Intracellular trafficking of photoreceptors during lightinduced signal transduction in plants

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1 COMMENTARY 475 Intracellular trafficking of photoreceptors during lightinduced signal transduction in plants Ferenc Nagy 1,2, Stefan Kircher 3 and Eberhard Schäfer 3, * 1 Plant Biology Institute, Biological Research Centre, H-6701 Szeged, PO Box 521, Hungary 2 Agricultural Biotechnology Centre, H-2101 Godollo, PO Box 411, Hungary 3 Institut für Biologie II/Botanik, Universität Freiburg, Schänzlestrasse 1, D Freiburg, Germany *Author for correspondence Journal of Cell Science 114, The Company of Biologists Ltd Summary Plants monitor changes in the ambient light environment by highly specialised photoreceptors, which include the red/far-red photoreversible phytochromes, the blue-lightabsorbing cryptochromes and phototropin and the so-farunidentified UVB photoreceptor(s). Light easily penetrates plant organs/tissues and reaches even the subcellular compartments of various cell types. Therefore, it is not surprising that the determination of the intracellular localisation of photoreceptors has been, for many years, a major, and often controversial, subject of plant photobiology and cell biology research. Phototropin, one of the blue-light photoreceptors of higher plants, controls phototropism by monitoring the direction of light, and it is localised in or at the plasmalemma. In contrast, the subcellular localisation of phytochromes changes dynamically and exhibits a very complex pattern. These photoreceptors are localised in the cytosol in darkgrown tissues. Irradiation, however, induces import of phytochromes into the nucleus. The import occurs in a light-quality- and light-quantity-dependent fashion and, as such, seems to be unique to higher plants. Light-induced accumulation of phytochromes in the nuclei correlates well with various physiological responses mediated by these photoreceptors. These observations indicate that lightdependent intracellular redistribution of phytochrome photoreceptors is one of the major regulatory steps in photomorphogenesis. Key words: Phytochrome, Nuclear import, Signalling Introduction Plants are sessile organisms; therefore optimal adaptation to changes in the natural environment is an essential strategy. Light is a dominant but broadly variable environmental factor. It is not only an energy source but also an essential signal for adaptation and regulation of plant growth and development. The most dramatic developmental switch occurs at the point of transition from skotomorphogenesis (development in darkness) to photomorphogenesis (development in light). Light-mediated signal transduction starts with the absorption of light by specialised photoreceptors. Several classes of photoreceptors that monitor the quality, quantity and the temporal and spacial pattern of light have evolved: the UVB photoreceptors (as yet unidentified), red/far-red photoreversible phytochromes (Clack et al., 1994), and the blue-light-absorbing proteins cryptochromes (Cashmore et al., 1999) and phototropin (Christie et al., 1998; Christie et al., 1999). Being an electromagnetic wave, light can easily penetrate plant tissues. Red and far-red light can even pass through compact organs, such as stems or leaves, and activate phytochromes deep within cells. Physiological experiments, in vivo spectroscopy, immunocytochemical studies and ubiquitous activities of phytochrome promoters in transgenic plants indicate that phytochromes are present in all tissue types examined. Therefore, each cell is likely to contain at least one type of phytochrome at any stage of development. Until 1999, however, the intracellular localisation of most of these photoreceptors, including phytochromes, remained largely obscure. In the case of phytochromes, a controversial debate about their exact intracellular localisation erupted nearly 20 years ago and has lasted until very recently. Phytochromes and cryptochromes were generally thought to be cytosolic, and this view became even more prevalent after the first sequence information became available. Phototropin was thought to be an exception and localised in/at the plasmalemma. Phototropin Darwin s pioneering work revealed that phototropism of plants is mediated by a blue-light-absorbing pigment; however, the photoreceptor involved, phototropin, was identified only a few years ago. Physiological and biochemical data showed a strong correlation between phototropism and a blue-light-mediated phosphorylation of a 120 kda protein associated with plasma membranes (Short and Briggs, 1994). Using a genetic approach, it has been shown (Huala et al., 1997) that the nonphototropic hypocotyl locus (NPH1) encodes a serine/ threonine kinase that contains two N-terminal domains characteristic of proteins mediating light-, oxygen- and voltage-dependent responses (LOV domains). Expression of NPH1 in baculovirus allowed the authors to demonstrate binding of flavin mononucleotide (FMN) to the LOV domains and blue-light-mediated autophosphorylation of the photoreceptor (Christie et al., 1998; Christie et al., 1999). However, it remains to be seen whether phototropin is a

