The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence

BIOPHYSICAL BASIS O F FLUORESCENCE EMISSION FROM CHLOROPLASTS . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 314 Fluorescence as a Reaction Competing in the Deactivation of Excited Chlorophyll . . . . . . . . . . ... . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 Lifetimes of Fluorescence . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 317 Origin of Fluorescence Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 1 Fluorescence of PS 11 and PS I at Ambient and Low Temperatures . . . . . . . . . . . . . . . . . . . 323 FLUORESCENCE INDUCTION AND PS II HETEROGENEITy 325 Fluorescence Transient from Fo to FM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 The FI Level and Inactive PS11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... .... . .. . . . .. . . . . . . . . . 326 Fluorescence Induction in High Ught .. . . . . . . . . 327 Rise in the Presence of DCMU and a/{3 Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 FLUORESCENCE QUENCHING 329 Resolution of Quenching Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ........ . .. . . . . . . . . . . . . . . 330 Mechanism of Energy·Dependent Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 1 Quenching Related t o State Transition . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . ......... . . .. .. . . . . . . . 334 Photoinhibitory Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 Further Quenching Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Physiological Aspects of Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 338 CONCLUSIONS AND PERSPECTiVES 341

Chlorophyll Fluorescence and Photosynthesis: The Basics

The use of chlorophyll fluorescence to monitor photosynthetic performance in algae and plants is now widespread. This review examines how fluorescence parameters can be used to evaluate changes in photosystem II (PSII) photochemistry, linear electron flux, and CO(2) assimilation in vivo, and outlines the theoretical bases for the use of specific fluorescence parameters. Although fluorescence parameters can be measured easily, many potential problems may arise when they are applied to predict changes in photosynthetic performance. In particular, consideration is given to problems associated with accurate estimation of the PSII operating efficiency measured by fluorescence and its relationship with the rates of linear electron flux and CO(2) assimilation. The roles of photochemical and nonphotochemical quenching in the determination of changes in PSII operating efficiency are examined. Finally, applications of fluorescence imaging to studies of photosynthetic heterogeneity and the rapid screening of large numbers of plants for perturbations in photosynthesis and associated metabolism are considered.

Chlorophyll fluorescence: a probe of photosynthesis in vivo

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Superoxide Radical and Superoxide Dismutases

Photon yields of oxygen evolution at saturating CO2 were determined for 44 species of vascular plants, representing widely diverse taxa, habitats, life forms and growth conditions. The photonyield values on the basis of absorbed light (φa) were remarkably constant among plants possessing the same pathway of photosynthetic CO2 fixation, provided the plants had not been subjected to environmental stress. The mean φa value ±SE for 37 C3 species was 0.106±0.001 O2·photon-1. The five C4 species exhibited lower photon yields and greater variation than the C3 species (φa=0.0692±0.004). The φa values for the two Crassulaceanacid-metabolism species were similar to those of C3 species. Leaf chlorophyll content had little influence on φa over the range found in normal, healthy leaves. Chlorophyll fluorescence characteristics at 77 K were determined for the same leaves as used for the photon-yield measurements. Considerable variation in fluorescence emission both at 692 nm and at 734 nm, was found 1) among the different species; 2) between the upper and lower surfaces of the same leaves; and 3) between sun and shade leaves of the same species. By contrast, the ratio of variable to maximum fluorescence emission at 692 nm (Fv/FM, 692) remained remarkably constant (The mean value for the C3 species was 0.832±0.004). High-light treatments of shade leaves resulted in a reduction in both φa and the Fv/FM, 692 ratio. The extent of the reductions increased with time of exposure to bright light. A linear relationship was obtained when φa was plotted against Fv/FM, 692. The results show that determinations of the photon yield of O2 evolution and the Fv/FM, 692 ratio can serve as excellent quantitative measures of photoinhibition of overall photosynthetic energy-conversion system and of photochemistry of photosystem II, respectively. This is especially valuable in field work where it is often impossible to obtain appropriate controls.

Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins.

Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone.

Energy Distribution in the Photochemical Apparatus of Photosynthesis

Responses of plant materials to radiation indicate that flowering and many other aspects of development are controlled by a reversible photoreaction1'2 involving two forms of a pigment, with action maxima near 660 and 735 mg. The photoreversible pigment can readily be changed from one to the other form, as indicated by response of the plant to irradiation in the region of the appropriate action maximum. Because the nature of the enzymatic action involved is still unknown, it appeared that direct observation of the pigment in the living material and an assay for its isolation would have to be based on spectrophotometric methods. The pigment should show a change in absorption at 655 and 735 mjA following conversion by radiation. The location of the pigment and its concentration in specific cells are evidenced by the photoinduced formation of anthocyanin, which depends upon energy transfer from both forms of the excited pigment.3 The concentration based on a molar absorptivity of 1 X 105 is estimated to be the order of 10-6 molar in the most effective cells and probably about 10-7 molar in the average tissue.4 A spectrophotometer suitable for detecting this low concentration of the pigment in tissue must measure absorption of radiation with high sensitivity in dense light-scattering material. Such measurements cannot be made with commercial instruments. Instrumentation.-The presence of the photoreversible pigment in intact tissue has been demonstrated with a recording, single-beam spectrophotometer. This spectrophotometer5 employs an end-window multiplier-type phototube placed directly behind the sample to collect a large fraction of the transmitted light. The sample is illuminated by monochromatic light from the exit slit of a double, prism monochromator. Spectral measurements can be made on light-scattering samples having optical densities between 0 and 6, with a sensitivity as high as 0.1 for full scale deflection. The noise level is equivalent to an optical density change of 0.002 for samples having an optical density less than 4. Since this is a single-beam instrument the recorded curve includes the spectral response of the instrument in addition to the absorption characteristics of the sample. The system response is sufficiently reproducible that valid difference spectra can be obtained by subtracting one recorded curve from another. When the spectral curve is very steep in the region of interest, an electrical compensation can be applied to alter the slope of the curve. This is achieved with a potentiometer geared to the wavelength drum which supplies an additional signal to the Y-axis. The compensation merely alters the system response to make it easier to compute difference spectra. A more useful instrument for assay of the pigment is one which measures directly the optical density difference between two fixed wavelengths. Such an instrument,

https://www.pnas.org/content/pnas/45/12/1703.full.pdf

Detection, assay, and preliminary purification of the pigment controlling photoresponsive development of plants.

Fluorescence quenching in Photosystem II of chloroplasts

— –A small computer on line with a single-beam spectrophotometer was used to obtain absorption spectra and higher derivatives (up to the fourth) of the absorption spectra. The spectral resolution was markedly enhanced in the higher derivative curves. The computer was also used to synthesize absorption spectra from Gaussian, Lorentzian and mixed Gaussian-Lorentzian band shapes in order to explore the higher derivative analysis of completely defined spectra.

Warren L. Butler

Papers

Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone.

Energy Distribution in the Photochemical Apparatus of Photosynthesis

Detection, assay, and preliminary purification of the pigment controlling photoresponsive development of plants.

Fluorescence quenching in Photosystem II of chloroplasts

Higher derivative analysis of complex absorption spectra