From Darkness into Light: Factors Controlling Photomorphogenesis

LIGHT PERCEPTION

There are two general classes of photomorphogenic mutants: those that display an etiolated, skotomorphogenic phenotype in the light and those that show aspects of a photomorphogenic phenotype when grown in the dark. Mutants that display an etiolated phenotype in the light identify positive regulatory components of photomorphogenesis. Koornneef et al. (1980) isolated a number of this type of mutant, called long hypocotyl or hy mutants, that fail to perceive light and produce long hypocotyls whether they are grown in light or darkness. Most of these are photoreceptor mutations, which collectively have shown that plants integrate signals perceived by numerous photoreceptors for normal photomorphogenic development. For example, the hy8 mutant is insensitive to far-red light, and the gene was found to encode the apoprotein of phytochrome A (renamed PHYA); the hy3 mutant is insensitive to red light, and the gene encodes the PHYB apoprotein (PHYB gene); and HY4, the mutant of which is insensitive to blue light, encodes the apoprotein of the blue light receptor CRY1. HY5 is the only gene in this group that is not directly related to photoreception; rather, it encodes a transcription factor that acts downstream of the other HY genes (see below).

It has long been suspected that hy1, hy2, and hy6 mutants are defective in chromophore biosynthesis or attachment (Parks and Quail, 1991; Chory, 1992). Phytochromes consist of an apoprotein with a thioether-linked chromophore called phytochromobilin. Apoproteins are encoded by five different genes in Arabidopsis (PHYA to PHYE), each of which has distinct spectral qualities. Phytochromobilin appears to be the chromophore partner of all of the phytochromes; thus, the hy1, hy2, and hy6 mutants are deficient in all of the phytochromes and in both red and far-red light responses. Genetic analysis suggests that the hy6 mutation lies at a locus distinct from hy1 and hy2 (Chory et al., 1989). The hy6 mutation has not been well studied since this initial report, perhaps because the mutant seed currently available are reported to carry a mutation in the HY1 gene (Muramoto et al., 1999). The HY1 gene was found to encode a heme oxygenase that localizes to chloroplasts and catalyzes the conversion of heme to a biliverdin precursor of phytochromobilin (Muramoto et al., 1999). Kohchi et al. (2001) show that the HY2 gene encodes phytochromobilin synthase, which is responsible for the next step in this pathway, the conversion of biliverdin to phytochromobilin. They demonstrate the catalytic activity of the recombinant protein and the chloroplast localization of a fusion protein consisting of the green fluorescent protein reporter fused to the putative phytochromobilin synthase chloroplast transit peptide.

Interestingly, the hy1 mutant phenotype is severe only at the seedling stage; later in development, the mutant plants appear relatively healthy. One explanation is that the plants have an alternate pathway for chromophore biosynthesis that is activated later in development. Consistent with this view, the Arabidopsis genome has other genes with similarities to HY1 (Muramoto et al., 1999; Kohchi et al., 2001). However, the hy2 mutant phenotype is less severe than that of hy1, even at the seedling stage, and no genes with similarities to HY2 have been found in the Arabidopsis genome (Kohchi et al., 2001). These observations might simply reflect differences in allele strength, and a definitive study of the phenotypic effects of null versus less severe mutant alleles is needed. Another interesting possibility is that the phytochrome signal transduction pathway overlaps with other developmental signal transduction pathways and the phytochrome pathway becomes relatively less critical to certain aspects of growth later in development, when other signals begin to act on many of the same downstream effectors. The identification of the HY1 and HY2 genes should help to resolve some of these questions.

LIGHT SIGNAL TRANSDUCTION

Mutants that exhibit aspects of a photomorphogenic phenotype when grown in the dark identify negative regulators of light signal transduction. These include the constitutive photomorphogenic/de-etiolated/fusca (cop/det/fus) group of mutants (reviewed in Chory, 1992; Howell, 1998; Hardtke and Deng, 2000). cop and det mutants are similar and highly pleiotropic; that is, the mutations cause a number of different phenotypic effects (in this case, phenotypic effects associated with photomorphogenesis, such as a shortened hypocotyl, opened and expanded cotyledons, and increased expression of certain light-regulated genes). The fus mutants, seedling-lethal mutants that accumulate anthocyanins in the cotyledons (a photomorphogenic characteristic), have been found to be null or severe alleles of various cop/det mutants. All of these mutations are recessive loss-of-function mutations that promote photomorphogenesis, suggesting that their normal function is to suppress photomorphogenesis in the dark.

