Role of Cold-Responsive Genes in Plant Freezing Tolerance

Arabidopsis COR Genes

Among the highly expressed cold-responsive genes of Arabidopsis are the COR genes, also designated LTI(low temperature induced),KIN (cold-inducible), RD(responsive to desiccation), and ERD(early dehydration-inducible; TableI; Thomashow, 1994). The COR genes comprise four gene families, each of which is composed of two genes that are physically linked in tandem array. The COR78, COR15, andCOR6.6 gene pairs encode newly discovered polypeptides, and the COR47 gene pair encodes homologs of LEA group II proteins (also known as dehydrins and LEA D11 proteins). Recent studies (see below) indicate that COR15a acts in concert with otherCOR genes to enhance freezing tolerance.

The COR15a gene of Arabidopsis is expressed in response to low temperature, drought, and ABA (Thomashow, 1994). As mentioned above, it encodes a 15-kD polypeptide that is targeted to the chloroplasts and processed to a 9.4-kD polypeptide designated COR15am. To determine whether COR15a might have a role in freezing tolerance, Artus et al. (1996) constructed transgenic plants that constitutively express the COR15am polypeptide and compared the freezing tolerance of chloroplasts in nonacclimated transgenic and wild-type plants. The results indicated that the COR15am-containing chloroplasts in transgenic plants were 1°C to 2°C more freezing tolerant than were the chloroplasts in wild-type plants that did not contain COR15am (cold acclimation increased chloroplast-freezing tolerance about 6°C). Moreover, the effects of COR15am were not limited to the chloroplasts. Protoplasts isolated from leaves of the nonacclimated transgenic plants that constitutively produced COR15am were about 1°C more freezing tolerant at freezing temperatures between −4°C and −8°C than were those isolated from nonacclimated wild-type plants.

The results of Artus et al. (1996) indicate a role forCOR15a in freezing tolerance. Moreover, they indicate thatCOR15a expression increases the cryostability of the plasma membrane. This conclusion comes from the fact that protoplast survival was measured by vital staining with fluorescein diacetate, a method that reports on retention of the semipermeable characteristics of the plasma membrane. However, unlike cold acclimation that increases protoplast survival over the range of −2°C to −8°C, expression ofCOR15a increased survival only over the temperature range of −4°C to −8°C (if anything, COR15a expression resulted in a slight decrease in protoplast survival between −2°C and −4°C). A possible explanation for this finding is thatCOR15a expression might prevent certain membrane lesions but not others. As noted earlier, the predominant form of membrane injury over the range of −2°C to −4°C appears to be expansion-induced lysis, whereas over the range of −4°C to −8°C, the predominant form of injury is freeze-induced lamellar-to-hexagonal II phase transitions (Steponkus and Webb, 1992; Steponkus et al., 1993). Thus, it is possible that constitutive expression of COR15a might defer the incidence of freeze-induced formation of hexagonal II phase lipids to a lower temperature, but have little or no effect on the incidence of expansion-induced lysis.

The mechanism by which COR15a stabilizes membranes against freeze-induced injury is not yet known. It seems unlikely that the COR15am protein has enzymatic activity, given its simple amino acid composition and primary structure. This, however, leaves open many possibilities. COR15am might interact directly with the chloroplast inner envelope and increase membrane cryostability. The location of COR15am in the chloroplast stroma is not necessarily inconsistent with protection of the plasma membrane, since formation of the hexagonal II phase is an interbilayer event that occurs largely between the plasmalemma and the chloroplast envelope. Decreasing the propensity of the chloroplast envelope to fuse with the plasma membrane could result in less damage to the plasma membrane. Experiments to detect a direct effect of COR15am on the stabilization of membranes, however, have yielded equivocal results (Uemura et al., 1996; Webb et al., 1996). Of course, COR15am may act indirectly to stabilize membranes. For example, it could potentially regulate the activity of other proteins that have roles in freezing tolerance, such as enzymes involved in sugar or lipid metabolism. Additional experiments are required to test these hypotheses.

Although constitutive expression of COR15a clearly enhances freezing tolerance at both the organelle (chloroplast) and cellular (protoplast) levels, the effects are modest (Artus et al., 1996). Moreover, unlike cold acclimation, COR15a expression alone does not result in a detectable increase in freezing survival of whole plants (Jaglo-Ottosen et al., 1998). These findings are not surprising given the results of genetic analyses indicating that freezing tolerance is a multigenic trait involving genes with additive effects (Thomashow, 1990). Indeed, multiple genes are activated by cold acclimation in Arabidopsis, including at least one member of each of the four COR gene pairs (Thomashow, 1994; Hughes and Dunn, 1996).

