A collaboration between the two groupsled by Drs. Jizong Wang and Xing Wang Deng at the Shandong Laboratory of Advanced Agricultural Sciences in Weifang /Beijing University Institute of Advanced Agricultural Scienceshas unraveled a novel mechanism of plant photomorphogenic signal transduction! The results have been published in the prestigious international journal "Cell" (https://doi.org/10.1016/j.cell.2024.09.005)

  

Sunlight serves not only as the energy source for plant growth but also as a crucial signal regulatingplant development processes. Phytochromesplay a pivotal role as the red/far-red light sensors in plants, acting as the "eyes" of plants. In the face of increasing global climate change and the gradual reduction of arable land resources, ensuring global food security poses a significant challenge. Over the past century, the continuous increase in crop yield has been attributed to the development of crop varieties that are more tolerant of high planting density. Future improvements in crop yield will also rely on the cultivation of new varieties that can withstand even denser planting. As the light perception mechanism and high-density productivity traits of plants in shaded environments are closely related to plant phytochromes, elucidating the response and signal transduction mechanisms of plant phytochromes will help enhance crop performance in dense planting conditions and improve their adaptability to complex environments, thereby contributing to food security.

Higher plants primarily encode two types of phytochromes, represented by phytochromeA (phyA) and phytochromeB (phyB). Among them, phyB is the predominant red-light receptor mediating reversible red-light responses. Phytochromes undergo reversible transitions between the red-light-absorbing state (Pr, ground state) and the far-red-light-absorbing state (Pfr, active state) through their chromophore moiety PΦB. Upon light activation, phyB in Arabidopsis can directly interact with a class of phytochrome-interacting-factors (PIFs), transmitting light signals and regulating downstream gene expression to promote photomorphogenesis. Therefore, phyB and PIFs constitute crucial signaling modules for plant response to the ambientlight environment. The eight members of PIFs in Arabidopsis (PIF1-8) all contain two important domains: an N-terminal active-PHYB binding motif (APB) that interacts with phyB-Pfr and a C-terminal bHLH domain that binds to DNA. Under dark conditions, phyB-Pr localizes in the cytoplasm, while PIF1/3/4/5 act in the nucleus to drive the developmental processes of etiolated seedlings (hypocotyl elongation, cotyledon closure). Once seedlings perceive red light, phyB-Pfrtranslocates into the nucleus, negatively regulates the aforementioned PIFs, inhibits hypocotyl elongation, promotes cotyledon opening, and maintains plant photomorphogenesis. Although the Vierstra team in the United States revealed the structure of phyB-Pr in a 2022 publication in Nature, the structural biology basis of phyB-Pfr and its recognition of PIFs, as well as the underlying regulatory mechanisms, remain largelyunknown.

On 23th September 2024, the research group led by Drs. Jizong Wang and Xing Wang Deng from the institute published a research article titled "Light-induced remodeling of phytochrome B enables signal transduction by phytochrome-interacting factor" in the journal Cell, unveiling the initial response mechanism for phyB light signal transduction that has been eagerly waiting for a long time.

This study elucidated the high-resolution cryo-electron microscopy structures (at resolutions of 3.1 Å and 3.2 Å, respectively) of the complex formed by the light-activated phyB-Pfrorthe constitutively activephyB^Y276H mutant, bound to the downstream signaling molecule PIF6. Based on structural analysis, spectroscopic characterization of phyB mutants, in vitro and semi-in vitro biochemical assays, as well as phenotype analysis of transgenic plants, the detailed molecular mechanism of photoactivation of phyB from Pr to Pfr was revealed. Additionally, a model of "induced-fit" between phyB and PIFs was proposed, filling a critical gap in the research of plant phytochromesignal transduction mechanisms.

