Lag1p and Lac1p are essential for the Acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisae.

Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1 Delta lac1 Delta cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA-independent ceramide synthesis activity. Quadruple mutant cells (lag1 Delta lac1 Delta ypc1 Delta ydc1 Delta) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1 Delta lac1 Delta cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation.

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.

Fatty acid alteration of plastidic and extra-plastidic membrane lipids in metribuzin-resistant photoautotrophic Chenopodium rubrum cells as compared to wild-type cells.

The fatty acid compositions of plastidic and extra-plastidic membrane lipids of two metribuzin-resistant cell lines L4 and L7 of Chenopodium rubrum were determined after growth in the absence and in the presence of the herbicide and compared with those of wild type cells. Fatty acid biosynthesis was markedly affected in all cell lines by metribuzin treatment. In the absence and in the presence of metribuzin alterations of the fatty acid composition of the various lipid classes were, as compared to wild type cells, generally lower in the highly resistant L4 cells than in the less resistant L7 cells. The two resistant cell lines demonstrated a higher degree of unsaturation within the plastidic monogalactosyldiacylglycerols (L4 cells also within plastidic digalactosyldiacylglycerols) and, particularly, within the predominantly extra-plastidic phosphatidylcholines (L7 cells also within the predominantly extra-plastidic phosphatidylethanolamines), whereas the degree of unsaturation was slightly altered in the plastidic phosphatidylglycerols. Within the two metribuzin-resistant cell lines, the highly resistant L4 cells differed from the less resistant L7 cells by increased alpha-linolenic acid/palmitic acid ratios in both the plastidic and extra-plastidic membrane lipids suggesting that particularly in L4 cells higher proportions of linolenate are formed as a result of selection pressure. On the other hand, the proportion of linoleate was increased predominantly in extra-plastidic membrane lipids of both L4 and L7 cells which explains a raise in linoleic acid/palmitic acid ratios in both cell lines as compared to wild-type cells. Moreover, in the absence of metribuzin decreased proportions of trans-3-hexadecenoic acid were found in phosphatidylglycerols of L4 and, particularly, of L7 cells as compared to the wild type cells. It is suggested that L4 and L7 cells--having multiple mutations in the psbA gene as observed earlier--are additionally characterized by increased degree of unsaturation of acyl moieties in various polar lipids, e.g. linoleoyl moieties in L4 and L7 cells as well as linolenoyl moieties particularly in highly resistant L4 cells. This increase gives rise to a change in membrane fluidity and may finally lead to increased metribuzin resistance.

Biochemical and molecular biological studies on infection (Ascochyta rabiei)-induced thaumatin-like proteins from chickpea plants (Cicer arietinum L.).

A pathogenesis-related protein induced by infection with Ascochyta rabiei was purified from intercellular washing fluid of chickpea (Cicer arietinum L.) leaves. Amino-terminal sequencing identified the protein, named PR-5a, as a thaumatin-like protein. The isoelectric point was determined with 6.5 and the molecular mass is 16 kDa. Therefore, chickpea PR-5a is the first dicot member of a TLP subgroup containing small TLPs with a molecular weight between 15 and 18 kDa. PR-5a shows no antifungal activity towards A. rabiei. Screening of a chickpea cDNA library led to the isolation of a cDNA clone (p5a-241) for this protein. A second cDNA clone (ELR112) encoding a TLP was isolated using differential hybridisation of cDNA libraries obtained from elicited and water treated cell suspension cultures of chickpea. The deduced protein (PR-5b) has a molecular mass of 22 kDa. PR-5b is postulated to be located in the vacuole due to the presence of a respective N-terminal signal peptide and a carboxy-terminal extension. Southern blot analyses showed that ELR112 and p5a-241 represent single copy genes. During fungal infection of chickpea plants expression of both genes proceeds much faster in an A. rabiei resistant cultivar than in a susceptible one.

Elicitor-induced defence reactions in cell suspension cultures of soybean cultivars.

