Summary
Purple carrots, the original domesticated carrots, accumulate highly glycosylated and acylated anthocyanins in root and/or petiole. Previously, a quantitative trait locus (QTL) for ...root‐specific anthocyanin pigmentation was genetically mapped to chromosome 3 of carrot. In this study, an R2R3‐MYB gene, namely DcMYB113, was identified within this QTL region. DcMYB113 expressed in the root of ‘Purple haze’, a carrot cultivar with purple root and nonpurple petiole, but not in the roots of two carrot cultivars with a purple root and petiole (Deep purple and Cosmic purple) and orange carrot ‘Kurodagosun’, which appeared to be caused by variation in the promoter region. The function of DcMYB113 from ‘Purple haze’ was verified by transformation in ‘Cosmic purple’ and ‘Kurodagosun’, resulting in anthocyanin biosynthesis. Transgenic ‘Kurodagosun’ carrying DcMYB113 driven by the CaMV 35S promoter had a purple root and petiole, while transgenic ‘Kurodagosun’ expressing DcMYB113 driven by its own promoter had a purple root and nonpurple petiole, suggesting that root‐specific expression of DcMYB113 was determined by its promoter. DcMYB113 could activate the expression of DcbHLH3 and structural genes related to anthocyanin biosynthesis. DcUCGXT1 and DcSAT1, which were confirmed to be responsible for anthocyanins glycosylation and acylation, respectively, were also activated by DcMYB113. The WGCNA identified several genes co‐expressed with anthocyanin biosynthesis and the results indicated that DcMYB113 may regulate anthocyanin transport. Our findings provide insight into the molecular mechanism underlying root‐specific anthocyanin biosynthesis and further modification in carrot and even other root crops.
The original domesticated carrots (
) are thought to have been purple, accumulating large quantities of anthocyanins in their roots. A quantitative trait locus associated with anthocyanin ...pigmentation in purple carrot roots has been identified on chromosome 3 and includes two candidate genes,
and
Here, we characterized the functions of
and
in carrots. Overexpression of
, but not
, in the orange carrot 'Kurodagosun' led to anthocyanin accumulation in roots. Knockout of
in the solid purple (purple periderm, phloem, and xylem) carrot 'Deep Purple' using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system resulted in carrots with yellow roots.
could activate the expression of its DcbHLH3 partner, a homolog of the anthocyanin-related apple (
) bHLH3, and structural genes in the anthocyanin biosynthetic pathway. We determined that the promoter sequence of
in nonpurple carrots was interrupted either by
, a nonfunctional tandem duplication of
, or by two transposons, leading to the transcriptional inactivation of
in nonpurple carrot roots. As a result, nonpurple carrots fail to accumulate anthocyanins in their roots. Our study supports the hypothesis that another genetic factor suppresses
expression in the phloem and xylem of purple peridermal carrot root tissues.
also regulated the glycosylation and acylation of anthocyanins by directly activating
and
We reveal the genetic factors conditioning anthocyanin pigmentation in purple versus nonpurple carrot roots. Our results also provide insights into the mechanisms underlying anthocyanin glycosylation and acylation.
SUMMARY
Carotenoids are important natural pigments that give bright colors to plants. The difference in the accumulation of carotenoids is one of the key factors in the formation of various colors in ...carrot taproots. Carotenoid cleavage dioxygenases (CCDs), including CCD and 9‐cis epoxycarotenoid dioxygenase, are the main enzymes involved in the cleavage of carotenoids in plants. Seven CCD genes have been annotated from the carrot genome. In this study, through expression analysis, we found that the expression level of DcCCD4 was significantly higher in the taproot of white carrot (low carotenoid content) than orange carrot (high carotenoid content). The overexpression of DcCCD4 in orange carrots caused the taproot color to be pale yellow, and the contents of α‐ and β‐carotene decreased sharply. Mutant carrot with loss of DcCCD4 function exhibited yellow color (the taproot of the control carrot was white). The accumulation of β‐carotene was also detected in taproot. Functional analysis of the DcCCD4 enzyme in vitro showed that it was able to cleave α‐ and β‐carotene at the 9, 10 (9′, 10′) double bonds. In addition, the number of colored chromoplasts in the taproot cells of transgenic carrots overexpressing DcCCD4 was significantly reduced compared with that in normal orange carrots. Results showed that DcCCD4 affects the accumulation of carotenoids through cleavage of α‐ and β‐carotene in carrot taproot.
Significance Statement
We analyzed seven carotenoid cleavage dioxygenase genes annotated from the carrot genome. Functional analysis revealed that DcCCD4 catalyzes the degradation of α‐ and β‐carotene to affect carotenoid accumulation. This work may provide a reference for generating new plant varieties with ideal color.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in ...carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using
Agrobacterium
-mediated genetic transformation. Four sites of
DcPDS
and
DcMYB113-like
genes were chosen as targets. Knockout of
DcPDS
in orange carrot ‘Kurodagosun’ resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency.
DcMYB113-like
was also successfully edited in purple carrot ‘Deep purple’, resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both
DcPDS
- and
DcMYB113-like
-knockout
T
0
plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.
