Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCP^O) into the red-shifted, active state (OCP^R) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCP^R is converted to OCP^O and dissociates from PBs; however, the mode and site of OCP^R/FRP interactions remain elusive. Recently, we have introduced the purple OCP^W288A mutant as a competent model for the signaling state OCP^R (Sluchanko et al., Biochim Biophys Acta 1858:1–11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCP^W288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCP^O, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCP^O, a substantial compaction of the OCP^W288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCP^W288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.     

Elucidation of the essential amino acids involved in the binding of the cyanobacterial Orange Carotenoid Protein to the phycobilisome

The Orange Carotenoid Protein (OCP) is a soluble photoactive protein involved in cyanobacterial photoprotection. It is formed by the N-terminal domain (NTD) and C-terminal (CTD) domain, which establish interactions in the orange inactive form and share a ketocarotenoid molecule. Upon exposure to intense blue light, the carotenoid molecule migrates into the NTD and the domains undergo separation. The free NTD can then interact with the phycobilisome (PBS), the extramembrane cyanobacterial antenna, and induces thermal dissipation of excess absorbed excitation energy. The OCP and PBS amino acids involved in their interactions remain undetermined. To identify the OCP amino acids essential for this interaction, we constructed several OCP mutants (23) with modified amino acids located on different NTD surfaces. We demonstrated that only the NTD surface that establishes interactions with the CTD in orange OCP is involved in the binding of OCP to PBS. All amino acids surrounding the carotenoid β1 ring in the OCP^R-NTD (L51, P56, G57, N104, I151, R155, N156) are important for binding OCP to PBS. Additionally, modification of the amino acids influences OCP photoactivation and/or recovery rates, indicating that they are also involved in the translocation of the carotenoid.     

Synthetic OCP heterodimers are photoactive and recapitulate the fusion of two primitive carotenoproteins in the evolution of cyanobacterial photoprotection

The orange carotenoid protein (OCP) governs photoprotection in the majority of cyanobacteria. It is structurally and functionally modular, comprised of a C‐terminal regulatory domain (CTD), an N‐terminal effector domain (NTD) and a ketocarotenoid; the chromophore spans the two domains in the ground state and translocates fully into the NTD upon illumination. Using both the canonical OCP1 from Fremyella diplosiphon and the presumably more primitive OCP2 paralog from the same organism, we show that an NTD‐CTD heterodimer forms when the domains are expressed as separate polypeptides. The carotenoid is required for the heterodimeric association, assembling an orange complex which is stable in the dark. Both OCP1 and OCP2 heterodimers are photoactive, undergoing light‐driven heterodimer dissociation, but differ in their ability to reassociate in darkness, setting the stage for bioengineering photoprotection in cyanobacteria as well as for developing new photoswitches for biotechnology. Additionally, we reveal that homodimeric CTD can bind carotenoid in the absence of NTD, and name this truncated variant the C‐terminal domain‐like carotenoid protein (CCP). This finding supports the hypothesis that the OCP evolved from an ancient fusion event between genes for two different carotenoid‐binding proteins ancestral to the NTD and CTD. We suggest that the CCP and its homologs constitute a new family of carotenoproteins within the NTF2‐like superfamily found across all kingdoms of life.     

Engineering the photoactive orange carotenoid protein with redox-controllable structural dynamics and photoprotective function

