Red and near infra-red signaling: Hypothesis and perspectives

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Journal of Photochemistry and Photobiology C: Photochemistry Reviews 13 (2012) 190– 203

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Review

Red and near infra-red signaling: Hypothesis and perspectives

Vladimir D. Kreslavskia, Irina R. Fominaa,b, Dmitry A. Losc, Robert Carpentierd,Vladimir V. Kuznetsovc, Suleyman I. Allakhverdieva,c,∗

a Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russiab Biosphere Systems International Foundation, Tucson, AZ 85755, USAc Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, Moscow 127276, Russiad Département de Chimie-Biologie, Université du Québec à Trois-Rivières C.P. 500, Trois-Rivières, Québec, G9A 5H7, Canada

a r t i c l e i n f o

Article history:Received 12 October 2011Received in revised form 11 January 2012Accepted 24 January 2012Available online 2 February 2012

Keywords:Cytochrome-c-oxidaseFlavoproteinsFree radicalsPhotoactive porphyrinsPhotoreceptorsRed lightNear infra-red lightSignaling systems

a b s t r a c t

The review covers some of the proposed cellular photoreceptors responsible for the effect of red andnear infra-red (NIR) light on mammalian cells, including cytochrome-c-oxidase, photoactive porphyrins,flavoproteins, and molecular oxygen. We do not discuss the clinical studies but consider animal models,especially fibroblasts. Several key hypotheses such as mitochondria signaling and free-radical concep-tion of the effects of red light and NIR light based on the changes in redox properties of photoreceptormolecules as well as membrane conception are examined. Special attention is paid to common mecha-nisms of light signaling in mammalian and plant organisms.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1912. Hypotheses and signaling systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

2.1. Photoacceptors and primary responses to red and NIR irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1932.2. Signal intermediates, second messengers and secondary processes of light signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1952.3. Role of cytochrome-c-oxidase in mitochondrial signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1962.4. Free radical conception of molecular–cellular mechanisms. H2O2 as a signaling molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

2.4.1. H2O2 as a signaling molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1983. Comparison of light signaling in animal and plant (photosynthesizing) cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1984. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2015. Perspectives (important points) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Abbreviations: cAMP, adenosine-3′ ,5′-cyclophosphate or 3′ ,5′-cyclo-AMP; Cytcox, cytochrome-c-oxidase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide;IP3, inositol-1,4,5-triphosphate; IR, infra-red; LEVL, low-energy visible light; LLLT, low level laser therapy; LPLI, low power laser irradiation; LPC, phospholipase C; LPO, lipidperoxidation; Ph, phytochrome; Px, peroxidase; NIR, near infra-red; ROS, reactive oxygen species; TF, transcription factor.

∗ Corresponding author at: Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.Tel.: +7 496 7731837;fax: +7 496 7330532.

E-mail addresses: [email protected], [email protected](S.I. Allakhverdiev).

1389-5567/$20.00 © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.jphotochemrev.2012.01.002


V.D. Kreslavski et al. / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 13 (2012) 190– 203 191

Vladimir D. Kreslavski is a senior researcher. He is thehead of group at the Laboratory of Ecology and Physiologyof Phototrophic Organisms at the Institute of Basic Bio-logical Problems, RAS, Pushchino, Moscow Region, Russia.The field of interests: photoreceptor signaling, molecu-lar mechanisms of plant stress resistance and acclimationof photosynthetic apparatus as well as the pathways ofphotosynthetic improvement.

Irina R. Fomina is a senior researcher at the Laboratory ofEcology and Physiology of Phototrophic Organisms, Insti-tute of Basic Biological Problems, RAS, Pushchino, MoscowRegion, Russia. The field of her interests: photosyntheticcarbon metabolism and strategy of its optimization incyanobacteria and higher plants.

Dmitry A. Los is a professor at the K.A. Timiryazev Insti-tute of Plant Physiology, Moscow, Russia. He obtained hisDr. Sci. degree in plant physiology and biochemistry in1997 (Moscow). His research interests are in the area ofstress sensors and transduction pathways in photosyn-thetic cells.

Robert Carpentier is professor at Universite du Que-bec a Trois-Rivieres, Quebec, Canada. He obtained hisPh.D. in biochemistry from Laval University (1983, Que-bec). He is editor of Journal of Photochemistry andPhotobiology B: Biology, associate editor of the JournalPhotosynthesis Research and was the chair of the XII-Ith International Congress on Photosynthesis (Montreal,2004). His research interests concern the influence ofenvironmental stresses on electron transport pathways inphotosystems I and II, and energy dissipation in photosyn-thesis.

Vladimir V. Kuznetsov is Director of the Institute of PlantPhysiology and the Head of the Laboratory of Adaptationof Plants (Moscow, Russia), Dr. Sci. in Biology, Profes-sor. His research interests are in the area of physiologyand biochemistry of plant stress, signaling, physiology oftransgenic plants. He is editor-in-chief of the Russian Jour-nal of Plant Physiology, the chairman of Scientific councilon Plant Physiology and Photosynthesis of the RussianAcademy of Sciences.

Suleyman I. Allakhverdiev is head of the Laboratory of“Controlled Photobiosynthesis” at the Institute of PlantPhysiology, Russian Academy of Sciences (RAS), Moscow,and Chief Research Scientist at the Institute of BasicBiological Problems, RAS, Pushchino, Moscow Region,Russia. He obtained his Dr. Sci. degree in plant physiologyand biochemistry from the Institute of Plant Physiology(2002, Moscow), and Ph.D. in physics and mathemat-ics (biophysics), from the Institute of Biophysics (1984,Pushchino). Earlier, he had graduated with a B.S./M.S., inphysics from the Department of Physics, Azerbaijan StateUniversity, Baku. Dr. Allakhverdiev has been guest-editor,as well as a member of the Editorial Board of more than 10

international journals. He also acts as a referee for major international journals andgrant proposals. He has authored (or co-authored) more than 300 papers. He hasorganized several international conferences on photosynthesis. His research inter-ests include the structure and function of photosystem II, water-oxidizing complex,artificial photosynthesis, hydrogen photoproduction, catalytic conversion of solarenergy, plant under environmental stresses, photoreceptor signaling.

1. Introduction

The first positive laser effect was demonstrated by Endre Mesterin Semmelweis University, Budapest, Hungary, who wanted to testif laser radiation might cause cancer in mice [1]. He shaved the dor-sal hair, and found that the hair grew back more quickly in the laser(694 nm) treated group than in the untreated one. During the lastdecades, low-energy visible light (LEVL) and near infra-red (NIR)light have been shown to stimulate several cell functions. Such“photobiostimulation” effects have been used successfully in sev-eral key areas of medicine and veterinary practice where low levellaser therapy (LLLT) plays an important role [2]. These are (i) woundhealing, tissue repair and prevention of tissue death; (ii) relief ofinflammation in chronic diseases and injuries with its associatedpain and edema; (iii) relief of neurogenic pain and some neurolog-ical problems [3]. One of the important fields of low-energy lightis the photodynamic therapy of swellings, and a second field forwhich outer sensitizers are not required is laser or light diode ther-apy [4,5]. This type of therapy is based on the stimulatory actionof laser or light diode irradiation in visible or near IR diapason ofwave lengths of 400–900 nm.

Today the following benefits for the low power laser irradiationhave been demonstrated [5,6]:

(1) cellular metabolic activation and increased functional activity(ATP synthesis is increased by up to 150%);

(2) stimulation of repair processes as a result of increased cell pro-liferation;

(3) anti-inflammatory effects;(4) activation of microcirculation and more efficient tissue

metabolism;(5) analgesic effects as a result of increased endorphin release;(6) immunostimulation with correction of cellular and humoral

immunity;(7) increased antioxidant activity in the blood;(8) stabilizing lipid peroxidation in cell membranes.

