The influence of elevated ozone on freezing ... - Wiley Online Library

New Phytol. (1994), 126, 327-335

The influence of elevated ozone on freezing tolerance of red spruce seedlings BY C. E. W A 1 T E \

D. H. D E H A Y E S \

J. REBBECK^ G. A. SCHIER^

AND A. H. JOHNSON^ ^ School of Natural Resources, The University of Vermont, Burlington, VT 05405, U.S.A. ^ U.S.D.A. Eorest Service, Northeastern Eorest Experiment Station, Delaware, OH 43015, U.S.A. ^Department of Geology, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. {Received 27 July 1993; accepted 14 October 1993) SUMMARY Laboratory cold-tolerance assessments were conducted over two seasons on red spruce (Picea rubens Sarg.) seedlings fumigated with various concentrations of ozone (O^) for one or two growing seasons in three independent experiments at three locations. Ozone fumigations were performed at either Boyce Thompson Institute in Ithaca, NY (BTI), the University of Maine in Orono, ME (UMO), or the CS Forest Service Research Laboratory in Delaware. OH (USFS). Acid mist treatments of either pH 3-0 or 4-2 were applied in combination with O^ treatments at USFS. Seedlings fumigated with moderate or high concentrations of Og were never significantly less cold tolerant than seedlings exposed to charcoal-filtered (CF) air or low 0^ concentrations. In fact, there was a tendency for seedlings fumigated with low concentrations of 0^ or CF air to be the least cold tolerant. USFSfumigated seedlings receiving the lowest O^ concentration (50/0 nl I"', day/night concentration) were least cold tolerant on six of the seven sampling dates and significantly less cold tolerant in October and January. In addition, UMO-fumigated seedlings receiving CF-air were significantly less cold tolerant in January than those receiving either ambient air (no chamber or non-filtered treatments) or elevated O3. Acid mist treatments had little influence on the cold tolerance of red spruce seedlings in autumn and early winter, but in January- 1990 seedlings exposed to pH 3-0 acid mist were approximately 6 °C less cold tolerant than those receiving pH 4 2 . Following the 1988-89 winter, freezing injury was evident on all seedlings fumigated at BTI, but differences between 3 x ambient O^ and CF treatments were not significant. Key words: Red spruce, ozone, cold tolerance, freezing injury, acid mist.

INTRODUCTION Winter injury has been implicated as a contributing factor to the reported decline of red spruce {Picea rubens Sarg.) throughout much of the northern part of its natural range (Johnson, Cook &Siccama, 1988; DeHayes et al., 19906; Wilkinson, 1990). Indeed, winter injury to current-year needles of red spruce m native and planted stands has been reported for many years (Curry & Church, 1952; Pomerleau, 1962; Morgenstern, 1969; Weiss et al., 1985; Johnson, Friedland & Dushoff, 1986) and has been particularly frequent during the past decade (Friedland et al., 1984; Johnson et al., 1988; DeHayes et al., 19906). Several recent studies have demonstrated that low temperature rather than foliar desiccation is the environmental factor responsible for injury to red spruce (see DeHayes, 1992 for detailed discussion).

Laboratory cold-tolerance studies also show that, although red spruce foliage is sufficiently cold tolerant in autumn (September to November) and spring (March to May) to escape freezing injury, it does not attain a midwinter depth of cold hardmess expected for boreal and montane species native to eastern North America (Sheppard, Smith & Cannell, 1989; DeHayes et al., 19906), In fact, maximum midwinter hardiness levels for red spruce are barely sufficient to protect foliage from mmimum temperatures commonly encountered in winter in the northern half of its range. Clearly, any natural or anthropogenic disturbance that decreases cold tolerance of red spruce by just a few degrees would likely result in an increased frequency of freezing mjury. Recent evidence suggests that tropospheric ozone (O3) may alter growth and production and allocation of carbohydrates in woody plants, and that both O3

