Perspectives on improving photosynthesis to increase crop yield.

Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase CO2 concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.

A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria, and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

New horizons for building pyrenoid-based CO2-concentrating mechanisms in plants to improve yields.

Many photosynthetic species have evolved CO2-concentrating mechanisms (CCMs) to improve the efficiency of CO2 assimilation by Rubisco and reduce the negative impacts of photorespiration. However, the majority of plants (i.e. C3 plants) lack an active CCM. Thus, engineering a functional heterologous CCM into important C3 crops, such as rice (Oryza sativa) and wheat (Triticum aestivum), has become a key strategic ambition to enhance yield potential. Here, we review recent advances in our understanding of the pyrenoid-based CCM in the model green alga Chlamydomonas reinhardtii and engineering progress in C3 plants. We also discuss recent modeling work that has provided insights into the potential advantages of Rubisco condensation within the pyrenoid and the energetic costs of the Chlamydomonas CCM, which, together, will help to better guide future engineering approaches. Key findings include the potential benefits of Rubisco condensation for carboxylation efficiency and the need for a diffusional barrier around the pyrenoid matrix. We discuss a minimal set of components for the CCM to function and that active bicarbonate import into the chloroplast stroma may not be necessary for a functional pyrenoid-based CCM in planta. Thus, the roadmap for building a pyrenoid-based CCM into plant chloroplasts to enhance the efficiency of photosynthesis now appears clearer with new challenges and opportunities.

The small subunit of Rubisco and its potential as an engineering target.

Rubisco catalyses the first rate-limiting step in CO2 fixation and is responsible for the vast majority of organic carbon present in the biosphere. The function and regulation of Rubisco remain an important research topic and a longstanding engineering target to enhance the efficiency of photosynthesis for agriculture and green biotechnology. The most abundant form of Rubisco (Form I) consists of eight large and eight small subunits, and is found in all plants, algae, cyanobacteria, and most phototrophic and chemolithoautotrophic proteobacteria. Although the active sites of Rubisco are located on the large subunits, expression of the small subunit regulates the size of the Rubisco pool in plants and can influence the overall catalytic efficiency of the Rubisco complex. The small subunit is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. Here we review our current understanding of the role of the small subunit and our growing capacity to explore its potential to modulate Rubisco catalysis using engineering biology approaches.

A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii.

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2 fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga Chlamydomonas that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant's phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

Demonstration of protein capture and separation using three-dimensional printed anion exchange monoliths fabricated in one-step.

Three-dimensional printing applications in separation science are currently limited by the lack of materials compatible with chromatographic operations and three-dimensional printing technologies. In this work, we propose a new material for Digital Light Processing printing to fabricate functional ion exchange monoliths in a single step. Through copolymerization of the bifunctional monomer [2-(acryloyloxy)ethyl] trimethylammonium chloride, monolithic structures with quaternary amine ligands were fabricated. The novel formulation was optimized in terms of protein binding and recovery, microporous structure, and its swelling susceptibility by increasing its cross-link density and employing cyclohexanol and dodecanol as pore forming agents. In static conditions, the material demonstrated a maximum binding capacity of 104.2 ± 10.6 mg/mL for bovine serum albumin, in line with commercially available materials. Its anion exchange behavior was validated by separating bovine serum albumin and myoglobin on a monolithic bed with Schoen gyroid morphology. The same column geometry was tested for the purification of C-phycocyanin from clarified as well as cell-laden Arthrospira platensis feedstocks. This represents the first demonstration of one-step printed stationary phases to capture proteins directly from solid-laden feedstocks. We believe that the material presented here represents a significant improvement towards implementation of three-dimensional printed chromatography media in the field of separation science.

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

Phycobiliproteins from extreme environments and their potential applications.

The light-harvesting phycobilisome complex is an important component of photosynthesis in cyanobacteria and red algae. Phycobilisomes are composed of phycobiliproteins, including the blue phycobiliprotein phycocyanin, that are considered high-value products with applications in several industries. Remarkably, several cyanobacteria and red algal species retain the capacity to harvest light and photosynthesise under highly selective environments such as hot springs, and flourish in extremes of pH and elevated temperatures. These thermophilic organisms produce thermostable phycobiliproteins, which have superior qualities much needed for wider adoption of these natural pigment-proteins in the food, textile, and other industries. Here we review the available literature on the thermostability of phycobilisome components from thermophilic species and discuss how a better appreciation of phycobiliproteins from extreme environments will benefit our fundamental understanding of photosynthetic adaptation and could provide a sustainable resource for several industrial processes.

Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.

The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

The pyrenoidal linker protein EPYC1 phase separates with hybrid Arabidopsis-Chlamydomonas Rubisco through interactions with the algal Rubisco small subunit.

