Pigment Binding Research Articles

Widespread adaptive evolution in angiosperm photosystems provides insight into the evolution of photosystem II repair.

Oxygenic photosynthesis generates the initial energy source that fuels nearly all life on Earth. At the heart of the process are the photosystems, which are pigment binding multi-protein complexes that catalyse the first step of photochemical conversion of light energy into chemical energy. Here, we investigate the molecular evolution of the plastid-encoded photosystem subunits at single-residue resolution across 773 angiosperm species. We show that despite an extremely high level of conservation, 7% of residues in the photosystems, spanning all photosystem subunits, exhibit hallmarks of adaptive evolution. Through in silico modelling of these adaptive substitutions, we uncover the impact of these changes on the predicted properties of the photosystems, focussing on their effects on co-factor binding and inter-subunit interface formation. By analyzing these cohorts of changes, we reveal that evolution has repeatedly altered the interaction between photosystem II and its D1 subunit in a manner that is predicted to reduce the energetic barrier for D1 turnover and photosystem repair. Together, these results provide insight into the trajectory of photosystem adaptation during angiosperm evolution.

Comparative proteomics elucidates the potential mechanism of heritable carapace color of three strains Chinese mitten crab Eriocheir sinensis

The carapace coloration is important for the environmental adaptation and reproductive behaviors of crustaceans. We selected red, green and white three carapace color strains of Chinese mitten crab (Eriocheir sinensis) strains. These three carapace colors have stable heritability, but the mechanism for their coloration remains unclear.Through histological observations, we have found significant differences in the composition of pigment cells and pigments within the inner membrane of the three color strains, which may be one of the reasons for the color variation. The levels of various carotenoids in both the shell and inner membrane tissues of red and green strains were significantly higher than those of the white strain, while there was no significant difference between the red and green strains. Proteomics studies have identified 2, 034 and 947 different proteins in the shell and inner membrane, respectively. In the shell, there were 18, 13 and 43 differential proteins between red and white strains, green and white strains and green and red strains, respectively. In the inner membrane, there were 44, 24 and 16 differential proteins between red and white strains, green and white strains and green and red strains, respectively. It is clear that the deposited quantity of carotenoids affects the shell formation of three color strains. Some members of the hemocyanin family showed significant variation among different strains. The study yielded two crustacyanin proteins, which were extracted from both the shell and membrane. Of the two proteins, only Crustacyanin-A1 expression showed a difference between the red and green shells strains. In conclusion, these results indicated that the carapace color formation of E. sinensis may be accomplished through pigment binding proteins (PBPs) and pigment cells, which enhance the understanding of color formation mechanism for crustacean.

Effect of decontamination materials on bond strength of saliva-contaminated CAD/CAM resin block and dentin.

The aim of this study was to evaluate the effect of decontamination agents against saliva contamination on the bonding of computer-aided design/computer-aided manufacturing (CAD/CAM) resin block. Three commercially available decontamination agents were used in this study. The samples were subjected to microtensile bond strength test. A saliva protein staining test was performed by the pigment binding method to investigate the effect of removing saliva protein. All the decontamination agents could significantly restore the bond strength from the saliva contamination, and KATANA Cleaner showed no significant difference with the control. From the results of the saliva protein staining test, KATANA Cleaner showed higher removal effect of saliva protein for CAD/CAM resin block surface than the other materials due to the surface active effect of MDP salt. It was suggested that cleaners containing MDP salt were more effective in removing artificial saliva contamination than cleaners containing other ingredients for CAD/CAM resin blocks.

In vitro reconstitution of complexes of stress HliA protein with pigments

Proteins similar to Hli (high light inducible) proteins of cyanobacteria are present in all photosynthetic eukaryotes and are necessary for survival in various stressful conditions, although their exact function is not fully understood. In current study, the recombinant stress-induced protein HliA of cyanobacterium Synechocystis was isolated and characterised for the first time. The synthetic gene of HliA protein was created and cloned into plasmid for expression of recombinant protein with Hisx6-tag at the C-terminus in bacteria. Recombinant HliA protein of Synechocystis was isolated by metal-affinity chromatography. The HliA protein was reconstituted with chlorophyll a and carotenoids. Using circular dichroism spectroscopy, it was shown that chlorophyll a and carotenoids interact in vitro with the HliA protein. The binding of pigments to the HliA protein favours the protective function of this protein. Apparently, Hli proteins are involved in the coordinated delivery of pigments for the biogenesis of photosynthetic complexes, thereby reducing the risk of accumulation of phototoxic free chlorophyll molecules. Current results are important for understanding the processes of photoprotection in either cyanobacteria or algae and higher plants.

