Potato Tuber Discs Research Articles

Effect of methyl jasmonate on arachidonic acid-induced resistance of potato to late blight

Methyl ester of jasmonic acid (Me-JA) influences the induced resistance of potato tubers to late blight caused byPhytophthora infestans. Treatment of potato tuber disk surfaces with Me-JA solution or exposure to an atmosphere containing Me-JA vapors (10−6–10−5 M) increased the rate of rishitin biosynthesis induced by arachidonic acid orP. infestans. Methyl jasmonate increased the sensitivity of potato tissue to arachidonic acid. As a result, in the presence of Me-JA, the protective properties of arachidonic acid were observed at lower concentrations than in the absence of Me-JA. In addition, Me-JA reduced the adverse effects of lipoxygenase inhibitors (salicylhydroxamic acid and esculetin) on the induced resistance of potato tubers to late blight. Therefore, the synergistic interaction of Me-JA and biogenic elicitors can be regarded as part of a mechanism of potato defense against diseases.

Influence of Ageing and Abscisic Acid on Potassium Uptake by Potato Tuber Tissues

The potassium uptake by potato tuber discs tissues freshly cut and after 24 h of ageing in the presence or not of abscisic acid was investigated. Uptake kinetics revealed a biphasic dependence on external K+ concentrations. At concentration less than 10 mM, uptake was mediated by a saturable component and a linear component became apparent at higher concentrations. At low K+ concentrations (lmM), the capacity of K+ uptake diminished by 2 times after ageing. Treatment of tissues with ABA increased the rate of K+ uptake. In both fresh and aged tissues the uptake was strongly enhanced by fusicoccin and decreased by several metabolic inhibitors and ATPase inhibitors, underlying the active nature of uptake and suggesting the involvement of a plasmalemma H+-ATPase in K+ transport system.

Stimulation of Potassium Uptake by Abscisic Acid in Potato Tuber Tissues. Relation with H+ and Ca2+ Fluxes

Previous results with potato tuber discs showed that a treatment with abscisic acid stimulated K+ uptake. In this investigation, we determine the relationship between increased K' uptake and H+extrusion, and Ca2+ fluxes by treating tissues with specific Ca2+ channel blocker (La3+), calmodulin (CaM) inhibitors (chlorpromazine and W7), and with Ca2+ ionophore (A23187). K+ uptake increased with increasing external pH whether tissues were treated with ABA or not. Treatment of tissues with La3+ inhibited K+ uptake, whereas CaM inhibitors have no effect. By contrast ABA and A23187 produced a synergistic effect, suggesting that ABA may act in part, on K+ uptake, like a Ca2+ agonist, in accord with Huddart's hypothesis.

Comparison of Assays for Measuring Sensitivity of Phytophthora infestans Isolates to Fungicides.

Pathogenic strains of Phytophthora infestans insensitive to phenylamide have been reported from around the world and are responsible for a lack of late blight control in both potatoes and tomatoes. In vivo laboratory assay methods used to determine the sensitivity of P. infestans to fungicides include floating of leaflet disks on fungicide suspension and the use of potato tuber disks. In our study, these two methods were compared with a new detached-leaf assay. The fungicides chlorothalonil (protective), oxadixyl and metalaxyl (systemic), and cymoxanil (locally systemic) were utilized. Phenylamide-insensitive and -sensitive genotypes of P. infestans were used as inoculum. Significant differences between preventive and systemic fungicides were found, and all assay methods differentiated between phenylamide-sensitive and -insensitive genotypes of P. infestans. Differences among assays were significant when comparing mean inhibitory values. There was no significant difference between the detached leaf and the tuber disk assay, but both methods were significantly different from the floating-leaflet disk assay.

