Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2004? 2004
27810471054
Original Article
Trade-off between photosynthesis and protection
T. Takashima
et al.
Plant, Cell and Environment (2004) 27, 1047–1054
Photosynthesis or persistence: nitrogen allocation in leaves
of evergreen and deciduous Quercus species
T. TAKASHIMA, K. HIKOSAKA & T. HIROSE
Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980–8578, Japan
ABSTRACT
Photosynthetic nitrogen use efficiency (PNUE, photosynthetic capacity per unit leaf nitrogen) is one of the most
important factors for the interspecific variation in photosynthetic capacity. PNUE was analysed in two evergreen
and two deciduous species of the genus Quercus. PNUE
was lower in evergreen than in deciduous species, which
was primarily ascribed to a smaller fraction of nitrogen
allocated to the photosynthetic apparatus in evergreen species. Leaf nitrogen was further analysed into proteins in the
water-soluble, the detergent-soluble, and the detergentinsoluble fractions. It was assumed that the detergentinsoluble protein represented the cell wall proteins. The
fraction of nitrogen allocated to the detergent-insoluble
protein was greater in evergreen than in deciduous leaves.
Thus the smaller allocation of nitrogen to the photosynthetic apparatus in evergreen species was associated with
the greater allocation to cell walls. Across species, the fraction of nitrogen in detergent-insoluble proteins was positively correlated with leaf mass per area, whereas that in
the photosynthetic proteins was negatively correlated.
There may be a trade-off in nitrogen partitioning between
components pertaining to productivity (photosynthetic
proteins) and those pertaining to persistence (structural
proteins). This trade-off may result in the convergence of
leaf traits, where species with a longer leaf life-span have a
greater leaf mass per area, lower photosynthetic capacity,
and lower PNUE regardless of life form, phyllogeny, and
biome.
Key-words : cell wall proteins; leaf life-span; leaf mass per
area; nitrogen partitioning; photosynthetic capacity; photosynthetic nitrogen use efficiency; protection; ribulose-1, 5bisphosphate carboxylase/ oxygenase.
INTRODUCTION
Within a species, leaf nitrogen is a determinant of photosynthetic capacity. There is a strong, positive correlation
between photosynthetic capacity and the nitrogen content
per unit leaf area. This may reflect the fact that about half
Correspondence: Hikosaka, Kouki. Fax: + 81 22 217 6699; e-mail:
[email protected]
© 2004 Blackwell Publishing Ltd
of leaf nitrogen is allocated to the photosynthetic apparatus
(Evans 1989; Evans & Seemann 1989). However, dependence of photosynthetic capacity on the nitrogen content
varies considerably among species (Field & Mooney 1986;
Evans 1989). Since the leaf nitrogen content has no clear
trend among species (Reich, Walters & Ellsworth 1991;
Reich et al . 1999), photosynthetic nitrogen use efficiency
(PNUE, photosynthetic capacity per unit nitrogen) may be
the most important factor for the interspecific difference in
photosynthetic capacity.
Many authors have studied PNUE as an inherent trait of
species to be related to their ecological characteristics. A
lower PNUE tends to be found in evergreen than in deciduous species (Field & Mooney 1986; Reich et al . 1995), in
stress-tolerant species (Chazdon & Field 1987; Poorter,
Remkes & Lambers 1990), in late-successional species
(Ellsworth & Reich 1996), in species that inhabit higher
altitudes (Westbeek et al . 1999; Hikosaka et al . 2002), and
in species with a long leaf life-span (Reich et al . 1991).
In studies of physiological mechanisms for the interspecific variation in PNUE, ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been focused upon because it is a
key enzyme of photosynthesis (Farquhar, von Caemmerer
& Berry 1980) and requires a large amount of nitrogen in
leaves (15–30% of leaf nitrogen; Evans 1989; Evans & Seemann 1989). It has been shown that species with a higher
PNUE allocate more nitrogen to RuBPCase (Hikosaka
et al . 1998; Poorter & Evans 1998; Westbeek et al . 1999;
Ripullone et al . 2003). Thus nitrogen allocation to photosynthetic proteins is one of responsible factors for interspecific variation in PNUE.
