Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation.

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.

Investigating the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles in atherosclerosis treatment.

BACKGROUND: Carthamus tinctorius L., a traditional herbal medicine used for atherosclerosis (AS), lacks a clear understanding of its therapeutic mechanisms. This study aimed to investigate the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles (CDNVs) in AS treatment. METHODS: CDNVs were isolated and characterized using improved isolation methods. Transmission electron microscopy, nanoparticle tracking analysis, and protein analysis confirmed their morphology, size, and protein composition. Small RNA sequencing was performed to identify the miRNA profile of CDNVs, and bioinformatics analysis was used to determine their potential biological roles. In vivo biodistribution and toxicity studies were conducted in mice to assess the stability and safety of orally administered CDNVs. The anti-atherosclerotic effects of CDNVs were evaluated in ApoE-/- mice through plaque burden analysis. The protective effects of CDNVs on ox-LDL-treated endothelial cells were assessed through proliferation, apoptosis, reactive oxygen species activation, and monocyte adhesion assays. miRNA and mRNA sequencing of CDNV-treated endothelial cells were performed to explore their regulatory effects and potential target genes. RESULTS: CDNVs were successfully isolated and purified from Carthamus tinctorius L. tissue lysates. They exhibited a saucer-shaped or cup-shaped morphology, with an average particle size of 142.6 ± 0.7 nm, and expressed EV markers CD63 and TSG101. CDNVs contained proteins, small RNAs, and metabolites, including the therapeutic compound HSYA. Small RNA sequencing identified 95 miRNAs, with 10 common miRNAs accounting for 72.63% of the total miRNAs. These miRNAs targeted genes involved in cell adhesion, apoptosis, and cell proliferation, suggesting their relevance in cardiovascular disease. Orally administered CDNVs were stable in the gastrointestinal tract, absorbed into the bloodstream, and accumulated in the liver, lungs, heart, and aorta. They significantly reduced the burden of atherosclerotic plaques in ApoE-/- mice and exhibited superior effects compared to HSYA. In vitro studies demonstrated that CDNVs were taken up by HUVECs, promoted proliferation, attenuated ox-LDL-induced apoptosis and ROS activation, and reduced monocyte adhesion. CDNV treatment resulted in significant changes in miRNA and mRNA expression profiles of HUVECs, with enrichment in inflammation-related genes. CXCL12 was identified as a potential direct target of miR166a-3p. CONCLUSION: CDNVs isolated from Carthamus tinctorius L. tissue lysates represent a promising oral therapeutic option for cardiovascular diseases. The delivery of miRNAs by CDNVs regulates inflammation-related genes, including CXCL12, in HUVECs, suggesting their potential role in modulating endothelial inflammation. These findings provide valuable insights into the therapeutic potential of CDNVs and their miRNAs in cardiovascular disease.

Identification of genes associated with fatty acid biosynthesis based on 214 safflower core germplasm.

BACKGROUND: Safflower (Carthamus tinctorius L.) is an oilseed crop with substantial medicinal and economic value. However, the methods for constructing safflower core germplasm resources are limited, and the molecular mechanisms of lipid biosynthesis in safflower seeds are not well understood. RESULTS: In this study, 11 oil-related quantitative traits and 50 pairs of InDel markers were used to assess the diversity of a collection of 605 safflower germplasms. The original safflower germplasm exhibited rich phenotypic diversity, with high variation for most of the phenotypic traits under investigation. Similarly, high genetic diversity was evaluated in the original germplasm, in which the mean Shannon's information index (I), observed heterozygosity (H0), and expected heterozygosity (He) were 0.553, 0.182, and 0.374, respectively. Four subgroups with strong genetic structures were identified and a core germplasm of 214 cultivars was constructed, which is well represented in the original germplasm. Meanwhile, differential expression analysis of the transcriptomes of high and low linoleic acid safflower varieties at two stages of seed development identified a total of 47 genes associated with lipid biosynthesis. High expression of the genes KAS II and SAD enhanced the synthesis and accumulation of oleic acid, while FAD genes like FAD2 (Chr8G0104100), FAD3, FAD7 and FAD8 promoted the consumption of oleic acid conversion. The coordinated regulation of these multiple genes ensures the high accumulation of oleic acid in safflower seed oil. CONCLUSIONS: Based on these findings, a core germplasm of 214 cultivars was constructed and 47 candidate genes related to unsaturated fatty acid biosynthesis and lipid accumulation were identified. These results not only provide guidance for further studies to elucidate the molecular basis of oil lipid accumulation in safflower seeds, but also contribute to safflower cultivar improvements.

