Denis J. Murphy

Mechanisms of lipid-body formation

Most organisms transport or store neutral lipids as lipid bodies - lipid droplets that usually are bounded by specific proteins and (phospho)lipid. Neutral-lipid bodies vary considerably in their morphology and are associated with an extremely diverse range of proteins. However, the mechanisms by which they are generated in plants, animals and microorganisms appear to share many common features: lipid bodies probably arise from microdomains of the endoplasmic reticulum (or the plasma membrane in prokaryotes) that contain lipid-biosynthesis enzymes, and their synthesis and size appear to be controlled by specific protein components.

Biogenesis and function of the lipidic structures of pollen grains

Abstract Pollen grains contain several lipidic structures, which play a key role in their development as male gametophytes. The elaborate extracellular pollen wall, the exine, is largely formed from acyl lipid and phenylpropanoid precursors, which together form the exceptionally stable biopolymer sporopollenin. An additional extracellular lipidic matrix, the pollen coat, which is particularly prominent in entomophilous plants, covers the interstices of the exine and has many important functions in pollen dispersal and pollen-stigma recognition. The sporopollenin and pollen coat precursors are both synthesised in the tapetum under the control of the sporophytic genome, but at different stages of development. Pollen grains also contain two major intracellular lipidic structures, namely storage oil bodies and an extensive membrane network. These intracellular lipids are synthesised in the vegetative cell of the pollen grain under the control of the gametophytic genome. Over the past few years there has been significant progress in elucidating the composition, biogenesis and function of these important pollen structures. The purpose of this review is to describe these recent advances within the historical context of research into pollen development.

The dynamic roles of intracellular lipid droplets: from archaea to mammals

During the past decade, there has been a paradigm shift in our understanding of the roles of intracellular lipid droplets (LDs). New genetic, biochemical and imaging technologies have underpinned these advances, which are revealing much new information about these dynamic organelles. This review takes a comparative approach by examining recent work on LDs across the whole range of biological organisms from archaea and bacteria, through yeast and Drosophila to mammals, including humans. LDs probably evolved originally in microorganisms as temporary stores of excess dietary lipid that was surplus to the immediate requirements of membrane formation/turnover. LDs then acquired roles as long-term carbon stores that enabled organisms to survive episodic lack of nutrients. In multicellular organisms, LDs went on to acquire numerous additional roles including cell- and organism-level lipid homeostasis, protein sequestration, membrane trafficking and signalling. Many pathogens of plants and animals subvert their host LD metabolism as part of their infection process. Finally, malfunctions in LDs and associated proteins are implicated in several degenerative diseases of modern humans, among the most serious of which is the increasingly prevalent constellation of pathologies, such as obesity and insulin resistance, which is associated with metabolic syndrome.

Oleosins prevent oil-body coalescence during seed imbibition as suggested by a low-temperature scanning electron microscope study of desiccation-tolerant and -sensitive oilseeds

Abstract. In order to clarify further the physiological role of oleosins in seed development, we characterized the oil-body proteins of several oilseeds exhibiting a range of desiccation sensitivities from the recalcitrant (Theobroma cacao L., Quercus rubra L.), intermediate (Coffea arabica L., Azadirachta indica A. Juss.) and orthodox categories (Sterculia setigera Del., Brassica napus L.). The estimated ratio of putative oleosins to lipid in oil bodies of Q. rubra was less than 5% of the equivalent values for rapeseed oil bodies. No oleosin was detected in T. cacao oil bodies. In A. indica cotyledons, oil bodies contained very low amounts of putative oleosins. Oil bodies both from C. arabica and S. setigera exhibited a similar ratio of putative oleosins to lipid as found in rapeseed. In C. arabica seeds, the central domain of an oleosin was partially sequenced. Using a low temperature field-emission scanning electron microscope, the structural stability of oil bodies was investigated in seeds after drying, storage in cold conditions and rehydration. Despite the absence or relative dearth of oleosins in desiccation-sensitive, recalcitrant oilseeds, oil bodies remained relatively stable after slow or fast drying. In A. indica seeds exposed to a lethal cold storage treatment, no significant change in oil-body sizes was observed. In contrast, during imbibition of artificially dried seeds containing low amounts of putative oleosins, the oil bodies fused to form large droplets, resulting in the loss of cellular integrity. No damage to the oil bodies occurred in imbibed seeds of Q. rubra, C. arabica and S. setigera. Thus the rehydration phase appears to be detrimental to the stability of oil bodies when these are present in large amounts and are lacking oleosins. We therefore suggest that one of the functions of oleosins in oilseed development may be to stabilize oil bodies during seed imbibition prior to germination.

