Primary Photochemical Activity Research Articles

UV light and adaptive divergence of leaf physiology, anatomy, and ultrastructure drive heat stress tolerance in genetically distant grapevines.

The genetic basis of plant response to light and heat stresses had been unveiled, and different molecular mechanisms of leaf cell homeostasis to keep high physiological performances were recognized in grapevine varieties. However, the ability to develop heat stress tolerance strategies must be further elucidated since the morpho-anatomical and physiological traits involved may vary with genotype × environment combination, stress intensity, and duration. A 3-year experiment was conducted on potted plants of Sardinian red grapevine cultivars Cannonau (syn. Grenache) and Carignano (syn. Carignan), exposed to prolonged heat stress inside a UV-blocking greenhouse, either submitted to low daily UV-B doses of 4.63 kJ m-2 d-1 (+UV) or to 0 kJ m-2 d-1 (-UV), and compared to a control (C) exposed to solar radiation (4.05 kJ m-2 d-1 average UV-B dose). Irrigation was supplied to avoid water stress, and canopy light and thermal microclimate were monitored continuously. Heat stress exceeded one-third of the duration inside the greenhouse and 6% in C. In vivo spectroscopy, including leaf reflectance and fluorescence, allowed for characterizing different patterns of leaf traits and metabolites involved in oxidative stress protection. Cannonau showed lower stomatal conductance under C (200 mmol m-2 s-1) but more than twice the values inside the greenhouse (400 to 900 mmol m-2 s-1), where water use efficiency was reduced similarly in both varieties. Under severe heat stress and -UV, Cannonau showed a sharper decrease in primary photochemical activity and higher leaf pigment reflectance indexes and leaf mass area. UV-B increased the leaf pigments, especially in Carignano, and different leaf cell regulatory traits to prevent oxidative damage were observed in leaf cross-sections. Heat stress induced chloroplast swelling, plastoglobule diffusion, and the accumulation of secretion deposits in both varieties, aggravated in Cannonau -UV by cell vacuolation, membrane dilation, and diffused leaf blade spot swelling. Conversely, in Carignano UV-B, cell wall barriers and calcium oxalate crystals proliferated in mesophyll cells. These responses suggest an adaptive divergence among cultivars to prolonged heat stress and UV-B light. Further research on grapevine biodiversity, heat, and UV-B light interactions may give new insights on the extent of stress tolerance to improve viticulture adaptive strategies in climate change hotspots.

Dorsoventral regulation of photosynthetic functions related to morphological and anatomical structures in field-grown sorghum

The rapid dorsoventral regulation of photosynthesis is important because the light conditions around both surfaces of field-grown leaves vary considerably. The photosynthetic asymmetry has been intensively studied, but structure-related explanations for asymmetry were not exactly clarified. This study aimed to investigate the photosynthetic asymmetry in field-grown Sorghum bicolor L. and analyze the impacts of dorsoventral structures on photosynthesis. We found that the photosynthetic asymmetry changed with light conditions, and adaxial surfaces had higher primary photochemical activity owing to higher mesophyll density, while abaxial surfaces had higher CO2-diffusion capacity due to higher stomatal density, substomatal cavities, and contact areas between bundle sheath and mesophyll cells. Moreover, abaxial surfaces could mainly use self-transmitted light (residual light intercepted by adaxial surface) to drive photoreaction which could partly retain high photosynthetic rates in the field. This study can improve our understanding on the photosynthetic dorsoventral regulation.

Photosynthesis and respiration in the needles of Pinus sibirica and Pinus pumila and their putative hybrids

