THE DEVELOPMENT OF PIGMENT GRANULES IN THE EYES OF WILD TYPE AND MUTANT DROSOPHILA MELANOGASTER

The eye pigment system in Drosophila melanogaster has been studied with the electron microscope. Details in the development of pigment granules in wild type flies and in three eye color mutants are described. Four different types of pigment granules have been found. Type I granules, which carry ommochrome pigment and occur in both primary and secondary pigment cells of ommatidia, are believed to develop as vesicular secretions by way of the Golgi apparatus. The formation of Type II granules, which are restricted to the secondary pigment cells and contain drosopterin pigments, involves accumulation of 60- to 80-A fibers producing an elliptical granule. Type III granules appear to be empty vesicles, except for small marginal areas of dense material; they are thought to be abnormal entities containing ommochrome pigment. Type IV granules are characteristic of colorless mutants regardless of genotype, and during the course of development they often contain glycogen, ribosomes, and show acid phosphatase activity; for these reasons and because of their bizarre and variable morphology, they are considered to be autophagic vacuoles. The 300-A particles commonly found in pigment cells are identified as glycogen on the basis of their morphology and their sensitivity to salivary digestion.     

The in vivo and in vitro Reconstitution of Pigment-protein Complexes,and its Implication in Acquiring a New System

Reconstitution is one of the most fundamental and powerful tools to investigate pigment—protein complexes, for example, light-harvesting complexes and reaction center complexes. Two reconstitution methods, in vitro and in vivo, have been applied to complexes. In vitro reconstitution methods were first developed using isolated proteins and pigments, and recently using over-expressed proteins. This method enables analysis of pigment binding, pigment stoichiometry, and protein flexibility when accepting extrinsic pigments, however, it has not yet been successfully applied to the core antenna system. In vivo reconstitution, which was developed using genetic modifications, is applicable even on core systems. In this Review, the in vivo reconstitution is mainly considered on the basis of the in vitro reconstitution, because the former was a recent development and will be expanded to many systems. When genes for a new pigment are acquired and expressed, the new pigment is incorporated into a pre-exiting complex(es) and becomes functional when it is accepted by this complex(es), or abandoned if it is not. This process is postulated to occur during the evolutionary process(es) of antenna and reaction centers, and it is now possible to reproduce this evolutionary developmental pathway(s). Several examples of in vivo reconstitution are given and considered from the viewpoint of evolutionary implication with regards to the antenna and reaction centers.This revised version was published online in October 2005 with corrections to the Cover Date.     

Pigment-binding properties of mutant light-harvesting chlorophyll-a/b-binding protein.

Light-harvesting chlorophyll-a/b-binding protein (LHCP), overexpressed in Escherichia coli, can be reconstituted with pigments to yield complexes that are structurally very similar to light-harvesting complex II (LHCII) isolated from thylakoids [Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta 181, 204-211]. In order to analyze which domains of the protein are involved in pigment binding, we reconstituted deletion mutants of LHCP with pigments and characterized the resulting complexes regarding their pigment composition and spectroscopic properties. Series of progressive deletions from either end of the protein revealed that most of the N-terminal and part of the C-terminal hydrophilic regions of LHCP are dispensible for pigment binding. In either deletion series, the deletions that completely abolished reconstitution could be narrowed down to segments of five amino acids that do not contain histidine, asparagine, or glutamine. All mutants either formed complexes with both pigment composition and spectroscopic properties very similar to those of light-harvesting complex II isolated from thylakoids, or they did not form any stable complexes at all. There is no indication of a segment of LHCP binding a subset of LHCII pigments. We conclude that the stabilization of LHCP-pigment complexes is highly synergetic rather than based on individual pigment-binding sites provided by the protein.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Fine Structure of the Retinal Pigment Epithelium of the River Lamprey (Lampetra fluviatilis,Cyclostomi)

The retinal pigment epithelium of Lampetra fluviatilis was studied by electron microscopy. The epithelial cells differ in many details from those of gnathostomes. The lateral cell membranes are difficult to distinguish. The smooth endoplasmic reticulum is well developed; in some animals undulated membrane complexes, comprising systems of tightly fused double membrane plates, are related to the endoplasmic reticulum. Myeloid bodies are common and well developed, but pigment granules are comparatively sparse. The intercellular space between pigment epithelium and photoreceptors is rather wide. There are only a few inclusion bodies with membranous contents. The importance of the pigment epithelium in the retinal metabolic exchange is discussed in view of the fine structure of the cells. Compared with that of hagfishes, the lamprey retina is well developed. However, any comparison must be made against the background of a diphyletic development of the two groups.     

