The Discovery Of Cryptochrome

Cryptochromes, first identified in Arabidopsis (Ahmad and Cashmore, 1993), have also been found in green algae (Small et al., 1995) and are now known to occur throughout the plant kingdom, including ferns (Kanegae and Wada, 1998) and mosses (Imaizumi et al., 2002; see Chapters 24, 25). Cryptochromes have also been described for animals, including flies (Emery et al., 1998; Stanewsky et al., 1998)

and humans (Hsu et al., 1996; Todo et al., 1996) where, as in plants, they play a role in circadian behaviour (see Chapter 26).

3.1 Cryptochromes of Arabidopsis

A description of the molecular properties of a blue light photoreceptor for plants came through the isolation of a T-DNA-tagged Arabidopsis mutant deficient in its response to blue light (Ahmad and Cashmore, 1993). This mutant was shown to be allelic to the previously described hy4 mutant (Koornneef et al., 1980). The mutant — now commonly referred to as the cryl mutant — has a long hypocotyl when grown under blue light, being attenuated in the blue light mediated inhibition of cell expansion that characterizes wild-type seedlings. By contrast, the mutant shows the normal response when grown under red or far-red light and similarly shows no distinct phenotype when grown in darkness; the mutant is selectively impaired in its response to blue light. The sequence of the CRY1 gene indicated that it was similar to those encoding proteins of the photolyase family (Ahmad and Cashmore, 1993). Furthermore, when expressed in insect cells the Arabidopsis CRY1 protein is observed to bind a flavin, also similar in this respect to photolyases (Lin et al., 1995). However, Arabidopsis CRY1 differs from photolyases in two significant ways: Firstly, and most significantly, CRY1 protein possesses no detectable photolyase activity. Secondly, Arabidopsis CRY1 is larger than E. coli photolyase, containing additional distinguishing amino acid residues at its C-terminus (Ahmad and Cashmore, 1993). We refer to this cryptochrome C-terminal domain as CCT and the N-terminal photolyase-like domain we refer to as CNT.

Ectopic overexpression of the CRY1 gene, in either transgenic tobacco or Arabidopsis plants, confers hypersensitivity to light (Lin et al., 1995, 1996). As with the loss of function cry1 alleles, this change in light sensitivity of the overexpressing plants was restricted to UV-A and blue light, and to a lesser extent to green light, consistent with the notion that CRY1 is a photoreceptor responding selectively to these wavelengths. The CRY1 overexpressing plants also show a dwarf phenotype when grown under white light, similar in this respect to that observed for plants overexpressing the phytochrome photoreceptors (Keller et al., 1989).

The cry1 mutants show a semidominant phenotype, reflecting haploid insufficiency (Koornneef et al., 1980; Ahmad and Cashmore, 1993). That is, heterozygous mutants exhibit a phenotype that is intermediate between that of the wild-type plants and the homozygous mutant. This observation simply indicates that both the activity of the photoreceptor and the severity of the associated phenotype are proportional to the amount of the photoreceptor.

As noted, photolyases are characterized by a second light-harvesting chromophore, commonly MTHF (Sancar 2003). plant cryptochromes also bind MTHF when expressed in E. coli (Malhotra et al., 1995), although when recombinant Arabidopsis CRY1 is isolated from insect cells it lacks this second chromophore (Lin et al., 1995). The precise identities of the cryptochrome chromophores in Arabidopsis, and their redox states and absorption properties, are of interest, as in the absence of this information no useful predictions or interpretations of action spectra are possible.

A second Arabidopsis cryptochrome gene, CRY2, was identified (Lin et al., 1996; Lin et al., 1998). The encoded CRY2 protein is light-labile, and in keeping with this, cry2 mutant Arabidopsis seedlings have elongated hypocotyls when grown under low intensity blue light, whereas light inhibition of hypocotyl growth is essentially the same as wild type when the seedlings are grown under blue light of intensity greater than 10 |imoles.meter-2.sec-1 (Lin et al., 1998). The most striking phenotypic feature of cry2 mutants is their altered flowering time (see later).

