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Plant Mol BiolDOI 10.1007/s11103-014-0256-z

Global changes in gene expression, assayed by microarray hybridization and quantitative RT-PCR, during acclimation of three Arabidopsis thaliana accessions to sub-zero temperatures after cold acclimation

Mai Q. Le · Majken Pagter · Dirk K. Hincha

Received: 2 April 2014 / Accepted: 7 October 2014 © Springer Science+Business Media Dordrecht 2014

Particularly photosynthesis-related genes are down-regulated and genes belonging to the functional classes of cell wall biosynthesis, hormone metabolism and RNA regulation of transcription are up-regulated. Collectively, these data pro-vide the first global analysis of gene expression during sub-zero acclimation and allow the identification of candidate genes for forward and reverse genetic studies into the molec-ular mechanisms of sub-zero acclimation.

Keywords Arabidospis thaliana · CBF regulon · Cold acclimation · Freezing tolerance · Transcription factor · Sub-zero acclimation

Introduction

Together with drought, cold is considered the most impor-tant factor limiting the distribution of plants on Earth. Freezing damage to native vegetation and crop plants is additionally a ubiquitous problem of major economic sig-nificance (Steponkus 1984). The freezing tolerance of most temperate plant species increases upon exposure to low, non-freezing temperatures over a period of days to weeks. This adaptive process is termed cold acclimation (Thom-ashow 1999; Xin and Browse 2000). Freezing tolerance beyond that resulting from exposure to low, non-freezing temperatures is conferred on cold acclimated plants by exposure to temperatures slightly below freezing but before freezing injury occurs. This is commonly referred to as sub-zero acclimation and has been described in a number of plant species, such as Medicago sativa (Castonguay et al. 1993; Monroy et al. 1993), Triticum aestivum (Herman et al. 2006) and other cereals (Livingston 1996), Agrostis (Espevig et al. 2011) and Arabidopsis thaliana (Le et al. 2008; Livingston et al. 2007).

Abstract During cold acclimation plants increase in freez-ing tolerance in response to low non-freezing temperatures. This is accompanied by many physiological, biochemical and molecular changes that have been extensively investi-gated. In addition, plants of many species, including Arabi-dopsis thaliana, become more freezing tolerant during expo-sure to mild, non-damaging sub-zero temperatures after cold acclimation. There is hardly any information available about the molecular basis of this adaptation. Here, we have used microarrays and a qRT-PCR primer platform covering 1,880 genes encoding transcription factors (TFs) to moni-tor changes in gene expression in the Arabidopsis acces-sions Columbia-0, Rschew and Tenela during the first 3 days of sub-zero acclimation at −3 °C. The results indicate that gene expression during sub-zero acclimation follows a tighly controlled time-course. Especially AP2/EREBP and WRKY TFs may be important regulators of sub-zero acclimation, although the CBF signal transduction pathway seems to be less important during sub-zero than during cold acclimation. Globally, we estimate that approximately 5 % of all Arabi-dopsis genes are regulated during sub-zero acclimation.

Mai Q. Le and Majken Pagter have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s11103-014-0256-z) contains supplementary material, which is available to authorized users.

M. Q. Le · M. Pagter · D. K. Hincha (*) Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam, Germanye-mail: hincha@mpimp-golm.mpg.de

M. Q. Le Hanoi University of Sciences, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

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Cold acclimation is a multigenic, quantitative trait and numerous studies have revealed the massive re-pro-gramming of the transcriptome and metabolome that are involved in this process (see Guy et al. 2008; Hincha et al. 2012; Thomashow 2010 for reviews). In Arabidopsis, changes in gene expression are initiated within 30 min of the transfer of plants to low temperatures. Some changes are transient, while others persist for days or weeks (Fowler and Thomashow 2002; Hannah et al. 2005), implying that cold acclimation is a highly regulated time-dependent response eventually leading to increased freezing toler-ance. Much of the emphasis in cold acclimation research has focused on the regulation of cold-responsive gene expression. The C-repeat Binding Factors (CBF) 1, 2, and 3 are the best characterized regulators of cold acclimation (Chinnusamy et al. 2007; Shinozaki et al. 2003, Thom-ashow 2010). All three CBFs are rapidly induced in the cold, coordinating the expression of the “CBF regulon” or down-stream genes, which have a large impact on freezing tolerance (Jaglo-Ottosen et al. 1998; Kasuga et al. 1999). However, transcript profiling data suggest the induction of multiple transcriptional pathways during cold acclimation (Chinnusamy et al. 2007; Fowler and Thomashow 2002; Hannah et al. 2005).

In contrast to cold acclimation, little is known about the molecular mechanisms underlying sub-zero acclimation. The physical trigger of sub-zero acclimation is not the crys-tallization of ice in the apoplastic spaces of the leaves or the resulting dehydration of the cells, but rather the reduc-tion in temperature itself, as the effect of sub-zero acclima-tion was indistinguishable in the absence or presence of ice crystallization (Le et al. 2008). Plants are well able to sense small temperature differences of 5 °C or less and an epige-netic sensing mechanism for such differences functions in the ambient temperature range (Kumar and Wigge 2010). How sub-zero temperatures are sensed, however, is still unclear.

In wheat, sub-zero acclimation results in several changes in gene expression, with predominant regulation of cold/dehydration-induced and photosynthesis-related genes (Herman et al. 2006). In different accessions of Arabidop-sis, we have previously shown that transfer of cold accli-mated plants to sub-zero conditions induces a moderate increase in the expression of CBF1–3 and five of their rep-resentative target COR genes (Le et al. 2008). However, the magnitude of the induction was significantly lower than after a shift of non-acclimated plants to cold acclimat-ing conditions and there was no correlation to changes in freezing tolerance. This suggests that the CBF signal trans-duction pathway and the associated target genes are less important during sub-zero acclimation than cold acclima-tion, and that the genetic and molecular basis of sub-zero acclimation is distinct from that of cold acclimation.

Many recent approaches to understand the genetic and molecular basis of complex quantitative traits in plants have focussed on the analysis of natural genetic variation (Weigel 2012). Arabidopsis thaliana is a geographically widely spread species and our previous study suggests that there is some genetic variability in the sub-zero acclima-tion response among different accessions (Le et al. 2008). In addition, several studies have documented considerable natural variation in the freezing tolerance and cold acclima-tion capacity of Arabidopsis at above freezing temperatures (Hannah et al. 2006; Zhen and Ungerer 2008a, b; Zuther et al. 2012), allowing investigations into genotype × environ-ment interactions.

The aim of the present study was to investigate the tran-scriptional regulation and to determine time-dependent global changes in gene expression during sub-zero accli-mation of three natural Arabidopsis accessions. We show that members of the APETELA2/Ethylene-response ele-ment binding protein (AP2/EREBP) and WRKY transcrip-tion factor (TF) families may be important transcriptional regulators during sub-zero acclimation. In addition, the data provide novel insights into plant responses to sub-zero temperature, some of which show similarities to cold accli-mation whereas others appear to be specific for sub-zero acclimation. Finally, this study provides new information on TFs and other functional classes of genes which may be molecular determinants of increased freezing tolerance dur-ing sub-zero acclimation.

Materials and methods

Plant material

Arabidopsis thaliana plants from the accessions Colum-bia-0 (Col-0), Rschew (Rsch) and Tenela (Te) were used in the experiments. The sources of the different seed stocks have been described (Schmid et al. 2006). Seeds used in the experiments were generated through single seed descent to assure genetic homogeneity of the plants (Törjek et al. 2003). Plants were grown in soil in the greenhouse at 16-h day length, with light supplementation to reach at least 200 µmol m−2 s−1, and a temperature of 20 °C dur-ing the day, 18 °C during the night until 42 days after sow-ing. For cold acclimation, plants were transferred to a 4 °C growth cabinet at 16-h day length with 90 µmol m−2 s−1 for an additional 14 days (Hannah et al. 2006; Zuther et al. 2012). For sub-zero acclimation, detached leaves from cold acclimated plants were transferred into glass tubes contain-ing 200 µl of distilled water and placed in a temperature-controlled silicon oil bath at −3 °C in darkness (Le et al. 2008). In the absence of active ice nucleation, the leaves stayed supercooled during the entire incubation period.

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Leaf freezing tolerance was determined using the elec-trolyte leakage method as described in detail in previous publications (Rohde et al. 2004; Thalhammer et al. 2014).

qRT-PCR analysis of the expression of genes encoding transcription factors

Transcript levels of most TFs known in Arabidopsis were analysed in cold acclimated leaves and leaves sub-zero acclimated at −3 °C for 1, 2, 3 or 8 h using a real-time qRT-PCR platform (Bieniawska et al. 2008; Czechowski et al. 2004, 2005). This platform contains 1,880 primer pairs on five 384-well plates to determine the abundance of tran-scripts from the majority of genes encoding TFs in Arabi-dopsis. The analysis included three independent biological replicates, giving a total of 45 samples.

Total RNA was isolated from a pool of three leaves from three different plants for each sample, using the RNeasy Midi Kit (Qiagen, Venlo, The Netherlands), and treated with Baseline-Zero DNase (Epicentre, Madison, WI, USA). RNA quantity and quality were determined using a Nan-odrop spectrophotomer (Nanodrop Technologies, Wilming-ton, DE, USA) and gel electrophoresis. Quantitative PCR with intron-specific primers (Zuther et al. 2012) was used to ascertain the absence of genomic DNA. cDNA was syn-thesized with SuperScript III reverse-transcriptase (Invitro-gen, Carlsbad, CA, USA) and oligo-dT18 primers. cDNA quality was checked using primers amplifying 3′ and 5′ regions of GAPDH (At1g13440) (Zuther et al. 2012). qRT-PCR was performed in 384-well plates with an ABI PRISM 7900 HT Sequence Detection System (Applied Biosys-tems, Foster City, CA, USA). Reactions contained 2.5 µl 2× Fast SYBR Green MasterMix (Applied Biosystems), 0.5 µl of diluted cDNA and 100 nM of each gene-specific primer in a total volume of 5 µl. To ensure accuracy, prim-ers were first added to each plate followed by a mastermix containing the cDNA and SYBR Green, and both steps were performed using an Evolution P3 pipetting robot (PerkinElmer, Zaventem, Belgium) [compare (Bieniawska et al. 2008)]. Ct values for TF genes were normalized by subtracting the mean Ct of three reference genes GAPDH, PDF2 and CACS that were included on each plate (Bie-niawska et al. 2008).

Microarray hybridization

Total RNA was isolated from a pool of three leaves from three different plants for each sample, using the RNeasy Mini Kit (Qiagen) from leaves of cold acclimated plants and leaves sub-zero acclimated for 8 h, 1 or 3 days at −3 °C. The experiment was performed in three independent biological replicates. RNA quality was checked with a Bio-analyzer (Aligent, Santa Clara, CA, USA). Biotin-labelled

aRNA was synthesized from 1 µg total RNA using the Mes-sageAmp II-Biotin Enhanced Single Round aRNA Amplifi-cation Kit (Ambion, Carlsbad, CA, USA). The quality of the biotin-labeled aRNA was checked with the Bioanalyzer. Arabidopsis ATH1 GeneChip microarrays, containing more than 22,500 probe sets representing approximately 24,000 genes (Redman et al. 2004) were hybridized and scanned at ATLAS Biolabs GmbH (Berlin, Germany). The microar-ray data have been deposited in the GEO database (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE55835.

Data analysis

With the data from the qRT-PCR measurements, a first step analysis was performed by principal components analysis (PCA) to determine the accuracy and reproducibility of the biological replicates using the pcaMethods Package in (R Development Core Team 2010) applied to the normalized Ct values. This analysis indicated that one of the Te cold acclimated (CA) replicates (replicate 1) was an outlier in this group that differed widely from the other 44 samples in this dataset (Online Resource 1) and was therefore excluded from further analysis. Finally, only TF genes with at least two replicate Ct values per accession and time point and with Ct values determined in ≥75 % of all samples were considered for further analysis. This resulted in 1,570 TF genes whose expression was analyzed in detail. A com-plete list of all respective expression values can be found in Online Resource 2. To detect differentially expressed TF genes, a two-way analysis of variance (ANOVA) type I SS was performed with the log2 transformed relative expres-sion values (i.e. the Ct values normalized to the expression of the reference genes) in R to compare the mean relative expression of a gene between accessions (Col-0, Rsch, Te) and different sub-zero treatment durations (i.e. between CA and 1, 2, 3 or 8 h of sub-zero acclimation). P values were corrected for multiple testing using the Benjamin–Hochberg method (Benjamini and Hochberg 1995). A P value cutoff of 0.05 was used to identify genes as differ-entially expressed. Enrichment of differentially expressed TF genes in particular TF families was tested for signifi-cance by applying Fisher tests with a Bonferroni correction for multiple tests using CorTo (http://www.usadellab.org). Hierarchical clustering (HCL) of TF genes differentially expressed between accessions using a Euclidian distance measure and complete linkage was performed with the MultiExperiment Viewer software v.4.4 (Saeed et al. 2003). HCL was based on the log2 fold changes in relative gene expression between cold-acclimated and sub-zero accli-mated samples.

The raw ATH1 hybridization intensities were imported into the Robin software (Lohse et al. 2010) to perform

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quality assessment and data normalization and to identify genes, which were differentially expressed between cold acclimated samples and samples subjected to different durations of sub-zero acclimation. PCA indicated that two Col-0 replicates sub-zero acclimated for 8 h and 3 days, respectively (replicate 3 in both cases), were outliers (see Online Resource 1). Hence, these replicates were excluded from further analysis. Data normalization was performed using the RMA method. Statistical analysis of differential gene expression between cold acclimated and sub-zero acclimated samples was carried out using a linear model-based approach, applying a 0.05 P values and log2 fold ratio greater than 1 and lower than −1 cut-off. P values were corrected for multiple testing using the Benjamin–Hochberg method (Benjamini and Hochberg 1995). Differ-ences in gene expression were visualized using PageMan software (Usadel et al. 2006). Enrichment of functional categories of the MapMan annotation bins among signifi-cantly differentially expressed genes was tested by apply-ing Fisher tests with a Bonferroni correction for multiple testing using CorTo (http://www.usadellab.org). The over-representation analyses in MapMan and CorTo provided slightly different results. Therefore, minor differences between the results visualized using PageMan and the cor-responding supplementary tables can be found.

Results

Transcriptional regulation during sub-zero acclimation

High-throughput qRT-PCR experiments allowed us to determine the transcript levels of 1,880 genes encoding TFs in Arabidopsis. After filtering, expression values of 1,570 genes were included in further analyses (Online Resource 2). As an initial step in the data analysis, PCA was used to identify the largest variance components in the relative expression values in leaf samples from cold acclimated plants or sub-zero acclimated leaves treated at −3 °C for 1–8 h. The first principal component (PC 1) separated the samples taken after cold acclimation and different durations of sub-zero acclimation (56 % of total variance), while PC 2 (9 % of total variance) separated the three accessions Col-0, Rsch and Te (Fig. 1). The distribution of cold accli-mated and sub-zero acclimated samples in each accession followed an order from cold acclimated to 1, 2, 3 and 8 h of sub-zero acclimation, indicating that changes in TF gene expression during sub-zero acclimation followed a strictly controlled time-course in all three accessions.

During this time course the relative expression of only one, very lowly expressed, TF gene was significantly affected by an interaction between the duration of sub-zero acclimation and accession. This gene, encoding a

protein belonging to the NAC-family, was up-regulated in Rsch, slightly down-regulated in Te and showed hardly any changes in Col-0 (Fig. 2, Online Resource 2). The expres-sion of 11 TF genes differed significantly with both the duration of sub-zero acclimation and between accession. Nine genes were up-regulated and one was down-regulated in all three accessions in response to sub-zero tempera-ture, but the extent of change differed between the acces-sions. In addition, the change in expression of one gene could not be determined in Rsch, while it was only slightly regulated in the two other accessions (Fig. 2). 27 TF genes showed a significant time-dependent response to sub-zero temperature, the majority an up-regulation (Fig. 2, Online Resource 3). The largest group of significantly regulated TF genes (436 members) consisted of genes whose expres-sion differed significantly between the accessions without a consistent effect of sub-zero acclimation. Hierarchical clustering grouped these genes into 19 clusters of varying size, corresponding to different expression patterns (Fig. 3, Online Resource 4). In addition, hierarchical clustering val-idated the results of the two-way ANOVA, showing clear differential expression among the accessions by separating the samples into three clusters, corresponding to the three accessions. Among the accessions, Col-0 and Rsch were clustered closer together and within the accessions, time points 1, 2 and 3 h were separated from 8 h.

To identify TF families whose members were pre-sent more than expected by chance among significantly

Fig. 1 Score plots from principal components analysis (PCA) of the relative expression of 1,570 genes encoding TFs in leaves of the Arabidopsis thaliana accessions Col -0 (square), Rsch (circle) and Te (triangle). Plants were cold acclimated at 4 °C for 2 weeks (black). Detached leaves were then sub-zero acclimated at −3 °C for 1 h (red), 2 h (blue), 3 h (green) or 8 h (grey) at −3 °C

Fig. 2 Expression of TF genes significantly affected by duration of sub-zero acclimation or duration of sub-zero acclimation and acces-sion in Arabidopsis thaliana accessions Co-0, Rsch and Te. Plants were cold acclimated at 4 °C for 2 weeks. Detached leaves were then sub-zero acclimated at −3 °C for 1, 2, 3 or 8 h. The top panel shows relative gene expression (2−ΔCt), the bottom panel the log2 fold change in relative gene expression between the cold-acclimated and sub-zero acclimated leaves

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regulated genes, we performed an overrepresentation analysis. Such families could be key regulators of sub-zero acclimation. The analysis showed that there was an over-representation of the AP2/EREBP family among regu-lated TFs (Table 1). This family contributed six (including DREB2A) of the 11 TF genes that were significantly regu-lated in response to both the duration of sub-zero acclima-tion and accession, and ten (including CBF2) of the 27 TF genes that showed a time-dependent response to sub-zero temperature. The WRKY family and TFs related to ethyl-ene metabolism were also overrepresented among TFs up-regulated during sub-zero acclimation in all three acces-sions. Among the 436 TF genes with significantly different expression between accessions, the members of 26 differ-ent families were highly enriched (Table 1).

Effects of sub-zero acclimation on global transcript profiles

In addition to the targeted analysis of TF gene expression during the early phase of sub-zero acclimation (up to 8 h), we also measured global changes in gene expression during the later stage of acclimation (8 h to 3 days) using micro-arrays. PCA of this data set showed that the greatest vari-ance (22 % of total variance in PC 1) was explained by the difference between samples taken after cold acclimation and different durations of sub-zero acclimation. Between the accessions, it was clear that Col-0 was most unlike the

other two, as it was separated by PC 2 that explained about 16 % of the total variance (Fig. 4).

The number of regulated genes increased with increas-ing time at sub-zero temperature. After 3 days, the expres-sion of 1,396, 879 and 1,089 genes in Col-0, Rsch and Te, respectively (about 6, 4 and 5 % of the total genes), was significantly affected by sub-zero temperature (Table 2). Different durations of sub-zero acclimation affected tran-script levels in different ways. Among those genes show-ing differential expression after 8 h of sub-zero acclimation 229, 356 and 233 were up-regulated, while 36, 79 and 18 were down-regulated in Col-0, Rsch and Te, respectively, compared to their corresponding levels at 4 °C. After 1 day of sub-zero acclimation the numbers of induced and repressed genes increased further in all accessions, whereas after 3 days the number of up-regulated genes decreased slightly in Rsch and increased further in Col-0 and Te, while the number of down-regulated genes continued to increase in all three accessions. The ratios of induced to repressed genes indicated that there were more up-regu-lated than down-regulated genes in all accessions after 8 h, but that after 3 days at sub-zero temperature up-regulation still predominated in Rsch and Te, while the changes were balanced in Col-0. Also, the ratio after 8 h was about twice and three times as high in Te as in Col-0 and Rsch, indicat-ing differences in global gene regulation at −3 °C between the accessions.

Fig. 3 Hierarchical clustering (HCL) of TF genes differ-entially expressed between accessions. Hierarchical trees were drawn, based on log2 fold change in relative expression of 436 genes between the cold-acclimated and sub-zero acclimated leaves of Arabidop-sis thaliana accessions Col-0, Rsch and Te. Plants were cold acclimated at 4 °C for 2 weeks. Detached leaves were then sub-zero acclimated at −3 °C for 1, 2, 3 or 8 h

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The overlap between genes significantly up- or down-regulated during sub-zero acclimation at different time points within accessions is presented in Fig. 5. The total number of up-regulated genes was 775, 881 and 1,011, increasing from Te, to Rsch and then Col-0. The percentages of up-regulated genes that were up-regulated at all time points increased in the opposite way. They were 9.2 % in Col-0, 13.2 % in Rsch and 16.4 % in Te. A total of 32 genes were commonly up-regulated by sub-zero temperature at all time points and in all accessions (Table 3). Eight of the 32 genes encode proteins with a putative role in cell wall biosynthesis or the modifi-cation of cell wall structure, indicating that the modification of cell wall properties is a major answer of Arabidopsis to

sub-zero temperature. Interestingly, there was also an early-responsive to dehydration (ERD) protein-related gene up-regulated in all three accessions. The ERD genes belong to the CBF regulon and play a role in increasing freezing tol-erance during cold acclimation at above-zero temperatures (Thomashow 1999). Another four of the consistently up-regu-lated genes encode TFs that were also tested in the qRT-PCR experiment. On the TF platform, three of the four genes were also significantly up-regulated during the first 8 h of expo-sure of leaves to sub-zero temperature. These were the AP2/EREBP genes At1g71450 and At3g50260, and the MYB gene At3g06490, whose expression was significantly affected by the duration of sub-zero acclimation and accession

Table 1 Transcription factor families which were significantly enriched in regulated genes during sub-zero acclimation of Arabidopsis thaliana accessions Columbia-0, Rschew and Tenela

Plants were cold acclimated at 4 °C for 2 weeks. Detached leaves were then sub-zero acclimated at −3 °C for 1, 2, 3 or 8 h. The expression of members of the families differed significantly between the accessions, over the duration of sub-zero acclimation, or in both cases

Duration of sub-zero acclimation and accession

Duration of sub-zero acclimation Accession

27.3.3 APETALA2/ethylene-responsive element binding protein family

27.3.3 APETALA2/ethylene-respon-sive element binding protein family

27.3.25 MYB domain transcription factor family

27.3.32 WRKY domain transcription factor family

27.3.32 WRKY domain transcription factor family

17.5 Hormone metabolism ethylene 27.3.24 MADS box transcription factor family

27.3.11 C2H2 zinc finger family

27.3.6 bHLH, Basic Helix-Loop-Helix family

27.3.3 APETALA2/Ethylene-responsive element binding protein family

27.3.22 HB, Homeobox transcription factor family

27.3.9 C2C2(Zn) GATA transcription factor family

27.3.20 G2-like transcription factor family, GARP

27.3.37 AS2,Lateral Organ Boundaries Gene Family

27.3.8 C2C2(Zn) DOF zinc finger family

27.3.35 bZIP transcription factor family

27.3.26 MYB-related transcription factor family

27.3.41 B3 transcription factor family

27.3.29 TCP transcription factor family

27.3.40 Aux/IAA family

27.3.27 NAC domain transcription factor family

27.3.23 HSF,Heat-shock transcription factor family

27.3.80 zf-HD

27.3.30 Trihelix, Triple-Helix transcription factor family

27.3.18 E2F/DP transcription factor family

27.3.5 ARR

27.3.14 CCAAT box binding factor family, HAP2

16.5.1.2 Secondary metabolism sulfur-containing glucosinolates regulation

17.5 Hormone metabolism ethylene

17.5.2 Hormone metabolism ethylene signal transduction

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(At1g71450) or only the duration of sub-zero acclimation (At3g50260 and At3g06490). The fourth TF gene belongs to the DOF family and its expression only differed signifi-cantly among accessions during the first 8 h as determined by qRT-PCR.

The total number of genes down-regulated during sub-zero acclimation was 797, 526 and 489 in Col-0, Rsch and Te, respectively (Fig. 5). Since only a small number of genes were down-regulated after 8 h in all accessions, the percent-ages of down-regulated genes that were down-regulated at all time points only comprised 2.6, 5.5 and 2.2 % of the total number of down-regulated genes in Col-0, Rsch and Te, respectively. Only two genes, a RING membrane-anchor 2 encoding gene (At4g28270) and a SAUR-like auxin-respon-sive protein family gene (At3g12830), were commonly down-regulated by sub-zero temperatures in all accessions.

Differences in transcript profiles during sub-zero acclimation between accessions

The overlap between genes that were induced or repressed during sub-zero acclimation at different time points among accessions is presented in Fig. 6. In Col-0 and Te, the majority of genes up-regulated at specific time points were also significantly up-regulated at the same time points in at least one other accession. The percentages of specifically up-regulated genes were 14.8 and 10.3 % in Col-0 or Te, respectively, after 8 h of exposure to sub-zero temperature but increased to 27.4 and 19.8 % after 3 days. In contrast, in Rsch the proportion of up-regulated genes whose expres-sion was also significantly increased in at least one other accession increased with longer sub-zero acclimation time, while the percentage of specifically up-regulated genes decreased from 30.9 % after 8 h to 11.7 and 12.5 % after 1 and 3 days, respectively. Of the down-regulated genes, about 52.8, 69.6 and 22.2 % were specifically regulated in Col-0, Rsch and Te, respectively, after 8 h. The percent-ages of specifically regulated genes were reduced after 1 and 3 days of sub-zero acclimation in Col-0 and Rsch, but increased to on average 30 % in Te.

Functional analysis of genes responsive to sub-zero acclimation

The genes represented on the ATH1 arrays were assigned to >1,000 functional categories using the MapMan hierarchi-cal ontology. To find out if in a given class the number of genes with significantly changed expression was higher or lower than expected by chance, an overrepresentation analy-sis was used and the significant changes are shown in Fig. 7. A complete overview of all significantly regulated bins and sub-bins is given in Online Resource 5. This analysis reveals physiological processes that are predominantly regulated at the level of gene expression during exposure to sub-zero temperature. Interestingly, in all three accessions more func-tional classes had an overrepresentation of down-regulated genes after 3 days than at any earlier time point. This was especially true for the class of photosynthesis (PS), indicat-ing that this process is only slowly down-regulated during sub-zero acclimation. Major CHO metabolism was overrep-resented among down-regulated genes in Rsch and Te after 3 days, but not in Col-0. Also, down-regulated genes were overrepresented in the secondary metabolism class in Col-0 and Te after 3 days, whereas only down-regulation of flavo-noid metabolism genes was overrepresented in Rsch (Online Resource 5). There were no overrepresented functional classes for down-regulated genes in Te after 8 h of sub-zero acclimation, but there was one in Rsch (hormone metabo-lism) and two in Col-0 (PS and hormone metabolism). In contrast, after 1 day of sub-zero acclimation the number of

Fig. 4 Score plots from principal components analysis (PCA) of the microarray results of the Arabidopsis thaliana accessions Col-0 (square), Rsch (circle) and Te (triangle). Plants were cold acclimated at 4 °C for 2 weeks (black). Detached leaves were then sub-zero accli-mated at −3 °C for 8 h (red), 1 day (blue) or 3 days (green)

Table 2 The numbers of up- and down-regulated genes and the ratio between up- and down-regulated genes following 8 h, 1 or 3 days of exposure of cold acclimated leaves of Arabidopsis thaliana acces-sions Columbia-0, Rschew and Tenela to sub-zero temperature (−3 °C)

Accession Treatment duration

No of genes Ratio

Up-regu-lated

Down-regu-lated

Regulated

Columbia-0 8 h 229 36 265 6.36

1 day 602 276 878 2.18

3 days 704 692 1,396 1.02

Rschew 8 h 356 79 435 4.51

1 day 592 261 853 2.26

3 days 504 375 879 1.34

Tenela 8 h 233 18 251 12.94

1 day 508 166 674 3.06

3 days 632 457 1,089 1.38

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functional classes with an overrepresentation of down-regu-lated genes had decreased to one (hormone metabolism) in Col-0 and none in Rsch, but increased to three (PS, second-ary metabolism and stress) in Te.

With up-regulated genes, the functional classes of cell wall biosynthesis, hormone metabolism and RNA regulation of transcription showed overrepresentation in all accessions at most time points. Genes involved in signaling were over-represented in Col-0 after 3 days at −3 °C and in Rsch at all time points, but not in Te. The functional class of transport was overrepresented after 3 days of sub-zero acclimation in all accessions. Under-represented functional classes were mainly DNA and protein synthesis in all accessions. Interest-ingly, the functional class stress showed no overrepresenta-tion among up-regulated genes (Online Resource 5).

The TF genes which were overrepresented among up-regulated genes during sub-zero acclimation belonged to the PHOR1, WRKY and AP2/EREBP TF families. A closer look at the significantly up-regulated members of the WRKY and AP2/EREBP families revealed a consider-able overlap with the members of these families that were significantly regulated in response to the duration of sub-zero acclimation and accession or only the duration of sub-zero acclimation on the TF platform (Online Resource 6). Hence, to a large extent the same members of these families were significantly up-regulated in the short term (1–8 h, TF platform) and in the long term (8 h to 3 days, ATH1 array)

during exposure to sub-zero temperatures. Only in Tenela, TF genes belonging to the MYB-related and CCAAT TF families were overrepresented among down-regulated genes after 1 and 3 days of sub-zero acclimation.

When counting the genes in these bins and sub-bins (not counting not assigned genes), RNA regulation of transcrip-tion was the group that had the highest number of up- and down-regulated genes in all accessions (Online Resource 7 and 8). In this group, the number of up-regulated genes increased from 8 h to 1 day and stayed constant from 1 to 3 days in all three accessions, whereas the number of down-regulated genes constantly increased over time. However, the number of up-regulated TF genes was consistently higher than the number of down-regulated TF genes. Some other groups of genes tended to share the same changes in expression patterns, such as secondary metabolism and hormone metabolism. In these groups, the number of up-regulated genes was also higher and stayed higher at all time points than the number of down-regulated genes.

Discussion

The expression of a large number of TFs varied signifi-cantly between the three accessions. This variation was not related to the acclimation state of the plants, but rather reflected transcript-level variability among the accessions.

Fig. 5 Venn diagrams showing overlap between genes dif-ferentially regulated by 8 h, 1 or 3 days of sub-zero acclimation at −3 °C in leaves of Arabidop-sis thaliana accessions Col-0, Rsch and Te. Genes up-regu-lated (top) and down-regulated (bottom) are shown separately for each accession. Genes are shown whose expression level differed significantly (adjusted P < 0.05) from the expression in leaves of plants cold acclimated at 4 °C for 2 weeks

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Transcript-level variability under stress generally seems to be large even between phylogenetically close genotypes, regardless of similarities in their stress tolerance (Sanchez 2013; van Leeuwen et al. 2007). This is in agreement with the large number of single-nucleotide and other polymor-phisms in the genome sequences of different Arabidopsis accessions (Cao et al. 2011) that can be expected to lead to expression differences.

Our previous study of sub-zero acclimation in Arabi-dopsis indicated that Col-0 is not able to increase its freez-ing tolerance significantly in response to up to 5 days of exposure to −3 °C following cold acclimation at 4 °C. In contrast, leaves of Te, Rsch and C24 increased their freez-ing tolerance significantly under these conditions (Le et al. 2008). On the other hand, an independent study using regrowth instead of electrolyte leakage measurements to

determine freezing tolerance had shown clear evidence for sub-zero acclimation in Col-0 (Livingston et al. 2007). Subsequent repetition of the electrolyte leakage experiment with Col-0, Rsch and Te showed that leaves of Col-0 are indeed able to increase their freezing tolerance in response to sub-zero temperature (Online Resource 9). Hence, 1 day of exposure of cold acclimated leaves to −3 °C was suf-ficient to induce a significant increase in freezing tolerance in all three accessions and after 3 days freezing tolerance of leaves of Rsch and Te was further increased. The absolute sub-zero acclimation capacity (i.e. the difference between the LT50 after cold acclimation and after subsequent sub-zero acclimation), however, was lower in Col-0 than in Rsch and Te, indicating natural variation for sub-zero accli-mation capacity. Despite these differences, the similar phe-notypic responses of the three accessions and the limited

Table 3 Genes commonly up-regulated by exposure of cold acclimated leaves of Arabidopsis thaliana accessions Columbia-0, Rschew and Tenela to sub-zero temperature (−3 °C) for 8 h, 1 or 3 days

Genes highlighted in italics encode proteins with a putative role in cell wall biosynthesis or the modification of cell wall structure, while genes highlighted in bold encode transcription factors

AGI ID Annotation

AT5G09440 EXORDIUM like 4

AT5G16910 Cellulose-synthase like D2

AT5G62360 Plant invertase/pectin methylesterase inhibitor superfamily protein

AT5G50450 HCP-like superfamily protein with MYND-type zinc finger

AT5G49900 Beta-glucosidase, GBA2 type family protein

AT5G12880 proline-rich family protein

AT3G59350 Protein kinase superfamily protein

AT3G52450 Plant U-box 22

AT3G50260 ABI4

AT4G38420 SKU5 similar 9

AT4G30290 Xyloglucan endotransglucosylase/hydrolase 19

AT4G30280 Xyloglucan endotransglucosylase/hydrolase 18

AT4G22470 Protease inhibitor/seed storage/lipid transfer protein (LTP) family protein

AT4G22590; AT4G22592 [AT4G22590, Haloacid dehalogenase-like hydrolase (HAD) superfamily protein];[AT4G22592, conserved peptide upstream open reading frame 27]

AT1G51820 Leucine-rich repeat protein kinase family protein

AT2G34090 Maternal effect embryo arrest 18

ATMG01360 Cytochrome oxidase

AT3G15440 RING/U-box superfamily protein

AT3G15450 Aluminium induced protein with YGL and LRDR motifs

AT3G06490 Myb domain protein 108

AT3G04010 O-Glycosyl hydrolases family 17 protein

AT3G05500 Rubber elongation factor protein

AT1G69570 Dof-type zinc finger DNA-binding family protein

AT1G71450 Integrase-type DNA-binding superfamily protein

AT1G19370 Unknown protein (TAIR:AT1G75140.1)

AT1G32860 Glycosyl hydrolase superfamily protein

AT1G22570 Major facilitator superfamily protein

AT1G75860 Unknown protein (TAIR:AT1G20100.1)

AT1G11960 ERD (early-responsive to dehydration stress) family protein

AT2G17480 Seven transmembrane MLO family protein

AT2G27500 Glycosyl hydrolase superfamily protein

AT2G35290 Unknown protein

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number of genotypes necessitates a cautious interpreta-tion of transcriptional differences between accessions with respect to their differential sub-zero acclimation ability.

Collectively, our gene expression data indicate a tem-porally highly regulated transcriptional response of Arabi-dopsis to sub-zero temperatures. PCA analyses revealed that both for the expression of TFs during the early phase of sub-zero acclimation and the global changes in gene expression at the later time points the largest variance in the data sets was related to the time the leaves were kept at −3 °C.

We identified 704, 504 and 632 up-regulated and 692, 375 and 457 down-regulated genes in Col-0, Rsch and Te, respectively, after 3 days of sub-zero acclimation, suggest-ing that about 5 % of the Arabidopsis genome is responsive to sub-zero temperature following cold acclimation. This is less than the more than 2,500 regulated genes detected over time during cold acclimation (Hannah et al. 2005), but no fold-change cut-off was applied in this study, making a direct comparison difficult. All accessions showed more up—than down-regulated genes at all times of sub-zero acclimation, indicating that the increase in freezing toler-ance may largely depend on transcriptional activation.

During cold acclimation at 4 °C the functional groups of genes that were most highly up-regulated were secondary

Fig. 6 Venn diagrams showing overlap between genes differen-tially regulated in leaves of the Arabidopsis thaliana acces-sions Col-0, Rsch and Ten after 8 h, 1 or 3 days of exposure to sub-zero temperature (−3 °C). Genes up-regulated (top) and down-regulated (bottom) are shown separately at each time point. Genes are shown whose expression level differed sig-nificantly (adjusted P < 0.05) from the expression in leaves of plants cold acclimated at 4 °C for 2 weeks

Fig. 7 PageMan display of coordinated changes of gene function cat-egories during different durations of sub-zero acclimation of leaves of cold acclimated plants of the Arabidopsis thaliana accessions Col-0, Rsch and Te. Normalized gene expression values were subjected to an overrepresentation analysis to identify functional categories that contained significantly more or less regulated genes than expected by chance. Blue color indicates significant enrichment of up- or down-regulated genes, red indicates significant depletion

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metabolism, transport and primary metabolism. On the other hand, genes in the functional groups photosynthe-sis, lipid metabolism, nucleotide metabolism, hormone and redox were mostly down-regulated (Hannah et al. 2005, 2006). After a shift from 4 °C to −3 °C, the signifi-cant overrepresentation of transport among up-regulated transcripts and of photosynthesis among down-regulated transcripts persisted in all accessions. Down-regulation of genes encoding components of the photosynthetic appa-ratus is in line with observations in sub-zero acclimating wheat (Herman et al. 2006). This down-regulation of pho-tosynthesis-related genes may also be due to experiments being performed in the dark. Evidence for the activation of senescence-associated pathways comes from the up-reg-ulation of WRKY22 in all accessions at one or more time points both in the qRT-PCR and microarray experimets. WRKY22 participates in a dark-induced leaf senescence signal transduction pathway (Zhou et al. 2011b).

Transcripts of secondary metabolism were overrepre-sented among up-regulated genes after 1 day of sub-zero acclimation, but overrepresented among down-regulated genes in all accessions after 3 days, indicating a fundamen-tal shift in secondary metabolism in the long term. Also, the overrepresentation of the functional group of RNA regulation of transcription was high after short-term expo-sure to sub-zero temperature, but was reduced after longer times. For Rsch and Te there was no overrepresentation of this functional class anymore after 3 days. Similarly, there were several other functional classes of genes that only showed over- or underrepresentation among up- or down-regulated transcripts either at early or at late time points of sub-zero acclimation. These data strongly indicate that the observed changes in gene expression are not primarily the result of dark-induced senescence, but are in fact part of a highly regulated, specific temperature response that leads to increased freezing tolerance.

It is noteworthy that in the microarray experiments RNA regulation of transcription was identified as the functional group that had the highest number of up- and down-regu-lated genes in all accessions, highlighting the extensive transcriptional regulation during sub-zero acclimation. However, hardly anything is known about transcriptional regulation of sub-zero-responsive genes and we there-fore used a dedicated qRT-PCR platform to identify sig-nal transduction pathways and candidate TFs that may be involved in sub-zero acclimation. The expression of 39 TF genes was significantly related to the time the leaves were kept at −3 °C, with the vast majority being up-regulated. Ten of the 11 TF genes whose expression differed signifi-cantly with both the duration of sub-zero acclimation and accession were differentially expressed in the same direc-tion in all accessions, indicating that the transcriptional changes of these TFs are generally important for increased

freezing tolerance in Arabidopsis at sub-zero temperatures. It is noteworthy that no components of the circadian clock are among the regulated TF genes. In time-courses such as those investigated here, it can be difficult to distinguish treatment effects from circadian regulation and often clock-related genes are among the apparently treatment-regulated genes. However, prior to sub-zero acclimation, the plants were cold acclimated for 14 days at 4 °C and this low-tem-perature treatment already leads to clock arrest (Bieniaw-ska et al. 2008; Espinoza et al. 2010). It seems reasonable to assume that at an even lower temperature and in the absence of a diurnal light–dark cycle the clock will remain in its arrested state and will therefore not have an influence on gene expression patterns.

The AP2/EREBP and WRKY families were overrep-resented among up-regulated TFs, indicating that these families play a key role in orchestrating the transcriptional changes observed during sub-zero acclimation. They were also overrepresented among regulated TFs on the ATH1 array, highlighting their functional importance both in the short and in the long term. The importance of members of the AP2/EREBP family in cold acclimation is well estab-lished (Medina et al. 2011; Thomashow 2010) and the pre-sent data suggest it may extend to sub-zero acclimation. The WRKY gene family also plays important roles in the regulation of transcriptional reprogramming associated with several plant stress responses, but is not known to play a crucial role in low temperature stress (Chen et al. 2012). In winter wheat, however, several WRKY TFs were sig-nificantly up-regulated when cold acclimated plants were slowly frozen to −10 °C (Skinner 2009), lending support to the proposition that members of the WRKY family are spe-cifically involved in transcriptional regulation at sub-zero temperatures.

Members of the AP2/EREBP family whose expression was found to be significantly up-regulated in response to either duration of sub-zero acclimation and accession or only duration using the qRT-PCR platform included, among others, CBF2, DDF1, DDF2, CBF4, ABI4 and DREB2A. Except for CBF2, these genes were addition-ally up-regulated in two or all accessions at one or more time points on the ATH1 array. CBF1–4 and DFF1–2 are closely related DREB1 genes that belong to the same sub-family of AP2/EREBP (Licausi et al. 2013). DREB1 and DREB2 specifically interact with a cis-acting CRT/DRE element, which is present in one or more copies in the pro-moters of many cold-inducible genes, but only CBF1–3 and DDF1 have a confirmed function in cold-responsive gene expression (Maruyama et al. 2009). CBF1–3 were all induced during the early phase of sub-zero acclimation, but the effect of time on gene expression was only significant for CBF2, while expression of CBF1 and CBF3 was only slightly above the significance threshold. On the ATH1

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array CBF1 was significantly up-regulated in all accessions after 8 h of sub-zero acclimation. Similar to our previous study (Le et al. 2008), the magnitude of the induction of the three genes was considerably lower during sub-zero accli-mation (approximately log2 2–4; i.e. 8- to 16-fold) than after a shift of non-acclimated plants to acclimation con-ditions (approximately 50 to 250-fold (Bieniawska et al. 2008; Vogel et al. 2005)). This may suggest that the three CBF genes share similar induction patterns under sub-zero conditions. Alternatively, the non-significant time depend-ence of the response of CBF1 and CBF3 may suggest a dif-ferent function of CBF2 than CBF1 and CBF3 at sub-zero temperature. Current models suggest a partially different regulation of CBF2 in comparison to CBF1 and CBF3 at above freezing temperatures (Medina et al. 2011; Thom-ashow 2010).

On the ATH1 array, RAP2.1 and RAP2.6, which also belong to the AP2/EREBP family, were significantly up-regulated in most accessions at the later time points, well after the induction of CBF1–3. RAP2.1 and RAP 2.6 are activated by the CBF TFs and thus presumably control parts of the CBF regulon (Fowler and Thomashow 2002). DDF1 is up-regulated in response to low temperature, drought, salt and heat in Arabidopsis, indicating that it is involved in regulating responses to several abiotic stresses (Kang et al. 2011). Less is known about DDF2, but a recent experiment showed that DDF2 expression is induced by salinity stress (Hong et al. 2013).

CBF4 transcripts accumulate in response to dehydration and abscisic acid (ABA) treatment (Haake et al. 2002) and the up-regulation of CBF4 therefore suggests that ABA is involved in mediating sub-zero acclimation. ABA plays a role in the cold acclimation process as well, via an ABA-dependent signal transduction pathway, which may inter-act with the CBF pathway (Chinnusamy et al. 2007; Hua 2009; Shinozaki et al. 2003; van Buskirk and Thomashow 2006). This ABA-dependent induction of cold-regulated genes may be mediated by CBF4, although ABA also induces CBF1–3 transcription (Knight et al. 2004). Another line of evidence indicating that ABA is involved in sub-zero signalling is the up-regulation of ABI4, WRKY40 and MYB108. ABI4 is a positive regulator of ABA-responses, while WRKY40 is a negative regulator of ABI4 and ABI5 (Liu et al. 2012) and MYB108 acts as a negative regulator of ABA biosynthesis (Cui et al. 2013).

The functional class ethylene metabolism was also sig-nificantly overrepresented among TFs whose expression changed with time at −3 °C. Although responsiveness to ethylene is not a universal feature of the AP2/EREBP pro-tein superfamily (Licausi et al. 2013), it is likely that the overrepresentation of ethylene metabolism is due to AP2/EREBP being the most significantly overrepresented TF family.

Interestingly, a number of members of the MYB and C2H2 zinc finger families, whose expression differed sig-nificantly with duration of sub-zero acclimation and acces-sion or only duration of sub-zero acclimation, are known to function in the CBF-dependent signaling pathway at above freezing temperatures. MYB15 can interact with ICE1, an upstream regulator of CBF3 and bind to the MYB recog-nition sequences in the promoters of CBFs, thereby func-tioning as a negative regulator of CBF expression (Agar-wal et al. 2006). ZAT12 controls a small regulon of genes involved in freezing tolerance and functions as a negative regulator of CBF2 expression (Vogel et al. 2005), while ZAT10 is part of the CBF regulon and itself a negative reg-ulator of the CBF-target gene RD29A (Zhou et al. 2011a). The up-regulation of several TFs associated with the CBF cold response pathway indicates that this extensive signal transduction network not only contributes to cold acclima-tion, but also to the added freezing tolerance gained during sub-zero acclimation.

Several reports suggest that different hormones may be directly involved in regulating the expression of cold-stress related genes, as well as being central in modulating plant growth and development under low temperature (Hannah et al. 2006; Hu et al. 2013; Kim et al. 2013). Jasmonate metabolism was highly overrepresented among up-reg-ulated genes in all accessions at one or more time points in the microarray experiments. Functionally, jasmonate positively regulates the CBF pathway in Arabidopsis (Hu et al. 2013) through JASMONATE ZIM-DOMAIN (JAZ) proteins, which physically interact with ICE1 and ICE2, thereby attenuating the expression of their regulons. ICE2 is the homolog of ICE1 influencing the expression of CBF1. JAZ9 was up-regulated under sub-zero temperature in leaves of all accessions and encodes one of the JAZ pro-teins that strongly interact with both ICE1 and ICE2. How-ever, neither JAZ7 nor JAZ8, whose transcript abundances also increased in sub-zero acclimating leaves, interact with ICE1 or ICE2.

Auxin metabolism was overrepresented among down-regulated genes in Col-0 and to a lesser extent Rsch during sub-zero acclimation. Auxin-induced genes are also over-represented among down-regulated genes in cold accli-mating plants. This is likely related to decreased growth in response to low temperature (Hannah et al. 2006).

Although there were many down-regulated genes during sub-zero acclimation, only two genes were down-regulated in all accessions and all time points. They encode a RING finger E3 ubiquitin ligase and an auxin-responsive family protein. On the other hand, several of the consistently up-regulated genes encode proteins with a putative role in cell wall biosynthesis or the modification of cell wall structure. This is in accordance with the overrepresentation of cell wall precursor synthesis and cell wall modification among

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up-regulated transcripts in our study and increased amounts of enzymes involved in cell wall modification in addition to structural cellular changes indicating increased deposition of xyloglucan material in the cell wall under sub-zero tem-peratures in wheat (Herman et al. 2006).

In conclusion, especially the TFs whose expression changed significantly over time in sub-zero acclimating plants are interesting candidates for further functional test-ing through forward and reverse genetic approaches. Also, determining which of the identified transcriptional changes are critical to the acquisition of additional freezing toler-ance during sub-zero acclimation is essential to increase our understanding of how plants obtain maximum freezing tolerance. This is not only of obvious scientific interest but also important with regard to winter survival of plants in both natural and agricultural environments.

Acknowledgments MQL was supported by a PhD fellowship from the Vietnamese Ministry of Education and Training and MP by a Post-doctoral fellowship from the Carlsberg Foundation (Denmark).

Conflict of interest The authors declare that they have no conflict of interest.

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