- © 2015 American Society of Plant Biologists. All rights reserved.
Abstract
The regulation of CONSTANS (CO) gene expression is crucial to accurately measure changes in daylength, which influences flowering time in Arabidopsis thaliana. CO expression is under both transcriptional and posttranslational control mechanisms. We previously showed that the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) physically interacts with CO in Arabidopsis. This interaction is required to precisely modulate the timing of CO accumulation and, consequently, to maintain low levels of FLOWERING LOCUS T expression during the first part of the day. The data presented here demonstrate that HOS1 is involved in the red light-mediated degradation of CO that takes place in the early stages of the daylight period. Our results show that phytochrome B (phyB) is able to regulate flowering time, acting in the phloem companion cells, as previously described for CO and HOS1. Moreover, we reveal that phyB physically interacts with HOS1 and CO, indicating that the three proteins may be present in a complex in planta that is required to coordinate a correct photoperiodic response in Arabidopsis.
INTRODUCTION
The timing of flowering to favorable seasons of the year is crucial for reproductive success of plants and is regulated by a number of environmental factors, including daylength (photoperiod), light quality, and temperature (Verhage et al., 2014). Genetic approaches in the model species Arabidopsis thaliana have shown how the underlying responses to changes in daylength are conferred and how they converge to create a robust seasonal response (Andrés and Coupland, 2012). Flowering time in this species, which flowers earlier under long-day (LD) conditions than under short-day (SD) conditions, is regulated by several interconnected pathways. Environmental cues such as daylength, low winter temperatures, and ambient temperature influence flowering through the photoperiod, vernalization, and temperature pathway, respectively. In contrast, endogenous signals are integrated in the control of flowering time based on plant age and via the autonomous and gibberellin pathways (Fornara et al., 2010). Information from the vernalization and autonomous pathways modulate the central floral repressor FLOWERING LOCUS C (FLC), a MADS box protein (Amasino, 2010). Eventually, the entire network converges in the regulation of FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 genes, which act as a switch of flowering initiation (Fornara et al., 2010).
Different core components of the photoperiod pathway, such as CONSTANS (CO) and FT, are widely conserved among plant species (Kobayashi and Weigel, 2007; Turck et al., 2008; Serrano et al., 2009). In Arabidopsis, the expression of the floral integrator gene FT is activated in the phloem companion cells of the leaves by CO, a B-box-type transcription factor (An et al., 2004; Jackson, 2009; Tiwari et al., 2010). Under LDs, a mobile signal translocates from the leaves to the shoot apex, promoting the floral transition. FT protein, and possibly TSF, are an essential part of this so called florigen (Corbesier et al., 2007; Jaeger and Wigge, 2007; Jang et al., 2009). The amount of FT strongly influences the timing of flowering in Arabidopsis (Andrés and Coupland, 2012; Golembeski et al., 2014).
Different layers of CO regulation at the transcriptional and posttranslational level are crucial for the ability of Arabidopsis to perceive and respond to photoperiod. The coincidence of CO expression with the light in LD conditions is essential for the promotion of flowering. In addition, different light qualities play antagonistic roles in the photoperiodic flowering response: while blue and far-red light (F-RL) promote flowering, red light (RL) delays it (Valverde et al., 2004). Circadian clock-regulated components, such as GIGANTEA (GI), CYCLING DOF FACTORS (CDFs), and the F-box protein FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1), regulate daily CO expression profiles (Imaizumi et al., 2005; Sawa et al., 2007; Fornara et al., 2009). Blue light promotes the binding of the E3 ubiquitin ligase FKF1 to GI; this interaction is necessary for the degradation of a family of CO repressors, the CDFs (Sawa et al., 2007). As a result, under inductive photoperiods, CO transcription peaks in the evening following the degradation of the CDFs at the end of a LD. A second peak of CO expression can be observed during the night and the early morning. In contrast, under SDs, the evening peak of CO expression disappears and only the night peak remains (Suárez-López et al., 2001; Song et al., 2013).
In addition, CO protein stability is controlled by various light signals during the day (Valverde et al., 2004; Jang et al., 2008). At night, the RING finger protein CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), together with members of the SUPPRESSOR OF PHYA-105 family (SPA1, SPA3, and SPA4), promote CO degradation (Laubinger et al., 2006; Jang et al., 2008). In the evening, blue light prevents CO proteolysis by COP1 (Jang et al., 2008; Liu et al., 2008; Yu et al., 2008). The photoreceptors Cryptochrome 1 (Cry1) and Cry2 use different mechanisms to repress COP1-SPA function in a blue light-dependent manner (Lian et al., 2011; H. Liu et al., 2011; Zuo et al., 2011). Recently, an additional role for the blue light photoreceptor FKF1 in CO regulation has been proposed, as it stabilizes CO protein in the evening under LD (Song et al., 2012).
In order to precisely regulate the timing of CO accumulation, an additional mechanism of CO degradation takes place in the morning (Jang et al., 2008). This mechanism depends on the RL photoreceptor phytochrome B (phyB), but the E3 ubiquitin ligase COP1 is not involved in this process (Valverde et al., 2004; Jang et al., 2008). Previous results from our group revealed that another RING finger protein, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1), participates in the control of photoperiodic flowering in Arabidopsis by negatively regulating CO abundance in the morning (Lazaro et al., 2012). The early flowering phenotype of hos1 mutants is due to the precocious upregulation of FT expression levels and is completely suppressed by mutations in the CO gene in the Landsberg erecta (Ler) background (Lazaro et al., 2012). Moreover, we showed that HOS1 physically interacts with CO in Arabidopsis and that the CO protein abnormally accumulates in the hos1 mutant during the early hours of the daylight period (Jung et al., 2012; Lazaro et al., 2012). Altogether, these observations suggest that a complex regulatory mechanism ensures that CO accumulates only at the end of a LD, allowing a correct seasonal response. In addition, CO activity in the early morning is inhibited by the TARGET OF EAT1 protein. This mechanism adds a new layer in the regulation of FT expression that ensures that flowering only takes place after the daylength reaches a threshold (Zhang et al., 2015).
The phytochrome family of photoreceptors (in Arabidopsis phyA to phyE) perceives the red and far-red wavelengths (Franklin and Quail, 2010). Phytochromes can exist in two photoreversible forms: a RL-absorbing Pr form (biologically inactive) and a F-RL-absorbing Pfr form (biologically active) (Rockwell et al., 2006). Photoactivation of the cytoplasmic-localized photoreceptors triggers their translocation into the nucleus, where several signaling components regulate different light responses (Kircher et al., 2002; Nagy and Schäfer, 2002; Casal, 2013). In the germination, hypocotyl elongation, and shade avoidance responses, nuclear Pfr phyB induces the rapid phosphorylation and degradation of negative regulators known as PHYTOCHROME-INTERACTING FACTORs (PIFs). Seven members of this subfamily of basic helix-loop-helix transcription factors (PIF1/PIF3-LIKE5 [PIL5], PIF3, PIF4, PIF5/PIL6, PIF6/PIL2, PIF7, and PIF8) have been shown to physically interact with phyB (Leivar and Quail, 2011; Casal, 2013). Moreover, phyB active form can be reverted to the inactive Pr form by F-RL or in the dark (D). More recently, it has been described that an additional receptor desensitization mechanism mediated by COP1 exists in Arabidopsis. A fraction of the Pfr form of phyB becomes degraded by COP1 in the nucleus, and the interaction between COP1 and phyB is enhanced by different PIFs (Jang et al., 2010). In addition, PIF3 has also been reported to recruit the LIGHT-RESPONSE BTB (LRB) proteins to the phyB-PIF3 complex and trigger the degradation of phyB and PIF3 itself (Ni et al., 2014). The LRB1 and LRB2 proteins are target recognition adaptors of multimeric E3 ubiquitin ligases, and mutations in both loci delay flowering in Arabidopsis (Christians et al., 2012). LRB proteins negatively influence phyB action by regulating its stability, thus providing another attenuation mechanism of phyB signaling (Christians et al., 2012; Ni et al., 2014).
