Handbook of Photosensory Receptors (BRIGGS:PHOTORECEPTORS O-BK) || Blue Light Sensing and Signaling by the Phototropins

13Blue Light Sensing and Signaling by the Phototropins

John M. Christie and Winslow R. Briggs

13.1Introduction

Plants are dependent on sunlight for photosynthesis. As a consequence, plants havethe ability to sample the surrounding light environment and use this information tooptimize their growth and development. In particular, wavelengths in the blue/UV-A region of the electromagnetic spectrum (from 320 to 500 nm) act to control a widerange of plant responses (Briggs and Huala, 1999; Lin, 2002). Some of these serve tomaximize photosynthetic potential in weak light, while others act to prevent damageto the photosynthetic apparatus in excess light. These responses include phototro-pism, light-induced stomatal opening, and chloroplast movement in response tochanges in light intensity (Christie and Briggs, 2001; Briggs and Christie, 2002; Ka-gawa and Wada, 2002; Liscum, 2002; Kagawa, 2003; Wada et al., 2003).

The phototropins (phot1 and phot2) are f lavoprotein photoreceptors that mediatethe three above-mentioned responses in Arabidopsis thaliana. In addition, the pho-totropins have been shown to control other blue light-activated processes, includingleaf expansion (Sakamoto and Briggs, 2002) and the rapid inhibition of hypocotylelongation in dark-grown seedlings (Folta and Spalding, 2001). Hence, phot1 andphot2 function to regulate a number of photoresponses in plants besides phototro-pism, after which they were originally named (Christie et al., 1999; Briggs et al.,2001a).

Our knowledge of the phototropins and how these photoreceptors function has in-creased dramatically since the isolation of the first phototropin gene back in 1997(Huala et al., 1997). In this chapter, we focus on some of the recent advances relatingto phototropins with respect to their structural and biochemical properties. We alsodescribe our current understanding of the signaling events that follow phototropinexcitation and how these events may be linked to particular phototropin-mediated re-sponses. The biophysical properties of the phototropins and their chromophore-binding domains are covered in more detail elsewhere in this book (Swartz andBogomolni, Chapter 14), as are structural studies of these domains (Crosson, Chap-ter 15).

Handbook of Photosensory Receptors. Edited by W. R. Briggs, J. L. SpudichCopyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN 3-527-31019-3

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13.2Phototropin Structure and Function

13.2.1Discovery of Phototropin

Much of our understanding of phototropin photoreceptors has come from geneticanalysis using the model plant Arabidopsis thaliana. Arabidopsis, or thale cress as it ismore commonly known, is not the most exciting plant to look at, but its small sizeand short life cycle combined with its plentiful seed production make it an ideal ge-netic tool for laboratory work. More importantly, Arabidopsis can be easily manipu-lated to generate mutants that show altered characteristics. And it was the isolationof Arabidopsis mutants altered in phototropism that eventually led to the cloning andcharacterization of the first phototropin gene.

The non-phototropic hypocotyl (nph) mutants of Arabidopsis show impaired stem(or hypocotyl) phototropism to low intensities of unilateral blue light (Liscum andBriggs, 1995). In particular, one class of nph mutants, the nph1 mutant, was found tolack the activity of a plasma membrane-associated protein that becomes phosphory-lated upon irradiation with blue light (Figure 13.1). The encoded protein, originallydesignated NPH1, was therefore hypothesized to represent a phototropic receptorthat undergoes autophosphorylation in response to blue light (Liscum and Briggs,1995). Later experiments confirmed this hypothesis and the NPH1 protein was re-named phototropin 1 (phot1) after its functional role in phototropism (Christie et al.,1999).

13 Blue Light Sensing and Signaling by the Phototropins

Figure 13.1 Blue light-induced kinase activityof phot1 in Arabidopsis membranes. Autoradi-ograph showing autophosphorylation of phot1in membrane protein extracts prepared from3-day-old dark-grown Arabidopsis seedlings.Protein extracts were prepared under a redsafe light and given a mock irradiation (D fordark) or a pulse of blue light (BL) prior to theaddition of radio-labeled ATP. In membrane

extracts prepared from wild-type seedlings(WT), phot1 undergoes autophosphorylationin response to blue light. This response islacking in the phot1 null mutant (previouslynph1). No phot1 kinase activity is detectable insoluble protein extracts from wild-typeseedlings, indicating that phot1 is membraneassociated.

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13.2.2Phot1: a Blue Light-activated Receptor Kinase

The NPH1 gene, or PHOT1 gene as it will now be called, encodes a serine/threonineprotein kinase (Figure 13.2 A). The kinase domain of phot1 is located at its C termi-nus and contains all 11 conserved subdomains typical of protein kinases (Hanks andHunter, 1995). When expressed in insect cells, phot1 undergoes autophosphorylationin response to blue light irradiation (Figure 13.2 B) implying that the heterologouslyexpressed protein is a functional photoreceptor kinase (Christie et al., 1998). Indeed,mutation of an essential aspartate residue within subdomain VII of the phot1 kinasedomain results in a loss of light-dependent autophosphorylation in the insect cell sys-tem, demonstrating that light-dependent phosphorylation of phot1 is mediated byphot1 itself and not some other kinase present in insect cells (Christie et al., 2002).Furthermore, recombinant phot1 binds the vitamin-B-related chromophore f lavin

13.2 Phototropin Structure and Function

Figure 13.2 Protein structures and kinase ac-tivites of Arabidopsis phot1 and phot2. (A)Schematic drawing of Arabidopsis phot1 andphot2 (996 and 915 amino acids respectively).The light-sensing LOV domains, each of whichbinds FMN are indicated. The serine/threo-nine kinase domains are shown in grey. A con-served α-helix at the C-terminal position ofLOV2 (Jα) is also shown. Displacement of thishelix in response LOV2 photoexcitation hasbeen proposed to result in activation of the C-terminal kinase domain (Harper et al., 2003).

(B) Blue light-induced autophosphorylationactivity of phot1 and phot2 expressed in insectcells. Insect cells expressing either phot1 orphot2 were grown in complete darkness for 3days. Soluble protein extracts were isolatedunder a red safe light. Protein samples weregiven either a mock irradiation (D) or irradiat-ed with blue light (BL) prior to the addition ofradio-labeled ATP. Samples were resolved byprotein gel electrophoresis and exposed to au-toradiography.

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monocleotide (FMN) and displays spectral characteristics consistent with the prop-erties of a photoreceptor for phototropism (Christie et al., 1998). Further details of thebiochemical and photophysiological properties of the light-activated kinase reactionhave been presented elsewhere (Briggs et al., 2001b).

The N-terminal region of phot1 contains a repeated motif of 110 amino acids thatbelongs to the large and diverse superfamily of PER/ARNT/SIM (PAS) domains (Tay-lor and Zhulin, 1999). PAS domains are found in proteins throughout nature and areoften associated with cofactor binding. However, the PAS domains of phot1 are mostclosely related to a subset of proteins within the PAS-domain superfamily that areregulated by external signals such as light, oxygen or voltage. Thus, the two PAS do-mains of phot1, with approximately 40% amino acid identity, were assigned theacronym LOV and named LOV1 and LOV2 respectively (Huala et al., 1997). As will bediscussed below, both LOV domains of phot1 and phot2 serve as binding sites for thechromophore FMN and are directly involved in light sensing (Christie et al., 1999; Sa-lomon et al., 2000).

13.2.3Phot2: a Second Phototropic Receptor

Homologs of phot1 have been identified in several plant species including oat, maize,rice, pea, and ferns (for the latter, see Nozue et al., 2000; Kagawa et al., 2004), andmore recently the unicellular green alga Chlamydomonas rheinhardti (Briggs et al.,2001a; Huang et al., 2002; Kasahara et al., 2002b). A second member of the Ara-bidopsis phototropin family, phot2, shows considerable homology to phot1 (Figure13.2 A) (Jarillo et al., 1998). Like phot1, phot2 contains two FMN-binding LOV do-mains and a kinase domain, and undergoes autophosphorylation in response to bluelight (Figure 13.2 B; Sakai et al., 2001; Christie et al., 2002). Both phot1 and phot2 con-tain a conserved α-helix, designated Jα, located at the C-terminal region of LOV2 (Fig-ure 13.2 A). This helical region has been proposed to couple photoexcitation of LOV2to activation of the C-terminal kinase domain (Harper et al., 2003) and will be dis-cussed in more detail later (Section 13.4.1).

