Journal of Experimental Botany, Vol. 52, No. 362, pp. 1817-1826,
September 1, 2001
© 2001 Oxford University Press
Original Papers |
Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues
1 Botanisches Institut, Universität Basel, Hebelstrasse 1, CH-4056 Basel, Switzerland
2 Max-Planck-Institute of Molecular Plant Physiology, D-14424 Potsdam, Germany
Received 23 March 2001; Accepted 7 June 2001
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Abstract |
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Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography–mass spectrometry (GC–MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE–PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT–PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.
Key words: Arabidopsis, trehalose, trehalose-6-phosphate phosphatase, trehalose-6-phosphate synthase, yeast complementation.
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Introduction |
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Trehalose (
-D-glucopyranosyl-1,1--D-glucopyranoside) is present in a large variety of microorganisms and invertebrate animals (Elbein, 1974
) where it can serve as a reserve carbohydrate and as a stress protectant (Crowe et al., 1984; Wiemken, 1990). The occurrence of trehalose has also been documented for the desiccation-tolerant plants, Myrothamnus flabellifolia (Bianchi et al., 1993; Drennan et al., 1993) and Selaginella lepidophylla (Adams et al., 1990), whereas reports on the occurrence of trehalose in many other plant species were ambiguous (for review see Müller et al., 1995a).
In yeast, trehalose is synthesized in a two-step process: First trehalose-6-phosphate is formed from UDP-glucose and glucose-6-phosphate in a reaction catalysed by trehalose-6-phosphate synthase (TPS), then trehalose-6-phosphate is hydrolysed to trehalose in a reaction catalysed by trehalose-6-phosphate phosphatase (TPP). The Saccharomyces cerevisiae genes for trehalose synthesis, ScTPS1 (encoding TPS) and ScTPS2 (encoding TPP), have been cloned (Bell et al., 1992; Vuorio et al., 1993; De Virgilio et al., 1993). Recently, a cDNA from Selaginella lepidophylla exhibiting homologies to ScTPS1 (SlTPS1; Zentella et al., 1999) and Arabidopsis cDNAs exhibiting homologies to ScTPS1 (AtTPS1; Blázquez et al., 1998) and to ScTPS2 (AtTPPA and AtTPPB; Vogel et al., 1998) were isolated. Transformation of the respective S. cerevisiae mutants with the Arabidopsis cDNAs resulted in the complementation of growth and in trehalose formation. These findings prompted speculation that trehalose may be endogenously produced in Arabidopsis. Since trehalose induces starch accumulation in the shoots of Arabidopsis seedlings and, at the same time, inhibits root elongation (Wingler et al., 2000; Fritzius et al., 2001), it is conceivable that endogenously formed trehalose acts as a signal molecule in the regulation of plant metabolism and development. In addition, it has been suggested that the precursor of trehalose, trehalose-6-phosphate, may play a role in the regulation of carbohydrate metabolism and of sugar sensing in plants (Goddijn and Smeekens, 1998).
Recently, a compound occurring in Arabidopsis was tentatively identified as trehalose (Müller et al., 2001). In the present study, sugar extracts from axenically grown plants were analysed by gas chromatography–mass spectrometry (GC–MS) in order to provide unequivocal evidence that trehalose synthesis occurs in Arabidopsis. In addition, full-length cDNAs of Arabidopsis genes exhibiting homologies to TPS and TPP genes were cloned. In order to analyse their enzymatic function and their involvement in trehalose synthesis, these cDNAs were expressed in the respective S. cerevisiae mutants as well as in tobacco protoplasts.
