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First published online February 24, 2003; 10.1105/tpc.007922 American Society of Plant Biologists Disruption Mutations of ADA2b and GCN5 Transcriptional Adaptor Genes Dramatically Affect Arabidopsis Growth, Development, and Gene Expression
a Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319 1 To whom correspondence should be addressed. E-mail triezenb{at}msu.edu; fax 517-353-9334
We previously identified Arabidopsis genes homologous with the yeast ADA2 and GCN5 genes that encode components of the ADA and SAGA histone acetyltransferase complexes. In this report, we explore the biological roles of the Arabidopsis ADA2b and GCN5 genes. T-DNA insertion mutations in ADA2b and GCN5 were found to have pleiotropic effects on plant growth and development, including dwarf size, aberrant root development, and short petals and stamens in flowers. Approximately 5% of the 8200 genes assayed by DNA microarray analysis showed changes of expression in the mutants, three-fourths of which were upregulated and only half of which were altered similarly in the two mutant strains. In cold acclimation experiments, C-repeat binding factors (CBFs) were induced in the mutants as in wild-type plants, but subsequent transcription of cold-regulated (COR) genes was reduced in both mutants. Remarkably, nonacclimated ada2b-1 (but not gcn5-1) mutant plants were more freezing tolerant than nonacclimated wild-type plants, suggesting that ADA2b may directly or indirectly repress a freezing tolerance mechanism that does not require the expression of CBF or COR genes. We conclude that the Arabidopsis ADA2b and GCN5 proteins have both similar and distinct functions in plant growth, development, and gene expression and may be components of both a common coactivator complex and separate complexes with distinct biological activities.
The regulation of gene expression at the level of transcription underlies many biological processes, such as growth and development, metabolic and physiological balances, and responses to the environment. In eukaryotes, chromatin plays a pivotal role in regulating gene expression (Narlikar et al., 2002  ). To activate gene expression, transcriptional activator proteins must overcome repressive chromatin structures. The changes in chromatin are accomplished by two broad classes of protein complexes. One class comprises the ATP-dependent chromatin-remodeling machines, which can alter nucleosome positions or conformations relative to the DNA (Peterson and Workman, 2000; Narlikar et al., 2002). The second class comprises complexes that covalently modify the histone proteins in the nucleosome by acetylation, phosphorylation, methylation, ADP-ribosylation, or ubiquitination (Strahl and Allis, 2000; Jenuwein and Allis, 2001; Berger, 2002).
The acetylation of specific Lys residues within the N-terminal tails of core histones, mediated by histone acetyltransferases (HATs), has been implicated in the regulation of transcription and other nuclear processes (Sterner and Berger, 2000
Our interest in the chromatin-remodeling factors in plants stems from our studies of the phenomenon of cold acclimation, the process whereby certain plants, including Arabidopsis, increase in freezing tolerance in response to low, nonfreezing temperatures (Thomashow, 1999
Previously, we reported results suggesting that transcriptional activation by Arabidopsis CBF1 might be mediated, at least in part, by homologs of the yeast Ada2 and Gcn5 proteins (Stockinger et al., 2001 In this report, we identify Arabidopsis mutants with T-DNA inserts in either the ADA2b or GCN5 gene and determine the effects of the mutations on plant growth, development, and gene expression. Our results indicate that ADA2b and GCN5 are important for normal plant growth and development and influence the low-temperature induction of the COR genes. Differences in the phenotypes of ada2b-1 and gcn5-1 mutant plants suggest that the wild-type proteins have distinct as well as common functions, a conclusion reinforced by gene expression profile analysis. Remarkably, nonacclimated ada2b-1 mutant plants were more freezing tolerant than nonacclimated wild-type plants, suggesting that ADA2b is involved in repressing a freezing tolerance pathway at warm temperature that is independent of the CBF cold response pathway.
