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The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates [Phaseolus vulgatis]
Abstract
The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO2 assimilation at known intercellular CO2 pressure, O2 pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O2, photosynthesis appeared RuBP-saturated at low CO2 pressure and RuBP-limited at high CO2 pressure. In 21 mbar (2%) O2, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.
... As A under elevated [CO 2 ] decreased in cpTPI-antisense plants, the pool sizes of the Calvin-Benson cycle and photorespiration metabolites were determined at pCa = 80 Pa in lines TPIAS4 and TPIAS7 and WT plants ( Figure 5). After A was equilibrated, leaves were immediately frozen using the rapid-kill system (Badger et al., 1984;Suzuki et al., 2012), followed by capillary electrophoresis-time-offlight mass spectrometry (CE-TOFMS) analysis. The pool sizes were presented as relative values to those of WT plants on a leaf area basis. ...
... These results strongly suggest that suppression of cpTPI (TPIAS4 and TPIAS7) were used as samples. The rate of CO 2 assimilation (A) in the uppermost and fully expanded leaves were monitored using a rapid-kill chamber (Badger et al., 1984;Suzuki et al., 2012) connected to an LI-6400XT at a photosynthetic photon flux density of 1,500-mmol quanta m -2 s -1 , a leaf temperature of 25 C, and ambient CO 2 partial pressure of 80 Pa. After A was in a steady-state, leaves were immediately frozen with liquid N 2 , followed by CE-TOFMS analysis. ...
... A was monitored using a rapid-kill chamber (Badger et al., 1984;Suzuki et al., 2012) connected to LI-6400XT (Li-Cor) under the conditions mentioned earlier, except that measurements were initiated at pCa = 80 Pa. Leaves were immediately frozen with liquid N 2 after A was in a steady state and stored at -80 C until analysis. ...
The availability of inorganic phosphate (Pi) for ATP synthesis is thought to limit photosynthesis at elevated [CO2] when Pi regeneration via sucrose or starch synthesis is limited. We report here another mechanism for the occurrence of Pi-limited photosynthesis caused by insufficient capacity of chloroplast triosephosphate isomerase (cpTPI). In cpTPI-antisense transgenic rice (Oryza sativa) plants with 55% to 86% reductions in cpTPI content, CO2 sensitivity of the rate of CO2 assimilation (A) decreased and even reversed at elevated [CO2]. The pool sizes of the Calvin-Benson cycle metabolites from pentose phosphates to 3-phosphoglycerate increased at elevated [CO2], whereas those of ATP decreased. These phenomena are similar to the typical symptoms of Pi-limited photosynthesis, suggesting sufficient capacity of cpTPI is necessary to prevent the occurrence of Pi-limited photosynthesis and that cpTPI content moderately affects photosynthetic capacity at elevated [CO2]. As there tended to be slight variations in the amounts of total leaf-N depending on the genotypes, relationships between A and the amounts of cpTPI were examined after these parameters were expressed per unit amount of total leaf-N (A/N and cpTPI/N, respectively). A/N at elevated [CO2] decreased linearly as cpTPI/N decreased before A/N sharply decreased, owing to further decreases in cpTPI/N. Within this linear range, decreases in cpTPI/N by 80% led to decreases up to 27% in A/N at elevated [CO2]. Thus, cpTPI function is crucial for photosynthesis at elevated [CO2].
... Information about the levels of CBC intermediates is surprisingly sparse. There were several studies of CBC intermediates levels under different conditions in the 1980s (Stitt et al., 1982a(Stitt et al., , 1983(Stitt et al., , 1984aBadger et al., 1984;Dietz and Heber, 1984;Sharkey, 1986, 1987;von Caemmerer and Edmondson, 1986;Sharkey and Seemann, 1989;Servaites et al., 1989;summarized in Supplementary (Cruz et al., 2008;Ma et al., 2014). Arnold and Nikoloski (2012) found poor agreement between the metabolite levels predicted by various CBC models and those measured in Arabidopsis under ambient CO2 and limiting irradiance ). ...
... Our results indicate that this increase in RuBP utilization is largely driven by increased RuBP in Arabidopsis, whilst further factors contribute in rice. Earlier studies also pointed to interspecies variation in the response of RuBP to rising irradiance; for example, RuBP levels rose progressively in bean leaves (Badger et al., 1984) and Chlamydomonas (Mettler et al., 2014), whereas RuBP plateaued at irradiances at which photosynthesis was still increasing in wheat (Perchorowicz et al., 1981), spinach (Dietz et al., 1984), and radish (von Caemmerer and Edmondson, 1986) leaves. The latter response resembles that in rice, and points to flux in these species being increased by removing Rubisco inhibitors and/or increasing Rubisco activation. ...
... There are also implications for CBC operation. The G6P pool typically contains a large part of the total P in photosynthetic metabolites [see Fig. 2 and Szecowka et al. (2013) for Arabidopsis, Dietz and Heber (1984), Stitt et al. (1984a, b), andGerhardt et al. (1987) for spinach, Stitt et al. (1983) for wheat, and Badger et al. (1984) for bean]. The small G6P pool in rice may allow larger cytosolic pools of triose-P and 3PGA, which may affect triose-P export from the chloroplast and energy shuttling via the triose-P/3PGA shuttle. ...
In C3 plants, CO2 diffuses into the leaf and is assimilated by the Calvin-Benson cycle in the mesophyll cells. It leaves Rubisco open to its side reaction with O2, resulting in a wasteful cycle known as photorespiration. A sharp fall in atmospheric CO2 levels about 30 million years ago have further increased the side reaction with O2. The pressure to reduce photorespiration led, in over 60 plant genera, to the evolution of a CO2-concentrating mechanism called C4 photosynthesis; in this mode, CO2 is initially incorporated into 4-carbon organic acids, which diffuse to the bundle sheath and are decarboxylated to provide CO2 to Rubisco. Some genera, like Flaveria, contain several species that represent different steps in this complex evolutionary process. However, the majority of terrestrial plant species did not evolve a CO2-concentrating mechanism and perform C3 photosynthesis. This thesis compares photosynthetic metabolism in several species with C3, C4 and intermediate modes of photosynthesis. Metabolite profiling and stable isotope labelling were performed to detect inter-specific differences changes in metabolite profile and, hence, how a pathway operates. The results obtained were subjected to integrative data analyses like hierarchical clustering and principal component analysis, and were deepened by correlation analyses to uncover specific metabolic features and reaction steps that were conserved or differed between species. The main findings are that Calvin-Benson cycle metabolite profiles differ between C3 and C4 species and between different C3 species, including a very different response to rising irradiance in Arabidopsis and rice. These findings confirm Calvin-Benson cycle operation diverged between C3 and C4 species and, most unexpectedly, even between different C3 species. Moreover, primary metabolic profiles supported the current C4 evolutionary model in the genus Flaveria and also provided new insights and opened up new questions. Metabolite profiles also point toward a progressive adjustment of the Calvin-Benson cycle during the evolution of C4 photosynthesis. Overall, this thesis point out the importance of a metabolite-centric approach to uncover underlying differences in species apparently sharing the same photosynthetic routes and as a valid method to investigate evolutionary transition between C3 and C4 photosynthesis.
... The model links pathway structure and key elements of enzyme kinetics, especially those of Rubisco, with the observed response of photosynthesis to irradiance and CO 2 supply. It identifies two basic states of the CBC; one in which photosynthesis is limited by Rubisco, for example in saturating light or low CO 2 , and one in which photosynthesis is limited by the rate of RuBP regeneration, for example in limiting light (Badger et al., 1984;Sharkey, 1985). The central role played by Rubisco in this model is fully consistent with its low k cat and high abundance (see above), and its kinetic characteristics (see below). ...
... This method was applied to study CBC regulation in isolated chloroplasts but was not applicable in intact cells or organisms because it is hardly possible to safely label them to steady state with 32 P. Use of analogous approaches with 14 CO 2 were precluded by rapid labelling of many metabolites outside the CBC, which interfered with reliable detection of CBC metabolites based on chromatographic mobility alone. For this reason, studies of CBC metabolite levels in leaves in the last decades of the 20th century used enzymatic metabolite assays (Stitt et al., 1980Badger et al., 1984;Dietz and Heber, 1984;von Caemmerer and Edmondson, 1986;Seeman andSharkey, 1986, 1987;Sharkey and Seeman, 1989;Servaites et al., 1989;compiled in Borghi et al., 2019). Enzymatic assays were only available for some metabolites, so these analyses were limited to RuBP and a handful of other intermediates [3PGA, triose-P, FBP, and fructose 6-phosphate (F6P), whereby FBP was not fully separated from SBP]. ...
... This means that even temporary changes in irradiance or CO 2 levels during the quenching process will lead to changes in metabolite levels. Various systems have been developed to circumvent this problem, including spraying algal suspension under ambient irradiance into very cold methanol (Bassham and Krause, 1969;Mettler-Altmann et al., 2014) and, for leaves, instantaneous freezing between two metal columns that have been pre-cooled to liquid N 2 temperature (Badger et al., 1984) and flooding of small containers with liquid N 2 (Arrivault et al., 2009(Arrivault et al., , 2017Szecowka et al., 2013;Ermakova et al., 2021;Xu et al., 2021). ...
Improving photosynthesis is a promising avenue to increase crop yield. This will be aided by better understanding of natural variance in photosynthesis. Profiling of Calvin-Benson cycle (CBC) metabolites provides a top-down strategy to uncover inter-species diversity in CBC operation. In a study of four C4 and five C3 species, principal components analysis separated C4 species from C3 species and also separated different C4 species. These separations were driven by metabolites that reflect known species-differences in their biochemistry and pathways. Unexpectedly, there was also considerable diversity between the C3 species. Falling atmospheric CO2 and changing temperature, nitrogen and water availability have driven evolution of C4 photosynthesis in multiple lineages. We propose that analogous selective pressures drove lineage-dependent evolution of the CBC in C3 species. Examples of species-dependent variation include differences in the balance between the CBC and the light reactions, and in the balance between regulated steps in the CBC. Metabolite profiles also reveal conserved features including inactivation of enzymes in low irradiance, and maintenance of CBC metabolites at relatively high levels in the absence of net CO2 fixation. These features may be important for photosynthetic efficiency in low light, fluctuating irradiance and when stomata close due to low water availability.
