Effects of elevated CO2 on growth,calcification, and spectral dependence of photoinhibition in the coccolithophore Emiliania huxleyi (Prymnesiophyceae)1

We studied the effects of elevated CO₂ concentrations on cell growth, calcification, and spectral variation in the sensitivity of photosynthesis to inhibition by solar radiation in the globally important coccolithophore Emiliania huxleyi. Growth rates and chlorophyll a content per cell showed no significant differences between elevated (800 ppmv) and ambient (400 ppmv) CO₂ conditions. However, the production of organic carbon and the cell quotas for both carbon and nitrogen, increased under elevated CO₂ conditions, whilst particulate inorganic carbon production rates decreased under the same conditions. Biometric analyses of cells showed that coccoliths only presented significant differences due to treatments in the central area width. Most importantly, the size of the coccosphere decreased under elevated CO₂ conditions. The susceptibility of photosynthesis to inhibition by ultraviolet radiation (UVR) was estimated using biological weighting functions (BWFs) and a model that predicts photosynthesis under photosynthetically active radiation and UVR exposures. BWF results demonstrated that the sensitivity of photosynthesis to UVR was not significantly different between E. huxleyi cells grown under elevated and present CO₂ concentrations. We propose that the acclimation to elevated CO₂ conditions involves a physiological mechanism of regulation and allocation of energy and metabolites in the cell, which is also responsible for altering the sensitivity to UVR. In coccolithophores, this mechanism might be affected by the decrease in the calcification rates.     

Evaluation of the potential to measure photosynthetic rates in C3 plants (Flaveria pringlei and Oryza sativa) by combining chlorophyll fluorescence analysis and a stomatal conductance model

The response curves of leaf photosynthesis to varying light, temperature and leaf-to-air vapour pressure deficit were measured in the C₃ plants Flaveria pringlei and Oryza sativa in normal air with a computerized open infrared gas analysis (IRGA) system, and the photochemical efficiency of photosystem II, described as (1–F,/F′_m) after Genty. Briantais & Baker (1989, Biochimica et Biophysica Acta 990, 87–92), was simultaneously measured with a modulated fluorometer. A model was written for rates of CO₂ fixation as a function of the true rate of O₂ evolution measured by fluorescene analysis (Jo₂), mesophyll conductance and intercellular CO₂ partial pressure. A second model was developed for rates of CO₂ fixation as a function of Jo₂, mesophyll conductance and stomatal conductance. In the latter case, leaf stomatal conductance was simulated using the stomatal model proposed by Leuning (1995, Plant, Cell and Environment 18 , 339–355). The rates of CO₂ fixation predicted from the models were similar to rates measured by IRGA. The results indicate that there is potential to measure CO₂ fixation in C₃ plants by combining the non-invasive measurement of Jo₂ by chlorophyll fluorescence analysis with the stomatal conductance model.     

Photoinhibition and xanthophyll cycle activity in bayberry (Myrica rubra) leaves induced by high irradiance

The effect of high irradiance (HI, photosynthetically active photon flux density of 1 300 μmol m⁻² s⁻¹) on net photosynthetic rate (P _N), chlorophyll fluorescence parameters, and xanthophyll cycle components were studied in fruit tree bayberry leaves. HI induced the photoinhibition and inactivation of photosystem 2 (PS2) reaction centres (RCs), which was characterized by decreased P _N, maximum yield of fluorescence after dark adaptation (F_m), photochemical efficiency of PS2 (F_v/F_m) and quantum yield of PS2 (Φ_PS2), and increased reduction state of Q_A (1-q_P) and non-photochemical quenching (NPQ). Initial fluorescence (F₀) showed a decrease after the first 2 h, and subsequently increased from the third hour exposure to HI. Furthermore, a greater increase in the ratio (F_i-F₀)/(F_p-F₀) which is an expression of the proportion of the Q_B non-reducing PS2 centres, whereas a remarked decrease in the slope of F_i to F_p which represents the rate of Q_A reduction was observed in leaves after HI exposure. Additionally, HI caused an increase in the pool size of the xanthophyll cycle pigments and sustained elevated contents of zeaxanthin (Z), antheraxanthin (A), and de-epoxidation state (DES) at the end of the irradiation period. During HI, decreased F_m, F_v/F_m, Φ_PS2, NPQ, slope of F_i to F_p, V+A+Z, and DES, and increased F₀, 1-q_P, ratio (F_i-F₀)/(F_p-F₀), and V were observed in dithiothreitol (DTT)-fed leaves compared to control ones under the same conditions. Hence photoinhibition caused by HI in bayberry was probably attributed to inactivation of PS2 RCs, and photoprotection from photodamage were mainly related to the xanthophyll cycle-dependent heat dissipation in excess photons.     

饱和白光引起的光系统II捕光复合体(LHCII)从反应中心复合体脱离不同于弱红光引起的状态1向状态2的转换

我们观测了不同光照预处理对拟南芥、小麦和大豆叶片光合作用和低温(77K) 叶绿素荧光参数F685、F735和F685/F735的影响.野生型拟南芥叶片光合作用对饱和光到有限光转变的响应曲线是V型,而缺乏叶绿体蛋白激酶的突变体STN7的这一曲线为L型. 饱和白光可以引起拟南芥叶片F685/F735的明显降低,但是F735没有明显增高,而弱红光可以导致拟南芥叶片F685/F735的明显降低和F735的明显增高,表明弱红光可以引起状态1向状态2的转变,同时伴随从光系统II脱离的LHC II与光系统I的结合,而饱和白光只能引起LHC II从光系统II反应中心复合体脱离.并且,低温叶绿素荧光分析结果证明,饱和白光可以引起大豆叶片LHC II脱离,但是不能引起小麦叶片LHC II脱离,而弱红光可以引起小麦叶片的这种状态转换,却不能引起大豆叶片的这种状态转换.因此,饱和白光引起的野生型拟南芥和大豆叶片的LHC II脱离不是一个典型的状态转换现象.     

Photoacclimation and Photoprotection of Juvenile Sporophytes of Macrocystis pyrifera (Laminariales,Phaeophyceae) Under High-light Conditions During Short-term Shallow-water Cultivation1

This study was designed to understand better if and how juvenile sporophytes of Macrocystis pyrifera can photoacclimate to high-light conditions when transplanted from 10 to 3 meters over 7 d. Acclimation of adult sporophytes to light regimes in the bathymetric gradient has been extensively documented. It primarily depends on photoacclimation and translocation of resources among blades. Among other physiological differences, juvenile sporophytes of M. pyrifera lack the structural complexity shown by adults. As such, juveniles may primarily depend on their photoacclimation capacities to maintain productivity and even avoid mortality under changing light regimes. However, little is known about how these mechanisms operate in young individuals. The capacity of sporophytes to photoacclimate was assessed by examining changes in their photosynthetic performance, pigment content, and bio-optical properties of the blade. Sporophytes nutritional status and oxidative damage were also determined. Results showed that juvenile sporophytes transplanted to shallow water were able to regulate light harvesting by reducing pigment concentration, and thus, absorptance and photosynthetic efficiency. Also, shallow-water sporophytes notably enhanced the dissipation of light energy as heat (NPQ) as a photoprotective mechanism. Generally, these adjustments allowed sporophytes to manage the absorption and utilization of light energy, hence reducing the potential for photo-oxidative damage. Furthermore, no substantial changes were found in the internal reserves (i.e., soluble carbohydrates and nitrogen) of these sporophytes. To our knowledge, these results are the first to provide robust evidence of photoprotective and photoacclimation strategies in juveniles of M. pyrifera, allowing them to restrict or avoid photodamage during shallow-water cultivation.     

RESPONSE OF THE PHOTOSYNTHETIC APPARATUS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) TO NITRATE,PHOSPHATE, OR IRON STARVATION1

The effects of nitrate, phosphate, and iron starvation and resupply on photosynthetic pigments, selected photosynthetic proteins, and photosystem II (PSII) photochemistry were examined in the diatom Phaeodactylum tricornutum Bohlin (CCMP 1327). Although cell chlorophyll a (chl a) content decreased in nutrient-starved cells, the ratios of light-harvesting accessory pigments (chl c and fucoxanthin) to chl a were unaffected by nutrient starvation. The chl a-specific light absorpition coefficient (a*) and the functional absorption cross-section of PSII (σ) increased during nutrient starvation, consistent with reduction of intracellular self-shading (i.e. a reduction of the “package effect”) as cells became chlorotic. The light-harvesting complex proteins remained a constant proportion of total cell protein during nutrient starvation, indicating that chlorosis mirrored a general reduction in cell protein content. The ratio of the xanthophylls cycle pigments diatoxanthin and diadinoxanthin to chl a increased during nutrient starvation. These pigments are thought to play a photo-protective role by increasing dissipation of excitation energy in the pigment bed upstream from the reaction centers. Despite the increase in diatoxanthin and diadinoxanthin, the efficiency of PSII photochemistry, as measured by the ration of variable to maximum fluorescence (F_v/F_m) of dark-adapted cells, declined markedly under nitrate and iron starvation and moderately under phosphate starvation. Parallel to changes in F_v/F_m were decreases in abundance of the reaction center protein D1 consistent with damage of PSII reaction centers in nutrient-starved cells. The relative abundance of the carboxylating enzyme, ribulose bisphosphate carboxylase/oxygenase (RUBISCO), decreased in response to nitrate and iron starvation but not phosphate starvation. Most marked was the decline in the abundance of the small subunit of RUBISCO in nitrate-starved cells. The changes in pigment content and fluorescence characteristics were typically reversed within 24 h of resupply of the limiting nutrient.     

