Localization of Cytochrome b-559 in the Chloroplast Thylakoid Membranes in Spinach.

Cytochrome b-559 was purified from spinach leaves and antibodies were made against it in rabbit. Using affinity-purified, monospecific antibodies, we have found that cytochrome b-559, which is closely associated with the primary photochemical activity of photosystem II, is localized exclusively in the grana thylakoids.

Fluorescence lifetimes in the bipartite model of the photosynthetic apparatus with alpha, beta heterogeneity in photosystem II.

Recent studies of the lifetime of fluorescence after picosecond pulse excitation of photosynthetic organisms revealed relatively complex decay kinetics that indicated a sum of three exponential components with lifetimes spanning the range from about 0.1-2.5 ns. These fluorescence lifetime data were examined in the context of a simple photochemical model for photosystem II that was used previously to account for fluorescence yield data obtained during continuous illumination. The model, which consists of a single fluorescing species of antenna chlorophyll and a reaction center, shows that, in general, the decay kinetics after pulse excitation should consist of the sum of two exponential decays. The model also shows that in going from open to closed reaction centers the lifetime of fluorescence may increase much more than the yield of fluorescence and surprisingly long fluorescence lifetimes can be obtained. However, conditions can be stated where fluorescence will decay essentially as a single component and with lifetime changes that are proportional to the yield changes. A heterogeneity was also introduced to distinguish photosystem II(alpha) units, which can transfer excitation energy among themselves but not the photosystem I, and photosystem II(beta) units, which can transfer energy to photosystem I but not to other photosystem II units. It is proposed that the rather complex fluorescence lifetime data can be accounted for in large part by the simple photochemical model with the alpha, beta heterogeneity in photosystem II.

Localization of phytochrome in oats by electron microscopy.

Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction.

Intracellular Localization of Nitrate Reductase in Neurospora crassa.

Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall-plasmalemma region and in the tonoplast membranes.

Localization of phytochrome in oats by electron microscopy.

Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction.

Intracellular Localization of Nitrate Reductase in Neurospora crassa.

Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall-plasmalemma region and in the tonoplast membranes.

Energy distribution in the photochemical apparatus of Porphyridium cruentum in state I and state II.

Fluorescence of Porphyridium cruentum in state I (cells equilibrated in light absorbed predominantly by Photosystem I) and in state II (cells equilibrated in light absorbed appreciably by Photosystem II) was examined to determine how the distribution of excitation energy was altered in the transitions between state I and state II. Low temperature emission spectra of cells frozen state I and state II confirmed that a larger fraction of the excitation energy is delivered to Photosystem II in state I. Low temperature measurements showed that the yield of energy transfer from Photosystem II to Photosystem I was greater in state II and calculations indicated that the photochemical rate constant for such energy transfer was approximately twice as large in state II. Measurements at low temperature also showed that the cross sections and the spectral properties of the photosystems did not change in the transitions between state I and state II. In agreement with predictions made from the parameters measured at low temperature, the action spectra for oxygen evolution measured at room temperature were found to be the same in state I and state II.

Energy transfer between photosystem II units in a connected package model of the photochemical apparatus of photosynthesis.

A model of the photochemical apparatus of photosynthesis, presented previously in a tripartite format, is used in a bipartite format to analyze energy transfer between photosystem II units. The model is used to develop analytical expressions for the photochemical properties of chloroplasts that include a term for the probability for energy transfer between photosystem II units. In particular, a normalized plot of the fluorescence of variable yield as a function of the closure of photosystem II reaction centers permits the evaluation of the energy transfer parameter. Data from a study by Melis [Melis, A. (1978) FEBS Lett. 95, 202-206] are used to estimate that the probability for energy transfer between photosystem II units in the alpha component of photosystem II was approximately 0.57 when the photosystem II reaction centers were all open and 0.83 when the centers were closed. This connected package model appears to provide a better description of the system than the matrix model that was used previously.

Effects of Chromatic Adaptation on the Photochemical Apparatus of Photosynthesis in Porphyridium cruentum.