A putative interspecific hybridization in Pinaceae family was investigated. Very rarely the physiological methods were involved in hybridization processes that occurs in the hybrid zones. It is well known that in most gymnosperms, the plastid genome is inherited from the paternal component while the mitochondrion is inherited from the maternal one. Therefore functioning pattern of organelles in the hybrid plant can suggest parent, from which they were inherited. The aim of this study was to indirectly establish the inheritance energy-transducing organelles (mitochondria, chloroplast) according to their functioning. Current year needles from Siberian Stone Pine (Pinus sibirica Du Tour) and Japanese Stone Pine (Pinus pumila (Pall.) Regel) as parent species and their putative hybrids were collected from Baikal Region. The photosynthesis rate was determined by using the spectrophotometer. The study of emission CO2 under dark respiration of needle was conducted with laser optical-acoustic gasanalyzer. The quantity was measured at 1, 2 and 3 hour after experiment start. The rate of the photoreduction ferricyanide potassium was characterized by the primary photochemical processes activity at the level of photosystem II. Comparison of pure species was shown that Japanese Stone Pine had higher functional activity of chloroplast as compared with Siberian Stone Pine in spite of the fact that they are growing in similar environment conditions. Two of three analyzed hybrids had decreased activity of their chloroplasts. Unfortunately, in this case we can't conclude if the chloroplasts were inherited from Siberian Stone Pine or from Japanese Stone Pine. Chloroplast activity of the third hybrid was approximately similar to that of Japanese Stone Pine suggesting that its chloroplasts were inherited from this parent. Consequently, the Siberian Stone Pine and the Japanese Stone Pine were maternal and paternal, respectively parents of the hybrid. Compared to the Siberian Stone Pine, the Japanese Stone Pine had higher dark respiration rate. The hybrid respiration rate was similar to that of the Japanese Stone Pine suggesting that the hybrid inherited mitochondria from this species. Our findings indicated that the P. pumila exhibited particular ecological plasticity and this phenomenon is attributable to its high potential activity of photosynthetic and respiratory processes. These physiological features explained the large transcontinental natural habitat of Japanese Stone Pine.

Changes in photosynthetic capacity and polypeptide patterns during natural senescence and rejuvenation of Cucurbita pepo L. (zucchini) cotyledons

Natural senescence of Cucurbita pepo (zucchini) cotyledons was accompanied by a gradual degradation of reserve proteins (globulins) and an intensive decrease in the content of both large subunit (LSU) and small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The net photosynthetic rate, the primary photochemical activity of PSII, estimated by the variable fluorescence (Fv)/maximal fluorescence (Fm) ratio (Fv/Fm) and the actual quantum yield of PSII electron transport in the light-adapted state (ΦPSII) also progressively decreased during natural senescence. In contrast, the fraction of the absorbed light energy, which is not used for photochemistry (LNU) increased with progression of senescence. The decline in the photosynthetic rate started earlier in ontogenesis compared with the down-regulation of the functional activity of PSII, thus suggesting the existence of protective mechanisms which maintain higher efficiency of the photochemical electron transport reactions of photosynthesis compared with the dark reactions of the Calvin cycle during earlier stages of natural senescence. Decapitation of the epicotyl above the senescing cotyledons resulted in full recovery of the polypeptide profile in the rejuvenated cotyledons. In addition, the photosynthetic rate increased reaching values that exceeded those measured in juvenile cotyledons. The photochemical efficiency of PSII also gradually recovered, although it did not reach the maximum values measured in the presenescent cotyledons.

Melittin-induced changes in thylakoid membranes: particle electrophoresis and light scattering study

Thylakoids were used as a model system to evaluate the effect of bee venom peptide melittin (Mt) on membrane surface charge. At neutral pH, thylakoid membrane surfaces carry excess negative electrical charge. Mt strongly altered the electrophoretic mobility (EPM) of ‘low-salt’ thylakoids and did not significantly change the EPM of ‘high-salt’ thylakoids. Mt increased the primary ionic-exchange processes across the ‘low-salt’ thylakoid membranes, while it did not affect those of ‘high-salt’ thylakoids. Mt decreased the proton gradient generation on the membranes at both ionic strengths, but it affected more strongly the ‘high-salt’ than that of ‘low-salt’ thylakoids. The primary photochemical activity of photosystem II, estimated by the ratio F v/ F m, was not influenced by the low Mt concentrations. It decreased only when chloroplasts had been incubated with higher Mt concentrations and this effect was better expressed in ‘low-salt’ than in ‘high-salt’ thylakoid membranes.

Electrokinetic properties of thylakoids in in vitro cultured Gypsophila paniculata plants

