Differences in sucrose-to-starch metabolism of Solanum tuberosum and Solanum brevidens

Plant Science 147 (1999) 81 – 88 www.elsevier.com/locate/plantsci

Differences in sucrose-to-starch metabolism of Solanum tuberosum and Solanum bre6idens Zso´fia Ba´nfalvi a,*, Attila Molna´r a, Lo´ra´nt Lakatos a, Holger Hesse b, Rainer Ho¨fgen c a Agricultural Biotechnology Center, H-2101, P.O. Box 411 Go¨do¨llo3 , Hungary Institut fu¨r Angewandte Genetik, Freie Uni6ersitat Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany c Max-Planck-Institut fu¨r Molekulare Pflanzenphysiologie, Karl-Liebknecht-Strasse 25, 14476 Golm, Germany b

Received 12 October 1998; received in revised form 28 May 1999; accepted 28 May 1999

Abstract Sucrose-to-starch metabolism of the tuberising species Solanum tuberosum and that of the non-tuberising Solanum bre6idens was studied using in vitro stem cuttings cultured under tuber inducing conditions. The shoots growing from axillary buds of S. bre6idens and the in vitro induced stolons and tubers of S. tuberosum were characterised with respect to their carbohydrate composition by measuring the glucose, fructose, sucrose and starch contents. Expression of the genes encoding vacuolar- and cell-wall-bound invertases, vascular- and tuber-specific sucrose synthases was studied by Northern blot hybridisation. Enzyme activity patterns of alkaline and acid invertases, sucrose synthase, hexokinase and ADP-glucose pyrophosphorylase were determined in both species. Major differences between S. bre6idens and S. tuberosum were detected in the starch content, in the expression of cell-wall-bound invertase (cwInv), tuber-specific sucrose synthase (SusyI) and in the activities of sucrose synthase and ADP-glucose pyrophosphorylase that were low in S. bre6idens. Genes homologous to the cwInv and SusyI were detected in S. bre6idens, however, expression of the SusyI, unlike in S. tuberosum, was not sucrose-inducible in the non-tuberising species. These data together with previous findings (Z. Ba´nfalvi, A. Molna´r, G. Molna´r, L. Lakatos, L. Szabo´, FEBS Lett. 383 (1996) 159–164) further support the idea that cell-wall-bound invertase, tuber-specific sucrose synthase and ADP-glucose pyrophosphorylase are important elements of regulatory and biosynthetic pathways resulting in storage starch synthesis and in the expression of tuber storage protein genes. The cwInv mRNA could be detected only in developing stolons and, thus, it can be a good molecular marker of stolon growth. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Solanum; Tuber development; Sucrose induction; Sucrose-to-starch metabolism

1. Introduction Tuber development is a genetically determined and complex process. When sufficient carbohydrates are available in photosynthetically active tissues, utilisation and storage processes are induced. S. tuberosum (potato) tubers originate from stolons that are lateral shoots borne at the basal nodes of the plant. Tuberisation, however, can also be induced in vitro. High levels of sucrose (6–12%) in the culture medium lead to rapid stolon development and tuber formation from ax* Corresponding author. Tel.: + 36-28-430-600; fax: + 36-28-430482. E-mail address: [email protected] (Z. Ba´nfalvi)

illary buds of S. tuberosum stem cuttings (reviewed in Ref. [1]). When single node segments of S. tuberosum are cultured on medium with 1–2% sucrose in the the dark etiolated shoots with reduced leaf growth develop from the axillary buds. High-sucrose medium, however, results in stolonlike shoots with 2-fold higher dry matter content [2]. Stolon growth is slower than shoot development and it stops on days 9–10 when the tuber morphogenesis and growth become visible [3]. Tuberisation is accompanied by starch accumulation and expression of starch synthesis and tuber storage protein genes [4]. Tuber initiation is characterised by a decline of alkaline and acidic invertases and an increase of sucrose synthase and

