Photoacoustic detection of photosynthetic activities in isolated broken chloroplasts

Methodology and demonstration how to utilize the photoacoustic technique in photosynthesis research are presented. Photoacoustic signals were obtained from suspensions of isolated broken chloroplasts. In the presence of strong, continuous (non-modulated) background light the signals were normally larger than without the background light. The effect of the background light was saturable and was absent when non-active (e.g. heat-treated) samples were used, showing that the normal smaller signal in the absence of background light is a genuine reflection of the loss of heat due to the competing photochemistry. The effect of the background light is to close the reaction-centers and hence to inhibit the photochemical process. The percent difference of the photoacoustic signal (± background light) is taken as a measure of the photochemical activity (‘photochemical loss’).

Initial results demonstrate the wavelength dependence of the ‘photochemical loss’. As expected there was a ‘red-drop’ decrease of the ‘photochemical loss’ for λ > 690 nm, when the cofactor methyl viologen was present. Surprisingly, however, there was a ‘red-rise’ increase for λ > 690 nm when no cofactor was present. These findings indicate that under the last conditions there is an unsuspected photoactivity of PS I which was not detected hitherto by the conventional techniques. The dependence on the background light intensity confirms this result. This photoactivity can be explained tentatively as a cyclic electron flow around PS I, present without any added cofactor.

Initial results on the modulation frequency dependence in the presence of electron acceptors are also demonstrated.     

Inhibition of the photosynthetic activities of isolated spinach chloroplasts by phosphonate compounds

The effects of three closely related phosphonate compounds on several photosynthetic activities of isolated chloroplasts were investigated. Phosphonoformic and phosphonopropionic acid were found to inhibit both CO₂ fixation and the reduction of 3-phosphoglyceric acid, with CO₂ fixation being more sensitive. In contrast, phosphonoacetic acid was only slightly inhibitory. The lack of inhibition appeared to be due to its inability to enter the stroma via the phosphate translocator. Measurements of changes in stromal metabolite levels following the inhibition of CO₂ fixation by either phosphonoformic or phosphonopropionic acid indicated that the activity of ribulose bisphosphate carboxylase/oxygenase was reduced. Studies with the isolated enzyme confirmed that both of these compounds were effective competitive inhibitors of the carboxylase activity of the enzyme.     

Choline, tetraalkylammonium and aminoalcohol as preserves of photosynthetic activities in isolated chloroplasts

Choline chloride, tetraalkylammonium chloride and aminoalcoholshow preservative effects on photosynthetic activities in spinachchloroplasts against deterioration after isolation, when oneof them is present in the medium used for isolating and storingchloroplasts within the concentration range between 0.1 and0.5 M. Any of these chemicals cause uncoupling to some extentif present in the reaction medium. (Received August 23, 1973; )     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

The mechanism of photosynthetic sulfate reduction by isolated chloroplasts

The reduction of sulfate by isolated spinach chloroplasts was studied. A reconstituted system of broken chloroplasts and of chloroplast extract reduced sulfate to sulfite in the light when ADP, NADP⁺, ferredoxin and glutathione were added. The chloroplast extract reduced sulfate to sulfite in the dark if supplemented with ATP and with reduced glutathione. Neither ferredoxin nor NADPH were needed for this reduction in the dark.

A sulfite reductase was purified from spinach leaves. Broken chloroplasts and sulfite reductase reduced sulfite to sulfide in the light when ferredoxin was added. NADP⁺ was not required for this reduction.

The results suggest that in chloroplasts a sulfate activated by ATP (phosphoadenosine phosphosulfate) is reduced to sulfite by a sulfhydryl compound and that sulfite is reduced to sulfide by a ferredoxin-dependent sulfite reductase.     

Inhibition of the photosynthetic capacity of isolated chloroplasts by ozone

Isolated spinach chloroplasts have been used as a model system for studying the interaction of ozone, a component of photochemical smog, with plant membranes. Ozone bubbled into a suspension of isolated chloroplasts inhibits electron transport in both photosystems without uncoupling ATP production. Photosystem I (reduced 2,6-dichlorophenolindolphenol → NADP⁺) is a little more sensitive than photosystem II (H₂O → 2,6-dichlophenolindolphenol). Ozone does not act as an energy transfer inhibitor, since the drop in ATP production and high energy intermediate (measured by amine-induced swelling) is nearly parallel to the decline in electron transport. A reasonable hypothesis is that ozone disrupts the normal pathway of energy flow from light-excited chlorophyll into the photoacts by a disruption of the components of the membrane but not a general disintegration of the membrane. In addition, ozone does not seem to penetrate into the grana region through the outer membrane of intact plastids, since ozone lowers the bicarbonate-supported O₂ evolution but does not affect the rate of ferricyanide reduction in the same plastids after osmotic disruption. This would indicate that the effect of ozone on green plants, at low concentrations, may be due to the interaction of ozone with the first membrane it contacts and not directly with internal metabolic processes.     

Effects of disalicylidenepropanediamines on photosynthetic electron transport of isolated spinach chloroplasts

The effects of disalicylidenepropanediamine (DSPD) and disulfo-disalicylidenepropanediamine (sulfo-DSPD) on the photosynthetic electron transport of isolated chloroplasts have been reexamined.     

Effects of ethephon on aging and photosynthetic activity in isolated chloroplasts

Chloroplasts, isolated from the primary leaves of 7-day-old seedlings, were incubated in vitro at 25°C with 2-chloroethylphosphonic acid (ethephon) under light (0.16 milliwatts per square centimeter) and dark conditions. Ethephon at 1 micromolar (0.1445 ppm), 0.1 and 1 millimolar, or 5 microliters ethylene promoted the deterioration of chloroplasts, increased proteolysis, and reduced the chlorophyll content and PSI and PSII during 72 hours under both light and dark conditions. The decline in PSI and PSII occurred prior to a measurable loss of chlorophyll. The loss of photosynthetic activity affected by ethephon was initiated prior to 12 hours of incubation. After 24 hours in light, 0.1 millimolar (1.445 ppm) epthephon significantly reduced PSI and PSII and promoted the total free amino acid liberation in isolated chloroplasts. In darkness the rate of loss of PSI activity was about 50% of that in light. After 24 hours, in light at 1 millimolar epthephon, PSII activity was 55% of the control, yet nearly 90% of the chlorophyll remained, which indicates that the loss of thylakoid integrity was promoted by ethephon. Ethylene injected in the chloroplast medium at 5 microliters (0.22 micromolar per milliliter) reduced PSI by nearly 50% of the initial in 12 hours. In leaf sections floated in 5 microliters per milliliter suspension medium, a 36% loss of chlorophyll of the control in 36 hours was observed. Cycloheximide at 0.5 millimolar masked the effect of 1 millimolar ethephon and maintained the initial chlorophyll content during the 72 hour period.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

Inhibition of photosynthetic carbon metabolism in isolated chloroplasts by iodoacetol phosphate

Carbon dioxide-dependent and 3-phosphoglycerate (PGA)-dependent O₂ evolution by isolated chloroplasts of wheat is inhibited by micromolar levels of iodoacetol phosphate (IAP). Loss of the activity is time-dependent and a higher concentration of PGA increases the half-time for inhibition (e.g. at 40 micromolar IAP the half-time is about 0.5 minutes at 1 millimolar PGA compared to 1.5 minutes at 10 millimolar PGA). A marked inhibition of NADP glyceraldehyde-3-P dehydrogenase was observed when chloroplasts were pretreated with micromolar levels of IAP, osmotically shocked, and several stromal enzymes assayed.     

Artificial increase of the light-harvesting ability of photosynthetic units in isolated chloroplasts

A synthetic fluorochromous lipid, rhodaminyl triglyceride (rhodaminyl TG), was intercalated into isolated thylakoid membranes of chloroplasts up to 30 molecules per 100 molecules of chlorophyll. As a result of fluorochrome presence, an absorption band appeared in a yellow-green spectrum region, its intensity being comparable with the red and blue chlorophyll bands. The energy absorbed by rhodaminyl TG was transferred through chlorophyll to the reaction centers of photosystems I and II, inducing an additional electron flow of about 30%. Therefore the exogenous fluorochrome dissolved in lipid matrix functions as an accessory pigment which significantly modifies the spectral sensitivity of the photosynthetic process. The energy transfer from rhodaminyl TG to chlorophyll occurs by mechanism of the inductive resonance type.Abbreviations rhodaminyl TG rhodaminyl triglyceride (rac-1,2-dioleoyl-3[11(3-rhodaminyl)amino-undelanoyl]glycerol) - Me₂SO dimethylsulfoxide - PS photosystem - PPC pigment-protein complex - F₀, F_m initial and maximal levels of chlorophyll fluorescence     

