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Uneven coloration is a common phenomenon in citrus fruit during the ripening stage, as affects the appearance and economic value of the fruit. The elevated expression of CsERF003 during the degreening process of both lemon and satsuma mandarin peels was reported. In this research, a similar performance of CsERF003 in the pericarp coloration process was also identified by transcriptome analysis of 'Fengjie 72–1' navel orange and Lane Late navel orange. However, the regulatory mechanism of CsERF003 is not clear yet. Overexpression of CsERF003 could deepen the color of citrus callus and promote peel degreening of Newhall navel orange, which was attributed to the upregulation of genes involved in chlorophyll degradation and carotenoid synthesis. Furthermore, CsERF003 acted as an activator to promote the expression of CsLCYE , but couldn't activate the expression of CsLCYB1 and CsLCYB2 ; CsERF003 could also bind to the promoter of CsSGR to activate its expression. Together, our findings shed light on the regulatory mechanism of CsERF003 in chlorophyll degradation and carotenoid accumulation, particularly in the α-branch of carotenoid metabolism. These insights offer valuable perspectives for the genetic enhancement of peel coloration in citrus. [Display omitted] • Overexpression of CsERF003 deepened the color of citrus callus and promoted peel degreening in Newhall navel orange. • CsERF003 activated the expression of CsLCYE and promoted the accumulation of lutein in the α-branch. • CsERF003 activated the expression of CsSGR and promoted the degradation of chlorophyll in the pericarp. [ABSTRACT FROM AUTHOR]

• 15 genotypes of F. hindsii were utilized to select the optimal genotypes for transformation. • The T0 generation produced highly chimeric transgenic plants. • the editing types of T1 generation more complex, which may be due to apomixis in citrus. • CRISPR/Cas9 vector was improved by using the endogenous U6 promoter in F. hindsii. The CRISPR/Cas9 editing system is presently regarded as one of the most efficient tools for genome editing and has been implemented in perennial fruit crops. However, the characteristics of transgenic plants and their subsequent generation have rarely been reported in fruit trees. In this study, 15 distinct preserved germplasm resources of Hongkong kumquat (Fortunella hindsii) were utilized to selecting suitable genotypes for genetic transformation. The shoot regeneration efficiency, seed number per fruit, and epicotyl diameter displaid a moderate positive correlation with transformation efficiency, with correlation coefficients of 0.6267, 0.5429, and 0.5388, respectively. The transcription factor RKD family gene FhRWP was knocked out in kumquat, resulting in the production of seven T0 and 34 T1 plants. Editing efficiency of T0 generation ranged from 1.62% to 68.11%, with a significant difference in editing activity among cases. Up to 23 editing types were detected in a single positive transformant, demonstrating that the high degree of citrus editing chimerism. Not all of T1 lines exhibited an increase in editing efficiency relative to T0. The descendants of T0-A2/A3 were all negative, while the other group of T1 lines showed both higher chimera and editing efficiencies of up to 92% than the others. Finally, the CRISPR/Cas9 vector was improved by using the endogenous U6 promoter in F. hindsii. This study provides novel insights into the application of the CRISPR/Cas9 system in asexual plants and serves as a reference for gene function verification in citrus and other species. [ABSTRACT FROM AUTHOR]

The fruit quality of 6 citrus cultivars growing in the same orchard was determined at ripening stage in both 2014 and 2015. We further measured the components of sugar (sucrose, fructose and glucose), organic acid (citric, malate and quinic acid), enzymes related to Glycolysis and Krebs cycle and mineral elements at 5 stages of fruit development in the second year. The results showed that at ripening stage of both years, ‘Newhall’ cultivar had higher TSS concentration and the TSS/TA ratio but lower TA concentration, while ‘Flame’ cultivar was exactly opposite. Sucrose and citric acid were the most accumulated compounds in fruit during the fruit development of 6 citrus cultivars. Fruit sucrose concentration increased from 9.26 mg·kg −1 at 60 DAFB to 50.92 mg·kg −1 at 180 DAFB, and the citric acid concentration increased from 1.41 mg·kg −1 at 60 DAFB to 29.87 mg·kg −1 at 90 DAFB or 29.02 mg·kg −1 at 120 DAFB then decreased till ripening (5.47 mg·kg −1 ). We found ACO was the key enzyme resulting in the difference of citric acid accumulation, but not quite clear in sucrose metabolism. The fruit mineral nutrient concentrations of 6 cultivars during the fruit development were 0.94–1.92% of N, 0.11–0.23% of P, 1.03–1.37% of K, 0.31–1.15% of Ca, 0.11–0.29% of Mg, 3.97–72.34 mg·Fe kg −1 , 1.93–10.64 mg·Mn kg −1 , 1.56–10.73 mg·Cu kg −1 , and 0.90–16.80 mg·Zn kg −1 . We also analyzed the relationship among each sugar, organic acid component and mineral nutrient in this study by curve estimation and PCA analysis. The results indicated that only Mg was significantly correlated with both sugar and organic acid component, negative and positive respectively. It suggested that the accumulation of sugar and organic acid might be related to the dynamic changes of fruit Mg concentrations of 6 citrus cultivars. [ABSTRACT FROM AUTHOR]

