Limnol. Oceanogr., 35(Z), 1990, 399-lO8 Q 1990, by the American Society of Limnology and Oceanography, Inc. Mechanism for the hydrogen sulfide-induced growth limitation in wetland macrophytes Marguerite S. Koch, Irving A. Mendelssohn, I and Karen L. McKee Laboratory for Wetland Soils and Sediments, Center for Wetland Resources, Louisiana State University, Baton Rouge 70803

Abstract Hydrogen sulfide, a phytotoxin that often accumulates in anoxic marine and freshwater marsh soils, suppressed the activity of alcohol dehydrogenase (ADH), the enzyme that catalyzes the terminal step in alcoholic fermentation, in the roots of two wetland macrophytes. This inhibition of root ADH activity with increasing sulfide concentration was associated with decreases in root total adenine nucleotide pool (ATP + ADP + AMP), the adenylate energy charge ratio (AEC), nitrogen uptake (percent recovery of rSNH,+-N) and growth (leaf elongation). These responses were species-specific with a greater negative impact in the freshwater marsh species that naturally inhabits low-sulfide environments. These findings lend support to the hypotheses that ADH activity, as a mcasurc of fermcntative metabolism, is important in maintaining the root energy status of wetland plants under hypoxic-anoxic conditions, that there is a significant negative effect of H,S on the anoxic production of energy in these roots, and that an important negative effect of H,S on plant growth is an inhibition of the energy-dependent process of N uptake.

Wetland macrophytes, including Spar- occur through an inhibition of NH,+ uptake tina altern~$oru, the dominant salt-marsh by some factor(s) associated with soil wa- species in North America, are characterized terlogging (Mendelssohn and Seneca 1980; by high rates of primary productivity. The Morris 1980). This hypothesis is supported importance of this carbon source to the en- by the finding that the shorter, less produc- ergy base of coastal and inland fisheries pro- tive form of S. alternifloru, which often in- duction has stimulated considerable re- habits more waterlogged regions of the search on the factors controlling plant growth marsh, is limited (Mendelssohn 1979a,b), and primary production in wetlands. Fac- yet the bulk soil contains higher concentra- tors most frequently cited as important in tions of NH4+ than is found in more pro- influencing marsh plant growth include sa- ductive areas (e.g. streamside or tall form) linity (Nestler 1977), tidal inundation (Mendelssohn 1979b; Delaune et al. 1983). (Odum and Fanning 1973), soil waterlog- In addition, half-saturation constants for ging (e.g. Linthurst and Seneca 1980; Howes NH,+ uptake are too low to explain the N et al. 198 I), and nutrient deficiencies (Men- limitation found in S. alternQ7ura marshes delssohn 1979aJ). (Morris 1980). Fertilization experiments have led to the Soil waterlogging may influence the in- consensus that N is the primary nutrient teractions of soils and roots in several ways. limiting salt-marsh primary production (e.g. Saturation of a soil with water reduces the Sullivan and Daiber 1974; Valiela and Teal rate of oxygen diffusion (Gambrel1 and Pat- 1974). Some investigators have suggested rick 1978) and thereby limits the amount that growth limitation of S. aZterniJoru may of oxygen available for plant root and mi- crobial respiration. Wetland plants must not only adapt to root anaerobiosis, but must r To whom correspondence should be addressed. also contend with the edaphic microbial Acknowledgments production of phytotoxins. When a soil is We thank D. M. Burdick, R. E. Turner, and R. Twil- flooded and oxygen becomes limiting, het- ley for reviewing the manuscript and K. Flynn and J. erotrophic facultatively and obligately an- Chupasko for technical assistance, aerobic microorganisms in the soil use in- This work was supported by funds from the Loui- siana Sea Grant Program (National Oceanic and At- organic ions as terminal electron acceptors mospheric Administration of the U.S. Department of to break down organic matter (Turner and Commerce). Patrick 1968). The flooded soil is thereby 399 400 Koch et al. rapidly converted through microbial res- house. Stems were separated carefully to piration to a biochemically reduced state avoid injury to roots and rhizomes. Each (Turner and Patrick 1968). plant was placed in a small1 plastic cup filled Hydrogen sulfide-a known phytotoxin with sand and watered with full-strength to wetland plants (Okajima and Takagi 1955; Hoaglands + Fe-EDTA nutrient solution Goodman and Williams 196 1; Koch and until enough root