Ozone-exposure responses of black cherry and red maple seedlings

Enuironmmtai and Expmmmlnl Bokztp Vol. 34, No. 4. pp. 355-362. 1994 Elsetier Science Ltd Printed in Great Britain

Pergamon 0098-8472(94)00028-X

OZONE-EXPOSURE

RESPONSES OF BLACK CHERRY RED MAPLE SEEDLINGS

AND

L. J. SAMUELSON* Tennessee

Valley Authority,

TVA Forestry Building, Ridgeway

Road, Norris TN 37828,

U.S.A.

(Receiued 2 3 March 1994; acceptedin reuisedfonn 16 June 1994)

Samuelson L. J. Ozone-exposure responses of black chewy and red maple seedlings. Environmental and Experimental Botany 34, 355-362, 1994.-Field foliar injury surveys have been used to identify ozone injury in deciduous tree species growing in forested areas in the southeastern United States. Whether ozone-induced foliar symptoms are indicative of leaf photosynthetic and growth reductions is not well understood. This research tested the hypothesis that ozone-induced foliar injury is accompanied by reductions in leaf gas exchange and growth in 1-year-old black cherry (&anus se-retina Ehrh.) and red maple (Acer rubram L.). Seedlings were grown under a 75% reduction of ambient light and fumigated with sub-ambient, ambient or twice-ambient concentrations of ozone on a 24-hr basis for one growing season (5 months). For both species, only leaves exposed to the twice-ambient ozone treatment displayed visible symptoms. Net photosynthesis and leaf conductance of red maple and black cherry leaves declined, and internal CO, concentrations increased in response to ozone treatment. Ozone reduced the height growth and the root/shoot ratio of black cherry, but ozone did not influence red maple growth. These results indicate that ozone-induced foliar injury may be accompanied by reductions in leaf gas exchange in black cherry and red maple, and by growth reductions in black cherry. Ky words: Net photosynthesis,

leaf conductance,

growth, ozone exposure.

INTRODUCTION

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356

L.J. SAMUELSON

mented for both red maple and black cherry,/4'17'22) but reported ozone effects on seedling growth are variable. Some studies have reported growth reductions in black cherry and red maple seedlings in response to ozone treatment. C5'17~In contrast, others have observed no influence of ozone treatment on red maple and black cherry seedling growth. (22~ Although growth and foliar injury responses to ozone have been previously examined in black cherry and red maple, the relationships among ozone concentration, foliar injury and leaf gas exchange have not been as well studied. The objectives of this research were to determine if elevated ozone treatment induced visible foliar symptoms, and if visible injury was accompanied by reductions in leaf gas exchange and growth of 1-year-old black cherry (Prunus serotina Ehrh.) and red maple (Acer rubrum L.). Red maple is considered a shade-tolerant species and black cherry, a more shade-intolerant species, may survive shade for 3 4 years in the seedling stage./~4'27/Therefore, both species were grown under a 75% reduction of ambient light to simulate a more natural understory light environment. Adaptation to low light may also influence the ozone sensitivity of leaf photosynthesis. (3'24'26/This research tested the hypothesis that ozone treatment influenced visible foliar health, leaf gas exchange and seedling growth. MATERIALS AND M E T H O D S

Ozone treatments Sub-ambient (charcoal-filtered), ambient or twice-ambient ozone treatments were delivered on a 24-hr basis to nine 3.0 m x 2.4 m open-top fumigation chambers arranged in three blocks (a total of three chambers for each ozone treatment) on the Tennessee Valley Authority Norris Dam Reservation at Norris, T N from 11 April to 31 August 1993. The ozone delivery and monitoring systems were previously described, i7)A computer-controlled monitoring and injection system was used to monitor the ozone concentrations within each chamber, and control the ozone concentration generated from oxygen gas and delivered to the twice-ambient ozone treatment. Ozone concentrations within each chamber were monitored every 7 min for 20 s using an ozone analyzer (Dasibi Model 1008AH, Dasibi Emdronmental Corp., Glendale, CA, U.S.A.) and sample air drawn continuously from

within the center of each chamber. An air exchange rate of 2.2 exchanges m i n - ~facilitated mixing and cooling within a chamber. Air temperature was monitored continuously within each chamber using temperature transducers (Analog Devices, Model AC2626, Norwood, MA, U.S.A.). Monthly average air temperatures, based on 7-hr means (1100-1700 hr), were a maximum of 3°C warmer than ambient air temperatures outside the fumigation chambers. When averaged over the growing season, air within the chambers was 1.6°C warmer than air outside the chambers. Monthly 7-hr (1 I00-1700 hr) ozone treatment means, SUM0 values (24-hr sum of all hourly average ozone concentrations), SUM06 values (24-hr sum of hourly averages equal to or above 60 ppb) and the number of hours equal to or above the 120 ppb National Ambient Air Quality Standard for ozone/25) are presented in Table 1.

