Research Group Hiltbrunner

Light Perception and Signalling in Plants

Institute of Biology II, Department of Molecular Plant Physiology, Faculty of Biology, University of Freiburg

Background and research projects

Phytochromes — Red/far-red light receptors in plants

Light plays a crucial role for growth and development of plants. Light drives photosynthesis and thereby provides energy and reducing power for carbon fixation and other metabolic processes. However, light is also a source of information, allowing plants to perceive and adapt to changes in the ambient environment. Phytochromes are red/far-red light sensing photoreceptors that control almost every step in the life cycle of plants, including seed germination, seedling establishment, growth and flowering (Legris et al., 2019).

Phytochromes can exist in two different states, the inactive Pr state and the physiologically active Pfr state (Fig. 1). Pr and Pfr have absorption peaks in red and far-red light, respectively, and reversibly convert into each other by absorption of light. Since the absorption spectra of Pr and Pfr overlap, a wavelength-dependent equilibrium between Pr and Pfr is established when phytochromes are exposed to light (Legris et al., 2019; Mancinelli, 1994).

Phytochrome diversity in seed plants

Seed plants contain several phytochromes, including phyA and phyB. These phytochromes are the result of gene duplication events early in the evolution of seed plants (Li et al., 2015; Wang et al., 2020). PhyB is most active under conditions that lead to high levels of Pfr, such as monochromatic red light and sunlight. In contrast, the physiological activity of phyA is highest when Pfr levels are low, such as in monochromatic far-red light, deep canopy shade and very weak light, or upon exposure to a short light stimulus (Legris et al., 2019). This difference between phyA and phyB is obvious in seedlings deficient in either phyA or phyB grown in monochromatic red or far-red light. In wildtype seedlings, hypocotyl growth is repressed in both red and far-red light compared to dark-grown seedlings, while this response is strongly reduced in red light-grown phyB mutant seedlings and entirely absent in phyA mutant seedlings grown in far-red light (Fig. 2A).

Response to canopy shade

In particular phyB also plays an import role in sensing canopy shade and potential competitors (Fig. 2B-D) (Legris et al., 2019; Huber et al., 2021). The red and far-red light content in sunlight is roughly equal, i.e. the red:far-red light ratio is approx. 1 (Fig. 2B). In canopy shade, however, the red:far-red ratio is strongly reduced, since photosynthetic pigments in shading leaves predominantly absorb in the red light range of the light spectrum, while far-red light is transmitted and reflected (Fig. 2D). Due to the reflection of far-red light, the red:far-red ratio is also reduced in proximity to potential competitors. Far-red light reflected from leaves of competitors leads to an increase of the far-red light content and therefore a reduced red:far-red light ratio. This in turn leads to inactivation of phyB and derepression of growth, allowing plants to outgrow competitors (Fig. 2E).

Phytochrome signal transduction – link between photoreceptor activation and gene expression

The expression of about 25% of the genes in Arabidopsis is regulated by light and phytochromes play an important role in this process. When activated by light, phytochromes are transported from the cytosol into the nucleus, where they interact with different downstream signalling components (Fig. 3A) (Legris et al., 2019; Klose et al., 2015). PIFs, COP1, SPAs and HY5 are well investigated components of phytochrome downstream signalling (Fig. 3B). PIFs are bHLH transcription factors that inhibit light responses. Light-activated phytochromes bind PIFs and repress their activity, thereby promoting responses to light. In parallel, phytochromes also inhibit COP1/SPA action in light-grown plants. COP1/SPA are part of an E3 ubiquitin ligase complex that targets positive factors of light signalling for degradation. Inhibition of COP1/SPA action in light therefore leads to the accumulation of HY5 and other transcription factors that promote light responses (Legris et al., 2019).

Research projects

Research projects in the lab aim at understanding phytochrome action at the level of the photoreceptor itself, at the level of downstream signalling, and in terms of their evolutionary origin.

Investigation of phytochromes using in vitro and in planta approaches

Although phytochromes have been identified decades ago, there are many aspects that (potentially) have a strong impact on physiological responses, but still have not been investigated in detail. This includes, for instance, effects of phytochrome dimerisation, thermal reversion, and formation of photobodies (see below) on phytochrome-mediated responses.

