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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, CAMTA3

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.

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.

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.

Summary 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.

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 β-sheet followed by a C-terminal α-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.

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.

Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. A fundamental goal of cold acclimation research is to identify genes with key roles in this response. Here we review results from our laboratory regarding the discovery of a family of transcriptional activators in Arabidopsis (Arabidopsis thaliana) that regulates the expression of freezing tolerance genes. Specifically, we have identified 3 genes that encode nearly identical transcriptional activators that bind to the CRT (C-repeat)/DRE (dehydration responsive element) DNA regulatory element present in the promoters of many cold- and drought-inducible genes, including those designated COR (cold-regulated). These regulatory genes, CBF1, CBF2 and CBF3 (CRT/DRE binding factor), are located in tandem array on chromosome 4. Overexpression of the CBF genes in Arabidopsis induces expression of the entire battery of known COR genes and increases freezing tolerance without a low temperature stimulus. We have, therefore, proposed that the CBF genes are ‘master switches’ that activate a regulon of genes involved in cold acclimation. Significantly, the CBF genes themselves are responsive to low temperature; the levels of CBF transcripts begin increasing within 15 min of transferring plants to low temperature followed by accumulation of COR gene transcripts at 2–4 h. The CBF genes do not appear to be subject to autoregulation as the promoter regions have no evident CRT/DRE elements and overexpression of CBF1 does not induce expression of CBF3. Thus, we have suggested that COR gene induction involves a two-step cascade of transcriptional activators: the first step, CBF induction, involving an unknown activator present at normal growth temperature and the second step, COR gene induction, involving the action of the CBF activators.

Cold-induced expression of the Arabidopsis COR (cold-regulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydration-responsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures induces COR gene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of the Arabidopsis genome along with genetic mapping experiments indicated that the three CBF genes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all three CBF genes increased within 15 min of transferring plants to low temperature, followed by accumulation of COR gene transcripts at about 2 h. CBF transcripts also accumulated rapidly in response to mechanical agitation. The promoter regions of the CBF genes do not contain the CRT sequence, CCGAC, and overexpression of CBF1 did not have a detectable effect on CBF3 transcript levels, suggesting that the CBF gene family is not subject to autoregulation. We propose that cold-induced expression of CRT/DRE-containing COR genes involves a low temperature-stimulated signalling cascade in which CBF gene induction is an early event.

Like many plants, Arabidopsis thaliana increases in freezing tolerance in response to low non-freezing temperatures, a phenomenon known as cold acclimation. Associated with cold acclimation are a number of biochemical changes including the expression of COR (cold-regulated) genes. Here we summarize recent progress we have made in understanding the function and regulation of these genes. One significant finding regarding COR gene function is that constitutive expression of COR15a in transgenic Arabidopsis plants enhances the freezing tolerance of both chloroplasts and protoplasts. These results provide the first direct evidence for a COR gene having a role in freezing tolerance. The precise mechanism of COR15a action is not yet know, but current results indicate the gene has a role in stabilizing membranes against freeze-induced damage. In regards to COR gene regulation, we have isolated a cDNA for CBF1, the first identified transcriptional activator that binds to the CRT (C-repeat)/DRE (drought responsive element), a cold- and drought-responsive DNA regulatory element present in the promoters of COR genes. Our working hypothesis is that CBF1 binds to the CRT/DRE sequence and participates in the regulation of COR genes in response to low temperature and drought.

Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 ( C -repeat/DRE B inding F actor 1 ). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1 , which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli . Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in 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.

