Circadian and Plastid Signaling Pathways Are Integrated to Ensure Correct Expression of the CBF and COR Genes during Photoperiodic Growth

HSP90, ZTL, PRR5 and HY5 integrate circadian and plastid signaling pathways to regulate CBF and COR expression.

Abstract

The circadian clock synchronizes a wide range of biological processes with the day/night cycle, and correct circadian regulation is essential for photosynthetic activity and plant growth. We describe here a mechanism where a plastid signal converges with the circadian clock to fine-tune the regulation of nuclear gene expression in Arabidopsis (Arabidopsis thaliana). Diurnal oscillations of tetrapyrrole levels in the chloroplasts contribute to the regulation of the nucleus-encoded transcription factors C-REPEAT BINDING FACTORS (CBFs). The plastid signal triggered by tetrapyrrole accumulation inhibits the activity of cytosolic HEAT SHOCK PROTEIN90 and, as a consequence, the maturation and stability of the clock component ZEITLUPE (ZTL). ZTL negatively regulates the transcription factor LONG HYPOCOTYL5 (HY5) and PSEUDO-RESPONSE REGULATOR5 (PRR5). Thus, low levels of ZTL result in a HY5- and PRR5-mediated repression of CBF3 and PRR5-mediated repression of CBF1 and CBF2 expression. The plastid signal thereby contributes to the rhythm of CBF expression and the downstream COLD RESPONSIVE expression during day/night cycles. These findings provide insight into how plastid signals converge with, and impact upon, the activity of well-defined clock components involved in circadian regulation.

In a wide range of organisms, the circadian clock synchronizes biological processes with the time of day. The circadian oscillator provides a robust internal rhythm that anticipates daily changes and optimizes the usage of resources with day/night cycles. In Arabidopsis (Arabidopsis thaliana), the clock consists of a repressilator with a series of transcription-translation feedback loops (Pokhilko et al., 2012), and up to 70% of the chloroplast and 36% of the nuclear genomes have been shown to be subject to circadian regulation (Harmer et al., 2000; Schaffer et al., 2001; Michael and McClung, 2003; Michael et al., 2008). Correct circadian regulation in plants is important for photosynthetic activity and growth by synchronizing gene expression, protein modification, and stomatal opening with the light/dark cycle (Hennessey and Field, 1991; Green et al., 2002; Dodd et al., 2005). The mechanisms and signaling pathways that connect the circadian clock with the regulation of photosynthetic activity in the chloroplasts are not well known; however, there are some suggested mechanisms by which the circadian oscillator can communicate timing information to the chloroplast (Dodd et al., 2014). For example, the core subunits of the plastid-encoded polymerase are encoded by the chloroplast genome, but the nucleus-encoded sigma factors are required for promoter specificity and the initiation of transcription (Schweer et al., 2010). The nuclear genome in Arabidopsis encodes six sigma factors (SIG1–SIG6), and their expression is circadian regulated; it was suggested that the circadian timing of the nucleus-encoded sigma factors in turn controls the timing of transcription of the photosynthesis genes encoded in the chloroplast (Noordally et al., 2013). This regulatory control of transcription could provide a way to communicate timing to the chloroplast. Furthermore, it has been suggested that the chloroplast itself is involved in the regulation of the circadian clock and that chloroplast retrograde signals can alter circadian rhythms (Hassidim et al., 2007). Thus, the close interaction between the circadian clock and chloroplast retrograde signaling systems could provide fine-tuning of photosynthetic gene expression and photosynthetic activity during the day.

Expression of the nucleus-encoded C-REPEAT BINDING FACTOR (CBF) transcription factors, CBF1, CBF2, and CBF3, is circadian regulated with a peak at the middle of the light period (Bieniawska et al., 2008; Kidokoro et al., 2009; Dong et al., 2011). The CBF pathway is important for cold acclimation and freezing tolerance in Arabidopsis, and it regulates the expression of more than 100 COLD RESPONSIVE (COR) genes (Fowler and Thomashow, 2002; Park et al., 2015). During unstressed photoperiodic conditions, PHYTOCHROME INTERACTING FACTOR7 (PIF7) was shown to negatively regulate CBF1 and CBF2 expression (Kidokoro et al., 2009). PIF7 is under circadian control and binds to the G-box element in the promoters of CBF1 and CBF2 and represses their expression. In contrast, CBF3 expression is not controlled by PIF7. The downstream COR genes, such as COR15a, COR47, and COR78, also have been shown to oscillate during the day and to peak just after the CBFs (Dong et al., 2011). COR15a encodes a cryoprotective protein that is targeted to the chloroplast stroma, where it decreases the propensity of the plastid membranes to form hexagonal II phase structures in response to low temperatures (Steponkus et al., 1998). In addition to low temperature and the circadian clock, COR15a expression has been shown to respond to conditions that alter the functional state of the chloroplast (Lin and Thomashow, 1992; Kleine et al., 2007). However, the mechanism by which the chloroplasts communicate the signal to regulate COR15a expression in the nucleus has remained unknown.

Several plastid signals have been reported to influence the transcription of nuclear genes encoding plastid-localized proteins in a process termed retrograde signaling (Rodermel, 2001; Fernández and Strand, 2008; Barajas-López et al., 2013a). One of the identified retrograde signals is linked to the tetrapyrrole biosynthetic pathway, and several reports have shown that perturbations of the tetrapyrrole pathway result in major changes of nuclear gene expression (Strand et al., 2003; Ankele et al., 2007; Pontier et al., 2007; Mochizuki et al., 2008; Moulin et al., 2008; Zhang et al., 2011). The copper response defect (crd) mutant has impaired activity of the cyclase complex, which converts magnesium protoporphyrin IX-methylester (Mg-ProtoIX-ME) and atomic oxygen to 3,8-divinyl protochlorophyllide in the chlorophyll branch of the tetrapyrrole pathway. As a result of this lesion, the crd mutant overaccumulates Mg-ProtoIX-ME and the upstream intermediate magnesium protoporphyrin IX (Mg-ProtoIX) that, in turn, triggers a plastid signal regulating nucleus-encoded genes (Tottey et al., 2003; Bang et al., 2008). In the wild type, Mg-ProtoIX and Mg-ProtoIX-ME were shown to accumulate transiently when plants were exposed to oxidative stress (Stenbaek et al., 2008; Kindgren et al., 2011; Zhang et al., 2011), and in response to the tetrapyrrole accumulation, the ATPase activity of cytosolic HEAT SHOCK PROTEIN90 (HSP90) proteins is inhibited, which leads to a change in nuclear gene expression (Kindgren et al., 2012). HSP90 is a molecular chaperone that interacts with proteins in their near-native state and is essential for maintaining the activity of signaling proteins (Young et al., 2001) as well as preventing the protein aggregation of freshly translated chloroplast preproteins (Fellerer et al., 2011). HSP90 also has been reported to be required for proper regulation of the circadian clock. A role for HSP90 was established in the direct control of maturation and stabilization of the F-box protein ZEITLUPE (ZTL; Kim et al., 2011). ZTL is an evening-phased clock component responsible for the proteasome-dependent degradation of TOC1 and PSEUDO-RESPONSE REGULATOR5 (PRR5), two evening-phased components of the circadian clock (Más et al., 2003; Kiba et al., 2007). PRR5 was shown to be a repressor of the CBF genes (Nakamichi et al., 2012). HSP90 effectively binds ZTL and prevents aggregation of the ZTL protein, providing a rhythm of ZTL and contributing to the oscillations of TOC1 and PRR5 (Kim et al., 2011).

