Oecologia
© Springer-Verlag 2004
10.1007/s00442-004-1500-z
Ecophysiology
Patterns of intramolecular carbon isotopic
heterogeneity within amino acids of
autotrophs and heterotrophs
William B. Savidge1
and Neal E. Blair2
(1) Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA 31411,
USA
(2) Department of Marine, Earth, and Atmospheric Sciences, North Carolina State
University, Raleigh, NC 27695, USA
William B. Savidge
Email: wsavidge@skio.peachnet.edu
Received: 7 February 2003 Accepted: 8 January 2004 Published online:
24 February 2004
Abstract A survey of the intramolecular C isotopic composition of a variety of
organisms was conducted to investigate the potential of intramolecular isotopic
measurements as a tracer of biological or geochemical processes. Based on a
consideration of inorganic C sources and enzymatic fractionations, contrasting
predictions were made for the relative 13C enrichments of the -carboxyl carbons fixed
by the anapleurotic ( )-carboxylation pathway during amino acid synthesis by
photoautotrophs and heterotrophs. To test the model predictions, the stable C isotopic
compositions of the acid hydrolyzable C fraction, the total amino acid -carboxyl C
fraction and the -carboxyl C of glutamate from a variety of autotrophic and
heterotrophic organisms were compared. The relative 13C enrichments of carboxyl
carbons in the bulk amino acid fraction and in glutamate conformed qualitatively to
model predictions. Macroalgal taxa possessed a significantly less enriched carboxyl C
fraction than did either C3 or C4 vascular plants, indicating the presence of a different carboxylation pathway operating in these organisms. In most multicellular heterotrophs,
the isotopic composition of the amino acid carboxyl carbons closely resembled that of
their food sources. Amino acids are apparently assimilated into tissue proteins directly
from their diets without significant metabolic modification. However, shifts in the
isotopic composition of the carboxyl C fractions in some organisms were detected that
were consistent with the occurrence of significant resynthesis of amino acids from nonamino acid precursors. Comparison of plant leaves and roots provided evidence of
environmentally controlled assimilate partitioning. Intramolecular isotopic measurements
of biological molecules provide unique insights into the origins and transformations of
bio-molecules.
Keywords Anapleurotic carboxylation - Tricarboxylic acid cycle - Carbon isotopes Glutamate - Phosphoenolpyruvate carboxylase
Introduction
The C isotopic composition of biological molecules reflects the physiological and
environmental circumstances under which they are synthesized. Brenna (2001) and
Hayes (2001) have noted that detailed information about the origins of bio-molecules lies
within the intramolecular (within molecule) distribution of C isotopes where there is
preserved evidence of biosynthetic pathways and metabolic fluxes during compound
synthesis. By analyzing the carbons at targeted positions within specific molecules, it
may be possible to infer biochemical or physiological properties of the source organism
that cannot readily be measured directly (e.g., Keeling and Nelson 2001). Intramolecular
isotopic information has some potentially unique and useful properties. Compounds with
long turnover times (e.g., structural proteins, lignins) may yield intramolecular isotopic
information integrated over an extended time period, whereas measurements of metabolic
intermediates (e.g., organic acids) may provide a more real time view of metabolic
state. Because some isotopic information may be recorded in stable molecules, there
exists the possibility for obtaining paleo-physiological information from geomolecules (Brenna 2001).
The earliest attempt to measure the natural abundance intramolecular distribution of 13C
and 12C in a biochemical was made by Abelson and Hoering in 1961. They compared the
isotopic composition of the -carboxyl carbons of amino acids obtained from cultured
microalgae and bacteria with the isotopic composition of the remaining carbons of the
molecules. Two patterns were observed. The carboxyl carbons of amino acids of algae
grown photoautotrophically were enriched in 13C by 11.6±8.6
relative to the
remaining carbons of the molecules, whereas the carboxyl carbons of algae (Chlorella
pyrenoidosa) or bacteria (Escherichia coli) grown heterotrophically on a minimal glucose
medium showed a much smaller intramolecular isotopic difference (–1.5±4.6 ). This
intramolecular 13C enrichment of carboxyl carbons in autotrophs was significantly greater
in amino acids derived from tricarboxylic acid (TCA) cycle intermediates (i.e., aspartate,
threonine, lysine, methionine, isoleucine, glutamate, proline and arginine) than it was in
amino acids arising from intermediates prior to the TCA cycle (15.9±7.5
7.4±7.6
; P<0.0003).
versus
The large intramolecular isotopic heterogeneities within amino acids found by Abelson
and Hoering (1961) suggest that it may be possible to use the relative distribution of 12C
and 13C within amino acids as an indicator of pathways of amino acid metabolism in
biological or geochemical samples. Because Abelson and Hoering s (1961) data were
collected using unicellular cultures grown under specific culture conditions, however, it
is unclear how robust the data set might be when extended to other taxa in natural
environments. As a first step toward evaluating potential applications of intramolecular
isotope measurements to the biochemistry and biogeochemistry of amino acids, a survey
of field-collected plants and animals was conducted in order to quantify the natural range
of intramolecular isotopic variation. Heterogeneity was assessed by comparing the C
isotopic composition of the total acid-hydrolyzable organic fraction with the isotopic
composition of the total amino acid carboxyl C fraction obtained by chemical
decarboxylation of amino acids in the hydrolysates. For a limited subset of the samples,
the isotopic composition of the -carboxyl C of glutamate was also measured. The data
were compared to the predictions of a simple conceptual model of isotopic fractionation
during amino acid synthesis by autotrophs and heterotrophs. The correspondence, or lack
thereof, between the observations and model predictions were used to draw inferences
about amino acid sources and metabolism in the sampled organisms.
Materials and methods
Sample analysis
Samples were collected for isotopic analysis from a variety of sources. Marine
macroinvertebrates, macroalgae [Ulva lactuca, Codium fragile, Enteromorpha
intestinalis (Chlorophyta), Dictyota sp. (Phaeophyta), and an unidentified species of
Rhodophyta], and Spartina alterniflora plants were collected from an intertidal mudflat
and a small fringing marsh at the University of North Carolina at Wilmington s research
lease property on Masonboro Sound near Wilmington, North Carolina (34°18 N, 77°54
W). A detailed description of the site can be found in Powell (1994). Terrestrial samples
(vascular plants and leptidopteran insects) were collected on or about the North Carolina
State University campus in Raleigh, North Carolina. Emergent freshwater aquatic
vascular plants (Pontederia sp. and Sagittara sp.) were obtained from muddy sand
sediments of Lake Hall, Florida, and from greenhouse cultures of Jeff Chanton and
Joanne Edwards at Florida State University. Freeze-dried Spirulina was obtained from a
commercial supplier of nutritional supplements (Lightforce, Santa Cruz, Calif.; T.
Bridges, personal communication). Anabaena oscillatoides samples were taken from Nfixing laboratory cultures maintained at the Institute of Marine Sciences at Morehead
City, North Carolina (T. Sharp, personal communication) Cultures of brewer s yeast
(Saccharomyces cervesae) were grown anaerobically at 20°C on a shaker table in sealed
2.5-l bottles on Wickerham s minimal glucose media (10 g glucose/l) (Atlas and Parks
1993) and harvested in stationary phase after 20 days. At the time of harvest, yields
differed substantially between the two cultures, with culture Y12A having a yield of 3.2 g
dry weight (dw)/l, and culture Y12B having a yield of 0.3 g dw/l. Additional yeast
sample material was obtained from a commercial preparation (Difco yeast extract).
Smaller and/or morphologically undifferentiated (e.g., algae) samples were freeze-dried
and ground to a powder in their entirety. However, for some larger and morphologically
complex samples, different parts of the organism were separated and analyzed
individually. The different portions were regarded as separate subsamples of an
individual organism for the purposes of statistical analysis.
Freeze-dried sample material was hydrolyzed in 6 N HCl for 2 h at 150°C, using a
procedure modified from Cowie and Hedges (1992). A subsample (50–150 l) of the
hydrolysate was pipetted into silver foil boats. Boat contents were dried under nitrogen
gas at 100°C and subsequently combusted in a Carlo Erba model 1108 elemental
analyzer. CO2 from the gas stream was trapped cryogenically and measured for its
13 12
C/ C ratio on a Finnegan Mat Delta E isotope ratio mass spectrometer (IRMS)
equipped with a modified inlet system for small sample volumes (Hayes et al. 1977).
13 12
C/ C ratios are reported relative to the PDB standard in the 13C notation (Craig 1953).
Combusted acetanilide standards averaged –0.04±0.02
relative to accepted values.
The acid hydrolysate was subsampled (generally 1–3 ml) for analysis of the isotopic
composition of the total carboxyl C fraction ( 13CCOO). The aliquot was dried by rotary
evaporation and then rehydrated in 0.5–1.5 ml of pH 2.5 phosphate buffer. The
decarboxylation reagent was a 0.4 M solution of ninhydrin (2,2 dihydroxy 1,3indandione) (Pierce Chemical) in the same 50 mM pH 2.5 phosphate buffer. A 0.5-ml
aliquot of the ninhydrin solution was transferred to a 3-ml serum vial (Wheaton) and
frozen. To the frozen reagent was added 0.5–1 ml of the rehydrated hydrolysate. The vial
was quickly capped with a greased (Apiezon L) red butyl rubber stopper (Wheaton) and
crimp-sealed. The ninhydrin reaction proceeds slowly at low temperatures (VanSlyke et
al. 1941); freezing the reagent prior to sample addition allowed the vial to be capped
without any loss of CO2. Capped vials were either re-frozen for <24 h prior to analysis or
analyzed immediately after capping.
The ninhydrin reaction was initiated by dropping the sealed vial in a boiling water bath
for 15–20 min. The reaction time was greater than required to bring the decarboxylation
to 100% completion (VanSlyke et al. 1941). After the vial was removed from the water
bath and allowed to cool, 0.1–0.2 ml of an anti-foaming agent (Antifoam-A, Sigma) was
injected through the stopper to prevent sample foaming during CO2 stripping. The sample
was bubbled with He gas, which was swept into a 1 m×2.2-mm internal diameter
stainless steel gas chromatography (GC) column packed with Unibeads 1S (Alltech). CO2
in the sample was quantified by GC, trapped cryogenically and transferred to a Pyrex
breakseal for isotopic analysis.
The CO2 collected, representing the pooled carboxyl carbons of all amino acids surviving
hydrolysis, was analyzed isotopically using the IRMS as described above. The isotopic
composition of blank-corrected HCO3- standards was –19.46±0.01
value of the standard was –19.5±0.1
.
. The accepted
Tests with casein standards showed no effect of hydrolysis time on the 13CCOO for
incubation times between 50 min and 5 h (r 2=0.015, P=0.66), or the isotopic
composition of the total hydrolyzable C fraction ( 13CHOC) for times between 50 min and
2 h (r 2=0.32, P=0.09). The C isotopic composition of CO2 derived from direct
combustion of casein standards (–29.79±0.29
; n=3) was not significantly different
from values obtained for combustion of casein hydrolysates (–29.71±0.13
; n=10).