0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 1 0 0 - 4

82

Z. Ba´nfal6i et al. / Plant Science 147 (1999) 81–88

fructokinase activities both in vivo and in vitro [5,2]. S. bre6idens is a close relative of S. tuberosum, but cannot form tubers. Studying single-node stem segments of this species under various conditions demonstrated in vitro responses similar to those detected in S. tuberosum, with the exception that no tubers were formed on this species [3]. Expression of the storage protein genes such as patatin and proteinase inhibitors did not occur in S. bre6idens. However, sucrose-mediated transcription of the genes involved in starch synthesis, i.e. ADPglucose pyrophosphorylase subunits (AGPaseB and S) and granule bound starch synthase (GBSS) could be detected in both species. Starch granules were present only in S. tuberosum [6]. This finding suggested that S. bre6idens, like S. tuberosum, is able to sense and respond to elevated levels of sucrose, but is unable to switch to tuber development and storage starch synthesis. The goal of the present study was to compare the sucrose-to-starch metabolism of S. bre6idens to that of the S. tuberosum and to identify elements that may be essential for the synthesis of storage starch.

2. Materials and methods

hybridisation was performed in 1M NaCl, 1% SDS, 10% dextran sulfate, 50 mM Tris–HCl (pH 7.5). After overnight incubation at 65°C, the filter was washed in distilled water for 2 min at room temperature and in 2×SSC, 1% SDS for 15 min at 65°C. Gene-specific probes for vacuolar (vacInv), and cell-wall-bound invertases (cwInv) were prepared by labelling the 647 bps EcoRI– XbaI fragment representing the 5% end of the vacInv and the 0.5 kb BamHI fragment of the cwInv clone, respectively. The invertase clones were kindly provided by Marcus Ebneth (Gatersleben, Germany). Sucrose synthase specific probes corresponding to the sus3 (vascular sucrose synthase; SusyIII) and sus4 (tuber-specific sucrose synthase; SusyI) genes described by Fu and Park [13] were obtained by labelling the 3% non-translated regions of the genes as cloned by Marcello Ehlers-Lourairo (Golm, Germany). The ubiquitin clone used as a control in Northern hybridisations was a gift from Jose J. Sanchez-Serrano (Madrid, Spain). Preparation of the Inv and ubiquitin probes were performed by random-primed labelling using kits and instructions from Amersham and Promega. The Susy probes were obtained by PCR amplification of the inserts cloned in the vector pBluescript, with pBluescript specific primers in the presence of [32P]dCTP in the reaction mix.

2.1. Plant material

2.3. Sucrose induction

Plants (S. tuberosum cv. Keszthelyi 855 and S. bre6idens) were vegetatively propagated from cuttings on RM salts [7] with 2% sucrose and 0.8% Bacto agar at 24°C with a 16 h photoperiod under 5000 lux intensity. Synchronised in vitro tuberisation was achieved in darkness as described by Ba´nfalvi et al. [3] in MS medium [7] supplemented with 8% sucrose and 2.5 mg/l 6benzylaminopurine.

Leaves were isolated from pot-grown plants and treated as described by Fu and Park [13].

2.2. DNA, RNA isolation and analysis The basic methods were according to Sambrook et al. [8]. Total RNA was extracted as described by Stiekema et al. [9] and subjected to electrophoresis on formaldehyde-agarose gels according to Logemann et al. [10]. Blotting and hybridisation conditions were as described by Amasino [11]. Genomic DNA from the leaves was prepared by using the method of Shure et al. [12]. Southern

2.4. Sugar and starch assays Micro-tuber discs, shoots or stolon slices (150 mg FW) were cracked into small bits at room temperature with a wooden stick at the bottom of micro-centrifuge tubes and extracted with 1 ml 90% ethanol at 70°C for 90 min. After centrifugation for 10 min at 13 000 rpm, the supernatant was used for the determination of glucose, fructose and sucrose as described by Stitt et al. [14]. The ethanol-insoluble material was homogenised in 400 ml 0.2 M KOH and incubated at 95°C for 1 h. After neutralization with acetic acid the samples were centrifuged at 14 000 rpm for 10 min and the supernatant was collected to measure the starch content using a commercially available kit (Boehringer Mannheim).

