Temperature modulation of CAMTA3 gene induction activity is mediated through the DNA binding domain.

The calmodulin-binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C-REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3334 , which includes the N-terminal CG-1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN-LIKE A1 (EXPL1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold-treated plants. Rather, domain-swapping experiments indicated that the CAMTA3 CG-1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG-1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C-terminal calmodulin-binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature.

CAMTA-Mediated Regulation of Salicylic Acid Immunity Pathway Genes in Arabidopsis Exposed to Low Temperature and Pathogen Infection.

Arabidopsis thaliana calmodulin binding transcription activator (CAMTA) factors repress the expression of genes involved in salicylic acid (SA) biosynthesis and SA-mediated immunity in healthy plants grown at warm temperature (22°C). This repression is overcome in plants exposed to low temperature (4°C) for more than a week and in plants infected by biotrophic and hemibiotrophic pathogens. Here, we present evidence that CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves the action of an N-terminal repression module (NRM) that acts independently of calmodulin (CaM) binding to the IQ and CaM binding (CaMB) domains, a finding that is contrary to current thinking that CAMTA3 repression activity requires binding of CaM to the CaMB domain. Induction of SA pathway genes in response to low temperature did not occur in plants expressing only the CAMTA3-NRM region of the protein. Mutational analysis provided evidence that the repression activity of the NRM was suppressed by action of the IQ and CaMB domains responding to signals generated in response to low temperature. Plants expressing the CAMTA3-NRM region were also impaired in defense against the bacterial hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000. Our results indicate that the regulation of CAMTA3 repression activity by low temperature and pathogen infection involves related mechanisms, but with distinct differences.

Genetic and physiological mechanisms of freezing tolerance in locally adapted populations of a winter annual.

PREMISE: Despite myriad examples of local adaptation, the phenotypes and genetic variants underlying such adaptive differentiation are seldom known. Recent work on freezing tolerance and local adaptation in ecotypes of Arabidopsis thaliana from Italy and Sweden provides an essential foundation for uncovering the genotype-phenotype-fitness map for an adaptive response to a key environmental stress. METHODS: We examined the consequences of a naturally occurring loss-of-function (LOF) mutation in an Italian allele of the gene that encodes the transcription factor CBF2, which underlies a major freezing-tolerance locus. We used four lines with a Swedish genetic background, each containing a LOF CBF2 allele. Two lines had introgression segments containing the Italian CBF2 allele, and two contained deletions created using CRISPR-Cas9. We used a growth chamber experiment to quantify freezing tolerance and gene expression before and after cold acclimation. RESULTS: Freezing tolerance was lower in the Italian (11%) compared to the Swedish (72%) ecotype, and all four experimental CBF2 LOF lines had reduced freezing tolerance compared to the Swedish ecotype. Differential expression analyses identified 10 genes for which all CBF2 LOF lines, and the IT ecotype had similar patterns of reduced cold responsive expression compared to the SW ecotype. CONCLUSIONS: We identified 10 genes that are at least partially regulated by CBF2 that may contribute to the differences in cold-acclimated freezing tolerance between the Italian and Swedish ecotypes. These results provide novel insight into the molecular and physiological mechanisms connecting a naturally occurring sequence polymorphism to an adaptive response to freezing conditions.

Genetic basis of photosynthetic responses to cold in two locally adapted populations of Arabidopsis thaliana.

Local adaptation is common, but the traits and genes involved are often unknown. Physiological responses to cold probably contribute to local adaptation in wide-ranging species, but the genetic basis underlying natural variation in these traits has rarely been studied. Using a recombinant inbred (495 lines) mapping population from locally adapted populations of Arabidopsis thaliana from Sweden and Italy, we grew plants at low temperature and mapped quantitative trait loci (QTLs) for traits related to photosynthesis: maximal quantum efficiency (Fv/Fm), rapidly reversible photoprotection (NPQfast), and photoinhibition of PSII (NPQslow) using high-throughput, whole-plant measures of chlorophyll fluorescence. In response to cold, the Swedish line had greater values for all traits, and for every trait, large effect QTLs contributed to parental differences. We found one major QTL affecting all traits, as well as unique major QTLs for each trait. Six trait QTLs overlapped with previously published locally adaptive QTLs based on fitness measured in the native environments over 3 years. Our results demonstrate that photosynthetic responses to cold can vary dramatically within a species, and may predominantly be caused by a few QTLs of large effect. Some photosynthesis traits and QTLs probably contribute to local adaptation in this system.

