Chemoecology 8:133 –139 (1998)
0937 – 7409/98/030133–07 $1.50 +0.20
© Birkhäuser Verlag, Basel, 1998
Biosynthetic origin of carbon-based secondary compounds: cause of
variable responses of woody plants to fertilization?
Erkki Haukioja1, Vladimir Ossipov2, Julia Koricheva1,3, Tuija Honkanen1, Stig Larsson3 and Kyösti Lempa1
1
Section of Ecology, Department of Biology, University of Turku, FIN-20014 Turku, Finland, e-mail: [email protected]
Laboratory of Physical Chemistry, Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
Department of Entomology, Swedish University of Agricultural Sciences, P.O. Box 7044, S-750 07 Uppsala, Sweden
2
3
Summary. We propose that variation in the responses
of carbon-based secondary compounds to fertilization
in woody plants has a biosynthetic cause. The synthesis
of phenylpropanoids and derived compounds (e.g., condensed tannins) competes directly with the synthesis of
proteins, and therefore with plant growth, because of a
common precursor, phenylalanine. In contrast, the
biosynthesis of terpenoids and of hydrolyzable tannins
proceeds presumably without direct competition with
protein synthesis. Therefore, accelerated plant growth
induced by fertilization may cause a reduction in concentrations of phenylpropanoids but may affect less or
not at all the levels of other classes of secondary
compounds. A meta-analysis based on fertilization experiments with 35 woody plant species supported the
predicted differences: fertilizing significantly decreased
concentrations of phenylpropanoids but not of terpenoids or hydrolyzable tannins.
Key words. carbon/nutrient balance hypothesis –
growth-defense trade-offs – growth/differentiation balance hypothesis – herbivory – meta-analysis
Introduction
Nutrient-deficient plants often display lower growth
rates and higher concentrations of carbon-based (nonnitrogen-containing) secondary compounds (CBSCs)
than conspecifics with access to ample nutrients (Bryant
et al. 1983; Coley et al. 1985). The negative correlation
between concentrations of CBSCs and plant growth
rate, or levels of nutrients in plant tissues, is assumed to
indicate a trade-off between plant growth and the production of defensive compounds, and represents the
cornerstone of the carbon/nutrient balance (CNB) hypothesis (Bryant et al. 1983; Tuomi et al. 1988) and the
growth/differentiation balance (GDB) hypothesis
(Loomis 1932; Lorio & Sommers 1986; Herms &
Mattson 1992). Both hypotheses state that growth processes dominate over the production of CBSCs, and
more carbon is left for CBSCs only when plant growth
Correspondence to: E. Haukioja
is restricted by a lack of mineral nutrients (emphasized
by the CNB hypothesis) or by any factor (according to
the GDB hypothesis). Conversely, plants growing
slowly because of low nutrient availability, but with
sufficient light for normal photosynthetic rates, will
have ‘extra’, ‘cheap’, or ‘cost-free’ carbon to allocate to
CBSCs. The above hypotheses have been tested by
manipulating carbon and nitrogen availability in fertilization or shading experiments, or by rearing plants
under CO2 -enriched atmosphere. Fertilization enhances
the availability of nutrients, whereas CO2 -enrichment
increases carbon availability. Shading decreases carbon
gain, and is therefore often assumed, like fertilization,
to raise the nitrogen/carbon ratio. Note that these tests
in practice introduce two usually implicit assumptions:
concentrations of CBSCs describe plant defensive commitments and the high concentrations of CBSCs in
nutrient deficient plants should hold irrespective of
which CBSCs are measured. But the tests have indicated that not all types of plant CBSCs respond alike to
the altered nutrient supply. Strong support for the
predictions of the CNB and GDB hypotheses has been
found for phenolics: levels of total phenolics usually
decrease after fertilizing (e.g., Tuomi et al. 1984;
Larsson et al. 1986; Bryant 1987; Bryant et al. 1987b),
although individual phenolics may show the opposite
response (Muzika et al. 1989; Reichardt et al. 1991;
Muzika 1993). Instead, fertilization effects on concentrations of terpenoids, the other major class of woody
plant CBSCs with putatively defensive properties, are
much less consistent (Muzika et al. 1989; Björkman et
al. 1991; McCullough & Kulman 1991; Rousi et al.
