Gibberellin Signaling through RGA Suppresses GCN5 Effects on Arabidopsis Developmental Stages.

Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5-6 mutants is reversed in the rga-t2;gcn5-6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.

DNA Barcoding of St. John's wort (Hypericum spp.) Growing Wild in North-Eastern Greece.

Plants of the genus Hypericum, commonly known as "St. John's wort" ("spathohorto" or "valsamo" in Greek), have been used since antiquity for their therapeutic properties. Wild-harvested Hypericum plants are still popular today in herbal medicines, commercially exploited due to their bioactive compounds, hypericin and hyperforin, which have antidepressant, antimicrobial and antiviral activity. Species identification of commercial products is therefore important and DNA barcoding, a molecular method that uses small sequences of organisms' genome as barcodes, can be useful in this direction. In this study, we collected plants of the genus Hypericum that grow wild in North-Eastern Greece and explored the efficiency of matK, and trnH-psbA regions as DNA barcodes for their identification. We focused on 5 taxa, namely H. aucheri, H. montbretii, H. olympicum, H. perforatum subsp. perforatum, and H. thasium, the latter a rare Balkan endemic species collected for the first time from mainland Greece. matK (using the genus-specific primers designed herein), trnH-psbA, and their combination were effectively used for the identification of the 5 Hypericum taxa and the discrimination of different H. perforatum subsp. perforatum populations. These barcodes were also able to discriminate Greek populations of H. perforatum, H. aucheri, H. montbretii, and H. olympicum from populations of the same species growing in other countries.

The histone acetyltransferase GCN5 and the transcriptional coactivator ADA2b affect leaf development and trichome morphogenesis in Arabidopsis.

MAIN CONCLUSION: The histone acetyltransferase GCN5 and associated transcriptional coactivator ADA2b are required to couple endoreduplication and trichome branching. Mutation of ADA2b also disrupts the relationship between ploidy and leaf cell size. Dynamic chromatin structure has been established as a general mechanism by which gene function is temporally and spatially regulated, but specific chromatin modifier function is less well understood. To address this question, we have investigated the role of the histone acetyltransferase GCN5 and the associated coactivator ADA2b in developmental events in Arabidopsis thaliana. Arabidopsis plants with T-DNA insertions in GCN5 (also known as HAG1) or ADA2b (also known as PROPORZ1) display pleiotropic phenotypes including dwarfism and floral defects affecting fertility. We undertook a detailed characterization of gcn5 and ada2b phenotypic effects in rosette leaves and trichomes to establish a role for epigenetic control in these developmental processes. ADA2b and GCN5 play specific roles in leaf tissue, affecting cell growth and division in rosette leaves often in complex and even opposite directions. Leaves of gcn5 plants display overall reduced ploidy levels, while ada2b-1 leaves show increased ploidy. Endoreduplication leading to increased ploidy is also known to contribute to normal trichome morphogenesis. We demonstrate that gcn5 and ada2b mutants display alterations in the number and patterning of trichome branches, with ada2b-1 and gcn5-1 trichomes being significantly less branched, while gcn5-6 trichomes show increased branching. Elongation of the trichome stalk and branches also vary in different mutant backgrounds, with stalk length having an inverse relationship with branch number. Taken together, our data indicate that, in Arabidopsis, leaves and trichomes ADA2b and GCN5 are required to couple nuclear content with cell growth and morphogenesis.

Synergistic action of GCN5 and CLAVATA1 in the regulation of gynoecium development in Arabidopsis thaliana.

In Arabidopsis thaliana the CLAVATA1 (CLV1) receptor and GENERAL CONTROL NON DEREPRESSIBLE 5 (GCN5) histone acetyltransferase both regulate inflorescence meristem size and affect the expression of the meristem-promoting transcription factor WUSCHEL (WUS). Single and multiple mutants of GCN5 and CLAVATA members, were analysed for their gynoecium development, using morphological, physiological, genetic and molecular approaches. The clv1-1gcn5-1 double mutants exhibited novel phenotypes including elongated gynoecia with reduced valves and enlarged stigma and style, indicating a synergistic action of CLAVATA signaling and GCN5 action in the development of the gynoecium. Reporter line and gene expression analysis showed that clv1-1gcn5-1 plants have altered auxin and cytokinin response, distribution and ectopic overexpression of WUS. WUS expression was found in the style of wild-type gynoecia stage 10-13, suggesting a possible novel role for WUS in the development of the style. CLV1 and GCN5 are regulators of apical-basal and mediolateral polarity of the Arabidopsis gynoecium. They affect gynoecium morphogenesis through the negative regulation of auxin biosynthesis and promotion of polar auxin transport. They also promote cytokinin signaling in the carpel margin meristem and negatively regulate it at the stigma. Finally, they synergistically suppress WUS at the centre of the gynoecium.

eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation.

