The Y locus encodes a REPRESSOR OF PHOTOSYNTHETIC GENES protein that represses carotenoid biosynthesis via interaction with APRR2 in carrot.

Little is known about the factors regulating carotenoid biosynthesis in roots. In this study, we characterized DCAR_032551, the candidate gene of the Y locus responsible for the transition of root color from ancestral white to yellow during carrot (Daucus carota) domestication. We show that DCAR_032551 encodes a REPRESSOR OF PHOTOSYNTHETIC GENES (RPGE) protein, named DcRPGE1. DcRPGE1 from wild carrot (DcRPGE1W) is a repressor of carotenoid biosynthesis. Specifically, DcRPGE1W physically interacts with DcAPRR2, an ARABIDOPSIS PSEUDO-RESPONSE REGULATOR2 (APRR2)-like transcription factor. Through this interaction, DcRPGE1W suppresses DcAPRR2-mediated transcriptional activation of the key carotenogenic genes phytoene synthase 1 (DcPSY1), DcPSY2, and lycopene ε-cyclase (DcLCYE), which strongly decreases carotenoid biosynthesis. We also demonstrate that the DcRPGE1W-DcAPRR2 interaction prevents DcAPRR2 from binding to the RGATTY elements in the promoter regions of DcPSY1, DcPSY2, and DcLCYE. Additionally, we identified a mutation in the DcRPGE1 coding region of yellow and orange carrots that leads to the generation of alternatively spliced transcripts encoding truncated DcRPGE1 proteins unable to interact with DcAPRR2, thereby failing to suppress carotenoid biosynthesis. These findings provide insights into the transcriptional regulation of carotenoid biosynthesis and offer potential target genes for enhancing carotenoid accumulation in crop plants.

Anthocyanin glucosylation mediated by a glycoside hydrolase family 3 protein in purple carrot.

Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP-sugar-dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl-glucose-dependent glucosyltransferase activity modifying cyanidin 3-xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3-type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells.

An anthocyanin activation gene underlies the purple central flower pigmentation in wild carrot.

Many organisms have complex pigmentation patterns. However, how these patterns are formed remains largely unknown. In wild carrot (Daucus carota subsp. carota), which is also known as Queen Anne's lace, one or several purple central flowers occur in white umbels. Here, we investigated the unique central flower pigmentation pattern in wild carrot umbels. Using wild and cultivated carrot (D. carota subsp. sativus L.) accessions, transcriptome analysis, protein interaction, stable transformation, and CRISPR/Cas9-mediated knockout, an anthocyanin-activating R2R3-myeloblastosis (MYB) gene, Purple Central Flower (DcPCF), was identified as the causal gene that triggers only central flowers to possess the purple phenotype. The expression of DcPCF was only detected in tiny central flowers. We propose that the transition from purple to nonpurple flowers in the center of the umbel occurred after 3 separate adverse events: insertion of transposons in the promoter region, premature termination of the coding sequence (caused by a C-T substitution in the open reading frame), and the emergence of unknown anthocyanin suppressors. These 3 events could have occurred either consecutively or independently. The intriguing purple central flower pattern and its underlying mechanism may provide evidence that it is a remnant of ancient conditions of the species, reflecting the original appearance of Umbelliferae (also called Apiaceae) when a single flower was present.

Molasses-based waste water irrigation: a friend or foe for carrot (Daucus carota L.) growth, yield and nutritional quality.

Management of molasses-based wastewater generated in yeast and sugar industries is a major environmental concern due to its high chemical oxygen demand and other recalcitrant substances. Several strategies have been used to reduce the inland discharge of wastewater but the results are not satisfactory due to high operating cost. However, reuse of molasses-based wastewater irrigation in agriculture has been a major interest nowadays to reduce the freshwater consumption. Thus, it is crucial to monitor the impacts of molasses-based waste water irrigation on growth, metabolism, yield and nutritional quality of crops for safer consumer's health. In present study, carrot seeds of a local cultivar (T-29) were germinated on filter paper in Petri dishes under controlled conditions. The germinated seeds were then transplanted into pots and irrigated with three different treatments normal water (T0), diluted molasses-based wastewater (T1), and untreated molasses-based wastewater (T2), in six replicates. Results revealed that carrot irrigated with untreated molasses-based waste water had exhibited significant reductions in growth, yield, physiology, metabolism, and nutritional contents. Additionally, accumulation of Cd and Pb contents in carrot roots irrigated with untreated molasses-based waste water exceed the permissible limits suggested by WHO and their consumption may cause health risks. While, diluted molasses-based waste water irrigation positively enhanced the growth, yield of carrot plants without affecting the nutritional quality. This strategy is cost effective, appeared as most appropriate alternative mean to reduce the freshwater consumption in water deficit regions of the world.

