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Plant red/far-red photoreceptor phytochromes are known as autophosphorylating serine/threonine kinases. However, the functional roles of autophosphorylation and kinase activity of phytochromes are largely unknown. We recently reported that the autophosphorylation of phytochrome A (phyA) plays an important role in regulating plant phytochrome signaling by controlling phyA protein stability. Two serine residues in the N-terminal extension (NTE) region were identified as autophosphorylation sites, and phyA mutant proteins with serine-to-alanine mutations were degraded in plants at a significantly slower rate than the wild-type under light conditions, resulting in transgenic plants with hypersensitive light responses. In addition, the autophosphorylation site phyA mutants had normal protein kinase activities. Collectively, our results suggest that phytochrome autophosphorylation provides a mechanism for signal desensitization in phytochrome-mediated light signaling by accelerating the degradation of phytochrome A.
Higher plants continually adapt to their light environments to promote photosynthesis for optimal growth and development. Natural light conditions are monitored by various plant photoreceptors, including red (R)/far-red (FR) photoreceptor phytochromes.1,2 Phytochromes are dimeric chromoproteins covalently linked to tetrapyrrole chromophore phytochromobilin, and exist as two photo-interconvertible species, red-light absorbing Pr and far-red-light absorbing Pfr forms. Phytochromes are biosynthesized as the Pr form in the dark, and are transformed to the Pfr form upon exposure to red light. This photoactivation of phytochromes induces a highly regulated signaling network for photomorphogenesis in plants.3,4 Recently, phosphorylation and dephosphorylation have been suggested to play important roles in phytochrome-mediated light signaling;5,6 for instance, a few phytochrome-associated protein phosphatases have been shown to act as positive regulators of phytochrome signaling.7–9 However, the functional roles of phytochrome phosphorylation remain to be explored.
Phytochromes are known as autophosphorylating serine/threonine protein kinases.10,11 To study phytochromes as autophosphorylating kinases, the autophosphorylation sites of phytochromes need to be determined first. We recently identified Serine-8 (Ser8) and Serine-18 (Ser18) residues in the 65 amino-acid-long N-terminal extension (NTE) region of oat (Avena sativa) phyA as autophosphorylation sites. These sites were consistent with the previously reported phosphorylation sites of phyA.12 The NTE is required for the full biological activity of phyA,13 and serine-to-alanine substitutions in the NTE region have been shown to result in increased biological activity.14 These results suggest that phosphorylation in the NTE region is involved in signal attenuation or desensitization in phytochrome signal transduction. However, it remains unclear how this substitution generates an attenuation signal. We recently showed that phyA with autophosphorylation site mutation were degraded in plants at a significantly slower rate than wild-type phyA under light conditions, suggesting that phytochrome autophosphorylation regulates the protein stability of phyA. Therefore, autophosphorylation can desensitize phytochrome signaling by promoting the protein degradation of phyA.
When the phosphorylation sites in the NTE region of monocot and dicot phyA were submitted to the NetPhos 2.0 server (www.cbs.dtu.dk/services/NetPhos), phosphorylatable serine residues other than Ser8 and Ser18 were predicted in the extreme N-terminus of phyA (Fig. 1A and B). Serine/threonine-rich sequences were present in the NTE of all species examined. In the case of oat phyA, a five-serine cluster (8–12aa) containing three highly phosphorylatable residues was identified in the NTE. Thus, to investigate the possible involvement of other serine residues in phytochrome autophosphorylation, we generated a deletion mutant (Δ6–12) of seven amino acid residues (6PASSSSS12) and a substitution mutant in which the five serine residues were replaced with alanine residues (S8-12A). In addition, we generated combination mutants bearing Δ6–12 or S8-12A and S18A (i.e., Δ6-12/S18A and S8-12A/S18A). Using purified mutant proteins, we confirmed that Δ6–12 and S8-12A mutants had reduced levels of autophosphorylation and that Δ6–12/S18A and S8-12A/S18A mutants had nearly no autophosphorylation (data not shown). Furthermore, transgenic Arabidopsis plants expressing these mutants exhibited hypersensitive light responses when compared to wild-type (Ler) or transgenic plants expressing wild-type oat phyA (Wt-OX6), similar to transgenic plants expressing the autophosphorylation site mutants (Fig. 1C and D). Considering that the serine-rich NTE of oat phyA is not necessary for chromophore binding but helps to regulate light responses,15,16 our results suggest that the NTE region is functionally important for autophosphorylation during phytochrome signaling.
