The Plant Cell, Vol. 9, 1225-1234, July 1997 O 1997 American Society of Plant Physiologists

Light Modulation of Vegetative Development

Joanne Choryl Plant Biology Laboratory, Salk lnstitute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037

INTRODUCTION

Plants sense many aspects of light in their environment, in- cluding its wavelength, duration, intensity, and direction. This information is used to optimize growth for the ambient light environment, thereby allowing the plant to function as an efficient photosynthetic machine throughout develop- ment. Light has particularly dramatic effects on the morpho- genesis of seedlings during the transition from heterotrophic (life under the ground) to photoautotrophic growth. During seedling development, light stimulates leaf and chloroplast differentiation, inhibits the rate of hypocotyl growth, and in- duces the expression of a large battery of nuclear- and chlo- roplast-encoded genes. Later in vegetative development, light sensing allows plants to properly time the transition to reproductive growth.

The light-dependent development of plants is a complex process involving the combined action of several photore- ceptor systems. These include the red/far-red light-absorb- ing phytochromes (Quail et al., 1995), the blue/UV-A light- absorbing cryptochromes (Ahmad and Cashmore, 1996), and distinct UV-A (Young et al., 1992) and UV-B (Beggs and Wellman, 1985; Christie and Jenkins, 1996) light photore- ceptors, about which little is known.

Plants use these photoreceptors to accurately sense and respond to light intensities that vary over seven to eight or- ders of magnitude (Kendrick and Kronenberg, 1994). For in- stance, some light responses, such as the induction of nuclear-encoded genes encoding the light-harvesting chlo- rophyll proteins of photosystem II (LHCB), can be initiated by fluences as low as 0.1 nmol m-2. These are classified as very-low-fluence (VLF) responses. Other responses (e.g., let- tuce seed germination), which cannot be initiated until the fluences reach 1 mmol m-2, are referred to as low-fluence (LF) responses. Lastly, responses such as stem growth inhi- bition and floral induction, which are elicited by prolonged or continuous irradiation by fluences >10 mmol m-2, are known as high-irradiation reactions (HIR). Phytochromes are responsible for VLF, LF, and HIR responses to red and far- red light (Mancinelli and Rabino, 1978; Kaufman et al., 1984). There are also distinct LF and high-fluence detection systems that mediate blue light responses (Warpeha and Kaufman, 1990).

1 E-mail chory@salk.edu; fax 61 9-558-6379.

The mechanism by which phytochromes and crypto- chromes regulate plant responses to these diverse light conditions is an area of intensive study. However, despite much work, no definitive evidence for the biochemical mechanism of action of either phytochrome or crypto- chrome is currently available. Nevertheless, molecular ge- netic studies using cloned genes and mutants have begun to elucidate the structure and function of individual photore- ceptors. Recently, analyses of Arabidopsis mutants have fa- cilitated the assignment of specific functions, such as the regulation of seed germination and plant development and the sensing of light of different intensities and wavelengths, to specific phytochromes and cryptochromes. Attempts to understand phytochrome function have focused on investi- gating its structure, and several domains of the phyto- chrome apoprotein have been implicated in its biological activity.

This review concentrates on recent advances in probing phytochrome and cryptochrome structure and function. Other reviews on the genetics and biochemistry of phyto- chrome and cryptochrome signal transduction have been published recently, and I refer the reader to these for addi- tional points of view (Terry et al., 1993; Vierstra, 1993; Jones and Edgerton, 1994; Quail et al., 1995; Ahmad and Cashmore, 1996; von Arnim and Deng, 1996).

PHYTOCHROME STRUCTURE

Phytochromes are soluble pigmented proteins of -1 25 kD. The prototypical phytochrome is a homodimer, each subunit of which contains a covalently linked linear tetrapyrrole chromophore, as shown in Figures 1 C to 1 E (Vierstra, 1993; Jones and Edgerton, 1994). The traditional view of phyto- chrome is that it mediates responses to red and far-red light through its ability to photointerconvert between two stable isomers, a red light-absorbing form, termed Pr (A,,, = 660 nm) and a far-red light-absorbing form termed Pfr (A,,, = 730 nm) (Figures 1A and 1B; Kendrick and Kronenberg, 1994). On the basis of physiological, genetic, and biochemi- cal studies, Pfr is thought to be the active form, although Pr may play a role in seed germination in some light conditions (Reed et al., 1994; Shinomura et al., 1994).

