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.     

Effect of Mastoparan on Phospholipase A2 Activity in Potato Tubers Treated with Fungal Elicitor

Participation of phospholipase (PL) A₂ in signal trans-ductionhas been reported to elicitate a resistance reaction in potatocells by inoculation of an incompatible race of Phytophthorainfestans, the late blight fungus, or by treatment with fungalelicitor hyphal wall components (HWC). Mastoparan, a genericG protein activator, has been shown to activate PLA in a G protein-dependentmanner in animal cells. We analyzed the effects of mastoparanand the inactive analog Mas-17 on PLA₂ activity in potato tubers.In healthy potato tubers, the activation of PLA₂ by mastoparanwas detected in the soluble fraction, but not micro-somal fraction.However, in potato tubers treated with HWC, PLA₂ activity wasstimulated by mastoparan in both soluble and microsomal fractions.Pretreatment of the microsomal fraction with neomycin, a PLCinhibitor, and staurosporine, a protein kinase inhibitor, inhibitedthe mastoparan-induced activation of PLA₂. This suggested thatthe PLA₂ activation in potato tubers by mastoparan was mediatedby the PLC pathway and protein phospho-rylation. We also examinedthe accumulation of potato phytoalexin rishitin. Mastoparanstimulated rishitin accumulation induced by HWC, but did notinduce the accumulation. This indicated that mastoparan mightactivate the signal transduction pathway in the resistance reactionsinduced in potato tubers. (Received March 12, 1998; Accepted August 6, 1998)     

Sucrose increases calcium-dependent protein kinase and phosphatase activities in potato plants.

The effect of sucrose on tuber formation, calcium-dependent protein kinase (CDPK) and phosphatase activities was analysed using in vitro cultured potato plants. In short treatments, sucrose induced CDPK and phosphatase activities. In long treatments, sucrose induced tuber formation in the absence of other tuber inducing stimuli. Sorbitol caused a minor increase in CDPK activity and affected plant morphology but did not induce tuber development. The addition of the protein kinase inhibitor Staurosporine precluded sucrose-induced tuberization. Altogether, our results suggest that phosphorylation/dephosphorylation events are involved in sucrose-induced tuber development.     

Calcium-dependent Protein Kinases are Involved in Potato Signal Transduction in Response to Elicitors from the Oomycete Phytophthora infestans

Plant response to pathogens involves an intricate network of signal transduction pathways. Here, potato cell cultures were used to study signal transduction in response to elicitors from Phytophthora infestans. Pretreatment of cells with Ser/Thr protein kinase inhibitors, EGTA, calmodulin antagonists or a channel blocker abolished the induction of two enzymes involved in defence responses, phenylalanine ammonia‐lyase (PAL) and peroxidase. Phosphatase inhibitors caused an increase of these activities in the absence of elicitors. Hyphal cell wall components (HWC) from an incompatible race (HWC 0) produced a rapid and transient increment of histone phosphorylation, whereas induction by HWC from a compatible race (HWC C) was less pronounced and more sustained. As activities were calcium‐dependent, a fraction enriched in calcium‐dependent protein kinases (CDPKs) was obtained by DEAE chromatography. Fractions from HWC 0‐ and HWC C‐treated cells presented higher kinase activity than that from untreated cells. Moreover, total activity was higher in the incompatible than in the compatible interaction. Activity was calcium‐dependent, partially inhibited by calmodulin antagonists and able to phosphorylate syntide‐2, a specific substrate of CDPKs. An in‐gel kinase assay showed the presence of a band of approximately 50 kDa whose activity was higher in HWC 0‐ than in HWC C‐treated cells and was not detected in control extracts. This report presents evidences of the differential activation of CDPKs in response to elicitors from different races of P. infestans, revealing that these protein kinases participate in the defence response to oomycete.     

Cloning, expression, and sequence conservation of pathogenesis-related gene transcripts of potato

Treatment of potato tuber disks with arachidonic acid elicits the accumulation of several mRNAs. cDNA clones corresponding to two of these mRNAs were isolated and characterized. Nucleotide sequence analysis reveals that both clones (pSTH-2 and pSTH-21) contain an open-reading frame coding for a 155-amino acid polypeptide. The polypeptides encoded by the two clones differ by only six amino acids and show a high degree of similarity with PR protein sequences from pea (approximately 42%) and parsley (approximately 37%). mRNAs corresponding to the two potato cDNA clones also accumulate in Solanum chacoense and in tomato following elicitor treatment. Maximum accumulation of the mRNAs corresponding to the two cDNA clones is reached 24 hr after elicitor treatment of the tuber disks. pSTH-2-related mRNAs also accumulate in tubers after wounding or treatment with eicosapentaenoic acid and are detected in potato and tomato leaves treated with a Phytophthora infestans mycelium homogenate. The presence of these conserved genes in species from three plant families and the similarity of their induction pattern suggest an important function during the plant defense response.     

