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Some effects of sugars and sugar phosphates on carbon fixation by isolated chloroplasts
Abstract
1. Carbon dioxide fixation by isolated pea chloroplasts was stimulated by the addition of intermediates of the Calvin photosynthesis cycle and by some related compounds. 2. Ribose 5-phosphate and fructose 1,6-diphosphate consistently produced the largest effects; free sugars such as erythrose and sedoheptulose and acids such as glycollate and glyoxylate were largely ineffective or even inhibitory. 3. Small effects were produced by fructose and ribose but not by their isomers, glucose and xylose. 4. Maximal rates in the presence of ribose 5-phosphate varied between 10 and 50mumoles of carbon dioxide fixed/mg. of chlorophyll/hr.
... Intact chloroplasts, in contrast to fragmented chloroplasts, will evolve oxygen and reduce carbon dioxide to carbohydrate (17). Some, but not all, intermediates of the photosynthetic carbon reduction cycle are known to stimulate photosynthesis in the intact chloroplast (4,5). It has also been shown that intermediates such as fructose 1,6-diP and ribose 5-P which can stimulate photosynthesis of the isolated chloroplast, in contrast to glucose 6-P which has little effect, can restore carbon dioxide assimilation or oxygen evolution inhibited by arsenite (4), iodoacetamide (4), or orthophosphate (8) but not by DCMU (3) or 2-heptyl-4-hydroxyquinoline-N-oxide (3). ...
... Chromatography was carried out as described in references 14 3Essentially dihydroxyacetone-P, some glyceraldehyde 3-P. and in that of Bucke et al. (5), chloroplasts were prepared in sorbitol. From these investigations, a general pattern arises that the photosynthetic carbon cycle intermediates can be divided into three classes according to their effects on the rate of photosynthetic CO2 fixation or 02 evolution and on their ability to affect reversal of certain inhibitors of photosynthesis: Class I. Maximal stimulation and reversal but, on occasion, Incubation was in the standard reaction mixture under N2 with a light intensity of 2600 ft-c. ...
... The restoration of 02 evolution and the observation that the usual pattern of distribution of radioisotope within the Calvin-Benson cycle intermediates reappears is taken as evidence that the chloroplast was functioning normally. Earlier, when CO2 assimilation was the only parameter of photosynthesis measured, it was thought that the class I intermediates bypassed the enzymic block by furnishing carbon skeletons (4,5). Clearly, this conclusion must now be modified. ...
The photosynthetic carbon reduction cycle intermediates can be divided into three classes according to their effects on the rate of photosynthetic CO2 evolution by whole spinach (Spinacia oleracea) chloroplasts and on their ability to affect reversal of certain inhibitors (nigericin, arsenate, arsenite, iodoacetate, antimycin A) of photosynthesis: class I (maximal): fructose 1, 6-diphosphate, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, ribose-5-phosphate; class 2 (slight): glucose 6-phosphate, fructose 6-phosphate, ribulose-1, 5-diphosphate; class 3 (variable): glycerate 3-phosphate. While class 1 compounds influence the photosynthetic rate, they do not lower the Michaelis constant of the chloroplast for bicarbonate or affect strongly other photosynthetic properties such as the isotopic distribution pattern. It was concluded that the class 1 compounds influence the chloroplast by not only supplying components to the carbon cycle but also by activating or stabilizing a structural component of the chloroplast.
... Among these metabolic pathways, carbon fixation in photosynthetic organisms [44] is closely related to plant photosynthesis. Moreover, NtSPS1 OE may enhance carbon fixation by increasing ribose 5-phosphate content (Tables S2 and S3), which affects CO 2 fixation in chloroplasts [45]. The increased CO 2 fixation rate could result from alterations in only part of the carbon reduction cycle utilizing ATP from the photochemical reactions to convert ribose 5-phosphate to ribose 5-diphosphate (the carboxylation reaction substrate) [46]. ...
Nicotiana tabacum solanesyl diphosphate synthase 1 (NtSPS1) is the key enzyme in solanesol biosynthesis. However, changes in the solanesol content, plant growth, photosynthesis, and metabolome of tobacco plants after NtSPS1 overexpression (OE) have not been previously reported. In the present study, these parameters, as well as photosynthetic gas exchange, chlorophyll content, and chlorophyll fluorescence parameters, were compared between NtSPS1 OE and wild type (WT) lines of tobacco. As expected, NtSPS1 OE significantly increased solanesol content in tobacco leaves. Although NtSPS1 OE significantly increased leaf growth, photosynthesis, and chlorophyll content, the chlorophyll fluorescence parameters in the leaves of the NtSPS1 OE lines were only slightly higher than those in the WT leaves. Furthermore, NtSPS1 OE resulted in 64 differential metabolites, including 30 up-regulated and 34 down-regulated metabolites, between the OE and WT leaves. Pathway enrichment analysis of these differential metabolites identified differentially enriched pathways between the OE and WT leaves, e.g., carbon fixation in photosynthetic organisms. The maximum carboxylation rate of RuBisCO and the maximum rate of RuBP regeneration were also elevated in the NtSPS1 OE line. To our knowledge, this is the first study to confirm the role of NtSPS1 in solanesol biosynthesis and its possible functional mechanisms in tobacco.
... Osterhout and Hass (1918) originally proposed that the time required for light-activation of Calvin cycle enzymes and the build-up of intermediates was the main cause for the induction period. Observation of shortened lag period by pre-illumination was supportive of the catalyst activation (Walker, 1976), and abolishment of the lag phase but with no effect on the linear rate by chloroplast penetrating sugar-Ps (triose-P, R5P) was seen as crucial evidence for the limitation in Calvin cycle intermediates during the lag period (Bamberger and Gibbs, 1965; Baldry et al., 1966b; Bucke et al., 1966). Since sugar-P content was directly involved in the manifestation of the lag period, we tested the effect of exogenous sugar-Ps added after heat-treatment on photosynthesis. ...
