Potato Tuber Discs Research Articles

Rapid Stimulation of 5-Lipoxygenase Activity in Potato by the Fungal Elicitor Arachidonic Acid

The activity of lipoxygenase (LOX) in aged potato tuber discs increased by almost 2-fold following treatment of the discs with the fungal elicitor arachidonic acid (AA). Enzyme activity increased above that in untreated discs within 30 min after AA treatment, peaked at 1 to 3 h, and returned to near control levels by 6 h. The majority of the activity was detected in a soluble fraction (105,000g supernatant), but a minor portion was also associated with a particulate fraction enriched in microsomal membranes (105,000g pellet); both activities were similarly induced. 5-Hydroperoxyeicosatetraenoic acid was the principal product following incubation of these extracts with AA. Antibodies to soybean LOX strongly reacted with a protein with a molecular mass of approximately 95-kD present in both soluble and particulate fractions whose abundance generally corresponded with LOX activity in extracts. LOX activity was not enhanced by treatment of the discs with nonelicitor fatty acids or by branched beta-glucans from the mycelium of Phytophthora infestans. Prior treatment of the discs with abscisic acid, salicylhydroxamic acid, or n-propyl gallate, all of which have been shown to suppress AA induction of the hypersensitive response, inhibited the AA-induced increment in LOX activity. Cycloheximide pretreatment, which abolishes AA elicitor activity for other responses such as phytoalexin induction, did not inhibit LOX activity in water- or elicitor-treated discs but enhanced activity similar to that observed by AA treatment. The elicitor-induced increase in 5-LOX activity preceded or temporally paralleled the induction of other studied responses to AA, including the accumulation of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase and phenylalanine ammonia lyase reported here. The results are discussed in relation to the proposed role of the 5-LOX in signal-response coupling of arachidonate elicitation of the hypersensitive response.

Characterization of elicitor-induction of sesquiterpene cyclase activity in potato tuber tissue

The olefin reaction products of the potato and tobacco sesquiterpene cyclases were partially separated from each other by argenation chromatography. Arachidonic acid-elicited potato tuber discs or cellulase-elicited tobacco cell suspension cultures metabolized the olefin reaction products of potato and tobacco sesquiterpene cyclases into different sets of compounds. These results provide evidence that the two olefin cyclase products are chemically different. The K m of the potato sesquiterpene cyclase for farnesyl diphosphate was 7μM and 1.0 mM for magnesium chloride. Inorganic diphosphate inhibited enzyme activity with an ID 50 of approximately 4 mM.

Regulation of Glycoalkaloid Accumulation in Potato Tuber Discs

The level of glycoalkaloids present in freshly cut potato tuber discs started to increase after 24 hours of incubation. This accumulation was inhibited by the sterol synthesis inhibitor, tridemorph, and was thus due to synthesis de novo. Concomitant to the accumulation of glycoalkaloids, there was an increase in the specific activity of a glycoalkaloid-specific enzyme, UDP-glucose:solanidine glucosyltransferase (solanidine-GT, EC 2.4.1). Time-course studies of enzyme activities in the discs showed that solanidine-GT activity increased at a low rate during the first 11 hours after slicing and at the highest rate between 11 and 16 hours. Other sterol-metabolizing enzymes exhibited different time-course curves:S-adenosyl-L-methionine:cycloartenol methyltransferase (cycloartenol-MT, EC 2.1.1.41) activity increased 5 hours after slicing and peaked at 11 hours. UDP-glucose:sterol glucosyltransferase (sterol-GT, EC 2.4.1) activity was not enhanced as a result of wounding. The accumulation of glycoalkaloids was not affected by light exposure or addition of abscisic acid, gibberellic acid or (2-chloroethyl)trimethylammonium chloride to the discs. Inhibition of ethylene synthesis or action gave a higher content of glycoalkaloids than in control discs, whereas discs incubated at high levels of ethylene had a very low glycoalkaloid content and also a lower activity of solanidine-GT than control discs.

Chitin-binding proteins in potato (Solanum tuberosum L.) tuber. Characterization, immunolocalization and effects of wounding.