Arabidopsis thaliana cold-regulated genes COR15a and COR6.6 encode 15- and 6.6-kD polypeptides, respectively. The COR15a polypeptide is known to be targeted to chloroplasts and, during import, to be processed to a 9.4-kD polypeptide designated COR15am. The COR6.6 polypeptide is thought to be located in the cytosol. The coding sequences for COR15am and COR6.6 were fused to the bacteriophage T7 promoter and expressed in Escherichia coli. The recombinant polypeptides COR15amr and COR6.6r were purified to near homogeneity using a combination of ammonium sulfate fractionation, ion-exchange chromatography, and adsorption chromatography on hydroxyapatite. COR15amr and the major species of COR15am in chloroplasts co-migrated on both two-dimensional O'Farrell gels and nondenaturing polyacrylamide gels. These data corroborate the site of COR15a processing and indicate no difference in charge or quaternary structure between COR15amr and the major species of COR15am in plants. In contrast, the migration patterns of COR6.6r and COR6.6 on two-dimensional gels suggest that a considerable portion of the COR6.6 population in plants is modified. In the accompanying papers (M.S. Webb, S.J. Gilmour, M.F. Thomashow, P.L. Steponkus [1996] Plant Physiology 111: 301–312; M. Uemura, S.J. Gilmour, M.F. Thomashow, P.L. Steponkus [1996] Plant Physiology 111:313–327), the effects of COR15amr and COR6.6r on the cryostability and lyotropic phase behavior of liposomes are examined.

Several cold-regulated (COR) polypeptides, which have little or no amino acid sequence identity with known proteins, are synthesized during cold acclimation of Arabidopsis thaliana. However, the function of the polypeptides has yet to be elucidated. The objective of this study was to determine if COR6.6 and COR15am influence the incidence of either freeze-induced fusion or freeze-induced leakage of small unilamellar vesicles (SUVs) composed of either a single species of phosphatidylcholine (either 1-palmitoyl-2-oleoyl-, dioleoyl-, or dilinoleoylphosphatidylcholine), a mixture of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and free sterols (1:1:1, mol:mol), or the total lipid extract of the plasma membrane of either nonacclimated or cold-acclimated rye leaves. When the SUVs were suspended in a dilute tris(hydroxymethyl)-aminomethane/2-(N-morpholino)ethanesulfonic acid buffer, both COR6.6 and COR15am invariably decreased the incidence of freeze-induced fusion regardless of the lipid composition. However, if the SUVs were suspended in a dilute solution of either sucrose or NaCl, the COR polypeptides had little or no effect on the incidence of freeze-induced fusion. Moreover, the COR polypeptides did not decrease the incidence of freeze-induced leakage[mdash]regardless of whether the SUVs were suspended in either the dilute buffer alone or with added sucrose or NaCl. In fact, with SUVs composed of a single species of phosphatidylcholine suspended in the dilute buffer, the COR polypeptides resulted in an anomalous increase in freeze-induced leakage. When considered collectively, these results suggest that neither COR6.6 nor COR15am has a direct cryoprotective effect on SUVs frozen in vitro.

The DNA sequences of cDNAs for two cor (cold-regulated) genes of Arabidopsis thaliana L. (Heyn) were determined. One cDNA (approximately 70% full-length) corresponds to a cor gene, designated cor47, that encodes a 47 kDa hydrophilic polypeptide. The data indicate that COR47 has amino acid sequence homology with Group II LEA (late embryogenesis abundant) proteins, a class of proteins that accumulate late in embryo development. DNA sequence analysis of a second cDNA (containing the complete protein coding sequence) indicates that it represents a cor gene, designated cor6.6, that encodes an alanine-rich 6.6 kDa hydrophilic polypeptide. COR6.6 is almost identical to KIN1, a cold-regulated Arabidopsis gene that has been suggested to have amino acid sequence similarities with type I fish antifreeze proteins (S. Kurkela, M. Franck, Plant Mol Biol 15: 137-144, 1990). Northern analysis indicated that transcripts for cor47 and cor6.6 do not accumulate to high levels in late-developing embryos or fresh mature seeds as is typical of lea gene transcripts. The similarities and differences between COR and LEA proteins are discussed as are their possible roles in freezing and drought tolerance.

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg 'erecta') and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.

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.

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.