material was present to Mendelssohn 1989)-may accumulate with conduct the experiment (2 weeks). Thirty- persistent soil waterlogging in natural five S. alterniflora plants with similar heights marshes due to the biochemical reduction (N 10-l 5 cm) were chosen for seven treat- of sulfate to sulfide by dissimilatory SO,*-- ments (n = 5). Fifty P. hemitomon plants reducing bacteria (e.g. Desulfivibrio) (Post- were selected for five treatments, but with gate 19 5 9). Concentrations of sulfide in ma- two plants (20-25 cm high) per experimen- rine :soils have been found to exceed 1 mM tal vessel (n = 5). The number of P. hemi- (e.g. Carlson and Forrest 1982; King et al. tomon plants, which produce relatively 1982:; Howes et al. 1985). This phytotoxin smaller amounts of roots, was doubled to has been shown to reduce growth in several ensure enough tissue for biochemical anal-, marine and freshwater marsh species (Joshi ysis. Plants were carefully removed from the et al. 1975; Ingold and Havill 1984) but the plastic cups and the sand gently washed off underlying physiological mechanism by before transfer to the experimental vessels.. which this growth inhibition occurs, partic- Experimental design-The experiments ularly in wetland plants, is not well under- were conducted in sealed hydroponic cul- stood. ture solution with sulfide concentrations of We present data supporting the suppo- 0.5, 1.0, 2.0, 3.0, and 4.0 mM for S. alter-, sition that the growth-limiting effect of HzS niflora and 0.5, 1.0, and 2.0 mM for P. on plants inhabiting waterlogged soil envi- hemitomon. In addition, a hypoxic treat- ronments results from an inhibition of the ment (0.0 mM sulfide level) was used to test anoxic generation of energy via alcoholic for the effect of low oxygen tension alone. fermentation and a concomitant reduction Well-aerated plants in bnffered culture so- in plant N uptake. This study investigates lution served as controls. The experiments the effects of aeration, hypoxia, and hypoxia were conducted (September 1988) as a com- plus sulfide on root ADH activity, root en- pletely random design (n = 5). Nutrient so- ergy status, nitrogen uptake, and leaf growth lution (full-strength Hoaglands + Fe-EDTA) of two wetland species: S. alterniJlora Loi- containing MES buffer (pH 7.0) and a ni- sel. (salt-marsh species) and Panicum hemi- trification inhibitor (N-Serve) was bubbled tom,on Schultes (freshwater marsh species). with N2 gas for 45 min and added to Erlen- The,se two macrophytes were chosen in or- meyer flasks (250 ml) tha.t were stopped im- der .to compare a salt-marsh species that in mediately. Plants were previously inserted nature is frequently exposed to high sulfide through holes in the stoppers and positioned concentrations (> 1 .O mM; Mendelssohn in the flasks to totally immerse the roots in and McKee 1988) to a freshwater marsh the nutrient solution. Flasks were then sealed species that rarely comes in contact with with a nontoxic silicone and wrapped in alu- high sulfide levels (~0.1 mM; McKee and minum foil to exclude light from the roots. Mendelssohn unpubl. data). The aerated treatment for both species was achieved by bubbling the media con- Met hods tinuously with air through a 0.1 -cm-diam- Plant source and pretreatment-Mono- eter tube. The hypoxic treatment consisted specific stands of S. alterniflora from a salt of deoxygenated nutrient solution created marsh (29”20’N, 90”4O’W) and P. hemito- by bubbling the culture media with N2 gas mon from a freshwater marsh (30”O’N, for 45 min before introducing the plants. 9OYO’W) were sampled in Terrebonne Par- The hypoxic sulfide treatments were ish, Louisiana. Several plugs with intact achieved by adding sulfide (Na,S * 9H20) to vegetation (15 x 50 cm) were extracted from the deoxygenated nutrient buffer solution. the marsh and transported to the green- After sulfide was added at the start of the H,S efect on wetland plants 401 experiment, r5NH,+ (99.0 atom% excess) Table 1. Average sulfide concentrations (mM) achieved in the treatment flasks during the 6-d (Spar- was added via syringe to achieve a final con- dina alterniflora) and 2-d (Panicum hemitomon) ex- centration of 5.0 ppm (357 PM) in the flask. periments. Sulfide concentrations measured in aerated A 5-ml portion of the medium from each and nonaerated (0.0 mM) flasks were below detection flask was extracted daily by syringe and im- limits ( 85%) and indicated no white stele were frozen with liquid nitrogen differential inhibition due to species or upon removal from the culture solution. treatment during analysis. 402 Koch et al.

