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AIR POLLUTANT - LOW TEMPERATURE INTERACTIONS IN TREES R.G. Alscher J.R. Cumming J. Fincher Virginia Polytechnic Institute and State University Blacksburg, VA 24061 Boyce Thompson Institute Ithaca, NY 14853 ABSTRACT Boyce Thompson Institute Ithaca, NY 14X53 Evidence is accumulating to suggest a causative role for ozone in winter injury of red spruce. This could be due to an impairment of the winter hardening process brought about by ozone. Hardening in conifers involves a complex series of physiological and ultrastructural adaptations. Ozone is known to affect both photosynthesis and carbohydrate allocation and to stimulate antioxidant production in actively growing crop species, but its metabolic effects on winter hardening in conifers have not been studied. Results from a dose-response study carried out on red spruce seedlings at Boyce Thompson Institute suggest that ozone exposure during the summer and fall leads to changes in carbohydrate metabolism associated with winter hardening and to cell damage during the late fall and early winter. Forest a uniform combine to growing at high elevations are any additional stress, such as pollutant decline at high elevations in both Europe and North America and the lack of causal agent have led to the suggestion that several interacting stresses cause loss of tree vigor (Johnson and Siccama, 1983; Blank, 1985~. Trees often functioning at their physiological limits such that exposure, may lead to tree mortality. In Western Europe, data accumulated over more than a decade in Finland indicate that conifers growing around industrial areas are more susceptible to winter injury in comparison to trees from relatively unpolluted environments (Huttenen et al., 1981; Huttenen and Soikkeli, 1984~. This pattern suggests that industrial pollutants are in some way altering cold tolerance of trees. Winter injury of declining trees has repeatedly been reported in the case of the German forests, and ozone has explicitly been proposed to play a central role in the decline phenomenon (Rehfuess, 1987~. In the United States, extensive evidence has been assembled for a change in the response of red spruce to temperature at high elevations over the past 20 years at Whiteface Mountain, NY, in the Adirondack Mountain Range. This change was found to be correlated in time with increased incidence of tree decline (Johnson et al., l9X6, 1987~. Recent data directly implicate ozone as a causative agent in decreased winter hardiness of spruce. An interaction between ozone and cold temperatures leading to visible injury has been reported in Norway spruce (Brown et al., 1987; Barnes and Davison, 1987~. Several clones that had experienced high ozone concentrations during the summer exhibited uniform browning and abscission of older year class needles. This pattern contrasts with decline symptoms in North America but is consistent with observed damage in Europe. In addition, clonal variation in response to ozone and cold temperature exposure suggests that there is a strong genetic component to resistance, the physiological basis of which is unknown. 341
342 The mechanisms) by which ozone caused this pattern of injury are not understood. Some hypotheses may be formulated, however, through the synthesis of what is known concerning metabolic effects of ozone, changes that underlie the winter hardening process, and physiological responses to cold temperatures. Ozone may impair plant metabolism through the formation of toxic free radicals in viva. Free radicals are metabolically neutralized through the action of a variety of antioxidants, which remove free radicals and their toxic by-products. Antioxidant compounds such as glutathione and SOD are produced in greater quantities by plant cells in response to oxidative stress (Lee and Bennett, 1982) and represent one possible resistance mechanism which may vary both within and among tree species. Mehlhorn et al. (1986) demonstrated increases in the levels of GSH and alpha-tocopherol in needles of spruce and fir as a consequence of long-term, low-level exposures to ozone. Only when the protective capacities of these mechanisms are overwhelmed will injury, such as lipid peroxidation, occur (Halliwell, 1981; Heath, 1987~. The winter hardening process in conifers involves a series of orchestrated physiological, histological, and biochemical events that prepare cells for exposure to cold temperatures. Among these are reductions in photosynthesis, increased hydrolysis of starch to form soluble sugars (Aronsson et al., 1976), an accumulation of antioxidants (Esterbauer and Grill, 1978), and vast ultrastructural changes (Soikkeli, 1978) as trees enter dormancy. The conversion of starch to soluble sugars in leaf tissue represents one mechanism by which plant cells increase their tolerance to freezing temperatures. Oligosaccharides such as sucrose and raffinose are known to protect membranes against the dehydrative effects of freezing (Quinn and Williams, 1985~. Raffinose in particular has been implicated as a cryoprotectant in Norway spruce. In spite of these changes in the cellular environment, damage due to cold temperatures does occur. Under conditions of relatively high light and low temperature, excess light is absorbed by antennae pigments. Reduced electron transport capacity of