Accumulation of a Complex of Pigments in Sorghum Mesocotyls in Response to Wounding

Sorghum mesocotyls upon mechanical injury with an abrasive (carborundum) or inoculation with the fungi Helminthosporium carbonum (non-pathogen) or Colletotrichum graminicola (pathogen) accumulate a methanol-soluble pigment complex with an absorption maximum around 480–490 nm. Spectral and thin-layer chromatographic analyses showed that the complexes which accumulated either in response to wounding or inoculation are similar. Thus, it is suggested that the accumulation of the pigmented phytoalexins in sorghum mesocotyls is a non-specific response of the tissues towards mechanical injury or fungal infection.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Degradation of carotenoids

The loss of pigments was assessed in detached leaves of Festuca pratensis Huds. kept in permanent darkness. Two genotypes, a normal yellowing cultivar Rossa and a non-yellowing mutant Bf 993 were compared with each other. Analysis of individual pigments, chlorophylls. β-carotene, lutein, violaxanthin and neoxanthin was performed using HPLC. In the non-yellowing genotype the high retention of chlorophylls was associated with an equally high retention of total carotenoids. Although the two genotypes differ markedly with regard to the rate of pigment loss, the ratios of yellow to green pigments did not change significantly during dark-induced senescence. At the end of the senescence period β-carotene was retained to a higher degree than the xanthophylls, particularly in the yellowing genotype. In the mutant leaves the ratio of chlorophyll a to b remained nearly constant, whereas in leaves of the normal genotype a preferential retention of chlorophyll b was observed towards the end of the senescence period. It is concluded that the thylakoids of the non-yellowing genotype retain all the principal components of protein-pigment complexes, i.e. chlorophylls, carotenoids and apoproteins. Possible explanations for the stability of these complexes in the mutant are discussed.     

Peridinin-chlorophyll-protein reconstituted with chlorophyll mixtures: preparation, bulk and single molecule spectroscopy

Reconstitution of the 16 kDa N-terminal domain of the peridinin-chlorophyll-protein, N-PCP, with mixtures of chlorophyll a (Chl a) and Chl b, resulted in 32 kDa complexes containing two pigment clusters, each bound to one N-PCP. Besides homo-chlorophyllous complexes, hetero-chlorophyllous ones were obtained that contain Chl a in one pigment cluster, and Chl b in the other. Binding of Chl b is stronger than that of the native pigment, Chl a. Energy transfer from Chl b to Chl a is efficient, but there are only weak interactions between the two pigments. Individual homo- and hetero-chlorophyllous complexes were investigated by single molecule spectroscopy using excitation into the peridinin absorption band and scanning of the Chl fluorescence, the latter show frequently well resolved emissions of the two pigments.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F(V)/F(M), whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F(V)/F(M) of approximately 0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

The genetics of cream coat color in dogs

Cream dogs of several breeds require a genotype of e/e at MC1R based on 27 individuals in this study. All Akita, Caucasian Mountain Dogs, German Shepherd Dogs, Miniature Schnauzer, and Puli with this genotype are cream, suggesting they are fixed at a second locus which causes the phaeomelanin pigmentation caused by this genotype to be diluted or pale. Conversely, although all Chinese Shar-Pei and Poodles that were cream had an e/e genotype at MC1R, not all dogs with this genotype are cream. Today, many Golden Retrievers and Labrador Retrievers with an e/e genotype are cream instead of the traditional yellow to golden color seen in the past. The second gene in these breeds must have multiple alleles, only one of which causes phaeomelanin pigment to be diluted or pale. Tyrosinase (TYR) and solute carrier family 45, member 2 (SLC45A2) have been shown to cause cream coat color in other species and were therefore investigated in dogs as candidate genes for this second locus. Although polymorphisms were detected in cDNA sequence from TYR and SLC45A2, no polymorphism was consistently associated with cream dogs or cosegregated with cream coat color in any of the families used in this study. A microsatellite was detected in a published BAC sequence (GenBank no. AAEX01017083) in intron 2 and was used to map SLC45A2 to CFA4.     