Phytochromes are obligate dimers. The current view is that phyA only forms homodimers, while phyB and other phytochromes form various homo- and heterodimers. We are using recombinant phytochromes expressed in E. coli to investigate properties of specific phytochrome homo- and heterodimers in vitro, as well as specific phytochrome higher order mutants to assess the physiological activity of phytochrome dimers in vivo.

Thermal reversion is a light-independent but temperature-dependent process by which phytochromes revert from the active Pfr to the inactive Pr state. In particular the physiological activity of phyB is strongly determined by thermal reversion. Thermal reversion competes with Pr→Pfr photoconversion and contributes to measuring the light intensity during the day. Thermal reversion also determines the persistence of phytochrome signalling after the onset of the dark phase and can play a role in measuring night-length. The rate of thermal reversion depends on the type of phytochrome, post-translational modifications and interacting proteins, such as PCH1 (Fig. 4). Since thermal reversion is accelerated at higher temperature, phytochromes also play a role as thermosensors under certain conditions (Klose et al., 2020). Using a wide range of phytochrome mutants or mutants deficient in proteins that affect thermal reversion, we are investigating the impact of thermal reversion on physiological responses controlled by phytochromes.

Further reading:

• Viczián, A., Ádám, , Staudt, A.-M., Lambert, D., Klement, E., Romero Montepaone, S., Hiltbrunner, A., Casal, J., Schäfer, E., Nagy, F., and Klose, C. (2020). Differential phosphorylation of the N-terminal extension regulates phytochrome B signaling. New Phytol. 225, 1635-1650. [PubMed] [doi: 10.1111/nph.16243]
• Klose, C., Nagy, F., and Schäfer, E. (2020). Thermal reversion of plant phytochromes. Mol. Plant 13, 386-397. [PubMed] [doi: 10.1016/j.molp.2019.12.004]
• Enderle, B., Sheerin, D.J., Paik, I., Kathare, P.K., Schwenk, P., Klose, C., Ulbrich, M.H., Huq, E., and Hiltbrunner, A. (2017). PCH1 and PCHL promote photomorphogenesis in plants by controlling phytochrome B dark reversion. Nat. Commun. 8, 2221. [PubMed] [doi: 10.1038/s41467-017-02311-8]
• Klose, C., Venezia, F., Hussong, A., Kircher, S., Schäfer, E., and Fleck, C. (2015). Systematic analysis of how phytochrome B dimerization determines its specificity. Nat. Plants 1, 15090. [PubMed] [doi: 10.1038/nplants.2015.90]

Phytochrome downstream signalling

A number of proteins that play a role in phytochrome downstream signalling have been identified through large-scale genetic screens. However, mutants deficient in proteins that act redundantly or that are essential for survival are hard to identify in genetic screens. We therefore used a yeast-two-hybrid screen to search for proteins that interact with light-activated full-length phytochromes and therefore are potential factors of phytochrome downstream signalling (Sheerin et al., 2015). We have already characterised a number of phytochrome-interacting proteins identified in this screen and confirmed that they play a role in phytochrome signalling. This includes PCH1 and PCHL, which delay thermal reversion of phyB and promote the formation of photobodies (Fig. 4) (Enderle et al., 2017), NOT9B, a component of the CCR4-NOT complex (Schwenk et al., 2021), ERF55 and ERF58, members of the AP2/ERF transcription factor family (Li et al., 2022), as well as COR27 and COR28 (Kahle et al., 2020). Research projects in the lab aim at understanding the molecular mechanism by which these proteins control light signalling downstream of phytochromes. Also other phytochrome-interacting proteins found in the yeast-two-hybrid screen are under investigation.