Isolation and Molecular Characterization of the ada2b-1 Mutant Plants bearing a disruption mutation of the Arabidopsis ADA2b gene were identified by screening a population of T-DNA insertional mutants from the University of Wisconsin Arabidopsis Knockout Facility (Krysan et al., 1996 , 1999). The initial allele that we acquired, which we designated ada2b-1, contains a T-DNA insertion in the fifth intron, 1243 bp downstream of the translational initiation codon (Figure 1A)
. DNA gel blot analysis revealed a single T-DNA insertion in ada2b-1 genomic DNA (Figure 1B). The progeny of heterozygous plants showed a segregation of the ada2b-1 phenotype (described below) of  1:3 ( 2 = 1.27, P > 0.05), indicating that the ada2b-1 mutation is a recessive loss-of-function mutation that segregates as a single nuclear locus. RNA gel blot analysis using a probe corresponding to the 5' half of the ADA2b gene revealed a truncated mRNA of 1 kb rather than the full-length ADA2b mRNA (Figure 1C), indicating that the transcript is terminated in the T-DNA in the ada2b-1 mutant. Reverse transcriptase–mediated (RT) PCR analysis using primers that span the T-DNA insertion confirmed the absence of full-length ADA2b mRNA (Figure 1D). However, transcripts corresponding to the 3' half of the ADA2b gene in the ada2b-1 mutants were detected by RNA gel blot analysis and RT-PCR (data not shown); these transcripts presumably initiated inside the T-DNA. We conclude that no full-length ADA2b transcripts are expressed in the ada2b-1 mutant and that this allele probably represents a null mutation.
Phenotypic Characterization and Complementation of the ada2b-1 Mutant Homozygous ada2b-1 plants showed pleiotropic effects at several stages of plant development. The ada2b-1 phenotype is characterized by small, dark green, twisted (curled) leaves, dwarf stature, and infertility (Figure 2) . The cotyledons of ada2b-1 seedlings were smaller, dark green, and epinastic (i.e., curled down) (Figures 2A and 2B). The hypocotyls of the homozygous mutant seedlings, with an average length of 5 mm at 9 days, were longer than the heterozygote or wild-type hypocotyls, which averaged 3 mm (Figure 2B). Histological analysis indicated that cells in the hypocotyl of ada2b-1 plants were longer than those of wild-type plants (data not shown). In contrast to the extended hypocotyls, the rosette leaves of the homozygous mutant plants were much smaller than the heterozygous or wild-type leaves (Figures 2A and 2D). Laser scanning confocal microscopy revealed that the palisade mesophyll leaf cells from ada2b-1 plants were smaller (approximately one-third by cross-sectional area) than corresponding cells of wild-type plants (Figures 2G and 2H), suggesting that the ada2b-1 dwarfism arises from decreased cell size rather than from decreased cell division. The root system in ada2b-1 plants was shorter than that in wild-type plants, with an increased number of secondary roots (Figure 2C) and with cells in the elongation zone being markedly smaller (data not shown). The inflorescence stems of mutant plants were much smaller than those of wild-type plants (Figures 2E and 2F). The floral organs, particularly petals and stamens, also were affected in ada2b-1 plants. The flowers opened prematurely, with small petals that curled outward and stamens that were markedly shorter than the carpels (Figures 2I and 2J). Cells in the stamen filament of ada2b-1 were shorter than wild-type filament cells (Figures 2K to 2N), although pollen morphology and production appeared normal (data not shown). Manual fertilization resulted in small siliques with few seeds. Thus, the infertility of homozygous mutant plants may be a consequence of the failure of the pollen to reach the stigma as a result of the short stamens.
To determine whether the mutant phenotypes were caused by the T-DNA insertion in the ADA2b gene, genetic complementation was performed by transforming ada2b-1 heterozygotes with a DNA fragment expressing both the wild-type ADA2b cDNA and an herbicide resistance marker gene from constitutive 35S promoters of Cauliflower mosaic virus. Transformed plants were screened by PCR to identify plants homozygous for the ada2b-1 allele and bearing the transgene. Introduction of the ADA2b cDNA in ada2b-1 plants rescued all of the ada2b-1 phenotypes described above, confirming that the T-DNA insertion was responsible for the observed phenotypes (Figure 2E). By contrast, constitutive expression of the ADA2a cDNA in homozygous ada2b-1 plants did not rescue any of the mutant phenotypes (data not shown).