Suspension cultured soybean (Glycine max [L.] Merr.) cells of four cultivars (Wilis, Lumut, Kalmit, Doko RC) were compared for their response to different fungal and bacterial elicitors. Cells were treated either with crude cell wall extracts of the fungal pathogens Phytophthora sojae (Pmg-elicitor) and Rhizoctonia solani (Riso-elicitor) or with two isolates of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg01/02) and a broad spectrum of antimicrobial defence reactions was measured. Cells of all four cultivars showed the same elicitor-induced rapid (H2O2 accumulation, alkalinization of the culture medium, peroxidative cross-linking of cell wall proteins) and slow (activation of phenylpropanoid metabolism, accumulation of phenolic compounds, induction of PR-proteins) defence responses. However, the reactivity of the cultivars was not identical in terms of time courses and intensities. Furthermore, the ability of the various elicitors to induce defence responses varied markedly. These differences indicate that (1) cells of the same species but of different cultivars are equipped with the same array of perception systems to recognise various stimuli but (2) the sensitivity of these perception systems or later steps in the signal transduction seem to be stimulated to a different extent in the analysed cultivars.

Cloning and characterization of eight cytochrome P450 cDNAs from chickpea (Cicer arietinum L.) cell suspension cultures.

Eight different P450 sequences were isolated from a cDNA library derived from cultured chickpea cells (cultivar ILC3279) elicited with a Phytophthora sojae (formerly megasperma) elicitor (Pmg-elicitor) by screening with heterologous and homologous probes. Screening with CYP73A1 from Helianthus tuberosus yielded several clones with one identical sequence. A full-length clone could be isolated and this sequence was assigned CYP73A19. Heterologous expression in yeast confirmed that CYP73A19 is the trans-cinnamic acid 4-hydroxylase of chickpea. Screening with a CYP81E2 polymerase chain reaction fragment from chickpea yielded a CYP81E2 full-length sequence and two almost identical CYP81E3 sequences, differing in only 16 out of 498 amino acids; both share more than 85% homology with the isoflavone 2'-hydroxylase from licorice (Glycyrrhiza echinata L.). Using CYP93A1 as a probe, it was possible to isolate a full-length member of the CYP93 family, CYP93C3, that shares more than 80% homology with isoflavone synthase from soybean. In addition, partial sequences CYP81E3, CYP81E4 and CYP81E5 were also found in this screening. The use of a CYP82A2 probe derived from BAC F10N7 from Arabidopsis thaliana yielded only one sequence, CYP76F1. Rescreening with CYP81E4 and CYP81E5 did not result in the isolation of any new P450 sequences. Northern blot experiments revealed that all but the CYP76F1 are induced rapidly and transiently in cell cultures upon elicitor treatment.

Genes expressed in Ascochyta rabiei-inoculated chickpea plants and elicited cell cultures as detected by differential cDNA-hybridization.

In response to the exogenous application of elicitors and attempted invasion by pathogens, plants exhibit a wide range of defense reactions. To understand the defense mechanisms at the level of gene activation and deactivation, differential screenings were performed to isolate cDNA clones which are differentially expressed in pathogen-inoculated resistant chickpea plants and elicitor-treated cell cultures. A plenty of genes were isolated and arranged in 5 groups, namely defense-related pathways, signal transduction pathways, regulation of gene expression, catabolic pathways and primary metabolism. Most of these genes were activated although several genes were also found to be suppressed. We discuss the plausible functions of cDNA products in plant defense responses. The cDNAs provide a variety of tools to investigate molecular mechanisms of defense responses and clearly reflect the massive genomic and metabolic changes which occur during manifestation of antimicrobial defense.

Characterization of cDNAs encoding two glycine-rich proteins in chickpea (Cicer arietinum L.): accumulation in response to fungal infection and other stress factors.

In chickpea plants infected with the pathogenic fungus Ascochyta rabiei [Pass.] Labr. several mRNAs for two glycine-rich proteins (GRPs) were identified by differential cDNA screening. The main part of the deduced amino acid sequences of the 14.6 kD GRP1 and the larger GRP2 consists of glycine-rich repetitive elements essentially as found for GRPs in other plants. Tyrosine residues in conserved positions inside these repetitive motifs suggest an involvement of the GRPs in a polymerization process by oxidative cross-linking, i.e. cell wall fortification. Both GRP transcripts are induced by infection with A. rabiei, showing a maximum of expression 5 days post infection. Wounding of leaves and the stress of water treatment (performed as a control) also seem to induce the accumulation of GRP transcripts.

Characterization and oxidative in vitro cross-linking of an extensin-like protein and a proline-rich protein purified from chickpea cell walls.

Two cell wall proteins from chickpea, known to be rapidly insolubilised by an elicitor-stimulated oxidative burst in-vivo, were purified from suspension cells. N-terminal protein sequencing revealed them as a proline-rich protein and an extensin-like protein. Oxidative cross-linking could be modelled in an in vitro system utilising horseradish peroxidase, H2O2 and the substrate proteins.

