Mitochondrial Respiratory Metabolism in Salt‐Stressed Barley ( Hordeum vulgare ): TCA Cycle Activation With Limited GABA Shunt Engagement

Plants maintain energy balance under salinity stress through increased respiration and energy use, processes also associated with reactive oxygen species generation. Although respiration imposes a high energy cost, mitochondrial respiration and the tricarboxylic acid (TCA) cycle activity are vital for ATP production and for providing electron donors that drive ion exclusion and ROS detoxification. This study examined the molecular basis of salinity-induced respiratory responses in barley using physiological, biochemical, metabolomic and proteomic analyses. Salt exposure resulted in sodium accumulation, decreased photosynthesis and biomass, and increased respiration. Metabolite profiling indicated activation of the TCA cycle, while proteomics showed increased abundance of all targeted TCA enzymes, including phosphoenolpyruvate carboxylase isoforms and succinate dehydrogenase. Enhanced pyruvate oxidation and accumulation of downstream metabolites are consistent with a central role for the classical TCA cycle in barley's salinity response. Conversely, reduced levels of 2-oxoglutarate and succinate, together with non-detection of key GABA shunt enzymes (SSADH, GDH), are consistent with limited GABA shunt contribution under the conditions examined, although we cannot exclude dynamic GABA cycling that does not result in net accumulation. The absence of detectable arginine and ornithine, unlike their salt-induced increase in wheat, further suggests that the GABA shunt may contribute less to barley's salinity response under these conditions. Overall, the combined metabolomic and proteomic evidence supports an interpretation that barley preferentially relies on enhanced mitochondrial respiration and the canonical TCA cycle under these experimental conditions, with a lesser contribution from GABA shunt metabolism than in wheat. As neither metabolite pool sizes nor protein abundances measure pathway flux directly, this is presented as an interpretation of the combined datasets rather than a demonstration of the relative fluxes through the two pathways. These results point to a species-specific divergence in respiratory and osmotic adjustment strategies under salinity and invite future investigation into how key compatible solutes such as glycine betaine, an alternative osmolyte with a known relationship to GABA metabolism, contribute to barley's salinity tolerance.