Isolation and Molecular Characterization of the gcn5-1 Mutant
Phenotypic Characterization and Complementation of the gcn5-1 Mutant Homozygous gcn5-1 plants were identified and confirmed by PCR. The phenotype of gcn5-1 homozygotes resembled the ada2b-1 phenotype in some respects, most notably in root growth, the dwarf size of rosette and adult plants, and tissue-specific deformities in flowers. By contrast, other phenotypic features were distinctly different, including hypocotyl elongation, leaf morphology, and the number of floral shoots. Specific features of the gcn5-1 phenotype, compared with those of wild-type and ada2b-1 plants, are detailed below. In seedlings and until the formation of the second pair of leaves, no morphological difference was apparent between gcn5-1 mutants and wild-type plants. In plate-grown gcn5-1 plants, the first pair of leaves had slightly longer petioles (data not shown). The second pair of leaves in the gcn5-1 plants initially was folded upward, suggesting that the abaxial cells had divided or expanded faster than the adaxial cells. When the leaves unfolded, they developed a serrated morphology (Figures 4A and 4B) . In contrast to ada2b-1 plants, gcn5-1 leaves were not dark green but instead were chlorotic (especially under increased light intensity). The size of the gcn5-1 palisade mesophyll cells was smaller than those in the wild type but larger than those in the ada2b-1 mutants (data not shown). The root development of gcn5-1 seedlings was similar to that of ada2b-1 plants (Figure 4C). The timing of the initiation of reproductive development was unaffected in the gcn5-1 mutants, but the growth of the inflorescence was strongly affected, resulting in dwarf plants (Figures 4D and 4E). Moreover, the gcn5-1 mutants displayed a loss of apical dominance. The number of secondary inflorescences increased from a typical wild-type number of two or three to an average in gcn5-1 plants of approximately six (Figures 4D and 4E). The flower morphology of the gcn5-1 mutants also was similar to that of the ada2b-1 mutants, with short stamens and petals (Figures 4F to 4H) and reduced fertility. As in ada2b-1 plants, cells in the stamen filament of gcn5-1 were shorter than corresponding cells in wild-type plants (Figures 4I to 4L). The gcn5-1 flowers showed a phenotypic gradient: early-arising flowers had shorter petals and stamens than late flowers (Figures 4G and 4H). The latest flowers produced small siliques that contained only a few seeds.
To determine whether the mutant phenotypes were caused solely by the T-DNA insertion in the GCN5 gene, genetic complementation was performed by transforming gcn5-1 heterozygous plants with the intact GCN5 cDNA linked to six repeats of the Myc epitope tag driven by a constitutive 35S promoter of Cauliflower mosaic virus. The expression of the Myc-tagged GCN5 transgene in plants homozygous for the gcn5-1 disruption allele rescued all gcn5-1 phenotypes, indicating that no other mutations were responsible for the phenotypes (Figure 4D).
Low-Temperature Induction of COR Genes Is Delayed in the ada2b-1 and gcn5-1 Mutants
The observed reduction in COR gene expression in the ada2b-1 and gcn5-1 mutants might have been attributable to either defects in transcriptional activation by the CBF proteins or reduced expression of the CBF genes themselves. To distinguish between these possibilities, we assessed the expression of three CBF genes (CBF1, CBF2, and CBF3) collectively by RNA gel blot analysis using as a probe a cDNA for the entire CBF1 sequence, which hybridizes with all three CBF transcripts (Gilmour et al., 1998 ). The results shown in Figure 5A indicate that CBF transcripts were induced by 1 h of cold treatment in both wild-type and mutant plants. In wild-type plants, CBF transcript levels remained high until gradually declining at 12 and 24 h. By contrast, CBF expression in ada2b-1 and gcn5-1 mutant plants showed a more rapid reduction, reaching a nadir at 8 h, before a second wave of expression evident in 12-h samples. This biphasic pattern of CBF expression in the mutant plants was reproduced in three separate experiments. We infer that the signal transduction pathway leading to initial CBF transcription is unimpaired in the ada2b-1 and gcn5-1 mutants but that some aspect of sustained CBF expression is altered.
Nonacclimated ada2b-1 Plants Display Freezing Tolerance
As expected, nonacclimated wild-type and gcn5-1 plants were killed by freezing temperatures. Remarkably, however, the nonacclimated ada2b-1 plants survived the freezing challenge without significant damage (Figure 6B). This experiment was repeated six times with similar results. Altogether, 86% of the nonacclimated ada2b-1 plants (n = 70) survived the freezing temperatures, in contrast to only 18% of the nonacclimated wild-type plants (n = 70). Given that neither the CBF nor the COR gene is expressed constitutively in the ada2b-1 plants (Figure 5), these results indicate that the freezing tolerance of the ada2b-1 mutants is achieved by a novel, undefined pathway.
