Environ. Sci. Technol. 1905, 19, 432-437
Effect of Ethylene and Related Hydrocarbons on Carbon Assimilation and Transpiration in Herbaceous and Woody S p e c i d Sheila A. Squler," George E. Taylor, Jr., Wllliam J. Selvldge, and Carla A. Gunderson Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
Although the hormonal effects of ethylene (CzH4) on plant growth and development are well documented, recent evidence suggests that carbon assimilation in some herbaceous species is highly sensitive to ethylene. Since ethylene is a common trace gas in many airsheds influenced by urban and industrial pollutant sources, elevated levels of ethylene may be affecting the productivity of some terrestrial vegetation. The objectives of this study were to investigate the resporisiveness of carbon assimilation to ethylene and related low molecular weight hydrocarbons in plant species of dissimilar growth and physiological features, to address the physiological mechanism of photosynthetic inhibition, and to estimate minimum Czy4 concentrations causing incipient effects on carbon assimilation. Of the four hydrocarbons studied only ethylene influenced carbon assimilation in a variety of species. The level of ethylene needed to elicit a change in carbon assimilation differed markedly among species. Estimated 5-h concentrations of ethylene required to influence carbon assimilatiori in ethylene-sensitive species ranged from 0.60 to 19.5 pmol/m3. The ethylene-induced inhibition of photosynthesis was correlated with a decline in stomatal conductance to HzO vapor. Introduction Low molecular weight hydrocarbons (i.e., carbon atom numbers 5 8) play a significant role in a humber of envirohmental issues relating to air quality, most notably the potential gldbal climate change and the formation of photochemical smog, aerosols, and acidic precipitation (1). The most common aliphatics include methane, ethane, ethylene, acetylene, propane, propylene, butanes, and pentanes (2). Of the reactive or non-methane hydrocarbons, ethylene (CZH4) is the most common, with concentrations 50.05 pmol/m3 in pristine areas (3), but often 2 orders of magnitude higher in urban airsheds or near major point sources ( 4 ) . The major anthropogenic sources of many low molecular weight hydrocarbons, including CZH4, are vehicular exhaust and volatization from petrochemical processes (5),while advanced energy technologies may be significant point sources in the future (6). The significance to terrestrial vegetation of elevated levels of low molecular weight hydrocarbons is not certain. With the exception of CzH4, little information is available regarding plant response to either short-term or long-term expoeure regimes. The hormonal effects of CzH4are well documented under controlled laboratory conditions, and threshold concentrations to induce many physiological/ biochemical characteristics of premature senescence lie between 0.4 and 4 hmol/m3 (7). Heck et al. (8) observed symptoms of hormonal CzH4 action (i.e., whole-plant senescence) in cotton (Gossypium hirsutum) after 30-day exposures to 25 pmol/m3, while Abeles and Heggestad (9) noted similar hormonal effects on a number of herbaceous species treated for 70 days to CzH4 concentrations 11.0 c t ~ ~ l / mIn 3 ,conjunction with air quality data and field 'Publication No. 2457, Environmental Sciences Division, ORNL. Address correspondence to this author at the Juniata College, Huntingdon, PA 16652. 432
Environ. Sci. Technol., Vol. 19, No. 5, 1985
studies, Abeles and Heggestad (9) concluded that CzH4 was an environmental stress influencing prihcipally urban vegetation. Some of the other hydrocarbon gases can affect plant growth at concentrations well above ambient levels ( l o ) ,and at least one compound, propylene, is reported to be a C2H4analogue (11). Early reports of C2H4action in terrestrial vegetation ihdicated that foliar gaseous exchange of carbon dioxide and water vapor were not affected by CzH4 exposure. For example, Pallaghy and Raschke (12) observed no change in stomatal conductance to HzO vapor in corn (Zea mays) and pea (Pisum satiuum) following exposure to CzH4 concentrations as high as 410 pmol/m3 (duration of exposure not specified). At a similar concentration Aharoni (13)reported no effect of CzH4 on the conductivity of the HzO diffusive path in 10 herbaceous species. However, a more recent study by Kays and Pallas (14) demonstrated pronounced and very rapid physiological effects from CzH4 exposure. Following only a 2-h exposure to 10 pmol/m3 C2H4, photosynthesis ( P N ) in peanut (Arachis hypogaea) was reduced as much as 33%. Further studies have demonstrated additional PNresponses among several herbaceous species (15, 16). The observation that C2H4can demonstrably affect carbon assimilation in a direct and immediate fashion (14) strengthens the proposal of Abeles and Heggestad (9) and suggests that the physiological mode of action may be a direct inhibition of carbon assimilation