The blue light-induced interaction of cryptochrome 1 with COP1 requires SPA proteins during Arabidopsis light signaling.

Plants constantly adjust their growth, development and metabolism to the ambient light environment. Blue light is sensed by the Arabidopsis photoreceptors CRY1 and CRY2 which subsequently initiate light signal transduction by repressing the COP1/SPA E3 ubiquitin ligase. While the interaction between cryptochromes and SPA is blue light-dependent, it was proposed that CRY1 interacts with COP1 constitutively, i.e. also in darkness. Here, our in vivo co-immunoprecipitation experiments suggest that CRY1 and CRY2 form a complex with COP1 only after seedlings were exposed to blue light. No association between COP1 and CRY1 or CRY2 was observed in dark-grown seedlings. Thus, our results suggest that cryptochromes bind the COP1/SPA complex after photoactivation by blue light. In a spa quadruple mutant that is devoid of all four SPA proteins, CRY1 and COP1 did not interact in vivo, neither in dark-grown nor in blue light-grown seedlings. Hence, SPA proteins are required for the high-affinity interaction between CRY1 and COP1 in blue light. Yeast three-hybrid experiments also show that SPA1 enhances the CRY1-COP1 interaction. The coiled-coil domain of SPA1 which is responsible for COP1-binding was necessary to mediate a CRY1-SPA1 interaction in vivo, implying that-in turn-COP1 may be necessary for a CRY1-SPA1 complex formation. Hence, SPA1 and COP1 may act cooperatively in recognizing and binding photoactivated CRY1. In contrast, the blue light-induced association between CRY2 and COP1 was not dependent on SPA proteins in vivo. Similarly, ΔCC-SPA1 interacted with CRY2, though with a much lower affinity than wild-type SPA1. In total, our results demonstrate that CRY1 and CRY2 strongly differ in their blue light-induced interaction with the COP1/SPA complex

The B-induced <i>in vivo</i>-association of COP1 with CRY1 requires SPA proteins, while COP1 associates with CRY2 independently of SPA.

<p>(<b>A</b>, <b>B</b>) Co-immunoprecipitation of CRY1 (<b>A</b>) and CRY2 (<b>B</b>) by YFP-COP1 in a <i>SPA</i> wild-type (<i>YFP-COP1</i>) or <i>spa</i> null background (<i>YFP-COP1 spaQn</i>). Seedlings were grown in darkness for 4 days (D) and subsequently transferred to blue light (B) of a fluence rate of 50 μmol m^-2 s^-1 for 1 h (<b>A</b>) or 5 min (<b>B</b>). Protein extracts were immunoprecipitated using α-GFP beads. YFP-COP1 was detected using α-GFP antibodies. YFP-COP1 expression is very low and frequently only detectable after immunoprecipitation. CRY1 and CRY2 were detected using α-CRY1 and α-CRY2 antibodies. Asterisks likely indicate phosphorylated CRY1 and CRY2, respectively. The samples shown in (<b>B</b>) were on the same membrane with one lane digitally removed in the center.</p

COP1 associates with CRY1 and CRY2 in a blue-light dependent manner <i>in vivo</i>.

<p>(<b>A</b>, <b>B</b>) Co-immunoprecipitation of CRY1 (<b>A</b>) and CRY2 (<b>B</b>) by YFP-COP1. Transgenic 35S::<i>YFP-COP1</i> seedlings were grown in darkness (D) for 4 days and subsequently transferred to blue light (B) of a fluence rate of 50 μmol m^-2 s^-1 for 1 h (<b>A</b>) or 5 min (<b>B</b>). Protein extracts were immunoprecipitated using α-GFP beads. YFP-COP1 was detected using α-GFP antibodies; CRY1 and CRY2 were detected using α-CRY1 and α-CRY2 antibodies. Asterisks likely indicate phosphorylated CRY1 and CRY2, respectively. All signals in <b>(A)</b> and <b>(B)</b> were from the same respective membrane. The YFP-COP1 signals <b>(A, B)</b> of the input samples were from longer exposures than those from the co-immunoprecipitates. The CRY1 signals <b>(A)</b> of the input samples were from shorter exposure than those from the co-immunoprecipitates.</p

Co-expression of SPA1 increases the interaction between COP1 and CRY1 in yeast three-hybrid experiments.

