Britta Förster

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.

A simple chlorophyll fluorescence parameter that correlates with the rate coefficient of photoinactivation of photosystem II.

A method of partitioning the energy in a mixed population of active and photoinactivated Photosystem II (PS II) complexes based on chlorophyll fluorescence measurements is presented. There are four energy fluxes, each with its quantum efficiency: a flux associated with photochemical electron flow in active PS II reaction centres (JPSII), thermal dissipation in photoinactivated, non-functional PS IIs (JNF), light-regulated thermal dissipation in active PS IIs (JNPQ) and a combined flux of fluorescence and constitutive, light-independent thermal dissipation (Jf,D). The four quantum efficiencies add up to 1.0, without the need to introduce an ‘excess’ term E, which in other studies has been claimed to be linearly correlated with the rate coefficient of photoinactivation of PS II (kpi). We examined the correlation of kpi with various fluxes, and found that the combined flux (JNPQ + Jf,D= Jpi) is as well correlated with kpi as is E. This combined flux arises from Fs/Fm′, the ratio of steady-state to maximum fluorescence during illumination, which represents the quantum efficiency of combined non-photochemical dissipation pathways in active PS IIs. Since Fs/Fm′ or its equivalent, Jpi, is a likely source of events leading to photoinactivation of PS II, we conclude that Fs/Fm′ is a simple predictor of kpi.

Cyanobacterial carboxysomes: microcompartments that facilitate CO2 fixation.

Carboxysomes are extraordinarily efficient proteinaceous microcompartments that encapsulate the primary CO2-fixing enzyme (ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) in cyanobacteria and some proteobacteria. These microbodies form part of a CO2-concentrating mechanism (CCM), operating together with active CO2 and HCO3- uptake transporters which accumulate HCO3- in the cytoplasm of the cell. Cyanobacteria (also known as blue-green algae) are highly productive on a global scale, especially those species from open-ocean niches, which collectively contribute nearly 30% of global net primary fixation. This productivity would not be possible without a CCM which is dependent on carboxysomes. Two evolutionarily distinct forms of carboxysome are evident that encapsulate proteobacterial RuBisCO form-1A or higher-plant RuBisCO form- 1B, respectively. Based partly on RuBisCO phylogeny, the two carboxysome types are known either as α-carboxysomes, found in predominantly oceanic cyanobacteria (α-cyanobacteria) and some proteobacteria, or as β-carboxysomes, found mainly in freshwater/estuarine cyanobacteria (β-cyanobacteria). Both carboxysome types are believed to have evolved in parallel as a consequence of fluctuating atmospheric CO2 levels and evolutionary pressure acting via the poor enzymatic kinetics of RuBisCO. The three-dimensional structures and protein components of each carboxysome type reflect distinct evolutionarily strategies to the same major functions: subcellular compartmentalization and RuBisCO encapsulation, oxygen exclusion, and CO2 concentration and fixation.

Photoinhibition: Then and Now

This perspective advocates a holistic view of photoinhibition from the molecule to the biosphere; a view that integrates many biophysical and biochemical processes in antennae and reaction centers of the photosystems that, when acting in concert, allow plants to respond to diverse and dynamic light conditions in many different environments. We take the general view that photoinhibition refers to a reduction in the efficiency of light use in the photosynthetic apparatus (Kok, 1956). Since the 1970s, biochemical, ecophysiological, and genetic studies of photosynthetic functions in strong light, in vivoand in situ, and their interactions with biotic and abiotic stresses, have significantly advanced our understanding of photoinhibition. We trace some origins of the idea then, that slow dark reactions, such as growth, CO2 assimilation, photorespiration, and photosynthetic electron transport, ultimately limit light use in photosynthesis, and thus determine whether light is in excess and the magnitude of “excitation pressure” in the photosynthetic apparatus at any moment. This and other ideas are followed through studies of photoacclimation in leaves of plants and algae from diverse terrestrial and marine environments. We highlight two currently interesting possibilities for the photoprotective dissipation of “excitation pressure” that reduce the efficiency of photosynthesis by changes in structure and function of antenna pigment-protein complexes and in the populations of functional and non-functional PS II centers. We conclude by briefly considering challenges presented nowby the discovery of “gain of function”, very high light resistant (VHLR) mutants of Chlamydomonas, by the accessory lutein-epoxide cycle, and by technologies for remote sensing of photoinhibition in the field.

Lutein from Deepoxidation of Lutein Epoxide Replaces Zeaxanthin to Sustain an Enhanced Capacity for Nonphotochemical Chlorophyll Fluorescence Quenching in Avocado Shade Leaves in the Dark

Leaves of avocado (Persea americana) that develop and persist in deep shade canopies have very low rates of photosynthesis but contain high concentrations of lutein epoxide (Lx) that are partially deepoxidized to lutein (L) after 1 h of exposure to 120 to 350 μmol photons m−2 s−1, increasing the total L pool by 5% to 10% (ΔL). Deepoxidation of Lx to L was near stoichiometric and similar in kinetics to deepoxidation of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). Although the V pool was restored by epoxidation of A and Z overnight, the Lx pool was not. Depending on leaf age and pretreatment, the pool of ΔL persisted for up to 72 h in the dark. Metabolism of ΔL did not involve epoxidation to Lx. These contrasting kinetics enabled us to differentiate three states of the capacity for nonphotochemical chlorophyll fluorescence quenching (NPQ) in attached and detached leaves: ΔpH dependent (NPQΔpH) before deepoxidation; after deepoxidation in the presence of ΔL, A, and Z (NPQΔLAZ); and after epoxidation of A+Z but with residual ΔL (NPQΔL). The capacity of both NPQΔLAZ and NPQΔL was similar and 45% larger than NPQΔpH, but dark relaxation of NPQΔLAZ was slower. The enhanced capacity for NPQ was lost after metabolism of ΔL. The near equivalence of NPQΔLAZ and NPQΔL provides compelling evidence that the small dynamic pool ΔL replaces A+Z in avocado to “lock in” enhanced NPQ. The results are discussed in relation to data obtained with other Lx-rich species and in mutants of Arabidopsis (Arabidopsis thaliana) with increased L pools.