rather than a long-term hormonal effect on plant growth processes. The determination of threshold levels of CzH4 causing incipient effects on PNrelative to reported CzH4 concentrations in the atmosphere is important in establishing the likelihood under natural conditions of a direct C2H4 effect on carbon assimilation. The objectives of this study were to (i) determine the responsiveness of PNin five plant species to a number of low molecular weight hydrocarbons at high exposure concentrations (screening investigation), (ii) for those hydrocarbon gases causing effects, determine the responsiveness of P, and transpiration (TR)at near-ambient concentrations, including estimates of the minimum concentration causing effects on P N , and (iii) investigate the physiological basis of changes in PNand T R . Materials and Methods Plant Material. Species were selected to include a range of growth habits (annual/herbaceous vs. woody/ perennials) and dissimilar carbon metabolism (C, vs. C4 assimilatory pathway). Herbaceous C3 (Glycine max L., cv. Davis; Nicotiana tobaccum L., cv. Bel W-3; Arachis hypogaea L., cv. Jumbo Virginia) and C4 (Zea mays L., cv. F1 hybrid Early Golden Giant) species were grown in Promix BX (Premier Brands, Inc., Rochelle, NY) in 1.0-L pots. After germination, seedlings were thinned to one per pot. Fraxinus pennsylvanica L. seedlings (green ash) were obtained as cold-stored, 1-year-oldplants (Forest Keeling Nursery, Elsberry, MO) and were potted in Promix BX in 2.5-L pots. Plants were watered daily and supplied weekly with a full complement of liquid fertilizer (Peters Fertilicter,W. R. Grace, Allentown, PA). Plants were gown a glasshouhe with the following environmental condi-
0013-936X/85/0919-0432$01 .sO/O
0 1985 American Chemical Society
Table I. Summary of Environmental Conditions in the Gas-Exchange Chambers condition
range
chamber volume, m3 air exchange, min air temperature, "C relative humidity, pmol/m3 photosynthetic photon flux density, pmol m-2 s-l photoperiod, h outlet COz concentration, pmol/m3 range of hydrocarbon concentrations, pmol/ms boundarv laver conductance to H,O vauor, cm/s
0.92 4-6 31-35 0.8-1.3 320-500 15 11.5-14.3 0-164 10
tions: mean day/night temperatures of 35/18 "C, 15-h photoperiod, photosynthetic photon flux density of 0-1600 pmol m-2 s-l, and relative humidity of 4540%. The natural photoperiod was extended to 15 h with HID sodium vapor lamps which provided 325 pmol m-2 s-l at bench height. Studies were conducted 5-7 weeks after germination for the herbaceous species and 4-5 months after bud break for green ash. Gaseous Exchange System. Exposures were conducted in an open gas-exchange system (17, 18)utilizing four matching, continuously stirred tank reactors. Operating conditions unique to this study are outlined in Table I. Hydrocarbon gases were dispensed from cylinders (Certified Grade Purity, Union Carbide Corp.), and the flow of pollutant gases to each chamber was regulated with rotameters. Hydrocarbon concentrations were monitored with a flame ionization method (Model 400 hydrocarbon analyzer, Beckman Instruments, Inc., Fullerton, CA) in which the ionization current was proportional to the rate at which carbon atoms of hydrocarbon origin entered the burner assembly. Calibration was achieved with methane and ethylene standards (Primary Standards, Union Carbide Corp.). Hydrocarbon concentrations were monitored at the outlet ports to each chamber, and the sample lines were heated and continuously exhausted. Concentrations of COz and HzO were monitored at each chamber's inlet and outlet ports using infrared gas analyzers (Beckman Instruments, Inc., Fullerton, CA) calibrated with a range of known gas concentrations (i.e., certified grade purity COz in nitrogen, Matheson, Morrow, GA; dilution of HzO-saturated air with dry nitrogen). The COz analysis was done after the HzOwas cryogenically trapped. Where necessary, estimates of whole-plant gas exchange rates of COz and H 2 0 were calculated as described in ref 17 and expressed as mmol m-2 h-' (projected leaf area). Experimental Protocol. Two types of exposures were conducted to address the first two objectives. In the first (objective l),representative C3 (G. max) and C4 (2.mays) herbaceous species were exposed for 6 h to 0,41, 103, or 164 pmol/m3 propylene (C3H6),butane (C4H10), ethane (CzH6),or ethylene (CzH4). These gases were selected on the basis of preliminary data (6) that indicated each hydrocarbon had some unique combination of chemical/ physical properties suspected of governing toxicity. Failure of a compound to elicit a response in this screening exercise was a basis for not pursuing subsequent studies (only CzH4 elicited a response). In the second type of exposure (objective 2), herbaceous (G. max, A. hypogaea, and N . tobacuum) and woody (F. pennsylvanica) species were exposed to CzH4 concentrations of 4, 10, 21, 41, or 82 pmol/m3. Exposures began 4 h after photoperiod initiation and continued uninterrupted for 28 h. Each combination of species and concentration was replicated at least twice. The logistics for each type of exposure were the same. Plants (8-16) were watered to drip point and transferred
to each of four chambers the afternoon prior to exposure. The top of the pot was covered with clear plastic to minimize the exchange of COz, HzO, and hydrocarbon gas between the soil and atmosphere. The following morning (i.e,, 14-h acclimation)before hydrocarbon exposures, rates of COz and HzO exchange were recorded for the plants in each chamber, These chamber-specific preexposure values were used to evaluate subsequent changes in COzand HzO exchange in both control and treated plants. A single hydrocarbon gas was added at different rates to the inlet plenum of three chambers, and the fourth served as a control. The COP,HzO, and hydrocarbon concentrations at inlet and/or outlet ports were recorded at 1-2-h intervals. For those situations in which PN was influenced by exposure to a hydrocarbon gas (i.e., CZH4), stomatal conductance to HzO vapor (g,) was measured to investigate the physiological basis of inhibition (objective 3). Studies were conducted in one of the chambers that was modified with hand-access ports to minimize disturbance of the chamber's environmental conditions during porometry measurements. Stomatal conductance of the abaxial surface (LI 1600 steady state porometer, Li Cor., Inc., Lincoln, NE) was measured before and during exposure in both control and treated plants of all five species except A. hypogaea, in which g, of the adaxial leaf surface was monitored (A. hypogaea is hyperstomatous). Two conductance measurements per leaf were taken on three leaves per plant. The growing and exposure conditions (timing of exposure relative to photoperiod) were comparable to that used for objective 1, except a single C2H4 concentration of 21 pmol/m3 (exceeded estimated threshold concentration to influence PN in most CzH4 sensitive species) was administered. Data Analysis. From each replicated exposure (i.e., combinations of plant species and hydrocarbon concentration), mean rates of COPand HzO exchange (expressed as percentages of initial rates) were calculated at common time intervals. The exchange rates of COz and HzO in control and treated plants were subsequently graphed as a function of time. For studies relating to objective 1, the rate of change over time in COz or HzO exchange for treated plants was expressed as a percentage of that which occurred in control plants at the same time interval. Statistical tests of hydrocarbon effects on PNand TRwere performed only on those data recorded at the last time interval. One-way analysis of variance was conducted on each combination of species and hydrocarbon gas, and the least significant difference was used to evaluate statistical significance between concentrations of a single gas. Stomatal conductance data were evaluated by using paired t tests. All statistical tests were conducted at a = 0.05. Results Rates of Carbon Assimilation (PN) and Transpiration (TR).BefoSe exposure and after acclimation, absolute PN rates ranged from 10.9 mmol of C02 m-2 h-l in F. pennsylvanica to 55.4 mmol of COz m-2 h-l in 2. mays (Table 11). In all five species, PNin control plants rose throughout the first day and, after 6-8 h, had risen 10-20%. On the second full day of exposure, PN rates continued to remain above the initial preexposure values. Transpiration rates ranged from 5.84 mol m-2 h-l in N. tobaccum to 9.18 mol m-2 h-' in G. max (Table 11). Objective 1. In 2. mays none of the four gases influenced PN at concentrations as high as 164 pmol/m3 (Table 111). At this highest concentration for each hydrocarbon gas, all mean PN rates in 2. mays were within f10% of that recorded in control plants. Carbon assimilation in G. man Environ. Sci. Technol., Vol. 19, No. 5, 1985
433
Table 11. Initial Preexposure Rates of Net Photosvnthesis and Transpiration (Mean f SD) net . photosynthesis, transpiration, mmol m-2 h-' mol m-2 h-'
Glycine rnax Zea mays Nicotiana tobaccum Arachis hypogaea Fraxinus pennsylvanica
36.2 f 11.7 55.4 f 11.8 51.1 f 21.4 22.3 i 5.3 10.9 4.0
9.18 f 1.94 8.80 f 0.98 5.84 f 1.15 8.34 f 1.11 6.39 i 1.86
*
50
t
0
100
Table 111. Mean Photosynthetic Rates (mmol m-* h-I) After 3-6 h of Exposure to Hydrocarbon Gasesa
C2H4/G. rnax
I
p
I
I
I
I
I
1
I
I
I
gas concentration, pmol/m3 species
Z . mays
G . max
41
102
164
62.6 51.5 52.6 82.5
60.9 52.6 54.3 79.8
60.9 53.7 54.5 75.9
63.2 52.7 56.7 77.0
C3H6
42.7 40.5 36.2 35.8
39.5 33.3 25.7 42.0
43.8 44.9 18.8 36.2
39.8 39.8 13.8 34.8
10.9 3.6
CZH, C2H4
19.4 8.3
13.6 8.0
8.0 8.0
6.2 4.9
6.5 1.2
C4H10 C2H6 C2H4 C.3H6 C4H10 C2H6
CZH4 A . hypogaea F. pennsylvanica
LSD
0
gas
NS NS NS NS NS NS
t o uu 0 c
yI-
100
z
> VI c 0
8 a
50 C2H4/N. tobaccum
OData for each combination of species and gas were analyzed via one-way ANOVA, and statistically significant F values are those with numeric LSD values.