Cultural history In early April 1993, bare root, 1-year-old red maple nursery stock seedlings of unknown parentage (Botanico Inc., McMinnville, TN, U.S.A.) were planted in 24-1 plastic pots filled with A horizon soil collected from an area adjacent to the site (Staser series: fine-loamy, mixed, thermic Cumulic Hapludoll). One-year-old black cherry seedlings grown from seed collected from trees growing at the northwest corner of the Great Smoky Mountains National Park in Tennessee were transplanted into the 24-1 pots with the soil described above. Three pots of each species were set into each chamber during the last week of April into high density foam bats to reduce soil heating. To simulate an understory environment, shade cloth (DeWitt, Knoxville Seed and Greenhouse Supply Corp., Knoxville, TN, U.S.A.) was used to reduce ambient photosynthetic photon flux density by approximately 75% (an average photosynthetic photon flux density of 476+ 196 pmol m -2 s ~ measured on a clear day in July between 1200 and 1300 hr). To avoid destructively sampling leaf tissue for measures of plant water relations, soil water status of each pot at a depth of 15 cm was monitored two times a week using time domain reflectometry (Trace System I, SoilMoisture Equipment Corp., Santa Barbara, CA, U.S.A.). Seedlings were watered when the volumetric soil water content fell below field capacity (25% volumetric water

OZONE EXPOSURE

357

Table 1. Month~ 7-hr means, SUM0 and SUM06 ozone values, and number 0fhr above 120 ppb for ozone treatments deliveredfrom 11 April to 31 August 1993

Ozone treatment

April

May

June

July

August

29 8 0 0

20 8 0 0

19 7 0 0

19 6 0 0

19 6 0 0

49 16 3 0

50 20 4 0

53 20 4 0

53 18 5 0

46 15 2 0

92 29 21 39

98 38 26 81

106 41 31 74

94 33 25 58

94 32 23 35

Sub-amblent

7-hr mean (ppb) SUM0 (ppm-h) SUM06 (ppm-h) No. hr > 120 ppb Ambient

7-hr mean (ppb) SUM0 (ppm-h) SUM06 (ppm-h) No. hr > 120ppb Twice-ambient

7-hr mean (ppb) SUM0 (ppm-h) SUM06 (ppm-h) No. hr > 120 ppb

content). Seedlings were watered on average two times a week to re-establish field capacity. Foliar injury O n 14 June, 12 July and 24 August, seedlings were evaluated in their respective chambers for stippling or chlorosis using the Horsfall-Barratt rating scale for foliar injury. (9)All leaves from randomly selected branches extending from the lower third of the main stem, middle third of the stem and upper third of the stem of each black cherry seedling, and all leaves along the main stem of each red maple seedling (branching on maple was minimal) were evaluated. Leaf chlorosis was defined as adaxial yellowing, and stippling was defined as dark, discrete groups of pigmented cells, i<' Assessment of visible injury was made on leaves of varying age due to indeterminate shoot growth (mainly on the dominant meristem) in black cherry and heterophyllous shoot growth in red maple./12/The first leaf flush was produced from mid- to late-April in black cherry and from late-April to early-May in red maple. Physiological and growth measurements Light-saturated net photosynthesis (Pn), leaf conductance (gl) and internal CO2 concentration (Ci)

of one fully expanded leaf from a lower, middle and upper stem section of each red maple seedling, and a leaf from a mid-branch location on branches extending from the lower, middle and upper third of each black cherry seedling (a total of 27 seedlings of each species at each session) were measured. All black cherry leaves selected for gas exchange measurement were produced in the first leaf flush. Leaf gas exchange measurements of red maple leaves were taken on first flush leaves from lower stem sections, second flush leaves from middle stem sections (produced during late May and early June) and third flush leaves from upper stem sections (produced in early July). Gas exchange measurements were made using a portable photosynthesis system (LI-6200, L I - C O R , Inc., Lincoln, NE, U.S.A.) on 27 29 July and 25 27 August, 1993 between 1000 and 1400 hr. A 75 W projector bulb (12V, Philips Co., Germany) provided an average photosynthetic flux density of 1300 #mol m 2 s-1 at the leaf surface. Seedlings were removed from the chambers for measurement and exposed to ambient conditions (averaged over both measurement sessions a relative humidity of 6 7 _ 6 % , air temperature of 29.7-I-2.4°C and CO~ concentration of 374+_21 #1 l-l). To monitor leaf water status during gas exchange

