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 K2HPO4/KH2PO4, 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 ddH2O 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 ddH2O 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 O2 production rate, H2O2 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 H2O2 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 H2O2 (Xilongs, China) and DMTU (a scavenger of H2O2; Macklin, China). After being cultured for 60 days on the media, the samples were used to evaluate H2O2 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.