High-light-inducible proteins HliA and HliB: pigment binding and protein-protein interactions.

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and β-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis

We observed differences in lhc classification in Chromista. We proposed a classification of the lhcf family with two groups specific to haptophytes, one specific to diatoms, and one specific to seaweeds. Identification and characterization of the Fucoxanthin and Chlorophyll a/c-binding Protein (FCP) of the haptophyte microalgae Tisochrysis lutea were performed by similarity analysis. The FCP family contains 52 lhc genes in T. lutea. FCP pigment binding site candidates were characterized on Lhcf protein monomers of T. lutea, which possesses at least nine chlorophylls and five fucoxanthin molecules, on average, per monomer. The expression of T. lutea lhc genes was assessed during turbidostat and chemostat experiments, one with constant light (CL) and changing nitrogen phases, the second with a 12 h:12 h sinusoidal photoperiod and changing nitrogen phases. RNA-seq analysis revealed a dynamic decrease in the expression of lhc genes with nitrogen depletion. We observed that T. lutea lhcx2 was only expressed at night, suggesting that its role is to protect \\cells from return of light after prolonged darkness exposure.

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins.

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Integrated Analysis of Transcriptome and Proteome to Reveal Pupal Color Switch in Papilio xuthus Butterflies.

Pupal color polyphenism in Papilio butterflies, including green, intermediate, or brown, is an excellent study system for understanding phenotypic plasticity. Previous studies suggested that development of brown pupae may be controlled by a hormone called pupal-cuticle-melanizing-hormone (PCMH) which is synthesized and secreted from brain-suboesophageal ganglion and prothoracic ganglion complexes (Br-SG-TG1) during the pre-pupa stage. However, detailed molecular mechanisms of neuroendocrine regulation in pupal color development remain unknown. In this study, we integrated the expression profiles of transcriptome and proteome at pre-pupa stages [2 h after gut purge (T1) and 3 h after forming the garter around the body (T2)] and pigmentation stages [10 h after ecdysis (T3) and 24 h after ecdysis (T4)] to identify important genes and pathways underlying the development of green and brown pupa in the swallowtail butterfly Papilio xuthus. Combined comparisons of each developmental stage and each tissue under green and brown conditions, a total of 1042 differentially expressed genes (DEGs) and 430 different abundance proteins (DAPs) were identified. Weighted gene co-expression network analysis (WGCNA) and enrichment analysis indicate that these DEGs were mainly related to oxidation-reduction, structural constituent of cuticle, and pigment binding. Soft clustering by Mfuzz and enrichment analysis indicate that these DAPs are mainly involved in tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. By homologous alignment, we further identified those genes encoding neuropeptides (51), GPCRs (116), G-proteins (8), cuticular proteins (226), chitinases (16), and chitin deacetylases (8) in the whole genome of P. xuthus and analyzed their expression profiles. Although we identified no gene satisfying with hypothesized expression profile of PCMH, we found some genes in the neuropeptide cascade showed differentially expressed under two pupal color conditions. We also found that Toll signaling pathway genes, juvenile hormone (JH) related genes, and multiple cuticular proteins play important roles in the formation of selective pupal colors during the prepupal-pupal transition. Our data also suggest that both green and brown pupa include complex pigment system that is regulated by genes involved in black, blue, and yellow pigments. Our results provide important insights into the evolution of pupal protective colors among swallowtail butterflies.

Biochemical and molecular properties of LHCX1, the essential regulator of dynamic photoprotection in diatoms.

Light harvesting is regulated by a process triggered by the acidification of the thylakoid lumen, known as nonphotochemical "energy-dependent quenching" (qE). In diatoms, qE is controlled by the light-harvesting complex (LHC) protein LHCX1, while the LHC stress-related (LHCSR) and photosystem II subunit S proteins are essential for green algae and plants, respectively. Here, we report a biochemical and molecular characterization of LHCX1 to investigate its role in qE. We found that, when grown under intermittent light, Phaeodactylum tricornutum forms very large qE, due to LHCX1 constitutive upregulation. This "super qE" is abolished in LHCX1 knockout mutants. Biochemical and spectroscopic analyses of LHCX1 reveal that this protein might differ in the character of binding pigments relative to the major pool of light-harvesting antenna proteins. The possibility of transient pigment binding or not binding pigments at all is discussed. Targeted mutagenesis of putative protonatable residues (D95 and E205) in transgenic P. tricornutum lines does not alter qE capacity, showing that they are not involved in sensing lumen pH, differently from residues conserved in LHCSR3. Our results suggest functional divergence between LHCX1 and LHCSR3 in qE modulation. We propose that LHCX1 evolved independently to facilitate dynamic tracking of light fluctuations in turbulent waters. The evolution of LHCX(-like) proteins in organisms with secondary red plastids, such as diatoms, might have conferred a selective advantage in the control of dynamic photoprotection, ultimately resulting in their ecological success.