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5'-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation.

Synergistic action of histone acetyltransferase GCN5 and receptor CLAVATA1 negatively affects ethylene responses in Arabidopsis thaliana.

GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is a histone acetyltransferase (HAT) and the catalytic subunit of several multicomponent HAT complexes that acetylate lysine residues of histone H3. Mutants in AtGCN5 display pleiotropic developmental defects including aberrant meristem function. Shoot apical meristem (SAM) maintenance is regulated by CLAVATA1 (CLV1), a receptor kinase that controls the size of the shoot and floral meristems. Upon activation through CLV3 binding, CLV1 signals to the transcription factor WUSCHEL (WUS), restricting WUS expression and thus the meristem size. We hypothesized that GCN5 and CLV1 act together to affect SAM function. Using genetic and molecular approaches, we generated and characterized clv gcn5 mutants. Surprisingly, the clv1-1 gcn5-1 double mutant exhibited constitutive ethylene responses, suggesting that GCN5 and CLV signaling act synergistically to inhibit ethylene responses in Arabidopsis. This genetic and molecular interaction was mediated by ETHYLENE INSENSITIVE 3/ EIN3-LIKE1 (EIN3/EIL1) transcription factors. Our data suggest that signals from the CLV transduction pathway reach the GCN5-containing complexes in the nucleus and alter the histone acetylation status of ethylene-responsive genes, thus translating the CLV information to transcriptional activity and uncovering a link between histone acetylation and SAM maintenance in the complex mode of ethylene signaling.

ADA2b and GCN5 Affect Cytokinin Signaling by Modulating Histone Acetylation and Gene Expression during Root Growth of Arabidopsis thaliana.

In Arabidopsis thaliana, the histone acetyltransferase GCN5 and the associated coactivator ADA2b regulate root growth and affect gene expression. The cytokinin signaling reporter TCS::GFP was introduced into gcn5-1, ada2b-1, and ada2a-2, as well as the ada2a-2ada2b-1 mutants. The early root growth (4 to 7 days post-germination) was analyzed using cellular and molecular approaches. TCS signal accumulated from the fourth to seventh days of root growth in the wild-type columella cells. In contrast, ada2b-1 and gcn5-1 and ada2a-2ada2b-1 double mutants displayed reduced TCS expression relative to wild type. Gene expression analysis showed that genes associated with cytokinin homeostasis were downregulated in the roots of gcn5-1 and ada2b-1 mutants compared to wild-type plants. H3K14 acetylation was affected in the promoters of cytokinin synthesis and catabolism genes during root growth of Arabidopsis. Therefore, GCN5 and ADA2b are positive regulators of cytokinin signaling during root growth by modulating histone acetylation and the expression of genes involved in cytokinin synthesis and catabolism. Auxin application in the roots of wild-type seedlings increased TCS::GFP expression. In contrast, ada2b and ada2ada2b mutant plants do not show the auxin-induced TCS signal, suggesting that GCN5 and ADA2b are required for the auxin-induced cytokinin signaling in early root growth.

Histone Acetyltransferase GCN5 Affects Auxin Transport during Root Growth by Modulating Histone Acetylation and Gene Expression of PINs.

