Different roles of alpha- and beta-branch xanthophylls in photosystem assembly and photoprotection

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively alpha-branch (chy1chy2lut5) or beta-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of beta-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.     

Pigment–Protein Complexes and the Number of Reaction Centers of the Photosystems in Pea Chlorophyll Mutants

We studied fluorescent and absorption properties of the chloroplasts and pigment–protein complexes isolated by gel electrophoresis from the leaves of pea, the parent cultivar Torsdag and mutants chlorotica 2004 and 2014. Specific fluorescence peaks of chlorophyll forms in individual complexes have been determined from the absorption and fluorescence spectra of the chloroplast chlorophyll and their second derivatives at 23 and –196°C. The mutant chlorotica 2004 proved to have an increased intensity of a long-wave band of the light-harvesting complex I at both 23°C (745 nm) and –196°C (728 nm). At the same time, this mutant manifested a decreased accumulation of the chlorophyll forms making up the nearest-neighbor antenna of the PS I reaction center (at 690, 697, and 708 nm). No spectral differences have been revealed between chlorotica 2014 mutant and the parent cultivar. Gel electrophoresis revealed the synthesis of all chlorophyll–protein complexes in both mutants. At the same time, analysis of photochemical activity of PS I and PS II reaction centers and calculations of their number and the size of the light-harvesting antenna have shown that the number of reaction centers in the PS I of chlorotica 2004 mutant is reduced by a factor of 1.7 because its chlorophyll a–protein complex is disturbed by the mutation. The primary effect of chlorotica 2014 mutation remains unclear. The proportional changes in the content of photosystem complexes in this mutant suggest that they are secondary and result from a 50% decrease in chlorophyll content.     

Importance of trans-Δ3-hexadecenoic acid containing phosphatidylglycerol in the formation of the trimeric light-harvesting complex in Chlamydomonas

Possible roles of trans-Δ³-hexadecenoic acid containing phosphatidylglycerol (PG) in the organisation of photosynthetic complexes were studied using two mutants of Chlamydomonas reinhardtii, mf1 and mf2, that totally lack this lipid and in which the level of the others remaining PG was consequently reduced to about 30% of the wild-type. Both the mutants have lost the capacity to stabilise the light-harvesting chlorophyll a/b–protein complex LHC II in a trimeric state and display an increased instability of the PS I light-harvesting-core complex after detergent mediated solubilisation. In this paper, we show that a very reduced growth rate of the mutant cells largely reduces the extent of these defects, allowing a significant formation of trimeric LHC II and a stabilisation of the PS I complex, in the absence of synthesis of trans-Δ³-hexadecenoic acid or of increased level of PG. These results seem to be at variance with the generally accepted role of trans-Δ³-hexadecenoic fatty acid (16:1(3t)) in the formation of the PS II light-harvesting antenna. On the other hand, they appear to be consistent with the observation that trimeric LHC II can be formed in the presence of 16:1(3t)-lacking PG in a mutant of Arabidopsis thaliana and in chloroplasts from cotyledons of some Orchideae. We conclude that 16:1(3t)-PG is indeed required for the stabilisation of the trimeric LHC II and of the PS I complex under conditions of high biosynthesis rate, and that it is not essential when these components of the photosynthetic membrane are synthesised at low rates.     

Photosystem Damage and Spatial Architecture of Thylakoids in Chloroplasts of Pea Chlorophyll Mutants

The effect of chlorophyll–protein complexes on the ultrastructure of chloroplasts was studied in the leaves of pea, the parent cultivar Torsdag and mutants chlorotica 2004 and 2014. The mutants were shown to accumulate 80 and 55% of chlorophyll, relative to the control, while the composition of the synthesized photosystem complexes was the same as in the parent cultivar Torsdag. The size of the light-harvesting antenna was similar to the control in the 2014 mutant but considerably increased (by 30%) in the 2004 mutant. These changes were due to a proportional decrease in the number of all complexes (by 40–45%) in the 2014 mutant. At the same time, the number of reaction center complexes of photosystem I (PS I) decreased by 50% while that of photosystem II (PS II) remained virtually constant in the 2004 mutant. A proportional decrease in the number of the PS I and PS II complexes in the chlorotica 2014 mutant was accompanied by a partial reduction of the entire chloroplast membrane system against the background of normal development of both granal and intergranal sites of thylakoids. Conversely, the loss of PS I reaction centers led mainly to the reduction of the intergranal sites of thylakoids in chloroplasts. This effect is attributed to the prevalence of PS I complexes in the intergranal thylakoids.     

