Journal of Experimental Botany, Vol. 54, No. 382, pp. 477-488,
January 1, 2003
© 2003 Oxford University Press
Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)
Received 8 April 2002; Accepted 6 September 2002
1 Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany
2 Present address: Kyushu National Agricultural Experiment Station, Nishigoshi, Kumamoto, 861-1192, Japan.
3 Present address: Plant Science Sweden AB, Herman Ehles Väg 4, 26831 Svalöv, Sweden.
4 Present address: Faculty of Biology, Yerevan State University, Alex Manoogian St. 1, 375025 Yerevan, Armenia.
5 To whom correspondence should be addressed. Fax: +49 39482 5515. e-mail: mohammad{at}ipk-gatersleben.de
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Abstract |
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To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.
Key words: Invertase, potato tuber, respiration, starch breakdown, sucrose isomerase, sucrose sensing.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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During potato tuber development four different developmental stages can be distinguished: (i) tuber induction, (ii) storage phase, (iii) dormancy, and (iv) sprouting. Molecular, biochemical and cell biological changes during tuberization have been studied in great detail. During the stolon-to-tuber transition, sucrose utilization changes from hydrolytic to sucrolytic breakdown (Appeldoorn et al., 1997; Hajirezaei et al., 2000). This process is associated with a switch from apoplastic to symplastic phloem unloading (Viola et al., 2001). Despite numerous studies on the effect of growth regulators on tuberization, endogenous tuberization factors have not been identified. Recent reports, however, suggest that sugars play a crucial role in the regulation of cellular functions (for a review see Pego et al., 2000). During seed development of Vicia faba, for instance, high glucose levels correlate with high metabolic activity whereas high sucrose levels correlate with starch accumulation (Borisjuk et al., 1998). In potato tubers, a similar situation has been hypothesized. By ectopic expression of a yeast-derived cytosolic invertase the hexose-to-sucrose ratio could be significantly increased (Sonnewald et al., 1997). As a consequence, the metabolic activity of growing potato tubers was shifted from starch synthesis towards glycolysis. This result suggested that either low sucrose or elevated cytosolic glucose levels would be responsible for the metabolic changes observed. To distinguish between both situations, Trethewey et al. (2001) expressed a bacterial sucrose phosphorylase in transgenic potato plants. Due to the phosphorolytic cleavage of sucrose, the formation of cytosolic glucose could be circumvented. Interestingly, glycolysis and respiration were induced in sucrose phosphorylase expressing potato tubers. On the basis of these results, the authors speculate that induction of glycolysis occurs via a glucose-independent mechanism. Although interesting, the data do not unequivocally prove that low levels of sucrose are sensed. Accelerated hexose metabolism and/or fructose signalling could similarly explain the results observed.
In contrast to numerous studies concerning metabolic regulation in growing potato tubers, much less is known about the regulatory processes during potato tuber dormancy. The onset of dormancy starts during tuber initiation and ends with the growth of a visible sprout. This period is defined as the time during which sprout growth will not occur even under otherwise favourable conditions. The length of the dormancy period is dependent on both the genetic background and the environmental conditions during tuber development. Post-harvest environmental conditions have only limited impact on the sprouting behaviour. Therefore, the period is classified as endodormancy (Lang et al., 1987). It is hypothesized that dormancy is regulated by the relative concentrations of growth promoters and inhibitors (Hemberg, 1985). Gibberellins and cytokinins are generally considered as growth promoters, whereas abscisic acid and ethylene are believed to inhibit sprout growth. Despite intensive studies, no unequivocal evidence for the phytohormonal control hypothesis could be obtained. During the sink (growing) to source (sprouting) transition of potato tubers, cellular metabolism shifts from a