Physiological and oxidative stress response of carrot (Daucus carota L.) to jumping plant-louse Bactericera trigonica Hodkinson (Hemiptera: Psylloidea) infestation.

BACKGROUND: Carrot is an important vegetable crop grown worldwide. The major economic problem in carrot cultivation is yellow disease caused by Bactericera trigonica, which induces biotic stress and has the greatest impact on crop productivity. Comprehensive studies on the mechanism of carrot defense response to biotic stress caused by B. trigonica infestation have yet to be conducted. METHODS: The changes in photosynthetic pigments, proline, TPC, H2O2 and MDA content, DPPH radical scavenging ability, and antioxidant enzyme activity of SOD, CAT, and POX in carrot leaves in response to insect sex (female and male), rapid response (during the first six hours), and long-term response to B. trigonica infestation were evaluated. RESULTS: The results of our study strongly suggest that B. trigonica infestation causes significant changes in primary and secondary metabolism and oxidative status of carrot leaves. Photosynthetic pigment content, TPC, and DPPH and CAT activities were significantly reduced in carrot leaves in response to insect infestation. On the other hand, proline, H2O2 content, and the activity of the antioxidant enzymes superoxide dismutase and peroxidase were increased in carrot leaves after B. trigonica infestation. The results indicate that B. trigonica attenuates and delays the oxidative stress responses of carrot, allowing long-term feeding without visible changes in the plant. Carrot responded to long-term B. trigonica infestation with an increase in SOD and POX activity, suggesting that these enzymes may play a key role in plant defense mechanisms. CONCLUSIONS: This is the first comprehensive study strongly suggesting that B. trigonica infestation causes significant changes in primary and secondary metabolism and an attenuated ROS defense response in carrot leaves that enables long-term insect feeding. The information provides new insights into the mechanisms of carrot protection against B. trigonica infestation.

CYP74B34 Enzyme from Carrot (Daucus carota) with a Double Hydroperoxide Lyase/Epoxyalcohol Synthase Activity: Identification and Biochemical Properties.

The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.

Transcriptome Sequencing Reveals the Mechanism of Auxin Regulation during Root Expansion in Carrot.

Carrot is an important vegetable with roots as the edible organ. A complex regulatory network controls root growth, in which auxin is one of the key players. To clarify the molecular mechanism on auxin regulating carrot root expansion, the growth process and the indole-3-acetic acid (IAA) content in the roots were measured in this experiment. It was found that the rapid expansion period of the root was from 34 to 41 days after sowing and the IAA content was the highest during this period. The root growth then slowed down and the IAA levels decreased. Using the transcriptome sequencing database, we analyzed the expression of IAA-metabolism-related genes and found that the expression of most of the IAA synthesis genes, catabolism genes, and genes related to signal transduction was consistent with the changes in IAA content during root expansion. Among them, a total of 31 differentially expressed genes (DEGs) were identified, including 10 IAA synthesis genes, 8 degradation genes, and 13 genes related to signal transduction. Analysis of the correlations between the DEGs and IAA levels showed that the following genes were closely related to root development: three synthesis genes, YUCCA10 (DCAR_012429), TAR2 (DCAR_026162), and AMI1 (DCAR_003244); two degradation genes, LPD1 (DCAR_023341) and AACT1 (DCAR_010070); and five genes related to signal transduction, IAA22 (DCAR_012516), IAA13 (DCAR_012591), IAA27 (DCAR_023070), IAA14 (DCAR_027269), and IAA7 (DCAR_030713). These results provide a reference for future studies on the mechanism of root expansion in carrots.

High-Pressure Processing of Fruit Smoothies Enriched with Dietary Fiber from Carrot Discards: Effects on the Contents and Bioaccessibilities of Carotenoids and Vitamin E.

