Freezing Tolerance Versus Freezing Avoidance

Strategies that allow plants to survive freezing temperatures have been placed into two major categories: freezing tolerance and freezing avoidance. Tissues displaying freezing tolerance respond to a low-temperature stress by the loss of cellular water to extracellular ice. This results in collapse of the cell wall (cytorrhysis) and increased concentration of the cell sap, which, in turn, lowers the freezing point. In contrast, tissues that avoid freezing stress but are still exposed to freezing temperatures do so by deep supercooling, a process in which cellular water is isolated from the dehydrative and nucleating effects of ice present outside the cell in extracellular spaces. It is noteworthy that, in many woody perennials, different tissues (bark and leaves versus xylem and buds) within the same plant respond in distinctly different ways to freezing temperatures that represent both aforementioned strategies.

Deep Supercooling of Xylem Tissues

Deep supercooling occurs in more than 240 species in 33 families of angiosperms and one family of gymnosperms (Quamme, 1991). Many deciduous tree fruit crops, such as apple, apricot, cherry, peach, pear, and plum, are known to exhibit deep supercooling. Living xy-lem tissues in most of these fruit crops avoid low temperature stress by deep supercooling. During the supercooling event, cellular water remains liquid within xylem parenchyma cells at very low temperatures by remaining isolated from heterogeneous ice nuclei and the nucleating effect of extracellular ice. Supercooled water is in a metastable condition and will form intracellular ice in response to a heterogeneous nucleation event or when the homogeneous nucle-ation temperature of water (-38°C) is reached. If and when intracellular freezing occurs, it always results in death of the tissue.

The freezing response of xylem tissues of fruit trees can be monitored using the technique of differential thermal analysis (DTA). Thermocouples are utilized to detect the heat of fusion produced by water in the samples as it undergoes a liquid to solid phase change. In DTA, sample temperatures are compared to a piece of freeze-dried tissue (reference) undergoing the same rate of cooling. This produces a flat baseline until the freezing of water within the sample tissue results in a difference in temperature between the sample and the reference. The sample-reference differential is visualized as a peak on a thermogram, hence the term "differential thermal analysis."

In thermograms of woody plants that exhibit deep supercooloing (Figure T1.1), the initial large peak is referred to as a high-temperature exotherm (HTE) and is believed to represent the freezing of bulk water contained within tracheary elements and extracellular spaces, whereas the peak occurring at very low temperatures (deep supercooling) is believed to represent the freezing of intracellular water contained within xylem parenchyma cells. The peak resulting from the freezing of deep supercooled water is referred to as a low-temperature exotherm (LTE). Typically, LTE on these thermograms is correlated with the death of xylem ray parenchyma cells. Because of this association with mortality, DTA has been extensively used to evaluate the degree of cold hardiness of stem tissues of fruit trees. DTA is

FIGURE T1.1. Freezing response of internodal xylem (debarked twig) of peach, flowering dogwood, and willow subjected to differential thermal analysis. HTE: high-temperature exotherm; LTE: low-temperature exotherm. Willow, a non-supercooling species, lacks LTE.

also used to detect seasonal changes in stem hardiness since woody plants display distinct seasonality of supercooling ability, in that it increases and decreases in fall and spring, respectively, and is greatest in winter. Many woody plants, including some fruit crops, do not exhibit deep supercooling. In these species, a typical DTA thermogram lacks LTE (Figure T1.1).

For deep supercooling to occur, a tissue must exhibit several features: (1) cells must be free of heterogeneous nucleating substances that are "active" at warm subfreezing temperatures; (2) a barrier must be present that excludes the growth of ice crystals into a cell; and, concomitantly, (3) a barrier to water movement must exist that prevents a "rapid" loss of cellular water to extracellular ice in the presence of a strong vapor pressure gradient. It is believed that physical properties of the cell wall largely account for the ability of xylem parenchyma cells to deep supercool (Wisniewski, 1995). In this regard, the use of colloidal gold particles of prescribed sizes and other apoplastic tracers have been used to study the porosity and perme ability of cell walls. Results of these studies indicate that the pit membrane (a thin portion of the cell wall that allows for the passage of solutes, as well as plasmodesmatal connections, between cells) and the associated amorphous layer, not the secondary wall, may play a limiting role in determining the ability of the cell wall to retain water against a strong vapor pressure gradient and the intrusive growth of ice crystals. Results from studies with peach xylem tissues indicate that by chemically or enzymatically altering the structure of the pit membrane, which is mainly composed of cellulose and pectic materials, the extent of deep supercooling can be reduced or eliminated (Wisniewski, 1995).

