HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis

Significance

In nature, plants are inevitably exposed to adverse conditions such as salinity, drought, and extreme temperatures. Recent reports suggest that environmental stresses critically affect the protein-folding capacity of endoplasmic reticula (ER), leading to ER stress. As the growth and development of plants significantly depend on light environment, the crosstalk between light signaling and ER stress response explained in current research can be a unique feature of plants. Our results suggest that light increases the ER stress sensitivity of plants and ELONGATED HYPOCOTYL 5, a positive regulator of light signaling, negatively regulates unfolded protein response gene expression in plant cells, which decreases the protein-folding capacity. The present study may form the basis for designing new strategies to increase stress tolerance of plants by tightly controlling light environment.

Abstract

Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box–like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression.

Because of their sessile lifestyle, plants have developed advanced mechanisms to cope with many forms of environmental stress conditions. Under changing environmental conditions, the adaptive responses of plants largely depend on the proper integration of light signaling and stress response pathways. Plants are equipped with multiple photoreceptors, including red/far-red light-absorbing phytochromes, blue/UV (UV)-A light-absorbing cryptochromes and phototropins, and the UV-B light-absorbing UV RESISTANCE LOCUS 8 (1, 2). The downstream component ELONGATED HYPOCOTYL 5 (HY5), a basic leucine zipper (bZIP) transcription factor (TF), mediates photoreceptor responses to promote photomorphogenesis (3). Recently, genome-wide gene expression analyses and chromatin immunoprecipitation (ChIP) studies have shown that HY5 is a higher hierarchical regulator of transcriptional networks for photomorphogenesis (4, 5). In particular, HY5 binds to the promoter of light-responsive genes featuring “ACGT-containing elements” such as the G-box (CACGTG), C-box (GACGTC), Z-box (ATACGGT), and A-box (TACGTA) (4, 6). Constitutive nuclear-localized HY5 plays an important role in integrating light signaling and several phytohormone and abiotic stress-related signaling pathways (7). For example, during seed germination and early seedling development, HY5 directly binds to G-box present in the promoter of ABA Insensitive 5 (ABI5) and activates its expression (8). Light and reactive oxygen species (ROS) signaling regulate deetiolation of seedlings, which is mediated by transcriptional modules regulated by direct binding of HY5 and phytochrome interacting factor (PIF) TFs to the G-box element present in the promoter of ROS-responsive genes (9). The cold acclimation response in Arabidopsis is positively regulated by HY5 through direct binding to Z-box and other cis-acting elements present in cold-inducible genes (10). These studies suggest that better performance of plant growth requires the proper integration of light and defense response against abiotic stressors, which is mediated through biding of HY5 to common cis-acting elements. Recent studies have reported that abiotic stresses, such as heat/cold shock, salt stress, oxidative stress, and osmotic pressure, can perturb endoplasmic reticulum (ER) homeostasis, causing the accumulation of misfolded proteins in ER and evoking ER stress in plants (11–14). These observations lead us to think about the role of light signals in ER stress response.

To mitigate ER stress, the level of protein-folding chaperones and ER-associated protein degradation (ERAD) machinery is increased, which is known as unfolded protein response (UPR) (15). Different membrane-associated TFs (MTFs) transduce stress signals to the nucleus. Typically, two MTFs, bZIP28 and bZIP60, play vital roles in ensuring cell survival during ER stress in plants (16, 17). These MTFs up-regulate genes encoding components of the ER protein-folding machinery, including luminal binding protein (BIP), calnexin (CNX), calreticulin (CRT), and protein disulfide isomerase (PDI). These genes share a consensus element in their promoters known as an ER stress response element (ERSE), which comprises two subelements: a CACG subelement that binds bZIP dimers and a CCAAT subelement that binds CCAAT box-binding factors (17, 18). However, proteins occasionally fail to mature in the ER and are exported from the ER for degradation by the ubiquitin–proteasome system as part of the ERAD pathway (15, 19). When plants are subjected to environmental stresses, the levels of such malformed proteins can exceed the capacity of the ER quality control and ERAD systems and leads to autophagy and programmed cell death to eliminate damaged cells (11, 20–23).

