Cyanide-resistant, alternative pathway respiration in guard cell protoplasts of Vicia faba

Abstract

The activity and capacity of cyanide-resistant or alternative pathway respiration was examined in Vicia faba L. guard and mesophyll cell protoplasts maintained either in darkness (dark adapted) or illuminated with blue light. Respiration rates by dark-adapted guard cell protoplasts were unaffected by titrating with salicylhydroxamic acid (0.1–2.0 mM), an inhibitor of alternative pathway respiration, suggesting the lack of activity of this pathway. In combination with 0.1 mM KCN, an inhibitor of cytochrome pathway respiration, salicylhydroxamic acid was effective in inhibiting total respiration rates. In contrast with guard cell protoplasts, salicylhydroxamic acid reduced rates of O2 consumption in dark-adapted mesophyll cell protoplasts by 25–30% of total respiration. Titrating the cytochrome pathway of guard cell protoplasts with KCN (10–140 μM) alone failed to reduce respiratory activity but was effective in combination with salicylhydroxamic acid. Addition of a proton ionophore, carbonyl cyanide m-chlorophenylhydraxone, to dark-adapted guard cell protoplast suspensions increased respiration by approximately 30 and 84% in the presence and absence of salicylhydroxamic acid, suggesting restriction of electron flow by adenylates. Illumination of guard cell protoplasts with blue light for 30 min increased the sensitivity of respiration to salicylhydroxamic acid and increased the activity of the alternative pathway over this time period to ~55% of total respiration. Blue light also increased the rate of uncoupled respiration by guard cell protoplasts treated with salicylhydroxamic acid compared with dark-adapted protoplasts. The results suggest that electron movement through either cytochrome or alternative pathways in guard cell mitochondria may be regulated during signal transduction of blue light. Key words: Vicia faba, guard cell protoplasts, alternative pathway, respiration, blue light.