... Information about the levels of CBC intermediates is surprisingly sparse. There were several studies of CBC intermediates levels under different conditions in the 1980s (Stitt et al., 1982a(Stitt et al., , 1984aBadger et al., 1984;Dietz and Heber, 1984;Sharkey, 1986, 1987;von Caemmerer and Edmondson, 1986;Sharkey and Seemann, 1989;Servaites et al., 1989;summarized in Supplementary Table S1 at JXB online; for details, see Discussion). However, for technical and partly conceptual reasons, these early studies focused on RuBP and a handful of other intermediates (3PGA, triose-P, FBP, and F6P). ...
... Our results indicate that this increase in RuBP utilization is largely driven by increased RuBP in Arabidopsis, whilst further factors contribute in rice. Earlier studies also pointed to interspecies variation in the response of RuBP to rising irradiance; for example, RuBP levels rose progressively in bean leaves (Badger et al., 1984) and Chlamydomonas (Mettler et al., 2014), whereas RuBP plateaued at irradiances at which photosynthesis was still increasing in wheat (Perchorowicz et al., 1981), spinach (Dietz et al., 1984), and radish (von Caemmerer and Edmondson, 1986) leaves. The latter response resembles that in rice, and points to flux in these species being increased by removing Rubisco inhibitors and/or increasing Rubisco activation. ...
... There are also implications for CBC operation. The G6P pool typically contains a large part of the total P in photosynthetic metabolites [see Fig. 2 and Szecowka et al. (2013) for Arabidopsis, Dietz and Heber (1984), Stitt et al. (1984a, b), and Gerhardt et al. (1987) for spinach, Stitt et al. (1983) for wheat, and Badger et al. (1984) for bean]. The small G6P pool in rice may allow larger cytosolic pools of triose-P and 3PGA, which may affect triose-P export from the chloroplast and energy shuttling via the triose-P/3PGA shuttle. ...
Metabolite profiles provide a top-down overview of the balance between the reactions in a pathway. We compared Calvin-Benson cycle (CBC) intermediate profiles in different conditions in Arabidopsis (Arabidopsis thaliana) and rice (Oryzasativa) to learn which features of CBC regulation differ and which are shared between these model eudicot and monocot C3 species. Principal component analysis revealed that CBC intermediate profiles follow different trajectories in Arabidopsis and rice as irradiance increases. The balance between sub-processes or reactions differed, with 3-phosphoglycerate reduction being favoured in Arabidopsis and ribulose 1,5-bisphosphate regeneration in rice, and sedoheptulose-1,7-bisphosphatase being favoured in Arabidopsis compared to fructose-1,6-bisphosphatase in rice. Photosynthesis rates rose in parallel with ribulose 1,5-bisphosphate levels in Arabidopsis, but not in rice. Nevertheless, some responses were shared between Arabidopsis and rice. Fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate were high or peaked at very low irradiance in both species. Incomplete activation of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase may prevent wasteful futile cycles in low irradiance. End-product synthesis is inhibited and high levels of CBC intermediates are maintained in low light or in low CO2 in both species. This may improve photosynthetic efficiency in fluctuating irradiance, and facilitate rapid CBC flux to support photorespiration and energy dissipation in low CO2.
... Under conditions of high CO 2 , the rate of photosynthesis is increased, but its potential rate is curbed by limited regeneration of ribulose-1,5-bisphosphate (RuBP; Farquhar et al., 1980). Thus, under these conditions, ATP, NADPH, and RuBP pool sizes are reduced (although their turnover is high; Badger et al., 1984;Dietz et al., 1984;Heber et al., 1986;von Caemmerer, 2000), leading to a reduced rate of DMADP synthesis and reduced isoprene emission (Rasulov et al., 2009b(Rasulov et al., , 2016. ...
... We suggest that the RuBP pool is the key sink of photosynthetic electron flow that determines the concentrations of ATP and reductive equivalents available for isoprene emission. Increases in CO 2 concentration under normal oxygen that reduced RuBP pool size (Badger et al., 1984;Laisk et al., 2002;Rasulov et al., 2016) and increased net assimilation rate resulted in lower isoprene emission rate, while decreases of oxygen concentration under ambient CO 2 that increased RuBP pool size (Badger et al., 1984;Laisk et al., 2002;Rasulov et al., 2016) and enhanced photosynthesis increased isoprene emission rate. In the case of feedback-limited conditions, under high CO 2 and low oxygen and low RuBP pool (Sharkey, 1990;Laisk et al., 2002;Rasulov et al., 2016), both photosynthesis and isoprene emission were reduced. ...
... We suggest that the RuBP pool is the key sink of photosynthetic electron flow that determines the concentrations of ATP and reductive equivalents available for isoprene emission. Increases in CO 2 concentration under normal oxygen that reduced RuBP pool size (Badger et al., 1984;Laisk et al., 2002;Rasulov et al., 2016) and increased net assimilation rate resulted in lower isoprene emission rate, while decreases of oxygen concentration under ambient CO 2 that increased RuBP pool size (Badger et al., 1984;Laisk et al., 2002;Rasulov et al., 2016) and enhanced photosynthesis increased isoprene emission rate. In the case of feedback-limited conditions, under high CO 2 and low oxygen and low RuBP pool (Sharkey, 1990;Laisk et al., 2002;Rasulov et al., 2016), both photosynthesis and isoprene emission were reduced. ...
Isoprene is synthesized via chloroplastic 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway and its synthesis is directly related to photosynthesis, except under high CO2 concentration when the rate of photosynthesis increases, but isoprene emission decreases. Suppression of MEP/DOXP pathway activity by high CO2 has been explained by limited supply of cytosolic substrate precursor, phosphoenolpyruvate (PEP) into chloroplast as the result of enhanced activity of PEP carboxylase (PEPC) or by limited supply of energetic and reductive equivalents. We tested the PEP-limitation hypotheses by feeding leaves with PEPC competitive inhibitors malate and diethyl oxalacetate (DOA) in strong isoprene emitter hybrid aspen (Populus tremula x P. tremuloides). Malate feeding resulted in inhibition of net assimilation, photosynthetic electron transport and isoprene emission rates, but DOA feeding did not affect any of these processes except at very high application concentrations. Both malate and DOA did not alter the sensitivity of isoprene emission to high CO2 concentration. Malate inhibition of isoprene emission was associated with enhanced chloroplastic reductive status that suppressed light reactions of photosynthesis ultimately leading to reduced isoprene substrate dimethylallyldiphosphate pool size. Additional experiments with altered O2 concentrations indicated that changes in isoprene emission rate in control and malate-inhibited leaves were associated with modifications in the share of ATP and reductive equivalent supply for isoprene synthesis. The results of this study collectively indicate that malate controls the chloroplast reductive status, and thereby alters isoprene emission, but does not support the hypothesis that cytosolic metabolite availability alters the response of isoprene emission to changes in atmospheric composition.
... Whilst our results show that Tre6P plays a major role, they do not exclude the possibility that further factors also exert feedback regulation on starch degradation in the light. For example, changes in light intensity might potentially affect starch metabolism via redox signaling (Glaring et al., 2012;Thormählen et al., 2013;Krasensky et al., 2014), or changes in energy supply or levels of phosphorylated intermediates (Stitt et al., 1982(Stitt et al., , 1983Badger et al., 1984;Dietz and Heber, 1984;Stitt et al., 2010;Borghi et al., 2019). However, we suspect that any such effects are minor, because starch accumulation responds in a qualitatively similar manner to a decrease in light intensity and a decrease in CO 2 concentration that have opposing effects on energy supply ( Figure 5). ...
... When photorespiration is rapid, starch may be degraded by phosphorylase to maintain levels of Calvin-Benson cycle intermediates (Weise et al., 2006(Weise et al., , 2011reviewed in Stitt et al., 2021). However, this is unlikely to contribute to the stimulation of starch degradation in response to moderate decreases in irradiance, as these lead to only small changes in the levels of phosphorylated intermediates (Stitt et al., 1983Badger et al., 1984;Dietz and Heber, 1984;Borghi et al., 2019). ...
Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the light and remobilize it to support maintenance and growth at night. Starch synthesis and degradation are usually viewed as temporally separate processes. Recently, we reported that starch is also degraded in the light. Degradation rates are generally low early in the day but rise with time. Here, we show that the rate of degradation in the light depends on time relative to dawn rather than dusk. We also show that degradation in the light is inhibited by trehalose 6-phosphate, a signal for sucrose availability. The observed responses of degradation in the light can be simulated by a skeletal model in which the rate of degradation is a function of starch content divided by time remaining until dawn. The fit is improved by extension to include feedback inhibition of starch degradation by trehalose 6-phosphate. We also investigate possible functions of simultaneous starch synthesis and degradation in the light, using empirically parameterized models and experimental approaches. The idea that this cycle buffers growth against falling rates of photosynthesis at twilight is supported by data showing that rates of protein and cell wall synthesis remain high during a simulated dusk twilight. Degradation of starch in the light may also counter over-accumulation of starch in long photoperiods and stabilize signaling around dusk. We conclude that starch degradation in the light is regulated by mechanisms similar to those that operate at night and is important for stabilizing C availability and signaling, thus optimizing growth in natural light conditions.
... Observing increased relative responses of compartment CO 2 to low RuBP concentrations in the model, where condensate pH is maximally decreased (SI Appendix, Fig. S4), we assessed the relative fitness of condensates and carboxysomes at subsaturating RuBP (50 μM) over HCO 3 − ranges in the model. Low cellular RuBP can generally be attributed to light-limited RuBP regeneration via the Calvin cycle in photoautotrophs (56). A concentration of 50 μM RuBP is approximately three times the K M RuBP of the tobacco Rubisco used here (Table 2) and supports ∼63% of the CO 2 -saturated rate for a condensed Rubisco (SI Appendix, Fig. S6). ...
... The relative response of Rubisco condensates and small carboxysomes to low RuBP supply suggests that low light may also provide conditions conducive to condensate and carboxysome evolution. Low light generally leads to low RuBP (56). In the model, such conditions lead to greater advantage of Rubisco condensates over large carboxysomes (SI Appendix, Fig. S15). ...
Significance
Rubisco is arguably the most abundant protein on Earth, and its catalytic action is responsible for the bulk of organic carbon in the biosphere. Its function has been the focus of study for many decades, but recent discoveries highlight that in a broad array of organisms, it undergoes liquid–liquid phase separation to form membraneless organelles, known as pyrenoids and carboxysomes, that enhance CO 2 acquisition. We assess the benefit of these condensate compartments to Rubisco function using a mathematical model. Our model shows that proton production via Rubisco reactions, and those carried by protonated reaction species, can enable the elevation of condensate CO 2 to enhance carboxylation. Application of this theory provides insights into pyrenoid and carboxysome evolution.
