Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.     

Participation of Chlorophyll b Reductase in the Initial Step of the Degradation of Light-harvesting Chlorophyll a/b-Protein Complexes in Arabidopsis

The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) is the most abundant membrane protein in green plants, and its degradation is a crucial process for the acclimation to high light conditions and for the recovery of nitrogen (N) and carbon (C) during senescence. However, the molecular mechanism of LHCII degradation is largely unknown. Here, we report that chlorophyll b reductase, which catalyzes the first step of chlorophyll b degradation, plays a central role in LHCII degradation. When the genes for chlorophyll b reductases NOL and NYC1 were disrupted in Arabidopsis thaliana, chlorophyll b and LHCII were not degraded during senescence, whereas other pigment complexes completely disappeared. When purified trimeric LHCII was incubated with recombinant chlorophyll b reductase (NOL), expressed in Escherichia coli, the chlorophyll b in LHCII was converted to 7-hydroxymethyl chlorophyll a. Accompanying this conversion, chlorophylls were released from LHCII apoproteins until all the chlorophyll molecules in LHCII dissociated from the complexes. Chlorophyll-depleted LHCII apoproteins did not dissociate into monomeric forms but remained in the trimeric form. Based on these results, we propose the novel hypothesis that chlorophyll b reductase catalyzes the initial step of LHCII degradation, and that trimeric LHCII is a substrate of LHCII degradation.     

Two Sweetclover (Melilotus alba Desr.) Mutants Temperature Sensitive for Chlorophyll Expression

The nonallelic sweetclover (Melilotus alba Desr.) mutants U371 (ch10/ch10 genotype) and U372 (ch11/ch11 genotype) are derived from the U389 (+/+ genotype) parental strain. Growth of the U389 strain at a temperature of 17 or 26[deg]C results in plants normally green in appearance. The U371 and U372 mutant plants grown at 26[deg]C are slightly to moderately chlorophyll (Chl) deficient and have decreased Chl b/a ratios. Growth of the mutants at 17[deg]C results in plants severely deficient in Chl a, with markedly reduced levels of carotenoids except for violaxanthin, and with negligible amounts of Chl b or apoproteins for the light-harvesting complex of photosystem II. If mutant plants grown at 17[deg]C are transferred to 26[deg]C, during the next 20 d the amount of Chl per fresh weight will increase 5-fold and both the Chl b/a ratio and the expression of the light-harvesting complex apoproteins will progressively increase. Studies of the U371 mutant during the temperature-induced greening demonstrate progressive changes in chloroplast ultra-structure and leaf carbon isotope fractionation that parallel the increases in Chl. Changes observed in the leaf carbon isotope fractionation in the mutant suggest that, in addition to the already known effects of various abiotic factors, structural and metabolic internal factors can also influence whether the limitation in CO2 fixation is at the level of diffusion or carboxylation. Such temperature-initiated progressive greening in these and similar mutants may make them useful tools to elucidate not only the biosynthesis and assembly of the photosynthetic apparatus, but also physiological phenomena such as the influence of light-driven energy production on the overall carbon isotope fractionation during photosynthesis.     

Pigment shuffling in antenna systems achieved by expressing prokaryotic chlorophyllide a oxygenase in Arabidopsis

The organization of pigment molecules in photosystems is strictly determined. The peripheral antennae have both chlorophyll a and b, but the core antennae consist of only chlorophyll a in green plants. Furthermore, according to the recent model obtained from the crystal structure of light-harvesting chlorophyll a/b-protein complexes II (LHCII), individual chlorophyll-binding sites are occupied by either chlorophyll a or chlorophyll b. In this study, we succeeded in altering these pigment organizations by introducing a prokaryotic chlorophyll b synthesis gene (chlorophyllide a oxygenase (CAO)) into Arabidopsis. In these transgenic plants (Prochlirothrix hollandica CAO plants), approximately 40% of chlorophyll a of the core antenna complexes was replaced by chlorophyll b in both photosystems. Chlorophyll a/b ratios of LHCII also decreased from 1.3 to 0.8 in PhCAO plants. Surprisingly, these transgenic plants were capable of photosynthetic growth similar to wild type under low light conditions. These results indicate that chlorophyll organizations are not solely determined by the binding affinities, but they are also controlled by CAO. These data also suggest that strict organizations of chlorophyll molecules are not essential for photosynthesis under low light conditions.     

