Reversible changes in structure and function of photosynthetic apparatus of pea (Pisum sativum) leaves under drought stress

The effects of drought on photosynthesis have been extensively studied, whereas those on thylakoid organization are limited. We observed a significant decline in gas exchange parameters of pea (Pisum sativum) leaves under progressive drought stress. Chl a fluorescence kinetics revealed the reduction of photochemical efficiency of photosystem (PS)II and PSI. The non-photochemical quenching (NPQ) and the levels of PSII subunit PSBS increased. Furthermore, the light-harvesting complexes (LHCs) and some of the PSI and PSII core proteins were disassembled in drought conditions, whereas these complexes were reassociated during recovery. By contrast, the abundance of supercomplexes of PSII-LHCII and PSII dimer were reduced, whereas LHCII monomers increased following the change in the macro-organization of thylakoids. The stacks of thylakoids were loosely arranged in drought-affected plants, which could be attributed to changes in the supercomplexes of thylakoids. Severe drought stress caused a reduction of both LHCI and LHCII and a few reaction center proteins of PSI and PSII, indicating significant disorganization of the photosynthetic machinery. After 7 days of rewatering, plants recovered well, with restored chloroplast thylakoid structure and photosynthetic efficiency. The correlation of structural changes with leaf reactive oxygen species levels indicated that these changes were associated with the production of reactive oxygen species.     

Association of chlorophyll a/c(2) complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c(2) proteins were found. The most common complexes have Chl a/c(2) complexes at both sides of the PSI core monomer and have dimensions of about 17x24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c(2) light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c(2) proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c(2) proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Chloramphenicol effects on chlorophyll degradation and photosystem I assembly in the chlorina CD3 wheat mutant

We previously reported that applications of chloramphenicol to the chlorina wheat mutant, CD3, decreased the leaf Chl a/b ratio and enhanced accumulations of LHC proteins and LHC complexes during greening (Duysen et al. 1985). We have now examined Chl degradation and the change in Chl a/b ratios in wheat leaves kept in the dark as a measure of LHC destruction. Chl b was stable in chloroplasts of the CD3 wheat kept in darkness up to 5 days. Chloramphenicol significantly increased Chl b accumulations and impaired Chl a degradation in both CD3 mutant and normal wheat relative to untreated plants. Our Chl data suggest that the chloramphenicol induced accumulation of the LHC complex in the mutant wheat results from enhanced processing of LHC into the membrane rather than impairment of LHC degradation. The photosystem I (PSI) fraction of the CD3 wheat mutant was examined relative to that of normal wheat after 3 days greening. PSI was deficient in 25, 26, 26.5 kD LHCI protein in the mutant but both wheats accumulated low quantities of the 27–29 kD LHCII protein as detected by Western blot analysis. Chloramphenicol enhanced accumulations of several LHCI proteins primarily near 25 kD in the mutant and the 27–29 kD LHCII protein in normal wheat. The fluorescence emission and absorbance spectra suggest that chloramphenicol enhances accumulations of dissociated LHC in the PSI preparation of normal and CD3 mutant wheat.A contribution of North Dakota Agricultural Experiment Station. Published with approval of the Director as Journal Paper Number 1563.     

Evidence of the supercomplex organization of photosystem II and light-harvesting complexes in <Emphasis Type="Italic">Nannochloropsis granulata</Emphasis>

