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.

Safflower yellow for injection enhances anti-coagulation of warfarin in rats: implications in pharmacodynamics and pharmacokinetics.

This study investigated whether Safflower Yellow for injection (SYI) would affect the anticoagulation of warfarin in rats.Wistar male rats were divided into six groups randomly and administered with SYI (9 mg/kg, intraperitoneal injection) in single-dose and steady-dose warfarin (0.2 mg/kg, oral gavage), respectively. The pharmacodynamic parameters of PT and APTT were measured by a coagulation analyser. R/S-warfarin concentration was measured by UHPLC-MS/MS, and pharmacokinetic parameters calculated using DAS 2.0 software.The single-dose study demonstrated that SYI, alone or co-administered with warfarin, could significantly increase PT, INR, and APTT values (p < 0.01). R-warfarin Cmax, AUC, and t1/2 values increased by 9.25% (p > 0.05), 25.96% (p < 0.01), and 26.17% (p < 0.01), respectively, whereas the CL/F value reduced by 22.22% (p < 0.01) in the presence of SYI. Meanwhile, S-warfarin Cmax, AUC, and t1/2 values increased by 37.41%, 32.11%, and 31.73% (all p < 0.01), respectively, whereas the CL/F value reduced by 33.33% (p < 0.01). The steady-dose study showed that PT, INR, APTT, and the concentrations of R/S-warfarin increased significantly when SYI was co-administered with warfarin (p < 0.01).SYI can enhance warfarin's anticoagulation intensity and decelerate its metabolism in rats.

Genetic diversity assessment of Palestinian safflower (Carthamus tinctorius L.) utilizing DAMD molecular markers.

The current knowledge about Palestinian safflower landraces is relatively limited in terms of phenotypic and molecular characterization, however, the purpose of this investigation was to determine the amount of genetic diversity in eighteen local safflower landraces using seven DAMD markers. The banding patterns for each primer were scored and compiled into a data matrix. Subsequently, the data matrix was analyzed using UPGMA cluster analysis to identify distinct genetic groups among the landraces. In total, 88 DNA fragments were found, and there were an average of 12.6 loci per assay unit observed. Resolving Power (RP) revealed an average of 7.09 was determined, with the highest RP value at 13.3. The dendrogram obtained from DAMD data divided the landraces into three main clusters, denoted as I, II and III. The first cluster (I) consisted of one genotype (PTUK.SA16). The second cluster (II) consisted of two genotypes (PTUK.SA13 and PTUK.SA10). The third cluster (III) was later partitioned into two distinct sub-clusters, which are III.a and III.b. Sub-cluster III.a comprised seven genotypes (PTUK.SA4, PTUK.SA9, PTUK.SA8, PTUK.SA7, PTUK.SA6, PTUK.SA5 and PTUK.SA3). While Sub-cluster III.b consisted of eight genotypes (PTUK.SA15, PTUK.SA18, PTUK.SA17, PTUK.SA14, PTUK.SA12, PTUK.SA2, PTUK.SA11, and PTUK.SA1). This research assess the genetic diversity of Palestinian safflower landraces using PCR-based DAMD markers. The remarkable level of polymorphism detected using DAMD markers demonstrated their effectiveness in distinguishing between Palestinian safflower genotypes.

CSM-CROPGRO model to simulate safflower phenological development and yield.

Crop simulation models are valuable tools for decision making regarding evaluation and crop improvement under different field conditions. CSM-CROPGRO model integrates genotype, environment and crop management portfolios to simulate growth, development and yield. Modeling the safflower response to varied climate regimes are needed to strengthen its productivity dynamics. The main objective of the study was to evaluate the performance of DSSAT-CSM-CROPGRO-Safflower (Version 4.8.2) under diverse climatic conditions. The model was calibrated using the field observations for phenology, biomass and safflower grain yield (SGY) of the year 2016-17. Estimation of genetic coefficients was performed using GLUE (Genetic Likelihood Uncertainty Estimation) program. Simulated results for days to flowering, maturity, biomass at flowering and maturity and SGY were predicted reasonably with good statistical indices. Model evaluation results elucidate phenological events with low root mean square error (6.32 and 6.52) and high d-index (0.95 and 0.96) for days to flowering and maturity respectively for all genotypes and climate conditions. Fair prediction of safflower biomass at flowering and maturity showed low RMSE (887.3 and 564.3 kg ha-1) and high d-index (0.67 and 0.93) for the studied genotypes across the environments. RMSE for validated safflower grain yield (101.8 kg ha-1) and d-index (0.95) depicted that model outperformed for all genotypes and growing conditions. Longer appropriate growing conditions at NARC-Islamabad took optimal duration to assimilate photosynthetic products lead to higher grain yield. Safflower resilience to different environments showed that it can be used as an alternate crop for different agroecological regions. Furthermore, CROPGRO-Safflower model can be used as tool to further evaluate inclusion of safflower in the existing cropping systems of studied regions.

