Potato Tuber Discs Research Articles

Inhibition of browning, phenoxyacetic acids and phenolic metabolism in potato tuber discs: a model system to study chemicals that control common scab

The browning of tuber discs was used as a model system to study the effect of substituted phenoxyacetic acids on tuber metabolism. Those substituted phenoxyacetic acids that reduce scab reduced browning but several compounds that do not affect scab also reduced browning. Evidence presented indicates that 3,5‐dichlorophenoxyacetic acid preferentially inhibited phenolic metabolism rather than generally inhibiting the changes that occur after cutting (wounding). It is suggested that the decrease in potato common scab by foliar sprays of 3,5‐D is due to a modified host metabolism which involves inhibition of phenolic biosynthesis but which has no effect on terpenoid biosynthesis.

The role of lignification in the resistance of plants to attack by pathogens

Reviews by Vance et al. (1980) and Friend (1981) contain details of and references to the earlier work on lignification in both cucumber and grasses that is briefly described below. The suggestion of Hijwegen in 1963 that active lignification could be a general resistance mechanism in plants arose from experiments in which it was found that lignin deposition was associated with the reaction of a resistant cultivar of cucumber to inoculation withCladosporium cucumerimum. More recently Hammerschmidt and Kuc (1982) have found that systemic resistance of cucumber, induced by pre-inoculation with eitherC. cucumerimum orColletotrichum lagenarium, was associated with the ability of the induced plants to produce lignin rapidly in response to a challenge inoculation with either fungus. They concluded that induced resistance may be similar to cultivar resistance. Hammerschmidt et al. (1982) also showed that induction inoculation gave enhanced peroxidase activity in protected plants, associated with the fast-moving anodic isoenzymes. Lignification, especially the formation of lignified papillae, seems to be a common reaction of grass plants to attempted infection by nonpathogens. This has been found in reed canary-grass,Phalaris arundinacea, and a range of grasses by Vance and Sherwood, and in wheat by Ride. In reed canary-grass, active lignification is associated with increases in the enzymes of phenolic acid biosynthesis, and in peroxidase with the appearance of one new isoenzyme. The inducible resistance response, together with the associated increases in enzyme levels, were inhibited by the protein synthesis inhibitor, cycloheximide. The difference in response of wounded wheat leaves to inoculation by the nonpathogenBotrytis cinerea and the pathogenSeptoria nodorum appears to be related to the speed of lignification. It was rapid in the former case, confining the fungus to the wounded area; in the latter, it was slower and the fungus was not restricted. Recent experiments indicate that the accumulation of phenolic compounds in cell walls is a major factor in the non-race-specific resistance of potato tuber discs toPhytophthora infestans and of the same cultivars toPhoma exigua var foveata (Ampomah, 1983; Ampomah and Friend, in preparation). Accumulation of these compounds, estimated either by the level of browning of discs or the intensity of blue-green staining with toluidine blue of the walls of the top layer of cells, was greater following inoculation of resistant than of susceptible cultivars. Treatment of discs with aminooxyacetic acid (AOA), which is an inhibitor of PAL activity in vivo, inhibited the deposition of the phenolic compounds and permitted the fungi to grow further through tuber tissue. After freezing the discs in liquid nitrogen and extracting with chloroform: methanol: water (CMW), the residue was extracted with phenol: acetic acid:water (PAW). The CMW extracts contained chlorogenic acid; its level was lower in inoculated than uninoculated tissue. δei measurements indicated that phenolic compounds were present both in the dialyzed PAW extract and in the PAW residue. There was always more phenolic material in both extract and residue from the inoculated discs of both resistant and susceptible cultivars than from uninoculated discs; the levels were always higher in the resistant than in the susceptible discs. The presence of quinic acid in the PAW extracts indicates the possibility that they contain oxidized, polymerized chlorogenic acid. Such material is absent from the PAW residue that contains esterified hydroxy cinnamic acids. We are, as yet, unable to determine whether these acids are esterified to lignin, to carbohydrate, or to the hydroxy-aliphatic acids in suberin.