Here, we report that CBF expression during photoperiodic conditions is regulated by the interplay between circadian and plastid signaling pathways. We demonstrate that the stability of ZTL, controlled by HSP90, is decreased in the crd mutant that overaccumulates tetrapyrroles. ZTL directly interacts with the transcription factor LONG HYPOCOTYL5 (HY5) and negatively regulates HY5. Low levels of ZTL result in HY5- and PRR5-mediated repression of CBF3 and PRR5-mediated repression of CBF1 and CBF2 expression. Thus, the plastid signal regulates the activity of HY5 and PRR5 via HSP90 and ZTL and thereby contributes to the changes in expression levels of CBF and COR during the day/night cycle. These findings provide novel insight into how plastid signals converge with, and impact upon, the activity of well-defined clock components involved in circadian regulation.

RESULTS

The Expression of COR and CBF Is Negatively Correlated with the Accumulated Levels of Tetrapyrroles in the crd Mutant

The expression of COR genes responds to the circadian clock as well as to abiotic stress and to treatments that alter the functional state of the chloroplast (Lin and Thomashow, 1992; Kleine et al., 2007; Nakayama et al., 2007; Dong et al., 2011). We examined the transcript levels for two COR genes, COR15a and COR47, in the wild type and in the tetrapyrrole-overaccumulating crd mutant. In accordance with earlier reports, COR15a and COR47 expression increased in the light period with a peak just before dusk (Fig. 1; Dong et al., 2011). In tobacco (Nicotiana tabacum) leaves, it was shown that tetrapyrrole levels also follow a diurnal oscillation pattern (Papenbrock et al., 1999). Also in Arabidopsis, the amounts of Mg-ProtoIX and Mg-ProtoIX-ME increased rapidly at the beginning of the light period, peaking in the middle of the day; thereafter, the levels of porphyrins decreased until dusk (Fig. 2). COR expression in the crd mutant, which overaccumulates Mg-ProtoIX and Mg-ProtoIX-ME, was significantly lower compared with that in the wild type, with the largest difference between the genotypes at 9 h after lights-on, Zeitgeber Time (ZT 9) (Fig. 1).

Figure 1.

COR expression is repressed in the crd mutant compared with the wild-type Columbia (Col). Plants were grown in short-day conditions (9/15 h light/dark), and 3-week-old plants were sampled every 3 h starting at dawn and analyzed for COR15a (At2g42540; A) and COR47 (At1g20440; B) expression. The last samples were collected 3 h into the dark period. Gene expression was normalized to ubiquitin-like protein (UBI; At4g36800) and related to the amount present in wild-type Col at ZT 0. Each data point represents the mean ± sd of at least three biological replicates.

Figure 2.

The diurnal oscillation of tetrapyrroles contributes to the circadian control of CBF3 expression. Plants were grown in short-day conditions (9/15 h light/dark), and 3-week-old plants were sampled every 3 h starting at dawn until 3 h into the dark period. After the dark period, the plants were kept in constant light for 27 h, and then another set of samples were collected every 3 h starting 3 h after the subjective dawn. Black bars on the x axis represent the true dark period, while gray bars represent the subjective dark period during the free-running period. A, Samples were analyzed for CBF3 (At4g25480) expression normalized to ubiquitin-like protein (At4g36800), and each time point was related to the amount present in wild-type Col at ZT 6. B and C, Mg-ProtoIX (B) Mg-ProtoIX-ME (C) contents. Each data point represents the mean ± sd of at least three biological replicates. FW, Fresh weight.

To investigate whether the misregulation of the COR genes in the crd mutant was due to a misregulation of the upstream CBF genes, we checked the expression of the CBF genes (CBF1 to CBF3) during the light/dark cycle (Fig. 2A; Supplemental Fig. S1). Analysis of CBF1 to CBF3 expression demonstrated that the crd mutant displayed repressed CBF expression compared with the wild type at ZT 6 (Fig. 2A; Supplemental Fig. S1). Taken together, these results suggest that the altered COR expression in the crd mutant is most likely not caused by a direct effect on the regulation of COR15a and COR47 expression but, rather, is the result of the misregulation of the CBF transcription factors.

The Plastid Signal Converges with the Circadian Control of CBF Expression

Under normal photoperiodic conditions, CBF3 expression has been shown to be regulated by components in the circadian clock, which also results in a rhythmic expression pattern of the COR genes (Fig. 1; Dong et al., 2011). Our results from the crd mutant suggest that a plastid signal triggered by the accumulation of tetrapyrroles during the day converges with the circadian control of CBF expression. Under constant light or in the dark, Mg-ProtoIX and Mg-ProtoIX-ME have been demonstrated conclusively to decrease to very low and stable levels without any rhythm (Papenbrock et al., 1999). Thus, in order to examine the regulation of CBF expression, and to dissect the circadian and diurnal signaling pathways, plants grown under photoperiodic conditions were transferred to constant light and CBF3 expression was investigated in the wild type and crd. The levels of Mg-ProtoIX were undetectable using our method following exposure to constant light, and Mg-ProtoIX-ME reached levels detected in the dark during the normal light/dark cycle in both the wild type and the crd mutant (Fig. 2, B and C). Furthermore, as observed previously, no oscillation pattern was detected for Mg-ProtoIX-ME following exposure to constant light (Fig. 2C).

The wild-type plants showed an oscillation of CBF3 expression during the day, peaking 6 h into the light period and then declining, correlating with the tetrapyrrole accumulation that peaked at the same time point (Fig. 2). Following exposure to constant light, the amplitude of CBF3 expression was reduced in the wild type (Fig. 2). This is commonly observed following shifts to constant light for clock-regulated genes and so also for CBF3 and the COR genes (Dong et al., 2011). However, the circadian oscillation of CBF3 expression was maintained in both the wild type and crd during the subjective day. Interestingly, the observed difference in CBF3 expression between the wild type and crd under diurnal conditions was diminished. The levels of Mg-ProtoIX-ME following exposure to constant light were similar to what was detected at ZT 12 under normal diurnal conditions, and at this time point there was no difference in CBF3 expression between the wild type and crd. These results suggest that the oscillation of tetrapyrrole levels during diurnal conditions contributes to the regulation of CBF3 expression.

The CBF cold-response pathway is of great importance during cold acclimation and the development of freezing tolerance. Our results indicate that the plastid signal triggered by tetrapyrrole accumulation during the day contributes to the regulation of CBF expression. In order to examine whether this plastid signal plays a role in response to low temperature, we performed a cold-stress experiment with wild-type plants and showed that, following a shift to 4°C, the amounts of Mg-ProtoIX and Mg-ProtoIX-ME decreased rapidly within the first 1.5 h in the cold and thereafter were maintained at very low but stable levels (Supplemental Fig. S2). This result is consistent with earlier reports demonstrating that the chlorophyll biosynthetic pathway is strongly inactivated and the enzymes in the pathway are inhibited by low temperatures (Mohanty et al., 2006). In contrast, CBF1 to CBF3 were strongly induced within the first 1.5 h following the shift to 4°C and thereafter decreased with exposure time, while COR15a expression showed a weak induction after 1.5 h but increased with time exposed to 4°C. The induction of CBF and COR expression by exposure to low temperatures is much greater compared with the diurnal changes in expression observed during warm control conditions (Supplemental Fig. S2; Fowler and Thomashow, 2002).