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Materials and methods |
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Plant material and growth conditions
Arabidopsis thaliana (L.) Heynh. (ecotypes Landsberg erecta and Ws-2) seeds were sterilized and germinated on vertically oriented Petri dishes on half-strength MS-medium (Sigma, Buchs, Switzerland) solidified with 1% (w/v) purified agar (Oxoid, Basingstoke, Hampshire, UK). To prevent the breakdown of trehalose by endogenous trehalase activity, 10 µM of the trehalase inhibitor validamycin A (Asano et al., 1987
) was added to the medium where indicated. The plants were grown in a daily cycle of 18 h light (80 µmol m-2 s-1) at 22 °C and 6 h darkness at 18 °C. Two weeks after germination, the plants were transferred into Phytacon plant cell culture vessels (Sigma, Buchs, Switzerland) with lids that allow ventilation. The plants were harvested 4 weeks after germination at between 7 h and 8 h into the photoperiod. Plants were also dark-treated for 24 h or illuminated for 31 h before harvest at midday.
Identification of trehalose in Arabidopsis
For the determination of trehalose in Arabidopsis plants (ecotype Ws-2), sugars were extracted, derivatized as described previously (Müller et al., 1995b) and quantified by capillary GC–FID using a Carlo Erba Mega 3500 gas chromatograph equipped with a flame ionization detector (Brechbühler, Zürich, Switzerland). For the identification of trehalose by GC–MS, complete inflorescences of plants grown axenically on medium containing 10 µM validamycin A were extracted in 80% ethanol at 80 °C and the extracts were freeze-dried. The sugars were then analysed by GC–MS (Fiehn et al., 2000). The peak corresponding to trehalose was compared to a trehalose standard. The total number of peaks in the chromatogram was analysed by the deconvolution of the mass spectra over time using the automated mass spectra deconvolution and identification software AMDIS (Stein, 1999).
RACE–PCR
Cloning of full-length cDNAs was based on EST sequences that had been found to exhibit homologies to conserved regions of other TPS genes (accession no. T76758, AtTPSA; accession no. T76390, AtTPSB; accession no. H37578, AtTPSC). For RACE–PCR (Frohman et al., 1988), total RNA was extracted from axenically grown Arabidopsis plants (ecotype Landsberg erecta) and reverse transcribed with EST specific primers (5'-RACE). Primers mapping to internal sequences served to amplify the cDNA ends. Two successive rounds of RACE–PCR were needed to reach the 5' ends. Both strands of the RACE products were sequenced using an ABI PRISM Big Dye Terminator Cycle Sequencing kit and an ABI PRISM 310 Genetic Analyser (Applied Biosystems, Foster City, USA). The predicted protein sequences were aligned using the PILEUP program from the Wisconsin Package Version 10.0 (GCG, Madison, Wisconsin). The degree of similarity between homologous regions of the proteins was calculated with the BESTFIT program (GCG). The obtained sequence information was used to design oligonucleotides flanking the 5' and the 3' ends of the open reading frames of AtTPSA, AtTPSB and AtTPSC. The corresponding pairs of oligonucleotides were used to amplify the full-length gene sequences using total Arabidopsis cDNA as a template. These amplified full-length gene sequences were then ligated into the yeast shuttle vector pFL61 (Minet et al., 1992) or the vector pDH51 and cloned in E. coli. As a positive control, the yeast ScTPS1 gene was amplified and cloned in a similar way. The pFL61-AtTPS1 construct was provided by M Blázquez (La Jolla, USA).
Yeast transformation
The S. cerevisiae tps1 (YSH 6.106.-1A) and tps2 (YSH 6.106.-8C) deletion mutants (Reinders et al., 1997) were transformed with the pFL61-based constructs as described earlier (Vogel et al., 1998). To select for transformants, the cells were first grown on minimal media supplemented with 2% galactose and 1% raffinose (Sgal/raf) at 28 °C. For complementation tests, transformants of the tps1 deletion mutant were then replica plated onto minimal plates with 2% glucose as a carbon source. Transformants of the tps2 deletion mutant were replica plated onto Sgal/raf medium and incubated at 38.6 °C.
Determination of trehalose in transformed yeast cells
For measuring trehalose formation during heat shock, the S. cerevisiae cells were grown in Sgal/raf liquid medium to a density of 5x106 to 2x107 cells ml-1 and transferred to 40 °C for 60 min. The cells were collected by filtration, resuspended in H2O and boiled for 10 min. Sugars in the extracts were determined by HPAEC–PED analysis using an anion-exchange column (CarboPac PA-10, Dionex, Olten, Switzerland) and a pulsed amperometric detector (Dionex) as described previously (De Virgilio et al., 1993).
