A Cytosolic Phospholipase A2 from Potato Tissues Appears to Be Patatin

Phospholipase (PL) A₂ is involved in signal transduction inthe resistance reaction that is induced in potato by inoculationof an incompatible race of Phytophthora infestans, the lateblight fungus, or by treatment with fungal elicitor hyphal wallcomponents (Kawakita et al. 1993). In this study, PLA₂ in thesoluble fraction from potato tuber was purified. The followingresults suggested that the enzyme was, in fact, patatin: (1)the molecular mass of the purified enzyme was 40 kDa, the sameas that of patatin; (2) the pI of the purified enzyme was approximately4.75, which corresponds to that of patatin; and (3) the amino-terminalamino acid sequence of the purified enzyme showed a high degreeof homology to that of patatin. Patatin is known as a storageprotein of the potato tuber and it has been shown to have esteraseactivity. However, other enzymatic activities and the function(s)of patatin are unknown. We investigated the PLA activities ofthe purified patatin. The PLA₂ activity of the patatin was muchhigher than the PLA₁ activity, even though the protein exhibitedboth activities. The PLA₂ activity of the enzyme was particularlyapparent when phosphatidylcholine with linoleic acid at thesn-2 position was used as substrate. Lower activity was observedwith phosphatidylcholine with palmitic acid, oleic acid andarachidonic acid at the sn-2 position. (Received October 5, 1995; Accepted February 9, 1996)     

Thrombin activates a membrane-associated calcium-independent PLA2 in ventricular myocytes

Activation of phospholipaseA₂(PLA₂) and accumulation oflysophosphatidylcholine contribute importantly to arrhythmogenesis during acute myocardial ischemia. We examined thrombinstimulation of PLA₂ activity inisolated ventricular myocytes. Basal and thrombin-stimulated cardiacmyocyte PLA₂ activity demonstrateda distinct preference for sn-1ether-linked phospholipids with arachidonate esterified at thesn-2 position. The majority ofPLA₂ activity was calcium independent and membrane associated. Thrombin stimulation ofmembrane-associated PLA₂ occurs ina time- and concentration-dependent fashion. An increase inPLA₂ activity was also observedusing the synthetic peptide SFLLRNPNDKYEPF (the tethered ligandgenerated by thrombin cleavage of its receptor). Bromoenol lactone, aselective inhibitor of calcium-independentPLA₂, completely blockedthrombin-stimulated increases inPLA₂ activity and arachidonic acidrelease. No significant inhibition of thrombin-inducedPLA₂ was observed followingpretreatment with mepacrine or dibucaine. These data confirm thepresence of high-affinity thrombin receptors on isolated cardiacmyocytes and demonstrate the specific activation of a uniquemembrane-associated, calcium-independentPLA₂ following thrombin receptorligation.

     

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

Biological activities of rishitin, an antifungal compound isolated from diseased potato tubers, and its derivatives

Rishitin, a norsesquiterpene alcohol, found in infected, resistantpotato-tuber tissue completely inhibited zoospore germinationand germtube elongation of Phytophthora infestans (MONT.) DEBARY at 10^–3M. There was little difference in sensitivityto rishitin among races of Phytophthora infestans. IAA-inducedelongation of Avcna coleoptile sections and GA₃-induced elongationof wheat leaf sections were also inhibited by rishitin. Theinhibition of IAA-induced elongation of Avena coleoptiles wasrelieved to some extent by increasing IAA concentration. However,little relief of the inhibition of GA₃-induced elongation ofwheat leaf sections was obtained by increasing GA₃ concentration.No plant injury was observed at this concentration of rishitin(10^–3M). Examination of a series of rishitin derivatives indicated thatthe hydroxyl group at C-3 is indispensable for antifungal activity.This activity was intensified by saturating the double bondbetween the rings of rishitin and/or that of the isopropenylgroup at C-7, though activity decreased when oxygenated functionalgroups were introduced into the side chain. Aromatization of the A ring did not lower biological activities.The antifungal activities of most rishitin derivatives almostparalleled their activities as plant growth retardants. However,some compounds without antifungal activity were active as growthretardants. ¹Studies on the phytoalexins (5). (Received August 14, 1968; )     