HY5 Binds to the Promoters of CBF1/2/3 and COR15a in Vitro

The promoters of the CBF genes show high sequence similarity, and a 65-bp fragment (−113 to −47 from the translation start) of CBF2 has been shown to be responsible for the circadian and cold regulation of transcription (Kidokoro et al., 2009). CBF1 and CBF2 have G-boxes (CACGTG; Kidokoro et al., 2009) close to the transcriptional start site in their promoters, while CBF3 has two Z-boxes (TACGTG). The promoter of COR15a includes several C-repeat elements, the binding site of the CBFs, as well as several G-boxes (Baker et al., 1994). Several transcription factors are able to bind the G-box and its variants, but we focused on the bZIP-type transcription factor HY5 (Chattopadhyay et al., 1998). HY5 responds to different light signals but has also been shown to respond to plastid signals (Ruckle et al., 2007; Kindgren et al., 2012). To study if HY5 interacts directly with the promoters of the CBF genes and COR15a, recombinant HY5 was expressed in Escherichia coli and purified under native conditions (Fig. 3A). Purified HY5 was detected on an SDS-polyacrylamide gel as a single band at approximately 22 kD, close to the predicted mass (18.5 kD + His tag). The purified protein had an A₂₆₀:A₂₈₀ ratio of 0.6, indicating negligible contamination of nucleic acids. Recombinant HY5 was incubated with 50-bp fluorescein-labeled fragments of each promoter (CBF1, −110 to −60 from the translation start; CBF2, −110 to −61; CBF3, −110 to −61; and COR15a, −176 to −126). The interaction between protein and probe was visualized after an EMSA, where a shifted band indicated a protein + DNA complex (Fig. 3B). Interaction between HY5 and the different promoter fragments could be seen in all cases, albeit a stronger binding affinity was found between HY5 and CBF1 and CBF3 compared with CBF2 and COR15a (Fig. 3B). The interaction was lost when another fragment of the CBF3 promoter that does not include a G-box or a Z-box element was incubated with HY5 (Fig. 3C). The affinity of HY5 to the CBF1 and CBF3 promoters also was examined with the addition of a nonlabeled competitor (Fig. 3, D and E). The disappearance of the shifted HY5-CBF1 and HY5-CBF3 complexes following the addition of a nonlabeled CBF3 promoter fragment was greater than that following the addition of a nonlabeled CBF1 promoter fragment. Thus, the competitor experiments showed that HY5 bound the promoter of CBF3 stronger than the promoter of CBF1. Thus, the in vitro assay showed that, while recombinant HY5 is able to recognize the promoters of CBF1 to CBF3 as well as the promoter of COR15a, it has the strongest affinity for CBF3.

Figure 3.

HY5 binds to COR15a and CBF1/2/3 promoters, with a preference for the CBF3 promoter. A, HY5 protein was expressed and purified as a His-tag fusion protein and used in electromobility shift assay (EMSA). B, Fluorescein-labeled probe (500 pm) was incubated with increasing concentrations of HY5 protein as indicated. C, Negative control for HY5 DNA binding using the CBF3 promoter sequence excluding the G-box and Z-box cis-elements. D and E, Competitor experiments with unlabeled CBF1 and CBF3 promoter fragments with labeled CBF1 probe (D) or CBF3 probe (E).

The Repression of CBF3 Is Mediated via HY5

In order to determine whether HY5 regulates the CBF genes in response to the tetrapyrrole-triggered plastid signal, 3-week-old plants were fed Mg-ProtoIX (Supplemental Fig. S3A) and checked for CBF1 to CBF3 and COR15a expression. To assess if tetrapyrrole feeding resulted in oxidative stress, we investigated the expression levels of the three marker genes for reactive oxygen species (ROS), FER1, GST5, and MAPK18 (Laloi et al., 2007). Following feeding, there was no change in expression of the ROS marker genes, indicating that no oxidative stress was induced by the treatment (Supplemental Fig. S3B). This suggests that the plastid signal is linked to the tetrapyrrole levels rather than ROS generated by their accumulation. The Mg-ProtoIX-incubated wild-type plants showed a significant repression of CBF1 to CBF3 and COR15a expression compared with the control plants (Fig. 4A; Supplemental Fig. S3C). This is similar to what has been reported for PHOTOSYNTHESIS-ASSOCIATED NUCLEAR GENE (PhANG) expression in earlier reports from Arabidopsis (Strand et al., 2003; Kindgren et al., 2012; Barajas-López et al., 2013b). In addition, CBF expression was repressed to less than 40% of control levels in response to norflurazon treatment in the wild type. This repression was absent in the genome uncoupled5 mutant that accumulated less tetrapyrroles compared with the wild type in response to the norflurazon treatment (Strand et al., 2003). In contrast to the wild type, the hy5 mutant did not show a repression of CBF3 or COR15a following Mg-ProtoIX feeding. However, the expression levels of CBF1 and CBF2 in hy5 were identical to those in the wild type following the feeding (Fig. 4A), implying that HY5 is only involved in the regulation of CBF3 expression. The result from the tetrapyrrole feeding is consistent with that from the EMSA, where the strongest affinity of HY5 was shown for CBF3 (Fig. 3).

Figure 4.

HY5 regulates CBF3 expression in response to the plastid signal. A, Plants were grown in short-day conditions (9/15 h light/dark), and 3-week-old plants were analyzed for CBF1 to CBF3 (At4g25490, At4g25470, and At4g25480) expression following 12 h of feeding with 50 µm Mg-ProtoIX. B, CBF3 (At4g25480) expression 6 h into the light period in wild-type Col, crd, hy5, and hy5 crd. CBF3 expression was significantly different in crd and hy5 compared with Col and in hy5 crd compared with crd. C, Plants were kept in constant light for 27 h, and then samples were collected 9 h after the subjective dawn at ZT 57 and analyzed for CBF3 (At4g25480) expression. Following constant light treatment, no significant difference could be observed between Col, crd, and hy5 gene expression. All gene expression data were normalized to ubiquitin-like protein (UBI; At4g36800) and related to the amount present in wild-type Col. Each data point represents the mean ± sd of at least three biological replicates. Statistical differences were calculated using Student’s t test: ***, P < 0.001.

