FORSCHUNG 45

CHIMIA 49 (1995) Nr. 3 (Miirz)

Chim;a 49 (1995) 45-68 quire specific molecules, pigments or dyes © Neue Schweizerische Chemische Gesellschaft (biochromes) or systems containing them, /SSN 0009-4293 to absorb the light energy. Photoprocesses and colors are essential for life on earth, and without these biochromes and the photophysical and photochemical interac- tions, life as we know it would not have The Function of Natural been possible [1][2]. a Colorants: The Biochromes ) 1.2. Notation The terms colorants, dyes, and pig- ments ought to be used in the following way [3]: Colorants are either dyes or pig- Hans-Dieter Martin* ments, the latter being practically insolu- ble in the media in which they are applied. Indiscriminate use of these terms is fre- Abstract. The colors of nature belong undoubtedly to the beautiful part of our quently to be found in literature, but in environment. Colors always fascinated humans and left them wonderstruck. But the many biological systems it is not possible trivial question as to the practical application of natural colorants led soon and at all to make this differentiation. The consequently to coloring and dyeing of objects and humans. Aesthetical, ritual and coloring compounds of organisms have similar aspects prevailed. This function of dyes and pigments is widespread in natl)re. been referred to as biochromes, and this The importance of such visual-effective dyes is obvious: they support communication seems to be a suitable expression for a between organisms with the aid of conspicuous optical signals and they conceal biological colorant, since it circumvents revealing ones, wl,1eninconspicuosness can mean survival. But the purpose of natural the difficulty to distinguish between dye pigments and biochromes is broader. Emergence and development of life on earth is and pigment. Notwithstanding these ob- inseparably associated with the radiating energy source, the sun. This led to the jections all terms will be used when dis- evolution of biochromes with various functions: collection and harvesting of light, cussing color in living systems [4][5]. transduction into chemical energy, transport of electrons or gases, photoreceptor processing of information and color discrimination, to mention only a few. Without 1.3. Function these pigments - with their photobiological, biochemical, and physiological reactions The functions of biochromes can be - life, as we know it, would not have been possible. Color is essential. Several times either biochemical and metabolic or bio- during the evolution protecting devices and mechanisms had to be invented against the physical and physiological, the visual-ef- destructive and damaging influence of reactive oxygen, light and UV radiation. To this fect functions belonging to the latter end nature employed accessory pigments as carotenoids and flavonoids. It is notable category .The connection between the two and interesting to recognize that human epidemiology and animal studies have groups is made by the light-perceptor func- indicated that cancer risk may be modified by changes in dietary habits or dietary tions [6]. These three main functions are components. Recent studies indicate that phytochemicals, among them the carotenoids presented in Fig. 1 and flavonoids, can inhibit tumor genesis at one or several stages.

1. Introduction

1.1. Life, Light, and Color Light from the sun has been associated ® Light Source with life since the very origin of life itself. This is born out by the fact that all organ- isms, from bacteria to man, exhibit some "IMetabaliC FunctionI form of photosensitivity to solar radiation. y According to Grotthus-Draper's Jaw of absorption photobiological phenomena re- ~ Response

~

*Correspondence: Prof. Dr. H.-D. Martin Institut fur Organische Chemie und Organism with Organism with Visual and/or Makromolekulare Chemie Lehrstuhl I other Photoreceptor Pigment Heinrich-Heine- Universitat Integumental Pigment Universitatsstrasse I D-40225 Dusseldorf IVisual-Effect Function I IPhotoreceptor Function I ") This article is an extended version of a lecture given at the' 12th International Color Symposium', Bad Homburg v.d.H., Germany, Fig. I. Visual-effect, photoreceptor and metabolic functions. IP: integumental pigment. R: photore- September 18-22, 1994. ceptor, M: metabolism. FORSCHUNG 46 CHIMIA 49 (1995) Nr. 3 (Miirz)

2. The Physical and Chemical Basis of tals form stacks of layers. The twisting of behind the retina in their eye that gives rise Natural Color them produces diffraction and interfer- to brilliant metallic reflection (tapetum ence. The iridescent color of scarab beetle lucidum in the choroid). The iridescent 2.1. Structural Colors Lomaptera jamesi and Plusiotis gloriosa scales of butterflies, e.g. Morpho, contain When a physical structure is capable of belongs to this category [7]. a variety of layered structures, and the producing color this may be called struc- under-scales are pigmented with melanin. tural color (the name 'schemochrome' has 2.1.2. Thin Film Inte1erence It is this structure of the scales with its also been suggested [4]). The majority of iridescent colors in vanes that is responsible for interference biological systems originates from this color, as shown schematically in Fig. 2 2.1 .1. Diffraction Gratings phenomenon.There is still a color change [6][7] (color prints Fig. 39-43). These colors are rather rare in nature. with the viewing angle, although not as They areon]y observable using direct light strong as with diffraction.The iridescence 2.1.3. Light Scattering and disappear when light becomes dif- coined the terms 'metallic or enameld' Rayleigh scattering is observed when fuse. The change of color with the angle of color. Outstanding examples are the beau- the responsible particles are smaller than viewing is strong, and the reflection is tiful colors caused by multiple film inter- the wavelength of light, so that good blue iridescent. Examples are the surface of ferences of butterfly wings and humming- scattering can be seen from particles as beetles Serica sericea and the indigo snake bird or peacock feathers as well as mother- large as 300 nm on down to sizes of I nm, Drymarchon corais (co]or print Fig. 38). of-pearl. Some nocturnal animals (cat, bush this is also called Tyndall blue. Most non- Cholesteric mesophases of! iquid crys- baby) have a reflecting layer-structure iridescent blue colors in animals are Tyn- dall blues: blue eye color in humans, bril- liant blue and purple areas in the faces, buttocks and genital areas of monkeys, Thickness Distance Stack of Angle of Wavelength blue color of bird feathers located on the of plate (P) between plates (P) plates incidence reflected and barbules, blue color of fishes and repti les colour (nm) usually based on guanine particles (color print Fig. 44). When the size of the scattering parti- 60° 405 violet cles becomes larger and eventually ex- 0.05 0.15 ceeds that of the wavelength of light, no 90° 454 blue-violet G D Rayleigh but Mie scattering is observed. White or whitish scattering arises in this 60° 464 blue way: snow, fog, mist, clouds, white hair, 0.10 0.10 white butterflies and moths, white and 90° 506 blue-green G D silver fish, white feathers. It should be mentioned here, that many green and pur- ple colors originate from blue and green Tyndall , 520 0.15 0.05 . 60° an additional yellow or red pigment, re- , . yellow-green G D 90° 559 specti vely [2] [4][7]. Fig. 2. Origin of butterfly wing interference. Structure of plates arises from the vanes in the iridescent 2.2. Chemical Color scales (modified after Fox [4]). 2.2.] . Localization of Biochromes The biochromes that will be discussed Table. Some Functional Localizations of Biochromes in the following exert their function in specialized organs, tissues, fluids, cells, pe ie. L alll.an n FunCli n organelles, and plastids. Some of these that are important for our discussion, are Ph I ) mhl Purple Ba ten a hmmalllphol1' listed in the Table. hlnr )SOIll , Ph to )'nthe:ls Green Bac~ nn Thylal-Olds Phot s nthesl. Pr chloron Energy Collection and Transduction: a) Thylalmlds Phot . ynthe " nnoba ten Lamellae, tubes, vesicles, called chromatophores, continuous with mem- Ph t • ynthcSls anobacteria, Red 19ae Phyc('blll'llme. brane (phototrophic purple bacteria); b) Higher Plant!.. Iga hl()mpla.\ts Phot . )nth 'sis Chlorosomes attached to but not continu- lembmn Light-Mediated IIal bactcnum ous with cytoplasmic membrane (pho- PI' t n Pump totrophic green bacteria); c) cytoplasmic membrane only (Heliobacteria); d) inter- IIgma. Paratla 'ellar PhOIOIU 'j, Euglena. hlam 'domona1> nal thylakoid membrane structure (Pro- Boo. lembran' chloron); e) lamellar multilayered thy]a- Pineal F \: C I 'f hange. Rh)'lhms mphlbl3n . Reptiles. Bird: koid membrane system especially preva- l~)t.'s 1 Ion Animnb lent towards the periphery of the cell (cy- Mllochondna Re pirati n ells anobacteria); f) phycobi]isomes (cyano- bacteria and red algae); g) chloroplasts hromoplasls. lIeunl 'S Isunl-ElTe t Higher Plants (higher plants and most algae); h) mem- OImal hromat ph r i uaJ- _ff t branes (Halobacterium). FORSCHUNG 47 CHI MIA 49 (1995) Nr, 3 (Marz)

Sensory Transduction: a) Stigma and paraflagellar swelling H Me S-¥roteln eyespot and plasma mem- (Euglena); b) Me Me H a brane (Chlamydomonas); c) plasma mem- M.o~eI I brane (Halobacterium); e) extra-ocular re- MeO ';:: H ceptors like median eye (arthropods), front o 7-10 organ (amphibians), parietal eye (reptiles) Quinone and pineal organ (birds and other);f) in- ae H a a (Ubiquinone) tegumental photoreceptors (bivalves, as- I COOMe cidians, insects); g) nerve cells (Aplysia, o~ echinoids, crinoids); h) visual organs and Porphyrin 8iIin eyes (eye cups, compound eyes, pinhole (Chlorophyll a ) (Phytochrome Pr(Z.syn» eyes, lens eyes). MeO

Metabolic Functions: OMe a) Chloroplasts (higher plants); b) mi- Carotenoid tochondria (respiration); c) blood, mus- (Spirilloxanthln) cle, root nodules (oxygen transport).

