Study of the Involvement of Phosphatidic Acid Formation in the Expression of Wound-Responsive Genes in Cotton.

Plants use phospholipase D (PLD, EC 3.1.4.4)/phosphatidic acid (PtdOH) for the transduction of environmental signals including those coming from wounding. Based on our previous findings suggesting that wound-induced PLDα-derived PtdOH can act as a local signaling molecule in cotton (Gossypium hirsutum), we show that wounding immediately increases local NADPH oxidase (NADPHox) and cellulose synthase A (CeSA) gene expression. After developing a novel fluorimetric assay for the investigation of n-butanol inhibitory effect on PLD activity, we show that only NADPHox gene upregulation is reduced when n-butanol is applied prior to wounding. This suggests that NADPHox is a possible downstream target of PLD function, while a different CeSA-involving response system may exist in cotton. Overall, this study provides new knowledge on signal-transduction mechanisms following wounding of cotton leaves.

Phospholipases Dα and δ are involved in local and systemic wound responses of cotton (G. hirsutum).

Phospholipases D (PLDs) catabolize structural phospholipids to produce phosphatidic acid (PtdOH), a lipid playing central role in signalling pathways in animal, yeast and plant cells. In animal cells two PLD genes have been studied while in model plant Arabidopsis twelve genes exist, classified in six classes (α-ζ). This underlines the role of these enzymes in plant responses to environmental stresses. However, information concerning the PLD involvement in the widely cultivated and economically important cotton plant responses is very limited. The aim of this report was to study the activity of conventional cotton PLD and its participation in plant responses to mechanical wounding, which resembles both biotic and abiotic stresses. PLDα activity was identified and further characterized by transphosphatidylation reaction. Upon wounding, cotton leaf responses consist of an acute in vitro increase of PLDα activity in both wounded and systemic tissue. However, determination of the in vivo PtdOH levels under the same wounding conditions revealed a rapid PtdOH formation only in wounded leaves and a late response of a PtdOH increase in both tissues. Εxpression analysis of PLDα and PLDδ isoforms showed mRNA accumulation of both isoforms in the wounded tissue, but only PLDδ exerts a high and sustainable expression in systemic leaves, indicating that this isoform is mainly responsible for the systemic wound-induced PtdOH production. Therefore, our data suggest that PLDα and PLDδ isoforms are involved in different steps in cotton wound signalling.

Genome-wide analysis of the phosphoinositide kinome from two ciliates reveals novel evolutionary links for phosphoinositide kinases in eukaryotic cells.

BACKGROUND: The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs) that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates. CONCLUSIONS/SIGNIFICANCE: Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs) not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory pathways of phosphoinositides may be more widespread than previously thought in unicellular organisms.

Emerging roles of phosphoinositide-specific phospholipases C in the ciliates Tetrahymena and Paramecium.

Phospholipases C (PLCs) that hydrolyze inositol phospholipids regulate vital cellular functions in both eukaryotic and prokaryotic organisms. The PLC superfamily consists of eukaryotic phosphoinositide-specific PLCs (PI-PLCs), bacterial PLCs and trypanosomal PLCs.1 PI-PLCs hydrolyze phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) to produce inositol-1,4,5-trisphosphate (Ins1,4,5P(3)) and constitute a hallmark feature of eukaryotic cells. In metazoa, this reaction is coupled to receptor signaling via specific PI-PLC isoforms and results in acute increase of cytosolic Ca(2+) levels by Ins1,4,5P(3)-sensitive Ca(2+) channels (IP(3)-receptors, IP3Rs).2 A striking result of many studies so far has been the presence of a single PI-PLC gene in all unicellular eukaryotes investigated, as opposed to expansion of PI-PLC isoforms in metazoa;3 this has suggested that a single housekeeping PI-PLC represents an archetypal and simplified form of PI-PLC signaling.3 Several studies however have noted a unique expansion of PI-PLC/IP3R pathway components in ciliates.4,5 In a recent paper we showed the presence of multiple functional PI-PLC genes in Tetrahymena thermophila and biochemical characterization, pharmacological studies and study of their expression patterns suggested that they are likely to serve distinct non-redundant roles.4 In this report we discuss these studies and how they advance our understanding of PI-PLC functions in ciliates.

Biochemical and genetic evidence for the presence of multiple phosphatidylinositol- and phosphatidylinositol 4,5-bisphosphate-specific phospholipases C in Tetrahymena.

Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], produce the Ca(2+)-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca(2+). One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca(2+), modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P(2) levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P(3)-Ca(2+) regulatory axis in ciliates.

Cold acclimation and low temperature resistance in cotton: Gossypium hirsutum phospholipase Dalpha isoforms are differentially regulated by temperature and light.

Phospholipase Dalpha (PLDalpha) was isolated from cultivated cotton (Gossypium hirsutum) and characterized. Two PLDalpha genes were identified in the allotetraploid genome of G. hirsutum, derived from its diploid progenitors, G. raimondii and G. arboreum. The genes contained three exons and two introns. The translated products shared a 98.6% homology and were designated as GrPLDalpha and GaPLDalpha. Their ORFs encoded a polypeptide of 807 amino acids with a predicted molecular mass of 91.6 kDa sharing an 81-82% homology with PLDalpha1 and PLDalpha2 from A. thaliana. A possible alternative splicing event was detected at the 5' untranslated region which, however, did not result in alternative ORFs. Cold stress (10 degrees C or less) resulted in gene induction which was suppressed below control levels (25 degrees C or 22 degrees C growth temperature) when plants were acclimated at 17 degrees C before applying the cold treatment. Differences in the expression levels of the isoforms were recorded under cold acclimation, and cold stress temperatures. Expression was light regulated under growth, acclimation, and cold stress temperatures. Characterization of the products of lipid hydrolysis by the endogenous PLDalpha indicated alterations in lipid species and a variation in levels of the signalling molecule phosphatidic acid (PA) following acclimation or cold stress.

A specific phospholipase C activity regulates phosphatidylinositol levels in lung surfactant of patients with acute respiratory distress syndrome.

Lung surfactant (LS) is a lipid-rich material lining the inside of the lungs. It reduces surface tension at the liquid/air interface and thus, it confers protection of the alveoli from collapsing. The surface-active component of LS is dipalmitoyl-phosphatidylcholine, while anionic phospholipids such as phosphatidylinositol (PtdIns) and primarily phosphatidylglycerol are involved in the stabilization of the LS monolayer. The exact role of PtdIns in this system is not well-understood; however, PtdIns levels change dramatically during the acute respiratory distress syndrome (ARDS) evolution. In this report we present evidence of a phosphoinositide-specific phospholipase C (PI-PLC) activity in bronchoalveolar lavage (BAL) fluid, which may regulate PtdIns levels. Characterization of this extracellular activity showed specificity for PtdIns and phosphatidylinositol 4,5-bisphosphate, sharing the typical substrate concentration-, pH-, and calcium-dependencies with mammalian PI-PLCs. Fractionation of BAL fluid showed that PI-PLC did not co-fractionate with large surfactant aggregates, but it was found mainly in the soluble fraction. Importantly, analysis of BAL samples from control subjects and from patients with ARDS showed that the PI-PLC specific activity was decreased by 4-fold in ARDS samples concurrently with the increase in BAL PtdIns levels. Thus, we have identified for the first time an extracellular PI-PLC enzyme activity that may be acutely involved in the regulation of PtdIns levels in LS.

Study of the Effect of Methyl Jasmonate Concentration on Aflatoxin B(1) Biosynthesis by Aspergillus parasiticus in Yeast Extract Sucrose Medium.

Aflatoxin B(1) (AFB(1)) is a carcinogenic metabolite produced by certain Aspergillus species on agricultural commodities. AFB(1) biosynthesis is affected by jasmonic acid and also by its methylester (MeJA), a plant growth regulator derived from linoleic acid. This study reports the effect of MeJA on the growth of A. parasiticus and AFB(1) output in yeast extract sucrose (YES) medium when added at three different concentrations; namely, 10(-2) M, 10(-4) M, and 10(-6) M. AFB(1) determination was performed by immunoaffinity and HPLC. MeJA at 10(-4) and 10(-6) M concentrations had no significant effect on mycelial growth but did affect AFB(1) production after the 7th day of incubation; on the 12th day, AFB(1) production was increased by 212.7% and 141.6% compared to the control samples (addition of 10(-6) M and 10(-4) M MeJA, resp.). Treatment of A. parasiticus cultures with 10(-2) M MeJA inhibited mycelial growth and AFB(1) production as well. These results suggest that the effect of MeJA on AFB(1) biosynthesis by A. parasiticus depends on the MeJA concentration used.

Deficiency in lysosomal enzyme secretion is associated with upregulation of phosphatidylinositol 4-phosphate in tetrahymena.

A variety of lower eukaryotes and certain mammalian cells are known to constitutively secrete lysosomal hydrolases. Recent studies in Tetrahymena have shown that phosphatidylinositol 3-phosphate regulates the proper secretion of lysosomal enzymes at the level of phagolysosome formation. We extend these findings by studying the secretion-deficient strain MS-1 of Tetrahymena thermophila, which possess phosphatidylinositol levels similar to wild type. However, steady-state levels of phosphatidylinositol 4-phosphate (PtdIns4P) were found to be doubled in this strain compared with wild type as shown by in vivo [3H]inositol labeling and high-performance liquid chromatography analysis. The increased PtdIns4P levels in MS-1 cells were unrelated to the upregulation of total phosphatidylinositol phosphate induced by hyperosmotic stress because this treatment resulted in a similar increase of PtdIns4P in MS-1 and wild-type cells. Hyperosmotic stress did not affect secretion in either of the two types of cells. On the other hand, under conditions of wortmannin-induced hypersecretion in wild-type cells, MS-1 cells developed a highly vacuolated phenotype while secretion was not induced. Importantly, comparative analysis of wild-type and MS-1 cells under wortmannin treatment showed that PtdIns4P levels were differentially regulated in the two strains. These results collectively suggest that PtdIns4P turnover in Tetrahymena is linked to lysosome secretion.

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum).

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.

Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum.

The role of phosphoinositide-specific phospholipase C (PI-PLC) signaling in the macrotubule-dependent protoplast volume regulation in plasmolyzed root cells of Triticum turgidum was investigated. At the onset of hyperosmotic stress, PI-PLC activation was documented. Inhibition of PI-PLC activity by U73122 blocked tubulin macrotubule formation in plasmolyzed cells and their protoplast volume regulatory mechanism. In neomycin-treated plasmolyzed cells, macrotubule formation and protoplast volume regulation were not affected. In these cells the PI-PLC pathway is down-regulated as neomycin sequesters the PI-PLC substrate, 4,5-diphosphate-phosphatidyl inositol (PtdInsP(2)). These phenomena were unaffected by R59022, an inhibitor of phosphatidic acic (PA) production via the PLC pathway. Taxol, a microtubule (MT) stabilizer, inhibited the hyperosmotic activation of PI-PLC, but oryzalin, which disorganized MTs, triggered PI-PLC activity. Taxol prevented macrotubule formation and inhibited the mechanism regulating the volume of the plasmolyzed protoplast. Neomycin partly relieved some of the taxol effects. These data suggest that PtdInspP(2) turnover via PI-PLC assists macrotubule formation and activation of the mechanism regulating the plasmolyzed protoplast volume; and the massive disorganization of MTs that is carried out at the onset of hyperosmotic treatment triggers the activation of this mechanism.

TNFalpha is a potent inducer of platelet-activating factor synthesis in adipocytes but not in preadipocytes. Differential regulation by PI3K.

Tumour necrosis factor alpha (TNFalpha) induces platelet-activating factor (PAF) synthesis in many inflammatory cells. Here, we investigate the possibility that TNFalpha stimulates PAF synthesis in rat adipocytes and preadipocytes and that phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2 (ERK1/2) are implicated in this process. Primary cultures were incubated with [3H]lyso-PAF and stimulated by TNFalpha in the presence or absence of wortmannin. We found that, although both cultures synthesized PAF at a similar basal rate, TNFalpha-induced PAF synthesis in adipocytes was 7-fold higher than in preadipocytes. This suggested a maturation of PAF-TNFalpha interrelationship during adipocyte differentiation. Wortmannin enhanced TNFalpha-dependent PAF synthesis in adipocytes but not in preadipocytes, indicating the negative control by PI3K in mature cells. PAF increase was due to the regulation of its biosynthesis since PAF-acetylhydrolase (PAF-AH) activity was TNFalpha- and wortmannin-independent. Our hypothesis is that PAF mediates TNFalpha inflammatory effects in both adipocytes and preadipocytes and that this pathway is enhanced during adipocyte differentiation, a mechanism which is highly active during the development of obesity.

D-3 phosphoinositides of the ciliate Tetrahymena: characterization and study of their regulatory role in lysosomal enzyme secretion.

Phosphatidylinositol 3-phosphate, PtdIns3P, is a phosphoinositide which is implicated in regulating membrane trafficking in both mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns3,5P2, a phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive lipid components of the ciliate Tetrahymena. Using HPLC analysis, PtdIns3P and PtdIns3,5P2 were found to comprise 16% and 30-40% of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1-10 microM) resulted in the depletion of PtdIns3P and PtdIns3,5P2 without any effect on D-4 phosphoinositides. Wortmannin was further used for the investigation of D-3 phosphoinositide involvement in the regulation of lysosomal vesicular trafficking. Incubation of Tetrahymena cells with wortmannin resulted in enhanced secretion of two different lysosomal enzymes without any change in their total activities. Experiments performed with a T. thermophila secretion mutant strain verified that the wortmannin-induced secretion is specific and it is not due to a diversion of lysosomal enzymes to other secretory pathways. Moreover, experiments performed with a phagocytosis-deficient T. thermophila strain showed that a substantial fraction of wortmannin-induced secretion was dependent on the presence of functional phagosomes/phagolysosomes.

Characterization and comparative analysis of Arabidopsis phosphatidylinositol phosphate 5-kinase 10 reveals differences in Arabidopsis and human phosphatidylinositol phosphate kinases.

Arabidopsis phosphatidylinositol phosphate (PtdInsP) kinase 10 (AtPIPK10; At4g01190) is shown to be a functional enzyme of the subfamily A, type I AtPtdInsP kinases. It is biochemically distinct from AtPIPK1 (At1g21980), the only other previously characterized AtPtdInsP kinase which is of the B subfamily. AtPIPK10 has the same K(m), but a 10-fold lower V(max) than AtPIPK1 and it is insensitive to phosphatidic acid. AtPIPK10 transcript is most abundant in inflorescence stalks and flowers, whereas AtPIPK1 transcript is present in all tissues. Comparative analysis of recombinant AtPIPK10 and AtPIPK1 with recombinant HsPIPKIalpha reveals that the Arabidopsis enzymes have roughly 200- and 20-fold lower V(max)/K(m), respectively. These data reveal one explanation for the longstanding mystery of the relatively low phosphatidylinositol-(4,5)-bisphosphate:phosphatidylinositol-4-phosphate ratio in terrestrial plants.