Stress Tolerance in Seagrass

1
Stress Tolerance in Seagrass Molly M. Mintz,
Kelly M. Major and Anne A. BoettcherDepartment
of Biological Sciences, University of South
Alabama, Mobile, AL 36688 USA
Abstract
A
Seagrasses constitute a group of aquatic
angiosperms with a worldwide distribution among
estuarine and shallow marine ecosystems. Thus,
these plants occupy nearshore environments that
are often affected by anthropogenic disturbance,
including exposure to high nutrient loads and
agricultural herbicides. Because seagrasses
represent a threatened and endangered habitat,
the potential impact of chemical exposure
coincident with other environmental stressors
(e.g., high salt, low light, high nutrients,
etc.) could be severe. This study examined how
well Thalassia testudinum (turtle grass Fig. 1A,
B), a seagrass native to the Gulf of Mexico,
tolerates high salinity and herbicide exposure
using Chlorophyll-a (Chl-a) fluorescence and heat
shock protein 70 (HSP 70) as indicators of plant
health. Plants were acclimated to 15 or 25 psu
and subsequently exposed to 0 or 1 µM
concentrations of dichlorophenyl dimethylurea
(DCMU). Measurements of fluorescence yield
(Fv/Fm) indicated that both high salinity and
herbicide exposure resulted in low photosynthetic
efficiency in T. testudinum Fv/Fm values
declined from ca. 0.750 to 0.320 following a
2-day exposure to high salt and DCMU. Low
fluorescence yields were attributed to 1) damage
to PSII as indicated by high values for initial
fluorescence (Fo) upon DCMU exposure (ca. 734)
and 2) reduction in pigment content under high
salt conditions. Values for Fm did not vary as a
function of salinity or herbicide. Preliminary
data suggest that high salinity, when combined
with herbicide exposure, acts synergistically to
negatively impact photosynthetic efficiency in
Thalassia. Future studies will include an
investigation into the role of HSP 70, a
time-course study of herbicide and salinity
effects, effects of different herbicide
concentrations and effects of other environmental
stressors on growth and productivity in seagrass
species.
HSP 70
B
Figure 4. Thalassia testudinum. HSP 70
expression in seagrass.
Preliminary Results
Measurements of fluorescence yield (Fv/Fm)
indicated that both high salinity and herbicide
exposure had significant negative effects on
photosynthetic efficiency in Thalassia testudinum
(p 0.037 and p lt0.0001, respectively Fig.
2A). Low values for Fv/Fm correspond to high
values for Fo upon DCMU exposure, regardless of
salinity (p lt0.0001 Fig. 2B). Values for Fm
did not vary as a function of salinity or
herbicide (Fig. 2C). Chlorophyll-a content
exhibited a relative decline with high salinity
(p 0.007) and herbicide exposure (p 0.041
Fig. 3A). However, Chl-b content was only
affected by salinity, exhibiting a reduction upon
acclimation to high salt (p 0.006 Fig. 3B).
Because Chl-a and Chl-b exhibited similar
responses to salinity, Chl-ab ratios did not
vary among treatments. As shown in Figure 4, T.
testudinum expressed the inducible form of heat
shock protein 70 (HSP 70).
C
A
A
Figure 2. Thalassia testudinum. Changes in (A)
fluorescence yield (Fv/Fm), (B) initial
fluorescence (Fo) and (C) maximum fluorescence
(Fm) as a function of salinity and herbicide
exposure. (Error bars denote SE n
5/treatment).
2
Preliminary Conclusions

• The overall health of T. testudinum was
  negatively
• affected upon exposure to either high salt or
  herbicide.
• Deterioration in fluorescence yield was
  attributed to
• i) damaging effect of DCMU on PSII
• ii) effect of high salinity on pigment content
• In contrast to previous studies on algae and
  other
• plants, exposure to one stressor does not seem to
• confer tolerance to a second stressor in T.
  testudinum.

B
B
A
A
B
Figure 1. Thalassia testudinum. (A) A view of
Thalassia plants in their native habitat
(http//faculty.washington.edu/gayaldo/images/thal
.jpg) (B) A line drawing depicting
morphological features of T. testudinum (http//ww
w.tbep.org/pics/turtlegrass.gif).
Materials and Methods
Planned Research
Plant Collection Thalassia testudinum plants
were collected in shallow water from Gulf Islands
National Seashore, Perdido Key, Florida, in late
June 2005, using a shovel to dig up cores. All
sediment and attached animals were removed and
plants were transferred to 5-gallon buckets
filled with seawater from the collection site.
Plants were transferred to tanks filled with
fresh seawater immediately upon return to the
lab. Chlorophyll Fluorescence Pigment
Content After two days exposure, the effects of
salinity (15 and 25 psu) and herbicide (1.0 µM
DCMU) were assessed by measuring Chlorophyll-a
(Chl-a) fluorescence using a Pulse Amplitude
Modulated (PAM) fluorometer (Dive PAM, Walz).
Fluorescence was measured on one randomly chosen
plant per tank (n 5 per treatment). A 1-cm
section (2 cm above the sheath) on the second
rank leaf of each plant was cleaned of epiphytes
and dark adapted for ten minutes prior to
determination of initial fluorescence (F0),
maximum fluorescence (Fm) and the fluorescence
yield (Fv/Fm). Immediately following
fluorescence yield measurements, the same 1-cm
portion of the second rank leaf was chopped with
a straight-edged razor and placed in 2.5 mL of
DMF (N, N-dimethlyforamide) for later pigment
analysis. Following a 7-day extraction period,
pigment concentrations (Chl-a -b) were
calculated on a per area basis using traditional
spectrophotometric methods and equations from
Porra et al. (1982). Heat Shock Protein
Analysis After four days of exposure to salinity
and DCMU, the remaining plants in each tank were
pooled for HSP 70 analysis. Proteins were
analyzed using 8 SDS-PAGE gel electrophoresis.
Western blotting was performed and proteins
identified with HSP 70-specific antibodies.
Following imaging, the blot was analyzed with a
PC and ADOBE Photoshop 3.0 software.

• Companion studies with Vallisneria americana
• Investigation into the role of HSP 70 in
  seagrass
• A time course study of herbicide and salinity
  effects

Literature Cited
Porra , R.J., Thompson, W.A. Kriedmann, P.E.
(1989) Biochimica Biophysica Acta. 975
384-394.
Figure 3. Thalassia testudinum. Changes in (A)
Chl-a and (B) Chl-b concentrations as a function
of salinity and herbicide exposure. (Error
denote SE n 5/treatment).
Acknowledgements
Funding for this research was provided by the
National Science Foundation REU Program. The
authors wish to acknowledge Dr. Julio Turrens for
all of his hard work and organizing the
University of South Alabamas summer REU program.
We would also like to thank Dr. Timothy Sherman
for his assistance and input regarding protein
analysis. Finally, many thanks to Emily Boone,
Melanie Caldwell, Nobuo Udea and Wesley Lumpkin
for their assistance with collections and
sampling related to this project.