Peter Jahns

The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II.

Photoprotection of photosystem II (PSII) is essential to avoid the light-induced damage of the photosynthetic apparatus due to the formation of reactive oxygen species (=photo-oxidative stress) under excess light. Carotenoids are known to play a crucial role in these processes based on their property to deactivate triplet chlorophyll (³Chl*) and singlet oxygen (¹O₂*). Xanthophylls are further assumed to be involved either directly or indirectly in the non-photochemical quenching (NPQ) of excess light energy in the antenna of PSII. This review gives an overview on recent progress in the understanding of the photoprotective role of the xanthophylls zeaxanthin (which is formed in the light in the so-called xanthophyll cycle) and lutein with emphasis on the NPQ processes associated with PSII of higher plants. The current knowledge supports the view that the photoprotective role of Lut is predominantly restricted to its function in the deactivation of ³Chl*, while zeaxanthin is the major player in the deactivation of excited singlet Chl (¹Chl*) and thus in NPQ (non-photochemical quenching). Additionally, zeaxanthin serves important functions as an antioxidant in the lipid phase of the membrane and is likely to act as a key component in the memory of the chloroplast with respect to preceding photo-oxidative stress. This article is part of a Special Issue entitled: Photosystem II.

Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases

Illumination changes elicit modifications of thylakoid proteins and reorganization of the photosynthetic machinery. This involves, in the short term, phosphorylation of photosystem II (PSII) and light-harvesting (LHCII) proteins. PSII phosphorylation is thought to be relevant for PSII turnover, whereas LHCII phosphorylation is associated with the relocation of LHCII and the redistribution of excitation energy (state transitions) between photosystems. In the long term, imbalances in energy distribution between photosystems are counteracted by adjusting photosystem stoichiometry. In the green alga Chlamydomonas and the plant Arabidopsis, state transitions require the orthologous protein kinases STT7 and STN7, respectively. Here we show that in Arabidopsis a second protein kinase, STN8, is required for the quantitative phosphorylation of PSII core proteins. However, PSII activity under high-intensity light is affected only slightly in stn8 mutants, and D1 turnover is indistinguishable from the wild type, implying that reversible protein phosphorylation is not essential for PSII repair. Acclimation to changes in light quality is defective in stn7 but not in stn8 mutants, indicating that short-term and long-term photosynthetic adaptations are coupled. Therefore the phosphorylation of LHCII, or of an unknown substrate of STN7, is also crucial for the control of photosynthetic gene expression.

Mechanism and regulation of the violaxanthin cycle: the role of antenna proteins and membrane lipids.

The violaxanthin cycle describes the reversible conversion of violaxanthin to zeaxanthin via the intermediate antheraxanthin. This light-dependent xanthophyll conversion is essential for the adaptation of plants and algae to different light conditions and allows a reversible switch of photosynthetic light-harvesting complexes between a light-harvesting state under low light and a dissipative state under high light. The photoprotective functions of zeaxanthin have been intensively studied during the last decade, but much less attention has been directed to the mechanism and regulation of xanthophyll conversion. In this review, an overview is given on recent progress in the understanding of the role of (i) xanthophyll binding by antenna proteins and of (ii) the lipid properties of the thylakoid membrane in the regulation of xanthophyll conversion. The consequences of these findings for the mechanism and regulation of xanthophyll conversion in the thylakoid membrane will be discussed.

Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis

The induction and relaxation of non-photochemical quenching (NPQ) under steady-state conditions, i.e. during up to 90min of illumination at saturating light intensities, was studied in Arabidopsis thaliana. Besides the well-characterized fast qE and the very slow qI component of NPQ, the analysis of the NPQ dynamics identified a zeaxanthin (Zx) dependent component which we term qZ. The formation (rise time 10-15min) and relaxation (lifetime 10-15min) of qZ correlated with the synthesis and epoxidation of Zx, respectively. Comparative analysis of different NPQ mutants from Arabidopsis showed that qZ was clearly not related to qE, qT or qI and thus represents a separate, Zx-dependent NPQ component.