Ultrastructure and development of the rhabdomeric eyes in Lineus viridis (Heteronemertea, Nemertea)

Nemerteans are undoubtedly members of the Spiralia, although their phylogenetic relationships are still a matter of debate. The apparently acoelomate organization suggests a relationship with the platyhelminths, whereas the blood-vascular system has been interpreted as an equivalent to coelomic cavities of annelids, indicating a close relation between annelids and nemerteans. Like other spiralians, most nemertean species are known to have one or several pairs of rhabdomeric and subepidermally situated eyes when adult. The development of these eyes as well as the mode in which the eyes are multiplied is as yet unknown. This is the first attempt to investigate eye formation in a nemertean. In the heteronemertean Lineus viridis (Müller, 1774) the everse rhabdomeric eyes are located deeply underneath the epidermis and consist of a few pigment cells that form a cup-like structure with interdigitating processes that contain numerous pigment granules. In hatchlings, the optical cavity contains processes of 12 sensory cells, each bearing a single cilium and various microvilli. The perikarya of these cells are located distally from the pigment cup. During further development the number of cells increases. Eye development starts with a small anlage situated underneath the epidermis, irrespective of whether this is the first eye or any additional one. The anlage consists of five unpigmented cells and three dendritic processes, each bearing apical microvilli and a single cilium. There is no evidence for an epidermal origin of the eyes. In L. viridis eye formation resembles that described in platyhelminths in which eyes also develop as cerebral derivatives. Although this result has the potential to influence the discussion on the position of Nemertea, the data have to be interpreted with care, since development of L. viridis is derived within the Nemertea.     

Angiotensin-converting enzyme gene I/D dimorphism does not play a major role in the susceptibility of Malaysian systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes systemic damage, involving auto-reactive antibodies and over-deposition of immune complexes. Susceptibility to SLE is believed to be multifactorial, and genetics is one of the proven etiological factors; it can affect SLE development, severity and prognosis. We investigated a possible association between the angiotensin-converting enzyme gene and susceptibility to SLE in the Malaysian population. PCR was employed for the determination of I/D dimorphism of this gene. The I allele was more frequent than the D allele in both the SLE patients (N = 170) and healthy controls (N = 190). However, there was no significant difference in the distribution of these two alleles between both groups studied (χ(2) = 0.284, P > 0.05). Interestingly, the DD homozygous genotype scored notably higher in the healthy control group (χ(2) = 7.568, P < 0.05), while the ID heterozygote was observed to be significantly associated with SLE (χ(2) = 11.143, P < 0.05). In conclusion, with respect to the Malaysian population, the DD genotype might play a protective role in the development of SLE while in contrast, those who carry the ID genotype might be at potential risk for onset of this disease.     

Functional Organization of Chlorophyll a and Carotenoids in the Alga, Nannochloropsis salina

Chlorophyll-protein complexes were isolated from a yellow-green alga, Nannochloropsis salina after mild detergent treatment and gel electrophoresis. Three different complexes were obtained which correspond to the three major kinds of chlorophyll-proteins isolated from spinach chloroplasts by the same procedure and previously identified as reaction center complexes for photosystems I and II and a light-harvesting complex. The analogy between the algal complexes and those from spinach was drawn from their absorption and fluorescence spectra and relative pigment content. The identities and amounts of the major carotenoids associated with each isolated complex were determined by HPLC. Although the reaction center complexes accounted for only 14% of the total chlorophyll, they were highly enriched in β-carotene, whereas the light-harvesting complex contained a high proportion of xanthophylls (mainly violaxanthin and vaucheriaxanthin-ester). Fluorescence excitation spectra of the algal membranes showed that one or both of the major xanthophylls may act as antenna pigment for photosynthesis.     