Preferential accumulation of apoproteins of the light-harvesting chlorophyll a/b-protein complex in greening barley leaves treated with 5-aminolevulinic acid

In order to study the coordinate accumulation of chlorophyll (Chl) and apoproteins of Chl-protein complexes (CPs) during chloroplast development, we examined changes in the accumulation of the apoproteins in barley (Hordeum vulgare L.) leaves when the rate of Chl synthesis was altered by feeding 5-aminolevulinic acid (ALA), a precursor of Chl biosynthesis. Pretreatment with ALA increased the accumulation of Chl a and Chl b 1.5- and 2.3-fold, respectively, after 12 cycles of intermittent light (2 min light followed by 28 min darkness). Apoproteins of the light-harvesting Chl a/b-protein complex of photosystem II (LHCII) were increased 2.4-fold with ALA treatment. However, apoproteins of the P700-Chl a-protein complex (CP1) and the 43-kDa apoprotein of a Chl a-protein complex of photosystem II (CPa) were not increased by ALA application. With respect to CPs themselves, LHCII was increased when Chl synthesis was raised by ALA feeding, whereas CP1 exhibited no remarkable increase. These results indicate that LHCII serves a role in maintaining the stoichiometry of Chl to apoproteins by acting as a temporary pool for Chl molecules.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll - CP chlorophyll-protein complex - CPa chlorophyll a-protein complex of PSII - CP1 P700-chlorophyll a-protein complex - LDS lithium dodecyl sulfate - LHCII light-harvesting chlorophyll a/b-protein complex of PSII This work was supported by the Grants-in-Aid for Scientific Research (04304004) from the Ministry of Education, Science and Culture, Japan.     

Protein synthesis by isolated Acetabularia chloroplasts. Synthesis of the two minor chlorophyll a complexes in vitro

Isolated chloroplasts of Acetabularia incorporate radioactive amino acids into more than 30 polypeptides in the light, including the apoprotein of the P700-chlorophyll a protein complex, the reaction centre core of photosystem I [Biochim. Biophys. Acta, 609. 107-120 (1980)]. In this paper it is shown that the apoproteins of the two minor chlorophyll a complexes, thought to be part of photosystem II reaction centre core, are also synthesized by isolated chloroplasts. Furthermore, they are integrated correctly into the thylakoid membrane in the absence of any cytoplasmic contribution, such that they can be isolated as chlorophyll-protein complexes indistinguishable from those already in the membrane. In contrast, the minor chlorophyll a + b complex 'CP 29' [Camm, E. L. and Green, B. R. (1980) Plant Physiol. 66, 428-432] and its dimers are not synthesized by isolated chloroplasts. In this they resemble the other chlorophyll a + b complex, the light-harvesting complex (LHC). It may be significant that the LHC, which is not essential for photosynthetic activity, is under nuclear control, while the reaction centre polypeptides, cytochrome b559, and cytochrome f, are synthesized on chloroplast ribosomes.     

Structural changes and lateral redistribution of photosystem II during donor side photoinhibition of thylakoids

The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.     

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.     

Mutants of Sweetclover (Melilotus alba) Lacking Chlorophyll b: Studies on Pigment-Protein Complexes and Thylakoid Protein Phosphorylation

Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorption spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid membranes lacking the three Chl b-containing pigment-protein complexes (AB-1, AB-2, and AB-3) while still containing A-1 and A-2, Chl a complexes derived from photosystems I and II, respectively. Complete solubilization and denaturation of the thylakoid proteins from this mutant revealed very little apoprotein from the Chl b-containing light-harvesting complexes, the major thylakoid proteins in normal plants. The normal and mutant sweetclover plants had active thylakoid protein kinase activities and numerous polypeptides were labeled following incubation with [γ-³²P]ATP. With the U374 mutant, however, there was very little detectable label co-migrating with the light-harvesting complex apoproteins on polyacrylamide gels. The Chl b-deficient chlorina-f2 mutant of barley (Hordeum vulgare) also had an active protein kinase activity capable of phosphorylating numerous polypeptides, including ones migrating with the same mobility as the light-harvesting complex apoproteins. These results indicate that the sweetclover mutants may be useful systems for studies on the function and organization of Chl b in thylakoid membranes of higher plants.     

Light-harvesting pigment-proteins of photosystem I in maize. Subunit composition and biogenesis

Three different pigment-binding proteins of the light-harvesting complex (LHC I) of maize photosystem I (PS I) have been isolated. Absorption and fluorescence excitation spectral analyses showed that each pigment-protein can transfer absorbed energy from its carotenoid and/or chlorophyll b components to chlorophyll alpha. Their apoproteins with apparent sizes of 24 (LHC Ia), 21 (LHC Ib), and 17 (LHC Ic) kDa have been purified to homogeneity. Differences in their pigment and amino acid compositions and in their reactions with antibodies demonstrate that the two smaller pigment-proteins are not proteolytically derived from the largest one. LHC Ib's apoprotein is particularly enriched in cysteine residues. None of the three apoproteins cross-reacted with antibodies raised against the major light-harvesting chlorophyll a/b-protein of photosystem II (LHC IIb) or against the PS I core complex (CC I) subunits. Studies of the biogenesis of PS I during greening of etiolated plants showed that all of the CC I subunits accumulated to a detectable level prior to the appearance of the 17-kDa subunit of LHC I, the accumulation of which preceded those of the 24- and 21-kDa subunits of LHC I. In addition, subunit VI of CC I is shown to be differentially expressed in mesophyll and bundle sheath cells; a slightly larger form of it accumulates in mesophyll than in bundle sheath thylakoids during plastid development.     