The effects of high-pressure processing (HPP) (450 MPa/600 MPa/3 min) on the carotenoid and vitamin E contents of smoothies made from strawberry, orange juice, banana and apple, and the same smoothies enriched with dietary fiber from discarded carrots were compared. The contents and bioaccessibilities of these compounds were also evaluated over the course of 28 days at 4 °C. The application of HPP in the formulations significantly increased the contents of ß-cryptoxanthin, α-carotene and ß-carotene and retained the contents of lutein, zeaxanthin and vitamin E compared to untreated samples. A decreasing trend in the content of each compound was observed with an increase in storage time. The application of HPP initially led to reductions in the bioaccessibility of individual compounds. However, overall, during storage, there was an increase in bioaccessibility. This suggests that HPP influences cell structure, favoring compound release and micelle formation. HPP is a sustainable method that preserves or enhances carotenoid extractability in ready-to-drink fruit beverages. Furthermore, the incorporation of dietary fiber from carrot processing discards supports circular economy practices and enhances the health potential of the product.

Metal content, bioaccumulation, translocation, and health risk assessment of root vegetables grown in KwaZulu-Natal small-scale farms of South Africa.

Metal uptake by vegetables is becoming a threat to the life of consumers. Therefore, continuous monitoring of metals in vegetables and soils is becoming a necessity. In this study, the occurrence of 18 metals in amadumbe (Colocasia esculenta L.), sweet potatoes (Ipomoea batatas L.), potatoes (Solanum tuberosum L.), and carrots (Daucus carrota L.) grown in small-scale South African agricultural farms was monitored using inductively coupled plasma-optical emission spectroscopy. All the 18 investigated elements were detected in soils and different vegetative plants parts. Bioaccumulation factors indicated the transfer of selected metals from soils into the plant roots. Toxic metals Cd, Cr, and Pb had their concentrations exceeding the maximum permissible levels set by the World Health Organization in the edible parts of all root vegetables. Cd and Pb varied between 18.89 and 19.19 mg kg-1 and 10.46 and 11.46 mg kg-1, respectively, while Cr remained constant at 16.78 mg kg-1. The exact metals together with As and Ni had their total hazard quotients exceeding the threshold value of 1, which indicated that the daily consumption of the investigated root vegetables is likely to pose health risks to both adults and children. Therefore, this study points out to a possibility of toxic health effects that could arise when these vegetables are consumed daily.

Development and carotenoid synthesis in dark-grown carrot taproots require PHYTOCHROME RAPIDLY REGULATED1.

Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange carrot (Daucus carota) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes, such as DcPSY1 and DcPSY2, reducing carotenoid accumulation. By means of RNA sequencing, in a previous analysis, we observed that carrot PHYTOCHROME RAPIDLY REGULATED1 (DcPAR1) is more highly expressed in the underground grown taproot compared with those grown in light. PAR1 is a transcriptional cofactor with a negative role in shade avoidance syndrome regulation in Arabidopsis (Arabidopsis thaliana) through the dimerization with PHYTOCHROME-INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here, we show that overexpressing AtPAR1 in carrot increases carotenoid production in taproots grown underground as well as DcPSY1 expression. The high expression of AtPAR1 and DcPAR1 led us to hypothesize a functional role of DcPAR1 that was verified through in vivo binding to AtPIF7 and overexpression in Arabidopsis, where AtPSY expression and carotenoid accumulation increased together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoid levels and in relative expression of key carotenogenic genes as well as impaired taproot development. These results suggest that DcPAR1 is a key factor for secondary root development and carotenoid synthesis in carrot taproot grown underground.

A MYB activator, DcMYB11c, regulates carrot anthocyanins accumulation in petiole but not taproot.

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.

Investigation of drought induced biochemical and gene expression changes in carrot cultivars.

BACKGROUND: Carrot is the most important vegetable in Apiaceae family, and it is consumed globally due to its high nutritional quality. Drought stress is major environmental constraint for vegetables especially carrot. Limited data is available regarding the mechanisms conferring drought tolerance in carrot. Methods and Results Eight commercial carrot cultivars were used in this study and subjected to drought stress under semi-controlled greenhouse conditions. Biochemical, antioxidant enzymatic activity and changes in transcript level of drought related genes was estimated, the gene expression analysis was done by using qRT-PCR in comparison with reference gene expression Actin (Act1). Results revealed that cultivars Coral Orange, Tendersweet and Solar Yellow were tolerant to drought stress, which was supported by their higher transcript levels of catalase gene (CAT), superoxide dismutase genes (Cu/ZN-SOD, Cu/Zn-SDC) in these cultivars. The downregulation of PDH1 gene (Proline dehydrogenase 1) was also observed that was associated with upregulation of proline accumulation in carrot plants. Moreover, results also suggested that PRT genes (Proline transporter genes) played a key role in drought tolerance in carrot cultivars. Conclusion Among the cultivars studied, Coral Orange showed overall tolerance to drought stress conditions, whereas cultivars Cosmic Purple and Eregli Black were sensitive based on their biochemical and gene expression levels. According to our knowledge, this is the first comparative study on drought tolerance in several carrot cultivars. It will provide a background for carrot breeding to understand biochemical and molecular responses of carrot plant to drought stress and mechanisms behind it.