1226 The Plant Cell

red light A responses

0.8

0.6

8

5: m e 0.4

n 4

0.2

C

Wavelength (nm)

N C

1ow ao - 2w 400 spectral inteqrity dimerization - .

His kinase homology - 4

regions required for biological activity

-H is-Leu-Gln-Tyr HOzC COzH / \

D Leu-Arg-Ala-Pro-His-Ser-Cys-His-Leu-Gln-Tyr

H H H

E

c c Figure 1. Summary of Current Knowledge Regarding Phytochrome

All higher plants examined, as well as many lower plants and algae, have multiple genes for phytochromes (Pratt, 1995; Quail et al., 1995). For example, in Arabidopsis, the apoprotein component of phytochrome is encoded by five genes, termed PHYA through PHYE (Sharrock and Quail, 1989; Clack et al., 1994). PHYA is a light-labile phyto- chrome, that is, it is proteolyzed after photoconversion to its Pfr form (Somers et al., 1991). By contrast, PHYB and PHYC appear to be light-stable phytochromes (Somers et al., 1991).

The higher plant phytochrome molecule can be divided roughly into two globular domains (Figures 1C and 1E; re- viewed in Jones and Edgerton, 1994; Quail et al., 1995). A highly conserved N-terminal domain of -600 amino acids binds the chromophore and retains the ability of native phy- tochrome to photoconvert between Pr and Pfr. The less well conserved C terminus is involved in the homodimerization of two monomers and the transduction of the light signal (Edgerton and Jones, 1992, 1993; Cherry et al., 1993). Elec- tron microscopy of pure phytochrome homodimers has re- vealed a tripartite arrangement comprising two globular N-terminal domains that are tethered to a central core com- posed of the two C-terminal domains (Figure 1 E; reviewed in Jones and Edgerton, 1994). These domains are separated by a protease-sensitive hinge region.

As shown in Figure lC, molecular genetic analyses have further subdivided the functional domains of phytochrome (reviewed in Vierstra, 1993; Elich and Chory, 1994; Jones and Edgerton, 1994; Quail et al., 1995). Two types of studies have been performed on the photochemically active N-ter- mina1 chromophore binding domain-overexpression in transgenic plants, and in vitro assembly of truncated or mu- tant recombinant phytochromes in yeast (Deforce et ai., 1991 ; Wahleithner et al., 1991 ; Cherry et al., 1993; Kunkel et al., 1993; Hill et al., 1994; Gartner et al., 1996). Earlier stud- ies had established that Cys-322 of oat PHYA was responsi- ble for chromophore binding and that the 70-kD N-terminal

(A) The two spectrally photointerconvertible forms of phytochrome, Pr and Pfr. Pfr has been correlated with induction of developmental responses. (B) The absorption spectrum of Pr and Pfr. Spectra are from purified oat PHYA (adapted from Kendrick and Kronenberg, 1994). (C) A prototypical phytochrome apoprotein. The figure delineates the sequences that mediate photosensory activity and those that are required for biological activity. The PAS repeats are indicated as P1 and P2. Amino acid numbering is based on Arabidopsis PHYB (adapted from Vierstra, 1993). Gray indicates the N-terminal domain, black the hinge region, and stippled the C-terminal domain. C, C terminus; N, N terminus. (D) Structure of the linear tetrapyrrole chromophore of a phyto- chrome. The image shows the thioether linkage that connects the chromophore to the Cys at amino acid position -320 (322 of oat PHYA). (E) The tripartite structure of a phytochrome homodimer. The N and C termini of each monomer are indicated (adapted from Jones and

Structure and Function. Edgerton, 1994).

Photoreceptors Regulate Development 1227

region was sufficient for photosensory function (Lagarias and Rapoport, 1980; Boylan et al., 1994). Transgenic plant and recombinant expression experiments have refined these original conclusions and established that the 40-kD region flanking Cys-322 is sufficient for chromophore attachment and photoreversibility (Cherry et al., 1993; Gartner et al., 1996). This work also showed that there is a blue shift of the Pfr absorption maximum and increased nonphotochemical reversion of Pfr to Pr when a 6- to 10-kD region at the N ter- minus is removed, suggesting that this region may be in- volved in establishing the proper chromophore-protein environment (Cherry et al., 1992).