Measurement of TPS activity
TPS activity was assayed with permeabilized S. cerevisiae cells. The cells were grown to a density of 5x106 to 2x107 cells ml-1, harvested, suspended in 200 mM Tricine (pH 7.0) containing 0.05% Triton X-100 and frozen in liquid nitrogen. After thawing for 3 min at 30 °C, the cells were washed four times with 200 mM K-phosphate buffer (pH 7.0) or 200 mM MES-KOH (pH 7.1). 60 µl of the resuspended cells (equivalent to 0.84 mg protein) was incubated at 35 °C with 5 mM UDP-[U-14C]glucose (5.4 MBq mmol-1), 10 mM glucose-6-phosphate, 12.5 mM MgCl2 and 2 mM DTT in a total volume of 240 µl. The reaction was also measured with ADP-glucose or UDP-galactose instead of UDP-glucose and with the addition of fructose-6-phosphate. After 20, 40 and 60 min, aliquots of 60 µl were boiled for 10 min. The reaction products were quantified by HPAEC–PED analysis as described above and by online-detection of radioactive compounds with a radio-chromatography detector (FLO-ONE\Beta Series A-500; Radiomatic, Meriden, Conneticut, USA). For the calculation of TPS activities, the rates of trehalose and trehalose-6-phosphate formation were added. Protein concentrations were quantified with a modified Lowry assay (Peterson, 1977).
Transient expression in tobacco protoplasts and assay of TPS activity
Tobacco (Nicotiana plumbaginifolia) protoplasts were isolated and aliquots of 106 cells were transformed with 10 µg of the pDH51-based constructs as described previously (Goodall et al., 1990). After the final wash, the protoplasts were resuspended and lysed in 50 mM K-phosphate buffer (pH 7.0). An equivalent of 600 000 protoplasts was assayed for trehalose-6-phosphate synthase activity by incubation at 30 °C with 1 mM UDP-[U-14C]glucose (27 MBq mmol-1), 2 mM glucose-6-phosphate, 12.5 mM MgCl2, 1 µM validoxylamine, and 1 mM DTT in a total volume of 240 µl. After 5, 15, 30, and 60 min, aliquots of 60 µl were boiled for 10 min and centrifuged at 20 000 g for 10 min. The radioactive reaction products in the supernatant were analysed after separation by TLC followed by phosphoimager analysis or by HPAEC-separation with online-detection of radioactive compounds using a radio-chromatography detector (FLO-ONE\Beta Series A-500; Radiomatic, Meriden, Conneticut, USA).
Quantitative RT–PCR
Total RNA was extracted from axenically grown Arabidopsis plants (ecotype Ws-2) using the Qiagen Rneasy kit (Qiagen, Basel, Switzerland). The RNA was treated with DNAse using the MessageClean kit (GenHunter, Nashville, TN) and reverse-transcribed using a reverse-transcription kit (Boehringer, Mannheim, Germany) with oligo-dT and random primers in the reaction. Quantitative PCR was carried out with an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Rotkreuz, Switzerland) using the SYBR Green PCR reagents (Applied Biosystems) according to the manufacturer's protocol. The genes tested, primers used and sizes of the amplified fragments were: AtTPS1 (accession no. Y08568) 5'-TTGAGGTCCCCGAAGTCAAAC-3' and 5'-TGCGGCCAACAATTTCATG-3', 255 bp; AtTPSA 5'-TGGATGTTCCCCTTCGCTT-3' and 5'-TTCTCATGCCGTAGCTGTTTCTC-3', 123 bp; AtTPSB 5'-TCCGTGTTAATCCGTGGAACA-3' and 5'-TCTTTGCACGCCCTTTGAAG-3', 175 bp; AtTPSC 5'-CGAGGAGGATCAATGAGCGTT-3' and 5'-TTGTGCGAGGCGATG-AATC-3', 222 bp; AtH3G (accession no. X60429) 5'-GATTTGCGTTTCCAGAGCCA-3' and 5'-CGAGCGAGCTGAATGTCTTTG-3', 143 bp; AtACT2 (accession no. U41998) 5'-GCAAGTCATCACGATTGGTGC-3' and 5'-GAACCACCGATCCAGACACTGT-3', 297 bp; AtCAB1 (accession no. X56062) 5'-GCTGTTGGCGTTTGTAGGATTC-3' and 5'-CAATGTTGTTGTGCCATGGATC-3', 107 bp. Before their use in quantitative PCR, all primer pairs were shown specifically to produce amplification products of the expected size. The PCR conditions were initial denaturation at 95 °C for 10 min, followed by cycles of 15 s at 95 °C, 30 s at 55 °C, and 60 s at 60 °C. Monitoring of the logarithmic increase in the amount of the PCR products showed that the amplification reactions were equally efficient for all samples. The relative amount of cDNA was calculated from the cycle number at which the amplified target reached a fixed threshold. All samples were analysed in duplicate. To account for possible contamination with genomic DNA, negative controls (no reverse transcriptase in the cDNA synthesis reaction) were included in the PCR analysis. For all samples, the amount of contaminating DNA was below 2%.