When the time course for CO2 fixation and O2 evolution in isolated intact spinach chloroplasts was examined, we found a prolonged lag time in the early phase of photosynthesis
after heat-treatment in the dark as well as an expected time-dependent decrease in the rate during the subsequent linear phase.
Because the lengthening of the lag period was generally attributed to the depletion of sugar phosphates in the chloroplasts,
we tested for the possible involvement of Calvin cycle intermediates in the change of the lag phase by heat-treatment When
triose phosphate was added to the heated chloroplasts, the lag time was re-shortened without the rate in the linear phase
being elevated to that measured in the control. Mg-ATP or triose phosphate plus oxaloacetate (previously known as protective
chemicals) prevented the lengthening of the lag time when added prior to heat-treatment. Quantification of some metabolites
in the chloroplasts confirmed that heavy losses had occurred for triose phosphate, fructose-1,6-bis-phosphate, glucose-6-phosphate,
and fructose-6-phosphate. However, the level of 3-phosphoglyceric acid was increased. The presence of Mg-ATP during heat-treatment
alleviated the losses of those sugar phosphates. Therefore, we conclude that the decrease in sugar phosphates in the chloroplasts,
as part of the negative effect from heat-treatment, is the primary cause of the lengthened lag time during the initial phase
of photosynthesis.
... Before the 1968 Photosynthesis Congress, just as we reached the magic 100 (Bucke et al. 1966), it was difficult to avoid some feeling of chagrin when we learned that we had been pipped at the post by Jensen and Bassham (1966) who made headlines in the New York Times with similarly high rates of CO 2 fixation. Moreover, their high rates were achieved without the requirement for 'catalytic' sugar phosphates which we used as a matter of routine to diminish the initial induction period before the attainment of maximum rate. ...
This is about a young man who wished to go to sea like his father and finished up, instead, in photosynthesis. It describes how he served his apprenticeship in England and the United States and how he was then lucky enough to find himself in the laboratory of Robin Hill, one of the all-time greats in this field. It discusses some of the events that led, via mitochondria in castor beans and carboxylating enzymes in Crassulacean plants, to the isolation of fully functional chloroplasts and the manner in which the first polarographic measurements of CO2-dependent O2 evolution contributed to present understanding of the movement of molecules through the chloroplast envelope. It describes some of the problems with materials and apparatus which were commonplace forty years ago and reflects on the advantages of working in foreign places and the pleasures of becoming a member of a truly international community.
... The inner envelope membrane of the higher plant chloroplast is selectively permeable to photosynthetic intermediates (Heber, 1974;Walker, 1974), and PGA, triose phosphates and Pi are specifically and rapidly transported across this metabolic barrier by a phosphate translocator (Heldt and Rapley, 1970). However, the effects of various photosynthetic carbon reduction cycle intermediates on carbon fixation by isolated C fragile chloroplasts (Table 2) are different from, and generally not as pronounced as those reported for isolated P/swm (Bucke, Walker and Baldry, 1966) or Spinacia (Bamberger and Gibbs, 1965) chloroplasts, particularly in their response to R5P, FBP and SBP. This suggests either that Codium fragile chloroplasts may not be as rapidly permeable to these metabolites as their higher plant counterparts, or that these metabolites function differently in the alga. ...
Chloroplasts of the siphonaceous marine alga Codium fragile exhibit different carbon fixation characteristics in vitro from those of higher plants. A simple extraction medium, 0.8 M osmoticum and a saturating light intensity of 25 W.m−2 produced maximum rates of 40–60 μmoles CO2 fixed mg. chlorophyll−1 h−1 at 20°C, depending on the age and condition of the parent tissue. No marked lag phase was apparent and fixation proceeded at a linear rate for up to 60 min.
Incubation with 10 mM PGA enhanced the fixation rate, as did 10 mM SBP, 10 mM FBP and 5 mM R5P to a lesser extent, whereas 6 PGlu, S7P, F6P, Ru5P and PEP produced no measureable effect. This lack of marked response to the addition of exogenous photosynthetic intermediates is correlated to a lack of induction by these chloroplasts.
Using the retention of the soluble stromal enzyme RuBP carboxylase as a criterion of intactness, freshly isolated C. fragile chloroplasts were approximately 80% intact, and were still 40% intact after 96 h storage in the dark on ice.
RuBP carboxylase was shown to resemble the higher plant carboxylase, existing in either an active or inactive form depending on the prevailing levels of RuBP and Mg2+, and to be metabolically regulated by PGA, 6PGlu, SBP, FBP and Pi. Maximum rates obtained with a chloroplast extract were 155 jumoles CO2 fixed mg chlorophyll−1 h−1, at 20°C.
Dan Arnon, Bob Whatley, Mary Belle Allen, and their colleagues, were the first to obtain evidence for ‘complete photosynthesis by isolated chloroplasts’ albeit at rates which were 1% or less of those displayed by the intact leaf. By the 1960s, partly in the hope of confirming full functionality, there was a perceived need to raise these rates to the same order of magnitude as those displayed by the parent tissue. A nominal figure of 100 μmol/mg·chlorophyll/h (CO2 assimilated or O2 evolved) became a target much sought after. This article describes the contributions that [Dick Jensen and Al Bassham [(1966) Proc Natl Acad Sci USA 56: 1095–1101], and my colleagues and I, made to the achievement of this goal and the way in which it led to a better understanding of the role of inorganic phosphate in its relation to the movement of metabolites across chloroplast envelopes.
The effect of chlorsulfuron [2-chloro-N-(4-methoxy-6-methyl-1,3,5-triazine-2-yl) aminocarbonyl benzene sulfonamide] was studied on different photochemical reactions in isolated chloroplasts of kidney beans Phaseolus vulgaris L). Chlorsulfuron (10⁻⁴ mol dm⁻³) inhibited both cyclic and non-cyclic photophosphorylation and C02 fixation by 15%, 22% and 17% respectively, whereas electron transport was stimulated by 10%. Although chlorsulfuron at high concentrations functions as an uncoupler of photophosphorylation in chloroplasts, it does not operate primarily as an inhibitor of photosynthesis.
