The Carthamus tinctorius L. genome sequence provides insights into synthesis of unsaturated fatty acids.

Domesticated safflower (Carthamus tinctorius L.) is a widely cultivated edible oil crop. However, despite its economic importance, the genetic basis underlying key traits such as oil content, resistance to biotic and abiotic stresses, and flowering time remains poorly understood. Here, we present the genome assembly for C. tinctorius variety Jihong01, which was obtained by integrating Oxford Nanopore Technologies (ONT) and BGI-SEQ500 sequencing results. The assembled genome was 1,061.1 Mb, and consisted of 32,379 protein-coding genes, 97.71% of which were functionally annotated. Safflower had a recent whole genome duplication (WGD) event in evolution history and diverged from sunflower approximately 37.3 million years ago. Through comparative genomic analysis at five seed development stages, we unveiled the pivotal roles of fatty acid desaturase 2 (FAD2) and fatty acid desaturase 6 (FAD6) in linoleic acid (LA) biosynthesis. Similarly, the differential gene expression analysis further reinforced the significance of these genes in regulating LA accumulation. Moreover, our investigation of seed fatty acid composition at different seed developmental stages unveiled the crucial roles of FAD2 and FAD6 in LA biosynthesis. These findings offer important insights into enhancing breeding programs for the improvement of quality traits and provide reference resource for further research on the natural properties of safflower.

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.

Response of photosynthetic characteristics and antioxidant system in the leaves of safflower to NaCl and NaHCO3.

KEY MESSAGE: Compared with NaCl, NaHCO3 caused more serious oxidative damage and photosynthesis inhibition in safflower by down-regulating the expression of related genes. Salt-alkali stress is one of the important factors that limit plant growth. NaCl and sodium bicarbonate (NaHCO3) are neutral and alkaline salts, respectively. This study investigated the physiological characteristics and molecular responses of safflower (Carthamus tinctorius L.) leaves treated with 200 mmol L-1 of NaCl or NaHCO3. The plants treated with NaCl treatment were less effective at inhibiting the growth of safflower, but increased the content of malondialdehyde (MDA) in leaves. Meanwhile, safflower alleviated stress damage by increasing proline (Pro), soluble protein (SP), and soluble sugar (SS). Both fresh weight and dry weight of safflower was severely decreased when it was subjected to NaHCO3 stress, and there was a significant increase in the permeability of cell membranes and the contents of osmotic regulatory substances. An enrichment analysis of the differentially expressed genes (DEGs) using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes identified significant enrichment of photosynthesis and pathways related to oxidative stress. Furthermore, a weighted gene co-expression network analysis (WGCNA) showed that the darkgreen module had the highest correlation with photosynthesis and oxidative stress traits. Large numbers of transcription factors, primarily from the MYB, GRAS, WRKY, and C2H2 families, were predicted from the genes within the darkgreen module. An analysis of physiological indicators and DEGs, it was found that under saline-alkali stress, genes related to chlorophyll synthesis enzymes were downregulated, while those related to degradation were upregulated, resulting in inhibited chlorophyll biosynthesis and decreased chlorophyll content. Additionally, NaCl and NaHCO3 stress downregulated the expression of genes related to the Calvin cycle, photosynthetic antenna proteins, and the activity of photosynthetic reaction centers to varying degrees, hindering the photosynthetic electron transfer process, suppressing photosynthesis, with NaHCO3 stress causing more pronounced adverse effects. In terms of oxidative stress, the level of reactive oxygen species (ROS) did not change significantly under the NaCl treatment, but the contents of hydrogen peroxide and the rate of production of superoxide anions increased significantly under NaHCO3 stress. In addition, treatment with NaCl upregulated the levels of expression of the key genes for superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), the ascorbate-glutathione cycle, and the thioredoxin-peroxiredoxin pathway, and increased the activity of these enzymes, thus, reducing oxidative damage. Similarly, NaHCO3 stress increased the activities of SOD, CAT, and POD and the content of ascorbic acid and initiated the glutathione-S-transferase pathway to remove excess ROS but suppressed the regeneration of glutathione and the activity of peroxiredoxin. Overall, both neutral and alkaline salts inhibited the photosynthetic process of safflower, although alkaline salt caused a higher level of stress than neutral salt. Safflower alleviated the oxidative damage induced by stress by regulating its antioxidant system.