In vitro cultured Gypsophila paniculata L. plants were used as a model to evaluate the effect of some cytokinins and anticytokinins on thylakoid surface charge. Influence of the cytokinins N-6-furfurylaminopurine (kinetin) and N1-(2-chloro-4-pyridyl)-N2-phenylurea (4-PU-30), cytokinin antagonists 2-chloro-4-cyclobutylamino-6-ethylamino-1,3,5-triazine and N-(4-pyridyl)-O-(4-chlorophenyl) carbamate on the pigment content, surface charge density (s ), fluorescence induction kinetics and millisecond-delayed light emission was studied. Our results showed that the chlorophyll (a+b) content significantly decreased after the 1st and the 2nd month of G. paniculata growth in the presence of the cytokinins kinetin and 4-PU-30. In our model system, cytokinins enhanced the number of open lateral buds and, as a consequence, more shoots per explant. Hence, chlorophyll synthesis was not inhibited but so-called ‘dilution of the pigments’ was available. Anticytokinins inhibited the formation of more than one shoot, and the chlorophyll content was not influenced significantly. The phenylurea cytokinin 4-PU-30 and anticytokinins increased the electrophoretic mobility, zeta potential and surface charge density of thylakoids after a longer time of treatment. Making thylakoid membranes more negatively charged, phenylurea cytokinin and anticytokinins increased the aggregation of the complexes and the energization of the membrane. Our results showed that plant growth regulators decreased the primary photochemical activity of photosystem II (estimated by the ratio Fv/Fm) and delayed fluorescence intensity in the 1st month. However, no significant changes were observed in these parameters in the 2nd month.

Chemical characterization of three summer cloud episodes at whiteface mountain

Abstract Cloudwater and aerosol samples were cullected during three 1987 summer events at Whiteface Mountain, New York. Dominant inorganic components in Cloudwater were SO42−, NO3−, H+, and NH4+. Maximum levels were 978, 256, 421, and 832,μeq l−1, respectively. pH values in Cloudwater ranged from 3.1 to 4.4. Large variations in concentrations were found, which can best be explained, on the basis of back-trajectory analysis, by different origins of air masses. A concave concentration versus a time profile for the ionic concentrations was observed, possibly as a result of dilution and concentration as liquid water content changed over the life of the cloud. Dominant components of the aerosol were NH4+ and SO42−. Changes in the gas-phase concentrations of SO2, O3, and H2O2 during a cloud event indicated a considerable S(IV) oxidation potential by H2O2. Free acidity (H+) was 32 – 81% of the total acidity. Carboxylic acid concentrations were dominated by formic acid (13.9 – 39.9μeq l−1), acetic acid (5.1 – 14.7 μeq l−1 oxalic acid (2.9 – 19.3 μeq l−1), malonic acid (3.9 – 14.7 μeq l−1, and pyruvic acid (2.4 – 8.6 μeq l−1); these acids have accounted for 7 – 15% of the dissolved organic carbon and up to 22% of the free acidity. Multivariate factor analysis yielded three components that accounted for 91 % of the variability of all the parameters: transported acid component, combination of fossil-fuel combustion and automobile emission, acid nontransported biogenic component. Primary pollutant concentrations and photochemical activity during long-range transport were the major factors in controlling the chemical species concentrations at the downwind site.

Chemical characterization of three summer cloud episodes at whiteface mountain

Cloudwater and aerosol samples were cullected during three 1987 summer events at Whiteface Mountain, New York. Dominant inorganic components in Cloudwater were SO 4 2−, NO 3 −, H +, and NH 4 +. Maximum levels were 978, 256, 421, and 832,μeq l −1, respectively. pH values in Cloudwater ranged from 3.1 to 4.4. Large variations in concentrations were found, which can best be explained, on the basis of back-trajectory analysis, by different origins of air masses. A concave concentration versus a time profile for the ionic concentrations was observed, possibly as a result of dilution and concentration as liquid water content changed over the life of the cloud. Dominant components of the aerosol were NH 4 + and SO 4 2−. Changes in the gas-phase concentrations of SO 2, O 3, and H 2O 2 during a cloud event indicated a considerable S(IV) oxidation potential by H 2O 2. Free acidity (H +) was 32 – 81% of the total acidity. Carboxylic acid concentrations were dominated by formic acid (13.9 – 39.9μeq l −1), acetic acid (5.1 – 14.7 μeq l −1 oxalic acid (2.9 – 19.3 μeq l −1), malonic acid (3.9 – 14.7 μeq l −1, and pyruvic acid (2.4 – 8.6 μeq l −1); these acids have accounted for 7 – 15% of the dissolved organic carbon and up to 22% of the free acidity. Multivariate factor analysis yielded three components that accounted for 91 % of the variability of all the parameters: transported acid component, combination of fossil-fuel combustion and automobile emission, acid nontransported biogenic component. Primary pollutant concentrations and photochemical activity during long-range transport were the major factors in controlling the chemical species concentrations at the downwind site.