328

C. E. Waite and others

and acidic cloudwater may directly or indirectly at greatest risk from freezing injury (DeHayes, influence the degree of cold tolerance and the 1992). Furthermore, results from different fumisusceptibility of trees to freezing injury. For ex- gation experiments have been in conflict. Neverample, visible foliar injury to year-old needles of theless, because O3 has been shown to reduce frost Norway spruce (Picea abies L. Karst.) was observed hardiness in some plants, and the frequency of both immediately following an autumn (November) frost potentially damaging O3 events and freezing injury subsequent to a summer O3 treatment (Brown, to red spruce have increased during the past 30 yr, it Roberts & Blank, 1987). Controlled freezing studies is plausible to consider 03-mediated freezing injury also revealed reduced or delayed autumn cold as an initiating factor in red spruce decline. This acclimation of year-old needles of Norway spruce paper examines the infiuence of summer and autumn (Barnes & Davison, 1988) and current-year foliage of fumigations with ambient or elevated concentrations Sitka spruce [Picea sitchensis (Bong) Carr.] (Lucas of O3 on subsequent development of freezing et al., 1988) seedlings exposed to O3 during the tolerance in current-year needles of red spruce previous growing season. However, frost hardiness seedlings. tests performed in December on previously O3fumigated Sitka spruce seedlings did not detect any evidence of reduced cold tolerance of Og-treated MATERIALS AND METHODS seedlings (Lucas et aL, 1988). Although the mech- Ozone exposures anism of an Og-induced delay in frost hardening In 1987, 1988, and 1989, potted red spruce seedlings has not been established, O3 has been shown to affect were exposed to differing concentrations of O3 for membrane permeability, enzyme activity, and photo- one or two growing seasons in three separate synthetic carbon reduction (Skarby & Sellden, 1984; fumigation experiments conducted independently at Freer-Smith & Mansfield, 1987), all of which could three different institutions. Because previous O3 potentially have a detrimental influence on cold experiments with red spruce had been inconsistent, acclimation and winter cold tolerance of forest trees. we chose a cooperative experimental approach Information on the influence of elevated O3 on among laboratories so that consistency in red spruce freezing tolerance of red spruce has been incon- cold tolerance responses could be examined. Arsistent. Fincher et al. (1989) observed mesophyll rangements were made for cold tolerance assessdisruption in early winter to red spruce seedlings ments to be made on red spruce plant material that exposed to elevated O3 for one season. However, had undergone O3 fumigations as part of ongoing similar injury was not detected following a second experiments at Boyce Thompson Institute in Ithaca, season of exposure (Fincher & Alscher, 1992). When NY (BTI), the University of Maine in Orono, ME red spruce seedlings were evaluated for the extent of (UMO), and at the U S Forest Service Research visible freezing injury in spring, Fincher et aL (1989) Laboratory in Delaware, OH (USFS). Each fureported that the influence of O3 on winter injury migation experiment was independently designed ranking and the number of shoots injured was not and, as a result, O3 treatments and seed sources and significant, although a slight effect of elevated O3 on ages of red spruce seedlings differed somewhat winter injur\' was evident when seedlings not among fumigation experiments. Because cold tolexhibiting winter injury symptoms were removed erance differences were compared among treatments from the data set. Furthermore, the correlations within each experiment rather than across experibetween visible freezing injury and measures of ments and each experimental data set was analysed cellular disruption were not significant. Amundson, separately, the different treatments and seedlings at Kohut & Laurence (1991 a) reported no significant each fumigation location posed no experimental, effect of O3 on cold tolerance of red spruce seedlings analytical, or interpretation problems. In fact, we in September to early November, following two believe the opportunity to interpret O3 response data seasons of exposures at concentrations up to 2 x am- across multiple experimental situations strengthens bient at Ithaca, NY. Finally, in a field exclusion our conclusions. study conducted on Whitetop Mountain, Virginia, At BTI, fumigations took place during summer DeHayes et al. (1991) found no significant difference and autumn 1987 and 1988 (1 June to 1 December, in autumn, winter, and spring cold tolerance of red 1987 and 1 June to 1 October, 1988). Ten 3-yr-old, spruce seedlings that were exposed to ambient O3 red spruce seedlings of Nova Scotia (NS) origin were (average 12 h concentration, 54 nl 1"'; 1 h maximum, exposed in open-top chambers to charcoal-filtered 163 nl 1"') and those exposed to reduced O3 (average air (CF) and ten were exposed to 3 x ambient O3 and maximum concentrations, 24 and 86 nl r \ levels at Ithaca (3X) in a completely randomized respectively). design with four chambers per treatment (8 To date, there have been no published reports of chambers total) and 2 or 3 seedlings per chamber. a seasonal assessment of the influence of O3 on the Fumigations were performed 8 to 11 h d"V2 h after developmental cold tolerance of red spruce, es- sunrise to 2 h before sunset) at BTI. Ambient levels pecially during winter when the species appears to be of O3 ranged from 20 to 60 nil"' and averaged

