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.     

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.     

A study of chlorophyll b in vivo

1. The absorption spectrum of chlorophyll b in vivo at 77°K is presented as the difference spectrum between preparations of spinach and chlorophyll b-free Vischeria stellata chloroplasts.

2. A shoulder on this spectrum around 662 nm is due to a component different from chlorophyll b. This component may well be identical with the chlorophyll a form, chlorophyll a (665).

3. The 77°K chlorophyll b absorption spectra in the nonfractionated photosyn-thetic pigment apparatus and in fractions mainly representing Photosystems 1 or 2 are not significantly different.

4. The aerobic irreversible photobleaching of chlorophyll b was studied in the intact pigment complex as well as in fractions mainly consisting of Photosystem 1 or 2. A two-step photobleaching was observed in all cases. The time-course of this bleaching was not significantly different for chlorophyll b in both fractions.

5. These results do not indicate that more than a single chlorophyll b complex occurs in vivo.     

Arrangement of the light-harvesting chlorophyll a/b protein complex in the thylakoid membrane

The transverse orientation of the light-harvesting chlorophyll a/b protein complex of Photosystem II (LHC II) in the thylakoid membrane of pea was investigated using surface radioiodination with Iodo-Gen^TM. The labelling effects on LHC II of four different membrane preparations were compared. One preparation was oriented right-side-out (intact thylakoids); two of them had an inside-out orientation exposing the lumenal surface (inside-out vesicles; PS II particles) and one had both sides of the membrane exposed (mechanically damaged thylakoids). It was found that LHC II could be iodinated only in membrane preparations with an exposed lumenal surface. Isolated apoproteins were chemically cleaved. Fragments analysis revealed a tyrosine residue located eight amino acids from the C-terminus as the single iodination site. It is concluded that the C-terminus of LHC II points towards the lumental side of the thylakoid. Differences in the labelling behaviour of the LHC apoproteins could be assigned to a heterogeneity in the C-terminal region in which the tyrosine residue is replaced by phenylalanine.     

Composition of the photosystems and chloroplast structure in extreme shade plants

Chloroplasts were isolated from leaves of three species of tropical rainforest plants, Alocasia macrorrhiza, Cordyline rubra and Lomandra longifolia; these species are representative of extreme “shade” plants. It was found that shade plant chloroplasts contained 4–5 times more chlorophyll than spinach chloroplasts. Their chlorophyll a/chlorophyll b ratio was 2.3 compared with 2.8 for spinach. Electron micrographs of leaf sections showed that the shade plant chloroplasts contained very large grana stacks. The total length of partitions relative to the total length of stroma lamellae was much higher in Alocasia than in spinach chloroplasts. Freeze-etching of isolated chloroplasts revealed both the small and large particles found in spinach chloroplasts.

Despite their increased chlorophyll content, low chlorophyll a/chlorophyll b ratio, and large grana, the shade plant chloroplasts were fragmented with digitonin to yield small fragments (D-144) highly enriched in Photosystem I, and large fragments (D-10) enriched in Photosystem II. The degree of fragmentation of the shade plant chloroplasts was remarkably similar to that of spinach chloroplasts, except that the subchloroplast fragments from the shade plants had lower chlorophyll a/chlorophyll b ratios than the corresponding fragments from spinach. The D-10 fragments from the shade plants had chlorophyll a/chlorophyll b ratios of 1.78-2.00 and the D-144 fragments ratios of 3.54–4.07. We conclude that Photosystems I and II of the shade plants have lower proportions of chlorophyll a to chlorophyll b than the corresponding photosystems of spinach. The lower chlorophyll a/chlorophyll b ratio of shade plant chloroplasts is not due to a significant increase in the ratio of Photosystem II to Photosystem I in these chloroplasts.

The extent of grana formation in higher plant chloroplasts appears to be related to the total chlorophyll content of the chloroplast. Grana formation may simply be an means of achieving a higher density of light-harvesting assemblies and hence a more efficient collection of light quanta.     

Structure and organization of System II photosynthetic units during the greening of a dark-grown Chlorella mutant

The formation and the organization of Photosystem II photosynthetic units during the greening of a dark-grown Chlorella vulgaris, mutant 5/520, have been investigated by analysing the kinetics of the “activation” of oxygen evolution and of the fluorescence induction.