In smaller extent the effects of low level laser irradiation andlight diodes are studied in plant cells and organisms. Laser light isalso able to initiate many processes in plants and seeds. In partic-ular, protective and repairing effects of He–Ne laser light (severalmW mm−2) were found in plants exposed to different intensities ofultraviolet-B radiation [7,8]. Oxidative stress was estimated by theconcentration of malondialdehyde in leaf discs and the rate of elec-trolyte leakage. On the other hand, high doses of low-intensity laserradiation (5-min exposure to the He–Ne laser light; � = 632.8 nm,I = 10 mW) were shown to induce lipid peroxidation (examined byan increase in the thiobarbituric acid reactive products) in wheatcallus culture [9]. Recently, the protective effect of low intensityred light against UV-B damage in the photosynthetic apparatus wasdemonstrated in leaves of Spinacia overacea L. sv. “Giant” [10].

Despite of the several positive reports obtained from exper-iments conducted in vitro, different animal models and clinicalstudies of laser and diode therapy remain controversial. This isdue mainly to the light dependence of the various parameters suchas specific properties and conditions of tissues and cells used inthe experiments as well as the complex dose, wavelength, treat-ment timing, etc. Besides, the mechanisms of photobiostimulativeprocesses are often unclear and still debated.

An important consideration involves the optical properties oftissue [11,12]. Both the absorption and scattering of light in tissueare wavelength dependent (both much higher in the blue region ofthe spectrum than the red). Water begins to absorb significantly atwavelengths greater than 1150 nm. Therefore although blue, greenand yellow light may have significant effects on cells growing inoptically transparent culture medium, the use of LLLT in animals


192 V.D. Kreslavski et al. / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 13 (2012) 190– 203

and patients almost exclusively involves red and near-infra-redlight (600–1100 nm).

The primary reactions by photoexcitation depend on type ofphotoreceptors. Type of photoreceptor is determined with thepresence of various chromophores which are sensitive to cer-tain wavelengths. In opposite to flavins and flavoproteins whichabsorb only blue and green light, porphyrins absorb also yellowand red light. Red and near infra-red light have been proposed tobe absorbed by cytochrome-c-oxidase (Cytcox). Catalase, COD andother enzymes containing porphyrin grope can be activated uponirradiation of red light, for example He–N laser [13]. They havehigh enough coefficients of absorption. As a result Cytcox can beactivated in response to moderate red or NIR light and porphyrin-containing enzymes to moderate red light. NADH-dehydrogenaseis active in violet-blue region etc. Oxygen molecule has in opticalregion several relatively narrow absorption bands possessing smallcoefficient of absorption due to optically forbidden transitions, par-ticularly close to wavelength of He–N laser (632.8 nm), also fewbands in NIR region [14]. It is suggested that very strong He–N laserinduces the formation of 1O2 in significant amounts [15].

Photon absorption modifies the molecular configuration of thephotoacceptor with a resulting alteration of a signaling cascade.Note that the reactions immediately following the changes inphotoacceptor condition constitute the primary reactions and theimplemented modifications of cellular functions are seen as sec-ondary reactions [16,17]. There are several hypothesis concerningthe nature of the primary reactions: induction of singlet-oxygenby the photoacceptor, alteration of redox properties, nitric oxidehypothesis, free-radical hypothesis [2,13,17–22] and generationof transient local heating [23]. In the case of plants, absorptionof red light with a maximum at 660 nm by the photoacceptor(phytochrome) leads to the formation of the physiologically theactive form of the phytochrome [22], which transducts light signalto the second messengers like Ca2+, cyclicAMP, inositol-1,4,5-triphosphate (IP3), etc. followed by a cascade of cell responses atthe biochemical and physiological levels.

It should be noted that the free-radical hypothesis of the actionof laser and light diode illumination [2,13,15,24–26] is one ofthe most important general concepts developed during the lastdecades. According to this hypothesis low energy light leadsto transient generation of oxygen reactive species (ROS), whichincreases the activity of antioxidant enzymes and the accumulationof low-molecular weight antioxidants and other protective com-pounds. Antioxidant enzymes (different peroxidases, catalase, andSOD) and also low-molecular antioxidants, such as carotenoids,anthocyanins, ascorbic acid, tocopherols, and glutathione, playa key role in the regulation of the content of various radicalsand peroxides both in animal and plant cells [27,28]. SOD bythe reaction of dismutation transforms two O2

•− radicals intoH2O2. Subsequently, endogenous peroxidase and oxidase enzymesconvert H2O2 to water, and catalase discomposes H2O2 to waterand dioxygen. In addition, carotenoids and tocopherols protectcells from generated 1O2.

During the nineties, it was proposed that low doses of laserirradiation (He–Ne laser with maximum radiation at 632.8 nm)can induce the production of singlet oxygen and other ROS.Small amounts of these ROS would activate some specific cellularfunctions [15,24,25]. This mechanism was based on photody-namic action of low-energy laser and light diode irradiation onbioorganic molecules such as porphyrins. Endogenous porphyrinswith a maximum of light absorption in the region of 600–700 nmwere proposed to be photosensitizers inducing the formationof 1O2 and other ROS. Firstly, through a process of intersystemcrossing, the excited singlet of a porphyrin can spin-flip into alower energy triplet state that reacts in an energy transfer reactionwith the ground triplet state of oxygen inducing the formation of

reactive singlet oxygen. According to rules of physical chemistry,the “relaxation” (excess energy loss) of singlet oxygen back tothe triplet state is “spin forbidden” and thus singlet oxygen hasa long lifetime for an energetically excited molecule, and musttransfer its excess energy to another molecule in order to relaxto the triplet state. Porphyrins are spread in many types of cells.They are found in leucocytes, fibroblasts, keratinocytes, plantcells, etc. Exited molecules of porphyrins induce the oxidizationof unsaturated fatty acids in lipid membranes. The intensificationof lipid peroxidation (LPO) processes then increases the ionicpermeability of cell membranes, including permeability to Ca2+-ions [13]. As a result of these processes, the synthesis of severalproteins and cytokines is enhanced and the rate of proliferationcan be increased [20,29,30]. A schematic representation of thisconcept is as follows [13]:

Exited photosensitizer molecule → ROS → LPO products

→ increase in [Ca2+] free → activation of intracellular

processes (priming of phagocytes, stimulation of cell

division, other effects) (1)

Another mechanism that results from the absorption of a lightquantum concerns the photoactivation of SOD [13,18] and otherantioxidant enzymes such as catalase [31]. It is known that thepH of the medium is lowered during an inflammatory process.Due to this pH decrease, the activity of many antioxidant enzymesalso decreases. He–Ne irradiation modifies the photoacceptormolecules that would lead to a rectification of the local pH, thusrestoring the activity of these enzymes [13].

Besides the above mechanisms, the possibility of heat-induced[32] and He–Ne laser light-induced [33] ROS generation due toreduction of dissolved air oxygen was demonstrated in bidis-tilled water. A 3–5 min laser (0.7 mWt mm−2) irradiation of waterinduced the formation of singlet oxygen and other ROS, especially,hydroxyl-radical and hydrogen peroxide [34].

A general cellular mechanism based on the hypothesis ofthe interaction of low intensity red and NIR light with com-ponents of mitochondrial electron transport chain, first of allcytochrome-c-oxidase, was also developed [31,34]. In our opinion,such mechanism can also operate in plant organisms [22]. A numberof studies provided experimental evidence [20,35,36] that NO wasinvolved in the mitochondrial mechanism of the cellular responseto LLLT in the red and IR regions of the spectrum.

The “transient local heating hypothesis” was offered based ona number of observations suggesting that the effects of lasertreatment on organisms involve not only a light effect, but alsoelectromagnetic, pressure, and temperature effects [6,37]. Accord-ing to this hypothesis, photophysic reactions are mainly the resultof local heating (by 0.1–0.3 ◦C) of the organism which induces a heatpower flow through the tissues. The temperature gradient beingmore pronounced in cell membranes, it would lead to an increasein ion transport due to opening of ion channels and flow-out of Na+

and K+ ions with their redistribution in different cell compartments.Here, note that effects of low light on biological tissue might

be dependent on the presence of coherency and grade of polariza-tion [6,38,39]. Coherence is one of the unique properties of laserradiation. Speckle patterns occur because of the interference of alarge number of elementary waves that arises when coherent lightis reflected from a rough surface, or when coherent light passesthrough a scattering medium (Fig. 1). The dimensions of thesespeckles at every occurrence of directed random interference areapproximately within the range of the light wavelength, �.

