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.

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.

Glutamate application maintains quality and antioxidant capacity of fresh-cut carrots by modulating GABA shunt, phenylpropanoid and ROS metabolism.

The effects of exogenous glutamate treatment on the quality attributes, γ-aminobutyric acid (GABA) shunt, phenylpropanoid pathway, and antioxidant capacity of fresh-cut carrots were investigated. Results showed that glutamate treatment suppressed the increases in lightness and whiteness values, inhibited the degradation of total carotenoids and maintained better flavor and taste in fresh-cut carrots. Moreover, glutamate treatment rapidly promoted the activities of glutamate decarboxylase and GABA transaminase, thus improving the GABA content. It also significantly enhanced the activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate coenzyme A ligase and promoted the accumulation of total phenolics as well as the main individual phenolic compounds, including chlorogenic and caffeic acid. In addition, glutamate application activated the reactive oxygen system-related enzyme including peroxidase, superoxide dismutase, ascorbate peroxidase, and catalase activities to maintain higher antioxidant capacity in fresh-cut carrots. These results demonstrated that exogenous glutamate treatment maintained better nutritional quality and alleviated color deterioration by accelerating the accumulation of GABA and phenolics and enhancing the antioxidant capacity in fresh-cut carrots.

Comparative analysis of the carrot miRNAome in response to salt stress.

Soil salinity adversely affects the yield and quality of crops, including carrot. During salt stress, plant growth and development are impaired by restricted water uptake and ion cytotoxicity, leading to nutrient imbalance and oxidative burst. However, the molecular mechanisms of the carrot plant response to salt stress remain unclear. The occurrence and expression of miRNAs that are potentially involved in the regulation of carrot tolerance to salinity stress were investigated. The results of small RNA sequencing revealed that salt-sensitive (DH1) and salt-tolerant (DLBA) carrot varieties had different miRNA expression profiles. A total of 95 miRNAs were identified, including 71 novel miRNAs, of which 30 and 23 were unique to DH1 and DLBA, respectively. The comparison of NGS and qPCR results allowed identification of two conserved and five novel miRNA involved in carrot response to salt stress, and which differentiated the salt-tolerant and salt-sensitive varieties. Degradome analysis supported by in silico-based predictions and followed by expression analysis of exemplary target genes pointed at genes related to proline, glutathione, and glutamate metabolism pathways as potential miRNA targets involved in salt tolerance, and indicated that the regulation of osmoprotection and antioxidant protection, earlier identified as being more efficient in the tolerant variety, may be controlled by miRNAs. Furthermore, potential miRNA target genes involved in chloroplast protection, signal transduction and the synthesis and modification of cell wall components were indicated in plants growing in saline soil.

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.

Restraining Quiescence Release-Related Ageing in Plant Cells: A Case Study in Carrot.

The blackening of cut carrots causes substantial economic losses to the food industry. Blackening was not observed in carrots that had been stored underground for less than a year, but the susceptibility to blackening increased with the age of the carrots that were stored underground for longer periods. Samples of black, border, and orange tissues from processed carrot batons and slices, prepared under industry standard conditions, were analyzed to identify the molecular and metabolic mechanisms underpinning processing-induced blackening. The black tissues showed substantial molecular and metabolic rewiring and large changes in the cell wall structure, with a decreased abundance of xyloglucan, pectins (homogalacturonan, rhamnogalacturonan-I, galactan and arabinan), and higher levels of lignin and other phenolic compounds when compared to orange tissues. Metabolite profiling analysis showed that there was a major shift from primary to secondary metabolism in the black tissues, which were depleted in sugars, amino acids, and tricarboxylic acid (TCA) cycle intermediates but were rich in phenolic compounds. These findings suggest that processing triggers a release from quiescence. Transcripts encoding proteins associated with secondary metabolism were less abundant in the black tissues, but there were no increases in transcripts associated with oxidative stress responses, programmed cell death, or senescence. We conclude that restraining quiescence release alters cell wall metabolism and composition, particularly regarding pectin composition, in a manner that increases susceptibility to blackening upon processing.

Population genomics identifies genetic signatures of carrot domestication and improvement and uncovers the origin of high-carotenoid orange carrots.

