Solanidine metabolism in potato tuber tissue slices and cell suspension cultures

Two metabolites have been isolated from potato tuber tissue slices or cell suspension cultures that have been incubated with labeled solanidine. Initially glucosyl solanidine is formed which subsequently is glycosylated to a diglucosyl solanidine.     

Selective measurement of starch synthesizing enzymes in permeabilized potato tuber slices

Osmotically permeabilized potato (Solanum tuberosum L.) tuber slices were used to study the biosynthesis of starch under semi in vivo conditions. Criteria to distinguish the various enzymes involved in starch biosynthesis were developed based on the characteristics of the enzymes in in vitro experiments. Branching enzyme activity was inhibited at pH 8.5 or higher, while the starch synthases functioned optimally between pH 8.8 and 9.1. Unprimed soluble starch synthase activity was only apparent in the presence of sodium citrate (0.4 molar or higher). Granulebound and primed soluble starch synthase were active in the absence of sodium citrate. Primed soluble starch synthase activity was susceptible to inhibition by 10 millimolar zinc sulfate, while granule-bound starch synthase activity was not. The incorporation of the Glc moiety of ADP-Glc into starch in tissue slices by the various starch synthases was consistent with in vitro data with respect to the affinity of the enzymes for substrate, the pH profile, the stimulation by citrate, and the inhibition by zinc sulfate. These data were used to determine the activity of each of the starch synthases in tissue slices: granule-bound and soluble starch synthase transferred 37 and 55 picomoles ADP-Glc per hour per milligram fresh weight into starch of permeabilized tissue slices at 30°C and pH 9.1. In the presence of 0.5 molar sodium citrate, at least 40 picomoles ADP-Glc per hour per milligram fresh weight as transferred into starch by unprimed soluble starch synthase activity.     

Enhancement of DNA polymerase activity in potato tuber slices

DNA polymerase was extracted from potato (Solanum tuberosumL.) tuber discs and the temporal correlation of its activitychange to DNA synthesis in vivo was examined during aging ofthe discs. Most of the DNA polymerase was recovered as a boundform in the 18,000?g precipitate. Reaction with the bound-formenzyme was dependent on the presence of four deoxynucleosidetriphosphates, Mg²⁺, and a template. "Activated" DNA and heat-denaturedDNA, but not native DNA, were utilized as templates. The polymeraseactivity was sensitive to SH reagents. Fresh discs, which donot synthesize DNA in vivo, contained a significant amount ofDNA polymerase and its activity increased linearly with timeuntil 48 hr after slicing and became four times that of freshdiscs after 72 hr, whereas the activity of DNA synthesis invivo increased with time and decreased after reaching a maximumat 30 hr. Cycloheximide inhibited the enhancement of polymeraseactivity. DNA polymerase from aged and fresh discs had identicalrequirements for deoxynucleotides and a template in their reactions,sensitivity to SH reagent, and affinity to thymidine triphosphate. (Received February 18, 1977; )     

Hydroxylation of dehydroepiandrosterone and its acetate with potato tuber slices

Evidence is presented for the view that the potato tuber tissue contains an unstable enzyme capable of hydroxylation of dehydroepiandrosterone and its acetate in low yield. The products of hydroxylation were isolated and identified. The possibility is discussed that auto-oxidation is a simultaneous reaction.     

Alternative pathway activities in aged potato tuber slices measured by three methods

To find a simple and reliable oxygen electrode-based method to estimate the values of alternative pathway activity (V_alt) and its contribution to total respiration V_alt/V_t) in aged potato (Solanum tuberosum L.) tuber slices, we compared conventional hydroxamate-inhibiting method, improved hydroxamate-inhibiting method with 2,6-dichlorophenol indophenol (DCPIP), and the oxygen isotope discrimination (OID) method. The values of V_alt and V_alt/V_t obtained with an improved hydroxamate-inhibiting method with DCPIP in 12-h- and 24-h-aged slices were about twice higher than those with the conventional hydroxamate-inhibiting method. Only a relatively small difference in the values of V_alt and V_alt/V_t obtained by the OID method and the improved hydroxamate-inhibiting method with DCPIP in 12-h and 24-h-aged slices was observed. These results indicated that the improved hydroxamate-inhibiting method with DCPIP could be considered as a new, simple, and reliable technique for the noninvasive assay of the AP activity.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 311–315.Original English Text Copyright © 2005 by Hou, Zhou, Kong, Liang, Zhang.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