1993). Similarly, elevated CO2 led to an increase in the
concentrations of phenolics, whereas no consistent
changes were observed in terpenoids (Peñuelas et al.
1997; Peñuelas & Estiarte 1998).
In this paper, we analyze the behaviors of woody
plant CBSCs following fertilization. We restrict the
analysis to fertilization experiments because they are
more numerous than manipulations of carbon
availability, and, although liable to various interpretations, have been much used as tests of the causalities
underlying trade-offs between growth and defense (see
Mole 1994). We show that the variable responses of
woody plant CBSCs to fertilization are linked to their
different biosynthetic origins. We first briefly describe
134
E. Haukioja et al.
the biosynthetic basis of different types of CBSCs and,
on the basis of their biosynthetic properties, make
predictions about their responses to fertilization. Finally, we test these predictions by means of meta-analysis of the data available in the literature.
Biosynthesis of carbon-based secondary compounds
Muzika & Pregitzer (1992) have suggested that the
availability of nitrogen should affect concentrations of
phenolics more strongly than those of terpenoids because phenolics are produced in the same shikimic acid
pathway as aromatic amino acids. Accordingly, there
should be a strong biochemical basis for a trade-off
between the synthesis of phenolics and of proteins.
Such a direct metabolic trade-off has been demonstrated for numerous plant species (see Margna 1990).
The most illuminating examples come from experiments
with soybeans (Hahlbrock et al. 1974) and maple cells
(Westcott & Henshaw 1976; Phillips & Henshaw 1977).
In maple cell cultures, condensed tannins accumulated
when net protein synthesis decreased after exhausting
the nitrogen medium. Doubling the medium nitrogen
delayed the onset of tannin production while adding
urea at the time of tannin accumulation inhibited the
process. Hence the biosynthesis of phenolics in plant
cells and tissues can be easily modified: phenolic production decreases at high nitrogen availability, and
increases under nitrogen deficiency.
In contrast to phenolics, terpenoid biosynthesis
takes place in the mevalonic acid pathway (Gershenzon
1994b), and therefore does not directly compete with
phenolic and protein synthesis of the shikimate pathway. Accordingly, the biosynthetic origin of terpenoids
does not indicate such a strong, direct trade-off with
protein synthesis as biosynthesis of phenolics does.
However, even phenolic compounds themselves
constitute a biosynthetically heterogeneous group, and
this also provides a possibility for the independent
testing of the importance of different biosynthetic
origins for the different responses of CBSCs to fertiliz-
CHEMOECOLOGY
ing. A large and diverse group of phenolics, phenylpropanoids and derived compounds (hydroxycinnamic
acids, flavonoids, condensed tannins and lignin) are
produced from phenylalanine, and therefore competes
directly with protein synthesis. However, the second
major group of phenolics, the hydrolyzable tannins, has
gallic acid as its precursor. Two origins for its synthesis
have been proposed: either from phenylalanine or from
dehydroshikimic acid, an intermediate compound of the
shikimate pathway (Ishikura et al. 1984; Waterman &
Mole 1989; Gross 1992; Ossipov et al. 1995) (for the
biosynthesis of phenolics in birch, see Fig. 1).
Therefore, depending on the relative strength of the
synthetic route via dehydroshikimic acid to gallic acid,
hydrolyzable tannins may or may not trade off directly
with protein synthesis. Note that the synthesis of hydrolyzable tannins via dehydroshikimic acid consumes
far less carbon and energy (as indicated by the use of
ATP; Fig. 1) than the synthesis of phenylpropanoids
(Ossipov et al. 1997).
To sum up, strictly for biosynthetic reasons we
predict that there should be a readily measurable tradeoff between the synthesis of proteins and phenylpropanoids, which might reflect in decreased concentrations of phenylpropanoids in response to fertilization. By the same logic, biosynthetic factors do not
predict a strong trade-off between protein synthesis and
the synthesis of terpenoids or hydrolyzable tannins.
Fertilization effects on carbon-based secondary
compounds of different biosynthetic origin: a
meta-analysis
A meta-analysis was conducted on the effects of fertilization on woody plants, to test whether responses
differ between those CBSCs whose biosynthesis proceeds through phenylalanine (phenylpropanoids) and
those whose biosynthesis proceeds only partially or not
at all (hydrolyzable tannins, terpenoids) via phenylalanine. The analysis included only studies reporting the
results of chemical assays; studies based on indirect
Fig. 1 Pathways of phenolics
and protein synthesis in birch
leaves. Modified from Nurmi et
al. (1996)
Vol. 8, 1998
Class of CBSC
Biosynthetic origin
N
d+
137
22
−0.518
−0.036
Class of phenolics
N
d+
phenylpropanoids
hydrolyzable tannins
89
35
−0.434
0.116
Class of phenylpropanoids
N
d+
condenensed tannins
flavonoids
lignin
hydroxycinnamic acids
62
18
5
2
−0.392
−0.425
−1.198
−0.022
phenolics
terpenoids
95%
CI
−0.691
−0.435
−0.344
0.364
95%
CI
−0.663
−0.289
−0.206
0.522
95%
CI
−0.695
−0.922
−2.196
−1.367
−0.089
0.072
−0.201
1.322
QB=4.71, df =1, P= 0.030*
135
Table 1 Effects of fertilization on
concentrations of general classes of
carbon-based
secondary
compounds, classes of phenolics and
classes of phenylpropanoid derivatives in the literature on woody
plants. Fertilization has a statistically significant effect if the 95% CI
of the cumulated effect size does not
overlap with zero. Among-class differences were tested by the term QB
QB= 5.37, df = 1, P=0.021*
QB =2.69. df = 3. P= 0.442
measurements of secondary compounds, such as numbers of resin droplets or resin flow, or on the proteinbinding capacity of plant extracts, were excluded,
because it is not possible from such indices to distinguish between the contributions of the above three
classes of CBSCs. We found forty studies (listed in the
Appendix), published during 1975-97 in eighteen different journals and covering a wide range of woody plants
(35 species, 21 genera) and various fertilizer types (N,
P, NP or NPK).
In many cases, the effects of different types and/or
concentrations of fertilizers on several woody plant
species or clones were tested in a single study, or
repeated measurements were reported for the same
plant individuals. To reduce the statistical problems
associated with the inclusion of such non-independent
comparisons (Abrami et al. 1988), we applied the following rules in selecting the data. (1) When different
concentrations of fertilizers were applied in a single
study, the treatment used in the analysis was the one
resulting in the greatest difference from the control. If
the aim is the falsification of our predictions this is a
conservative practice. (2) When plants had been sampled repeatedly at different phenological stages, the
data used were those concerning mature fully expanded
leaves, which are believed to have more stable levels of
secondary compounds than expanding leaves. This
practice may lead to conservative estimates if plants
exhibit the inverse relationship between nitrogen
availability and allocation to carbon-based defenses
only during periods of growth (Lerdau et al. 1995). (3)
When secondary compounds were analyzed in different
parts of the same plant (e.g., leaves, stems, roots), we
used the data on foliar chemistry only. (4) When several
clones/seed lots of the same species were tested, and the
results of the treatments varied among clones, we randomly selected one clone per study for the analysis.
Our final database consisted of 211 comparisons
between concentrations of various CBSCs in fertilized
and control plants. For each comparison, an estimate
of the magnitude of the treatment effect (effect size, d)
was calculated as the difference between the means of
the experimental and control groups divided by the
pooled standard deviation, and weighted by a correction term eliminating small sample size bias (Gurevitch
& Hedges 1993). The mean effect sizes (d+) for each
class of CBSCs were calculated and compared using the
MetaWin statistical program (Rosenberg et al. 1997)
and the mixed effects model of meta-analysis (Gurevitch & Hedges 1993). The two major classes of CBSCs,
terpenoids and phenolics, demonstrated significantly
different responses to fertilization (Table 1). As predicted by different biosynthetic origins of the classes of
CBSCs, fertilizing reduced the concentrations of phenolic compounds but did not significantly affect those of
terpenoids. These differences were also observed in
studies simultaneously measuring the concentrations of
the two classes of compounds (e.g., Muzika 1993; Hartley et al. 1995; Iason et al. 1996; Lawler et al. 1997;
Honkanen et al. 1999). When we split the group of
phenolic compounds into phenylpropanoids and hydrolyzable tannins, the difference between the two
classes of CBSCs was again significant; as predicted,
only the concentrations of phenylpropanoids decreased
in response to fertilizing (Table 1).
Because phenylpropanoids themselves are structurally and biosynthetically a relatively heterogeneous
group, we also tested whether different classes of
phenylpropanoids differ in their responses to fertilization. We did not find significant heterogeneity among
classes of phenylpropanoids (Table 1). However, only
condensed tannins and lignin significantly responded to
fertilization whereas flavonoids and hydroxycinnamic
acids did not. Due to the relatively scanty data, the
136
E. Haukioja et al.
possibility of variation in responses of different classes
of phenylpropanoids to fertilizing cannot be excluded.
Discussion
The observed differences in behaviors of the three
classes of CBSCs cannot be directly predicted on the
basis of either the CNB or the GDB hypothesis.
Rather, the results of meta-analysis support our predictions made solely on the basis of the biosynthetic
origins of CBSCs. Trade-offs between growth and defense seem to be regularly detectable only in cases when
growth and defense directly compete over the same
precursor (phenylalanine). In that case, plants seem to
sequentially allocate resources to growth and defense,
as reflected in the tendency for condensed tannins to
accumulate in mature leaves (Feeny & Bostock 1968;
Faeth 1985; Baldwin et al. 1987; Ossipov et al. 1997). In
contrast, plants can combine active growth with high
concentrations of those classes of CBSCs which do not
directly compete with protein synthesis. For instance,
high concentrations of hydrolyzable tannins and of
some terpenoids occur primarily in young rapidly growing tissues, and their concentrations quickly drop when
the leaves mature (e.g., Ikeda et al. 1977; Crankshaw &
Langenheim 1981; Faeth 1985; Baldwin et al. 1987;
Potter & Kimmerer 1989; Ossipov et al. 1997).
Naturally, biosynthetic origin represents only one of
several potential differences among the three classes of
CBSCs, and other factors may contribute to their different responses to fertilization. In the following, four
proposed alternative explanations are discussed.
Reichardt et al. (1991) pointed out that some types
of CBSCs, particularly monoterpenoids, undergo rapid
turnover and that their static concentration is a poor
predictor of production rate. Therefore, the CNB and
GDB hypotheses may adequately predict the responses
of the end products of secondary metabolism, but they
may fail to predict the responses of compounds with
rapid turnover. However, at least some reported cases
of monoterpene turnover are artefects (Mihaliak et al.
1991) and many monoterpenes do not undergo rapid
turnover (Gershenzon et al. 1993). Therefore we tested
whether monoterpenes respond differently from higher
terpenoids which are known not to undergo rapid
turnover, and found no difference in responses to fertilization between monoterpenoids and terpenoids with
higher molecular weights (QB=0.497, P= 0.581).
Unlike phenolics, terpenoids in woody plants are
usually stored in complex multicellular compartments,
such as resin ducts, the construction of which might
also be limited by the availability of nitrogen. It has
therefore been suggested that the production of terpenoids may generally be limited more by the number
and size of storage compartments than by the availability of carbon for terpenoid synthesis (Björkman et al.
1991; Gershenzon 1994a, b). While this reasoning may
explain why terpenoids do not respond to fertilization,
it cannot account for differences in the responses of
foliage phenylpropanoids and hydrolyzable tannins.
