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Article

Transcriptomic Analysis Reveals the Impact of Interstock on Vesicle Granulation in ‘Hainan Qingyou’ Pomelo (Citrus maxima) Fruit

by
Chengchao Yang
1,2,†,
Chengkun Yang
1,2,†,
Haibo Li
1,2 and
Chengdong Jiang
1,2,*
1
School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
2
Hainan Provincial Key Laboratory of Quality Regulation of Tropical Horticultural Crops, Hainan University, Haikou 570228, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(10), 1230; https://doi.org/10.3390/horticulturae11101230 (registering DOI)
Submission received: 25 August 2025 / Revised: 4 October 2025 / Accepted: 10 October 2025 / Published: 12 October 2025
(This article belongs to the Collection Recent Scientific Developments in Genetics, Genomics, and Breeding of Fruit Trees)
Browse Figures
Versions Notes

Abstract

‘Hainan Qingyou’ (Citrus maxima) Pomelo is one of the predominant local cultivars cultivated in Hainan Province, renowned for its high economic value and strong market competitiveness. However, during cultivation, it was observed that the fruit quality of ‘Hainan Qingyou’ grafted onto a ‘Sanhong’ interstock deteriorated, predominantly manifesting as vesicle granulation. This study was therefore conducted to investigate this phenomenon using ‘Sanhong’ Honey Pomelo as the interstock. Fruit quality indicators were measured, and pulp transcriptomic analysis was performed during the expansion and maturation stages. The results indicated that fruits grafted onto ‘Sanhong’ interstock (SHZ) exhibited increased peel thickness, yellower peel, reduced edible rate, higher pulp firmness, decreased total soluble solids (TSS), increased total acid content, and reduced total antioxidant capacity at maturity, all contributing to diminished fruit quality. Additionally, SHZ fruit accumulated higher lignin content in the pulp, leading to vesicle granulation, which severely compromised marketability. Transcriptomic analysis identified 42 structural genes involved in lignin biosynthesis in ‘Hainan Qingyou’ pulp, including 5 PAL, 2 C4H, 2 4CL, 6 CAD, 15 PER, 2 HCT, 1 C3′H, 1 CCoAOMT, 1 CCR, 1 COMT, 2 CSE, and 1 F5H genes. Most of these genes were highly expressed in SHZ fruit at maturity, with expression levels significantly higher than those in fruit grafted onto ‘Hainan Qingyou’ interstock (QYZ). The interstock also affected hormone signaling pathways. Weighted gene co-expression network analysis (WGCNA) identified transcription factors such as MYB, MIKC, ERF, and bZIP as key regulators involved in pulp lignin biosynthesis. This study provides insights into the effects of rootstocks on citrus fruit quality and offers valuable information for cultivar improvement in pomelo orchards.

1. Introduction

Top-grafting, a form of grafting, is widely used in various fruit trees [1,2,3,4,5]. However, not all top-grafting combinations are suitable due to factors such as scion–rootstock interactions [6]. Incompatible rootstocks may adversely affect the scion, leading to poor shoot development and reduced vitality [7]. They can also significantly impact fruit quality, resulting in thicker peel, reduced sugar content, and altered taste [7,8]. Studies on citrus have shown that rootstock–scion incompatibility may also cause vesicle granulation, severely reducing market value [9].
Vesicle granulation in citrus can occur both pre- and post-harvest [10], primarily characterized by deteriorated flavor, reduced juice sac moisture, and hardened texture, significantly impairing commercial quality [11]. External factors may include abnormal temperatures, intense light, and altitude [12], while internal factors may involve techniques, mineral levels, and rootstock–scion affinity [12,13]. Although the mechanism of citrus vesicle granulation is not fully understood, existing studies consistently associate it with increased lignin accumulation in juice sacs [14,15,16]. The lignin biosynthesis pathway is well-established, proceeding through the phenylpropanoid pathway and involving structural genes such as phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), shikimate O-hydroxycinnamoyltransferase (HCT), coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD); these genes work together to positively regulate the synthesis of lignin monomers [17,18,19,20].
‘Hainan Qingyou’ pomelo (pummelo), introduced in the early 21st century, is a cultivar of Malaysian pummelo. Known for its abundant juice and unique flavor, it is highly popular in the market and is currently the most competitive pomelo variety in Hainan [21]. In the 1990s, Shatian and Guanxi honey pomelo (Sanhong) were also introduced to Hainan, but their fruit quality was significantly inferior to that of their origin regions, resulting in low market competitiveness and economic value [21]. Currently, some ‘Sanhong’ pomelo orchards use top-grafting to convert to ‘Hainan Qingyou’ to enhance market competitiveness. However, field observations indicate that the ‘Sanhong’ interstock adversely affects the fruit quality of ‘Hainan Qingyou’, though the underlying mechanisms remain unclear.
This study evaluated the impact of the ‘Sanhong’ and ‘Hainan Qingyou’ interstock on ‘Hainan Qingyou’ fruit quality by measuring external and internal quality parameters and analyzing transcriptomic data to identify differentially expressed genes. The findings provide important insights into the causes of vesicle granulation in ‘Hainan Qingyou’ and offer guidance for cultivar improvement in ‘Sanhong’ pomelo orchards.

