Measurement of the fluorescence lifetime of chlorophyll a in vivo

New measurements have been made of fluorescence lifetime (τ) of chlorophyll a in the algae Chlorella pyrenoidosa, Porphyridium cruentum, Anacystis nidulans, and in spinach chloroplast. τ-values of 0.6 and 0.7 nsec were obtained with green plants. Anacystis and Porphyridium gave a τ of 0.5 nsec. The previously described two stage decay of fluorescence in vivo in these organisms could not be confirmed. This observation could have been caused by a second wave of light emission from the exciting hydrogen lamp (not detected in earlier work). The lifetimes found in this study (calculated, as before, by the method of convolution integrals) were close to those found by other observers for “low” excitation intensities; the value first reported from this laboratory (1.0-1.7 nsec) may have corresponded to “high” excitation intensity.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Events: Lifetime of the Excited State In Vivo: II. Bacteriochlorophyll in Photosynthetic Bacteria at Room Temperature

Lifetime of the excited state (τ) of bacteriochlorophyll (BChl) in photosynthetic bacteria, measured with a mode-locked argon laser (oscillating at 488 nm; mode locked at 56 MHz) as light source, ranged from 0.3 to 2.5 nsec. These τ values are reported with a precision of ±0.1 nsec. The value of τ at high exciting light intensity (I) was two to three times that at low intensity. For young cultures of green bacterium Chloropseudomonas ethylicum, τ ranged from 0.5 (low I) to 1.0 nsec (high I); for those of the purple bacterium Rhodospirillum rubrum, from 0.4 (low I) to 1.0 nsec (high I); and for those of the BChl b-containing Rhodopseudomonas viridis, from 1.0 (low I) to 2.5 nsec (high I). These data provide information regarding the efficiencies of the photochemical process in these bacteria. Quantum yield (ø) of BChl fluorescence, calculated from ø = τ/τ₀ (where τ₀ is the intrinsic lifetime of fluorescence), ranges from 2-6% at low intensities to 6-14% at high intensities.     

Studies on the Second Emerson Effect in the Hill Reaction in Algal Cells

This paper shows that the “second Emerson effect”¹ exists not only in photosynthesis, but also in the quinone reduction (Hill reaction), in Chlorella pyrenoidosa and Anacystis nidulans. The peaks at 650 mμ, 600 mμ, 560 mμ, 520 mμ, and 480 mμ, observed in the action spectrum of this effect in the Hill reaction in Chorella, are attributable to chlorophyll b; the occurrence of an additional peak at 670 mμ, 620 mμ, and of two (or three) peaks in the blueviolet region suggests that (at least) one form of chlorophyll a contributes to it. In analogy to suggestions made previously in the interpretation of the Emerson effect in photosynthesis, these results are taken as indicating that excitation by light preferentially absorbed by one (or two) forms of chlorophyll a (Chl a 690 + 700), needs support by simultaneous absorption of light in another form of chlorophyll a (Chl a 670)—directly or via energy transfer from chlorophyll b—in order to produce the Hill reaction with its full quantum yield. In Anacystis, the participation of phycocyanin in the Emerson effect in the Hill reaction is revealed by the occurrence, in the action spectrum of this effect, of peaks at about 560 mμ, 610 mμ, and 640 mμ; a peak at 670 mμ, due to Chl a 670, also is present.     

Inhibition of Photosynthesis in Certain Algae by Extreme Red Light

This paper shows that in Porphyridium cruentum and in Chlorella pyrenoidosa (but apparently not in Anacystis nidulans) “extreme red” light (> 720 mμ) can inhibit photosynthesis produced by “far red” light (up to 720 mμ). From the action spectrum of this phenomenon, it appears that an unknown pigment with an absorption band around 745 mμ must be responsible for it.     