Redistribution and abnormal activity of phospholipase A(2) isoenzymes in postinfarct congestive heart failure

Cardiacsarcolemmal (SL) cis-unsaturated fatty acid sensitivephospholipase D (cis-UFA PLD) is modulated by SLCa²⁺-independent phospholipase A₂(iPLA₂) activity via intramembrane release ofcis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca²⁺ concentration and contractileperformance of the cardiomyocyte, changes in iPLA₂ activitymay contribute to abnormal function of the failing heart. We examinedPLA₂ immunoprotein expression and activity in the SL andcytosol from noninfarcted left ventricular (LV) tissue of rats in anovert stage of congestive heart failure (CHF). Hemodynamic assessmentof CHF animals showed an increase of the LV end-diastolic pressure withloss of contractile function. In normal hearts, immunoblot analysisrevealed the presence of cytosolic PLA₂ (cPLA₂)and secretory PLA₂ (sPLA₂) in the cytosol, withcPLA₂ and iPLA₂ in the SL. IntracellularPLA₂ activity was predominantly Ca²⁺independent, with minimal sPLA₂ activity. CHF increasedcPLA₂ immunoprotein and PLA₂ activity in thecytosol and decreased SL iPLA₂ and cPLA₂immunoprotein and SL PLA₂ activity. sPLA₂activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA₂isoenzymes, suggesting that PLA₂-mediated bioprocesses arealtered in CHF.

     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

Tuberization in Potato at High Temperatures: Gibberellin Content and Transport from Buds

Warm temperatures (35°C day/30°C night) which inhibittuberization in potato (Solanum tuberosum L., cv. Sebago) increasedgibberellin activity in crude extracts from buds, but not frommature leaves, as determined by the lettuce hypocotyl bioassay.Changes in the growth of tubers and stolons indicate the occurrenceof basipetal movement of GA₃ applied to the terminal bud ora mature leaf. ¹⁴C labelling from GA₃ or mevalonic acid injectedjust below the terminal bud was recovered in the lower shoot,stolons and tubers, but the amount transported was greater atcool temperatures (20/15°C). It is concluded that high temperaturespromote the synthesis of gibberellin in the buds rather thantransport to the stolons. Solanum tuberosum L., potato, tuberization, gibberellin     

Effects of enzymatically digested microsome fractions on red-light-enhanced pelletability of pea phytochrome in vitro in the presence of calcium ion

The 130,000 ?g supernatant of Sephadex G-50 filtrate and a 78,000?g microsomal fraction were prepared separately from homogenatesof etiolated pea (Pisum sativum L. cv. Alaska) shoots. The pelletabilityof phytochrome increased ca. 10-fold by exposure of the filtrateto red light and mixing the filtrate with the microsomal fractionin the dark at ca. 0?C. The increase of pelletable phytochromewas inhibited by 84% when the microsomal fraction digested withtrypsin was used. Phospholipase C (Clostridium welchii) digestionof the microsome inhibited no more than 13% of the pelletability.Although phospholipase A₂ (Crotalus terrificus terrificus) digestioninhibited 43% of the pelletability, addition of defatted albuminduring the enzymatic digestion completely restored the levelof the pelletability. The decrease of RNA content of the microsomalfractions by ribonuclease A digestion did not result in a proportionalinhibition of the pelletability. These results indicate thatproteinaceous component in the microsomal fraction is essentialfor phytochrome pelletability in vitro, that RNA and polar headsof phospholipids are unlikely to be the partner for the binding,and that products of phospholipase A₂ digestion inhibit thepelletability partially. (Received September 12, 1979; )     

A 51 kDa Protein Kinase of Potato Activated with Hyphal Wall Components from Phytophthora infestans

