To obtain potato plants expressing CgS_Δ90 we transformed the cultivar Désirée with a construct containing the CgS_Δ90 cDNA driven by the constitutive CaMV 35S promoter, and fused to a chloroplast transit peptide at the 5' end for targeting, and three copies of heamagglutinin (3HA) at the 3' end for immunological detection 12. Thirty kanamycin-resistant lines were obtained. PCR analysis using CgS_Δ90-specific primers detected the presence of the transgene in 27 lines. The expression levels of CgS_Δ90 were determined at both transcript and protein levels by RNA- and protein-blot analysis using anti-HA monoclonal antibody for detection of the CgS_Δ90 fusion protein. Three lines, C1, C2, and C3, showing the highest CgS_Δ90 transcript- and CgS_Δ90 protein levels were selected for further studies (Figure 2).

To increase the incorporation of free methionine into a storage protein, co-transformation experiment was carried out using two Agrobacterium strains. One of them contained a plasmid with the CgS_Δ90 gene 12, while the other carried a construct for constitutive expression of the methionine-rich storage protein, 15-kD β-zein fused to 3HA 15. This protein naturally accumulates in ER-derived protein bodies, and is stable in seed- and non-seed tissues 16171819. After transformation, 25 independent kanamycin-resistant potato lines were obtained. Co-transformed lines were selected by PCR analysis using CgS_Δ90- and zein-specific primers. Six lines were identified in which both transgenes were present. Two of these lines, designated C+Z1 and C+Z2, expressed both transgenes at high levels (Figure 2). The anti-HA recognised the CgS_Δ90 and zein fusion proteins both in leaves and tubers. The level of expression, however, was higher in leaves than in tubers (Figure 2).

It was shown that modification of the methionine pathway by over-expressing the TS protein in potato caused phenotypic changes 4. To investigate the effect of expressing the CgS_Δ90 protein on the phenotype of potato non-transformed Désirée plants and the selected transgenic lines were transferred into soil and cultivated under greenhouse conditions. After six weeks, the plant material was evaluated and scored based on macroscopic phenotypic alterations. Transgenic line C3 was phenotypically almost indistinguishable from the non-transformed plants. The other 26 lines, however, exhibited serious alterations such as severe growth retardation and changes in leaf architecture. The strongest phenotypical alterations were displayed by lines C1 and C2 with no composite leaves at all. Lines C+Z1 and C+Z2 developed composite leaves, which, however, were smaller than those of the wild-type plants (Figure 3a). Due to severe growth retardation, a 40- to 60% reduction in tuber yield was also found (Figure 3b). Interestingly, we observed that the colour of the tubers' skin was also changed. The amounts of anthocyanin pigments were measured spectrophotemetrically in the skin of the non-transformed Désirée and its transgenic derivatives. Except of line C3, which was similar to the non-transformed control, a 4- to 5-fold reduction in the anthocyanin content of the skin of transgenic tubers was found compared to the control (Figure 4).

To investigate whether simultaneous expression of CgS_Δ90 and zein changes the amino acid content of tubers, extracts of tuber flesh were analysed using a technology based on gas chromatography-mass spectrometry (GC-MS). The results are shown in Figure 5. We found that the amount of free methionine increased almost 2-fold in line C+Z1 and 6-fold in line C+Z2. In addition, the amount of serine and isoleucine was also 2- to 3-fold higher in C+Z2 tubers compared to wild-type. Elevated levels of these two amino acids, however, were not observed in the C+Z1 line. Although some variation in the levels of individual amino acids was observed in a fully independent second and third experiment, the changes detected in methionine, serine and isoleucine content were always repeatable. The total free amino acid content of line C+Z1 was not significantly different from that of the control, while a 1.4-fold increase was found in line C+Z2 (Figure 5a).

The protein content of tubers was also analysed. Because the 15-kD β-zein is an ethanol-soluble protein, the amino acid constitutions of proteins were determined not only in the water-, but also in the ethanol-soluble fraction. In the water-soluble fraction no significant difference between the transgenic lines and the control was detected (data not shown). In contrast, the amount of methionine in the ethanol-soluble fraction was much higher in the double transgenic than in the control plants, and, in particular, line C+Z2 displayed an about 2-fold higher level than line C+Z1. In sum, expression of zein resulted in an approximately 2-fold increase in the total ethanol-soluble protein content of tubers (Figure 5b).

