A Novel Function for Arabidopsis CYCLASE1 in Programmed Cell Death Revealed by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Analysis of Extracellular Matrix Proteins.

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 Arabidopsis cell 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 CYCLASE1 gene 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 CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout 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.

Transcriptomic analysis comparing stay-green and senescent Sorghum bicolor lines identifies a role for proline biosynthesis in the stay-green trait.

Sorghum bicolor is an important cereal crop grown on the arid and semi-arid regions of >98 different countries. These regions are such that this crop is often subjected to low water conditions, which can compromise yields. Stay-green sorghum plants are able to retain green leaf area for longer under drought conditions and as such have higher yields than their senescent counterparts. However, the molecular and physiological basis of this drought tolerance is yet to be fully understood. Here, a transcriptomic approach was used to compare gene expression between stay-green (B35) and senescent (R16) sorghum varieties. Ontological analysis of the differentially expressed transcripts identified an enrichment of genes involved with the 'response to osmotic stress' Gene Ontology (GO) category. In particular, delta1-pyrroline-5-carboxylate synthase 2 (P5CS2) was highly expressed in the stay-green line compared with the senescent line, and this high expression was correlated with higher proline levels. Comparisons of the differentially expressed genes with those that lie in known stay-green qualitative trait loci (QTLs) revealed that P5CS2 lies within the Stg1 QTL. Polymorphisms in known cis-elements were identified in the putative promoter region of P5CS2 and these could be responsible for the differences in the expression of this gene. This study provides greater insight into the stay-green trait in sorghum. This will be greatly beneficial not only to improve our understanding of drought tolerance mechanisms in sorghum, but also to facilitate the improvement of future sorghum cultivars by marker-assisted selection (MAS).

Transcriptomic analysis of Sorghum bicolor responding to combined heat and drought stress.

BACKGROUND: Abiotic stresses which include drought and heat are amongst the main limiting factors for plant growth and crop productivity. In the field, these stress types are rarely presented individually and plants are often subjected to a combination of stress types. Sorghum bicolor is a cereal crop which is grown in arid and semi-arid regions and is particularly well adapted to the hot and dry conditions in which it originates and is now grown as a crop. In order to better understand the mechanisms underlying combined stress tolerance in this important crop, we have used microarrays to investigate the transcriptional response of Sorghum subjected to heat and drought stresses imposed both individually and in combination. RESULTS: Microarrays consisting of 28585 gene probes identified gene expression changes equating to ~4% and 18% of genes on the chip following drought and heat stresses respectively. In response to combined stress ~20% of probes were differentially expressed. Whilst many of these transcript changes were in common with those changed in response to heat or drought alone, the levels of 2043 specific transcripts (representing 7% of all gene probes) were found to only be changed following the combined stress treatment. Ontological analysis of these 'unique' transcripts identified a potential role for specific transcription factors including MYB78 and ATAF1, chaperones including unique heat shock proteins (HSPs) and metabolic pathways including polyamine biosynthesis in the Sorghum combined stress response. CONCLUSIONS: These results show evidence for both cross-talk and specificity in the Sorghum response to combined heat and drought stress. It is clear that some aspects of the combined stress response are unique compared to those of individual stresses. A functional characterization of the genes and pathways identified here could lead to new targets for the enhancement of plant stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of these abiotic stress types.

Proteomics reveals new insights into the role of light in cadmium response in Arabidopsis cell suspension cultures.

Light plays an important role in plant growth, development, and response to environmental stresses. To investigate the effects of light on the plant responses to cadmium (Cd) stress, we performed a comparative physiological and proteomic analysis of light- and dark-grown Arabidopsis cells after exposure to Cd. Treatment with different concentrations of Cd resulted in stress-related phenotypes such as cell growth inhibition and decline of cell viability. Notably, light-grown cells were more sensitive to heavy metal toxicity than dark-grown cells, and the basis for this appears to be the elevated Cd accumulation, which is twice as much under light than dark growth conditions. Protein profiles analyzed by 2D DIGE revealed a total of 162 protein spots significantly changing in abundance in response to Cd under at least one of these two growing conditions. One hundred and ten of these differentially expressed protein spots were positively identified by MS/MS and they are involved in multiple cellular responses and metabolic pathways. Sulfur metabolism-related proteins increased in relative abundance both in light- and dark-grown cells after exposure to Cd. Proteins involved in carbohydrate metabolism, redox homeostasis, and anti-oxidative processes were decreased both in light- and dark-grown cells, with the decrease being lower in the latter case. Remarkably, proteins associated with cell wall biosynthesis, protein folding, and degradation showed a light-dependent response to Cd stress, with the expression level increased in darkness but suppressed in light. The possible biological importance of these changes is discussed.

UDP-glucose pyrophosphorylase is a novel plant cell death regulator.