Natural variation in the C-repeat binding factor cold response pathway correlates with local adaptation of Arabidopsis ecotypes.

The natural range of Arabidopsis thaliana (Arabidopsis) encompasses geographical regions that have greatly differing local climates, including harshness of winter temperatures. A question thus raised is whether differences in freezing tolerance might contribute to local adaptation in Arabidopsis. Consistent with this possibility is that Arabidopsis accessions differ in freezing tolerance and that those collected from colder northern latitudes are generally more tolerant to freezing than those collected from warmer southern latitudes. Moreover, recent studies with Arabidopsis genotypes collected from sites in Sweden (SW) and Italy (IT) have established that the two accessions are locally adapted, that the SW ecotype is more tolerant of freezing than the IT ecotype, and that genetic differences between the two ecotypes that condition local adaptation and freezing tolerance map to a region that includes the C-repeat binding factor (CBF) locus. The CBF locus includes three genes - CBF1, CBF2 and CBF3 - that are induced by low temperature and encode transcription factors that regulate a group of more than 100 genes, the CBF regulon, which impart freezing tolerance. Here we show that cold induction of most CBF regulon genes is lower in IT plants compared with SW plants, and that this is due to the IT CBF2 gene encoding a non-functional CBF2 protein. The non-functional IT CBF2 protein also contributes to the lower freezing tolerance of the IT plants compared with the SW plants. Taken together, studies on the SW and IT ecotypes provide evidence that natural variation in the CBF pathway has contributed to adaptive evolution in these Arabidopsis populations.

Regulation of the Arabidopsis CBF regulon by a complex low-temperature regulatory network.

Exposure of Arabidopsis thaliana plants to low non-freezing temperatures results in an increase in freezing tolerance that involves action of the C-repeat binding factor (CBF) regulatory pathway. CBF1, CBF2 and CBF3, which are rapidly induced in response to low temperature, encode closely related AP2/ERF DNA-binding proteins that recognize the C-repeat (CRT)/dehydration-responsive element (DRE) DNA regulatory element present in the promoters of CBF-regulated genes. The CBF transcription factors alter the expression of more than 100 genes, known as the CBF regulon, which contribute to an increase in freezing tolerance. In this study, we investigated the extent to which cold induction of the CBF regulon is regulated by transcription factors other than CBF1, CBF2 and CBF3, and whether freezing tolerance is dependent on a functional CBF-CRT/DRE regulatory module. To address these issues we generated transgenic lines that constitutively overexpressed a truncated version of CBF2 that had dominant negative effects on the function of the CBF-CRT/DRE regulatory module, and 11 transcription factors encoded by genes that were rapidly cold-induced in parallel with the 'first-wave' CBF genes, and determined the effects that overexpressing these proteins had on global gene expression and freezing tolerance. Our results indicate that cold regulation of the CBF regulon involves extensive co-regulation by other first-wave transcription factors; that the low-temperature regulatory network beyond the CBF pathway is complex and highly interconnected; and that the increase in freezing tolerance that occurs with cold acclimation is only partially dependent on the CBF-CRT/DRE regulatory module.

Photoperiodic regulation of the C-repeat binding factor (CBF) cold acclimation pathway and freezing tolerance in Arabidopsis thaliana.