net synthesis of reserve compounds to net degradation. During this process, starch and protein breakdown outweighs their synthesis leading to the formation of soluble sugars and amino acids. As the transport sugar, sucrose is formed in parenchyma cells and transported via the phloem system towards the developing sprout. Within the developing sprout sucrose is hydrolysed and utilized to support growth and development. Since visible sprout growth precedes detectable starch degradation, the initial growth phase is most likely initiated by preformed sucrose. This assumption has recently been supported by increasing the sucrose content of tubers by tuber-specific expression of a cytosolic pyrophosphatase, which leads to accelerated sprouting (Farre et al., 2001; discussed in Sonnewald, 2001). Due to increased sucrose demand in developing sprouts, soluble sugar levels decrease in storage parenchyma cells. Falling sugar levels might serve as a signal to trigger starch breakdown to provide sufficient assimilates for sprout growth. Based on this hypothesis, it is tempting to speculate that sucrose levels may act as sink signals to adapt reserve mobilization in storage parenchyma cells according to sink demand. To test this hypothesis, transgenic potato plants with altered sucrose levels were created. To inhibit the phloem transport of sucrose, a cytosolic yeast-derived invertase was expressed in phloem cells of transgenic potato plants. Tubers harvested from these transgenic plants were analysed for their sprouting behaviour, carbohydrate content and rate of respiration. sucrose levels dramatically decreased in tuber tissue due to invertase activity. In agreement with the hypothesis that low sucrose levels would trigger starch breakdown, accelerated starch turnover in stored potato tubers was found. Irrespective of the observed reserve mobilization, sprout growth of transgenic tubers was strongly delayed. Since sucrose hydrolysis leads to an accelerated hexose metabolism, analysis of these plants did not allow differences between sucrose and hexose effects to be distinguished. To circumvent enhanced hexose metabolism, the sucrose content was decreased by tuber-specific expression of an apoplastic sucrose isomerase from Erwinia rhapontici (Börnke et al., 2002b). The enzyme catalyses the reversible conversion of sucrose into palatinose which is not metabolizable by plant cells. As a consequence of sucrose isomerase activity, a nearly quantitative conversion of sucrose into palatinose was observed. By contrast with invertase-expressing plants, hexose metabolism was not stimulated. The investigation of starch levels during potato tuber storage revealed accelerated starch breakdown. This observation strongly suggests that sucrose levels are responsible for the regulation of metabolic processes during the sink-to-source transition of potato tubers.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Plants, bacterial strains and media
Potato plants (Solanum tuberosum) were obtained through Vereinigte Saatzuchten eG (cv. Desiree) or Bioplant (cv. Solara), Ebstorf, Germany. Plants in tissue culture were grown under a 16/8 h light/dark period on Murashige and Skoog medium (Murashige and Skoog, 1962) containing 2% sucrose. Plants used for biochemical analysis were grown in individual pots (diameter 20 cm, depth 13 cm) in the greenhouse. E. coli strain XL1-blue (Stratagene, La Jolla) was cultivated using standard techniques (Sambrook et al., 1989). Agrobacterium tumefaciens strain C58C1 containing pGV2260 (Debleare et al., 1985) was cultivated in YEB medium (Verveliet et al., 1975).
Enzymes and reagents
Enzymes and biochemicals were purchased from Boehringer (Mannheim, FRG) or Sigma (Deisenhofen, FRG).
Plasmid construction and potato transformation
To obtain phloem-specific cytosolic invertase expression the truncated suc2 gene from Saccharomyces cerevisiae encoding the mature invertase was placed under transcriptional control of the rolC promoter from Agrobacterium rhizogenes as described in Lerchl et al. (1995). Construction of the chimeric sucrose isomerase gene has been described in Börnke et al. (2002a). Direct transformation of Agrobacterium tumefaciens strain C58C1:pGV2260 was performed as described by Höfgen and Willmitzer (1988). Potato transformation using Agrobacterium-mediated gene transfer was performed as described by Rocha-Sosa et al. (1989).