Safflower CtFLS1-Induced Drought Tolerance by Stimulating the Accumulation of Flavonols and Anthocyanins in Arabidopsis thaliana.

Flavonol synthase gene (FLS) is a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily and plays an important role in plant flavonoids biosynthetic pathways. Safflower (Carthamus tinctorius L.), a key source of traditional Chinese medicine, is widely cultivated in China. Although the flavonoid biosynthetic pathway has been studied in several model species, it still remains to be explored in safflower. In this study, we aimed to elucidate the role of CtFLS1 gene in flavonoid biosynthesis and drought stress responses. The bioinformatics analysis on the CtFLS1 gene showed that it contains two FLS-specific motifs (PxxxIRxxxEQP and SxxTxLVP), suggesting its independent evolution. Further, the expression level of CtFLS1 in safflower showed a positive correlation with the accumulation level of total flavonoid content in four different flowering stages. In addition, CtFLS1-overexpression (OE) Arabidopsis plants significantly induced the expression levels of key genes involved in flavonol pathway. On the contrary, the expression of anthocyanin pathway-related genes and MYB transcription factors showed down-regulation. Furthermore, CtFLS1-OE plants promoted seed germination, as well as resistance to osmotic pressure and drought, and reduced sensitivity to ABA compared to mutant and wild-type plants. Moreover, CtFLS1 and CtANS1 were both subcellularly located at the cell membrane and nucleus; the yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assay showed that they interacted with each other at the cell membrane. Altogether, these findings suggest the positive role of CtFLS1 in alleviating drought stress by stimulating flavonols and anthocyanin accumulation in safflower.

Highly Promiscuous Flavonoid Di-O-glycosyltransferases from Carthamus tinctorius L.

Safflower (Carthamus tinctorius L.) has been recognized for its medicinal value, but there have been limited studies on the glycosyltransferases involved in the biosynthesis of flavonoid glycosides from safflower. In this research, we identified two highly efficient flavonoid O-glycosyltransferases, CtOGT1 and CtOGT2, from safflower performing local BLAST alignment. By constructing a prokaryotic expression vector, we conducted in vitro enzymatic reactions and discovered that these enzymes were capable of catalyzing two-step O-glycosylation using substrates such as kaempferol, quercetin, and eriodictyol. Moreover, they exhibited efficient catalytic activity towards various compounds, including flavones (apigenin, scutellarein), dihydrochalcone (phloretin), isoflavones (genistein, daidzein), flavanones (naringenin, glycyrrhizin), and flavanonols (dihydrokaempferol), leading to the formation of O-glycosides. The broad substrate specificity of these enzymes is noteworthy. This study provides valuable insights into the biosynthetic pathways of flavonoid glycosides in safflower. The discovery of CtOGT1 and CtOGT2 enhances our understanding of the enzymatic processes involved in synthesizing flavonoid glycosides in safflower, contributing to the overall comprehension of secondary metabolite biosynthesis in this plant species.

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.

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.

Carthamus Tinctorius L. (Safflower) extracts inhibit expression of metastatic genes of MDA-MB-231 breast cancer cells.

Breast cancer is the most common type of cancer in women and the second  cause of cancer-related death after lung cancer. Although the common methods used in the treatment of breast cancer are chemotherapy, radiotherapy and surgery, the search for alternative treatments continues.  The leading  alternative treatments are medicinal plants which actually inspire the production of many cancer drugs. In this study, the proliferative and metastatic effects of Carthamus tinctorius L., known for its many therapeutic properties, on metastatic breast cancer were investigated. Here, intending to evaluate the the content and actions of different extracts of safflower leaves extracts were prepared by extracting in water, alcohol and oil and analysed by FTIR. Their antioxidant effect was tested and then the extracts were applied to metastatic breast cancer cells. FTIR spectrums of all three extracts have revealed the presence of organic compounds.  It is found that all extracts but mostly the oil extract has antioxidant property. MTT assay, wound healing assay and gene expression analysis were performed to assess the antiproliferative and anti metastatic effects of the extracts on breast cancer cells. It is found that, there is no significant antiproliferative effect of extracts on MDA-MB-231 cells except the alcohol extract. However, all safflower extracts, especially the oil extract, significantly reduced the metastatic potential of breast cancer cells. It is concluded that safflower contents are potent chemicals which inhibit the cellular mechanisms underlying the spreading of cancer cells and further analysis may lead to new initiatives in drug design research.

