Expression of threonine synthase from
Solanum tuberosum
L. is
not metabolically regulated by photosynthesis-related signals or by
nitrogenous compounds
A.P. Casazza
a, A. Basner
a, R. Ho¨fgen
a, H. Hesse
b,*
aMax-Planck-Institut fu¨r Molekulare Pflanzenphysiologie,Abt. Willmitzer,Am Mu¨hlenberg1,14476 Golm, Germany bFreie Uni6ersita¨t Berlin,Institut fu¨r Biologie,Angewandte Genetik,Albrecht-Thaer-Weg 6,14195Berlin, Germany
Received 7 February 2000; received in revised form 27 March 2000; accepted 27 March 2000
Abstract
Although the control of carbon fixation and nitrogen assimilation has been studied in detail, little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper the isolation of a cDNA encoding threonine synthase is reported (TS; EC 4.2.99.2) from a leaf l ZAP II-library of Solanum tuberosum L. and the transcriptional regulation of the respective gene expression in response to metabolic changes. The pattern of expression of TS by feeding experiments of detached petioles revealed that TS expression is regulated neither by photosynthesis-related metabolites nor by nitrogenous compounds. The present study suggests that the regulation of the conversion of aspartate to threonine is not controlled at the transcript level of TS. The nucleotide and deduced amino acid sequences of potato TS show homology to other known sequences fromArabidopsis thaliana
and microorganisms. TS is present as a low copy gene in the genome of potato as demonstrated in Southern blot analysis. When cloned into a bacterial expression vector, the cDNA did functionally complement the Escherichia colimutant strain Gif41. TS transcript was found in all tissues of potato and was most abundant in flowers and source leaves. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Chloroplast; O-Phosphohomoserine; Threonine synthase; Threonine biosynthesis;Solanum tuberosum
www.elsevier.com/locate/plantsci
1. Introduction
Threonine, like all members of the aspartate family of amino acids, is synthesised through a branched pathway mainly located in the chloro-plast [1]. Threonine represents one of the essential amino acids in the diet of monogastric animals, which determines to some extent the nutritional value of crops. In plants, the regulation of the threonine concentration in the cell is subject to tight control mechanisms. Threonine synthase (TS; EC 4.2.99.2) is a pyridoxal 5%-phosphate
(PLP)-de-pendent enzyme and catalyses the last step of threonine formation, converting O-phosphoho-moserine (OPH) to threonine and inorganic phos-phate. While in bacteria and fungi homoserine is the intermediate branch point leading to synthesis of either threonine or methionine, in plants phos-phohomoserine is the last common intermediate used to synthesise threonine or methionine. There-fore, in plants TS competes with cystathionine gamma-synthase, the first enzyme required for subsequent methionine biosynthesis, for phospho-homoserine. TS activity is activated by high con-centrations of S-adenosylmethionine (SAM) and inhibited by cysteine [2 – 4]. SAM in turn is synthe-sised directly from methionine, and therefore in-creasing levels of methionine will increase SAM concentration and subsequently TS activity, thereby diverting phosphohomoserine from the Abbre6iations: TS, threonine synthase; PLP, pyridoxalphosphate.
The nucleotide sequence data reported will appear in the EMBL Database under the accession number AF082894.
* Corresponding author. Tel.: +49-30-83856797; fax: + 49-30-83854345.
E-mail address:[email protected] (H. Hesse).
equilibrium towards synthesis of threonine at the expense of methionine and SAM synthesis. Fungal and bacterial TSs as not being branch point en-zymes are not activated by SAM [5].
TSs were purified from Neurospora crassa [6] and from Escherichia coli [7]. The corresponding gene was isolated from a number of bacteria [7 – 12] and from Saccharomyces cer6isiae [13]. In plants, partial purification [14,2,3] and the com-plete purification of TS from Arabidopsis thaliana [15] were reported. In addition, a cDNA was isolated from A. thaliana by functional comple-mentation [4]. However, this clone was not full length as the methionine start site was lacking. The missing information could be provided by isolating a genomic clone, identical to the previous described cDNA [16]. Here the isolation and func-tional characterisation of a full-length cDNA en-coding TS from potato is reported.