If multiple COR genes act in concert to increase freezing tolerance, then expression of the entire COR gene “regulon” would presumably increase freezing tolerance more than expressing COR15a alone. This hypothesis was recently tested by Jaglo-Ottosen et al. (1998). Expression of the entire battery ofCOR genes was accomplished by overexpressing the Arabidopsis transcriptional activator CBF1 (Stockinger et al., 1997). CBF1 binds to a DNA regulatory element, the CRT/DRE, which stimulates transcription in response to both low temperature and water deficit (Yamaguchi-Shinozaki and Shinozaki, 1994). The element is present in the promoters of COR15a, COR78,COR6.6, COR47, and presumably other yet-to-be-identified COR genes. Jaglo-Ottosen et al. (1998)found that constitutive overexpression of CBF1 induces expression of COR6.6, COR15a, COR47,and COR78 in nonacclimated Arabidopsis plants. Moreover, it results in an increase in freezing tolerance that is greater than that which occurs upon expressing COR15a alone. Indeed, overexpression of CBF1 increased freezing tolerance at the whole-plant level. Taken together, the results of COR15a andCBF1 overexpression indicate that the Arabidopsis CRT/DRE regulon includes freezing-tolerance genes that have roles in cold acclimation.

Spinach and Cabbage Cryoprotectins

Almost 25 years ago, Volger and Heber (1975) reported that cold-acclimated spinach and cabbage synthesize polypeptides that are highly effective in protecting isolated thylakoid membranes against freeze-thaw damage in vitro. These putative cryoprotective polypeptides were detected only in cold-acclimated plants, suggesting that they were encoded by COR genes. Subsequent studies by Hincha et al. (1990) indicated that the cryoprotective polypeptides act to protect membranes against freeze-induced damage by reducing membrane permeability during freezing and increasing membrane expandability during thawing. A significant limitation in all of these studies, however, was that only partially purified protein preparations were used. Thus, it was unclear whether the cryoprotective activity detected was due to a single protein or to multiple polypeptides. However, from the enrichment procedures used, it was evident that the polypeptides, like the COR polypeptides, were very hydrophilic and remained soluble upon boiling.

A significant advance in the study of the spinach and cabbage cryoprotective proteins was recently made by Sieg et al. (1996), who purified a single cryoprotective protein from cold-acclimated cabbage that is effective in protecting isolated thylakoids against freeze-thaw damage in vitro. This protein, which was designated cryoprotectin, has a mass of 7 kD, remains soluble upon boiling, and appears to be encoded by a cold-inducible gene (the protein is present in cold-acclimated plants but not in nonacclimated plants). Unfortunately, there is no information concerning the amino acid sequence of cryoprotectin, and thus, it is unknown whether it is related to any of the hydrophilic polypeptides encoded by the cold-responsive genes described above. Future studies will hopefully reveal more about the nature of cryoprotectin and its mode of action in vitro and provide direct evidence about whether it has a role in protecting membranes against freezing injury in vivo.

LEA Proteins

The HVA1 gene of barley, which encodes a LEA group III protein (also known as LEA D7 protein), is expressed during cold acclimation, in aleurone layers late in embryogenesis, and in seedlings in response to ABA and water deficit (Hong et al., 1988). Although there is no direct evidence that HVA1 expression during cold acclimation contributes to increased freezing tolerance, there is recent evidence that the gene confers tolerance to dehydration stress.Xu et al. (1996) reported that expression of HVA1 in transgenic rice results in increased tolerance to both water deficit and high-salinity stress. Given the relationship between dehydration tolerance and freezing tolerance, HVA1 is a strong candidate for being a freezing-tolerance gene.

LEA D113 proteins may also have roles in freezing tolerance. Imai et al. (1996) recently demonstrated that expression of the tomatoLe25 gene in yeast increases both the freezing and high salinity tolerance of the cells. Interestingly, the protein did not impart tolerance to high concentrations of sorbitol, indicating that the Le25 protein does not protect cells against low water potentials per se. Regardless, homologs of Le25 seem to be good candidates for being freezing-tolerance genes. In tomato, which is a chilling-sensitive plant that does not cold acclimate, Le25is expressed at very low levels, if at all, in response to low temperature (Cohen et al., 1991). Whether homologs of Le25are expressed at high levels at low temperature in plants that cold acclimate remains to be determined.

Plant AFPs

A recent exciting development in cold acclimation research was the finding that plants, like certain fish and insects, synthesize AFPs in response to low temperature (Urrutia et al., 1992; Griffith et al., 1997). The hallmark characteristic of these proteins is “thermal hysteresis” activity: the proteins decrease the temperature at which ice is formed but do not affect the melting point of the solution. This effect results from AFP's binding to the surface of ice nuclei and inhibiting ice crystal growth. In addition, AFPs affect the shape of the ice crystals that form when temperatures decrease below the freezing point of the solution and are potent inhibitors of ice recrystallization (they inhibit the coalescing of small ice crystals into large ice crystals).