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Results |
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Identification of trehalose in Arabidopsis
When extracts from axenically grown Arabidopsis plants were analysed for sugars by GC–FID, a peak with the same retention time as trehalose was regularly detected. The amount of this compound increased when the plants were grown in the presence of validamycin A, which has been shown to inhibit the activity of trehalase in Arabidopsis (Müller et al., 2001
). For example, assuming the detected compound was trehalose, its content rose from 0.14 mg g-1 DW in the flowers of plants grown in the absence of validamycin A to 0.66 mg g-1 DW in the presence of validamycin A (for a more detailed analysis of trehalose contents in Arabidopsis see Müller et al., 2001).
To confirm the identity of this compound as trehalose, sugars were analysed by GC–MS. For this analysis, whole inflorescences (including the flowers, stems and cauline leaves) of plants grown axenically in the presence of validamycin A were extracted. In three independent experiments, trehalose contents in the inflorescences as measured by GC–FID (Fig. 1) ranged between 0.11 and 0.28 mg g-1 DW (0.20±0.05 mg g-1 DW; mean±SE). No trehalose was found in concentrated growth medium, showing that the trehalose peak was not due to a contamination of any of the reagents used in the analysis. When the polar fraction of extracts from the experiment yielding the highest trehalose contents was analysed by GC–MS, more than 700 components were found after mass spectra deconvolution. The retention time of one of the major peaks corresponded precisely with the retention time of trehalose in an external reference chromatogram. Despite the extreme complexity of the plant chromatogram, this peak was unambiguously identified as trehalose by comparison with the trehalose mass spectrum (Fig. 2). High m/z fragments are highly characteristic for trehalose, since most of these fragments are not present in other disaccharides.
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Cloning and sequencing of TPS homologues from Arabidopsis
Full-length Arabidopsis cDNAs were amplified by the RACE method (Frohmann et al., 1988) based on the sequence of previously published Arabidopsis ESTs with homologies to microbial TPS genes. Three different full-length sequences were obtained and the respective genes were named AtTPSA, AtTPSB and AtTPSC. In the meantime, all three sequences were found on BACs from Arabidopsis chromosomes, confirming that they originated from Arabidopsis. AtTPSA is located on chromosome 1 (accession no. AC068143), AtTPSB is located on chromosome 2 (accession no. AC005724), and AtTPSC is located on chromosome 1 (accession no. AC003671). Full-length sequences of all three genes were translated and aligned with other plant and microbial TPS and TPP proteins. The proteins encoded by AtTPSA, AtTPSB and AtTPSC show homologies to TPS and TPP proteins (Fig. 3; Table 1). In contrast to the E. coli EcOTSA and the S. cerevisiae ScTPS1, the plant proteins have an additional C-terminal part homologous to the S. cerevisiae TPP, ScTPS2 (Fig. 3A). This phosphatase-like part is also present in the Selaginella lepidophylla SlTPS1 (Zentella et al., 1999). In contrast to other TPS proteins, AtTPSA, AtTPSB and AtTPSC possess a variable insert of 12, 15 or 13 amino acids, respectively, between amino acid position 405 and 406 of ScTPS1 (Fig. 3B).