Effect of cadmium on glutathione reductase in potato tubers

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl2 changed glutathione reductase (GR) activity depending on cadmium ions concentrations, kind of tuber and time of incubation. The increase of GR activity at 10 and 100 µmol·dcm−3 of CdCl2 solutions was marked in less resistant tissues of cv. Bintje after 24 hrs, and was slight in more resistant tissues of cv. Bzura after 72 hrs. At 1 mmol·dcm−3 concentration of CdCl2 rapid and total inactivation in both kind of tissues was observed, which disappeared after a few days. However this elevation was faster in more resistant tissues. These inhibition effects come from the inactivation process of GR by cadmium. The values of KI for cadmium and KM for GSSG of GR from potato tuber tissues indicated that enzyme from more resistant tissues possessed lower affinity to toxic metal and higher affinity to substrate.

Induction of the Maize GapC4 Promoter in Transgenic Potato under Anaerobiosis and in Erwinia carotovora-Inoculated Tuber Tissue

The induction pattern of the GapC4 promoter from maize in transgenic potato has been analyzed by fusion to the β-glucuronidase (gus) gene. Under anaerobic conditions this promoter confers high level expression not only in leaves, stems, and roots but also in tubers. After inoculation of potato tuber disks with Erwinia carotovora subsp. atroseptica, β-glucuronidase (GUS) activity could be detected in macerated tissue as well as in surrounding intact tissue. In mock controls no induction was detected, ruling out any induction due to an overall limitation in oxygen in the experimental system. In addition, it could be proven that no diffusion of GUS protein from macerated into intact tissue occurred. The promoter was shown to be aerobically induced even in the absence of live bacteria by incubation with purified Erwinia spp. pectolytic enzymes alone. Therefore, promoter induction seems to be mediated by a mobile factor instead of by limitation in oxygen. These results demonstrate that the maize GapC4 promoter is suitable for directing foreign genes encoding antibacterial proteins in transgenic potato.

Partial characterization of potato F0F1‐ATPase Γ‐subunit cDNA and its regulation during fungal infection and elicitor treatment

Mitochondrial F0F1‐ATP synthase from dicotyledonous plants contains six different subunits named α, β, Γ, Δ, Δ′ and ɛ. A key subunit in the coupling mechanism is the Γ‐subunit, which appears as a single copy in the complex interacting with multiple α and β subunits that contain the catalytic sites. One 0.8‐kb potato cDNA clone sharing 88% homology with the 3′‐end of sweet potato Γ‐ATPase has been isolated by reverse transcriptase‐polymerase chain reaction (RT‐PCR) amplification from potato leaves infected with the pathogenic fungus Phytophthora infestans. Northern blot experiments revealed that the potato Γ‐subunit mRNA level increased 2‐ and 3‐fold in infected potato leaves 48 and 72 h after infection, respectively. The enzyme activity of F0F1‐ATPase was 2‐fold higher in 48‐h‐infected potato leaves than in mock‐inoculated ones. The fungal elicitors eicosapentanoic acid (EPA) and glucans from P. infestans cell wall were used in induction experiments on potato tuber discs. After 48 h of treatment, the Γ‐ATPase mRNA levels were respectively increased by 1.8, 1.4 and 2.2‐fold in EPA, glucans and EPA plus glucan treatments. The present results show that the Γ‐subunit mRNA levels of F0F1‐ATPase are up‐regulated during biological stress conditions and elicitor treatments in potato and correlate with the increase of respiration rate and metabolic activity of mitochondria observed during infection processes in plants.

Effect of cytochalasin D on defense responses of potato tuber discs treated with hyphal wall components of Phytophthora infestans

Cytoplasmic aggregation, the rapid translocation of cytoplasm and organelles to the site of fungal penetration of a cell, occurs in potato tissue after penetration of an incompatible race of Phytophthora infestans, the late blight fungus, or treatment with hyphal wall components (HWC) prepared from P. infestans. This phenomenon is thought to be one of the defense reactions of plant against pathogens, but the exact function of cytoplasmic aggregation is currently unknown. To evaluate the relationship between cytoplasmic aggregation and defense reactions, we suppressed the cytoplasmic aggregation by treatment with cytochalasin D, an inhibitor of the polymerization of actin, and examined the effect of inhibition on certain defense responses caused by HWC treatment. In potato tuber discs treated simultaneously with HWC and cytochalasin D, a delay in the induction of phenylalanine ammonia-lyase mRNA, decrease in the accumulation of PR (pathogenesis-related) proteins and partial suppression of cell death were observed, while the generation of superoxide anions was not affected. These results suggested that cytoplasmic aggregation was essential for the timely induction of defense reactions manifested at late resistant stage.

Plant regeneration from tuber discs of potato (Solanum tuberosum L.) using 6-benzylaminopurine (BAP)

Shoots, roots and callus were formed from tuber discs of potato, cultivar Desiree, when grown in vitro on the basal medium of Murashige & Skoog (1962) (MS) supplemented with 2,4-D and/or BAP. Callus was formed in MS medium with 1 mg l−1 BAP plus 0.5 mg l−1 2,4-D, callus and roots were formed in MS with 1 mg l−1 BAP plus more than 0.5 mg l−1 2,4-D and shoots were formed directly on tuber discs cultured on MS medium with 1 mg l−1 BAP without the addition of 2,4-D.

Anti-idiotypic antibodies can mimic the ability ofPhytophthora infestansto induce the accumulation of transcripts associated with defence responses in potato

The initial infection process involving adhesion/recognition events between plants and fungal pathogens is essential for the establishment of pathogenesis. The basis of the specificity betweenPhytophthora infestansand potato is not yet known. It could lie in the recognition between extracellular matrix and plasma membrane components of the penetrating structures of the fungus and host cells during the earliest stages of infection processes. We have used polyclonal antibodies (AZandAC) obtained after immunizing rabbits with either 107zoospores (Z) or germinating cysts (C) ofP. infestansto detect putative fungal components located on the surface of invasive structures.AZrecognized several bands, whileACrecognized three polypeptides of approximately 87, 73 and 45 kDa in western blots. Zoospore suspensions preincubated either withAZorACwere unable to infect potato leaves. Since it was already known that some anti-idiotypic (anti-IT)antibodies may resemble the antigen, thus mimicking its structure,anti-ITantibodies (a-AZanda-AC)were generated in mice and used as inducers on potato tuber discs. In northern blotsa-AZtreatments increased 1.5-fold the glyceraldehyde-3-P dehydrogenase (GAPDH) and 1.8-fold the phenylalanine ammonia-lyase (PAL) transcript levels, while an increase of 1.8 and 1.7-fold for GAPDH and PAL, respectively, was observed when the inducer wasa-AC.Both PAL and GAPDH transcript levels have previously been shown to be modulated by fungal and elicitor treatments. These results show that the anti-idiotypic antibodies are able to mimic the pathogen components, leading to the accumulation of transcripts related to defence responses in potato–P. infestansinteraction.

The Use of Biological Assays to Evaluate Botanicals

The Department of Medicinal Chemistry and Molecular Pharmacology’s laboratory has adopted four “bench top” bioassays which do not require higher animals to screen and direct the fractionation of botanical extracts in drug discovery efforts. These are: 1. The brine shrimp lethality test (BST) (a general bioassay), 2. The inhibition of crown gall tumors on discs of potato tubers (an antitumor bioassay), 3. The inhibition of frond proliferation in duckweed (a bioassay for herbicides and plant growth stimulants), and 4. The yellow fever mosquito larvae lethality test (a bioassay for pesticides). The materials and procedures for those bioassays will be briefly described. The authors’ results in applying these simple methods in the discovery of Annonaceous acetogenins will be presented to illustrate their successful use. The BST is especially suggested as an inexpensive, simple, and rapid means of standardization of bioactivity in heterogeneous botanical products.