The CBF (C-repeat binding factor) pathway has a major role in plant cold acclimation, the process whereby certain plants increase in freezing tolerance in response to low nonfreezing temperatures. In Arabidopsis thaliana, the pathway is characterized by rapid cold induction of CBF1, CBF2, and CBF3, which encode transcriptional activators, followed by induction of CBF-targeted genes that impart freezing tolerance. At warm temperatures, CBF transcript levels are low, but oscillate due to circadian regulation with peak expression occurring at 8 h after dawn (zeitgeber time 8; ZT8). Here, we establish that the CBF pathway is also regulated by photoperiod at warm temperatures. At ZT8, CBF transcript levels in short-day (SD; 8-h photoperiod) plants were three- to fivefold higher than in long-day plants (LD; 16-h photoperiod). Moreover, the freezing tolerance of SD plants was greater than that of LD plants. Genetic analysis indicated that phytochrome B (PHYB) and two phytochrome-interacting factors, PIF4 and PIF7, act to down-regulate the CBF pathway and freezing tolerance under LD conditions. Down-regulation of the CBF pathway in LD plants correlated with higher PIF4 and PIF7 transcript levels and greater stability of the PIF4 and PIF7 proteins under LD conditions. Our results indicate that during the warm LD growing season, the CBF pathway is actively repressed by PHYB, PIF4, and PIF7, thus mitigating allocation of energy and nutrient resources toward unneeded frost protection. This repression is relieved by shortening day length resulting in up-regulation of the CBF pathway and increased freezing tolerance in preparation for coming cold temperatures.

Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade.

Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.

Roles of CAMTA transcription factors and salicylic acid in configuring the low-temperature transcriptome and freezing tolerance of Arabidopsis.

Previous studies in Arabidopsis thaliana established roles for CALMODULIN BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) in the rapid cold induction of CRT/DRE BINDING FACTOR (CBF) genes CBF1 and CBF2, and the repression of salicylic acid (SA) biosynthesis at warm temperature. Here we show that CAMTA1 and CAMTA2 work in concert with CAMTA3 at low temperature (4°C) to induce peak transcript levels of CBF1, CBF2 and CBF3 at 2 h, contribute to up-regulation of approximately 15% of the genes induced at 24 h, most of which fall outside the CBF pathway, and increase plant freezing tolerance. In addition, CAMTA1, CAMTA2 and CAMTA3 function together to inhibit SA biosynthesis at warm temperature (22°C). However, SA levels increase in Arabidopsis plants that are exposed to low temperature for more than 1 week. We show that this chilling-induced SA biosynthesis proceeds through the isochorismate synthase (ICS) pathway, with cold induction of ICS1 (which encodes ICS), and two genes encoding transcription factors that positively regulate ICS1 - CBP60g and SARD1 -, paralleling SA accumulation. The three CAMTA proteins effectively repress the accumulation of ICS1, CBP60g and SARD1 transcripts at warm temperature but not at low temperature. This impairment of CAMTA function may involve post-transcriptional regulation, as CAMTA transcript levels did not decrease at low temperature. Salicylic acid biosynthesis at low temperature did not contribute to freezing tolerance, but had a major role in configuring the transcriptome, including the induction of 'defense response' genes, suggesting the possible existence of a pre-emptive defense strategy programmed by prolonged chilling temperatures.

Transcription factors that directly regulate the expression of CSLA9 encoding mannan synthase in Arabidopsis thaliana.

Mannans are hemicellulosic polysaccharides that have a structural role and serve as storage reserves during plant growth and development. Previous studies led to the conclusion that mannan synthase enzymes in several plant species are encoded by members of the cellulose synthase-like A (CSLA) gene family. Arabidopsis has nine members of the CSLA gene family. Earlier work has shown that CSLA9 is responsible for the majority of glucomannan synthesis in both primary and secondary cell walls of Arabidopsis inflorescence stems. Little is known about how expression of the CLSA9 gene is regulated. Sequence analysis of the CSLA9 promoter region revealed the presence of multiple copies of a cis-regulatory motif (M46RE) recognized by transcription factor MYB46, leading to the hypothesis that MYB46 (At5g12870) is a direct regulator of the mannan synthase CLSA9. We obtained several lines of experimental evidence in support of this hypothesis. First, the expression of CSLA9 was substantially upregulated by MYB46 overexpression. Second, electrophoretic mobility shift assay (EMSA) was used to demonstrate the direct binding of MYB46 to the promoter of CSLA9 in vitro. This interaction was further confirmed in vivo by a chromatin immunoprecipitation assay. Finally, over-expression of MYB46 resulted in a significant increase in mannan content. Considering the multifaceted nature of MYB46-mediated transcriptional regulation of secondary wall biosynthesis, we reasoned that additional transcription factors are involved in the CSLA9 regulation. This hypothesis was tested by carrying out yeast-one hybrid screening, which identified ANAC041 and bZIP1 as direct regulators of CSLA9. Transcriptional activation assays and EMSA were used to confirm the yeast-one hybrid results. Taken together, we report that transcription factors ANAC041, bZIP1 and MYB46 directly regulate the expression of CSLA9.

