Continuous turnover of carotenes and chlorophyll a in mature leaves of Arabidopsis revealed by 14CO2 pulse-chase labeling

Abstract

Carotenoid turnover was investigated in mature leaves of Arabidopsis (Arabidopsis thaliana) by 14CO2 pulse-chase labeling under control-light (CL; 130 micromol photons m(-2) s(-1)) and high-light (HL; 1,000 micromol photons m(-2) s(-1)) conditions. Following a 30-min 14CO2 administration, photosynthetically fixed 14C was quickly incorporated in beta-carotene (beta-C) and chlorophyll a (Chl a) in all samples during a chase of up to 10 h. In contrast, 14C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the 14C fractions incorporated in beta-C and Chl a, whereas long-term HL acclimation significantly enhanced 14C labeling of Chl a but not beta-C. Consequently, the maximal 14C signal ratio between beta-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing alpha-carotene (alpha-C) together with reduced amounts of beta-C, remarkably high 14C labeling was found for alpha-C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum 14C ratios between carotenes and Chl a were 1:2 for alpha-C:Chl a and 1:5 for beta-C:Chl a in CL-grown lut5 plants, suggesting high turnover of alpha-C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.

Figure 1.

F_v/F_m in leaves of Arabidopsis wild-type plants during different light treatments. Plants acclimated and subjected to 130 μmol photons m⁻² s⁻¹ (CL; black circles), plants acclimated and subjected to 1,000 μmol photons m⁻² s⁻¹ (HL; white triangles), and plants acclimated to CL and transferred to HL at 0 h (CL→HL; white circles) were used. All data are means ± se (n = 6). Error bars are shown when they are larger than the symbols.

Figure 2.

Changes in carotenoid contents of wild-type leaves during different light treatments. A to C, CL plants. D to F, CL→HL plants. G to I, HL plants. Carotenoid contents are given relative to the Chl a contents (mmol mol⁻¹ Chl a). Chl contents per unit of leaf area did not change during the experiment for both Chl a and Chl b (data not shown), with average ratios of Chl a to Chl b of 3.8 ± 0.03 in the CL and CL→HL plants and 4.77 ± 0.06 in the HL plants. All data are means ± se (n = 3). Error bars are shown when they are larger than the symbols.

Figure 3.

A and B, Changes in incorporated ¹⁴C radioactivity in Chl a (A) and β-C (B) in leaves of wild-type plants under CL, HL, and CL→HL conditions. C, Ratio between ¹⁴C radioactivities of the two pigments (β-C/Chl a). Detached leaves were subjected to ¹⁴CO₂ administration under CL for 30 min (pulse period) and subsequently exposed to either CL or HL for up to 10 h (chase period, starting at 0 h). The ¹⁴C radioactivities of Chl a (A) and β-C (B) were normalized to the Chl a content measured in the same samples (Bq μg⁻¹ Chl a). Asterisks above the symbols indicate significant differences between the CL and HL plants at each time point; no significant difference was found between data of the CL and CL→HL plants. Plus signs above the symbols show significant differences between the time points within each treatment (i.e. after 3-, 6-, or 10-h chase compared with 0.5 h). ** P < 0.01, * and ⁺ P < 0.05. All data are means ± se (n = 3–5).

Figure 4.

F_v/F_m in leaves of lut5 mutants during HL exposure. Plants grown in CL were transferred to HL at 0 h, as in the CL→HL treatment of the wild-type plants in Figure 1. All data are means ± se (n = 6). For comparison, the data of the wild-type CL→HL plants are also shown (dotted line).

Figure 5.

Changes in carotenoid contents of lut5 leaves during HL exposure. Plants grown in CL were transferred to HL at 0 h, as in the CL→HL treatment of the wild-type plants in Figure 2, D to F. Carotenoid contents are given relative to the Chl a contents (mmol mol⁻¹ Chl a). Chl contents per unit of leaf area did not change during the experiment for both Chl a and Chl b (data not shown), with an average ratio of Chl a to Chl b of 4.17 ± 0.05. Data are means ± se (n = 3). Error bars are shown when they are larger than the symbols.

Figure 6.

A and B, Changes in incorporated ¹⁴C radioactivity in Chl a (A) and α-C and β-C (B) in leaves of lut5 mutants under CL→HL conditions. C, Ratios between ¹⁴C radioactivity of carotenes and Chl a (β-C/Chl a or α-C/Chl a). The experimental protocol was as described for the CL→HL treatment of wild-type plants (compare with Fig. 3). The ¹⁴C radioactivities of Chl a or α-C and β-C were normalized to the Chl a contents measured in the same samples (Bq μg⁻¹ Chl a). Plus signs above the symbols indicate significant differences between the time points within each treatment (i.e. after 3-, 6-, or 10-h chase compared with 0.5 h). ⁺⁺ P < 0.01, ⁺ P < 0.05. All data are means ± se (n = 3).

Figure 7.

Radio-HPLC analysis of photosynthetic pigments. A, A chromatogram (detection at 440 nm) of pigments extracted from a leaf of wild-type Arabidopsis grown in CL and exposed to HL for 3 h. Peak 1, V; peak 2, N; peak 3, A; peak 4, L; peak 5, Z; peak 6, Chl b; peak 7, Chl a; peak 8, β-C. AU, Arbitrary units. B, Simultaneous radiogram of ¹⁴C-labeled compounds. Dotted lines indicate the expected positions of pigment peaks in the radiogram, with a 20-s offset compared with the corresponding peaks in the chromatogram due to the sequential detection by the UV-visible light and radio detectors. Radioactivity was hardly detectable at peak positions 2, 5, and 6. C and D, Saponification eliminated Chl peaks 6 and 7 in the chromatogram (C) as well as the prominent peak 7 in the corresponding radiogram (D). E and F, Lack of xanthophyll pigments at peaks 1 to 4 concomitant with pronounced accumulation of Z at peak 5 in lut2/npq2 mutants (E) did not eliminate wild-type levels of radiolabeling at less than 40 min (F). G and H, Appearance of peak 9 (α-C) at the expense of peak 8 (β-C) in lut5 mutants (G) was accompanied by the appearance of a new peak in the radiogram at the expected position of α-C together with a smaller peak of β-C (H).