Although pectin-mediated regulation of deep supercooling may account for the aforementioned observations, many fundamental questions must still be resolved. For example, how do species that supercool differ from those that do not? How do we account for seasonal shifts in deep supercooling? If pectin degradation (or lack thereof) is a key determinant of seasonal changes in supercooling ability, do changes in the activity and/or amount of pectin-degrading enzymes parallel these seasonal shifts in supercooling ability? Furthermore, it has been reported that apple, peach, and some other species do not exhibit homogeneous freezing responses, in that their thermograms show multiple LTEs (in apple) or bimodal peaks (in peach). How are these complex freezing behaviors regulated? In-depth investigations aimed at answering these questions will indicate whether this trait can be manipulated in a manner that will enhance cold hardiness in tree fruit crops.

Extraorgan Freezing and Deep Supercooling of Dormant Buds

The response of dormant buds of tree fruit to freezing temperatures is different from that of other portions of the tree and varies with species. The pattern of freezing in the dormant floral buds of apple and pear begins with the initiation of freezing of extracellular water within the bud scales and subtending stem tissues. The introduction of ice into the bud tissue results in the establishment of a water potential gradient. Consequently, water migrates from the shoot or floral apex to the sites of extracellular ice in response to the water potential gradient. Thus, the floral primordium is isolated from the mechanical damage caused by the presence of large ice crystals. This response to freezing temperatures has been described as "extraorgan freezing"

(Sakai, 1979). When buds are killed, mortality results from the dehy-drative stress rather than the low temperature or the presence of ice. Extraorgan freezing is characteristic of most cold-hardy species, and vegetative buds of all temperate fruit species respond in this manner.

In other fruit species, however, not all the water from floral tissue migrates to the ice in bud scales. Instead, a portion of water remains supercooled within the floral tissue. Deep supercooling of flower buds has been observed in several Prunus species (Quamme, 1991). For these species, as with deep supercooling of xylem tissues, two distinct exotherms are detected when buds are subjected to DTA. The HTE is associated with the freezing of water in the bud scales and subtending stem tissue, and the LTE is associated with the freezing of intracellular, deep supercooled water contained within the floral tissue. The LTE is correlated with the degree of cold hardiness of tissue and is used extensively as an evaluation tool. In species containing multiple flowers within a single flower bud, each floret freezes as an independent unit. This is demonstrated by the appearance of multiple LTEs obtained using DTA. This is true for sweet cherry and sour cherry (Quamme, 1995).

As in the case of xylem parenchyma cells, in order for deep supercooling of buds to occur, a barrier to water movement and ice propagation must exist. The nature of this barrier and how deep supercooling in buds is regulated are not fully understood. However, research with peach flower buds suggests that the loss of deep supercooling (during the spring when the buds begin to break dormancy and progressively lose cold hardiness) is associated with the development of vascular continuity between the flower and stem axis (Ashworth, 1984). The functional strand of xylem between the developing bud primordium and the subtending shoot serves as a conduit for the rapid spread of ice into the primordium, and deep supercooling can no longer occur. Whether similar events occur in other fruit species is not known.

Equilibrium Freezing

In contrast to xylem tissue of some species and some dormant buds, which avoid freezing stress by supercooling, bark and leaf tissues of temperate fruit trees undergo "equilibrium freezing" and con-comitantly tolerate the extracellular ice formation and the dehy-drative stress that result from the loss of cellular water to extracellular ice. Equilibrium freezing in plant cells occurs during slow cooling rates (1 to 2°C per hour), when ice formation is initiated at high subzero temperatures (-1 to -4°C) in the extracellular spaces due to a nucleation event. This occurs because (1) the extracellular solution has a higher (warmer) freezing point than the intracellular solution or cell sap and (2) efficient nucleators such as dust or bacteria are prevalent in the extracellular environment. Once the tissue temperature drops below the freezing point of the cell sap, the internal vapor pressure becomes higher than that of extracellular ice. The formation of this gradient results in the movement of cellular water to extracellular ice crystals, which then increase in size. A gradual or slow cooling allows the diffusion of cellular water to ice at a speed sufficient to increase the solute concentration of the cell sap as rapidly as the temperature drops. This allows the chemical potential of the cell sap to be in equilibrium with that of the ice, hence, the term "equilibrium freezing." As a result of this type of freezing, the leaf and bark cells of fruit trees undergo dehydrative stress (due to the loss of cellular water), low temperature stress per se, mechanical stress (due to the presence of large ice crystals and cell wall collapse), and toxic stress (due to the increased concentration of solutes in the cell sap).

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