Circadian rhythm response and its effect on photosynthetic characteristics of the Lhcb family genes in tea plant.

BACKGROUND: The circadian clock, also known as the circadian rhythm, is responsible for predicting daily and seasonal changes in the environment, and adjusting various physiological and developmental processes to the appropriate times during plant growth and development. The circadian clock controls the expression of the Lhcb gene, which encodes the chlorophyll a/b binding protein. However, the roles of the Lhcb gene in tea plant remain unclear. RESULTS: In this study, a total of 16 CsLhcb genes were identified based on the tea plant genome, which were distributed on 8 chromosomes of the tea plant. The promoter regions of CsLhcb genes have a variety of cis-acting elements including hormonal, abiotic stress responses and light response elements. The CsLhcb family genes are involved in the light response process in tea plant. The photosynthetic parameter of tea leaves showed rhythmic changes during the two photoperiod periods (48 h). Stomata are basically open during the day and closed at night. Real-time quantitative PCR results showed that most of the CsLhcb family genes were highly expressed during the day, but were less expressed at night. CONCLUSIONS: Results indicated that CsLhcb genes were involved in the circadian clock process of tea plant, it also provided potential references for further understanding of the function of CsLhcb gene family in tea plant.

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.

CsPAT1, a GRAS transcription factor, promotes lignin accumulation by antagonistic interacting with CsWRKY13 in tea plants.

Lignin is an important component of plant cell walls and plays crucial roles in the essential agronomic traits of tea quality and tenderness. However, the molecular mechanisms underlying the regulation of lignin biosynthesis in tea plants remain unclear. CsWRKY13 acts as a negative regulator of lignin biosynthesis in tea plants. In this study, we identified a GRAS transcription factor, phytochrome A signal transduction 1 (CsPAT1), that interacts with CsWRKY13. Silencing CsPAT1 expression in tea plants and heterologous overexpression in Arabidopsis demonstrated that CsPAT1 positively regulates lignin accumulation. Further investigation revealed that CsWRKY13 directly binds to the promoters of CsPAL and CsC4H and suppresses transcription of CsPAL and CsC4H. CsPAT1 indirectly affects the promoter activities of CsPAL and CsC4H by interacting with CsWRKY13, thereby facilitating lignin biosynthesis in tea plants. Compared with the expression of CsWRKY13 alone, the co-expression of CsPAT1 and CsWRKY13 in Oryza sativa significantly increased lignin biosynthesis. Conversely, compared with the expression of CsPAT1 alone, the co-expression of CsPAT1 and CsWRKY13 in O. sativa significantly reduced lignin accumulation. These results demonstrated the antagonistic regulation of the lignin biosynthesis pathway by CsPAT1 and CsWRKY13. These findings improve our understanding of lignin biosynthesis mechanisms in tea plants and provide insights into the role of the GRAS transcription factor family in lignin accumulation.

The high-quality genome of Cryptotaenia japonica and comparative genomics analysis reveals anthocyanin biosynthesis in Apiaceae.

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.

DcGST1, encoding a glutathione S-transferase activated by DcMYB7, is the main contributor to anthocyanin pigmentation in purple carrot.

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.

Telomere-to-telomere carrot (Daucus carota) genome assembly reveals carotenoid characteristics.

Carrot (Daucus carota) is an Apiaceae plant with multi-colored fleshy roots that provides a model system for carotenoid research. In this study, we assembled a 430.40 Mb high-quality gapless genome to the telomere-to-telomere (T2T) level of "Kurodagosun" carrot. In total, 36 268 genes were identified and 34 961 of them were functionally annotated. The proportion of repeat sequences in the genome was 55.3%, mainly long terminal repeats. Depending on the coverage of the repeats, 14 telomeres and 9 centromeric regions on the chromosomes were predicted. A phylogenetic analysis showed that carrots evolved early in the family Apiaceae. Based on the T2T genome, we reconstructed the carotenoid metabolic pathway and identified the structural genes that regulate carotenoid biosynthesis. Among the 65 genes that were screened, 9 were newly identified. Additionally, some gene sequences overlapped with transposons, suggesting replication and functional differentiation of carotenoid-related genes during carrot evolution. Given that some gene copies were barely expressed during development, they might be functionally redundant. Comparison of 24 cytochrome P450 genes associated with carotenoid biosynthesis revealed the tandem or proximal duplication resulting in expansion of CYP gene family. These results provided molecular information for carrot carotenoid accumulation and contributed to a new genetic resource.

Generating colorful carrot germplasm through metabolic engineering of betalains pigments.