Since the serine-to-alanine site mutants showed significantly reduced autophosphorylation, it may be questioned whether the reduced autophosphorylation is caused by a decrease in the protein kinase activity of phyA. Thus, we investigated the phospho-transfer activities of the site mutants (Fig. 2). For this experiment, we used GST-fused phytochrome-interacting factor 3 (PIF3) as a phytochrome kinase substrate. Wild-type oat phyA, as well as S599A, showed autophosphorylation and also phospho-transfer activity on PIF3, as previously reported.9 Both S8A and S18A mutants showed a phospho-transfer activity similar to that of wild-type oat phyA, suggesting that these mutants retained normal protein kinase activities. Therefore, the hypersensitive light responses of the transgenic plants expressing the autophosphorylation site mutants and the slow degradation of the phyA mutant proteins cannot be attributed to a loss of protein kinase activity. In addition, the S8A mutant and the S18A mutant exhibited a much greater reduction in autophosphorylation in the presence of a substrate than in its absence (Fig. 2), suggesting the occurrence of competitive phosphorylation between the substrate and the phytochrome itself. However, further studies will be required to establish whether the autophosphorylation is intermolecular or intramolecular.
Despite extensive studies of phytochrome-interacting proteins, there has been no report of a protein kinase that phosphorylates phytochromes. On the other hand, a few protein phosphatases bind and dephosphorylate phytochromes, including flower-specific phytochrome-associated protein phosphatase (FyPP),7 phytochrome-associated protein phosphatase 5 (PAPP5),8 and phytochrome-associated protein phosphatase type 2C (PAPP2C).9 It is possible that phytochrome phosphorylation is regulated by autophosphorylation and protein phosphatase activity. Furthermore, previous studies with PAPP5 showed that phytochrome stability is increased in PAPP5-overexpression lines and decreased in papp5 knockout lines, suggesting that phytochrome phosphorylation is involved in the regulation of phytochrome stability.8 Based on these results, it has been suggested that phosphorylated phytochrome mediates low flux signaling, while unphosphorylated or dephosphorylated phytochrome mediates high flux signaling with enhanced photoresponsiveness.17 In addition, a recent report showed that phosphorylated phyA preferentially associates with the COP1/SPA1 complex, an E3 ligase involved in phyA degradation.18 This report further suggested that phyA phosphorylation was important for its interaction with the COP1/SPA1 complex and showed that the phosphorylated form of phyA was enriched during co-immuno-precipitation with COP1. Therefore, these results suggest that autophosphorylation site mutants of phyA are not efficiently degraded, possibly because of a decreased interaction between the mutants and the COP1/SPA1 complex.
Based on our results and previous reports, we provide a model for the functional role of phyA autophosphorylation (Fig. 3). In the dark, phyA proteins are synthesized and accumulate as the Pr form in the cytosol, which consists of autophosphorylated phyA and unphosphorylated phyA. Upon illumination, the Pr form is photoactivated to the Pfr form, which is then translocated into the nucleus where it regulates gene expression for photomorphogenesis. At the same time, the Pfr form is degraded via the ubiquitin/26S proteasome protein degradation pathway. At this point, the phosphorylated Pfr form undergoes more rapid degradations than unphosphorylated phyA or dephosphorylated phyA species by protein phosphatases, because the phosphorylated phyA proteins are more efficiently degraded by the ubiquitin/26S proteasome through the enhanced interaction with the COP1/SPA1 complex. This rapid degradation of autophosphorylated phyA might facilitate the efficient desensitization of the phyA signal. If phyA activity is not properly attenuated upon illumination, plants could over-respond to light by eliciting too many photomorphogenic signaling events and/or could become desensitized to subsequent changes in light quality or quantity. Therefore, this regulation may provide an efficient means of rapidly controlling the number of active phyA photoreceptors available to initiate signaling events, and thereby improve the response of plants to fluctuating light environments.
This work was supported in part by the Plant Diversity Research Center of 21st Century Frontier Research Program (grant no. PF06302-02); the MEST/NRF to the Environment Biotechnology National Core Research Center (NCRC) (grant no. 20090091493); and the BioGreen 21 Program (Code # 20080401034014), Rural Development Administration, Republic of Korea.
Previously published online: www.landesbioscience.com/journals/psb/article/11898