2 476 JOURNAL OF CELL SCIENCE 114 (3) A) PHYA synthesis light VLFR signaling P r P fr HIR gene regulation photomorphogenesis destruction B) PHYB synthesis light LFR signaling gene P r P fr cr regulation photomorphogenesis dark reversion Fig. 1. Modes of action of (A) PHYA and (B) PHYB. Red-light-absorbing (Pr) and far-red-light-absorbing forms (Pfr) of PHYA and PHYB are shown. Signal transduction competes with proteolytic degradation of Pfr (PHYA) or dark reversion of Pfr back to Pr (PHYB). Four different modes of action leading to regulation of gene expression can be distinguished: (1) the very-low-fluence response (VLFR), a response that is saturated at about 1% of Pfr and involves PHYA; (2) the high irradiation response (HIR), a response to continuous irradiation with maximal responsiveness in far-red (~720 nm) light that also involves PHYA; (3) the low fluence response (LFR), a response to red light pulses that can be reverted by far-red light and involves PHYB; and (4) the continuous red light response (cr), a response that can not be induced by red light pulses (not discussed in this review) and involves PHYB. membrane-associated protein, as was inferred by the early biochemical studies. Jarillo et al. recently identified a new NPH1-like protein, NPL1 (Jarillo et al., 1998), that shows significant structural similarity to NPH1. We speculate that NPL1 and NPH1 together are probably the most important photoreceptors controlling phototropism in higher plants. Cryptochromes Ahmad and Cashmore (Ahmad and Cashmore, 1993) cloned the HY4 gene, identifying the first member of the long-sought blue-light-absorbing photoreceptor family. HY4 encodes the cryptochrome 1 (CRY1) photoreceptor. CRY1 displays striking similarity to photolyases; however, it has a unique C-terminal extension and lacks photolyase activity. FADH is its catalytically active chromophore; the second chromophore could be either a pterin or a deazoflavin (Malhotra et al., 1995). The second member of this subclass of blue-light photoreceptor, CRY2, has also been identified and contains a different C-terminal extension (Lin et al., 1998). Results of biochemical studies employing CRY1- overexpressor lines were interpreted as indicating that this photoreceptor is cytosolic (Lin et al., 1996a; Lin et al., 1996b). In contrast, experiments on transiently transformed plant cells expressing the CRY1-GFP fusion protein indicated that CRY1 is localised in the nucleus in the dark (Cashmore et al., 1999). Moreover, analysis of the cellular distribution of the CRY2-GUS or CRY2-GFP fusion proteins in transgenic plants showed that the CRY2 photoreceptor is constitutively localised in the nucleus (Kleiner et al., 1999; Guo et al., 1999). A more complex picture is emerging in lower plants, especially in fern gametophytes. Microbeam irradiation indicates that fern homologues of higher-plant CRY photoreceptors are present in the cytosol but also associated with the nucleus (Wada and Sugai, 1994). Recent studies showed that at least two members of the CRY family in Adiantum capillus-veneris are indeed localised in the nucleus and that the nucleo/cytoplasmic distribution of these receptors is not influenced by light (Imaizumi et al., 2000). Phytochromes In Arabidopsis, phytochromes are encoded by five genes (PHYA-PHYE) (Clack et al., 1994). PHYA (and probably all phytochrome types) binds an open chain tetrapyrol (phytochromobilin), which is covalently linked by a thioetherlinkage to the apoprotein, as a functional chromophore (Lagarias et al., 1980; Kunkel et al., 1995; Kunkel et al., 1996). In general, the photosensory function of phytochromes is based on their capacity to perform reversible interconversion between the red-light-absorbing Pr form and the far-red-light-absorbing Pfr form following sequential absorption of red and far-red light. However, the different phytochromes are not only encoded by different genes but also have different modes of action (Fig. 1). PHYA is the most specialised phytochrome. It is responsible for the very low fluence rate response (VLFR) and the high