A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.

To explore the complete biosynthesis process of flavonoid glycosides in safflower, specifically the key glycosyltransferase that might be involved, as well as to develop an efficient biocatalyst to synthesize flavonoid glycosides, a glycosyltransferase CtUGT4, with flavonoid-O-glycosyltransferase activity, was identified in safflower. The fusion protein of CtUGT4 was heterologously expressed in Escherichia coli, and the target protein was purified. The recombinant protein can catalyze quercetin to form quercetin-7-O-glucoside, and kaempferol to form kaempferol-3-O in vitro, and a series of flavones, flavonols, dihydroflavones, chalcones, and chalcone glycosides were used as substrates to generate new products. CtUGT4 was expressed in the tobacco transient expression system, and the enzyme activity results showed that it could catalyze kaempferol to kaempferol-3-O-glucoside, and quercetin to quercetin-3-O-glucoside. After overexpressing CtUGT4 in safflower, the content of quercetin-3-O-rutinoside in the safflower florets increased significantly, and the content of quercetin-3-O-glucoside also tended to increase, which preliminarily confirmed the function of CtUGT4 flavonoid-O-glycosyltransferase. This work demonstrated the flavonoid-O-glycosyltransferase function of safflower CtUGT4 and showed differences in the affinity for different flavonoid substrates and the regioselectivity of catalytic sites in safflower, both in vivo and in vitro, providing clues for further research regarding the function of UGT genes, as well as new ideas for the cultivation engineering of the directional improvement of effective metabolites in safflower.

Identification and Characterization of CtUGT3 as the Key Player of Astragalin Biosynthesis in Carthamus tinctorius L.

Safflower (Carthamus tinctorius L.) is a multipurpose economic crop that is distributed worldwide. Flavonoid glycosides are the main bioactive components in safflower, but only a few UDP-glycosyltransferases (UGT) have been identified. Three differentially expressed UGT genes related with the accumulation of 9 flavonoid O-glycosides were screened from metabolomics and transcriptome analysis. Safflower corolla protoplasts were used to confirm the glycosylation ability of UGT candidates in vivo for the first time. The astragalin content was significantly increased only when CtUGT3 was overexpressed. CtUGT3 also showed flavonoid 3-OH and 7-OH glycosylation activities in vitro. Molecular modeling and site-directed mutagenesis revealed that G15, T136, S276, and E384 were critical catalytic residues for the glycosylation ability of CtUGT3. These results demonstrate that CtUGT3 has a flavonoid 3-OH glycosylation function and is involved in the biosynthesis of astragalin in safflower. This study provides a reference for flavonoid biosynthesis genes research in nonmodel plants.

Genome-wide identification and comprehensive analysis of WRKY transcription factor family in safflower during drought stress.

The WRKY family is an important family of transcription factors in plant development and stress response. Currently, there are few reports on the WRKY gene family in safflower (Carthamus tinctorius L.). In this study, a total of 82 CtWRKY genes were identified from the safflower genome and could be classified into 3 major groups and 5 subgroups based on their structural and phylogenetic characteristics. The results of gene structure, conserved domain and motif analyses indicated that CtWRKYs within the same subfamily maintained a consistent exon/intron organization and composition. Chromosomal localization and gene duplication analysis results showed that CtWRKYs were randomly localized on 12 chromosomes and that fragment duplication and purification selection may have played an important role in the evolution of the WRKY gene family in safflower. Promoter cis-acting element analysis revealed that the CtWRKYs contain many abiotic stress response elements and hormone response elements. Transcriptome data and qRT-PCR analyses revealed that the expression of CtWRKYs showed tissue specificity and a strong response to drought stress. Notably, the expression level of the CtWRKY55 gene rapidly increased more than eightfold under drought treatment and rehydration, indicating that it may be a key gene in response to drought stress. These results provide useful insights for investigating the regulatory function of the CtWRKY gene in safflower growth and development, as well as identifying key genes for future molecular breeding programmes.

