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.     

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.     

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 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 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.     

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 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.     

The cupric ion as an inhibitor of photosynthetic electron transport in isolated chloroplasts

Strong inhibition of uncoupled photosynthetic electron transport by Cu²⁺ in isolated spinach chloroplasts was observed by measuring changes in O₂ concentration in the reaction medium. Inhibition was dependent not only on the concentration of the inhibitor, but also on the ratio of chlorophyll to inhibitor. Binding of Cu²⁺ to the chloroplast membranes resulted in removal of Cu²⁺ from solution. When chloroplasts were exposed to preincubation in light, there was increased inhibition as a result of Cu²⁺ binding to inhibitory sites. Preincubation in the dark resulted in Cu²⁺ binding to noninhibitory sites and decreased inhibition. The degree of inhibition was lower at low light intensities than at high light intensities.     

High resolution imaging of photosynthetic activities of tissues, cells and chloroplasts in leaves

Through imaging of chlorophyll fluorescence, it is possible to produce parameterized fluorescence images that estimate the operating quantum efficiency of photosystem II (PSII) photochemistry and which can be used to reveal heterogeneous patterns of photosynthetic performance within leaves. The operating quantum efficiency of PSII photochemistry is dependent upon the effective absorption cross-section of the light-harvesting system of PSII and the photochemical capacity of PSII. The effective absorption cross-section is decreased by the process of down-regulation, which is widely thought to operate within the pigment matrices of PSII and which results in non-photochemical quenching of chlorophyll fluorescence. The photochemical capacity is non-linearly related to the proportion of PSII centres in the 'open' state and results in photochemical quenching of chlorophyll fluorescence. Examples of heterogeneity of the operating quantum efficiency of PSII photochemistry during the induction of photosynthesis in maize leaves and in the chloroplast populations of stomatal guard cells of a leaf of Tradescantia albifora are presented, together with analyses of the factors determining this heterogeneity. A comparison of the operating quantum efficiency of PSII photochemistry within guard cells and adjacent mesophyll cells of Commelina communis is also made, before and after stomatal closure through a change in ambient humidity.     

Photosynthetic carbon metabolism in isolated pea chloroplasts: metabolite levels and enzyme activities

We report here that enzyme activation precedes the rise in metabolite levels, which appear to limit photosynthetic CO2 fixation during induction in pea leaf chloroplasts. Therefore light activation may be required for the build-up of photosynthetic intermediates and hence for photosynthesis in isolated chloroplasts. Analysis of metabolite levels and the known kinetic properties of the chloroplast enzymes indicates that the reductive pentose phosphate cycle is subject to control which fluctuates between several points during induction and when CO2 fixation is maximal. The transketolase-aldolase-catalyzed reactions around sedoheptulose-biphosphatase appear to provide a simple and effective primary control for photosynthetic CO2 fixation. When substrate levels and enzyme active site concentrations are taken into account, there is insufficient glyceraldehyde 3-phosphate dehydrogenase, aldolase, and transketolase activity to support photosynthetic CO2 fixation at observed rates. These results suggest that there may be direct transfer of glyceraldehyde 3-phosphate among these enzymes in the pea chloroplast.     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

Inhibition of photosynthetic electron transport in isolated spinach chloroplasts by two 1,3,4-thiadiazolyl derivatives

Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) are two new promising herbicides for selective weed control in corn (Zea mays L.) and sugarcane (Saccharum officinarum L.), respectively. The effects of these two compounds on various photochemical reactions of isolated spinach (Spinacia oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5, 5, and 500 micromolar. Buthidazole and tebuthiuron at concentrations higher than 0.5 micromolar inhibited uncoupled electron transport from water to ferricyanide or to methyl viologen very strongly. Photosystem II-mediated transfer of electrons from water to oxidized diamonodurene, with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited 34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 micromolar. Inhibition of photosystem I-mediated transfer of electrons from diaminodurene to methyl viologen with 3,4-dichlorophenyl-1,1-dimethylurea (DCMU) blocking photosystem II was insignificant with either herbicide at all concentrations tested. Transfer of electrons from catechol to methyl viologen in hydroxylamine-washed chloroplasts was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at 0.5 micromolar. The data indicate that the inhibition of electron transport by both herbicides is primarily at the reducing side of photosystem II. However, since catechol is an electron donor at the oxidizing side of photosystem II, between water and chlorophyll a₆₈₀, and lower inhibition levels were observed in the last study (catechol to methyl viologen), it may be that there is also a small inhibition of the mechanism of water oxidation by both herbicides.