Nitrogen isotope analysis-Plant tissue These sulfide treatments reduced average was ground as described above and ana- leaf growth in S. altern@ora and P. hemi- lyzed for nitrogen content (TKN) by a semi- tomon by 42 and 200% (Fig. Id). micro-Kjeldahl method (Buresh et al. 1982). ADH activity-Under hypoxic condi- The samples were oxidized with NaOBr, and tions, ADH activity was increased fourfold lSN content was determined with a DuPont in S. alterniflora and twofold in P. hemi-, 21-6 14 isotope ratio mass spectrometer ac- tomon roots compared to the aerated treat- cording to Hauck (1982). Atom% 15N was ment which exhibited limited ADH activity determined with the ratio of the 28 (14N: (Fig. lb). At redox potentials of +60 mV 14N) and 29 (14N : 15N) peak heights, as well for S. alternijlora and + 93 mV in P. hemito- as t.he 30 (15N: 15N) peak heights when man, root ADH activity was stimulated (Fig. atom% 15N was >5.0. Atom% 15N excess la,b). Sulfide concentrations of 1.0 mM in was determined by subtracting the atom% P. hemitomon and 2.0 mM in S. altermiflora ‘*N of unlabeled NH,+ (0.349) from the cal- culture solution resulted, however, in sig- culat;ed atom% 15N of each sample. This nificant inhibition (P < 0.05) of root ADH excess was multiplied by the total weight of activity. This inhibition occurred even N in the plant tissue to determine atom% though addition of sulfide caused the Eh of lsN tissue recovery. Total percent recovery the culture solution to decline (Fig. la,b). in th;e root and leaf was determined by di- With an increase in sulfide level from 0.0 viding 15N in the tissue by that added to the to 4.0 mM, S. alterniflora root ADH activ- culture media and multiplying by 100. ity decreased (slope = --0.46; P < 0.01) Skztistical analysis-Analysis of variance after 6 d. Although ADH activity was sig- (ANtOVA; proc GLM), regression (proc nificantly inhibited in P. hemitomon roots REG), and contrast (proc GLM) were per- exposed to 1.0 and 2.0 mM sulfide, it was Formed with the SAS statistical package (SAS higher at 0.5 mM sulfide relative to the non- 198;!). Statistically significant differences aerated (0.0 mM sulfide) treatment. The between treatment means were found with shorter exposure time for P. hemitomon (2 Duncan’s multiple range test (ANOVA). All d) may be the reason for the lack of inhi- means are significantly different at the 0.05 bition of ADH activity at 0.5 mM sulfide level unless otherwise stated. compared to S. alterniflora (6 d). Adenine nucleotides and AEC ratio-To- Results tal adenine nucleotide concentrations de- Redox potential-- Redox values were high creased in both species when ADH activi- in the aerated treatment (+400 to +700 ties increased in the hypoxic roots (Fig. lc), mV), but significantly lower (0 to + 125 mV) but the AEC ratio-an indicator of meta- in the treatment that was initially bubbled bolic activity describing the equilibrium be- with N2 gas (Fig. la). An increase in sulfide tween ATP-generating and -utilizing reac- concentration in the hypoxic culture solu- tions (Atkinson 1968)-was maintained tion from 0.0 to 4.0 mM for S. alterniJora during root hypoxia in S. alterniflora. Al- and from 0.0 to 2.0 mM for P. hemitomon though P. hemitomon root AEC ratio was resulted in a linear decrease in Eh from +60 lower under hypoxic conditions, multiple to -170 mV(R 2 == 0.84) and from +93 to range testing showed no significant differ- - 1080mV (R2 = 0.87) (Fig. la). ence between the aerated and nonaerated Plant growth-Leaf elongation was sig- (0.0 mM sulfide) treatment. nificantly slower in the anoxic treatment (0.0 Neither species was able to maintain its mM sulfide) compared with the aerated total adenylate pool or AEC ratio when sul- treatment for both species (Fig. Id). Leaf fide was present in the culture media (Fig. elongation was further reduced by adding lc). After 2 d of exposure to sulfide treat- sulfide to the culture media. The freshwater ments, P. hemitomon showed a significant marsh species, P. hcmitomon, however, was reduction in total adenylate concentrations significantly affected at a lower concentra- and AEC at sulfide concentrations of 1.0 tion of sulfide (1.0 mM) than was the salt mM and above (Fig. lc). Spartina alterni- marsh species, S. alterniflora (2.0 mM). flora also exhibited significantly reduced ad- H2S efect on wetland pIants 403

SPARTINA AiTERNlFLORA PANICVM HEMITOMON (a)

250 P s 5 5

w” y" 0

T-== .250- A 0 0.5 1.0 2.0 (b) 1? ." 3wl

A 0 0.5 1 .o 2.0 3.0 4.0 (cl OAm

(d) I.0 IT m LEAFEUlNOATION

0.6 0 IsN RECOVERY z 2 12 E-r 0.6 2

S" e 0.4 ii 4" 6 ,z w H J 0.2

00 0 A 0 OS 1.0 2.0 3.0 4.0 A 0 0.5 1.0 2.0

SULFIDE (mM) SULFIDE (mM) Fig. 1. Sparfina a/tern&a culture solution experiment (6 d) in response to the seven treatments applied [aerated (A), hypoxic (0.0 mM sulfide), and hypoxic with sulfide additions of 0.5, 1.O, 2.0, 3.0, and 4.0 mMJ and P. hemitomon experiment (2 d) with five treatments applied [aerated (A), hypoxic (0.0 mM sulfide), and hypoxic with sulfide additions of 0.5, 1.0, and 2.0 mM]. a. Changes in oxidation-reduction potentials (Eh) in the culture solutions. b. Alcohol dehydrogenase activity (ADH) in the roots. c. Total adenine nucleotide pool (ATP + ADP + AMP) and adenylate energy charge ratio (AEC) in the roots. d. Daily leaf elongation rates, averaged over the experimental period and total r5NH,+-N percent recovery in the roots. Error bars represent the standard errors of the means (n = 5). Koch et al.

Table 2. Leaf rsNH,+ recovery in Spartina alter- lation of reduced inorganic and organic niJora and Fanicum hemitomon. Values represent the compounds in the soil. The redox potential mean 3: SE (n = 5). Asterisk: significantly different means (P < 0.05; Duncan’s multiple range test). (Eh), which is a measure of the intensity of soil reduction, decreases from a highly ox- Leaf “N recovery (W) [Sulfidcl - idized state (+ 700 mV) when the soil is Treatment (mW s. olterniJlora P. hemitomon drained to a moderately (+ 100 mV) or Aerated 0.0 27.17k3.00 28.28+8.14 strongly reduced state (- 300 mV) when Nonaerated 0.0 28.31k3.19 26.1Yt2.77 flooded. In the aerated culture solution, the Nonaerated 0.5* 25.4Ok4.71 12.82k3.49 Eh for both species (Fig. la) was high and Nonaerated 1.0* 32.32k3.57 6.61zkl.47 occurred within the range (+ 400 to + 700 Nonaerated 2.0* 26.5 1k4.04 1 l.lS-+ 1.75 Nonaerated 3.0 22.79k7.14 - mV) characteristic of a well-drained soil Nonaerated 4.0 30.84k7.30 - where oxygen is available and utilized as the preferred electron acceptor (Turner and Patrick 1968). Bubbling the culture solution enylate concentrations and AEC, but at with N, gas resulted in a significantly lower higher (3.0 and 4.0 mM) sulfide levels (Fig. redox status that was indicative of anoxic lc). conditions (Fig. la). In the natural environ- Recovery of “IV in root and leaf tissue- ment, sulfide accumulates in strongly re- N uptake in the roots-a metabolic func- ducing soil conditions where sulfate is avail- tion dependent on energy production-was able (e.g. through inputs of seawater) for found1 to be limited by the hypoxic and sul- reduction by bacteria (Postgate 1959). The fide treatments in S. alternijlora and P. addition of Na,S to the culture solution suc- hemitomon (P < 0.01). Hypoxic conditions cessfully produced a range of sulfide treat- in the culture solution containing 357 PM ment levels (Table 1) similar to that en- 15NH,+ significantly reduced the total 15N% countered in salt marshes (Delaune et al. recovery in the roots of S. alterniflora (5%) 1983; Howes et al. 1985; Mendelssohn and and 1”. hemitomon (7%) compared to the McKee 1988; McKee and Mendelssohn un- aerated roots (Fig. Id). Multiple range tests publ. data). The increase in sulfide concen- showled a further reduction of the percent tration resulted in a linear decrease in Eh 15N recovery in the roots at sulfide levels (Fig. 1a). Similar negative relationships be- z 3.0 mM in S. alterniflora (4-5%) and 1.1 .O tween sulfide concentrations and redox po- mM in P. hemitomon (3-5%) relative to tentials have been found in wetland soils in unaerated roots without sulfide exposure the field (Delaune et al. 1983; Mendelssohn (Fig. 1d). These results are supported by lin- and McKee 1988) and in laboratory soil sus- ear c(ontrast analysis that showed a highly pension experiments (Connell and Patrick signilicant difference (P < 0.0~1) in root 15N 1968). recovery between unaerated controls and sul- Plants exposed to an anoxic root envi- fide-amended plants of both species. Re- ronment must have a means for internal coveq of S. alterniflora leaf 15N was not oxygen transport if they are to maintain signincantly different as a function of treat- aerobic root respiration. Although most ment, but differences were highly significant wetland plants adapted to anoxic conditions (P < 0.0 1) among the aerated, hypoxic, and possess aerenchyma or airspace tissue sulfide-amended P. hemitomon plants (Ta- (Armstrong 1979), this mechanism is not ble 2). always sufficient to maintain completely aerobic metabolism in the roots (see Craw- Discussion ford 1982; Mendelssohn and Burdick 1988). When drained soils are flooded, oxygen Even a species with an extensive aerenchy- diffusion is reduced 10,000 times (Gambrel1 ma system such as S. aIterniJlora may suffer and Patrick 1978). This slow rate of diffu- root oxygen deficiencies when exposed to sion combined with the respiratory de- extremely waterlogged conditions (Men- mands of plant roots and soil microorgan- delssohn et al. 198 1). Root-cell hypoxia can isms results in little or no oxygen available result in a switch to anoxic metabolism as externally to plant roots and the accumu- indicated by an increase in the activity of H,S efect on wetland plants alcohol dehydrogenase (ADH) - the en- (1984) and Pezeshki et al. (1988) found no zyme catalyzing the terminal step in alco- evidence of NH4+ uptake inhibition due to holic fermentation (Mendelssohn and Bur- anaerobiosis in stirred sediment suspen- dick 1988). Spartina alterniflora has been sions. Morris (1984) demonstrated a 60% found to increase root ADH activity in re- decrease in root-specific uptake rates (V,,,) sponse to a hypoxic root environment in of NH,+ by S. alterniflora, however, when the laboratory (Mendelssohn and McKee exposed to anoxic conditions in hydroponic 1987), as well as to soil waterlogging in the culture. Morris and Dacey (1984) further field (Mendelssohn et al. 198 1; Mendels- found that NH,+ uptake was reduced at low sohn and McKee 1988). Similarly, we found concentrations of root oxygen where aero- ADH activity to be stimulated fourfold in bic respiration was inhibited. S. alterniflora roots and twofold in P. hemi- A decreased energy status in the hypoxic tomon roots in the hypoxic treatment com- roots cannot explain the inhibition of N up- pared to the aerated treatment, which ex- take by P. hemitomon and S. alterniJlora hibited limited ADH activity (Fig. 1b). This because both species maintained a relative- response indicated a root oxygen deficiency ly high AEC ratio in this treatment (Fig. in plants grown in the hypoxic media and 1c,d). Accelerated glycolysis, however, may the potential for alcoholic fermentation result in a deficiency of carbon skeletons (Smith and ap Rees 1979; Keeley 1979; Pe- available for nitrogen metabolism. The end rata et al. 1988) (Fig. lb). product of alcoholic fermentation, ethanol, A change from aerobic to anoxic root me- apparently readily diffuses from the roots of tabolism may have serious consequences, S. aIternij7ora (Mendelssohn et al. 198 1; particularly with respect to energy (ATP) Mendelssohn and McKee 1987), as well as production in the roots. Hypoxia does not other wetland macrophytes (Bertani et al. appear to cause a root energy deficiency in 1980), and would represent a major carbon higher plants, however, if alcoholic fermen- drain from the plant, particularly if glucose tation is stimulated and maintained at suf- consumption is substantially increased. ficiently high levels (Mendelssohn et al. Thus, even though stimulation of ADH ac- 1981; Saglio et al. 1983; Mendelssohn and tivity appeared to aid in maintenance of McKee 1987). The maintenance of a high root energy status in S. alternzjlora and P. AEC during hypoxia could occur through hemitomon, it may have resulted in a de- accelerated consumption of glucose (Pas- ficiency of carbon skeletons for NH,+ assim- teur effect) or by decreased ATP utilization ilation. The effect of hypoxia on NH4+ up- (Mendelssohn and Burdick 1988). The take is supported by the significantly lower maintenance of root energy status during leaf elongation rate relative to the plants in hypoxia was most apparent in S. alternQlora the aerated treatment (Fig. Id). Further de- (Fig. lc). Although the total adenylate con- creases in NH,+ uptake and leaf elongation centration and AEC ratio in P. hemitomon occurred when sulfide was added to the hy- roots under hypoxia were lower than that poxic culture solution (Fig. Id). in well-aerated roots, the differences were Nutrient uptake requires energy. There- not significant (Fig, lc). fore, the decline of the root AEC ratios of The significant reduction in percent 15N S. alterniflora and P. hemitomon may have recovery in the hypoxic roots indicated that been an important factor in reducing root decreased aeration did affect nitrogen up- 15NH4+ uptake when sulfide was added to take by S. alterniflora and P. hemitomon the culture media. This interpretation is (Fig. Id). Soil waterlogging and hypoxia of supported by the fact that the same sulfide culture solutions have been demonstrated levels that suppressed AEC resulted in sig- to reduce nutrient uptake in nonwetland nificantly lower N uptake in the roots (Fig. plant species (Drew and Sisworo 1977, 1979; lc,d). Percent recovery of 15N was lower in Trought