Rewriting the chapter more than 10 years after its original publication (Giles 1977) is not made easy by the exciting changes that have occurred in closely associated fields over that time. To a large degree the uptake of foreign DNA, using Agrobacterium tumefaciens as a vector has eclipsed organelle uptake studies in the past few years. This trend is understandable because of the increased flexibility and range of genetic manipulations possible using the Agrobacterium system, since it effectively allows the manipulation of the whole genome rather than the plastoms alone. Chloroplast uptake and exchanges, however, continue to have a useful role in studies involving nuclear-chloroplast interaction, the physiology of isolated plastids and experiments investigating the transfer of herbicide resistance, particularly to the triazines, between varieties and species.
Fruit Effect on Photosynthesis and Respiration
Several investigations about fruit effects on photosynthesis and respiration, mainly of Citrus madurensis (Lour.) and Solanum melongena (L) plants are reviewed.
Fruiting plants had higher photosynthetic rates (mg CO2 · dm−2 · h−1) than non fruiting ones, particularly during stages of most intensive fruit growth. The photosynthetic rates could be stimulated by increasing fruit temperatures.
No differences in dark respiration rates of fruiting and non fruiting plants were observed. However photorespiration was considerably higher in non fruiting plants as compared to fruiting ones. Leaves of non fruiting plants accumulated more sugars and starch in the leaves during the light periods than fruiting ones.
The gaseous diffusive resistance of leaves was higher in non fruiting as compared to fruiting ones.
The higher photosynthetic efficiency of fruiting plants is probably controlled by several mechanisms, some of which were discussed in the paper.
Increasing levels ofC02havebeenshowntostimulate the rateofphotosynthesis, eliminate theoxygeninhibition ofpho- tosynthesis (Warburg effect), anddecrease glycolate formation inisolated spinach chloroplasts. Ribose5-phosphate andfruc- tose1,6-diphosphate atconcentrations of5to10IMalsostimu- latetherateofplastid photosynthesis andeliminate theWar- burgeffect. Incontrast totheeffect ofhighCO2levels, these sugarphosphates havelittle effect onglycolate formation. Evi- denceispresented toshowthatthelevel ofintermediates of thephotosynthetic carbonreduction cycle may influence the Warburgeffect invivo.Itispostulated thattheformation of glycolate isnotthecausal factor oftheWarburg effect.
Thephotosynthetic carbon reduction cycle intermediates can bedivided intothreeclasses according totheir effects on the rateofphotosynthetic C02evolution bywholespinach (Spinacia oleracea) chloroplasts andontheir ability toaffect reversal of certain inhibitors (nigericin, arsenate, arsenite, iodoacetate, an- timycin A)ofphotosynthesis: class I(maximal): fructose 1,6- diphosphate, dihydroxyacetone phosphate, glyceraldehyde-3- phosphate, ribose-5-phosphate; class2 (slight): glucose 6-phosphate, fructose 6-phosphate, ribulose-1, 5-diphosphate; class3 (variable): glycerate 3-phosphate. Whileclass1 compoundsinfluence thephotosynthetic rate,theydo not lowertheMichaelis constant ofthechloroplast forbicarbonate oraffect strongly otherphotosynthetic properties suchasthe isotopic distribution pattern. Itwasconcluded thattheclass 1compounds influence thechloroplast bynotonlysupplying components tothecarboncycle butalsobyactivating orsta- bilizing astructural componentofthechloroplast.
This chapter discusses the preparation of higher plant chloroplasts, concentrates on the possible sources of difficulty in separation of intact chloroplasts from spinach, and mentions of some relevant matters that have become clearer in the recent past. Fully functional chloroplasts may be readily separated from spinach protoplasts. Chloroplasts isolated from young pea shoots have different permeability characteristics to those from mature spinach. For culture preparation in the case of peas, seeds are soaked overnight in aerated water and germinated, in vermiculite, in flat trays at a density of 125 gm (dry weight) per tray (38 × 23 × 8 cm). The combination of low light and high temperature is avoided, but in relatively strong light (>50 W/m 2) temperatures of 20–25° and photoperiods of 9–12 hours, good material can be grown in 9–11 days, by that time, the shoots of a variety are 3–4 cm high. For spinach, any one of a number of sugars or sugar alcohols may be used as osmotica. Good chloroplasts can be prepared in the absence of Mg, but the presence of Mg together with EDTA appears to be beneficial.
CO2 exchange, 14CO2 fixation and 14C labelled products of Chlorella vulgaris (strain 211-11f) were studied during the photosynthetic induction period at +10° and +25°C after a dark period of 40 min and 40 sec. The algae were grown under normal aerated conditions (0.03 vol.-% CO2) at +27°C. Transient changes in CO2 uptake, measured with an infrared gas analyzer, could be observed only after a dark period of >3 min; no such changes occurred after a dark period of 40 sec. The autoradiographic studies of the kinetics of the appearance of labelled products at +10° and +25°C showed that after a long dark period (40 min) at the beginning of illumination 14CO2 was incorporated into malate, aspartate and 3-phosphoglycerate. Under these conditions, the intermediates of the Calvin cycle were labelled after 30 sec (+25°C) or 2 min (+10°C) of photosynthesis. After a dark period of 40 sec (at +10° and +25°C), however, 14C incorporation into malate and aspartate was rather low at the beginning of illumination; moreover, the intermediates of the Calvin cycle appeared earlier and were more strongly labelled after this short dark period. The results are discussed with reference to the influence of intermediates on the formation of the transient changes of CO2 uptake in Chlorella.
