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First published online August 26, 2005; 10.1104/pp.105.066373 Plant Physiology 139:474-484 (2005) © 2005 American Society of Plant Biologists Diurnal and Seasonal Variation of Isoprene Biosynthesis-Related Genes in Grey Poplar Leaves1Forschungszentrum Karlsruhe GmbH, Institut für Meteorologie und Klimaforschung, Atmosphärische Umweltforschung, 82467 Garmisch-Partenkirchen, Germany
Transcript levels of mRNA from 1-deoxy-D-xylulose 5-phosphate reductoisomerase (PcDXR), isoprene synthase (PcISPS), and phytoene synthase (PcPSY) showed strong seasonal variations in leaves of Grey poplar (Populus x canescens [Aiton] Sm.). These changes were dependent on the developmental stage and were strongly correlated to temperature and light. The expression rates of the genes PcDXR and PcISPS were found to be significantly correlated to each other, whereas the expression of the PcPSY gene showed a different seasonal pattern. Protein concentration and enzyme activity of PcISPS showed distinct seasonal patterns peaking in late summer, whereas highest transcription levels of PcISPS were observed in early summer. Moreover, correlation between PcISPS protein concentration and enzyme activity changed, in particular in autumn, when PcISPS protein levels remained high while enzyme activity declined, indicating posttranslational modifications of the enzyme. The positive correlation between dimethylallyl diphosphate levels and PcISPS protein content was found to be consistent with the demonstrated synchronized regulation of PcDXR and PcISPS, suggesting that metabolic flux through the 1-deoxy-D-xylulose 5-phosphate pathway and isoprene emission capacity are closely intercoordinated. Transcript levels of PcISPS showed strong diurnal variation with maximal values before midday in contrast to PcDXR, whose gene expression exhibited no clear intraday changes. During the course of a day, in vitro PcISPS activities did not change, whereas leaf dimethylallyl diphosphate levels and isoprene emission showed strong diurnal variations depending on actual temperature and light profiles on the respective day. These results illustrate that the regulation of isoprene biosynthesis in Grey poplar leaves seems to happen on transcriptional, posttranslational, and metabolic levels and is highly variable with respect to seasonal and diurnal changes in relation to temperature and light.
Isoprene (2-methyl-1,3-butadiene) is a volatile organic compound naturally emitted by many tree species and has a significant influence on atmospheric chemistry (Thompson, 1992  ; Biesenthal et al., 1997; Derwent et al., 1998). Different hypotheses are discussed to explain the functional role of isoprene emission for the plant itself, either to prevent leaf metabolic processes from thermal (Sharkey and Singsaas, 1995; Singsaas et al., 1997; for overview, see Sharkey and Yeh, 2001) and oxidative stress (Loreto and Velikova, 2001; Loreto et al., 2001) or to serve as an overflow mechanism for excess of carbon intermediates or photosynthetic energy (Logan et al., 2000; Rosenstiel et al., 2004).
Isoprene emission is controlled at least at two different levels. Intraday variations of isoprene emissions can be explained on the one hand by the synthesis of isoprene synthase (ISPS) substrate, mainly originating from recently fixed CO2. On the other hand, they are caused by the temperature dependence of ISPS activity (Eisenreich et al., 2001
So far, the unraveling of the control of isoprene formation has been mainly focused on biochemical and physiological investigations. They show that long-term, seasonal variations in isoprene emission capacity (Monson et al., 1994
The identification of the regulatory steps of the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway is of major importance in the study of isoprene biosynthesis. Currently, not much is known about the regulation of genes involved in isoprene biosynthesis. Very recently, Wiberley et al. (2005)
In plastids, isopentenyl diphosphate and dimethylallyl diphosphate (DMADP) are formed via the DOXP pathway. The first step is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which is encoded by the DXS gene. The following conversion of DOXP to methyl erythritol 5-phosphate is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the product of the DXR gene. DOXP is a precursor not only for isoprenoids but also for thiamine diphosphate and pyridoxal phosphate (Julliard and Douce, 1991 However, detailed information on transcriptional regulation at different stages of plastidic isoprenoid biosynthesis during leaf development and the growing season is still lacking. In particular, comprehensive analysis of gene expression of ISPS related to protein level, enzyme activity, concentration of DMADP, and isoprene emission has not been investigated.