Unraveling the functional characterization of a jasmonate-induced flavonoid biosynthetic CYP45082G24 gene in Carthamus tinctorius.

The cytochrome P450 superfamily of monooxygenases plays a major role in the evolution and diversification of plant natural products. The function of cytochrome P450s in physiological adaptability, secondary metabolism, and xenobiotic detoxification has been studied extensively in numerous plant species. However, their underlying regulatory mechanism in safflower still remained unclear. In this study, we aimed to elucidate the functional role of a putative CtCYP82G24-encoding gene in safflower, which suggests crucial insights into the regulation of methyl jasmonate-induced flavonoid accumulation in transgenic plants. The results showed that methyl jasmonate (MeJA) was associated with a progressive upregulation of CtCYP82G24 expression in safflower among other treatment conditions including light, dark, and polyethylene glycol (PEG). In addition, transgenic plants overexpressing CtCYP82G24 demonstrated increased expression level of other key flavonoid biosynthetic genes, such as AtDFR, AtANS, and AtFLS, and higher content of flavonoid and anthocyanin accumulation when compared with wild-type and mutant plants. Under exogenous MeJA treatment, the CtCYP82G24 transgenic overexpressed lines showed a significant spike in flavonoid and anthocyanin content compared with wild-type and mutant plants. Moreover, the virus-induced gene silencing (VIGS) assay of CtCYP82G24 in safflower leaves exhibited decreased flavonoid and anthocyanin accumulation and reduced expression of key flavonoid biosynthetic genes, suggesting a possible coordination between transcriptional regulation of CtCYP82G24 and flavonoid accumulation. Together, our findings confirmed the likely role of CtCYP82G24 during MeJA-induced flavonoid accumulation in safflower.

Complete Mitogenome and Phylogenetic Analysis of the Carthamus tinctorius L.

Carthamus tinctorius L. 1753 (Asteraceae), also called safflower, is a cash crop with both edible and medical properties. We analyzed and reported the safflower mitogenome based on combined short and long reads obtained from Illumina and Pacbio platforms, respectively. This safflower mitogenome mainly contained two circular chromosomes, with a total length of 321,872 bp, and encoded 55 unique genes, including 34 protein-coding genes (PCGs), 3 rRNA genes, and 18 tRNA genes. The total length of repeat sequences greater than 30 bp was 24,953 bp, accounting for 7.75% of the whole mitogenome. Furthermore, we characterized the RNA editing sites of protein-coding genes located in the safflower mitogenome, and the total number of RNA editing sites was 504. Then, we revealed partial sequence transfer events between plastid and mitochondria, in which one plastid-derived gene (psaB) remained intact in the mitogenome. Despite extensive arrangement events among the three mitogenomes of C. tinctorius, Arctium lappa, and Saussurea costus, the constructed phylogenetic tree based on mitogenome PCGs showed that C. tinctorius has a closer relationship with three Cardueae species, A. lappa, A. tomentosum, and S. costus, which is similar to the phylogeny constructed from the PCGs of plastid genomes. This mitogenome not only enriches the genetic information of safflower but also will be useful in the phylogeny and evolution study of the Asteraceae.

Phylogenomic investigation of safflower (Carthamus tinctorius) and related species using genotyping-by-sequencing (GBS).

