New horizons for building pyrenoid-based CO2-concentrating mechanisms in plants to improve yields

Abstract

Many photosynthetic species have evolved CO2-concentrating mechanisms (CCMs) to improve the efficiency of CO2 assimilation by Rubisco and reduce the negative impacts of photorespiration. However, the majority of plants (i.e. C3 plants) lack an active CCM. Thus, engineering a functional heterologous CCM into important C3 crops, such as rice (Oryza sativa) and wheat (Triticum aestivum), has become a key strategic ambition to enhance yield potential. Here, we review recent advances in our understanding of the pyrenoid-based CCM in the model green alga Chlamydomonas reinhardtii and engineering progress in C3 plants. We also discuss recent modeling work that has provided insights into the potential advantages of Rubisco condensation within the pyrenoid and the energetic costs of the Chlamydomonas CCM, which, together, will help to better guide future engineering approaches. Key findings include the potential benefits of Rubisco condensation for carboxylation efficiency and the need for a diffusional barrier around the pyrenoid matrix. We discuss a minimal set of components for the CCM to function and that active bicarbonate import into the chloroplast stroma may not be necessary for a functional pyrenoid-based CCM in planta. Thus, the roadmap for building a pyrenoid-based CCM into plant chloroplasts to enhance the efficiency of photosynthesis now appears clearer with new challenges and opportunities.

Figure 1

Overview of inorganic carbon uptake in the Chlamydomonas CCM. A, At ambient levels of CO₂ (300–500 ppm, 0.03–0.05%, 10–18 µM CO₂), extracellular inorganic carbon (Ci, i.e. CO₂ and hydrated Ci, such as ${\text{HCO}}_3^{-}$) uptake is thought to be driven by drawdown of CO₂ into the chloroplast through rapid conversion of CO₂ to bicarbonate (${\text{HCO}}_3^{-}$) by LCIB, which is dispersed throughout the stroma in a complex with LCIC. ${\text{HCO}}_3^{-}$ is then transported into the lumen of thylakoid tubules traversing the pyrenoid by bestrophin-like channels (BST1-3) on the pyrenoid periphery. Carbonic anhydrase 3 (CAH3) located in the lumen within the pyrenoid converts ${\text{HCO}}_3^{-}$ to CO₂, which diffuses into the surrounding Rubisco-EPYC1 matrix. CO₂ not assimilated by Rubisco is converted back to ${\text{HCO}}_3^{-}$ by LCIB. Periplasmic carbonic anhydrases 1 and 2 (CAH1/2) and the plasma membrane CO₂ channel low CO₂-inducible protein 1 (LCI1) assist with inward CO₂ diffusion (Fujiwara et al., 1990). Font sizes for CO₂ and ${\text{HCO}}_3^{-}$ represent their relative concentration. B, At sub-ambient CO₂ levels (<200 ppm, <0.03%, <7 µM CO₂), the CCM transitions to an active ${\text{HCO}}_3^{-}$ uptake system that relies on the ${\text{HCO}}_3^{-}$ channels HLA3 protein and LCIA at the plasma membrane and chloroplast envelope, respectively. The LCIB/C complex relocalizes the pyrenoid periphery, and may interact with BST1-3 to rapidly recapture leaked CO₂ as ${\text{HCO}}_3^{-}$ for re-uptake into the thylakoid lumen.