A new fluorescence band F689 in photosystem II revealed by picosecond analysis at 4-77 K: Function of two terminal energy sinks F689 and F695 in PS II

We performed picosecond time-resolved fluorescence spectroscopy in spinach photosystem II (PS II) particles at 4, 40, and 77 K and identified a new fluorescence band, F689. F689 was identified in addition to the well-known F685 and F695 bands in both analyses of decay-associated spectra and global Gaussian deconvolution of time-resolved spectra. Its fast decay suggests the energy transfer directly from F689 to the reaction center chlorophyll P680. The contribution of F689, which increases only at low temperature, explains the unusually broad and variable bandwidth of F695 at low temperature. Global analysis revealed the three types of excitation energy transfer/dissipation processes: (1) energy transfer from the peripheral antenna to the three core antenna bands F685, F689, and F695 with time constants of 29 and 171 ps at 77 and 4 K, respectively; (2) between the three core bands (0.18 and 0.82 ns); and (3) the decays of F689 (0.69 and 3.02 ns) and F695 (2.18 and 4.37 ns). The retardations of these energy transfer rates and the slow F689 decay rate produced the strong blue shift of the PS II fluorescence upon the cooling below 77 K.     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

Disentangling two non-photochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77 K

Diatoms, which are primary producers in the oceans, can rapidly switch on/off efficient photoprotection to respond to fast light-intensity changes in moving waters. The corresponding thermal dissipation of excess-absorbed-light energy can be observed as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Fluorescence-induction measurements on Cyclotella meneghiniana diatoms show two NPQ processes: qE₁ relaxes rapidly in the dark while qE₂ remains present upon switching to darkness and is related to the presence of the xanthophyll-cycle pigment diatoxanthin (Dtx). We performed picosecond fluorescence measurements on cells locked in different (quenching) states, revealing the following sequence of events during full development of NPQ. At first, trimers of light-harvesting complexes (fucoxanthin–chlorophyll a/c proteins), or FCPa, become quenched, while being part of photosystem II (PSII), due to the induced pH gradient across the thylakoid membrane. This is followed by (partial) detachment of FCPa from PSII after which quenching persists. The pH gradient also causes the formation of Dtx which leads to further quenching of isolated PSII cores and some aggregated FCPa. In subsequent darkness, the pH gradient disappears but Dtx remains present and quenching partly pertains. Only in the presence of some light the system completely recovers to the unquenched state.     

On the approaches applied in formulation of a kinetic model of photosystem II: Different approaches lead to different simulations of the chlorophyll a fluorescence transients

Chlorophyll a fluorescence rise (O-J-I-P transient) was in literature simulated using models describing reactions occurring solely in photosystem II (PSII) and plastoquinone (PQ) pool as well as using complex models which described, in addition to the above, also subsequent electron transport occurring beyond the PQ pool. However, there is no consistency in general approach how to formulate a kinetic model and how to describe particular reactions occurring even in PSII only. In this work, simple kinetic PSII models are considered always with the same electron carriers and same type of reactions but some reactions are approached in different ways: oxygen evolving complex is considered bound to PSII or “virtually” separated from PSII; exchange of doubly reduced secondary quinone PSII electron acceptor, Q_B, with PQ molecule from the PQ pool is described by one second order reaction or by two subsequent reactions; and all possible reactions or only those which follow in logical order are considered. By combining all these approaches, eight PSII models are formulated which are used for simulations of the chlorophyll a fluorescence transients. It is shown that the different approaches can lead to qualitatively different results. The approaches are compared with other models found elsewhere in the literature and therefore this work can help the readers to better understand the other models and their results.     

Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: The qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20-200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700⁺ and oxidized plastocyanin in the 20-200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP⁺-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 μmol photons m⁻² s⁻¹ did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to ‘close’ all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q_A a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q_B-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.     

In intact leaves, the maximum fluorescence level (FM) is independent of the redox state of the plastoquinone pool: A DCMU-inhibition study

The effects of DCMU (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea) on the fluorescence induction transient (OJIP) in higher plants were re-investigated. We found that the initial (F₀) and maximum (F_M) fluorescence levels of DCMU-treated leaves do not change relative to controls when the treatment is done in complete darkness and DCMU is allowed to diffuse slowly into the leaves either by submersion or by application via the stem. Simultaneous 820 nm transmission measurements (a measure of electron flow through Photosystem I) showed that in the DCMU-treated samples, the plastoquinone pool remained oxidized during the light pulses whereas in uninhibited leaves, the F_M level coincided with a fully reduced electron transport chain. The identical F_M values with and without DCMU indicate that in intact leaves, the F_M value is independent of the redox state of the plastoquinone pool. We also show that (i) the generally observed F₀ increase is probably due to the presence of (even very weak) light during the DCMU treatment, (ii) vacuum infiltration of leaf discs leads to a drastic decrease of the fluorescence yield, and in DCMU-treated samples, the F_M decreases to the I-level of their control (leaves vacuum infiltrated with 1% ethanol), (iii) and in thylakoid membranes, the addition of DCMU lowers the F_M relative to that of a control sample.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems

Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems’ activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30-40% for both photoreactions—P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core-membrane linker allophycocyanin APC-L_CM is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-L_CM may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.