Effect of Sodium Chloride on Primary Photochemical Activities in Cotyledonary Leaves of Brassica juncea

Summary The effect of varying concentrations of sodium chloride (NaCl) (0, 100, 150 and 200 mM) on seedling growth, pigment composition and the primary photochemical activities in cotyledonary leaves of Brassica juncea cv. DIRA 367 was investigated. NaCl-stress resulted in a considerable decrease in growth of the seedlings without causing any significant change in dry weight. While, chlorophyll a content was higher in cotyledonary leaves of seedlings raised in high salt containing media, chlorophyll b content did not show any significant variation till 11th day of growth. Thus the high Chl a/Chl b ratio in NaCl treatments reflects changes in the photosynthetic antenna size. The carotenoid content in 11 day old cotyledonary leaves, on the other hand, was much reduced at high concentrations of NaCl. NaCl treatments caused an increase in water oxidation capacity, as measured through H2O → MV (whole chain) or H2O → PDOX (PS II) photoreactions, by 1.2 to 2.0 fold. But, PS I mediated photochemical activities showed no or only a minor increase by NaCl treatment. The enhancement in photochemical activities noted even in presence of uncouplers (NH4Cl and methyl amine) suggest that the NaCl induced enhancement of primary photochemical activity is not linked to the uncoupling of electron transport from photophosphorylation. Also, the studies on the interactive effect of in vivo ageing and salinity on the pigment composition and primary photochemical activities suggest that ageing and NaCI-stress result in partly independent and partly interactive changes in the pigment composition and photochemical activities.

Q A-depletion and reconstitution of a reaction center preparation from the photosynthetic bacterium Rhodopseudomonas viridis

Isolated RC's from Rps. viridis were reversibly depleted of both UQ (Q B) and MK (Q A). The photochemical activity was eliminated and restored upon the depletion and reconstitution of the Q A. Four methods for depleting Q A from the RC's were studied and their results were compared. The best way allowed us for the first time to prepare Q A-depleted RC's from Rps. viridis that retained only 10% of primary photochemical activity, which could be restored up to 85% by addition of vitamin K 1.

Photochemical electron transfer reactions in the acceptor complex of reaction centers of Rhodopseudomonas spheroides treated with sodium dodecyl sulfate

We have compared some photochemical properties of the reaction-center complex of Rhodopseudomonas spheroides (wild-type) treated with various amounts of either sodium dodecyl sulfate (SDS) or dodecyl dimethylamine N-oxide. In the presence of the latter, the native structure and activity of the reaction center are preserved even at high concentrations of detergent. In contrast, SDS denatures the protein. It does this by a cooperative process, as shown by the sigmoidal relationship between primary photochemical activity and SDS concentration. SDS binds to the reaction center at up to about 6 g SDS/g protein. The most notable modifications brought about by low SDS concentrations are: a slowing down of rereduction of P+ by Q-B from 1 s-1 to 0.25 s-1 and, to a lesser extent, of P+ by Q-A; the reoxidation of Q-A and Q-B by N,N,N',N'-tetramethyl-1,4-phenylenediamine is accelerated as an exponential function of SDS concentration. In this case Q-A become more accessible to external acceptors but not Q-B. It is thought that these changes are mainly due to progressive binding of SDS resulting in the unfolding of the protein, probably accompanied by the loss of the metal. Herbicides partly protect the reaction center against SDS denaturation, but their efficiency to block electron transfer between QA and QB is greatly reduced in the altered reaction centers present in the transition region of the denaturation curve. It is concluded that in the early region of the SDS denaturation curve, no dissociation of the reaction center in subunits occurs. In the transition region, it appears that the reaction center reaches the critical state in which mainly dissociation of polypeptide H from the complex of chains L and M and loss of QA take place.

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.

Stoichiometry of system I and system II reaction centers and of plastoquinone in different photosynthetic membranes

The concentrations of photochemical centers and of plastoquinone were measured in several kinds of photosynthetic membranes by optical difference spectroscopy. Photosystem I reaction centers were measured from the light-induced absorbance change at 700 nm (oxidation of the primary electron donor, P700). Photosystem II reaction centers were estimated from the light-induced absorbance change at 325 nm (reduction of the primary electron acceptor, Q). Spinach chloroplasts and membrane fractions obtained by French press treatment, mature and developing pea chloroplasts, and blue-green algal membranes were investigated. No loss of primary photochemical activity occurred during fractionation of the chloroplasts. The results indicated a large variability in the ratio of system II to system I reaction centers (from 0.43 to 3.3) in different photosynthetic membranes. Oxygen-evolving plants may change the ratio of their photosystems in response to environmental light conditions. The amount of photoreducible plastoquinone was also measured at 263 nm. In spinach chloroplasts, seven to eight plastoquinone molecules were found per reaction center of system II. Most of the plastoquinone pool was associated with the grana. However, the ratio of chemically determined plastoquinone to chlorophyll was similar in the grana and stroma thylakoids.