Z. Ba´nfal6i et al. / Plant Science 147 (1999) 81–88

2.5. Enzyme assays Extracts were prepared to assay enzyme activities by homogenising micro-tuber discs, shoots or stolon slices (1 g FW) in 3 ml extraction buffer, as described by Dancer et al. [15], on ice with a mortar and pestle. Clear supernatants were obtained by centrifugation in micro-centrifuge tubes at 14 000 rpm for 10 min at 4°C. Desalting of the samples was achieved by applying 500 ml supernatant to a NAP-5 (Pharmacia) column equilibrated with extraction buffer at 4°C. The column was eluted with 1 ml extraction medium. Aliquots of 100 ml were frozen in liquid N2 and stored at − 70°C until required. Alkaline and soluble acid invertases were assayed exactly as described by Zrenner et al. [16]. Hexokinase (glucokinase) was measured at room temperature in 50 mM Tris–HCl (pH 8.0), 4 mM MgCl2, 2.5 mM ATP, 0.33 mM NAD + , 2.5 U/ml glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides and 10–100 ml of the protein extract in a final volume of 300 ml. The reaction was started with glucose at a concentration of 1 mM. Sucrose synthase activities were determined according to Dancer et al. [15]. AGPase was assayed using the method of Mu¨llerRo¨ber et al. [17]. Reactions were carried out in microtiter plates. NAD + reduction was followed spectrophotometrically at 340 nm.

83

Glucose, fructose, sucrose and starch content of the S. bre6idens shoots, S. tuberosum stolons and micro-tubers were analysed after 10 and 15 days of induction. Relatively high levels of soluble sugars were detected in S. bre6idens shoots (50, 30 and 30–70 mg/g FW glucose, fructose and sucrose, respectively) and in S. tuberosum stolons where the dominant sugar was glucose (80 mg/g FW). Tubers were characterised by high levels of starch (80–170 mg/g FW). A slight increase in starch content (from 10 to 30 mg/g FW) was detected in potato stolons by day 15 (Fig. 1).

3. Results

3.1. Carbohydrate analysis The carbohydrate contents of S. tuberosum and S. bre6idens were compared under in vitro tuber inducing conditions. Shoots of 2-month-old cultures were harvested and single-node segments with a resting axillary bud were obtained after the leaves were removed. The stem segments were explanted in MS medium containing 8% sucrose and were incubated further in the dark. In the case of S. tuberosum tuberising shoots, stolons were developed from the axillary buds with visible swellings by day 10 that resulted in micro-tubers by day 15. Axillary buds of S. bre6idens also formed shoots, although the rate of elongation was slower than in S. tuberosum. No tubers or tuber-like structures were detected on S. bre6idens.

Fig. 1. Carbohydrate content of S. bre6idens shoots, S. tuberosum stolons and tubers developed from axillary buds of in vitro stem cuttings under tuber inducing conditions in constant darkness. All stolons were tuberising but the tubers were separated and measured independently. Samples were taken 10 and 15 days after induction. Each extract was prepared from at least 10 – 15 explants. The data are the mean9 S.E. of 4 – 5 independent experiments. High S.E. is derived from the individual experiments and not from the assay. Repeating the assay with the same extract resulted in less than 10% deviation in each case (data not shown).

84

Z. Ba´nfal6i et al. / Plant Science 147 (1999) 81–88

Fig. 2. Northern hybridisation of invertases and sucrose synthases in S. tuberosum and in S. bre6idens. Samples of total RNA (20 mg) isolated from stolons, shoots, and tubers (tub.) developed from axillary buds 5, 10 and 15 days after transferring single node stem segments to MS medium containing 8 or 2% sucrose, were subjected to electrophoresis and probed with the gene-specific probes cwInv, SusyI, vacInv, and SusyIII. Ubiquitin was used as a probe to show that approximately equal amounts of RNA were loaded in each lane. 18S RNA is also shown as a control.

