-cell junction, which showed that ubiquitinated proteins played an important role in these cellular structures (Fig. 3b). In the molecular functions, including the glutathione transferase activity, antioxidant activity and glutathione peroxidase activity were most significantly enriched (Fig. 3c). Furthermore, the pathway related to glutathione metabolism was enriched using the KEGG functional analysis (Fig. 3d and Supplementary Table S7). Above data suggest that protein lysine ubiquitination is involved in antioxidant enzyme system, which appears to associate with the occurrence of pomelo juice sac granulation. Analysis of ubiquitinated antioxidant enzymes and ROS contents in granulated juice sacs of pomelo Our results showed that antioxidant enzymes, including SOD, CAT, GST and GPX, were significantly increased at ubiquitination level in granulated juice sacs of pomelo (Fig. 4a, Table 1). However, the activities of the four enzymes were decreased in granulated juice sacs of pomelo (Fig. 4b). In addition, two key antioxidants reduced glutathione (GSH) and ascorbic acid (AsA) in the GSH-AsA cycle were determined. The results showed that GSH and AsA contents decreased (Fig. 4c). Furthermore, the results of ROS data demonstrated that superoxide anion (O 2 ) and H 2 O 2 contents increased obviously, and hydroxyl radical ( OH) scavenging capacity decreased in granulated juice sacs (Fig. 4d). Above results indicated that the ROS scavenging abilities were overall decreased in granulated juice sacs of pomelo. Table 1 Differentially ubiquitinated antioxidant enzymes in granulated juice sacs of pomelo Protein accession Granulation/Control Ratio P value Regulated Type KEGG Gene XP-006471806.1 2.270 0.01869 Up SOD XP-006473789.1 1.688 0.03944 Up CAT XP-006479908.1 1.901 0.00574 Up GST XP-006480546.1 2.976 0.00510 Up GST XP-006483268.1 1.798 0.04714 Up GST XP-006484982.1 1.786 0.02446 Up GST XP-006476598.1 2.456 0.00759 Up GPX XP-006494185.1 2.550 0.01281 Up GPX Note: Control: normal juice sacs, Granulation: Granulated juice sacs. Lignin accumulation in response to H 2 O 2 and DMTU treatments in granulated juice sacs of pomelo We found through ROS data analysis that increased H 2 O 2 may be correlated with the juice sac granulation of pomelo due to its signalling role. This raises the question of how H 2 O 2 affects the juice sac granulation. To verify H 2 O 2 activating the key gene expressions in lignin biosynthesis pathway, the juice sacs of pomelo treated with H 2 O 2 and DMTU were continuously cultured on the culture medium for 60 days. The degree of juice sac granulation increased obviously after H 2 O 2 treatment compared with control, and the juice sacs became rough, cloudy and yellow. However, there was no distinct granulation observed in the juice sacs treated with DMTU, and the juice sacs showed transparent (Fig. 5a). In addition, the contents of H 2 O 2 and lignin in the juice sacs increased after H 2 O 2 treatment, and inhibited by DTMU treatment (Fig. 5b). qRT-PCR results indicated that the expressions of key genes CsPAL , Cs4CL , CsCAD and CsPOD in lignin biosynthesis of the juice sacs treated with H 2 O 2 were significantly upregulated, while the gene expressions of the juice sacs treated with DTMU were downregulated (Fig. 5C). Similarly, the activities of phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) in juice sacs treated with H 2 O 2 increased, while the enzyme activities in juice sacs treated with DTMU decreased (Fig. 5D). These results confirm that H 2 O 2 can induce the increases of the relative enzyme activities by up-regulating the expression of the key genes in lignin synthesis pathway, promoting the accumulation of lignin and finally leading to the pomelo juice sac granulation. Discussion In recent years, juice sac granulation, an important physiological disorder affecting the quality of citrus fruits, has been wildly concerned. Previous studies reported that the contents of cell wall components in granulated juice sacs had been changed, in which lignin was specifically accumulated [20, 21]. Lignin is a phenolic polymer deposited in plant secondary cell walls. The change of lignin contents is connected with the expressions of the genes related to lignin biosynthesis pathway such as PAL , C4H , 4CL , CCR , CAD and POD , which plays a vital role in the process of lignin biosynthesis in fruits [2, 22]. In the present study, we identified the genes of PALs , 4CL , HCT , COMTs , CCoAMTs , CCR , CADs and PODs in lignin biosynthesis pathway, and the expression levels of the most genes were significantly upregulated in granulated juice sacs. These results were consistent with the change of lignin content in this study, indicating that the genes regulate the lignin biosynthesis in granulated juice sacs of pomelo. Therefore, lignin accumulation is an important cause of juice sac granulation, which declines pomelo fruit quality [23]. To our knowledge, investigations of ubiquitinome in response to physiological disorders have not been reported in any fruit plants. Protein ubiquitination has been shown to play important roles in diverse biological processes including gene expression, protein activity, stress response, secondary metabolism in plants [24,25]. In the present study, 5,361 ubiquitination sites were identified on 1,990 proteins in pomelo juice sacs. And the ubiquitination sites and proteins in pomelo juice sacs are more than those of most reported plants such as tea and maize [26,27,28], indicating an essential role of ubiquitinated proteins in pomelo juice sacs. Additionally, we found that ubiquitination motifs share common features in dicotyledonous plants. Furthermore, 314 proteins in the granulated juice sacs were defined as DUPs, 96.49% of which were upregulated, indicating that these upregulated DUPs might be related to the granulation of juice sacs. Moreover, GO and KEGG analyses showed that the upregulated DUPs such as antioxidant enzymes were significantly enriched in the glutathione transferase activity, antioxidant activity, glutathione peroxidase activity and glutathione metabolism, implying an essential role in the granulated juice sacs of pomelo. Increasing evidences show that antioxidant enzymes play crucial role in the regulation of ROS metabolism in plant cells. The enzymes are essential for scavenging ROS, maintaining the dynamic balance of ROS in the cells [29]. Antioxidants such as AsA and GSH are also effective in preventing ROS generation and accumulation in plants [30]. We notice that numerous protein/enzyme activities can be negatively regulated by ubiquitination. For example, increasing the level of ubiquitination of Nrf2 inhibits the activity of ARE, leading to the accumulation of intracellular ROS [31]. Many receptor tyrosine kinases (RTKs) are negatively regulated by ubiquitination [32]. In the present study, we found that the genes expression levels of SOD , CAT, GST and GPX mined from transcriptome were significantly upregulated, and the relative enzymes screened from ubiquitinome were obviously increased in abundance while the enzyme activity were significantly reduced in granulated juice sacs. These results suggest that the enzyme activities are negatively affected by ubiquitination modification, resulting in a decrement of the enzyme activities in granulated juice sacs of pomelo. However, there is no instance where the ubiquitination levels of antioxidant enzymes have been directly related to the enzyme activities, which needs further study. Furthermore, we found that antioxidant GSH and AsA contents decreased in the granulated juice sacs. Additionally, O 2 production rate and H 2 O 2 content increased, and OH scavenging capacity were also found to be decreased in the granulated juice sacs. Taken together, these results suggest that the total ROS scavenging ability is reduced, leading to the increase of ROS in the granulated juice sacs. Although ROS are highly toxic substances, many investigations have verified that ROS such as H 2 O 2 and O 2 are vital signaling messengers and playing a vital role in controlling a variety of biological processes within plant cells [33]. Lignin is a phenolic polymer synthesized within cell walls of plants [34]. Peroxidases and/or laccases located in cell walls activate lignin monomer to form the lignin polymers [33]. In addition, many previous studies reported that ROS signaling might be important in the plant lignification [35,36]. H 2 O 2 is fairly stable compared to other ROS in plant cells, and is thus considered as the dominant ROS involved in both cellular signaling and the lignification process [37]. Previous studies indicate that H 2 O 2 is necessary for lignin biosynthesis in postharvest bamboo shoots. The endogenous H 2 O 2 acts as a crucial signaling molecule activating the enzyme activities of PAL, C4H, and 4CL, which promotes the lignin biosynthesis [38]. Moreover, the PuPOD2 and PuLAC2 response to H 2 O 2 is modulated at the gene transcription level, which induces the expressions of these two genes regulating lignin biosynthesis in pear calli [39]. Similarly, in the present study, the endogenous H 2 O 2 content was found to be accumulated in the granulated juice sacs of pomelo, and the key gene expressions levels of CsPAL , Cs4CL , CsCAD and