Light Intensity-Induced Changes in cab mRNA and Light Harvesting Complex II Apoprotein Levels in the Unicellular Chlorophyte Dunaliella tertiolecta

During a transition from high growth irradiance (700 micromoles quanta per square meter per second) to low growth irradiance (70 micromoles quanta per square meter per second), the unicellular marine chlorophyte Dunaliella tertiolecta Butcher increases the cellular pool size of the light-harvesting complex of photosystem II (LHC II). We showed that the increase in LHC II apoproteins and in chlorophyll content per cell is preceded by an approximately fourfold increase in cab mRNA. The increase in cab mRNA is detectable within 1.5 hours following a shift from high to low light intensity. An increase in the relative abundance of cab mRNA was also found following a shift from high light to darkness and from high light to low light in the presence of gabaculine, a chlorophyll synthesis inhibitor. However, the LHC II apoproteins did not accumulate in the latter experiments, suggesting that LHC II apoprotein synthesis is coupled to chlorophyll synthesis at or beyond translation. We propose that changes in energy balance brought about by a change in light intensity may control a regulatory factor acting to repress cab mRNA expression in high light.     

Development at Cold-Hardening Temperatures : The Structure and Composition of Purified Rye Light Harvesting Complex II

Light harvesting complex II (LHCII) was purified from cold-hardened (RH) and nonhardened winter rye (RNH) (Secale cereale L. cv Puma) employing a modified procedure of JJ Burke, CL Ditto, CJ Arntzen (Arch Biochem Biophys 187: 252-263). Triton X-100 solubilization of thylakoid membranes followed by three successive precipitations with 100 mm KCl and 10 mm MgCl₂ resulted in yields of up to 25% on a chlorophyll (Chl) basis and a purity of 90 to 95%, based on polypeptide analysis within 4 hours. Polypeptide and pigment analyses, 77 K fluorescence emission and room temperature absorption spectra indicate the LHCII obtained by this modified method is comparable to LHCII obtained by other published methods. Comparison of purified RH and RNH LHCII indicated no significant differences with respect to polypeptide, amino acid, Chl, and carotenoid compositions as well as no differences in lipid content. However, RH LHCII differed from RNH LHCII specifically with respect to the fatty acid composition of phosphatidyldiacylglycerol only. RH LHCII exhibited a 54% lower trans-Δ³-hexadecenoic acid level associated with PG and a 60% lower oligomeric LHCII:monomeric LHCII (LHCII₁:LHCII₃) than RNH LHCII. Both RH and RNH LHCII exhibited a 5-fold enrichment in PG specifically. Complete removal of PG by enzymic hydrolysis resulted in a significant reduction in the oligomeric content of both RH and RNH LHCII such that LHCII₁:LHCII₃ of RH and RNH LHCII preparations were the same. This confirms that this specific compositional change accounts for the structural differences between RH and RNH LCHII observed in situ and in vitro.     

Identification and Partial Characterization of the Denaturation Transition of the Light Harvesting Complex II of Spinach Chloroplast Membranes

Differential scanning calorimetry was employed to investigate the structure of spinach (Spinacia oleracea) chloroplast membranes. In a low ionic strength Hepes-buffered medium, major calorimetric transitions were resolved at 42.5°C. (A), 60.6°C (B), 64.9°C (C₁), 69.6°C (C₂), 75.8°C (D), 84.3°C (E), and 88.9°C (F). A lipid melting transition was also commonly seen at 17°C in scans starting at lower temperatures. The D transition was demonstrated by four independent methods to derive from denaturation of the light harvesting complex associated with photosystem II (LHC-II). Evidence for this conclusion was as follows: (a) the endotherm of the isolated LHC-II (74.0°C) was very similar to that of D (75.8°C); (b) the denaturation temperature of the 27 kilodalton LHC-II polypeptide determined in intact chloroplast membranes by thermal gel analysis was identical to the temperature of the D transition at pH 7.6 and after destabilization by shifting the pH to 6.6 or by addition of Mg²⁺; (c) analysis of the stability of the LHC-II complex by electrophoresis in native gels demonstrated that the complex dissociates during the D transition, both at pH 7.6 and 6.6; and (d) the 77 Kelvin fluorescence maximum of LHC-II in chloroplasts was seen to shift to lower wavelengths (indicating gross denaturation of LHC-II), at the temperature of the D transition when examined at either of the above pHs. With this identification, five of the eight major endotherms of the chloroplast membrane have now been assigned.     