To test the genetic interaction between HY5 and CRD, we generated a hy5 crd double mutant. The hy5 single mutant showed increased CBF3 expression compared with the wild type at ZT 6 (Fig. 4B). CBF1 and CBF2 expression was similar to that in the wild type in the hy5 mutant, supporting the tetrapyrrole feeding experiment indicating that HY5 is only involved in the repression of CBF3 (Fig. 4, A and B). The tetrapyrrole levels in the hy5 mutant were similar to wild-type levels, excluding any direct tetrapyrrole effect in the mutant (Table I). If HY5 is downstream of the plastid signal, the hy5 crd double mutant should release the suppressed CBF3 expression in the crd mutant. The hy5 crd double mutant showed significantly higher CBF3 expression compared with the crd single mutant (Fig. 4B). However, hy5 is not completely epistatic to crd, suggesting that another component also is involved in the regulation of CBF3 expression. The tetrapyrrole levels in the hy5 crd double mutant were close to the levels detected for the crd single mutant (Table I); hence, the rescue of the phenotype in the hy5 crd mutant with regard to CBF3 expression cannot be explained by changed tetrapyrrole levels. We also tested CBF3 expression in the hy5 mutant following exposure to constant light at ZT 57 (Fig. 4C), when the accumulated levels of tetrapyrroles were similar to those found in the dark under normal diurnal conditions (Fig. 2C). Thus, when the oscillation of tetrapyrrole levels was lost, the hy5 mutant showed CBF3 expression similar to the wild type. Taken together, these results suggest that HY5 is responding to elevated tetrapyrrole levels and represses specifically CBF3 expression during diurnal conditions.

Table I. Mg-ProtoIX and Mg-ProtoIX-ME contents in hy5 and hy5 crd.

Plants were grown in short-day conditions (9/15 h light/dark, 22°C/18°C, and 150 µE m⁻² s⁻¹), and 3-week-old plants were sampled 6 h into the light period and analyzed for Mg-ProtoIX and Mg-ProtoIX-ME contents. Each data point represents the mean ± sd of at least three biological replicates.

Plant	Mg-ProtoIX	Mg-ProtoIX-ME
	pmol g−1 fresh wt
Col	109.4 ± 21.7	94.0 ± 16.1
crd	146.9 ± 14.4	837.2 ± 94.5
hy5	102.0 ± 12.5	65.5 ± 13.2
hy5 crd	188.0 ± 20.1	1,168.2 ± 71.7

HSP90 Responds to the Accumulated Tetrapyrroles and Inhibits HY5

The accumulation of tetrapyrroles inhibits the ATPase activity of HSP90, and it has been shown that HY5 responds to the inactivation of HSP90 (Kindgren et al., 2012). In order to examine if HSP90 plays a role in the photoperiodic control of CBF3 and COR15a expression, we investigated CBF3 and COR15a expression in two independent Col-hsp90-RNAi lines with 30% of the wild-type HSP90 expression levels left (Kindgren et al., 2012; Fig. 5A). CBF3 expression in the Col-hsp90-RNAi lines was significantly lower in the RNA interference (RNAi) lines compared with the wild type, supporting the described connection between the plastid signal and HSP90 (Fig. 5A). We also incubated crd and hy5 with the specific inhibitor of HSP90, geldanamycin (GDA). GDA binds specifically to the ATP-binding site of HSP90 (Whitesell et al., 1994) and has been shown to clearly inhibit the ATPase activity of HSP90 (Avila et al., 2006). Wild-type seedlings showed a clear repression of CBF3 expression following GDA treatment, confirming that HSP90 activity is involved in the regulation of CBF3 expression (Fig. 5B). Similar to the wild type, crd also showed a repression of CBF3 expression following GDA treatment compared with control conditions (Fig. 5B). In contrast, the hy5 mutant was insensitive to the GDA treatment with regard to CBF3 expression, suggesting that HY5 acts downstream of HSP90. However, CBF1 and CBF2 expression was repressed similar to the wild type in hy5 following GDA treatment, supporting that HY5 specifically regulates CBF3 expression (Supplemental Fig. S3D).

Figure 5.

HSP90 responds to the plastid signal and regulates HY5. A, Plants were grown in short-day conditions (9/15 h light/dark), and 3-week-old plants were analyzed for CBF3 (At4g25480) expression 6 h into the light period in Col and two independent hsp90-RNAi lines in the wild-type background. The expression data were normalized to ubiquitin-like protein (UBI; At4g36800) and related to the amount present in wild-type Col. CBF3 expression was significantly different in the Col-hsp90 lines compared with Col. Each data point represents the mean ± sd of at least three biological replicates. B, Plants were grown for 10 d in short-day conditions and analyzed for CBF3 (At4g25480) expression following 24 h of feeding with 80 µm GDA. The gene expression data were normalized to ubiquitin-like protein (At4g36800) and related to the amount present in the Murashige and Skoog (MS) control for each genotype. Each data point represents the mean ± se of at least three biological replicates. Statistical differences were calculated using Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

HY5 Interacts Physically with ZTL in Vivo

HSP90 has been shown to be required for the proper function of the circadian clock through maturation and stabilization of the clock protein ZTL (Kim et al., 2011). Possibly, ZTL could be the connection between HSP90 and HY5 and, thereby, constitute the clock component involved in the photoperiodic control of CBF3 and COR15a expression. As described previously by Kim et al. (2011) Col-hsp90-RNAi lines with reduced HSP90 levels showed reduced protein levels of ZTL, which also was seen in our lines (Supplemental Fig. S5). To test the putative link between the plastid signal and ZTL, ZTL protein levels were determined in wild-type and crd plants during the light period and 3 h after dusk (Fig. 6A). The result showed a significant reduction of ZTL protein levels in the crd mutant compared with the wild type at all time points investigated (Fig. 6B). In addition, the ZTL protein levels reached the highest amounts in the wild type once the levels of tetrapyrroles had decreased at ZT 9 and ZT 12 (Figs. 2 and 6A). It has been shown previously that ZTL protein levels oscillate with a peak at the end of the light period and that GIGANTEA is essential to establish and sustain oscillations of ZTL by a direct protein-protein interaction (Kim et al., 2007). Our data also suggest that the oscillations of tetrapyrrole levels and their inactivation of HSP90 contribute to the level of ZTL protein.

Figure 6.

ZTL is involved in the regulation of CBF3 expression. A, Plants were grown in short-day conditions (9/15 h light/dark), and 3-week-old plants of Col and crd were analyzed for ZTL protein levels every 3 h starting at dawn and the last samples were collected 3 h into the dark period. Representatives of three blots from three individual experiments are shown. B, Relative ZTL protein levels in Col and crd. Protein levels in Col were set as 100% for each blot, and protein levels in crd were compared with Col at the same time point and adjusted according to the α-tubulin loading control levels. Means of the relative levels in three independent experiments are presented. ZTL protein levels were significantly lower in crd compared with Col at all time points, as demonstrated by Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. C, Co-IP interaction was identified between ZTL and HY5 protein using Arabidopsis protoplasts. The blots presented are representative of three individual experiments. The images of negative controls and expressed ZTL and HY5 proteins are cut from the same blot, as indicated by the center lines, and merged for the illustration. D, Plants were grown in short-day conditions, and 3-week-old plants of Col, crd, hy5, prr5-1, ztl-3, ZTL-OX, ztl crd, ztl hy5, and ztl prr5 were analyzed for CBF3 (At4g25480) expression 6 h into the light period. Gene expression was normalized to ubiquitin-like protein (UBI; At4g36800) and related to the amount present in wild-type Col. Each data point represents the mean ± sd of at least three biological replicates. CBF3 expression was significantly different in crd, hy5, prr5, ztl, and ZTL-OX compared with Col and in ztl hy5 and ztl prr5 compared with ztl, as demonstrated by Student’s t test: ***, P < 0.001.

