Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors

Golonka, David; Fischbach, Patrick; Jena, Siddhartha G.; Kleeberg, J; Essen, Lars‐Oliver; Toettcher, Jared E.; Zurbriggen, Matías D.; Möglich, Andreas

doi:10.1038/s42003-019-0687-9

Cited by 22 publications

(31 citation statements)

References 77 publications

(103 reference statements)

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“…This finding indicates that DrRR binds to the DrBphP HK, favoring the Pr state conformation. Interestingly, this contrasts the Arabidopsis thaliana phytochrome B, where the binding of the phytochrome-interacting factor (PIF) stabilizes the Pfr state 36 .…”

Section: The Spectral Properties Of Drbphp Are Affected By Drrrmentioning

confidence: 95%

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

Multamäki

Nanekar

Morozov

et al. 2020

Preprint

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Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this longstanding conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics. radiodurans (DrBphP), light induces extensive structural changes in the photosensory module that are relayed to the output module 29 .In terms of enzymatic activity, the dark-adapted Pr state exhibited higher kinase activity than the Pfr state in cyanobacterial phytochrome Cph1, 27,30,31 , similar to other bacteriophytochromes 32 . In particular, the bacteriophytochrome from Agrobacterium fabrum (AgP1) displays histidine kinase activity in its resting Pr state 28,33 ; in the Pfr state, the autophosphorylation and phosphotransfer reactions are down-regulated by 2-fold and 10-fold, respectively 28 . This kinase activity of the AgP1 has been shown to control bacterial conjugation 34 . By contrast, no kinase activity has been demonstrated for DrBphP, notwithstanding close sequence homology and the elaborate structural changes this receptor undergoes under light 22,29 . Despite the eminent role of DrBphP as a paradigm for photoreception, the enzymatic activity and the physiological role of this model phytochrome have hence remained enigmatic.Here, we unravel this long-standing puzzle by studying the enzymatic activity and interactions of DrBphP and AgP1, as two canonical bacteriophytochromes with HK effector domains. By pursuing an integrated biochemical and structural strategy, we show that despite close homology, AgP1 acts as a histidine kinase whereas DrBphP functions as a light-activated phosphatase. Our biochemical and structural data pinpoint two key residues proximal to the catalytic histidine that affect the balance between the kinase and phosphatase activities. Together, the two phytochromes provide soluble, lightcontrollable systems with opposite activities for the study and application of two-component signaling.

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Section: The Spectral Properties Of Drbphp Are Affected By Drrrmentioning

confidence: 95%

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

Multamäki

Nanekar

Morozov

et al. 2020

Preprint

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“…As positive controls, known interaction pairs were transfected (PhyA/FHL; PhyB/PIF6). Exchange of media and other cell‐handling was done under 522 nm safe light, to prevent inadvertent activation of the light‐sensitive systems (Baaske et al ., 2018; Golonka et al ., 2019).…”

Section: Methodsmentioning

confidence: 99%

“…The split transcription factor system for light controlled gene expression in eukaryotic cells was based on a previously reported setup (Müller et al ., 2013; Müller et al ., 2014; Golonka et al ., 2019). For testing of COR27 and COR28, At PIF6 (1–100) was replaced by the corresponding coding sequences by AQUA cloning (Beyer et al ., 2015).…”

Section: Methodsmentioning

confidence: 99%

“…CHO‐K1 cells (5 × 10 4 ) were transfected using polyethylenimine (PEI; Polysciences Inc. Europe, Hirschberg, Germany; no. 23966‐1) (Baaske et al ., 2018; Golonka et al ., 2019). DNA (0.75 µg) was diluted in 50 μl OptiMEM (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and mixed with a PEI/OptiMEM mix (2.5 μl PEI solution in 50 μl OptiMEM).…”

Section: Methodsmentioning

confidence: 99%

“…As positive controls, known interaction pairs were transfected (PhyA/FHL; PhyB/PIF6). Exchange of media and other cell-handling was done under 522 nm safe light, to prevent inadvertent activation of the lightsensitive systems (Baaske et al, 2018;Golonka et al, 2019). For SEAP activity measurement, the supernatant of transfected cells was transferred to 96-well round-bottom microtitre plates and incubated at 68°C for 1 h to inactivate endogenous phosphatases.…”

Section: M2h Assaymentioning

confidence: 99%

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

et al. 2020

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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, farred 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.

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Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives

Fan

Skeeters

et al. 2021

Advanced Biology

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In a transparent medium, lensbased optical microscopy could focus a coherent light beam (e.g., laser) into a tiny spot, whose dimension is comparable to the size of the wavelength of the light. The diameter of the smallest beam waist is about half the size of the wavelength, which is referred to as the diffraction limit. Thus, for visible light, the theoretical diffraction limit is between 200 and 400 nm, much smaller than the size of a single cell (Figure 1A). However, in biological tissues that significantly scatter and absorb visible light, the spatial resolution could be compromised. In multicellular organisms, light absorption limits the penetration depth. The scattering of the light by the opaque biological tissues would expand the volume of light stimulation and reduce the spatial resolution.

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Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors

Cited by 22 publications

References 77 publications

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

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

Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives

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