Circadian clock-associated 1 and late elongated hypocotyl regulate expression of the C-repeat binding factor (CBF) pathway in Arabidopsis.

The C-Repeat Binding Factor (CBF) cold-response pathway has a prominent role in cold acclimation, the process whereby certain plants increase tolerance to freezing in response to low nonfreezing temperatures. In Arabidopsis, the CBF pathway is characterized by rapid induction of the C-Repeat Binding Factor 1 (CBF1), CBF2, and CBF3 genes, which encode transcriptional activators, followed by induction of the CBF-targeted genes known as the "CBF regulon." Expression of the CBF regulon results in an increase in freezing tolerance. Previous studies established that CBF1, CBF2, and CBF3 are subject to circadian regulation and that their cold induction is gated by the circadian clock. Here we present the results of genetic analysis and ChIP experiments indicating that both these forms of regulation involve direct positive action of two transcription factors that are core components of the clock, i.e., Circadian Clock-Associated 1 (CCA1) and Late Elongated Hypocotyl (LHY). In plants carrying the cca1-11/lhy-21 double mutation, cold induction of CBF1, CBF2, and CBF3 was greatly impaired, and circadian regulation of CBF1 and CBF3 was essentially eliminated; circadian regulation of CBF2 continued, although with significantly reduced amplitude. Circadian regulation and cold induction of three CBF regulon genes, i.e., COld-regulated Gene15a (COR15A), COR47, and COR78, also were greatly diminished in plants carrying the cca1-11/lhy-21 double mutation. Furthermore, the cca1-11/lhy-21 double mutation resulted in impaired freezing tolerance in both nonacclimated and cold-acclimated plants. These results indicate that CCA1/LHY-mediated output from the circadian clock contributes to plant cold tolerance through regulation of the CBF cold-response pathway.

Cis-regulatory code of stress-responsive transcription in Arabidopsis thaliana.

Environmental stress leads to dramatic transcriptional reprogramming, which is central to plant survival. Although substantial knowledge has accumulated on how a few plant cis-regulatory elements (CREs) function in stress regulation, many more CREs remain to be discovered. In addition, the plant stress cis-regulatory code, i.e., how CREs work independently and/or in concert to specify stress-responsive transcription, is mostly unknown. On the basis of gene expression patterns under multiple stresses, we identified a large number of putative CREs (pCREs) in Arabidopsis thaliana with characteristics of authentic cis-elements. Surprisingly, biotic and abiotic responses are mostly mediated by two distinct pCRE superfamilies. In addition, we uncovered cis-regulatory codes specifying how pCRE presence and absence, combinatorial relationships, location, and copy number can be used to predict stress-responsive expression. Expression prediction models based on pCRE combinations perform significantly better than those based on simply pCRE presence and absence, location, and copy number. Furthermore, instead of a few master combinatorial rules for each stress condition, many rules were discovered, and each appears to control only a small subset of stress-responsive genes. Given there are very few documented interactions between plant CREs, the combinatorial rules we have uncovered significantly contribute to a better understanding of the cis-regulatory logic underlying plant stress response and provide prioritized targets for experimentation.

DNA binding by the Arabidopsis CBF1 transcription factor requires the PKKP/RAGRxKFxETRHP signature sequence.