... Observing increased relative responses of compartment CO 2 to low RuBP concentrations in the 388 model, where condensate pH is maximally decreased (Fig. S2), we assessed the relative fitness of condensates at sub-saturating RuBP (50 µM) over HCO 3 ranges in the model. Low cellular RuBP can 390 generally be attributed to light-limited RuBP regeneration via the Calvin Cycle (45). A concentration of 50 µM RuBP is approximately three-times the K M RuBP of the tobacco Rubisco used here (Table 2) 392 and supports ≈63% of the CO 2 -saturated rate for a condensed Rubisco (Fig. S4). ...
... The relative response of Rubisco condensates to low RuBP supply suggests that low light may also provide conditions conducive to condensate evolution. Low light generally leads to low RuBP (45). 458 ...
Membraneless organelles containing the enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), formed via liquid-liquid phase separation, are a common feature of organisms utilizing CO 2 concentrating mechanisms (CCMs). In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while microalgae have evolved liquid-like Rubisco condensates known as pyrenoids. In both cases, CO 2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the function of carboxysomes to the generation of elevated CO 2 within the organelle via a co-localized carbonic anhydrase (CA), and inwardly diffusing HCO 3 ⁻ . Here we present a novel concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction, and have evaluated this in a reaction-diffusion, compartment model to investigate functional advantages of Rubisco condensation and how these may lead to carboxysome evolution. Applying diffusional resistance to reaction species within a modelled condensate allows Rubisco-derived protons to drive the conversion of HCO 3 ⁻ to CO 2 via co-condensed CA, enhancing both localized [CO 2 ] and Rubisco rate. Protonation of RuBP and PGA plays an important role in modulating internal pH and CO 2 generation. Application of the model to potential evolutionary intermediate states prior to contemporary carboxysomes revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully-formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO 2 and low light environments.
... It has been thought that the reported concentration of NADPH (0.25-0.5 mM) 43) was too low compared with the concentration of the RuBisCO protomer in chloroplasts (4 mM). FBP and phosphate are reported to be present at approximately 5.4 and 12 mM, respectively, in chloroplasts 45,46) , and may similarly maintain the activated state of RuBisCO as does NADPH. [37][38][39] The minimum required amount of RuBisCO activase hexamer required for 1 μmol m −2 of the RuBisCO holoenzyme in chloroplasts ranges from approximately 0.04 to 0.06 μmol m −2 RuBisCO activase, as discussed above. ...
... 60,61) Also, reported stromal concentrations of RuBP have sometimes exceeded 6 mM. 45,62) A similar photosynthesis-promoting effect has also been observed when the NAD kinase-2 gene was introduced into chloroplasts of rice via Agrobacterium. The introduction of NAD kinase-2 caused the concentration of NADPH to increase to levels 1.5 times higher than those in the non-transformant and the concentration of RuBP to increase 2.7-fold. ...
Since the discovery of its role in the CO2 fixation reaction in photosynthesis, RuBisCO has been one of the most extensively researched enzymes in the fields of biochemistry, molecular biology, and molecular genetics as well as conventional plant physiology, agricultural chemistry, and crop science. In addition, the RuBisCO and RuBisCO-like genes of more than 2000 organisms have been sequenced during the past 20 years. During the course of those studies, the origin of the RuBisCO gene began to be discussed. Recent studies have reported that the RuBisCO gene emerged in methanogenic bacteria long before photosynthetic organisms appeared. The origin of similar early genes might have allowed this gene to overcome changes in global environments during ancient and recent eras and to participate in the fixation of 200 GT of CO2 annually. In this review, I focus on several points that have not been discussed at length in the literature thus far.
... As isohydric species, P. nigra responds to drought by stomatal closure which impedes CO 2 uptake causing low intercellular CO 2 concentrations, C i (Sade et al., 2012). With decreasing C i , PGI inhibitor concentrations reportedly decrease, and the chloroplastic PGI reaction shifts towards equilibrium (Badger et al., 1984;Dietz & Heber, 1984;Dietz, 1985). Concomitantly increasing G6P concentrations can be expected to cause increasing G6PD activity and anaplerotic flux (see last paragraph). ...
As the central carbon uptake pathway in photosynthetic cells, the Calvin–Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast‐localized oxidative branch of the pentose phosphate pathway) into the Calvin–Benson cycle was proposed recently.
Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO2 concentrations, Ca. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations.
We report deuterium fractionation signals at H¹ and H² of starch glucose. Below a Ca change point, these signals increase with decreasing Ca consistent with modelled fractionations by anaplerotic flux. Under standard conditions (Ca = 450 ppm corresponding to intercellular CO2 concentrations, Ci, of 328 ppm), we estimate negligible anaplerotic flux. At Ca = 180 ppm (Ci = 140 ppm), more than 10% of the glucose‐6‐phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway.
In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin–Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5‐bisphosphate under source‐limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build‐up source strength; and counteract oxidative stress.
... A high-O 2 and low-CO 2 environment, however, promotes photorespiration [4]. Additionally, a CO 2 -free atmosphere inhibits both the activity of Rubisco [53] and the regeneration of its substrate RuBP [54], leading to underestimates of photorespiration. ...
Photorespiration, or C2 photosynthesis, is generally considered a futile cycle that potentially decreases photosynthetic carbon fixation by more than 25%. Nonetheless, many essential processes, such as nitrogen assimilation, C1 metabolism, and sulfur assimilation, depend on photorespiration. Most studies of photosynthetic and photorespiratory reactions are conducted with magnesium as the sole metal cofactor despite many of the enzymes involved in these reactions readily associating with manganese. Indeed, when manganese is present, the energy efficiency of these reactions may improve. This review summarizes some commonly used methods to quantify photorespiration, outlines the influence of metal cofactors on photorespiratory enzymes, and discusses why photorespiration may not be as wasteful as previously believed.
... These sensors are often combined with additional detector systems, such as online mass spectrometers, to provide information on the relative contributions of photorespiratory and non-photorespiratory sources of CO 2 loss, which in turn inform efforts at improving crop productivity [15,39,52,53]. Gas exchange measurements are the primary input into biochemical models of photosynthesis, which have yielded significant insights into the coupling of photosynthetic electron flow to chemical energy production through the carbon reduction cycle [4,26,87]. The accurate estimation of carbon assimilation is therefore central to understanding the photosynthetic response of plants to changes in CO 2 concentration (i.e. ...
Background
We report a method to estimate carbon assimilation based on isotope ratio-mass spectrometry (IRMS) of ¹³ CO 2 labeled plant tissue. Photosynthetic carbon assimilation is the principal experimental observable which integrates important aspects of primary plant metabolism. It is traditionally measured through gas exchange. Despite its centrality in plant research, gas exchange performs poorly with rosette growth habits typical of Arabidopsis thaliana , mutant lines with limited biomass, and accounts poorly for leaf shading.
Results
IRMS-based carbon assimilation values from plants labeled at different light intensities were compared to those obtained by gas exchange, and the two methods yielded similar values. Using this method, we observed a strong correlation between ¹³ C content and labeling time (R ² = 0.999) for 158 wild-type plants labeled for 6 to 42 min. Plants cultivated under different light regimes showed a linear response with respect to carbon assimilation, varying from 7.38 nmol ¹³ C mg ⁻¹ leaf tissue min ⁻¹ at 80 PAR to 19.27 nmol ¹³ C mg ⁻¹ leaf tissue min ⁻¹ at 500 PAR. We applied this method to examine the link between inhibition of the 2 C -methyl- d -erythritol-4-phosphate (MEP) pathway and suppression of photosynthesis. A significant decrease in carbon assimilation was observed when metabolic activity in the MEP pathway was compromised by mutation or herbicides targeting the MEP pathway. Mutants affected in MEP pathway genes 1-DEOXY- d -XYLULOSE 5-PHOSPHATE SYNTHASE (DXS) or 1-HYDROXY-2-METHYL-2-(E)-BUTENYL 4-DIPHOSPHATE SYNTHASE (HDS) showed assimilation rates 36% and 61% lower than wild type. Similarly, wild type plants treated with the MEP pathway inhibitors clomazone or fosmidomycin showed reductions of 52% and 43%, respectively, while inhibition of the analogous mevalonic acid pathway, which supplies the same isoprenoid intermediates in the cytosol, did not, suggesting inhibition of photosynthesis was specific to disruption of the MEP pathway.
Conclusions
This method provides an alternative to gas exchange that offers several advantages: resilience to differences in leaf overlap, measurements based on tissue mass rather than leaf surface area, and compatibility with mutant Arabidopsis lines which are not amenable to gas exchange measurements due to low biomass and limited leaf surface area. It is suitable for screening large numbers of replicates simultaneously as well as post-hoc analysis of previously labeled plant tissue and is complementary to downstream detection of isotopic label in targeted metabolite pools.
... The stable semiquinones of flavoenzymes can be oxidized by O2 with a high rate [133]. In leaves, the photoreduction rate of O2 was estimated to be 18-26 μmol O2 (m −2 of leaf area) s −1 [146] which gives a rate of O2 •-production of 240-350 μmol (mg Chl) −1 h −1 assuming 0.6 mmol Chl (m leaf area) −2 [147]. Thus, flavoenzymes may contribute to the high rates of O2 photoreduction in chloroplasts. ...
Oxygen is a natural acceptor of electrons in the respiratory pathway of aerobic organisms and in many other biochemical reactions. Aerobic metabolism is always associated with the formation of reactive oxygen species (ROS). ROS may damage biomolecules but are also involved in regulatory functions of photosynthetic organisms. This review presents the main properties of ROS, the formation of ROS in the photosynthetic electron transport chain and in the stroma of chloroplasts, and ROS scavenging systems of thylakoid membrane and stroma. Effects of ROS on the photosynthetic apparatus and their roles in redox signaling are discussed.
... Closer analysis of these "lipid-remodeling" irradiance responsive genes reveals that especially genes involved in phosphate-dependent lipid-remodeling are accession-specifically up-regulated upon the irradiance increase ( Figures 4 and 5). Phosphate is an essential cofactor for photosynthesis and after an irradiance increase there is an increase in the pool size of phosphorylated intermediates accompanying the increased assimilation rate, requiring that more inorganic phosphate (Pi) be made available (Badger, Sharkey, & von Caemmerer, 1984;Caemmerer & Edmondson, 1986;Sivak & Walker, 1988;Walker & Sivak, 1986). A decrease in Pi such as occurs in end-product (or sink) limitation of photosynthesis, is associated with an overall decrease in assimilation rate (Paul & Foyer, 2001;Williams, 1998). ...