2. Materials and Methods

2.1. Plant Materials

Pomelo fruit samples were collected from a commercial orchard in Lingao County, Hainan Province, China. The trees were 10 years old and grafted onto Citrus maxima var. aurantifolia rootstock, with either ‘Hainan Qingyou’ (Citrus maxima ‘Hainan Qing You’, QYZ) or ‘Sanhong’ pomelo (Citrus maxima ‘Sanhong You’, SHZ) as the interstock. The scion was ‘Hainan Qingyou’, grafted five years prior. Five healthy trees of each interstock type with moderate growth were randomly selected. Fifteen fruits were randomly harvested at the expansion stage (80 days after flowering) and the mature stage (130 days after flowering). Peel and pulp were quickly separated in the field using a sharp knife, flash-frozen in liquid nitrogen, and transported to the laboratory. Five randomly selected fruits constituted one biological replicate, with three replicates per group. An additional set of samples was collected for physiological index determination using the same method.

2.2. Fruit Quality Measurement

Fruit longitudinal and transverse diameters and peel thickness were measured using a digital vernier caliper (Shengli, 5150, Shenzhen, China). The fruit shape index was calculated as the ratio of longitudinal to transverse diameter. Peel and pulp weights were measured using an electronic balance to determine the edible rate. After removing the peel and segment membranes, pulp firmness was measured using a TA.XT Plus texture analyzer (Bosin Tech, Shanghai, China) with a 2 mm probe inserted vertically to a depth of 5 mm. Settings were: pre-test speed 2 mm/s, test speed 4 mm/s, post-test speed 3 mm/s, and interval time 2 s [22]. Soluble solids content (SSC), titratable acidity (TA), and sugar–acid ratio were determined using a citrus sugar–acid meter (ATAGO, PAL-BX/ACID 1, Tokyo, Japan). For each biological replicate, all metrics were measured using two fruits sampled separately (resulting in a total of 6 fruits per treatment per stage). Total flavonoid and phenol contents were measured spectrophotometrically [23]. Ascorbic acid (AsA) content was determined using the fast blue salt B method (Comin, ASA-2-W, Suzhou, China) [22]. Lignin staining was performed using the phloroglucinol method (Yuanye, R30443, Shanghai, China) according to the manufacturer’s instructions. The lignin content in the pulp was determined using a spectrophotometric method with a commercial assay kit (Comin, MZS-2-G, Suzhou, China), following the manufacturer’s instructions. The staining results of the fruit flesh were examined using an optical microscope (Novel, N-117M, Ningbo, China) at a 40× magnification. For each biological replicate, all metrics were measured using one fruits sampled separately (resulting in a total of 3 fruits per treatment per stage).

2.3. Pulp Antioxidant Capacity Assay

Glutathione reductase (GR) activity was measured using the NADPH colorimetric method (Comin, GR-2-W, Suzhou, China). Polyphenol oxidase (PPO) activity was determined using the catechol method (Solarbio, BC0190, Beijing, China). Superoxide dismutase (SOD) activity was assayed using the nitroblue tetrazolium (NBT) method (Comin, SOD-2-Y, Suzhou, China). Hydroxyl radical scavenging capacity was measured using the o-phenanthroline method (Comin, QZQ-2-G, Suzhou, China). Total antioxidant capacity (T-AOC) was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method (Comin, DPPH-2-D, Suzhou, China) and the ferric reducing antioxidant power (FRAP) assay (Comin, FRAP-2-G, Suzhou, China) [22]. For each biological replicate, all metrics were measured using one fruits sampled separately (resulting in a total of 3 fruits per treatment per stage).