CsPOD in lignin biosynthesis pathway was remarkably higher compared with normal juice sacs. Additionally, we found that the change trend of H 2 O 2 contents was consistent with lignin accumulation in pomelo juice sacs. Notably, the signaling role of H 2 O 2 on regulating these gene expressions leaded to lignin accumulation in the granulated juice sacs, which was verified using tissue culture by adding exogenous H 2 O 2 and DTMU. This result indicates that the expressions of lignin synthesis genes CsPAL , Cs4CL , CsCAD and Cs POD are upregulated by H 2 O 2 signalling, and the activities of the relative enzymes are increased, which contributes to lignin accumulation in the granulated juice sacs of pomelo fruits. As an inhibitor of H 2 O 2 production, DTMU can reduce granulation process in the juice sacs. This result was consistent with the previous report [40]. Taken together, we suggest that increased H 2 O 2 activates the gene expressions and enzyme activities in lignin synthesis pathway, which eventually leads to lignification in the granulated juice sacs of pomelo. Conclusion The present study is the first report focusing on the molecular basis at the level of transcriptome and ubiquitinome regarding to the juice sac granulation of pomelo. We found there were two ways of molecular regulation on the lignification in the granulated juice sacs of pomelo. Firstly, the lignification is modulated by upregulated gene expressions in lignin biosynthesis pathway, which leads to lignin accumulation and induces the juice sac granulation. Secondly, the lignification is also regulated by deceased activities of antioxidant enzymes due to ubiquitination modification, which results in the increase of endogenous H 2 O 2 content in the granulated juice sacs. H 2 O 2 might act as a signalling molecule to activate the gene expressions and enzyme activities in the lignin biosynthesis pathway, promoting lignin accumulation and inducing juice sac granulation of pomelo. Our findings may provide new leads for further pomelo production in mitigating juice sac granulation and improving fruit quality. Materials and methods Plant materials Guanxi pomelo ( Citrus grandis L. Osbeck) fruits were collected at maturity (about 215 days after anthesis) in an orchard of Pinghe County, Fujian Province, China. The juice sacs were divided into control group (normal juice sacs) and granulation group (granulated juice sacs). Juice sacs from the samples of control and granulation in each fruit were collected and frozen using liquid nitrogen, and stored at -80°C for RNA extraction, transcriptome sequencing, ubiquitinome analysis, lignin content determination and enzyme activity assay. Three biological replicates were used for all the above experiments. Lignin content determination As described by Shi et al. [41], 8 g juice sacs were ground to powder and homogenized in 15 ml wash buffer (100mM K 2 HPO 4 /KH 2 PO 4 , 0.5% Triton X-100, 0.5% PVP-K30; pH 7.8), and then washed on a shaker for 15 min at room temperature. Subsequently, the mixture was centrifuged at 15,000g for 15 min at 4°C and the supernatant was discarded (Eppendorf, Germany). The washing process was repeated three times. Then the precipitates were washed three times with ddH 2 O and dried in a vacuum at 60°C overnight. The dried powder was resuspended in 2 ml of 1.0 M NaOH and centrifuged at 15,000g for 20 min at 4°C. 0.1 ml HCl was added to the supernatant (500 μl), incubated to precipitate the lignin thioglycolic acid and centrifugated at 15,000g for 15 min at 4°C. The precipitate was then dissolved in 1.0M NaOH (1:100 ml v/v) and was measured at 280 nm by ultraviolet spectrophotometry (U-T3C, China). RNA extraction, cDNA library construction and RNA sequencing Total RNA was extracted according to the manufacturer's instructions (TianGen, China) from pomelo juice sacs. The cDNA library construction and RNA sequencing were performed by APTBIO (Shanghai, China). To obtain a clear raw read, the Illumina HiSeq sequencing platform was used to sequence the libraries. After being filtered by HISAT2, all of the clean reads have been mapped to the Citrus sinensis reference genome. The featureCounts software is then used to calculate expression values of FPKM for the genes in each sample. Quantitative real-time PCR (qRT-PCR) As the manufacturer’s instructions (TianGen, China), total RNA was extracted from pomelo juice sacs, and the RNA was synthesized into cDNA by the commercial kit (Vazyme, China). The qRT-PCR was carried out using qRT-PCR kit (Vazyme, China) with a light cycler 96 system (Roche, Switzerland). The actin gene has been used as an internal standard, and the 2 −ΔΔCt method has been applied for qRT-PCR data analysis [42]. Supplementary Table S8 listed all primer sequences. For each sample, a total of three separate biological replications were carried out. Protein extraction Proteins of pomelo juice sacs were extracted according to previous descriptions [43]. Pomelo juice sacs (0.5 g) were ground to powder using liquid nitrogen and resuspended in lysis buffer (10 mM dithiothreitol, 1% protease inhibitor cocktail, 50 μM PR-619, 3 μM TSA, 50 mM NAM), and then sonicated on ice for 3 times with a high-intensity ultrasonic processor (Scientz, China). Subsequently, an equal volume of Tris-saturated phenol (pH 8.0) was added, and the supernatant was collected by centrifugation at 20,000g for 15 min at 4°C. Next, the supernatant was precipitated overnight by adding 0.1 M ammonium acetate/methanol. Then, the precipitates were washed 3 times with cold acetone, then discarded the supernatant after centrifugation at 15,000g for 20 min at 4°C. The precipitates were resuspended in urea and the protein concentration was determined using the BCA kit (Beyotime, China) according to manufacturer’s instructions. Trypsin digestion of proteins Tryptic digestion for the protein samples of pomelo juice sacs was performed as described previously [44]. The protein solution was reduced with 5 mM dithiothreitol (DTT) for 30 min at 56°C and alkylated with 11 mM iodoacetamide (IAM) for 15 min at room temperature in darkness. Then 200 mM TEAB was added to dilute the protein. Trypsin was added, and the mass ratio of trypsin to protein was 1:50 and 1:100, respectively. The first digestion was done overnight, and the second one for 4 h. Three biological replicates were performed. Affinity enrichment of ubiquitinated proteins Ubiquitinated proteins of pomelo juice sacs were enriched as described previously [44]. For enrichment of lysine-ubiquitinated peptides, the tryptic peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and incubated with prewashed anti-K-ε-GG antibody beads (Jingjie, China) overnight at 4°C with moderate oscillation. Subsequently, the beads were washed with IP buffer 4 times and with ddH 2 O twice. The beads were eluted three times with 0.1% trifluoroacetic acid (w/v). The eluted fractions were collected and dried in a vacuum overnight. Following the manufacturer’s instructions, the resulting peptides were cleaned using C18 ZipTips (Millipore). LC-MS/MS analysis For LC-MS/MS analysis, ubiquitinated peptides of pomelo juice sacs were conducted as previously studied [45]. The ubiquitinated peptides dissolved into solvent A (0.1% formic acid, 2% acetonitrile) and the peptides were separated by gradient in 8% - 80% solvent B (0.1% formic acid, 90% acetonitrile). The peptides were then separated by an EASYnLC 1200 UPLC system (Thermo Scientific) and injected into the NSI Ion Source for ionisation and then analyzed by mass spectrometry on an Orbitrap Exploris™ 480 (Thermo Fisher Scientific). Database search and bioinformatic analysis The levels of gene expressions were calculated by FPKM (Fragments Per Kilobase of exon model per Million mapped fragments). Using a threshold of p < 0.05 and |fold-change| >1.5 or |fold-change| < 1/1.5, the DEGs in pomelo juice sacs were identified. The ubiquitinated proteins and sites have been identified using Andromeda search engine on Max Quant (v.1.5.2.8). The obtained MS/MS data was searched against the Citrus sinensis uniprot database sequences concatenated with the reverse decoy database. The DUPs or modified Kub sites in pomelo juice sacs were identified with a threshold of p < 0.05 and |fold-change| >1.5 or |fold-change| < 1/1.5. GO and KEGG public databases were used to annotate all the transcripts and ubiquitinated proteins. Determination of the indexes related to ROS metabolism By manufacturer's instructions, the antioxidant enzyme activities were measured with SOD, CAT, GST and GPX commercial assay kits (Solarbio, China), and the absorbances were determined at 560 nm, 240 nm, 412 nm and 340 nm, respectively. The antioxidant contents were detected using GSH and AsA commercial assay kits (Solarbio, China), and the absorbances were determined at 265 nm and 412 nm, respectively. The O 2 production rate, H 2 O 2 content, and hydroxyl radical OH scavenging capacity were detected using commercial assay kits (Solarbio, China), and the absorbance were determined at 530 nm, 415nm and 536 nm, respectively. Pomelo juice sacs with H 2 O 2 and DMTU treatments The juice sacs of the Guanxi pomelo were collected at 150 days after anthesis in 2023 and cultured on murashige and skoog medium with concentration