Red Drop in the Quantum Yield of Fluorescence of Sonicated Algae

The change of the quantum yield of fluorescence, Φ, with the frequency of exciting light, was investigated in Chlorella, Anacystis, and Porphyridium suspensions, and in sonicates from these cells prepared under aerobic and anaerobic conditions. In case of Chlorella, sonicates were made in acid and in alkaline media (pH 4.65 and 7.80). In the alkaline medium, a drop of Φ towards the longer waves was found to begin at 1.466 × 10⁴ cm^-1 (682 nm) in sonicates, and in suspension. In the acid medium, the drop began at 1.471 × 10⁴ cm^-1 (680 nm), 1.418 × 10⁴ cm^-1 (705 nm), and 1.389 × 10⁴ cm^-1 (720 nm) in suspension, anaerobic sonicate, and aerobic sonicate, respectively. The results indicate that the cause of the change in the red drop is preferential destruction of a long-wave component of chlorophyll a (such as Chl a 693). The amount of this component remaining after sonication is larger in alkaline than in acid sonicates. With Anacystis and Porphyridium, only alkaline suspensions (pH 7.80) could be used for sonication, because in acid medium, the phycobilin-chlorophyll complex is rapidly broken and phycobilin extracted from the cell. In Anacystis, the red drop begins at 1.562 × 10⁴ cm^-1 (640 nm) and 1.538 × 10⁴ cm^-1 (650 nm) in suspension and sonicate, respectively; in Porphyridium, it starts at 1.550 × 10⁴ cm^-1 (645 nm) in both cases. These results suggest that sonication in alkaline medium (pH 7.80) destroys some Chl a 693 in Anacystis, but not in Porphyridium.     

Alteration of Components of Chlorophyll-Protein Complexes and Distribution of Excitation Energy Between the Two Photosystems in Two New Rice Chlorophyll b-less mutants

Two new yellow rice chlorophyll (Chl) b-less (lack) mutants VG28-1 and VG30-5 differ from the other known Chl b-less mutants with larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small sub-unit and smaller amounts of Chl a. We investigated the altered features of Chl-protein complexes and excitation energy distribution in these two mutants, as compared with wild type (WT) rice cv. Zhonghua 11 by using native mild green gel electrophoresis and SDS-PAGE, and 77 K Chl fluorescence in the presence of Mg²⁺. WT rice revealed five pigment-protein bands and fourteen polypeptides in thylakoid membranes. Two Chl b-less mutants showed only CPI and CPa pigment bands, and contained no 25 and 26 kDa polypeptides, reduced amounts of the 21 kDa polypeptide, but increased quantities of 32, 33, 56, 66, and 19 kDa polypeptides. The enhanced absorption of CPI and CPa and the higher Chl fluorescence emission ratio of F685/F720 were also observed in these mutants. This suggested that the reduction or loss of the antenna LHC1 and LHC2 was compensated by an increment in core component and the capacity to harvest photon energy of photosystem (PS) 1 and PS2, as well as in the fraction of excitation energy distributed to PS2 in the two mutants. 77 K Chl fluorescence spectra of thylakoid membranes showed that the PS1 fluorescence emission was shifted from 730 nm in WT rice to 720 nm in the mutants. The regulation of Mg²⁺ to excitation energy distribution between the two photosystems was complicated. 10 mM Mg²⁺ did not affect noticeably the F685/F730 emission ratio of WT thylakoid membranes, but increased the ratio of F685/F720 in the two mutants due to a reduced emission at 685 nm as compared to that at 720 nm.     

Relationship between the Absorption and Emission Spectra and the "Red Drop" in the Action Spectra of Fluorescence In Vivo

The Stepanov equation, relating the intensity of emission, f_e($\bar{v}$), at a given frequency, and that of absorption, k($\bar{v}$), at the same frequency, is applied, in its modified form (see equation 3 in text) to suspensions of Chlorella, Porphyridium, and Anacystis and to chlorophyll solutions. This application can reveal whether the yield of fluorescence, Φ($\bar{v}$), is constant, or changes with frequency. In Chlorella (green alga) a sharp drop of Φ($\bar{v}$) is indicated towards the lower frequencies (longer waves), beginning around $\bar{v}$ = 1.48 × 10⁴cm^-1 (680 mμ); the Φ($\bar{v}$) function calculated from the Stepanov equation is in fair agreement with the directly determined action spectrum for the excitation of chlorophyll fluorescence in this organism. In Porphyridium (red alga) and Anacystis (blue-green alga) application of the Stepanov equation supports the conclusions, derived from direct measurements, of a much earlier “red drop” of the fluorescence excitation spectra. Direct measurements suggest that the drop in Porphyridium may begin at about 1.53 × 10⁴cm^-1 (654 mμ); in Anacystis, it may begin already above 1.57 × 10⁴cm^-1 (<637mμ). These results confirm the relation, postulated earlier by Duysens and others, between the action spectra of photosynthesis and of chlorophyll a fluorescence in algal cells. The relation of these findings to spectroscopic evidence, suggesting the existence of two main chlorophyll a components in vivo, in green as well as in red and blue-green algae, is discussed.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

Photoacclimation in the Red Alga Porphyridium cruentum: Changes in Photosynthetic Enzymes, Electron Carriers, and Light-Saturated Rate of Photosynthesis as a Function of Irradiance and Spectral Quality

