Organization of the photosynthetic apparatus of the chlorina-f2 mutant of barley using chlorophyll fluorescence decay kinetics

The time-resolved chlorophyll fluorescence emission of higher plant chloroplasts monitors the primary processes of photosynthesis and reflects photosynthetic membrane organization. In the present study we compare measurements of the chlorophyll fluorescence decay kinetics of the chlorophyll-b-less chlorina-f2 barley mutant and wild-type barley to investigate the effect of alterations in thylakoid membrane composition on chlorophyll fluorescence. Our analysis characterizes the fluorescence decay of chlorina-f2 barley chloroplasts by three exponential components with lifetimes of approx. 100 ps, 400 ps and 2 ns. The majority of the chlorophyll fluorescence originates in the two faster decay components. Although photo-induced and cation-induced effects on fluorescence yields are evident, the fluorescence lifetimes are independent of the state of the Photosystem-II reaction centers and the degree of grana stacking. Wild-type barley chloroplasts also exhibit three kinetic fluorescence components, but they are distinguished from those of the chlorina-f2 chloroplasts by a slow decay component which displays cation- and photo-induced yield and lifetime changes. A comparison is presented of the kinetic analysis of the chlorina-f2 barley fluorescence to the decay kinetics previously measured for intermittent-light-grown peas (Karukstis, K. and Sauer, K. (1983) Biochim. Biophys. Acta 725, 384–393). We propose that similarities in the fluorescence decay kinetics of both species are a consequence of analogous rearrangements of the thylakoid membrane organization due to the deficiencies present in the light-harvesting chlorophyll $\text{a}\text{b}$ complex.     

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.     

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

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

Linear dichroism of microalgae,developing thylakoids and isolated pigment-protein complexes in stretched poly (vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields.Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b (648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl $\text{a}\text{b}$ ratio of approx. 6 and the LD spectrum was positive with a maximum at 690–694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack Chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.     

Pigment protein complexes and functional properties of tetratype resulting from crosses between CP1 and CP2 less Chlamydomonas mutants

A chlorophyll b-less mutant of Chlamydomonas reinhardtii (Pg ₂₇) was isolated after UV irradiation of the wild type cells. This photosynthetically competent mutant totally lacks chlorophyll b and the CP₂ chlorophyll-protein complex. However, SDS-PAGE, proteolytic digestions and immunodetections demonstrated that the 24–25 Kd apoproteins of the lacking CP₂ complex are still present in thylakoids of the Pg₂₇ mutant. It is concluded that this CP₂-less mutant is affected in the biosynthesis pathway of chlorophyll b.This CP₂-less mutant was crossed with a CP₁-less mutant (Fl₅) Fluorescence emission spectra and fluorescence inductions in the presence of DCMU were analysed in the resulting (cp ₂ ^– , cp ₁ ⁺ ), (cp ₂ ⁺ , cp ₁ ^– ), (cp ₂ ⁺ , cp ₁ ⁺ ), cp ₂ ^– , cp ₁ ^– )tetratype. Differences in PS 2 optical cross section and in the relative amplitude or localisation of fluorescence emission peaks fit well with a quadripartite model where PS1 and PS2 would each correspond to a reaction centre core complex (CP₁ and CP₂ respectively) associated to a light harvesting antenna (LHC1 and LHC2 respectively). The occurrence of energy transfers from PS1 peripheral antenna to PS2 in the Fl ₅ mutant shows that, in absence of CP₁, at least a part of its associated PS1 light harvesting antenna migrates in the PS2 containing appressed thylakoids.Abbreviations Chl Chlorophyll - LHC Light harvesting chl a/b complex - CP₂ Predominant form of LHC or SDS polyacrylamide gels - WT Wild type - DM Double mutant (cp ₁ ^– , cp ₂ ^– ) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - DOC-PAGE Deoxycholate polyacrylamide gel electrophoresis     

Evolutionary Changes in Chlorophyllide a Oxygenase (CAO) Structure Contribute to the Acquisition of a New Light-harvesting Complex in Micromonas

Chlorophyll b is found in photosynthetic prokaryotes and primary and secondary endosymbionts, although their light-harvesting systems are quite different. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which is a Rieske-mononuclear iron oxygenase. Comparison of the amino acid sequences of CAO among photosynthetic organisms elucidated changes in the domain structures of CAO during evolution. However, the evolutionary relationship between the light-harvesting system and the domain structure of CAO remains unclear. To elucidate this relationship, we investigated the CAO structure and the pigment composition of chlorophyll-protein complexes in the prasinophyte Micromonas. The Micromonas CAO is composed of two genes, MpCAO1 and MpCAO2, that possess Rieske and mononuclear iron-binding motifs, respectively. Only when both genes were introduced into the chlorophyll b-less Arabidopsis mutant (ch1-1) was chlorophyll b accumulated, indicating that cooperation between the two subunits is required to synthesize chlorophyll b. Although Micromonas has a characteristic light-harvesting system in which chlorophyll b is incorporated into the core antennas of reaction centers, chlorophyll b was also incorporated into the core antennas of reaction centers of the Arabidopsis transformants that contained the two Micromonas CAO proteins. Based on these results, we discuss the evolutionary relationship between the structures of CAO and light-harvesting systems.     