Safflower (Carthamus tinctorius, Asteraceae) is a source of high-quality edible oil growing in moisture-limited environments. Despite its economic importance, the relationships to close wild species in Carthamus and the presence and relationships of ecotypes within safflower are still not fully clarified. Here we use genotyping-by-sequencing to identify the wild progenitor of C. tinctorius, infer phylogenetic relationship within the series Carthamus and identify groups of closely related lineages within cultivated safflower. Phylogenetic and population genomic analyses found C. palaestinus to be the closest relative and single progenitor of C. tinctorius, which confirms the Levant as the area of domestication of the crop. Flow cytometry showed all analyzed samples of C. oxyacantha, C. palaestinus and C. tinctorius to be diploid (2n = 2x = 24) with 2C genome sizes of 2.4-2.7 pg. Analyses of a set of 114 worldwide distributed safflower accessions arrived at two to five genetic groups, which showed, however, no correlation with the geographic origins of these accessions. From this, we conclude that the trade of safflower seeds resulted in multiple introductions of genotypes from the Levant into other areas with suitable climate conditions for the plant, as well as exchange of genotypes among these areas.

The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L.

BACKGROUND: Safflower (Carthamus tinctorius L.) is an important economic crop and a traditional medicinal material rich in flavonoids, which can alleviate cardiovascular and cerebrovascular pathologies. Thus, many candidate genes involved in safflower flavonoid biosynthesis have been cloned. However, owing to the lack of a homologous gene expression system, research on gene function is limited to model plants. Therefore, a gene function identification protocol for safflower must be established. RESULTS: In the present study, using safflower callus as the experimental material, Agrobacterium and biolistic transient expression systems were established. In the Agrobacterium transient expression system, the highest transformation rate was obtained at the original Agrobacterium concentration of OD600 0.4, infiltration concentration of OD600 0.6, infection for 20 min, co-culture for 3 days, and acetosyringone concentration of 100 µmol·L-1. In the biolistic transient expression system, the highest transformation efficiency was observed at helium pressure of 1,350 psi, vacuum degree of -0.8 bar, flight distance of 6.5 cm, one round of bombardment, plasmid concentration of 3 µg·shot-1, and gold particle concentration of 100 µg·shot-1. Further, these two transient expression systems were used for the functional analysis of CtCHS1 as an example. After overexpression, relative CtCHS1 expression increased, particularly in Agrobacterium-transformed calli. Additionally, the contents of some flavonoids were altered; for instance, naringenin and genistein levels were significantly increased in Agrobacterium-transformed calli, whereas luteolin, luteolin-7-O-rutinoside, and apigenin derivative levels were significantly decreased in biolistic-transformed calli. CONCLUSION: Using safflower callus as the experimental material, highly efficient Agrobacterium and biolistic transient expression systems were successfully established, and the utility of both systems for investigating gene function was demonstrated. The proposed safflower callus transient expression systems will be useful for further functional analyses of flavonoid biosynthetic genes in safflower.

Safflower Flavonoid 3'5'Hydroxylase Promotes Methyl Jasmonate-Induced Anthocyanin Accumulation in Transgenic Plants.

Flavonoids are the most abundant class of secondary metabolites that are ubiquitously involved in plant development and resistance to biotic and abiotic stresses. Flavonoid biosynthesis involves multiple channels of orchestrated molecular regulatory factors. Methyl jasmonate (MeJA) has been demonstrated to enhance flavonoid accumulation in numerous plant species; however, the underlying molecular mechanism of MeJA-induced flavonoid biosynthesis in safflower is still not evident. In the present study, we revealed the underlying molecular basis of a putative F3'5'H gene from safflower imparting MeJA-induced flavonoid accumulation in transgenic plants. The constitutive expression of the CtF3'5'H1 gene was validated at different flowering stages, indicating their diverse transcriptional regulation through flower development in safflower. Similarly, the CtF3'5'H1-overexpressed Arabidopsis plants exhibit a higher expression level, with significantly increased anthocyanins and flavonoid content, but less proanthocyanidins than wild-type plants. In addition, transgenic plants treated with exogenous MeJA revealed the up-regulation of CtF3'5'H1 expression over different time points with significantly enhanced anthocyanin and flavonoid content as confirmed by HPLC analysis. Moreover, CtF3'5'H1- overexpressed Arabidopsis plants under methyl violet and UV-B irradiation also indicated significant increase in the expression level of CtF3'5'H1 with improved anthocyanin and flavonoid content, respectively. Noticeably, the virus-induced gene silencing (VIGS) assay of CtF3'5'H1 in safflower leaves also confirmed reduced anthocyanin accumulation. However, the CtF3'5'H1 suppression in safflower leaves under MeJA elicitation demonstrated significant increase in total flavonoid content. Together, our findings confirmed that CtF3'5'H1 is likely mediating methyl jasmonate-induced flavonoid biosynthesis in transgenic plants via enhanced anthocyanin accumulation.