In contrast, only a few signaling components have been identified in the regulation of the floral transition by phyB. In response to RL, phyB downregulates CO mRNA levels through factors such as PHYTOCHROME AND FLOWERING TIME1 (PFT1). PFT1 is an activator of CO transcription that has been proposed to act as a negative regulator of phytochrome signaling (Cerdán and Chory, 2003; Wollenberg et al., 2008; Iñigo et al., 2012). In addition, several factors have been shown to interact with phyB and regulate photoperiodic flowering. The PHYTOCHROME-DEPENDENT LATE FLOWERING (PHL) nuclear protein accelerates flowering by suppressing the inhibitory effect of phyB on flowering time (Endo et al., 2013). PHL interacts physically with phyB in a RL-dependent manner and, in the presence of active phyB, PHL interacts with CO as well (Endo et al., 2013). The VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2 transcription factors are required for proper regulation of flowering time and cold stress responses in Arabidopsis (Yasui et al., 2012; Celesnik et al., 2013; Nakai et al., 2013). The voz1 voz2 double mutant plants display a late flowering phenotype under LD conditions, which is suppressed by mutations in PHYB (Yasui et al., 2012; Celesnik et al., 2013). Moreover, interaction of VOZ proteins with phyB was shown to occur in the cytoplasm under F-RL, and the VOZ proteins are degraded in the nucleus under F-RL and D conditions in a phytochrome-dependent manner (Yasui et al., 2012). Therefore, it has been proposed that the partial translocation of VOZ proteins from the cytoplasm to the nucleus may mediate the initial step of the phyB signal transduction pathway that regulates flowering (Yasui et al., 2012).
However, the molecular mechanism mediating the RL-induced degradation of CO that regulates photoperiodic flowering in Arabidopsis remains to be elucidated. A recent report has shown that intermittent cold treatments trigger the degradation of CO through a HOS1-mediated ubiquitination process (Jung et al., 2012). Interestingly, cold-induced regulation of CO abundance occurs independently of phyB because the effects of cold on CO degradation are maintained in the phyB-9 background (Jung et al., 2012, 2014). Here, we present evidence indicating that the degradation of CO under RL is prevented in the hos1 mutant at normal growth temperatures; therefore, we propose that HOS1 is involved in the RL-mediated degradation of CO. Complementation studies reveal that phyB plays a key role in the vascular tissue to regulate flowering time, similarly to CO and HOS1 (An et al., 2004; Lazaro et al., 2012). Moreover, we demonstrate that phyB physically interacts in vivo with HOS1 and CO, suggesting that the mechanism by which CO is degraded under RL involves the formation of a complex between these proteins that may be essential to modulate a correct photoperiodic response in Arabidopsis.
RESULTS
HOS1 and PHYB Regulate Flowering Time Additively
Previous results have shown that both the hos1-3 and phyB-9 mutants display an early flowering phenotype under LD conditions compared with Col (Goto et al., 1991; Reed et al., 1993; Lazaro et al., 2012; Lee et al., 2012), and our results show that the hos1 mutant flowers, on average, when it has two fewer leaves than phyB (Figures 1A and 1B). Under SD photoperiods, the early flowering phenotype of hos1 mutant is accentuated compared with phyB (Figures 1A and 1C). Interestingly, the double mutant hos1 phyB flowers even earlier than any of the single mutants both in LD and SD, revealing an additive role of HOS1 and PHYB genes in the regulation of flowering time (Figures 1A to 1C).
HOS1 and PHYB Regulate Flowering Time Additively.
(A) to (C) Flowering time phenotype of Col, hos1-3 and phyB-9 single mutants, and hos1-3 phyB-9 double mutant plants. Plants in (A) were grown for 18 d in LD conditions and for 60 d in SD. Data in (B) and (C) are shown as mean ± se and were derived from two experiments in which a minimum of 20 plants were scored for each genotype. Student’s t test *P < 0.01 when compared with hos1-3 phyB-9.
(D) and (E) Flowering time phenotype of Col and hos1-3, phyB-9, and co-10 double and triple mutant combinations. Plants in (D) were grown for 42 d in LD conditions. Data in (E) are shown as mean ± se and were derived from two experiments in which a minimum of 15 plants were scored for each genotype. Student’s t test *P < 0.01 when compared with co-10.
To assess if the early flowering phenotype of both hos1 and phyB mutants depends on the presence of a functional CO gene, we obtained double and triple mutants combining mutant alleles of HOS1, phyB, and CO. This analysis allowed us to confirm that the double mutant phyB-9 co-10 showed the same flowering time as co-10 in LDs, confirming an epistasis of CO gene over phyB (Iñigo et al., 2012). On the other hand, the double mutant hos1-3 co-10 and the triple mutant hos1-3 co-10 phyB-9 showed that hos1 mutation is still able to accelerate co late flowering phenotype in all combinations tested (Figures 1D and 1E), indicating that HOS1 has an additional role in flowering time control besides regulating CO. This is consistent with previous data demonstrating that HOS1 is required to maintain high expression levels of the floral repressor FLC (Lee et al., 2001; Lazaro et al., 2012; Jung et al., 2013). We analyzed FLC transcript levels in the single, double, and triple mutant plants and observed that the hos1 mutation consistently reduces FLC expression in every combination analyzed compared with the wild type (Supplemental Figure 1). Hence, we conclude that the regulatory effect of HOS1 over FLC could account for the additive flowering phenotype observed in the hos1 phyB double mutant plants, as well as for the acceleration of the late flowering phenotype of co in the hos1 co combinations.
We also analyzed the genetic relationship between HOS1 and the genes encoding the VOZ transcription factors. The voz1 voz2 double mutant plants show a delay on flowering time under LD conditions that is suppressed by phyB mutation (Yasui et al., 2012; Celesnik et al., 2013). Taking into account the involvement of HOS1 and VOZ proteins in common biological processes (Lee et al., 2001; Lazaro et al., 2012; Yasui et al., 2012; Celesnik et al., 2013; Nakai et al., 2013), we decided to generate the triple mutant hos1-3 voz1-1 voz2-1 and analyze its flowering time. The triple mutant showed an intermediate phenotype in LDs between the early flowering time of hos1 and the late flowering time of the voz1-1 voz2-1 double mutant (Supplemental Figure 2). This result could indicate that HOS1 and the VOZ proteins do not regulate the floral transition through the same pathway, although we cannot rule out the possibility that a genetic interaction might be masked by the photoperiod-independent effect of HOS1 on flowering time (Supplemental Figure 1).
Regulation of FT Expression by HOS1 and phyB Depends on CO
Previous results from our group showed that the early flowering phenotype of hos1 mutant requires a functional FT gene and that HOS1 is required to repress the expression of FT in the first part of the daylight period under LDs (Lazaro et al., 2012), when the involvement of phyB in the modulation of CO stability has been proposed (Valverde et al., 2004; Jang et al., 2008). In a time-course expression analysis over a 24-h period, we observed that the hos1-2 mutant showed an unusual peak of FT expression in the subjective morning, mainly at Zeitgeber time 4 (ZT4), but also at ZT8, when CO transcript levels are barely detectable in the mutant (Lazaro et al., 2012). In order to investigate the behavior of the FT transcript in plants lacking HOS1, phyB, and/or CO, we performed expression analyses in the hos1, phyB, and co double and triple mutant combinations. Seedlings were grown in LD for 11 d and harvested at ZT4 and ZT16. During the early stage of daylight (ZT4), mutations in HOS1, phyB, or both cause an increase in FT expression compared with the wild type (Figure 2A). As we previously showed for the hos1-2 mutant (Ler background), the hos1-3 mutant (Col) displays high FT levels at this time point (Lazaro et al., 2012). In addition, when these mutations were combined with a mutation in CO, FT expression was reduced to the levels observed in Col plants, or even lower in the case of phyB co double mutant (Figure 2A). In contrast, during the subjective evening (ZT16), only the phyB-9 single mutant and the hos1-3 phyB-9 double mutant showed higher FT expression than Col, while all the mutant combinations affected in CO showed a clear decrease in FT levels compared with the wild type (Figure 2B).