3.2. Gene expression studies Previously it has been shown that under tuberinducing conditions the expression of the tuber storage protein genes did not occur in S. bre6idens, however, sucrose mediated transcription of the starch-synthesis genes, AGPaseB, S and GBSS, could be detected in this species also. The highest amount of AGPase and GBSS mRNAs in S. bre6idens was found 5 days after induction, while they were further increased in time in the case of S. tuberosum [6]. Tuberisation is paralleled by a switch from an invertase-sucrolytic to a sucrose synthase-sucrolytic system [2,5]. Therefore, expression of the genes encoding different types of invertases and sucrose synthases in S. bre6idens was also investigated. Fig. 2 shows that the cell-wall-bound invertase (cwInv) and the tuber-specific sucrose synthase (SusyI) are highly expressed only in S. tuberosum.

Large amounts of cwInv transcript can be detected in stolons under tuber-forming conditions in vitro. The maximum expression is between 5–10 days after induction, but then declines completely. By day 15 no cwInv mRNA at all can be detected in the stolons. Developing tubers have no detectable amount of cwInv mRNA. On the other hand, activation of cwInv transcription is mediated by sucrose. cwInv mRNA was found only in S. tuberosum stolons grown from axillary buds at high (8% w/v) sucrose concentration in the medium but not in shoots developed from them in the same medium at low (2% w/v) sucrose concentration. The expression pattern of SusyI is different from that of the cwInv. Although SusyI is also sucrose-inducible and transcribed in stolons, unlike cwInv, the maximum amount of steady-state SusyI mRNA is present in developing tubers. Much less differences in expression were found in the case of vacuolar invertase (vacInv) and vascular sucrose synthase (SusyIII). Transcripts of these genes were detectable in stolons and shoots as well as in the induced micro-tubers of S. tuberosum and in the axillary shoots of S. bre6idens. The intensity of hybridisation, however, in the case of S. tuberosum was slightly dependent on the sucrose concentration used in the medium, namely, more vacInv and SusyIII mRNA was present in the stolons (8% sucrose) than in the shoots (2% sucrose). The Northern hybridisations showed less vacInv and SusyIII mRNA in the developing tubers than in the stolons. However, concerning the intensity of hybridisation of the individual samples to the ubiquitin probe as a control and the high deviations found in repeated experiments (Figs. 1 and 5), these differences in mRNA amounts might not be significant. The lack of strong hybridisation signals in Northern blots with the probes cwInv and SusyI in S. bre6idens could be due either to a lack of homolog genes or to different regulation of gene expression in S. bre6idens as compared to that in S. tuberosum. To distinguish between these two possibilities Southern hybridisations were performed with cwInv and SusyI specific probes. Hybridisations in both cases were detected in S. bre6idens. Thus, the lack of hybridising mRNA in Northern blots was not due to weak homology between the cwInv and SusyI genes of the two species, but rather was due to the lack of gene expression under tuber inducing conditions in S.

Z. Ba´nfal6i et al. / Plant Science 147 (1999) 81–88

85

tion. Fig. 4 shows that an even lower amount of transcript was present in this species at high than at low sucrose concentration. This result suggests that regulation of the SusyI gene expression in S. bre6idens is different to that observed in S. tuberosum.

3.3. Enzyme acti6ities

Fig. 3. Southern hybridisation of cwInv and SusyI to the genomic DNA of S. tuberosum and S. bre6idens. E, EcoRV; H, HindIII.

bre6idens. The copy number of the genes, however, might be different since fewer hybridising bands were visible in S. bre6idens than in S. tuberosum (Fig. 3). SusyI mRNA is mainly present in developing tubers, but its expression is not restricted to this organ [18]. An increase in sucrose concentration leads to an increase in SusyI mRNA in potato leaves [13]. Sucrose inducibility of SusyI in S. bre6idens leaves was tested by Northern hybridisa-

Fig. 4. Expression of the SusyI in detached leaves of S. tuberosum and S. bre6idens. Samples of total RNA (20 mg) isolated from tuber and detached leaves incubated in MS basal medium ( − ) or that supplemented with 200 mM sucrose ( +).