~OH Bioluminescence: Retinoid a) Bacteria (Photobacterium); b) light (Vitamin A) organs (luminescent animals)

Visual-Effect Functions: H 0 HO OHHoae 0 a a) Chloroplasts, chromoplasts, vacu- oles (higher plants); b) chromatophores or O-Il.~{) -f-O-eo V ~ ~ ~ pigments dispersed in epidermis, feathers, ~NJ ~J~ hair (animals). Indol Derivative C~ (Violocein) ~ Eumelanlns , (Sepiomelanin) 2.2.2. The Basic Chromophores H The number of known classes of bio- HOOC 2 chromes is surprisingly small. This may NO be due to the general law of biochemical ~ I s}t-- 2frN\s ~ sX1I ~ NHHa austerity which governs the exploitation l; ~N eaOH "'"N Q of materials by living organisms [6]. ~ocB~ ~ ~ OH a It has been argued that there are good Trlchoslderln Flavonoid Phaeomelonlns HO reasons forthis photobiological economy. (Quercetin) Since light absorption ofbiochromes leads primarily to excited states that can be N NrCoaH ~ extremely harmful if not deactivated and IX HN N-?,O r?"Y0H lead to highly toxic species like superox- HO D~ =S S ?=' I N ~ IN many of the important biochromes are not ::::,.. ~ N-l .,:;7, "" Me N ~ ~ active in their S I state but deactivate radi- o 0 0 ationless to the ground state. Moreover it pterin Isoalloxazine Ommatin is precisely this group of chromophores (Xanthopterin) (Riboflavin) (Xanthommotin) that frequently act as protecting pigments against light or agressive molecules. The most important general chemical struc- N_ N tures are presented in Fig. 3. H20_0=t>==Q0 H N - G~~?eOOH H 0 NH2 3. The Evolution of Natural Dyes W

HO OH 3.1. The Radiation Environment Phenazine Betalain Indigoid Life must have evolved by adapting to (Iodinine) (Betanidin) (Indigoidine) the visible part of the electromagnetic spectrum from ca. 380 nm to ca. 760 nm. Fig, 3. Classes of ch romopho res FORSCHUNG 48

CHIMIA 49 (1995) Nr. 3 (MUrz)

The solar light intensity, i.e., the photon Nature developed chromophores and of a 'photobiological spectrum' that con- irradiance in quanta per sand cm2, inci- pigments for all spectroscopically rele- tains important chromophores and dyes dent on the surface of the earth, is given in vant wavelengths. Not only structures of distributed over the whole range of bio- Fig. 4. UV Radiation below 300 nm is chromophores were modified but wave- logical meaningful radiation [10]. In Fig. largely absorbed by ozone in the upper length adaptation was also achieved by S a selection of those molecules is given atmosphere. In water high light absorp- using the modulation by the apoprotein. displaying their absorption or emission in tion and light scattering modify the situa- The apoprotein associated with the chro- dependence on the solar spectrum. tion completely. With increasing depth mophore of a photoreceptor can signifi- there is not only a considerable light inten- cantly modify the wavelength of light ab- 3.2. The Phylogeny of Biochromes sity decrease but also a shift in the spectral sorbed by the latter. The wavelenght of The formation of the earth began more distribution. The least absorbed part is light absorbed by the photobiologic ally than 4.5 billion years ago. The first fossil- green and blue-green. The light conditions active molecule determines the spectral ized evidence of living cells is found in are of eminent importance for the natural response of an organism to solar radiation. rocks from Australia and South Africa selection of biochromes. This evolutionary adaptation is the basis which date back 3.5 billion years (photo- synthetic cyanobacteria, stromatolites). The early atmosphere of the primordial earth was anoxic, devoid of significant amounts of oxygen and hence constituted a reducing environment with changing amounts of H20, CH4, H2, N2, NH3, HCN, CO, CO2 etc. Energy sources were UV radiation (UV-A, -B, -C), lightning dis- charges, radioactivity and thermal energy from volcanic activities. If gaseous mix- tures resembling those thought to be present on the pristine earth are irradiated with UV light or excited by electrical discharges, electrons or heat (Stanley MiLLer-type of experiments, Hodgson, Ponnamperuma, 1968) important molecules are formed: amino acids, carboxylic acids, sugars, ad- enine, purines, pyrimidines, and porphy- rins [1][11-13].

Porphyrins: The cyclic tetrapyrroles are of very ancient origin. According to the above- mentioned experiments it is very likely Fig. 38. The indigo snake Drymarchon corais. An example for structural colors due to diffraction that the origin of porphyrins precedes that gratings. With kind permission of Droemer-Knaur-Kindler [83). of life. Porphyrins have been components ofliving systems from the very beginning. The formation of uroporphyrin from suc- cinic acid and glycine is thermodynami- cally favored. Porphyrins combine two a) b) important properties: ability to absorb light R and to undergo reversible redox reactions 4 when associated with metal ions. This might have been the start of photosynthe- sis [14]. 3 Plerins and Flavins: 2 Much the same holds for the pteridin and isoalloxazine system. They probably existed during prebiotic evolution, too. 1 Their importance is in their function as part offlavoproteins and prosthetic groups in redox reactions, blue light perception and DNA repair mechanisms. The remov- 300 400 500 600 700 800 400 500 600 700 al of toxic oxygen using bioluminescent Wavelength[nniI Wavelength [nm1 systems is also considered to have been of evolutionary value [15]. It has been speculated that flavin-me- Fig. 4. a) Radiantfluxdensity R (in 1Ol4q S-I cm-2 nm-I) of solar radiation on the suiface of the earth; diated photoresponses (phototaxis, photo- b) R in various depths ofa crater lake (modified after [9]) phobic reactions) evolved after those that FORSCHUNG 49 CHIMIA 49 (1995) Nr. J (Mtirz) were modulated by the quinone stentorin (a hypericin) and pterins in order to enable R phototrophic bacteria and other not to move too far away from the light source [13]. DNA photo lyase, containing f1avins, must have played an eminent role on the primitive earth and later on, and this ex- plains why this enzyme is stable towards oxygen whereas the bacterialluciferase is extremely sensitive. The occurrence of the 5-deazaflavin chromophore in the enzyme

F42o-NADP oxidoreductase of methane- producing archaea gives also a hint as to !! ! ! the early appearance of this biochrome two billion years ago. It seems that this coenzyme F420(acting as electron carrier) together with methanopterin (acting as C I carrier) and coenzyme F430 constitute a very first of redox active coenzymes which 300 400 500 600 700 800 900 1000 A. differs in many respects from that of eu- Fig. 5. The photobiological spectrum showing important chromophores together with the solar bacteria (Fig. 6) [11]. The anaerobic respi- spectrum. R: Solar radiant flux density on the surface of the earth, 1\.:Wavelength [nm] (after [10]) ration leading from CO2 to CH4 can then be simply formulated as:

Rib-®-LaC-GIU-Ctu F420 F4:JO I , C02+4H2~~~CH4+2H20 H0'(yNyNyO Pterin 0~ANH Caroteno ids: Bacterioruberin Coenzyme F420 (oxidized) In the early period of life on the pri- mordial earth organisms depended on anaerobic fermentation for the inefficient production of ATP. Archaebacteria con- tain in their membranes hydrocarbon chains mostofwhich are derivatives of the isoprenoid compound squalene, i.e. they have branched chains in contrast to un- branched fatty acids of modern mem- branes. In addition extremely halophilic Methanopterin Archaebacteria (Halobacteria) possess higher C50 carotenoids, e.g. bacterioru- berin (Fig. 6). It is, therefore, not difficult Coenzyme F430 to imagine that a first primitive photore- ceptor like bacterial rhodopsin may have Fig. 6. Some evolutionary ancient chromophores found in Archaebacteria evolved from the primitive membrane structure in which it is located [13]. The Eubacteria Archaea Eukarya simultaneous occurrence of three bacteri- al rhodopsins in some Halobacteria sug- gests a possible sequence of appearance: sensory rhodopsins (responsible for pho- totaxis and photophobia) evolved first, then a light-driven chloride pump halo- rhodopsin was invented (in order to main- tain a high intracellular salt concentra- tion), and later the light-driven proton pump bacteriorhodopsin was developed (in order to pump protons across the mem- brane and to drive ATP synthesis). When oxygenic photosynthesis evolved one of the key functions of carotenoids was to protect aerobic photosynthetic organisms against destruction by photodynamic sen- sitization. Aerobic photosynthesis would not exist without the coevolution of caro- Fig. 7. The universal tree of life as determined from comparative ribosomal RNA sequencing [11] FORSCHUNG so

C'HIMIA 49 (1995) Nr. 3 (Man)

tenoids alongside the chlorophylls in pho- IFermentation I Time tosynthetic organelles [16].