The role of coherency and polarization of light in its biologicaleffects has been examined by the usage of laser and light emittingdiode sources of the same integral intensity. There is only small



Infl

uen

ces

La ser ligh t

Wound healin g

Acceleratio n of

inflamm atory

process Pain influence

Increase of

endothelial

cells and

kerat inoc ytes

Enhancement

of serum

respon se

factor

Enhancement

of SOD levels

Decrea sed C-f iber

activity

Incre ased nerve cell

acti on potent ial

Incre ased of

serotonin

level in blood

Enhance d

synthesis of

endorphin

Bradykinin

decrea se Increase of

procol lagen

synthesis in

fibroblast s

Incre ase i n

number in

mast cells

Sec

on

dary

mec

han

ism

s

Triggers an

imm uno logic al

chain reaction

Activation of

macroph ages

Increased rec eptor act ivit y on cell

membrane s

Influences the permeability of cell membranes,

which eff ect s Ca2+, Na+ and K+ as well as the proton

gradient over the mi tochondria membra nes

Increa se of ATP- asa

and act ivation of

cAMP an d en zymes

Pri

mary

mec

han

ism

s

Local dif ferences in intensit y create tempe rature -

and pres sure gr adients across cell membra nes. The

genera tion of organell e membrane po tentials

induces a dipole moment on th e bar shaped lip ids.

Polari zati on can enhance these ef fects of ligh t

Point s of high la ser light

intensi ty ap pear

Areas of high difference

in light absorption Volumes of p artial ly

polarized light are f ormed

Diff use scatte ring of la ser light in tiss ue

induce s interf erenc e an d spec kle formati on

Fig. 1. Possible effects of monochromatic, coherent polarized low power radiation on mammalian tissue.Modified from [6].

amount of studies which compare the effects of these sources undersimilar conditions.

The conclusion can be done from the studies on effects ofcoherency that illumination of biological tissues by coherent laserlight unavoidably leads to strong intensity gradients of the radia-tion in the tissue due to speckle formation. In case of the use oflight-emitting diodes the speckle formation was absent [6]. Theselaser-specific speckles cause a spatially nonhomogeneous depo-sition of light energy, and lead to statistically nonhomogeneousphotochemical processes, an increase in temperature, changes inlocal pressure, deformation of cellular membranes, etc. (Fig. 1).Finally, inter- and intracellular gradient forces appear whose actionmay significantly influence the pathways and rates of biologicalprocesses. In contrast to the photochemical action of light, it is not

accompanied by photon absorption and has a universal character –it depends weakly on the radiation wavelength, but requires a highdegree of coherence. However, the effects of biostimulation are notspecific to laser application; the effects of red and near-infra redlight are also demonstrated by using incoherent sources such aslight emitting diodes [6,39].

2. Hypotheses and signaling systems

2.1. Photoacceptors and primary responses to red and NIRirradiation

The most common deactivation pathway upon absorption ofa light quantum by a photoacceptor molecule in living tissues is



internal conversion, which is a transformation of part of absorbedlight energy into heat. Another pathway is the emission of a lightquantum of longer wavelength by the exited molecule; this isfluorescence. The third pathway can be called photochemical.Because the energy of photons in visible and infra-red regions hasinsufficient power, covalent bonds in photoacceptor moleculescannot be broken. On the other hand, the absorbed energy isenough for the formation of excited singlet state, which can betransformed into the long-lived triplet state of the chromophore. Inthis state the chromophore is able to transfer its excitation energyto the ground triplet state of molecular oxygen thus transformingit to its reactive species, singlet oxygen. The chromophore tripletstate may also undergo an electron transfer to oxygen forming thesuperoxide anion-radical. Electron transfer reactions are highlyimportant in the mitochondrial respiratory chain, where theprincipal chromophores involved in laser therapy are thought tobe located [16,20,30].

It is known that if light absorption converses the molecule intoan electron-exited state, the redox-properties of this molecule arechanged [40]. As a result of the transformation, the dissociation of aweekly bound ligand, for instance NO or CO, from a binding site ona metal containing cofactor of an enzyme is possible. Especially, thekey photoreceptor of the mitochondrial respiratory chain, Cytcox,can change its conformation and redox-state due to dissociationof NO bound to the iron-containing and copper-containing redoxcenters of the enzyme. In turn, the change in the redox-state ofCytcox modifies the rate of electron transfer in the respirationchain [20].

One of the key questions is what kind of photoreceptors isresponsible for the effect of biostimulation or more exactly forphotoregulation of cell processes. In a search for chromophoresresponsible for photobiostimulation, endogenous porphyrins,mitochondrial membrane cytochromes, and flavoproteins werefound to be suitable candidates for visible light and NIR lightphotoacceptors [2,20,41]. Between photoacceptors, a primary pho-toacceptor, Cytcox, the terminal enzyme of the mitochondrialelectron transport chain [42] was examined the most [34,43–48].The Cytcox contains as absorbing chromophores two iron centers,heme a and heme a3 (also referred to as cytochromes a and a3),and two copper centers, CuA and CuB, one zinc and one magne-sium [17,42]. Fully oxidized Cytcox has both iron atoms in theFe(III) oxidation state and both copper atoms in the Cu(II) oxidationstate, while fully reduced Cytcox has the iron in Fe(II) and copperin Cu(I) oxidation states. Absorption spectra obtained for Cytcoxin different oxidation states were found to be very similar to theaction spectra for biological responses to low-intensity laser light[3,45,47,48]. These and other [49,50] studies provided strong evi-dence to conclude that low-intensity red and NIR light are actingon cells through this photoacceptor. However, one should take intoaccount that fully oxidized or fully reduced Cytcox cannot be con-sidered as a primary photoacceptor but only its intermediate forms(partially reduced or mixed-valence enzyme) [17].

Flavins are small and water soluble photosensitizers whichabsorb throughout the UV-A-blue spectrum, with peaks at 370 and450 nm [51]. Flavins are available in cells primarily as flavin ade-nine dinucleotide (FAD) and flavin mononucleotide (FMN). Mostflavin molecules in the cell are present as flavoproteids, especiallyNADH-dehydrogenase, as part of complex I of the mitochondrialrespiratory chain. Flavins can be responsible for the photosensiti-zation of oxyradicals in cells [41]. On excitation to a triplet state byUV-A-blue light, flavins (FAD and FMN) and flavoproteins can bereduced by cellular reducing agents, which then can result in theproduction of H2O2 [52,53].

This hypothesis was tested in the study of Hockberger et al.[54] by using fluorescence microscopy. The authors demonstratedthat violet-blue light stimulated H2O2 production in peroxisomes

and mitochondria in cultured mouse, monkey, and human cellsand that a likely mechanism of production was activation offlavin-containing oxidases. The findings propose that violet-blue light initiates photoreduction of flavins, which activatesflavin-containing oxidases in mitochondria and peroxisomes,resulting in H2O2 production.

Thus, upon irradiation these chromophores can photosensitizethe formation of ROS, which together with photoinduced dissocia-tion of NO and changes in the redox state of the photoacceptor canplay a key role in the modification of cell functions and activities[13,20,21,55].

The above concepts were the most developed during theexamination of the mitochondria hypothesis which suggests theinteraction between laser light and the components of the elec-tron transport chains in mitochondria, including the absorption ofred and near infra-red light by Cytcox [35,43,56,57]. In contrast tored and NIR light absorption by complex I, flavoproteins such asNADH-dehydrogenase can work as photoacceptors in the violet-blue spectral region [41].