Cells of Porphyridium cruentum were grown in different colors of light which would be absorbed primarily by chlorophyll (Chl) (red and blue light) or by the phycobilisomes (green or two intensities of cool-white fluorescent light), and samples of these cells were frozen to -196 C for measurements of absorption and fluorescence emission spectra. Cells grown in the high intensity white light had least of all of the photosynthetic pigments, a higher ratio of carotenoid/Chl, but essentially the same ratio of phycobilin to Chl as cells grown in the low intensity white light. The ratio of photosystem II (PSII) to photosystem I (PSI) pigments was affected by light quality; the ratios of phycobilin to Chl and of short wavelength (PSII) Chl to long wavelength (PSI) Chl were both greater in the cells grown in red or blue light.Light quality also exerted a strong influence on the structural and functional organization of the photochemical apparatus. Data on the relative optical cross-sections of PSI and PSII as a function of excitation wavelength indicate that cells grown in light absorbed primarily by the phycobilisomes package a large fraction of their Chl into PSI (PSI Chl/PSII Chl approximately 20), whereas cells grown in light absorbed by Chl distribute their Chl much more equitably (PSI Chl/PSII Chl approximately 1.5). In both types of cells the phycobilisomes transfer their excitation energy predominantly to PSII Chl with little or no direct energy transfer to PSI, but the yield of energy transfer from PSII to PSI is approximately twice as large for cells grown in the phycobilin wavelengths of light. These differences in functional organization and energy distribution account for the physiological expressions of chromatic adaptation. The effects of chromatic adaptation on O(2) evolution can be predicted from our calculations of energy distribution between PSI and PSII for cells grown in the different colors of light.

The relationship between the lifetime and yield of the 735 nm fluorescence of chloroplasts at low temperatures.

The lifetime and relative yield of the 735 nm fluorescence of chloroplasts, over a range of low temperatures (-60 to -196 degrees C) where the yield of fluorescence changes markedly, were found to be directly proportional. It is concluded that the species of chlorophyll responsible for the 735 nm fluorescence, C-705, is present over the entire temperature range but is less fluorescent at the higher temperatures because of greater energy transfer to P-700. It is also concluded from attempts to measure the rise-time of the 735 nm fluorescence at -196 degrees C that the rise-time is less than 50 ps.

Competition between the 735 nm fluorescence and the photochemistry of Photosystem I in chloroplasts at low temperature.

Fluorescence emission spectra of chloroplasts, initially frozen to--196 degrees C, were measured at various temperatures as the sample was allowed to warm. The 735 nm emission band attributed to fluorescence from Photosystem I was approx. 10-fold greater at--196 degrees C than at--78 degrees C. The initial rate of photooxidation of P-700 was also measured at--196 degrees C and--78 degrees C and was found to be approximately twice as large at the higher temperature. It is proposed that the 735 nm emission band is fluorescence from a long wavelength form of chlorophyll, C-705, which acts as a trap for excitation energy in the antenna chlorophyl system of Photosystem I. Furthermore, it is proposed that C-705 only forms on cooling to low temperatures and that the temperature dependence of the 735 nm emission is the temperature dependence for the formation of C-705. C-705 and P-700 compete to trap the excitation energy in Photosystem I. It is estimated from the data that at--78 degrees C P-700 traps approx. 20 times more energy than C-705 while, at--196 degrees C, the two traps are approximately equally effective. By analogy, the 695 nm fluorescence which also appears on cooling to--196 degrees C is attributed to traps in Photosystem II which form only on cooling to temperatures near--196 degrees C.

Low temperature spectral properties of subchloroplast fractions purified from spinach.

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F(I), F(II), and F(III), corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at -196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F(II) (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at -196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F(I), F(II), and F(III) at -196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F(I) showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F(II) and F(III) in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F(I) and F(II) caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at -196 C with an increase in the yield of fluorescence especially in the case of F(I).

Tripartite and bipartite models of the photochemical apparatus of photosynthesis.

Tripartite and bipartite models for the photochemical apparatus of photosynthesis are presented and examined. It is shown that the equations for the yields of fluorescence from the different parts of the photochemical apparatus of the tripartite model transform into the simple equations of the bipartite formulation when the probability for energy transfer from the light-harvesting chlorophyll a/b complex to photosystem II is unity. The nature of the 695 and 735 nm fluorescence bands which appear in the emission spectrum of chloroplasts at low temperature is examined. It is proposed that these bands are due to fluorescence from energy-trapping centres which form in the antenna chlorophyll of photosystem II and photosystem I on cooling to low temperature. Even though these fluorescence emissions can be regarded as low temperature artifacts since they are not present at physiological temperatures, they nevertheless are proportional to the excitation energy in the two photosystems and can be used to monitor energy distribution in the photochemical apparatus. However, the question of their artifactual nature is crucial to the interpretation of fluorescence-lifetime measurements at low temperature.

Does the rate of cooling affect fluorescence properties of chloroplasts at -196 degrees C?

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to -196 degrees C rapidly, as very thin samples adsorbed into substrates whick were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum.

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of porphyridium cruentum which had been frozen to -196 degrees C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at -196 degrees C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants.

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.