Accumulating evidence suggests that different abiotic stresses induce UPR and that the stress response is integrated with light signaling. Moreover, it has recently been suggested that light enhances UPR through the activation of BIP2 expression; however, the molecular mechanism underlying this process remains unclear (24). In this study, we suggest that light critically enhances the ER stress sensitivity and that a key component of light signaling, HY5, acts as a negative regulator of the UPR signaling pathway.

Results

The ER Stress Response in Plants Is Critically Affected by Light Intensity.

To investigate the effect of light on the ER stress response, we germinated wild-type (WT) Arabidopsis seeds in continuous darkness or white light on a medium containing different tunicamycin (Tm) concentrations. Plants grown in the dark exhibited skotomorphogenic development and plant growth was unaffected by Tm treatment. In contrast, plants grown under white light exhibited photomorphogenic development, and plant growth was seriously affected by Tm treatment. The stronger the light intensity, the more severe was the growth inhibition caused by Tm addition. For example, compared with seedlings grown under 20 µmol m⁻²⋅s⁻¹ light, those grown under 100 µmol m⁻²⋅s⁻¹ light showed greater Tm sensitivity (Fig. 1A). A significant reduction of the relative fresh weight was observed with increased light intensity in presence of Tm (Fig. 1B). We examined the inhibitory effects of Tm and DTT on primary root elongation. Severe growth inhibition was evident in presence of Tm and DTT with increasing light intensity (Fig. 1 C and D and SI Appendix, Fig. S1). Thus, the combination of ER stress and light synergistically inhibited root growth, demonstrating the significant role of light in enhancing the ER stress sensitivity. Interestingly, the fresh weight of seedlings showed significant recovery in presence of 0.2 mM tauroursodexycholic acid (TUDCA), a chemical chaperone under the Tm-treated condition at 100 µmol m⁻²⋅s⁻¹ light condition, implying that UPR relieves the Tm sensitivity (SI Appendix, Fig. S2). To confirm roles of UPR, we compared expression of UPR marker genes such as BIP3, CRT1, and CNX1 with or without Tm treatment under different light intensities. We found that expression of the UPR marker genes was induced under the Tm-treated condition. Moreover, the UPR gene expression was higher in the dark condition, whereas it decreases by increasing the light intensity (SI Appendix, Fig. S3).

Fig. 1.

Light enhances the ER stress sensitivity of Arabidopsis. (A) Phenotypes of 2-wk-old WT (Col-0) seedlings grown on MS medium containing 10 or 20 ng/mL of Tm or DMSO in continuous darkness (intensity = 0 µmol m⁻²⋅s⁻¹) or light (intensity = 20, 100, or 150 µmol m⁻²⋅s⁻¹). (B) Relative fresh weight of plants (mean ± SEM; n = 3) treated as indicated in A. Fresh weights of seedlings grown in the Tm-untreated conditions were set at 100%. (C) Primary root length phenotype of 7-d-old WT seedlings grown on MS medium containing 25 or 50 ng/mL of Tm or DMSO in continuous light (intensity = 20, 100, or 150 µmol m⁻²⋅s⁻¹). Root lengths of seedlings grown in the Tm-untreated conditions were set at 100%. (White scale bar, 20 mm.) (D) Relative root length of plants (mean ± SEM; n ≥ 20) treated as indicated in C. (B and D) Statistics by t test are shown; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and NS, no significance.

HY5 Positively Mediates the ER Stress Sensitivity.