Betalains are tyrosine-derived plant pigments exclusively found in the Caryophyllales order and some higher fungi and generally classified into two groups: red-violet betacyanins and yellow-orange betaxanthins. Betalains attract great scientific and economic interest because of their relatively simple biosynthesis pathway, attractive colors and health-promoting properties. Co-expressing two core genes BvCYP76AD1 and BvDODA1 with or without a glycosyltransferase gene MjcDOPA5GT allowed the engineering of carrot (an important taproot vegetable) to produce a palette of unique colors. The highest total betalains content, 943.2 µg·g-1 DW, was obtained in carrot taproot transformed with p35S:RUBY which produces all of the necessary enzymes for betalains synthesis. Root-specific production of betalains slightly relieved tyrosine consumption revealing the possible bottleneck in betalains production. Furthermore, a unique volcano-like phenotype in carrot taproot cross-section was created by vascular cambium-specific production of betalains. The betalains-fortified carrot in this study is thus anticipated to be used as functional vegetable and colorful carrot germplasm in breeding to promote health.

Overexpressing CsPSY1 Gene of Tea Plant, Encoding a Phytoene Synthase, Improves α-Carotene and ß-Carotene Contents in Carrot.

Tea plants (Camellia sinensis (L.) O. Kuntze) belong to Theaceae family, in the section Thea. Tea plants are widely distributed in subtropical and tropical regions in the word. α-carotene and ß-carotene in the tea leaves belong to carotenoids, which are associated with the aroma and color of the tea. Phytoene synthase (PSY) is a rate-limiting enzyme in carotenoids biosynthesis. We identified three CsPSY genes in 'Shuchazao', named CsPSY1, CsPSY2, and CsPSY3. Structural analysis of three CsPSY genes showed that CsPSY1 had a longer intro structure. The cis-acting elements of CsPSYs promoter were mainly associated with light-responsiveness, abiotic stress-responsiveness, and hormone-responsiveness. CsPSY1 exhibited expression in all tissues of the tea plants, whereas CsPSY2 and CsPSY3 were trace expression levels in all tissues. The positive expression of CsPSY1 under hormonal and abiotic stresses suggested its role in plant development and defense responses. The amino acid sequence of CsPSY1 was highly conserved in eight tea cultivars. The recombinant vector pCAMBIA1301-CsPSY1 was constructed to stabilize the overexpression of CsPSY1 in carrot. The contents of α-carotene and ß-carotene in transgenic carrot callus were significantly increased. This study provides a foundational basis for further research on the function of CsPSYs and carotenoids accumulation in tea plants.

AeWRKY32 from okra regulates anthocyanin accumulation and cold tolerance in Arabidopsis.

Okra (Abelmoschus esculentus L.) is a tropical crop species, and its growth and development are severely affected by cold stress. Recent studies have identified a potential association between WRKY transcription factors and the cold response mechanism of crops. In this study, the AeWRKY32 transcription factor that encodes 482 amino acids was amplified from A. esculentus, and its expression level was found to be the highest in the okra flower. AeWRKY32 localized to the nucleus and displayed transcriptional activation capability. Under normal conditions, overexpression of AeWRKY32 induced anthocyanin accumulation, with higher expression levels of AtCHS1, AtCHI4, AtF3H1, and AtDFR2 in transgenic Arabidopsis. Under cold stress, anthocyanin levels were further elevated in transgenic Arabidopsis plants. At the same time, AeWRKY32 overexpression promoted ABA biosynthesis, inhibited H2O2 and O2- generation, induced stomatal closure, reduced electrolyte leakage, and thus improved the cold resistance of transgenic Arabidopsis. Furthermore, under cold stress, the expression profiles of AtCOR413, AtCOR15B, AtCBF1, and AtCBF2 were upregulated in transgenic Arabidopsis. Overall, our study provides evidence that AeWRKY32 serves as a crucial regulator in both anthocyanin accumulation and cold tolerance of transgenic Arabidopsis. Our findings could provide insights into the molecular mechanism linking AeWRKYs to plant cold tolerance.

Effect of betanin synthesis on photosynthesis and tyrosine metabolism in transgenic carrot.

BACKGROUND: Betalain is a natural pigment with important nutritional value and broad application prospects. Previously, we produced betanin biosynthesis transgenic carrots via expressing optimized genes CYP76AD1S, cDOPA5GTS and DODA1S. Betanin can accumulate throughout the whole transgenic carrots. But the effects of betanin accumulation on the metabolism of transgenic plants and whether it produces unexpected effects are still unclear. RESULTS: The accumulation of betanin in leaves can significantly improve its antioxidant capacity and induce a decrease of chlorophyll content. Transcriptome and metabolomics analysis showed that 14.0% of genes and 33.1% of metabolites were significantly different, and metabolic pathways related to photosynthesis and tyrosine metabolism were markedly altered. Combined analysis showed that phenylpropane biosynthesis pathway significantly enriched the differentially expressed genes and significantly altered metabolites. CONCLUSIONS: Results showed that the metabolic status was significantly altered between transgenic and non-transgenic carrots, especially the photosynthesis and tyrosine metabolism. The extra consumption of tyrosine and accumulation of betanin might be the leading causes.