Regulation of FT Expression by HOS1 and phyB Depends on CO.
FT expression analysis in the hos1, co, and phyB mutant combinations. Eleven-day-old seedlings grown in LDs were harvested at ZT4 (A) and ZT16 (B), and FT expression was detected by RT-qPCR. Relative expression levels were normalized to UBC21 expression. Each data point was derived from three biological replicates and is shown as mean ± se of three technical replicates.
The variation detected in FT expression in the different mutant combinations analyzed correlates with the flowering time phenotypes shown in Figure 1. In addition, these results corroborate that HOS1 regulation of FT expression depends on CO and that the additive flowering phenotype observed in the hos1 phyB and hos1 co double mutants, as well as in the hos1 phyB co triple mutant, could be explained by the effect of HOS1 on additional flowering time pathways mediated by FLC (Supplemental Figure 1).
Mutations in HOS1 Affect Several Developmental Processes Regulated by RL
Light quality regulates many developmental processes in plants such as flowering time, shade avoidance, hypocotyl elongation, and seed germination (Kami et al., 2010; Casal, 2013). In order to gain a deeper insight in the implication of HOS1 in the RL-dependent regulation of several of these developmental transitions, we analyzed the phenotypic alterations present in the hos1 mutant and the hos1 phyB double mutant.
As previously described, phyB mutant plants grown under continuous RL displayed a conspicuous early flowering phenotype (Reed et al., 1993; Endo et al., 2013), indicating that this wavelength delays flowering time in Arabidopsis. We have also grown Col, hos1, and the double hos1 phyB mutant under continuous RL, and we observed that hos1 flowering time is not as delayed as in the wild type, but later than in the phyB mutant (Figure 3A). In addition, the hos1 phyB double mutant flowers earlier than any of the single parents. Both results indicate that HOS1 is mediating RL signal transduction in the flowering response, although the early flowering phenotype of hos1 mutants under RL is not totally dependent on PHYB.
Mutations in HOS1 Affect Several Developmental Processes Regulated by RL.
(A) Flowering time phenotype of Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant plants grown in continuous RL. Data are shown as mean ± se. A minimum of 15 plants were scored for each genotype. Student’s t test *P < 0.01 when compared with hos1-3 phyB-9.
(B) Hypocotyl length in Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seedlings grown in continuous RL or D for 5 d.
(C) Quantification of the percentage of inhibition of hypocotyl elongation in Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seedlings. Data are shown as mean ± se and were derived from three experiments in which a minimum of 25 seedlings were scored for each genotype.
(D) and (E) Germination phenotype of Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seeds after 5 d in D.
(F) and (G) Germination phenotype of Col, hos1-3, pil5-2, and hos1-3 pil5-2 double mutant seeds after 2 d in D. Arrows mark emerging radicles from pil5-2 mutant seeds.
(H) and (I) Germination phenotype of Col, hos1-3, phyB-9, and hos1-3 phyB-9 double mutant seeds after 2 d in continuous RL.
(J) Germination phenotype of Col, hos1-3, phyB-9, hos1-3 phyB-9, pil5-2, and hos1-3 pil5-2 stratified seeds after 5 d in WL LDs (viability). Data are shown as mean ± se and were derived from three experiments in which a minimum of 50 seeds were scored for each genotype.
To check the effect of HOS1 in hypocotyl elongation, we grew Col, hos1, phyB, and hos1 phyB mutant seedlings in continuous RL for 5 d. In accordance with the inhibitory effect of RL on this process, wild-type seedlings displayed hypocotyls ∼60% shorter than in D conditions, while phyB mutants were completely insensitive to RL, showing no inhibition of hypocotyl elongation under this wavelength compared with D. We found that RL inhibits hypocotyl growth in hos1 to a smaller extent than in Col, ∼50%, which is consistent with previous observations (MacGregor et al., 2013) and that this phenotype totally depends on a functional PHYB gene because the double mutant hos1 phyB is as insensitive to RL as phyB (Figures 3B and 3C). These findings suggest that HOS1 participates in the PHYB-dependent pathway that controls hypocotyl elongation under RL conditions.
In contrast to the repressive role of RL on the floral transition and hypocotyl elongation, this light wavelength promotes seed germination, as shown by the failure to germinate of the phyB mutant under short pulses of RL or D conditions (Shinomura et al., 1994; Hennig et al., 2002; Oh et al., 2004). Unexpectedly, when we performed germination assays, we observed that hos1 mutant seeds showed a high germination rate in the D (on average 50% of the seeds germinate), conditions in which Col seeds do not germinate at all (Figures 3D to 3G). As expected, phyB seeds are completely incompetent to germinate in D conditions, while the double mutant hos1 phyB displayed a low germination rate compared with the hos1 mutant after 5 d in the D (Figures 3D and 3E). Still, some of the double mutant seeds (an average of 9%) germinated in D conditions, indicating that the hos1 germination phenotype depends to some extent on the PHYB gene.
To further investigate the role of HOS1 in the regulation of seed germination, we obtained the double mutant hos1-3 pil5-2. PIL5, also known as PIF1, has been proposed to act as a molecular switch that connects light perception to hormonal pathways that regulate seed germination (Oh et al., 2004; Lau and Deng, 2010). Previous studies indicate that some phytochromes activate seed germination by promoting the degradation of PIL5 protein (Oh et al., 2006); therefore, we investigated whether HOS1 could be involved in the same repressing mechanism as PIL5. As shown in Figures 3F and 3G, HOS1 and PIL5 act additively regarding seed germination because almost every seed of the double mutant hos1-3 pil5-2 germinated after 2 d in D, while the germination rate of the hos1 and the pil5 single mutants was around 56 and 33%, respectively.
We also examined the germination rate of hos1 and hos1 phyB under continuous RL. In accordance with the role of RL in promoting seed germination, almost every Col and hos1 mutant seed germinated after 2 d under this light condition (Figures 3H and 3I). As expected, phyB mutant seeds showed lower germination rates than Col and hos1, but still, three out of four seeds germinated, probably due to the effect of other RL-absorbing phytochromes. Interestingly, the loss of HOS1 overcomes the incompetence to germinate of one-quarter of the phyB mutant seeds, as almost every hos1 phyB seed germinated under RL (Figures 3H and 3I). To rule out that these results could be due to low seed viability, we confirmed that the germination rate in every seed batch used was 100% after stratifying the seeds and exposing them to white light (WL) LDs for 5 d (Figure 3J).
Altogether, these observations led us to conclude that HOS1 is involved in repressing seed germination in D and RL conditions. The hos1 mutant shows a constitutive activation of seed germination even in the absence of light, and the effect of PHYB in seed germination under continuous RL is fully dependent on a functional HOS1 gene. Furthermore, our results demonstrate that HOS1 is involved in the regulation of an array of developmental responses influenced by RL.
The Level and Subcellular Localization of HOS1 Are Independent of Light Conditions
Since HOS1 is necessary for the regulation of several developmental processes that are also controlled by RL, we decided to test whether HOS1 protein stability or subcellular localization is modulated by light. For that, we studied HOS1 protein regulation in WL, D, and RL conditions in transgenic lines harboring a 35S:HOS1-GFP construct in the hos1-3 mutant background. We previously confirmed that these transgenic plants showed a delay in flowering time when compared with hos1-3, indicating that the fusion protein was functional (Lazaro et al., 2012). In order to study the accumulation of HOS1 protein, we collected transgenic seedlings grown for 10 d in LD for the WL samples and for 9 d in LD plus 1 d in D conditions or continuous RL for the D and RL samples, respectively. All samples were harvested at the same time in the early morning. Immunoblot analysis using an anti-GFP antibody revealed that HOS1 protein levels were higher in all the conditions tested when we added the proteasome inhibitor MG-132 during the extraction process (Figures 4A and 4B). This result indicates that HOS1 protein is regulated posttranslationally by degradation through the ubiquitin-proteasome proteolytic pathway. In addition, we observed in MG-132-treated samples that HOS1 levels were lower in RL than in WL (ZT4), probably due to the degradation of the protein once it has accomplished its function (Figures 4A and 4B). This is a common feature in the regulation of E3 ubiquitin ligases activity to avoid its overfunctioning (Jurado et al., 2008; Yan et al., 2013).
