Dynamics of Photosystem II during short and long term response to light intensity: a biochemical and biophysical study

La fotosintesi è il processo attraverso il quale le piante assorbono l'energia solare per convertirla in energia chimica e, infine, in biomassa. Durante questo processo, varie funzioni chiave sono svolte dai fotosistemi, PSI e PSII, i quali sono straordinarie macchine per l'uso dell'energia solare, combinando l'alta efficienza quantica e la presenza di meccanismi inducibili al fine di evitare fotoinibizione. L'organizzazione peculiare dei fotosistemi è determinante per la loro funzione. Entrambi i fotosistemi sono costituiti da due porzioni: un complesso core e i complessi antenna (famiglia di proteine LHC). Le proteineLHC svolgono un ruolo fondamentale nella fotosintesi , essendo coinvolte nella raccolta della luce e nella fotoprotezione. Le proteine antenna del PSII, le subunità Lhcb, sono responsabili per il meccanismo di dissipazione termica di energia di eccitazione in eccesso (NPQ, quenching non-fotochimico). Chiarire i dettagli molecolari di induzione di NPQ nelle piante superiori è una grande sfida: nel mio lavoro di dottorato di ricerca, ho studiato in particolare la riorganizzazione dei domini proteici all'interno delle membrane tilacoidali dopo trattamento con un eccesso di luce, verificando la sua importanza per il pieno funzionamento durante NPQ.Photosynthesis is the process by which plants absorb solar energy and convert it to chemical energy and finally biomass. During this process, several key functions are carried out by Photosystems. PSI and PSII represent extraordinary machines for solar energy use, combining high quantum efficiency and the presence of inducible mechanisms in order to avoid photoinhibition which unavoidably derives from performing photosynthesis in oxygenic environment. The peculiar organization of Photosystems is determinant for function. Both Photosystems are composed by two moieties: a core complex and the antenna system (LHC protein family). LHC proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. Among other photoprotecting mechanisms, antenna proteins of PSII, so-called Lhcb subunits, are responsible for the mechanism of thermal dissipation of excitation energy in excess (NPQ, non-photochemical quenching). Elucidating the molecular details of NPQ induction in higher plants has proven to be a major challenge. In my phD work, I investigated the reorganization of the protein domains inside grana membranes upon high light treatment and verified its importance for full functioning of NPQ. Below the main results obtained are summarized. Section A. Zeaxanthin modulates energy quenching properties of monomeric Lhcb antenna proteins. Among photosynthetic pigments, a special role is played by zeaxanthin (Zea), which is only accumulated under excess light. We studied the dynamics of xanthophylls binding to Lhcb proteins upon exposure of leaves to excess light. We found that Lhcb6 undergoes faster Zea accumulation than any other thylakoid protein so far described. We then studied in vitro modulation of Lhcb6 (CP24) functional properties by studying the effects of binding different xanthophyll species by using several spectroscopic techniques. The results suggest for Lhcb6 a special role in binding Zea and enhancing photoprotection under excess light. The Lhcb6 subunit is a recent addition to the photosynthetic apparatus of viridiplantae, being absent in algae and first appearing in mosses, together with the adaptation to the highly stressful conditions typical of land environment. Consistently, it is involved in several regulation mechanisms, as evidenced by genetic (de Bianchi, S. et al. 2008, Kovacs, L. et al. 2006) and biochemical analysis (Ballottari, M. et al. 2007). Previously it was reported to be involved in Non-Photochemical Quenching (NPQ) (Ahn, T. K. et al. 2008, Avenson, T. J. et al. 2008). In the second part of this section we focuses on fluorescence quenching and compared the effect of aggregation, which has been proposed to occur in vivo during NPQ due to a conformational change allowing energy transfer from Chl a excited states to the short lived carotenoid S1 excited state (Ruban, A. V. et al. 2007). This aggregation-dependent quenching (ADQ) has been proposed as an alternative to charge transfer quenching (CTQ) (Ahn, T. K. et al. 2008) mechanism proposed by other groups, including our laboratory. We studied the properties, particularly dependence on zeaxanthin binding, of ADQ using time-resolved and steady state spectroscopy. We obtained evidence that monomeric Lhcb proteins undergo ADQ even better than trimeric LHCII for which this mechanism was originally proposed. In these proteins the amplitude of the process is enhanced by zeaxanthin, while this is not the case for LHCII. Nevertheless, when LHCII is mixed with Lhcb6, this provides zeaxanthin-deppendent enhancement. This result complements previous studies of CTQ, which localized the quenching site to Chl 603, Chl 609, and Zea in carotenoid-binding site L2 (Ahn, T. K. et al. 2008) and suggests that two different types of quenching may occur in Lhcb proteins. Section B. Membrane dynamics during NPQ: PsbS and zeaxanthin-dependent reorganization of Photosystem II is controlled by dissociation of a pentameric supercomplex. Antenna subunits heve been shown to host the site of energy quenching, while the trigger of the mechanism is mediated by PsbS (Bonente, G. et al. 2008), a PSII subunit involved in transducing the signal of over-excitation consisting into lumen acidification (Li, X. P. et al. 2002, Li, X. P. et al. 2004). In this section we investigate the molecular mechanism by which PsbS regulates light harvesting efficiency. We showed that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins, Lhcb4 and Lhcb6 and the trimeric LHCII-M (namely Band 4 Complex - B4C). We demonstrated that the dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light. Direct observation of grana membranes upon treatment with excess light for different timelengths by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes, reducing average distances between PSII core complexes. This phenomenon was reversible upon dark relaxation. We interpret these results proposing that the dissociation of B4C makes quenching sites, possibly Lhcb4 and Lhcb6, available for the switch to an energy-quenching conformation. Section C. New insights on the role of the monomeric Lhcb4 antenna subunit. PSII is surrounded by a external antenna system composed by trimeric LHCII and monomeric minor antenna complexes. Several evidences suggests that Lhcb4, in particular, is a key factor in both light harvesting and photoprotection (Ballottari, M. et al. 2007) or under chronic excitation of PSII (Morosinotto, T. et al. 2006), b) is able to perform charge transfer quenching (Ahn, T. K. et al. 2008). We characterized the function of Lhcb4 subunits in Arabidopsis thaliana. In order to determine the function of Lhcb4 in A. thaliana, we have constructed knock-out mutants lacking one or more Lhcb4 isoforms and analyzed their performance in photosynthesis and photoprotection. The absence of Lhcb4 also caused a destabilization of PSII supercomplexes, modifying antenna system organization. The distribution of PSII complexes within grana membranes is affected in koLhcb4 and LHCII enriched domains are formed. PSII quantum efficiency and NPQ activity were affected, and photoprotection efficiency under high light conditions was impaired in koLhcb4 plants with respect to either WT or mutants depleted of any other Lhcb subunit. Electron microscopy analysis reveal that PSII supercomplex from koLhcb4 plants bears a hole in its structure. We conclude that Lhcb4 is a fundamental component of PSII which is essential for maintenance of both the function and structural organization of this photosystem. Section D. Chlorophyll b reductase affected the regulation of antenna complexes during light stress. The molecular mechanism of antenna complexes breakdown is largely unknown and it is still unclear whether chlorophyll degradation precedes the degradation of the protein moiety or whether protein degradation is the first event. Recently chlorophyll b reductase mutant has been isolated (Kusaba, M. et al. 2007). This enzyme is responsible for the first step of chlorophyll degradation pathway, the conversion of Chlorophyll (Chl) b in Chl a and the mutant is called “stay-green”, because of PSII antenna retention upon leaf senescence induction (Horie, Y. et al. 2009). We characterized the response of Chl b reductase ko mutant to acclimation in high light. The mutant showed a slower antenna size reduction with respect to WT. This enzyme is upregulated during HL acclimation. In vitro assay with recombinant Chl b reductase demonstrated that its activity is higher when zeaxanthin, which accumulate during stress, is bound to PSII antenna complexes