Figure 2

Rubisco condensation occurs through interactions between the Rubisco small subunit and the RBM. A, LLPS of Chlamydomonas Rubisco through multivalent interactions between the Rubisco small subunit (SSU) and the linker protein EPYC1. The model structure of EPYC1(49–72)-bound Rubisco was from Protein Data Bank entry 7JFO (figure made in ChimeraX). B, The Rubisco-binding motifs (RBMs) of EPYC1 and the two α-helices of the CrSSU interact through key residues that facilitate salt-bridge interactions (left) and the formation of a hydrophobic pocket (right). C, The sequence diversity of RBMs within and between pyrenoid-localized proteins from Chlamydomonas. For RBMs from EPYC1 (top), the squared and circled amino acid residues form salt-bridge interactions and the hydrophobic pocket, respectively (as shown in B). The core motif is boxed, and residues that putatively interact with CrSSU are bold and colored according to chemical properties. Shaded background indicates the level of conservation where darker shading indicates that the residue is more highly conserved. Numbers indicate the location of the motifs in the mature peptide. D, Strategies to achieve LLPS of plant Rubisco include the modification of plant Rubisco SSUs to generate a hybrid plant Rubisco compatible with EPYC1 (top right), or the generation of a linker protein with synthetic RBMs compatible with plant Rubisco SSUs (bottom right).

Figure 3

Hypothetical evolutionary pathways to pyrenoids from condensation. Model simulations propose that free Rubisco (A) and carbonic anhydrase (CA) could proceed to a phase separated condensate of Rubisco in the presence of a condensing protein factor/linker (e.g. EPYC1 in the Chlamydomonas pyrenoid, or CsoS2/CcmM in α/β-carboxysomes) with CA in close external proximity (B) or co-condensed inside the condensate (C) (Long et al., 2021). Due to the net proton release during Rubisco carboxylation and subsequent decrease in internal pH, co-condensation with CA would favor conversion of CO₂ to ${\text{HCO}}_3^{-}$, and thus elevate CO₂. Although the condensate could partially restrict outward diffusion, other models suggest that the condensate would have to be large (i.e. >3 µm in radius) or be surrounded by a diffusion barrier (Fei et al., 2022). Both models indicate that evolution of Rubisco condensation is feasible in the absence of additional Ci uptake components (e.g. LCIA and HLA3). Following condensation, pyrenoid evolution could proceed in several ways, including development of a starch sheath (D) to restrict diffusion of CO₂ out of condensate, and/or a traversing thylakoid membrane that could allow regulatory re-localization of CA to within the condensate when required (E) (i.e. when the CCM is induced), and, in some cases, both combined (F). A comprehensive array of the diversity of pyrenoid architectures is illustrated in Barrett et al. (2021).

Figure 4

Expression of a selection of CCM genes under three different CO₂ concentrations and over dark/light diel cycles. A, Absolute values for transcript levels in fragments per kilobase per million (FPKM) at above ambient CO₂ (5%), ambient CO₂ (0.03%–0.05%), and sub-ambient CO₂ (0.01%–0.02%). Notably, the CCM genes highlighted here show a wide range of transcript abundances. B, Relative transcript abundance of each CCM gene. Data for A and B were derived from Fang et al. (2012). C, Relative transcript abundances in cultures grown at ambient CO₂ (0.04%) over dark/light diel cycles (units refer to hours) (Strenkert et al., 2019). Genes are color coded according to localization and/or role in CCM function.

Figure 5

Pathway for engineering a pyrenoid-based CCM into C3 plant chloroplasts. A, A six-step strategy is shown for incorporating a minimal CCM into a C3 plant as based on the model proposed by Fei et al. (2022). For each step, the predicted leaf CO₂ assimilation rate values are based on normalized CO₂ fixation flux estimates, using an arbitrary starting value of 10 µmol CO₂ m⁻² s⁻¹ for a wild-type C3 plant, and energy cost (i.e. additional ATP per CO₂ fixed beyond that typically used in the CBB cycle). New additions in each subsequent step are highlighted in bold. A functional minimal CCM (i.e. step 6) is predicted to increase CO₂ assimilation rates by three-fold with an energetic cost of 1.3 ATP per CO₂ fixed. These values assume that native plant CA is excluded from the Rubisco matrix from step 1 and that CO₂ assimilation rates are not limited by RuBP regeneration or triose phosphate utilization limitations. B, Schematic of a reconstituted minimal pyrenoid-based CCM in a C3 plant mesophyll cell chloroplast. Elements shown include Rubisco, native CA, LCIB, EPYC1, lumenal CA (e.g. CAH3) and starch or thylakoid tethers.