Genome-wide analysis and transcriptional reprogrammings of MYB superfamily revealed positive insights into abiotic stress responses and anthocyanin accumulation in Carthamus tinctorius L.

The MYB transcription factors comprise one of the largest superfamilies in plants that have been implicated in the regulation of plant-specific metabolites and responses to biotic and abiotic stresses. Here, we present the first comprehensive genome-wide analysis and functional characterization of the CtMYB family in Carthamus tinctorius. A total of 272 CtMYBs were identified and classified into 12 subgroups using comparative phylogenetic analysis with Arabidopsis and rice orthologs. The overview of conserved motifs, gene structures, and cis elements as well as the expression pattern of CtMYB genes indicated the diverse roles of these transcription factors during plant growth, regulation of secondary metabolites, and various abiotic stress responses. The subcellular localization and transactivation analysis of four CtMYB proteins indicated predominant localization in the nuclei with enhanced transcriptional activation in yeast. The expression of CtMYB63 induced with various abiotic stress conditions showed upregulation in its transcription level. In addition, the expression analysis of the core structural genes of anthocyanin biosynthetic pathway under drought and cold stress in CtMYB63 overexpressed transgenic lines also supports the notion of CtMYB63 transcriptional reprogramming in response to abiotic stress by upregulating the anthocyanin biosynthesis. Together, our findings revealed the underlying regulatory mechanism of CtMYB TF network involving enhanced cold and drought stress tolerance through activating the rapid biosynthesis of anthocyanin in C. tinctorius. This study also presents useful insights towards the establishment of new strategies for crop improvements.

The Comprehensive Evaluation of Safflowers in Different Producing Areas by Combined Analysis of Color, Chemical Compounds, and Biological Activity.

In the present study, a new strategy including the combination of external appearance, chemical detection, and biological analysis was proposed for the comprehensive evaluation of safflowers in different producing areas. Firstly, 40 batches of safflower samples were classified into class I and II based on color measurements and K-means clustering analysis. Secondly, a rapid and sensitive analytical method was developed for simultaneous quantification of 16 chromaticity-related characteristic components (including characteristic components hydroxysafflor yellow A, anhydrosafflor yellow B, safflomin C, and another 13 flavonoid glycosides) in safflowers by ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (UPLC-QTRAP®/MS2). The results of the quantification indicate that hydroxysafflor yellow A, anhydrosafflor yellow B, kaempferol, quercetin, and safflomin C had significant differences between the two types of safflower, and class I of safflower had a higher content of hydroxysafflor yellow A, anhydrosafflor yellow B, and safflomin C as the main anti-thrombotic components in safflower. Thirdly, chemometrics methods were employed to illustrate the relationship in multivariate data of color measurements and chromaticity-related characteristic components. As a result, kaempferol-3-O-rutinoside and 6-hydroxykaempferol-3-O-ß-d-glucoside were strongly associated with the color indicators. Finally, anti-thrombotic analysis was used to evaluate activity and verify the suitability of the classification basis of safflower based on the color measurements. It was shown that brighter, redder, yellower, more orange-yellow, and more vivid safflowers divided into class I had a higher content of characteristic components and better anti-thrombotic activity. In summary, the presented strategy has potential for quality evaluation of other flower medicinal materials.

Mobile genomic element diversity in world collection of safflower (Carthamus tinctorius L.) panel using iPBS-retrotransposon markers.

Safflower (Carthamus tinctorius L.) is a multipurpose crop of dry land yielding very high quality of edible oil. Present study was aimed to investigate the genetic diversity and population structure of 131 safflower accessions originating from 28 different countries using 13 iPBS-retrotransposon markers. A total of 295 iPBS bands were observed among which 275 (93.22%) were found polymorphic. Mean Polymorphism information content (0.48) and diversity parameters including mean effective number of alleles (1.33), mean Shannon's information index (0.33), overall gene diversity (0.19), Fstatistic (0.21), and inbreeding coefficient (1.00) reflected the presence of sufficient amount of genetic diversity in the studied plant materials. Analysis of molecular variance (AMOVA) showed that more than 40% of genetic variation was derived from populations. Model-based structure, principal coordinate analysis (PCoA) and unweighted pair-group method with arithmetic means (UPGMA) algorithms clustered the 131 safflower accessions into four main populations A, B, C, D and an unclassified population, with no meaningful geographical origin. Most diverse accessions originated from Asian countries including Afghanistan, Pakistan, China, Turkey, and India. Four accessions, Turkey3, Afghanistan4, Afghanistan2, and Pakistan24 were found most genetically distant and might be recommended as a candidate parents for breeding purposes. The findings of this study are most probably supported by the seven similarity centers hypothesis of safflower. This is a first study to explore the genetic diversity and population structure in safflower accessions using the iPBS-retrotransposon markers. The information provided in this work will therefore be helpful for scientists interested in safflower breeding.

