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Journal of Experimental Botany, Vol. 50, No. 333, pp. 477–486, April 1999 Impact of invertase overexpression on cell size, starch granule formation and cell wall properties during tuber development in potatoes with modified carbon allocation patterns Eva Tauberger1, Susanne Hoffmann-Benning2, Helga Fleischer-Notter1, Lothar Willmitzer1 and Joachim Fisahn1,3 1 Max Planck Institut für molekulare Pflanzenphysiologie, Karl Liebknechtstr. 25, D-14476 Golm, Germany 2 Institut für Genbiologische Forschung, i.L. Ihnestr. 63, D-14195 Berlin, Germany Received 8 June 1998; Accepted 25 September 1998 Abstract Introduction Transgenic potato tubers that overexpressed either a cytosolic or an apoplastic invertase in the wild type or AGPase antisense background were used to analyse the effect of invertase activity on cell expansion, starch granule formation and turgor pressure during tuber development. Although the transgenic plants did not develop a visible phenotype in aerial regions the size and number of tubers were significantly modified in the various lines. Transmission electron and light microscopy were performed to monitor starch grain size and number, cell size and cell wall thickness. Water potential, osmotic pressure, and, indirectly, turgor pressure were determined during the final stages of tuber development. Glucose levels were high in transgenic tubers that overexpressed a yeastderived invertase. The number of starch grains per cell was almost identical in all transgenic lines. However, the amount of starch was modified in the transgenics as compared to the wild type. As expected, the size of starch grains was reduced in all lines that expressed an AGPase antisense mRNA. These results indicate that invertase activity and glucose levels do not affect initiation of starch grain formation during the early stages of tuber development, but growth of starch corns in the later stages of tuber maturation. Sucrose is the major form of translocated carbon in most plant species. Therefore, it is a major substrate for plant growth and generation of turgor pressure. In potato there is strong evidence that sucrose is unloaded from the phloem into tuber storage parenchyma cells through plasmodesmata (symplastic unloading, Oparka and Prior, 1988; Oparka et al., 1992). The initial cleavage of sucrose in potato tubers occurs mostly through sucrose synthase and to a smaller extent through invertase (Pressey, 1969; ap Rees and Morrell, 1990; Ross and Davies, 1992). It was reported that the activity of sucrose synthase was low in small tubers, but increased to a peak before declining at the end of the growing season (Pressey, 1969; Tsay and Kuo, 1980). To investigate the role of sucrose cleavage during tuber development on a cellular basis, transgenic potato plants were used which overexpressed a yeast-derived invertase in the cytosol or the apoplast in a wild-type (Büssis et al., 1997; Sonnewald et al., 1997) or AGPase antisense background. Invertase has also been discussed to be involved in the control of phloem unloading and rapid growth of sink organs. In terms of a model described by Roitsch and Tanner (1996) sucrose would be released from the sieve elements into the apoplast and subsequently split by invertase. Uptake of hexoses into the sink cells would then be mediated by hexose transport proteins. A number of reports indicate that in organs which grow by cell elongation, acid invertase is the dominant enzyme for Key words: Potato, tuber, development, starch, osmotic pressure, water potential. 3 To whom correspondence should be addressed. Fax: +49 331 977 2301. E-mail [email protected] Abbreviations: AGPase, ADP-glucose-pyrophosphorylase; TEM, transmission electron microscopy. © Oxford University Press 1999 478 Tauberger et al. Fig. 1. Developmental stages of a potato tuber. Growth of potato tubers was divided into six classes which are depicted in the photograph. Stage one was the earliest stage of tuber development and characterized by a thickening of the stolon. Mature tubers were obtained in stage 6. sucrose breakdown, whereas in non-growing sink tissue sucrose synthase predominates (Sung et al., 1989). In dark-grown seedlings of sunflower as well as in bean internodes and pea epicotyls the specific activities of invertase were closely associated with the rate of cell elongation (Maclachland et al., 1970; Morris and Arthur, 1985; Kutschera and Köhler, 1994). The potential importance of invertase during growth and development is also indicated in transgenic plants overexpressing yeast invertase in various compartments of Arabidopsis (von Schaewen et al., 1990), tobacco ( Heinecke et al., 1994; Hoffmann-Benning et al., 1997; Sonnewald et al., 1997), potato (Büssis et al., 1997; Sonnewald et al., 1997), and