Enriched biochars with silicon and calcium nanoparticles mitigated salt toxicity and improved safflower plant performance.

Modifying biochar with nano-nutrients is one of the most effective methods in improving the efficiency of biochar in reducing the adverse effects of environmental stresses such as salinity on plant growth and productivity. The possible effects of solid biochar, nano-silicon dioxide enriched biochar, nano-calcium carbonate enriched biochar, and combined application of these enriched biochars on physiological performance of safflower (Carthamus tinctorius L.) were evaluated under different levels of salt stress (non-saline, 6 and 12 dSm-1). Salt stress increased sodium content, reactive oxygen species generation, and antioxidant enzymes activity, but decreased potassium, calcium, magnesium, iron, zinc, silicon, photosynthetic pigments, leaf water content, and seed yield (by about 36%) of safflower plants. The addition of biochar forms to the saline soil improved growth (up to 24.6%) and seed yield (up to 37%) of safflower by reducing sodium accumulation (by about 32%) and ROS generation and enhancing nutrient uptake, photosynthetic pigments, and water contents of leaves. The combined forms of enriched biochars were the best treatment on reducing salt stress effects on safflower plants. Therefore, application of enriched biochars has a high potential to reduce the harmful effects of salt stress on plants.

Enriching biochar with calcium and silicon nanoparticles is a practical method to improve the ability of biochar to adsorb and immobilize harmful elements such as sodium in the rhizosphere. This enrichment enhanced safflower plant growth and physiological efficiency under salt stress by reducing sodium absorption and increasing the availability of beneficial nutrients.

A Method for Real-Time Recognition of Safflower Filaments in Unstructured Environments Using the YOLO-SaFi Model.

The identification of safflower filament targets and the precise localization of picking points are fundamental prerequisites for achieving automated filament retrieval. In light of challenges such as severe occlusion of targets, low recognition accuracy, and the considerable size of models in unstructured environments, this paper introduces a novel lightweight YOLO-SaFi model. The architectural design of this model features a Backbone layer incorporating the StarNet network; a Neck layer introducing a novel ELC convolution module to refine the C2f module; and a Head layer implementing a new lightweight shared convolution detection head, Detect_EL. Furthermore, the loss function is enhanced by upgrading CIoU to PIoUv2. These enhancements significantly augment the model's capability to perceive spatial information and facilitate multi-feature fusion, consequently enhancing detection performance and rendering the model more lightweight. Performance evaluations conducted via comparative experiments with the baseline model reveal that YOLO-SaFi achieved a reduction of parameters, computational load, and weight files by 50.0%, 40.7%, and 48.2%, respectively, compared to the YOLOv8 baseline model. Moreover, YOLO-SaFi demonstrated improvements in recall, mean average precision, and detection speed by 1.9%, 0.3%, and 88.4 frames per second, respectively. Finally, the deployment of the YOLO-SaFi model on the Jetson Orin Nano device corroborates the superior performance of the enhanced model, thereby establishing a robust visual detection framework for the advancement of intelligent safflower filament retrieval robots in unstructured environments.

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.

Growth performance, meat quality and hematological parameters of broiler chickens fed safflower seed.

