Redesigning the photosynthetic light reactions to enhance photosynthesis - the PhotoRedesign consortium.

In this Perspective article, we describe the visions of the PhotoRedesign consortium funded by the European Research Council of how to enhance photosynthesis. The light reactions of photosynthesis in individual phototrophic species use only a fraction of the solar spectrum, and high light intensities can impair and even damage the process. In consequence, expanding the solar spectrum and enhancing the overall energy capacity of the process, while developing resilience to stresses imposed by high light intensities, could have a strong positive impact on food and energy production. So far, the complexity of the photosynthetic machinery has largely prevented improvements by conventional approaches. Therefore, there is an urgent need to develop concepts to redesign the light-harvesting and photochemical capacity of photosynthesis, as well as to establish new model systems and toolkits for the next generation of photosynthesis researchers. The overall objective of PhotoRedesign is to reconfigure the photosynthetic light reactions so they can harvest and safely convert energy from an expanded solar spectrum. To this end, a variety of synthetic biology approaches, including de novo design, will combine the attributes of photosystems from different photoautotrophic model organisms, namely the purple bacterium Rhodobacter sphaeroides, the cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana. In parallel, adaptive laboratory evolution will be applied to improve the capacity of reimagined organisms to cope with enhanced input of solar energy, particularly in high and fluctuating light.

Extracellular CahB1 from Sodalinema gerasimenkoae IPPAS B-353 Acts as a Functional Carboxysomal ß-Carbonic Anhydrase in Synechocystis sp. PCC6803

Cyanobacteria mostly rely on the active uptake of hydrated CO2 (i.e., bicarbonate ions) from the surrounding media to fuel their inorganic carbon assimilation. The dehydration of bicarbonate in close vicinity of RuBisCO is achieved through the activity of carboxysomal carbonic anhydrase (CA) enzymes. Simultaneously, many cyanobacterial genomes encode extracellular α- and ß-class CAs (EcaA, EcaB) whose exact physiological role remains largely unknown. To date, the CahB1 enzyme of Sodalinema gerasimenkoae (formerly Microcoleus/Coleofasciculus chthonoplastes) remains the sole described active extracellular ß-CA in cyanobacteria, but its molecular features strongly suggest it to be a carboxysomal rather than a secreted protein. Upon expression of CahB1 in Synechocystis sp. PCC6803, we found that its expression complemented the loss of endogenous CcaA. Moreover, CahB1 was found to localize to a carboxysome-harboring and CA-active cell fraction. Our data suggest that CahB1 retains all crucial properties of a cellular carboxysomal CA and that the secretion mechanism and/or the machinations of the Sodalinema gerasimenkoae carboxysome are different from those of Synechocystis.

An ancient function of PGR5 in iron delivery?

In all phototrophic organisms, the photosynthetic apparatus must be protected from light-induced damage. One important mechanism that mitigates photodamage in plants is antimycin A (AA)-sensitive cyclic electron flow (CEF), the evolution of which remains largely obscure. Here we show that proton gradient regulation 5 (PGR5), a key protein involved in AA-sensitive CEF, displays intriguing commonalities - including sequence and structural features - with a group of ferritin-like proteins. We therefore propose that PGR5 may originally have been involved in prokaryotic iron mobilization and delivery, which facilitated a primordial type of CEF as a side effect. The abandonment of the bacterioferritin system during the transformation of cyanobacterial endosymbionts into chloroplasts might have allowed PGR5 to functionally specialize in CEF.

Enhancing photosynthesis at high light levels by adaptive laboratory evolution.

Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants.

PGRL2 triggers degradation of PGR5 in the absence of PGRL1.

In plants, inactivation of either of the thylakoid proteins PGR5 and PGRL1 impairs cyclic electron flow (CEF) around photosystem I. Because PGR5 is unstable in the absence of the redox-active PGRL1, but not vice versa, PGRL1 is thought to be essential for CEF. However, we show here that inactivation of PGRL2, a distant homolog of PGRL1, relieves the need for PGRL1 itself. Conversely, high levels of PGRL2 destabilize PGR5 even when PGRL1 is present. In the absence of both PGRL1 and PGRL2, PGR5 alters thylakoid electron flow and impairs plant growth. Consequently, PGR5 can operate in CEF on its own, and is the target of the CEF inhibitor antimycin A, but its activity must be modulated by PGRL1. We conclude that PGRL1 channels PGR5 activity, and that PGRL2 triggers the degradation of PGR5 when the latter cannot productively interact with PGRL1.

Introduction of the Carotenoid Biosynthesis α-Branch Into Synechocystis sp. PCC 6803 for Lutein Production.

Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous ß-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD730 (1.03 ± 0.47 mmol mol-1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system.

Evidence that cyanobacterial Sll1217 functions analogously to PGRL1 in enhancing PGR5-dependent cyclic electron flow.