We further investigated the role of the ArabidopsisCBF regulatory genes in cold acclimation, the process whereby certain plants increase in freezing tolerance upon exposure to low temperature. The CBF genes, which are rapidly induced in response to low temperature, encode transcriptional activators that control the expression of genes containing the C-repeat/dehydration responsive element DNA regulatory element in their promoters. Constitutive expression of either CBF1 orCBF3 (also known as DREB1b andDREB1a, respectively) in transgenic Arabidopsis plants has been shown to induce the expression of target COR(cold-regulated) genes and to enhance freezing tolerance in nonacclimated plants. Here we demonstrate that overexpression ofCBF3 in Arabidopsis also increases the freezing tolerance of cold-acclimated plants. Moreover, we show that it results in multiple biochemical changes associated with cold acclimation:CBF3-expressing plants had elevated levels of proline (Pro) and total soluble sugars, including sucrose, raffinose, glucose, and fructose. Plants overexpressing CBF3 also had elevated P5CS transcript levels suggesting that the increase in Pro levels resulted, at least in part, from increased expression of the key Pro biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthase. These results lead us to propose that CBF3 integrates the activation of multiple components of the cold acclimation response.

Constitutive expression of the co ld- r egulated COR15a gene of Arabidopsis thaliana results in a significant increase in the survival of isolated protoplasts frozen over the range of −4.5 to −7°C. The increased freezing tolerance is the result of a decreased incidence of freeze-induced lamellar-to-hexagonal II phase transitions that occur in regions where the plasma membrane is brought into close apposition with the chloroplast envelope as a result of freeze-induced dehydration. Moreover, the mature polypeptide encoded by this gene, COR15am, increases the lamellar-to-hexagonal II phase transition temperature of dioleoylphosphatidylethanolamine and promotes formation of the lamellar phase in a lipid mixture composed of the major lipid species that comprise the chloroplast envelope. We propose that COR15am, which is located in the chloroplast stroma, defers freeze-induced formation of the hexagonal II phase to lower temperatures (lower hydrations) by altering the intrinsic curvature of the inner membrane of the chloroplast envelope.

Cold acclimation in plants is associated with the expression of COR (cold-regulated) genes that encode polypeptides of unknown function. It has been widely speculated that products of these genes might have roles in freezing tolerance. Here we provide direct evidence in support of this hypothesis. We show that constitutive expression of COR15a , a cold-regulated gene of Arabidopsis thaliana that encodes a chloroplast-targeted polypeptide, enhances the in vivo freezing tolerance of chloroplasts in nonacclimated plants by almost 2°C, nearly one-third of the increase that occurs upon cold acclimation of wild-type plants. Significantly, constitutive expression of COR15a also affects the in vitro freezing tolerance of protoplasts. At temperatures between −5 and −8°C, the survival of protoplasts isolated from leaves of nonacclimated transgenic plants expressing COR15a was greater than that of protoplasts isolated from leaves of nonacclimated wild-type plants. At temperatures between −2 and −4°C, constitutive expression of COR15a had a slight negative effect on survival. The implications of these data regarding possible modes of COR15a action are discussed.

Cold acclimation of Arabidopsis thaliana includes the expression of cold-regulated (COR) genes and the accumulation of COR polypeptides. The hydration characteristics of two COR polypeptides, COR6.6 and COR15am, have been determined and their effects on the dehydration-induced liquid crystalline-to-gel and lamellar-to-hexagonal II phase transitions in phospholipid mixtures have been examined. After dehydration at osmotic pressures between 8 and 150 MPa, the water content of the COR polypeptides was less than that of bovine serum albumin, with COR15am the least hydrated: bovine serum albumin > COR6.6 > COR15am. Neither COR6.6 nor COR15am altered the dehydration-induced gel lamellar -> fluid lamellar phase transition temperature of either dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine (DOPC). In multilamellar vesicles of dioleoylphosphatidylethanolamine:DOPC (1:1, mol:mol) prepared by either freeze-thaw or reverse-phase evaporation methods, neither COR6.6, COR15am, nor bovine serum albumin altered the incidence of the dehydration-induced formation of the inverted hexagonal phase as a function of osmotic pressure. However, a specific ultrastructural alteration[mdash] the formation of a striated surface morphology in the lamellar domains[mdash]was observed in mixtures of dioleoylphosphatidylethanolamine:DOPC that were dehydrated in the presence of COR15am. Nevertheless, neither COR6.6 nor COR15am appears to participate in a specific protein-phospholipid interaction that alters the dehydration-induced phase behavior of phospholipid vesicles.