Ozone and cold tolerance in red spruce 3 5 ^ 0 nl P at the exposure site throughout the treatment period. Upon completion of the O3 treatments in 1988, the seedlings were moved to Burlington, VT where they acclimated and overwintered under ambient conditions. Minimum ambient temperatures during the 1988-89 winter at Burlington reached - 2 2 ° , - 2 4 ° , and - 2 3 °C, during December, January, and February, respectively. At UMO, 3-yr-old red spruce seedlings of NS origin were exposed to one of five O3 treatments in an open-top chamber experiment following a randomized complete block design with three replications. Each plot of each treatment (chamber) was represented by 28 seedlings. From 9 June to 14 October 1988, seedlings were exposed to one of five O3 treatments (4 chamber and 1 non-chatnber) for 8 h d"' (09.00 to 17.00 h EST). The treatments were: (1) ambient ozone (no chamber) (NC), (2) charcoalfiltered air (CF), (3) non-filtered air (NF), (4) nonfiltered air with the addition of 75 nl 1"^ O3 (NF + 75), and (5) non-filtered air with the addition of 150 n! 1"' O3 (NF' -I-150). Ambient levels of O3 at the treatment site ranged from 10 to 80 nil"' and averaged 20—30 nl r^ during the treatment period. Prior to winter, the chambers w'ere removed and the perimeter of undisturbed plots of seedlings were surrounded with snow fencing and hay bales to protect the roots from freezing damage. The seedlings then acclimated and overwintered at the University of Maine under ambient conditions. Minimum ambient temperatures during the 1988-89 winter at Orono reached —19 °C in December and — 21 °C in Januar\' and February. At USFS, 72 potted, 4-yr-old, red spruce seedlings of Maine (ME) or New Hampshire (NH) origin were exposed indoors in continuously stirred tank reactor (CSTR) chambers to day (16 h)/night (8 h) O3 levels of 50/0, 100/50. or 150/100 nl I"' from 5 May to 5 September, 1989. These seedlings also received weekly treatn:ients (40 min wk"-^; 12-7 mm) with an acid mist solution (SO4^":NO2", 2:1) of either pH 3-0 or 4 2. The randomized complete block design consisted of a 3 (O3) x 2 (acid mist) factorial arrangement of treatments replicated three times with four seedlings per O.j-acidic mist combination per replicate. Photoperiod was regulated to correspond to natural daylength at 45° north latitude at an intensity of 225 fiE m"^ s"\ throughout the treatment period. In early September, the seedlings were transported to Burlington, VT where they were placed in an insulated rack raised 0 5 m above the ground and individual pots were surrounded with bark mulch. The seedlings acclimated and overwintered under ambient conditions. During winter 1989-90, December was an extremely cold month in Burlington and the entire northeastern US. Minimum ambient temperatures in Burlington reached a low of — 27 °C and remained below freezing for the

329 entire month. The average temperature for December 1989 was — 14°C. Minimum ambient temperatures during January and February at Burlington were —17°, and —21 °C> respectively. Laboratory cold-tolerance assessments Laboratory cold-tolerance assessments of currentyear needles were conducted on eleven dates, over two winters, to determine the extent and nature of cold-tolerance differences among red spruce seedlings associated with O3 fumigation treatments. On the morning of each assessment, current-year shoots were collected from each of 2—4 seedlings per treatment per replicate (10-12 seedlings per treatment) for seedlings treated at BTI (assessed during winter 1988-89) and USFS (assessed during winter 1989-90), Shoots were placed in sealed plastic bags and transported immediately to the laborator\'. Shoots from seedlings fumigated at UMO (assessed during winter 1988-89) were collected from each of six seedlings per treatment per replicate (18 seedlings per treatment) one day prior to assessment, packed in an insulated cooler with ice packs, and sent via overnight delivery to Burlington, VT for coldtolerance testing. A previous study had shown no change in cold tolerance for red spruce foliage samples stored under these conditions (DeHayes, Waite & Ingle, 1990a), In the laboratory, currentyear needles were excised from shoots and 0 5 g of needles from each replicate of each treatment were placed in seven separate glass test tubes and each test tube of needles assigned at random to 1 of 7 subfreezing test temperatures. Prior to freezing, all test tubes were placed in a 4 °C cooler for 2 h to equilibrate. Following equilibration, racks of test tubes representing each of the seven subfreezing test temperatures were placed in a computer-controlled freezing cabinet and temperature was lowered at the rate of 3 °C h " \ Each preselected test temperature was maintained for 0-5 h after which time racks of test tubes representing that test temperature were placed in insulated boxes, remo\'ed from the freezer, and thawed in a 4 °C cooler. After thawing, 10 ml of deionized water was added to each test tube. Following equilibration (20-22 h), tissue injury was assessed using electrolyte leakage. Original field fumigation designs were maintained throughout the laboratory studies. Field freezing injury In early March 1989, mottling and discolouration of current-year needles, indicative of the early stages of freezing injury commonly observed on red spruce, was seen on some of the red spruce seedlings that had been fumigated at BTI. This injury became more pronounced with time and on 9 May 1989, percent visible injury to current-year needles was recorded for each of the BTl-treated seedlings.

330

C E. Waite and others

Table 1. Critical temperatures during autumn, winter, and early spring (1989-90) of current-year needles from red spruce seedlings fumigated at the US Forest Service Research Laboratory in Delaware, Ohio {USFS) zoith three (day/night) concentrations of ozone, in combination with tzvo add mist treatments, during the 1989 growing season Critical temperatures (°C) on Ozone treatment

1989

(nl 1-')

25 Sep

50/0

-9 -9 -9 21

100/50 150/100 CV (%) Treatment comparisons 50/0 vs. mean of

ns

100/50, 150/100 100/50 vs. 150/100

ns

1990 16 0ct

30Oct

13Nov

18 Dec

22 Jan

19 Mar

-10 -11 -14 16

-20 -24 -24 9

-39 -41 -41

-43 -49 -46 14

-40 -46 -48 9

^26 -30 -32 15

*

«#

ns

ns

«

ns

ns

ns

ns

ns

ns

10

; * * / ' ^ 0 ' 0 1 ; ns. not significant. Seedlings were treated with ozone and acid mist at USFS, but overwintered in Burlington, Vermont. Coefficients of variation (CV) provide a relative measure of error variance on each sampling date.

