Heiko Lokstein

Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal DeltapH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1 approximately lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.

Kinetic Studies on the Xanthophyll Cycle in Barley Leaves (Influence of Antenna Size and Relations to Nonphotochemical Chlorophyll Fluorescence Quenching)

Xanthophyll-cycle kinetics as well as the relationship between the xanthophyll de-epoxidation state and Stern-Volmer type nonphotochemical chlorophyll (Chl) fluorescence quenching (qN) were investigated in barley (Hordeum vulgare L.) leaves comprising a stepwise reduced antenna system. For this purpose plants of the wild type (WT) and the Chl b-less mutant chlorina 3613 were cultivated under either continuous (CL) or intermittent light (IML). Violaxanthin (V) availability varied from about 70% in the WT up to 97 to 98% in the mutant and IML-grown plants. In CL-grown mutant leaves, de-epoxidation rates were strongly accelerated compared to the WT. This is ascribed to a different accessibility of V to the de-epoxidase due to the existence of two V pools: one bound to light-harvesting Chl a/b-binding complexes (LHC) and the other one not bound. Epoxidation rates (k) were decreased with reduction in LHC protein contents: kWT > kmutant >> kIML plants. This supports the idea that the epoxidase activity resides on certain LHC proteins. Irrespective of huge zeaxanthin and antheraxanthin accumulation, the capacity to develop qN was reduced stepwise with antenna size. The qN level obtained in dithiothreitol-treated CL- and IML-grown plants was almost identical with that in untreated IML-grown plants. The findings provide evidence that structural changes within the LHC proteins, mediated by xanthophyll-cycle operation, render the basis for the development of a major proportion of qN.

Changes in the Composition of the Photosynthetic Apparatus in the Galactolipid-Deficient dgd1 Mutant of Arabidopsis thaliana

The glycerolipid digalactosyl diacylglycerol (DGDG) is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus. Here we employ a genetic approach based on the dgd1 mutant of Arabidopsis thaliana to investigate the function of DGDG in thylakoid membranes. The primary defect in the genetically well-characterized dgd1 mutant resulted in a 90% reduction of the DGDG content. The mutant showed a decreased photosystem II (PSII) to photosystem I ratio. In vivo room- and low-temperature (77 K) chlorophyll fluorescence measurements with thylakoid preparations are in agreement with a drastically altered excitation energy allocation to the reaction centers. Quantification of pigment-binding apoproteins and pigments supports an altered stoichiometry of individual pigment-protein complexes in the mutant. Most strikingly, an increase in the amount of peripheral light-harvesting complexes of PSII relative to the inner antenna complexes and the PSII reaction center/core complexes was observed. Regardless of the severe alterations in thylakoid organization, photosynthetic oxygen evolution was virtually not compromised in dgd1 mutant leaves.

The gun4 gene is essential for cyanobacterial porphyrin metabolism.

Ycf53 is a hypothetical chloroplast open reading frame with similarity to the Arabidopsis nuclear gene GUN4. In plants, GUN4 is involved in tetrapyrrole biosynthesis. We demonstrate that one of the two Synechocystis sp. PCC 6803 ycf53 genes with similarity to GUN4 functions in chlorophyll (Chl) biosynthesis as well: cyanobacterial gun4 mutant cells exhibit lower Chl contents, accumulate protoporphyrin IX and show less activity not only of Mg chelatase but also of Fe chelatase. The possible role of Gun4 for the Mg as well as Fe porphyrin biosynthesis branches in Synechocystis sp. PCC 6803 is discussed.

The phospholipid-deficient pho1 mutant of Arabidopsis thaliana is affected in the organization, but not in the light acclimation, of the thylakoid membrane.