Plants have evolved several mechanisms for sensing increased irradiance, involving signal perception by photoreceptors (cryptochromes), and subsequent biochemical (reactive oxygen species, ROS) and metabolic clues to transmit the signals. This results in the increased expression of heat‐shock response genes and of the transcription factor LONG HYPOCOTYL 5 (HY5, mediated by the cryptochrome photoreceptor 1, CRY1). Here, we show the existence of another response pathway in Arabidopsis. This pathway evokes the SPX1‐mediated expression activation of the transcription factor PHR1 and leads to the expression of several galactolipid biosynthesis genes. Gene expression analysis of accessions Col‐0, Ga‐0, and Ts‐1, showed activated expression of the SPX1/PHR1‐mediated gene expression activation pathway acting on galactolipids biosynthesis genes in both Ga‐0 and Col‐0, but not in Ts‐1. The activation of the SPX1/PHR1‐mediated response pathway can be associated with lower photosynthesis efficiency in Ts‐1, compared to Col‐0 and Ga‐0. Besides the accession‐associated activation of the SPX1/PHR1‐mediated response pathway, comparing gene expression in the accessions showed stronger activation of several heat responsive genes in Ga‐0, and the opposite in Ts‐1, when compared to Col‐0, in line with the differences in their efficiency of photosynthesis. We conclude that natural variation in activation of both heat responsive genes and of galactolipids biosynthesis genes contribute to the variation in photosynthesis efficiency in response to irradiance increase.
... In addition, people in charge of the harvest should be trained and aware that plant samples should be frozen or quenched as quickly as possible in liquid nitrogen to stop enzymatic reactions and oxidation degradation, in order to maintain metabolite concentrations as closely as possible to in vivo levels [168,169]. For photosynthetic leaves, frozen clamps [170] or specialized devices are used [171] to limit the variation of certain metabolite concentrations in response to the light and/or temperature variations during the sampling. These variations particularly affect phosphorylated compounds with short life-time or primary metabolites such as organic acids [172]. ...
The study of plant metabolism impacts a broad range of domains such as plant cultural practices, plant breeding, human or animal nutrition, phytochemistry and green biotechnologies. Plant metabolites are extremely diverse in terms of structure or compound families as well as concentrations. This review attempts to illustrate how NMR spectroscopy, with its broad variety of experimental approaches, has contributed widely to the study of plant primary or specialized metabolism in very diverse ways. The review presents recent developments of one-dimensional and multi-dimensional NMR methods to study various aspects of plant metabolism. Through recent examples, it highlights how NMR has proved to be an invaluable tool for the global characterization of sample composition within metabolomic studies, and shows some examples of use for targeted phytochemistry, with a special focus on compound identification and quantitation. In such cases, NMR approaches are often used to provide snapshots of the plant sample composition. The review also covers dynamic aspects of metabolism, with a description of NMR techniques to measure metabolic fluxes – in most cases after stable isotope labelling. It is mainly intended for NMR specialists who would be interested to learn more about the potential of their favourite technique in plant sciences and about specific details of NMR approaches in this field. Therefore, as a practical guide, a paragraph on the specific precautions that should be taken for sample preparation is also included. In addition, since the quality of NMR metabolic studies is highly dependent on approaches to data processing and data sharing, a specific part is dedicated to these aspects. The review concludes with perspectives on the emerging methods that could change significantly the role of NMR in the field of plant metabolism by boosting its sensitivity. The review is illustrated throughout with examples of studies selected to represent diverse applications of liquid-state or HR-MAS NMR.
... In most cases, the O 2 mole fraction used is 1% or 2% and the major effect is a decrease in Rubisco-catalysed oxygenation, regardless of potential effects on respiratory metabolism (e.g. Badger et al. 1984). However, day respiration (i.e. ...
The response of underground plant tissues to O2 limitation is currently an important topic in crop plants since adverse environmental conditions (e.g. waterlogging) may cause root hypoxia and thus compromise plant growth. However, little is known on the effect of low O2 conditions in leaves, probably because O2 limitation is improbable in these tissues under natural conditions, unless under complete submersion. Nevertheless, an O2-depleted atmosphere is commonly used in gas exchange experiments to suppress photorespiration and estimate gross photosynthesis. However, the nonphotosynthetic effects of gaseous O2 depletion, particularly on respiratory metabolism, are not well documented. Here, we used metabolomics obtained under contrasting O2 and CO2 conditions to examine the specific effect of a changing O2 mole fraction from ambient (21%) to 0%, 2% or 100%. In addition to the typical decrease in photorespiratory intermediates (glycolate, glycine and serine) and a build-up in photosynthates (sucrose), low O2 (0% or 2%) was found to trigger an accumulation of alanine and change succinate metabolism. In 100% O2, the synthesis of threonine and methionine from aspartate appeared to be stimulated. These responses were observed in two species, sunflower (Helianthus annuus L.) and Arabidopsis thaliana (L.) Heynh. Our results show that O2 causes a change in the oxygenation?:?carboxylation ratio and also alters postphotosynthetic metabolism: (i) a hypoxic response at low O2 mole fractions and (ii) a stimulation of S metabolism at high O2 mole fractions. The latter effect is an important piece of information to better understand how photorespiration may control S assimilation.
... 5,6 The use of a copper piston pre-cooled with liquid nitrogen through the upper window of the gas exchange cuvette allowed continuous illumination during rapid freezing and thus capturing an in vivo snapshot of a leaf's metabolic status. 7,8 A similar principle was followed when harvesting spinach leaves by pouring liquid nitrogen into the cuvette containing the leaves. 9 Alternatively, plants with smaller leaves than spinach (for example Arabidopsis) were sampled by pouring liquid nitrogen directly onto the Arabidopsis' rosette, placed in the cuvette, avoiding shading the rosette. ...
Plants, as well as other aerobic organism constantly produce reactive oxygen species (ROS). At regulated low concentrations ROS may serve as signal molecules while in excessive amounts these may cause oxidative damage to biomolecules. Actual cellular concentrations are controlled by a network of various antioxidants, and acclimation to stress conditions is achieved by a dynamic balance of ROS production and neutralization. Accordingly, plant stress physiology studies generally include an array of methods testing the occurrence of ROS as well as evaluating antioxidant capacities. The aim of the present work is to provide an overview of these methods, with special emphasis on avoiding errors that can possibly lead to either inaccurate data or misinterpretations of otherwise correct measurements.
... Significant work has been done on mesophyll conductance though there remain important questions about this factor (Sharkey 2012). The effects of Rubisco, RuBP regeneration capacity and R day were extensively explored in the ' 'Farquhar model'' published in 1980(Farquhar et al. 1980) and followed up by several papers comparing the underlying biochemistry with the predictions of the model (von Caemmerer and Farquhar 1981Badger et al. 1984von Caemmerer et al. 1985). ...
Main conclusion:
The triose phosphate use limitation was studied using long-term and short term changes in capacity. The TPU limitation caused increased proton motive force; long-term TPU limitation additionally reduced other photosynthetic components. Photosynthetic responses to CO2 can be interpreted primarily as being limited by the amount or activity of Rubisco or the capacity for ribulose bisphosphate regeneration, but at high rates of photosynthesis a third response is often seen. Photosynthesis becomes insensitive to CO2 or even declines with increasing CO2, and this behavior has been associated with a limitation of export of carbon from the Calvin-Benson cycle. It is often called the triose phosphate use (TPU) limitation. We studied the long-term consequences of this limitation using plants engineered to have reduced capacity for starch or sucrose synthesis. We studied short-term consequences using temperature as a method for changing the balance of carbon fixation capacity and TPU. A long-term and short-term TPU limitation resulted in an increase in proton motive force (PMF) in the thylakoids. Once a TPU limitation was reached, any further increases in CO2 was met with a further increase in the PMF but no increase or little increase in net assimilation of CO2. A long-term TPU limitation resulted in reduced Rubisco and RuBP regeneration capacity. We hypothesize that TPU, Rubisco activity, and RuBP regeneration are regulated so that TPU is normally in slight excess of what is required, and that this results in more effective regulation than if TPU were in large excess.
Background
Photosynthesis close interacts with respiration and nitrogen assimilation, which determine the photosynthetic efficiency of a leaf. Accurately quantifying the metabolic fluxes in photosynthesis, respiration and nitrogen assimilation benefit the design of photosynthetic efficiency improvement. To accurately estimate metabolic fluxes, time-series data including leaf metabolism and isotopic abundance changes should be collected under precisely controlled environments. But for isotopic labelled leaves under defined environments the, time cost of manually sampling usually longer than the turnover time of several intermediates in photosynthetic metabolism. In this case, the metabolic or physiological status of leaf sample would change during the sampling, and the accuracy of metabolomics data could be compromised.
Results
Here we developed an i ntegrated isotopic l abeling and freeze s ampling a pparatus (ILSA), which could finish freeze sampling automatically in 0.05 s. ILSA can not only be used for sampling of photosynthetic metabolism measurement, but also suit for leaf isotopic labeling experiments under controlled environments ([CO 2 ] and light). Combined with HPLC–MS/MS as the metabolic measurement method, we demonstrated: (1) how pool-size of photosynthetic metabolites change in dark-accumulated rice leaf, and (2) variation in photosynthetic metabolic flux between rice and Arabidopsis thaliana .
Conclusions
The development of ILSA supports the photosynthetic research on metabolism and metabolic flux analysis and provides a new tool for the study of leaf physiology.
- As the central carbon uptake pathway in photosynthetic cells, the Calvin-Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e., through the chloroplast-localised oxidative branch of the pentose phosphate pathway) into the Calvin-Benson cycle was proposed recently.
- Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO2 concentrations, Ca. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations.
- We report deuterium fractionation signals at H1 and H2 of starch glucose. Below a Ca change point, these signals increase with decreasing Ca consistent with modelled fractionations by anaplerotic flux. Under standard conditions (Ca=450 ppm corresponding to intercellular CO2 concentrations, Ci, of 328 ppm), we estimate negligible anaplerotic flux. At Ca=180 ppm (Ci=140 ppm), more than 10% of the glucose 6-phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway.
- In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin-Benson cycle in vivo. We propose the flux may help to (i) maintain high levels of ribulose 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength and (ii) counteract oxidative stress.
