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1.
The aim of this work was to investigate the extent to which starch synthesis in potato (Solanum tuberosum L.) tubers is controlled by the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27; AGPase). In order to do this, fluxes
of carbohydrate metabolism were measured in tubers that had reduced AGPase activity as a result of the expression of a cDNA
encoding the B subunit in the antisense orientation. Reduction in AGPase activity led to a reduction in starch accumulation,
and an increase in sucrose accumulation. The control coefficient of AGPase on starch accumulation in intact plants was estimated
to be around 0.3. The fluxes of carbohydrate metabolism were measured in tuber discs from wild-type and transgenic plants
by investigating the metabolism of [U-14C]glucose. In tuber discs, the control coefficient of AGPase over starch synthesis was estimated as 0.55, while the control
coefficient of the enzyme over sucrose synthesis was −0.47. The values obtained suggest that AGPase activity exerts appreciable
control over tuber metabolism in potato.
Received: 24 February 1999 / Accepted: 8 April 1999 相似文献
2.
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. 总被引:36,自引:0,他引:36 下载免费PDF全文
Transgenic potato plants were created in which the expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited by introducing a chimeric gene containing the coding region of one of the subunits of the AGPase linked in an antisense orientation to the CaMV 35S promoter. Partial inhibition of the AGPase enzyme was achieved in leaves and almost complete inhibition in tubers. This resulted in the abolition of starch formation in tubers, thus proving that AGPase has a unique role in starch biosynthesis in plants. Instead up to 30% of the dry weight of the transgenic potato tubers was represented by sucrose and up to 8% by glucose. The process of tuber formation also changed, resulting in significantly more tubers both per plant and per stolon. The accumulation of soluble sugars in tubers of antisense plants resulted in a significant increase of the total tuber fresh weight, but a decrease in dry weight of tubers. There was no significant change in the RNA levels of several other starch biosynthetic enzymes, but there was a great increase in the RNA level of the major sucrose synthesizing enzyme sucrose phosphate synthase. In addition, the inhibition of starch biosynthesis was accompanied by a massive reduction in the expression of the major storage protein species of potato tubers, supporting the idea that the expression of storage protein genes is in some way connected to carbohydrate formation in sink storage tissues. 相似文献
3.
The aim of this work was to investigate the effect of exogenous sugars on the extent to which starch synthesis in potato ( Solanum tuberosum L.) is controlled by adenosine 5'-diphosphoglucose pyrophosphorylase (EC 2.7.7.27; AGPase). Tuber discs were incubated in the presence of a range of concentrations of glucose and sucrose, and metabolic fluxes measured following the supply of [U-14C]glucose and measurement of the specific radioactivity of the hexose phosphate pool. In the presence of glucose there was a marked increase in the flux through glucose-phosphorylating hexokinase, and at high concentrations of external glucose this led to a stimulation of the rate of starch and sucrose synthesis relative to those measured in the presence of sucrose. In the presence of glucose the ratio of the rate of starch synthesis to the rate of glycolysis was higher than in the presence of sucrose. Similar effects of glucose were observed at two stages of tuber development. We conclude that the presence of glucose perturbs the carbohydrate metabolism of tuber discs so that starch synthesis is favoured. In order to determine the extent to which AGPase controls flux, we measured fluxes in wild-type plants and transgenic plants with reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. In the presence of sucrose a reduction in AGPase activity had a greater impact on the rate of starch synthesis than in the presence of glucose. The flux control coefficient of AGPase over starch synthesis was higher in the presence of sucrose (0.7-0.9) than in the presence of glucose (0.4-0.6). Conversely, the impact of reduced AGPase activity on the rate of sucrose synthesis was lower in the presence of sucrose than glucose. In the presence of 200 mM sucrose the flux control coefficient of AGPase over the rate of sucrose synthesis was not significantly different from zero. This demonstrates that the nature of the sugar supplied to potato tuber discs can have a major influence on the distribution of control within metabolism. These data were also used to investigate the relationship between demand for ATP and the rate of hexose phosphate entry into glycolysis. A very strong correlation between ATP demand and glycolytic flux was demonstrated. 相似文献
4.
Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply 总被引:19,自引:0,他引:19 下载免费PDF全文
Tiessen A Hendriks JH Stitt M Branscheid A Gibon Y Farré EM Geigenberger P 《The Plant cell》2002,14(9):2191-2213
5.
Water stress stimulates sucrose synthesis and inhibits starch and cell-wall synthesis in tissue slices of growing potato
(Solanum tuberosum L. cv. Desirée) tubers. Based on the analysis of fluxes and metabolites, Geigenberger et al. (1997, Planta 201: 502–518)
proposed that water deficits up to −0.72 MPa stimulate sucrose synthesis, leading to decreased starch synthesis as a result
of the resulting decline of phosphorylated metabolite levels, whereas more-severe water deficits directly inhibit the use
of ADP-glucose. Potato plants with decreased expression of adenosine 5′-diphosphoglucose pyrophosphorylase (AGPase) have been
used to test the prediction that the contribution of AGPase to the control of starch synthesis should decrease in severely
water-stressed tuber material. Freshly cut slices from wild-type and antisense tubers were incubated at a range of mannitol
concentrations (20, 300 and 500 mM) and the metabolism of [14C]glucose was analysed. A 86–97% reduction of AGPase activity led to a major but non-stoichiometric inhibition of starch accumulation
in intact growing tubers attached to the plant (40–85%), and an inhibition of starch synthesis in non-stressed tuber slices
incubated in 20 mM mannitol (60–80%). The inhibition of starch synthesis was accompanied by a 2- to 8-fold increase in the
levels of sugars in intact tubers and a 2- to 3-fold stimulation of sucrose synthesis in tuber slices, whereas respiration
and cell-wall synthesis were not significantly affected. The strong impact of AGPase on carbon partitioning in non-stressed
tubers and tuber slices was retained in slices subjected to moderate water deficit (300 mM mannitol, corresponding to −0.72 MPa).
In discs incubated in 500 mM mannitol (corresponding to −1.2 MPa) this response was modified. A 80–97% reduction of AGPase
resulted in only a 0–40% inhibition of starch synthesis. Further, the water stress-induced stimulation of sucrose synthesis
was abolished in the transformants. The results provide direct evidence that the contribution of AGPase to the control of
starch synthesis can be modified by environmental factors, leading to a lower degree of control during severe water deficits.
There was also a dramatic decrease in the labelling of cell-wall components in wild-type tuber slices incubated with 300 or
500 mM mannitol. The water stress-induced inhibition of cell-wall synthesis occurred independently of AGPase expression and
the accompanying changes in starch and sucrose metabolism, indicating a direct inhibition of cell-wall synthesis in response
to water stress.
Received: 24 February 1999 / Accepted: 28 May 1999 相似文献
6.
Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.) 总被引:18,自引:0,他引:18
Rita Zrenner Marcel Salanoubat Lothar Willmitzer Uwe Sonnewald 《The Plant journal : for cell and molecular biology》1995,7(1):97-107
Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants. 相似文献
7.
Tauberger E; Hoffmann-Benning S; Fleischer-Notter H; Willmitzer L; Fisahn J 《Journal of experimental botany》1999,50(333):477-486
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 yeast-derived
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. 相似文献
8.
Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1 总被引:5,自引:0,他引:5
McKibbin RS Muttucumaru N Paul MJ Powers SJ Burrell MM Coates S Purcell PC Tiessen A Geigenberger P Halford NG 《Plant biotechnology journal》2006,4(4):409-418
Transgenic potato ( Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene ( SnRK1 ) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%−167% compared with that in the wild-type. Glucose levels were decreased, at 17%−56% of the levels of the wild-type, and the starch content showed an increase of 23%−30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, α-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%–60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers. 相似文献
9.
Antisense Repression of Hexokinase 1 Leads to an Overaccumulation of Starch in Leaves of Transgenic Potato Plants But Not to Significant Changes in Tuber Carbohydrate Metabolism 下载免费PDF全文
Jon Veramendi Ute Roessner Andreas Renz Lothar Willmitzer Richard N. Trethewey 《Plant physiology》1999,121(1):123-134
Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis. 相似文献
10.
11.
