Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48℃ in the dark for 30 min or gradually elevated temperature from 30℃ to 80℃ at 0.5℃/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of Fo-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3℃ lower in the Chl b-less mutants (48℃) than in the wild type (51℃). At temperature up to about 45℃, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastruc-ture in the wild type showed no obvious alteration. After 55℃ exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chl _b-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a cer-tain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the in-ternal oxidative stress produced at severely high temperature.     

Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyllb-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48°C in the dark for 30 min or gradually elevated temperature from 30°C to 80°C at 0.5°C/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H₂O₂ accumulation. There were different patterns of F_o-temperature curves between the Chlb-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3°C lower in the Chlb-less mutants (48°C) than in the wild type (51°C). At temperature up to about 45°C, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chlb-less mutants, but chloroplast ultrastructure in the wild type showed no obvious alteration. After 55°C exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chlb-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a certain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chlb-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chlb-less mutants showed a tendency for higher thermosensitivity, and loss of Chlb in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the internal oxidative stress produced at severely high temperature.     

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.Abbreviations CP chlorophyll-protein - LHC the chlorophyll a-b binding light-harvesting complex - LHC-II subunit a the Lhcb4/5 gene product - subunit b the Lhcb1 gene product - subunit c Lhcb2 the gene product - subunit d the Lhcb3 gene product - subunit e the Lhcb6 gene product - PMSF phenylmethane sulphonyl fluoride - RC reaction center - Q_A the primary quinone electron acceptor of Photosystem-II - P700 the reaction center of PS I     

Photosynthetic responses of Oryza sativa L. seedlings to cadmium stress: physiological,biochemical and ultrastructural analyses

In the present study, photosynthetic responses induced by cadmium stress in chlorophyll biosynthesis, photochemical activities, the stability of thylakoid membranes chlorophyll-protein complexes and the chloroplast ultrastructure of the cereal crop Oryza sativa L. were characterized. Cadmium inhibited the biosynthesis of chlorophyll by interfering with activity of δ-aminolevulinic acid dehydratase in the rice seedlings. For the photochemical activities analyses, the extent of the decrease in photosystem II activity was much greater than that in the PS I activity. The variations in the chlorophyll a fluorescence parameters also indicated that cadmium toxicity drastically affected the photochemistry of PS II. Biochemical analyses by BN-PAGE and protein immunoblot showed that cadmium toxicity considerably affected the stability of PS II-core, cytb ₆ /f, RuBisCO, PSI + LHCI and LHCII (Trimeric). We observed the rate of the thylakoid membranes protein degradation, was mainly at the level of RbcL, PsaA, Lhca1 and D1. In addition, the damages to chloroplast structure and thylakoid stacking analyzed by transmission electron microscopy were indicative of general disarray in the photosynthetic functions exerted by cadmium toxicity. These results are valuable for understanding the biological consequences of heavy metals contamination particularly in soils devoted to organic agriculture.     

Chlorophyll-protein complexes of a Codium species,including a light-harvesting siphonaxanthin-Chlorophylla ab-protein complex,an evolutionary relic of some Chlorophyta

Eight chlorophyll-protein complexes were isolated from thylakoid membranes of a Codium species, a marine green alga, by mild SDS-polyacrylamide gel electrophoresis. CP 1a¹, CP 1a², CP 1a³ and CP 1a⁴ were partially dissociated Photosystem (PS) I complexes, which in addition to the core reaction centre complex, CP 1, possessed PS I light-harvesting complexes containing chlorophyll (Chl) a, Chl b and siphonaxanthin. LHCP¹ and LHCP³ are orange-brown green chlorophyll $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 0.66) that contain siphonaxanthin and its esterified form, siphonein. CP a and CP 1, the core reaction centre complexes of PS II and PS I, respectively, had similar spectral properties to those isolated from other algae or higher plants. These P-680- or P-700-Chl a-proteins are universally distributed among algae and terrestrial plants; they appear to be highly conserved and have undergone little evolutionary adaptation. Siphonaxanthin and siphonein which are present in the Codium light-harvesting complexes of PS II and PS I are responsible for enhanced absorption in the green region (518 and 538 nm). Efficient energy transfer from both xanthophylls and Chl b to only Chl a in Codium light-harvesting complexes, which have identical fluorescence emission spectra at 77 K to those of the lutein-Chl $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 1.2) of most green algae and all higher plants, proved that the molecular arrangement of these light-harvesting pigments was maintained in the isolated Codium complexes. The siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ allow enhanced absorption of blue-green and green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats. Since there is a variable distribution of lutein, siphonaxanthin and siphonein in marine green algae and siphonaxanthin is found in very ancient algae, these novel siphonein-siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ may be ancient light-harvesting complexes which were evolved in deep water algae.     