Light-Induced Transformation of Amyloplasts into Chloroplasts in Potato Tubers

The transformation of amyloplast into chloroplasts in potato (Solanum tuberosum L.) tuber tissue can be induced by light. Excised potato tuber discs illuminated with white light of 3000 lux began to synthesize chlorophyll after a lag period of 1 day, and continued to synthesize chlorophyll for 3 weeks. In this paper we present evidence, based on ultracentrifugal sedimentation and immunoprecipitation, that the light-mediated synthesis of Ribulose-1,5-bisphosphate carboxylase began 1 day after illumination with white light. When illuminated the chloroplasts isolated from light-grown potato tuber tissue incorporated [(35)S]methionine into polypeptides, one of which has been identified as the large subunit of Ribulose-1,5-bisphosphate carboxylase. These chloroplasts are functional as determined by O(2) evolution in the Hill reaction.

Alternative pathway respiration in vivo of potato tuber callus grown at various temperatures

During incubation of potato tuber discs (Solanum tuberosum L. cv. Bintje) on a callus‐inducing medium at 28°C, a high capacity of in vivo alternative pathway respiration develops (75% of uninhibited respiration is azide‐resistant). When callus induction takes place at 8°C, only 45% of respiration is resistant to azide. In the lag phase of growth the activity of alternative pathway is low. during the exponential growth phase the activity reaches its maximal rate. This in vivo activity is of the same size at both culture temperatures. As a consequence a greater part of alternative pathway capacity is operating in uninhibited respiration during growth at low temperatures.

Comparison of the effects of auxin and fusicoccin on phosphate absorption by aged potato tuber discs

Auxin (IAA, 5 × 10−5M) partially prevents the increase in the rate of phosphate uptake during ageing of potato tuber discs (Solatium tuberosum L. cv. Bintje), whereas fusicoccin (FC, 10−5 M) stimulates it. After the development of enhanced phosphate transport capacity, the response to fusicoccin is greater than with fresh discs. Complementary experiments on K+ (86Rb) absorption show that FC also slightly enhances the rate of K+ uptake, while IAA has no much effect. It is suggested that IAA acts specifically on the development of a mechanism which occurs during the ageing period, while FC action may be more directly linked to the system of phosphate transport itself.

Inhibition of Phytoalexin Synthesis in Arachidonic Acid-Stressed Potato Tissue by Inhibitors of Lipoxygenase and Cyanide-Resistant Respiration

Arachidonic acid-stressed potato tuber discs synthesized the phytoalexin rishitin. This synthesis was inhibited by salicylhydroxamic acid (SHAM), and to a lesser extent by tetraethylthiuram disulfide (disulfiram). Disulfiram was less effective apparently because it was inactivated in the tuber discs. Disulfiram and SHAM both inhibited cyanide-resistant respiration of whole potato discs and lipoxygenase extracted from these discs. When low disulfiram concentrations were used, the lipoxygenase inhibition was quickly overcome, again because the disulfiram apparently was inactivated by oxidation.

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.

Stability of Polysome-Associated mRNA in Potato Tuber Cells during Aging of Tissue Discs

The stability of polysome-associated mRNA in potato tuber discs in the early stage of aging was examined by pulse-chase labeling experiments and the change in the translational capacity of the RNA was studied using a wheat germ translation system. The incorporation of pulse-fed 3H-uridine into polysomal RNA was not arrested immediately after the addition of actinomycin D to the tissue, but increased by 25% during 4 hr of chasing. The radioactivity in the polysomal RNA then decreased by only 30% of the value at the 4th hr during the next 9 hr in the presence of actinomycin D. The remaining radioactivity in the polysomal RNA was stable at least for 18 hr. The proportion of radioactivity in polyadenylated RNA to that in non-polyadenylated RNA did not vary appreciably during the chasing period. Non-polyadenylated RNA of high molecular weight degraded faster than that of low molecular weight, but polyadenylated RNA did not show such size-selective degradation. The translational capacity of the polysomal RNA also decreased by about 23% within 9 hr during the period of inhibited RNA synthesis. In vivo experiments of 14C-leucine incorporation into proteins in the absence of RNA synthesis suggested that stable polysome-associated mRNA was actually functioning in the cells. SDS-polyacrylamide gel electrophoresis of the in vitro translation products indicated that mRNA coding for polypeptides with relatively high molecular weights turned over slightly faster than those for low molecular weight polypeptides.