The first domesticated carrots were thought to be purple carrots rich in anthocyanins. The anthocyanins biosynthesis in solid purple carrot taproot was regulated by DcMYB7 within P3 region containing ...a gene cluster of six DcMYBs. Here, we described a MYB gene within the same region, DcMYB11c, which was highly expressed in the purple pigmented petioles. Overexpression of DcMYB11c in ‘Kurodagosun’ (KRDG, orange taproot carrot with green petioles) and ‘Qitouhuang’ (QTHG, yellow taproot carrot with green petioles) resulted in deep purple phenotype in the whole carrot plants indicating anthocyanins accumulation. Knockout of DcMYB11c in ‘Deep Purple’ (DPPP, purple taproot carrot with purple petioles) through CRISPR/Cas9‐based genome editing resulted in pale purple phenotype due to the dramatic decrease of anthocyanins content. DcMYB11c could induce the expression of DcbHLH3 and anthocyanins biosynthesis genes to jointly promote anthocyanins biosynthesis. Yeast one‐hybrid assay (Y1H) and dual‐luciferase reporter assay (LUC) revealed that DcMYB11c bound to the promoters of DcUCGXT1 and DcSAT1 and directly activated the expression of DcUCGXT1 and DcSAT1 responsible for anthocyanins glycosylation and acylation, respectively. Three transposons were present in the carrot cultivars with purple petioles but not in the carrot cultivars with green petioles. We revealed the core factor, DcMYB11c, involved in anthocyanins pigmentation in carrot purple petioles. This study provides new insights into precise regulation mechanism underlying anthocyanins biosynthesis in carrot. The orchestrated regulation mechanism in carrot might be conserved across the plant kingdom and useful for other researchers working on anthocyanins accumulation in different tissues.
Summary statement
Here, we described a MYB gene, DcMYB11c, which was highly expressed in the purple pigmented petioles. The orchestrated regulation mechanism in carrot might be conserved across the plant kingdom and useful for other researchers working on anthocyanins accumulation in different tissues.
SUMMARY
The color of purple carrot taproots mainly depends on the anthocyanins sequestered in the vacuoles. Glutathione S‐transferases (GSTs) are key enzymes involved in anthocyanin transport. ...However, the precise mechanism of anthocyanin transport from the cytosolic surface of the endoplasmic reticulum (ER) to the vacuoles in carrots remains unclear. In this study, we conducted a comprehensive analysis of the carrot genome, leading to the identification of a total of 41 DcGST genes. Among these, DcGST1 emerged as a prominent candidate, displaying a strong positive correlation with anthocyanin pigmentation in carrot taproots. It was highly expressed in the purple taproot tissues of purple carrot cultivars, while it was virtually inactive in the non‐purple taproot tissues of purple and non‐purple carrot cultivars. DcGST1, a homolog of Arabidopsis thaliana TRANSPARENT TESTA 19 (TT19), belongs to the GSTF clade and plays a crucial role in anthocyanin transport. Using the CRISPR/Cas9 system, we successfully knocked out DcGST1 in the solid purple carrot cultivar ‘Deep Purple’ (‘DPP’), resulting in carrots with orange taproots. Additionally, DcMYB7, an anthocyanin activator, binds to the DcGST1 promoter, activating its expression. Compared with the expression DcMYB7 alone, co‐expression of DcGST1 and DcMYB7 significantly increased anthocyanin accumulation in carrot calli. However, overexpression of DcGST1 in the two purple carrot cultivars did not change the anthocyanin accumulation pattern or significantly increase the anthocyanin content. These findings improve our understanding of anthocyanin transport mechanisms in plants, providing a molecular foundation for improving and enhancing carrot germplasm.
Significance Statement
These findings improve our understanding of anthocyanin transport mechanisms in plants, providing a molecular foundation for improving and enhancing carrot germplasm.
SUMMARY
The accumulation of carotenoids, such as xanthophylls, lycopene, and carotenes, is responsible for the color of carrot (Daucus carota subsp. sativus) fleshy roots. The potential role of ...DcLCYE, encoding a lycopene ε‐cyclase associated with carrot root color, was investigated using cultivars with orange and red roots. The expression of DcLCYE in red carrot varieties was significantly lower than that in orange carrots at the mature stage. Furthermore, red carrots accumulated larger amounts of lycopene and lower levels of α‐carotene. Sequence comparison and prokaryotic expression analysis revealed that amino acid differences in red carrots did not affect the cyclization function of DcLCYE. Analysis of the catalytic activity of DcLCYE revealed that it mainly formed ε‐carotene, while a side activity on α‐carotene and γ‐carotene was also observed. Comparative analysis of the promoter region sequences indicated that differences in the promoter region may affect the transcription of DcLCYE. DcLCYE was overexpressed in the red carrot ‘Benhongjinshi’ under the control of the CaMV35S promoter. Lycopene in transgenic carrot roots was cyclized, resulting in the accumulation of higher levels of α‐carotene and xanthophylls, while the β‐carotene content was significantly decreased. The expression levels of other genes in the carotenoid pathway were simultaneously upregulated. Knockout of DcLCYE in the orange carrot ‘Kurodagosun’ by CRISPR/Cas9 technology resulted in a decrease in the α‐carotene and xanthophyll contents. The relative expression levels of DcPSY1, DcPSY2, and DcCHXE were sharply increased in DcLCYE knockout mutants. The results of this study provide insights into the function of DcLCYE in carrots, which could serve as a basis for creating colorful carrot germplasms.