1. 1. The existence during the early stages of the greening of a stationary photosynthesis demonstrates the presence of active Photosystem II at these initial stages, which are integrated in a functional whole, leading to overall photosynthesis.

2. 2. The rise-time of oxygen evolution has been measured using far-red and green light in order to estimate the relative number of chlorophylls per unit. The amount of chlorophyll a remains relatively constant during the greening, while the progressive addition of chlorophyll b causes the size of the units to increase approx. 2-fold.

3. 3. The induction kinetics of the fluorescence are exponential during the early phases of greening and later become distinctly sigmoidal; this suggests that the first units synthesized on the surface of the membrane are isolated from each other by obstacles preventing electronic excitation transfers and that such obstacles which might correspond to some distance between such units, can disappear at later stages, allowing energy transfers to occur.

These observations suggest that the Photosystem II units represent organized functional entities. They apparently consist of a relatively constant number of chlorophyll a molecules, which during the greening is complemented progressively by the addition of chlorophyll b.     

Protein-protein interactions of the light-harvesting chlorophyll a/b protein. II. Evidence for two stages of cation independent association

In a previous paper, we observed a two-stage cation-independent association of the light-harvesting chlorophyll a/b protein from spinach chloroplasts based on concentration-dependent changes in the sedimentation coefficient. The two stages of association occurred between (2–4) and (4–7) μg/ml chlorophyll. In this paper, we provide further evidence for this association.

This includes: (1) A decrease in the number of divalent cation binding sites in the second stage of association. (2) A corresponding decrease in the extent of the cation-dependent association. (3) A positive deviation from Beer's law for chlorophyll b for both stages of the cation-independent association and a positive deviation for chlorophyll a for the second stage of association only. (4) A change in the fluorescence emission of both chlorophyll a and b. The change for chlorophyll b was observed for both steps of association whereas that for chlorophyll a was observed for the second step of association only. Therefore, the first stage of association affects only chlorophyll b whereas the second stage alters the environment of both chlorophyll a and b. (5) In addition, divalent cations quenched chlorophyll fluorescence. However, the quenching which required 200–300 μM divalent cation for half-maximal effects was related neither to divalent cation binding nor to the divalent cation-induced association of the protein.     

The P-700-chlorophyl a-protein complex and two major light-harvesting complexes of Acrocarpia paniculata and other brown seaweeds

Acrocarpia paniculata thylakoids were fragmented with Triton X-100 and the pigment-protein complexes so released were isolated by sucrose density gradient centrifugation. Three main chlorophyll-carotenoid-protein complexes with distinct pigment compositions were isolated.

1. (1) A P-700-chlorophyll a-protein complex, with a ratio of 1 P-700: 38 chlorophyll a: 4 ta-carotene molecules, had similar absorption and fluorescence characteristics to the chlorophyll-protein complex 1 isolated with Triton X-100 from higher plants, green algae and Ecklonia radiata.

2. (2) An orange-brown complex had a chlorophyll a : c₂ : fucoxanthin molar ratio of 2 : 1 : 2. This complex had no chlorophyll c₁ and contained most of the fucoxanthin present in the chloroplasts. This pigment complex is postulated to be the main light-harvesting complex of brown seaweeds.

3. (3) A green complex had a chlorophyll a : c₁ : c₂ : violaxanthin molar ratio of 8 : 1 : 1 : 1. This also is a light-harvesting complex.

The absorption and fluorescence spectral characteristics and other physical properties were consistent with the pigments of these three major complexes being bound to protein. Differential extraction of brown algal thylakoids with Triton X-100 showed that a chlorophyll c₂-fucoxanthin-protein complex was a minor pigment complex of these thylakoids.     

Isolation and properties of particles containing the reactioncenter complex of Photosystem II from spinach chloroplasts

By density gradient centrifugation of the 80000 × g supernatant of digitonintreated spinach chloroplasts two main green bands and one minor green band were obtained. The purification and properties of the particles present in the main bands, which were shown to be derived from Photosystem I and Photosystem II, have been described previously; those of the particles in the minor fraction will be described in the present paper.