Engineering oil production in rapeseed and other oil crops

The first transgenic crop with a modified seed compositionlauric-oil rapeseed as cultivated for commercial use in 1995, and many additional transgenic rapeseed varieties, expressing a variety of novel seed-oils or proteins, are under development. Research advances in Arabidopsis molecular genetics, and the emerging Brassica-Arabidopsis genome relatedness, will enable the radical manipulation of many key agronomic traits in rapeseed, ranging from greatly improved seed-oil yield and quality, to improved disease resistance, pod shattering and canopy architecture. Many of these advances will be directly applicable to other oil crops, including high-yielding tropical perennials such as oil palm.

Arabidopsis Peptide Methionine Sulfoxide Reductase2 Prevents Cellular Oxidative Damage in Long Nights

Peptide methionine sulfoxide reductase (PMSR) is a ubiquitous enzyme that repairs oxidatively damaged proteins. In Arabidopsis (Arabidopsis thaliana), a null mutation in PMSR2 (pmsr2-1), encoding a cytosolic isoform of the enzyme, exhibited reduced growth in short-day conditions. In wild-type plants, a diurnally regulated peak of total PMSR activity occurred at the end of the 16-h dark period that was absent in pmsr2-1 plants. This PMSR activity peak in the wild-type plant coincided with increased oxidative stress late in the dark period in the mutant. In pmsr2-1, the inability to repair proteins resulted in higher levels of their turnover, which in turn placed an increased burden on cellular metabolism. This caused increased respiration rates, leading to the observed higher levels of oxidative stress. In wild-type plants, the repair of damaged proteins by PMSR2 at the end of the night in a short-day diurnal cycle alleviates this potential burden on metabolism. Although PMSR2 is not absolutely required for viability of plants, the observation of increased damage to proteins in these long nights suggests the timing of expression of PMSR2 is an important adaptation for conservation of their resources.

Biosynthesis of seed storage products during embryogenesis in rapeseed, Brassica napus

Summary The timing of the synthesis of storage oils and storage proteins during embryogenesis in field-grown rapeseed, Brassica napus L. has been examined. For the first two weeks after anthesis the embryos grew by cell division until they were about 1 mm in length. Storage oil accumulation was apparent even in very young embryos but only became significant when cell division ceased and cell expansion commenced at 2-3 weeks after anthesis. The most rapid phase of oil deposition was between 4 and 6 weeks after anthesis. The bulk of the storage oil, which accumulated initially as large oil-bodies of 2-4pm diameter, was deposited before the onset of storage protein synthesis. The major seed storage proteins, cruciferin and napin, were synthesised from week 5 and the maximal rate of synthesis was between weeks 5 and 7 after anthesis. The hydrophobic protein, oleosin, which accounted for 20% of total protein in mature seeds, was synthesised between 7 and 10 weeks after anthesis. It has recently been shown that this protein is the major component of the osmiophilic membrane which develops around the storage oil-bodies at this stage of embryogenesis [Murphy et al. (1989) Biochem. J. 258,285-293]. The acquisition of a proteinaceous membrane by the oil-bodies coincided with a reduction in their average diameter from 2-4Im to 0.3 -1 p,m. It is proposed that there are three major phases in storage product formation in rapeseed embryos, (i) oil deposition into large storage bodies, (ii) polar protein deposition into vacuoles, (iii) oleosin synthesis and assembly onto oil-bodies causing their reduction in size. The significance of these results for the regulation of storage product synthesis in oilseeds is discussed.

