A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll a/b-protein complex but not in its apoproteins

The mutant pg 113, derived from Chlamydomonas reinhardii, arg₂⁻ mt⁺ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl a/b-protein complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12–58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E⁶⁵VIHARWAM⁷³ led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159–166] is proposed.Abbreviations BSA bovine serum albumin faction V - ELIPs early light-inducible proteins - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LHCP light-harvesting chlorophyll a/b-protein - LHC IIb light-harvesting complex associated with Photosystem II - pLHCP precursor to LHCP - Rubisco ribulose 1,5-biphosphate carboxylase-oxygenase - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis     

Evidence for the role of the light-harvesting chlorophyll protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II_α and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II_α centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II_α component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II_α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II_α and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II_α and PS II_β to the fluorescence induction kinetics. PS II_α characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly

The biogenetic interdependence of light-harvesting chlorophyll (Chl) a/b proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP II) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesis/assembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.     

Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.     

Polypeptide composition and spectral properties of light-harvesting chlorophyll ab-protein complexes from intact and trypsin-treated chloroplast thylakoid membranes

The polypeptide composition and spectral properties of isolated light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complexes from intact and trypsin-treated thylakoid membranes of Hordeum vulgare and Vicia faba are compared. The LHCP complexes consist of four distinct polypeptides with molecular weights between 21 000 and 25 000 occurring in equal relative amounts in the whole polypeptide spectra of thylakoid membranes. It is shown indirectly that the two major polypeptides very probably belong to different chlorophyll-proteins. The loss of a small segment from both polypeptides during trypsin digestion of thylakoids does not substantially alter the spectral properties and cation-mediated aggregation of isolated LHCP complexes.     

A hydrophobic, carboxy-proximal region of a light-harvesting chlorophyll a/b protein is necessary for stable integration into thylakoid membranes.

Proteins synthesized as soluble precursors in the cytoplasm of eukaryotic cells often cross organellar membrane barriers and then insert into lipid bilayers. One such polypeptide, the light-harvesting chlorophyll a/b-binding protein (LHCP), must also associate with pigment molecules and be assembled into the photosystem II light-harvesting complex in the chloroplast thylakoid membrane. A study of the import of mutant LHCPs into isolated chloroplasts has shown that a putative alpha-helical membrane-spanning domain near the carboxy terminus (helix 3) is essential for the stable insertion of LHCP in the thylakoid. Protease digestion experiments are consistent with the carboxy terminus of the protein being in the lumen. This report also shows that helix 3, when fused to a soluble protein, can target it to the thylakoids of isolated, intact chloroplasts. Although helix 3 is required for the insertion of LHCP and mutant derivatives into the thylakoid, the full insertion of helix 3 itself requires additionally the presence of other regions of LHCP. Thus, LHCP targeting and integration into thylakoid membranes requires a complex interaction involving a number of different domains of the LHCP polypeptide.     

Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli

A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146–150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

Stage-dependent localization of LHCP II apoprotein in the Golgi of synchronized cells of Euglena gracilis by immunogold electron microscopy

We have localized LHCP II apoprotein in the Golgi and thylakoids of Euglena gracilis Klebs var. bacillaris Cori and strain Z Pringsheim by electron microscopy using a specific antibody and protein A-gold. Using synchronized cells (light, 14 h:dark, 10 h) we show that thylakoids are always immunoreactive. There is no reaction in the Golgi at 0 h (the beginning of the light period) but immunoreaction appears in the Golgi soon thereafter, rises to a peak at 8 h and declines to zero by 16 h (2 h into the dark period). The peak in immunoreaction in the Golgi immediately precedes the peak in cellular ¹⁴C-labeling of thylakoid LHCP II apoprotein seen by Brandt and von Kessel (Plant Physiol. (1983) 72, 616), supporting our suggestion that processing in the Golgi precedes deposition of LHCP apoprotein in the thylakoids. Substitution of preimmune serum for antiserum eliminates the immunoreaction in the Golgi, and thylakoids of synchronized cells of mutant Gr₁BSL which lacks LHCP II apoprotein show no immunoreaction in the Golgi or thylakoids at any stage. Random observations indicate that the compartmentalized osmiophilic structure (COS) shows an immunoreaction with anti-LHCP II apoprotein antibody at 1 h into the light period (when the Golgi is not immunoreactive) and at 10 h into the light period (when the Golgi is fully reactive), suggesting that the COS remains immunoreactive throughout the cell cycle.     

