A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the enzyme Rubisco in specialized protein micro-compartments called carboxysomes. To date, chloroplastic expression of carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2. This result demonstrates the formation of α-carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-carboxysomes in chloroplasts.

HighlightBroad variations in the CO2 fixation kinetics of diatom Rubisco indicate novel mechanistic diversity and large differences in their carbon-concentrating mechanism.

New Jersey 08854-8020 (P.M.)Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL S ) produced tobacco Rst transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L s S t ). The tobacco Rst plants required CO 2 (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K m , for CO 2 and CO 2 /O 2 selectivity of the L s S t enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L s S t content were sufficient, tobacco Rst plants grew to maturity without CO 2 supplementation. When grown under a high pCO 2 , the tobacco Rst seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L s S t content in tobacco Rst leaves was 4-to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO 2 and growth illumination CO 2 assimilation in mature tobacco Rst leaves remained limited by Rubisco activity and its rate (approximately 11 mmol m 22 s 21 ) was half that of tobacco controls. 35 S-methionine labeling showed the stability of assembled L s S t was similar to tobacco Rubisco and measurements of light transient CO 2 assimilation rates showed L s S t was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco Rst growth primarily stem from reduced rbcL S mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L s S t synthesis.

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...

19Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for 20 improving agricultural crop productivity. Here we reveal high diversity in the kinetic 21 response between 10°C to 37°C by Rubisco from C3-and C4-species within the grass tribe 22Paniceae. The CO2-fixation rate (kcat C ) for Rubisco from the C4-grasses with NADP-malic 23 enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic 24 pathways was two-fold greater than the kcat C of Rubisco from NAD-ME species over all 25 temperatures. The decline in the response of CO2/O2 specificity with increasing 26 temperature was slower for PCK and NADP-ME Rubisco -a trait which would be 27 advantageous in the warmer climates they inhabit relative to the NAD-ME grasses. 28Variation in the temperatures kinetics of Paniceae C3-Rubisco and PCK-Rubisco were 29 modelled to differentially stimulate C3-photosynthesis above and below 25°C under current 30 and elevated CO2. Identified are large subunit amino acid substitutions that could account 31 for the catalytic variation among Paniceae Rubisco. Incompatibilities with Paniceae 32Rubisco biogenesis in tobacco however hindered their mutagenic testing by chloroplast 33 transformation. Circumventing these bioengineering limitations is critical to tailoring the 34 properties of crop Rubisco to suit future climates. 35Concerns about how escalating climate change will influence ecosystems are particularly 36 focused on the consequences to global agricultural productivity where increases are 37 paramount to meet the rising food and biofuel demands. Strategies to improve crop yield 38 by increasing photosynthesis have largely focused on overcoming the functional 39 inadequacies of the CO2-fixing enzyme Rubisco. A competing O2-fixing reaction by 40Rubisco produces a toxic product whose recycling by photorespiration consumes energy 41 and releases carbon. The frequency of the oxygenation reaction increases with temperature. 42To evade photorespiration many plants from hot, arid ecosystems have evolved C4 43 photosynthesis that concentrates CO2 around Rubisco that also facilitates improved plant 44water, light and nitrogen use. Here we show extensive catalytic variation in Rubisco from 45Paniceae grasses that align with the biochemistry and environmental origins of the different 46 C4 plant subtypes. We reveal opportunities for enhancing crop photosynthesis under 47 current and future CO2 levels at varied temperatures. 48The realization of the dire need to address global food security has heightened the need for 49 new solutions to increase crop yields 1 . Field tests and modelling analyses have highlighted 50 how photosynthetic carbon assimilation underpins the maximal yield potential of crops 2 . 51This has increased efforts to identify solutions to enhance photosynthetic efficiency and 52 hence plant productivity 3 . Particular attention is being paid to improving the rate at which 53 ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) can fix CO2 54 (refs...

494I.495II.496III.496IV.499V.499VI.501VII.501VIII.502IX.505X.506507References507 Summary The uncertainty of future climate change is placing pressure on cropping systems to continue to provide stable increases in productive yields. To mitigate future climates and the increasing threats against global food security, new solutions to manipulate photosynthesis are required. This review explores the current efforts available to improve carbon assimilation within plant chloroplasts by engineering Rubisco, which catalyzes the rate‐limiting step of CO2 fixation. Fixation of CO2 and subsequent cycling of 3‐phosphoglycerate through the Calvin cycle provides the necessary carbohydrate building blocks for maintaining plant growth and yield, but has to compete with Rubisco oxygenation, which results in photorespiration that is energetically wasteful for plants. Engineering improvements in Rubisco is a complex challenge and requires an understanding of chloroplast gene regulatory pathways, and the intricate nature of Rubisco catalysis and biogenesis, to transplant more efficient forms of Rubisco into crops. In recent times, major advances in Rubisco engineering have been achieved through improvement of our knowledge of Rubisco synthesis and assembly, and identifying amino acid catalytic switches in the L‐subunit responsible for improvements in catalysis. Improving the capacity of CO2 fixation in crops such as rice will require further advances in chloroplast bioengineering and Rubisco biogenesis.

No abstract

Ribonuclease J is an essential enzyme, and the Bacillus subtilis ortholog possesses both endoribonuclease and 59 / 39 exoribonuclease activities. Chloroplasts also contain RNase J, which has been postulated to participate, as both an exo-and endonuclease, in the maturation of polycistronic mRNAs. Here we have examined recombinant Arabidopsis RNase J and found both 59 / 39 exoribonuclease and endonucleolytic activities. Virus-induced gene silencing was used to reduce RNase J expression in Arabidopsis and Nicotiana benthamiana, leading to chlorosis but surprisingly few disruptions in the cleavage of polycistronic rRNA and mRNA precursors. In contrast, antisense RNAs accumulated massively, suggesting that the failure of chloroplast RNA polymerase to terminate effectively leads to extensive symmetric transcription products that are normally eliminated by RNase J. Mung bean nuclease digestion and polysome analysis revealed that this antisense RNA forms duplexes with sense strand transcripts and prevents their translation. We conclude that a major role of chloroplast RNase J is RNA surveillance to prevent overaccumulation of antisense RNA, which would otherwise exert deleterious effects on chloroplast gene expression.