Considering the abundance of disordered structures in full-length PIF proteins, there are great challenges in the in vitro reconstitution and assembling of phyB-PIF full-length protein complexes. Given the growing body of literature suggesting that the APB motif (the N-terminal 100 amino acids) of PIFs is sufficient for their interaction with phyBin vivo and in vitro, the researchers selected PIF6-APB (PIF6-100, consisting of only the N-terminal 100 amino acids) with the strongest binding ability to assemble the phyB-PIF complex and prepare cryo-electron microscopy samples. Ultimately, thehigh-resolution cryo-electron microscopy structures of the phyB-Pfr-PIF6 and phyB^Y276H-908-PIF6 complexes, lacking the HKRD domain (containing only the N-terminal 908 amino acids), were successfully resolved. Structural analysis revealed that phyB-Pfr undergoes extensive conformational rearrangements upon light activation, with its photosensory module (PSM) transitioning from a "head-to-tail" dimer in Pr state to a "head-to-head" dimer in Pfr state, with PIF6-APB monomer binding on one side of the phyB-Pfr dimer interface, forming a phyB-PIF6 trimer. Furthermore, the structure of the phyB^Y276H-908-PIF6 complex is nearly identical to that of the phyB-Pfr-PIF6 complex (Figure 1).

 

Figure 1 The asymmetric trimeric structures of phyB-Pfr-PIF6 (B) and phyB^Y276H-PIF6 (C).

 

In order to elucidate the structural rearrangement of photoactivatedphyB, the authors conducted a detailed comparison between the previously reported structure of phyB-Pr and the phyB-Pfr structure obtained in this study. They observed that the chromophore molecule PΦB undergoes a 180˚ flip in the D-ring upon red light absorption, establishing a new interaction network with a series of amino acids within the pocket (Figure 2A, left). This ultimately leads to a conformational transition of the tongue-like protrusion within the PHY domain from a β-sheet to an α-helix (Figure 2A, right). Structural analysis reveals that serine S584 plays a crucial role in stabilizing the α-helical conformation of the tongue-like structure (Figure 2A, right). Further biochemical assays and phenotype analysis of transgenic plants demonstrate the vital role of S584 in maintaining the activated state of phyB-Pfr and the phyB signaling pathway (Figure 2B).

Figure 2 The conformational transition and functional validation of the light-induced PΦB molecule (A, left) and the tongue-like protrusion structure in the PHY domain (A, right). The functional validation of these structures is depicted in panel (B).

 

In phyB-Pr, the C-terminal domain PAS2 and HKRD interact with the N-terminal domains GAF and PHY to maintain a "head-to-tail" dimeric conformation. However, in phyB-Pfr, when the tongue-like protrusion of the PHY domain transitions into an α-helix, it directly clashes with the PAS2 domain in phyB-Pr, disrupting the extensive interactions between PAS2 and PHY domains (Figure 3A, left; Figure 3B, left). This further disrupts the intramolecular interactions between HKRD and PHY domains (Figure 3A, left; Figure 3B, middle), as well as between HKRD and GAF domains (Figure 3A, right; Figure 3B, right), completely breaking the "head-to-tail" structure in phyB-Pr and ultimately activating phyB. Additionally, the truncated form of phyB lacking the HKRD domain (phyB-908, consisting of only the N-terminal 908 amino acids) tends to exist as a monomer after light activation (Figure 3C), further supporting that the "head-to-tail" dimer, which was originally maintained by interactions between the N-terminal and C-terminal modules, is disrupted upon light activation, resulting in the formation of phyB-Pfr.

Figure 3 The conformational transition of the tongue-like protrusion structure in the PHY domain, which drives the rearrangement from phyB-Pr to phyB-Pfr (A, B). The biochemical validation of this transition is depicted in panel (C).

 

After light activation, phyB-Pfr utilizes its N-terminal NTE domain and the "head-to-head" dimer formed by the overall N-terminal domain to recognize and bind to both the N-terminal and C-terminal regions of PIF6-APB (Figure 4A). The N-terminal region of PIF6-APB extensively interacts with the NTE domain of phyB-Pfr by forming a β-hairpin structure (PIF6-APB-β) (Figure 4B). It should be noted that the other phyB molecule within the phyB-Pfr dimer does not possess a stable NTE domain, indicating that the stable structure of the NTE domain is induced by the binding of PIF6-APB. Additionally, the C-terminal region of PIF6-APB forms an α-helix structure (PIF6-APB-α) and interacts extensively with the N-terminal domains of both phyB-Pfr molecules (Figure 4C), confirming the role of PIF6-100 in promoting dimerization of phyB-908-Pfr (Figure 3C). Furthermore, the authors employed a series of biochemical and spectroscopic experiments, as well as the phenotype characterization of transgenic plants, to delineate the importance of the NTE domain and other N-terminal domains in mediating the dimerization of phyB-Pfr and its interaction with PIF6.

