Genome-wide analysis of BAHD gene family and in vivo characterization of HQT-like genes involved in chlorogenic acid synthesis in tobacco (Nicotiana tabacum L.)

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Abstract
In plants, the diverse enzymes known as BAHD acyltransferases use acyl coenzyme A (acyl-CoA) as their substrate and fulfill crucial biological functions. In this study, 94 NtBAHD genes were identified in the tobacco ( Nicotiana tabacum ) genome. These genes displayed considerable diversity in their physicochemical properties, phylogenetic relationships, gene structures, and conserved motifs. Chlorogenic acid (CGA), which is the predominant polyphenol found in tobacco leaves, is mainly synthesised via the hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) pathway. Four HQT genes ( NtHQT1 – NtHQT4 ) were identified in the BAHD gene family of N. tabacum . Of these, NtHQT1 and NtHQT2 were highly expressed in leaves, while NtHQT3 and NtHQT4 were root-specific. Following heterologous expression in vitro , three of the four HQT proteins (NtHQT2, NtHQT3, and NtHQT4) were obtained in soluble form; however, only NtHQT2 and NtHQT3 exhibited enzymatic activity. Downregulation of NtHQT1 , NtHQT2 , NtHQT3 , and NtHQT4 significantly reduced both total polyphenol and chlorogenic acid (CGA) content, whereas the levels of rutin, scopoletin, and lignin remained unchanged. Furthermore, the expression of NtHQT genes was differentially induced under abiotic and biotic stresses, including drought, cold, and Phytophthora nicotianae infection. In contrast, their responses to various phytohormones exhibited distinct expression profiles. These results suggest that whereas in vitro assays identified NtHQT2 and NtHQT3 as the catalytically active isoforms, all four NtHQT genes contribute to chlorogenic acid (CGA) biosynthesis and environmental adaptation. Trial registration This study does not involve clinical trials or human participants and, as such, does not require clinical trial registration.

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