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.

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.

CBF-dependent and CBF-independent regulatory pathways contribute to the differences in freezing tolerance and cold-regulated gene expression of two Arabidopsis ecotypes locally adapted to sites in Sweden and Italy.

Arabidopsis thaliana (Arabidopsis) increases in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. The CBF regulatory pathway, which contributes to cold acclimation, includes three genes-CBF1, CBF2 and CBF3-encoding closely-related transcription factors that regulate the expression of more than 100 genes-the CBF regulon-that impart freezing tolerance. Here we compare the CBF pathways of two Arabidopsis ecotypes collected from sites in Sweden (SW) and Italy (IT). Previous studies showed that the SW ecotype was more freezing tolerant than the IT ecotype and that the IT ecotype had a nonfunctional CBF2 gene. Here we present results establishing that the difference in CBF2 alleles contributes to the difference in freezing tolerance between the two ecotypes. However, other differences in the CBF pathway as well as CBF-independent pathways contribute the large majority of the difference in freezing tolerance between the two ecotypes. The results also provided evidence that most cold-induced CBF regulon genes in both the SW and IT ecotypes are coregulated by CBF-independent pathways. Additional analysis comparing our results with those published by others examining the Col-0 accession resulted in the identification of 44 CBF regulon genes that were conserved among the three accessions suggesting that they likely have important functions in life at low temperature. The comparison further supported the conclusion that the CBF pathway can account for a large portion of the increase in freezing tolerance that occurs with cold acclimation in a given accession, but that CBF-independent pathways can also make a major contribution.

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.

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.

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.

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.

Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria.

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.

Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities.

When Arabidopsis is exposed to low temperature a small gene family encoding transcription factors known as CBF1, CBF2, and CBF3 (also referred to as DREB1b, DREB1c, and DREB1a, respectively) is rapidly induced followed by expression of CBF-targeted genes, the CBF regulon, which act to bring about an increase in freezing tolerance. The CBF1, 2 and 3 proteins, though highly similar in amino acid sequence, are not identical, raising the question of whether the proteins have the same functions. Here we explored this issue by comparing the effects that overexpression of each CBF gene had on Arabidopsis growth and development, proline and sugar composition, freezing tolerance and gene expression. Taken together, the results support the conclusion that the CBF1, 2 and 3 transcriptional activators have redundant functional activities.

Cold signalling associated with vernalization in Arabidopsis thaliana does not involve CBF1 or abscisic acid.

In genotypes of Arabidopsis that exhibit a winter-annual flowering habit, floral induction in response to extended cold exposure (vernalization) is mediated by repression of the flowering-inhibitor gene FLC. We are interested in identifying components of the cold signal transduction pathway leading to FLC repression. We examined the potential involvement of two factors that are known to play roles in plant cold responses: (1) CBF1, a cold-responsive transcription factor that is involved in activating the cold acclimation response, and (2) the phytohormone abscisic acid (ABA), which has traditionally been associated with plant cold responses. We introduced a transgene driving constitutive expression of CBF1 into a winter-annual genotype of Arabidopsis. In transgenic lines expressing CBF1 mRNA to high levels, FLC mRNA expression was not repressed, and flowering was not accelerated relative to control plants. We also introduced mutations that compromise ABA biosynthesis or sensitivity into a winter-annual genotype and found that the vernalization response was not affected. Finally, we found that presumed increases in ABA levels, as a result of direct application of the hormone or severe water stress, were insufficient to substitute for cold to induce flowering. Taken together, these findings indicate that vernalization involves a pathway that is distinct from cold-response mechanisms involving CBF1, cold-regulated genes under CBF1 control, and ABA.