Response of Carrot (Daucus carota L.) to Multi-Contaminated Soil from Historic Mining and Smelting Activities.

A pot experiment was undertaken to investigate the effect of Cd, Pb and Zn multi-contamination on the physiological and metabolic response of carrot (Daucus carota L.) after 98 days of growth under greenhouse conditions. Multi-contamination had a higher negative influence on leaves (the highest Cd and Zn accumulation) compared to the roots, which showed no visible change in terms of anatomy and morphology. The results showed the following: (i) significantly higher accumulation of Cd, Zn, and Pb in the multi-contaminated variant (Multi) compared to the control; (ii) significant metabolic responses-an increase in the malondialdehyde content of the Multi variant compared to the control in the roots (by 20%), as well as in the leaves (by 53%); carotenoid content in roots decreased by 31% in the Multi variant compared with the control; and changes in free amino acids, especially those related to plant stress responses. The determination of hydroxyproline and sarcosine may reflect the higher sensitivity of carrot leaves to multi-contamination in comparison to roots. A similar trend was observed for the content of free methionine (significant increase of 31% only in leaves); (iii) physiological responses (significant decreases in biomass, changes in gas-exchange parameters and chlorophyll a); and (iv) significant changes in enzymatic activities (chitinase, alanine aminopeptidase, acid phosphatase) in the root zone.

Effect of Extraction Methods on the Antioxidant Potential and Cytotoxicity of the Combined Ethanolic Extracts of Daucus carota L., Beta vulgaris L., Phyllanthus emblica L. and Lycopersicon esculentum against Gastric Adenocarcinoma Cells.

Frequent consumption of fruits and vegetables in the daily diet may alleviate the risk of developing chronic diseases. Daucus carota L. (carrot), Beta vulgaris L. (beetroot) Phyllanthus emblica L. (amla), and Lycopersicon esculentum M (tomatoes) are traditionally consumed functional foods that contain a high concentration of antioxidants, ascorbic acid, polyphenols, and numerous phytochemicals. This study assessed how three distinct preparation methods affect the phenolic, flavonoid, carotenoid, and ascorbic acid contents, antioxidant level, and cytotoxicity of the combined fruit extract. The fruit samples were taken in the ratio of carrot (6): beetroot (2): tomato (1.5): amla (0.5) and processed into a lyophilized slurry (LS) extract, lyophilized juice (LJ) extract, and hot-air oven-dried (HAO) extract samples. The sample extracts were assessed for their phytoconstituent concentrations and antioxidant and cytotoxic potential. The total phenolic content in LS, LJ, and HAO extracts was 171.20 ± 0.02, 120.73 ± 0.02, and 72.05 ± 0.01 mg gallic acid equivalent/100 g, respectively and the total flavonoid content was 23.635 ± 0.003, 20.754 ± 0.005, and 18.635 ± 0.005 mg quercetin equivalent/100 g, respectively. Similarly, total ascorbic acid content, carotenoids, and antioxidant potential were higher in the LS and LJ extracts than in HAO. Overall, the LS extract had a substantially higher concentration of phytochemicals and antioxidants, as well as higher cytotoxic potential, compared to the LJ and HAO extracts. The LS extract was tested in the MKN-45 human gastric cancer cell line to demonstrate its effective antioxidant potential and cytotoxicity. Hence, lyophilization (freezing) based techniques are more effective than heat-based techniques in preserving the phytoconstituents and their antioxidant and cytotoxic potential.

The genetic control of polyacetylenes involved in bitterness of carrots (Daucus carota L.): Identification of QTLs and candidate genes from the plant fatty acid metabolism.