dormant tissue impairs the transfer of reductant to acceptors such as NADP (Oquist, 1982, 1983, 1986~. Instead, molecular oxygen will serve as an electron acceptor and consequently will be reduced to superoxide in the Mehler reaction. The photoscavenging cycle will act to remove the superoxide and attendant molecular species, and antioxidant compounds will be consumed in the course of this process. If- it continues beyond the limit of antioxidant capacity, the oxidation of labile components such as photosynthetic pigments will follow. Oxidation of this kind does in fact occur. For example, photobleaching of up to half of the total chlorophyll content of Scots pine has been reported to occur under natural conditions in northern Sweden in early spring (Oquist, 1986~. Shade needles show less bleaching than do needles in full sun, as would be expected from the mechanism of injury involved. Increased photo-oxidation of chlorophyll, as well as other free radical injury that occurs at low temperatures and high light in conifers, may be observed under conditions where free radicals are being, or have been, generated by an air pollutant such as ozone. This damage may be due to the exhaustion of antioxidant mechanisms of plant cells brought about by the joint stress of ozone during the growing and/or hardening season and by low temperatures, which occur during the winter months. During the winter, when de novo production of antioxidants may be low, plants that have been previously depleted of antioxidant reserves by ozone may incur damage when protective and repair mechanisms are insufficient to protect tissue further from cold injury. We have biochemical and histological evidence that ozone exposure both altered winter hardening processes and increased cellular damage during early winter frosts of
343 red spruce seedlings. Four-year-old seedlings were exposed to charcoal filtered air or to doses of ozone ranging from ambient to 4X ambient in Ithaca, NY during the summer and fall of 1987. In December there were no visible symptoms on any of the current year foliage. However, histological examination of these needles showed that there was damage to mesophyll cells in needles exposed to ozone. This damage included vesiculation, and at its most extreme involved total disruption of cells with breakage of cell walls and leakage of contents into intercellular spaces. In needles with patches of damage, nearby cells appeared to be undergoing the normal transition, characterized by alterations of chloroplasts, lack of starch, and increased tannin in vacuoles. Disruption of this nature was not seen earlier in the season prior to freezing temperatures. An examination of the patterns of seasonal changes in carbohydrate levels in the seedlings revealed that exposure to ozone also resulted in a shift in the time course for starch mobilization in the late summer and fall. Early frost susceptibility and winter dieback of ozone-treated spruce foliage was observed by Amundson and Cumming (unpublished). Interacting influences on cold tolerance in conifers of growth regulating substances, oxidative stress, and seasonal cycling in response to environmental cues remain to be understood. The combined influences on winter hardening in conifers of growth regulating substances and oxidative stress remain to be elucidated. SUMMARY AND DISCUSSION: METABOLIC ~THRESHOLDS" FOR STRESS We have accumulated biochemical, physiological, and histological data suggesting that exposure to ozone increases the susceptibility of red spruce foliage to cold temperatures. The patterns can be fit into a scenario where prior exposure to ozone overloads the cell's antioxidant resistance mechanisms past some critical level or "threshold," beyond which the cells can no longer tolerate the processes associated with exposure to low temperatures, e.g., free radical production. Were this threshold to be established experimentally, it might be possible to use it as an indicator or "marker" of air pollution stress on coniferous forest tress. It is important to keep in mind that a concept such as this is useful only when implemented with discretion. A tree that is severely stressed with respect to nutrition or water will most probably have a lower threshold for the additional stress of oxidizing air pollutants than one which is not. As a consequence, the need for further research to establish the impact of interacting stresses on the physiology and metabolism of forest trees should be apparent. Oxidative pollutant and cold temperature stresses may further interact if changes in the timing of physiological events associated with the hardening process occur because of pollutant exposure and such alterations increase the susceptibility of foliage directly. Alternatively, these changes could influence the period of time during which the foliage is vulnerable to cold temperatures. Again, further experimentation is required before any evaluation of these various possibilities can be made. REFERENCES Aronsson, A., T. Ingestad, and L. Lars-Gorau. . .. . . . . ~ . 1976. Carbohydrate metabolism and frost naralness In pine and spruce seeollogs at different photoperiods and thermoperiods. Physiol. Plant. 36:127-132. Barnes, J.D., and A.W. Davison. 1987. The influence of ozone on the winter hardiness of Norway spruce (Picea abies L.~. New Phytol. 108: 159-166.