was not responsive to either C4HI0or C.&, but both C2H4 and c3H6exhibited statistically significant effects on PN (Table 111). The lowest (41 pmol/m3) and highest C2H4 concentrations reduced P N by 29% and 62% in G. max, respectively. In the same species exposed to C3H6,the only PNrate that was statistically different from the others was that at 41 pmol/m3, in which P N was 17% above that in control plants. Thus, a consistent concentration-dependent response in G. max was evident only for C2H4. In the remaining two species,PNwas responsive to C2H4 exposure. In A. hypogaea, all concentrations markedly inhibited PN, and rates ranged from 32% (164 pmol/m3) to 70% (41 pmol/m3) of that in control plants, while in F. pennsyluanica, a statistically significant reduction in PNwas recorded only at the highest concentration (Table 111). Objective 2. The objective of this study was to determine the responsiveness of P N and TR to near ambient concentrations of CzH4. In G. max exposed to CzH4,P N declined at all concentrations >4 pmol/m3 relative to that in control plants (Figure la). By 1600 h on day 1, P N was demonstrably inhibited only at the highest concentration (21 pmol/m3), but by 1200 h on the second day P N was depressed at all concentrations. While P N in control plants at the end of exposure had risen nearly 20% above initial rates, PNin C2H4-exposed plants either remained unchanged (4 and 10 pmol/m3) or was depressed to a level 50% below the initial rate (21 pmol/m3). Carbon assimilation was more rapidly affected by C2H4 exposure in A. hypogaea; within 3.5-5 h, P N was less than the initial rate at concentrations 1 4 pmol/m3, while P N in control plants had risen 8-12% (Figure lb). This C2H4-inducedinhibition of P N continued through day 1. By 1200 h of day 2, P N at 4 pmol/m3 of C2H4 was comparable to that in control plants while that in the two highest concentrations (10 and 21 pmol/m3) was only 40-50% of the initial rate. In N . tobaccum, the effects of C2H4 on PNwere apparent within 2-3 h although a higher range of concentrations 434
Environ. Sci. Technol., Vol. 19, No. 5, 1985
50 0
I
41
I
I
I
t
C,H4 / F pennsylvanlco
I
I
1
I
I
I
0800
\ZOO
1600
0800
I200
I
(600
TIME ( h )
Flgure 1. Response of photosynthesis (as a percentage of initial value) as a function of time and CzH, concentration in 0 . max (a), A . hypogaea (b), N. fobaccum (c), and F. pennsyhanica (d). Numbers after each response line are CzH, concentrations (pmoi/rn3).
than used for A. hypogaea was needed to induce effects (Figure IC). Within 3.5 h of exposure, P N in control plants rose 15% but declined to rates of 50-80% of the initial values at C2H4 levels of 41 and 82 pmol/m3, respectively. In both C2H4 exposures, P N remained depressed throughout day 1,and by 1600 h all PNrates were substantially below that of control plants. Carbon assimilation on day 2 rose 20% in control plants, while P N was 130% of the initial rate at C2H4 concentrations 2 2 1 pmol/m3. The effects of C2H4 on PNin F. pennsyluanica (Figure Id) were not as pronounced as those recorded in the herbaceous species. At the end of day 1, P N at concentrations 110 pmol/m3 was below that in control plants although the percentage of inhibition remained constant through day 2. To estimate minimum C2H4 concentrations required to inhibit carbon assimilation, the change in P N in each species was expressed relative to its respective controls at the same time interval and plotted as a function of the natural log of the product of concentration and time (18). The data for each species were subsequently analyzed by using linear regression techniques (Figure 2). With the exception of F. pennsyluanica, the regression coefficients (i.e., slopes) were significantly different from zero, and the regression accounted for 280% of the variation in PN. Estimates of minimum C2H4 concentrations needed to just
I
I
I
I 1 1 1 1 1 1 1
I 1 1 1 1 1 1 1
I
I
I I
I
W
1
Table IV. Stomatal Conductance to HzO Vapor (g,) in Control and CzH4-TreatedPlants (Mean SD)
*
I
species/ treatment, pmol/m3
leaf conductance, cm/s initial final
change, %
N . tobaccum 0.34 (0.07) 0.20 (0.03)
0
0.19
21
(0.05) 0.19 (0.06)
0.18 (0.09) 0.11 (0.02)
0.21 (0.01) 0.21 (0.04)
0.21 (0.02) 0.19 (0.03)
0.20 (0.02) 0.20 (0.01)
0.18 (0.03) 0.19 (0.04)
-10
0.18 (0.03) 0.28 (0.05)
0.28 (0.05) 0.17 (0.06)
+58"
21 N. taboccum I
0
I I I I Ill1
IO'
I
-\ I
I I
I 03
CONCENTRATION
X
TIME
Flgure 2. Regression of the change in photosynthesis (as a percentage of that in contrd plants) as a function of natural logarithm of the product of C&14 concentration (pmol/m3) and time (h). Symbols are as follows: G. max (O),N . tobaccum (A),A . hypcgaea (0),and F . pennsyhanica I
I
1
I
-5 -42"
G. mal: 0 21
(A). 200,
-54"
F. pennsylvanica
I IiI11111
IO2
-19
0.42 (0.04) 0.43 (0.03)
0
0
-10
2.mays 0 21
-5
A. hypogaea 0
21
z+
a4
o
;
200,
,
I
I
I
I
1
-41"
" Indicates statistically significant difference between initial and final leaf conductance values (paired t test). ( N . tobaccum, F. pennsylvanica, and A. hypogaea).
0
I
I
1
1
0800
1200
1600
0800
I
(200
TIME OF DAY (h)
Figure 3. Response of transpiration (as a percentage of initial value) as a function of time and CpH, concentration in A . hypogaea and N . fobaccum , Numbers after each response line are C2H4concentrations (~.~mol/m~).
inhibit PN(incipient inhibition) following a 5-h exposure were 0.60 (F.pennsylvanica), 1.4 (A. hypogaea), 2.8 (G. max), and 19.5 (N. tobaccum) pmol/m3. For this time interval (5 h) among all species the mean concentration for incipient inhibition of PNwas 6.1 pmol/mS, and 1.6 pmol/m3 if only the more C2H4 responsive species were considered. Transpiration ( TR).The only species exhibiting a marked and consistent TRresponse as a function of C2H4 exposure were A. hypogaea and N . tobaccum (Figure 3a,b). In A. hypogaea, TR rates by 1200 h on day 2 were 70% (10 pmol/m3) and 90% (21 pmol/m3) of initial rates, while that in the controls and 4 pmol/m3 C2H4 rose nearly 40%. Transpiration in N . tobaccum a t the end of each day at C2H4concentrations 1 2 1 pmol/m3 was less than that in control plants, and on the second day TR ranged from 50 to 100% of initial rates in C2H4-exposedplants but nearly 160% in control plants. Stomatal Conductance to H 2 0 Vapor (gs).With the exception of A. hypogaea, g, in control plants (0.0 pmol/m3 C2H4) of all species either remained unchanged (G. max) or declined over time (Table IV)although the decline never exceeded 19% (N.tobaccum). In A. hypogaea, g, increased more than 50% over time in control plants. In all species exposed to C2H4, g, declined over time although the change was statistically significant in only three of the five species
Discussion The first objective of this study was to determine the responsiveness of PN to four hydrocarbon gases. Two of the gases, C2H6 and C4H10, are reported to be innocuous (19,20) although the physicochemical properties of C4H10 suggest that the gas is readily taken up by vegetation (6). Propylene is thought to be a C2H4 analogue and at high concentrations produces symptoms indicative of hormonal action (19). Reported C2H4 effects on PN are variable, with both substantial inhibition (14) and no demonstrable change (12) observed. Our results confirm the innocuous nature of C2HG and C4H10 in 2. mays and G. max, and C3H6exhibited no consistent effects on PN in the same two species at concentrations 1164 pnol/m3. These results for C3H6agree with that of Kays and Pallas (14). The responsiveness of PN to trace C2H4 levels has been confirmed in several species, and the ambivalent nature of different reports in the literature appears to be a consequence of interspecific variation in the responsiveness of PN to C2H4. Carbon assimilation in 2. mays exhibited no response to C2H4 concentrations