A Cinnamate 4-HYDROXYLASE1 from Safflower Promotes Flavonoids Accumulation and Stimulates Antioxidant Defense System in Arabidopsis.

C4H (cinnamate 4-hydroxylase) is a pivotal gene in the phenylpropanoid pathway, which is involved in the regulation of flavonoids and lignin biosynthesis of plants. However, the molecular mechanism of C4H-induced antioxidant activity in safflower still remains to be elucidated. In this study, a CtC4H1 gene was identified from safflower with combined analysis of transcriptome and functional characterization, regulating flavonoid biosynthesis and antioxidant defense system under drought stress in Arabidopsis. The expression level of CtC4H1 was shown to be differentially regulated in response to abiotic stresses; however, a significant increase was observed under drought exposure. The interaction between CtC4H1 and CtPAL1 was detected using a yeast two-hybrid assay and then verified using a bimolecular fluorescence complementation (BiFC) analysis. Phenotypic and statistical analysis of CtC4H1 overexpressed Arabidopsis demonstrated slightly wider leaves, long and early stem development as well as an increased level of total metabolite and anthocyanin contents. These findings imply that CtC4H1 may regulate plant development and defense systems in transgenic plants via specialized metabolism. Furthermore, transgenic Arabidopsis lines overexpressing CtC4H1 exhibited increased antioxidant activity as confirmed using a visible phenotype and different physiological indicators. In addition, the low accumulation of reactive oxygen species (ROS) in transgenic Arabidopsis exposed to drought conditions has confirmed the reduction of oxidative damage by stimulating the antioxidant defensive system, resulting in osmotic balance. Together, these findings have provided crucial insights into the functional role of CtC4H1 in regulating flavonoid biosynthesis and antioxidant defense system in safflower.

Effects of Temperature and Salt Stress on the Expression of delta-12 Fatty Acid Desaturase Genes and Fatty Acid Compositions in Safflower.

The regulation of microsomal (e.g., FAD2) and plastidial (e.g., FAD6) oleate desaturases by cold, heat and salt stress were investigated. Gene expression levels and fatty acid compositions were determined in the roots, stems and leaves of safflower following stress treatments. A safflower plastidial oleate desaturase gene, CtFAD6, was cloned, and oleic acid desaturation was confirmed in Synechococcus sp. strain PCC7942. The results showed that temperature regulated oleate desaturation at the transcriptional level, and this regulation pattern was tissue-specific. CtFAD2-1, CtFAD2-2 and CtFAD6 were significantly induced under cold and heat stress in young leaves, and CtFAD2-2 and CtFAD6 were slightly induced in young stems. In contrast, CtFAD2-1, CtFAD2-11 and CtFAD2-10 were sensitive to salt stress in all safflower tissues (roots, stem and leaves). CtFAD6 was insensitive to salt and was slightly induced in leaves only.

The safflower MBW complex regulates HYSA accumulation through degradation by the E3 ligase CtBB1.

The regulatory mechanism of the MBW (MYB-bHLH-WD40) complex in safflower (Carthamus tinctorius) remains unclear. In the present study, we show that the separate overexpression of the genes CtbHLH41, CtMYB63, and CtWD40-6 in Arabidopsis thaliana increased anthocyanin and procyanidin contents in the transgenic plants and partially rescued the trichome reduction phenotype of the corresponding bhlh41, myb63, and wd40-6 single mutants. Overexpression of CtbHLH41, CtMYB63, or CtWD40-6 in safflower significantly increased the content of the natural pigment hydroxysafflor yellow A (HYSA) and negatively regulated safflower petal size. Yeast-two-hybrid, functional, and genetic assays demonstrated that the safflower E3 ligase CtBB1 (BIG BROTHER 1) can ubiquitinate CtbHLH41, marking it for degradation through the 26S proteasome and negatively regulating flavonoid accumulation. CtMYB63/CtWD40-6 enhanced the transcriptional activity of CtbHLH41 on the CtDFR (dihydroflavonol 4-reductase) promoter. We propose that the MBW-CtBB1 regulatory module may play an important role in coordinating HYSA accumulation with other response mechanisms.