General Control Non-Derepressible 5 (GCN5) is a histone acetyltransferase that targets multiple genes and is essential for the acetylation of Lysine residues in the N-terminal tail of histone H3 in Arabidopsis. GCN5 interacts with the transcriptional coactivator Alteration/Deficiency in Activation 2b (ADA2b), which enhances its activity functioning in multiprotein complexes, such as the Spt-Ada-Gcn5-Acetyltransferase complex (SAGA). Mutations in GCN5 and ADA2b result in pleiotropic phenotypes, including alterations in the growth of roots. Auxin is known to regulate root development by modulating gene expression patterns. Auxin moves polarly during plant growth via the Pin-formed (PIN) auxin efflux transport proteins. The effect of GCN5 and ADA2b on auxin distribution at different stages of early root growth (4 to 7 days post-germination) was studied using the reporter lines DR5rev::GFP and PIN1::PIN1-GFP. In wild-type plants, auxin efflux transporter PIN1 expression increases from the fourth to the seventh day of root growth. The PIN1 expression was reduced in the roots of gcn5-1 and ada2b-1 compared to the wild type. The expression of PIN1 in ada2b-1 mutants is confined only to the meristematic zone, specifically in the stele cells, whereas it is almost abolished in the elongation zone. Gene expression analysis showed that genes associated with auxin transport, PIN1, PIN3 and PIN4, are downregulated in gcn5-1 and ada2b-1 mutants relative to the wild type. As a result, auxin accumulation was also reduced in gcn5-1 and ada2b-1 compared to wild-type roots. Furthermore, acetylation of Lysine 14 of histone H3 (H3K14) was also affected in the promoter and coding region of PIN1, PIN3 and PIN4 genes during root growth of Arabidopsis in gcn5 mutants. In conclusion, GCN5 acts as a positive regulator of auxin distribution in early root growth by modulating histone H3 acetylation and the expression of auxin efflux transport genes.

Arabidopsis thaliana transcriptional co-activators ADA2b and SGF29a are implicated in salt stress responses.

The transcriptional co-activator ADA2b is a component of GCN5-containing complexes in eukaryotes. In Arabidopsis, ada2b mutants result in pleiotropic developmental defects and altered responses to low-temperature stress. SGF29 has recently been identified as another component of GCN5-containing complexes. In the Arabidopsis genome there are two orthologs of yeast SGF29, designated as SGF29a and SGF29b. We hypothesized that, in Arabidopsis, one or both SGF29 proteins may work in concert with ADA2b to regulate genes in response to abiotic stress, and we set out to explore the role of SGF29a and ADA2b in salt stress responses. In root growth and seed germination assays, sgf29a-1 mutants were more resistant to salt stress than their wild-type counterparts, whereas ada2b-1 mutant was hypersensitive. The sgf29a;ada2b double mutant displayed similar phenotypes to ada2b-1 mutant with reduced salt sensitivity. The expression of several abiotic stress-responsive genes was reduced in ada2b-1 mutants after 3 h of salt stress in comparison with sgf29a-1 and wild-type plants. In the sgf29a-1;ada2b-1 double mutant, the salt-induced gene expression was affected similarly to ada2b-1. These results suggest that under salt stress the function of SGF29a was masked by ADA2b and perhaps SGF29a could play an auxiliary role to ADA2b action. In chromatin immunoprecipitation assays, reduced levels of histone H3 and H4 acetylation in the promoter and coding region of COR6.6, RAB18, and RD29b genes were observed in ada2b-1 mutants relative to wild-type plants. In conclusion, ADA2b positively regulates salt-induced gene expression by maintaining the locus-specific acetylation of histones H4 and H3.

The Histone Acetyltransferase GCN5 and the Associated Coactivators ADA2: From Evolution of the SAGA Complex to the Biological Roles in Plants.

Transcription of protein-encoding genes starts with forming a pre-initiation complex comprised of RNA polymerase II and several general transcription factors. To activate gene expression, transcription factors must overcome repressive chromatin structure, which is accomplished with multiprotein complexes. One such complex, SAGA, modifies the nucleosomal histones through acetylation and other histone modifications. A prototypical histone acetyltransferase (HAT) known as general control non-repressed protein 5 (GCN5), was defined biochemically as the first transcription-linked HAT with specificity for histone H3 lysine 14. In this review, we analyze the components of the putative plant SAGA complex during plant evolution, and current knowledge on the biological role of the key components of the HAT module, GCN5 and ADA2b in plants, will be summarized.

The Transcriptional Adaptor Protein ADA3a Modulates Flowering of Arabidopsis thaliana.

