UV Chapter 7 RuBP

Ribulose-1,5-bisphosphate

RuBisCO derived from spinach (Spinacia oleracea) and alfalfa (Medigaco sativa) have particularly been used to produce bioactive peptides and hydrolysates.

From: Trends in Food Science & Technology, 2017

Related terms:

Chloroplast

Carbonic Anhydrase

Photorespiration

Photosystem

Cyanobacteria

Carboxysome

Oxygenase

Carboxylation

Nested Gene

Carbon Dioxide Fixation

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PHOTOSYNTHESIS AND PARTITIONING | C3 Plants

A.S. Raghavendra, in Encyclopedia of Applied Plant Sciences, 2003

Regulation

C3 photosynthesis is regulated in multiple ways, but is primarily initiated by illumination. One of the most important phenomena in the pathway is the light activation of key enzymes, through either dithiol-reduction of cysteines on protein or through changes in the microenvironment (e.g., magnesium or pH levels) of the stroma. Then, changes in substrate availability and metabolite flux rapidly set in motion an efficient autocatalysis of the Calvin cycle. In a long-term mode, the levels and turnover of rubisco protein also modulate the capacity of carbon fixation in the Calvin cycle. For example, limitation of nitrogen availability decreases the levels of rubisco protein and restricts the photosynthetic carbon assimilation in the leaves.

Regulation of Rubisco

The activity of rubisco, the first enzyme in the Calvin cycle, is highly regulated. Rubisco is inactive in the dark and is converted to an active form on illumination, which catalyzes fixation of CO2. Activation of rubisco is the result of carbamylation, which involves the binding of CO2 and Mg2+ to a lysine residue near the catalytic site.

Rubisco is active only when lysine-201 reacts with CO2 near the catalytic site to form a carbamate and allows the binding of the Mg2+ ion. Carbamylation changes the conformation of the large subunit and activates the enzyme, while the active conformation is stabilized by the formation of a complex with Mg2+. Carbamylation is essential for rubisco activation, as the noncarbamylated rubisco binds RuBP too tightly to allow catalysis.

Another protein, rubisco activase, is also involved in mediating the light activation of rubisco. On illumination, rubisco activase releases the inhibitor compounds, such as 2-carboxyarabinitol 1-phosphate or CA1P, which are bound to the active site of rubisco; otherwise, for example in darkness, these inhibitors prevent activation (carbamylation) of the enzyme. Rubisco activase itself is activated in light by utilizing ATP produced from photosynthetic electron transport. Rubisco activase and rubisco activation provide another mechanism of strong regulation by light of carbon assimilating reactions of photosynthesis.

CA1P, which occurs naturally in the leaves of several plants, is a strong inhibitor of rubisco. The affinity of rubisco for CA1P is much stronger than that for RuBP, the substrate. As a result, CAP, which accumulates in leaves during the night, inactivates rubisco by blocking the binding sites. During the day (or on illumination), the bound CA1P is released from rubisco, and this process is further accelerated by rubisco activase. However, the physiological role of CA1P is still debated, as it is not found in all plant species.

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Why C4 Photosynthesis?

Rowan F. Sage, in C4 Plant Biology, 1999

II The Problem with Rubisco

Rubisco evolved early in the history of life, more than 3 billion years ago (Hayes, 1994). The CO2 content of the atmosphere at this time was orders of magnitude greater than now, and O2 was rare (Fig. 2A; Kasting, 1987; Holland, 1994). In this environment, oxygenase activity was uncommon, probably less than one oxygenation per billion carboxylations (Fig. 2C). Atmospheric O2 level remained low and was unable to support an oxygenation rate that was more than 1% of the carboxylation rate until approximately 2 billion years ago. At this time, atmospheric O2 began to rise, eventually surpassing 200 mbar (20%) about 0.6 billion years ago (Kasting, 1987; Berner and Canfield, 1989; Holland, 1994). Atmospheric CO2 level continuously declined prior to 1 billion years ago, yet at the advent of the first land plants some 450 million years ago, atmospheric CO2 was still high enough to saturate Rubisco and minimize oxygenase activity (Fig. 2C). Coal-forming forests of the Carboniferous period (360 to 280 million years ago) contributed to a reduction in CO2 partial pressures to less than 500 mbar and a rise in O2 partial pressures to more than 300 mbar (Berner and Canfield, 1989; Berner, 1994). As a consequence, Rubisco oxygenase activity is predicted to have become significant (>20% of the carboxylation rate at 30°C) for the first time in nonstressed plants at the prevailing atmospheric conditions (Fig. 2B,D). After the Carboniferous period, CO2 levels are modeled to have risen to more than five times current levels for about 200 million years, and the rate of RuBP oxygenation was again a small percentage of the carboxylation rate. Over the past 100 million years, atmospheric CO2 levels declined from more than 1,000 μbar, eventually falling below 200 μbar during the Pleistocene epoch (2 to 0.01 million years ago). In the last 15 million years, Rubisco oxygenase activity is modeled to have risen above 20% of carboxylase activity at 30°C, eventually surpassing 40% of carboxylase activity at the low CO2 levels (180 μbar) experienced during the late Pleistocene (Fig. 2D). It is only after atmospheric CO2 levels are low enough to allow the rate of RuBP oxygenation to exceed 20% to 30% of the carboxylation potential that C4 plants appear in the fossil record (Cerling, Chapter 13). No evidence exists for C4 photosynthesis during the Carboniferous (Cerling, Chapter 13).

Figure 2. The modeled change in atmospheric CO2 and O2 partial pressures over (A) the past 4 billion years; and (B) the past 600 million years (according to Berner, 1994). C and D present the modeled change in Rubisco oxygenase activity (v0) to carboxylase activity (vc) at 30°C for the corresponding CO2 and O2 levels presented in A and B, respectively, calculated assuming a spinach C3-type Rubisco according to Jordan and Ogren, 1984.

(adapted from Kasting, 1987, and Berner and Canfield, 1989)

Above 200 mbar oxygen, CO2 partial pressures of less than 500 μbar pose two problems. First, as a substrate for Rubisco carboxylation, CO2 availability becomes strongly limiting, reducing the turnover of the enzyme in vivo and imposing a limitation on photosynthesis by reducing the capacity of Rubisco to consume RuBP (Fig. 3A; Sage, 1995). Second, O2 competition becomes significant at warmer temperature (>30°C), and this causes a high rate of photorespiration (Fig. 3B; Sharkey, 1988; Sage, 1995). Whereas a CO2-substrate deficiency occurs only when the capacity for Rubisco to consume RuBP limits the rate of CO2 assimilation in plants, photorespiration is inhibitory regardless of whether Rubisco capacity or the capacity of the leaf to regenerate RuBP limits photosynthesis (Sharkey, 1985). At current CO2 levels, photorespiration can reduce photosynthesis by more than 40% at warmer temperatures (Sharkey, 1988; Ehleringer et al., 1991).

Figure 3. The modeled response of (A) Rubisco activity (as a percent of Vmax) as a function of stromal CO2 concentration, and (B) the percent of photorespiratory inhibition of photosynthesis (0.5v0/vc, × 100%). Modeled according to Sage (1995) using equations from Farquhar and von Caemmerer (1982). Ca indicates atmospheric CO2 content corresponding to indicated chloroplast CO2 concentrations. (Note: at sea level, μmol mol−1 ≅ μbar.)

The rise in photorespiratory potential triggered by the increase in the atmospheric O2:CO2 over the past 50 million years created high evolutionary pressure for dealing with the consequences of Rubisco oxygenation (Ehleringer et al., 1991). In all higher photosynthetic organisms, however, an elaborate biochemical edifice was already built around Rubisco, such that substantial barriers likely prevented the evolution of a novel carboxylase to replace Rubisco. Not only would the new carboxylase be required, but the accompanying biochemistry to regenerate acceptor molecules and process photosynthetic products would likely have to change as well. Such new photosynthetic systems would then have to compete against preexisting Rubisco-based systems, which though inefficient, would have had the advantage of working reasonably well and of being integrated into the associated cellular, organismal, and ecological systems. To a large degree, the potential for RuBP oxygenation represents a systematic constraint around which evolution must work, an evolutionary spandrel sensu Gould and Lewontin (1979) (Somerville et al., 1983). In a situation similar to that of a restoration architect who is constrained by preexisting structures, evolution is constrained by preexisting enzymes, genes, and regulatory systems in dealing with novel challenges (Gould and Lewontin, 1979). In plants and algae, the evolutionary response to declining atmospheric CO2 levels was to modify existing leaf physiology to create CO2 concentrating systems ("CO2 pumps") that were coupled to preexisting Rubisco-based biochemistry. In land plants, the most elaborate and successful of these modifications is C4 photosynthesis.

