Light is essential for plants as an energy source as well as a critical signal that influences plant developmental processes, including germination, de-etiolation, phototropism, vegetative growth, and reproductive development, during the entire lifecycle (Kami et al., 2010). Upon exposure to light, plants undergo photomorphogenesis, which entails cotyledon opening, leaf expansion, and shoot development (Fankhauser and Chory, 1997). The response of plants to a wide spectrum of light wavelengths (from far-red to UV-B light) allows for proper development and growth (Kendrick and Kronenberg, 1994). Plants are receptive to red/far-red light through phytochromes; blue light via cryptochromes, phototropins, and ZEILUPE (ZTL)/FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1)/LOV KELCH PROTEIN2 (LKP2), and to UV-B light via UV-B RESISTANCE8 (UVR8; Mawphlang and Kharshiing, 2017).

Phytochromes are synthesized in the inactive Pr (red absorbing) form and are converted to the active Pfr (far-red absorbing) form upon light absorption. A high R:FR ratio increases the photo-equilibrium, that is, the ratio of active form (Pfr) to total phytochrome (Pr + Pfr), whereas the photo-equilibrium is decreased under a low R:FR ratio condition (Legris et al., 2019). Some environmental factors, such as far-red light, darkness, and high temperatures, accelerate reversion in the Pr form (Legris et al., 2016, 2019). In Arabidopsis, five phytochromes (phyA to phyE) have been identified and classified into two types according to the triggered responses under specific light conditions (Kircher et al., 2002; Franklin and Quail, 2010). PhyA is a type I phytochrome that is light-labile and recognizes prolonged far-red light (FR high irradiance response, FR-HIR) and very low fluence response (VLFR), as occurs in soil and deep shade. Type II is composed of phyB to phyE, which are light-stable; phyB is the most abundant than phyC-phyE (Sharrock and Clack, 2002). Type II phytochromes are activated under relatively high red/far-red ratio (FR low fluence response, LFR) light conditions and red light-dependent high irradiance responses (R-HIRs; Shinomura et al., 1996; Hirschfeld et al., 1998). Because of these differences, the action spectra are different, although the absorption spectra of types I and II are similar (Eichenberg et al., 2000; Klose et al., 2015). The far-red light-induced leaf senescence is regulated by antagonistic roles of phyA and phyB (Lim et al., 2018).

PhyB is the most abundant phytochrome in plants grown under normal light and is widespread in plant organs and tissues, acting to synchronize plant growth (Sharrock and Clack, 2002; Ha et al., 2018; Oh et al., 2018). In the root, shoot-driven light signals or stem-piped light activates phyB to trigger light responses (Lee et al., 2017). In addition, phyB acts as a thermosensor in temperature response (Jung et al., 2016). Thus, phyB combines temperature information with light signals in various morphogenic and adaptive responses, including photomorphogenesis, thermomorphogenesis, cold tolerance, drought tolerance, thermotolerance, and biotic stress tolerance, to maintain plant fitness (González et al., 2012; De Wit et al., 2013; Kim et al., 2021). PhyB has two domains, a photosensory domain (N-term) and kinase domain (C-term). The N-terminal domain of phyB is important for signal transduction, and the C-terminal domain attenuates phyB activity. Based on the truncated protein complementation experiment, it was well defined that the N-terminal domain of phyB can transduce the signal in the nucleus (Matsushita et al., 2003).