Photosystem reaction centers and structural organization of chloroplasts in chlorotica mutants of Pisum sativum L

Chlorophyll-protein complexes of photosystems (PS), as well as ultrastructural arrangement of chloroplasts in pea leaves of the primary cultivar Torsdag and mutants chlorotica 2004 and 2014 were studied. It was shown that both mutants accumulated 80 and 55% of chlorophyll, respectively, and were able to synthesize all of four types of photosystems complexes. The value of the light-harvesting antenna in mutant 2014 was close to the control one, and in mutant 2004 it increased significantly (by 30%). These changes were caused by a proportional decrease, 40-50%, of any complexes in mutant 2014, whereas the number of PS I reaction centers in mutant 2004 decreased by 50% and the reaction centers of PS II complexes were almost completely retained. It was established that the proportional decrease of PS I and PS II complexes in mutant chlorotica 2014 was followed by a partial reduction of the entire membrane system in chloroplasts, but with a good development of both granal and intergranal sites of thylakoids. On the contrary, the loss of complexes of PS I reaction centers in mutant chlorotica 2004 led to a reduction of unstacked sites of thylakoids in chloroplasts. It was concluded that the disturbance of the lateral orientation of the membrane system of chloroplasts is associated with the loss of complexes of reaction centers of PS I, which is predominantly localized in unstacked sites of thylakoids.     

Orientation of photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I₁₁₀, PS I₆₅, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I₆₅ (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F₇₃₅ and also as the Q_Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl-b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.     

Spectral and functional comparisons between the carotenoids of the two antenna complexes of Rhodopseudomonas capsulata

The spectral and functional properties of carotenoids associated with each of the two light-harvesting complexes of the Rhodopseudomonas capsulata photosynthetic antenna system have been distinguished by studying mutants lacking one or the other complex. In mutants containing only the light-harvesting I complex (LH-I), the absorption spectrum of the carotenoids is blue-shifted compared to wild type. Carotenoid absorption in mutants possessing only the light-harvesing II complex (LH-II) complex is red-shifted. The circular dichroism spectrum of carotenoids in each complex is also distinctive. Although carotenoids in each complex function with approximately the same efficiency in harvesting and transmitting light energy for photosynethesis, only the carotenoids associated with LH-II undergo an electrochromic bandshift upon generation of a transmembrane potential. These observations are interpreted to indicate that both the orientation of carotenoid molecules with respect to the plane of the membrane, and the immediate electrochemical environment of these molecules differ in the two light-harvesting complexes.     

Structural and functional organization of the peripheral light-harvesting system in Photosystem I

This review centers on the structural and functional organization of the light-harvesting system in the peripheral antenna of Photosystem I (LHC I) and its energy coupling to the Photosystem I (PS I) core antenna network in view of recently available structural models of the eukaryotic Photosystem I–LHC I complex, eukaryotic LHC II complexes and the cyanobacterial Photosystem I core. A structural model based on the 3D homology of Lhca4 with LHC II is used for analysis of the principles of pigment arrangement in the LHC I peripheral antenna, for prediction of the protein ligands for the pigments that are unique for LHC I and for estimates of the excitonic coupling in strongly interacting pigment dimers. The presence of chlorophyll clusters with strong pigment–pigment interactions is a structural feature of PS I, resulting in the characteristic red-shifted fluorescence. Analysis of the interactions between the PS I core antenna and the peripheral antenna leads to the suggestion that the specific function of the red pigments is likely to be determined by their localization with respect to the reaction center. In the PS I core antenna, the Chl clusters with a different magnitude of low energy shift contribute to better spectral overlap of Chls in the reaction center and the Chls of the antenna network, concentrate the excitation around the reaction center and participate in downhill enhancement of energy transfer from LHC II to the PS I core. Chlorophyll clusters forming terminal emitters in LHC I are likely to be involved in photoprotection against excess energy.     

Excitation energy transfer in thylakoid membranes from Chlamydomonas reinhardtii lacking chlorophyll b and with mutant Photosystem I

Energy trapping in Photosystem I (PS I) was studied by time-resolved fluorescence spectroscopy of PS II-deleted Chl b-minus thylakoid membranes isolated from site-directed mutants of Chlamydomonas reinhardtii with specific amino acid substitutions of a histidine ligand to P₇₀₀. In vivo the fluorescence of the PS I core antenna in mutant thylakoids with His-656 of PsaB replaced by asparagine, serine or phenylalanine is characterized by an increase in the lifetime of the fast decay component ascribed to the energy trapping in PS I (25 ps in wild type PS I with intact histidine-656, 50 ps in the mutant PS I with asparagine-656 and 70 ps in the mutant PS I with phenylalanine-656). Assuming that the excitation dynamics in the PS I antenna are trap-limited, the increase in the trapping time suggests a decrease in the primary charge separation rate. Western blot analysis showed that the mutants accumulate significantly less PS I than wild type. Spectroscopically, the mutations lead to a decrease in relative quantum yield of the trapping in the PS I core and increase in relative quantum yield of the fluorescence decay phase ascribed to uncoupled chlorophyll–protein complexes which suggests that improper assembly of PS I and LHC in the mutant thylakoids may result in energy uncoupling in PS I.     

Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas

Recent results obtained by electron microscopic and biochemical analyses of greening Chlamydomonas reinhardtii y1 suggest that localized expansion of the plastid envelope is involved in thylakoid biogenesis. Kinetic analyses of the assembly of light-harvesting complexes and development of photosynthetic function when degreened cells of the alga are exposed to light suggest that proteins integrate into membrane at the level of the envelope. Current information, therefore, supports the earlier conclussion that the chloroplast envelope is a major biogenic structure, from which thylakoid membranes emerge. Chloroplast development in Chlamydomonas provides unique opportunities to examine in detail the biogenesis of thylakoids.Abbreviations Rubisco ribulose bisphosphate carboxylase/oxygenase - CAB Chl a/b-binding (proteins) - Chlide chlorophyllide - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - Pchlide protochlorophyllide     

Photosystem stoichiometry and state transitions in a mutant of the cyanobacterium Synechococcus sp. PCC 7002 lacking phycocyanin

Phycobilisomes (PBS) function as light-harvesting antenna complexes in cyanobacteria, red algae and cyanelles. They are composed of two substructures: the core and peripheral rods. Interposon mutagenesis of the cpcBA genes of Synechococcus sp. PCC 7002 resulted in a strain (PR6008) lacking phycocyanin and thus the ability to form peripheral rods. Difference absorption spectroscopy of whole cells showed that intact PBS cores were assembled in vivo in the cpcBA mutant strain PR6008. Fluorescence induction measurements demonstrated that the PBS cores are able to deliver absorbed light energy to photosystem (PS) II, and fluorescence induction transients in the presence of DCMU showed that PR6008 cells could perform a state 2 to state 1 transition with similar kinetics to that of the wild-type cells. Thus, PBS core assembly, light-harvesting functions and energy transfer to PS I were not dependent upon the assembly of the peripheral rods. The ratio of PS II:PS I in the PR6008 cells was significantly increased, nearly twice that of the wild-type cells, possibly a result of long-term adaptation to compensate for the reduced antenna size of PS II. However, the ratio of PBS cores:chlorophyll remained unchanged. This result indicates that approximately half of the PS II reaction centers in the PR6008 cells had no closely associated PBS cores.     

Pigment protein complexes and the concept of the photosynthetic unit: Chlorophyll complexes and phycobilisomes

The photosynthetic unit includes the reaction centers (RC 1 and RC 2) and the light-harvesting complexes which contribute to evolution of one O₂ molecule. The light-harvesting complexes, that greatly expand the absorptance capacity of the reactions, have evolved along three principal lines. First, in green plants distinct chlorophyll (Chl) a/b-binding intrinsic membrane complexes are associated with RC 1 and RC 2. The Chl a/b-binding complexes may add about 200 additional chromophores to RC 2. Second, cyanobacteria and red algae have a significant type of antenna (with RC 2) in the form of phycobilisomes. A phycobilisome, depending on the size and phycobiliprotein composition adds from 700 to 2300 light-absorbing chromophores. Red algae also have a sizable Chl a-binding complex associated with RC 1, contributing an additional 70 chromophores. Third, in chromophytes a variety of carotenoid-Chl-complexes are found. Some are found associated with RC 1 where they may greatly enhance the absorptance capacity. Association of complexes with RC 2 has been more difficult to ascertain, but is also expected in chromophytes. The apoprotein framework of the complexes provides specific chromophore attachment sites, which assures a directional energy transfer whithin complexes and between complexes and reaction centers. The major Chl-binding antenna proteins generally have a size of 16–28 kDa, whether of chlorophytes, chromophytes, or rhodophytes. High sequence homology observed in two of three transmembrane regions, and in putative chlorophyll-binding residues, suggests that the complexes are related and probably did not evolve from widely divergent polyphyletic lines.Abbreviations APC allophycocyanin - B phycoerythrin-large bangiophycean phycoerythrin - Chl chlorophyll - L_CM linker polypeptide in phycobilisome to thylakoid - FCP fucoxanthin Chl a/c complex - LHC(s) Chl-binding light harvesting complex(s) - LHC I Chl-binding complex of Photosystem I - LHC II Chl-binding complex of Photosystem II - PC phycocyanin - PCP peridinin Chl-binding complex - P700 photochemically active Chl a of Photosystem I - PS I Photosystem I - PS II Photosystem II - RC 1 reaction center core of PS I - RC 2 reaction center core of PS II - R phycoerythrin-large rhodophycean phycoerythrin - sPCP soluble peridinin Chl-binding complex     

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b ₆/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.Abbreviations ENDOR electron nuclear double resonance - ESEEM electron spin echo envelope modulation - LHC light harvesting complex - PSI Photosystem I - PS II Photosystem II - P₆₈₀ primary electron donor in PS II - P₇₀₀ primary electron donor in PS I     

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.     