Photosynthesis requires various photoprotective mechanisms for survival of organisms in high light. In cyanobacteria exposed to high light, the Orange Carotenoid Protein (OCP) is reversibly photoswitched from the orange (OCP^O) to the red (OCP^R) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCP^R accumulation implicates restructuring of a compact dark-adapted OCP^O state including detachment of the N-terminal extension (NTE) and separation of protein domains, which is reversed by interaction with the Fluorescence Recovery Protein (FRP). OCP phototransformation supposedly occurs via an intermediate characterized by an OCP^R-like absorption spectrum and an OCP^O-like protein structure, but the hierarchy of steps remains debatable. Here, we devise and analyze an OCP variant with the NTE trapped on the C-terminal domain (CTD) via an engineered disulfide bridge (OCP_CC). NTE trapping preserves OCP photocycling within the compact protein structure but precludes functional interaction with PBs and especially FRP, which is completely restored upon reduction of the disulfide bridge. Non-interacting with the dark-adapted oxidized OCP_CC, FRP binds reduced OCP_CC nearly as efficiently as OCP^O devoid of the NTE, suggesting that the low-affinity FRP binding to OCP^O is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCP_CC indicates that OCP_CC binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCP^R-like intermediate in the OCP photocycle and shows effective uncoupling of spectral changes from functional OCP photoactivation, enabling redox control of its structural dynamics and function.     

Binding of red form of Orange Carotenoid Protein (OCP) to phycobilisome is not sufficient for quenching

The Orange Carotenoid Protein (OCP) is responsible for photoprotection in many cyanobacteria. Absorption of blue light drives the conversion of the orange, inactive form (OCP^O) to the red, active form (OCP^R). Concomitantly, the N–terminal domain (NTD) and the C–terminal domain (CTD) of OCP separate, which ultimately leads to the formation of a quenched OCP^R–PBS complex. The details of the photoactivation of OCP have been intensely researched. Binding site(s) of OCP^R on the PBS core have also been proposed. However, the post–binding events of the OCP^R–PBS complex remain unclear. Here, we demonstrate that PBS–bound OCP^R is not sufficient as a PBS excitation energy quencher. Using site–directed mutagenesis, we generated a suite of single point mutations at OCP Leucine 51 (L51) of Synechocystis 6803. Steady–state and time–resolved fluorescence analyses demonstrated that all mutant proteins are unable to quench the PBS fluorescence, owing to either failed OCP binding to PBS, or, if bound, an OCP–PBS quenching state failed to form. The SDS–PAGE and Western blot analysis support that the L51A (Alanine) mutant binds to the PBS and therefore belongs to the second category. We hypothesize that upon binding to PBS, OCP^R likely reorganizes and adopts a new conformational state (OCP^3rd) different than either OCP^O or OCP^R to allow energy quenching, depending on the cross–talk between OCP^R and its PBS core–binding counterpart.     

Photoactivation and relaxation studies on the cyanobacterial orange carotenoid protein in the presence of copper ion

Photosynthesis starts with absorption of light energy by light-harvesting antenna complexes with subsequent production of energy-rich organic compounds. However, all photosynthetic organisms face the challenge of excess photochemical conversion capacity. In cyanobacteria, non-photochemical quenching (NPQ) performed by the orange carotenoid protein (OCP) is one of the most important mechanisms to regulate the light energy captured by light-harvesting antennas. This regulation permits the cell to meet its cellular energy requirements and at the same time protects the photosynthetic apparatus under fluctuating light conditions. Several reports have revealed that thermal dissipation increases under excess copper in plants. To explore the effects and mechanisms of copper on cyanobacteria NPQ, photoactivation and relaxation of OCP in the presence of copper were examined in this communication. When OCP^o (OCP at orange state) is converted into OCP^r(OCP at red state), copper ion has no effect on the photoactivation kinetics. Relaxation of OCP^r to OCP^o, however, is largely delayed—almost completely blocked, in the presence of copper. Even the addition of the fluorescence recovery protein (FRP) cannot activate the relaxation process. Native polyacrylamide gel electrophoresis (PAGE) analysis result indicates the heterogeneous population of Cu²⁺-locked OCP^r. The Cu²⁺-OCP binding constant was estimated using a hyperbolic binding curve. Functional roles of copper-binding OCP in vivo are discussed.     

Chemical activation of the cyanobacterial orange carotenoid protein

The effects of the Hofmeister series of ions on the activation of the orange carotenoid protein (OCP) from the inactive orange form to the active red form were tested. Kosmotropes led to lower OCP activation, whereas chaotropes led to greater OCP activation. Concentrations of thiocyanate exceeding 1.5 M dark activate the orange carotenoid protein to its red form. This chemically activated OCP was studied by UV–vis and circular dichroism spectroscopies. The chemically-activated OCP quenches the fluorescence of phycobilisomes in vitro, to a level comparable to that of the light-activated OCP.     

The purple Trp288Ala mutant of Synechocystis OCP persistently quenches phycobilisome fluorescence and tightly interacts with FRP

In Cyanobacteria, the Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) are central to the photoprotective mechanism consisting in regulated quenching of phycobilisome (PBs) fluorescence. Due to a transient and flexible nature of the light-activated red quenching form, OCP^R, which is obtained from the stable dark-adapted orange form, OCP^O, by photoconversion, the detailed mechanism of photoprotection remains unclear. Here we demonstrate that our recently described W288A mutant of the Synechocystis OCP (hereinafter called OCP^W288A) is a fully functional analogue of the OCP^R form which is capable of constitutive PBs fluorescence quenching in vitro with no need of photoactivation. This PBs quenching effect is abolished in the presence of FRP, which interacts with OCP^W288A with micromolar affinity and an apparent stoichiometry of 1:1, unexpectedly, implying dissociation of the FRP dimers. This establishes OCP^W288A as a robust model system providing novel insights into the interplay between OCP and FRP to regulate photoprotection in cyanobacteria.     

Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection

The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCP^O, to an active red form, OCP^R. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCP^R. A comparison of the spectroscopic properties of the RCP with OCP^R indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCP^R form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.     

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.     

Role of inter-domain cavity in the attachment of the orange carotenoid protein to the phycobilisome core and to the fluorescence recovery protein

Using molecular modeling and known spatial structure of proteins, we have derived a universal 3D model of the orange carotenoid protein (OCP) and phycobilisome (PBS) interaction in the process of non-photochemical PBS quenching. The characteristic tip of the phycobilin domain of the core-membrane linker polypeptide (L_CM) forms the attachment site on the PBS core surface for interaction with the central inter-domain cavity of the OCP molecule. This spatial arrangement has to be the most advantageous one because the L_CM, as the major terminal PBS-fluorescence emitter, accumulates energy from the most other phycobiliproteins within the PBS before quenching by OCP. In agreement with the constructed model, in cyanobacteria, the small fluorescence recovery protein is wedged in the OCP’s central cavity, weakening the PBS and OCP interaction. The presence of another one protein, the red carotenoid protein, in some cyanobacterial species, which also can interact with the PBS, also corresponds to this model.     

Ultrafast spectroscopy tracks carotenoid configurations in the orange and red carotenoid proteins from cyanobacteria

A quenching mechanism mediated by the orange carotenoid protein (OCP) is one of the ways cyanobacteria protect themselves against photooxidative stress. Here, we present a femtosecond spectroscopic study comparing OCP and RCP (red carotenoid protein) samples binding different carotenoids. We confirmed significant changes in carotenoid configuration upon OCP activation reported by Leverenz et al. (Science 348:1463–1466. doi:  10.1126/science.aaa7234, 2015) by comparing the transient spectra of OCP and RCP. The most important marker of these changes was the magnitude of the transient signal associated with the carotenoid intramolecular charge-transfer (ICT) state. While OCP with canthaxanthin exhibited a weak ICT signal, it increased significantly for canthaxanthin bound to RCP. On the contrary, a strong ICT signal was recorded in OCP binding echinenone excited at the red edge of the absorption spectrum. Because the carbonyl oxygen responsible for the appearance of the ICT signal is located at the end rings of both carotenoids, the magnitude of the ICT signal can be used to estimate the torsion angles of the end rings. Application of two different excitation wavelengths to study OCP demonstrated that the OCP sample contains two spectroscopically distinct populations, none of which is corresponding to the photoactivated product of OCP.     