of 100 μ mol L -1 H 2 O 2 (Xilongs, China) and DMTU (a scavenger of H 2 O 2 ; Macklin, China). After being cultured for 60 days on the media, the samples were used to evaluate H 2 O 2 and lignin contents, lignin biosynthesis gene expressions and enzyme activities. Determination of enzyme activities related to lignin biosynthesis PAL, 4CL, CAD and POD activities were measured by commercial assay kits (Solarbio, China), and the absorbances were determined at 290 nm, 333 nm, 340 nm and 470 nm, respectively. Statistical analysis Gene expression and ubiquitinated protein profiles were processed with Excel, SPSS 26.0 and GraphPad Prism 8.0. Duncan’s multiple comparison test or Student's t -test is used to calculate the statistical significance of the difference, and P < 0.05 is considered to be significant. Declarations Acknowledgments We thank Prof. Dr. Wei Chen (Fujian Agriculture and Forestry University, China) for critical review of the manuscript. Authorship contribution LL: conceptualization; YC, WW and QC: writing - original draft; ZT: methodology; JH, RH, JZ and XD: performed some of the experiments; MZ: provided experimental materials; PW: funding acquisition. Funding This work was funded by the earmarked fund for China Agriculture Research System (CARS-26). Ethics approval and consent to participate We declare that the Guanxi pomelo ( Citrus grandis L. Osbeck) fruits bureau of agriculture and rural affairs of Pinghe County gave permission for sampling on this land. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Insititute of Genetics and Breeding in Horticultural Plants, College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, China. 2 Bureau of Agriculture and Rural Affairs of Pinghe County, Pinghe, China Data availability The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/, PRJNA1077242. References Xiang N, Zhao Y, Zhang B, Gu Q, Chen W, Guo X. Volatiles accumulation during young pomelo ( Citrus maxima (Burm.) Merr.) fruits development. Int J Mol Sci. 2022;23(10):5665. Jia N, Liu J, Sun Y, Tan P, Cao H, Xie Y, Wen B, Gu T, Liu J, Li M, Huang Y, Lu J, Jin N, Sun L, Xin F, Fan B. Citrus sinensis MYB transcription factors CsMYB330 and CsMYB308 regulate fruit juice sac lignification through fine-tuning expression of the Cs4CL1 gene. Plant Sci. 2018;277:334-343. 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Shi M, Liu X, Zhang H, He Z, Yang H, Chen J, Feng J, Yang W, Jiang Y, Yao JL, Deng CH, Xu J. The IAA- and ABA-responsive transcription factor CgMYB58 upregulates lignin biosynthesis and triggers juice sac granulation in pummelo. Hortic Res. 2020;7(1):139. Li X, Huang H, Rizwan HM, Wang N, Jiang J, She W, Zheng G, Pan H, Guo Z, Pan D, Pan T. Transcriptome analysis reveals candidate lignin-related genes and transcription factors during fruit development in pomelo ( Citrus maxima ). Genes (Basel). 2022;13(5):845. Lin W, Li Y, Luo C, Huang G, Hu G, He X. Proteomic analysis of ubiquitinated proteins in 'Xiangshui' lemon Citrus limon (L.) pistils after self- and cross-pollination. J Proteomics. 2022;264. Zhu LY, Cheng H, Peng GQ, Wang SS, Zhang ZG, Ni ED, Fu XD, Zhuang CX, Liu ZX, Zhou H. Ubiquitinome profiling reveals the landscape of ubiquitination regulation in rice young panicles. Ge nomics Proteomics Bioinformatics. 2020;18(3):305-320. Berger N, Demolombe V, Hem S, Rofidal V, Steinmann L, Krouk G, Crabos A, Nacry P, Verdoucq L, Santoni V. Root membrane ubiquitinome under short-term osmotic stress. Int J Mol Sci. 2022;23(4):17. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFig.1.tif Supplementary Fig 1.Heatmap of lignin biosynthesis genes in control and granulation group. SupplementaryFig.2.tif Supplementary Fig 2. the expression level of 10 DEGs related to antioxidant enzyme and lignin biosynthesis was based on the FPKM value. FPKM values were the means of three replications. SupplementaryTable18.xlsx Additional file 2: Supplementary Table 1. List of primers used for qRT-PCR in pomelo juice sacs. Supplementary Table 2.Comparison of the changes in transcript levels of DEGs in pomelo juice sacs. Supplementary Table 3. GO Enrichment of DEGs in the transcriptome of pomelo juice sacs. Supplementary Table 4. KEGG Enrichment of DEGs in the transcriptome of pomelo juice sacs. Supplementary Table 5. DEGs of antioxidant enzymes system and lignin biosynthesis pathway in pomelo juice sacs. Supplementary Table 6. Comparison of the changes in protein levels of DUPs in pomelo juice sacs. Supplementary Table 7. GO Enrichment of DUPs in the ubiquitinome of pomelo juice sacs. Supplementary Table 8. KEGG Enrichment of DUPs in the ubiquitinome of pomelo juice sacs. Cite Share Download PDF Status: Published Journal Publication published 11 May, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 26 Mar, 2024 Reviews received at journal 18 Mar, 2024 Reviewers agreed at journal 05 Mar, 2024 Reviewers invited by journal 05 Mar, 2024 Editor invited by journal 16 Feb, 2024 Editor assigned by journal 16 Feb, 2024 Submission checks completed at journal 16 Feb, 2024 First submitted to journal 15 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3958230\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":273225360,\"identity\":\"ed855ae7-3e6b-417f-bf9e-f0f84b90708e\",\"order_by\":0,\"name\":\"Luning LIU\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Fujian Agriculture and Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Luning\",\"middleName\":\"\",\"lastName\":\"LIU\",\"suffix\":\"\"},{\"id\":273225361,\"identity\":\"ac914fd9-ffc1-4007-a4e2-e078a753e971\",\"order_by\":1,\"name\":\"YiRan 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sacs.\\u003c/p\\u003e\\n\\u003cp\\u003ePhenotype characteristics of normal and granulated juice sacs (\\u003cstrong\\u003ea\\u003c/strong\\u003e), lignin content (\\u003cstrong\\u003eb\\u003c/strong\\u003e) were shown.\\u003c/p\\u003e\\n\\u003cp\\u003eControl, normal juice sacs; Granulation, Granulated juice sacs. The asterisks represented a significant difference at \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 level by Student's \\u003cem\\u003et\\u003c/em\\u003e-test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/b3264aa8b003e979edd0dab2.png\"},{\"id\":51294290,\"identity\":\"3376882c-d451-4b4d-b6b5-c636aa7b81e8\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 03:16:00\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2022009,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFunctional enrichment analysis of genes in the transcriptome of pomelo juice sacs.\\u003c/p\\u003e\\n\\u003cp\\u003eGO enrichment analysis (\\u003cstrong\\u003ea\\u003c/strong\\u003e), KEGG enrichment analysis (\\u003cstrong\\u003eb\\u003c/strong\\u003e) were shown.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/c6be4bfc0c8bcb4e279b712b.png\"},{\"id\":51294292,\"identity\":\"5b1a419f-c84e-4431-9114-05bc282f6cb5\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 03:16:00\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3707108,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFunctional enrichment analysis of proteins in the ubiquitinome of pomelo juice sacs. \\u0026nbsp;GO enrichment analysis of biological processes (a), GO enrichment analysis of cellular components (\\u003cstrong\\u003eb\\u003c/strong\\u003e), GO enrichment analysis of molecular functions (\\u003cstrong\\u003ec\\u003c/strong\\u003e), KEGG enrichment analysis (\\u003cstrong\\u003ed\\u003c/strong\\u003e) were shown.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/5c082d3473971a715c27d9fa.png\"},{\"id\":51294278,\"identity\":\"3a742fad-aec1-4fbc-bb27-f67a4774f917\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 03:15:59\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1836920,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eChanges of antioxidant enzyme activities, antioxidant contents and ROS indexes in granulated juice sacs of pomelo. Gene expression levels and abundances of ubiquitinated antioxidant enzymes in pomelo juice sacs (\\u003cstrong\\u003ea\\u003c/strong\\u003e), SOD, CAT, GST and GPX activities (\\u003cstrong\\u003eb\\u003c/strong\\u003e), GSH and AsA contents (\\u003cstrong\\u003ec\\u003c/strong\\u003e), O\\u003csub\\u003e2\\u003c/sub\\u003e\\u003cimg width=\\\"4\\\" height=\\\"46\\\" src=\\\"file:///C:/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_e01/AC/Temp/msohtmlclip1/01/clip_image002.gif\\\"/\\u003e\\u0026nbsp; and H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and scavenging capacity of \\u003cimg width=\\\"4\\\" height=\\\"46\\\" src=\\\"file:///C:/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_e01/AC/Temp/msohtmlclip1/01/clip_image004.gif\\\"/\\u003eOH (\\u003cstrong\\u003ed\\u003c/strong\\u003e) were shown. Different asterisks represented a significant difference at \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 level by Student's \\u003cem\\u003et\\u003c/em\\u003e-test. Control, normal juice sacs; Granulation, Granulated juice sacs.