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Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell–cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsiscell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which had no known function hitherto, is a negative regulator of cell death and regulates pathogen-induced symptom development in Arabidopsis.

In plants, fatty acid and complex lipid synthesis requires the correct spatial and temporal activity of many gene products. Quantitative northern analysis showed that mRNA for the biotin carboxylase subunit of heteromeric acetyl-coenzyme A carboxylase, fatty acid synthase components (3-oxoacyl-acyl carrier protein [ACP] reductase, enoyl-ACP reductase, and acyl-ACP thioesterase), and stearoyl-ACP desaturase accumulate in a coordinate manner during Brassica napus embryogenesis. The mRNAs were present in a constant molar stoichiometric ratio. Transcript abundance of mRNAs for the catalytic proteins was found to be similar, whereas the number of ACP transcripts was approximately 7-fold higher. The peak of mRNA accumulation of all products was between 20 and 29 d after flowering; by 42 d after flowering, the steady-state levels of all transcripts fell to about 5% of their peak levels, which suggests that the mRNAs have similar stability and kinetics of synthesis. Biotin carboxylase was found to accumulate to a maximum of 59 fmol mg(-1) total RNA in embryos, which is in general agreement with the value of 170 fmol mg(-1) determined for Arabidopsis siliques (J.S. Ke, T.N. Wen, B.J. Nikolau, E.S. Wurtele [2000] Plant Physiol 122: 1057-1071). Embryos accumulated between 3- and 15-fold more transcripts per unit total RNA than young leaf tissue; the lower quantity of leaf 3-oxoacyl-ACP reductase mRNA was confirmed by reverse transcriptase-polymerase chain reaction. This is in conflict with analysis of B. napus transcripts using an Arabidopsis microarray (T. Girke, J. Todd, S. Ruuska, J. White, C. Benning, J. Ohlrogge [2000] Plant Physiol 124: 1570-1581) where similar leaf to seed levels of fatty acid synthase component mRNAs were reported.

In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3‐oxoacyl‐ACP reductase. Using a novel radio‐chemical assay for 3‐oxoacyl‐ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post‐emergence, but slowed after this point even though 3‐oxoacyl‐ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis.

In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3-oxoacyl-ACP reductase. Using a novel radio-chemical assay for 3-oxoacyl-ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post-emergence, but slowed after this point even though 3-oxoacyl-ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis.

The yeast Candida cloacaeis capable of growing on alkanes and fatty acids as sole carbon sources. Transfer of cultures from a glucose medium to one containing oleic acid induced seven proteins of Mr102,000, 73,000, 61,000, 54,000, and 46,000 and two in the region ofMr45,000 and repressed a protein ofMr64,000. The induction of theMr73,000 protein reached a 7-fold maximum 24 h after induction. The protein was confirmed by its enzyme activity to be a long-chain fatty-acid alcohol oxidase (LC-FAO) and purified to homogeneity from microsomes by a rapid procedure involving hydrophobic chromatography. An internal peptide of 30 amino acids was sequenced. A 1100-base pair cDNA fragment containing the LC-FAO peptide coding sequence was used to isolate a single exon genomic clone containing the full-length coding sequence of an LC-FAO (fao1). The fao1gene product was expressed inEscherichia coliand was translated as a functional long-chain alcohol oxidase, which was present in the membrane fraction. In addition, full-length coding sequences for a Candida tropicalisLC-FAO (faoT) and a second C. cloacaeLC-FAO (fao2) were isolated. The DNA sequences obtained had open reading frames of 2094 (fao1), 2091 (fao2), and 2112 (faoT) base pairs. The derived amino acid sequences of fao2and faoTshowed 89.4 and 76.2% similarities to fao1. Thefao1gene is much more highly induced on alkane than isfao2. Although this study describes the first known DNA sequences encoding LC-FAOs from any source, there are unassignedArabidopsissequences and an unassignedMycobacteriumsequence in the GenBankTMData Bank that show strong homology to the described LC-FAO sequences. The conservation of sequence between yeast, plants, and bacteria suggests that an as yet undescribed family of long-chain fatty-acid oxidases exists in both eukaryotes and prokaryotes.

Sequence analysis of several cDNAs encoding the phasin protein of Ralstonia eutrophaindicated that the carboxyl terminus of the resulting derived protein sequence is different from that reported previously. This was confirmed by: (1) sequencing of the genomic DNA; (2) SDS‐PAGE and peptide analysis of wild‐type and recombinant phasin; and (3) mass spectrometry of wild‐type phasin protein. The results have implications for the model proposed for the binding of this protein to polyhydroxyalkanoic acid granules in the bacterium.

Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia colihas been determined to 2.1 Å resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 Å to 2.1 Å to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel β-sheet flanked on each side by three α-helices with a further helix lying at the C terminus of the β-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.

A major wall protein of suspension-cultured cells of French bean has been isolated and characterised. It can be prepared from walls or the culture filtrate and in composition it is particularly rich in proline, valine and glutamic acid/glutamine and contains appreciable amounts of hydroxyproline. The N-terminus shows some glycosylation, while following chemical deglycosylation the first 38 residues were found to be identical to those of proline-rich proteins from soybean. However, the composition of the highly purified Mr-42000 bean protein differs considerably from the soybean proteins and must contain its own specific domains. An antibody was raised and used to demonstrate the inducibility of the Mr-42000 bean protein in response to elicitor action. The protein was found to be mainly localised in the intercellular spaces of the cortical cells of bean hypocotyls and at the wall-plasmalemma interface of xylem vessels, another potentially accessible compartment for pathogens. Following wounding, the protein was found to be generally distributed in the wall of epidermal and cortical cells of the hypocotyls. The Mr-42000 protein is cross reactive with antibodies raised to glycoproteins of the Rhizobium infection thread and the chitin-binding hydroxyproline-rich glycoprotein, potato lectin. These common epitopes together with the previously demonstrated chitin-binding properties of the bean protein indicate a role in host-microbial interactions. Furthermore, the Mr-42000 protein itself bound to the growing hyphal tips of the bean pathogen, Colletotrichum lindemuthianum.

Cytoskeletons prepared from carrot protoplasts often retain the nucleus, which is associated with bundles of 7 nm fibrils. These were also seen in cytoskeletons from oil palm, maize and Italian rye grass cells. By examining such cytoskeletons by immunofluorescence, negative staining and stereo scanning electron microscopy it appears that the bundles could be part of a more delicate system of fibrils distal to the nucleus. Optical diffraction of the bundles, staining with heavy meromyosin, drug treatments and phalloidin-staining all confirm that the bundles are not composed of actin. Bundles could not be isolated from such cytoskeletons in quantities sufficient for analysis, but by disrupting protoplasts prepared from stationary-phase cells it was possible to purify fibrillar bundles that appeared in the lysate. These were similar in size and properties to the nucleus-associated bundles. The former were shown by sodium dodecyl sulphate/polyacrylamide gel electrophoresis to be composed of several proteins and were sensitive to reducing agents. Since they are not composed of actin, the possibility was explored that these fibrils are related to the P-proteins of phloem sieve tube and accessory cells.