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Temperature-Responsive Flowering in Arabidopsis thaliana. Jeong Hwan Lee .... For permissions, please email: [email protected]. 1802 Plant ...... ated by amplifying the coding regions of HOS1 and FVE by PCR using Pfusion ...
Plant and Cell Physiology Advance Access published September 7, 2012

Running title: HOS1 in ambient temperature-responsive flowering

Corresponding author: Ji Hoon Ahn, Ph.D.

Creative Research Initiatives Division of Life Sciences, Korea University Anam dong 5 ga, Seongbuk-Gu, Seoul 136-701 Korea

E-mail: [email protected] Phone: +82-2-3290-3451 Fax: +82-2-927-9028

Subject areas: (1) growth and development (2) environmental and stress responses

Number of black and white figures: 6 Color figures: 2

© The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected] 1

The E3 ubiquitin ligase HOS1 Regulates Low Ambient Temperature-Responsive Flowering in Arabidopsis thaliana Jeong Hwan Lee1, Jae Joon Kim1, Soo Hyun Kim1, Hyun Jung Cho1, Joonki Kim1, and Ji Hoon Ahn1,* 1

Creative Research Initiatives, Division of Life Sciences, Korea University, Seoul 136-701,

Korea

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Abstract

Ubiquitin-dependent proteolysis regulates multiple aspects of plant growth and development, but little is known about its role in ambient temperature-responsive flowering. In addition to being regulated by day length, the onset of flowering in many plants can also be delayed by low ambient temperatures. Here, we show that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1), which encodes an E3 ubiquitin ligase, controls flowering time in response to ambient temperatures (16°C and 23°C) and intermittent cold. hos1 mutants flowered early, and were insensitive to ambient temperature, but responded normally to vernalization and gibberellic acid. Genetic analyses suggested that this ambient temperatureinsensitive flowering was independent of FLOWERING LOCUS C (FLC). Also, FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) expression was upregulated in hos1 mutants at both temperatures. The ft tsf mutation almost completely suppressed the early flowering of hos1 mutants at different temperatures, suggesting that FT and TSF are downstream of HOS1 in the ambient temperature response. A lesion in CO did not affect the ambient temperature-insensitive flowering phenotype of hos1-3 mutants. In silico analysis showed that FVE was spatio-temporally co-expressed with HOS1. A HOS1–GFP fusion colocalized with FVE–GFP in the nucleus at both 16°C and 23°C. HOS1 physically interacted with FVE and FLK in yeast two-hybrid and coimmunoprecipitation assays. Moreover, hos1 mutants were insensitive to intermittent cold. Collectively, our results suggest that HOS1 acts as a common regulator in the signaling pathways that control flowering time in response to low ambient temperature.

Keywords: Ambient temperature, Cross-talk, E3 ubiquitin ligase, Flowering time, HOS1, Intermittent cold

Abbreviations: Gibberellic acid, GA; Quantitative reverse transcription-polymerase chain reaction, qPCR; Ubiquitin, Ub

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Introduction

Because environmental factors such as light and temperature change constantly, plants have evolved the ability to adapt to fluctuations in their external environment, particularly during development (Nicotra et al. 2010). One important example of this plasticity is the flowering response to ever-changing environmental cues, which is an important trait for successful reproduction. Extensive genetic and physiological studies in Arabidopsis thaliana revealed that floral inductive/inhibitory stimuli are mediated by five major pathways (Baurle and Dean 2006; Boss et al. 2004; Fornara et al. 2010; Wellmer and Riechmann 2010), which in turn are integrated by two floral activators, FLOWERING LOCUS T (FT) (Kardailsky et al. 1999; Kobayashi et al. 1999) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) (Lee et al. 2000; Samach et al. 2000). The integrated signals ultimately lead to the activation of the floral meristem identity genes APETALA1 (AP1) (Weigel and Meyerowitz 1994) and LEAFY (LFY) (Weigel et al. 1992), and the induction of flowering. In addition to being regulated by day length, flowering is significantly modulated by changes in growth temperature (Lee et al. 2008; Penfield 2008; Samach and Wigge 2005). In Arabidopsis, several genetic screens using known flowering time mutants and wild accessions have revealed that the plant’s response to low ambient temperature (>15ºC or non-stress temperature) is mediated through FCA, FVE, and SHORT VEGETATIVE PHASE (SVP) (Blázquez et al. 2003; Lee et al. 2007), whereas the plant’s response to high ambient temperature is modulated by FLOWERING LOCUS M (FLM) and PHYTOCHROME INTERACTING FACTOR4 (PIF4) (Balasubramanian et al. 2006; Kumar et al. 2012). Recent reports have also shown that some microRNAs (miRNAs) affect ambient temperatureresponsive flowering (Kim et al. 2012; Lee et al. 2010). These ambient temperature signals ultimately regulate FT expression. Distinct, but partially overlapping pathways mediate the response to more severe temperature conditions in plants. Intermittent cold (a short-term cold treatment during the day) as well as more extreme temperature conditions (i.e., cold and heat stress) affect flowering (Kim et al. 2004; Seo et al. 2009). Overexpression of CRT/DRE binding factor 1 (CBF1), CBF2, and CBF3, which are involved in response to low temperature, results in late flowering (Gilmour et al. 2004), and loss- and gain-of-function mutants of CALMODULIN-LIKE24 (CML24), a potential Ca2+ sensor whose expression increases in response to heat stress, are characterized by alterations in their flowering time (Delk et al. 2005; Tsai et al. 2007). Recent reports have suggested that various temperature signals in different temperature 4

regimes have substantial crosstalk and are integrated through a subset of genes that modulate flowering in plants. FVE, which has been classified into the autonomous and thermosensory pathways (Blázquez et al. 2003; Koornneef et al. 1991), regulates intermittent cold response and flowering time by modulating the chromatin state of COLD-REGULATED 15a (COR15a) and FLOWERING LOCUS C (FLC), respectively (Jeon and Kim 2011; Kim et al. 2004). SOC1 and FLC, the key players within the vernalization pathway, function in the intermittent cold-sensing pathway independently of FVE (Seo et al. 2009). The loss of SVP activity results in a flowering response that is insensitive to intermittent cold, similar to the response shown by flc mutants, suggesting crosstalk between the thermosensory pathway and the intermittent cold-sensing pathway. Genome-wide studies have revealed that nearly 6% of the Arabidopsis proteome is involved in the ubiquitin (Ub)-26S proteasome pathway (Smalle and Vierstra 2004; Vierstra 2009), with the Arabidopsis genome containing more than 1,400 different E3 Ub ligase genes. Ub ligases are classified into four groups according to their mechanisms of action and subunit compositions: (1) REALLY INTERESTING NEW GENE (RING), (2) Homology to E6-AP Carboxyl Terminus (HECT), (3) U-BOX, and (4) cullin-RING ligases. The RING E3 Ub ligases plays an active role in a wide array of physiological functions in plants, including those involved in the regulation of flowering time, such as the photoperiodic regulation of CONSTANS (CO) protein stability via CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) and SUPPRESSOR OF PHYTOCHROME A (SPA) protein (Henriques et al. 2009; Laubinger et al. 2006). Under long-day (LD) conditions, COP1 interacts with the CCT domain of CO during the night, leading to the degradation of CO, while COP1 and SPA proteins cooperate to negatively regulate CO stability under short-day (SD) conditions. Thus, the control of the turnover of signaling components via the Ub-26S proteasome pathway is very important for plant growth and development. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (HOS1) was originally identified as a key regulator of the transcription of genes responsive to low temperature (4ºC) (Ishitani et al. 1998; Lee et al. 2001). In hos1 mutants, low temperature enhances the induction of CBF transcription factors, leading to increased levels of their downstream genes. HOS1 encodes a RING E3 Ub ligase that physically interacts with INDUCER OF CBF EXPRESSION 1 (ICE1) to mediate the ubiquitination of ICE1, thereby inducing the degradation of ICE1 under cold stress (Dong et al. 2006). HOS1 protein has also recently been found to modulate photoperiodic flowering by negatively regulating the levels of CO protein, particularly during daytime (Lazaro et al. 2012). At normal growth 5

temperatures, hos1 mutant plants also show a weak early flowering phenotype via reduced FLC expression, suggesting that the ubiquitination of target(s) by HOS1 plays a role in the control of flowering time. However, little is known of the role of HOS1 in the regulation of low ambient temperature-responsive flowering. We report here that Arabidopsis HOS1 regulates low ambient temperature- and intermittent cold-responsive flowering. Loss-of-function mutants of HOS1 showed ambient temperature-insensitive flowering. A lesion in HOS1 resulted in the upregulation of FT and TWIN SISTER OF FT (TSF), and the downregulation of FLC at both 23°C and 16°C. Genetic analyses revealed that ambient temperature-insensitive flowering of hos1-3 mutants was independent of FLC, and that both FT and TSF are downstream genes of HOS1 in ambient temperature-responsive flowering. Also, hos1 mutation strongly suppressed the late flowering of fve-4 and flk-1 mutants at different ambient temperatures. However, the loss of CO activity did not affect the ambient temperature-insensitive flowering of hos1-3 mutants. HOS1 colocalized with FVE in the nucleus. Yeast two-hybrid and coimmunoprecipitation analyses revealed that HOS1 physically interacted with FVE as well as FLK in vitro and in vivo. Finally, hos1-3 mutants were not able to inhibit flowering in response to intermittent cold. Based on our results, we propose that HOS1 plays an important role in the modulation of flowering time in response to low ambient temperature.

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Results

hos1 mutants show ambient temperature-insensitive flowering

To find regulators of the response to ambient temperatures, we examined the flowering phenotypes of known flowering time mutants at different ambient growth temperatures, as described in our earlier study (Lee et al. 2007). We measured the time of flowering as the number of leaves present at bolting and compared this number at two ambient temperatures, 23°C and 16°C. We found that hos1-1 mutants in the C24 background (Ishitani et al. 1998) exhibited an early-flowering phenotype that was not substantially altered at different ambient temperatures under LD conditions (10.9 and 13.0 leaves at 23°C and 16°C, respectively; cf. C24 plants = 13.7 and 26.3 leaves at 23°C and 16°C, respectively) (Fig. 1A), indicating that the flowering of hos1-1 mutants was non-responsive to different ambient temperatures. As the C24 ecotype is considered to have a weak FLC allele (Gazzani et al. 2003) and it has not been widely used for genetic interaction studies, we chose to study a T-DNA allele of HOS1 in the Columbia background. We identified SALK_069312 [recently renamed hos1-3 (Lazaro et al. 2012)], which contains a single T-DNA insertion in the fifth exon of the HOS1 gene (+1912 relative to the ATG start codon) (Fig. 1B). Quantitative reverse transcription-polymerase chain reaction (qPCR) analyses showed that HOS1 mRNA was nearly absent in the homozygous hos1-3 mutants (Fig. 1C), suggesting that hos1-3 is a strong loss-of-function mutation. Flowering time measurements at different ambient temperatures revealed that hos1-3 mutants showed an early flowering phenotype at both 23°C and 16°C under LD conditions (10.4 and 12.9 leaves at 23°C and 16°C, respectively; cf. Col plants = 15.5 and 29.7 leaves at 23°C and 16°C, respectively), similar to that of hos1-1 mutants (Fig. 1A, D). These observations indicated that the ambient temperature-insensitive flowering phenotype of hos1 mutants that we observed was not allele-specific. Other than the flowering time phenotype, there were no apparent defects in the overall morphology and architecture of vegetative and reproductive organs from hos1-3 mutants when grown under LD conditions at 23°C (Supplementary Fig. S1). Since most flowering time mutants generally respond to a low ambient temperature (i.e., show delayed flowering at 16°C) (Lee et al. 2007), we chose to study the flowering phenotype of hos1 mutants in response to this low ambient temperature. We examined the spatial expression patterns of HOS1 in both the shoot apex, which is the site of floral primordium emergence, and the leaf, which is the site of the initial perception

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of photoperiodic conditions, of wild-type plants. qPCR analysis revealed that HOS1 expression was not significantly altered in the leaf and shoot apex regions at the different temperatures tested (Fig. 1E). By contrast, the expression levels of FLC and FT, two temperature-responsive genes (Blázquez et al. 2003), were significantly higher and lower, respectively, at 16°C, indicating that HOS1 mRNA levels were not altered at different ambient temperatures.

HOS1 is not regulated by the vernalization and gibberellic acid pathways HOS1 expression under various floral inductive stimuli was also investigated to determine the mechanism by which HOS1 regulates flowering time. HOS1 expression in wild-type plants remained unaltered by vernalization treatment (Fig. 2A), whereas the expression levels of FLC, which is an important integrator gene within the vernalization pathway, decreased. Similarly, gibberellic acid (GA) treatment did not change HOS1 expression levels in wild-type plants (Fig. 2A), whereas it did increase the expression level of SOC1, a GA-responsive gene during floral induction (Fig. 2A). This analysis indicates that HOS1 mRNA expression was not altered by vernalization or GA treatment. The response of hos1-3 plants to vernalization and GA treatments was similar to that of wild-type plants (Fig. 2B). Both the vernalization and GA treatments accelerated flowering in the hos1-3 mutants to a similar extent, demonstrating that hos1-3 mutants responded to the vernalization and GA treatments. These results suggest that HOS1 is unlikely to be regulated by the vernalization and GA pathways, which is consistent with a recent finding by Lazaro et al. (2012), and support the notion that HOS1 mainly functions in ambient temperatureresponsive flowering. The near-absence of any effect of ambient temperatures on the early flowering phenotype of hos1-3 plants (Fig. 1) led us to examine the expression of flowering time genes in hos1-3 plants grown at 23°C and 16°C under LD conditions. The results of the qPCR analysis indicated that the expression of both FT and TSF, which are direct targets of FLC (Helliwell et al. 2006; Michaels et al. 2004), was upregulated in hos1-3 mutants at both temperatures (Fig. 3A), while the expression of SOC1, another downstream target of FLC (Helliwell et al. 2006; Searle et al. 2006), was only marginally upregulated. The expression levels of FCA, FVE, FLK, and FY, which are important regulators of FLC in the autonomous pathway, did not change (Supplementary Fig. S2).

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Because SVP, SPL3, ELF3, TFL1, and miR172 genes were known to act in the ambient temperature-responsive flowering (Kim et al. 2012; Lee et al. 2010; Lee et al. 2007; Strasser et al. 2009), we analyzed their expression levels in hos1-3 mutants. No significant alteration in SVP, SPL3, ELF3, TFL1, and pri-miR172 expression was observed in hos1 mutants (Fig. 3B). In addition, the expression levels of SPL4, SPL5, SPL9, and FUL were not affected by hos1 mutation. Collectively, these results suggest that the early flowering phenotype of hos1-3 plants at both 23°C and 16°C is likely to be due to the combined action of FT, TSF, and FLC.

HOS1 regulates FT and TSF in temperature-responsive flowering independent of FLC To determine whether the reduced expression level of FLC is the main cause of the temperature-insensitive early flowering phenotype of hos1-3 mutants, we examined hos1-3 flc-3 double mutants. We compared the flowering time of hos1-3 flc-3 double mutants with that of hos1-3 single mutants at 23°C and 16°C under LD conditions. The hos1-3 flc-3 double mutants flowered earlier than either of the parental single mutants at 23°C (Fig. 4A), suggesting that HOS1 acts – at least partially – in parallel with FLC in regulating flowering time. Interestingly, the responsiveness of hos1-3 flc-3 double mutants to ambient temperature was similar to that of hos1-3 mutants, which is in sharp contrast to the normal responsiveness of flc-3 mutants to different ambient temperatures, indicating that the flc mutation did not affect the ambient temperature-insensitive flowering of the hos1-3 mutants. This suggests that the ambient temperature-insensitive flowering of hos1-3 plants is genetically independent of FLC. Because the expression levels of FT and TSF, the output genes within the thermosensory pathway (Lee et al. 2007), were upregulated in hos1-3 mutants (Fig. 3A), we examined the genetic relationship between HOS1, FT, and TSF. The hos1-3 ft-10 double mutants flowered later than the hos1-3 mutants at both 23°C and 16°C (Fig. 4A), which indicates that the early flowering of the hos1-3 mutants was partially suppressed by the ft mutation. Interestingly, the temperature response of the hos1-3 ft-10 double mutants was similar to that of the ft-10 mutants, which is consistent with a role of FT downstream of the thermosensory pathway (Lee et al. 2007). In contrast, the flowering times of the hos1-3 tsf-1 double mutants at the different ambient temperatures were almost identical, similar to the response of hos1-3 single mutants (Fig. 4A). This suggests that increased FT activity by hos1 mutation causes the ambient temperature-insensitivity of hos1-3 tsf-1 double mutants, because the flowering phenotype of

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tsf-1 single mutants was similar to that of wild-type plants at different temperatures. Lesions in both FT and TSF genes strongly, but not completely, delayed the flowering of hos1-3 mutants at 23°C and 16°C (45.0 and 57.5 leaves in hos1-3 ft-10 tsf-1 triple mutants, respectively) (Fig. 4A). Furthermore, the temperature response of the hos1-3 ft-10 tsf-1 triple mutants was similar to that of the ft-10 tsf-1 double mutants. The strong suppression of the flowering time phenotype of hos1-3 mutants by ft and tsf mutations at different temperatures suggests that FT and TSF are downstream of HOS1 in ambient temperature-responsive flowering. The expression of FT and TSF was investigated in hos1-3 flc-3 double mutants to determine the effect of the hos1 mutation on FT and TSF expression when FLC activity is absent. The expression levels of FT and TSF were increased in hos1-3 single mutants at both temperatures (Fig. 4B). The increase in FT and TSF expression was stronger in hos1-3 mutants than in flc-3 mutants, suggesting that HOS1 exerts a stronger effect on FT and TSF expression than on FLC expression. In particular, an additive increase in the expression levels of FT and TSF was obviously seen in hos1-3 flc-3 double mutants at both 23°C and 16°C (Fig. 4B). Taken together with the genetic analysis, this result supports the notion that HOS1 regulates FT and TSF expression, at least in part, in an FLC-independent manner for the regulation of ambient temperature-responsive flowering. Because some autonomous pathway mutants showed ambient temperature-insensitive flowering (Blázquez et al. 2003; Lee et al. 2007), we also tested the genetic interactions among HOS1, FVE, and FLK by crossing hos1-3 mutants with fve-4 or flk-1 mutants. The hos1-3 fve-4 double mutants flowered with 15.4 and 21.5 leaves at 23°C and 16°C, respectively (Fig. 4C). The number of leaves produced in hos1-3 flk-1 double mutants (14.2 and 20.1 leaves at 23°C and 16°C, respectively) was lower than that produced in flk-1 mutants. However, temperature responses of two double mutants were similar to those of their parental lines. This result indicated that hos1-3 mutation strongly, but not completely, suppressed the late flowering of fve-4 and flk-1 mutants at both temperatures and further suggested that HOS1 acts in a genetic pathway with FVE and FLK to control ambient temperature-responsive flowering.

A lesion in CO does not alter the ambient temperature-insensitive flowering phenotype of hos1-3 mutants

It was recently shown that HOS1 regulates CO activity in the photoperiodic control of

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flowering (Lazaro et al. 2012). CO is also known to be a direct activator of FT and TSF (Jang et al. 2009; Samach et al. 2000; Yamaguchi et al. 2005). Thus, to test the effect of CO on ambient temperature-responsive flowering, we investigated the flowering phenotype of hos1-3 co-10 double mutants at 23°C and 16°C under LD conditions. hos1-3 co-10 double mutants flowered with 24.3 and 33.6 leaves at 23°C and 16°C, respectively (Fig. 5A), indicating that flowering of hos1-3 co-10 double mutants was apparently accelerated at 16°C. Thus, flowering of hos1-3 co-10 double mutants was almost ambient temperature-insensitive, as similarly seen in hos1-3 mutants. In contrast, co-10 mutants showed ambient temperatureresponsive flowering (30.1 and 55.2 leaves at 23°C and 16°C, respectively). This indicated that the loss of CO activity did not alter the ambient temperature-insensitive flowering phenotype of hos1-3 mutants. Expression analysis to test the effect of the co mutation on FT and TSF upregulation in hos1-3 co-10 double mutants revealed that hos1 mutation caused an increase in FT and TSF expression and a decrease in FLC expression in the co mutant background at both temperatures (Fig. 5B). Collectively, these results suggested that a lesion in CO does not alter the effect of HOS1 on ambient temperature-responsive flowering.

HOS1 protein interacts with FVE and FLK proteins

Protein–protein interactions between E3 Ub ligases and their respective proteins targeted for degradation require co-expression in both time and space for the formation of a protein complex (De Bodt et al. 2009). We searched a public microarray database (AtGenExpress ME00319) for genes that showed spatial and temporal co-expression patterns (similarity index >80%) with HOS1 (Schmid et al. 2005) and identified 79 genes (Fig. 6A; Supplementary Table S1). Subsequent comparison of their expression patterns with known flowering time genes led to the identification of FVE (Fig. 6B). To confirm co-localization of HOS1 and FVE, the subcellular localization patterns of HOS1–GFP and FVE–GFP were examined in onion epidermal cells incubated at 23°C and 16°C. HOS1-GFP was localized in both the cytoplasm and the nucleus at 23°C and 16°C (Fig. 6C). This subcellular localization of HOS1 in the nucleus at 23°C provides an explanation for the reduced level of ICE1 protein observed at warm temperatures by Dong et al. (2006). At 4°C, HOS1-GFP was predominantly localized in the nucleus (Supplementary Fig. S3), consistent with an earlier observation (Lee et al. 2001). By contrast, FVE protein was located in the nucleus at all of the temperatures examined (Fig. 6C; Supplementary Fig. S3). It can

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therefore be concluded that the subcellular localization patterns of HOS1 and FVE proteins overlap at both 23°C and 16°C. The co-localization of HOS1–GFP with FVE–GFP suggested the possibility that HOS1 interacts with FVE. A yeast two-hybrid assay was therefore performed to test whether HOS1 protein does interact with FVE. Positive β-galactosidase (β-gal) activity was observed when HOS1 protein as bait was combined with FVE protein as prey (Fig. 6D). β-gal activity was also observed when ICE1, which is known to interact with HOS1 protein (Dong et al. 2006), was used as a positive control. In contrast, β-gal activity was not observed when the negative control bait (pGADT7) was used. The results of this assay demonstrate that HOS1 can interact with FVE in this yeast two-hybrid assay. Since FVE functions within the autonomous pathway and its loss-of-function allele showed a similar flowering phenotype of insensitivity to ambient temperature changes, we extended the yeast two-hybrid assay using proteins that act in the autonomous pathway. Among the autonomous pathway proteins, we found that FLK protein also interacted with HOS1 (Fig. 6D), whereas FLD protein did not. To confirm the yeast two-hybrid assay and test whether HOS1 and FVE/FLK proteins directly interact in vivo, a reciprocal coimmunoprecipitation analysis was performed using HOS1-HA and FVE-GFP or FLK-GFP proteins. 35S::HOS1-HA was transiently expressed with 35S::FVE-GFP or 35S::FLK-GFP constructs in Arabidopsis mesophyll protoplasts. Protein extract was first immunoprecipitated with anti-GFP antibody and then the precipitated proteins were analyzed by western blot using the anti-HA antibody, and vice versa. A band with the expected size of the HOS1-HA protein was detected from the anti-GFP immunoprecipitates (Fig. 6E, left). The in vivo interaction between HOS1 and FVE or FLK was also revealed in the anti-HA immunoprecipitates (Fig. 6E, right). However, no band was observed from the anti-GFP or anti-HA immunoprecipitates when only one construct was expressed. Taken together with the genetic analysis (Fig. 4C), these results suggest that HOS1 protein physically interacts with FVE and FLK to control ambient temperature-responsive flowering.

Effects of HOS1 mutations on the flowering response to intermittent cold treatment The results of our experiments suggest that HOS1 is involved in ambient temperatureresponsive flowering (16°C), which is in accordance with previous reports that HOS1 functions in the cold response (2.0 and A260/A280 >1.8) were used for subsequent qPCR experiments. To remove possible genomic DNA contamination, we treated RNA samples with DNaseI (New England Biolabs, MA) for 60 min at 37°C. RNA samples (1–2 µg) were used for cDNA synthesis in accordance with the manufacturer’s instructions (Roche Applied Science, NJ). The qPCR primers were designed using SciTools at Integrated DNA Technologies (http://www.idtdna.com) with the criterion of a melting temperature (TM) of 62±0.5°C. Specific amplification of PCR products was visualized in a 12% polyacrylamide gel. The qPCR analysis was carried out in 384-well plates in a LightCycler 480 system (Roche Applied Science, NJ) using Roche SYBR Green Master mixture (Roche Applied Science, NJ). The cycling program consisted of pre-denaturation for 3 min at 94°C, followed by 45 cycles of denaturation for 10 s at 94°C, annealing for 10 s at 60-62°C, and elongation for 10 s at 72°C. Melting curve analysis was performed from 65°C to 97°C to assess the specificity of the qPCR products. For the qPCR analysis, the ‘Eleven Golden Rules for Quantitative RT-PCR’ were followed (Udvardi et al. 2008) to ensure a reproducible and accurate measurement of transcript levels. Samples for qPCR were harvested at Zeitgeber time (ZT) 16, at which point FT transcript levels are high (Corbesier et al. 2007). Two reference genes (AT1G13320/AT2G28390) that are stably expressed at 23°C and 16°C (Hong et al. 2010) were used for quantification. All qPCR experiments were carried out in two or three biological replicates (independently harvested samples on different days) with three technical triplicates, each with similar results. The results from biological triplicates are shown. Oligonucleotide primers used for qPCR are listed (Supplementary Table S2).

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Determination of relative abundance of transcripts

The procedure used for determining the relative abundance of transcripts has been described in detail elsewhere (Hong et al. 2010). Threshold cycle (Ct) and PCR efficiency of the primers used were calculated using LinRegPCR (Ramakers et al. 2003). Relative abundance of the transcripts was calculated by the statistical formula from geNorm (Vandesompele et al. 2002). The coefficient of variation (Cv) was calculated from three technical replicates according to the following formula: Cv = 100 × (standard deviation of Ct/average of Ct). The Ct and Cv values of each sample were then examined. If the Cv value of a sample was >2.0%, which indicated a reaction that deviated very significantly from the mean of three technical replicates, it was considered to be an outlier and thus excluded from further analysis. A >1.5fold upregulation or downregulation was considered to be significant.

Subcellular localization analysis

Constructs for the subcellular localization studies were generated by amplifying the coding regions of HOS1 and FVE by PCR using Pfusion DNA polymerase (New England Biolabs, MA), cloning the amplified coding regions into a Gateway entry vector, and subsequently recombining the resulting vector into destination vectors (pGWB5 or pGWB6) harboring the 35S promoter and GFP using Gateway LR clonase II Enzyme mix (Invitrogen, CA). A particle bombardment system (PDS-1000/He; Bio-Rad, CA) was used for the delivery of DNA-coated gold particles into onion epidermal cells, as previously reported (Lee et al. 2007). After 17–18 h of incubation at 23°C, 16°C, or 4°C, the subcellular localization pattern was observed by fluorescence microscopy (Carl Zeiss, Germany). The oligonucleotide primers used for cloning are listed in Supplementary Table S2.

Yeast two-hybrid assay For the yeast two-hybrid analysis, open reading frames (ORFs) of FVE, FLK, FLD, and ICE1 were amplified via PCR and cloned into pGADT7 or pGBKT7 (Clontech, CA). In the case of HOS1 protein, the C-terminal portion (from 450 to 842 amino acid residues) of HOS1 protein was used, as previously reported (Dong et al. 2006). The yeast two-hybrid assay was performed as described by Lee et al. (2012).

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Coimmunoprecipitation assay For the coimmunoprecipitation experiment, the full-length ORF of HOS1 was cloned into a vector harboring the 35S promoter and HA. Full-length ORFs of FVE and FLK were cloned into a vector harboring the 35S promoter and GFP (provided by Dr. I. Hwang, POSTECH, Korea). Recombination reactions were used for cloning in accordance with the manufacturer’s instructions (Clontech, CA). The oligonucleotide primers used for cloning are listed (Supplementary Table S2). A coimmunoprecipitation analysis was performed using wild-type (Col) Arabidopsis mesophyll protoplasts, as described previously (Yoo et al. 2007a). HOS1GFP protein was transiently co-expressed with FVE-HA or FLK-HA proteins in protoplasts by

polyethylene

glycol-mediated

transformation.

After

17–18

h

incubation,

immunoprecipitation was carried out using anti-GFP or anti-HA monoclonal antibodies [sc9996 (Santa Cruz Biotechnology) and 2-2.2.14 (Pierce), respectively]. Immunoprecipitated proteins were released in 2X SDS sample buffer, separated by SDS-PAGE, and detected by western blot using anti-HA or anti-GFP monoclonal antibodies.

Accession numbers Arabidopsis Genome Initiative gene identifiers were as follows: CBF3 (AT4G25480); COR15a (AT2G42540); FD (AT4G35900); FLC (AT5G10140); FLD (AT3G10390); FLK (AT3G04610); FT (AT1G65480); FVE (AT2G19520); HOS1 (AT2G39810); ICE1 (AT3G26744); PP2AA3 (AT1G13320); SAND family protein (AT2G28390); SOC1 (AT2G45660); SVP (AT2G22540); TSF (AT4G20370).

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Supplementary data are available at PCP online.

Funding

This work was supported by the Creative Research Initiatives of the National Research Foundation for the Ministry of Education, Science and Technology (R16-2008-106-01000-0 to J.H. Ahn) and by the BK 21 program (to J.H. Lee, J.J. Kim, S.H. Kim, and H.J. Cho).

Acknowledgments

We thank Dr. S.Y. Yoo, Dr. S.C. Bahn, and K.E. Kim for their technical assistance.

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References

Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653-657. Balasubramanian, S., Sureshkumar, S., Lempe, J. and Weigel, D. (2006) Potent induction of Arabidopsis thaliana flowering by elevated growth temperature. PLoS Genet 2: e106. Baurle, I. and Dean, C. (2006) The timing of developmental transitions in plants. Cell 125: 655-664. Blázquez, M.A., Ahn, J.H. and Weigel, D. (2003) A thermosensory pathway controlling flowering time in Arabidopsis thaliana. Nat Genet 33: 168-171. Boss, P.K., Bastow, R.M., Mylne, J.S. and Dean, C. (2004) Multiple pathways in the decision to flower: enabling, promoting, and resetting. Plant Cell 16 Suppl: S18-31. Corbesier, L., Vincent, C., Jang, S., Fornara, F., Fan, Q., Searle, I., Giakountis, A., Farrona, S., Gissot, L., Turnbull, C. and Coupland, G. (2007) FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316: 1030-1033. De Bodt, S., Proost, S., Vandepoele, K., Rouze, P. and Van de Peer, Y. (2009) Predicting protein-protein interactions in Arabidopsis thaliana through integration of orthology, gene ontology and co-expression. BMC Genomics 10: 288. Delk, N.A., Johnson, K.A., Chowdhury, N.I. and Braam, J. (2005) CML24, regulated in expression by diverse stimuli, encodes a potential Ca2+ sensor that functions in responses to abscisic acid, daylength, and ion stress. Plant Physiol 139: 240-253. Dong, C.H., Agarwal, M., Zhang, Y., Xie, Q. and Zhu, J.K. (2006) The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci U S A 103: 8281-8286. Fornara, F., de Montaigu, A. and Coupland, G. (2010) SnapShot: Control of flowering in Arabidopsis. Cell 141: 550, 550 e551-552. Gazzani, S., Gendall, A.R., Lister, C. and Dean, C. (2003) Analysis of the molecular basis of flowering time variation in Arabidopsis accessions. Plant Physiol 132: 1107-1114. Gilmour, S.J., Fowler, S.G. and Thomashow, M.F. (2004) Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Mol Biol 54: 767-781. Helliwell, C.A., Wood, C.C., Robertson, M., James Peacock, W. and Dennis, E.S. (2006) The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. Plant J 46: 183-192. 22

Henriques, R., Jang, I.C. and Chua, N.H. (2009) Regulated proteolysis in light-related signaling pathways. Curr Opin Plant Biol 12: 49-56. Hong, S.M., Bahn, S.C., Lyu, A., Jung, H.S. and Ahn, J.H. (2010) Identification and testing of superior reference genes for a starting pool of transcript normalization in Arabidopsis. Plant Cell Physiol 51: 1694-1606. Ishitani, M., Xiong, L., Lee, H., Stevenson, B. and Zhu, J.K. (1998) HOS1, a genetic locus involved in cold-responsive gene expression in arabidopsis. Plant Cell 10: 1151-1161. Jang, S., Torti, S. and Coupland, G. (2009) Genetic and spatial interactions between FT, TSF and SVP during the early stages of floral induction in Arabidopsis. Plant J 60: 614625. Jeon, J. and Kim, J. (2011) FVE, an Arabidopsis homologue of the retinoblastoma-associated protein that regulates flowering time and cold response, binds to chromatin as a large multiprotein complex. Mol Cells. Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen, S.K., Nguyen, J.T., Chory, J., Harrison, M.J. and Weigel, D. (1999) Activation tagging of the floral inducer FT. Science 286: 1962-1965. Kim, D.H., Doyle, M.R., Sung, S. and Amasino, R.M. (2009) Vernalization: winter and the timing of flowering in plants. Annu Rev Cell Dev Biol 25: 277-299. Kim, H.J., Hyun, Y., Park, J.Y., Park, M.J., Park, M.K., Kim, M.D., Lee, M.H., Moon, J., Lee, I. and Kim, J. (2004) A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana. Nat Genet 36: 167-171. Kim, J.J., Lee, J.H., Kim, W., Jung, H.S., Huijser, P. and Ahn, J.H. (2012) The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis. Plant Physiol 159: 461-478. Knight, H., Thomson, A.J. and McWatters, H.G. (2008) Sensitive to freezing6 integrates cellular and environmental inputs to the plant circadian clock. Plant Physiol 148: 293303. Kobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M. and Araki, T. (1999) A pair of related genes with antagonistic roles in mediating flowering signals. Science 286: 1960-1962. Koornneef, M., Hanhart, C.J. and van der Veen, J.H. (1991) A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol Gen Genet 229: 57-66. Kraft, E., Stone, S.L., Ma, L., Su, N., Gao, Y., Lau, O.S., Deng, X.W. and Callis, J. (2005) Genome analysis and functional characterization of the E2 and RING-type E3 ligase 23

ubiquitination enzymes of Arabidopsis. Plant Physiol 139: 1597-1611. Kumar, S.V., Lucyshyn, D., Jaeger, K.E., Alos, E., Alvey, E., Harberd, N.P. and Wigge, P.A. (2012) Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 484: 242-245. Laubinger, S., Marchal, V., Le Gourrierec, J., Wenkel, S., Adrian, J., Jang, S., Kulajta, C., Braun, H., Coupland, G. and Hoecker, U. (2006) Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability. Development 133: 3213-3222. Lazaro, A., Valverde, F., Pineiro, M. and Jarillo, J.A. (2012) The Arabidopsis E3 Ubiquitin Ligase HOS1 Negatively Regulates CONSTANS Abundance in the Photoperiodic Control of Flowering. Plant Cell. Lee, H., Suh, S.S., Park, E., Cho, E., Ahn, J.H., Kim, S.G., Lee, J.S., Kwon, Y.M. and Lee, I. (2000) The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes & Dev 14: 2366-2376. Lee, H., Xiong, L., Gong, Z., Ishitani, M., Stevenson, B. and Zhu, J.K. (2001) The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo--cytoplasmic partitioning. Genes Dev 15: 912-924. Lee, H., Yoo, S.J., Lee, J.H., Kim, W., Yoo, S.K., Fitzgerald, H., Carrington, J.C. and Ahn, J.H. (2010) Genetic framework for flowering-time regulation by ambient temperatureresponsive miRNAs in Arabidopsis. Nucleic Acids Res 38: 3081-3093. Lee, J.H., Lee, J.S. and Ahn, J.H. (2008) Ambient temperature signaling in plants: an emerging field in the regulation of flowering time. Journal of Plant Biology 51: 321326. Lee, J.H., Park, S.H. and Ahn, J.H. (2012) Functional conservation and diversification between rice OsMADS22/OsMADS55 and Arabidopsis SVP proteins. Plant Sci 185186: 97-104. Lee, J.H., Yoo, S.J., Park, S.H., Hwang, I., Lee, J.S. and Ahn, J.H. (2007) Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. Genes & Dev 21: 397-402. Lim, M.H., Kim, J., Kim, Y.S., Chung, K.S., Seo, Y.H., Lee, I., Hong, C.B., Kim, H.J. and Park, C.M. (2004) A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C. Plant Cell 16: 731-740. 24

Michaels, S.D. and Amasino, R.M. (1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11: 949-956. Michaels, S.D., Himelblau, E., Kim, S.Y., Schomburg, F.M. and Amasino, R.M. (2004) Integration of Flowering Signals in Winter-Annual Arabidopsis. Plant Physiol. Nicotra, A.B., Atkin, O.K., Bonser, S.P., Davidson, A.M., Finnegan, E.J., Mathesius, U., Poot, P., Purugganan, M.D., Richards, C.L., Valladares, F. and van Kleunen, M. (2010) Plant phenotypic plasticity in a changing climate. Trends Plant Sci 15: 684-692. Penfield, S. (2008) Temperature perception and signal transduction in plants. The New phytologist 179: 615-628. Ramakers, C., Ruijter, J.M., Deprez, R.H. and Moorman, A.F. (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339: 62-66. Samach, A., Onouchi, H., Gold, S.E., Ditta, G.S., Schwarz-Sommer, Z., Yanofsky, M.F. and Coupland, G. (2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288: 1613-1616. Samach, A. and Wigge, P.A. (2005) Ambient temperature perception in plants. Curr Opin Plant Biol 8: 483-486. Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel, D. and Lohmann, J.U. (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet 37: 501-506. Searle, I., He, Y., Turck, F., Vincent, C., Fornara, F., Krober, S., Amasino, R.A. and Coupland, G. (2006) The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes & Dev 20: 898-912. Seo, E., Lee, H., Jeon, J., Park, H., Kim, J., Noh, Y.S. and Lee, I. (2009) Crosstalk between cold response and flowering in Arabidopsis is mediated through the flowering-time gene SOC1 and its upstream negative regulator FLC. Plant Cell 21: 3185-3197. Smalle, J. and Vierstra, R.D. (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 55: 555-590. Stone, S.L., Hauksdottir, H., Troy, A., Herschleb, J., Kraft, E. and Callis, J. (2005) Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol 137: 13-30. Strasser, B., Alvarez, M.J., Califano, A. and Cerdan, P.D. (2009) A complementary role for ELF3 and TFL1 in the regulation of flowering time by ambient temperature. Plant J 25

58: 629-640. Tsai, Y.C., Delk, N.A., Chowdhury, N.I. and Braam, J. (2007) Arabidopsis potential calcium sensors regulate nitric oxide levels and the transition to flowering. Plant Signal Behav 2: 446-454. Udvardi, M.K., Czechowski, T. and Scheible, W.R. (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20: 1736-1737. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034. Vierstra, R.D. (2009) The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol 10: 385-397. Weigel, D., Alvarez, J., Smyth, D.R., Yanofsky, M.F. and Meyerowitz, E.M. (1992) LEAFY controls floral meristem identity in Arabidopsis. Cell 69: 843-859. Weigel, D. and Glazebrook, J. (2002) Arabidopsis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Weigel, D. and Meyerowitz, E.M. (1994) The ABCs of floral homeotic genes. Cell 78: 203209. Wellmer, F. and Riechmann, J.L. (2010) Gene networks controlling the initiation of flower development. Trends Genet 26: 519-527. Yamaguchi, A., Kobayashi, Y., Goto, K., Abe, M. and Araki, T. (2005) TWIN SISTER OF FT (TSF) acts as a floral pathway integrator redundantly with FT. Plant Cell Physiol 46: 1175-1189. Yoo, S.D., Cho, Y.H. and Sheen, J. (2007a) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2: 1565-1572. Yoo, S.K., Chung, K.S., Kim, J., Lee, J.H., Hong, S.M., Yoo, S.J., Yoo, S.Y., Lee, J.S. and Ahn, J.H. (2005) CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis. Plant Physiol. 139: 770-778. Yoo, S.Y., Kim, Y., Kim, S.Y., Lee, J.S. and Ahn, J.H. (2007b) Control of flowering time and cold response by a NAC-domain protein in Arabidopsis. PLoS ONE 2: e642.

Figure 1. Ambient temperature-insensitive flowering of hos1 mutants under long-day (LD) conditions. (A) Total leaf numbers of hos1-1 (C24 background) and hos1-3 (Col 26

background) mutants at 23°C and 16°C. (B) Gene structure and position of mutations (indicated by inverted triangles) of HOS1 alleles used in this study. Closed boxes and lines indicate exons and introns, respectively. Three amplicons used for the detection of HOS1 mRNA are shown as lines. (C) The near-absence of HOS1 mRNA expression revealed by testing three amplicons (Fig. 1B) in hos1-3 (SALK_069312) mutants under LD conditions. (D) Phenotype of hos1-3 mutants grown at 23°C and 16°C. Photographs were taken when each plant flowered at each temperature. (E) qPCR analysis of HOS1, FLC, and FT expression in the leaf (L) and the shoot apical regions (SA) of 8-day-old wild-type seedlings grown at 23°C and 16°C. The expression of each gene in the leaf of wild-type seedlings grown at 23°C was set to one. Error bars indicate standard deviation.

Figure 2. Expression of HOS1 and the flowering time of hos1-3 mutants in response to vernalization and GA treatment. (A) Effects of vernalization (VRN) and gibberellic acid (GA) treatments on HOS1 mRNA expression in wild-type plants determined by qPCR. FLC and SOC1 expression was used as the positive control for the vernalization and GA treatments, respectively. (B) Effects of vernalization and GA treatments on flowering time of hos1-3 mutants grown at 23°C under SD conditions. HOS1 expression levels in wild-type plants were set to one. Error bars indicate standard deviation.

Figure 3. The expression levels of flowering time genes in hos1-3 mutants grown at 23°C and 16°C under LD conditions. (A) qPCR analysis of FT, TSF, FLC, and SOC1 expression in 8-day-old seedlings of wild-type (Col) and hos1-3 plants. (B) qPCR analysis of SVP, SPLs, ELF3, TFL1, FUL, and pri-miR172a expression in 8-day-old seedlings of wild-type (Col) and hos1-3 plants. The expression level of each gene in wild-type seedlings at 23°C was set to one. Error bars indicate standard deviation.

Figure 4. The genetic interactions among HOS1, FLC, FT, TSF, FLK, and FVE under LD conditions. (A) Total leaf number of double and triple mutants carrying hos1-3 mutation grown at 23°C and 16°C. (B) qPCR analysis of FT and TSF expression in 8-day-old seedlings of wild-type (Col), hos1-3, flc-3, and hos1-3 flc-3 plants grown at 23°C and 16°C. The expression level of each gene in wild-type seedlings at 23°C was set to one. (C) Flowering time of hos1-3 fve-4 and hos1-3 flk-1 double mutants grown at 23°C and 16°C. Error bars indicate standard deviation.

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Figure 5. Independent role of CO in the ambient temperature-insensitive flowering of hos1-3 mutants under LD conditions. (A) Total leaf number of hos1-3 co-10 mutants grown at 23°C and 16°C. (B) qPCR analysis of FT, TSF, and FLC expression in 8-day-old seedlings of wild-type (Col), hos1-3, co-10, and hos1-3 co-10 plants grown at 23°C and 16°C. The expression level of each gene in wild-type seedlings at 23°C was set to one. Error bars indicate standard deviation.

Figure 6. Protein–protein interaction between HOS1 and FVE/FLK proteins in vitro and in vivo. (A) Expression levels of genes that showed similar expression patterns with HOS1 in microarray data (AtGenExpress ME00319). Solid black line indicates the expression of HOS1. (B) Venn diagram identifying FVE, which shows a similar spatio-temporal expression pattern as HOS1. (C) Subcellular localization of HOS1 and FVE proteins at different ambient temperatures. Onion epidermal cells were bombarded with 35S::HOS1-GFP, 35S::FVE-GFP, and 35S::GFP, then incubated overnight at 23°C and 16°C. 4', 6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. Bars, 200 µm. (D) Yeast two-hybrid analysis using HOS1 proteins. Note that β–gal activity was observed with FVE and FLK proteins as well as with ICE1 as a positive control. Transformed yeast cells were assayed on selective SD/-Trp/Leu medium (bottom panel) and SD/-Trp/-Leu/-His/X-α-gal medium (top panel). (E) The in vivo interaction between HOS1 and FVE or FLK proteins. Arabidopsis mesophyll protoplasts were used for coimmunoprecipitation analysis. Arrows denote the expected size of HOS1-HA (lane 1, 2), FLK-GFP (lane 6), and FVE-GFP (lane 7) proteins. Asterisks indicate a nonspecific band.

Figure 7. Effect of intermittent cold treatment on HOS1. (A) Total leaf numbers of wildtype (Col), hos1-3, flk-1, fve-4, and fld-1 mutants treated with intermittent cold. (B) qPCR analysis of HOS1, FVE, FLK, FLC, SOC1, CBF3, and COR15a expression in wild-type plants with or without intermittent cold treatment. Error bars indicate standard deviation.

Figure 8. A proposed model for the role of HOS1 as an integrator in the low temperature response. Low ambient temperature and intermittent cold cause alterations in the activity of HOS1 protein, which in turn may negatively regulate FVE and FLK at the post-transcriptional level by ubiquitination. This low temperature signaling relay regulates the expression of

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FT/TSF and FLC. Thus, it is likely that this intricate crosstalk constitutes a fine-tuning protective mechanism against premature flowering in inappropriate seasons, such as early spring or late fall. Intermittent cold and cold stresses mediated by HOS1 induce cold resistance and cold acclimation, respectively. Black and gray colors represent genetic pathways found in this study and those reported in previous studies, respectively. Arrows represent promotion effects, whereas T-bars indicate repression effects.

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Figure 1. Ambient temperature-insensitive flowering of hos1 mutants under long-day (LD) conditions. (A) Total leaf numbers of hos1-1 (C24 background) and hos1-3 (Col background) mutants at 23°C and 16°C. (B) Gene structure and position of mutations (indicated by inverted triangles) of HOS1 alleles used in this study. Closed boxes and lines indicate exons and introns, respectively. Three amplicons used for the detection of HOS1 mRNA are shown as lines. (C) The near-absence of HOS1 mRNA expression revealed by testing three amplicons (Fig. 1B) in hos1-3 (SALK_069312) mutants under LD conditions. (D) Phenotype of hos1-3 mutants grown at 23°C and 16°C. Photographs were taken when each plant flowered at each temperature. (E) qPCR analysis of HOS1, FLC, and FT expression in the leaf (L) and the shoot apical regions (SA) of 8day-old wild-type seedlings grown at 23°C and 16°C. The expression of each gene in the leaf of wild-type seedlings grown at 23°C was set to one. Error bars indicate standard deviation. 136x127mm (300 x 300 DPI)

Figure 2. Expression of HOS1 and the flowering time of hos1-3 mutants in response to vernalization and GA treatment. (A) Effects of vernalization (VRN) and gibberellic acid (GA) treatments on HOS1 mRNA expression in wild-type plants determined by qPCR. FLC and SOC1 expression was used as the positive control for the vernalization and GA treatments, respectively. (B) Effects of vernalization and GA treatments on flowering time of hos1-3 mutants grown at 23°C under SD conditions. HOS1 expression levels in wildtype plants were set to one. Error bars indicate standard deviation. 93x55mm (300 x 300 DPI)

Figure 3. The expression levels of flowering time genes in hos1-3 mutants grown at 23°C and 16°C under LD conditions. (A) qPCR analysis of FT, TSF, FLC, and SOC1 expression in 8-day-old seedlings of wild-type (Col) and hos1-3 plants. (B) qPCR analysis of SVP, SPLs, ELF3, TFL1, FUL, and pri-miR172a expression in 8-dayold seedlings of wild-type (Col) and hos1-3 plants. The expression level of each gene in wild-type seedlings at 23°C was set to one. Error bars indicate standard deviation. 138x91mm (300 x 300 DPI)

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Re Figure 4. The genetic interactions among HOS1, FLC, FT, TSF, FLK, and FVE under LD conditions. (A) Total leaf number of double and triple mutants carrying hos1-3 mutation grown at 23°C and 16°C. (B) qPCR analysis of FT and TSF expression in 8-day-old seedlings of wild-type (Col), hos1-3, flc-3, and hos1-3 flc-3 plants grown at 23°C and 16°C. The expression level of each gene in wild-type seedlings at 23°C was set to one. (C) Flowering time of hos1-3 fve-4 and hos1-3 flk-1 double mutants grown at 23°C and 16°C. Error bars indicate standard deviation. 75x174mm (300 x 300 DPI)

Figure 5. Independent role of CO in the ambient temperature-insensitive flowering of hos1-3 mutants under LD conditions. (A) Total leaf number of hos1-3 co-10 mutants grown at 23°C and 16°C. (B) qPCR analysis of FT, TSF, and FLC expression in 8-day-old seedlings of wild-type (Col), hos1-3, co-10, and hos1-3 co-10 plants grown at 23°C and 16°C. The expression level of each gene in wild-type seedlings at 23°C was set to one. Error bars indicate standard deviation. 153x56mm (300 x 300 DPI)

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Figure 6. Protein–protein interaction between HOS1 and FVE/FLK proteins in vitro and in vivo. (A) Expression levels of genes that showed similar expression patterns with HOS1 in microarray data (AtGenExpress ME00319). Solid black line indicates the expression of HOS1. (B) Venn diagram identifying FVE, which shows a similar spatio-temporal expression pattern as HOS1. (C) Subcellular localization of HOS1 and FVE proteins at different ambient temperatures. Onion epidermal cells were bombarded with 35S::HOS1-GFP, 35S::FVEGFP, and 35S::GFP, then incubated overnight at 23°C and 16°C. 4', 6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. Bars, 200 µm. (D) Yeast two-hybrid analysis using HOS1 proteins. Note that β–gal activity was observed with FVE and FLK proteins as well as with ICE1 as a positive control. Transformed yeast cells were assayed on selective SD/-Trp/-Leu medium (bottom panel) and SD/-Trp/-Leu/-His/X-α-gal medium (top panel). (E) The in vivo interaction between HOS1 and FVE or FLK proteins. Arabidopsis mesophyll protoplasts were used for coimmunoprecipitation analysis. Arrows denote the expected size of HOS1-HA (lane 1, 2), FLK-GFP (lane 6), and FVE-GFP (lane 7) proteins. Asterisks indicate a non-specific band. 122x114mm (300 x 300 DPI)

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Re Figure 7. Effect of intermittent cold treatment on HOS1. (A) Total leaf numbers of wild-type (Col), hos1-3, flk-1, fve4, and fld-1 mutants treated with intermittent cold. (B) qPCR analysis of HOS1, FVE, FLK, FLC, SOC1, CBF3, and COR15a expression in wild-type plants with or without intermittent cold treatment. Error bars indicate standard deviation. 78x105mm (300 x 300 DPI)

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Re Figure 8. A proposed model for the role of HOS1 as an integrator in the low temperature response. Low ambient temperature and intermittent cold cause alterations in the activity of HOS1 protein, which in turn may negatively regulate FVE and FLK at the post-transcriptional level by ubiquitination. This low temperature signaling relay regulates the expression of FT/TSF and FLC. Thus, it is likely that this intricate crosstalk constitutes a fine-tuning protective mechanism against premature flowering in inappropriate seasons, such as early spring or late fall. Intermittent cold and cold stresses mediated by HOS1 induce cold resistance and cold acclimation, respectively. Black and gray colors represent genetic pathways found in this study and those reported in previous studies, respectively. Arrows represent promotion effects, whereas T-bars indicate repression effects. 83x113mm (300 x 300 DPI)