The ability of overexpressed deletion derivatives of phyto- chrome to enhance hypocotyl growth inhibition has been used as an assay for phytochrome function (Figure 1C). This assay has implicated both the N and C termini of phyto- chrome in signaling (Çherry et al., 1992, 1993; Boylan et al., 1994; Emmler et al., 1995). Deletion analysis of oat PHYA suggests that the N-terminal 52 amino acids are required for far-red light signaling in Arabidopsis but that they play no ap- parent role in white or red light signaling (Boylan et al., 1994). Analagous studies in tobacco, however, suggest that this re- gion is also required for signaling in white light (Cherry et al., 1992). It is not clear how to resolve this discrepancy, although species differences may account for the contradictory results.

Additional evidence for the functional importance of the phytochrome N terminus comes from experiments showing that multiple Ser-to-Ala substitutions at the N terminus of PHYA or deletion of this region results in enhanced activity in the hypocotyl growth inhibition assay (Stockhaus et al., 1992; Jordan et al., 1996). Although the roles of these Ser residues in the biological activity of phytochrome are not known, it has been established that this Ser-rich region is phosphorylated by an endogenous kinase activity that copurifies with PHYA (Wong et al., 1986). One attractive hy- pothesis is that the Ser residues are involved in the desensi- tization of stimulated phytochrome. Thus, mutating or deleting them would result in a phytochrome whose signal could not be dampened.

In addition to the N-terminal 100 amino acids, the 35 resi- dues at the C terminus of oat PHYA are required for its ac- tivity in transgenic plants (Cherry et al., 1993), although deletion of these 35 residues has no effect on spectral prop- erties or dimerization. Furthermore, a region at the N-termi- na1 end of the C-terminal domain of PHYA (amino acids 61 7 to 686) appears to be important in phytochrome’s regulatory function because deletion of this region results in a photoac- tive dimer with no biological activity in the transgenic plant assay (Boylan et al., 1994).

The enhanced hypocotyl growth inhibition assay has also been used to analyze functional domains in PHYB (Wagner et al., 1996b). As was found in the PHYA studies, both N- and C-terminal domains of PHYB are required for its function. One surprising result from the PHYB studies was that over- expression of full-length Arabidopsis PHYB as well as either the N- or C-terminal domain of PHYB interferes with endog-

enous PHYA activity in LF far-red light (Wagner et al., 1996b). No interference was seen in red light, suggesting that overexpression of these domains does not interfere with PHYB activity. That PfrB is responsible for this interference was shown by overexpressing a mutated phyB (Cys-357-+ Ser), which cannot bind the chromophore. This phyB does not interfere with PHYA function (Wagner et al., 199613).

Some of these results can be explained by the observa- tion that the leve1 of the phytochrome chromophore is limit- ing to endogenous PHYA in the overexpressing lines. However, overexpression of the C terminus does not affect chromophore availability, nor does this deletion derivative heterodimerize inappropriately with PHYA. As such, Wagner et al. (1996b) favor the alternative possibility that overex- pression of PHYB interferes with endogenous PHYA activity because of competition for a common signal transduction component.

Further support for this idea comes from the analysis of transgenic Arabidopsis plants containing reciproca1 N- and C-terminal domain swaps of rice PHYB with oat PHYA, in which it was shown that the C-terminal halves of PHYA and PHYB are reciprocally interchangeable (Wagner et al., 1996a). Although these experiments are subject to the ca- veat that heterologous phytochromes were used in the ex- change, this result is intriguing enough to be examined in greater detail, for example, by using homologous phyto- chromes in the appropriate mutant background.

Extensive sequence analysis of >20 phyA and phyB ethyl methanesulfonate-induced point mutations has identified a region of -1 60 amino acids (residues 680 to 840) that appears to be involved in phytochrome signaling (Reed et al., 1994; Quail et al., 1995; Wagner and Quail, 1995; Xu et al., 1995; Bradley et al., 1996). This region overlaps with one of those whose importance was implied in the deletion experiments discussed above. The mutant proteins result in plants with elongated hypocotyls, although in each case the phyto- chrome apoprotein accumulates to normal levels and ho- modimerizes. In addition, some of the mutant proteins can bind the chromophore and also exhibit normal photoreversibility.