Regulation of Orange Carotenoid Protein Activity in Cyanobacterial Photoprotection

Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP^r) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP^r. This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP^r, enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm.Full sunlight is dangerous for plants, algae, and cyanobacteria. It can cause oxidative damages leading to the destruction of the photosynthetic apparatus and to cell death. A short-term photoprotective mechanism developed by oxygenic photosynthetic organisms is the reduction of excitation energy being funneled into the photochemical reaction centers by dissipating excess energy as heat at the level of the antennae (Niyogi and Truong, 2013). In plants and green algae, this mechanism involves the membrane chlorophyll antennae, the light-harvesting complex (for review, see Horton et al., 1996; Horton and Ruban, 2005; Jahns and Holzwarth, 2012), and in cyanobacteria, the extramembrane phycobiliprotein-containing antennae, the phycobilisomes (PBSs; for review, see Kirilovsky and Kerfeld, 2012; Kirilovsky, 2014). Despite these differences in composition and structure of their antennae, carotenoids have an essential role in both plants and cyanobacteria. In plants, high irradiance leads to acidification of the lumen that triggers conformational changes in the light-harvesting complexes and in their organization in the membrane, switching the light-harvesting complex into an effective energy-dissipating form. In cyanobacteria, high irradiance photoactivates a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), that acts as the stress sensor and the energy quencher. In both cases, changes in pigment-pigment interactions (carotenoid-chlorophyll, carotenoid-bilin, chlorophyll-chlorophyll) enable thermal dissipation of excitation energy via three different possible mechanisms: excitation energy transfer (Ruban et al., 2007; Berera et al., 2013), charge transfer (Holt et al., 2005; Tian et al., 2011), or excitonic interactions between the pigments (Bode et al., 2009).The study of the photoactivation of the OCP and its interaction with the phycobilisome is essential to elucidate the mechanism of energy quenching in cyanobacterial photoprotection. The OCP is a soluble 35-kD protein constituted by an α-helical N-terminal domain (residues1–165) and an α-helix/β-sheet C-terminal domain (residues 190–317; Kerfeld et al., 2003; Wilson et al., 2010; Fig. 1A). A flexible linker of 25 amino acids connects both domains. The ketocarotenoid 3′-hydroxyechinenone (3′-hECN), having a carbonyl (keto) group in one of the rings and a hydroxyl group in the other one, spans both domains of the protein, with the carbonyl group residing in a hydrophobic pocket of the C-terminal domain. Tyr-201 and Trp-288 interact via hydrogen bonds to the carotenoid keto group. In the dark, the OCP is orange (OCP^o). Absorption of blue-green light by the carotenoid induces conformational changes in the carotenoid and in the protein converting the orange form into the active red form (OCP^r; Wilson et al., 2008; Fig. 1C). The photoconversion reaction has a very low quantum yield, and the rate of OCP^r accumulation largely depends on light intensity (Wilson et al., 2008). Thus, accumulation of the red form occurs only under high irradiance (Wilson et al., 2008). Both OCP^o and OCP^r are energetically suitable to quench PBS fluorescence and excitation energy (Polívka et al., 2013; Niedzwiedzki et al., 2014), but only OCP^r is able to bind to the PBS and dissipate most of the excess energy as heat (Gwizdala et al., 2011). In OCP^o, strong interactions exist between the N- and C-terminal