Nonphotochemical reduction of the plastoquinone pool in sunflower leaves originates from chlororespiration

Abstract

We investigated the relationship between nonphotochemical plastoquinone reduction and chlororespiration in leaves of growth-chamber-grown sunflower (Helianthus annuus L.). Following a short induction period, leaves of previously illuminated sunflower showed a substantially increased level of minimal fluorescence following a light-to-dark transition. This increase in minimal fluorescence was reversed by far-red illumination, inhibited by rotenone or photooxidative methyl viologen treatment, and stimulated by fumigation with CO. Using flash-induced electrochromic absorption-change measurements, we observed that the capacity of sunflower to reduce plastoquinone in the dark influenced the activation state of the chloroplast ATP synthase, although chlororespiratory transmembrane electrochemical potential formation alone does not fully explain our observations. We have added several important new observations to the work of others, forming, to our knowledge, the first strong experimental evidence that chlororespiratory, nonphotochemical plastoquinone reduction and plastoquinol oxidation occur in the chloroplasts of higher plants. We have introduced procedures for monitoring and manipulating chlorores-piratory activity in leaves that will be important in subsequent work aimed at defining the pathway and function of this dark electron flux in higher plant chloroplasts.

Figure 1

Changes in apparent F_o emission after a light-to-dark transition in sunflower leaves with different light-acclimation treatments. In all cases, fluorescence emission was measured from the leaves following 5 min of preillumination under 1000 μmol quanta m⁻² s⁻¹ green light. The sunflower plants had been light acclimated in a growth chamber at 650 μmol quanta m⁻² s⁻¹ PPFD for 4 h (○), light acclimated for 4 h and then treated with 10% CO for 65 s (•), or dark adapted for 10 h in the presence (▪) or absence (□) of CO.

Figure 2

Reversal of the increase in the apparent F_o in light-acclimated sunflower leaves by far-red illumination. Leaves were exposed to growth chamber light (650 μmol quanta m⁻² s⁻¹ PPFD) for 4 h, and the apparent F_o was measured following a preillumination treatment with (•) or without (○) CO. CO and preillumination conditions are the same as described in Figure 1. The effects of far-red light on a dark-adapted leaf (10 h) that was preilluminated for 5 min with 1000 μmol quanta m⁻² s⁻¹ green light are also depicted (□). The beginning of the 10-s far-red light pulses (approximately 730 nm, 10 μmol quanta m⁻² s⁻¹) is indicated by arrows.

Figure 3

Photooxidative MV treatment prevents the increase in the apparent F_o following a light-to-dark transition in sunflower leaf discs. Leaves were light acclimated under 4 h of growth-chamber light (650 μmol quanta m⁻² s⁻¹ PPFD) before sampling. The control (○) and MV-treated (▪) leaf discs were preilluminated for 5 min under 1000 μmol quanta m⁻² s⁻¹ green actinic light as before. The leaf discs were floated in an aqueous 100 μm MV solution during the preillumination period. CO fumigation had virtually no effect in MV-treated leaf discs (•). MV added in the dark (i.e. no photooxidative preillumination) had very little effect on the postillumination increase in apparent F_o (□).

Figure 4

Rotenone inhibits the postillumination increase of apparent F_o in sunflower leaf discs. Leaves used for experiments were exposed to 4 h of growth-chamber light (650 μmol quanta m⁻² s⁻¹ PPFD) prior to the introduction of the inhibitor. The leaf discs were floated on distilled water (○) or on a 200 μm rotenone solution (□) during the preillumination period (i.e. 5 min at 1000 μmol quanta m⁻² s⁻¹). At 800 s after the light-to-dark transition, the rotenone-treated leaf was fumigated with 10% CO (•).

Figure 5

Postillumination changes in the fast-relaxation time constant (τ_fast) for the single-turnover flash-induced electrochromic change measured at 518 nm in sunflower leaves. Changes in τ_fast were measured in leaves light acclimated for 4 h under 650 μmol quanta m⁻² s⁻¹ PPFD and then preilluminated at 1000 μmol quanta m⁻² s⁻¹ for 5 min with (•) and without (○) CO added. Results are also shown for a leaf that was preilluminated after a 10-h dark-adaptation period (▪).

Figure 6

Changes in apparent F_o (A) and F_m quenching (F_m − F_m′/F_m; B) measured with saturation pulses in sunflower leaves exposed to 650 μmol quanta m⁻² s⁻¹ PPFD for 4 h (○) and 11 h (□). Saturation pulses (3500 μmol quanta m⁻² s⁻¹) were 400 ms long and given at 240-s intervals. Leaves were preilluminated for 5 min at 1000 μmol quanta m⁻² s⁻¹ as before. A leaf sample that was dark adapted for 10 h and then preilluminated is included for comparison (▵).