3 Intracellular redistribution of phytochromes 477 irradiance response (HIR) (Furuya and Schäfer, 1996). The extraordinary responsiveness of PHYA allows it to control germination of seeds buried in the soil and to induce germination when seeds are exposed to star light (Hartmann et al., 1998). The other phytochromes control, to different extents, the classical red/far-red reversible induction, the so-called lowfluence-rate responses (LFRs) and responses to continuous redlight (Furuya and Schäfer, 1996). All phytochromes are synthesised in the biologically inactive, red-light-absorbing Pr forms. The half-life of the physiologically active form of PHYA is extremely short, and proteolytic degradation of the Pfr form is believed to be responsible for termination of signalling. In the case of the light-stable phytochromes, especially PHYB, termination of signalling is not yet understood. Regulation of the dark reversion of the Pfr to the Pr form is thought to be the most likely mechanism, since proteolytic degradation of the Pfr form of PHYB clearly cannot play a major role (Fig. 1). Computational analysis of phytochrome sequences and structures from a variety of species, including ferns, mosses and algae, indicates that they are cytosolic: no motif, domain or even secondary structure that could facilitate insertion into or association with any type of membrane has been identified. This contrasts with in vivo observations of lower plants, in which microbeam irradiation has revealed a very localised response and an action dichroism for phytochrome-mediated growth responses and chloroplast re-orientation (Kraml, 1994). These observations suggest that phytochromes associate, at least in lower plants, with the plasma membrane and that this probably involves as-yet-unknown protein-protein interactions. In higher plants, early immunocytochemical analysis of the subcellular distribution of PHYA demonstrated only cytosolic localisation of the photoreceptor. Therefore, until recently, in the absence of any other indication, phytochromes of higher plants were thought to be localised in the cytosol and/or associated with cell membranes. protein is localised in the cytosol in the dark and that accumulation of the tagged photoreceptor in the nucleus is a light-driven process. Nuclear translocation is induced by repeated pulses of or continuous red light and reversed by farred light. After light treatment, PHYB-GFP is detectable mainly in the form of speckles, and these speckles are present only in nuclei (Fig. 2). Upon transfer to dark, the structures gradually dissolve, and nuclei become diffusely stained and finally free of any detectable fluorescence, which indicates that the PHYB-GFP fusion protein has disappeared. Intracellular localization of PHYA in light and darkness PHYA is a highly specialised photoreceptor that has physiology and biochemistry markedly different from those of other phytochromes. Therefore, it was expected that the lightdependent intracellular redistribution of PHYA, if it occurs and plays a role in PHYA-mediated signalling, would exhibit characteristics quite different from those of PHYB. It has been demonstrated (Kircher et al., 1999; Kim et al., 2000) that lightdependent intracellular redistribution of PHYA indeed takes place, and PHYA, similarly to PHYB, is imported into the nucleus. These studies clearly established that the PHYA-GFP fusion protein is a functional photoreceptor. In darkness it is localised exclusively in the cytosol, and light induces its translocation to the nucleus (Fig. 2). The kinetic, light-quality and -quantity requirements of the redistribution of PHYA are sharply different from those of PHYB. Import of the PHYA-GFP fusion protein is an order of Intracellular localization of PHYB in light and darkness PHYB is the most ancient type of phytochrome present in higher plants. Given the observations outlined above, it was therefore fairly surprising when, in 1996, Sakamoto and Nagatani reported the enrichment of PHYB in nuclear fractions isolated from light-grown trabidopsis seedlings (Sakamoto and Nagatani, 1996). They also showed that a fusion protein consisting of the C-terminal part of the A. thaliana PHYB fused to GUS is constitutively localised to nuclei in transgenic plants. Despite the potential importance of these data, they were largely ignored. However, recent studies by Nagatani and co-workers (Yamaguchi et al., 1999) and ourselves (Kircher et al., 1999), which showed that chimeric proteins consisting of full-length PHYB fused to GFP can complement PHYB mutations, substantiated the earlier findings. These later reports showed convincingly that the PHYB-GFP fusion protein is a biologically active photoreceptor, that the PHYB contains a functional NLS and that the NLS motif is localised in the C-terminal part of the photoreceptor. More importantly, these reports documented unambiguously that the PHYB-GFP fusion Fig. 2. Localisation of PHYA-GFP (a and b) and PHYB-GFP fusion proteins (c and d) in Arabidopsis seedlings grown for 7 days darkness and kept in darkness (a and c) irradiated for 24 hours with far-red light (b) or irradiated for 6 hours with red light (d), before analysis by epifluorescence microscopy. Nuclei (nu) and selected plastids (pl) are indicated.