Effect of light quality on the composition and function of thylakoid membranes in atriplex triangularis

Light quality was shown to exert well-coordinated regulatory effects on the composition and function of the thylakoid membranes as well as on the photosynthetic rates of intact leaves from Atriplex triangularis grown in continuous blue, white and red lights (50 μE · m^?2 · s^?1). The higher photosynthetic rates in plants grown in blue light, as compared to those in white and red lights, resulted from marked changes in both light-harvesting complexes and electron carriers. The concentrations of electron carriers such as atrazine binding sites, plastoquinone, cytochromes b and f and P-700 on a chlorophyll basis were markedly increased in Atriplex grown in blue light; and the apparent light-harvesting antenna unit sizes of Photosystems I and II were greatly reduced. Consequently, the electron transport capacities of Photosystems I and II were also increased as was the coupling factor CF₁ activity. Atriplex grown in red light had lower photosynthetic rates than those grown in blue or white light by incorporating changes in the composition and function of the thylakoids in a direction opposite to those caused by growth in blue light. When these regulatory effects of light quality were compared with those of light quantity [6,7], it is clear that $\text{Chl}\text{a}\text{Chl}\text{b}$ ratios, electron transport capacities of Photosystems I and II, concentrations of plastoquinone, atrazine binding sites, coupling factor CF₁ activity and the apparent antenna unit size of Photosystem II are more affected by light quantity, whereas light quality has a greater influence on the concentration of P-700, the apparent antenna unit size of Photosystem I and the overall photosynthetic rates of intact leaves.     

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

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

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)     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C₃ plants. Agranal bundle sheath chloroplasts ($\text{Chl}\text{a}\text{Chl}\text{b}\text{> 5.0}$) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl $\text{a}\text{b}$ light-harvesting complex (LHC), and most of the cyt b₅₅₉. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl $\text{a}\text{b}$ LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P₇₀₀ are closely coordinated. Thus, it is likely that both cyt f and P₇₀₀ are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II_αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II_β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

Thf1 interacts with PS I and stabilizes the PS I complex in Synechococcus sp. PCC7942

Thylakoid formation1 protein (Thf1) is a multifunctional protein that is conserved in all photosynthetic organisms. In this study, we used the model cyanobacterium Synechococcus sp. PCC7942 (hereafter Synechococcus) to show that the level of Thf1 is altered in response to various stress conditions. Although this protein has been reported to be involved in thylakoid formation, the thylakoid membrane in the thf1 deletion strain (ΔThf1) was not affected. Compared with the WT, ΔThf1 showed reduced PS II activity, with increased levels of D1 under high light (HL) conditions, which was resulted from blocked D1 degradation by the FtsH protease and thus inhibits PS II repair. PS I was found to be more seriously affected than PS II in ΔThf1, even under low light conditions, suggesting that PS I damage could be the primary effect of thf1 deletion in Synechococcus. Further analysis revealed that the ΔThf1 mutant had a lower PS I subunit content and lower PS I stability under HL conditions. Further sucrose gradient fractionation of the membrane protein complexes and crosslinking and immunoblot analysis indicated that Thf1 interacts with PS I. Together, our results reveal that Thf1 interacts with PS I and thereby stabilizes PS I in Synechococcus.     