Replacement of Histidines of Light Harvesting Chlorophyll a/b Binding Protein II Disrupts Chlorophyll-Protein Complex Assembly

Eukaryotic light harvesting proteins (LHCPs) bind pigments and assemble into complexes (LHCs) that channel light energy into photosynthetic reaction centers. The structures of several prokaryotic LHCPs are known and histidines are important for the binding of the associated pigments. It has been difficult to predict how the eukaryotic LHCPs associate with pigments as the structure of the major LHCP of photosystem II is not yet known. While each LHCPII binds approximately 13 chlorophylls the protein contains only three histidines, one in each putative transmembrane helix. Experiments that use isolated pea (Pisum sativum L.) chloroplasts and mutant LHCPII synthesized in vitro show that the substitution of either an alanine or an arginine for each histidine residue inhibits some aspect of LHCII assembly. The histidine of the first membrane helix, but not the second or third, may be involved in the transport across the chloroplast envelope. No histidine alone is essential for the insertion of LHCP into thylakoid membranes, yet arginine substitutions are more inhibitory than those of alanine. The histidine replacements have their most pronounced effect on the assembly of LHCP into LHCII.     

A Femtosecond Visible/Visible and Visible/Mid-Infrared Transient Absorption Study of the Light Harvesting Complex II

Light harvesting complex II (LHCII) is the most abundant protein in the thylakoid membrane of higher plants and green algae. LHCII acts to collect solar radiation, transferring this energy mainly toward photosystem II, with a smaller amount going to photosystem I; it is then converted into a chemical, storable form. We performed time-resolved femtosecond visible pump/mid-infrared probe and visible pump/visible probe absorption difference spectroscopy on purified LHCII to gain insight into the energy transfer in this complex occurring in the femto-picosecond time regime. We find that information derived from mid-infrared spectra, together with structural and modeling information, provides a unique visualization of the flow of energy via the bottleneck pigment chlorophyll a604.     

The Light Harvesting System of the Diatom Cyclotella cryptica. Isolation and Characterization of the Main Light Harvesting Complex and Evidence for the Existence of Minor Pigment Proteins*

The main chlorophyll a/c light harvesting complex of the diatom Cyclotella cryptica was isolated by sucrose density gradient centrifugation. It consisted of two polypeptides of Mrs 18000 and 22000. Both polypeptides and fragments thereof, obtained by formic acid treatment, were blocked at their N-ter-mini. An antiserum raised against the two subunits selectively immunolabeled the thylakoid within the chloroplasts. The subunits were nuclear encoded and could be immunoprecipitated from poly (A)⁺ RNA as precursor proteins in the Mr range of 20000 to 24000. The existence of minor chlorophyll protein complexes and their possible function in light climate adaptation processes was investigated in cells adapted to low light and high light conditions. Low light grown cells contained more fucoxanthin and less β-carotene relative to chlorophyll a than high light adapted cells. The xanthophyll cycle pigments diatoxanthin and diadinoxanthin increased five-fold relative to chlorophyll a under high light conditions. Western-immunoblotting experiments with antisera raised against several chlorophyll a/b and chlorophyll a/c antenna complexes demonstrated that, beside the dominating chlorophyll a/c light harvesting complex, minor antenna complexes might exist, which, in part, seem to react to the light climate applied.     

Spectral Engineering Via Complex Patterns of Circular Nano-Object Miniarrays: II. Concave Patterns Tunable by Integrated Lithography Realized by Circularly Polarized Light

Plasmonics - The use of circularly polarized beams in interferometric illumination of colloid sphere monolayers enables the direct fabrication of rectangular patterns composed of circular nanohole...     

Modeling of the N-terminal Section and the Lumenal Loop of Trimeric Light Harvesting Complex II (LHCII) by Using EPR

The major light harvesting complex II (LHCII) of green plants plays a key role in the absorption of sunlight, the regulation of photosynthesis, and in preventing photodamage by excess light. The latter two functions are thought to involve the lumenal loop and the N-terminal domain. Their structure and mobility in an aqueous environment are only partially known. Electron paramagnetic resonance (EPR) has been used to measure the structure of these hydrophilic protein domains in detergent-solubilized LHCII. A new technique is introduced to prepare LHCII trimers in which only one monomer is spin-labeled. These heterogeneous trimers allow to measure intra-molecular distances within one LHCII monomer in the context of a trimer by using double electron-electron resonance (DEER). These data together with data from electron spin echo envelope modulation (ESEEM) allowed to model the N-terminal protein section, which has not been resolved in current crystal structures, and the lumenal loop domain. The N-terminal domain covers only a restricted area above the superhelix in LHCII, which is consistent with the “Velcro” hypothesis to explain thylakoid grana stacking (Standfuss, J., van Terwisscha Scheltinga, A. C., Lamborghini, M., and Kühlbrandt, W. (2005) EMBO J. 24, 919–928). The conformation of the lumenal loop domain is surprisingly different between LHCII monomers and trimers but not between complexes with and without neoxanthin bound.     

Polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids oriented in polyvinyl alcohol films

The polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids in unstretched and stretched polyvinyl alcohol films were measured. The intensity ratios of fluorescence bands at 674 nm, 700 nm, 730 nm and 750 nm, and the polarized fluorescence excitation spectra are strongly dependent on light polarization and film stretching. In stretched films, thylakoids exhibit predominantly 674 nm emission. The ratio of photoacoustic signal to absorption is different for light polarized parallel and perpendicular to film stretching. This difference is large in the region of chlorophyll a and carotenoids absorption in which the fluorescence excitation spectra are also strongly dependent on light polarization and film stretching. The observed spectral changes are explained by reorientation of pigment molecules influencing the yield of excitation transfer between different pigments.     

In Vivo Identification of Photosystem II Light Harvesting Complexes Interacting with PHOTOSYSTEM II SUBUNIT S

Light is the primary energy source for photosynthetic organisms, but in excess, it can generate reactive oxygen species and lead to cell damage. Plants evolved multiple mechanisms to modulate light use efficiency depending on illumination intensity to thrive in a highly dynamic natural environment. One of the main mechanisms for protection from intense illumination is the dissipation of excess excitation energy as heat, a process called nonphotochemical quenching. In plants, nonphotochemical quenching induction depends on the generation of a pH gradient across thylakoid membranes and on the presence of a protein called PHOTOSYSTEM II SUBUNIT S (PSBS). Here, we generated Physcomitrella patens lines expressing histidine-tagged PSBS that were exploited to purify the native protein by affinity chromatography. The mild conditions used in the purification allowed copurifying PSBS with its interactors, which were identified by mass spectrometry analysis to be mainly photosystem II antenna proteins, such as LIGHT-HARVESTING COMPLEX B (LHCB). PSBS interaction with other proteins appears to be promiscuous and not exclusive, although the major proteins copurified with PSBS were components of the LHCII trimers (LHCB3 and LHCBM). These results provide evidence of a physical interaction between specific photosystem II light-harvesting complexes and PSBS in the thylakoids, suggesting that these subunits are major players in heat dissipation of excess energy.Photosynthetic organisms exploit sunlight energy to support their metabolism. However, if absorbed in excess, light can produce harmful reactive oxygen species (Li et al., 2009; Murchie and Niyogi, 2011). In a natural environment, light intensity is highly variable and can rapidly change from being limited to being in excess. To survive and thrive in such a variable habitat, plants evolved multiple strategies to modulate their light use efficiency to limit reactive oxygen species formation when exposed to excess illumination while maintaining the ability to harvest light efficiently when required (Li et al., 2009; Murchie and Niyogi, 2011; Ruban, 2015). Among these different protection processes, the fastest, called nonphotochemical quenching (NPQ), is activated in a few seconds after a change in illumination, and it leads to the thermal dissipation of excess absorbed energy. NPQ is a complex phenomenon with different components that are distinguished according to their activation/relaxation time scale (Demmig-Adams et al., 1996; Szabó et al., 2005; Niyogi and Truong, 2013). The primary and fastest NPQ component, called qE (for energy-quenching component) or feedback deexcitation, depends on the generation of a pH gradient across the thylakoid membranes (Niyogi and Truong, 2013). In land plants, qE activation requires the presence of a thylakoid protein called PHOTOSYSTEM II SUBUNIT S (PSBS; Li et al., 2000, 2004). The Arabidopsis (Arabidopsis thaliana) PSBS-depleted mutant psbs KO (Li et al., 2000) is unable to activate qE and also showed reduced fitness when exposed to natural light variations in the field, supporting a major role for this protein in responding to illumination intensity fluctuations (Li et al., 2000; Külheim et al., 2002). Mutational analyses showed that the PSBS role in qE strictly depends on the presence of two protonable Glu residues, which are most likely involved in sensing the pH decrease in the lumen (Li et al., 2004). Despite several studies, however, the precise molecular mechanism by which PSBS controls NPQ induction remains debatable, and contrasting hypotheses have been presented (for review, see Ruban et al., 2012). PSBS has been hypothesized to bind pigments and to be directly responsible for energy dissipation based on its sequence similarity with LIGHT HARVESTING COMPLEX (LHC) proteins (Li et al., 2000; Aspinall-O’Dea et al., 2002). An alternative hypothesis instead suggested that PSBS is unable to bind pigments (Funk et al., 1995; Crouchman et al., 2006; Bonente et al., 2008a) and that it plays an indirect role in NPQ by modulating the PSII antenna protein transition from light harvesting to an energy dissipative state (Betterle et al., 2009; Johnson et al., 2011). This transition has been suggested to depend on the control of the macroorganization of the PSII-LHCII supercomplexes that are present in the grana membranes (Kiss et al., 2008; Betterle et al., 2009; Kereïche et al., 2010; Johnson et al., 2011). Consistent with this hypothesis, it was recently demonstrated that PSBS is able to induce a dissipative state in isolated LHCII proteins in liposomes (Wilk et al., 2013), suggesting that its interactions with antenna proteins play a key role in its biological activity. However, the precise identity of PSBS interactors (Teardo et al., 2007; Betterle et al., 2009), the PSBS oligomerization state (Bergantino et al., 2003), and its localization within PSII supercomplexes (Nield et al., 2000; Haniewicz et al., 2013) remain unclear or at least controversial, limiting the current understanding of PSBS molecular mechanisms.The moss Physcomitrella patens has recently emerged as a valuable model organism in which to study NPQ. As in the model angiosperm Arabidopsis, PSBS accumulation modulates NPQ amplitude and protects plants from photoinhibition under strong light in P. patens (Li et al., 2000; Alboresi et al., 2010; Zia et al., 2011; Gerotto et al., 2012). PSBS-mediated NPQ in P. patens also showed zeaxanthin dependence as in other plants (Niyogi et al., 1998; Pinnola et al., 2013). The moss P. patens has another protein involved in NPQ, LHCSR, which is typically found in algae and is different from proteins found in vascular plants (Peers et al., 2009; Bailleul et al., 2010; Gerotto and Morosinotto, 2013). Even if LHCSR is present in P. patens, LHCSR- and PSBS-dependent NPQ mechanisms were shown to be independent and to have an additive effect without any significant functional synergy (Gerotto et al., 2012).Previous data also demonstrated the possibility of achieving strong overexpression of PSBS in P. patens (Gerotto et al., 2012), which, however, was never observed in Arabidopsis (Li et al., 2002). This property was exploited in this work to overexpress a His-tagged PSBS isoform, which was afterward purified in its native state from dark-adapted thylakoid membranes. Several PSII antenna proteins were copurified with PSBS and identified by mass spectrometry analyses, demonstrating that they interact physically in dark-adapted thylakoid membranes. Components of LHCII trimers (LHCB3 and LHCBM) appear to be major, but not exclusive, components of PSBS interactors.     