Gene Expression Profiles in the ada2b-1 and gcn5-1 Mutants Of the affected genes, 293 (or 73%) were induced and 108 (or 27%) were repressed at least threefold in the mutant plants (Figures 7A and 7C) . Of the induced genes, 149 (51%) were increased in both mutants, whereas 117 (40%) were induced only in gcn5-1 and 27 (9%) were upregulated solely in ada2b-1. Of the repressed genes, 38 (35%) were repressed in both mutants, whereas 43 (40%) were decreased only in ada2b-1 and 27 (25%) were downregulated in gcn5-1.
Approximately 85% of the genes exhibiting significantly higher or lower expression levels in ada2b-1 or gcn5-1 could be assigned to groups according to known or putative functions (Figures 7B and 7D; see also supplemental data online), whereas the remaining genes were characterized as encoding unknown or unclassified proteins. Genes whose expression was increased in ada2b-1 or gcn5-1 mutant plants (Figure 7B; see also supplemental data online) represent a wide range of biological functions, including cellular metabolism (20%), signal transduction (14%), transcription regulation (14%), defense against pathogens (14%), various abiotic stresses (5%), and senescence (4%). The remaining 15% include genes putatively involved in transport, protein destination, cellular organization, energy, or cell growth and development. Genes exhibiting significantly lower expression levels in ada2b-1 and gcn5-1 than in wild-type plants also represent a range of biological functions (Figure 7D; see also supplemental data online). Interestingly, transcripts of genes involved in energy and particularly in photosynthesis accumulated to lower levels in gcn5-1 than in ada2b-1 or wild-type plants.
In yeast, the results of both biochemical and genetic analyses indicate that ADA2 and GCN5 have a common set of functions: the yeast Ada2 and Gcn5 proteins are components of the same multiple-protein complexes, ADA and SAGA, and yeast strains carrying ada2 and gcn5 null mutations have very similar phenotypes (Berger et al., 1992 ; Marcus et al., 1994; Grant et al., 1997, 1998a). Here, we describe the effects of mutations in ADA2b and GCN5 on gene expression and on growth and development in Arabidopsis. Our results indicate that the Arabidopsis ADA2b and GCN5 genes have both common and distinct functions, suggesting that the plant ADA2b and GCN5 proteins are likely to be components of a common coactivator complex, as in yeast, as well as being components of separate complexes with distinct biological activities.
Arabidopsis Ada2b and Gcn5 Proteins Have Common and Distinct Functions
In other respects, however, the phenotypes of the ada2b-1 and gcn5-1 mutants are quite distinct, suggesting that the corresponding proteins also have separate biological functions. For instance, some features of the ada2b-1 phenotype are similar to those of auxin-overproducing mutants, including elongated hypocotyls, small and epinastic cotyledons, reduced leaf expansion rates, increased number of lateral roots, and dwarf plants (Boerjan et al., 1995 Gene expression patterns in the ada2b-1 and gcn5-1 mutants also support the conclusion that ADA2b and GCN5 might function in both the same and distinct coactivator complexes. We analyzed the presence of some 8200 transcripts in leaf tissue from wild-type and mutant plants and observed changes in relative expression levels for some 400 genes in the ada2b-1 and gcn5-1 mutants using rather stringent criteria. Of these genes, nearly half (187 of 401) were affected similarly in both mutants. Thus, these genes might depend on coactivator complexes that contain both ADA2b and GCN5. However, a significant number of genes were affected more significantly in one mutant than in the other (Figure 7). For instance, 27 transcripts that were more abundant in ada2b-1 than in wild-type plants did not show a similar increase in gcn5-1 plants. Conversely, 117 transcripts that were more abundant in gcn5-1 than in wild-type plants did not show a similar increase in ada2b-1 plants. Although in a few cases these distinctions may be rather subtle (if transcript levels barely miss the cutoff criterion), many genes are affected markedly by the ada2b-1 mutation but not by the gcn5-1 mutation (or vice versa), indicating that the ADA2b and GCN5 proteins have distinct biological activities. As with any such microarray analysis, our results cannot distinguish between direct and indirect contributions of ADA2b and GCN5 to the expression levels of specific genes. Moreover, although some of the changes in gene expression in ada2b-1 and gcn5-1 mutant plants are likely to be responsible for the dramatic phenotypes observed, the converse also may be true—that is, some gene expression changes may be the consequence rather than the cause of the pronounced phenotypes. More direct biochemical assays, such as chromatin immunoprecipitation or promoter-based microarray analysis, will be necessary to distinguish between these possibilities. The analysis reported here was limited to a single time point under a single set of growth conditions. Other differences in gene expression are likely to be revealed by examining these mutants at earlier or later times in development, by analyzing additional tissues, and by testing the effects of various growth and stress conditions.