A question arises, then: why do some species allocate less
nitrogen to photosynthetic proteins to have low PNUE? As
nitrogen is an element that limits plant growth in many
natural and agricultural ecosystems, the ecological significance of low PNUE is a puzzling question. It has been
hypothesized that low PNUE species compensate for their
low productivity by a long leaf life-span (Small 1972; Berendse & Aerts 1987; Aerts & Chapin 2000). To persist for
a long time, leaves may need to be physically tough (Reich
et al . 1991; Wright & Cannon 2001). Cell walls play an
important role in mechanical toughness of plant tissues. It
is known that cell walls accumulate a significant amount of
nitrogenous compounds up to 10% of cell wall materials
(Lamport & Northcote 1960; Lamport 1965; Reiter 1998).
We may hypothesize that species with longer leaf life-span
1047
1048 T. Takashima et al.
invest more nitrogen in cell walls to increase leaf toughness
at the expense of PNUE.
In the present study, we examined nitrogen allocation in
evergreen and deciduous species that belong to the genus
Quercus . First we studied nitrogen allocation to photosynthetic proteins as has been done in previous studies (e.g.
Hikosaka et al . 1998; Poorter & Evans 1998). Next we
devised a new approach, in which leaf proteins were
divided into three fractions; water-soluble, detergentsoluble, and detergent-insoluble. The water-soluble fraction
includes soluble enzymes in stroma and cytosol (Evans &
Seemann 1989). The detergent-soluble fraction includes
membrane-associated proteins (Evans & Seemann 1989).
We assumed the detergent-insoluble fraction to represent
cell wall proteins, which are further assumed to contribute
to leaf mechanical toughness. We tested the hypothesis that
evergreen species have a lower PNUE with a larger content
of cell wall proteins than deciduous species.
MATERIALS AND METHODS
The genus Quercus includes both evergreen and deciduous
species. They are important tree species in temperate forests
in Asia (Miyawaki 1987). We used two evergreen species,
Quercus acuta and Q. glauca , and two deciduous species,
Q. serrata and Q. crispula . All four are tall trees that can
dominate in temperate forests. Deciduous species extend
to higher latitudes than evergreen species but they coexist
in ecotonal areas between the warm and the cool temperate
region in Japan (Kurosawa, Tateishi & Kajita 1995).
Seeds were collected in autumn 2000 at the Botanical
Garden of Tohoku University (35∞15¢ N, 140∞51¢ E) except
for Q. crispula that was collected in a forest in Aomori
Prefecture (40∞30¢ N, 140∞56¢ E). They were stored in a
refrigerator until germination next spring (2001). Plants
were grown at the experimental garden of Tohoku University (35∞15¢ N, 140∞51¢ E). Each plant was transplanted into
a 1 L pot filled with washed river sand. Two growth irradiances and two nutrient availabilities were applied to obtain
a variation in the leaf nitrogen content; high- and low-light
were 90% (under a transparent plastic sheet) and 30%
(plus neutral shading with shade cloth) of full sunlight,
respectively, and high- and low-nutrient availability were
created by adding 20 mL of the commercial nutrient solution (Hyponex, N : P : K = 5 : 10 : 5; Murakami-bussan,
Kamigori, Japan) that contained 35 and 3.5 mM nitrogen,
respectively.
Photosynthetic rates were determined for fully expanded
young leaves with an open gas exchange system (Li-6400;
LiCor, Lincoln, NE, USA). In August, when the photosynthetic capacity was highest, photosynthetic rates were measured at a leaf temperature of 25 ∞C, photosynthetic photon
flux density of 2000 mmol m-2 s-1, and vapour pressure deficit of less than 1 kPa. First, photosynthetic rates were
determined at air CO2 partial pressure of 36 Pa (regarded
as photosynthetic capacity) and then at various air CO2
partial pressures.
From leaves used for photosynthetic measurements, leaf
discs, 1 cm in diameter, were punched out excluding midrib.
Three of them were dried at 70 ∞C for more than 48 h and
used for determination of leaf mass per area (LMA) and
nitrogen content (NC analyser; Shimadzu, Kyoto, Japan).
Other discs were frozen in liquid nitrogen and stored at
-80 ∞C.
Contents of chlorophyll (chl) and RuBPCase were determined from the frozen leaf discs. One leaf disc was powdered in liquid nitrogen in a mortar with a pestle and
homogenized in 1 mL of 100 mM Na-phosphate buffer
(pH 7.5) with 0.4 M sorbitol, 2 mM MgCl2, 10 mM NaCl,
5 mM iodo acetate, 1% polyvinylpyrroridone, 5 mM phenylmethyl sulfonyl fluoride, and 5 mM dithiothreitol. The
homogenate was filtered with 20 mm mesh. The chl concentration in the filtrate was determined with 80% acetone
(Porra, Thompson & Kriedemann 1989). The RuBPCase
concentration was determined according to Hikosaka et al .
(1998). The filtrate was applied to sodium dodecyl sulfate
(SDS; a detergent) polyachrylamide gel electrophoresis.
The gel was stained with Coomassie Brilliant Blue R-250.
The band of the large subunit of RuBPCase was extracted
with formamide for spectrophotometric determination of
RuBPCase. Calibration curves were obtained with RuBPCase purified from Spinacia oleracea .
The water-soluble, SDS-soluble, and SDS-insoluble fractions were isolated from another leaf disc. The leaf disc was
homogenized in 1 mL of the phosphate buffer as mentioned above. The mortar was washed with 3 mL of the
phosphate buffer, which was added to the homogenate.
The homogenate was centrifuged at 15 000 g for 30 min
and the supernatant was regarded as the water-soluble
fraction. The phosphate buffer that contained 3% SDS was
added to the pellet and heated at 90 ∞C for 5 min. The
mixture was centrifuged at 4500 g for 10 min. This procedure was repeated four times. The supernatants obtained
through this process were collected (SDS-soluble fraction).
The final pellet was regarded as the SDS-insoluble fraction.
Soluble proteins were precipitated with 10% (water-soluble) or 20% (SDS-soluble) trichloroacetic acid (TCA) and
washed with ethanol. After hydrolysis of the precipitated
proteins by 0.316 mmol Ba(OH)2 with 200 mL water in an
autoclave (120 ∞C, 0.12 MPa) for 15 min, protein content in
each fraction was determined with the ninnhydrin method
(McGrath 1972). Calibration curves were made with bovine
serum albumin.
The photosynthetic apparatus was divided into three categories: (1) RuBPCase; (2) bioenergetics (other Calvin
cycle enzymes, ATP synthase, and electron carriers); and
(3) light-harvesting (photosystem I and II) (Hikosaka &
Terashima 1995; Niinemets & Tenhunen 1997). Nitrogen in
RuBPCase (N r) was calculated assuming that nitrogen concentration in RuBPCase is 16% (Hikosaka & Terashima
1995). Nitrogen in bioenergetics (N b) was estimated from
gas exchange characteristics. The maximum rate of electron
transport in chloroplasts (Jmax) was determined from CO2
response curve of photosynthesis according to a biochemical model of photosynthesis (Farquhar et al. 1980). We
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1047–1054
Trade-off between photosynthesis and protection 1049
assumed that nitrogen in bioenergetics is proportional to
Jmax, where the ratio of Jmax to the cytochrome f content
is 156 mmol mol-1 s-1 (Niinemets & Tenhunen 1997) and
nitrogen in bioenergetics per unit cytochrome f is
9.53 mol mmol-1 (Hikosaka & Terashima 1995). Nitrogen in
light harvesting (N h) was calculated assuming 37.1 mol
mol-1 chl (Evans & Seemann 1989). Nitrogen in the watersoluble, SDS-soluble, and SDS-insoluble fractions was
estimated assuming 16% nitrogen in proteins.
RESULTS
In each species, photosynthetic capacity (Pmax, the lightsaturated rate of photosynthesis at 25 ∞C and 36 Pa CO2
partial pressure) was strongly correlated with the leaf nitrogen content per unit area (N L) (Fig. 1a). These correlations
were not affected by growth conditions in each species
(P > 0.05, ANCOVA). They were not different within each
leaf habit (deciduous or evergreen) but were different
between different leaf habits: evergreen species had a significantly lower Pmax at a given N L than deciduous species
(Table 1). Photosynthetic nitrogen use efficiency (Pmax/N L)
was higher in deciduous (mean and standard deviation
were 135 ± 17 mmol mol-1 s-1 for Q. serrata and 134 ± 16 for
Q. crispula ) than in evergreen species (83 ± 23 for Q. acuta
and 85 ± 9 for Q. glauca ).
Pmax was strongly correlated with the RuBPCase content
in each species (Fig. 1b) as well as with N L. Evergreen species had a significantly lower Pmax especially at higher RuBPCase contents (Table 1). However, the difference between
evergreen and deciduous species was smaller in Pmax–RuBPCase than in Pmax–N L relationships (Fig. 1), which indicates that allocation of nitrogen to RuBPCase is a primary
factor for the difference in the Pmax–N L relationship.