Veramendi J Fernie AR Leisse A Willmitzer L Trethewey RN 《Plant molecular biology》2002,49(5):491-501
Potato plants (Solanum tuberosum L. cv. Désirée) transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 2 exhibited altered enzyme activities and expression of hexokinase 2 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed an 11-fold variation in leaf (from 48% of the wild-type activity in antisense transformants to 446% activity in sense transformants) and an 8-fold variation in developing tubers (from 35% of the wild-type activity in antisense transformants to 212% activity in sense transformants). Despite the wide range of hexokinase activities, no substantial change was found in the fresh weight yield, starch, sugar and metabolite levels of transgenic tubers. However, both potato hexokinases 1 and 2 were able to complement the hyposensitivity of antisense hexokinase 1 Arabidopsis transgenic plants to glucose. In an in vitro bioassay of seed germination in a medium with high glucose levels, double transformants showed the same sensitivity to glucose as that of the wild-type ecotype, displaying a stunted phenotype in hypocotyls, cotyledons and roots. 相似文献
12.
Expression of a bacterial sucrose phosphorylase in potato tubers results in a glucose-independent induction of glycolysis 总被引:7,自引:1,他引:6
R. N. Trethewey A. R. Fernie A. Bachmann H. Fleischer-Notter P. Geigenberger & L. Willmitzer 《Plant, cell & environment》2001,24(3):357-365
Sugars are not only metabolic substrates: they also act as signals that regulate the metabolism of plants. Previously, we found that glycolysis is induced in transgenic tubers expressing a yeast invertase in the cytosol but not in those expressing invertase in the apoplast. This suggests that either the low level of sucrose, the increased formation of cytosolic glucose or the increased levels of metabolites downstream of the sucrose cleavage is responsible for the induction of glycolysis in storage organs. In order to discriminate between these possibilities, we cloned and expressed a bacterial sucrose phosphorylase gene from Pseudomonas saccharophila in potato tubers. Due to the phosphorolytic cleavage of sucrose, formation of glucose was circumvented, thus allowing assessment of the importance of cytosolic glucose – and, by implication, flux through hexokinase – in glycolytic induction. Expression of sucrose phosphorylase led to: (i) a decrease in sucrose content, but no decrease in glucose or fructose; (ii) a decrease in both starch accumulation and tuber yield; (iii) increased levels of glycolytic metabolites; (iv) an induction of the activities of key enzymes of glycolysis; and (v) increased respiratory activity. We conclude that the induction of glycolysis in heterotrophic tissues such as potato tubers occurs via a glucose‐independent mechanism. 相似文献
13.
Adenine nucleotides are of general importance for many aspectsof cell function, but their role in the regulation of biosyntheticprocesses is still unclear. It was previously reported thatdecreased expression of plastidial adenylate kinase, catalysingthe interconversion of ATP and AMP to ADP, leads to increasedadenylate pools and starch content in transgenic potato tubers.However, the underlying mechanisms were not elucidated. Here,it is shown that decreased expression of plastidial adenylatekinase in growing tubers leads to increased rates of respiratoryoxygen consumption and increased carbon fluxes into starch.Increased rates of starch synthesis were accompanied by post-translationalredox-activation of ADP-glucose pyrophosphorylase (AGPase),catalysing the key regulatory step of starch synthesis in theplastid, while there were no substantial changes in metabolicintermediates or sugar levels. A similar increase in post-translationalredox-activation of AGPase was found after supplying adenineto wild-type potato tuber discs to increase adenine nucleotidelevels. Results provide first evidence for a link between redox-activationof AGPase and adenine nucleotide levels in plants. Key words: Adenylate kinase, ADPglucose pyrophosphorylase, plastid, redox-regulation, potato, respiration, starch
Received 18 September 2007; Revised 12 November 2007 Accepted 13 November 2007 相似文献
14.
15.