Programmed cell death (PCD) is an essential process that functions in plant organ sculpture, tissue differentiation, nutrient recycling, and defense against pathogen attack. A full understanding of the mechanism of PCD in plants is hindered by the limited identification of protein components of the complex signaling circuitry that underpins this important physiological process. Here we have used Arabidopsis thaliana and fumonisin B1 (FB1) to identify proteins that constitute part of the PCD signaling network. We made an inadvertent, but important observation that exogenous sucrose modulates FB1-induced cell death and identified sucrose-induced genes from publicly available transcriptomic data sets for reverse genetic analyses. Using transfer-DNA gene knockout plants, UDP-glucose pyrophosphorylase 1 (UGP1), a sucrose-induced gene, was demonstrated to be a critical factor that regulates FB1-induced PCD. We employed 2D-DiGE to identify proteomic changes preceding PCD after exposure of Arabidopsis to FB1 and used UGP1 knockout plants to refine the analysis and isolate downstream candidate proteins with a putative PCD regulatory function. Our results reveal chloroplasts as the predominantly essential organelles in FB1-induced PCD. Overall, this study reveals a novel function of UGP1 as a cell death regulator and provides candidate proteins likely recruited downstream in the activation of plant PCD.

Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis.

Phospholipid biosynthesis is essential for the construction of most eukaryotic cell membranes, but how this process is regulated in plants remains poorly understood. Here, we show that in Arabidopsis thaliana, two Mg(2+)-dependent phosphatidic acid phosphohydrolases called PAH1 and PAH2 act redundantly to repress phospholipid biosynthesis at the endoplasmic reticulum (ER). Leaves from pah1 pah2 double mutants contain ~1.8-fold more phospholipid than the wild type and exhibit gross changes in ER morphology, which are consistent with massive membrane overexpansion. The net rate of incorporation of [methyl-(14)C]choline into phosphatidylcholine (PC) is ~1.8-fold greater in the double mutant, and the transcript abundance of several key genes that encode enzymes involved in phospholipid synthesis is increased. In particular, we show that PHOSPHORYLETHANOLAMINE N-METHYLTRANSFERASE1 (PEAMT1) is upregulated at the level of transcription in pah1 pah2 leaves. PEAMT catalyzes the first committed step of choline synthesis in Arabidopsis and defines a variant pathway for PC synthesis not found in yeasts or mammals. Our data suggest that PAH1/2 play a regulatory role in phospholipid synthesis that is analogous to that described in Saccharomyces cerevisiae. However, the target enzymes differ, and key components of the signal transduction pathway do not appear to be conserved.

Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthase beta-subunit as a novel plant cell death regulator.

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase ß-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase ß-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.

Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation.

Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14)C]acetate, but stimulated that from [U-14C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27±0.06 for Block A and 0.73±0.06 for Block B. The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.

Proteomics reveals a role for the RNA helicase crhR in the modulation of multiple metabolic pathways during cold acclimation of Synechocystis sp. PCC6803.

One of the earliest and largest transcriptional responses that occur during exposure of Synechocystis sp. PCC6803 to cold is the induction of the crhR RNA helicase transcript. We show that crhR deletion results in failure to cold acclimate: there is reduced growth at 24 °C and marked impairment of growth at 20 °C. 2D-DIGE, using five biological replicates, was used to analyze the proteomic differences between the wild-type and ΔcrhR strains grown at (1) 34 °C and (2) following transfer from 34 to 24 °C (cold-acclimation). Sixteen significantly differentially expressed proteins were identified between the two strains grown at 34 °C. Forty-three distinct proteins were identified that responded to cold-acclimation of the wild-type and 34 proteins for the mutant, with only 26 proteins common to both. A large proportion of the proteomic responses (76.5%) could not be predicted from published transcriptomic data. Only modest similarity is observed between proteomic and transcriptomic responses (r = 0.54-0.70). We propose functions for three previously hypothetical proteins. We suggest molecular targets for CrhR action and identify downstream regulated events in metabolism.

Components of complex lipid biosynthetic pathways in developing castor (Ricinus communis) seeds identified by MudPIT analysis of enriched endoplasmic reticulum.

Ricinoleic acid is a feedstock for nylon-11 (N11) synthesis which is currently obtained from castor (Ricinus communis) oil. Production of this fatty acid in a temperate oilseed crop is of great commercial interest, but the highest reported level in transgenic plant oils is 30%, below the 90% observed in castor and insufficient for commercial exploitation. To identify castor oil-biosynthetic enzymes and inform strategies to improve ricinoleic acid yields, we performed MudPIT analysis on endoplasmic reticulum (ER) purified from developing castor bean endosperm. Candidate enzymes for all steps of triacylglycerol synthesis were identified among 72 proteins in the data set related to complex-lipid metabolism. Previous reported proteomic data from oilseeds had not included any membrane-bound enzyme that might incorporate ricinoleic acid into oil. Analysis of enriched ER enabled determination of which protein isoforms for these enzymes were in developing castor seed. To complement this data, quantitative RT-PCR experiments with castor seed and leaf RNA were performed for orthologues of Arabidopsis oil-synthetic enzymes, determining which were highly expressed in the seed. These data provide important information for further manipulation of ricinoleic acid content in oilseeds and peptide data for future quantification strategies.