Recent genomic studies have shown that light induces massive reprogramming of the plant transcriptome and that this process is primarily regulated by one key TF, HY5 (5). Thus, to determine the involvement of HY5 in UPR signaling, an hy5 mutant, an HY5 overexpression line P_35S:yellow fluorescent protein (YFP or Y)-HY5/hy5, and WT plants were germinated under long-day conditions on a medium supplemented with (15 or 30 ng/mL) or without (control) Tm. Under control conditions, hy5 mutants exhibited an elongated hypocotyl, whereas P_35S:Y-HY5/hy5 plants showed hypocotyl growth similar to that of WT and lacked an inhibitory phenotype (SI Appendix, Fig. S4). However, when the growth medium was supplemented with Tm, the effect was clear (Fig. 2 A–C). Under stress conditions, hy5 mutant growth was less affected, evident in the larger proportion of greenish-big (G-B) plants and smaller proportion of yellowish-small (Y-S) plants compared with WT plants. In contrast, P_35S:Y-HY5/hy5 plants were more sensitive to Tm-induced ER stress than WT and hy5 plants. We also obtained another hy5 mutant, hy5-1 (Landsberg erecta; Ler) and c-hy5, a complementation line of the mutant with HY5 under its own promoter (25). When we compared phenotypes of the wild type (Ler), hy5-1, and c-hy5 plants grown under different light intensity conditions (20 or 150 µmol m⁻²⋅s⁻¹) with or without Tm treatment, hy5-1 was strongly tolerant of ER stress and the tolerance level was reverted in c-hy5 to a level similar to Ler (SI Appendix, Fig. S5). Consistently, our electrolyte leakage assay revealed that Tm-induced cell death was enhanced in P_35S:Y-HY5/hy5 plants, but reduced in hy5 mutants, compared with that in WT plants (Fig. 2D). Taken together, these findings suggest that hy5 mutation conferred ER stress tolerance, whereas HY5 overexpression enhanced ER stress sensitivity in Arabidopsis.

Fig. 2.

HY5 positively mediates the ER stress sensitivity. (A) Phenotypes of 2-wk-old WT, hy5, and P_35S:Y-HY5/hy5 seedlings grown on MS medium containing 15 or 30 ng/mL of Tm or DMSO under LD (16-h light/8-h dark) conditions. (B) Percentage of G-B, G-S, and Y-S plants (mean ± SEM; n = 3) treated as indicated in A. (C) Relative fresh weight of plants (mean ± SEM; n = 3) treated as indicated in A. Fresh weights of seedlings grown in the Tm-untreated conditions were set at 100%. (D) WT, hy5, and P_35S:Y-HY5/hy5 seedlings were grown on MS medium for 2 wk and treated with or without 5 µg/mL of Tm for 6 h. They were used for ion leakage (mean ± SEM; n = 3) after 24-h recovery. (B–D) Statistics by t test are shown; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and NS, no significance.

The tolerant phenotype of the hy5 mutant and sensitivity of P_35S:Y-HY5/hy5 plants prompted us to investigate whether HY5 regulates genes downstream of UPR in plants. We compared the expression of genes downstream of UPR in WT, hy5, and P_35S:Y-HY5/hy5 plants under normal and ER stress conditions. The expression of UPR marker genes such as BIP3, CRT1, and PDI10 was induced by Tm treatment in WT plants; however, the induction of these marker genes occurred to a lesser degree in P_35S:Y-HY5/hy5 plants compared with that in WT plants, whereas to a higher degree in hy5 mutant plants compared with that in WT plants (Fig. 3), suggesting that HY5 acted as a negative regulator for the expression of genes downstream of UPR.

Fig. 3.

HY5 suppresses the expression of UPR marker genes. qRT-PCR analysis of transcript levels of BIP3, CRT1, and PDI10 in WT, hy5, and P_35S:Y-HY5/hy5 plants at the indicated time points after Tm treatment. Error bars denote ±SEM.

HY5 Binds to the Promoter of the BIP3 Gene.

It has been established that HY5 binds to G-box (CACGTG) in the promoters of light-responsive genes (4). We examined the promoter sequence of the BIP3 gene and found that it contains two G-box–like sequences within the ERSE1 and ERSE2 motifs (SI Appendix, Fig. S6). Thus, to test the hypothesis that HY5 directly binds to the two ERSE motifs of the BIP3 promoter, we conducted in vitro electrophoretic mobility shift assays (EMSAs). For this experiment, the G-box of the RBCS1A promoter was used as the positive control (6). Probes containing ERSE1 and ERSE2 from the BIP3 promoter and the G-box of the RBCS1A promoter were designed and labeled with biotin (SI Appendix, Table S1 and Fig. S6). Recombinant proteins, maltose-binding protein (MBP) alone and MBP fused to HY5 (MBP-HY5), produced in Escherichia coli were incubated with the biotin-labeled probes. MBP-HY5, but not MBP alone, caused a mobility shift of the three probes (Fig. 4A). Furthermore, the addition of unlabeled ERSE competitors diminished the intensities of the shifted bands, indicating that HY5 bound specifically to both ERSE motifs and the control G-box (Fig. 4B).