2. Materials and methods
2.1. Bacterial strains and plants
E. coli XL-1-blue (Stratagene) was cultured us-ing standard techniques [17]. For complementa-tion, E. coli mutant GIF41 (thrC 1001 thi-1 relA spoT1) was used, (kindly provided by Mary Berlyn,E. coli Genetic Stock Center). For growth studies of the complemented mutants under re-strictive conditions M9-medium was used accord-ing to Sambrook et al. [17]. The medium was supplemented with 30 mM thiamine and 2 mM threonine was used as a positive control. The solid medium contained 1.5% agarose (w/v, Gibco). Plates were incubated at 37°C.
Potato plants (cv. De´sire´e, Saatzucht Fritz Lange KG, Bad Schwartau, Germany) were culti-vated under 16 h light/8 h dark regiment in soil in a greenhouse.
2.2. Isolation of TS cDNA
Approximately 2.5×105 pfu of a Solanum
tuberosum cDNA library in l-ZAP II XR derived
from flower mRNA (kindly provided by M. Klein, MPI-MOPP, Germany) was screened at low strin-gency using a PCR amplified fragment of TS from A. thaliana genomic DNA. Primers (AtTS-N: 5% -TCT CCT CCG CCA CCG CCC CG-3%; AtTS-C:
5%-TAA GTG TAT TAC TTC CTA AG-3%) used for PCR correspond to the region of the published sequence from A. thaliana [4]. Screening was per-formed at 42°C in Denhardt’s buffer according to Sharrock et al. [18] containing 25% formamide. Filters were washed for 20 min in 3×SSC, 0.1% SDS at 45°C. Further screening was performed according to the manufacturer’s protocol (Strata-gene, La Jolla, CA).
2.3. Nucleic acid manipulation
Both strands of the isolated cDNA insert were sequenced with T7 polymerase (Pharmacia) from a set of subclones (all in pBluescript SK−) using synthetic oligonucleotides. All other methods were performed according to Sambrook et al. [17].
Plant material was harvested in the morning from potato wildtype plants grown in the green-house and immediately frozen in liquid nitrogen prior to storage at −80°C. Total RNA from potato leaves, stems, roots, tubers and flowers was prepared according to Logemann et al. [19]. From each tissue 40 mg of total RNA per lane were separated on a denaturing agarose gel (1.2%) con-taining 15% formaldehyde. After blotting, the RNA was fixed to the membranes (Porablot NY plus, Macherey & Nagel, Germany) by incubation at 80°C for 2 h.
To analyse the influence of metabolites onStTS expression, petioles of 8-week-old potato plants were cut while submerged under water and incu-bated in medium containing phosphate, phospho-homoserine, threonine, phospho-homoserine, asparagine, glutamine, oxalacetate, and sucrose, respectively. Incubation was done under constant light at 25°C. Genomic DNA isolation from plant tissue was done according to Dellaporta et al. [20]. Genomic DNA was digested with the restriction enzymes Eco RI, Hind III, PstI,BamHI, SalI, andXho I and fractionated on 1.0% agarose gels, then blot-ted onto nylon membranes. Membranes (Porablot NY plus, Macherey & Nagel, Germany) were in-cubated for 2 h at 80°C.
SDS and 1% (w/v) BSA. Subsequent hybridisation with the probe was carried out overnight at the same conditions.
The membranes were washed twice with 0.5× SSC, 0.1% SDS at 65°C for 20 min and exposed to a X-ray film at −80°C.
2.4. Recombinant DNA manipulations
In order to complement E. coli mutant Gif41 [21], the isolated TS-cDNA was cloned into the expression vector pQE30 (Qiagen, Germany).