Thus, the first event of mitochondrial signaling can be asexample an activation of Cytcox by red light and NIR light. Thenext step is an enhancement of mitochondrial potential, ATP andROS, leading to biochemical and cellular changes at macroscopiclevel such as enhanced cell proliferation or accelerated woundhealing [30,49,58]. The modulation of several elements of themitochondrial retrograde signaling (�� m, ROS, Ca2+, NO•, pHi,fission–fusion homeostasis of mitochondria) by the irradiationwere reviewed recently [20]. Cell redox systems such as ox/redglutathione or ox/red ascorbate, activation of MAP-kinase cascadeand transcription factors (TFs) can be involved in the furthersteps of the light signal transduction (see below) [21]. Note thatin case of plant hormonal receptors, for example cytokinins, itis suggested that the receptors, which are located in the innermembranes or in the plasma membrane, are the sensory histidinekinases. In Arabidopsis, the activated phosphate from receptorsis transmitted to the corresponding genes by special proteinstransmitters/phototransmitters [59].

The cyanobacterium Synechocystis possesses six histidinekinases that resemble plant phytochromes and may function aslight photoreceptors: hik35, hik3, hik1, hik44, hik24 [60,61] Thehistidine kinase Hik35 expressed in Escherichia coli produced theproduct with the features of plant phytochrome [62]. However,inactivation of hik35 did not lead to significant changes in theexpression of genes that are under RL and FRL control [63]. Thisallows suggest that Hik35 is not the key element, which controlsthe response of cells to RL and FRL. However, the response to RLor FRL can be under control of (Hik33), which is multifunctionalhistidine kinase (Fig. 2) and reviewed as stress histidine kinase. Itmight be that Hik35 and/or other potential light-sensory histidinekinases may sense H2O2, which can be produced under moderateor high dose of RL.

Another type of photoreceptors is mobile plant photoreceptorssuch as phytochromes. Phytochrome is a photo-isomerizable sen-sory chromoprotein that exists in a red light-absorbing form anda far-red light-absorbing form. The phytochromes of Arabidopsisand other higher plants have a phytochromobilin chromophore,whereas the phytochromes of green algae and cyanobacteria havea phycocyanobilin chromophore. Red light (660 nm) promotes theformation of the active form of phytochrome, which translocatesinto the nucleus [22]. Here, the phytochrome is able to regulatethe transcription of nuclear genes directly involved in growth andphotosynthetic reactions.

Activated TFs, such as OxyR of eubacteria, may participate in thephytochrome signal transduction chain [64]. The TFs, due to thepresence of thiol groups and ferrous-sulfur clusters, may changetheir structure and properties and undergo the transition from



hliA hliB hliC sigD feoB slr154 4 sll148 3 ssr2016 sll154 1 slr168 7

ndhD2 fus ycf39 crtP slr0616 slr1747 slr0400 slr0401 sll0815 sll1770 ssl3446sll1911 sll0086

hliA hliB hliC slr1544 sll14 83 slr1687 ftsH prg5 sds abfB sll0330

hliA hliB hliC sigD feoB slr1544 sll1483 ssr201 6

hliA hliB hliC sigD feoB slr1544 sll1483 ssr2016 sll1541 slr1687 ssl344 6 fabGgloA

hliA hliB hliC slr1544 sll1483 ssr2016 fabG ftsH psbA pgk sodB gpx2 hspA pho-re g

Cold H2O2NaCl Sorbitol

Hik3 3 – multifunctio nal histi dine kinase

Red Lig ht?

? ? Rre26 Rre31

Fig. 2. Possible role of multifunctional histidine kinase33 in low power red lighteffects in plant tissues. Rre26 and Rre31 – are response regulators.

Modified from [146].

inactive form into active form. Here, the changes in localizationof TFs may be observed.

Thus, light has a physiological significance for plants as an ener-getic and regulatory factor. However, light sensitivity might be acommon property of higher animals and be important for their life[47]. A possible scheme of primary stages of low intensity red andNIR light action on mammalian and plant organisms is presentedin Fig. 3.

2.2. Signal intermediates, second messengers and secondaryprocesses of light signaling

There exists ample evidence that acting at lower concentrations,ROS as well as NO [66] can be purposely produced within cells toserve as signaling molecules [67,68]. Another second messengerboth in plant and animal cells is cAMP [20,22].

An increase in mitochondrial respiration was shown to stimu-late ROS generation in experiments with orange-red and infra-redlow level laser light (see ref. [19,20,46]). It is important to note thatboth stimulation and inhibition of the respiratory chain can resultin enhanced ROS generation [69]. Mitochondria have the capacityto communicate with the nucleus through structural changes in theorganelle itself, e.g. by changes in fission–fusion homeostasis in adynamic mitochondrial network (for review see [70]). Structuralchanges in mitochondria can lead, through the endoplasmic retic-ulum, to changes in other organelles leading to the accumulation offree calcium (Cacyt

2+) and cAMP in the cell [20,70]. It was supposedthat these second messengers can activate some redox-sensitiveTFs (Fig. 4) and by this way induce the expression of genes respon-sible for DNA and RNA synthesis and, finally, increase the rate of cellproliferation, which was observed in cell cultures [47]. The involve-ment of the redox-sensitive transcription factor NF-kB and anothertranscription factor, AP-1, in cellular signaling in irradiated cellshas also been suggested theoretically (Fig. 4) [20].

TFs may change their activity and specificity due to the followingevents [71]:

(1) direct oxidation/reduction of the certain amino acid groups(mainly – SH-groups) in TFs;

(2) changes in redox-state of various buffer cell systems;(3) changes in subcellular location of TFs under oxidative stress

induced by ROS. It is also suggested the direct action of ROSon the activity of transcription factor NF-kB [72,73]. A modelof transcriptional regulation by redox-sensitive transcriptionfactor Yap1, which is a homolog of bacterial TF – OxyR, hadbeen proposed on a basis of yeast system [71].

Activation of G-

proteins, cAMP,

binding to TFs

Formation of

active form of

phytochr ome

Generation of

singlet O2

Photodynamic

eff ect

O2 → single t O2

Trans ient ROS

generation by

chromophore

Changes in

enzyme acti vity

S0

S1

Photoacceptor

Heat di ssipation and buildup of

local temperat ure and press ure

gradients across cell

memb ran es

Heating of

chromophores

Changes in

redox-pote ntial of

chromoph ore

NO-liberation

Fig. 3. Primary stages of low intensity red and NIR light action on mammalian and plant organisms. The scheme is based on a number of reviews [6,13,21,22,37,65].



Fig. 4. A schematic view explaining the putative mitochondrial retrograde signalingpathways after absorption visible and IR-A radiation (marked hv) by the photoac-ceptor, cytochrome c oxidase. Arrows ↑ and ↓ indicate an increase or decreasein the values, brackets [] indicate concentration. FFH = changes in mitochondrialfusion–fission homeostasis; AP-1 = activator protein-1 (TF); NF-kB = nuclear factorkappa B (redox-sensitive TF). Experimentally proved (continuous lines) and the-oretically suggested (dotted lines) pathways are shown. Em, plasma membraneelectrical potential; � m, mitochondrial membrane potential; NADH-ase, NADH-dehydrogenase; Cytcox, cytochrome c oxidase; Eh, cellular redox potential; pHi,intracellular pH.

Adapted from [20] with some modifications [22].

Cell proliferation, especially its initial phase, adhesion of cellsto a matrix, and DNA and RNA synthesis rates are known cellu-lar responses to LLLT largely studied in series of work (see ref.[20,21]). One of the important positive effects of these responsesis an increased resistance of cells to the action of environmentalstressors like UV-radiation [74].