Preparation and analysis of samples for enzyme activities
To measure enzyme activities, 100–200 mg potato tuber slices were homogenized in 0.5 ml 100 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (Hepes)-KOH, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 5 mM mercaptoethanol, 15% glycerine, and 0.1 mM Pefabloc phosphatase inhibitor. After centrifugation for 10 min, 13 000 rpm at 4 °C, the supernatant was frozen immediately for further analysis. Invertase and Sucrose synthase activities were determined as described in Hajirezaei et al. (1994). Amylase activities were measured using the megazyme kit (Megazyme Ireland). The procedure is based on the cleavage of a bond within the blocked p-nitrophenyl maltosaccharide substrate. The non-blocked reaction product containing p-nitrophenyl substituent is instantly cleaved to glucose and free p-nitrophenyl by the excess quantities of glucoamylase and 
-glucosidase. The phenolate colour is developed on the addition of Trizma base. For the measurement, 15 µl of extract was added to a buffer containing 100 mM MES-KOH, pH 6.2, 1 mM EDTA, and 1% mercaptoethanol. The reaction was started by adding 15 µl of substrate and the mixture was incubated at 37 °C for 30–60 min. After incubation, the reaction was stopped by adding 250 µl 1% Tris solution (w/v). The developed colour was detected at 405 nm using an Elisa Reader (Tecan Deutschland GmbH). Starch phosphorylase was determined as described in Steup (1990). An aliquot of extract was added to a buffer containing 25 mM imidazol-HCl, pH 6.9, 5 mM MgCl2, 1 mM EDTA, 5 mM sodium molybdate, 0.6 mM NAD, 2.5 µM glucose-1,6-bisphosphate, 0.4 U phosphoglucomutase, 0.4 U glucose-6-P dehydrogenase, and 0.1% soluble starch. The reaction was started by adding of 2 mM sodium dihydrogen phosphate, pH 7.0 and the increase of NAD was detected at 340 nm.
Preparation and determination of invertase activity and sugar contents in vascular tissues and parenchyma cells
To verify phloem-specific expression of invertase, vascular tissue and parenchyma from the central pith were collected and used for the measurement of soluble invertase activity and sugar contents. To harvest different tissues, tuber cross-sections (2 mm thickness) were placed on a light box and vascular tissue was cut as 1 mm strips along the slices. For parenchyma preparation, samples were collected from the middle part of the same cross-sectioned tuber. All samples were frozen immediately for further analysis. Soluble invertase activity and sugar measurement were done as described in Hajirezaei et al. (1994).
Histochemical staining of invertase
Staining of endogenous and yeast invertase was performed using an enzyme-coupled assay. The procedure is based on the cleavage of sucrose by invertase to glucose and fructose. In the presence of oxygen, the glucose produced is converted to gluconolactone and hydrogen peroxide by glucose oxidase. The oxidized coloured diaminobenzidine is visualized by adding diaminobenzidine as substrate and peroxidase. For the assay tuber discs (thickness: 2 mm, diameter 2–3 cm) were washed three times with bi-distilled water and incubated for 2 h at 37 °C in a buffer containing 0.38 M sodium phosphate, pH 6.0, 0.1 mM DTT, 0.3 mg ml–1 diaminobenzidine (DAB, Sigma, Germany), 0.25 mg ml–1 horseradish peroxidase (Sigma, Germany), 100 mM purest sucrose and 0.02 mg ml–1 glucose oxidase. After incubation the buffer was replaced by the same buffer without DTT and a brown colour developed after 1–3 h. As the control, discs were preincubated at 80 °C for 30 min (data not shown).
Metabolite determination
Metabolites were extracted essentially as described in Jelitto et al. (1992). 50–300 mg tissue material was frozen immediately in liquid nitrogen. After homogenizing, the frozen material was ground to a fine powder, 1.5 ml of 16% (w/v) trichloroacetic acid (TCA) in diethylether (4 °C) was added and the tissue further homogenized. After incubating the extract on dry ice for 15 min, 0.8 ml of 16% TCA (w/v) in water containing 5 mM EGTA (4 °C) was added to the homogenate, which was then left for an additional 3 h at 4 °C. Following centrifugation for 5 min at 15 000 rpm, the water phase was washed 3–4-fold with 600 µl water-saturated ether each time and thereafter neutralized with 5 M KOH/1 M triethanolamine.
The concentrations of metabolites and ATP/ADP were determined photometrically as in Stitt et al. (1989) using a dual wavelength spectral photometer (Sigma-ZWS II, Biochem). The recovery of small, representative amounts of each metabolite through the extraction has been documented (Hajirezaei et al., 1994).