BACKGROUND: Falcarinol-type polyacetylenes (PAs) such as falcarinol (FaOH) and falcarindiol (FaDOH) are produced by several Apiaceae vegetables such as carrot, parsnip, celeriac and parsley. They are known for numerous biological functions and contribute to the undesirable bitter off-taste of carrots and their products. Despite their interesting biological functions, the genetic basis of their structural diversity and function is widely unknown. A better understanding of the genetics of the PA levels present in carrot roots might support breeding of carrot cultivars with tailored PA levels for food production or nutraceuticals. RESULTS: A large carrot F2 progeny derived from a cross of a cultivated inbred line with an inbred line derived from a Daucus carota ssp. commutatus accession rich in PAs was used for linkage mapping and quantitative trait locus (QTL) analysis. Ten QTLs for FaOH and FaDOH levels in roots were identified in the carrot genome. Major QTLs for FaOH and FaDOH with high LOD values of up to 40 were identified on chromosomes 4 and 9. To discover putative candidate genes from the plant fatty acid metabolism, we examined an extended version of the inventory of the carrot FATTY ACID DESATURASE2 (FAD2) gene family. Additionally, we used the carrot genome sequence for a first inventory of ECERIFERUM1 (CER1) genes possibly involved in PA biosynthesis. We identified genomic regions on different carrot chromosomes around the found QTLs that contain several FAD2 and CER1 genes within their 2-LOD confidence intervals. With regard to the major QTLs on chromosome 9 three putative CER1 decarbonylase gene models are proposed as candidate genes. CONCLUSION: The present study increases the current knowledge on the genetics of PA accumulation in carrot roots. Our finding that carrot candidate genes from the fatty acid metabolism are significantly associated with major QTLs for both major PAs, will facilitate future functional gene studies and a further dissection of the genetic factors controlling PA accumulation. Characterization of such candidate genes will have a positive impact on carrot breeding programs aimed at both lowering or increasing PA concentrations in carrot roots.

MYB-6 and LDOX-1 regulated accretion of anthocyanin response to cold stress in purple black carrot (Daucus carota L.).

AIM: Anthocyanin, an essential ingredient of functional foods, is present in a wide range of plants, including black carrots. The current investigation was carried out to analyse the effect of cold stress on the expression of major anthocyanins and anthocyanin biosynthetic pathway genes, MYB6 and LDOX-1. METHODS AND RESULTS: Five cultivated carrot genotypes belonging to the eastern group, having anthocyanin pigment, were used in the current study. The qRT-PCR analysis revealed that relative gene expression of transcription factor MYB-6 and LDOX1gene was highly expressed upon cold stress compared to non-stress samples. High-performance liquid chromatography-based quantification of Cyanidin 3-O-glucoside (Kuromanin chloride), Ferulic acid, 3,5-Dimethoxy-4-hydroxycinnamic acid (Sinapic acid), and Rutin revealed a significant increase in these major anthocyanins in response to cold stress when compared to control plants. CONCLUSION: We conclude that MYB6 and LDOX1 gene expression increases upon cold stress, which induces accumulation of major anthocyanins in purple black carrot and suggests a possible cross-link between cold stress and anthocyanin biosynthesis in purple black carrot.

Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation.

KEY MESSAGE: We have developed and validated an efficient protocol for producing gene-edited carrot plants that do not result in the stable incorporation of foreign DNA in the edited plant's genome. We report here a method for producing transgene-free, gene-edited carrot (Daucus carota subs. sativus) plants. With this approach, PEG-mediated transformation is used to transiently express a cytosine base editor and a guide RNA in protoplasts to induce targeted mutations in the carrot genome. These protoplasts are then cultured under conditions that lead to the production of somatic embryos which subsequently develop into carrot plants. For this study, we used the Centromere-Specific Histone H3 (CENH3) gene as a target for evaluating the efficiency with which regenerated, edited plants could be produced. After validating sgRNA performance and protoplast transformation efficiency using transient assays, we performed two independent editing experiments using sgRNAs targeting different locations within CENH3. In the first experiment, we analyzed 184 regenerated plants and found that 22 of them (11.9%) carried targeted mutations within CENH3, while in the second experiment, 28 out of 190 (14.7%) plants had mutations in CENH3. Of the 50 edited carrot lines that we analyzed, 43 were homozygous or bi-allelic for mutations in CENH3. No evidence of the base editor expression plasmid was found in the edited lines tested, indicating that this approach is able to produce transgene-free, gene-edited lines. The protocol that we describe provides an efficient method for easily generating large numbers of transgene-free, gene-edited carrot plants.

Characterization of Abscisic Acid and Ethylene in Regulating the White Blush in Fresh-Cut Carrots.