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The Biochemistry of C4 Photosynthesis

Ryuzi Kanai, Gerald E. Edwards, in C4 Plant Biology, 1999

6 RuBP Carboxylase-Oxygenase

Ribulose-1,5-bisphosphate carboxylase—oxygenase (Rubisco, EC 4.1.1.39) of C4 plants, like that of C3 plants, consists

of eight large subunits of 53.5 kDa and eight small subunits of 13 kDa (L8S8). Kinetic properties show some difference between C3 and C4 plants (Yeoh et al., 1980; 1981). The range of Km(CO2) values for the enzyme among C3 species (12–25 μM) is lower than those in C4 (28–34 μM), although variation of Km (RuBP) values (15–82 μM in C4) is not related to the photosynthetic pathway of higher plants. Among the C4 grasses, the Km(CO2) values of Rubisco in PEP-CK type (28–41, mean 35 μM) are significantly lower than those in NAD- and NADP-ME types (41–63, mean 53 μM). Molecular activities of some C4 Rubiscos (2280–4020, mean of 3240 mol CO2 mol enzyme−1 min−1) are about twofold higher than those from C3 higher plant species (Seemann et al., 1984). These higher Vmax values of Rubisco in C4 plants accompanied by higher Km(CO2) may be an evolutionary change that allows a high activity per unit Rubisco protein under high levels of CO2 in BSC in the light. However, there are no clear differences between the C4 and C3 Rubisco in their specificity factor (Srel), that is, the relative specificity to react with CO2 versus O2, which is based on Vmax values and Michaelis constants for the two gases. An improved measurement of the specificity factor of Rubisco resulted in a value of 79 mol/mol for maize leaves, which is marginally smaller than those of five C3 higher plant species (82–90 mol/mol) (Kane et al., 1994).

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Agricultural and Related Biotechnologies

H. Ashida, A. Yokota, in Comprehensive Biotechnology (Second Edition), 2011

4.13.2.1 Effects of Enzymatic Properties of RuBisCO on Leaf Photosynthesis

In photosynthetic organisms, RuBisCO enzymes can be classified into two forms on the basis of their amino acid sequence and three-dimensional (3D) structure [1, 7]. Form I RuBisCOs consist of eight 50–55 kDa large subunits and eight 12–18 kDa small subunits, and comprise two subclasses: green- and red-type RuBisCOs [1]. Green-type RuBisCOs are found in plants, green algae, cyanobacteria, and photosynthetic bacteria, whereas red-type RuBisCOs are found in phototrophic bacteria and nongreen eukaryotic algae such as red algae. Form II RuBisCOs consist of only large subunits and are found in photosynthetic bacteria, chemoautotrophic bacteria, and eukaryotic dinoflagellates. In both forms of RuBisCO, the large subunit assembles into a dimer to form a common minimum catalytic unit. The active site is located at the inter-dimer interface between the N-terminal domain of one large subunit and the C-terminal domain of the neighboring large subunit. The enzymatic properties of RuBisCOs from different sources vary. Among green-type RuBisCOs, the maximum catalytic turnover rate of the carboxylase reaction (kcatc) is 2.9–4.2/s/site for RuBisCO from plants, 2.3–5.4/s/site for RuBisCO from green algae, 2.6–13.2/s/site for RuBisCO from cyanobacteria, and 1.9–7.1/s/site for RuBisCO from bacteria [1, 4]. On the other hand, kcatc is 1.3–3.4/s/site for red-type RuBisCOs from nongreen eukaryotic algae and 2.0–3.2/s/site for phototrophic bacterial red-type RuBisCOs. Form II RuBisCOs have kcatc values ranging from 3.5 to 7.3/s/site.

The CO2/O2 specificity factor of RuBisCO, Srel determines the relative rates of the carboxylase reaction to oxygenase reaction at given CO2 and O2 concentrations. The Srel value has a special meaning in kinetic parameters of RuBisCO and is calculated by the equation (kcatc/Kmc)/(kcato/Kmo), where kcatc,kcato,Kmo, and Kmo are the catalytic turnover rates and Michaelis constants for CO2 and O2, respectively [1]. RuBisCOs with high Srel values have lower reactivity for O2, and are relatively specialized for catalysis of the carboxylase reaction. Among green-type RuBisCOs, Srel values are 70–100 among plant RuBisCOs, 54–83 among green algal RuBisCOs, 35–56 among cyanobacterial RuBisCOs, and 26–53 among bacterial RuBisCOs (Figure 1) [1, 4]. On the other hand, in the red-type RuBisCO lineage, Srel is 40–75 for RuBisCOs from phototrophic bacteria, 100–110 for RuBisCOs from diatoms, 130 and 144 for RuBisCOs from the red algae Porphyra and Porphyridium, respectively, and 225 and 238 for RuBisCOs from Cyanidium and Galdieria partita, respectively [8, 9]. The species variation in the Srel value suggests that enzymatic properties of RuBisCO have acclimated to the atmospheric and environmental conditions that each photosynthetic organism has encountered [1, 4]. For example, RuBisCOs with lower Srel values are found in photosynthetic bacteria that live in anaerobic conditions, and in cyanobacteria that employ CCMS to prevent oxygen inhibition [4, 5]. By contrast, RuBisCOs of land plants have evolved higher Srel values to adapt to the present atmospheric conditions, in which the concentration of O2 (21%) is much higher than that of CO2 (0.038%).

Figure 1. Variations in relative specificity of RuBisCOs.

Adapted from Roy H and Andrews TJ (2000) RuBisCO: Assembly and mechanism. In: Leegood RC, Sharkey TD, and von Caemmerer S (eds.) Photosynthesis: Physiology and Metabolism, vol. 9, pp. 53–83. Dordrecht: Kluwer Academic, Uemura K, Anwaruzzaman, Miyachi S, and Yokota A (1997) Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic red algae with a strong specificity for CO2 fixation. Biochemical and Biophysical Research Communications 233: 568–571, and Uemura K, Suzuki Y, Shikanai T, et al. (1996) A rapid and sensitive method for determination of relative specificity of RuBisCO from various species by anion-exchange chromatography. Plant and Cell Physiology 37: 325–331.

Another interesting point is that the Srel value varies depending on the phylogenetic lineage of the primary sequence of the catalytic subunit (Figure 1). In contrast to RuBisCOs from photosynthetic organisms in the lineage that includes the cyanobacterial enzyme, red-type RuBisCOs generally show greater diversity in their Srel values. In particular, the RuBisCO from the red alga Galdieria partita has the highest Srel value (238) of all RuBisCOs examined to date [8]. RuBisCOs in this lineage originated from the Alcaligenes enzyme, and the Galdieria enzyme has the highest Srel value. The molecular mechanism that underlies this diversity among RuBisCOs in this lineage is of great interest, but is still poorly understood.

How does an increase in RuBisCO's Srel value affect plant photosynthesis in vivo? The CO2 assimilation rate in plant leaves can be simulated using the model described by Farquhar et al. and RuBisCO's kinetic parameters [3, 10, 11]. There are two phases in this model: the RuBisCO activity-limiting phase at lower CO2 concentrations and the RuBP regeneration-limiting phase at higher CO2 concentrations. The latter phase may be further separated into two phases: the RuBP regeneration phase and the Pi availability limiting phase. The transition point is predicted by the intersection of the two phases. The calculated CO2 assimilation rate at each given intercellular CO2 partial pressure is consistent with measured values of the CO2-exchange rate in living plant leaves. We simulated the CO2 assimilation rates of plant leaves in which RuBisCO was assumed to be improved by doubling the Srel value of wild-type RuBisCO in tobacco (Figure 2). This can be achieved by increasing kcatc or Kmo twofold, or by halving Kmc or kcato. All improved RuBisCOs were predicted to increase CO2 assimilation rate over the entire range of intercellular CO2 partial pressures, and to decrease the CO2 compensation point from 50 to 30 μbar (Figure 2). In particular, improving the carboxylase reaction's kinetic parameters kcatc and Kmc remarkably increased the CO2 assimilation rate at low CO2 concentrations. In other words, transgenic plants expressing the improved RuBisCO required lower CO2 concentrations to support the same CO2 assimilation rate as the wild type and, therefore, were not as vulnerable to water loss that occurs when stomata are open. As discussed above, the RuBP carboxylation and the subsequent metabolism of fixed CO2 do not consume all of the solar energy captured on thylakoid membranes. RuBP oxygenation and metabolism of the products from that reaction consume an appreciable amount of the captured solar energy. Thus, we should not aim to just eliminate the oxygenase activity from plant RuBisCOs without improving the RuBP carboxylation reaction, as this reaction is required to consume surplus energy [2]. The surplus energy resulting from a decrease in RuBP-oxygenation could be dissipated if the RuBP carboxylation efficiency is increased to a level where the improved CO2 fixation compensates for the excess energy originally consumed by RuBP-oxygenation. Thus, improving RuBisCO's carboxylation efficiency is expected to increase the CO2 assimilation rate and also improve water-use efficiency in plants. These are desirable outcomes, but one may wonder what strategies are available to improve RuBisCO?