Figure 2 shows nitrogen contents in photosynthetic proteins. In each species, the content of RuBPCase nitrogen
(N r) was positively correlated with N L irrespective of
growth conditions (ANCOVA, P > 0.05) (Fig. 2a). N r was significantly higher in deciduous than in evergreen species
when compared at a common N L (Table 1). Similar tenden-
cies were observed for the relationship between the nitrogen content in bioenergetics (N b) and N L (Fig. 2b; Table 1).
The relationship between nitrogen in light harvesting (N h)
and N L differed depending on growth irradiance: leaves
grown at the low irradiance had a higher content of chl and
thus N h (Fig. 2c & d). When compared at the same growth
irradiance, deciduous species had a significantly higher N h
at a given N L (Table 1). Owing to the higher N h, the nitrogen content in the photosynthetic apparatus (N r + N b + N h)
was slightly higher in leaves grown at the low irradiance
(data not shown).
When N b and N h (high and low growth irradiance separately) were plotted against N r, there was no significant
difference in the regression line among species (ANOVA,
P > 0.05; data not shown), suggesting that four species had
a similar nitrogen allocation within the photosynthetic
apparatus.
Figure 3 shows allocation of nitrogen to three protein
fractions. The content of water-soluble proteins was positively correlated with N L across different growth conditions
(Fig. 3a). Deciduous species had significantly higher contents of water-soluble proteins than evergreen species when
compared at the same N L (Table 1). The content of SDSsoluble proteins were also correlated with N L (Fig. 3b) but
there was no significant difference among the four species
(Table 1). The content of SDS-insoluble proteins was relatively stable against N L in each species (Fig. 3c) with
evergreen species having a significantly higher content of
SDS-insoluble proteins (Table 1). No significant difference
was found in total protein (water-soluble + SDS-soluble +
SDS-insoluble) between species (Fig. 3d). The total protein
accounted for 71–77% of leaf nitrogen.
Figure 4 shows allocation and use of nitrogen as a function of LMA. Evergreen leaves had a larger LMA and a
lower PNUE (Pmax/N L) (Fig. 4a). Across evergreen and
deciduous species, the fraction of leaf nitrogen allocated to
RuBPCase decreased with increasing LMA (Fig. 4b) while
that to SDS-insoluble proteins increased (Fig. 4c).
Figure 5 summarizes nitrogen allocation in leaves of each
species. Results obtained from leaves grown under high
Figure 1. Photosynthetic capacity as a
function of the content of leaf nitrogen (a)
and ribulose-1,5-bisphosphate carboxylase
(RuBPCase) (b) per unit leaf area in Quercus acuta (), Q. glauca (), Q. serrata (),
and Q. crispula ().
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1047–1054
1050 T. Takashima et al.
Table 1. Regression and correlation coefficients
Slope
Pmax–NL
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Pmax–RuBPCase content
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Nr–NL
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Ne–NL
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Nh–NL in high light
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Intercept
Correlation
coefficient (r)
P < 0.05
109 a
109 a
173 a
172 a
-2.00 a
-2.35 a
-2.96 b
-3.18 b
0.85
0.79
0.92
0.92
P < 0.01
4.61 a
4.37 a
8.35 b
7.61 ab
1.88 a
2.13 a
-1.75 b
-0.36 b
0.84
0.75
0.96
0.89
0.231
0.219
0.234
0.231
P < 0.001
-0.0060 a
-0.0052 a
-0.0015 b
-0.0018 b
0.91
0.81
0.95
0.93
0.0905
0.0819
0.0908
0.0912
P < 0.001
-0.0043 a
-0.0036 a
-0.0023 b
-0.0022 b
0.90
0.95
0.89
0.96
0.110
0.134
0.124
0.099
P < 0.001
-0.0021 a
-0.0047 a
-0.0012 b
-0.0009 b
0.88
0.92
0.95
0.92
ns
ns
ns
Nh–NL in low light
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Water soluble N–NL
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
SDS-soluble N–NL
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
SDS-insoluble N–NL
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Total protein N–NL
Among four species
Q. acuta
Q. glauca
Q. serrata
Q. crispula
Correlation
coefficient (r)
Slope
Intercept
ns
0.126
0.120
0.133
0.141
P < 0.001
0.0004 a
0.0011 a
0.0024 b
0.0015 b
0.95
0.76
0.91
0.90
0.399
0.272
0.319
0.400
P < 0.01
-0.0078 a
0.0035 ab
0.0042 bc
-0.0024 c
0.62
0.32
0.84
0.88
0.142
0.024
0.293
0.230
ns
0.0109
0.0235
0.0010
0.0037
0.40
0.04
0.74
0.69
ns
ns
ns
-0.0278
-0.0555
-0.0233
-0.0137
P < 0.001
0.0154 a
0.0180 a
0.0078 b
0.0072 b
ns
ns
0.0184
0.0473
0.0141
0.0079
0.513
0.240
0.589
0.616
-0.42
-0.49
-0.62
-0.27
0.62
0.20
0.88
0.85
Difference in regression coefficients among four species was tested with the analysis of covariance ( ANCOVA) according to Sokal & Rohlf
(1981). Significance for intercepts was tested only when there was no significant difference for slopes. When there was a significant difference
among four species, significance between two species (shown as alphabets) was tested according to Rice (1989) (Sequential Bonferroni test, a
< 0.05) except for the relationship between water-soluble nitrogen and leaf nitrogen content (P < 0.05). Significance for intercepts was tested
only when there was no significant difference for slopes.
light and high nutrient conditions are shown. ‘Other protein
nitrogen’ was calculated as nitrogen in the water- and SDSsoluble protein minus nitrogen in the photosynthetic
apparatus. ‘Other nitrogen’ was calculated as the residual.
Nitrogen allocated to the photosynthetic apparatus was
smaller in evergreen species (about 30%) than in deciduous
species (40%) and the difference was partly counterbalanced by greater allocation of nitrogen to SDS-insoluble
proteins.
DISCUSSION
Partitioning of leaf nitrogen
PNUE was lower in evergreen than in deciduous species
(Figs 1a & 4a), which is in accord with earlier studies (Field
& Mooney 1986; Reich et al. 1995; Ripullone et al. 2003).
Lower PNUE in evergreen species was associated with a
smaller allocation of nitrogen to the photosynthetic apparatus (Fig. 2). Similar trends have been observed across
species with different PNUE (Hikosaka et al. 1998; Poorter
& Evans 1998; Westbeek et al. 1999; Ripullone et al. 2003).
Thus the present result confirms the generality of the contribution of nitrogen allocation to the interspecific difference in PNUE.
We divided leaf proteins into three fractions (Fig. 3). The
water-soluble fraction has been termed as ‘soluble protein’
in previous studies, of which about one-half was represented by RuBPCase (Terashima & Evans 1988; Hikosaka
& Terashima 1996). This was also the case in the present
study (Figs 2a & 3a). The amount of the water-soluble protein was greater in deciduous species (Fig. 3a; Table 1). The
SDS-soluble fraction was presumed to mostly consist of
membrane-associated proteins such as thylakoid components (Evans & Seemann 1989). This fraction was not significantly different between deciduous and evergreen
species (Fig. 3b). Then the smaller content of photosynthetic proteins in evergreen species (Fig. 2) suggests that
evergreens invest more nitrogen in the SDS-soluble proteins that are not involved in photosynthesis.
A remarkable difference was found in SDS-insoluble
proteins: evergreen species allocated two-fold more nitrogen to SDS-insoluble proteins than deciduous species
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1047–1054
Trade-off between photosynthesis and protection 1051
Figure 2. The content of nitrogen allocated to RuBPCase (a), bioenergetics (b),
and light-harvesting in high-light grown
leaves (c) and in low-light grown leaves (d).
Symbols are as in Fig. 1.
(Fig. 3c). It is surprising that some leaves of evergreen species invest nitrogen into the SDS-insoluble proteins in an
amount that is comparable with that invested into RuBPCase (Fig. 4). Since water and SDS remove soluble and
membrane-associated proteins, we assumed the SDS-insoluble fraction to represent cell wall proteins that are tightly
bound to cell walls (Reiter 1998). The amount of cell wall
proteins presented here would be minimal because the SDS
Figure 3. The content of protein nitrogen
in the water-soluble (a), the SDS-soluble
(b), and the SDS-insoluble fraction, and the
content of total protein nitrogen (d). Symbols are as in Fig. 1.
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1047–1054
1052 T. Takashima et al.
acids (8.5%, Evans & Seemann 1989; 15%, Chapin et al.
1986; 5–10%, Chapin 1989). Chapin et al. (1986) showed
that several percent of N was invested in free amino acids
and lipids. Thus these compounds may explain about 20%
of leaf nitrogen. Furthermore, some species are known to
accumulate secondary compounds such as alkaloids and
cyanogenic glycosides (Burns, Gleadow & Woodrow 2002).
However, the accumulation is species-dependent and there
seems no report for the species that we studied. It should
be noted that the protein content might have been underestimated in the present study. Proteins that are soluble to
TCA and ethanol were not determined. It is known that
cell wall proteins are difficult to extract completely: 10–
20% of cell wall proteins have been suggested to be hardly
solubilized by KOH at 25 ∞C (Lamport 1965). Although
hydrolysis by Ba(OH)2, which we used in the present study,
is considered to be more effective for extraction than KOH
solution, it is probable that some of the tightly bound proteins was not extracted. Furthermore, the ninhydrin
method might underestimate proline and hydroxyproline
(Yemm & Cocking 1955), which are rich in cell wall proteins (Lamport 1965; Showalter 1993). These proteins, however, would explain a few percent of leaf nitrogen.
Ecological significance
Nitrogen allocation to the SDS-insoluble fraction was
strongly correlated with LMA irrespective of species and
growth conditions (Fig. 4c). This is in accord with our recent
study showing a positive correlation between the contents
of cell wall proteins and LMA across leaves of Polygonium
cuspidatum plants germinated at different times (Onoda,
Hikosaka & Hirose 2004). Terashima et al. (1995) observed
that leaf thickness was similar in evergreen and deciduous
Figure 4. Photosynthetic nitrogen use efficiency (photosynthetic
capacity per unit nitrogen) (a), the fraction of RuBPCase nitrogen
(b), and the fraction of the SDS-insoluble protein nitrogen (c) as
a function of leaf mass per area. Symbols are as in Fig. 1.
treatment might remove proteins that were weakly associated with cell walls. These results suggest that leaves invest
a considerable amount of nitrogen in cell walls and that
evergreen species allocate more nitrogen to cell walls than
deciduous species, which necessarily decreases their PNUE
(Fig. 4).
In the present study, 71–77% of leaf nitrogen was found
to be allocated to proteins (Fig. 3d). This value seems
slightly lower than that reported for several Alaskan tundra
species (75–80% for Eriophorum vafinatum , Chapin,
Shaver & Kedrowski 1986; 75–88% for several species with
different life forms, Chapin 1989). Where is nitrogen allocated to other than proteins? Several authors suggested
that a significant amount of nitrogen is invested in nucleic
Figure 5. Nitrogen allocation in leaves grown at high light and
high nitrogen availability. Mean of three to four leaves.
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1047–1054
Trade-off between photosynthesis and protection 1053
Quercus species, while the cell wall thickness of mesophyll
cells was greater in an evergreen than in a deciduous Quercus species. Thus the higher LMA in evergreen species is
attributable to a higher leaf density resulting from a greater
cell wall thickness. Variation in cell wall thickness might
have produced the strong correlation between LMA and
nitrogen allocation to SDS-insoluble proteins.
Why did evergreen species have a greater amount of cell
wall proteins? It has been suggested that the greater LMA
contributes to mechanical toughness of leaves (Reich et al.
1991; Wright & Cannon 2001). Mechanical protection is
important for maintaining leaves for a long period (Coley,
Bryant & Chapin 1985; Reich et al. 1991). For example,
Coley (1983) showed that leaf toughness was the trait that
was most highly correlated with levels of herbivory across
46 species in a tropical forest. Evergreen Quercus species
must make their leaves tough enough to maintain leaves
for longer than 2 years (Hikosaka 2004). On the other hand,
leaves of deciduous species have a shorter leaf life-span (5–
6 months at the site of seed collection). They can allocate
less nitrogen for leaf toughness and more for photosynthesis to have a high PNUE (Fig. 4).
The present results may explain a convergence of leaf
traits across species. Regardless of life form, phyllogeny,
and biome, species with a shorter leaf life-span have a
smaller LMA, higher nitrogen concentration per unit leaf
mass, higher Pmax, and higher PNUE (Reich et al. 1991,
1999; Reich, Walters & Ellsworth 1997; Wright, Reich &
Westoby 2001; Wright et al. 2004). Species with a smaller
LMA invest more nitrogen in the photosynthetic apparatus
and thus have higher growth rates (Poorter et al. 1990),
leading to a competitive success at high nutrient availabilities. Species with a greater LMA, on the other hand, invest
more nitrogen in cell walls, leading to reduction in the
amount of photosynthetic proteins and thus to slow growth
rates. However, they have a longer leaf life-span and mean
residence time of nitrogen in leaves, which may be advantageous at low nutrient availabilities (Aerts & van der Peijl
1993; Aerts & Chapin 2000). The convergence of leaf traits
across species may result from a trade-off in nitrogen partitioning within the leaf: one for photosynthetic capacity
and the other for persistence of leaves.
ACKNOWLEDGMENTS
We thank K Sato for kind help in growing plants and Y
Onoda and Y Yasumura for comments. This study was supported in part by grants from Japan Ministry of Education,
Science, Sports, and Culture.
REFERENCES
Aerts R. & Chapin F.S. III (2000) The mineral nutrition of wild
plants revisited: a re-evaluation of processes and patterns.
Advances in Ecological Research 30, 1–67.
Aerts R. & van der Peijl M.J. (1993) A simple model to explain
the dominance of low-productive perennials in nutrient-poor
habitats. Oikos 66, 144–147.
Berendse F. & Aerts R. (1987) Nitrogen use efficiency: a biologically meaningful definition? Functional Ecology 1, 293–296.
Burns A.E., Gleadow R.M. & Woodrow I.E. (2002) Light alters
the allocation of nitrogen to cyanogenic glycosides in Eucalyptus
cladocalyx. Oecologia 133, 288–294.
Chapin F.S. III (1989) The cost of tundra plant structures: evaluation of concepts and currencies. American Naturalist 133, 1–19.
Chapin F.S. III, Shaver G.R. & Kedrowski R.A. (1986) Environmental controls over carbon, nitrogen and phosphorus fraction
in Eriophorum vaginatum in Alaskan tussock tundra. Journal of
Ecology 74, 167–195.
Chazdon R.L. & Field C.B. (1987) Determinants of photosynthetic
capacity in six rainforest Piper species. Oecologia 73, 222–230.
Coley P.D. (1983) Herbivory and defensive characteristics of tree
species in a low land tropical forest. Ecological Monographs 53,
209–233.
Coley P.D., Bryant J.P. & Chapin F.S. III (1985) Resource availability and plant anti-herbivore defense. Science 230, 895–899.
Ellsworth D.S. & Reich P.B. (1996) Photosynthesis and leaf nitrogen in five Amazonian tree species during early secondary succession. Ecology 77, 581–594.
Evans J.R. (1989) Photosynthesis and nitrogen relationships in
leaves of C3 plants. Oecologia 78, 9–19.
Evans J.R. & Seemann J.R. (1989) The allocation of protein nitrogen in the photosynthetic apparatus: costs, consequences and
control. In Photosynthesis (ed. W.R. Brigs), pp. 183–205. Alan
R. Liss, New York, USA.
Farquhar G.D., von Caemmerer S. & Berry J.A. (1980) A biochemical model of photosynthetic CO2 assimilation in leaves of
C3 species. Planta 149, 78–90.
Field C. & Mooney H. (1986) The photosynthesis-nitrogen relationship in wild plants. In On the Economy of Form and Function (ed. T.J. Givnish), pp. 25–55. Cambridge University Press,
Cambridge, UK.
Hikosaka K. (2004) Leaf canopy as a dynamic system. Ecophysiology and optimality in leaf turnover. Annals of Botany (in press).
Hikosaka K., Hanba Y.T., Hirose T. & Terashima I. (1998) Photosynthetic nitrogen-use efficiency in woody and herbaceous
plants. Functional Ecology 12, 896–905.
Hikosaka K., Nagamatsu D., Ishii H.S. & Hirose T. (2002) Photosynthesis-nitrogen relationships in species at different altitudes
on Mount Kinabalu, Malaysia. Ecological Research 17, 305–313.
Hikosaka K. & Terashima I. (1995) A model of the acclimation of
photosynthesis in the leaves of C3 plants to sun and shade with
respect to nitrogen use. Plant, Cell and Environment 18, 605–
618.
Hikosaka K. & Terashima I. (1996) Nitrogen partitioning among
photosynthetic components and its consequence in sun and
shade plants. Functional Ecology 10, 335–343.
Kurosawa T., Tateishi Y. & Kajita T. (1995) Flora of Aobayama,
wild vascular plants in the botanical garden of Tohoku University, Sendai, Japan. Ecological Review 23, 111–170.
Lamport D.T.A. (1965) The protein component of primary cell
walls. Advances in Botanical Research 2, 151–218.
Lamport D.T.A. & Northcote D.H. (1960) Hydroxyproline in primary cell walls of higher plants. Nature 188, 665–666.
McGrath R. (1972) Protein measurement by ninhydrin determination of amino acids released by alkaline hydrolysis. Analytical
Biochemistry 49, 95–102.