Cyanobacterial Production of Biopharmaceutical and Biotherapeutic Proteins

Efforts to express human therapeutic proteins in photosynthetic organisms have been described in the literature. Regarding microalgae, most of the research entailed a heterologous transformation of the chloroplast, but transformant cells failed to accumulate the desired recombinant proteins in high quantity. The present work provides methods and DNA construct formulations for over-expressing in photosynthetic cyanobacteria, at the protein level, human-origin bio-pharmaceutical and bio-therapeutic proteins. Proof-of-concept evidence is provided for the design and reduction to practice of "fusion constructs as protein overexpression vectors" for the generation of the bio-therapeutic protein interferon alpha-2 (IFN). IFN is a member of the Type I interferon cytokine family, well-known for its antiviral and anti-proliferative functions. Fusion construct formulations enabled accumulation of IFN up to 12% of total cellular protein in soluble form. In addition, the work reports on the isolation and purification of the fusion IFN protein and preliminary verification of its antiviral activity. Combining the expression and purification protocols developed here, it is possible to produce fairly large quantities of interferon in these photosynthetic microorganisms, generated from sunlight, CO2, and H2O

Perspectives of cyanobacterial cell factories

Cyanobacteria are prokaryotic photosynthetic microorganisms that can generate, in addition to biomass, useful chemicals and proteins/enzymes, essentially from sunlight, carbon dioxide, and water. Selected aspects of cyanobacterial production (isoprenoids and high-value proteins) and scale-up methods suitable for product generation and downstream processing are addressed in this review. The work focuses on the challenge and promise of specialty chemicals and proteins production, with isoprenoid products and biopharma proteins as study cases, and the challenges encountered in the expression of recombinant proteins/enzymes, which underline the essence of synthetic biology with these microorganisms. Progress and the current state-of-the-art in these targeted topics are emphasized

The Geomagnetic Field (GMF) Is Required for Lima Bean Photosynthesis and Reactive Oxygen Species Production

