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1.
Michael M. Burrell Peter J. Mooney Margaret Blundy Dawn Carter Fiona Wilson John Green Keith S. Blundy Tom ap Rees 《Planta》1994,194(1):95-101
The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of 14CO2 production from [1-14C]-and [6-14C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO2 production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO2 production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP
fructose-2,6-bisphosphate
- FW
freshweight
- GUS
-glucuronidase
- PFK(ATP)
6-phosphofructokinase
- PFK(PPi)
pyrophosphate: fructose-6-phosphate 1-phosphotransferase 相似文献
2.
Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers 总被引:21,自引:0,他引:21
Cold storage of potato (Solanum tuberosum L.) tubers is known to cause accumulation of reducing sugars. Hexose accumulation has been shown to be cultivar-dependent and proposed to be the result of sucrose hydrolysis via invertase. To study whether hexose accumulation is indeed related to the amount of invertase activities, two different approaches were used: (i) neutral and acidic invertase activities as well as soluble sugars were measured in cold-stored tubers of 24 potato cultivars differing in the cold-induced accumulation of reducing sugars and (ii) antisense potato plants with reduced soluble acid invertase activities were created and the soluble sugar accumulation in cold-stored tubers was studied. The cold-induced hexose accumulation in tubers from the different potato cultivars varied strongly (up to eightfold). Large differences were also detected with respect to soluble acid (50-fold) and neutral (5-fold) invertase activities among the different cultivars. Although there was almost no correlation between the total amount of invertase activity and the accumulation of reducing sugars there was a striking correlation between the hexose/sucrose ratio and the extractable soluble invertase activitiy. To exclude the possibility that other cultivar-specific features could account for the obtained results, the antisense approach was used to decrease the amount of soluble acid invertase activity in a uniform genetic background. To this end the cDNA of a cold-inducible soluble acid invertase (EMBL nucleicacid database accession no. X70368) was cloned from the cultivar Desirée, and transgenic potato plants were created expressing this cDNA in the antisense orientation under control of the constitutive 35S cauliflower mosaic virus promotor. Analysis of the harvested and cold-stored tubers showed that inhibition of the soluble acid invertase activity leads to a decreased hexose and an increased sucrose content compared with controls. As was already found for the different potato cultivars the hexose/sucrose ratio decreased with decreasing invertase activities but the total amount of soluble sugars did not significantly change. From these data we conclude that invertases do not control the total amount of soluble sugars in coldstored potato tubers but are involved in the regulation of the ratio of hexose to sucrose.The authors are grateful to Heike Deppner and Christiane Prüßner for tuber harvest and technical assistance during the further analysis. We thank Andrea Knospe for taking care of tissue culture, Birgit Schäfer for patient photographic work, Hellmuth Fromme and the greenhouse personnel for attending plant growth and development and Astrid Basner for elucidating the sequence of clone INV-19. The work was supported by the Bundesministerium für Forschung und Technologie (BMFT). 相似文献
3.
Dennis A. Wardale 《Phytochemistry》1980,19(2):173-177
Three varieties of potato were used to investigate the activity of lipolytic acyl hydrolase, (LAH) and lipoxygenase, (LOX), for a short period after harvest. Both enzymes displayed very low levels of activity during the first few days, followed by an increase in later storage, with the hydrolase activity of Désirée tubers remaining low. An inverse relationship was found between the total LOX activity and the percentage of activity obtained in a particulate form. Only when the total LOX content was below 0.7 units (μmol/g/min fr. wt), was it possible to obtain a highly active particulate fraction. LAH particulate activity was dependent upon both enzymes remaining low. Protoplasts were isolated by the use of cell-degrading enzymes. When the total LOX activity in the tubers was low, 50% of this activity could be obtained in intact protoplasts. Once the LOX concentration in the tubers had risen, fewer intact protoplasts were isolated. No particulate activity of either enzyme was found when these protoplasts were lysed. The two lipid-degrading enzymes were not located in the amyloplasts. 相似文献
4.
A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species 总被引:2,自引:2,他引:0
Secondary metabolites, latex/mucilagenous secretions, polysaccharides, and proteins interfere with the extraction of high-quality,
restrictable total cellular DNA from sweet potato [Ipomoea batatas (L.) Lamk.] and related species. A method for the DNA extraction is described which overcomes these problems. 相似文献
5.
Ascrobate free-radical reductase (EC 1.6.5.4) from potato tubers was purified to apparent homogencity by a method which included ammonium-sulfate precipitation, gel filtration and chromatography on diethylaminoethyl cellulose and hydroxylapatite. Gel filtration and gel electrophoresis showed that the purified enzyme was monomeric with a molecular weight of about 42 000. Enzyme activity was heat lable and severely inhibited by thiol reagents. The Km values for enzyme substrates were estimated.Abbreviations AFR
ascorbate free radical
- AsA
ascorbic acid
- DE-32(52)
diethylaminoethyl cellulose
- Tricine
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine 相似文献
6.
Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity 总被引:8,自引:0,他引:8
A combination of chromatography on DE-52 cellulose, Cibacron Blue agarose, Mono Q anion exchanger and gel filtration was used to resolve different hexose-phosphorylating enzymes from growing sink potato tubers (Solanum tuberosum L.). Three enzymes (fructokinases: FK1, FK2 and FK3) are active with fructose and inactive with glucose, and three (hexokinases: HK1, HK2 and HK3) are active with glucose but not with fructose. Elution from DE-52 columns showed that the relative abundance of the six activities changes, depending on the organ and on the developmental stage. FK1 and FK2 were present at high activities in tubers but at very low activity in leaves; conversely FK3 was present at very low activity in tubers but at high activity in leaves. During storage of potato tuber, and also during sprouting, there was a decrease of FK1 and FK2. In contrast, glucose-phosphorylating activity was very low in growing tubers. During storage and sprouting the activity of the glucose-phosphorylating enzymes rose, until they exceeded FK1 and FK2. This was due particularly to an increase of HK1, whereas HK2 declined relative to HK1, and HK3 was always negligible. These changes in the pattern of hexose-phosphorylating enzyme forms are compared with the changing metabolic fluxes and pools of hexose sugars in potato tubers. It is concluded that organ- and development-specific changes in the abundance of the various enzyme forms contribute to the regulation of hexose metabolism in the potato.Abbreviations DTT
dithiothreitol
- FK
fructokinase
- FPLC
fast protein liquid chromatography
- HK
hexokinase
- Susy
sucrose synthase
- UDPGlc
uridine-5-diphosphoglucose
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). We are grateful to Professor E. Beck (Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, FRG) for providing laboratory facilities, and to Professor L. Willmitzer and Dr. U. Sonnewald (Institut für Genbiologische Forschung, Berlin, FRG) and Professor H.W. Heldt and Dr. D. Heineke (Institut für Biochemie der Pflanze, Universität Göttingen, FRG) for discussion. 相似文献
7.
Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates 总被引:11,自引:0,他引:11
Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and 13C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV
cauliflower mosaic virus
- NMR
nuclear magnetic resonance
We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing 13C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work. 相似文献
8.
A method of DNA extraction is given which is suitable for use with birch (Betula spp.) material collected from natural populations. Such material is frequently old and insect-damaged, and contains high
levels of polyphenols. The method relies on repeated extraction of the material with a high molarity urea phosphate buffer,
and yields DNA suitable for RFLP analysis. 相似文献
9.
Labelling experiments in which high-specific-activity [U-14C]sucrose or [U-14C]hexoses were injected into potato (Solanum tuberosum L. cv. Desiree) tubers showed that within 1 d of detaching growing tubers from their mother plant, there is an inhibition
of starch synthesis, a stimulation of the synthesis of other major cell components, and rapid resynthesis of sucrose. This
is accompanied by a general increase in phosphorylated intermediates, an increase in UDP-glucose, and a dramatic decrease
of ADP-glucose. No significant decline in the extracted activity of enzymes for sucrose degradation or synthesis, or starch
synthesis is seen within 1 d, nor is there a significant decrease in sucrose, amino acids, or fresh weight. Over the next
7 d, soluble carbohydrates decline. This is accompanied by a decline in sucrose-synthase activity, hexose-phosphate levels,
and the synthesis of structural cell components. It is argued that a previously unknown mechanism acting at ADP-glucose pyrophosphorylase
allows sucrose-starch interconversions to be regulated independently of the use of sucrose for cell growth. 相似文献
10.
The control of bud dormancy in potato tubers 总被引:5,自引:0,他引:5
Potato (Solanum tuberosum L.) tuber buds normally remain dormant through the growing season until several weeks after harvest. In the cultivar Majestic, this innate dormancy persisted for 9 to 12 weeks in storage at 10° C, but only 3 to 4 weeks when the tubers were stored at 2° C. At certain stages, supplying cytokinins to tubers with innately dormant buds induced sprout growth within 2 d. The growth rate was comparable to that of buds whose innate dormancy had been lost naturally. Cytokinin-treatment did not accelerate the rates of cell division and cell expansion in buds whose innate dormancy had already broken naturally. Gibberellic acid did not induce sprout growth in buds with innate dormancy. We conclude that cytokinins may well be the primary factor in the switch from innate dormancy to the non-dormant state in potato tuber buds, but probably do not control the subsequent sprout growth.Abbreviations tio 6ade
6-(4-hydroxy-3-methylbut-trans-2-enyl amino)purine, zeatin
- tio6ado
6-(4-hydroxy-3-methylbut-trans-2-enyl amino)-9--D-ribofuranosyl purine, zeatin riboside 相似文献
11.