The surface of fresh-cut carrots is apt to white blush, however the physiological and molecular mechanism for this process is not yet fully understood. In this study, exogenous abscisic acid (ABA) and ethylene separately promoted and inhibited the white-blush formation after three days after treatment, respectively. Metabolome analysis found that white-blush components mainly consist of p-hydroxyphenyl lignin and guaiacyl lignin. Transcriptome analysis found an increase in the whiteness values was consistent with the higher expression of genes encoding O-methyltransferase, trans-anol O-methyltransferase, bergaptol O-methyltransferase, caffeic acid 3-O-methyltransferase, phenylalanine ammonia-lyase, and ferulate-5-hydroxylase, together with the lower expression of genes encoding cinnamic acid 4-hydroxylase caffeoyl-CoA O-methyltransferase and 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase. In conclusion, ABA plays an important role in lignin biosynthesis essential to the formation of white blush in fresh-cut carrots. This is the first report that uncovers the physiological and molecular causes of white blush in fresh-cut carrots, providing a basis for white-blush control in fresh-cut carrots.

Genome-Wide Identification and Evolution Analysis of R2R3-MYB Gene Family Reveals S6 Subfamily R2R3-MYB Transcription Factors Involved in Anthocyanin Biosynthesis in Carrot.

The taproot of purple carrot accumulated rich anthocyanin, but non-purple carrot did not. MYB transcription factors (TFs) condition anthocyanin biosynthesis in many plants. Currently, genome-wide identification and evolution analysis of R2R3-MYB gene family and their roles involved in conditioning anthocyanin biosynthesis in carrot is still limited. In this study, a total of 146 carrot R2R3-MYB TFs were identified based on the carrot transcriptome and genome database and were classified into 19 subfamilies on the basis of R2R3-MYB domain. These R2R3-MYB genes were unevenly distributed among nine chromosomes, and Ka/Ks analysis suggested that they evolved under a purified selection. The anthocyanin-related S6 subfamily, which contains 7 MYB TFs, was isolated from R2R3-MYB TFs. The anthocyanin content of rhizodermis, cortex, and secondary phloem in 'Black nebula' cultivar reached the highest among the 3 solid purple carrot cultivars at 110 days after sowing, which was approximately 4.20- and 3.72-fold higher than that in the 'Deep purple' and 'Ziwei' cultivars, respectively. The expression level of 7 MYB genes in purple carrot was higher than that in non-purple carrot. Among them, DcMYB113 (DCAR_008994) was specifically expressed in rhizodermis, cortex, and secondary phloem tissues of 'Purple haze' cultivar, with the highest expression level of 10,223.77 compared with the control 'DPP' cultivar at 70 days after sowing. DcMYB7 (DCAR_010745) was detected in purple root tissue of 'DPP' cultivar and its expression level in rhizodermis, cortex, and secondary phloem was 3.23-fold higher than that of secondary xylem at 110 days after sowing. Our results should be useful for determining the precise role of S6 subfamily R2R3-MYB TFs participating in anthocyanin biosynthesis in carrot.

Carrot DcALFIN4 and DcALFIN7 Transcription Factors Boost Carotenoid Levels and Participate Differentially in Salt Stress Tolerance When Expressed in Arabidopsis thaliana and Actinidia deliciosa.

ALFIN-like transcription factors (ALs) are involved in several physiological processes such as seed germination, root development and abiotic stress responses in plants. In carrot (Daucus carota), the expression of DcPSY2, a gene encoding phytoene synthase required for carotenoid biosynthesis, is induced after salt and abscisic acid (ABA) treatment. Interestingly, the DcPSY2 promoter contains multiple ALFIN response elements. By in silico analysis, we identified two putative genes with the molecular characteristics of ALs, DcAL4 and DcAL7, in the carrot transcriptome. These genes encode nuclear proteins that transactivate reporter genes and bind to the carrot DcPSY2 promoter in yeast. The expression of both genes is induced in carrot under salt stress, especially DcAL4 which also responds to ABA treatment. Transgenic homozygous T3 Arabidopsis thaliana lines that stably express DcAL4 and DcAL7 show a higher survival rate with respect to control plants after chronic salt stress. Of note is that DcAL4 lines present a better performance in salt treatments, correlating with the expression level of DcAL4, AtPSY and AtDXR and an increase in carotenoid and chlorophyll contents. Likewise, DcAL4 transgenic kiwi (Actinidia deliciosa) lines show increased carotenoid and chlorophyll content and higher survival rate compared to control plants after chronic salt treatment. Therefore, DcAL4 and DcAL7 encode functional transcription factors, while ectopic expression of DcAL4 provides increased tolerance to salinity in Arabidopsis and Kiwi plants.