Activity patterns of the enzymes most characteristic for tuber development (e.g. alkaline and acid invertases, sucrose synthase, AGPase) and that of the hexokinase implicated in sugar sensing in higher plants [19] were determined in S. bre6idens shoots growing under tuber inducing conditions and compared to those obtained in tuberising stolons and developing micro-tubers of S. tuberosum. High levels of alkaline and acid invertase activities (300 and 2000 nmol/min/g FW, respectively), independent of induction time, were detected in S. bre6idens shoots. The invertase activities of S. tuberosum stolons and tubers were also high on day 10, but decreased over those time intervals when the activities of hexokinase and sucrose synthase were increased in the tubers. Hexokinase and sucrose synthase activities were low in S. bre6idens shoots (3–10 nmol/min/g FW) as in the S. tuberosum stolons (0–3 nmol/min/g FW). Interestingly, the activity of AGPase was very high not only in tubers but also in stolons (400 nmol/min/g FW), while it was relatively low (100 nmol/min/g FW) in the S. bre6idens shoots (Fig. 5).

4. Discussion Using an in vitro tuberisation system, several differences in sucrose-to-starch metabolism of a tuberising and a non-tubersing Solanum species, S. tuberosum and S. bre6idens, respectively, were detected. In the presence of 8% sucrose in the medium, shoots with relatively high amounts of sucrose developed from the axillary buds of S. bre6idens, although high alkaline and acid invertase activities were detected at the same time in this tissue. Acid invertases are thought to function in the vacuole and in the apoplast of the plant cell, while alkaline invertases are cytoplasmic. Since no expression of cwInv was detected in S. bre6idens but the vacInv mRNA was present in the shoots,

86

Z. Ba´nfal6i et al. / Plant Science 147 (1999) 81–88

Fig. 5. Enzyme activities detected in S. bre6idens shoots and in S. tuberosum stolons and tubers developed from axillary buds of in vitro stem cuttings under tuber inducing conditions in darkness. The samples were taken from the same tissues used for carbohydrate analysis (see Fig. 1 for the legend).

it is reasonable to conclude that the invertase activities in S. bre6idens are due to the vacuolar and cytoplasmic invertases. In contrast, cwInv has probably the major acid invertase activity in potato stolons. This is indicated by the high level of transient expression of cwInv on days 5–10 that coincided with the high acid invertase activities detected on day 10 with a decline on day 15. It is well established that high acid invertase activity is common in rapidly elongating tissues [5], thus the expression pattern of cwInv might be associated with the stolon development that is stopped on day 10 when tuber growth started.

Sucrose synthase constitutes the predominant route of sucrose breakdown after tuber initiation [20]. The SusyI gene of the potato is sucrose inducible [13]. In agreement with the highest amount of mRNA, the highest sucrose synthase activity was also detected in developing tubers in our experiments. In contrast, we found that the SusyI gene of S. bre6idens was not sucrose inducible. No increase in SusyI expression was detected either in the axillary shoots developed at high sucrose concentration in the medium or in sucrose-treated detached leaves of S. bre6idens. In this later case even a lower amount of SusyI mRNA was present than in the control.

