ABSTRACT Plant phytochromes are red/far-red photochromic photoreceptors that act as master regulators of development, controlling the expression of thousands of genes. Here, we describe the

crystal structures of four plant phytochrome sensory modules, three at about 2 Å resolution or better, including the first of an A-type phytochrome. Together with extensive spectral data,

these structures provide detailed insight into the structure and function of plant phytochromes. In the Pr state, the substitution of phycocyanobilin and phytochromobilin cofactors has no

structural effect, nor does the amino-terminal extension play a significant functional role. Our data suggest that the chromophore propionates and especially the phytochrome-specific domain

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CONTENT BEING VIEWED BY OTHERS STRUCTURAL INSIGHT INTO PIF6-MEDIATED RED LIGHT SIGNAL TRANSDUCTION OF PLANT PHYTOCHROME B Article Open access 22 May 2025 RED LIGHT-INDUCED STRUCTURE CHANGES

IN PHYTOCHROME A FROM _PISUM SATIVUM_ Article Open access 02 February 2021 THE STRUCTURE OF _ARABIDOPSIS_ PHYTOCHROME A REVEALS TOPOLOGICAL AND FUNCTIONAL DIVERSIFICATION AMONG THE PLANT

PHOTORECEPTOR ISOFORMS Article 08 June 2023 DATA AVAILABILITY The three-dimensional structural data that support the findings of this study have been deposited in wwPDB with the accession

codes 6TBY, 6TC5, 6TL4 and 6TC7. The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information files.

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Appl. Crystallogr._ 42, 339–341 (2009). CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank SFB 1078 for funding (subprojects B6 to P.H. and B8 to J.H.). The X-ray diffraction

measurements were carried out at BL14.1 at BESSY II (Helmholtz-Zentrum Berlin für Materialien und Energie (HZB)) and at ID30-A3 at ESRF with support from CALIPSOplus (Grant Agreement 730872

of the EU Framework Programme HORIZON 2020) and HZB. We thank L.-O. Essen (University of Marburg) for collaboration in the early stages of the project, W. Wende (Giessen) for cooperation in

measuring the CD spectra and C. Lang (Giessen) for expert technical assistance. We dedicate this paper to the memory of Winslow Briggs. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Institut für Pflanzenphysiologie, Justus-Liebig-Universität, Gießen, Germany Soshichiro Nagano, Kaoling Guan, Sintayehu Manaye Shenkutie & Jon Hughes * BESSY II, Helmholtz-Zentrum Berlin

für Materialien und Energie, Berlin, Germany Christian Feiler & Manfred Weiss * Institut für Chemie, Sekr. PC14, Technische Universität, Berlin, Germany Anastasia Kraskov, David Buhrke 

& Peter Hildebrandt Authors * Soshichiro Nagano View author publications You can also search for this author inPubMed Google Scholar * Kaoling Guan View author publications You can also

search for this author inPubMed Google Scholar * Sintayehu Manaye Shenkutie View author publications You can also search for this author inPubMed Google Scholar * Christian Feiler View

author publications You can also search for this author inPubMed Google Scholar * Manfred Weiss View author publications You can also search for this author inPubMed Google Scholar *

Anastasia Kraskov View author publications You can also search for this author inPubMed Google Scholar * David Buhrke View author publications You can also search for this author inPubMed 

Google Scholar * Peter Hildebrandt View author publications You can also search for this author inPubMed Google Scholar * Jon Hughes View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS S.N., G.K. and S.M.S. designed, cloned, expressed, purified and crystallized the holophytochrome constructs. S.N. solved the structures with

suggestions from C.F. and M.W. S.N. and J.H. interpreted the structures. A.K., D.B. and P.H. measured and interpreted the vibrational spectra. J.H. wrote the manuscript with the

participation of P.H. and in discussion with all authors. J.H. devised and co-ordinated the project. CORRESPONDING AUTHOR Correspondence to Jon Hughes. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 RR AND IR DIFFERENCE SPECTRA OF PHYA AND PHYB CONSTRUCTS. Left, _Sorghum bicolor_; Right _Glycine max_. ABOVE: RR spectra of

the Pr states (blue traces) and their photoconversion products (red traces) obtained upon 670 and 750 nm irradiation at ambient temperature. All spectra were measured at 90 K with 1064 nm

excitation. The spectral regions labelled are indicative of (i) the methine bridge configurations and conformations (C=C stretching modes of the _A-B_ and _C-D_ methine bridges at _ca_.