Figure 2. Effect of RuBisCO's properties on photosynthetic CO2 assimilation rates. Photosynthetic CO2 assimilation rate (A) calculated according to Farquhar et al. [10]. Examples are from Andrews and Whitney [11]. CO2 assimilation rate in RuBisCO activity-limited phase at lower CO2 pressure is expressed as follows: A = [RuBisCO]·Kcatc·(pc·sc − 0.5·[O2]/Srel)/{pc·sc + Kmc (1 + [O2]/Kmo)} – Rd, where [RuBisCO] is the mole number of active sites of RuBisCO per unit leaf area, pc the intercellular CO2 partial pressure, sc the CO2 solubility in water (0.0334 M/bar), [O2] the O2 concentration in the chloroplast (252 μM), and Rd the day respiration rate (1 μ mol m−2 s−1). CO2 assimilation rate in RuBP regeneration-limited phase at higher CO2 pressure was obtained using the following equation: A = J·(pc·sc − 0.5·[O2]/Srel)/4·(pc·sc + [O2]/Srel) − Rd, where J is the photosynthetic electron flux of 120 (μ mol m−2 s−1). (a) Kinetic parameters of wild-type tobacco RuBisCO used in the simulation were as follows: 3.4/s/site for Kcatc, 10.7 μM for Kmc, 0.87/s/site for Kcato, and 295 μM for Kmo(solid line). To double Srel, kinetic parameters were changed as follows: Kcatc (closed squares) or Kmo (opened circles) were doubled, or Kmc (closed circles) or Kcato (opened squares) were halved. (b) CO2 assimilation rates with red algal RuBisCO from Griffithsia monilis (closed triangles). Kinetic parameters were as follows: 2.6/s/site for Kcatc, 2.6 μM for Kmc, 710 μM for Kmo, and 167 for Srel. In the case of wild-type tobacco RuBisCO (solid line), data are the same as in (a). In all calculations, the mole number of active sites of RuBisCO per unit leaf area was set to 20 μ mol.

(b) Adapted from Andrews TJ and Whitney SM (2003) Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants. Archives of Biochemistry and Biophysics 414: 159–169.

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Photosynthetic Carbon Dioxide Fixation

Thomas D. Sharkey, in Reference Module in Life Sciences, 2020

Oxygenation/photorespiration

Carboxylation by rubisco involves making RuBP susceptible to chemical attack by carbon dioxide. Unfortunately, the carboxylation step is inherently susceptible to competition from oxygen (Bowes et al., 1971). When rubisco "fixes" oxygen instead of CO2, a series of reactions are required to deal with the consequences of making 2-phosphoglycolate from the top two carbons of RuBP. Two-phosphoglycolate is an extremely potent inhibitor of triose phosphate isomerase (Anderson, 1971; Flügel et al., 2017; Li et al., 2019).

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The Origins of C4 Genes and Evolutionary Pattern in the C4 Metabolic Phenotype

Russell K. Monson, in C4 Plant Biology, 1999

E The Evolution of Differential Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) in C4 Leaves

In C4 photosynthesis, expression of Rubisco is up-regulated in bundlesheath cells and suppressed in mesophyll cells. Rubisco is a large, multimeric protein consisting of eight small subunits and eight large subunits, with the large subunits carrying the active sites for catalysis. The gene for the large subunit (typically represented as rbcL) is present as a single copy per chloroplast genome (Gutteridge and Gatenby, 1995). rbcL is actively transcribed in C4 mesophyll cell chloroplasts, despite the absence of mature Rubisco protein (Schäffner and Sheen, 1991; Boinski et al., 1993). The suppression of Rubisco in C4 mesophyll cells is due to posttranscriptional processes. Roth et al. (1996) have demonstrated a probable role for the nuclear gene (bundle sheath defective 2 Bsd2) in the posttranscriptional control of rbcL transcript accumulation and/or translation in mesophyll and bundle-sheath cells. Although the involvement of nuclear regulation in control over chloroplast gene expression is known from several plant systems, the nature of the control mechanisms is unclear (Mayfield et al., 1995).

More is known about differential expression of the Rubisco small subunit. The small subunit (typically represented as rbcS) is encoded from a nuclear, multigene family (2–12 gene members) (Gutteridge and Gatenby, 1995). The differential expression of rbcS is controlled by a combination of (1) a promoter sequence that flanks the transcription start site and regulates the light-stimulated increase in rbcS expression in bundle-sheath cells (Bansal et al., 1992; Viret et al., 1994), (2) a sequence within the 3′ transcribed region of the gene that is photoactivated and interacts with the promoter sequence upstream from the transcription start site and suppresses transcription in mesophyll cells (Viret et al., 1994), and (3) probable posttranscription suppression of rbcS mRNA in mesophyll cells (Schäffner and Sheen, 1991). The suppresser activity of the 3′ transcribed sequence most likely involves an interaction with trans-acting elements unique to C4 mesophyll cells (Fig. 7).

Figure 7. Regulatory interactions among different sequences of the RuBP carboxylase/oxygenase (Rubisco) gene in C4 plants. The absence of Rubisco expression in mesophyll cells is accomplished through interactions between a 3′ repression sequence and the promoter, an interaction that appears to be regulated by light availability. In bundle-sheath cells, the 3′ repression sequence is inactive, and a light-inducible sequence that overlaps the promoter and transcribed region is active.

Insertion of the rbcS promoter from C4 maize bundle sheath cells into Cs rice mesophyll cells results in normal C3-like expression (Matsuoka et al., 1994). In addition, insertion of an rbcS promoter from C3 wheat mesophyll cells into C4 maize mesophyll protoplasts results in normal C3-like expression (Schäffner and Sheen, 1991). Thus, the promoter of the rbcS gene, including those sequences that affect the light dependency of expression, is similar in both C3 and C4 monocots. It may be that evolution of the differential expression of Rubisco occurred on the appearance of the 3' suppresser sequence concomitant with trans-acting elements unique to C4 mesophyll cells. Alternatively, the 3′ sequence may have always been present in the C3 rbcS gene, but only became active as a suppresser in C4 mesophyll on the appearance of the unique trans-acting factor(s).

There are obvious differences in the regulatory systems for rbcS between C4 dicots and monocots (Schäffner and Sheen, 1991). The rbcS promoter from dicot C3 or C4 species is not active when fused to a reporter gene and inserted into maize mesophyll protoplasts. The chimeric gene that is constructed through this process lacks the 3′ suppresser sequence that would normally inhibit expression in mesophyll cells. However, the rbcS promoter is active when inserted into dicot mesophyll cells, including those from the C4 dicot F. trinervia (Martineau et al., 1989; Schäffner and Sheen, 1991). Conversely, the rbcS promoter from monocot C3 and C4 species is only active in monocot cells, but not dicot cells (Schäffner and Sheen, 1991). In dicots, GT-box and G-box motifs upstream from the TATA box have been shown to exert strong regulation in the expression of dicot rbcS (Dean et al., 1989; Ueda et al., 1989) but are inactive in monocot cells (Schäffner and Sheen, 1991). Different sequences upstream from the TATA box appear to be active in monocot rbcS promoters, including a highly conserved GAACGGT constitutive element and unique light-sensitive elements that are absent from the dicot promoters that have been examined (though there is good reason to believe that dicot rbcS promoters contain light-sensitive elements of a different nature; Schäffner and Sheen, 1991). On the basis of these observations, it appears that monocot- and dicotspecific promoter sequences evolved prior to the evolution of C4 photosynthesis in these groups, and have been retained in the C4 systems of modern taxa.

Evolutionary modification has also occurred in the kinetic affinity and turnover capacity of the rbcL active site of Rubisco with respect to CO2 (Seemann et al., 1984). In a survey of eight monocot C4 species, three dicot C4 species, four C3 dicot species, and one C3 monocot species, it was discovered that C4 species from both groups exhibited CO2-saturated specific activities (CO2 assimilation rate per unit mass of enzyme) that were twice those of C3 species from both groups. The improvement in turnover capacity of the C4 Rubisco comes with a potential cost, however, in that the Km(CO2) for C4 species is almost double that of C3 species (Yeoh et al., 1981). Thus, affinity of the active site for CO2 has decreased because of increased turnover capacity. It is likely that this tradeoff is due to the coupled dependence of substrate/product binding on properties of the active site. Increases in the turnover capacity of an active site (the capacity to release product) is probably dependent on weakening the binding interaction between product molecules and certain active site moieties (Gutteridge et al., 1995). Such weakening may also decrease the binding affinity of substrate for the active site, resulting in a higher apparent Km. In the case of Rubisco, selection for increases in turnover number in C4 leaves probably arose as a result of its exposure to relatively high bundle-sheath CO2 concentrations, a situation that would favor faster turnover capacity while avoiding the need for tight substrate binding such as might be required at the lower CO2 concentrations characteristic of C3 leaves.

