Rubisco kinetic adaptations to extreme environments.

Photosynthetic and chemosynthetic extremophiles have evolved adaptations to thrive in challenging environments by finely adjusting their metabolic pathways through evolutionary processes. A prime adaptation target to allow autotrophy in extreme conditions is the enzyme Rubisco, which plays a central role in the conversion of inorganic to organic carbon. Here, we present an extensive compilation of Rubisco kinetic traits from a wide range of species of bacteria, archaea, algae, and plants, sorted by phylogenetic group, Rubisco type, and extremophile type. Our results show that Rubisco kinetics for the few extremophile organisms reported up to date are placed at the margins of the enzyme's natural variability. Form ID Rubisco from thermoacidophile rhodophytes and form IB Rubisco from halophile terrestrial plants exhibit higher specificity and affinity for CO2 than their non-extremophilic counterparts, as well as higher carboxylation efficiency, whereas form ID Rubisco from psychrophile organisms possess lower affinity for O2. Additionally, form IB Rubisco from thermophile cyanobacteria shows enhanced CO2 specificity when compared to form IB non-extremophilic cyanobacteria. Overall, these findings highlight the unique characteristics of extremophile Rubisco enzymes and provide useful clues to guide next explorations aimed at finding more efficient Rubiscos.

Singular adaptations in the carbon assimilation mechanism of the polyextremophile cyanobacterium Chroococcidiopsis thermalis.

Cyanobacteria largely contribute to the biogeochemical carbon cycle fixing ~ 25% of the inorganic carbon on Earth. However, the carbon acquisition and assimilation mechanisms in Cyanobacteria are still underexplored regardless of being of great importance for shedding light on the origins of autotropism on Earth and providing new bioengineering tools for crop yield improvement. Here, we fully characterized these mechanisms from the polyextremophile cyanobacterium Chroococcidiopsis thermalis KOMAREK 1964/111 in comparison with the model cyanobacterial strain, Synechococcus sp. PCC6301. In particular, we analyzed the Rubisco kinetics along with the in vivo photosynthetic CO2 assimilation in response to external dissolved inorganic carbon, the effect of CO2 concentrating mechanism (CCM) inhibitors on net photosynthesis and the anatomical particularities of their carboxysomes when grown under either ambient air (0.04% CO2) or 2.5% CO2-enriched air. Our results show that Rubisco from C. thermalis possess the highest specificity factor and carboxylation efficiency ever reported for Cyanobacteria, which were accompanied by a highly effective CCM, concentrating CO2 around Rubisco more than 140-times the external CO2 levels, when grown under ambient CO2 conditions. Our findings provide new insights into the Rubisco kinetics of Cyanobacteria, suggesting that improved Sc/o values can still be compatible with a fast-catalyzing enzyme. The combination of Rubisco kinetics and CCM effectiveness in C. thermalis relative to other cyanobacterial species might indicate that the co-evolution between Rubisco and CCMs in Cyanobacteria is not as constrained as in other phylogenetic groups.

Carbon assimilation in upper subtidal macroalgae is determined by an inverse correlation between Rubisco carboxylation efficiency and CO2 concentrating mechanism effectiveness.

Seaweeds have a wide ecophysiological and phylogenetic diversity with species expressing different Rubisco forms that frequently coexist with biophysical CO2 concentrating mechanisms (CCMs), an adaptation that overcomes the low CO2 availability and gas diffusion in seawater. Here, we assess the possible coevolution between the Rubisco catalysis and the type and effectiveness of CCMs present in six upper subtidal macroalgal species belonging to three phylogenetic groups of seaweeds. A wide diversity in the Rubisco kinetic traits was found across the analyzed species, although the specificity factor was the only parameter explained by the expressed Rubisco form. Differences in the catalytic trade-offs were found between Rubisco forms, indicating that ID Rubiscos could be better adapted to the intracellular O2  : CO2 ratio found in marine organisms during steady-state photosynthesis. The biophysical components of the CCMs also differed among macroalgal species, resulting in different effectiveness to concentrate CO2 around Rubisco active sites. Interestingly, an inverse relationship was found between the effectiveness of CCMs and the in vitro Rubisco carboxylation efficiency, which possibly led to a similar carboxylation potential across the analyzed macroalgal species. Our results demonstrate a coevolution between Rubisco kinetics and CCMs across phylogenetically distant marine macroalgal species sharing the same environment.