The kinetic relationship between the C-550 absorbance change, the reduction of Q( ΔA320) and the variable fluorescence yield change in chloroplasts at room temperature

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolated, envelopefree chloroplasts in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isosbestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differential extinction coefficient Δϵ(540–550) is calculated to be approximately 5 mM −1 · cm −1. The kinetics of the C-550 absorbance change, occurring upon the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical conditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The light-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers. It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Thus, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at room temperature.

The involvement of iron and ubiquinone in electron transfer reactions mediated by reaction centers from photosynthetic bacteria

Reaction centers from Rhodopseudomonas sphaeroides strain R-26 were prepared with varying Fe and ubiquinone (Q) contents. The photooxidation of P-870 to P-870 + was found to occur with the same quantum yield in Fe-depleted reaction centers as in control samples. The kinetics of electron transfer from the initial electron acceptor (I) to Q also were unchanged upon Fe removal. We conclude that Fe has no measurable role in the primary photochemical reaction. The extent of secondary reaction from the first quinone acceptor (Q A) to the second quinone acceptor (Q B) was monitored by the decay kinetics of P-870 + after excitation of reaction centers with single flashes in the absence of electron donors, and by the amount of P-870 photooxidation that occurred on the second flash in the presence of electron donors. In reaction centers with nearly one iron and between 1 and 2 ubiquinones per reaction center, the amount of secondary electron transfer is proportional to the ubiquinone content above one per reaction center. In reaction centers treated with LiClO 4 and o-phenanthroline to remove Fe, the amount of secondary reaction is decreased and is proportional to Fe content. Fe seems to be required for the secondary reaction. In reaction centers depleted of Fe by treatment with SDS and EDTA, the correlation between Fe content and secondary activity is not as good as that found using LiClO 4. This is probably due in part to a loss of primary photochemical activity in samples treated with SDS; but the correlation is still not perfect after correction for this effect. The nature of the back reaction between P-870 + and Q − B was investigated using stopped flow techniques. Reaction centers in the P-870 + Q − B state decay with a 1-s half-time in both the presence and absence of o-phenanthroline, an inhibitor of electron transfer between Q − B and Q B. This indicates that the back reaction between P-870 + and Q − A is direct, rather than proceeding via thermal repopulation of Q − A. The P-870 + Q − B state is calculated to lie at least 100 mV in free energy below the P-870 + Q − A state.

Development of the Primary Photochemical Apparatus of Photosynthesis during Greening of Etiolated Bean Leaves

Seven-day-old dark-grown bean leaves were greened under continuous light. The amount of chlorophyll, the ratio of chlorophyll a to chlorophyll b, the O(2) evolving capacity and the primary photochemical activities of Photosystem I and Photosystem II were measured on the leaves after various times of greening. The primary photochemical activities were measured as the photo-oxidation of P(700), the photoreduction of C-550, and the photo-oxidation of cytochrome b(559) in intact leaves frozen to -196 C. The results indicate that the reaction centers of Photosystem I and Photosystem II begin to appear within the first few minutes and that Photosystem II reaction centers accumulate more rapidly than Photosystem I reaction centers during the first few hours of greening. The very early appearances of the primary photochemical activity of Photosystem II was also confirmed by light-induced fluorescence yield measurements at -196 C.

