Development of the photosynthetic apparatus during light-dependent greening of a mutant of Chlorella fusca

The formation of chlorophyll, cytochrome f, P-700, ribulose bisphosphate carboxylase as well as photosynthesis and Hill reaction activities were tested during the light-dependent greening process of the Chlorella fusca mutant G 10. Neither chlorophyll nor protochlorophyllide was detected in the darkgrown cells. When transferred to light the mutant cells developed chlorophyll and established its photosynthetic capacity after a short lag phase. In the in vivo absorption spectra a spectral shift of the red absorption peak position from 674 to 680 nm was indicated during the first 3 h of greening. Cytochrome f was already present in the dark-grown cells, but during the greening phase a threefold increase in the cytochrome f content could be seen. At the early stages of greening a characteristic primary oscillation in the content of cytochrome f was observed. P-700 was lacking in the dark and during the first 30 min of illumination. From the first to the second h of light a forced synthesis of P-700 took place and the time-course curve for the ratios of P-700/chlorophyll rose to a sharp maximum. The synthesis of P-700 started together with photosystem I activity and showed similar kinetics. We found the simultaneous appearance of photosystem II, photosystem I, and photosynthetic activities 30 min after the beginning of the illumination. Based on chlorophyll content they attained maximum activity after 2 h of light, but at this time photosystem I capacity proved to be remarkably higher than photosynthetic and photosystem II activities. Highest carboxylase activity existed in darkgrown cells. During the greening process the activity of the enzyme decreased continuously. After 2 h of illumination chlorophyll synthesis partially served to increase the size of the photosynthetic unit, which consequently led to a decrease in the light energy needed to saturate photosynthesis and also to a decrease of photosynthetic rate based on chlorophyll content.Abbreviations Chl chlorophyll - Cyt f cytochrome f - DPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - GSH glutathione - LH light-harvesting - PS photosystem - RuBP ribulose bisphosphate     

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ～1.9, ～1.5, and 1.1–1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments — carotenoids, Chl b, and phycobilins — were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.     

建兰叶艺品种光合色素含量及叶绿素荧光特性分析

以建兰栽培品种‘八宝奇珍’Cymbidium ensifolium ‘Ba Bao Qi Zhen’为材料,对其正常绿色叶片及黄化变异叶片的光合色素含量、叶绿素荧光参数进行比较。结果表明,‘八宝奇珍’黄化变异叶片的叶绿素总量、叶绿素a 和叶绿素b 含量均显著低于正常绿色叶片,且随着黄化面积的增大呈现递减趋势;黄化变异叶片的初始荧光量(Fo)、最大荧光产量(Fm)、Kautsky 诱导效应最大荧光(Fp)、稳态光适应光化学淬灭系数(qP)以及光适应稳态荧光产量(Ft_Lss)均显著低于正常绿色叶片;PSⅡ原初光能转化效率(Fv/Fm)与正常绿色叶片无明显差异;稳态非光化学荧光淬灭系数(NPQ)高于正常绿色叶片。     

Greening under High Light or Cold Temperature Affects the Level of Xanthophyll-Cycle Pigments, Early Light-Inducible Proteins, and Light-Harvesting Polypeptides in Wild-Type Barley and the Chlorina f2 Mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5°C or 20°C and continuous illumination varying from 50 to 800 μmol m⁻² s⁻¹. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20°C and 250 μmol m⁻² s⁻¹. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5°C or 20°C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5°C or 20°C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : II. Plastid Differentiation during Greening of a Dark-Grown Algal Mutant (Chlamydomonas reinhardi)

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6–8 hr in the absence of cell division. Photosynthetic activity (O₂ evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.     

Photothermal Spectra of Thylakoids Isolated from Cucumber Cotyledons at Various Stages of Greening

The character of interaction between carotenoids (Cars) and chlorophylls (Chls) in thylakoids isolated from cucumber cotyledons at three stages of greening (3, 6, and 24 h of irradiation with 120 µmol m^–2 s^–1) was studied. The shapes of the steady state photoacoustic spectra were changed with the change in time of greening and with the frequency of radiation modulation. The shapes show that changes not only in the contents of various pigments but also in pigment interactions with surrounding occur and that processes of thermal deactivation characterised by different kinetics take place. Slow processes of thermal deactivation are in most cases due to deactivation of triplet states. Long living triplet states are very often engaged in photochemical reactions that can destroy the tissue. Analysis of the time-resolved photothermal spectra shows that at later stage of greening, the chlorophyll (Chl) molecules are better shielded against photo-destruction because Cars more efficiently quench their triplet states. The yield of formation of the pigment triplet states measured by the time resolved photothermal method, always at the same energy absorbed by pigment mixture, declined during sample greening. The decay time of the slow component of pigment thermal deactivation, due predominantly to deactivation of the triplet state of Chl, decreases with the increase of time of greening from 6.2 µs for the 3-h sample to 1.5 µs for the 24 h sample. The energy taken by Cars from Chls is dissipated into heat, therefore the steady state and quick thermal deactivation values increased during the greening process. The Cars/Chls ratio in the thylakoids decreased during greening approximately 2 fold. Hence at a later phase of greening the Cars can quench the triplet states of Chls more efficiently than at an earlier phase of greening.     

Triplet-triplet energy transfer in Peridinin-Chlorophyll a-protein reconstituted with Chl a and Chl d as revealed by optically detected magnetic resonance and pulse EPR: Comparison with the native PCP complex from Amphidinium carterae

The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at ∼ 620 and ∼ 640 nm in the Triplet-minus-Singlet (T − S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.     

Mimicry by cytokinin of phytochrome-regulated inhibition of chloroplast development in etiolated cucumber cotyledons

A study of the kinetics of chlorophyll (Chl) synthesis in cotyledons of etiolated cucumber seedlings ( Cucumis sativus L . cv. Delilah) treated with 5×10^-5 M -ben-zyladenine (BA) showed that cytokinin, like a red light pulse, could inhibit as well as promote pigment accumulation depending on the length of the dark period following induction. Spraying intact, dark-grown seedlings with BA, 24 h prior to white light exposure, eliminated the lag phase in Chl synthesis, while treatment with hormone 72 h before greening not only delayed the onset of synthesis, but it also reduced the amount of Chl accumulated after 24 h continuous white light. Impairment of Chl formation was correlated with inhibited regeneration of protochlorophyll and delayed appearance of the light harvesting Chl alb polypeptide. Application of σ-aminolevulinic acid (15 m M ) 2 h before white light exposure shortened the lag phase in Chl synthesis in control as well as in inhibited cotyledons, but the adverse effect of the red light and BA treatments on long-term Chl accumulation (24 h) was not reversed. Application of glutamate did not stimulate Chl production. Simultaneous treatment with hormone and red light 72 h before greening enhanced their separate inhibitory effects on Chl synthesis, but when given together 24 h prior to white light, their promotive effects on pigment accumulation were not additive.     

A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria

Fluorometric determination of the chlorophyll (Chl) content of cyanobacteria is impeded by the unique structure of their photosynthetic apparatus, i.e., the phycobilisomes (PBSs) in the light-harvesting antennae. The problems are caused by the variations in the ratio of the pigment PC to Chl a resulting from adaptation to varying environmental conditions. In order to include cyanobacteria in fluorometric analysis of algae, a simplified energy distribution model describing energy pathways in the cyanobacterial photosynthetic apparatus was conceptualized. Two sets of mathematical equations were derived from this model and tested. Fluorescence of cyanobacteria was measured with a new fluorometer at seven excitation wavelength ranges and at three detection channels (650, 685 and 720 nm) in vivo. By employing a new fit procedure, we were able to correct for variations in the cyanobacterial fluorescence excitation spectra and to account for other phytoplankton signals. The effect of energy-state transitions on the PC fluorescence emission of PBSs was documented. The additional use of the PC fluorescence signal in combination with our recently developed mathematical approach for phytoplankton analysis based on Chl fluorescence spectroscopy allows a more detailed study of cyanobacteria and other phytoplankton in vivo and in situ.     

