Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C 4 photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO 2 -fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C 3 ancestors, C 4 plants combine a faster rubisco with a biochemical CO 2 -concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C 4 -rubisco that can be transplanted into C 3 -rubisco to improve carboxylation rate (V C ). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C 3 ), Flaveria floridana (C 3 -C 4 ), and Flaveria bidentis (C 4 ), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C 4 ( 309 Ile; faster V C , lower CO 2 affinity) and C 3 ( 309 Met; slower V C , higher CO 2 affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V C in C 3 crops. Coengineering a catalytically faster C 3 rubisco and a CO 2 -concentrating mechanism within C 3 crop species could enhance their efficiency in resource use and yield.CO 2 assimilation | rbcL mutagenesis | gas exchange | chloroplast transformation T he future uncertainties of global climate change and estimates of unsustainable population growth have increased the urgency of improving crop yields (1). One possible solution is to "supercharge" photosynthesis by improving the C 3 cycle (2, 3). Although a simple idea, this is a complex challenge that involves several possible alternatives. Many of these alternatives focus on enhancing the performance of the CO 2 -fixing enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (rubisco), which catalyses the first step in the synthesis of carbohydrates. Despite its pivotal role, rubisco is a slow catalyst, completing only one to four carboxylation reactions per catalytic site per second in plants (4, 5). Moreover CO 2 not only is fixed through a complex catalytic process but also must compete with O 2 . The oxygenation of RuBP produces 2-phosphoglycolate, whose recycling by photorespiration requires energy and results in the futile loss of fixed carbon [∼30% of fixed CO 2 in many C 3 plants (6)].To compensate for rubisco's catalytic limitations, plants invest as much as 25% of their leaf nitrogen in rubisco (7). This value is much lower in C 4 plants, where a biochemical CO 2 -concentrating mechanism (CCM) elevates CO 2 around rubisco. This optimized microenvironment allows rubisco to operate close to its maximal activity, reducing O 2 competition. This CCM has enabled C 4 plants to evolve rubiscos with substantially im...

The threat to global food security of stagnating yields and population growth makes increasing crop productivity a critical goal over the coming decades. One key target for improving crop productivity and yields is increasing the efficiency of photosynthesis. Central to photosynthesis is Rubisco, which is a critical but often rate-limiting component. Here, we present full Rubisco catalytic properties measured at three temperatures for 75 plants species representing both crops and undomesticated plants from diverse climates. Some newly characterized Rubiscos were naturally "better" compared to crop enzymes and have the potential to improve crop photosynthetic efficiency. The temperature response of the various catalytic parameters was largely consistent across the diverse range of species, though absolute values showed significant variation in Rubisco catalysis, even between closely related species. An analysis of residue differences among the species characterized identified a number of candidate amino acid substitutions that will aid in advancing engineering of improved Rubisco in crop systems. This study provides new insights on the range of Rubisco catalysis and temperature response present in nature, and provides new information to include in models from leaf to canopy and ecosystem scale.

Summary Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco‐containing pyrenoid that is needed to minimise CO 2 retro‐diffusion for CCM operating efficiency.Rubisco in Arabidopsis was re‐engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface‐exposed algal SSU α‐helices.Leaves of Arabidopsis rbcs mutants expressing ‘pyrenoid‐competent’ chimeric Arabidopsis SSUs containing the SSU α‐helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α‐helices are catalytically neutral.The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild‐type levels of the hybrid Rubisco were similar to those of wild‐type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild‐type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.

Summary Sub‐Saharan Africa is projected to see a 55% increase in food demand by 2035, where cassava (Manihot esculenta) is the most widely planted crop and a major calorie source. Yet, cassava yield in this region has not increased significantly for 13 yr. Improvement of genetic yield potential, the basis of the first Green Revolution, could be realized by improving photosynthetic efficiency. First, the factors limiting photosynthesis and their genetic variability within extant germplasm must be understood. Biochemical and diffusive limitations to leaf photosynthetic CO2 uptake under steady state and fluctuating light in 13 farm‐preferred and high‐yielding African cultivars were analyzed. A cassava leaf metabolic model was developed to quantify the value of overcoming limitations to leaf photosynthesis. At steady state, in vivo Rubisco activity and mesophyll conductance accounted for 84% of the limitation. Under nonsteady‐state conditions of shade to sun transition, stomatal conductance was the major limitation, resulting in an estimated 13% and 5% losses in CO2 uptake and water use efficiency, across a diurnal period. Triose phosphate utilization, although sufficient to support observed rates, would limit improvement in leaf photosynthesis to 33%, unless improved itself. The variation of carbon assimilation among cultivars was three times greater under nonsteady state compared to steady state, pinpointing important overlooked breeding targets for improved photosynthetic efficiency in cassava.