After purification, these particles show Photosystem II activity but are devoid of Photosystem I activity. They have a high chlorophyll a/chlorophyll b ratio and are enriched in β-carotene and cytochrome b₅₅₉. At liquid nitrogen temperature, photoreduction of C550 and photooxidation of cytochrome-b₅₅₉ can be observed. At room temperature, cytochrome b₅₅₉ undergoes slight photooxidation.

These properties indicate that this particle may be the reaction-center complex of Photosystem II. It is suggested that, in vivo, the Photosystem II unit is made up of a reaction-center complex and an accessory complex, the latter being found in one of the main green bands of the density gradient.     

New chlorophyll-b forms in a chlorophyll-detergent photosynthetic model system

The absorption spectra of chlorophyll b in Triton X-100 micelles at room temperature are superpositions of components with absorption maxima at 640.8, 648.9, 659.5, 669.6, 682.1 and 695.7 nm, obtained from Gaussian analysis of the spectra. The last four forms strongly overlap the chlorophyll a forms of this system obtained with maxima at 659.3, 667.6, 674.3, 680.8, 686.5, 692.8, 701.9, 713.6 and 722.0 nm.

Since the in vivo chlorophyll a forms practically coincide with the forms found in this system, the possible existence of in vivo overlapping chlorophyll b and a forms eventually should be taken into consideration. In this case, however, the Gaussian analysis of in vivo absorption bands in itself in the proper spectrum range cannot discriminate between chlorophyll a and b components.     

Photochemical systems in mesophyll and bundle sheath chloroplasts of C4 plants

1. The agranal bundle sheath chloroplasts of Sorghum bicolor possess very low Photosystem II activity compared with the grana-containing mesophyll chloroplasts.

2. Sorghum mesophyll chloroplasts have a chlorophyll (chl) and carotenoid composition similar to that of spinach chloroplasts. In contrast, the sorghum bundle sheath chloroplasts have a higher chl a/chl b ratio; they are enriched in β-carotene and contain relatively less xanthophylls as compared to sorghum mesophyll or spinach chloroplasts.

3. Sorghum mesophyll chloroplasts with 1 cytochrome f, 2 cytochrome b₆ and 2 cytochrome b-559 per 430 chlorophylls have a cytochrome composition similar to spinach chloroplasts. Sorghum bundle sheath chloroplasts contain cytochrome f and cytochrome b₆ in the same molar ratios as for the mesophyll chloroplasts, but cytochrome b-559 is barely detectable.

4. The chl/P700 ratios of mesophyll chloroplasts of S. bicolor and mesophyll and bundle sheath chloroplasts of Atriplex spongiosa are similar to that of spinach chloroplasts suggesting that these chloroplasts possess an identical photosynthetic unit size to that of spinach. The agranal bundle sheath chloroplasts of S. bicolor possess a photosynthetic unit which contains only about half as many chlorophyll molecules per P700 as found in the grana-containing chloroplasts.

5. The similarity of the composition of the bundle sheath chloroplasts of S. bicolor with that of the Photosystem I subchloroplast fragments, together with their smaller photosynthetic unit and low Photosystem II activities suggests that these chloroplasts are highly deficient in the pigment assemblies of Photosystem II.     

Energy transfer in the photochemical apparatus of flashed bean leaves

Fluorescence and energy transfer properties of bean leaves greened by brief, repetitive xenon flashes were studied at −196 °C. The bleaching of P-700 has no influence on the yield of fluorescence at any wavelength of emission. The light-induced fluorescence yield changes which are observed in both the 690 and 730 nm emission bands in the low temperature fluorescence spectra are due to changes in the state of the Photosystem II reaction centers. The fluorescence yield changes in the 730 nm band are attributed to energy transfer from Photosystem II to Photosystem I. Such energy transfer was also confirmed by measurements of the rate of photooxidation of P-700 at −196 °C in leaves in which the Photosystem II reaction centers were either all open or all closed. It is concluded that energy transfer from Photosystem II to Photosystem I occurs in the flashed bean leaves which lack the light-harvesting chlorophyll a/b protein.     