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/69fa85215af4c03d1a41ee27.png\"},{\"id\":51294271,\"identity\":\"ecec4bac-b592-46bb-ba69-60ac9fba8cc7\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 03:15:56\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3878002,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eJuice sac granulation can be affected by H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003eand DTMU treatments through the regulation of lignin contents in pomelo fruits. Phenotype characteristics after H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and DTMU treatments in pomelo juice sacs (\\u003cstrong\\u003ea\\u003c/strong\\u003e), the contents of H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and lignin (\\u003cstrong\\u003eb\\u003c/strong\\u003e), the expression levels of lignin biosynthesis pathway genes (\\u003cstrong\\u003ec\\u003c/strong\\u003e), PAL, 4CL, CAD and POD activities (\\u003cstrong\\u003ed\\u003c/strong\\u003e) were shown. Different letters in different treatments represented a significant difference at \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 level by Duncan’s test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/d9b40c405dd288f907719f69.png\"},{\"id\":56488273,\"identity\":\"eafef14e-07ca-461c-b569-2f22ce353492\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 21:31:08\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2096907,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/ff2aa503-0097-4d2f-9cac-8f0bb812f0e4.pdf\"},{\"id\":51294293,\"identity\":\"154f358d-1a33-4797-8fd7-f5cde135718e\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 03:16:01\",\"extension\":\"tif\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":32096060,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Fig 1.\\u003c/strong\\u003eHeatmap of lignin biosynthesis genes in control and granulation group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SupplementaryFig.1.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/a8ed27e5aaef9aabe22961f5.tif\"},{\"id\":51294294,\"identity\":\"9769accf-1082-4338-a637-d2ad69306a80\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 03:16:02\",\"extension\":\"tif\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":28758556,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary\\u003c/strong\\u003e \\u003cstrong\\u003eFig 2.\\u003c/strong\\u003e the expression level of 10 DEGs related to antioxidant enzyme and lignin biosynthesis was based on the FPKM value. FPKM values were the means of three replications.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SupplementaryFig.2.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/ca7d6e8753eac4c542c59db2.tif\"},{\"id\":51294279,\"identity\":\"a2a607ee-617c-4942-ad33-3e877fc35e10\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 03:15:59\",\"extension\":\"xlsx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1272458,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 2: Supplementary Table 1.\\u003c/strong\\u003e List of primers used for qRT-PCR in pomelo juice sacs. \\u003cstrong\\u003eSupplementary Table 2.\\u003c/strong\\u003eComparison of the changes in transcript levels of DEGs in pomelo juice sacs. \\u003cstrong\\u003eSupplementary Table 3\\u003c/strong\\u003e. GO Enrichment of DEGs in the transcriptome of pomelo juice sacs. \\u003cstrong\\u003eSupplementary Table 4.\\u003c/strong\\u003e KEGG Enrichment of DEGs in the transcriptome of pomelo juice sacs. \\u003cstrong\\u003eSupplementary Table 5.\\u003c/strong\\u003e DEGs of antioxidant enzymes system and lignin biosynthesis pathway in pomelo juice sacs. \\u003cstrong\\u003eSupplementary Table 6.\\u003c/strong\\u003e Comparison of the changes in protein levels of DUPs in pomelo juice sacs. \\u003cstrong\\u003eSupplementary Table 7.\\u003c/strong\\u003e GO Enrichment of DUPs in the ubiquitinome of pomelo juice sacs. \\u003cstrong\\u003eSupplementary Table 8.\\u003c/strong\\u003e KEGG Enrichment of DUPs in the ubiquitinome of pomelo juice sacs.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SupplementaryTable18.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3958230/v1/2305d3bbadc73e4d03fe2257.xlsx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"A multilevel investigation to reveal the regulatory mechanism of lignin accumulation in juice sac granulation of pomelo\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003ePomelo is a citrus fruit, native to China and now widely grown around the world, with high nutritional value [1]. Juice sac granulation, one of the serious physiological disorders in citrus fruits, occurs during harvest and post-harvest storage of the fruits. The occurrence of pomelo juice sac granulation degrades fruit quality [2].\\u003c/p\\u003e\\n\\u003cp\\u003eRecently, there have been several transcriptome researches regard to the juice sac granulation of citrus. Yao et al. [3] reported that the changes in gene expressions based on the transcriptome datasets in the metabolism of sugar and organic acid might be related to juice sac granulation by modulating cell wall components in post-harvest Ponkan (\\u003cem\\u003eCitrus reticulata Blanco\\u003c/em\\u003e cv. Ponkan). In addition, Yao et al. [4] revealed the molecular basis of the granulation of juice sacs in navel orange (\\u003cem\\u003eCitrus sinensis\\u003c/em\\u003e L. Osbeck) through the analyses of metabolome, transcriptome and methylome profilings. Kang et al. [5] indicated that the accumulated cell wall components such as lignin, cellulose and protopectins were closely related to the juice sac granulation by comparing the transcriptome profiles and physiological properties in different juice sacs of Huyou fruit (\\u003cem\\u003eCitrus changshanensis\\u003c/em\\u003e). Ubiquitination is a crucial post-translational modification (PTM) of protein, which is important for regulating protein localization, functions and interactions in biological cells [6]. Previous proteomic investigations indicate that tens-of-thousands of ubiquitination sites on thousands of proteins are identified. It appears that most proteins will be ubiquitinated at some point in their cellular lifetime. The degradation of large number proteins in cells are controlled by ubiquitin-proteasome system [7]. The increasing evidence shows that protein ubiquitination involves virtually all cellular processes and almost all events in the entire life cycle of plants [8]. Lu et al. [9] reported the ubiquitinome of senescing rose petals were changed, suggesting that protein degradation and autophagy, transport of substances and brassinosteroid signaling played important roles in petal senescence. He et al. [10] found that protein ubiquitination played a widely regulating role in rice seed germination by analyzing the ubiquitinome profiles. Cheng et al. [11] highlighted that protein ubiquitination, especially the proteins related to primary metabolism, involved in responding to post-harvest pathogen infection of sweet orange fruit. Furthermore, several investigations reported that protein ubiquitination played crucial roles in fruit ripening and quality. The degradation of ubiquitinated MADS-box proteins was involved in modulating fruit development and ripening of bananas [12]. Ubiquitinated MdbHLH3 regulated apple fruit ripening and quality by promoting ethylene biosynthesis [13]. Auxin receptor TIR1, as positive regulator of auxin signaling, coordinated tomato fruit development and ripening by depredating auxin repressors via the ubiquitin/26S proteasome pathway [14].\\u003c/p\\u003e\\n\\u003cp\\u003ePrevious studies indicated that ROS and cell wall compositions are important intracellular factors affecting juice sac granulation of citrus fruit [15,16]. ROS play a role in plant growth and development, acting both as crucial signal transduction molecules and on the other as toxic by-products that accumulate in cells under various stresses [17]. Superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and glutathione peroxidase (GPX) are antioxidant enzymes acting as ROS scavengers. It has been reported that excessive ROS is scavenged via increasing the activities of SOD and CAT in post-harvest Majia pomelo fruits coated with chitosan, which delays the juice sac granulation [18]. Moreover, the increased cell wall components such as lignin, pectin, and cellulose are closely associated with juice sac granulation of citrus [19]. Among them, the lignin accumulation is considered to be the most important factor causing juice sac granulation [20].\\u003c/p\\u003e\\n\\u003cp\\u003eHowever, PTM such as protein ubiquitination related to the juice sac granulation of pomelo has not been reported. Although many studies of transcriptome and proteome have been carried out in granulated juice sacs of citrus fruits, we are not very clear about the molecular regulatory ways regarding to pomelo juice sac granulation. Therefore, it is necessary to screen genes and ubiquitinated proteins on a large scale by transcriptome and ubiquitinome profiles analyses in order to reveal the molecular basis of the juice sac granulation in pomelo.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003ch1\\u003ePhenotypic characteristics and lignin content in granulated juice sacs of pomelo\\u003c/h1\\u003e\\n\\u003cp\\u003ePomelo juice sac granulation obviously influences fruit quality. The results of the observation showed that the phenotypic characteristics of pomelo granulated juice sacs were markedly different from those of normal juice sacs in pomelo (Fig.1a). The normal juice sacs were transparent and it was possible for the juice sacs to spill out after being cut. However, the granulated juice sacs became rough, hard, cloudy white or yellow. In addition, the lignin content was significantly increased by 2.48 times in the granulated juice sacs compared with normal ones (Fig. 1b). These results indicate that the phenotype characteristics and quality of juice sacs has been changed in the granulated pomelo.