The volatile profiles of fruit peels and juice sacs from 108 citrus accessions representing seven species were analyzed. Using GC–MS 162 and 107 compounds were determined in the peels and juice sacs, respectively. In the peels, monoterpene alcohols were accumulated in loose-skin mandarins; clementine tangerines and papedas were rich in sesquiterpene alcohols, sesquiterpenes, monoterpene alcohols and monoterpene aldehydes. β -pinene and sabinene were specifically accumulated in 4 of 5 lemon germplasms. Furthermore, concentrations of 34 distinctive compounds were selected to best represent the volatile profiles of seven species for HCA analysis, and the clustering results were in agreement with classic citrus taxonomy. Comparison of profiles from different growing seasons and production areas indicated that environmental factors play important roles in volatile metabolism. In addition, a few citrus germplasms that accumulated certain compounds were determined as promising breeding materials. Notably, volatile biosynthesis via MVA pathway in C. ichangensis ‘Huaihua’ was enhanced. [ABSTRACT FROM AUTHOR]

Due to the furanocoumarin compounds in the fruit, the production and consumption of grapefruit have been affected in the past decades since the ‘grapefruit juice effect’ was declared. To provide elite germplasm and obtain knowledge for future citrus breeding programs, the contents of 4 furanocoumarin monomers (FCMs) in the juice sacs from 73 citrus germplasms were evaluated using ultra-performance liquid chromatography. 6′,7′-Dihydroxybergamottin and bergamottin were dominant in all the tested grapefruits, while there were some pomelos with dominant epoxybergamottin, and some with dominant 6′,7′-dihydroxybergamottin and bergamottin. The contents of FCMs were low or below detection in sweet oranges, mandarins, lemons and trifoliate oranges. The results also show that the dominant patterns of FCMs are genotype-related, and crossing and selection are effective approaches to alter FCM profiles in citrus breeding. Furthermore, the contribution of pomelo as a parent to grapefruit regarding their FCM profiles was discussed. [ABSTRACT FROM AUTHOR]

• Eleven PMFs in 116 citrus accessions were determined by UPLC–ESI-QTOF-MS/MS and HPLC–DAD. • The spatiotemporal and species-specific distribution of PMFs in citrus was revealed. • The clustering of eleven PMFs by HCA agreed with classic citrus taxonomy. • Wild or semi-wild mandarins contained large amounts of total or specific PMFs. • PMF accumulation may be a phenotype inherited from mandarin ancestors. Citrus fruits are the main dietary source of polymethoxylated flavones (PMFs) with significant effects on consumer health. In this study, eleven main PMFs were evaluated in the fruit flavedo or leaves of 116 citrus accessions via UPLC–DAD–ESI-QTOF-MS/MS combined with HPLC–DAD analysis, which revealed significant species-specific and spatiotemporal characteristics. All Citrus reticulata and their natural or artificial hybrids were found to have detectable PMFs, especially in the fruit flavedo of the wild or early-cultivated mandarins at early fruit development stages. However, PMFs were not detected in citrons, pummelos, kumquats, trifoliata oranges, papedas, Chinese box oranges and 'Mangshanyegan'. The results enlightened that PMF accumulation only in mandarins and mandarin hybrids is a phenotype inherited from mandarin ancestors. This study provides a comprehensive PMF profile in various citrus germplasms and will benefit future functional citrus breeding practices aimed at designing plants rich in total or specific PMFs for health benefits. [ABSTRACT FROM AUTHOR]

• This study focused on the observation of chromoplast ultrastructure in citrus flesh. • Carotenoid showed tissue-specific accumulation in sub-divided tissues of grapefruits. • Lycopene accumulation was closely related to crystalline chromoplasts formation. Citrus flesh color due to the accumulation of carotenoids in chromoplasts is one of the most important traits for breeding. However, the relationship between carotenoids and chromoplast ultrastructure in citrus is largely unclear. Thus, in this study fourteen representative citrus cultivars including those with yellow, orange, and red flesh were comparatively analyzed by light microscopy, exhibiting three types of chromoplasts (i.e. globular, fusiform and crystalloid). Significant differences in carotenoid content and cell wall thickness were found in sub-divided tissues of three grapefruits by high performance liquid chromatography and transmission electron microscopy. Based on our findings, it could be inferred that the accumulation of lycopene was closely related to the formation of crystalline chromoplasts. In the meantime, 'Flame' grapefruit with high content of carotenoids and cells with thin cell wall would have greater bio-accessibility, which indicated that 'Flame' grapefruit was superior to 'Star Ruby' grapefruit in the processing of citrus pulp juice. In conclusion, bio-accessibility influenced by chromoplasts and cell wall should be taken into consideration in citrus nutritional breeding, and differences in carotenoid accumulation and chromoplast morphology with tissue-specific characteristics would provide a new insight for further research. [ABSTRACT FROM AUTHOR]