Developing a simple and rapid method for cell-specific transcriptome analysis through laser microdissection: insights from citrus rind with broader implications.

BACKGROUND: With the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed. RESULTS: We omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 µm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach. CONCLUSIONS: We developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues.

Myo-inositol oxygenase CgMIOX3 alleviates S-RNase-induced inhibition of incompatible pollen tubes in pummelo.

Pummelo (Citrus grandis L. Osbeck) exhibits S-RNase-based self-incompatibility (SI), during which S-RNase cytotoxicity inhibits pollen tubes in an S-haplotype specific manner. The entry of S-RNase into self-pollen tubes triggers a series of reactions. However, these reactions are still poorly understood in pummelo. In the present study, we used S-RNases as baits to screen a pummelo pollen cDNA library and characterized a myo-inositol oxygenase (CgMIOX3) that physically interacts with S-RNases. CgMIOX3 is highly expressed in pummelo pollen tubes and its down-regulation leads to a reduction in pollen tube growth. Upon entering pollen tubes, S-RNases increase the expression of CgMIOX3 and enhance its activity by directly binding to it in an S-haplotype-independent manner. CgMIOX3 improves pollen tube growth under oxidative stress through ascorbic acid accumulation and increases the length of self-pollen tubes. Furthermore, over-expression of CgMIOX3 increases the relative length of self-pollen tubes growing in the style of petunia (Petunia hybrida). This study provides intriguing insights into the pumelo SI system, revealing a regulatory mechanism mediated by CgMIOX3 that plays an important role in the resistance of pollen tubes to S-RNase cytotoxicity.

A transcriptional cascade involving BBX22 and HY5 finely regulates both plant height and fruit pigmentation in citrus.

Dwarfing is a pivotal agronomic trait affecting both yield and quality. Citrus species exhibit substantial variation in plant height, among which internode length is a core element. However, the molecular mechanism governing internode elongation remains unclear. Here, we unveiled that the transcriptional cascade consisting of B-BOX DOMAIN PROTEIN 22 (BBX22) and ELONGATED HYPOCOTYL 5 (HY5) finely tunes plant height and internode elongation in citrus. Loss-of-function mutations of BBX22 in an early-flowering citrus (Citrus hindsii "SJG") promoted internode elongation and reduced pigment accumulation, whereas ectopic expression of BBX22 in SJG, sweet orange (C. sinensis), pomelo (C. maxima) or heterologous expression of BBX22 in tomato (Solanum lycopersicum) significantly decreased internode length. Furthermore, exogenous application of gibberellin A3 (GA3) rescued the shortened internode and dwarf phenotype caused by BBX22 overexpression. Additional experiments revealed that BBX22 played a dual role in regulation internode elongation and pigmentation in citrus. On the one hand, it directly bound to and activated the expression of HY5, GA metabolism gene (GA2 OXIDASE 8, GA2ox8), carotenoid biosynthesis gene (PHYTOENE SYNTHASE 1, PSY1) and anthocyanin regulatory gene (Ruby1, a MYB DOMAIN PROTEIN). On the other hand, it acted as a cofactor of HY5, enhancing the ability of HY5 to regulate target genes expression. Together, our results reveal the critical role of the transcriptional cascade consisting of BBX22 and HY5 in controlling internode elongation and pigment accumulation in citrus. Unraveling the crosstalk regulatory mechanism between internode elongation and fruit pigmentation provides key genes for breeding of novel types with both dwarf and health-beneficial fortification in citrus.

Adventitious embryonic causal gene FhRWP regulates multiple developmental phenotypes in citrus reproduction.

Citrus is a model plant for studying adventitious embryos, a form of asexual reproduction controlled by a single dominant gene, RWP. This gene has been identified as the causal gene for nucellar embryogenesis, but its function has not yet been fully understood. In this study, we used the fast-growing Fortunella hindsii as a system to explore chromatin accessibility during the nucellar embryony initiation, emphasizing elevated chromatin accessibility in polyembryonic (PO) genotypes compared to monoembryonic ones (MO). Notably, a higher level of accessible chromatin was observed in one allele of the promoter region of FhRWP, consistent with increased expression of the allele carrying the causal structural variant. By independently performing RNAi and gene editing experiments on PO genotypes, we found the downregulation of FhRWP expression could reduce the number of nucellar embryos, while its knockout resulted in abnormal axillary bud development. In overexpression experiments, FhRWP was identified as having the unique capability of inducing the embryogenic callus formation in MO stem segments, possibly through the regulation of the WUS-CLV signaling network and the ABA and cytokinin pathway, marking the inaugural demonstration of FhRWP's potential to reignite somatic cells' embryogenic fate. This study reveals the pleiotropic function of RWP in citrus and constructs a regulatory network during adventitious embryo formation, providing a new tool for bioengineering applications in plant regeneration.

