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.

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.

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.

Lignin is crucial to the formation of fruit texture quality. Here, we aimed to explore the relationship between lignin metabolism and fruit texture by investigating the lignin content, total phenols and their related enzyme activities among three Chinese olive (Canarium album (Lour.) Raeusch) genotypes. Our results showed that lignin deposition moved from the exocarp to the flesh in Chinese olive fruit. The lignin, total phenols and enzyme activities were all different between the three Chinese olive cultivars at each developmental stage. The lignin content was positively correlated with the PAL, 4CL and POD activities. These results demonstrated that lignin metabolism was regulated through the related enzyme activities. Therefore, our findings may provide insight to facilitate further improvement in fruit texture quality in Chinese olive.

To gain a deeper understanding of the mechanisms by which cushioning packaging preserves the quality of Chinese olive fruits during cold chain transportation and extends their shelf life, this study simulated cold chain conditions and investigated the effects of cushioning packaging on the physiology, antioxidant capacity, and secondary metabolites of fruits during a 20-day shelf life. The results indicated that the decay rate in cushioning-packaging-treated fruit was 75% lower than that in the unbuffered packaging fruit at day 20 of shelf life. Simultaneously, cushioning packaging treatment mitigated the damage severity of the cell membrane structure and kept the cell membrane permeability at a low level, which was 15.34% lower than that in the unbuffered packaging fruit at day 20 of shelf life. Additionally, cushioning packaging effectively restrained the increases in malondialdehyde (MDA) content and alleviated the decline in chlorophyll and total flavonoid contents. It kept a balance among reactive oxygen species (ROS), antioxidant levels, and antioxidant enzyme activities, thereby reducing mechanical-damage-induced decay rates in Chinese olive fruits during the shelf life. Furthermore, metabolome analysis of Chinese olives during the shelf life was performed comparing those without buffered packaging to those with buffered packaging. The metabolome analysis found that the flavonoid biosynthetic pathway exhibited a higher accumulation of chrysin, neohesperidin, naringenin chalcone, sakuranetin, quercetin, catechin, and naringenin metabolites in cushion-packaging treatment compared to those without cushioning treatment. Furthermore, within the phenylalanine metabolic pathway, the accumulation of phenylalanine, p-coumaraldehyde, p-coumaric acid, coniferin and caffeoyl quinic acid metabolites was significantly higher in buffered-packaging groups compared to those without buffering. Together, these findings suggest that cushioning packaging can effectively sustain the integrity of cell membranes and enhance the shelf-life quality of Chinese olive fruits by regulating the balance of ROS and mitigating oxidative stress during cold chain transportation.