Organization of chlorophyll-protein complexes of Photosystem I in Chlamydomonas reinhardii

The polypeptide pattern, chlorophyll-protein complexes, fluorescence emission spectra and light intensity required for saturation of electron flow via Photosystem (PS) II and PS I in a pale-green photoautotrophic mutant, y-lp, were compared to those of the parent strain, Chlamydomonas reinhardii y-1 cells. The mutant exhibits a 686 nm fluorescence yield at 25°C and 77 K 2–6-fold higher than that of the parent strain cells, and is deficient in thylakoid polypeptides 14, 17.2, 18 and 22 according to the nomenclature of Chua (Chua, N.-H. (1980) Methods Enzymol. 60C, 434–446). All chlorophyll-protein complexes ascribed to PS II and the CP I complex were present in both type of cells. However, a chlorophyll-protein complex CP Ia containing — in the parent strain — the 66–68 kDa polypeptides of CP I and the four above-mentioned polypeptides, was absent in the mutant. It was previously reported that a chlorophyll-protein complex, CP O, obtained from C. reinhardii contains five polypeptides, namely, 14, 15, 17.2, 18 and 22 (Wollman, F.A. and Bennoun, P. (1982) Biochim. Biophys. Acta 680, 352–360). A CP O-like complex was present also in the mutant y-lp cells but it contains only one polypeptide, 15. Energy transfer from PS II to PS I was not impaired in the mutant, although a 4-fold higher light intensity was required for the saturation of PS I electron flow in the y-lp cells as compared with the parent strain. No difference was found in the light saturation curves for PS II activity between the mutant and parent strain cells. Based on these and additional data (Gershoni, J.M., Shochat, S., Malkin, S. and Ohad, I. (1982) Plant Physiol. 70, 637–644), it is concluded that the chlorophyll-protein complexes of PS I in Chlamydomonas comprise a reaction center-core antenna complex containing the 66–68 kDa polypeptides (CP I), a connecting antenna consisting of four polypeptides (14, 17.2, 18 and 22), and a light-harvesting antenna containing one polypeptide, 15. These appear to be organized as a complex, CP Ia. The interconnecting antenna is deficient in the y-lp mutant and thus the CP Ia complex is unstable and energy is not transferred from CP O to CP I. The effective cross-section of PS I antenna is thus reduced and a high fluorescence is emitted at 686 nm.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal ΔpH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1≈lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.     

Fluorescence,chlorophyll shape and number of photosystem reaction centers I and II in chlorotica mutants of Pisum sativum L

The fluorescent and absorbing properties of chloroplasts and pigment-protein complexes isolated by gel electrophoresis from pea leaves of the cultivar Torsdag and the mutants chlorotica 2004 and 2014 were studied. From the absorption and fluorescence spectra of chlorophylls and their 2nd derivatives, the range of their changes in the native state at 23 degrees C and specific maxima of fluorescence and the forms of chlorophyll of individual complexes at -196 degrees C were found. It was found that in mutant chlorotica 2004 the intensity of fluorescence of long-wave band at 745 nm (23 degrees C) and the maximum--at 728 nm (-196 degrees C) belonging to the light-harvesting complex I increased. Nevertheless, the accumulation of the chlorophyll forms in this mutant at 690, 697 and 708 nm, which make an antenna of reaction centers of photosystem (PS) I decreased. No spectral differences from the spectrum of the wild type were found in mutant chlorotica 2014, except for a weakening of interaction between the complexes of PS I and PS II. It was shown by gel electrophoresis that both mutants were capable of synthesizing any chlorophyll-protein complexes. However, the analysis of the photochemical activity of reaction centers of PS I and PS II as well as calculations of the value of the photosynthetic unit and the number of reaction centers of the photosystems enabled us to conclude that the quantity of the reaction centers of PS I in the mutant chlorotica 2004 was 1.7 times lower due to disturbance of mutations in biosynthesis or the formation of the chlorophyll a-protein complex of PS I. No primary effect of mutation of chlorotica 2014 was established. Proportional changes of all parameters in this mutant gave us the ground to consider them as secondary ones, which are caused by a decrease in chlorophyll content by half.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

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.