Thermal hysteresis activity has been detected in the cell sap of cold-acclimated plants representing more that 20 species, including both dicotyledonous and monocotyledonous plants (Griffith et al., 1997). Griffith and colleagues (Antikainen and Griffith, 1997; Griffith et al., 1997) have shown that in cereals, including winter and spring rye, winter and spring wheat, winter barley, and spring oats, AFPs accumulate in the apoplastic fluid during cold acclimation (Fig.1). AFPs do not accumulate in cold-treated maize or tobacco, which are freezing-sensitive plants that do not cold acclimate (Fig. 1). Moreover, they do not accumulate to detectable levels in the apoplastic fluids of all plants that cold acclimate. In particular, AFP activity was not detected in the apoplastic fluids of spinach and spring canola and was found at very low levels in apoplastic fluids of winter canola (B. napus) and kale (Fig. 1), all of which are dicotyledonous plants that can survive freezing below −14°C after cold acclimation. It is interesting that cold-acclimated kale does appear to synthesize significant levels of AFPs (Antikainen and Griffith, 1997). Thus, perhaps in kale and other dicots AFPs are primarily present intracellularly.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Antifreeze activity in apoplastic fluids from leaves of cold-acclimated (CA) and nonacclimated (NA) plants. Antifreeze activity was determined by observing the morphology of ice crystals grown in solution. All crystals were photographed at the same magnification. The bar shown on the CA tobacco sample represents 17 μm. These results are reprinted with permission fromAntikainen and Griffith (1997).

Six AFPs ranging in molecular mass from 16 to 35 kD accumulate in the apoplastic fluid of winter rye during cold acclimation (Antikainen and Griffith, 1997). Surprisingly, these AFPs are related to pathogenesis-related proteins, an intriguing situation given that winter cereals have been reported to be more resistant to fungal diseases after cold acclimation (Griffith et al., 1997). N-terminal amino acid sequencing and immunoblot analyses have established that two of the AFPs are endochitinase-like proteins, two are β-1,3-glucanase-like proteins, and two are thaumatin-like proteins (Antikainen and Griffith, 1997). One of the rye AFPs has been biochemically purified and shown to have both endochitinase and antifreeze activity (the protein alters the shape of ice crystals and has a low level of thermal hysteresis activity). In contrast, a purified endochitinase from tobacco, a freezing-sensitive plant, was found to be devoid of antifreeze activity. In addition, endochitinase-, glucanase-, and thaumatin-like proteins are present in cell extracts of nonacclimated rye plants, but no antifreeze activity can be detected. Thus, antifreeze activity is not an inherent property of these proteins. Instead, it appears that there are either specific isozymes of the rye pathogenesis-related proteins that have antifreeze activity and/or posttranslational modifications convert the pathogenesis-related proteins to forms that have antifreeze activity.

It is not yet certain that the plant AFPs contribute to freezing tolerance. There are no mutants of rye or other plants, for instance, that do not produce the AFPs that can be compared with wild-type plants for freezing tolerance. However, given their known activities and accumulation during cold acclimation, it seems highly probable that they have roles in cold acclimation. The results of Marentes et al. (1993) are consistent with this notion. These investigators found that leaves from cold-acclimated rye plants that had the apoplastic proteins removed by washing were less freezing tolerant than control leaves that had not been washed free of apoplastic proteins.

How might the AFPs enhance freezing tolerance? In general, it would not appear to involve inhibition of ice formation. Indeed, the herbaceous plants in which the AFPs have been shown to exist tolerate freezing, they do not avoid it. Moreover, the thermal hysteresis activity of the plant AFPs is quite low (Griffith et al., 1997). Cell sap and apoplastic fluids from cold-acclimated plants produce only a few tenths of a degree of thermal hysteresis. A 67-kD AFP purified from cold-acclimated bittersweet nightshade (Solanum dulcamara) produces only about 0.3°C of thermal hysteresis at a concentration of 10 to 30 mg mL⁻¹ (Duman, 1994). In contrast, certain insects that avoid freezing synthesize AFPs that can produce as much as 7°C of thermal hysteresis at similar protein concentrations. The plant AFPs, however, are potent inhibitors of ice recrystallization, a property that occurs over a much larger temperature range than thermal hysteresis. In the case of apoplastic AFPs, it is conceivable that this property, or the effects that AFPs have on ice crystal shape, might mitigate against physical damage caused by ice and thereby enhance freezing tolerance. Intracellular AFPs, which do not come into direct contact with ice, might have fundamentally different roles. For instance, it has been demonstrated that certain AFPs from fish can inhibit the ice-nucleating activity of the ice-nucleation proteins of Erwinia herbicola(Parody-Morreale et al., 1988). The ability to neutralize potential ice nuclei within plant cells could help prevent intracellular ice formation, a potential problem if temperatures quickly decrease below 0°C, resulting in supercooling of both the intracellular and extracellular fluids. Testing models for how plant APFs might contribute to freezing tolerance is clearly an important objective for the coming years.