Spencer M. Whitney

Advancing our understanding and capacity to engineer nature’s CO2 sequestering enzyme, Rubisco

There is a growing impetus in developing novel strategies to address global concerns regarding food security. As crop productivity gains through traditional breeding begin to lag and arable land becomes scarcer, it seems that we are heading for unsustainable global populations. It has been

Analysis of carboxysomes from Synechococcus PCC7942 reveals multiple Rubisco complexes with carboxysomal proteins CcmM and CcaA.

In cyanobacteria, the key enzyme for photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral microcompartments called carboxysomes. Cyanobacteria with Form IB Rubisco produce β-carboxysomes whose putative shell proteins are encoded by the ccm-type genes. To date, very little is known of the protein-protein interactions that form the basis of β-carboxysome structure. In an effort to identify such interactions within the carboxysomes of the β-cyanobacterium Synechococcus sp. PCC7942, we have used polyhistidine-tagging approaches to identify at least three carboxysomal subcomplexes that contain active Rubisco. In addition to the expected L8S8 Rubisco, which is the major component of carboxysomes, we have identified two Rubisco complexes containing the putative shell protein CcmM, one of which also contains the carboxysomal carbonic anhydrase, CcaA. The complex containing CcaA consists of Rubisco and the full-length 58-kDa form of CcmM (M58), whereas the other is made up of Rubisco and a short 35-kDa form of CcmM (M35), which is probably translated independently of M58 via an internal ribosomal entry site within the ccmM gene. We also show that the high CO2-requiring ccmM deletion mutant (ΔccmM) can achieve nearly normal growth rates at ambient CO2 after complementation with both wild type and chimeric (His6-tagged) forms of CcmM. Although a significant amount of independent L8S8 Rubisco is confined to the center of the carboxysome, we speculate that the CcmM-CcaA-Rubisco complex forms an important assembly coordination within the carboxysome shell. A speculative carboxysome structural model is presented.

Plastome-encoded bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco

The efficiency with which crop plants use their resources of light, water, and fertilizer nitrogen could be enhanced by replacing their CO2-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), with more efficient forms, such as those found in some algae, for example. This important challenge has been frustrated by failure of all previous attempts to substitute a fully functional, foreign RubisCO (efficient or inefficient) into higher plants. This failure could be caused by incompatibility between the plastid-encoded large subunits and the nucleus-encoded small subunits or by inability of the foreign RubisCO subunits to fold or assemble efficiently in the plastid. Mismatch between the regulatory requirements of the foreign RubisCO and conditions in the chloroplast also might render the substituted enzyme inactive but, previously, it has not been possible to test this. To answer the general question of whether a foreign RubisCO can support photosynthesis in a plant, we used plastid transformation to replace RubisCO in tobacco with the simple homodimeric form of the enzyme from the α-proteobacterium, Rhodospirillum rubrum, which has no small subunits and no special assembly requirements. The transplastomic plants so obtained are fully autotrophic and reproductive but require CO2 supplementation, consistent with the kinetic properties of the bacterial RubisCO. This establishes that the activity of a RubisCO from a very different phylogeny can be integrated into chloroplast photosynthetic metabolism without prohibitive problems.

The cyanobacterial CCM as a source of genes for improving photosynthetic CO2 fixation in crop species