358

L.J. SAMUELSON

measurement, leaf water potentials were measured on a leaf from each seedling using a pressure chamber (PMS Co., Corvallis, OR, U.S.A.). All seedlings (a total of 27 for each species) were harvested on 1 September and dry weight by component was determined. Seasonal height and diameter growth increment was calculated from measurements of stem diameter and height on 30 April and 1 September. Stem diameter was measured 2.54 cm above the ground line.

Statistical analysis For each species and each date, contigency table analysis and the Chi-square test statistic/~5/ were used to determine whether the presence of any foliar injury in response to ozone treatment was statistically dependent on leaf location. This analysis does not establish causal relationships or separate means with respect to criteria. For those leaves with injury, a one-way analysis of variance (ANOVA) was used to test whether leaf location influenced average leaf injury. The A N O V A was performed on arc-sine transformed injury scores to satisfy heterogeneity of variance assumptions. Gas exchange responses were averaged by date of measurement, chamber, species and leaf location. Since the cumulative effect of ozone was monitored on the same seedlings over time, a repeated measures design C~/ was used to test for interactions between date of measurement and ozone treatment or location. Since no significant interactions among date and other main effects were detected, leaf gas exchange data were averaged across dates. A randomized block factorial design/HI with three blocks was then used to test for ozone treatment and location effects, and interactions. When the F-test of ozone treatment effects indicated a trend in population means, linear and quadratic orthogonal contrasts/~/were used to detect trends in response to ozone treatment. Leaf location means were compared using Tukey's H S D test. Seedling growth responses to ozone were averaged by chamber and species. A randomized block design ~ ~)was used to test for ozone treatment effects on seedling height and diameter growth, and biomass. Initial seedling height measured in April was used as a covariate in the analysis of biomass components since initial seedling size varied within a species. Orthogonal contrasts were used to detect trends in the growth responses to ozone treatment.

Treatment effects were considered significant at the 0.10 level.

RESULTS AND D I S C U S S I O N

Evidence of visible injury was detected for black cherry and red maple leaves on the 12 July screening session. Red maple and black cherry leaves were diagnosed with chlorosis and stippling injury, respectively, in response to treatment with twiceambient levels of ozone (Table 2). Seedlings exposed to sub-ambient or ambient ozone treatment displayed no foliar injury symptoms at any time. Leaf chlorosis in red maple and leaf stippling in black cherry have been observed previously in response to ozone treatments of varying concentration and duration.~' ~7,22/ For red maple and black cherry seedlings treated with twice-ambient ozone, the percentage of sampled leaves with injury symptoms in both July and August was statistically dependent on leaf location (Table 2). O f those leaves with injury symptoms, greater average injury scores were observed for older red maple leaves (first flush) growing lower on the stem than younger leaves (third flush) growing closer to the apical meristem (Table 2). Greater foliar injury in older red maple leaves was likely due to a longer duration of ozone exposure than leaves produced later in the growing season. Leaves measured for gas exchange at each specified stem or branch location were selected based on average injury levels in Table 2 so that the same average injury level was measured on each seedling exposed to the twice-ambient ozone treatment. Red maple leaves selected for measurement had foliar injury levels of 25% on the lower stem, 12% on the middle stem and 0% on the upper stem during the July gas exchange measurement session. During the August session, red maple leaves selected for measurement had 50% injury scores on the lower stem, 12% injury scores on the middle stem and 0% scores on the upper stem. Black cherry leaves selected for gas exchange measurements from the three branch locations had visible injury levels of 1-3% during the July session and 1-6% during the August session. Although black cherry and red maple leaves treated with sub-ambient and ambient ozone showed no foliar injury at either gas exchange measurement session, care was taken to measure

O Z O N E EXPOSURE

359

Table 2. Percentage of aU sampled leaves with injury (%injured leaves), and the minimum, maximum and mean percentage injury scores for black cherry and red maples leaves with visible injury in the twice-ambient ozone concentration. Leaves were sampledfiom lower, middle and upper seedling locations. Obsoved significance values for Chi-square tests of independence age between percentage of leaves with injury and location, and for F-tests of mean injury response to location are indicated in parentheses 12 July %injured leaves