2.4. RNA Extraction, cDNA Synthesis, and RT-qPCR Analysis

RNA was extracted and sequenced (RNA-seq) from three biological replicates of ‘Hainan Qingyou’ fruit at both expansion and mature stages from each rootstock. RNA was extracted using ethanol precipitation and CTAB-PBIOZOL methods. Concentration was measured using a Qubit 4.0 Fluorometer/MD Microplate Reader (Life Technologies, Carlsbad, CA, USA), and integrity was assessed using a Qsep400 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were constructed by Wuhan Maiwei Biotechnology Co., Ltd. (Wuhan, China) on the Illumina sequencing platform. Poly(A)+ mRNA was enriched using oligo (dT) beads, fragmented, and reverse-transcribed using random hexamers to synthesize first-strand cDNA. Second-strand cDNA was synthesized using buffer, dNTPs, and DNA polymerase. After purification, double-stranded cDNA was end-repaired, adenylated, and ligated with sequencing adapters. Fragment size selection was performed using DNA purification beads, followed by PCR enrichment to obtain the final cDNA library. After quality control, libraries were pooled based on effective concentration and sequencing requirements, and 150 bp paired-end reads were generated on the Illumina platform. Clean reads were aligned to the reference genome using HISAT2. Differential gene expression analysis was performed using DESeq2 with thresholds of |log2 fold change| > 1 and p < 0.05 [24].
Differentially expressed genes (DEGs) were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases, and enrichment analysis was performed using the hypergeometric distribution. Weighted gene co-expression network analysis (WGCNA) was performed using the Metware Cloud platform (https://cloud.metware.cn, accessed on 18 March 2025). The gene filtering threshold was set to 0, the merge cut height for modules was 0.25, and the minimum module size was 50. All other parameters were set to their defaults [25]. CgActin was used as the reference gene [26]. qPCR primers were designed using Primer 3.0 (https://www.primer3plus.com/, accessed on 16 May 2025) (Table S1) [27], and cDNA synthesis and RT-qPCR were performed according to Shi et al. [28].

2.5. Heatmap and Statistical Analysis

Heatmaps were generated using TBtools_v2.357 [29], with row-wise data normalized to a 0–1 range. Independent sample t-tests were performed using IBM SPSS 27.0. Significance levels were set at p < 0.05, p < 0.01, and p < 0.001, denoted by *, **, and ***, respectively.

3. Results

3.1. Effects of Different Interstocks on Fruit Size and Edible Rate at Maturity

At maturity, QYZ fruit had greener peel, while SHZ fruit exhibited yellower peel. Peel thickness was significantly lower in QYZ than in SHZ (Figure 1A). There were no significant differences in transverse diameter or fruit shape index between QYZ and SHZ, but the longitudinal diameter was significantly greater in SHZ (Figure 1B–D). Peel thickness was significantly higher in SHZ, being 1.64 times that of QYZ (Figure 1E). Although single fruit weight did not differ significantly, peel weight was significantly lower and pulp weight higher in QYZ, resulting in a significantly higher edible rate in QYZ than in SHZ (Figure 1F–I). These results indicate that the ‘Sanhong’ interstock adversely affects the edible rate of ‘Hainan Qingyou’ fruit at maturity.

3.2. Effects of Different Interstocks on Internal Fruit Quality

At maturity, pulp firmness in QYZ was only 41.44% of that in SHZ and significantly lower (Figure 2A). SSC and sugar–acid ratio were significantly higher in QYZ, while TA was significantly lower (Figure 2B–D). AsA, total flavonoids, and total phenols were measured at both developmental stages. AsA was significantly higher in SHZ at the expansion stage but lower at maturity. Total flavonoids showed no significant difference at either stage. Total phenol content was significantly higher in SHZ at the expansion stage but not significantly different at maturity (Figure 2E–G). The lignin content in the pulp of SHZ and QYZ fruits showed no significant difference at the developmental stage. However, at maturity, the lignin content in SHZ pulp was significantly higher than that in QYZ (Figure 2H). Lignin staining showed no staining in either QYZ or SHZ pulp at the expansion stage, indicating low lignin content. At maturity, QYZ pulp remained unstained, while SHZ pulp was stained bright red, indicating substantial lignin accumulation (Figure 2I). These results suggest that reduced TSS accumulation, increased acid content, and lignin accumulation in SHZ pulp lead to hardening and quality deterioration.

3.3. Effects of Different Interstocks on Pulp Antioxidant Indicators

GR and POD activities were similar between QYZ and SHZ, but both were significantly higher in SHZ at both developmental and mature stages (Figure 3A,B). SOD activity was significantly higher in SHZ at the expansion stage but not significantly different at maturity (Figure 3C). Hydroxyl radical scavenging capacity was significantly lower in QYZ at the expansion stage but higher at maturity (Figure 3D). T-AOC, measured by both DPPH and FRAP methods, was consistently higher in QYZ pulp at both stages (Figure 3E,F). These findings demonstrate that divergence between SHZ and QYZ fruit had already emerged during the developmental phase, even though their flesh lignin levels did not yet differ significantly.

3.4. Transcriptome Overview

RNA-seq yielded 53,224,956–81,688,50446,463,006–54,199,384 raw reads per sample. After filtering, 51,406,600–78,976,222 clean reads were obtained, corresponding to 7.71–11.85 Gb of clean data. Error rates were 0.01% for all samples. Q20, Q30, and GC contents were 99.19–99.33%, 97.34–97.84%, and 44.60–45.28%, respectively (Table S2). PCA showed clear separation among QYZ-D, QYZ-M, SHZ-D, and SHZ-M samples, with replicates clustering together. PC1 and PC2 explained 34.5% and 30.32% of the variance, respectively (Figure S1).

