Pascale Jolivet

Structure and function of seed lipid body-associated proteins

Many organisms among the different kingdoms store reserve lipids in discrete subcellular organelles called lipid bodies. In plants, lipid bodies can be found in seeds but also in fruits (olives, ...), and in leaves (plastoglobules). These organelles protect plant lipid reserves against oxidation and hydrolysis until seed germination and seedling establishment. They can be stabilized by specific structural proteins, namely the oleosins and caleosins, which act as natural emulsifiers. Considering the putative role of some of them in controlling the size of lipid bodies, these proteins may constitute important targets for seed improvement both in term of oil seed yield and optimization of technological processes for extraction of oil and storage proteins. We present here an overview of the data on the structure of these proteins, which are scarce, and sometimes contradictory and on their functional roles.

Plant organelle proteomics: collaborating for optimal cell function.

Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear-encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter-plant and inter-tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in-depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in cell, their proper function and evolution.

Protein composition of oil bodies from mature Brassica napus seeds.

Seed oil bodies (OBs) are intracellular particles storing lipids as food or biofuel reserves in oleaginous plants. Since Brassica napus OBs could be easily contaminated with protein bodies and/or myrosin cells, they must be purified step by step using floatation technique in order to remove non‐specifically trapped proteins. An exhaustive description of the protein composition of rapeseed OBs from two double‐zero varieties was achieved by a combination of proteomic and genomic tools. Genomic analysis led to the identification of sequences coding for major seed oil body proteins, including 19 oleosins, 5 steroleosins and 9 caleosins. Most of these proteins were also identified through proteomic analysis and displayed a high level of sequence conservation with their Arabidopsis thaliana counterparts. Two rapeseed oleosin orthologs appeared acetylated on their N‐terminal alanine residue and both caleosins and steroleosins displayed a low level of phosphorylation.

Crop seed oil bodies: from challenges in protein identification to an emerging picture of the oil body proteome

Oleaginous seeds store lipids in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids bound by proteins embedded in a phospholipid monolayer. OB proteins are well conserved in plants and have long been grouped into only two categories: structural proteins or enzymes. Recent work, however, which identified other classes of proteins associated with OBs, clearly shows that this classification is obsolete. Proteomics‐mediated OB protein identification is facilitated in plants for which the genome is sequenced and annotated. However, it is not clear whether this knowledge can be dependably transposed to less well‐characterized plants, including the well‐established commercial sources of seed oil as well as the many others being proposed as novel sources for biodiesel, especially in Africa and Asia. Toward an update of the current data available on OB proteins this review discusses (i) the specific difficulties for proteomic studies of organelles; (ii) a 2012 census of the proteins found in seed OBs from various crops; (iii) the oleosin composition of OBs and their role in organelle stability; (iv) PTM of OB proteins as an emerging field of investigation; and finally we describe the emerging model of the OB proteome from oilseed crops.

Oil body proteins sequentially accumulate throughout seed development in Brassica napus

Despite the importance of seed oil bodies (OBs) as enclosed compartments for oil storage, little is known about lipid and protein accumulation in OBs during seed formation. OBs from rapeseed (Brassica napus) consist of a triacylglycerol (TAG) core surrounded by a phospholipid monolayer embedded with integral proteins which confer high stability to OBs in the mature dry seed. In the present study, we investigated lipid and protein accumulation patterns throughout seed development (from 5 to 65 days after pollination [DAP]) both in the whole seed and in purified OBs. Deposition of the major proteins (oleosins, caleosins and steroleosins) into OBs was assessed through (i) gene expression pattern, (ii) proteomics analysis, and (iii) protein immunodetection. For the first time, a sequential deposition of integral OB proteins was established. Accumulation of oleosins and caleosins was observed starting from early stages of seed development (12-17 DAP), while steroleosins accumulated later (~25 DAP) onwards.

Evidence for sulphite oxidase activity in spinach leaves

The present paper provides evidence that spinach chloroplasts possess a sulphite oxidase activity coupled with oxygen consumption and reduction of ferricyanide. This activity is associated with thylakoids and solubilized by non-ionic biological detergents. The pH and temperature dependencies of sulphite oxidase activity solubilized by Triton X-100 from spinach thylakoids were consistent with those of an intrinsic membrane protein. This isolated activity was insensitive towards radical scavengers (mannitol, mannose and fructose) and catalase, and was inhibited only with very high concentrations of superoxide dismutase. Thus, observed sulphite oxidation was not induced through the photosynthetic electron transport system, but achieved via a thylakoid membrane enzymic system showing a sulphite oxidase activity. Kinetic parameters of thylakoid sulphite oxidase were measured and compared with those of other sulphite oxidases.