THE chloroplasts isolated by Hill1 in the 1930s were capable of rapid rates of oxygen evolution when provided with an artificial electron acceptor (oxidant) but did not retain the ability to assimilate CO2 at rates detectable by the methods then available. Nearly 20 yr later the ability of isolated chloroplasts to fix CO2 was demonstrated by Arnon, Alien and Whatley2 but even then the rates were only 3-6% of those supported by the intact plant. By 1966, when the rates of fixation by chloroplasts isolated in sugar media first matched those of intact leaves3,4, it had been established5,6 that high rates of carbon assimilation were only achieved by chloroplasts with intact envelopes. It is unlikely that all the earlier difficulties were caused by the unintentional isolation of damaged chloroplasts but, except for some early work by Arnon7 (in which chloroplasts were supplemented with cytoplasmic malic enzyme), it has certainly not been possible to show CO2-dependent oxygen evolution by envelope-free chloroplasts8 and rapid rates (comparable with the Hill reaction) have not been previously reported. Such high rates have now been observed in a reconstituted system containing ATP, ferredoxin, NADP (nicotinamide adenine dinucleotide phosphate), R5P (ribose-5-phosphate), CE (soluble components released from osmotically shocked chloroplasts) and envelope-free chloroplasts.
Using isolated spinach chloroplasts capable of high rates of photosynthesis with 14CO2, we have investigated the effects on these rates of the additions of metabolic intermediate compounds of the carbon reduction cycle, of additions of certain cofactors, and the additions of pyrophosphate and of phosphate. Following a pre-illumination period of 3 min to overcome the induction period, the rates of 14CO2 photosynthesis during the first 10 min are essentially the same as would be found for spinach leaves in vivo, when allowance is made for the fraction of chloroplasts (30% or less) that have lost their structural integrity. During this 10-min period, no metabolic intermediate compounds or cofactors were found which appreciably stimulated the rate, except that added inorganic pyrophosphate is required to achieve the maximum rate. After 20 min the rates were always less, but could be somewhat sustained by the addition of fructose 1,6-diphosphate, and to a lesser degree by addition of NADP+. This stimulation could not be replaced or enhanced (in the highly active preparations) by ribose 5-phosphate. It is proposed that the activity of the carboxylation enzyme, ribulose-diphosphate carboxylase (EC 4.1.1.39), is the rate-limiting factor after 20 min. Thus, it appears that the stimulatory effects of added fructose 1,6-diphosphate and NADP+ may be exerted in some way on the carboxylation reaction itself rather than in the regeneration of the carboxylation substrate. The rates were not sharply dependent on inorganic phosphate concentration, but the maximum rate was found with about 1.0 mM added inorganic phosphate.
RECENTLY developed techniques have led to the isolation of intact chloroplasts which will assimilate carbon dioxide at rates approaching those achieved by the intact plant1-6. This has made it possible to measure the associated oxygen evolution, polarographically, in aerobic conditions7. Normally, oxygen evolution parallels carbon dioxide fixation7,8. On illumination, both start slowly and accelerate gradually until a maximum rate is reached after several minutes. This induction phase is thought to reflect corresponding changes in the concentration of carbon cycle intermediates as these increase autocatalytically to a steady state level2-4,7-11. If 3-phosphoglycerate (PGA) is provided as a substrate the initial lag is virtually eliminated7 and the kinetics then approximate to those observed in the Hill reaction12 in which an artificial hydrogen acceptor reacts more directly with the photochemical system. This was held7 to be consistent with the view13 that the 3-phosphoglycerate is the immediate precursor of the hydrogen acceptor (1,3-diphosphoglycerate) in the carbon cycle. If this is correct, it would follow that the oxygen evolution observed when PGA is added as a substrate7 should not be dependent on carbon dioxide-bicarbonate (Fig. 1). Conversely, any stimulation of oxygen evolution by triose phosphate should cease in the absence of carbon dioxide. Experiments which substantiate these conclusions are reported here.
In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PPi), required for maximum activation depended on the pH, the concentration of orthophosphate (Pi) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.
Low concentration of hydrogen peroxide strongly inhibit CO2 fixation of isolated intact chloroplasts (50% inhibition at 10(-5) M hydrogen peroxide). Addition of catalase to a suspension of intact chloroplasts stimulates CO2 fixation 2--6 fold, indicating that this process is partially inhibited by endogenous hydrogen peroxide formed in a Mehler reaction. The rate of CO2 fixation is strongly increased by addition of Calvin cycle intermediates if the catalase activity of the preparation is low. However, at high catalase activity addition of Calvin cycle intermediates remains without effect. Obviously the hydrogen peroxide formed at low catalase activity leads to a loss of Calvin cycle substrates which reduces the rate of CO2 fixation. 3-Phosphoglycerate-dependent O2-evolution is not influenced by hydrogen peroxide at a concentration (5x10(-4) M) which inhibits CO2 fixation almost completely. Therefore the inhibition site of hydrogen peroxide cannot be at the step of 3-phosphoglycerate reduction. Dark CO2 fixation of lysed chloroplasts in a hypotonic medium is not or only slightly inhibited by hydrogen peroxide (2,5x10(-4) M), if ribulose-1,5-diphosphate, ribose 5-phosphate or xylulose 5-phosphate were added as substrates. However, there is a strong inhibition of CO2 fixation by hydrogen peroxide, if fructose 6-phosphate together with triose phosphate are used as substrates. This indicates that hydrogen peroxide interrupts the Calvin cycle at the transketolase step, leading to a reduced supply of the CO2-acceptor ribulose 1,5-diphosphate.
Glycerate-3-P inhibits CO2 fixation of isolated spinach chloroplats at concentrations higher than 1 mM but does not inhibit O2 evolution. Glycerate-3-P inhibition of photosynthesis is not overcome by higher bicarbonate concentrations.
Homogenates of dark-pretreated leaves yield two particulate fractions in density gradient centrifugation: one contains chlorophyll (chloroplasts) while a second fraction contains ribulose-1, 5-bisphophate carboxylase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and catalase. Addition of a microbody-rich pellet to chloroplasts isolated from dark-pretreated plants largely enhances both oxygen evolution and CO2-fixation into organic compounds. The pathway of CO2 reduction may be part of a membrane system which, under suitable conditions, may separate from the chloroplast as a distinct cytoplasmic entity, having physical properties similar to those of microbodies.
