Changes in cyclic-AMP phosphodiesterase activity during the formation of crown-gall tumors on potato discs

Cyclic-AMP phosphodiesterase activity was found in crown-galltumor tissue obtained from the inoculation of potato discs withAgrobacterium tumefacicns strain B6. Consistently more enzymeactivity was found, in this tumor tissue than in the correspondingnormal tissue. This difference in enzyme activity was observedwhen strains other than B6 were used to induce tumors, or whennormal and tumor potato tissue grown in culture were examinedfor phosphodiesterase activity. (Received January 27, 1978; )     

Inhibition of crown-gall tumor formation on potato discs by cyclic-AMP and prostaglandins E1 and E2

Cyclic-AMP was found to inhibit the induction of crown-galltumors on potato discs by as much as 60%. This inhibition couldbe obtained when cyclic-AMP was added up to 10 hr after inoculationof the potato discs with a tumor inducing strain of Agrobacteriumtumefaciens. Similar results were obtained with prostaglandinsE¹ and E². Some of the implications of these results will bediscussed. (Received September 30, 1976; )     

The effect of plant growth regulators on formation of crown gall tumors on potato tuber discs

The effect of several plant growth regulators on the number of tumors developing on potato tuber discs (Solatium tuberosum L. cv. Radka) inoculated withAgrobacterium tumefaciens, strain C 58 was studied. The plant growth regulators used in appropriate range of concentrations stimulated the formation of tumors byA. tumefaciens. Naphthaleneacetic acid (NAA) was most active in concentration of 10⁻⁴ mg ml⁻¹. Kinetin gave a biphasic response with optimal promotions of tumor initiation at 10⁻⁴ − 2 × 10⁻³ mg ml⁻¹. High kinetin concentration (10⁻¹ mg ml⁻¹) inhibited the formation of tumors completely. Indoleacetic acid (IAA) stimulated the initiation of tumors in the same range of concentrations as kinetin, except that very high concentrations did not inhibit but enhanced tumor formation. 2,4-diehlorphenoxyacetic acid (2,4-D) showed a biphasic response with maxima in 10⁻⁴ mg ml⁻¹ and 10⁻¹ mg ml⁻¹. All the tumors scored for nopaline production showed nopaline synthase activity independently whether their formation was stimulated by l0⁻¹ mg ml⁻¹ IAA or they were initiated without any treatment by plant growth regulators.     

Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea

cis-Zeatin riboside was identified in transfer-RNA hydrolysates from both normal and crown-gall tissue of Vinca rosea L. The trans-isomer was associated exclusively with the crowngall transfer-RNA. The importance of these observations is discussed in relation to biosynthesis of free cytokinins.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - TMSi trimethylsilyl - tRNA transfer RNA - ZR zeatin riboside     

Sucrose uptake and partitioning in discs derived from source versus sink potato tubers

The uptake of sucrose into isolated discs cut from sink (growing) and source (sprouting) potato (Solanum tuberosum L.) tuber tissue was studied. The uptake of sucrose into sink-tuber discs demonstrated biphasic kinetics. The large saturable component was inhibited by incubation of the discs with p-chloromercuribenzene sulfonic acid (PCMBS) whilst both the saturable and linear components were inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP). By contrast, in source-tuber discs, the linear component represented the majority of sucrose taken up, the saturable component playing only a minor role. In source discs, only the saturable component of uptake was inhibited by either PCMBS or CCCP. A large proportion (up to 25%) of sucrose taken up into sink-tuber discs was converted to starch but as the tubers aged the proportion of sucrose converted to starch decreased to the level found in source-tuber discs (approx. 3%). By contrast with sink-tuber discs (see Oparka and Wright, 1988b, Planta 175, 520–526) sucrose uptake into source discs was insensitive to turgor and demonstrated an uptake pattern similar to that of CCCP-treated sink tissue. It is proposed that exogenous sucrose is taken into the storage parenchyma of sink-tuber discs by both a carrier-mediated and a diffusional process. By contrast, uptake into the storage parenchyma of source-tuber discs appears to be essentially diffusional. The turgor sensitivity of sucrose uptake into sink-tissue discs may be mediated via the plasmalemma H⁺-ATPase. As the tuber ages the sucrose-uptake activity decreases and the capacity of the storage parenchyma to synthesise starch is lost. The data are discussed in relation to the in-vivo mechanisms of sucrose transport in storage tissues.     