This study was therefore conducted in Grey poplar (Populus x canescens [Aiton] Sm.) leaves to examine whether transcript levels of PcDXR and PcISPS follow diurnal and seasonal changes. Another aim was to determine if these changes are related to diurnal and seasonal variations of PcISPS activity and the level of its substrate DMADP. In addition, we investigated their relationship to the diurnal variations of isoprene emission and seasonal variation of phytoene synthase gene (PcPSY) expression (encoding a key enzyme in carotenoid biosynthesis [Von Lintig et al., 1997
Isolation and Functional Analysis of PcDXR cDNA from Grey Poplar To isolate a full-length cDNA clone encoding DXR from Grey poplar, initially a 309-bp segment was amplified by PCR using heterologous oligonucleotide primers for conserved sequences of other known DXR genes from plants. The resulting sequence of the PCR product showed highest homology to DXR from Lycopersicon esculentum Mill. (EMBL AF331705: 82.03% identity) on the nucleic acid level. Using this segment as a probe for hybridization with a poplar cDNA library, a cDNA clone was isolated harboring an open reading frame (ORF) of 1,724 bp with high homology to other known DXR genes, e.g. Arabidopsis (Arabidopsis thaliana; EMBL AF148852: 86.23%), Catharanthus roseus L. G. Don (EMBL AF250235: 86.23%), L. esculentum (EMBL AF331705: 84.75%), and Zea mays (EMBL AJ297566: 82.20%). The deduced amino acid sequence consisted of 472 amino acids and a putative molecular mass of about 51 kD. The EMBL accession number of the complete cDNA is AJ574852.
The coding sequence, including the sequence for the putative plastidic transit peptide at the 5'-end, was amplified by PCR, introducing Gateway recombination sequences, and cloned into the expression vector pDEST17 to transform Escherichia coli BL21-Star. The heterologously expressed PcDXR enzyme was purified under native conditions. Protein extracts were assayed indirectly for PcDXR activity by measuring spectrophotometrically the oxidation of NADPH to NADP. The apparent PcDXR activity of the heterologously expressed protein was 6.87 mkat kg–1 protein. Enzyme specificity was further tested by the addition of fosmidomycin (1 mM), a competitive inhibitor of DXR, to the enzyme assay. This application resulted in an inhibition of approximately 85% of PcDXR activity, confirming data on DXR from other species (e.g. the cyanobacterium Synechococcus leopoliensis; Miller et al., 2000
The present molecular biological and biochemical data on poplar show a clear seasonal variation (Fig. 1) of PcDXR and PcISPS transcript levels, PcISPS protein amount, enzyme activity, and total leaf DMADP levels dividing the growing season into four periods: springtime, early summer, late summer, and autumn.
During the first 4 weeks of the growing season, transcript levels of PcISPS and PcDXR were low. However, levels increased very rapidly during the first strong temperature increase at beginning of June, such that values 8-fold higher were found (Fig. 1A). Over the early summer period, transcript levels of both genes remained high but fluctuated strongly, correlating with temperature and light variations (Fig. 1, A and B). In late summer and autumn, the daily mean air temperature and transcript levels correlate most obviously (Fig. 1A). The variation among individual trees was smaller in these periods as compared to in early summer. Regression analysis (Table I) reveals that transcript levels of PcDXR and PcISPS are significantly correlated to each other, indicating a synchronized regulation of gene expression. Also, expression levels of both genes showed a positive linear correlation (P  0.002) to actual mean air temperature values on the respective sampling days (Table I). Moreover, PcISPS gene expression correlated with light period means of PPFD.