In potato tuber tissue, treatment of fungal elicitor, hyphalwall components (HWC) induces various plant defense reactions.As treatment of protein kinase inhibitor prior to HWC treatmentblocks some of defense reactions induced by HWC, involvementof protein kinases in plant defense induction is proposed. Here,we demonstrate HWC-induced activation of a 51-kDa protein kinase(abbreviated p51-PK) using myelin basic protein as a substratein potato tuber discs. The activity of p51-PK was not detectedin the absence of phosphatase inhibitor, NaF, and p51-PK wasimmuroprecipitated with antibody against phosphotyrosine. Pretreatmentof phospholipase C inhibitor, neomycin, and GTP-binding proteinactivator, mastoparan, partially inhibited the HWC-induced activationof p51-PK, suggesting possible involvement of phospholipaseC and GTP-binding protein in the activation of p51-PK. Exogenouslysupplied elicitors, salicylic acid and arachidonic acid, whichare known to induce various defense responses in potato plants,also activated the protein kinase showing the same migrationas p51-PK on SDS-PAGE and different activation patterns. Theseresults implied that p51-PK might be involved in several signaltransduction pathways leading to plant defense responses initiatedby different stimuli. (Received January 11, 1999; Accepted May 25, 1999)     

Effect of wounding or infection by Phytophthora infestans on the contents of terpenoids in potato tubers

Inoculation of the potato cut surfaces with an incompatiblerace of Phytophthora infestans induced an accumulation of rishitin,but only a trace occurred when infected by a compatible race.When the tuber was cut, a large amount of steroid-glycoalkaloids(solanine) accumulated in the cut surface tissue, although onlya trace was found in intact tissue. Only a small amount of solanine,if any, was contained in the wound periderm tissue. Most ofthe solanine seemed to be distributed in tissue neighbouringthe newly formed meristematic tissue zone. Distribution of solanineas a function of distance from the cut surface was exponential.Infection of the cut surface by an incompatible race of Phytophthorainfestans reduced the accumulation of solanine. The higher theconcentration of zoospore used, the less the solanine content.It has been reported that the higher the concentration of zoosporesused for inoculation of the cut surface, the less the numberof renewed meristematic cells in the wound tissue. In experimentsusing fresh and aged tubers, a good correlation between thenumber of renewed meristematic cells and solanine content wasfound. The accumulation of solanine in the wound tissue andits reduction due to infection by an incompatible race may berelated to renewed meristematic cells formation and its reductioncaused by the infection. No drastic change in carotenoids or sterol contents was found2 days after cutting or inoculation, when the tubers were cut,or cut and then infected by the incompatible race. ¹ Studies on the phytoalexin (No. 10). (6) in References constitutes"Studies on the phytoalexin No. 9". ² Present adress: Plant Pathology Laboratory, Faculty of Agriculture,Nagoya University, Chikusa-ku, Nagoya, 464, Japan. (Received April 1, 1972; )     

Host-Pathogen Interactions: XV. Fungal Glucans Which Elicit Phytoalexin Accumulation in Soybean Also Elicit the Accumulation of Phytoalexins in Other Plants

A β-glucan isolated from the mycelial walls of Phytophthora megasperma var. sojae and a glucan purified from yeast extract stimulate the accumulation of phytoalexins in red kidney bean, Phaseolus vulgaris, and stimulate the accumulation of the phytoalexin, rishitin, in potato tubers, Solanum tuberosum. These glucans have previously been shown to be potent elicitors of glyceollin accumulation in soybean, Glycine max.

Treatment of kidney bean cotyledons with the glucan elicitors resulted in the accumulation of at least five fungistatic compounds. These compounds migrate during thin layer chromatography identically to the fungistatic compounds which accumulate in kidney beans which have been inoculated with Colletotrichum lindemuthianum, a fungal pathogen of kidney beans.

Potatoes accumulate as much as 29 micrograms of rishitin per gram fresh weight following exposure to the glucan from Phytophthora megasperma var. sojae and as much as 19.5 micrograms of rishitin per gram fresh weight following exposure to yeast glucan. Potatoes accumulated 28 micrograms of rishitin per gram fresh weight following inoculation with live Phytophthora megasperma var. sojae.

     

Induction of 3-Hydroxy-3-methylglutaryl CoA Reductase in Potato Tubers after Slicing, Fungal Infection or Chemical Treatment, and Some Properties of the Enzyme

Intact tubers of potato (Solanum tuberosum L. cv. Irish Cobblerand an interspecific hybrid between S. tuberosum and S. demissumcv. Rishiri) contain a very low activity of 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase. The activity increased first in responseto slicing, and again in response to additional treatments suchas inoculation with an incompatible race of Phytophthora infestans,application of a hyphal wall component of the fungus or HgCl₂solution, and then decreased. Both the first and the secondincreases in activity in response to slicing and additionaltreatment with a hyphal wall component to elicit phytoalexinproduction were inhibited by blasticidin S. Properties of HMG-CoAreductase induced by slicing and by additional treatment withHgCl₂ or fungal inoculation were investigated. ² Present address: Faculty of Home Economics, Nagoya Women'sUniversity, Shioji-cho, Mizuho, Nagoya 467, Japan.     