Fig. 4.

HY5 binds to the promoter of BIP3 gene and undergoes 26S proteasome-mediated degradation in ER stress. (A) EMSA of the binding of the recombinant MBP-HY5 to ERSE1 and ERSE2 motifs of the BIP3 promoter and G-box motif of the RBCS1A promoter (positive control) in a concentration-dependent manner. (B) Respective unlabeled probes (with the same sequence as the biotin-labeled probes) were used as competitors. (A and B) SuperScript numbers, 1X–3X represent the increasing concentration of unlabeled probes. (C) Schematic representation of the BIP3 promoter with the location of the two ERSE motifs. P1 and P2 represent the respective primer positions used for ChIP-qPCR. (D) ChIP-qPCR analyses of HY5 binding to the BIP3 promoter. ChIP assays were performed on hy5 and P_35S:Y-HY5/hy5 seedlings treated with Tm (+Tm) or DMSO (−Tm) for 12 h. DNA–protein complexes were immunoprecipitated using antibodies against GFP and rabbit IgG (negative control). ChIP DNA was quantified by qRT-PCR with primers specific to the ERSE motifs (P1 and P2) and TA3 promoter (control; mean ± SEM; n = 3 technical replicates). (E) Immunoblotting analysis of P_35S:Y-HY5/hy5 seedlings expressing YFP-HY5 treated with 5 µg/mL of Tm with or without 50 µM of MG132 for the indicated time. (Upper) Representative Western blot. (Lower) Ponceau S staining of same blot, which served as a loading control. (F) Quantification of relative abundance (fold) of YFP-HY5 protein levels compared with control conditions. The data are shown as means ± SEM from three independent biological repeats. (B and D) Statistics by t test are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and NS, no significance.

To confirm the binding of HY5 to particular ERSE sequences in vivo, we performed ChIP experiments using P_35S:Y-HY5/hy5 seedlings. After the immunoprecipitation of protein–DNA complexes using an anti-GFP antibody, the DNA fragments were quantified by quantitative real-time PCR (qRT-PCR) (Fig. 4C). Primers for the TA3 promoter were used as the negative control (26). The occupancy of HY5 was remarkably high in the “P1” and “P2” regions, which contain ERSE1 and ERSE2 of the BIP3 promoter, respectively, compared with that in negative control site in P_35S:Y-HY5/hy5 plants but not in the hy5 mutants (Fig. 4D). These results confirm that HY5 directly associated with the ERSE motifs of the BIP3 promoter in vitro and in vivo and that the binding of HY5 to the BIP3 promoter was reduced under the ER stress. To know the reason behind reduction of the binding affinity of HY5 in ER stress compared with normal conditions, we investigated the levels of HY5 protein in 10-d-old P_35S:Y-HY5/hy5 seedlings in the presence or absence of Tm with or without supplementation of the proteasome inhibitor MG132 for 12 h. The total proteins were extracted and subjected to immunoblotting analysis (Fig. 4E). Under control light conditions, HY5 protein level was relatively stable, showing no significant difference. The HY5 protein was clearly degraded by Tm treatment, and this Tm-induced degradation of HY5 was significantly inhibited by MG132 treatment (Fig. 4F), indicating that the HY5 protein was subjected to 26S proteasome-mediated degradation under ER stress conditions.

HY5 Competes with bZIP28 to Bind to the ERSE Motifs in the BIP3 Promoter.

