The rubisco chaperone BSD2 may regulate chloroplast coverage in maize bundle sheath cells

In maize (Zea mays), Bundle Sheath Defective2 (BSD2) plays an essential role in Rubisco biogenesis and is required for correct bundle sheath (BS) cell differentiation. Yet, BSD2 RNA and protein levels are similar in mesophyll (M) and BS chloroplasts, although Rubisco accumulates only in BS chloroplasts. This raises the possibility of additional BSD2 roles in cell development. To test this hypothesis, transgenic lines were created that overexpress and underexpress BSD2 in both BS and M cells, driven by the cell type-specific Rubisco Small Subunit (RBCS) or Phosphoenolpyruvate Carboxylase (PEPC) promoters or the ubiquitin promoter. Genetic crosses showed that each of the transgenes could complement Rubisco deficiency and seedling lethality conferred by the bsd2 mutation. This was unexpected, as RBCS-BSD2 lines lacked BSD2 in M chloroplasts and PEPC-BSD2 lines expressed half the wild-type BSD2 level in BS chloroplasts.We conclude that BSD2 does not play a vital role inM cells and that BS BSD2 is in excess of requirements for Rubisco accumulation. BSD2 levels did affect chloroplast coverage in BS cells. In PEPC-BSD2 lines, chloroplast coverage decreased 30% to 50%, whereas lines with increased BSD2 content exhibited a 25% increase. This suggests that BSD2 has an ancillary role in BS cells related to chloroplast size. Gas exchange showed decreased photosynthetic rates in PEPC-BSD2 lines despite restored Rubisco function, correlating with reduced chloroplast coverage and pointing to CO2 diffusion changes. Conversely, increased chloroplast coverage did not result in increased Rubisco abundance or photosynthetic rates. This suggests another limitation beyond chloroplast volume, most likely Rubisco biogenesis and/or turnover rates

Synthetic biology and opportunities within agricultural crops

Conventional breeding techniques have been integral to the development of many agronomically important traits in numerous crops. The adoption of modern biotechnology approaches further advanced and refined trait development and introduction beyond the scope possible through conventional breeding. However, crop yields continue to be challenged by abiotic and biotic factors that require the development of traits that are more genetically complex than can be addressed through conventional breeding or traditional genetic engineering. Therefore, more advanced trait development approaches are required to maintain and improve yields and production efficiency, especially as climate change accelerates the incidence of biotic and abiotic challenges to food and fibre crops. Synthetic biology (SynBio) encompasses approaches that design and construct new biological elements (e.g., enzymes, genetic circuits, cells) or redesign existing biological systems to build new and improved functions. SynBio ‘upgrades’ the potential of genetic engineering, which involves the transfer of single genes from one organism to another. This technology can enable the introduction of multiple genes in a single transgenic event, either derived from a foreign organism or synthetically generated. It can also enable the assembly of novel genomes from the ground up from a set of standardised genetic parts, which can then be transferred into the target cell or organism. New opportunities to advance breeding applications through exploiting SynBio technology include the introduction of new genes of known function, artificially creating genetic variation, topical applications of small RNAs as pesticides and potentially speeding up the production of new cultivars with elite traits. This review will draw upon case studies to demonstrate the potential application of SynBio to improve crop productivity and resistance to various challenges. Here, we outline specific solutions to challenges including fungal diseases, insect pests, heat and drought stress and nutrient acquisition in a range of important crops using the SynBio toolkit

Expression of a CO2-permeable aquaporin enhances mesophyll conductance in the C4 species setaria viridis

A fundamental limitation of photosynthetic carbon fixation is the availability of CO2. In C4 plants, primary carboxylation occurs in mesophyll cytosol, and little is known about the role of CO2 diffusion in facilitating C4 photosynthesis. We have examined the expression, localization, and functional role of selected plasma membrane intrinsic aquaporins (PIPs) from Setaria italica (foxtail millet) and discovered that SiPIP2;7 is CO2-permeable. When ectopically expressed in mesophyll cells of S. viridis (green foxtail), SiPIP2;7 was localized to the plasma membrane and caused no marked changes in leaf biochemistry. Gas-exchange and C18O16O discrimination measurements revealed that targeted expression of SiPIP2;7 enhanced the conductance to CO2 diffusion from the intercellular airspace to the mesophyll cytosol. Our results demonstrate that mesophyll conductance limits C4 photosynthesis at low pCO2 and that SiPIP2;7 is a functional CO2 permeable aquaporin that can improve CO2 diffusion at the airspace/mesophyll interface and enhance C4 photosynthesis

Rht18 semidwarfism in wheat is due to increased GA 2-oxidaseA9 expression and reduced GA content