The Level and Subcellular Localization of HOS1 Are Independent of Light Conditions.
(A) Immunodetection of HOS1-GFP fusion protein in hos1-3 transgenic seedlings in different light conditions. The WL samples were grown in LDs for 10 d. The RL and D samples were grown in LDs for 9 d and then transferred to RL or D for 24 h, respectively. Protein extraction and immunoprecipitation were performed in the presence or absence of the proteasome inhibitor MG-132.
(B) Quantification of HOS1-GFP protein levels in the three different light conditions tested. Data are shown as mean ± se of two biological replicates.
(C) and (D) Confocal images of 7-d-old transgenic plants containing a HOS1-GFP construct in hos1-3 mutant background. All seedlings were grown in LDs, treated with F-RL for 30 min, and kept in D overnight. The RL image was taken from seedlings further exposed to RL for 4 h.
(C) Arabidopsis roots were stained with propidium iodide.
(D) Arabidopsis leaf petioles. The chloroplast autofluorescence and the bright field are merged with the GFP signal. Bar = 25 μm.
Previously, we have shown that the fusion protein HOS1-GFP is localized in the nuclear envelope of Arabidopsis root cells (Lazaro et al., 2012). This observation is in agreement with the finding that HOS1 interacts with the RNA EXPORT FACTOR1 (RAE1) nucleoporin (Tamura et al., 2010). To analyze if the subcellular localization of HOS1 changed under RL conditions, we grew transgenic seedlings for 7 d in LD. We then exposed the plants to F-RL for 30 min and kept them in the D overnight. Half of the plates were then exposed to RL for 4 h, and the rest were kept in D. Confocal microscope images of Arabidopsis roots and leaf petioles showed that the HOS1-GFP fusion protein did not change its subcellular localization under RL conditions and could be clearly observed in the nuclear envelope in the different light conditions used (Figures 4C and 4D). In addition, we analyzed HOS1-GFP localization in etiolated seedlings and we concluded that HOS1 also accumulates in the nuclear envelope in plants that have never been exposed to the light (Supplemental Figure 3). These results led us to conclude that RL does not directly regulate either HOS1 accumulation or nuclear localization.
phyB Acts in the Phloem to Regulate Flowering Time
Photoperiod is perceived in the leaves, which are composed of different tissues, such as the epidermis, the mesophyll, and the vascular bundles. It has been reported that the expression of a PHYB-GFP fusion protein in Arabidopsis mesophyll cells of phyB-9 mutants substantially delayed flowering (Endo et al., 2005). However, some essential components of the photoperiodic flowering pathway are known to act in the vascular tissue, including CO and HOS1 (An et al., 2004; Lazaro et al., 2012). To test whether the presence of phyB in other leaf tissues may also regulate floral transition, we analyzed flowering time in transgenic phyB-9 mutant lines expressing a PHYB-YFP construct under the control of the phloem companion cell SUCROSE H+ SYMPORTER2 (SUC2) promoter (Imlau et al., 1999), the β-CARBONIC ANHYDRASE1 (βCA1) promoter (Hu et al., 2010), which preferentially directs PHYB-YFP expression in mesophyll cells, and the constitutive 35S promoter (Casson and Hetherington, 2014). In agreement with previous reports, the presence of phyB in the mesophyll delayed phyB-9 flowering time (Figures 5A, 5B, and 5E). The constitutive expression of PHYB under the control of the 35S promoter also delayed the early flowering time phenotype of phyB-9 (Figures 5A, 5B, and 5D). Interestingly, our results reveal that the expression of PHYB in the phloem companion cells also substantially delays flowering time and causes a partial complementation of the early-flowering phenotype of the phyB-9 mutant, similar to that displayed by the βCA1:PHYB-YFP lines (Figures 5A to 5C), indicating that phyB acts in the phloem to regulate flowering time.
phyB Acts in the Phloem to Regulate Flowering Time.
(A) and (B) Flowering time phenotype of Col, phyB-9 mutant, SUC2:PHYB-YFP phyB-9, 35S:PHYB-YFP phyB-9, and βCA1:PHYB-YFP phyB-9 transgenic plants grown for 21 d in LD conditions. Data in (B) are shown as mean ± se and were derived from two experiments in which a minimum of 25 plants were scored for each genotype. Student’s t test *P < 0.01 when compared with phyB-9.
(C) to (E) Confocal images of cotyledons of SUC2:PHYB-YFP phyB-9, 35S:PHYB-YFP phyB-9, and βCA1:PHYB-YFP phyB-9 transgenic plants. Bar = 100 μm.
(F) to (H) Expression analysis in Col, the phyB-9 mutant, and the SUC2:PHYB-YFP phyB-9, 35S:PHYB-YFP phyB-9, and βCA1:PHYB-YFP phyB-9 transgenic plants. Eleven-day-old seedlings grown in LDs were harvested at ZT16, and YFP (F), PHYB (G), and FT (H) expression was detected by RT-qPCR. Relative expression levels were normalized to UBC21 expression. Each data point was derived from three biological replicates and is shown as mean ± se of three technical replicates.
As previously reported, YFP expression levels are much higher in the 35S:PHYB-YFP lines than when PHYB-YFP is expressed only in the phloem or the mesophyll (Figure 5F) (Casson and Hetherington, 2014). Similar results were obtained when PHYB expression was determined by RT-qPCR in these lines (Figure 5G). Two independent transgenic lines that were analyzed per promoter construct behaved similarly (Casson and Hetherington, 2014). Moreover, the level of FT expression in the SUC2:PHYB-YFP and the βCA1:PHYB-YFP transgenic lines was the same as that observed in Col and correlates with the flowering time observed in these lines (Figure 5H). These results show that when phyB is present in the phloem or the mesophyll, FT expression is reduced compared with the phyB-9 mutant. The expression of this floral integrator is slightly lower in the 35S:PHYB-YFP plants than in the other transgenic lines studied and Col, but considerably lower than in the phyB-9 mutant (Figure 5H).
HOS1, CO, and phyB Proteins Interact in Planta
Previous results showed that HOS1 and CO proteins physically interact in planta (Jung et al., 2012; Lazaro et al., 2012). To explore the possibility of a direct interaction between phyB and HOS1 or CO, we expressed the C-terminal part of HOS1 (cHOS1), the C-terminal part of phyB (cPHYB), and full-length CO and COP1 in yeast cells to perform two-hybrid experiments (Yu et al., 2008). Based on the evidence that the C-terminal domain of phyB is directly involved in interactions with signaling partners (Quail et al., 1995; Ni et al., 1998) and that only cHOS1 interacts with CO in yeast (Jung et al., 2012), we used these fragments instead of the full-length proteins in the yeast two-hybrid assays. As shown in Figure 6A, we observed that when we coexpressed cHOS1 together with either CO or cPHYB, yeast cells could grow in selective medium, revealing a protein interaction between them, while the coexpression of cHOS1 and COP1 did not allow yeast cells to grow, meaning that no direct interaction takes place between these two E3 ubiquitin ligases. In addition, CO and cPHYB proteins also interacted in yeast cells that could grow in medium supplemented with 10 mM 3-amino-1,2,4-triazole (3-AT) (Figure 6A).
HOS1, CO, and phyB Proteins Interact in Yeast Two-Hybrid Assays and in Planta.
(A) Yeast two-hybrid assay between CO and the C-terminal part of HOS1 (cHOS1), CO and cPHYB, cPHYB, and cHOS1, and cHOS1 and COP1. Physical interactions are detected for CO and cHOS1, as well as for CO and cPHYB, on minimal growth medium without Leu, Trp, His, and Ade (-LWHA), supplemented with 10 mM 3-AT. A physical interaction is also detected for cPHYB and cHOS1 on minimal growth medium -LWHA.
(B) and (C) Co-IP experiments in agroinfiltrated N. benthamiana plants.
(B) HOS1-GFP and HA-PHYB fusion proteins were produced alone or in combination in wild tobacco plants, and protein extracts were incubated with GFP-Trap under RL. Immunodetection was performed with anti-HA and anti-GFP antibodies.
(C) CO-TAP and HA-PHYB fusion proteins were produced alone or in combination in wild tobacco plants, and protein extracts were incubated with IgG Sepharose under RL. Immunodetection was performed with anti-HA and anti-protein A antibodies.
To further investigate interactions between these proteins and to determine if they also occur in planta, we performed in vivo coimmunoprecipitation (co-IP) assays in wild tobacco (Nicotiana benthamiana) plants infiltrated with CO-TAP or HOS1-GFP and HA-PHYB fusion proteins. We detected a physical interaction between HOS1 and phyB, as well as between CO and phyB, in protein extracts incubated under RL conditions (Figures 6B and 6C). As expected, the immunoprecipitation assays showed no interactions in plants infiltrated only with HOS1, CO, or phyB. We also performed the co-IP assays in protein extracts incubated in D and, although the interaction was slightly weaker, we could also pull down the phyB protein with HOS1-GFP, indicating that the physical interactions detected in wild tobacco are not dependent on the light treatment of the protein extracts (Supplemental Figure 4). The fact that phyB physically interacts with CO and HOS1 when they are coexpressed in wild tobacco leaves could indicate that CO, HOS1, and phyB may be present in a complex in planta.
It had been reported that a fraction of the light-activated form of phyB is degraded by the E3 ubiquitin ligase COP1 in the nucleus (Jang et al., 2010; Ni et al., 2014). To check if HOS1 could also be involved in the regulation of phyB abundance, we analyzed phyB levels in total protein extracts using an anti-phyB antibody (Somers et al., 1991). We could not detect any change in phyB levels when we compared Col, the hos1 mutant, and the HOS1-overexpressing plants, suggesting that HOS1, in contrast to COP1, is not involved in a phyB desensitization mechanism (Supplemental Figure 5).
HOS1 Regulates CO Stability under RL Conditions
CO protein stability has been shown to play a central role in the control of photoperiodic flowering, and previous results showed that CO degradation is impaired in the phyB mutant under RL (Valverde et al., 2004). Given that HOS1 interacts with phyB and CO, we used a 35S:CO-LUC construct to analyze the stability of CO protein in RL conditions. One representative line expressing the CO-LUC fusion protein was crossed into the hos1-3 mutant background. The plants overexpressing the CO-LUC fusion were early flowering, but the 35S:CO-LUC/hos1-3 plants flowered even earlier than the 35S:CO-LUC/Col plants, and earlier than the hos1-3 mutant, indicating that the CO-LUC construct was functional (Supplemental Figure 6). To carry out the analysis, we grew transgenic seedlings for 8 d in LD conditions and detected luminescence at ZT3 and ZT13 (WL ZT3 and WL ZT13). The D and RL images in Figure 7 show plants grown for 7 d in LD and then kept in D conditions or exposed to RL for 24 h. We checked that CO mRNA levels were similar in Col and hos1-3 seedlings overexpressing the CO-LUC fusion, and this allowed us to presume that the changes that might be observed in CO protein accumulation were not due to the transcriptional regulation of CO (Supplemental Figure 7).
HOS1 Regulates CO Stability under RL Conditions.
(A), (B), (D), and (E) Noninvasive in vivo luciferase imaging of 35S:CO-LUC/Col and 35S:CO-LUC/hos1-3 transgenic seedlings.
(A) and (B) WL ZT3 and WL ZT13 pictures show 8-d-old seedlings grown in LDs, 3 and 13 h after the lights were turned on, respectively.
(C) Quantification of the luciferase activity in LDs in both genetic backgrounds expressed as counts per second (cps) per plant.
(D) and (E) D and RL pictures show 7-d-old seedlings grown in LDs and then transferred to D or RL for 24 h. The color scale in the right side of each picture refers to the counts per second.
(F) Quantification of the luciferase activity in D and RL conditions in both genetic backgrounds expressed as counts per second per plant. All quantifications were done at the same time in the early morning. Each data point was derived from three biological replicates and is shown as mean ± se of three technical replicates.
As expected from our previous results, plants grown in LD conditions showed higher levels of CO protein in hos1 mutant background than in the wild type at ZT3 (Lazaro et al., 2012) (Figures 7A and 7C). Moreover, consistent with the previously described daily pattern of CO accumulation, at the end of a LD (ZT13), CO protein levels in Col were much higher than they were in the first part of the day, and only slight differences are observed between the wild type and hos1 (Figures 7B and 7C). In contrast, when the plants were kept in D for 24 h, CO expression was very low in both genetic backgrounds (Figures 7D and 7F). This result is in accordance with the previously reported role of COP1 in CO degradation that takes place during the night (Jang et al., 2008; Liu et al., 2008). Interestingly, when the plants were exposed to RL for 24 h, we could clearly detect higher CO accumulation in the hos1 mutant background than in Col (Figures 7E and 7F), indicating that HOS1, like phyB, regulates CO stability under RL.
The hos1 Mutation Does Not Further Upregulate FT Expression in phyB-Deficient Plants under RL Conditions
To determine if HOS1 and phyB could be acting together in the regulation of flowering time in RL, we analyzed FT transcript levels in Col and in mutant plants lacking HOS1, phyB, or both proteins. Seedlings were grown for 8 d in LD conditions and exposed to continuous RL for 24 h. In accordance with the role of RL in CO degradation, we did not detect significant FT expression in Col plants, and as expected, the impaired degradation of CO in the hos1 and the phyB mutant under RL led to the upregulation of FT transcript to detectable levels (Figure 8). Moreover, we found that FT expression was not further upregulated in the hos1 phyB double mutant compared with hos1 and phyb single mutants under RL conditions (Figure 8), suggesting that HOS1 and phyB regulate FT expression through the same genetic mechanism.
The hos1 Mutation Does Not Further Upregulate FT Expression in phyB-Deficient Plants under RL Conditions.
FT expression analysis in Col, the hos1 and the phyB single mutants, and the hos1 phyB double mutant. Eight-day-old seedlings grown in LDs where transferred to continuous RL for 24 h and FT expression was detected by RT-qPCR. Relative expression levels were normalized to UBC21 expression. Each data point was derived from three biological replicates and is shown as mean ± se of three technical replicates.
Altogether, our results indicate that HOS1 is involved in the regulation of CO accumulation in the early stage of the daylight period and that it also participates in the RL-mediated degradation of CO required to maintain low levels of FT expression during the early part of the day in LDs. Given that phyB interacts with HOS1 and CO, these three proteins may be part of functional complex(es) that could be indispensable to properly modulate the photoperiodic response in Arabidopsis.
DISCUSSION
HOS1 was initially described as a negative regulator of low temperature responses, but additional evidence suggested that the function of this E3 ubiquitin ligase was not restricted to cold acclimation (Ishitani et al., 1998; Lee et al., 2001; Dong et al., 2006). Previous results obtained in our group demonstrated that HOS1 is required for proper regulation of photoperiodic flowering in Arabidopsis by modulating the stability of the CO protein in the subjective morning under LDs (Lazaro et al., 2012). HOS1 also mediates CO degradation at cold temperatures in the evening under LDs (Jung et al., 2012). The cold-induced degradation of CO protein was significantly reduced in hos1 mutants, which exhibited cold-insensitive early flowering. Notably, the effects of cold stress on CO degradation were maintained in phyB mutants, showing that the HOS1-mediated degradation of CO protein at low temperatures was independent of phyB (Jung et al., 2012). Here, we show that HOS1 is involved in the RL-mediated degradation of CO, which regulates photoperiodic flowering in Arabidopsis. We demonstrate that the degradation of CO under RL is substantially reduced in the hos1 mutant at normal growth temperature (Figure 7). Moreover, FT expression is not further upregulated in the hos1 phyB double mutant compared with the single mutants under RL (Figure 8) or in the first part of the day (ZT3) under LD photoperiods (Figure 2), suggesting that HOS1 and phyB regulate FT expression through the same genetic mechanism.
Recent studies indicate that HOS1 also plays nonproteolytic roles in gene expression regulation (Jung et al., 2014; MacGregor and Penfield, 2015). We and other groups have shown that HOS1 is required for full activation of the floral repressor gene FLC and that the early flowering phenotype of hos1 mutants partially depends on FLC (Ishitani et al., 1998; Lee et al., 2001, 2012; Lazaro et al., 2012; Jung et al., 2013) (Supplemental Figure 1). FLC also plays a role in flowering time control under short-term cold stress, conditions in which flowering is delayed (Seo et al., 2009). The effects of intermittent cold treatments on FLC expression and flowering time are strongly diminished in hos1 mutants (Seo et al., 2009; Jung et al., 2013), indicating that the activation of the FLC gene by cold stress is mediated by HOS1 (Jung et al., 2014). Recently, it has been shown that HOS1 binds to FLC chromatin and that this binding is significantly induced by cold stress (Jung et al., 2013). Arabidopsis flowering under cold stress is also mediated by FVE (Kim et al., 2004). The fve mutant exhibits enhanced freezing tolerance and late flowering due to FLC upregulation (Ausín et al., 2004; Kim et al., 2004). HOS1 interacts with FVE and HISTONE DEACETYLASE6 (HDA6) and inhibits the binding of HDA6 to FLC chromatin, resulting in increased histone acetylation levels in the FLC locus and subsequent activation of FLC expression. Therefore, HOS1 activates FLC transcription through chromatin remodeling under short-term cold stress conditions (Jung et al., 2013). HOS1 also binds the microRNA gene MIR168b and promotes its transcription (Wang et al., 2015). MicroRNA 168a/b targets ARGONAUTE1 (AGO1) transcript to regulate AGO1 protein level, which is important for the function of all microRNAs in Arabidopsis. Given that HOS1 can function through both proteolytic and nonproteolytic modes of action, and based on the wide range of genes that are influenced by mutations in the HOS1 gene (MacGregor et al., 2013), it is evident that HOS1 plays versatile roles in gene expression regulation (Jung et al., 2014).
Since HOS1 mediates responses to both RL and cold, it has been proposed that this protein could function as a molecular hub that integrates light and temperature signals to modulate cellular processes that regulate plant development (Jung et al., 2014; MacGregor and Penfield, 2015). However, how these environmental cues influence HOS1 activity at the transcriptional or protein level remains unknown. In our previous work, we studied HOS1 expression pattern over a 24-h period in LDs and concluded that HOS1 transcript did not show a diurnal oscillation in the Ler background (Lazaro et al., 2012). In this study, we also addressed the effect of different light qualities in the regulation of HOS1 protein levels or subcellular localization. Our results demonstrate that there is no difference in HOS1 accumulation between LD-grown or D-treated seedlings overexpressing HOS1 and that plants exposed to RL for 24 h show a slight decrease in HOS1 protein levels compared with seedlings grown in LDs (Figures 4A and 4B). Moreover, our previous results revealed that a HOS1-GFP fusion protein localizes in the nuclear envelope of Arabidopsis root cells (Lazaro et al., 2012). This is consistent with previous observations showing that HOS1 interacts with components of the nuclear pore complex, such as RAE1 and the nucleoporin Nup43 (Tamura et al., 2010). The nuclear pore complex plays an essential role in gene expression regulation by mediating nucleocytoplasmic mRNA export. Moreover, it has been reported that polyadenylated mRNAs accumulate to a high level in the nucleus of HOS1-deficient mutants and that HOS1 is required to maintain circadian periodicity over a wide range of light and temperature environments through the promotion of mRNA export between the nucleus and the cytoplasm (MacGregor et al., 2013). In this work, we demonstrate that HOS1 nuclear localization does not change in response to RL or D treatment in different tissues (Figures 4C and 4D). However, it is still possible that HOS1 activity might be differentially regulated by light; additional experiments will have to be performed to explore this point.
RL regulates a number of developmental processes, including seed germination, hypocotyl elongation, chloroplast development, shade avoidance, and flowering (Goto et al., 1991; Chory et al., 1996; Li et al., 2011). Distinct phyB signaling pathways regulate hypocotyl elongation and floral initiation (Botto and Smith, 2002; Cerdán and Chory, 2003), although there are some components involved in phyB signaling shared by several developmental processes regulated by RL. Our results indicate that HOS1 promotes the destabilization of CO protein during the first part of the daylight period and that HOS1 participates in the RL-dependent degradation of CO, which precisely regulates photoperiodic flowering in Arabidopsis (Figures 7 and 8). Moreover, we have determined that HOS1 also plays a role in hypocotyl elongation in response to RL, corroborating previous observations (MacGregor et al., 2013), and that the effect of HOS1 on this response depends on PHYB (Figures 3B and 3C). Our data also revealed that this E3 ubiquitin ligase acts as a repressor of seed germination in D and RL conditions (Figures 3D, 3E, 3H, and 3I). Particularly under RL, the effect of PHYB is fully dependent on HOS1, suggesting that both loci could act in the same genetic pathway to regulate seed germination. PIL5/PIF1 is also involved in the light-mediated control of seed germination (Oh et al., 2004), but our results indicate that it works independently of HOS1 in this developmental transition (Figures 3F and 3G). Interestingly, HOS1 acts antagonistically to phyB in seed germination, but HOS1 and phyB function as repressors of the floral transition and hypocotyl elongation. This is also the case for PIF4, which has the opposite effect to phyB on growth responses, but acts in the same way as phyB in stomatal index responses (Casson et al., 2009; Casal, 2013).
Altogether, these results suggest that HOS1 and phyB proteins participate in common molecular regulatory mechanisms. In contrast to previous observations (Endo et al., 2005), we have shown that phyB is able to regulate flowering time in the phloem companion cells (Figure 5). We observed a complementation of the early flowering phenotype of the phyB-9 mutant in the SUC2:PHYB-YFP phyB-9 line, as well as the downregulation of FT expression to Col levels (Figure 5). Moreover, we detected physical interactions between HOS1 and phyB when we coexpressed both proteins in yeast or in plant cells (Figure 6). Also, we demonstrated that phyB binds to CO in yeast two-hybrid assays and in co-IP experiments in N. benthamiana (Figure 6). In addition, previous results obtained in our group revealed that HOS1 and CO proteins interact physically in Arabidopsis (Lazaro et al., 2012). The fact that CO, HOS1, and phyB regulate flowering time in the same leaf tissue, together with the physical interactions detected in co-IP experiments, supports that the three proteins may be present in a complex in planta (An et al., 2004; Lazaro et al., 2012). How this complex is shaped in response to environmental cues remains to be elucidated. It is well established that phyB moves to the nucleus in response to light (Kircher et al., 2002), whereas HOS1 and CO seem to be constitutively nuclear localized (Robson et al., 2001; Lazaro et al., 2012). It is tempting to speculate that HOS1 could assist in the translocation of phyB to the nucleus. Alternatively, it is also conceivable that light-activated phyB might mediate CO recognition by HOS1 in the nucleus. Thus, under RL, the three proteins are nuclear localized, enabling the formation of a complex that could modulate the photoperiodic flowering response in Arabidopsis.
As described above, the light-activated form of phyB is degraded in the nucleus after ubiquitination by the E3 ubiquitin ligase COP1 (Jang et al., 2010; Ni et al., 2014). In vitro pull-down assays showed that the N-terminal region of phyB strongly interacts with COP1, whereas weak binding was detected with the C-terminal region (Jang et al., 2010). In this work, we have shown that the C-terminal fragment of HOS1 interacts with the C-terminal domain of phyB in yeast cells (Figure 6) and that HOS1 is not able to bind COP1 (Figure 6). In addition, our results indicate that HOS1 is not involved in phyB degradation (Supplemental Figure 5), and we previously demonstrated that HOS1 and COP1 regulate flowering time additively (Lazaro et al., 2012). Altogether, these data suggest that HOS1 participates in phyB signaling, while COP1 mediates a receptor desensitization mechanism.
Light signaling pathways control CO protein activity to induce FT under favorable conditions for flowering (Song et al., 2013). We demonstrated that HOS1 regulation of FT expression depends on CO (Figure 2) and that the additive flowering phenotype observed in the hos1 phyB and hos1 co double mutants, as well as in the hos1 phyB co triple mutant, could be due to the effect of HOS1 on an additional flowering time pathway mediated by FLC (Supplemental Figure 1). The regulation of FT by HOS1 seems to be crucial in the first part of the day, when an unusual peak of FT expression can be observed in the hos1 mutant at ZT3 (Figure 2) (Lazaro et al., 2012). In contrast, in the late-flowering phl-1 mutant, FT expression is substantially reduced compared with Col at ZT15 (Endo et al., 2013). Endo and coworkers performed a co-IP assay to test if PHL could interact with both phyB and CO. The co-IP was performed with tagged versions of PHL and phyB proteins extracted from Arabidopsis transgenic lines combined with extracts containing CO-tdTomato prepared from wild tobacco leaves. The results obtained indicate that PHL can bridge phyB and CO in a RL-dependent manner (Endo et al., 2013), indicating that phyB and CO should be present in the same plant tissue, although no direct interaction was demonstrated for phyB and CO. Interestingly, we present sound evidence supporting the notion that phyB and CO, and also phyB and HOS1, physically interact in planta (Figure 6), and we reveal that phyB regulates flowering time in the phloem (Figure 5). Moreover, we observed that the hos1 mutation does not further upregulate FT expression in PHYB-deficient plants under RL conditions (Figure 8). Therefore, HOS1 and phyB might prevent precocious flowering by mediating the degradation of CO protein in the morning (Valverde et al., 2004; Lazaro et al., 2012), whereas PHL would stabilize it in the afternoon (Endo et al., 2013), shaping the accumulation of CO protein during the daylight period in LDs and contributing to the fine-tuning of the seasonal flowering response in Arabidopsis.
According to the current photoperiodic flowering model, red and blue light have antagonistic functions in the regulation of the flowering activator CO. Direct regulation of CO stability by Cry1, Cry2, and FKF1 has recently been reported (Lian et al., 2011; B. Liu et al., 2011; Zuo et al., 2011; Song et al., 2012). However, how RL regulates CO degradation remains unclear. The results presented here, together with previous observations (Lazaro et al., 2012), allow us to propose that CO, HOS1 and phyB could be at least transiently bound in a complex in planta and that HOS1 mediates the RL-mediated degradation of CO in Arabidopsis. RL regulates flowering time in a variety of LD and SD species. Indeed, pea (Pisum sativum) phyB mutants flower earlier in SDs, rice (Oryza sativa) phyB mutants flower early under both photoperiodic conditions, and sorghum (Sorghum bicolor) genotypes lacking PHYB mainly flower earlier in LD (Weller and Reid, 1993; Childs et al., 1997; Takano et al., 2005). Because HOS1, CO, and phyB homologs are found in all plant species, the interaction revealed here may be generally present in all flowering plants. The existence of this protein complex provides a framework that will help elucidate how flowering time is regulated by environmental signals, particularly by RL.
METHODS
Plant Material and Growing Conditions
The Arabidopsis thaliana mutant seed stocks used were in the Col genetic background and were obtained from the ABRC of Ohio State University (Columbus, OH) and personal donations. The monogenic mutants used in this work were described previously: hos1-3 (Lazaro et al., 2012), co-10 (Laubinger et al., 2006), phyB-9 (Reed et al., 1998), pil5-2 (Penfield et al., 2005), and voz1-1 and voz2-1 (Yasui et al., 2012).
Plants were grown in plastic pots containing a mixture of substrate and vermiculite (3:1). For in vitro culture, seedlings were grown in MS (Murashige and Skoog) medium supplemented with 0.8% (w/v) agar, with or without 1% (w/v) sucrose. Controlled environmental conditions were provided in growth chambers at 22°C and 70% relative humidity. Plants were illuminated with cool-white fluorescent lights (∼120 μmol m–2 s–1). LD conditions consisted of 16 h of light followed by 8 h of D; SD conditions consisted of 8 h of light followed by 16 h of D. Continuous RL treatments consisted of a flow rate of 32 μmol m–2 s–1 and F-RL consisted of a flow rate of 5 μmol m–2 s–1 (E-30LEDL3 monochromatic growing chamber; Percival Scientific).
Phenotypic Analysis
Flowering Time Measurement
Plants were grown on soil under LD or SD photoperiods or continuous RL. Total leaf number was scored as the number of leaves in the rosette (excluding cotyledons) plus the number of cauline leaves in the inflorescence at the time of opening of the first flower (Koornneef, 1991). A minimum of 15 plants were scored for each genotype. Data are shown as mean ± se.
Hypocotyl Measurement
Seeds were plated in MS medium supplemented with 0.8% (w/v) agar, stratified, and exposed to WL for two and a half hours before keeping them under continuous RL for 5 d. The inhibition of hypocotyl elongation was calculated as the ratio between the hypocotyl length in RL and the hypocotyl length in D. The mean and se were calculated from three independent replicates.
Germination Rate Experiments
Seeds were sterilized with ethanol, plated on MS medium supplemented with 0.8% (w/v) agar, and kept in D or continuous RL from imbibition until germination was quantified. The percentage of germination was calculated as the ratio between germinated and nongerminated seeds normalized to the viability. In all experiments, the viability (germination in WL after stratification) of each genotype was 100%. The mean and se were calculated from three independent replicates.
RNA Extraction and RT-qPCR
Plants were grown in MS medium supplemented with 1% (w/v) sucrose and 0.8% (w/v) agar and harvested at ZT4 and ZT16 after 11 d in LDs. Total RNA was isolated using the E.Z.N.A. Plant RNA Kit (OMEGA Bio-Tek) and treated with RQ1 DNase (Promega). Retrotranscription to cDNA was performed using M-MLV RT (Invitrogen). RT-qPCR analyses were performed using FastStart Universal SYBR Green Master (Roche) in a LightCycler 480 (Roche). Primers already described for analyzing the expression of FT, FLC, PHYB, YFP, and the UBIQUITIN-CONJUGATING ENZYME21 (UBC21) genes were used (Czechowski et al., 2005; Chiang et al., 2009; Morris et al., 2010; Strange et al., 2011; Crevillén et al., 2013; Casson and Hetherington, 2014). Each data point was derived from three biological replicates and is shown as mean ± se.
Confocal Microscope Images
Previously described mutant hos1-3 plants containing a 35S:HOS1-GFP construct were grown in MS medium supplemented with 1% (w/v) sucrose and 1% (w/v) plant agar in Petri dishes placed vertically (Lazaro et al., 2012). Seven-day-old transgenic plants grown in LDs were treated with F-RL for 30 min and kept in D overnight. Half of the plates were further exposed to RL for 4 h, and the other half were kept in D. Roots were stained with propidium iodide before the confocal microscopy images were taken (Leica SP8).
Protein Extraction and Immunodetection
Protein extraction of Arabidopsis seedlings and wild tobacco (Nicotiana benthamiana) leaves was performed using co-IP buffer (50 mM Na-phosphate, pH 7.4, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 2 mM Na3VO4, and 2 mM NaF) plus Complete protease inhibitor cocktail tablets (Roche) and 50 μM proteasome inhibitor MG-132 (Peptide Institute) (Song et al., 2012). Plant tissue was ground with liquid nitrogen, resuspended in co-IP buffer, and centrifuged twice to obtain the total soluble protein extract.