There are several systems that regulate cell metabolism. Amongthem, changes of redox systems, processes of phosphorylation anddephosphorylation as well as variations of pro/antioxidant ratioare the most important. The mechanism can involve an increaseof Cytcox activity [20], which is accompanied by increased ratesof electron transport and hence, respiration. Enhancement of elec-tron transport can lead to the increased production of ATP and ROS[75]. It was shown recently [76] that IR-A radiation (760–1440 nm)elicits a retrograde signaling response in normal human skin fibrob-lasts. Broad band radiation (760–1440 nm) induced the formationof mitochondrial-derived ROS ([ROS]m) in cultured human dermalfibroblasts. The increase in [ROS]m lead to a rise of the intracellularredox potential, Eh (Fig. 4).

The light-induced increase in proton gradient and ATP synthesiscan lead to a higher activity of the Na+/H+ and Ca2+/Na+ antiporters,and of different ATP driven ion carriers (for example, Na+/K+ ATPaseas well as Ca2+ pumps) and trigger Ca2+ transport. ATP is the sub-strate for adenyl cyclase, hence, the modulation of ATP level leads tochanges in cAMP. Both Ca2+ and cAMP are the key second messen-gers and seem to be included in the red light transduction chain ofmammalian cells [20]. Other messengers in the light transductionchain might be H2O2 and nitric oxide, NO (see ref. [13,20]).

2.3. Role of cytochrome-c-oxidase in mitochondrial signaling

Together with photosynthesis, mitochondrial respiration isan important source of energy in plant cells. However, in plant

cells, there is little information concerning the role of mitochon-drial components in light signal transduction. The regulation ofexpression and activity of enzymes of mitochondrial respirationchain can be one of the key elements that link photosynthesis andrespiration. There are observations that the activity of rotenoneinsensitive NADH-dehydrogenase from plant mitochondria isunder phytochrome control and the enzyme is activated with redlight (�m = 660 nm) (Fig. 4; Fomenko, personal communication).It might be suggested that plant mitochondria plays an importantrole in light signal transduction in plant cells and a cascade ofsignaling events between the mitochondria and the nucleus maybe found in the future. However, this hypothesis is evidencedmainly on the basis of model systems and sometimes the approachcannot explain the presence of a log-phase in the development oflight-induced effects.

On the other hand, it was suggested as early as 1981 thatphotosensitivity might be a common mitochondrial property inhigher animals [77]. According to that, Karu [78–80] suggested thatthe universality of the low power laser effects and the possibilityof using different wavelengths for irradiation are accounted for bythe fact that the primary photoacceptors of monochromatic visibleand NIR radiation are the respiratory chain components. Indeed,photoirradiation enhanced the activities of NADH: ubiquinoneoxidoreductase, ubiquinol: ferricytochrome-c-oxidoreductaseand ferrocytochrome-c: oxygen oxidoreductase of red liver mito-chondria after irradiation by an argon-dye laser at a wavelengthof 660 nm (0.6 J/cm2, 1.2 J/cm2, 2.4 J/cm2 and 4.8 J/cm2, P < 0.05)(see [81]).

In contrast to photodynamic therapy and UV-A therapy, pho-tobiomodulation uses light to affect the activity of one or moreendogenous enzyme photoreceptors, which likely initiate cell sig-naling pathways and alter cell and tissue metabolism as well as cellproliferation.

Although the mechanisms underlying the therapeutic ben-efits of photobiomodulation are still incompletely understood,an important first step in understanding this phenomenon hascome mainly from the finding that Cytcox, the terminal acceptorof the mitochondrial electron transport chain, is a photorecep-tor that mediates many positive effects of photobiomodulation[17,46,47,82]. Evidence that mitochondrial Cytcox is the primaryphotoreceptor for photobiomodulation initially came from thefinding that most of the light absorbed by cells is absorbed by mito-chondrial Cytcox (and, to a lesser extent, by other mitochondrialcomponents) [83], and from a determination of the action spec-trum on NIR light on cell proliferation and cell attachment [17,47].Note that the single most important molecule in cells and tissuethat absorbs light between 630 and 900 nm is Cytcox (responsiblefor more than 50% of the absorption greater than 800 nm).

Karu and co-workers [43,48] examined a number of the actionspectra of different biological processes such as proliferation, adhe-sion of cells to matrix, and DNA and RNA synthesis rates. Theseaction spectra summarized in a review by Hamblin and Demidova[3] were measured at various light intensities and duration fordifferent wavelengths. The effects of several exogenous chemicalcompounds were also examined [35,48]. In the red region of thespectrum two absorption bands of Cytcox were found with max-imums at about 620 and 680 nm as well as several bands in NIRregion [43]. The following wavelength intervals in the LLLT actionspectra were described: (1) 613.5–623.5 nm, (2) 667.5–683.7 nm,(3) 750.7–772.3 nm, (4) 812.5–846.0 nm [48]. The action spectrawere compared with the absorption spectra of cell monolayers inthe 600–860 nm region [47] and examination showed the similarityof the main bands in both cases.

As a photoreceptor, mitochondrial Cytcox of the respiratorychain in eukaryotic cells represents the first step in an intracellularphoto-signaling pathway, and mediates the transfer of electrons



from cytochrome c to molecular oxygen. It is likely that its enzy-matic activities are responsible for initiation of a signaling cascadein response to light in the vision and IR region [3,17,47,84–86].Until recently, mitochondrial Cytcox was thought to have only oneenzymatic activity: the reduction of oxygen to water. This reactionoccurs under normoxic conditions and involves the addition of4 electrons and 4 protons to diatomic oxygen, according to thefollowing reaction: 4H+ + 4e− + 2O2 → H2O. This reaction has beendesignated Cytcox/H2O [87]. During the Cytcox/H2O reaction,oxygen is reduced by a series of one-electron transfers which canalso generate O2

•−, H2O2 and other ROS. Recently, mitochondrialCytcox was discovered to have a second enzymatic activity: thereduction of nitrite to nitric oxide. This activity has been calledCytcox/NO [87].

Cytcox contains four redox-active cofactors (2): two iron cen-ters, heme a and heme a3 and two redox-active copper centers,CuA (Cu–Cu) and CuB as well as magnesium center, which can bepossible visible light accepting chromophores [42,88]. The enzymeprovides the final electron sink by accepting electrons from reducedcytochrome c and passing them sequentially through CuA, heme aand finally to its active site, composed of heme a3 and CuB, where O2is reduced to water [84,85,88,89]. Together with fully oxidized andreduced states there are many intermediate mixed-valence formsof the enzyme and other coordinate ligands such as CO, CN, and for-mate can be involved [11], which may be distinguished kineticallyand their structures assigned by spectroscopic methods [88,90].

It is also suggested that [91] electron transfer from heme a to theO2 reduction site initiates the proton pump mechanism by beingkinetically linked to an internal vectorial proton transfer.

Cytochrome c oxidase −cyt c → CuA → heme a

→ heme a3-CuB → O2 (2)

According to spectroscopic data available (in region of red to NIR)in literature on Cytcox it is suggested that the band 825 nm belongsto oxidized CuA, the 760 nm band-to the reduced CuB, the 680 nmband-to the reduced CuA [43]. The 350–500 nm bands are likely thesuperposition of several bands. On a base of the analysis of actionspectra of cellular monolayers it is also suggested that the primaryphotoacceptor for red-near IR region is a mixed valence form ofCytcox.

The intrinsic rate of electron equilibration between the twochemically identical hemes of mitochondrial Cytcox, a key stepin respiratory oxygen reduction. Pilet et al. [92] recorded opticalabsorbance changes after CO photolysis in the fully reduced andmix-valent states of Cytcox on the timescale up to 4 ns. Accordingto their data the time of electron transfer from cytochrome c to CuA– 12–16 �s and from CuA to heme a – 50–100 �s. Electron redistri-bution between the two hemes according to the new equilibriumoccurs in 1.2 ns.

