Benefits of Adding Physiological
Measurements to Long-term
Monitoring Efforts
Calcium Depletion and Red Spruce / Sugar
Maple Decline as Examples
USDA Forest Service
Paul Schaberg
Kelly Baggett
John Bennink
Brynne Lazarus
Paula Murakami
The University of Vermont
Rubenstein School, Burlington, VT
Donald DeHayes
Gary Hawley
Cathy Borer
Tammy Coe
Michelle Turner
Brett Huggett
Josh Halman
and many students…
Winter Injury
of Red Spruce
Cold tolerance of several north temperate conifers
(sampled in Wolcott, VT on March 3, 1998)
Species
Red spruce
Mean cold tolerance (ºC)
-38.1
White pine
-59.5
Eastern hemlock
-61.0
White spruce
-90.0
Red pine
-90.0
DeHayes et al. 2001
Mechanisms for acid-induced
freezing injury of red spruce
Acid deposition (H+)
H+ displacement
of membrane-associated Ca
(-20%, P<0.05)
Foliar Ca leaching
(2-8X increase, P<0.05)
Membrane destabilization
(+20%, P<0.05)
& reduction in messenger Ca
Reduced cold tolerance
(-10%, P<0.05)
Increased susceptibility
to freezing injury
DeHayes et al. 1999, Schaberg et al. 2000, Schaberg et al. 2001
Unresolved issues…
•Relevance to field?
•Soil Ca depletion?
•Just cold tolerance?
•Just red spruce?
Providing a Broader Context
Field verification
Soil-based Ca depletion
Stress response systems
Other tree species
2003 – high winter injury year
Historic Context of 2003 Winter Injury
100
Mean % current-year foliage injured
90
80
Colebrook, NH
70
Mt. Moosilauke, NH
60
Whiteface Mtn., NY
50
40
30
20
10
0
1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004
Year
2003 sampling: 185 plots at 28 sites in VT,
NH, eastern NY, and Northwestern MA
Lazarus et al. 2004
2003 sampling: 185 plots at 28 sites in VT,
NH, eastern NY, and Northwestern MA
 96% dominant and co-
dominant trees effected
 65% current-year
foliage lost
 56% buds killed
Lazarus et al. 2004
Statistical spatial analysis significantly greater injury at:
• Higher elevations
• Western longitudes
• West-facing plots
Lazarus et al. 2006
Providing a Broader Context
Field verification
Soil-based Ca depletion
Stress response systems
Other tree species
HBEF watershed 1 (wollastonite) compared to
watershed 6 (reference)
Wollastonite
Treated Oct. 1999
(CaSiO3)
Reference
Red Spruce Winter injury
90
**
Winter injury of current-year foliage (%)
80
Reference
Ca-addition
70
60
50
*
Slide 15\
40
ns
30
20
10
0
All
Dominant and codominant
Intermediate, suppressed &
understory
Tree crown class
Hawley et al. 2006
Providing a Broader Context
Field verification
Soil-based Ca depletion
Stress response systems
Other tree species
Environmental Change/Stress Signal
Temperature extremes, fungal elicitors, wounding, drought, ozone, etc.
Signal transduction: Ca
moves from area of
high concentration (e.g.,
extracellular mCa or
organelle stores) to the
low Ca cytosol.
Ca
Cytoplasmic Ca binds to specific
proteins (e.g. calmodulin).
2+
Ca2+
Ca2+
vacuole
cell membrane
protein
Ca2+
Denzyme activity, genetic
transcription, etc.
changes in cell physiology
cell wall
Plant Adaptation to Environmental Change/Stress
Alterations in cold tolerance, stomatal conductance,
antimicrobial defense, carbon allocation, etc.
Our Overarching Hypothesis
Acid Rain, Calcium Depletion
and
Immune Dysfunction
HBEF watershed 1 (wollastonite) compared to
watershed 6 (reference)
Wollastonite
Treated Oct. 1999
(CaSiO3)
Reference
Red Spruce Winter injury
90
**
Winter injury of current-year foliage (%)
80
Reference
Ca-addition
70
60
50
*
Slide 15\
40
ns
30
20
10
0
All
Dominant and codominant
Intermediate, suppressed &
understory
Tree crown class
Hawley et al. in press
Foliar Cold Tolerance
Hubbard Brook Watershed
Cold Tolerance ( oC)
Ca addition
0
-10
-20
-30
-40
-50
-60
Reference
*
P = 0.0431
Red Spruce Foliar Cold Tolerance
Halman et al. in prep.
Carbohydrate Metabolism






Cell energy relations
Sugars important to cold tolerance
Osmotic control during freezing
Act as an “antifreeze” agent
Direct cryoprotection of biomolecules
Influenced by Ca deficiency
Foliar Carbohydrates
Sugar Concentration (mg/g)
Total Sugars - 2005 Foliage
90
85
80
75
70
65
60
55
50
45
40
Reference
*
*
Ca-add
*
*
Nov. 2005
Feb. 2006
Fig. 2. Total sugar levels in current-year foliage were significantly
greater in WS1 than in WS6 in both fall and winter collections; * = (p< 0.05)
Individual Sugars
Table 1. Individual sugar concentrations (mean ± SE, n = 30) in current-year foliage
(mg g-1). Three major sugars tested had significantly greater means between
watersheds from the fall collection. Data collected from winter collection
showed significant differences between watersheds in sucrose and glucose.
Sugar
November
February
Reference
Ca-addition
Reference
Ca-addition
Fructose
24.56 ± 0.76**
27.75 ± 0.73**
25.74 ± 1.20
28.01 ± 1.18
Sucrose
4.14 ± 0.76
5.30 ± 0.74
2.83 ± 0.42**
4.00 ± 0.42**
Glucose
39.62 ± 0.83**
43.47 ± 0.78**
39.85 ± 1.48*
44.18 ± 1.42*
Stachyose
0.232 ± 0.03*
0.308 ± 0.03*
3.41 ± 0.19
3.56 ± 0.19
Note: * = p<0.1; ** = p<0.05
Antioxidant Enzymes

Enzymes responsible for scavenging harmful
reactive oxygen species

Inadequate function can result in cellular and
membrane dysfunction, and/or cell death

Assaying ascorbate peroxidase (APX) – activity
dependent upon Ca availability
APX Activity (µmoles ascorbate
min-1 mg-1 protein)
Seasonal APX Activity
30
25
Ca-add
*
Reference
20
*
15
10
5
0
Nov. 2005
Feb. 2006
Fig. 4. APX activity (mean ± SE, n = 30) from current-year foliage. Winter APX activity
is elevated in Ca-addition watershed yet relatively unchanged in reference. * = p < 0.1
Providing a Broader Context
Field verification
Soil-based Ca depletion
Stress response systems
Other tree species
Field Examples
Ca Depletion and Tree Decline
•
•
•
•
•
Red Spruce Winter Injury
Sugar Maple Decline
Flowering Dogwoods and Anthracnose
Norway spruce freezing injury
Hemlock Woolly Adelgid Infestation
Sugar Maple Decline
• Nutritional predisposition
(D cations – Ca, Mg)
• Disproportionate decline
following secondary
stress
Mechanism of Maple Decline?
Soil Ca depletion
mCa disruption
Impairment of
stress response
system
Maple decline
Secondary
stresses
drought
insects
disease
pollutants
etc.
NAMP and HHS Plots
Predicted High
Ca (6)
Predicted Low
Ca (8)
6 sugar maples per site
84 total trees
Till Source Model: Source Envelope
Glacial
Movement
-Courtesy of Scott
Bailey et al.
32 km
60°
Designated site
• 26 HHS & NAMP sites were selected for TSM predictions.
• Latitudinal and longitudinal coordinates were used to generate a
source envelope.
• 8 sites predicted to contain low levels of Ca, 6 were predicted to have
high levels of Ca.
16000
800
a
a
Soil Ca
-1
Foliar Ca
12000
600
b
400
0
High
8000
4000
b
200
Low
Foliar Ca (mg · kg )
-1
Soil Ca (mg · kg )
1000
0
TSM Ca classes
Schaberg et al 2006
Schaberg et al., 2006
% Branch dieback
25
20
a
Ca (P = 0.021)
Mg (P = 0.052)
Al (P = 0.035)
a
15
a
b
b
b
10
5
0
Low
Moderate
Published foliar range
Schaberg et al 2006
Schaberg et al., 2006
Basal growth (cm2)
160
120
80
r = 0.53, P = 0.050
40
0
0
5000
10000
15000
20000
-1
Foliar Ca (mg · kg )
Schaberg et al 2006
Schaberg et al., 2006
Hubbard Brook Experimental Forest
•
12 (45m x 45m) plots
• Control plots – marginal Ca nutrition
Randomized Ca or Al additions – begun 1995
• Sugar Maple Dominant Species
•
•
NuPert Plots
Total Foliar Cation Analysis
-1
Mean foliar Ca (mg • kg ) (±SE)
Foliar Calcium Levels
10000
a
8000
Minimum level for
healthy sugar maple
Kolb and McCormick (1993)
b
6000
b
4000
2000
0
Calcium
Control
Aluminum
Treatment
Treatment means with different letters are significantly different (P ≤ 0.05)
based on orthogonal contrasts: Ca vs. Al and Control; Al vs. Control
Crown Health Evaluation
Percent Branch Dieback
decreased
dieback
7
Mean % branch dieback
rating (±SE)
increased
dieback
2
X = 6.92, p = 0.0314
6
5
4
3
2
1
0
Calcium
Control
Treatment
Aluminum
Tree Increment Growth
5.00
a
Basal Area Increment (BAI) Ratio
Ca
Control
4.00
Al
a
a
a
2.00
Treatments
Began
b
b
a
1.00
a
a
3.00
b
b
b
b
b
b
b
b
b
b
b
b
0.00
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Year
Tree Increment Growth
5.00
a
Basal Area Increment (BAI) Ratio
Ca
Control
4.00
Al
a
a
a
3.00
2.00
Treatments
Began
Ice
Storm
1.00
b
b
a
a
a
b
b
b
b
b
b
b
b
b
b
b
b
0.00
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Year
Wound Closure Amount Results
Treatment means for wound closure amounts
(mm ± SE)
Ca addition
Wound
Closure 0.615±0.313
Amount
Control
Al addition
-0.303±0.26 0.060 ±0.25
P-values
Ca vs.
Control
Control and Al
vs. Al
0.0414
0.3598
• Differences associated w/ Ca fertilization were most pronounced
in wounds lower of the tree stem (100cm-130cm, P = 0.0016)
Huggett et al. submitted to CJFR
*
SugarMaple
Maple Shoot
Shoot Cold
Cold Tolerance
Sugar
Tolerance
Site
Ca
Site Soil
Casoil
Status
High
Low
-25
o
Cold
C)
Tolerance((oC)
Coldtolerance
-26
-27
-28
-29
-30
-31
-32
-33
-34
-35
***
P = 0.0076
March 8, 2006
Providing a Broader Context
Field verification
Soil-based Ca depletion
Stress response systems
Other tree species
Synergies: Physiology and Monitoring
Gain information about the
relevance of experimental
evidence to trees/forests in
the “real world”
Synergies: Physiology and Monitoring
Physiological measurements can
add a mechanistic understanding
to changes in forest health and
productivity noted via monitoring
Questions?