: Plants evolved in the presence of the Earth's magnetic field (or geomagnetic field, GMF). Variations in MF intensity and inclination are perceived by plants as an abiotic stress condition with responses at the genomic and metabolic level, with changes in growth and developmental processes. The reduction of GMF to near null magnetic field (NNMF) values by the use of a triaxial Helmholtz coils system was used to evaluate the requirement of the GMF for Lima bean (Phaseolus lunatus L.) photosynthesis and reactive oxygen species (ROS) production. The leaf area, stomatal density, chloroplast ultrastructure and some biochemical parameters including leaf carbohydrate, total carbon, protein content and δ13C were affected by NNMF conditions, as were the chlorophyll and carotenoid levels. RubisCO activity and content were also reduced in NNMF. The GMF was required for the reaction center's efficiency and for the reduction of quinones. NNMF conditions downregulated the expression of the MagR homologs PlIScA2 and PlcpIScA, implying a connection between magnetoreception and photosynthetic efficiency. Finally, we showed that the GMF induced a higher expression of genes involved in ROS production, with increased contents of both H2O2 and other peroxides. Our results show that, in Lima bean, the GMF is required for photosynthesis and that PlIScA2 and PlcpIScA may play a role in the modulation of MF-dependent responses of photosynthesis and plant oxidative stress

Heterologous Leader Sequences in Fusion Constructs Enhance Expression of Geranyl Diphosphate Synthase and Yield of β-Phellandrene Production in Cyanobacteria ( Synechocystis).

Fusion constructs as protein overexpression vectors proved to be critical in the heterologous expression of terpene synthases in cyanobacteria. The concept was recently applied to the heterologous overexpression of the β-phellandrene synthase (β- PHLS) from plants, fused to the highly expressed endogenous cpcB gene encoding the β-subunit of phycocyanin. Overexpressed CpcB*PHLS fusion proteins enhanced the heterologous yield of C10H16 β-phellandrene hydrocarbons production in Synechocystis. This work extended the concept of fusion constructs as protein overexpression vectors by showing that highly expressed heterologous genes could also serve as leader sequences for protein overexpression in cyanobacteria. Examined are the kanamycin nptI and chloramphenicol cmR resistance cassettes, both of which are overexpressed in Synechocystis. Evidence showed a dual purpose of the nptI gene, as a leader sequence fused to a heterologous geranyl-diphosphate synthase ( GPPS), promoting its expression, while at the same time serving as a selectable marker for the screening of transformants. The work further showed that enhanced GPPS expression increased the yield of β-phellandrene in Synechocystis transformants harboring the β- PHLS gene. Moreover, the research evaluated the expression efficacy of a DNA fragment comprising 87 nucleotides from the 5' end of the cmR gene in fusion with the GPPS gene. This short fusion construct substantially increased the intracellular geranyl-diphosphate synthase level, suggesting that "short-stretch" cmR leader sequences can be used to drive a higher expression level of heterologous biosynthetic genes, while avoiding undesirable internal recombinations, as these sequences are shorter than the threshold of 200 bp, commonly assumed to be the threshold of high efficiency recombinations

Photosynthetic generation of heterologous terpenoids in cyanobacteria.

The work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene β-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. "Fusion constructs as protein overexpression vectors" were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and β-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation

Phycocyanin Fusion Constructs for Heterologous Protein Expression Accumulate as Functional Heterohexameric Complexes in Cyanobacteria

: Overexpression of heterologous proteins from plants, bacteria, and human as fusion constructs in cyanobacteria has been documented in the literature. Typically, the heterologous protein "P" of interest is expressed as a fusion with the abundant CpcB β-subunit of phycocyanin (PC), which was placed in the leader sequence position. The working hypothesis for such overexpressions is that CpcB*P fusion proteins somehow accumulate in a soluble and stable form in the cytosol of the cyanobacteria, retaining the activity of the trailing heterologous "P" protein of interest. The present work revealed a substantially different and previously unobvious picture, comprising the following properties of the above-mentioned CpcB*P fusion constructs: (i) the CpcB*P proteins assemble as functional (α,β*P)3CpcG heterohexameric discs, where α is the CpcA α-subunit of PC, β*P is the CpcB*P fusion protein, the asterisk denotes fusion, and CpcG is the 28.9 kDa PC disc linker polypeptide CpcG1. (ii) The (α,β*P)3CpcG1 complexes covalently bind one open tetrapyrrole bilin co-factor per α-subunit and two bilins per β-subunit. (iii) The (α,β*P)3CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (α,β*P)3CpcG1 heterohexamer to the PSII reaction center, enhancing the rate of photochemical charge separation and electron transfer activity in this photosystem. (iv) In addition to the human interferon α-2 and tetanus toxin fragment C tested in this work, we have shown that enzymes such as the plant-origin isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase, and geranyl linalool synthase are also overexpressed, while retaining their catalytic activity in the respective fusion construct configuration. (v) Folding models for the (α,β*P)3CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (α,β)3 compact disc. Elucidation of the fusion construct configuration and function will pave the way for the rational design of fusion constructs harboring and overexpressing multiple proteins of scientific and commercial interest