(i) Sucrose-phosphate synthase (SPS) was purified 40-fold from stored potato (Solanum tuberosum L.) tubers to a final specific activity of 33–70 nkat·(mg protein)–1 via batch elution from diethylaminoethyl (DEAE)-sephacel, polyethylene glycol (PEG) precipitation and Mono Q anion-exchange chromatography. (ii) Immunoblotting revealed a major and a minor band with molecular weights of 124.8 kDa and 133.5 kDa, respectively. Both bands were also present in extracts prepared in boiling SDS to exclude proteolysis. No smaller polypeptides were seen, except when the preparations were incubated before application on a polyacrylamide gel. (iii) The enzyme preparation was activated by glucose-6-phosphate and inhibited by inorganic phosphate. Both effectors had a large effect on the K
m (fructose-6-phosphate) and the K
m (uridine-5-diphosphoglucose) with phosphate acting antagonistically to glucose-6-phosphate. (iv) Preincubation of potato slices with low concentrations of okadaic acid or microcystin resulted in a three- to fourfold decrease in the activity of SPS when the tissue was subsequently extracted and assayed. The decrease was especially marked when the assay contained low concentrations of substrates and glucose-6-phosphate, and inorganic phosphate was included. Preincubation with mannose or in high osmoticum resulted in an increase of SPS activity. (v) Analogous changes were observed in germinating Ricinus communis L. seedlings. After preincubation of the cotyledons in glucose, high SPS activity could be measured, whereas okadaic acid, omission of glucose, or addition of phosphate or sucrose led to a large decrease of SPS activity in the selective assay. (vi) It is argued that SPS from non-photosynthetic tissues is regulated by metabolites and by protein phosphorylation in an analogous manner to the leaf enzyme.Abbreviations Fru6P
fructose-6-phosphate
- Glc6P
glucose-6-phosphate
- Pi
inorganic phosphate
- PGI
phosphoglucose isomerase
- PP2A
phosphoprotein phosphatase 2A
- PEG
polyethyleneglycol
- SPS
sucrose-phosphate synthase
- UDPGlc
uridine-5-diphosphoglucose
This work was supported by the Deutsche Forschungsgemeinschaft, the BMFT and Sandoz AG, Basel, Switzerland. We are grateful to Prof. E. Beck (Pflanzenphysiologie, Bayreuth, Germany) for providing us with laboratory facilities, and to Dr. U. Sonnewald (Institut für Genbiologische Forschung, Berlin, Germany) for many discussions and providing us with unpublished data. 相似文献
12.
Domenico Carputo Teodoro Cardi Tiberio Chiari Giovanni Ferraiolo Luigi Frusciante 《Plant Cell, Tissue and Organ Culture》1995,41(2):151-158
The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.Abbreviations BA
6-benzylaminopurine
- GA3
gibberellic acid
- NAA
naphthaleneacetic acid
- MS
Murashige & Skoog (1962)
- 2,4-d
dichlorophenoxyacetic acid 相似文献
13.
A filtrate, prepared from potato tuber by grinding in an isotonic medium, has been separated into a particulate and a ‘soluble’ fraction by ultracentrifugation. Following dialysis and lyophilization, both fractions catalysed the oxidation of l-DOPA, with approximately 30% of the l-DOPA: oxygen-oxidoreductase (EC 1.14.18.1; DOPA oxidase) activity being associated with the particulate fraction. When dithiothreitol (DTT, 10?2M was included in the grinding medium, much lower yields of DOPA oxidase were obtained and 80% appeared to be associated with the particulate fraction. DTT proved to be a powerful inhibitor of DOPA oxidase. With concentrations of DTT causing only partial inhibition, the kinetics of the inhibited rate of dopachrome formation from l-DOPA were complex. When oxygen consumption was measured inhibition was not transient. The degree of inhibition was inversely related to the DOPA oxidase activity, indicating interaction of a product of this activity with DTT. Direct determination of -SH groups in DTT using 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) showed that they were all oxidised during the initial phase of inhibition of dopachrome formation. It is concluded that the first phase of inhibition involves oxidation of DTT by an intermediate between l-DOPA and dopachrome. The second phase of inhibition also appeared to require -SH groups initially, since trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT) caused very little inhibition at all. 相似文献
14.
The aim of this work was to study the cold-lability of phosphofructokinase from tubers of Solanum tuberosum cv Record, a variety that exhibits low temperature sweetening. The enzyme was purified by affinity chromatography and samples were examined by differential scanning calorimetry. Power-time curves were recorded for cooling and warming between 293 and 265 K. This revealed an exothermic dissociation, centred on 286 K, as the temperature was lowered. The latter temperature is close to that at which the tubers start to sweeten. It is suggested that hydrophobic interactions that contribute to the stability of the active configuration of the oligomeric enzyme are weakened at low temperatures, and that this causes spontaneous dissociation and consequent loss of activity of the enzyme. The results are discussed in relation to low temperature sweetening of potatoes. 相似文献
15.