Chlamydomonas reinhardtii LHCSR1 and LHCSR3 proteins involved in photoprotective non-photochemical quenching have different quenching efficiency and different carotenoid affinity

Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, their limited biomass productivity represents a bottleneck that needs to be overcome to meet the applicative potential of these organisms. One of the domestication targets for improving their productivity is the proper balance between photoprotection and light conversion for carbon fixation. In the model organism for green algae, Chlamydomonas reinhardtii, a photoprotective mechanism inducing thermal dissipation of absorbed light energy, called Non-photochemical quenching (NPQ), is activated even at relatively low irradiances, resulting in reduced photosynthetic efficiency. Two pigment binding proteins, LHCSR1 and LHCSR3, were previously reported as the main actors during NPQ induction in C. reinhardtii. While previous work characterized in detail the functional properties of LHCSR3, few information is available for the LHCSR1 subunit. Here, we investigated in vitro the functional properties of LHCSR1 and LHCSR3 subunits: despite high sequence identity, the latter resulted as a stronger quencher compared to the former, explaining its predominant role observed in vivo. Pigment analysis, deconvolution of absorption spectra and structural models of LHCSR1 and LHCR3 suggest that different quenching efficiency is related to a different occupancy of L2 carotenoid binding site.

Anatomical Distribution of Ochronotic Pigment in Alkaptonuric Mice is Associated with Calcified Cartilage Chondrocytes at Osteochondral Interfaces

Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl’s staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c Hgd−/−, Hgd tm1a−/−), using Schmorl’s staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (p = 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.

How water-soluble chlorophyll protein extracts chlorophyll from membranes

Water-soluble chlorophyll proteins (WSCPs) found in Brassicaceae are non-photosynthetic proteins that bind only a small number of chlorophylls. Their biological function remains unclear, but recent data indicate that WSCPs are involved in stress response and pathogen defense as producers of reactive oxygen species and/or Chl-regulated protease inhibitors. For those functions, WSCP apoprotein supposedly binds Chl to become physiologically active or inactive, respectively. Thus, Chl-binding seems to be a pivotal step for the biological function of WSCP. WSCP can extract Chl from the thylakoid membrane but little is known about the mechanism of how Chl is sequestered from the membrane into the binding sites. Here, we investigate the interaction of WSCP with the thylakoid membrane in detail. The extraction of Chl from the thylakoid by WSCP apoprotein is a slow and inefficient reaction, because WSCP presumably does not directly extract Chl from other Chl-binding proteins embedded in the membrane. WSCP apoprotein interacts with model membranes that contain the thylakoid lipids MGDG, DGDG or PG, and can extract Chl from those. Furthermore, the WSCP-Chl complex, once formed, no longer interacts with membranes. We concluded that the surroundings of the WSCP pigment-binding site are involved in the WSCP-membrane interaction and identified a ring of hydrophobic amino acids with two conserved Trp residues around the Chl-binding site. Indeed, WSCP variants, in which one of the Trp residues was exchanged for Phe, still interact with the membrane but are no longer able to extract Chl.

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs.

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology.

The pigment binding behaviour of water-soluble chlorophyll protein (WSCP).

Water-soluble chlorophyll proteins (WSCPs) are homotetrameric proteins that bind four chlorophyll (Chl) molecules in identical binding sites, which makes WSCPs a good model to study protein-pigment interactions. In a previous study, we described preferential binding of Chl a or Chl b in various WSCP versions. Chl b binding is preferred when a hydrogen bond can be formed between the C7 formyl of the chlorin macrocycle and the protein, whereas Chl a is preferred when Chl b binding is sterically unfavorable. Here, we determined the binding affinities and kinetics of various WSCP versions not only for Chl a/b, but also for chlorophyllide (Chlide) a/b and pheophytin (Pheo) a/b. Altered KD values are responsible for the Chl a/b selectivity in WSCP whereas differences in the reaction kinetics are neglectable in explaining different Chl a/b preferences. WSCP binds both Chlide and Pheo with a lower affinity than Chl, which indicates the importance of the phytol chain and the central Mg2+ ion as interaction sites between WSCP and pigment. Pheophorbide (Pheoide), lacking both the phytol chain and the central Mg2+ ion, can only be bound as Pheoide b to a WSCP that has a higher affinity for Chl b than Chl a, which underlines the impact of the C7 formyl-protein interaction. Moreover, WSCP was able to bind protochlorophyllide and Mg-protoporphyrin IX, which suggests that neither the size of the π electron system of the macrocycle nor the presence of a fifth ring at the macrocycle notably affect the binding to WSCP. WSCP also binds heme to form a tetrameric complex, suggesting that heme is bound in the Chl-binding site.

