In the present study, the fruit physiology and sugar-acid ratio of 24 pomelo cultivars grown in ten different locations of the subtropical region of China were measured. The contents of soluble sugars and organic acids were quantified using high-performance (HPLC-MS) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), respectively. The results revealed that the physiological and basic quality attributes of 24 pomelo cultivars, including fruit weight, fruit width, fruit length, peel thickness, number of segments, pulp weight, pulp color, soluble solids, and Vitamin C, ranged between 264.63–1945.85 g, 8.60–19.56 g, 7.40–20.70 g, 0.46–3.33 mm, 11–18.66, 210.25–1351.66 g, 8.59–15.14 Brix°, and 34.79–84.58 mg/100 g, respectively. Soluble sugars, i.e., fructose, glucose, and sucrose, ranged between 16.25–24.25, 16.17–24.22, and 19.90–55.28 mg/g, respectively. Similarly, Organic acids, i.e., pyruvate, fumaric acids, succinic acid, tartaric acid, quinic acid, citric acid, malic acid, and cis-aconitic acid, in 24 pomelo cultivars ranged between 0.48–1.84, 0.02–0.45, 0–0.05, 0.01–0.1, 0–0.14, 3.01–11.85, 0.18–1.42, and 0.01–0.16 mg/g, respectively. The pomelo cultivars ‘Hongzuanmi’, ‘Minihong’, and ‘Hangwanmi’ exhibited maximum contents of citric acid and pyruvate and showed ultimately excessive organic acids. Overall, the ‘Guanximi’ and its budding cultivars, i.e., ‘Hongroumi’, ‘Huangjinmi’, and ‘Sanhongmi’, had the best quality fruits having maximum sugar-acid ratio. Correlation analysis showed that total soluble sugars had a significantly positive correlation with sucrose contents, while citric acids, malic acid, and pyruvate were positively correlated with total organic acids. The determined sugar-acid profile of pomelo cultivars provides the basis for future elucidation of key mechanisms regulating sugars and acids biosynthesis in pomelo.

Overaccumulation of lignin (a physiological disorder known as granulation) often occurs during fruit ripening and postharvest storage in pomelo (Citrus grandis). It causes an unpleasant fruit texture and taste. Previous studies have shown that lignin metabolism is closely associated with the process of juice sacs granulation. At present, the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate NAC transcription factor, CgNAC043, that is involved in the regulation of lignin biosynthesis in Citrus grandis, which has homologs in Arabidopsis and other plants. We used the fruit juice sacs of ‘San hong’ as the material, the staining for lignin with HCl−phloroglucinol of fruit juice sacs became dark red from the various developmental stages at 172 to 212 days post anthesis (DPA). The RT-qPCR was used to analyze the gene expression of CgNAC043 and its target gene CgMYB46 in fruit sacs, it was found that the expression trend of CgNAC043 was basically same as CgMYB46, which increased gradually and peaked at 212 DPA. The expression level of CgNAC043 in juice sacs obtained away from the core was the lowest, while those near the core and granulated area were highly expressed. The transcriptional activation activity of CgNAC043 and CgMYB46 was analyzed by a yeast two-hybrid system, with only CgNAC043 showing transcriptional activation activity in Y2H Gold yeast. A transformation vector, p1301- CgNAC043, was transformed into the mesocarp of ‘San hong’ by Agrobacterium-mediated transformation. Results showed that the expression of transcription factors CgMYB58 and CgMYB46 are all upregulated. Further experiments proved that CgNAC043 not only can directly trans-activate the promoter of CgMYB46 but also trans-activate the promoters for the lignin biosynthesis-related genes CgCCoAOMT and CgC3H by dual luciferase assay. We isolated the CgNAC043 gene in pomelo and found CgNAC043 regulates target genes conferring the regulation of juice sacs granulation.