Properties of thylakoids and thylakoid particles derived from structurally different chloroplasts

Structurally and functionally different tobacco chloroplasts were subjected to digitonin treatment and subsequent fractional centrifugation. The light-harvesting $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b-}\text{protein}$ complex was found to be enriched in the most dense fraction regardless of the presence of grana in the original preparation. It is suggested that isolated thylakoid membranes and fragments thereof which contain sufficient light-harvesting protein may, under appropriate ionic conditions, form aggregates even when they originate from unstacked thylakoid systems. Comparative studies of fluorescence properties and polypeptide composition of the thylakoids suggest that the light-harvesting protein does not contribute significantly to the fluorescence spectrum of isolated chloroplasts as long as this protein is intimately associated with the Photosystem II (PS II) pigment-protein complex responsible for the 685 nm emission. While the PS II-deficient mutant chloroplasts of the variegated tobacco variety NC 95 lacked both the 685 nm fluorescence component and two or three PS II proteins, one of these proteins was found to be very prominent in our chlorophyll b-deficient mutant thylakoids which also displayed an intense 685 nm fluorescence peak. This correlation supports the contention that a 45 kdalton polypeptide is an apoprotein of pigments associated with the PS II reaction center.     

IMMUNOLOGICAL AND ULTRASTRUCTURAL CHARACTERIZATION OF THE PHOTOSYNTHETIC COMPLEXES OF THE PROCHLOROPHYTE PROCHLOROCOCCUS (OXYCHLOROBACTERIA)

Ultrastructural features and immunological properties of some thylakoid proteins were examined in two strains of the prochlorophyte Prochlorococcus and compared to those of other photosynthetic prokaryotes and eukaryotes. Both strains exhibited two or three rows of tightly appressed thylakoidal membranes, located at the cell periphery. However, thylakoids were concentrically arranged in the strain from the Sargasso Sea (SARG) and horseshoe-shaped in the Mediterranean isolate (CCMP 1378). Although lacking phycobilisomes, both cell types shared with cyanobacteria the presence of carboxysome-like structures and glycogen granules as storage compounds. The main thylakoid polypeptides separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis were characterized by Western blotting using several antibodies. The 30-kDa polypeptide of the light-harvesting complex (LHC) of Prochlorococcus showed a weak positive immunological cross-reaction with an antibody raised against the 32-kDa apoprotein of the LHC of the prochlorophyte Prochlorothrix hollandica. In contrast, it showed no immunological relationships with the chlorophyll a/b (Chl a/b) LHCs of green algae and higher plants. Protein membranes from Prochlorococcus strongly cross-reacted with antibodies raised against reaction center polypeptides of photosystems II and I (PSs II and I) of other photosynthetic organisms, confirming the high degree of conservation of these basic compounds of the photosynthetic machinery during evolution. Immunolocalization of thylakoid proteins showed that the LHC proteins, the major PS II reaction center proteins (CP 43 and D2), and the PS I reaction center proteins were equally distributed within the thylakoid membranes in contrast to the segregation observed in higher plants and green alga thylakoids. We also identified ribulose-1, 5-bisphosphate carboxylase/oxygenase in the carboxysomes. These results suggest that Prochlorococcus is more closely related to cyanobacteria than to green plastids even though it contains Chl b.     

Regulation of the Stoichiometry of Thylakoid Components in the Photosynthetic System of Cyanophytes: Model Experiments Showing that Control of the Synthesis or Supply of Chl a can Change the Stoichiometric Relationship between the Two Photosystems

Regulation of the assembly of the photosystem I (PS I) complexin response to the light regime in the photosynthetic systemof cyanophytes was studied in Synechocystis PCC 6714. The relationshipbetween the assembly of the PS I complex and synthesis of Chla was examined by model experiments in which synthesis of Chla was controlled by two inhibitors, gabaculine (GAB) and 2,2'-dipyridyl(DP). Both inhibitors caused a change to a lower ratio of PSI to PS II even under light that normally induces a high ratioof PS I to PS II. The change in stoichiometry induced by theseinhibitors was suppressed when protein synthesis was inhibitedby chloram-phenicol, similarly to the change in the stoichiometryinduced by light that excites mainly PS I (PS I light). Comparisonof the levels of PS I, PS II and Cyt b₆-f complexes per cellindicated that a selective suppression of the assembly of thePS I complex was induced by the inhibitors: the stoichiometricrelationship among PS I, PS II and Cyt b₆-f complexes was identicalto that induced by PS I light or white light of high intensity.GAB induced a decrease in size of the phycobilisome also, whileDP did not, similarly to PS I light. The results indicate thatthe ratio of PS I to PS II can be changed by the control ofsynthesis of Chl a. They also suggest that control of the synthesisor supply of Chl a probably exerted at site(s) in or after theprocess of the Mg-protoporphyrin branch, is involved in themechanism of regulation of the assembly of the PS I complexin cyanophytes. (Received September 7, 1989; Accepted November 20, 1989)     