Molecular Characterization of a Stereoselective and Promiscuous Flavanone 3-Hydroxylase from Carthamus tinctorius L.

Flavanone 3-hydroxylases (F3Hs) belong to the 2-oxoglutarate-dependent dioxygenase family and play an important role in plant flavonoid biosynthesis. However, the stereoselective catalytic mechanism and substrate promiscuity of this type of enzyme are not well understood. In this study, we identified and biochemically characterized CtF3H1, an F3H from Carthamus tinctorius, a plant used in traditional Chinese medicine that exhibits high stereoselectivity and substrate promiscuity toward structurally diverse (2S)-flavanones. Isothermal titration calorimetry revealed that CtF3H1 exhibits distinctly different binding behaviors with (2S)-flavanone (2S-naringenin) and (2R)-flavanone (2R-naringenin), and these differences govern its stereoselectivity. An investigation of the structure-activity relationships between the enzyme and its substrates demonstrated that 7-OH and/or 4'-OH are necessary for regio- and stereoselective 3-hydroxylation of (2S)-flavanones. Homology modeling and molecular docking combined with site-directed mutagenesis identified the amino acid residues necessary for hydroxylation. These findings demonstrate the potential versatility of CtF3H1 in regio- and stereohydroxylation and provide molecular insights into the catalytic mechanism of F3H for further enzyme engineering.

Genome-Wide Identification of MADS-Box Family Genes in Safflower (Carthamus tinctorius L.) and Functional Analysis of CtMADS24 during Flowering.

Safflower is an important economic crop with a plethora of industrial and medicinal applications around the world. The bioactive components of safflower petals are known to have pharmacological activity that promotes blood circulation and reduces blood stasis. However, fine-tuning the genetic mechanism of flower development in safflower is still required. In this study, we report the genome-wide identification of MADS-box transcription factors in safflower and the functional characterization of a putative CtMADS24 during vegetative and reproductive growth. In total, 77 members of MADS-box-encoding genes were identified from the safflower genome. The phylogenetic analysis divided CtMADS genes into two types and 15 subfamilies. Similarly, bioinformatic analysis, such as of conserved protein motifs, gene structures, and cis-regulatory elements, also revealed structural conservation of MADS-box genes in safflower. Furthermore, the differential expression pattern of CtMADS genes by RNA-seq data indicated that type II genes might play important regulatory roles in floral development. Similarly, the qRT-PCR analysis also revealed the transcript abundance of 12 CtMADS genes exhibiting tissue-specific expression in different flower organs. The nucleus-localized CtMADS24 of the AP1 subfamily was validated by transient transformation in tobacco using GFP translational fusion. Moreover, CtMADS24-overexpressed transgenic Arabidopsis exhibited early flowering and an abnormal phenotype, suggesting that CtMADS24 mediated the expression of genes involved in floral organ development. Taken together, these findings provide valuable information on the regulatory role of CtMADS24 during flower development in safflower and for the selection of important genes for future molecular breeding programs.

Cloning and expression analysis of HY5 transcription factor gene of safflower in response to light signal.

The flower of the safflower (Carthamus tinctorius L.) is a traditional Chinese medicine that can improve cerebral blood flow due to its enrichment in flavonoids. Light is one of the main environmental factors that affects safflower growth and flavonoid synthesis. Elongated hypocotyl 5 (HY5) plays an important role in plants' light signal transduction. However, no study of HY5 in safflower has been conducted. In this study, a 462-bp sequence of CtHY5 was successfully cloned. The expression pattern of CtHY5 in different safflower tissues and the expression patterns of CtHY5 and CtCHS1 in full-blooming flowers that were treated under different light intensities were studied. The subcellular localization and the overexpression of CtHY5 were carried out as well. CtHY5 has a DNA-binding region belonging to the basic leucine zipper transcription factor family. CtHY5 was specifically expressed in flowers. The expression level of CtHY5 first increased and then decreased with increasing light intensity, which was similar to the expression pattern of CtCHS1. The subcellular localization study was implemented in safflower protoplasts and the YFP fluorescence was observed in nucleus. The overexpression analysis initially verified the promotion effect of CtHY5 to the expression of CtCHS1 and the content of flavonoids.

Genome-wide analysis of CtNF-YB and lipid synthesis regulation of CtNF-YB12 in Carthamus tinctorius L.

KEY MESSAGE: The nuclear Factor YB of Carthamus tinctorius L. increased the content of unsaturated fatty acids by regulating the expression of genes involved in fatty acid synthesis and oil accumulation. Safflower (Carthamus tinctorius L.) seed oil is rich in linoleic acid and is widely used in food and medicine. Therefore, key genes regulating oil synthesis were mined through genetic engineering to provide genetic resources for improving oil content. Based on the conserved domain of the NF-YB, we screened and identified 14 CtNF-YB transcription factors in the safflower genome and divided them into three subfamilies through phylogenetic analysis. Regulatory motif analysis of the CtNF-YB promoter revealed specific cis-regulatory elements related to abiotic stress, growth, and development. Expression analysis of CtNF-YB family genes showed that non-Leafy Cotyledon 1(non-LEC1) genes were highly expressed in roots, leaves, and flowers; Leafy Cotyledon 1(LEC1) genes were highly expressed during early seed development; and Dr1-like genes were highly expressed in roots, stems, and leaves. CtNF-YB12 was identified as a LEC1 transcription factor based on phylogeny and BLAST alignment. Heterologous CtNF-YB12 expression in Arabidopsis thaliana increased seed pod length and seed size. Moreover, CtNF-YB12 overexpression increased the oil content of seeds, upregulated genes involved in fatty acid biosynthesis and glycolysis, and altered the content of unsaturated fatty acids, including oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), as well as of sucrose, fructose, and glucose. CtNF-YB12 may increase the oil content by regulating key enzyme genes of oil synthesis, so it can be used as a reliable target.

[Functional characterization and enzymatic properties of flavonoid glycosyltransferase gene CtUGT49 in Carthamus tinctorius].

Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 µmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.

[Identification of Carthamus tinctorius NAC gene family and analysis of drought stress response].

The NAC(NAM/ATAF/CUC) transcription factors are members of the largest transcriptional gene family in plants and play an essential role in the response of plants to drought stress. To identify the number and function of the NAC gene family in Carthamus tinctorius, the present study adopted bioinformatics methods to identify NAC gene family members based on the whole genome data of C. tinctorius, and analyzed their physicochemical properties, chromosomal location, phylogenetic relationship, gene structure, conserved domain, and conserved motif. Meanwhile, the real-time fluorescence-based quantitative RT-PCR(qRT-PCR) was used to analyze the transcription level of four NAC genes under drought stress in different time. The results showed that C. tinctorius contained 87 NAC genes unevenly distributed on 11 chromosomes, while no NAC gene was found on chromosome 12. The encoded proteins were 103-974 amino acids and the number of CDS ranged from 3 to 9. According to the phylogenetic relationships, 87 NAC genes were clustered into17 subfamilies. The analysis of conserved domains and motifs revealed that most of the genes contained five conserved subdomains, A-E and motif2 was the most conserved among NAC genes. The expression pattern analysis showed that the transcription levels of four NAC genes related to drought resistance were all up-regulated after drought stress treatment for different time, suggesting that these four NAC genes may be related to drought resistance of C. tinctorius. This study is expected to provide a theoretical basis for further functional analysis of NAC transcription factors in C. tinctorius and references for the cultivation of drought-tolerant C. tinctorius varieties.