▪ Abstract Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. In this review, recent advances in determining the nature and function of genes with roles in freezing tolerance and the mechanisms involved in low temperature gene regulation and signal transduction are described. One of the important conclusions to emerge from these studies is that cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury. In addition, a family of Arabidopsis transcription factors, the CBF/DREB1 proteins, have been identified that control the expression of a regulon of cold-induced genes that increase plant freezing tolerance. These results along with many of the others summarized here further our understanding of the basic mechanisms that plants have evolved to survive freezing temperatures. In addition, the findings have potential practical applications as freezing te...

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PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms

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 Binding Factor 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.

https://www.pnas.org/content/pnas/94/3/1035.full.pdf

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

In plants, low temperature and dehydration activate a set of genes containing C-repeat/dehydration-responsive elements in their promoter. It has been shown previously that the Arabidopsis CBF/DREB1 transcription activators are critical regulators of gene expression in the signal transduction of cold acclimation. Here, we report the isolation of an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. Overexpression of CBF4 in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation. As a result, the transgenic plants are more tolerant to freezing and drought stress. Because of the physiological similarity between freezing and drought stress, and the sequence and structural similarity of the CBF/DREB1 and the CBF4 proteins, we propose that the plant's response to cold and drought evolved from a common CBF-like transcription factor, first through gene duplication and then through promoter evolution.

Transcription Factor CBF4 Is a Regulator of Drought Adaptation in Arabidopsis

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.

https://www.pnas.org/content/pnas/93/23/13404.full.pdf

Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance

Dehydrins are characterized by the consensus KIKEKLPG amino acid sequence found near the carboxy terminus, and usually repeated from one to many times within the protein. A synthetic peptide containing this consensus sequence was used to produce specific antibodies that recognize dehydrins in a wide range of plants. This range covered two families of monocots, viz. Gramineae (Hordeum vulgare L., Triticum aestivum L., Zea mays L., Oryza sativa L.) and Liliaceae (Allium sativa L.), and five families of dicots, Malvaceae (Gossypium hirsutum L.), Solanaceae (Lycopersicon esculentum L.), Brassicaceae (Raphanus sativus L.), Fabaceae (Vigna unguiculata L.), and Cucurbitaceae (Cucumis sativus L.). Two families of gymnosperms, Pinaceae (Pinus edulis Engelm.) and Ginkgoaceae (Ginkgo biloba L.), were also included. For several plants in which dehydrin cDNA and genomic clones have previously been characterized, it now appears that the dehydrin family of proteins is larger, and the regulation of dehydrin expression much more complex, than earlier studies have shown.

A view of plant dehydrins using antibodies specific to the carboxy terminal peptide.

Spinach (Spinacia oleracea L) seedlings exposed to low nonfreezing temperatures (0–10° C) that promote cold acclimation, synthesize a variety cold-acclimation proteins and at the same time acquire a greater ability to withstand cellular dehydration imposed by the freezing of tissue water Two of these proteins (160 and 85 kDa) become more abundant over time at low temperature In addition, a small decline in tissue water status from a maximally hydrated state also appears to be associated with an initiation of the accumulation of these proteins at a noninductive temperature Imposing a severe water stress on young seedlings grown at 25° C by withholding water leads to substantial accumulation of the 160- and 85-kDa proteins, and maximal induction of freezing tolerance This evidence implies that responses to cold acclimation and water stress involve common mechanisms, and further establishes the linkage of these two proteins with stresses having an osmotic component

Chris Smelser

Papers

Hydration-state-responsive proteins link cold and drought stress in spinach.