4 478 JOURNAL OF CELL SCIENCE 114 (3) FR/R FHY1 SPA1 FAR1 EID1 PHYA pr PHYA pfr R Cytosol Nucleus PHYA pfr PIFX LRE 1 RED1 mrna photomorphogenesis PHYB pr PHYB pfr PHYB pfr PIF3 LRE 2 mrna Fig. 3. A model depicting molecular components and cellular processes involved in PHYA- and PHYB-mediated phototransduction in higher plants. PHYA pr and PHYB pr represent red-light-absorbing forms of phytochrome-a and phytochrome-b, respectively. PHYA pfr and PHYB pfr represent far-red-light-absorbing forms of phytochrome A and phytochrome B, respectively. FHY1, SPA1, FAR1 and EID1 are shown to effect PHYA-mediated signalling; RED1 is known to modulate PHYB-mediated signalling; PIF3 binds to different LREs (cis-acting light regulatory elements) required for light-induced transcription of target genes and interacts selectively with the pfr form of PHYB. PIFX is a postulated nuclear protein that is involved in PHYA-mediated phototransduction. magnitude faster, easily detectable within a minute and induced by pulses of or continuous far-red light. Although PHYA-GFP imported into the nucleus forms speckles, these speckles are smaller and more numerous when compared with those formed by PHYB-GFP. In addition, the speckles are not restricted to nuclei: they are easily detectable in the cytosol, and formation of these structures in the cytosol precedes their appearance in the nucleus. The PHYA GFP-containing speckles formed in the cytosol are reminiscent of sequestered areas of phytochrome (SAPs) thought to represent intermediates or products of ubiquitin-mediated proteolytic degradation of PHYA (Speth et al., 1986). Intracellular localization of PHYC-PHYE To analyse intracellular localisation of the other three members of the phytochrome photoreceptor family, we chose an approach similar to that used for PHYA and PHYB, analysing the nucleo/cytoplasmic distribution of PHYC-GFP, PHYD- GFP and PHYE-GFP fusion proteins in transgenic tobacco and Arabidopsis seedlings. Interpretation of the emerging data seems to be rather difficult for several reasons. First, in sharp contrast to PHYA-GFP and PHYB-GFP, the PHYC-GFP, PHYD-GFP and PHYE-GFP fusion proteins are always detectable in the nuclei in dark-grown seedlings in all lines tested so far. Import of these proteins into nuclei might therefore be light independent and constitutive. Second, in etiolated seedlings, staining of the nuclei is always diffuse: speckles do not form. Finally, red-light treatment induces formation of speckles in the nuclei (which can be reversed by far-red light) but does not significantly affect the diffuse fluorescence. The kinetics of the appearance/disappearance of these speckles is, however, comparable to that of PHYB GFPcontaining speckles. These results might indicate that PHYC- PHYE, in contrast to PHYA and PHYB, can be imported into the nucleus in their biologically inactive Pr (dark) form, and yet the speckle formation is dependent on the presence of the Pfr form (Kircher et al., unpublished data). Why might the regulation of PHYC-PHYE import into the nucleus be so different? Is the import of these photoreceptors indeed regulated differentially or is there an active retention mechanism that ensures efficient cytosolic retention of PHYA and PHYB but not that of PHYC-PHYE in the dark? If so, what is the biological function of PHYC-PHYE accumulated in nuclei in the dark, and why is speckle formation a strictly lightinduced phenomenon for all phytochromes? Conclusions and perspectives The localisation of photoreceptors in algae, ferns and mosses is still not clear, but we expect that a specific mechanism to recruit photoreceptors to the plasma membrane exists.