Relationship among lipoperoxides, jasmonates and indole-3-acetic acid formation in potato tuber after wounding

Plant responses to biotic and abiotic stress can be mediated by oxidised products and in this study we analysed the relation among some of them and the growth factor indole-3-acetic acid (IAA). The plant material used was potato tuber sliced below bud and incubated for different lengths of time before analysis. Wounding in potato tuber leads, in a very short time (0-30 min), to the generation of lipid hydroperoxides (LOOH) from polyunsaturated fatty acids (PUFA). These reactive species could cause a subsequent increase of 9 and 13-lipoxygenase (LOX, E.C.1.13.12.12.), analysed by RT-PCR and spectrophotometric assay, LOOH, Jasmonates and IAA all quantified by GC-MS analysis. The activation of 9 and 13-LOX, using different timing, leads to the formation of LOOH with a subsequent generation of jasmonates and IAA as highlighted by the addition on the potato tuber slices of salicylhydroxamic acid (SHAM), an inhibitor of LOX activity. A correlation between jasmonates and IAA resulted by testing their reciprocal influence during wounding in potato tuber. The relationship occurring among each hormone analysed during wounding underlines the fact that the jasmonates level can be regulated in situ and this can suggest a role for these compounds in potato tuber which has been underestimated up to now.     

Relationship among lipoperoxides,jasmonates and indole-3-acetic acid formation in potato tuber after wounding

Plant responses to biotic and abiotic stress can be mediated by oxidised products and in this study we analysed the relation among some of them and the growth factor indole-3-acetic acid (IAA). The plant material used was potato tuber sliced below bud and incubated for different lengths of time before analysis. Wounding in potato tuber leads, in a very short time (0–30?min), to the generation of lipid hydroperoxides (LOOH) from polyunsaturated fatty acids (PUFA). These reactive species could cause a subsequent increase of 9 and 13-lipoxygenase (LOX, E.C.1.13.12.12.), analysed by RT-PCR and spectrophotometric assay, LOOH, Jasmonates and IAA all quantified by GC–MS analysis. The activation of 9 and 13-LOX, using different timing, leads to the formation of LOOH with a subsequent generation of jasmonates and IAA as highlighted by the addition on the potato tuber slices of salicylhydroxamic acid (SHAM), an inhibitor of LOX activity. A correlation between jasmonates and IAA resulted by testing their reciprocal influence during wounding in potato tuber. The relationship occurring among each hormone analysed during wounding underlines the fact that the jasmonates level can be regulated in situ and this can suggest a role for these compounds in potato tuber which has been underestimated up to now.     

Expression and activation of RAS and mitogen-activated protein kinases in macrophages treated in vitro with cisplatin: regulation by kinases, phosphatases and Ca2+/calmodulin.

Cisplatin (cis-dichlorodiammineplatinum II), a potent antitumour compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the exact mechanism by which cisplatin activates these cells is poorly characterized and attempts are being made to understand this mechanism. Previous studies from this laboratory have shown that Lyn, a protein tyrosine kinase of the src family, and nuclear factor (NF)-kappaB are involved in cisplatin-induced macrophage activation. Recent studies suggest that the RAS and mitogen-activated protein (MAP) kinases function as a connecting link between activated lyn and NF-kB, which raises the possibility of their involvement in cisplatin-induced macrophage activation. Therefore, this study was undertaken to investigate the effect of cisplatin treatment on the expression/activation of RAS (a low molecular weight GTP-binding protein) and MAP kinases in murine peritoneal macrophages. The underlying mechanism of expression/activation of RAS and MAP kinases in cisplatin-treated macrophages was also investigated. Immunoblotting and immune-complex kinase assays revealed that cisplatin treatment of macrophages leads to increased expression/activation of RAS and MAP kinases, with optimal expression/activation at 15 min of treatment. Using a battery of specific inhibitor/modulators of different signalling molecules, this study shows that expression and activation of MAP kinases are two unrelated processes. It was also observed that kinase (protein tyrosine and protein kinase C) inhibitor and Ca2+/calmodulin antagonist inhibit expression/activation of RAS/MAP kinases in macrophages, whereas phosphatases (protein tyrosine and serine/threonine) inhibitor up-regulate these kinases.     

p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.     

