Dimethylsulphoniopropionate (DMSP) in Spartina alterniflora Loisel

ELSEVIER

Aquatic Botany48 (1994) 239-259

Dimethylsulphoniopropionate (DMSP) in Spartina alterniflora Loisel. M a r i n u s L. O t t e *'1, J a m e s T. M o r r i s Department of Biological Sciences, Universityof South Carolina, Columbia, SC29208, USA Accepted 15 February 1994

Abstract Dimethylsulphoniopropionatc (DMSP) in saltmarsh grasses of the genus Spartina has been suggested to act as a compatible osmolyte or as an intermediary product of a sulphide detoxification mechanism. Investigations of Spartina alterniflora Loisel. plants collected from salinity and sulphide gradients along a South Carolina fiver showed that DMSP concentrations were not correlated with either salinity or sulphide concentrations in soil porewater. This suggested that DMSP is neither a compatible osmolyte nor involved in sulphide detoxification. Greenhouse experiments were also performed, investigating the effects of sulphate, sulphide and ammonium nitrate on DMSP concentrations in the plants. Only ammonium nitrate affected DMSP concentrations, which decreased in the shoots upon increasing nitrogen amendments. It is suggested that nitrogen stimulates biomass production, leading to dilution of DMSP concentrations. Ammonium nitrate amendments also decreased the fraction of total sulphur in the shoots allocated to DMSP, which ranged between 36 and 86%. The data suggest that nitrogen plays a key role in determining DMSP concentrations in plants of the genus Spartina and that DMSP is not involved in a sulphide detoxifying mechanism. Alternative hypotheses for the functions of DMSP in Spartina alterniflora are Suggested.

1. Introduction Dimcthylsulphoniopropionate (DMSP), also known as dimcthylpropiothetin (DMPT; Greene, 1962), is a tertiary sulphur compound found in many, mostly *Correspondingauthor. ~Presentaddress: Departmentof Botany,UniversityCollegeDublin, Belfield,Dublin 4, Ireland. 0304-3770/94/$07.00 © 1994 ElsevierScienceB.V. All rights reserved SSD10304-3770 (94) 00387-2

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marine, algae (e.g. Dickson et al., 1980; Karsten et al., 1990) and in some higher plant species, namely in the halophytic grasses Spartina anglica Hubbard (Larher et al., 1977; Van Diggelen et al., 1986) and Spartina alterniflora Loisel. (Dacey et al., 1987), and in the dicotyledonous halophyte Melanthera biflora (L.) Wild (Allaway et al., 1984). DMSP is the precursor of dimethylsulphide (DMS) (Cantoni and Anderson, 1956). The chemical similarity between DMSP and quaternary ammonium compounds, such as glycinebetaine, which are known to act as osmolytes in plants, has led researchers to speculate on the osmoregulatory function of DMSP in Spartina species (Wyn Jones and Gorham, 1983; Dacey et al., 1987). Indeed, research on certain algae suggests that DMSP can act as an osmolyte (Dickson et al., 1980; Reed, 1983) and Dacey et al. (1987) found a positive correlation between soil salinity and DMSP concentrations in S. alterniflora. In contrast, Van Diggelen et al. (1986) did not find an effect of salinity on DMSP concentrations in S. anglica in greenhouse experiments. Their experiments indicated that DMSP concentrations in this species are positively affected by sulphide concentrations in the growth medium, leading them to suggest that production of DMSP and the subsequent formation of volatile DMS is a mechanism to avoid build-up of toxic sulphide concentrations in Spartina species. Although sulphate and sulphide differ in mobility and toxicity, it might be expected that increased availability of sulphate would have the same increasing effect on DMSP concentrations as sulphide, because plants reduce sulphate to sulphide in order to incorporate it in sulphur-containing compounds (Goldhaber and Kaplan, 1974, Rennenberg, 1984). However, in the research by Van Diggelen et al. (1986) sulphate increased the total sulphur content, but did not affect DMSP concentrations in plants. S. alterniflora is found along a salinity gradient on the banks of the Cooper River, South Carolina, from saline at the mouth to freshwater more than 50 km upstream (Bradley et al., 1990). The sulphide concentrations in porewater also decrease with increasing distance from the mouth of the river, reflecting the decreasing marine influence (Morris et al., 1991 ). However, the sulphide gradient does not exactly follow the salinity gradient. This situation allowed us to investigate the effects of salinity and sulphide on DMSP in S. alterniflora under natural conditions. If DMSP has a function in either osmoregulation or sulphide detoxification, concentrations of this compound were expected to correlate with either salinity or sulphide concentrations in the soil porewater. To test these hypotheses plants were collected on a monthly to bimonthly basis from March to September 1992, and analysed for DMSP. In addition, greenhouse experiments were performed to investigate the effects of sulphate and nitrogen supply on DMSP in S.

alterniflora. As was emphasised by Rennenberg (1984), a close relation exists between sulphur and nitrogen metabolism, since most sulphur taken up is used for protein synthesis. The nitrogen status of plants may therefore affect DMSP concentrations. Dacey et al. (1987 ) reported lower DMSP concentrations in nitrogen-fertilised plants compared to unfertilised plants, and recently Gr~ine and Kirst

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(1992) showed that nitrogen deficiency induced increased concentrations of DMSP in the alga Tetraselmis subcordiformis (Wille) Butcher. In a greenhouse experiment the effect of sulphate on DMSP was investigated. IfDMSP concentrations in S. alterniflora depend on sulphur supply, high amendments of sodium sulphate should increase DMSP concentrations in the plants. Alternatively, nitrogen supply to the plants may affect DMSP concentrations. This was investigated by growing plants on different concentrations of ammonium nitrate, and in another experiment on combined concentrations of sulphide and ammonium nitrate. The latter experiment was designed to also identify interactions between nitrogen supply to the plants and sulphide in the growth medium. 2. Materials and methods

2. I. DMSP concentrations in S. alterniflora along the Cooper River 2.1.1. Sampling of plants S. alterniflora plants (three replications) were collected within 2 m of the dialysis vials (see 'Sampling and analysis of porewater') from ten sites along the Cooper River estuary, South Carolina (Fig. 1 ), in March, April, May, June, July and September 1992. The sampling sites were all flooded during high tides, i.e. at least twice a day. The plants were transported to the laboratory on ice and stored in a freezer at - 18 ° C. Before analysis, plants were washed with tap water, and algal growth on the surface of the leaves was wiped off with a paper tissue. Live roots and green leaves were analysed for DMSP as described below. S. alterniflora and Spartinafoliosa Trin. plants were also collected in June 1992, from the San Francisco bay area (Petaluma River, San Pablo Bay) by Dr. D.R. Strong. They were sent overnight to South Carolina by Federal Express at ambient temperatures. The leaves of the plants were analysed for DMSP immediately after arrival in our laboratory. At ambient temperatures the turnover rate of DMSP to DMS is so low (see 'Discussion') as not to have affected the DMSP concentrations in the plants during transport.