Influence of Growth Temperature on Respiratory Characteristics of Mitochondria from Callus-Forming Potato Tuber Discs

The uninhibited respiration of mitochondria, isolated from potato tuber discs (Solanum tuberosum L. cv. Bintje) incubated on a callus-inducing medium at 28 degrees C, is higher than that of mitochondria from tissue incubated at 8 degrees C. This respiration is composed of a CN-sensitive and a CN-resistant part. The capacity of the CN-resistant alternative oxidase pathway is larger in mitochondria from 28 degrees C tissue than in mitochondria from 8 degrees C tissue (35% and 8% of uninhibited respiration, respectively). The alternative pathway is operative both in mitochondria from 28 degrees C tissue and 8 degrees C tissue.The observed difference in uninhibited respiration, is not only caused by lower values of respiration via the alternative pathway in mitochondria from 8 degrees C tissue, but also by lower values of respiration via the cytochrome pathway.A positive correlation has been demonstrated between the incubation temperature (ranging from 4-37 degrees C) and the relative capacity of respiration via alternative pathway in the mitochondria. Induction of alternative pathway is not directly correlated with growth (in terms of increase in fresh weight) of the potato tuber discs.

The effect of the different components of the inhibitor β complex in peelings of resting potato tubers on the respiration and uptake of inorganic phosphate

The four different inhibitors or groups of inhibitors, called I, II, IV and V, which could be separated from the inhibitor β complex from peelings of resting potatoes (Solanum tuberosum L., variety Majestic) by thin layer chromatography in chloroform: 96% acetic acid (95:5 v/v), stimulated the oxygen uptake of discs of potato tubers during the first 4 to 5 h after addition of the substances. Number I was less active, the other three more active. Furthermore II, IV, and V significantly inhibited the uptake of inorganic phosphate by potato discs measured 24 and 5 h after addition of the substances, so that they seem to be uncouplers of oxidative phosphorylation.

Polyamines and wounded storage tissues – Inhibition of RNase activity and solute leakage

The “protective” effect of polyamines and related metabolites on wounded storage tissues was investigated in two experimental systems: RNase activity of potato (Solanum tuberosum L.) tuber discs, and betacyanin leakage from beet root (Beta vulgaris L.) discs. Potato tuber discs evince a dramatic rise in RNase activity 24 h after excision, and this rise is completely blocked by the polyamines, spermidine and spermine. The effect is concentration‐dependent, and several precursors of polyamines are also effective to various degrees in inhibiting the rise in RNase activity. Polyamines inhibit both the wound‐induced RNase and the rise in RNase activity which may result from other senescence‐linked events. Betacyanin leakage from beet root discs is enhanced upon aging of discs and in response to various treatments which may cause membrane damage. Spermidine and spermine effectively inhibit betacyanin leakage which results from aging at 25°C, from incubation at higher temperatures (35–40°C), from freezing of discs, or from treatment of discs with RNase or protease. It is suggested that polyamines decrease RNase activity and solute leakage in sliced storage tissues by affecting primary wound‐induced destabilization of cell membranes.

Biosynthesis, Deposition, and Partial Characterization of Potato Suberin Phenolics

