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DMSP AND ITS ROLE AS AN ANTIOXIDANT IN THE SALT MARSH MACROPHYTE SPARTINA ALTERNIFLORA
Impact/Purpose:
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.
Description:
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.