Effect of ageing on sterol metabolism in potato tuber slices

When fresh potato tuber slices were incubated with [1-¹⁴C]-sodium acetate, cycloartenol was heavily labelled but no radioactivity was recovered in 24-methylene cycloartanol and free sterols. If potato slices were aged for 0–24 hr before feeding with radioactive acetate, a rapid increase of the label in the sterol precursors and the free sterols was observed. The free sterol content was 5 × higher after ageing for 24 hr. Isofucosterol synthesis was especially stimulated. The synthesis of sterols during the ageing process seems to be related to the appearance of a cycloartenol C24-methylase and may be linked to a biogenesis of membranes.Nomenclature: (1) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-cholest-24-en 3β-ol; (2) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-ergost-24(28)-en 3β-ol; (3) 4α,14α-dimethyl 9β,19β-cyclo 5α-ergost 24(28)-en 3β-ol; (4) 4α, 14α-dimethyl 5α-ergosta 8.24(28)-dien 3β-ol; (5) 4α-methyl 5α-ergosta 7,24(28)-dien 3β-ol; (6) ergosta 5,24(28)-dien 3β-ol; (7) stigmasta 5,Z-24(28)-dien 3β-ol; (8) (24R)-24 methyl cholest 5-en 3β-ol; (9) (24R)-24 ethyl cholest 5-en 3β-ol; (10) (24S)-24 ethyl cholesta 5,E-22(23)-dien 3β-ol; (11) cholest 5-en 3β-ol.     

Inhibition of nitrate accumulation and in vivo nitrate reductase activity in potato tuber slices by dimethysuIfoxide

Dimethylsulfoxide at concentrations of 0.5 to 5.0% inhibitednitrate reductase activity and the accumulation of nitrate in"fresh" and "aged" potato tuber slices. Fresh slices were moresensitive to concentrations of 0.5 to 2.5% and suppression ofenzyme activity paralleled a decline in NO₃ accumulation. With aged slices concentrations of 0.5 to 2.5% progressivelysuppressed enzyme activity without affecting NO₃ accumulation.These results are discussed in relation to the known effectsof dimethylsulfoxide on the permeability of biological membranesand protein structure. (Received November 29, 1978; )     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

Absolute structure of N-p-coumaroyloctopamine in elicitor-treated potato tuber tissue

Treatment of potato tuber tissue with beta-1,3-oligoglucosaccharide causes an accumulation of N-p-coumaroyloctopamine (1). In order to determine the absolute structure of 1 in potato, optically active 1 was synthesized from (R)-octopamine which had been obtained from the racemic mixture by the fractional crystallization. By comparing the chromatographic behavior of synthetic and naturally-occurring samples with a chiral HPLC analysis, the absolute configuration of 1 in potato was determined to be S. This indicates that the absolute configuration of the octopamine moiety of 1 is opposite to that of octopamine formed in animal tissues.     

Phenol metabolism, phytoalexins, and respiration in potato tuber tissue treated with Fatty Acid

Potato (solanum tuberosum L. cv Katahdin) tuber discs treated with arachidonic acid become necrotic and accumulate sesquiterpenoid phytoalexins. The arachidonic acid also causes increases in both phenylalanine ammonia lyase and lignin, but no change in total alcohol-soluble phenols. Linoleic acid does not alter any of these parameters. A high concentration of nonanoic acid promotes both necrosis and accumulation of low levels of phytoalexins, but decreased levels of phenols, phenylalanine ammonia lyase, and lignin. The respiration of the control discs and those treated with linoleic acid declines by 24 hours after treatment, but the respiration of arachidonic acid-treated discs remains constant for at least 48 hours.     

Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry

A new method is presented in which gas chromatography coupled to mass spectrometry (GC-MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and specific manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modification and GC-MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70-140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a first example this method has been applied to soil- and in vitro-grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ significantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modified in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.     

Induction of deoxyribonucleic Acid synthesis in potato tuber slices: role of protein synthesis

Timing of protein synthesis which is a prerequisite to DNA synthesis induced in potato tuber tissue (Solanum tuberosum L.) by cut injury has been studied using cycloheximide. The induction of DNA synthesis which was measured by incorporation of ³H-thymidine was completely inhibited when the inhibitor was applied to the tuber discs immediately after slicing. When the application of cycloheximide was delayed for 6 hours or more after slicing, DNA synthesis was observed but its rate was reduced to 20% of control. The inhibitory effect of cycloheximide, however, rapidly decreased when the inhibitor was applied at 6 or less hours immediately prior to determination of DNA synthesis. The effect of cycloheximide on the incorporation of ¹⁴C-leucine suggests that the change in the effect of cycloheximide on the induction of DNA synthesis is not due to incomplete inhibition of protein synthesis. Cycloheximide did not have significant effects on either uptake or phosphorylation of ³H-thymidine in the discs. Inhibition of both protein and DNA synthesis by cycloheximide was reversed by washing and further incubation of the discs. Almost no qualitative difference was detected by buoyant density analysis between DNA formed under inhibition of protein synthesis of the later stage and DNA synthesized under normal conditions. These results suggest that DNA synthesis induced in potato tuber tissue by cut injury requires continuous synthesis of new protein molecules in a characteristically programmed sequence.     