Dynamics of Xanthophyll-Cycle Activity in Different Antenna Subcomplexes in the Photosynthetic Membranes of Higher Plants (The Relationship between Zeaxanthin Conversion and Nonphotochemical Fluorescence Quenching).

The generation of nonphotochemical quenching of chlorophyll fluorescence (qN) in the antenna of photosystem II (PSII) is accompanied by the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin. The function of zeaxanthin in two mechanisms of qN, energy-dependent quenching (qE) and photoinhibitory quenching (qI), was investigated by measuring the de-epoxidation state in the antenna subcomplexes of PSII during the generation and relaxation of qN under varying conditions. Three different antenna subcomplexes were separated by isoelectric focusing: Lhcb1/2/3, Lhcb5/6, and the Lhcb4/PSII core. Under all conditions, the highest de-epoxidation state was detected in Lhcb1/2/3 and Lhcb5/6. The kinetics of de-epoxidation in these complexes were found to be similar to the formation of qE. The Lhcb4/PSII core showed the most pronounced differences in the de-epoxidation state when illumination with low and high light intensities was compared, correlating roughly with the differences in qI. Furthermore, the epoxidation kinetics in the Lhcb4/PSII core showed the most pronounced differences of all subcomplexes when comparing the epoxidation after either moderate or very strong photoinhibitory preillumination. Our data support the suggestion that zeaxanthin formation/epoxidation in Lhcb1-3 and Lhcb5/6 may be related to qE, and in Lhcb4 (and/or PSII core) to qI.

The Arabidopsis Thylakoid Protein PAM68 Is Required for Efficient D1 Biogenesis and Photosystem II Assembly

This work shows that PAM68 is a previously unknown PSII assembly factor that is required for efficient accumulation of photosystem II complexes in Arabidopsis thaliana and that is involved in early steps of PSII assembly in Synechocystis and Arabidopsis. Photosystem II (PSII) is a multiprotein complex that functions as a light-driven water:plastoquinone oxidoreductase in photosynthesis. Assembly of PSII proceeds through a number of distinct intermediate states and requires auxiliary proteins. The photosynthesis affected mutant 68 (pam68) of Arabidopsis thaliana displays drastically altered chlorophyll fluorescence and abnormally low levels of the PSII core subunits D1, D2, CP43, and CP47. We show that these phenotypes result from a specific decrease in the stability and maturation of D1. This is associated with a marked increase in the synthesis of RC (the PSII reaction center-like assembly complex) at the expense of PSII dimers and supercomplexes. PAM68 is a conserved integral membrane protein found in cyanobacterial and eukaryotic thylakoids and interacts in split-ubiquitin assays with several PSII core proteins and known PSII assembly factors. Biochemical analyses of thylakoids from Arabidopsis and Synechocystis sp PCC 6803 suggest that, during PSII assembly, PAM68 proteins associate with an early intermediate complex that might contain D1 and the assembly factor LPA1. Inactivation of cyanobacterial PAM68 destabilizes RC but does not affect larger PSII assembly complexes. Our data imply that PAM68 proteins promote early steps in PSII biogenesis in cyanobacteria and plants, but their inactivation is differently compensated for in the two classes of organisms.

Kinetic correlation of recovery from photoinhibition and zeaxanthin epoxidation

The generation of non-photochemical fluorescence quenching under photoinhibitory illumination and its relaxation under subsequent low light illumination in leaves from intermittent-light-grown pea (Pisum sativum L.) plants (IML-plants) has been investigated. In parallel, we studied (i) the activity of the xanthophyll cycle with emphasis on zeaxanthin formation and reconversion to violaxanthin and (ii) the degradation rate of D1 protein. In comparison to control plants grown in continuous light, IML-plants were much more susceptible to photoinhibition as determined from the increase of slowly (halftimes > 20 min) relaxing quenching (qI) of variable chlorophyll fluorescence. The relaxation (recovery) kinetics of qI (under weak light) in both types of plant depended on the photon flux density, temperature and duration of pre-illumination. The recovery time generally increased with an increasing degree of qI. In IML-plants, relaxation of qI was kinetically closely related to the epoxidation of zeaxanthin. At high degrees of photosystem II inhibition the kinetics resembled those of D1 degradation. The results are discussed in terms of the mechanisms of photosystem II inactivation in vivo.