1. After an initial lag, isolated spinach chloroplasts evolved O2 in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate.2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O2 were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.3. With CO2 as the sole added substrate the extent of the initial lag in O2 evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO2.4. There was also a requirement for phosphate by chloroplasts evolving O2 in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO2. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O2 evolution which normally followed the addition of 3-phosphoglycerate.5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.
1.1. Whole spinach chloroplasts incorporated 32P into organic compounds in the light in the presence of ribose 5-phosphate, without added ADP or bicarbonate. This incorporation was inhibited by antimycin A.2.2. The simultaneous addition of ADP and ribose 5-phosphate resulted in a higher 32P incorporation than with either compound added alone, providing that residual bicarbonate was present to allow some carboxylation to occur: photophosphorylation seems to be stimulated under carboxylating conditions.3.3. The CO2-fixation activities in the presence of ribose 5-phosphate were identical whether antimycin was present or not, when the phosphate concentration was low. Under these conditions, a relative accumulation of phosphoglyceric acid was observed in all cases, but in the presence of antimycin, this accumulation appeared sooner. In the presence of high phosphate concentration chloroplasts accumulated only triose phosphates: the phosphoglyceric acid accumulation seems to be related to a lack of ATP. This observation allows the assumption that antimycin inhibits photophosphorylation even under conditions where the CO2 fixation activity is enhanced or unaffected.4.4. A working hypothesis is proposed to account for the stimulation of carboxylation linked to the inhibition of photophosphorylation: bicarbonate would be absorbed by chloroplasts at the expense of a high-energy intermediate.Résumé1.1. En absence d'ADP ou de bicarbonate ajouté, les chloroplastes entiers d'épinard incorporent du 32P à la lumière en présence de ribose-5-phosphate. Cette incorporation est inhibée par l'antimycine.2.2. L'addition simultanée d'ADP + ribose-5-phosphate entraîne une incorporation de 32P plus importante qu'en présence de chacun de ces composés ajouté séparément, à condition que la présence de bicarbonate résiduel permettre une carboxylation non négligeable: le fonctionnement de la carboxylation semble entraîner une stimulation de la phosphorylation.3.3. Les activités de fixation du CO2 sont identiques en présence de ribose-5-phosphate ou de ribose-5-phosphate + antimycine, lorsque la concentration en phosphate est faible. Dans ces conditions, on observe toujours une accumulation relative d'acide phosphoglycérique, cette accumulation étant plus précoce en présence d'antimycine. En présence de concentrations en phosphate élevées, les chloroplastes n'accumulent que des trioses-phosphate: l'accumulation d'acide phosphoglycérique semble liée à un déficit en ATP. Cette observation permet de supposer que l'antimycine inhibe la photophosphorylation même lorsque l'activité de fixation du CO2 est stimulée ou inchangée.4.4. Pour rendre compte de la stimulation de la carboxylation liée à une inhibition de la phosphorylation, l'hypothèse d'une absorption de bicarbonate aux dépens d'un intermédiaire riche en énergie est proposée.
Light-dependent O2 evolution by isolated chloroplasts was first observed by Haberlandt in 1888 (1) and confirmed by Ewart in 1896 (2) but for all practical purposes the quantified study of in
vitro photosynthesis started with the experiments of Robert Hill (3, 4). Following his development of a spectroscopic method for measuring O2 (5), he prepared chloroplasts by crushing leaves in a solution containing 0.29 M sucrose and 0.033 M orthophosphate (3, 4, 6). He illuminated these with myoglobin in the presence of an aqueous extract derived from an acetone leaf powder. In his own words “It was a very thrilling moment when I saw the spectrum of oxymyoglobin. Then later on a sad disappointment: the presence or absence of CO
2
made no difference. This was really lucky, however, because if CO
2
had ‘worked’ I might well have got no further” (7). Because his chloroplasts did not “work” with CO2, Hill pursued the contribution made to his reaction by the acetone powder and showed that the oxygen produced corresponded to the reduction of a hydrogen or electron acceptor. In so doing he laid the foundations on which much of our present understanding of photosynthetic electron transport rests. It is not the aim of this article to attempt to evaluate the contribution made to photosynthesis by the army of workers who have followed Hill’s example and started their laboratory day by grinding leaves in a variety of media.
The chloroplast always seems to be faced with reconciling the irreconcilable. While evolving oxygen, it must simultaneously produce an intermediate more reducing than hydrogen. While reducing NADP it can simultaneously generate ATP, an achievement which, when first reported, seemed almost as remarkable as making water flow uphill. No less striking in their own way are two seemingly conflicting roles in carbon metabolism. On the one hand, the chloroplast must operate its carbon cycle as an autocatalytic breeder reaction, while on the other, it must export elaborated carbon and chemical energy to its cellular environment. In order to export, it must produce more than it uses, but it can only do this by returning newly synthesised intermediates to the cycle. Conversely, in order to satisfy the needs of the cell, it must release newly made products to the cytoplasm. Clearly, these processes could not be efficiently accomplished unless it were possible to strike a delicate balance between recycling, export and internal storage. Precisely how this is done is still largely a matter for speculation, but this Chapter will attempt to show how contemporary work has provided a factual basis for conjecture.
After a brief discussion of my graduate work at Duke University, I describe a series of investigations on redox proteins at the University of California, Berkeley. Starting with ferredoxin from fermentative bacteria, the Berkeley research fostered experiments that uncovered a pathway for fixing CO2 in bacterial photosynthesis. The carbon work, in turn, opened new vistas, including the discovery that thioredoxin functions universally in regulating the Calvin-Benson cycle in oxygenic photosynthesis. These experiments, which took place over a 50-year period, led to the formulation of a set of biological principles and set the stage for research demonstrating a role for redox in the regulation of previously unrecognized processes extending far beyond photosynthesis.