Improving photosynthesis is considered a major and feasible option to dramatically increase crop yield potential. Increased atmospheric CO2 concentration often stimulates both photosynthesis and crop yield, but decreases protein content in the main C3 cereal crops. This decreased protein content in crops constrains the benefits of elevated CO2 on crop yield and affects their nutritional value for humans. To support studies of photosynthetic nitrogen assimilation and its complex interaction with photosynthetic carbon metabolism for crop improvement, we developed a dynamic systems model of plant primary metabolism, which includes the Calvin-Benson cycle (CBC), the photorespiration pathway (PRP), starch synthesis, glycolysis-gluconeogenesis, the tricarboxylic acid (TCA) cycle, and chloroplastic nitrogen assimilation. This model successfully captures responses of net photosynthetic CO2 uptake rate (A), respiration rate, and nitrogen assimilation rate to different irradiance and CO2 levels. We then used this model to predict inhibition of nitrogen assimilation under elevated CO2. The potential mechanisms underlying inhibited nitrogen assimilation under elevated CO2 were further explored with this model. Simulations suggest that enhancing the supply of α-ketoglutarate (2-OG) is a potential strategy to maintain high rates of nitrogen assimilation under elevated CO2. This model can be used as a heuristic tool to support research on interactions between photosynthesis, respiration, and nitrogen assimilation. It also provides a basic framework to support the design and engineering of C3 plant primary metabolism for enhanced photosynthetic efficiency and nitrogen assimilation in the coming high-CO2 world.
Respiration in the light (RL) releases CO2 in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since RL lowers net carbon fixation, understanding RL could help improve plant carbon-use efficiency and models of crop photosynthesis. Although RL was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to RL, we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during 13CO2 labeling time course, gas exchange and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO2 sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N2; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that RL releases 5.2 μmol CO2 g−1 FW hr−1 of CO2, which is relatively consistent with a value of 9.3 μmol CO2 g−1 FW hr−1 measured by CO2 gas exchange. The results indicated that ≤10% of RL results from TCA cycle reactions, which are widely considered to dominate RL. Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >93% of CO2 released by RL.
This chapter discusses the control of photosynthetic sucrose formation. In most species, starch and sucrose are the principal end products of photosynthesis. It is clear that the formation of both carbohydrates is highly regulated biochemically. The interdependence of chloroplast and cytosolic metabolism implies that coordination of the fluxes and conditions in these compartments is a precondition for rapid photosynthesis. This interaction is defined by the stoichiometry of carbon and phosphate flow during steady-state photosynthesis. Chloroplasts convert three CO2 and one Pi to one molecule of triose-P, and a continuation of photosynthesis depends on the triose-P being removed and more Pi becoming available. This is achieved by exporting triose-P from the chloroplast, in exchange for Pi. However, the rate of this exchange must be coordinated with the rate of CO2 fixation, because only one-sixth of the triose-P may be removed, representing the net gain of carbon in one turn of the Calvin cycle.
D-ribulose 1,5-bisphosphate carboxylase-oxygenase's (Rubisco) central role in photosynthesis and photorespiration makes it a likely candidate for regulation, though whether it is more or less regulated than other photosynthetic enzymes remains to be seen. Rubisco's activity in vivo certainly seems to be tightly controlled, very probably by a multiplicity of mechanisms. This chapter discusses the recent advances in the understanding of Rubisco, its mechanisms of catalysis and regulation, the synthesis and assembly of its subunits, and the role of interactions between them. The only function that the glycolate pathway seems to serve is to salvage three-quarters of the carbon diverted from photosynthesis by RuBP oxygenase as phosphoglycolate. In doing so, it consumes energy in the form of ATP and reducing equivalents. Such energy consumption may be advantageous in some circumstances. For example, it may dissipate excess photosynthetic reductant under photo-inhibitory conditions associated with CO2 limitation. Rubisco stands at the interface between the inorganic and organic phases of the biosphere's carbon cycle, catalyzing the only reaction by which atmospheric CO2 may be acquired by living organisms.
Rubisco is the primary carboxylase of the photosynthetic process, the most abundant enzyme in the biosphere, and also one of the best-characterized enzymes. Rubisco also functions as an oxygenase, a discovery made 50 years ago by Bill Ogren. Carboxylation of ribulose bisphosphate (RuBP) is the first step of the photosynthetic carbon reduction cycle and leads to the assimilation of CO2, whereas the oxygenase activity necessitates the recycling of phosphoglycolate through the photorespiratory carbon oxidation cycle with concomitant loss of CO2. Since the discovery of Rubisco’s dual function, the biochemical properties of Rubisco have underpinned the mechanistic mathematical models of photosynthetic CO2 fixation which link Rubisco kinetic properties to gas exchange of leaves. This has allowed assessments of global CO2 exchange and predictions of how Rubisco has and will shape the environmental responses of crop and global photosynthesis in future climates. Rubisco’s biochemical properties, including its slow catalytic turnover and poor affinity for CO2, constrain crop growth and therefore improving its activity and regulation and minimising photorespiration are key targets for crop improvement.
During photosynthesis, plants fix CO2 from the atmosphere onto ribulose-bisphosphate, producing 3-phosphoglycerate, which is reduced to triose phosphates (TPs). The TPs are then converted into the end products of photosynthesis. When a plant is photosynthesizing very quickly, it may not be possible to commit photosynthate to end products as fast as it is produced, causing a decrease in available phosphate and limiting the rate of photosynthesis to the rate of triose phosphate utilization (TPU). The occurrence of an observable TPU limitation is highly variable based on species and especially growth conditions, with TPU capacity seemingly regulated to be in slight excess of typical photosynthetic rates the plant might experience. The physiological effects of TPU limitation are discussed with an emphasis on interactions between the Calvin-Benson cycle and the light reactions. Methods for detecting TPU-limited data from gas exchange data are detailed and the impact on modeling of some physiological effects are shown. Special consideration is given to common misconceptions about TPU.
The appearance of the C⁴photosynthetic pathway in the Earth’s flora represents one of the most impressive and curious examples of evolutionary diversification and biogeographic expansion in the history of life (Ehleringer and Monson, 1983). This complex pathway, involving novel patterns of biochemical compartmentation and anatomical design, has evolved with independent but convergent patterns approximately fifty times during the relatively short geological span of 12–15 million years (Kellogg, 1999; Monson, 1999; Sage, 2004; Christin et al., 2007). The appearance of C4photosynthesis has changed the nature of photosynthetic productivity and ecosystem structure on Earth, both regionally and globally. Grassland ecosystems emerged in southwestern Asia, Africa and North America during the mid- to late-Miocene (5–10 Ma) and continued through the Pliocene, (~3Ma), with many of these systems dominated by C4species (Cerling, 1999; Beerling and Osborne, 2006). During the appearance of C⁴ grasslands, the trophic structures of grazed ecosystems were completely revised, resulting in the emergence of novel mammalian lineages (Cerling et al., 1993; Wang et al., 1994; MacFadden and Cerling, 1996; Ehleringer et al., 1997). Arguably, there is not a better example in the history of life to illustrate the tightly integrated nature of evolutionary novelty and ecological impact, as that shown in C⁴ photosynthesis. Clearly, C⁴ photosynthesis, though present in only 8,000 of the estimated 250,000 higher plant species, deserves a significant role in the discussion of plant biology.
Rubisco can be catalytically competent only after a specific lysyl residue within the active site has been carbamylated. Before carbamylation can occur, any inhibitory ligands bound at the site must be released, and this process is facilitated by another enzyme, Rubisco activase. It has been shown in vitro that Rubisco activase needs to hydrolyse ATP to function and is inhibited by ADP, and so presumably is sensitive to the chloroplast ATP/ADP ratio (1). However, there are indications that activase is also regulated by transthylakoid pH gradient (∆pH) and electron transport through PSI (2).
The carboxylation of ribulose 1,5-bisphosphate (RuBP) by the enzyme RuBP carboxylase is the primary CO2 fixing reaction of photosynthesis. It is therefore reasonable to assume that any factor which changes the rate of photosynthesis must ultimately do so through an effect on this reaction. The reaction rate may be influenced by the concentration of the two substrates (CO2 and RuBP), the activity of RuBP carboxylase present, or by inhibitors of the reaction. For example, limitation of photosynthesis by CO2 is easily determined based on the response of photosynthetic rate to CO2 concentration in the intercellular spaces. It is clear that photosynthesis is limited by CO2 at CO2 concentrations around ambient, and this observation is consistent with kinetic data for RuBP carboxylase and CO2in
vitro.
Robert Hill’s [1] first isolation of chloroplasts opened the door to the study of in vitro photosynthesis on a major scale and much of our present understanding of electron, transport, photophosphorylation and transport of metabolites between the chloroplast and the cellular environment derives from work on thylakoids and intact chloroplasts. In pursuing this approach ourselves we have been conscious for many years of the need to relate “in vitvo information” to the physiology of the cell and the leaf.
Carbon dioxide is assimilated by plants in the process of photosynthesis. Virtually all life depends on this process. The measurement of carbon dioxide assimilation is one method for studying photosynthesis which is adaptable both to reductionist questions such as what specific biochemical process limits the overall rate of photosynthesis and to ecophysiological questions such as how does water stress affect photosynthesis. The techniques required by reductionists and ecophysiologists are similar and are the subject of this chapter. Other descriptions of these methods will also be useful (Bloom et al. 1980; Field et al. 1982 and in press; Ball 1987; Field and Mooney in press).
The light environment in forest understory habitats is highly variable because of the punctuation of the very low diffuse light background by sunflecks lasting from fractions of a second to minutes. While they may strike a leaf for only a small fraction of a day, the importance of sunflecks is underscored by field observations showing that 40 to 60% of the daily assimilation (A) may be due to them (1,2). In this study, the responses of leaves to transient light changes designed to simulate sunflecks were investigated.
The carboxylation of RuBP is a key process in the photosynthesis of C3 plants being rate-limiting in many cases. The kinetic properties of RuBP carboxylase have been extensively studied (1), but as in vitro conditions never exactly imitate the conditions in a living cell, it would be interesting to obtain information about the kinetic parameters of the carboxylation process in intact leaves.
Recently, we analyzed induction phenomena in maize leaves and indicated that i) stomatal conductance does not limit photosynthesis during induction period [(1), also Furbank & Walker (2)], ii) during an initial phase of induction, light activation of pyruvate, Pi dikinase, Nadp-malate dehydrogenase, and FBPase plays an iirportant role in increasing the rate of CO2 assimilation (3), and iii) build-up of metabolites in the C4 cycle and Rpp pathway is required to reach a steady-state photosynthesis (4). During the course of these studies we found that there was a transient peak in the level of RuBP during the initial phase of induction of photosynthesis in maize leaves (4). The purpose of this study are to assess the possible mechanism behind the transient peak of RuBP which occurs during the induction of photosynthesis and to measure metabolite levels in a steady-state photosynthesis under various conditions in maize leaves.