We have found that anthocyanin content of the transgenic potato tubers expressing CgS_Δ90 at high level was strongly reduced compared to the wild-type (Figure 4). Anthocyanins are flavonoid-type secondary metabolites whose synthesis starts with a reaction catalysed by PAL. It is known that the transcription of PAL has a differential regulation during plant development and by environmental cues [20, and references therein]. We assumed that a reduction in PAL transcription could abolish the synthesis of anthocyanins and tested the PAL mRNA levels in both tuber skin and flesh on RNA-blots (Figure 6a). The PAL transcript level was four times higher in skin than in flesh in wild type tubers. The amount of PAL mRNA was 1.5- and 2-fold lower, respectively, in C+Z1 and C+Z2 tuber skins than in the control. A 7-fold decrease in the amount of PAL mRNA was detected in tuber flesh of lines C+Z1 and C+Z2 compared to the control (Figure 6b). There was a positive correlation between the anthocyanin content and level of PAL expression in tuber skin (Figure 6c).

In order to obtain potato plants with high methionine content in their tubers, we co-expressed the CgS_Δ90 and the 15-kD β-zein genes in the Désirée variety. We have found that the soluble methionine content of the obtained transgenic tubers was 2- to 6-fold higher compared to the wild-type, and that the transgenic plants displayed abnormal phenotypes, such as altered leaf morphology and growth, and changes in skin colour of tubers. Di et al. 7 expressed the full-length CgS of Arabidopsis in potato, which also resulted in a maximum of 6-fold elevation of methionine accumulation in tubers. All plants expressing the full-length CgS were phenotypically normal and indistinguishable from the non-transformed potato plants while our lines expressing CgS_Δ90 alone or in combination with zein were phenotypically abnormal. It was demonstrated earlier that Arabidopsis and the T₁ generation of transgenic tobacco plants over-expressing CgS_Δ90 display an abnormal phenotype including stunted growth, slow development rate, narrow and curly leaves, and, in tobacco only, a high emission rate of the methionine catabolic product, dimethylsulfide 612. Activity of the full-length CgS of Arabidopsis is regulated by methionine via SAM 91011, while that of the potato is not 414. Thus we concluded that irrespective of the mode of regulation of the endogenous CgS over-expression of CgS_Δ90 results in phenotypic aberrations.

Methionine belongs to the aspartate family of amino acids (Figure 7). Interestingly, the C+Z2 tubers, which accumulated six times more soluble methionine than the control tubers, had 2- to 3-fold higher isoleucine and serine content too. Isoleucine is derived from threonine by deamination, and serine, which is converted to O-acetylserine, is important in sulphur assimilation (Figure 7). Thus it is likely that a fine mechanism of regulation exists, keeping the balance between the rate of synthesis of certain end- and start compounds in the aspartate-derived amino acid metabolism of potato. Rébeillé et al. 21 demonstrated that the expression of methionine γ-lyase (MGL) was induced in Arabidopsis cells in response to high methionine levels and that the production of S-methylcysteine and isoleucine was directly associated to the function of MGL (Figure 7). This step in methionine catabolism may also exist in potato and is responsible for the increased level of isoleucine in C+Z2 tubers.

In contrast, no significant difference in the levels of amino acids belonging to the aspartate family was observed between the wild-type and C+Z1 tubers. A huge variation depending on the intensity of transgene expression was detected also in the amino acid content of the leaves and siliques of transgenic Arabidopsis plants co-suppressed for CgS 5.

The 15-kD β-zein protein of maize has already been successfully expressed in tobacco and alfalfa. Expression of zein did not result in phenotypical changes in transgenic plants 15161718. Compared with plants expressing zein alone, transgenic alfalfa plants co-expressing zein and CgS contained enhanced level of zein concurrently with a reduction in the soluble methionine content 22. To some extent, similar effect was detected in tobacco, in which, however, even the reduced amount of soluble methionine was accompanied by an abnormal phenotype 15. We have observed similar phenomenon in potato. Co-expression of the 15-kD β-zein could not overcome the phenotypical changes caused by CgS_Δ90 in potato.

Golan et al. 15 reported that expression of either CgS, or zein, or both resulted in increased amount of methionine incorporated in the water-soluble protein fraction of tobacco leaves. Major differences in the composition of water-soluble seed storage proteins were also reported for transgenic rice seeds expressing a methionine-rich storage protein 23. In contrast, we found no alteration in the amino acid content of the water-soluble proteins in the C+Z potato tubers while a 2- to 6-fold increase in methionine content was observed in the zein-containing ethanol-soluble protein fraction. Considering, however, that 99% of tuber proteins are water-soluble (data not shown) the nutritional value of tubers could not be significantly improved by the 2-fold increase detected in the amount of ethanol-soluble proteins.

Tubers of the potato cv. Désirée are characterised by red skin caused by the accumulation of anthocyanin pigments. Surprisingly, we have found that high constitutive expression of CgS_Δ90 results in a 4- to 5-fold reduction in anthocyanin level of the tuber skin.

The anthocyanins are exclusively accumulated in the vacuole, although they are synthesised in the cytosol in the phenylpropanoid biosynthesis pathway that is initiated by deamination of phenylalanine catalysed by PAL (Figure 7). It is known that several environmental factors including light, wounding, or pathogen attack can affect anthocyanin levels of tubers 24. Previous publications reported that repression of 14-3-3 protein synthesis or that of the chalcone synthase, chalcone isomerase, and dihydroflavonol reductase, enzymes involved in anthocyanin synthesis consecutive to PAL, decreased the amount of anthocyanins present in tuber skin 2526. We found a 7-fold reduction in the amount of PAL mRNA in flesh and a 1.5- to 2-fold reduction in skin of C+Z tubers compared to controls. In all species analysed to date, the common denominators in the regulation of anthocyanin biosynthesis genes are transcription factors with MYB or HLH domains and a WD40 protein in mutual interaction 27. Co-ordinated, sucrose-specific modulation of the anthocyanin pathway is evidenced in Arabidopsis. However, this is likely achieved through the last few steps and does not influence PAL transcription 28. Arabidopsis plants treated with exogenous cytokinins accumulate anthocyanin pigments. Deikman and Hammer 29 reported that PAL is controlled by the cytokinin benzyladenine posttranscriptionally. In contrast, using another cytokinin, isopentenyl adenosine, for treatment of Arabidopsis plants Németh et al. 30 detected higher amount of PAL mRNA in treated than in control plants. Assuming that transcriptional regulation of PAL by cytokinins exists in potato, a reduction in cytokinin level may explain the reduced amounts of PAL mRNA in the C+Z transgenic tubers compared to controls. The cytokinin-binding protein S-adenosylhomocysteine hydrolase (SAHH) that regulates biological methylation reactions through a control of the intracellular SAM/SAH ratio 2 may contribute to the alteration of cytokinin level in C+Z transgenic plants. Previously, it was found that anthocyanins accumulate in sulfur-starved Arabidopsis plants. The SAM decreased (3–30-fold) under sulphur deficiency, whereas SAH remained unchanged, the SAM/SAH ratio decreased accordingly 3132. Analysis of antisense SAHH tobacco plants showed that they contained excess levels of cytokinin 33. High activity of the CgS_Δ90 may increase the SAM level and SAHH activity reducing thereby the cytokinin level and transcription of PAL gene. Further biochemical and transcriptional investigations would help to elucidate the current hypothetical model (Figure 7), including the possible involvement of cytokinins in regulation of PAL expression in potato tubers.

Solanum tuberosum cv. Désirée was vegetatively propagated from single-node stem segments in tissue culture at 24°C under a 16 h light/8 h dark regime on MS or RM medium 34. Transgenic potato lines were generated by leaf transformation according to Dietze et al. 35. Agrobacterium tumefaciens strain C58C1 containing pGV2260 36 was grown by standard techniques. The binary plasmid pZP111 carrying the deleted form (Δ358–447 bp) of CgS 12 and the same plasmid with a 15-kD β-zein insert 15 were introduced into Agrobacterium by triparental mating 37. Co-transformation was carried out by simultaneous infection of leaf explants by the two A. tumefaciens strains in equal cell density and volume. Six-week-old transgenic and control plants were transferred from tissue culture to pots and grown further under greenhouse conditions at 18–28°C for molecular and metabolite analysis as well as for yield tests. Tuberisation of 30 plants/line was tested in three independent experiments.

