Effects of growth and measurement light intensities on temperature dependence of CO2 assimilation rate in tobacco leaves

Effects of growth light intensity on the temperature dependence of CO₂ assimilation rate were studied in tobacco (Nicotiana tabacum) because growth light intensity alters nitrogen allocation between photosynthetic components. Leaf nitrogen, ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) contents increased with increasing growth light intensity, but the cyt f/Rubisco ratio was unaltered. Mesophyll conductance to CO₂ diffusion (g_m) measured with carbon isotope discrimination increased with growth light intensity but not with measuring light intensity. The responses of CO₂ assimilation rate to chloroplast CO₂ concentration (C_c) at different light intensities and temperatures were used to estimate the maximum carboxylation rate of Rubisco (V_cmax) and the chloroplast electron transport rate (J). Maximum electron transport rates were linearly related to cyt f content at any given temperature (e.g. 115 and 179 µmol electrons mol^?1 cyt f s^?1 at 25 and 40 °C, respectively). The chloroplast CO₂ concentration (C_trans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurred increased with leaf temperature and was independent of growth light intensity, consistent with the constant ratio of cyt f/Rubisco. In tobacco, CO₂ assimilation rate at 380 µmol mol^?1 CO₂ concentration and high light was limited by RuBP carboxylation above 32 °C and by RuBP regeneration below 32 °C.     

Producing fast and active Rubisco in tobacco to enhance photosynthesis

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L₈S₈ hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO₂. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Introducing a proteobacterial Rubisco with a greater carboxylation rate and a higher content of active sites into tobacco chloroplasts supports photosynthesis and growth at high CO2 concentrations.

IN A NUTSHELL Background: Rubisco is the key enzyme responsible for fixing CO₂. However, due to its intrinsically low catalytic turnover rate, Rubisco represents the ultimate rate-limiting step in plant photosynthesis. Improving Rubisco carboxylation and assembly in plants has been a long-standing challenge in crop engineering to meet the pressing need for increased global food production. There is mounting interest in replacing endogenous plant Rubisco with active non-native Rubisco candidates from other organisms to enhance photosynthetic carbon fixation. Question: The folding and assembly of Rubisco in chloroplasts are intricate processes that usually require a series of ancillary factors. Seeking a new Rubisco variant that can be produced in chloroplasts with a high yield and high catalytic performance, without the requirement for cognate assembly factors and activases, could help improve carbon fixation in crop plants. Finding: In this work, we introduced a Rubisco from a proteobacterium into tobacco chloroplasts to replace native tobacco Rubisco. In the proteobacteria, Rubisco is naturally encapsulated at a high density within a CO₂-fixing protein organelle, the carboxysome. The foreign Rubisco derived from bacteria formed efficiently and was functional in chloroplasts without the need for exogenous chaperones. Intriguingly, the chloroplast-expressed bacterial Rubisco supported the autotrophic growth of transgenic plants at a similar rate to wild-type plants at 1% CO₂. Next Step: The successful production of functional bacterial Rubisco represents a step toward installing faster, highly active Rubisco, functional carboxysomes, and eventually active CO₂ concentration mechanisms into chloroplasts to improve Rubisco carboxylation, with the intent of enhancing crop photosynthesis and crop yield on a global scale.     

Carbon dioxide fixation in the light and in the dark by isolated spinach chloroplasts

Factors affecting CO₂ fixation in the spinach (Spinacia oleracea) chloroplast were investigated. Free magnesium ions are shown to be highly inhibitory for photosynthetic CO₂ fixation in isolated intact spinach chloroplasts. The pH optimum for CO₂ fixation is about 8.5 but is dependent upon the reaction medium. Conditions are defined under which chloroplasts illuminated in the absence of CO₂ accumulate ribulose 1,5-diphosphate, and fix CO₂ in a subsequent dark period when high magnesium ion concentrations are provided. The regulation of photosynthetic CO₂ assimilation by these factors is discussed.     

Rising atmospheric CO2 concentration inhibits nitrate assimilation in shoots but enhances it in roots of C3 plants

We have proposed that rising atmospheric CO₂ concentrations inhibit malate production in chloroplasts and thus impede assimilation of nitrate into protein in shoots of C₃ plants, a phenomenon that will strongly influence primary productivity and food security under the environmental conditions anticipated during the next few decades. Although hundreds of studies support this proposal, several publications in 2018 and 2019 purport to present counterevidence. The following study evaluates these publications as well as presents new data that elevated CO₂ enhances root nitrate assimilation in wheat and Arabidopsis while it inhibits shoot nitrate assimilation.     