The aim of the current study was to determine the effects of dietary supplementation of safflower seed (SS) on the growth performance and hematological parameters of broiler birds along with the physicochemical, textural and sensory attributes of chicken meat. A total of 200 male chickens (7-days-old) were distributed into 5 groups (40 chickens in each) with 5 replicates of 8 chicks in a 42-day experiment. Each group was allocated to one of 5 dietary treatments, i.e., 0, 2.5, 5, 7.5, and 10% SS. The experimental diets were formulated for starter (7 to 21 days) and finisher (22 to 42 days) phases. Inclusion of SS in the diet improved growth performances in treatment groups between 7 and 42 days. The highest and lowest body weights were observed at the 5% SS and 0% SS levels, respectively. The physicochemical attributes of breast and thigh meat were found (P > 0.05) except for crude fat. The crude fat was significantly (P < 0.05) increased with increasing levels of SS in the diet. The inclusion of SS in the diet did not negatively impact the textural properties, i.e., hardness, cohesiveness, springiness, gumminess, chewiness, and shear force of breast and thigh meat. There was no significant difference in the sensory parameters of cooked chicken meat with increasing levels of SS in the diet. The results demonstrated a significant (P < 0.01) improvement in hematological parameters in the blood samples of broiler chickens fed diet supplemented with various levels of SS for five weeks. These findings suggest that, SS may be used as an oil seed for broiler chicken feed.

Use of hydroponics-based evaluation for phenotyping tolerance/susceptibility to the aphid, Uroleucon compositae and inheritance analysis of aphid tolerance in a global germplasm collection of Carthamus tinctorius L. (Safflower).

Carthamus tinctorius L. (Safflower) is an important oilseed crop that is cultivated globally. Aphids are a serious pest of safflower and cause significant yield losses of up to 80% due to their ability to multiply rapidly by parthenogenesis. In this study, we report the identification of an aphid-tolerant accession in safflower following screening of a representative global germplasm collection of 327 accessions from 37 countries. Field-based screening methods gave inconsistent and ambiguous results for aphid tolerance between natural and controlled infestation assays and required ~ 3 months for completion. Therefore, we used a rapid, high-throughput hydroponics-based assay system that allows phenotyping of aphid tolerance/susceptibility in a large number of plants in a limited area, significantly reduces the time required to ~ 45 days and avoids inconsistencies observed in field-based studies. We identified one accession out of the 327 tested germplasm lines that demonstrated aphid tolerance in field-based natural and controlled infestation studies and also using the hydroponics approach. Inheritance analysis of the trait was conducted using the hydroponics approach on F1 and F2 progeny generated from a cross between the tolerant and susceptible lines. Aphid-tolerance was observed to be a dominant trait governed by a single locus/gene that can be mobilized after mapping into cultivated varieties of safflower. The hydroponics-based assay described in this study would be very useful for studying the molecular mechanism of aphid-tolerance in safflower and can also be used for bioassays in several other crops that are amenable to hydroponics-based growth. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01467-0.

α-Glucosidase, butyrylcholinesterase and acetylcholinesterase inhibitory activities of phenolic compounds from Carthamus tinctorius L. flowers: In silico and in vitro studies.

Chemical investigation of Carthamus tinctorius L. flowers resulted in isolation of seven metabolites that were identified as; p-Hydroxybenzoic acid (1), trans hydroxy cinnamic acid (2), kaempferol-6-C-glucoside (3), astragalin (4), cartormin (5), kaempferol-3-O-rutinoside (6), and kaempferol-3-O-sophoroside (7). Virtual screening of the isolated compounds against human intestinal α-glucosidase, acetylcholinesterase, and butyrylcholinesterase was carried out. Additionally, the antioxidant activity of the bioactive compounds was assessed. Compounds 1 and 5 exhibited moderate binding affinities to acetylcholinesterase (binding energy -5.33 and -4.18 kcal/mol, respectively), compared to donepezil (-83.33kcal/mol). Compounds 1-7 demonstrated weak affinity to butyrylcholinesterase. Compounds 2 and 4 displayed moderate binding affinity to human intestinal α-glucosidase,compared to Acarbose (reference compound), meanwhile compound 2 exhibited lower affinity. Molecular dynamic studies revealed that compound 4 formed a stable complex with the binding site throughout a 100 ns simulation period. The in-vitro results were consistent with the virtual experimental results, as compounds 1 and 5 showed mild inhibitory effects on acetylcholinesterase (IC50s 150.6 and 168.7 µM, respectively). Compound 4 exhibited moderate α-glucosidase inhibition with an IC50 of 93.71 µM. The bioactive compounds also demonstrated notable antioxidant activity in ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], ORAC (oxygen radical-absorbance capacity), and metal chelation assays, suggesting their potential in improving dementia in Alzheimer's disease (AD) and mitigating hyperglycemia.