3.5. Differential Gene Expression Analysis

A total of 5395 DEGs were identified between QYZ-D vs. SHZ-D (4511) and QYZ-M vs. SHZ-M (1395), with 511 common DEGs (Figure S2). GO annotation categorized DEGs into biological process (BP), cellular component (CC), and molecular function (MF). In both comparisons, the most annotated terms were “cellular process” in BP (2219 and 704 genes, respectively), “cellular anatomical entity” in CC (3015 and 939 genes), and “binding” in MF (2118 and 628 genes) (Figure S3). KEGG analysis showed that in SHZ-D vs. QYZ-D, most DEGs were enriched in “Plant-pathogen interaction” (279 genes). In SHZ-M vs. QYZ-M, most DEGs were enriched in “Metabolic pathways” (252 genes) (Figure 4). Notably, no lignin biosynthesis-related pathways were enriched in SHZ-D vs. QYZ-D, whereas “Phenylpropanoid biosynthesis” and “Phenylalanine, tyrosine and tryptophan biosynthesis” were enriched in SHZ-M vs. QYZ-M (Figure 4), consistent with the high lignin content in SHZ fruit during late development.

3.6. Expression Profiles of Key Structural Genes in Lignin Biosynthesis

We identified 42 key structural genes involved in pulp lignin biosynthesis, including 5 PAL, 2 C4H, 2 4CL, 6 CAD, 15 PER, 2 HCT, 1 C3′H, 1 CCoAOMT, 1 CCR, 1 COMT, 2 CSE, and 1 F5H genes. Most showed the highest expression in SHZ at maturity, significantly exceeding levels in QYZ. At the expansion stage, expression was generally low but higher in SHZ than in QYZ. For example, Cg1g021310-CAD expression in SHZ-M was 179.20, 17.96 times that in QYZ-M, and 4.63 times higher in SHZ-D than in QYZ-D. This trend was more pronounced in upstream genes like PAL and C4H, all of which showed higher expression in SHZ at the same stage. These findings suggest that lignin biosynthesis initiates during the expansion stage but accelerates toward maturity (Figure 5).

3.7. Expression Profiles of Hormone Signaling Pathway Genes

In the SHZ-M vs. QYZ-M comparison, 62 DEGs were identified in hormone signaling pathways. Compared to QYZ, SHZ showed upregulation of 1 AUX1, 1 SAUR, 1 TMK1/4, and 1 AHA1/2 in auxin signaling; downregulation of 4 AUX/IAA, 4 SAUR, 1 TAA1, and 1 AHA1/2; in cytokinin signaling, 1 CRE1, 1 AHP, and 2 B-ARR were downregulated, while 1 AHP and 1 A-ARR were upregulated; in gibberellin signaling, most DEGs were upregulated, including 2 GID1, 1 DELLA, and 5 bHLH, with only 3 GID1 downregulated; in abscisic acid signaling, 1 PYR/PYL and 1 ABF were upregulated; in ethylene signaling, 1 ERF1/2 was upregulated; all DEGs in brassinosteroid signaling were upregulated, including 6 BAK1 and 3 BRI1; in jasmonic acid signaling, 1 JAZ was upregulated and 1 MYC2 downregulated; in salicylic acid signaling, 2 TGA genes were upregulated and 1 PR-1 downregulated (Figure 6).

3.8. WGCNA and Key Transcription Factor Screening

WGCNA was performed to identify key candidate TFs regulating lignin biosynthesis in ‘Hainan Qingyou’ pulp. DEGs were grouped into 12 modules based on expression pattern similarity (Figure 7A, Table S4). Using a correlation threshold of >0.7 or <−0.7, the blue module showed strong positive correlation with 11 key structural genes, while green and black modules showed strong negative correlation (Figure 7A, Table S4). Cg1g021310-CCR had the strongest positive correlation with the blue module and the strongest negative correlation with the green module; novel.1178-COMT had the strongest negative correlation with the black module (Figure 7A). Hub genes identified included 1 OFP, 2 MYB, 1 MIKC, and 1 Dof in the blue module; 1 ARF, 3 ERF, 1 LIM, and 1 HRT in the green module; and 1 bZIP, 1 MIKC, 1 C2H2, 1 BPC, 1 HD-ZIP, and 1 LOB in the black module (Figure 7B).

3.9. RT-qPCR Validation

To validate RNA-seq results, 8 DEGs from the lignin biosynthesis pathway were analyzed by qPCR. Most genes, such as Cg2g026340-4CL, Cg1g021310-CCR, and Cg6g001770-PAL, showed high consistency between qPCR and RNA-seq. Some genes, like Cg8g003850-CAD and Cg2g038950-CAD, showed slight discrepancies but similar expression trends (Figure 8). These results confirm the reliability of RNA-seq for gene expression analysis.