Plant growth and adaptation to various environmental changes depend primarily on the proper integration of light and stress response signaling. The abiotic and biotic stress responses are linked in the ER. Therefore, the ER is a key organelle to control multiple stress responses (Park and Park, 2019). The ER-resident luminal binding proteins (BiPs) play a role in maintaining ER homeostasis as protein-folding chaperones and UPR signaling transducers (Liu et al., 2007; Iwata et al., 2008). BiPs monitor the accumulation of misfolded proteins in response to excessive demands on the protein-folding capacity of the ER and communicate with the nucleus through two arms of the UPR signaling pathway in plants (Howell, 2013; Bao et al., 2019). One arm includes membrane-associated basic leucine zipper (bZIP) transcription factors, bZIP17 and bZIP28, which are transported to the Golgi and processed by site 1 and site 2 proteases to release their transcription factor domains toward the nucleus (Liu et al., 2007). The bZIP17 functions with bZIP28 in non-canonical UPR signaling under stress conditions (Kim et al., 2018). The other arm is the RNA splicing factor, inositol requiring enzyme1 (IRE1). Under ER stress, IRE1 is activated and participates in bZIP60 mRNA splicing to allow nuclear transport (Deng et al., 2011). In the nucleus, processed bZIP17, bZIP28, and spliced bZIP60 form a homodimer as well as a heterodimer to activate the ER stress response (Liu and Howell, 2010). The UPR signaling is associated with the plant development and stress response in the same manner (Bao et al., 2019).

Severe or prolonged ER stress can lead to PCD, called vacuolar cell death in the plant (Howell, 2013). The developmental and cell death domain (DCD)-containing asparagine-rich proteins (NRPs) function as signal transducers of PCD derived from prolonged ER stress. In Arabidopsis, NRP1 and NRP2 are involved in ER stress-induced PCD. Overexpression of BiP attenuates the ER stress-induced NRP1 and NRP2-mediated PCD (Reis et al., 2016). Metacaspases are cysteine proteases and are involved in the regulation of PCD. The nine metacaspase genes (MC1 to MC9) are identified and show different expression patterns depending on the tissues and stress responses. Among them, MC8 is strongly upregulated by oxidative stresses caused by UVC and hydrogen peroxide (H₂O₂; He et al., 2008). Light is an essential factor for plant growth and development, but it induces the production of reactive oxygen species (ROS), leading to PCD. High-light stress induces the expression of OXI1 encoding a serine/threonine kinase, which accelerates the cell death response under ER stress conditions (Shumbe et al., 2016; Beaugelin et al., 2020).

A previous study defined that light enhances the ER stress sensitivity of plants, and the light signaling is coupled with ER stress response through (UPR) signaling in the nucleus. The molecular mechanism of crosstalk between light signaling and UPR is well characterized by the ELONGATED HYPOCOTYL 5 (HY5) as a negative regulator of UPR, which competes with bZIP28 to bind to the BiP3 promoter (Nawkar et al., 2017). Although UPR activation is required to enhance protein-folding capacity to reduce light-induced ER stress, a positive regulator of UPR in light response has not yet been identified. Phytochromes may be one of the candidates for that. Especially, phyB plays an important role in abiotic and biotic challenges for plant environmental adaptation (Kim et al., 2021). Therefore, we hypothesized that phyB is involved in light-induced ER stress response.

Light is an essential factor for plant growth and development throughout life. Light signaling is closely related to ER stress response, but few studies have been conducted on it. Previous studies have shown that ELONGATED HYPOCOTYL 5 (HY5), a positive regulator of light signaling, negatively regulates UPR by competing with bZIP28 for binding to the ER stress response element (ERSE) on the promoter (Nawkar et al., 2017). Our results revealed that the red light photoreceptor phyB positively regulates the ER stress response under light conditions, unlike HY5. These results suggest that light signaling components with antagonistic functions tightly regulate the ER stress response, resulting in normal plant development.

ER is important for maintaining cellular homeostasis in cellular processes, such as plant development and environmental adaptation. Therefore, plant development and various environmental stresses affect the induction of ER stress responses. In this study, we want to understand the role of phyB with ER stress response according to photo-equilibrium. To minimize the secondary effects caused by developmental difference, 10-day-old typically grown plants were transferred to high concentration (100 and 200 ng/ml) of TM-containing media for a short time (3 days) in various light conditions: red, far-red, red:far-red (1:1), and dark (Figure 4). Under red light conditions, phyB-5 displayed a leaf yellowing phenotype in TM dose-dependent manner, which was not the case in the PBG (Figure 4A). For quantitative phenotype analysis, we compared the change of relative chlorophyll contents per unit of fresh weight. Although chlorophyll contents were reduced in TM dose-dependent manner, their fresh weight was not significantly changed under our experiment condition (data not shown). It might indicate that plant developmental difference was minimized, and our observations were derived from the cellular process. This is consistent with the previous research showing that ER stress attributes to chlorosis without a fresh weight reduction (McCormack et al., 2015). Furthermore, leaf senescence response induces the reduction of chlorophyll content, and especially far-red light induces a more robust leaf senescence response than darkness in Arabidopsis (Lim et al., 2018). In our experimental conditions, the yellowing phenotypes of leaves were not shown in far-red light, so the leaf senescence response did not affect the phenotype. Therefore, our observations could be mainly caused by ER stress response.

