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

Background → short introduction to phytochromes
Research projects:

Project 1 – Molecular mechanisms of phytochrome signalling
Project 2 – Interaction of light and other environmental cues
Project 3 – Light regulation of symbiotic nitrogen fixation in Lotus japonicus
Project 4 – Phytochrome-controlled responses in Physcomitrella patens and evolution of light signalling

Phytochromes — Red/far-red light receptors important for regulation of plant development

Fig. 1: Phytochrome mutant seedlings in D, R, and FR — **Fig. 1: Phytochrome mutant seedlings.** *Arabidopsis* wildtype (WT, Col-0), *phyA-211*, and *phyB-9* seedlings were grown for 4 days in the dark (D), red (R) or far-red light (FR).

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 (Kami, 2010). Plants contain several classes of photoreceptors to detect different aspects of their light environment, including light intensity, quality, and direction. The red and far-red light-sensing phytochromes are important photoreceptors involved in control of growth and development. There are five phytochromes in the model plant Arabidopsis thaliana (phyA-phyE), of which phyA and phyB play a dominant role in regulation of seed germination, de-etiolation, induction of flowering, and responses to canopy shade (Li, 2011; Ballaré, 2017; Casal, 2012). PhyB is the primary receptor for red light (R), while far-red light (FR), weak light and short light signals are perceived by phyA (Fig. 1) (Viczián, 2017; Sheerin, 2017). Phytochrome downstream signalling relies on the PHY INTERACTING FACTORS (PIFs) and the COP1/SPA E3 ubiquitin ligase complex, which both negatively regulate light responses (Fig. 2). In light-grown plants, phys inactivate PIFs by inhibiting their binding to target promoters and promoting their degradation (Lee, 2017; Pham, 2018). Light-activated phyA and phyB also bind specific members of the SPA protein family to destabilise them and inhibit their direct interaction with COP1, which results in downregulation of COP1/SPA's E3 ubiquitin ligase activity (Hoecker, 2017). In the dark, ELONGATED HYPOCOTYL 5 (HY5), CONSTANS (CO), and other transcription factors involved in e.g. light regulation of hypocotyl growth, anthocyanin biosynthesis and induction of flowering are ubiquitinated and targeted for degradation by COP1/SPA (Lau, 2012; Menon, 2016). Downregulation of COP1/SPA activity by light-activated phys allows these transcription factors to accumulate and regulate the expression of target genes.

Fig. 2: Model for phytochrome signalling — **Fig. 2: Model for phytochrome-mediated regulation of light-controlled gene expression.** See text above for details.

Project 1: Molecular mechanisms of phytochrome signalling

There are numerous current and past research projects that aim at understanding the molecular mechanisms underlying phytochrome signalling through PIFs and COP1/SPA (Fig. 2). In contrast, other, COP1/SPA- and PIF-independent pathways that also might play a role in light signalling have not been investigated in as much detail. To identify novel phytochrome signal transduction pathways, we performed a yeast two hybrid (Y2H) screen for proteins interacting with light activated phyA. In this screen we identified well established phyA binding proteins (e.g. PIF3 and FHY1) (Ni, 1998, Hiltbrunner, 2005), novel phyA interactors working through COP1/SPA (e.g. SPA1, PCH1) (Sheerin, 2015; Enderle, 2017) and proteins that have not been linked to COP1/SPA and/or PIFs. These proteins might be factors in COP1/SPA and PIF-independent phytochrome downstream signalling pathways. Part of these proteins are involved in hormone signalling pathways and therefore might play a role in integration of light and hormone signalling, while the function of other phytochrome-interacting proteins identified in the Y2H screen is still unknown. Current research projects in the lab investigate the function of these proteins.
Current funding: DFG HI-1369/9-1; FNR (Anne-Marie Staudt); DFG HI-1369/10-1
Past funding: DFG HI-1369/6-1; DFG HI-1369/7-1; BIOSS, EXC 294 (project C20); LGFG (Beatrix Enderle); Carl-Zeiss foundation (Nikolai Kahle); CSC (Zenglin Li)

Fig. 3: Karrikin promotes photomorphogenesis. — **Fig. 3: Karrikin enhances light signalling.** *Arabidopsis* wildtype and *kai2* mutant seedlings were grown for 4 days in red light. Wildtype seedlings were grown in presence or absence of karrikin (+KAR/-KAR).

Project 2: Interaction of light and other environmental cues

Karrikins are components in smoke of bush fires that strongly affect growth and development of plants (Flematti, 2015; Morffy, 2016). Karrikins promote seed germination and seedling establishment in fire-following species by enhancing the sensitivity to light. As such, karrikins play an important role in fire ecosystems. However, karrikins even promote germination and early seedling development in species not adapted to fire such as Arabidopsis (Fig. 3), suggesting that the karrikin signalling pathway is conserved in all plants (Waters, 2017). Interestingly, seedlings lacking the karrikin receptor KAI2 are less sensitive to light even in the absence of external karrikins (Fig. 3). Therefore, it has been proposed that an unknown endogenous karrikin-like compound, referred to as KAI2 ligand (KL), is present in plants and promotes light responses by activating the karrikin signalling pathway (Sun, 2016; De Cuyper, 2017). The putative KL can be considered a new plant hormone that interacts with light signalling through unknown molecular mechanisms. In this project we are investigating potential mechanisms for the integration of signals perceived by phytochromes and the karrikin/KL receptor KAI2.
Current funding: Velux Stiftung (project 1160)

Project 3: Light regulation of symbiotic nitrogen fixation in Lotus japonicus

Fig. 4: Symbiotic nitrogen fixation. — **Fig. 4: Light regulation of symbiotic nitrogen fixation.** Nitrogen fixation is an energy-demanding process, but light is not not only required to provide the energy for nitrogen fixation, it also acts as a signal that regulates nodulation and symbiotic nitrogen fixation.