4. Discussion

Grafting involves the formation of new vascular tissues at the scion–rootstock junction, facilitating exchange of materials and signals. Scion–rootstock interactions significantly affect photosynthetic efficiency, mineral uptake, and fruit quality [30,31]. For example, interspecific grafted blueberries exhibit higher glucose, maltose, raffinose, inositol, and galactinol contents than intraspecific grafts [32]. However, not all combinations improve fruit quality, as compatibility is crucial [33,34,35]. Citrus scions grafted onto trifoliate orange (Poncirus trifoliata) produced higher-quality fruit than those on red tangerine (Citrus × tangerina) [36]; The dwarfing hybrid rootstock Forner-Alcaide 418 ((Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf.) × Citrus deliciosa Ten. & Pasq.) has been reported to result in a lower external color index in ‘Navel’ orange (Citrus sinensis [L.] Osbeck var. brasiliensis Tanaka) compared to other dwarfing hybrid rootstocks [37]. In a study by Sau et al., ‘Nagpur’ mandarin (Citrus reticulata Blanco) grafted onto kumquat (Fortunella sps.) and ‘Rough’ lemon (Citrus × limon) rootstocks exhibited higher juice content and lower juice sac granulation, whereas grafting onto ‘Gandharaj’ (Citrus × lemon Burn.) led to reduced juice content and increased juice sac granulation [38]. McCollum et al. investigated the influence of different rootstocks on ‘Marsh’ grapefruit (Citrus × paradisi) and found that fruits from trees grafted onto ‘Sour’ orange (Citrus × aurantium) had the highest total soluble solids (TSS) and acidity. In contrast, those grafted onto ‘Carrizo’ citrange (Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf.) showed the lowest acidity, while the lowest TSS content was observed in fruits grafted onto ‘Smooth Flat Seville’ orange (Citrus aurantium putative hybrid) [39]. In this study, the ‘Sanhong’ rootstock significantly reduced edible rate, AsA content, and TSS, while increasing firmness, flavonoids, and lignin content, leading to quality deterioration and vesicle granulation, greatly reducing market value. This suggests that ‘Sanhong’ is incompatible as a rootstock for ‘Hainan Qingyou’, and top-grafting ‘Sanhong’ orchards to ‘Hainan Qingyou’ is not recommended.
Vesicle granulation severely impacts citrus fruit quality. Previous studies attribute it to variety, tree age, fruit size, peel structure, pollination methods, irrigation, fertilization, storage conditions, and rootstock type [9,40]. For example, ‘Kinnow’ (Citrus reticulata) grafted onto ‘Sohsarkar’ (Citrus reticulata) had a 38.3% granulation incidence, which dropped to 5.9% when grafted onto ‘Troyer’ (Citrus × aurantium) [13]. Granulation reduces juice sac moisture, hardens texture, and impairs flavor, closely associated with increased lignin content [9]. In navel orange (Citrus × sinensis), many DEGs encoding monolignol biosynthesis genes, including 4 PAL, 2 4CL, 3 HCT, 4 CCoAOMT, 1 CCR, and 10 COMT, were upregulated during granulation [15]; in pomelo, lignin synthesis during maturation involved CgPAL, CgC4H, Cg4CL, CgC3H, CgCCoAOMT, and CgLAC [41]; in ‘Ponkan’ citrus (Citrus reticulata), two CAD genes were significantly upregulated during granulation [14]. This study found higher lignin content in SHZ fruit and identified key lignin biosynthesis genes, including 5 PAL, 2 C4H, 2 4CL, 6 CAD, 15 PER, 2 HCT, 1 C3′H, 1 CCoAOMT, 1 CCR, 1 COMT, 2 CSE, and 1 F5H, most highly expressed in SHZ fruit at maturity. Thus, the ‘Sanhong’ interstock upregulates lignin biosynthesis genes, increasing pulp lignin content and causing vesicle granulation. The elevated lignin levels, typically associated with plant stress responses, suggest that the ‘Sanhong’ interstock may have imposed certain stresses on the scion and fruit compared to the QYZ fruit. Previous studies have shown that incompatible rootstocks can induce water deficit in fruits, leading to thicker peel, reduced juice content, and juice sac granulation in citrus—findings that align with the results observed in this study [39].
Plant hormones, particularly auxin and ABA, may regulate lignin synthesis in citrus vesicle granulation. Pre- or post-harvest application of auxinic regulators reduces granulation incidence, e.g., 2,4-D in ‘Hongju’ and pomelo, and NAA in ‘Kaula’ citrus (Citrus reticulata) [9]. ABA plays a key role in non-climacteric fruit ripening [42,43] and is closely associated with granulation in various citrus types. Rootstocks can differentially affect water uptake. Potential water deficit induces ABA and other hormonal signals, which in turn stimulate lignin synthesis as a defense mechanism. This process can ultimately lead to juice sac granulation [1]. ABA content increases during granulation in ‘Ponkan’ and blood orange, correlating positively with severity [9,44]. Recent studies show that exogenous ABA accelerates lignin synthesis, while IAA inhibits it [9]. In this study, most auxin signaling genes were downregulated in SHZ; ABA signaling regulator PYR/PYL was downregulated, while ABF was upregulated, suggesting that auxin and ABA signaling are involved in lignin synthesis in SHZ fruit. These findings inspire further work, such as using exogenous auxin to alleviate or inhibit granulation in SHZ fruit, requiring additional experimentation.
Interestingly, many genes in GA and BR signaling pathways were also upregulated in SHZ fruit. Although studies on these hormones in citrus granulation are lacking, references from other species exist. GA signaling positively regulates lignin synthesis in rice to prevent seed shattering [45]; exogenous GA significantly reduced lignin content in ramie (Boehmeria nivea) leaves [46]. In Arabidopsis, GA regulates xylem formation in the hypocotyl during flowering [47] and promotes polar auxin transport to specify xylem identity from cambial stem cells, regulating xylem-phloem ratio [48]. In three peanut varieties, the difference in plant height was due to transcriptomically identified differential expression of key brassinosteroid biosynthesis and signaling genes (e.g., AhCPD, AhBRI1, AhBAK1), which correspondingly affected BR concentration, stem growth, and lignin accumulation [49]. The increased lignin content in SHZ fruit may be regulated by hormone crosstalk, warranting further investigation.
Transcription factors regulate pulp lignin synthesis by controlling structural gene expression. This study identified 18 TFs as key regulators, including 3 MYBs (positive regulators). Previous studies report TFs increasing lignin content in citrus: transient overexpression of CsMYB330 (ortholog of AtMYB58/63) activated Cs4CL1, increasing lignin content [50]; CgMYB58 upregulated at least 19 lignin biosynthesis genes (including CgPAL1, CgPAL2, Cg4CL1, and CgC3H), and transient and stable expression confirmed its role in increasing lignin in pomelo [41]; CsMYB85 directly binds the CsMYB330 promoter to activate its expression and lignin biosynthesis genes [51]; CgNAC043 directly activates CgCCoAOMT and CgC3H promoters, promoting granulation [52]. These results indicate that TFs play crucial regulatory roles in SHZ pulp granulation.
While this study provides initial evidence for the role of the ‘Sanhong’ and ‘Hainan Qingyou’ interstock in regulating fruit quality through hormonal and lignin pathways, it is important to consider its scope. The findings are based on data collected from a single orchard over one growing season, focusing on two key developmental stages, with a sample size of n = 3 per group. Consequently, the generalizability of these results to other environmental conditions, orchard management practices, or larger populations requires further investigation. To robustly validate these conclusions, we have outlined a clear future research plan. This includes: (1) expanding the study to include orchards in different geographical regions with distinct soil and climate profiles; and (2) testing the effects of a broader range of interstocks and rootstock combinations to determine the specificity and broader applicability of the proposed mechanism. Such efforts will be crucial for translating these findings into practical horticultural applications.