Acclimation of the photosynthetic apparatus to changes in the light environment was studied in the unicellular red alga Porphyridium cruentum (American Type Culture Collection No. 50161). Absolute or relative amounts of four photosynthetic enzymes and electron carriers were measured, and the data were compared with earlier observations on light-harvesting components (F.X. Cunningham, Jr., R.J. Dennenberg, L. Mustárdy, P.A. Jursinic, E. Gantt [1989] Plant Physiol 91: 1179-1187; F.X. Cunningham, Jr., R.J. Dennenberg, P.A. Jursinic, E. Gantt [1990] Plant Physiol 93: 888-895) and with measurements of photosynthetic capacity. P_max, the light-saturated rate of photosynthesis on a chlorophyll (Chl) basis, increased more than 4-fold with increase in growth irradiance from 6 to 280 μeinsteins·m⁻²·s⁻¹. Amounts of ferredoxin-NADP⁺ reductase, ribulose-1,5-bisphosphate carboxylase, and cytochrome f increased in parallel with P_max, whereas numbers of the light-harvesting complexes (photosystem [PS] I, PSII, and phycobilisomes) changed little, and ATP synthase increased 7-fold relative to Chl. The calculated minimal turnover time for PSII under the highest irradiance, 5 ms, was thus about 4-fold faster than that calculated for cultures grown under the lowest irradiance (19 ms). A change in the spectral composition of the growth light (irradiance kept constant at 15 μeinsteins·m⁻²·s⁻¹) from green (absorbed predominantly by the phycobilisome antenna of PSII) to red (absorbed primarily by the Chl antenna of PSI) had little effect on the amounts of ribulose-1,5-bisphosphate carboxylase, ATP synthase, and phycobilisomes on a Chl, protein, or thylakoid area basis. However, the number of PSI centers declined by 40%, cytochrome f increased by 40%, and both PSII and ferredoxin-NADP⁺ reductase increased approximately 3-fold on a thylakoid area basis. The substantial increase in ferredoxin-NADP⁺ reductase under PSI light is inconsistent with a PSI-mediated reduction of NADP as the sole function of this enzyme. Our results demonstrate a high degree of plasticity in content and composition of thylakoid membranes of P. cruentum.     

Mechanism of the light state transition in photosynthesis. IV. Picosecond fluorescence spectroscopy of Anacystis nidulans and Porphyridium cruentum in state 1 and state 2 at 77 K

The sequential energy-transfer pathway through the phycobilin pigments to chlorophyll a was investigated as a function of the state transition in the cyanobacterium Anacystis nidulans and the red alga Porphyridium cruentum. The fluorescence decay kinetics of the phycobilin pigments and chlorophyll a were determined for cells frozen at 77 K in state 1 and state 2 using a single-photon timing fluorescence spectroscopy apparatus with picosecond resolution. Time-resolved 77 K fluorescence emission spectra were also obtained for both species in state 1 and state 2. In both A. nidulans and P. cruentum the transition to state 1 was accompanied by a large increase in the apparent fluorescent lifetime of chlorophyll a associated with PS II (emission peak at 695 nm). There were smaller increases in the lifetime of the terminal phycobilin emitter (685 nm) in both species and no change in phycocyanin (645 nm) or allophycocyanin (660 nm). Time-resolved spectra showed sequential emission from phycocyanin, allophycocyanin, the terminal phycobilin emitter and chlorophyll a. Spectral red shifts were observed with time for all emission peaks with the exception of the terminal phycobilin emitter. In A. nidulans this peak showed a small blue shift with time. The results are interpreted as evidence for an effective uncoupling of PS II chlorophyll a from subsequent energy transfer to PS I chlorophyll a upon transition to state 1. Our recently proposed model for the mechanism of the state transition in phycobilisome-containing organisms is discussed in terms of a decrease in the energy transfer overlap between PS II chlorophyll a and PS I chlorophyll a in state 1.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