The Arabidopsis genome encodes two homologs of the yeast ADA2 gene, both of which are expressed at the mRNA level in plant roots, leaves, and flowers (Stockinger et al., 2001
Disruption or knockout mutations of GCN5 have been described previously only in budding yeast (Berger et al., 1992
Ada2b and Gcn5 Contribute to Cold-Regulated Gene Expression
The induction of COR gene expression during low-temperature treatment was modestly diminished or delayed in ada2b-1 and gcn5-1 plants compared with wild-type plants. The reduced COR gene expression does not result from an inability to induce CBF gene expression (Figure 5), although the curious decrease in apparent CBF expression at 8 h of cold treatment might reflect a transient difficulty in maintaining CBF expression. If COR gene expression depends exclusively on the CBF regulatory proteins, then our observations suggest that transcriptional activation by CBF proteins is only partially dependent on the ADA2b and GCN5 coactivators. This partial dependence might reflect the existence of other mechanisms for transcriptional activation, perhaps by direct recruitment of basal transcription factors or the RNA polymerase II holoenzyme. A yeast gcn5 mutant strain also showed a delay in induced gene expression, associated with slow remodeling of chromatin at the PHO5 promoter (Barbaric et al., 2001
In another Arabidopsis mutant, sfr6, the expression of CBF genes (but not COR genes) was induced by low temperature (Knight et al., 1999
Enhanced Freezing Tolerance in ada2b-1 Plants
Freezing tolerance in warm-grown Arabidopsis plants has been observed previously in at least two circumstances. First, constitutive expression of CBF proteins themselves can result in COR gene expression and freezing tolerance in warm-grown plants (Jaglo-Ottosen et al., 1998
What, then, makes ada2b-1 plants freezing tolerant? Some of the transcription factors upregulated in nonacclimated ada2b-1 plants, including ERF5 and ERF6, WRKY33 and WRKY53, ZAT10 and ZAT12, and other putative zinc-finger proteins, also are upregulated in cold-acclimated wild-type plants (Fowler and Thomashow, 2002
Positive and Negative Regulation by Arabidopsis ADA2b and GCN5
These findings suggest that ADA2b and GCN5 can function as both positive and negative regulators of transcription. This hypothesis is consistent with a recent report that the yeast SAGA complex plays dual roles, acting to repress and activate specific target genes in different growth conditions (Ricci et al., 2002
Plant Materials and Growth Conditions Arabidopsis thaliana ecotype Wassilewskija-2 was grown in controlled-environment chambers at 22°C under constant illumination with cool-white fluorescent lights (100 to 150 µmol·m-2·s-1) in Bacto planting mix (Michigan Peat, Houston, TX). Plants used for low-temperature experiments were grown on Gamborg's B5 medium (Gibco BRL) for 14 days at 22°C and then placed at 5°C under continuous light (20 to 60 µmol·m-2·s-1).
Identification of T-DNA Mutants
Complementation of the T-DNA Mutants The full-length ADA2b cDNA was subcloned into a binary vector, pKVG1 (35S promoter, bar1 selection), which was derived from pCAMBIA 3301 (CAMBIA, Canberra, Australia), using the restriction sites BamHI and SacI to create pKVA31. The 6Myc epitope-tagged full-length GCN5 cDNA (pKVB32) was subcloned into a binary vector, pKVB24 (35S promoter, bar1 selection), using the restriction sites ClaI and SacI to create pKVB34. The binary vectors pKVA31 and pKVB34 were introduced into Agrobacterium tumefaciens strain GV3101 and used to transform mutant plants heterozygous at the ada2b-1 locus and the gcn5-1 locus, respectively, by the floral-dip method (Clough and Bent, 1998 ). T1 plants were selected in saturated soil with 200 µg/L BASTA. PCR methods were used to distinguish mutant plants homozygous at the ada2b-1 or gcn5-1 locus from wild-type plants in T1-transformed populations.