Composition and role of tapetal lipid bodies in the biogenesis of the pollen coat of Brassica napus

Abstract. The composition of the two major lipidic organelles of the tapetum of Brassica napus L. has been determined. Elaioplasts contained numerous small (0.2–0.6 μm) lipid bodies that were largely made up of sterol esters and triacylglycerols, with monogalactosyldiacylglycerol as the major polar lipid. This is the first report in any species of the presence of non-cytosolic, sterol ester-rich, lipid bodies. The elaioplast lipid bodies also contained 34- and 36-kDa proteins which were shown by N-terminal sequencing to be homologous to fibrillin and other plastid lipid-associated proteins. Tapetosomes contained mainly polyunsaturated triacylglycerols and associated phospholipids plus a diverse class of oleosin-like proteins. The pollen coat, which is derived from tapetosomes and elaioplasts, was largely made up of sterol esters and the C-terminal domains of the oleosin-like proteins, but contained virtually no galactolipids, triacylglycerols or plastid lipid-associated proteins. The sterol compositions of the elaioplast and pollen coat were almost identical, consisting of stigmasterol > campestdienol > campesterol > sitosterol ≫ cholesterol, which is consistent with the majority of the pollen coat lipids being derived from elaioplasts. These data demonstrate that there is substantial remodelling of both the lipid and protein components of elaioplasts and tapetosomes following their release into the anther locule from lysed tapetal cells, and that components of both organelles contribute to the formation of the lipidic coating of mature pollen grains.

Differential presence of oleosins in oleogenic seed and mesocarp tissues in olive (Olea europaea) and avocado (Persea americana)

Abstract Olive drupes accumulate triacylglycerol (TAG) in the fleshy mesocarp of the fruit and also in the endosperm and embryo tissue of their seeds. Ultrastructural analysis has shown that, whereas the TAG in the seed tissues is stored in small, relatively regular oil bodies with diameters in the region of 0.5–2.0 μm, it is present in large, irregular oil bodies of 10–20 μm in the mesocarp. The fatty acid profiles of the TAG in oil bodies isolated from seed and mesocarp tissues are very similar, but oil bodies from these tissues differ dramatically in their protein content. Oil bodies from olive seed endosperm and embryo tissues contain about 10% (w/w) protein, whereas no significant protein was detected in oil bodies from mesocarp tissue of olive or avocado. Major polypeptides of 22 kDa and 50 kDa were purified from olive seed oil bodies and antibodies were raised against them. The 22-kDa oil-body protein was demonstrated to be an oleosin, based on its exclusive localisation on the surface of oil bodies of mature seed tissues, as shown by immunogold electron microscopy. It is concluded that oleosins, such as the 22-kDa polypeptide in olive, are present in the long-term storage oil bodies from the embryo and endosperm tissues of the seed and are absent from oil bodies of the mesocarp.

The importance of non-planar bilayer regions in photosynthetic membranes and their stabilisation by galactolipids

Photosynthetic membranes contain considerable regions of high surface curvature, notably at their margins, where the average radius of curvature is about 10 nm. The proportion of total membrane lipid in the outer and inner thylakoid margin monolayers is estimated at 21% and 13%, respectively. The major thylakoid lipid, monogalactosyldiacylglycerol, is roughly cone‐shaped and will not form complete lamellar bilayer phases, even in combination with other thylakoid lipids. It is proposed that this galactolipid plays a role in: (a) stabilising regions of concave curvature in thylakoids; and (b) packaging hydrophobic proteins in planar bilayer regions by means of inverted micelles. This model predicts substantial asymmetries in the distribution of lipids both across and along the thylakoid bilayer plane.