While phot1 mutants show impaired hypocotyl phototropism to low intensities ofblue light (Liscum and Briggs, 1995), they retain phototropic responsiveness to high-intensity blue light (Sakai et al., 2000; Sakai et al., 2001), demonstrating the presenceof a second phototropic photoreceptor in Arabidopsis. The curvature response to highlight intensities is severely impaired in the phot1phot2 double mutant (Sakai et al.,2001). Thus, both phot1 and phot2 mediate phototropism, but phot2 functions onlyunder high light intensities. The functional activity of phot2 under high light inten-sities most likely requires differential gene expression; PHOT2 gene expression indark-grown seedlings is induced by light (Jarillo et al., 2001b; Kagawa et al., 2001) andis mediated by the red/far-red light receptor phytochrome A (phyA; Tepperman et al.,2001). In contrast to phot2, phot1 appears to be constitutively expressed and func-tions as the primary phototropic receptor in etiolated Arabidopsis because phot2 sin-gle mutants exhibit normal phototropic curvature to both low and high intensities of


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blue light (Sakai et al., 2001), indicating that phot1 functions over a broad range oflight conditions.

13.2.4Phototropins: Photoreceptors for Movement and More

Phototropism is not the only form of photomovement found in plants. Photomove-ment can also occur at the cellular level. The opening of stomata (pores in the epi-dermis) in response to blue light allows the plant to regulate CO2 uptake for photo-synthesis. Recent genetic studies have shown that this response is controlled redun-dantly by phot1 and phot2 (Kinoshita et al., 2001). But unlike the case for phototro-pism, phot1 and phot2 contribute equally to stomatal opening by working across thesame light-intensity range.

In addition to the cellular level, photomovement in plants can also occur at the sub-cellular level. For instance, chloroplasts move in response to changes in blue light in-tensity (Kagawa, 2003; Wada et al., 2003). Like phototropism and stomatal opening,chloroplast movement is a process that helps to regulate the photosynthetic efficien-cy of a plant. At low light intensities, chloroplasts redistribute themselves within thecell to maximize light capture for photosynthesis. This process is often referred to asthe low-light accumulation response. On the contrary, chloroplasts under high-lightconditions reorganize themselves to avoid the potentially damaging effects of excesslight (Kasahara et al., 2002a) by a process known as the high-light avoidance response.Genetic analysis with phototropin-deficient mutants has shown that phot1 and phot2overlap in function to control the low-light accumulation response (Sakai et al., 2001).Yet, the high light avoidance response is controlled exclusively by phot2 (Jarillo et al.,2001b; Kagawa et al., 2001).

It is now apparent through the study of phototropin-deficient mutants that the pho-totropins are not only involved in controlling phototropism, stomatal opening andchloroplast movement in Arabidopsis. These photoreceptors are now associated withcontrolling other growth responses to blue light as well as phototropism, includingleaf expansion (Sakamoto and Briggs, 2002) which is partially induced by blue light(van Volkenburgh, 1999). The leaves of some plant species can follow the position ofthe sun throughout the day, in a process known as solar tracking. Solar tracking isregulated by blue light (Yin, 1938) and it is postulated that the phototropins may alsofunction to control this photomovement response (Briggs and Christie, 2002).

It is worth noting that phot1 and phot2 not only overlap in function, but also ex-hibit distinct functional roles: phot2 acts as the sole photoreceptor for the chloroplasthigh-light avoidance response (Jarillo et al. 2001b; Kagawa et al. 2001), and phot1alone has been shown to mediate the rapid inhibition of hypocotyl elongation by bluelight (Folta and Spalding, 2001). More recently, phot1 activity has been reported tocontrol the destabilization of specific nuclear and chloroplast transcripts in Ara-bidopsis. Both Lhcb and rbcL transcripts, although transcribed in different subcellularcompartments, are destabilized by high-intensity blue light (Folta and Kaufman,2003). The blue light-dependent destabilization of these transcripts is impaired inphot1 mutants, demonstrating a role for phot1 in regulating the level of chloroplast


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and nuclear-encoded transcripts during plant growth and development. A summaryof all known phototropin-mediated responses identified to date is shown in Figure13.3.

A major task for researchers in the future will be to unravel the signaling eventsthat couple photoreceptor activation to specific blue light-induced responses, such asphototropism and chloroplast movement. Yet significant progress in this area has al-ready begun. Before we address what is known regarding these downstream events,we brief ly review our current knowledge of the photochemical and biochemicalmechanisms associated with phototropin receptor activation by blue light.


Figure 13.3 Range of physiological and bio-logical responses mediated by phot1 andphot2. When phot1 and phot2 are activated byblue light (BL) they overlap in function to me-diate several responses. These are shown inthe center and include, from left to right: pho-totropism, stomatal opening, chloroplast ac-cumulation movement, leaf expansion and

possibly solar tracking. By contrast, thechloroplast avoidance movement is only con-trolled by phot2. Similarly, phot1 alone plays arole in mediating the rapid inhibition ofhypocotyl elongation by blue light and thedestabilization of Lhcb (and rbcS) transcriptsin response to high intensity blue light treat-ment.

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13.2.5Overview of Phototropin Activation

Both phot1 and phot2 are hydrophilic proteins but have been shown to localize to,and co-purify with the plasma membrane in Arabidopsis (Sakamoto & Briggs, 2002;Harada et al., 2003) and other plant species (Briggs et al., 2001b). Although the natureof their association with the plasma membrane is unknown, it is likely that phot1 andphot2 undergo post-translational modification and/or bind a protein cofactor to fa-cilitate membrane interaction.

For convenience, phototropin activation can be viewed as a series of events begin-ning with the absorption of blue light by the LOV domains (Figure 13.4 A). In thedark or ground state, the phototropin receptor remains unphosphorylated and inac-tive. Upon illumination, light sensing by LOV2 is considered to result in a proteinconformational change that involves a highly conserved α-helical region, designatedJα (Figure 13.4 B). Evidence for this light-induced protein structural change will bediscussed later (Section 13.4.1). Although the exact role of LOV1 in regulating pho-totropin activity is not fully understood (see Section 13.3.3), the displacement of Jα inresponse to LOV2 photoexcitation is hypothesized to lead to activation of the C-ter-minal kinase domain, which in turn results in autophosphorylation of the photore-ceptor protein (Figure 13.4 C). At least for phot1, autophosphorylation has beenshown to occur on multiple serine residues (Palmer et al., 1993; Short et al., 1994; Sa-lomon et al., 1996).


Figure 13.4 Overview of phototropin activa-tion by blue light. (A) In the dark or groundstate, the phototropin receptor is unphospho-rylated and inactive. Upon irradiation, the LOVdomains detect blue light. (B) Photoexcitationof LOV2 results in a protein conformationalchange that involves the displacement of ahighly conserved α-helix from the surface ofLOV2 (Jα). This protein structural change is

hypothesized to lead to activation of the C-ter-minal kinase domain. The function of LOV1 ispresently unknown. (C) Activation of the ki-nase domain consequently leads to autophos-phorylation of the photoreceptor protein. It isunknown whether autophosphorylation is in-volved in receptor signaling or whether pho-totropin initiates signaling via substrate phos-phorylation.

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The above description outlining the photochemical and biochemical mechanismsassociated with phototropin receptor activation is clearly oversimplified. In the fol-lowing sections, we review in more detail what is known regarding each of these re-action steps, but it is important to note that there are many aspects associated withthis light-driven molecular switch that remain to be addressed. For example, it ispresently unknown whether light-mediated autophosphorylation of the phototropinsplays a role in receptor signaling or is involved in some other function, say receptordesensitization, or both. It is also unknown whether phot1 and phot2 can phospho-rylate an interacting substrate. Nevertheless, mutant alleles of PHOT1 and PHOT2carrying single amino acid substitutions in the kinase domain have been identifiedindicating that kinase activity is essential for signaling (Huala et al., 1997; Kagawa etal., 2001). We will discuss the subject of phototropin kinase activation in more detailafter addressing the photochemical mechanisms associated with light sensing by thephototropin LOV domains.