Intraspecific variability of carbon isotope discrimination and its correlation with grain yield in safflower: prospects for selection in a Mediterranean climate.

The goals of the present study were to obtain a first estimate of intraspecific variability of carbon isotope discrimination (Δ) in safflower, a thistle-like herbaceous plant, and to determine the statistical relationship between Δ and grain yield as well as its components in a collection of 45 accessions of different origins. Grain yield and aboveground biomass, harvest index, average grain weight, and Δ (measured on the bulk leaf organic matter) were investigated in experimental field conditions. A large variability was noted for all traits but a principal component analysis (PCA) allowed to identify several homogeneous groups of accessions. Average grain yield per plant varied between 1 and 39 g. Δ varied between 21.3 and 25.2 ‰, i.e. a large variation of 3.9 ‰. In our experiment, the variation of Δ was not significantly related to that of grain yield in the whole accession sample. However, we found contrasting trends for this relation within accession groups. These initial results motivate further experiments to assess more in depth correlation between Δ and yield in safflower and are encouraging regarding the possibility of using Δ as an effective selection index in safflower to obtain genotypes that efficiently consume water. This study also highlighted one accession that combines the two characters required in the Mediterranean regions, i.e. high yield performance and high water-use efficiency.

The complete chloroplast genome sequence of Safflower (Carthamus tinctorius L.).

Safflower (Carthamus tinctorius L.) is a traditional medical plants of Asia. In this study, the complete chloroplast genome of safflower was presented. The total genome size was 153,675 bp in length, containing a pair of inverted repeats (IRs) of 25,407 bp, separated by large single copy (LSC) and small single copy (SSC) of 83,606 bp and 19,156 bp, respectively. Overall GC content of the genome was 37.4%. The chloroplast genome harbored 127 annotated genes, including 89 protein coding genes, 30 tRNA genes and 8 rRNA genes. A total of 7 of these genes were duplicated in the inverted repeat regions. Twelve genes contained one intron.

[Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector].

Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids.

Genetic assessment of safflower (Carthamus tinctorius L.) collection with microsatellite markers acquired via pyrosequencing method.

A genetic evaluation of safflower germplasm collections derived from different geographical regions and countries will provide useful information for sustainable conservation and the utilization of genetic diversity. However, the molecular marker information is limited for evaluation of genetic diversity of safflower germplasm. In this study, we acquired 509 putative genomic SSR markers for sufficient genome coverage using next-generation sequencing methods and characterized thirty polymorphic SSRs in safflower collection composed of 100 diverse accessions. The average allele number and expected heterozygosity were 2.8 and 0.386, respectively. Analysis of population structure and phylogeny based on thirty SSR profiles revealed genetic admixture between geographical regions contrary to genetic clustering. However, the accessions from Korea were genetically conserved in distinctive groups in contrast to other safflower gene pool. In conclusion, these new genomic SSRs will facilitate valuable studies to clarify genetic relationships as well as conduct population structure analyses, genetic map construction and association analysis for safflower.

A large and functionally diverse family of Fad2 genes in safflower (Carthamus tinctorius L.).