Succinate 3.3. The Evolution of Photosynthetic Pigment Systems ATP t NADH \Jf~ UQ Photosynthesis and Obviously electron and proton trans- "Photosystem I" porting pigment systems were invented twice: the Archaebacteria and the Euhac- teria developed different strategies. The eubacterial way was more successful in the long run [12]. It is very useful to discuss functional Photosynthesis and pigment systems in relation to the phylo- "Photosystem II" hv- 8Chl ATPase genetic tree representing the three su- perkingdoms of equal status: the Archae- Succinate bacteria, the Euhacteria and the Eukary-

ATP! ATP otes (Fig. 7). Rhodopsin is so widely dis- NADHUUQ~ tributed in and outside the animal king- dom that it goes back at least to the com- et L e~ Green Sulphur bacteria mon ancestor of the Archaebacteria and BChl*~BChl Eukaryotes and probably to the common Purple bacteria ancestor of all three superkingdoms. Rho- IPhotosynthesis and two Photosystems dopsin was found to be but one member of a family of homologous receptor proteins. Chi" The geneforthe mammalian ,B-adrenergic receptor, which senses the hormoneepine- S Chi" hV) ~ Fig. 8. The evolution phrine (adrenaline) shows clear sequence of oxygenic photosyn- hvl~ NADH homology with the gene for bovine rho- thesis showing the de- Chi dopsin. Rhodopsin could have originated velopment of pigments 2e Chi as an early experiment in photosynthesis, like quinones (UQ), 8 H20/"'" a role it retained only in the Archaebacte- haeteriochlorophylls '-..,.. 2H+ + 1/2 02 (Behl) and chloro- ria [17]. phylls (Chi), (modified Cyanobacteria Chlorophyll-based photosynthesis after [ 121[ 18]) arose only once, early in the eubacterial line (see Fig. 7). Eukaryotic chloroplasts Time before Oxygen % originated as photosynthetic endosymbi- present Pigments Function In onts [17]. (billions of Atmosphere The early, very first primitive organ- years) isms carried out some form of anaerobic metabolism, e.g. 4.5 - - -

4.0 Porphyrins Fermentation Anoxic Flavlns, Purines Chemolithotrophy Quinones Anoxygenic as a chemolithotrophic example and fer- Carotenes Photosynthesis mentation, e.g. Retinoids 3.5 Bacterloch lorophyll Chlorophyll a NADH + Pyruvic acid ~ ~ Succinic acid ~ 3.0 Oxygenic as a chemoorganotrophic example. Photosynthesis Accessory Pigments A highlight in metabolic evolution 2.5 Xanthophylls 0.1 would have been the utilization of already Chlorophyll b available porphyrins and retinoids/carote- 2.0 Phycoblllns 1 noids as photoreceptors in simple photo- synthetic devices. 1.5 Fig. 8 gives an impression how the 10 !1 0 sequence starting with fermentation and 1.0 i arriving at two light reactions could have Flavonoids Photo protection Phytochrome Chemical defense 20 evolved in the porphyrin receptor line. 0.6 Melanins Photoreceptor The rhodopsin line of Archaebacteria was not pursued any more but, instead, rho- Fig. 9. Approximate Visual-Effect dopsins became most important sensory evolution of natural 0.0 pigments and hio- photoreceptors in Eukaryotes. chromes The first organisms using chlorophyll FORSCHUNG 51

CHI MIA 49 (19~5) Nr. .llMiirl\

were probably eubacteria. They evolved later than the rhodopsin-based Archae- Thylakoid Membrane bacteria (see below). Chlorophyll b, the xanthophylls, and the phycobilins were hll invented during the transition from a re- Stroma Fig. 10. Structural mod- ducing to an oxidizing atmosphere. They (out) el ofphotosynthetic thyl- all need oxygen in one or several steps of akoid membrane in green their biogenesis [18]. plants and algae. LHCP: Flavonoids are universally present in light-harvesting chloro- all species of land plants. They also occur phyll a,b-protein, P: re- in two species of relatively advanced green action center. PQ: plas- algae families whose ancestors are be- toquinone. bo• f: cyto- lieved to have been the progenitors ofland chromes, FeS: iron-sul- phur protein, Fd: ferre- plants over 400 million years ago. Phyto- doxin. FAD: flavopro- chrome is considered to be a typical pig- Photosystem TI Photo system I tein, CFo' CF1: coupling ment of higher plants although some phy- factors of ATP synthase, tochrome effects have also been reported PC: plastocyanin [211. in green and red algae. Rapid evolution of f1avonoids occurred during the Devonian period (400 million years ago) and in the mid-Cretaceous (130 million years ago). The original rise of flavonoid diversity e was due to their importance in protection m b of plants against light, herbivores and phy- r topathogens, the post-Cretaceous devel- a n opment reflected their association with e the co-evolving diverse pollinators, birds - and insects [19][20] (Fig. 9). b

Fig. II. Light-harvesting 4. The Functions complexes. a) LHC IT of A ~ higher plants with lX-he- 4.1. Energy Transducing Photorecep- 100 licesA,B,C.D.luteincar- tors otenoids and Chi a.b. b) 4.1 .1 . Energy Collection 50 Phycobi Iisome. PE: phy- The photosynthetic membranes con- coerythrin, PC: phyco- cyanin,APC: allophyco- tain all the pigments and other compo- cyanin. Absorption spec- nents ofthe light-gathering apparatus. The 400 500 600 700 nm 400 500 600 700 nm tra a) ChI a,b in ether, E location of these membranes within the [mM-1 cnrll, b) phyco- cell differs between prokaryotes and eu- biIiprotei ns [20][2111231. karyotes. The chloroplasts of eukaryotes possess stacked thylakoids forming grana. Within the thylakoid membrane, chloro- creasing order of excitation: with decreas- ChI b ~ ChI a ~ ChI a ~ Chi a (spec., PS II) phyll molecules are associated in com- ing distance from the reaction center the 650 nm -660 11m 670 11m 680 11m plexes consisting of 200-300 molecules. absorption maximum of transmitting clus- Only few of these are involved in photo- ters shifts to longer wavelengths, i.e. in a Red Algae and Cyanobacteria: chemistry: the reaction center chlorophyll. bathochromic way. This is nicely demon- Extramembrane light-collecting sys- The more numerous other chlorophylls strated by inspection of important Iight- tems, the phycobilisomes, contain bilin are light-harvesting or antenna molecules. harvesting complexes and systems. pigments: phycoerythrin (PE), phycocy- The structural model of a thylakoid mem- anin (PC) and allophycocyanin (APC). brane including all membrane complexes Higher Plants and Green Algae: The transfer of energy to the RC is pre- and units is shown in Fig 10. Some of Light-harvesting pigments are chloro- cisely in that order as shown in Fig. 1/h by these functions are being discussed be- phylls a and b (ChI a, ChI b). The reaction the fine structure of phycobi Iisomes low. center (RC) contains ChI a. The structure [20][21]. The chromophoric molecules in light- of the light-harvesting complex II (LHC harvesting complexes function as anten- II) is known [22]. There are three trans- PE ~ PC ~ APC ~ APC B ~ Chi a (spec, PS II) nas, receiving radiation of defined wave- membrane - helices, two of them are held 570 nm 630 nm 650 nm 670 nm 670 nl11 length and transfer the energy to the reac- together by ion pairs. In the center of the tion center with high efficiency. This vec- complex Chi a is in close contact with ChI Purple and Green Bacteria: torial energy transfer is intimately de- b for rapid energy transfer, and with two The pigment complexes contain bacte- pendent on the fine structure, order and carotenoids (lutein) that prevent the for- riochlorophylls a, b (purple), and a, c, d, e symmetry of the collecting system. mation of toxic singlet oxygen (Fig. II a). (green) [24]. The sequence of absorbing and trans- The sequence of energy transfer is from The sequence of transfer in purple bac- ferring molecules is energetically in de- the antenna to the RC: teria is [21]: FORSCHUNG 52 CHIMIA 49 (1995) Nr. 3 (Mtirz)

B 800-820 -) B 800-850 -) B 870 -) RC (870) rophyll cannot absorb green light. Chloro- that finally led to a changed spectral ab- phyll b is the main accessory pigment in sorption were minor redox reactions and Within the chlorosomes of green bac- higher plants and is the result of oxidation rearrangements but with the mentioned teria the direction of energy transfer is: of a short side chain in chlorophyll a. consequences. Fig. 13 represent the basic Many carotenoids have oxidized func- structures in the chlorophyll series. B 750 ~ B 790 ~ B 804 ~ RC (840) tional groups and absorb in the middle of the spectrum (like phycobilins, ChI b) and 4.1.2. Energy Transfer Light-collection and harvesting de- serve as accessory pigments. Only the Electronic energy transfer can be dis- pends strongly on the position of the ab- bacteriochlorophylls explored the IR part cussed considering different limiting cas- sorption band. The different spectra of the of the spectrum with bacteriochlorophyll es [28]. Depending on the magnitude of pigments tum out to be a consequence of b going farthest (BChl b: 835-850, 1020- donor-acceptor interaction energy three different conditions at those periods when 1040 nm). The reason is that these bac- cases arise, defined as strong interaction they evolved (Fig. 9). The following hy- teria could utilize the spectral window in case, weak interaction case and very weak potheses may give a plausible explanation water-absorption between 970 and] ]90 interaction case. The Forster mechanism, [ 18J. nm [25]. i.e., the long-range transfer via dipole in- The first photosynthetic organisms There is a tendency for organisms liv- teractions is based upon the very weak could have been planktonic Archaebacte- ing deep underwater to contain a greater interaction model. ria operating with bacteriorhodopsin and proportion of phycoerythrin because it is It is now generally accepted that sin- absorbing light (570 nm) right in the mid- better at absorbing green light. In brown glet-singlet energy transfer between chlo- dle of the visible spectrum. Halobacteri- algae the carotenoid fucoxanthin is very rophylls occurs by the Forster mecha- um halobium is an extant, present-day efficient in transmitting energy to the re- nism. The typical distances of neighbor- example. action center. With its absorption maxima ing chromophores are 1-2 nm (10-20 A). All green and yellow light would have at 421, 446, and 475 nm it operates suc- The rate of energy transfer obeys the equa- been absorbed. Light was used to transport cessfully together with chlorophyll c in tion given in Fig. 14. protons and for the production of ATP. harvesting light in the blue and green Here k is dependent on dipole orienta- Thechlorophylls and bacteriochlorophylls range [2]][26J. tion, CPo is the donor fluorescence quan- absorb strongly UV, violet, and blue light Spirilloxanthin, a carotenoid typical of tum yield, n is the refractive index of the as well as red or IR light. The bacterio- purple bacteria, has a chain of 13 conju- solvent, No is Avogadro's number, r is the chlorophylls are now used by purple and gated double bonds. This renders the mol- donor-acceptor distance, To is the donor green bacteria, the first chlorophyll a con- ecule an ideal light collector in the useful fluorescence lifetime, and J is the spectral taining organisms were probably Eubac- spectral range 450 ... 550 nm (absorption overlap. Carotenoid-chlorophyll energy teria. Since they lived in the deep mud maxima at ca. 460,490, and 530 nm) [21]. transfer has an efficiency which can be as under water, rich in organic matter, they All this happened probably in the tran- high as 100% [29]. Carotenoids have an received only red and blue light. The green sition from a reducing to an oxidizing intense absorption band that is due to the and yellow portion had been fi Itered out atmosphere. The carotenoids (see Fig. II a) transition from the ground to a second by the purp1ecoloredArchaebacteria. This as the lutein molecules in LHC II had a exited singlet state S2 (symmetry 1Bu). could have led to theevolution ofthe green dual role: light-harvesting function and a The first excited singlet stateS I is nollo be chlorophyll a (Fig. 12). protective function, namely to quench tri- seen in conventional absorption spectra The phycobilins as accessory collect- plet chlorophyll excited states. since the transition is forbidden (symme- ing pigments evolved later because chlo- The chemical structural modifications try IAg). However, the lifetimes of S I and