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Expression analysis of AtTPSA, AtTPSB and AtTPSC
Probably due to the low expression of AtTPSA, AtTPSB and AtTPSC, no signal was obtained by Northern blot analysis. Therefore, the expression of these genes was analysed by quantitative RT-PCR (Table 2). AtTPSA, AtTPSB, AtTPSC, and AtTPS1 were expressed in all organs tested (rosette leaves, roots, stems, and flowers). Expression levels of all four TPS genes were similar in the rosette leaves and the stems. In the flowers, expression varied considerably between different experiments (data not shown), probably due to different developmental stages harvested. In the roots, the expression of all TPS genes was lower than in the shoot organs. The expression of AtTPS1 was consistently lower in rosettes of dark-incubated plants than in rosettes of illuminated plants. Expression of the chlorophyll a/b-binding protein gene, AtCAB1, of the histone gene, AtH3G, and of the actin gene, AtACT2, was used as control. As expected, the expression levels of AtACT2 and AtH3G were similar in all organs (between 34% and 188%), while the expression of AtCAB1 was highest in the leaves and stems, lower in the flowers and very low in the roots, and strongly reduced in by dark incubation.
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Expression in the S. cerevisiae tps1 deletion mutant
The function of the Arabidopsis genes was analysed by complementation studies with S. cerevisiae mutants. For this purpose, the RACE–PCR products were ligated into the yeast shuttle vector pFL61 behind a strong phosphoglycerate kinase promoter (Minet et al., 1992) and cloned in E. coli. Whereas this step was repeatedly successful for AtTPSA and AtTPSC, no transformants were obtained for constructs containing AtTPSB, indicating that AtTPSB might be toxic in E. coli. pFL61-based constructs containing AtTPSA, AtTPSC, AtTPS1 or ScTPS1 were transformed into the S. cerevisiae tps1 mutant. Due to the lack of TPS activity, this mutant is unable to grow on glucose, although it does grow on galactose. After transformation with the S. cerevisiae ScTPS1 or the Arabidopsis AtTPS1 cDNAs, growth on glucose was restored (Fig. 4). In contrast, growth on glucose was not complemented by expression of AtTPSA or AtTPSC. In addition, whereas expression of ScTPS1 and AtTPS1 restored the ability to accumulate trehalose after heat shock (Table 3), no trehalose was produced in heat-shocked cells transformed with the AtTPSA or AtTPSC cDNAs. In vitro activities of TPS were assayed with permeabilized S. cerevisiae cells. While ScTPS1 restored TPS activity (Table 3), TPS activities were below the detection limit in cells transformed with any of the Arabidopsis cDNAs (AtTPS1, AtTPSA or AtTPSC). Furthermore, no activity was detected when ADP-glucose or UDP-galactose (instead of UDP-glucose) or fructose-6-phosphate was added, when MES-KOH instead of K-phosphate was used as a buffer system or when the activity was assayed with protein extracts instead of permeabilized cells. When GST-fusions of AtTPSA and AtTPSC were expressed in the tps1 mutant, fusion proteins of the correct size were detected (data not shown), indicating that the Arabidopsis proteins were expressed and not degraded in the yeast cells.
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Expression in the S. cerevisiae tps2 deletion mutant
Since the C-terminal parts of AtTPSA and AtTPSC show homologies to TPP proteins (Table 1
; Fig. 3), AtTPSA and AtTPSC were also expressed in the S. cerevisiae tps2 mutant, which cannot grow at high temperatures. As expected, expression of the S. cerevisiae TPP, ScTPS2, and of the Arabidopsis TPP, AtTPPA, complemented growth at 38.6 °C (Fig. 5). Growth at this temperature was, however, not restored after transformation with the AtTPSA, AtTPSC or AtTPS1 cDNAs. In addition, trehalose formation after heat shock was only restored by transformation with ScTPS2 and AtTPPA, but not by transformation with the AtTPSA, AtTPSC or AtTPS1 cDNAs (data not shown).
