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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Contribution of Different Carbon Sources to Isoprene Biosynthesis in Poplar Leaves¹

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.)

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
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 x 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.

Light controls isoprene emission through the production of photosynthetic metabolites and the supply of ATP/NADPH to the chloroplastidic deoxyxylulose-5-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 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 x 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 xylem-derived 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

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

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.

View larger version (28K): [in this window] [in a new window]   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.

It has been proposed (Karl et al., 2002a, 2002b; Affek and Yakir, 2003) that additional carbon for isoprene 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.

View larger version (17K): [in this window] [in a new window]   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 (±SD) are shown; different letters indicate significant differences at P < 0.05 as calculated with Student's t test.

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 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 µL L^{–1 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.

View larger version (12K): [in this window] [in a new window]   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 µmol m–2 s–1) for 3 more days. During light exposure 1 leaf was placed into a cuvette and fumigated with 360 µL L–1 13CO2. Data shown are means of 4 leaves (±SD) exposed to 13CO2. Different letters indicate significant differences at P < 0.05 as calculated with one-way ANOVA.

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 m71⁺ and m72⁺ 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 (m69⁺) 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 xylem-derived 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, xylem-transported plus atmospheric ¹³CO₂ (Fig. 1B; see arrows in Fig. 1A); this can be taken as indirect evidence for a contribution of internal carbon sources.

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 (m74⁺) emitted by poplar leaves and a successive appearance of, in particular, unlabeled (m69⁺), as well as single- (m70⁺) and double-¹³C labeled (m71⁺) molecules. The half-lives (_1/2) of the more ¹³C labeled isoprene molecules m73⁺ and m74⁺ were approximately 3 to 6 min, while the _1/2 of the less ¹³C labeled isotopes were significantly longer. A similar rapid exchange of ¹³C to ¹²C in isoprene carbon has also been observed by Karl et al. (2002a) in aspen and oak leaves. However, the _1/2 of isoprene isotopes during the rapid washout of ¹³C 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. While the partially and fully labeled isotopes slowly disappeared, (see Table I: _1/2 of m72⁺: 148.3 min; _1/2 of m73⁺: 197.8; and _1/2 of m74⁺: 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.

View this table: [in this window] [in a new window]   Table I. Half-lives 1/2 [min] of isoprene isotope masses from the experiment shown in Figure 3

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 ¹³C incorporation into isoprene (Fig. 3B). The observed increase was mainly due to a transiently enhanced emission of the isoprene isotopes m71⁺ and m72⁺ (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 m70⁺ and m71⁺ increased, indicating an enhanced use of leaf internal carbon sources. Due to the higher emission of m70⁺ and m71⁺, the portion of ¹³C of total carbon emitted as isoprene significantly increased to approximately 5% (Fig. 4 ). A comparable 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.

View larger version (18K): [in this window] [in a new window]   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 = 4 experiments (±SD) 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.

The use of [U-¹³C]Glc fed to poplar leaves via the xylem for isoprene formation (Fig. 1A) suggests that ¹³C 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 (m70⁺) and double (m71⁺) labeled isoprene molecules increased within 7 to 10 min, accompanied by a reduced emission of mass m69⁺ (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).

View larger version (14K): [in this window] [in a new window]   Figure 6. Ratio of isoprene isotopes m71+ to m70+ during feeding of 13C 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 = 4 experiments (±SD) 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).

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.

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 m71⁺ compared to the emission of m70⁺. In particular, the significant increase of the isotope ratio of m71⁺ to m70⁺ (Fig. 5 ) from 0.23 ± 0.06 to 0.34 ± 0.06 is a strong indication for a direct incorporation of a C₂ fragment via the proposed mechanism.

View larger version (23K): [in this window] [in a new window]   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 (m69+, [12C]isoprene; m70+, isoprene with one 13C; m71+, isoprene with two 13C).

View larger version (23K): [in this window] [in a new window]   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.

Equivalent feeding experiments with 5 mM [3-¹³C]Glc (Fig. 8 ) slightly increased the emission of single labeled isoprene m70⁺, whereas no incorporation of higher labeled ¹³C isotopes, in particular of the double labeled isotope m71⁺, were detected (see Fig. 5). The unchanged m71⁺ to m70⁺ 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.

View larger version (21K): [in this window] [in a new window]   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 (m69+, [12C]isoprene; m70+, isoprene with one 13C; m71+, isoprene with two 13C).

	CONCLUSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
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

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials

For all experiments 6-month-old hybrid poplar plants (Populus x canescens) were used. Seedlings were amplified by micropropagation under sterile conditions as described by Leplé et al. (1992). Cultivation proceeded in plastic pots (12 x 10 x 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 L^–1 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 ± 3°C and 70% ± 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 µmol m^–2 s^–1 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 ± 7 µL L^–1 CO₂ of natural C isotope signature (1.1% ¹³CO₂; Messer, Austria) or 360 µL L^–1 of 99% ¹³CO₂ (Cambridge Isotope Laboratories, Andover, MA) at a flow rate of 2 L min^–1. 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.

Measurement of Stable ¹³C Isotopes of Isoprene with PTR-MS

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 ¹³C isotopes of isoprene, which were detected at protonated isotope masses 69⁺ (¹²C₅H₉⁺), 70⁺ (¹³C¹²C₄H₉⁺), 71⁺ (¹³C₂¹²C₃H₉⁺), 72⁺ (¹³C₃¹²C₂H₉⁺), 73⁺ (¹³C₄¹²C₁H₉⁺), and 74⁺ (¹³C₅H₉⁺), respectively. At m73⁺ a notable background due to the H₃O⁺(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 ± 0.8 µL L^–1 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 L^–1. 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 ± 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 (m69⁺), 5 (m70⁺), 10 (m71⁺), 10 (m72⁺), 10 (m73⁺), and 20 (m74⁺) 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 m70⁺, two ¹³C in m71⁺), 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 µL 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 (±SD) 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.

	ACKNOWLEDGMENTS

 
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.

	FOOTNOTES

 
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 (Atmosphären-Forschungsprogramm 2000).

² These authors contributed equally to the paper.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037374.

^* Corresponding author; e-mail armin.hansel{at}uibk.ac.at; fax 43–512–507–2932.

	LITERATURE CITED

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Affek HP, Yakir D (2003) Natural abundance carbon isotope composition of isoprene reflects incomplete coupling between isoprene synthesis and photosynthetic carbon flow. Plant Physiol 131: 1727–1736[Abstract/Free Full Text]

Anderson MD, Che P, Song J, Nikolau BJ, Syrkin-Wurtele E (1998) 3-Methylcroronyl coenzyme A carboxylase is a component of the mitochondrial leucine catabolic pathway in plants. Plant Physiol 118: 1127–1138[Abstract/Free Full Text]

Biesenthal TA, Wu Q, Shepson PB, Wiebe HA, Anlauf KG, MacKay GI (1997) A study of relationships between isoprene, its oxidation products, and ozone, in the lower Fraser Valley, BC. Atmos Environ 31: 2049–2058[CrossRef]

Brüggemann N, Schnitzler J-P (2002a) Diurnal variation of dimethylallyl diphosphate concentrations in oak (Quercus robur L.) leaves. Physiol Plant 115: 190–196[CrossRef][Medline]

Brüggemann N, Schnitzler J-P (2002b) Comparison of isoprene emission, intercellular isoprene concentration and photosynthetic performance in water-limited oak (Quercus pubescens Willd. and Quercus robur L.) saplings. Plant Biol 4: 456–463[CrossRef]

Brüggemann N (2002) Untersuchungen zur Regulation der Isoprenbildung bei Eichen. PhD thesis. University of Freiburg, Schriftenreihe des Fraunhofer-Instituts Atmosphärische Umweltforschung, Garmisch-Partenkirchen, Band 70-2002, Aachen, Germany: Shaker Verlag

Chameides WL, Lindsay RW, Richardson J, Kiang CS (1988) The role of biogenic hydrocarbons in urban photochemical smog: Atlanta as a case study. Science 241: 1473–1475[Abstract/Free Full Text]

Delwiche C, Sharkey TD (1993) Rapid appearance of ¹³C in biogenic isoprene when ¹³CO₂ is fed to intact leaves. Plant Cell Environ 16: 587–591[CrossRef]

Eisenreich W, Rohdich F, Bacher A (2001) Deoxyxylulose phosphate pathway of terpenoids. Trends Pharmacol Sci 6: 78–84

Flügge U-I (1999) Phosphate translocators in plastids. Annu Rev Plant Physiol Plant Mol Biol 50: 27–45[CrossRef][Web of Science]

Funk JL, Jones CG, Lerdau MT (1999) Defoliation effects on isoprene emission from Populus deltoides. Oecologia 118: 333–339[CrossRef]

Guenther AB, Hewitt CN, Erickson D, Fall R, Geron C, Graedel T, Harley P, Klinger L, Lerdau M, McKay WA, et al. (1995) A global model of natural volatile organic compound emissions. J Geophys Res 100: 8873–8892[CrossRef][Web of Science]