Diverse light-harvesting complexes (LHCs) have been found in photosynthetic microalgae that originated from secondary endosymbiosis involving primary red algae. However, the associations between LHCs and photosystem I (PSI) and photosystem II (PSII) in these microalgae are not fully understood. Eustigmatophyta is a red algal lineage that appears to have a unique organization in its photosynthetic machinery, consisting of only chlorophyll a and carotenoids that are atypical compared with other closely related groups. In this study, the supramolecular organization of pigment–protein complexes in the eustigmatophyte alga, Nannochloropsis granulata was investigated using Clear Native (CN) PAGE coupled with two-dimensional (2D) SDS-PAGE. Our results showed two slowly migrating green bands that corresponded to PSII supercomplexes, which consisted of reaction centers and LHCs. These green bands were also characterized as PSII complexes by their low temperature fluorescence emission spectra. The protein subunits of the PSII–LHC resolved by 2D CN/SDS-PAGE were analyzed by mass spectrometry, and four different LHC proteins were identified. Phylogenetic analysis of the identified LHC protein sequences revealed that they belonged to four different Lhc groups; (1) stress-related Lhcx proteins, (2) fucoxanthin chlorophyll a/c-binding Lhcf proteins, (3) red-shifted Chromera light-harvesting proteins (Red-CLH), and (4) Lhcr proteins, which are commonly found in organisms possessing red algal plastids. This is the first report showing evidence of a pigment–protein supercomplex consisting of PSII and LHCs, and to identify PSII-associated LHC proteins in Nannochloropsis.     

Absence of lutein, violaxanthin and neoxanthin affects the functional chlorophyll antenna size of photosystem-II but not that of photosystem-I in the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii double mutant npq2 lor1 lacks the beta, epsilon-carotenoids lutein and loroxanthin as well as all beta,beta-epoxycarotenoids derived from zeaxanthin (e.g. violaxanthin and neoxanthin). Thus, the only carotenoids present in the thylakoid membranes of the npq2 lor1 cells are beta-carotene and zeaxanthin. The effect of these mutations on the photochemical apparatus assembly and function was investigated. In cells of the mutant strain, the content of photosystem-II (PSII) and photosystem-I (PSI) was similar to that of the wild type, but npq2 lor1 had a significantly smaller PSII light-harvesting Chl antenna size. In contrast, the Chl antenna size of PSI was not truncated in the mutant. SDS-PAGE and Western blot analysis qualitatively revealed the presence of all LHCII and LHCI apoproteins in the thylakoid membrane of the mutant. The results showed that some of the LHCII and most of the LHCI were assembled and functionally connected with PSII and PSI, respectively. Photon conversion efficiency measurements, based on the initial slope of the light-saturation curve of photosynthesis and on the yield of Chl a fluorescence in vivo, showed similar efficiencies. However, a significantly greater light intensity was required for the saturation of photosynthesis in the mutant than in the wild type. It is concluded that zeaxanthin can successfully replace lutein and violaxanthin in most of the functional light-harvesting antenna of the npq2 lor1 mutant.     

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS-PAGE and BN/BN-PAGE. Two supercomplexes of 1060 and approximately 1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1-4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.     

Monomeric light harvesting complexes enhance excitation energy transfer from LHCII to PSII and control their lateral spacing in thylakoids

Proper assembly of plant photosystem II, in the appressed region of thylakoids, allows for both efficient light harvesting and the dissipation of excitation energy absorbed in excess. The core moiety of wild type supercomplex is associated with monomeric antennae that, in turn, bind peripheral trimeric LHCII complexes. Acclimation to light environment dynamics involves structural plasticity within PSII-LHCs supercomplexes, including depletion in LHCII and CP24. Here, we report on the acclimation of NoM, an Arabidopsis mutant lacking monomeric LHCs but retaining LHCII trimer. Lack of monomeric LHCs impaired the operation of both photosynthetic electron transport and state transitions, despite the fact that NoM underwent a compensatory over-accumulation of the LHCII complement compared to the wild type. Mutant plants displayed stunted growth compared to the wild type when probed over a range of light conditions. When exposed to short-term excess light, NoM showed higher photosensitivity and enhanced singlet oxygen release than the wild type, whereas long-term acclimation under stress conditions was unaffected. Analysis of pigment-binding supercomplexes showed that the absence of monomeric LHCs did affect the macro-organisation of photosystems: large PSI-LHCII megacomplexes were more abundant in NoM, whereas the assembly of PSII-LHCs supercomplexes was impaired. Observation by electron microscopy (EM) and image analysis of thylakoids highlighted impaired granal stacking and membrane organisation, with a heterogeneous distribution of PSII and LHCII compared to the wild type. It is concluded that monomeric LHCs are critical for the structural and functional optimisation of the photosynthetic apparatus.     