As mentioned above, the absorption bands in the red and infra-red regions of LLLT action spectra were considered to be mostlyrelated with the spectrum of Cytcox that is agreed with the par-ticipation of Cytcox in photobiostimulation. Additional evidencefor the involvement of mitochondrial Cytcox in photobiomodula-tion has come from several studies with neuronal cells and tissues[82,93]. Thus, more direct evidence for the involvement of Cytcoxin photobiomodulation comes from studies on neuronal cell death[93]. These studies examined whether inhibitors of mitochondrialCytcox could compete with NIR treatment. The results from thesestudies indicated that NIR light could protect neuronal cells frominduced cell death by potassium cyanide, a potent Cytcox inhibitor.These studies, done under normoxic conditions, also revealed thatthe most effective wavelengths paralleled the NIR absorption spec-trum of oxidized Cytcox.

Pastore et al. [94] found increased activity of Cytcox and anincrease in polarographically measured oxygen uptake after illu-mination of mitochondrial suspension with HeNe (2 J cm−2). Amajor stimulation in the proton pumping activity was found inilluminated mitochondria. Moreover, an increased oxidation ofcytochrome c and increased rate of electron transfer were foundin vitro in samples of the purified Cytcox enzyme illuminated withHe–Ne laser [34].

It is suggested that irradiation intensifies the electron transferstage within the Cytcox and makes more electrons available for thereduction of dioxygen in the catalytic center heme a3-CuB site [84].

The question about a mechanism of the primary reactions uponthe irradiation of biological objects by red and near IR is one of themost important. It is known that the electron excitation changes theredox-properties of absorbing molecules [40,95]. Karu [79,80] sug-gested that photoexcitation of redox-active centers in the moleculeof Cytcox had an influence on redox-state of the centers and therate of electron transfer in the molecule and enhances its func-tional activity. This leads to acceleration of electron transfer inelectron–transport chain of mitohondrion and is a base of the mech-anism of low-power laser therapy at the cellular level [[84], see alsohttp://www.isan.troitsk.ru/dls/Review articles.html]. According todata available LLLT produces a shift in overall cell redox potentialin the direction of greater oxidation [19].

It is suggested that redox-sensitive CuA and CuB centers, andalso hemes a and a3 seem to be responsible for the redox-shift [17].The modes of action of respective redox control initiating biolog-ical molecules are quite diverse and include reduction–oxidationof thiol groups, iron–sulfur centers, hemes and flavins (reviewedby [96]). Similar mechanism of changes in redox-state of car-riers as a result of their photoexcitation is often viewed inphotosynthetic processes. Well known examples related to pho-tosynthesis are the regulation of Calvin cycle enzymes by thethioredoxin system (reviewed by [97,98]) or the mechanismsinvolved in scavenging oxygen radicals under light stress, such asthe ascorbate–glutathione cycle (reviewed by [99]).

Thus, the hypothesis mentioned by Karu and Afanasyeva [43]that the primary acceptor of low level laser light is a Cytcoxmolecule in intermediate redox-state is well supported. In 2004,experimental evidence of a novel mitochondrial-signaling path-way was found in mammalian cells irradiated by red or near-IRlight [100]. The absorption of photons by molecules leads toelectronically excited states, and consequently to changes in redox-properties of these components. This can lead to an acceleration ofelectron transfer reactions [81].

Although early studies identified mitochondrial Cytcox as anendogenous photoreceptor for photobiomodulation, the cellularand molecular mechanisms underlying photobiomodulation havenot been so far clear. Three recent findings provide important newinsight [86]. First, nitric oxide has been implicated in the effectof photoiostimulation. Second, Cytcox has been shown to have anew enzymatic activity – the reduction of nitrite to nitric oxide.This nitrite reductase activity is elevated under hypoxic condi-tions but also occurs under normoxia. And third, low intensitylight enhances nitric oxide synthesis by Cytcox without altering itsability to reduce oxygen. From these findings, it is supposed thatCytcox functions in photobiomodulation by producing nitric oxide,a signaling molecule which can then function in both intra- andextracellular signaling pathways [86].

Nitric oxide can regulate respiration by binding to CuB [44] ofthe catalytic center of Cytcox and, thus, inhibit its activity. Red orNIR light changes the redox-state of the chromophore CuB, thuschanging the conformation of Cytcox and decreasing the bound NOto the catalytic center of Cytcox [101]. During tissue pathologicalstates the binding of NO to Cytcox can be enhanced. Under irradi-ation NO is liberated and the respiratory activity increases; this is



the “NO hypothesis” [35]. Karu [35] has also demonstrated a role ofNO in the activation of HeLa cells attachment to a glass matrix usingthe NO donor sodium nitroprusside (SNP). The addition of SNP to acellular suspension of HeLa cells eliminated the red light effect onthe attachment of the cells.

The generation of NO often leads to the production of ROS andtriggers redox signaling and mitochondrial retrograde signalingvia changes in �� m [101]. Here, NO can modulate mitochondrialrespiration by direct binding to Cytcox. The role of NO in plantmitochondria is reviewed in details in [86,87,102]..

2.4. Free radical conception of molecular–cellular mechanisms.H2O2 as a signaling molecule

Today, the idea of a free-radical regulation of protein synthesisincluding light-induced changes in the balance of antioxi-dant/oxidant is generally recognized [103]. The production of ROSin animal tissues by sunlight has been studied considerably, firstof all its adverse effects observed during aging and skin cancer[67,104]. One of the main sources of sunlight-induced damage isultraviolet light. However, some ROS can be generated also whena tissue is illuminated with visible light [41,105]. The levels of ROSproduced in this manner is usually low and do not cause significantdamage of cells, except at high doses of illumination [105].

It has been proposed that ROS production is one of the mainmechanisms by which low intensity visible light affect cell culturesand animal organisms [106]. The hypothesis that ROS mediate theeffects of visible light is supported by the findings that low levels ofROS have various non-destructive effects on cells, and are naturallyproduced as signal messengers [28,67]. In addition, antioxidantshave been reported to inhibit the effect of visible light on cells[107,108], which is in accordance with the concept of a role of ROSproduction in visible light effects.

2.4.1. H2O2 as a signaling moleculeROS are extremely reactive molecules that have high affin-

ity to membranes, DNA, or proteins in plant cells. The ETC ofchloroplast, mitochondria, and ER can all leak electrons to O2,resulting in the formation of ROS [109,110]. It is not surprisingthat until recently ROS were still viewed mainly as toxic cellularmetabolites in plant and animal organisms. However, increas-ing lines of evidence support the idea that ROS also function assignaling molecules that mediate protective responses to vari-ous stimuli [111], including laser and diode red and NIR light[2,13]. According to this hypothesis low energy light leads to tran-sient generation of ROS and development of weak oxidative stress,hence, to induction of the activity of antioxidant enzymes andaccumulation of low-molecular antioxidants and other protectivecompounds.

Among ROS, H2O2 seems to be the best suited to play the role ofsignaling molecule due to its higher stability and longer half-life. IfH2O2 serves as a stress signal, the fluctuation of H2O2 level in plantsshould spatially and temporally reflect changes in the environment.Indeed, an oxidative burst is a common response of plants to bothbiotic and abiotic stresses such as pathogens, elicitors, wounding,and environmental stresses [112]. H2O2 has also been regarded asa regulator of the expression of some genes in cells [113]. Thesegenes encode antioxidants, cell rescue/defense proteins, and sig-naling proteins such as kinase, phosphatase, and TFs. The lastsare key elements in H2O2 signaling both in plant and animal cells[22,76,114]. Histidine kinase Hik34, characterized earlier as regu-lator of gene expression under heat [115], salt and hyperosmoticstresses [116], regulates also expression of htpG under oxidativestress.

Low energy laser light (light diodes) in infra-red and NIR regionapplied to living systems (organisms and cell cultures) was shown

to generate ROS by several experiments [13,105]. In this case,the photoreceptors can be porphyrins and their derivatives (forexample, chlorophyll and its precursors). Absorbing a lightquantum in the red region (these compounds usually have along-wavelength band at 630–640 nm) these photoreceptors cantransform into their triplet-exited state. Then, in a photosensi-bilization reaction, they pass their excitation energy to oxygenthat transforms from its basic triplet state into the active sin-glet state. Similar way of energy transduction takes place withcytochromes containing, like hemoglobin, a Fe-porphyrin complexas photoacceptor that activates certain redox-sensitive TFs [20](see Fig. 3). Because of TFs activation, synthesis DNA and RNAcan be stimulated and, by this way, cell proliferation increases asobserved in cell cultures. It should be noted, however, that not allauthors agree with such scheme of cytochrome action.