Arabidospis thaliana alters gene expression and increases in freezing tolerance in response to low non-freezing temperatures. One of our long range goals has been to determine whether certain COR (cold-regulated) genes that encode hydrophilic, “boiling-soluble” polypeptides have a role in freezing tolerance; another has been to determine the mechanism(s) by which A. thaliana senses low temperature and alters gene expression. Most of our work on COR gene function has focused on COR15. We have found that this gene encodes a 15 kD polypeptide that is targeted to the stromal compartment of chloroplasts. During import, the polypeptide is processed to a hydrophilic 9 kD polypeptide, designated COR15am, that shares a low degree of amino acid sequence similarity with group III LEA (late-embryogenesis-abundant) proteins Transgenic A. thaliana plants that constitutively produce COR15am have been created and tested for freezing tolerance. The data indicate that COR15am enhances the freezing tolerance of chloroplasts in non-acclimated plants by abundant 2°C, nearly one-third of the increase that occurs upon cold acclimation of both transgenic and wild type plants. Constitutive expression of COR15a was also found to affect freezing tolerance at the cellular level: at freezing temperatures between -6 and -8 °C, the survival of protoplasts isolated from leaves of non-acclimated plants expressing CORl5a was greater than that of protoplasts isolated from leaves of plants not expressing CORl5a. The implications of these data regarding possible COR15a functions are discussed. In regarding to gene regulation, the promoters of COR15a and COR78 were found to be up-regulated in response to low temperature, drought and ABA. An examination of COR gene expression in the A. thaliana abi (ABA-insensitive) mutants indicated that low temperature and ABA regulation involve independent pathways. Gene fusion experiments established that COR15a has a functional DRE (drought-regulatory-element) that imparts cold- and drought-regulated gene expression in tobacco. Gel retardation studies indicated that nuclear extracts prepared from both cold-acclimated and non-acclimated Arabidopsis plants contain a protein(s) that binds to the COR15a DRE and that the avidity of the protein(s) for the element is not significantly altered with cold acclimation.

We have previously shown that changes in gene expression occur in Arabidopsis thaliana. L. (Heyn) during cold acclimation (SJ Gilmour, RK Hajela, MF Thomashow [1988] Plant Physiol 87: 745-750). Here we report the isolation of cDNA clones of four cold-regulated (cor) genes from Arabidopsis and examine their expression in response to low temperature, abscisic acid (ABA), water stress, and heat shock. The results of Northern analysis indicated that the transcript levels for the four cor genes, represented by clones pHH7.2, pHH28, pHH29, and pHH67, increased markedly between 1 and 4 hours of cold treatment, reached a maximum at about 8 to 12 hours, and remained at elevated levels for as long as the plants were kept in the cold (up to 2 weeks). Returning cold acclimated plants to control temperature resulted in the levels of the cor transcripts falling rapidly to those found in nonacclimated plants; this occurred within 4 hours for the transcripts represented by pHH7.2 and pHH28, and 8 hours for those represented by pHH29 and pHH67. Nuclear run-on transcription assays indicated that the temperature-regulated expression of the cor genes represented by pHH7.2, pHH28, and pHH29 was controlled primarily at the posttranscriptional level while the cor gene represented by pHH67 was regulated largely at the transcriptional level. Northern analysis also indicated that the levels of cor gene transcripts increased in response to both ABA application and water stress, but not to heat shock. The possible significance of cor genes being regulated by both low temperature and water stress is discussed.