Tubers of potato (Solanum tuberosum L.) contain a number of chitin-binding proteins which have possible functions in defence against pathogens. A major protein of the tuber is the chitin-binding lectin which has been further characterized with respect to its antigenicity and N-terminal amino acid sequence. By using an antiserum monospecific for tuber lectin in unwounded potato the protein was found in the cytoplasm and vacuole, unusually for a hydroxyproline-rich glycoprotein, but consistent with its soluble nature in subcellular extracts. Little increased synthesis of the lectin precursor or the post-translationally modified form could be demonstrated in excised potato tuber discs. However, after wounding there is increased synthesis of another hydroxyproline-containing glycoprotein of Mr 57,000, which binds to chitin and shares common epitopes with the lectin. In comparison with the tuber lectin, this novel glycoprotein contains less hydroxyproline, but from its overall composition it is clearly not an underhydroxylated form of the tuber lectin. It differed in its N-terminal amino acid sequence and was much less glycosylated, although arabinose was still present. Synthesis of the Mr-57,000 polypeptide began after the initial burst of protein synthesis and increased, reaching a peak at 24 h after wounding. The protein was produced with its enzymes of post-translational modification, prolyl hydroxylase and arabinosyltransferase, concomitantly with the marker enzymes for wounding, phenylalanine ammonia-lyase and membrane-bound phenol oxidase and peroxidase.

Expression of PVX coat protein gene under the control of extensin-gene promoter confers virus resistance on transgenic potato plants

Tuber discs of potato (Solanum tuberosum L.) cultivars Desirée and Gracia were infected by Agrobacterium tumefaciens carrying a binary vector with the coat protein gene of potato virus X controlled by the carrot extensin gene long-transcript promoter. Several transgenic potato plants have been obtained by direct regeneration of shoots on culture medium with kanamycin used for selection. The presence of the coat protein gene was proved by Southern hybridization in several transformants. Its low but detectable expression level was shown by Northern and Western analysis. Ethephon treatment resulted in a five-fold increase in the amounts of the coat protein mRNA. The majority of transformants exhibited reduced accumulation of virus RNA in inoculated leaves. Potentials in the use of an ethylene-inducible promoter in the production of virus-resistant transgenic plants will be discussed.

Oxazolomycin Esters, Specific Inhibitors of Plant Transformation

Oxazolomycin diacetate, dipropionate, monobutyrate and dibutyrate were derived from oxazolomycin, a product of Streptomyces sp. KBFP-2025. These esters were potent inhibitors of crown gall formation on potato tuber disks upon infection with Agrobacterium tumefaciens. They showed neither antibacterial nor phytotoxic activity, whereas oxazolomycin showed both antibacterial and phytotoxic activities. Further, they had no inhibitory activity against A. tumefaciens on the potato tuber disk. The inhibitory activity of these esters against crown gall formation seems to be due to specific inhibition of the transformation of plants with A. tumefaciens.

The use of a sterol inhibitor to investigate changes in the rate of synthesis of 2,3-oxidosqualene in elicitor-treated potato tuber tissue

U18666A an inhibitor of 2,3-oxidosqualene: cycloartenol cyclase, was used to investigate whether elicitation of sesquiterpenoid phytoalexins in potato tuber discs by arachidonic acid alters the in vivo flow of substrate through 2,3-oxidosqualene. The level of 2,3-oxidosqualene increased 10-fold in potato tuber discs treated with a 10·0 μ m concentration of U18666A. The same concentration of U18666A inhibited steroid glycoalkaloid accumulation by 90%, but had no effect on the accumulation rishitin and lubimin elicited by arachidonic acid. In potato tuber discs treated with U18666A, 2,3-oxidosqualene accumulated to lower levels in arachidonic acid-elicited potato tuber discs than in non-elicited discs. The application of arachidonic acid also decreased the incorporation of radioactivity from [ 3H]mevalonate into squalene and 2,3-oxidosqualene in potato tuber discs treated with U18666A. These data provide evidence that suppression of squalene synthetase activity accounts, at least in part, for the reduction of sterol biosynthesis which occurs during the elicitation of sesquiterpenoid phytoalexin accumulation in potato tuber discs.

Induction of sesquiterpene cyclase and suppression of squalene synthetase activity in elicitor-treated or fungal-infected potato tuber tissue

Arachidonic acid, a fungal elicitor from Phytophthora infestans, caused a 10-fold increase in sesquiterpene cyclase activity in potato tuber discs 36 h after application as compared to discs treated with water, whereas squalene synthetase activity increased 10-fold in discs treated with water 48 h after slicing. Arachidonic acid suppressed squalene synthetase activity in wounded potato tuber tissue 6 h after application, and the enzyme's activity remained low for the next 78 h. A similar elicitation of sesquiterpene cyclase activity and suppression of squalene synthetase activity was observed after inoculation with Helminthosporium carbonum, a non-pathogen of potato. There was a similar time course of induction of sesquiterpene cyclase activity after inoculation with either a compatible or incompatible race of Phytophthora infestans, whereas marked suppression of squalene synthetase activity occurred later after inoculation with a compatible race of P. infestans as compared to that which occurred after inoculation with an incompatible race. These results support the concept of co-ordinated regulation of sesquiterpene cyclase and squalene synthetase in potato during the elicitation of sesquiterpenoid phytoalexin accumulation.

Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome

picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract. Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus. Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus. This sequence, denoted pgl, has extensive homology to polygalacturonase genes from several organisms and inhibited the induction of the picA promoter when present in multiple copies in A. tumefaciens. DNA sequence analysis indicated at least two long open reading frames (ORFs) in the picA region. S1 nuclease mapping was used to identify the transcription initiation site of picA. Mutation of ORF1, but not ORF2, of the picA locus was responsible for an increased aggregation of A. tumefaciens, forming "ropes" in the presence of pea root cap cells. In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild-type control, a further indication that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant cell extracts.

Synergistic interactions between phenylamide fungicides and respiration inhibitors in controlling phenylamide-resistant populations ofPhytophthora infestans on potato tuber disks

The efficacy of mixtures of respiration inhibitors and phenylamide fungicides (oxadixyl and metalaxyl) in controlling late blight was investigated using potato tuber disks. Results showed that uncoupling agents(e.g. DNP), inhibitors of ATP formation(e.g. oligomycin), ionophores(e.g, valinomycin) and inhibitors that block specific carriers in the electron transport chain(e.g. rotenone, antimycin A) increased significantly the control efficacy of both metalaxyl and oxadixyl towards populations ofPhytophthora infestons when applied to tuber slices pretreated with sublethal doses of both fungicides. When applied alone at the same doses, respiration inhibitors did not inhibit the development of the late blight fungus. Increased control efficacy of mixtures was not noted against phenylamide-sensitive populations of the fungus.

Concurrent Synthesis and Degradation of Alcohol Dehydrogenase in Elicitor-Treated and Wounded Potato Tubers

The accumulation of alcohol dehydrogenase (ADH) in arachidonic acid-elicited potato (Solanum tuberosum L.) tuber discs was studied. In accordance with our previous report of the accumulation of Adh mRNA beginning 2 hours after elicitor treatment (DP Matton, CP Constabel, N Brisson [1990] Plant Mol Biol 14: 775-783), immunoprecipitation of ADH from in vivo labeled discs indicated that ADH synthesis occurred as early as 12 hours after treatment. However, levels of ADH activity and protein, as shown by enzyme assay and immunoblot, did not rise in parallel but decreased during the first 24 hours of treatment. After 24 hours, ADH activity and protein began to increase, reaching a several-fold increase at 96 hours after elicitation. Water-treated control discs showed a similar though delayed and less pronounced pattern. These results imply a turnover of ADH following elicitor treatment of potato tuber discs. As shown by nondenaturing gel electrophoresis, the synthesis and degradation involved the same ADH isozyme.

Alcohol dehydrogenase gene expression in potato following elicitor and stress treatment

A cDNA clone corresponding to a mRNA that rapidly accumulates during the hypersensitive-like response induced by elicitor treatment of potato (Solanum tuberosum L.) tuber was characterized. The clone encodes a polypeptide (Mr = 41,097) having 83%-85% amino acid identity with known plant alcohol dehydrogenase sequences (ADH; EC 1.1.1.1). The identity of the clone was confirmed by measuring the ADH enzyme activity in extracts of Escherichia coli transformed with the cDNA clone. In potato tuber disks, a wide range of stresses, including treatment with fatty acid elicitors, salicylic acid, UV light and anaerobiosis, was shown to induce accumulation of Adh transcripts. In stems, a high constitutive level of Adh transcripts could be detected in 4-week old plants, but not in 8-week old plants. However, the mRNA could be induced to accumulate in stems of 8-week old plants by treatment with arachidonic acid elicitor or by anaerobiosis. Induction in leaves was also obtained during anaerobiosis and after treatment with a Phytophthora infestans mycelial homogenate.