Previous studies have shown that the core CACG element of the ERSE motif is important for bZIP28 binding, whereas the CCAAT element is critical for nuclear factor Y (NF-Y) binding (SI Appendix, Fig. S7) (17, 18). Because HY5 binding to the BIP3 promoter was reduced under ER stress conditions (Fig. 4D), we tested the possibility that bZIP28 exerts a negative effect on HY5 binding to the ERSE motifs. To confirm this hypothesis, we focused on the binding sites in the ERSE motif. We performed an EMSA assay with various point-mutated DNA probes containing ERSE1 and ERSE2 motifs of the BIP3 promoter (SI Appendix, Table S1 and Fig. S7). HY5 strongly bound to the WT ERSE1 and ERSE2 motifs (Fig. 5A; lane 2, both panels); however, nucleotide base substitutions in the bZIP binding elements of the ERSE motifs significantly prevented mobility shifting (lane 4, both panels). In contrast, nucleotide base substitution in the NF-Y binding sites (lane 3, both panels) and nucleotide base addition or deletion (lanes 5 and 6, both panels) had no effect on mobility shifting, indicating that HY5 bound to the bZIP28 binding site but not to the NF-Y binding sites.

Fig. 5.

HY5 competes with bZIP28 to bind to the ERSE motifs of the BIP3 promoter. (A) EMSA of the binding of MBP-HY5 to the core CACG subelement (bZIP28 binding site) present in ERSE motifs of the BIP3 promoter. Different ERSE probes (WT, M1–M4 shown in SI Appendix, Fig. S7) were altered by multiple substitutions (M1 and M2), or the length of spacers (M3 and M4) was altered. The black arrowhead shows the free probe. (B) EMSA showing the competition between MBP-HY5 and MBP-bZIP28ΔC to bind to the ERSE motifs of the BIP3 promoter. Superscript numbers 1X–3X represent the increasing protein concentration. The black arrowhead shows the free probe. (C) Schematic representation of constructs used in the transient transcription assay in tobacco leaves. A reporter vector P_BIP3:LUC containing the BIP3 promoter (942 bp upstream of the start codon) driving LUC and P_35S:GUS was used as an internal control. P_35S:Y-HY5 and P_35S:HA-bZIP28 were used as effector constructs. (D) Activation of P_BIP3:LUC by different combinations of effectors. (Lower) Superscript numbers indicate the ratio of OD₆₀₀ of Agrobacterium used in coinfiltration. The activation value of P_BIP3:LUC under control conditions without any effectors was set at 1 (mean ± SEM; n = 3).

Using the EMSA assay and ERSE1 probe, we tested whether HY5 and bZIP28 compete with each other to bind to the ERSE motifs (Fig. 5B). The mobility of the probe was shifted in a concentration-dependent manner by MBP-HY5 and MBP-bZIP28 (lanes 3–8) but was unaffected by MBP alone (lanes 1–2). When the amount of MBP-bZIP28 was fixed and increasing amounts of MBP-HY5 were added, the formation of the MBP-bZIP28-probe complex was dramatically decreased, whereas that of the MBP-HY5-probe complex increased in a concentration-dependent manner (lanes 9–11). Moreover, MBP-bZIP28 was able to replace MBP-HY5 for the formation of the complex with probe (SI Appendix, Fig. S8). However, the addition of MBP alone did not affect the formation of the MBP-bZIP28-probe complex (compare lanes 8 and 12 in Fig. 5B). These suggest that bZIP28 and HY5 specifically competed to bind to the ERSE motifs. In conclusion, HY5 and bZIP28 shared binding sites in the BIP3 promoter and competed to bind to the ERSE motifs in vitro.

We performed a transient transcription assay in tobacco leaves to investigate whether HY5 functions in the regulation of BIP3 gene expression in vivo. We constructed luciferase (LUC) reporter plasmids in which LUC reporter-gene expression was driven by the BIP3 promoter containing the ERSE motifs (Fig. 5C). Increased expression of the reporter gene in response to Tm treatment was confirmed to validate the functionality of the construct. As shown in Fig. 5D, transiently expressed bZIP28 activated BIP3 expression, consistent with previous reports (18). HY5 alone, however, had no effect on BIP3 expression, regardless of Tm treatment. Interestingly, the coexpression of HY5 and bZIP28 dramatically repressed bZIP28-activated reporter gene expression, confirming the negative role of HY5 on bZIP28-driven transcriptional activation.

