Hydroxycinnamoyl-CoA shikimate / quinate hydroxycinnamoyl transferase CtHCT061 enhances lignin biosynthesis and drought tolerance in safflower (Carthamus tinctorius L.)

Lignin fortifies cell walls and improves drought resilience, yet the molecular drivers of lignification in safflower ( Carthamus tinctorius L.) are poorly defined. Hydroxycinnamoyl-CoA shikimates/quinate hydroxycinnamoyl transferase (HCT) is one of the key regulatory enzymes in the lignin biosynthesis pathway. In this study, we aimed to characterize the role of a specific CtHCT gene in lignin biosynthesis and drought response by mining safflower expression atlas comprising 79 HCT genes. The spatiotemporal expression bias identified a candidate CtHCT061 gene encoding a BAHD acyltransferase containing conserved HXXXD and DFGWG motifs. Furthermore, Agrobacterium-mediated over-expression of CtHCT061 in Arabidopsis evidently enhanced drought tolerance by decreasing MDA and H₂O₂ levels by ∼35 % and ∼40 %, respectively, and increasing proline accumulation by ∼45 %. Similarly, transient overexpression of CtHCT061 in safflower also showed improved drought resilience by increasing lignin content (∼30–40 %) and upregulating key lignin biosynthesis genes (CtCCR, ∼8-fold; CtCAD, ∼9-fold), concurrently mitigating oxidative stress and increasing proline accumulation. On the contrary, Silencing CtHCT061 via VIGS led to a > 60 % reduction in CtCCR and CtCAD transcript levels, a ∼35 % decrease in lignin accumulation, and stunted growth, highlighting its critical role in modulating drought resilience. These findings positioned CtHCT061 as a key regulator of BAHD-mediated lignin flux for engineering drought resilient plants and promoting sustainable agriculture in the future. A schematic model illustrating the role of CtHCT061 in enhancing drought tolerance in safflower through lignin biosynthesis. Under drought stress conditions, the CtHCT061 gene is significantly upregulated. The increased expression of CtHCT061 positively influences the lignin biosynthetic pathway by promoting the transcription of downstream genes, particularly CtCCR and CtCAD. This regulatory cascade leads to enhanced lignin accumulation, contributing to a more robust root system and ultimately resulting in enhanced drought tolerance This model highlights CtHCT061 as a key positive regulator in the lignin biosynthesis-mediated drought stress response in safflower • CtHCT061 enhances lignin deposition in safflower and Arabidopsis. • CtHCT061 improves drought tolerance via antioxidants system. • Silencing CtHCT061 impairs lignin gene expression and stress tolerance. • CtHCT061 acts as a key regulator of phenylpropanoid metabolism in roots.