Light Harvesting and Utilization by Phytoplankton

In this study we use a model based on target theory to analyzesteady-state photosynthesis-irradiance relationships in continuouslight. From the average turnover time () of photosynthetic units(PSU_O2), numerical analyses of the model coefficients, and measurementsof the light field and cell absorptivity, apparent absorptioncrosssections of photosystem II (PSII) were determined for three species of marine unicellular algaegrown at different irradiance levels. These cross-sections generally,but not always, increased with decreased growth irradiance.Additionally, the ratios of photosystem I/photosystem II reactioncenters were calculated from measurements of oxygen flash yieldsand chlorophyll/P₇₀₀ ratios. From the ratios of the reactioncenters, cell absorptivity and the apparent absorption cross-sectionof photosystem II, the apparent absorption cross-sections ofphotosystem I (PSI) were also calculated. Finally, on the basis of our calculated absorptioncross-sections, we estimated the minimum quantum requirementsfor O₂ evolution. Our results suggest that the absorption cross-sectionsof PS I and PS II vary independently and the minimum quantumrequirements for O₂ vary by more than twofold, increasing from9.1 to 20.6 quanta/O₂, as growth irradiance increases. The increasein quantum requirement corresponds to larger apparent cross-sectionsfor photosystem I and higher carotenoid/chlorophyll ratios. (Received October 15, 1985; Accepted July 17, 1986)     

Comparison of the Thermodynamic Landscapes of Unfolding and Formation of the Energy Dissipative State in the Isolated Light Harvesting Complex II

