Functional analysis of NtPDX2 in Nicotiana tabacum L. associated with stem development

Vitamin B₆ is a water-soluble vitamin that is essential for all living organisms in their life activities. Among its forms, pyridoxal 5'-phosphate (PLP) is the primary metabolically active form of Vitamin B₆, which usually plays a crucial role in the metabolism of proteins, fatty acids, and carbohydrates. To date, although the molecular functions of genes involved in vitamin B₆ biosynthesis, including <i>Pdx1</i>, <i>Pdx2</i>, <i>Pdx3</i>, and <i>Sos4</i>, have been reported in various plants, no studies have yet explored the functions of <i>NtPDX1</i> and <i>NtPDX2</i> in tobacco. This study used the <i>Nicotiana tabacum</i> L. as material to clone the CDS sequence of the <i>NtPDXs</i>. Through bioinformatics analysis, we predicted the phylogenetic relationships and functions of these genes; the subcellular localization of <i>NtPDX2</i> was found to be in the cytoplasmic structures. By conducting both constitutive overexpression and homozygous knockout studies of the <i>NtPDX2</i>, we observed a significant increase in vitamin B₆ content in the stem tissues of overexpressing plants (up to 150%), while knockout plants showed a decrease to 60%. This led to changes in agronomic traits such as plant height and stem thickness in tobacco plants. The overexpressing plants exhibited a significant increase in height (100.93 cm) and stem thickness (13.64 cm), whereas the knockout plants were shorter in height (73.10 cm) and had thinner stems (10.83 cm). By integrating transcriptome sequencing technology with molecular biology methods, we aim to elucidate the molecular mechanisms underlying the role of <i>NtPDX2</i> in tobacco growth and development, thereby providing new genetic resources and a theoretical foundation for the cultivation of new tobacco varieties with superior quality for flue-cured tobacco.