Organization and Stability of Polypeptides Associated with the Chlorophyll a-b Light-Harvesting Complex of Photosystem-II

In the oxygen-evolving photosystem-II (PSII) of higher plantchioroplasts and green algae, most of the light-harvesting functionis performed by the chlorophyll (Chl) a-b-protein complex (LHC-II).On the average, the LHC-II contains about 210 Chl (a+b) moleculesper PSII reaction center. The polypeptide composition, copynumber and organization of assembly in the LHC-II complex arenot fully understood at present. This work utilized the chlorinaf2 mutant of barley (lacking Chl b and having a LHC-II antennaof only 13 Chl a molecules) to determine the organization andstability of assembly of proteins in the LHC-II. High-resolutionSDS-PAGE and immunoblot analysis showed the presence of fourmain constitutive polypeptides in the wild-type LHC-II (termedhere subunits a, b, c and d) with molecular masses in the range30–25 kDa. Of those, only subunit d (a 25 kDa polypeptide)was found to occur at an equal copy number per PSII reactioncenter in both wild-type and in the Chl b-less chlorina f2 mutant.All other subunits were either absent or existed in much loweramounts in the mutant. Subunit d is a polypeptide constituentof the major Chl-protein subcomplex (CPII) of the LHC-II. Itis stably incorporated in the thylakoid membrane in the absenceof Chl b and probably binds the 13 Chl a molecules in the residualLHC-II antenna of the chlorina f2 mutant. We propose that, ofall LHC-II polypeptides, subunit d is most proximal to the PSIIcore and may serve as a linker in the process of excitationenergy transfer from the bulk LHC-II to the PSII reaction centerin chloroplasts. (Received February 25, 1992; Accepted May 12, 1992)     

Spectral features and structure of chloroplasts under an early block of chlorophyll synthesis

A xantha mutant of cotton (Gossypium hirsutum L.) with blocked synthesis of 5-aminolevulinic acid in light accumulates 30 times less chlorophyll than the parental strain. Formation of the chloroplast membrane system in the mutant stops at very early stages, mostly vesicles and single short thylakoids. The mutant plastid membranes contain only light-harvesting chlorophyll-a/b-protein complexes I and II with fluorescence maxima at 728 and 681 nm, respectively. Thus, an early block of chlorophyll synthesis impairs the formation and function of photosystem reaction centers and retards the development of the chloroplast membrane system at the stage of proplastids.     

Distribution of light excitation in an intact leaf between the two photosystems of photosynthesis. Changes in absorption cross-sections following state 1-state 2 transitions

Using the photoacoustic technique, state 1-state 2 transitions were studied in an intact leaf by direct monitoring of modulated oxygen evolution, excited by modulated light. States 1 and 2 were characterized by the extent of immediate enhancement of the modulated oxygen evolution — ‘Emerson enhancement’ — and the concomitant fluorescence quenching, resulting from the addition of continuous far-red light (greater than 700 nm), absorbed primarily in Photosystem I (light 1). The extent of Emerson enhancement as well as the saturation curve of this effect by far-red light are very sensitive and quantitative indicators for the ratio of light excitation distributed between Photosystems I and II. The enhancement ratios at 650 nm light, a typical light 2, were in a range 1.4–1.8 in state 1, while values as low as 1.06 were observed in state 2. During the transition from state 2 to state 1, monitored in presence of modulated light 2 and background continuous light 1, the modulated oxygen yield increased considerably, indicating a major increase in excitation flux into Photosystem II. Conversely, with modulated light 2 alone in state 1, the modulated oxygen evolution yield was smaller than in state 2, indicating a decrease of the excitation flux in Photosystem I. In a typical example, of the transition to state 1, the fraction of light absorbed by Photosystem II, β, increased from 0.46 to 0.64, while that absorbed by Photosystem II, α, decreased from 0.43 to 0.36. State 1-state 2 transitions, thus, reflect reciprocal changes in the cross-sections of the two photosystems for light absorption. There is no evidence for the operation of a ‘spill-over’ mechanism. The enhancement effect displayed maxima at 480 and 650 nm, related to chlorophyll-b absorption, as well as another band at 500–550 nm. In a chlorophyll-b-less barley mutant, state 1-state 2 transitions, as monitored by modulated oxygen evolution, were absent, and the resulting enhancement corresponded to state 2. These observations are consistent with the model that the light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ complex plays a role in regulating the distribution of light to the photosystems. It is probable that this complex migrates reversibly in the thylakoid membrane in such a way that it is mainly associated with Photosystem II in state 1, but is more evenly distributed in the two photosystems in state 2.     