Miyawaki A. (1987) The Vegetation of Japan, Vol. 8. Tohoku. Shibundo Publishers, Tokyo, Japan.
Niinemets Ü & Tenhunen J.D. (1997) A model separating leaf
structural and physiological effects on carbon gain along light
gradients for the shade tolerant species Acer saccharum. Plant,
Cell and Environment 20, 845–866.
Onoda Y., Hikosaka K. & Hirose T. (2004) Allocation of nitrogen
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1047–1054
1054 T. Takashima et al.
to cell walls decreases photosynthetic nitrogen-use efficiency.
Functional Ecology 18, 419–425.
Poorter H. & Evans J.R. (1998) Photosynthetic nitrogen-use efficiency of species that differ inherently in specific area. Oecologia
116, 26–37.
Poorter H., Remkes C. & Lambers H. (1990) Carbon and nitrogen
economy of 24 wild species differing in relative growth rate.
Plant Physiology 94, 621–627.
Porra R.J., Thompson W.A. & Kriedemann P.E. (1989) Determination of accurate extinction coefficients and simultaneous
equations for assaying chlorophyll a and b extracted with four
different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica
et Biophysica Acta 975, 384–394.
Reich P.B., Ellsworth D.S., Walters M.B., Vose J.M., Gresham
C., Volin J.C. & Bowman W.D. (1999) Generality of leaf
trait relationships: a test across six biomes. Ecology 80, 1955–
1969.
Reich P.B., Kloeppel B.D., Ellsworth D.S. & Walters M.B. (1995)
Different photosynthesis-nitrogen relations in deciduous hardwood and evergreen coniferous tree species. Oecologia 104, 24–
30.
Reich P.B., Walters M.B. & Ellsworth D.S. (1991) Leaf lifespan as
a determination of leaf structure and function among 23 Amazonian tree species. Oecologia 86, 16–24.
Reich P.B., Walters M.B. & Ellsworth D.S. (1997) From tropics to
tundra: Global convergence in plant functioning. Proceedings of
Natural Academy of Science of the USA 94, 13730–13734.
Reiter W.D. (1998) The molecular analysis of cell wall components. Trends in Plant Science 3, 27–32.
Rice W.R. (1989) Analyzing tables of statistical tests. Evolution
43, 223–225.
Ripullone F., Grassi G., Lauteri M. & Borghetti M. (2003) Photosynthesis-nitrogen relationships: interpretation of different patterns between Pseudotsuga menziesii and Populus ¥
eueoamericana in a mini-stand experiment. Tree Physiology 23,
137–144.
Showalter A.M. (1993) Structure and function of plant cell wall
proteins. Plant Cell 5, 9–23.
Small E. (1972) Photosynthetic rates in relation to nitrogen recycling as an adaptation to nutrient deficiency in peat bog plants.
Canadian Journal of Botany 50, 2227–2233.
Sokal R.R. & Rohlf F.J. (1981) Biometry, 2nd edn. Freeman, New
York, USA.
Terashima I. & Evans J.R. (1988) Effects of light and nitrogen
nutrition on the organization of the photosynthetic apparatus in
spinach. Plant and Cell Physiology 29, 143–155.
Terashima I., Ishibashi M., Ono K. & Hikosaka K. (1995) Three
resistances to CO2 diffusion: leaf-surface water, intercellular
spaces and mesophyll cells. In: Photosynthesis: from Light to
Biosphere, Vol. V (ed. P. Mathis), pp. 537–542. Kluwer Academic Press, Dordrecht, The Netherlands.
Westbeek H.M.H., Pons T.L., Cambridge M.L. & Atkin O.K.
(1999) Analysis of differences in photosynthetic nitrogen use
efficiency of alpine and lowland Poa species. Oecologia 120, 19–
26.
Wright I.J. & Cannon K. (2001) Relationships between leaf
lifespan and structural defences in a low-nutrient, sclerophyll
flora. Functional Ecology 15, 351–359.
Wright I.J., Reich P.B. & Westoby M. (2001) Strategy shifts in leaf
physiology, structure and nutrient content between species of
high- and low-rainfall and high- and low-nutrient habitats. Functional Ecology 15, 423–434.
Wright I.J., Reich P.B., Westoby M., et al. (2004) The leaf economics spectrum worldwide. Nature 428, 821–827.
Yemm E.W. & Cocking E.C. (1955) The determination of aminoacids with ninhydrin. Analyst 80, 209–213.
Received 2 December 2003; received in revised form 18 March 2004;
accepted for publication 13 April 2004
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1047–1054