2.1.2. Sampling and analysis of porewater At every sampling site along the Cooper River, three dialysis units were placed at approximately 10 cm depth in the soil within 0.5 m of each other, top down. These units consisted of 25 ml glass scintillation vials capped with 45/an nylon screen (Nytex) and filled with deionised water. Vials were replaced during every sampling. The time the vials stayed in the soil was generally between 4 and 6 weeks. Controlled laboratory trials indicated that water in the vials reaches equilibrium with the surrounding porewater within a few hours to 3 weeks depending on the permeability of the sediment. The sulphides in dialysis vials were stabilised within 2 h of collection in the field. In the interim, the membranes of the vials were kept covered with sediment to keep them air-locked, stored in plastic

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'~~)]j

/

INPOPOLI'S

DAM

1 "I"

Fig. 1. Map of the Cooper River, South Carolina, showing sampling sites (1-10). Distances of the sites to the mouth of the river (km) as measured along the river are listed in Table 2.

Zip-Loc bags and kept on ice. To preserve sulphides, 5 ml 10 mM zinc acetate solution was added to a 5 ml sample. This procedure precipitates sulphides as their zinc salts and makes them less sensitive to oxidation. Zinc precipitates were found to be stable for several weeks. Determination of sulphides was carded out following a modified method after Cline (1969). To 5 ml pretreated sample (diluted if necessary), 0.4 ml dye solution (2 g N,N-dimethyl-p-phenylenediamine sulphate + 3 g ferric chloride (FeCI3"6H20) in 500 ml cold 50% HCI) was added, incubated at room temperature for at least 30 min and absorbance measured at 670 nm using a Perkin-Elmer Lambda 3 UV/VIS spectrophotometer. Chloride concentrations in porewater were determined by eoulometric titration using a Haake Biichler Digital Chloridometer. Within-site variation, i.e. for the three replications at any site at any date, was typically less than 10% for both sulphide and chloride concentrations.

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2.2. Greenhouse experiments 2.2.1. Plant material For the experiments described in this paper plants were used that had been pregrown in the greenhouse for some time. S. alterniflora plants either originated from the Great Sippewissett Saltmarsh, Massachusetts ('Sippewissett stock'), originally collected in summer 1981 and propagated from cuttings from then onwards, or from the mouth of the Cooper River ('Cooper River stock'), South Carolina, originally collected in September 1991. Plants were kept on a mixture of pot soil and sand ( 1:10 w/w). Stocks were kept and experiments performed in a greenhouse at the university campus. Temperatures ranged from 24 to 35°C, and light intensity and light period followed natural sunlight outside the greenhouse, depending on season and time of the day. Table 1 Set-up of greenhouse experiments

Experiment 1. Effects of sulphate Plant stock Substrate Treatments Number of plants per treatment Harvests

'Sippewissett' Pot soil-sand mixture ( l: l 0 w / w ) Tapwater, 90 mM NaC1, 45 mM Na~SO4 15 Five plants each on t = 0 (shoots only), t = 8 (shoots only) and t = 68 days (main shoots, young shoots and roots)

Experiment 2. Effects of ammonium nitrate Plant stock Substrate Treatments Number of plants per treatment Harvests

'Cooper River' Coarse sand 0, 0.5, 2 and 5 mM NH4NO3 10 Five plants each on t = 36 and t = 71 days (shoots and roots)

Experiment 3. Effects of ammonium nitrate and sulphide Plant stock Substrate Treatments

'Cooper River' Coarse sand According to the set-up below. Concentrations of ammonium nitrate (N) and sodium sulphide (S) in treatment solutions, and treatment codes as used hereafter in the text. Nitrogen (N) Sulphur

0 mM

0.5 mM

2 mM

(s)

Number of plants per treatment Harvests

0 mM 0NOS 0.SNOS 0.5 mM 0N0.5S 0.5N0.5S l mM" 0NIS 0.5N1S 5 All plants on t = 56 days (shoots and roots )

aFrom t= 23 days onwards 2 mM sodium sulphide instead of 1 mM was used.

2NOS 2N0.5S 2N1S

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The experiments were all carried out during spring and summer 1992, on sand with plants grown in pots of 9 cm diameter. The treatments were applied as simple solutions of the treatment salts instead of balanced nutrient solutions, to prevent interactions between ions in the growth medium (e.g. precipitation of sulphides with metals such as iron), so that effects could be clearly allocated to the treatments. It was assumed that the sand culture provided enough additional nutrients for the duration of the experiments and no indications of nutrient deficiency were observed in plants in any of the experiments. The plants were grown from cuttings and did not develop rhizomes during the experiments. The shoots consisted almost entirely of leaves. All greenhouse experiments were carried out using fully randomised set-ups (Table 1 ). Further particulars of the experiments are described below.

2.2.2. Experiment 1. Effects of sulphate The pots were placed on trays ( 13 cm diameter, 2 cm height) on a table and submerged once a week in the respective treatment solutions up to 2 cm above the soil level in the pots (five pots at a time in 5 1 solution) for 15 rain, so that the soil became saturated with the treatment solution. Both sodium chloride and sodium sulphate solution contained equal concentrations of sodium (90 mM). The sulphate concentration was about 1.5 times that of seawater (Stumm and Morgan, 1981 ). The sodium chloride treatment was a control to assure that any effect of the sodium sulphate treatment could be attributed to sulphate, not to sodium.

2.2.3. Experiment 2. Effects of ammonium nitrate The concentrations of ammonium nitrate were chosen based on field observations (Smart and Barko, 1980; Morris et al., 1991 ) and on treatments previously used by Van Diggelen et al. (1986). Treatments were applied as described for Experiment 1.