Carbon Dioxide, Oxygen and Ethylene Effects on Potato Tuber Dormancy Release and Sprout Growth

The possible roles of oxygen and carbon dioxide treatments inthe presence or absence of ethylene on tuber dormancy releasein potato (Solanum tuberosumL.) were examined. Using two gascompositions (I: 60% CO₂–20% O₂–20% N₂and II: 20%CO₂–40% O₂–40% N₂), the phase of tuber dormancyand previous storage temperature were demonstrated to be importantparameters for dormancy release by these gas mixtures. Gas Icaused decreased abscisic acid (ABA) levels within 24 h regardlessof previous storage temperature, although this effect was reversible.Exogenous C₂H₄, an effective dormancy release agent, also causeddecreased ABA levels within 24 h. It also enhanced dormancyrelease and further promoted ABA losses by gas I. Gas II treatmentled to slight reductions in ABA levels that were further decreasedby C₂H₄. Sprout length was modelled successfully by multipleregression analysis in terms of glucose and ABA levels withinthe apical eye tissues of Russet Burbank tubers immediatelyafter, and regardless of, previous gas treatments or storagetemperatures. Solanum tuberosum,potato, abscisic acid, ethylene, carbon dioxide, oxygen, dormancy.     

Detoxification of sesquiterpene phytoalexins byGibberella pulicaris (Fusarium sambucinum) and its importance for virulence on potato tubers

Summary Gibberella pulicaris (Fusarium sambucinum) is a major cause of dry-rot of stored potatoes (Solanum tuberosum) worldwide. The ability of field strains ofG. pulicaris to cause dry-rot is correlated with their ability to detoxify sesquiterpene phytoalexins produced by potato. All highly virulent field strains can detoxify the sesquiterpenes rishitin and lubimin. Meiotic recombinational analysis indicates that rishitin detoxification can be controlled at two or more loci. High virulence has been associated with one of these loci, designatedRiml. Detoxification of rishitin and lubimin comprises a complex pattern of reactions involving epoxidation, dehydrogenation, and cyclization. To date, seven lubimin metabolites and one rishitin metabolite have been characterized. Genes for rishitin and lubimin detoxification are being cloned fromG. pulicaris in order to more rigorously analyze the role and regulation of sesquiterpene metabolism in potato dry-rot. Our results indirectly support a role for sesquiterpene phytoalexins in resistance of potato tubers to dry-rot and may enhance research on alternative control strategies for this economically important potato disease.     

Comparative subcellular distribution of apyrase from animal and plant sources. Characterization of microsomal apyrase

1. Apyrase (ATP: diphosphohydrolase) has been found in the microsomal fraction of rat salivary gland, mammary gland and uterus. 2. This enzyme, already described in plant tissue, is mainly present as a soluble polypeptide in tubers of Solanum tuberosum. 3. A fraction of this enzyme is associated with the microsomal fraction with a higher specific activity than the soluble one, for either ATP or ADP as substrate. 4. Apyrase bound to microsomes from rat and potato tissues was characterized in its substrate specificity and effect of inhibitors. 5. The Km values for ATP and ADP, optimum pH and metal ion requirement were determined. 6. A characteristic common to the microsomal and soluble apyrases is the stimulatory effect of a potato activator protein of soluble plant apyrase. 7. The microsomal-bound apyrase from rat and potato tissues were solubilized and subjected to size-exclusion chromatography. 8. The mammary gland and salivary gland apyrases eluted as molecular aggregates, in contrast to the uterus and potato enzyme.     