To further test the putative link between HY5 and ZTL, we determined if there is a direct physical interaction between ZTL and HY5. In an in vivo coimmunoprecipitation (Co-IP) assay, ZTL-Myc and HY5-HA fusion proteins were transiently coexpressed in Arabidopsis protoplasts, and Co-IP was performed using an anti-cMyc monoclonal antibody bound to protein G-coated magnetic beads. Inputs and Co-IP fractions were detected by immunoblot analysis with anti-c-Myc and anti-HA antibodies, respectively (Fig. 6C). HY5 was successfully detected in the ZTL-Myc immunoprecipitate, whereas only a background band could be detected in the control and single transformations. Thus, a direct physical interaction between ZTL and HY5 was observed in Arabidopsis protoplasts.

ZTL, HY5, and PRR5 Regulate CBF3 and COR15a Expression during Photoperiodic Conditions

We investigated CBF3 and COR15a expression in the ztl-3 mutant, the double mutants ztl crd and ztl hy5, and the ZTL-OX line (Fig. 6D). CBF3 and COR15a expression in the ztl mutant was very low, which indicates that ZTL is required for proper expression under warm growth conditions (Figs. 6D and 7A). In contrast, the ZTL-OX line showed higher CBF3 expression compared with the wild type (Fig. 6D). The low expression levels observed in the ztl mutant cannot be explained by changes in the circadian period; CBF3 and COR15a expression was lower compared with wild-type expression in ztl at all time points investigated in the light (Supplemental Fig. S6). In addition, the expression of CBF1 and CBF2 also was lower in the ztl mutant compared with the wild type (Supplemental Fig. S4). Furthermore, the ztl crd double mutant did not show any additive effect for CBF3 expression compared with the ztl single mutant, suggesting that CRD and ZTL are genetically linked (Fig. 6D). The hy5 ztl double mutant showed higher CBF3 and COR15a expression compared with the ztl single mutant (Figs. 6D and 7A). However, similar to the hy5 crd double mutant, hy5 is not fully epistatic to ztl, suggesting that another component is indeed involved in the regulation of CBF expression. PRR5 is a repressor of the CBF genes (Nakamichi et al., 2012) and also is negatively regulated by ZTL through ubiquitin-mediated degradation (Kiba et al., 2007). Thus, a higher level of PRR5 in the ztl mutant could be partly responsible for the dramatic decrease in CBF3 expression in the mutant (Fig. 6D). The prr5 single mutant also displayed a phenotype similar to hy5, with significantly higher CBF3 and COR15a expression levels compared with the wild type (Figs. 6D and 7A). In addition, similar to hy5, the prr5 mutant was less sensitive to the GDA treatment with regard to CBF3 expression (Supplemental Fig. S3E). However, in contrast to hy5, CBF1 and CBF2 expression was higher compared with the wild type in the prr5 mutant (Supplemental Fig. S4). The prr5 ztl double mutant displayed higher CBF3 and COR15a expression levels compared with the ztl single mutant, but again, prr5 is not fully epistatic to ztl, suggesting that HY5 and PRR5 act in concert to repress CBF3 expression in response to the plastid signal.

Figure 7.

HSP90, ZTL, and HY5 regulate COR15a in response to the plastid signal. A, Plants were grown in short-day conditions (9/15 h light/dark), and 3-week-old-plants were analyzed for COR15a (At2g42540) expression 9 h into the light period. Gene expression data were normalized to ubiquitin-like protein (UBI; At4g36800) and related to the amount present in wild-type Col. Each data point represents the mean ± sd of at least three biological replicates. COR15a expression was significantly different in crd, hy5, Col-hsp90-1, Col-hsp90-3, ztl, and prr5 compared with Col, in hy5 crd compared with crd, and in ztl crd, ztl hy5, and ztl prr5 compared with ztl. Statistical differences were calculated using Student’s t test: *, P < 0.05; and ***, P < 0.001. B, COR15a protein levels were analyzed 9 h into the light period. Coomassie Blue-stained gels are shown for loading controls. The blots are representative from at least three individual experiments with the same relative protein levels.

The protein levels of COR15a also were investigated (Fig. 7B), and protein abundance in the wild type and the mutants correlated with the observed gene expression profiles, with the high amounts of COR15a protein in the hy5 and prr5 mutants and low amounts in all other single mutants compared with the wild type. However, a higher amount of COR15a protein was found in the hy5 crd, hy5 ztl, and prr5 ztl double mutants compared with the crd and ztl single mutants (Fig. 7B). Taken together, these results indicate that HSP90 and ZTL, together with HY5 and PRR5, respond to the plastid signal and regulate CBF3 and downstream COR15a expression during photoperiodic conditions.

HY5, a Putative Target for ZTL-Mediated Degradation

ZTL and HY5 were shown to interact physically in vivo (Fig. 6C). ZTL is responsible for the proteasome-dependent degradation of TOC1 and PRR5 (Más et al., 2003; Kiba et al., 2007). Possibly, ZTL also targets HY5 for degradation. HY5 protein levels were determined in wild-type, ztl, and ZTL-OX plants during the light period and 3 h after dusk. The results from three trials indicated possibly higher HY5 protein levels in the ztl mutant and suggested slightly lower HY5 protein levels in the ZTL-OX line compared with the wild type (Supplemental Fig. S7C). To test the possibility that the degradation of HY5 may be mediated by ZTL, we measured HY5 half-life under white light in etiolated seedlings of the wild type, ztl, and ZTL-OX (Somers et al., 2004). We followed the protocol to determine protein half-life using cycloheximide (CHX) described by Kiba et al. (2007), where the seedlings were exposed to white light for 11 h and then treated with CHX and incubated under light conditions. In parallel to the CHX treatment, a mock experiment was performed. Samples were collected and HY5 levels were detected (Fig. 8A; Supplemental Fig. S7D). In the mock samples, no change in the HY5 protein level was found during the time period for any of the genotypes (Supplemental Fig. S7D). The level of HY5 protein was reduced significantly compared with the control at 9 and 12 h after the CHX treatment in wild-type plants, suggesting some level of degradation (Fig. 8B). In ztl, the HY5 half-life was extended and the protein levels were almost unchanged during the entire experiment (Fig. 8). In contrast, the degradation of HY5 was enhanced slightly in the ZTL-OX line (Fig. 8), where the level of HY5 protein was reduced significantly compared with the control at 6, 9, and 12 h after the CHX treatment. These results are similar to what was seen for PRR5 (Kiba et al., 2007), but the effects of variation in ZTL levels on HY5 turnover are much less impactful, suggesting that ZTL may be only one of multiple factors in the regulation of HY5 protein levels.

Figure 8.

HY5 protein is potentially subject to ZTL-mediated degradation. A, Five-day-old etiolated plants were exposed to white light for 11 h and then incubated with 100 µm CHX. Samples were collected at the indicated time points and analyzed for HY5 protein levels. α-Tubulin (α-TUB) was used as a loading control. Three representative blots from individual experiments are shown. B, Quantification of the results obtained in A. Each data point was normalized to the loading control levels and related to the amount present at ZT 0 for each genotype. Data points represent means ± sd of at least three independent blots and experiments. HY5 protein levels were significantly lower in wild-type (WT) Col after 9 and 12 h of incubation compared with Col at 0 h and in ZTL-OX after 6, 9, and 12 h of incubation compared with ZTL-OX at 0 h, as demonstrated by Student’s t test: **, P < 0.01; and ***, P < 0.001.