tomato (Dickinson et al., 1991). Cleavage of sucrose can affect turgor pressure. This was demonstrated in a recent study by Hoffmann-Benning et al. (1997). In particular, transgenic tobacco plants that overexpressed a yeast-derived invertase in the vacuole exhibited a severe increase in turgor pressure within epidermis and mesophyll cells. In contrast, turgor and osmotic pressure remained largely constant during rapid stem elongation in darkness ( Kutschera, 1991a, b). Since stem elongation is accompanied by a corresponding accumulation of soluble sugars, it was proposed that uptake of hexoses into expanding cells may counter the dilution of the vacuolar contents caused by water uptake. Hence internal osmotic pressure and thus cell turgor could be maintained by the uptake of sugars. However, sugars not only function as substrates for growth but affect sugar-sensing systems that initiate changes in gene expression ( Koch, 1996). The realization that these genes are also affected by exogenous stimuli (Sturm and Chrispeels, 1990) and endogenous hormones further complicates our understanding of the mechanisms for the regulation of sugar metabolism, turgor and growth. Recently it has become apparent that sugars affect the expression of certain genes involved in many essential processes such as photosynthesis, glycolysis, sucrose and starch metabolism, defense mechanisms, cell cycle regulation, and growth (Sheen, 1994; Koch, 1996). The aim of the present investigation was to elucidate the relationship between sucrose cleavage and cell size, starch granule formation, and turgor pressure during potato tuber development. Genetic engineering provides a novel approach to modulate the degree of sucrose cleavage and thus growth and development of specific plant organs. Several lines of transgenic plants were used that overexpressed an invertase which was targeted either to the cytosol or to the apoplast in a wild-type or an AGPase antisense background. All transgenic plants showed no phenotype in aerial parts, but changes in tuber development and size and in starch and sugar accumulation. Materials and methods Plant material Wild-type and transgenic plants were grown in the greenhouse at a 25 °C day (14 h) and a 20 °C night temperature and watered twice a day as described previously (Fisahn et al., 1995). The relative humidity was 65%. Wild type (Solanum tuberosum cv. Desirée) were obtained from Saatzucht Fritz Lange (Bad Schwartau, Germany). U-IN-1: transgenic plants expressed an apoplastic invertase from Sacchoromyces cerevisiae (Sonnewald et al., 1991). U-IN-2: transgenic lines expressed a cytosolic invertase from Sacchoromyces cerevisiae (Sonnewald et al., 1997). B-AGP-93: transgenic potato expressed ADP glucosepyrophosphorylase antisense mRNA (Müller-Röber et al., 1992). H-AI: AGPase antisense plant with a second transgene coding for an invertase from Sacchoromyces cerevisiae that was targeted to the apoplast (Trethewey et al., 1998). H-IN: AGPase antisense plant with a second transgene coding for an invertase from Sacchoromyces cerevisiae that was targeted to the cytosol ( Trethewey et al., 1998). The genes of the transgenic plants were under the transcriptional control of the class I patatin promotor (B33) which shows tuber specific expression. Tuber sample collection Experiments were performed on the tuber developmental stages shown in Fig. 1. Samples for microscopy and biochemical analysis were taken in the morning. The protocol for sample collection is depicted in Fig. 2. To reduce variations due to different potato tuber areas equivalent samples were taken from all stages of tuber development: In stages 1–3 cross-sections of 1 mm were taken and the outer half viewed in the microscope. In stages 4–6 samples were taken with a cork-borer (5 mm diameter), discarded the outermost mm and used the next 1 cm of the resulting cylinder for biochemical and the next 1–2 mm for microscopical analysis. Microscopy Samples were collected as described above. Quarters of the resulting discs were fixed and embedded as described in Hoffmann-Benning et al. (1994). Ultrathin sections (80 nm) were stained with lead citrate and viewed in a Philips 400 transmission electron microscope (TEM ) at the Max Planck Effects of invertase overexpression on tuber development 479 tubers than the wild type and U-IN-2 plants more but smaller ones. The latter result has also been found by Sonnewald et al. (1997). Supertransformants expressing both invertase and an AGPase antisense construct had tuber sizes and numbers intermediate between wild type and AGPase antisense plants. Tuber developmental stages Tubers of different lines were analysed during several stages of development ( Fig. 1). State 1 was characterized by a thickening of the stolon. The later phases were divided into five