The increase in photosynthetic CO2 uptake observed when O2 concentration is decreased (e.g. from 21 % to 2 % or so) can be easily explained in terms of the supression of photorespiratory metabolism. Oxygen, however, plays other roles, and some effects brought about by changes in oxygen concentration cannot be identified so easily. Oxygen is involved in the Mehler reaction [1] (pseudocyclic electron transport, in which O2 rather than NADP acts as the terminal electron acceptor) and some O2 is required for the maintenance of an appropriate redox state of the electron transport chain [2]. Oxygen also seems to play a major role in a less well understood process, the xanthophyll cycle [3,4]. Also, it is still a matter of argument at what rate dark respiration proceeds in the light. It is likely that the relative importance of these many roles of O2 varies with the plant material and environmental conditions.
The rate of whole leaf photosynthetic carbon fixation is ultimately dependent upon the capacity of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) to carboxylate RuBP. This capacity is modulated by the concentration of substrates (RuBP, CO2 and O2), the concentration of enzyme, its activity and the interaction of these factors with environmental conditions. Changes in the photosynthetic rate of a leaf which result from alterations in environmental conditions (i.e. light intensity, O2 concentration) apparently stimulate a cascade of biochemical events which result in a new equilibrium being reached between the capacity of the leaf to generate metabolic intermediates and/or products and its capacity to utilize them. Of considerable interest is the apparent balancing of the in vivo activity of RuBPCase with the potential of the entire photosynthetic apparatus to support a particular rate of CO2 fixation. The mechanisms by which the CO2 fixation capacity of RuBPCase is regulated in response to varying environmental conditions is the subject of the research and discussion presented here.
The identity of enzymes present in soluble extracts of spinach (Spinacia oleracea) chloroplasts that are capable of hydrolysing fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate has been investigated using antibodies against purified spinach chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11). The activity of purified fructose-1,6-bisphosphatase, which can exist in a less active oxidised form or a more active reduced form as well as total fructose-1,6-bisphosphatase in stromal extracts is inhibited completely by the antiserum. Apparently, only a single enzyme, which can exist in an oxidised or reduced form, is responsible for hydrolysis of fructose-1,6-bisphosphate in the chloroplast. Purified chloroplast fructose-1,6-bisphosphatase can also exhibit sedoheptulose-1,7-bisphosphatase activity, but only when reduced. Oxidised chloroplast stromal extracts contain little or no sedoheptulose-1,7-bisphosphatase activity whereas reduced extracts contain sedoheptulose-1,7-bisphosphatase activity. Antiserum against fructose-1,6-bisphosphatase does not inhibit sedoheptulose-1,7-bisphosphatase activity detectable at pH 8 or less with 2 mM Mg2+ bur substantially inhibits (up to 60%) the sedoheptulose-1,7-bisphosphatase activity at higher pH or Mg2+ concentration, i.e. conditions under which the chloroplast fructose-1,6-bisphosphatase exhibits sedoheptulose-1,7-bisphosphatase activity. Apparently, the chloroplast stroma contains at least two enzyme species capable of hydrolysing sedoheptulose-1,7-bisphosphate, a specific sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) and the chloroplast fructose-1,6-bisphosphatase.
Plants face constant change in their environment. Part of their success in dealing with such change lies with the ability of carbon metabolism to be responsive to changes in the relationship between the supply of CO2 or the products of electron transport and the demand for assimilated carbon. Metabolic adjustment in the leaf must occur both in the short-term (e.g. to fluctuations in temperature or light, such as sunflecks), and during longer-term changes in environmental conditions (e.g. acclimation to temperature or to sun and shade). At a time of increasing atmospheric CO2 and rapid progress in the genetic manipulation of crop plants, it is becoming more important than ever for the plant biochemist to understand the impact of environmental and other changes on the regulation of the metabolism of carbon, nitrogen, etc. in plants.
Glyphosate (Glp), (N-phosphonomethyl)glycine, is a nonselective, broad spectrum herbicide discovered in 1971 (Baird et al. 1971) and introduced in 1974 (Franz et al. 1997). Effectiveness, along with outstanding environmental and safety qualities, has made Glp one of the most successful commercial herbiides. Essentially nontoxic to mammals, birds, fish, insects, and most bacteria, the herbicide is readily broken down in soil to ammonia, carbon dioxide and inorganic phosphate (Franz et al. 1997; Giesy et al. 2000; Williams et al. 2000). Glp was one of the first commercially important herbicides whose site of action was characterized as a single, defined target enzyme in plants, and is the only herbicide known to inhibit 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) (phosphoenolpyruvate: 3 phosphoshikimate 1-carboxyvinyl transferase; E.C. 2.5.1.19) which catalyzes the penultimate reaction of the shikimate (Shk) pathway (Amrhein et al. 1980, 1982; Steinrücken and Amrhein 1980; Gruys and Sikorski 1999) in certain bacteria and plants. Because of these unique favorable characteristics, Glp has become and is likely to continue to be one of the most widely used and studied herbicides in the world (Woodburn 2000).
Fluxes of trace gases, water and energy - the ‘breathing of the biosphere’ - are controlled by a large number of interacting physical, chemical, biological and ecological processes. in this interdisciplinary book, the authors provide the tools to understand and quantitatively analyse fluxes of energy, organic compounds such as terpenes, and trace gases including carbon dioxide, water vapour and methane. it first introduces the fundamental principles affecting the supply and demand for trace gas exchange at the leaf and soil scales: Thermodynamics, diffusion, turbulence and physiology. it then builds on these principles to model the exchange of water, carbon dioxide, terpenes and stable isotopes at the ecosystem scale. Detailed mathematical derivations of commonly used relations in biosphere-atmosphere interactions are provided for reference in appendices. An accessible introduction for graduate students and a key resource for researchers in related fields, such as atmospheric science, hydrology, meteorology, climate science, biogeochemistry and ecosystem ecology.
Photosynthesis in C3 plants is usually inhibited by air levels of O2. Because C3 plants have no accessory metabolic pathways for concentrating CO2, the characteristics of photosynthesis such as oxygen sensitivity, measured with intact leaves can be readily interpreted at the biochemical level. This paper will discuss the biochemical basis for O2 sensitivity of C3 photosynthesis, the predictions of O2 sensitivity which follow from known mechanisms, and the metabolic information which O2 sensitivity measurements can give.
Understanding how photosynthesis responds to the environment is crucial for improving plant production and maintaining biodiversity in the context of global change. Covering all aspects of photosynthesis, from basic concepts to methodologies, from the organelle to whole ecosystem levels, this is an integrated guide to photosynthesis in an environmentally dynamic context. Focusing on the ecophysiology of photosynthesis how photosynthesis varies in time and space, responds and adapts to environmental conditions and differs among species within an evolutionary context the book features contributions from leaders in the field. The approach is interdisciplinary and the topics covered have applications for ecology, environmental sciences, agronomy, forestry and meteorology. It also addresses applied fields such as climate change, biomass and biofuel production and genetic engineering, making a valuable contribution to our understanding of the impacts of climate change on the primary productivity of the globe and on ecosystem stability.
Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP⁺ ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO2 assimilation rates decreased with decreasing ribulose 1,5-bisphosphate concentrations. In anti-b/f plants, CO2 assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.
The effect of changing the oxygen concentration from 21 to 2% on photosynthesis of wheat, sunflower, and soybean was investigated. At low CO2 concentrations and low light intensities, a stimulation of photosynthesis was observed in 2% oxygen compared with the rate in 21% O2. At high CO2 concentrations and high light intensities, a temporary inhibition of photosynthesis was observed when the oxygen concentration was changed from 21 to 2%. In wheat and sunflower, this inhibition was observed at progressively lower CO2 concentrations as temperatures were decreased. In soybean only, a slight inhibition of photosynthesis was observed at higher temperatures. In some cases, especially in plants grown under a low light intensity, a long-lasting (> 45 min) inhibition of photosynthesis was observed. In most cases, however, the inhibition lasted only for several minutes and final rates of photosynthesis in 2% O2 were equal to or greater than the rate of photosynthesis in 21% O2. The stimulatory effect of oxygen on photosynthesis at high CO2 concentrations and high light intensities could possibly be due to a regulation of the oxidation–reduction state of the electron transport chain and the maintenance of phosphorylation
Various aspects of the biochemistry of photosynthetic carbon assimilation in C3 plants are integrated into a form compatible with studies of gas exchange in leaves. These aspects include the kinetic properties of ribulose bisphosphate carboxylase-oxygenase; the requirements of the photosynthetic carbon reduction and photorespiratory carbon oxidation cycles for reduced pyridine nucleotides; the dependence of electron transport on photon flux and the presence of a temperature dependent upper limit to electron transport. The measurements of gas exchange with which the model outputs may be compared include those of the temperature and partial pressure of CO2(p(CO2)) dependencies of quantum yield, the variation of compensation point with temperature and partial pressure of O2(p(O2)), the dependence of net CO2 assimilation rate on p(CO2) and irradiance, and the influence of p(CO2) and irradiance on the temperature dependence of assimilation rate.
In limiting light the activation of ribulose-1,5-bisphosphate (RuP(2)) carboxylase [3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] in leaf extracts of 7- to 8-day-old wheat seedlings changed proportionally with the photosynthetic rate of the intact plants. Higher rates of photosynthesis, induced by increasing irradiances, were accompanied by an increase in activation of the leaf RuP(2) carboxylase, while RuP(2) levels remained unchanged. The degree of activation varied from 20% to 60% of full activation at irradiances of 225-1650 muE/m(2).s (photosynthetically active radiation; E = einstein, 1 mol of photons). Between 225 muE/m(2).s and darkness, activation approached 50% while RuP(2) levels dropped more than 90%. During steady-state photosynthesis, levels of the substrate RuP(2) were 250-300 nmol/mg of chlorophyll in the leaves and were similar at all irradiances above 225 muE/m(2).s (25% of light saturation). When velocities of the carboxylase in leaf extracts were corrected for CO(2) levels estimated to exist within the leaf, they compared favorably with the photosynthetic rates of the intact seedlings. Comparison of CO(2) exchange rate, RuP(2) level, and activation of the carboxylase indicates that light limitation of photosynthesis can be due to two factors: the availability of RuP(2) in dark to dim light and activation of the RuP(2) carboxylase in dim light and higher irradiances.
A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis.
The partial pressure of CO(2) inside leaves of several species was measured directly. Small gas exchange chambers were clamped above and below the same section of an amphistomatous leaf. A flowing gas stream through one chamber allowed normal CO(2) and water vapor exchange. The other chamber was in a closed circuit consisting of the chamber, an infrared gas analyzer, and a peristaltic pump. The CO(2) in the closed system rapidly reached a steady pressure which it is believed was identical to the CO(2) pressure inside the leaf, because there was no flux of CO(2) across the epidermis. This measured partial pressure was in close agreement with that estimated from a consideration of the fluxes of CO(2) and vapor at the other surface.