13.3LOV Domain Structure and Function

13.3.1Light Sensing by the LOV Domains

A detailed account of the photochemical properties of the phototropin LOV domainsis described elsewhere in this book (Swartz and Bogomolni, Chapter 14) as is a de-tailed account of LOV-domain structure (Crosson, Chapter 15). Therefore, related as-pects of the phototropin LOV domains that have been covered already will be men-tioned only brief ly.

The LOV1 and LOV2 domains from several phototropin proteins have been pro-duced, either singly or in tandem, in Escherichia coli (Christie et al., 1999; Kasahara etal., 2002b). LOV-domain fusion proteins purified from E. coli are highly f luorescentand function as binding sites for the chromophore FMN (Christie et al., 1999). FMNbinding is non-covalent and each LOV domain binds one molecule of FMN. Thespectral properties of recombinant LOV1 and LOV2 fusion proteins are similar tothose of the full-length photoreceptor protein expressed in insect cells (Christie et al.,1998; Kasahara et al., 2002b), showing absorption in the blue/UV-A regions of thespectrum that closely matches the action spectrum for phototropism (Baskin andIino, 1987).

Salomon et al. (2000) were the first to show that LOV domains expressed and pu-rified from E. coli function as light sensors. When irradiated with blue light, bothLOV1 and LOV2 undergo a complex spectral change (Figure 13.5), characteristic ofthat known to accompany the formation of a stable bond between the C(4a) carbon ofthe f lavin isoalloxazine ring and a cysteine residue within the f lavoprotein (Miller etal., 1990). These light-induced spectral changes are fully reversible in darkness, indi-cating that the photoproduct generated has the ability to regenerate back to the initialground state.


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All phototropin LOV domains identified to date contain a cysteine residue situatedwithin a highly conserved motif, GRNCRFLQ (Crosson et al., 2003). This cysteinecorresponds to residue 39 within the 110 amino-acid stretch of LOV1 and LOV2, andhas been designated Cys39 for convenience (Salomon et al., 2000; Swartz et al., 2001;Kasahara et al., 2002b; Christie et al., 2002). Replacement of Cys39 with either alanineor serine results in a loss of photochemical reactivity of LOV1 and LOV2 (Salomon etal., 2000), implying that the photochemical reaction involved requires the formationof a covalent bond between the FMN chromophore and the side chain of Cys39 (Fig-ure 13.5). Formation of this bond, known as a f lavin-cysteinyl adduct, has subse-quently been confirmed by a plethora of biophysical studies (Salomon et al., 2001;Swartz et al., 2001; Holzer et al., 2002; Iwata et al., 2002; Ataka et al., 2003; Kottke etal., 2003; Kennis et al., 2003) and protein crystallography (Crosson and Moffat, 2001,2002; Federov et al., 2003). Indeed, the crystal structures of LOV1 and LOV2 providea snapshot of the domains at the atomic level. Both domains are very similar in struc-ture and resemble a molecular hand, holding the f lavin chromophore tightly within

13.3 LOV Domain Structure and Function

Figure 13.5 Schematic representation of LOV-domain photochemistry. Blue light drives theformation of a covalent bond between theFMN chromophore and a conserved cysteineresidue within the LOV domain (designatedCys39). This process is self-contained and isfully reversible in darkness. The graph (centre)illustrates the light-induced absorbancechanges observed for the LOV2 domain of

phot1 over several seconds. The uppermostspectrum at 450 nm represents the proteinsample in the dark state. The sample is thenirradiated with blue light and the absorptionspectra recorded at 1-s intervals. The light-in-duced absorbance changes detected are char-acteristic of the formation of a f lavin-cysteinyladduct. Adapted from Salomon et al. (2000).

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its grasp with the sulfhydryl group of Cys39 located close to the C(4a) position of theFMN isoalloxazine ring. (For simplicity, the cysteine sulfur in Figure 13.5 is repre-sented as a sulfhydryl group but there is the possibility that it may exist as a thiolateanion as well. Swartz and Bogomolni discuss this matter in detail in Chapter 14)

13.3.2LOV is all Around

Crosson and Moffat (2001) were the first to obtain the crystal structure of a pho-totropin LOV domain. For their crystallographic studies, they used the LOV2 domainfrom a novel photoreceptor from the fern Adiantum capillus-veneris, designated phy-tochrome 3 (phy3), which is a chimera of the red/far-red light photoreceptor phy-tochrome and phototropin (Nozue et al., 1998; Nozue et al., 2000). Phy3 has beenshown to be required for red light-activated phototropism and chloroplast relocationin Adiantum, both of which are regulated by red and blue light in this organism(Kawai et al., 2003).

The phy3 LOV2-domain structure closely resembles that of other PAS domains, in-cluding PYP, HERG, and FixL (Crosson and Moffat, 2001). Its protein fold compris-es five anti-parallel β-sheets and four α-helices that form a central pocket to accom-modate the FMN chromophore. The crystal structure of Adiantum phy3 LOV2 is verysimilar to that recently obtained for the LOV1 domain of Chlamydomonas phot(Federov et al., 2003). However, the side chain of Cys39 in Chlamydomonas phot LOV1appears to exist in two conformations instead of one as was found in Adiantum phy3LOV2. It should be noted that the LOV1 domain structure of Chlamydomonas photwas solved under liquid nitrogen temperatures. In contrast, the structure of Adi-antum phy3 LOV2 was solved at room temperature, which may account for the dif-ference observed. A more detailed description of LOV domain structure can be foundelsewhere in this book (Crosson, Chapter 15).

From the structure of Adiantum phy3 LOV2, Crosson and Moffat (2001; 2002) wereable to pinpoint 10 amino acid residues, in addition to Cys39, that come into contactwith the FMN chromophore via hydrogen bonding or Van der Waals forces. Threeother proteins containing a single LOV domain, with these exact same residues, havebeen found in Arabidopsis (Crosson et al., 2003): ZTL/ADO (Somers et al., 2000; Jar-illo et al., 2001a), FKF1 (Nelson et al., 2000), and LKP2 (Schultz et al., 2001) all ofwhich are associated with a regulation of the circadian clock. Mutants at the ZTL/ADO locus show a much-lengthened period for their circadian rhythms (Somers etal., 2000; Jarillo et al., 2001a), whereas overexpression of LKP2 results in arrhythmicphenotypes for several circadian responses and causes a loss of the photoperiodiccontrol of f lowering (Schultz et al., 2001). Similarly, an absence of FKF1 results in alate-f lowering phenotype and alters the expression of clock-regulated genes (Nelsonet al., 2000). Although none of these findings establish these encoded proteins asphotoreceptors, the presence of a canonical, phototropin-like LOV domain in each ofthese proteins and their close interaction with both circadian responses and pho-toperiodism support this possibility. In fact, Imaizumi et al. (2003) have shown thatthe LOV domains of ZTL/ADO, FKF1 and LKP2 all bind FMN and undergo a blue


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light-activated photochemical reaction identical to that of the phototropin LOV do-mains (Salomon et al., 2000). Curiously, all three LOV domains fail to revert back tothe dark state, in dramatic contrast to the phototropin LOV domains. Whether thisfailure to recover to the dark state is functionally relevant or physiologically mean-ingful remains to be determined, but demonstration of photochemical reactivity lead-ing to the formation of a f lavin-cysteinyl adduct provides strong support for the hy-pothesis that these proteins function as blue-light receptors in Arabidopsis. Schultzdiscusses these putative photoreceptors in the context of circadian rhythms and day-length responses in detail in Chapter 16.