\\u003c/p\\u003e\\n\\u003ch2\\u003eIdentification and functional annotation of differentially expressed genes (DEGs) in granulated juice sacs of pomelo\\u003c/h2\\u003e\\n\\u003cp\\u003eThe transcriptome data showed that at least 23,247 genes were detected in one sample, of which 5,990 DEGs were identified (Supplementary Table S1). Gene Ontology (GO) enrichment showed that the entries related to antioxidant enzymes such as oxidoreductase activity, hydrogen peroxide catabolic process and oxidoreductase activity were significantly enriched (Fig. 2A and Supplementary Table S2). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that DEGs was enriched to 22 pathways (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05), among which phenylpropanoid biosynthesis pathway is the most significant one (Fig. 2b and Supplementary Table S3). Furthermore, the genes encoding antioxidant enzymes including \\u003cem\\u003eSOD[Fe]\\u003c/em\\u003e, \\u003cem\\u003eSOD[Mn]\\u003c/em\\u003e, \\u003cem\\u003eCAT\\u003c/em\\u003e, \\u003cem\\u003eGSTs\\u003c/em\\u003e and GPX were also identified (Supplementary Table S5), which were notably upregulated in granulated juice sacs (Fig. 4A). In addition, the lignin biosynthesis genes \\u003cem\\u003ePALs\\u003c/em\\u003e, \\u003cem\\u003e4CL\\u003c/em\\u003e, \\u003cem\\u003eHCT\\u003c/em\\u003e, \\u003cem\\u003eCCoAMTs\\u003c/em\\u003e, \\u003cem\\u003eCCR\\u003c/em\\u003e, \\u003cem\\u003eCADs\\u003c/em\\u003e and \\u003cem\\u003ePODs\\u003c/em\\u003e in phenylpropanoid pathway were identified (Supplementary Table S4), among which \\u003cem\\u003ePALs\\u003c/em\\u003e, \\u003cem\\u003e4CL\\u003c/em\\u003e, \\u003cem\\u003eCADs\\u003c/em\\u003e and \\u003cem\\u003ePODs\\u003c/em\\u003e were significantly upregulated in granulated juice sacs (Supplementary Fig. S1) that was consistent with the data of lignin contents (Fig. 1B). Above results indicate that antioxidant enzymes and lignin biosynthesis genes are closely related to the physiological changes in granulated juice sacs of pomelo. To verify the validation of transcriptome data, we selected 10 genes in antioxidant enzyme genes and lignin biosynthesis genes as candidate genes for quantitative real-time PCR (qRT-PCR) validation. The results confirmed the gene expression trends were consistent with DEGs data (Supplementary Fig. S2), showing that our transcriptome result is true and reliable.\\u003c/p\\u003e\\n\\u003ch2\\u003eIdentification and functional annotation of deferentially ubiquitinated proteins (DUPs) in granulated juice sacs of pomelo\\u003c/h2\\u003e\\n\\u003cp\\u003eSupplementary Table S5 showed that 505 ubiquitination sites in 314 DUPs were identified in granulated juice sacs. The DUPs were annotated by GO enrichment analysis and divided into three categories including biological processes, cellular components and molecular functions (Fig. 3 and Supplementary Table S6). \\u0026nbsp;In the biological processes, the DUPs were found to be involved in a large number of processes, such as purine ribonucleoside metabolic, purine nucleoside biosynthetic, adenosine metabolic, tricarboxylic acid metabolic and citrate metabolic processes (Fig. 3a). In the cellular components, most DUPs were enriched in the cytosol, symplast, plasmodesma, and cell-cell junction, which showed that ubiquitinated proteins played an important role in these cellular structures (Fig. 3b). In the molecular functions, including the glutathione transferase activity, antioxidant activity and glutathione peroxidase activity were most significantly enriched (Fig. 3c). Furthermore, the pathway related to glutathione metabolism was enriched using the KEGG functional analysis (Fig. 3d and Supplementary Table S7). Above data suggest that protein lysine ubiquitination is involved in antioxidant enzyme system, which appears to associate with the occurrence of pomelo juice sac granulation.\\u003c/p\\u003e\\n\\u003ch2\\u003eAnalysis of ubiquitinated antioxidant enzymes and ROS contents in granulated juice sacs of pomelo\\u003c/h2\\u003e\\n\\u003cp\\u003eOur results showed that antioxidant enzymes, including SOD, CAT, GST and GPX, were significantly increased at ubiquitination level in granulated juice sacs of pomelo (Fig. 4a, Table 1). However, the activities of the four enzymes were decreased in granulated juice sacs of pomelo (Fig. 4b). In addition, two key antioxidants reduced glutathione (GSH) and ascorbic acid (AsA) in the GSH-AsA cycle were determined. The results showed that GSH and AsA contents decreased (Fig. 4c). Furthermore, the results of ROS\\u0026nbsp;data demonstrated that superoxide anion (O\\u003csub\\u003e2\\u003c/sub\\u003e ) and H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e contents increased obviously, and hydroxyl radical ( OH) scavenging capacity decreased in granulated juice sacs (Fig. 4d). Above results indicated that the ROS scavenging abilities were overall decreased in granulated juice sacs of pomelo.\\u003c/p\\u003e\\n\\u003cp\\u003eTable 1 Differentially ubiquitinated antioxidant enzymes in granulated juice sacs of pomelo \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"20.615384615384617%\\\"\\u003e\\n \\u003cp\\u003eProtein accession\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"31.53846153846154%\\\"\\u003e\\n \\u003cp\\u003eGranulation/Control Ratio\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.692307692307692%\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eP\\u003c/em\\u003e value\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"20.307692307692307%\\\"\\u003e\\n \\u003cp\\u003eRegulated Type\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.846153846153847%\\\"\\u003eKEGG Gene\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"20.615384615384617%\\\"\\u003e\\n \\u003cp\\u003eXP-006471806.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"31.53846153846154%\\\"\\u003e\\n \\u003cp\\u003e2.270\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.692307692307692%\\\"\\u003e\\n \\u003cp\\u003e0.01869\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"20.307692307692307%\\\"\\u003e\\n \\u003cp\\u003eUp\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.846153846153847%\\\"\\u003e\\n \\u003cp\\u003eSOD\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"20.615384615384617%\\\"\\u003e\\n \\u003cp\\u003eXP-006473789.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"31.53846153846154%\\\"\\u003e\\n \\u003cp\\u003e1.688\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n 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\\u003ctd width=\\\"20.307692307692307%\\\"\\u003e\\n \\u003cp\\u003eUp\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.846153846153847%\\\"\\u003e\\n \\u003cp\\u003eGST\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"20.615384615384617%\\\"\\u003e\\n \\u003cp\\u003eXP-006484982.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"31.53846153846154%\\\"\\u003e\\n \\u003cp\\u003e1.786\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.692307692307692%\\\"\\u003e\\n \\u003cp\\u003e0.02446\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"20.307692307692307%\\\"\\u003e\\n \\u003cp\\u003eUp\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.846153846153847%\\\"\\u003e\\n \\u003cp\\u003eGST\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"20.615384615384617%\\\"\\u003e\\n \\u003cp\\u003eXP-006476598.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"31.53846153846154%\\\"\\u003e\\n \\u003cp\\u003e2.456\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.692307692307692%\\\"\\u003e\\n \\u003cp\\u003e0.00759\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"20.307692307692307%\\\"\\u003e\\n \\u003cp\\u003eUp\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.846153846153847%\\\"\\u003e\\n \\u003cp\\u003eGPX\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"20.615384615384617%\\\"\\u003e\\n \\u003cp\\u003eXP-006494185.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"31.53846153846154%\\\"\\u003e\\n \\u003cp\\u003e2.550\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.692307692307692%\\\"\\u003e\\n \\u003cp\\u003e0.01281\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"20.307692307692307%\\\"\\u003e\\n \\u003cp\\u003eUp\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.846153846153847%\\\"\\u003e\\n \\u003cp\\u003eGPX\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eNote: Control: normal juice sacs, Granulation: Granulated juice sacs.