Restriction of Chlorophyll Synthesis Due to Expression of Glutamate 1-Semialdehyde Aminotransferase Antisense RNA Does Not Reduce the Light-Harvesting Antenna Size in Tobacco

The formation of 5-aminolevulinate is a key regulatory step in tetrapyrrole biosynthesis. In higher plants, glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes the last step in the sequential conversion of glutamate to 5-aminolevulinate. Antisense RNA synthesis for GSA-AT leads to reduced GSA-AT protein levels in tobacco (Nicotiana tabacum L.) plants. We have used these transgenic plants for studying the significance of chlorophyll (Chl) availability for assembly of the light-harvesting apparatus. To avoid interfering photoinhibitory stress, plants were cultivated under a low photon flux density of 70 [mu]mol photons m-2 s-1. Decreased GSA-AT expression does not seem to suppress other enzymic steps in the Chl pathway, indicating that reduced Chl content in transgenic plants (down to 12% of the wild-type level) is a consequence of reduced GSA-AT activity. Chl deficiency correlated with a drastic reduction in the number of photosystem I and photosystem II reaction centers and their surrounding antenna on a leaf area basis. Different lines of evidence from the transgenic plants indicate that complete assembly of light-harvesting pigment-protein complexes is given preference over synthesis of new reaction center/core complexes, resulting in fully assembled photosynthetic units with no reduction in antenna size. Photosynthetic oxygen evolution rates and in vivo Chl fluorescence showed that GSA-AT antisense plants are photochemically competent. Thus, we suggest that under the growth conditions chosen during this study, plants tend to maintain their light-harvesting antenna size even under limited Chl supply.     

Resolution of the light-harvesting chlorophyll a/b-protein of vicia faba chloroplasts into two different chlorophyll-protein complexes.

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll a/b-protein was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll a/b ratio of 0.9 and is associated with polypetides of 24,000 and 23,000 daltons. The 24,000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23,000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll a/b ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23,000. The two 23,000 dalton polypeptides occurring in both complexes are not identical.     

Association of chlorophyll a/b-binding Pcb proteins with photosystems I and II in Prochlorothrix hollandica

Action spectra for photosystem II (PSII)-driven oxygen evolution and of photosystem I (PSI)-mediated H(2) photoproduction and photoinhibition of respiration were used to determine the participation of chlorophyll (Chl) a/b-binding Pcb proteins in the functions of pigment apparatus of Prochlorothrix hollandica. Comparison of the in situ action spectra with absorption spectra of PSII and PSI complexes isolated from the cyanobacterium Synechocystis 6803 revealed a shoulder at 650 nm that indicated presence of Chl b in the both photosystems of P. hollandica. Fitting of two action spectra to absorption spectrum of the cells showed a chlorophyll ratio of 4:1 in favor of PSI. Effective antenna sizes estimated from photochemical cross-sections of the relevant photoreactions were found to be 192+/-28 and 139+/-15 chlorophyll molecules for the competent PSI and PSII reaction centers, respectively. The value for PSI is in a quite good agreement with previous electron microscopy data for isolated Pcb-PSI supercomplexes from P. hollandica that show a trimeric PSI core surrounded by a ring of 18 Pcb subunits. The antenna size of PSII implies that the PSII core dimers are associated with approximately 14 Pcb light-harvesting proteins, and form the largest known Pcb-PSII supercomplexes.     

Photosynthetic acclimation: structural reorganisation of light harvesting antenna--role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins

In order to carry out photosynthesis, plants and algae rely on the co-operative interaction of two photosystems: photosystem I and photosystem II. For maximum efficiency, each photosystem should absorb the same amount of light. To achieve this, plants and green algae have a mobile pool of chlorophyll a/b-binding proteins that can switch between being light-harvesting antenna for photosystem I or photosystem II, in order to maintain an optimal excitation balance. This switch, termed state transitions, involves the reversible phosphorylation of the mobile chlorophyll a/b-binding proteins, which is regulated by the redox state of the plastoquinone-mediating electron transfer between photosystem I and photosystem II. In this review, we will present the data supporting the function of redox-dependent phosphorylation of the major and minor chlorophyll a/b-binding proteins by the specific thylakoid-bound kinases (Stt7, STN7, TAKs) providing a molecular switch for the structural remodelling of the light-harvesting complexes during state transitions. We will also overview the latest X-ray crystallographic and electron microscopy-derived models for structural re-arrangement of the light-harvesting antenna during State 1-to-State 2 transition, in which the minor chlorophyll a/b-binding protein, CP29, and the mobile light-harvesting complex II trimer detach from the light-harvesting complex II-photosystem II supercomplex and associate with the photosystem I core in the vicinity of the PsaH/L/O/P domain.     