Beside the LOV domain, ZTL/ADO, FKF1 and LKP2 share no structural homolo-gy with the phototropins. Rather they have an F-box (related to targeting other pro-teins for degradation) located at the C-terminal region of the protein followed by 6kelch-domain repeats that form a propeller-like structure thought to be involved inprotein-protein interactions. Indeed, ZTL has been reported to modulate circadianclock function by targeting TOC1, a key component of the circadian oscillator, fordegradation (Mas et al., 2003). Interestingly, the LOV domains of Arabidopsis ZTL/ADO, FKF1, and LKP2, unlike the phototropin LOV domains, contain an additionalamino acid insert of 9–11 residues hypothesized to accommodate a larger f lavin co-factor such as FAD rather than FMN (Crosson et al., 2003). Nonetheless, as men-tioned above, the LOV domains of ZTL/ADO, FKF1 and LKP2 have been reported tobind FMN (Imaizumi et al., 2003), at least when expressed in E. coli.

In addition to plants, single LOV-domain containing proteins have also been iden-tified in fungi (Crosson et al., 2003). The LOV domain of WC-1, a photoreceptor formany but not all blue light responses in the filamentous fungus Neurospora crassa(Froehlich et al., 2002; He et al., 2002), binds f lavin adenine dinucleotide (FAD). Asecond LOV-domain containing protein in Neurospora, VIVID (VVD), serves as thephotoreceptor that allows the fungus to adapt to changes in light intensity(Schwerdtfeger and Linden, 2003). Like the LOV domains of ZTL/ADO, FKF1, andLKP2, the LOV domains of WC-1 and VVD contain an 11-amino acid insert that is notfound in phototropin LOV domains. Other than the LOV domain, WC-1 and VVD donot share any sequence homology either with each other or with the phototropins.WC-1 is a zinc-finger protein (Ballario et al., 1996), whereas VVD is a relatively smallcytosolic protein with no homology to proteins of known function (Heinzten et al.,2001).

When expressed and purified from E. coli, VVD binds a f lavin chromophore thatforms a f lavin-cysteinyl adduct when irradiated with blue light (Schwerdtfeger andLinden, 2003). Like the phototropin LOV domains, the conserved photoactive cys-teine is essential for the photochemical reactivity and function of VVD in Neurospora.Intriguingly, VVD is able to bind both FAD and FMN when expressed in E. coli.Whether VVD exhibits this f lexibility in chromophore binding in Neurospora has yetto be determined. Nevertheless, Cheng et al. (2003) have demonstrated that the LOVdomain of VVD can partially replace the function of the WC-1 LOV domain, sug-gesting that these domains are, at least in part, functionally interchangeable. Furtherinformation regarding these and other fungal photoreceptors is discussed elsewherein this book (Dunlap and Loros, Chapter 18).


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Single LOV domain-containing proteins have even been identified in bacteria(Crosson et al., 2003). These proteins typically contain a single LOV domain coupledto a specific output domain, such as a histidine kinase, a phosphodiesterase, or aSTAS domain (Crosson et al., 2003). STAS domains are generally found in bacterialsulfate transporters and antisigma factor antagonists (Aravind and Koonin, 2000).YtvA, a LOV-STAS protein from Bacillus subtilis has been shown to bind FMN and un-dergoe a blue light-activated photocycle analogous to that of the phototropin LOVdomains (Losi et al., 2002). Though the presence of a LOV domain with its charac-teristic photocycle strongly implies that YtvA functions as a photoreceptor, the effectsof blue light on Bacillus subtilis, a non-photosynthetic soil organism, are unknown.Nonetheless, the presence of LOV domain-containing proteins throughout variouskingdoms of life indicate that this functional light-sensing mechanism is not just re-stricted to plants but has been conserved throughout evolution.

13.3.3Are Two LOVs Better than One?

Although single LOV domain-containing proteins have been identified in plants,fungi and bacteria, the phototropins are the only proteins identified to date that pos-sess two LOV domains (Briggs et al., 2001a). So why do phototropins possess twoLOV domains? Are two LOV domains better than one? At present, the significance oftwo chromophore-binding sites within the phototropin molecule is not fully under-stood. Energy transfer occurring between LOV1 and LOV2 seems unlikely given thattheir respective chromophores are tightly bound within the LOV domain apoprotein(Crosson and Moffat, 2001; 2002; Federov et al., 2003). Similarly, the absorption max-ima of LOV1 and LOV2 are only a few nm apart (Salomon et al., 2000), an arrange-ment that would not favour unidirectional energy transfer. Recent studies, however,have uncovered some insights into the functions of LOV1 and LOV2.

Christie et al. (2002) have used the Cys39Ala mutation, which blocks LOV domainphotochemistry, to investigate the individual roles of LOV1 and LOV2 in regulatingphototropin function in Arabidopsis. In brief, the photochemical activity of LOV2 isrequired to mediate phot1 kinase activity and to elicit phot1-mediated hypocotyl pho-totropism in response to low intensities of unilateral blue light. LOV1 photochem-istry, on the other hand, plays at most a minor light-sensing role in regulating thephotochemical reactivity of phot1 and is not sufficient to elicit phot1-mediatedhypocotyl phototropism under low light conditions or phot1 kinase activity. Thus, atleast for phototropism, LOV2 is essential for phot1 function in Arabidopsis. Similarly,initial studies suggest that phot2, like phot1, operates through a mechanism by whichLOV2 acts as the principal light-sensing domain (Christie et al., 2002). Indeed, Ka-gawa et al. (2004) have recently shown that the LOV2 domain of phot2, in the absenceof LOV1, is sufficient to mediate the chloroplast avoidance movement in Adiantum.It is worth noting, however, that the LOV1 domain of phot2 is still able to mediate asmall degree of light-activated autophosphorylation (Christie et al., 2002). This situa-tion is not apparent for phot1. It will now be important to establish whether the LOV1


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and LOV2 domains of phot2 mediate autophosphorylation on the same or differentamino acid residues.

Although the above findings demonstrate that LOV2 plays an important role inregulating phototropin activity, the exact role of LOV1 remains unclear. The LOV1domain of oat phot1a has been reported to self-dimerize, whereas the LOV2 domaindoes not (Briggs et al., 2001b; Salomon et al., 2004). Likewise, the LOV domain ofWC-1 from Neurospora has been shown to homodimerize in vitro (Ballario et al.,1998). LOV1 may therefore play a role in receptor dimerization. If so, receptor di-merization may be affected by light, and in turn, control the sensitivity of a pho-totropin-associated signaling complex (Liscum and Stowe-Evans, 2000). Alternative-ly, LOV1 may be involved in regulating phototropin-activated processes other thanphot1-mediated phototropism and the phot2-induced chloroplast avoidance move-ment. Evidently, further work is needed to understand the exact role of LOV1 in con-trolling phototropin function and elucidate whether two LOVs are indeed better thanone.

LOV1 and LOV2 have also been shown to exhibit different photosensitivities andreaction kinetics (Salomon et al., 2000; Kasahara et al., 2002b), consistent with theirapparently distinct light-sensing roles in regulating photoreceptor function (Christieet al., 2002). Though studies of individual LOV domains have been instrumental inuncovering the primary mechanisms associated with phototropin photochemistry(Briggs and Christie, 2002; see Swartz and Bogomolni, Chapter 14), photochemicalstudies have shown that bacterially expressed fusion proteins containing both LOVdomains (designated LOV1+LOV2) possess photochemical properties that moreclosely resemble those of full-length phot1 and phot2 expressed in insect cells (Kasa-hara et al., 2002b). Hence, truncated LOV1+LOV2 fusion proteins represent the moreappropriate model system to study phototropin photochemistry in relation to the full-length photoreceptor proteins.