\\u003c/p\\u003e\\n\\u003ch2\\u003eLignin accumulation in response to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and DMTU treatments in granulated juice sacs of pomelo\\u003c/h2\\u003e\\n\\u003cp\\u003eWe found through ROS data analysis that increased H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e may be correlated with the juice sac granulation of pomelo due to its signalling role. This raises the question of how H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e affects the juice sac granulation. To verify H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e activating the key gene expressions in lignin biosynthesis pathway, the juice sacs of pomelo treated with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and DMTU were continuously cultured on the culture medium for 60 days. The degree of juice sac granulation increased obviously after H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u0026nbsp;\\u003c/sub\\u003etreatment compared with control, and the juice sacs became rough, cloudy and yellow. However, there was no distinct granulation observed in the juice sacs treated with DMTU, and the juice sacs showed transparent (Fig. 5a). In addition, the contents of H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and lignin in the juice sacs increased after H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e treatment, and inhibited by DTMU treatment (Fig. 5b). qRT-PCR results indicated that the expressions of key genes \\u003cem\\u003eCsPAL\\u003c/em\\u003e, \\u003cem\\u003eCs4CL\\u003c/em\\u003e, \\u003cem\\u003eCsCAD\\u003c/em\\u003e and \\u003cem\\u003eCsPOD\\u003c/em\\u003e in lignin biosynthesis of the juice sacs treated with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e were significantly upregulated, while the gene expressions of the juice sacs treated with DTMU were downregulated (Fig. 5C). Similarly, the activities of phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) in juice sacs treated with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e increased, while the enzyme activities in juice sacs treated with DTMU decreased (Fig. 5D). These results confirm that H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e can induce the increases of the relative enzyme activities by up-regulating the expression of the key genes in lignin synthesis pathway, promoting the accumulation of lignin and finally leading to the pomelo juice sac granulation.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn recent years, juice sac granulation, an important physiological disorder affecting the quality of citrus fruits, has been wildly concerned. Previous studies reported that the contents of cell wall components in granulated juice sacs had been changed, in which lignin was specifically accumulated [20, 21]. Lignin is a phenolic polymer deposited in plant secondary cell walls. The change of lignin contents is connected with the expressions of the genes related to lignin biosynthesis pathway such as \\u003cem\\u003ePAL\\u003c/em\\u003e, \\u003cem\\u003eC4H\\u003c/em\\u003e, \\u003cem\\u003e4CL\\u003c/em\\u003e, \\u003cem\\u003eCCR\\u003c/em\\u003e, \\u003cem\\u003eCAD\\u003c/em\\u003e and \\u003cem\\u003ePOD\\u003c/em\\u003e, which plays a vital role in the process of lignin biosynthesis in fruits [2, 22]. In the present study, we identified the genes of \\u003cem\\u003ePALs\\u003c/em\\u003e, \\u003cem\\u003e4CL\\u003c/em\\u003e, \\u003cem\\u003eHCT\\u003c/em\\u003e, \\u003cem\\u003eCOMTs\\u003c/em\\u003e, \\u003cem\\u003eCCoAMTs\\u003c/em\\u003e, \\u003cem\\u003eCCR\\u003c/em\\u003e, \\u003cem\\u003eCADs\\u003c/em\\u003e and \\u003cem\\u003ePODs\\u0026nbsp;\\u003c/em\\u003ein lignin biosynthesis pathway, and the expression levels of the most genes were significantly upregulated in granulated juice sacs. These results were consistent with the change of lignin content in this study, indicating that the genes regulate the lignin biosynthesis in granulated juice sacs of pomelo. Therefore, lignin accumulation is an important cause of juice sac granulation, which declines pomelo fruit quality [23]. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTo our knowledge, investigations of ubiquitinome in response to physiological disorders have not been reported in any fruit plants. Protein ubiquitination has been shown to play important roles in diverse biological processes including gene expression, protein activity, stress response, secondary metabolism in plants [24,25]. In the present study, 5,361 ubiquitination sites were identified on 1,990 proteins in pomelo juice sacs. And the ubiquitination sites and proteins in pomelo juice sacs are more than those of most reported plants such as tea and maize [26,27,28], indicating an essential role of ubiquitinated proteins in pomelo juice sacs. Additionally, we found that ubiquitination motifs share common features in dicotyledonous plants. Furthermore, 314 proteins in the granulated juice sacs were defined as DUPs, 96.49% of which were upregulated, indicating that these upregulated DUPs might be related to the granulation of juice sacs. Moreover, GO and KEGG analyses showed that the upregulated DUPs such as antioxidant enzymes were significantly enriched in the glutathione transferase activity, antioxidant activity, glutathione peroxidase activity and glutathione metabolism, implying an essential role in the granulated juice sacs of pomelo.\\u003c/p\\u003e\\n\\u003cp\\u003eIncreasing evidences show that antioxidant enzymes play crucial role in the regulation of ROS metabolism in plant cells. The enzymes are essential for scavenging ROS, maintaining the dynamic balance of ROS in the cells [29]. Antioxidants such as AsA and GSH are also effective in preventing ROS generation and accumulation in plants [30]. We notice that numerous protein/enzyme activities can be negatively regulated by ubiquitination. For example, increasing the level of ubiquitination of Nrf2 inhibits the activity of ARE, leading to the accumulation of intracellular ROS [31]. Many receptor tyrosine kinases (RTKs) are negatively regulated by ubiquitination [32]. In the present study, we found that the genes expression levels of \\u003cem\\u003eSOD\\u003c/em\\u003e, \\u003cem\\u003eCAT,\\u003c/em\\u003e \\u003cem\\u003eGST\\u003c/em\\u003e and \\u003cem\\u003eGPX\\u003c/em\\u003e mined from transcriptome were significantly upregulated, and the relative enzymes screened from ubiquitinome were obviously increased in abundance while the enzyme activity were significantly reduced in granulated juice sacs. These results suggest that the enzyme activities are negatively affected by ubiquitination modification, resulting in a decrement of the enzyme activities in granulated juice sacs of pomelo. However, there is no instance where the ubiquitination levels of antioxidant enzymes have been directly related to the enzyme activities, which needs further study. Furthermore, we found that antioxidant GSH and AsA contents decreased in the granulated juice sacs. Additionally, O\\u003csub\\u003e2\\u003c/sub\\u003e production rate and H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e content increased, and OH scavenging capacity were also found to be decreased in the granulated juice sacs. Taken together, these results suggest that the total ROS scavenging ability is reduced, leading to the increase of ROS in the granulated juice sacs. Although ROS are highly toxic substances, many investigations have verified that ROS such as H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and O\\u003csub\\u003e2\\u003c/sub\\u003e are vital signaling messengers and playing a vital role in controlling a variety of biological processes within plant cells [33].\\u003c/p\\u003e\\n\\u003cp\\u003eLignin is a phenolic polymer synthesized within cell walls of plants [34]. Peroxidases and/or laccases located in cell walls activate lignin monomer to form the lignin polymers [33]. In addition, many previous studies reported that ROS signaling might\\u0026nbsp;be\\u0026nbsp;important\\u0026nbsp;in\\u0026nbsp;the plant lignification [35,36]. H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e is fairly stable compared to other ROS in plant cells, and is thus considered as the dominant ROS involved in both cellular signaling and the lignification process [37]. Previous studies indicate that H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e is necessary for lignin biosynthesis in postharvest bamboo shoots. The endogenous H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e acts as a crucial signaling molecule activating the enzyme activities of PAL, C4H, and 4CL, which promotes the lignin biosynthesis [38]. Moreover, the \\u003cem\\u003ePuPOD2\\u003c/em\\u003e and \\u003cem\\u003ePuLAC2\\u003c/em\\u003e response to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e is modulated at the gene transcription level, which induces the expressions of these two genes regulating lignin biosynthesis in pear calli [39]. Similarly, in the present study, the endogenous H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e content was found to be accumulated in the granulated juice sacs of pomelo, and the key gene expressions levels of \\u003cem\\u003eCsPAL\\u003c/em\\u003e, \\u003cem\\u003eCs4CL\\u003c/em\\u003e, \\u003cem\\u003eCsCAD\\u003c/em\\u003e and \\u003cem\\u003eCsPOD\\u003c/em\\u003e in lignin biosynthesis pathway was remarkably higher compared with normal juice sacs. Additionally, we found that the change trend of H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e contents was consistent with\\u0026nbsp;lignin accumulation in pomelo juice sacs.