A DNA fragment encoding the mature TS protein was amplified by polymerase chain reac-tion generating a BamHI site at its 5%-end and an Asp 718 at its 3%-end. Oligonucleotides TS-TP 5% -GAG AGA GGA TCC TCT CAT AAT TTT TCA GCC AGG and universal primer 3%-AGT CAC GAC GTT GTA AAA CG (TIB MOL-BIOL, Berlin, Germany) were used and amplified fragments encoding for the full length protein (approximately 1.3 kb) but omitting the transit peptide were ligated into the pCR II cloning vec-tor (Invitrogene) and subsequently into pQE30 via Bam HI and Asp 718.
Sequence analysis was performed with the help of the programs of the Wisconsin Genetics Group (GCG Package, Version 8.1; [22]).
3. Results
3.1. Isolation and characterisation of the TS cDNA
A cDNA of TS was isolated by screening 2.5× 105pfu from a potato leafl-ZAP II-library with a PCR derived DNA probe encoding a fragment of the TS gene fromA. thaliana [4]. The sequence of the PCR fragment was identical to the sequence of Arabidopsis TS previously described [4]. The iso-lated potato cDNA, designated StTS, was fully sequenced. The cDNA is 1.707 kb in length with an open reading frame of 1.557 kb. The cDNA encodes for 519 amino acids with a predicted molecular mass of approximately 57.4 kDa. A sequence comparison of the predicted amino acid sequence for the potato TS with the deduced sequences from A. thaliana showed highly con-served sequence stretches (Fig. 1) with an identity of 83.9% (88.7% similarity). The homology
de-creased when StTS was compared to TS from Bacillus subtilis (35.5% identity), Corynebacterium glutamicum (22.9%), E. coli (23.9%), Pseudomonas aeruginosa (18.2%) and Saccharomyces cere6isiae (21.4%).
In plants, the enzyme was shown to be exclu-sively confined to the chloroplast [1]. Curien et al. [4] purified TS from A. thaliana. Its N-terminal sequence was determined and resulted in a se-quence of 15 residues (T-A-D-G-N-N-I-K-A-P-I-E-T-A-V) indicating the possible start of the mature protein and that TS is synthesised with an N-terminal presequence. There is no identical se-quence present in the deduced peptide sese-quence of potato TS. Thus, the start point of the mature protein cannot be determined exactly. The de-duced amino acid residues from the start methion-ine to position 42 show typical features of a transit peptide being high in serine content and contain-ing positively charged or hydrophobic side chains [23,24].
The mature protein probably consists of 477 residues with a Mr of approximately 52.5 kDa. The consensus sequence of the pyridoxal phos-phate-binding domain — VSTAHGLK6 FTQSKI — was identified and extends over position 462 – 476 with a lysine residue at position 470 as the likely site of cofactor binding via a Schiff Base.
3.2. Genomic DNA analysis for StTS homologous
sequences
Genomic DNA blot analysis was used to esti-mate the copy number of theStTS gene(s) present in the genome of potato. Fig. 2 shows that in each lane only very few DNA fragments hybridise with the StTS probe. This suggests that the StTS gene may represent a low-copy number gene in the potato genome.
3.3. Functional expression of the recombinant potato TS in E. coli
trans-formed into the TS-deficient E. coli mutant Gif41 (Fig. 3). When plated on minimal medium without threonine, the Gif41 grew in the presence of StTS. No growth was monitored when Gif41 was trans-formed with pQE30. From this data it was con-cluded that expression of the plantStTS cDNA is able to functionally complement the E. coli mutant restoring its ability to grow on minimal medium.
