The Dynamics of Energy Dissipation and Xanthophyll Conversion in Arabidopsis Indicate an Indirect Photoprotective Role of Zeaxanthin in Slowly Inducible and Relaxing Components of Non-photochemical Quenching of Excitation Energy

Abstract

The dynamics of non-photochemical quenching (NPQ) of chlorophyll fluorescence and the dynamics of xanthophyll conversion under different actinic light conditions were studied in intact leaves of Arabidopsis thaliana. NPQ induction was investigated during up to 180 min illumination at 450, 900, and 1,800 μmol photons m^-2 s^-1 (μE) and NPQ relaxation after 5, 30, 90, or 180 min of pre-illumination at the same light intensities. The comparison of wild-type plants with mutants affected either in xanthophyll conversion (npq1 and npq2) or PsbS expression (npq4 and L17) or lumen acidification (pgr1) indicated that NPQ states with similar, but not identical characteristics are induced at longer time range (15-60 min) in wild-type and mutant plants. In genotypes with an active xanthophyll conversion, the dynamics of two slowly (10-60 min) inducible and relaxing NPQ components were found to be kinetically correlated with zeaxanthin formation and epoxidation, respectively. However, the extent of NPQ was independent of the amount of zeaxanthin, since higher NPQ values were inducible with increasing actinic light intensities without pronounced changes in the zeaxanthin amount. These data support an indirect role of zeaxanthin in pH-independent NPQ states rather than a specific direct function of zeaxanthin as quencher in long-lasting NPQ processes. Such an indirect function might be related to an allosteric regulation of NPQ processes by zeaxanthin (e.g., through interaction of zeaxanthin at the surface of proteins) or a general photoprotective function of zeaxanthin in the lipid phase of the membrane (e.g., by modulation of the membrane fluidity or by acting as antioxidant). The found concomitant down-regulation of zeaxanthin epoxidation and recovery of photosystem II activity ensures that zeaxanthin is retained in the thylakoid membrane as long as photosystem II activity is inhibited or down-regulated. This regulation supports the view that zeaxanthin can be considered as a kind of light stress memory in chloroplasts, allowing a rapid reactivation of photoprotective NPQ processes in case of recurrent light stress periods.

Keywords: energy dissipation; non-photochemical quenching; photoinhibition; photosynthesis; xanthophyll cycle; zeaxanthin.

Figure 1

NPQ induction. The induction of NPQ during 180 min of illumination at three different actinic light intensities (450, 900, and 1,800 μE of white light) was determined for (A) WT, (B) pgr1, (C) L17, (D) npq4, (E), npq2, and (F) npq1 plants. During the whole measurements, detached leaves were placed on wet paper in a temperature-controlled cuvette (20°C) under permanent supply with ambient air. Mean values ± SE of 3–6 independent measurements are shown.

Figure 2

Differences in NPQ induction upon increase of light intensities. To visualize the differences in NPQ induction upon increase of the actinic light intensity from either 450 to 900 μE or from 900 to 1,800 μE, the differences of the respective curves shown in in Figure 1 were plotted for all genotypes. (A) WT, (B) pgr1, (C) L17, (D) npq4, (E), npq2, and (F) npq1 plants.

Figure 3

Zx synthesis. The synthesis of Zx during 180 min of illumination at three different actinic light intensities (450, 900, and 1,800 μE of white light) is shown for (A) WT, (B) pgr1, (C) L17, (D) npq4 plants. Detached leaves were placed on water in temperature-controlled cuvette (20°C). At indicated time, leaves were rapidly frozen in liquid N₂ and the pigment composition was determined by HPLC analyses. The Zx content in % of the total VAZ-pool size (= sum of Vx, Ax and Zx) is shown. Data represent mean values ± SE of 3–4 independent measurements.

Figure 4

Comparison of NPQ induction and Zx synthesis. The time course of NPQ induction and Zx synthesis is compared for the four genotypes with an active xanthophyll cycle for the three actinic light intensities of 450, 900, and 1,800 μE: (A–C) WT, (D–F) pgr1, (G–I) npq4, and (J–L) L17. The data were taken from Figure 1 (NPQ) and Figure 3 (Zx). The determined Pearson's correlation coefficient r is indicated in each panel.

Figure 5

Comparison of the kinetics of NPQ induction and Zx synthesis in WT and L17 plants. The data for NPQ induction (open circles) and Zx synthesis (filled circles) are compared for WT plants (A,B) and L17 plants (C,D) at 900, (A,C) and 1,800 μE (B,D). For direct comparison, the data for Zx synthesis were fitted to match the amplitudes of the slowly developing (> 2 min) NPQ components, only. The data were taken from Figure 1 (NPQ) and Figure 3 (Zx). The determined Pearson's correlation coefficient r is indicated in each panel.

Figure 6

NPQ relaxation. The dark relaxation of NPQ after pre-illumination at three different actinic light intensities (450, 900, and 1,800 μE of white light) for 5, 30, 90, and 180 min was determined for (A) WT, (B) pgr1, (C) L17, (D) npq4, (E), npq2, and (F) npq1 plants. During the whole measurements, detached leaves were placed on wet paper in a temperature-controlled cuvette (20°C) under permanent supply with ambient air. Mean values of 3–6 independent measurements are shown. For clarity, error bars, which ranged from 0.1 to 0.3 in all cases, are not shown.

Figure 7

Zx epoxidation. The reconversion of Zx to Vx after pre-illumination at three different actinic light intensities (450, 900, and 1,800 μE of white light) for 5, 30, 90, and 180 min was determined for WT (A–C), pgr1 (D–F), npq4 (G–I), and L17 (J–L) plants. During the whole experiment, detached leaves were placed on water in temperature-controlled cuvette (20°C). At indicated time, leaves were rapidly frozen in liquid N₂ and the pigment composition was determined by HPLC analyses. The Zx content in % of the total VAZ-pool size (= sum of Vx, Ax and Zx) is shown. Data represent mean values of 3–4 independent measurements. For clarity, error bars, which ranged from 1 to 5% in all cases, are not shown.

Figure 8

Comparison of NPQ relaxation and Zx epoxidation after 90 min of pre-illumination. The time course of NPQ relaxation and Zx epoxidation after 90 min of pre-illumination at the three actinic light intensities of 450, 900, and 1800 μE is compared for the four genotypes with an active xanthophyll cycle: (A–C) WT, (D–F) pgr1, (G–I) npq4, and (J–L) L17. The data were taken from Figure 6 (NPQ) and Figure 7 (Zx). The determined Pearson's correlation coefficient r is indicated in each panel.

Figure 9

Comparison of the kinetics of NPQ relaxation and Zx epoxidation after 90 min of pre-illumination. The data for NPQ relaxation (open circles) and Zx epoxidation (filled circles) after 90 min of pre-illumination at the three actinic light intensities of 450, 900, and 1,800 μE are compared for the four genotypes with an active xanthophyll cycle: (A–C) WT, (D–F) pgr1, (G–I) npq4 and (J–L) L17. For direct comparison, the data for Zx epoxidation were fitted to match the amplitudes of the slowly relaxing (> 2 min) NPQ components, only. The data were taken from Figure 6 (NPQ) and Figure 7 (Zx). The dotted lines in panels (A–C) and (J–L) indicate the NPQ amplitudes after relaxation of qE. The determined Pearson's correlation coefficient r is indicated in each panel.