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.

Supportive effect of naringenin on NaCl-induced toxicity in Carthamus tinctorius seedlings.

In the present study, we used exogenous naringenin (0.5 mM) pretreatment before the stress (25 mM NaCl) on the growth and tolerance of safflower seedlings under non-salinity conditions and salinity conditions. Our results showed that salinity treatment significantly declined the biomass, leaf relative water content, chlorophyll content, K+ content, and K+/Na+ ratio by 28%, 28%, 12%, 36%, and 56%, respectively, as compared to untreated control. The results obtained in the present study showed the beneficial effects of the pretreatment of naringenin in safflower seedlings under non-salinity conditions concerning increasing plant biomass, total phenolic compound, radical scavenging activity (RSA), soluble sugar content, proline, glutathione, enzymatic antioxidants, and K+ content. Nevertheless, naringenin pretreated plants showed a clear increment in the values of biomass, RSA, total phenolic compound, and catalase enzyme activity parameters under salinity stress. Salinity stress caused ionic phytotoxicity and oxidative stress by enhancing Na+ content, H2O2 accumulation, malondialdehyde (MDA), and antioxidants. However, naringenin alleviated salt-induced oxidative stress by decreasing H2O2 and MDA content in the leaves and improving the catalase activity in treated plants. Generally, it could be concluded pretreatment of naringenin before stress could partly diminish NaCl-caused oxidative stress in safflower seedlings, probably due to improvement in enzymatic and non-enzymatic antioxidants and reduced cell membrane damage.

We report for the first time that applying exogenous naringenin pretreatment before the stress could improve growth and diminish NaCl-caused oxidative stress in safflower seedlings, probably due to the improvement in enzymatic and non-enzymatic antioxidants and reduced cell membrane damage. This implies that applying exogenous naringenin pretreatment before the stress is a promising approach for sustainable crop production under salinity stress.

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.

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.

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.

Identification and functional characterization of safflower cysteine protease 1 as negative regulator in response to low-temperature stress in transgenic Arabidopsis.

MAIN CONCLUSION: We performed genome-wide and heterologous expression analysis of the safflower cysteine protease family and found that inhibition of CtCP1 expression enhanced plant cold resistance. Cysteine protease (CP) is mainly involved in plant senescence and stress responses. However, the molecular mechanism of endogenous cysteine protease inhibition in plant stress tolerance is yet unknown. Here, we report the discovery and functional characterization of a candidate CP1 gene from safflower. The conserved structural topology of CtCPs revealed important insights into their possible roles in plant growth and stress responses. The qRT-PCR results implied that most of CtCP genes were highly expressed at fading stage suggesting that they are most likely involved in senescence process. The CtCP1 expression was significantly induced at different time points under cold, NaCl, H2O2 and PEG stress, respectively. The in-vitro activity of heterologously expressed CtCP1 protein showed highest protease activity for casein and azocasein substrates. The expression and phenotypic data together with antioxidant activity and physiological indicators revealed that transgenic plants inhibited by CtCP1-anti showed higher tolerance to low temperature than WT and CtCP1-OE plants. Our findings demonstrated the discovery of a new Cysteine protease 1 gene that exerted a detrimental effect on transgenic Arabidopsis under low-temperature stress.

Molecular Characterization of an Isoflavone 2'-Hydroxylase Gene Revealed Positive Insights into Flavonoid Accumulation and Abiotic Stress Tolerance in Safflower.

Flavonoids with significant therapeutic properties play an essential role in plant growth, development, and adaptation to various environments. The biosynthetic pathway of flavonoids has long been studied in plants; however, its regulatory mechanism in safflower largely remains unclear. Here, we carried out comprehensive genome-wide identification and functional characterization of a putative cytochrome P45081E8 gene encoding an isoflavone 2'-hydroxylase from safflower. A total of 15 CtCYP81E genes were identified from the safflower genome. Phylogenetic classification and conserved topology of CtCYP81E gene structures, protein motifs, and cis-elements elucidated crucial insights into plant growth, development, and stress responses. The diverse expression pattern of CtCYP81E genes in four different flowering stages suggested important clues into the regulation of secondary metabolites. Similarly, the variable expression of CtCYP81E8 during multiple flowering stages further highlighted a strong relationship with metabolite accumulation. Furthermore, the orchestrated link between transcriptional regulation of CtCYP81E8 and flavonoid accumulation was further validated in the yellow- and red-type safflower. The spatiotemporal expression of CtCYP81E8 under methyl jasmonate, polyethylene glycol, light, and dark conditions further highlighted its likely significance in abiotic stress adaption. Moreover, the over-expressed transgenic Arabidopsis lines showed enhanced transcript abundance in OE-13 line with approximately eight-fold increased expression. The upregulation of AtCHS, AtF3'H, and AtDFR genes and the detection of several types of flavonoids in the OE-13 transgenic line also provides crucial insights into the potential role of CtCYP81E8 during flavonoid accumulation. Together, our findings shed light on the fundamental role of CtCYP81E8 encoding a putative isoflavone 2'-hydroxylase via constitutive expression during flavonoid biosynthesis.

Temporal transcriptome profiling of developing seeds reveals candidate genes involved in oil accumulation in safflower (Carthamus tinctorius L.).

BACKGROUND: The investigation of molecular mechanisms involved in lipid metabolism plays a critical role for the genetic engineering of safflower (Carthamus tinctorius L.) to increase the oil accumulation level or to change the oil composition. Although transcript sequences are currently available for the leaves and flowers of safflower, a wide range scan of temporal transcripts at different stages of seed development has not been conducted for safflower. RESULTS: In this study, temporal transcriptome sequencing was executed at 10, 14, 18, and 22 days after flowering (DAF) to uncover the molecular networks concerned in the biosynthesis of unsaturated fatty acids (USFAs). The results revealed that the biosynthesis of fatty acids is a dominant cellular process from 10 to 14 DAF, while degradation mainly happens after 18 DAF. Significant expression changes of two genes, stearoyl-[acyl-carrier-protein] 9-desaturase gene (SAD) from 10 to 14 DAF and oleate desaturase (FAD2-1) from 14 to 18 DAF, were detected at the transcriptomic levels, and the temporal expression patterns revealed by the transcriptomic analysis were confirmed using quantitative real-time PCR experiments. In addition, 13 candidate transcription factors (TFs) involved in regulating the expression level of the FAD2-1 gene were identified. CONCLUSIONS: These results create a link between fatty acid biosynthesis and gene expression at different developmental stages of the seeds, provide insight into the underlying lipid metabolism, and meanwhile lay an important foundation for the genetic engineering of safflower varieties. We have identified novel candidate genes, including TFs, that are worthy of further exploration.

Rapid screening and identification of anticoagulation component from carthami flos by two-dimensional thrombin affinity chromatography combined with HPLC-MS/MS.

Carthami flos, commonly known as Honghua in China, is the dried floret of safflower and widely acknowledged as a blood stasis promoting herb. The study aimed at investigating the relationship between thrombin and carthami flos through a high-performance thrombin affinity chromatography combined with a high-performance liquid chromatography-tandem mass spectrometry system. First, thrombin was immobilized on the glutaraldehyde-modified amino silica gel to prepare the thrombin affinity stationary phase, which was packed into a small column (1.0 × 2.0 mm, id) for recognizing the anticoagulant active components of carthami flos. The target component was enriched and analyzed by the high-performance liquid chromatography-tandem mass spectrometry system. Finally, hydroxysafflor yellow A was screened out and identified as the active component. The anticoagulant effects of hydroxysafflor yellow A were analyzed by anticoagulant experiments in vitro, and the interaction of hydroxysafflor yellow A with thrombin was investigated by the molecular docking method. The results proved that hydroxysafflor yellow A (30 µg/mL, 0.05 mM) and carthami flos extract (30 µg/mL) could prolong activated partial thrombin time and thrombin time by 50 and 11%, respectively. Moreover, hydroxysafflor yellow A exhibits a good hydrogen bond field and stereo field matching with thrombin. Overall, it was concluded that hydroxysafflor yellow A might exert an anticoagulation effect by interacting with thrombin and thus could be potential anticoagulant drugs for the prevention and treatment of venous thrombosis.

Changes in soil properties and salt tolerance of safflower in response to biochar-based metal oxide nanocomposites of magnesium and manganese.

This original research was performed to assess the possible effects of solid biochar (25 g biochar kg-1 soil) and biochar-based nanocomposites (BNCs) of magnesium oxide (25 g BNC-MgO kg-1 soil), manganese oxide (25 g BNC-MnO biochar kg-1 soil) and combined use of these nanocomposites (12.5 g BNC-MgO + 12.5 g BNC-MnO kg-1 soil) on soil properties and salinity (non-saline, 6 and 12 dSm-1) tolerance of safflower plants (Carthamus tinctorius L.). Application of biochar, particularly BNCs increased the pH and cation exchange capacity of soil, and the contents of water, potassium, calcium, magnesium, manganese, chlorophyll (a & b), nutrients uptake, water use efficiency and plant growth. Sodium adsorption ratio (SAR), exchangeable sodium percentage (ESP) of soil, sodium absorption rate of plants and osmolyte production (soluble carbohydrates and proteins, proline and glycine betaine) under 6 and 12 dSm-1 salinities were decreased by biochar and BNCs treatments. Sodium sorption capacity of BNCs was much higher than the solid biochar, which reflected the superiority of BNCs in decreasing sodium uptake by plants. The combined application of BNC-MgO + BNC-MnO proved to be the preferable treatment for decreasing salt toxicity in safflower. Biochar and BNCs improved root and shoot growth by lowering SAR, ESP, sodium absorption rate of plants and osmotic stress under saline conditions. These results conclude that BNCs can enrich the plant cells with nutrients, increase the nutrients absorption rate and maintain the plant tissue water content at an optimum level to improve plant growth under salt stress.

Enhanced thioredoxin, glutathione and Nrf2 antioxidant systems by safflower extract and aceglutamide attenuate cerebral ischaemia/reperfusion injury.

A large number of reactive oxygen species (ROS) aggravate cerebral damage after ischaemia/reperfusion (I/R). Glutathione (GSH), thioredoxin (Trx) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) represent three major antioxidant systems and play vital roles in affecting each other in eliminating ROS. Identification of drugs targeting triple antioxidant systems simultaneously is vital for inhibiting oxidative damage after cerebral I/R. This study investigated the protective effect of safflower extract and aceglutamide (SAAG) against cerebral I/R injury through modulating multiple antioxidant systems of GSH, Trx and Nrf2 and identified each role of its component acegluatminde (AG) and safflower extract (SA) on these systems. Safflower extract and aceglutamide and its two components decreased neurological deficit scores, infarction rate, apoptosis and oxidative damage after cerebral I/R while enhanced cell viability, decreased reactive oxygen species and nitric oxide level in H2 O2 -induced PC12 cell model. Importantly, compared to its two components, SAAG demonstrated more effective enhancement of GSH, Nrf2 and Trx systems and a better protection against cerebral I/R injury. The enhanced antioxidant systems prevented ASK1 activation and suppressed subsequent p38 and JNK cascade-mediated apoptosis. Moreover, inhibition of Trx and Nrf2 systems by auranofin and ML385 abolished SAAG-mediated protection, respectively. Thus, enhanced triple systems by SAAG played a better protective role than those by SA or AG via inhibition of ASK1 cascades. This research provided evidence for the necessity of combination drugs from the perspective of multiple antioxidant systems. Furthermore, it also offers references for the study of combination drugs and inspires novel treatments for ischaemic stroke.