Light-induced absorbance changes in chloroplasts mediated by Photosystem I and Photosystem II at low temperature

Stable light-induced absorbance changes in chloroplasts at −196 °C were measured across the visible spectrum from 370 to 730 nm in an effort to find previously undiscovered absorbance changes that could be related to the primary photochemical activity of Photosystem I or Photosystem II. A Photosystem I mediated absorbance increase of a band at 690 nm and a Photosystem II mediated absorbance increase of a band at 683 nm were found. The 690-nm change accompanied the oxidation of P 700 and the 683-nm increase accompanied the reduction of C-550. No Soret band was detected for P 700. A specific effort was made to measure the difference spectrum for the photooxidation of P 680 under conditions (chloroplasts frozen to −196 °C in the presence of ferricyanide) where a stable, Photosystem II mediated EPR signal, attributed to P 680 + has been reported. The difference spectra, however, did not show that P 680 + was stable at −196 °C under any conditions tested. Absorbance measurements induced by saturating flashes at −196 °C (in the presence or absence of ferricyanide) indicated that all of the P 680 + formed by the flash was reduced in the dark either by a secondary electron donor or by a backreaction with the primary electron acceptor. We conclude that P 680 + is not stable in the dark at −196 °C: if the normal secondary donor at −196 °C is oxidized by ferricyanide prior to freezing, P 680 + will oxidize other substances.

Extraction and Reconstitution of Photosystem II

Hill activity (oxygen evolution with ferricyanide as the electron acceptor), light-induced absorbance changes at liquid nitrogen temperature associated with the primary activity of photosystem II, and fluorescence yield changes at both low temperature and room temperature were measured with lyophilized spinach chloroplasts before and after extraction with hexane and reconstitution with beta-carotene and plastoquinone A. Extraction eliminated the Hill activity, and both beta-carotene and plastoquinone A were required for maximal restoration of activity to the reconstituted chloroplasts.Extraction also eliminated the light-induced absorbance changes at -196 C due to the photoreduction of C-550 and photooxidation of cytochrome b(559), and reconstitution with beta-carotene and plastoquinone A restored the low temperature photoreactions. However, only beta-carotene was essential for the restoration of the photoreactions. Cytochrome b(559) was modified, as a result of the extraction, to a lower redox potential, autooxidizable form and remained as such after reconstitution with beta-carotene. The beta-carotene-restored chloroplasts showed the photoreduction of C-550 but not the photooxidation of cytochrome b(559) because the cytochrome was already oxidized. When beta-carotene-reconstituted chloroplasts were suspended in buffer containing ascorbate prior to freezing, the cytochrome b(559) was reduced and could be photooxidized by irradiation at low temperature. After reconstitution with beta-carotene plus plastoquinone A the cytochrome b(559) was partially restored to its original high potential form and was in the reduced state so that both the photoreduction of C-550 and the photooxidation of cytochrome b(559) occurred on irradiation of the beta-carotene plus plastoquinone A-reconstituted chloroplasts. Reconstitution with plastoquinone A alone had essentially no effect on restoring the photoreactions.The fluorescence yield of dark-adapted lyophilized chloroplasts at -196 C showed an irreversible increase of about 2.5-fold during irradiation. After extraction the fluorescence yield of the chloroplasts was high (at the maximal light-induced level of the lyophilized control chloroplasts) and showed very little change in the light. Reconstitution with beta-carotene alone restored some fluorescence quenching which was relieved by irradiation at low temperature. Reconstitution with plastoquinone A alone restored a high degree of quenching, but this quenching was not relieved by light at low temperature. Fluorescence emission spectra at -196 C showed that the fluorescence of variable yield in the lyophilized and beta-carotene-reconstituted chloroplasts involved only the 680 and 695 nm emission bands but not the larger 730 nm emission band, whereas the irreversible quenching in plastoquinone A-reconstituted chloroplasts involved all wavelengths of emission. Extraction of the chloroplasts also eliminated the sharp 695 nm emission band at low temperature, and reconstitution with beta-carotene partially restored it.The fluorescence yield changes at room temperature differed from the low temperature measurements in that the strong fluorescence quenching restored to the plastoquinone A-reconstituted chloroplasts was relieved by light and reappeared in the dark. Thus plastoquinone A appeared to be much more effective than beta-carotene in restoring the fluorescence of variable yield in room temperature measurements. However, it is argued from the results at low temperature that the quenching in plastoquinone A-reconstituted chloroplasts, which is probably due to the oxidized form of the quinone, is nonspecific and a different quenching mechanism from that which obtains in normal chloroplasts.The results suggest that extraction with hexane removes plastoquinone A, which interrupts electron transport, and beta-carotene, which disrupts the primary photochemical activity of photosystem II. Reconstitution of the extracted chloroplasts with beta-carotene alone restores C-550 and the primary photochemical activity of photosystem II, and when the photosystem II reaction centers are restored the additional requirement of plastoquinone A for the Hill reaction can be demonstrated.