BACKGROUND: The application and nutritional value of vegetable oil is highly dependent on its fatty acid composition, especially the relative proportion of its two major fatty acids, i.e oleic acid and linoleic acid. Microsomal oleoyl phosphatidylcholine desaturase encoded by FAD2 gene is known to introduce a double bond at the Δ12 position of an oleic acid on phosphatidylcholine and convert it to linoleic acid. The known plant FAD2 enzymes are encoded by small gene families consisting of 1-4 members. In addition to the classic oleate Δ12-desaturation activity, functional variants of FAD2 that are capable of undertaking additional or alternative acyl modifications have also been reported in a limited number of plant species. In this study, our objective was to identify FAD2 genes from safflower and analyse their differential expression profile and potentially diversified functionality. RESULTS: We report here the characterization and functional expression of an exceptionally large FAD2 gene family from safflower, and the temporal and spatial expression profiles of these genes as revealed through Real-Time quantitative PCR. The diversified functionalities of some of the safflower FAD2 gene family members were demonstrated by ectopic expression in yeast and transient expression in Nicotiana benthamiana leaves. CtFAD2-1 and CtFAD2-10 were demonstrated to be oleate desaturases specifically expressed in developing seeds and flower head, respectively, while CtFAD2-2 appears to have relatively low oleate desaturation activity throughout the plant. CtFAD2-5 and CtFAD2-8 are specifically expressed in root tissues, while CtFAD2-3, 4, 6, 7 are mostly expressed in the cotyledons and hypocotyls in young safflower seedlings. CtFAD2-9 was found to encode a novel desaturase operating on C16:1 substrate. CtFAD2-11 is a tri-functional enzyme able to introduce a carbon double bond in either cis or trans configuration, or a carbon triple (acetylenic) bond at the Δ12 position. CONCLUSIONS: In this study, we isolated an unusually large FAD2 gene family with 11 members from safflower. The seed expressed FAD2 oleate Δ12 desaturase genes identified in this study will provide candidate targets to manipulate the oleic acid level in safflower seed oil. Further, the divergent FAD2 enzymes with novel functionality could be used to produce rare fatty acids, such as crepenynic acid, in genetically engineered crop plants that are precursors for economically important phytoalexins and oleochemical products.

Effects of feeding roasted safflower seeds (variety IL-111) and fish oil on dry matter intake, performance and milk fatty acid profiles in dairy cattle.

UNLABELLED: Safflower seed has the highest concentration of linoleic acid among 80 oilseeds but little information exists on the effective use of SS for lactation cows. It was hypothesised that a diet supplemented with an Iranian SS variety (IL-111) in combination with fish oil (FO) would result in higher concentrations of trans-18:1 (including vaccenic acid) and conjugated linoleic acids in milk fat than feeding an unsupplemented control diet. Our objective was to determine the effects of feeding diets containing: (i) CONTROL: (C); (ii) 25 g of roasted SS IL-111 (RSS); (iii) 20 g FO and (iv) 25 g RSS + 10 g FO (RSS + FO) per kilogram of dietary DM on feed intake, ruminal fermentation, milk production and fatty acid profile. Eight multiparous Holstein cows were used in a replicated 4 × 4 Latin square design study. The experiment had four periods of 21 days. Milk Fat percentage was lower (p < 0.01) with FO supplementation and averaged 19.0 and 21.5 g/kg milk with FO and RSS + FO compared with 30.3 and 32.5 g/kg with C and RSS. Feed intake also was lower (p < 0.01) with FO vs. C (23.1 vs. 24.5 kg/day) but feeding RSS resulted in greater feed intake compared with other treatments (26 kg/day). Despite lower feed intake with FO, milk production did not change from controls but feeding RSS + FO resulted in greater milk yield than controls (42.6 vs. 39.3 kg/day). Ruminal pH was greater (p < 0.01) in cows fed FO than other treatments. Supplemental FO alone or in combination with RSS resulted in dramatic increases (p < 0.01) in c9,t11-18:2 in milk fat (12.7 and 13.2 g/day vs. 5.8 and 7.02 with C and RSS). It was surprising to note that 25 g/kg RSS can improve feed intake.

Salt-induced modulation in inorganic nutrients, antioxidant enzymes, proline content and seed oil composition in safflower (Carthamus tinctorius L.).

