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.

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 ß-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,ß*P)3CpcG heterohexameric discs, where α is the CpcA α-subunit of PC, ß*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,ß*P)3CpcG1 complexes covalently bind one open tetrapyrrole bilin co-factor per α-subunit and two bilins per ß-subunit. (iii) The (α,ß*P)3CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (α,ß*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, ß-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,ß*P)3CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (α,ß)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.

Recombinant Protein Stability in Cyanobacteria.

The living cell possesses extraordinary molecular and biochemical mechanisms by which to recognize and efficiently remove foreign, damaged, or denatured proteins. This essential function has been a barrier to the overexpression of recombinant proteins in most expression systems. A notable exception is the overexpression in E. coli of recombinant proteins, most of which, however, end-up as "inclusion bodies", i.e., cytoplasmic aggregates of proteins that are inaccessible to the cell's proteasome. "Fusion constructs as protein overexpression vectors" proved to be unparalleled in their ability to cause substantial accumulation of recombinant proteins from plants, animals, and bacteria, as soluble proteins in unicellular cyanobacteria. Recombinant protein levels in the range of 10-20% of the total cellular protein can be achieved. The present work investigated this unique property in the context of recombinant protein stability in Synechocystis sp. PCC 6803 by developing and applying an in vivo cellular tobacco etch virus cleavage system with the objective of separating the target heterologous proteins from their fusion leader sequences. The work provides new insights about the overexpression, cellular stability, and exploitation of transgenes with commercial interest, highly expressed in a cyanobacterial biofactory. The results support the notion that eukaryotic plant- and animal-origin recombinant proteins are unstable, when free in the cyanobacterial cytosol but stable when in a fusion configuration with a highly expressed cyanobacterial native or heterologous protein. Included in this analysis are recombinant proteins of the plant isoprenoid biosynthetic pathway (isoprene synthase, ß-phellandrene synthase, geranyl diphosphate synthase), the human interferon protein, as well as prokaryotic proteins (tetanus toxin fragment C and the antibiotic resistance genes kanamycin and chloramphenicol). The future success of synthetic biology approaches with cyanobacteria and other systems would require overexpression of pathway enzymes to attain product volume, and the work reported in this paper sets the foundation for such recombinant pathway enzyme overexpression.

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.

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 ß-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 ß-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.

Design of a highly thermostable hemicellulose-degrading blend from Thermotoga neapolitana for the treatment of lignocellulosic biomass.

The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-ß-galactanase and an α-l-arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (kcat values of ˜177 and ˜133 s-1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases - endo-1,4-ß-xylanase (XynA), endo-1,4-ß-mannanase (ManB/Man5A) and ß-glucosidase (GghA) - to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases.

Downregulation of the CpSRP43 gene expression confers a truncated light-harvesting antenna (TLA) and enhances biomass and leaf-to-stem ratio in Nicotiana tabacum canopies.

MAIN CONCLUSION: Downregulation in the expression of the signal recognition particle 43 (SRP43) gene in tobacco conferred a truncated photosynthetic light-harvesting antenna (TLA property), and resulted in plants with a greater leaf-to-stem ratio, improved photosynthetic productivity and canopy biomass accumulation under high-density cultivation conditions. Evolution of sizable arrays of light-harvesting antennae in all photosynthetic systems confers a survival advantage for the organism in the wild, where sunlight is often the growth-limiting factor. In crop monocultures, however, this property is strongly counterproductive, when growth takes place under direct and excess sunlight. The large arrays of light-harvesting antennae in crop plants cause the surface of the canopies to over-absorb solar irradiance, far in excess of what is needed to saturate photosynthesis and forcing them to engage in wasteful dissipation of the excess energy. Evidence in this work showed that downregulation by RNA-interference approaches of the Nicotiana tabacum signal recognition particle 43 (SRP43), a nuclear gene encoding a chloroplast-localized component of the photosynthetic light-harvesting assembly pathway, caused a decrease in the light-harvesting antenna size of the photosystems, a corresponding increase in the photosynthetic productivity of chlorophyll in the leaves, and improved tobacco plant canopy biomass accumulation under high-density cultivation conditions. Importantly, the resulting TLA transgenic plants had a substantially greater leaf-to-stem biomass ratio, compared to those of the wild type, grown under identical agronomic conditions. The results are discussed in terms of the potential benefit that could accrue to agriculture upon application of the TLA-technology to crop plants, entailing higher density planting with plants having a greater biomass and leaf-to-stem ratio, translating into greater crop yields per plant with canopies in a novel agronomic configuration.

