Experimental evidence for ascorbate-dependent electron transport in leaves with inactive oxygen-evolving complexes

Abstract

Previously, we showed that in barley (Hordeum vulgare) leaves with heat-inactivated oxygen-evolving complexes, photosystem II (PSII) has access to a large pool of alternative electron donors. Based on in vitro data, we proposed that this donor was ascorbate, yet this hypothesis has not been substantiated in vivo. In this paper, with the aid of chlorophyll a fluorescence induced by short (5-ms) light pulses and 820-nm absorbance transient measurements on wild-type and ascorbate-deficient (vtc2-1) mutant leaves of Arabidopsis (Arabidopsis thaliana), we show that in heat-treated leaves the rate of electron donation to PSII as well as the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive electron transport toward photosystem I depend on the ascorbate content of the leaves: upon ascorbate treatment, the donation half-time in the wild type and the mutant decreased from 25 to 22 ms and from 55 to 32 ms, respectively. Thermoluminescence measurements show that Tyr(Z)(+) is involved in the electron transfer from ascorbate to PSII. These data and the similar ascorbate dependencies of the heat-treated and the tris(hydroxymethyl)aminomethane-treated thylakoid membranes, with maximal donation half-times of about 16 ms, show that ascorbate is capable of supporting a sustained electron transport activity in leaves containing inactivated oxygen-evolving complexes. This alternative electron transport appears to be ubiquitous in the plant kingdom and is present in the green alga Chlamydomonas reinhardtii, and its rate depends on the physiological state of the plants and on environmental conditions. Our data suggest that ascorbate, as an alternative PSII electron donor, plays a physiological role in heat-stressed plants.

Figure 1.

Fast Chl a fluorescence transients (OJIP curves) of untreated and heat-treated (49°C, 40 s) wild-type (WT) and Asc-deficient vtc2-1 Arabidopsis mutant plants measured at 3,500 μmol m⁻² s⁻¹ photon flux density. The approximate positions of the different steps of the OJIP transient and of the K peak are indicated in parentheses. The curves are averages of eight to 10 measurements. The excitation light was produced by three 650-nm LEDs, and the Chl a fluorescence was measured at wavelengths above 700 nm. a.u., Arbitrary units.

Figure 2.

Dependence of the recovery of the K peak of the fast Chl a fluorescence transient in heat-treated wild-type and Asc-deficient Arabidopsis plants (vtc2-1 mutant) on the dark interval between the light pulses. A, Fast Chl a fluorescence transients measured on heat-treated (49°C, 40 s) wild-type (WT) leaves during 5-ms light pulses that were spaced 2.3 to 200 ms apart, as indicated. The fluorescence curves are averages of eight to 10 measurements. a.u., Arbitrary units. B and C, Regeneration of the K peak (calculated as F₂₀μ_s/F₃₀₀μ_s) as a function of dark intervals between the light pulses in heat-treated wild-type and vtc2-1 plants in the absence (B) and the presence (C) of externally supplied Asc (incubation of leaves in 20 mm Asc for 2 h in low light). The points are mean values from four to eight individual measurements that were fitted with exponentials for the determination of the half-recovery time. The error of the fitting was 5% to 6%.

Figure 3.

Light-induced 820-nm absorbance transients in wild-type (WT) and Asc-deficient Arabidopsis plants (vtc2-1 mutant). Heat treatment (49°C, 40 s), DCMU treatment (200 μm), and the addition of Asc (20 mm) were carried out as described in “Materials and Methods.” The kinetics were measured on dark-adapted samples during continuous illumination with blue light of 1,800 μmol m⁻² s⁻¹ photon flux density. The traces are averages of four to six measurements. a.u., Arbitrary units.

Figure 4.

Regeneration of the K peak of the fast Chl a fluorescence (fl.) transient after multiple 5-ms light pulses in thylakoid membranes isolated from heat-treated leaves (A) and in Tris-washed thylakoids (B) in the presence of 0, 6, and 50 mm Asc, as indicated. The dark interval between the consecutive light pulses was 200 ms. The Chl content was adjusted to 20 μg mL⁻¹. The traces are averages of four to six measurements. a.u., Arbitrary units.

Figure 5.

Regeneration of the K peak (calculated as F₂₀μ_s/F₃₀₀μ_s) of Tris-washed thylakoids and thylakoid membranes isolated from heat-treated (48°C, 40 s) barley leaves as a function of Asc concentration (A) and the dark interval between the light pulses (B). In A, the dark interval between the light pulses was 200 ms. In B, the Asc concentration was 50 mm. The Chl content was adjusted to 20 μg mL⁻¹. The data points are mean values from four to six measurements. The K_m value (A) and the half-recovery time (B) were determined by fitting the data points with exponentials. The error of the fitting was 5% to 6%.

Figure 6.

TL glow curves of heat-treated (48°C, 40 s) leaves, control thylakoid membranes, and thylakoids isolated from heat-treated barley leaves in the absence and presence of 50 mm Asc. The curves are averages of three measurements. a.u., Arbitrary units.

Figure 7.

The effect of moderate heat stress (39°C, 15 min) on the electron transport of wild-type and vtc2-1 Arabidopsis leaves. A, Fast Chl a fluorescence transients (OJIP curves) of untreated and heat-treated leaves measured at 3,500 μmol m⁻² s⁻¹ photon flux density. The curves are averages of eight to 10 measurements. WT, Wild type; a.u., arbitrary units. B, Amplitudes of the B TL band of untreated and heat-treated wild-type and mutant plants; mean and se values from 10 to 12 measurements are shown. C, Light-induced 820-nm absorbance transients on control and heat-treated wild-type and vtc2-1 leaves. The kinetics were measured with a Dual-PAM-100 instrument on dark-adapted samples during continuous illumination with red light of 1,950 μmol m⁻² s⁻¹ photon flux density for 1 s. The traces are averages of six to seven transients. D, Amounts of P₇₀₀⁺ in continuous red light of 95 μmol m⁻² s⁻¹ photon flux density, relative to the maximum oxidizable amounts, induced by saturating pulses of 10,000 μmol m⁻² s⁻¹ photon flux density, in untreated and heat-stressed wild-type and vtc2-1 leaves. Average traces and se values were calculated from six to seven 820-nm absorbance transient measurements.