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Photosynthetic Efficiency Improvement

Rebecca A. Slattery, Donald R. Ort, in Reference Module in Life Sciences, 2020

Improved Rubisco activase

Before Rubisco can catalyze carboxylation of RuBP, the enzyme must first be activated. Activation occurs through the carbamylation of a lysine residue in the catalytic site of Rubisco, which is then stabilized by Mg2+ binding. However, sugar phosphates, such as D-xylulose-1,5-bisphosphate (XuBP) or even Rubisco's substrate RuBP, can bind to the catalytic site of the non-activated enzyme, thereby blocking activation. In addition, XuBP and other sugar phosphates, such as 2-carboxy-D-arabinitol 1-phosphate (CA1P), D-glycero-2,3-pentodiulose-1,5-bisphosphate (PDBP), 2-carboxytetritol-1,4-bisphosphate (CTBP), and 3-ketoarabinitol-1,5-bisphosphate (KABP), can bind Rubisco after activation, thereby inhibiting catalysis of the activated enzyme. Rubisco activase (Rca) is required to sustain the activated state of Rubisco by facilitating the removal of these inhibitors from its catalytic site (Wang and Portis, 1992; Portis, 2003). Although levels of Rca are not normally limiting in steady-state conditions, higher levels are sometimes associated with increased yield (Yin et al., 2014). In addition, higher levels are also beneficial for more rapid induction of photosynthesis in fluctuating light environments (Yamori et al., 2012). However, if higher levels of Rca occur at the expense of Rubisco levels, this can be detrimental to photosynthesis in steady-state conditions (Fukayama et al., 2012). Thus, fine-tuning the regulation of Rca activation may offer a solution for more rapid activation of Rubisco upon low-to-high light transitions that is predicted to significantly increase diurnal canopy carbon gain (Carmo-Silva et al., 2015). To realize this potential, the sensitivity of Rca to high temperatures (Feller et al., 1998) will necessitate greater thermotolerance to maintain photosynthetic rates in warming climates.

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Regulation of the C4 Pathway

Richard C. Leegood, Robert P. Walker, in C4 Plant Biology, 1999

A Ribulose-l,5-bisphosphate Carboxylase–Oxygenase

All the C4 acid decarboxylases release CO2 for fixation by ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) in the bundle sheath. The absence of carbonic anhydrase in bundle-sheath cells is of critical importance in ensuring that this CO2 (which is the inorganic carbon substrate for Rubisco) is not converted to bicarbonate (Furbank and Hatch, 1987). The regulation of Rubisco in C4 plants is a neglected area when compared with its C3 counterparts. The activity of Rubisco is considerably lower in C4 plants, but its specific activity is higher. Rubisco from C4 plants has a Km(CO2) (28–63 μM), which is appreciably higher than that of C3 and CAM plants (8–26 μM) but comparable to those of aquatic plants with CO2 concentrating mechanisms (Yeoh et al., 1980,1981). However, the specificity factor of Rubisco from C4 plants is little different from that of Rubisco in C3 plants. Analysis of other enzymes of the Benson–Calvin cycle in C4 plants shows that most are rather similar to their C3 counterparts, so that the fundamental features of regulation are unlikely to be different in the bundle-sheath chloroplasts of C4 plants (Ashton et al., 1990).

There are two mechanisms of regulating Rubisco in C3 plants (Portis, 1992). The first mechanism is a change in carbamylation state, catalyzed by Rubisco activase. The second is regulation by the naturally occurring inhibitor of Rubisco, carboxyarabinitol-l-P, which binds tightly to the enzyme in darkened leaves, rendering Rubisco inactive. There is evidence that most C4 plants regulate Rubisco by changes in carbamylation state between light and dark (Sage and Seemann, 1993), although there are a few exceptions (such as maize) in which such light–dark modulation of total Rubisco activity is very weak (Vu et al., 1984; Usuda, 1985; Sage and Seemann, 1993), possibly because the activity of the CO2 pump may limit photosynthesis at low light intensities (Sage and Seemann, 1993). There is little evidence for regulation of Rubisco activity by carboxyarabinitol-l-P in most C4 plants, although it may be present (Sage and Seemann, 1993). However, guinea grass, P. maximum (PEP-CK type), showed a 2.5-fold increase in Rubisco activity on illumination (Vu et al., 1984) and had a CA1P content that was 44% of Rubisco active site content (Moore et al., 1991). The parent sugar, 2′-carboxyarabinitol is also present in leaves of maize and P. maximum (Moore et al., 1992), suggesting that CA1P is metabolized in the leaves of these species. However, this mode of regulation may operate more as a light–dark switch, rather than as a means of modulating Rubisco activity at different light intensities (Sage and Seemann, 1993).

Flaveria bidentis (NADP-ME type) has been transformed to reduce the amount of Rubisco by antisense (Furbank et al., 1996; von Caemmerer et al., 1997). In ambient CO2 and saturating light, even modest reductions in Rubisco led to substantial reductions in the photosynthetic rate (see also Edwards et al., 1988), indicating substantial control of the rate of CO2 assimilation (Table I). Thus, despite the high bundle-sheath CO2 concentrations attained in C4 plants, there is only just enough Rubisco in leaves of Flaveria to support maximum rates of photosynthesis. Transformants showed a decrease in the CO2-saturated rate of photosynthesis, but no change in the initial slope of the assimilation rate versus intercellular concentration of CO2. It has been suggested that, unlike C3 plants, this initial slope is largely determined by the kinetic characteristics and activity of PEP-C (Edwards and Walker, 1983; Collatz et al., 1992) and, consistent with this, mutants of Amaranthus edulis with less PEP-C, or leaves treated with an inhibitor of PEP-C, show a decrease in the initial slope (Jenkins et al., 1989a; Dever et al., 1995). This means that reduced Benson–Calvin cycle turnover in the Rubisco transformants does not affect PEP-C activity, indicating an imbalance in the operation of mechanisms that coordinate the C3 and C4 cycles. There was no effect of a decrease in Rubisco on the photosynthetic rate at low light intensities (Furbank et al., 1996), indicating that at low light, C4 photosynthesis is limited by RuBP regeneration, as in C3 plants (von Caemmerer and Farquhar, 1981) or by the rate of regeneration of PEP, both of which would be dependent on the provision of ATP and NADPH by electron transport.

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Photosynthesis

J.R. Bowyer, R.C. Leegood, in Plant Biochemistry, 1997

(ii) Regulation of Rubisco

In the 1970s it was discovered that, in vitro, Rubisco was activated by preincubation with CO2 and Mg2+ to form an active carbamylated enzyme. This carbamylation occurs on Lys-201, and the CO2 molecule concerned is different from the CO2 molecule involved in catalysis.

E-lys + CO2 + Mg2+ → E · CO2 · Mg2+

The carbamylation state of Rubisco was also shown to change in vivo, in isolated chloroplasts or in leaves. However, carbamylation in vitro occurred only in the presence of millimolar concentrations of CO2, whereas in vivo the concentration of CO2 in the leaf cell would only be about 10 μM. How, then, could Rubisco possibly be carbamylated in vivo? The answer came from a mutant of Arabidopsis thaliana isolated in the early 1980s (Portis, 1992). This mutant was able to survive when grown in high CO2, but not in air, because it was unable to carbamylate its Rubisco. This mutation was shown to be due to the absence of an enzyme, Rubisco activase, which enhances the carbamylation of Rubisco in the presence of physiological concentrations of CO2 (Ka(CO2) =4 μM). Binding of RuBP to the active site prevents carbamylation. The activase appears to be involved in removing bound RuBP from the active site to allow carbamylation (Wang & Portis, 1992).

A second enigma concerning the regulation of Rubisco emerged in the early 1980s. It was found that in some plants, such as soybean, Rubisco in extracts from darkened leaves could not be activated by CO2 and Mg2+, whereas Rubisco could readily be activated in leaf extracts made from plants in the middle of the day (Vu et al., 1983). It was subsequently shown that this was due to the presence of a tight-binding inhibitor, 2-carboxyarabinitol-1-P (CA1P) (Kd 32 nM), which is an analog of the transition state intermediate, 3-keto-2-carboxyarabinitol bisphosphate (Fig. 2.25). Rubisco activase may also be involved in removing bound CA1P from the enzyme.

