▪ 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

Molecular studies of drought stress in plants use a variety of strategies and include different species subjected to a wide range of water deficits. Initial research has by necessity been largely descriptive, and relevant genes have been identified either by reference to physiological evidence or by differential screening. A large number of genes with a potential role in drought tolerance have been described, and major themes in the molecular response have been established. Particular areas of importance are sugar metabolism and late-embryogenesis-abundant (LEA) proteins. Studies have begun to examine mechanisms that control the gene expression, and putative regulatory pathways have been established. Recent attempts to understand gene function have utilized transgenic plants. These efforts are of clear agronomic importance.

The molecular basis of dehydration tolerance in plants

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . ......... 187 ICE FORMATION IN PLANTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Intracellular Freezing . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . ...... .. .. . . . . . . . . . . . . . . . . . . 189 Extracellular Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 GENETICS OF FREEZING TOLERANCE . . . . . . . . .. . .. . . ... . ........ 192 Overwintering. . . .. . . . . . . ... . . . . . . .. . . . . . . . . . . . . . .. . . . .... . . . . . .. . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . .. 192 Cold Acclimation. . . . . ..... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 CO LD ACCLIMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Inducible Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Kinetics of Cold Acclimation and Deacclimation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 Protein Metabolism . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Candidate Genes for Upregulation during Cold Acclimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 CONCLUDING REMA RKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Cold Acclimation and Freezing Stress Tolerance: Role of Protein Metabolism

Numerous studies are reviewed of the plasma membrane in relation to freezing injury and cold acclimation. An attempt is made to explore the nature of freezing injury of plants on which to formulate a mechanism(s) of injury and how the biochemical changes occurring during cold acclimation can be accurately assessed. Although it is likely that all cellular membranes are susceptible to freezing injury, there has been a tendency to establish a hierarchy of injury to various cellular membranes. Nevertheless, it is the plasma membrane that plays a central role in the behavior of the a cell during a freeze-thaw cycle. 247 references.

Role of the Plasma Membrane in Freezing Injury and Cold Acclimation

Lipid-transfer proteins (LTP) are basic, 9-kDa proteins present in high amounts (as much as 4% of the total soluble proteinss) in higher plants. LTPs can enhance the in vitro transfer of phospholipids between membranes and can bind acyl chains. On the basis of these properties, LTPs were thought to participate in membrane biogenesis and regulation of the intracellular fatty acid pools. However, the isolation of several cDNAs and genes revealed the presence of a signal peptide indicating that LTPs could enter the secretory pathway. They were found to be secreted and located in the cell wall. Thus, novel roles were suggested for plant LTPs: participation in cutin formation, embryogenesis, defense reactions against phytopathogens, symbiosis, and the adaptation of plants to various environmental conditions. The validity of these suggestions needs to be determined, in the hope that they will elucidate the role of this puzzling family of plant proteins.

Lipid-transfer proteins in plants

Plant Freezing and Damage

Temperature is expected to affect all plant processes. Consistent with this, expression of a large number of specific mRNAs and proteins is up-regulated during cold-acclimation. Their possible functions are outlined, encompassing a wide range of processes, some possibly related to other winter stresses besides cold. Some of the cold-responsive sequences are associated with constitutive or stress-related metabolism, others are probably protective, some may influence freezing, and many are of as yet unknown function. While many of the sequences code for intracellular proteins, a significant number code for apoplastic proteins with a variety of possible functions. Transient influx of calcium into the cytosol appears to be a key step in the response to cold, and also to many other stresses and signals. Similarly, the cold-responsive promoter element identified so far is also responsive to drought and salt. However, how cold is sensed, and any cold-specific aspects of the cold signal transduction pathway, are, so far, unknown. What is clear is that, at least in the model plant Arabidopsis thaliana, cold-, desiccation-, salt- and ABA-triggered signal transduction pathways, and possibly others, run partly in parallel and partly intersect. This may be partly explained by a need for an integrated winter-response. The total number of genes which are cold-responsive and the quantity of resources which this implies are used in this way, indicate that many must have a positive role in acclimation. Experiments which modify membrane lipid unsaturation or solute accumulation, achieved by transformation of plants to express exotic or heterologous genes or by other means, confirm that these factors affect chill- or freezing-tolerance. A transgenic test has shown that one cold-up-regulated gene of previously unknown function contributes to freezing-tolerance, but the small effect re-emphasises the probably cumulative nature of the contributions of many cold-up-regulated sequences to acclimation. On the other hand, mutant analysis indicates some genes may make a comparatively larger contribution. Transformation of alfalfa to overexpress a superoxide dismutase gene increased cold-tolerance and drought-resistance and demonstrated that improvements in field-survival of stresses is possible by transgenic means. Over-expression of a transcription factor, CBF1, conferred freezing-tolerance on Arabidopsis, showing that manipulation of the signal transduction pathway could be an important method for modifying cold-tolerance.