Nitrogen availability is a limiting factor for plant growth. Legumes like Lotus japonicus circumvent this problem by symbiotic interaction with nitrogen-fixing rhizobia. Nevertheless, legumes need to balance the benefits and costs of this interaction, as the symbiosis is an energy demanding process. Therefore, legumes tightly control the formation of root nodules, where the symbiosis takes place (Fig. 4) (Nishida, 2018). Experiments done in the 1960s to 90s show that end-of-day far-red treatments reduce the root nodule number in pea, soybean, and southern pea plants inoculated with Rhizobia; this effect is red/far-red light reversible, suggesting that it is dependent on phytochrome (Lie, 1969; Kasperbauer, 1987; Kasperbauer, 1994). Furthermore, nodulation is also reduced in canopy shade, which is characterised by a low red:far-red light ratio detected by phytochromes, and also short-day conditions reduce the number of root nodules (Kasperbauer, 1994; Suzuki, 2011; Casal, 2012). In CIBSS project C1 we investigate how environmental conditions such as light and temperature control nodulation in the model legume Lotus japonicus. We want to characterise photoreceptors and downstream signalling factors involved in environmental control of nodulation and identify the postulated shoot-to-root signal that communicates light and temperature conditions in the environment to roots.
Current funding: CIBSS, EXC 2189 (project C1)

Fig. 5: Phylogenetic tree for phytochromes. — **Fig. 5: Phylogenetic tree for phytocrhomes.** Seed plants (e.g. *Arabidopsis thaliana*, rice), mosses (*Physcomiitrella patens*, *Ceratodon purpureus*), and most ferns (e.g. *Adiantum capillus-veneris*) contain small phytochrome gene families that evolved through independent duplications from a single phytochrome gene in the last common ancestor of plants.

Fig. 6: Physcomitrella patens grown under different light conditions. — **Fig. 6: Light regulates growth of *Physcomitrella patens* plants.** *Physcomitrella patens* gametophores were grown for 11 days in red (R) or far-red light (FR) or in the dark (D). Scale bar, 500 μm. From Possart, 2013 (modified).

Project 4: Phytochrome-controlled responses in Physcomitrella patens and evolution of light signalling

Phytochromes are ubiquitous in the plant kingdom and derive from a single phytochrome gene in the last common ancestor of plants (Li, 2015). Independent gene duplications in the course of evolution resulted in small phytochrome coding gene families in most species, for instance PHYA-PHYE in Arabidopsis thaliana, PHYA-C in rice, and PHY1-PHY4 and PHY5a/b/c in Physcomitrella patens (Fig. 5). Similar to seed plants, phytochromes regulate growth and development in mosses and other early diverging land plants (Fig. 6). Diverse functions for cytosolic or plasma membrane localised phytochromes have been described in mosses, ferns and algae (Hughes, 2013). Like in seed plants, phytochromes are also transported from the cytosol into the nucleus when activated by light (Fig. 7) (Tsuboi, 2012; Possart, 2013; Inoue, 2016). Furthermore, several light signalling components identified in Arabidopsis thaliana are also present in Physcomitrella patens. Some of the Physcomitrella proteins, e.g. COP1, PIF, and FHY1, are active in Arabidopsis, while others, including SPA proteins, appear to have species-specific functions and therefore are not interchangeable between Arabidopsis and Physcomitrella (Possart, 2013; Ranjan, 2014; Possart, 2017; Xu, 2017). Using a bioinformatic approach, we identfied potential target genes of PIFs in Physcomitrella. Currently, we are investigating the regulation of these genes under different light conditions and in different mutant strains that we generated. Interestingly, PIFs are much more stable in Physcomitrella than in Arabidopsis, where light-induced targeting for degradation is an important mechanism for the inactivation of PIFs (Lee, 2017). Physcomitrella PIFs expressed in Arabidopsis are hyperactive and we hypothesise that this might be due to different mechanisms required for the inactivation of PIFs in Arabidopsis and Physcomitrella, i.e. the mechanism involved in inactivation of PIFs in Physcomitrella might not be present in Arabidopsis (Possart, 2017; Xu, 2017).
Current funding: CSC (Jinhong Yuan); DFG HI 1369/8-1 (SPP 2237)
Past funding: HFSP Research Grant RGP0025/2013; DFG HI 1369/2-1; CSC and LGFG (Tengfei Xu)

Fig. 7: PHY1 nuclear accumulation in light — **Fig. 7: Light induced nuclear accumulation of PHY1 in *Physcomitrella patens*.** Transgenic *Physcomitrella patens* plants (upper panel: gametophores; lower panel: protonema) expressing YFP-tagged PHY1 were dark-adapted and exposed to white light. From Possart, 2013 (modified).