<p><b>(A)</b> Yeast two-hybrid assay analyzing the interaction between CRY1 and COP1. (<b>B</b>) Yeast three-hybrid assay analyzing the CRY1-COP1 interaction in the presence or absence of co-expressed SPA1. (<b>C</b>) Yeast three-hybrid assay analyzing the CRY1-SPA1 interaction in the presence or absence of co-expressed COP1. Co-transformed yeast cells were grown in darkness for 24 h and exposed to B (50 μmol m^-2 s^-1) or kept in darkness for 24 h before measuring ß-galactosidase activity. Error bars represent the SEM of three biological replicates. Asterisks indicate significant differences between the indicated comparisons (** <i>p</i> < 0.01, *** <i>p</i> < 0.001, n.s. not significant at <i>p</i> = 0.05).</p

A SPA1 deletion-protein defective in COP1-interaction shows no or a strongly reduced <i>in vivo</i>-association with CRY1 and CRY2, respectively.

<p>(<b>A</b>) ΔCC SPA1-HA lacking the coiled-coil domain fails to interact with COP1 <i>in vivo</i>. Seedlings were grown in darkness for 4 d. <b>(B, C)</b> Co-immunoprecipitation of CRY1 (<b>B</b>) and CRY2 (<b>C</b>) by SPA1-HA and ΔCC SPA1-HA. Seedlings were grown in darkness for 4 days (D) and subsequently transferred to blue light (B) of a fluence rate of 50 μmol m^-2 s^-1 for 1 h (<b>B</b>) or 5 min (<b>C</b>). HA-tagged proteins were immunoprecipitated using α-HA-coupled beads. SPA1-HA and ΔCC SPA1-HA were detected using α-HA antibodies. CRY1 and CRY2 were detected using α-CRY1 and α-CRY2 antibodies. Asterisks likely indicate phosphorylated CRY1 and CRY2, respectively. Images in (<b>B</b>) separated by a vertical bar represent the same membrane which was exposed for different periods of time.</p

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 DN 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

A fast and simple LC-MS-based characterization of the flavonoid biosynthesis pathway for few seed(ling)s

Background: (Pro)anthocyanidins are synthesized by the flavonoid biosynthesis pathway with multi-layered regulatory control. Methods for the analysis of the flavonoid composition in plants are well established for different purposes. However, they typically compromise either on speed or on depth of analysis. Results: In this work we combined and optimized different protocols to enable the analysis of the flavonoid biosynthesis pathway with as little as possible biological material. We chose core substances of this metabolic pathway that serve as a fingerprint to recognize alterations in the main branches of the pathway. We used a simplified sample preparation, two deuterated internal standards, a short and efficient LC separation, highly sensitive detection with tandem MS in multiple reaction monitoring (MRM) mode and hydrolytic release of the core substances to reduce complexity. The method was optimized for Arabidopsis thaliana seeds and seedlings. We demonstrate that one Col-0 seed/seedling is sufficient to obtain a fingerprint of the core substances of the flavonoid biosynthesis pathway. For comparative analysis of different genotypes, we suggest the use of 10 seed(lings). The analysis of Arabidopsis thaliana mutants affecting steps in the pathway revealed foreseen and unexpected alterations of the pathway. For example, HY5 was found to differentially regulate kaempferol in seeds vs. seedlings. Furthermore, our results suggest that COP1 is a master regulator of flavonoid biosynthesis in seedlings but not of flavonoid deposition in seeds. Conclusions: When sample numbers are high and the plant material is limited, this method effectively facilitates metabolic fingerprinting with one seed(ling), revealing shifts and differences in the pathway. Moreover the combination of extracted non-hydrolysed, extracted hydrolysed and non-extracted hydrolysed samples proved useful to deduce the class of derivative from which the individual flavonoids have been released

Additional file 1: Figure S1. of The functional divergence between SPA1 and SPA2 in Arabidopsis photomorphogenesis maps primarily to the respective N-terminal kinase-like domain

The coiled-coil and WD-repeat domains of SPA1 do not provide higher stability to the chimeric SPA2 protein in light-grown seedlings. A, B. SPA-HA protein levels in 4-day-old T2 DS_212-HA (A) or DS_221-HA (B) transgenic spa1 spa2 spa3 mutant seedlings. Seedlings were grown in darkness (D) for 4 days and subsequently transferred to 0.35 μmol m−2 s−1 FR for 30 min. All transgenes were expressed under the control of the SPA2 promoter. SPA-HA was detected using an α–HA antibody. HSC70 levels served as a loading control. (PDF 245 kb

Additional file 3: Table S1. of The functional divergence between SPA1 and SPA2 in Arabidopsis photomorphogenesis maps primarily to the respective N-terminal kinase-like domain

Primer sequences. (PDF 32 kb