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature.

The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 degrees C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 degrees C to 38 degrees C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.

Differential proteomic analysis using iTRAQ reveals changes in thylakoids associated with Photosystem II-acquired thermotolerance in Synechocystis sp. PCC 6803.

Growth temperature has a marked influence on the thermotolerance of photosystem II (PSII), which is the most heat-sensitive component of photosynthesis. Using Synechocystis sp. PCC 6803 we have established that thylakoids isolated from cells grown at 38 degrees C have a greater degree of thermotolerance than those isolated from cells grown at 25 degrees C. Reconstitution experiments using Triton X-100 protein extracts of these thylakoids added to Triton-treated thylakoid membranes further indicated that the 38 degrees C Triton extract contains proteins that are directly capable of enhancing PSII thermotolerance. We have used 4-plex iTRAQ, extensive off-line fractionation and sample re-injection to comprehensively identify the differences between these two preparations that may be responsible for the observed effects on PSII thermotolerance. This has resulted in the reproducible identification of 168 proteins out of a total of 385 distinct proteins. Our results have identified 15 proteins whose levels are increased in extracts that result in increased thermotolerance of PSII and 33 proteins whose levels decrease. Notably, components of the cytochrome b(6)/f and NADH dehydrogenase complexes, crucial components in electron transport, are approximately twofold more abundant in 38 degrees C thylakoid extracts. The possible biological importance of these changes is discussed.

The effects of extracellular adenosine 5'-triphosphate on the tobacco proteome.

Extracellular adenosine 5'-triphosphate (eATP) is emerging as an important plant signalling compound capable of mobilising intracellular second messengers such as Ca(2+), nitric oxide, and reactive oxygen species. However, the downstream molecular targets and the spectrum of physiological processes that eATP regulates are largely unknown. We used exogenous ATP and a non-hydrolysable analogue as probes to identify the molecular and physiological effects of eATP-mediated signalling in tobacco. 2-DE coupled with MS/MS analysis revealed differential protein expression in response to perturbation of eATP signalling. These proteins are in several functional classes that included photosynthesis, mitochondrial ATP synthesis, and defence against oxidative stress, but the biggest response was in the pathogen defence-related proteins. Consistent with this, impairment of eATP signalling induced resistance against the bacterial pathogen Erwinia carotovora subsp. carotovora. In addition, disease resistance activated by a fungal pathogen elicitor (xylanase from Trichoderma viride) was concomitant with eATP depletion. These results reveal several previously unknown putative molecular targets of eATP signalling, which pinpoint eATP as an important hub at which regulatory signals of some major primary metabolic pathways and defence responses are integrated.

Extracellular ATP is a regulator of pathogen defence in plants.

In healthy plants extracellular ATP (eATP) regulates the balance between cell viability and death. Here we show an unexpected critical regulatory role of eATP in disease resistance and defensive signalling. In tobacco, enzymatic depletion of eATP or competition with non-hydrolysable ATP analogues induced pathogenesis-related (PR) gene expression and enhanced resistance to tobacco mosaic virus and Pseudomonas syringae pv. tabaci. Artificially increasing eATP concentrations triggered a drop in levels of the important defensive signal chemical salicylic acid (SA) and compromised basal resistance to viral and bacterial infection. Inoculating tobacco leaf tissues with bacterial pathogens capable of activating PR gene expression triggered a rapid decline in eATP. Conversely, inoculations with mutant bacteria unable to induce defence gene expression failed to deplete eATP. Furthermore, a collapse in eATP concentration immediately preceded PR gene induction by SA. Our study reveals a previously unsuspected role for eATP as a negative regulator of defensive signal transduction and demonstrates its importance as a key signal integrating defence and cell viability in plants.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channels.

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Structural studies of fatty acyl-(acyl carrier protein) thioesters reveal a hydrophobic binding cavity that can expand to fit longer substrates.