Characterization of photosynthetic protein complexes in conchocelis and blades of Pyropia yezoensis (Rhodophyta)

The red alga Pyropia yezoensis is an economically important marine crop whose life cycle includes two distinct stages: conchocelis and blades. These two types of thalli live in significantly different environments and have distinct shapes and physiological features. The photosynthetic apparatus of red algae, especially the light harvesting antenna, is unique. However, little is known about the photosynthetic apparatus of P. yezoensis. We studied the composition and organization of thylakoid membranes in conchocelis and blades of P. yezoensis and found that the photosynthetic complexes differed between the two stages. Conchocelis had much higher photosystem I (PSI)/ photosystem II and PSI/Cytochrome b6/f ratios compared with blades. Additionally, a macromolecular protein complex that bound with lutein and Chlorophyll a was detected in conchocelis but not in blades. The number and types of light harvesting complex subunits associated with PSI and the corresponding pigment binding also varied between conchocelis and blades. These differences are an important strategy that allows the two P. yezoensis stages to adapt to various environments.

ONE-HELIX PROTEIN1 and 2 Form Heterodimers to Bind Chlorophyll in Photosystem II Biogenesis.

Members of the light-harvesting complex protein family participate in multiple processes connected with light sensing, light absorption, and pigment binding within the thylakoid membrane. Amino acid residues of the light-harvesting chlorophyll a/b-binding proteins involved in pigment binding have been precisely identified through x-ray crystallography experiments. In vitro pigment-binding studies have been performed with LIGHT-HARVESTING-LIKE3 proteins, and the pigment-binding ability of cyanobacterial high-light-inducible proteins has been studied in detail. However, analysis of pigment binding by plant high-light-inducible protein homologs, called ONE-HELIX PROTEINS (OHPs), is lacking. Here, we report on successful in vitro reconstitution of Arabidopsis (Arabidopsis thaliana) OHPs with chlorophylls and carotenoids and show that pigment binding depends on the formation of OHP1/OHP2 heterodimers. Pigment-binding capacity was completely lost in each of the OHPs when residues of the light-harvesting complex chlorophyll-binding motif required for chlorophyll binding were mutated. Moreover, the mutated OHP variants failed to rescue the respective knockout (T-DNA insertion) mutants, indicating that pigment-binding ability is essential for OHP function in vivo. The scaffold protein HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244) is tethered to the thylakoid membrane by the OHP heterodimer. We show that HCF244 stability depends on OHP heterodimer formation and introduce the concept of a functional unit consisting of OHP1, OHP2, and HCF244, in which each protein requires the others. Because of their pigment-binding capacity, we suggest that OHPs function in the delivery of pigments to the D1 subunit of PSII.

Enrichment Analysis of Differentially Expressed Genes during Endosperm Grouting Periods in Non-waxy and Waxy Foxtail Millets

The content of amylose and amylopectin is an important trait affecting cooking and eating quality in millet. During process of kernel grouting, the synthesis of starch involves different pathways and components in non-waxy and waxy millets. Immature grouting grains of waxy millet ‘Gonggu68’ and non-waxy millet ‘Chigu4’ (grouting period 1 and 5 days) were used to analyze their transcriptome sequences by using Illumina Hiseq4000. The results showed that: (1) GBSSⅠ enzymes activity of waxy and non-waxy millet was low-high-low. There were some differences between the two activities. 665 upregulated differentially expressed genes were screened on day 5 and day 1 during grouting period in waxy cultivar ‘Gonggu68’, there were 431 more upregulated genes than downregulated genes. There were 97 more up-regulated genes than down-regulated genes in non-waxy cultivar ‘Chigu4’ on day 5 and day 1 in grouting period. (2) In the A 2 -VS-A 1 waxy comparison group, the differential genes were mainly GO enriched in 7 functions such as the seed oil body biogenic function, 17-β-ketosteroid reductase activity function and so on. it was mainly enriched in biological processes and molecular functions. In the B 2 -VS-B 1 non-waxy comparison group, the differential genes were mainly GO enriched in 8 functions such as the light capturing function and the pigment binding function in the light system I for non-waxy millet. (3) Differentially expressed genes were mainly KEGG enriched in caffeine metabolism pathway, linoleic acid metabolism pathway, anthocyanin biosynthesis pathway, aflatoxin biosynthesis pathway in waxy A 2 -VS-A 1 , but which were mainly KEGG enriched in the synergy-antenna protein pathway, the linoleic acid metabolic pathway, the caffeine metabolic pathway, the brassinosteroid biopathway in non-waxy B 2 -VS-B 1 . These two comparative groups were enriched Caffeine metabolism pathway and linoleic acid metabolism pathway appeared in the process. (4) Three ( SSII-3 , PHO1 , AS ) and four ( PHO1-1 , AS , AGP16 , WAXY ) genes with significant differences and related to waxy and non-waxy millet were screened. With Actin (Si001873) as the internal reference gene, the above seven differentially expressed genes were verified by qRT-PCR, which was consistent with the transcriptome results, indicating that the differentially expressed genes were related to waxy or non-waxy endosperm.

