The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants. We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al., 1992, Plant Physiol Biochem 30: 579–584). The role of this compound in the induction of this and of other defence reactions is further studied in this paper. Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var. nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX. Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ. Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect. Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ. However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes. Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction. The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions. Received: 4 July 1996 / Accepted: 23 November 1996     

Involvement of phospholipases C and D in early response to SAR and ISR inducers in Brassica napus plants.

Phospholipid signaling is an important component in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as in responses to environmental stresses, including pathogen attack. We investigated the involvement of both phospholipase C (PLC, EC 3.1.4.11) and D (PLD, EC 3.1.4.4) in early responses to the treatment of Brassica napus plants with the chemical inducers of systemic acquired resistance (SAR): salicylic acid (SA), benzothiadiazole (BTH), and with the inducer mediating the induced systemic resistance (ISR) pathway, methyl jasmonate (MeJA). Rapid activation (within 0.5-6 h treatment) of the in vitro activity level was found for phosphatidyl inositol 4,5 bisphosphate (PIP2)-specific PLC (PI-PLC) and three enzymatically different forms of PLD: conventional PLDalpha, PIP2-dependent PLD beta/gamma, and oleate-stimulated PLDdelta. The strongest response was found in case of cytosolic PIP2-dependent PLD beta/gamma after BTH treatment. PLDdelta was identified in B. napus leaves and was very rapidly activated after MeJA treatment with the highest degree of activation compared to the other PLD isoforms. Interestingly, an increase in the amount of protein was observed only for PLDgamma and/or delta after ISR induction, but later than the activation occurred. These results show that phospholipases are involved in very early processes leading to systemic responses in plants and that they are most probably initially first activated on post translational level.     

Effect of ethanol on ATP-induced phospholipases C and D and serine base exchange in glioma C6 cells

The effect of extracellular ATP, a nucleotide receptor agonist in the central nervous system, was investigated in glioma C6 cells on the intracellular Ca2+ level and the formation of phosphatidylethanol and phosphatidic acid in the presence and absence of ethanol (150 mM). In the cells prelabeled with [14C]palmitic acid, 100 microM ATP induced both the hydrolysis and the transphosphatidylation reactions leading to the formation of [14C]phosphatidic acid; addition of ethanol generated [14C]phosphatidylethanol. However, ATP-mediated increase in the level of [14C]phosphatidic acid was not inhibited by ethanol. Furthermore, ethanol augmented ATP-induced transient and sustained increase in the intracellular Ca2+ concentration, whereas ethanol alone did not produce any change in the intracellular Ca2+ level. These results indicate that in glioma C6 cells, ATP induces activation of polyphosphoinositide-specific phospholipase C and phospholipase D and that ethanol enhances this effect. In the present investigation we have also shown that long-term (2 days) ethanol treatment, at concentration relevant to chronic alcoholism (100 mM), decreased the incorporation of [14C]serine into phosphatidylserine. Since the effect of ethanol on ATP-induced activities of phospholipase C and phospholipase D and on serine base-exchange in glioma C6 cells differs significantly from that in cultured neuronal cells, these results may contribute to a better understanding of the mechanisms of ethanol action in cells of glial origin.     

The involvement of phospholipases C and D in the asymmetric division of subsidiary cell mother cells of Zea mays

In the present study, the involvement of phospholipase C and D (PLC and PLD) pathways in the asymmetric divisions that produce the stomatal complexes of Zea mays was investigated. In particular, the polar organization of microtubules (MTs) and actin filaments (AFs) and the process of asymmetric division were studied in subsidiary cell mother cells (SMCs) treated with PLC and PLD modulators. In SMCs treated with butanol-1 (but-1), which blocks phosphatidic acid (PA) production via PLDs, AF-patch formation laterally to the inducing guard cell mother cell (GMC) and the subsequent asymmetric division were inhibited. In these SMCs, cell division plane determination, as expressed by MT preprophase band (MT-PPB) formation, was not disturbed. Exogenously applied PA partially relieved the but-1 effects on SMCs. In contrast to SMCs, but-1 did not affect the symmetric GMC division. Inhibition of the PLC catalytic activity by neomycin or U73122 resulted in inhibition of asymmetric SMC division, while AF-patch and MT-PPB were organized as in control SMCs. These data show that the PLC and PLD signaling pathways are involved in the transduction and/or perception of the inductive stimulus that is emitted by the GMCs and induces the polar AF organization and asymmetric SMC division. In contrast, division plane determination in SMCs, as expressed by MT-PPB formation, does not depend on PLC and PLD signaling pathways.     

Involvement of nuclear phosphatidylinositol-dependent phospholipases C in cell cycle progression during rat liver regeneration

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI-phospholipase C (PI-PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI-PLC beta(1) isoform is exclusively localized at the chromatin level, PI-PLC beta(1) co-localizes with DNA replication sites much more than PI-PLC gamma(1), which is also present at the nuclear envelope. These findings and the increased amount of PI-PLC gamma(1) occurring after the peak of DNA replication suggest that PI-PLC beta(1) and gamma(1) play different roles in cell cycle progression during regenerating liver. The increased activity of PI-PLC beta(1) constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI-PLC gamma(1) should be involved in G2/M phase transition through lamin phosphorylation.     

Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells

The signaling events generated by a cold exposure are poorly known in plants. We were interested in checking the possible activation of enzymes of the phosphoinositide signaling pathway in response to a temperature drop. In Arabidopsis suspension cells labeled with (33)PO(4)(3-), a cold treatment induces a rapid increase of phosphatidic acid (PtdOH) content. This production was due to the simultaneous activation of phospholipase C (through diacylglycerol kinase activity) and phospholipase D, as monitored by the production of inositol triphosphate and of transphosphatidylation product, respectively. Moreover, inhibitors of the phosphoinositide pathway and of diacylglycerol kinase reduced PtdOH production. Enzyme activation occurred immediately after cells were transferred to low temperature. The respective contribution of both kind of phospholipases in cold-induced production of PtdOH could be estimated. We created conditions where phospholipids were labeled with (33)PO(4)(3-), but with ATP being nonradioactive. In such conditions, the apparition of radioactive PtdOH reflected PLD activity. Thus, we demonstrated that during a cold stress, phospholipase D activity accounted for 20% of PtdOH production. The analysis of composition in fatty acids of cold-produced PtdOH compared with that of different phospholipids confirmed that cold-induced PtdOH more likely derived mainly from phosphoinositides. The addition of chemical reagents modifying calcium availability inhibited the formation of PtdOH, showing that the cold-induced activation of phospholipase pathways is dependent on a calcium entry.     

Real-time electrochemical detection of extracellular nitric oxide in tobacco cells exposed to cryptogein, an elicitor of defence responses

It was previously reported that cryptogein, an elicitor of defence responses, induces an intracellular production of nitric oxide (NO) in tobacco. Here, the possibility was explored that cryptogein might also trigger an increase of NO extracellular content through two distinct approaches, an indirect method using the NO probe 4,5-diaminofluorescein (DAF-2) and an electrochemical method involving a chemically modified microelectrode probing free NO in biological media. While the chemical nature of DAF-2-reactive compound(s) is still uncertain, the electrochemical modified microelectrodes provide real-time evidence that cryptogein induces an increase of extracellular NO. Direct measurement of free extracellular NO might offer important new insights into its role in plants challenged by biotic stresses.     

Involvement of the small GTPase Rac in the defense responses of tobacco to pathogens

During the hypersensitive response (HR), plants accumulate reactive oxygen species (ROS) that are likely generated at least in part by an NADPH oxidase similar to that found in mammalian neutrophils. An essential regulator of mammalian NADPH oxidase is the small GTP-binding protein Rac. To investigate whether Rac also regulates the pathogen-induced oxidative burst in plants, a dominant negative form of the rice OsRac1 gene was overexpressed in tobacco carrying the N resistance gene. Following infection with Tobacco mosaic virus (TMV), DN-OsRacl plants developed smaller lesions than wild-type plants, accumulated lower levels of lipid peroxidation products, and failed to activate expression of antioxidant genes. These results, combined with the demonstration that superoxide and hydrogen peroxide levels were reduced in DN-OsRacl tobacco developing a synchronous HR triggered by transient expression of the TMV p50 helicase domain or the Pto and AvrPto proteins, suggest that ROS production is impaired. The dominant negative effect of DN-OsRacl could be rescued by transiently overexpressing the wild-type OsRac1 protein. TMV-induced salicylic acid accumulation also was compromised in DN-OsRacl tobacco. Interestingly, while systemic acquired resistance to TMV was not impaired, nonhost resistance to Pseudomonas syringae pv. maculicola ES4326 was suppressed. Thus, the effect DN-OsRac1 expression exerts on the resistance signaling pathway appears to vary depending on the identity of the inoculated pathogen.     

Study of phospholipases D and C in maturing and germinating seeds of Brassica napus

Different forms of phospholipase D (dependent on and independent of the presence of phosphatidylinositol 4,5-bisphosphate, PIP(2)) were identified in maturing and germinating seeds of Brassica napus. Both forms were present in cytosolic and membrane fractions of maturing seeds. PIP(2)-dependent activity increased continuously during seed germination, while PIP(2)-independent activity appeared mostly at the very beginning of seed maturation. PIP(2)-dependent activity was detected mainly in the plasma-membrane fraction. Phosphatidylinositol-specific phospholipase C (PI-PLC) was found only in membrane fractions of both types of developing rape seed tissues. The increasing activities of PLC and PIP(2)-dependent PLD were mainly detected in hypocotyls of seedlings. Some biochemical characteristics of both described enzymes are also presented.     

Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco

A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat-stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor-encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide-associated complex domain. The peaT1 gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway.     

Effects of phospholipases C and D on ordering of channel proteins in the mitochondrial outer membrane

The effects of phospholipases C and D on the state of order of the channels in the outer membranes of Neurospora mitochondria have been investigated by negative-stain electron microscopy and optical diffraction. Unlike the situation with phospholipase A2, treatment of the isolated membranes with phospholipase C or D does not induce crystallization of the channels in the membrane plane. Furthermore, treatment of already-formed periodic arrays of outer membrane channels with either phospholipase C or D causes loss of long-range order in the arrays. The latter result suggests that zwitterionic phospholipids may play an important role in stabilizing the periodic arrays of the channel-forming protein in this membrane.     

Identification of phosphatidylcholine-selective and phosphatidylinositol-selective phospholipases D in Madin-Darby canine kidney cells.

Intact cells and cell-free systems were employed to characterize phospholipase D (PLD) activity in Madin-Darby canine kidney (MDCK) cells. In cells prelabeled with [3H]glycerol, 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited phosphatidylcholine (PC) hydrolysis by PLD, as shown by the prolonged formation of [3H]phosphatidylethanol (PEt) and an accompanying decrease in [3H]PC. In contrast, bradykinin elicited rapid formation of [3H]PEt (approximately 1 min) accompanied by a decrease in [3H]phosphatidylinositol (PI). When the agonists were administered simultaneously, [3H]PEt formation was biphasic. In cells prelabeled with [3H] choline, at times less than 1 min, bradykinin failed to induce significant change in [3H]choline release. Bradykinin-induced formation of [3H]PEt in the [3H]glycerol-labeled cells was strictly dependent on extracellular Ca2+, whereas TPA-induced formation of [3H]PEt did not require extracellular Ca2+. Cell-free assays for PLD were used to assess the enzyme location, substrate specificity, and cofactor requirements. The PC-PLD activity (PEt formation) against [3H]stearoyl-PC was primarily localized in the 440 x g pellet (membrane- and nuclear-associated), preferred PC as a substrate, required detergent, and was not influenced by Ca2+ at low concentrations but was inhibited by Ca2+ in excess of 0.5 mM. The PI-PLD activity against [3H]stearoyl-PI was found largely in the 100,000 x g supernatant (cytosol), was strictly Ca(2+)-dependent, and did not require detergent. From these data, we conclude that MDCK cells contain two PLD subtypes: 1) a membrane-associated, PC-selective enzyme that responds to TPA resulting in prolonged hydrolysis of PC (the PC-PLD is Ca(2+)-independent, but requires detergent); 2) a cytosolic, PI-selective enzyme that responds rapidly but transiently to bradykinin (the PI-PLD requires Ca2+ but not detergent).     

Short communication. Differential regulation of a glucosyl transferase gene homologue during defence responses in tobacco

The regulation of a glucosyl transferase homologue which is rapidly induced during defence response in tobacco was investigated. Expression of the gene is induced in reasons to wounding and during a hypersensitive response to TMV, but regulation differs in these two responses.Key words: Plant defence, signalling, glucosyl transferase.