Recently, Lagarias et al. (1995) pointed out that this region of phytochrome contains two direct repeats that are also found in the photoactive yellow protein of the purple bacte- rium Ectothiorhodospira halophila (Baca et al., 1994; Borgstahl et al., 1995), and related sequences have been described in some members of the bacterial sensor kinase family. This motif also shares some similarity with the Drosphila period and single-minded loci and a subunit of the aromatic hydro- carbon receptor encoded by the arnt locus (Crews et al., 1988; Hoffman et al., 1991 ; Baylies et al., 1992; Huang et al., 1993). This domain, known as PAS (for per, grnt, and gim; Figure 1 C), is involved in protein-protein interactions; it may be of consequence that a similar sequence has been identi- fied in a region of phytochrome that is predicted to be in- volved in signaling.

Potential insight into the evolutionary origin of phyto- chrome has recently become available with the discovery of

1228 The Plant Cell

a chromatic adaptation regulator from the cyanobacterium Fremyella diplosiphon that exhibits sequence similarity to both the N- and C-terminal domains of higher plant phyto- chromes (Kehoe and Grossman, 1996). A gene with even higher identity to phytochrome has been found through the Synechocystis sp PCC6803 genome sequencing project, which is being performed by the Kazusa DNA Research In- stitute (Kazusa, Japan; sequence data and open reading frame [ORF] identification are available from their web site at http:www. kazusa.or.jp/cyano/cyano.html). The genome of this cyanobacterium contains at least one ORF whose N terminus exhibits -30% amino acid identity to the N-termi- na1 chromophore binding domain of plant phytochromes. Furthermore, this ORF also contains an -25Gamino acid C-terminal domain that shows strong similarity to bacterial his- tidine kinases (Kehoe and Grossman, 1996). Very recently, it has been shown that recombinant PCC6803 “phytochrome” can attach the chromophore and has similar spectral prop- erties to the higher plant phytochromes (Hughes et al., 1997; K.-C. Yeh and J.C. Lagarias, personal communication). More- over, the purified protein can autophosphorylate as well as phosphorylate a protein that is encoded in the same operon (K.-C. Yeh and J.C. Lagarias, personal communication).

These data have broad implications for the mechanism by which phytochrome transmits its signal. Plant phytochromes also possess a C-terminal domain with similarity to histidine kinases that has been implicated in biological activity (Schneider-Poetsch and Braun, 1991 ; Schneider-Poetsch, 1992). However, unlike the cyanobacterial homolog, this domain in plant phytochromes is separated from the chro- mophore binding domain by the region including the PAS- like repeats mentioned above. Thus, although we remain hopeful that work on cyanobacteria will lead to insights into phytochrome action, it seems likely that eukaryotic phyto- chromes have evolved unique mechanisms that are not shared with their prokaryotic counterparts.

PHYTOCHROME FUNCTIONS DURING DEVELOPMENT

Throughout the life cycle of a plant, the phytochrome photo- receptors play a vital role in the plant’s adaptation to its light environment. Phytochromes control seed germination, seed- ling deetiolation &e., cotyledon, leaf, and root growth promo- tion and stem growth inhibition), gene expression, chloroplast differentiation, floral induction or suppression, and senes- cence (reviewed in Chory, 1991, 1993). In addition, phyto- chromes interact with the gravity-sensing apparatus to control gravitropism (Parks et al., 1996) and are responsible for sensing the proximity of nearby plants (Smith, 1995). The quality of light and the photoperiod also reflect diurna1 and seasonal time, and phytochromes contribute to the time- keeping mechanism of plants (see Kreps and Kay, 1997, in this issue). How are these widely divergent processes regu- lated by phytochromes? 1s a particular phytochrome re-

sponsible for a specific response? Molecular genetic studies clearly show that the diversity of phytochrome-regulated re- sponses is brought about through the combined action of severa1 phytochromes contributing to the control of a single developmental process.