Transcript levels of PcPSY, encoding a key enzyme in carotenoid biosynthesis (Von Lintig et al., 1997 ), were one order of magnitude lower and different concerning the seasonal trend when compared with transcript levels of PcDXR and PcISPS (Fig. 1B). High transcript levels were present during leaf development, decreasing for 3 weeks and rising again with the temperature increase at beginning of June. Over the summer period, the gene expression pattern of PcPSY was different than that of PcDXR and PcISPS. In autumn, however, the values varied in a similar way to PcDXR and PcISPS with respect to temperature. No correlation of PcPSY transcript levels with seasonal changes of light and temperature was detectable (Table I). Amounts of PcISPS protein and enzyme activity (Fig. 1C) also showed a strong seasonal dependency, which can again be divided into four periods. During leaf development in May, only minimal amounts of PcISPS but no PcISPS activity were detectable. This observation coincides with the very low PcISPS transcript levels at that time point. Parallel to the strong increase of PcISPS transcript copy numbers in June, the amount of PcISPS protein increased constantly, reaching values of approximately 10 ng ISPS µg protein–1. However, PcISPS activity became detectable but remained constant at low values of approximately 0.5 nmol m–2 s–1. During late summer, the amount of PcISPS protein and PcISPS activity increased strongly within a few days. Following this increase, the level of PcISPS protein remained constant until the last sampling in October 2004. In contrast to this, PcISPS activity decreased in autumn in parallel to the decline of photosynthetic pigments (Fig. 1D). Determination of the turnover number (kcat [s–1]) of PcISPS confirms that protein content and enzyme activity of ISPS are not strictly related to each other (Fig. 1C). In early summer, the kcat was low (approximately 0.004 s–1), whereas in late summer, when protein and activity levels reached their maximum, a mean kcat of 0.022 s–1 was determined. In autumn, the kcat of ISPS dropped again to approximately 0.007 s–1 due to the decline of PcISPS activity.
Midday levels of total leaf DMADP concentration (Fig. 1D) correlate well with changes in PcISPS protein (Fig. 1C; see also Table I), with low DMADP contents, and with PcISPS protein (activity) levels during springtime. All values increased over summer, and DMADP as well as PcISPS protein concentrations remained high in autumn. Total leaf DMADP amount was found to be highly variable (Figs. 3 and 4), correlating with light and temperature during daytime, even if the values represent the total amount of DMADP of the leaves not distinguishing between cytosolic and plastidic pools. Hence, the present midday levels of DMADP only represent snapshots depending on the intrinsic climatic and physiological conditions at the sampling date (see also Brüggemann and Schnitzler, 2002b
The importance of ISPS activity for the magnitude of isoprene emission is shown in Figure 2. In the experiment, mature poplar leaves had been acclimated in leaf cuvettes to different leaf temperatures (15°C–38°C) under saturating PPFD. The comparison of isoprene emission rate and ISPS activity adjusted to the respective leaf temperature using the temperature profile of poplar ISPS activity (Schnitzler et al., 2005 ) demonstrates the clear dependence of isoprene emission on ISPS activity actually present in the individual leaf.
Daily Variation of Transcript Levels of PcDXR and PcISPS in Relation to PcISPS Activity and Isoprene Emission
On two consecutive days in August 2001 (Fig. 3) and 2002 (Fig. 4), diurnal changes in isoprene emission, transcript levels of important genes in the DOXP pathway, and PcISPS activity were acquired twice. Emission measurements were performed on two trees, while leaf material for the molecular and biochemical characterization was collected from four different comparable trees. Five sampling points each day (predawn, morning, noon, afternoon, and night) were chosen for analysis (for experimental details, see Mayrhofer et al., 2004 Isoprene emission showed a typical daily variation with a light- and temperature-dependent increase in the morning and a decline of the emission rate during the night (Figs. 3B and 4B). A similar trend could be observed for the leaf DMADP pool. Maximum daily values were reached by noon with 74 to 93 pmol mg dry weight–1 in 2001 (Fig. 3B) and 115 to 212 pmol mg dry weight–1 in 2002 (Fig. 4B).
Transcript levels of PcISPS showed a remarkable intraday variation with a clear maximum in the morning on all four investigated days, although the absolute amounts varied from day to day (Figs. 3A and 4A). In 2001, relative transcript levels of PcISPS peaked on August 7th at approximately 0.2 copies Normalization of maximum (set to 1) and minimum (set to 0) levels of each day (Fig. 5) revealed that PcISPS transcript levels significantly peak in the morning and decrease continuously.