Stage-Specific State I-State II Transitions during the Cell Cycle of Euglena gracilis

In synchronized Euglena gracilis (light-dark regime of 14:10 hours) the successive formation of the photosynthetic apparatus during cell ontogeny is correlated with large changes in photosynthetic efficiency (P Brandt, B von Kessel 1983 Plant Physiol 72: 616-619; B Kohnke, P Brandt 1984 Biochim Biophys Acta 766: 156-160). This observation led us to investigate the functional association of the chlorophyll a/b light-harvesting protein complex (LHCP) with photosystem I or II, because changes in energy flow to photosystem I or II and in energy transfer between the two photosystems can be a reason for these alterations. As criterion for the association of the LHCP with photosystem I or II, state transitions were determined after 15 minutes preillumination using wave-lengths of 725 or 620 nanometers. The state transitions were determined from measurements of fluorescence induction at room temperature, and fluorescence kinetics at 77 K. According to the obtained data (a) mobile LHCP is present only between the 6th and the 10th hour of the light-time of the cell cycle and (b) this functional relation of the LHCP to photosystem I only at this stage of Euglena chloroplast development is not accompanied by a decrease in stacking. A model for the organization of the newly inserted LHCP within the photosynthetic apparatus of E. gracilis is discussed.     

Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana

The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de-etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a: b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full-green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light-harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green-gel system increased by 20%. (3) The ratio of the light-harvesting complex II apoproteins (LHCP) to 47-kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10-20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.     

Synthesis and Breakdown of the Apoproteins of Light-Harvesting Chlorophyll a/b Proteins in Chlorophyll b-deficient Mutants of Rice

Turnover, in the light, of apoproteins of light-harvesting chlorophylla/6-proteins for Photo-system I and II (LHC-I and LHC-II, respectively)was studied with the wild-type and three chlorophyll 6-deficientmutants of rice. (1) Synthesis of the 24 and 25 kDa apoproteinsof LHC-II and the 20 and 21 kDa apoproteins of LHC-I was examinedby incubating leaf segments with [³⁵S]-methionine. The threerice mutants, chlorina 2, which totally lacks chlorophyll b,and chlorina 11 and 14, which are partially deficient in chlorophyllb, synthesized the apoproteins as rapidly as did the wild typerice. (2) Pulse-chase experiments showed that breakdown of theapoproteins proceeded slowly, such that only a small proportionof the newly synthesized apoproteins was lost during the 48h of the chase in normal rice leaves. By contrast, large fractionsof the labelled apoproteins were rapidly degraded within thefirst several hours of the chase period in the chlorina mutants.The greater the deficiency in chlorophyll b of the mutant, thelarger were the rate and extent of the protein breakdown. Thisresult indicates that chlorophyll b is needed to stabilize theapoproteins of LHC-II and LHC-I. (3) However, even in chlorina2, there were small fractions of the apoproteins with lifetimesas long as those of apoproteins in the wild-type rice, suggestingthat the newly synthesized apoproteins are partially protectedby a factor(s) other than chlorophyll b. (4) The rate of turnoverof the apoproteins was significantly reduced in the dark andstrongly inhibited by prior treatment of leaf segments withchloramphenicol. (Received November 24, 1988; Accepted March 17, 1989)     

Chlorophyll-protein-complexes of thylakoids of wild type and chlorophyll b mutants of Arabidopsis thaliana

Pigment-protein-complexes of two chlorophyll b deficient mutants of Arabidopsis and from the wild type were separated electrophoretically. Light-harvesting proteins were absent in the chlorophyll b free mutant ch¹ and their amount was reduced in the mutant ch² which has a reduced content of chlorophyll b. The ratio of CPa:CP I increased with decreasing chlorophyll b content which indicated that the stoichiometry of photosystem II to photosystem I is not constant.Abbreviations Chl chlorophyll - CPa chlorophyll a-protein - CP I P-700 chlorophyll a-protein - LHCP light-harvesting chlorophyll a/b-protein - PAGE polyacrylamide gel electrophoresis - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate