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The low temperature response pathways for cold acclimation and vernalization are
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independent
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Donna M. Bond1,2, Elizabeth S. Dennis1, and E. Jean Finnegan1,*
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CSIRO, Plant Industry, Canberra, Australia
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Current Address: Department of Plant Sciences, University of Cambridge, Cambridge,
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United Kingdom
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Corresponding author: Jean Finnegan
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CSIRO Plant Industry
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PO Box 1600
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Canberra, ACT 2601
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Australia
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Phone: 61-2-6246-5307
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Fax : 61-2-6246-5000
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e-mail: [email protected]
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[email protected]
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[email protected]
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ABSTRACT
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Vernalization is the promotion of flowering in response to the prolonged cold of winter.
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To survive sub-zero winter temperatures, plants must first acclimate to low, non-
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freezing temperatures, (cold acclimation). Induction of VERNALIZATION
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INSENSITIVE 3 (VIN3), the first gene in the vernalization pathway, is initiated within
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the same time frame as the induction of genes in the cold acclimation pathway raising
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the question of whether there are common elements in the signal transduction pathways
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that activate these two responses to cold. We show that none of the signalling
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components required for cold acclimation, including the “master regulator”
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INDUCTION OF CBF EXPRESSION1 (ICE1) or HIGH EXPRESSION OF
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OSMOTICALLY RESPONSIVE GENE1 (HOS1), which has been described as a link
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between cold acclimation and vernalization, play a role in VIN3 induction. We also
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show that the hormone ABA does not modulate VIN3 induction, consistent with earlier
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reports that ABA signalling plays no role in the vernalization response. The cold
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acclimation pathway is activated at 12°C, at which temperature there is no induction of
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VIN3 expression. Taken together our data demonstrate that the responses to low
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temperatures leading to cold acclimation and vernalization are controlled by distinct
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signalling pathways.
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Key words: ABA, Ca2+ signalling, CVP2, FLC, GCN5, inositol triphosphate (IP3), PLC1,
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SAC9, SAL1, VIN3
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INTRODUCTION
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There are two well characterised responses of plants to low temperatures: cold acclimation
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and vernalization. A short-term exposure to low, non-freezing temperatures is sufficient to
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induce cold acclimation resulting in increased tolerance to freezing conditions (reviewed in
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Thomashow 1999; Chinnusamy, Zhu & Zhu 2006; Thomashow 2010). In contrast, an
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extended period at low temperatures, such as occurs in winter, is required to promote the
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transition from vegetative to reproductive growth in the more favourable conditions of spring,
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a response known as vernalization (reviewed in Kim et al. 2009). While the biochemical
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events and signal transduction pathway leading to cold acclimation are relatively well
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characterised, little is known of the upstream events that activate the vernalization response.
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Induction of VIN3, the first known gene in the vernalization pathway (Sung and Amasino
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2004), is initiated within the same time frame as genes in the cold acclimation pathway (Bond
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et al. 2009b; Finnegan et al. 2010) suggesting that there may be common elements in the
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events leading to gene activation in these two cold responsive pathways.
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The cold acclimation pathway (Fig. 1) is initiated when plants sense low temperatures
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through membrane rigidification; this is followed closely by an influx of Ca2+ into the cytosol
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and the production of IP3, which can then signal a further release of Ca2+ from intracellular
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stores (Knight, Trewavas & Knight 1996; Ruelland et al. 2002). ABA has also been
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implicated in cold-signalling and it too is thought to mediate a Ca2+ signal (Chen, Li &
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Brenner 1983). The cold-sensing and signalling pathways activate the constitutively
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expressed MYC transcription factor, ICE1, through sumoylation and phosphorylation.
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Activated ICE1 up-regulates the expression of the three CRT/DRE BINDING FACTOR
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(CBF) transcription factors (Chinnusamy et al. 2003; Miura et al. 2007). CBF1, 2 and 3 are
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induced within 15 minutes of exposure to low temperatures and transcript levels increase
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over a 2 hour period and before declining as cold acclimation is initiated. CBF1, 2 and 3 are
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transcriptional activators with partially redundant activities that induce a group of genes
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known as the “CBF regulon” including the COLD-REGULATED (COR) genes that confer
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freezing tolerance (Gilmour et al. 1998; Shinwari et al. 1998). The term COR refers to four
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gene families, each of which comprises two genes arranged in tandem; at least one of each
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gene pair is induced by low temperatures while the other gene is induced by conditions such
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as water deficit (Thomashow 1999). Induction of COR6.6, COR15a, COR47 and COR78
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occurs within 4 hours of cold exposure and continues until acclimation leads to down-
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regulation of the COR genes (Hajela et al. 1990; Gilmour et al. 1998; Zarka et al. 2003). It is
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thought that the COR proteins act by protecting the cell from dehydration and freezing injury
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during the acclimation process (Thomashow 1999). In addition to the COR genes, CBFs also
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regulate genes involved in phosphoinositide metabolism, osmolyte protection, reactive
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oxygen species (ROS) detoxification, membrane transport and many other genes with known
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or presumed roles in cellular protection (Chinnusamy, Zhu & Zhu 2007).
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As plants acclimate to cold, the cold-sensing mechanisms become desensitized (Zarka et al.
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2003); HOS1, a RING E3 ligase, mediates the ubiquitination and proteasomal degradation of
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ICE1, and thus negatively regulates the CBF genes and the cold acclimation response (Dong
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et al. 2006). Although the CBF and COR genes play a dominant role in cold acclimation,
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there are many other genes that show altered expression in response to low temperatures
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(Hannah, Heyer & Hincha 2005; Lee, Henderson & Zhu 2005). Transcriptome analysis of an
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ice1 mutant indicates that approximately 40% of genes that respond to low temperatures are
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primary or secondary targets of ICE1 (Lee et al. 2005; Chinnusamy, Zhu & Zhu 2007).
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Vernalization is a quantitative response to low temperatures; short periods of cold exposure
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are less effective at promoting flowering time than longer periods (Sheldon et al. 2006). The
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most upstream event of the vernalization pathway identified is the induction of VIN3 in
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response to low temperatures; VIN3 expression continues to increase until plants are returned
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to warmer temperatures when VIN3 expression is repressed to basal levels (Sung and
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Amasino 2004; Bond et al. 2009b; Finnegan et al. 2010). VIN3 is required for the
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vernalization-mediated epigenetic repression of FLOWERING LOCUS C (FLC) (Sung and
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Amasino 2004), the central regulator of flowering time in Arabidopsis (Michaels and
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Amasino 1999; Sheldon et al. 1999). The quantitative induction of VIN3 in response to low
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temperatures suggests that the mechanisms regulating its expression are capable of measuring
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the duration of cold exposure.
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Little is known of the molecular events leading to induction of VIN3 in response to low
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temperatures. VIN3 chromatin is marked with tri-methylation of lysine 27 on histone H3
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(H3K27me3), a hallmark of PRC2 mediated repression, in both non-vernalized and
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vernalized plants (Zhang et al. 2007; Finnegan et al. 2010; Kim, Zografos & Sung 2010).
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PRC2 activity is required to maintain VIN3 in a repressed state under normal growth
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conditions, and when plants are exposed to low temperatures PRC2 activity modulates the
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rate of VIN3 induction (Finnegan et al. 2010). The chromatin modifying proteins, EARLY
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FLOWERING IN SHORT DAYS (EFS) and the RNA POLYMERASE ASSOCIATED
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FACTOR1 (PAF1) complex, are essential for full induction of VIN3 in response to cold
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(Finnegan et al. 2010; Kim et al. 2010), but to date no other transcriptional regulators of
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VIN3 have been identified. The VIN3 promoter contains binding sites for MYC (5’-
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CANNTG-3’) and MYB (5’-[T/C]AAC[T/G]G-3’ or 5’-CC[T/A]ACC-3’) transcription
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factors (Meshi and Iwabuchi 1995; Bond et al. 2009a). Both motifs are also found in the
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promoters of the CBF genes (Shinwari et al. 1998) and are required for their repression at
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22°C and activation at 4°C, respectively (Agarwal et al. 2006; Chinnusamy et al. 2003). In
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addition, the VIN3 promoter contains a low temperature response element (LTRE); a similar
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element in the COR15a promoter is required for its induction (Baker, Wilhelm &
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Thomashow 1994). Treatment with the protein synthesis inhibitor, cycloheximide, does not
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block induction of VIN3 by low temperatures (Bond et al. 2009c). Consistent with this, the
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transcriptional regulators GCN5, ADA2a and ADA2b, which bind the cold-induced
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transcription factor CBF1 to form a SAGA(Spt-Ada-Gcn5 Acetyltransferase)-like complex
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that up-regulates the COR genes, play no role in VIN3 induction (Bond et al. 2009b).
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However, there have been no reports describing any role for other components of the cold-
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acclimation pathway in VIN3 induction.
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Here we investigate whether there are components in common between the signal
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transduction pathways that regulate the cold acclimation and vernalization pathways by using
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mutants in key genes of the well characterised cold acclimation pathway including the
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“master regulator” ICE1 (Chinnusamy et al. 2003), and HOS1, which has previously been
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described as a link between cold acclimation and vernalization (Ishitani et al. 1998; Lee et al.
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2001). We also ask whether the hormone ABA and the signalling molecules IP3 and Ca2+
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play any role in induction of VIN3. Finally we determine whether there is a common
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temperature threshold for activation of the two pathways.
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MATERIALS AND METHODS
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Plant material
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The Arabidopsis ecotypes Col and C24 were used unless otherwise stated. The characterised
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mutants and their relevant controls are given in Table 1.
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Plant growth conditions
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Seeds were surface sterilized with a bleach:ethanol solution (1:3, v/v) and plated onto 150mm
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Petri dishes containing MS agar medium (Murashige and Skoog 1962) with 3% (w/v)
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sucrose. After a 2-day stratification period at 4°C to break dormancy, seeds were exposed to
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long day (LD) growth conditions (16-hour days with cool white fluorescent light at an
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intensity of ~170 μmol m-2 s-1) at 22°C; these conditions, which are termed ambient, are those
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used for the 0 day time point in all experiments.
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Short- and long-term (vernalization) cold treatments
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For short- and long-term cold treatments, 14-day-old seedlings were exposed to 4°C or 12°C
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under low light conditions for the specified times. Seedlings were transferred to 4°C or 12°C
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at noon, 6 hours after the start of the LD due to circadian regulation of the cold acclimation
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response (Fowler, Cook & Thomashow 2005). For post-vernalization treatments, plates were
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returned to ambient growth conditions for the specified time.
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ABA treatment
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Fourteen-day-old seedlings were transferred to 90mm Petri dishes containing 15 mL liquid
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MS media (3% sucrose, v/v). Plates were sealed and placed under ambient growth conditions
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with gentle shaking for 24 hours to allow the seedlings to adapt to liquid conditions. ABA
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(100 µM final concentration; Sigma-Aldrich, Sydney, NSW, Australia) was added to the
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liquid media; plates were sealed and grown under ambient conditions with gentle shaking for
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24 hours. Control samples were treated the same but without ABA.
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Caffeine treatment
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Caffeine experiments were as described by Baxter-Burell et al. (2003) with a few minor
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alterations. Fourteen-day-old seedlings were transferred to 90mm petri dishes containing
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fresh MS agar (3% sucrose, w/v) supplemented with 5 mM Caffeine (Sigma-Aldrich).
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Control seedlings were transferred to fresh MS agar (3% sucrose, w/v). Plates were sealed
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and placed at ambient conditions for 24 hours.
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Flowering time analysis
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To assess the response to vernalization, 14-day-old seedlingsm were placed under vernalizing
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conditions for 28 days; at the end of the treatment plates were transferred to ambient
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conditions until seedlings flowered. Flowering time was measured by counting the rosette
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and cauline leaves at flowering, and the days from germination to floral bud formation.
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Vernalized seedlings were compared to non-vernalized controls grown under ambient
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conditions. Each experiment included at least 2 biological replicates, each containing 21
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seedlings. Statistical significance of flowering time between genotypes was determined using
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the Mann Whitney U test.
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RNA extraction and cDNA synthesis
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RNA was extracted from approximately 100 mg of whole seedlings, aerial tissues or roots
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using the RNeasy Plant Mini Kit according to the manufacturer’s instructions (Qiagen,
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Doncaster, VIC, Australia). Three micrograms of RNA was used for 20 μL cDNA synthesis
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reaction using SuperScript II (Invitrogen, Carlsbad, CA, USA) with an oligodT primer,
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following the protocol outlined by the supplier.
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Quantitative real-time PCR (qPCR )
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The sequences of the primers used in this study are given in Supplementary Table 1. For all
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gene expression analyses, at least 2 biological replicates were assessed. Procedures for qPCR
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were similar to those previously described (Bond et al. 2009b). Quantification of each primer
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set and cDNA template was performed in triplicate and the average relative concentration
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was determined using the three replicates. A “no template” control was included for each
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primer pair to ensure that the results were not influenced by primer dimer formation, or
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contamination from other sources. Data was normalized by determining the ratio of the
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concentration for the gene of interest and the control gene, FORMALDEHYDE
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DEHYDROGENASE (FDH; At5g43940)..
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Statistical analysis of expression data
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The normalized gene expression data was then Log10-transformed and entered into the
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GenStat (12th Edition, VSN International, Hemel Hempstead, UK) statistical suite as
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described previously (Bond et al. 2009b; Bond et al. 2009c).
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RESULTS AND DISCUSSION
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VIN3 induction does not require ICE1, the “master regulator” of the cold acclimation
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pathway
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VIN3 induction is not blocked by cycloheximide suggesting that VIN3 is activated by a
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constitutively expressed protein that is post-translationally modified in response to low
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temperatures (Bond et al. 2009c). The constitutively expressed MYC transcription factor,
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ICE1, is one such candidate for regulating VIN3 induction particularly as there are putative
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MYC binding sites in the VIN3 promoter (Bond et al., 2009a). Mutations in ICE1 block the
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expression of CBF3, and reduce the level of CBF1 and CBF2 induction during cold exposure,
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leading to a significant reduction in freezing tolerance (Chinnusamy et al. 2003). A large
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percentage of the other cold-induced genes are not induced or show a much reduced
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induction in ice1 mutants. Many of these genes are transcription factors, suggesting that ICE1
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acts as a master regulator of cold acclimation by affecting a cascade of cold-responsive genes
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(Chinnusamy et al. 2003). The induction of VIN3 by low temperatures was not compromised
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in the ice1-1 mutant (Fig. 2a) indicating that ICE1 does not play a role in activating VIN3
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expression.
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HOS1 does not regulate VIN3 expression during cold exposure
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Numerous cold-responsive genes are super-induced in the hos1-1 mutant indicating that
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HOS1 is a negative regulator of low temperature-induced gene expression (Ishitani et al.
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1998; Lee et al. 2001). Furthermore, the hos1-1 mutant has been described as being
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“constitutively vernalized” as it displays an early flowering phenotype that correlates with
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lower levels of FLC expression (Ishitani et al. 1998; Lee et al. 2001). This raises the
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possibility that VIN3, a cold responsive gene required for the vernalization-mediated
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repression of FLC, could be responsible for the “constitutively vernalized” phenotype of
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hos1-1. VIN3 expression in the hos1-1 mutant was comparable to wild-type, both in plants
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grown at 22°C (ambient) and at low temperatures (Fig. 2b). This indicates that HOS1 does
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not regulate VIN3, and that the reported decrease in FLC expression and associated early
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flowering phenotype of hos1-1 is independent of VIN3 activity.
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In contrast to previous reports, we found that FLC expression in hos1-1 grown under ambient
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temperatures was comparable to that in wild-type plants, and hos1-1 did not flower early
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under these conditions (Fig. 2c and Fig. S1a,b). In response to low temperatures, FLC
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expression was slightly lower in hos1-1 compared to wild-type seedlings at all time points
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(Fig. 2c), but this was at the lower limits of statistical significance and was much smaller than
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the difference reported by Lee et al. (2001). After 4 weeks vernalization, hos1-1 flowered
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earlier than wild-type when the days to flowering were measured, but with the same number
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of leaves (Fig. S1a,b). As HOS1 is involved in the response to multiple abiotic stresses the
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differences in FLC expression and flowering time of hos1-1 observed between groups could
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be associated with differences in growth conditions.
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VIN3 is constitutively expressed in the fiery1-1 (fry1-1) mutant of SAL1, and is hyper-
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induced by low temperatures
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SAL1 is a bifunctional enzyme with both inositol polyphosphate-1-phosphatase and 3’(2’),5’-
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bisphosphate nucleotidase activities. The inositol polyphosphate 1-phosphatase activity is
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proposed to mediate IP3 breakdown by dephosphorylating the inositol 1,4-bisphosphate and
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inositol 1,3,4-trisphosphate intermediates (Majerus 1992), whereas the nucleotidase activity
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against 2’-phophoadenosine 5’-phosphate and 3’-phosphoadenosine 5’-phophosulfate has
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been implicated in sulfate metabolism and post-transcriptional gene silencing (Quintero,
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Garciadeblas & Rodriguez 1996; Dichtl, Stevens & Tollervey 1997; Gy et al. 2007). Several
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mutant alleles of SAL1 have been identified in different forward genetic screens; these
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mutants show pleiotropic phenotypes, although the phenotypes vary between the different
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mutant alleles (Quintero et al. 1996; Xiong et al. 2001; Xiong et al. 2004; Gy et al. 2007;
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Kim and von Arnim 2009; Wilson et al. 2009; Robles et al. 2010)
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The fry1-1 allele of SAL1, in ecotype C24, accumulates IP3 to a level approximately ten times
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that seen in wild-type plants (Xiong et al. 2001). The fry1-1 mutant displays constitutive
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expression of COR47 and COR78 under ambient temperatures, perhaps due to the
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constitutively high IP3 levels (Xiong et al. 2001). Tolerance to cold stress, as measured by
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cold-induced electrolyte leakage, is compromised in fry1-1 and yet stress-responsive genes,
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such as ADH1, are de-repressed at ambient temperatures and show enhanced transcription
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when exposed to cold (Fig. S2a; Xiong et al. 2001). VIN3 was also de-repressed at ambient
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temperatures and showed a more rapid induction when plants were placed at low
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temperatures (Fig. 3a). After 1 day of cold exposure, VIN3 expression in fry1-1 seedlings was
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similar to the level attained in wild-type plants after 14 days at low temperatures. The
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induction of VIN3 is not quantitative in fry1-1 seedlings, but reaches a plateau after 7 days at
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low temperatures (Fig. 3a). After 21 days at low temperatures, VIN3 expression was
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comparable in the fry1-1 and wild-type seedlings. When seedlings were returned to ambient
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temperatures, VIN3 expression was repressed to pre-vernalized levels in both fry1-1 and
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wildtype seedlings (Fig. S2b).
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Constitutive expression of VIN3 does not promote flowering in plants grown under ambient
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temperatures (Sung and Amasino 2004), however, the effects of constitutive VIN3 expression
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during low temperatures has not been reported. The enhanced expression of VIN3 in the fry1-
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1 mutant did not alter the kinetics of FLC repression in response to low temperatures (Fig.
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3b). The fry1-1 mutant flowered slightly later than wild-type C24 seedlings grown under LD
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conditions irrespective of whether total leaf number or days to flowering were measured, and
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showed a similar response to vernalization as wild-type plants (Fig 3c, S2c).
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Accumulation of IP3 is not sufficient to induce VIN3 expression at ambient
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temperatures or enhance induction in response to low temperatures
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The mutation in fry1-1 causes premature termination of translation, suggesting that both
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enzymatic activities of SAL1 are missing in these plants (Xiong et al. 2001). IP3 is an
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important signalling molecule in the cold acclimation pathway (Xiong et al. 2001),
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suggesting that the enhanced accumulation of IP3 in fry1-1 plants may be associated with
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expression of VIN3 at ambient temperatures and enhanced induction after short periods of
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cold. Mutation of two other genes, SUPPRESSOR OF ACTIN domain phosphoinositide
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phosphatase 9 (SAC9) and COTYLEDON VASCULAR PATTERN2 (CVP2) has been
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associated with increased accumulation of IP3. Mutants of sac9 show a systemic stressed
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phenotype, including over-expression of stress-induced genes, elevated accumulation of
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ROS, and are dwarfed slow growing plants (Williams et al. 2005). Roots from sac9-1 mutant
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plants accumulate a three-fold higher level of IP3 than wild-type roots, although no
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differences in IP3 levels were observed in shoots. CVP2 encodes an inositol polyphosphate 5’
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phosphatase, which is expressed in developing vascular cells in several plant organs (Carland
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and Nelson 2004). Levels of IP3 are approximately three-fold higher in a cvp2-1 mutant,
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which shows increased sensitivity to ABA.
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We used these mutants to test the effect of constitutive IP3 accumulation on VIN3 expression.
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The level of VIN3 expression at ambient temperatures and the kinetics of VIN3 induction at
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low temperatures in both sac9-1 and cvp2-1 mutants mirrored that seen in wild-type plants
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(Fig. 3d). There was no increase in VIN3 expression in sac9-1 roots compared to wild-type
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roots even though IP3 levels were shown to be elevated in roots of the sac9-1 mutant (Fig. 3e;
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Williams et al. 2005). In contrast to fry1-1, there was no change in ADH1 expression in either
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sac9-1 or cvp2-1 mutants grown under ambient or low temperatures (Fig. S3).
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As a further test of the role of IP3 on VIN3 expression, we measured VIN3 induction in
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transgenic plants constitutively expressing a plasma membrane localised mammalian type 1
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inositol 5-phosphatase, which is specific for IP3 hydrolysis (Mitchell et al. 1996; De Smedt et
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al. 1997). In these plants the basal level of IP3 is 5% lower than wild-type under normal
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conditions and remains below basal wild-type levels even in response to stress (Perera et al.
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2006; Perera et al. 2008). There was no change in the kinetics of VIN3 induction when these
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transgenic plants were grown at low temperatures (Fig. S4).
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Soluble IP3 is released by the activity of PHOSPHOLIPASE C (PLC), which cleaves
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phospholipid phosphatidylinositide (4,5) bisphosphate to generate the lipid, diacylglycerol,
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and IP3. PLC activity is enhanced by cold exposure of Arabidopsis suspension cells in a Ca2+
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dependent manner (Ruelland et al. 2002). PLC1 expression is low under normal conditions
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but is induced by environmental stresses including salinity, dehydration and low
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temperatures, suggesting that it plays a role in signalling the response to changing
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environmental conditions (Hirayama et al. 1995). Blocking the release of IP3 in a plc1 mutant
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had no effect on VIN3 induction (Fig. S5). Taken together, these data suggest that the
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elevated levels of IP3 seen in fry1-1 did not cause the enhanced expression of VIN3.
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VIN3 expression is not affected in the altered expression of APX2 (alx8) mutant allele of
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SAL1
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To investigate whether the 3’(2’),5’-bisphosphate nucleotidase activity of SAL1 is required
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for repression of VIN3 we used alx8, another allele of SAL1 in ecotype Col (Wilson et al.
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2009). The alx8 mutation is a point mutation leading to a single amino acid substitution
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(G217D); this substitution may decrease protein stability as no SAL1 protein was detected in
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alx8 seedlings, suggesting that alx8 can be considered to be a null mutant (Wilson et al.
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2009). In contrast to fry1-1, alx8 showed the same VIN3 expression in response to cold as its
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wild-type parent, Col (compare Fig. 3a,f). Microarray experiments showed that only about
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50% of the differentially expressed genes in fry1-1 and alx8 mutants are in common (Wilson
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et al. 2009), suggesting that the difference in genetic background between fry1-1 and alx8
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(C24 and Col, respectively) has a significant effect on the phenotype. As the sac9-1 and
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cvp2-1 mutants are also in a Col background, it seems likely that elevated VIN3 expression
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seen in fry1-1 is a secondary effect of this mutation in the C24 background rather than being
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caused by elevated levels of IP3 per se.
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Exogenous ABA does not induce VIN3 at ambient temperatures
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The phytohormone ABA increases in response to low temperatures and is thought to play a
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role in the signal transduction of the cold response (Chen, Li & Brenner 1983; Lång and
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Palva 1992). Exogenous ABA does induce the expression of the three CBF genes, but to a
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lesser extent than cold exposure (Knight et al. 2004). In contrast, exogenous ABA did not
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induce VIN3 expression at ambient temperatures (Fig. 4a), although the expression of another
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cold-regulated gene, ADH1, was induced (Fig. 4a; Dolferus et al. 1994). These observations
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are consistent with early reports showing that the cold-signalling associated with
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vernalization does not require ABA (Chandler, Martinez-Zapater & Dean 2001; Liu et al.
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2002).
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Elevated Ca2+ does not induce VIN3 expression
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The elevation in cytosolic free Ca2+ levels is one of the earliest known plant responses to low
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temperatures (Örvar et al. 2000; Sangwan et al. 2001). Caffeine treatment, which stimulates
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an immediate increase in cytosolic Ca2+ levels and induction of ADH1 expression (Baxter-
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Burrell et al. 2003), was used to assess whether VIN3 induction could be regulated by a Ca2+
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signal during cold exposure.
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Treatment of C24 seedlings with 5 mM caffeine did not induce VIN3 expression at 22°C (Fig.
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4b), suggesting the cold induced expression of VIN3 is independent of Ca2+ release.
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Consistent with previous reports, ADH1 expression was induced (Fig. 4b) confirming the
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efficacy of the caffeine treatment. Similarly, CBF3 expression was increased in response to
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caffeine, but there was no increase in CBF1 & 2 transcript levels (Fig. 4c). Three of the 4
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COR genes (COR6.6, COR15a and COR47) showed no response to caffeine, while
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expression of the remaining gene, COR78, increased about four-fold (Fig. 4c). The caffeine
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treatment was done at 22ºC and so it is likely that the relatively small induction of the CBF
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genes and the downstream COR genes observed in response to caffeine (compare Fig. 4c,
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Fig. 5b,c and Fig. S6a,b) is due to the low stability of CBF transcripts at warm temperatures
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(Zarka et al. 2003).
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Cold acclimation is initiated at temperatures that do not activate the vernalization
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pathway
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The threshold temperature at which the accumulation of transcripts for the CBF genes and
14
downstream target, COR15a, becomes detectable is 14ºC, indicating that cold acclimation is
15
initiated at temperatures ranging from 14ºC to below zero (Zarka et al. 2003). Even though
16
the cold acclimation pathway is activated at 14ºC, transcripts accumulate to a higher level in
17
plants grown at 4ºC. The temperature at which the Arabidopsis vernalization pathway
18
becomes active has not been determined.
19
20
There was no change in VIN3 expression after 21 days at 12ºC, whereas the gene is induced
21
rapidly at 4ºC (Fig. 5a; Bond et al. 2009b; Finnegan et al. 2010). Consistent with previous
22
reports, we found that CBF3 and COR15a were induced when plants were grown at 12ºC,
23
although the pattern of expression differed from that seen when plants were grown at 4ºC
24
(Fig. 5b,c). For both genes, the level of transcripts was lower when plants were grown at
25
12ºC than at 4ºC, and continued to accumulate gradually over 21 days at 12ºC (Fig 5b,c). We
17
1
found no accumulation of CBF1 and CBF2 transcripts when plants were placed at 12ºC (not
2
shown). Although it has been reported that all CBF genes were expressed at 14ºC (Zarka et
3
al. 2003), it is likely that only CBF3 transcript levels were elevated because the probe used to
4
detect expression did not discriminate between the different CBF genes. These observations
5
suggest that the sensitivity of the sensory mechanisms to cold may differ between the
6
vernalization pathway and at least some genes of the cold acclimation pathway.
7
8
Concluding remarks
9
Acclimation to low temperatures is an important biochemical and physiological process that
10
improves the tolerance of plants to freezing temperatures (Thomashow 1999). Cold
11
acclimation is an early step in the process of vernalization, which for many plants involves
12
exposure to sub-freezing temperatures (Sung and Amasino 2005). Plants acclimate to the cold
13
within a 24 hour period, and after this time the cold-sensing mechanism becomes desensitized
14
to low temperatures resulting in the down-regulation of the CBF and COR genes (Gilmour et
15
al. 1998; Zarka et al. 2003). In contrast to the genes involved in cold acclimation, VIN3
16
induction, which is initiated within the first 24 hours, continues as the time at low
17
temperature increases (Sung and Amasino 2004; Bond et al. 2009b; Finnegan et al. 2010).
18
The increase of VIN3 expression correlates with the repression of FLC, promoting the
19
transition to flowering (Sung and Amasino 2004). Only two genes with links to both the cold
20
response and flowering time pathways have been identified, the autonomous pathway
21
member FVE, a retinoblastoma-associated protein, and LOV1, a NAC-domain protein;
22
however, neither protein affects the vernalization response of Arabidopsis (Ausín et al. 2004;
23
Kim et al. 2004; Yoo et al. 2007).
24
18
1
Our data indicate that cold acclimation and vernalization use different pathways to signal
2
changes in gene expression. Firstly, none of the key genes in the cold acclimation pathway
3
affect VIN3 induction. Secondly, VIN3 expression was not induced at 22°C in the presence of
4
caffeine, which stimulates an influx of cytosolic Ca2+ (Baxter-Burrell et al. 2003). Thirdly,
5
application of the phytohormone ABA, which is thought to contribute to the cold acclimation
6
response by stimulating expression of some cold-responsive genes (Chen et al. 1983; Lång
7
and Palva 1992), did not induce VIN3 expression. Finally, the cold acclimation pathway is
8
initiated at temperatures that do not stimulate VIN3 induction.
9
10
How then is VIN3 expression regulated by low temperatures? Possible scenarios include the
11
turnover of a constitutively expressed, cold-labile repressor. This scenario would be
12
consistent with the hyperinduction of VIN3 in plants treated with the protein synthesis
13
inhibitor, cycloheximide, at 4°C. We are unable to test this hypothesis as inhibitors of the
14
proteasome, such as MG132 and ALLN, do not block protein degradation over the time
15
frame required to measure VIN3 induction (not shown). Given that the VIN3 promoter
16
contains putative MYC binding motifs and yet it is not regulated by the MYC transcription
17
factor, ICE1, it seems likely that one of the other 160 bHLH MYC, ICE1-like, transcription
18
factors in the Arabidopsis genome may contribute to its cold-induced expression. The
19
transcription factor that activates VIN3 expression may require a post-translational
20
modification, such as phosphorylation, for VIN3 induction to occur. If the kinase that
21
modifies this factor is more active at low temperatures than the phosphatase that
22
dephosphorylates this factor, then cold treatment would lead to induction of VIN3. Further
23
research is required to determine the events leading to the induction of VIN3.
24
25
ACKNOWLEDGEMENTS
19
1
The authors thank Prof. Jian-Kang Zhu (University of California, Riverside), Prof. Barry
2
Pogson (Australian National University), Dr Imara Perera (North Carolina State University),
3
Dr Timothy Nelson (Yale University) and Dr Mary Williams (Harvey Mudd College) for
4
their generous gift of seed stocks and Xiaomei Wallace for excellent technical assistance
5
6
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27
1
Table 1 Cold mutants used in the present study.
Name
Locus
Source
Allele
Ecotype
References
HOS1
At2g39810
J.-K. Zhu
hos1-1
C24
Ishitani et al. 1998;
(University of
Lee et al. 2001
California,
Riverside)
ICE1
At3g26744
J.-K. Zhu
ice1-1
(University of
#
Col-gl1 +
Chinnusamy et al.
CBF3pro::LUC
(2003)
fry1-1
C24
Xiong et al. (2001)
alx8
Col
Rossel et al. (2004);
California,
Riverside)
SAL1
At5g63980
ABRC
(Alonso et al.
2003)
SAL1
At5g63980
B. Pogson
(Australian
Rossel et al. (2006);
National
Wilson et al. (2009)
Univeristy)
CVP2
At1g05470
T. Nelson
cvp2-1
Col
(Yale
Carland and Nelson
(2004)
University
SAC9
At3g59770
M. Williams
sac9-1
Col
Williams et al.
(2005)
(Harvey
Mudd
College)
PLC1
At5g58670
ABRC
plc1
Col
Hirayama et al.
28
(Alonso et al.
2003)
1
2
#
(SALK_
025769)
Control was also obtained from J.-K. Zhu.
(1995)
29
1
FIGURE LEGENDS
2
Figure 1. Steps of the cold acclimation and its possible connection with the vernalization
3
pathway. As described in the text, plants probably sense low temperatures through membrane
4
rigidification which is thought to stimulate the influx of Ca2+ into the cytosol and the
5
production of IP3, which can then signal the release of Ca2+ from intracellular stores. Cold
6
also induces endogenous ABA whose action is thought to involve a Ca2+ mediated signal
7
transduction. Activation of protein kinases by the cold-induced Ca2+ signature might be part
8
of this pathway. Constitutively expressed ICE1 is activated by cold stress through
9
sumoylation and phosphorylation. Active ICE1 then induces expression of the CBF3 (with
10
less effect on CBF1 and CBF2). The CBF transcription factors regulate the expression of a
11
suite of genes known as the “CBF regulon” including the COR genes that confer freezing
12
tolerance. HOS1 mediates the ubiquitination and proteasomal degradation of ICE1 and, thus,
13
negatively regulates the CBF regulon. SAL1 is a negative regulator of cold-responsive genes
14
via catabolism of IP3, production of which increases at low temperatures through the activity
15
of PLC. Solid arrows indicate stages of the cold acclimation and signalling pathways that
16
have been experimentally determined and/or are widely accepted. Broken arrows that lead to
17
“VIN3?” indicate the stages of the cold acclimation and signalling pathways investigated in
18
this study. P = Phosphorylation; S = Sumoylation; MYC = MYC-recognition promoter motif
19
where ICE1 binds; CRT/DRE = C-repeat/drought responsive element in the promoters of
20
genes in the CBF regulon where CBF transcription factors bind. The arrows on the solid bars
21
indicate the start of transcription.
22
23
Figure 2. ICE1 and HOS1 do not regulate the expression of VIN3 in response to low
24
temperatures. (a) qPCR measuring the expression of VIN3 relative to FDH in CBF3pro::LUC
25
ICE1/ICE1 (solid line) and ice1-1/ice1-1 (dashed line) seedlings in response to cold exposure
30
1
(4°C) for 1, 7, 14 and 21 days. All samples are compared to 14-day-old non-vernalized
2
control seedlings (0 days). Error bars represent Standard Error of the Difference of the means
3
(S.E.D.). The F test p-values for the terms in the ANOVA analysis suggested significant
4
effects for the time of cold treatment (p < 0.001), but not the seedling genotype or their
5
interaction. (b) qPCR measuring the expression of VIN3 relative to FDH in C24 (solid line)
6
and hos1-1 (dashed line) seedlings in response to 1, 7, 14 and 21 days of cold exposure. All
7
samples are compared to 14-day-old non-vernalized seedlings (0 days). Error bars represent
8
the S.E.D. The F test p-values for the terms in the ANOVA analysis suggested significant
9
effects for the time of cold treatment (p < 0.001), but not the genotype or their interaction. (c)
10
As for (b) but measuring the expression of FLC. The F test p-values for the terms in the
11
ANOVA analysis suggested significant effects for the time of cold treatment (p < 0.001), and
12
the genotype of the seedlings (p = 0.02) but not their interaction.
13
14
Figure 3. The fry1-1 mutation in SAL1 de-regulates VIN3 expression. (a) qPCR measuring
15
the expression of VIN3 relative to FDH in C24 (solid line) and fry1-1 (dashed line) seedlings
16
in response to 1, 7, 14 and 21 days of cold exposure. All samples are compared to 14-day-old
17
non-vernalized control seedlings (0 days). Error bars represent S.E.D. The F test p-values for
18
the terms in the ANOVA analysis suggested significant effects for the time of cold treatment
19
(p < 0.001), the seedling genotype (p < 0.001) and their interaction (p = 0.002). (b) qPCR
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measuring the expression of FLC relative to FDH in C24 (solid line) and fry1-1 (dashed line)
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seedlings in response to 1, 7 and 28 days of cold exposure (shaded grey), followed by 22°C
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for 1, 3 and 14 days (white area). All samples are compared to 14-day-old non-vernalized
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seedlings (0 days). Error bars represent S.E.D. The F test p-values for the terms in the
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ANOVA analysis suggested significant effects for the time of cold treatment (p < 0.001), but
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not the seedling genotype or their interaction. (c) Flowering time (total leaf number) of C24
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1
(light grey) and fry1-1 (dark grey) seedlings after exposure to 4°C for 28 days (Vernalized),
2
compared to control seedlings (Non-vernalized). Error bars represent standard deviation. (d)
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qPCR measuring the expression of VIN3 relative to FDH in Col (solid line, filled symbol),
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cvp2-1 (dashed line) and sac9-1 (solid line, open symbol) seedlings in response to 1, 3, 7 and
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21 days of cold exposure. All samples are compared to 14-day-old non-vernalized seedlings
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(0 days). Error bars represent S.E.D. The F test p-values for the terms in the ANOVA
7
analysis suggested significant effects for the time of cold treatment (p < 0.001), but not the
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seedling genotype or their interaction. (e) qPCR measuring the expression of VIN3 relative to
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FDH in Col (light grey) and sac9-1 (dark grey) shoot and root tissue from 14-day-old non-
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vernalized seedlings. Error bars represent S.E.D. (f) qPCR measuring the expression of VIN3
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relative to FDH in Col (solid line) and alx8 (dashed line) seedlings in response to 1, 7, 14
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and 21 days of cold exposure. All samples are compared to 14-day-old non-vernalized
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seedlings (0 days). Error bars represent S.E.D. The F test p-values for the terms in the
14
ANOVA analysis suggested significant effects for the time of cold treatment (p < 0.001), but
15
not the seedling genotype or their interaction.
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Figure 4. VIN3 expression in response to chemical alterations of cold-signalling molecules
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ABA and Ca2+. (a) qPCR measuring the expression of VIN3 and ADH1 relative to FDH in
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C24 seedlings in response to 100 µM ABA (dark grey), compared to control conditions (light
20
grey). Error bars represent S.E.D. For ADH1, the F test p-values for the terms in the ANOVA
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analysis suggested significant effects for the ABA treatment (p = 0.004). (b) qPCR
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measuring the expression of VIN3 and ADH1 relative to FDH in C24 seedlings in response to
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5 mM Caffeine (dark grey), compared to control conditions (light grey). Error bars represent
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S.E.D. For ADH1, the F test p-values for the terms in the ANOVA analysis suggested
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significant effects for the caffeine treatment (p = 0.002). (c) qPCR measuring the expression
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1
of CBF1, CBF2, CBF3, COR6.6, COR15a, COR47 and COR78 relative to FDH in C24
2
seedlings in response to 5 mM Caffeine (dark grey), compared to control conditions (light
3
grey). Error bars represent S.E.D. For CBF3, the F test p-values for the terms in the ANOVA
4
analysis suggested significant effects for the caffeine treatment (p = 0.012). For COR78, the
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F test p-values for the terms in the ANOVA analysis suggested significant effects for the
6
caffeine treatment (p = 0.009).
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8
Figure 5. Unlike the cold acclimation pathway, VIN3 expression is not induced
9
when plants are grown at 12°C. (a) qPCR measuring the expression of VIN3 relative
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to FDH in Col seedlings in response to 4°C (solid line) and 12°C (dashed line) for
11
1, 7, and 21 days. All samples are compared to 14-day-old non-vernalized seedlings
12
(0 days). Error bars represent S.E.D. The F test p-values for the terms in the
13
ANOVA analysis suggested significant effects for the time of 4°C cold treatment (p
14
< 0.001). (b) As for (a) but measuring the expression of CBF3. Error bars represent
15
S.E.D. The F test p-values for the terms in the ANOVA analysis suggested
16
significant effects for the time of 4°C cold treatment (p < 0.001) and 12°C cold
17
treatment (p = 0.014). (c) As for (a) but measuring the expression of COR15a. Error
18
bars represent S.E.D. The F test p-values for the terms in the ANOVA analysis
19
suggested significant effects for the time of 4°C cold treatment (p = 0.024) and
20
12°C cold treatment (p = 0.022).
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