Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O₂•⁻) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O₂•⁻ accumulation pattern was different. For PA shoots, the increase in O₂•⁻ concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O₂•⁻ concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O₂•⁻ concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment.