SEARCH

SEARCH BY CITATION

Keywords:

  • Arabidopsis;
  • cold acclimation;
  • CBF/DREB1;
  • freezing tolerance;
  • low temperature transcriptome;
  • ZAT12

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

The CBF cold response pathway has a prominent role in cold acclimation. The pathway includes action of three transcription factors, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), that are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert to increase plant-freezing tolerance. The results of transcriptome profiling and mutagenesis experiments, however, indicate that additional cold response pathways exist and may have important roles in life at low temperature. To further understand the roles that the CBF proteins play in configuring the low temperature transcriptome and to identify additional transcription factors with roles in cold acclimation, we used the Affymetrix GeneChip® containing probe sets for approximately 24 000 Arabidopsis genes to define a core set of cold-responsive genes and to determine which genes were targets of CBF2 and 6 other transcription factors that appeared to be coordinately regulated with CBF2. A total of 514 genes were placed in the core set of cold-responsive genes, 302 of which were upregulated and 212 downregulated. Hierarchical clustering and bioinformatic analysis indicated that the 514 cold-responsive transcripts could be assigned to one of seven distinct expression classes and identified multiple potential novel cis-acting cold-regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were assigned to the CBF2 regulon. An additional nine cold-induced genes and 15 cold-repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-induced genes that were most highly upregulated (induced over 15-fold), 19 genes (84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2 and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to the CBF and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12 regulon in cold acclimation. In addition, ZAT12 downregulated the expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Many plants increase in freezing tolerance in response to low non-freezing temperature, a phenomenon known as ‘cold acclimation’ (Smallwood and Bowles, 2002; Thomashow, 1999). Cold acclimation in Arabidopsis involves action of the CBF cold response pathway (Thomashow, 2001). The pathway includes the CBF1, CBF2, and CBF3 genes (Gilmour et al., 1998; Jaglo et al., 2001; Medina et al., 1999), also known as DREB1b, DREB1c, and DREB1a, respectively (Liu et al., 1998), which encode transcriptional activators that bind to the C-repeat (CRT)/dehydration response element (DRE) regulatory element present in the promoters of COR and other cold-responsive genes (Baker et al., 1994; Gilmour et al., 1998; Stockinger et al., 1997; Yamaguchi-Shinozaki and Shinozaki, 1994). Transcripts for CBF1, 2, and 3 accumulate rapidly (within 15 min) upon exposing plants to low temperature followed by induction of the CBF-targeted genes known as the CBF regulon. Constitutive expression of the CBF genes results in constitutive expression of the CBF regulon and increased freezing tolerance without a low-temperature stimulus (Gilmour et al., 2000, 2004; Jaglo-Ottosen et al., 1998; Liu et al., 1998). The freezing tolerance conferred by the CBF regulon involves the production of cryoprotective polypeptides such as COR15a (Artus et al., 1996; Steponkus et al., 1998) and the accumulation of compatible solutes such as sucrose, raffinose, and proline (Gilmour et al., 2000, 2004; Nanjo et al., 1999).

The CBF cold response pathway is currently the best understood genetic system with a role in cold acclimation. However, it does not appear to be the sole pathway with a role in freezing tolerance. The eskimo1 mutant of Arabidopsis described by Xin and Browse (1998) is constitutively more freezing tolerant than wild-type plants, but the COR genes are not constitutively expressed indicating that the mutation activated a freezing tolerance pathway outside of the CBF system. Similarly, ada2 mutants of Arabidopsis (ADA2 encodes a transcriptional adaptor protein) are constitutively more freezing tolerant than wild-type plants, but COR genes are not constitutively induced suggesting that the ADA2 protein is involved in inhibiting expression of a freezing tolerance pathway that is distinct from the CBF cold response pathway (Vlachonasios et al., 2003).

To more fully understand the role of the CBF cold response pathway in cold acclimation, investigators have examined the changes that occur in the Arabidopsis transcriptome in response to low temperature and overexpression of the CBF transcription factors. Fowler and Thomashow (2002) surveyed the expression of about 8000 Arabidopsis genes in response to low temperature. The results indicated that extensive changes occur in the transcriptome during cold acclimation. In particular, 306 (approximately 4%) genes were found to be either up- or down-regulated at least threefold in response to low temperature. However, only 12% of these genes could be assigned to the CBF regulon (i.e., were both cold- and CBF-responsive); at least 28% of the cold-responsive genes were not affected by expression of the CBF transcription factors, including 15 encoding known or putative transcription factors. Thus, it was concluded that cold acclimation is associated with the activation of multiple low temperature regulatory pathways. Similar conclusions have been reached by others studying the Arabidopsis low temperature transcriptome (Kreps et al., 2002; Seki et al., 2001, 2002).

Here we further explored the regulation of the low temperature transcriptome of Arabidopsis. Using the Affymetrix GeneChip® containing probe sets for approximately 24 000 genes, we defined a core set of cold-responsive genes and determined whether they were targets of CBF2 or six other transcription factors that appeared to be coordinately regulated with CBF2. We conclude that the majority of genes that are most highly induced in response to low temperature are part of the CBF2 regulon; that the ZAT12 transcription factor participates in the induction and repression of cold-responsive genes; and that the ZAT12 regulon contributes to an increase in freezing tolerance. The results also indicate that certain cold-responsive genes are members of both the ZAT12 and CBF2 regulons and that ZAT12 has a role in a negative regulatory circuit that dampens expression of the CBF cold response pathway.

Identification of a core set of cold-responsive genes

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Our first objective was to identify a core set of cold-responsive genes that could be used to further our understanding of the low temperature regulons and regulatory networks of Arabidopsis. Our intention was not to identify all genes that were cold-responsive, but to identify a set of genes that were reproducibly cold-responsive using two common laboratory conditions, plants grown on soil in pots and plants grown on plates containing solid culture medium. This was accomplished using the Arabidopsis Affymetrix GeneChip® ATH1 array, which contains probes for approximately 24 000 genes, to compare the transcriptomes of plants grown in soil and on plates at ‘warm’ temperatures (22°C soil, 24°C plates) with those of plants that had been transferred to low temperature (4°C) for 1 h, 24 h, and 7 days. In the plate experiments both root and shoot tissue was harvested, while in the soil experiments, shoot tissue was harvested. Pooled RNA from multiple samples at each time point was labeled and hybridized to the arrays. A probe set was designated as being upregulated if, in both biological samples for a given time point, the detection algorithm assigned a call of ‘present’ in the cold-treated sample, the change algorithm assigned a call of ‘increased,’ and the signal log ratio was greater than or equal to 1.3. A probe set was designated as being downregulated if, in both biological samples for a given time point, the detection algorithm assigned a call of ‘present’ in the warm sample, the change algorithm assigned a call of ‘decreased,’ and the signal log ratio was less than or equal to −1.3. The signal log ratio cut-offs used corresponded to approximately a 2.5-fold change.

Using these criteria, 1295 probe sets were cold-responsive in the plate experiments and 938 probe sets were cold-responsive in the soil experiments (the complete ‘raw’ data sets for these experiments are posted at the TAIR website ‘http://www.arabidopsis.org’ and the cold-responsive probe sets are listed in Tables S1–S4). In the plate experiments, 673 probe sets were upregulated, 627 were downregulated and five were both up- and down-regulated at different times in the experiment. In the soil experiments, 557 probe sets were upregulated, 382 were downregulated and one was both up- and down-regulated at different times in the experiment. A comparison of the probe sets that were cold-responsive in the plate and soil experiments indicated that 302 were upregulated in both experiments and that 212 were downregulated in both experiments (Figure 1a; Tables S5 and S6), with the largest number of changes occurring at 24 h (Figure 1b). These changes in gene expression were statistically significant at P < 0.05, with 95% (490/514) statistically significant at P < 0.005 (see Experimental procedures). Many of the probe sets that were categorized as not being responsive in both soil- and plate-grown plants showed a corresponding increase or decrease in both experiments, but did not meet the strict criteria used for designating cold-responsiveness in both experiments. Some probe sets, however, were only responsive in either the soil or plate experiments. The reason for these differences is currently unknown, but is likely to reflect, in part, the differences in the culture conditions and tissues harvested in the experiments. Exploring these differences will be the subject of future study. The goal accomplished here was the identification of 514 probe sets that were cold-responsive in four experiments using two different culture conditions; that is, the identification of a robust set of cold-responsive genes that could be used in deciphering the low temperature regulatory network of Arabidopsis. We refer to these genes as a ‘cold standard’ (COS) set of cold-responsive genes.

image

Figure 1. Low temperature-responsive probe sets. (a) Probe sets differentially regulated (2.5-fold cutoff) at low temperature in plants grown in soil (soil) and on solid medium (plates). (b) Number of probe sets with altered accumulation at 1, 24, and 168 h after transfer to low temperature in both soil and solid medium.

Download figure to PowerPoint

Members of the COS gene set can be assigned to one of seven expression clusters

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

The 514 COS genes were subjected to hierarchical clustering based on their relative transcript levels in plants after 1 h, 24 h, and 7 days of cold treatment (Figure 2a). Each gene was assigned to one of seven expression clusters (Figure 2b). Expression clusters I and II comprised downregulated transcripts and clusters III–VII comprised upregulated transcripts. The major difference between clusters I and II was that the transcripts in the former cluster continued to decrease in levels between 24 h and 7 days whereas those in cluster II showed some recovery over this time interval. A major difference between the upregulated transcripts in clusters III and IV, compared with those in clusters V, VI, and VII, was that those in the former two clusters were delayed in response; that is, there was little increase in the levels of the transcripts in clusters III and IV after 1 h of cold treatment whereas there was an increase for the transcripts in clusters V, VI, and VII. In addition, a distinguishing feature of cluster V was that the cold-response for these transcripts was transient in nature; transcript levels increased dramatically by 1 h, but returned to near-‘un-stressed’ levels by 24 h.

image

Figure 2. Hierarchical cluster and expression profiles of the 514 COS genes. (a) Hierarchical cluster analysis of the 514 COS genes. Log of ratio values for each probe set responding to low temperature at 1, 24, and 168 h was used to generate the cluster (see Experimental procedures). Downregulated probe sets (n = 212, green) consisted of two sub-clusters (yellow and orange side bars). Upregulated probe sets (n = 302, red) comprised five sub-clusters (red, blue, green, navy, and light blue side bars). (b) Expression profiles for the genes in each cluster are presented in a graph format. The graphs plot log of ratio values over time (h).

Download figure to PowerPoint

The clustering of genes with similar expression patterns raises the possibility of using bioinformatic approaches to identify potential cis-acting regulatory elements involved in coordinate gene regulation (reviewed in Rombauts et al., 2003). In this regard, MotifSampler (Thijs et al., 2002) was used to search for 8 bp sequences that were significantly overrepresented in the promoter regions of the COS genes that comprised the different expression clusters. Those elements that were enriched fivefold or more (when compared with all genes on the array) are listed in Table 1. No potential cold-regulatory elements were identified for the genes in clusters I and II, the cold-repressed genes, but many were identified for the upregulated genes in clusters III–VII. Most of these potential regulatory sequences were novel. However, an analysis of the genes comprising cluster III led to the identification of the motif krCCGAC which contains the CRT/DRE element, A/GCCGAC, the element to which the CBF transcriptional activators bind (Sakuma et al., 2002). The sequence A/GCCGAC was present within 1 kb upstream of 123 (53%) of the 233 genes included in the cluster (as shown below, most of the genes assigned to the CBF regulon were members of this cluster). In addition, a potential jasmonic acid-responsive element along with three other novel elements, having in common the sequence CGCGT, were enriched in cluster V.

Table 1.  Potential low temperature regulatory elementsa
ClassMotifPlantCAREClass (%)GeneChip (%)ClassMotifPlantCAREClass (%)GeneChip (%)
  1. aAll 8 bp motifs overrepresented in the promoters of a given class of transcripts are shown. The motifs were queried to PlantCARE (http://oberon.fvms.ugent.be:8080/PlantCARE/index.html) to determine whether they were similar to any known cis-element. The percentage of promoters in each class containing the motifs and for all transcripts on the GeneChip was calculated.

Class INone   CBF2 and cold upACCGACrTCRT/DRE281
Class IINone   krCCGACCRT/DRE606
Class IIIkrCCGACCRT/DRE336rCCGACnTCRT/DRE554
Class IVCAATGAGGNovel230.8GACCGACCRT/DRE242
GTGATCACNovel80.4GCCGACCRT/DRE405
GnATTGACNovel394GyCGrCCRT/DRE4710
TGTATACANovel232CCGACrTCRT/DRE444
Class VAGCGCGTGNovel160.9CBF2 and cold downAGnCGnCTNovel403
mrCGCGTGNovel222CACmACACNovel254
mrCGCGTkNovel253CAAGTTGrNovel504
AGCCGmCCNovel90.5CGAwCyAGNovel252
ACCGmGTNovel223CTTTGCCTNovel252
rCCGCsTJERE254TTCnGAGTNovel505
Class VIGAGAArGGNovel254Zat12 and cold upTCsnCTCsNovel509
CTCTCACTNovel383GCATTGACNovel440.8
kTrGCTGTNovel504yCTCTTCANovel446
yTkCCTCTNovel6312CAATGmkGUnnamed443
AGkyTACGNovel382TGAGGTCANovel220.7
yAnCTTCCNovel6311rsAATGAGNovel566
Class VIIGsCTCGTGNovel130.8Zat12 and cold downmCAACTTsNovel568
rAGnCGACNovel386AwGAkGwCOCS6711
AACGCGTTNovel130.5ACAwmTTCNovel6712
GCTTCGTCNovel61TATCCAAANovel405
GCyTCGTCNovel61GAmTAAGANovel335
CArAGsCkNovel386TAyGCCAGNovel130.4

The CBF2 regulon comprises 93 COS genes

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Previous transcriptome profiling experiments that surveyed about one-third of the Arabidopsis transcriptome indicated that: (i) the CBF transcription factors controlled expression of about 30 cold-regulated genes (Fowler and Thomashow, 2002; Seki et al., 2001); and (ii) that there were no obvious differences in the genes that were affected by overexpression of either CBF1, 2, or 3 (Gilmour et al., 2004). Here we extended these previous findings by profiling two independent transgenic lines overexpressing CBF2 using the ‘full genome’ ATH1 Affymetrix array. Probe sets were designated CBF2-upregulated if, in both biological samples, the detection algorithm assigned a call of ‘present’ in the transgenic line, the change algorithm assigned a call of ‘increased,’ and the signal log ratio was greater than or equal to 1.3. A probe set was labeled as downregulated by CBF2 if, in both biological samples, the detection algorithm assigned a call of ‘present’ in the wild type sample, the change algorithm assigned a call of ‘decreased,’ and the signal log ratio was less than or equal to −1.3.

Of the 302 COS genes that were upregulated in response to low temperature, 85 were also upregulated in response to CBF2 expression at control temperature (Table S7). These genes were assigned to the CBF regulon. Analysis of the promoter regions of these genes using MotifSampler identified six versions of the CRT/DRE element (Table 1). Alignment of all six motifs revealed a consensus of A/GCCGACnT. Additionally, when the sequence 1 kb upstream of these genes was examined for longer motifs (n = 10), the most conserved sequence in the CBF regulon was found to be krCCGACnTm. Further analysis indicated that of these 85 genes, 68 (80%) had at least one potential CRT/DRE element (A/GCCGAC) within 1 kb upstream of the start of the protein coding sequence and thus, were likely to be direct targets of the CBF transcription factors. All but three of these 68 genes fell into expression cluster III (Figure 3), the expression cluster for which the bioinformatic analysis described above identified the CRT/DRE element. The 17 genes that were both cold- and CBF-regulated, but did not have potential CRT/DRE elements within 1 kb of the start of the coding sequence, might have active CRT/DRE elements elsewhere within or surrounding the gene coding sequence or might have been targets of transcription factors that were upregulated in response to CBF2; such factors include RAP2.1 (At1g46768) (Okamuro et al., 1997), a putative zinc finger (At5g04340), a CONSTANS B-box zinc finger (At2g47890), and a protein related to squamosa promoter-binding protein (At1g76580).

image

Figure 3. Expression profiles of the COS genes and those that comprise the CBF2 and ZAT12 regulons. The expression profiles for all of the COS genes are presented in the panel labeled ‘Cold.’ In the panels labeled CBF2 and ZAT12 are the cold-regulation profiles for those genes that comprise the CBF2 and ZAT12 regulons, respectively. In each graph, log of ratio values are plotted versus time (h).

Download figure to PowerPoint

Of the 212 genes that were downregulated in response to low temperature, eight (4%) were downregulated in response to CBF2 overexpression (Table S8). These genes, which belong to expression cluster II (Figure 3), were also assigned to the CBF regulon. The promoter regions of these genes did not have potential CRT/DRE elements, but an analysis using MotifSampler identified a number of potential novel elements (Table 2). We conclude that these genes are not likely to be direct targets of the CBF transcription factors, but could be repressed by the expression of genes that are members of the CBF regulon.

Table 2.  Chi-square analysis of expected and observed changes in gene expression in response to low temperature and transcription factor overexpression
TransgenicTotal changesa Total possibleb Expectedc ObserveddChi-square P-value
UpDownUpDownUpDownUpDownUpDown
  1. aThe number of probe sets up- or down-regulated by expression of each transcription factor (see Experimental procedures).

  2. bThe number of probe sets that could possibly be up- or down-regulated by each transcription factor. Every probe set on the GeneChip had an equal probability of being upregulated in a transgenic line, but only those probe sets ‘present’ in the control samples could be downregulated and this number fluctuated between the various controls.

  3. cThe number of probe sets expected to be regulated by a transcription factor and the cold (see Experimental procedures).

  4. dThe actual number of probe sets that were both cold- and transcription factor-regulated.

CBF21514322 74612 72620.7858<0.0001<0.0001
ZAT124715822 74613 3200.62.5915<0.0001<0.0001
RAV1836322 74614 8841.10.9100.70.7
STZ581122 74613 9220.80.2200.45
MYB7328222 74614 1980.40000.9n/a
CZF2362422 74614 1980.50.41110.9

The effects of CBF2 overexpression were not limited to cold-responsive genes. Transcripts for 66 cold-non-responsive genes were upregulated and 35 were downregulated by CBF2 overexpression. Assignment of many of these genes to the cold-non-responsive class, however, was due to the strict criteria used to designate a gene as being cold-responsive. In fact, many of these transcripts were increased or decreased in the cold, but not to the same level used for selecting the COS transcripts. There were, however, some CBF2-regulated transcripts that showed no sign of cold-responsiveness. The altered expression of these genes likely resulted from the secondary effects that CBF2 overexpression has on plant growth and development, including stunted growth and a delay in flowering (Gilmour et al., 1998; Liu et al., 1998). A question thus raised was whether it was valid to assign a gene to the CBF regulon based on the criterion of the gene being both cold- and CBF2-responsive. Statistical analysis supported such assignments. In particular, the transcript levels for 151 genes were upregulated and 43 genes downregulated in the CBF2 overexpressing plants which corresponded, respectively, to 0.66 and 0.34% of the total genes assayed on the array. Thus, by chance, one would expect two of the 302 cold-induced COS genes to be upregulated in response to CBF2 overexpression and less than one of the 212 cold-repressed COS genes to be downregulated in response to low temperature (Table 2). Instead, 85 of the cold-induced genes and eight of the cold-repressed genes were up- and down-regulated, respectively, in response to CBF2 overexpression (Table 2). The large differences between expected and observed genes argued strongly against the hypothesis that the observed regulation of the COS genes by both cold and CBF2 was the result of chance (P < 0.0001).

CBF2 regulates a majority of highly induced COS genes

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

The results presented above indicated that the CBF regulon included a significant portion (28%) of the cold-induced COS genes. Further analysis indicated that of the genes that were most highly induced in response to low temperature, the large majority were members of the CBF regulon. Shown in Figure 4 is a ranking of the 302 cold-induced transcripts ordered from the most highly induced to the least highly induced based on the average fold-change at 24 h. All 13 transcripts that increased 25-fold or more in response to low temperature were induced in response to CBF2 expression. Of those transcripts that increased more than 15-fold, 84% increased in response to CBF2 expression. In contrast, about 90% of the transcripts that accumulated less than fivefold in response to low temperature fell outside of the CBF regulon.

image

Figure 4. CBF2 and ZAT12 induce a majority of the most highly cold-induced transcripts. (a) Cold-upregulated transcripts were ranked by average fold-change at 24 h and colored red if they were responsive to CBF2, green if responsive to ZAT12, and blue if responsive to both CBF2 and ZAT12. (b) Cold-upregulated transcripts were placed into classes defined by fold-change and the percentage of transcripts responsive to CBF2, ZAT12, both, or neither (not assigned) are indicated. The number of transcripts in each fold-change class is indicated above each bar.

Download figure to PowerPoint

Candidate transcription factors for regulating COS gene expression

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

The experiments described above indicated that 70% of the cold-induced COS genes, and more than 95% of the cold-repressed COS genes, remained unassigned to a regulon. The challenge was to determine which of the more than 1700 transcription factors encoded by the Arabidopsis genome (Riechmann, 2002) might have roles in controlling expression of these genes. We hypothesized that transcription factors that were coordinately regulated with CBF2 might be involved in regulating expression of these genes. Genes encoding transcription factors that were candidates for being coordinately regulated with CBF2 included six that were previously identified as being upregulated within 1 h of transferring plants to low temperature (Fowler and Thomashow, 2002). These genes were ZAT12, RAV1, MYB73, STZ/ZAT10, and two genes that we designated CZF1 (cold induced zinc finger proteins) (At2g40140) and CZF2 (At5g04340) [CZF2 is likely to correspond to ZAT6 (Meissner and Michael, 1997) designated from a partial sequence of the gene]. Northern analysis confirmed that transcripts for these six genes increased within 1 h of exposing plants to low temperature (Figure 5a). Gene fusion experiments indicated that the promoter regions (1 kb upstream of the ATG) of each of these genes, with the exception (for unknown reasons) of RAV1, were responsive to low temperature (D.G. Zarka, J.T. Vogel, H.A. Van Buskirk and M.F. Thomashow, unpublished data).

image

Figure 5. Expression of transcription factors that are candidates for configuring the low temperature transcriptome. RNA was isolated from plants that were exposed to low temperature, mechanical agitation or treatment with cycloheximide for various times and the transcript levels for the indicated genes were determined by RNA blot hybridization. eIF4a was used as a loading control on each blot and a representative blot is shown. (a) Transcript accumulation in response to low temperature. Plants were grown at 24°C and then exposed to 4°C for the indicated times. (b) Transcript accumulation in response to mechanical agitation (MA) and treatment with cycloheximide (CHX). Plants were grown at 24°C and then treated with MA or CHX for the times indicated (see Experimental procedures for details).

Download figure to PowerPoint

Additional experiments indicated that transcripts for all six genes, like those for CBF2 (Zarka et al., 2003), increased in response to both mechanical agitation and treatment with cycloheximide (Figure 5b). This was not true of all cold-inducible genes that encode transcription factors. For instance, RAP2.1, RAP2.7 (At2g28550), CZF3 (At4g38960), and HPPBF-2a (At3g47500), which are induced after 2 h of cold treatment (Figure 5a), were not induced in response to mechanical agitation or treatment with cycloheximide (Figure 5b). The link between cold, mechanical, and cycloheximide regulation of CBF2 is not known. However, two regulatory regions within the CBF2 promoter, designated ICEr1 (induction of CBF expression region 1) and ICEr2, have been shown to be involved in gene induction by each of these three stimuli, indicating a regulatory link between them (Zarka et al., 2003). A question thus raised was whether ICEr1 or ICEr2 sequences were present in the promoters of ZAT12, MYB73, ZAT10, CZF1, and CZF2. Searches indicated that the promoters of each of these genes had four sequences that overlapped with ICEr1, and a fifth sequence that overlapped with ICEr2 (Figure 6).

image

Figure 6. Potential regulatory sequences common to the promoter regions of CBF2, ZAT12, ZAT10/STZ, MYB73, CZF1, and CZF2. (a) Motifs that overlap the CBF2 ICEr1 or ICEr2 sequences that are conserved in the promoter regions of ZAT12, ZAT10/STZ, MYB73, CZF1, and CZF2 (see Experimental procedures for details). Base pairs of the motifs that are perfectly conserved in all the promoters analyzed are underlined. The sequences were queried to PlantCARE (http://oberon.fvms.ugent.be:8080/PlantCARE/index.html) to determine whether they were similar to known cis-acting regulatory elements. All promoters in the Arabidopsis genome were examined for the presence of these motifs and the percentage containing the motif is given. (b) Position of motifs aligned to the ICEr1 and ICEr2 sequences.

Download figure to PowerPoint

Transgenic plants that constitutively expressed RAV1, MYB73, ZAT10, CZF2, or ZAT12 under control of the CaMV 35S promoter were generated and the transcriptomes profiled to determine whether any of these transcription factors controlled expression of COS genes. The results with plants overexpressing RAV1, MYB73, ZAT10/STZ, or CZF2, using one GeneChip per line, indicated that the expression of few, if any, COS genes were affected by these transcription factors (Northern analysis indicated that the transcript levels for each of the transgenes were equal to or greater than the maximum level for the corresponding endogenous gene attained upon cold treatment; data not shown). RAV1 upregulated one COS transcript (At1g35140), STZ/ZAT10 upregulated two COS transcripts (At4g22470 and At4g12490), and CZF2 upregulated one COS transcript (At4g12490) and downregulated one COS transcript (At2g40610). These effects were confirmed by Northern analysis (not shown). However, the chance of observing up- or down-regulation of the COS genes by this group of transcription factors was no greater than expected by random occurrence; that is, the extent to which COS genes were affected by expression of the transcription factors was no greater than that observed for non-COS genes (Table 2). Thus, no COS genes could be confidently assigned to regulons for RAV1, MYB73, STZ/ZAT10, or CZF2. As described in the next section, COS genes could be assigned to the ZAT12 regulon.

The ZAT12 regulon comprises 24 COS genes

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Transgenic plants that constitutively expressed ZAT12 under control of the CaMV 35S promoter were generated (Figure 7) and the transcriptome profiled using the Affymetrix array. An examination of two independent transgenic lines indicated that 24 COS genes were responsive to ZAT12 overexpression (Tables S9 and S10). Of the 302 COS genes that were upregulated in response to low temperature, nine were also upregulated in response to ZAT12 overexpression and fell into expression clusters III and IV (Figure 3). Of the 212 COS genes that were downregulated by low temperature, 15 were also downregulated by ZAT12 overexpression and fell into expression clusters I and II (Figure 3). Significantly, seven of the ZAT12-responsive genes were also responsive to CBF2 overexpression (Figure 8), indicating the possibility that these two transcription factors coordinately regulate expression of these COS genes.

image

Figure 7. ZAT12 transcript levels in transgenic Arabidopsis plants. Transgenic lines expressing ZAT12 under control of the CaMV 35S promoter (#2, #8, and #15) or carrying an empty vector (V) were grown at 24°C and the levels of ZAT12 transcripts determined by RNA blot analysis. For comparison, wild-type (WT) plants were exposed to low temperature (4°C) for 0, 2, and 24 h and ZAT12 transcripts were determined by RNA blot analysis. eIF4a was used as a loading control.

Download figure to PowerPoint

image

Figure 8. Regulation of COS genes by CBF2 and ZAT12. Cold-responsive transcripts are indicated by boxes. Arrows indicate transcripts also responsive to CBF2 or ZAT12 expression.

Download figure to PowerPoint

A number of genes that were not cold-regulated were responsive to ZAT12 expression; 38 were upregulated and 143 were downregulated. As with CBF2 overexpression, assignment of many of these genes to the cold-non-responsive class was due to the strict criteria used to designate a gene as being cold-responsive and a number were either increased or decreased to a lower extent than the COS genes. Expression of the remaining genes was likely a consequence of the effects that ZAT12 expression had on growth and development (Figure 9). ZAT12-overexpressing plants were smaller than wild-type plants and had curled leaves, short petioles, short bolts, and downward-pointing siliques. As with the CBF2 overexpression experiments, the question raised was whether it was valid to assign a gene to the ZAT12 regulon based on the criterion of the gene being both cold- and ZAT12-responsive. Again, statistical analysis supported such assignments. Chi-square analysis indicated that it was highly unlikely for a given gene to be both cold- and ZAT12-responsive by chance (P < 0.0001) (Table 2). Thus, the 24 COS genes that were responsive to ZAT12 were assigned to the ZAT12 regulon.

image

Figure 9. Effect of ZAT12 expression on plant morphology and freezing tolerance. (a) Six-week-old wild-type (WT) and ZAT12-overexpressing (35S::ZAT12) plants. (b) Magnified view of 6-week-old ZAT12-overexpressing plants. (c) Mature 11-week-old ZAT12 overexpressing plants. A U.S. dime (17 mm) is shown for size comparison. (d) Effect of freezing on non-acclimated 10-day-old WT and ZAT12-expressing plants. (e) Magnified view of non-acclimated ZAT12 plant after freezing. (f) Table of results of multiple freeze tests, including the percentage of plants scored as surviving (% survival), the number of plants examined (n), and the P-value from a t-test.

Download figure to PowerPoint

The binding site for ZAT12 is unknown, and thus we were unable to tentatively assign genes to being direct targets for the ZAT12 protein. However, bioinformatic analysis of the promoter regions of the genes that were upregulated in response to both cold and ZAT12 expression identified multiple novel sequences that were overrepresented, three of which had CATTG as a core sequence suggesting that this may be a part of the ZAT12 binding site (Table 1). Three additional novel elements were detected in the genes that were downregulated in response to both cold and ZAT12.

ZAT12 negatively regulates CBF gene expression

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

The level of CBF1, 2, and 3 transcripts peaks at about 2 h after shifting plants to low temperature and then, as the COR gene transcripts begin to accumulate, the CBF transcripts begin to decrease. We were interested to determine whether ZAT12 might have a role in this downregulation of the CBF gene expression as the ZAT12 protein can function as a transcriptional repressor (Hiratsu et al., 2002) and its induction kinetics (Figure 5) were consistent with this possibility. To test this hypothesis, we compared the kinetics of low temperature induction of the CBF1-3 genes in wild-type plants and in transgenic plants overexpressing ZAT12. The results indicated that the CBF1, 2, and 3 transcripts accumulated to much lower levels in response to low temperature in the ZAT12-overexpressing plants when compared with the control plants (Figure 10). Transcript levels of the CBF-targeted genes COR78 and COR6.6, however, were only slightly lower in the ZAT12 transgenic plants.

image

Figure 10. ZAT12 overexpression dampens cold induction of CBF1, 2, and 3. Wild-type (WT) or ZAT12-overexpressing (35S::ZAT12) plants were grown at 24°C and then exposed to 4°C for the times indicated. RNA was isolated and CBF1, 2, and 3 transcript levels were determined by RNA blot hybridization using gene-specific probes. rDNA was used as a loading control. (a) Transcript accumulation in response to low temperature. Plants were grown at 24°C and then exposed to 4°C for the indicated times. (b) Quantitative transcript accumulation in response to low temperature. The highest transcript level after normalization was set to 100 and all other values are shown proportionally.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

The objectives of this study were to determine more completely the role of the CBF transcriptional activators in configuring the low temperature transcriptome and to identify additional transcription factors that participate in this process (the functional roles of the cold-responsive genes identified in this study will be considered elsewhere). To begin, we used the ‘full genome’ Affymetrix GeneChip® array to define a core set of cold-responsive genes that were reproducibly up- or down-regulated in response to low temperature in plants grown both in soil and on solid culture medium, two commonly used growth conditions. The experiments resulted in the identification of 514 such cold-responsive genes, referred to as COS (‘cold standard’) genes, 302 of which were upregulated and 212 downregulated at least 2.5-fold after either 1 h, 24 h or 7 days of exposing plants to low temperature. This gene set does not contain all cold-responsive genes as the tissue harvested from the soil-grown plants was primarily foliar, whereas both root and shoot tissue was harvested from the plate-grown plants. In addition, many genes were not designated as COS genes due to the strict signal log ratio cutoff imposed in the analysis. Over 80% of the upregulated genes that were designated as being ‘present,’‘increased’ and had a signal log ratio of ≥1.3 in both replicates for a given growth condition, were also ‘present’ and ‘increased’ under the other growth condition. The same situation held for approximately 70% of the downregulated genes. However, the objective here was not to identify all cold-responsive genes, but to generate a robust gene set that could be used in efforts to decipher the low temperature regulatory network of Arabidopsis. We believe that the COS gene set should have considerable utility in this regard.

To assess the extent to which the low temperature transcriptome falls under control of the CBF1-3 transcription factors, we determined which cold-induced and cold-repressed COS genes were upregulated and downregulated, respectively, at least 2.5-fold in response to constitutive CBF2 expression. Those genes that were both cold- and CBF2-responsive were assigned to the CBF regulon. The analysis indicated that 93 of the 514 COS genes were members of the regulon, 85 of which were cold-induced. Significantly, these 85 genes included the majority of those genes that were the most highly induced in response to low temperature; that is, 84% of the genes induced more than 15-fold, and 50% of those induced between five- and 10-fold, were found to belong to the CBF regulon. In addition, greater than 50% (37/67) of the COS genes that continued to be upregulated after 7 days of cold treatment were members of the CBF regulon. Taken together, these results indicate that the CBF transcription factors have a prominent role in regulating the expression of those cold-responsive genes that are both highly and stably induced in response to low temperature.

Maruyama et al. (2004) presented results identifying genes that are members of the CBF/DREB1 regulon. In these experiments, both the Affymetrix AtGenome1 GeneChip and a RIKEN Arabidopsis full-length cDNA array (Seki et al., 2001) were used. The two arrays combined represented approximately 12 000 different genes. Of these, 38 were found to be responsive to both low temperature and DREB1A/CBF3 overexpression. In our experiments, 22 of these 38 genes were also assigned to the CBF regulon. Of the 16 genes that we did not assign to the CBF regulon, one was not represented on the array that we used (there was no probe for the KIN2 gene) and seven others fell short of the 2.5-fold cutoff that we used in one or more samples from either the cold-treated plants or plants overexpressing CBF2. The remaining eight genes (At4g14000, At4g15910, At5g17460, At1g29390, At2g43510, At1g27730, At2g31360, and At2g33830) showed ‘no change’ in our experiments in both transgenic lines overexpressing CBF2. One possibility that could potentially explain this difference in the two studies is that CBF2 and DREB1A/CBF3 might have somewhat different target gene specificities. Although this possibility cannot be ruled out at present, the recent findings of Gilmour et al. (2004) argue against it. In particular, these investigators used the Affymetrix AtGenome1 GeneChip to compare the transcriptomes of transgenic Arabidopsis lines overexpressing either CBF1, 2, or 3, and found no qualitative differences in target gene specificity between the three transcription factors. Other possible explanations for the observed differences include differences in the probe sets used to represent genes in the Affymetrix AtGenome1 GeneChip and the Affymetrix Arabidopsis ATH1 GeneChip, potential cross hybridization of probes on the cDNA arrays, and differences in growth conditions.

The CBF proteins are transcriptional activators that bind to the CRT/DRE DNA regulatory element. Thus, it was not surprising that of the 85 CBF regulon genes that were cold-induced, 68 (80%) had at least one putative CRT/DRE element within the 1 kb upstream region of the gene coding sequence. It is likely that most, if not all, of these genes are direct targets of the CBF transcription factors. The other 17 cold-induced genes that belong to the CBF regulon might have active CRT/DRE elements elsewhere within or surrounding their coding sequences. Alternatively, these genes could be targets of one or more of the transcription factors that belong to the CBF regulon, such as RAP2.1 (Table S7), or their regulation might be affected by expression of other CBF regulon genes. Similarly, downregulation of the eight CBF regulon genes is likely to involve the action of regulatory or other proteins induced by the CBF transcription factors as the CBF1-3 proteins have not been reported to act as repressors.

Previous transcript profiling and mutagenesis experiments suggested the existence of cold regulatory pathways in addition to the CBF cold response pathway in Arabidopsis and that such putative pathways might have roles in freezing tolerance (Fowler and Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002; Vlachonasios et al., 2003; Xin and Browse, 1998). Here we confirm this hypothesis. A new low temperature regulatory pathway, the ZAT12 cold response pathway, was defined and evidence presented indicating that it has a role in cold acclimation. In particular, we show that ZAT12 is induced in parallel with the CBF1-3 transcription factors; that it regulates expression of 24 COS genes, nine of which were cold-induced genes and 15 cold-repressed; and that expression of the ZAT12 regulon results in a limited, but reproducible, increase in freezing tolerance. Additionally, the results indicated that the ZAT12 cold response pathway interacts in at least two fundamental ways with the CBF cold response pathway. First, there is overlap in the genes that comprise the ZAT12 and CBF regulons (Figure 8); four genes were upregulated and three downregulated in response to both transcription factors and low temperature. Thus, the ZAT12 and CBF cold response pathways appear to coordinately regulate the expression of certain COS genes. Moreover, constitutive ZAT12 expression was found to dampen the induction of the CBF1-3 genes in response to low temperature (Figure 10). Thus, the ZAT12 regulon appears to be involved in negative regulation of the CBF cold response pathway.

Precisely how ZAT12 controls expression of the 24 COS genes and suppresses induction of the CBF1-3 genes remains to be determined. However, ZAT12 has an EAR (ERF-associated amphiphilic repression)-like repressor domain (Ohta et al., 2001) and can act as a repressor (Hiratsu et al., 2002). Thus, a simple scenario would be that ZAT12 directly represses expression of CBF1-3 and the 15 downregulated COS genes that belong to the ZAT12 regulon, the consequences of which include induction of the cold-induced COS genes that belong to the ZAT12 regulon. It must be stressed, however, that this scenario is highly speculative and that its central tenant, that ZAT12 binds to a cis-acting regulatory element in promoters of the cold- and ZAT12-repressed genes, cannot be assessed at present as the putative DNA binding site of the ZAT12 protein has not been reported.

In addition to a role for ZAT12 in cold acclimation reported here, ZAT12 has been implicated in other multiple abiotic stress responses. Transcripts for ZAT12 have been shown to increase in response to high light (Iida et al., 2000), wounding (Chen et al., 2002; Cheong et al., 2002; Rizhsky et al., 2004), low-oxygen (Klok et al., 2002), hydrogen peroxide (Desikan et al., 2001), heat, and treatment with paraquat (Rizhsky et al., 2004). Moreover, overexpression of ZAT12 in transgenic Arabidopsis plants has been shown to result in increased tolerance to high light (Iida et al., 2000) and oxidative stress caused by application of paraquat (Rizhsky et al., 2004). As both high light (Demmigadams and Adams, 1992) and cold (Wise and Naylor, 1987) are associated with the formation of reactive oxygen species, the role of the ZAT12 regulon may be to help plants cope with oxidative stress. In this regard, it is of interest that ZAT12 expression resulted in downregulation of transcripts encoding a putative l-ascorbate oxidase (At5g21100; Table S10). This downregulation could account, at least in part, for the increased levels of ascorbic acid, a potent antioxidant (Noctor and Foyer, 1998; Sanmartin et al., 2003), observed in cold-acclimated Arabidopsis plants (Cook et al., 2004). In addition, ZAT12 expression resulted in accumulation of transcripts encoding arginine decarboxylase (At4g34710; Table S9), which is involved in the production of putrescine, a polyamine that appears to have protective roles against abiotic stress, including oxidative stress (Bouchereau et al., 1999; Ye et al., 1997).

Transgenic Arabidopsis plants overexpressing ZAT12 have been described in two previous studies (Iida et al., 2000; Rizhsky et al., 2004) in which different effects on plant morphology were noted. Rizhsky et al. (2004) reported that ZAT12 overexpression had no effect on plant growth and development while Iida et al. (2000) found that ZAT12-overexpressing plants had leaves that were thicker, rounder, and darker green than the control plants. The ZAT12-expressing plants in our study had curled leaves, short petioles, short bolts, and downward-pointing siliques. A simple explanation for these different observations is that the levels of ZAT12 expression attained in the three studies were different. It is more difficult, however, to explain the differences observed in the makeup of the ZAT12 regulon reported by Rizhsky et al. (2004) and that reported here. Rizhsky et al. (2004) reported upregulated expression of 67 genes using an SLR cutoff of 0.8. Of these, only two were upregulated in our experiments (‘P’, ‘I’ in both ZAT12 lines) and of these only one was upregulated at least 2.5-fold. Moreover, of the nine genes that we found to be upregulated in response to both low temperature and ZAT12 expression, none were found to be induced in response to ZAT12 by Rizhsky et al. (2004). No data are available for a comparison of downregulated transcripts.

While the CBF and ZAT12 regulons include the majority of the highly and stably expressed cold-regulated genes, most of the COS genes remain to be assigned to regulons. Determining which of the approximately 1700 transcription factors encoded by the Arabidopsis genome have roles in regulating expression of these genes is now the challenge. As hypothesized above, transcription factors that are upregulated in parallel with the CBF1-3 transcription factors seem to be logical candidates, and proved to be true for ZAT12. However, we were unable to assign COS genes to regulons controlled by any of the other genes encoding known or putative transcription factors that were upregulated along with the CBF genes; namely RAV1, MYB73, STZ/ZAT10, or CZF2. One interpretation of these results is that these transcription factors simply do not have a role in configuring the low temperature transcriptome. However, the results obtained with DREB2a (Liu et al., 1998) suggest that it may be premature to draw this conclusion. DREB2a is a dehydration-induced gene that encodes an AP2-domain transcription factor that binds to the CRT/DRE element. Expression of the DREB2a protein in transient assays activates expression of reporter genes containing the CRT/DRE sequence. However, constitutive overexpression of the DREB2a gene in transgenic plants does not result in constitutive expression of CRT/DRE-containing genes. Thus, it has been proposed that the DREB2a protein must undergo post-translational modification induced by dehydration stress to be active (Liu et al., 1998). Similarly, the RAV1, MYB73, STZ/ZAT10, and CZF2 proteins could be activated at low temperature to regulate expression of bona fide target genes. Another possibility is that the proteins cannot work alone, but must function in conjunction with other cold-regulated proteins.

A final point regards the potential cis-acting regulatory elements that were identified for the COS genes. In using MotifSampler (Thijs et al., 2002) to analyze the promoter regions of COS genes included in the seven cold-regulated expression clusters (Figure 2), we were able to identify a number of potential cis-acting regulatory elements involved in cold-regulated gene expression. Most of these potential elements were novel. However, the fact that the CRT/DRE element was identified as a potential element for genes in expression cluster III, which includes the large majority of CBF regulon genes (Figure 3), lends credence to the hypothesis that at least some of the sequences are active. If this can be verified, the elements could then be used in biochemical and genetic approaches to identify additional transcription factors involved in shaping the low temperature transcriptome.

Constructs and plant transformation

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Plasmids were constructed using polymerase chain reaction (PCR) and standard molecular biological techniques (Sambrook and Russell, 2001). Primers used to amplify the coding regions of each transcription factor from either genomic DNA or cDNA contained sites for unique restriction enzymes. PCR fragments were cloned into the pGEM-T easy vector (Promega, Madison, WI, USA). The inserts were excised using restriction enzymes for the sites present in the primers and then cloned into a plant expression vector. The following primers and restriction enzymes were used: ZAT12 SpeI 5′-ACTAGTCAGAAGAAAAATGGTTGCGATA-3′, BamHI 5′-GGATCCGAAAAATTCAAAGAATGAGAGAAACA-3′; RAV1 XbaI 5′-CTAGACAGATTAAATGGAATCGAGTAGC-3′, BamHI 5′-GGATCCGAGTTGTTACGAGGCGTGAA-3′; CZF2 XbaI 5′-TCTAGATCTTCAAGATAATGGCACTTGAAA-3′, BamHI 5′-GGATCCTCCTAGGTTTATGTTTAGGGTTTCTC-3′; MYB73 XbaI 5′-TCTAGATAAAAAGATCCGGCGATGTC-3′, BamHI 5′-GGATCCCACTCTACTCCATCTTCCCAAT-3′; STZ/ZAT10 XbaI 5′-TCTAGACGAGAGACAAGAAATCCTCAGAA-3′, BamHI 5′-GGATCCTTTCCTTAAAGTTGAAGTTTGACC-3′.

The DNAs were transferred to Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col-0 or gl1 Col-PRL) via a whole plant dipping method similar to that described by Clough and Bent (1998). Seed germination on medium containing kanamycin (50 mg l−1) (Sigma-Aldrich, St Louis, MO, USA) was used to identify plants containing transferred DNA. Expression of the transgene was monitored via RNA blot analysis and T3 lines homozygous for the transferred DNA were used in all experiments.

Plant growth and experimental treatments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Arabidopsis thaliana constitutively expressing ZAT12, RAV1, MYB73, CZF2, or STZ/ZAT10 (this study) or CBF2 (Gilmour et al., 2004) were grown in controlled environment chambers at 22°C under constant illumination from cool-white fluorescent lights (approximately 100 μmol m−2 sec−1) in Baccto planting mix (Michigan Peat, Houston, TX, USA). Pots were subirrigated with deionized water as needed. Plants were also grown on solid agar medium which contained Gamborg's B5 nutrients (Caisson Laboratories, Inc., Rexburg, ID, USA) and 0.8% phytagar (Life Technologies Inc., Gaithersburg, MD, USA) at 24°C under constant illumination from cool-white fluorescent lights (approximately 100 μmol m−2 sec−1). Seeds were stratified 4 days at 4°C before being grown at warm temperatures.

Experiments were performed on seedlings that were 10–12 days old. Treatments with cycloheximide (Sigma-Aldrich) were performed by growing seedlings on filter papers that had been placed on top of the agar so that the seedlings could be lifted off the plate with minimal damage or mechanical stress. The seedlings were then floated on a solution of cycloheximide (10 μg ml−1) in covered dishes for various times. Mechanical treatment involved tapping plates on a bench top for 15 min before harvesting tissue. All transgenic lines for the transcriptome analysis were grown in plates on solid media. Cold shock treatments involved transfer of plants (in the plate experiment, 10-day-old plants were used with approximately 100 per plate; in the soil experiment 18-day-old plants had been grown one plant per pot, 50 per flat) to a 4°C chamber with constant dim light (approximately 25 μmol m−2 sec−1). Two biological duplicates of both the plate and soil experiments were performed for profiling.

Affymetrix GeneChip hybridization and data collection

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

RNA isolation, probe labeling, hybridization, and data collection were performed as described (Fowler and Thomashow, 2002) with the following modifications. For the plate-grown transgenic and wild-type Arabidopsis, approximately 100 plants grown on a single plate were collected and total RNA was extracted using the Qiagen RNeasy Plant Mini Kits (Qiagen Inc., Valencia, CA, USA) with modifications. To obtain adequate and consistent yields with the kit, the amount of starting plant tissue was doubled. Subsequently the amount of extraction buffer (RLT) was also doubled. The remaining procedure was performed as described in the Qiagen manual. Biotinylated target RNA was prepared from 16 μg of total RNA equally pooled from two to four plates. For the soil-grown plants, aerial parts of 12 plants were harvested from various parts of a flat for each time point. Total RNA was extracted from two seedlings at once as described above. All samples for a given time point were then pooled and precipitated. Biotinylated target RNA was prepared from 16 μg of total RNA. The samples were hybridized to the Affymetrix Arabidopsis ATH1 GeneChip (Affymetrix, Santa Clara, CA, USA) representing approximately 24 000 Arabidopsis genes. Two biological replicates were analyzed per growth condition, for a total of four samples per time point.

The results from the microarray experiments have been submitted to TAIR (http://www.arabidopsis.org) according to MAIME guidelines under the accession numbers ME00320, ME00321, ME00322, and ME00323.

Affymetrix GeneChip data analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

The Affymetrix Microarray Suite 5.0 statistical algorithms were used for microarray data analysis. Output from all GeneChip hybridizations was scaled globally such that its average intensity was equal to an arbitrary target intensity of 500 allowing comparisons between GeneChips. Signal (gene expression) and signal log ratios (SLR) were calculated from the GeneChip fluorescence intensity data. The software was also used to determine whether each gene was present or absent (Detection) and whether the SLR represented a genuine change in mRNA accumulation (Change). SLRs and ‘change’ calls were determined for each transgenic line or cold sample compared with its corresponding wild-type sample. Microsoft Access was used as the database management software and to filter and query the data.

Probe sets that met the following criteria were selected for further analysis. Those determined to be upregulated by the cold treatment were selected as having, at any time point, an absolute call of present in both cold samples (soil or plates), difference calls of increase, and SLRs of at least 1.3 for both change comparisons. Those determined to be downregulated by the cold treatment were selected as having, at any time point, an absolute call of present in both warm samples, difference calls of decrease, and SLRs of at least −1.3 for both change comparisons. Those probe sets meeting the above criteria in both the soil and plate experiments at any time point were analyzed. Statistically significant changes in mRNA abundance were determined using the statistical package with GeneSpring 6.0 (Silicon Genetics, Redwood City, CA, USA). The GeneChip data were imported into GeneSpring and normalized to respective control samples as specified by the manufacturer for single color array data. The 3178 transcripts with reliably altered levels (probe sets with an absolute call of ‘present’ in all four cold samples and four difference calls of ‘increase’ for a given time point or an absolute call of ‘present’ in all four warm samples and four difference calls of ‘decrease’ for a given time point) were analyzed using anova (P-value cutoff 0.05 or 0.005) and the Benjamini and Hochberg False Discovery Rate multiple testing correction.

Genes upregulated by CBF2 (lines E2 and E24) or ZAT12 (lines p15-8 and p15-15) expression were selected as having an absolute call of present in both transgenic samples, difference calls of increase, and an SLR of at least 1.3 for both change comparisons between wild-type and transgenic lines. Those determined to be downregulated were selected as having an absolute call of present in both wild-type samples, difference calls of decrease, and an SLR of at least −1.3 for both change comparisons between wild-type and CBF transgenic lines. Probe sets changing in the RAV1, MYB73, CZF2, or STZ/ZAT10 expressing lines were selected in the same manner, but with data from only one GeneChip experiment per line.

Statistical analysis of the probe sets changing in a given overexpression line compared with the cold-responsive transcripts was performed by chi-square analysis using Yate's correction, χ2 = Σ(|O − E| − 0.5)2/E. The expected number of cold-responsive probe sets was calculated by dividing the number of probe sets changing in a transgenic line by the total number of possible probe sets that could change in the cold and multiplying by the number of cold-responsive probe sets (302 or 212). Every probe set on the GeneChip had an equal probability of being upregulated in a transgenic line, but only those probe sets ‘present’ in the control samples for each transgenic line could be downregulated, hence this number could fluctuate between various controls.

Hierarchical clustering was performed with GeneSpring 6.0, using data normalized as described previously. Hierarchical clustering was performed using the log of ratios (i.e., the ratio of the signal to the control, not their logs) of both the soil and plate experiment data using a standard correlation (separation ratio of 1 and a minimum distance of 0.001).

Whole plant freeze test

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Arabidopsis plants were grown on plates as described above. Cold- and non-acclimated plants were placed at −2°C in the dark for 3 h after which freezing of the plates was nucleated with ice chips. The plants were incubated an additional 21 h at −2°C, followed by −5°C for 24 h. The temperature was then raised to 4°C for 24 h. The plants were allowed to recover at 24°C for 48 h in continuous light and then scored for survival. A t-test was used to assess difference in the mean survival between control and experimental plants. In most experiments, the control and test plants were grown on separate plates (data presented in Figure 9f), but similar results were obtained when control and test plants were grown on the same plate (Figure 9d).

RNA blot analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

RNA blots were prepared (10 μg of total RNA was loaded) and hybridized as described (Hajela et al., 1990) using normal high stringency wash conditions (Stockinger et al., 1997). Quantitation of mRNA abundance was performed with a Molecular Imager FX Pro multi-imager system (Bio-Rad Laboratories, Hercules, CA, USA).

Probes for CBF (Gilmour et al., 1998), COR6.6, COR78 (Hajela et al., 1990), eIF4a (Taylor et al., 1993), ZAT12 (Fowler and Thomashow, 2002), 25S rDNA (Delseny et al., 1983), RAP2.1 and RAP2.7 (Okamuro et al., 1997) were prepared as previously described. Probes for MYB73, STZ/ZAT10, CZF1, CZF2, CZF3, and HPPBF-2a were PCR-amplified from cDNAs using the primer pairs used in ‘Constructs and Plant Transformation’ except for the following: CZF1 (At2g40140) 5′-TCTAGACACTTTTGAATCACAGGcAAGA-3′, 5′-GGATCCGCTTCTTATGCCACAATCTGC-3′; CZF3 (At4g38960) 5′-TCTAGAGAAAAAGCAAGATGCGGATT-3′, 5′-GGATCCTTATCACTTCTCAGACTCTCG-3′; HPPBF-2a 5′-TCTAGAAAAATGATGATGGAGACTAGAGA-3′, 5′-AGATCTCATATGTAACTCTAAATCTGTTCATGG-3′. The RAV1 probe was isolated from an AclI and BamHI digest using an AclI site present in the RAV1 coding region and the BamHI site in the PCR primer used for amplifying the coding region. Probes for genes up- or down-regulated in the cold and in one of the single GeneChip experiments profiling transgenic lines were performed with restriction enzyme digests of ESTs available from the Arabidopsis Biological Resource Center at Ohio State University (Columbus). Probes were labeled with 32P by random priming (Feinberg and Vogelstein, 1983).

Motif analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Motif searches were performed with the command line version of MotifSampler (v3.0) (http://www.esat.kuleuven.ac.be/thijs/Work/MotifSampler.html). The dataset searched was the 500 bp locus upstream sequence downloaded from TAIR (http://www.arabidopsis.org) on March 17, 2003. Arabidopsis intergenic regions were used as a background model with the order set to 3. The motif length was set to 8 bp, with the prior probability of finding one motif instance set to 0.3. No limit was set for the number of motif instances that could be found per sequence, the maximum overlap between different motifs was set to 1, the number of motifs per run to be found was set to three, and the total number of runs was set to 100. The highest scoring motifs were then ranked by MotifRanking using Arabidopsis intergenic sequence as a background model and the Kullback–Lieber distance to score the top 10 motifs. Those motifs greater than fivefold overrepresented in a class compared with the rest of the promoters on the GeneChip were analyzed. The number of motifs present in the upstream regions of Arabidopsis genes was determined using Patmatch at TAIR or a set of custom PERL scripts. All PERL scripts are available upon request. All motifs were queried at PlantCARE (Lescot et al., 2002) to determine whether they were similar to any known sequence.

MotifSampler was also used to identify motifs present in the 1000 bp locus upstream sequence (downloaded from TAIR) of CZF1, CZF2, MYB73, ZAT12, STZ/ZAT10, and the ICE regions (including flanking sequences) of the CBF2 promoter. The ICE regions searched were ICEr1: atgggtcaaaGGACACATGTCAGAttctcagtga and ICEr2: agagacagaaACTCCGcgttcgaccc (Zarka et al., 2003). The defined ICE regions are capitalized. Only motifs common among all the promoters were analyzed.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

We thank Dr Rebecca Grumet, Dr Marianne Huebner, and Dr Vince Melfi for their help with statistical analysis and Dr Gert Thijs for helpful advice with MotifSampler. GeneChip hybridization and scanning was performed by Dr Annette Thelen in the Genomics Technology Support Facility at Michigan State University. We thank Kurt Stepnitz and Marlene Cameron for help with photography and graphics and Dr Sarah Gilmour for critical reading of this manuscript. ESTs were obtained from the Arabidopsis Biological Resource Center (ABRC). J.T.V. was a recipient of a US Department of Education Graduate Assistantship in Areas of National Need (GAANN) fellowship. This study was supported in part by a grant from the NSF Plant Genome Project (DBI 0110124). A project description, databases, and other information regarding the project can be found on the web at http://aztec.stanford.edu/cold/index.html. Research in the Thomashow laboratory was also funded in part by grants from the DOE (DEFG0291ER20021) and the Michigan Agricultural Experiment Station.

Supplementary Material

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Table S1 Probe sets upregulated by low temperature in the plate experiment

Table S2 Probe sets downregulated by low temperature in the plate experiment

Table S3 Probe sets upregulated by low temperature in the soil experiment

Table S4 Probe sets downregulated by low temperature in the soil experiment

Table S5 Probe sets upregulated by low temperature in both the plate and soil experiments

Table S6 Probe sets downregulated by low temperature in both the plate and soil experiments

Table S7 Probe sets upregulated in response to CBF2 expression

Table S8 Probe sets downregulated in response to CBF2 expression

Table S9 Probe sets upregulated in response to ZAT12 expression

Table S10 Probe sets downregulated in response to ZAT12 expression

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information
  • Artus, N.N., Uemura, M., Steponkus, P.L., Gilmour, S.J., Lin, C. and Thomashow, M.F. (1996) Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proc. Natl Acad. Sci. USA, 93, 1340413409.
  • Baker, S.S., Wilhelm, K.S. and Thomashow, M.F. (1994) The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol. Biol. 24, 701713.
  • Bouchereau, A., Aziz, A., Larher, F. and Martin-Tanguy, J. (1999) Polyamines and environmental challenges: recent development. Plant Sci. 140, 103125.
  • Chen, W., Provart, N.J., Glazebrook, J. et al. (2002) Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell, 14, 559574.
  • Cheong, Y.H., Chang, H.S., Gupta, R., Wang, X., Zhu, T. and Luan, S. (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol. 129, 661677.
  • Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735743.
  • Cook, D., Fowler, S., Fiehn, O. and Thomashow, M.F. (2004) A prominent role for the CBF cold response pathway in configuring the low temperature metabolome of Arabidopsis. Proc. Natl Acad. Sci. USA, 101, 1524315248.
  • Delseny, M., Cooke, R. and Penon, P. (1983) Sequence heterogeneity in radish nuclear ribosomal RNA genes. Plant Sci. Lett. 30, 107119.
  • Demmigadams, B. and Adams, W.W. (1992) Photoprotection and other responses of plants to high light stress. Annu. Rev. Plant Phys. 43, 599626.
  • Desikan, R.A.H., Mackerness, S., Hancock, J.T. and Neill, S.J. (2001) Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol. 127, 159172.
  • Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 613.
  • Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell, 14, 16751690.
  • Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M. and Thomashow, M.F. (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J. 16, 433442.
  • Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D. and Thomashow, M.F. (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124, 18541865.
  • Gilmour, S., Fowler, S. and Thomashow, M. (2004) Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Mol. Biol. 54, 767781.
  • Hajela, R.K., Horvath, D.P., Gilmour, S.J. and Thomashow, M.F. (1990) Molecular-cloning and expression of COR (cold-regulated) genes in Arabidopsis thaliana. Plant Physiol. 93, 12461252.
  • Hiratsu, K., Ohta, M., Matsui, K. and Ohme-Takagi, M. (2002) The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers. FEBS Lett. 514, 351354.
  • Iida, A., Kazuoka, T., Torikai, S., Kikuchi, H. and Oeda, K. (2000) A zinc finger protein RHL41 mediates the light acclimatization response in Arabidopsis. Plant J. 24, 191203.
  • Jaglo, K.R., Kleff, S., Amundsen, K.L., Zhang, X., Haake, V., Zhang, J.Z., Deits, T. and Thomashow, M.F. (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol. 127, 910917.
  • Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O. and Thomashow, M.F. (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science, 280, 104106.
  • Klok, E.J., Wilson, I.W., Wilson, D., Chapman, S.C., Ewing, R.M., Somerville, S.C., Peacock, W.J., Dolferus, R. and Dennis, E.S. (2002) Expression profile analysis of the low-oxygen response in Arabidopsis root cultures. Plant Cell, 14, 24812494.
  • Kreps, J.A., Wu, Y., Chang, H.S., Zhu, T., Wang, X. and Harper, J.F. (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol. 130, 21292141.
  • Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouze, P. and Rombauts, S. (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325327.
  • Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10, 13911406.
  • Maruyama, K., Sakuma, Y., Kasuga, M., Ito, Y., Seki, M., Goda, H., Shimada, Y., Yoshida, S., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2004) Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. Plant J. 38, 982993.
  • Medina, J., Bargues, M., Terol, J., Perez-Alonso, M. and Salinas, J. (1999) The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiol. 119, 463470.
  • Meissner, R. and Michael, A.J. (1997) Isolation and characterisation of a diverse family of Arabidopsis two and three-fingered C2H2 zinc finger protein genes and cDNAs. Plant Mol. Biol. 33, 615624.
  • Nanjo, T., Kobayashi, M., Yoshiba, Y., Kakubari, Y., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1999) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett. 461, 205210.
  • Noctor, G. and Foyer, C.H. (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249279.
  • Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H. and Ohme-Takagi, M. (2001) Repression domains of class II ERF transcriptional repressors share an essential motif for active repression. Plant Cell, 13, 19591968.
  • Okamuro, J.K., Caster, B., Villarroel, R., Van Montagu, M. and Jofuku, K.D. (1997) The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc. Natl Acad. Sci. USA, 94, 70767081.
  • Riechmann, J.L. (2002) Transcriptional regulation: A genomic overview. In The Arabidopsis Book (Somerville, C.R. and Meyerowitz, E.M., eds). Rockville, MD: American Society of Plant Biologists doi/10.1199/tab.0085, http://www.aspb.org/publications/arabidopsis/.(last accessed: 1 October 2004)
  • Rizhsky, L., Davletova, S., Liang, H. and Mittler, R. (2004) The zinc finger protein ZAT12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J. Biol. Chem. 279, 1173611743.
  • Rombauts, S., Florquin, K., Lescot, M., Marchal, K., Rouze, P. and van de Peer, Y. (2003) Computational approaches to identify promoters and cis-regulatory elements in plant genomes. Plant Physiol. 132, 11621176.
  • Sakuma, Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2002) DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration and cold-inducible gene expression. Biochem. Biophys. Res. Commun. 290, 9981009.
  • Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
  • Sanmartin, M., Drogoudi, P.A., Lyons, T., Pateraki, I., Barnes, J. and Kanellis, A.K. (2003) Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone. Planta, 216, 918928.
  • Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P., Hayashizaki, Y. and Shinozaki, K. (2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. Plant Cell, 13, 6172.
  • Seki, M., Narusaka, M., Ishida, J. et al. (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J. 31, 279292.
  • Smallwood, M. and Bowles, D.J. (2002) Plants in a cold climate. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 831847.
  • Steponkus, P.L., Uemura, M., Joseph, R.A., Gilmour, S.J. and Thomashow, M.F. (1998) Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc. Natl Acad. Sci. USA, 95, 1457014575.
  • Stockinger, E.J., Gilmour, S.J. and Thomashow, M.F. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl Acad. Sci. USA, 94, 10351040.
  • Taylor, C.B., Bariola, P.A., DelCardayre, S.B., Raines, R.T. and Green, P.J. (1993) RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc. Natl Acad. Sci. USA, 90, 51185122.
  • Thijs, G., Marchal, K., Lescot, M., Rombauts, S., De Moor, B., Rouze, P. and Moreau, Y. (2002) A Gibbs sampling method to detect overrepresented motifs in the upstream regions of coexpressed genes. J. Comput. Biol. 9, 447464.
  • Thomashow, M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant Phys. 50, 571599.
  • Thomashow, M.F. (2001) So what's new in the field of plant cold acclimation? Lots! Plant Physiol. 125, 8993.
  • Vlachonasios, K.E., Thomashow, M.F. and Triezenberg, S.J. (2003) Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression. Plant Cell, 15, 626638.
  • Wise, R.R. and Naylor, A.W. (1987) Chilling-enhanced photooxidation – evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. Plant Physiol. 83, 278282.
  • Xin, Z. and Browse, J. (1998) Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc. Natl Acad. Sci. USA, 95, 77997804.
  • Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell, 6, 251264.
  • Ye, B., Muller, H.H., Zhang, J. and Gressel, J. (1997) Constitutively elevated levels of putrescine and putrescine-generating enzymes correlated with oxidant stress resistance in Conyza bonariensis and wheat. Plant Physiol. 115, 14431451.
  • Zarka, D.G., Vogel, J.T., Cook, D. and Thomashow, M.F. (2003) Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiol. 133, 910918.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a core set of cold-responsive genes
  6. Members of the COS gene set can be assigned to one of seven expression clusters
  7. The CBF2 regulon comprises 93 COS genes
  8. CBF2 regulates a majority of highly induced COS genes
  9. Candidate transcription factors for regulating COS gene expression
  10. The ZAT12 regulon comprises 24 COS genes
  11. The ZAT12 regulon contributes to freezing tolerance
  12. ZAT12 negatively regulates CBF gene expression
  13. Discussion
  14. Experimental procedures
  15. Constructs and plant transformation
  16. Plant growth and experimental treatments
  17. Affymetrix GeneChip hybridization and data collection
  18. Affymetrix GeneChip data analysis
  19. Whole plant freeze test
  20. RNA blot analysis
  21. Motif analysis
  22. Acknowledgements
  23. Supplementary Material
  24. References
  25. Supporting Information

Table S1. Probe sets up-regulated by low temperature in the plate experiment.

Table S2.  Probe sets down-regulated by low temperature in the plate experiment.

Table S3.  Probe sets up-regulated by low temperature in the soil experiment.

Table S4.  Probe sets down-regulated by low temperature in the soil experiment.

Table S5.  Probe sets up-regulated by low temperature in both the plate and soil experiments.

Table S6.  Probe sets down-regulated by low temperature in both the plate and soil experiments.

Table S7. Probe sets up-regulated in response to CBF2 expression.

Table S8.  Probe sets down-regulated in response to CBF2 expression.

Table S9.  Probe sets up-regulated in response to ZAT12 expression.

Table S10.  Probe sets down-regulated in response to ZAT12 expression.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
TPJ_2288_sm_TableS1.pdf754KSupporting info item
TPJ_2288_sm_TableS10.pdf82KSupporting info item
TPJ_2288_sm_TableS2.pdf864KSupporting info item
TPJ_2288_sm_TableS3.pdf942KSupporting info item
TPJ_2288_sm_TableS4.pdf345KSupporting info item
TPJ_2288_sm_TableS5.pdf217KSupporting info item
TPJ_2288_sm_TableS6.pdf208KSupporting info item
TPJ_2288_sm_TableS7.pdf89KSupporting info item
TPJ_2288_sm_TableS8.pdf40KSupporting info item
TPJ_2288_sm_TableS9.pdf39KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.