Hansel A, Jordan A, Holzinger R, Prazeller P, Vogel W, Lindinger W (1995) Proton transfer reaction mass spectrometry: on-line trace gas analysis at ppb level. Int J Mass Spectrom 149: 609–619[CrossRef]

Heizmann U, Kreuzwieser J, Schnitzler J-P, Brüggemann N, Rennenberg H (2001) Assimilate transport in the xylem sap of pedunculate oak (Quercus robur) seedlings. Plant Biol 3: 132–138[CrossRef]

Karl T, Fall R, Rosenstiel TN, Prazeller P, Larsen B, Seufert G, Lindinger W (2002a) On-line analysis of the ¹³CO₂ labeling of leaf isoprene suggests multiple subcellular origins of isoprene precursors. Planta 215: 894–905[CrossRef][Web of Science][Medline]

Karl T, Curtis AJ, Rosenstiel TN, Monson RK, Fall R (2002b) Transient releases of acetaldehyde from tree leaves – products of a pyruvate overflow mechanism? Plant Cell Environ 25: 1121–1131[CrossRef]

Kelly GJ, Latzko E (1995) Photosynthesis. In HD Behnke, U Lüttge, K Esser, JW Kadereit, M Runge, eds, Progress in Botany: Structural Botany, Physiology, Genetics, Taxonomy, Geobotany, Vol 56. Springer-Verlag, Berlin, pp 134–163

Kesselmeier J, Staudt M (1999) Biogenic volatile organic compounds (VOC): an overview on emission, physiology and ecology. J Atmos Chem 33: 23–88

Kreuzwieser J, Kühnemann F, Martis A, Rennenberg H, Urban W (2000) Diurnal pattern of acetaldehyde emission by flooded poplar trees. Physiol Plant 108: 79–86[CrossRef]

Kreuzwieser J, Graus M, Wisthaler A, Hansel A, Rennenberg H, Schnitzler J-P (2002) Xylem-transported glucose as additional carbon source for leaf isoprene formation in Quercus robur. New Phytol 156: 171–178[CrossRef]

Leplé JC, Brasileiro ACM, Michel MF, Delmotte F, Jouanin L (1992) Transgenic poplars: expression of chimeric genes using four different constructs. Plant Cell Rep 11: 137–141

Lindinger W, Hansel A, Jordan A (1998) Proton-transfer-reaction mass spectrometry (PTR-MS): on-line monitoring of volatile organic compounds at pptv levels. Chem Soc Rev 27: 347–354

Loreto F, Ciccioli P, Cecinato A, Brancaleoni E, Frattoni M, Fabozzi C, Tricoli D (1996) Evidence of the photosynthetic origin of monoterpenes emitted by Quercus ilex. L. leaves by C¹³ labeling. Plant Physiol 110: 1317–1322[Abstract]

Monson RK, Harley PC, Litvak ME, Wildermuth M, Guenther AB, Zimmerman PR, Fall R (1994) Environmental and developmental controls over the seasonal pattern of isoprene emission from aspen leaves. Oecologia 99: 260–270[CrossRef][Web of Science]

Sanadze GA (1991) Isoprene effect: light-dependent emission of isoprene by green parts of plants. In GA Sanadze, ed, Trace Gas Emissions by Plants. Academic Press, San Diego, pp 135–152

Sanadze GA, Dzhaiani GI, Tevzadaze IM (1972) Incorporation into the isoprene moelcule of 13CO2 assimilated during photosynthesis. Sov Plant Physiol 19: 17–20

Schulze-Siebert D, Schultz G (1987) Full autonomy in isopenoid synthesis in spinach chloroplasts. Plant Physiol Biochem 25: 145–153

Sharkey TD, Singsaas EL, Vanderveer PJ, Geron C (1996) Field measurements of isoprene emission from trees in response to temperature and light. Tree Physiol 16: 649–654

Sharkey TD, Yeh S (2001) Isoprene emission from plants. Annu Rev Plant Phys Plant Mol Biol 52: 407–436[CrossRef][Web of Science][Medline]

Stitt M, Heldt HW (1981) Simultaneous synthesis and degradation of starch in spinach chloroplasts in the light. Biochim Biophys Acta 638: 1–11[CrossRef]

Thompson AM (1992) The oxidizing capacity of the Earth's atmosphere: probable past and future changes. Science 256: 1157–1165[Abstract/Free Full Text]

Zimmer W, Brüggemann N, Emeis S, Giersch C, Lehning A, Steinbrecher R, Schnitzler J-P (2000) Process-based modeling of isoprene emission by oaks. Plant Cell Environ 23: 585–597[CrossRef]

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