Characterization of green mutants in Fremyella diplosiphon provides insight into the impact of phycoerythrin deficiency and linker function on complementary chromatic adaptation

Functions of phycobiliprotein (PBP) linkers are less well studied than other PBP polypeptides that are structural components or required for the synthesis of the light-harvesting phycobilisome (PBS) complexes. Linkers serve both structural and functional roles in PBSs. Here, we report the isolation of a phycoerythrin (PE) rod-linker mutant and a novel PE-deficient mutant in Fremyella diplosiphon. We describe their phenotypic characterization, including light-dependent photosynthetic pigment accumulation and photoregulation of cellular morphology. PE-linker protein CpeE and a novel protein impact PE accumulation, and thus PBS function, primarily under green light conditions.     

Antioxidant capacity of a 3-deoxyanthocyanidin from soybean.

Soybean cotyledons directly exposed to UV-C (190-280 nm) contained a colored pigment in those areas of the epidermis directly exposed to UV-C. Ethanolic extracts from UV-C irradiated cotyledons showed a significant peak at 532 nm at pH=10, but not seen at pH=6, successive changes in pH were accompanied by reversible changes in the spectra. The identity of the pigment isolated from soybean cotyledons was established as apigeninidin by comparing the features of standard of a apigeninidin (from sorghum) previously characterized by FAB-MS, UV, HPLC, 1H NMR, and IR spectroscopy. To characterize antioxidant activity of this compound, its ability to scavenge radical species in vitro was tested. In the concentration range tested (up to 200 microg ml (-1)), apigeninidin did not show any scavenger activity towards hydroxyl radical, quinones or NO. However, ascorbyl radical and lipid radicals were effectively quenched in a dose-dependent manner. Overall, UV-C radiation triggers molecular signals that lead in soybean cotyledons to the synthesis and accumulation of an antioxidant pigment, apigeninidin, that shows scavenger activity against ascorbyl and lipid radicals in in vitro studies.     

Pigment-pigment interactions and secondary structure of reconstituted algal chlorophyll a/b-binding light-harvesting complexes of Chlorella fusca with different pigment compositions and pigment-protein stoichiometries

Earlier we have shown by in vitro reconstitution experiments that the pigment composition of the chlorophyll alb-binding light-harvesting complex of the green alga Chlorella fusca could be altered in a relatively broad range (Meyer and Wilhelm 1993). In this study we used these reconstituted complexes of different pigment loading to analyze the excitonic interactions between the pigment molecules and the secondary structure by means of circular dichroism spectra in the visible and the far UV spectral regions, respectively. We found that, in contrast to the expectations, the pigment composition and pigment content hardly affected the circular dichroism spectra in the visible spectral region. Reconstituted complexes, independent of their pigment composition, exhibited the most characteristic circular dichroism bands of the native light-harvesting complex, even if one polypeptide bound only 3 chlorophyll a, 3 chlorophyll b and 1–2 xanthophyll molecules. Full restoration of the protein secondary structure, however, could not be achieved. The -helix content depended significantly on the pigment composition as well as on the pigment-protein ratio of the reconstituted complexes. Further binding of pigments resulted in restoration of the minor excitonic circular dichroism bands, the amplitudes of which depended on the pigment content of the reconstituted complexes. These data suggest that in the reconstitution of light-harvesting complexes a central cluster of pigment molecules plays an important role. Further binding of pigments to the peripheral binding sites appeared also to stabilize the protein secondary structure of the reconstituted complexes.Abbreviations CD- circular dichroism - LHC- chlorophyll a/b light-harvesting complex(es) - LHC II- light-harvesting complex(es) of Photosystem II of higher plants - LHCP- light-harvesting Chl a/b-binding protein(s) - PP- polypeptide(s)