Determination of soluble sugars and starch
Soluble sugars and starch were quantified in tuber samples extracted with 80% ethanol, 20 mM Hepes-KOH, pH 7.5 as described in Hajirezaei et al. (2000). Palatinose content was measured using Dionex HPLC system (Sunnyvale, CA, USA) as described in Börnke et al. (2002a). The quantification of palatinose content was carried out on a peak area basis.
Measurement of respiration rate in whole potato tubers
Gas exchange measurements were carried out using an infrared gas analysis in an open system (Compact minicuvette System CMS-400, Walz GmbH, Effeltrich, Germany). Whole tubers were enclosed in a standard chamber MK-022/A and the release of CO2 was monitored continuously. Chamber temperature and dew point temperature of the air entering the chamber were adjusted to 20 °C and 13 °C, respectively. Measurements were done at a gas flow rate of 1500 cm3 s–1 and ambient CO2 concentration of about 100 µmol mol–1. CO2 evolution rate referred to tuber fresh weight is given as nmol CO2 g–1 s–1.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Phloem-specific expression of cytosolic yeast-derived invertase in transgenic potato plants
A chimeric gene consisting of the rolC promoter, the truncated suc2 gene from yeast encoding the mature invertase protein, and the octopine synthase polyadenylation signal (Lerchl et al., 1995) was used to obtain phloem-specific expression of invertase in transgenic potato plants. After Agrobacterium tumefaciens-mediated gene transfer 95 regenerated plants (subsequently named DIN8) were screened for enzymatic activity in roots using an invertase activity gel assay (von Schaewen et al., 1990; data not shown). Thirty preselected plants were multiplied in tissue culture and duplicates transferred to the greenhouse. No detrimental effects on plant growth or development were observed for greenhouse-grown DIN8 plants (data not shown). DIN8 plants had a similar size, growth rate, branching pattern of the shoot, leaf appearance, timing of flowering, and tuber yield as compared to untransformed control plants. For a detailed biochemical analysis, three independent DIN8 lines (numbers 30, 87 and 90) were selected and amplified in tissue culture.
Histochemical detection of invertase activity in potato tuber cross-sections verifies phloem-restricted expression of the chimeric gene
To verify the cell-specific invertase expression, a histochemical invertase assay was performed. Cross-sections of potato tubers were collected and washed three times with bi-distilled water to remove endogenous soluble sugars. Subsequently, slices were incubated in a sucrose-containing buffer and invertase activity was visualized by the formation of a brown colour (see materials and methods). A slight staining of the endogenous invertase was detected throughout control tubers (Fig. 1A). As shown in Fig. 1B, invertase activity of transgenic plants was strongly enhanced in vascular tissues, as compared to untransformed controls (Fig. 1A). This result was confirmed by measuring invertase activity in tissue samples enriched for vascular tissue or containing mainly parenchyma cells. As shown in Fig. 1C acid invertase activity of parenchyma cells was only slightly elevated (lines 87 and 90) or unaltered (line 30) in transgenic tuber samples, as compared to untransformed controls. In vascular tissue-enriched samples, however, invertase activity of transgenic tubers was more than 10-fold higher than in control extracts (Fig. 1C). Tissue-specific activity of invertase was further supported by measuring carbohydrate levels in vascular tissue-enriched and parenchyma samples. Cell-specific expression of invertase was expected to result in an increased hexose-to-sucrose ratio in phloem cells. As shown in Fig. 2, hexose content of vascular tissue-enriched samples from transgenic tubers was strongly increased (up to 10-fold) at the expense of sucrose which strongly decreased as compared to untransformed tubers. In parenchyma cells, the contents of hexoses remained unchanged while sucrose content decreased only slightly in transformed tubers compared to untransformed tubers (Fig. 2). The slightly reduced sucrose content in parenchyma cells may be explained by phloem-specific invertase activity acting as a strong sucrose sink, rather than unspecific invertase expression. Comparing these results with results obtained from transgenic potato plants expressing cytosolic invertase under the control of a tuber-specific promoter (Sonnewald et al., 1997) strongly supports the phloem-specific expression of the yeast-derived invertase. Tuber-specific invertase expression leads to a massive increase of glucose (10–20-fold) and a dramatic decrease (down to 4%) of sucrose in parenchyma cells (Sonnewald et al., 1997).