Intact chloroplasts of wheat (Triticum aestivum) were isolated from mesophyll protoplasts. With decreasing concentrations of bicarbonate from 10 to 0.3 millimolar (pH 8.0), the optimal concentration of orthophosphate (Pi) for photosynthetic O(2) evolution decreased from a value of 0.1 to 0.2 millimolar to 0 to 0.025 millimolar. The extremely low Pi optimum for photosynthesis at the low bicarbonate levels of 0.3 millimolar was increased by lowering the O(2) concentration from 253 (21% gas phase) to 72 micromolar (6% gas phase). The relative amount of glycolate and dihydroxyacetone phosphate (DHAP) synthesized under high and low levels of bicarbonate and varying levels of Pi was determined. At low levels of bicarbonate, glycolate was the main product, whereas at high bicarbonate levels, DHAP was the main product. Most of the DHAP and glycolate was found in the extrachloroplastic fraction.The rate of photosynthesis at low levels of bicarbonate (0.3 millimolar) was as high as 75 to 95% of that at high levels of bicarbonate (10 millimolar) at the respective optimal levels of Pi. At low bicarbonate levels, and without Pi, there was little lag in photosynthetic O(2) evolution upon illumination in comparison to that of high bicarbonate levels and optimal levels of Pi. It is proposed that conditions which favor glycolate synthesis allow photosynthesis to continue without a depletion of internal Pi, whereas consumption of Pi may occur in the chloroplast during the net synthesis of organic phosphates under high levels of bicarbonate and without addition of Pi. At low bicarbonate levels, the extreme susceptibility of photosynthesis to inhibition by Pi may be due to excessive export of carbon from the chloroplast in the form of both glycolate and triose phosphate.
The compartmentation of cellular energy relations during dark-light and light-dark transitions was studied by means of a newly developed technique to fractionate oat (Avena sativa L., var. Arnold) mesophyll protoplasts. Using an improved microgradient system with hydrophobic and hydrophilic layers of increasing density, a pure plastid pellet (up to 90% of total chloroplasts) could be separated from an interphase of only slightly contaminated mitochondria (70 to 80% of total mitochondria), and a cytoplasmic supernatant could be obtained within 60 seconds. Appropriate controls indicate that, under the conditions employed, metabolic interconversions of adenylates can be kept to a minimum and, thus, be determined and corrected for. Cross contamination of the fractions, as well as liberation of organelles to the supernatant, was assessed by specific markers, and the metabolite levels recorded were corrected accordingly. Using this technique, we found that, during dark-light transition, the chloroplastic and cytosolic ATP exhibits a rapid increase, while the mitochondrial ATP level decreases. In all compartments, ADP levels mirror alterations of the ATP pool in the opposite way, at least to some extent. To compensate fully for the rise in ATP, chloroplastic and mitochondrial AMP levels change accordingly, indicating that, due to the more or less unchanged level of total adenylates, there is no net flux of adenylates between the compartments. In contrast to the organelles, no AMP could be detected within the cytosol. When the light is turned off, a decrease of ATP coincides between chloroplast stroma and the cytosol for only about 30 seconds. Under prolonged dark treatment, cytosolic ATP rises again, while stroma ATP levels exhibit a further decrease. After about 60 seconds of darkness, the cytosolic ATP level is back to its initial value. This obviously is due to the immediate rise in mitochondrial ATP upon darkening, which cumulates after about 60 seconds; then, caused by an ATP/ADP exchange with the cytosol, it levels off again at the state before changing the conditions, as soon as the cytosolic ATP is also back to its original level. All of these events are closely mirrored by the change in the ATP/ADP ratio and the energy charge within the compartments. While the values for chloroplasts exhibit considerable differences between dark and light, those calculated for mitochondria and the cytosol exhibit only transient changes. These are limited to about 60 seconds of undershoot or overshoot, with respect to the cytosol, and then return to nearly the levels observed before changing the conditions. Adenylate kinase was found to be exclusively associated with chloroplasts (90% of total activity level) and mitochondria. Isotonic liberation of vacuoles did not point toward a significant association of adenylates with this compartment.The results are discussed with respect to an effective collaboration between photosynthetic and oxidative phosphorylation in order to keep the cytosolic energy state at a constant, preset value.
In cotton the rate of CO2 assimilation, at O2 partial pressures up to 200 mbar, increased to a maximum and then declined as the intercellular partial pressure of CO2 was increased. The specific intercellular partial pressure of CO2 at which rate of assimilation began to decline depended on the environmental conditions. At 19 mbar partial pressure of O2 the decline occurred at CO2 partial pressure >390 µbar. At 200 mbar partial pressure of O2 it occurred at CO2 partial pressure > 534 µbar. O2 increased the CO2 partial pressure required for inhibition but it did not appear to affect the steepness of the decline of rate of assimilation with further increase in partial pressure of CO2 once the decline became apparent. The decline was more readily observed at low temperature and low O2 partial pressure, and in plants grown at low light and NO3- levels. It was also observed in cowpea and sunflower. Changes in quantum efficiency in cotton at high and low CO2 concentrations were observed. At ambient CO2 concentration (300 µbar), the quantum yields measured at 19 and 200 mbar partial pressure of O2 were 0.072 ± 0.0003 and 0.053 ± 0.0060 mol CO2 per mol absorbed quanta, respectively. In contrast, at 900 µbar CO2 partial pressure the respective values were 0.050 ± 0.0023 and 0.070 ± 0.0006 mol CO2 per mol absorbed quanta. The nature of the inhibition of CO2 assimilation by high partial pressure of CO2 is discussed.
The salts of several weak acids have been used to render the envelope permeable to protons. In order to investigate the role of stromal pH changes in the light regulation of CO2 fixation, formate, octanoate, nitrite, and glyoxylate have been tried as tools to reverse the light-dependent alkalization of the stroma. For this purpose, the decrease of the stromal pH in illuminated spinach chloroplasts, as caused by the addition of these substances or by instantaneous lowering of the pH in the medium, has been compared with the corresponding decrease of CO2 fixation and the change of stromal metabolite levels. It appears from out data that formate and octanoate are suited best to obtain a specific inhibition of CO2 fixation by lowering the stromal pH. The measurement of the corresponding metabolite levels indicates that this inhibition is primarily due to an inhibition of fructose- and sedoheptulose bisphosphatase. It is concluded that these two enzymes are important regulatory steps for the light control of CO2 fixation.
A series of experiments is presented investigating short term and long term changes of the nature of the response of rate of CO2 assimilation to intercellular p(CO2). The relationships between CO2 assimilation rate and biochemical components of leaf photosynthesis, such as ribulose-bisphosphate (RuP2) carboxylase-oxygenase activity and electron transport capacity are examined and related to current theory of CO2 assimilation in leaves of C3 species. It was found that the response of the rate of CO2 assimilation to irradiance, partial pressure of O2, p(O2), and temperature was different at low and high intercellular p(CO2), suggesting that CO2 assimilation rate is governed by different processes at low and high intercellular p(CO2). In longer term changes in CO2 assimilation rate, induced by different growth conditions, the initial slope of the response of CO2 assimilation rate to intercellular p(CO2) could be correlated to in vitro measurements of RuP2 carboxylase activity. Also, CO2 assimilation rate at high p(CO2) could be correlated to in vitro measurements of electron transport rate. These results are consistent with the hypothesis that CO2 assimilation rate is limited by the RuP2 saturated rate of the RuP2 carboxylase-oxygenase at low intercellular p(CO2) and by the rate allowed by RuP2 regeneration capacity at high intercellular p(CO2).
Experiments were performed with intact chloroplasts and leaf cell protoplasts isolated from spinach. The light-dependent decrease in (H+) in the chloroplast stroma counteracts carbon reduction and is offset at low light intensities by a large decrease in NADP and a significant increase in ratios. Excess accumulation of NADPH and/or ATP permits 3-phosphogly cerate reduction to occur. With increasing light intensity, NADP levels and ratios increased. High rates of photosynthesis were observed at high and at low ratios. Levels of dihydroxyacetone phosphate were dramatically increased in the light. In chloroplasts, this permitted conversion to ribulose bisphosphate which on carboxylation yields 3-phosphoglycerate. The light-dependent alkalization of the chloroplast stroma is known to be responsible for phosphogly cerate retention in the chloroplasts. A high chloroplast ratio of phosphogly cerate to dihydroxyacetone phosphate aids carbon reduction. Measured ratios of dihydroxyacetone phosphate to phosphogly cerate were averages between low chloroplast ratios and high cytosolic ratios. They were far higher, even under low-intensity illumination, than dark ratios. Since cytosolic NADH levels are known to increase much less in the light than cytosolic dihydroxyacetone phosphate levels, the large increase in the ratio of didydroxyacetone phosphate to phosphogly cerate must considerably increase cytosolic phosphorylation potentials even at very low light intensities. It is proposed that this increase is communicated to the mitochondrial adenylate system, and inhibits dark respiratory activity, giving rise to the Kok effect. The extent of stroma alkalization, the efficiency of metabolite shuttles across the chloroplast envelope, and rates of cytosolic ATP consumption are proposed to be factors determining whether and to what extent the Kok effect can be observed. Light activation of chloroplast enzymes was slow at low and fast at high light intensities. This contrasts to low NADP levels at low and usually higher levels at high light intensities. Maximum enzyme activation was observed far below light saturation of photosynthesis, and light activation of enzymes was often less pronounced at very high than at intermediate light intensities.
This chapter describes photosynthetic carbon reduction cycle. Organic carbon is derived from atmospheric carbon dioxide (CO2) by photosynthetic carbon assimilation. In this process, CO2 is joined to an existing acceptor in such a way that a new carboxyl group is formed. For this process to continue, it is necessary that the CO2 acceptor is regenerated and, in for the plants to grow, it is necessary that the amount of this acceptor is increased. Unlike the Krebs cycle, the formulation of the photosynthetic carbon reduction cycle (PCR cycle) owes everything to the availability of radioactive CO2. The PCR cycle can be divided into three main aspects—carboxylation, reduction, and regeneration. The carboxylase also functions as an oxygenase; however, in high CO2, it catalyzes the addition of one molecule of CO2 to one molecule of ribulose-l,5-P2 with the formation of two molecules of 3-phosphoglycerate (PGA). Accordingly, the fixation of three molecules of CO2 leads to the synthesis of six molecules of PGA. These are then reduced to triose phosphate at the expense of NADPH. One molecule of triose phosphate can be regarded as product. The remaining five are rearranged to regenerate the CO2 acceptor. The net product of the cycle is, in the first instance, triose phosphate, and this is one of the major products of photosynthesis by isolated chloroplasts.
Protoplasts were isolated from wheat and spinach leaves and after photosynthesis in the presence of fractionated into a chloroplast and a nonchloroplast fraction. The kinetics of the distribution of labeled metabolities between the fractions indicated carbon flow from the chloroplasts into the cytosol and the vacuole. After 10 min of photosynthesis, more than 90% of the assimilated soluble carbon was outside the chloroplasts. Different metabolite levels indicated intracellular metabolite compartmentation and metabolite gradients. During photosynthesis, gradient coupling facilitated uphill export of triosephosphate from the chloroplasts into the cytosol. The driving force appeared to be an opposite phosphate gradient. Different ratios of phosphoglycerate to triosephosphate inside and outside the chloroplasts indicated the existence of a transenvelope pH gradient. Sucrose appeared to be synthesized outside the chloroplasts and is probably exported into the vacuole. The behavior of malate also suggested transfer into the vacuole. The malate/ aspartate ratio was higher outside the chloroplasts that inside suggesting import of reducing equivalents into the chloroplasts through the dicarboxylate translocator.
The light activation of photosynthesis has been investigated in spinach palisade cell protoplasts. 1.(1) After a short induction period, maximal rates of photosynthesis are achieved.2.(2) [14C] Bicarbonate initially labels anionic compounds in the chloroplast and then in the extrachloroplast compartments. These pools saturate within 2–4 min and radioactivity accumulates mainly in sucrose in the extrachloroplast compartment, in starch and in cationic compounds.3.(3) Enzymic determinations were made of metabolite levels during the induction period in the chloroplast and extrachloroplast compartments. There is no general build-up of intermediates. Perturbations of individual intermediates occurred, consistent with the activation of specific enzymes.4.(4) It is suggested that fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase may limit flux in the Calvin cycle during induction.5.(5) The onset of sucrose synthesis is not accompanied by accumulation of intermediates in the cytosol. It is suggested that sucrose phosphate synthase or sucrose phosphate phosphatase is activated.6.(6) Measurements of metabolites in whole leaves during a 24 h illumination cycle confirmed that substrates are not depleted during the dark period, and that the onset of photosynthesis is not accompanied by a rise in intermediate levels.7.(7) It is concluded that the causes of the induction lag in protoplasts can differ from those in isolated chloroplasts.
Equations are developed to describe the reactions of ribulose 1,5-biphosphate carboxylase—oxygenase with ribulose biphosphate (RuP2), carbon dioxide, and oxygen. It is predicted that at the high concentrations of enzyme sites found in vivo there will be a large proportion of the total RuP2 bound to the enzyme. The kinetic characteristics of the in vivo reactions with RuP2 are predicted to be analogous to those which would occur in the presence of a tight-binding substrate. Equations are developed which are applicable when the enzyme is only partially activated by CO2 and Mg2+. The response of carboxylase velocity to CO2 concentration is sigmoidal when Mg2+ concentration is low.
Anions of several weak acids inhibited photosynthesis in isolated spinach chloroplasts. Inhibition was drastic at low pH and weak or absent at high pH. Glyoxylate was particularly effective and inhibition decreased in the order: glyoxylate, nitrite, glycerate, formate, hydroxypyruvate, glycolate, propionate, acetate, pyruvate. These anions operated as indirect proton shuttles across the chloroplast envelope. They compensated active proton fluxes into the medium, minimized gradients in proton activity across the chloroplast envelope, and so prevented light-dependent stroma alkalization. This caused inhibition of sugar bisphosphatases which are known to be pH-regulated. At concentrations that caused potosynthesis inhibition, the proton shuttles were not effective in decreasing the proton gradient across the thylakoids. Some anions also inhibited fructose-bisphosphatase directly, when present at concentratins higher than needed for photosynthesis inhibition.
Ribulose-1,5-bisphosphate caryboxylase-oxygenase is activated by CO2 and Mg2+ in a process distinct from catalysis. The effect of chloroplast metabolites as they separately influenced either activation or catalysis of tobacco carboxylase was examined. Of the 28 metabolites examined, 13 effected activation of the carboxylase. The strongest positive effectors were NADPH, gluconate-6-P, glycerate-2-P, and glycerate-3-P. Negative effectors included ribose-5-P, fructose-6-P, glucose-6-P, and pyrophosphate. The concentration of CO2 or Mg2+ necessary to produce half-maximal activation is defined as Kact. NADPH and gluconate-6-P decreased the Kact(CO2) from 43 to 7.4 and 3.5 μm, respectively (pH 8.0, 5 mm MgCl2). They also decreased the Kact(M.g2+), but had little affect on the affinity of the enzyme for CO2 during the catalytic process. Increasing Mg2+ concentration decreased the Kact(CO2) and increasing CO2 concentration decreased the Kact-(Mg2+). NADP+ and gluconate-6-P also affected the pH profile of activation, shifting it toward lower pH values. Changes in activation had no effect on the pH profile for catalysis of CO2 fixation. Effectors influenced ribulose-1,5-bisphosphate oxygenase in a manner analogous to the carboxylase. At air levels of O2 and CO2, the ratio of carboxylase to oxygenase activity was not changed by the presence of effectors, including hydroxylamine.
The activated and catalytically competent form of ribulose-1,5-bisphosphate carboxylase is a ternary complex of enzyme-activator CO2 x Mg. The effectors of NADPH and 6-phosphogluconate promoted activation by formation of a rapid equilibrium quaternary complex of enzyme-activation CO2 x Mg x effector; i.e., the effectors did not activate the enzyme per se but promoted the basic activation process by stabilizing the activated enzyme-activator CO2 x Mg complex. Kinetic and gel filtration studies showed that the effectors stabilized the binding of the activator CO2 and MG2+ (or Mn2+), thereby decreasing the rate of deactivation. Binding studies indicated the presence of one 6-phosphogluconate binding site per protomer. The binding of 6-phosphogluconate and NADPH to the enzyme-activator CO2 x Mg complex was (a) completely prevented when the catalytic site for ribulose bisphosphate was occupied by the transition-state analogue, 2-carboxyarabinitol 1,5-bisphosphate, and (b) competitively diminished in the presence of 3-phosphoglycerate, the product of the carboxylation reaction. NADPH, 6-phosphogluconate, and 3-phosphoglycerate acted as linear competitive inhibitors of carboxylation with respect to ribulose bisphosphate. These results demonstrate that the effectors elicit their response through interaction at the catalytic site for ribulose bisphosphate and that their effect is secondary to the basic CO2-Mg2+-dependent activation reaction. An enzyme molecule cannot be simultaneously catalytically competent (capable of binding and carboxylating ribulose bisphosphate) and activated by an effector, since the latter involves occupancy of the ribulose bisphosphate binding site.
In order to study the relative contributions of the autocatalytic increase in the level of substrates and the light activation of enzymes to the control of the induction phase or “lag” in wheat chloroplasts, we measured the light-induced reductive activation of fructose 1,6-bisphosphatase, phosphoglycerate kinase, NADP+-dependent glyceraldehyde-phosphate dehydrogenase, ribulose 1,5-bisphosphate carboxylase, and phosphoribulokinase in isolated chloroplasts. Each was rapidly activated to levels more than adequate to support the maximum rate of photosynthesis. Induction in wheat chloroplasts is characterized by a period of about 1 min during which no O2 is evolved. If small quantities of intermediates such as dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerate (PGA) are added, maximum rates of photosynthesis are achieved within the first minute of illumination. The presence of PGA did not affect the activation of any of the above-mentioned enzymes. Each of the enzymes was therefore capable of sustaining maximum rates of photosynthesis in the presence of PGA, even though there was no O2 evolution from those chloroplasts incubated with CO2 alone as substrate. The inclusion of PGA did not give rise to abnormally high levels of DHAP, FBP, or fructose 6-phosphate in the stroma. We conclude that the levels of substrates or cofactors are the principal, if not the sole, determinants of the rate of photosynthetic carbon assimilation during induction in wheat chloroplasts.
The levels of ribulose 1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), glycolate, glycine, and serine were measured in soybean leaflets during photosynthesis in atmospheres ranging from 1 to 60% O(2) and from 0 to 500 microliters per liter CO(2.)The RuBP level remained constant as CO(2) concentration was decreased in atmospheres containing 20 or 60% O(2), but increased as CO(2) concentration was decreased in atmospheres containing 1% O(2.) PGA levels decreased at CO(2) concentrations near or below the CO(2) compensation point under all O(2) concentrations. The glycolate pool at 300 microliters per liter CO(2) increased slightly with increasing O(2) concentration, but remained nearly constant at very low CO(2). The serine pool showed no measurable change over the range of CO(2) or O(2) concentrations tested. The glycine pool did not change significantly with varying CO(2) concentration but increased linearly with increasing O(2) concentration.Measured RuBP levels indicate an RuBP concentration less than the estimated concentration of RuBP carboxylase/oxygenase active sites. The constant RuBP pool size in 20% O(2), however, indicates that RuBP level does not limit photosynthesis or photorespiration any more at 50 microliters per liter CO(2) than at 450 microliters per liter.
On the regulation of CO2 fixation by light. In: Photo-synthesis IV : Regulation of carbon metabolism Regulation of photosynthesis by sink activity -the missing link
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A flexible system of en-zymic analysis Inhibition of photosyn-thesis by low oxygen concentrations
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Lowry, O.H., Passoneau, J.V. (1972) A flexible system of en-zymic analysis. Academic Press, New York London McVetty, P.B.E., Canvin, D.T. (1981) Inhibition of photosyn-thesis by low oxygen concentrations. Can. J. Bot. 59, 721-725
Phosphate re Photosynthetic intermediates and gas exchange in bean leaves 313 quirement for the light activation of ribulose-l,5-bisphos-phate carboxylase in intact spinach chloroplasts
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A bio-chemical model of photosynthetic CO 2 assimilation of C 3 plants
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Farquhar, G.D., Caemmerer, S. von (1982) Modelling of pho-tosynthesis to environmental conditions. In: Encyclopedia of plant physiology, N.S., vol. 12B: Physiological plant ecol-ogy II. Water relations and carbon assimilation, pp. 549-587, Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H., eds. Springer, Berlin Heidelberg New York Flfigge, U.I., Freisl, M., Heldt, H.W. (1980) The mechanism of the control of carbon fixation by the pH in the chloro-plast stroma. Planta 149, 48-51
A flexible system of enzymic analysis
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On the regulation of CO2 fixation by light
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