Influence of ethanol on alternative oxidase in mitochondria from callus-forming potato tuber discs

Ethanol, when added to the incubation medium of callus-forming potato tuber discs, inhibits callus growth and causes an increase of the mitochondrial antimycin-A resistant respiration, expressed as a percentage of state III-respiration. This increase in resistance to antimycin-A is the result of a poor development of the cytochrome pathway in tissue discs treated with ethanol. The development of the antimycin-A resistant alternative oxidase sensitive to chelator is about the same for treated and untreated discs. The respiratory control (RC) ratio of the mitochondrial respiration increases after addition of a chelator, which inhibits the alternative pathway. The RC ratio of the uninhibited mitochondrial respiration appears to be inversely related to the capacity of the alternative pathway, when mitochondrial preparations with different capacities to transfer electrons via the alternative path are compared. From the experimentally observed relation between RC-ratio and alternative oxidase capacity, it was concluded that at least half of the capacity of the alternative path is used in uninhibited state IV respiration.     

Gibberellic acid regulation of adventitious shoot formation from tuber discs of potato

Summary The formation of adventitious shoots from potato tuber discs explanted onto a modified Murashige and Skoog (MS) medium containingN ⁶-benzylaminopurine (BAP) (3.0 mg/l), and α-naphthaleneacetic, acid (NAA) (0.01 mg/l), was affected by gibberellic acid (GA). The presence of GA in the explant medium was required for shoot formation and 3×10⁻¹⁰ M GA appeared optimum. However, microscopic examination of the tissue protuberances on the surface of the tuber discs from which shoots arose revealed that GA inhibited the formation of shoot meristems. Tuber discs cultured for 6 wk on MS medium containing BAP and NAA without GA did not initiate adventitious shoots that could be determined visually, but microscopic examination of the tissue protuberances revealed the presence of numerous shoot meristems. Subsequent transfer of these tuber discs to medium with GA but without BAP or NAA resulted in the formation of shoots from 100% of the recultrued dises. Thus it appears that although GA inhibits shoot meristem initiation from potato tuber discs, it is required for shoot development once meristems are initiated. This is Journal Paper 8297 of the Purdue University Agricultural Experiment Station. The research was supported by Purdue University Agricultural Experiment Station Program Improvement Funds. Potato tubers were supplied by Wm. Gehring Farms, Inc., Rensselaer, Indiana.     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Hormonal control of 5-bromodeoxyuridine (BUdR) tolerance in crown-gall tumors and habituated tissues

We have found that the unorganized tobacco crown-gall tumor, isolated in our laboratory, and Braun's teratomatic tumor both exhibit the capacity to grow in the presence of high toxic concentrations of BUdR (up to 10^-3 mol·l^-1). Double cytokinin-auxin habituated tissues also exhibit a constitutive BUdR-tolerance. In cytokinin-habituated tissues the BUdR-tolerance seems to depend on the degree of cytokinin autonomy. The presence of exogenous cytokinin significantly increases BUdR-tolerance, and the presence of BUdR suppresses the inhibitory effect of exogenous cytokinin upon habituated tissues. The results clearly show that the disposition of a cell for transient BUdR tolerance requires at least active expression of genes for cytokinin synthesis and that the tolerance becomes permanent when the auxin system is turned on. We conclude that BUdR-tolerance is connected with the establishment of a certain hormonal regulator pattern, depending on the state of cellular differentiation.     

Factors affecting shoot initiation from tuber discs of potato (Solanum tuberosum)

Discs of cortical and perimedullary tissue from tubers of potato (Solanum tuberosum L. cv. Superior) formed adventitious shoots when cultured on a medium containing Murashige and Skoog's salts, myo-inositol, 100 mg/l; folic acid, 0.5 mg/l; D-biotin, 0.05 mg/l; gibberellic acid (GA₃), 0.5 mg/l; thiamine ˙ HCl, 0.1 mg/l; glycine, 2.0 mg/l; pyridoxine ˙ HCl, 0.5 mg/l; nicotinic acid, 0.5 mg/l; sucrose, 25 g/l; casein hydrolysate, 1 g/l; Bacto agar, 9.0 g/l; and a cytokinin [N⁶-benzylaminopurine (BAP), N⁶-γ,γ-dimethylallylaminopurine (2iP), or N⁶-furfurylaminopurine (kinetin)]. Discs of pith tissue either failed to survive or produced callus but did not undergo morphogenesis. Cytokinin was essential for explant survival, while BAP at 3.0 mg/l was most efficacious in promoting shoot initiation. Auxin was not essential for shoot initiation or development; however, a low concentration (0.03 mg/l) of α-naphthaleneacetic acid (NAA) stimulated both explant survival and the number of shoots produced per disc. Indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) did not stimulate shoot initiation. GA₃ was essential for both shoot initiation and subsequent shoot development. The highest incidence of morphogenesis (over 100 shoots in 12 weeks) occurred from tuber discs cultured at 18°C constant and under a photon flux density of 60 μE m^-2s^-1. No difference in regenerative ability was found in explants taken from source tubers stored for 0 to 20 weeks at 4°C. A histological examination indicated that shoots developed from small clusters of meristematic cells which were initiated from within small protuberances on the surface of the tuber disc 3 weeks after transfer.     

