Measuring photosynthetic parameters in individual algal cells by Fast Repetition Rate fluorometry

A Single Cell Fast Repetition Rate (SCFRR) fluorometer was developed to measure the quantum yield of photochemistry, the functional absorption cross section of PS II and the kinetics of electron transport on the acceptor side of PS II in individual algal cells. These parameters are used to quantify the cell-specific photosynthetic performance in natural phytoplankton assembledges in aquatic ecosystems. The SCFRR technique measures chlorophyll fluorescence transients induced by a precisely controlled series of excitation flashlets that cumulatively saturate PS II within 120 s. To meet the requirement in the analysis for single algal cells, the measurements are conducted in micro volumes, such that the probability of probing more than one cell at a time is vanishingly low. We designed a novel, computer-controlled hydromechanical system to deliver a portion of the sample into the measuring chamber and, following measurement, remove it into one of six sorting containers. The fluorescence signal is induced by a series of high frequency flashlets obtained from high luminosity blue light-emitting diodes and is acquired by a novel red-sensitive PMT-based detection system exhibiting both high sensitivity and a very wide dynamic range. The wide dynamic range of the detector allows SCFRR measurements for a wide variety of cell sizes ranging from 1 to 100 m equivalent spherical diameter. The compact and light-weight design makes the SCFRR Fluorometer applicable for both laboratory and field studies.     

春季太湖梅梁湾水体中藻类光量子产额的变化规律

藻类光量子产额是表征藻类对环境因素响应的重要参数之一.利用中国科学院太湖生态网络站栈桥头测定的水体初级生产力及水下辐照度数据,计算了4个观测时段藻类的光量子产额,结果表明,自表层至30 cm处,藻类的光量子产额随深度基本呈现线性增加的趋势,但随深度的进一步增加,光量子产额增加的趋势变缓,直至接近于平直状态(在到60～80 cm之间),该最大值也基本为0.12 mol(O2)/mol(光子),且该处的光量子产额是表层的10倍左右.春季,在太湖梅梁湾60 cm以上,光并不是藻类生长的限制条件,而在60 cm以下,由于光的衰减,使得光成为限制其光合作用的条件.     

一些海洋浮游植物量子产值的研究

现场实验以及用硅藻、金藻和绿藻所做的实验表明,在光饱和深度以下,随着深度的增加,浮游植物的量子产懂具有不变、增加和下降3种垂直变化趋势。偏离理论模式的后两种结果可能是由浮游植物的光强适应(Light-shade adaptation)等原因引起。还探讨了量子产值作为初级生产力模型和光利用效率模型中的参数的问题。     

D,L-甘油醛处理引起的小麦叶片表观光合量子效率的降低（英文）

D,L-甘油醛(磷酸核酮糖激酶抑制剂,10mmol/L)处理小麦旗叶1 h可降低叶片净光合速率和表观量子效率.同时,光系统Ⅱ光化学效率(△F/Fm′)、电子传递速率(ETR)和单位叶面积ATP含量均降低,而胞间二氧化碳浓度(Ci)和叶绿素荧光非光化学猝灭(NPQ)增加.这些结果说明,D,L-甘油醛引起的小麦旗叶表观量子效率降低是由于光合碳同化受阻对光合电子传递的反馈抑制.     

Estimation of Photorespiration Rate by Simultaneous Measurements of CO2, Gas Exchange Rate,and Chlorophyll Fluorescence Quenching in the C3 Plant Vigna Radiata (L.) Wilczek and the C4 Plant Amaranthus Mongostanus L.

So far the photorespiration rate (R _P) in a leaf has been determined as the difference between the net photosynthetic rates (P _N) measured in 21 % O₂ air (P _N21%) and 3 % O₂ air (P _N3%). In the C₃ plant Vigna radiata and the C₄ plant Amaranthus mongostanus L., P _N and chlorophyll fluorescence quenching in leaves were monitored simultaneously. R _P of leaves in situ was estimated as termed R _PE from the electron transport rates through photosystem 2 (PS2), and compared with R _PO (P _N3% – P _N21%). In V. radiata R _PO was 11.9 µmol(CO₂) m^–2 s^–1 and the ratio of R _PO to P _N21% was 42.2 %, whereas the ratio of R _PE to P _N21% was 25.7 %. This suggests that R _PO may be over-estimated for the real R _P in normal air. In A. mongostanus, P _N was almost not changed with a decrease in O₂ concentration from 21 to 3 %, whereas the quantum yield of PS2 was evidently affected by the change in O₂ concentration. This fact shows the presence of photorespiration in this C₄ species, where R _PE was equivalent to 3.8 % of P _N21%.     