A phosphatidyl choline exchange protein isolated from germinated castor bean endosperms

A phospholipid exchange protein (PLEP) functioning between theendoplasmic reticulum and the mitochondrion was purified fromthe cytosolic fraction of germinated castor bean endosperms.In the protein fraction eluted from Sephadex G-100 column, theexchange rate reached 7.3µg phospholipids exchanged/mgprotein/15 min, which was 60-fold that of pota to tuber PLEP.The lipid transfer by this protein was specific for phosphatidylcholine and the transfer rate from microsomes to mitochondriawas as high as that from mitochondria to microsomes. Castorbean PLEP transferred phospholipid from castor bean microsomesto mitochondria from other sources such as potato tubers, cauliflowerinflorescences, pumpkin hypocotyls and rat livers, and to liposomes,but not to Avena etioplasts. In addition, it transferred phospholipidfrom potato microsomes to potato mitochondria. (Received November 17, 1978; )     

Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

The present study illustrates elements ofthe signal cascades involved in the activation of taurine effluxpathways in myotubes derived from skeletal muscle cells. Exposingprimary skeletal muscle cells, loaded with ¹⁴C-taurine, to1) hypotonic media, 2) the phospholipaseA₂ (PLA₂) activator melittin, 3)anoxia, or 4) lysophosphatidyl choline (LPC) causes anincrease in ¹⁴C-taurine release and a concomitantproduction of reactive oxygen species (ROS). The antioxidants butulatedhydroxy toluene and vitamin E inhibit the taurine efflux after cellswelling, anoxia, and addition of LPC. The muscle cells possess twoseparate taurine efflux pathways, i.e., a swelling- andmelittin-induced pathway that requires 5-lipoxygenase activity foractivation and a LPC-induced pathway. The two pathways aredistinguished by their opposing sensitivity toward the anion channelblocker DIDS and cholesterol. These data provide evidence forPLA₂ products and ROS as key mediators of the signalcascade leading to taurine efflux in muscle.

     

Letters to the Editor

The following is the abstract of the article discussed in thesubsequent letter:

Mitchell, Claire H., Jin Jun Zhang, Liwei Wang, andTim J. C. Jacob. Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. Am. J. Physiol. 272 (Cell Physiol. 41): C212-C222, 1997.Thewhole cell recording technique was used to examine an outwardlyrectifying chloride current activated by hypotonic shock in bovinepigmented ciliary epithelial (PCE) cells. Removal of internal andexternal Ca²⁺ did not affect the activation of thesecurrents, but they were abolished by the phospholipase C inhibitorneomycin. The current was blocked by5-nitro-2-(3-phenylpropylamino)benzoic acid,4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in avoltage-dependent manner, but tamoxifen, dideoxyforskolin, andquinidine did not affect it. This blocking profile differs from that ofthe volume-sensitive chloride channel in neighboring nonpigmentedciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob. J. Physiol. Lond. 491: 743-755, 1996), and thisdifference implies that the volume responses of the two cell types aremediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTPS) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent blockof the GTPS-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells incalphostin C, an inhibitor of protein kinase C (PKC). Removal ofCa²⁺ did not affect activation of the current by GTPSbut extended the duration of the response. Inhibition of phospholipaseA₂ (PLA₂) with p-bromophenacyl bromideprevented the activation of the hypotonically induced current and alsoinhibited the current once activated by hypotonic solution. Thefindings imply that the hypotonic response in PCE cells is mediated byboth phospholipase C (PLC) and PLA₂. Both phospholipasesgenerate arachidonic acid, and, in addition, the PLC pathway regulatesthe PLA₂ pathway via a PKC-dependent phosphorylation ofPLA₂.

     

The nature of gibberellin-induced dormancy in aerial tubers of Begonia evansiana

Four kinds of GA, GA₁, GA₂, GA₃, and GA₄, all inhibited sproutingin aerial tubers of Begonia evansiana. The sprout-inhibiting action of GA increased with incubationin a high O₂ concentration, and decreased in a low O₂ Concentration. Inhibition of sprouting by GA was reduced by incubation in thepresence of p-nitrophenol, resorcinol, salicylaldoxime, 2, 4-dinitrophenol,sodium azide and cycloheximide. The higher activity of polyphenol oxydase was observed in ahomogenate of GA treated tubers. Existence of counteracting two systems which were activatedby GA was considered. (Received January 13, 1972; )