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Phloem-specific expression of cytosolic invertase results in non-sprouting potato tubers
Recently, it was shown that removal of cytosolic pyrophosphate changed the sprouting behaviour of transgenic potato tubers dramatically (Hajirezaei and Sonnewald, 1999). It was postulated that the inhibition of sprouting was most likely due to reduced sucrose export and its subsequent utilization (Hajirezaei and Sonnewald 1999). This assumption is supported by the finding that removal of PPi in phloem cells of transgenic tobacco plants resulted in photoassimilate accumulation in leaves and reduced growth of tobacco plants (Lerchl et al., 1995; Geigenberger et al., 1996). It was concluded that inorganic pyrophosphate was essential for long-distance sucrose transport. Based on these observations it was expected that phloem restricted sucrose hydrolysis would result in a disconnection between storage parenchyma cells and the developing bud. Thereby, sprout growth should be inhibited. As expected a strong delay in sprouting was observed in the case of DIN8 tubers (Fig. 3B–D). After 5 months of storage, control tubers showed well-developed sprouts (Fig. 3A) whereas DIN8 tubers did not develop sprouts exceeding the 1–2 mm stage. Even after prolonged storage (8 months) transgenic tubers did not develop sprouts larger than 3 mm (data not shown). The observation suggests that the initiation of sprouting (indicative for the end of the dormancy period) is independent of phloem-bound sucrose transport, whereas sprout growth is highly dependent on phloem-mediated sucrose supply.
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Irrespective of decreased sprout growth, starch breakdown and dry matter loss is highly accelerated in low sucrose-containing invertase-expressing tubers during storage
Based on the results discussed, it was speculated that, due to the cell-specific manipulation of sucrose hydrolysis, reserve mobilization of tuber parenchyma cells would be delayed as compared to wild-type tubers. To test this assumption, potato tubers from transgenic and wild-type plants were harvested from 60-d-old plants before the onset of flowering. Subsequently, samples for carbohydrate analysis were taken from freshly harvested tubers (growing) and tubers stored for 10, 16, and 28 weeks at room temperature. As shown in Fig. 4, glucose, fructose and starch levels did not significantly differ between growing DIN8 and wild-type tubers. However, sucrose levels were strongly decreased in growing DIN8 tubers (Fig. 4C). During storage, sucrose levels of DIN8 and wild-type tubers gradually decreased. Irrespective of the time-dependent decrease of sucrose, DIN8 sucrose levels always remained lower as compared to the wild type. During storage, hexose levels of DIN8 tubers remained comparable to the wild type controls. These moderate changes of soluble sugars would have been expected because of the cell-specific expression of invertase. Analysing for starch accumulation, however, revealed a different picture. In growing potato tubers, no obvious difference in the amount of starch accumulation between transgenic and wild-type tubers was detectable, however, this changed drastically during storage. While starch breakdown was hardly significant in the case of wild-type tubers, DIN8 tubers were found to degrade large amounts of starch (Fig. 4D). This accelerated starch breakdown was paralleled by a significant loss of dry matter (Fig. 4E).
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Accelerated starch breakdown of DIN8 tubers is not paralleled by significant changes in the activity of starch-degrading enzymes
To investigate whether the accelerated starch breakdown of DIN8 tubers would correlate with changes in enzyme activities, enzymes involved in sucrose and starch mobilization were measured in DIN8 and wild-type tubers stored for 5 months at room temperature. As shown in Fig. 5 sucrose synthase activity increased 1.4–1.7-fold in DIN8 tubers, as compared to control tubers. A comparison of starch-degrading enzymes showed a slight decrease of starch phosphorylase, unaltered
-amylase and moderately increased ß-amylase activities. Although, slight changes in enzyme activities could be detected, it is unlikely that these changes are responsible for the major changes in starch turnover.