additional classes. Mature tubers were developed in stadium 6. Sugar analysis Fig. 2. Schematic diagram of the protocol that was applied for sample collection. For investigation of the tuber developmental stages 1–3, cross-sections were used to measure parameters of interest. To obtain samples from developmental stages 4–6, a cork-borer was radially inserted into growing tubers. Institute for Molecular Biology, Berlin. Measurements of cell wall thickness were obtained from these micrographs. Osmotic pressure 1 cm slices of all tuber stages were homogenized for 15–20 s and centrifuged at 13 000 rpm for 1 min. The osmotic pressure of 50 ml supernatant or appropriate dilutions was determined using a microliter osmometer (Osmomat 030, Gonotech, Berlin, Germany; Hoffmann-Benning et al., 1997). Water potential measurements 1 mm slices of stages 4–6 were collected with a cork-borer with opening diameter of 5 mm. Water potential was measured using a Wescor-H33T dew point microvoltmeter and C52 sample chamber (Logan, UT, USA). Sucrose and hexose concentrations Samples were immediately frozen, extracted in 80% ethanol at 80 °C for 1 h and sugars measured as described (Stitt et al., 1989; Sonnewald et al., 1991). All measurements are presented as mean values plus standard deviation. Means were calculated from at least 20 repeats. Results Phenotypes and number of tubers No visible phenotype evolved in the aerial regions of all transgenic lines that were investigated in the present study. They all resembled the wild type. However, significant differences between the transgenic lines emerged in the number and the size of tubers ( Fig. 3; Table 1). AGPase antisense plants were characterized by a higher amount of tubers, but, of smaller size. U-IN-1 plants (apoplastic localization) had fewer, but bigger As expected, sugar analysis resulted in high levels of glucose in transgenic plants that overexpressed a yeastderived invertase ( Fig. 4). This increase started to be visible in stage 3 except for U-IN-2 plants where changes were less obvious until stage 4. When the glucose-tosucrose ratio was calculated (not shown) a significant increase was seen from stage 3 onwards when the invertase was localized in the apoplast. When it was targeted to the cytoplasm changes were seen starting in stage 4. Thus the effect of invertase activity on glucose levels was stronger and visible earlier when the enzyme was localized in the apoplast. However, cytoplasmic targeting led to an equally high or even higher glucose content and to elevation in osmolality during the last developmental stage and the harvest experiment. Osmolarity Osmolarity showed significant differences in late stages of tuber development (Table 2). In particular, osmolality was reduced in AGPase antisense plants and increased in both cytoplasmic invertase lines in stage 6 of tuber development. Apoplastic invertase plants showed significant differences compared to the wild type in the early stages of tuber development, probably due to the different compartmentation of the sugars or due to other osmotically active substances participating in the osmotic adjustment (data not shown). Microscopy Cross-sections of comparable tuber regions of transgenic and wild-type plants were made to analyse the effect of invertase activity and glucose accumulation on cell and starch grain size and growth in the various stages of tuber development. One representative set of photographs is shown in Fig. 5 (stage 5). A summary of the results concerning cell size and starch grain size is shown in Figs 6 and 7. In the early stages of tuber development cells remained small, indicating that cell division is one important factor leading to tuber growth. During that 480 Tauberger et al. Fig. 3. Characteristic display of the amounts and sizes of tubers that were obtained from the lines investigated in the present study. The highest number of tubers was developed by the AGP-93 transgenic lines. time the number of starch grains fluctuated (Fig. 8). In stages 3–6 the number of starch grains was reduced in all lines, while the cell diameter increased. This result indicated that cells located within the sampled regions ceased to divide and tissue grew by cell expansion. The number of starch grains remained almost constant after one last division indicating that initiation of starch grains had ceased. Parallel to cell size, starch grain size and cell wall thickness were monitored using electron micrographs (Figs 7, 9). Size of starch grains increased continuously during tuber development in almost all plant lines. However, clear differences were visible in the various lines. While starch grains of wild-type plants increased continuously, those in AGPase antisense plants showed no more enlargement from stage 3 onwards. Plants expressing invertase in the