and Drew 1980). Previous work, the roots of both species after a brief ex- however, has yielded conflicting results re- posure to sulfide treatments, whereas this garding the effect of hypoxia on nutrient response was seen in leaf tissue only in P. uptake in S. alternijlora. Delaune et al. hemitomon (Table 2). The absence of an Koch et al, effecl. of sulfide on S. alternijlora leaf r5N also alIect alternate anoxic pathways through recovery in this experiment may have been inhibition of a key enzyme, ADH. Men- caused by the slower growth rate of this delssohn et al. (198 1) found depressed root species relative to that of P. hemitomon (Fig. ADH activity within the die-back zone of Id). A longer term study may thus be need- a S. alterniflora marsh where Eh indicated ed to show significant differences in N trans- strongly reducing conditions, but higher en- port to the leaf tissue as a result of sulfide zyme activities in adjacent zones where the toxicity in S. alterniflora. soil was less reduced; they concluded that Species-specific differences in pattern of ADH activity in the die-back zone was in- response to sulfide indicated that the fresh- hibited by a soil toxin such as H,S. water marsh species, P. hemitomorz, was Since sulfide addition caused a significant more sensitive to the presence of this phy- decrease in culture solution Eh (P < 0.01; totoxin than was the salt-marsh species, S. Fig. la) and, hence, an increase in the oxy- alternzJora. At sulfide concentrations of 1 .O gen demand (reducing power) of the rooting mM and above, root energy status, NH4+ media, these species were simultaneously uptake, and leaf growth of P. hemitomon exposed to two potential stresses: sulfide were more negatively affected relative to toxicity and root oxygen deficiency. At- aerat’ed controls. These results support ear- tempts to separate the effects of redox po- lier findings that showed similar differences tential and sulfide in culture solution by in sulfide tolerance between these two species adding reducing agents have been unsuc- (K0c.h and Mendelssohn 1989). Work with cessful (Ingold and Havill 1984). A decrease other wetland macrophytes has also shown in Eh due to sulfide additions would be ex- differential tolerance of sulfide and coinci- pected to cause stimulation in root ADH dence of sensitivity with species distribu- activity at least equal to that in hypoxic tion iin the field (Havill et al. 1985). rooting media because oxygen would bc The precise mechanism whereby sulfide limiting in all cases. Instead, ADH activity exerts an effect on plant growth is not well was significantly inhibited in both species unde:rstood. A decrease in the activity of at low redox conditions where sulfide con- root metallo-enzymes has been postulated centrations were high (Fig. la,b). At low as the phytotoxic effect of HzS (Allam and sulfide concentrations (0.5 mM), however, Hollis 1972; Havill et al. 1985; Pearson and a stimulatory effect on ADH activity was Havill 1988) primarily because important observed in P. hemitomon. Pearson and oxidalses such as cytochrome oxidase - the Havill (1988) also showed an increase in terminal enzyme in the electron transport ADH activity in several wetland and non- chain of aerobic respiration-are inhibited wetland macrophytes when 0.10 mM sul- by sulfide. Wetland macrophytes growing in fide was added to a culture solution (+ 190 anoxrc, waterlogged soils, where H,S can mV). It is possible that these low sulfide accumulate often exhibit anoxic root me- concentrations increased the oxygen de- tabolism., however, which is believed to mand of the culture solution, but were not contribute to energy production (e.g. Mo- high enough to inhibit ADH activity. Thus, quot et al. 1981; Saglio et al. 1983, 1988). although our results indicate the possibility Thercfore, the inhibitory effect of sulfide on of a sulfide effect at low concentrations aerobic respiration (i.e. cytochrome oxi- through a further increase in oxygen de- dase) alone may not be sufficient to explain mand (decrease in Eh) of the rooting media, the growth inhibition and lethal response, they also support a toxic effect at higher i.e. “(die-back,” found in natural and man- concentrations, as evidenced by significant aged wetland systems where sulfide accu- inhibition in the root ADH activity. Con- mulates (Okajima and Takagi 1955; Men- comitant decreases in root energy status (Fig. delssohn and McKee 1988). The significant lc) suggest the possibility that as ADH ac- inhibition of ADH activity in S. alternzjlora tivity was suppressed by H2S the resultant and P. hemitomon roots at sulfide concen- decrease in anoxic root metabolism, as well trations of 2.0 and 1.0 mM, respectively as any limited aerobic respiration, caused (Fig. 1b), supports the idea that in addition root adenylate levels and energy charge to to aerobic respiratory enzymes, H,S may decline. It is apparent that although hypoxia H2S e#ect on wetland plants 407 alone did not cause a significant decrease in bedause of the suppression of anoxic, as well the energy status of P. hemitomon and S. as aerobic, energy production. alternzjZoraroots, hypoxia plus sulfide (2 1 .O or 2.0 mM, respectively) did. A decrease in 15N recovery and leaf elon- References gation with increasing sulfide concentration ALLAM, A. I., AND J. P. HOLLIS. 1972. Sulfide inhi- (Fig 1d) provides direct evidence of sulfide- bition of oxidases in rice roots. Phytopathology induced inhibition of N uptake in a fresh- 62: 634-639. water and a salt-marsh species. Whether the ARMSTRONG,W. 1979. Aeration in higher plants. Adv. effect of sulfide on NH4+ uptake (Fig. Id) Bot. Res. 7: 225-332. ATKINSON. D. E. 1968. Energy charge of adenylate was indeed mediated through an inhibition pool as a regulatory parameter: Interaction with of alcoholic fermentation and ATP produc- feedback modifiers. Biochemistrv 7: 4030-4034. tion is not conclusive and requires further BERTANI, A., I. BRAMBILLA,AND F. MENEGUS. 1980. investigation. Nonetheless, our data sup- Effect of anaerobiosis on rice seedlings: Growth, port the hypothesis that sulfide limits the metabolic rate, and fate of fermentation products. J. Exp. Bot. 31: 325-331. potential for root anoxic energy production BURESH,R. J., E. R. AUSTIN, AND E. T. CRASWELL. and contributes to the growing body of in- 1982. Analytical methods in 15N research. Fert. formation that implicates sulfide as a major Res. 3: 37-82. growth-limiting factor for wetland plant CARLSON.P. R.. AND H. FORREST. 1982. &take of dissdlved sulfide by Spartina alterncjloia: Evi- species. S&l sulfide concentrations may, dence from natural sulfur isotope ratios. Science therefore, be acting in concert with other 216: 158-165. factors (e.g. salinity) in exerting control of CONNELL,W. E., AND W. H. PATRICK,JR. 1968. Sul- macrophyte species distribution in wet- fate reduction in soil: Effects of redox potential lands. and pH. Science 159: 86-87. CRAWFORD,R. M. M. 1982. Physiological responses to flooding, p. 453-477. In Encyclopedia of plant physiology: v. 12B. Springer. - - Conclusions DELALNE. R. D.. C. J. SMITH. AND W. H. PATRICK.JR. Sulfide toxicity has been implicated as a 1983: Relationships oi marsh elevation, redox potential, and sulfide lo Spartina alterniflora pro- causative factor in the die-back of European ductivity. Soil Sci. Sot. Am. J. 47: 930-935. and North American salt marshes (Good- -- AND M. D. TOLLEY. 1984. The effect man and Williams 196 1; Ingold and Havill of’sedime;t redox potential on nitrogen uptake, 1984; Mendelssohn and McKee 1988). Al- anaerobic root respiration and growth of Sparfina though H,S is known to reduce growth in alterniflora Loisely Aquat. Bat.-1% 223-250. DREW, M. C., AND E. J. SISWORO. 1977. Early effects plants, we are only beginning to understand of flooding on nitrogen deficiency and leaf chlo- how this phytotoxin affects metabolic func- rosis in barley. New Phytol. 79: 567-57 1. tions in marine, brackish, and freshwater -, AND -. 1979. The development of wa- wetland plant species. Under aerobic con- terlogging damage in young barley plants in rela- ditions in the root, sulfide can bind with tion to plant nutrient status and changes in soil properties. New Phytol. 82: 301-314. metallo-enzymes (Allam and Hollis 1972; GAMBRELL, R. P., AND W. H. PATRICK, JR. 1978. 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