344 Blank, L.W. 1985. A new type of forest decline in Germany. Nature. 314: 311-314. Brown, K.A., T.M. Roberts, and L.W. Blank. 1987. Interaction between ozone and cold sensitivity in Norway spruce: a factor contributing to the forest decline in Central Europe? New Phytol. 105: 149-155. Esterbauer, H., and D. Grill. 1978. Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiol. 61: 1 19-121. Heath, R.L. 1987. Biochemistry of ozone attack on the plasma membrane of plant cells. Rec. Adv. in Phytochem. 21: 29-54. Huttunen, S., L. Karenlampi, and K. Kolari. 1981. Changes in osmotic potential and some related physiological variables in needles of polluted Norway spruce (Picea abies). Ann. Bot. Fennici. 18: 63-71. Huttunen, S., and S. Soikkeli. 1984. Pp. 117-128 in Gaseous Air Pollutants and Plant Metabolism (Koziol, M.J., Whatley, F.R., eds.) Botany School, University of Oxford, Oxford, UK. Johnson, A.H., and T.G. Siccama. 1983. Acid deposition and forest decline. Environ. Sci. Technology 17: 294a-305a. Johnson, A.H., A.~. Friedland, and J. Dushoff. 1986. Recent and historic red spruce mortality: Evidence of climatic influence. Water Air Soil Pollut. 30: 319-330. Johnson, A.H., E.R. Cook, and T.G. Siccama. 1988. Relationships between climate and red spruce growth and decline. Proc. Nat. Acad. Sci. (in press). Lee, E.H., and J.H. Bennett. 1982. Superoxide dismutase. A possible protective enzyme against ozone injury in snap beans (Phaseolus vulgaris L.~. Plant Physiol. 69: 1444- 1449. Mehlhorn, H., G. Seufert, A. Schmidt, and K.J. Kunert. 1986. Effect of SO2 and O3 on production of antioxidants in conifers. Plant Physiol. 82: 336-338. Oquist, G. 1982. Seasonally induced changes in Acyl lipids and fatty acids of chloroplast thylakoids of Pinus silvestris. Plant Physiol. 69: 869-875. Oquist, G. 1983. Effects of low temperature on photosynthesis. Plant, Cell and Environ. 6: 281-300. Oquist, G. 1986. Effects of winter stress on chlorophyll organization and function in Scots pine. J. Plant Physiol. 122: 169-179. Quinn, P.J., and W.P. Williams. 1985. Pp. 1-48 in Photosynthetic Mechanisms and the Environment (Barber, J. and Baker, N.R. eds.) Vol. 6. Amsterdam, New York and Oxford. Rehfuess, K.E. 1987. Perceptions on forest diseases in Central Europe. Forestry. 60~1~: 1-11.
345 Soikkeli, S. 1978. Seasonal changes in mesophyll ultrastructure of needles of Norway spruce (Picea abies). Can. J. Bot. 56: 1932-1940.
344 Blank, L.W. 1985. A new type of forest decline in Germany. Nature. 314: 311-314. Brown, K.A., T.M. Roberts, and L.W. Blank. 1987. Interaction between ozone and cold sensitivity in Norway spruce: a factor contributing to the forest decline in Central Europe? New Phytol. 105: 149-155. Esterbauer, H., and D. Grill. 1978. Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiol. 61: 1 19-121. Heath, R.L. 1987. Biochemistry of ozone attack on the plasma membrane of plant cells. Rec. Adv. in Phytochem. 21: 29-54. Huttunen, S., L. Karenlampi, and K. Kolari. 1981. Changes in osmotic potential and some related physiological variables in needles of polluted Norway spruce (Picea abies). Ann. Bot. Fennici. 18: 63-71. Huttunen, S., and S. Soikkeli. 1984. Pp. 117-128 in Gaseous Air Pollutants and Plant Metabolism (Koziol, M.J., Whatley, F.R., eds.) Botany School, University of Oxford, Oxford, UK. Johnson, A.H., and T.G. Siccama. 1983. Acid deposition and forest decline. Environ. Sci. Technology 17: 294a-305a. Johnson, A.H., A.~. Friedland, and J. Dushoff. 1986. Recent and historic red spruce mortality: Evidence of climatic influence. Water Air Soil Pollut. 30: 319-330. Johnson, A.H., E.R. Cook, and T.G. Siccama. 1988. Relationships between climate and red spruce growth and decline. Proc. Nat. Acad. Sci. (in press). Lee, E.H., and J.H. Bennett. 1982. Superoxide dismutase. A possible protective enzyme against ozone injury in snap beans (Phaseolus vulgaris L.~. Plant Physiol. 69: 1444- 1449. Mehlhorn, H., G. Seufert, A. Schmidt, and K.J. Kunert. 1986. Effect of SO2 and O3 on production of antioxidants in conifers. Plant Physiol. 82: 336-338. Oquist, G. 1982. Seasonally induced changes in Acyl lipids and fatty acids of chloroplast thylakoids of Pinus silvestris. Plant Physiol. 69: 869-875. Oquist, G. 1983. Effects of low temperature on photosynthesis. Plant, Cell and Environ. 6: 281-300. Oquist, G. 1986. Effects of winter stress on chlorophyll organization and function in Scots pine. J. Plant Physiol. 122: 169-179. Quinn, P.J., and W.P. Williams. 1985. Pp. 1-48 in Photosynthetic Mechanisms and the Environment (Barber, J. and Baker, N.R. eds.) Vol. 6. Amsterdam, New York and Oxford. Rehfuess, K.E. 1987. Perceptions on forest diseases in Central Europe. Forestry. 60~1~: 1-11.
