Co-action of COP1, SPA and cryptochrome in light signal transduction and photomorphogenesis of the moss Physcomitrium patens.

The Arabidopsis COP1/SPA ubiquitin ligase suppresses photomorphogenesis in darkness. In the light, photoreceptors inactivate COP1/SPA to allow a light response. While SPA genes are specific to the green lineage, COP1 also exists in humans. This raises the question of when in evolution plant COP1 acquired the need for SPA accessory proteins. We addressed this question by generating Physcomitrium Ppcop1 mutants and comparing their visible and molecular phenotypes with those of Physcomitrium Ppspa mutants. The phenotype of Ppcop1 nonuple mutants resembles that of Ppspa mutants. Most importantly, both mutants produce green chloroplasts in complete darkness. They also exhibit dwarfed gametophores, disturbed branching of protonemata and absent gravitropism. RNA-sequencing analysis indicates that both mutants undergo weak constitutive light signaling in darkness. PpCOP1 and PpSPA proteins form a complex and they interact via their WD repeat domains with the VP motif of the cryptochrome CCE domain in a blue light-dependent manner. This resembles the interaction of Arabidopsis SPA proteins with Arabidopsis CRY1, and is different from that with Arabidopsis CRY2. Taken together, the data indicate that PpCOP1 and PpSPA act together to regulate growth and development of Physcomitrium. However, in contrast to their Arabidopsis orthologs, PpCOP1 and PpSPA proteins execute only partial suppression of light signaling in darkness. Hence, additional repressors may exist that contribute to the repression of a light response in dark-exposed Physcomitrium.

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light.

In Arabidopsis, although studies have demonstrated that phytochrome A (phyA) and phyB are involved in blue light signaling, how blue light-activated phytochromes modulate the activity of the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-SUPPRESSOR OF PHYA-105 (SPA1) E3 complex remains largely unknown. Here, we show that phyA responds to early and weak blue light, whereas phyB responds to sustainable and strong blue light. Activation of both phyA and phyB by blue light inhibits SPA1 activity. Specifically, blue light irradiation promoted the nuclear import of both phytochromes to stimulate their binding to SPA1, abolishing SPA1's interaction with LONG HYPOCOTYL 5 (HY5) to release HY5, which promoted seedling photomorphogenesis.

PEAPOD repressors modulate and coordinate developmental responses to light intensity in Arabidopsis.

Higher plants adapt to different light intensities by altering hypocotyl elongation, stomatal density, seed size, and flowering time. Despite the importance of this developmental plasticity, knowledge of the underlying genetic and molecular mechanisms modulating and coordinating responses to light intensity remains incomplete. Here, I report that in Arabidopsis the PEAPOD (PPD) repressors PPD1 and PPD2 prevent exaggerated responses to light intensity. Genetic and transcriptome analyses, of a ppd deletion mutant and a PPD1 overexpression genotype, were used to identify how PPD repressors modulate the light signalling network. A ppd1/ppd2 deletion mutant has elongated hypocotyls, elevated stomatal density, enlarged seed, and delayed flowering, whereas overexpression of PPD1 results in the reverse. Transcription of both PPD1 and PPD2, upregulated in low light and downregulated in higher light, is activated by PHYTOCHROME INTERACTING FACTOR 4. I found PPDs modulate light signalling by negative regulation of SUPPRESSOR OF phyA-105 (SPA1) transcription. Whereas PPDs coordinate many of the responses to light intensity - hypocotyl elongation, flowering time, and stomatal density - by repression/de-repression of SPA1, PPD regulation of seed size occurs independent of SPA1. In conclusion PPD repressors modulate and coordinate developmental responses to light intensity by altering light signal transduction.

COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling.

Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development. Phytochromes can sense the light environment and contribute to measuring day length; thereby, they allow plants to respond and adapt to changes in the ambient environment. Two well-characterized signalling pathways act downstream of phytochromes and link light perception to the regulation of gene expression. The CONSTITUTIVELY PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA-105 (COP1/SPA) E3 ubiquitin ligase complex and the PHYTOCHROME INTERACTING FACTORs (PIFs) are key components of these pathways and repress light responses in the dark. In light-grown seedlings, phytochromes inhibit COP1/SPA and PIF activity and thereby promote light signalling. In a yeast-two-hybrid screen for proteins binding to light-activated phytochromes, we identified COLD-REGULATED GENE 27 (COR27). COR27 and its homologue COR28 bind to phyA and phyB, the two primary phytochromes in seed plants. COR27 and COR28 have been described previously with regard to a function in the regulation of freezing tolerance, flowering and the circadian clock. Here, we show that COR27 and COR28 repress early seedling development in blue, far-red and in particular red light. COR27 and COR28 contain a conserved Val-Pro (VP)-peptide motif, which mediates binding to the COP1/SPA complex. COR27 and COR28 are targeted for degradation by COP1/SPA and mutant versions with a VP to AA amino acid substitution in the VP-peptide motif are stabilized. Overall, our data suggest that COR27 and COR28 accumulate in light but act as negative regulators of light signalling during early seedling development, thereby preventing an exaggerated response to light.

Mutations in the N-terminal kinase-like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis.

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark-grown plants. While both COP1 and the four SPA proteins contain coiled-coil and WD-repeat domains, SPA proteins differ from COP1 in carrying an N-terminal kinase-like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N-terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N-terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N-terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase-like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase-like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase-like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N-terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N-terminal domain. Together, these results uncover an important, but complex role of the SPA1 N-terminus in the suppression of photomorphogenesis.