In silico,in vitro and in vivo approach in understanding the functional relationship between ergosterol and Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is one of the key enzymes involved in assimilation of CO₂ in chloroplasts. Phylloplane microfungi and their metabolites have been reported to affect the physiology of host plants, particularly, their photosynthesis. However, information is lacking on the effect of these microflora on the physiology of chloroplasts. The current study emphasized the impact of two dominant phylloplane fungi, Aspergillus niger and Fusarium oxysporum, on activity of Rubisco in tomato chloroplasts. Ergosterol, which is a component of only fungal cell membranes and is not synthesized by plants, have been demonstrated to elicit activity of Rubisco. In the present study, it was demonstrated through in silico, in vitro, and in vivo approaches. Results demonstrated that the fungal metabolites, which contained ergosterol, could double Rubisco activity. Maximum carboxylation rate of Rubisco increased also in ergosterol-treated plants. Michaelis-Menten constant of Rubisco was also slightly affected. Ergosterol was found also to influence and enhance the binding of CO₂ and ribulose-1,5-bisphosphate to Rubisco. Therefore we can postulate that the physiology of the chloroplast is probably influenced by phylloplane microfungi.     

Prolonged Exposure of Tobacco to a Low Oxygen Atmosphere to Suppress Photorespiration Decreases Net Photosynthesis and Results in Changes in Plant Morphology and Chloroplast Structure

Air-grown tobacco (Nicotiana tabacum L.) plants were transferred for one week into a low oxygen atmosphere (2 kPa O₂, LO) to study both immediate and long-term effects of the suppression of photorespiration on net photosynthetic rate (P_N), plant morphology, and chloroplast ultrastructure. The P_N and the leaf conductance for CO₂ increased upon exposure of attached tobacco leaves to LO. These results may suggest that under LO, external CO₂ is used to consume the radiant energy normally utilized in photorespiration by net CO₂ assimilation at the expense of an increased rate of transpiration. The increase in the coefficient of nonphotochemical fluorescence quenching indicates that under LO, (surplus) radiant energy is also dissipated as heat. Prolonged LO-treatment of tobacco resulted in a decrease in the P_N (measured in air) and in a reduction in the number of starch grains in the chloroplasts. Concomitantly, large lipid globuli appeared in the chloroplasts and the distance between the thylakoids forming the grana decreased. These changes in the ultrastructure of chloroplasts may have contributed to the decline in the P_N. The LO-treated plants were considerably smaller than the control plants maintained in air. This appears to have resulted from a reduction in the rate of leaf area expansion at the expense of an increase in the specific mass of the leaves. This long-term response to LO-treatment may allow the plants to conserve water. This revised version was published online in September 2006 with corrections to the Cover Date.     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO₂ assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO₂, and 25°C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO₂ partial pressures (p_i). In control leaves, Rubisco activity only limited the rate of CO₂ assimilation below a p_i of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p_i. The unchanged conductance and lower CO₂ assimilation resulted in a higher p_i, which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.     

Mesophyll conductance in leaves of Japanese white birch (Betula platyphylla var. japonica) seedlings grown under elevated CO2 concentration and low N availability

To test the hypothesis that mesophyll conductance (g_m) would be reduced by leaf starch accumulation in plants grown under elevated CO₂ concentration [CO₂], we investigated g_m in seedlings of Japanese white birch grown under ambient and elevated [CO₂] with an adequate and limited nitrogen supply using simultaneous gas exchange and chlorophyll fluorescence measurements. Both elevated [CO₂] and limited nitrogen supply decreased area‐based leaf N accompanied with a decrease in the maximum rate of Rubisco carboxylation (V_c,max) on a CO₂ concentration at chloroplast stroma (C_c) basis. Conversely, only seedlings grown at elevated [CO₂] under limited nitrogen supply had significantly higher leaf starch content with significantly lower g_m among the treatment combinations. Based on a leaf anatomical analysis using microscopic photographs, however, there were no significant difference in the area of chloroplast surfaces facing intercellular space per unit leaf area among treatment combinations. Thicker cell walls were suggested in plants grown under limited N by increases in leaf mass per area subtracting non‐structural carbohydrates. These results suggest that starch accumulation and/or thicker cell walls in the leaves grown at elevated [CO₂] under limited N supply might hinder CO₂ diffusion in chloroplasts and cell walls, which would be an additional cause of photosynthetic downregulation as well as a reduction in Rubisco activity related to the reduced leaf N under elevated [CO₂].     