Cyanobacterial Acclimation to Photosystem I or Photosystem II Light

The organization and function of the photochemical apparatus of Synechococcus 6301 was investigated in cells grown under yellow and red light regimes. Broadband yellow illumination is absorbed preferentially by the phycobilisome (PBS) whereas red light is absorbed primarily by the chlorophyll (Chl) pigment beds. Since PBSs are associated exclusively with photosystem II (PSII) and most of the Chl with photosystem I (PSI), it follows that yellow and red light regimes will create an imbalance of light absorption by the two photosystems. The cause and effect relationship between light quality and photosystem stoichiometry in Synechococcus was investigated. Cells grown under red light compensated for the excitation imbalance by synthesis/assembly of more PBS-PSII complexes resulting in high PSII/PSI = 0.71 and high bilin/Chl = 1.30. The adjustment of the photosystem stoichiometry in red light-grown cells was necessary and sufficient to establish an overall balanced absorption of red light by PSII and PSI. Cells grown under yellow light compensated for this excitation imbalance by assembly of more PSI complexes, resulting in low PSII/PSI = 0.27 and low bilin/Chl = 0.42. This adjustment of the photosystem stoichiometry in yellow light-grown cells was necessary but not quite sufficient to balance the absorption of yellow light by the PBS and the Chl pigment beds. A novel excitation quenching process was identified in yellow light-grown cells which dissipated approximately 40% of the PBS excitation, thus preventing over-excitation of PSII under yellow light conditions. It is hypothesized that State transitions in O₂ evolving photosynthetic organisms may serve as the signal for change in the stoichiometry of photochemical complexes in response to light quality conditions.     

The Effect of Diuron and 5-Aminolevulinic Acid on the Maintenance of Stable Chlorophyll Content in Greening Barley Leaves

Chlorophyll (Chl) accumulation and delayed luminescence of PSII were compared in greening barley leaves pretreated and untreated with diuron (DCMU) in the etiolated state, and reactions of two photosystems were studied in the plastids isolated from the pretreated and untreated leaves. The effect of treatment in light of post-etiolated leaves after 40-h illumination with 5-aminolevulinic acid (ALA), on the content of Chl and its precursor, protochlorophyllide (PChld) was also studied. The pretreatment of etiolated leaves with DCMU did not affect the rate of greening and the stable level of Chl content in barley. ALA, when introduced to leaves after the termination of Chl accumulation, increased PChld, but not Chl level. We suppose that the primary cause of greening cessation in etiolated leaves is the inhibition and cessation of the synthesis of apoproteins of pigment–protein complexes. The exhaustion of binding sites for newly synthesized Chl molecules leads to their retention in the so-called retroinhibitory pool of Chl, thus resulting in the inhibition of ALA synthesis by a negative feedback mechanism.     

Light intensity regulates the accumulation of the major light-harvesting chlorophyll-protein in greening seedlings

Etiolated pea (Pisum sativum [L.] cv Progress 9) and barley (Hordeum vulgare [L.] cv Boone) seedlings greened under either low (40 microeinsteins per square meter per second) or high (550 microeinsteins per square meter per second) intensity light were analyzed for chlorophyll (Chl) content and the levels of mRNA and protein for the major light-harvesting chlorophyll (Chl)-protein of photosystem II (LHC-II). Low intensity plants accumulated Chl more rapidly than high intensity plants. Both single radial immunodiffusion analysis and mild sodium dodecyl sulfate-polyacrylamide gel electrophoresis green gels showed that low intensity plants also accumulated LHC-II protein more rapidly than high intensity plants, following a kinetic pattern similar to the total Chl data. In contrast, LHC-II mRNA levels appeared to be independent of LHC-II protein levels although pea and barley LHC-II mRNA exhibited different light intensity responses. The absence of coordination between LHC-II mRNA and protein levels suggested that the biosynthesis of LHC-II in greening seedlings is not limited by mRNA. A correlation (better than the 0.01 significance level) between LHC-II protein accumulation and Chl accumulation was found for both pea and barley. The accumulation of LHC-II protein was not linked to the development of photosynthetic electron transport. These results and the similar effect of light intensity on Chl content and LHC-II protein levels suggested that the availability of Chl may limit LHC-II protein accumulation in greening seedlings.     

Development of Photosynthetic Activity following Anaerobic Germination in Rice-Mimic Grass (Echinochloa crus-galli var oryzicola)

Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24-hour midtreatment of air before light.

Development of photosynthetic activity in etiolated shoots, determined by CO₂ gas exchange, ¹⁴CO₂ uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development.

     

Chloroplast biogenesis at cold-hardening temperatures. Development of photosystem I and photosystem II activities in relation to pigment accumulation

Chloroplast biogenesis during continuous illumination at either low, cold-hardening temperatures (5°C) or non-hardening temperatures (20°C) was examined by monitoring the etioplast-chloroplast transformation with respect to pigment accumulation and the development of PSI- and PSII-associated electron transport activities in winter rye (Secale cereale L. cv Puma). Generally, chlorophyll and carotenoid accumulation during greening at 20°C were characterized by rapid initial rates in contrast to pronounced, initial lag times during biogenesis at 5°C. Although greening temperature had no effect on the sequential appearance of PSI relative to PSII, greening temperature significantly altered the pattern of appearance of PSI relative to chlorophyll accumulation. Thylakoid biogenesis under continuous illumination at 20°C imposed a pattern whereby the development of PSI activity was antiparallel to chlorophyll accumulation. In contrast, the development of PSI activity under continuous illumination at 5°C was paralllel to chlorophylll accumulation. These developmental patterns were independent of the temperature experienced during etiolation. However, rye seedlings etiolated at 20°C and subsequently subjected to continuous illumination at 5°C exhibited a 70% reduction in the maximum PSII activity (100 mol DCPIP reduced.mg Chl^-1.h^-1) attained relative to that observed for similar etiolated seedlings greened at 20°C (300 mol DCPIP reduced.mg Chl^-1.h^-1). This low temperature-induced inhibition could be alleviated by an initial 2 h exposure to continuous light at 20°C prior to greening to 5°C. Rye seedlings etiolated at 5°C attained similar maximal PSII activities (300 mol DCPIP reduced.mg Chl^-1.h^-1) regardless of the greening temperature. We suggest that the altered kinetics for pigment accumulation, the low temperature-induced change in the pattern for the appearance of PSI activity relative to chlorophyll accumulation and the differential sensitivity of 20° and 5° etiolated seedlings to greening temperature reflect an alteration in membrane organization incurred as a consequence of thylakoid assembly at low temperature.Abbreviations RH cold-hardened rye - RNH non-hardened rye - MV methylviologen - ASC ascorbate - Chl chlorophyll - DCPIP dichlorophenol indophenol     

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559_HP). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.     

Developmental changes in energy dissipation in etiolated wheat seedlings during the greening process

We studied the developmental changes in photosynthetic and respiration rates and thermal dissipation processes connected with chloroplasts and mitochondria activity in etiolated wheat (Triticum aestivum L., var. Irgina) seedlings during the greening process. Etioplasts gradually developed into mature chloroplasts under continuous light [190 μmol(photon) m^?2 s^?1] for 48 h in 5-day-dark-grown seedlings. The net photosynthetic rate of irradiated leaves became positive after 6 h of illumination and increased further. The first two hours of de-etiolation were characterized by low values of maximum (F_v/F_m) and actual photochemical efficiency of photosystem II (PSII) and by a coefficient of photochemical quenching in leaves. F_v/F_m reached 0.8 by the end of 24 h-light period. During greening, energy-dependent component of nonphotochemical quenching of chlorophyll fluorescence, violaxanthin cycle (VXC) operation, and lipoperoxidation activity changed in a similar way. Values of these parameters were the highest at the later phase of de-etiolation (4–12 h of illumination). The respiration rate increased significantly after 2 h of greening and it was the highest after 4–6 h of illumination. It was caused by an increase in alternative respiration (AP) capacity. The strong, positive linear correlation was revealed between AP capacity and heat production in greening tissues. These results indicated that VXC in chloroplasts and AP in mitochondria were intensified as energy-dissipating systems at the later stage of greening (after 4 h), when most of prolamellar bodies converted into thylakoids, and they showed the greatest activity until the photosynthetic machinery was almost completely developed.     

Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings

Cytokinin promotes morphological and physiological processes including the tetrapyrrole biosynthetic pathway during plant development. Only a few steps of chlorophyll (Chl) biosynthesis, exerting the phytohormonal influence, have been individually examined. We performed a comprehensive survey of cytokinin action on the regulation of tetrapyrrole biosynthesis with etiolated and greening barley seedlings. Protein contents, enzyme activities and tetrapyrrole metabolites were analyzed for highly regulated metabolic steps including those of 5-aminolevulinic acid (ALA) biosynthesis and enzymes at the branch point for protoporphyrin IX distribution to Chl and heme. Although levels of the two enzymes of ALA synthesis, glutamyl-tRNA reductase and glutamate 1-semialdehyde aminotransferase, were elevated in dark grown kinetin-treated barley seedlings, the ALA synthesis rate was only significantly enhanced when plant were exposed to light. While cytokinin do not stimulatorily affect Fe-chelatase activity and heme content, it promotes activities of the first enzymes in the Mg branch, Mg protoporphyrin IX chelatase and Mg protoporphyrin IX methyltransferase, in etiolated seedlings up to the first 5 h of light exposure in comparison to control. This elevated activities result in stimulated Chl biosynthesis, which again parallels with enhanced photosynthetic activities indicated by the photosynthetic parameters F _V/F _M, J _CO2max and J _CO2 in the kinetin-treated greening seedlings during the first hours of illumination. Thus, cytokinin-driven acceleration of the tetrapyrrole metabolism supports functioning and assembly of the photosynthetic complexes in developing chloroplasts.     

Changes in the photosynthetic pigments in bean leaves during the first photoperiod of greening and the subsequent dark-phase. Comparison between old (10-d-old) leaves and young (2-d-old) leaves

Chlorophyll and carotenoid variations of 2-d-old and 10-d-old bean leaves (Phaseolus vulgaris var Red Kidney) were analyzed by HPLC during the first photoperiod of greening (16 h light + 8 h dark). The HPLC method used is suitable for the separation of cis- and trans-carotenoid isomers, Pchlide a and Chlide a as well as their esters. The main results are (1) before illumination the composition of the carotenoid pool is similar at the two developmental stages; (2) non-illuminated 2-d-old leaves are devoid of Pchlide a ester; (3) chlorophyll and carotenoid accumulation in 2-d-old leaves presented a lag phase twice longer than observed in 10-d-old ones; (4) Chlide a seems directly esterified to Chl a in 2-d-old leaves whereas esterification requires four steps in 10-d-old leaves and, (5) the kinetics of Chl and carotenoid accumulation are different at the two investigated developmental stages.     

In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation

First successful $\text{in vitro}$ synthesis of functional photosynthetic phosphorylating membrane is reported. Etioplasts, highly enriched in cytoplasmic and plastid proteins, isolated from etiolated Cucumber cotyledons pretreated with kinetin and gibberellic acid, and illuminated in a cofactor fortified medium showed commencement of chlorophyll (Chl) synthesis immediately after illumination from exogenous δ-aminolevulinic acid, while photosystem I (PS I) activity commenced 15 min after the onset of illumination. When cotyledons pretreated with kinetin and gibberellic acid were illuminated directly, there was a lag phase of 30 min before the commencement of Chl synthesis and PS I activity developed after 1 h of illumination. In plastids developed both $\text{in vivo}$ and $\text{in vitro}$, the electron flow from dichlorophenolindophenol to methyl-viologen was coupled to phosphorylation as observed by an increase in the electron transport rate on the addition of uncouplers. Analysis of polypeptide profiles of the greening plastids $\text{in vitro}$ showed the disappearance of many higher molecular weight proteins during greening. Polypeptides of molecular weight 32, 20.5, 19.5 K absent in etioplasts appeared as distinct bands after 4 h of greening $\text{in vitro}$.     

Reduction kinetics of the photo-oxidized chlorophyll a+II in the nanosecond range: Measurements of the absorption changes at 688 nm under repetitive flash excitation

The 688 nm absorption changes (ΔA₆₈₈), indicating the photochemical turnover of chlorophyll a_II (Chl a_II) have been investigated under repetitive laser flash excitation conditions in spinach chlorplasts. It was found that under steady state conditions about 50–60% of the photo-oxidized primary donor of Photosystem II (PS II), Chl a⁺_II, becomes re-reduced with a biphasic kinetics in the nanosecond time scale with half-life times of about 50 ns and 400 ns. The remaining Chl a⁺_II becomes re-reduced in the microsecond range.