While the LOV1+LOV2 fusion proteins of phot1 and phot2 exhibit similar approx-imate quantum efficiencies, their times for dark regeneration differ significantly(Kasahara et al., 2002b). Dark-regeneration kinetics for phot1 are far slower thanthose observed for phot2. The reason for this discrepancy is unclear. However, giventhat the full-length photoreceptor proteins expressed in insects also show this differ-ence in dark regeneration kinetics (Kasahara et al., 2002b), it seems likely that thisphenomenon has some functional significance. Notably, autophosphorylation ofphot1 in vitro has been shown to possess a memory for a light pulse when subse-quently transferred to darkness prior to the addition of radio-labeled ATP (Short etal., 1992, Palmer et al., 1993; Salomon et al., 1996; Christie et al., 1998). This memo-ry capability of phot1 for a light pulse is only lost in darkness after 10 minutes ormore, which closely corresponds to the kinetics of dark regeneration observed forphot1 photochemistry (Kasahara et al., 2002b) and may ref lect a return of the pho-toactivated system to its initial ground state. On the other hand, the rapid recovery ob-served for phot2 would be expected to yield steady-state levels of photoproduct muchlower than those of phot1 at a given f luence rate of blue light. As a result, higher lightintensities would be required to drive phot2 to the same photostationary equilibriumas phot1.


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Whether the difference in dark regeneration kinetics observed for phot1 and phot2relates to their physiological functions remains to be determined. Phot1 and phot2have been reported to exhibit different photosensitivities in activating chloroplast ac-cumulation movement in response to low light intensities, with phot2 requiring ahigher light threshold for activity than phot1 (Kagawa and Wada, 2000; Sakai et al.,2001). Still, the mechanisms associated with phototropin recovery in vivo are un-doubtedly more complex than a simple reversal of LOV-domain photochemistry. Asdiscussed below, phototropin recovery for light-activated phosphorylation in vivo in-volves dephosphorylation of the photoreceptor protein by an as yet unidentified pro-tein phosphatase rather than degradation and resynthesis of the photoreceptor pro-tein.

13.4From Light Sensing to Receptor Activation

13.4.1LOV Connection

It is generally accepted that light sensing by the LOV domains results in a proteinconformational change that somehow leads to activation of the C-terminal kinase do-main (Crosson et al., 2003). How then does light absorption and subsequent f lavin-cysteinyl adduct formation lead to such a structural change(s) and kinase activation?The photoexcited crystal structure of the Adiantum phy3 LOV2, compared to that ofthe ground state shows only minor, light-induced protein changes, all within thevicinity of the FMN chromophore (Crosson and Moffat, 2002). This is not too sur-prising given the probable structural constraints of the crystal lattice. In contrast, so-lution difference infrared absorbance spectroscopy (FTIR) indicates that photoactiva-tion of purified LOV2 is accompanied by changes in the LOV domain apoprotein(Swartz et al., 2002; Iwata et al., 2003; see Swartz and Bogomolni, Chapter 14;Crosson, Chapter 15). In addition, circular dichroism measurements using purifiedLOV2 from oat phot1a suggest that light-induced f lavin-cysteinyl adduct formation isfollowed by a loss of α-helicity (Corchnoy et al., 2003). But the greatest detail so far re-garding light-induced protein structural changes associated with LOV2 has comefrom solution nuclear magnetic resonance (NMR) spectroscopy. Using NMR, Harp-er et al. (2003) have identified an amphipathic α-helix located just C-terminal ofLOV2 whose structure is altered in response to light (Figure 13.4). This 20-amino acidregion, designated Jα, associates with the surface of LOV2 in the dark. The interac-tion between the Jα helix and LOV2 is disrupted following illumination and f lavin-cysteinyl adduct formation. This structural mechanism has been proposed to couplephotoexcitation of LOV2 to activation of the C-terminal kinase domain (Harper et al.,2003).

The structural consequences of LOV2 photoexcitation may serve as a light-inducedactivator of the C-terminal kinase domain. Alternatively, LOV2 may serve as an au-toinhibitor, repressing kinase activity in the dark whereupon photoexcitation would


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function to relieve this repression. A similar PAS/kinase domain interaction mecha-nism has been proposed for regulating the activities of the bacterial oxygen sensor,FixL (Gong et al., 1998), and the novel eukaryotic protein kinase, PAS kinase (Rutteret al., 2001). Intriguingly, collective alignment of LOV1 or LOV2 domains from allphototropins identified to date reveals that peptide sequences which can form the Jα-helix are only found associated with LOV2 and not LOV1 (K. Gardner, personal com-munication). Thus, the structural consequences accompanying LOV1 photoexcita-tion are likely to differ from those following photactivation of LOV2, consistent withthe apparent distinct functionality observed for these two LOV domains (Christie etal., 2002).

13.4.2Phototropin Autophosphorylation

While a specific role for LOV1 remains to be elucidated, light activation of the pho-totropins ultimately results in autophosphorylation of the photoreceptor protein. Thekinase domain of plants phototropins belongs to the family of cAMP-dependent pro-tein kinases (Hanks and Hunter, 1995), although it seems unlikely that light-activat-ed phototropin autophosphorylation requires cAMP. In particular, the phototropinkinase domain is a member of the AGC-VIII subfamily of protein kinases (Watson,2000). There are two differences between the AGC-VIII subfamily of kinases and themajority of other protein kinases (Watson, 2000). First, the AGC-VIII family containsa DFD amino-acid motif instead of a DFG motif in subdomain VII. The Asp of theDFG motif is required for chelating Mg2+, an ion necessary for phosphate transfer(Hanks and Hunter, 1995) and is essential for phototropin kinase activity (Christie etal., 2002). Second, there is an additional peptide sequence between subdomains VIIand VIII in the phototropin kinase domains. The nature of this region varies de-pending on the kinase, but subdomain VIII is typically involved in the recognition ofpeptide substrates (Hanks and Hunter, 1995).

As mentioned previously, autophosphorylation, at least for phot1, has been shownto occur on multiple serine residues (Palmer et al., 1993; Short et al., 1994; Salomonet al., 1996). Consistent with phosphorylation on multiple sites, phot1 from severalplant species has been reported to show reduced electrophoretic mobility after bluelight irradiation (Short et al., 1993; Liscum and Briggs, 1995; Knieb et al., 2004). A re-cent study by Salomon et al. (2003) has identified eight serine residues within oatphot1a that become phosphorylated upon illumination. Two of these sites (Ser27,Ser30) are located upstream of LOV1, near the N terminus of the protein. The re-maining six sites (Ser274, Ser300, Ser317, Ser325, Ser332, and Ser349) are located inthe peptide region between LOV1 and LOV2. Salomon et al. (2003) also demonstrat-ed that phot1 autophosphorylation in vivo is f luence dependent: the two serineresidues situated at the N terminus are phosphorylated in response to low f luencesof blue light, whereas the other sites are phosphorylated either at intermediate orhigh f luences. This hierarchical pattern of phosphorylation is only observed whenblue light irradiation is performed in vivo. No such f luence discrimination is detect-ed when the blue light irradiation is given in vitro, suggesting that the f luence de-

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pendency for phot1 autophosphorylation in vivo depends on the presence of an addi-tional factor(s).

At least in vivo, the biochemical consequences of low-f luence phosphorylationcould be quite different from those arising from high-f luence phosphorylation. Sa-lomon et al. (2003) have proposed, as Briggs (1996) had earlier, that low-f luence phos-phorylation of phot1 might initiate signaling by modifying the interaction status be-tween the receptor and a specific signaling partner. In fact, phot1a from Vicia faba(broad bean) has recently been reported to interact with a 14-3-3 protein whose bind-ing is dependent on the phosphorylation of a particular serine residue within the pro-tein (Kinoshita et al., 2003). This interaction will be discussed in more detail below(Section 13.5.3). The high-f luence-activated phosphorylation sites, on the contrary,have been proposed to play some other role e.g. receptor desensitization (Briggs,1996; Christie and Briggs, 2001; Briggs and Christie, 2002; Liscum, 2002; Salomon etal., 2003). Site-directed mutagenesis of these sites and their effect on photoreceptorfunction will help to provide information on the role of autophosphorylation in pho-totropin activation and signaling.

So far, there is no evidence to indicate that phototropins initiate signaling throughactivation of a phosphorylation cascade. However, a truncated version of phot2 com-prising only the LOV2 domain and the C-terminal kinase domain is able to comple-ment the chloroplast high-light avoidance response in a phot2 mutant of Adiantum(Kagawa et al., 2004). Given that the sites of phototropin autophosphorylation are lo-cated before LOV2 (Salomon et al., 2003), it is tempting to speculate that phot2 usessome means other than autophosphorylation to bring about this response. Could thissignaling mechanism involve the phosphorylation of an as yet unidentified interact-ing partner? Further studies are now required to test such a hypothesis.