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.

Low light intensity elongates period and defers peak time of photosynthesis: a computational approach to circadian-clock-controlled photosynthesis in tomato.

Photosynthesis is involved in the essential process of transforming light energy into chemical energy. Although the interaction between photosynthesis and the circadian clock has been confirmed, the mechanism of how light intensity affects photosynthesis through the circadian clock remains unclear. Here, we propose a first computational model for circadian-clock-controlled photosynthesis, which consists of the light-sensitive protein P, the core oscillator, photosynthetic genes, and parameters involved in the process of photosynthesis. The model parameters were determined by minimizing the cost function ( [Formula: see text]), which is defined by the errors of expression levels, periods, and phases of the clock genes (CCA1, PRR9, TOC1, ELF4, GI, and RVE8). The model recapitulates the expression pattern of the core oscillator under moderate light intensity (100 µmol m -2 s-1). Further simulation validated the dynamic behaviors of the circadian clock and photosynthetic outputs under low (62.5 µmol m-2 s-1) and normal (187.5 µmol m-2 s-1) intensities. When exposed to low light intensity, the peak times of clock and photosynthetic genes were shifted backward by 1-2 hours, the period was elongated by approximately the same length, and the photosynthetic parameters attained low values and showed delayed peak times, which confirmed our model predictions. Our study reveals a potential mechanism underlying the circadian regulation of photosynthesis by the clock under different light intensities in tomato.

Lycopene ε-cyclase mediated transition of α-carotene and ß-carotene metabolic flow in carrot fleshy root.

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 ß-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.

AgMYB5, an MYB transcription factor from celery, enhanced ß-carotene synthesis and promoted drought tolerance in transgenic Arabidopsis.

BACKGROUND: Water shortage caused by global warming seriously affects the yield and quality of vegetable crops. ß-carotene, the lipid-soluble natural product with important pharmacological value, is abundant in celery. Transcription factor MYB family extensively disperses in plants and plays regulatory roles in carotenoid metabolism and water scarcity response. RESULTS: Here, the AgMYB5 gene encoding 196 amino acids was amplified from celery cv. 'Jinnanshiqin'. In celery, the expression of AgMYB5 exhibited transactivation activity, tissue specificity, and drought-condition responsiveness. Further analysis proved that ectopic expression of AgMYB5 increased ß-carotene content and promoted drought tolerance in transgenic Arabidopsis thaliana. Moreover, AgMYB5 expression promoted ß-carotene biosynthesis by triggering the expression of AtCRTISO and AtLCYB, which in turn increased antioxidant enzyme activities, and led to the decreased contents of H2O2 and MDA, and the inhibition of O2- generation. Meanwhile, ß-carotene accumulation promoted endogenous ABA biosynthesis of transgenic Arabidopsis, which resulted in ABA-induced stomatal closing and delayed water loss. In addition, ectopic expression of AgMYB5 increased expression levels of AtERD1, AtP5CS1, AtRD22, and AtRD29. CONCLUSIONS: The findings indicated that AgMYB5 up-regulated ß-carotene biosynthesis and drought tolerance of Arabidopsis.

Advances in engineering the production of the natural red pigment lycopene: A systematic review from a biotechnology perspective.

BACKGROUND: Lycopene is a natural red compound with potent antioxidant activity that can be utilized both as pigment and as a raw material in functional food, and so possesses good commercial prospects. The biosynthetic pathway has already been documented, which provides the foundation for lycopene production using biotechnology. AIM OF REVIEW: Although lycopene production has begun to take shape, there is still an urgent need to alleviate the yield of lycopene. Progress in this area can provide useful reference for metabolic engineering of lycopene production utilizing multiple approaches. KEY SCIENTIFIC CONCEPTS OF REVIEW: Using conventional microbial fermentation approaches, biotechnologists have enhanced the yield of lycopene by selecting suitable host strains, utilizing various additives, and optimizing culture conditions. With the development of modern biotechnology, genetic engineering, protein engineering, and metabolic engineering have been applied for lycopene production. Extraction from natural plants is the main way for lycopene production at present. Based on the molecular mechanism of lycopene accumulation, the production of lycopene by plant bioreactor through genetic engineering has a good prospect. Here we summarized common strategies for optimizing lycopene production engineering from a biotechnology perspective, which are mainly carried out by microbial cultivation. We reviewed the challenges and limitations of this approach, summarized the critical aspects, and provided suggestions with the aim of potential future breakthroughs for lycopene production in plants.

