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Costs of Defense and a Test of the Carbon-Nutrient
Balance and Growth-Differentiation Balance Hypotheses
for Two Co-Occurring Classes of Plant Defense
Tara Joy Massad
1
*
¤
, Lee A. Dyer
2
, Gerardo Vega C.
2
1Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, Louisiana, United States of America, 2Department of Biology, University of Nevada,
Reno, Nevada, United States of America
Abstract
One of the goals of chemical ecology is to assess costs of plant defenses. Intraspecific trade-offs between growth and
defense are traditionally viewed in the context of the carbon-nutrient balance hypothesis (CNBH) and the growth-
differentiation balance hypothesis (GDBH). Broadly, these hypotheses suggest that growth is limited by deficiencies in
carbon or nitrogen while rates of photosynthesis remain unchanged, and the subsequent reduced growth results in the
more abundant resource being invested in increased defense (mass-balance based allocation). The GDBH further predicts
trade-offs in growth and defense should only be observed when resources are abundant. Most support for these
hypotheses comes from work with phenolics. We examined trade-offs related to production of two classes of defenses,
saponins (triterpenoids) and flavans (phenolics), in Pentaclethra macroloba (Fabaceae), an abundant tree in Costa Rican wet
forests. We quantified physiological costs of plant defenses by measuring photosynthetic parameters (which are often
assumed to be stable) in addition to biomass. Pentaclethra macroloba were grown in full sunlight or shade under three
levels of nitrogen alone or with conspecific neighbors that could potentially alter nutrient availability via competition or
facilitation. Biomass and photosynthesis were not affected by nitrogen or competition for seedlings in full sunlight, but they
responded positively to nitrogen in shade-grown plants. The trade-off predicted by the GDBH between growth and
metabolite production was only present between flavans and biomass in sun-grown plants (abundant resource conditions).
Support was also only partial for the CNBH as flavans declined with nitrogen but saponins increased. This suggests saponin
production should be considered in terms of detailed biosynthetic pathway models while phenolic production fits mass-
balance based allocation models (such as the CNBH). Contrary to expectations based on the two defense hypotheses, trade-
offs were found between defenses and photosynthesis, indicating that studies of plant defenses should include direct
measures of physiological responses.
Citation: Massad TJ, Dyer LA, Vega C. G (2012) Costs of Defense and a Test of the Carbon-Nutrient Balance and Growth-Differentiation Balance Hypotheses for
Two Co-Occurring Classes of Plant Defense. PLoS ONE 7(10): e47554. doi:10.1371/journal.pone.0047554
Editor: Martin Heil, Centro de Investigacio
´n y de Estudios Avanzados, Mexico
Received March 12, 2012; Accepted September 17, 2012; Published October 24, 2012
Copyright: ß2012 Massad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by an Environmental Protection Agency STAR Fellowship; support was also provided by the National Science Foundation grant,
CHE 0849369, and the Organization for Tropical Studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tmassad77@gmail.com
¤ Current address: Program on the Global Environment, University of Chicago, Chicago, Illinois, United States of America
Introduction
Herbivory and neighboring plant competition for resources are
two of the most important biotic forces affecting plant distributions
and fitness [1]. Competition, resource availability, and herbivory
can affect levels of defensive compounds in plants, since chemical
defense is a plastic response. Production of secondary metabolites
is often associated with reduced fitness in terms of lower growth
and reproduction [2–10]. This trade-off between investment in
plant defense versus growth and reproduction is termed an
allocation cost [10,11]. However, comparisons between defense
and growth or reproduction may be insufficient to quantify the
costs of defense because natural selection may strongly favor
reductions in trade-offs between such important activities as
growth, reproduction, and defense. Physiological parameters can
be more useful than growth rates for quantifying the cost of plant
defenses [12–16,8,10] (but see [17]). Physiological costs, such as
reductions in photosynthetic enzymes or the biosynthesis of other
proteins required for primary metabolism are said to arise from
‘metabolic competition’ between defense production and primary
metabolic functions [18]. Further examination of physiological
costs is important for determining the mechanisms underlying
allocation costs and for understanding interactions between
pathways leading to primary and secondary metabolites. In
addition, despite the notable contributions of induced defense
literature to understanding costs of chemical defense, it may be
particularly interesting to study costs in constitutive defenses to
understand the baseline value plants place on tissue retention.
