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Flavonoid 3′-hydroxylase (F3’H) enzyme is essential in determining the flavonoids B-ring hydroxylation pattern. It is mainly implicated in the biosynthetic pathway of cyaniding-based anthocyanins, flavonols, and flavan-3-ols. However, the evolution and regulatory mechanism of these important flavonoid hydroxylases have not been systematically investigated in safflower (Carthamus tinctorius L.). In this study, we identified 22 duplicatedCtF3'H-encoding genes from safflower through genome-wide prediction and conservation analysis. Phylogenetic analysis revealed the pattern of conservation and divergence of CtF3'Hs encoding proteins and their homologs from different plant species. The distribution of conserved protein motifs and cis-regulatory units suggested several structural components that could be crucial in deciphering the final function of CtF3'H proteins. Furthermore, the results of RNA-seq and qRT-PCR assay in different flowering tissues suggested differential expression level of CtF3’H genes during flower development. Based on the unique homology of CtF3’H5 with flavonoid 3’ hydroxylases from other plant species, further validation of CtF3’H5 was carried out. The transient expression of CtF3’H5 in onion epidermal cells implied that the subcellular localization of the fusion construct containing CtF3’H5 and GFP was predominantly detected in the plasma membrane. Similarly, the prokaryotic expression and western blot hybridization of CtF3’H5 demonstrated the detection of a stable 50.3kD target protein. However, more efforts are needed to further extend the functional validation of CtF3’H5 in safflower. This study provides a fundamental gateway for future functional studies and understanding the genetic evolution of F3'Hs in plants.

Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) is mainly associated with monolignol biosynthesis, a central precursor to producing guaiacyl and syringyl lignins in plants. However, the explicit regulatory mechanism of HCT-mediated monolignol biosynthesis in plants still remained unclear. Here, the genome-wide analysis of the HCT gene family in Carthamus tinctorius as a target for understanding growth, development, and stress-responsive mechanisms was investigated. A total of 82 CtHCT genes were identified and characterized. Most of the CtHCTs proteins demonstrated the presence of two common conserved domains, including HXXXD and DFGWG. In addition, the conserved structure of protein motifs, PPI network, cis-regulatory units, and gene structure analysis demonstrated several genetic determinants reflecting the wide range of functional diversity of CtHCT-encoding genes. The observed expression analysis of CtHCT genes in different flowering stages under normal conditions partially highlighted their putative roles in plant growth and development pathways. Moreover, CtHCT genes appeared to be associated with abiotic stress responses as validated by the expression profiling in various flowering phases under light irradiation and MeJA treatment. Altogether, these findings provide new insights into identifying crucial molecular targets associated with plant growth and development and present practical information for understanding abiotic stress-responsive mechanisms in plants.

BackgroundCysteine protease (CP) plays an important role in plant senescence. In this study, the whole genome evolution information of CPs was analyzed by using safflower, and the function of CtCP1 under l abiotic stress was analyzed.Results25 CPs members were identified in the safflower genome and divided into 9 subfamilies. Gene structure analysis showed that the possible evolutionary conservatism and functional similarity of the same family members. qRT-PCR at different florescence showed that the expression of CPs gene was the highest in the decline period, and CtCP1 gene changed significantly under abiotic stress. We cloned the qRT-PCR of CtCP1, at different florescence and stress, which showed that the expression of CtCP1 was the highest in the decline stage and low temperature. In order to study the function of CtCP1 gene, we obtained the overexpression CtCP1 line (OE) and the inhibition CtCP1 expression line (Anti) in Arabidopsis thaliana. The results of transgenic lines under low temperature stress showed that inhibition of CtCP1 expression enhanced the resistance of Carthamus tinctorius to low temperature, and overexpression of CtCP1 weakened the resistance of Carthamus tinctorius to low temperature.ConclusionWe have identified the cysteine protease genome of safflower and CtCP1 gene expression under abiotic stress

Cytochrome P450s are widely known as an important class of enzymes involved in multi-dimensional metabolic reactions which facilitate both primary and secondary metabolism in plants. Recent advances in genome sequencing of new plant species have greatly influenced our knowledge of the evolution of gene families. Herein, we present the extensive genome-wide identification study and early experimental groundwork of CtCYP81E subfamily extracted from safflower genome. The evolutionary divergence and several other molecular aspects of CtCYP81E enzymes were described with the help of phylogenetic reconstruction and robust in silico analysis. A total of 15 CtCYP81E candidate enzymes were identified and clustered together with A-type CYP71 clan of the model plant. The detail overview of their gene structures organization, conserved signatures motif, cis regulatory elements, Go functional categorization and protein-protein interaction network, respectively suggested novel insights for physiological and biosynthetic implications. Following multiple recombinant DNA approaches combined with the development of GPF fusion, heterologous expression, and transcriptional regulation network of CtCYP81E8 under normal and fluctuating environments, further functional validation was performed. The transient expression system using onion epidermal cells revealed the candidate protein's subcellular position to cell membrane. Similarly, the biochemical assay of recombinant CtCYP81E8 protein, effectively produced during heterologous expression, verified 2,4-dimethylphenol activity over different time periods. Moreover, the results of RNA-transcriptomic data and qRT-PCR analysis of 15 CtCYP81Es at different flowering stages indicated a differential expression levels defining their potential roles during safflower metabolite biosynthesis.Consequently, the transcriptional regulation of CtCYP81E exploited with various stress conditions indicated considerable susceptibility against these environmental drifts. Furthermore, the correlation analysis of CtCYP81E8 transcription and metabolite accumulation pattern in wild and mutant safflower lines also suggested positive outcomes during flower development. Although presumably, these results may be helpful in determining the fundamental idea of transcriptional regulation channels that strategically turn on the secondary metabolic pool of plant system in response cute environmental falls.