A knowledge of the structures of acyl chain loaded species of the acyl carrier protein (ACP) as used in fatty acid biosynthesis and a range of other metabolic events, is essential for a full understanding of the molecular recognition at the heart of these processes. To date the only crystal structure of an acylated species of ACP is that of a butyryl derivative of Escherichia coli ACP. We have now determined the structures of a family of acylated E. coli ACPs of varying acyl chain length. The acyl moiety is attached via a thioester bond to a phosphopantetheine linker that is in turn bound to a serine residue in ACP. The growing acyl chain can be accommodated within a central cavity in the ACP for transport during the elongation stages of lipid synthesis through changes in the conformation of a four alpha-helix bundle. The results not only clarify the means by which a substrate of varying size and complexity is transported in the cell but also suggest a mechanism by which interacting enzymes can recognize the loaded ACP through recognition of surface features including the conformation of the phosphopantetheine linker.

Isolation and fractionation of the endoplasmic reticulum from castor bean (Ricinus communis) endosperm for proteomic analyses.

This chapter describes the preparation and isolation of highly purified endoplasmic reticulum (ER) from the endosperm of developing and germinating castor bean (Ricinus communis) seeds to provide a purified organelle fraction for differential proteomic analyses. The method uses a two-step ultracentrifugation protocol first described by Coughlan (1) and uses sucrose density gradients and a sucrose flotation step to yield purified ER devoid of other contaminating endomembrane material. Using a combination of one dimensional (1D) and two dimensional (2D) gel electrophoresis the complexity and reproducibility of the protein profile of the purified organelle is evaluated prior to detailed proteomic analyses using mass spectrometry based techniques.

Candida yeast long chain fatty alcohol oxidase is a c-type haemoprotein and plays an important role in long chain fatty acid metabolism.

The industrial yeasts Candida tropicalis or Candida cloacae are able to grow on a variety of long chain alkanes and fatty acids as the sole carbon source. The complete oxidation of these substrates involves two sequential oxidative pathways: omega-oxidation, comprising the P450 alkane oxidase, a flavin-dependent membrane-bound long chain fatty alcohol oxidase [FAO] and a possible separate aldehyde oxidase [F.M. Dickinson, C. Wadforth, Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis, Biochem. J. 282 (1992) 325-331], and the beta-oxidation pathway, which utilises acylCoA substrates. We recently purified the membrane-bound long chain fatty alcohol oxidase FAO1 and confirmed it is also a c-type haemoprotein. Multiple isoforms may exist for many of these long chain fatty alcohol oxidases and the in vivo requirements for individual genes with respect to specific substrates are still being elucidated. In vitro reconstitution experiments have demonstrated that in Candida maltosa, the cytochrome P450 52A3 gene product can completely oxidise alkanes to dicarboxylic acids [U. Scheller, T. Zimmer, D. Becher, F. Schauer, W. Schunck, Oxygenation Cascade in Conversion of n-Alkanes to, -Dioic Acids Catalyzed by Cytochrome P450 52A3, J. Biol. Chem. 273 (1998) 32528-32534], potentially obviating requirements for a long chain alcohol oxidase. Here, we directly determine in vivo the role of the long chain alcohol oxidase (FAOT) in C. tropicalis, grown on a variety of substrates, followed by gene deletion. The faot double knockout has no detectable faot activity and is incapable of growth on octadecane, but it grows well on oleic acid, palmitic acid and shorter chain alkanes/fatty acids. A spontaneous mutation[s] may have occurred in the faot double gene knockout of C. tropicalis resulting in its inability to grow on oleic acid and hexadecane. The mutations demonstrate that different pathways of octadecane, hexadecane, oleic acid and palmitic acid utilisation exist in C. tropicalis.

Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals.

Seed oil from castor bean (Ricinus communis) contains high amounts of hydroxy fatty acid rich triacylglycerols (TAGs) that can serve as raw material for production of bio-based products such as nylon, cosmetics, lubricants, foams, and surfactants. Diacylglycerol acyltransferase (DGAT) catalyses the terminal reaction in the acyl-CoA dependent Kennedy pathway of triglyceride biosynthesis. There is still some debate whether there are three or four enzymes in yeast that have DGAT activity and catalyse the synthesis of TAG but of these the DGAT2 homologue Dga1 contributes in a major way to TAG biosynthesis. Here we report on the cloning of a cDNA for DGAT2 from castor bean and prove its biological activity following expression in yeast and enzymatic assays using diricinolein as the acceptor and ricinoleoyl-CoA as the donor. Previous reports of DGAT in castor have focussed on DGAT1 which has little amino acid sequence homology to DGAT2. Expressional studies demonstrate that DGAT2 is 18-fold more highly expressed in seeds than in leaves and shows temporal specific expression during seed development. In contrast, DGAT1 shows little difference in expression in seeds versus leaves. We conclude that in castor bean DGAT2 is more likely to play a major role in seed TAG biosynthesis than DGAT1.

X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site.

Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps. We have determined the first crystal structures of an acylated form of ACP from E. coli, that of butyryl-ACP. Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and beta-mercaptoethylamine moieties of the acylated 4'-phosphopantetheine group in one of our crystal forms. In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.