Functional characterization of Elaeis guineensis cinnamyl alcohol dehydrogenase 2

The phenylpropanoid pathway produces numerous essential compounds that contribute to structural support, defense, and adaptation. Among the genes in this pathway, cinnamyl alcohol dehydrogenase (CAD) is one of the most crucial. Previous studies have shown that the oil palm genome contains seven CAD genes, designated as EgCAD1-EgCAD7. Among them, EgCAD1 and EgCAD2 were classified as bona fide CADs. Since the function of the EgCAD2 remains unclear, this study aimed to clarify its role. This study aimed to investigate the role of EgCAD2 in relation to lignin biosynthesis. Transgenic tobacco plants carrying the EgCAD2 and GUS genes were generated by the Agrobacterium-mediated transformation method. The transgenic plants were verified by Polymerase Chain Reaction (PCR), and the PCR-positive lines were subjected to segregation analysis to identify the homozygous T2 lines. The expression of the transgenes was then evaluated using semi-quantitative Reverse Transcription PCR (RT-PCR). Next, the CAD enzyme activity in the selected transgenic lines was analyzed and compared with that of wild-type tobacco. The transgenic lines were then assessed for visible morphological changes, total lignin, and flavonoid contents. Additionally, the lignin composition was analyzed using High-Performance Liquid Chromatography (HPLC), and the cell wall surface structure was examined using Field Emission Scanning Electron Microscopy (FESEM). The transgenic plants were also evaluated for their response to salt and drought stress. A total of 22 CAD2OE lines and nine GUS-OE lines were successfully generated and verified. Three CAD2-OE lines (P1, U1, W5) and one GUS-OE line (GUS1), which contain a single transgene integration locus, were selected to produce homozygous T2 generation. RTPCR analysis indicated that the EgCAD2 or GUS genes was present in the T2 generation and was abundantly expressed in the selected transgenic lines. Furthermore, the increase in CAD activity was accompanied by notable morphological changes, including reduced height, shorter internodes, altered flower coloration, and delays in germination and flowering. Moreover, histochemical staining showed enhanced lignin deposition in the transgenic lines compared to the wild-type. Additionally, an increase in xylem cell wall thickness was observed in the transgenic lines. Elevated lignin accumulation (70120% increment) in the transgenic lines was confirmed by the acetyl bromide assay. HPLC analysis revealed that the transgenic plants have a higher level of S- and H-units of lignin. Surprisingly, overexpression of EgCAD2 resulted in a reduction in total flavonoid content by ~41-51%. Additionally, the CAD-OE transgenic lines exhibited higher sensitivity to salt stress but responded similarly to the control plants under drought conditions. In this study, the thickening of the xylem cell wall likely reduced the efficiency of phytohormone diffusion, leading to a delay in flowering time. Meanwhile, the delayed germination time may result from increased lignin accumulation in the seed coat, which inhibits the imbibition process. Furthermore, the reduction in flavonoid content is likely caused by the overexpression of the lignin biosynthetic gene, which leads to a shift in metabolic flux, directing more resources toward lignin production rather than flavonoid production. As flavonoid levels decrease, osmoprotectant levels also decline, making the plant more sensitive to salt stress. In conclusion, the functional analysis of EgCAD2 suggests that it plays a key role in lignin biosynthesis, affecting plant growth and development, as well as osmotic stress tolerance.