Sarah J. Gilmour

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.

cDNA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana

The DNA sequences of cDNAs for twocor (cold-regulated) genes ofArabidopsis thaliana L. (Heyn) were determined. One cDNA (approximately 70% full-length) corresponds to acor gene, designatedcor47, that encodes a 47 kDa hydrophilic polypeptide. The data indicate that COR47 has amino acid sequence homology with Group II LEA (lateembryogenesisabundant) 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 acor gene, designatedcor6.6, that encodes an alanine-rich 6.6 kDa hydrophilic polypeptide. COR6.6 is almost identical to KIN1, a cold-regulatedArabidopsis 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 forcor47 andcor6.6 do not accumulate to high levels in late-developing embryos or fresh mature seeds as is typical oflea gene transcripts. The similarities and differences between COR and LEA proteins are discussed as are their possible roles in freezing and drought tolerance.

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

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.

Effects of COR6.6 and COR15am polypeptides encoded by COR(Cold-Regulated) genes of Arabidopsis thaliana on the freeze-induced fusion and leakage of liposomes.

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.

Effects of COR6.6 and COR15am Polypeptides Encoded by COR (Cold-Regulated) Genes of Arabidopsis thaliana on Dehydration-Induced Phase Transitions of Phospholipid Membranes

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.

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.

Function and regulation of Arabidopsis thaliana COR (cold-regulated) genes

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.

CAMTA-mediated regulation of salicylic acid immunity pathway genes in arabidopsis exposed to low temperature and pathogen infection

CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves action of a repression module that acts independently of calmodulin binding, a finding that challenges current models. 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.

Regulation of Plant Gene Expression in Response to Low Temperature

In 1985, Guy et al. (1985) showed that spinach alters gene expression in response to low nonfreezing temperatures. Results since then have established that cold-regulated gene expression is a common feature of higher plants (Thomashow, 1993). It occurs in chilling tolerant species that cold acclimate (i. e., increase in freezing tolerance in response to low nonfreezing temperatures); chilling tolerant species that do not cold acclimate; and in chilling sensitive species. A basic question raised is how do plants sense low temperature and alter gene expression? Are the sensing and regulatory mechanisms the same in both chilling tolerant and chilling sensitive plants? Also, towards the practical end of crop improvement, can we manipulate these sensing and regulatory systems to increase the chilling and freezing tolerance of agronomically important plants? These and related questions are now being addressed in a number of laboratories. Here, we summarize some of the findings that we have made on cold-regulated gene expression in Arabidopsis thaliana, a chilling tolerant plant that cold acclimates.

Cold Acclimation in Arabidopsis thaliana: Function and Regulation of COR Genes

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.