13.4.3Phototropin Recovery

As mentioned earlier, it is generally viewed that the phototropins are unphosphory-lated in the dark or ground state and that light activation results in autophoshoryla-tion of the photoreceptor protein. Autophosphorylation of phot1 in vivo has beenshown to return to its unphosphorylated or inactive state in darkness following a sat-urating pulse of blue light (Short and Briggs 1990; Hager and Brich, 1993; Salomonet al., 1997a; Kinoshita et al., 2003). Moreover, the recovered photoreceptor system canbe rephosphorylated in response to a second blue light pulse (Hager et al., 1993; Sa-lomon et al., 1997a; Kinoshita et al., 2003). These findings therefore demonstrate thatthe phot1, and most likely phot2, have the ability to regenerate back to the non-phos-phorylated form.

How do the phosphorylated receptors regenerate back to the dark or inactive state?One possibility is that the phosphorylated form of the receptor is degraded and is, atthe same time, accompanied by de novo synthesis of the unphosphorylated form. In-deed, prolonged exposure of phot1 to blue light has been shown to result in a grad-ual decrease in phot1 protein levels in dark-grown Arabidopsis seedlings (Sakamotoand Briggs, 2002). However, this decrease in phot1 protein levels in response to con-


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tinuous blue light irradiation is apparent only after many hours. In contrast, the re-covery of phot1 to its non-phosphorylated form in vivo occurs 20–90 minutes after ablue light pulse (Short and Briggs 1990; Hager and Brich, 1993; Salomon et al., 1997a;Kinoshita et al., 2003). Thus, the reduction of phot1 protein levels in response to aprolonged light exposure most likely represents a long-term adaptation process.

Further studies now indicate that phototropin recovery in vivo involves dephos-phorylation of the photoreceptor system in addition to a dark reversal of LOV-domainphotochemistry as discussed previously. Using a specific antibody to Arabidopsisphot1, Knieb et al. (2004) have confirmed that phot1 from several plants species ex-hibits reduced electrophoretic mobility after blue light irradiation, first noted byShort et al. (1993), and commonly associated with protein phosphorylation (Beebeand Corbin, 1986). The mobility shift observed for phot1 corresponds to an increasesize of 2–3 kDa. This size increase is transient and gradually reverts back to the orig-inal size in darkness within 60–90 min, consistent with the protein being dephos-phorylated. Similarly, Salomon et al. (2003) have shown that the recovery of unphos-phorylated oat phot1a in vivo occurs in a f luence-dependent manner; sites that arephosphorylated by high f luences are dephosphorylated first followed by those thatare phosphorylated at intermediate and low f luences respectively. This f luence-de-pendent decline in phosphorylation most likely ref lects dephosphorylation of phot1by an as yet unidentified protein phosphatase rather than a degradation and resyn-thesis of the photoreceptor protein. Whether dephosphorylation of phot1 representsthe rate-limiting step in the recovery for light-activated autophosphorylation in vivoremains unknown.

Blue light irradiation has been reported to result in a rapid reduction (within min-utes) in the association of phot1 with the plasma membrane (Sakamoto and Briggs,2002; Knieb et al., 2004). The significance of this partial redistribution of phot1 iscurrently unknown. Whether it represents the dissociation of a phototropin-associat-ed signaling complex (Liscum and Stowe-Evans, 2000) or is somehow involved inphotoreceptor signaling requires further investigation. Resolving the relationshipsbetween receptor dephosphorylation and photoproduct decay within the LOV do-mains and how these events are linked to intracellular movements like those ob-served for phot1 upon excitation will help to improve our understanding of the short-term processes associated with phototropin recovery. Incidentally, the decrease inphot1 protein levels mentioned above in response to continuous blue light irradiationis reported to coincide with a reduction in PHOT1 transcript levels (Kanagae et al.,2000; Sakamoto and Briggs, 2002; Elliot et al., 2004). This light-induced decrease inPHOT1 mRNA levels is dependent on the photoactivation of phytochrome (Elliot etal., 2004).

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13.5Phototropin Signaling

13.5.1Beyond Photoreceptor Activation

While much progress has been made in elucidating the photochemical and bio-chemical properties of the phototropins, less information is known regarding thedownstream signaling events that follow phototropin activation. The ultimate chal-lenge for researchers will be to identify these components and determine how theseare connected to particular phototropin-mediated responses. Exciting progress in thisarea has already begun. In the following section, we brief ly discuss recent insightsthat have been made regarding phototropin signaling and how these relate to pho-toreceptor localization and certain phototropin-mediated responses.

13.5.2Phototropism

Phototropism is important for germinating seedlings, whereby the emerging shootmust grow towards the light in order to survive. Generally, shoots show positive pho-totropism; movement towards a light source, whereas roots exhibit negative pho-totropic movement. Positive phototropism is mediated by an increase in growth onthe shaded side of the stem resulting from an accumulation of the growth hormoneauxin (Iino, 2001).

At present, very little is known regarding how phototropin activation by blue lightleads to an accumulation of auxin in the shaded side of the stem to bring about a cur-vature response. The model currently favored begins with an establishment of a lightgradient across the hypocotyl in response to a directional light stimulus (Iino, 2001).This results in an unequal activation of photoreceptors across the phototropicallystimulated organ. Indeed, Salomon et al. (1997b; 1997c) have shown that unilateral ir-radiation induces a gradient in phot1 autophosphorylation across oat coleoptiles,with a higher level of phosphorylation on the irradiated side than on the shaded side.The biochemical gradient produced somehow leads to a lateral accumulation of aux-in on the shaded side of the stem (Iino, 2001). Although Cholodny and Went first in-troduced this hypothesis over 70 years ago with respect to the oat coleoptile (Iino,2001), it has proved markedly difficult to demonstrate as a general model. Recentsupport for this mechanism has come from the use of an auxin-sensitive gene re-porter, designated DR5::GUS, whose activity correlates with auxin measurements.This reporter has been used successfully to demonstrate the establishment of an aux-in gradient across a phototropically stimulated Arabidopsis hypocotyl (Friml et al.,2002).

Further support for the Cholodny-Went model of lateral auxin movement in dicotshas recently come from the identification of a putative auxin transporter in Ara-bidopsis that is localized to the plasma membrane at the outer lateral side of hypocotylendodermal cells (Friml et al., 2002). PIN3 is a member of the PIN-FORMED or PIN


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family of putative auxin eff lux carriers of which there are eight members in Ara-bidopsis (Friml and Palme, 2002). Mutants lacking PIN3 exhibit reduced phototro-pism, suggesting that PIN3 (in conjunction with some other PIN family member)may function to bring about a lateral movement of auxin to the shaded side of thestem. How the activity of such a transporter is controlled by the phototropins is notknown. Yet, a lateral relocalization of PIN3 has been shown to occur in roots follow-ing a gravity stimulus (Friml et al., 2002), raising the possibility that phototropin ac-tivation could modulate PIN3 distribution in such a way as to bring about a lateralasymmetry of PIN3 across the stem in response to a phototropic stimulus.

Given the known role of auxin in phototropism (Koller, 2000; Iino, 2001), it is per-haps not surprising that phot1 is found in cells associated with polar auxin transport(Sakamoto and Briggs, 2002). The main mode of active auxin transport in stems ispolar or unidirectional, a movement from cell to cell from the tip of the stem (whereit is predominantly synthesized) to the base (Iino, 2001). Polar auxin transport, un-like lateral auxin movements, can be measured readily and has been attributed to theaction of specific auxin inf lux and eff lux carriers (Friml, 2003). Members of the AUXsubfamily of amino-acid permeases have been identified as candidate auxin-inf luxcarriers (Swarup et al., 2000). AUX1 is localized to the apical surface of root cells, con-sistent with its role in controlling auxin inf lux (Swarup et al., 2001). PIN1, on the oth-er hand, a member of the PIN family, is localized to the basal side of the cell in rootsand shoots consistent with its role in auxin eff lux (Gälweiler et al., 1998). The asym-metric distribution of these two types of putative transporters is thought to determinethe polarity of auxin f low.

Sakamoto and Briggs (2002) have examined the intracellular distribution of phot1in Arabidopsis by fusing the photoreceptor to green f luorescent protein (GFP). Thenative PHOT1 promoter was used to drive expression of phot1-GFP in the transgeniclines obtained. In cells associated with polar auxin transport, phot1 is more stronglylocalized to the plasma membrane adjacent to the apical and basal walls, rather thanthe side walls, placing the receptor in an ideal location to inf luence the activity or dis-tribution of auxin inf lux and eff lux carriers. How this mode of transport is connect-ed to the proposed lateral accumulation of auxin on the shaded side of a phototropi-cally stimulated stem is not known. However, inhibitors of polar auxin transport havebeen shown to block phototropism (Friml et al., 2002) suggesting that this mode oftransport is required to generate the differential auxin distribution associated withphototropism. Support for this conclusion has come from recent experimentsdemonstrating that the amplitude of phototropic curvature in Arabidopsis is inf lu-enced by changes in the basal distribution of PIN1 (Noh et al., 2003). This alterationin PIN1 distribution is reported to involve AtMDR1, a member of a different putativetransporter family associated with polar auxin transport (Noh et al., 2001; Noh et al.,2003). However, the role of AtMDR1 in regulating phototropism and auxin transportis poorly understood at present and requires further investigation.

To date, only two proteins have been shown to interact with phototropin (phot1).One is the scaffold-type protein, NPH3 (Motchoulski and Liscum, 1999). NPH3 is anovel protein containing several protein-protein interacting motifs and has beenshown to interact with phot1 in vitro. The second is a 14-3-3 protein, which will be dis-

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cussed in the following section. NPH3 was first identified through the isolation ofphototropism mutants of Arabidopsis (Liscum and Briggs, 1995; 1996). Indeed, NPH3appears to be essential for phototropism since nph3 mutants exhibit impairedhypocotyl phototropism to both low and high intensities of unilateral blue light with-out affecting phot1 autophosphorylation (Liscum and Briggs, 1995; 1996; Motchoul-ski and Liscum, 1999; Sakai et al., 2000). Like phot1 and phot2, NPH3 is associatedwith the plasma membrane and most likely serves as a bridge to bring componentsof a phot1 photoreceptor complex together (Liscum and Stowe-Evans, 2000). A pro-tein closely related to NPH3, designated root phototropism 2 (RPT2), has been iso-lated from a separate genetic screen (Sakai et al., 2000). In contrast to NPH3, RPT2gene expression is enhanced at increased light intensities similar to the situationfound for PHOT2 gene expression (Jarillo et al., 2001b; Kagawa et al., 2001; Tepper-man et al., 2001). Thus, RPT2 in conjunction with phot2 seems to play a role in me-diating phototropic curvature under high-light conditions.

13.5.3Stomatal Opening

Phot1 has been shown, by means of GFP f luorescence, to localize at or near the plas-ma membrane of stomatal guard cells (Sakamoto and Briggs, 2002), consistent withits role in blue light-induced stomatal opening. While epidermal strips from phot1and phot2 single mutants show blue light-induced stomatal opening (Kinoshita et al.,2001), the phot1phot2 double mutant fails to exhibit this response, demonstrating thatboth phototropins redundantly mediate blue light-induced stomatal opening in Ara-bidopsis (Kinoshita et al., 2001). In addition, stomatal guards cells of the phot1phot2double mutant fail to extrude protons in response to blue light treatment (Kinoshitaet al., 2001). Proton extrusion is essential for stomatal opening and is known to resultfrom activation of the plasma membrane proton-ATPase (Schroeder et al., 2001). Inbrief, proton extrusion by guards cells creates an electrochemical gradient that drivesK+ and Cl– uptake which in turn causes a rise in osmotic potential, resulting in waterinf lux, swelling of guard cells, and consequent stomatal opening. In fact, blue lightactivates the guard cell proton-ATPase via phosphorylation of its C terminus (Ki-noshita and Shimizaki, 1999), providing a tentative connection between blue light-in-duced proton extrusion and phototropin activation. Could the proton-ATPase be a po-tential substrate for phototropin kinase activity? This vital question still remains to beaddressed.

Recent biochemical studies provide a further connection between phototropin ac-tivation and the plasma membrane proton-ATPase. Kinoshita and Shimizaki (2002)have shown that the guard-cell proton-ATPases, VHA1 and VHA2 from Vicia faba(broad bean) bind a 14-3-3 protein at their C terminus when phosphorylated in re-sponse to blue light. 14-3-3 proteins belong to a highly conserved protein family thatgenerally bind to phosphorylated target proteins and play a central role in regulatingsignaling in eukaryotic cells (Sehnke et al., 2002). While the role of 14-3-3 binding inthe activation of the proton-ATPase is not clear, Kinoshita et al. (2003) have gone onto show that phot1a from broad bean also binds a 14-3-3 protein after undergoing au-


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tophosphorylation by blue light. This binding is reversible and like phot1 autophos-phorylation recovers to a basal level in darkness approximately 15 minutes followinga blue light pulse, indicating that 14-3-3 binding to phot1 is dependent on autophos-phorylation.

The binding of a 14-3-3 protein to phot1a from broad bean requires phosphoryla-tion of Ser358 situated between LOV1 and LOV2 (Kinoshita et al., 2003), equivalentto Ser325 in oat phot1a, a residue that is phosphorylated in response to intermediatef luences of blue light (Salomon et al., 2003). Therefore, consistent with the mecha-nism proposed by Briggs (1996) and Salomon et al. (2003), autophosphorylation ofphot1 in response to low and/or intermediate f luence rates of blue light may initiatesignaling by binding a 14-3-3 protein. 14-3-3 binding may in turn allow the photore-ceptor system to associate with or modify the activity of the guard cell proton-ATPase.Studies are now required to clarify the significance and functional consequences of14-3-3 binding to light-activated phot1 and determine its role in activation of the plas-ma membrane proton-ATPase. For instance, it is unknown whether the 14-3-3 pro-tein that binds phosphorylated phot1 is the same one that binds to the phosphorylat-ed proton-ATPase. Interestingly, Kinoshita et al. (2003) observed that blue light-de-pendent 14-3-3 binding to phot1 is not restricted to stomatal guard cells. The authorsfound that light-induced 14-3-3 binding to phot1 was also detectable in dark-grownseedlings and mature leaves of broad bean and other plant species, including Ara-bidopsis, indicating that 14-3-3 binding may represent a common event associatedwith phototropin signaling.

13.5.4Chloroplast Movement

The location of phot1 close to the plasma membrane of Arabidopsis mesophyll cells(Sakamoto and Briggs, 2002) is consistent with its role in mediating blue light-acti-vated chloroplast movement (Kagawa and Wada 2000, Kagawa et al., 2001, Kagawa,2003; Wada et al., 2003). Similarly, phot2 has been reported to co-purify with the plas-ma membrane from Arabidopsis leaves (Harada et al., 2003). However, the mecha-nisms coupling phototropin activation at the plasma membrane to chloroplast move-ments are poorly understood. Similarly, it is not yet known whether chloroplast ac-cumulation and chloroplast-avoidance movements share the same signals. All thesame, recent genetic studies indicate that both chloroplast accumulation and chloro-plast-avoidance movements in Arabidopsis are mediated through changes in the cy-toskeleton.

Many experiments involving the use of cystoskeletal inhibitors have implicated arole for actin filaments in mediating chloroplast movement (Wada et al., 2003). In ad-dition, the isolation of mutants impaired in their chloroplast avoidance response hasled to the identification of a novel actin-binding protein, designated chloroplast un-usual positioning 1 (CHUP1). CHUP1 is required for the positioning of chloroplaststo both low and high light intensities in Arabidopsis (Kasahara et al., 2002a; Oiwaka etal. 2003). Mutants lacking CHUP1 exhibit aberrant chloroplast positioning and light-induced movement compared to wild-type plants, in that chloroplasts are constantly


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gathered at the bottom of palisade cells. CHUP1 contains multiple functional do-mains some of which are involved in mediating protein-protein interactions. One do-main located in the N-terminal half of the protein is an actin-binding motif and hasbeen shown to bind F-actin in vitro (Oikawa et al., 2003). Therefore, CHUP1 mostlikely interacts with the actin-based cytoskeleton in vivo. The extreme N-terminal re-gion of CHUP1 contains a hydrophobic segment that on its own confers the ability totarget GFP into the chloroplast envelope (Oikawa et al., 2003), suggesting thatCHUP1 could function at the periphery of the chloroplast outer membrane. In con-trast to Arabidopsis phot1 and phot2 mutants, which are specifically defective inchloroplast accumulation movement and/or chloroplast avoidance movement, chup1mutants are altered in both the positioning and movement of chloroplasts. Hence,CHUP1 most likely represents an essential component of the machinery required forchloroplast positioning and movement, rather than being directly associated withphototropin signaling.

A possible signal connecting photoreceptor activation at the plasma membrane tothe chloroplasts is the versatile intracellular messenger, calcium (Wada et al., 2003).Several approaches have been used to demonstrate that blue light irradiation leads toan increase in cytosolic calcium concentrations [Ca2+]cyt, in Arabidopsis. Baum et al.(1999) were the first to report a blue light-induced elevation of [Ca2+]cyt in Arabidopsisseedlings transformed with the gene encoding the Ca2+-luminescent protein, ae-quorin. This response is both rapid and transient (rises and falls over a period of 80seconds) and is severely attenuated in a null mutant of phot1. Harada et al. (2003)have recently extended these aequorin measurements and demonstrated that phot2,in addition to phot1, mediates a rapid blue light-dependent increase in [Ca2+]cyt inArabidopsis leaves. Moreover, Stoelzle et al. (2003) have used patch-clamping tech-niques to identify a phototropin-activated plasma membrane Ca2+ channel in Ara-bidopsis mesophyll cells.

Using various pharmacological agents, Harada et al. (2003) were able to show thatphot1 and phot2 can mediate an inf lux of Ca2+ from the apoplast through the activa-tion of a Ca2+ channel(s) at the plasma membrane, whereas phot2 alone can induce arelease of Ca2+ from intracellular stores via phospholipase C-mediated phosphoinosi-tide signaling. A separate study involving microelectrode impalement also suggeststhat phot2 mediates Ca2+ movement from intracellular stores whereas phot1 activa-tion exclusively results in an inf lux of Ca2+ from the apoplast (Babourina et al., 2002).It is worth noting that phot1 and phot2 induce an increase in [Ca2+]cyt in Arabidopsisleaves with different photosensitivities (Harada et al., 2003) similar to that found forthe activation of chloroplast accumulation movement, in which phot2 requires ahigher light threshold for activity than phot1 (Kagawa and Wada, 2000; Sakai et al.,2001). These observations thus provide a tenuous link between calcium and pho-totropin-mediated chloroplast movements in Arabidopsis.

Although studies from several plant species provide evidence for an involvement ofCa2+ in eliciting chloroplast movements (Wada et al., 2003), none of the above find-ings conclusively demonstrate a role for Ca2+ in phototropin-induced chloroplastmovement in Arabidopsis. Indeed, such blue light-induced Ca2+ increases are not on-ly restricted to leaves but are also found in hypocotyls (Babourina et al., 2002; Stoel-


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zle et al., 2003), indicating that Ca2+ may act as an intracellular messenger in otherphototropin-mediated responses. Research is now required to establish how the bluelight-induced Ca2+ f luxes described above are connected to specific phototropin-me-diated responses. Nonetheless, a recent study by Folta et al. (2003) indicates that theearly transient rise in [Ca2+]cyt is associated with at least one, but not all phot1-medi-ated responses in Arabidopsis. These studies are described in the following section.

13.5.5Rapid Inhibition of Hypocotyl Growth by Blue Light

Transfer of dark-grown seedlings to blue light causes an inhibition of hypocotylgrowth. This growth inhibition response can be separated into two distinct phases: arapid, transient response occurring within a few minutes after the onset of blue lighttreatment and a slow response that continues for many hours (Spalding, 2000; Parkset al., 2001). The phot1 single mutant has been shown to lack the rapid growth-inhi-bition response (Folta and Spalding, 2001). Evidently, phot2 plays no role in the rap-id inhibition of hypocotyl growth as phot1 mutants completely lack this response (Fol-ta and Spalding, 2001). These findings are consistent with the expression pattern ofPHOT2, whereby transcripts are not abundant in dark-grown seedlings but increaseupon illumination (Jarillo et al., 2001b; Kagawa et al., 2001; Tepperman et al., 2001).By contrast, the slow growth inhibition response is mediated by the cryptochromes,a second family of blue light receptors in plants (Lin and Shalitin, 2003; seeBatschauer, Chapter 10; Cashmore, Chapter 11).

Using the Ca2+-specific chelator BAPTA Folta et al. (2003) were able to demonstratethat the rapid increase in [Ca2+]cyt levels in response to blue light treatment is associ-ated with phot1-mediated inhibition of hypocotyl growth. Equivalent concentrationsof BAPTA were found to be effective in preventing both the rise in [Ca2+]cyt and phot1-mediated hypocotyl growth inhibition by blue light. More importantly, the samechelator treatment did not impair phot1-mediated phototropism in Arabidopsis, indi-cating that the rise in [Ca2+]cyt is a signaling step in the transduction process linkingphot1 activation to hypocotyl growth inhibition and not phototropism. The signalingpathway for phot1-mediated inhibition of hypocotyl growth also differs from that forphot1-mediated phototropism in that it does not require the phot1-interacting pro-tein, NPH3 (Folta and Spalding, 2001). Once again, these findings suggest that thesignal transduction pathways for these two phototropin-mediated responses are dis-tinct. NPH3, however, is not only involved in phototropism as this protein, in addi-tion to phot1, is required for the blue light-dependent destabilization of Lhcb and rcbStranscripts in Arabidopsis (Folta and Kaufman, 2003). It now remains to be seenwhether other phototropin-mediated responses such as light-induced chloroplastmovement have a strong dependence on rapid changes in [Ca2+]cyt.


300

13.6Future Prospects

Although exciting progress has been made in the last few years since the discovery ofthe phototropins, most of this progress has concerned the photochemical and struc-tural properties of the phototropin LOV domains. Much work remains to be done toimprove our understanding of how these photoreceptors function to bring about a di-verse range of responses. More information is required to understand how these bluelight-activated kinases initiate signaling and to identify the cellular events down-stream of phototropin activation. At present, it is still unknown why the phototropinscontain two LOV domains with different photochemical properties, and how thesedifferences relate to their physiological functions. Moreover, in contrast to phot1, verylittle is known regarding the subcellular distribution of phot2. Clearly, there are manyissues still to be addressed.

Even so, it is now apparent through the study of phototropin-deficient mutants thatthese blue light receptors not only function to regulate phototropism, after which theywere first named, but activate a wide range of blue light responses. More extraordi-nary, and by some weird twist of fate, is the recent discovery that the LOV domainsare actually involved in love; phototropin from Chlamydomonas has been shown toserve as the photoreceptor mediating various steps in the cycle for sexual reproduc-tion (Huang and Beck, 2003). Perhaps further analysis of phototropin-deficient mu-tants will uncover other exciting roles for these blue light receptors. Mutants lackingphot1 and phot2 have been reported to exhibit residual phototropism under highlight intensities of unilateral blue light (Sakai et al., 2001). Similarly, Talbott et al.(2003) have recently shown that the phot1phot2 double mutant still exhibits blue light-induced stomatal opening under certain light conditions. Whether these residualphotoresponses can be attributed to a leaky phot2 mutation or to cryptochrome orphytochrome action or some other novel photoreceptor system awaits further inves-tigation.

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Handbook of Photosensory Receptors (BRIGGS:PHOTORECEPTORS O-BK) || Blue Light Sensing and Signaling by the Phototropins