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PubMed1999

A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A.

Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2000

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate.

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.

Antisense Elements (Genetics)↗
PubMed1998

Quantitative analysis of anti-emetic principle in the tubers of Pinellia ternata by enzyme immunoassay.

A crude globulin fraction was isolated from the tubers of Pinellia ternata (Thunb.) Breit. Tubers of P. ternata are one of the well known Chinese traditional medicines. It is used on account of its anti-emetic effect. 6KDP was separated by means of gel filtration and chromatography on CM-Toyopearl 650 M from the crude globulin fraction. 6KDP is a glycoprotein of M.W. 6 kDa. 6KDP has an anti-emetic activity and hemagglutination activity. Both activities dropped during heat treatment. For the purpose to evaluate the quality of the powdered tubers of P. ternata, the quantitative analysis by means of EIA was established. The contents of 6KDP varied from 5.75 to 8.30%. The assay demonstrated a low cross-reaction with the crude globulin fraction of Colocasia antiquorum, a member of the same family as P. ternata. 6KDP is one of major proteins in the tubers of P. ternata. Thus 6KDP is a marker for quantitative analysis of the tubers of P. ternata.

Antiemetics↗
PubMed2000

Transgenic potato (Solanum tuberosum) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke (Cynara scolymus) roots.

The ability to synthesize high molecular weight inulin was transferred to potato plants via constitutive expression of the 1-SST (sucrose:sucrose 1-fructosyltransferase) and the 1-FFT (fructan: fructan 1-fructosyltransferase) genes of globe artichoke (Cynara scolymus). The fructan pattern of tubers from transgenic potato plants represents the full spectrum of inulin molecules present in artichoke roots as shown by high-performance anion exchange chromatography, as well as size exclusion chromatography. These results demonstrate in planta that the enzymes sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1-fructosyltransferase are sufficient to synthesize inulin molecules of all chain lengths naturally occurring in a given plant species. Inulin made up 5% of the dry weight of transgenic tubers, and a low level of fructan production also was observed in fully expanded leaves. Although inulin accumulation did not influence the sucrose concentration in leaves or tubers, a reduction in starch content occurred in transgenic tubers, indicating that inulin synthesis did not increase the storage capacity of the tubers.

Asteraceae↗
PubMed2002

Control of photoperiod-regulated tuberization in potato by the Arabidopsis flowering-time gene CONSTANS.

Photoperiod controls several responses throughout the plant life cycle, like germination, flowering, tuber formation, onset of bud dormancy, leaf abscission, and cambium activity. From these processes, flowering has been most extensively studied, especially in Arabidopsis thaliana. Photoperiod sensing by the function of photoreceptors and the circadian clock appears to regulate flowering time via Arabidopsis CONSTANS (AtCO), a putative transcription factor that accelerates flowering in response to long days. The genetic factors controlling plant photoperiodic responses other than flowering are little known. However, interspecific grafting experiments demonstrated that the flower-inducing (florigen) and tuber- inducing (tuberigen) signals are functionally exchangeable. Here we show that constitutive overexpression in potato of the Arabidopsis flowering-time gene AtCO impairs tuberization under short-day inductive conditions; AtCO overexpressing lines require prolonged exposure to short days to form tubers. Grafting experiments using these lines indicated that AtCO exerts its inhibitory effect on tuber formation by acting in the leaves. We propose that a conserved photoperiodic functional module may be involved in controlling distinct photoperiod-regulated evocation responses in different species. This module would involve the action of CONSTANS in the production of the elusive and long-distance acting florigen-tuberigen signal(s) in the leaves.

Arabidopsis↗
PubMed1996

Review on tropical root and tuber crops. I. Storage methods and quality changes.

The root tuber crops, including cassava, sweet potato, yams, and aroids, enjoy considerable importance as a vegetable, staple food, or raw material for small-scale industries at a global level, particularly in the less developed tropical countries. The perishability and postharvest loses of root and tuber crops are the major constraints in the utilization of these crops. Several simple, low-cost traditional methods are being followed by farmers in different parts of the world to store different root and tuber crops in the fresh state. An account of different storage practices and constraints is reviewed in this article. Some of these methods have been studied and evaluated by different research workers. Several modern techniques, including refrigerated cold storage, freezing, chemical treatments, wax coating, and irradiation, for storing fresh tropical tubers are also reviewed. The pre- and postharvest factors to be considered for postharvest storage of different root and tuber crops are incorporated into the review.

Africa↗
PubMed2005

Relationship among lipoperoxides, jasmonates and indole-3-acetic acid formation in potato tuber after wounding.