2.2. 4. Experiment 3. Effects of ammonium nitrate and sulphide The pots were placed in larger pots ( 12 cm diameter) lined with black horticultural plastic to prevent solutions from running out. Solutions were saturated with nitrogen by bubbling the gas through them for several hours before and after adding the salts. The pH was set at 7.5. On the first treatment day the pots were submerged with solution (0.61 per pot) so that the sand surface was just covered. Every Monday, Wednesday and Friday solutions were refreshed. The pH was found not to drop below 6.5 in any of the solutions. Because no effect on growth (as indicated by shoot length) was found in the 1 mM sulphur treatments compared with the control (0NOS), the concentrations were increased to 2 mM from t= 23 days onward.

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2.2.5. All experiments Plants from all three experiments were washed with water after harvest and, if necessary, stored in a freezer at - 18 °C until further analysis. Plant parts were separated, weighed and analysed for DMSP as described below.

2.3. Analysis of DMSP in plant material DMSP in plants was analysed following a modified procedure after Van Diggelen et al. (1986). About 0.3 g fresh weight of plant material (roots or the widest parts of the leaves in the case of shoots) was submerged in liquid nitrogen. The plant material was then transferred into 25 ml vials and 5 ml 4.25 M NaOH solution added. Vials were sealed with Teflon-lined grey butyl septa (Wheaton) with crimp caps. For standards, known amounts of pure DMSP were added to 5 ml NaOH solution. DMSP was kindly supplied by the Department of Ecology and Ecotoxicology, Vrije Universiteit, Amsterdam, The Netherlands, and was originally synthesised as the hydrochloride salt by Dr. D.M. Dickson following Larher et al. (1977). Incubation time was at least 16 h. It was found that DMS was still liberated from the samples when using shorter incubation periods (as used by other researchers; e.g. Van Diggelen et al. (1986), Weber et al. ( 1991 ) ). Also, shorter incubation periods appeared to overestimate DMSP concentrations in samples, because the rate of DMS formation during the first 3 h of incubation is faster from plant samples than from synthetic DMSP in standards (Fig. 2). Headspace gas was analysed for DMS by injecting 0.1 ml (standards and leaf samples) or 0.5 ml (root samples) using a gas-tight syringe into a Carle Analyti~oo~ ,~ 80

-"E 80 -t1)/? II / Jl;

I--"'0 ....

=_ 2o "ll/ n-

o

I

•

14

16

Standard. Sample -U I

~ 0

2

4

6

8

10

12

18

20

22

(h) Fig. 2. Dimethylsulphide (DMS) evolution from plant samples and standards as a function of incubation time. Standards contained 6.45 #tool DMSP (i.e. 0.5 mi of a solution containing 12.9 #mol DMSP m1-1 in 4.5 ml 4.25 M NaOH in a 25 ml vial). Samples (in 25 ml vials) contained 0.2320.285 g fresh plant material in 5 ml 4.25 M NaOH solution. For standards the reading after incubation for 21 h without intermediate puncturingof the septa was set at 100%, whereas for samples the highest reading was set at 100%. Sample - N: sample not submerged in liquid nitrogen prior to incubation. Sample + N: sample submerged in liquid nitrogen prior to incubation. Number of replications was 2. Standard deviation of standards was less than 4% of the mean reading. DMSP concentrations in samples - N was calculated to be 23.0 (SD=2.5) and in samples + N 28.8 (SD=0.3) indicating a higher recovery when samples are submerged in liquid nitrogen prior to incubation. Time

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cal Gas Chromatograph equipped with a glass column (3.2 m m × 2 m) packed with 0. 1% SP-1000 on 80/100 Carbopack C and flame ionisation detector. Column temperature was 100 ° C and carder gas was helium at a flow rate of 20 ml min-1. Retention time for DMS typically was about 1.9 min. The method assumes that all DMSP is converted to DMS and that DMS distributes over gas and liquid phases according to Henry's Law (Przyjazny et al., 1983; Dacey et al., 1984; Dacey and Blough, 1987).

2.4. Analysis of plant samples for carbon (C), nitrogen (N) and sulphur (S) Shoots 9f plants from greenhouse Experiment 3 (effects of ammonium nitrate and sulphide), grown in the 0NOS, ON 1S, 2NOS and 2N 1S treatments, were dried at 50°C to constant weight, thoroughly ground using a mortar and pestle, and analysed for total carbon, nitrogen and sulphur. The plants from the other, intermediate treatments were not analysed for carbon, nitrogen and sulphur. The analyses were all carried out by the Microanalytical Laboratory, Department of Chemistry, University College Dublin, Ireland. Total carbon and nitrogen were determined using a Carlo-Erba 1106 element analyser. Total sulphur was determined by oxygen flask combustion followed by titration with Ba(C104)2.

2. 5. Statistics Statistical analysis was performed following Sokal and Rohlf ( 1981 ) using SAS for Mainframe (Statistical Analysis Systems Institute Inc., 1985) and Statview for Macintosh. Data were log-transformed before statistical analysis to obtain homogeneity of variances. Further details on statistical analysis are given in the text.

3. Results

3.1. DMSP concentrations in S. alterniflora along the Cooper River The full set of data on salinity and sulphide concentrations in riverbank sediments along the Cooper River will be published elsewhere. In this paper some representative data are shown that illustrate the gradients in salinity and sulphide concentrations in porewater in riverbank sediments (Fig. 3). Over the period April to October 1992 the average standard deviation (SD) for all sulphide data was 42% of the mean (n = 143 ). For salinity (chloride concentrations) this was 7% (n = 104). Salinity was high at the mouth of the river, low at the upstream end. Sulphide concentrations showed great variability, but were generally higher between 5 and 20 km from the mouth of the river compared with the other sampiing sites. Note that between approximately 25 and 35 km upstream from the mouth still considerable salinity levels were detected, while sulphide concentrations were close to zero.

M.L. Otte, J. T. Morris/Aquatic Botany 48 (1994) 239-259

247 4OO

4000

Sulfide (p.M) Salinity (mM CI) 3000

I

,300 A

o

\\

.-¢

,200

2000 "0

tl)

1000

'

,0 0

10

20

30

Distance

40

50

100

o 60

(km)

Fig. 3. Salinity (mM C1- ) and sulphide concentrations (#M S 2- ) in porewater in the riverbanks of the Cooper River, as a function of distance from the mouth of the river (km). Data represent averages ( n = 8-10 months) of means per month (number of replications is 3 ) of data collected from February to October 1992. Bars indicate standard deviations.

