Functional Characterization of Haloarchaeal UDP-Glucose 4-Epimerase and 3-Hydroxybutyryl Dehydrogenase Genes in Nicotiana Tabacum

Abstract

The objective of the study: Abiotic stress significantly threatens plant survival. In response to challenging conditions, plants have evolved various resistance mechanisms. Recently, genetic strategies have become increasingly prevalent in efforts to enhance abiotic stress tolerance and develop stress-resistant plant varieties. In this study, we employed heterologous expression of two metabolism- related haloarchaeal genes in the Nicotiana tabacum genome to improve its salt and drought tolerance mechanisms. Experimental procedure(s): The U2642 and CL116 genes encoding 3-hydroxybutyryl-CoA dehydrogenase and UDP-glucose 4-epimerase, respectively, were subcloned into the pIPKb004 vector under the control of Cauliflower mosaic virus 35S (CaMV35S) promoter using the Gateway cloning system. The recombinant vectors were introduced into the N. tabacum through an Agrobacterium- mediated gene transfer procedure. Both wild-type (WT) and transgenic plants were cultivated under conditions of 175 mM NaCl and 200 mM mannitol, alongside a control group grown without stress. The transgenic plants were evaluated based on morphological characteristics, germination rates, gene expression levels, proline and malondialdehyde (MDA) accumulation, and antioxidant enzyme activities. Key results: The transgenic lines exhibited enhanced tolerance to abiotic stress, particularly drought, as demonstrated by higher germination rates, increased proline accumulation, reduced MDA production, and elevated antioxidant enzyme activities compared to WT plants. Conclusions: The metabolism-related genes from haloarchaea conferred significant resistance to abiotic stress when heterologously expressed in tobacco, highlighting their potential as promising candidates for developing stress-tolerant crops.