Plant responses to biotic and abiotic stress can be mediated by oxidised products and in this study we analysed the relation among some of them and the growth factor indole-3-acetic acid (IAA). The plant material used was potato tuber sliced below bud and incubated for different lengths of time before analysis. Wounding in potato tuber leads, in a very short time (0-30?min), to the generation of lipid hydroperoxides (LOOH) from polyunsaturated fatty acids (PUFA). These reactive species could cause a subsequent increase of 9 and 13-lipoxygenase (LOX, E.C.1.13.12.12.), analysed by RT-PCR and spectrophotometric assay, LOOH, Jasmonates and IAA all quantified by GC-MS analysis. The activation of 9 and 13-LOX, using different timing, leads to the formation of LOOH with a subsequent generation of jasmonates and IAA as highlighted by the addition on the potato tuber slices of salicylhydroxamic acid (SHAM), an inhibitor of LOX activity. A correlation between jasmonates and IAA resulted by testing their reciprocal influence during wounding in potato tuber. The relationship occurring among each hormone analysed during wounding underlines the fact that the jasmonates level can be regulated in situ and this can suggest a role for these compounds in potato tuber which has been underestimated up to now.

Cyclopentanes↗
PubMed2002

Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum L. cv. SR1) axillary buds establishes a role for cytokinins in tuberization and sink formation.

The developmental characteristics of a transgenic tobacco line (BIK62) expressing the ipt cytokinin-biosynthetic gene under the control of a tagged promoter were analysed. In situ hybridization and cytokinin immunocytochemistry revealed that the ipt gene was mainly expressed in the axillary buds after the floral transition. The ipt-expressing axillary buds presented morphological alterations such as short and narrow scale-leaflets, and swollen internodes filled with starch grains, giving rise to short and tuberized lateral branches. In addition, the modification of the endogenous cytokinin balance in the axillary meristems resulted in a fast rate of leaf initiation and cytokinins accumulated mostly in the lateral zones of the reactivated axillary meristems, suggesting a role in leaf organogenesis. Cell cycle analysis revealed that the reactivated axillary meristems were characterized by predominant S+G2 nuclei. Terminal internodes displayed low levels of hexose and sucrose concomitant with starch accumulation. Extracellular invertases (EC 3.1.26) were also present in higher amounts in the tuberizing internodes compared to the axillary buds of wild-type tobacco. These results underline the role of cytokinins in cell cycle regulation and in the creation of a sink--source effect. They also provide new information about cytokinin involvement in the process of tuberization and their overproduction in axillary buds giving rise to tuberized lateral branches in a naturally non-tuberizing species.

Alkyl and Aryl Transferases↗
PubMed1998

Vacuolar membrane lesions induced by a freeze-thaw cycle in protoplasts isolated from deacclimated tubers of Jerusalem artichoke (Helianthus tuberosus L.).

The processes of freezing injury in Jerusalem artichoke (Helianthus tuberosus L.) tubers were studied using protoplasts isolated from cold-acclimated and deacclimated tubers. Prior to freezing, protoplasts were preloaded with 10 microM fluorescein diacetate (FDA) in an isotonic sorbitol solution. After freeze-thawing at various temperatures, cell viability was evaluated under a fluorescence microscope. In cold-acclimated tubers, more than 80% of protoplasts survived freezing to -20 degrees C. By contrast, in deacclimated tubers, the cell survival abruptly declined after freezing to temperatures below -5 degrees C. Thus, freezing tolerance differed significantly between protoplasts isolated from cold-acclimated and deacclimated tubers. Two distinct types of cell injury, which were caused by either damage to plasma membrane (cell-lysis type) or by damage to the vacuolar membrane (abnormal-staining type), were observed, depending on the cold hardiness and freezing temperature. In the cells of the abnormal-staining type, shrinkage of the central vacuolar space and simultaneous acidification of the cytoplasmic space were characteristically observed immediately before complete cell-rehydration during thawing. The decrease in freezing tolerance of protoplasts after deacclimation was suggested to be due mainly to destabilization of the vacuolar membrane by freeze-induced dehydration stress.

Freezing↗
PubMed1996

Changes in Calcium-Dependent Protein Kinase Activity during in Vitro Tuberization in Potato.

A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 [mu]M) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 [mu]M). The kinase was inhibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [[gamma]-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.