DMSP concentrations in the shoots ofS. alterniflora ranged from 9 to 48 #mol g-~ fresh weight and in the roots from 0.3 to 7.9 #tool g-1 fresh weight (Table 2). Standard deviations for DMSP concentrations ranged between 5 and 133% of the mean (average 39%, n = 49 ) and between 6 and 67% (average 28%, n = 49 ) in roots and shoots, respectively. The shoot/root ratios for DMSP concentrations ranged from 3.6 to 67. DMSP concentrations from the shoots and roots of individual plants were weakly correlated (r2=0.191, n=49, P<0.01, log-transformed data), but the mean concentrations pooled for all sampling dates showed similar spatial patterns for shoots and roots along the fiver (Fig. 4). Dry weight/ fresh weight ratios in the shoots did not vary consistently with distance from the mouth of the fiver and were on average 0.25 (SD=0.04, n = 3 0 ) and 0.33 (SD = 0.08, n = 26) in April and September, respectively. There was no consistent pattern in DMSP concentrations in the roots or the shoots of the plants with distance from the mouth of the river, and no significant correlation (at P < 0.05 ) was shown with the gradients in salinity or sulphide concentrations in porewater (correlations for mean values for all sampling dates (n = 10): Sulphide-DMSP roots, r 2 = 0.074, P= 0.447; SUlphide-DMSP shoots, r 2 = 0.003, P= 0.8926; Salinity-DMSP roots, r2=0.396, P=0.069; Salinity-DMSP shoots, r2=0.053, P = 0 . 5 5 0 ) (also compare patterns for porewater sulphide concentrations and salinity (Fig. 3 ) with patterns for DMSP concentrations in roots and shoots of the plants (Fig. 4 ) ). When tested by one-way analysis of variance for every sampling date separately, concentrations in roots or shoots generally differed significantly between sites. In most cases, however, the concentrations in plants collected at

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248

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%. 0

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M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994) 239-259 A

249

100

0

E a.

10

fo

0

i

i

i

i

10

20

30

40

50

60

Distance from mouth of river (km)

Fig. 4. Average of means (pooled for sampling dates) of DMSP concentrations in shoots and roots of

Spartina alterniflora along the Cooper River, as a function of distance from the mouth of the river (km). Table 3 Experiment 1: effects of sulphate on DMSP in S. alterniflora Treatment

Control Sodium chloride Sodium sulphate

t= 0

t= 8

t = 68

Shoot

Shoot

Main shoot

Young shoots

Roots

24 (8) 28 (6) 25 ( l l )

32 (19) 25 (7) 26 (10)

37 (7) 29 (8) 37 (7)

36 (22) 33 (10) 36 (13)

1.5 (0.7) 2.3 (1.3) 1.4 (0.9)

Concentrations of dirnethylsulphoniopropionate in plant parts ofS. alterniflora (/anol g- ~fresh weight) treated with tap water (Control), 90 mM NaC! (sodium chloride) or 45 mM Na2SO4 (sodium sulphate) at the start of the experiment (t = 0), after 8 days (t = 8) and after 68 days at the end of the experiment (t = 68 ). Mean values and standard deviations between parentheses.

upstream Site 10 were not significantly different from the downstream Sites 1 or 2 (Tukey's studentised range test). When tested by two-way analysis of variance, concentrations in both shoots and roots differed significantly between sites ( P < 0.001 ) and between sampling dates ( P < 0.01 ), and there was a significant interaction between the factors site and sampling date (P<0.001), indicating again that the effect of sites was not consistent at all sampling dates.

3.2. Greenhouse experiments 3.2.1. Experiment 1. Effects of sulphate Growth of the plants (shoot length and weight, data not shown) was not significantly affected by the treatments. DMSP concentrations in the roots were lower than in the main and young shoots (Table 3). The DMSP concentrations in the shoots did not vary with time, or with treatment (two-way analysis of variance). DMSP concentrations in young shoots and roots did not differ significantly between treatments either (one-way analysis of variance).

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Table 4 Experiment 2: effects of ammonium nitrate on DMSP in S. alterniflora N-treatment (mM)

n

g fw

g dw

Roots

Shoots

Roots

Shoots

2.0 2.5 3.7 5.0

-

0.55 (0.20) 0.68 (0.22) 1.00 (0.39) 1.34 (0.64)

1.08 (0.37) a 1.13 (0.27) a 1.22 (0.57) a 1.89 (1.40) a

1.10 (0.50) a 0.87 (0.21) a 2.16 (1.54) a 2.49 (1.87) a

First harvest (1 month after start of experiment) 0 0.5 2 5

5 5 5 5

2.3 1.5 2.0 2.2

(I.0) a (1.1) a (0.7) ~ (1.7) ~

(0.7)" (0.8) a (1.5) ab (2.4) b

Second harvest (2 months after start of experiment) 0 0.5 2 5

5 3 4 5

4.6 (1.7) 5.0 (1.3) 5.4 (2.7) 10 (8)

~ ~ ~ ~

Results of two-way analys& of variance Source N-treatment Harvest Interaction

n.s. *** n.s.

** ** n.s.

Mean biomass of roots and shoots ofS. alterniflora plants grown on 0, 0.5, 2 or 5 mM ammonium nitrate, standard deviations between parentheses, n, number of replications; fw, fresh weight; dw, dry weight; -, not calculated; ~, unreliable data (see text). Statistical analysis: one-way analysis of variance and Tukey's test for comparisons of means were carried out with data based on fresh weights for the first harvest and with data based on dry weights for the second harvest (see text); values with different letters (superscript) within one column per harvest are significantly different at P< 0.05. Results of two-way analysis of variance: *P< 0.05; **P<0.01; ***P< 0.001; n.s., not significant at P=0.05.

3.2.2. E x p e r i m e n t 2. Effects o f a m m o n i u m nitrate Fresh weight o f the plants (Table 4 ) was affected b y the treatments. D r y w e i g h t / fresh weight ratios o f t h e s h o o t s were n o t affected b y the t r e a t m e n t s a n d were 0.27 ( _+0.07, n = 12 ). R o o t weights at the first h a r v e s t were n o t significantly different. It is preferable to d e t e r m i n e D M S P c o n c e n t r a t i o n s b a s e d o n fresh weight, b e c a u s e the p r o c e d u r e u s e d for D M S P analysis uses fresh p l a n t m a t e r i a l a n d c o n c e n t r a t i o n s b a s e d o n d r y weights t h e r e f o r e h a v e to be c a l c u l a t e d indirectly using d r y w e i g h t / f r e s h w e i g h t ratios. H o w e v e r , b e c a u s e o f e x t r e m e h e a t the d a y before the p l a n t s were h a r v e s t e d for the s e c o n d time, s o m e plants, p a r t i c u l a r l y the larger ones, h a d d r i e d out. F r e s h weights o f the plants, the s h o o t s in particular, were t h e r e f o r e n o l o n g e r reliable a n d c o m p a r a b l e . D r y w e i g h t / f r e s h weight ratios f o r the s h o o t s v a r i e d b e t w e e n 0.27 a n d 0.77. T h e s e ratios d i d n o t v a r y in the r o o t s a n d were 0.22 ( _+0.02, n = 17). T h e r e f o r e , tests for significance o f differences b e t w e e n t r e a t m e n t s w i t h i n - h a r v e s t s w e r e c a r r i e d o u t with d a t a b a s e d o n fresh weights for the first h a r v e s t a n d o n d r y weights f o r the s e c o n d harvest. F o r c o m p a r i s o n b e t w e e n h a r v e s t s ( t w o - w a y analysis o f v a r i a n c e ) , d a t a b a s e d o n fresh

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251

Table 5

Experiment 2: effects of ammonium nitrate on DMSP in S. alterniflora Total amount of DMSP (pmol)

N-treatment n Concentration DMSP (mM) /tmol g - ~ fw

/tmol g - t dw

Roots

Roots

Shoots

Roots

Shoots

Shoots

First harvest (1 month after start of experiment) 0 0.5 2 5

5 5 5 5

2.2 3.6 1.1 1.3

(2.4)" (2.2)" (0.2)" (1.0)"

67 (20)" 43 (12) a 18 (5) b 24(4) b

-

249 158 67 88

(73) (43) (19) (17)

169 82 75 62

(76)" (8) ~ (34) ~ (27) b

4.2 4.7 2.1 2.5

(3.6)" (2.8)" (1.1)" (2.2) a

126 102 63 113

(24) a (23) ~ (14) b (49) ab

176 70 160 192

(89) a (10)" (127)" (196)"

Secondharvest ~ monthsafterstartofexpe~men 0 0 0.5 2 5

5 3 4 5

3.7 1.7 1.0 0.9

(2.2) (1.6) (1.1) (0.3)

-

17 13 5 4

(10)" (4) ~ (5) ~ (1) b

21 15 5 9

(16)" (6)" (3)" (8)"

Results of two-way analysis of variance Source

N-treatment ** Harvest

Interaction

n.s. n.s.

*** ** n.s.

n.s. *** n.s.

n.s. n.s. n.s.

Mean concentrations and total amounts (concentration × biomass) of DMSP in roots and shoots of S. alterniflora plants grown on 0, 0.5, 2 or 5 mM ammonium nitrate, standard deviations between

parentheses, n, number of replications; fw, fresh weight; dw, dry weight; -, not calculated. Statistical analysis: one-way analysis of variance and Tukey's test for comparisons of means were carried out with data based on fresh weights for the first harvest and with data based on dry weights for the second harvest (see text); values with different letters (superscript) within one column per harvest are significantly different at P<0.05. Results of two-way analysis of variance: *P<0.05; **P<0.01; ***P< 0.001; n.s., not significant at P = 0.05.

weights were used for roots, whereas data based on dry weights were used for shoots. For the latter tests, biomass and concentration data were transformed using dry weight/fresh weight ratios. Differences between treatments for biomass data from the second harvest were not significant. Root and shoot biomass increased between harvests, and shoot biomass varied significantly between treatments. DMSP concentrations in the shoots at the first harvest were affected by the treatments, whereas concentrations in roots were not (Table 5). Concentrations in shoots decreased with increasing ammonium nitrate amendments. Total amounts of DMSP in the shoots (i.e. concentration × biomass) were lower in the 2 mM treatment compared with the 0 mM control. From the second harvest, concentrations in both roots and shoots decreased with increasing ammonium nitrate amendments. Total amounts of DMSP in roots and shoots were not affected by the treatments. As was shown previously (Table 2), concentrations of DMSP in roots are lower than in shoots (Table 5). DMSP concentrations in shoots were significantly different between

252

M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994)239-259

Table 6 Experiment 3: effects of ammonium nitrate and sulphide on DMSP in S. alterniflora Treatment

0NOS 0N0.5S 0N1S 0.5NOS 0.5N0.5S 0.5N1S 2NOS 2N0.5S 2NIS

Roots

Shoots

fw (g)

DMSP conc. (#mol g-~)

D M S Ptotal (#mol)

fw (g)

DMSP conc. (#mol g-~)

DMSPtotal (#mol)

2.5 (1.4) 4.3 (2.5) 3.1 (1.4) 2.8 (1.8) 3.8 (1.2) 3.4 (1.6) 3.9 (2.5) 3.6 (2.5) 5.0 (3.7)

3.3 (1.8) 2.7 (1.4) 3.5 (0.4) 2.4 (1.1) 2.0 (0.9) 2.6 (1.6) 2.7 (1.2) 2.4 (1.7) 2.7 (2.2)

8.0 (5.9) 11.1 (5.7) 11.2 (5.6) 6.5 (4.1) 7.7 (4.6) 8.2 (5.1) 9.8 (5.7) 8.7 (8.1) 16.2 (17.0)

1.7 (1.0) 2.4 (0.4) 2.3 (0.4) 3.6 (1.7) 4.9 (1.5) 5.7 (2.7) 4.6 (2.5) 6.0 (2.8) 6.1 (2.9)

41 (11) 34 (8) 38 (15) 19 (10) 16 (6) 26 (8) 17 (7) 14 (8) 15 (6)

68 (46) 82 (20) 86 (42) 61 (33) 82 (54) 145 (76) 69 (34) 77 (47) 95 (50)

n.s. n.s. n.s.

n.s. n.s. n.s.

n.s. n.s. n.s.

*** n.s. n.s.

*** n.s. n.s.

n.s. n.s. n.s.

Sign~cance N S N*S

Means and standard deviations (between parentheses) of fresh weight (fw), DMSP concentrations (based on fresh weight, DMSP conc.) and total DMSP content (DMSP total) of roots and shoots. Number of replications is 5. Significanceof differences between treatments as tested by two-wayanalysis of variance is indicated below each column. N, source of variation is ammonium nitrate treatment; S, source of variation is sodium sulphide treatment; N'S, interaction between N and S; n.s., not significant at P= 0.05, ***P<0.001. harvests. The non-significant interaction indicates that the differences between harvests were consistent for all treatments, i.e. concentrations decreased between the first a n d second harvest. In contrast to the total a m o u n t o f D M S P in the shoots, the a m o u n t s in the roots increased, reflecting the increase in biomass o f the roots between harvests.

3.2.3. Experiment 3. Effects o f a m m o n i u m nitrate and sulphide Fresh weight o f the shoots o f the plants (Table 6) was higher in the a m m o n i u m nitrate treated plants c o m p a r e d with the control plants, but was not affected by sulphide treatments. Fresh weight o f the roots o f the plants was not affected by any o f the treatments. D M S P concentrations a n d total D M S P content (biomass × D M S P c o n c e n t r a t i o n ) in the roots were not affected by the treatments,

M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994) 239-259

253

60' II

50' A m []

0

E

[]

40'

u

mm

3

m

0

m

30'

~m m

m

[]

L

[]

u

20'

mmu m

t3

m I

"

m

m

m

10'

m

B

nl

E B

0

!

|

2

4

m

m

|

6

8

10

FW shoot (g)

Fig. 5. Correlation between DMSP concentrations (#mol g- mfresh weight) in shoots of Spartina alterniflora as a function of shoot biomass. (Data of all separate plants from Experiment 3: effects of ammonium nitrate and sulphide on DMSP in S. alterniflora. )

Table 7 Experiment 3: effects of ammonium nitrate and sulphide on DMSP in shoots ofS. alternifiora Treatment

n

C (g 100g - l )

N (g 100g - l )

S (g 100g -~ )

DMSP (/~molg-1 )

DMSP/S (mol mol -~ )

0NOS

3

0N1S

5

2NOS

5

2N1S

5

41.7 (0.8) 43.5 (1.2) 43.2 (0.8) 42.6 (0.5)

0.87 (0.10) 1.01 (0.19) 2.49 (0.42) 2.64 (0.26)

0.41 (0.10) 0.45 (0.06) 0.33 (0.04) 0.46 (0.13)

103 (34) 105 (40) 49 (20) 49 (19)

0.86 (0.43) 0.78 (0.36) 0.47 (0.17) 0.36 (0.19)

Significance (two-way analysis of variance) N

n.s.

***

n.s.

**

*

S

n.s.

n.s.

*

N*S

*

n.s.

n.s.

n.s. n.s.

n.s. n.s.

Mean and standard deviation (between parentheses) of concentrations (based on dry weight) of C, N, S and DMSP, and DMSP/S ratio, n, number of replications. Significance of differences between treatments as tested by two-way analysis of variance is indicated below each column. N, source of variation is ammonium nitrate treatment; S, source of variation is sodium sulphide treatment; N'S, interaction between N and S; n.s., not significant at P = 0.05; *P< 0.05; **P< 0.01; ***P<0.001.

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M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994) 239-259

but in the shoots DMSP concentrations decreased with increasing ammonium nitrate concentration in the growth medium (Table 6). This was also reflected in the inverse and what appears to be a non-linear relation between DMSP concentrations and biomass in the shoots (Fig. 5 ). Total DMSP content of the shoots was not affected by the treatments. Total carbon, nitrogen and sulphur content of the shoots of the plants was determined in the 0NOS, 0N1 S, 2NOS and 2N1S treatments only (Table 7). Total nitrogen content was higher in the 2N treatments compared with the ON treatments. Total sulphur content was higher in the 1S treatments compared with the 0S treatments. Carbon content of the plants was not consistently affected by the treatments. The significant interaction for carbon content may reflect the changes in total nitrogen and sulphur content, which will have affected the relative contribution of carbon to total biomass. Two-way analysis of variance of this restricted number of data gave the same outcome as for the whole set (Table 5 ): DMSP concentrations in the 2N treatments were lower than in the ON treatments (note that in Table 6 DMSP concentrations are based on fresh weight, whereas in Table 7 they are based on dry weights). The amount of sulphur in the shoots allocated to DMSP (calculated as the DMSP/S ratio in mol DMSP per mol S) ranged between 36 and 86% and was significantly lower in the 2N treated plants compared with the ON treated plants. Treatment with sulphide did not affect the DMSP/S ratio in the shoots of the plants. 4. Discussion

IfDMSP would act as a compatible solute analogous to glycinebetaine, concentrations in the plants would be expected to increase with increasing salinity of the porewater (Cavalieri and Huang, 1981; Cavalieri, 1983). The correlation of DMSP concentrations with salinity in the study of Dacey et al. (1987) seemed to support that hypothesis. However, Van Diggelen et al. (1986) showed that DMSP concentrations in S. anglica did not respond to salinity when the plants were grown in nutrient solution. The same authors found that DMSP concentrations in S. anglica increased with increasing concentrations of sulphide in the growth medium and it was suggested that DMSP is produced to store excess sulphur, reducing toxic levels of sulphide in the plants. If this hypothesis is true, then DMSP concentrations in the plants should increase with increasing sulphide concentrations in the growth medium, since the turnover rate of DMSP to DMS and acrylate is low, according to the following calculation: given a concentration of DMSP in the aboveground biomass of the plants of about 20/zmol g- 1fresh weight (about 8 gmol g-1 dry weight) (this study), a maximum aboveground biomass for S. alterniflora in the south-eastern USA of about 700 g dry weight m -2 (Morris and Haskin, 1990) and a sulphur emission rate (as DMS) from Spartina stands of about 2.87 g m -2 per year (Steudler and Peterson, 1984), the turnover rate for DMSP to DMS can be estimated.to be 1 per 0.6 year. This estimate was also supported by Kiene and Service (1991 ). Sulphides in porewater and D M S P concentrations in plants along the Cooper

M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994) 239-259

255

River showed large variation in this study. Although sulphide concentrations and salinity in porewater differed significantly between the extremes of Sites 2 and 10 (data for March-September 1992: sulphides at Site 2 1540+ t890, at Site 10 6.2 +_3.3, paffed t-test on log-transformed data, P<0.01; salinity at Site 2 285 +_40, at Site 10 not detectable ( < 1 mM C1), no test possible for lack of variance in Site 10 data), DMSP concentrations in plants from those sites were not different, except in roots in September 1992 (see Table 2). Since DMSP concentrations in S. alterniflora along the Cooper River did not correlate overall with the salinity or the sulphide gradient (compare also Figs. 3 and 4), neither of the above-mentioned hypotheses is supported by the field observations, i.e. neither salinity nor sulphide concentrations appear to determine DMSP concentrations in the plants. The greenhouse experiment with sulphate (Experiment 1 ) described here esseutially repeated an experiment performed by Van Diggelen et al. (1986) and confirmed their findings: DMSP concentrations in S. alterniflora are not affected by sulphate concentrations. Total sulphur was not determined in our experiment, but Van Diggelen et al. (1986) showed that total sulphur in Spartina plants increased with increasing concentrations of sulphate in the growth medium. Spartina appears to take up both sulphate and sulphide (Carlson and Forrest, 1982). Apparently, a low supply of sulphur to the plants, as in the control treatments in the greenhouse experiments described here, is sufficient to allow them to synthesise DMSP at levels comparable to those encountered in the field. Dacey et al. (1987) and Gr6ne and Kirst ( 1992 ) suggested that nitrogen supply could affect DMSP concentrations in the plants. Pilot samples from the field station of the University of South Carolina at the Belle W. Baruch Institute (data not shown) supported observations of Dacey et al. (1987), that nitrogen-fertilised plants contained lower DMSP concentrations than unfertilised plants. Van Diggelen et al. (1986) failed to find an effect of nitrate supply on DMSP concentrations in Spartina, but this could be explained because both growth solutions with and without nitrate in their experiment contained ammonium, which may have been a sufficient supply of nitrogen in both treatments. The results from greenhouse Experiment 2 in this study indicate that nitrogen supply to the plants affects DMSP concentrations by affecting growth. Although total DMSP in the shoots was significantly lower in the plants treated with 2 mM ammonium nitrate at the first harvest, it did not vary consistently with increasing nitrogen supply to the plants, and at the second harvest total amounts of DMSP in the shoots did not vary. From this experiment it appears that in the shoots the effect of nitrogen supply lies in the stimulation of growth, increasing biomass, but not DMSP production. As a result DMSP concentrations in the shoots are diluted in the nitrogen treated plants compared with the controls. The DMSP concentrations in the roots decreased significantly with increasing nitrogen amendments at the second harvest. This could be explained by the same phenomenon as in the shoots, namely dilution by biomass production. However, total DMSP content of the roots increased between the first and second harvest, paralleling the increase in biomass. This may indicate that concentrations of DMSP in the roots are regulated within a relatively narrower range than in the shoots. The amount of variation in DMSP

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M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994) 239-259

concentrations in the plants created through variation in nitrogen supply in this experiment is comparable to the variation encountered in the field. It may therefore be that most variation in DMSP concentrations in Spartina plants under field conditions can be explained by differences in growth, which is particularly affected by nitrogen supply (Morris, 1982 ). The results from greenhouse Experiment 3, combining the effects of nitrogen supply and sulphide in the growth medium, support the findings from Experiment 2. Increased nitrogen supply stimulated growth of the shoots. This resulted in lower DMSP concentrations, because production of DMSP (as indicated by the total DMSP content) was not stimulated by nitrogen supply. Although total sulphur content of the plants increased with increasing sulphide in the growth medium, DMSP concentrations were not affected by sulphide treatments. The fraction of total sulphur in the shoots allocated to DMSP was up to 86% of total sulphur content, which is higher than the previously reported 50% (Van Diggelen et al., 1987 ), and is an indication of the unique sulphur metabolism in these plants (Ernst, 1990). The DMSP/S ratio was significantly lower in the high nitrogen (2N) treatments than in the control (ON) treatments, but was not affected by the sulphide treatment. Therefore, apart from the dilution effect on DMSP concentrations due to enhanced growth of the plants upon increased nitrogen supply, DMSP concentrations also appear to be lowered by a decrease in the amount of sulphur being allocated to DMSP. This may be the result of less methionine being converted to DMSP. Gr6ne and Kirst ( 1992 ) suggested that methionine is one of the precursors of DMSP and that "methionine availability, the surplus of methionine production over all non-DMSP-related consumption processes, controls the size of the DMSP pool". It may be that relatively more methionine is consumed by increased synthesis of proteins with increasing supply of nitrogen to the plants. Our results indicate that the hypothesis that DMSP in Spartina is the product of a sulphide-detoxifying mechanism (Havill et al., 1985; Van Diggclen et al., 1986; Ernst, 1990) is incorrect. The increase of DMSP concentrations with increasing sulphide concentrations in the study of Van Diggelen et al. (1986) is more likely the result of dilution/concentration effects, similar to the response to nitrogen supply observed in our experiments. In contrast to our findings, growth of the plants in the study of Van Diggelen et al. ( 1986 ) decreased with increasing sulphide treatment, which in turn may have led to higher DMSP concentrations without increasing the production rate of DMSP. The same type of interaction may explain the observations of Dacey ct al. (1987) that DMSP concentrations in S. alterniflora plants correlated with salinity of the soil. Uptake of nitrogen by S. alterniflora decreases with increasing salinity (Morris, 1984), which may have affected growth and DMSP concentrations as described above. That no correlations of DMSP concentrations in plants with either salinity or sulphide were identified along the Cooper River in this study suggests that nitrogen supply to the plants varied strongly along that fiver. This needs further investigation. Where the functions of DMSP in Spartina are concerned, several alternative hypotheses can be formulated. It is remarkable that some species of the genus

M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994) 239-259

257

Table 8 Concentrations of DMSP in leaves of Spartina species (/zmol g - i fresh weight)

S. alterniflora Loisel. (Cooper River; this study ) S. alterniflora Loisel. (two samples) (San Francisco bay, June 1992; this study) S. foliosa Trin. (two samples) (San Francisco bay, June 1992; this study) S. anglica Hubbard (The Netherlands; Van Diggelen et al., 1986) S. cynosuroides (L.) Roth (Cooper River; this study) S. patens (Aiton) Muhl. (North Inlet; this study)

