Heiko Härtel

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.

The pgp1 Mutant Locus of Arabidopsis Encodes a Phosphatidylglycerolphosphate Synthase with Impaired Activity

Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants. Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation. In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol. Details on the plant-specific pathways in different organelles are scarce. Here, we describe a phosphatidylglycerol biosynthesis-deficient mutant of Arabidopsis, pgp1. The overall content of phosphatidylglycerol is reduced by 30%. This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (EC 2.7.8.5) isoform encoded by a gene on chromosome 2. The mutant shows an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoform. Mutant plants are pale green, and their photosynthesis is impaired. This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants.

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.

Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio.

A gene of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 that is homologous to the conserved chloroplast open reading frame orf184 has been cloned and sequenced. The nucleotide sequence of the gene predicts a protein of 184 amino acids with a calculated molecular mass of 21.5 kD and two membrane-spanning regions. Amino acid sequence analysis showed 46 to 37% homology of the cyanobacterial orf184 with tobacco orf184, rice orf185, liverwort orf184, and Euglena gracilis orf206 sequences. Two orf184-specific mutants of Synechocystis sp PCC 6803 were constructed by insertion mutagenesis. Cells of mutants showed growth characteristics similar to those of the wild type. Their pigment composition was distinctly different from the wild type, as indicated by an increase in the phycocyanin-to-chlorophyll ratio. In addition, mutants also had a two- to threefold increase in photosynthetic electron transfer rates as well as in photosystem II-to-photosystem I ratio-a phenomenon hitherto not reported for mutants with altered photosynthetic characteristics. The observed alterations in the orf184-specific mutants provide strong evidence for a functional role of the orf184 gene product in photosynthetic processes.

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.

Digalactosyldiacylglycerol synthesis in chloroplasts of the Arabidopsis dgd1 mutant

Galactolipid biosynthesis in plants is highly complex. It involves multiple pathways giving rise to different molecular species. To assess the contribution of different routes of galactolipid synthesis and the role of molecular species for growth and photosynthesis, we initiated a genetic approach of analyzing double mutants of the digalactosyldiacylglycerol (DGDG) synthase mutantdgd1 with the acyltransferase mutant,act1, and the two desaturase mutants,fad2 and fad3. The double mutants showed different degrees of growth retardation: act1,dgd1 was most severely affected and growth of fad2,dgd1 was slightly reduced, whereas fad3,dgd1 plants were very similar to dgd1. In act1,dgd1, lipid and chlorophyll content were reduced and photosynthetic capacity was affected. Molecular analysis of galactolipid content, fatty acid composition, and positional distribution suggested that the growth deficiency is not caused by changes in galactolipid composition per se. Chloroplasts of dgd1 were capable of synthesizing monogalactosyldiacylglycerol, DGDG, and tri- and tetragalactosyldiacylglycerol. Therefore, the reduced growth ofact1,dgd1 and fad2,dgd1 cannot be explained by the absence of DGDG synthase activity from chloroplasts. Molecular analysis of DGDG accumulating in the mutants during phosphate deprivation suggested that similarly to the residual DGDG ofdgd1, this additional lipid is synthesized in association with chloroplast membranes through a pathway independent of the mutations, act1, dgd1,fad2, and fad3. Our data imply that the severe growth defect of act1,dgd1 is caused by a reduced metabolic flux of chloroplast lipid synthesis through the eukaryotic and prokaryotic pathway as well as by the reduction of photosynthetic capacity caused by the destabilization of photosynthetic complexes.

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.

Can digalactosyldiacylglycerol substitute for phosphatidylcholine upon phosphate deprivation in leaves and roots of Arabidopsis

To explore the role of digalactosyldiacylglycerol (DGDG) in plants the dgd1 mutant of Arabidopsis thaliana was grown in the presence and absence of inorganic phosphate. Phosphate deficiency in the dgd1 mutant causes a strong decrease in all phospholipids accompanied by an increase in DGDG and sulpholipid. Moreover, a significant DGDG accumulation was found in roots upon phosphate deprivation as well. Our data indicate that DGDG accumulation upon phosphate deprivation is due to the activation of a specific eukaryotic dgd1-independent biosynthetic pathway. We propose that DGDG may substitute for phosphatidylcholine upon phosphate deprivation.