High frequency of tetraploidy in Agrobacterium-mediated transformants regenerated from tuber discs of diploid potato lines

Summary Variations in the ploidy level of 69 transgenic potato (Solanum tuberosum L.) plants regenerated from the tuber discs of 17 diploid lines were studied: 24 plants (35%) were diploid, the other 45 plants (65%) were tetraploid. Seventy-eight control regenerants obtained without Agrobacterium inoculation showed a relatively low tendency to tetraploidization (35%). The results obtained suggested that chromosome doubling occurred frequently in diploid potato lines during the tissue culture process for regeneration. Putative somaclonal changes in in vitro-formed tuber proteins were detected in three out of six transformants by electrophoretic analysis.     

Ribosomal RNA synthesis in newly sliced discs of potato tuber

A burst of ribosomal RNA synthesis is induced in potato tissue by slicing, and continues at a decreasing rate for about 12 hours. Ribosomal RNA synthesis in potato discs is sensitive to puromycin, in contrast to non-ribosomal RNA synthesis. Thus, the influence of puromycin on total RNA synthesis is significant only during the first 12 hours following slicing. The function of RNA made after 12 hours in a puromycin-insensitive manner is unknown. However, it is apparently unrelated to protein synthesis, since it has been shown that total inhibition of RNA synthesis by addition of actinomycin D to potato tissue after 12 hours of aging has no effect upon protein synthesis during the ensuing 12 hours.     

Regulation of the activity of the alternative oxidase in callus forming discs from potato tubers

Callus-forming discs from potato tubers lose 80% of their starch during one month of incubation on nutrient medium containing either 0, 3 or 6% (w/v) sucrose. The content of soluble sugar in the discs varies from 5 mg (incubated without sucrose) to 22 mg (on 3% sucrose) and 40 mg (on 6% sucrose) per g fresh weight. The activity of the cytochrome pathway (V_cyt) increases during the first week of incubation on all media. Thereafter V_cyt decreases again on 0% sucrose medium, while it remains constant on 3 and 6% sucrose media. Alternative pathway capacity (V_alt), absent in freshly sliced tissue, shows a sharp increase during the first days of incubation, independent of the sucrose concentration in the medium. This capacity further increases during prolonged incubation on 3 and 6% sucrose but decreases on 0% sucrose. The in vivo activity of the alternative pathway (the participation in uninhibited respiration, ?V_alt) varies with the sucrose concentration and with the culture time. In tissue incubated for 2-3 weeks on 6% sucrose as much as 45% of the electrons are transfered to oxygen via the alternative pathway. In this tissue the factor Q (the part of the alternative pathway capacity that is operative) is about 0.8, while in tissue incubated on 0 and 3% sucrose media p generally does not exceed 0.5. When chloramphenicol, an inhibitor of mitochondrial protein synthesis, is added to the medium together with 3% sucrose, the increase in V_yet does not occur, while the induction of V_alt during the first week of incubation is the same as without chloramphenicol. A greater part of the alternative pathway capacity becomes operative in this tissue, leading to values of Q of almost 1 after prolonged incubation. Apparently, incubation on high sugar medium leads to extra participation in respiration of the energetically inefficient alternative oxidase pathway Excess sugar leads to wasteful respiration suggesting that the alternative oxidase functions as an ‘energy overflow’.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Effect of ethylene on DNA synthesis in potato tuber discs

The effect of ethylene on DNA synthesis in potato tuber discsinduced by cutting was examined. Continuous presence of ethylenein the ambient atmosphere of the slices lowered the rate ofinduced DNA synthesis by about 50%, but did not alter the timecourse pattern of development of DNA synthesis. RNA and proteinsyntheses were not affected. The inhibitory effect on DNA synthesiswas observed at as low as 0.01 µl/liter and was due tothe specific action of ethylene, not to a non-specific actionof gaseous hydrocarbons. Ethylene also decreased the numberof cells which could synthesize DNA. The results of ethylenetreatment of various durations at various times after cuttingindicate that a process prerequisite for DNA synthesis and susceptibleto ethylene action starts at about 6 hr after cutting and continuesfor only a limited period. (Received July 5, 1976; )