Molecular cloning and analysis of four potato tuber mRNAs

Tuberization in potato is a complex developmental process involving the expression of a specific set of genes leading to the synthesis of tuber proteins. We here report the cloning and analysis of mRNAs encoding tuber proteins. From a potato tuber cDNA library four different recombinants were isolated which hybridized predominantly with tuber mRNAs. Northern blot hybridization experiments showed that three of them, pPATB2, p303 and p340, can be regarded as tuber-specific while the fourth, p322, hybridizes to tuber and stem mRNA. Hybrid-selected in vitro translation and nucleotide sequence analysis indicate that pPATB2 and p303 represent patatin and the proteinase inhibitor II mRNA respectively. Recombinant p322 represents an mRNA encoding a polypeptide having homology with the soybean Bowman-Birk proteinase inhibitor while p340 represents an mRNA encoding a polypeptide showing homology with the winged bean Kunitz trypsin inhibitor. In total, these four polypeptides constitute approximately 50% of the soluble tuber protein. Using Southern blot analysis of potato DNA we estimate that these mRNAs are encoded by small multigene families.     

Differential formation of octadecadienoic acid and octadecatrienoic acid products in control and injured/infected potato tubers

Lipoxygenases in plants have been implicated in the activation of defense responses against injury/infection. Pathogen-derived polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and their metabolites have been shown to elicit defense responses against pathogen infection in plants. However, not much is known about the role of host-derived fatty acids and their metabolites in plant defense responses. In this study, isolation and characterisation of endogenous lipoxygenase metabolites formed in potato tubers in response to injury/infection was undertaken. While 9-hydroperoxyoctadecadienoic acid (9-HPODE), derived from octadecdienoic acid (linoleic acid) is the major lipoxygenase product formed in control potato tubers, 9-hydroperoxyoctadecatrienoic acid (9-HPOTrE), derived from octadecatrienoic acid (alpha-linolenic acid) is the major lipoxygenase product formed in potato tubers in response to injury or infection with Rhizoctonia bataticola. As a result, the relative ratio of 9-HPODE to 9-HPOTrE showed a shift from 4:1 in control to 1:2 and 1:4.5 in injured and infected potato tubers respectively. From this study, it is proposed that lipoxygenase metabolites of octadecadienoic acid may be involved in physiological responses under control conditions, while octadecatrienoic acid metabolites are mediating the defense responses. This forms the first report on the differential formation of endogenous lipoxygenase products in potato tubers under control and stress conditions.     

Identification of Chitinase and Osmotin-Like Protein as Actin-Binding Proteins in Suspension-Cultured Potato Cells

Cytoplasmic aggregation is an early resistance-associated eventthat is observed in potato tissues either after penetrationof an incompatible race of Phytophthora infestans, the potatolate blight fungus, or after treatment with hyphal wall components(HWC) prepared from P. infestans. In potato cells in suspensionculture, the number of cells with cytoplasmic aggregation increasedupon treatment with HWC, but such an increase was suppressedby treatment with cytochalasin D prior to treatment with HWC.This result suggested that cytoplasmic aggregation in culturedpotato cells might be connected with the association of actinfilaments. To identify the molecular basis of cytoplasmic aggregation,we purified actin and actin-related proteins by affinity chromatographyon a column of immobilized DNase I from cultured potato cellsand isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysisof the amino-terminal amino acid sequences indicated that the43 kDa, 32 kDa and 22 kDa proteins were potato actin, basicchitinase and osmotin-like protein, respectively. This conclusionwas supported by the results of Western blotting analysis ofthe 43 kDa and 32 kDa proteins with antibodies against actinand basic chitinase. Binding analysis with actin coupled toactin-specific antibodies and biotinylated actin suggested thatthe 32 kDa and 22 kDa proteins had actin-binding activity. Inaddition, examination of biomolecular interactions using anoptical biosensor confirmed the binding of chitinase to actin.These results imply the possibility that basic chitinase andosmotin-like protein might be involved in cytoplasmic aggregation,hereby participating in the potato cell's defense against attackby pathogen. (Received June 11, 1996; Accepted January 27, 1997)     

