Lipid Body Protein Research Articles

Genetic merit of sires for ad libitum residual feed intake affects feed efficiency of restricted-fed heavy pigs but not body weight gain tissue composition.

The study aimed at evaluating how sires, classified for their additive genetic effects on residual feed intake (RFI) of ad libitum-fed progeny, influence growth performance, tissue accretion, and gain composition in restricted-fed offspring (96-168 kg body weight, BW). A total of 416 purebred C21 Goland pigs, offspring of 23 sires, were randomly allocated to three feeding groups: ad libitum, restricted medium-protein, or restricted low-protein. Empty BW, body lipid mass and body protein mass were estimated from individual BW and backfat measures using literature equations. Residuals of a linear regression of average daily feed intake on average empty BW, body lipid and protein daily gains were used as estimates of individual RFI in ad libitum-fed pigs. Additive genetic effects of sires on RFI of ad libitum-fed pigs were estimated with a linear animal model and solutions of the model were used to allocate the sires to low- (LRFI), medium- (MRFI), or high-RFI (HRFI) groups. Restricted-fed progeny of LRFI sires exhibited reduced average daily feed intake (-3%) compared to MRFI and HRFI progeny. This indicates that LRFI progeny make a more efficient use of energy intake and implies that variation in RFI across families, assessed under ad libitum feeding, is related to the across-family variation in feed efficiency detected under restricted feeding. LRFI progeny exhibited also a lower feed conversion ratio (-11%), partially resulting from of a 3% increase in growth rate compared with HRFI. Thus, LRFI progeny consumed less feed, while growing at a similar or slightly higher rate than MRFI or HRFI. No significant differences across sire classes were observed for daily tissue accretion and gain composition. Hence, we can hypothesise that efficient sires would not affect carcass leanness of heavy pig progeny fed restricted.

Genome-Wide Identification and Expression Profiling of B3 Transcription Factor Genes in Prunus armeniaca

The B3 superfamily, which belongs to the plant-specific transcription factors, is widely involved in multiple biological processes. In apricot (Prunus armeniaca), the classification, structure, and function of the B3 superfamily are not yet clear. In this study, a total of 75 B3 genes were identified from the apricot genome. The apricot B3 superfamily can be divided into five subfamilies, i.e., REM, ARF, ABI3, RAV, and HSI, and genes in the same subfamily have similar structures. The distribution of B3 genes on chromosomes presents a clustering phenomenon. Tandem duplication is the main mode of apricot B3 family expansion, and gene duplication mainly occurs in the REM and ARF subfamilies. Many B3 genes originated from a common ancestor of Arabidopsis and apricot before lineage divergence, and ancestor genes expanded fewer times in apricot than in Arabidopsis. Gene ontology analysis showed that apricot B3 genes were closely related to vernalization, flower development, and shoot system development. PaABI3-1 and PaABI3-2 might play a positive regulation role in the transcription of PaOleosin, which encodes a lipid body protein. This study lays a foundation for the further study of the B3 superfamily function in apricot, especially the specific functions of the ABI3 subfamily in apricot kernel oil storage.

Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa

BackgroundLobosphaera incisa (L. incisa) is an oleaginous microalga that stores triacylglycerol (TAG) rich in arachidonic acid in lipid bodies (LBs). This organelle is gaining attention in algal research, since evidence is accumulating that proteins attached to its surface fulfill important functions in TAG storage and metabolism.ResultsHere, the composition of the LB proteome in L incisa was investigated by comparing different cell fractions in a semiquantitative proteomics approach. After applying stringent filters to the proteomics data in order to remove contaminating proteins from the list of possible LB proteins (LBPs), heterologous expression of candidate proteins in tobacco pollen tubes, allowed us to confirm 3 true LBPs: A member of the algal Major Lipid Droplet Protein family, a small protein of unknown function and a putative lipase. In addition, a TAG lipase that belongs to the SUGAR DEPENDENT 1 family of TAG lipases known from oilseed plants was identified. Its activity was verified by functional complementation of an Arabidopsis thaliana mutant lacking the major seed TAG lipases.ConclusionsHere we describe 3 LBPs as well as a TAG lipase from the oleaginous microalga L. incisa and discuss their possible involvement in LB metabolism. This study highlights the importance of filtering LB proteome datasets and verifying the subcellular localization one by one, so that contaminating proteins can be recognized as such. Our dataset can serve as a valuable resource in the identification of additional LBPs, shedding more light on the intriguing roles of LBs in microalgae.

Daily walnut consumption effects lipid profile and body protein in Korean subjects with metabolic syndrome

The objective of current study was to assess the gender differences on the lipid profiles, apoprotein B, and body composition among the subjects with metabolic syndrome after daily walnut intake (45 g) for 16 weeks. 89 participants (44 men, 40±6.2yr) with metabolic syndrome were screened if they meet at least three metabolic syndrome components out of five diagnosis criteria (i.e., waist circumstance ≥90cm for men and ≥85cm for women, blood pressure 130/85mmHg or higher, HDL‐cholesterol ≤40mg/dL for men and ≤50mg/dL for women, triglyceride level ≥150mg/dL, and fasting blood glucose ≥100mg/dL). Blood lipid profiles (consisting of triglyceride, total cholesterol, HDL‐cholesterol, and LDL‐cholesterol), apoprotein B, fasting blood sugar, anthropometric measurement, bioimpedance analysis, and blood pressure were measured at baseline and week 16. The significance of the differences between baseline and week 16 were analyzed using the paired t‐test or Wilcoxon signed‐rank test, as appropriate (p<0.05). Body protein of male and all subjects at week 16 was significantly higher compared to the baseline (p=0.026, p=0.039, respectively), whereas no significance among female subjects (p=0.876). Male showed significantly increased basal metabolic rate at week 16 (p=0.017), but not significant among female subjects (p=0.581) and all subjects (p=0.12). Waist circumstance among female and all subjects were significantly lower compared to baseline (p=0.036 and 0.026, respectively), and marginally significant among male subjects (p=0.050). LDL‐cholesterol and total cholesterol levels were significantly decreased among male subjects (p=0.011 and 0.003, respectively), whereas those among female subjects were not significantly different (p=0.307, and 0.072, respectively). Triglyceride level was significantly decreased among female subjects compared with baseline (p=0.007) by Wilcoxon test. The positive change of apoprotein B among male and all subjects were significant compared with the baseline (p=0.001 and 0.003, respectively), whereas those among female subjects were not significant (p=0.282). The results suggest that daily walnut consumption for 16 weeks may increase body protein and basal metabolic rate, and decrease the levels of LDL‐cholesterol, cholesterol and apoprotein B among male subjects with metabolic syndrome. Also, daily walnut intake may decrease waist circumstance and triglyceride level among female subjects with metabolic syndrome.IRB(SMWU‐1508‐BR‐051‐01)Support or Funding InformationCalifornia Walnut Commission

Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

Abiotic stress in oleaginous microalgae enhances lipid accumulation and is stored in a specialised organelle called lipid droplets (LDs). Both the LDs and body are enriched with major lipid droplet protein (MLDP). It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of S. quadricauda under the salt stress of 10mM concentration is about 0.174μ and in control, the SGR is 0.241μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17). The dry biomass content also decreased drastically at 50mM salt-treated cells (129mg/L) compared to control (236mg/L) on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

Comparative Genomics of the Lipid-body-membrane Proteins Oleosin, Caleosin and Steroleosin in Magnoliophyte, Lycophyte and Bryophyte

Lipid bodies store oils in the form of triacylglycerols. Oleosin, caleosin and steroleosin are unique proteins localized on the surface of lipid bodies in seed plants. This study has identified genes encoding lipid body proteins oleosin, caleosin and steroleosin in the genomes of five plants: Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Selaginella moellendorffii and Physcomitrella patens. The protein sequence alignment indicated that each oleosin protein contains a highly-conserved proline knot motif, and proline knob motif is well conserved in steroleosin proteins, while caleosin proteins possess the Dx[D/N]xDG-containing calcium-binding motifs. The identification of motifs (proline knot and knob) and conserved amino acids at active site was further supported by the sequence logos. The phylogenetic analysis revealed the presence of magnoliophyte- and bryophyte-specific subgroups. We analyzed the public microarray data for expression of oleosin, caleosin and steroleosin in Arabidopsis and rice during the vegetative and reproductive stages, or under abiotic stresses. Our results indicated that genes encoding oleosin, caleosin and steroleosin proteins were expressed predominantly in plant seeds. This work may facilitate better understanding of the members of lipid-body-membrane proteins in diverse organisms and their gene expression in model plants Arabidopsis and rice.

Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-beta-glucanases to reopen signal conduits and release dormancy in Populus.

In trees, production of intercellular signals and accessibility of signal conduits jointly govern dormancy cycling at the shoot apex. We identified 10 putative cell wall 1,3-β-glucanase genes (glucan hydrolase family 17 [GH17]) in Populus that could turn over 1,3-β-glucan (callose) at pores and plasmodesmata (PD) and investigated their regulation in relation to FT and CENL1 expression. The 10 genes encode orthologs of Arabidopsis thaliana BG_ppap, a PD-associated glycosylphosphatidylinositol (GPI) lipid-anchored protein, the Arabidopsis PD callose binding protein PDCB, and a birch (Betula pendula) putative lipid body (LB) protein. We found that these genes were differentially regulated by photoperiod, by chilling (5°C), and by feeding of gibberellins GA(3) and GA(4). GA(3) feeding upregulated all LB-associated GH17s, whereas GA(4) upregulated most GH17s with a GPI anchor and/or callose binding motif, but only GA(4) induced true bud burst. Chilling upregulated a number of GA biosynthesis and signaling genes as well as FT, but not CENL1, while the reverse was true for both GA(3) and GA(4). Collectively, the results suggest a model for dormancy release in which chilling induces FT and both GPI lipid-anchored and GA(3)-inducible GH17s to reopen signaling conduits in the embryonic shoot. When temperatures rise, the reopened conduits enable movement of FT and CENL1 to their targets, where they drive bud burst, shoot elongation, and morphogenesis.

High water solubility and fold in amphipols of proteins with large hydrophobic regions: Oleosins and caleosin from seed lipid bodies

Seed lipid bodies constitute natural emulsions stabilized by specialized integral membrane proteins, among which the most abundant are oleosins, followed by the calcium binding caleosin. These proteins exhibit a triblock structure, with a highly hydrophobic central region comprising up to 71 residues. Little is known on their three-dimensional structure. Here we report the solubilization of caleosin and of two oleosins in aqueous solution, using various detergents or original amphiphilic polymers, amphipols. All three proteins, insoluble in water buffers, were maintained soluble either by anionic detergents or amphipols. Neutral detergents were ineffective. In complex with amphipols the oleosins and caleosin contain more beta and less alpha secondary structures than in the SDS detergent, as evaluated by synchrotron radiation circular dichroism. These are the first reported structural results on lipid bodies proteins maintained in solution with amphipols, a promising alternative to notoriously denaturing detergents.

Lipid bodies in innate immune response to bacterial and parasite infections

Lipid bodies (also known as lipid droplets, adiposomes) are dynamic organelles with key roles in regulating storage and turnover of lipids in different cells and organisms. The emerging role of lipid bodies as inflammatory organelles raises lipid body status to critical regulators of different inflammatory and infectious diseases and key markers of cell activation. Notably, lipid body biogenesis is highly regulated and is cell and stimuli specific. Lipid body structural features, including lipid and protein composition may vary according to the cell type, activation state and inflammatory environment and thus may determine different cellular functions for lipid bodies. Here we will review the morphological and structural aspects of lipid bodies, the regulated mechanisms of formation, as well as lipid body functions in cells involved in the innate immune response during bacterial and parasite infections.

Eukaryotic Lipid Body Proteins in Oleogenous Actinomycetes and Their Targeting to Intracellular Triacylglycerol Inclusions: Impact on Models of Lipid Body Biogenesis

Bacterial neutral lipid inclusions are structurally related to eukaryotic lipid bodies. These lipid inclusions are composed of a matrix of triacylglycerols (TAGs) or wax esters surrounded by a monolayer of phospholipids. Whereas the monolayers of lipid bodies from animal and plant cells harbor specific classes of proteins which are involved in the structure of the inclusions and lipid homoestasis, no such proteins are known to be associated with bacterial lipid inclusions. The present study was undertaken to reveal whether the mammalian lipid body proteins perilipin A, adipose differentiation-related protein, and tail-interacting protein of 47 kDa (TIP47), which comprise the so called PAT family proteins, and the maize (Zea mays L.) oleosin are targeted to prokaryotic TAG bodies in vivo. When fused to enhanced green fluorescent protein, all proteins except the oleosin were mainly located at the surfaces of lipid inclusions when heterologously expressed in the recombinant actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc(2)155. A more detailed intracellular distribution analysis of TIP47 in recombinant R. opacus cells by immunocytochemical labeling of ultrathin cryosections and freeze fracture replicas revealed a substantial amount of TIP47 protein also pervading the cores of the inclusions. We discuss the impact of these results on the current model of lipid body biogenesis in prokaryotes.

Body composition of breeding gilts in response to dietary protein and energy balance from thirty kilograms of body weight to completion of first parity1

The relationships among BW, backfat depth, and body physical and chemical composition were evaluated in response to dietary protein and DE balance in breeding gilts from 30 kg of BW to weaning of the first litter. Large White (sire) x Landrace (dam) F1 hybrid (White; n = 75) and Landrace (sire) x (Meishan x Large White; dam) (Meishan; n = 19) hybrid gilts were received at 30 kg of BW. Five gilts were taken as the initial slaughter group at 30 kg of BW, and the remaining gilts were fed diets differing in total lysine to DE ratio, high (H) vs. low (L), from 30 kg of BW to mating (rearing), and during gestation and lactation, allowing factorial investigation of dietary treatment effects and interactions during rearing, gestation, and lactation. Gilts were slaughtered at approximately 50 and 90 kg of BW, and at mating, farrowing, and weaning. Gilts fed L diets during rearing were lighter at mating (117.9 vs. 133.6 kg of BW, P = 0.035) due to a reduction in gain (592 vs. 720 g/d, P = 0.002) and a restriction in protein accretion (83 vs. 117 g/d, P = 0.001). During rearing, lipid accretion did not differ between L- and H-fed gilts (208 vs. 198 g/d, P = 0.60), but the ratio of lipid to protein accretion was about 1.5-fold greater in L-fed gilts, where lipid mass expressed as a percentage of BW was increased at mating (26.0 vs. 21.9%, P = 0.005). Effects of L diets on lipid accretion during rearing were transient; no residual effects on body lipid mass (P > 0.17) were found at farrowing or weaning. Overall, Meishan hybrids carried greater lipid mass (P < 0.001) than White hybrid gilts. Whereas the rate of body lipid and protein accretion and body lipid and protein mass can be nutritionally influenced and can vary according to growth stage, reproductive status, and genotype, this study established that body protein mass expressed as a proportion of the lipid free empty BW remains inflexible. A value for this measure of 0.188 +/- 0.0052 was found in White and Meishan hybrid gilts ranging from 28 to 203 kg of BW and 3 to 36 mm backfat depth, covering growth, pregnancy, and lactation, and offered diets differing in protein and energy balance. Body protein mass can be predicted as approximately 0.2 of the lipid free empty BW once body lipid mass is estimated accurately from physical measurements, such as backfat depth (P2, mm) and BW (kg), by regression using lipid (kg) = - 8.14 (SE, 1.302) + 0.167 (SE, 0.010) BW + 0.883 (SE, 0.065) P2 (residual SD = 3.51; R2 = 0.912).

Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis.

The B3 domain protein LEAFY COTYLEDON2 (LEC2) is required for several aspects of embryogenesis, including the maturation phase, and is sufficient to induce somatic embryo development in vegetative cells. Here, we demonstrate that LEC2 directly controls a transcriptional program involved in the maturation phase of seed development. Induction of LEC2 activity in seedlings causes rapid accumulation of RNAs normally present primarily during the maturation phase. Several RNAs encode proteins with known roles in maturation processes, including seed-storage and lipid-body proteins. Clustering analyses identified other LEC2-induced RNAs not previously shown to be involved in the maturation phase. We show further that genes encoding these maturation RNAs all possess in their 5' flanking regions RY motifs, DNA elements bound by other closely related B3 domain transcription factors. Our finding that recombinant LEC2 specifically binds RY motifs from the 5' flanking regions of LEC2-induced genes provides strong evidence that these genes represent transcriptional targets of LEC2. Although these LEC2-induced RNAs accumulate primarily during the maturation phase, we show that a subset, including AGL15 and IAA30, accumulate in seeds containing zygotes. We discuss how identification of LEC2 target genes provides a potential link between the roles of LEC2 in the maturation phase and in the induction of somatic embryogenesis.

Appearance of smaller lipid bodies and protein kinase activation in the lipid body fraction are induced by an increase in the nitrogen source in the Mortierella fungus.

We studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 micro m, which are usually found at a C/N ratio of 38 in this fungus. To determine factors regulating lipid body size, we compared lipid body fractions from fungal cells cultured at different C/N ratios. We found some differences in polypeptide profiles between lipid body fractions from fungal cells cultured at different C/N ratios for 2 days when the lipid bodies were enlarged at a C/N ratio of 38. We then compared the phosphorylation of lipid body proteins, since protein phosphorylation plays a pivotal role in various aspects of signal transduction. In vitro phosphorylation in the lipid body fraction indicated that protein kinase activity toward endogenous and exogenous substrates such as histone IIIS, VIIS, and myelin basic protein increased in the lipid body fraction at a C/N ratio of 9. Further analysis by in-gel protein kinase assay indicated the presence of at least three activated protein kinases with molecular masses of 75, 72, and 42 kDa, which were also autophosphorylated. These results indicate the presence of nutrient-regulated protein kinases and increased phosphorylation in lipid bodies, which correlate with the appearance of smaller lipid bodies in this fungus. Further studies to characterize these protein kinases at the molecular level should provide new insights into the link between nutrient sensing and lipid storage.

Role of lipid bodies and lipid-body proteins in seeds and other tissues

Summary Lipid bodies are present in most plant cells and not just in storage tissues, such as seeds, as is often assumed. There is a growing number of proteins that are associated with lipid bodies in different plant tissues. In seeds, the most common class of lipid-body protein is the oleosins, which are found on the surfaces on lipid bodies in all desiccation tolerant plants but are absent from lipid bodies in the seeds of desiccation-sensitive plants, e.g. cocoa or neem. The absence of oleosins in these seeds is not problematic during dehydration but leads to a fatal coalescence of lipid bodies after germination. A class of anther-specific proteins has a precursor form that contains an oleosin-like hydrophobic domain and is initially localised on lipid bodies in the tapetum. However, the oleosin-like domain is cleaved off after release into the anther locule and the mature proteins, which are the major protein components of the pollen coat, contain no oleosin-like sequences. Therefore these proteins, which have been named «pollenins», use their oleosin-like domain as a novel form of cleavable targeting sequence. A third type of lipid-body protein is caleosin, which also has endoplasmic reticulum associated isoforms and may be involved in lipid-body biogenesis and membrane trafficking. Analysis of the expression patterns of oleosin and caleosin genes/proteins revealed an unexpected localisation in young root tips, as confirmed by expressing promoter-GUS constructs in transgenic plants. Several other classes of storage product-related genes hitherto believed to seed-specific are also transiently expressed in root tips following seed germination. We discuss the implications of these findings for root development and the use of «seed-specific» gene promoters in general.

New insights into the mechanisms of lipid-body biogenesis in plants and other organisms

A comparative approach has been used to study the role of several lipid-body-binding proteins in plants and animals. Caleosins are a newly discovered class of calcium-binding lipid-body proteins found in plants and fungi, which we now report to have separate endoplasmic reticulum- and lipid-body-associated isoforms. We also compare the lipid-body targeting of oleosin from plants and the core protein of the hepatitis C virus when they were expressed separately in lipid-accumulating animal cell lines. This is a novel and powerful approach to investigating the factors that determine the lipid-body targeting of a wide range of proteins.

Caleosins: Ca2+-binding proteins associated with lipid bodies.

We have previously identified a rice gene encoding a 27 kDa protein with a single Ca2+-binding EF-hand and a putative membrane anchor. We report here similar genes termed caleosins, CLO, in other plants and fungi; they comprise a multigene family of at least five members in Arabidopsis (AtClo1-5). Northern hybridization demonstrated that AtClo2-4 mRNAs levels were low in various tissues, while AtClo1 mRNA levels were high in developing embryos and mature seeds. Analysis of transgenic Arabidopsis plants expressing the GUS reporter under control of the AtClo1 promoter showed strong levels of expression in developing embryos and also in root tip cells. Antibodies raised against AtCLO1 were used to detect caleosin in cellular fractions of Arabidopsis and rapeseed. This indicated that caleosins are a novel class of lipid body proteins, which may also be associated with an ER subdomain.

Cucumber cotyledon lipoxygenase during postgerminative growth. Its expression and action on lipid bodies.

In cucumber (Cucumis sativus), high lipoxygenase-1 (LOX-1) activity has been detected in the soluble fraction prepared from cotyledons of germinating seeds, and the involvement of this enzyme in lipid turnover has been suggested (K. Matsui, M. Irie, T. Kajiwara, A. Hatanaka [1992] Plant Sci 85: 23-32; I. Fuessner, C. Wasternack, H. Kindl, H. Kühn [1995] Proc Natl Acad Sci USA 92: 11849-11853). In this study we have investigated the expression of the gene lox-1, corresponding to the LOX-1 enzyme. LOX-1 expression is highly coordinated with that of a typical glyoxysomal enzyme, isocitrate lyase, during the postgerminative stage of cotyledon development. In contrast, although icl transcripts accumulated in tissue during in vitro senescence, no accumulation of lox-1 mRNA could be observed, suggesting that lox-1 plays a specialized role in fat mobilization. LOX-1 is also known to be a major lipid body protein. The partial peptide sequences of purified LOX-1 and lipid body LOX-1 entirely coincided with that deduced from the lox-1 cDNA sequence. The data strongly suggest that LOX-1 and lipid body LOX-1 are derived from a single gene and that LOX-1 can exist both in the cytosol and on the lipid bodies. We constructed an in vitro oxygenation system to address the mechanism of this dual localization and to investigate the action of LOX-1 on lipids in the lipid bodies. LOX-1 cannot act on the lipids in intact lipid bodies, although degradation of lipid body proteins, either during seedling growth or by treatment with trypsin, allows lipid bodies to become susceptible to LOX-1. We discuss the role of LOX-1 in fat mobilization and its mechanism of action.

Characterization of a 14 kDa component with low expression in a unique Nod+ Fix- Bradyrhizobium japonicum

A pronase-sensitive, 14 kDa component of bacteroids of Bradyrhizobium japonicum I-110ARS was identified and characterized using monoclonal antibodies. This component was weakly synthesized or was missing in bacteroids of a unique Nod- Fix- mutant, ARS2525. Both the 14 kDa component and poly-β-hydroxybutyrate (PHB) were located in the same protein peak after sucrose density gradient separation of lysed bacteroids. Thirty-five-day-old bacteroids of B. japonicum I-110ARS contained up to 10 times more PHB than B. japonicum ARS2525 bacteroids. Immunocytochemistry of ultra-thin nodule sections showed that the component was associated with PHB granules in the bacteroids, possibly with the limiting membrane around the granules. This observation implies that the component may be similar to other storage lipid body proteins. Whether low synthesis of the 14 kDa component or low PHB content was a cause, a consequence or related to the Fix- phenotype was not established.

Induction of accumulation and degradation of the 18.4-kDa oleosin in a triacylglycerol-storing cell culture of anise (Pimpinella anisum L.)

Two closely related anise cell-culture lines, Pa15 and Pa19, differ considerably in growth rate, potential to form somatic embryoids, triacylglycerol (TAG) storage and pattern of lipid-body proteins. Line Pa15 grows very fast (doubling rate: 3 d), mainly as single cells, exhibits a low potential for somatic embryogenesis and its TAG content is relatively low (5–20 mg TAG per g dry weight). In contrast, the line Pa19 shows lower growth rates (doubling rate: 8 d), tends to form clusters of somatic cells, has a higher TAG content (100–150 mg TAG per g dry weight), and somatic embryoids are easily induced. Under defined culture conditions, the TAG content of the line Pa19 can be increased to approximately 70% of that of ripe anise seeds (150 and 220 mg TAG per g dry weight, respectively). Polyclonal antibodies prepared against the most abundant protein (relative molecular mass 18.4 kDa) from the lipid-body fraction of anise seeds (Radetzky et al. 1993, Planta 191, 166–172) react also with a 18.4-kDa protein from the lipid-body fraction of cells of the Pa19 culture. In contrast, only fairly low levels of the 18.4-kDa oleosin were detected in Pal5. Limited sucrose supply in the medium resulted in TAG degradation and the concomitant decrease in the amount of immunodetectible 18.4-kDa protein in the Pa19 cell culture. Treatment with sorbitol, or abscisic acid and sorbitol in combination, enhanced TAG contents and also the amount of immunostained 18.4-kDa protein in the cell culture Pa19, whereas no effect was found on either TAG content or 18.4-kDa protein in the cell-culture line Pa15. The 18.4-kDa protein can be classified as an oleosin, a proposal which is supported by the similarity in molecular mass compared with other known oleosins, its occurrence in the lipid-body fraction and the fact that its amount correlates with the TAG content. The results of this study indicate that the Pa19 cell culture provides a valid model system for investigations of lipid storage and mobilization in higher-plant cells.

Transient occurrence of lipoxygenase and glycoprotein gp49 in lipid bodies during fat mobilization in anise seedlings

The patterns of lipid-body proteins prepared at different stages of germination from seedlings of anise (Pimpinella ansium L.) were investigated. During the stage of fat mobilization, a set of proteins is synthesized de novo and transferred to existing lipid bodies. Analysis of the protein constituents, detected either by protein staining, concanavalin-A/peroxidase staining or immunoreaction on Western blots showed three distinct protein species: a lipoxygenase firmly integrated into the lipid bodies, a glycoprotein gp50 only extractable from the lipid bodies by treatment with sodium dodecyl sulfate and a glycoprotein gp49 solubilized by diethyl-ether treatment of lipid bodies. Both the lipoxygenase immunodetected on Western blots and gp49 stained with concanavalin-A/ peroxidase conjugate occurred transiently in the lipidbody fraction, reaching maximum concentrations between days 6 and 13 of germination. This behavior was in contrast to the decreasing level of the 18.4-kDa oleosin already present at the beginning of germination.