BACKGROUND: Safflower (Carthamus tinctorius L.) has gained considerable ground as a potential oil-seed crop. However, its yield and oil production are adversely affected under saline conditions. The present study was conducted to appraise the influence of salt (NaCl) stress on yield, accumulation of different inorganic elements, free proline and activities of some key antioxidant enzymes in plant tissues as well as seed oil components in safflower. Two safflower accessions differing in salt tolerance (Safflower-33 (salt sensitive) and Safflower-39 (salt tolerant)) were grown under saline (150 mmol L(-1) ) conditions and salt-induced changes in the earlier-mentioned physiological attributes were determined. RESULTS: Salt stress enhanced leaf and root Na(+) , Cl(-) and proline accumulation and activities of leaf superoxide dismutase, catalase and peroxidase, while it decreased K(+) , Ca(2+) and K(+) /Ca(2+) and Ca(2+) /Na(+) ratios and seed yield, 100-seed weight, number of seeds, as well as capitula, seed oil contents and oil palmitic acid. No significant effect of salt stress was observed on seed oil α-tocopherols, stearic acid, oleic acid or linoleic acid contents. Of the two safflower lines, salt-sensitive Safflower-33 was higher in leaf and root Na(+) and Cl(-) , while Safflower-39 was higher in leaf and root K(+) , K(+) /Ca(2+) and Ca(2+) /Na(+) and seed yield, 100-seed weight, catalase activity, seed oil contents, seed oil α-tocopherol and palmitic acid. Other attributes remained almost unaffected in both accessions. CONCLUSION: Overall, high salt tolerance of Safflower-39 could be attributed to Na(+) and Cl(-) exclusion, high accumulation of K(+) and free proline, enhanced CAT activity, seed oil α-tocopherols and palmitic acid contents.

Introgression potential between safflower (Carthamus tinctorius) and wild relatives of the genus Carthamus.

BACKGROUND: Safflower, Carthamus tinctorius, is a thistle that is grown commercially for the production of oil and birdseed and recently, as a host for the production of transgenic pharmaceutical proteins. C. tinctorius can cross with a number of its wild relatives, creating the possibility of gene flow from safflower to weedy species. In this study we looked at the introgression potential between different members of the genus Carthamus, measured the fitness of the parents versus the F1 hybrids, followed the segregation of a specific transgene in the progeny and tried to identify traits important for adaptation to different environments. RESULTS: Safflower hybridized and produced viable offspring with members of the section Carthamus and species with chromosome numbers of n = 10 and n = 22, but not with n = 32. The T-DNA construct of a transgenic C. tinctorius line was passed on to the F1 progeny in a Mendelian fashion, except in one specific cross, where it was deleted at a frequency of approximately 21%. Analyzing fitness and key morphological traits like colored seeds, shattering seed heads and the presence of a pappus, we found no evidence of hybrid vigour or increased weediness in the F1 hybrids of commercial safflower and its wild relatives. CONCLUSION: Our results suggest that hybridization between commercial safflower and its wild relatives, while feasible in most cases we studied, does not generate progeny with higher propensity for weediness.

Development, polymorphism, and cross-taxon utility of EST-SSR markers from safflower (Carthamus tinctorius L.).

Due to their highly polymorphic and codominant nature, simple-sequence repeat (SSR) markers are a common choice for assaying genetic diversity and genetic mapping. In this paper, we describe the generation of an expressed-sequence tag (EST) collection for the oilseed crop safflower and the subsequent development of EST-SSR markers for the genetic analysis of safflower and related species. We assembled 40,874 reads into 19,395 unigenes, of which 4,416 (22.8%) contained at least one SSR. Primer pairs were developed and tested for 384 of these loci, resulting in a collection of 104 polymorphic markers that amplify reliably across 27 accessions (3 species) of the genus Carthamus. These markers exhibited a high level of polymorphism, with an average of 6.0 +/- 0.4 alleles per locus and an average gene diversity of 0.54 +/- 0.03 across Carthamus species. In terms of cross-taxon transferability, 50% of these primer pairs produced an amplicon in at least one other species in the Asteraceae, and 28% produced an amplicon in at least one species outside the safflower subfamily (i.e., lettuce, sunflower, and/or Gerbera). These markers represent a valuable resource for the genetic analysis of safflower and related species, and also have the potential to facilitate comparative map-based analyses across a broader array of taxa within the Asteraceae.