Data analysis

RESIILTS

Upon completion of laboratory- freezing procedures Influence of O3 on cold tolerance a critical temperature (7^), defined as the highest Cold-tolerance assessments performed over two temperature at which statistically significant freezing seasons on red spruce seedlings fumigated at three injury can be detected, was calculated separately for independent locations, with O3 concentrations up to each treatment and replicate combination for each 166 nl r^ (ambient-I-150 nl I"-*), revealed no signififumigation experiment on each sampling date cant negative influence of O3 on cold tolerance (DeHayes & Williams, 1989). In previous studies, (Tables 1, 2). On none of the 11 sampling dates were critical temperatures have been shown to be strongly seedlings fumigated with moderate or high concencorrelated with field freezing injury for several trations of O3 significantly less cold tolerant than species (Kolb, Steiner & Barbour, 1985; DeHayes & those exposed to CF air or low O3 concentrations. In Williams, 1989), and have been effective in dis- fact, there was a tendency for seedlings exposed to cerning cold-tolerance differences between fertilizer low O3 concentrations or CF air to be the least cold treatments (DeHayes, Ingle & Waite, 1989), between tolerant. For instance, for seedlings fumigated at different-aged needles of red spruce and balsam fir USFS, those receiving the lowest O3 treatment [Abies balsamea (L.) Mill.] (DeHayes et aL, 19906), (50 nl r^ during day, 0 nl 1"' at night) were least cold and between different tissues of pitch pine (Pinus tolerant on 6 of 7 dates on which cold tolerance was rigida Mill.) (Berrang & Steiner, 1986). For detailed assessed, and significantly less cold tolerant than one descriptions of freezing procedures, tissue injury or both of the higher O3 treatments on 16 and 30 assays, and critical temperature calculation, refer to October, and 22 January (Table 1). For seedlings DeHayes & Williams (1989) and DeHayes et al. fumigated at UMO, those exposed to CF air were (\99Qa). among the least cold tolerant in December and Critical temperature data from each fumigation significantly less cold tolerant in January than those experiment on each sampling date were subjected to exposed to either elevated O3 concentration or analysis of variance to determine the significance of ambient air (Table 2). These data collectively differences among O3, and for the USFS-treated provide no evidence for reduced cold tolerance seedlings, acid mist treatments and the acid mist x O3 associated with increasing O3 concentration and, in interaction. Because O3 concentrations are con- fact, appear to contradict the general opinion and sidered quantative treatments, orthogonal contrasts some studies that suggest elevated O3 reduces cold were used to discern the nature of treatment tolerance in red spruce. differences. Field freezing injury data were examined by analysis of variance to determine the significance of differences in injury between the two O3 treat- Infiuence of acid mist on cold tolerance ments. Acid mist treatments did not significantly influence

Ozone and cold tolerance in red spruce

331

Table 2. Critical temperatures during winter and early spring (1988-89) of current-year needles from red spruce seedlings fumigated with various ozone concentrations at the University of Maine (UMO) or Boyce Thompson Institute (BTI)

Ozone treatment UMO No chamber (NC) Non-filtered air (NF) Charcoal-filtered air (CF) CV(%) Treatment comparisons CF vs. mean of N F + 75, N F + 1 5 0 N F + 75 vs. N F + 1 5 0 N F fi. mean of CF, NF-i-75, N F + 1 5 0 NC vs. mean of NF, CF, NF + 75. NF+150 BTI 3 X ambient (3X) Charcoal-filtered air (CF) CV (%) Treatment comparisons 3X vs. C F

Critical temperatures {°C) on „ _ ^ 19 Dec 88 23 Jan 89 27 Feb 89

27 Mar 89

—36 -36 —36 -36 —30 -30 -34 -30 20 20

-—49 49 —49 -49 -—38 38 -46 -45 9 9

—36 —40 —42 -42 -42 10

ns

#

ns

ns ns

ns

—

—

as

-37 -43 U

-47 -48 11

-42 -40

ns

ns

ns

22

-38 -35 11

ns

*P ^ 0-05; ns, not significant. Coefficients of variation (CV) provide a relative measure of error variance on each sampling date.