Induction of <i>N</i>-<i>p</i>-Coumaroyloctopamine in Potato Tuber Disks by β-1, 3-Glucooligosaccharide

ジャガイモ疫病菌由来の菌糸細胞壁成分で処理したジャガイモ塊茎ディスクに誘導されるフェノール性化合物として, N-p-クマロイルオクトパミンを同定した. 本化合物は, ディスクを褐藻類由来の市販β-1,3-グルコオリゴサッカライドであるラミナリンで処理した際にも, 濃度依存的に誘導された. 誘導量の最大レベルは組織重1gあたり約150μg (約500ナノモル) に及んだ. またラミナリンより誘導活性は劣るものの, β-1,3-グルコヘプタサッヵライド (7量体) に有意な活性が認められた. 一方, これまでにジャガイモでエリシター活性が認められているアラキドン酸は, 本アミド化合物の誘導に関してはあまり有効ではなかった. 以上のようなジャガイモ組織の反応は, 植物に対し代謝調節活性を示すオリゴ糖類の作用機構の解析に役立つものと期待される.

Nitrogen recycling during phenylpropanoid metabolism in sweet potato tubers

In the first step of the phenylpropanoid metabolic pathway, L-phenylalanine (L-Phe) is deaminated to form E-cinnamate, in a conversion catalyzed by phenylalanine ammonia-lyase (PAL; EC 4.3.1.5). The metabolic fate of the ammonium ion (NH4+) produced in this reaction was investigated in sweet potato (Ipomoea batatas) tuber discs. [15N]-Labeled substrates including L-Phe, in the presence or absence of specific enzyme inhibitors, were administered to sweet potato discs in light under aseptic conditions. 15N-Nuclear magnetic resonance spectroscopic analyses revealed that the 15NH4+ liberated during the PAL reaction is first incorporated into the amide nitrogen of L-glutamine (L-Gln) and then into L-glutamate (L-Glu). These results extend our previous observations in pine and potato that PAL-generated NH4+ is assimilated by the glutamine synthetase (GS; EC 6.3.1.2)/glutamate synthase (GOGAT; EC 1.4.1.13) pathway, with the NH4+ so formed ultimately being recycled back to L-Phe via L-Glu as aminoreceptor and donor.

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.

The Appearance of Metalaxyl-Resistant Isolates of Phytophthora infestans in Delaware.

Prior to 1995, late blight of potatoes (Solanum tuberosum L.) caused by Phytophthora infestans (Mont.) de Bary had not been reported from Delaware for decades. In that year, the fungus was isolated from potato foliage collected from fields in the Dover area that sustained severe foliage loss due to the disease. To establish pathogenicity, potato and tomato plants grown in the greenhouse were inoculated with the isolates obtained and Koch's postulates were completed. Analyses to determine mating type, genotype at two allozyme loci (2), Gpi and Pep, and sensitivity to metalaxyl were also completed. Measurements were made every 5 days and EC50 values were calculated for radial mycelial growth on metalaxyl-amended agar, for leaflets inoculated with P. infestans floated in metalaxyl solutions, and for inoculated potato tuber disks deposited on filter paper saturated with metalaxyl (1). Isolates were pathogenic to potatoes and tomatoes, were A2 mating type with Gpi 100/111/122 and Pep 100/100 (same as US-8), and displayed resistance to metalaxyl up to 140 mg/l.

First Report from Morocco of Phytophthora infestans Isolates with Metalaxyl Resistance.

Late blight of potato (Solanum tuberosum L.) caused by Phytophthora infestans (Mont.) de Bary first appeared in Africa in 1941. It has been observed sporadically in Morocco for decades but only recently became a major problem. Significant losses have been recorded in the last two growing seasons in spite of the use of various disease control programs that included combinations of systemic and protectant fungicides. Phytophthora infestans was cultured from diseased foliage collected from commercial potato fields near Larache, Morocco. Isolates were analyzed to determine pathogenicity on several potato and tomato cultivars, mating type, genotype at two allozyme loci (2), and relative sensitivity to metalaxyl. Responses of the isolates to metalaxyl were assayed by mycelial radial growth on metalaxyl-amended agar, by floating leaves inoculated with P. infestans on metalaxyl solutions, and via potato tuber disks placed on filter paper saturated with metalaxyl solutions (1). Koch's postulates were completed; all isolates were pathogenic to potato and tomato cultivars tested, are consistent with the A1 mating type, and have the same allozyme pattern (Gpi 100/100, Pep 92/100) as US-6 genotype. All tests indicated resistance to metalaxyl up to 250 ppm.