wild-type background exhibited starch grain deposition similar to that of wild-type plants. Plants with the invertase expressed in the AGPase antisense background developed starch grains similar to B-AGP plants. Cell wall diameter of all lines increased during tuber maturation (Fig. 9). Significant increases in cell wall diameter were developed in the transgenic lines U-IN-1 and 2 as compared to the wild type in the final stage 6 of tuber development. Water relations Water potential and turgor pressure fluctuated during the last stages of tuber development ( Table 2). Turgor pressure was calculated from these water potential and osmotic pressure determinations according to Taiz and Table 1. Average number of tubers per genotye Genotype Wild type B-AGP-93 U-IN-1 U-IN-2 H-AI H-IN Number of tubers 8±2 28±4 7±2 9±2 12±2 15±2 Effects of invertase overexpression on tuber development 481 Fig. 4. Sugar distributions of all potato lines that were investigated, exhibited characteristic patterns in the individual developmental stages. Glucose levels were high in the later stages of tuber development in plants with invertase overexpression. U-IN-1: transgenic plants expressed an apoplastic invertase from Sacchoromyces cerevisiae. U-IN-2: transgenic lines expressed a cytosolic invertase from Sacchoromyces cerevisiae. B-AGP-93: transgenic potato expressed ADP glucose-pyrophosphorylase antisense mRNA. H-AI: AGPase antisense plant with a second transgene coding for an invertase from Sacchoromyces cerevisiae that was targeted to the apoplast. H-IN: AGPase antisense plant with a second transgene coding for an invertase from Sacchoromyces cerevisiae that was targeted to the cytosol. Table 2. Water potential and osmolality of transgenic plants in the developmental stages 4–6 Stage Wild type U-IN-1 U-IN-2 H-AI H-IN B-AGP Water potential (MPa) 4 5 6 −0.4±0.08 −0.4±0.1 −0.7±0.11 −0.4±0.1 −0.4±0.1 −0.5±0.08 −0.6±0.1 −0.7±0.08 −0.5±0.1 −0.5±0.1 −0.4±0.11 −0.6±0.08 −0.3±0.11 −1.0±0.1 −0.5±0.11 −0.2±0.1 −0.5±0.08 −0.5±0.1 Osmolality (mOsmol kg−1 fw) 4 5 6 400±12 370±15 340±18 420±15 370±12 360±10 350±10 440±10 460±9 370±13 300±15 310±12 330±18 300±13 400±9 250±10 300±12 241±18 Zeiger (1991). Because of the fluctuations in water potential and osmotic pressure turgor from stages 4 to 6 was averaged. As a results, apoplastic invertase lines exhibited the highest turgor pressure values ( Table 3). These values were significantly higher than those of the wild type ( Table 3). All other lines were reduced in turgor. for an individual developmental stage of a tuber existed, this classification depended on the subjective estimation of the experimenter. However, similar fluctuations in morphometric parameters during tuber development were reported by Sanz et al. (1996). Analysis of scatter Discussion Most of the measurements exhibited a rather big scatter between individual stages. Therefore, major sources of errors are due to the classification of developing tubers into individual categories. Since no objective parameters The neccesity for assimilate partitioning during tuber development has been well established (Oparka et al., 1992). However, little attention has been paid to the interaction between sugar availability, cell division and 482 Tauberger et al. Fig. 5. Characteristic sample of cross-sections ( light microscopy) of stage 5 of tuber development. (A) Wild type, (B) apoplastic invertase, (C ) cytosolic invertase, (D) AGPase antisense, (E ) apoplastic invertase in AGPase antisense background, (F ) cytosolic invertase in AGPase antisense background. expansion as well as cell wall properties during tuber development ( Xu et al., 1998). A perfect tool to investigate the role of these parameters was provided through the regeneration of transgenic plants affected in sucrose partitioning (Sonnewald et al., 1997). Those plants showed changes in final tuber size due to overexpression of a yeast invertase in different cell compartments ( Fig. 3). These changes were probably a consequence of modified sugar distribution. The role of various cell wall and osmotic parameters on starch grain and tuber development was investigated. Sugar analysis Fig. 6. Diameters of individual cells were obtained from light micrographs as shown in Fig. 4. Cell diameter increased from stage 3 onwards in all lines indicating that tubers are mainly growing by cell expansion. U-IN-1: transgenic plants expressed an apoplastic invertase from Sacchoromyces cerevisiae. U-IN-2: transgenic lines expressed a cytosolic invertase from Sacchoromyces cerevisiae. B-AGP-93: transgenic potato expressed ADP glucose-pyrophosphorylase antisense mRNA. H-AI: AGPase antisense plant with a second transgene coding for an invertase from Sacchoromyces cerevisiae that was targeted to the apoplast. H-IN: AGPase antisense plant with