The genetics of plant light responses have been most in- tensively studied in Arabidopsis (reviewed in Chory, 1993; Deng, 1994; Reed and Chory, 1994; von Arnim and Deng, 1996). Mutations have been identified in three of the five Arabidopsis phytochrome apoprotein genes-PHYA (Dehesh et al., 1993; Nagatani et al., 1993; Parks and Quail, 1993; Whitelam, 1993), PHYB (Koornneef et al., 1980; Reed et al., 1993), and PHYD (Aukerman et al., 1997)-as well as two in- volved in the biosynthesis of the linear tetrapyrrole chro- mophore (HYI , HY2; Koornneef et al., 1980; Parks and Quail, 1991).

With the exception of the phyD mutants, these -phyto- chrome mutants were identified in screens for seedlings identified on the basis of their inability to restrict hypocotyl growth in response to light of different wavelengths. Analy- sis of these mutants indicates that PHYA and PHYB each mediate overlapping subsets of phytochrome responses (summarized in Figures 2A and 2B), with their individual con- tributions depending on the light conditions. For instance, phyA mutants fail to germinate in far-red light (Reed et al., 1994; Shinomura et al., 1994) and germinate poorly under VLF light over a wide spectral range (from 350 to 750 nm; Shinomura et al., 1996). By contrast, phyB mutants germi- nate poorly in response to red light (Shinomura et al., 1994). Similarly, phyA mutants fail to restrict hypocotyl growth in far-red light, whereas phyB mutants are insensitive to red light (Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993). Both of these phytochromes also contribute to the induction of flowering in different ways. phyB mutants flower early in all photoperiods (Goto et al., 1991; Reed et al., 1994), whereas phyA mutants have a reduced accelera- tion of flowering in response to night breaks or day-length extensions (Reed et al., 1994).

In other cases, it is clear that these three phytochromes contribute redundantly to a response. One example is the redundant role that PHYA and PHYB play in LHCB gene in- duction by a red light pulse (Reed et al., 1994). Moreover, the effects of phyA and phyD mutations on the inhibition of hypocotyl elongation by red light are most apparent in the phyB mutant background; however, phyD single mutants show slight defects in red light responses in the presence of wild-type PHYB (Aukerman et al., 1997).

The phenotypes of phytochrome-overexpressing and phytochrome-deficient mutant seedlings suggest that PHYA and PHYB can activate a shared signal transduction path- way, perhaps by interactions with a common downstream component. Although this may be true for some responses, each of these photoreceptors also regulates a specific sub- set of responses, which suggests that there may also be signaling components that interact uniquely with individual phytochromes. This latter idea has been reinforced by the

Photoreceptors Regulate Development 1229

PfrA

dark reversion

destruction

seed germinationLHCB inductionfloral inductionchloroplast development

hypocotyl growth

BPfrB seed germination

LHCB inductioncotyledon expansionchloroplast development

floral inductionhypocotyl growth

seed germination

CRY1UV-A.B.G CHS induction

cotyledon expansion

hypocotyl growth

Figure 2. Summary of Developmental Pfeeesses Regulated by PHYA, PHYB, and CRY1.

(A) PHYA-mediated responses.(B) PHYB-mediated responses.(C) CRY1-mediated responses.Arrows indicate an inductive process, whereas T-bars indicate negative interactions. Wavelengths of light are abbreviated as follows: B, blue; G,green; R, red; FR, far red. CHS, chalcone synthase gene.

recent report that PHYB migrates to the nucleus in responseto light, suggesting that this phytochrome may function inthe nucleus (Sakamoto and Nagatani, 1996). The potentialnuclear localization of PHYB is in contrast to that of PHYA,which appears to be cytoplasmically localized (Kendrick andKronenberg, 1994).

PHYTOCHROMES AS PHOTON COUNTERS

Phytochrome mutants have also been useful in efforts to de-termine which phytochromes are responsible for the VLF,LF, and HIR responses. Several studies using phyA, phyB,

1230 The Plant Cell

or phyA phyB double mutants clearly support the notion that PHYA is the photoreceptor responsible for the VLF re- sponses that control seed germination, hypocotyl growth in- hibition, and LHCB expression (Botto et al., 1996; Shinomura et al., 1996; Mazzella et al., 1997; F. Hamazato and M. Furuya, personal communication). Moreover, these studies indicate that PHYA induces LHCB expression and seed germination over a broad spectrum of VLF light, including wavelengths in UV, blue, green, red, and near far-red (Shinomura et al., 1996). These PHYA-mediated responses are not photore- versible. Thus, PHYA-mediated responses to VLF light are not prototypical phytochrome responses. PHYA also ap- pears to mediate the HIR of Arabidopsis that is responsible for hypocotyl growth inhibition (Parks and Quail, 1993). Thus, PHYA appears to operate via two different action modes: HIR (under continuous far-red light) and VLF response (under multiple wavelengths of light).