DISCUSSION

We describe a mechanism where retrograde signals triggered by diurnal oscillations in tetrapyrrole levels converge with the circadian clock to fine-tune nuclear gene expression under photoperiodic conditions. CBF expression is under circadian control, and the oscillation of CBF expression peaks 6 h into the light period (Fig. 2; Supplemental Fig. S1). Accumulation of the tetrapyrroles, Mg-ProtoIX and Mg-ProtoIX-ME, also showed a strong rhythmic pattern where the tetrapyrrole levels increased rapidly at the beginning of the light period and thereafter decreased until dusk (Fig. 2; Papenbrock et al., 1999). The rhythmic patterns of tetrapyrrole accumulation and CBF expression indicate that, when the tetrapyrrole levels peak, a repression of CBF expression is triggered (Fig. 2). This is supported by the results with the tetrapyrrole-overaccumulating mutant crd, which showed the same rhythmic circadian pattern of CBF expression as the wild type but with strongly repressed CBF expression levels. The diurnal oscillations of Mg-ProtoIX and Mg-ProtoIX-ME are completely abolished when plants are transferred to constant light (Fig. 2), and the activity of enzymes responsible for tetrapyrrole biosynthesis is inhibited when plants are exposed to continuous light (Papenbrock et al., 1999). Thus, the accumulation of Mg-ProtoIX and Mg-ProtoIX-ME is not regulated by any clock-controlled mechanisms and requires daily light and dark changes in order to maintain the oscillation pattern. When the plants were exposed to constant light, the circadian regulation of CBF3 expression was maintained in both the wild type and crd, but the observed difference in expression levels under diurnal conditions between the wild type and crd was abolished (Fig. 2). Thus, under constant light conditions, the role of the tetrapyrrole-triggered plastid signal is diminished and does not contribute to the regulation of CBF3, which then was maintained only by the circadian clock components.

Several possible mechanisms have been proposed to explain the regulation of CBF expression under warm growth conditions, including activation by LHY and CCA1 (Espinoza et al., 2010; Dong et al., 2011) and inhibition by PRR5, PRR7 and PRR9 (Nakamichi et al., 2009). Overexpression of CBF3 has been shown to affect vegetative growth and flowering time (Liu et al., 1998; Kasuga et al., 1999; Gilmour et al., 2000), suggesting that CBF3 is involved in processes other than the response to low temperatures. This is further supported by the downstream COR genes that respond not only to cold; COR15a expression, for example, also is altered in plants with defective chloroplasts and altered levels of tetrapyrroles (Nakayama et al., 2007; Bang et al., 2008; Dong et al., 2011). The promoters of CBF1 and CBF2 have G-box elements close to the transcriptional start, and the transcription factor PIF7 has been reported to bind to the G-box element in CBF2 and to function as a repressor of CBF1 and CBF2 expression under circadian control (Kidokoro et al., 2009). However, instead of a conserved G-box element, CBF3 contains Z-box elements close to the transcriptional start. Both the G-box and Z-box elements respond to light signals and have been shown to contain the core ACGT enriched in genes responding to the plastid signal triggered by tetrapyrrole accumulation (Strand et al., 2003). HY5 was described previously to respond to plastid signals (Ruckle et al., 2007; Kindgren et al., 2012), and in the experiments we report here, HY5 was confirmed to bind to the promoter fragments of CBF1, CBF2, COR15a, and CBF3, but with the strongest affinity for CBF3 (Fig. 3). In support of the biochemical data from the EMSA studies, the hy5 mutant showed higher CBF3 expression compared with the wild type, while CBF1 and CBF2 expression in the hy5 mutant was similar to that in the wild type (Fig. 4; Supplemental Fig. S4). Thus, the preferred binding of HY5 to the CBF3 promoter was shown in vivo, where HY5 functions as a repressor of CBF3 expression, but the interaction of HY5 to the promoters of CBF1 and CBF2 in vitro has no or little effect on CBF1 and CBF2 expression in vivo. Furthermore, the hy5 crd double mutant released the strong repression of CBF3 and COR15a expression shown by the crd single mutant (Figs. 4 and 7). Greater abundance of the COR15a protein also was found in both hy5 and hy5 crd mutants compared with the wild type (Fig. 7). Similar to what was observed for crd, the difference in CBF3 expression under diurnal conditions between the wild type and hy5 was abolished when the plants were exposed to constant light conditions, suggesting that HY5 responds to the plastid signal triggered by the diurnal changes in tetrapyrrole levels (Fig. 4). Thus, the repression of CBF3 expression in response to the plastid signal triggered in the crd mutant most likely involves HY5. However, the analysis of the double hy5 crd mutant suggested that other components must be involved in the regulation of CBF3 expression. PRR5 was shown to be a repressor of the CBF genes (Nakamichi et al., 2012), and similar to the hy5 mutant, prr5 showed higher CBF3 expression compared with the wild type. In contrast to hy5, CBF1 and CBF2 expression also was higher compared with the wild type in prr5 (Fig. 6; Supplemental Fig. S4).

HY5 was shown to be part of a regulatory system including HSP90 proteins, which are modified by the accumulation of tetrapyrroles in response to oxidative stress (Kindgren et al., 2012). Oxidative stress results in reduced flux through the tetrapyrrole biosynthetic pathway and a significant accumulation of the chlorophyll intermediates Mg-ProtoIX and Mg-ProtoIX-ME (Stenbaek et al., 2008; Kindgren et al., 2011; Zhang et al., 2011). Reduced flux through the tetrapyrrole pathway and the accumulation of Mg-ProtoIX were shown to inhibit the ATPase activity of HSP90, which in turn resulted in reduced expression of PhANGs via HY5 (Kindgren et al., 2012). CBF3 and COR15a expression also is dependent on functional HSP90, and both expression levels and protein content were reduced compared with the wild type in two independent HSP90 RNAi lines (Figs. 5 and 7). In addition, treatment with the inhibitor of HSP90 GDA resulted in a clear repression of CBF3 expression in the wild type and crd, confirming that HSP90 activity is involved in the regulation of CBF3 expression (Fig. 5B). In the hy5 and prr5 mutants, CBF3 expression was insensitive to GDA treatment, supporting that HY5 and PRR5 act downstream of HSP90. However, CBF1 and CBF2 expression was repressed similar to that in the wild type in hy5 following GDA treatment (Supplemental Fig. S3), which argues for a specific regulation of CBF3 by HY5.