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Hexose-phosphates and glycolytic intermediates accumulate in stored DIN8 tubers
In order to investigate the consequences of phloem-specific yeast invertase expression on pathways involved in sucrose degradation, starch synthesis and glycolysis, concentrations of different intermediates in growing tubers and tubers stored for 5 months at room temperature were measured. As shown in Table 1, in growing DIN8 tubers a reduction of uridinediphosphate glucose (UDPGlc) was found which, along with fructose, is the product of the cleavage of sucrose by sucrose synthase. In contrast to growing tubers, the UDPGlc level did not change significantly in stored tubers of DIN8 lines compared to control tubers. The three hexose phosphates, glc6P, fru6P and glc1P showed similar behaviour in growing and stored tubers of DIN8 lines. Transgenic tubers contained between 2.2–5-fold higher levels of hexose phosphates compared with control tubers (Table 1). Similar responses were found for the concentrations of the glycolytic intermediates 3-phosphoglycerate (3PGA) and phosphoenolpyruvate (PEP). While in growing tubers of DIN8 lines, the level of 3PGA increased 2.1–2.6-fold, stored DIN8 tubers contained 1.5–2.0 times higher levels. The concentration of PEP increased up to 3.0-fold in growing DIN8 tubers and up to 1.8-fold in stored Din8 tubers compared to control tubers. Pyruvate content of growing DIN8 tubers was indistinguishable from wild-type controls, but increased 2.0–3.2-fold in stored DIN8 tubers. The levels of ATP and ADP did not differ significantly between DIN8 and wild-type tubers.
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The rate of respiration is strongly enhanced in DIN8 tubers
According to these results, starch degradation of DIN8 tubers is highly accelerated which leads to a substantial loss of dry matter. In parallel, sprout growth was strongly inhibited. This conflicting observation indicates that excess carbohydrates are most likely channelled towards alternative pathways. The increased content of glycolytic intermediates suggests that the flux through glycolysis and possibly respiration may be enhanced. To test this assumption, the respiration rate of intact growing and stored tubers was measured. As shown in Fig. 6 the rate of respiration markedly decreases from growing to stored tubers, irrespective of the genetic background. Nevertheless, DIN8 tubers are characterized by an elevated respiration rate, compared with controls. In growing tubers, the rate of respiration of DIN8 tubers was found to be only moderately increased. This difference became more pronounced during storage. During storage, respiration rates of DIN8 tubers were found to be 2–4-fold higher, compared with controls (Fig. 6). This suggests that, in the absence of sprout growth, excess carbohydrates are utilized to fuel the respiratory chain.
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Isomerization of sucrose as an alternative tool to decrease sucrose levels in potato tubers
The results discussed so far indicate that (i) phloem-mediated sucrose transport is required for sprout growth and (ii) that low sucrose may act as a signal to communicate sink demand and to regulate reserve mobilization in source tubers. On the basis of the results a working hypothesis has been developed which is summarized in Fig. 7. According to the model, sucrose is transported via the phloem system from the storage parenchym to the developing sprout. As a consequence, the sucrose content decreases in parenchyma cells which may act as a ‘sink signal’ to stimulate reserve mobilization. Phloem-specific expression of invertase leads to four major changes as compared with untransformed controls: (i) due to sucrose hydrolysis, sucrose levels decline, (ii) nutrient supply of the developing sprout is blocked, (iii) the rate of respiration is stimulated, and (iv) starch breakdown is accelerated. Since invertase expression leads to severe changes in sprout development, it cannot be ruled out that unknown ‘sink signals’ exist, regulating starch breakdown and respiration in storage parenchyma cells. In order to substantiate the results obtained with the phloem-specific invertase-expressing potato lines, it was decided to use a second set of experiments employing an additional transgenic approach. the generation of transgenic potato plants expressing a chimeric sucrose isomerase gene from Erwinia rhapontici under the control of the tuber-specific patatin class I B33 promoter (Börnke et al., 2002b) has recently been described. The gene product was targeted to the apoplasm where it catalyses the conversion of sucrose into the non-metabolizable sucrose isomer palatinose. Biochemical analyses revealed a nearly quantitative conversion of sucrose into palatinose within these transgenic tubers. Palatinose production only slightly affected starch accumulation, resulting in an only marginally decreased starch content (Börnke et al., 2002b). The common feature of both invertase-expressing and sucrose isomerase-expressing potato tubers is the drastically reduced sucrose content. However, since palatinose is not further metabolized its accumulation does not provide precursors for any further enzymatic reactions which is in contrast to the more global metabolic impact of sucrose cleavage by invertase and the subsequent release of hexoses which preferentially feed into the glycolytic pathway. The comparison of both transgenic genotypes would allow for a clear dissection of metabolic effects mediated by a reduced sucrose content per se and of effects exerted by some downstream metabolism. In order to test this hypothesis, three previously described sucrose isomerase-expressing potato lines (Börnke et al., 2002b) were subjected to a thorough biochemical analysis.