Proteomic analysis of the potato tuber life cycle

The tuber of potato (Solanum tuberosum) is commonly used as a model for underground storage organs. In this study, changes in the proteome were followed from tuberization, through tuber development and storage into the sprouting phase. Data interrogation using principal component analysis was able to clearly discriminate between the various stages of the tuber life cycle. Moreover, five well-defined protein expression patterns were found by hierarchical clustering. Altogether 150 proteins showing highly significant differences in abundance between specific stages in the life cycle were highlighted; 59 of these were identified. In addition, 50 proteins with smaller changes in abundance were identified, including several novel proteins. Most noticeably, the development process was characterized by the accumulation of the major storage protein patatin isoforms and enzymes involved in disease and defense reactions. Furthermore, enzymes involved in carbohydrate and energy metabolism and protein processing were associated with development but decreased during tuber maturation. These results represent the first comprehensive picture of many proteins involved in the tuber development and physiology.     

Endogenous enzyme reactions closely related to photon emission in the plant defense response

Lipoxygenase (LOX) and peroxidase (POD) reactions, which are involved in the production of reactive oxygen and radical species, are shown to be associated with ultraweak photon emission in plant defense mechanisms. These enzyme reactions induced high-level ultraweak photon emission in an in vitro reaction system. The application of LOX to sweet potato slices caused photon emission directly in plants. LOX substrate promoted photon emission in chitosan-treated sweet potato, and LOX inhibitor markedly suppressed this emission. Therefore, a LOX-related pathway, including LOX and other downstream reactions, is principally associated with photon emission in plant defense mechanisms.     

Alternaric acid stimulates phosphorylation of His-tagged RiCDPK2, a calcium-dependent protein kinase in potato plants

Calcium-dependent protein kinases (CDPK) are an essential component of plant defense mechanisms against pathogens. We investigated the effect of alternaric acid, a host-specific toxin produced by the plant fungal pathogen Alternaria solani (Pleosporaceae), on a putative plasma membrane and cytosolic kinase RiCDPK2 of potato (Solanum tuberosum) and on hypersensitive cell death of host potato cells. Alternaric acid, in the presence of Ca(2+) and Mg(2+), stimulated in vitro phosphorylation of His-tagged RiCDPK2, a Ca(2+)-dependent protein kinase found in potato plants. We concluded that Ca(2+) and Mg(2+) play an important role in the interaction between alternaric acid and RiCDPK2. Based on our observations, alternaric acid regulates RiCDPK2 kinase during the infection process in an interaction between host and A. solani, leading to the inhibition of hypersensitive cell death in the host. We suggest that alternaric acid is a primary determinant by which A. solani stimulates CDPK activity in the host, suppressing hypersensitive cell death.     

Glucose and Stress Independently Regulate Source and Sink Metabolism and Defense Mechanisms via Signal Transduction Pathways Involving Protein Phosphorylation

In higher plants, sugars are required not only to sustain heterotrophic growth but also to regulate the expression of a variety of genes. Environmental stresses, such as pathogen infection and wounding, activate a cascade of defense responses and may also affect carbohydrate metabolism. In this study, the relationship between sugar- and stress-activated signal transduction pathways and the underlying regulatory mechanism was analyzed. Photoautotrophically growing suspension culture cells of Chenopodium rubrum were used as a model system to study the effects of the metabolic regulator D-glucose and of different stress-related stimuli on photosynthesis, sink metabolism, and defense response by analyzing the regulation of mRNAs for representative enzymes of these pathways. Glucose as well as the fungal elicitor chitosan, the phosphatase inhibitor endothall, and benzoic acid were shown to result in a coordinated regulatory mechanism. The mRNAs for phenylalanine ammonia-lyase, a key enzyme of defense response, and for the sink-specific extracellular invertase were induced. In contrast, the mRNA for the Calvin cycle enzyme ribulose bisphosphate carboxylase was repressed. This inverse regulatory pattern was also observed in experiments with wounded leaves of C. rubrum plants. The differential effect of the protein kinase inhibitor staurosporine on mRNA regulation demonstrates that the carbohydrate signal and the stress-related stimuli independently activate different intracellular signaling pathways that ultimately are integrated to coordinately regulate source and sink metabolism and activate defense responses. The various stimuli triggered the transient and rapid activation of protein kinases that phosphorylate the myelin basic protein. The involvement of phosphorylation in signal transduction is further supported by the effect of the protein kinase inhibitor staurosporine on mRNA levels.