Photosynthetic efficiencies in plants are restricted by the CO2-fixing enzyme Rubisco but could be enhanced by introducing a CO2-concentrating mechanism (CCM) from green algae, such as Chlamydomonas reinhardtii (hereafter Chlamydomonas). A key feature of the algal CCM is aggregation of Rubisco in the pyrenoid, a liquid-like organelle in the chloroplast. Here we have used a yeast two-hybrid system and higher plants to investigate the protein-protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1), a linker protein required for Rubisco aggregation. We showed that EPYC1 interacts with the small subunit of Rubisco (SSU) from Chlamydomonas and that EPYC1 has at least five SSU interaction sites. Interaction is crucially dependent on the two surface-exposed α-helices of the Chlamydomonas SSU. EPYC1 could be localized to the chloroplast in higher plants and was not detrimental to growth when expressed stably in Arabidopsis with or without a Chlamydomonas SSU. Although EPYC1 interacted with Rubisco in planta, EPYC1 was a target for proteolytic degradation. Plants expressing EPYC1 did not show obvious evidence of Rubisco aggregation. Nevertheless, hybrid Arabidopsis Rubisco containing the Chlamydomonas SSU could phase separate into liquid droplets with purified EPYC1 in vitro, providing the first evidence of pyrenoid-like aggregation for Rubisco derived from a higher plant.

CRISPR/Cas in Arabidopsis: overcoming challenges to accelerate improvements in crop photosynthetic efficiencies.

The rapid and widespread adoption of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technologies has allowed genetic editing in plants to enter a revolutionary new era. In this mini review, we highlight the current CRISPR/Cas tools available in plants and the use of Arabidopsis thaliana as a model to guide future improvements in crop yields, such as enhancing photosynthetic potential. We also outline the current socio-political landscape for CRISPR/Cas research and highlight the growing need for governments to better facilitate research into plant genetic-editing technologies.

Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis.

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 CO2 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.

Progress and challenges of engineering a biophysical CO2-concentrating mechanism into higher plants.

Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.

Lack of fructose 2,6-bisphosphate compromises photosynthesis and growth in Arabidopsis in fluctuating environments.

The balance between carbon assimilation, storage and utilisation during photosynthesis is dependent on partitioning of photoassimilate between starch and sucrose, and varies in response to changes in the environment. However, the extent to which the capacity to modulate carbon partitioning rapidly through short-term allosteric regulation may contribute to plant performance is unknown. Here we examine the physiological role of fructose 2,6-bisphosphate (Fru-2,6-P2 ) during photosynthesis, growth and reproduction in Arabidopsis thaliana (L.). In leaves this signal metabolite contributes to coordination of carbon assimilation and partitioning during photosynthesis by allosterically modulating the activity of cytosolic fructose-1,6-bisphosphatase. Three independent T-DNA insertional mutant lines deficient in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP), the bifunctional enzyme responsible for both the synthesis and degradation of Fru-2,6-P2 , lack Fru-2,6-P2 . These plants have normal steady-state rates of photosynthesis, but exhibit increased partitioning of photoassimilate into sucrose and have delayed photosynthetic induction kinetics. The F2KP-deficient plants grow normally in constant environments, but show reduced growth and seed yields relative to wildtype plants in fluctuating light and/or temperature. We conclude that Fru-2,6-P2 is required for optimum regulation of photosynthetic carbon metabolism under variable growth conditions. These analyses suggest that the capacity of Fru-2,6-P2 to modulate partitioning of photoassimilate is an important determinant of growth and fitness in natural environments.

Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components.

Many eukaryotic green algae possess biophysical carbon-concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO2 concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H(14) CO3 (-) uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild-type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.

Engineering highly productive cyanobacteria towards carbon negative emissions technologies.

Cyanobacteria are a diverse and ecologically important group of photosynthetic prokaryotes that contribute significantly to the global carbon cycle through the capture of CO2 as biomass. Cyanobacterial biotechnology could play a key role in a sustainable bioeconomy through negative emissions technologies (NETs), such as carbon sequestration or bioproduction. However, the primary issues of low productivities and high infrastructure costs currently limit the commercialisation of such applications. The isolation of several fast-growing strains and recent advancements in molecular biology tools now offer promising new avenues for improving yields, including metabolic engineering approaches guided by high-throughput screening and metabolic models. Furthermore, emerging research on engineering coculture communities could help to develop more robust culturing systems to support broader NET applications.

Comparison of power output by rice (Oryza sativa) and an associated weed (Echinochloa glabrescens) in vascular plant bio-photovoltaic (VP-BPV) systems.

Vascular plant bio-photovoltaics (VP-BPV) is a recently developed technology that uses higher plants to harvest solar energy and the metabolic activity of heterotrophic microorganisms in the plant rhizosphere to generate electrical power. In the present study, electrical output and maximum power output variations were investigated in a novel VP-BPV configuration using the crop plant rice (Oryza sativa L.) or an associated weed, Echinochloa glabrescens (Munro ex Hook. f.). In order to compare directly the physiological performances of these two species in VP-BPV systems, plants were grown in the same soil and glasshouse conditions, while the bio-electrochemical systems were operated in the absence of additional energy inputs (e.g. bias potential, injection of organic substrate and/or bacterial pre-inoculum). Diurnal oscillations were clearly observed in the electrical outputs of VP-BPV systems containing the two species over an 8-day growth period. During this 8-day period, O. sativa generated charge ∼6 times faster than E. glabrescens. This greater electrogenic activity generated a total charge accumulation of 6.75 ± 0.87 Coulombs for O. sativa compared to 1.12 ± 0.16 for E. glabrescens. The average power output observed over a period of about 30 days for O. sativa was significantly higher (0.980 ± 0.059 GJ ha(-1) year(-1)) than for E. glabrescens (0.088 ± 0.008 GJ ha(-1) year(-1)). This work indicates that electrical power can be generated in both VP-BPV systems (O. sativa and E. glabrescens) when bacterial populations are self-forming. Possible reasons for the differences in power outputs between the two plant species are discussed.