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

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.