In terms of physiological costs, photosynthesis is among the
most important variables to quantify as it forms the foundation of
a plant’s carbon budget. Studies combining measures of plant
defense and photosynthesis can also help clarify two prominent
mass-balance based hypotheses of secondary metabolite pro-
duction. The carbon-nutrient balance hypothesis (CNBH) [19]
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and the growth-differentiation balance hypothesis (GDBH) [11]
were formulated to address differences in defense concentrations
among individuals within a species; both hypotheses stem from the
assumption that an imbalance in nutrients and carbon will allow
plants to invest excess resources in defense as growth becomes
limited before photosynthesis. Plants that produce nitrogen-
containing defensive compounds (N-based defenses) are expected
to increase their production of defenses when available nitrogen is
more abundant than carbon; likewise, plants capable of synthe-
sizing carbon-based secondary metabolites (C-based defenses)
should increase production when fixed carbon exceeds require-
ments for growth [11,19]. Nitrogen-rich enzymes and nitrogen-
containing precursors are involved in the production of what are
termed C-based defenses [20–23], however, so this classification of
defenses as C- or N-based may be an oversimplification and
confound interpretation of responses to resources in the framework
of the CNBH or GDBH. There has, in fact, been much debate as
to the utility of the CNBH [24,25], and it has also been
erroneously applied [26]. Nonetheless, the empirical support for
this hypothesis shows predicted patterns of phenotypic changes in
defenses for temperate woody [27,28], herbaceous [29], and
tropical [30–33] species.
The GDBH is more detailed than the CNBH and predicts
a negative correlation between growth and defense under
conditions of moderate to high resource availability [11]. The
GDBH is difficult to test because: 1) a broad range of resource
availability must be included in studies, 2) most variables assessed
are merely correlates of the plastic physiological processes that are
part of the hypothesis (e.g., biomass is often a proxy for resource
allocation to growth, but it can include tissues and compounds
important in defense and storage as well), and 3) it is difficult to
ensure the maintenance of experimental resource conditions
throughout a plant’s growth [34]. Despite these challenges,
valuable insights on trade-offs and priorities in plant resource
allocation can be gained from studies addressing aspects of the
GDBH [35–37].
A key postulate of the CNBH and the GDBH is that defenses
will increase under conditions of limited growth when photosyn-
thesis continues to function at normal levels. This mechanistic
aspect of the hypotheses is difficult to test, yet some studies have
measured photosynthesis, growth, and defense simultaneously.
Results from these studies show a variety of patterns. Light can
increase photosynthesis and N-based defenses but decrease C-
based defenses [38]; available nitrogen can increase photosynthesis
and monoterpene production (except during the leaf expansion
stage) [39], and high nitrogen can have inverse effects on
photosynthesis (positive) and phenolic defenses (negative) [40,41].
In addition, the down-regulation of genes important to photosyn-
thesis has been shown to accompany herbivore induced up-
regulation of defenses in Nicotiana attenuata (Solanaceae) [42,43],
although resource conditions mediate changes in transcription
such that they do not always correspond to equivalent changes in
the products encoded for [43]. Nevertheless, the paradigm persists
that growth is more sensitive to a plant’s resource environment
than is photosynthesis, and decreased growth with concomitant
increases in defenses has been documented many times [11,33,44–
47]. The sensitivity of photosynthesis to environmental conditions
and the connection between photosynthesis and growth and
defense production merit more empirical study.
Here we present experimental results quantifying saponin
(terpenoid) and flavan (phenolic) production in a neotropical tree,
Pentaclethra macroloba Kuntze (Fabaceae: Mimosoideae), a shade-
tolerant species with nitrogen-fixing root nodules [48] that
produces high levels of saponins which function as an antiherbi-
vore defense [49,50] as well as flavonoids. Saponins are a class of
glycosylated triterpenoid, steroid, or steroidal alkaloid C-based
compounds produced primarily via the mevalonic acid pathway
[51], and flavans are flavonoids known to serve as plant defenses in
a related genus, Inga [52,53]. Most studies addressing the CNBH
and GDBH have focused on phenolics [54], making studies of
other classes of defense important. Terpenoids are especially
interesting because they are produced by the mevalonic acid
pathway, and defenses from this pathway do not fit predictions of
the CNBH and GDBH as well as the phenolics produced via the
shikimic acid pathway [20,54–56].
We tested the hypothesis that saponin production in P. macroloba
seedlings incurs both physiological (photosynthetic) and allocation
(biomass) costs. We measured saponin and flavan production
under different light regimes in response to changes in nutrients
and plant density to test ecological predictions made by the CNBH
and GDBH. Tests of the CNBH and GDBH have been criticized
for not measuring complete costs of secondary defenses [57]; by
quantifying relationships between two separate classes of defense,
as well as photosynthesis and growth, we do not escape this
criticism, but attempt to provide a more complete measure of these
trade-offs.
Materials and Methods
We collected seeds of Pentaclethra macroloba from multiple
individuals distributed throughout the forest of La Tirimbina
Rainforest Center, Sarapiqui, Heredia, Costa Rica (10u23 N,
84u8 W) in January 2008. Pentaclethra macroloba was selected for this
study because of its dominance in tropical forests where it is found
[58], its diverse defensive chemistry, and the ease with which seeds
can be found and propagated. La Tirimbina contains 345 ha of
tropical wet forest (sensu [59]) with an average of 4000 mm annual
precipitation, 26uC mean annual temperature, and an average day
length of 12 hours.
Planting Design
One hundred eighty seeds were planted in 60 6-liter pots
with 1.5 kg of sterile peat moss (Berger BM4: Sphagnum peat
moss (coarse), dolomitic and calcitic lime, initial fertilizer charge,
wetting agent; pH 5.4–6). A general fertilizer containing 25%
phosphate, 41% potassium, 0.02% boron, 8.27% sulfur, 0.1%
iron, 0.05% copper, 0.05% magnesium, 0.05% zinc, 0.001%
molybdenum, and 25.459% inert ingredients (Miller Chemical
and Fertilizer Corporation) was added to each pot at
a concentration of 0.35 g/kg soil. Three levels of nitrogen
fertilizer (urea: (NH
2
)
2
CO) were also applied: low = 0.002% N,
intermediate = 0.004% N, and high = 0.008% N (20 pots per
treatment). Seeds were planted alone (30 pots) and in
competition pots (30 pots–5 seeds per pot). Half of the pots
were then placed in full sunlight (,1175 PAR (mmol/m
2
/s)) and
half at 24% full sunlight (,282 PAR) in a shadehouse at La
Tirimbina (Figure 1). The plants in full sun were exposed to
natural rain, and those in the shadehouse were watered
regularly to ensure they received adequate moisture. The
seedlings never appeared water-stressed. Only one shadehouse
was available at the research station, so seedlings exposed to low
light were grown together. Therefore, the two levels of the light
treatment were analyzed as separate experiments to avoid
pseudoreplication. Within each light regime, each combination
of the nitrogen and competition treatments was replicated five
times, with an individual pot as a replicate. Fertilizer was
applied a second time in May 2008. Seedlings were routinely
examined, and aboveground herbivore damage was not
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detected, so it is very unlikely that induction of defenses affected
the data. Results indicate the competition treatment increased
available nitrogen rather than decreasing it (because P. macroloba
has N-fixing root nodules), and other work shows legumes can
enhance the performance of neighboring plants [60].
Seedling Measurements
Seedling height (cm), leaf area (cm
2
), the light saturated rate of
photosynthesis (A
max
;mmol CO
2
/m
2
/s), and dark respiration
(mmol CO
2
/m
2
/s) were measured for each replicate after six
months of growth. Leaf samples were also collected at this time for
chemistry analyses. The area of all the leaves on each seedling was
measured as the length and width of the leaves multiplied together
(cm
2
); the leaves are bipinnately compound, so this measurement
was used to compare leaf sizes but not to determine actual leaf
area. For pots with competition, the average height and leaf area
of individuals in the same pot were used in analyses. Plant biomass
was determined using regression equations from field collected
seedlings (sun n = 10; shade n = 14). PAR at the seedlings was
measured between 11:00 and 13:00, and the shade collected plants
had an average PAR of 20% while the sun collected plants had an
average PAR of 84%. The height and leaf area of the collected
seedlings was measured, and the stems and leaves of the seedlings
were then oven dried at 40 degrees Celsius for 72 hours and
weighed. Regressions of aboveground biomass by stem height plus
leaf area were then created (sun plants R
2
= 0.76, P= 0.001; shade
plants R
2
= 0.55, P= 0.002). The resulting regression formulas
were used to calculate aboveground biomass for the experimental
seedlings.
A
max
and dark respiration were measured with a LI-COR 6400
gas exchange system (LI-COR, Nebraska, USA), and only one
individual was measured in pots with competition. The third leaf
from the apical meristem was measured for consistency in leaf age.
Measurements were made between 7:00 and 13:00 hours. Leaves
were clamped into an airtight cuvette with a red-blue LED light
source. Incoming CO
2
was set to 380 mmol/mol from a CO
2
cartridge. Light response curves were made from darkness to 10,
25, 50, 100, 150, 200 mmol/s and continued in increments of
200 mmol/s until an asymptote was reached. Leaves were given
120 seconds to adjust to each light level, and the CO
2
differential
was recorded when flow rate, CO
2
and humidity were constant.
The flow rate was set to 550 mmol/s, and humidity was between
Figure 1. Schematic of experimental design. Seeds were planted individually or in competition with four conspecific neighbors and growth at
low, intermediate, or high nitrogen levels. There were ten replicate pots per nitrogen x competition combination, five of which were grown in
a shadehouse, and five of which were grown in full sunlight. The two light levels were analyzed as separate experiments (a). The photograph shows
a sun-grown (left) and shade-grown plant (right) side by side (b).
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65 and 75%. All necessary permits and permissions were obtained
for the described field studies.
Chemical Analyses
We collected and air-dried leaves for saponin and flavan
quantification. In preparation for chemical extraction, leaf samples
were dried overnight in an oven at low temperature and ground to
a coarse powder. We utilized a new isolation and quantification
procedure for saponin content [61]. One hundred milligrams of
dry leaf powder were measured into a centrifuge tube and
compounds were extracted from the leaf material in 30 ml of 80%
ethanol with stirring. The samples were then centrifuged and the
extracted compounds plus solvent were separated from the leaf
material and dried. The process was repeated to extract any
remaining compounds from the plant material. The dried samples
were then dissolved in 15 ml methanol and defatted by shaking the
solution with hexanes. The hexane layer was pipetted-off and the
process was repeated. The defatted methanol layer was dried, and
the samples were dissolved in 20 ml water. This solution was
centrifuged to separate any remaining leaf material from the
dissolved sample.
C-18 SepPak cartridges (Waters Corp., Massachusetts, USA)
were then preconditioned with 15 ml acetone followed by 15 ml
water. The water with dissolved sample was passed through the
cartridge, and the elution was dried. The cartridge was then
sequentially eluted with 20 ml each of 35%, 60%, and 100%
methanol. The 35% and 60% methanol elutions were also dried.
The 100% methanol elution was transferred directly to a pre-
weighed scintillation vial and dried. The dried samples were
redissolved in methanol, transferred to pre-weighed scintillation
vials, and dried a final time. Samples were stored in the freezer.
The water elution contains sugars and organic acids. The 35%
elution contains flavans, the 60% layer is comprised of flavones,
and the 100% layer contains saponins [61] (Lokvam, pers.
comm.). Subsequent work demonstrated that the 60% elution
contains mostly sapongenins (saponins without the attached
polysaccharides), so it was not used in further flavonoid analyses.
Samples were completely dried overnight in an oven at low
temperature. Vials with samples were then weighed to determine
the mass of each class of compound contained in the leaf material.
The weights of samples from the elutions were used in analyses.
HPLC was utilized to examine five samples from the 100%
elutions to confirm the presence of saponins and purity of the
samples. The HPLC system consisted of a Hitachi LaChrom Elite
HPLC System (Hitachi High Technologies America, California,
USA) with a diode array detector (DAD; Hitachi High Technol-
ogies America) and an evaporative light scattering detector (ELSD;
SEDEX 55 Evaporative Light-Scattering Detector; S.E.D.E.R.E.,
Alfortville, France). The column was packed with C8 coated beads
(2650 mm; 3 mm particle size; 100 A
˚pore size; Advanced
Chromatography Systems, South Carolina, USA). Dry samples
from the 100% methanol elution were dissolved in 250 ml 100%
methanol and 10 ml were run on the HPLC at a gradient of
MeOH:H
2
O (1:1) to 100% methanol over 40 minutes with
a 0.25 ml/min flow rate. The DAD absorption was set between
225–400 nm. The ELSD detector was maintained at 40uC and the
pressure was at 2.3 bar. Nitrogen was the nebulizing gas.