By contrast, PHYB requires -1000-fold more light to elicit seed germination and the induction of LHCB expression (Reed et al., 1994; F. Hamazato and M. Furuya, personal communication). It does so in a blue light- or red light-spe- cific manner that is far-red light reversible. When considered together with the studies of red light-induced hypocotyl growth inhibition, these studies indicate that PHYB is re- sponsible for the LF response to red light in Arabidopsis. Moreover, PHYB is a “traditional” phytochrome in terms of its far-red light photoreversible properties.

These data suggest that there are three major pathways of phytochrome activity in etiolated seedlings: one mediated by PfrA in VLF light, one by PfrB in LF red light, and one by an unknown active component of PHYA in HIR. To further complicate matters, there are interactions between these three pathways. For instance, the hypocotyl growth inhibi- tion in high-irradiance far-red light that is mediated by PHYA can be amplified by the PfrB that is formed after a red light pulse (Botto et al., 1996). PHYA and PHYB can also counter each other’s activity. This is seen as a negative effect of PHYA on WrB-induced seed germination or hypocotyl growth inhibition. PHYA can also antagonize the activity of PHYB during shade avoidance behavior (Smith, 1995). These ob- servations of multiple, synergistic, and opposing interac- tions between PHYA and PHYB, together with the different subcellular localizations of PHYA and PHYB, make it difficult to formulate models of the precise mechanisms by which PHYA and PHYB initiate the signal transduction cascades that trigger the varied responses of plants to light (see also Trewavas and Malhó, 1997, in this issue).

BLUE LIGHT PERCEPTION BY CRYPTOCHROMES AND PHYTOCHROMES

Responses specific to blue light are widespread throughout the plant and animal kingdoms. In higher plants, these responses include phototropism, inhibition of hypocotyl

elongation, expansion of cotyledons, stomatal opening, and induction of gene expression (reviewed in Ahmad and Cashmore, 1996). Some of these responses-inhibition of hypocotyl elongation, expansion of cotyledons, and induc- tion of gene expression-overlap those regulated by phyto- chromes. These latter blue light responses are mediated by a 75-kD soluble flavoprotein called cryptochrome 1 (CRYl), which is encoded by the HY4 gene of Arabidopsis (Ahmad and Cashmore, 1993).

The hy4 mutants were isolated during the screens for long hypocotyl mutants described above (Koornneef et al., 1980); however, unlike the phytochrome apoprotein, chromophore, or signaling mutants, hy4 mutants are deficient specifically in blue and UV-A light-mediated hypocotyl growth inhibition (Figure 2C; Koornneef et al., 1980; Ahmad et al., 1995). In ad- dition, they show insensitivity to green light-induced hypo- cotyl growth inhibition. In continuous blue light, hy4 mutants also accumulate approximately threefold less anthocyanin than do wild-type plants. Moreover, the mRNA for chalcone synthase does not accumulate further if hy4 seedlings are grown in continuous red light to saturate the phytochrome response and then are transferred to blue light (Figure 2C; Ahmad et al., 1995). Also, stomatal opening and phototro- pism responses are normal in hy4 mutants, suggesting that these responses are regulated by a different blue light photo- receptor(s) (see Trewavas and MalhÓ, 1997, in this issue).

The HY4 gene was isolated by gene tagging and shown to encode a unique protein that combines the activities of two previously identified proteins (Ahmad and Cashmore, 1993). The N-terminal 500 amino acids share significant sequence identity (30% over the entire region) to type I DNA photol- yases; the identity is particularly high (80%) in the region of photolyase that is involved in chromophore binding. The C terminus of HY4 shows significant sequence relatedness (30% identity over a stretch of 86 amino acids) to rat smooth muscle tropomyosin. Although the significance of this ob- servation is unknown, molecular analysis of a total of 23 mu- tant alleles of HY4 has shown that the N- and C-terminal domains are both required for CRYl activity (Ahmad and Cashmore, 1993; Ahmad et al., 1995).