Regenerated plants were screened for insertion of the transgene(s) by polymerase chain reaction. Genomic DNA was prepared from leaves as described by Shure et al. 38. The 5'-gcaacatcggtgttgcacag-3'/5'-cagtgcttgcacacatccca-3' and the 5'-gatggtcatcgtctcgtcg-3'/gtagtagggcaggaatggca-3' primer pairs were used to test the presence of the CgS_Δ90 and the 15-kD β-zein genes in the potato genome, respectively.

Expression of the genes was tested by RNA-blot analysis. Total RNA was extracted from leaves using the method of Stiekema et al. 39 and separated on formaldehyde-agarose gel according to Logemann et al. 40. Blotting and hybridisation was carried out under stringent conditions according to Sambrook et al. 41. Radioactive DNA probes were generated by PCR, using the above described primers and ³²P-labelled dCTP from the CgS_Δ90 and 15-kD β-zein cDNA as templates. The phenylalanine ammonia-lyase cDNA [NCBI: BE344057] was labelled by the random priming method 41.

Concentration of the anthocyanin pigments was measured with the simplified method of Toguri et al. 42. Freshly harvested potato tubers of four-month-old plants grown in soil in greenhouse were peeled. Anthocyanin pigments were extracted from 1 g skin with 10 ml 1% HCl in methanol overnight at 4°C. Concentrations of the chloride forms of the anthocyanin pigments were determined spectrophotometrically by measuring the absorbance at 540 nm.

100 mg leaf or tuber tissue was grinded in liquid nitrogen. The obtained powder was vigorously mixed with 400 μl of 0.1 M Na-phosphate buffer, pH 7.8, followed by centrifugation at 13000 rpm for 30 min at 4°C. Concentration of total water-soluble proteins in the supernatant was determined by the method of Bradford 43. The ethanol-soluble protein fraction was extracted from the pellet with 400 μl solution containing 70% ethanol and 1% 2-mercapto-ethanol by incubation at 65°C for 30 min with shaking at 1000 rpm. Extracts were centrifuged at 13000 rpm for 30 min at 4°C.

An aliquot containing 50 μg protein from the water- or ethanol-soluble fraction was dried. The dried material was dissolved in 10 μl loading buffer, and proteins were resolved by SDS-PAGE on 14% polyacrylamide gel for 250 Vh. After separation, proteins were transferred to nitrocellulose membrane and screened with Mono HA11 (Covance) using the HRP-based enhanced chemiluminescence (ECL) system (Amersham). HRP-linked anti-mouse IgG (Amersham) was used as secondary antibody.

For the analysis of free amino acids, polar metabolite fractions were extracted from 30 mg of potato tubers according to Nikiforova et al. 32.

For protein-bound amino acid analysis an aliquot containing 200 μg protein was transferred into a fresh Eppendorf tube from the water-soluble protein extract. The same volume was transferred from the ethanol-soluble fraction into a separate tube. Both samples were dried in a Speed-vac. 100 μl 6 N HCl solution was added to the dried samples, and incubated at 100°C with shaking at 500 rpm for 16 h. The solvent was then evaporated at 100°C. To the dry material 75 μl methanol, 25 μl chlorophorm and 100 μl water were added and mixed by vortexing. The mixtures were centrifuged at 13000 rpm for 5 min at 4°C, and the upper phase was dried in a Speed-vac.

Derivatization and metabolite analysis of soluble and protein-derived amino acids was carried out as described by Nikiforova et al. 32. Ribitol was added as an internal standard. Samples were injected in splitless mode (1 μl/sample) and analysed in a quadrupole-type GC-MS system (Finnigan Trace/DSQ, Thermo Electron Corp.). The chromatograms and mass spectra were evaluated by using the XCALIBUR software (Thermo Electron Corp.) and the NIST 2.0 library.