Table 3. Critical temperatures during autumn, winter., and early spring (1989-90) of current-year needles from red spruce seedlings fumigated at the US Forest Service Research Laboratory in Delaware, Ohio (USFS) with acid mist solutions of differing pH, in combination with three [day/night) ozone concentrations, during the 1989 growing season Critical temperatures (°C) on 1990

1989 Acid mist treatment (pH) 3-0 4-2 CV (%)

25Sep -9 —9

14

16 Oct

30 Oct

13 Nov

18 Dec

22 Jan

19 Mar

-12 -12 21

-23 -23 14

-40 -40 5

-47 -45 12

-42 -48 14

-29 -30 9

No significant difference in cold tolerance was found between seedlings receiving pH 3 0 and pH 4-2 acid mist. Seedlings were treated with ozone and acid mist at USFS, but over-wintered in Burlington. Vermont. Coefficients of variation (CV) provide a relative measure of error variance on each sampling date.

thecoldtoleranceof red spruce seedlings throughout autumn and early winter. However, on 22 January 1990, cold tolerance difTerences associated with acid mist treatments approached statistical significance (treatment differences at P ^^ 0-106). On that date, seedhngs exposed to acid mist at pH 3-0 were approximately 6 °C less cold tolerant than those receiving pH 4-2 (Table 3). A significant (P ^ 0-006) Oo X acid mist (pH) interaction was detected on 19 * VF / March 1990. The interaction resulted when seedlings treated with high O3 (150/100 nl I"') and acid mist of low pH (3-0) were found to be 10 °C less cold

tolerant than those receiving the less acidic mist (pH 42) and the same O3 concentration (Fig. 1). However seedlings receiving the other two O3 concentrations (50/0, 100/50 nil"') in combination with pH 3.0 acid mist were approximately 5 °C more cold tolerant than those receiving pH 4-2 acid mist and Og at those same two levels, , Influence of O, on freezing injury ^ 3 ^ Visible freezing injury (browning) to current-year needles of red spruce seedhngs, fumigated at BTI for

332

CE.

Waite and others 0

-40 50/0

100/50 Day/night ozone concentration (nl

150/100

Figure 1. The nature of a significant {P < 0'006) acid mist x ozone (O^) interaction in a March 1990 coldtolerance assessment, illustrating the differential cold-tolerance response of red spruce seedlings exposed to a range of acid mist and O3 treatment combinations during the 1989 growing season at the US Forest Service Research Laboratory in Delaware, Ohio. Acid mist at pH 3 0 ( • ) and pH 4 2 (A).

two seasons and overwintered in Burlington, VT during the 1988-89 winter, was quantified in early May 1989. The average percent injury to CF and 3X treatments were 75 and 19-5'^o. respectively. However, with the exception of two seedlings in the 3X treatment, each of which sustained injury to 60 ^^ of their current-year needles, injury to individual seedlings ranged from 5 to 15 % in both treatments. Injury was evident in all seedlings in both O3 treatments, and difierences between CF and 3X treatments were not statistically significant. The lack of a significant difference between CF and 3Xtreated seedlings in visible freezing damage supports laboratory' results from assessments performed during early winter through to early spring, which indicated no significant reduction in cold tolerance associated with elevated O3 concentration (Table 2). DISCUSSION

The collective results from these three independent O3 fumigation studies, using multiple seed sources and exposing red spruce seedlings to varying O3 levels for up to two growing seasons, revealed no evidence of reduced cold tolerance associated with elevated O3 concentrations at anytime during autumn, winter, or spring. Laboratory results are supported by field data collected in spring 1989 indicating no significant difference in visible freezing injury among the same CF and 3X-treated seedlings (BTI) that were assessed for cold tolerance. Because of the independent nature of the Og fumigations, these results are not simply a function of a particular fumigation study, facility, or environment. An examination of coefficients of variation, a relative measure of error variance, associated with each coldtolerance data set suggests that error variances were not unusually high and were probably not responsible for the absence of differences on most sampling

dates (Tables 1-3). Coefficients of variation generally ranged from 9 to 21 % over the course of the study. These coefficients are typical of values in other experiments where significant differences in cold tolerance were consistently detected among fertilizer treatments, difTerent-aged foliage, acidic cloud water treatments, or provenances (DeHayes et al., 1989, 19906, 1991, 1992). Other studies have also failed to demonstrate any negative influence of supplemental O3 on red spruce physiology (Alscher et al., 1989; Kohut et al., 1990; Patton, Jensen & Schier, 1991; Fincher & Alscher, 1992). Amundson et al. (1991 a) found no significant effect of elevated O3 on cold tolerance or foliar carbohydrate levels of red spruce seedlings fumigated for two growing seasons with O3 concentrations up to 2 X ambient levels at BTI. Jensen et al. (1987) and Rebbeck, Jensen & Greenwood (1992) found that growth rates of red spruce seedlings were not adversely affected by O3 concentrations up to 166 nl 1 ^ However, following two consecutive seasons of O3 exposures at BTI with concentrations up to 3 X ambient, Amundson et al. (1991 b) reported a decline in total shoot biomass and trends toward reduced starch and sugar contents with increasing O3 exposures to red spruce seedlings. The tendency on some dates for reduced cold tolerance associated with low O3 or CF air in the USFS and U M O fumigations is neither easily explainable nor unique. Reduced cold tolerance associated with relatively low O3 concentrations has been previously observed. For example, Wang (personal communication) found lower cold tolerance levels during autumn in 10-yr-old Douglas fir trees [Pseudotsuga menziesii (Mirb.) Franco] receiving CF air than those receiving elevated O3 concentrations up to 250 nl r ' or NF air. In addition, examples of apparent growth stimulation attributed to O3, when compared to trees grown in CF air or