Histone acetylation is directly related to gene expression. In yeast, the acetyltransferase general control nonderepressible-5 (GCN5) targets histone H3 and associates with transcriptional co-activators alteration/deficiency in activation-2 (ADA2) and alteration/deficiency in activation-3 (ADA3) in complexes like SAGA. Arabidopsis thaliana has two genes encoding proteins, designated ADA3a and ADA3b, that correspond to yeast ADA3. We investigated the role of ADA3a and ADA3b in regulating gene expression during flowering time. Specifically, we found that knock out mutants ada3a-2 and the double mutant ada3a-2 ada3b-2 lead to early flowering compared to the wild type plants under long day (LD) conditions and after moving plants from short days to LD. Consistent with ADA3a being a repressor of floral initiation, FLOWERING LOCUS T (FT) expression was increased in ada3a mutants. In contrast, other genes involved in multiple pathways leading to floral transition, including FT repressors, players in GA signaling, and members of the SPL transcriptional factors, displayed reduced expression. Chromatin immunoprecipitation analysis revealed that ADA3a affects the histone H3K14 acetylation levels in SPL3, SPL5, RGA, GAI, and SMZ loci. In conclusion, ADA3a is involved in floral induction through a GCN5-containing complex that acetylates histone H3 in the chromatin of flowering related genes.

Two Arabidopsis orthologs of the transcriptional coactivator ADA2 have distinct biological functions.

Histone acetylation is an example of covalent modification of chromatin structure that has the potential to regulate gene expression. Gcn5 is a prototypical histone acetyltransferase that associates with the transcriptional coactivator Ada2. In Arabidopsis, two genes encode proteins that resemble yeast ADA2 and share approximately 45% amino acid sequence identity. We previously reported that plants harboring a T-DNA insertion in the ADA2b gene display a dwarf phenotype with developmental defects in several organs. Here we describe T-DNA insertion alleles in the ADA2a gene, which result in no dramatic growth or developmental phenotype. Both ADA2a and ADA2b are expressed in a variety of plant tissues; moreover, expression of ADA2a from a constitutive promoter fails to complement the ada2b-1 mutant phenotype, consistent with the hypothesis that the two proteins have distinct biochemical roles. To further probe the cellular roles of ADA2a and ADA2b, we studied the response of the transcriptional coactivator mutants to abiotic stress. Although ada2b seedlings display hypersensitivity to salt and abscisic acid and altered responses to low temperature stress, the responses of ada2a seedlings to abiotic stress generally parallel those of wildtype plants. Intriguingly, ada2a;ada2b double mutant plants display an intermediate, gcn5-like phenotype, suggesting that ADA2a and ADA2b each work independently with GCN5 to affect genome function in Arabidopsis.

The histone acetyltransferase GCN5 affects the inflorescence meristem and stamen development in Arabidopsis.

A central question in biology is to understand how gene expression is precisely regulated to give rise to a variety of forms during the process of development. Epigenetic effects such as DNA methylation or histone modification have been increasingly shown to play a critical role in regulation of genome function. GCN5 is a prototypical histone acetyltransferase that participates in regulating developmental gene expression in several metazoan species. In Arabidopsis thaliana, plants with T-DNA insertions in GCN5 (also known as HAG1) display a variety of pleiotropic effects including dwarfism, loss of apical dominance, and floral defects affecting fertility. We sought to determine when during early development floral abnormalities first arise. Using scanning electron microscopy, we demonstrate that gcn5-1/hag1-1 and gcn5-5/hag1-5 mutants display overproliferation of young buds and development of abnormal structures around the inflorescence meristem. gcn5 mutants also display defects in stamen number and arrangement at later stages. This analysis provides temporal and spatial information to aid in the identification of GCN5 target genes in the developing flower. Preliminary studies of putative targets using reverse transcriptase PCR suggest that the floral meristem identity gene LEAFY is among factors upregulated in gcn5-1 mutants.

Expression of hydroxytyrosol and oleuropein biosynthetic genes are correlated with metabolite accumulation during fruit development in olive, Olea europaea, cv. Koroneiki.

Olive tree is one of the most valuable crops cultivated for its oil that is rich in antioxidants. The beneficial effects of oleuropein and hydroxytyrosol (HT), the most abundant and the most powerful antioxidant respectively, as well as tyrosol, HT's precursor molecule, are well studied however their biosynthetic pathways are not yet clarified. The transcriptome analysis of the young olive fruit, cultivar "Koroneiki", revealed transcripts of all the enzymes used to reconstitute the biosynthetic pathway of tyrosol and HT in other organisms. We also identified transcripts of the genes that encode for enzymes involved in the secologanin biosynthesis, oleuropein's precursor molecule. Following the transcriptome analysis, the relative expression of the transcripts was monitored during fruit development and compared to the concentration of the 3 metabolites they synthesize at the same developmental stages. The highest expression levels, accompanied by the maximum concentration of the three metabolites, was found in the young olive fruit. The correlation between the expression profile and the metabolites' concentration indicates that the transcripts were correctly identified and the synthesis of the compounds is regulated at a transcriptional level. Interestingly, HT showed a sudden increment in the final developmental stage of the black mature fruit that is attributed to oleuropein catabolism.