Juice sac granulation (a physiological disorder) leads to large postharvest losses of pomelo (Citrus maxima). Previous studies have shown that juice sac granulation is closely related to lignin accumulation, while the molecular mechanisms underlying this disorder remain elusive in pomelo. Our results showed that the lignin content in NC (near the core) and FC (far away from the core) juice sacs overall increased from 157 DPA (days post anthesis) to 212 DPA and reached a maximum at 212 DPA. Additionally, the lignin content of NC juice sacs was higher than that of FC juice sacs. In this study, we used transcriptome-based weighted gene co-expression network analysis (WGCNA) to address how lignin formation in NC and FC juice sacs is generated during the development of pomelo. After data assembly and bioinformatic analysis, we found a most correlated module (black module) to the lignin content, then we used the 11 DEGs in this module as hub genes for lignin biosynthesis. Among these DEGs, PAL (phenylalanine ammonia lyase), HCT (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase), 4CL2 (4-coumarate: CoA ligase), C4H (cinnamate 4-hydroxylase), C3′H (p-coumarate 3-hydroxylase), and CCoAOMT1 (caffeoyl CoA 3-Omethyltransferase) were the most distinct DEGs in granulated juice sacs. Co-expression analysis revealed that the expression patterns of several transcription factors such as MYB, NAC, OFP6, and bHLH130 are highly correlated with lignin formation. In addition, the expression patterns of the DEGs related to lignin biosynthesis and transcription factors were validated by qRT-PCR, and the results were highly concordant with the RNA-seq results. These results would be beneficial for further studies on the molecular mechanism of lignin accumulation in pomelo juice sacs and would help with citrus breeding.

Phenolics in the Chinese olive (Canarium album (Lour.) Raeusch) fruit significantly affect its flavor and quality. The shikimate pathway is a bridge connecting primary metabolism and secondary metabolism through which fixed carbon can be transformed into phenolics. In this study, we aimed to reveal the relationship between the shikimate pathway and phenolic compound biosynthesis. Three Chinese olive fruits (cv. Tanxiang (TX), Changying (CY) and Lingfeng (LF)) with distinct flavor were utilized as materials. The results of this study showed that the synthesis and accumulation of quinate and gallate were active in the Chinese olive fruit. The accumulation amount of phenolic compounds was significantly different among the three cultivars. TX contained the highest content of ellagate, (iso)corilagin, conjugated quercetin and conjugated kaempferol; CY contained the highest content of conjugated luteolin; and LF contained the lowest content of ellagate, conjugated gallate, hyperin, conjugated quercetin, conjugated kaempferol and conjugated luteolin during fruit development. The expression of 3-dehydroquinate/shikimate dehydrogenase gene-4 (DHD/SDH-4), 3-dehydroquinate synthase gene (DHQS), chorismate synthase gene (CS) and Chorismate mutase gene-1 (CM-1) and shikimate content increased with the maturing of fruit. The gene 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase gene-1 (DAHPS-1) was most expressed in TX, while barely expressed in LF during fruit development. The expression of CM-1 was highest in CY. Chorismate mutase gene-2 (CM-2) expression was higher in TX and CY during late fruit development. The cultivars with higher expression of DAHPS-1 and Chorismate mutase genes (CMs) accumulated more phenolic compounds in fruit. DAHPS-1 and CMs are proposed as key genes for polyphenolic synthesis in the Chinese olive fruit. These results proved that shikimate metabolism had a positive effect on the phenols’ synthesis. Our study provides new insight into the regulatory mechanism of the biosynthesis and accumulation of phenolic compounds in the fruit of Chinese olive.

The granulation of pomelo (Citrus maxima) juice sacs severely compromises fruit quality and is closely associated with lignin accumulation, a process catalyzed by peroxidases (PODs). Analysis of ‘Sanhong’ pomelo juice sacs collected 175–215 days after flowering revealed that bound peroxidase (BPOD) activity paralleled changes in lignin content, suggesting a potential role for BPOD in lignin biosynthesis. A total of 71 CmPOD genes were identified in the pomelo genome through integrated HMMER and BLAST analyses. Among them, CmPOD52 was selected for functional characterization based on its alkaline peroxidase properties, absence of a CE domain, predicted extracellular localization, and gradually increasing expression pattern revealed by RT-qPCR. Its transient overexpression in ‘Sanhong’ pomelo juice sacs for 36 h increased BPOD activity 2.06-fold (p < 0.01) compared to the empty vector control, indicating that CmPOD52 may be a BPOD gene. The recombinant CmPOD52 protein was expressed in a prokaryotic system, purified, and used in enzymatic assays with sinapyl alcohol as the substrate. The recombinant CmPOD52 protein, assayed at 272 nm with controls (substrate-only blank and heat-inactivated protein), showed an activity of 13.67 ± 0.9 U. The experimental group showed new products, identified by mass spectrometry as sinapyl alcohol dimers, thus suggesting that the recombinant protein catalyzes the dehydrogenation and polymerization of sinapyl alcohol monomers. This study identified CmPOD52, a gene potentially involved in lignin polymerization in pomelo juice sacs, offering a key candidate for further in vivo validation.