3.4. Expression analysis of TS mRNA in potato
To determine the expression pattern of StTS, total RNA was isolated from different potato tissues of potato grown under greenhouse condi-tions. In Northern blot experiments using the potato StTS cDNA as a probe a single 1.8 kb transcript was detected which matches the size of the isolated cDNA. Analysis of TS expression in
Fig. 1. Sequence alignment of StTS. The amino acid sequence of StTS was aligned with the deduced sequences fromArabidopsis thaliana (AtTS; L41666, AB027151), Bacillus subtilis (PsthrC; X04603), Corynebacterium glutamicum (CgthrC; X56037), Es
Fig. 2. Southern blot analysis of DNA fromSolanum tubero
-sum using the full-length cDNA StTS as a probe. Approxi-mately 10 mg of DNA were digested with the respective restriction enzyme (EcoRI, Hind III,Pst I,Bam HI,Sal I, andXhoI). The positions of the size markers given in kb are shown on the right.
expression was observed in sink leaves, stems, stolons and roots. Notably, the lowest levels of expression were detected in tubers.
3.5. Transcriptional regulation of TS by carbon supply and nitrogenous compounds
Synthesis of amino acids requires both carbon and nitrogen. To analyse the influence of metabo-lites on the expression ofStTSin vegetative tissue, feeding experiments of detached leaves were per-formed. One wanted to evaluate whether the availability the carbon sources of sucrose and oxalacetate or the availability of compounds re-lated to nitrogen assimilation, respectively, exerted an effect on the expression of StTS. To this end, detached petiols of potato were infiltrated with MES-buffer containing EDTA to avoid callose formation (Fig. 5, lane 1), inorganic phosphate (Fig. 5, lane 2), phosphohomoserine (Fig. 5, lane 3), threonine (Fig. 5, lane 4), homoserine (Fig. 5, lane 5), asparagine (Fig. 5, lane 6), glutamine (Fig. 5, lane 7), oxalacetate (Fig. 5, lane 8), or sucrose
Fig. 4. Expression of the StTS gene. Total RNA (40 mg) isolated from various tissues was separated on a 1.2% formaldehyde/agarose gel, blotted onto a nylon membrane, and fixed at 80°C. Hybridisation was carried out at 60°C to the randomly labeled cDNA as a probe. To quantify the hybridisation signals the blots were hybridised with the 28S rRNA probe as an internal loading control. Relative values of expression were calculated from data obtained by phosphor-imager scanning.
Fig. 3. Complementation of a threonine synthase (TS)-defi-cientEscherichia colimutant. Plasmids pQE30 (empty vector) and StTS (harbouring the corresponding genes cloned in pQE30) were transformed into theE.colimutant Gif41 and plated onto M9 minimal medium with threonine (left) or without threonine (right). Concentration of threonine in the medium was 2 mM. Plates were incubated at 37°C.
Fig. 5. RNA blot analysis of threonine synthase (TS) gene
StTS expression in petiols incubated with different metabo-lites after 24 h in constant light. Total RNA (40 mg) was separated by electrophoresis on a formaldehyde gel, blotted onto a nylon membrane and hybridised to the full sizeStTS
cDNA. Petiols were incubated with buffer (lane 1; 10 mM MES, 1 mM EDTA, pH 6.5) as control or with buffer supplemented with 0.5 mM phosphate (lane 2), 5 mM phos-phohomoserine (lane 3; containing 0.5 mM phosphate), 5 mM threonine (lane 4), 5 mM homoserine (lane 5), 5 mM as-paragine (lane 6), 5 mM glutamine (lane 7), 5 mM oxalacetate (lane 8) or 10% sucrose (lane 9), respectively.
sion of the StTS cDNA, which lacks the putative transit peptide. The Southern blot analysis pre-dicts a low copy number of TS genes in the genome of S. tuberosum using both stringent and non-stringent hybridisation and washing condi-tions, respectively (data not shown for non-strin-gent conditions).