Journal Article↗
PubMed1998

High-temperature perturbation of starch synthesis is attributable to inhibition of ADP-glucose pyrophosphorylase by decreased levels of glycerate-3-phosphate in growing potato tubers

To investigate the short-term effect of elevated temperatures on carbon metabolism in growing potato (Solanum tuberosum L.) tubers, developing tubers were exposed to a range of temperatures between 19 degreesC and 37 degreesC. Incorporation of [14C]glucose (Glc) into starch showed a temperature optimum at 25 degreesC. Increasing the temperature from 23 degreesC or 25 degreesC up to 37 degreesC led to decreased labeling of starch, increased labeling of sucrose (Suc) and intermediates of the respiratory pathway, and increased respiration rates. At elevated temperatures, hexose-phosphate levels were increased, whereas the levels of glycerate-3-phosphate (3PGA) and phosphoenolpyruvate were decreased. There was an increase in pyruvate and malate, and a decrease in isocitrate. The amount of adenine diphosphoglucose (ADPGlc) decreased when tubers were exposed to elevated temperatures. There was a strong correlation between the in vivo levels of 3PGA and ADPGlc in tubers incubated at different temperatures, and the decrease in ADPGlc correlated very well with the decrease in the labeling of starch. In tubers incubated at temperatures above 30 degreesC, the overall activities of Suc synthase and ADPGlc pyrophosphorylase declined slightly, whereas soluble starch synthase and pyruvate kinase remained unchanged. Elevated temperatures led to an activation of Suc phosphate synthase involving a change in its kinetic properties. There was a strong correlation between Suc phosphate synthase activation and the in vivo level of Glc-6-phosphate. It is proposed that elevated temperatures lead to increased rates of respiration, and the resulting decline of 3PGA then inhibits ADPGlc pyrophosphorylase and starch synthesis.

Journal Article↗
PubMed1976

Potato tuber callus: validation as a biochemical tool.

Callus was initiated from explants of tubers of the Norchip cultivar of Solanum tuberosum L. and grown on medium with a single carbon source and without addition of coconut milk, protein hydrolysate, or amino acid. Callus samples were harvested at intervals and compared to mature tubers for which there was good biochemical knowledge.The amino acid spectrum, the glycoalkaloid content, and the properties of the isolated invertase and sucrose synthetase were similar in callus and in tuber. Significantly the level of sucrose synthetase varied with the age of the developing callus just as it did with the age of the developing tuber. Of greater significance, levels of reducing sugars and invertase varied with the age of developing callus and also with time and temperature of storage after the callus has ceased growth. Similar changes occur in intact tubers.Callus and tuber biochemistry differed in the amount of deposited starch and in the absence of potato invertase inhibitor.

Journal Article↗
PubMed1981

Correlation between Changes in Mitochondrial Membranes of Artichoke Tubers and Their Hardening and Dormancy.

Spin labeling studies using mitochondrial membranes of Jerusalem artichoke (Helianthus tuberosus L.) showed that the decrease during winter in the temperatures of the upper and lower lipid transitions correlated with the development of freezing hardiness of the tubers. The killing temperature for tuber tissue reached a minimum of -12 C, about 5 C degrees lower than the lower transition. Freeze-hardiness decreased when the lower transition increased at the time of sprouting.Low temperature storage was not required to induce freeze-hardiness or to lower the transitions. These changes occurred in tubers under field conditions and at a constant growth temperature of 25 +/- 1 C. In both conditions, tuber dormancy preceded the mitochondrial membrane changes.In young nondormant tubers, cessation of growth by storage at 0 or 4 C and the induction of dormancy with abscisic acid led to decreased temperatures of the upper and lower transitions.Changes in Arrhenius activation energy of succinate oxidase were correlated with the seasonal changes in the upper and lower transitions confirming that these were indeed a reflection of altered thermal responses of mitochondrial membranes, which might be part of a general mechanism in this plant of insuring membrane integrity during the freeze-thaw process. In contrast with chilling injury, there is no precise correlation between the temperature of the lower transition and the temperature below which freezing injury occurs.

Journal Article↗
PubMed1984

Increased mitochondrial DNA and RNA polymerase activity in ethylene-treated potato tubers.

A purified mitochondrial fraction was isolated from potato (Solanum tuberosum L.) tubers respiring normally at 23 degrees C or at an accelerated rate in response to treatment with ethylene (10 microliters per liter).A pronounced increase in various mitochondrial enzymic activities was observed in response to exposure of the whole tubers to ethylene. Cytochrome c oxidase activity increased more than 50%, DNA polymerase activity increased about 2-fold, and RNA polymerase activity increased 2.5-fold. Moreover, DNA or RNA polymerase activities of mitochondria isolated from tubers not treated with ethylene were not affected by ethylene treatment in vitro. Respiratory control ratios decreased from 2.84 to 1.50 with increasing periods of ethylene treatment from 0 to 15 hours. None of these changes were observed in untreated tubers. It is concluded that the stimulation of respiration by ethylene in potato tubers is accompanied in vivo by an enhancement of mitochondrial enzymic activity of both membrane-associated enzymes which participate in the mitochondrial oxidative electron transport as well as soluble enzymes which are not directly involved in respiration.

Journal Article↗
PubMed1988

Hypoxic stress inhibits the appearance of wound-response proteins in potato tubers.

Potato (Solanum tuberosum L.) tubers respond to environmental stresses by alterations of macromolecular synthesis. In an aerobic environment tubers respond rapidly to wounding by synthesizing a set of proteins, the most prominent of which display apparent molecular weights of 78, 48, 38, and 31 kilodaltons. These proteins become intensely labeled by [(35)S]methionine within 2 hours of wounding. The 78 kilodalton polypeptide has been identified by immunoprecipitation as phenylalanine ammonia-lyase. By contrast, tubers incubated in hypoxic conditions for a period as short as 1.5 hours exhibit significantly reduced incorporation of amino acids such that newly synthesized polypeptides are not detected. However, a second set of proteins is synthesized by wounded tubers after prolonged incubation in a hypoxic environment. One peptide of this set is precipitated by an antibody directed against aldolase; several of these proteins may be enzymes of glycolysis necessary for anaerobic metabolism. The results indicate that there is a complex regulatory mechanism which allows mature potato tubers to respond to changes in the environment.

Journal Article↗
PubMed1990

Superoxide Dismutase, Catalase, and alpha-Tocopherol Content of Stored Potato Tubers.

Activated oxygen or oxygen free radical mediated damage to plants has been established or implicated in many plant stress situations. The extent of activated oxygen damage to potato (Solanum tuberosum L.) tubers during low temperature storage and long-term storage is not known. Quantitation of oxygen free radical mediated damage in plant tissues is difficult. However, it is comparatively easy to quantitate endogenous antioxidants, which detoxify potentially damaging forms of activated oxygen. Three tuber antioxidants, superoxide dismutase, catalase, and alpha-tocopherol were assayed from four potato cultivars stored at 3 degrees C and 9 degrees C for 40 weeks. Tubers stored at 3 degrees C demonstrated increased superoxide dismutase activities (up to 72%) compared to tubers stored at 9 degrees C. Time dependent increases in the levels of superoxide dismutase, catalase, and alpha-tocopherol occurred during the course of the 40 week storage. The possible relationship between these increases in antioxidants and the rate of activated oxygen production in the tubers is discussed.

Journal Article↗
PubMed1992

Sucrose Metabolism in Tubers of Potato (Solanum tuberosum L.): Effects of Sink Removal and Sucrose Flux on Sucrose-Degrading Enzymes.

Excision of developing potato (Solanum tuberosum L.) tubers from the mother plant, followed by storage at 10 degrees C, resulted in a rapid, substantial decrease in sucrose synthase activity and considerable increases in hexose content and acid invertase activity. A comparison of the response of three genotypes, known to accumulate different quantities of hexoses in storage, showed that both sucrose synthase activity and the extent to which activity declined following excision were similar in all cases. However, there was significant genotypic variation in the extent to which acid invertase activity developed, with tubers accumulating the highest hexose content also developing the highest extractable activity of invertase. Similar effects were found in nondetached tubers when growing plants were maintained in total darkness for a prolonged period. Furthermore, supplying sucrose to detached tubers through the cut stolon surface prevented the decline in sucrose synthase activity. Maltose proved to be ineffective. Western blots using antibodies raised against maize sucrose synthase showed that the decline in sucrose synthase activity was associated with the loss of protein rather than the effect of endogenous inhibitors. Although there were indications that maintaining a flux of sucrose into isolated tubers could prevent the increase in acid invertase activity, the results were not conclusive.

Journal Article↗
PubMed1994

Formation and Deposition of Amylose in the Potato Tuber Starch Granule Are Affected by the Reduction of Granule-Bound Starch Synthase Gene Expression.

The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via antisense RNA in Agrobacterium rhizogenes-transformed potato plants. Analysis of starch production and starch granule composition in transgenic tubers revealed that reduction of GBSS activity always resulted in a reduction of the production of amylose. Field experiments, performed over a 2-year period, showed that stable inhibition of GBSS gene expression can be obtained. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. In plants showing inhibition of GBSS gene expression, the reduced amylose content in tuber starch was not a consequence of a lower amylose content throughout the entire starch granule. Starch granules of transgenic tubers were found to contain amylose at a percentage similar to wild-type starch in a core of varying size at the hilum of each granule. This indicated that reduced GBSS gene expression results in amylose formation in a restricted zone of the granules. The size of this zone is suggested to be dependent on the GBSS protein level. During development of the granules, the available GBSS protein is thought to become limiting, resulting in the formation of starch that lacks amylose. RNA gel blot analysis of tuber tissue showed that inhibition of GBSS gene expression resulted in a reduced GBSS mRNA level but did not affect the expression level of other starch synthesizing enzymes. Antisense RNA could only be detected in leaf tissue of the transgenic plants.

Journal Article↗