The functional divergence between SPA1 and SPA2 in Arabidopsis photomorphogenesis maps primarily to the respective N-terminal kinase-like domain.

BACKGROUND: Plants have evolved complex mechanisms to adapt growth and development to the light environment. The COP1/SPA complex is a key repressor of photomorphogenesis in dark-grown Arabidopsis plants and acts as an E3 ubiquitin ligase to ubiquitinate transcription factors involved in the light response. In the light, COP1/SPA activity is inhibited by photoreceptors, thereby allowing accumulation of these transcription factors and a subsequent light response. Previous results have shown that the four members of the SPA family exhibit partially divergent functions. In particular, SPA1 and SPA2 strongly differ in their responsiveness to light, while they have indistinguishable activities in darkness. The much higher light-responsiveness of SPA2 is partially explained by the much stronger light-induced degradation of SPA2 when compared to SPA1. Here, we have conducted SPA1/SPA2 domain swap experiments to identify the protein domain(s) responsible for the functional divergence between SPA1 and SPA2. RESULTS: We have individually swapped the three domains between SPA1 and SPA2 - the N-terminal kinase-like domain, the coiled-coil domain and the WD-repeat domain - and expressed them in spa mutant Arabidopsis plants. The phenotypes of transgenic seedlings show that the respective N-terminal kinase-like domain is primarily responsible for the respective light-responsiveness of SPA1 and SPA2. Furthermore, the most divergent part of the N-terminal domain was sufficient to confer a SPA1- or SPA2-like activity to the respective SPA protein. The stronger light-induced degradation of SPA2 when compared to SPA1 was also primarily conferred by the SPA2 N-terminal domain. At last, the different affinities of SPA1 and SPA2 for cryptochrome 2 are defined by the N-terminal domain of the respective SPA protein. In contrast, both SPA1 and SPA2 similarly interacted with COP1 in light-grown seedlings. CONCLUSIONS: Our results show that the distinct activities and protein stabilities of SPA1 and SPA2 in light-grown seedlings are primarily encoded by their N-terminal kinase-like domains. Similarly, the different affinities of SPA1 and SPA2 for cry2 are explained by their respective N-terminal domain. Hence, after a duplication event during evolution, the N-terminal domains of SPA1 and SPA2 underwent subfunctionalization, possibly to allow optimal adaptation of growth and development to a changing light environment.

Characterization of PIF4 Phosphorylation by SPA1.

The PHYTOCHROME INTERACTING FACTORs (PIFs) play pivotal roles in regulating thermo- and photo-morphogenesis in Arabidopsis. One of the main hubs in thermomorphogenesis is PIF4, which regulates plant development under high ambient temperature along with other PIFs. PIF4 enhances its own transcription and PIF4 protein is stabilized under high ambient temperature. However, the mechanisms of thermo-stabilization of PIF4 are less understood. Recently, it was shown that SUPPRESSOR OF PHYA-105 1 (SPA1) can function as a serine/threonine kinase to phosphorylate PIF4 in vitro, and the phosphorylated form of PIF4 is more stable under high ambient temperature conditions. In this chapter, we describe the in vitro kinase assay of PIF4 by SPA1. In principle, this protocol can be applied for other putative substrates and kinases.

Red-light-dependent interaction of phyB with SPA1 promotes COP1-SPA1 dissociation and photomorphogenic development in Arabidopsis.

Arabidopsis phytochromes (phyA-phyE) are photoreceptors dedicated to sensing red/far-red light. Phytochromes promote photomorphogenic developments upon light irradiation via a signaling pathway that involves rapid degradation of PIFs (PHYTOCHROME INTERACTING FACTORS) and suppression of COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) nuclear accumulation, through physical interactions with PIFs and COP1, respectively. Both phyA and phyB, the two best characterized phytochromes, regulate plant photomorphogenesis predominantly under far-red light and red light, respectively. It has been demonstrated that SPA1 (SUPPRESSOR OF PHYTOCHROME A 1) associates with COP1 to promote COP1 activity and suppress photomorphogenesis. Here, we report that the mechanism underlying phyB-promoted photomorphogenesis in red light involves direct physical and functional interactions between red-light-activated phyB and SPA1. We found that SPA1 acts genetically downstream of PHYB to repress photomorphogenesis in red light. Protein interaction studies in both yeast and Arabidopsis demonstrated that the photoactivated phyB represses the association of SPA1 with COP1, which is mediated, at least in part, through red-light-dependent interaction of phyB with SPA1. Moreover, we show that phyA physically interacts with SPA1 in a Pfr-form-dependent manner, and that SPA1 acts downstream of PHYA to regulate photomorphogenesis in far-red light. This study provides a genetic and biochemical model of how photoactivated phyB represses the activity of COP1-SPA1 complex through direct interaction with SPA1 to promote photomorphogenesis in red light.

Rap signaling in normal lymphocyte development and leukemia genesis.

Although Rap GTPases of the Ras family remained enigmatic for years, extensive studies in this decade have revealed diverse functions of Rap signaling in the control of cell proliferation, differentiation, survival, adhesion, and movement. With the use of gene-engineered mice, we have uncovered essential roles of endogenous Rap signaling in normal lymphocyte development of both T- and B-lineage cells. Deregulation of Rap signaling, on the other hand, results in the development of characteristic leukemia in manners highly dependent on the contexts of cell lineages. These results highlight crucial roles of Rap signaling in the physiology and pathology of lymphocyte development.