333

Ozone and cold tolerance in red spruce

low O3 concentrations, have been reported in red Strimbeck & Johnson, 1992). The inconsistent spruce (Jensen et a}., 1987; Cumming, personal response reported here was not surprising given the communication), yellow poplar (Liriodendron tulip- low frequency of application and short duration of ifera L,), and sugar maple (Acer saccharum Marsh.) the acid mist treatments. Seedlings received acid mist at pH 3 0 or 4 2 for only 40 min wk"' for about {Kress & Skelly, 1982). As a possible explanation for the reduced cold 15 wk. In contrast, the studies of Fowler era/. (1989) tolerance associated with low O3 or CF air, weand Cape et al. (1991), in which seedlings were considered the possibility that moderate or high exposed to simulated acid mist solutions of pH as levels of O3 may reduce stomatal conductance, which low as 2-5 for 1-5 h wk"^ for 20-23 wk, demonstrated in turn could limit foliar uptake of Oy at these levels reduced cold tolerance in red spruce seedlings and minimize potential associated impacts on cold exposed to these simulated acidic mists. Also, tolerance. This conceivably could explain the ap- DeHayes et al. (1991) reported reduced cold tolparent reductions in cold tolerance associated with erance of red spruce seedlings exposed to ambient the low O3 (USFS) and CF-treated seedlings (UMO) acidic cloudwater with a mean pH of 3 44 and an on some dates. However, there is no empirical average frequency of cloud cover of 3 5 % , and evidence to support this explanation and the poss- jacobson et al. (1990) found that frequent interibility seems unlikely. In fact, Taylor et al. (1986) mittent exposures of red spruce foliage to acidic mist reported higher rates of CO^ assimilation and caused injury. Given the ephemeral nature of the transpiration, indicative of higher stomatal con- acid mist treatments in this study, the trend toward ductance, in red spruce seedlings exposed to mod- reduced cold tolerance in January for seedlings erate levels of O3 in episodic exposures. Further- receiving acid mist at pH 30 and in March for more, if stomata closed in response to moderate or seedlings receiving acid mist at low p H (3"0) in high levels of O3, a reduction in growth, photo- combination with high O3 (150/100 nl 1"^) is notesynthetic rate, and/or biomass accumulation might worthy. Previous work has suggested that the loss of be expected. Although we did not measure these just a few degrees of cold tolerance during midwinter parameters, most studies to date Vv^ith red spruce that may result in an increased frequency of freezing used similar O3 fumigation treatments have been injury in red spruce (DeHayes et al., 19906; unable to demonstrate such reductions (Alscher DeHayes, 1992). The significant acid mist x O3 et al., 1989; Kohut et al., 1990; Patton et al., 1991 ; interaction is a further indication of physiological perturbation resulting at least in part from acid m.ist Rebbeck el al., 1992; Fincher & Alscher, 1992). An alternative explanation for the apparent re- treatments. On 11 separate dates over two seasons (1988-89 duction in cold tolerance among seedlings receiving the 50/0 nl T' (USFS) or CF air (UMO) treatments and 1989-90) we assessed the autumn, winter, and involves the antioxidant systems in red spruce spring cold tolerance of red spruce seedlings exposed (Amundson et al., 1991 b). It has been suggested that to a variet\' of ambient and elevated O3 concentraantioxidants help retard the oxidative degradation of tions. During this time we found no evidence that O3 both proteins and lipids in cell components, in- significantly reduced cold tolerance of these seedcluding cell membranes (Mehlhorn et al., 1986). If lings during any season, including midwinter, a time the antioxidant systems of red spruce were triggered when red spruce may be particularly susceptible to at some threshold concentration above 50/0 nl 1" , it freezing injury. Others have suggested that effects of is possible that seedlings receiving the higher rates of O3 on red spruce may be subtle, cumulative, and O3 (100/50, 150/100 n i r ^ at C S F S ; NF-f75, require multiple seasons of exposure to become NF-|-150nir^ at UMO) were afforded sufficient evident due to red spruce's inherently low rate of protection from eel! membrane degradation by stomatal conductance and effective antioxidant sysantioxidants to mitigate any reduction in cold tems (Alscher et al., 1989; Kohut et al., 1990; tolerance. Although content of" foliar antioxidants in Amundson et aL, 19916). This study does address red spruce has been shown to be responsive to O3 effects of O3 exposures on red spruce seedlings for up concentration, direct evidence of their actual role to two growing seasons and indicates no significant and capacity to protect trees from pollutants has not negative effects on developmental cold tolerance at been established (Hausladen et al., 1990; Mc- concentrations up to 166 nll"^ Furthermore, labLaughlin & Kohut, 1992). For now, this explanation oratory results are supported by actual field freezing injury data. can only be presented as a possibility'. The lack of a significant influence of acid mist on cold tolerance in the USFS fumigation is contrary to the findings of others, who have demonstrated a consistent reduction in cold tolerance of red spruce associated with both simulated and ambient acidic cloud water (Fowler et al., 1989; Eamus & Fowler, 1990; Cape et al., 1991; DeHayes et al., 1991; Vann,