Crop yields need to nearly double over the next 35 years to keep pace with projected population growth. Improving photosynthesis, via a range of genetic engineering strategies, has been identified as a promising target for crop improvement with regard to increased photosynthetic yield and better water-use efficiency (WUE). One approach is based on integrating components of the highly efficient CO(2)-concentrating mechanism (CCM) present in cyanobacteria (blue-green algae) into the chloroplasts of key C(3) crop plants, particularly wheat and rice. Four progressive phases towards engineering components of the cyanobacterial CCM into C(3) species can be envisaged. The first phase (1a), and simplest, is to consider the transplantation of cyanobacterial bicarbonate transporters to C(3) chloroplasts, by host genomic expression and chloroplast targeting, to raise CO(2) levels in the chloroplast and provide a significant improvement in photosynthetic performance. Mathematical modelling indicates that improvements in photosynthesis as high as 28% could be achieved by introducing both of the single-gene, cyanobacterial bicarbonate transporters, known as BicA and SbtA, into C(3) plant chloroplasts. Part of the first phase (1b) includes the more challenging integration of a functional cyanobacterial carboxysome into the chloroplast by chloroplast genome transformation. The later three phases would be progressively more elaborate, taking longer to engineer other functional components of the cyanobacterial CCM into the chloroplast, and targeting photosynthetic and WUE efficiencies typical of C(4) photosynthesis. These later stages would include the addition of NDH-1-type CO(2) pumps and suppression of carbonic anhydrase and C(3) Rubisco in the chloroplast stroma. We include a score card for assessing the success of physiological modifications gained in phase 1a.

Using Deubiquitylating Enzymes as Research Tools

Ubiquitin is synthesized in eukaryotes as a linear fusion with a normal peptide bond either to itself or to one of two ribosomal proteins and, in the latter case, enhances the yield of these ribosomal proteins and/or their incorporation into the ribosome. Such fusions are cleaved rapidly by a variety of deubiquitylating enzymes. Expression of heterologous proteins as linear ubiquitin fusions has been found to significantly increase the yield of unstable or poorly expressed proteins in either bacterial or eukaryotic hosts. If expressed in bacterial cells, the fusion is not cleaved due to the absence of deubiquitylating activity and can be purified intact. We have developed an efficient expression system, utilizing the ubiquitin fusion technique and a robust deubiquitylating enzyme, which allows convenient high yield and easy purification of authentic proteins. An affinity purification tag on both the ubiquitin fusion and the deubiquitylating enzyme allows their easy purification and the easy removal of unwanted components after cleavage, leaving the desired protein as the only soluble product. Ubiquitin is also conjugated to epsilon amino groups in lysine side chains of target proteins to form a so-called isopeptide linkage. Either a single ubiquitin can be conjugated or other lysines within ubiquitin can be acceptors for further conjugation, leading to formation of a branched, isopeptide-linked ubiquitin chain. Removal of these ubiquitin moieties or chains in vitro would be a valuable tool in the ubiquitinologists tool kit to simplify downstream studies on ubiquitylated targets. The robust deubiquitylating enzyme described earlier is also very useful for this task.

Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants.

Transgenic manipulation of the photosynthetic CO2-fixing enzyme, ribulose bisphosphate carboxylase/oxygenase (Rubisco) in higher plants provides a very specific means of testing theories about photosynthesis and its regulation. It also encourages prospects for radically improving the efficiencies with which photosynthesis and plants use the basic resources of light, water, and nutrients. Manipulation was once limited to variation of the leafs total content of Rubisco by transforming the nucleus with antisense genes directed at the small subunit. More recently, technology for transforming the small genome of the plastid of tobacco has enabled much more precise manipulation and replacement of the plastome-encoded large subunit. Engineered changes in Rubiscos properties in vivo are reflected as profound changes in the photosynthetic gas-exchange properties of the leaves and the growth requirements of the plants. Unpredictable expression of plastid transgenes and assembly requirements of some foreign Rubiscos that are not satisfied in higher-plant plastids provide challenges for future research.

Heat stress causes inhibition of the de novo synthesis of antenna proteins and photobleaching in cultured Symbiodinium

