Lutein accumulation in the absence of zeaxanthin restores nonphotochemical quenching in the Arabidopsis thaliana npq1 mutant

Abstract

Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid DeltapH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and alpha-carotene than the wild type. szl1 contains a point mutation in the lycopene beta-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene beta-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.

Figure 1.

Carotenoid Biosynthetic Pathway in Plants. The block in xanthophyll metabolism in the npq1 mutant, which lacks zeaxanthin due to a defect in the violaxanthin deepoxidase gene, is indicated by the symbol “npq1.”

Figure 2.

Screening for Suppressors of npq1 by Video Imaging of Chlorophyll Fluorescence Quenching. Mutagenized npq1 plants (M2 generation) on agar medium were exposed to 800 μmol photons m⁻² s⁻¹ for 1 min. In this false-color image of NPQ, the wild type appears red and an npq1 mutant appears blue, whereas the szl1 npq1 suppressor appears greenish red.

Figure 3.

HPLC Analysis of Pigments in the Wild Type, npq1, szl1, and szl1 npq1. (A) Comparison of pigment profile of the wild type, npq1, and szl1 npq1 after exposure to high light (1000 μmol photons m⁻² s⁻¹) for 30 min. (B) Overlay of HPLC traces of a szl1 single mutant before (LL) and after (HL) treatment with high light. Neo, neoxanthin; Vio, violaxanthin; An, antheraxanthin; Zea, zeaxanthin; Chl a, chlorophyll a; Chl b, chlorophyll b; α-car, α-carotene; β-car, β-carotene.

Figure 4.

Characteristics of Wild-Type, npq1, szl1, and szl1 npq1 Plants. (A) Growth of the four Arabidopsis strains. Plants were grown in LL (150 μmol photons m⁻² s⁻¹) with a short-day photoperiod (10 h light and 14 h dark). Plants were photographed at an age of 4 weeks. (B) Xanthophyll cycle pigment pool size (V+A+Z) and lutein levels of the four Arabidopsis strains. Plants were grown as described in (A). At the end of the dark period, whole plants were exposed to HL (2000 μmol photons m⁻² s⁻¹) for 30 min, and leaf samples were taken and analyzed by HPLC. Data were normalized to chlorophyll a and shown as the means ± sd (n = 9).

Figure 5.

NPQ Induction Curves in the Wild Type, Homozygous npq1, szl1, and szl1 npq1, and Heterozygous szl1/SZL1 npq1/npq1. NPQ was measured during 5 min of illumination with HL (1200 μmol photons m⁻² s⁻¹), followed by relaxation in the dark for 5 min. Data are presented as the means ± sd (n = 4).

Figure 6.

Molecular Genetic Analysis of szl1. (A) Sequence and position of the szl1 allele of the LCYB gene. The szl1 npq1 suppressor has a single base substitution from G to A, which changes the Gly residue (position 451) in a predicted transmembrane helix (black box) to a Glu. This Gly is invariant in the seven available plant LCYB enzymes, including Arabidopsis, maize, rice (Oryza sativa), tobacco (Nicotiana tabacum), tomato, citrus (Citrus sinensis), and papaya (Carica papaya). Dots in the szl1 npq1 sequence indicate identity to the wild-type sequence. (B) Cosegregation analysis of szl1 and NPQ phenotype. The szl1 npq1 suppressor was backcrossed to the npq1 parent to obtain F2 progeny. Lanes 1 to 16 are the first 16 plants among the total of 48 F2 progeny that were tested. The PCR fragments amplified from the genomic DNA of F2 progeny were digested with EcoRI and separated on agarose gel.

Figure 7.