Figure 4 The specific recognition of PIF-APB by the N-terminal domains of phyB-Pfr (A), as well as the NTE domain's specific recognition of the N-terminal β-hairpin structure of PIF-APB (B). The dimeric phyB-Pfr also exhibits specific recognition of the C-terminal α-helix structure of PIF-APB (C).

 

The structural alignment of the two phyB-Pfrprotomers from phyB-Pfr dimerreveals the formation of an asymmetric dimer in the phyB-PIF6 complex(Figure 5A). These asymmetries result in the lack of sufficient space on the opposite interface of the phyB-Pfr dimer to accommodate proper interactions with both PIF6-APB-β and PIF6-APB-α (Figure 5B). To further assess the interaction modes of phyB with PIF-APB and PIF-FL in solution, the authors conducted pull-down and Co-IP assays. The biochemical data demonstrated that the APB motifs of PIF3 and PIF6, as well as the full-length proteins of PIF1 and PIF3, can form complexes with the phyB-Pfr dimer at a molar ratio of 1:2 (2:4).

Figure 5 Depicting that the asymmetric phyB-Pfr dimer can only bind to a single PIF6-APB monomer.

 

In summary, this study reveals the complex structure of the "head-to-head" activated phyB dimer recognizing and binding to PIF6-APB, answered a critical question in the field of phytochromeresearch regarding how light induces conformational changesof phyB and transduces PIF signals (Figure 6). This work provides a blueprint at the molecular level for the modification of light sensitivity traits in crops and the development of optogenetic tools for controlling gene expression related to phyB signaling.

 

Figure 6 A schematic model illustrating the structural mechanism of red light-induced activation of phyB and transduction of PIF signals.

The corresponding authors for this paper are Professors Jizong Wang and Xing Wang Dengfrom the School of Advanced Agricultural Sciences, Peking University, and the Peking University Institute of Advanced Agricultural Sciences. The co-first authors of the paper are MrZhengdongWang and MsYanping Song, doctoral candidates from the Academy for Advanced Interdisciplinary Studies, Peking University, as well as MsWenfeng Wang, MsDidi Zhao, and Dr. XiaoliLin from the Peking University Institute of Advanced Agricultural Sciences. Dr. Jun Zhao, Dr. Cheng Chi, and Senior Engineer MrBin Xu from the Peking University Institute of Advanced Agricultural Sciences, and doctoral candidate Ms.Meng Shen from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, also made significant contributions to this research. The sample preparation, screening, and data collection were carried out at the Cryo-Electron Microscopy FacilityPlatform of the Institute.

This study received funding support from the National Natural Science Foundation of China, the National Key Research and Development Program of China, the Key Research and Development Program of Shandong Province, China,the Shandong Provincial Science and Technology Innovation Fund, the Young Elite Scientists Sponsorship Program by CAST, the Taishan Scholars Program of Shandong Province, the Peking University Institute of Advanced Agricultural Sciences, the Shandong Laboratory of Advanced Agricultural Sciences in Weifang, National Key Laboratory of Wheat Improvement, and the StateKey Laboratory of Protein and Plant Gene Research, Peking University.

Introduction to the Cryo-Electron Microscopy Facility Platform

The Cryo-Electron Microscopy Facility Platform is one of the seven key public platforms established by the Peking University Institute of Advanced AgriculturalSciences. The platform started the installation of cryo-electron microscopy and related equipment in November 2021, underwent debugging in March 2022, and official operation in September 2022. Since its operation for two years, the platform has supported researchers from within and outside the institute in publishing multiple high-level works in journals such as Nature (5 papers) and Cell (2 papers). It can facilitate various electron microscopy data collection and sample preparation tasks, including single-particle protein 3D structure analysis and cryo-electron tomography reconstruction, among others. This paper (the second paper in Cell) is the first published work in a premier journal with the Institute being the first author's affiliation. Much of the work for this achievement was carried out in the Institute, and authors would like to express their gratitude for the significant support from the various levels of the Shandong provincial government and the Peking University Institute of Advanced Agricultural Sciences/the Shandong Laboratory of Advanced Agricultural Sciences in Weifang. Currently, the platform is open for reservations to professional users, providing electron microscopy technologies to assist cutting-edge research in the field of life sciences.