To confirm the antagonistic relationship between HY5 and bZIP28, we created a hy5 bzip28 double mutant by crossing hy5 and bzip28 single mutants (SI Appendix, Fig. S9) and analyzed the genetic interaction between HY5 and bZIP28 for UPR. We examined expression of UPR marker genes such as BIP3 and PDI10 in WT, hy5, bzip28, and hy5 bzip28 plants over the course of Tm treatment (SI Appendix, Fig. S9). qRT-PCR showed that the UPR gene expression was lower in bzip28 and hy5 bzip28 mutants than in WT plants; moreover, bzip28 and hy5 bzip28 plants exhibited similar levels of the expression, implying that the negative roles of HY5 in the UPR were mostly bZIP28 dependent. Furthermore, the hy5 mutant showed a much higher induction of the UPR genes compared to the other plants. These findings suggest that HY5 negatively regulates the UPR gene expression and antagonistically acts on the bZIP28-mediated gene activation.

Discussion

Light is one of the main factors required for proper plant growth and development, from seed germination to flowering and seed maturation (2, 27). Recently, crosstalk in plants between light signaling and stress responses has become a hot research topic. However, to date, the influence of light on the ER stress response has not been studied. To analyze the involvement of light in the ER stress response in plants, we investigated the effect of ER stress in WT Arabidopsis in the presence or absence of light. We found that light is required for the ER stress-induced growth inhibition of plants and that increasing light intensity enhances the sensitivity of plants to ER stress. Based on these results, we attempted to identify the molecular links between light signaling and UPR.

In this study, we found that a positive regulator of light signaling, HY5, negatively regulates UPR. The loss of function of HY5 rendered plants tolerant to ER stress induced by Tm treatment. Previously, it has been established that ER stress evoked by environmental conditions activates the two arms of UPR and ∼0.7% of the genome is up-regulated as a part of UPR in Arabidopsis (28). The activity of bZIP28 and bZIP60 is required for the induction of UPR and stress-tolerance phenotype in plants (29, 30). Because HY5 is a well-known TF, we hypothesized that it plays an important role in UPR by regulating gene expression. In fact, our qRT-PCR data suggest that the tolerance of the hy5 mutant to ER stress conditions was due to the elevated expression of UPR marker genes in these plants. In conclusion, these results are consistent with those of recent studies suggesting that HY5 is a critical component in the integration of light signaling with different defense responses against stress conditions, an important event for plant survival under adverse conditions (10, 31, 32).

As shown by our EMSA results, HY5 specifically bound to the bZIP28 binding sites of ERSE1 and ERSE2 in the BIP3 promoter. Furthermore, the binding of bZIP28 to the ERSE motif was reduced when the HY5 protein was present in excess and vice versa. This analysis illustrates the competition between HY5 and bZIP28 for binding to the BIP3 promoter, emphasizing the importance of the concentrations of particular TFs. This idea is also supported by our transient transcription assay in tobacco, which showed that HY5 inhibited transcriptional activation by bZIP28. Previously, it has been reported that bZIP28 forms a transcriptional complex with NF-Y TFs, which is important for full induction of the BIP3 gene (18). Recently, bZIP28 has been shown to be involved in formation of a transcriptional complex with COMPASS-like components, which are involved in histone H3K4 trimethylation (H3K4me3), which correlates with active gene expression (33). Moreover, it is also known that histone modification plays an important role in HY5-mediated gene expression (4, 34). Thus, it is likely to form such transcriptional complexes with bZIP28 under ER stress conditions, which can interfere with HY5 binding to the ERSE motifs. Our ChIP assay also supports this notion, as the binding of HY5 is decreased under ER stress conditions (Fig. 4). Our results imply that HY5 exerted an antagonistic effect on bZIP28, but we cannot exclude the possibility that HY5 affects activity of other TFs, such as bZIP60, NAC062, and NAC103, which regulate the expression of UPR marker genes required for ER stress tolerance (35–38).