Involvement of CgHSFB1 in the regulation of self-incompatibility in 'Shatian' pummelo.

As self-incompatibility is a major issue in pummelo breeding and production, its mechanism in citrus was analyzed to improve breeding efficiency and reduce production costs. Rutaceae belongs to S-RNase type of gametophytic self-incompatibility. While the function of S-RNase/SLF and the mechanism of self-incompatibility have been studied extensively, the transcriptional regulation of S-RNase has been less studied. We performed transcriptome sequencing with the styles of 'Shatian' pummelo on the day of anthesis and 1-5 days before anthesis, and found that the transcript level of S-RNase gradually decreased with flower development. By analyzing differentially expressed genes and correlation with the expression trend of S-RNase, we identified a candidate gene, CgHSFB1, and utilized biochemical experiments such as yeast one-hybrid assay, electrophoretic mobility shift assay and dual-luciferase assay, as well as transient transformation of citrus calli and Citrus microcarpa and demonstrated that CgHSFB1 could directly bind to the S1-RNase promoter and repress the expression of S1-RNase, which is involved in the pummelo self-incompatibility response. In contrast, CgHSFB1 did not bind to the promoter of S2-RNase, and there was specificity in the regulation of S-RNase.

CsERF003 enhanced peel coloration by promoting both chlorophyll degradation and carotenoid accumulation in citrus.

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.

An S-locus F-box protein as pollen S determinant targets non-self S-RNase underlying self-incompatibility in Citrus.

Self-incompatibility (SI) is a crucial mechanism that prevents self-fertilization and inbreeding in flowering plants. Citrus exhibits SI regulated by a polymorphic S-locus containing an S-RNase gene and multiple S-locus F-box (SLF) genes. It has been documented that S-RNase functions as the pistil S determinant, but there is no direct evidence that the SLF genes closely linked with S-RNase function as pollen S determinants in Citrus. This study assembled the genomes of two pummelo (Citrus grandis) plants, obtained three novel complete and well-annotated S-haplotypes, and isolated 36 SLF or SLF-like alleles on the S-loci. Phylogenetic analysis of 138 SLFs revealed that the SLF genes were classified into 12 types, including six types with divergent or missing alleles. Furthermore, transformation experiments verified that the conserved S6-SLF7a protein can lead to the transition of SI to self-compatibility by recognizing non-self S8-RNase in 'Mini-Citrus' plants (S7S8 and S8S29, Fortunella hindsii), a model plant for citrus gene function studies. In vitro assays demonstrated interactions between SLFs of different S haplotypes and the Skp1-Cullin1-F-box subunit CgSSK1 protein. This study provides direct evidence that SLF controls the pollen function in Citrus, demonstrating its role in the 'non-self recognition' SI system.

Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

Cytochrome P450 CitCYP97B modulates carotenoid accumulation diversity by hydroxylating ß-cryptoxanthin in Citrus.

Carotenoids in plant foods provide health benefits by functioning as provitamin A. One of the vital provitamin A carotenoids, ß-cryptoxanthin, is typically plentiful in citrus fruit. However, little is known about the genetic basis of ß-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 ß-cryptoxanthin. We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of ß-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 ß-ring of ß-cryptoxanthin in a heterologous expression system. In planta experiments provided further evidence that CitCYP97B negatively regulates ß-cryptoxanthin content. Using the sequenced Citrus accessions, we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B, thereby altering ß-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 ß-cryptoxanthin-biofortified products.

Transcription factor CrWRKY42 coregulates chlorophyll degradation and carotenoid biosynthesis in citrus.

Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for the coloration and nutritional value of fruits. However, the synergistic regulation of these 2 processes at the transcriptional level remains largely unknown. In this study, we identified a WRKY transcription factor, CrWRKY42, from the transcriptome data of the yellowish bud mutant "Jinlegan" ([Citrus unshiu × C. sinensis] × C. reticulata) tangor and its wild-type "Shiranui" tangor, which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 directly bound to the promoter of ß-carotene hydroxylase 1 (CrBCH1) and activated its expression. The overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in carotenoid biosynthesis, including phytoene desaturase (CrPDS) and lycopene ß-cyclase 2 (CrLCYB2). In addition, CrWRKY42 could bind to the promoters of NONYELLOW COLORING (CrNYC) and STAY-GREEN (CrSGR) and activate their expression, thus promoting chlorophyll degradation. The overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expression of genes involved in both pathways. Our data revealed that CrWRKY42 acts as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening.

Multiomics integrated with sensory evaluations to identify characteristic aromas and key genes in a novel brown navel orange (Citrus sinensis).

'Zong Cheng' navel orange (ZC) is a brown mutant of Lane Late navel orange (LL) and emits a more pleasant odor than that of LL. However, the key volatile compound of this aroma and underlying mechanism remains unclear. In this study, sensory evaluations and volatile profiling were performed throughout fruit development to identify significant differences in sensory perception and metabolites between LL and ZC. It revealed that the sesquiterpene content varied significantly between ZC and LL. Based on aroma extract dilution and gas chromatography-olfactometry analyses, the volatile compound leading to the background aroma of LL and ZC is d-limonene, the orange note in LL was mainly attributed to octanal, whilst valencene, ß-myrcene, and (E)-ß-ocimene presented balsamic, sweet, and herb notes in ZC. Furthermore, Cs5g12900 and six potential transcription factors were identified as responsible for valencene accumulation in ZC, which is important for enhancing the aroma of ZC.

Phased genomics reveals hidden somatic mutations and provides insight into fruit development in sweet orange.

Although revisiting the discoveries and implications of genetic variations using phased genomics is critical, such efforts are still lacking. Somatic mutations represent a crucial source of genetic diversity for breeding and are especially remarkable in heterozygous perennial and asexual crops. In this study, we focused on a diploid sweet orange (Citrus sinensis) and constructed a haplotype-resolved genome using high fidelity (HiFi) reads, which revealed 10.6% new sequences. Based on the phased genome, we elucidate significant genetic admixtures and haplotype differences. We developed a somatic detection strategy that reveals hidden somatic mutations overlooked in a single reference genome. We generated a phased somatic variation map by combining high-depth whole-genome sequencing (WGS) data from 87 sweet orange somatic varieties. Notably, we found twice as many somatic mutations relative to a single reference genome. Using these hidden somatic mutations, we separated sweet oranges into seven major clades and provide insight into unprecedented genetic mosaicism and strong positive selection. Furthermore, these phased genomics data indicate that genomic heterozygous variations contribute to allele-specific expression during fruit development. By integrating allelic expression differences and somatic mutations, we identified a somatic mutation that induces increases in fruit size. Applications of phased genomics will lead to powerful approaches for discovering genetic variations and uncovering their effects in highly heterozygous plants. Our data provide insight into the hidden somatic mutation landscape in the sweet orange genome, which will facilitate citrus breeding.

Molecular regulation of oil gland development and biosynthesis of essential oils in Citrus spp.

Secretory structures in terrestrial plants serve as reservoirs for a variety of secondary metabolites. Among these, the secretory cavity of the Rutaceae family is notable for containing essential oils with a wide range of applications. However, the molecular basis underlying secretory cavity development is unknown. Here, we reveal a molecular framework for Citrus oil gland formation. Using genetic mapping and genome editing, we demonstrated that this process requires LATE MERISTEM IDENTITY1 (LMI1), a key regulator of leaf serration. A conserved GCC box element of the LMI1 promoter recruits DORNROSCHEN-like (DRNL) for transcriptional activation. This DRNL-LMI1 cascade triggers MYC5 activation, facilitating the development of oil glands and the biosynthesis of essential oils. Our findings spotlight cis-regulatory divergence within leaf shape genes, propelling novel functional tissue formation.

The transcriptional regulatory module CsHB5-CsbZIP44 positively regulates abscisic acid-mediated carotenoid biosynthesis in citrus (Citrus spp.).