The increase in the PcISPS transcript level was not reflected directly in an increase of ISPS activity. In fact, in vitro activity of PcISPS did not change dramatically during the course of the day. During the first sampling period in 2001, in vitro ISPS activities were slightly enhanced at night (Figs. 3C and 4C). In contrast to PcISPS expression, the relative transcription rates of PcDXR of all 4 d investigated gave no clear evidence for a higher/induced transcription during light periods (Figs. 3A, 4A, and 5). In fact, PcDXR expression rates were found to oscillate over the day, but in three of the four days values increased to the maximum at the latest sampling point of the day (Fig. 5).
Several studies (Monson et al., 1994 ; Kempf et al., 1996; Funk et al., 2005) have observed a strong seasonal variation of isoprene emission rates, with increases in springtime that rapidly decline in autumn. Kuzma and Fall (1993) and Lehning et al. (2001) correlated these changes mainly to the seasonal development of ISPS activity, adjusting the constitutive isoprene emission capacity (Fall, 1999). This study on poplar, which employed molecular biological and biochemical experiments, also showed clear seasonal variation during the growing season. Gene expression rates of isoprenoid biosynthesis-related genes in poplar leaves are highly variable over the growing season, with temperature and light as the most obvious factors controlling transcript levels in fully developed leaves.
During leaf expansion, transcript levels of PcISPS and PcDXR were low; however, levels increased very rapidly after approximately 4 weeks at the beginning of June. The delayed increase in transcript level of poplar ISPS is consistent with recent data on kudzu leaves, where it was found that ISPS gene expression also increased after a certain lag phase (Wiberley et al., 2005
This assumption is confirmed by the study of Lehning et al. (2001)
ISPS gene expression in poplar undergoes a strong diurnal variation with maximum levels before midday, when the temperature has not necessarily reached its daily maximum. This observation confirms an earlier study from Arimura et al. (2004) It has to be analyzed in future experiments to which extent gene expression of PcISPS is triggered by photoperiod and light intensity. In addition, the role of temperature has to be elucidated in this process. Does temperature influence the intraday variation, or does it serve as an important trigger that determines the maximum level of transcript? The significant correlation between daily mean temperatures and PcISPS transcript levels observed during the long-term study supports this hypothesis. In addition to the seasonal variation of PcISPS transcript level, a strong seasonal dependence of PcISPS and enzyme activity, which divided the growing season into four periods, could be demonstrated. Indeed, transcript, protein, and enzyme activity levels were not completely identical over the growing season. Therefore, no statistically significant correlation between both parameters could be found, indicating that besides transcriptional control other parameters influence isoprene biosynthesis in poplar leaves.
Changes of ISPS activity over the growing season have also been reported for different species by Kuzma and Fall (1993)
There did not appear to be a strict correlation between the enzyme activity and the PcISPS protein levels in poplar leaves over the growing season. Therefore, different apparent turnover numbers (kcat [s–1]) of PcISPS were determined. In contrast to Silver and Fall (1995) The changes in the apparent kcat values indicate that effects on the protein level influence PcISPS activity. Consequently, a variable portion of active and inactive PcISPS protein is building up in the leaves over the vegetation period, in particular in autumn after the onset of senescence. At that time, ISPS protein levels remained high while ISPS activity was declining.