The CBF/DREB1 transcriptional activators are key regulators of plant freezing tolerance. They are members of the AP2/ERF multi-gene family, which in Arabidopsis comprises about 145 members. Common to these proteins is the AP2/ERF DNA-binding domain, a 60-amino-acid fold composed of a three-stranded beta-sheet followed by a C-terminal alpha-helix. A feature that distinguishes the CBF proteins from the other AP2/ERF proteins is the presence of "signature sequences," PKKP/RAGRxKFxETRHP (abbreviated PKKPAGR) and DSAWR, which are located immediately upstream and downstream, respectively, of the AP2/ERF DNA-binding domain. The signature sequences are highly conserved in CBF proteins from diverse plant species suggesting that they have an important functional role. Here we show that the PKKPAGR sequence of AtCBF1 is essential for its transcriptional activity. Deletion of the sequence or mutations within it greatly impaired the ability of CBF1 to induce expression of its target genes. This impairment was not due to the mutations eliminating CBF1 localization to the nucleus or preventing protein accumulation. Rather, we show that this loss of function was due to the mutations greatly impairing the ability of the CBF1 protein to bind to its DNA recognition sequence, the CRT/DRE element. These results establish that the ability of the CBF proteins to bind to the CRT/DRE element requires amino acids that extend beyond the AP2/ERF DNA-binding domain and raise the possibility that the PKKPAGR sequence contributes to determining the DNA-binding specificity of the CBF proteins.

A comparison of the low temperature transcriptomes and CBF regulons of three plant species that differ in freezing tolerance: Solanum commersonii, Solanum tuberosum, and Arabidopsis thaliana.

Solanum commersonii and Solanum tuberosum are closely related plant species that differ in their abilities to cold acclimate; whereas S. commersonii increases in freezing tolerance in response to low temperature, S. tuberosum does not. In Arabidopsis thaliana, cold-regulated genes have been shown to contribute to freezing tolerance, including those that comprise the CBF regulon, genes that are controlled by the CBF transcription factors. The low temperature transcriptomes and CBF regulons of S. commersonii and S. tuberosum were therefore compared to determine whether there might be differences that contribute to their differences in ability to cold acclimate. The results indicated that both plants alter gene expression in response to low temperature to similar degrees with similar kinetics and that both plants have CBF regulons composed of hundreds of genes. However, there were considerable differences in the sets of genes that comprised the low temperature transcriptomes and CBF regulons of the two species. Thus differences in cold regulatory programmes may contribute to the differences in freezing tolerance of these two species. However, 53 groups of putative orthologous genes that are cold-regulated in S. commersonii, S. tuberosum, and A. thaliana were identified. Given that the evolutionary distance between the two Solanum species and A. thaliana is 112-156 million years, it seems likely that these conserved cold-regulated genes-many of which encode transcription factors and proteins of unknown function-have fundamental roles in plant growth and development at low temperature.

A role for circadian evening elements in cold-regulated gene expression in Arabidopsis.

The plant transcriptome is dramatically altered in response to low temperature. The cis-acting DNA regulatory elements and trans-acting factors that regulate the majority of cold-regulated genes are unknown. Previous bioinformatic analysis has indicated that the promoters of cold-induced genes are enriched in the Evening Element (EE), AAAATATCT, a DNA regulatory element that has a role in circadian-regulated gene expression. Here we tested the role of EE and EE-like (EEL) elements in cold-induced expression of two Arabidopsis genes, CONSTANS-like 1 (COL1; At5g54470) and a gene encoding a 27-kDa protein of unknown function that we designated COLD-REGULATED GENE 27 (COR27; At5g42900). Mutational analysis indicated that the EE/EEL elements were required for cold induction of COL1 and COR27, and that their action was amplified through coupling with ABA response element (ABRE)-like (ABREL) motifs. An artificial promoter consisting solely of four EE motifs interspersed with three ABREL motifs was sufficient to impart cold-induced gene expression. Both COL1 and COR27 were found to be regulated by the circadian clock at warm growth temperatures and cold-induction of COR27 was gated by the clock. These results suggest that cold- and clock-regulated gene expression are integrated through regulatory proteins that bind to EE and EEL elements supported by transcription factors acting at ABREL sequences. Bioinformatic analysis indicated that the coupling of EE and EEL motifs with ABREL motifs is highly enriched in cold-induced genes and thus may constitute a DNA regulatory element pair with a significant role in configuring the low-temperature transcriptome.

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.