a second transgene coding for an invertase from Sacchoromyces cerevisiae that was targeted to the cytosol. Due to the correlation that emerges from the measurements depicted in Figs 1 and 4, initiation of tubers might involve high levels of glucose. In all lines investigated the glucose levels, even those of the wild type were significantly higher than fructose and sucrose (Ross et al., 1994; Visser et al., 1994). Because of these high glucose levels in the wild type it has to be assumed that an endogenous invertase or sucrose synthase is highly active in this phase of tuber initiation. Although the transgenic lines expressed an additional invertase, no increase in glucose levels were detected. These results suggest that either the B33 promotor is not active in the early stages of tuber development or that sufficient, endogeneous invertase activity is already present in this early stage of tuber initiation ( Visser et al., 1994). High levels of glucose have been reported to induce the expression of several genes (Sheen, 1994; Koch, 1996). It might be speculated that a defined level of glucose is required for the initiation of tuber formation. Although other compounds have been proposed to induce tuber initiation, as for example, jasmonic acid or tuberonic acid ( Koda and Okazawa, 1988; Vreugdenhil and Struik, 1989; Yoshihara et al., 1989), high glucose levels might addidionally be required to induce tuberization. Common to all lines that were investigated is a reduction of glucose in the second stage of tuber development ( Fig. 4). This reduction in glucose is paralleled by an increase in sucrose (Ross et al., 1994). Therefore, it seems most likely that the B33 promotor is not active in these early stages of tuber development, as sucrose should be cleaved more efficiently in the lines overexpressing invertase ( Visser et al., 1994). In later stages of tuber Effects of invertase overexpression on tuber development 483 Fig. 7. Diameter of starch grains. Starch grains of wild-type plants and invertase lines in the wild-type background increased during the final stages of tuber development. Starch grains of AGPase antisense lines did not expand in these late stages of tuber maturation. development (stages 4–6) a pronounced effect of the transgenic invertase became apparent. Invertase activity as reflected by the glucose-to-sucrose ratio started to be obvious in tuber developmental stage 3. At this point the initiation of starch grains had already started in all plants ( Visser et al., 1994). When comparing Figs 4 and 7 it might be suggested that starch grain growth could be modified by the kinds of sugars available. Higher availability of glucose led to a reduced starch grain size in some transgenic lines while their number remained comparable to that of wild-type plants ( Fig. 7, 8). Tuber growth Tubers grew by cell division and cell expansion ( Xu et al., 1998). This was confirmed by the fact that the cell size did not increase even though tubers grew bigger (Fig. 6). After stage 3 all sets of transgenic plants showed cell enlargement at almost the same rate as wild-type plants even though the sugar distribution and osmolarity exhibited big variations. Tubers were growing in different Fig. 8. Number of starch grains per cell. Almost identical amounts of starch grains were observed in the cells of mature tubers. numbers and to different sizes. The tuber size decreased in plants with invertase overexpression when the glucoseto-sucrose ratio increased. This leads to the conclusion that invertase activity and subsequent glucose accumulation affect cell expansion rather than cell division. Cell wall Cell wall thickness was affected to some degree by the genetic manipulations ( Fig. 9). However, the increased amount of glucose was possibly stored mainly in the vacuole of transgenic lines, since synthesis of cell wall constituents represents a minor pathway for carbon allocation. Previous reports indicated that cell wall constituents amount to approximately 4% of the dry weight (Hoff and Castro, 1969). Similar to our results a significant increase in cell wall diameter was described for the leaves of transgenic plants that overexpressed a vacuolar invertase (HoffmannBenning et al., 1997). In addition, these vacuolar invertase lines exhibited a strong elevation in turgor pressure. 