Leaf development, temperature and light stress control of the expression of early light-inducible proteins (ELIPs) in Vitis vinifera L.

Early light inducible proteins (ELIPs) are thylakoid proteins transiently induced by light. They are found in many species, most of which are annual plants. Studies on perennial plants are scarce and in grapevine almost nil. Because grapevines normally grow in sites with high radiation, we aimed to characterize the ELIP expression to evaluate whether they have a role in photoprotection in this species. ELIP expression was characterized in leaves at different temperatures, times of induction and natural light intensities. Very low expression was found in mature leaves. In young leaves, ELIP expression was high but began at temperatures higher than 13 °C. Maximal expression was obtained at 30 °C after 4 h of induction at 1000 μmol PAR m⁻² s⁻¹. At this intensity, the level of photoinhibition was also maximal. We conclude that in grapevine leaves, ELIP expression is developmentally dependent, occurring mainly in developing leaves, Finally, our results showed that the light intensity dependence of the ELIP expression correlated with the extent of photoinhibition indicating that ELIP induction is controlled by the strength of light stress and suggesting a role of these proteins in photoprotection.     

Rapid light-response curves of chlorophyll fluorescence in microalgae: relationship to steady-state light curves and non-photochemical quenching in benthic diatom-dominated assemblages

Rapid light-response curves (RLC) of variable chlorophyll fluorescence were measured on estuarine benthic microalgae with the purpose of characterising its response to changes in ambient light, and of investigating the relationship to steady-state light-response curves (LC). The response of RLCs to changes in ambient light (E, defined as the irradiance level to which a sample is acclimated to prior to the start of the RLC) was characterised by constructing light-response curves for the RLC parameters α _RLC, the initial slope, ETR_m,RLC, the maximum relative electron transport rate, and E _k,RLC, the light-saturation parameter. Measurements were carried out on diatom-dominated suspensions of benthic microalgae and RLC and LC parameters were compared for a wide range of ambient light conditions, time of day, season and sample taxonomic composition. The photoresponse of RLC parameters was typically bi-phasic, consisting of an initial increase of all parameters under low ambient light (E < 21–181 μmol m⁻² s⁻¹), and of a phase during which α _RLC decreased significantly with E, and the increase of ETR_m,RLC and E _k,RLC was attenuated. The relationship between RLC and LC parameters was dependent on ambient irradiance, with significant correlations being found between α _RLC and α, and between ETR_m,RLC and ETR_m, for samples acclimated to low and to high ambient irradiances, respectively. The decline of α _RLC under high light (Δα _RLC) was strongly correlated (P < 0.001 in all cases) with the level of non-photochemical quenching (NPQ) measured before each RLC. These results indicate the possibility of using RLCs to characterise the steady-state photoacclimation status of a sample, by estimating the LC parameter E _k, and to trace short-term changes in NPQ levels without dark incubation.     

Application of fast repetition rate fluorometry to phytoplankton photosynthetic parameters in freshwaters

Fast repetition rate fluorometry (FRRF) was successfully applied to various studies in modern oceanography. In this study, for the first time, the seasonality of phytoplankton photosynthetic parameters in a deep alpine lake was observed using FRRF in combination with the traditional ¹⁴C incubation technique. Special attention was given to the differences in photosynthetic behaviour during mixed and stratified conditions, characterised especially during summer by a deep chlorophyll maximum (DCM) dominated by the filamentous cyanobacterial species Planktothrix rubescens. Maximum light-utilisation efficiency (α*_14C) was in the range of 0.01–0.03 mgC (mg Chl-a)⁻¹ h⁻¹ (μmol phot. m⁻² s⁻¹)⁻¹, while maximum quantum yields for carbon fixation (Φ_C,max) varied from 0.01–0.07 molC (mol phot.)⁻¹. Higher values occurred during thermal stratification indicating acclimation of the phytoplankton assemblage. These findings were supported by FRRF-based estimates, although cyanobacterial blooms could not be characterised by FRRF-excitation due to methodological deficiencies. In general, however, instantaneous photosynthetic rates measured by FRRF-excitation correlate well at sub-saturating light-intensities with conventional ¹⁴C-uptake rates, although they operate on different time-scales.     

Cyclic electron flow around Photosystem II in vivo

The oxygen flash yield (Y_O2) and photochemical yield of PS II (_{PS II}) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, Y_O2 and _{PS II} followed each other, suggesting a close coupling between the oxidation of water and Q_A reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432–443). Following a light-to-dark transition, however, the relationship between Q_A reduction and the fraction of PS II reaction centers capable of evolving O₂ became temporarily uncoupled. _{PS II} recovered to the preillumination levels within 5–10 s, while the Y_O2 required up to 60 s to recover under aerobic conditions. The recovery of Y_O2 was independent of the redox state of Q_A, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (_{PS II}). The hysteresis between Y_O2 and the reduction of Q_A during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting enzyme (agents) - Chl chlorophyll - cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_O minimum fluorescence yield in the dark-adapted state - F_I minimum fluorescence yield under ambient irradiance or during transition from the light-adapted state - F_M maximum fluorescence yield in the dark-adapted state - F_M maximum fluorescence yield under ambient irradiance or during transition from light-adapted state - F_V, F_V variable fluorescence (F_V=F_M–F_O ; F_V=F_M–F_I) - FRR fast repetition rate (fluorometer) - _{PS II} quantum yield of Q_A reduction (_{PS II}=(F_M – F_O)/F_M or _{PS II})=(F_M= – F_I=)/F_M=) - LHCII Chl a/b light harvesting complexes of Photosystem II - OEC oxygen evolving complex of PS II - P680 reaction center chlorophyll of PS II - PQ plastoquinone - POH₂ plastoquinol - PS I Photosystem I - PS II Photosystem II - RC II reaction centers of Photosystem II - PS II the effective absorption cross-section of PHotosystem II - TL thermoluminescence - Y_O2 oxygen flash yield The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

EFFECTS OF TEMPERATURE ON GROWTH,LIGHT ABSORPTION,AND QUANTUM YIELD IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

The effects of growth temperature on the marine chlorophyte Dunaliella tertiolecta Butcher were studied to provide a more mechanistic understanding of the role of environmental factors in regulating bio-optical properties of phytoplankton. Specific attention was focused on quantities that are relevant for modeling of growth and photosynthesis. Characteristics including chlorophyll a (chl z)-specific light absorption (a*_ph(λ)), C:chl a ratio, and quantum yield for growth (φ_μ) varied as functions of temperature under conditions of excess light and nutrients. As temperature increased over the range examined (12°-28°C), intracellular concentrations of chl a increased by a factor of 2 and a*_ph(λ) values decreased by more than 50% at blue to green wavelengths. The lower values of a*_ph(λ) were due to both a decrease in the abundance of accessory pigments relative to chl a and an increase in pigment package effects arising from higher intracellular pigment concentrations. Intracellular pigment concentration increased as a consequence of higher cellular pigment quotas combined with lower cell volume. At high growth temperatures, slightly more light was absorbed on a per-cell-C basis, but the dramatic increases in growth rate from μ= 0.5 d^?1 at 12° C to μ= 2.2 d^?1 at 28°C were primarily due to an increase in φ_μ (0.015–0.041 mol C (mol quanta)^?1). By comparison with previous work on this species, we conclude the effects of temperature on a*_ph(λ) and φ_μ are comparable to those observed for light and nutrient limitation. Patterns of variability in a*_ph(λ)and φ_μ as a function of growth rate at different temperatures are similar to those previously documented for this species grown at the same irradiance but under a range of nitrogen-limited conditions. These results are discussed in the context of implications for bio-optical modeling of aquatic primary production by phytoplankton.     

Photosynthetic properties of leaves of a yellow green mutant of wheat compared to its wild type

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O₂ evolution of the leaf tissue in saturating light and CO₂ was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO₂ assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.Abbreviations cyt f cytochrome f - chl chlorophyll - PS II photosystem II - Pn_max maximum photosynthesis - RuBCase Ribulose, 1-5,bisphosphate carboxylase