Constitutive antisense inhibition of the cytosolic isoform of phosphoglucomutase in the potato (Solanum tuberosum L.) results in restriction of photosynthesis, growth inhibition and modified tuber morphology, and a severe restriction of tuber starch synthesis. Here we describe the consequences of the tuber-specific expression of an Escherichia coli phosphoglucomutase in the cytosol. Analysis of [14C]glucose metabolism by tuber discs isolated from wild type and transformants revealed that the rates of sucrose and starch synthesis were unaltered but that the rate of glycolysis was depressed in the transgenics. The transformant tubers also contained dramatically reduced amino acid content and significantly higher levels of ADP, but were characterized by elevated levels of Krebs cycle intermediates and an unaltered rate of respiration. In addition to these metabolic consequences of the overexpression of the E. coli enzyme, we observed morphological changes in tubers, with the transformants having a smaller number of larger tubers which exhibited delayed rates of sprouting with respect to the wild type. These results are discussed with respect to current models of the regulation of central plant metabolism and tuber dormancy. 相似文献
16.
Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers 总被引:17,自引:1,他引:16
Peter Geigenberger Ralph Reimholz Uta Deiting Uwe Sonnewald Mark Stitt 《The Plant journal : for cell and molecular biology》1999,19(2):119-129
Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation. 相似文献
17.
Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase. 相似文献
18.
The contribution of plastidial phosphoglucomutase to the control of starch synthesis within the potato tuber 总被引:1,自引:0,他引:1
The aim of this work was to evaluate the extent to which plastidial phosphoglucomutase (PGM) activity controls starch synthesis within potato (Solanum tuberosum L. cv. Desirée) tubers. The reduction in the activity of plastidial PGM led to both a correlative reduction in starch accumulation and an increased sucrose accumulation. The control coefficient of plastidial PGM on the accumulation of starch was estimated to approximate 0.24. The fluxes of carbohydrate metabolism were measured by investigating the metabolism of [U-14C]glucose in tuber discs from wild-type and transgenic plants. In tuber discs the control coefficient of plastidial PGM over starch synthesis was estimated as 0.36, indicating that this enzyme exerts considerable control over starch synthesis within the potato tuber. 相似文献
19.
Inorganic pyrophosphate (PPi) is an enzyme involved in sugar metabolism in potato tubers. In our previous study, we isolated an inorganic pyrophosphatase (PPase) gene from potato and obtained the transgenic potato plants transformed with the sense and antisense PPase genes respectively. In the present experiment, the physiological indexes, tuber dormancy, and sprouting characteristics of the transgenic potatoes were analyzed and evaluated. The result showed that the PPase activity and the inorganic phosphate content of tubers were lower in the antisense transgenic plant lines but were higher in the sense transgenic plant lines, compared with wild-type tubers. Soluble sugars, such as glucose, fructose and sucrose increased in transgenic plants that had overexpression of the sense PPase gene, but decreased in the antisense transgenic plant lines, compared with wild-type tubers. Tuber sprouting time of the antisense transgenic plants were delayed for 2 and 3 weeks and reached the 100 % sprouting rate only after 14 and 16 weeks storage compared with the wild-type when tubers are stored under 25 and 4 °C, respectively. In contrast, tuber sprouting time of the sense transgenic plants was earlier by approximately 2 weeks than that of wild-type tubers under these storage temperatures. 相似文献
20.
Viola R Pelloux J van der Ploeg A Gillespie T Marquis N Roberts AG Hancock RD 《Plant, cell & environment》2007,30(8):973-983
To gain greater insight into the mechanism of dormancy release in the potato tuber, an investigation into physiological and biochemical changes in tuber and bud tissues during the transition from bud dormancy (immediately after harvest) to active bud growth was undertaken. Within the tuber, a rapid shift from storage metabolism (starch synthesis) to reserve mobilization within days of detachment from the mother plant suggested transition from sink to source. Over the same period, a shift in the pattern of [U-(14)C]sucrose uptake by tuber discs from diffuse to punctate accumulation was consistent with a transition from phloem unloading to phloem loading within the tuber parenchyma. There were no gross differences in metabolic capacity between resting and actively growing tuber buds as determined by [U-(14)C]glucose labelling. However, marked differences in metabolite pools were observed with large increases in starch and sucrose, and the accumulation of several organic acids in growing buds. Carboxyfluorescein labelling of tubers clearly demonstrated strong symplastic connection in actively growing buds and symplastic isolation in resting buds. It is proposed that potato tubers rapidly undergo metabolic transitions consistent with bud outgrowth; however, growth is initially prevented by substrate limitation mediated via symplastic isolation. 相似文献