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After multiplication in tissue culture the plants were transferred to the greenhouse. As observed before (Börnke et al., 2002b) sucrose isomerase-expressing plants were indistinguishable from control plants with respect to size, growth rate, overall appearance, and tuber yield. The plants were allowed to set tubers and after harvest a portion was directly used for biochemical analyses, whereas the remaining tubers were stored at room temperature before analysis. As described earlier (Börnke et al., 2002b) expression of the Erwinia rhapontici sucrose isomerase within the apoplasm of transgenic tubers evoked a nearly quantitative shift of sucrose into palatinose (Fig. 8). Starch content was only slightly decreased in growing transgenic tubers. The picture changed distinctly when tubers stored for 80 d were used for the analyses. Hexose content was now elevated compared with control tubers whereas sucrose levels remained lower in the transgenics. The difference in starch content between control and transgenic tubers was now much more pronounced with starch levels being 66% in line 5, 56% in line 12 and 60% in line 26, respectively, lower.
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Palatinose production has no impact on metabolite contents in transgenic potato tubers
In order to investigate the impact of sucrose conversion into palatinose on sucrose degradation and glycolysis, the contents of different intermediates of these pathways was determined. In contrast to the phloem-specific invertase-expression, sucrose isomerase-expression had no impact on UDPGlc levels in freshly harvested tubers, whereas stored tubers showed a reduction of this metabolite up to 42% compared with the control tubers (Fig. 9A). Hexose phosphate levels increased in freshly harvested transgenic tubers, while, in stored transgenic tubers, the levels decreased up to 45% compared with the control. 3-PGA content remained unchanged in both growing and stored tubers.
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Stored potato tubers expressing sucrose isomerase differ in respiration rate from the untransformed control
Although the levels of glycolytic intermediates in palatinose-accumulating potato tubers remain largely unaltered, starch breakdown is heavily accelerated during storage. To find out whether this would affect the respiration rate a similar experiment as described for the invertase-expressing lines was conducted. Respiration rates of the untransformed control and that of sucrose isomerase-expressing lines did not differ during growth and dramatically decreased during storage. However, after 80 d of storage the respiration rates of transgenic tubers were significantly higher than those of control tubers (Fig. 0
).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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To study the possible regulatory role of sucrose in potato tuber metabolism, two transgenic approaches were followed. The first approach, phloem-specific expression of cytosolic invertase, aimed to block the phloem transport of sucrose. As a consequence, tuber sprouting was strongly impaired. Surprisingly, reserve mobilization was found to be highly accelerated, even in the absence of any visible sprout growth. Based on this result, it was speculated that metabolic signals rather than real sink demand would trigger starch breakdown. Hexose metabolism or sucrose levels were selected as possible candidates for metabolic signals. To distinguish between both possibilities, transgenic plants engineered to express a bacterial sucrose isomerase were included in these studies. Due to isomerase activity, sucrose is converted to palatinose, which leads to a depletion of sucrose. In contrast to sucrose hydrolysis (as catalysed by invertase), sucrose isomerization does not lead to enhanced hexose metabolism. Using these transgenic plants allowed starch turnover in low sucrose-containing tubers to be studied without significant changes in hexose metabolism. In agreement with a prominent role of sucrose in the regulation of reserve mobilization, accelerated starch breakdown in sucrose isomerase-expressing tubers was observed. Based on this result, it was concluded that low sucrose levels trigger starch mobilization in stored potato tubers.
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Acknowledgements |
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We wish to thank Andrea Knospe and Sybille Freist for doing plant transformation, Enk Geyer and the greenhouse personnel for taking care of the greenhouse plants and H Ernst for photographic work. Furthermore, we are grateful to Hannelore Apel for the measurement of respiration rate and to Ulrike Schlereth for the excellent technical assistance.
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References |
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