The Coevolution of RuBisCO, Photorespiration, and Carbon Concentrating Mechanisms in Higher Plants

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) is the carbon-fixing enzyme present in most photosynthetic organisms, converting CO2 into organic matter. Globally, photosynthetic efficiency in terrestrial plants has become increasingly challenged in recent decades due to a rapid increase in atmospheric CO2 and associated changes toward warmer and dryer environments. Well adapted for these new climatic conditions, the C4 photosynthetic pathway utilizes carbon concentrating mechanisms to increase CO2 concentrations surrounding RuBisCO, suppressing photorespiration from the oxygenase catalyzed reaction with O2. The energy efficiency of C3 photosynthesis, from which the C4 pathway evolved, is thought to rely critically on an uninterrupted supply of chloroplast CO2. Part of the homeostatic mechanism that maintains this constancy of supply involves the CO2 produced as a byproduct of photorespiration in a negative feedback loop. Analyzing the database of RuBisCO kinetic parameters, we suggest that in genera (Flaveria and Panicum) for which both C3 and C4 examples are available, the C4 pathway evolved only from C3 ancestors possessing much lower than the average carboxylase specificity relative to that of the oxygenase reaction (SC/O=SC/SO), and hence, the higher CO2 levels required for development of the photorespiratory CO2 pump (C2 photosynthesis) essential in the initial stages of C4 evolution, while in the later stage (final optimization phase in the Flaveria model) increased CO2 turnover may have occurred, which would have been supported by the higher CO2 levels. Otherwise, C4 RuBisCO kinetic traits remain little changed from the ancestral C3 species. At the opposite end of the spectrum, C3 plants (from Limonium) with higher than average SC/O, which may be associated with the ability of increased CO2, relative to O2, affinity to offset reduced photorespiration and chloroplast CO2 levels, can tolerate high stress environments. It is suggested that, instead of inherently constrained by its kinetic mechanism, RuBisCO possesses the extensive kinetic plasticity necessary for adaptation to changes in photorespiration that occur in the homeostatic regulation of CO2 supply under a broad range of abiotic environmental conditions.

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcatC, up 27%), carboxylation efficiency (kcatC/Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).

Abscisic acid as a factor in regulation of photosynthetic carbon metabolism of pea seedlings

The influence of abscisic acid (ABA) on carbon metabolism and the activity of ribulosebisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases in 8-day-old pea seedlings was investigated. It was endeavoured to correlate the changes observed in metabolic processes with the endogenous ABA level. In plants treated with ABA incorporation of labeled carbon into sucrose, glucose, fructose and sugar phosphates was depressed, while ¹⁴C incorporation into starch, ribulose and malic acid was enhanced. The activity of RuBP carboxylase was considerably lowered, whereas that of PEP carboxylase was slightly increased. It is considered that inhibition of photosynthesis due to the action of ABA is caused to a great extent by the obstruction of the C-3 pathway and reduced activity of RuBP carboxylase, whereas (β-carboxylation was not blocked.

Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses the key reaction by which inorganic carbon may be assimilated into organic carbon. Phylogenetic analyses indicate that there are three classes of bona fide RubisCO proteins, forms I, II and III, which all catalyse the same reactions. In addition, there exists another form of RubisCO, form IV, which does not catalyse RuBP carboxylation or oxygenation. Form IV is actually a homologue of RubisCO and is called the RubisCO-like protein (RLP). Both RubisCO and RLP appear to have evolved from an ancestor protein in a methanogenic archaeon, and comprehensive analyses indicate that the different forms (I, II, III and IV) contain various subgroups, with individual sequences derived from representatives of all three kingdoms of life. The diversity of RubisCO molecules, many of which function in distinct milieus, has provided convenient model systems to study the ways in which the active site of this protein has evolved to accommodate necessary molecular adaptations. Such studies have proven useful to help provide a framework for understanding the molecular basis for many important aspects of RubisCO catalysis, including the elucidation of factors or functional groups that impinge on RubisCO carbon dioxide/oxygen substrate discrimination.

Limitations to photosynthesis at different temperatures in the leaves of Citrus limon

The response of CO2 assimilation rate (A) to the intercellular partial pressure of CO2 (Ci) was measured on intact lemon leaves over a range of temperatures (10 to 40ºC). The A/Ci response shows how change in the leaf temperature alters the activity of ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) and RuBP regeneration via electron transport. The rate of A reached a maximum of 7.9 to 8.9 µmol m-2 s-1 between 25 and 30ºC, while dark respiration (Rd) increased with temperature from 0.4 µmol m-2 s-1 at 10ºC to 1.4 µmol m-2 s-1 at 40ºC. The maximum rates of carboxylation (Vc,max) and the maximum rates of electron transport (Jmax) both increased over this temperature range from 7.5 to 142 µmol m-2 s-1 and from 23.5 to 152 µmol m-2 s-1, respectively. These temperature responses showed that A can be limited by either process depending on the leaf temperature, when Ci or stomatal conductance are not limiting. The decrease in A associated with higher temperatures is in part a response to the greater increase in the rate of oxygenation of RuBP compared with carboxylation and Rd at higher temperatures. Although A can in theory be limited at higher Ci by the rate of triose-phosphate utilization, this limitation was not evident in lemon leaves.