Changes in Rubisco Kinetics during the Evolution of C4 Photosynthesis in Flaveria (Asteraceae) Are Associated with Positive Selection on Genes Encoding the Enzyme

Abstract

Rubisco, the primary photosynthetic carboxylase, evolved 3-4 billion years ago in an anaerobic, high CO(2) atmosphere. The combined effect of low CO(2) and high O(2) levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO(2) and O(2), leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C(4) photosynthesis largely solves this problem by facilitating a high CO(2)/O(2) ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcat(CO2) and lower CO(2)/O(2) specificity (S(C/O)) is well supported, but the structural basis for the differences between C(3) and C(4) Rubiscos is not clear. Flaveria (Asteraceae) includes C(3), C(3)-C(4) intermediate, and C(4) species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C(3) to C(4) photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C(4) Rubiscos from the ancestral C(3) species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic "C(4)" SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus.

Stability-activity tradeoffs constrain the adaptive evolution of RubisCO

“…However, A281 packs against S321 and G322 at the end of the strand, which leads to loop 6, and destabilization of this interaction may have a long-range effect on the dynamics at the active site. Another frequently positively selected mutation, M309I, also identified in some previous phylogenetic studies (20,24), lies at the interface of the two C-terminal domains within a dimer and also close to the junction between N-and C-terminal domains within each subunit. This mutation has been demonstrated to act as a catalytic switch between C 3 -like and C 4 -like properties (i.e., decreasing specificity for CO 2 over O 2 and increasing the turnover) in Flaveria species and in chimeric enzymes consisting of large subunits from Flaveria and tobacco small subunits (21).…”

“…The high concentration of CO 2 at the site of RubisCO in C 4 plants allows a lower specificity ratio of CO 2 /O 2 and therefore an increase in turnover rate and thus efficiency (17,18). Experimental studies of RubisCOs from very closely related C 3 and C 4 species within the Flaveria, Atriplex, and Neurachne genera showed that very few changes may be necessary to modify enzymatic properties in response to the modification of the metabolic context (19,20). Indeed, in the Flaveria context, a single mutation (M309I) has been identified as key in modifying specificity and increasing turnover (21); it remains unclear as to how this observation applies to a wider range of plants and what the contributions are of other observed mutations to adaptation.…”

“…Indeed, in the Flaveria context, a single mutation (M309I) has been identified as key in modifying specificity and increasing turnover (21); it remains unclear as to how this observation applies to a wider range of plants and what the contributions are of other observed mutations to adaptation. Comparative sequence analysis of a broader range of plant species does suggest that, in general, adaptation of RubisCO to C 4 metabolism involves a larger number of amino acid changes found to be under positive selection (19,20). Here, we investigate the role of mutations in the adaptation of a large group of plants, focusing in particular on the constraints imposed by stability requirements, which have been previously shown to be important in the directed evolution of enzymes.…”

Adaptive signals in algal Rubisco reveal a history of ancient atmospheric carbon dioxide

“…In land plants, it has been established that positive selection in rbcL emerges coincident with the development of a C 4 CCM which elevates CO 2 to almost saturation at the site of Rubisco [55,56]. This relaxes pressure for Rubisco to have a high affinity for CO 2 and therefore allows an increase in V c , which results in increased photosynthetic efficiency as the plant requires less nitrogen to achieve a given CO 2 [4,6,20] and K c (dark blue bars) [6,21] with standard error bars and number of species measured (n) are shown.…”

“…This relaxes pressure for Rubisco to have a high affinity for CO 2 and therefore allows an increase in V c , which results in increased photosynthetic efficiency as the plant requires less nitrogen to achieve a given CO 2 [4,6,20] and K c (dark blue bars) [6,21] with standard error bars and number of species measured (n) are shown. [55,56]. By analogy, therefore, positive selection in Rubiscos of the Haptophyta and diatoms is likely to have occurred also in response to emergence of CCMs as both Haptophyta and diatoms are thought to possess them [57].…”