Further reading:

• Li, Z., Sheerin, D.J., von Roepenack-Lahaye, E., Stahl, M., and Hiltbrunner, A. (2022). The phytochrome interacting proteins ERF55 and ERF58 repress light-induced seed germination in Arabidopsis thaliana. Nat. Commun. 13, 1656. [PubMed] [doi: 10.1038/s41467-022-29315-3]
• Schwenk, P., Sheerin, D.J., Ponnu, J., Staudt, A.-M., Lesch, K.L., Lichtenberg, E., Medzihradszky, K.F., Hoecker, U., Klement, E., Viczián, A., and Hiltbrunner, A. (2021). Uncovering a novel function of the CCR4-NOT complex in phytochrome A-mediated light signalling in plants. eLife 10, e63697. [PubMed] [doi: 10.7554/eLife.63697]
• Kahle, N., Sheerin, D.J., Fischbach, P., Koch, L.-A., Schwenk, P., Lambert, D., Rodriguez, R., Kerner, K., Hoecker, U., Zurbriggen, M.D., and Hiltbrunner, A. (2020). COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling. Plant J. 104, 1038-1053. [PubMed] [doi: 10.1111/tpj.14979]
• Enderle, B., Sheerin, D.J., Paik, I., Kathare, P.K., Schwenk, P., Klose, C., Ulbrich, M.H., Huq, E., and Hiltbrunner, A. (2017). PCH1 and PCHL promote photomorphogenesis in plants by controlling phytochrome B dark reversion. Nat. Commun. 8, 2221. [PubMed] [doi: 10.1038/s41467-017-02311-8]
• Sheerin, D.J., Menon, C., zur Oven-Krockhaus, S., Enderle, B., Zhu, L., Johnen, P., Schleifenbaum, F., Stierhof, Y.-D., Huq, E., and Hiltbrunner, A. (2015). Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex. Plant Cell 27, 189-201. [PubMed] [doi: 10.1105/tpc.114.134775]

Evolution and diversification of phytochromes and phytochrome signalling pathways

Canonical plant phytochromes originated in a common ancestor of extant streptophytes (Li et al., 2015; Wang et al., 2020; Rockwell and Lagarias, 2020). While liverworts and hornworts typically have a single phytochrome, independent gene duplications resulted in small phytochrome families in seed plants, ferns and mosses (Fig. 5A). Functional diversification into phytochromes specifically sensing red or far-red light is well documented for seed plants, but has not been explored for other land plants. In a recent study, we have addressed this question for moss phytochromes and found diversification into phytochromes that specifically sense either red or far-red light and phytochromes that contribute to responses under both light conditions (Fig. 5A, B) (Yuan et al., 2023). In contrast, the functional diversification of fern phytochromes has not yet been investigated. Moreover, the molecular determinants underlying functional diversification into either red or far-red light-specific phytochromes are still largely unknown, and it is unclear if factors that determine the wavelength specificity of phytochromes are conserved across mosses, ferns and seed plants. In a project within the DFG-funded Priority Programme SPP 2237 "MAdLand: Molecular Adaptation to Land", we aim to clarify these questions and identify the molecular basis for the functional diversification of phytochromes.

Similar to phytochromes in seed plants, also phytochromes in the moss Physcomitrium patens and the liverwort Marchantia polymorpha accumulate in the nucleus when activated by light (Fig. 5C) (Possart and Hiltbrunner, 2013; Inoue et al., 2016). Despite some similarities in phytochrome downstream signalling in seed plants, mosses and liverworts, it is clear that also important mechanistic differences exist (Inoue et al., 2017). In our research, we aim at understanding common and lineage-specific mechanisms.

Further reading:

• Yuan, J., Xu, T., and Hiltbrunner, A. (2023). Phytochrome higher order mutants reveal a complex set of light responses in the moss Physcomitrium patens. New Phytol. 239, 1035-1050. [PubMed] [doi: 10.1111/nph.18977]
• Possart, A., Xu, T., Paik, I., Hanke, S., Keim, S., Hermann, H.-M., Wolf, L., Hiß, M., Becker, C., Huq, E., Rensing, S.A., and Hiltbrunner, A. (2017). Characterization of phytochrome interacting factors from the moss Physcomitrella patens illustrates conservation of phytochrome signaling modules in land plants. Plant Cell 29, 310-330. [PubMed] [doi: 10.1105/tpc.16.00388]
• Possart, A. and Hiltbrunner, A. (2013). An evolutionarily conserved signaling mechanism mediates far-red light responses in land plants. Plant Cell 25, 102-114. [PubMed] [doi: 10.1105/tpc.112.104331]