- Chi b 8Chl~ - CHO \c,~ BChl a (R = Farnesyl) \ e,g BChl b

E _ n*------n* - n *------n*

___---it 02 -it 02------

-it b2 ------* b2 Chlorophyll a (R = Phytyl) Chi ° BChl b 400 500 600 700 nm

Fig. 12. Spectral adaptation of light-harvesting pigments. The spectral Fig. 13. Structure ofchlorophylls and bacteriochlorophylls. Frontier orbit- window first used and closed by bacteriorhodopsin BR had to be opened als of Chi a and BChl a, showing the bathochromicity in the latter chromo- using chlorophyll (Chi) a and bacteriochlorophylls a-g. Later, in the course phore [27]. of oxygen evolution, BR disappeared and the window was used by acces- sory pigments: carotenoids, Chi b, phycoerythrin PE, phycocyanin PC and allophycocyanin APC [18]. FORSCHUNG 53

CHIMIA 49 (1995) Nr. .1(Marl)

S2 are different: S I - 10-11 s, S2 - 10-13 s. Both S I and S2 states participate in the 9000 (In10) K2 CPo above-mentioned singlet energy transfer k Forster . J [30]. ET - 128 1\5n4 No To r6 4.1.3. Energy Transduction The third major role of chlorophylls - beside energy collection and energy trans- fer - is light-energy transduction, i.e. the lnt. 0*- 0 A-A* transfer of an electron from a specially oriented chlorophyll molecule to an elec- tron acceptor. The reaction centers of purple bacteria Fo ,'\ (e.g. Rhodobacter sphaeroides and Rho- I \ ,. \ /' dopseudomonas viridis) have been inves- ,... I \/" I \.l tigated using X-ray diffraction [3 I]. The I I• analysis revealed that there is an appro- rv ximate two-fold rotation symmetry that 11 relates the bound bacteriochlorophylls, bacteriophaephytins and quinones. The carotenoid is the only chromophore in the Fig. 14. Rate of energy transfer according to RC that does not conform to this approx- rv the Forster equation 11 imate two-fold symmetry. The reaction and exptanation of sequence after excitation of one bacterio- spectral overtop J [281 chlorophyll b of the special pair is shown in Fig. 15. The role of the carotinoid is usually the quenching of chlorophyll (the primary donor) triplet states [31]. This is true in Rb. ~hll sphaeroides where spheroidene, a carote- noid with 10 conjugated double bonds, 3.5 ps accepts triplet energy in ca. 30 ns. The exception to this rule is Rps. viridis, the 4 ps main carotenoid of which, 1,2-dihydrone- urosporene, does not quench the triplet state of the primary donor (Fig. 15) [29]. It Fig. IS. Reaction cen- ter of Rps. viridis 121] is remarkable that cis-configurations of [31]. P-960: special carotenoids are selected by the RC for the pair, BChl: bacterio- ps photo-protective functions. This is attrib- 200 chlorophyll b, V-Behl: utable to the unique isomerization proper- 'Voyeur' molecule, Bphe: bacteriophae- ties of the cis-molecules. On the other 10 IJ.s hand trans-configurations prevail in the phytin b, MQ: menaqui- LHC because of better energy transfer none, UQ: ubiquinone, Cyt c: cytochrome c, abilities [30]. The energy transduction is e Car: 1,2-dihydroneuro- finally brought about by the ubiquinone sporene. which enters in reduced form the electron and proton transport chain (Fig. 10).

4.1.4. Light-driven Ion Pumps The purple membrane of Halobacteri- Cytoplasm um halobium contains a pigment of which retinal is the chromophore. This bacteri- Asp 96 eOOH orhodopsin functions as a light-driven pro- ton pump. A second retinal pigment of halobacteria, called halorhodopsin, acts Lys 216 as a chloride pump. The purpose of both pumping systems is as follows: Halobac- teria are equipped with the regular metab- Asp 85 eo06 olism for energy transduction as in Eubac- Fig. 16. a) The seven teria. But upon oxygen limitation the syn- a transmembrane a-he- Extracellular thesis of bacteriorhodopsin is induced. lices of bacteriorllO- / Side dopsin. b) Light-in· When light is present, retinal-based pho- hv duced proton tramlo- tosynthesis replaces oxidative metabolism b cation. Asp: Aspartic [32]. acid (after (33)). FORSCHUNG 54

CHIMIA 49 (1995) Nr. 3lMtif1.)

One absorbed photon pumps one pro- acid residues (Asp 96 and 85) act as donor ChI hv) IChl* --? 3Chl* ton across the bacterial membrane. The and acceptor for protons during the Iight- 3Chl* + 302 --? Chi + '02* resulting electrochemical pH-gradient induced transport [33]. drives ATP synthesis. In order to counter- The produced singlet oxygen will then act extracellular osmotic pressure the bac- 4.2. Photoprotection wreak havoc in all organelles. Caroten- teria have to accumulate a large concen- oids protect the system efficiently provid- tration of potassium chloride. This is 4.2.1. Quenching of Excited States ed that they are bound in proximity to the achieved by the chloride pump halorho- If cells of photosynthetic organisms chlorophylls. Apart from transferring en- dopsin. The rhodopsin pigments belong to without carotenoids (e.g. mutants like Rh. ergy this protection is a most essential an evolutionary very old family of mem- sphaeroides R-26) are illuminated in the function of carotenoids in oxygenic, aero- brane proteins having seven transmem- presence of oxygen they sensitize their bic photosynthesis [31]. brane helices. The process of proton trans- own death [29]. The process responsible On the other hand there is also a direct location is shown in Fig. J 6. Two aspartic for the killing is interaction of carotenoids with singlet oxygen

102* + Car --? 302 + 3Car* 3Car* --? Car + heat

H There are some carotenoids which un- Violaxantbin dergo rapid, light-induced changes in their

OH concentration. In higher plants, ferns, mosses, green and brown algae these are violaxanthin, antheraxanthin, and zeaxan-

HO thin. In other algae (diatomeae, chryso- Antberaxanthin phyceae, xanthophyceae, dinophyceae, and other) the two carotenoids diatoxan- OH thin and diadinoxanthin are important. The conversion of violaxanthin into zeaxanthin, the de-epoxidation sequence, HO Zeaxantbin as well as the reverse, the epoxidation OH sequence, is a true cycle since the forward and the back reaction sequences are cata- lyzed by two different enzymes. The de- epoxidation is favored under excessive Diadinoxanthin light and a low pH (optimum 5.1), and it uses ascorbate. The epoxidation occurs under limiting light, a higher pH (opti- mum 7.5), and needs oxygen as well as NADPH. What is the function of this xan- thophyll cycle? Fig. 17. The Cllrotell- Diatoxantbin An excess of excitation energy could aids involved in the two xanthophyll cycles of potentially result in the accumulation of higher plants and al· The Xanthophyll Cycle Carotenoids excitation energy. This, in turn, could gae 134][35] lead to the damaging and destructive spe- cies singlet oxygen 102, superoxide anion O2'- and hydrogen peroxide H202. How- IPhotochemical Fluorescence Quenching I ever, the accumulation of excess excita- tion energy is counteracted: thermal dissi- Photochemistry pation of excess energy occurs directly within the system. There is strong evi- 1 dence that zeaxanthin is involved in this II Chi "'I Intersystem Crossine. 13chI'* I thermal energy dissipation, thus protect- ing the photosynthetic apparatus against