Histone dynamics and roles of histone acetyltransferases during cold-induced gene regulation in Arabidopsis.

In Arabidopsis, CBF transcription factors bind to and activate certain cold-regulated (COR) gene promoters during cold acclimation. Consistent with the prevailing model that histone acetylation and nucleosomal depletion correspond with transcriptionally active genes, we now report that H3 acetylation increases and nucleosome occupancy decreases at COR gene promoters upon cold acclimation. Overexpression of CBF1 resulted in a constitutive increase in H3 acetylation and decrease in nucleosome occupancy, consistent with the constitutive activation of COR gene expression. Overexpression of a truncated form of CBF2 lacking its transcriptional activation domain resulted in a cold-stimulated increase in H3 acetylation, but no change in nucleosomal occupancy or COR gene expression, indicating that histone acetylation is congruent with but not sufficient for cold-activation of COR gene expression. Plants homozygous for T-DNA disruption alleles of GCN5 (encoding a histone acetyltransferase) or ADA2b (a GCN5-interacting protein) show diminished expression of COR genes during cold acclimation. Contrary to expectations, H3 acetylation at COR gene promoters was stimulated upon cold acclimation in ada2b and gcn5 plants as in wild type plants, but the decrease in nucleosome occupancy was diminished. Thus, GCN5 is not the HAT responsible for histone acetylation at COR gene promoters during cold acclimation. Several other HAT mutant plants were also tested; although some do affect COR gene expression, none affected histone acetylation. Therefore, H3 acetylation at the COR gene promoters is not solely dependent on any of the HATs tested.

Arabidopsis CAMTA Transcription Factors Regulate Pipecolic Acid Biosynthesis and Priming of Immunity Genes.

The Arabidopsis thaliana Calmodulin-binding Transcription Activator (CAMTA) transcription factors CAMTA1, CAMTA2, and CAMTA3 (CAMTA123) serve as master regulators of salicylic acid (SA)-mediated immunity, repressing the biosynthesis of SA in healthy plants. Here, we show that CAMTA123 also repress the biosynthesis of pipecolic acid (Pip) in healthy plants. Loss of CAMTA123 function resulted in the induction of AGD2-like defense response protein 1 (ALD1), which encodes an enzyme involved in Pip biosynthesis. Induction of ALD1 resulted in the accumulation of high levels of Pip, which brought about increased levels of the SA receptor protein NPR1 without induction of NPR1 expression or requirement for an increase in SA levels. Pip-mediated induction of ALD1 and genes regulating the biosynthesis of SA-CBP60g, SARD1, PAD4, and EDS1-was largely dependent on NPR1. Furthermore, Pip-mediated increase in NPR1 protein levels was associated with priming of Pip and SA biosynthesis genes to induction by low levels of SA. Taken together, our findings expand the role for CAMTA123 in regulating key immunity genes and suggest a working model whereby loss of CAMTA123 repression leads to the induction of plant defense genes and initiation of SAR.

Cold-Induced CBF-PIF3 Interaction Enhances Freezing Tolerance by Stabilizing the phyB Thermosensor in Arabidopsis.

Growth inhibition and cold-acclimation strategies help plants withstand cold stress, which adversely affects growth and survival. PHYTOCHROME B (phyB) regulates plant growth through perceiving both light and ambient temperature signals. However, the mechanism by which phyB mediates the plant response to cold stress remains elusive. Here, we show that the key transcription factors mediating cold acclimation, C-REPEAT BINDING FACTORs (CBFs), interact with PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) under cold stress, thus attenuating the mutually assured destruction of PIF3-phyB. Cold-stabilized phyB acts downstream of CBFs to positively regulate freezing tolerance by modulating the expression of stress-responsive and growth-related genes. Consistent with this, phyB mutants exhibited a freezing-sensitive phenotype, whereas phyB-overexpression transgenic plants displayed enhanced freezing tolerance. Further analysis showed that the PIF1, PIF4, and PIF5 proteins, all of which negatively regulate plant freezing tolerance, were destabilized by cold stress in a phytochrome-dependent manner. Collectively, our study reveals that CBFs-PIF3-phyB serves as an important regulatory module for modulating plant response to cold stress.