Physiological adaptation to a newly observed low light intensity state in intact leaves,resulting in extreme imbalance in excitation energy distribution between the two photosystems

In intact leaves, a new physiological state is obtained reversibly at low light intensity (typically 1 W / m²), in which oxygen evolution yield, monitored by the photoacoustic method, approaches zero. In this ‘low-light’ state, irradiation with far-red (λ > 700 nm) background light immediately restores the normal oxygen yield, resulting in an unusually high Emerson enhancement ratio. Quantitative analysis of the enhancement ratio and the saturation curve of enhancement by far-red light shows that in the new state, short wavelength excitation does not reach PS I reaction centers, resulting in an extreme imbalance between the two photosystems. We suggest that adaptation to the low-light state occurs through loss of excitonic interaction between antennae of PS I and their reaction-centers. It appears also that the ‘far-red’ absorbing pigments do not participate in the disconnection and remain closely attached to the reaction centers of PS I. Their number is estimated to be less than 30 per reaction center. The disconnection of the antennae from the reaction center appears to be reversed by readaptation to ‘normal’ light levels, as well as by a brief preillumination with broad band (400–600 nm) light, acting as a trigger. In the last case, the transition to high oxygen yield state is transient. The quantum requirement of this recovery process is very small (approx. 10 hv / reaction center). The adaptation times after switching from higher to lower intensities and vice versa are in the range of minutes. The fluorescence yield remains virtually constant during adaptation to the low-light state in contrast to expectations, suggesting the possibility of cyclic electron flow around PS II in this state. In a chlorophyll-b-less barley mutant, which lacks the light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ protein (LHC) (and possibly the newly discovered light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ protein associated with PS I (LHC-I)), the ‘low-light’ state was absent. These results are consistent with the hypothesis that these antennae complexes participate directly in the adaptation to low light intensities.     

Further Chemical and Morphological Characterization of Chloroplast Membranes from a Chlorophyll b-less Mutant of Hordeum vulgare

A comparative study of peptide composition and freeze-fracture morphology of chloroplast membranes from a chlorophyll b-less mutant and a normal barley plant (Hordeum vulgare L.) is reported in this work. Using a high resolution, discontinuous sodium dodecyl sulfate—acrylamide gel electrophoretic system, we show that the mutant chloroplast membranes not only completely lack the 25-kilodalton peak, which accounts for about 18% of the chloroplast membrane protein in the normal plant, but also exhibit gross reduction in other components at 27.5- and 20-kilodalton regions. Despite such extensive deletions in the peptide composition of the mutant chloroplast lamellae, no alteration could be detected in either density or size of the intramembranous particles, visualized by freeze-fracturing.     

Vibrational mode frequency calculations of chlorophyll-<Emphasis Type="Italic">d</Emphasis> for assessing (P740<Superscript>+</Superscript>-P740) FTIR difference spectra obtained using photosystem I particles from <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Acaryochloris marina is an oxygen-evolving organism that utilizes chlorophyll-d for light induced photochemistry. In photosystem I particles from Acaryochloris marina, the primary electron donor is called P740, and it is thought that P740 consist of two chlorophyll-d molecules. (P740⁺-P740) FTIR difference spectra have been produced, and vibrational features that are specific to chlorophyll-d (and not chlorophyll-a) were observed, supporting the idea that P740 consists chlorophyll-d molecules. Although bands in the (P740⁺-P740) FTIR difference spectra were assigned specifically to chlorophyll-d, how these bands shifted, and how their intensities changed, upon cation formation was never considered. Without this information it is difficult to draw unambiguous conclusions from the FTIR difference spectra. To gain a more detailed understanding of cation induced shifting of bands associated with vibrational modes of P740 we have used density functional theory to calculate the vibrational properties of a chlorophyll-d model in the neutral, cation and anion states. These calculations are shown to be of considerable use in interpreting bands in (P740⁺-P740) FTIR difference spectra. Our calculations predict that the 3¹ formyl C–H mode of chlorophyll-d upshifts/downshifts upon cation/anion formation, respectively. The mode intensity also decreases/increases upon cation/anion formation, respectively. The cation induced bandshift of the 3¹ formyl C–H mode of chlorophyll-d is also strongly dependant on the dielectric environment of the chlorophyll-d molecules. With this new knowledge we reassess the interpretation of bands that were assigned to 3¹ formyl C–H modes of chlorophyll-d in (P740⁺-P740) FTIR difference spectra. Considering our calculations in combination with (P740⁺-P740) FTIR DS we find that the most likely conclusions are that P740 is a dimeric Chl-d species, in an environment of low effective dielectric constant (∼2–8). In the P740⁺ state, charge is asymmetrically distributed over the two Chl-d pigments in a roughly 60:40 ratio. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Influence of the colonizing substrate on diatom assemblages and implications for bioassessment: a mesocosm experiment