(ZeaXnniliin ) the adverse effects of excessive light. The precise mechanism, however, is not yet ( Antheraxanthin ) known. One suggestion is that the singlet excited state of chlorophyll is quenched Violaxanthin by the carotenoid. The diverse protecting Excessive Limiting functions are summarized in Fig. 17 and TJight Light 18 [34-40]. Fig. 18. The protecting Ascorbate NADPH,Oz The accumulation of large amounts of role ofca rotenoids. Chi: 5.1 pH 7.5 Chlorophyll, Car: caro- carotenoids in algae under intensive solar tenoid, FI: tluorescence I Non- photochemical Fluorescence Qnenching I I Triplet- Triplet Energy Transfer I radiation is well-known. Snow algae oc- [2911351. cupy a unique habitat in high altitude and FORSCHUNG 55

nIlMI"~9 (1astaxanthin esters in extra- ~ H chloroplastic lipid globules produce the Meyy RX;' "(0 HoyyN).C;Nyo Me~ NH V, NH characteristic red pigmentation typical of C 2 H 0 some snow algae, e.g. Chlamydomonas o nivalis. Consequently, these compounds FADH2 5-Deazaflavin Metbenyltetrabydrofolate greatly reduce the amount of light availa- Pterin Derivative ble for absorption by the light-harvesting pigment-protein complexes, thus poten- e e .8 tially limiting photoinhibition and photo- hv NoNWW~ Pterin * - Flavin-H2* - HN NH damage caused by intense solar radiation Light Harvesting Energy Transfer Electron Transf.r oA ..Ao 5H tH [41]. Much the same occurs in Euglena sanguinea, a freshwater flagellate, when cells are irradiated with UV-B radiation. ·e• M.{ This suggests that the formed carotenoid Flavin -H2 -- ;t r Cleavage is a photoprotective pigment [42]. o YN N 0 H H Fig. 19. Chrollloplwres Electron Back Transfer of DNA plwtolYlise re- 4.2.2. Photoreactivation pair en::.yme. Suggested DNA Pbotolyase Repair Enzyme Photoreactivation is a process in which mechanism of thymine an enzyme is activated by long-wave UV dimercleavage [811151. or visible radiation in order to bind and cleave dimers of pyrimidine bases, i.e. dimers of thymine. This photorepair of UV-induced DNA damage is a vital func- Visual System in the green alga Chlamydomonas tion that is present in microorganisms, fungi, plants, and animals including man. Phototectic Rate Irradiation with far UV-light induces for- hv [~m s-l] mation of pyrimidine dimers. Upon ab- I Photon Counting Intenslty- sorption of light of wavelengths between Response. 300 and 600 nm, a DNA photolyase splits ! Curve the cyclobutane ring that was formed by Pattern Detection cycloaddition of adjacent pyrimidines ! Irradiance (photons m-2s-1] [8][ 15][26]. Bohavlour Modification The enzyme isolated from E. coli con- Sensitivity Rhodopsin tains both flavin (FADH2) and a pterin m2slphoton derivative (5,10-methenyl-tetrahydro- Phototaxis Action Spectrum folylpolyglutamate) absorbing at 390 nm of Chlamydomonas 0~'_·m in the protein-bound state. The in vivo action spectrum for photoreactivation is at 380 nm. The pterin chromophore causes a Fig. 20. Phototaxis in 621 500 413 nm fIuorescence at 470 nm. Some other repair 2.0 2.5 3.0 eV the green alga Chlamy- enzymes of the DNA photolyase type con- domonas [17] tain not a pterin but two special flavin chromophores. Streptomyces griseus and Methanobacte rium thermoautotrophicum successfully in their environments [8]. The respond to the absorption spectrum of the possess FADH2 together with 8-hydroxy- most important chromophores for sensory molecules reponsible for that behavior 5-deazaflavin which has already been transducing pigments are retinal, tetrapy- [I]. For any particular light source, thresh- mentioned in Fig. 6. The action spectrum rrole, flavin, pterin, and hypericin pig- old is the lowest intensity that produces a has a peak at 436 nm. The deazaflavin ments. Absent from this list are particular- reponse [17]. The phenomenon is shown chromophore is an evolutionary very an- ly the carotenoids and hemes. The reason explaining phototaxis of the green alga cient type (at least 2 billion years old) and is that the latter two pigment groups show Chlamydomonas reinhardtii. Photomotion had been preserved throughout the plant efficient radiationJess deactivation to the comprises phototaxis (stimulus-oriented and animal kingdom [15]. The mecha- ground state without any significant pho- directed movement relative to radiation nism of repair is not completely under- toreceptor photochemistry. On the other direction), photokinesis (dependence of stood but a working hypothesis is given in hand these biochromes have their own rate [ms-I] on photon irradiance [mol S-I Fig. 19 [8]. important function as precursors for reti- m-2] ), and photophobic reactions (move- nals or as protecting dyes. One ofthe most ment responses to changes of light inten- 4.3. Sensory or Signal Transducing important means for identifying the kinds sity or photon irradiances) [12][26]. The Photoreceptors of molecules in a photosensitive system is prerequisite for a phototactic movement is 4.3.1. Photomotion (Tactic, Kinetic, by obtaining the behavioral action spec- the existence of a photoreceptor with cor- Phobic) trum. The action spectrum is the relative responding pigments. A photoreceptor mo- Besides providing essential energy for response that a plant or animal makes to a lecule must meet stringent requirements. life on earth, light provides signals and light stimulus of different wavelengths. It must absorb a photon with high proba- images that enable organisms to manage The action spectrum obtained should cor- bility and it must pass the information on FORSCHUNG 56 CHI MIA 49 (1995) Nr. 3 (Mo,z)

with high efficiency. A visual photorecep- Chloroplastmem. Stigma Layers with tor system must also detect patterns and Membrane, I /I~otenoids should modify its behavior. Thus, we have Chloroplast three criteria for visual photoreceptor sys- tems: photon counting, pattern detection and behavior modification [17]. In Fig. 20 the responses and the action spectrum for phototactic rate of Chlamydomonas are given, showing the rhodopsin-type action Chlamydomonas I I spectrum of this particular photoreceptor Light perpendicular on Surface - [17]. The arrangement of the pigment sys- tem (eyespot pigments and photoreceptor Response Blue light pigments) in the cells of Euglena and action spectrum Receptor EuglenaI Chlamydomonas is an excellent example Flavin I of Euglena pterin of combined pigment function (Fig. 21). phototaxis In Chlamydomonas the eyespot (stigma) is composed of several layers of caroten- oid pigment formed in the chloroplast. The carotenoid layers are spaced in such a nm 400 500 600 way that the eyespot becomes a tiny mirror which is due to constructive interference Fig. 21. Photoreceptor apparatus of the green alga Chlamydomonas and the euglenoid Euglena and of light reflected from each surface of the function of the eyespot (stigma) [15][17][26][43] layers. The photoreceptor pigment, rho- dopsin, used for phototaxis forms a patch in the plasma membrane over the eyes pot [17][26][43]. hll * ~ $ In Euglena gracilis the eyespot is not OH 0 OH R-OH - R-OH - R-O +H part of the chloroplast, but its pigments are also carotenoids. The flagellum contains 1 e the photoreceptor pigment in its paraflag- hll R-O + hll HO Me ~ ellar body. Euglena uses two receptor pig- HO Me ments: a flavin receptor for phototaxis and a pterin receptor for photophobic response and negative phototaxis [] 5]. ! A novel class of photoreceptor pig- OH 0 OH ments was discovered in the protozoan Stentorcoeruleus. These pigments, which Hypericin (Stentor in) employ hypericin as chromophore, are stentorin and blepharismin. They serve as photoreceptors for phototaxis and photo- phobic responses [8].The remarkable fact Fig. 22. Hypericin (Stentorin) in the protozoan Stentor and mechanism of photophobic response, i.e. is that hypericin acts as photosensitizer for ciliary reversal [45] photodynamic action. Hypericin is also a product of higher plants, the Hypericace- ae, and particularly of Hypericum perfa- Response ratum. Hypericin is responsible for the Phototropism toxicity of these plants towards animals or (St. John's wort). Hypericin acts in ani- A mals as photosensitizer which brings about photooxidation and causes inflammations, oedema and even death [44]. Despite of this phototoxicity the ciliate Stentor has developed the capacity, i.e. the step-up photophobic response (light-avoiding) and negative phototaxis (away from light source), to survive the damaging radiation environment [8][ I0][26]. Hypericin, sten- torin I and II absorb around 590-620 nm and the action spectra of Stentor for pho- tophobic response are similar. The prima- 350 400 450 500 nm ry photochemical process may be a proton Fig. 23. Phototropism action spectrum (Avena, oats) and comparison with riboflavin and f3-carotene dissociation in the excited state (Fig. 22). [12] The resulting pH gradient triggers a Ca++ FORSCHUNG 57

CfIlMIA 4~ (1995) Nr. J (M,irz)

Fig. 39. The word 'color' was written using letters of the butterfly alphabet. Beautiful patterns on butterfly wings form letters and numbers. They have been observed and photographed by K.B. Sandved. With kind permission of Sandved Photography [82]. influx which is finally responsible for cil- iary reversal [45] (color prints Figs. 45 and 46).

4.3.2. Blue Light Photoreceptors (Cryptochrome) The assignment of blue light photore- ceptors to f1avins, pterins or carotenoids is a serious problem. Often the lack of a near UV peak in the action spectra has been taken as evidence that a carotene functions as receptor. This conclusion, however, is unwarranted, because f1avins and f1avo- proteins are known that are atypical, i.e., they lack a near UV peak [15]. An additional problem is that action spectra can depend on light intensity. The most probable candidates for blue light receptors are f1avins and pterins.

4.3.3. Phototropism A growth response involving bending Fig. 40. Colors of butterflies are caused by thinjilm interference. This monarch Danaus plexipus is or curving of a plant toward or away from distasteful. Birds avoid them. With kind permission of Droemer-KnQur-Kindler [84]. an external light stimulus is called photo- tropism. Photoreceptor for phototropism of the coleoptiles of oats (Avena sativa) is a flavin (Fig. 23). The earlier discussed carotenes are not photoreceptors but may function as screening pigments. Since the shoot bends in the direction of irradiance differences between the flanks of an or- gan, screening pigments wi II increase irra- diance differences and enhance photo- tropic sensitivity.