Nuclear DNA assay in solving issues related to ancestry of the domesticated diploid safflower (Carthamus tinctorius L.) and the polyploid (Carthamus) taxa, and phylogenetic and genomic relationships in the genus Carthamus L. (Asteraceae).

The multipronged nuclear DNA assay by random amplified polymorphic DNA (RAPD) fingerprinting, ribosomal DNA repeat unit length polymorphism, internal transcribed sequence (ITS) RFLP, and comparative sequence analysis of ITS and external transcribed sequence (ETS) regions of the 29 accessions belonging to 18 Carthamus taxa including five unverified species was undertaken to obtain new information on (1) interrelationships among botanical varieties of cultivated safflower, C. tinctorius, and phylogenetic relationships (2) among the safflower and its close relatives and (3) that of Carthamus species and subspecies. The root tip cells of the 12 accessions contained 24 chromosomes followed by 64, 44 and 20 chromosomes in 9, 6 and 2 accessions, respectively. Barring C. lanatus, the accessions within each taxon had the same zygotic number. The present results strongly support the view that the wild C. palaestinus (2n=24) and the cultivated C. tinctorius (2n=24) are closely related. With few exceptions, all DNA based dendrograms support three lineages within the genus. One lineage is constituted by C. arborescens (2n=24) alone. The present data indicates that because of unique composition of its nuclear genome vis-à-vis other Carthamus taxa, C. arborescens should be placed in a separate subgenus. The two remaining lineages, constituted by the taxa with 2n=24, and the taxa with 2n=20, 2n=44 and 2n=64, respectively should be given the rank of two taxonomic sections in the other subgenus. The present study also demonstrates that none of the present taxa with 2n=24 have contributed to the origin of polyploid taxa. Carthamus boisserii (2n=20) and C. glaucus ssp. anatolicus (2n=20) are more likely to be one of the diploid progenitor of C. lanatus ssp. creticus (2n=64), C. lanatus (2n=44), C. lanatus ssp. lanatus (2n=44) and C. lanatus ssp. montanus (2n=44), and C. lanatus ssp. turkestanicus (2n=64), respectively. Within Lanatus species complex, constituted by C. lanatus, C. lanatus ssp. lanatus, C. lanatus ssp. montanus, C. lanatus ssp. turkestanicus and C. lanatus ssp. creticus, high proportion of autapomorphic characters and low number of synapomorphies in the ITS and ETS sequences suggest a relatively recent diversification of the taxa within the species complex. Carthamus lanatus ssp. creticus (2n=64) and C. lanatus ssp. turkestanicus (2n=64) within the complex deserve species rank. This analysis provided evolutionary relatedness of the five unverified taxa with the known Carthamus taxa.

[Study on HPLC fingerprint of water-soluble constituents of Carthamus tinctorius].

OBJECTIVE: To establish the HPLC-fingerprint of the water-soluble constituents of Carthamus tinctorius. METHODS: 18 samples of Carthamus tinctorius from different producing areas were determined by Agilent 1100 DAD-HPLC under the chromatographic conditions: column by SinoChrom ODS-BP (250 mm x 4.6 mm, 5 microm), methanol-0.7% H3PO4 water with gradient elution, column temperature 30 degrees C, flow rate by 1 ml/min, wavelength 280 nm, and inject volume 20 microl. RESULTS: The HPLC-fingerprint of the water-soluble constituents of Cartharnus tinctorius was established on the basis of 10 bitch of drugs from Xinjiang according to SPSS analysis. CONCLUSION: A reliable method is provided for the quality identification of Carthamus tinctorius.

[Determination of the saflor yellow-A in Carthamin tinctorius].

The content of the saflor yellow-A in Carthamin tinctorius medicinal materials was determined by the HPLC method. The C18-ODS(150 x 4.5 mm) column and methanol-acetonitrile-0.7% H3PO4(26:2:72) as a mobile phase were used. The flow rate was 1.0 ml/min and the detective wavelength was 403 nm. The average recovery was 100.72% and the relative standard deviation (RSD) was 1.73%.