ACKNOWLEDGEMENTS

We thank Ruth Alscher, Robert Amundson, Jonathan Cumming. and Leonard Weinstein for the red spruce seedlings originating from BTI; Keith Jensen, Roy Patton and Mary Ann Keyser for their effort in arranging for and

334

C E. Waite and others

conducting the O^ fumigations at USFS and John Austin for his technical assistance. We also thank Jonathan Cumming, Lucy Sheppard, and Jenny Wolfenden for their thoughtful reviews of this manuscript. This research was supported by funds provided hy the Northeastern Forest Fxperiment Station, Spruce-Fir Research Cooperative within the joint US Environmental Protection Agency-USDA Forest Service Forest Response Program and the Andrew W. Mellon Foundation. The Forest Response Program is part of the National Acid Precipitation Assessment Program. This paper has not been subject to EPA or Forest Service review and should not be construed to represent the policies of either agency.

exposure on injury in seedlings of red spruce (Picea rubens Sarg.). NetL- Phytologist 120: 49-59. Fincher J, Cumming JR, Alscher RG, Rubin G, Weinstein L. 1989, Long-term ozone exposure affects winter hardiness of red spruce (Picea rubens Sarg.) seedlings. Neti: Phytologist 113: 85-96. Fowler D, Cape JN, Deans JD, Leith ID, Murray MB, Smith RI, Sheppard LJ, Unsworth MH. 1989. EflFects of acid mist on the frost hardiness of red spruce seediings. New Phvtologist 113: 321-335. Freer-Smith PH, Mansfield TA, 1987, Tbe combined effects of low temperature and SO^-l-NO.j pollution on the new season's growth and water relations of Picea sitchensis. New Phytologist 106: 237-250. Friedland AJ, Gregory RA, Karenlampi L, Johnson AH. 1984. Winter damage to foliage as a factor in red spruce decline. Canadian Journal of Forest Research 14: 963-965. Hausladen A, Madamancbi NR, Fellows S, Alscher RG, Amundson RG. 1990, Seasonal changes in antioxidants in red spruce as affected by ozone. New Phytologist 115: 447-458. Jacobson JS, Bethard T, Heller LI, Lassoie JP. 1990. Response of Picea rubens seedlings to intermittent mist varying REFERENCES in acidity, and in concentration of sulfur- and nitrogenAlscher RG, Amundson RG, Cumming JR, Fellows S, containing pollutants. Physiologia Plantarum 78: 595-601. Fincher J, Rubin G, Van Leuken P, Weinstein LH. 1989. Jensen KF, Patton RL, Schier GA, Loats KV. 1987. Effect of Seasonal changes in the pigments, carbohydrates and growth of simulated acid rain and ozone on red spruce seedlings: an red spruce as affected by ozone. Neii> Phytologist 113: 211-223. interim report In: Proceedings of the US/FRG research Amundson RG, Alscher RG, Fellows S, Rubin G, Fincher J, symposium : effects of atmospheric pollutants on the spruce-fir Van Leuken P, Weinstein LH, 19916. Seasonal changes in forests of the Ba.ttern United States and Ihe Federal Republic of the pigments, carbohydrates and growth of red spruce as Germany. 19-23 October 1987, Burlington. Vermont. United affected by exposure to ozone for two growing seasons. New States Department of Agriculture Forest Service, Nortbeastern Phytologist 118: 127-137. Forest Experitnent Station, Broomail, Pennsylvania. General Amundson RG, Kohut, RJ, & Laurence, JA. (1991a. Mineral Technical Report NE-120, 4 1 3 ^ 1 5 . nutrition, carbohydrate content and cold tolerance of foliage of Johnson AH, Cook ER, Siccama TG. 1988, Climate and red potted red spruce exposed to ozone and simulated acidic spruce growth and decline in the northern Appalachians. precipitation treatments. Water, Air, and Soil Pollution 54: Proceedings of the National Academy of Science, USA 85: 17.S-182. 5369-5373. Barnes JD, Davison AW. 1988. The influence of ozone on the Johnson AH, Friedland AJ, Dushoff JG. 1986, Recent and winter hardiness of Norway spruce [Picea abies (L.) Karst.]. historic red spruce mortality: evidence of climatic influence. AW Phytologist 108: 159-166. Water. Air, and Soil Pollution 30; 319-330. Berrang PC, Steiner KC. 1986, Seasonal changes in the cold Kohut RJ, Laurence JA, Amundson RG, Raba RM, Meltolerance of pitch pine. Canadian Journal of Forest Research 16: konian JJ. 1990. Effects of ozone and acidic precipitation on 408-410. the growth and photosynthesis of red spruce after tuo years of Brown KA, Roberts TM, Blank LW. 1987. Interaction exposure. Water, Air and Soil Pollution 51: 277-286. between ozone and coid sensitivity in Norway spruce: a factor Kolb TE, Steiner KC, Barbour HF. 1985. Seasonal and genetic contributing to the forest decline in central Europe? Netu variations in loblolly pine cold tolerance. Forest Science 31: Phytologist 105: 149-155. 