5. Conclusions

This study found that using ‘Sanhong’ as an interstock adversely affects ‘Hainan Qingyou’ fruit quality, manifesting as increased peel thickness, yellower peel, reduced edible rate, higher pulp firmness, decreased TSS, increased acidity, and reduced antioxidant capacity at maturity. Additionally, it increased lignin content in SHZ pulp, causing vesicle granulation and severely reducing marketability. Transcriptomic analysis identified 42 structural genes involved in lignin biosynthesis, including 5 PAL, 2 C4H, 2 4CL, 6 CAD, 15 PER, 2 HCT, 1 C3′H, 1 CCoAOMT, 1 CCR, 1 COMT, 2 CSE, and 1 F5H genes. Most were highly expressed in SHZ at maturity, far exceeding levels in QYZ. The interstock affected hormone signaling. Using WGCNA, key candidate TFs such as MYB, MIKC, ERF, and bZIP were identified for their role in regulating pulp lignin biosynthesis. This study not only provides insights into how interstocks affect citrus fruit quality, but also opens up promising prospects for compatibility identification and the selection of superior progeny lines.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11101230/s1, Figure S1: PCA analysis; Figure S2: Differential expression gene (DEGs) Venn diagram analysis; Figure S3: GO analysis of differentially expressed genes (DEGs); Figure S4: Melting Curves of qPCR Primers; Table S1: qRT-PCR primer information; Table S2: Statistics on the quality and output of the RNA-Seq; Table S3: The FPKM values of lignin synthesis pathway genes in different SHZ and QYZ fruits; Table S4 WGCNA module gene classification.