Semidwarfing genes have improved crop yield by reducing height, improving lodging resistance, and allowing plants to allocate more assimilates to grain growth. In wheat (Triticum aestivum), the Rht18 semidwarfing gene was identified and deployed in durum wheat before it was transferred into bread wheat, where it was shown to have agronomic potential. Rht18, a dominant and gibberellin (GA) responsive mutant, is genetically and functionally distinct from the widely used GA-insensitive semidwarfing genes Rht-B1b and Rht-D1b. In this study, the Rht18 gene was identified by mutagenizing the semidwarf durum cultivar Icaro (Rht18) and generating mutants with a range of tall phenotypes. Isolating and sequencing chromosome 6A of these "overgrowth"mutants showed that they contained independent mutations in the coding region of GA2oxA9. GA2oxA9 is predicted to encode a GA 2-oxidase that metabolizes GA biosynthetic intermediates into inactive products, effectively reducing the amount of bioactive GA (GA1). Functional analysis of the GA2oxA9 protein demonstrated that GA2oxA9 converts the intermediate GA12 to the inactive metabolite GA110. Furthermore, Rht18 showed higher expression of GA2oxA9 and lower GA content compared with its tall parent. These data indicate that the increased expression of GA2oxA9 in Rht18 results in a reduction of both bioactive GA content and plant height. This study describes a height-reducing mechanism that can generate new genetic diversity for semidwarfism in wheat by combining increased expression with mutations of specific amino acid residues in GA2oxA9

Reconstructing CO2 fixation from the past : analysis of Rubisco evolution could inform how to engineer a better enzyme

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is the central CO2-fixing enzyme of photosynthesis and often limits the rate of carbon assimilation for key agricultural crops. Rubisco is catalytically feeble because of its slow catalytic speed (1 to 9 catalytic cycles per second), low affinity for CO2, and low specificity for CO2 compared with O2. These catalytic parameters strongly influence crop water and nitrogen use required for efficient growth of photosynthetic organisms. The decline in CO2 and the concomitant rise of O2 in Earth’s atmosphere led to the independent evolution of carbon-concentrating mechanisms across lineages of photosynthetic organisms to improve Rubisco catalysis by increasing CO2 around the active site while effectively suppressing oxygenation. On page 155 of this issue, Schulz et al. report the ancestral reconstruction of Rubisco to predict early isoforms and the functional adaptation that enabled sufficient carbon fixation in the face of rising O2 in Earth’s atmosphere

Mix-and-match Rubisco subunits

Rubisco is a complex, ubiquitous protein composed of eight large and eight small subunits. By assembling Rubisco in bacterial cells, the specific characteristics of individual small subunits have been investigated with a view to engineering more efficient carbon fixation

Rubisco, the imperfect winner : it's all about the base

Rubisco catalysis is complex and includes an activation step through the formation of a carbamate at the conserved active site lysine residue and the formation of a highly reactive enediol that is the key to its catalytic reaction. The formation of this enediol is both the basis of its success and its Achilles' heel, creating imperfections to its catalytic efficiency. While Rubisco originally evolved in an atmosphere of high CO2, the earth's multiple oxidation events provided challenges to Rubisco through the fixation of O2 that competes with CO2 at the active site. Numerous catalytic screens across the Rubisco superfamily have identified significant variation in catalytic properties that have been linked to large and small subunit sequences. Despite this, we still have a rudimentary understanding of Rubisco's catalytic mechanism and how the evolution of kinetic properties has occurred. This review identifies the lysine base that functions both as an activator and a proton abstractor to create the enediol as a key to understanding how Rubisco may optimize its kinetic properties. The ways in which Rubisco and its partners have overcome catalytic and activation imperfections and thrived in a world of high O2, low CO2, and variable climatic regimes is remarkable

Rubisco engineering by plastid transformation and protocols for assessing expression

The assimilation of CO2 within chloroplasts is catalyzed by the bifunctional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multigene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master line and analyzing leaf Rubisco content

Plastid transformation for Rubisco engineering and protocols for assessing expression

The assimilation of CO2 within chloroplasts is catalyzed by the bi-functional enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multi-gene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long-term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master-line and analyzing leaf Rubisco content

Effects of leaf age during drought and recovery on photosynthesis, mesophyll conductance and leaf anatomy in wheat leaves

Summary statement: Mesophyll conductance (gm) was negatively correlated with wheat leaf age but was positively correlated with the surface area of chloroplasts exposed to intercellular airspaces (Sc). The rate of decline in photosynthetic rate and gm as leaves aged was slower for water-stressed than well-watered plants. Upon rewatering, the degree of recovery from water-stress depended on the age of the leaves, with the strongest recovery for mature leaves, rather than young or old leaves. Diffusion of CO2 from the intercellular airspaces to the site of Rubisco within C3 plant chloroplasts (gm) governs photosynthetic CO2 assimilation (A). However, variation in gm in response to environmental stress during leaf development remains poorly understood. Age-dependent changes in leaf ultrastructure and potential impacts on gm, A, and stomatal conductance to CO2 (gsc) were investigated for wheat (Triticum aestivum L.) in well-watered and water-stressed plants, and after recovery by re-watering of droughted plants. Significant reductions in A and gm were found as leaves aged. The oldest plants (15 days and 22 days) in water-stressed conditions showed higher A and gm compared to irrigated plants. The rate of decline in A and gm as leaves aged was slower for water-stressed compared to well-watered plants. When droughted plants were rewatered, the degree of recovery depended on the age of the leaves, but only for gm. The surface area of chloroplasts exposed to intercellular airspaces (Sc) and the size of individual chloroplasts declined as leaves aged, resulting in a positive correlation between gm and Sc. Leaf age significantly affected cell wall thickness (tcw), which was higher in old leaves compared to mature/young leaves. Greater knowledge of leaf anatomical traits associated with gm partially explained changes in physiology with leaf age and plant water status, which in turn should create more possibilities for improving photosynthesis using breeding/biotechnological strategies