Effects of low and high levels of magnesium on the response of sunflower plants grown with ammonium and nitrate

The effect of the nitrogen source (ammonium and nitrate) and its interaction with magnesium on various physiological processes was studied in sunflower plants (Helianthus annuusL.). Plants were grown in hydroponic culture with nitrate (5 mM) or ammonium (5 mM) and four concentrations of magnesium (0.1, 0.8, 5 and 10 mM). After 2 weeks, growth, gas exchange and fluorescence parameters, soluble carbohydrates, free amino acids, soluble protein and mineral elements were determined. Ammonium nutrition resulted in a reduction of dry matter accumulation, as well as in a decrease in the CO₂ assimilation. Moreover, ammonium-fed plants showed a greater content of free amino acids, soluble protein, Rubisco and anions, and a lower cation content, mostly Mg²⁺. The presence of high levels of Mg²⁺ in the nutrient solution containing NH₄ ⁺ resulted in a stimulation of growth and CO₂ assimilation to the levels observed in nitrate-fed plants. The lower photosynthetic rate of ammonium-fed plants grown with low level of magnesium does not seem to be due to a lower photosynthetic pigment content, or a deficiency in Photosystem II activity, or to lower Rubisco content. Hence, Rubisco activity or other enzymes involved in CO₂ fixation could have been affected in ammonium-fed plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m⁻² s⁻¹ was followed, the slow increase in CO₂ assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO₂ assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO₂ assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

Changes in photosynthesis and other chloroplast traits in lanceolate leaflet isoline of soybean

Lanceolate leaflet soybean (Glycine max L. Merrill) has been known to photosynthesize more CO₂ per unit leaf area than normal leaflet soybean. The exact reason for this increase in photosynthetic rate is still unclear. The present study was undertaken to investigate the leaf photosynthetic rate and other physiological traits in relation to chloroplast of lanceolate leaflet soybean. Ontogenic changes in apparent photosynthesis (AP) were related primarily to variations in the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit leaf area, and only secondarily to difference in specific activity of the said enzyme. Moreover, lanceolate leaflet consistently maintained a higher leaf AP, higher Rubisco activity, and more chloroplasts per unit leaf area basis than did normal leaflet soybean throughout leaf ontogeny. However, lanceolate soybean tended to have lower AP and Rubisco activity on a chloroplast basis. The superiority of leaf AP and other leaf physiological traits, expressed on a leaf area basis, in lanceolate leaflet soybean is associated with a corresponding increase in chloroplast number.     

Effects of salt-tolerant rootstock grafting on ultrastructure,photosynthetic capacity,and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-scavenging system in chloroplasts of cucumber seedlings under NaCl stress

Plant growth, photosynthetic parameters, chloroplast ultrastructure, and the ascorbate-glutathione cycle system in chloroplasts of self-grafted and rootstock-grafted cucumber leaves were investigated. Grafted plants were grown hydroponically and were exposed to 0, 50, and 100 mM NaCl concentrations for 10 days. Under NaCl stress, the hydrogen peroxide (H₂O₂) content in cucumber chloroplasts increased, the chloroplast ultrastructure was damaged, and the gas stomatal conductance, intercellular CO₂ concentration, as well as shoot dry weight, plant height, stem diameter, leaf area, and leaf relative water content were inhibited, whereas these changes were less severe in rootstock-grafted plants. The activities of ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2), and dehydroascorbate reductase (DHAR EC 1.8.5.1) were higher in the chloroplasts of rootstock-grafted plants compared with those of self-grafted plants under 50 and 100 mM NaCl. Similar trends were shown in leaf net CO₂ assimilation rate and transpiration rate, as well as reduced glutathione content under 100 mM NaCl. Results suggest that rootstock grafting enhances the H₂O₂-scavenging capacity of the ascorbate–glutathione cycle in cucumber chloroplasts under NaCl stress, thereby protecting the chloroplast structure and improving the photosynthetic performance of cucumber leaves. As a result, cucumber growth is promoted.     

Effects of Cerium on Key Enzymes of Carbon Assimilation of Spinach Under Magnesium Deficiency

The mechanism of the fact that cerium improves the photosynthesis of plants under magnesium deficiency is poorly understood. The main aim of the study was to determine the role of cerium in the amelioration of magnesium deficiency effects in CO₂ assimilation of spinach. Spinach plants were cultivated in Hoagland’s solution. They were subjected to magnesium deficiency and to cerium chloride administered in the magnesium-present Hoagland’s media and magnesium-deficient Hoagland’s media. The results showed that the chlorophyll synthesis and oxygen evolution was destroyed, and the activities of Rubisco carboxylasae and Rubisco activase and the expression of Rubisco large subunit (rbcL), Rubisco small subunit (rbcS), and Rubisco activase subunit (rca) were significantly inhibited, then plant growth was inhibited by magnesium deficiency. However, cerium promotes the chlorophyll synthesis, the activities of two key enzymes in CO₂ assimilation, and the expression of rbcL, rbcS, and rca, thus leading to the enhancement of spinach growth under magnesium-deficient conditions.     