That CRYl is indeed a blue light flavin-type photoreceptor has been shown convincingly by three lines of evidence. First, four independent point mutations have been identified in a region within or immediately adjacent to the conserved chromophore binding motif found in all type I photolyases and in CRYl (Ahmad and Cashmore, 1993; Ahmad et al., 1995). Disruption of this region of Escherichia coli photol- yase completely inactivates the enzyme.

Second, analysis of recombinant CRYl expressed in and purified from insect and bacterial cells shows that the re- combinant protein from insect cells binds flavin adenine di- nucleotide (FAD) noncovalently and, in its oxidized state, has an absorption spectrum expected for a blue/UV-A light photoreceptor (Lin et al., 199513). In addition, when CRYl is photoreduced under anaerobic conditions, the neutra1 radi- cal flavosemiquinone (FADH) is unusually stable, which re-

Photoreceptors Regulate Development 1231

sults in a redox intermediate that also absorbs green light. This is consistent with the observed insensitivity to green light seen in hy4 mutants. In bacterial cells, the recombinant CRYl protein binds a pterin, methenyltetrahydrofolate (MTHF), in addition to FAD (Malhotra et al., 1995). This pterin chromophore is most likely responsible for the majority of the blue light-absorbing properties of CRYl. Indeed, an al- lele of hy4 with normal absorption in green light but impaired action in blue light has a lesion close to the site of attach- ment of the MTHF chromophore.

Lastly, overexpression of CRYl in transgenic tobacco results in seedlings with increased sensitivity to blue, UV-A, and green light compared with that of wild-type seedlings (Lin et al., 1995a). These data prove that CRYI is indeed a flavoprotein blue light photoreceptor that contributes to the control of an- thocyanin synthesis and hypocotyl growth inhibition by light.

But which is the photoreceptor(s) that controls blue light- regulated stomatal opening, cotyledon expansion, LHCB gene induction, and phototropism? A perusal of the litera- ture suggests that Arabidopsis expresses at least four, per- haps more, distinct blue/UV-A light receptors. One of these may already have been identified with the cloning of a sec- ond gene for a CRYl -type protein, designated CRY2 (Lin et al., 1996). Overexpression of CRY2 in transgenic Arabidop- sis seedlings results in plants with larger cotyledons than those of the wild type when grown in blue light (C. Lin and A. Cashmore, personal communication). This suggests that CRY2 may control blue light-induced cotyledon expansion during seedling deetiolation, a hypothesis that has been tested by screening for mutants with reduced cotyledon size in continuous blue light. This screen has resulted in the iso- lation of two cry2 putative mutants that accumulate no CRY2 protein (C. Lin, personal communication). Further studies of these cry2 mutants should provide evidence re- garding the biological functions of CRY2 and its interactions with other blue light photoreceptors.

Severa1 papers postulate a role for a 120-kD plasma membrane protein early in the transduction chain controlling phototropism in blue, UV-A, and green light (Khurana et al., 1989; Reymond et al., 1992; Liscum and Briggs, 1995). This protein is rapidly phosphorylated after treatment with blue light, indicating a role for protein phosphorylation in the sig- na1 transduction pathway controlling phototropism. The pro- tein is missing or severely reduced in phototropic mutants of Arabidopsis at the NONPHOTOTROPIC HYPOCOnL- 1 (NPH7) locus but is present in the wild type and in hy4 mu- tants (Khurana et al., 1989; Liscum and Briggs, 1995). It has been suggested that NPHl is the blue light receptor control- ling phototropism because the lesion in one of the nphl al- leles (nph7-2, previously called JK224) affects phototropic sensitivity to blue light but not to UV-A or green light, whereas the three remaining alleles affect phototopism in all wavelengths tested. Furthermore, NPHl may encode a re- ceptor with a flavin chromophore, because the phosphoryla- tion reaction is inhibited by flavin antagonists. Positional cloning of the NPHl locus, which is currently under way,

should open up new avenues of research in blue light per- ception and signaling.