Alkaline nitrobenzene oxidation of the polymeric materials from wound-healed potato (Solanum tuberosum L. var. White Rose) tuber tissue liberated p-hydroxybenzaldehyde, vanillin, and minor amounts of syringaldehyde as determined by gas chromatography/mass spectrometry. The aromatic aldehydes were derived only from periderm. The amounts of aromatic aldehydes liberated were used as a measure of the deposition of phenolic suberin components. Phenolic deposition began after about 2 days of wound healing; after 8 days the amounts of p-hydroxybenzaldehyde released by nitrobenzene oxidation leveled off at 5 milligrams per gram dry weight and after 12 days vanillin liberation reached a maximum at 7.5 milligrams per gram dry weight. The time course of deposition of the phenolic polymeric material is analogous to that reported for the deposition of the aliphatic components of suberin and therefore these results are consistent with the proposed structure of suberin. Experiments with radiolabeled l-phenylalanine and cinnamic acid indicated that exogenous phenylalanine was less efficient than cinnamic acid as a precursor of suberin phenolics. Nitrobenzene oxidation of radiolabeled suberin preparations gave three major labeled fractions: a diethyl ether-soluble fraction containing aromatic aldehydes ( approximately 20%), an ethyl acetate-soluble fraction containing unknown compounds ( approximately 15%), and a condensed phenolic fraction ( approximately 10%). Thin-layer and gas-liquid chromatographic analysis of the ether fraction showed that the major labeled components were vanillin and p-hydroxybenzaldehyde. The condensed tannin fraction revealed the presence of several labeled macromolecular phenolic fractions. Elution profiles of the condensed tannin fraction from tissues suberized for different periods of time were essentially identical, suggesting qualitative similarity of deposition and polymerization of suberin phenolics throughout the duration of wound healing. Chlorogenic acid accumulation in wound healing potato tuber discs was measured by high-performance liquid chromatography. The level of this compound reached 130 micrograms per disk after 11 days and did not decline even after the deposition of suberin ceased, revealing no precursor role for this acid in suberization.

Interaction of Agrobacterium with potato lectin and concanavalin A and its effect on tumor induction in potato

Fluorescent-labeled concanavalin A, a d-glucose- and d-mannose-specific lectin from jackbean seeds, adhered to cells of Agrobacterium tumefaciens strains B6M and NT-1 (B6) (oncogenic on potato) and NT- I (nononcogenic on potato), but not to cells of 19 other Agrobacterium strains. The mean number of radioiodinated concanavalin A molecules bound per bacterial cell was 27 600 for B6M, 30 000 for NT-1 (B6) and 50 600 for NT-1. Concanavalin A precipitated lipopolysaccharides from strains B6M, NT-1, 2R and B695 (intact cells of the latter 2 strains did not bind the lectin). Lipopolysaccharides from strains B6 and C-58 and extracellular polysaccharides from all 6 strains were not precipitated by the lectin. When supplied at 3 × 10 8 molecules per bacterial cell, concanavalin A specifically reduced the number of tumors produced on potato by strain B6M by about 25%, but had no significant effect on the tumorigenicity of strain B6. Potato lectin, which is specific for oligomers of N-acetyl- d-glucosamine and was purified from potato tubers, did not adhere to cells of any of the 22 Agrobacterium strains. The lectin did not precipitate lipopolysaccharides or extracellular polysaccharides from 6 strains, and it did not influence the ability of strain B6 to produce tumors on potato tuber discs.

Effect of coronatine on the induction of cell wall degrading enzymes in potato tuber discs.

Activities of cellulase, polygalacturonase (PG) and polymethylgalacturonase (PMG) in potato tuber discs treated with coronatine, an extracellular toxin produced by Pseudomonas syringae pv. atropurpurea (Reddy and Godkin) Young et al., were determined as an approach to the mechanism of hypertrophy induced by the toxin. The optimum pHs of cellulase, PG and PMG were around 5.0, 4.0 and 5.0, respectively. All these enzyme activities were considerably increased by coronatine treatment. Cellulase, PG or PMG was little altered, partially inhibited or slightly enhanced, respectively, by the addition of Ca++. The hypertrophy induced by coronatine can be partly explained in terms of the loosening of cell wall due to the increased activities of cellulase, PG and PMG.