The role of lipid peroxidation in aluminium toxicity in soybean cell suspension cultures.

The primary reactions leading to Al toxicity in plant cells have not yet been elucidated. We used soybean (Glycine max [L.] Merr.) cell suspension cultures to address the question whether lipid peroxidation plays an important role in Al toxicity. Upon transfer to an Al-containing culture medium with a calculated Al3+ activity of 15 microM soybean cells showed a distinct and longtime increase in lipid peroxidation within 4 h. At the same time a drastic loss of cell viability was observed. Butylated hydroxyanisole (BHA) and N,N'-diphenyl-p-phenylenediamine (DPPD), two lipophilic antioxidants, were able to almost completely suppress lipid peroxidation in Al-treated cells at a concentration of 20 microM. This effect was dose-dependent for DPPD and was observed at minimum concentrations of 1-2 microM. When lipid peroxidation was suppressed by DPPD or BHA cell viability remained high even in the presence of toxic Al concentrations. These results suggest that Al-induced enhancement of lipid peroxidation is a decisive factor for Al toxicity in suspension cultured soybean cells.

The reaction center-LH1 antenna complex of Rhodobacter sphaeroides contains one PufX molecule which is involved in dimerization of this complex.

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides wild-type cells. PufX is associated with the reaction center-light harvesting 1 (RC-LH1) core complex and plays a key role in lateral ubiquinone/ubiquinol transfer. We have determined the PufX/RC stoichiometry by quantitative Western blot analysis and RC photobleaching. Independent of copy number effects and growth conditions, one PufX molecule per RC was observed in native membranes as well as in detergent-solubilized RC-LH1 complexes which had been purified over sucrose gradients. Surprisingly, two gradient bands with significantly different sedimentation coefficients were found to have a similar subunit composition, as judged by absorption spectroscopy and protein gel electrophoresis. Gel filtration chromatography and electron microscopy revealed that these membrane complexes represent a monomeric and a dimeric form of the RC-LH1 complex. Since PufX is strictly required for the isolation of dimeric core complexes, we suggest that PufX has a central structural role in forming dimeric RC-LH1 complexes, thus allowing efficient ubiquinone/ubiquinol exchange through the LH1 ring surrounding the RC.

Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

A new psbA mutation yielding an amino-acid exchange at the lumen-exposed site of the D1 protein.

In eight metribuzin-resistant photoautotrophic cell cultures of Chenopodium rubrum (Thiemann and Barz, 1994 a, b) sequence analyses of a part of the psbA gene coding for the photosystem-II D1 protein had revealed different double and triple mutations within the herbicide binding niche of the protein (Schwenger-Erger et al., 1993). Two pairs of the examined cell lines carried identical mutations within this part of the protein, although their growth performance and their herbicide resistance patterns were different, both at the cellular and the thylakoid level. Starting from the known part of the psbA gene we have amplified the remaining psbA sequences using inverse polymerase chain reaction. Thus the complete sequence of the coding part of the gene was elucidated. After sequence analyses we found an additional amino acid exchange at the position 184 (ile-->asn) of the D1 protein in cell line L1. Metabolic consequences of this mutation are discussed. Partial sequence analyses of the psbD gene of the herbicide resistant cell culture lines revealed no mutation within that part of the D2 protein, which is in direct contact with the D1 protein.

cDNA cloning and gene expression of three small GTP-binding proteins in defense response of chickpea.

The cDNA clones encoding rab type (INR134 and ELR19) and rac type (ELR26) small GTP-binding proteins were isolated from Ascochyta rabiei-inoculated chickpea leaves and the elicitor-treated cell cultures. Rac type ELR26 showed enhanced expression in inoculated leaves indicating correlation with the defense response.

A functional GTPase domain, but not its transmembrane domain, is required for function of the SRP receptor beta-subunit.

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRalpha and SRbeta, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRbeta. This notion is supported (a) by Srp102p's sequence similarity to SRbeta; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRalpha homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.

Characterization and cloning of cutinase from Ascochyta rabiei.