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.

Multi-omics analyses reveal the importance of chromoplast plastoglobules in carotenoid accumulation in citrus fruit.

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.

Transposable elements cause the loss of self-incompatibility in citrus.

Self-incompatibility (SI) is a widespread prezygotic mechanism for flowering plants to avoid inbreeding depression and promote genetic diversity. Citrus has an S-RNase-based SI system, which was frequently lost during evolution. We previously identified a single nucleotide mutation in Sm-RNase, which is responsible for the loss of SI in mandarin and its hybrids. However, little is known about other mechanisms responsible for conversion of SI to self-compatibility (SC) and we identify a completely different mechanism widely utilized by citrus. Here, we found a 786-bp miniature inverted-repeat transposable element (MITE) insertion in the promoter region of the FhiS2-RNase in Fortunella hindsii Swingle (a model plant for citrus gene function), which does not contain the Sm-RNase allele but are still SC. We demonstrate that this MITE plays a pivotal role in the loss of SI in citrus, providing evidence that this MITE insertion prevents expression of the S-RNase; moreover, transgenic experiments show that deletion of this 786-bp MITE insertion recovers the expression of FhiS2-RNase and restores SI. This study identifies the first evidence for a role for MITEs at the S-locus affecting the SI phenotype. A family-wide survey of the S-locus revealed that MITE insertions occur frequently adjacent to S-RNase alleles in different citrus genera, but only certain MITEs appear to be responsible for the loss of SI. Our study provides evidence that insertion of MITEs into a promoter region can alter a breeding strategy and suggests that this phenomenon may be broadly responsible for SC in species with the S-RNase system.

The kiwifruit amyloplast proteome (kfALP): a resource to better understand the mechanisms underlying amyloplast biogenesis and differentiation.

The biogenesis and differentiation (B&D) of amyloplasts contributes to fruit flavor and color. Here, remodeling of starch granules, thylakoids and plastoglobules was observed during development and ripening in two kiwifruit (Actinidia spp.) cultivars - yellow-fleshed 'Hort16A' and green-fleshed 'Hayward'. A protocol was developed to purify starch-containing plastids with a high degree of intactness, and amyloplast B&D was studied using label-free-based quantitative proteomic analyses in both cultivars. Over 3000 amyloplast-localized proteins were identified, of which >98% were quantified and defined as the kfALP (kiwifruit amyloplast proteome). The kfALP data were validated by Tandem-Mass-Tag (TMT) labeled proteomics in 'Hort16A'. Analysis of the proteomic data across development and ripening revealed: 1) a conserved increase in the abundance of proteins participating in starch synthesis/degradation during both amyloplast B&D; 2) up-regulation of proteins for chlorophyll degradation and of plastoglobule-localized proteins associated with chloroplast breakdown and plastoglobule formation during amyloplast differentiation; 3) constitutive expression of proteins involved in ATP supply and protein import during amyloplast B&D. Interestingly, two different pathways of amyloplast B&D were observed in the two cultivars. In 'Hayward', significant increases in abundance of photosynthetic- and tetrapyrrole metabolism-related proteins were observed, but the opposite trend was observed in 'Hort16A'. In conclusion, analysis of the kfALP provides new insights into the potential mechanisms underlying amyloplast B&D with relevance to key fruit quality traits in contrasting kiwifruit cultivars.

Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.).

Carotenoids directly influence citrus fruit color and nutritional value, which is critical to consumer acceptance. Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality. Despite the well-established carotenoid biosynthetic pathways, the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood. Our previous studies have reported that the Myc-type basic helix-loop-helix (bHLH) transcription factor (TF) regulates citrus proanthocyanidin biosynthesis. Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli, but its regulatory mechanism is still unclear. In the present study, we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR, CsHDS, CsHDR, CsPDS, CsLCYE, CsZEP, and CsNCED2, which was accompanied by changes in the contents of abscisic acid and gibberellin. The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR, CsHDS, and CsHDR, the key metabolic enzymes of the methylerythritol 4-phosphate (MEP) pathway, thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes. In addition, CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes, CsPDS, CsLCYE, CsZEP, and CsNCED2, directly promoting carotenoid biosynthesis. This study reveals a novel network of carotenoid metabolism regulated by CsTT8. Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.