[Research progress in chemical constituents and pigment extraction process of Carthami Flos].

Carthami Flos(flowers of Carthamus tinctorius) with the effects of activating blood, dredging meridians, dissipating stasis, and relieving pain is one of the commonly used traditional Chinese medicines for promoting blood circulation and resolving stasis in clinical practice. So far, more than 210 compounds in Carthami Flos have been isolated and reported, including quinochalcones(safflower yellow pigments and red pigments), flavonoids, spermidines, alkaloids, polyacetylenes, and organic acids. Safflower yellow pigments, as the main water-soluble active components of Carthami Flos, is commonly obtained by the water extraction method, while red pigments are commonly obtained by the alkali extraction and acid precipitation method. In recent years, natural deep eutectic solvents as green solvents have demonstrated promising application prospects in the extraction and separation of pigments from Carthami Flos. This review systematically summarizes the chemical constituents of Carthami Flos and analyzes the extraction process of pigment components from Carthami Flos, aiming to provide a reference for further utilization of Carthami Flos resources.

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.

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.

Fatty acid correlations with HOMA-IR and HOMA-% ß are differentially dictated by their serum free and total pools and flaxseed oil supplementation.

Objective. The intent of the present study was to test two hypotheses. The primary hypothesis was that there would be differences between blood serum individual free fatty acids (SIFFA) and serum individual total fatty acids (SITFA) in terms of their different relationships (correlations) to each of homeostatic model assessment-individual insulin resistance (HOMA-IR) and homeostatic model assessment-individual insulin resistance-percentage ß-cell function (HOMA-% ß) remaining in human type 2 diabetic patients with pre-flaxseed oil (FXO) and pre-safflower oil (SFO) administration. The secondary hypothesis was that FXO (rich in alpha-linolenic acid, ALA) supplementation would alter these correlations differently in the SIFFA and STIFFA pools in comparison with the placebo SFO (poor in ALA). Methods. Patients were recruited via a newspaper advertisement and two physicians. All patients came to visit 1 and three months later to visit 2. At visit 2, the subjects were randomly assigned (double-blind) to flaxseed or safflower oil (placebo) treatment for three months until visit 3. Results. There were pre-intervention differences in the SIFFA and STIFA pool's relationships with each of HOMA-IR and HOMA-% ß. These relatioships remained either unchanged or became significant after intervention (treatment or placebo). There was a negative correlation found between HOMA-IR and serum free ALA (SFALA) mol % after FXO. Serum total ALA (STALA) mol % had no significant correlations with HOMA-IR and HOMA- % ß before and after flaxseed oil administration. Conclusions. The SIFFA and SITFA pools have different relationships with HOMA-IR and HOMA-% ß for each of pre- and post-intervention. It is concluded that the data support both the primary and the secondary hypotheses indicating that they are correct.

Safflower yellow and its main component hydroxysafflor yellow A alleviate hyperleptinemia in diet-induced obesity mice through a dual inhibition of the GIP-GIPR signaling axis.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone secreted by K cells in the small intestine and is considered an obesity-promoting factor. In this study, we systematically investigated the anti-obesity effects of intragastric safflower yellow (SY)/hydroxysafflor yellow A (HSYA) and the underlying mechanism for the first time. Our results showed that intragastric SY/HSYA, rather than an intraperitoneal injection, notably decreased serum GIP levels and GIP staining in the small intestine in diet-induced obese (DIO) mice. Moreover, intragastric SY/HSYA was also first found to significantly suppress GIP receptor (GIPR) signaling in both the hypothalamus and subcutaneous White adipose tissue. Our study is the first to show that intragastric SY/HSYA obviously reduced food intake and body weight gain in leptin sensitivity experiments and decreased serum leptin levels in DIO mice. Further experiments demonstrated that SY treatment also significantly reduced leptin levels, whereas the inhibitory effect of SY on leptin levels was reversed by activating GIPR in 3 T3-L1 adipocytes. In addition, intragastric SY/HSYA had already significantly reduced serum GIP levels and GIPR expression before the serum leptin levels were notably changed in high-fat-diet-fed mice. These findings suggested that intragastric SY/HSYA may alleviate diet-induced obesity in mice by ameliorating hyperleptinemia via dual inhibition of the GIP-GIPR axis.