Metabolism of elemental sulphur and oxidation of sulphite by wheat and spinach chloroplasts

Abstract Wheat or spinach chloroplasts were fed with 35 S-labelled elemental sulphur (S 0 ). 35 S-labelled compounds appearing thereafter were analysed. Five 35 S-compounds were recovered, thiosulphate, sulphite, sulphate, cysteine and glutathione. The effect of environmental conditions was also studied. The measurement of S 0 metabolism rate was compared to the recovery of the introduced S 0 into other S compounds. The specific radioactivity of thiosulphate and sulphate was determined. The results obtained have permitted the elucidation of the precise biochemical pathway of S 0 oxidation. The oxidation of sulphite in wheat and spinach chloroplasts occurs via both non-enzymatic (in particular the operation of the photosynthetic electron transport chain) and enzymatic processes (sulphite oxidase activity).

New insights into the structure of apolipoprotein B from low-density lipoproteins and identification of a novel YGP-like protein in hen egg yolk.

Apoproteins of low-density lipoproteins (LDL) and soluble proteins (livetins) contained in hen egg yolk plasma have been demonstrated as being essential to the interfacial and emulsifying properties of yolk. The knowledge of their structure is necessary to better understand these properties. Purified protein fractions were separated by SDS-PAGE or 2D-PAGE and identified through the LC-MS/MS of their trypsin peptides. Hen blood apolipoprotein B gives rise to nine different apoproteins in LDL after maturation and proteolysis. Among these apoproteins, two protein fragments appeared to be less accessible to proteases and could be enriched in beta-sheets and firmly associated with lipids. Plasma soluble proteins were constituted by approximately 45% of yolk immunoglobulins with a high heterogeneity of the variable regions of both heavy and light chains, 41% of glycoproteins constituted by YGP42 and YGP40, 14% of albumins, and one new minor protein we called YGP30, showing 75% similarity to YGP40.

Characterization of an exocellular protein phosphatase with dual substrate specificity from the yeast Yarrowia lipolytica

In previous work, the major endocellular protein phosphatase activity has been identified in the secretory yeast Yarrowia lipolytica as a PP2A. The aim of the present work was to seek the presence of one protein phosphatase excreted in the exocellular medium and to study its activity during yeast growth in media supplemented or not supplemented with inorganic phosphate. Protein phosphatase was purified and activity was assayed by following the dephosphorylation of three substrates, [32P]casein, phosphotyrosine and a synthetic tyrosine-phosphorylated peptide. Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency. After several purification steps, the enzyme preparation presents an apparent electrophoretic homogeneity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyrosyl activities. The kinetic properties exclude contamination by a copurified protein and it is concluded that the two activities are carried by the same single proteic species. It was characterized by gel filtration as a 33 kDa protein with one single subunit demonstrated by SDS-PAGE. An absolute requirement for reducing-agents is observed suggesting that the enzyme contains at least one essential reactive cysteinyl residue. Optimum pH value is 6.1, apparent K(m) for phosphotyrosine was calculated to be 760 microM and Hill coefficient 3.2 indicating a rather high cooperativity. These results showed that the involvement of alkaline and/or acid phosphatase was unlikely. In conclusion, a protein phosphatase distinct from endocellular PP2A is secreted by Yarrowia lipolytica and characterized as a phosphotyrosine protein phosphatase with associated phosphoseryl/threonyl activity.

Quantitative determination of glutamate in a Rhodophyceae (Chondrus crispus) and four Phaeophyceae (Fucus vesiculosus, Fucus serratus, Cystoseira elegans, Cystoseira barbata)

A study was made to find a better method of analyzing the glutamate pool in seaweeds than the use of HPLC, which provides unsatisfactory results with material rich in alginates and salts. A method recommended elsewhere (Inglis A, Bartone N, Finlayson J, 1988, J. Biochem. Biophys. Methods 15: 249–254) for physiological fluids has been assayed and improved for algal samples. It consisted of the addition of lithium acetate before the phenylisothiocyanate derivatization, omission of one drying step and extraction of the derivative with heptane before chromatographic analysis. Neither salt nor alginates interfered with analysis.