Sugar is a primary determinant of citrus fruit flavour, but undergoes varied accumulation processes across different citrus varieties owing to high genetic variability. Sucrose phosphate synthase (SPS), a key enzyme in glucose metabolism, plays a crucial role in this context. Despite its significance, there is limited research on sugar component quality and the expression and regulatory prediction of SPS genes during citrus fruit development. Therefore, we analysed the sugar quality formation process in ‘Kiyomi’ and ‘Succosa’, two citrus varieties, and performed a comprehensive genome-wide analysis of citrus CsSPSs. We observed that the accumulation of sugar components significantly differs between the two varieties, with the identification of four CsSPSs in citrus. CsSPS sequences were highly conserved, featuring typical SPS protein domains. Expression analysis revealed a positive correlation between CsSPS expression and sugar accumulation in citrus fruits. However, CsSPS expression displays specificity to different citrus tissues and varieties. Transcriptome co-expression network analysis suggests the involvement of multiple transcription factors in shaping citrus fruit sugar quality through the regulation of CsSPSs. Notably, the expression levels of four CsWRKYs (CsWRKY2, CsWRKY20, CsWRKY28, CsWRKY32), were significantly positively correlated with CsSPSs and CsWRKY20 might can activate sugar accumulation in citrus fruit through CsSPS2. Collectively, we further emphasize the potential importance of CsWRKYs in citrus sugar metabolism, our findings serve as a reference for understanding sugar component formation and predicting CsSPS expression and regulation during citrus fruit development.

In autumn and spring, citrus leaves with a Ponkan (Citrus reticulata Blanco cv. Ponkan) genetic background (Harumi, Daya, etc.) are prone to abnormal physiological chlorosis. The effects of different degrees of chlorosis (normal, mild, moderate and severe) on photosynthesis and the chlorophyll metabolism of leaves of Citrus cultivar (Harumi) were studied via field experiment. Compared with severe chlorotic leaves, the results showed that chlorosis could break leaf metabolism balance, including reduced chlorophyll content, photosynthetic parameters, antioxidant enzyme activity and enzyme activity related to chlorophyll synthesis, increased catalase and decreased enzyme activity. In addition, the content of chlorophyll synthesis precursors showed an overall downward trend expected for uroporphyrinogen III. Furthermore, the relative expression of genes for chlorophyll synthesis (HEMA1, HEME2, HEMG1 and CHLH) was down-regulated to some extent and chlorophyll degradation (CAO, CLH, PPH, PAO and SGR) showed the opposite trend with increased chlorosis. Changes in degradation were more significant. In general, the chlorosis of Harumi leaves might be related to the blocked transformation of uroporphyrinogen III (Urogen III) to coproporphyrinogen III (Coprogen III), the weakening of antioxidant enzyme system activity, the weakening of chlorophyll synthesis and the enhancement in degradation.

During fresh fruit consumption, sensory texture is one factor that affects the organoleptic qualities. Chemical components of plant cell walls, including pectin, cellulose, hemicellulose and lignin, play central roles in determining the textural qualities. To explore the genes and regulatory pathways involved in fresh citrus’ perceived sensory texture, we performed mRNA-seq analyses of the segment membranes of two citrus cultivars, Shiranui and Kiyomi, with different organoleptic textures. Segment membranes were sampled at two developmental stages of citrus fruit, the beginning and end of the expansion period. More than 3000 differentially expressed genes were identified. The gene ontology analysis revealed that more categories were significantly enriched in ‘Shiranui’ than in ‘Kiyomi’ at both developmental stages. In total, 108 significantly enriched pathways were obtained, with most belonging to metabolism. A detailed transcriptomic analysis revealed potential critical genes involved in the metabolism of cell wall structures, for example, GAUT4 in pectin synthesis, CESA1, 3 and 6, and SUS4 in cellulose synthesis, CSLC5, XXT1 and XXT2 in hemicellulose synthesis, and CSE in lignin synthesis. Low levels, or no expression, of genes involved in cellulose and hemicellulose, such as CESA4, CESA7, CESA8, IRX9 and IRX14, confirmed that secondary cell walls were negligible or absent in citrus segment membranes. A chemical component analysis of the segment membranes from mature fruit revealed that the pectin, cellulose and lignin contents, and the segment membrane’s weight (% of segment) were greater in ‘Kiyomi’. Organoleptic quality of citrus is easily overlooked. It is mainly determined by sensory texture perceived in citrus segment membrane properties. We performed mRNA-seq analyses of citrus segment membranes to explore the genes and regulatory pathways involved in fresh citrus’ perceived sensory texture. Transcriptomic data showed high repeatability between two independent biological replicates. The expression levels of genes involved in cell wall structure metabolism, including pectin, cellulose, hemicellulose and lignin, were investigated. Meanwhile, chemical component contents of the segment membranes from mature fruit were analyzed. This study provided detailed transcriptional regulatory profiles of different organoleptic citrus qualities and integrated insights into the mechanisms affecting citrus’ sensory texture.