Author Contributions

Conceptualization, C.J., C.Y. (Chengchao Yang) and C.Y. (Chengkun Yang); methodology, C.Y. (Chengchao Yang) and C.Y. (Chengkun Yang); software, C.Y. (Chengchao Yang) and H.L.; validation, C.Y. (Chengchao Yang), C.Y. (Chengkun Yang) and H.L.; formal analysis, C.Y. (Chengchao Yang); investigation, C.J. and C.Y. (Chengchao Yang); resources, C.J.; data curation, C.Y. (Chengchao Yang) and H.L.; writing—original draft preparation, C.Y. (Chengchao Yang) and C.Y. (Chengkun Yang); writing—review and editing, C.J. and C.Y. (Chengkun Yang); visualization, C.Y. (Chengchao Yang); supervision, C.J.; project administration, C.J.; funding acquisition, C.J. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Key Research and Development Program of Hainan Province (grant numbers: RZ2300000808).

Data Availability Statement

Raw RNA-seq data have been deposited in Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/, accessed on 22 February 2025) CNCB-NGDC under accession number PRJCA037359: CRR1687143 (QYZ-D-1), CRR1687144 (QYZ-D-2), CRR1687145 (QYZ-D-3), CRR1687149 (QYZ-M-1), CRR1687150 (QYZ-M-2), CRR1687151 (QYZ-M-3), CRR1687155 (SHZ-D-1), CRR1687156 (SHZ-D-2), CRR1687157 (SHZ-D-3), CRR1687161 (SHZ-M-1), CRR1687162 (SHZ-M-2) and CRR1687163 (SHZ-M-3).