Our data suggest that HY5 protein level is regulated in ER stress conditions through the 26S proteasomal degradation system, which is similar to light/dark regulation of the HY5 level during the deetiolation process (3). Previous studies also suggest that the abundance of HY5 protein is regulated by either low temperature or short heat shock through the 26S proteasome-mediated degradation system (10, 31). The proteosomal degradation of HY5 and proteolytic cleavage of bZIP28 fine tunes the balance between the two proteins to compete for binding the ERSE motifs and in turn regulate UPR genes. Nearly 50% degradation of HY5 protein suggests that it may have other functions that should be identified in future studies.

Considering these results together, it is possible to propose a hypothetical model that explains the roles of HY5 and bZIP28 in mediating crosstalk between light signaling and UPR (Fig. 6). Under normal light conditions, HY5 binds to not only G-box elements present in the promoter of light-response genes to regulate photomorphogenesis, but also to the ERSE motifs present in the promoters of UPR genes to reduce their expression to basal levels. Under these conditions, bZIP28 is anchored to the ER membrane through its transmembrane domain. Under ER stress, bZIP28 is activated by proteolysis and the activated bZIP28∆C translocates to the nucleus, where it competes with HY5 to bind to the ERSE motifs present in the promoters of UPR genes. Under these conditions, active bZIP28∆C accumulates in the nucleus, whereas HY5 is removed by the 26S proteasome system, resulting in the binding of bZIP28∆C to the ERSE motifs and the up-regulation of UPR genes. This study may contribute to a better understanding of crosstalk between light signaling and UPR in plants.

Fig. 6.

Schematic model showing HY5-mediated crosstalk between light signaling and UPR. Under normal light condition, HY5 positively regulates light-responsive genes and directly binds to the ERSE motifs present in the promoter of UPR genes to repress their expression. At the same time, bZIP28 is anchored to the ER membrane in a dormant form. Under ER stress conditions, bZIP28 is translocated to the Golgi apparatus, where it is proteolytically processed, after which it translocates to the nucleus, where it competes with HY5 to bind to the ERSE motifs. The HY5 protein is subjected to 26S proteasomal degradation, resulting in higher levels of active bZIP28 in the nucleus, which turn on UPR for plant survival.

Materials and Methods

Plant Materials and Growth Conditions.

Arabidopsis WT, T-DNA mutants, and transgenic lines were prepared in the Columbia (Col-0) ecotype background. We obtained the homozygous T-DNA lines hy5 (SALK_096651C) and bzip28 (SALK_132285) from the Arabidopsis Biological Resource Center, The Ohio State University, Columbus, OH. Double-mutant hy5 bzip28 was generated by genetic crossing using hy5 female and bzip28 male parents, and homozygous lines were confirmed by PCR genotyping (SI Appendix, Fig. S9). We obtained mutant seeds hy5-1 and P_HY5:HY5-Y/hy5-1 (c-hy5) in Landsberg erecta (Ler) background as generous gifts from R. Ulm, University of Geneva, Geneva, Switzerland (25). Seedlings were grown on half-strength Murashige and Skoog (MS) medium containing 2% (wt/vol) sucrose and 0.25% (wt/vol) Phytagel (Sigma-Aldrich), pH 5.7. Details on growth conditions, stress treatments for seedlings, and other methods used in this study are described in SI Appendix, SI Materials and Methods.

Construction of Transgenic Plants.

All DNA constructs and transgenic plants prepared in this study are described in SI Appendix, SI Materials and Methods.

Measurement of Electrolyte Leakage.

Details are in SI Appendix, SI Materials and Methods.

EMSA and ChIP Assay.

Details are in SI Appendix, SI Materials and Methods. All primer sequences are listed in SI Appendix, Table S2.

Immunoblotting Analysis.

Details are in SI Appendix, SI Materials and Methods.

Transient Transcription Assay.

Details are in SI Appendix, SI Materials and Methods.

Total RNA Isolation and Quantitative Real-Time PCR Analysis.

Details are in SI Appendix, SI Materials and Methods. All primer sequences are listed in SI Appendix, Table S2.

Statistical Analysis.

Statistical significances were determined using Student’s t test. P values were calculated using GraphPad QuickCalcs (available online at www.GraphPad.com/).