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.

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis “Xuegan” plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.

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.

MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.