Objective definition of rosette shape variation using a combined computer vision and data mining approach.

Computer-vision based measurements of phenotypic variation have implications for crop improvement and food security because they are intrinsically objective. It should be possible therefore to use such approaches to select robust genotypes. However, plants are morphologically complex and identification of meaningful traits from automatically acquired image data is not straightforward. Bespoke algorithms can be designed to capture and/or quantitate specific features but this approach is inflexible and is not generally applicable to a wide range of traits. In this paper, we have used industry-standard computer vision techniques to extract a wide range of features from images of genetically diverse Arabidopsis rosettes growing under non-stimulated conditions, and then used statistical analysis to identify those features that provide good discrimination between ecotypes. This analysis indicates that almost all the observed shape variation can be described by 5 principal components. We describe an easily implemented pipeline including image segmentation, feature extraction and statistical analysis. This pipeline provides a cost-effective and inherently scalable method to parameterise and analyse variation in rosette shape. The acquisition of images does not require any specialised equipment and the computer routines for image processing and data analysis have been implemented using open source software. Source code for data analysis is written using the R package. The equations to calculate image descriptors have been also provided.

DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çesme-Karaburun Peninsula (Turkey).

The plant family Labiatae (Lamiaceae) is known for its fine medicinal and aromatic herbs like lavender, mint, oregano, sage and thyme and is a rich source of essential oils for the food, pharmaceutical and cosmetic industry. Besides its great economic importance, the Labiatae family contributes significantly to the endemic flora of Greece and Turkey. Owing to its economic and biological significance and to the difficult identification based on morphological characters of several of its taxa, the Labiatae family is an ideal case for developing DNA barcodes. The purpose of this study is to evaluate the utility of DNA barcoding on a local scale in discriminating Labiatae species in Chios Island (Greece) and the adjacent Çesme-Karaburun Peninsula (Turkey). We chose three cpDNA regions (matK, rbcL, trnH-psbA) that were proposed by previous studies and tested them either as single region or as multiregion barcodes based on the criteria determined by Consortium for the Barcode of Life (CBOL). Our results show that matK and trnH-psbA taken as useful in discriminating species of the Labiatae, for the species we examined, as any multiregion combination. matK and trnH-psbA could serve as single-region barcodes for Labiatae species contributing to the conservation and the trade control of valuable plant resources.

The Arabidopsis ortholog of the YEATS domain containing protein YAF9a regulates flowering by controlling H4 acetylation levels at the FLC locus.

Histone acetylation and complexes associated with this process are directly involved in chromatin regulation and gene expression. Among these, NuA4 complex is directly involved in acetylation of histone H4, H2A and H2A.Z. In yeast, the NuA4 complex contains the catalytic subunit, the histone acetyltransferase ESA1, and several associated components including YAF9. In this report we explored the biological role of YAF9a in Arabidopsis thaliana. Homozygous yaf9a-1 and yaf9a-3 mutants show early flowering phenotypes. Moreover, yaf9a-1 mutants displayed reduced expression of the flowering repressor FLC, whereas the expression of the flowering activators FT and SOC1 was induced in comparison to wild-type plants. Using chromatin immunoprecipitation assays with H4 tetra-acetylated antibodies we observed a positive correlation with gene expression profile of FLC and FT in yaf9a-1 mutants under long days. We therefore conclude that YAF9a in Arabidopsis is a negative regulator of flowering by controlling the H4 acetylation levels in the FLC and FT chromatin.

The role of transcriptional coactivator ADA2b in Arabidopsis abiotic stress responses.

Plant growth and crop production can be greatly affected by common environmental stresses such as drought, high salinity and low temperatures. Gene expression is affected by several abiotic stresses. Stress-inducible genes are regulated by transcription factors and epigenetic mechanisms such as histone modifications. In this Mini-Review, we have explored the role of transcriptional adaptor ADA2b in Arabidopsis responses to abiotic stress. ADA2b is required for the expression of genes involved in abiotic stress either by controlling H3 and H4 acetylation in the case of salt stress or affecting nucleosome occupancy in low temperatures response.

Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression.

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.