Heterologous Leader Sequences in Fusion Constructs Enhance Expression of Geranyl Diphosphate Synthase and Yield of ß-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 ß-phellandrene synthase (ß- PHLS) from plants, fused to the highly expressed endogenous cpcB gene encoding the ß-subunit of phycocyanin. Overexpressed CpcB*PHLS fusion proteins enhanced the heterologous yield of C10H16 ß-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 ß-phellandrene in Synechocystis transformants harboring the ß- 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.

Deletion of the chloroplast LTD protein impedes LHCI import and PSI-LHCI assembly in Chlamydomonas reinhardtii.

Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.

The STN8 kinase-PBCP phosphatase system is responsible for high-light-induced reversible phosphorylation of the PSII inner antenna subunit CP29 in rice.

Reversible phosphorylation of thylakoid light-harvesting proteins is a mechanism to compensate for unbalanced excitation of photosystem I (PSI) versus photosystem II (PSII) under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high-light (HL) response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in HL, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of AtPBCP, antagonist of AtSTN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P-CP29. Based on these data, we propose that the activities of the OsSTN8 kinase and the antagonistic OsPBCP phosphatase, in addition to being involved in the repair of photo-damaged PSII, are also responsible for the HL-dependent reversible phosphorylation of the inner antenna CP29.

High light-dependent phosphorylation of photosystem II inner antenna CP29 in monocots is STN7 independent and enhances nonphotochemical quenching.

Phosphorylation of the photosystem II antenna protein CP29 has been reported to be induced by excess light and further enhanced by low temperature, increasing resistance to these stressing factors. Moreover, high light-induced CP29 phosphorylation was specifically found in monocots, both C3 and C4, which include the large majority of food crops. Recently, knockout collections have become available in rice (Oryza sativa), a model organism for monocots. In this work, we have used reverse genetics coupled to biochemical and physiological analysis to elucidate the molecular basis of high light-induced phosphorylation of CP29 and the mechanisms by which it exerts a photoprotective effect. We found that kinases and phosphatases involved in CP29 phosphorylation are distinct from those reported to act in State 1-State 2 transitions. In addition, we elucidated the photoprotective role of CP29 phosphorylation in reducing singlet oxygen production and enhancing excess energy dissipation. We thus established, in monocots, a mechanistic connection between phosphorylation of CP29 and nonphotochemical quenching, two processes so far considered independent from one another.

Magnetic nanoparticles from Magnetospirillum gryphiswaldense increase the efficacy of thermotherapy in a model of colon carcinoma.

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.

Enhancement of non-photochemical quenching in the Bryophyte Physcomitrella patens during acclimation to salt and osmotic stress.

Drought and salt stress are major abiotic constraints affecting plant growth worldwide. Under these conditions, the production of reactive oxygen species (ROS) is a common phenomenon taking place mainly in chloroplasts, peroxisomes, mitochondria and apoplasts, especially when associated with high light stress. ROS are harmful because of their high reactivity to cell components, thereby leading to cytotoxicity and cell death. During the Ordovician and early Devonian period, photosynthetic organisms colonized terrestrial habitats, and the acquisition of desiccation tolerance has been a major component of their evolution. We have studied the capacity for acclimation to drought and salt stress of the moss Physcomitrella patens, a representative of the early land colonization stage. Exposure to high concentrations of NaCl and sorbitol strongly affects chloroplast development, the Chl content and the thylakoid protein composition in this moss. Under sublethal conditions (0.2 M NaCl and 0.4 M sorbitol), the photosynthetic apparatus of P. patens responds to oxidative stress by increasing non-photochemical quenching (NPQ). Surprisingly, the accumulation of PSBS and LHCSR, the two polypeptides essential for NPQ in P. patens, was not up-regulated in these conditions. Rather, an increased NPQ amplitude correlated with the overaccumulation of zeaxanthin and the presence of the enzyme violaxanthin de-epoxidase. These results suggest that the regulation of excess energy dissipation through control of PSBS and LHCSR is mainly driven by light conditions, while osmotic and salt stress act through acclimative regulation of the xanthophyll cycle. We conclude that regulation of the xanthophyll cycle is an important anticipatory strategy against photoinhibition by high light.