First report of Fusarium verticillioides causing safflower root rot in Sinaloa, Mexico.

Safflower cultivation is of great socioeconomic importance worldwide. Production is intended for the extraction of oil from the seeds. In 2021 Mexico ranked fifth in world production with approximately 52,553.28 tons (SIAP, 2021). In April 2022, in the north-central zone of Sinaloa, Mexico, diseased plants were reported in fields planted with safflower. Symptoms included chlorotic plants, necrosis and rot in vascular bundles, dwarfed plants and reflexed plants bent towards the ground. The disease caused estimated losses of 15% of seed production, with respect to the production obtained from the previous year in the safflower fields surveyed. Twenty-five plants with symptoms were sampled to isolate the pathogen. Plants were cut at the base of the stem near the roots and roots cut into 5 mm2 pieces. Tissue samples were superficially disinfected by immersing in 70% alcohol for 10 sec, 2% sodium hypochlorite for 1 min, washed in sterile water, and placed on potato dextrose agar (PDA) at 28 ºC for 7 days in the dark. Twelve monosporic isolates derived from the PDA culture were morphologically characterized. Abundant white aerial mycelium and small pink to dark violet pigments in the center of the culture were observed. From 10-day-old cultures grown on carnation leaf agar medium microconidia and macroconidia were produced. Microconidia were hyaline, had zero to two septa, and were oval or ellipsoidal, 4.6 to 14 x 1.8 to 4.2 µm (n = 40). The macroconidia were hyaline, were slightly curved with three to five septa, and measured from 26 to 69 x 3 to 6.1 µm (n = 40). No chlamydospores were observed. According to the morphological characteristics, the isolates were identified as Fusarium verticillioides (Leslie and Summerell, 2006). DNA was extracted from one isolate and the Translation Elongation Factor 1-α (EF1) gene was amplified and sequenced (O'Donnell et al. 2010). The sequence obtained from isolate FV3CARCULSIN with 645 base pairs was submitted to NCBI GenBank with accession number OQ262963. The BLAST search revealed 100% similarity with F. verticillioides isolate 13 (KM598773) (Lizárraga et al. 2015). Identification in FUSARIUM ID resulted in a 99.85% similarity with isolate F. verticillioides CBS 131389 (MN534047) (Yilmaz et al. 2021). A phylogenetic tree, made with sequences of the EF1 gene, revealed that FV3CARCULSIN was most closely related to F. verticillioides (100% bootstrap). Pathogenicity tests were carried out on safflower plants (cv. Oleico) grown in sterile vermiculite. Plants were inoculated with a conidial suspension (1 × 105 conidia/ml) obtained from FV3CARCULSIN grown on PDA for 7 days. A total of 45 plants were inoculated by drenching the roots with 20 ml of inoculum when the plants were 20 days old. Fifteen plants served as negative controls without inoculation. Plants were kept for 60 days in greenhouse conditions; however, after 45 days the plants began to die. The assay was conducted twice. Rotting and necrosis was observed in the roots of the plants. The pathogen was reisolated from the tissue of all the plants with symptoms and identified as F. verticillioides using morphological characteristics and EF1 sequences, completing Koch's postulates. No symptoms were observed in control plants after 60 days. This is the first report of root rot in safflower caused by F. verticillioides in Mexico. The fungus has been reported in maize (Figueroa et al. 2010), but it is unknown if it could be the same pathogen of safflower. Identification of the pathogen is important for implementing management methods to reduce yield losses and for additional studies on the impact of the disease on oil quality extracted from safflower seeds.

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 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.