Coral bleaching, caused by heat stress, is accompanied by the light-induced loss of photosynthetic pigments in in situ symbiotic dinoflagellate algae (Symbiodinium spp.). However, the molecular mechanisms responsible for pigment loss are poorly understood. Here, we show that moderate heat stress causes photobleaching through inhibition of the de novo synthesis of intrinsic light-harvesting antennae [chlorophyll a–chlorophyll c2–peridinin–protein complexes (acpPC)] in cultured Symbiodinium algae and that two Clade A Symbiodinium species showing different thermal sensitivities of photobleaching also show differential sensitivity of this key protein synthesis process. Photoinhibition of photosystem II (PSII) and subsequent photobleaching were observed at temperatures of >31°C in cultured Symbiodinium CS-73 cells grown at 25–34°C, but not in cultures of the more thermally tolerant control Symbiodinium species OTcH-1. We found that bleaching in CS-73 is associated with loss of acpPC, which is a major antennae protein in Symbiodinium. In addition, the thermally induced loss of this protein is light-dependent, but does not coincide directly with PSII photoinhibition and is not caused by stimulated degradation of acpPC. In cells treated at 34°C over 24 h, the steady-state acpPC mRNA pool was modestly reduced, by ≈30%, whereas the corresponding synthesis rate of acpPC was diminished by >80%. Our results suggest that photobleaching in Symbiodinium is consequentially linked to the relative susceptibility of PSII to photoinhibition during thermal stress and occurs, at least partially, because of the loss of acpPC via undefined mechanism(s) that hamper the de novo synthesis of acpPC primarily at the translational processing step.

The Catalytic Properties of Hybrid Rubisco Comprising Tobacco Small and Sunflower Large Subunits Mirror the Kinetically Equivalent Source Rubiscos and Can Support Tobacco Growth

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcLS) produced tobaccoRst transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (LsSt). The tobaccoRst plants required CO2 (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, Km, for CO2 and CO2/O2 selectivity of the LsSt enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf LsSt content were sufficient, tobaccoRst plants grew to maturity without CO2 supplementation. When grown under a high pCO2, the tobaccoRst 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 LsSt content in tobaccoRst leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO2 and growth illumination CO2 assimilation in mature tobaccoRst leaves remained limited by Rubisco activity and its rate (approximately 11 μmol m−2 s−1) was half that of tobacco controls. 35S-methionine labeling showed the stability of assembled LsSt was similar to tobacco Rubisco and measurements of light transient CO2 assimilation rates showed LsSt was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobaccoRst growth primarily stem from reduced rbcLS mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted LsSt synthesis.

Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species

Coral bleaching caused by heat stress is accompanied by photoinhibition, which occurs under conditions where the rate of photodamage to photosystem II (PSII) exceeds the rate of its repair, in the symbiotic algae (Symbiodinium spp.) within corals. However, the mechanism of heat stress-induced photoinhibition in Symbiodinium still remains poorly understood. In the present work, we have investigated the effect of elevated temperature on the processes associated with the repair of photodamaged PSII in cultured Symbiodinium (OTcH-1 and CS-73). Severe photoinhibition was observed at temperature exceeding 32 °C in Symbiodinium CS-73 cells grown at 25–34 °C but not in cultures of the more thermally tolerant Symbiodinium OTcH-1. After photoinhibition treatment by strong light, photodamaged PSII was repaired close to initial levels under low light at 25 °C in both OTcH-1 and CS-73. However, the repair was strongly inhibited by increased temperature exceeding 31 °C in CS-73 but only weakly in OTcH-1. We found that inhibition of the repair process in CS-73 is attributed to impairment of both protein synthesis-dependent and -independent repair processes and is at least partially caused by suppression of the de novo synthesis of thylakoid membrane proteins and impairment of the generation of ΔpH across the thylakoid membrane, respectively. Our results suggest that acceleration of photoinhibition by moderate heat stress is attributed primarily to inhibition of the repair of photodamaged PSII and that the photoinhibition sensitivity of Symbiodinium to heat stress is determined by the thermal sensitivity of the PSII repair processes.

Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

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 C4 photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C3 ancestors, C4 plants combine a faster rubisco with a biochemical CO2-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 C4-rubisco that can be transplanted into C3-rubisco to improve carboxylation rate (VC). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C3), Flaveria floridana (C3-C4), and Flaveria bidentis (C4), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C4 (309Ile; faster VC, lower CO2 affinity) and C3 (309Met; slower VC, higher CO2 affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco VC in C3 crops. Coengineering a catalytically faster C3 rubisco and a CO2-concentrating mechanism within C3 crop species could enhance their efficiency in resource use and yield.