globular domains, including salt bridges between residues Trp-277-Asn-104 and Arg-155-Glu-244 (Kerfeld et al., 2003; Wilson et al., 2010). Upon photoactivation, these bonds are broken, leading to the solvent exposure of Arg-155, which plays an essential role in OCP binding to PBS (Wilson et al., 2012; Fig. 1C). The PBSs from Synechocystis sp. PCC 6803 (used in this work and hereafter simply referred to as Synechocystis) are formed by a core of allophycocyanin (APC) trimers. These trimers are organized in three cylinders from which rods containing phycocyanin hexamers radiate (for review, see Grossman et al., 1993; MacColl, 1998; Adir, 2005). OCP^r binds to one APC trimer, and its open structure allows the interaction between the OCP carotenoid and one APC bilin (Wilson et al., 2012). The first site of energy and fluorescence quenching is an APC trimer emitting at 660 nm (Tian et al., 2011, 2012, 2013; Takahashi et al., 2013). Once OCP^r is attached to PBS, thermal dissipation increases and less energy arrives at both photosystems (Wilson et al., 2006; Rakhimberdieva et al., 2010; Gorbunov et al., 2011). When the light becomes less intense, full antenna capacity is required. The Fluorescence Recovery Protein (FRP) is essential for this process. FRP accelerates the OCP^r to OCP^o dark conversion and facilitates OCP detachment from PBS (Boulay et al., 2010; Gwizdala et al., 2011; Sutter et al., 2013). The active FRP is a nonchromophorylated dimer that interacts with the C-terminal domain of the OCP^r (Sutter et al., 2013).Open in a separate windowFigure 1.A and B, Structure of the OCP from Synechocystis sp. PCC 6803 (Protein Data Bank identifier: 3MG1). The OCP monomer is represented in the orange state. The N-terminal arm (residues 1–22; red) interacts with the C-terminal domain (residues 196–315; sky blue). The Pro-22 and the Asp-6 are marked in blue. The N-terminal domain (residues 22–165) is green in the figure, and the linker between N-terminal and C-terminal domains is colored in violet. C, Model of photoactivation. Upon light absorption, the orange OCP^o is converted into the active red OCP^r. Changes in the carotenoid conformation induce conformational changes in the C-terminal domain, leading to the breakage of the interactions between the N-terminal and C-terminal domains and the opening of the protein.Previously, it has been demonstrated that the N-terminal globular domain of the OCP (green in Fig. 1, A and B) is a constitutively active energy quencher (Leverenz et al., 2014). Thus, its interaction with the C-terminal globular domain is essential for inhibiting OCP binding to PBS and energy quenching under low irradiance. This process must be tightly regulated. Little is known about this regulation. One possibility is that the N-terminal arm of the protein (red in Fig. 1, A and B), which in OCP^o interacts with the C-terminal globular domain, could have a role in this regulation.According to the OCP structure Asp-6, could form a hydrogen bond with Arg-229, which could stabilize the closed form of OCP^o. Pro-22 is located at the bent junction between the N-terminal arm and the N-terminal globular domain. It has been proposed that a cis-trans Pro isomerization could be involved in OCP photoactivation (Gorbunov et al., 2011), suggesting that Pro-22 isomerization could help the movement of the N-terminal arm and its detachment from the C-terminal domain during OCP photoactivation. In this work, we studied the effect of deleting the N-terminal arm and the mutations Asp-6-Leu and Pro-22-Val on photoactivity and OCP interaction with PBS and FRP (for the position of the N-terminal arm in the structure of OCP^o, see in Fig. 1, A and B).     

