Secondary fluorescence kinetics of spinach leaves in relation to the onset of photosynthetic carbon assimilation

When spinach leaves are re-illuminated, after dark periods of 90 s or less, an initial fluorescence peak is observed which rapidly gives way to a much lower terminal value. After 2 min or more in the dark, however, there is a secondary rise, at about 50–70 s, which then gives way, more slowly, to approximately the same low terminal value as before. The secondary rise is eliminated or disguised by feeding D,L-glyceraldehyde (a specific inhibitor of photosynthetic carbon assimilation) and by manose, 2-deoxyglucose and glucosamine, all of which are believed to sequester cytoplasmic orthophosphate. This secondary rise in fluorescence is discussed in relation to photosynthetic induction and the manner in which these compounds may modulate fluorescence by their effect on the availability of orthophosphate and their consequent impact on the adenylate status of the stroma.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - CCCP carbonylcyanidchlorophenylhydrazon     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

Energy-dependent quenching of dark-level chlorophyll fluorescence in intact leaves

A new type of modulation fluorometer was used in the study of energy-dependent chlorophyll fluorescence quenching (q_E) in intact leaves. Under conditions of strong energization of the thylakoid membrane (high light intensity, absence of CO₂) not only variable fluorescence, F_V, but also dark-level fluorescence, F_O, was quenched, leading to definition of a quenching coefficient, q_O. Information on q_O was shown to be essential for correct determination of photochemical (q_Q) and energy dependent quenching (q_E) by the saturation pulse method. The relationship between q_E and q_O was analysed over a range of light intensities at steady state conditions. q_E was found to consist of two components, the second of which is linearly correlated with q_O. q_O and the second component of q_E are interpreted to reflect the state 1 — state 2 shift caused by LHC II phosphorylation.     

Nondestructive determination of nitrogen and chlorophyll content in olive tree leaves and the relation with photosynthesis and fluorescence parameters

For Tunisian olive tree orchards, nitrogen deficiency is an important nutritional problem, in addition to the availability of water. Establishment of relationships between nutrients such as nitrogen and ecophysiological parameters is a promising method to manage fertilisation at orchard level. Therefore, a nitrogen stress experiment with one-year-old olive trees (Olea europaea L. ‘Koroneiki’ and ‘Meski’) was conducted with trees respectively subjected to four nitrogen supply regimes (23.96 meq l⁻¹, 9.58 meq l⁻¹, 4.79 meq l⁻¹ and 0 meq l⁻¹ NO₃ ⁻¹).     

Photosynthesis and photoinhibition in leaves of chlorophyll b-less barley in relation to absorbed light

The response of photosynthesis to absorbed light by intact leaves of wild-type ( Hordeum vulgare L. cv. Gunilla) and chlorophyll b -less barley ( H. vulgare L. cv. Dornaria, chlorina-f2²⁸⁰⁰) was measured in a light integrating sphere. Up to the section where the light response curve bends most sharply the responses of the b -less and wild-type barley were similar but not identical. Average quantum yield and convexity for the mutant light response curves were 0.89 and 0.90, respectively, times those of the wild-type barley. The maximum quantum yield for PSII photochemistry was also 10% lower as indicated by fluorescence induction kinetics (F_v/F_m). Just above the region where the light curve bends most sharply, photosynthesis decreased with time in the mutant but not in the wild-type barley. This decrease was associated with a decrease in F_v/F_m indicating photoinhibition of PSII. This photoinhibition occurred in the same region of the light response curve where zeaxanthin formation occurs. Zeaxanthin formation occurred in both the chlorophyll b -less and wild-type leaves. However, the epoxidation state was lower in the mutant than in the wild-type barley. The results indicate that chlorophyll b -less mutants will have reduced photosynthetic production as a result of an increased sensitivity to photoinhibition and possibly a lowered quantum yield and convexity in the absence of photoinhibition.     

Extended depth-of-focus imaging of chlorophyll fluorescence from intact leaves

Imaging dynamic changes in chlorophyll a fluorescence provides a valuable means with which to examine localised changes in photosynthetic function. Microscope-based systems provide excellent spatial resolution which allows the response of individual cells to be measured. However, such systems have a restricted depth of focus and, as leaves are inherently uneven, only a small proportion of each image at any given focal plane is in focus. In this report we describe the development of algorithms, specifically adapted for imaging chlorophyll fluorescence and photosynthetic function in living plant cells, which allow extended-focus images to be reconstructed from images taken in different focal planes. We describe how these procedures can be used to reconstruct images of chlorophyll fluorescence and calculated photosynthetic parameters, as well as producing a map of leaf topology. The robustness of this procedure is demonstrated using leaves from a number of different plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Correlation between chlorophyll fluorescence and photoacoustic signal transients in spinach leaves

Chlorophyll fluorescence and photoacoustic transients from dark adapted spinach leaves were measured and analyzed using the saturating pulse technique. Except for the first 30 s of photosynthetic induction, a good correlation was found between photoacoustically detected oxygen evolution at 35 Hz modulation frequency and electron flow calculated from the fluorescence quenching coefficients q_P and q_N. The induction kinetics of the photothermal signal, i.e., the photoacoustic signal at 370 Hz, reveal a fast (t _r <10 ms) and a slow (t _r 1 s) rise component. The fast component is suggested to be composed of the minimal thermal losses in photosynthesis and thermal losses from non-photosynthetic processes. The slow phase is attributed to variable thermal losses in photosynthesis. The variable thermal losses were normalized by measuring the minimal photothermal signal (H₀) in the dark-adapted state and the maximal photothermal signal (H_m) during a saturating light pulse. The kinetics of the normalized photochemical loss (H-H₀)/(H_m-H₀) obtained from high-frequency PA measurements were found to correlate with the kinetics of oxygen evolution measured at low frequency.Abbreviations F_m maximum fluorescence - F₀ initial fluorescence - F_v variable fluorescence - H photothermal signal - I in-phase - LED light emitting diode - PA photoacoustic - PL photochemical loss - Q quadrature - q_N non-photochemical quenching - q_P photochemical quenching - VCLS voltage controlled light source     

Chlorophyll a fluorescence and carbon assimilation in developing leaves of light-grown cucumber

The development of photochemical activity and carbon assimilation in light-grown cucumber (Cucumis sativus L. cv Natsusairaku) leaves was studied to determine the pattern of acquisition and its relationship to leaf growth and expansion. Measurements of chlorophyll a fluorescence showed that leaves acquire photochemical function over a period of 6 or more days, and gas exchange studies showed increases in carbon assimilation over a parallel time period. As leaves expand and mature, they undergo a sequential, three-step series of changes in fluorescence response. The initial kinetics show the absence of wholly functional quenching mechanisms. Dynamic imaging of fluorescence kinetics showed that a temporal series of changes occurred within defined areas of individual developing leaves. The spatial acquisition of photochemical activity in leaves was basipetal as is their directional expansion, development of air spaces and stomata, and the cessation of imported carbon.     

Analysis of chlorophyll a fluorescence and proteomic differences of rice leaves in response to photooxidation

This study investigated the effects of increased sunlight on photooxidation of rice leaf mutant 812HS and its wild-type 812S under field conditions. Light is important for plant growth and development. However, when the absorbed energy exceeds the capacity of utilization of photosynthesis, it leads to the accumulation of singlet oxygen molecules and other reactive oxygen species, which causes oxidative damage. Chlorophyll a fluorescence was applied to examine photosystem II photochemistry. The results demonstrated that intensive light had a negative influence on plant photosynthetic processes. However, the electron transport chain was inhibited and energy dissipation was increased, which can minimize photooxidative damage to the optical system. Meanwhile, proteomic analysis showed that the differential expression of proteins in response to photooxidation participated in multiple pathways, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit, RuBisCO large chain precursor, RuBisCO activase, flavodoxin-like quinone reductase 1, l-ascorbate peroxidase S, oxygen-evolving complex protein 1, and glycolate oxidase. The results indicated that photooxidation induced a response in the rice via the stress-related pathway. The aforementioned proteins, identified by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), may be very useful in comprehending how plants respond to photooxidation and can be used as characteristics of stress-induced signals. The results of chlorophyll fluorescence parameter analysis demonstrated the negative influence of intense light on plant photosynthetic processes. This was evidenced by the dissipation of excessive energy and the suppression of the electron transport chain to minimize photooxidative damage to the proteins. Future studies should compare the proteomic difference with parallel gene expression and metabolite profiles.     

Resolution of components of non-photochemical chlorophyll fluorescence quenching in barley leaves

Non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves has been analysed by monitoring its relaxation in the dark, by applying saturating pulses of light. At least three kinetically distinct phases to qN recovery are observed, which have previously been identified (Quick and Stitt 1989) as being due to high-energy state quenching (fast), excitation energy redistribution due to a state transition (medium) and photoinhibition (slow). However, measurements of chlorophyll fluorescence at 77 K from leaf extracts show that state transitions only occur in low light conditions, whereas the medium component of qN is very large in high light. The source of that part of the medium component not accounted for by a state transition is discussed.Abbreviations ATP adenosine 5-triphosphate - DCMU 3[3,4-dichlorophenyl]-1,1 dimethylurea - pH trans-thylakoid pH gradient - F_o, F_m room-temperature chlorophyll fluorescence yield with all reaction centres open, closed - F_v variable fluorescence = F_m–F_o - LHC II Light harvesting complex II - PS I, PS II Photosystem I, II - P700, P680 primary donor in photosystem I, II - qP photochemical quenching of variable fluorescence - qN non-photochemical quenching of variable fluorescence - qN_e, qN_t, qN_i non-photochemical quenching due to high energy state, state transition, photoinhibition - qN_f, qN_m, qN_s components of qN relaxing fast, medium, slow - q_r quenching of r relative to the dark state - tricine N-tris[hydroxymethyl]methylglycine - r ratio of fluorescence maximum from photosystem II to that from photosystem I at 77 K     

快速叶绿素荧光动力学及其在植物抗逆生理研究中的应用

快速叶绿素荧光动力学是研究光合原初反应的无损探针,可监测光合反应的多个事件,并能反应植物的生理状况,可以应用于环境物理及化学条件胁迫对植物的影响(如化肥及除草剂的使用;CO2、O2、O3浓度的变化及温度、光强度胁迫等);也应用于农业生产(如确定耕作方式及日常管理;除草剂、杀虫剂及激素的使用;品种选优等);更是光合生理研究的便捷工具。介绍了快速叶绿素荧光动力学中的JIP-test分析方法,并从光、水分、温度、盐等多个方面介绍了JIP-test方法在植物抗逆生理研究中的应用。     

Measurement of chlorophyll fluorescence within leaves using a fibreoptic microprobe

Abstract. The distribution of chlorophyll fluorescence was measured within leaves of Medicago saliva with a fibre optic microprobe. Leaves were irradiated with broad band blue light (1000 μmol m⁻²s⁻¹) and chlorophyll fluorescence was measured at 688 nm. The amount of fluorescence measured within the leaf depended upon the direction in which the probe was inserted. When the probe was advanced directly through the leaf from the shaded towards the irradiated surface, the maximum amount of detected fluorescence occurred near the boundary between the palisade and spongy mesophyll. When the probe was advanced through the leaf from the opposite direction maximum detected fluorescence was at the boundary between the epidermis and palisade. These results appear to be a consequence of the blue light gradient, which declined exponentially within the palisade but was counterbalanced by increasing chlorophyll content within the leaf. Modelling indicates that the measured distribution of chlorophyll fluorescence can be explained by relatively uniform emission of fluorescence throughout the palisade layer, indicating that the chloroplasts may be photosynthetically specialized to their light environment within the leaf.     

Chilling injury and recovery in detached and attached leaves measured by chlorophyll fluorescence

Smillie, R. M., Nott, R., Hetherington, S. E. and Öyustt, G. 1987. Chilling injury and recovery in detached and attached leaves measured by chlorophyll fluorescence Chilling injury was compared in detached and attached leaves chilled at 0 or 0.5°C by measuring the decrease in induced chlorophyll fluorescence in vivo. The fluorescence parameter measured was F_R, the maximal rate of rise of induced chlorophyll fluorescence emission after irradiating dark-adapted leaves. The plants used were bean, Phaseolus vulgaris L. cv. Pioneer, and maize, Zea mays L. cvs hybrid GH 390 and Northern Belle. Leaves were detached and placed on wet paper and covered with thin polyethylene film to prevent water loss during chilling. Leaves left attached on plants were treated similarly. When chilled in this way at 100% relative humidity, the chilling-induced decrease in F_R was the same in detached and attached leaves. For the attached leaves, the same result was obtained whether just a single leaf was chilled or the whole plant. Expression of chilling injury was greatest in fully turgid leaves and comparisons can be invalid unless the water status of the detached and attached leaves are the same. Problems arising from diurnal fluctuations in water potential of plants grown in a glasshouse were circumvented by placing leaves on the wet filter paper under polyethylene film prior to chilling, which allowed high water potentials to be regained, or mist sprays in the glasshouse were employed. Determinations of the time course for changes in F_R of maize (cv. Northern Belle) during chilling at 0°C showed that F_R decreased exponentially, at the same rate (time to 50% decrease in F_R was 9.3 h) in detached and attached leaves. Chilling injury was largely reversible for the first 20 h of chilling stress as both detached and attached leaves recovered their pre-chilling values of F_R after a further 20 h at 20°C in darkness. Leaves chilled for 48 h showed partial recovery, while those chilled for 72 h did not recover. Recovery was impeded by light. Inability to recover from chilling as indicated by measurements of F_R was paralleled by the incidence of visible symptoms of injury. It is concluded that detached and attached leaves behave similarly during chilling and short-term recovery, provided a similarity in treatments is rigorously maintained.     

The effect of sulphite on chlorophyll fluorescence and sucrose metabolism in poplar leaves

Sulphite at concentrations from 0.5 to 5.0 mM was supplied to illuminated, detached poplar (Populus deltoides Bartr. ex Marsh) leaves via the transpiration stream. Chlorophyll a fluorescence parameters, the contents of fructose-2,6-bisphosphate (Fru2,6BP) and starch, and extractable specific activity of sucrose-phosphate synthase (SPS), sucrose synthase (SuSy), acid invertase (AI), neutral invertase (NI), ATP-dependent fructose-6-phosphate 1-phosphotransferase (PFK) and pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) were measured. Chlorophyll fluorescence parameters appeared to be unaffected by sulphite. Application of ≥ 1.0 mM sulphite led to an increase in the content of Fru2,6BP and starch. There was also a decline in the activity of SPS, NI and PFK. On the other hand, the influence of sulphite on the activity of AI and PFP was negligible. Specific activity of SuSy was inhibited by 1.0 and 2.5 mM but activated by 5.0 mM of sulphite. On the basis of the results obtained in the present study, we postulate that sulphite at concentrations ≥ 1.0 mM inhibits primarily sucrose synthesis, favours starch accumulation and has an indirect effect on the sucrolytic activities in poplar leaves.     

Effects of prolonged freezing stress on the photosynthetic apparatus of moderately hardy leaves as assayed by chlorophyll fluorescence kinetics

Abstract Moderately frost-hardy leaves of the wintergreen broadleaf woody shrubs Pyracantha coccinea and Ligustrum ovalifolium and the winter annual herb Spinacia oleracea were subjected to extended freezing stress up to 15 d at temperatures 2–8°C above the mean lethal temperature (LT₅₀). After thawing, the fast kinetics of in vivo chlorophyll fluorescence of photosystem II (PSII) and the potential of linear photosynthetic electron transport of isolated thylakoid membranes was measured at room temperature. The lower the minimum freezing temperature and the longer the time of exposure, the greater was the suppression of the fluorescence signals of the leaves and decrease of the electron transport capacity of the thylakoid membranes. The pattern of inactivation of PSII -mediated electron flow, i.e. inhibition of photoreaction to photochemistry and/or electron donation to the photochemical reaction, during long-term freezing at temperatures somewhat above the LT₅₀ of the leaves was similar to that observed earlier after relatively brief exposure of leaves and isolated thylakoid membranes to more severe freezing stress. As injury occurred during freezing in complete darkness, it is likely that prolonged winter stress under natural environmental conditions causes changes in the photosynthetic apparatus of moderately hardy leaves which are not due to photoinhibition.     