Here an improved carrot reference genome and resequencing of 630 carrot accessions were used to investigate carrot domestication and improvement. The study demonstrated that carrot was domesticated during the Early Middle Ages in the region spanning western Asia to central Asia, and orange carrot was selected during the Renaissance period, probably in western Europe. A progressive reduction of genetic diversity accompanied this process. Genes controlling circadian clock/flowering and carotenoid accumulation were under selection during domestication and improvement. Three recessive genes, at the REC, Or and Y2 quantitative trait loci, were essential to select for the high α- and ß-carotene orange phenotype. All three genes control high α- and ß-carotene accumulation through molecular mechanisms that regulate the interactions between the carotenoid biosynthetic pathway, the photosynthetic system and chloroplast biogenesis. Overall, this study elucidated carrot domestication and breeding history and carotenoid genetics at a molecular level.

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.

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.

Feeding an unsalable carrot total-mixed ration altered bacterial amino acid degradation in the rumen of lambs.

The objective of this study was to determine the influence of a total-mixed ration including unsalable carrots at 45% DM on the rumen microbiome; and the plasma, rumen and liver metabolomes. Carrots discarded at processing were investigated as an energy-dense substitute for barley grain in a conventional feedlot diet, and improved feed conversion efficiency by 25%. Here, rumen fluid was collected from 34 Merino lambs at slaughter (n = 16 control; n = 18 carrot) after a feeding period of 11-weeks. The V4 region of the 16S rRNA gene was sequenced to profile archaeal and bacterial microbe communities. Further, a comprehensive, targeted profile of known metabolites was constructed for blood plasma, rumen fluid and biopsied liver metabolites using a gas chromatography mass spectrometry (GC-MS) metabolomics approach. An in vitro batch culture was used to characterise ruminal fermentation including gas and methane (CH4) production. In vivo rumen microbial community structure of carrot fed lambs was dissimilar (P < 0.01; PERMANOVA), and all measures of alpha diversity were greater (P < 0.01), compared to those fed the control diet. Unclassified genera in Bacteroidales (15.9 ± 6.74% relative abundance; RA) were more abundant (P < 0.01) in the rumen fluid of carrot-fed lambs, while unclassified taxa in the Succinivibrionaceae family (11.1 ± 3.85% RA) were greater (P < 0.01) in the control. The carrot diet improved in vitro ruminal fermentation evidenced as an 8% increase (P < 0.01) in DM digestibility and a 13.8% reduction (P = 0.01) in CH4 on a mg/ g DM basis, while the control diet increased (P = 0.04) percentage of propionate within total VFA by 20%. Fourteen rumen fluid metabolites and 27 liver metabolites were influenced (P ≤ 0.05) by diet, while no effect (P ≥ 0.05) was observed in plasma metabolites. The carrot diet enriched (impact value = 0.13; P = 0.01) the tyrosine metabolism pathway (acetoacetic acid, dopamine and pyruvate), while the control diet enriched (impact value = 0.42; P ≤ 0.02) starch and sucrose metabolism (trehalose and glucose) in rumen fluid. This study demonstrated that feeding 45% DM unsalable carrots diversified bacterial communities in the rumen. These dietary changes influenced pathways of tyrosine degradation, such that previous improvements in feed conversion efficiency in lambs could be explained.

Genome-wide identification of MATE, functional analysis and molecular dynamics of DcMATE21 involved in anthocyanin accumulation in Daucus carota.

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs, DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota.

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.

Biochemical Characterization of a Flavone Synthase I from Daucus carota and its Application for Bioconversion of Flavanones to Flavones.

In this study, we studied the biochemical characterization of flavone synthase I from Daucus carota (DcFNS I) and applied it with flavonoid 6-hydroxylase from Scutellaria baicalensis (SbCYP) to convert flavanones to flavones. The recombinant DcFNS I was expressed in the form of the glutathione-S-transferase fusion protein. Rather than taxifolin, naringenin, pinocembrin, and eriodictyol were accepted as substrates. The optimal temperature and pH for reaction in vitro were 35 °C and 7.5, respectively, and 2-oxoglutarate was essential in the assay system. Co2+, Cu2+, Mn2+, Ni2+, and Zn2+ were not substitutes for Fe2+. EDTA and pyruvic acid inhibited the activity, except for Fe3+. Kinetic analysis revealed that the Vmax and kcat values of the recombinant DcFNS I against naringenin were 0.183 nmol mg-1 s-1 and 0.0121 s-1, and 0.175 nmol mg-1 s-1 and 0.0116 s-1 against pinocembrin. However, the recombinant DcFNS I had a higher affinity for naringenin than pinocembrin, with kM values for each of 0.076 mM and 0.174 mM respectively. Thus, it catalyzed naringenin more efficiently than pinocembrin. Subsequently, using an Escherichia coli and Saccharomyces cerevisiae co-culture system, we successfully converted naringenin and pinocembrin to scutellarein and baicalein respectively. In a synthetic complete medium, the titers of scutellarein and baicalein reached 5.63 mg/L and 0.78 mg/L from 200 mg/L precursors.