1600–1650 cm−1), (ii) pyrrole nitrogen protonation state (N-H in-plane bending modes of rings _B_ and _C_ at _ca_. 1550 – 1580 cm−1) and (iii) the _C-D_ methine bridge torsion

(hydrogen-out-of-plane [HOOP] mode at _ca_. 795 - 825 cm−1). The broad feature at _ca_. 1460 cm−1 is largely due to non-resonant Raman bands of the protein. The high intensity of this

feature relative to the RR bands of the chromophore indicates that the latter experience a low resonance enhancement. BELOW: IR “photoproduct minus Pr” difference spectra obtained upon

irradiation with 670 and 750 nm at ambient temperature. The positive signals indicated by black lines and labels refer to the photoproduct, whereas the grey lines and labels mark the signals

of the Pr state. Representative spectra based on at least two samples are shown. Spectra for each sample were measured several times. Each spectrum is based on 1000 separate FT scans.

EXTENDED DATA FIG. 2 SB.PHYB(PG)-PCB AND -PΦB STRUCTURES ARE ALMOST IDENTICAL. ABOVE: Superimposition of peptide chains with chromophores. The N- and C-termini, the two unresolved loops and

the somewhat deviant R234-D236 regions are labelled. Inset: superimposition of the PCB (cyan) and P(B (green) _D_ rings. BELOW: Chemical structures of PCB, PΦB, and the incorrect model used

in 4OUR (PDB cofactor codes CYC, O6E and 2VO, respectively). Note that the uncharged structures are shown, whereas in both Pr and Pfr holoprotein states all four cofactor nitrogens are

protonated. EXTENDED DATA FIG. 3 GM.PHYA(PG)-PCB 2.1 Å STRUCTURE (PDB 6TC7) OF SUBUNIT B. PCB (cyan), PAS (slate), GAF (gold), PCB (cyan), waters (red spheres). PW, pyrrole water. Subunit A

(grey) is superimposed. EXTENDED DATA FIG. 4 GM.PHYA(PG)-PCB 2.1 Å STRUCTURE (PDB 6TC7) OF SUBUNIT B INCLUDING SIDE CHAINS AND HYDROGEN BONDING NETWORK. Note that the weak (3.1 Å) _D_-ring

carbonyl – H370 hydrogen bond in subunit B is somewhat stronger (3.0 Å) in subunit A. PCB (cyan), PAS (slate), GAF (gold), PCB (cyan), waters (red spheres). PW, pyrrole water. EXTENDED DATA

FIG. 5 PUTATIVE CLASS I NLS SPECIFIC TO A-TYPE PLANT PHYTOCHROMES. ABOVE. Superimposition of Gm.phyA(PG)-PCB subunit B (GAF domain, gold) with subunit A (grey) superimposed. Although the

more mobile N-terminal section of the “380s” loop is missing (gold dashes), the final triad of the putative NLS (R360-R362) is resolved. BELOW. Alignment of the “380s” loop region in plant

phytochromes (from Mathews _et al_. 47). sm, _Selaginella martensii_; cp, _Ceratodon purpureus_; ac, _Adiantum caperis-veneris_; atA-D, _Arabidopsis thaliana_ PHYA-D; cpA, _Curcurbita pepo_

PHYA; psA, _Pisum sativum_ PHYA; stA/B, _Solanum tuberosum_ PHYA/B, asA-D, _Avena sativa_ PHYA; osA/B, _Oryza sativa_ PHYA/B; zmA, _Zea mais_ PHYA. The K(R/K)K(R/K) consensus is boxed red.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–3, Tables 1 and 2, and references. REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS

ARTICLE CITE THIS ARTICLE Nagano, S., Guan, K., Shenkutie, S.M. _et al._ Structural insights into photoactivation and signalling in plant phytochromes. _Nat. Plants_ 6, 581–588 (2020).

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