The effects of chilling stress on the chlorophyll fluorescence of leaves

The effects of temperature lowering on the steady state chlorophyllfluorescence of leaves was investigated. Leaves from chill-sensitivespecies responded to a greater degree than did leaves from chill-resistantspecies. Arrhenius plots of fluorescence change vs temperatureyielded a straight line with no breaks that would have indicateda membrane lipid phase change. We measured the fluorescencechanges at high and low excitation intensitites and monitoredfluorescence at 690 and 735 nm. The low temperature inducedfluorescence increase and the ratio of 690 to 735 nm fluorescencewere generally greater at lower light intensities. Treatmentwith DCMU reduced the effects of temperature on fluorescence.However, the effect of low intensity pulsing light on the fluorescenceyield of DCMU-treated leaves proved to be temperature dependent.At a low pulse rate (2 msec pulse, 5 pps) a logarithmic increasein fluorescence as a function of temperature was noted. Chill-sensitivespecies proved to be more responsive than chill-resistant species.Slight differences between hardened and unhardened materialwere noted. While the causal factors for the differential sensitivityof species to temperature lowering were not investigated, fluorescencemonitoring has proved to be a convenient and accurate methodfor investigating chill-sensitivity in leaves. ¹ Contribution from the Missouri Agricultural Experiment Station.Journal Series Number 7791.² Present address: Florigen Greenhouses, 1351 E. Silver LakeRoad, Traverse City, Michigan 49684, U.S.A.³ Present address: Department of Forestry, Iowa State University,Ames, Iowa 50011, U.S.A. (Received March 14, 1977; )     

Effects of chill stress on prompt and delayed chlorophyll fluorescence from leaves

This paper describes the utilization of a portable solid state device for the simultaneous measurement of prompt and delayed fluorescence transients in leaves from a variety of species subjected to temperature lowering. The induction transients of the two phenomena were not identical as the peak in prompt fluorescence yield always preceded that of delayed fluorescence. Temperature lowering delayed the occurrence of peak fluorescence, increased prompt fluorescence yield, decreased delayed fluorescence yield, and caused the occurrence of a new, more rapid delayed fluorescence transient. Leaves from all species had qualitatively the same type of induction curves although the response to temperature differed between species. The delayed fluorescence yield of chill-sensitive species was reduced to a greater extent than that of chill-insensitive species. Cold hardening leaf material did not greatly change the fluorescence response to temperature lowering. Arrhenius plots showed a linear relationship between delayed fluorescence yield and temperature. There were no breaks that would suggest membrane lipid phase changes. The data indicate that thylakoid membranes of chill-sensitive species are less capable of maintaining a light-induced high energy state at low temperatures than are thylakoid membranes of chill-resistant species.     

Effects of exogenous putrescine on gas-exchange characteristics and chlorophyll fluorescence of NaCl-stressed cucumber seedlings

The effects of 10 mM putrescine (Put) treated by spraying on leaves on growth, chlorophyll content, photosynthetic gas-exchange characteristics, and chlorophyll fluorescence were investigated by growing cucumber plants (Cucumis sativus L. cv. ChangChun mici) using hydroponics with or without 65 mM NaCl as a salt stress. Salt stress caused the reduction of growth such as leaf area, root volume, plant height, and fresh and dry weights. Furthermore, net photosynthesis rate (P _n), stomatal conductance (g _s), intercellular CO₂ concentration (C _i), and transpiration rate (T _r) were also reduced by NaCl, but water use efficiency (WUE; P _n/T _r) showed a tendency to be enhanced rather than reduced by NaCl. However, Put alleviated the reduction of P _n by NaCl, and showed a further reduction of C _i by NaCl. The reduction of g _s and T _r by NaCl was not alleviated at all. The enhancement of WUE by NaCl was shown to have no alleviation at day 1 after starting the treatment, but after that, the enhancement was gradually reduced till the control level. Maximum quantum efficiency of PSII (F _v/F _m) showed no effects by any conditions based on the combination of NaCl and Put, and in addition, kept constant values in plants grown in each nutrient solution during this experimental period. The efficiency of excitation energy capture by open photosystem II (PSII) (F _v′/F _m′), actual efficiency of PSII (Φ_PSII), and the coefficient on photochemical quenching (qP) of plants with NaCl were reduced with time, and the reduction was alleviated till the control level by treatment with Put. The F _v′/F _m′, Φ_PSII, and qP of plants without NaCl and/or with Put showed no variation during the experiment. Non-photochemical quenching of the singlet excited state of chlorophyll a (NPQ) showed quite different manner from the others as mentioned above, namely, continued to enhance during the experiment.