De Novo Synthesis and Degradation of Lx and V Cycle Pigments during Shade and Sun Acclimation in Avocado Leaves

The photoprotective role of the universal violaxanthin cycle that interconverts violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) is well established, but functions of the analogous conversions of lutein-5,6-epoxide (Lx) and lutein (L) in the selectively occurring Lx cycle are still unclear. We investigated carotenoid pools in Lx-rich leaves of avocado (Persea americana) during sun or shade acclimation at different developmental stages. During sun exposure of mature shade leaves, an unusual decrease in L preceded the deepoxidation of Lx to L and of V to A+Z. In addition to deepoxidation, de novo synthesis increased the L and A+Z pools. Epoxidation of L was exceptionally slow, requiring about 40 d in the shade to restore the Lx pool, and residual A+Z usually persisted overnight. In young shade leaves, the Lx cycle was reversed initially, with Lx accumulating in the sun and declining in the shade. De novo synthesis of xanthophylls did not affect α- and β-carotene pools on the first day, but during long-term acclimation α-carotene pools changed noticeably. Nonetheless, the total change in α- and β-branch carotenoid pools was equal. We discuss the implications for regulation of metabolic flux through the α- and β-branches of carotenoid biosynthesis and potential roles for L in photoprotection and Lx in energy transfer to photosystem II and explore physiological roles of both xanthophyll cycles as determinants of photosystem II efficiency.

Very high light resistant mutants of Chlamydomonas reinhardtii : Responses of Photosystem II, nonphotochemical quenching and xanthophyll pigments to light and CO 2

We have isolated very high light resistant nuclear mutants (VHLR) in Chlamydomonas reinhardtii, that grow in 1500–2000 μmol photons m−2 s−1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHLR mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L*) from which another class of VHLR mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHLR mutants was independent of CO2 availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 μmol photons m−2 s−1) was strongly depressed when all genotypes were grown in 5% CO2. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO2, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid ΔpH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO2 at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L* and VHLR mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO2, possibly because they were unable to maintain sufficiently high ΔpH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHLR phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHLR mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.

Transplastomic integration of a cyanobacterial bicarbonate transporter into tobacco chloroplasts

Improving global yields of agricultural crops is a complex challenge with evidence indicating benefits in productivity are achieved by enhancing photosynthetic carbon assimilation. Towards improving rates of CO2 capture within leaf chloroplasts, this study shows the versatility of plastome transformation for expressing the Synechococcus PCC7002 BicA bicarbonate transporter within tobacco plastids. Fractionation of chloroplast membranes from transplastomic tobBicA lines showed that ~75% of the BicA localized to the thylakoid membranes and ~25% to the chloroplast envelope. BicA levels were highest in young emerging tobBicA leaves (0.12 μmol m–2, ≈7mg m–2) accounting for ~0.1% (w/w) of the leaf protein. In these leaves, the molar amount of BicA was 16-fold lower than the abundant thylakoid photosystem II D1 protein (~1.9 μmol m–2) which was comparable to the 9:1 molar ratio of D1:BicA measured in air-grown Synechococcus PCC7002 cells. The BicA produced had no discernible effect on chloroplast ultrastructure, photosynthetic CO2-assimilation rates, carbon isotope discrimination, or growth of the tobBicA plants, implying that the bicarbonate transporter had little or no activity. These findings demonstrate the utility of plastome transformation for targeting bicarbonate transporter proteins into the chloroplast membranes without impeding growth or plastid ultrastructure. This study establishes the span of experimental measurements required to verify heterologous bicarbonate transporter function and location in chloroplasts and underscores the need for more detailed understanding of BicA structure and function to identify solutions for enabling its activation and operation in leaf chloroplasts.

From ecophysiology to phenomics: some implications of photoprotection and shade–sun acclimation in situ for dynamics of thylakoids in vitro

Half a century of research into the physiology and biochemistry of sun–shade acclimation in diverse plants has provided reality checks for contemporary understanding of thylakoid membrane dynamics. This paper reviews recent insights into photosynthetic efficiency and photoprotection from studies of two xanthophyll cycles in old shade leaves from the inner canopy of the tropical trees Inga sapindoides and Persea americana (avocado). It then presents new physiological data from avocado on the time frames of the slow coordinated photosynthetic development of sink leaves in sunlight and on the slow renovation of photosynthetic properties in old leaves during sun to shade and shade to sun acclimation. In so doing, it grapples with issues in vivo that seem relevant to our increasingly sophisticated understanding of ΔpH-dependent, xanthophyll-pigment-stabilized non-photochemical quenching in the antenna of PSII in thylakoid membranes in vitro.

Characterisation of cyanobacterial bicarbonate transporters in E. coli shows that SbtA homologs are functional in this heterologous expression system.

Cyanobacterial HCO3 - transporters BCT1, SbtA and BicA are important components of cyanobacterial CO2-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six β-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO3 - transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants.