Contribution of Different Carbon Sources to Isoprene
Biosynthesis in Poplar Leaves1
Jörg-Peter Schnitzler2, Martin Graus2, Jürgen Kreuzwieser2, Ulrike Heizmann,
Heinz Rennenberg, Armin Wisthaler, and Armin Hansel*
Forschungszentrum Karlsruhe GmbH Institut für Meteorologie und Klimaforschung, Atmosphärische
Umweltforschung (IMK-IFU), D–82467 Garmisch-Partenkirchen, Germany (J.-P.S.); Institut für Ionenphysik,
Leopold-Franzens-Universität Innsbruck, A–6020 Innsbruck, Austria (M.G., A.W., A.H.); and Institut für
Forstbotanik und Baumphysiologie, Professur für Baumphysiologie, Albert-Ludwigs-Universität Freiburg,
D–79110 Freiburg i. Br., Germany (J.K., U.H., H.R.)
This study was performed to test if alternative carbon sources besides recently photosynthetically fixed CO2 are used for
isoprene formation in the leaves of young poplar (Populus 3 canescens) trees. In a 13CO2 atmosphere under steady state
conditions, only about 75% of isoprene became 13C labeled within minutes. A considerable part of the unlabeled carbon may be
derived from xylem transported carbohydrates, as may be shown by feeding leaves with [U-13C]Glc. As a consequence of this
treatment approximately 8% to 10% of the carbon emitted as isoprene was 13C labeled. In order to identify further carbon
sources, poplar leaves were depleted of leaf internal carbon pools and the carbon pools were refilled with 13C labeled carbon by
exposure to 13CO2. Results from this treatment showed that about 30% of isoprene carbon became 13C labeled, clearly
suggesting that, in addition to xylem transported carbon and CO2, leaf internal carbon pools, e.g. starch, are used for isoprene
formation. This use was even increased when net assimilation was reduced, for example by abscisic acid application. The data
provide clear evidence of a dynamic exchange of carbon between different cellular precursors for isoprene biosynthesis, and an
increasing importance of these alternative carbon pools under conditions of limited photosynthesis. Feeding [1,2-13C]Glc and
[3-13C]Glc to leaves via the xylem suggested that alternative carbon sources are probably derived from cytosolic pyruvate/
phosphoenolpyruvate equivalents and incorporated into isoprene according to the predicted cleavage of the 3-C position of
pyruvate during the initial step of the plastidic deoxyxylulose-5-phosphate pathway.
Isoprene (2-methyl-1,3-butadiene), an unsaturated
C-5 hydrocarbon, is emitted in vast amounts from
photosynthesizing leaves of many plant species, particularly by trees (Kesselmeier and Staudt, 1999). With
a global atmospheric carbon flux of approximately 450
million tons of carbon per year, isoprene emissions are
a major contributor to the total biogenic volatile
organic compound (BVOC) flux of 1,200 million tons
of carbon per year (Guenther et al., 1995). Current
interest in understanding the biochemical and physiological mechanisms controlling isoprene formation in
plants comes from the important role isoprene plays in
atmospheric chemistry. Isoprene rapidly reacts with
hydroxyl radicals in the atmosphere (Thompson,
1992). In the presence of nitric oxides (NOX), the
oxidation of isoprene contributes significantly to the
formation of ozone, a dominant tropospheric air
1
This work was supported by the German Federal Ministry of
Education and Research (BMBF), BEWA2000 (Biogenic emissions
of volatile organic compounds from forest ecosystems), a subproject
of the national joint research project AFO2000 (AtmosphärenForschungsprogramm 2000).
2
These authors contributed equally to the paper.
* Corresponding author; e-mail [email protected]; fax 43–
512–507–2932.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.037374.
152
pollutant (Biesenthal et al., 1997). Moreover, isoprene
also contributes to the regulation of tropospheric
hydroxyl radicals concentration and thus plays an
important role in determining the abundance of atmospheric methane, an important greenhouse gas
(Chameides et al., 1988).
Light controls isoprene emission through the production of photosynthetic metabolites and the supply
of ATP/NADPH to the chloroplastidic deoxyxylulose5-phosphate (DOXP) pathway (Eisenreich et al., 2001;
Sharkey and Yeh, 2001). Isoprene emission is also
strongly dependent on temperature, increasing exponentially up to a maximum emission at approximately
40°C with subsequent rapid decrease with further
temperature increase (Zimmer et al., 2000). This temperature dependency strongly differs from that of net
assimilation that generally exhibits a broad temperature optimum at approximately 30°C in plants of temperate climates. The portion of fixed carbon emitted as
isoprene therefore increases rapidly with temperatures above 30°C and under conditions in which
assimilation is virtually absent, for example under
water limitation, reaching maximum losses of 15% to
20% of photosynthetically fixed carbon (Sharkey et al.,
1996; Brüggemann and Schnitzler, 2002a, 2002b).
Exposure of plants to 13CO2 showed that instantaneously assimilated carbon is the primary carbon source
for isoprene formation (Sanadze, 1991; Delwiche and
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Alternative Carbon Sources for Isoprene Biosynthesis
Sharkey, 1993; Karl et al., 2002a), accounting for 70%
to 80% of the carbon in isoprene. Variations in diurnal
isoprene fluxes that cannot be explained by temperature and light, however, for example under severe
water stress (Brüggemann and Schnitzler, 2002b)
or midday depression of photosynthesis (Zimmer
et al., 2000), have led to the suggestion that alternative
carbon sources may also contribute to the production
of isoprene (Sharkey and Yeh, 2001). Monson et al.
(1994) suggested that changes in chloroplastidic carbon
pools, particularly in starch, may explain the seasonal
correlation between isoprene emission rates and photosynthesis in aspen (Populus tremuloides Micheaux).
Moreover, experiments on aspen by Funk et al. (1999)
indicated that in addition to the direct use of photosynthetically fixed carbon, isoprene formation is controlled by the whole-plant carbon allocation pattern.
Recent work with pedunculate oak (Quercus robur;
Heizmann et al., 2001) and poplar hybrids (Populus 3
canescens; U. Heizmann, personal communication)
revealed that high amounts of sugars can be allocated
to the leaves via the transpiration stream, which, in the
case of oak, can even exceed the daily carbon yield by
net assimilation under conditions of restricted photosynthesis (Heizmann et al., 2001; Brüggemann and
Schnitzler, 2002b). These results indicated a significant
cycling of carbon and raised the question as to whether
this extrachloroplastidic carbon pool may serve as an
additional source for isoprene formation, particularly
when photosynthesis is limited.
Recently, Kreuzwieser et al. (2002) provided first
direct evidence of an extrachloroplastidic carbon
source—other than atmospheric CO2—for leaf isoprene formation. Feeding [U-13C]Glc to excised oak
leaves via the petioles led to a rapid emission of single
and double 13C labeled isoprene molecules, amounting
to 3% to 8% of total carbon emitted as isoprene. This
finding is consistent with 13CO2 feeding (Delwiche and
Sharkey, 1993; Karl et al., 2002a) and 13C discrimination experiments (Affek and Yakir, 2003), suggesting
that approximately 10% to 20% of carbon in isoprene is
extrachloroplastidic but not from photorespiratory
carbon (Karl et al., 2002a, 2002b). Alternative carbon
for isoprene formation cannot, however, originate
from xylem-transported carbon compounds alone
but may also originate from additional carbon sources,
such as the breakdown of starch (Monson et al., 1994;
Kelly and Latzko, 1995) or mitochondrial respiration
(Anderson et al., 1998).
This study aimed to quantify additional leaf-internal
carbon sources, beside photoassimilates and xylemderived sugars, that contributed to isoprene formation
in poplar leaves. For this purpose, leaf internal carbon
pools were labeled by 13CO2 via photosynthesis and the
contribution of this carbon pool to isoprene formation
was quantified with on-line proton-transfer-reaction
mass spectrometry (PTR-MS). In addition to this,
feeding experiments with [1,2-13C]Glc and [3-13C]Glc
were performed to trace and quantify the transition of
the 13C label from cytosolic pyruvate/phosphoenol-
pyruvate (PEP) equivalents into the chloroplastidic
DOXP-pathway.
RESULTS AND DISCUSSION
Exposure of Poplar Leaves to 13CO2 Causes Fast But
Incomplete 13C Labeling of Emitted Isoprene
Isoprene emission is closely related to photosynthesis, with 13CO2 labeling being an effective way to
demonstrate the dynamic use of photoassimilates for
isoprene biosynthesis in intact leaves (Sanadze et al.,
1972; Delwiche and Sharkey, 1993; Karl et al., 2002a,
2002b). In this study with poplar, however, about 20%
to 30% of isoprene carbon atoms remained unlabeled
when intact leaves were exposed to 13CO2 (data not
shown), even under nonstressed conditions with net
assimilation rates (Fig. 3) in the same ranges as found
in other experiments with poplar hybrids from the
same line and in the same age (Kreuzwieser et al.,
2000). Our data are consistent with previous 13CO2
feeding experiments, which demonstrated that isoprene (Delwiche and Sharkey, 1993; Karl et al., 2002a)
and monoterpenes (Loreto et al., 1996) become rapidly
labeled but that approximately 10% to 20% of the
carbon must be derived from sources other than
atmospheric CO2. All of these findings are consistent
in supporting the view that readily available carbon
pools, which are not directly linked to recently assimilated carbon, exist in leaves to enable formation of the
direct isoprene precursor dimethylallyl diphosphate.
Alternative Carbon Sources for Isoprene Biosynthesis
It has been proposed (Karl et al., 2002a, 2002b; Affek
and Yakir, 2003) that additional carbon for isoprene
Figure 1. Portion of 13C from total carbon emitted as isoprene. Poplar
trees were preexposed either (a) to 12CO2 or (b) to 13CO2 (for detail, see
‘‘Materials and Methods’’). a, 13C labeling from xylem sap-derived
[U-13C]Glc and subsequent feeding of 13CO2. b, 13C labeling from
internal 13C pools in 13CO2 pretreated leaves and additional feeding of
[U-13C]Glc and 13CO2. Each value was taken approximately 30 min
after treatment change when conditions had been stabilized. Means of
four independent experiments (6SD) are shown; different letters indicate
significant differences at P , 0.05 as calculated with Student’s t test.
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Schnitzler et al.
Figure 2. Starch concentration in poplar leaves. Trees were kept in
darkness at room temperature and ambient 12CO2 conditions for 2 d and
were then exposed to permanent irradiation (PPFD of 800 mmol m22
s21) for 3 more days. During light exposure 1 leaf was placed into
a cuvette and fumigated with 360 mL L21 13CO2. Data shown are means
of 4 leaves (6SD) exposed to 13CO2. Different letters indicate significant
differences at P , 0.05 as calculated with one-way ANOVA.
sources were labeled with 13CO2 and their contribution
to isoprene formation was determined under natural
atmospheric CO2. The procedure for labeling carbon
pools with 13C was as follows: Having first kept the
trees in darkness for 48 h in order to deplete leaf internal C pools (in particular starch), individual leaves of
the trees were subsequently exposed to 360 mL L21
13
CO2 in cuvettes during a continuous irradiation
period of 3 d. Continuous irradiation was chosen in
order to prevent starch synthesis degradation occurring in poplar leaves during night. Starch concentrations in the 13CO2 experiment decreased to about 40%
of the initial levels (Fig. 2, bars); thus, a remarkable
unlabeled pool of starch remained and was still
present during exposure to 13CO2. During the light
period the starch concentrations recovered to the
control levels.
formation could be derived from (1) chloroplastidic
breakdown of starch, occurring simultaneously to
starch biosynthesis, (2) refixation of respiratory carbon, or (3) influx of cytosolic precursors (pyruvate/
PEP) into the chloroplast.
Xylem Transported Carbon
This study investigated whether xylem transported
carbohydrates are used as carbon sources for isoprene
biosynthesis. For this purpose, excised poplar leaves
were fed 5 mM [13C]Glc via the xylem. Within minutes,
the xylem-transported [13C]Glc was used as a carbon
source for isoprene formation, with the portion of 13C
of total carbon emitted as isoprene amounting to approximately 4% to 10% at stabilized conditions 30 min
after treatment (Fig. 1A). The temporal evolution
and rate of isoprene labeling via the xylem sap was
quite similar to that previously reported for oak
(Kreuzwieser et al., 2002). It confirmed the contribution of xylem-derived carbon to isoprene formation.
In addition to [U-13C]Glc fed via the xylem, poplar
leaves were exposed to 13CO2. Together with the xylem
transported carbohydrates, still only 72% 6 10% of the
carbon emitted as isoprene was labeled with 13C some
30 min after treatment change (Fig. 1), indicating that
other carbon sources for isoprene biosynthesis must
exist in poplar leaves.
Leaf Internal Carbon Pools
Besides photoassimilates and xylem-derived sugars
for isoprene formation in poplar leaves, quantification
of additional leaf internal carbon pools was made.
These leaf internal carbon pools as potential carbon
Figure 3. Effects of 13CO2 and 12CO2 fumigation on (a) the emission of
total isoprene and its different isotopes and (b) the portion of 13C from
total carbon emitted as isoprene (solid line) and net assimilation
(dashed line; measured only during 12CO2 exposure). Poplar leaves
were pretreated as described in legend of Figure 2 and treated in this
experiment as indicated by the vertical lines. (1) 13CO2 exposure was
replaced by exposure to 12CO2; (2) leaf was cut and the leaf petiole
placed into a solution containing 5 mM of unlabeled Glc (the increased
incorporation of 13C is due to reduced assimilation after cutting the
leaf); (3) Glc solution was replaced by 5 mM [U-13C]Glc; and (4) 12CO2
exposure was replaced by 13CO2. Typical results of four independent
replicates are shown.
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Alternative Carbon Sources for Isoprene Biosynthesis
Table I. Half-lives t 1/2 [min] of isoprene isotope masses from the
experiment shown in Figure 3
Half-lives (t 1/2) were determined from the time evolution of individual isoprene isotopes according to following equations: (a)
NðtÞ 5 N0 e2lt ; (b) t1=2 5 lnð2Þ=l, where N0 is the isotope concentration at t 5 0.
13
C- Labeled
Isoprene
m721
m731
m741
Rapid Washout of
C in Isoprene after
Exposure to 12CO2
13
t 1/2 min
15.7
6.3
3.4
SD;
n54
11.1
2.7
2.0
Slow Washout of 13C in Isoprene
Related to Endogenous
Labeled 13C Pools
t 1/2 min
148.3
197.8
213.0
SD;
n54
62.6
93.7
149.4
After this treatment, emission measurements during
exposure to 360 mL L21 13CO2 were started (Fig. 3).
Total isoprene emission of poplar leaves thereby
ranged from 5 to 12 nmol m22 s21 (e.g. Fig. 3A). These
emissions were dominated by the isotope mass m741,
amounting to approximately 4 nmol m22 s21 (Fig. 3A).
Emissions of the isoprene isotopes m731 (approximately 3.5 nmol m22 s21), m721 (approximately
1.7 nmol m22 s21), m711 (0.7 nmol m22 s21), m701
(0.2 nmol m22 s21), and m691 (0.1 nmol m22 s21) were
significantly lower. Switching from 13CO2 exposure to
12
CO2 exposure caused a drastic exchange of isotope
distribution. The total isoprene emission rates (sum of
all isoprene isotopes), however, did not change. During exposure to 12CO2 the portion of 13C of total carbon
emitted as isoprene still amounted to approximately
30% 6 12% (see also Fig. 1B). This labeling of isoprene
must have been derived from starch or other leaf
internal carbon pools labeled during the pretreatment
with 13CO2. Such values fit to actual measurements of
the natural abundance of carbon isotope composition
(d13C), which demonstrated that 9% to 28% of isoprene
carbon was contributed from alternative, slow turnover carbon source(s) (Affek and Yakir, 2003) for three
different isoprene-emitting species.
The nature of the additional carbon source(s) for
isoprene formation is, however, still unclear. The use of
starch for isoprene formation requires its partial breakdown to occur simultaneously to its synthesis, a feature
which has been described for spinach chloroplasts
(Stitt and Heldt, 1981) but has yet to be demonstrated
for poplar. Another more likely explanation, which is
supported by the observed rapid changes in the isoprene isotopes m711 and m721 when net assimilation
is fluctuating (see below), is the assumption that
cytosolic sugar and carbon compounds and plastidic
carbon compounds, which do not directly participate
in photosynthetic carbon reduction, act as an alternative carbon for isoprene formation.
Further tests were conducted in order to establish
whether [U-13C]Glc fed to leaves via the petioles
increases the rate of 13C labeling of isoprene in addition to its labeling by leaf internal 13C labeled carbon
pools. Additional feeding of [U-13C]Glc counteracted
the slow continuous washout of the 13C label and led
to a small transient increase in emissions of double and
triple labeled isotopes (Fig. 3A). The concurrent emission of the unlabeled isotope species (m691) dropped.
The effect of additional 13C via the xylem in this study
with 13CO2 pretreated plants, however, was minimal
compared to the exclusive feeding of [U-13C]Glc
shown in Figure 1A. This strongly indicates that the
cytoplasmic pool of glycolytic intermediates was
widely labeled with 13C and that additional xylemderived 13C only slightly affected cytosolic 13C sources
that contributed to isoprene formation.
At the end of the experiments shown in Figure 3,
the 13C supply from leaf internal carbon sources,
xylem-transported [U-13C]Glc, plus atmospheric 13CO2
resulted in an overall 13C labeling rate of the isoprene molecules of 85%. This indicates that a complete
labeling of the isoprene molecule was not obtained.
The gap of approximately 15% unlabeled carbon in
isoprene could be due to (1) the incomplete removal of
unlabeled starch (see Fig. 2) at the beginning of 13CO2
fumigation, (2) an incomplete exchange of carbon in
pools with low turn over rates, or (3) the fact that
mature poplar leaves fumigated with 13CO2 received
unlabeled carbon compounds via xylem and phloem.
Inconsistent with the finding of an approximately 30%
use of alternative carbon sources for isoprene formation (Fig. 1B), a considerably lower 13C amount was
found in the isoprene emitted from leaves labeled by
xylem-transported Glc plus atmospheric 13CO2 (Fig.
1A) than from leaves labeled by leaf internal, xylemtransported plus atmospheric 13CO2 (Fig. 1B; see
Figure 4. The portion of 13C from total carbon emitted as isoprene.
Poplar leaves were excised and the cut ends of the petioles were fed via
the xylem with 5 mM of either [U-13C]Glc (light gray bar), [1,2-13C]Glc
(medium gray bar), or [3-13C]Glc (dark gray bar) without (a) or with (b)
addition of 0.25 mM ABA. Values were taken at stabilized conditions
30 min after treatment change. Means of n 5 4 experiments (6SD) are
shown; different letters (capital letters for [U-13C]glc, lowercase letters
for [1,2-13C]glc) indicate significant differences at P , 0.05 as
calculated with Student’s t test; n.d., not determined.
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Schnitzler et al.
While the partially and fully labeled isotopes slowly
disappeared, (see Table I: t 1/2 of m721: 148.3 min; t 1/2
of m731: 197.8; and t 1/2 of m741: 213.0 min) the portion
of unlabeled and single labeled isoprene molecules
increased constantly. This slow exchange of 13C in
isoprene probably demonstrates the dilution of alternative carbon source(s) with unlabeled carbon from
actual photosynthetic carbon fixation feeding into the
DOXP pathway.
Transiently Decreased Net Assimilation Causes
Enhanced Incorporation of Alternative Carbon
Sources into Emitted Isoprene
Figure 5. Emission of isoprene and its different isotopes. a, The portion
of 13C from total carbon emitted as isoprene (solid line) as well as net
assimilation (b, dashed line) as a consequence of [U-13C]Glc application. A leaf was placed into the cuvette and treated as indicated by the
vertical lines. 1, Leaf was cut from the intact plant and the cut end was
inserted into 5 mM [U-13C]Glc; 2, 0.25 mM ABA was applied in addition
to 5 mM [U-13C]Glc. Data shown represent a typical result of 4
independent replicates under similar experimental conditions.
arrows in Fig. 1A); this can be taken as indirect
evidence for a contribution of internal carbon sources.
After switching from 13CO2 to 12CO2 exposure (Fig.
3) the leaves were cut from the plants and 5 mM
[12C]Glc was immediately applied via the petioles.
This manipulation caused a transient decrease in
photosynthesis (Fig. 3B) accompanied by increased
13
C incorporation into isoprene (Fig. 3B). The observed
increase was mainly due to a transiently enhanced
emission of the isoprene isotopes m711 and m721 (Fig.
3A). This antidromic trend directly reflects the dynamic use of alternative carbon sources for isoprene
formation under limited net assimilation.
The incomplete coupling between net assimilation
and isoprene production (Affek and Yakir, 2003) could
be confirmed when, in addition to [U-13C]Glc, 0.25 mM
abscisic acid (ABA) was fed and net assimilation
reduced due to ABA induced stomatal closure. Under
this condition the emission rate of partially labeled
isotopes m701 and m711 increased, indicating an
enhanced use of leaf internal carbon sources. Due to
the higher emission of m701 and m711, the portion of
13
C of total carbon emitted as isoprene significantly
increased to approximately 5% (Fig. 4). A comparable
Fast and Slow Turnover in Isoprene Labeling after
Changing from 13CO2 Exposure to 12CO2 Exposure
and Vice Versa
Following the changes of natural carbon isotope
abundance (Fig. 3A) from 13CO2 to 12CO2, there was
a rapid disappearance of fully labeled isoprene molecules (m741) emitted by poplar leaves and a successive
appearance of, in particular, unlabeled (m691), as well
as single- (m701) and double-13C labeled (m711) molecules. The half-lives (t 1/2) of the more 13C labeled isoprene molecules m731 and m741 were approximately 3
to 6 min, while the t 1/2 of the less 13C labeled isotopes
were significantly longer. A similar rapid exchange of
13
C to 12C in isoprene carbon has also been observed by
Karl et al. (2002a) in aspen and oak leaves. However, the
t 1/2 of isoprene isotopes during the rapid washout of
13
C found in the present experiment were somewhat
longer.
The fast exchange in isoprene isotopes caused by
switching from 13CO2 to 12CO2 exposure was followed
by much slower exchange rates over the next 3 h.
Figure 6. Ratio of isoprene isotopes m711 to m701 during feeding of
13
C labeled Glc. Prior feeding (black bars). Feeding of 5 mM [U-13C]Glc,
[1,2-13C]Glc or [3-13C]Glc, respectively (gray bars). Means of n 5 4
experiments (6SD) are shown; different letters indicate significant
differences at P , 0.05 as calculated with Student’s t test (capital
letters for [U-13C]Glc, lowercase letters for [1,2-13C]Glc, Greek letters
for [3-13C]Glc).
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Alternative Carbon Sources for Isoprene Biosynthesis
Figure 7. Emission of isoprene and its different isotopes (a), the portion
of 13C from total carbon emitted as isoprene (solid line) as well as net
assimilation (b, dashed line) as a consequence of [1,2-13C]Glc application. A leaf was placed into the cuvette and treated as indicated by
the vertical lines. 1, Leaf was cut from the intact plant and the cut end
was inserted into 5 mM [1,2-13C]Glc; 2, 0.25 mM ABA was applied in
addition to 5 mM [1,2-13C]Glc, Data shown represent a typical result of
four independent replicates under similar experimental conditions
(m691, [12C]isoprene; m701, isoprene with one 13C; m711, isoprene
with two 13C).
Single labeling of isoprene (m701) can be explained
by refixation of 13CO2 released by intercellular decarboxylation, e.g. during mitochondrial respiration
(Anderson et al., 1998), photorespiration, or decarboxylation of pyruvate during formation of DOXP, which
is the first intermediate of the plastidic isoprenoid
pathway (Eisenreich et al., 2001). Double 13C labeled
isoprene (m711) can result from plastidic pyruvate
after refixation of 13CO2 but also from fully labeled
pyruvate, decomposed from Glc during glycolysis and
transferred as PEP by the PEP/Pi antiporter system
(Flügge, 1999) into the chloroplast.
The fast occurrence of a fully-labeled isoprene isotope after exposure to 13CO2 (Fig. 3) and vice versa (its
rapid washout after changing to 12CO2) is an indication
either (1) for a rapid chloroplastidic efflux of recently
fixed triose phosphate and reimport of the carbon
skeleton as pyruvate by the above mentioned antiporter system or (2) for a substantial chloroplastidic
formation of pyruvate. Previous work has already
shown that isolated chloroplasts possess the full autonomy for isoprenoid biosynthesis (Schulze-Siebert and
Schultz 1987). To which extent pyruvate originates
from one or the other source has to be elucidated in
further experiments.
increase in 13C utilization has been observed for oak
leaves (Kreuzwieser et al., 2002), when leaf temperature exceeded 35°C to 37°C and net assimilation
uncoupled from isoprene formation.
Feeding of 13C Labeled Glucose via the Xylem Indicates
a Direct Incorporation of a C2 Fragment into Isoprene
The use of [U-13C]Glc fed to poplar leaves via the
xylem for isoprene formation (Fig. 1A) suggests that
13
C enters the chloroplast either as CO2 from
mitochondrial respiration or as metabolic intermediate (e.g. pyruvate/PEP) from the cytosol (Fig. 6;
Kreuzwieser et al., 2002). When excised poplar leaves
were fed with 5 mM [U-13C]Glc, the emission of single
(m701) and double (m711) labeled isoprene molecules
increased within 7 to 10 min, accompanied by a reduced emission of mass m691 (Fig. 6A). As a consequence, the portion of 13C of total carbon emitted as
isoprene increased to approximately 4% (Fig. 6B). Total
emission of isoprene remained unaffected by this
treatment, whereas for net assimilation a transient
decrease was observed while cutting the petiole (Fig.
6B, as also seen in Fig. 3B).
Figure 8. Emission of isoprene and its different isotopes (a), the portion
of 13C from total carbon emitted as isoprene (solid line) as well as net
assimilation (b, dashed line) as a consequence of [3-13C]Glc application. A leaf was placed into the cuvette and treated as indicated by the
vertical line: 1, Leaf was cut from the intact plant and the cut end was
inserted into 5 mM [3-13C]Glc. Data shown represent a typical result of
four independent replicates under similar experimental conditions
(m691, [12C]isoprene; m701, isoprene with one 13C; m711, isoprene
with two 13C).
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Pyruvate-derived carbons in isoprene originate
from the transketolase-like DOXP synthase reaction
in which pyruvate is linked to glyceraldehyde-3-phosphate under cleavage of the 3-C position of pyruvate
(Eisenreich et al., 2001).
To test whether cytosolic Glc loaded from xylem-sap
contributes as pyruvate to this enzymatic step, we fed
leaf petioles with Glc (5 mM) labeled with 13C at either
the C-position 1 and 2 or the C-position 3 (Fig. 7). As
shown in Figure 7A, feeding of [1,2-13C]Glc remarkably increased the portion of the labeled isotope m711
compared to the emission of m701. In particular, the
significant increase of the isotope ratio of m711 to
m701 (Fig. 5) from 0.23 6 0.06 to 0.34 6 0.06 is a strong
indication for a direct incorporation of a C2 fragment
via the proposed mechanism.
The portion of 13C of total carbon emitted as isoprene increased to a level of approximately 4% due to
enhanced emission of isotopes m701 and m711
(Fig. 7B). Application of 0.25 mM ABA to these leaves
caused a reduction of net assimilation (Fig. 7) and an
enhanced incorporation of a double-labeled C2 fragment into isoprene.
Equivalent feeding experiments with 5 mM
[3-13C]Glc (Fig. 8) slightly increased the emission of
single labeled isoprene m701, whereas no incorporation of higher labeled 13C isotopes, in particular of the
double labeled isotope m711, were detected (see Fig.
5). The unchanged m711 to m701 ratio shows that the
probability of a double labeling of isoprene from
refixed 13CO2 seems to be very low. However, feeding
experiments with 13CO2 (F. Loreto, personal communication) showed a relationship between the fraction
of unlabeled isoprene and the fraction of refixed
respiratory CO2, indicating that this may be an alternative source of carbon for isoprene influencing the
completeness of labeling under 13CO2 atmosphere.
The portion of 13C of total carbon emitted as isoprene increased to a level of approximately 2% (Fig.
4A; Fig. 8B). Under the assumption that the probability of double incorporation of 13C from refixed CO2 is
marginal, the increase of the emission of m711 after
[1,2-13C]Glc feeding was used as a measure for the
contribution of cytosolic pyruvate, that itself is derived from xylem-transported Glc. The estimation
showed that under steady state conditions approximately 4% of the pyruvate fragment in the isoprene
molecule originates from xylem-transported Glc. This
value increased up to approximately 9% with the
application of ABA. The range of 4% to 9% indicates
that the cytosolic pool of glycolytic intermediates is
large and tends to be diluted by a continuous glycolytic flux from 12C sugar pools or triose-phosphates
released from the chloroplast, also diluting pyruvate
carbon.
CONCLUSION
The present investigations with poplars clearly
support the idea that beside photosynthetically fixed
CO2 other carbon sources are used for isoprene
formation. These carbon sources contribute to about
20% to 30% of the carbon atoms incorporated into
isoprene. The alternative sources of carbon become
even more important under conditions of limited
photoassimilation of CO2, e.g. upon stomatal closure.
The nature of these carbon sources is quite heterogeneous; about one-third seems to be derived from
xylem transported carbon compounds such as carbohydrates. The rest could be provided either from
starch degradation or from other carbon containing
compounds present in the leaves. Leaf internal cytosolic carbon compounds, as well as carbohydrates
transported via the xylem into the leaves, are probably
delivered to the chloroplast as C3 compounds, as suggested from labeling experiments with [1,2-13C]Glc
and [3-13C]Glc. Future studies should follow two
strategies. First, the interactions between chloroplasts
and the cytosol should be investigated in order to
understand which carbon compounds are shifted
between the two compartments and how these processes are regulated. Second, the nature of carbon
sources other than CO2 and xylem transported Glc
used for isoprene formation should be identified.
MATERIALS AND METHODS
Plant Materials
For all experiments 6-month-old hybrid poplar plants (Populus 3 canescens) were used. Seedlings were amplified by micropropagation under sterile
conditions as described by Leplé et al. (1992). Cultivation proceeded in plastic
pots (12 3 10 3 10 cm) filled with a mixture of perlite, sand, and commercial
potting soil (Floradur Type 1, Floragard, Oldenburg, Germany; 2/1/1; v/v/v)
as a substrate. Plants were fertilized every 2 weeks with 100 mL of a nutrient
solution containing 6 g L21 of a complete fertilizer (Hakaphos Blau; Bayer,
Leverkusen, Germany) and were watered daily with tap water. Plants were
kept under long-day conditions (16 h light exposure) at day and night
temperatures and relative humidity of 20°C 6 3°C and 70% 6 10%,
respectively.
Cuvette Measurements of Photosynthetic
Gas Exchange
Photosynthetic gas exchange was measured using the dynamic cuvette
system described by Kreuzwieser et al. (2002). Light was provided by an
artificial cool light source (KL2500 LCD, Schott, Germany) with mean
intensities of approximately 800 mmol m22 s21 photosynthetic photon flux
density (PPFD) at leaf level. Leaf temperatures were held constant at
approximately 32°C. The cuvette contained sensors for cuvette air temperature and relative humidity (1400-104, Walz, Effeltrich, Germany), leaf temperature (NiCr-Ni temperature transmitters GNTP, Greisinger Electronic
GmbH, Regenstauf, Germany), and PPFD (LI-190SA, LI-COR, Lincoln, NE).
It was flushed continuously with synthetic air containing either 358 6 7 mL L21
CO2 of natural C isotope signature (1.1% 13CO2; Messer, Austria) or 360 mL L21
of 99% 13CO2 (Cambridge Isotope Laboratories, Andover, MA) at a flow rate of
2 L min21. Relative air humidity was kept constant at approximately 50% (see
Kreuzwieser et al., 2002). One fully expanded leaf was placed into the cuvette
for gas exchange measurements. The photosynthetic gas exchange was monitored using a differential infrared absorption analyzer (Li-6262, LI-COR).
To feed the plant with 13C labeled Glc, the cuvette leaves were cut and the
petioles were then immediately placed into aquatic solutions containing either
5 mM [U-13C]Glc, [1,2-13C]Glc, or [3-13C]Glc (Cambridge Isotope Laboratories).
Similar Glc concentrations in the xylem sap of poplar had been found recently
by U. Heizmann (personal communication) under the same conditions as in
the present work.
158
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Alternative Carbon Sources for Isoprene Biosynthesis
Measurement of Stable
with PTR-MS
13
C Isotopes of Isoprene
The PTR-MS technique has been described in great detail elsewhere
(Hansel et al., 1995; Lindinger et al., 1998). The volume mixing ratios (VMRs)
of isoprene and other volatile organic compounds present in the air stream
from the cuvette system were measured with the Innsbruck PTR-MS in an
analog set-up, as described by Kreuzwieser et al. (2002). The total residence
time in the inlet line was less than 2 s, which is far less than the gas exchange
time (,1 min.) of the cuvette (Brüggemann, 2002).
In this study, the PTR-MS technique was used for on-line monitoring of the
13
C isotopes of isoprene, which were detected at protonated isotope masses
691 (12C5H91), 701 (13C12C4H91), 711 (13C212C3H91), 721 (13C312C2H91), 731
(13C412C1H91), and 741 (13C5H91), respectively. At m731 a notable background
due to the H3O1(H2O)3 cluster ions was always present. This enhanced
background caused a somewhat higher detection limit for the 13C412C1H8
isotope but was not critical for the interpretation of the results. The entire
(background corrected) ion signal at these mass-to-charge ratios was converted into VMRs of the given isotope of isoprene. The PTR-MS instrument
was calibrated for isoprene using a calibration standard 7.9 6 0.8 mL L21
isoprene, in N2 (Messer, Griesheim, Germany), which was diluted with
humidified synthetic air (50% relative humidity) to provide isoprene VMRs
in the range of 0.6 to 69 nL L21. The linearity of the PTR-MS instrument was
better than 2%, which was basically equal to the accuracy of the flow dilution
system. The accuracy of the isoprene measurements correspond to the error in
the gas standard, which is 6 10%.
The stable 13C isotopes of isoprene (indicated as mass) and several other
volatile organic compounds were measured on a time shared basis for
5 (m691), 5 (m701), 10 (m711), 10 (m721), 10 (m731), and 20 (m741) s,
respectively, once every 75 s. VMRs of the individual isoprene isotopes were
converted to isoprene emission rates from the known flow through the cuvette
system and the net surface area of plant material in the cuvette. The
percentage rate of 13C labeling was calculated by summing all 13C atoms
present in the detectable isoprene isotopes (e.g. one 13C in m701, two 13C in
m711), relating it to the overall sum of (12C and 13C atoms) isoprene carbon
and multiplying by 100.
Determination of Starch
Starch was analyzed colorimetrically using a commercial test kit (Boehringer, Ingelheim, Germany). For extraction, 50 mg of powdered (under liquid
N2) plant material was added to 1 mL dimethyl sulphoxide (25% [w/v] HCl
80:20, [v/v]). After 30 min of incubation at 60°C, samples were centrifuged
(5 min at 12,000g) and then 200 mL supernatant was added to 1.2 mL ice-cold
0.2 M citrate buffer (pH 10.6). This solution was used for analyses.
Statistical Analysis
Data shown in figures are means (6SD) of four independent experiments
per treatment. Statistical analysis was performed with SPSS for Windows NT
(release 8.0.0; SPSS, Chicago). Statistical significant differences of means at P ,
0.05 were calculated using one-way ANOVA or Student’s t test and are
indicated by different letters above bars. Letters of the same type (uppercase,
lowercase, Greek) above bars indicate the means that have been compared.
ACKNOWLEDGMENT
Armin Wisthaler thanks the Verein zur Förderung der wiss. Ausbildung
und Tätigkeit von Südtirolern an der Landesuniversität Innsbruck for postdoctoral support.
Received December 8, 2003; returned for revision March 19, 2004; accepted
March 23, 2004.
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