Xanthophyll cycle and energy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light

The possible role of zeaxanthin formation and antenna proteins in energy-dependent chlorophyll fluorescence quenching (qE) has been investigated. Intermittent-light-grown pea (Pisum sativum L.) plants that lack most of the chlorophyll a/b antenna proteins exhibited a significantly reduced qE upon illumination with respect to control plants. On the other hand, the violaxanthin content related to the number of reaction centers and to xanthophyll cycle activity, i.e. the conversion of violaxanthin into zeaxanthin, was found to be increased in the antenna-protein-depleted plants. Western blot analyses indicated that, with the exception of CP 26, the content of all chlorophyll a/b-binding proteins in these plants is reduced to less than 10% of control values. The results indicate that chlorophyll a/b-binding antenna proteins are involved in the energy-dependent fluorescence quenching but that only a part of qE can be attributed to quenching by chlorophyll a/b-binding proteins. It seems very unlikely that xanthophylls are exclusively responsible for the qE mechanism.

Ion antiport accelerates photosynthetic acclimation in fluctuating light environments

Many photosynthetic organisms globally, including crops, forests and algae, must grow in environments where the availability of light energy fluctuates dramatically. How photosynthesis maintains high efficiency despite such fluctuations in its energy source remains poorly understood. Here we show that Arabidopsis thaliana K+ efflux antiporter (KEA3) is critical for high photosynthetic efficiency under fluctuating light. On a shift from dark to low light, or high to low light, kea3 mutants show prolonged dissipation of absorbed light energy as heat. KEA3 localizes to the thylakoid membrane, and allows proton efflux from the thylakoid lumen by proton/potassium antiport. KEA3’s activity accelerates the downregulation of pH-dependent energy dissipation after transitions to low light, leading to faster recovery of high photosystem II quantum efficiency and increased CO2 assimilation. Our results reveal a mechanism that increases the efficiency of photosynthesis under fluctuating light.

In-depth analysis of the distinctive effects of norflurazon implies that tetrapyrrole biosynthesis, organellar gene expression and ABA cooperate in the GUN-type of plastid signalling.

Application of norflurazon (NF) damages plastids, induces photobleaching and represses expression of the nuclear LHCB1.2 gene encoding a light-harvesting protein. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of NF. The mutants gun2, gun4 and gun5 exhibit perturbations in tetrapyrrole biosynthesis, but gun1 is defective in organellar gene expression (OGE). How gun mutations affect nuclear gene expression (NGE) and why the signals elicited by the two types evoke the same response remains unknown. Here we show that the carotenoid biosynthesis inhibitors amitrole and flurochloridone can replace NF in gun assays, whereas novel tetrapyrrole pathway mutations do not provoke a gun phenotype. Changes in haem levels also do not account for LHCB1.2 derepression in NF-treated gun mutants. Pigment measurements indicated that gun mutants are not resistant to NF, but gun2, gun4 and gun5 retain low levels of lutein, as well as of neoxanthin and violaxanthin, the precursors of abscisic acid (ABA). This might explain the enhanced ABA sensitivity of gun4 and gun5 plants found in germination assays. Metabolite profiling and analyses of reactive oxygen species and cellular redox state failed to suggest a link between gun mutations and altered LHCB1.2 expression. However, in contrast to NF-treated wild-type plants, gun mutants retain to a marked extent the capability to express the plastome-encoded proteins AtpB and RbcL. This, together with the finding that application of ABA can partially restore LHCB1.2 expression in NF-treated wild-type plants, supports the view that tetrapyrrole, OGE and ABA signalling are interconnected.