484 Tauberger et al. induce a pronounced effect on the water potential and the osmolality. What is not detected is a clear correlation between water potential and osmolality. It might be expected that a reduction in osmolality corresponded with an increase in water potential. Moreover, a positive correlation between water potential and osmotic potential was observed in the U-IN-2 line between stages 5 and 6. Unloading and turgor Fig. 9. Cell wall thickness increased in all plant lines. Increases were more pronounced in the late stages of tuber development. Water relations Water potentials became more negative in the developmental stage 6 as compared to stage 4 in almost all lines. Only the transgenic lines that overexpress a cytosolic invertase ( U-IN-2) deviate from this pattern. During tuber development from stages 4–6 the osmolality declined in the wild type, the apoplastic invertase in the AGPase antisense background and B-AGPase antisense lines ( Table 2). In the final stage 6 of tuber development the water potential of the wild type is more negative than in all other transgenic lines ( Table 2). However, the osmolality of wild-type tubers in this developmental stage is intermediate to the osmolalities of the transgenic lines. Therefore, our experiments on these transgenic plant lines indicate that modifications in sugar allocation patterns The observed levels of glucose are in line with a symplastic unloading of sucrose from the phloem (Oparka et al., 1992). Immediate conversion of sucrose in glucose increases the sink strength of the growing stolon. Apoplastic unloading seems unfavourable under these conditions of high glucose, since the hexose carrier would then have to transport hexoses against a very high concentration gradient (Roitsch and Tanner, 1996). An increase in sink strength by invertase overexpression should enable an efficient unloading of sucrose into these sinks. Therefore, it should be expected that cytoplasmic invertase overexpressing tubers should have the highest sink strength. However, the uncontrolled increase in glucose due to an additional invertase could induce a closure of plasmodesmata and thus inhibit further unloading. This potential closure of plasmodesmata could explain the size of tubers that expressed invertase in an AGPase antisense background. High turgor pressure would result in a closure of plasmodesmata and thus inhibit further unloading. Experiments performed by Oparka et al. (1991) provided direct evidence that plasmodesmata function as pressure-sensitive valves. These workers demonstrated that raising the turgor pressure between two cells resulted in a closure of plasmodesmata. Apoplastic invertase plants exhibited the highest turgor, whereas cytosolic invertases had the lowest turgor with the wild type in between ( Table 3). This finding might be explained by the assumption of symplastic unloading and leakage of sucrose into the apoplast as follows (Fig. 10). Cleavage of sucrose in the apoplast increases the osmotic potential of the apoplast. Therefore, plasmodesmata will not close, even at high glucose concentration in the cytoplasm, since they are turgor regulated. When finally the level for closure is reached these cells contain much more osmotic sugars than the wild type. The turgor is higher since hexoses are subsequently taken up from the apoplast due to hexose transport proteins. Because of Table 3. Average of turgor pressure (MPa) in tuber developmental stages 4–6 Turgor pressure (MPa) Wild type U-IN-1 U-IN-2 H-AI H-IN B-AGP 0.43±0.05 0.55±0.04 0.30±0.03 0.30±0.03 0.30±0.04 0.33±0.03 Effects of invertase overexpression on tuber development 485 thickness of the cell walls. Turgor pressure was highest in the apoplastic invertase plants which developed the biggest tubers. Acknowledgements We thank Professor Dr U Sonnewald (Gatersleben, Germany) and Dr Müller-Röber (Golm, Germany) for the generous gift of some of the transgenic potato plants. References Fig. 10. Schematic diagram of turgor pressure generation due to invertase overexpression. Sucrose enters tuber parenchyma cells via plasmodesmata. In the upper panel a leakage of sucrose into the apoplast has been indicated. Apoplastic invertase will cleave sucrose and thus increase the osmotic potential of the apoplast. In the lower panel, leakage of sucrose into the apoplast is reduced as a consequence of overexpression of cytosolic invertase. this active uptake of hexoses into the cytoplasm the turgor can gain high values since the amounts of apoplastic glucose are much smaller than in the cytoplasm Cytosolic invertase plants have an additional mechanism to cleave sucrose ( Fig. 10). Therefore, the leakage of sucrose into the apoplast is further reduced. The threshold for plasmodesmatal closure will thus be obtained at relatively low glucose concentrations in the cytoplasm. The wild type would be intermediate, since small amounts of sucrose can leak into the apoplast thus slightly increasing the osmotic potential of the apoplast. An AGPase antisense background provides similar conditions as are found in the cytosolic invertase plants. Elevated levels of sugars are always present in the cytoplasm of these transgenic plants resulting in an early closure of plasmodesmata. 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