\\u0026nbsp;Notably, the signaling role of H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e on regulating these gene expressions leaded to lignin accumulation in the granulated juice sacs, which was verified using tissue culture by adding exogenous H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and DTMU. This result indicates that the expressions of lignin synthesis genes \\u003cem\\u003eCsPAL\\u003c/em\\u003e, \\u003cem\\u003eCs4CL\\u003c/em\\u003e, \\u003cem\\u003eCsCAD\\u003c/em\\u003e and Cs\\u003cem\\u003ePOD\\u003c/em\\u003e are upregulated by H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u0026nbsp;\\u003c/sub\\u003esignalling, and the activities of the relative enzymes are increased, which contributes to lignin accumulation in the granulated juice sacs of pomelo fruits. As an inhibitor of H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u0026nbsp;\\u003c/sub\\u003eproduction, DTMU can reduce granulation process in the juice sacs. This result was consistent with the previous report [40]. Taken together, we suggest that increased H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e activates the gene expressions and enzyme activities in lignin synthesis pathway, which eventually leads to lignification in the granulated juice sacs of pomelo.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThe present study is the first report focusing on the molecular basis at the level of transcriptome and ubiquitinome regarding to the juice sac granulation of pomelo. We found there were two ways of molecular regulation on the lignification in the granulated juice sacs of pomelo. Firstly, the lignification is modulated by upregulated gene expressions in lignin biosynthesis pathway, which leads to lignin accumulation and induces the juice sac granulation. Secondly, the lignification is also regulated by deceased activities of antioxidant enzymes due to ubiquitination modification, which results in the increase of endogenous H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e content in the granulated juice sacs. H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e might act as a signalling molecule to activate the gene expressions and enzyme activities in the lignin biosynthesis pathway, promoting lignin accumulation and inducing juice sac granulation of pomelo. Our findings may provide new leads for further pomelo production in mitigating juice sac granulation and improving fruit quality.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003ch2\\u003ePlant materials\\u003c/h2\\u003e\\n\\u003cp\\u003eGuanxi pomelo (\\u003cem\\u003eCitrus grandis\\u003c/em\\u003e L. Osbeck) fruits were collected at maturity (about 215 days after anthesis) in an orchard of Pinghe County, Fujian Province, China. The juice sacs were divided into control group (normal juice sacs) and granulation group (granulated juice sacs). Juice sacs from the samples of control and granulation in each fruit were collected and frozen using liquid nitrogen, and stored at -80\\u0026deg;C for RNA extraction, transcriptome sequencing, ubiquitinome analysis, lignin content determination and enzyme activity assay. Three biological replicates were used for all the above experiments.\\u003c/p\\u003e\\n\\u003ch2\\u003eLignin content determination\\u003c/h2\\u003e\\n\\u003cp\\u003eAs described by Shi et al. [41], 8 g juice sacs were ground to powder and homogenized in 15 ml wash buffer (100mM K\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e/KH\\u003csub\\u003e2\\u003c/sub\\u003ePO\\u003csub\\u003e4\\u003c/sub\\u003e, 0.5% Triton X-100, 0.5% PVP-K30; pH 7.8), and then washed on a shaker for 15 min at room temperature. Subsequently, the mixture was centrifuged at 15,000g for 15 min at 4\\u0026deg;C and the supernatant was discarded (Eppendorf, Germany). The washing process was repeated three times. Then the precipitates were washed three times with ddH\\u003csub\\u003e2\\u003c/sub\\u003eO and dried in a vacuum at 60\\u0026deg;C overnight. The dried powder was resuspended in 2 ml of 1.0 M NaOH and centrifuged at 15,000g for 20\\u0026thinsp;min at 4\\u0026deg;C. 0.1\\u0026thinsp;ml HCl was added to the supernatant (500 \\u0026mu;l), incubated to precipitate the lignin thioglycolic acid and centrifugated at 15,000g for 15 min at 4\\u0026deg;C. The precipitate was then dissolved in 1.0M NaOH (1:100 ml v/v) and was measured at 280 nm by ultraviolet spectrophotometry (U-T3C, China).\\u003c/p\\u003e\\n\\u003ch2\\u003eRNA extraction, cDNA library construction and RNA sequencing\\u003c/h2\\u003e\\n\\u003cp\\u003eTotal RNA was extracted according to the manufacturer\\u0026apos;s instructions (TianGen, China) from pomelo juice sacs. The cDNA library construction and RNA sequencing were performed by APTBIO (Shanghai, China). To obtain a clear raw read, the Illumina HiSeq sequencing platform was used to sequence the libraries. After being filtered by HISAT2, all of the clean reads have been mapped to the Citrus sinensis reference genome. The featureCounts software is then used to calculate expression values of FPKM for the genes in each sample.\\u003c/p\\u003e\\n\\u003ch2\\u003eQuantitative real-time PCR (qRT-PCR)\\u003c/h2\\u003e\\n\\u003cp\\u003eAs the manufacturer\\u0026rsquo;s instructions (TianGen, China), total RNA was extracted from pomelo juice sacs, and the RNA was synthesized into cDNA by the commercial kit (Vazyme, China). The qRT-PCR was carried out using qRT-PCR kit (Vazyme, China) with a light cycler 96 system (Roche, Switzerland). The actin gene has been used as an internal standard, and the 2\\u003csup\\u003e\\u0026minus;\\u0026Delta;\\u0026Delta;Ct\\u003c/sup\\u003e method has been applied for qRT-PCR data analysis [42]. Supplementary Table S8 listed all primer sequences. For each sample, a total of three separate biological replications were carried out.\\u003c/p\\u003e\\n\\u003ch2\\u003eProtein extraction\\u003c/h2\\u003e\\n\\u003cp\\u003eProteins of pomelo juice sacs were extracted according to previous descriptions [43]. Pomelo juice sacs (0.5 g) were ground to powder using liquid nitrogen and resuspended in lysis buffer (10 mM dithiothreitol, 1% protease inhibitor cocktail, 50 \\u0026mu;M PR-619, 3 \\u0026mu;M TSA, 50 mM NAM), and then sonicated on ice for 3 times with a high-intensity ultrasonic processor (Scientz, China). Subsequently, an equal volume of Tris-saturated phenol (pH 8.0) was added, and the supernatant was collected by centrifugation at 20,000g for 15 min at 4\\u0026deg;C. Next, the supernatant was precipitated overnight by adding 0.1 M ammonium acetate/methanol. Then, the precipitates were washed 3 times with cold acetone, then discarded the supernatant after centrifugation at 15,000g for 20 min at 4\\u0026deg;C. The precipitates were resuspended in urea and the protein concentration was determined using the BCA kit (Beyotime, China) according to manufacturer\\u0026rsquo;s instructions.\\u003c/p\\u003e\\n\\u003ch2\\u003eTrypsin digestion of proteins\\u003c/h2\\u003e\\n\\u003cp\\u003eTryptic digestion for the protein samples of pomelo juice sacs was performed as described previously [44]. The protein solution was reduced with 5 mM dithiothreitol (DTT) for 30 min at 56\\u0026deg;C and alkylated with 11 mM iodoacetamide (IAM) for 15 min at room temperature in darkness. Then 200 mM TEAB was added to dilute the protein. Trypsin was added, and the mass ratio of trypsin to protein was 1:50 and 1:100, respectively. The first digestion was done overnight, and the second one for 4 h. Three biological replicates were performed.\\u003c/p\\u003e\\n\\u003ch2\\u003eAffinity enrichment of ubiquitinated proteins\\u003c/h2\\u003e\\n\\u003cp\\u003eUbiquitinated proteins of pomelo juice sacs were enriched as described previously [44]. For enrichment of lysine-ubiquitinated peptides, the tryptic peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and incubated with prewashed anti-K-\\u0026epsilon;-GG antibody beads (Jingjie, China) overnight at 4\\u0026deg;C with moderate oscillation. Subsequently, the beads were washed with IP buffer 4 times and with ddH\\u003csub\\u003e2\\u003c/sub\\u003eO twice. The beads were eluted three times with 0.1% trifluoroacetic acid (w/v). The eluted fractions were collected and dried in a vacuum overnight. Following the manufacturer\\u0026rsquo;s instructions, the resulting peptides were cleaned using C18 ZipTips (Millipore).\\u003c/p\\u003e\\n\\u003ch2\\u003eLC-MS/MS analysis\\u003c/h2\\u003e\\n\\u003cp\\u003eFor LC-MS/MS analysis, ubiquitinated peptides of pomelo juice sacs were conducted as previously studied [45]. The ubiquitinated peptides dissolved into solvent A (0.1% formic acid, 2% acetonitrile) and the peptides were separated by gradient in 8% - 80% solvent B (0.1% formic acid, 90% acetonitrile). The peptides were then separated by an EASYnLC 1200 UPLC system (Thermo Scientific) and injected into the NSI Ion Source for ionisation and then analyzed by mass spectrometry on an Orbitrap Exploris\\u0026trade; 480 (Thermo Fisher Scientific).