Exogenous biotin modulates non-stomatal photosynthetic limitation under salinity in in vitro–grown Dizygostemon riparius

This study investigates whether exogenous biotin (vitamin B7) mitigates salinity-induced physiological limitations in Dizygostemon riparius , a medicinal species endemic to Maranhão, Brazil, grown in vitro. Salinity (50 mM NaCl) significantly restricts shoot growth, reduces shoot dry mass, and decreases leaf number. Gas exchange analyses demonstrate that the salinity-induced decline in photosynthesis is driven predominantly by non-stomatal limitations, as evidenced by reduced carboxylation efficiency and intrinsic water use efficiency, indicating biochemical constraints on carbon fixation. Biotin supplementation (16 mg L⁻¹) partially alleviates these effects, restoring photosynthetic performance and attenuating growth reductions under salinity. In contrast, photosynthetic pigment composition, maximum quantum efficiency of photosystem II (Fv/Fm), and leaf micromorphology remain stable across treatments, indicating that salinity does not induce photochemical impairment or structural damage. Untargeted metabolomic analysis revealed that salinity extensively reprograms primary metabolism, altering tricarboxylic acid cycle intermediates while promoting the accumulation of osmoprotective and secondary metabolites, consistent with metabolic adjustment to osmotic stress. Although biotin restored photosynthetic performance, it did not reverse the salt-induced metabolic reprogramming, suggesting that its protective effects arise from improved metabolic efficiency rather than changes in the stress-associated metabolome. These findings demonstrate that biotin modulates salinity responses primarily at the metabolic level, sustaining carbon assimilation and water use efficiency without anatomical or photochemical adjustment. The results highlight the sensitivity of D. riparius to salinity and identify biotin as a potential metabolic regulator of its photosynthetic resilience in vitro.