Migration of Electronic Energy from Chlorophyll b to Chlorophyll a in Solutions

Absorption, emission, and fluorescence excitation spectra of pure solutions of chlorophyll a (Chl a) and chlorophyll b (Chl b) in diethyl ether and of equimolecular mixed solutions of the two pigments, were determined at room temperature as functions of concentration (in the range from 5 × 10^-6 M to 4 × 10^-3 M) and of wavelength of the exciting light (in the regions 380-465 and 550-650 nm). The efficiency of energy transfer from Chl b to Chl a, derived from these data, was found to depend on the wavelength of exciting light. Furthermore, the transfer efficiency calculated from sensitization of Chl a fluorescence by Chl b was substantially smaller than that calculated from quenching of Chl b fluorescence by Chl a. Both these effects are tentatively explained as evidence of superposition of a “fast” energy transfer (taking place before the Boltzmann distribution of vibrational energy had been reached) upon the “delayed” transfer, which takes place after vibrational equilibration. The first-named mechanism is made possible by overlapping of the absorption bands of the two pigments; the second, by overlapping of the emission band of Chl b and the absorption band of Chl a. The first mechanism can lead to repeated transfer of excitation energy between pigment molecules, the second only to a one-time transfer from the donor to the acceptor. Both mechanisms could be of the same, second-order type, with the transfer rate proportional to r^-6. An alternative is for the fast mechanism to be of the first order, with the transfer rate proportional to r^-3, but spectroscopic evidence seems to make this alternative less probable.     

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F₀ level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λ_max^em ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ_max^em = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ_max^em = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ_max^em = 685 nm) derives from dead chlorophyll. (b) At the F_max level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F₀ level, from closed PS II α-centers (τ ≈ 2.2 ns, λ_max^em = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ_max^em = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).     

Interaction Effects of Light,Temperature and Nutrient Limitations (N,P and Si) on Growth,Stoichiometry and Photosynthetic Parameters of the Cold-Water Diatom Chaetoceros wighamii

Light (20-450 μmol photons m^-2 s^-1), temperature (3-11°C) and inorganic nutrient composition (nutrient replete and N, P and Si limitation) were manipulated to study their combined influence on growth, stoichiometry (C:N:P:Chl a) and primary production of the cold water diatom Chaetoceros wighamii. During exponential growth, the maximum growth rate (~0.8 d^-1) was observed at high temperture and light; at 3°C the growth rate was ~30% lower under similar light conditions. The interaction effect of light and temperature were clearly visible from growth and cellular stoichiometry. The average C:N:P molar ratio was 80:13:1 during exponential growth, but the range, due to different light acclimation, was widest at the lowest temperature, reaching very low C:P (~50) and N:P ratios (~8) at low light and temperature. The C:Chl a ratio had also a wider range at the lowest temperature during exponential growth, ranging 16-48 (weight ratio) at 3°C compared with 17-33 at 11°C. During exponential growth, there was no clear trend in the Chl a normalized, initial slope (α*) of the photosynthesis-irradiance (PE) curve, but the maximum photosynthetic production (P_m) was highest for cultures acclimated to the highest light and temperature. During the stationary growth phase, the stoichiometric relationship depended on the limiting nutrient, but with generally increasing C:N:P ratio. The average photosynthetic quotient (PQ) during exponential growth was 1.26 but decreased to <1 under nutrient and light limitation, probably due to photorespiration. The results clearly demonstrate that there are interaction effects between light, temperature and nutrient limitation, and the data suggests greater variability of key parameters at low temperature. Understanding these dynamics will be important for improving models of aquatic primary production and biogeochemical cycles in a warming climate.     

Action spectra for nitrate and nitrite assimilation in blue-green algae

Action spectra for the assimilation of nitrate and nitrite have been obtained for several blue-green algae (cyanobacteria) with different accessory pigment composition. The action spectra for both nitrate and nitrite utilization by nitrate-grown Anacystis nidulans L-1402-1 cells exhibited a clear peak at about 620 nanometers, corresponding to photosystem II (PSII) C-phycocyanin absorption, the contribution of chlorophyll a (Chl a) being barely detectable. The action spectrum for nitrate reduction by a nitrite reductase mutant of A. nidulans R2 was very similar. All these action spectra resemble the fluorescence excitation spectrum of cell suspensions of the microalgae monitored at 685 nanometers—the fluorescence band of Chl a in PSII. In contrast, the action spectrum for nitrite utilization by nitrogen-starved A. nidulans cells, which are depleted of C-phycocyanin, showed a maximum near 680 nanometers, attributable to Chl a absorption. The action spectrum for nitrite utilization by Calothrix sp. PCC 7601 cells, which contain both C-phycoerythrin and C-phycocyanin as PSII accessory pigments, presented a plateau in the region from 550 to 630 nanometers. In this case, there was also a clear parallelism between the action spectrum and the fluorescence excitation spectrum, which showed two overlapped peaks with maxima at 562 and 633 nanometers. The correlation observed between the action spectra for both nitrate and nitrite assimilation and the light-harvesting pigment content of the blue-green algae studied strongly suggests that phycobiliproteins perform a direct and active role in these photosynthetic processes.     