Arabidopsis mutants deleted in the light-harvesting protein Lhcb4 have a disrupted photosystem II macrostructure and are defective in photoprotection.

The role of the light-harvesting complex Lhcb4 (CP29) in photosynthesis was investigated in Arabidopsis thaliana by characterizing knockout lines for each of the three Lhcb4 isoforms (Lhcb4.1/4.2/4.3). Plants lacking all isoforms (koLhcb4) showed a compensatory increase of Lhcb1 and a slightly reduced photosystem II/I ratio with respect to the wild type. The absence of Lhcb4 did not result in alteration in electron transport rates. However, the kinetic of state transition was faster in the mutant, and nonphotochemical quenching activity was lower in koLhcb4 plants with respect to either wild type or mutants retaining a single Lhcb4 isoform. KoLhcb4 plants were more sensitive to photoinhibition, while this effect was not observed in knockout lines for any other photosystem II antenna subunit. Ultrastructural analysis of thylakoid grana membranes showed a lower density of photosystem II complexes in koLhcb4. Moreover, analysis of isolated supercomplexes showed a different overall shape of the C2S2 particles due to a different binding mode of the S-trimer to the core complex. An empty space was observed within the photosystem II supercomplex at the Lhcb4 position, implying that the missing Lhcb4 was not replaced by other Lhc subunits. This suggests that Lhcb4 is unique among photosystem II antenna proteins and determinant for photosystem II macro-organization and photoprotection.

Identification of the chromophores involved in aggregation-dependent energy quenching of the monomeric photosystem II antenna protein Lhcb5.

Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light-harvesting complex II in ADQ and exclusively monomeric Lhcb proteins in charge transfer quenching. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin, a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than light-harvesting complex II during aggregation and that this is enhanced by binding to zeaxanthin, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll 612 and 613, in close contact with lutein bound at site L1, are important facilitators of ADQ.

Dynamics of zeaxanthin binding to the photosystem II monomeric antenna protein Lhcb6 (CP24) and modulation of its photoprotection properties.

Lhcb6 (CP24) is a monomeric antenna protein of photosystem II, which has been shown to play special roles in photoprotective mechanisms, such as the Non-Photochemical Quenching and reorganization of grana membranes in excess light conditions. In this work we analyzed Lhcb6 in vivo and in vitro: we show this protein, upon activation of the xanthophyll cycle, accumulates zeaxanthin into inner binding sites faster and to a larger extent than any other pigment-protein complex. By comparative analysis of Lhcb6 complexes violaxanthin or zeaxanthin binding, we demonstrate that zeaxanthin not only down-regulates chlorophyll singlet excited states, but also increases the efficiency of chlorophyll triplet quenching, with consequent reduction of singlet oxygen production and significant enhancement of photo-stability. On these bases we propose that Lhcb6, the most recent addition to the Lhcb protein family which evolved concomitantly to the adaptation of photosynthesis to land environment, has a crucial role in zeaxanthin-dependent photoprotection.

Light-induced dissociation of an antenna hetero-oligomer is needed for non-photochemical quenching induction.

PsbS plays a major role in activating the photoprotection mechanism known as "non-photochemical quenching," which dissipates chlorophyll excited states exceeding the capacity for photosynthetic electron transport. PsbS activity is known to be triggered by low lumenal pH. However, the molecular mechanism by which this subunit regulates light harvesting efficiency is still unknown. Here we show that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins (CP29 and CP24) and the trimeric LHCII-M. Dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light, strongly suggesting that protein subunits catalyzing the reaction of heat dissipation are buried into the complex and thus not available for interaction with PsbS. Consistently, we showed that knock-out mutants on two subunits participating to the B4C complex were strongly affected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpreted these results to mean that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions.