Rubisco catalyses the entry of almost all CO₂ into the biosphere and is often the rate-limiting step in plant photosynthesis and growth. Its notoriety as the most abundant protein on Earth stems from the slow and error-prone catalytic properties that require plants, cyanobacteria, algae and photosynthetic bacteria to produce it in high amounts. Efforts to improve the CO₂-fixing properties of plant Rubisco has been spurred on by the discovery of more effective isoforms in some algae with the potential to significantly improve crop productivity. Incompatibilities between the protein folding machinery of leaf and algae chloroplasts have, so far, prevented efforts to transplant these more effective Rubisco variants into plants. There is therefore increasing interest in improving Rubisco catalysis by directed (laboratory) evolution. Here we review the advances being made in, and the ongoing challenges with, improving the solubility and/or carboxylation activity of differing non-plant Rubisco lineages. We provide perspectives on new opportunities for the directed evolution of crop Rubiscos and the existing plant transformation capabilities available to evaluate the extent to which Rubisco activity improvements can benefit agricultural productivity.

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosynthetic carbon assimilation, as it catalyses the conversion of inorganic carbon into organic carbon. Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substrate specificity. Improving the catalytic performance of RuBisCO is one of the key routes for enhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalytic properties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understanding of functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPR‐Cas9 technology to randomly edit five rbcS genes in rice (OsrbcS1–5), generating a series of knockout mutants. The mutations of predominant rbcS genes in rice photosynthetic tissues, OsrbcS2–5, conferred inhibited growth, delayed heading and reduced yield in the field conditions, accompanying with lower RuBisCO contents and activities and significantly reduced photosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations. In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lower sugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of the mutated RuBisCO enzyme in one rbcs2,3,5 mutant line uncovered no significant differences from the wild‐type protein, indicating that the mutations of rbcS did not compromise the protein assembly or the structure. Our findings generated a mutant pool with genetic diversities, which offers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice. The multiplex genetic engineering approach of this study provides an effective and feasible strategy for RuBisCO modification in crops, further facilitate the photosynthesis improvement and sustainable crop production.

Although Rubisco is the most abundant enzyme globally, it is inefficient for carbon fixation because of its low turnover rate and limited ability to distinguish CO2 and O2, especially under high O2 conditions. To address these limitations, phytoplankton, including cyanobacteria and algae, have evolved CO2-concentrating mechanisms (CCM) that involve compartmentalizing Rubisco within specific structures, such as carboxysomes in cyanobacteria or pyrenoids in algae. Engineering plant chloroplasts to establish similar structures for compartmentalizing Rubisco has attracted increasing interest for improving photosynthesis and carbon assimilation in crop plants. Here, we present a method to effectively induce the condensation of endogenous Rubisco within tobacco (Nicotiana tabacum) chloroplasts by genetically fusing superfolder green fluorescent protein (sfGFP) to the tobacco Rubisco large subunit (RbcL). By leveraging the intrinsic oligomerization feature of sfGFP, we successfully created pyrenoid-like Rubisco condensates that display dynamic, liquid-like properties within chloroplasts without affecting Rubisco assembly and catalytic function. The transgenic tobacco plants demonstrated comparable autotrophic growth rates and full life cycles in ambient air relative to the wild-type plants. Our study offers a promising strategy for modulating endogenous Rubisco assembly and spatial organization in plant chloroplasts via phase separation, which provides the foundation for generating synthetic organelle-like structures for carbon fixation, such as carboxysomes and pyrenoids, to optimize photosynthetic efficiency.

crassulacic acid metabolism (CAM), which facilitates the initial fixation of CO 2 into oxaloacetate. PEPC catalyzes the conversion of phosphoenolpyruvate (PEP) and bicarbonate (HCO 3 − ) to oxaloacetate, which is then converted to malate [ 106 ]. This step is critical for C 4 plants because it effectively concentrates CO 2 near Rubisco, thereby increasing carboxylation efficiency and minimizing photorespiration. In plants, CAM-PEPC also plays a vital role in the temporal separation of carbon fixation and carbon assimilation, allowing these plants to conserve water. The insertion of C 4 -specific enzymes, such as PEPC, into C 3 plants, aims to increase photosynthetic efficiency by increasing the internal CO 2 concentration around Rubisco. The overexpression of PEPC has been investigated in various C 3 plants, with promising results. Transgenic O. sativa plants overexpressing the Z. mays PEPC gene exhibited increased photosynthetic rates and biomass production under high-CO 2 conditions. This improvement was due to the increased availability of CO 2 in the Calvin cycle, which increased the overall efficiency of photosynthesis. CAM photosynthesis has attracted significant interest because of its water-use efficiency. Approximately 90% of higher plants undergo C 3 photosynthesis, and approximately 3% of the remaining plants are C 4 plants. Approximately 6% of CAM plants, similar to C 4 plants, have evolved several times. C 4 and CAM plants enrich carbon dioxide molecules around RUBISCO for carbon assimilation reactions, but while C 4 plants have a CO 2 concentration mechanism (CCM)that spatially separates the Calvin cycle and electron transport chains of the photosynthetic pathways, CAM plants use CCM to temporarily separate the photosynthetic pathways. The genomes and important genes involved in improving water-use efficiency in CAM plants have been well studied. For example, a study by VanBuren et al. (2015) sequenced the pineapple genome, revealing the genetic basis