Ascochyta rabiei, the causal agent of Ascochyta blight on chickpea plants, secretes a cutinase in the culture filtrate when it is induced by cutin or hydroxylated fatty acids. This cutinase is the main esterase in the culture fluids. The enzyme was purified to homogeneity by three successive chromatographic steps. It showed an apparent molecular weight of 22 kD in SDS-PAGE and cleaved ester bonds of 3H-labelled cutin or p-nitrophenylbutyrate with maximal activities around pH 8. As a serine esterase, cutinase is strongly inhibited by organophosphorous compounds and the most effective inhibitor 2,3,5-trichloropyridine-6-(O-methyl-O-n-butyl)-phosphateester++ + (MAT 9564) shows a Ki value of 0.8 nM. The cutinase gene was cloned from a genomic cosmid library by screening with two oligonucleotides directed against cutinase consensus peptides. The gene was subcloned to a 1.7 Kb SaII/HindIII-insert and sequenced. The cutinase gene codes for a 223 amino acid protein with strong homology to other fungal cutinase sequences. The purified cutinase is encoded by a single copy gene.

Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe.

Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions. All strains converted glycitein and daidzein to 6,7,4'-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4'-trihydroxyisoflavone. Three strains transformed daidzein to 7,8,3',4'-tetrahydroxyisoflavone and 6,7,3',4'-tetrahydroxyisoflavone. In addition, two strains formed 6,7,8,4'-tetrahydroxyisoflavone from daidzein. Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3',4'-tetrahydroxyisoflavone. The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation.

Degradation of stachyose, raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi.

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of alpha-galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean alpha-protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the alpha-galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.

Role of PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 1. PufX is required for efficient light-driven electron transfer and photophosphorylation under anaerobic conditions.

The pufX gene is essential for photoheterotrophic growth of the purple bacterium Rhodobacter sphaeroides. In order to analyze the molecular function of the PufX membrane protein, we constructed a chromosomal pufX deletion mutant and phenotypically compared it to a pufX+ control strain and to two suppressor mutants which are able to grow photosynthetically in the absence of pufX. Using this genetic background, we confirmed that PufX is required for photoheterotrophic growth under anaerobic conditions, although all components of the photosynthetic apparatus were present in similar amounts in all strains investigated. We show that the deletion of PufX is not lethal for illuminated pufX- cells, suggesting that PufX is required for photosynthetic cell division. Since chromatophores isolated from the pufX- mutant were found to be unsealed vesicles, the role of PufX in photosynthetic energy transduction was studied in vivo. We show that PufX is essential for light-induced ATP synthesis (photophosphorylation) in anaerobically incubated cells. Measurements of absorption changes induced by a single turnover flash demonstrated that PufX is not required for electron flow through the reaction center and the cytochrome bc1 complex under anaerobic conditions. During prolonged illumination, however, PufX is essential for the generation of a sufficiently large membrane potential to allow photosynthetic growth. These in vivo results demonstrate that under anaerobic conditions PufX plays an essential role in facilitating effective interaction of the components of the photosynthetic apparatus.

Role of the PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 2. PufX is required for efficient ubiquinone/ubiquinol exchange between the reaction center QB site and the cytochrome bc1 complex.

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides because it is required for multiple-turnover electron transfer under anaerobic conditions [see accompanying article; Barz, W. P., Francia, F., Venturoli, G., Melandri, B. A., Verméglio, A., & Oesterhelt, D. (1995) Biochemistry 34, 15235-15247]. In order to understand the molecular role of PufX, light-induced absorption spectroscopy was performed using a pufX- mutant, a pufX+ strain, and two suppressor mutants. We show that the reaction center (RC) requires PufX for its functionality under different redox conditions than the cytochrome bc1 complex: When the kinetics of flash-induced reduction of cytochrome b561 were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome bc1 complex only at high redox potential (Eh > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. In contrast, PufX is required for multiple turnover of the RC only under reducing conditions: When the Q pool was partially oxidized in vivo using oxygen or electron acceptors like dimethyl sulfoxide or trimethylamine N-oxide, the deletion of PufX had no effect on light-driven electron flow through the RC. Flash train experiments under anaerobic in vivo conditions revealed that RC photochemistry does not depend on PufX for the first two flash excitations. Following the third and subsequent flashes, however, efficient charge separation requires PufX, indicating an important role of PufX for fast Q/QH2 exchange at the QB site of the RC. We show that the Q/QH2 exchange rate is reduced approximately 500-fold by the deletion of PufX when the Q pool is nearly completely reduced, demonstrating an essential role of PufX for the access of ubiquinone to the QB site. The fast ubiquinone/ubiquinol exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in structurally organizing a functional photosynthetic apparatus.