CHEMOECOLOGY
None of these groups require complex storage compartments and are either stored in vacuoles or bound to cell
wall fractions (Strack 1997).
The absence of terpenoid responses to fertilization
has sometimes been attributed to terpene production
being under strong genetic control (e.g., Hanover 1966;
Merk et al. 1988; Muzika et al. 1989; Barton et al.
1991). However, substantial variation in monoterpene
production has been demonstrated between seasons
(Nerg et al. 1994) and with regard to the availability of
light (Gref & Tenow 1987) and water (Kainulainen et
al. 1992; Doran & Bell 1994). Moreover, several classes
of phenylpropanoids are also considered to be strongly
genetically controlled (e.g., McDougal & Parks 1986;
Yazdani & Leberton 1991; Orians et al. 1996) but
nevertheless show a significant response to fertilization.
Another explanation for different responses to fertilization might be found in differences in the energetic
costs of synthesizing various classes of CBSCs. However, terpenoids and hydrolyzable tannins occupy opposite ends of the cost range, with phenylpropanoids
intermediate (Gershenzon 1994b; Ossipov et al. 1997).
The fact that concentrations of the intermediately
costly class of CBSCs decrease after fertilization while
neither of the extremes is affected implies that fertilization responses cannot be explained in terms of resources or energetic costs only.
Summarizing, while several factors may contribute
to the lack of terpenoid response to fertilization, the
observed differences between responses of terpenoids
and phenolics and between those of phenylpropanoids
and hydrolyzable tannins are consistent with predictions based on their biosynthetic origin. Therefore, we
suggest the following modifications of the CNB and
GDB hypotheses. First, a negative correlation between
growth and synthesis of CBSCs in woody plants is most
likely to occur when the secondary compounds directly
share a common precursor with protein synthesis. Second, in addition to the availability of carbon 6s. nutrients, the regulation of their use must be crucial as well
(Koricheva et al. 1999). The trade-off between allocation to growth vs CBSCs holds; an important determinant, however, seems to be which CBSCs to combine
with active growth, not simply whether to allocate
carbon to growth or to CBSCs. Therefore, in woody
plants realistic hypotheses must explicitly allow both
sequential and simultaneous allocation to CBSCs and
to plant growth.
Acknowledgements
We want to thank Christer Björkman, Pirjo Elamo,
Riitta Julkunen-Tiitto, Kari Karhu, Antti Kause,
Markku Keinänen, Bill Mattson, Pekka Niemelä, Matti
Rousi and Jorma Tahvanainen for critical comments
on previous versions of the manuscript. G.R. Iason, P.
Kainulainen and D.W. Ross generously provided additional data not available in their articles. The study was
financed by the Academy of Finland and the Swedish
Council of Forestry and Agricultural Research.
Vol. 8, 1998
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Appendix
The plant species, plant developmental stage, compounds measured, methods employed and references for
the studies used in the meta-analysis. Compounds: TP
– total phenolics, CT – condensed tannins, HT –
hydrolyzable tannins; F – flavonoids, HCA – hydroxycinnamic acids, L – lignin, TT – total terpenes, MT
– monoterpenes, ST – sesquiterpenes, RA – resin
.
acids. Methods: FD – Folin-Denis method, FC –
Folin-Ciocalteu method, PB – Prussian Blue method,
PA – proanthocyanidin assay (butanol-HCl), VA –
vanillin assay, RM – rhodanine method, PI – potassium iodate method, SN – sodium nitrite method,
AH – acid hydrolysis, GC – gas chromatography,
HPLC – high performance liquid chromatography,
HPTLC – high performance thin-layer chromatography.
Vol. 8, 1998
Biosynthetic origin
Plant species
Developmental
stage
Compounds
measured
Methods
employed
Reference
Abies grandis
Abies grandis
Abies grandis
Acer saccharum
Alnus crispa
A6icennia germinans
Betula, pubescens ssp.
tortuosa
Betula, pubescens ssp.
tortuosa
Betula resinifera
Betula resinifera
Calluna 6ulgaris
Calluna 6ulgaris
seedling
seedling
seedling
seedling
juvenile
seedling
mature
MT, ST
TP, HCA, F
TP, F, HCA, MT, ST
CT, HT
TP, CT
TP, CT, HT
TP
GC
FC, HPLC
FC, HPLC, GC
PA, RM
FD, VA
FD, PA, PI
FD
Muzika et al. (1989)
Muzika & Pregitzer (1992)
Muzika (1993)
Kinney et al. (1997)
Reichardt et al. (1987a)
McKee (1995)
Tuomi et al. (1984)
juvenile
TP, CT
FD, PA
Ruohomäki et al. (1996)
juvenile
juvenile
mature
mature
TP, CT
CT
TP, CT, L
TP, CT, L
FD, VA
PA
PB, PA, AH
PB, PA, AH
Cornus florida
Eucalyptus tereticornis
Fagus syl6atica
Inga oerstediana
Laguncularia racemosa
Liriodendron tulipifera
Miconia barbiner6is,
M. gracilis,
M. ner6osa
Pinus banksiana
Pinus syl6estris
Pinus syl6estris
Pinus syl6estris
Pinus syl6estris
seedling
seedling
mature
seedling
seedling
seedling
cuttings
TP,
TP,
TP
TP,
TP,
TP,
TP,
FD, PA, SN
FC, PA, AH
FD
FD, PA
FD, PA, PI
FD, PA, SN
FD, PA, PI
Bryant et al. (1987a)
Bryant et al. (1993)
Iason et al. (1993)
Iason & Hester (1993);
Hartley & Gardner (1995);
Hartley et al. (1995)
Dudt & Shure (1994)
Lawler et al. (1997)
Balsberg-Påhlsson (1992)
Nichols-Orians (1991)
McKee (1995)
Dudt & Shure (1994)
Denslow et al. (1987)
juvenile
mature
mature
juvenile
seedling
MT
MT, ST
TP
RA
TP, MT, RA
GC
GC
FD
GC
FC, GC
mature
seedling
cuttings
TP, MT, RA
TP, CT
TP, CT, HT
FC, GC
FD, PA
FD, PA, PI
McCullough & Kulman (1991)
Hiltunen et al. (1975)
Rousi et al. (1987)
Björkman et al. (1991)
Holopainen et al. (1995);
Kainulainen et al. (1996)
Honkanen et al. (1999)
Ross & Berisford (1990)
Denslow et al. (1987)
seedling
juvenile
seedling
seedling,
juvenile
seedling
mature?
seedling
mature
seedling
cuttings from
mature plants
cuttings from
mature plants
seedlings
cuttings
F
TP, CT, F
CT, F
TP, TT
GC
FD, PA, HPLC
PA, HPTLC
FC, GC
Reichardt et al. (1991)
Bryant et al. (1987b)
Kinney et al. (1997)
Joseph et al. (1991, 1993)
TP, CT, HT
TP, CT
CT, HT
TP, CT
TP, CT, HT
TP, CT
FD, PA, PI
FC, PA
PA, RM
FD, PA
FD, PA, PI
FD, PA, VA
Hunter & Schultz (1995)
Glyphis & Puttick (1989)
Kinney et al. (1997)
Feller (1995)
McKee (1995)
Bryant (1997)
TP
FD
Waring & Price (1988)
CT, F
F
PA, HPLC, GC
HPLC
Julkunen-Tiitto et al. (1993)
Hakulinen et al. (1995)
Pinus syl6estris
Pinus taeda
Piper arieianum,
P. culebranum,
P. urostachyum
Populus balsamifera
Populus tremuloides
Populus tremuloides
Pseudotsuga menziesii
Quercus prinus, A. rubra
Quercus coccifera
Quercus rubra
Rhizophora mangle
Rhizophora mangle
Salix alaxensis
Salix lasiolepis
Salix myrsinifolia
Salix myrsinifolia
CT, HT
CT, L
CT
CT, HT
CT, HT
CT, HT
139