For a limited subset of samples, the hydrolysate was subsampled for the 13C of the
carboxyl C of glutamate ( 13CGLU). The aliquot was dried and rehydrated in a 50 mM
(pH 5.5) phosphate buffer. The solution pH was re-adjusted as needed to 5.5 by addition
of NaOH or HCl. One to two milliliters of the solution was pipetted into a 3-ml serum
vial containing approximately 0.2 mg (>4 units) glutamic decarboxylase enzyme (Sigma
G 3757) and 2 mg of its pyridoxal-5-phosphate cofactor (Sigma P 9255). The vials were
incubated in a heated water bath at 37°C for 20 min. After incubation, vials were injected
with 0.2–0.3 ml of anti-foaming solution and stripped of CO2 in a He stream. The CO2
was quantified by GC, trapped cryogenically, and analyzed by IRMS. Yields were
estimated by the recovery of CO2 from glutamate spikes added to sample hydrolysates.
Spike recoveries averaged 103±6%. The high recovery efficiencies indicate that
reconstituted hydrolysate did not inhibit the enzymatic decarboxylation of glutamate and
that any isotope effects associated with the decarboxylation reaction were not expressed.
Blanks (n=23) were obtained by submitting sample-free acid to hydrolysis and carrying
the acid through the entire analytical procedure. Sample data were corrected by
subtraction of the acid blanks. Blank-corrected yields of glutamate carboxyl carbons from
hydrolysates of casein standards (Eastman Kodak technical grade casein) averaged
1.16±0.06
mol/mg (n=8). Gordon and Whittier (1966) estimated the glutamate content
of whole casein to be 1.08
mol/mg. The precision of seven replicate measurements of
13
CGLU obtained from casein hydrolysates was ±0.3
.
For the purpose of this study the intramolecular isotopic heterogeneity of a sample (
13
C) was defined as the 13C of the total carboxyl C fraction obtained from the ninhydrin
decarboxylation reaction (or the glutamic decarboxylase reaction) minus the 13C of the
total hydrolyzable C obtained by combustion, i.e., 13CCOO– 13CHOC.
Statistical analysis
The statistical analysis of the data set was designed to accomplish two goals: (1) estimate
confidence limits for data pooled at the level of sample, (sample) type , (metabolic)
group and (synthetic) class level; and (2) test for differences in the relative 13C
13
enrichment of amino acid carboxyl carbons (
C) at each level. Data were analyzed
13
13
13
separately for each quantity,
CHOC,
CCOO and
CCOO, at each data level using a
mixed-model ANOVA (SAS PROC MIXED). At each level of analysis the remaining
variables were nested as random variables within the fixed variable of interest, reflecting
the hierarchical nature of the data. Estimates of least square sample means and SEs were
obtained from the analysis. df s were calculated using the Satterthwaite approximation
(Verbeke and Molenberghs 1997). Because of the imbalance of the data set and the
13
statistical approach employed in this study, the value of
CCOO calculated for a
sample is not necessarily identical to the arithmetic difference between 13CHOC and
13
CCOO measured for the same sample. Confidence intervals are calculated as a linear
combination of variance components. Pairwise contrasts at each level of analysis for
13
CCOO were conducted where a significant (P<0.05) fixed-factor effect was detected. P values of pairwise comparisons were corrected for multiple testing using the TukeyKramer adjustment (Westfall et al. 1999).
13
A slightly different statistical approach was required for the analysis of
CGLU. For
13
13
most samples,
CHOC and
CGLU were not obtained from the same hydrolysate. As a
result, differences in the two quantities were not simply the difference between two point
13
estimates, as they were for
CCOO, but were the differences between least square
means obtained from separate data analyses. The resultant error estimate was obtained by
propagating SEs of the two estimates (Lyon 1970). Among-group comparisons of
13
CGLU were undertaken using a Kruskal-Wallis nonparametric ANOVA. Post hoc
pairwise comparisons were calculated using the procedure outlined in Sprent and
Sweeton (2001) and corrected for multiplicity by Hochberg s method (Westfall et al.
1999).
Results
Total ninhydrin-reactive amino acids
Autotrophs
The data for the total carboxyl C fraction of autotrophs are given in Table 1 and Fig. 1.
Both the C3 and C4 vascular plants display very similar 13C enrichments in the total
13
amino acid carboxyl C fraction relative to bulk C. The mean
CCOO for C3 plants is
7.1±0.8
(n=7). The mean
13
CCOO for the pooled C4 (Spartina alterniflora) plants
is 7.5±0.5
(n=19). The means are not significantly different. In contrast, the
macroalgal samples show lesser enrichment of 13C in their amino acid carboxyl carbons,
13
with a mean
CCOO of 3.0±0.7
(n=12). The mean of the pooled macroalgal
samples is significantly less than that of either the C3 or C4 vascular plants (P=0.008 and
P=0.0001, respectively; Table 2).
Table 1 Summary of least-square means and SEMs obtained for the isotopic
composition of the total hydrolyzable C fraction ( 13CHOC), the isotopic composition of
13
the total carboxyl C fraction ( 13CCOO ),
CCOO, 13C of the carboxyl C of glutamate
13
( 13CGLU) and
CGLU for samples pooled by group. Note that data expressed in italics
were obtained by error propagation rather than from the statistical models (see text). P values are given for overall differences among groups
13C
HOC
SEM
P<0.0001
13C
COO
SEM
P<0.0001
13C
COO
SEM
P<0.0001
13C
GLU
SEM
13C
GLU
P=0.036
SEM
P=0.08
–19.1
1.8
–15.0
1.9
3.0
0.7
–14.1
3.4
8.6
3.2
Cyanobacteria –13.9
3.2
–2.6
3.6
10.7
1.8
11.0
4.2
25.0
4.0
C3 plant leaves –24.9
1.9
–18.2
19.7 7.1
0.8
–16.1
4.2
11.7
4.0
C4 plant leaves –13.3
4.4
–5.9
4.2
7.5
0.6
0.0
5.5
14.2
5.3
C3 plant roots
–27.0
3.1
–23.4
3.2
3.9
1.1
C4 plant roots
–11.5
4.4
–12.3
4.2
–0.2
0.6
–8.0
5.5
3.2
5.3
Unicellular
heterotrophs
–16.1
3.2
–15.6
3.4
–0.3
1.5
–15.0
4.0
0.5
3.9
Marine
heterotrophs
–16.0
1.1
–11.8
1.2
4.4
0.5
–6.8
4.1
8.8
3.9
Terrestrial
heterotrophs
–29.0
2.2
–21.7
2.3
7.7
0.8
Macroalgae
Fig. 1 Comparison of the isotopic composition of the total hydrolyzable C ( 13CHOC)
fraction and the total amino acid carboxyl C ( 13CCOO) fraction for autotroph samples.
13
Error bars represent 1 SE. Diagonal lines represent the mean
CCOO for each group
13
relative to
CHOC=0
Table 2 P-values for pairwise comparisons of
significance is given in Table 1
Autotrophs
13
Cyanobacteri C3
C4
C3
a
leaves leaves roots
CCOO among groups. Overall test
Heterotrophs
C4
roots
Yeast Marine
Terrestria
l
Autotrophs
Macroalgae
Cyanobacteri
a
Heterotrophs
Cyanobacteri C3
C4
C3
C4
Terrestria
Yeast Marine
a
leaves leaves roots roots
l
0.000 0.999
0.551
0.005
0.008
0.0331
0.8117 0.0017
1
1
8
0.696
0.042 <0.000 0.000
0.761
0.03
4
9
1
4
C3 leaves
C4 leaves
C3 roots
C4 roots
Yeast
Marine
heterotrophs
1
0.8493
0.276 <0.000
0.001 0.1007 0.9999
7
1
0.07
<0.000 0.000
0.001
1
1
0.04
0.371
1
6
1
1
0.1221
<0.000
<0.0001
1
0.08
0.0003
0.02
The isotopic composition of the carboxyl C fraction of the cultured N-fixing
cyanobacterium Anabaena oscillatoides lies well away from the remaining data,
including that of the cyanobacterium Spirulina. Abelson and Hoering (1961) found that
the carboxyl C fraction of the cyanobacterium Anacystis nidulans was the most enriched
in 13C among the samples they analyzed. Anacystis was also obtained from a N-fixing
culture.
Invertebrate heterotrophs
13
Among the marsh-dwelling organisms,
CCOO (4.4±0.5 ) (n=28) appears to be
intermediate between that of the macroalgae and Spartina alterniflora found at that site
(Fig. 2; Tables 1, 2). The bulk isotopic composition of the leptidopterans (n=9) was very
similar to that of their food sources (Petroselinum and Catalpa). There is little change in
13
the
CCOO between plant food, caterpillar and pupa tissues (Fig. 3). The absolute and
relative 13C enrichment of the carboxyl carbons declines substantially within the newly
emerged adult (Fig. 3), although the difference is not significant.
Fig. 2 Comparison of the 13CHOC fraction and the 13CCOO for heterotroph samples.
13
Error bars represent 1 SE. Diagonal lines represent the mean
CCOO for each
13
autotrophic group relative to
CCOO=0 for comparison with Fig. 1. For abbreviations,
see Fig. 1
Fig. 3 Isotopic composition of lepidopteran samples. Error bars represent 1 SE. For
abbreviations, see Fig. 1
Yeasts
The
13
CCOO of the three yeast samples were all near zero. The mean enrichment
13
present in the pooled yeast samples (
CCOO=–0.3±1.5 ) was significantly less than
13
that of all autotrophic groups (Table 2). No differences between
CCOO of yeasts and
plant roots were detected. Carboxyl C enrichments in yeasts were less than those
observed in insect samples, but not in multicellular marine heterotrophs (P=0.08;
Table 2).
Plant roots
Although an integral part of autotrophic organisms, plant roots are entirely heterotrophic
(Flores et al. 1993). They are dependent on exogenous nutrient sources (C from the plant
s leaves via the phloem, inorganic salts from the surrounding medium) to generate energy
and form biomass. The roots of the salt marsh plant Spartina alterniflora display amino
13
acid carboxyl C enrichments near zero (
CCOO=–0.1±0.6 ) (n=14). The pooled
data for roots from Sagittaria sp. and Pontederia sp. indicate significant enrichments of
13
the amino acid carboxyl C fraction relative to hydrolyzable C (
CCOO=3.8±1.1 )
13
(n=4). The means are significantly different (P=0.04; Table 2). The
CCOO of
Spartina roots is significantly less than that of the leaves, but no significant differences
were detected between the roots and leaves of Pontederia or Sagittaria (Table 2).
-Carboxyl C of glutamate
13
13
CGLU was greater than
CCOO in all but two samples (one Spartina root sample
and one Ulva sample) (Fig. 4). In the case of the Spartina root sample, the observed
depletion of 13C in the pooled carboxyl C fraction is extremely large and may represent
an experimental error. The greatest enrichments were seen for the glutamate carboxyl
13
carbons of the cyanobacteria Anabaena and Spirulina.
CGLU for Anabaena was
32.8±0.9
. Although
13
CCOO of the Spirulina sample (
13
CCOO=2.6±2.6
) was
13
relatively small,
CGLU (= 17.1±3.1 ) was greater than for all autotrophs other
than Anabaena. The smallest carboxyl C enrichments were found in the yeast samples.
13
The relative ordering of the different groups by
CGLU was identical to their ordering
13
by
CCOO.
13
13
Fig. 4
CCOO (black bars) and
CGLU (gray bars). Samples: 1: Enteromorpha; 2:
unidentified red alga; 3–5: Ulva; 6: Anabaena; 7: Spirulina; 8: Catalpa; 9: Juglans nigra;
10–12: Spartina alterniflora leaves; 13–15 Spartina alterniflora roots; 16–17: yeast
cultures; 18: commercial yeast extract; 19–20: Mercenaria; 21: Uca. Groups: 1–5 =
macroalgae, 6–7 = cyanobacteria, 8–9 = C3 plants, 10–12 = C4 plants, 13–15 = C4 plant
roots, 16–18 = yeasts, 19–21 = marine heterotrophs
13
Significant differences in
CGLU among metabolic groups were detected by the
Kruskal-Wallis test. The results of the multiple comparisons are given in Table 3. Among
13
the autotrophs, macroalgae had significantly smaller
CGLUs than either the
cyanobacteria or C4 vascular plant leaves. Yeast and Spartina root samples had smaller
13
CGLUs than did the multicellular marine heterotrophs or any autotrophic groups
other than the macroalgae.
Table 3 Results of post-hoc test following Kruskall-Wallis test of difference in location
13
by group for
CGLU
Autotrophs
Heterotrophs
Macroalgae
Cyanobacteria
C3 leaves
Cyanobacteria C3 leaves C4 leaves C4 roots Yeast Marine
**
NS
**
NS
**
NS
NS
NS
***
**
NS
NS
**
**
NS
Autotrophs
C4 leaves
Heterotrophs
Cyanobacteria C3 leaves C4 leaves C4 roots Yeast Marine
**
**
NS
C4 roots
NS
Yeast
**
**
NS P>0.05, * P <0.05, ** P <0.01, *** P <0.001
Discussion
Models of intramolecular isotope enrichment
The photosynthetic plant cell can be regarded as an open isotopic system, with CO2
entering the cell as a reactant and fixed C and unfixed CO2 exiting as products (O Leary
1981). Most of the inorganic C is fixed by RUBISCO, but additional C must be fixed by
anapleurotic pathways to form oxaloacetate to replace TCA cycle intermediates drawn
off for amino acid synthesis. In C3 plants, both RUBISCO and anapleurotic enzymes ( carboxylases) fix inorganic C from a common intracellular dissolved inorganic C (DIC)
pool. The isotopic difference in the C fixed by the two pathways reflects the differences
in isotopic fractionations in their enzymatic reactions (Melzer and O Leary 1987).
Assuming an isotopic equilibrium between intracellular CO2 and HCO3-, the predicted
isotopic difference between RUBISCO-fixed C and anapleurotically fixed C is ~35
(Melzer and O Leary 1987) for plants using phosphoenolpyruvate (PEP) carboxylase
(PEPC) as a -carboxylase. PEP carboxykinase (PEPCK) has been identified as an
alternative carboxylase in some algal taxa (Kremer and Kuppers 1977; Holdsworth and
Bruck 1977; Kremer 1981; Reiskind et al. 1988; Raven and Farquhar 1990; DescolasGros and Oriol 1992). PEPCK (12k /13k=1.024–1.040; Arnelle and O Leary 1992) has a
substantially larger isotope effect than PEPC (12k/13k=1.0022; Schmidt and Winkler 1979;
O Leary 1981), and thus the intramolecular isotopic heterogeneity is expected to be
much smaller in the case of PEPCK carboxylations. Given the range in isotope effects for
PEPCK carboxylations reported by Arnelle and O Leary (1992) for Chloris gayana,
anapleurotically fixed C may be enriched in 13C by as much as 5
much as 11
relative to RUBISCO-fixed C (Fig. 5).
or depleted by as
Fig. 5 Conceptual model of fractionations associated with the initial fixation of inorganic
C by RUBISCO and -carboxylases in C3 autotrophs. Atmospheric CO2 diffuses into the
cell where it re-equilibrates between CO2 and HCO3-. Phosphoenolpyruvate (PEP)
carboxylase (PEPC) fractionation is expressed relative to its HCO3- substrate. The
substrate of PEP carboxykinase (PEPCK) and RUBISCO fixation reactions is CO2. The
relative isotopic composition of C fixed by the two pathways is independent of the net
fractionation of RUBISCO-fixed organic C expressed relative to the external CO2 pool
In C4 plants the situation is complicated by the complex carboxylation
decarboxylation carboxylation photosynthetic fixation pathway (Fig. 6). Intracellular
DIC is fixed initially by PEPC in the mesophyll and appears at the C-4 position of
malate. Decarboxylation of the malate in the bundle sheath leaves a highly enriched
residual malate fraction that is free to enter the mitochondria to replenish C drawn off by
amino acid synthesis. The C-4 of malate appears in part at the C-1 (carboxyl C) position
of glutamate and its derivatives and (via fumarase randomization) the C-1 position of
aspartate and its derivatives. The enrichment ultimately expressible within the residual C4 of malate (and within TCA cycle products) is a function of the isotope effect and
efficiency of the enzymatic decarboxylation within the bundle sheath—which establishes
the absolute level of malate C-4 enrichment relative to its source—and to the efficiency
of C fixation by RUBISCO—which establishes the 13C depletion of bulk organic C
resulting from photosynthesis.
Fig. 6 Conceptual model of fractionations associated with the initial fixation of inorganic
C by RUBISCO and -carboxylases in C4 autotrophs. HCO3- is fixed by PEPC to form
malate, which is shuttled to the bundle sheath and decarboxylated. The decarboxylation
reaction as presented here has a 12k /13k ~1.03 and is assumed to be nearly quantitative.
CO2 generated by the reaction has approximately the same isotopic composition as the
malate C4 fixed within the mesophyll. The residual unreacted malate in the bundle sheath
13
is proportionally 13C-enriched at the C-4 position, and has a
C of ~30 relative to
13
the bundle sheath CO2. The C-enriched residual malate can be transported into the
bundle sheath mitochondria for synthesis of 13C-enriched Kreb s cycle amino acids. CO2
is fixed by RUBISCO to form the bulk organic C of the plant. Given the high efficiency
of the RUBISCO fixation process (~80%), the intrinsic isotopic fractionation of the
RUBISCO reaction is only slightly expressed, and the isotopic composition of bulk plant
tissues are only modestly depleted relative to atmospheric CO2
In both C3 and C4 photosynthesis the relative 13C enrichment of the initial products of
anapleurotic carboxylation are subject to modification by other reactions within and
around the TCA cycle. Abelson and Hoering (1961) found considerable variation in the
13
C enrichment of carboxyl carbons among amino acids within organisms and within
amino acids among organisms, even within a single amino acid family. Since the
carboxyl C does not participate directly in any other reactions during amino acid
synthesis, variations in its isotopic composition must arise from other post-fixation
processes such as peptide bond formation/scission (Silfer et al. 1992) or secondary
decarboxylation reactions (Edens et al. 1997). Significant dilution of anapleurotically
fixed C by C derived from acetyl-CoA may occur within the TCA cycle as well. Rollin et
al. (1995) showed that the extent of labeling of TCA cycle intermediates is inversely
proportional to dilution by acetyl-CoA. The net effect of these processes will be to
attenuate and broaden the initial anapleurotic carboxylation signal within the amino acid
pool.
In heterotrophs (and autotrophs metabolizing in darkness), C flux through the TCA cycle
is devoted largely to respiration rather than amino acid synthesis. The primary C source
of the TCA cycle is acetyl-CoA, which is expected to be 13C depleted relative to bulk C
(DeNiro and Epstein 1977; Monson and Hayes 1982). The DIC substrate for anapleurotic
carboxylation is likely to consist primarily of respired C. The isotopic composition of
respired C has been shown to be similar to, or somewhat 13C depleted, relative to bulk C
during heterotrophic metabolism (e.g., DeNiro and Epstein 1978; Blair et al. 1985; Lin
and Ehleringer 1997; Breteler et al. 2002). Any isotopic fractionation of the DIC source
during anapleurotic C fixation will further deplete the (carboxyl C) product in 13C. There
is thus no known mechanism for generating significant 13C enrichment at the -carboxyl
C position of TCA cycle amino acids in heterotrophs via anapleurotic fixation.
Autotrophs
The open fractionation models predict a constant isotopic offset between anapleurotically
fixed and RUBISCO-fixed C independent of the 13C of the bulk C (Figs. 5, 6). The
prediction is borne out by the macroalgal data, where the slope of a regression of
13
13
CCOO versus
CHOC (m=0.92) is not significantly different from 1 (P>0.7). The C3
vascular plants also appear to fit the model predictions, although the trend is anchored
by a single observation, the data point for the C3 seagrass, Halodule wrightii.
13
13
The ratio of
CGLU/
CCOO=2.2±0.6 for all autotrophs and is consistent with an
~50% dilution of amino acids derived from TCA cycle intermediates bearing 13Cenriched carboxyl carbons by amino acids synthesized via other pathways (
13
13
CCOO~
CHOC). Although the relative abundances of different amino acids are
variable among organisms, the proportion of the total amino acid pool that is synthesized
from TCA cycle intermediates is ~45–55% for a wide variety of organisms (e.g., de la
Cruz and Poe 1975; Boyd 1979; Lee et al. 1993; Ramos-Elduroy et al. 1997). The
13
13
CGLU/
CCOO ratio supports the assumption that the total carboxyl C isotope data
can be used as a proxy for anapleurotically fixed C.
The observed 13C enrichment of glutamate carboxyl carbons, which are derived directly
from anapleurotic C fixation, in C3 vascular plants are much less than the 35
predicted by the model. The mean enrichment is 11.7±4.0 , or 33±11% of the
theoretical value. Loss of the initial anapleurotic carboxylation signal reflects post-
fixation isotopic dilution and scrambling during cellular metabolism. Similar results were
reported by Melzer and O Leary (1987, 1991). They found that the 13C enrichment of the
C-4 of aspartate (homologous to the C-1 of glutamate) in the C3 plants tobacco, soybeans
and spinach was approximately 50% of the 35
considerations.
predicted from theoretical
13
The close correspondence between
C in C3 and C4 vascular plants in this study
seems to be a fortuitous coincidence. The mechanisms proposed for generating the
observed intramolecular isotopic heterogeneities are sufficiently different that similar
isotopic outcomes cannot be predicted a priori.
The significant difference between
13
is hypothesized to reflect the dominant
CCOO for C3 vascular plants versus macroalgae
-carboxylating enzyme active in each taxonomic
group. Vascular plants and cyanobacteria use PEPC for -carboxylation (Owttrim and
Coleman 1986; Raven and Farquhar 1990). In contrast, brown macroalgae (Phaeophyta)
employ PEPCK as a -carboxylase (Kremer 1979; Reiskind et al. 1988). Green
macroalgae (Chlorophyta) and red macroalgae (Rhodophyta) have been reported to use
PEPC (Raven and Farquhar 1990), but measurements of enzyme activity have indicated
that PEPCK rather than PEPC is the predominantly active carboxylase (Kremer 1979;
Kremer 1981; Reiskind et al. 1988). Several algal species, including the chlorophyte Ulva
lactuca, have shown evidence of simultaneous PEPC and PEPCK activity (Kremer and
Kuppers 1977). Given that vascular plants employ PEPC as a -carboxylase, the
13
significantly smaller
CCOO of macroalgae suggests that they may employ an
alternative -carboxylase—presumably PEPCK—that expresses a larger isotope effect
relative to intracellular inorganic C.
13
The
CGLU data follow the same trend, but the differences between vascular plants
and algae are not significant (Tables 1, 3). Abelson and Hoering (1961) analyzed
individual amino acids of the red macroalga Gracilaria sp. and five other unicellular
autotrophs (Chlorella, Anacystis, Scenedesmus, Chromatium, and Euglena). The
13
CGLU of Gracilaria (10.9 ) was smaller than that of any of the other taxa, and the
average 13C enrichment of all TCA cycle amino acids analyzed was significantly less
13
than that of four-fifths of the other species investigated. The
CGLU of the
chlorophyte Chlorella pyrenoidosa (12.3
Gracilaria.
) was not significantly different from that of
13
Expression of
C is expected to be maximized in culture when light is continuously
available. Naturally occurring phototrophs experience oscillating light-dark cycles; thus
potentially half of the amino acid synthesis could take place under heterotrophic (dark)
conditions. Comparison of Abelson and Hoering s (1961) laboratory data with those of
this study indicates greater intramolecular isotopic heterogeneity in cultured unicellular
autotrophs than in field-collected samples regardless of potential -carboxylation
13
pathways. Average
CCOO of five cultured photoautotophs analyzed by Abelson and
13
Hoering (1961) was 12.3±3.3 . The values for
CGLU were 19±7.3 . Both
values are greater than equivalent values obtained from this study (Table 1). The isotopic
differences are hypothesized to be a result of contrasting physiological demands and
biochemical fluxes within algae under field and culture conditions. Direct comparison of
cultured algal samples between this study and Abelson and Hoering s (1961) yield more
13
similar results.
CCOO for the cultured cyanobacterium Anacystis nidulans measured
by Abelson and Hoering (1961) was 16.1
;
Anabaena oscillatoides in this study was 18.5±0.7
were equivalently enriched:
13
CGLU~28
13
CGLU=32.8±2.4
13
CCOO of the cultured cyanobacterium
. Carboxyl carbons of glutamate
(A. oscillatoides);
(A. nidulans).
Heterotrophs
Invertebrates
The data for marine and terrestrial invertebrate heterotrophs conflict with the model
prediction that they will have small intramolecular isotopic heterogeneities within their
13
amino acids. Instead,
CCOO of most of the marine organisms appears to be
intermediate between that of vascular marsh plants and macroalgae, and the insect larvae,
with one exception, are isotopically indistinguishable from their plant food sources
(Figs. 2, 3). The close correspondence of the isotopic composition of the amino acid
suites of consumers and likely food items indicates that these organisms are simply
translocating dietary amino acids into their tissues without any significant metabolic
modification—a phenomenon known as metabolic routing (Kreuger and Sullivan
1984; Gannes et al. 1998). Dietary amino acids in non-ruminant animals are employed
preferentially in protein synthesis (Ambrose and Norr 1993; Gannes et al. 1997).
Carbohydrates are used to generate amino acid C skeletons only under conditions of
protein deficiency (Ambrose and Norr 1993). In addition, all multicellular heterotrophs
have essential amino acid requirements that must be met from the diet. As a result, the
extent to which heterotrophic reworking can modify the bulk carboxyl C signature is
constrained by the proportional abundances of essential versus non-essential amino acids.
Nevertheless, there are several samples that depart substantially from the majority and
that begin to approach the range of microbial heterotrophs that are synthesizing a full
suite of amino acids. These six data points are identified by intersections of their error
bars with the line of no carboxyl C enrichment in Fig. 2. Two hypotheses may be
proposed to explain these anomalous data points: (1) either they represent organisms that
are feeding on an isotopically distinct resource like, for example, heterotrophic bacteria
or PEPCK-utilizing algae; or (2) represent organisms that are somehow metabolically
distinct from the majority of the animals sampled. The hypothesis of diet must be
rejected. There is nothing about feeding mode, sampling location, or taxon (two bivalves,
a decapod crustacean, a lepidopteran larva, a polychaete, and a bryozoan) that would
separate the anomalous samples from the others and suggest that they might have access
to an isotopically distinct food source. That leaves the alternative hypothesis that the
organisms in question differ physiologically from the majority. It is proposed that the
unusual isotopic composition of these animals indicates that they have either not been
feeding or had been feeding slowly for some time prior to collection. Visual observation
supports the hypothesis in at least one instance, a mussel (Geukensia demissa) obtained
from high in the marsh. The animal had a noticeably small tissue volume in relation to its
shell volume, as if it had undergone a period of degrowth (e.g., Russell-Hunter 1985).
13
The data taken from the butterfly samples illustrate the feeding hypothesis.
CCOO of
13
the organism represents a weighted average of the
CCOO of amino acids assimilated
13
from the diet and the
CCOO of amino acids synthesized in situ from non-keto acid
precursors (e.g., O Brien et al. 2002). The similarity between the isotopic composition of
the food source and the Papilio and Ceratomia larvae (Fig. 3) suggests that the extent of
the de novo heterotrophic synthesis is small. During the pupal phase larval protein
reserves are consumed to support the production of adult structures and reproductive
products (Wheeler et al. 2000). Changes in the intramolecular isotopic composition of
amino acids indicate that storage reserves are not simply re-routed to new tissues, but are
13
subject to extensive metabolic reworking. The large change (–7±3.9 ) in
CCOO of
Papilio during metamorphosis is consistent with a near complete turnover and
replacement of the non-essential TCA cycle amino acids of autotrophic (dietary) origin
amino acids synthesized heterotrophically in situ during the non-feeding pupal phase.
It may be argued by analogy that the isotopic composition of the carboxyl carbons of the
marine invertebrates is under similar control. Under normal conditions, demand for
new amino acid C skeletons for protein synthesis is met by absorption of dietary amino
acids. The molecular and intramolecular isotopic composition of the amino acids of the
heterotrophs closely resembles that of their plant food. During times of fasting, amino
acid demand must be met by mobilization of non-amino acid reserves (Ambrose and Norr
1993; Nelson et al. 1998). Replacement or recycling of autotrophically derived nonessential amino acids by heterotrophically synthesized amino acids will shift the amino
acid carboxyl C pool toward more 13C depleted values. There may be a continuum
between a metabolic routing strategy during nutrient replete conditions and a
metabolic scrambling strategy during times of nutrient deprivation in invertebrates. The
isotopic composition of the carboxyl carbons of the non-essential amino acids arising
from TCA cycle precursors (i.e., aspartate, glutamate and proline) may be uniquely
sensitive indicators of the relative contributions of diet and de novo synthesis of amino
acids to heterotroph tissues.
Heterotrophs
Yeasts
Unlike the multicellular heterotrophs, the heterotrophic yeasts conform qualitatively to
the predictions of the conceptual model: the isotopic composition of the -carboxyl C
fraction is very similar to that of the total organic C which serves as the source for the
inorganic C fixed by the anapleurotic carboxylation reaction. The results for the yeast
cultures are similar to those reported by Abelson and Hoering (1961) for E. coli or
13
Chlorella pyrenoidosa grown on minimal media. Although
CGLU in yeast is more
variable than
13
CCOO among the three samples, the mean
13
CGLU (0.5±3.9
) is
13
13
not different for E. coli (
CGLU=2.2 ) or Chlorella (
CGLU=0.9 ) (Abelson
and Hoering 1961). In yeast culture Y12B both the pooled carboxyl C fraction and the
glutamate carboxyl C are depleted in 13C relative to the hydrolyzable C, and the depletion
in the glutamate carboxyl C is a little more than twice that of the pooled fraction. Thus in
instances of both 13C enrichment and depletion the shift relative to total C is driven by
changes in the isotopic composition of TCA cycle amino acids.
Heterotrophs
Plant roots
Spartina root metabolism is analogous to that of the heterotrophic yeast cultures
synthesizing a full suite of amino acids from a minimal (carbohydrate plus inorganic N)
medium. Consistent with the predictions of the metabolic models for heterotrophs, the
roots of the salt marsh plant Spartina alterniflora display amino acid carboxyl C
enrichments near zero (
13
CCOO=–0.1±0.6
).
13
CGLU is also not significantly
different from zero, although the uncertainty of the estimate is much greater (3.2±5.3
). Root and shoot amino acid pools appear to be separate in Spartina, with no significant
contribution of autotrophically synthesized leaf-derived amino acids to root tissues
(Savidge and Blair 2004). In contrast, the data for roots from Sagittaria sp. and
Pontederia sp., both of which are emergent freshwater vascular plants, exhibit significant
13
C enrichments in the amino acid carboxyl C fraction relative to total hydrolyzable
organic C. In Sagittaria and Pontederia the situation is more analogous to that of
invertebrate heterotrophs. The isotopic composition of the amino acids in these plant
roots reflects, at least in part, inputs of autotrophically synthesized amino acids from
photosynthesizing tissues (Gleixner et al. 1998; Savidge and Blair 2004).
13
The difference in
CCOO between Spartina and the freshwater plants is hypothesized
to be related to the form of N utilized by the plant and its influence on the location of
amino acid synthesis within the plant (Savidge and Blair, submitted). Both Pontederia
and Sagittaria have been shown to produce oxidized rhizospheres (Chen and Barko 1988;
Calhoun and King 1997), but oxygen transport in Spartina alterniflora is rarely sufficient
to oxygenate the surrounding sediment (Mendelssohn et al. 1981; Howes and Teal 1994;
Mendelssohn and Morris 2000). In instances in which the rhizosphere is well-oxidized,
DIN may be taken up by roots predominantly as NO3-. NO3- can then be transported to
the leaves as the anion where it is assimilated into autotrophically synthesized amino acid
skeletons and re-exported to the roots. In contrast, where rhizosphere oxygenation is
poor, N is taken up primarily as NH4+. The NH4+ is assimilated within the roots using
locally (=heterotrophically) synthesized amino acid C skeletons and transported to the
leaves as an amide (Savidge and Blair 2004).
Conclusions
The intramolecular distribution of isotopes provides a qualitatively different isotopic
level of isotopic information about the origins and transformation of molecules within
organisms (Abelson and Hoering 1961; Brenna 2001). Measurement of intramolecular
isotopic heterogeneity has potential application as a uniquely specific tracer of material
fluxes from intracellular to ecosystem scales. In this project, some gross patterns in the
behavior of one particular class of compounds have been outlined. The results of the
survey of the intramolecular isotopic heterogeneity of the total amino acid pool and of
glutamate confirm that the observations of Abelson and Hoering (1961) can be broadly
applied to a variety of autotrophic and heterotrophic organisms in their natural
environment—with the caveat that intramolecular isotopic data must be interpreted
within the total metabolic context of the organism and not simply as the products of
specific reaction pathways. Among autotrophs, the primary determinants of carboxyl C
13
C enrichment appear to be the relative isotope effects associated with RUBISCO and
-carboxylating enzymes (or decarboxylating enzymes in the case of C4 plants), and the
extent to which secondary metabolic processes attenuate the initial signal. Within
heterotrophs, the dominant control lies in the relative importance of de novo synthesis
versus assimilation of dietary amino acids. Dietary signals are likely to dominate under
normal circumstances. Plant roots reflect a combination of processes in that their amino
acids can be obtained by translocation from photosynthesizing tissues or by in situ
heterotrophic production. The balance between the two sources varies with species and
environment (Savidge and Blair 2004).
Acknowledgements This research was partially supported by ACS-PRF grant 00–7972.
We thank Andy Liepens and Shannon Sullivan for their assistance in the laboratory, and
two anonymous reviewers and J. R. Ehleringer for their critiques of an earlier draft of this
manuscript.
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