In biochemistry and cell biology, understanding the molecular mechanisms by which physiological processes are regulated is regarded as an ultimate goal. In higher plants, one of the most widely investigated regulatory processes occurs in the light harvesting complexes (LHCII) of the chloroplast thylakoid membranes. Under limiting photon flux densities, LHCII harvests sunlight with high efficiency. When the intensity of incident radiation reaches levels close to the saturation of the photosynthesis, the efficiency of light harvesting is decreased by a process referred to as nonphotochemical quenching (NPQ), which enhances the singlet-excited state deactivation via nonradiative dissipative processes. Conformational rearrangements in LHCII are known to be crucial in promoting and controlling NPQ in vitro and in vivo. In this article, we address the thermodynamic nature of the conformational rearrangements promoting and controlling NPQ in isolated LHCII. A combined, linear reaction scheme in which the folded, quenched state represents a stable intermediate on the unfolding pathway was employed to describe the temperature dependence of the spectroscopic signatures associated with the chlorophyll fluorescence quenching and the loss of secondary structure motifs in LHCII. The thermodynamic model requires considering the temperature dependence of Gibbs free energy difference between the quenched and the unquenched states, as well as the unfolded and quenched states, of LHCII. Even though the same reaction scheme is adequate to describe the quenching and the unfolding processes in LHCII monomers and trimers, their thermodynamic characteristics were found to be markedly different. The results of the thermodynamic analysis shed light on the physiological importance of the trimeric state of LHCII in stabilizing the efficient light harvesting mode as well as preventing the quenched conformation of the protein from unfolding. Moreover, the transition to the quenched conformation in trimers reveals a larger degree of cooperativity than in monomers, explained by a small characteristic entropy (ΔH_q = 85 ± 3 kJ mol⁻¹ compared to 125 ± 5 kJ mol⁻¹ in monomers), which enables the fine-tuning of nonphotochemical quenching in vivo.     

Changes of Freeze-Fracture Ultrastructure and Light Harvesting Chlorophyll Protein Complex in Thylakoid Membranes During Ontogeny of Maize

Freeze-fracture electron microscopy enables us to observe and count the freeze-fracture particles which correspond to the different functional components of thylakoid membranes. The present paper reports the observation on freeze-fracture ultrastructure of thylakoid membranes and the analysis of proteins by the SDS-polyacrylamide gel electrophoresis within the membranes from differenly located leaves of maize. In the past, we found that the leaies subtending the ear of maize had a much higher chlorophyll content, a lower chlorophyll a/b ratio and more staking thylakoid membranes and provided the photosynthetic energy used to fill the maize seeds more than that of other leaves. Recently, we have further found that the particle densities of all four faces of thylakoid membranes from the ear leaf were the highest, than those, successively, from the terminal leaf, and the fifth leaf (from the base of the plant). The particle densities on all four fracture faces of thylakoid membranes isolated from the ear leaves of maize were significantly higher than those from the terminal leaves with the increases of 19% in EFs, 28% in PFs and 20% in PFu. Increases in particle densities on the PFs, EFs and PFu faces result in increased densities of LHCP II, PSⅡ and PSI reactions centres, respectively. It is significant that this supramolecular architecture of the ear leaves is consistent with our analytical results of the SDS-polyacrylamide gel electrophoresis within the membranes (a detailed report in another paper). The contents of major polypeptides of 21 kD (LHCP Ⅰ) and 25 kD (LHCP Ⅱ) in thylakoid membranes from the ear leaves were more than those from the terminal leaves. The characteristics of both supramolecular architecture and polypeptide components are in favour of absorbing, transferring, distributing and conversing light energy in the course of photosynthesis of the ear leaves in maize.     

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using ³²P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.     

Light Harvesting and State Transitions in Cyanobacteria

Cyanobacteria are oxygenic phototrophic prokaryotes and are considered to be the ancestors of chloroplasts. Their photosynthetic machinery is functionally equivalent in terms of primary photochemistry and photosynthetic electron transport. Fluorescence measurements and other techniques indicate that cyanobacteria, like plants, are capable of redirecting pathways of excitation energy transfer from light harvesting antennae to both photosystems. Cyanobacterial cells can reach two energetically different states, which are defined as “State 1” (obtained after preferential excitation of photosystem I) and “State 2” (preferential excitation of photosystem II). These states can be distinguished by static and time resolved fluorescence techniques. One of the most important conclusions reached so far is that the presence of both photosystems, as well as certain antenna components, are necessary for state transitions to occur. Spectroscopic evidence suggests that changes in the coupling state of the light harvesting antenna complexes (the phycobilisomes) to both photosystems occur during state transitions. The finding that the phycobilisome complexes are highly mobile on the surface of the thylakoid membrane (the mode of interaction with the thylakoid membrane is essentially unknown), has led to the proposal that they are in dynamic equilibrium with both photosystems and regulation of energy transfer is mediated by changes in affinity for either photosystem.