Optimization of ascorbate and glutathione assays for woody perennial bud tissues: overcoming matrix effects and storage instability.

Quantifying ascorbate (ASC) and glutathione (GSH) in woody perennial tissues presents significant methodological challenges due to complex tissue matrices and the lability of these redox-active metabolites. We adapted established enzymatic microplate assays for ASC and GSH quantification to mature dormant grapevine buds, a structure containing protective scales, hairs and lignified components. Key optimizations included: (i) use of 1 M HClO₄ rather than 0.2 M HCl for effective enzyme inactivation and matrix disruption; (ii) sample-to-solvent ratios of 1:10 to 1:15; and (iii) extended centrifugation to effectively sediment organic components. Standard addition experiments revealed significant matrix effects, with recovery of ~80% for ASC and 65% for GSH. The optimized method successfully determined >90% reduction status for both metabolites in mature buds. Critically, we observed severe ASC instability during frozen storage, with ~80% loss of ASC after 4 months at -80 °C, while GSH remained stable. These findings underscore the necessity of immediate analysis for ASC quantification in woody tissues and provide a validated protocol for studying redox metabolism in perennial bud tissues.