Cryptochromes have now been characterized for several additional plant species including tomato (Ninu et al., 1999; Weller et al., 2001) and rice (Matsumoto et al., 2003). In both cases, as in Arabidopsis, these cryptochromes apparently play a role in blue light mediated de-etiolation and photomorphogenesis.

3.2 Cryptochromes of algae, mosses and ferns

Cryptochromes have been described for algae (Small et al., 1995), and ferns (Kanegae and Wada, 1998), and recently for mosses (Imaizumi et al., 2002). In the fern Adiantum capillus-veneris, spore germination is regulated by blue light, and two of the five cryptochromes described for this fern are thought to be involved in this process. Two CRY genes have been described for the moss Physcomitrella patens, and disruption of these genes confers an increase in auxin sensitivity in a blue light specific manner (Imaizumi et al., 2002). In the green alga Chlamydomonas, there are blue light specific responses in addition to the phototactic response that is mediated by a rhodopsin-like photoreceptor residing with the eye spot (Deininger et al., 1995). Whether CRY is the photoreceptor mediating any of these other blue light responses, has not been determined.

3.3 Drosophila cryptochrome

A Drosophila cryptochrome was identified and characterized by two independent approaches. A mutant was identified in a screen involving transgenic flies expressing a luciferase reporter driven by the promoter of PERIOD (PER), a gene that performs an integral role in the central oscillator of the Drosophila circadian clock (Stanewsky et al., 1998). This cry mutant exhibited arrhythmic luciferase expression. In a second approach, the Drosophila gene was identified as encoding a protein with sequence similarity to both photolyases and the mammalian and Arabidopsis CRY sequences, prompting the investigators to entertain the possibility that this gene encoded a Drosophila blue light photoreceptor (Emery et al., 1998). In support of this proposal, transcription of the fly CRY gene was observed to be under circadian control and influenced by PER and TIM genes; furthermore, circadian photosensitivity was enhanced in CRY overexpressing strains.

3.4 Mammalian cryptochromes

The earlier-mentioned 6-4 photolyases were first identified in Drosophila (Todo, Takemori et al., 1993). It was of interest to determine if a related sequence existed in mammals, as it had long been believed that mammals lacked any photolyase activity; lesions in mammalian DNA being repaired by an excision repair process. Screening of a human cDNA library led to the identification of a sequence with similarities to the fly 6-4 photolyase gene (Todo et al., 1996). However, expression of this human sequence produced a protein that, although it bound both a flavin and a pterin, possessed no detectable photolyase activity (Hsu et al., 1996). By analogy with the Arabidopsis CRY genes it was proposed that this human photolyase-like gene encoded a blue light photoreceptor; furthermore, it was postulated that this photoreceptor might serve to entrain human circadian rhythms (Miyamoto and Sancar 1998).

Cryptochromes have now been identified in a wide variety of animals, including zebrafish (Cermakian et al., 2002; Hirayama et al., 2003). Here, as in mammals (see later), the CRY1 protein is involved in circadian repression of CL0CK:BMAL1-mediated transcription (Hirayama et al., 2003).

3.5 Bacterial and related cryptochromes

Cryptochromes, initially thought to be restricted to eukaryotic organisms, have recently been described for bacteria (Hitomi et al., 2000; Brudler et al., 2003). A photolyase-like sequence from Synechocystis was demonstrated to lack detectable photolyase activity, and hence was designated as a cryptochrome. This sequence was given the name CRY DASH. A crystal structure of the Synechocystis CRY was determined and microarray gene expression studies showed that some genes were upregulated in a mutant lacking the CRY DASH (Brudler et al., 2003). As the protein showed DNA binding properties it was speculated that it might function as a transcriptional repressor.

A related CRYDASH/CRY3 gene was described for Arabidopsis (Brudler et al., 2003; Kleine et al., 2003). The nuclear encoded CRY3 protein shows the unusual property of being localized to both mitochondria and chloroplasts (Kleine et al., 2003).

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