Identification and characterization of the AtBOR4 ortholog in Gypsophila Perfoliata L. genome

Boron is an essential micronutrient for plants. Boron toxicity is a limiting factor for agricultural production in arid and semi-arid regions with low precipitation. The reclamation of soils containing toxic levels of boron is extremely challenging. To address this issue, the present study aims to investigate the genetic resources of Gypsophila perfoliata L., a species well adapted to such environments, and to elucidate its boron tolerance mechanisms. The genes identified in this species are expected to contribute to the development of boron-tolerant plant varieties and to phytoremediation efforts. In line with these objectives, existing cDNA libraries prepared from RNA isolated from 6-hour samples of G. perfoliata exposed to 500 mg/L boron were transferred into Saccharomyces cerevisiae BY4743Δatr1 yeast strains via electroporation. In a yeast functional screening assay using the root cDNA library, colonies displaying boron tolerance were selected on medium containing 80 mM boric acid, a concentration considered toxic for yeast. The selected colonies were verified through spot assays and serial dilution analyses on media containing increasing concentrations of boric acid. Following plasmid isolation and re-transformation of yeast strains, only the strain designated PKB26 retained its tolerance. The PKB26 strain was capable of surviving in media containing up to 300 mM boric acid. Sequencing analysis revealed that the cDNA responsible for tolerance in the plasmid carried by PKB26 shares 72,9% homology with the Arabidopsis thaliana HCO₃⁻ transporter family gene (BOR4). This cDNA was named GpBOR4 (Gypsophila perfoliata BOR4). To determine its subcellular localization, the GpBOR4 gene was fused to the EGFP gene and expressed in BY4743Δatr1 yeast. Confocal microscopy showed that the protein localizes to the plasma membrane. Intracellular boron measurements demonstrated that yeast strains carrying GpBOR4 accumulated less boron than control strains lacking the gene. For further characterization, the GpBOR4 gene was introduced into Arabidopsis thaliana bor4 mutant and Col-0 (WT) lines via Agrobacterium-mediated floral dip transformation. The development of transgenic plants under toxic boron conditions was monitored, and transgene expression was quantified by qPCR. Introduction of GpBOR4 using the GFPGUSPlus vector conferred survival at 10 mM boron in three independent transgenic Col-0 lines. Southern blot analysis was performed to determine the copy number of GpBOR4 in the G. perfoliata genome. Expression analysis revealed that GpBOR transcript levels in G. perfoliata increased nearly seven-fold at 12 hours under boron stress. Overall, these findings suggest that the identification of a gene responsible for boron efflux in the highly boron-tolerant G. perfoliata may significantly contribute to the improvement of boron-sensitive plant species and to strategies for mitigating boron toxicity.