In accordance with this assumption, Schnitzler et al. (2005)
There are two possibilities as to how the activation of plastidic PcISPS could be achieved. Activation can be driven by a light-dependent shift of stromal conditions (pH, Mg2+ concentration) to the optimal range of PcISPS activity (for details about poplar PcISPS, see Schnitzler et al., 2005
DMADP levels on the daily scale show that dark-adapted leaves contained approximately 30% of the maximum DMADP level of light-adapted leaves, as was found by Fisher et al. (2001)
The significant positive correlation between PcISPS protein and leaf DMADP levels indicates that the metabolic flux through the plastidic DOXP pathway (Eisenreich et al., 2001
With respect to this assumption, future studies will have to ascertain how the different fluxes of metabolic intermediates during certain stages of leaf ontogenesis are regulated on a molecular and/or biochemical level to sustain the individual demands of the different branches of plastidic isoprenoid biosynthesis. Several investigations support that DXS and DXR play an important role in the control of plant isoprenoid biosynthesis. To date, the rate-limiting step is unknown, although molecular engineering revealed that overexpression of DXS (Estévez et al., 2001 Although the present data manifest that gene expression of PcDXR and PcISPS vary in relation to day-to-day changes of temperature and light intensity, further efforts are needed to clarify the linkage between gene expression of PcDXS, PcDXR, and PcISPS, actual protein levels, and enzyme activities. In particular, laboratory studies under controlled conditions are necessary to analyze the chronological interrelation of gene expression, protein biosynthesis, posttranslational modifications of PcISPS protein, and protein turnover to manifest isoprene emission rates.
Plant Material and Experimental Design
Intraday experiments were performed in special greenhouses (solar domes) with 4-month-old Grey poplar plants (Populusxcanescens [Aiton] Sm.), cultivated by micropropagation (Leplé et al., 1992 Temperature experiments under controlled conditions were performed in addition. Poplar leaves were adjusted to different leaf temperatures (15°C–38°C) under saturating PPFD (400–700 µmol photons m–2 s–1) in leaf cuvettes. After reaching a stable leaf temperature, gas exchange and isoprene emission rates were measured for 45 to 60 min. Leaves from inside the cuvette were then rapidly removed and shock frozen in liquid N2 for biochemical analysis. The seasonal study was performed with 4-year-old poplar plants of the same origin. In spring 2001, saplings were planted in 10-L pots containing commercial garden soil and cultivated in the garden of the Institut für Meteorologie und Klimaforschung, Atmosphärische Umweltforschung in Garmisch-Partenkirchen, Germany. Plants were irrigated regularly, trimmed each spring, and fertilized in May by applying 20 g Osmocote (Spiess). From May to October 2004, mixed leaf samples were taken from three trees simultaneously in intervals of 1 to 3 weeks. Sampling was performed exactly at 12:30 PM CET by shock freezing four comparable mature leaves per tree in liquid nitrogen. During the entire growing season, half-hour means of air temperature (HP-100-A; Imko) and PPFD (quantum sensor Li-190SA; LI-COR) were monitored.
Photosynthetic gas exchange and rates of isoprene emission of leaves were measured in real time with a leaf cuvette system on twigs of two plants simultaneously during each sampling period according to Mayrhofer et al. (2004)
ISPS activity was assayed as previously described by Lehning et al. (1999)
The procedure for the determination of DMADP and photosynthetic pigments was as described by Brüggemann and Schnitzler (2002b)
Quantification of ISPS protein was performed according to Schnitzler et al. (2005) The ELISA was set up according to the QIAexpress and Assay Handbook (Qiagen). Ninety-six-well microtiter plates (Greiner) with high protein binding capacity were precoated overnight at 4°C with anti-PcISPS-IgG diluted 1:500 in phosphate-buffered saline (PBS; 50 mM NaPi, pH 7.2, 140 mM NaCl). Before the next step and in between all following steps, the wells were washed four times with 200 µL PBS-Tween (0.1% [v/v] in PBS) for 1 min each. In the second step, residual protein binding sites were blocked with BSA (0.2% [w/v] in PBS) for 1 h. For binding of ISPS, protein samples with a total of 2.5 µg protein in 200 µL PBS per well were incubated at room temperature for 1 h and carefully agitated (Titramax 100; Heidolph Instruments). Thereafter, the plates were incubated for 1 h with the second anti-PcISPS-IgG antibody, which was conjugated with HRP. After the final washing, each well was filled with 200 µL of a ready-to-use staining solution containing the HRP substrate tetra-methylbenzidine. The plates were incubated at room temperature for 45 min. After that time the staining intensity was enhanced by the addition of 10 µL 2 N H2SO4. The resulting yellow-colored complex was analyzed with a plate reader (Tecan Spectra Image; SLT) at 450 nm. For calibration, purified 6x His-tagged PcISPS protein was used in a range of 10 to 250 ng protein well–1. Each sample and standard was analyzed in triplicates.