9-48 6.8/7.2 7.6/8.2 7.1-21.7 ND ND

ND, not detectable ( < 0.05/zmol g- 1fresh weight).

contain DMSP, whereas others that may be found growing in the same habitat (e.g.S. alterniflora versus Spartina cynosuroides) do not (Table 8). The fact that species that do contain DMSP generally grow in lower lying areas of the saltmarsh compared with the species that do not contain DMSP, with generally higher salinity and sulphide concentrations in the soil, suggests that the compound may still be involved in salt or sulphide tolerance. Possibly, the mere presence of DMSP in the plants decreases the osmotic potential to a constantly lower 'baseline' compared with other species, giving it an advantage under conditions of fluctuating salinities, because the plants would have to invest less energy in syntbesising and degrading compatible solutes, such as glycinebetaineand proline. However, DMSP could be a multifunctional compound and could also function as: ( 1) a methylating compound as suggested by Challenger et al. (1957) and Weber ct al. ( 1991 ). (2) A herbivore deterrent: some field observations (unpublished data by M.L. Otte, B. Haskin and J.T. Morris, 1992) from North Inlet (South Carolina) suggest that rice rats prefer as food plant parts of S. alterniflora that are low in DMSP. Nakajima (1989) suggested that laboratory rats preferred water containing low concentrations of DMSP (0.1 or 0.5 mM) over water with high concentrations (5 mM). A preference for food with low concentrations of DMSP could be due to its flavour or taste, or to its potentially toxic degradation product acrylic acid (Sieburth, 1960). (3) An intermediate compound in the synthesis of other compounds such as acrylic acid. This would mean that dimethyl sulphide could just be a by-product.

Acknowledgements The authors wish to thank Marie-Jos6 Ettema, Shawn Coffman, Shepard McAninch, Dionne J. Allison and Amy Mozingo for their assistance. The Department of Ecology and Ecotoxicology of the Vrije Universiteit, Amsterdam, The Nctbeflands, and Dr. David M. Dickson are acknowledged for supplying and

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M.L. Otte, J.T. Morris/Aquatic Botany 48 (1994) 239-259

synthesising DMSP, respectively. Dr. D.R. Strong is acknowledged for providing plants from the San Francisco bay area. This project was supported by the South Carolina Seagrant Consortium.

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Larher, F., Hamelin, J. and Steward, G.R., 1977. L'acide dim6thylsulfonium-3-propan6ique de Spartina anglica. Phytochemistry, 16:2019-2020. Morris, J.T., 1982. A model of growth responses by Spartina alterniflora to nitrogen limitation. J. Ecol., 70: 25-42. Morris, J.T., 1984. Effects of oxygen and salinity on ammonium uptake by Spartina alterniflora Loisel. and Spartina patens (Aiton) Muhl. J. Exp. Mar. Biol. Ecol., 78: 87-98. Morris, J.T. and Haskin, B., 1990. A 5-yr record of aerial primary production and stand characteristics of Spartina alterniflora. Ecology, 71: 2209-2217. Morris, J.T., Huang, Y.H., Bradley, P.M. and McAninch, S.W., 1991. Distribution of major ions along a salt gradient in the Cooper River, South Carolina, in relation to community structure. In: Abstracts of the 1 lth International Estuarine Research Conference, San Francisco. Estuarine Research Federation, Crownsville, MD, p. 96. Nakajima, K., 1989. Effects of high concentrations of dimethylthetin, dimethyl-fl-propiothetin and vitamin U on young rats. Mere. Koshien Univ., 17: 1-8. Przyjazny, A., Janicki, W., Chrzanowski, W. and Staszewski, R., 1983. Headspace gas chromatographic determination of distribution coefficients of selected organosulphur compounds and their dependence on some parameters. J. Chromatogr., 280: 249-260. Reed, R.H., 1983. Measurements and osmotic significance of dimethylsulphoniopropionate in marine macro-aloe. Mar. Biol. Lett., 4: 173-181. Rennenberg, H., 1984. The fate of excess sulphur in higher plants. Annu. Rev. Plant Physiol., 35:121153. Sieburth, J.M., 1960. Acrylic acid, an 'antibiotic' principle in Phaeocystis blooms in Antarctic waters. Science, 132: 676-677. Smart, R.M. and Barko, J.W., 1980. Nitrogen nutrition and salinity tolerance ofDistichlis spicata and Spartina alterniflora. Ecology, 61: 630-638. Sokal, R.R. and Rohlf, F j . , 1981. Biometry, 2nd edn. W.H. Freeman, San Francisco. Statistical Analysis Systems Institute Inc., 1985. SAS User's Guide: Statistics. Statistical Analysis Systems Institute Inc., Cary, NC. Steudler, P.A. and Peterson, B.J., 1984. Contribution of gaseous sulphur from saltmarshes to the global sulphur cycle. Nature, 311: 455-457. Stumm, W. and Morgan, J.J., 1981. Aquatic Chemistry, 2nd edn. Wiley, New York. Van Diggelen, J., Rozema, J., Dickson, D.M. and Broekman, R., 1986. ~3-Dimethyisulphoniopropionate, proline and quaternary ammonium compounds in Spartina anglica in relation to sodium chloride, nitrogen and sulphur. New Phytol., 103: 573-586. Van Diggelen, J., Rozema, J. and Broekman, R., 1987. Growth and mineral relations of sahmarsh species on nutrient solutions containing various sodium sulphide concentrations. In: A.H.L. Huiskes, C.W.P.M. Biota and J. Rozema (Editors), Vegetation Between Land and Sea. W. Junk, Dordrecht, pp. 260-268. Weber, J.H., Billings, M.R. and Kalke, A.M., 1991. Seasonal methyltin and (3-dimethylsulphonio) propionate concentrations in leaf tissue of Spartina alterniflora of the Great Bay estuary (NH). Estuarine Coast. Shelf. Sci., 33: 549-557. Wyn Jones, R.G. and Gorham, J., 1983. Osmoregnlation. In: O. Lange, P.S. Nobel, C.B. Osmond and H. Ziegler (Editors), Encyclopedia of Plant Physiology N.S., Vol. 12c: Physiological Plant Ecology III. Springer, Berlin, pp. 35-58.