The temperature response of CO2 assimilation, photochemical activities and Rubisco activation in Camelina sativa, a potential bioenergy crop with limited capacity for acclimation to heat stress

The temperature optimum of photosynthesis coincides with the average daytime temperature in a species’ native environment. Moderate heat stress occurs when temperatures exceed the optimum, inhibiting photosynthesis and decreasing productivity. In the present study, the temperature response of photosynthesis and the potential for heat acclimation was evaluated for Camelina sativa, a bioenergy crop. The temperature optimum of net CO₂ assimilation rate (A) under atmospheric conditions was 30–32?°C and was only slightly higher under non-photorespiratory conditions. The activation state of Rubisco was closely correlated with A at supra-optimal temperatures, exhibiting a parallel decrease with increasing leaf temperature. At both control and elevated temperatures, the modeled response of A to intercellular CO₂ concentration was consistent with Rubisco limiting A at ambient CO₂. Rubisco activation and photochemical activities were affected by moderate heat stress at lower temperatures in camelina than in the warm-adapted species cotton and tobacco. Growth under conditions that imposed a daily interval of moderate heat stress caused a 63?% reduction in camelina seed yield. Levels of cpn60 protein were elevated under the higher growth temperature, but acclimation of photosynthesis was minimal. Inactivation of Rubisco in camelina at temperatures above 35?°C was consistent with the temperature response of Rubisco activase activity and indicated that Rubisco activase was a prime target of inhibition by moderate heat stress in camelina. That photosynthesis exhibited no acclimation to moderate heat stress will likely impact the development of camelina and other cool season Brassicaceae as sources of bioenergy in a warmer world.     

Activation of Rubisco controls CO<Subscript>2</Subscript> assimilation in light: a perspective on its discovery

CO₂ fixation during photosynthesis is regulated by the activity of ribulose bisphosphate carboxylase (Rubisco). This conclusion became more apparent to me after CO₂-fixation experiments using isolated spinach chloroplasts and protoplasts, purified Rubisco enzyme, and intact leaves. Ribulose bisphosphate (RuBP) pools and activation of Rubisco were measured and compared to ¹⁴CO₂ fixation in light. The rates of ¹⁴CO ₂ assimilation best followed the changes in Rubisco activation under moderate to high light intensities. RuBP pool sizes regulated ¹⁴ ₂ assimilation only in very high CO₂ levels, low light and in darkness. Activation of Rubisco involves two separate processes: carbamylation of the protein and removal of inhibitors blocking carbamylation or blocking RuBP binding to carbamylated sites before reaction with CO₂ or O₂. This revised version was published online in June 2006 with corrections to the Cover Date.     

One crop breeding cycle from starvation? How engineering crop photosynthesis for rising CO2 and temperature could be one important route to alleviation

Global climate change is likely to severely impact human food production. This comes at a time when predicted demand for primary foodstuffs by a growing human population and changing global diets is already outpacing a stagnating annual rate of increase in crop productivity. Additionally, the time required by crop breeding and bioengineering to release improved varieties to farmers is substantial, meaning that any crop improvements needed to mitigate food shortages in the 2040s would need to start now. In this perspective, the rationale for improvements in photosynthetic efficiency as a breeding objective for higher yields is outlined. Subsequently, using simple simulation models it is shown how predicted changes in temperature and atmospheric [CO₂] affect leaf photosynthetic rates. The chloroplast accounts for the majority of leaf nitrogen in crops. Within the chloroplast about 25% of nitrogen is invested in the carboxylase, Rubisco, which catalyses the first step of CO₂ assimilation. Most of the remaining nitrogen is invested in the apparatus to drive carbohydrate synthesis and regenerate ribulose-1:5-bisphosphate (RuBP), the CO₂-acceptor molecule at Rubisco. At preindustrial [CO₂], investment in these two aspects may have been balanced resulting in co-limitation. At today''s [CO₂], there appears to be over-investment in Rubisco, and despite the counter-active effects of rising temperature and [CO₂], this imbalance is predicted to worsen with global climate change. By breeding or engineering restored optimality under future conditions increased productivity could be achieved in both tropical and temperate environments without additional nitrogen fertilizer. Given the magnitude of the potential shortfall, better storage conditions, improved crop management and better crop varieties will all be needed. With the short time-scale at which food demand is expected to outpace supplies, all available technologies to improve crop varieties, from classical crop breeding to crop genetic engineering should be employed. This will require vastly increased public and private investment to support translation of first discovery in laboratories to replicated field trials, and an urgent re-evaluation of regulation of crop genetic engineering.