The antagonistic roles of light signaling components are well defined in leaf senescence (Lim et al., 2018). Leaf senescence is accelerated by PhyB and inhibited by PhyA under far-red light conditions. Furthermore, PBG5 (PHYB-GFP overexpression in Col-0) showed an accelerated leaf senescence phenotype under far-red light conditions compared to Col-0 (Lim et al., 2018), indicating that phyB Pr (known as the inactive form) inhibits leaf senescence, in contrast to phyB Pfr (known as the active form). Thus, phytochromes could have two regulation modes depending on photo-equilibrium in the cellular response. The phyB-5 plants displayed an opposite ER stress-sensitive phenotype under red light and dark conditions (Figure 4). This result supports the conception that phytochromes may also function in the Pr form, which is known as the inactive form. In Arabidopsis, five phytochromes (phyA to phyE) could have antagonistic functions in two forms: Pfr and Pr. Red and far-red light signaling will be fine-tuned for proper plant growth and environmental adaptation according to the photo-equilibrium of the five phytochromes.

The subcellular behavior of phyB is dynamic, that is, a translocation between the cytosol and nucleus, as well as nuclear body formation depending on environmental conditions (Legris et al., 2019). PhyB induced ER stress response genes in the nucleus via the N-terminal domain (Figure 5). There are two possibilities for the subcellular localization of phyB under ER stress conditions. First, the nuclear translocation of phyB may be necessary to induce the expression of ER stress response genes. Second, nuclear body formation affects the integration of light signaling and ER stress response. Thus, phyB subcellular behavior may be dynamic to integrate the light signaling and ER stress response for plant development and environmental stress adaptation.

Phytochromes regulate thermomorphogenesis as thermosensors in Arabidopsis (Jung et al., 2016). Recent research has revealed that a low R:FR ratio increases heat stress tolerance. PhyB inactivation, which increases the abundance of phytochrome-interacting factors (PIFs), enhances heat stress tolerance by shifting the fatty acid composition from unsaturated to saturated forms (Arico et al., 2019). In plants, high-temperature exposure leads to the accumulation of unfolded proteins in the ER, causing an ER stress response (Neill et al., 2019). Phytochromes can integrate thermosensory signaling into the ER stress response. We anticipate that understanding the integration of various phytochrome-dependent signaling pathways, including light, temperature, and different stress responses, will help overcome the challenges of growing crops in conditions introduced by global warming.

In this study, we showed that phyB is required for the induction of ER stress response. The phyB-impaired mutant phyB-5 was insensitive to TM-induced ER stress (Figure 6). On the other hand, PhyB-overexpressing plants (PBG) were sensitive to this and highly induced gene expression of the ER stress response, UPR signaling, and PCD. Interestingly, the ER stress-sensitive phenotype of phyB-5 under red light conditions was diminished by far-red light and darkness. The N-terminus of phyB is involved in signal transduction of the ER stress response in the nucleus. Taken together, our results demonstrated that phyB integrates red light-mediated signaling with UPR in the nucleus to prevent ER stress as a positive regulator.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

GA, IJ, and W-YK designed the research. GA, IJ, J-YC, SJ, G-IS, and MJ performed the experiments. GA, J-YC, MK, SL, and W-YK analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.

This research was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean Government (MSIT-2020R1A2C3014814 to W-YK) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A1A01059532 to GA).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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