4.3.4. Photomorphogenesis Three pigment systems are indispen- sable for the development of chloroplasts and chlorophyll: protochlorophyllid, phy- tochrome and blue light receptors. Proto- chlorophyllid is transformed into chloro- phyll a in two steps. The first is a light- requiring reaction for the reduction of pyrrole ring D, the second one is esterifi- cation of the propionic acid substituent. It is remarkable that protochlorophyllid Fig. 41. The butterfly Limenitis archippus may be looked at as a mimic of the monarch, but it is bad- serves as the photoreceptor for its own tasting, too. An example for Mullerian mimicry [49]. With kind permission of Droemer-Knllur- conversion to chlorophyll a [24]. Phyto- Kindler [84]. FORSCHUNG 58

CHIMIA 49 (1995) Nr. 3 (Marz)

Fig. 42./ridescent colors of butterflies originatefrom the layered structure Fig. 43. Nocturnal animals enhance vision using a reflecting layer (tapetum of scales. With kind permission of Droemer-Knaur-Kindler [85]. luddum) behind the retina. The layer consists of golden-yellow crystalls of riboflavin [2]: the bush-baby galago. With kind permission of Droemer- Knaur-Kindler [86].

chrome is a chromoprotein and the red light receptor in plants [12][46][47]. It has been called the 'visual pigment of plants' [47]. The photochromic phytochrome sys- tem has a conspicuous analogy to rho- dopsin and bacteriorhodopsin: the prima- ry photoprocess is a very fast « 100 ps) (E/Z)-isomerization of the chromophore which leads to subsequent changes of the surrounding protein and triggers such im- portant processes as pattern formation, induction of germination, induction of flowering, synthesis of chlorophyllid and many other. Since phytochrome absorbs either red or far-red light (Pr and Prr), it responds to light quantity and to light quality via the ratio P/Prr or Pr/Ptot, re- spectively (Fig. 24) [12].

4.3.5. Photoperiodism Many growth and developmental phe- nomena are controlled by the dai Iy radiant exposure period. A critical day lenght is decisive: day lenghts longer than the crit- icallenght are called long-day, otherwise short-day. The photoperiodical regulation of flowering has been well investigated. One can distinguish between short-day, long-day, and day-neutral plants. The phy- tochrome system enables the plant to sense whether it is in light or in darkness, but the actual measuring of the time lapse be- tween the moment the plant senses the onset of darkness and the moment it senses the next exposure to light depends on an internal clock. Fig. 25 shows the effect of day lenghts on flowering (color prints Figs. 47 and 48). In animals there are also processes that are regulated by light and correspond to Fig. 44. Colors offishes (e.g. Hippocampus hudsonius) caused by light scattering based on guanine diurnal circadian or longer-period rhythms. particles (blue, silvery) or on carotenoids (yellow, red) or based on both effects (green). Melanin in The light is detected either by eyes or by melanophores contributes to dark and black [2]. With kind permission of Droemer-Knaur-Kindler other extra-retinal and extra-ocular pho- [87]. FORSCHUNG S9

CHI MIA -19(1995) Nr..1 (Marl)

Figs. 45 and 46. Hypericin is responsiblefor the toxicity of plants like Hypericum perforatum (St. Johns wort). The same chromophore acts as photoreceptor in the protozoan Stentor coeruleus. With kind permission of Ecomed [88] and Droemer-Knaur-Kindler [89].

Long-day Night Day Short-day Plant Plant 660nm +- critical lenght'- 730 nm + ';9"{ + r

+ rtr I Phytochrome Pr Phytochrome Pfr + rtrr Protochlorophyllid A.n 380, 666 nm i-m 400, 730 nm + rtrrfr _11- • + I 1 I 0 12 24 Hours

+ Flowering No Flowering

Fig. 24. Protochlorophyllid (PChl), the precursor of chlorophyll, and the Fig. 25. Reactions of long-day and short-day plants. r: red tlash, fr: far red photochromic phytochrome P/Pjrsystem. Control of chloroplast formation tlash [49]. Long-day plants tlower when the night is shorter than the.critical by light and two pigment system. ChI: Chlorophyll, ALA: aminolevulinic length. A long night with a red tlash is seen as a short one. A second far red acid, Th: thylakoids, LHCP: light-harvesting chlorophyll protein [48]. Two tlash will abolish the effect of the first tlash. photo processes are needed for formation of chloroplasts and thylakoids: excitation of Pr as well as photoconversion of PChI. FORSCHUNG 60 CHI MIA 49 (1995l Nr. 3 (Marz)

Figs. 47 and 48. Euphorbia pu\cherrima and Hyoscyamus niger are examples for shon-day and long-day plants. resp. Hyoscyamus requires about 10 h day-length (depending on temp.) or more to flower. With kind permission of Urania-Verlag [90] and Ecomed [91].

Fig. 49. Alder trees (alnus)form rOOT-nodules that are induced by nitrogen- Fig. 50. Flatfishes. like Bothus. show excellent camouflage due to cryptic fixing actinomycetes similar to legumes and Rhizobium where flavonoids coloration. With kind permission of Belser-Verlag [93). activate nodulation genes. With kind permission of Urania-Verlag [92].

Fig. 51. The praying mantid Hymenopus coronatus Lives onflowers of an Fig. 52. Pterins. not carotenoids. are responsible for yellow colors of orchid which is extremely similar in shape and color. With kind permission wasps; Xallthopterin in Paravespula germanica. With kind permission of of Droemer-Knaur-Kindler [94). Droemer-Knaur-Kindler [95]. FORSCHUNG 61 CHIMIA 49 (1995) Nr. 3 (M"rz) toreceptors like median eyes, pineal gland, front organ, parietal eye or even parts of the integument.

4.3.6. Vision Two basic eye structures are the re- fracting eye with a large single lens (ver- tebrates) and the compound eye with a large number of small photoreceptor units called ommatidia (invertebrates, arthro- pods). The photoreceptor cells are of spe- cial interest: rods and cones in vertebrates, and rhabdoms in arthropods. They contain the visual pigments, the rhodopsins, which employ retinal or structural modifications of it as photoreceptors (Fig. 26). Four retinal aldehydes (and the corresponding alcohols) are known: AI-A4 [32]. The rhodopsin class is of very ancient origin Fig. 53. In mostfamilies ofCentrospermae (Caryophyllales), with the exception oftheCaryphyllaceae occurring in prokaryotic and lower eu- and Molluginaceae, yellow and violet pigments, the betalains, occur instead of the usual flower karyotic organisms. It is assumed that the pigments anthocyanins: Bougainvillea. With kind permission of Urania-Verlag [96]. rhodopsin polypetide chains thread their way back and forth through the membrane with total of seven transmembrane hel ical segments [8][32][50]. All retinal AI based pigments are called rhodopsins, and those using retinal A2 are called porphyropsins. Xanthopsins are then derived from retinal A3 [32]. In Fig. 26 two structural models are given; one for bovine rhodopsin (cf Fig. 16), and one for the fly visual pigment which has two chromophores. The photo- chemistry of visual pigments and bacteri- orhodopsins is involved but fairly well understood [8][32][33]. In Halobacterium halobium four rho- dopsin pigments have been discovered, all of which reside in the plasma membrane: Bacleriorhodopsin (photocycle, proton pump function), Halorhodopsin (photocyc1e, chloride pump function), Sensory rhodopsin I (photocycle, photo- taxis function, attraction by green light, repulsion by UV light), Sensory rhodopsin 1/ (photocycle, repul- sion by blue-green light, 'phoborho- dopsin'). The function of the above mentioned two visual pigments, Fly xanthopsin (photocycle, thermostable intermediate, sensitizer, vision function), Bovine rhodopsin (photocycle, vision func- tion), is shown in Fig. 27. In the vertebrate retina the rod cells are responsible for scotopic or night vision, i.e. detection of low light intensities. In apparent contrast to the constant occur- rence of rhodopsin in terrestrial verte- brates are the scotopic pigments offishes. These pigments are either bathochromi- Fig. 54. The orchid Ophrys insectifera mimics afemale thread-waisted wasp Gorytes mystaceus and cally or hypsochromically shifted from allures the male insects. An example for mimicry of plants [97]. With kind permission of O. Danesch 500 nm in accordance with the nature of [98]. FORSCHUNG 62 CHIMIA 49 (1995) Nr. 3lMtirz) their photic habitat. Nature employs two Fig. 28 and reflects the light conditions there is some suitable protective mecha- methods: a) varying the structure of the shown in Fig. 4. nism [2]. The protective role of carot- individual opsin (opsin shift) and b) vary- enoids in photosynthesis has been dis- ing the chromophore, either Al or A2 ret- 4.4. Screening and Protection Against cussed (see Fig. 18). But carotenoids also inal. In some fishes the retina has both AI Radiation play an important part in non-photosyn- and A2 pigments, the proportion changing Light is used by living organisms in so thetic organisms, particularly in microor- with season (A2 winter/autumn and AI many beneficial ways that it is easy to ganisms. The mechanisms for deactiva- spring/summer). In certain amphibia lar- overlook the fact that light energy can also tion of singlet oxygen were already men- vae have porphyropsin (ca. 520 nm) and be damaging. Various natural pigments, tioned. The screening effect of caroten- later a change occurs in metamorphosis to e.g. porphyrins and hypericin, can act as oids in phototropism was also discussed. ca. 500 nm [51]. The visual adaptation to sensitizer for light-catalyzed damage, es- Cyanobacteria (e.g. ChLorogLoeopsis) water quality and water depth is shown in pecially in the presence of oxygen, unless have developed a passive UV -A sunscreen, the pigment scytonemin. It is a yellow- brown dye of cyanobacterial extracellular sheaths, and was found in species thriving in habitats exposed to intense solar radia- tion. High light intensities (up to 250 mmol photon m-2s-l) promoted the synthesis of scytonemin in cultures of cyanobacteria. UV -A (320-400 nm) was very effective in eliciting scytonemin synthesis. In acetone scytonemin absorbs strongly at 385 nm with a long tail extending to the IR. Other absorptions are at 250 and 280-320 nm.The dye is probably a sunscreen far more an- cient than plant flavonoids and animal melanins [52]. Lichens are mutualistic symbiotic as- sociations between ascomycetes and cer- tain genera of green algae or cyanobacte- ria. The protective surface of a lichen, the upper cortex, contains compounds that absorb incident light. They function as screen and control the sun-light intensity for the algae which are located below the upper cortex. Sun-exposed lichens have larger quantities of screening compounds than species growing at shady sites. The screening pigments can reduce the trans- parency of the upper cortex by as much as 50%. In addition there is a selective UV absorption in some pigments (Fig. 29). Usninic acid and parietin absorb con- siderable UV proportions. The colorless atranorin acts possibly as accessory pig- ment that sensitizes photosynthetic active chlorophylls [53][54]. In animals, protection against light is usually afforded by a screening layer of pigment which either absorbs all light or filters out harmful rays. Dark pigments as melanin are often utilized. The black slug Arion ater accumulates an amount of mel- anin proportional to the amount of photo- dynamic free porphyrin in the integument [2]. Free porphyrins occur in man when there is a deficiency in uroporphyrinogen III cosynthetase and large amounts of uro- porphyrinogen I accumulate (erythropoi- etic porphyria). Injected J3-carotene can be Figs. 55 and 56. Cephalopod molluscs (e.g. Sepia officinalis) are famous examples for efficient color deposited in dermal tissues where it ab- changes. In addition they produce a very dark brown pigment, sepiomelanin, in their defensive ink. sorbs light and protects against photooxi- With kind permission of Droemer-Knaur-Kindler [99). dation [2]. FORSCHUNG 63