Deletion of the tobacco plastid psbA gene triggers an upregulation of the thylakoid-associated NAD(P)H dehydrogenase complex and the plastid terminal oxidase (PTOX)

We have constructed a tobacco psbA gene deletion mutant that is devoid of photosystem II (PSII) complex. Analysis of thylakoid membranes revealed comparable amounts, on a chlorophyll basis, of photosystem I (PSI), the cytochrome b6f complex and the PSII light-harvesting complex (LHCII) antenna proteins in wild-type (WT) and Δ psbA leaves. Lack of PSII in the mutant, however, resulted in over 10-fold higher relative amounts of the thylakoid-associated plastid terminal oxidase (PTOX) and the NAD(P)H dehydrogenase (NDH) complex. Increased amounts of Ndh polypeptides were accompanied with a more than fourfold enhancement of NDH activity in the mutant thylakoids, as revealed by in-gel NADH dehydrogenase measurements. NADH also had a specific stimulating effect on P700⁺ re-reduction in the Δ psbA thylakoids. Altogether, our results suggest that enhancement of electron flow via the NDH complex and possibly other alternative electron transport routes partly compensates for the loss of PSII function in the Δ psbA mutant. As mRNA levels were comparable in WT and Δ psbA plants, upregulation of the alternative electron transport pathways (NDH complex and PTOX) occurs apparently by translational or post-translational mechanisms.     

Loss of a single chlorophyll in CP29 triggers re-organization of the Photosystem II supramolecular assembly

In land plants, both efficient light capture and photoprotective dissipation of chlorophyll excited states in excess require proper assembly of Photosystem II supercomplexes PSII-LHCs. These include a dimeric core moiety and a peripheral antenna system made of trimeric LHCII proteins connected to the core through monomeric LHC subunits. Regulation of light harvesting involves re-organization of the PSII supercomplex, including dissociation of its LHCII-CP24-CP29 domain under excess light. The Chl a603-a609-a616 chromophore cluster within CP29 was recently identified as responsible for the fast component of Non-Photochemical Quenching of chlorophyll fluorescence. Here, we pinpointed a chlorophyll-protein domain of CP29 involved in the macro-organization of PSII-LHCs. By complementing an Arabidopsis knock-out mutant with CP29 sequences deleted in the residue binding chlorophyll b614/b3-binding, we found that the site is promiscuous for chlorophyll a and b. By plotting NPQ amplitude vs. CP29 content we observed that quenching activity was significantly reduced in mutants compared to the wild type. Analysis of pigment-binding supercomplexes showed that the missing Chl did hamper the assembly of PSII-LHCs supercomplexes, while observation by electron microscopy of grana membranes highlighted the PSII particles were organized in two-dimensional arrays in mutant grana partitions. As an effect of such array formation electron transport rate between Q_A and Q_B reduced, likely due to restricted plastoquinone diffusion. We conclude that chlorophyll b614, rather being part of pigment cluster responsible for quenching, is needed to maintain full rate of electron flow in the thylakoids by controlling protein-protein interactions between PSII units in grana partitions.     

Structural analysis and comparison of light-harvesting complexes I and II

Photosynthesis is a fundamental biological process involving the conversion of solar energy into chemical energy. The initial photochemical and photophysical events of photosynthesis are mediated by photosystem II (PSII) and photosystem I (PSI). Both PSII and PSI are multi-subunit supramolecular machineries composed of a core complex and a peripheral antenna system. The antenna system serves to capture light energy and transfer it to the core efficiently. Both PSII and PSI in the green lineage (plants and green algae) and PSI in red algae have an antenna system comprising a series of chlorophyll- and carotenoid-binding membrane proteins belonging to the light-harvesting complex (LHC) superfamily, including LHCII and LHCI. However, the antenna size and subunit composition vary considerably in the two photosystems from diverse organisms. On the basis of the plant and algal LHCII and LHCI structures that have been solved by X-ray crystallography and single-particle cryo-electron microscopy we review the detailed structural features and characteristic pigment properties of these LHCs in PSII and PSI. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.     