An analysis of the effect of gibberellic acid on adventitious shoot formation and development from tuber discs of potato: Robert L. Jarret and Paul M. Hasegawa, Purdue University, Department of Horticulture, West Lafayette, IN 47907

An analysis of the effect of gibberellic acid on adventitious shoot formation and development from tuber discs of potato: Robert L. Jarret and Paul M. Hasegawa, Purdue University, Department of Horticulture, West Lafayette, IN 47907

Influence of auxin on phosphate absorption and metabolism of phosphorylated compounds in aged potato tuber discs (Solanum tuberosum)

Ageing of potato tuber discs markedly increases the rate of phosphate uptake. This increase is partially prevented by the presence of indoleacetic acid (IAA: 50 μM) in the ageing medium. 32P distribution among the various phosphorylated fractions (P1, organic soluble phosphate, acid‐insoluble phosphate) was carried out after 24 h of ageing in the presence of IAA. An equal inhibition of the labelling rates of each of the different fractions is induced by the hormone. No important effect on respiration and ATP content was observed. Moreover, IAA neither changes the total phospholipid content nor the relative distribution of 32P between the components. These results support the hypothesis that IAA acts specifically on the development of uptake mechanisms during the ageing period.

Changes in phenylalanine ammonia‐lyase levels in excised potato tuber tissue: effects of the gaseous environment and desensitization to cinnamate repression by in situ pre‐incubation

Abstract. The rise in phenylalanine ammonia‐lyase (PAL) activity following excision of potato tuber discs is antagonized by increasing partial pressures of CO2 This inhibition is potentiated by depleting the atmospheric ethylene level. We suggest that the previously observed suppression of PAL appearance by in situ incubation of excised discs in reassembled tubers may be related to an internal atmosphere relatively rich in CO2 and of low ethylene content. The transition to an oscillatory time course of PAL activity that follows transfer of discs from in situ incubation to air appears to be accompanied by the development of enzyme activity becoming desensitized to repression by exogenous cinnamate. The concentration dependence of cinnamate uptake is not significantly altered by in situ pre‐incubation of tuber discs.

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

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.

Changes in Electrophoretic Pattern of Ribonucleases During Aging of Potato Tuber Slices

Summary Polyacrylamide gel electrophoresis of potato tuber extracts allows the separation of five bands of ribonuclease activity. Deoxyribonuclease activity is confined to a single gel zone. All bands with ribonuclease activity appear in the electrophoretic pattern of supernatant of 100,000 g, which shows more than 90 per cent of total activity. However, the possibility of solubilization of particulate ribonuclease during tissue homogenization is not precluded. Aging of potato tuber disks causes a great increase in the activity of ribonuclease, while deoxyribonuclease activity is not significantly changed during the first 24 hr of incubation. During aging of disks the electrophoretic pattern of ribonuclease shows a considerable increase of activity in only one of the bands, with a simultaneous decrease in another. The remaining bands show no considerable variations. It has been possible to separate the enzyme corresponding to the band showing increased activity. The enzyme was isolated from aged tissue and partially purified by precipitation with ammonium sulfate, dialysis and Sephadex chromatography. It is a ribonuclease with pH optimum of 5.5, the activity of which is neither affected by Ca 2+ nor Mg 2+ ions, nor by ethylene diamine tetraacetate. This ribonuclease has no phosphodiesterase activity and is inactive against native or denatured DNA.

The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue

During the first 24 h of in vitro incubation of excised potato tuber (Solanum tuberosum L.) discs, the appearance of phenylalanine ammonia-lyase (PAL; EC 3.4.1.5) and the accumulation of chlorogenic acid are both stimulated by infection with Phytophthora infestans (Mont.) de Bary. Whereas in control tissue the level of PAL reached a stable plateau value after 40 h, in infected tissue it subsequently rose again, in one experiment, as the fungal mycelium developed. In the infected but not the control tissue, the level of chlorogenic acid subsequently fell to about to about 20% of its maximum after 50 h. The time courses of increases in cinnamic acid 4-hydroxylase (CA4H; EC 1.14.13.11; 0-60 h) and of caffeic acid acid o-methyltransferase (COMT; EC 2.1.1.42; 0-160 h) are not altered by fungal infection. If the discs are restored to the tuber environment immediately after excision, by placing them inside a "host tuber", the activity of PAL as well as those of CA4H and COMT remained at the constant low endogenous level for at least 60 h, irrespective of whether the discs had first been inoculated with P. infestans. The increase in PAL may not be an obligatory feature of the P. infestans/potato compatible interaction but dependent on an underlying wound response. The experiments provide further evidence that PAL is the rate limiting step of chlorogenic acid biosynthesis in potato tuber discs.