IDENTIFICATION OF THE PAINTING MATERIALS OF A UNIQUE EASEL PAINTING BY MAHMOUD SA'ID

The present study aims at examining a unique easel painting by the well-knownEgyptian painter, Mahmoud Sa'id. It was painted over four years and displayed atthe Agricultural Museum in Egypt. It experienced many deterioration phenomena.The study utilized some non-destructive and non-invasive analytical techniques,i.e. MA-XRF, SEM-EDX, ATR-FTIR, and GC-FID in addition to XRD and SM.Due to the case-study painting multilayers technique, the results of MA-XRF andEDX mapping and XRD patterns revealed using Ultramarine (Na,Ca)8 (AlSiO4)6(SO4,S,Cl)2, yellow ochre (Fe2O3.H2O), red ochre (Fe2O3), and a mixture ofgraphite (C) and bone black (C,Ca5(OH)(PO4)3) as a coloring pigments in theblue, yellow, reddish brown and black samples, respectively. Moreover, Barite(BaSO4) and Zincite (ZnO) were used in the ground layer with Hydrocerussite(Pb3(CO3)2(OH)2) and Sphalerite (ZnS) in lightening colors. Linseed oil wasasserted as the pigment binding media, while the binding media of the groundlayer was not confirmed. Furthermore, fragility, brittleness, cleavages, peeling,cracks, macro cracks, stains, and ancient fault restoration were detected in thecase-study painting. Accordingly, it urgently requires treatment.

A Supramolecular Approach for Modulated Photoprotection, Lysosomal Delivery, and Photodynamic Activity of a Photosensitizer.

Prompted by a knowledge of the photoprotective mechanism operating in photosystem supercomplexes and bacterial antenna complexes by pigment binding proteins, we have appealed to a boxlike synthetic receptor (ExBox·4Cl) that binds a photosensitizer, 5,15-diphenylporphyrin (DPP), to provide photoprotection by regulating light energy. The hydrophilic ExBox4+ renders DPP soluble in water and modulates the phototoxicity of DPP by trapping it in its cavity and releasing it when required. While trapping removes access to the DPP triplet state, a pH-dependent release of diprotonated DPP (DPPH22+) restores the triplet deactivation pathway, thereby activating its ability to generate reactive oxygen species. We have employed the ExBox4+-bound DPP complex (ExBox4+⊃DPP) for the safe delivery of DPP into the lysosomes of cancer cells, imaging the cells by utilizing the fluorescence of the released DPPH22+ and regulating photodynamic therapy to kill cancer cells with high efficiency.

Encapsulation of Photosystem I in Organic Microparticles Increases Its Photochemical Activity and Stability for Ex Vivo Photocatalysis.

Photosystem I (PSI) is a pigment binding multisubunit protein complex involved in the light phase of photosynthesis, catalyzing a light-dependent electron transfer reaction from plastocyanin to ferredoxin. PSI is characterized by a photochemical efficiency close to one, suggesting its possible application in light-dependent redox reaction in an extracellular context. The stability of PSI complexes isolated from plant cells is however limited if not embedded in a protective environment. Here we show an innovative solution for exploiting the photochemical properties of PSI, by encapsulation of isolated PSI complexes in PLGA (poly lactic-co-glycolic acid) organic microparticles. These encapsulated PSI complexes were able to catalyze light-dependent redox reactions with electron acceptors and donors outside the PLGA microparticles. Moreover, PSI complexes encapsulated in PLGA microparticles were characterized by a higher photochemical activity and stability compared with PSI complexes in detergent solution, suggesting their possible application for ex vivo photocatalysis.