Z. Ba´nfal6i et al. / Plant Science 147 (1999) 81–88

Sucrose synthase is a major determinant of sink strength in tubers (see Ref. [21] for a review). Antisense reduction of sucrose synthase in tubers resulted in a strong accumulation of reducing sugars and an inhibition of starch accumulation. The changes in carbohydrate content were accompanied by the lack of expression of tuber storage protein genes as well as that of AGPaseB and S [16]. The characteristics of the S. bre6idens shoots resemble those of the potato tubers with greatly reduced levels of sucrose synthase activity due to an antisense construct, viz. high level of soluble sugars, low amount of starch, and lack of the patatin and storage protein gene expression. The AGPaseB and S genes, however, are activated in S. bre6idens [6] even if their expression is transient and the activity of AGPase in the shoots remains low. These data, together with previous findings [6], suggest that the feed-back mechanism which reduces the amount of mRNAs from genes involved in starch synthesis might rely on the lack of the metabolic channeling of sucrose via sucrose synthase in S. bre6idens. Southern hybridisation showed that S. bre6idens carries a gene homologous to the SusyI gene of S. tuberosum. The patatin gene of S. tuberosum is also present in a homologous form in S. bre6idens [22]. However, this gene, like SusyI, is not sucroseinducible in S. bre6idens [23]. This finding may correlate with the lack of sucrose-inducible promoter elements in SusyI and patatin genes of S. bre6idens that were detected in SusyI (sus4 ) and the classI patatin genes of S. tuberosum [24,25]. Another possibility is that the sucrose signal transduction pathway or one of its trans-acting element differs in S. bre6idens from that present in potato. Hexokinases are implicated in the sugar sensing mechanism in higher plants (see Ref. [19] for a review). The hexokinase activity of S. bre6idens shoots is low, although this activity is even lower in S. tuberosum stolons and is increased only in tubers. An alternative to hexokinase signalling might be the apoplastic invertase-sucrose synthase pathway. When Chenopodium rubrum was supplied with the non-mobilizable glucose analogue 6-deoxyglucose (which is not a substrate for hexokinase) induction by glucose could be mimicked [26,27]. Since very characteristic differences in cwInv and SusyI mRNA levels were detected between S. tuberosum stolons and S. bre6idens shoots developed from axillary buds at high sucrose con-

87

centration and between S. tuberosum stolons (8% sucrose) and axillary shoots (2% sucrose) we speculate that the apoplastic invertase-sucrose synthase pathway might be important in stolon development. Comparison of alternative sink organ systems, such as tomato fruit, allows a generalisation of the observations made for the tuber system. During the stage of starch accumulation in tomato fruit development sucrose synthase, fructokinase, AGPase and starch synthase are co-ordinately regulated [28]. The same co-ordinated regulation of sucrose synthase, hexokinase and AGPase was found in potato micro-tubers. Suprisingly, however, AGPase activity was as high in the stolons as in the developing micro-tubers, although the hexokinase and sucrose synthase were relatively inactive in the stolons. The starch content of the stolons was, however, low. These data suggest a dual role for AGPase: transient starch synthesis with rapid turnover in the stolons, and storage starch synthesis in the developing tubers. However, more studies are needed to investigate this hypothesis.

Acknowledgements We are grateful to M. Ebneth, M. EhlersLourario and J. Sanchez-Serrano for the invertase, sucrose synthase and ubiquitin clones. We thank R. Trethewey and J. Veramendi for discussions, X. Sztanko´ for technical assistance, G. Taka´cs for the photos and J. Lloyd for critical reading of the manuscript. A.M. was a PhD student of the Eo¨tvo¨s Lora´nd University, Budapest. This work was supported by Volkswagen-Stiftung I/71 757.

References [1] E.E. Ewing, Cuttings as simplified models of the potato plant, in: P.H. Li (Ed.), Potato Physiology, Academic Press, New York, 1985, pp. 154 – 207. [2] N.J.G. Appeldoorn, S.M. de Bruijn, E.A.M. KootGronsveld, R.G.F. Visser, D. Vreugdenhil, L.H.W. van der Plas, Developmental changes of enzymes involved in conversion of sucrose to hexose-phosphate during early tuberisation of potato, Planta 202 (1997) 220 – 225. [3] Z. Ba´nfalvi, A. Molna´r, Z. Kostya´l, L. Lakatos, G. Molna´r, Comparative studies on potato tuber development using an in vitro tuber induction system, Acta Biol. Hung. 48 (1977) 77 – 86.