The StTS gene encodes for a protein with a molecular mass of 57 kDa. The amino terminal sequence displays an increased number of serine and threonine residues being common features of chloroplast transit peptides. This is consistent with the observation that TS enzyme activity was found in the plastids of plants [1,4]. The transit peptides of cytoplasmatically synthesised but chloroplast-localised proteins are usually removed by a specific protease. For the deduced potato protein one can-not predict the actual cleavage site because of the presence several potential cleavage consensus se-quences [24]. Curien et al. [4] demonstrated in Western blot analysis that antibodies raised against recombinant TS protein from Arabidopsis reacted with a single polypeptide of 55 kDa in Arabidopsis leaf extracts.
For complementation of an auxotrophic E. coli mutant a truncated StTS cDNA was expressed that encodes a peptide starting with amino acid 69 to ensure omission of the putative transit peptide. This fragment proved to be functional for comple-mentation of the thrC deficient E. coli strain Gif41.
As shown in Northern blot experiments, the expression of the potatoStTSgene is differentially regulated in a tissue specific manner. StTS tran-script is most abundant in source leaves and in flowers. Less expression is detected in sink leaves, stems, stolons and roots and the lowest level of expression is identified in tubers. However, threonine biosynthesis does occur in all plant tis-sues and is not restricted to source organs.
Metabolic regulation has been described for sev-eral genes involved in the assimilation of carbon and nitrogen. Carbon partitioning between carbo-hydrates and amino acids is subject to a complex metabolic regulation system triggered by light, photosynthesis-related metabolites, and nitroge-nous compounds [25 – 27]. It has been found that at high sugar but limiting nitrogen levels, carbohy-drates accumulate preferentially, whereas under conditions of high carbon and high nitrogen levels, a larger proportion of the carbon is shifted to-wards production of amino acids and proteins. (Fig. 5, lane 9). Total RNA was extracted and
subjected to Northern-blot analysis using StTS cDNA as a probe. As shown in Fig. 5, none of the treatments resulted in any significant modulation of StTS expression. Marginal changes in steady-state mRNA levels of StTS observed in indepen-dent experiments were probably due to minor variations in RNA loading.
4. Discussion
ver-Nitrogen is primarily assimilated into glutamine and glutamate. Via a transamination reaction as-partate is formed, which can either be converted to asparagine by asparagine synthase or diverted to synthesis of the amino acids lysine, threonine, methionine and isoleucine, the so-called aspartate family pathway of amino acids. This pathway is initiated through the activity of aspartate kinase competing with asparagine synthase for their com-mon substrate, aspartate [28,27]. The metabolism of glutamine, glutamate, aspartate and asparagine is subject to a coordinated metabolic regulation. Whereas the expression of glutamine synthetase was shown to be stimulated by light [29,27], the expression of asparagine synthase was repressed by light and sucrose but stimulated at night by nitrogen [30,31,27,32]. In the present study the expression ofStTSwas followed in the presence of various metabolites in Northern blot experiments. They revealed that StTS expression is not regu-lated at the level of transcription. Neither photo-synthesis-related metabolites such as sucrose, oxalacetate and phosphate nor nitrogenous com-pounds or intermediates/products such as phos-phohomoserine, threonine, homoserine, asparagine, glutamine were able to influence gene expression. Therefore, it is most likely that TS is post-transcriptionally regulated by metabolites, e.g. for SAM [15,33] or by the amount of protein which was published by Muhitch [34]. Here the expression of deregulated TS from E. coli in to-bacco yielded a more than fivefold increase in free threonine indicating that protein amount and en-zymatic properties regulate threonine synthesis but not the availability of precursors. Taken together these results, it appears that TS regulates the synthesis of a single step in the pathway but the major control point of flux towards the synthesis of aspartate derived amino acids is aspartate ki-nase as demonstrated by Zhu-Shimoni and Galili [35].
Acknowledgements
We thank Simone Kaiser for technical assis-tance and Josef Bergstein for the photographic work. We thank Dr Bernd Laber (Hoechst AgrEvo GmbH, Germany) for providing the sub-strate phosphohomoserine. We thank Professor Lothar Willmitzer and Dr Georg Leggewie for critical reading the manuscript.
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