A Component of the Cryptochrome Blue Light Signaling Pathway

The ability to perceive the quantity and quality of light in the environment is important for the normal growth and development of many organisms. Plant have evolved highly sophisticated light-sensing mechanisms that are intricately associated with fundamental processes such as photosynthesis, photomorphogenesis, photoperiodic control of flowering, phototropism, and entrainment of circadian rhythms, all of which enable the optimal use of light in a sessile organism. Aside from the light-harvesting complexes that participate in photosynthesis, multiple photoreceptors participate in these processes, and principal among these are the red/far-red light–sensing phytochromes and the blue light–sensing phototropins and cryptochromes. Cryptochrome and phytochrome signaling pathways interact to regulate photomorphogenic and photoperiodic responses, including hypocotyl elongation and flowering time, whereas phototropins principally regulate short-term movement light responses, such as phototropism, chloroplast relocation, and stomatal opening. Plant photoreceptors are the most well characterized in Arabidopsis, which contains genes for five different phytochromes (PHYA to PHYE), two phototropins (PHOT1 and PHOT2), and two cryptochromes (CRY1 and CRY2).

Cryptochromes are flavoproteins that are similar to and likely evolutionarily derived from DNA photolyases, enzymes that are activated by blue/UV-A light to bind to and mediate the repair of pyrimidine dimers induced by the exposure of DNA to UV-B light. However, cryptochromes lack DNA photolyase activity and appear to have evolved to perform other functions related to the light control of development (Cashmore et al., 1999; reviewed by Lin, 2002). Cryptochrome function appears to overlap extensively with that of the phytochromes, but very little is known about the cryptochrome signal transduction pathway. In this issue of The Plant Cell, Møller et al. (pages 1111–1119) show that the Ser/Thr phosphatase AtPP7 likely functions as a positive regulator of blue light signaling in Arabidopsis (Figure 1).

Figure 1.

AtPP7 Is a Positive Regulator of Blue Light Signaling.

The long-hypocotyl phenotype of antisense AtPP7 seedlings (L5 and L7) under blue light is very similar to that of the hy4 mutant, which is disrupted in the CRY1 gene. Ler and Col indicate the wild-type photomorphogenic response. The phenotype is specific to blue light in cry1/hy4 mutants and antisense AtPP7 plants, suggesting that AtPP7 functions as a positive regulator of blue light signaling in Arabidopsis.

CRYPTOCHROME FUNCTION

Most plant species studied, including Arabidopsis, tomato, barley, the fern Adantium, and the moss Physcomitrella, have at least two cryptochrome genes. Arabidopsis CRY1 and CRY2 may have distinct but overlapping functions in photomorphogenesis and the photoperiodic control of flowering time. cry1 mutant seedlings fail to undergo photomorphogenesis and exhibit the characteristic etiolated appearance of dark-grown seedlings when grown under blue light, but they appear normal under white or red light (Ahmad and Cashmore, 1993). cry1 mutants have been reported to flower later than wild-type plants, but evidently this is not a marked feature of the cry1 phenotype. CRY2 may be involved in photomorphogenesis, because cry2 mutant seedlings also show a short hypocotyl phenotype under blue light and the cry1 cry2 double mutant exhibits a more severe deetiolation phenotype than either of the single mutants (Mockler et al., 1999). However, cry2 mutants have been studied much more extensively for defects in the regulation of flowering time. In addition, the independently isolated late-flowering mutant fha1 was found to carry a mutation in CRY2 (Guo et al., 1998), and a quantitative trait locus that affects flowering time in natural populations of Arabidopsis was mapped to CRY2 (El-Din El-Assal et al., 2001). Therefore, the evidence suggests principal functions for CRY1 and CRY2 in photomorphogenesis and the photoperiodic control of flowering, respectively, with some degree of overlap (reviewed by Lin, 2002).

AtPP7 FUNCTIONS IN CRYPTOCHROME SIGNALING

Phosphorylation/dephosphorylation reactions have long been recognized as one of the basic mechanisms of signal transduction (Bowler and Chua, 1994), and it is reasonable to hypothesize that light activation of the photoreceptor domain leads to the phosphorylation of cryptochrome. It has been shown that AtCRY2 undergoes blue light–dependent phosphorylation that triggers photomorphogenic responses (Shalitin et al., 2002) and that recombinant phyA protein phosphorylates AtCRY1 in vitro (Ahmad et al., 1998).