The pho1 mutant of Arabidopsis has been shown to respond to the phosphate deficiency in the leaves by decreasing the amount of phosphatidylglycerol (PG). PG is thought to be of crucial importance for the organization and function of the thylakoid membrane. This prompted us to ask what the consequences of the PG deficiency may be in the pho1 mutant when grown under low or high light. While in the wild-type, the lipid pattern was almost insensitive to changes in the growth light, PG was reduced to 45% under low light in the mutant, and it decreased further to 35% under high light. Concomitantly, sulfoquinovosyl diacylglycerol (SQDG) and to a lesser extent digalactosyl diacylglycerol (DGDG) increased. The SQDG increase correlated with increased amounts of the SQD1 protein, an indicator for an actively mediated process. Despite of alterations in the ultrastructure, mutant thylakoids showed virtually no effects on photosynthetic electron transfer, O2 evolution and excitation energy allocation to the reaction centers. Our results support the idea that PG deficiency can at least partially be compensated for by the anionic lipid SQDG and the not charged lipid DGDG. This seems to be an important strategy to maintain an optimal thylakoid lipid milieu for vital processes, such as photosynthesis, under a restricted phosphate availability.

RELATIONSHIP BETWEEN QUENCHING OF MAXIMUM AND DARK-LEVEL CHLOROPHYLL FLUORESCENCE IN VIVO: DEPENDENCE ON PHOTOSYSTEM II ANTENNA SIZE

Abstract The effects of nonphotochemical quenching were quantified separately applying the Stem-Volmer formalism for dark level (SV 0 ) and maximum chlorophyll fluorescence yield (SV N ) in barley leaves comprising a step-wise altered Photosystem II (PS II) antenna size. Proportions of overall SV N can be attributed to distinct sites of the photosynthetic apparatus: (i) the bulk light-harvesting complex of PS II (LHC II), (ii) the inner LHC II antenna, and (iii) the reaction center/core complex of PS II. The fraction of SV N which exerts an effect on SV 0 appeared to arise almost exclusively from the inner LHC II antenna. A strong linear correlation between SV 0 and violaxanthin de-epoxidation points to an intrinsic relationship of both. The results are in line with the notion of a regulatory function of the inner LHC II antenna, thus controlling excitation energy delivery from the bulk LHCII to the PS II-core complex.

Comparison of chlorophyll fluorescence quenching in leaves of wild-type with a chlorophyll-b-less mutant of barley (Hordeum vulgare L.)

Abstract A pulse amplitude-modulated fluorometric technique was employed to separate photochemical (qP) and non-photochemical (qN) chlorophyll fluorescence quenching in attached leaves of wild-type and a chlorophyll-b-less mutant of barley ( Hordeum vulgare L.). Whereas a significantly higher qN developed in the wild type in the intensity range at which the photosynthetic light response became non-linear, there were virtually no differences in qP until pronounced photoinhibition was apparent. On monitoring the “dark” recovery of the maximum ( F M ) and dark level ( F 0 ) fluorescence yield, three distinct kinetic phases were resolved, which are ascribed to relaxation of high energy state quenching (qE), state 1-state 2 transitions (qT) and quenching due to photoinhibition (qI). The results provide evidence for heterogeneity of qE. The major part of qE (related to a fast-relaxing phase of F M quenching) is strongly reduced in mutant leaves. A fast-relaxing phase of F 0 quenching, readily observed in wild-type leaves under high light, is absent in the mutant under the same conditions. Hence, qE appears to be associated with the photosystem II light-harvesting complex (LHC II). A medium component of “dark” relaxation kinetics was observed in both mutant and wild-type leaves. However, it appears attributable to qT only in the latter at low light. Supported by the finding that there was no difference in xanthophyll pool size (on a chlorophyll a basis) between wild type and mutant, under high light conditions the medium phase may reflect a slower-relaxing portion of qE, probably due to xanthophyll conversion. Under supersaturating light the very slowly relaxing qI component became dominating, affecting mutant leaves to a considerably greater extent. The results stress the key role of LHC II not only as a solar energy collector but also in protecting the photosynthetic apparatus from adverse effects of excess excitation input.