926-932. Cape JN, Leith ID, Fowler D, Murray MB, Sheppard LJ, Kress LW, Skelly JM. 1982. Response of several eastern forest Eamus D, Wilson RHF 1991. Sulphate and ammonium in tree species to cbronic doses of O2one and nitrogen dioxide. mist impair the frost hardening of red spruce seedlings. Nevj Plant Disease 66: 1149-1152. Phytologist 118: 119-126. Lucas PW, Cottam DA, Sheppard LJ, Francis BJ. 1988. Curry JR, Church TW. 1952. Observation.s on winter dr\'ing of Growth responses and delayed winter hardening in Sitka spruce conifers in tbe Adirondacks. Journal of Forestry 50: 114—116. following summer exposure to ozone. New Phytologist 108: DeHayes DH. 1992. Winter injury and developmental cold 495-504, tolerance of red spruce. In: Eager C, Adams MB, eds. The McLaughlin SB, Kohut RJ. 1992, Carbon allocation and ecology and decline of red spruce in the Eastern United States, associated proce.sses. In : Eager C, Adams MB, eds. The ecology New York: Springer-Verlag, 295-337. and decline of red spruce tn the Eastern United States, New York: DeHayes DH, Ingle MA, Waite CE, 1989. Nitrogen fertilSpringer-Verlag. 338-382, ization enhances cold tolerance of red spruce seedlings. Mehlhorn H, Seufert G, Schmidt A, Kunert KJ. 1986. Effect Canadian Journal of Forest Research 19:8: 1037-1043. of SOj and Og on production of antioxidants in conifers. Plant DeHayes DH, Thornton FC, Waite CE, Ingle MA. 1991. Physiology 82: 336-338, Ambient cloud deposition reduces cold tolerance of red spruce Morgenstern EK, 1969. Winter drying of red spruce provenseedlings. Canadian Journal of Forest Research Zl\ 1292-1295. ances related to introgressive hybridization with black spruce. DeHayes DH, Waite CE, Ingle MA. 1990a. Storage temBi-Monthly Research Notes. Canadian Fore.st Service IS: 34—36. perature and duration influence coid tolerance of red spruce Patton RL, Jensen KF, Schier GA. 1991. Responses of red foliage. Forest Science 36:4: 1153-1158. spruce seedlings to ozone and acid deposition. Canadian Journal DeHayes DH, Waite CE, Ingle MA, Williams MW, 19906. of Forest Research 2 1 : 1354-1359, Winter injury susceptibility and cold tolerance of current and Pomerleau R. 1962. Severe winter browning of red spruce in year-old needles of red spruce trees from several provenances. soutbeastern Quebec, Bi-Monthly Report, Canadian DepartFore.st Science 36:4: 982-994. ment of Forest Entomology and Pathology 18: 3, DeHayes DH, Williams MW. 1989. Critical temperature: A Rebbeck J, Jensen KF, Greenwood MS, 1992. Ozone effects quantitative method of assessing cold tolerance. United States on the growtb of grafted mature and juvenile red spruce. Department of Agriculture Forest Service General Technical Canadian Journal of Forest Research 22: 756-760, Report NE-134. Sheppard LJ, Smith RI, Cannell MGR. 1989. Frost hardiness Eamus D, Fowler D. 1990. Photosynthetic and stomatal of Picea rubens growing in spruce decline regions of the conductance responses to acid mist of red spruce seedlings. Appalachians. Tree Physiology 5: 25-37. Plant, Cell and Environment 13: 349-357. Skarby L, Sellden G. 1984. Tbe effects of ozone on crops and Fincher J, Alscher RG. 1992. The effect of iong-term ozone forests. Ambio 13: 68-72,

Ozone and cold tolerance in red spruce Taylor GE, Norby RJ, McLaughlin SB, Johnson AH, Turner RS. 1986. Carbon dioxide assimilation and growth of red spruce (Picea rubem Sarg.) seedlings in response to ozone, precipitation chemistrj', and soil type. Oecologia 70: 163-171. Vann DR, Strirabeck GR, Johnson AH. 1992. Effects of ambient levels of airborne chemicals on freezing resistance of red spruce foliage. Forest Ecology and Management 51: 69-79. Weiss MJ, McCreery L, Miller I, O'Brien JT, Miller-Weeks

335 M. 1985. Red spruce and balsam fir decline and mortality. Interim Report, Uniled States Department of Agriculture Forest Service Northeastern Area, Broomall, Pennsylvania. Wilkinson RC. 1990. Effects of winter injury on basal area and height growth oi 30-year-old red spruce from 12 provenances growing in northern Nev, Hampshire. Canadian Journal of Forest Research 20: 1616-1622.