How does H2O2 exert its influence on gene activity? One ofthe mechanisms may comprise a redox regulation of transcriptionactivity by changes in H2O2 concentration [113]. Formation of smallamount of ROS can cause the transformation of many TFs into anactive state in which they connect with gene promoters and inducethe transcription and translation, including antioxidant proteins.GSH and GR are important factors participating in the redox stateregulation of plant cells [113] and, likely, animal cells.

Involvement of the redox-sensitive TFs such as nuclear factor kB(NF-kB) [72,73] and AP-1 (activator protein-1; a complex composedof jun and fos gene products) [117] in cellular signaling in irradi-ated cells has been suggested (see Fig. 4). Activation of antioxidantenzymes directly by red light absorption, due to changes in theredox-state of the photoreceptors in enzymes such as SOD or cata-lase may also be possible [13]. Involvement of various TFs such as agroup of PIF and CPRF1, CPRF2, GBF1 in red light signal transductionchain was also suggested for plant cells [22].

The induction of the activity of antioxidant enzymes and accu-mulation of low-molecular weight antioxidants can be suggested toresult from developing weak oxidative stress. The work of Qi et al.[7,8] demonstrated that red light irradiation in the dozes whichlead to decrease of oxidative stress induced by UV-B, decreasesmalondialdehyde concentration as compared with control level,and increases the activity of the antioxidant enzymes such asSOD, peroxidases and catalase as well as the concentration ofascorbic acid and UV-B-absorbing pigments. It was suggested thatincrease in antioxidant activity under RL is part of the protectiveand reactivation effects of red light in plants [7]. These con-cepts are reflected in the scheme of Fig. 5. In case of plant cells,H2O2 may participate in the phytochrome-controlled transduc-tion pathway (Fig. 4). Irradiation with red light (660 nm) or far-redlight (710–720 nm) can induce the formation of the active phy-tochrome form following the H2O2 burst. According to Fig. 5, RLacting via the phytochrome system generates an oxidative burst,inducing an enhancement of the activity of antioxidant enzymesand stress-proteins and accumulation of low-molecular protectivecompounds.

3. Comparison of light signaling in animal and plant(photosynthesizing) cells

The involvement of signal molecules in secondary cellular sig-naling in response to LPLI is reviewed in details in a recent paperof Gao and Xing [21]. Thus, we will examine only the points whichare interesting to compare with relevant light signaling processes inplant cells. First of all, it should be noted that many components ofthe receptor signal transduction chains and their regulation path-ways in animal cells were also found in the light signal transductionchains of plant cells. However, there are some differences in thecomposition and function of these elements. Hence, in many cases,it is difficult to make unambiguous conclusions.



Red ligh t Visible li ght

Phytoch rome Photor eceptors

Low po ol of RO S

Regula�on of pr otein synthesis

Stressor

Recep tors

High pool of ROS

Photosynthe�capparatus

PA damage Increasing PA stress-resistance

Oxida�vestress

Ac�va�on ofprotectorysystems

Increasin g ac�vity ofan�oxidant enzy mes (SOD,

AsP etc )

Enhanced synthes isof low-molecula r

compounds

Increas ing poo lof protec�veprot eins

Weak stre ss

Fig. 5. A general scheme illustrating the regulation of stress-resistance of the photosynthetic apparatus and whole plant to abiotic stressor as a result of pre-irradiation ofplants with low intensity red light and visible light. The arrows show the pathway of light signal transduction. The possible intermediates of light signaling – ROS, Ca2+,TFs etc. The scheme is presented on a base of our [10,22] and literature data [7,8,20,59,113]. PA – photosynthetic apparatus. Phytochrome, means PhyB involved in highirradiance response [22].

A specific photoreceptor, phytochrome, function in higherplants. The photoreceptor exists in two photoconvertible con-formations, denoted red light-absorbing (PR form) and far-redlight-absorbing (PFR form) and can translocate from cytoplasmto nucleus. Active state (PFR) is reached only when phytochromeis irradiated with red light, while phytochrome becomes to beinactive when irradiated with far-red light. Photoconversionbetween PR and PFR constitutes a unique light-regulated molec-ular switch that operates in various signal transduction cascades[22]. Photoreceptors similar to the phytochromes of higher plants,including two photoreversible states, were also found in greenalgae and cyanobacteria [118–120]. The question about such pho-toreceptors in animal organisms is open [121]. However, we cansuggest a similarity between plant (bacterial) phytochromes andanimal sensory kinases. An unusual phytochrome-like histidineprotein kinase (CikA) was examined in Synechococcus elonga-tus PCC7942 [122]. Most of the bacterial phytochromes have ahistidine-kinase module [123]. Further, low intensity red and nearinfra-red light affect respiration in animal cells [20,21] and greenalga Dunaliella T. [124]. Although Ruyters [124] suggested thatactive form of phytochrome is involved in the light-stimulatedrespiration, mitochondria photoreceptors such as Cytcox andNADH-dehydrogenase in mitochondria of plant cells could beinvolved in red light signaling.

The general scheme adopted for the Cytcox as photoacceptor inmitochondria signaling pathway is shown in Fig. 4. Comparison ofFig. 4 with the schematic diagram of primary stages of transductionof the light signaling in plant cells presented earlier in [22] demon-strates many similar features of the pathways occurring both inmammalian and plant cells. LPLI is thought to be absorbed by mito-chondrial respiratory chain components and induces an increase in

mitochondrial membrane potential (� m), ATP and ROS. The mito-chondria changes are transduced to other organella and cytoplasm[70] and lead to an increase in cyclic AMP (GMP), as well as a celloxidative burst and changes in [Cain

2+] [17,20,57,125,126]. This isaccompanied by an alteration of cellular homeostasis parameterssuch as pHi and the redox state of the cell. Second messengers suchas cAMP and Ca2+ can activate the expression of redox-sensitiveTFs like NF-kB and AP-1 (Fig. 2). Due to their activation, synthesis ofDNA and RNA can be induced and, hence, enhance cell proliferationand other cell responses [20]. Analogous second messengers suchas cAMP (cGMP), Cain

2+, components of phosphoinositide cycle, orand signaling proteins-TFs can induce the changes in the processesof ionic exchange and photomorphogenetic responses associatedwith the pathways of low power light signal transduction in plantcells [22].

Lets examine the LPLI signaling in more details. The extracellularsignal-regulated protein kinase (ERK) pathway and MAPK-cascadesappear to play an important role in growth-factor-induced pro-liferation, differentiation and cellular transformation [127]. It wassuggested that He–Ne laser would induce the phosphorylation oftyrosine protein kinase receptor (TPKR), which likely activates theMAPK/ERK pathway, and promote the proliferation of skeletal mus-cle cells [128]. However, LPLI has no effects on the cytoplasmictumor necrosis factor alpha (TNF-alpha) receptor.

Irradiation by low power laser can induce an oxidative burst andextra amounts of ROS [107,129,130]. Mitochondrial ROS generationwas detected in studies of LPLI effects upon the action of orange-red and infra-red light (for review see [20,29,46]. Various proteinkinases can be activated by ROS [131]. Tyrosine phosphorylationin proteins constitutes a small fraction of total protein phospho-rylation. However, it plays a key role in the regulation of basic



cell processes in various organisms [132]. Non-receptor tyrosinekinases, particularly the Src kinases are important in regulatingcell proliferation, attachment and survival [133]. ROS, which caninduce the activity of the Src kinases [129,134], are suggested to beinvolved in LPLI signaling [21].