CA1P appears to be present in all plants, but it is probably only important in regulation of Rubisco when present in the large amounts found in legumes (soybean, Phaseolus vulgaris), tomato and sunflower. It is, nevertheless, present in small amounts in leaves of plants such as spinach, wheat or maize (Moore et al., 1991). CA1P is gradually degraded with increasing photon flux density (PFD) and is rapidly degraded on exposure to saturating light (Vu et al., 1983; Kobza & Seemann, 1989). At present, comparatively little is known about the mode of synthesis and degradation of this inhibitor. However, carboxyarabinitol has been found to be present in large amounts in leaves of a range of species (Moore et al., 1992), and carboxyarabinitol fed to leaves of plants highly active in CA1P metabolism is readily converted into CA1P in the dark. CA1P is then degraded to carboxyarabinitol in the light, suggesting the operation of a metabolic cycle between the two (Moore & Seemann, 1992). A CA1P phosphatase activity has been purified (Holbrook et al., 1989).

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Ribulosebisphosphate Carboxylase

Rubisco evolved in the photosynthetic bacteria billions of years ago in an atmosphere, which was richer in CO2 and depleted in O2 in comparison with the present atmosphere.

From: Encyclopedia of Biological Chemistry (Second Edition), 2013

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Chloroplast

Photosystem

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Adenosine Triphosphate

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Organellar and Metabolic Processes

Katia Wostrikoff, David B. Stern, in The Chlamydomonas Sourcebook, 2009

I. Introduction

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) catalyzes the addition of gaseous carbon dioxide to ribulose-1,5-bisphosphate (RuBP), generating two molecules of 3-phosphoglyceric acid (3-PGA), and is thus the key enzyme in CO2 assimilation. Rubsico is capable of a competing oxygenation reaction, which generates one molecule of 3-PGA and one of 2-phosphoglycolate from RuBP. In Chlamydomonas as in higher plants, the enzyme is a hexadecamer composed of eight large subunits (LS) and eight small subunits (SS), encoded by a single chloroplast gene (rbcL) and two adjacent nuclear genes (RBCS1/2), respectively. The number of the RBCS genes varies among plants but generally constitutes a small multigene family. Rubisco is mainly localized to the pyrenoid in Chlamydomonas.

Because of its high abundance and central metabolic role, Rubisco has been studied in many cellular contexts. Its high abundance necessitates a considerable diversion of amino acids and energy to its synthesis, and the many physiological stimuli that impinge upon the regulation of photosynthesis, require that Rubisco abundance and activity be controlled. Thus, Rubisco is discussed in several other chapters of this volume. The photorespiratory glycolate pathway, which is initiated through Rubisco oxygenase activity, is discussed in more detail in Chapter 8. This same chapter also discusses the carbon concentrating mechanism and how Rubisco may access its substrates under different growth conditions, particularly limiting CO2, whereas Chapter 7 discusses relevance of Rubisco to hydrogen production. Chapter 29 discusses the translational regulation of LS synthesis in response to SS limitation and oxidative stress; topics which are summarized in section VII. Rubisco is also mentioned in the context of nutrient stress and transition metal deficiency, where it is subject to repression; these are referred to in section VI. Rubisco proteolysis is discussed in Chapter 19.

While Rubisco has been studied in many organisms, and thousands of rbcL genes have been sequenced for phylogenetic purposes (Kapralov and Filatov, 2007), Chlamydomonas has been particularly useful for several reasons. First, the rbcL gene can be readily manipulated by chloroplast transformation, leading to facile structure-function analyses. Second, the two RBCS genes are tightly linked and mutants exist with a deletion covering both. This is thus the only eukaryote for which there are mutants that stably and totally lack RBCS expression (Khrebtukova and Spreitzer, 1996; Dent et al., 2005). Third, culture conditions can be changed rapidly and uniformly. This allows a relatively uniform population to be examined for Rubisco regulation under a variety of stress conditions. This chapter focuses on studies in Chlamydomonas, while other aspects of Rubisco have been reviewed elsewhere. These include its potential to increase CO2 storage in trees and diatoms (Millard et al., 2007; Roberts et al., 2007), choice of model system for studying Rubisco compartmentalization in C4 plants (Brown et al., 2005), and a series of reviews on Rubisco activity, assembly, and manipulation (Chollet and Spreitzer, 2003).

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Photosynthetic Carbon Dioxide Fixation

Thomas D. Sharkey, in Reference Module in Life Sciences, 2020

Activation/carbamylation

Rubisco must undergo post-translational modification in order to be active. A specific lysine in the active site is carboxylated, forming a carbamate, after which a metal ion, normally Mg2+, is bound to create the active site for carboxylation (Lorimer et al., 1976). Uncarbamylated rubisco will bind RuBP very tightly but this RuBP cannot undergo catalysis. This makes RuBP a dead-end inhibitor for uncarbamylated rubisco (Fig. 7). Under normal conditions carbamate formation is faster than the binding of RuBP but because RuBP binds so tightly to uncarbamylated rubisco, eventually nearly all rubisco would be bound to RuBP and so be inactive. This situation is reversed by an enzyme called rubisco activase that removes RuBP (and other molecules) from uncarbamylated rubisco (Bhat et al., 2017; Salvucci et al., 1985; Mueller-Cajar, 2017). Because carbamylation is faster, most rubisco can be carbamylated and active as long as rubisco activase continually removes RuBP from uncarbamylated rubisco. On the other hand, when rubisco capacity exceeds the capacity of other processes in photosynthesis, it may be advantageous to let the rubisco capacity decline to keep it in line with the rest of the photosynthetic reactions. Rubisco is found to be deactivated in low light, when starch and sucrose synthesis limits photosynthesis, and at high temperature, when photorespiration rates may be deleteriously high.

Fig. 7. Activation of rubisco by formation of a carbamate. Two protons are lost making carbamate formation much faster at high pH (Panel A). Panel B shows carboxylation resulting in carbamate formation (EC) of free enzyme by addition of CO2 is faster (thicker arrow) than binding RuBP (thinner arrow). The dead-end enzyme-plus-RuBP (ER) would accumulate if not for the action of rubisco activase (Rca, a AAA+ enzyme) that uses ATP to modify protein structure, presumably allowing the release of RuBP. EC can bind Mg2+ to become ECM and then bind RuBP to become ECMR, the catalytic form. Note that the CO2 used for carbamylation is different from the CO2 that is added to RuBP. The curved lines denote the catalytic cycle in which RuBP is converted to two PGAs by addition of CO2. The ECM complex can stay intact through many catalytic cycles.

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Photosynthetic Efficiency Improvement

Rebecca A. Slattery, Donald R. Ort, in Reference Module in Life Sciences, 2020

Better Rubisco

Rubisco catalyzes the carboxylation of the 5-carbon sugar RuBP to ultimately yield the net production of two molecules of glycerate 3-phosphate (G3P), which leaves use to produce carbohydrates that are the building blocks for the vast array of molecules and compounds that plants make. As the most abundant protein in the biosphere (Bar-On and Milo, 2019), the inefficiency of Rubisco represents a major limitation to photosynthesis. Not only is the enzyme catalytically slow, it also has a relatively low specificity for CO2 versus O2, likely a consequence of having evolved when atmospheric CO2 levels were high and O2 levels were low or absent. This presages the oxygenation of RuBP, which then requires the energetically costly photorespiratory pathway to recycle the inhibitory byproducts (see below). While there is genetic variability in Rubisco specificity there is seemingly an inherent tradeoff between Rubisco catalytic rate and specificity. Nevertheless, genetic diversity in some of the world's major crops and their wild relatives may provide new materials for improving Rubisco kinetics and specificity (Orr et al., 2016), which will be even more important as temperatures rise and the specificity for CO2 over O2 in solution declines and the solubility of CO2 declines to a greater degree than the solubility of O2. However, there has so far been only limited success in transplanting foreign Rubisco into different species to take advantage of this genetic diversity. But the expression of Rubisco genes in the chloroplast genome as well as co-transformation with Rubisco chaperone proteins holds promise (Bracher et al., 2017; Conlan et al., 2019), as does the long-sought after complete expression and assembly of functional higher plant Rubisco in E. coli (Aigner et al., 2017). Efforts to re-engineer Rubisco for improved kinetic properties have to date been unsuccessful even with the guidance of a high-resolution atomic structure of the Rubisco enzyme complex.

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Photosynthesis

R.C. Leegood, in Encyclopedia of Biological Chemistry (Second Edition), 2013

Photorespiration

Rubisco is an inefficient enzyme. It has a slow catalytic turnover rate, and about half the soluble protein in leaves is Rubisco, making it the most abundant protein in nature:

RuBP+O2RuBP oxygenase⟶glycerate-3-P+glycollate-2-P

Rubisco also catalyzes a side reaction with oxygen, an inevitable consequence of its reaction mechanism. Rubisco evolved in the photosynthetic bacteria billions of years ago in an atmosphere, which was richer in CO2 and depleted in O2 in comparison with the present atmosphere. The ratio of oxygenation to carboxylation by Rubisco depends upon the relative concentrations of CO2 and O2 and oxygenation increases as the temperature increases. Photorespiration is the process by which two molecules of glycollate-2-P, which is not a metabolite of the Benson–Calvin cycle, are retrieved and converted into two molecules of an amino acid, glycine (C2), and then into one molecule of glycerate-3-P (C3). This involves shuttling of metabolites among the chloroplasts, cytosol, peroxisomes, and mitochondria. During this process, one-quarter of the carbon in glycollate-2-P is lost as CO2 and ammonia are liberated. Photorespiration is therefore a wasteful process because it both reduces carbon gain and dissipates photosynthetic energy (because CO2 is first fixed and released again). There is, therefore, a selection pressure on plants to reduce the rate of photorespiration by means of CO2-concentrating mechanisms, so as to improve their carbon economy. This is particularly strong when high rates of photorespiration are favored, as at high temperatures. However, a future doubling of ambient CO2 will reduce photorespiration by about 50%, and it would be completely eliminated by a fivefold rise in atmospheric CO2.

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Exorphins

Masaaki Yoshikawa, in Handbook of Biologically Active Peptides (Second Edition), 2013

Rubiscolins

Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is a key enzyme in photosynthesis catalyzing corbondioxide fixation. Rubisco is ubiquitus for photosynthetic organisms and is regarded as the most abundant protein on earth., From a nutritional point of view, the large subunit of Rubisco has an exceptionally ideal composition of essential amino acids among plant proteins. Therefore, plant Rubisco is expected to be a large source of food protein in the future. We found the Tyr-Pro-Leu sequence, which satisfies the requirements for opioid activity as described above, in the conserved region in the large subunit of plant Rubisco. We then synthesized the corresponding sequences, Tyr-Pro-Leu-Asp-Leu-Phe and Tyr-Pro-Leu-Asp-Leu. Both peptides were active in the MVD assay; IC50 values were 24.4 and 50 µM, respectively.26 We named them Rubiscolins-6 and -5, respectively. Their opioid activities were very weak in the GPI and µ receptor binding assays. Results from the receptor binding assay also suggested that rubiscolins are δ-selective opioid peptides.

We examined the conditions for the enzymatic release of rubiscolins from spinach Rubisco by using a model peptide and partially purified protein. Rubiscolins-6 and -5 were released by the action of pepsin, pancreatic elastase, and leucine aminopeptidase. The yield of rubiscolin-5 was higher than that of rubiscolin-6.

In certain algae species, the residue corresponding to Leu3 of rubiscolin-6 is replaced by Ile. Tyr-Pro-Ile-Asp-Leu-Phe is about 4 times more active than Tyr-Pro-Leu-Asp-Leu-Phe in the MVD assay.28 The Asp residue in Rubiscolin is essential for δ-opioid activity; it could not be replaced with Glu. Replacement of Leu3 or Ile3 with Phe or Trp also results in a reduction of δ-opioid activity. The rubiscolin sequence has been present in Rubisco far longer than the appearance of δ-receptors in animals. The sequence might be essential for enzymatic activity. It may be attractive to speculate that the δ-receptor in animals has evolved to fit the rubiscolin sequence in Rubisco. However, this possibility might be remote. We think that the rubiscolin sequence might have affinity for the δ-receptor in animals just by chance. This might be the case for most plant-derived peptide sequences acting on animal receptors. In this sense, these bioactive peptides could be regarded as those obtained from a sort of random library.

Rubiscolin-6 showed antinociceptive activity in mice after oral administration at a dose of 300 mg/kg. Rubiscolin-6 stimulated acquisition of memory after oral administration at doses of 100/mgkg in a passive avoidance experiment.27 In the elevated-plus maze test in mice, rubisolin-6 showed anxiolytic activity after oral administration at a dose of 30 mg/kg. Thus, rubiscolin-6 is about 3 times more potent than gluten exorphin-A5 in vivo.

Both µ and δ opioids have been reported to inhibit memory consolidation, while κ opioid stimulated it. It is an interesting observation that food-derived δ opioid such as gluten exorphin and rubiscolin stimulate memory consolidation after oral administration.

Rubiscolin-6 stimulated food intake on a normal diet after oral administration in mice at a dose of 0.3 mg/kg in a δ-receptor-dependent manner, followed by activation of prostaglandin D2 and neuropeptide Y systems (see Kanako et al., 2012, Mol Nutr Food Res). However, it suppressed food intake on a high fat diet via a different mechanism. Recently, rubiscolin-6 was reported to suppress the escape reaction of the American cockroach, which might suggest the protective role of the peptide for plants against insects.9

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C4 Plants

Rowan F. Sage, Tammy L. Sage, in Encyclopedia of Biodiversity (Second Edition), 2013

Diffusion Barriers Preventing CO2 Loss

Compartmentalization of Rubisco and the decarboxylating enzymes in an inner compartment contributes to CO2 concentration in two ways. First, the compartmentalization localizes Rubisco to the immediate vicinity of the deacarboxylation reaction. Second, it serves to trap the CO2 near Rubisco before it can escape back into the mesophyll and intercellular air spaces. This trapping ability requires the presence of a diffusion barrier to slow CO2 efflux. In numerous grasses, the diffusion barrier is a thick BS wall impregnated with the waxy compound suberin. In other grasses and the dicot C4 lineages, chloroplasts and the decarboxylating enzymes are located on the centripetal (inner) end of the BS cells, near the vascular tissue, and a large vacuole separates them from the outer edge of the BS cell. CO2 diffuses 10,000 times slower through water than air, so a large vacuole can serve as an effective diffusion barrier.

In the terrestrial single-celled C4 species, a large vacuole between the site of decarboxylation and the intercellular air space slows CO2 efflux (Figure 4).

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Photosynthesis

Darrell Fleischman, in Cell Physiology Source Book (Fourth Edition), 2012

IVB Photorespiration and C4 Plants

Not only does ribulose 1,5-bisphosphate carboxylase/oxygenase have a low affinity for CO2, it also catalyzes a competing reaction in which O2 rather than CO2, is added to ribulose-1,5-bisphosphate. The products are 3-phosphoglycerate and phosphoglycolate.

(51.5)Ribulose 1,5-bisphosphate+O2→ribulosebis-phosphate carboxylase/oxygenase3-phospoglycerate+phosphoglycolate

This reaction seems to serve no useful purpose. Many plants grow much faster in a CO2-enriched atmosphere in which the carboxylation reaction can compete more effectively with the oxygenation reaction. It appears that in the 2 billion years since plants began to fill the atmosphere with oxygen while removing CO2 from it, they have been unable to modify the enzyme so that its affinity for CO2 is increased or its affinity for O2 is decreased significantly. Molecular biologists are now trying to accomplish that task.

Part of the carbon appearing in phosphoglycolate is rescued. In a series of reactions occurring in peroxisomes and in mitochondria, two molecules of phosphoglycolate are converted to one molecule of glycerate, which is returned to the chloroplast and re-phosphorylated. But one carbon atom is lost as CO2 and ATP and O2 are consumed. This process is known as photorespiration. It is not coupled to ATP formation and the net result is waste of ATP and fixed carbon.

A number of plants, including corn, sugarcane and crabgrass, partially avoid photorespiration by concentrating CO2 in the cells that contain the Calvin cycle enzymes, so that carboxylation can compete more effectively with oxygenation. In these plants, the Calvin cycle enzymes are located in the chloroplasts of the bundle sheath cells, which surround the vascular bundles deep within the leaves (see Fig. 51.5). CO2 is first captured in the mesophyll cells, which lie near the leaf surface, by carboxylation of phospho-enolpyruvate (PEP):

(51.6)PEP+CO2undefined→PEP carboxylaseoxaloacetate+Pi

The carboxylation is catalyzed by PEP carboxylase, an enzyme that has a high affinity for CO2 and does not catalyze an oxygenation reaction. Oxaloacetate is next reduced to malate (or transaminated to form aspartate in some plants). The malate or aspartate is transferred to the bundle sheath cells through fibers known as plasmodesmata. Some of these can be seen at the arrows in Fig. 51.5. Malate is oxidatively decarboxylated to pyruvate in the bundle sheath cells in a reaction that also generates NADPH. The CO2 that is released enters the Calvin cycle in the bundle sheath cells (Fig. 51.8). Pyruvate returns to the mesophyll cells where it is again transformed to PEP. The net result is the transfer of CO2 and NADPH to the bundle sheath cells.

FIGURE 51.8. The mechanism used by C4 plants to concentrate CO2 and NADPH in bundle sheath cells. CO2 is captured in the mesophyll cells by carboxylation of phosphoenolpyruvate. Malate is transferred to the bundle sheath cells where CO2 is released and NADPH is formed to enter the Calvin cycle. Pyruvate returns to the mesophyll cells.

The CO2-concentrating mechanism consumes energy, since ATP is converted to adenosine monophosphate (AMP) and phosphate. Nevertheless, plants that use it are often referred to as efficient plants because avoidance of photorespiration more than compensates for this expense. Such plants are usually known as C4 plants because of the involvement of four-carbon acids. Many C4 plants are native to tropical areas, where the C4 pathway is especially advantageous since high temperatures and bright sunlight encourage photorespiration.

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Photosynthetic Carbon Dioxide Fixation

M.J. Paul, in Encyclopedia of Biological Chemistry (Second Edition), 2013

Carboxylation Phase

Carboxylation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (Rubisco), which can constitute up to 50% of the soluble protein in a leaf and is probably the Earth's most abundant protein. The combination of CO2 with RuBP, a five-carbon compound, yields two molecules of the three-carbon compound 3-PGA. Many enzymes will bind to molecules in addition to the ones they have primary affinity for, if they are present at high enough concentrations. Present-day atmospheric O2 concentration is 1000 times higher than that of CO2, and oxygenation of RuBP also occurs. This produces one molecule of 3-PGA and one molecule of phosphoglycolate (glycolate 2-P). The formation of phosphoglycolate represents carbon lost to the Calvin cycle and a series of reactions serve to return this carbon by converting phosphoglycolate to 3-PGA in a process termed as 'photorespiration' because CO2 is evolved (Figure 1). These reactions involve the peroxisome where glycolate is converted into glycine, and mitochondria where glycine is converted into serine where CO2 and ammonia are evolved. Serine then returns to the peroxisome for conversion to 3-PGA and exports back to the chloroplast. Ammonia is reassimilated through a reaction with glutamate and ATP to form glutamine catalyzed by glutamine synthetase in chloroplasts. Glutamate synthetase then catalyzes the formation of glutamate from glutamine and α-ketoglutarate. Oxygenation of Rubisco and the retrieval of carbon from phosphoglycolate are wasteful of carbon and ATP and are also costly in terms of investment in the catalytic machinery required. Early evolution of Rubisco occurred when there was no oxygen in the atmosphere. When oxygen appeared more than a billion years later, the complexity of the Rubisco protein may have made it too difficult to eliminate oxygenase activity linked to phosphoglycolate production.

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Solar-Driven Hydrogen Production in Green Algae

Steven J. Burgess, ... Peter J. Nixon, in Advances in Applied Microbiology, 2011

5 Rubisco mutants

Mutant CC-2803, which lacks Rubisco, a key enzyme of the Calvin cycle involved in the fixation of CO2, has a light-sensitive phenotype and a dramatically reduced rate of photosynthesis. Consequently, cultures go anaerobic and produce H2 in sealed containers even in the presence of sulfur (Hemschemeier et al., 2008a).

Control of the Calvin cycle or Rubisco activity therefore represents a potentially novel method of inducing H2 production (Marín-Navarro et al., 2010) by removing the major sink of electrons for reduced ferredoxin generated by the light reactions. This could be achieved through inducible control of Rubisco or Calvin cycle enzyme expression or control of CO2 supply to carbon-concentrating mutants (Spalding, 2008).

Rubisco may also make an interesting target to decrease the specificity of carboxylation to oxygenation reactions as has previously been demonstrated (Chen et al., 1988; Genkov et al., 2006; Satagopan and Spreitzer, 2004), which would result in a higher respiratory rate as well as reduced flux through the Calvin cycle.

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Organellar and Metabolic Processes

William Zerges, Charles Hauser, in The Chlamydomonas Sourcebook, 2009

4. rbcL translational repression during high light stress

Translation of the rbcL mRNA, which encodes the Rubisco large subunit, is repressed during high light stress. This has been linked to an oxidative stress response, because it was induced under low light when ROS production was photosensitized by methyl viologen, and it was prevented by the antioxidant ascorbate (Shapira et al., 1997; Irihimovitch and Shapira, 2000). Glutathione appears to be the redox sensor that mediates this response. Repression of rbcL translation is exerted, at least in part, during the entry to, or early in the elongation phase because the mRNA shifts from large to small polysomes and monosomes during high light stress (Cohen et al., 2005).

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Terms and conditions

Ribulose-1,5-bisphosphate

RuBisCO derived from spinach (Spinacia oleracea) and alfalfa (Medigaco sativa) have particularly been used to produce bioactive peptides and hydrolysates.

From: Trends in Food Science & Technology, 2017

Related terms:

Chloroplast

Photorespiration

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Plant Physiology and Development

A.S. Raghavendra, ... R.B. Bapatla, in Encyclopedia of Applied Plant Sciences (Second Edition), 2017

Rubisco

Rubisco is a unique and interesting enzyme, mediating the first and key reaction of photosynthetic CO2 assimilation: conversion of one molecule of RuBP and one of CO2 into two molecules of PGA. Besides the carboxylation reaction, Rubisco reacts with oxygen to form one molecule of 2-phosphoglycolate and one of PGA. The plants recover part of the carbon diverted into phosphoglycolate through the process of photorespiration and lose part of carbon as CO2. Photorespiration also helps in the recycling of nitrogen and dissipation of excess energy and reductants (ATP and NADPH) in the chloroplasts. A major drawback of Rubisco is its very slow catalytic capacity. The turnover number (Kcat) of the enzyme is in the range of 3–10 mol s−1, while most of the enzymes have >1000 mol s−1 (for e.g., carbonic anhydrase can be as high as 500 000 mol s−1). As a consequence large amounts of Rubisco (up to 50% of the total soluble protein) are present in chloroplasts to ensure the carbon flux.

The activity of Rubisco is highly regulated. Rubisco is inactive in the dark and is converted to an active form on illumination, which catalyzes fixation of CO2. Activation of Rubisco is the result of carbamylation, which involves the binding of CO2 and Mg2+ to a lysine residue near the catalytic site (Figure 2). Rubisco is active only when lysine-201 reacts with CO2 near the catalytic site to form a carbamate and allows the binding of the Mg2+. Carbamylation changes the conformation of the large subunit and activates the enzyme, while the active conformation is stabilized by the formation of a complex with Mg2+. Carbamylation is essential for Rubisco activation, as the noncarbamylated Rubisco binds RuBP too tightly to allow catalysis.

Figure 2. The reactions and regulation of Rubisco. The inactive Rubisco (E) reacts with CO2 and forms the carbamate, gets stabilized by Mg2+, and becomes active (E.CO2.Mg2+). The active enzyme functions as both carboxylase and oxygenase. The product of the carboxylase reaction, PGA, is metabolized in the Calvin cycle. One of the products of oxygenase, P-glycolate, is metabolized through photorespiration. However, analogues of RuBP in the stroma, can bind to the Rubisco, blocking the active site on the enzyme. For example, 2-carboxy-D-arabinitol 1-phosphate (CA1P) binds tightly to Rubisco and makes it inactive. Thus, Rubisco is activated in light in two process: (1) changes in the stromal microenvironment (increase in pH, Mg2+ levels, and Calvin cycle metabolites); and (2) by Rubisco activase, which itself is activated in light through ATP.

Another protein, Rubisco activase, is also involved in mediating the light activation of Rubisco. On illumination, Rubisco activase releases the inhibitor compounds, such as 2-carboxyarabinitol 1-phosphate (CA1P), which are bound to the active site of Rubisco; otherwise, for example, in darkness, these inhibitors prevent activation (carbamylation) of the enzyme. Rubisco activase itself is activated in light by utilizing ATP produced from photosynthetic electron transport. Rubisco activase and Rubisco activation provide another mechanism of strong regulation by light of carbon assimilating reactions of photosynthesis.

CA1P, which occurs naturally in the leaves of several plants, is a strong inhibitor of Rubisco. The affinity of Rubisco for CA1P is much stronger than that for RuBP, the substrate. As a result, CA1P, which accumulates in leaves during the night, inactivates Rubisco by blocking the binding sites. During the day (or on illumination), the bound CA1P is released from Rubisco, and this process is further accelerated by Rubisco activase. However, the physiological role of CA1P is still debated, as it is not found in all plant species.

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Plant Physiology and Development

M. Pribil, D. Leister, in Encyclopedia of Applied Plant Sciences (Second Edition), 2017

CO2 Fixation (CB) and Photorespiration

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme involved in photosynthetic carbon fixation, as it catalyzes the conversion of atmospheric CO2 into organic compounds. In the first step two molecules of 3-phosphoglycerate (3-PGA) are produced, one of which is required for the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate to which the enzyme attaches the CO2. In every third reaction cycle one molecule of 3-PGA is diverted to sustain the biosynthesis of sugars and other high-energy compounds (Figure 1). Under CO2 limitation and saturating light conditions, the CO2 fixation step by RuBisCO is regarded as the rate-limiting step in the photosynthetic process. Moreover, RuBisCO displays a significant affinity for molecular oxygen and also catalyzes an oxygenation reaction that further diminishes the overall efficiency of photosynthesis. At current atmospheric CO2 concentrations and temperatures (25 °C), about one-third of the carbohydrate formed during C3 photosynthesis is consumed by this photorespiration reaction. In this energetically wasteful process RuBisCO reacts with molecular oxygen to form one molecule of 3-PGA and one of 2-phosphoglycolate (PG). The latter acts as an inhibitor of CB enzymes and must be converted back into 3-PGA in an energy-intensive conversion reaction (photorespiration) that requires shuttling between three additional cell compartments/organelles – the cytosol, peroxisomes, and mitochondria – and produces CO2. Furthermore, the extent of loss of fixed CO2 gradually increases with a rise in ambient temperature.

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Light, Hormones, and Cell Signaling Pathways

Donald E. Fosket, in Plant Growth and Development, 1994

Regulation of RUBISCO gene expression is complex

RUBISCO is a very important photosynthetic enzyme. It is the first enzyme in the Calvin cycle. It is a large multimeric protein composed of two different subunit polypeptides, known simply as the large subunit (LSU) and the small subunits (SSUs). The functional RUBISCO protein consists of eight copies each of these two different polypeptides. The gene encoding the LSU, designated rbcL, is in the chloroplast genome, and the genes encoding the small subunits, rbcS, are present in the nuclear genomes of angiosperms as small multigene families containing 4 to 13 members.

The synthesis of the functional RUBISCO protein is regulated at several levels. There is a single rbcL gene in the chloroplast genome, but each leaf mesophyll cell will have several hundred copies of the rbcL gene, as there are several hundred chloroplasts per cell. In contrast, there are up to a dozen rbcS genes in the nucleus, but most of the SSUs in a particular tissue will be derived from two or three of these. In Petunia, rbcS is encoded by six genes, but they do not exhibit coordinate expression. One rbcS gene accounts for 47% of all the transcript in the leaf, whereas the other genes are expressed at levels that range from 2 to 23% of the total. Despite these differences, the two subunits are accumulated in approximately equal amounts in the chloroplast. Expression of the rbcS genes is regulated by light, tissue and cell type differences, and development. In contrast, the rbcL genes probably are expressed constitutively, although the LSU does not accumulate unless the SSU peptide is present so that the complete RUBISCO protein complex is formed. Neither subunit is stable unless it is part of a RUBISCO molecule.

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PHOTOSYNTHESIS AND PARTITIONING | C3 Plants

A.S. Raghavendra, in Encyclopedia of Applied Plant Sciences, 2003

Regulation of Rubisco

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Why C4 Photosynthesis?

Rowan F. Sage, in C4 Plant Biology, 1999

II The Problem with Rubisco

(adapted from Kasting, 1987, and Berner and Canfield, 1989)

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The Biochemistry of C4 Photosynthesis

Ryuzi Kanai, Gerald E. Edwards, in C4 Plant Biology, 1999

6 RuBP Carboxylase-Oxygenase

Ribulose-1,5-bisphosphate carboxylase—oxygenase (Rubisco, EC 4.1.1.39) of C4 plants, like that of C3 plants, consists

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Photosynthesis and respiration

Wataru Yamori, in Plant Factory (Second Edition), 2020

12.5 Photorespiration

RuBisCO facilitates CO2 fixation (carboxylation) in the Calvin–Benson cycle, but at the same time, it also fixes O2 (oxygenation). The latter reaction is the first step of photorespiration. Photorespiration involves the movement of metabolites among chloroplasts, peroxisomes, and mitochondria in a metabolic pathway that fixes O2, releases CO2, and consumes ATP. Therefore, it reduces photosynthetic efficiency (Sharkey, 1988). Respiration and photorespiration both release CO2 in the mitochondria. However, respiratory and photorespiratory processes are well separated with respect to both carbon intermediates and compartmentation.

Similar to photosynthesis, photorespiration is a dynamic process, the rate of which can be modulated by light intensity, internal CO2/O2 concentrations, or leaf temperature (Jordan and Ogren, 1984). Generally, the photorespiratory rate increases under strong light, high temperature, and low humidity when stomata close and the CO2 concentration in the leaf decreases. It has been proposed that photorespiration could prevent over-reduction of the chloroplast stroma under conditions of high light and low CO2 when stomata are closed at midday to prevent water loss (Takahashi and Badger, 2011).

Photorespiration is inherently difficult to measure, although its rate is substantially higher than that of mitochondrial respiration. Net photosynthesis is estimated by the difference between gross photosynthesis and the sum of photorespiration and dark respiration in the mitochondria: net photosynthesis = gross photosynthesis − (photorespiration + dark respiration). The rate of photorespiration is about 25% of the photosynthetic rates in moderate nonstressed conditions, but under stress conditions, it could be 50% or more of the photosynthetic rate. Due to the substantial reductions in photosynthetic efficiency by photorespiration and the fact that increases in CO2 concentration reduce photorespiration, increasing CO2 concentration can enhance plant growth both by its effect as a substrate for photosynthesis and by its effect on suppressing photorespiration (Ainsworth and Long, 2005), if other environmental factors are not limiting.

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Rising Atmospheric Carbon Dioxide and Plant Responses: Current and Future Consequences

Amit Kumar Mishra, ... Madhoolika Agrawal, in Climate Change and Agricultural Ecosystems, 2019

11.3.3.4 Effect of CO2 on RuBisCO

In C3 plants, RuBisCO is a rate-limiting enzyme used in the process of photosynthesis and is comprised of about 56% of all soluble protein and 26% of total leaf nitrogen (N) (Makino and Osmond, 1991). According to Mae et al. (1983), the amount of RuBisCO in the leaves is the outcome of the equilibrium between its production and degradation. In plant cells, RuBisCO synthesis is regulated by transcriptional, post-transcriptional, and translational processes (Moore et al., 1999; Stitt and Krapp, 1999). RuBisCO is promptly generated during the development of leaves followed by a progressive degradation (Suzuki et al., 2001). The synthesis of RuBisCO and its degradation are influenced by environmental factors, such as light intensity, soil nitrogen, CO2, and O3. Under EC, changes in leaf N status are strongly associated to a diminution in RuBisCO content and photosynthetic acclimation. Under elevated CO2, Makino et al. (2000) reported a loss of 30% in RuBisCO before it begins to confine photosynthesis. The suppression of RuBisCO synthesis takes place when an imbalance occurs between supply and utilization of carbohydrates under EC (Moore et al., 1998, 1999). Approximately 80%–90% of RuBisCO is formed just before the full expansion of leaf blades in cereal crops (Suzuki et al., 2001; Feller et al., 2008; Seneweera and Norton, 2011). Similarly, upregulation in rbcS and rbcL mRNA occurs during leaf expansion and reaches a maximum a few days before full expansion, while little RuBisCO is synthesized after full expansion.

According to Ludewig and Sonnewald (2000), the downregulation of photosynthetic genes under EC is evident only in senescing leaves and no association was noticed between gene transcripts and soluble sugars. In monocots, the degradation of RuBisCO is constantly preponderant after complete expansion of the leaf blade leading to a prompt decrease in RuBisCO content. This might be a modifying recovery mechanism in relation to nutrient remobilization for the development of sinks as RuBisCO characterizes a significant store of N as well as a role in its metabolism. However, the mechanism by which EC speeds up degradation of RuBisCO is not well understood. Under elevated CO2, the activities of antioxidative enzymes like superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX) are lower (Pritchard et al., 2000; Vurro et al., 2009). These enzymes are well-known to combat highly reactive oxygen species (ROS). Under elevated CO2, a decrease in the activities of antioxidative enzymes may lead to an upregulation in ROS levels in the chloroplast, which could possibly contribute to the degradation of RuBisCO.

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Agricultural and Related Biotechnologies

H. Ashida, A. Yokota, in Comprehensive Biotechnology (Second Edition), 2011

4.13.2.1 Effects of Enzymatic Properties of RuBisCO on Leaf Photosynthesis

Figure 1. Variations in relative specificity of RuBisCOs.

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