Biophysical studies on subcellular particles VI. Photosynthetic activities in isolated spinach chloroplasts after transient warming

Isolated spinach chloroplasts were warmed for a period (typically5 min) at temperatures ranging from 15? to 55?C. Some generalfeatures of the after-effects of transient warming on photosyntheticactivities and some physico-chemical properties were surveyedunder various conditions. Fecy reduction was stimulated when chloroplasts were warmedat about 40?C and was inhibited at higher temperatures. Thetemperature for maximum stimulation shifted to a lower temperaturewith prolongation of warming time, depending on the pH of thewarmed suspension of chloroplasts, and was the same that atwhich photophosphorylation activity fell to zero. The decrease in photosynthetic activities is discussed in connectionwith possible modification of the membrane construction in thylakoidsand is ascribed to the loss of coupling efficiency. (Received August 5, 1972; )     

Biophysical studies on subcellular particles IV. The light scattering response and photosynthetic activities of spinach chloroplasts treated with n-alkanes

A suspension of isolated spinach chloroplasts was shaken withone of a series of liquid n-alkanes, from hexane to dodecane,for several sec. Physical and biological activities of alkane-treatedchloroplasts were compared with those of untreated ones. Thelight scattering response, the light-induced pH shift, the ferricyanide-Hillreaction and noncyclic phosphorylation were inhibited by shakingwith alkanes, with the exception of undecane and dodecane whichtemporarily stimulated the latter two reactions. The degreeof inhibition was inversely proportional to the chain-lengthof the n-alkane. This inhibition by alkane treatment was observed in the lightscattering response much more strongly than in any other reaction.Thus, we postulated that the light scattering response is attributableto changes in some internal structures that are sensitive topenetrated n-alkane molecules. Electronmicroscopy of alkane-treated chloroplasts revealed thatmost of the thylakoid sacs were swollen while partitions apparentlyremained intact. The degree of swelling was also inversely proportionalto the chain-length of the n-alkane. (Received April 22, 1971; )     

Biophysical studies on subcellular particles V. Effects of temperature on the ferricyanide-Hill reaction, the light-induced pH shift and the light scattering response of isolated spinach chloroplasts

Effects of temperature on the ferricyanide-Hill reaction, thelight-induced pH shift (JpH) and the light scattering response(4LS) of isolated spinach chloroplasts were studied at temperaturesranging from 5 to 60°C. Activities which produce JpH and JLS under actinic illuminationdecreased with a rise in the reaction temperature above 30°Cand disappeared at 50°C. The ferricyanide-Hill reactionat pH 6.0 was stimulated by raising the reaction temperaturefrom 5 to 35°C, was slowed at higher temperatures and wasinactivated at 50°C. 4LS activity at temperatures below20°C depended on the activity of electron transport. With a rise in temperature of a suspension of chloroplasts whichexhibited JLS under actinic illumination, the scattering intensitydecreased. The decrease in scattering intensity was limited,at most, to an extent equal to ALS. The behavior of JLS whichdiffered from that of basal scattering is discussed in connectionwith a mechanism of JLS formation other than the shrinkage-swellingcycle. When activities were measured at 25°C with chloroplastswarmed transiently (2 min) at various temperatures (25–60°C),the dependence of JLS on the wanning temperature was identicalwith that on the reaction temperature, while the activitiesof both ferricyanide reduction and JpH formation were higherthan those measured at the corresponding reaction temperature.This suggested that the mechanism for 4LS formation could beirreversibly altered by a warming treatment and that it wasless stable against heat than the mechanisms of ferricyanidephotoreduction and 4pH formation. That is, transiently warmedchloroplasts showed a similar behavior to alkane-treated chloroplasts.The similarity is discussed in relation to the activity changethrough the possible and common cause of disordering of theorganized structures of lipophilic components in the lamellarmembranes. (Received June 5, 1971; )     

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.     

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).     

Relation between photosynthetic and phenolase activities in spinach chloroplasts

During O°-storage of class I chloroplasts from spinach leaves, activation of phenolase strongly correlates with the inactivation of photosynthetic re     

Influence of light on the heat sensitivity of the photosynthetic apparatus in isolated spinach chloroplasts

The most heat-sensitive functions of chloroplasts in Spinacia oleracea L. including the stromal carboxylation reaction, the light-induced electrical field gradient across the thylakoid membrane, as well as the overall photosynthetic CO₂ fixation were less affected by heat if chloroplasts were heated in the light: 50% inactivation occurred around 35°C in the dark and around 40°C in the light. Relative low light intensities were sufficient to obtain optimal protection against heat. In contrast, the light-induced ΔpH across the thylakoid membrane, the photophosphorylation, and the photochemical activity of photosystem II which were less sensitive to heat in the dark (50% inactivation above 40°C) were not protected by light. Photosystem II even was destabilized somewhat by light.

The effect of light on the heat sensitivity of the water-splitting reaction was dependent on the pH in the medium. Protection by light only occurred at alkaline pH, in which case heat sensitivity was high (50% inactivation at 33°C in the dark and at 38°C in the light). Protection was prevented by uncouplers. At pH 6.8 when the heat sensitivity was low in any case (50% inactivation at 41°C in the dark), light had no further protecting effect.

Protection by light has been discussed in terms of light-induced transport of protons from the stroma to the thylakoid space and related ion fluxes.

     

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

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 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.     

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.     

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.     

Phenols,phenoloxidase, and photosynthetic activity of chloroplasts isolated from sugar cane and spinach

Summary Sugar cane chloroplasts isolated in simple media possessed little photochemical activity, but showed rapid O₂ uptake, independent of light. A similar rapid consumption of O₂ was observed with brei prepared from cane leaves. This was not observed in brei of spinach leaves. Authentic polyphenols and cane leaf extracts stimulated the consumption of O₂ by cane preparations and inhibited photosynthesis in chloroplasts isolated from spinach. Chlorogenic acid and caffeic acid were the major o-diphenols in extracts of cane leaves. These compounds inhibited reactions associated with CO₂ fixation by the photosynthetic carbon reduction cycle. Assimilation of CO₂ due to phosphoenol pyruvate carboxylase activity was less sensitive to inhibition by o-diphenols. Mechanisms are discussed whereby o-diphenols may inhibit cane chloroplasts during their isolation.     

Inhibition of photosynthetic carbon dioxide fixation in isolated spinach chloroplasts exposed to reduced osmotic potentials

Reduced osmotic potentials inhibited the rate of CO₂ fixation by isolated intact spinach (Spinacia oleracea) chloroplasts. This inhibition was observed immediately after transfer of chloroplasts from a solution containing 0.33 m sorbitol to higher sorbitol concentrations, and the depressed rate remained constant. The inhibited CO₂ fixation could not be attributed to a decreased rate of photosynthetic electron transport, since NADP reduction was unaffected by subjecting the chloroplasts to low potentials. It could also not result from restricted permeability to CO₂, as CO₂ concentrations had no effect on the relative inhibition induced by the lowered potential.