Møller et al. set out to examine a potential role in light signal transduction for the novel Ser/Thr phosphatase AtPP7 by antisense inhibition of AtPP7 gene expression in Arabidopsis. They found that transgenic seedlings with highly reduced AtPP7 transcript levels showed loss of the photo-morphogenic response specifically in re-sponse to blue light irradiation, a phenotype that is strikingly similar to that of the cry1 mutant (Figure 1), suggesting that AtPP7 may function as a positive regulator of cryptochrome signaling. Several antisense lines with reduced AtPP7 transcript levels exhibited a similar phenotype (with varied levels of severity). As a check against the possible contamination of antisense seed stocks with cry1 mutant seeds or a secondary mutation in the CRY1 gene, CRY1 was sequenced and found to be identical to wild-type sequence in the antisense lines. The authors also analyzed the expression of CRY1, CRY2, and several light-regulated genes in antisense AtPP7 lines compared with wild-type and cry1 mutant seedlings. There was little to no effect on the expression of CRY1 or CRY2 in the AtPP7 antisense lines, and light-induced effects on the expression of light-regulated genes were similar in AtPP7 antisense and cry1 mutant plants, suggesting that AtPP7 may act downstream of CRY1 and/or CRY2.

SIMILARITIES BETWEEN PLANTS AND ANIMALS

Analyses of CRY gene sequences from animals and plants have led to the hypothesis that plant and animal cryptochromes evolved independently from divergent photolyase ancestral sequences (Cashmore et al., 1999). Specifically, human and Drosophila CRY gene sequences are more similar to a group of photolyases called (6-4) photolyases [including Arabidopsis (6-4) photolyase gene sequences] than they are to plant CRY genes. The work of Møller et al. nonetheless suggests an intriguing parallel between animal and plant cryptochrome signal transduction pathways in that AtPP7 is related to the PP5/RdgC subfamily of Ser/Thr phosphatases, various members of which have been associated with light signaling in human and Drosophila.

In animals, the major photoreceptor that functions in vision is rhodopsin. Animal cryptochromes are highly expressed in the retina and are believed to function in the entrainment of circadian rhythms (Cashmore et al., 1999). Zhao and Sancar (1997) used the yeast two-hybrid assay to show that human CRY2 interacts specifically with PP5 and Tpr1 proteins and that the interaction of CRY2 with PP5 inhibited its phosphatase activity. Møller et al. found that AtPP7 did not interact with AtCRY1 in a yeast two-hybrid assay (interaction with AtCRY2 was not tested). Interestingly, both PP5 and Trp1 contain the tetratricopeptide repeat (Tpr) motif, which is known to mediate protein–protein interactions, whereas AtPP7 does not contain this motif.

AtPP7 also is similar to the PP5 phosphatase RdgC, which is associated with light-induced signaling in Drosophila. Mutations in RdgC cause retinal degeneration in Drosophila as a result of sustained photoexcitation (Steele et al., 1992). Another human Ser/Thr phosphatase, hPP7, is homologous with Drosophila RdgC and AtPP7 and is expressed specifically in human retinal cells (Huang and Honkanen, 1997). RdgC, hPP7, and AtPP7 all contain EF hand calcium binding motifs at the C-terminal end of the putative phosphatase catalytic domain, although the precise role of calcium in regulating the activity of these proteins is unclear. Kutuzov et al. (1998) showed that recombinant AtPP7 phosphatase activity was stimulated by Mn²⁺ but not by Ca²⁺ alone. Interestingly, the activity of recombinant hPP7 was found to be dependent on Mn²⁺ and to be stimulated further by Ca²⁺ (Huang and Honkanen, 1997).

Møller et al. showed that AtPP7 expression in wild-type plants was highest in leaves compared with stems and flowers and very low to absent in roots. Kutuzov et al. (1998) reported the preliminary observation that AtPP7 was expressed most highly in guard cells. Clearly, much work remains in discovering AtPP7 target proteins and the precise role of this phosphatase in cryptochrome signaling. However, the work of Møller et al. has opened an exciting new avenue in light signaling.