Molecular analysis of acclimation to cold

Freezing of barley ( Hordeum vulgare ), Hordeum murinum , and Holcus lanatus was studied using infrared video thermography. In the field, ice could enter H. lanatus leaves through hydathodes. In laboratory tests with barley, initially 0.4% of the leaf water froze, spreading in alternate strips of high and low freezing intensity longitudinally at 1 to 4 cm s −1 , and simultaneously spreading laterally at 0.3 cm s −1 . Similar results were obtained in the field with H. lanatus . A distinct second, more intense, freezing event spread slowly from the margins of the leaves toward the midrib. Organs of uprooted barley tested in the laboratory froze in this order: nucleated leaf, roots, older leaves, younger leaves, and secondary tillers. When ice spread from one leaf to the rest of the plant the crown delayed spread to the roots and other leaves. There was a longer delay above than below −2°C, helping to protect the crown from freezing during mild frosts. Initial spread of freezing was not damaging. However, the initial spread is a prerequisite for the second freezing event, which can cause damage. The route of the initial spread of ice may be extracellular, drawing water from more gel-like parts of the cell wall.

Freezing of Barley Studied by Infrared Video Thermography

Tissues expressing mRNAs of three cold-induced genes, blt101, blt14, and blt4.9, and a control gene, elongation factor 1alpha, were identified in the crown and immature leaves of cultivated barley (Hordeum vulgare L. cv Igri). Hardiness and tissue damage were assessed. blt101 and blt4.9 mRNAs were not detected in control plants; blt14 was expressed in control plants but only in the inner layers of the crown cortex. blt101 was expressed in many tissues of cold-acclimated plants but most strongly in the vascular-transition zone of the crown; blt14 was expressed only in the inner layers of the cortex and in cell layers partly surrounding vascular bundles in the vascular-transition zone; expression of blt4.9, which codes for a nonspecific lipid-transfer protein, was confined to the epidermis of the leaf and to the epidermis of the older parts of the crown. None of the cold-induced genes was expressed in the tunica, although the control gene was most strongly expressed there. Thus, the molecular aspects of acclimation differed markedly between tissues. Damage in the vascular-transition zone of the crown correlated closely with plant survival. Therefore, the strong expression of blt101 and blt14 in this zone may indicate a direct role in freezing tolerance of the crown.

Localization of Expression of Three Cold-Induced Genes,blt101, blt4.9, and blt14, in Different Tissues of the Crown and Developing Leaves of Cold-Acclimated Cultivated Barley

Roger S. Pearce

Papers

Plant Freezing and Damage

Molecular analysis of acclimation to cold

Freezing of Barley Studied by Infrared Video Thermography

Localization of Expression of Three Cold-Induced Genes,blt101, blt4.9, and blt14, in Different Tissues of the Crown and Developing Leaves of Cold-Acclimated Cultivated Barley

An abscisic‐acid‐responsive, low temperature barley gene has homology with a maize phospholipid transfer protein