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of ‘Sanhong’ interstock on fruit size and edible rate of ‘Hainan Qingyou’ at maturity. (A) Fruit appearance at maturity; (B) longitudinal diameter; (C) transverse diameter; (D) fruit shape index; (E) peel thickness; (F) single fruit weight; (G) peel weight; (H) pulp weight; (I) edible rate. Note: SHZ: ‘Hainan Qingyou’ fruit grafted onto ‘Sanhong’ interstock; QYZ: ‘Hainan Qingyou’ fruit grafted onto ‘Hainan Qingyou’ interstock. Statistical significance (Student’s t-test) is indicated as follows: ** p < 0.01, *** p < 0.001, ns p ≥ 0.05. n = 6.
Figure 1. Effects of ‘Sanhong’ interstock on fruit size and edible rate of ‘Hainan Qingyou’ at maturity. (A) Fruit appearance at maturity; (B) longitudinal diameter; (C) transverse diameter; (D) fruit shape index; (E) peel thickness; (F) single fruit weight; (G) peel weight; (H) pulp weight; (I) edible rate. Note: SHZ: ‘Hainan Qingyou’ fruit grafted onto ‘Sanhong’ interstock; QYZ: ‘Hainan Qingyou’ fruit grafted onto ‘Hainan Qingyou’ interstock. Statistical significance (Student’s t-test) is indicated as follows: ** p < 0.01, *** p < 0.001, ns p ≥ 0.05. n = 6.
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Figure 2. Effects of ‘Sanhong’ interstock on internal quality of ‘Hainan Qingyou’ fruit. (A) Pulp firmness; (B) soluble solids content; (C) total organic acid content; (D) sugar–acid ratio; (E) AsA content; (F) total flavonoid content; (G) total phenol content; (H) lignin content; (I) lignin staining experiment. Note: SHZ: ‘Hainan Qingyou’ grafted onto ‘Sanhong’ interstock; QYZ: ‘Hainan Qingyou’ grafted onto ‘Hainan Qingyou’ interstock; D: developmental stage; M: mature stage. Statistical significance (Student’s t-test) is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, ns p ≥ 0.05. (A–D): n = 6; (E–H): n = 3.
Figure 2. Effects of ‘Sanhong’ interstock on internal quality of ‘Hainan Qingyou’ fruit. (A) Pulp firmness; (B) soluble solids content; (C) total organic acid content; (D) sugar–acid ratio; (E) AsA content; (F) total flavonoid content; (G) total phenol content; (H) lignin content; (I) lignin staining experiment. Note: SHZ: ‘Hainan Qingyou’ grafted onto ‘Sanhong’ interstock; QYZ: ‘Hainan Qingyou’ grafted onto ‘Hainan Qingyou’ interstock; D: developmental stage; M: mature stage. Statistical significance (Student’s t-test) is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, ns p ≥ 0.05. (A–D): n = 6; (E–H): n = 3.
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Figure 3. Effects of ‘Sanhong’ interstock on antioxidant enzyme activities and antioxidant capacity in ‘Hainan Qingyou’ pulp. (A) GR activity; (B) POD activity; (C) SOD activity; (D) hydroxyl radical scavenging capacity; (E) total antioxidant capacity (DPPH methods); (F) total antioxidant capacity (FRAP methods). Note: SHZ: ‘Hainan Qingyou’ grafted onto ‘Sanhong’ interstock; QYZ: ‘Hainan Qingyou’ grafted onto ‘Hainan Qingyou’ interstock; D: developmental stage; M: mature stage. Statistical significance (Student’s t-test) is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, ns p ≥ 0.05. n = 3.
Figure 3. Effects of ‘Sanhong’ interstock on antioxidant enzyme activities and antioxidant capacity in ‘Hainan Qingyou’ pulp. (A) GR activity; (B) POD activity; (C) SOD activity; (D) hydroxyl radical scavenging capacity; (E) total antioxidant capacity (DPPH methods); (F) total antioxidant capacity (FRAP methods). Note: SHZ: ‘Hainan Qingyou’ grafted onto ‘Sanhong’ interstock; QYZ: ‘Hainan Qingyou’ grafted onto ‘Hainan Qingyou’ interstock; D: developmental stage; M: mature stage. Statistical significance (Student’s t-test) is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, ns p ≥ 0.05. n = 3.
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Figure 4. KEGG enrichment analysis of differentially expressed genes in the (A) SHZ-D vs. QYZ-D and (B) SHZ-M vs. QYZ-M comparison groups.
Figure 4. KEGG enrichment analysis of differentially expressed genes in the (A) SHZ-D vs. QYZ-D and (B) SHZ-M vs. QYZ-M comparison groups.
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Figure 5. Expression patterns of differentially expressed genes (DEGs) involved in lignin biosynthesis in SHZ and QYZ pulp. The heatmap colors represent FPKM values from low (blue) to high (red).
Figure 5. Expression patterns of differentially expressed genes (DEGs) involved in lignin biosynthesis in SHZ and QYZ pulp. The heatmap colors represent FPKM values from low (blue) to high (red).
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Figure 6. Expression patterns of DEGs associated with plant hormone signaling pathways. Upregulated genes in SHZ are marked with red boxes, downregulated with blue boxes.
Figure 6. Expression patterns of DEGs associated with plant hormone signaling pathways. Upregulated genes in SHZ are marked with red boxes, downregulated with blue boxes.
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Figure 7. (A) Weighted gene co-expression network analysis (WGCNA) of DEGs. Module–trait correlations and corresponding p-values (in parentheses) are shown. Left: genes clustered into 12 modules based on expression patterns. Right: color scale indicates module–trait correlations from −1 (blue) to 1 (red). (B) Hub genes in blue, green, and black modules visualized using Cytoscape_v3.10.2. Hub TFs are shown as large circles, other genes as small nodes. Numbers in parentheses indicate connectivity; circle size and color intensity reflect connectivity. Note: For each module, the numbers without parentheses represent the p-values for the correlation between module genes and the trait, while those in parentheses represent the corresponding q-values (FDR).
Figure 7. (A) Weighted gene co-expression network analysis (WGCNA) of DEGs. Module–trait correlations and corresponding p-values (in parentheses) are shown. Left: genes clustered into 12 modules based on expression patterns. Right: color scale indicates module–trait correlations from −1 (blue) to 1 (red). (B) Hub genes in blue, green, and black modules visualized using Cytoscape_v3.10.2. Hub TFs are shown as large circles, other genes as small nodes. Numbers in parentheses indicate connectivity; circle size and color intensity reflect connectivity. Note: For each module, the numbers without parentheses represent the p-values for the correlation between module genes and the trait, while those in parentheses represent the corresponding q-values (FDR).
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Figure 8. Expression of candidate genes analyzed by qPCR and RNA-seq, n = 3.
Figure 8. Expression of candidate genes analyzed by qPCR and RNA-seq, n = 3.
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Yang, C.; Yang, C.; Li, H.; Jiang, C. Transcriptomic Analysis Reveals the Impact of Interstock on Vesicle Granulation in ‘Hainan Qingyou’ Pomelo (Citrus maxima) Fruit. Horticulturae 2025, 11, 1230. https://doi.org/10.3390/horticulturae11101230

AMA Style

Yang C, Yang C, Li H, Jiang C. Transcriptomic Analysis Reveals the Impact of Interstock on Vesicle Granulation in ‘Hainan Qingyou’ Pomelo (Citrus maxima) Fruit. Horticulturae. 2025; 11(10):1230. https://doi.org/10.3390/horticulturae11101230

Chicago/Turabian Style

Yang, Chengchao, Chengkun Yang, Haibo Li, and Chengdong Jiang. 2025. "Transcriptomic Analysis Reveals the Impact of Interstock on Vesicle Granulation in ‘Hainan Qingyou’ Pomelo (Citrus maxima) Fruit" Horticulturae 11, no. 10: 1230. https://doi.org/10.3390/horticulturae11101230

APA Style

Yang, C., Yang, C., Li, H., & Jiang, C. (2025). Transcriptomic Analysis Reveals the Impact of Interstock on Vesicle Granulation in ‘Hainan Qingyou’ Pomelo (Citrus maxima) Fruit. Horticulturae, 11(10), 1230. https://doi.org/10.3390/horticulturae11101230

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