Biophysical studies on subcellular particles VII. Combined effects of alcohol and heat on photosynthetic activities in isolated spinach chloroplasts

Isolated spinach chloroplasts were warmed for 5 min at temperaturesranging between 20? and 55?C in the presence of short-chainaliphatic alcohols, and their photosynthetic activities wereassayed. Tmax, the temperature at which ferricyanide reduction is stimulatedto a maximum extent, was lowered with an increase in the concentrationof alcohol or the chain-length of n-alcohols. Values also differedamong the structural isomers of an alcohol. The Tmax shift wasobserved only when alcohol was present in a chloroplast suspensionduring warming treatment, indicating that heat and alcohol wereacting together to lower the Tmax, at which the phosphorylationactivity fell to zero. The combined effects of alcohol and heat are discussed in connectionwith the lipophilic construction of thylakoids through the partitionco-efficient of individual alcohols between water and n-octylalcohol. (Received August 5, 1972; )     

Effects of long-chained aliphatic amines on photosynthetic reactions in isolated spinach chloroplasts

The effect of six long-chained aliphatic amines on ¹⁴CO₂-reduction, electron transport and the 515 nm absorbance change and shrinkage in isolated intact and broken chloroplasts from spinach ( Spinacia oleracea L. cv. Weibulls Medania) was investigated. Five of the six investigated amines affected photosynthesis in intact chloroplasts by inhibiting ¹⁴CO₂-reduction. In broken chloroplasts the same amines uncoupled electron transport. When added to intact chloroplasts the five amines induced a light-dependent oxygen uptake leading to (he formation of hydrogen peroxide. The oxygen uptake was not due to the amines acting as Mehler reactants. The mode of action, different from that of simple aliphatic amines, was an effect on membrane integrity, first affecting the membrane potential. At higher amine concentrations a more general effect on the ion conditions in the thylakoids was evident.     

The effects of digitonin on photochemical activities of isolated chloroplasts

The addition of digitonin to chloroplasts stimulated the rate of oxygen evolution followed by a gradual inhibition. The effect of digitonin was dependent on the digitonin to chlorophyll ratio and on temperature and time. The initial stimulation of oxygen evolution appeared to be a result of uncoupling as digitonin did not stimulate oxygen evolution by uncoupled chloroplasts. The stimulatory effect occurred more rapidly at high digitonin to chlorophyll ratios but the extent of stimulation was low and inhibition occurred soon after addition of the detergent. The inhibition of electron flow by digitonin was due to a site of action near photosystem II which resembled the inhibition reported for tris buffer and resulted in photobleaching. However, digitonin inhibition could not be recovered by washing with reducing agents and was only partially recovered by the addition of artificial electron donors to photosystem II. Electron flow mediated by photosystem I was unaffected by the addition of digitonin but was decreased when the chloroplasts were separated by subsequent centrifuging. This suggested that digitonin solubilizes photosystem I components which remain active in the soluble form.     

Photosynthesis in trees: organization of chlorophyll and photosynthetic unit size in isolated gymnosperm chloroplasts

Chloroplasts have been isolated in high yield from several gymnosperms and from two deciduous trees. The organization of chlorophyll in the chloroplasts of these woody species is basically similar to that in angiosperm crop plants and green algae. The tree chloroplasts contain two chlorophyll proteins, the P700-chlorophyll a-protein and the major light-harvesting chlorophyll a/b-protein, the size, spectral characteristics, and function of which are the same as the equivalent complexes previously isolated from other classes of green plants. All the gymnosperms have chlorophyll/P700 ratios (photosynthetic unit sizes) 1.6 to 3.8 times larger than that typically found in crop plants; the deciduous trees have units of intermediary size. The presence of fewer but larger photosynthetic units in the woody species can partially account for their lower photosynthetic rate and explains why their photosynthetic processes saturate at lower light intensities. Chloroplasts of shade needles have large units containing a greater proportion of the light-harvesting chlorophyll a/b-protein than those of sun needles.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.