Phospholipid synthesis in aging potato tuber tissue

The effect of activation (“aging”) of potato tuber slices on their phospholipid metabolism was investigated. Aged slices were incubated with ¹⁴C labeled choline, ethanolamine, methionine, serine, and acetate. In all cases, the incorporation of radioactivity into the lipid fraction increased with the length of time the slices were aged. This incorporation was shown to be true synthesis and not exchange between precursors and existing phospholipids.

The increased incorporation of labeled choline into lipids was mainly due to an increase in its uptake by the tissue, the presence of actidione during aging prevented this increased uptake. The increase in the incorporation of labeled acetate into lipids resulted from the development of a fatty acid synthetase during aging. In the case of ethanolamine, both its uptake into the tissue and its incorporation into the lipid fraction increased.

The phospholipids formed from these precursors were identified by paper and thin-layer chromatography. The major compound formed from choline was lecithin, while phosphatidylethanolamine and a small amount of lecithin were formed from ethanolamine.

     

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; )     

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; )     

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.     

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.     

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.     

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.     

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.     

Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices.

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase (GART) and 5-aminoimidazole ribonucleotide synthetase (AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5'-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho-d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8-(14)C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.     

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; )     

Utilization of stored messenger RNA during early aging of Jerusalem artichoke tuber slices

Using dissociation in 0.8 M KCl, it was established that in freshly excised Jerusalem artichoke (Helianthus tuberosus L.) tuber slices less than 8% of the ribosomes were in polysomes. The first hour of aging in water was the period of most rapid polysome accumulation; over 32% of the ribosomes carried nascent polypeptide chains at the end of this time. Thereafter polysome accumulation continued to increase, but more gradually. While synthesis of high-molecular-weight RNA (presumed mRNA) was inhibited more than 95% by -amanitin during the first hour of aging, the inhibitor had no effect on polysome formation. As determined by [³H]polyuridylic acid hybridization, unaged cells contained polyadenylated RNA with a size range of 6–30S. The amount of polyadenylated RNA did not change during the first hour of aging. In control cells in water the in-vivo rate of protein synthesis increased exponentially during the first 4 h of aging without a comparable increase in polysomes. In -amanitintreated tissues a similar increase in protein synthesis was not observed despite the presence of near control levels of polysomes. It is suggested that early polysome formation depends on stored mRNA. Inhibition of mRNA synthesis by -amanitin prevents the normal development of an enhanced rate of protein synthesis which is not directly related to numbers of ribosomes in polysomes.Abbreviations Poly(A) polyadenylic acid - Poly(A)+RNA polyadenylated RNA - Poly(U) polyuridylic acid - TCA trichloroacetic acid     

Biochemical studies on mitochondria formed during aging of sliced potato tuber tissue

Respiratory activities in mitochondria of potato tuber tissueincreased about 3-fold after aging sliced tissue for 1 day.Increased activity was insensitive to cyanide. The number ofmitochondrial particles also increased during aging. It seemslikely that newly formed mitochondria are heavier than pre-existingones and that respiration in the former is insensitive to cyanide. (Received January 26, 1970; )     

Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices

Summary A biochemical and cytochemical study has been made of the distribution of -glycerophosphatase (EC 3.1.3.2) activity in mature and differentiating phloem cells of Nicotiana tabacum L. and the pH dependence and kinetics of -glycerophosphate hydrolysis of homogenates of fresh leaf midveins and midveins fixed in formaldehyde-gluteraldehyde. -glycerophosphatase showed two peaks of activity at pH 5.5 and 6.2. Enzyme saturation kinetics were exhibited by both fresh and fixed tissue homogenates. At a substrate concentration of 2 mM, 65% of the enzyme activity survived fixation. Specimens for cytochemical localization were incubated with 2 mM -glycerophosphate at pH 5.5 and 6.2. Specimens showed consistent patterns of reaction product deposition. Little or no reaction product was deposited in controls incubated without substrate or with substrate plus 0.01 M fluoride. -glycerophosphatase activity in the phloem and xylem is considerably higher than in surrounding tissue. Dense localization of reaction product was demonstrated on the vacuolar membranes, the inner membranes of mitochondria, and the dictysomes of phloem parenchyma and companion cells. The plasma membrane and endoplasmic reticulum cisternae of these cells were usually free of reaction products. Enzyme activity in mature sieve elements was associated with the parietal and stacked systems of endoplasmic reticulum and with the P-protein. There was inconsistency of staining of P-protein in mature sieve elements although the association of reaction products with the P-protein appeared to show a correlation with maturity and dispersal. The P-protein bodies of differentiating sieve elements showed no reaction product deposition. The distribution of -glycerophosphatase activity has been compared with that previously recorded for ATPase activity in the phloem of Nicotiana tabacum.     

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; )