Water permeability of chlorella cell membranes by nuclear magnetic resonance: measured diffusion coefficients and relaxation times

Measurement by two nuclear magnetic resonance (NMR) techniques of the mean residence time τ_a of water molecules inside Chlorella vulgaris (Beijerinck) var. “viridis” (Chodot) is reported. The first is the Conlon and Outhred (1972 Biochim Biophys Acta 288: 354-361) technique in which extracellular water is doped with paramagnetic Mn²⁺ ions. Some complications in application of this technique are identified as being caused by the affinity of Chlorella cell walls for Mn²⁺ ions which shortens the NMR relaxation times of intra- and extracellular water. The second is based upon observations of effects of diffusion on the spin echo of intra- and extracellular water. Echo attenuation of intracellular water is distinguished from that of extracellular water by the extent to which diffusive motion is restricted. Intracellular water, being restricted to the cell volume, suffers less echo attenuation. From the dependence of echo amplitude upon gradient strength at several values of echo time, the mean residence time of intracellular water can be determined. From the mean residence time of intracellular water, the diffusional water permeability coefficient of the Chlorella membrane is calculated to be 2.1 ± 0.4 × 10⁻³ cm sec⁻¹.     

Thermal Acclimation of Photosynthetic Electron Transport Activity by Thylakoids of Saxifraga cernua

Thermal acclimation by Saxifraga cernua to low temperatures results in a change in the optimum temperature for gross photosynthetic activity and may directly involve the photosynthetic apparatus. In order to test this hypothesis photosynthetic electron transport activity of S. cernua thylakoids acclimated to growth temperatures of 20°C and 10°C was measured in vitro. Both populations exhibited optimum temperatures for whole chain and PSII electron transport activity at temperatures close to those at which the plants were grown. Chlorophyll a fluorescence transients from 10°C-acclimated leaves showed higher rates in the rise and subsequent quenching of variable fluorescence at low measuring temperatures; 20°C-acclimated leaves showed higher rates of fluorescence rise at higher measuring temperatures. At these higher temperatures, fluorescence quenching rates were similar in both populations. The kinetics of State 1-State 2 transitions in 20°C- and 10°C-acclimated leaf discs were measured as changes in the magnitude of the fluorescence emission maxima measured at 77K. Leaves acclimated at 10°C showed a larger F730/F695 ratio at low temperatures, while at higher temperatures, 20°C-acclimated leaves showed a higher F730/F695 ratio after the establishment of State 2. High incubation temperatures also resulted in a decrease in the F695/F685 ratio for 10°C-acclimated leaves, suggesting a reduction in the excitation transfer from the light-harvesting complex of photosystem II to photosystem II reaction centers. The relative amounts of chlorophyll-protein complexes and thylakoid polypeptides separated electro-phoretically were similar for both 20°C- and 10°C-acclimated leaves. Thus, photosynthetic acclimation to low temperatures by S. cernua is correlated with an increase in photosynthetic electron transport activity but does not appear to be accompanied by major structural changes or different relative amounts in thylakoid protein composition.     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Action spectrum for an enhancement of endogenous respiration by light in chlorella

The oxygen consumption of a starved chlorophyll-free, yellow mutant of Chlorella vulgaris is enhanced by very small amounts of blue light (λ 450 mμ); a saturation level is reached at about 500 ergs cm⁻² sec⁻¹. At that intensity the respiration is about 3 times greater than in the dark. An action spectrum for the enhancement of respiration shows 2 peaks around λ 450 and 375 mμ. Flavins and cis-carotenoids are discussed as the pigments involved.     

Studies of Colloidal Chlorophyll in Aqueous Dioxane

The preparation and properties of a colloidal state of pure chlorophyll a in aqueous dioxane are described. The red absorption maximum is at 685± 1 mμ, depending on buffer concentration. The typical 672 mμ colloid (obtained by diluting an acetone solution with water) can be converted directly to the 685 mμ colloid by the addition of 1 M dioxane. The 672 → 685 mμ conversion is irreversible and is second order with respect to both 672 colloid and dioxane. It is shown that the formation of the 685 mμ colloid of chlorophyll a requires the Mg atom; no dioxane species is obtained with pheophytin or ethyl pheophorbide. Furthermore, of the transition metal salts of chlorophyll, Cu, Co, Ni, and Zn, only the Zn salt interacts with dioxane.