88

Z. Ba´nfal6i et al. / Plant Science 147 (1999) 81–88

[4] S. Prat, W.B. Frommer, R. Ho¨fgen, et al., Gene expression during tuber development in potato plants, FEBS Lett. 286 (1990) 334–338. [5] H.A. Ross, H.V. Davies, L.R. Burch, R. Viola, D. Mc-Rae, Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum), Physiol. Plant. 90 (1994) 748–756. [6] Z. Ba´nfalvi, A. Molna´r, G. Molna´r, L. Lakatos, L. Szabo´, Starch synthesis-, and tuber storage protein genes are differently expressed in Solanum tuberosum and in Solanum bre6idens, FEBS Lett. 383 (1996) 159–164. [7] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant. 15 (1962) 473–497. [8] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. [9] W.J. Stiekema, F. Heidkamp, W.G. Dirkse, et al., Molecular cloning and analysis of four tuber mRNA, Plant Mol. Biol. 11 (1988) 255–269. [10] J. Logemann, J. Schell, L. Willmitzer, Improved method for the isolation of RNA from plant tissues, Anal. Biochem. 163 (1987) 16–20. [11] R.M. Amasino, Acceleration of nucleic acid hybridization rate by polyethyleneglycol, Anal. Biochem. 152 (1986) 304–307. [12] M. Shure, S. Wessler, N. Fedoroff, Molecular identification and isolation of the Waxy locus of maize, Cell 35 (1983) 225–233. [13] H. Fu, W.D. Park, Sink and vascular-associated sucrosesynthase functions are encoded by different gene classes in potato, Plant Cell 7 (1995) 1369–1385. [14] M. Stitt, R. MacLilley, R. Gerhardt, H.W. Heldt, Metabolite levels in specific cells and subcellular compartments of plant leaves, Methods Enzymol. 174 (1989) 518–552. [15] J.E. Dancer, W.-D. Hatzfeld, M. Stitt, Cytosolic cycles regulate the turnover of sucrose in heterotrophic cell-suspension cultures of Chenopodium rubrum L, Planta 182 (1990) 223–231. [16] R. Zrenner, M. Salanoubat, L. Willmitzer, U. Sonnewald, Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato (Solanum tuberosum L.) plants, Plant J. 7 (1995) 97–107.

.

[17] B. Mu¨ller-Ro¨ber, U. Sonnewald, L. Willmitzer, Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes, EMBO J. 11 (1992) 1229 – 1238. [18] M. Salanoubat, G. Belliard, The steady-state level of potato sucrose synthase mRNA is dependent on wounding, anaerobiosis and sucrose concentration, Gene 84 (1989) 181 – 185. [19] J. Jyan-Chyan, J. Sheen, Sugar sensing in higher plants, Trends Plant Sci. 2 (1997) 208 – 214. [20] S. Morrell, T. ap Rees, Sugar metabolism in developing tubers of Solanum tuberosum, Phytochemistry 25 (1986) 1579 – 1585. [21] K. Herbers, U. Sonnewald, Molecular determinants of sink strength, Curr. Opin. Plant Biol. 1 (1998) 207–216. [22] D.J. Hannapel, Differential expression of potato tuber protein genes, Plant Physiol. 94 (1990) 919 – 925. [23] Z. Ba´nfalvi, Z. Kostya´l, E. Barta, Solanum bre6idens possesses a non-sucrose-inducible patatin gene, Mol. Gen. Genet. 245 (1994) 517 – 522. [24] H. Fu, S.Y. Kim, W.D. Park, High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5% and 3% flanking sequences and the leader intron, Plant Cell 7 (1995) 1387 – 1394. [25] C. Grierson, J.S. Du, M.T. Zabal, et al., Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene, Plant J. 5 (1994) 815 – 826. [26] T. Roitsch, M. Bittner, D.E. Godt, Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analog and tissue-specific expression suggest a role in sink – source regulation, Plant Physiol. 108 (1995) 285 – 294. [27] D.E. Godt, A. Riegel, T. Roitsch, Regulation of sucrose synthase expression in Chenopodium rubrum: characterization of sugar induced expression in photoautotrophic suspension cultures and sink tissue specific expression in plants, J. Plant Physiol. 146 (1995) 231 – 238. [28] A.A. Schaffer, M. Petreikov, Sucrose-to-starch metabolism in tomato fruit undergoing transient starch accumulation, Plant Physiol. 113 (1997) 739 – 746.