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.     

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.     

Effects of selective inactivation of individual genes for low-molecular-mass subunits on the assembly of photosystem II,as revealed by chloroplast transformation: the <Emphasis Type="Italic"> psbEFLJ </Emphasis>operon in <Emphasis Type="Italic"> Nicotiana tabacum</Emphasis>

Photosystem (PSII) is a supramolecular polypeptide complex found in oxygenic photosynthetic membranes, which is capable of extracting electrons from water for the reduction of plastoquinone. An intriguing feature of this assembly is the fact that it includes more than a dozen low-mass polypeptides of generally unknown function. Using a transplastomic approach, we have individually disrupted the genes of the psbEFLJoperon in Nicotiana tabacum, which encode four such polypeptides, without impairing expression of downstream loci of the operon. All four mutants exhibited distinct phenotypes; none of them was capable of photoautotrophic growth. All mutants bleached rapidly in the light. Disruption of psbEand psbF, which code for the alpha and beta apoproteins of cytochrome b(559), abolished PSII activity, as expected; Delta psbL and Delta psbJ plants displayed residual PSII activity in young leaves. Controlled partial solubilisation of thylakoid membranes uncovered surprisingly severe impairment of PSII structure, with subunit and assembly patterns varying depending on the mutant considered. In the Delta psbL mutant PSII was assembled primarily in a monomeric form, the homodimeric form was preponderant in Delta psbJ, and, unlike the case in Delta psbZ, the thylakoids of both mutants released some PSII supercomplexes. On the other hand, Photosystem I (PSI), the cytochrome b(6)f complex, ATP synthase, LHCII, and CP24/CP26/CP29 antennae were present in near wild-type levels. The data are discussed in terms of their implications for structural, biogenetic and functional aspects of PSII.     

Association of chlorophyll a/c2 complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c₂ proteins were found. The most common complexes have Chl a/c₂ complexes at both sides of the PSI core monomer and have dimensions of about 17 × 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c₂ light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c₂ proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c₂ proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Supercomplexes of plant photosystem I with cytochrome b6f,light-harvesting complex II and NDH

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b₆f (Cytb₆f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb₆f at the antenna side of PSI. The rectangular-shaped Cytb₆f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1–3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb₆f, may improve regulation of electron transport by the control of binding partners and distances in small domains.     

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.     

A model for the 77 K excited state dynamics in Chlamydomonas reinhardtii in state 1 and state 2

The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core. In a simultaneous analysis of the data from all strains under S1 or S2 conditions a unified model for the excited state dynamics at 77 K was created. This yielded the completely resolved LHCII-LHCI-PSI and LHCII-PSII dynamics and quantified the state transitions. In WT cells the fraction of light absorbed by LHCII connected to PSII decreases from 45% in S1 to 29% in S2, while it increases from 0% to 16% for LHCII connected to PSI. Thus (16/45 =) 36% of all LHCII is involved in the state transition. In the mutant strains deficient in the PSI core, the red most species peaking at 716 nm disappears completely, indicating that this far red Chl pigment is located in the PSI core. In the mutant strain deficient in the PSII core, red shifted species with maxima at 684 and 686 nm appear in the LHCII antenna. LHCII-684 is quenched and decays with a rate of (310 ps)^? 1.     

Supramolecular Assembly and Function of Subunits Associated with the Chlorophyll a-b Light-Harvesting Complex II (LHC II) in Soybean Chloroplasts

