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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Carotenoids in plant foods provide health benefits by functioning as provitamin A. One of the vital provitamin A carotenoids, β-cryptoxanthin, is typically plentiful in citrus fruit. However, little is known about the genetic basis of β-cryptoxanthin accumulation in citrus. Here, we performed a widely targeted metabolomic analysis of 65 major carotenoids and carotenoid derivatives to characterize carotenoid accumulation in Citrus and determine the taxonomic profile of β-cryptoxanthin. We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of β-cryptoxanthin accumulation through a genome-wide association study. We identified a causal gene, CitCYP97B, which encodes a cytochrome P450 protein whose substrate and metabolic pathways in land plants were undetermined. We subsequently demonstrated that CitCYP97B functions as a novel monooxygenase that specifically hydroxylates the β-ring of β-cryptoxanthin in a heterologous expression system. In planta experiments provided further evidence that CitCYP97B negatively regulates β-cryptoxanthin content. Using the sequenced Citrus accessions, we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B, thereby altering β-cryptoxanthin accumulation in fruit. Hybridization/introgression appear to have contributed to the prevalence of two cis-element variations in different Citrus types during citrus evolution. Overall, these findings extend our understanding of the regulation and diversity of carotenoid metabolism in fruit crops and provide a genetic target for production of β-cryptoxanthin-biofortified products., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Domesticated citrus varieties are woody perennials and interspecific hybrid crops of global economic and nutritional importance. The citrus fruit "hesperidium" is a unique morphological innovation not found in any other plant lineage. Efforts to improve the nutritional quality of the fruit are predicated on understanding the underlying regulatory mechanisms responsible for fruit development, including temporal control of chlorophyll degradation and carotenoid biosynthesis. Here, we investigated the molecular basis of the navel orange (Citrus sinensis) brown flavedo mutation, which conditions flavedo that is brown instead of orange. To overcome the limitations of using traditional genetic approaches in citrus and other woody perennials, we developed a strategy to elucidate the underlying genetic lesion. We used a multi-omics approach to collect data from several genetic sources and plant chimeras to successfully decipher this mutation. The multi-omics strategy applied here will be valuable in driving future gene discovery efforts in citrus as well as in other woody perennial plants. The comparison of transcriptomic and genomic data from multiple genotypes and plant sectors revealed an underlying lesion in the gene encoding STAY-GREEN (SGR) protein, which simultaneously regulates carotenoid biosynthesis and chlorophyll degradation. However, unlike SGR of other plant species, we found that the carotenoid and chlorophyll regulatory activities could be uncoupled in the case of certain SGR alleles in citrus and thus we propose a model for the molecular mechanism underlying the brown flavedo phenotype. The economic and nutritional value of citrus makes these findings of wide interest. The strategy implemented, and the results obtained, constitute an advance for agro-industry by driving opportunities for citrus crop improvement., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Carotenoids are important antioxidant components in the human diet. To develop carotenoid-rich agricultural products by genetic intervention, understanding the genetic basis of carotenoids variation is essential. In this study, we constructed a high-density integrated genetic map with 3817 molecular markers using specific locus amplified fragment (SLAF) sequencing from a C. reticulata × P. trifoliata F 1 pseudotestcross population. A total of 17 significant quantitative trait loci (QTLs) distributed on Chromosomes (Chr) 2, 3, 5, 6, and 9 were detected to determine the carotenoid variation in the population. In particular, three QTL colocalizations for multiple carotenoid constituents were observed on Chr 2, 3, and 9, one of which was located on Chr2:34,654,608-35430715 accounted for 20.1-25.4% of the variation of luteoxanthin, auroxanthin, lutein, violaxanthin, and total carotenoid content. Overall, this study provides a genetic foundation for marker-assisted selection (MAS) breeding of nutritionally enhanced citrus fruit.

In plants, the carotenoid cleavage dioxygenase 4 (CCD4) could target on plastoglobules and cleave specific carotenoids, producing apocarotenoids and volatile compounds. These compounds are important for color and aroma formation in fruits and flowers. In this study, five CCD4 gene members (CCD4a, b, c, d, and e) were investigated in different citrus species including mandarin, pummelo, and sweet orange. Sequence analysis showed that the CCD4 genes from all the species examined exhibited extensive allelic variability (including SNPs and frame-shift mutations). Furthermore, the distribution of the CCD4 allelic mutation sites supported our previous hypothesis that the sweet orange originated from the hybridization of mandarin and pummelo. A derived cleaved amplified polymorphic sequence (dCAPs) marker was then successfully developed based on the allelic polymorphism of CCD4c, providing an ideal molecular marker for studying the genetic relationship between citrus species. Quantitative RT-PCR analysis identified differential expression patterns for the CCD4 genes in tissues/organs, and CCD4b was shown to have a high-level expression in citrus fruit flavedos (especially those with a deep orange-reddish color). HPLC-based detection of a key component (i.e., β-citraurin) for orange-reddish flavedo formation in different citrus revealed a positive correlation between CCD4b expression levels and the presence of β-citraurin, suggesting that CCD4b may be responsible for β-citraurin biosynthesis in flavedo. In summary, this study not only reinforced the anticipated roles of CCD4 genes in flavedo color formation in citrus, but also provided new information about gene expression patterns, allelic polymorphism characteristics, and sequence variability for this gene subfamily.

Summary: Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA‐mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism‐related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid‐driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5‐CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5‐CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5‐CsbZIP44 precisely modulates ABA signal‐mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops. [ABSTRACT FROM AUTHOR]

SUMMARY: Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non‐photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty‐four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG‐localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi‐feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops. [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]