To isolate the DXR gene from Grey poplar, a Oligonucleotide primers (Roth) were designed for conserved homologous regions of the deduced amino acid sequences of DXR genes from Oryza sativa (EMBL AF367205), Zea mays (EMBL AJ297566), Arabidopsis (Arabidopsis thaliana; EMBL AF148852), Lycopersicon esculentum (EMBL AF331705), and Mentha piperita (EMBL AF116825). The forward oligonucleotide primer [5' gac atc gtc gc(agct) ga(ag) aa(tc) cc(agct) g 3'] and the reverse oligonucleotide primer [5' gct atg tcc ttc cc(agct) gc(tc) tc(agt) at(agct) gc 3'] were used in PCR (PCR conditions: hot start at 95°C for 30 s; annealing, two cycles at 48°C for 30 s followed by 40 cycles at 52°C; extension at 72°C for 30 s; and denaturation 30 s at 95°C) to produce a 300-bp product. After cloning and sequencing, the amplified product was used for screening of the cDNA library. The product was labeled by the incorporation of digoxigenin-labeled nucleotides and used for hybridization according to the DIG Nucleic Acid labeling and detection kit (Roche Diagnostics).
PCR was performed with Grey poplar cDNA using degenerated oligonucleotide primer [forward oligonucleotide primer, 5' (ac)(ag) a a(ag)(ag) gc(gt) (ag) t(act) tgg gc(at) at 3'; reverse oligonucleotide primer, 5' tct c(acg) g c(cgt)(at) (ct)(ag) t c(ag) a aga ac 3'] designed by the alignment of diverse PSY gene sequences from the literature. Two clones of the 549-bp amplicon were sequenced from both directions, resulting in a unique sequence (EMBL AJ889824) with high homology to characterized PSY genes as the ones from Daucus carota (EMBL AB032797), Citrus unshiu Marc. (EMBL AF220218), Tagetes erecta (EMBL AF252015), and Citrus sinensis L. Osbeck (EMBL AY669084).
Cycle sequencing dideoxy chain termination reactions with Big Dye Terminators (Applied Biosystems) were performed for both cDNA strands of all DNA segments investigated, using universal forward and reverse oligonucleotide primers (Invitrogen) or sequence-specific oligonucleotide primers. The sequences were analyzed by using an ABI PRISM System 310 (Applied Biosystems).
Cloning of the full-length PcDXR cDNA into an expression vector was performed using Gateway technology (Invitrogen). The PcDXR sequence including start and stop codons was amplified from the isolated cDNA clone by PCR using primers with attB sites (forward oligonucleotide primer, 5' g ggg aca agt ttg tac aaa aaa gca ggc ttg atg gca ctt aat att cta tct cca g 3'; reverse oligonucleotide primer, 5' gg gga cca ctt tgt aca aga aag ctg ggt tca agc aaa aac agg act tgg 3'). The underlined nucleotides are gene specific; italicized nucleotides have been integrated to get an ORF. PCR was performed with 1 unit Platinum Taq Polymerase High Fidelity (Invitrogen). PCR conditions were as follows: hot start at 96°C for 3 min; annealing, four cycles at 55°C for 45 s followed by 19 cycles at 65°C; extension at 72°C for 2 min; and denaturation 45 s at 96°C. The amplicon was recombined into the vector DONR 221 (Invitrogen) by BP recombination reaction according to the Gateway protocol and transformed into E. coli Top 10 (Invitrogen). After verification of the sequence, the ORF segment was transferred into pDEST17 expression vector with an N-terminal His-encoding region (Invitrogen) by a LR recombination reaction according to the Gateway protocol and transformed into E. coli BL21-Star (Invitrogen) for protein expression.