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.

Phosphodiesterase 4 mRNA Level Suppression is Important for Extract of Black Carrot to Protect Against Hepatic Injury Induced by Ethanol.

Excessive alcohol use often results in alcoholic liver disease (ALD). An early change in the liver due to excessive drinking is hepatic steatosis, which may ultimately progress to hepatitis, liver fibrosis, cirrhosis, and liver cancer. Among these debilitating processes, hepatic steatosis is reversible with the appropriate treatment. Therefore, it is important to find treatments and foods that reverse hepatic steatosis. Black carrot has antioxidant and anti-inflammatory effects. In this study, we examined the effectiveness of black carrot extract (BCE) on hepatic steatosis in in vivo and in vitro ethanol-induced liver injury models. For the in vivo experiments, serum aminotransferase activities enhanced by ethanol- and carbon tetrachloride were significantly suppressed by the BCE diet. Furthermore, morphological changes in the liver hepatic steatosis and fibrosis were observed in the in vivo ethanol-induced liver injury model, however, BCE feeding resulted in the recovery to an almost normal liver morphology. In the in vitro experiments, ethanol treatment induced reactive oxygen species (ROS) levels in hepatocytes at 9 h. Conversely, ROS production was suppressed to control levels and hepatic steatosis was suppressed when hepatocyte culture with ethanol were treated with BCE. Furthermore, we investigated enzyme activities, enzyme protein levels, and messenger RNA levels of alcohol dehydrogenase (ADH), cytochrome p450 2E1 (CYP2E1), and aldehyde dehydrogenase (ALDH) using enzyme assays, western blot, and quantitative reverse transcription-polymerase chain reaction analyses. We found that the activities of ADH, CYP2E1, and ALDH were regulated through the cAMP-PKA pathway at different levels, namely, translational, posttranslational, and transcriptional levels, respectively. The most interesting finding of this study is that BCE increases cAMP levels by suppressing the Pde4b mRNA and PDE4b protein levels in ethanol-treated hepatocytes, suggesting that BCE may prevent ALD.

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.

The phytochrome-interacting factor DcPIF3 of carrot plays a positive role in drought stress by increasing endogenous ABA level in Arabidopsis.

The phytochrome-interacting factor (PIF) subfamily of basic helix-loop-helix (bHLH) transcription factors plays a critical role in plant growth and development. However, there has been no detailed report on the PIFs in carrot. In this study, we present the identification and characterization of DcPIF gene family in carrot (Daucus carota L.). Phylogenetic analysis indicated that PIFs from carrot and other five plant species could be divided into four groups supported by similar gene structure and motif analysis. Expression profiles showed that all DcPIF genes were tissue-specific and could be induced by drought or abscisic acid (ABA) treatment except DcPIF7.1, among which DcPIF3 was the most responsive. The DcPIF3-overexpressed Arabidopsis plants exhibited more tolerance to drought stress, with higher antioxidant capacity and lower malondialdehyde content after drought treatment than wild type plants. Further stress tolerance assays revealed that DcPIF3 plays a positive role in drought stress by increasing endogenous ABA level and promoting the expression of ABA-related genes. Our results can enrich the understanding of DcPIF family genes and lay a foundation for further investigation of DcPIF3 function to defend against drought stress in carrot.

Carrot protoplasts as a suitable method for protein subcellular localization.

Carrot (Daucus carota) is a useful plant model for the study of carotenoid biosynthesis, specifically in roots which are enriched in carotenoids. Carrot genome and transcriptome sequences, complemented by optimized methods for carrot transformation, contribute to a comprehensive toolbox for exploring pathway regulation. To expand the repertoire of tools available for the study of D. carota, we present protocols for the isolation of protoplasts from D. carota cell suspension cultures and polyethylene glycol (PEG)-mediated transformation. To obtain carrot protoplasts, in vitro somatic embryogenesis from epicotyls is induced. The somatic embryogenic tissue that develops is transferred to liquid medium to obtain a suspension of cells which are homogenized and incubated with cell-wall degrading enzymes to release protoplasts. For transfection, protoplasts are incubated with a plasmid encoding a protein of interest prior to examination of protein localization by light microscopy. As an example, we demonstrate nuclear localization of a carrot transcription factor, DcAREB3.