Sugars and organic acids are the main factors determining the flavor of citrus fruit. The WRKY transcription factor family plays a vital role in plant growth and development. However, there are still few studies about the regulation of citrus WRKY transcription factors (CsWRKYs) on sugars and organic acids in citrus fruit. In this work, a genome-wide analysis of CsWRKYs was carried out in the citrus genome, and a total of 81 CsWRKYs were identified, which contained conserved WRKY motifs. Cis-regulatory element analysis revealed that most of the CsWRKY promoters contained several kinds of hormone-responsive and abiotic-responsive cis-elements. Furthermore, gene expression analysis and fruit quality determination showed that multiple CsWRKYs were closely linked to fruit sugars and organic acids with the development of citrus fruit. Notably, transcriptome co-expression network analysis further indicated that three CsWRKYs, namely, CsWRKY3, CsWRKY47, and CsWRKY46, co-expressed with multiple genes involved in various pathways, such as Pyruvate metabolism and Citrate cycle. These CsWRKYs may participate in the metabolism of fruit sugars and organic acids by regulating carbohydrate metabolism genes in citrus fruit. These findings provide comprehensive knowledge of the CsWRKY family on the regulation of fruit quality.

Loss of quality in citrus fruit is a common occurrence during postharvest storage due to oxidative stress and energy consumption. In recent years, glycine betaine (GB) has been widely applied to postharvest horticulture fruit. This study aimed to investigate the effect of GB treatment (10 mM and 20 mM) on the quality and antioxidant activity of ‘Huangguogan’ fruit during postharvest storage at room temperature. Our results indicated that both 10 mM and 20 mM treatments effectively reduced weight and firmness losses and maintained total soluble solid (TSS), titratable acidity (TA), and ascorbic acid contents. Additionally, GB treatment significantly increased the activity of antioxidant enzymes, maintained higher levels of total phenols and total flavonoids, and led to slower accumulation of H₂O₂. A transcriptome analysis conducted at 28 days after treatment (DAT)identified 391 differentially expressed genes (DEGs) between 20 mM GB (GB-2) and the control (CK) group. These DEGs were enriched in various pathways, particularly related to oxygen oxidoreductase, peroxidase activity, and flavonoid biosynthesis. Overall, the application of GB proved beneficial in enhancing the storability and extending the shelf life of ‘Huangguogan’ fruit.

Xyloglucan endotransglycosylase (XET) genes are widely distributed in most plants, but the codon usage bias of XET genes has remained uncharacterized. Thus, we analyzed the codon usage bias using 4500 codons of 20 XET genes to elucidate the genetic and evolutionary patterns. Phylogenetic and hierarchical cluster analyses revealed that the 20 XET genes belonged to two groups. The closer the genetic distance, the more similar the codon usage preference. The codon usage bias of most XET genes was weak, but there was also some codon usage bias. AGA, AGG, AUC, and GUG were the top four codons (RSCU > 1.5) in the 20 XET genes. CitXET had a stronger codon usage bias, and there were eight optimal codons of CitXET (i.e., AGA, AUU, UCU, CUU, CCA, GCU, GUU, and AAA). The RSCU values underwent a correspondence analysis. The two main factors affecting codon usage bias (i.e., Axes 1 and 2) accounted for 54.8% and 17.6% of the total variation, respectively. Multiple correspondence analysis revealed that XET genes were widely distributed, with Group 1 genes being closer to Axis 1 than Group 2 genes, which were closer to Axis 2. Codons with A/U at the third codon position were distributed closer to Axis 1 than codons with G/C at the third codon position. PgXET, ZmXET, VlXET, VrXET, and PcXET were biased toward codons ending with G/C. In contrast, CitXET, DpXET, and BrpXET were strongly biased toward codons ending with A/U, indicating that these XET genes have a strong codon usage bias. Translational selection and base composition (especially A and U at the third codon position), followed by mutation pressure and natural selection, may be the most important factors affecting codon usage of 20 XET genes. These results may be useful in clarifying the codon usage bias of XET genes and the relevant evolutionary characteristics.