Studies on enzymes involved in DNA synthesis and thymine nucleotide formation in potato tuber slices

Activity changes of several enzymes involved in DNA synthesiswere investigated in potato tuber tissue in which DNA synthesiswas induced by slicing. Nucleoside phosphotransferase activityincreased only slightly during aging of the tissue discs. Thymidinemonophosphate (TMP) kinase activity increased about 36% afteraging for 24 hr. Protein synthesis in an early stage of agingwas necessary for the activity increase. A 2.7-fold increasewas observed in DNA polymerase activity after aging for 36 hr.The activity increase was due to continuous synthesis of enzymeprotein. In vivo examination of TMP synthetase suggests thatits activity does not necessarily increase before full developmentof DNA synthesis. It was concluded that among the enzymes examined,TMP kinase activity may increase shortly after slicing to supporta massive supply of thymidine triphosphate and the increasedactivity of DNA polymerase may contribute to the active synthesisof DNA in aged discs. (Received February 18, 1977; )     

Control of root and shoot-bud formation from potato tuber tissue cultured in vitro

Comparative studies on the control of root and shoot-bud formation and plant regeneration have been undertaken in discs (1 × 6 mm in diameter) excised from tubers of potato ( Solanum tuberosum L. cv. Irish Cobbler) cultured in vitro. The results clarified that the optimal culture conditions for shoot-bud formation were quite different from those for root formation and, in conclusion, that (1) shoot-buds were produced when cultured in modified White's medium containing 0.25 M mannitol with 2.3 μ M zeatin and 0.57 μ M indole-3-acetic acid (IAA) at 20°C under relatively high light irradiation, while (2) roots were readily formed when cultured in modified White's medium containing 29 m M sucrose with 4.7 μ M kinetin plus 1.7 μ M IAA at 30°C in darkness.     

Reduction of water permeability in potato tuber slices by cyanide, ammonia, 2,4-dinitrophenol, and oligomycin and its reverse by adenosine 5'-triphosphate and cytidine 5'-triphosphate

Five millimolar KCN reduced water permeability in 1-millimeter thick slices of potato tuber (Solanum tuberosum L.). One-tenth millimolar ATP and CTP prevented or reversed the reduced permeability. UTP and GTP were not effective. Five millimolar ammonium carbonate or 0.1 millimolar 2,4-dinitrophenol also reduced water permeability, but ATP and CTP were only partially effective in reversing the reduced permeability. Oligomycin, 5 micrograms per milliliter, reduced water permeability, and the reduction was reversed by ATP and CTP. ATP and CTP appear to be involved in maintaining the structure of water pathways into the cell.     

Effect of temperature on starch synthesis in potato tuber tissue and in amyloplasts

A sharp temperature optimum is observed at 21.5°C when the incorporation of [¹⁴C]sucrose into starch is measured with discs cut from developing tubers of potato (Solanum tuberosum L. cv Desirée). By contrast, increasing temperatures over the range 9 to 31°C only enhance release of ¹⁴C to respiratory CO₂ and incorporation of ¹⁴C into the ethanolsoluble fraction. By comparison, starch synthesis in discs from developing corms of cocoyam (Colocasia esculenta L. Schott) is increased by raising the temperature from 15 to 35°C. The significance of a relatively low temperature optimum for starch synthesis in potato is discussed in relation to the yield limitations imposed by continuously high soil temperatures. Amyloplasts isolated from protoplasts prepared from developing potato tubers contain activities of alkaline pyrophosphatase, NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, and phosphoglucomutase in addition to ADP-glucose-pyrophosphorylase, starch phosphorylase and starch synthase. Cell-free amyloplasts released by thinly slicing developing potato tubers synthesize starch from [¹⁴C]triose-phosphate generated from [¹⁴C]fructose-1,6-bisphosphate in the reaction medium. This starch synthesis is inhibited by addition of 10 millimolar inorganic phosphate and requires amyloplast integrity, suggesting the operation of a triose-phosphate/inorganic phosphate exchange carrier at the amyloplast membrane. The temperature optimum at 21.5°C observed with tissue discs is not observed with amyloplasts.     

The synthesis and properties of polysomal RNA in potato tuber slices during the early stage of aging

The synthesis, molecular size, and coding properties of polysome-associatedpolyadenylated RNA[poly(A)(+)RNA]and non-polyadenylated RNA[poly(A)(–)RNA] were investigated in potato tuber discsduring the early stage of aging. Tissue discs were labeled for6 hr with ³H-uridine in the presence of 5-fluorouracil to suppressrRNA synthesis, and polysomal RNA was isolated from the discs.Poly(A)(+)RNA accounted for 70% of the radioactivity in polysomalRNA and had a molecular size ranging from 6S to 30S with a peakat about 15S, when measured by formamide-polyacrylamide gelelectrophoresis. The rest of the radioactivity was in poly(A)(–)RNAwhich had nearly the same range in molecular size, but had noconspicuous peaks on the gel. The polysomal RNA could programthe synthesis of a wide variety of polypeptides in a cell-freetranslation system of wheat germ. Seventy percent of the translationalcapacity of polysomal RNA was attributed to poly(A)(+)RNA. Theelectrophoretic behaviour of the majority of the products frompoly(A)(+)RNA was similar to that of products from poly(A)(–)RNA,but the former could program the synthesis of five polypeptidesin addition to those translated from the latter. There was atendency for poly(A)(–)RNA to be a more efficient messengerfor large polypeptides. ¹Present address: Department of Agricultural Chemistry, Facultyof Horticulture, Chiba University, Matsudo 271, Japan. (Received November 16, 1979; )