Min

24 August

Injury (%) Max Mean

%injured leaves

Min

Injury (%) Max Mean

Species

Location

Black cherry

lower middle upper

74.1 16.1 20.7 (<0.001)

1 1 1

25 1 6

4.2 1.0 2.3 (0.435)

93.9 50.0 60.0 (<0.001)

1 1 1

75 75 75

11.5 4.4 9.7 (0.492)

Red maple

lower middle upper

100.0 81.8 8.3 (< 0.001)

1 3 1

75 25 1

39.3a 9.6b 1.0c (< 0.001)

97.0 87.3 29.2 (0.011)

12 1 1

75 50 12

45.9a 19.0b 3.7b (0.011)

Table 3. Black cheW and red maple leaf net photosynthesis (Pn), leaf conductance (gl) and internal CO 2 concentration (Ci) in response to ozone treatment and leaf location. The standard error of the mean is indicated in parentheses. Different lowercase letters indicate differences among means when P ~< 0.10 (total d f = 2739r each species) Black cherry Treatment

Ozone (ppb) sub-ambient ambient twice-ambient Contrasts (P > F) linear quadratic Location upper middle lower P > F

Pn

gl

5.3 (0.4) 5.4 (0.4) 4.1 (0.2)

132 (14) 94 (9) 117 (9)

Red maple Ci

(/~molm 2s-1) (mmolm 2s l) (#11-1)

< 0.001 0.038 5.9 (0.4)" 4.8 (0.3) b 4.0 (0.3) ~ <0.001

Pn gl (#molm 2s-l) ( m m o l m - 2 s

281 (3) 270 (5) 299 (2)

6.4 (0.3) 4.9 (0.5) 4.8 (0.5)

165 (7) 94 (11) 115 (9)

0.091 0.028

< 0.001 0.523

0.925 0.004

0.078 < 0.001

132 (11) 4 110 (10) ab 100 (13) b

280 (5) 282 (5) 287 (6)

6.1 (0.3) a 5.4 (0.5) au 4.5 (0.5) ~

140 (9)~ 124 (17)~t, 111 (12) b

0.061

leaves of the same age and location as leaves measured in the twice-ambient ozone treatment. N o significant ozone t r e a t m e n t and leaf location interactions were observed for leaf gas exchange of either species. T h e Pn and gl of black cherry leaves increased from lower to u p p e r b r a n c h locations (Table 3). Y o u n g e r red maple leaves growing closer

0.428

0.022

0.054

Ci l) (pll ~)

283 (3) 274 (5) 290 (6) 0.034 0.888 274 (4) 286 (6) 287 (6) 0.124

to the apical meristem h a d greater Pn and gl than older leaves p r o d u c e d lower on the stem (Table 3). Although leaf location influenced leaf Pn a n d gl, location did not influence leaf response to ozone. Light-saturated Pn a n d gl of black cherry and red m a p l e leaves from different leaf locations decreased in response to ozone t r e a t m e n t (Table 3). L e a f gas

360

L.J. SAMUELSON

exchange responses to ozone were not likely due to an ozone effect on leaf water status, since no significant influence of ozone treatment on leaf water potential was observed for either species (average leaf water potentials were - 0 . 6 9 MPa, - 0.54 MPa and - 0.64 MPa, respectively, for subambient, ambient and twice-ambient ozone treated red maple seedlings, and - 0 . 7 5 MPa, - 0 . 8 9 MPa and - 1 . 0 MPa, respectively, for sub-ambient, ambient and twice-ambient ozone treated black cherry seedlings). The significant increase in Ci of black cherry and red maple leaves in response to ozone treatment (Table 3) indicates that ozone reduced photosynthesis not only through reductions in gl, but also through reductions or limitations in leaf biochemical processes within the mesophyll. Mesophyll processes that may be affected by ozone include light-harvesting, RuBP carboxylase oxygenase activity and RuBP regeneration./3'~8'2~! Variation in the ozone response within a species may be explained by differences in ozone exposure, plant genetic material and environmental con-

ditions among studies. For example, leaf shading has been shown to influence leaf physiological responses to ozone in some species. Ozone had a greater influence on the net photosynthesis, quantum yield and dark respiration of unshaded compared to shaded leaves of Populus L., a shade-intolerant species./24'26'In contrast, Acersaccharum Marsh., a shade-tolerant species, showed greater leaf ozone sensitivity when grown in a more shaded environ° ment./2426! Leaf physiological responses to ozone in red maple and black cherry grown with a 75% reduction of light may therefore differ from the responses of seedlings exposed to ozone in a more open, unshaded environment. For black cherry, foliar injury, and reductions in Pn and gl in response to ozone treatment were accompanied by reductions only in height growth and the root/shoot ratio (Table 4). Leaf injury and reductions in root, leaf and total biomass were previously reported for black cherry seedlings in response to ozone./17/ The significant increase in branch weight in response to ozone (Table 4) suggests that normal carbon allocation patterns were