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38 degrees C : Evidence for Proteolytic Control

The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38°C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b-deficient strain pg-113 at the same rate in the dark or the light at 38°C, an indication that light did not affect translation of LHCP mRNA. Membrane-associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established, the specific activity of this peptidase in pg-113 cells was only 10 to 15% of the level in y-1 cells.     

Membrane adhesion in reconstituted proteoliposomes containing the light-harvesting chlorophyll ab-protein complex: The role of charged surface groups

The light-harvesting chlorophyll $\text{a}\text{b}$-protein complexes (LHCP) of spinach, pea, and barley thylakoids apparently contain four nonidentical polypeptide subunits of between 29,000 and 23,000 daltons on highly resolving sodium dodecyl sulfate-polyacrylamide gradient gels. Trypsin treatment of the spinach complex degraded at least the two major subunits by approximately 2000 daltons and resulted in a three-subunit pattern on gels. Proteoliposomes reconstituted with LHCP and the chloroplast diacyl lipids aggregated markedly in the presence of cations but vesicles containing LHCP prepared from trypsin-treated thylakoids did not. Amino acid analysis of native- and trypsin-treated LHCP indicated that the fragment(s) released by trypsin, which is essential for cation-induced stacking of thylakoids, contains lysine and arginine, but not aspartate or glutamate, and is thus cationic. Carboxyl groups on the surface of LHCP were charge neutralized using a water-soluble carbodiimide (1-ethyl-(3-dimethylaminopropyl)carbodiimide) plus [¹⁴C]glycine ethyl ester. Only two or three sites were labeled per 26,000-dalton polypeptide equivalent and only a minor fraction of this (22–24%) was located in the surface fragment(s) released by trypsin. Both LHCP and LHCP proteoliposomes, after carboxyl modification, aggregated avidly at low salt concentrations. The findings suggest that exposed anionic groups on the surface of LHCP contribute to an electrostatic repulsive force between membranes which must be attenuated, either by cation binding or chemical neutralization, before membrane-membrane adhesion can occur. In line with this the binding of Mn²⁺ by LHCP (approximately four Mn²⁺ bound/26,000-dalton polypeptide equivalent) was sharply decreased after carboxyl modification.     

Modulation of the light-harvesting chlorophyll antenna size in Chlamydomonas reinhardtii by TLA1 gene over-expression and RNA interference

Truncated light-harvesting antenna 1 (TLA1) is a nuclear gene proposed to regulate the chlorophyll (Chl) antenna size in Chlamydomonas reinhardtii. The Chl antenna size of the photosystems and the chloroplast ultrastructure were manipulated upon TLA1 gene over-expression and RNAi downregulation. The TLA1 over-expressing lines possessed a larger chlorophyll antenna size for both photosystems and contained greater levels of Chl b per cell relative to the wild type. Conversely, TLA1 RNAi transformants had a smaller Chl antenna size for both photosystems and lower levels of Chl b per cell. Western blot analyses of the TLA1 over-expressing and RNAi transformants showed that modulation of TLA1 gene expression was paralleled by modulation in the expression of light-harvesting protein, reaction centre D1 and D2, and VIPP1 genes. Transmission electron microscopy showed that modulation of TLA1 gene expression impacts the organization of thylakoid membranes in the chloroplast. Over-expressing lines showed well-defined grana, whereas RNAi transformants possessed loosely held together and more stroma-exposed thylakoids. Cell fractionation suggested localization of the TLA1 protein in the inner chloroplast envelope and potentially in association with nascent thylakoid membranes, indicating a role in Chl antenna assembly and thylakoid membrane biogenesis. The results provide a mechanistic understanding of the Chl antenna size regulation by the TLA1 gene.     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b₆/f-less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light-dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III-S at comparable rates. However, the redox-controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b₆/f-less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b₆/f complex inhibited both the electron flow and duroquinol-activated LHCII phosphorylation, but had no effect on the duroquinol-dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox-activated protein kinase(s). A quinone-binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b₆/f complex.     

Isolation and Characterization of the Thylakoid Membranes from the NaCl-Resistant (NaClr) Mutant Strain of the Cyanobacterium Anabaena variabilis

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl^r mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl^r mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl^r mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl^r mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl^r mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl^r mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl^r mutant strain also showed morphological variations under NaCl stress. Received: 14 April 2000 / Accepted: 23 May 2000