Although the physiological nature of cyclic photophosphorylation is now well documented (MACLACHLAN & PORTER 1959, FORTI & PARISI 1963, URBACH & SIMONIS 1964, WIESSNER & GAFFRON 1964, TANNER et al. 1965, 1966, WIESSNER 1965, NULTSCH 1966, 1967, JESCHKE 1967, RAVEN 1967, RAMIREZ et al. 1968, ZANETTI 1969, GIMMLER 1970, MIGINIAC-MASLOW 1971) there is a divergence of views concerning its involvement in CO2 assimilation. A need for cyclic photophosphorylation seemed to arise from the requirement of an excess of ATP over NADPH (in a ratio of 3 to 2) for the assimilation of CO2 to the level of carbohydrate (CALVIN & BASSHAM 1962) - a requirement that cannot be met by noncyclic photophosphorylation alone which produces ATP and NADPH in a ratio of 1 to 1 (ref. ARNON et al 1958, JAGENDORF 1958, AVRON & JAGENDORF 1959, STILLER & VENNESLAND 1962, TURNER et al 1962, DEL CAMPO et al 1968). Consistent with this conclusion were CO2 assimilation experiments with broken chloroplasts in which sugar phosphates were formed only when both cyclic and noncyclic photophosphorylation were operating in a proper balance (TREBST et al 1959).
One of the most significant developments in the field of plant tissue culture during. recent years has been the isolation, culture and fusion of protoplasts (Coking, 1972). The techniques are especially important because of their far-reaching implications in studies of plant improvement by cell modification and somatic hybridization (see Bajaj, 1974a). Isolated protoplasts also offer a means of tackling various fundamental and pragmatic research problems in experimental plant biology. This can be realized mainly because of the totipotent nature of plant cells. The literature accumulated so far has revealed that protoplasts in culture can be regenerated into an entire plant; they can be induced to undergo intra- and interspecific fusion to form a somatic hybrid, and also to take up foreign organelles and genetic materials. In addition to general protoplast culture and somatic hybridization studies referred to in this article, protoplasts have been used to investigate various problems in plant physiology (Boulware and Camper, 1972; Bayer, 1973; Hess and Endress, 1973; Kanai and Edwards, 1973; Börner, 1973; Birecka and Miller, 1974; Hall and Cocking, 1974; Hoffmann and Kull, 1974; Gutierrez
et al., 1974; Ku et al., 1974; Podbielkowska et al., 1975; Shepard and Totten, 1975), radiobiology (Ohyama
et al.,1974; Galun and Raveh, 1975; Howland, 1975; Howland
et al., 1975), virology and pathology (Cocking, 1966; Aoki and Takebe, 1969; Hibi and Yora, 1972; Coutts, 1973; Honda
et al., 1973; Burgess
et al., 1974; Sarkar
et al., 1974; Zaitlin and Beachy, 1974; Birecka et al., 1975a, b; Pelcher et al.,1975), cytogenetics (Carlson, 1973 a, Chaleff and Carlson, 1974) and cell modification and uptake studies (Davey and Cocking, 1972; Hess, 1973a, b; Hoffmann, 1973; Hoffmann and Hess, 1973; Blaschek
et al., 1974; Giles, 1974; Holl
et al., 1974).
Investigations of the effects of fruit (mainly citrus) on growth, flower formation, water consumption, nutrient uptake, photosynthesis, and respiration of plants are reviewed. The main emphasis is on photosynthetic efficiency of leaves as affected and perhaps regulated by fruit.
At present, man’s existence rests almost entirely on photosynthetic carbon assimilation. In the higher plant, carbon dioxide moves inwards across the limiting membranes (envelopes) of the chloroplast, and the elaborated carbon required for plant growth and development is exported. It has been known for many years that sucrose is an important “end-product” of photosynthesis and that in most plants it is the principal compound to be moved from tissue to tissue (Arnold, 1968). Since the reductive pentose phosphate pathway (RPPP) or Benson-Calvin cycle (Bassham and Calvin, 1957) is located within the chloroplast1 (see e. g. Gibbs, 1971), it seemed very likely that sucrose would be synthesized within this organelle and transported from it to the rest of the plant (see e. g. Davies, 1974). During the past decade it has become increasingly evident that this assumption is incorrect (Bird et al., 1974; Walker, 1976a). At least for C3 species there is compelling evidence that the major metabolite exported from the chloroplast is triose phosphate (Heber, 1974; Walker, 1974 and 1976 a). In its simplest form the RPPP can be written $$3{\rm{C}}{{\rm{O}}_{\rm{2}}} + {{\rm{P}}_{\rm{i}}} + {{\rm{H}}_{\rm{2}}}{\rm{O}} \to {\rm{triose}}\,{\rm{phosphate}} + 3{{\rm{O}}_{\rm{2}}}$$ (1) If triose phosphate is to be exported and photosynthesis is to continue, it follows that there must be a stoichiometric import of orthophosphate. Contemporary theory therefore not only locates photosynthesis within the chloroplast, but proposes that equation (1) also represents the most important exchange of metabolites across the chloroplast envelope. In this way, the availability of cytoplasmic orthophosphate must influence events within the chloroplast.
In the two centuries that have elapsed since the discovery of photosynthesis, no period has surpassed the last 25 years in richness of discovery and conceptual advances. It so happens that the progress made in the last quarter of a century can be assessed with considerable accuracy, for at midcentury, in July 1950, the Society for Experimental Biology held a Symposium on Carbon Dioxide Fixation and Photosynthesis at Sheffield, England, and the Proceedings of that Symposium (published in 1951) provide a reliable record of some of the main experimental and conceptual realities that characterized photosynthesis research up to the mid-twentieth century. This article will use the Sheffield Symposium as a point of departure for tracing progress in the next quarter of a century and will give special emphasis to the experimental and conceptual advances in the areas of photosynthetic electron transport and photophosphorylation in chloroplasts. No attempt will be made to cover all of the vast literature that has accumulated in these areas in the last 25 years. Such an attempt would be neither practical nor desirable; individual topics are covered in separate chapters of this volume. The emphasis here will be on developments that led to changes in concepts and perspectives.