Synthesis of protective proteins such as antioxidative enzymesas well NO synthase is likely stimulated by the activation of proteinkinases due to formation of ROS [13]. Specificity of cell responses isprobably expressed at the level of cell signaling [20]. We supposethat ROS might be produced upon additional irradiation of plantswith light diodes or helium–neon laser under conditions of artifi-cial illumination. Such short-time formation of extra H2O2 after 2 hirradiation of spinach leaves with red light diodes (�m – 660 nm;I = 1.5 W m−2) was shown in our study (data not shown). Anotherpathway examined in [21] is shown below (3):

LPLI → PLC → PIP2 → DAG → PKC → NF-kB → Gene exp ression

→

→

→

IP3 Ca2+

IkB

(3)

According to the above pathway, LPLI is involved in TPKR/PLC-gamma/PKC signaling pathway. Activated TPKR could activatePLC-gamma/PKC pathway in the process of cell proliferation [21].PKCs are involved in different cell functions, such as the cellularproliferation, differentiation, and apoptosis in many types of cells[135,136]. Note that PKC was first described as a Ca2+ activatedphospholipid-dependent serine/threonine protein kinase.

The activated PLC could catalyze the hydrolysis of somephospholipids and then increase the concentration of second mes-sengers such as diacylglycerol (DAG) and inositol triphosphate (IP3)in the cytoplasm. IP3 is able to causes the release Ca2+ into the cyto-plasm, which then interacts with DAG to activate PKCs (for reviewsee [21]). The nuclear TF kappa B (NF-kB) belongs to the Rel familyof DNA-binding proteins and is present in the cytoplasm in an inac-tive state as a dimmer bound with the inhibitory IkB protein [137].A variety of external stimuli, such as ROS, several cytokines, orphorbol esters can activate NF-kB by phosphorylation of IkB usingIkB kinases (IKK). NF-kB is activated and induces the expression ofdifferent genes.

A similar pathway involving components of phosphatidylinos-itol signaling system and protein kinase C, the second messengerssuch as cAMP, Ca2+ and various signaling proteins (SPs) during lightsignal transduction in plant cells was suggested earlier [22] (4) and(5):

P → P* → G-protein → PIP2→ IP3 → Ca in2+

→ CaM → SPs

↓ ↓

DAG → PKC → SPs → SIO (CR) → metabolic r esponse (4)

or

P → P∗ → G-protein → ADC → cAMP → cAMP-dependent PK

→ phosphorylation of signaling proteins → activation of

systems of ion exchange → increase in free [Ca2+]

→ metabolic response (5)

Here, P is phytochrome, P* is active form of phytochrome (PFR);DAG, PIP2, IP3 and PLC-phospholipase C are components of phos-phoinositol cycle; calmodulin (CaM), PK – protein kinases, PKC isprotein kinase C. Signaling proteins (SPs) and elements of endo-plasmatic reticulum (E are suggested to be involved in the lightsignaling. SIE are systems of ion exchange, CR – cell response. Inthis case PFR is associated with the plasma membrane or localizedneat it.

H2O2

OxyR

SOH

SHAc�ve for m

Inac�ve formOxyR

SH

SH

OxyR

S

S

Fig. 6. Proposed scheme of regulation of the bacterial transcription factor OxyR.Inactive form contains thiol groups (SH), the key ones are localized in two cysteineresidues. The H2O2 oxidizes SH-groups with formation of SOH-group and then disul-fide bond; the OxyR transforms into an active form (modified from [71]). Hik33 –multifunctional histidine kinase 33.

It is suggested that both Ca2+ and cyclic nucleotides like cGMPare secondary messengers in the system of phytochrome signaling[138–141].

The question arises: how the phytochrome modulates the con-centration of cyclic nucleotides or Ca2+? One of the possibilitiesconcerns the interaction of the phytochrome with G-proteins [142]that could activate further such enzymes as guanylate cyclase orphospholipase C. As mentioned, phospholipase C catalyzes thehydrolysis of PIP2 into two secondary messengers, IP3 and DAG.Formation of IP3 results in an increase of cytosolic calcium, andDAG activates protein kinases [143,144].

Note that the TFs, both in the nucleus and cytosol, play an impor-tant role in the transduction of environmental signals, including redand NIR light signals. Following the generation of ROS in the cyto-sol, a transduction signal could be transmitted into the genomeand induce the expression of corresponding genes by TFs suchas bacterial OxyR [64]. Owing to the presence of SH-groups andFe–S-clusters, the later can change its structure from its inactive toactive form (Fig. 6). The model of transcription/regulation involv-ing redox-sensitive molecules was also proposed in yeast cells

where a redox-sensitive TF Yap 1, homolog to bacterial TF OxyR,was described [71]. Like phytochrome, Yap1 can be present in anactive form in the nucleus and in an inactive form in the cytosol[71]. It might be suggested that the H2O2 induced by low energy-light can change the conformation of this protein by the inductionof disulfide bond formation between separately localized cysteineresidues, resulting in Yap1 transport into the nucleus and induc-tion of antioxidant genes. The model was supported by experimentswith yeast mutants deficient in Yap1 which were not able to induceantioxidant genes in the presence of H2O2 (cited on [71]).

Thus, there is a similarity between some light signal pathwaysin both types of cells. However, concrete pathways might havedifferent types of signaling proteins, second messengers, or othercomponents.



4. Conclusion

Red light plays a regulatory role both in plants and ani-mal organisms inducing changes in many processes of growth,development and cell metabolism. Infra-red light can also affectthe cell metabolism of various organisms.

The processes of photoinduced changes in cell metabolism(photobiostimulation) might be triggered via several primarymechanisms including changes in redox properties of photoaccep-tors or generation of singlet oxygen or superoxide anion-radical.This can involve the interaction of photoacceptors with oxygen, aswell as the dissipation of the absorbed energy by the photoacceptoras heat, changes in pro-/antioxidant ratio in cells, or even alterationof the temperature and pressure gradients at the local level due toabsorbed light energy.

The mechanisms by which low energy red light protects thephotosynthetic apparatus can be important to understand themechanisms of laser effects on mammalian and human organisms.The plant and animal cells probably have common photoreceptors,for example, chromoproteins containing a porphyrin chromophore.The mitochondrial respiratory chain, through which the protectiveaction of low energy red light could be realized [20], is present inevery eukaryotic cell.

The research of photoreceptors like to red light-sensitive chro-moprotein – phytochrome can be also important for appliedaims. Recently, American researchers built the switch using phy-tochrome from Arabidopsis thaliana, a plant in the mustard family.In plant cells, due to red light absorption the apoprotein of chro-moprotein changes its shape when exposed to red light but returnsto its normal state in far-red light. The introduced photoreceptorcaused mouse cells to move in response to laser light. Such cells canbe trained to follow a light beam or stop on command (for moredetails visit [145]).

5. Perspectives (important points)

1. It is suggested that phytohormones, in particular cytokinins, caninduce genomic activity according to the principle of transcrip-tional cascades [59] where a small quantity of quickly activatedgenes, coded transcriptional factors, or their modulators, leadto changes in expression of a large set of genes taking part inthe realization of global physiological programs. We suggest thatsuch concept is universal and might be involved in low intensityred and IR light effects.

2. The appearance of strong micro gradients in ions, temperatureetc. due to monochromatic, polarized light like laser should beresearched in more details.

3. The idea that red and near infra-red mitochondria signalingcould be universal for both animal and plant cells should bestrongly considered.

4. Phytochrome photoreceptors are widely spread in higher plants.They are also discovered in various bacteria [118,123]. Manyphotomorphogenetic processes, which are under phytochromecontrol, possess red/far-red reversibility. The discovery of suchphotoreceptors in animal organisms should prompt furtherresearch.

5. The general comprehension of the role of low amounts of ROS inred and NIR signaling is also an important focus point.

Acknowledgments

This work was supported by grants from the Russian Founda-tion for Basic Research, Russian Ministry of Science and Education(no: 16.740.11.0176), Molecular and Cell Biology Programs of theRussian Academy of Sciences, and by BMBF (no: 8125) BilateralCooperation between Germany and Russia.

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