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Expression in tobacco protoplasts
To exclude the possibility that the lack of TPS activity of AtTPSA and AtTPSC was due to unsuitable conditions in the yeast cells, their function was also studied in tobacco protoplasts. Whereas transformation with the positive control, pDH51-ScTPS1, resulted in the formation of trehalose-6-phosphate, no formation of trehalose-6-phosphate was found in protoplasts transformed with pDH51-AtTPSA or pDH51-AtTPSC (Fig. 6). No conversion of trehalose-6-phosphate into trehalose could be detected in the protoplasts transformed with pDH51-ScTPS1.
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Discussion |
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Occurrence of trehalose in Arabidopsis
Until recently, most higher plants, such as Arabidopsis, were not considered to form trehalose (Müller et al., 1995
a). However, the discovery of an Arabidopsis TPS gene, AtTPS1 (Blázquez et al., 1998), and of two TPP genes, AtTPPA and AtTPPB (Vogel et al., 1998), suggested that Arabidopsis has the potential for trehalose synthesis.
Here, it is shown that trehalose, the product of the TPS and TPP reactions, does occur in Arabidopsis (Figs 1, 2). This finding confirms results of other studies in which chromatographic techniques were used for measuring trehalose in plants: for example, trehalose was found in tobacco plants grown hydroponically in the presence of validamycin A (Goddijn et al., 1997), and in a salt-stressed rice plant (Garcia et al., 1997). In Arabidopsis, a compound that increased in the presence of validamycin A was tentatively identified as trehalose (Müller et al., 2001). To provide unambiguous evidence that trehalose occurs in plants it was, however, necessary to identify trehalose using GC–MS or NMR analysis. Recently, trehalose was identified by GC–MS analysis in soil-grown potato tubers (Roessner et al., 2000). In the present study, axenically grown Arabidopsis plants were used to determine trehalose by GC–MS analysis in order to rule out that micoorganisms were the source of trehalose. Unless axenically grown Arabidopsis plants contain seed-borne microbial endophytes, an involvement of microorganisms in the formation of the trehalose found in this study can be excluded. One of the reasons why the occurrence of trehalose in Arabidopsis has not been described earlier, is probably the cleavage of trehalose by the endogenous trehalase activity of Arabidopsis (Müller et al., 2001). When trehalase activity was inhibited by validamycin A, the content of trehalose rose to easily detectable amounts.
The detection of trehalose in higher plants, such as Arabidopsis, raises the question about its function (Müller et al., 1999). In view of its low abundance, it probably does not play a role as a stress protectant. However, there are indications that trehalose and/or trehalose-6-phosphate act as signal molecules in the regulation of plant metabolism and development. The results obtained by the expression of microbial TPS proteins in plants, by inhibition of trehalase activity by validamycin A and by external supply of trehalose support this hypothesis (Wagner et al., 1986; Müller et al., 1998, 2000; Pilon-Smits et al., 1998; Romero et al., 1997; Wingler et al., 2000; Fritzius et al., 2001).
Involvement of the individual TPS homologues in trehalose synthesis
Having established that trehalose synthesis occurs in Arabidopsis, it was interesting to investigate where the Arabidopsis TPS and TPP homologues are expressed and which of them catalyse reactions of trehalose synthesis. Since AtTPSA, AtTPSB, AtTPSC, and AtTPS1 are expressed in all organs of Arabidopsis plants (Table 2), they could be simultaneously involved in trehalose synthesis. To analyse their function, the open reading frames of AtTPSA and AtTPSC were amplified by PCR and cloned. Complementation studies with yeast mutants (Figs 4, 5; Table 3) confirm that AtTPS1 (Blázquez et al., 1998) encodes a functional TPS, while no enzymatic function could be ascribed to AtTPSA and AtTPSC.