The COP1 gene product occupies a central position in the regulation of photomorphogenesis. COP1 appears to suppress the activity of downstream factors that promote photomorphogenesis by tagging them for proteolytic degradation. Interaction with COP1 has been definitively shown for HY5, a bZIP DNA binding transcriptional activator that promotes photomorphogenesis in the light (Osterlund et al., 2000). Consistent with a primary role in promoting photomorphogenesis, all of the photoreceptor hy mutants have abnormally low levels of HY5 in the light, and in wild-type seedlings, HY5 levels are positively correlated with light intensity and degree of hypocotyl elongation. COP1 is localized to the nucleus in the dark, where it appears to target HY5 for proteasome-mediated degradation; it is excluded from the nucleus and routed to the cytoplasm in the light, thus allowing the nuclear accumulation of HY5 and the consequent promotion of photomorphogenesis (Osterlund et al., 2000).

Most of the COP/DET/FUS genes have been found to encode components of what has been named the COP9 signalosome, or CSN (Wei and Deng, 1999). The CSN is a complex of eight subunits that are similar to the subunits that make up the lid of the 26S proteasome, a protein degradation complex responsible for the degradation of ubiquitinated proteins that are present in all eukaryotes. The CSN itself also is present in animal as well as plant cells, and the CSN and the 26S proteasome lid are probably derived from a common ancestral complex (Wei and Deng, 1999). At least eight of the cop/det/fus mutants are “CSN” mutants, which fail to accumulate the complex and likely encode CSN structural components. The exact function of the CSN is unknown, but evidence suggests that it may be related to protein degradation. Deshaies and Meyerowitz (2000) propose a model for CSN function wherein the CSN recognizes and delivers COP1-targeted proteins to the proteasome for degradation, either functioning in place of the proteasome lid for COP1-targeted proteins or perhaps delivering COP1-targeted proteins to the lid of the intact proteasome. COP1 contains a RING finger domain and WD-40 repeats, both of which have been shown to be involved in the activity of ubiquitin ligases, thus offering the attractive possibility that COP1 is a ubiquitin ligase that targets proteins for interaction with the CSN and the proteasome via ubiquitination (Deshaies and Meyerowitz, 2000).

On the basis of the hypothesis that COP1 is a key negative regulator of photomorphogenesis with multiple protein–protein interaction domains, Yamamoto et al. (1998) set out to identify COP1-interacting proteins (CIPs) by screening an Arabidopsis cDNA expression library using ³²P-labeled COP1 protein as a probe. They identified CIP7 as a positive regulator of anthocyanin biosynthesis and chlorophyll accumulation during photomorphogenesis and as a potential target for COP1-mediated repression. In this issue, Yamamoto et al. (2001) characterize CIP4, another potential target of COP1 and a positive regulator of chlorophyll accumulation and hypocotyl elongation during photomorphogenesis. Thus, a clearer picture of COP1 emerges, one of a protein that acts in the nucleus and interacts specifically with transcriptional activators of photomorphogenesis to target them for degradation. Consistent with this view, Cao et al. (2000) identified CR88 as another possible target of COP1. The cr88 mutant is a chlorate-resistant mutant that displays a long-hypocotyl phenotype in red light and reduced expression of several light-induced genes. Although a direct interaction between COP1 and CR88 has not been shown, Cao et al. (2000) demonstrated that CR88 acts downstream of COP1, in a branch separate from HY5, to promote the greening process during photomorphogenesis.

The approach used by Yamamoto et al. (1998, 2001) may also work to identify upstream factors that interact with COP1 and/or factors involved directly with COP1 function. For example, CIP1 is a cytosolic protein associated with the cytoskeleton; thus, it might be involved in the localization of COP1 to the cytoplasm in the light (Matsui et al., 1995; Hardtke and Deng, 2000). Another CIP that has been described is CIP8, which is a RING finger protein that interacts with the COP1 RING finger domain (Torii et al., 1999). RING finger interactions are a feature of E3 ubiquitin ligase complexes; thus, CIP8 offers another tantalizing piece of evidence for a similar role for COP1. Further characterization of these and other CIPs should help to define COP1 function and its mechanism of action more clearly.