Significance Statement
In this study, we found that the accumulation of lycopene and carotenes in carrot (Daucus carota subsp. sativus) root was related to the expression of the lycopene ε‐cyclase gene DcLCYE. Catalytic activity analysis in Escherichia coli and gene function verification in transgenic carrot demonstrated that DcLCYE could alter the metabolic flow of carotenes.
Carrots (
L.), among the most important root vegetables in the Apiaceae family, are cultivated worldwide. The storage root is widely utilized due to its richness in carotenoids, anthocyanins, dietary ...fiber, vitamins and other nutrients. Carrot extracts, which serve as sources of antioxidants, have important functions in preventing many diseases. The biosynthesis, metabolism, and medicinal properties of carotenoids in carrots have been widely studied. Research on hormone regulation in the growth and development of carrots has also been widely performed. Recently, with the development of high-throughput sequencing technology, many efficient tools have been adopted in carrot research. A large amount of sequence data has been produced and applied to improve carrot breeding. A genome editing system based on CRISPR/Cas9 was also constructed for carrot research. In this review, we will briefly summarize the origins, genetic breeding, resistance breeding, genome editing, omics research, hormone regulation, and nutritional composition of carrots. Perspectives about future research work on carrots are also briefly provided.
In higher plants, salicylic acid (SA) plays important roles in inducing resistance to biotic and abiotic stresses. Tomato yellow leaf curl virus (TYLCV) causes a highly devastating viral disease in ...plants, particularly in tomato. However, the roles of SA in inducing tomato plant resistance to TYLCV remain unclear.
In this study, we investigated whether the exogenous application of SA can improve the resistance of tomato plants to TYLCV in two tomato cultivars, resistant 'Zhefen-702' and susceptible 'Jinpeng-1'. The impacts of SA on the accumulation of ascorbic acid (AsA) and biosynthetic gene expression, the activity of some important reactive oxygen species (ROS)-scavenging enzymes, and the expression patterns of stress-related genes were also determined. Results indicated that SA can effectively regulate the accumulation of AsA, especially in 'Jinpeng-1'. Similarly, the expression patterns of most of the AsA biosynthetic genes showed a negative relationship with AsA accumulation in the resistant and susceptible tomato cultivars. In the two tomato cultivars, the activities of ascorbate peroxidase (APX) and peroxidase (POD) in the SA + TYLCV treated plants were increased during the experiment period except at 14 days (APX in 'Jinpeng-1' was also at 4 days) post infected (dpi) with TYLCV. Simultaneously, the activity of SOD was reduced in 'Jinpeng-1' and increased in 'Zhefen-702' after treatment with SA + TYLCV. SA can substantially induce the expression of ROS-scavenging genes at different extents. From 2 to 10 dpi, the virus content in the SA + TYLCV treated plants was remarkably lower than those in the TYLCV treated plants in 'Jinpeng-1'and Zhefen-702'.
The above results suggest that SA can enhance tomato plant resistance by modulating the expression of genes encoding for ROS-scavenging players, altering the activity of resistance-related enzymes, and inducing the expression of pathogenesis-related genes to produce systemic acquired resistance. Simultaneously, these results confirm that SA is a resistance-inducing factor against TYLCV infection that can be effectively applied in tomato plants.
SUMMARY
Cryptotaenia japonica, a traditional medicinal and edible vegetable crops, is well‐known for its attractive flavors and health care functions. As a member of the Apiaceae family, the ...evolutionary trajectory and biological properties of C. japonica are not clearly understood. Here, we first reported a high‐quality genome of C. japonica with a total length of 427 Mb and N50 length 50.76 Mb, was anchored into 10 chromosomes, which confirmed by chromosome (cytogenetic) analysis. Comparative genomic analysis revealed C. japonica exhibited low genetic redundancy, contained a higher percentage of single‐cope gene families. The homoeologous blocks, Ks, and collinearity were analyzed among Apiaceae species contributed to the evidence that C. japonica lacked recent species‐specific WGD. Through comparative genomic and transcriptomic analyses of Apiaceae species, we revealed the genetic basis of the production of anthocyanins. Several structural genes encoding enzymes and transcription factor genes of the anthocyanin biosynthesis pathway in different species were also identified. The CjANSa, CjDFRb, and CjF3H gene might be the target of Cjaponica_2.2062 (bHLH) and Cjaponica_1.3743 (MYB). Our findings provided a high‐quality reference genome of C. japonica and offered new insights into Apiaceae evolution and biology.
Significance Statement
Our results indicated how the genome of C. japonica provided a useful model for studying evolutionary trajectory and biological properties in Apiaceae species. Our findings are helpful for enriching genome research of Apiaceae family.
PDF