Although diatoms are important bioindicators of ecological quality, their ecological traits are still not well understood. A major issue is that of substrate preferences, which may result in differences in production, and assemblage structure and composition, and which should therefore be taken into account for ecological quality assessment studies. Thus, in this work, the periphyton grown on sand and ceramic tiles in indoor controlled channels were compared to understand whether substrate differences lead to differences in: periphyton production (chlorophyll-a), chlorophyll-b and c concentrations, diatom assemblages (diversity-Shannon-Wiener, cell density, taxonomic composition, trait proportions), and ecological quality assessments (IPS-‘Indice de Polluosensibilité Spécifique’). A combined inoculum of periphyton from four Portuguese streams was introduced to the running channels (six sand and six tile) and left to colonize for 35 days. Epilithic (tiles) and epipsammic (sand) assemblages were sampled at days 14 and 35. We verified that there were no differences in chlorophyll-a concentration over time and between substrates. On both sampling occasions, the epipsammic assemblages had higher concentration of chlorophyll-c and diatom density but without significant differences over time in each substrate. The taxonomic composition was different between substrates and over time. However, these differences were not reflected in ecological quality assessment. The diversity was also similar between substrates in both sampling occasions, but it was higher at day 14. Mobile and stalked species were more abundant over the entire study and differed significantly between substrates, with the epipsammic assemblages presenting higher abundances of both traits. We concluded that the colonizing substrate influences diatom assemblages but not the ecological quality assessment.     

Photosynthetic characteristics and antioxidant enzyme system in high-chlorophyll rice Gc mutant

The physiological photosynthetic characteristics and antioxidant enzyme system of the high-chlorophyll rice (Oryza sativa L.) mutant (Gc) and its wild type (Zhenshan 97B) were compared and analyzed. Resulting data showed that the total chlorophyll (Chl) and Chl b contents in the Gc mutant were significantly increased by 19.0 and 81.7%, respectively, while the increase in Chl a and thylakoid membrane protein contents was insignificant. The net photosynthetic rate (P _N) was significantly higher in the mutant; stomatal conductance, intercellular CO₂ concentration, and transpiration rate decreased significantly, and water-use efficiency increased significantly, indicating the higher photochemical efficiency of the mutant. The chlorophyll fluorescence parameters: electron transport rate and effective quantum yield of PSII photochemistry of the mutant were significantly higher than those of Zhenshan 97B. The nonphotochemical quenching of the mutant under light adaptation increased by 52.3%. The enzymatic activity of superoxide dismutase, peroxidas, and catalase in the mutant roots and leaves were all higher than those for the wild-type plants. It is believed that the higher activity of antioxidant enzymes in the mutant may be an important factor making difficult the photo-inactivation of Chl, and thus, increasing the content of Chl, especially Chl b.     

Reduction of the chloroplast membrane system caused by disorders in early stages of chlorophyll biosynthesis

A chlorophyll-deficient xantha mutant of cotton (Gossypium hirsutum L.) was examined with respect to development and structural organization of the chloroplast membrane system as affected by disruption of early stages of chlorophyll biosynthesis in the light. The analysis of early chlorophyll precursors showed that the mutant is unable to synthesize 5-aminolevulinic acid (5-ALA) in the light. The disorders in early stages of chlorophyll biosynthesis arrested the development of chloroplast membrane system at the stage of vesicles and single thylakoids. The accumulation of 2–5% chlorophyll in the mutant was related to the formation of light-harvesting chlorophyll-a/b-protein complexes I and II, whereas pigment-protein complexes composing reaction centers of photosystem I and photosystem II were lacking. It is concluded that the chloroplast membrane system in the mutant with impaired 5-ALA synthesis is incapable of development and is even reduced upon long-term growing under light.     

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.     

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.     

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.     

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll <Emphasis Type="Italic">b</Emphasis>-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 μmol m⁻² s⁻¹). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (F_v/F_m), quantum efficiency of PS2 electron transport (Φ_PS2), photochemical quenching (q_P), as well as rate of photochemistry (P_rate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (D_rate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–q_P (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 μmol m⁻² s⁻¹) and high, HI (700 μmol m⁻² s⁻¹) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.     

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.