Site of non-photochemical quenching of the phycobilisome by orange carotenoid protein in the cyanobacterium Synechocystis sp. PCC 6803

In cyanobacteria, the thermal dissipation of excess absorbed energy at the level of the phycobilisome (PBS)-antenna is triggered by absorption of strong blue-green light by the photoactive orange carotenoid protein (OCP). This process known as non-photochemical quenching, whose molecular mechanism remains in many respects unclear, is revealed in vivo as a decrease in phycobilisome fluorescence. In vitro reconstituted system on the interaction of the OCP and the PBS isolated from the cyanobacterium Synechocystis sp. PCC 6803 presents evidence that the OCP is not only a photosensor, but also an effecter that makes direct contacts with the PBS and causes dissipation of absorbed energy. To localize the site(s) of quenching, we have analyzed the role of chromophorylated polypeptides of the PBS using PBS-deficient mutants in conjunction with in vitro systems of assembled PBS and of isolated components of the PBS core. The results demonstrated that L(CM), the core-membrane linker protein and terminal emitter of the PBS, could act as the docking site for OCP in vitro. The ApcD and ApcF terminal emitters of the PBS core are not directly subjected to quenching. The data suggests that there could be close contact between the phycocyanobilin chromophore of L(CM) and the 3'-hydroxyechinenone chromophore present in OCP and that L(CM) could be involved in OCP-induced quenching. According to the reduced average life-time of the PBS-fluorescence and linear dependence of fluorescence intensity of the PBS on OCP concentration, the quenching has mostly dynamic character. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Effects of simulated drought stress on carotenoid contents and expression of related genes in carrot taproots

Carotenoids are liposoluble pigments found in plant chromoplasts that are responsible for the yellow, orange, and red colors of carrot taproots. Drought is one of the main stress factors affecting carrot growth. Carotenoids play important roles in drought resistance in higher plants. In the present work, the carotenoid contents in three different-colored carrot cultivars, ‘Kurodagosun’ (orange), ‘Benhongjinshi’ (red), and ‘Qitouhuang’ (yellow), were determined by ultra-high-performance liquid chromatography (UPLC) after 15% polyethylene glycol (PEG) 6000 treatment. Real-time fluorescence quantitative PCR (RT-qPCR) was then used to determine the expression levels of carotenoid synthesis- and degradation-related genes. Increases in β-carotene content in ‘Qitouhuang’ taproots under drought stress were found to be related to the expression levels of DcPSY2 and DcLCYB. Increases in lutein and decreases in α-carotene content in ‘Qitouhuang’ and ‘Kurodagosun’ under PEG treatment may be related to the expression levels of DcCYP97A3, DcCHXE, and DcCHXB1. The expression levels of DcNCED1 and DcNCED2 in the three cultivars significantly increased, thus suggesting that NCED genes could respond to drought stress. Analysis of the growth status and carotenoid contents of carrots under PEG treatment indicated that the orange cultivar ‘Kurodagosun’ has better adaptability to drought stress than the other cultivars and that β-carotene and lutein may be involved in the stress resistance process of carrot.

     

The orange carotenoid protein in photoprotection of photosystem II in cyanobacteria

Photoprotective mechanisms have evolved in photosynthetic organisms to cope with fluctuating light conditions. Under high irradiance, the production of dangerous oxygen species is stimulated and causes photo-oxidative stress. One of these photoprotective mechanisms, non photochemical quenching (qE), decreases the excess absorbed energy arriving at the reaction centers by increasing thermal dissipation at the level of the antenna. In this review we describe results leading to the discovery of this process in cyanobacteria (qE(cya)), which is mechanistically distinct from its counterpart in plants, and recent progress in the elucidation of this mechanism. The cyanobacterial photoactive soluble orange carotenoid protein is essential for the triggering of this photoprotective mechanism. Light induces structural changes in the carotenoid and the protein leading to the formation of a red active form. The activated red form interacts with the phycobilisome, the cyanobacterial light-harvesting antenna, and induces a decrease of the phycobilisome fluorescence emission and of the energy arriving to the reaction centers. The orange carotenoid protein is the first photoactive protein to be identified that contains a carotenoid as the chromophore. Moreover, its photocycle is completely different from those of other photoactive proteins. A second protein, called the Fluorescence Recovery Protein encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in dislodging the red orange carotenoid protein from the phycobilisome and in the conversion of the free red orange carotenoid protein to the orange, inactive, form. This protein is essential to recover the full antenna capacity under low light conditions after exposure to high irradiance. This article is part of a Special Issue entitled: Photosystem II.     

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10^?5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.