Statistical Analyses
Shade-grown plants and sun-grown plants were treated as two
experiments and analyzed separately to avoid problems with
pseudoreplication. Biomass and photosynthesis variables (A
max
and dark respiration) were analyzed together using multivariate
analysis of variance (MANOVA) followed by profile analysis with
fertilizer, competition, and their interaction as independent
variables; when interactions were not significant, main effects
models were run. Primary (sugars) and secondary (flavans and
saponins) metabolite production were likewise analyzed together
with MANOVA and profile analysis. MANOVA analyzes the
response of multiple dependent variables to experimental treat-
ments, and profile analysis tests for differences in the magnitude or
direction of response of different dependent variables. Three
outliers were removed from dark respiration for the shade-grown
plants for normality. A
max
was square-root transformed for
normality in the sun-grown plant dataset, and biomass was log-
transformed in the sun dataset as well. Sugars and flavans were
square-root transformed in both the shade and sun datasets.
We used structural equation models [62] to test hypothesized
relationships between photosynthesis (A
max
), biomass, flavans,
saponins, and planting treatments. Models were sequentially run
testing a priori hypotheses of treatment effects and relationships
between response variables, and the best fitting model is presented.
The SAS Calis (Covariance Analysis of Linear Structural
Equations) procedure was utilized to determine the fit of the
models. The Calis procedure uses normal theory maximum
likelihood procedures to estimate fit, and parameter vectors are
estimated iteratively with a nonlinear optimization algorithm to
Table 1. MANOVA and profile analysis results for the
response of Pentaclethra macroloba photosynthesis, biomass,
and carbon-based metabolites (sugars, flavans, and saponins)
to light, fertilizer, and competition.
Photosynthesis and biomass
Factor df MANOVA F
P
Profile F
P
Sun plants
Fertilizer 2 0.2 0.8 2.4 0.1
Competition 1 0.05 0.8 2.8 0.1
Error 23
Shade plants
Fertilizer 2 0.97 0.4 0.8 0.5
Competition 1 8.0 0.01 0.4 0.5
Error 23
Metabolites
Factor df MANOVA F
P
Profile F
P
Sun plants
Fertilizer 2 0.2 0.8 0.2 0.8
Competition 1 19.5 0.0002 19.6 0.0002
Error 21
Shade plants
Fertilizer 2 0.8 0.5 1.1 0.4
Competition 1 0.2 0.6 0.03 0.9
Fert. * comp. 2 5.7 0.01 0.7 0.5
Error 17
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optimize a goodness of fit function. Chi-square values are
calculated for the maximum likelihood goodness of fit to determine
the fit of the models. P-values greater than 0.05 indicate a good fit
of the data to the model. We accepted the model with the highest
P-value as the best description of the relationships between
variables. All analyses were done with SAS 9.1 (SAS Institute Inc.
2003).
Results
Photosynthesis and biomass of the shade-grown plants were
highest with competition, but dark respiration was slightly higher
without competition (Table 1; Figure 2a). The fertilizer treatment
did not have an effect on the response variables. Neither
photosynthesis, respiration, nor biomass of plants grown in the
sun changed with the competition or fertilizer treatments (Table 1;
Figure 2b).
The interaction between fertilizer and competition was signif-
icant for shade-plant metabolite production (Table 1). Sugars were
higher in plants with competition and low or intermediate levels of
fertilizer. They were lowest also with competition but with high
levels of fertilizer. The two groups of secondary metabolites
responded in the opposite direction to increased nitrogen. Flavans
were highest in low nitrogen conditions (no competition, low
fertilizer) and lowest in conditions of high nitrogen (competition
and high fertilizer levels). In contrast, saponins were highest with
competition and high fertilizer levels and lowest without compe-
tition and with low fertilizer levels (Figure 2c).
Metabolites of sun-grown plants were affected by competition
such that sugars and flavans were all higher without competition
(low nitrogen), and saponins were higher with competition
(Table 1; Figure 2d).
The best-fitting structural equation models differed for plants
grown in the sun or the shade (Figure 3). In both datasets,
competition increased A
max
and saponins, but decreased levels of
flavans. Higher levels of nitrogen in the competition treatment
likely allowed for increased photosynthesis and saponin production
by providing nitrogen necessary for enzymatic processes. This
increase in N may have instigated a diversion of C from flavan
production to processes or pools that were limited under lower N
conditions (negative effect of competition on flavans). Competition
did not have a direct effect on biomass, however. The effect of
competition in the sun was not quite significant for A
max
(t-value of
relationship = 1.55; a significant relationship is described by a t-
value $1.96), but the best-fitting model included this pathway.
The best-fitting model for shade-grown plants also included
a slightly non-significant causal pathway indicating fertilizer
increased saponin levels (t-value = 1.92). Both sun and shade
plants showed evidence of a trade-off between photosynthesis and
Figure 2. Means (SE) of photosynthesis, dark respiration, biomass, and carbon-based metabolites. Values are from Pentaclethra
macroloba seedlings grown in shade (a, c) or full sunlight (b, d) with and without competition.
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saponin production, and a negative relationship was also present
between A
max
and flavans in shade-grown plants. Flavans in
shade-grown plants were also positively correlated with biomass,
contrary to expectations of a growth-defense trade-off. Plants
grown in the sun showed the opposite pattern, and flavans and
biomass were negatively correlated. Perhaps full sunlight pro-
moted increased growth (positive relationship between photosyn-
thesis and biomass), creating demands on pathways of plant
allocation that limited production of flavans. When the correlation
between A
max
and biomass was included in the model for shade-
grown plants, the relationship was negative, although weak
(PE = 20.02), and the model fit less well. The shade plant model
including the relationship between saponins and biomass also fit
less well, and the correlation between saponins and biomass was
negative (PE = 20.07).
In summary, in sun-grown plant photosynthesis and biomass
were positively correlated as were saponins and biomass. In the
shade, both these relationships were negative. In contrast, flavans
and biomass were negatively related in the sun, and their
relationship was positive in the shade. Light therefore seems to
be the limiting factor which, when abundant, allows for positive
relationships between saponins and biomass and A
max
and
biomass or, when restricted, leads to a situation in which trade-
offs between these processes and pools become apparent. Flavans,
however, show a trade-off with growth only under full sun
conditions.
Discussion
Trade-offs between growth and defense differed with the light
conditions seedlings were grown under, and the GDBH [11] and
CNHB [18] were supported only by comparisons between flavans
and biomass. The GDBH predicts growth and defenses will be
positively correlated when resources are limited and negatively
correlated when resources are abundant. As expected, we found
that growth and defense were positively correlated in the shade,
while the predicted trade-off between flavans and biomass became
apparent in the sun. Both the CNBH and the GDBH assume that
growth is limited before photosynthesis, allowing excess resources
to accumulate and serve in defense production. By measuring
photosynthesis, growth, and two classes of defense, however, we
uncovered trade-offs between photosynthesis and defense when
biomass and defenses were positively correlated. A similar trade-
off between defense and photosynthesis rather than defense and
growth was found for an imide (a N-based defense) in Piper
cenocladum [63]. This is consistent with the hypothesis that costs of
defense are not only manifested in growth and reproduction but
exist at a physiological level. One important caveat is that we do
not have data on root biomass. Overall results may change with
the inclusion of information on allocation to below-ground growth;
however, because P. macroloba have N-fixing root nodules and were
grown in pots, differences in below-ground biomass were probably
minimal.
The correlations between secondary metabolites and biomass
suggest that flavans or saponins are not costly to a plant, except
under conditions of full sunlight (contrary to expectations, costs
should be most evident when resources are limited) [47]. However,
including physiological data showed relationships between de-
fenses and photosynthesis were negative under both shade and full
sun. The trade-off between photosynthesis and defense production
may occur because defense production, regardless of a compound’s
classification as C- or N-based, requires nitrogen for enzymes
involved in the metabolic pathways. The majority of nitrogen in
a plant is contained in Rubisco, the primary enzyme in
photosynthesis, which accounts for roughly 25% of leaf nitrogen
in C
3
plants. Rubisco content increases with leaf nitrogen and is
sometimes, but not always, produced in excess of photosynthetic
requirements as a means of nitrogen storage [64]. It is therefore
possible that leaf nitrogen in P. macroloba is sufficiently limited, such
that trade-offs between different cellular demands for nitrogen
exist. In addition, flavan production decreased with presence of
neighboring plants (higher nitrogen), following predictions of the
CNBH and the GDBH that investments in C-based defenses
decline as nitrogen availability increases.
In the shade experiment, a positive correlation between growth
and defense was present for flavans. This relationship changed in
the full sun, and flavans were negatively correlated with biomass.
Initially, it would seem the negative relationship is due to increased
growth at high light. Biomass was greater, however, in shade
grown plants while dark respiration and photosynthesis were
higher in the sun. Pentaclethra macroloba is a shade-adapted plant, so
the increase in respiration may result from metabolic processes
necessary to avoid photoinhibition, and the trade-off with biomass
may be related to an underlying relationship with respiration.
Including dark respiration in the structural equation model for
Figure 3. Interactions between experimental treatments and
trade-offs in photosynthesis, growth, and defense production.
Path diagrams showing causal relationships (single headed arrows) and
correlations (double headed arrows) between competition, fertilizer,
photosynthesis (A
max
), flavans, saponins, and biomass in Pentaclethra
macroloba seedlings grown in the shade (A; x
2
= 0.8, df = 5, P= 0.98) or
the sun (B; x
2
= 0.02, df = 1, P= 0.89). Bullets indicate negative relation-
ships and arrows indicate positive relationships. Numbers are the
standardized parameter estimates for relationships between variables.
All relationships were significant with t-values .1.96 except where
smaller t-values are indicated.
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sun-grown plants yielded a significant model (x
2
= 6.0, df = 3,
P= 0.1), and flavans and respiration were negatively related while
biomass and respiration were positively correlated. Flavan levels
were at their highest in plants grown with full sunlight and no
competition; this increased production could result from a greater
need for the defensive role of flavonoids as UV-B protectants (e.g.,
[65]), and flavonoid production increases in full sunlight in other
species as well [66].
Unlike flavans, triterpenoid saponin levels did not fit predictions
of the two defense hypotheses, increasing with nitrogen and having
a positive relationship with biomass. Phenolics are the class of
secondary metabolites most often found to fit predictions of the
CNBH [17,54,67–69], and it has been suggested that the CNBH
and GDBH are more relevant to phenolics because they are
produced via the shikimic acid pathway which competes directly
with protein synthesis (growth) for nitrogen via metabolism of
phenylalanine [54,70], while terpenoids are produced by different
biosynthetic pathways. Biosynthesis of saponins is initiated via the
mevalonic acid and methylerythritol phosphate pathways [51,70],
which do not experience a direct trade-off with growth based on
available nitrogen [71,72]. Our data suggest saponins and
photosynthesis compete for nitrogen before carbon is divided
between growth and ‘excess’ carbohydrates (as per [54]). This may
explain why fewer data from terpenoid studies fit predictions of the
CNBH and GDBH.
Gershenzon speculated that the CNBH would apply to
terpenoids only when they are substrate limited [20], but our
data suggest saponin production was more limited by nitrogen
resources required for synthesis rather than carbon required as
a substrate, and this was also true in the shade for flavans. Overall,
we found restricted support for the GDBH and the CNBH but
have demonstrated that investigations of costs of defense should
focus on the physiological level where many trade-offs appear to
take place. In spite of context dependent support of the GDBH
and CNBH based on terpenoids and phenolics, the appropriate
application of these hypotheses should continue to guide
experiments that enhance a clear understanding of plant defensive
investments. Basic and applied ecology will benefit from advances
in studies that document costs of defense against parasites, and
further investigations of interactions between resource availability
and physiological trade-offs will demonstrate the strength of both
ecological and evolutionary influences on investments in defense–
issues of particular contemporary importance due to rapid changes
in carbon and nitrogen availability in the environment.
Acknowledgments
Massad and Dyer would like to dedicate this work to their co-author,
Gerardo Vega, who sadly passed away before publication. His extensive
knowledge of tropical forests helped many researchers over the years.
Special thanks to John Lokvam for sharing his chemical analysis methods
and the Coley/Kursar laboratory for sharing their laboratory facilities. We
would also like to thank Ryan Massad and several EarthWatch volunteers
for their assistance in measuring the plants. Jeffrey Chambers and Karen
Holl provided valuable comments on this manuscript. La Tirimbina
Rainforest Center generously provided facilities for the experiment, and
the Max Planck Institute for Biogeochemistry enabled collection of biomass
data. Finally, we would like to thank the Organization for Tropical Studies
for supporting the work.
Author Contributions
Conceived and designed the experiments: TJM LAD. Performed the
experiments: TJM GVC. Analyzed the data: TJM. Contributed reagents/
materials/analysis tools: LAD TJM. Wrote the paper: TJM LAD.
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