The chlorophyll (Chl) a-b light harvesting complex II (LHC II)contains more than 80% of the light-harvesting pigments of photosystemII (PS II) in chloroplasts. The supramolecular assembly andfunction of this auxiliary antenna system was investigated inChi b-deficient and Chi b-less mutant chloroplasts from soybeanand barley plants, and in their wild-type counterparts. Fourdistinct LHC II polypeptides were resolved by SDS-PAGE (subunitsa, b, c and d), having apparent molecular masses of 29, 28,27.2 and 26.8 kDa, respectively. The analysis of LHC II subunitcomposition in different developmental stages of the PS II unitin soybean (3>Chla/Chlbb>6), indicated the associationof specific subunits with the LHC H-inner and LHC II-peripheralin the chloroplast. The amount of subunit a in PS II was constantover a broad range of Chl a/Chl b ratios, suggesting that thissubunit is closely associated with the PS II-core complex. Subunitd also appeared to be constant over a wide range of Chl a/Chlb ratios, suggesting close association with the LHC II-inner.The PS II content in subunits b and c increased with the PSII antenna development in soybean but the ratio of b/c remainedconstant in all developmental stages and equal to 2 :1. Subunita was present in the Chl b-less chlorina f2 mutant of barleygrown under continuous illumination but was absent under intermittentillumination. The results suggest that each subunit binds 13-15Chl molecules. A working hypothesis is presented on the PS IIantenna development and LHC II subunit composition in soybeanchloroplasts. (Received October 11, 1988; Accepted January 19, 1989)     

Characterisation of senescence-induced changes in light harvesting complex II and photosystem I complex of thylakoids of Cucumis sativus cotyledons: age induced association of LHCII with photosystem I

Structure and function of chloroplasts are known to after during senescence. The senescence-induced specific changes in light harvesting antenna of photosystem II (PSII) and photosystem I (PSI) were investigated in Cucumis cotyledons. Purified light harvesting complex II (LHCII) and photosystem I complex were isolated from 6-day non-senescing and 27-day senescing Cucumis cotyledons. The chlorophyll a/b ratio of LHCII obtained from 6-day-old control cotyledons and their absorption, chlorophyll a fluorescence emission and the circular dichroism (CD) spectral properties were comparable to the LHCII preparations from other plants such as pea and spinach. The purified LHCII obtained from 27-day senescing cotyledons had a Chl a/b ratio of 1.25 instead of 1.2 as with 6-day LHCII and also exhibited significant changes in the visible CD spectrum compared to that of 6-day LHCII, indicating some specific alterations in the organisation of chlorophylls of LHCII. The light harvesting antenna of photosystems are likely to be altered due to aging. The room temperature absorption spectrum of LHCII obtained from 27-day senescing cotyledons showed changes in the peak positions. Similarly, comparison of 77K chlorophyll a fluorescence emission characteristics of LHCII preparation from senescing cotyledons with that of control showed a small shift in the peak position and the alteration in the emission profile, which is suggestive of possible changes in energy transfer within LHCII chlorophylls. Further, the salt induced aggregation of LHCII samples was lower, resulting in lower yields of LHCII from 27-day cotyledons than from normal cotyledons. Moreover, the PSI preparations of 6-day cotyledons showed Chl a/b ratios of 5 to 5.5, where as the PSI sample of 27-day cotyledons had a Chl a/b ratio of 2.9 suggesting LHCII association with PSI. The absorption, fluorescence emission and visible CD spectral measurements as well as the polypeptide profiles of 27-day cotyledon-PSI complexes indicated age-induced association of LHCII of PSII with PSI obtained from 27-day cotyledons. We modified our isolation protocols by increasing the duration of detergent Triton X-100 treatment for preparing the PSI and LHCII complexes from 27-day cotyledons. However, the PSI complexes isolated from senescing samples invariably proved to have significantly low Chl a/b ratio suggesting an age induced lateral movement and possible association of LHCII with PSI complexes. The analyses of polypeptide compositions of LHCII and PSI holocomplexes isolated from 6-day control and 27-day senescing cotyledons showed distinctive differences in their profiles. The presence of 26-28 kDa polypeptide in PSI complexes from 27-day cotyledons, but not in 6-day control PSI complexes is in agreement with the notion that senescence induced migration of LHCII to stroma lamellae and its possible association with PSI. We suggest that the migration of LHCII to the stroma lamellae region and its possible association with PSI might cause the destacking and flattening of grana structure during senescence of the chloroplasts. Such structural changes in light harvesting antenna are likely to alter energy transfer between two photosystems. The nature of aging induced migration and association of LHCII with PSI and its existence in other senescing systems need to be estimated in the future.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.