Four enzyme activities catalyzing the following oxidative steps in the gibberellin (GA) biosynthetic pathway have been extracted from spinach (Spinacia oleracea L.) leaves after exposure to 8 long days: GA12 → GA53 → GA44 → GA19 → GA20. Two of these, GA53 oxidase and GA19 oxidase, were separable from the other two, GA44 oxidase and GA12 13-hydroxylase, by anion exchange high performance liquid chromatography (HPLC). Apparent molecular weights of the four enzymes as determined by gel filtration HPLC are: GA12 13-hydroxylase, 28,400; GA53 oxidase, 42,500; GA44 oxidase, 38,100; GA19 oxidase, 39,500. GA44 oxidase was purified approximately 100-fold in 0.3% yield by a combination of ammonium sulfate fractionation, anion exchange HPLC, phenyl-Sepharose chromatography and gel filtration HPLC.

Gibberellins (GAs) A17, A19, A20, A29, A44, 2βOH-GA44 (tentative) and GA29-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA19 which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA1, GA8, GA20, GA29, GA8-catabolite and GA29-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA8, GA20, GA29 and GA29-catabolite, and the presence of GA1 was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA1 in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA20, GA29 and GA29-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10(2)- to 10(3)-fold less than the levels in developing seeds. The 2-epimer of GA29 is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA1 (trace), GA4 (trace), GA8 (trace), GA12, GA17, GA20 (tentative) (trace), GA25, GA34, GA48, 18-hydroxy-GA4, 12β-hydroxy-GA9, and 18-hydroxy-GA34 (tentative). A hydrolyzed butanol extract contained GA17, GA20, GA48, and 18-hydroxy-GA34 (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA1, GA3 (possibly a contaminant), GA17, GA19, GA20, GA34, GA48, and 18-hydroxy-GA34 (tentative). A hydrolyzed butanol extract contained GA34, GA48, and 18-hydroxy-GA34 (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1β-Hydroxylated GAs previously found in wheat were not found in barley in this study.

[(13)C, (3)H]Gibberellin A20 (GA20) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [(13)C, (3)H]Gibberellin A20 was metabolized to [(13)C, (3)H]GA29-catabolite and [(13)C, (3)H]GA1 by the normal, and to [(13)C, (3)H]GA29 and [(13)C, (3)H]GA1 by the dwarf-5 mutant. In the dwarf-1 mutant, [(13)C, (3)H]GA20 was metabolized to [(13)C, (3)H]GA29 and [(13)C, (3)H]GA29-catabolite; no evidence was found for the metabolism of [(13)C, (3)H]GA20 to [(13)C, (3)H]GA1. [(13)C, (3)H]Gibberellin A8 was not found in any of the feeds. In all feeds no dilution of (13)C in recovered [(13)C, (3)H]GA20 was observed. Also in the dwarf-5 mutant, the [(13)C]label in the metabolites was apparently undiluted by endogenous [(13)C]GAs. However, dilution of the [(13)C]label in metabolites from [(13)C, (3)H]GA20 was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA20 to GA1.

Cell-free extracts capable of converting [14C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [14C]-labeled GAs, prepared enzymically from [14C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA12 → GA53 → GA44 → GA19 → GA20. The pH optima for the enzymic conversions of [14C]GA53, [14C]GA44 and [14C]GA19 were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe2+, α-ketoglutarate and O2 for activity, and ascorbate stimulated the conversion of [14C]GA53 and [14C]GA19. Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA53 and GA19 are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA44 remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA20 content of spinach plants in LD than in SD.

The induction of α-amylase by exogenously supplied gibberellin A1 (GA1) and GA4 in embryoless caryopses of Hordeum vulgare (cv. Himalaya) was determined indirectly by measuring reducing sugars released from the endosperm. The presence of the inhibitors of GA biosynthesis, 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo 1618), Ancymidol, 2-chloroethyl trimethyl ammonium chloride (CCC) or (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl)pentan-3-ol (PP333) did not inhibit α-amylase production by either GA1 or GA4.

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24 degrees C, increased in freezing tolerance from an initial 50% lethal temperature (LT(50)) of about -3 degrees C to an LT(50) of about -6 degrees C after 24 hours at 4 degrees C; LT(50) values of -8 to -10 degrees C were achieved after 8 to 9 days at 4 degrees C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A(+)) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4 degrees C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4 degrees C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.