HPLC Analysis of Products Formed from Lycopene in E. coli Expressing the Arabidopsis Wild-Type ε-cyclase and Wild-Type or Mutant β-Cyclase. Carotenoid pigment composition was examined in cultures of E. coli containing the plasmids and genes indicated above and to the left. The Arabidopsis wild-type and mutant copy of β-cyclase were cloned directly in the pAC-LYC plasmid to give the plasmid pAC-BETA-At and pAC-BETA-At-szl1, respectively (see Methods). Carotenoids were extracted with acetone from equal numbers of cells (based on A₆₀₀), and pigments were separated by HPLC and detected by absorbance at 445 nm. α-car, α-carotene; β-car, β-carotene. (A) pAC-LYC plus the Arabidopsis wild-type β-cyclase. (B) pAC-LYC plus the Arabidopsis mutant β-cyclase. (C) pAC-LYC plus the Arabidopsis wild-type β-cyclase and ε-cyclase. (D) pAC-LYC plus the Arabidopsis mutant β-cyclase and wild-type ε-cyclase.

Figure 8.

PSII and PSI Protein Levels in LL-Grown Wild Type, npq1, szl1, and szl1 npq1. Thylakoid protein samples were loaded on the basis of total protein (5 μg lane⁻¹), and immunoblot analysis was performed with polyclonal antibodies directed against each of the indicated proteins. D1 is a PSII reaction center protein; PsbS is a PSII protein that is essential for qE; Lhcb1, Lhbc2, and Lhcb3 are components of LHCII trimers; Lhcb4, Lhcb5, and Lhcb6 (also called CP29, CP26, and CP24, respectively) are monomeric, minor antenna proteins of PSII; PsaF is a PSI reaction center protein; Lhca1 is a PSI antenna protein. For comparison to mutant samples, dilutions were made from wild-type samples. (A) Immunoblot analysis of D1, PsbS, and Lhcb protein levels in the four genotypes. (B) Quantification of Lhcb4, Lhcb5, and Lhcb6 protein levels in szl1 and szl1 npq1. (C) Quantification of PsaF and Lhca1 protein levels in szl1 and szl1 npq1.

Figure 9.

TA Spectroscopy of szl1 npq1 Thylakoids. (A) TA spectrum from 880 to 1040 nm of szl1 npq1 mutant thylakoids with qE (red line) or without qE (black line) at 15 ps delay after pump pulse. The blue line shows the difference kinetics between the red and the black lines, and the blue dotted line is the spectrum of a β-carotene radical cation (β-Car^+•) for comparison (Tracewell and Brudvig, 2003). Data are presented as the means ± se (n = 5). (B) TA kinetics probed at 950 nm. (C) TA kinetics probed at 1000 nm.

Figure 10.

TA Spectroscopy of LHC Complexes. (A) Difference NIR-TA spectrum (from 880 to 1040 nm) between CP24 complexes binding lutein in both L1 and L2 sites (CP24-LL) and CP24 with violaxanthin in site L2 (CP24-LV). Each point represents the difference between the ΔA value obtained at 20 ps delay after pump pulse in CP24-LL and the corresponding value in CP24-LV. Data are presented as the means ± se (n = 5). (B) Difference NIR-TA spectrum (from 880 to 1040 nm) between CP29 complexes binding lutein in both L1 and L2 sites (CP29-LL) and CP29 with violaxanthin or neoxanthin in site L2 (CP29-LNV) . Each point represents the difference between the ΔA value obtained at 20 ps delay after pump pulse in CP29-LL and the corresponding value in CP29-LNV. Data are presented as the means ± se (n = 5). (C) TA kinetics probed at 920 nm of CP24-LL (red trace) and CP24-LV (black trace). Difference kinetic trace is reported in blue with rise and decay times indicated. (D) TA kinetics probed at 920 nm of CP29-LL (red trace) and CP29-LNV (black trace). Difference kinetic trace is reported in blue with rise and decay times indicated.

Figure 11.

Light-Induced Spectral Absorbance Changes in Leaves. Intact leaves of wild type (squares), szl1 (circles), szl1 npq1 (triangles), and npq1 (inverted triangles) were illuminated with ∼1150 μmol photons m⁻² s⁻¹ red light for 10 min to induce qE, and absorbance changes (from 460 to 563 nm) were measured during a 1-min dark interval. The qE spectrum was calculated as the difference in absorbance between 10 and 60 s after illumination to eliminate contributions from the electrochromic shift. The dashed lines indicate the peak positions of ∼530 to 535 nm in the wild type and ∼525 to 530 nm in the szl1, szl1 npq1, and npq1 mutants.