Table 4. Black che~ and red maple seedling height and diameter growth, and biomass responses to ozone treatment. Biomass means for each species are adjustedJbr variation in initial height. The standard error of the mean is indicated in parentheses (total d f = 9for each

species) Ozone treatment Growth parameter

sub-ambient

Black cherry

height (cm) diameter (mm) root (g) stem (g) branch (g) leaf(g) total (g) root/shoot

Red maple

height (cm) diameter (mm) root (g) stem (g) branch (g) leaf(g) total (g) root/shoot

Species

P> F quadratic

ambient

twice-ambient

linear

43.4 (4.8) 3.7 (0.2) 71.8 (5.3) 37.2 (3.2) 23.2 (2.2) 31.7 (4.6) 163.9 (10.4) 0.74 (0.0t)

40.4 (4.6) 2.9 (1.2) 77.0 (5.1) 40.1 (3.1) 35.4 (2.2) 36.1 (4.5) 188.6 (10.1) 0.72 (0.01)

30.9 (1.9) 2.9 (0.10) 68.5 (4.9) 40.5 (2.9) 27.6 (2.1) 41.8 (2.9) 178.7 (9.6) 0.64 (0.01)

0.071 0.972 0.312 0.934 0.077 0.416 0.522 0.018

0.085 0.657 0.892 0.504 0.062 0.305 0.234 0.069

47.3 (12.5) 2.5 (0.4) 19.3 (2.0) 20.5 (1.8) 11.9 (0.5) 20.7 (1.6) 72.4 (5.5) 0.36 (0.01)

50.3 (4.2) 3.0 (0.7) 21.3 (2.0) 23.5 (1.8) 11.4 (0.5) 21.3 (1.6) 78.9 (5.4) 0.37 (0.01)

47.3 (4.2) 2.2 (0.6) 15.6 (2.1) 2o.2 (1.9) 10.4 (0.5) 17.1 (1.7) 63.4 (5.6) 0.33 (0.01)

0.842 0.403 0.142 0.309 0.280 0.171 0.142 0. I 16

0.452 0.933 0.743 0.6o4 0.198 0.510 0.860 0.602

OZONE EXPOSURE disrupted. Existing data indicate that carbon m a y be retained in leaves and stems rather than transported to roots in seedlings exposed to ozone. (1'6'23/ Only a trend (P= 0.116) towards a reduced r o o t / shoot ratio was observed in red maple, otherwise no significant influence of ozone treatment on diameter or height growth, and biomass was detected. In contrast, reduced stem diameter and dry weight biomass in response to ozone treatment has been previously reported for red maple. 15/ In conclusion, black cherry and red maple seedlings grown in a shaded environment expressed visible foliar injury and reductions in leaf Pn and gl in response to ozone treatment. While field research has utilized visual leaf symptoms to indicate ozone injury in hardwood species,/2/it is unclear if visible symptoms correspond to declines in leaf gas exchange. Although these results were obtained under more controlled experimental conditions, this study confirms the potential for reductions in black cherry and red maple seedling leaf gas exchange with the identification of visible ozone-induced foliar injury by field studies. Acknowledgement~Funding for this project was provided by the Tennessee Valley Authority and the Electrical Power Research Institute. This research was supported in part by an appointment to the Research Participation Program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Tennessee Valley Authority. The author wishes to thank P. A. Mays for field assistance, G.S. Edwards for project support, and two anonymous referees for manuscript review. REFERENCES 1. Adams M. B., Edwards N. T., Taylor G. E. and Skaggs B. L. (1990) Whole-plant 14C-photosynthate allocation in Pinus taeda: seasonal patterns at ambient and elevated ozone levels. Can. J. For. Res. 20, 152 158. 2. Chappelka A. H, Hildebrand E., SkellyJ. M., Mangis D. and RenfroJ. R. (1992) Effects of ambient ozone concentrations on mature eastern hardwood trees growing in the Great Smoky Mountains National Park and Shenandoah National Park. In Proc.A WMA Meetings, Kansas City, Missouri. 3. Darrall N. M. (1989) The effect of air pollutants on physiological processes in plants. Pl. Cell Envir. 12, 1 30.

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