▪ 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

Plant growth is greatly affected by drought and low temperature. Expression of a number of genes is induced by both drought and low temperature, although these stresses are quite different. Previous experiments have established that a cis-acting element named DRE (for dehydration-responsive element) plays an important role in both dehydration- and low-temperature-induced gene expression in Arabidopsis. Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique. The two cDNA libraries were prepared from dehydrated and cold-treated rosette plants, respectively. The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence similarity, except in the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively. Both the DREB1A and DREB2A proteins specifically bound to the DRE sequence in vitro and activated the transcription of the b-glucuronidase reporter gene driven by the DRE sequence in Arabidopsis leaf protoplasts. Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration. Overexpression of the DREB1A cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. These transgenic plants also revealed freezing and dehydration tolerance. In contrast, overexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused growth retardation of the transgenic plants. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.

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Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis.

Plant growth and productivity are greatly affected by environmental stresses such as drought, high salinity, and low temperature. Expression of a variety of genes is induced by these stresses in various plants. The products of these genes function not only in stress tolerance but also in stress response. In the signal transduction network from perception of stress signals to stress-responsive gene expression, various transcription factors and cis-acting elements in the stress-responsive promoters function for plant adaptation to environmental stresses. Recent progress has been made in analyzing the complex cascades of gene expression in drought and cold stress responses, especially in identifying specificity and cross talk in stress signaling. In this review article, we highlight transcriptional regulation of gene expression in response to drought and cold stresses, with particular emphasis on the role of transcription factors and cis-acting elements in stress-inducible promoters.

Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses.

Agricultural productivity worldwide is subject to increasing environmental constraints, particularly to drought and salinity due to their high magnitude of impact and wide distribution. Traditional breeding programs trying to improve abiotic stress tolerance have had some success, but are limited by the multigenic nature of the trait. Tolerant plants such as Craterostigma plantagenium, Mesembryanthemum crystallinum, Thellungiella halophila and other hardy plants could be valuable tools to dissect the extreme tolerance nature. In the last decade, Arabidopsis thaliana, a genetic model plant, has been extensively used for unravelling the molecular basis of stress tolerance. Arabidopsis also proved to be extremely important for assessing functions for individual stress-associated genes due to the availability of knock-out mutants and its amenability for genetic transformation. In this review, the responses of plants to salt and water stress are described, the regulatory circuits which allow plants to cope wit...

Drought and Salt Tolerance in Plants

Environmental stresses come in many forms, yet the most prevalent stresses have in common their effect on plant water status. The availability of water for its biological roles as solvent and transport medium, as electron donor in the Hill reaction, and as evaporative coolant is often impaired by environmental conditions. Although plant species vary in their sensitivity and response to the decrease in water potential caused by drought, low temperature, or high salinity, it may be assumed that all plants have encoded capability for stress perception, signaling, and response. First, most cultivated species have wild relatives that exhibit excellent tolerance to abiotic stresses. Second, biochemical studies have revealed similarities in processes induced by stress that lead to accumulated metabolites in vascular and nonvascular plants, algae, fungi, and bacteria (Csonka, 1989; Galinski, 1993; Potts, 1994). These metabolites include nitrogen-containing compounds (proline, other amino acids, quaternary amino compounds, and polyamines) and hydroxyl compounds (sucrose, polyols, and oligosaccharides) (McCue and Hanson, 1990). Accumulation of any single metabolite is not restricted to taxonomic groupings, indicating that these are evolutionarily old traits. Third, molecular studies have revealed that a wide variety of species express a common set of genes and similar proteins (for example, Rab-related proteins and dehydrins) when stressed (Skriver and Mundy, 1990; Vilardell et al., 1994). Although functions for many of these genes have not yet been unequivocally assigned, it is likely, based on their characteristics, that these proteins play active roles in the response to stress. Learning about the biochemical and molecular mechanisms by which plants tolerate environmentat stresses is necessary for genetic engineering approaches to improving crop performance under stress. By investigating plants under stress, we can learn about the plasticity of metabolic pathways and the limits to their functioning. Also, questions of an ecological and evolutionary nature need investigation. Are the genes that confer salt tolerance on halophytes and/or drought tolerance on xerophytes evolutionarily ancient genes that have been selected against in saltand drought-sensitive plants (glycophytes) for the sake of productivity? Or have some species obtained nove1 genes in their evolutionary history that have enabled them to occupy stressful environments? How will the

Adaptations to Environmental Stresses.

Previous nuclear run-on experiments indicated that the cor15a (cold-regulated) gene of Arabidopsis thaliana L. (Heyn) has a cold-inducible promoter (Hajela et al., Plant Physiol 93: 1246-1252, 1990). The data presented here indicate that the 5' region of cor15a between nucleotides -305 and +78 (relative to the start of transcription) contains a cis-acting element(s) that can impart cold-regulated gene expression. Histochemical staining experiments indicated that the cor15a promoter is inactive, or very weakly active, in most of the tissues and organs of plants grown at normal temperature and that it becomes activated throughout most of the plant in response to low temperature. Notable exceptions to this general pattern include constitutive activity of the promoter in anthers of control grown plants and apparent inactivity of the promoter in the roots and ovaries of cold-treated plants. Histochemical staining experiments also indicated that low temperature regulation of cor15a does not involve the synthesis of a regulatory molecule that can spread throughout the plant and induce cor gene expression at normal growth temperature. Finally, gene fusion experiments indicated that the 5' region of cor15a between nucleotides -305 and +78, in addition to imparting cold-regulated gene expression, can impart ABA- and drought-regulated gene expression.

The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression.

We previously described a cold-regulated (cor) gene from Arabidopsis thaliana, cor15a (formerly cor15), that encodes a 15 kDa polypeptide that is targeted to chloroplasts (C Lin and M Thomashow, Plant Physiol 99 (1992): 519–525) Here we describe an apparent homologue of cor15a, cor15b cor15b is located immediately downstream from cor15a (within 1 kb) and has the same relative 5′ to 3′ orientation as cor15a The predicted coding regions for the two genes are 82% identical at the nucleic acid sequence level Transcripts for cor15b, like those of cor15a, increase dramatically in response to low temperature and exogenous application of ABA However, only cor15a transcripts accumulate to high levels in response to drought The cor15a/cor15b gene pair is the third case in which A thaliana cold-regulated genes have been found to exist as tandem gene pairs with the members of each pair being differentially regulated

Arabidopsis thaliana cor15b , an apparent homologue of cor15a , is strongly responsive to cold and ABA, but not drought

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.

Kathy S. Wilhelm

Papers

The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression.

Arabidopsis thaliana cor15b , an apparent homologue of cor15a , is strongly responsive to cold and ABA, but not drought

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