Publisher Summary In higher plants, the process of photosynthesis occurs within specific membrane bounded organelles called “chloroplasts.” All the chloroplasts exhibit three major structural regions— namely, highly organized internal sac-like flat compressed vesicles called “thylakoids,” an amorphous background rich in soluble proteins called “stroma,” and a pair of outer membranes known as the “envelope.” The envelope essentially renders functional and structural integrity to the chloroplast. This chapter discusses the structure, isolation, chemical composition, and origin of the higher plant chloroplast envelope. The chapter examines the multiple functions of this important membranous system involved in the regulation of the inflow of raw materials for photosynthesis and the outflow of photosynthetic products. The chloroplast envelope of higher plants is a permanent structure and consists of two morphologically and topologically distinct membranes separated by a region about 10–20 nm thick, which appears electron-translucent. The structure of both envelope membranes is consistent with the lipid-globular protein mosaic model of membrane structure as proposed by Singer and Nicolson.
Photosynthetic carbon assimilation by isolated chloroplasts was first demonstrated by Arnon et al. in 1954. Work in the same laboratory also showed that the amount of CO2 fixed was diminished by osmotic shock and that photosynthesis could be “reconstituted” by mixing osmotically shocked chloroplasts with “chloroplast extract”. At first, only slow rates of CO2 fixation by intact chloroplasts were reported, but improvements in technique eventually led to rates which equalled those of the parent tissue, (Bucke et al., 1966; Jensen and Bassham, 1966).
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.
When leaf discs from spinach plants (Spinacia oleracea L.) maintained in the dark for several days were subsequently illuminated, the decrease of incorporated (14)CO2 measured under steady state conditions was found to be accompanied by an altered fixation pattern. Substances found to contain a significantly lower label, were malate and aspartate. In contrast, an enhanced incorporation of radioactivity was observed for those substances known to be formed during light respiration. Since the same tendencies were obtained at higher CO2 concentrations and after the removal of the lower epidermis, a new metabolic situation rather than an impaired CO2 supply, was considered to be responsible for the altered turnover of intermediates. The constant ratio in the labelling of intermediates formed in the chloroplasts and the known localization of enzymes involved in the formation of C-4 components led us to conclude that primarily the activity of cytoplasmic enzymes is influenced by dark.
The fixation pattern of radioactive labelled photosynthetic intermediates was followed under steady state conditions during prolonged dark starvation of spinach plants (Spinacia oleracea L.). It is suggested that the considerable increase of radioactive dihydroxyacetonephosphate is correlated with a specific leakage of the outer chloroplast envelope induced by dark starvation. The primary fixation product, phosphoglyceric acid, followed the same decreasing tendency as observed for the net CO2 fixation. In contrast, the relative label in other intermediates is the same as in the controls. When after several days of dark starvation the plants were again transferred into light, a regeneration of the CO2 fixation accompanied by the appearance of a normal fixation pattern was observed. Since the regeneration was prevented by the addition of lincomycin, the net increase is considered to be due to a new protein synthesis rather than a reactivation.
Glutaraldehyde fixation in 0.33 M sorbitol without any buffer reveals changes in the staining properties of the envelopes of chloroplasts of pea plants kept in the light or in the dark prior to fixation. After dark pretreatment the outer double membrane of the chloroplast does not adsorb heavy metals, resulting in a "white" unstained rim instead of the usual membrane. All other membranes of the cell, including chloroplast grana, are not affected and stain normally. Light pretreatment of the plants allows the usual staining of the outer membrane of the chloroplats. Fixation carried out in the medium usually used to isolate intact CO2 fixing chloroplasts (sorbitol+buffer+ions) reverses the above process and results in unstained envelopes of chloroplasts from preilluminated leaves, while the envelopes of chloroplasts from leaves kept in the dark stain normally. Glutaraldehyde-fixed chloroplats isolated from preilluminated leaves show a very basic isoelectric point during electrofocusing, while fixed chloroplasts from predarkened tissue exhibit an isoelectric point at about pH 7.
Net photosynthetic rates are closely related with the net matter production of a plant. For this reason it is of considerable interest to examine the machanism regulating the net photosynthetic rate. It has often been supposed that the assimilate concentration in the leaves could play a role in this regulation. In order to obtain information about the significance of the sucrose and starch contents of leaves for the regulation of the net photosynthetic rate, experimental data obtained from eggplants bearing and lacking fruit were compared. All investigations were carried out in a control chamber. The results of these investigations were:
1.During the period of illumination, the net photosynthetic rate of the eggplants without fruit decreased to a considerably greater degree than did that of the eggplants bearing fruit. This stronger decrease in the net photosynthetic rate has been found to be independent of whether the fruit was excised just prior to the beginning of the measurement or whether the fruit set was prevented by removal of the flowers.2.The removal of the fruit immediately before the beginning of the photosynthetic measurement did not influence the starch content of the leaves. On the other hand, the prevention of fruit set by removal of the flowers resulted in higher starch contents of the leaves as compared with those plants bearing a fruit.3.The removal of the fruit prior to the beginning of the photosynthetic measurement led to a considerable increase in the sucrose content of the leaves. On the other hand, the prevention of fruit set by removal of the flowers did not influence the sucrose content of the leaves.
The results of the investigations clearly indicated that the carbohydrates examined here are of no significance in the regulation of the net photosynthetic rate.
Induction of photosynthetic carbon assimilation in spinach leaves in saturating CO2 has been studied in relation to such factors as the length of the preceding dark period, intensity of pre-illumination, irradiance and the phosphate status of the leaf, through measurements of O2 evolution and of changes in leaf metabolites. (i) In low light, induction comprised two phases, an oxygen burst and a slow rise to the steady-state rate. Both the oxygen burst and induction (measured as induction gain or loss) correlated with the amount of glycerate-3-P present in the leaf prior to illumination. (ii) The induction period in dark-adapted leaves was dependent on the irradiance, being greater at higher irradiance. In the early stages of induction at different irradiances metabolite levels rather than the rate of electron transport appeared to limit the rate of photosynthesis. (iii) The induction period was extended in leaves from plants grown in low phosphate or where phosphate was sequestered by the use of 2-deoxyglucose while the induction period was shortened by feeding phosphate to phosphate-deficient leaves. Phosphate deficiency was characterized by low glycerate-3-P in the dark and a slow build-up to lower steady-state levels of metabolites in the light, with the absence of characteristic peaks in the contents of glycerate-3-P, Fru-1,6-P2 and the ratio during induction. These observations serve to emphasise that the conversion of existing metabolite pools and metabolite build-up are important determinants of the induction period in leaves.