After transformation of S. cerevisiae with the AtTPS1 cDNA, trehalose formation in the heat-shocked tps1 mutant was detected by HPAEC-PED analysis. Previously, trehalose formation in this system had only been demonstrated using a coupled enzymatic assay (Blázquez et al., 1998), which is not necessarily specific. Although expression of AtTPS1 restored trehalose formation in vivo, it did not result in measurable in vitro activity of TPS. Much lower in vitro activities compared with the in vivo rates of trehalose plus trehalose-6-phosphate formation have also been reported for ScTPS1 in S. cerevisiae mutants with deletions of other components of the TPS complex (ScTPS2, ScTPS3 or ScTSL1; Reinders et al., 1997). This indicates that the TPS activity is underestimated in the in vitro assay, when no TPS complex can be formed. For AtTPS1 this might be due to a lack of interaction with the S. cerevisiae proteins.
In contrast to AtTPS1, expression of AtTPSA and AtTPSC did not restore trehalose formation and did not complement growth of the S. cerevisiae tps1 mutant on glucose. Fusion proteins of AtTPSA or AtTPSC with GST were also expressed in S. cerevisiae. In this case, fusion proteins of the expected size were readily detected (data not shown), indicating that AtTPSA or AtTPSC were correctly expressed at the protein level. Moreover, the lack of trehalose formation was probably not due to an incompatibility of AtTPSA and AtTPSC function with specific conditions in the S. cerevisiae cells, since expression of AtTPSA and AtTPSC in tobacco protoplasts did not result in trehalose or trehalose-6-phosphate synthesis either (Fig. 6). ScTPS1, in contrast, was also active in the tobacco protoplasts, showing that tobacco protoplasts are a suitable system for studying the function of yeast enzymes. Taken together, the results suggest that AtTPSA and AtTPSC probably do not encode functional TPS enzymes.
As the C-terminal parts of AtTPSA and AtTPSC show homologies to functional TPP proteins, it was also attempted to complement the S. cerevisiae tps2 mutant lacking TPP. However, neither growth at 38.6 °C nor trehalose formation could be restored. Similarly, the Selaginella lepidophylla SlTPS1 probably does not exhibit TPP activity, even though it contains a C-terminal domain homologous to microbial TPP proteins (Zentella et al., 1999).
The results presented here suggest that trehalose is indeed formed in Arabidopsis plants and that the first step of trehalose synthesis is catalysed by AtTPS1. The function of AtTPSA and AtTPSC, on the other hand, remains unclear. Since AtTPSA and AtTPSC also show homologies to ScTPS3 and ScTSL1, two regulatory proteins of the S. cerevisiae TPS complex (Vuorio et al., 1993; Reinders et al., 1997), they might present regulatory proteins of a putative plant TPS complex. The cloning of the full-length cDNAs makes it possible to manipulate the expression of AtTPSA and AtTPSC in Arabidopsis in order to analyse their function in planta.
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Acknowledgments |
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We thank M Blázquez (La Jolla, USA) for providing the AtTPS1 clone. This work was supported by grants of the Swiss National Science Foundation (3100-042535.94 to A Wiemken and 3100-040837.94 to T Boller). We also thank Novartis Agribusiness Biotechnology Research Inc. for financial support.
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Notes |
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3 Present address: Kantonales Laboratorium Basel-Stadt, Kontrollstelle für Chemie- und Biosicherheit, Missionsstrasse 60, Postfach, 4012 Basel, Switzerland.
4 Present address and to whom correspondence should be sent: Department of Biology, University College London, Gower Street, London WC1E 6BT, UK. Fax: +44 20 7679 7096. E-mail: a.wingler{at}ucl.ac.uk
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Abbreviations |
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GC–FID, gas chromatography–flame ionization detection; GC–MS, gas chromatography–mass spectrometry; EST, expressed sequence tag; HPAEC, high-performance anion exchange chromatography; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; TPP, trehalose-6-phosphate phosphatase; TPS, trehalose-6-phosphate synthase.
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References |
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