Recently, it also was demonstrated that HSP90 is involved in maturation of the circadian clock-associated F-box protein ZTL (Kim et al., 2011). Treatment with the inhibitor GDA or RNAi-mediated depletion of cytosolic HSP90 reduced the levels of ZTL and lengthened the circadian period, consistent with ztl loss-of-function alleles (Kim et al., 2011). Thus, maturation of ZTL by HSP90 was shown to be essential for the proper function of the circadian clock. With F-box proteins such as ZTL as clients, HSP90 has a unique and central role in proteostasis and thereby could control many different cellular responses. Similar to crd and the HSP90 RNAi lines, ztl displayed lower expression levels for CBF3 and COR15a compared with the wild type (Figs. 6 and 7). Furthermore, CBF3 expression was similar in the ztl crd double mutant compared with the ztl single mutant (Fig. 6D). Thus, no enhanced suppression of expression could be found in the double mutant compared with the ztl single mutant, supporting the suggestion that CRD and ZTL are genetically linked and that ZTL is downstream of tetrapyrrole accumulation. In addition, the protein levels of ZTL were significantly lower at all time points investigated in the crd mutant compared with the wild type (Fig. 6), supporting the demonstrated inhibition of HSP90 activity by accumulated tetrapyrroles (Kindgren et al., 2012). It is clear that both HY5 and ZTL are linked to HSP90 function. In addition, in a Co-IP assay using Arabidopsis protoplasts, HY5 was found to interact directly with ZTL (Fig. 6), emphasizing the connection between these two components. The F-box protein ZTL is responsible for the proteasome-dependent degradation of TOC1 and PRR5 (Más et al., 2003; Kiba et al., 2007). It is clear that PRR5 is involved in the regulation of CBF expression during photoperiodic growth (Figs. 6 and 7). In addition, similar to PRR5, increased stability of HY5 was seen in the ztl mutant, suggesting that HY5 possibly is a target for ZTL-mediated degradation. Thus, the effects seen in ztl with severely repressed expression of CBF3 could be a response to higher levels of HY5 and PRR5. It is unlikely that TOC1 would contribute to CBF regulation, since no effect on CBF1 to CBF3 expression was reported in the toc mutants. Putative target genes for TOC1, however, do include HY5 (Huang et al., 2012), but HY5 was not differentially expressed in overexpressors of TOC1 or in the toc1-2 mutant (Legnaioli et al., 2009). HY5 was not found to be regulated by PRR5 (Nakamichi et al., 2012), indicating that a direct effect of PRR5 on HY5 levels also is unlikely in the ztl mutant. Thus, CBF expression is regulated by a combination of HY5 and PRR5 during photoperiodic growth.

The regulation of ZTL by the plastid signal via HSP90 is most likely the site of interaction between the clock and the plastid signaling pathways. In addition to the impaired retrograde signaling, the crd mutant has a pale phenotype and reduced growth (Tottey et al., 2003; Bang et al., 2008). Thus, it is possible that the plastid signal triggered in the crd mutant could be more complex than the described overaccumulation of tetrapyrroles. However, connections between plastid and circadian signaling pathways have been suggested in the literature, and mutations in CHLOROPLAST RNA BINDING (CRB) resulted in an increased amplitude of expression of CCA1 and LHY. The crb plants showed altered chloroplast morphology and impaired chlorophyll biosynthesis, suggesting an involvement of the chloroplast, and tetrapyrrole biosynthesis in particular, in the regulation of the circadian clock in Arabidopsis (Hassidim et al., 2007). It has been shown that CCA1 and LHY bind to the promoters of CBF1 to CBF3 to activate their transcription (Dong et al., 2011). In addition, CCA1 and HY5 together positively regulate LHCB gene expression (Andronis et al., 2008). Hence, it is possible that HY5 impacts the CCA1 regulation of CBFs, positively or negatively depending on the chloroplast signaling status. PRR5 and PIF7 could potentially interact as repressors of CBF1 and CBF2 expression. However, the difference in CBF1 and CBF2 expression between the wild type and the pif7 mutant was only found when the plants were exposed to constant light; under light/dark cycles, no difference in CBF expression could be detected (Kidokoro et al., 2009; Lee and Thomashow, 2012).

We propose here a model for integration between plastid and circadian signaling pathways (Fig. 9). This cross talk would provide a mechanism for fine-tuning gene expression during light/dark cycles. It was suggested that HSP90 activity is under diurnal or circadian control (Kim et al., 2011), and we have shown that HSP90 responds to a plastid signal triggered by the accumulation of tetrapyrroles (Kindgren et al., 2012). Accumulation of tetrapyrroles demonstrated a strong rhythmic pattern (Fig. 2) and thus could explain the phase-specific differences in HSP90 activity (Kim et al., 2011). When tetrapyrrole levels are elevated, HSP90 is inactivated, and as a consequence, there is a significant reduction in ZTL levels (Fig. 6). Low levels of ZTL would potentially result in increased protein levels of HY5 and PRR5 and a repression of CBFs (Fig. 9). HY5 and PRR5 act in concert to repress CBF3, and PRR5 represses CBF1 and CBF2. The plastid signal thereby contributes to the rhythm of CBF expression during day/night cycles. These findings provide a mechanism by which plastid signals converge with and impact on the activity of well-defined clock components involved in circadian regulation, and our results provide further evidence that the functional state of the chloroplast is an important factor that affects the circadian system. The close interaction between the circadian clock and chloroplast retrograde signaling systems could fine-tune photosynthetic activity under photoperiodic conditions.

Figure 9.

Model for the regulation of CBF expression under photoperiodic conditions. We propose a model for integration between plastid and circadian signaling pathways. HSP90 responds to the plastid signal triggered by tetrapyrrole accumulation, and its activity is inhibited. The accumulation of tetrapyrroles demonstrated a strong rhythmic pattern, and when the tetrapyrrole levels are elevated, HSP90 is inactivated. A consequence of HSP90 inactivation is a significant reduction in ZTL levels. Low levels of ZTL would result in increased levels of HY5 and PRR5 and a repression of CBFs. HY5 and PRR5 act in concert to repress CBF3 and PRR5 represses CBF1 and CBF2. The plastid signal thereby contributes to the rhythm of CBF and COR expression during day/night cycles.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants were grown for 3 weeks on soil in short-day conditions (9/15-h light/dark and 22°C/18°C) with a light intensity of 150 µmol photons m⁻² s⁻¹. All mutants were in the Col background and described elsewhere: crd1 (Ankele et al., 2007), hy5-1 (Maxwell et al., 2003; Kleine et al., 2007), Col-hsp90-RNAi lines (Kindgren et al., 2012), ztl-3 (Jarillo et al., 2001), and prr5-1 (Eriksson et al., 2003). For the constant light experiments, plants were kept in constant 150 µmol photons m⁻² s⁻¹ light and 22°C. For the Mg-ProtoIX feeding experiments, plants were grown on soil in short-day conditions for 3 weeks and then transferred to either 1× MS or 1× MS + 50 µm Mg-ProtoIX solution and kept in continuous low light (20 µmol photons m⁻² s⁻¹). Roots were rinsed with water before incubation. The feeding was started at 9 am, and plants were sampled 12 h later. To avoid contamination of Mg-ProtoIX in solution, only green tissue was sampled for real-time PCR and HPLC analysis. Cold treatment was performed in 4°C, short-day conditions (9/15 h), and 150 µmol photons m⁻² s⁻¹ light. Plants for GDA feeding were grown on 1× MS + 1% Suc plates for 10 d in short-day conditions and then incubated with 1× MS or 1× MS + 80 µm GDA + 0.01% Triton X-100 solution for 24 h. The feeding was started at ZT 6, and fresh GDA solution was added after 3 and 18 h. For the CHX experiment, seeds were plated on 1× MS plates, exposed to 3 h of 150 µmol photons m⁻² s⁻¹ light, and then grown in the dark for 5 d. Etiolated plants were exposed to 11 h of 150 µmol photons m⁻² s⁻¹ light before starting the incubation with 100 µm CHX solution in 1× MS + 0.01% Triton X-100 or mock treatment with 1× MS + 0.01% Triton X-100. Plants were incubated shaking at 100 rpm for 12 h.

RNA Isolation, Complementary DNA Synthesis, and Real-Time PCR

Total RNA was isolated using the EZNA Plant RNA mini kit, and DNase treatment was performed using Thermo Scientific DNase I, RNase-free according to the manufacturer’s instructions. Using the iScript cDNA Synthesis Kit (Bio-Rad), complementary DNA (cDNA) was synthesized from 0.5 µg of total RNA according to the manufacturer’s instructions. cDNA was diluted 10-fold, and 3 µL of the diluted cDNA was used in a 10-µL iQ SYBR Green Supermix reaction (Bio-Rad). All reactions were performed in three technical replicates using primers for the genes COR15a, COR47, CBF1, CBF2, CBF3, FER1, GST5, and MAPK18, and relative gene expression was normalized to the expression level of ubiquitin-like protein (Supplemental Table S1). Quantitative real time-PCR was run in the CFX96 real-time system (Bio-Rad) and monitored using the CFX Manager (Bio-Rad). The adjustment of baseline and threshold was done according to the manufacturer’s recommendations. Data were analyzed using LinRegPCR (Pfaffl, 2001; Ramakers et al., 2003), and the relative abundance of all transcripts amplified was normalized to the constitutive expression level of ubiquitin-like protein.

HPLC Analysis

HPLC analyses were done according to the method described by Mochizuki et al. (2008). Leaf material was homogenized in acetone:0.1 m NH₄OH (9:1, v/v). Column eluent was monitored by UV light detection, and tetrapyrroles were identified and quantified using authentic standards. Mg-ProtoIX and Mg-ProtoIX-ME were purchased from Frontier Scientific.

Expression and Purification of HY5

A fragment of the coding region of HY5 was amplified from Arabidopsis (Col) genomic DNA (Supplemental Table S1). The fragment was cloned into pDONR207 using Gateway technology (Invitrogen) according to the manufacturer’s instructions. The cloned fragment was sequenced to confirm that no mistakes were made during the PCR. For expression, the fragment was subsequently cloned into pETG-10K vector, creating a His-HY5 fusion protein. The protein was expressed in Escherichia coli Rosetta2 cells. Transformed cells were grown in Luria-Bertani medium at 37°C until the optical density at 600 nm reached 0.4 and transferred to 16°C for 30 min. Expression was induced by adding isopropylthio-β-galactoside to a final concentration of 0.2 mm. The induced culture was grown at 16°C for 4 h and harvested. The pellet was dissolved in ice-cold lysis buffer (50 mm Tris, pH 7.2, 500 mm NaCl, 10 mm imidazole, and 7 mm β-mercaptoethanol), and cells were destroyed by homogenization. After centrifugation (13,000g, 15 min, and 4°C), the supernatant was loaded onto a nickel-nitrilotriacetic acid agarose column (Bio-Rad). The protein was purified according to the manufacturer’s instructions. Eluted protein was dialyzed overnight at 4°C into dialysis buffer (50 mm Tris, pH 7.2, 500 mm NaCl, 50% glycerol, and 7 mm β-mercaptoethanol).

EMSA

EMSAs were done according to Schallenberg-Rüdinger et al. (2013). Briefly, purified protein was incubated with 500 pm labeled DNA oligonucleotide (Sigma) for 30 min at 25°C. Final binding reactions included 10% glycerol, 1× THE, pH 7.2, 200 mm NaCl, 5 mm dithiothreitol, 0.1 mg mL⁻¹ bovine serum albumin, 0.5 mg mL⁻¹ heparin, and 8 units of RNAseOUT. Samples were loaded onto a prerun 5% 1× THE native polyacrylamide gel, and a constant voltage of 100 V was applied. Gels were visualized in a Typhoon scanner (GE Healthcare).

Western-Blot Analysis

To determine the levels of COR15a protein in the plants, 3-week-old plants grown in short-day conditions were sampled 9 h into the light period. A total of 20 mg of ground plant material was used for protein extraction with 200 µL of extraction buffer (10% [w/v] SDS, 20% [v/v] glycerol, 0.2 m Tris-HCl, pH 6.8, 0.05% [w/v] Bromophenol Blue, 10 mm β-methanol, and 5 mm dithiothreitol). The samples were loaded onto a 16% gel and separated by SDS-PAGE. Equal protein loading was determined by Coomassie Blue staining, and the COR15a antibody (Nakayama et al., 2007) was used for detection by western blotting. For ZTL protein levels, plants were grown for 3 weeks in short-day conditions and sampled 0, 3, 6, and 9 h into the light period and also 3 h into the dark period. The samples were prepared as described above and separated on a 12% gel, and ZTL antibody (Kim et al., 2011) was used for detection by western blotting. To determine HY5 protein levels in Col and ztl during short-day conditions, plants were grown for 7 d on MS medium + 1% Suc plates and sampled 0, 3, 6, and 9 h into the light period and also 3 h into the dark period. The samples were prepared as described above and separated onto a 12% gel, and two different HY5 antibodies (Santa Cruz Biotechnology and a gift from Dr. X.W. Deng) were used for detection by western blotting. In contrast to the western blot presented by Kleine et al. (2007) using the Santa Cruz Biotechnology HY5 antibody, where seedlings were used, we used mature plants. In the mature plants, the hy5-1 mutant contains some HY5 protein, demonstrating that it is not a complete null allele. Equal protein loading was determined by the detection of α-tubulin levels on the same membrane as the protein of interest. Quantification of the ZTL and HY5 protein levels was done using the program ImageJ, and each data point was normalized to the loading control levels on each blot.

Co-IP Assay

Expression constructs were generated for the assay by cloning full-length coding sequences of ZTL and HY5 into pRT104_3Myc and pRT104_3HA vector, respectively. Arabidopsis Landsberg cell cultures were grown at constant 12/12-h light/dark, 23°C/23°C, with a light intensity of 150 μmol m⁻² s⁻¹. Protoplasts were isolated and transformed with ZTL-Myc or HY5-HA constructs and analyzed as described by Shaikhali et al. (2012).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Expression of CBF1 and CBF2 in Col and crd.

• Supplemental Figure S2. Tetrapyrrole contents in 3-week-old plants following a shift to 4°C.

• Supplemental Figure S3. Tetrapyrrole levels following Mg-ProtoIX feeding.

• Supplemental Figure S4. Expression analysis of CBF1 to CBF3 in Col, hy5, prr5, and ztl.

• Supplemental Figure S5. ZTL protein contents in two independent HSP90 RNAi lines.

• Supplemental Figure S6. Expression analysis of CBF3 and COR15 during a day/night cycle in Col and ztl.

• Supplemental Figure S7. HY5 protein contents in Col, ztl, and ZTL-OX under control conditions.

• Supplemental Table S1. Primers used in real-time PCR analysis and for cDNA synthesis.

Glossary

Mg-ProtoIX-ME

magnesium protoporphyrin IX-methylester

Mg-ProtoIX

magnesium protoporphyrin IX

EMSA

electromobility shift assay

ROS

reactive oxygen species

RNAi

RNA interference

GDA

geldanamycin

Co-IP

coimmunoprecipitation

CHX

cycloheximide

MS

Murashige and Skoog

cDNA

complementary DNA

Col

Columbia