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDatabase search and bioinformatic analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe levels of gene expressions were calculated by FPKM (Fragments Per Kilobase of exon model per Million mapped fragments). Using a threshold of \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 and |fold-change| \\u0026gt;1.5 or |fold-change| \\u0026lt; 1/1.5, the DEGs in pomelo juice sacs were identified. The ubiquitinated proteins and sites have been identified using Andromeda search engine on Max Quant (v.1.5.2.8). The obtained MS/MS data was searched against the Citrus sinensis uniprot database sequences concatenated with the reverse decoy database. The DUPs or modified Kub sites in pomelo juice sacs were identified with a threshold of \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 and |fold-change| \\u0026gt;1.5 or |fold-change| \\u0026lt; 1/1.5. GO and KEGG public databases were used to annotate all the transcripts and ubiquitinated proteins.\\u003c/p\\u003e\\n\\u003ch2\\u003eDetermination of the indexes related to ROS metabolism\\u003c/h2\\u003e\\n\\u003cp\\u003eBy manufacturer\\u0026apos;s instructions, the antioxidant enzyme activities were measured with SOD, CAT, GST and GPX commercial assay kits (Solarbio, China), and the absorbances were determined at 560 nm, 240 nm, 412 nm and 340 nm, respectively.\\u003c/p\\u003e\\n\\u003cp\\u003eThe antioxidant contents were detected using GSH and AsA commercial assay kits (Solarbio, China), and the absorbances were determined at 265 nm and 412 nm, respectively.\\u003c/p\\u003e\\n\\u003cp\\u003eThe O\\u003csub\\u003e2\\u003c/sub\\u003e production rate, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e content, and hydroxyl radical OH scavenging capacity were detected using commercial assay kits (Solarbio, China), and the absorbance were determined at 530 nm, 415nm and 536 nm, respectively.\\u003c/p\\u003e\\n\\u003ch2\\u003ePomelo juice sacs with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and DMTU treatments\\u003c/h2\\u003e\\n\\u003cp\\u003eThe juice sacs of the Guanxi pomelo were collected at 150 days after anthesis in 2023 and cultured on murashige and skoog medium with concentration of 100\\u0026thinsp;\\u0026mu;\\u0026nbsp;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e (Xilongs, China) and DMTU (a scavenger of H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e; Macklin, China). After being cultured for 60 days on the media, the samples were used to evaluate H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and lignin contents, lignin biosynthesis gene expressions and enzyme activities.\\u003c/p\\u003e\\n\\u003ch2\\u003eDetermination of enzyme activities related to lignin biosynthesis\\u003c/h2\\u003e\\n\\u003cp\\u003ePAL, 4CL, CAD and POD activities were measured by commercial assay kits (Solarbio, China), and the absorbances were determined at 290 nm, 333 nm, 340 nm and 470 nm, respectively.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStatistical analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGene expression and ubiquitinated protein profiles were processed with Excel, SPSS 26.0 and GraphPad Prism 8.0. Duncan\\u0026rsquo;s multiple comparison test or Student\\u0026apos;s \\u003cem\\u003et\\u003c/em\\u003e-test is used to calculate the statistical significance of the difference, and \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 is considered to be significant.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Prof. Dr. Wei Chen (Fujian Agriculture and Forestry University, China) for critical review of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthorship contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLL: conceptualization; YC, WW and QC: writing - original draft; ZT: methodology; JH, RH, JZ and XD: performed some of the experiments; MZ: provided experimental materials; PW: funding acquisition.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was funded by the earmarked fund for China Agriculture Research System (CARS-26).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe declare that the Guanxi pomelo (\\u003cem\\u003eCitrus grandis\\u003c/em\\u003e L. Osbeck) fruits bureau of agriculture and rural affairs of Pinghe County gave permission for sampling on this land.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;Not applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;Competing interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;The authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor details\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003e1\\u003c/sup\\u003e Insititute of Genetics and Breeding in Horticultural Plants, College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, China. \\u003csup\\u003e2\\u003c/sup\\u003e Bureau of Agriculture and Rural Affairs of Pinghe County, Pinghe, China\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/, PRJNA1077242.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eXiang N, Zhao Y, Zhang B, Gu Q, Chen W, Guo X. Volatiles accumulation during young pomelo (\\u003cem\\u003eCitrus maxima\\u003c/em\\u003e (Burm.) Merr.) fruits development. Int J Mol Sci. 2022;23(10):5665.\\u003c/li\\u003e\\n \\u003cli\\u003eJia N, Liu J, Sun Y, Tan P, Cao H, Xie Y, Wen B, Gu T, Liu J, Li M, Huang Y, Lu J, Jin N, Sun L, Xin F, Fan B. Citrus sinensis MYB transcription factors \\u003cem\\u003eCsMYB330\\u003c/em\\u003e and \\u003cem\\u003eCsMYB308\\u003c/em\\u003e regulate fruit juice sac lignification through fine-tuning expression of the \\u003cem\\u003eCs4CL1\\u003c/em\\u003e gene. Plant Sci. 2018;277:334-343.\\u003c/li\\u003e\\n \\u003cli\\u003eYao S, Cao Q, Xie J, Deng L, Zeng K. 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Int J Mol Sci. 2022;23(4):17.\\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\":\"info@researchsquare.com\",\"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\":\"Antioxidant enzyme, H2O2, juice sac granulation, lignin, pomelo, ROS, transcriptome, ubiquitinome\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3958230/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3958230/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eGranulation of juice sacs is a physiological disorder, which affects pomelo fruit quality. Here, the transcriptome and ubiquitinome of the granulated juice sacs were analyzed in Guanxi pomelo. We found that lignin accumulation in the granulated juice sacs was regulated at transcription and protein modification levels. In transcriptome data, we found that the genes in lignin biosynthesis pathway and antioxidant enzyme system of the granulated juice sacs were significantly up-regulated. However, in ubiquitinome data, we found that ubiquitinated antioxidant enzymes increased in abundance but the enzyme activities decreased after the modification, which gave rise to reactive oxygen species (ROS) contents in granulated juice sacs. This finding suggests that ubiquitination level of the antioxidant enzymes is negatively correlated with the enzyme activities. Increased H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e is considered to be a signalling molecule to activate the key gene expressions in lignin biosynthesis pathway, which leads to the lignification in granulated juice sacs of pomelo. This regulatory mechanism in juice sac granulation of pomelo was further confirmed through the verification experiment using tissue culture by adding H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e or dimethylthiourea (DMTU). Our findings suggest that scavenging H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and other ROS are important for reducing lignin accumulation, alleviating juice sac granulation and improving pomelo fruit quality.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A multilevel investigation to reveal the regulatory mechanism of lignin accumulation in juice sac granulation of pomelo\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-02-19 03:15:42\",\"doi\":\"10.21203/rs.3.rs-3958230/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-03-26T16:07:26+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-03-19T02:38:51+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"34466991-2a87-4bd7-a800-3ef4b56f4ec0_SNPRID\",\"date\":\"2024-03-05T13:48:58+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-03-05T09:48:05+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2024-02-16T14:20:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-02-16T14:14:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-02-16T14:14:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"BMC Plant Biology\",\"date\":\"2024-02-15T09:19:33+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"29d27ffb-1fb4-4024-a3d2-70dd21cb481a\",\"owner\":[],\"postedDate\":\"February 19th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-05-14T21:26:27+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-3958230\",\"link\":\"https://doi.org/10.1186/s12870-024-05095-4\",\"journal\":{\"identity\":\"bmc-plant-biology\",\"isVorOnly\":false,\"title\":\"BMC Plant Biology\"},\"publishedOn\":\"2024-05-11 21:17:39\",\"publishedOnDateReadable\":\"May 11th, 2024\"},\"versionCreatedAt\":\"2024-02-19 03:15:42\",\"video\":\"\",\"vorDoi\":\"10.1186/s12870-024-05095-4\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12870-024-05095-4\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3958230\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3958230\",\"identity\":\"rs-3958230\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}