The activity of a trimeric G-protein in the plasma mem- brane of etiolated pea buds is also regulated by LF blue light (Short et al., 1992). This response appears to be distinct from phototropism because G-protein activity and the 120- kD protein phosphorylation event occur in different tissues (discussed in Warpeha et al., 1992). Instead, fluence re- sponse studies suggest that this G-protein may be involved in the blue light activation of severa1 nuclear-encoded genes, including LHCB (Marrs and Kaufman, 1991 ; Warpeha et al., 1991; Gao and Kaufman, 1994). The blue light induc- tion of LHCB expression is unlikely to be under the control of HY4 because LHCB transcripts accumulate in hy4 mu- tants in response to blue light. In addition to this LF blue light photosynthetic gene regulatory system, there is a high- fluence blue/UV-A light-signaling system, which requires at least 10-fold more light for induction, that regulates the ex- pression of the psbDa-psbC promoter in the chloroplast (Christopher and Mullet, 1994). The requirement for high- fluence blue light indicates that additional photosensory fac- tors, distinct from those mediating responses to LF light, are involved in the perception of light intensity and in the regula- tion of photosynthetic gene expression.

As discussed above, phytochrome also acts as a blue light receptor for seed germination, hypocotyl growth inhibi- tion, and LHCB induction in Arabidopsis (Shinomura et al., 1996; F. Hamazato and M. Furuya, personal communica- tion). Using phyA, phyf3, and phyA phyB double mutants, it has been shown that PHYA is the most sensitive blue light receptor for the induction of LHCB expression, with a threshold fluence of -1 mmol m-* (F. Hamazato and M. Furuya, personal communication). PHYB and an additional phytochrome of unknown identity contribute to an LF blue light induction of LHCB (threshold fluence of 103 mmol m-*), which shows far-red light reversibility. In similar studies of seed germination, it was shown that PHYA photoirreversibly triggers germination under VLF irradiations of light from 300 to 780 nm (which includes UV-A, blue, green, red, and near- infrared light) (Shinomura et al., 1996). Moreover, phyA null mutants have elongated hypocotyls in continuous blue light, suggesting that PHYA contributes to the blue light-induced hypocotyl growth inhibition response (C. Fankhauser, M. Neff, and J. Chory, unpublished data). Whether this synergy is due to the combined action of PHYA and CRYl remains to be determined. Thus, it seems likely that some of the ef- fects of blue and UV light reported in the literature might re- sult from the action of phytochromes rather than distinct blue/UV-A light photoreceptors.

FUTURE PERSPECTIVES

During the past few years, many significant advances have been made in plant photoreceptor genetics and biochemistry.

1232 The Plant Cell

Despite this progress, a glaring gap in our knowledge re- mains the failure to identify signal transduction components that act early in the photoreceptor action pathways, particu- larly those that may interact directly with a photoreceptor. Studies with mutants deficient in phytochromes suggest that there may be multiple interacting components of vary- ing specificity. Clearly, new approaches are needed to eluci- date the mode of action of phytochromes and cryptochromes in order to understand the complexities of the signal trans- duction networks that integrate light perception with the large number of downstream responses. One such ap- proach is a biochemical approach designed to use the CRY7, PHYA, and PHYB clones in experiments to identify proteins with which these molecules interact. In addition, the isolation and characterization of extragenic suppressors that specifically suppress the defects of a particular photorecep- tor may lead to the genetic characterization of components that act early in the photoreceptor action pathways. The fact that significant amounts of recombinant phytochromes and cryptochromes can be purified will also advance efforts to reveal the atomic structure of these photoreceptors. The re- sults from these experiments and others will then allow us to construct models of the mechanisms by which photons control plant development. I look forward to many break- throughs in the study of the mechanisms of photoreceptor action and signal amplification during the next few years.

ACKNOWLEDGMENTS

I thank Tedd Elich for the sequence comparisons with the cyanobac- teria1 phytochrome and Cindy Doane and Tedd Elich for helping me assemble Figure 1. I am grateful to Tedd Elich, Christian Fankhauser, and Michael Neff for thoughtful comments on the manuscript and to Masaki Furuya, Clark Lagarias, Chentao Lin, and Bob Sharrock for sharing unpublished data. Work on photomorphogenesis in my lab- oratory is supported by the National lnstitutes of Health (Grant No. GM52413), the National Science Foundation (Grant No. MCB9631390), the U.S. Department of Energy (Grant No. DOE-ER13993), the U.S. Department of Agriculture (Grant No. 96-35301 -3153), and the Samuel Roberts Noble Foundation.

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