DNA extraction from a previously recalcitrant plant genus 总被引:2,自引:2,他引:0
Numerous DNA extraction methods failed to remove contaminants that interfere with restriction digests ofCuphea DNA. The method described here removes those contaminants and maintains relatively high DNA yields. The primary purification
process consists of washing the DNA with phenol while it is complexed with CTAB and dissolved in 1 M NaCl. 相似文献
16.
The demonstration of activity of p-coumaryl CoA ligase in extracts of aged potato disks proved difficult owing to the presence of extremely high levels of apyrase which caused rapid hydrolysis of ATP, a co-factor for ligase activity. This problem was largely overcome by including an inhibitor of apyrase, sodium fluoride in the ligase assay and by initiation of the reaction with ATP. A method for the separation of apyrase and p-coumaryl CoA ligase by chromatography on DEAE-cellulose is described. p-Coumaryl CoA ligase was not detectable in freshly prepared disks of potato tubers. However on ageing in the light a large increase in the activity of this enzyme occurs, The enzyme of aged potato disks shows high activity with p-coumaric, ferulic, caffeic and with m- and p-methoxycinnamic acids. However the affinity of the enzyme for the methoxy derivatives is much lower than for cinnamic acids bearing free hydroxyl groups. 相似文献
17.
Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers 总被引:10,自引:0,他引:10
The substrate dependence and product inhibition of three different fructokinases and three different hexokinases from growing potato (Solanum tuberosum L.) tubers was investigated. The tubers contained three specific fructokinases (FK1, FK2, FK3) which had a high affinity for fructose K
m=64, 90 and 100 (M) and effectively no activity with glucose or other hexose sugars. The affinity for ATP (K
m=26, 25 and 240 M) was at least tenfold higher than for other nucleoside triphosphates. All three fructokinases showed product inhibition by high fructose (K
i=5.7, 6.0 and 21 mM) and were also inhibited by ADP competitively to ATP. Sensitivity to ADP was increased in the presence of high fructose, or fructose-6-phosphate. In certain conditions, the K
i (ADP) was about threefold below the K
m (ATP). All three fructokinase were also inhibited by fructose-6-phosphate acting non-competitively to fructose (K
i=1.3 mM for FK2). FK1 and FK2 showed very similar kinetic properties whereas FK3, which is only present at low activities in the tuber but high activities in the leaf, had a generally lower affinity for ATP, and lower sensitivity to inhibition by ADP and fructose. The tuber also contained three hexokinases (HK1, HK2, HK3) which had a high affinity for glucose (K
m=41, 130 and 35 M) and mannose but a poor affinity for fructose (K
m=11, 22 and 9 mM). All three hexokinases had a tenfold higher affinity for ATP (K
m=90, 280 and 560 M) than for other nucleoside triphosphates. HK1 and HK2 were both inhibited by ADP (K
i=40 and 108 M) acting competitively to ATP. HK1, but not HK2, was inhibited by glucose-6-phosphate, which acted non-competitively to glucose (K
i=4.1 mM). HK1 and HK2 differed, in that HK1 had a narrower pH optimum, a higher affinity for its substrate, and showed inhibition by glucose-6-phosphate. The relevance of these properties for the regulation of hexose metabolism in vivo is discussed.Abbreviations FK
fructokinase
- Fru6P
fructose-6-phosphate
- Glc6P
glucose-6-phosphate
- HK
hexokinase
- NTP
nucleoside triphosphate
- Pi
inorganic phosphate
- UDPGlc
uridine-5-diphosphoglucose
This work was supported by the Deutsche Froschungsgemeinschaft (SFB 137). We are grateful to Professor E. Beck (Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, FRG) for providing laboratory facilities. 相似文献
18.
19.
The aim of this work was to compare the coldlability of phosphofructokinase (EC 2.7.1.11) from tubers of potato cultivars
(cvs.) known to differ in their propensity to accumulate sugars at low temperature. When stored at 4°C for six weeks, the
sugar content of tubers ofSolanum tuberosum L. cv. Record doubled whereas the amount of sugar in tubers of cv. Brodick and an advanced breeding clone (13676) decreased
slightly. Tubers from each line contained four forms of phophofructokinase. Over the range 12°–16°C the temperature coefficients
of the four forms of phosphofructokinase from cvs. Record and Brodick were similar. In cv. Record the temperature coefficients
of three of the enzyme forms were significantly higher at 2°–6°C than at 12°–16°C, whereas those from cv. Brodick were unchanged.
These results are consistent with the proposal that inactivation of phosphofructokinase at low temperature results in the
accumulation of hexose phosphates leading to increased sucrose synthesis. 相似文献
20.