A functional homolog of mammalian protein kinase C participates in the elicitor-induced defense response in potato.

The elicitor-induced activation of the potato pathogenesis-related gene PR-10a is positively controlled by a protein kinase(s) that affects the binding of the nuclear factors PBF-1 (for PR-10a binding factor-1) and PBR-2 to an elicitor response element (ERE). In this study, we have identified a kinase that has properties similar to the conventional isoenzymes of the mammalian protein kinase C (PKC) family. the treatment of potato tuber discs with specific inhibitors of PKC abolished the elicitor-induced binding of the nuclear factor PBF-2 to the ERE. This correlated with a reduction in the accumulation of the PR-10a protein. In contrast, treatment of the tuber discs with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, led to an increase in binding of PBF-2 to the ERE and the corresponding increase in the level of the PR-10a protein, mimicking the effect seen with the elicitor arachidonic acid. Biochemical characterization of proteins extracted from the particulate fraction of potato tubers demonstrated that a kinase belonging to the conventional isoforms of PKC is present. This was confirmed by immunoprecipitation with antibodies specific to the conventional isoforms of human PKC and in-gel kinase assays. The ability of the immunoprecipitates to phosphorylate the alpha-peptide (a PKC specific substrate) in the presence of the coactivators calcium, phosphatidylserine, and TPA strongly suggested that the immunoprecipitated kinase is similar to the kinase characterized biochemically. Finally, the similar effects of the various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato.

Metabolic Control Analysis of glycolysis in tuber tissue of potato (Solanum tuberosum): explanation for the low control coefficient of phosphofructokinase over respiratory flux.

We have applied Metabolic Control Analysis (MCA) in an attempt to determine the distribution of glycolytic flux control between the steps of glycolysis in aged disks of potato tuber under aerobic conditions, using concentrations of glycolytic metabolites in tuber tissue from a range of transgenic potato plants and published enzyme kinetic data. We modelled the substrate and effector kinetics of potato tuber phosphofructokinase (PFK) by reanalysing published results. Despite the scarcity of reliable kinetic data, our results are in agreement with experimental findings namely that, under the conditions described, PFK has little control over glycolytic flux. Furthermore our analysis predicts that under these conditions far more control lies in the dephosphorylation of phosphoenolpyruvate and/or in the steps beyond. We have validated the results of our analysis in two ways. First, predictions based on calculated concentration control coefficients from the analysis show generally good agreement with observed metabolite deviation indices discussed in the preceding paper [Thomas, Mooney, Burrell, and Fell (1997) Biochem. J. 322, 111-117]. Second, sensitivity analysis of our results shows that the calculated control coefficients are robust to errors in the elasticities used in the analysis, of which relatively few need to be known accurately. Experimental and control analysis results agree with previous predictions of MCA that strong co-operative feedback inhibition of enzymes serves to move flux control downstream of the inhibiting metabolite. We conclude that MCA can successfully model the outcome of experiments in the genetic manipulation of enzyme amounts.

Ultrasound-mediated stable transformation of potato tuber discs

Plasmid DNA (pBARGUS), containing the selectable bar gene for resistance to the herbicide Basta, was delivered into potato tuber discs via ultrasonication. Transformed plants were identified by their ability to grow on a medium containing 1mg phosphinothricin/l. Southern hybridization and plant resistance to the application of Basta indicated that a functional bar gene had integrated into potato chromosomal DNA.

Purification and characterization of hydroxycinnamoyl-coenzyme A: ?-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for ω-hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to ω-hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of ω-hydroxy fatty acids which are formed in vitro.