CHI MIA 49 (1995) Nr. 3 (Marl)

Naphthoquinones in echinoderms have also been discussed as photoprotectors. The protecting role of f1avonoids during the evolution of terrestrial plants has been discussed in Sect. 3.3 [19][55]. Flavo- no ids, as apigenin, naringenin or querce- tin, absorb strongly around 290 and 350 nm. They efficiently screen plants and animals (guinea pig) against UV-B radia- tion and photooxidation [56-58].

4.5. Accessory Pigments in Vision The accessory visual pigments can be classified in three groups: screening pig- ments, color discriminating pigments and reflecting pigments. Screening pigments in vertebrates are melanins that are present in various tis- sues. Melanin absorbs stray light of all wavelength. In invertebrate eyes screen- ing pigments in the outer zones of receptor units absorb all light that is not directed along the axis of the ommatidium. The Fig. 57. The sea hare Aplysia dactylomela uses a defensive purple ink which contains the tetrapyrrole pigments can be melanin, ommochromes biliprotein aplysioviolin, that is the methyl ester of phycoerythrobilin [2]. With kind permission of and pterins. One example has been given Droemer-Knaur-Kindler [100]. in Fig. 27. Color discriminating dyes occur in oil- droplet light filters in inner segments of receptor cells of reptiles and birds. They contain carotenoids. Reflecting pigments serve to increase the sensitivity of the eye. In some cases a reflecting layer lies behind the receptor layer of diurnal and nocturnal animals. In bush-baby (galago) the dye is riboflavin, emitting light at 520 nm, and in the deep sea fish Malacosteus the pigment is astax- anthin [2)[39] (color print Fig. 43).

4.6. The Metabolic Transport Functions Electron, ion (proton), and gas (oxy- gen) transport phenomena are indispensa- ble metabolic processes. Many pigments are involved and have been adapted to these specific functions. Electron and proton transport were discussed in the context of photosynthesis (Fig. 10). The respiratory chain in the mitochondria is Fig. 58. This purple sea urchin Sphaerechinus granularis is colored by J,4-naphthoquinones.With similar. As an example of the versatile kind permission of Belser-Verlag [101]. transport function of an involved dye, the quinone pool with the Q-cycle is shown in Fig. 30. confined to root-nodule cells containing for Rhizobium is carefully controlled by Q-Cycles playa part in both photosyn- the symbiotic nitrogen-fixing bacteria. the Oz-binding protein leghemoglobin. In thetic electron transport and in the respira- A very interesting function of flavo- the process of gene expression the Nod tory chain. Using this cycle ubiquinone or noids has been found in this process of D gene encodes a regulatory protein plastoquinone is able to carry two protons nitrogen fixation [11]. One of the most that controls expression of other nod ge- across the membrane while transferring important plant bacterial interactions is nes. Following interaction with inducer one electron to cytochrome. that between leguminous plants and bac- molecules, the conformation of the Nod The vital, oxygen-transporting blood teria of the genera Rhizobium, or between D-protein changes (Fig. 31). This presum- pigment of most animals is hemoglobin, other plants, e.g. alder trees (alnus) and ably initiates transcription of other nod in muscles it is the related myoglobin. actinomycetes. Infection of the roots of a genes. The inducers are in most cases Leghemoglobin has been indentified in leguminous plant with Rhizobium leads to f1avonoids that are secre~ed by the roots leguminous plants. There its presence is the formation of root nodules. The oxygen of leguminous plants in order to trigger FORSCHUNG 64

CHIMIA 49 (1995) Nr. 3 (Mtirzl

nescence had been adopted as a conven- ient mechanism to detoxify the system of molecular oxygen [15]. Later, other applications evolved, e.g. camouflage. An impressive example for camouflage is bioluminescence of fishes (or symbiotic bacteria in fishes) that is directed downward and intensity-adapted to the surface-light intensity. Thus, seen from below, the fish vanishes in surface- light [9]. The dinoflagellate Gonyaulax polyedra displays the interesting phenom- enon of circadian rhythms of biolumines- cence and aggregation ('red tides') [65]. The real mechanism of production of excited and emitting states can be rather involved as is shown for bioluminescence of Renilla (an anthozoan coelenterate) and Photohacterium (Fig. 32).

4.8. Visual-Effect Functions One important function of integumen- tal pigments is in the contrasting roles of crypsis (camouflage) and semasis (adver- tisement). A classification of integumen- tal color schemes is given in Fig. 33 [6]. The aim and purpose of cryptic colors is to conceal. This can be done by adapta- tion to environmental color and shapes, by countershading and shape-disruption (so- matolysis). A species may also imitate an inconspicuos object (mimesis) (color prints Figs. 50 and 51). Sematic coloration is either repulsive or attractive. Aposematic colors warn the viewer that the colored species is either dangerous or unpalatable. Episematic co- lors attract the sexual mate or allure in- sects and birds for pollination and seed dispersal [2] (color prints Figs. 52 and 53). Pseudosematic colors mimic the se- matic or aposematic pattern of another species (Fig. 34). This mimicry can com- prise [66-68]: a) MuLLerian mimicry (model and mimic are dangerous or unpalatable, color prints Figs. 40 and 4]), Fig. 59. Thefurocoumarines ofHeracleum manlegazzianum are well-knownfor their photosensitizing b) Batesian mimicry (the mimic is un- properties. With kind permission of Urania-Verlag [102]. dangerous and palatable), c) Peckham's mimicry (the mimic is the predator or deceiver that has an advan- gene expression [J 1][60] (color print Fig. (Fig. 4), it is understandable that marine tage by cheating and misleading, also 49). bioluminescence is blue-green as a rule called aggressive mimicry, color prints [2][61-64]. Figs. 51 and 54). 4.7. Bioluminescence The chromophores (two of them are Many animals change their appear- Bioluminescence, the 'cold light', has shown in Fig. 3) stem from various classes ance often very rapidly, in response to been observed in marine bacteria, dino- of heterocyclic and aliphatic compounds. environmental changes. These physiolog- flagellates, some fungi and particularly in The mechanism may be generalized as ical or chromomotor color changes occur animals. The light, which is emitted, is in reptiles, amphibians, fishes, and many used in courtship displays, shoaling and luciferin (substrate) + luciferase (enzyme) invertebrates. There are two principal communication, differentiation of the sex- + O2 ~ product * ~ product + hv mechanisms that are regulated by the pitu- es, finding and attracting prey, distracting itary gland and the pineal gland. The one predators and camouflage. Since light in This dependence on oxygen has led to mechanism consists of aggregation and the depths of water tends to be blue-green the opinion that during evolution biolumi- dispersion of pigment granules (e.g. mel- FORSCHUNG 65

CHIMIA 49 (199S) Nr. 3lMlirz)

Rhodop,in 498 ~rno Ibv Retinal A1 Photo 555 ~ 6 p' Batho S43 ~CHO I 42o,

3,4-Dldehydro-Retinal A2 BSI 478 I 225o, Lurn; 497 ~C110 II.M- - -- ~ 10", 300 400 500 600 Meta I 478 3-Hydroxy-Retinal A3 • H(I) ~ I I rn, Mela 0380

HO~Ho~OH I too, allatrans-Rctinal A4 R HO ~ + Opsin

~CHO a HOU-~-- b

Fig. 26. The four retinal chromophores AJ-A4' a) Model of the seven ex- Fig. 27. a) Fly photo receptors, R: visual pigment. M: thermostable interme- helices of bovine rhodopsin, b) model of fly visual pigment with ll-cis- diate which is photoreconverted to R by red stray light, S: sensitizer for UV retinal A3 and a sensitizing chromophore all-trans-retinol A3' light; b) Photoreaction and sequence of bovine rhodopsin (after [32))

m H a a

M M. Fresh Water "" I I M..;p~ Me ~ OM. OM. 195 Brackish Water o Scytonemin Usninic acid Parietin ( Physcion ) Littoral

Epipelagic Zone ~~. HO a I HO 0 ~I ~I 200 HO OH Me Mesopelagic Zone CHO w'H 0 WOH 1000 460 480 500 520 540 nm Atranorin Apigenin Naringenin

Visual Pigment Absorptions Screening Pigments

Fig. 28. Wavelengths for visual pigments of teLeosts in freshwater and Fig. 29. Screening pigments seawater in various depths (after [9]). Littoral: zone of the sea where light gets to the sea bed. Pelagial: open water zone with epipelagic zone (0-200 m) and mesopelagic zone (200-1000 m).