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.     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II     

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     

Fluorescence emission spectra and thylakoid protein kinase activities of three higher plant mutants deficient in chlorophyll b

The properties of three higher plant mutants having less than normal amounts of chlorophyll b were compared with their respective wild-types. These mutants included the chlorophyll-b-lacking U374 sweet clover (Melilotus alba) and chlorina-f2 barley (Hordeum vulgare) as well as the chlorophyll b-deficient CD3 wheat (Triticum aestivum). Fluorescence emission spectra from leaves of the sweet-clover mutant at 77 K show great similarity to the previously published spectrum of the barley mutant; rather than the predominant long-wavelength emission at approx. 740 nm in the wild-type plants, an emission maximum at approx. 720 nm is observed. The wheat mutant, containing reduced but measurable amounts of chlorophyl b, had 77 K long-wavelength fluorescence emissions at both 720 and 740 nm. These data indicate that these PS-I-derived fluorescence emissions are strongly influenced by the presence of antennae components. When examined for the ability to perform a light-induced State 1-State 2 transition in vivo, none was detected in the U374 sweet clover, whereas the CD3 wheat was capable of this process. The phosphorylation of endogenous polypeptides in isolated thylakoid membranes was examined using [γ-³²P]ATP as substrate for the thylakoid protein kinase activities. All three mutants had higher thylakoid protein kinase activity than the respective normal plants on a chlorophyll basis. The response of the mutant and normal sweet clover thylakoid protein kinase activities to ATP concentration was essentially identical. In contrast, the thylakoid protein kinase activities in the barley and wheat mutants appeared to saturate at markedly lower ATP concentrations than in the respective normal plants. These data suggest that the chlorina-f2 and CD3 mutants may be lacking one of the thylakoid protein kinases normally present in wild-type plants and that mutants lacking chlorophyll b may be of at least two different types.     

LHC II protein phosphorylation in leaves of Arabidopsis thaliana mutants deficient in non-photochemical quenching

Phosphorylation of the light-harvesting chlorophyll a/b complex II (LHC II) proteins is induced in light via activation of the LHC II kinase by reduction of cytochrome b₆f complex in thylakoid membranes. We have recently shown that, besides this activation, the LHC II kinase can be regulated in vitro by a thioredoxin-like component, and H₂O₂ that inserts an inhibitory loop in the regulation of LHC II protein phosphorylation in the chloroplast. In order to disclose the complex network for LHC II protein phosphorylation in vivo, we studied phosphorylation of LHC II proteins in the leaves of npq1-2 and npq4-1 mutants of Arabidopis thaliana. In comparison to wild-type, these mutants showed reduced non-photochemical quenching and increased excitation pressure of Photosystem II (PS II) under physiological light intensities. Peculiar regulation of LHC II protein phosphorylation was observed in mutant leaves under illumination. The npq4-1 mutant was able to maintain a high amount of phosphorylated LHC II proteins in thylakoid membranes at light intensities that induced inhibition of phosphorylation in wild-type leaves. Light intensity-dependent changes in the level of LHC II protein phosphorylation were smaller in the npq1-2 mutant compared to the wild-type. No significant differences in leaf thickness, dry weight, chlorophyll content, or the amount of LHC II proteins were observed between the two mutant and wild-type lines. We propose that the reduced capacity of the mutant lines to dissipate excess excitation energy induces changes in the production of reactive oxygen species in chloroplasts, which consequently affects the regulation of LHC II protein phosphorylation.