Nucleotide sequence of a pathogenesis-related gene of potato

Environmental stresses cause rapid and specific changes in plant metabolism. Among the various metabolic activities stimulated in potato during the defense response are that of enzymes required for phytoalexin synthesis [2], chitinases and 1,3-fl-glucanases [7], as well as phenylalanine ammonia-lyase and 4-coumarate CoA-ligase [3, 5]. Treatment of potato tuber disks with arachidonic or eicosapentaenoic acid elicits the accumulation of several mRNAs [8]. cDNA clones corresponding to two of these mRNAs (pSTH-2 and pSTH-21) were isolated and characterized [9]. They were found to accumulate in tubers upon wounding and elicitor treatments. pSTH-2 related mRNAs also accumulate in stems upon wounding and in potato and tomato leaves treated with a mycelial homogenate from the late blight fungus Phytophthora infestans [9]. Proteins encoded by these cDNAs (pSTH-2 and pSTH-21) were found to be highly similar to pea pathogenesis-related (PR) protein pi49 and pI 176 [4], to parsley PR protein PRI-1 and PR1-3 [ 10], and to Betvl, the major pollen allergen from white birch [ 1]. The two potato cDNA clones were used to screen a genomic library made in the vector 2 EMBL 3. One hybridizing clone was characterized and shown to contain a gene corresponding to pSTH-21. Its nucleotide sequence is presented in Fig. 1. The gene is made of two exons that are 100~o identical to the pSTH-21 cDNA clone, including the 5' and 3' untranslated regions [9]. The intervening sequence is 347 bp long and interrupts the coding region at amino acid 57. As in the case for many plant genes, no AAUAAA consensus polyadenylation signal sequence can be found in the vicinity of the poly(A) addition site [6]. Multiple transcription start sites have been identified by reverse transcription of polyadenylated RNA from elicitortreated tuber disks, using as a primer a synthetic oligonucleotide complementary to nucleotides 957 to 971. The fact that STH-21 is part of a multigene family [8, 9] could explain this result. Sequences homologous to TATA and CAAT boxes are located at position 22 (TATAAATA) and 117 (CAAAT), respectively, from the most upstream transcription start site. Identical sequences are also located 27 and 67/102bp upstream of the transcription start site of the parsley PRI-1 gene [10].

Wound-induced phenylalanine ammonia-lyase in potato (Solanum tuberosum) tuber discs. Significance of glycosylation and immunolocalization of enzyme subunits

1. Excised discs of potato (Solanum tuberosum) tuber were incubated with [3H]fucose and extracts were prepared and incubated with an antibody to phenylalanine ammonia-lyase. Analysis of the resulting immunoprecipitated proteins by SDS/PAGE showed [3H]mannose- and [3H]fucose-labelled bands with Mr values corresponding to those of phenylalanine ammonia-lyase subunits. 2. When potato discs were incubated with [3H]sugars in the presence of tunicamycin, an inhibitor of N-linked protein glycosylation, incorporation of radioactivity from [3H]mannose into the immunoprecipitated enzyme subunits was virtually eliminated, whereas that from [3H]fucose was only marginally inhibited. 3. Tunicamycin reduced the level of extractable phenylalanine ammonia-lyase activity induced in excised potato tuber discs. Kinetic analysis revealed that the Vmax value of the enzyme in crude extracts from tunicamycin-treated tissue was reduced, whereas the apparent Km values were unaffected. 4. Immunoprecipitation of the enzyme labelled in vivo with [35S]methionine showed that tunicamycin did not inhibit the synthesis of the enzyme protein per se, nor did it increase the degradation of the enzyme protein. 5. Immunoprecipitation of the enzyme labelled in vitro with [14C]nitromethane showed that tunicamycin did not affect the introduction of the dehydroalanine residue into the active site. 6. These results are consistent with the following hypothesis: tunicamycin inhibits the N-linked glycosylation of phenylalanine ammonia-lyase which, in turn, results in imperfect folding of the enzyme protein. The orientation of the active site is changed in such a way that the affinity of the enzyme for its substrate is unaffected, whereas the catalytic activity of the enzyme is reduced. 7. Both optical- and electron-microscopic immunolocalization studies with antibody to phenylalanine ammonia-lyase showed increased deposition of silver granules in cells in sections of potato discs in which induction of the enzyme was allowed to occur compared with cells from newly wounded tissue. The enzyme was located in the cytoplasm, and was possibly membrane-associated.

Mannose Feeding and Its Effect on Starch Synthesis in Developing Potato Tuber Discs

Le mannose augmente la capacite de disques de tubercules de pomme de terre a synthetiser et accumuler l'amidon a partir de saccharose 14 C. Le role du Pi dans la synthese d'amidon via le controle allosterique de l'ADP-glucose pyrophosphorylase est egalement apprehende

Differential ethylene production by potato tuber tissue inoculated with a compatible or an incompatible race ofPhytophthora infestans

Synthesis of ethylene in Kennebec potato tuber discs was measured at various time intervals after inoculation with zoospores of race 0 (incompatible) or race 1,2,3,4 (compatible) ofPhytophthora infestans (Mont.) de Bary. A burst of ethylene production was observed within 6 hr after inoculation with race 0, but not with race 1,2,3,4. This response may have been due to recognition of the incompatible race 0 during host penetration. Additional ethylene was synthesized concomitant with symptom appearance in both compatible and incompatible combinations.

The Genetic Engineering of Two Commercial Potato Cultivars for Resistance to Potato Virus X

We efficiently transformed the commercial cultivars of potato (Solanum tuberosum) Bintje, Desiree and Escort after optimizing the conditions for regeneration from potato tuber discs. For transformation, tuber discs were cocultivated with Agrobacterium tumefaciens using a disarmed binary vector system. This system allowed the introduction of a chimaeric gene encoding the coat protein (CP) of potato virus X (PVX) into two cultivars most susceptible to this virus, Bintje and Escort. Five transgenic plant lines with expression levels of CP higher than 0.1% of soluble leaf protein were analyzed for resistance to a challenging inoculation with PVX (1 μg/ml). We observed a delay in symptom development as well as a drastic reduction in the accumulation of virus. Furthermore, we found a correlation between the expression level of the CP–gene and the reduction in virus accumulation. Cytogenetic analysis of 62 independently obtained transgenic lines showed the normal tetraploid number of chromosomes (2n = 4x = 48) in 97 percent of the examined plants. Phenotypically all these plants appeared normal. One plant line exhibited an abnormal phenotype and contained about the octaploid number of chromosomes (4n ≈ 8x = 96). These results and preliminary data on morphological characteristics, as these are determined in the official variety registration procedure, indicate that potato cultivars can be genetically engineered to contain new desirable traits with preservation of their intrinsic properties.

Latency of Infection in Unripe Avocados by Colletotrichum gloeosporioides is Not Due to Inadequate Enzyme Potential

Abstract Colletotrichum gloeosporioides produced exo‐pectin lyase and protease in a) liquid cultures with incorporated washed cell wall material from unripe or ripe avocado and b) autoclaved immature fruit. The activity of exo‐pectin lyase and protease produced in liquid cultures incorporating washed cell walls from immature fruits was almost the same as when washed cell walls from ripe fruits were incorporated. Ripe fruit tissue rotted by the fungus contained exo‐pectin lyase, endo‐polygalacturonase (endo‐PG) and protease. The endo‐PG was found to be endogenous to avocado fruit, and had a pH optimum of 5.5. The pH optima of exo‐pectin lyase and protease were 8.5 and 7.5 respectively in all three enzyme preparations. All these enzyme preparations completely macerated avocado fruit tissue discs in vitro in less than 3 h of incubation but not potato tuber discs. Neither immature nor ripe fruit contained substances, proteinaceous or otherwise, which could inhibit the exo‐pectin lyase or protease activity of these preparations. The results indicated that C. gloeosporioides possesses sufficient enzyme potential to invade cell walls of unripe fruit and that the fruit tissue does not have a mechanism to inactivate such enzymes.

Osmotically-induced mass transfer in plant storage tissues: A mathematical model. Part II

In Part I of the present communication, a mathematical model incorporating cellular membrane characteristics along with other structural and biophysical features was proposed for the description of water and solute fluxes in complex cellular structures of plant storage tissues subjected to osmotic dehydration. This second part gives the results of comparative studies performed to validate the model. In addition the influence of various cell and tissue properties on the kinetics of mass transport occurring in discs of beet roots and potato tubers is presented. The simulations confirm that the cell membrane represents the major resistance to mass transfer in such systems.

Osmotically-induced mass transfer in plant storage tissues: A mathematical model. Part I

A mathematical model incorporating cell membrane characteristics for the simulation of water and solute fluxes in complex cellular structures is developed. The overall transport of material in the supporting matrix is considered using relationships associated with an extended form of the second-degree Fickian equation. The interaction of the cells with their immediate environment is described by transport relationships based on irreversible thermodynamics. Because of the complexity of biological tissue structures, a simplified geometrical analogue of the actual cellular matrix is used. This analogue represents the main features of the actual tissue, i.e. apoplastic and symplastic transport, cell volume changes, tissue shrinkage and internal volumetric rearrangements. The mathematical model has been used to simulate movement of water and diffusible permeating and ron-permeating solutes in discs of beet roots and potato tubers. The influence of various cellular and tissue properties on the dynamics of mass transport phenomena occurring in the cellular structures was studied. These simulations will be presented in Part II of this study.