The fractionation of plant photoactive pigment-protein complexes I and II

The pigment-protein complexes enriched with Photosystem I (PPC-I) and Photosystem II (PPC-II) were obtained using sievorptive chromatography on DEAE-Sephadex column. Both types of complexes contain Chlorophyll a, β-carotene and minor quantities of Chl b. Red absorbance maxima are located at 676 nm and 673 nm for PPC-I and PPC-II, respectively. The degrees of reaction centre enrichment were measured by the method of differential spectrophotometry: PPC-I has one P-700 per 35 bulk Chl a molecules, PPC-II contains one P-680 per 18 bulk Chl a molecules. The yield of PPC-II is 7–10 times lower than that of PPC-I. After one chromatographic procedure the amount of P-680 in PPC-I preparation does not exceed 7% of that of P-700, the amount of P-700 in PPC-II preparation 2% of that of P-680. The product of PPC-II degradation was studied.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll a/b-protein complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl a/b-protein complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl a/b-protein complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Observations on Photosystem II mutants of Scenedesmus: Pigments and proteinaceous components of the chloroplasts

Nine mutants of the green alga, Scenedesmus obliquus, which are blocked in the Photosystem II portion of photosynthesis were analyzed for possible deletion or alteration of (1) various components of the photosynthetic electron transport system, (2) of chloroplast lipids, (3) of total chlorophyll or of the chlorophyll a/chlorophyllb ratio, and (4) of their content of carotenes and carotenoids. No changes in content or activity of ferredoxin, ferredoxin-NADP⁺ reductase, plastocyanin, cytochrome c-552, and the membrane-bound b-type or c-type cytochromes were observed. The most consistent differences noted between the mutant strains and the wild-type strain were in the molar ratio of chlorophyll/plastoquinone A, the total chlorophyll content, and a decreased content of - and β-carotene with a concomitant increase of carotenoids. The loss of Photosystem II activity in these mutant strains, as observed either with whole cells or with isolated chloroplast fragments, may be accounted for by their decreased content of plastoquinone A. Their decreased chlorophyll content and altered carotene/xanthophyll ratio also suggests possible alteration of chloroplast membrances resulting in increased internal oxidation of the photosynthetic pigments.     

C-550 in photosystem II subchloroplast particles

The photoreactions mediated by Photosystem II at low temperatures were examined in subchloroplast particles enriched in Photosystem II to determine if the Photosystem II activity was independent of C-550 in such particle preparations. Two types of Photosystem II particle preparations were tested, one enriched in chlorophyll b and the other purified further to eliminate the chlorophyll b. The Photosystem II-mediated photoreactions at low temperature were the same in both of the Photosystem II particle preparations as they were in normal chloroplasts. C-550 acted as if it were the primary electron acceptor of Photosystem II in all of the experiments performed.     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

Excitation energy transfer in the light-harvesting chlorophyll

The “light-harvesting chlorophyll a/b · protein” described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600–700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitation dependence of the fluorescence polarization shows a minimum polarization of 1.9 % at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8 % at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a doublet structure in the chlorophyll b absorption band which suggests an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the S₀→S₁ transition moments of the chlorophyll molecules within the protein.     

Further data on the identity of a pigment absorbing around 640 nm in Ulva lactuca

A direct relationship exists between the amount of a pigment complex, absorbing around 640 nm, removed from Ulva lactuca chloroplast fragment preparations by mild acetone extraction, and the quantity of a pigment component, absorbing around 633 nm, occurring in the extract. It is concluded that both spectral components refer to one and the same pigment.

Evidence that this pigment is identical with chlorophyll a is presented. In view of earlier data, this result supports the suggestion that the reaction center pigment of Photosystem II consists of chlorophyll a.     

The relationship between Q, C-550 and cytochrome b 559 in photoreactions at −196° in chloroplasts

Absorbance changes and fluorescence yield changes induced by irradiating spinach chloroplasts with red light at −196° were measured as a function of the redox potential of the chloroplast suspension. Absorbance changes at 546 nm indicate the photoreduction of C-550 and changes at 556 nm indicate the photooxidation of cytochrome b 559. The changes of fluorescence yield indicate the photoreduction of Q, the fluorescence quencher of chlorophylla a in Photosystem II. The titration curves for all three changes were essentially the same and showed the same midpoint potential. In other experiments as well, it was found that when C-550 is in the reduced state the fluorescence yield of the chloroplasts is high and the low-temperature photooxidation of cytochrome b 559 is blocked. These data indicate that C-550 may be equivalent to Q and that cytochrome b 559 serves as the electron donor for the photoreduction of C-550 at low temperature.