2002 Progress Report: DMSP and its Role as an Antioxidant in the Salt Marsh Macrophyte Spartina alterniflora

EPA Grant Number: R827072C029
Subproject: this is subproject number 029 , established and managed by the Center Director under grant R827072
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Alabama Center For Estuarine Studies (ACES)
Center Director: Shipp, Robert L.
Title: DMSP and its Role as an Antioxidant in the Salt Marsh Macrophyte Spartina alterniflora
Investigators: Kiene, Ronald P. , Husband, Joseph D.
Institution: University of South Alabama
EPA Project Officer: Packard, Benjamin H
Project Period: January 1, 2001 through December 31, 2003
Project Period Covered by this Report: January 1, 2001 through December 31, 2002
RFA: Alabama Center For Estuarine Studies (ACES) (1999) RFA Text |  Recipients Lists
Research Category: Targeted Research

Objective:

The objective of this research project is to test the hypothesis that the osmolyte dimethylsulfoniopropionate (DMSP) and its degradation products, dimethylsulfide (DMS) and dimethylsulfoxide (DMSO), function as antioxidants in the smooth cordgrass Spartina alterniflora. Field and nursery-grown plants will be used to examine natural and experimentally imposed stresses. We focus on the responses of S. alterniflora to natural environmental stress factors, including elevated salinity, high solar radiation, nitrogen limitation, and iron limitation. Additionally, we selectively will impose oxidative stress by administration of the herbicides paraquat and dichlorophenyldimethyl urea (DCMU). Plant responses to be monitored will include DMSP dynamics (along with its degradation products, DMS, DMSO, and methanesulfonic acid), reactive oxygen scavenging enzymes (peroxidase and superoxide dismutase), and the quantum yield of photosystem II. The data gathered will be used to assess whether DMSP production confers advantages to S. alterniflora living in salt marsh habitats.

Progress Summary:

The production of DMSP by S. alterniflora is well known, but no previous studies had looked for potential oxidation products of DMSP in the tissues of this important salt marsh macrophyte. Our initial efforts focused on adapting assays to measure DMSO in plant tissues. The method proved to work well, and for the first time we have demonstrated that DMSO is present in the leaves of field-collected S. alterniflora. Not only is DMSO present, but its intracellular concentrations were relatively high, in the low mM range, and equivalent to 5-20 percent of the DMSP concentrations. At these concentrations, DMSO could indeed function as a radical scavenger and an antioxidant. This is an important and very exciting finding. In plants from the natural environment, the ratio of DMSO:DMSP was higher in segments of necrotic tissue (0.16) than healthy tissue (0.06) from the same leaf. In addition, we found higher concentrations of DMSO in the spring and summer, when insolation and photosynthetic activity are at a maximum. These periods may correspond to the highest oxidative stress. Furthermore, we found that intracellular DMSP concentrations decreased along the length of leaves from the ligule to the tip. DMSO, in contrast, showed the opposite trend, being highest near the leaf tip. As S. alterniflora leaves tend to senesce from the tip, this further argues that DMSP oxidation may be promoted in stressed tissues.

Direct evidence that DMSP may be involved in oxidative stress response was obtained from experiments in which selected leaves of nursery-grown plants were treated with the herbicide paraquat (methylviologen). Paraquat is known to promote excessive production of reactive oxygen species (ROS) in plants exposed to light. Application of paraquat to leaves held in sunlight caused visible leaf chlorosis within 24 hours. Intracellular DMSP and DMSO concentrations were significantly higher in leaves treated with the paraquat (34.3 µmol/ml and 3.25 µmol/ml leaf water, respectively) compared to untreated control leaves (14.4 µmol/ml and 2.54 µmol/ml leaf water) from the same plant. These findings suggest that S. alterniflora may be able to rapidly increase synthesis of DMSP in response to oxidative stress. Our findings further suggest that DMSP is oxidized during times of stress, such as that caused by natural tissue necrosis or by administration of an oxidative stressor such as paraquat. It remains to be demonstrated, however, whether translocation of DMSP (or DMSO) into stressed areas is responsible for the localized increases that we observed.

We have carried out some tests showing that DMS release from chlorotic or necrotic (i.e., stressed) tissues of S. alterniflora leaves is significantly higher than from healthy green leaves. DMS is a cleavage product of DMSP, and can be produced via the enzyme DMSP lyase. These findings suggest that the stressed leaves are consuming more DMSP to produce DMS. Some of this DMS is volatilized from the leaves, but some is further oxidized to yield DMSO, which explains why higher DMSO was found in chlorotic (yellow) leaf sections and near the leaf tips. At present, we do not know whether the S. alterniflora plants are the source of the DMSP lyase enzyme that may be cleaving DMSP into DMS, or whether microbes (bacteria or fungi) are involved. This will be a subject for further study in the year to come. Preliminary tests for DMSP lyase activity in healthy green leaf sections of S. alterniflora failed to show significant activity. We did find, however, that leaf extracts prepared for these lyase assays rapidly consumed DMS in some cases. Thus, we demonstrated that S. alterniflora tissues have the capability to consume DMS (presumably by oxidation). More tests on potential lyase activity and DMS consumption are planned.

S. alterniflora is native to the East and Gulf Coasts of North America, but it recently has invaded various estuaries on the West Coast. While valued on the East Coast as a stabilizing force on shorelines and as a major source of organic matter and habitat, on the West Coast S. alterniflora is considered an unwelcome invader. There is growing evidence that S. alterniflora is hybridizing with the native West Coast species S. foliosa, providing a further threat to the ecological balance in West Coast marshes. On the European side of the Atlantic, S. alterniflora has crossed with the native species S. maritima, producing the successful hybrid S. anglica. Given the ability of S. alterniflora and its congenerics to dominate some ecosystems, and its apparent ability to invade new areas and cross with closely related species, we need to understand what makes this species so successful in colonizing euryhaline coastal margins. What specific adaptation(s) allows S. alterniflora to dominate under the harsh estuarine conditions where stressors include variable salinity, aerial desiccation, high light exposure, soil/sediment anoxia, sulfide, and anthropogenic pollutants? We believe the high concentration of DMSP in S. alterniflora may help explain this dominance.

Future Activities:

We will continue to measure seasonal patterns of DMSP and its oxidation products in natural populations of S. alterniflora throughout the 2002-2003 project period. We will carry out tests using antibiotics and antifungal agents to evaluate whether leaf microbes might be involved in DMSP transformations. Further work on assaying the potential DMSP lyase activity in S. alterniflora tissues will be conducted. To aid in this work, we have initiated a side project involving isolation and purification of DMSP lyase enzyme(s) from the green macroalga Enteromorpha intestinalis. This macroalga has very high DMSP lyase activity, and provides a good opportunity to procure a purified, functional protein. If we can acquire a pure DMSP lyase, we can obtain the N-terminal amino acid sequence and thereby infer the DNA sequence encoding this enzyme. We then would be able to use this information to generate DNA probes that would allow us to test S. alterniflora (and other organisms) for this important enzyme.

Methods are under development to measure methanesulfonic acid (MSA) in leaf tissue samples. MSA is a potential oxidation product of DMSP via DMS and DMSO, and it is a difficult compound to measure in a saline matrix. If we can measure MSA and observe its production in vivo, this would provide unambiguous confirmation that DMSP and its degradation products are scavenging ROS in S. alterniflora.

Other methods under development are assays for ROS scavenging enzymes, including peroxidase and superoxide dismutase. We will use these assays, together with pulsed amplitude modulated fluorometry, to assess the level of oxidative stress experienced by plants.

More experimental work is planned to examine the effects of direct oxidative stressors on DMSP production and dynamics in the S. alterniflora leaves. We will repeat experiments with paraquat and also test DCMU, another herbicide expected to promote oxidative stress in the plants.

Nursery-grown plants will be used for a series of experiments examining how common stresses, including elevated salinity, N-limitation, and iron limitation, affect oxidative stress and DMSP dynamics in the S. alterniflora plants.

Finally, we will investigate the hypothesis that the high concentrations of DMSP found in S. alterniflora confer increased resistance to oxidative stress for this species. To test whether DMSP provides S. alterniflora with greater resistance to oxidative stress (relative to the non-DMSP containing congener S. patens) time series measurements will be made of oxidative stress in paraquat-treated S. alterniflora and S. patens, in parallel with measurements of the concentration of DMSP and its oxidation products in S. alterniflora. A PAM-fluorometer will be used to assess in situ oxidative stress in the paraquat-treated plants.

Journal Articles:

No journal articles submitted with this report: View all 1 publications for this subproject

Supplemental Keywords:

osmolyte, dimethylsulfoniopropionate, DMSP, dimethylsulfide, DMS, dimethylsulfoxide, DMSO, smooth cordgrass, Spartina alterniflora, field-grown plants, nursery-grown plants, stress factors, salinity, solar radiation, nitrogen limitation, iron limitation, oxidative stress, herbicides, paraquat, dichlorophenyldimethy urea, DCMU, reactive oxygen scavenging enzymes, quantum yield, photosystem II, risk, assessment, indicators, environmental stress, antioxidant, Alabama, AL, ecosystem, ecosystem protection, ecology, ecological effects, ecological indicators, environmental exposure, geographic area, environmental chemistry, chemistry, chemical engineering, salt marsh habitat., RFA, Scientific Discipline, ECOSYSTEMS, Water, Ecosystem Protection/Environmental Exposure & Risk, estuarine research, Aquatic Ecosystems & Estuarine Research, Ecology, Ecosystem/Assessment/Indicators, Ecosystem Protection, Restoration, Aquatic Ecosystem, Aquatic Ecosystems, Ecological Monitoring, Terrestrial Ecosystems, Ecology and Ecosystems, Aquatic Ecosystem Restoration, Ecological Indicators, coastal ecosystem, eutrophication, water use, nursery habitats, estuaries, watersheds, nutrients, osmolyte dimethylsufoniopropionate, fisheries, aquatic plants, submerged aquatic vegetation, dimethylsulfoxide, ecosystem, environmental indicators, water quality, estuarine waters, seagrass

Relevant Websites:

http://www.southalabama.edu/aces/ Exit

Progress and Final Reports:

Original Abstract
  • 2001
  • Final

  • Main Center Abstract and Reports:

    R827072    Alabama Center For Estuarine Studies (ACES)

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