Immunodetection of HOS1-GFP Fusion Protein in hos1-3 Transgenic Seedlings
We grew plants in MS medium supplemented with 1% (w/v) sucrose and 0.8% (w/v) agar for 10 d in LD photoperiod for the WL sample and 9 d in LDs plus 24 h of D or continuous RL for the D and RL samples, respectively. Proteins were extracted with and without the proteasome inhibitor MG-132. Extracts were incubated with GFP-Trap_A (ChromoTek) for 2 h at 4°C, agarose beads were washed three times with co-IP buffer, and proteins bound to the anti-GFP antibody were separated by SDS-PAGE. Immunodetection was performed with a monoclonal anti-GFP antibody diluted 1/1000 (Roche catalog number 11814460001). Coomassie Brilliant Blue staining was used to quantify the amount of proteins in each sample. The mean and se were calculated from two biological replicates.
Co-IP Experiments in N. benthamiana
We used a pCTAPi-CO construct (Song et al., 2012) and the 35S:HOS1-GFP construct for the co-IP experiments in wild tobacco. The full-length cDNA of PHYB was obtained by PCR and cloned using Gateway recombinant technologies (Life Technologies) in the pEarlyGate201 vector. We used 3-week-old N. benthamiana plants to produce CO-TAP, HOS1-GFP, and HA-PHYB fusion proteins. Infiltration was performed in 10 mM MES, pH 5.6, 10 mM MgCl, and 150 μM acetosyringone, at an optical density of 0.3 for each of the Agrobacterium tumefaciens cultures transformed with the single constructs. Wild tobacco leaves were collected 3 d after agroinfiltration. Protein extracts were incubated with either GFP-Trap_A (ChromoTek) or IgG Sepharose 6 Fast Flow (GE Healthcare) for 1 h in RL or D conditions at room temperature. Beads were washed three times with co-IP buffer and resuspended in Laemmli buffer. Proteins were resolved in 8% SDS-PAGE gels. As an input, 0.2 to 2.5% of the total extract was loaded. Immunodetections were performed with monoclonal antibodies anti-GFP diluted 1/1000 (Roche catalog number 11814460001) and anti-HA clone 3F10 diluted 1/3000 (Roche catalog number 11867423001), and anti-protein A diluted 1/5000 (Sigma-Aldrich catalog number P3775), using a SuperSignal West Femto Trial Kit (Thermo Scientific).
Immunodetection of phyB in Total Protein Extracts
Immunodetection was performed with a 1:1000 dilution of the monoclonal anti-phyB B1 antibody (Somers et al., 1991).
Yeast Two-Hybrid Assays
Yeast two-hybrid interaction analyses were conducted in the pJ69-4α Saccharomyces cerevisiae strain. The C-terminal part of the protein encoded by the HOS1 cDNA (amino acids 457 to 927) was obtained by PCR and cloned using Gateway recombinant technologies (Life Technologies) to generate the GBD or GAD fusion constructs (Jung et al., 2012). The C-terminal part of phyB contains amino acids 645 to 1210 (Ni et al., 1998). cHOS1 was cloned both in the pGAD and the pGBT8 vectors, cPHYB was cloned in the pGADT7 and pGBKT7 vectors, and CO and COP1 were cloned in the pGAD and the pGBKT7 vectors, respectively. Selection was performed on synthetic complete minimal medium without Leu and Trp, or without Leu, Trp, His, and Ade, supplemented with 10 mM 3-AT if required.
Luciferase Activity Assays
The 35S:CO-LUC construct was transformed in Col plants and homozygous lines were established. Several independent transgenic plants exhibiting early flowering phenotype were selected and one representative line was crossed with hos1-3 plants. For noninvasive in vivo luciferase (LUC) imaging, seedlings were grown in MS plates for 8 d in LD conditions and sprayed with 200 μM luciferin (Biosynth) 3 and 13 h after dawn. Both the D and RL images were taken after growing seedlings for 7 d in LDs and then keeping them in D or treating them with RL for 24 h. Image acquisition and quantification of luciferase activity were performed with NightOWL II (Berthold Technologies) and IndiGO software provided by the camera manufacturer. Luciferase activity is expressed as counts per second per plant. The mean and se are calculated from three biological replicates.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: HOS1 (At2g39810), PHYB (At2g18790), CO (At5g15840), VOZ1 (At1g28520), VOZ2 (At2g42400), FT (At1g65480), FLC (At5g10140), UBC21 (At5g25760), PIL5 (At2g20180), and COP1 (At2g32950).
Supplemental Data
Supplemental Figure 1. FLC expression in the hos1, co, and phyB mutant combinations.
Supplemental Figure 2. Flowering time of hos1 voz1 voz2 triple mutant plants.
Supplemental Figure 3. Confocal images of etiolated transgenic plants expressing the 35S:HOS1-GFP construct in the hos1-3 mutant background.
Supplemental Figure 4. Co-IP assays in protein extracts incubated in D from N. benthamiana plants infiltrated with HOS1-GFP and HA-PHYB constructs.
Supplemental Figure 5. Immunodetection of phyB in protein extracts from Col, the phyB-9 and the hos1-3 mutant, and HOS1-GFP transgenic seedlings grown in LDs for 10 days.
Supplemental Figure 6. Flowering time of transgenic Col and hos1-3 plants expressing the 35S:CO-LUC construct.
Supplemental Figure 7. CO expression in Col and hos1-3 plants expressing the 35S:CO-LUC construct.
Acknowledgments
voz1 and voz2 mutants were kindly provided by T. Kohchi (Kyoto University, Kyoto, Japan), the transgenic plants expressing PHYB-YFP under the control of the SUC2, βCA1, and 35S promoters were kindly donated by Casson (University of Sheffield, UK), the C-terminal clone of phyB was kindly provided by S. Prat (CNB, CSIC, Madrid, Spain), the full-length COP1 cDNA was kindly donated by V. Rubio (CNB, CSIC, Madrid, Spain), the pCTAPi-CO construct was kindly donated by T. Imaizumi (University of Washington, Seattle, WA), the 35S:CO-LUC construct was kindly provided by H.-Q. Yang (Shanghai Institute for Biological Sciences, Chinese Academy of Science, Shanghai, China), and the anti-phyB antibody was kindly provided by Peter Quail (University of California, Berkeley, CA). We thank L. Narro (CBGP, UPM-INIA, Madrid, Spain) for establishing homozygous pil5-2 mutant lines from the SALK collection (SALK_131872) and J.C. del Pozo (CBGP, UPM-INIA, Madrid, Spain) and S. Prat (CNB, CSIC, Madrid, Spain) for helpful discussions. We also thank M. Albani (University of Cologne, Germany) for her kind help. This work was supported by Grants BIO2010-15589 and BIO2013-43098R to J.A.J. and M.P. from the Spanish Ministerio de Ciencia e Innovación and Ministerio de Economia y Competitividad, respectively.
AUTHOR CONTRIBUTIONS
A.L. performed all the experiments, except for the expression analysis in Figure 5, which was done by A.M. A.L., M.P., and J.A.J. designed the research, analyzed data, and wrote the article.
Footnotes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: José A. Jarillo (jarillo{at}inia.es).
↵1 Current address: Botanical Institute, University of Cologne, Zülpicher Str. 47B, 50674 Cologne, Germany; Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany.
Glossary
- LD
- long-day
- SD
- short-day
- F-RL
- far-red light
- RL
- red light
- Ler
- Landsberg erecta
- D
- dark
- WL
- white light
- 3-AT
- 3-amino-1,2,4-triazole
- co-IP
- coimmunoprecipitation
- MS
- Murashige and Skoog
- Received June 15, 2015.
- Revised August 12, 2015.
- Accepted August 22, 2015.
- Published September 15, 2015.
References
