The NAD(P)H dehydrogenase in barley thylakoids is photoactivatable and uses NADPH as well as NADH

Abstract

An improved light-dependent assay was used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of barley (Hordeum vulgare L.). The enzyme was sensitive to rotenone, confirming the involvement of a complex I-type enzyme. NADPH and NADH were equally good substrates for the dehydrogenase. Maximum rates of activity were 10 to 19 &mgr;mol electrons mg-1 chlorophyll h-1, corresponding to about 3% of linear electron-transport rates, or to about 40% of ferredoxin-dependent cyclic electron-transport rates. The NDH was activated by light treatment. After photoactivation, a subsequent light-independent period of about 1 h was required for maximum activation. The NDH could also be activated by incubation of the thylakoids in low-ionic-strength buffer. The kinetics, substrate specificity, and inhibitor profiles were essentially the same for both induction strategies. The possible involvement of ferredoxin:NADP+ oxidoreductase (FNR) in the NDH activity could be excluded based on the lack of preference for NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the diaphorase activity of FNR but not the NDH activity. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible.

Figure 1

The thylakoidal NDH activity in isolated thylakoids of barley is NAD(P)H bispecific. The assay used in this study for measuring the oxidation of NAD(P)H with MeV as the electron acceptor is absolutely light dependent and thus specific for thylakoidal NDH activity. The NDH was activated by illuminating the thylakoids in the presence of the assay reagents for 45 min before measurement.

Figure 2

Inhibition of light-dependent NADPH (○) and NADH (•) oxidation by rotenone in isolated thylakoids. The thylakoids were activated by preillumination for 45 min. The activity of thylakoids in the absence of rotenone was 19 μmol electrons mg⁻¹ Chl h⁻¹ for the oxidation of NADPH and 20 μmol electrons mg⁻¹ Chl h⁻¹ for the oxidation of NADH.

Figure 3

TTFA as an inhibitor of NADPH oxidation by isolated thylakoids or by FNR. The activity (± sd) was determined as light-dependent NADPH oxidation by thylakoids in the presence of MeV (○), as NADPH oxidation in the dark by thylakoids in the presence of duroquinone (▴), or as NADPH oxidation by isolated FNR in the presence of duroquinone (▪). The light-dependent assay was performed with thylakoids activated by preillumination for 45 min. The activity of thylakoids in the absence of TTFA was 15 ± 2 μmol electrons mg⁻¹ Chl h⁻¹ for the light-dependent assay and 36 ± 7 μmol electrons mg⁻¹ Chl h⁻¹ for the duroquinone-dependent assay. The activity of FNR in the absence of TTFA was 91 ± 10 μmol electrons nmol⁻¹ FNR h⁻¹.

Figure 4

Thylakoidal NDH activity (± sd) as a function of NADPH concentration. The thylakoids were activated by preillumination for 45 min. The curve shows a fit obtained by hyperbolic regression. Inset, Eadie-Hofstee plot of the data.

Figure 5

Light activation of the thylakoidal NDH. The thylakoids were illuminated in the presence of the assay reagents for the time periods indicated under the open bars. After illumination, the samples were kept in the dark and rates of NADPH oxidation (± sd) were determined at different time points. The numbers under the shaded bars indicate the total time from onset of the experiment, i.e. the sum of the illumination and subsequent dark periods.

Figure 6

Induction of NDH activity. The assay mixtures contained low concentrations of monovalent cations: 48 mm Na⁺ (♦); or high concentrations of mono- and/or divalent cations: 240 mm Na⁺ (▿), 48 mm Na⁺/9.6 mm Ca²⁺ (⋄), or 48 mm Na⁺/9.6 mm Mg²⁺ (○). The thylakoids were incubated in the dark in the presence of the respective assay reagents for 60 min, after which time the thylakoids were illuminated for the specified times before measurement of NADPH oxidation (± sd).

Figure 7

Model of cyclic electron transport in thylakoid membranes of barley, showing the site of action of inhibitors and artificial acceptors (italics) used in this study. A, B, C, D, and E, Subunits of PSI; DQ, duroquinone; FQR, antimycin-insensitive Fd:quinone reductase; and Pc, plastocyanin.