Purification of the His-tagged protein was performed according to the QIAexpress Type IV protocol (Qiagen), except that E. coli cells were disrupted by a French pressure cell press (SLM Instruments) at 140 MPa and 0°C to 4°C as described by Miller et al. (2000)
After affinity chromatography on a Ni-NTA column according the manufacturer's instructions (Qiagen), 2.5 mL of eluate was desalted on a PD-10 column (Pharmacia) into 3.5 mL assay buffer (150 mM Tris/HCl, pH 7.0, with 5 mM MgCl2, 1 mM thiamine diphosphate, 5 mM
Total RNA from fresh leaves was isolated with Tri-Reagent (Sigma) according to the manufacturer's protocol. From leaves stored for more than 1 year at –80°C, total RNA was prepared with Qiagen RNeasy minikit (Qiagen) following the Qiagen standard protocol. The amount and purity of isolated RNA were determined spectrophotometrically. For first-strand cDNA synthesis, 3 µg of total RNA was reverse transcribed using oligo(dT) primers and Superscript II reverse transcriptase (Invitrogen) in a total volume of 20 µL according to the manufacturer's protocol. cDNA was stored at –20°C prior to analysis.
For quantitative PCR measurements of the transcription rates of PcISPS (EMBL AJ294819), PcDXR, and PcPSY, the following oligonucleotide primer sets were used: for PcISPS, forward 5' ttt gcc tac ttt gcc gtg gtt caa aac 3' and reverse 5' tcc tca gaa atg cct ttt gta cgc atg 3'; for PcDXR, forward 5' gca tat gtc ttt tcc agc ttc tat tgc 3' and reverse 5' gga ata gta ggt tgc gca ggc 3'; for PcPSY, forward 5' atg cat cac ata tca cac cca aa 3' and reverse 5' ctc cta gca tct tct cca aca tct c 3'. The resulting PCR segment lengths were 197 bp (PcISPS), 66 bp (PcDXR), and 379 bp (PcPSY), respectively. SYBR Green was used as a fluorescent marker for the increasing amount of double-stranded DNA. The assays contained 12.5 µL 2x SYBR Green PCR Master Mix (Applied Biosystems), 300 nM of each primer, and 5 µL of total cDNA (diluted 7-fold) in a final volume of 25 µL. After a "hot start" (10 min, 95°C), 45 PCR cycles were performed with a 15-s melting step at 95°C and a 1-min annealing/extension step at 60°C on a GeneAmp 5700 sequence detection system (Applied Biosystems). For internal normalization, transcription rates of poplar PcTUB (EMBL AY353093) with forward primer 5' gat ttg tcc ctc gcg ctg t 3' and reverse primer 5' tcg gta taa tga ccc ttg gcc 3' were determined and used.
Statistical and correlation analysis was performed with SPSS for Windows NT (release 8.0.0) and Sigmaplot 2000 for Windows (Version 6.10), both programs from SPSS. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AJ574852 and AJ889824.
We greatly acknowledge the provision of a PcTUB fragment of poplar by S. Kopriva (University of Freiburg, Germany, now Norwich Research Park, UK), and the critical reading of the manuscript by B. Miller (University of Freiburg, Germany). We further acknowledge the stylistic proofreading by I.H. Franke-Whittle (University of Innsbruck, Austria). Received May 30, 2005; returned for revision June 20, 2005; accepted June 20, 2005.
1 This work was supported by national (joint research project Atmosphären-Forschungsprogramm 2000 [Bundesministerium für Bildung und Forschung], research group "Poplar: A Model to Address Tree-Specific Questions" [Deutsche Forschungsgemeinschaft]) and international (EU Marie Curie Research Training Networks ISONET) research programs. 
2 Present address: Department of Microbiology, University of Innsbruck, Technikerstr. 25, 6020 Innsbruck, Austria. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.066373. * Corresponding author; e-mail robert.fischbach{at}imk.fzk.de; fax 49–8821–73573.
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-tubulin (PcTUB)–1, whereas during the second day maximum values of approximately 0.6 copies PcTUB–1 were reached. The relative PcISPS transcript level in the morning was therefore on average at least 6 times higher than the corresponding predawn value. In 2002, relative expression levels of PcISPS were 9 times higher than in 2001 with a relative transcript level of approximately 5.3 copies PcTUB–1, within the peak of the second day. 
-terpinene synthase from Thymus vulgaris (Alonso and Croteau, 1991
-phage cDNA library (Miller et al., 2001
