Kang Ma

Safflower (Carthamus tinctorius L.) is a cash crop grown worldwide. Its seeds and flowers are primarily processed for edible oil and medicinal raw materials, while stems and leaves are discarded as agricultural waste. Recently, plant-derived exosome-like nanoparticles (PELNs) have gained growing research interest in food nutrition owing to their exceptional biocompatibility and relatively low cost for large-scale production. This study aimed to explore the potential functional value of safflower leaf agricultural waste by isolating safflower-derived PELNs. Firstly, a protocol was established for the isolation and purification of safflower (Carthamus tinctorius L.)-derived exosome-like nanoparticles (Ct-ELNs) from safflower leaves using sucrose density gradient ultracentrifugation. NTA analysis revealed that the 30–45% sucrose fraction enriched with Ct-ELNs exhibited the most uniform particle size, highest particle concentration, and optimal purity. Transmission electron microscopy confirmed typical PELNs ultrastructure: disc- or cup-shaped vesicles surrounded by bilayer lipid membranes. FM4-64 fluorescence suggests time-dependent association and likely cellular uptake of labeled Ct-ELNs by IPEC-J2 cells. Cellular assays demonstrated that Ct-ELNs elevated IPEC-J2 cell metabolic activity under matched protein-normalized dosing conditions. The 30–45% sucrose fraction showed the most favorable physicochemical profile and preliminary in vitro protective effects, including improved IPEC-J2 cell metabolic activity and modulation of Enterotoxigenic Escherichia coli (ETEC)-induced inflammation-related gene expression. Overall, this study established an optimized isolation protocol for Ct-ELNs derived from safflower leaves. These data indicate that safflower-derived Ct-ELNs confer preliminary cytoprotective transcriptional regulatory effects on intestinal epithelial cells under in vitro culture conditions.

Background: Safflower (Carthamus tinctorius L.) is a high-value economic crop with broad applications in agriculture, industry, and traditional medicine. Seed germination and seedling establishment are critical stages in the safflower life cycle, as they directly influence subsequent seedling establishment, survival, and plant growth. However, the transcriptomic dynamics and regulatory mechanisms underlying these processes remain largely unexplored, and the functional roles of long non-coding RNAs (lncRNAs) in this context are also poorly understood. Methods: In this study, transcriptome sequencing was performed across five developmental stages from seed germination to seedling establishment in safflower, followed by a comprehensive transcriptomic analysis and lncRNA identification. Results: Transcriptome sequencing identified a total of 3027 lncRNAs, including 940 natural antisense transcript (NAT)-pair-associated lncRNAs, which were classified into the divergent, convergent, and chimeric categories. Among these, 767 lncNATs were differentially expressed. Further analysis identified 542 NAT pairs in which both the protein-coding gene and its corresponding lncNAT exhibited a differential expression across the five developmental stages. A functional enrichment analysis of the predicted target genes of these lncRNAs suggested their involvement in photosynthesis and hormone-related responses. An enrichment analysis of differentially expressed genes (DEGs) across developmental stages further revealed the significant enrichment of photosynthesis and plant hormone signal transduction-related pathways, suggesting that these pathways are closely associated with safflower seed germination and seedling establishment. A further analysis of photosynthesis-related genes, particularly the expression patterns of LHC family members, suggested that Stage 3 may represent an important developmental transition associated with the optimization of the light-harvesting capacity during early seedling establishment, whereas plant-hormone-related genes are involved in regulating seed germination and subsequent leaf growth during seedling establishment. In addition, a weighted gene co-expression network analysis (WGCNA) identified candidate transcription factors associated with photomorphogenesis and plant hormone responses in safflower. Conclusions: This study advances our understanding of the regulatory mechanisms underlying safflower seed germination and subsequent growth and provides valuable molecular resources for future safflower breeding programs.

Safflower (Carthamus tinctorius L.) is an economically important crop, yet its production is severely threatened by fungal diseases including Botrytis cinerea. The molecular mechanism underlying disease resistance in safflower remains largely unclear. Extracellular vesicles (EVs), as vital carriers for cross-kingdom communication and transport, play crucial roles in plant antifungal defense. Lipid transfer proteins (LTPs), members of the pathogenesis-related protein 14 family, have also been shown to be key players in plant disease resistance. The promising resistance-related candidate gene CtLTP8 was previously identified via genome-wide association study (GWAS). In this study, a genome-wide analysis of the LTP gene family in safflower was performed. EVs were isolated from the apoplastic washing fluid of B. cinerea-infected safflower leaves, and proteomic analysis was performed. Numerous proteins associated with disease resistance, including CtLTP8, were detected by proteomic profiling. CtLTP8 was found to be present in EVs through molecular biological experiments. Moreover, stable overexpression of CtLTP8 in safflower significantly increased resistance to B. cinerea. In summary, this study characterized the disease resistance-related proteome of safflower EVs, and verified the presence of CtLTP8 in EVs and its antifungal function, providing valuable gene resources and theoretical support for safflower disease-resistance breeding and research on EV-mediated plant immune mechanisms.

Safflower ( Carthamus tinctorius ), a versatile economic crop of the Asteraceae family used for both medicinal and oil purposes, is widely cultivated worldwide. During its growth period, particularly during the harvesting phase, safflower is susceptible to fungal infections, leading to reduced product quality and yield. Currently, there is a lack of research on candidate genes associated with safflower disease resistance, which hinders the breeding of disease‐resistant varieties of safflower. The disease severity index of 499 safflower germplasm lines during the flowering period was analysed through genome‐wide association studies (GWAS), and a highly correlated TLP (thaumatin‐like proteins) family disease resistance candidate gene, CtTLP13 , was identified. Subsequently, transcriptomic and proteomic analyses were conducted to characterise the molecular features of safflower under fungal infection, and it was confirmed that CtTLP13 can respond to the biotic stress when safflower is infected by Botrytis cinerea . CtTLP13 overexpression in Arabidopsis and safflower enhanced safflower disease resistance; in vitro experiments confirmed its inhibition of B. cinerea growth and spore germination. We isolated safflower extracellular vesicles (EVs), verified CtTLP13 localisation within them and showed that CtTLP13‐carrying safflower EVs are taken up by B. cinerea and inhibit the fungal growth. Overall, this study identified a valuable disease resistance gene ( CtTLP13 ) in safflower. We also investigated the role of safflower EVs in disease resistance, and the results showed that safflower EVs exhibit anti‐ B. cinerea activity.