It was the development of enzymic methods of protoplast isolation in the 1960s that allowed the preparation of large numbers of isolated protoplasts. An isolated protoplast may be defined as a plant cell that has had its outer wall removed; therefore the only boundary between the cell contents and the external environment is the plasma membrane. The removal of the rigid cell wall causes the protoplast to take on a spherical conformation in liquid culture (conformation of minimum energy) and also opens the way to technologies that cannot be applied to other plant cells.
The role of cyclic photophosphorylation in photosynthetic CO2 assimilation was investigated in isolated, intact chloroplasts capable of high rates of CO2 fixation. ATP produced by endogenous cyclic photophosphorylation was found to play an important role in shortening the lag period in CO2 assimilation and in the formation of sugar phosphates. Inhibition of either cyclic or noncyclic photophosphorylation severely lowered total CO2 fixation but gave contrasting patterns of products formed: increased sugar phosphates and decreased phosphoglycerate when noncyclic photophosphorylation was inhibited and decreased sugar phosphates and increased phosphoglycerate when cyclic photophosphorylation was inhibited. Antimycin A, which inhibits ferredoxin-catalyzed cyclic photophosphorylation in broken chloroplasts, was also found to inhibit endogenous cyclic photophosphorylation in intact chloroplasts with a resultant decrease in total CO2 assimilation and the characteristic shift toward increased phosphoglycerate and decreased sugar phosphate formation. The addition of ATP to chloroplasts inhibited by antimycin A quadrupled the rate of CO2 fixation and restored the products to their original pattern. These results support the view that the ATP produced by cyclic photophosphorylation is essential in sustaining a high rate of CO2 assimilation and maintaining a high ATP:NADPH ratio that favors the conversion of phosphoglycerate to carbohydrate.
Photochemical ATP formation by isolated chloroplasts was coupled with a reduction of ferricyanide or TPN. Esterification of two moles of orthophoshate was coupled with the formation of two moles of TPNH2 and the evolution of one mole of oxygen.
1.1. Photosynthetic esterification of inorganic phosphate into adenosine triphosphate, and reduction of CO2 to the level of carbohydrate, hitherto found to occur only in whole chloroplasts, have now been observed with chloroplasts broken by treatment with water.2.2. Broken chloroplasts retained only two of the three groups of enzymes contained in whole chloroplasts, namely, those controlling the photolysis of water and photosynthetic phosphorylation. At least some of the enzymes concerned in reduction of CO2 were leached out by treating the chloroplasts with water, with the result that CO2 fixation was completely abolished.3.3. On addition of the requisite cofactors, the capacity of broken chloroplasts for photosynthetic phosphorylation was the same as that of whole chloroplasts.4.4. The restoration to broken chloroplasts of the full capacity for photosynthetic CO2 fixation of whole chloroplasts required the addition of pyridine nucleotides, adenosine triphosphate, and the soluble enzymes removed by water treatment of whole chloroplasts.5.5. An additional several-fold increase in the rate of CO2 fixation by the reconstituted broken chloroplast system was obtained by the further addition of one of several compounds, principally phosphorylated sugars. This has resulted in a level of CO2 fixation by broken chloroplasts which is much higher than that obtained with whole chloroplasts.
The rate of reduction of oxidants by illuminated chloroplasts is greatly stimulated by the presence of high concentrations of many anions. This stimulation is associated with, and quantitatively related to, a decrease in the ability of the chloroplasts to phosphorylate adenosine diphosphate. Phosphate, pyrophosphate, arsenate, citrate, lactate, malonate, and malate are particularly effective in uncoupling electron transport from phosphorylation. Acetate and butryate uncouple but also produce rapidly increasing inhibitions. Oxalate, tartrate, and bicarbonate are intermediate uncouplers. Sulfate and chloride are themselves feeble uncouplers but interact in a complex fashion with other anions, sometimes causing thereby large rate increases. The dependence of various chloroplast preparations on ferric oxalate, chloride ions, or CO2 should be reconsidered in the light of these general anion effects.Zwitter ions in the form of neutral amino acids uncouple only very slightly and then only at the highest concentrations. In order to reduce anion uncoupling to a minimum, we have prepared a neutral amino acid with appropriate dissociation constants to serve as a buffer for chloroplast reactions. Reacting tris(hydroxymethyl)aminomethane with monochloroacetic acid gave tris(hydroxymethyl)methylglycine, pK's 7.95 and about 2.3 at 25 °C. This substance is much less inhibitory at high concentrations than is the parent tris(hydroxymethyl)aminomethane. It also produces negligible uncoupling at any concentration up to 0.05 molar.
1. Induction periods in carbon dioxide fixation by isolated pea chloroplasts were shortened by small quantities of Calvin-cycle intermediates. The additional fixation was larger than that which would have followed direct stoicheiometric conversion into ribulose 1,5-diphosphate. 2. When chloroplasts were illuminated in the absence of added substrates (other than carbon dioxide) soluble products were formed in the medium that stimulated fixation by fresh chloroplasts. 3. The induction periods were lengthened by washing the chloroplasts. Addition of catalytic quantities of Calvin-cycle intermediates then decreased the induction periods to their previous values. 4. The induction period was extended by a decrease in temperature but was largely unaffected by a decrease in light-intensity that was sufficient to decrease the maximum rate. 5. It is concluded that the lag periods are a consequence of the loss of Calvin-cycle intermediates, such as sugar phosphates, through the intact chloroplast envelope and that these losses can be made good by new synthesis from carbon dioxide in the reactions of the Calvin cycle.
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