RNA Analysis
Confocal Microscopy and Scanning Electron Microscopy Flowers from the mutant and wild-type plants were fixed at 4°C for 2 h in 4% glutaraldehyde buffered with 0.1 M sodium phosphate, pH 7.4. After a brief rinse in that buffer, samples were dehydrated in an ethanol series (25, 50, and 75%) for 10 to 15 min and with three 10-min changes in 100% ethanol. Samples were dried in a Balzers critical point dryer (Balzers, Liechtenstein) using liquid carbon dioxide as the transitional fluid and then mounted on aluminum stubs using adhesive tabs. Samples were coated with gold (20 nm thickness) for 9 min in an Emscope Sputter Coater (model SC 500; Ashford, Kent, UK) purged with argon before being examined with a JEOL-35S scanning electron microscope.
Whole Plant Freezing Test
Microarray Experiments Hybridization to the GeneChip Arabidopsis Genome Array (Affymetrix, Santa Clara, CA), scanning, and data analysis were performed at the Michigan State University Genomic Technology Support Facility according to standard Affymetrix protocols. In all GeneChip experiments, hybridization signals are scaled such that the average intensity from all genes is set equal to an arbitrary intensity (which in our case was 1500). By scaling all experiments to the same target intensity, comparisons can be made between the various GeneChips. In our experiments, the scaling factors required to achieve the target intensity ranged from 7 to 28. Two biological replicates of each type of plant (wild type, ada2b-1, or gcn5-1) were obtained, permitting four pair-wise comparisons of wild-type and mutant expression patterns.
Several criteria were applied to identify genes whose expression in mutant plants was different from that in wild-type plants. The gene must have been expressed in one or both of the samples (P or M in the Affymetrix nomenclature; approximately 5400 genes met this criterion). The differences in corrected hybridization signals must have been threefold or greater in at least three of the four pair-wise comparisons. The differences in signal strength must have been designated as either increased (I or MI) or decreased (D or MD) by the Affymetrix software. Within any replicate experimental pair (i.e., wild type versus wild type, ada2b-1 versus ada2b-1, or gcn5-1 versus gcn5-1), only 2% of the genes met these criteria. These differences presumably represent random experimental noise, because few of genes that were identified as different in one replicate pair were determined to be different in the other replicate pairs. Thus, by requiring that differences between wild-type and mutant signals be discerned in three of four pair-wise comparisons, the likelihood of a given gene appearing in our lists by random chance is remote. (Applying the binomial theorem, only Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.
We thank Annette Thelen of the Michigan State University Genomic Technology Support Facility and Sarah Fowler for advice and assistance with Affymetrix microarrays, Joanne Whallon and Shirley Owens for assistance with confocal and electron microscopy, Megan Rich for assistance with plant husbandry, and Kurt Stepnitz for photographic help. We also thank Min-Hao Kuo, Amy Hark, and Yaopan Mao for comments on the manuscript. This work was supported by a grant from the National Science Foundation (MCB 9728462) to M.F.T. and S.J.T., by funds from the Michigan Agricultural Experiment Station and Department of Energy (DEFG 0291ER20021) to M.F.T., and by funds from the Michigan State University Department of Biochemistry and Molecular Biology to S.J.T.
Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.007922. Received September 27, 2002; accepted January 3, 2003.
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Abstract
INTRODUCTION
, a constitutively expressed gene, was used to normalize signals from the COR and CBF genes.
100, were considered in the comparisons; on average, 
1-pyrolline-5-carboxylase synthetase (P5CS), the first enzyme in Pro synthesis, and have 30-fold higher levels of Pro as well as 2-fold higher levels of total soluble sugars than do wild-type plants. Again, we surmise that the freezing tolerance of ada2b-1 likely arises from a different mechanism, because our microarray data indicate that expression of the P5CS gene is not increased in ada2b-1 plants. We conclude that the loss of ADA2b may result in the activation of a freezing tolerance mechanism that differs markedly from the precedents established for the CBF and ESK1 signal transduction pathways.