In plants and cyanobacteria, the PGR5 protein contributes to cyclic electron flow around photosystem I. In plants, PGR5 interacts with PGRL1 during cyclic electron flow, but cyanobacteria appear to lack PGRL1 proteins. We have heterologously expressed the PGR5 and PGRL1 proteins from the plant Arabidopsis in various genetic backgrounds in the cyanobacterium Synechocystis. Our results show that plant PGR5 suffices to re-establish cyanobacterial cyclic electron flow (CEF), albeit less efficiently than the cyanobacterial PGR5 or the plant PGR5 and PGRL1 proteins together. A mutation that inactivates Arabidopsis PGR5 destabilises the protein in Synechocystis. Furthermore, the Synechocystis protein Sll1217, which exhibits weak sequence similarity with PGRL1, physically interacts with both plant and cyanobacterial PGR5 proteins, and stimulates CEF in Synechocystis. Therefore, Sll1217 partially acts as a PGRL1 analogue, the mode of action of PGR5 and PGRL1/Sll1217 proteins is similar in cyanobacteria and plants, and PGRL1 could have evolved from a cyanobacterial ancestor.

The role of KDEL-tailed cysteine endopeptidases of Arabidopsis (AtCEP2 and AtCEP1) in root development.

Plants encode a unique group of papain-type cysteine endopeptidases (CysEP) characterized by a C-terminal KDEL endoplasmic reticulum retention signal (KDEL-CysEP) and an unusually broad substrate specificity. The three Arabidopsis KDEL-CysEPs (AtCEP1, AtCEP2, and AtCEP3) are differentially expressed in vegetative and generative tissues undergoing programmed cell death (PCD). While KDEL-CysEPs have been shown to be implicated in the collapse of tissues during PCD, roles of these peptidases in processes other than PCD are unknown. Using mCherry-AtCEP2 and EGFP-AtCEP1 reporter proteins in wild type versus atcep2 or atcep1 mutant plants, we explored the participation of AtCEP in young root development. Loss of AtCEP2, but not AtCEP1 resulted in shorter primary roots due to a decrease in cell length in the lateral root (LR) cap, and impairs extension of primary root epidermis cells such as trichoblasts in the elongation zone. AtCEP2 was localized to root cap corpses adherent to epidermal cells in the rapid elongation zone. AtCEP1 and AtCEP2 are expressed in root epidermis cells that are separated for LR emergence. Loss of AtCEP1 or AtCEP2 caused delayed emergence of LR primordia. KDEL-CysEPs might be involved in developmental tissue remodeling by supporting cell wall elongation and cell separation.

Enhancing (crop) plant photosynthesis by introducing novel genetic diversity.

Although some elements of the photosynthetic light reactions might appear to be ideal, the overall efficiency of light conversion to biomass has not been optimized during evolution. Because crop plants are depleted of genetic diversity for photosynthesis, efforts to enhance its efficiency with respect to light conversion to yield must generate new variation. In principle, three sources of natural variation are available: (i) rare diversity within extant higher plant species, (ii) photosynthetic variants from algae, and (iii) reconstruction of no longer extant types of plant photosynthesis. Here, we argue for a novel approach that outsources crop photosynthesis to a cyanobacterium that is amenable to adaptive evolution. This system offers numerous advantages, including a short generation time, virtually unlimited population sizes and high mutation rates, together with a versatile toolbox for genetic manipulation. On such a synthetic bacterial platform, 10 000 years of (crop) plant evolution can be recapitulated within weeks. Limitations of this system arise from its unicellular nature, which cannot reproduce all aspects of crop photosynthesis. But successful establishment of such a bacterial host for crop photosynthesis promises not only to enhance the performance of eukaryotic photosynthesis but will also reveal novel facets of the molecular basis of photosynthetic flexibility.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

Involvement of Arabidopsis thaliana endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 (AtCEP1) in powdery mildew-induced and AtCPR5-controlled cell death.

Programmed cell death (PCD) is a prerequisite for successful development and it limits the spread of biotrophic pathogens in a rapid hypersensitive response at the site of infection. KDEL-tailed cysteine endopeptidases (KDEL CysEP) are a subgroup of papain-type cysteine endopeptidases expressed in tissues undergoing PCD. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. We have previously shown that AtCEP1 is a factor of basal resistance to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum, and is expressed in spatiotemporal association with the late fungal development on Arabidopsis leaves. The endoplasmic reticulum-localized proenzyme of AtCEP1 was further visualized at the haustorial complex encased with callose. The AtCPR5 gene (CONSTITUTIVE EXPRESSION OF PR GENES 5) is a regulator of expression of pathogenesis related genes. Loss of AtCPR5 leads to spontaneous expression of chlorotic lesions which was associated with enhanced expression of AtCEP1. We used the atcpr5-2 mutant plants and the atcep1 atcpr5-2 double mutants harboring a non-functional reporter (PCEP1::pre-pro-3xHA-EGFP-KDEL) for visualization of AtCEP1 promoter activity. We found the specific up-regulation of AtCEP1 in direct neighborhood of spreading leaf lesions thus likely representing cells undergoing PCD. Furthermore, we found a strong resistance of atcpr5 mutant plants against infection with E. cruciferarum. Loss of AtCEP1 had no obvious influence on the strong resistance of atcpr5-2 mutant plants against infection with E. cruciferarum. However, the area of necrotic leaf lesions associated with E. cruciferarum colonies was significantly larger in atcpr5-2 as compared to atcep1 atcpr5-2 double mutant plants. The presence of AtCEP1 thus contributes to AtCPR5-controlled PCD at the sites of powdery mildew infection.