Symbiotic Interaction

I-p-I-a-n-t-R-o-o-ts-I~I-R-h-i-Z-O-b-iu-m-

OH I Nod D - Protein HO •• OH

Conformational OH 0 Change Luteolin Q-Cycle nod factor ~ Nodule Formation -c Induction of genes Fig. 30. Q-Cycleofthe respiratory chain. Ubiquinone Q carries two protons across the membrane and transfers one electron. The other electron is

always in the cycle. Q-:-: semiquinone radical, bS62' bs66: heme b in cyto- Fig. 31. Molecular signalling between roots of leguminous plants and chrome b (after [59]). Rhizobia in the soil (after [60)) FORSCHUNG 66 CHI MIA 49 (1995) Nr. 3 (M'rz) anin), and is utilized by the chameleon as mobacterium violaceum synthesizes the biochrome classes, the following have to an example. antibiotic pigment violacein [71]. In plants be mentioned: A (retinol), E (tocopherol),

The other mechanism, used by cepha- the phytoalexins play the part of defense K (menaquinone), Q (ubiquinone), B2 (ri- lopods (squid, octopus), expands or con- compounds that are produced only when boflavin, folic acid), B12 (cobalamine). tracts the whole chromatophore with the the cell is being attacked. Phytoalexins Epidemiological studies indicate that aid of radial muscle fibres [2][9] (color belong to various classes, among them the the frequent and high intake of fresh veg- prints Figs. 55 and 56). f1avonoids and isoflavonoids. Isoflavonoids etables and fruits is associated with lower have been studied mainly because of this cancer incidence and that high plasma 4.9. Chemical Defense ability. The furocoumarines act against her- levels of ascorbic acid, a-tocopherol, ~ Many dyes of organisms have destruc- bivores. They have strong photosensitiz- carotene, vitamin A and certain phyto- tive properties towards the tissues of other ing properties (color prints Figs. 45 and chemicals are inversely related to cancer species and may, therefore, used for de- 57-59). incidence [72]. Obviously humans ingest fensive or protective purpose (Fig. 35). large numbers of naturally occurring anti- Many quinones are produced as defense 4.10. Vitamin, Antimutagenic and mutagens and anticarcinogens in food. chemicals, antibacterial and antiviral com- Anticarcinogenic Functions The US Food and Drug Administration pounds. Examples are the benzoquinones Many of the so far discussed classes of has cautiously acknowledged thatthe 'pub- of the bombardier beetle, the naphthoqui- biochromes have contributed to the metab- licly available evidence does indicate that none juglone of the walnut tree and hyper- olism and life functions of man. In partic- diets rich in fruits and vegetables, which icin of St. John's wort [2]. The function of ular they afford vital compounds as vita- are low in fat and high in vitamin A (as ~ many pigments of fungi, however, is not mins, coenzymes, prosthetic groups etc. carotene), vitamin C and fiber are associ- yet understood [69]. Aspergillus flavus Among the water- and liposoluble vita- ated with a decreased risk of several types produces the toxic aflatoxins [70]. Chro- mins, that can be ranked among the various of cancer [73]'. Among the phytochemi-

Binding protein) c.- (Binding protein) Colour Scheme Function ( Luciferin -- Luciferin Ca++ Camouflage Crypsis o. To escape detection by FMN (008)- Enzyme Lucirerase (Luciferase) (Binding Protein) predators Luciferin + Ca + + b. To ovoid detection by prey I R'-CHO o (LUciferin - Monoanion * ) Semasis Advertising, Signalling -L- Luciferase + CO 2 Aposemasis Warning Creen FP Green FP ::xx;r;C * HO Episemasis Attraction l o. Directed towards opposite Luciferin - Monoanion ) _ sex EneflO' Luciferase _ LIght Light ( b. Luring prey Trsmfer Green FP * 509 nm 490 nm

a b Pseudosemasis Mimicking Semasis o. Pseudooposemosis Bioluminescence b. Pseudoepisemosis

Fig. 32. a) Renilla bioluminescene, b) bacterialluminescene. (after [25](64)). Fig. 33. Classification afintegumental color schemes (after [6)) FP: fluorescent protein.

Sematic Patterns

Benzoquinones Juglone Aflatoxin B2 Bombardier beetles Walnut Aspergillus

Mullerian Mimicry Sex, Pollination, Dispersal (Warning) 1~0 Bataslan Mimicry Peckham's Mimicry

(Warning) (aggressive, luring) OH ~I ~ H H ISignallmitation I Violacein Phaseollin (lsoOavonoid ) Psora len Model- Receiver- Mimic Chromobacterium Plants Apiaceae

Fig. 34. Mimicry and sematic patterns Fig. 35. Defense chemicals FORSCHUNG 67

CHIMIA 49( 1995) NT. 3 (Mlirz) cals that show inhibitory effect on chem- ically induced carcinogenesis are [72][74]: Vitamins (A, E, C) Carotenoids (,B-carotene) !Interventlon I Presumable Chemopreventive Agent Chlorophyll Flavonoids (quercetin, rutin, tangeretin, nobiletin) -----Vltamln C, E, Gallotannins Green tea, tea catechlns Carotenolds Ellagitannins The mode of interaction of these dyes and phytochemicals is speculative butthey intervene at one or the other stage of the Coumarin, Umbeillferone, carcinogenesis (Fig. 36). In cell cultures Eplcatechln gallate of carcinogen-initiated fibroplasts, a num- ber of retinoids and carotenoids can re- versibly inhibit progression to the trans- formed state. The retinoids acted during the promotion stage as chemopreventive, not as chemotherapeutic, agents, reversi- bly suppressing transformation after initi- ation but before expression of the trans- formed phenotype. The anti carcinogenic action of retinoids and carotenoids is close- ly correlated with enhanced gap-junction cell-to-cell communication and with in- Fig. 36. Multistage carcinogenesis model and possible points of intervention by chemopreventive creased synthesis of the gap-junction pro- phytochemicals, pigments, and vitamins and some presumable agents [72-78] tein, connexin [77] [78]. Retinoids are of particular interest in oncology. They exert their antitumor ac- tivity through inhibition of cell prolifera- Retinol-Prealbumin-RBP tion, induction of cell differentiation and suppression of oncogene expression. In animal experiments, retinoids have pre- Prealbumin RBP ventive and therapeutic effects on prema- + lignant and malignant lesions [79-81]. A possible machanism for vitamin A (retin- CellMembrane~ ol) and tretinoin (all-trans-retinoic acid) -;J77777777T j I I I I II I III activity is given in Fig. 37 [80]. III

Retinol :;;;===~ all-trans-Retinoic Acid Recei ved: September 30, 1994 / [I] J.J. Wolken, 'Photo processes, Photorecep- 9-cis-RA tors, and Evolution', Academic Press, New ROL-CRBPI \ CRABP I-RA York ,1975. [2] G. Britton, 'The Biochemistry of Natural ROL- CRBP II j CRABP II- RA Pigments', Cambridge University Press, London, ]983. [3] H. Zollinger, 'Color Chemistry', VCH, Weinheim, 1991. [4] D.L. Fox, 'Animal Biochromes and Struc- tural Colors', University ofCalifomiaPress, Berkeley, 1976. [5] D.L. Fox, 'Biochromy', University of Cal- ifornia Press, Berkeley, 1979. [6] A.E. Needham, 'The Significance of Zoo- chromes', Springer Verlag, Berlin, ]974. [7] K. Nassau, 'The Physics and Chemistry of Color', John Wiley & Sons, New York, 1983. Proliferation ~ntlation [8] B.D. Lipson, B.A. Horwitz, Mod. Cell. BioI. 1991, 10, I. [9] R. Wehner, W. Gehring, 'Zoologie', 22nd Fig. 37. Vitamin A activity and possible mechanism for cell proliferation and cell differentiation. edn., Georg Thieme Verlag, Stuttgart, ]990. CRBP: cellular retinol binding protein, CRABP: cellular retinoic acid binding protein, RBP: retinol [10] P.S. Song, S. Suzuki, J.D. Kim, J.H. Kim, binding protein, RAR: retinoic acid receptor ex, {3,y, RXR: retinoid X receptor, RA: retinoic acid, ROL: in' Photoreceptor Evolution and Function' , retinol [79-8\]. FORSCHUNG 68

CHI MIA 49 (1995) Nr. 3 (Man)

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