The role of light-harvesting complex II in excess excitation energy dissipation: An in-vivo fluorescence study on the origin of high-energy quenching

Abstract A functionally intact light-harvesting complex II (LHC II) was recently inferred to be involved in the induction of high-energy dependent chlorophyll (Chl) fluorescence quenching (qE) in leaves (Lokstein et al., J. Photochem. Photobiol. B: Biol., 19 (1993) 217–225). The present study was performed to further elucidate this finding and, in particular to analyze the role of the xanthophyll cycle for qE formation. Measurements of modulated Chl fluorescence using the saturating flash technique and of xanthophyll cycle activity were performed in barley leaves of wild type (WT) and a Chl-b-less mutant with reduced LHC II ( chlorina 3613 ) in the absence and presence of the violaxanthin (V) de-epoxidase inhibitor dithiothreitol (DTT). The results can be summarized as follows: (a) The content of xanthophyll cycle-constituents in mutant leaves is lower on a leaf area basis (but significantly higher when related to Chl a content); the mutant has a substantially higher ability to convert V into antheraxanthin (A) and zeaxanthin (Z) when compared with the WT. In both genotypes in response to DTT treatment only low levels of A accumulated and Z formation was almost completely suppressed. (b) The photochemical efficiency of photosystem (PS) II expressed as the ratio of variable to maximum fluorescence, F V / F M , in dark-adapted leaves is virtually the same in WT and the mutant and remains invariant to treatment with DTT. (c) Nearly complete abolishment of the ‘fast’ relaxation phase of non-photochemical quenching (qNf) was observed after DTT treatment. Suppression of qNf (as an approximate measure of qE) accompanied by the disappearance of a ‘rapid’ phase of F 0 quenching relaxation provides strong evidence for qE being an antenna associated phenomenon. (d) A ‘medium’ phase of qN relaxation (qNm) was not influenced by DTT treatment at all light intensities investigated. Thus, the results do not indicate a significant contribution of persisting xanthophyll-dependent (A + Z) quenching to qNm. The phenomenon of qNm can not be explained by chloroplast movements. The data obtained support the hypothesis that ΔpH-mediated synergistic operation of both, structural rearrangement of LHC II and xanthophyll-cycle activity, is required for efficient qE formation in vivo.

Photophysical properties of Prochlorococcus marinus SS120 divinyl chlorophylls and phycoerythrin in vitro and in vivo

Prochlorococcus marinus SS120 is an ecologically important and biochemically intriguing marine cyanobacterium. In addition to divinyl chlorophylls (DV‐Chls) a and b it possesses a particular form of phycoerythrin (PE), but no other phycobilins and therefore no complete phycobilisomes. Here, a spectroscopic characterisation of these DV‐Chls and PE is provided. Comparison of fluorescence quantum yields, excited state lifetimes and absorption characteristics indicate similar light‐harvesting properties of the DV‐Chls as their monovinyl counterparts. PE, which is present only in tiny amounts, was purified and considerably enriched. A phycourobilin to phycoerythrobilin ratio of 3:1 chromophores per (αβ) PE monomer is suggested. The in vitro fluorescence lifetime of PE is 1.74 ns. In vivo time‐resolved fluorescence measurements with synchrotron radiation were used to investigate the possible role of PE in light‐harvesting. The fluorescence decay time for PE is about 550 ps, indicating an unusually slow excitation energy transfer. The decay time slowed to 1 ns after addition of glycerol to cell cultures. The contribution of PE to total light‐harvesting capacity was estimated to be about one (αβ) PE monomer per 330 DV‐Chl b molecules. Thus, the capacity of PE to function primarily as a photosynthetic light‐harvesting pigment in P. marinus SS120 is low.

Light-harvesting antenna function of phycoerythrin in Prochlorococcus marinus

Prochlorococcus marinus strain CCMP 1375 is the sole prokaryote to possess phycoerythrin in addition to (divinyl-)chlorophyll a/b binding antenna complexes. Here we demonstrate, employing a spectrofluorimetric assay, that phycoerythrin serves a light-harvesting antenna function (transfers energy to chlorophylls).