Correlative adaptation between Rubisco and CO2-concentrating mechanisms in seagrasses.

Submerged angiosperms sustain some of the most productive and diverse ecosystems worldwide. However, their carbon acquisition and assimilation mechanisms remain poorly explored, missing an important step in the evolution of photosynthesis during the colonization of aquatic environments by angiosperms. Here we reveal a convergent kinetic adaptation of Rubisco in phylogenetically distant seagrass species that share catalytic efficiencies and CO2 and O2 affinities up to three times lower than those observed in phylogenetically closer angiosperms from terrestrial, freshwater and brackish-water habitats. This Rubisco kinetic convergence was found to correlate with the effectiveness of seagrass CO2-concentrating mechanisms (CCMs), which probably evolved in response to the constant CO2 limitation in marine environments. The observed Rubisco kinetic adaptation in seagrasses more closely resembles that seen in eukaryotic algae operating CCMs rather than that reported in terrestrial C4 plants. Our results thus demonstrate a general pattern of co-evolution between Rubisco function and biophysical CCM effectiveness that traverses distantly related aquatic lineages.

Improving photosynthesis through the enhancement of Rubisco carboxylation capacity.

Rising human population, along with the reduction in arable land and the impacts of global change, sets out the need for continuously improving agricultural resource use efficiency and crop yield (CY). Bioengineering approaches for photosynthesis optimization have largely demonstrated the potential for enhancing CY. This review is focused on the improvement of Rubisco functioning, which catalyzes the rate-limiting step of CO2 fixation required for plant growth, but also catalyzes the ribulose-bisphosphate oxygenation initiating the carbon and energy wasteful photorespiration pathway. Rubisco carboxylation capacity can be enhanced by engineering the Rubisco large and/or small subunit genes to improve its catalytic traits, or by engineering the mechanisms that provide enhanced Rubisco expression, activation and/or elevated [CO2] around the active sites to favor carboxylation over oxygenation. Recent advances have been made in the expression, assembly and activation of foreign (either natural or mutant) faster and/or more CO2-specific Rubisco versions. Some components of CO2 concentrating mechanisms (CCMs) from bacteria, algae and C4 plants has been successfully expressed in tobacco and rice. Still, none of the transformed plant lines expressing foreign Rubisco versions and/or simplified CCM components were able to grow faster than wild type plants under present atmospheric [CO2] and optimum conditions. However, the results obtained up to date suggest that it might be achievable in the near future. In addition, photosynthetic and yield improvements have already been observed when manipulating Rubisco quantity and activation degree in crops. Therefore, engineering Rubisco carboxylation capacity continues being a promising target for the improvement in photosynthesis and yield.

Evolutionary trends in RuBisCO kinetics and their co-evolution with CO2 concentrating mechanisms.

RuBisCO-catalyzed CO2 fixation is the main source of organic carbon in the biosphere. This enzyme is present in all domains of life in different forms (III, II, and I) and its origin goes back to 3500 Mya, when the atmosphere was anoxygenic. However, the RuBisCO active site also catalyzes oxygenation of ribulose 1,5-bisphosphate, therefore, the development of oxygenic photosynthesis and the subsequent oxygen-rich atmosphere promoted the appearance of CO2 concentrating mechanisms (CCMs) and/or the evolution of a more CO2 -specific RuBisCO enzyme. The wide variability in RuBisCO kinetic traits of extant organisms reveals a history of adaptation to the prevailing CO2 /O2 concentrations and the thermal environment throughout evolution. Notable differences in the kinetic parameters are found among the different forms of RuBisCO, but the differences are also associated with the presence and type of CCMs within each form, indicative of co-evolution of RuBisCO and CCMs. Trade-offs between RuBisCO kinetic traits vary among the RuBisCO forms and also among phylogenetic groups within the same form. These results suggest that different biochemical and structural constraints have operated on each type of RuBisCO during evolution, probably reflecting different environmental selective pressures. In a similar way, variations in carbon isotopic fractionation of the enzyme point to significant differences in its relationship to the CO2 specificity among different RuBisCO forms. A deeper knowledge of the natural variability of RuBisCO catalytic traits and the chemical mechanism of RuBisCO carboxylation and oxygenation reactions raises the possibility of finding unrevealed landscapes in RuBisCO evolution.