Morphological characteristics, anatomical structure, and dynamic change of ascorbic acid under different storage conditions of celery.

Ascorbic acid (AsA) is a crucial antioxidant in vegetables. Celery (Apium graveolens L.) is a vegetable of Apiaceae and is rich in AsA. Till now, the effects of different storage conditions on celery morphological characteristics, anatomical features, and antioxidant accumulation are unclear. Here, the celery cvs. 'Sijixiaoxiangqin' and 'Liuhehuangxinqin' were selected as experimental materials, and the two celery plants grown for 65 days were harvested from soils and stored in light at room temperature (25 °C), darkness at low temperature (4 °C), and darkness at room temperature (25 °C) for 0, 6, 24, 30, 48, and 54 h, respectively. The results showed that celery in darkness had better water retention capacity than celery in light. Morphological changes in celery mesophyll, leaf veins, and petioles were the least in darkness at low temperature (4 °C). The weight loss rate and wilting degree in darkness at low temperature (4 °C) were the lowest, and the AsA content remained at a high level. The expression patterns of GDP-D-mannose pyrophosphorylase (AgGMP) and L-galactose dehydrogenase (AgGalDH) were similar to the change of AsA content. The results indicated that low temperature and dark was the optimized storage condition for 'Sijixiaoxiangqin' and 'Liuhehuangxinqin' celery. AgGMP and AgGalDH genes may play an important role in the accumulation of AsA in celery. This paper will provide potential references for prolonging the shelf life of celery and other horticultural crops.

Transcriptome-wide identification of NAC (no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon) transcription factors potentially involved in salt stress response in garlic.

Soil salinity has been an increasing problem worldwide endangering crop production and human food security. It is an ideal strategy to excavate stress resistant genes and develop salt tolerant crops. NAC (no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon) transcription factors have been demonstrated to be involved in salt stress response. However, relevant studies have not been observed in garlic, an important vegetable consumed in the world. In this study, a total of 46 AsNAC genes encoding NAC proteins were identified in garlic plant by transcriptome data. Phylogenetic analysis showed that the examined AsNAC proteins were clustered into 14 subgroups. Motif discovery revealed that the conserved domain region was mainly composed of five conserved subdomains. Most of the genes selected could be induced by salt stress in different tissues, indicating a potential role in salt stress response. Further studies may focus on the molecular mechanisms of the AsNAC genes in salt stress response. The results of the current work provided valuable resources for researchers aimed at developing salt tolerant crops.

Differential hydroxylation efficiency of the two non-heme carotene hydroxylases: DcBCH1, rather than DcBCH2, plays a major role in carrot taproot.

Carotene hydroxylase plays an important role in catalyzing the hydroxylation of carotene to xanthopylls, including two types: non-heme carotene hydroxylase (BCH type) and heme-containing cytochrome P450 hydroxylase (P450 type). Two BCH-encoding genes were annotated in the carrot genome. However, the role of BCHs and whether there are functional interactions between the duplicated BCHs in carrot remains unclear. In this study, two BCH encoding genes, DcBCH1 and DcBCH2, were cloned from carrot. The relative expression level of DcBCH1 was much higher than that of DcBCH2 in carrot taproots with different carotene accumulation levels. Overexpression of DcBCH1 in 'KRD' (high carotene accumulated) carrot changed the taproot color from orange to yellow, accompanied by substantial reductions in α-carotene and ß-carotene. There was no obvious change in taproot color between transgenic 'KRD' carrot overexpressing DcBCH2 and control carrot. Simultaneously, the content of α-carotene in the taproot of DcBCH2-overexpressing carrot decreased, but the content of ß-carotene did not change significantly in comparison with control carrot. Using the CRISPR/Cas9 system to knock out DcBCH1 in 'KRD' carrot lightened the taproot color from orange to pink-orange; the content of α-carotene in the taproot increased slightly, while the ß-carotene content was still significantly decreased, compared with control carrot. In DcBCH1-knockout carrot, the transcript level of DcBCH2 was significantly increased. These results indicated that in carrot taproot, DcBCH1 played the main function of BCH enzyme, which could hydroxylate α-carotene and ß-carotene; DcBCH1 and DcBCH2 had functional redundancy, and these two DcBCHs could partially compensate for each other.

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.