5 Intracellular redistribution of phytochromes 479 Whether this is completely lost in higher plants or whether there are still traces of this type of regulation mediating some of the known light-dependent membrane responses (such as regulation of ion channels in guard cells and pulvini) remains to be elucidated. In higher plants, all photoreceptors, except NPH1-like receptors, are imported into nuclei, and their nucleo/ cytoplasmic partitioning is regulated by light, albeit to different degrees. It is well documented that light, in a quality- and quantity-dependent fashion, induces transport of PHYA-GFP and PHYB-GFP into nuclei and that the import is accompanied by spot formation within the nucleus. What is the biological relevance, if any, of these speckles? Recent experiments show that PHYB and CRY2 physically interact in these spots (Más et al., 2000). In addition, Quail and co-workers have provided compelling evidence that PHYB interacts in vitro with a variety of nuclear proteins, mainly transcription factors. These interactions are light reversible, specific to the physiologically active Pfr form of PHYB (Ni et al., 1998; Ni et al., 1999), and one of these interacting proteins, PIF3, an HLH-type factor, binds to the promoters of several but not all light-regulated genes tested (Martinez-Garcia et al., 2000). Taken together these results indicate that, at least for some PHYB-mediated responses, a very short signal transduction chain can be envisaged: light absorption leads to Pfr formation, which promotes import of the protein into the nucleus and interaction with promoter-bound transcription factors. Do the nuclear speckles whose appearance is strictly light induced and which are quite different for the different phytochromes, by analogy with PHYB-CRY2, reflect different functional complexes that contain, for example, PHYB-PIF3? We were currently trying to answer these questions. After light-to-dark transitions, the phytochrome-containing speckles dissolve, producing a diffuse staining that is followed by a complete loss of fluorescence in the nucleus. Is this due to degradation even for the stable phytochromes PHYB- PHYE or to export into the cytosol? Notwithstanding these and many more questions, it is safe to say that light-qualityand light-quantity-dependent import of the different phytochromes into the nuclei is a key regulatory step in controlling the transition from skoto- to photo-morphogenesis (Fig. 3). Such a conclusion is further supported by the fact that the specific functions of the different phytochromes clearly manifest themselves at the level of light-dependent import into the nuclei. The wavelength- and fluence-rate dependence of PHYA and PHYB nuclear import correlates well with the established physiological functions of these receptors. PHYA regulates VLFR and far-red HIR, and nuclear import of PHYA is also induced by VLFR and far-red high irradiance (Kim et al., 2000). Likewise, PHYB mediates the low fluence rate response (LFR), and nuclear import of PHYB is also induced by LFR (Gil et al., 2000). It follows that, in far-red light, only import of PHYA into the nuclei is observed, whereas in red light the appearance of PHYA in the nuclei is only transient; thus, PHYB cannot be a physiological far-red-light-activated photoreceptor, and PHYA cannot be a red-light-activated photoreceptor. The work in Freiburg was supported by funding from the Graduiertenkolleg, SFB 388, Human Frontier Science Programme to E.S and a Humboldt Stiftung Award to F.N. Work in Hungary was supported a Howard Hughes International Scholarship ( ) and by Human Frontier Science Programme and OTKA T grants to F.N. References Ahmad, M. and Cashmore, A. R. (1993). 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