Ion distribution measured by electron probe X-ray microanalysis in apoplastic and symplastic pathways in root cells in sunflower plants grown in saline medium

Little is known about how salinity affects ions distribution in root apoplast and symplast. Using x-ray microanalysis, ions distribution and the relative contribution of apoplastic and symplastic pathways for delivery of ions to root xylem were studied in sunflower plants exposed to moderate salinity (EC=6). Cortical cells provided a considerably extended Na+ and Cl- storage facility. Their contents are greater in cytoplasm (root symplast) as compared to those in intercellular spaces (root apoplast). Hence, in this level of salinity, salt damage in sunflower is not dehydration due to extracellular accumulation of sodium and chloride ions, as suggested in the Oertli hypothesis. On the other hand, reduction in calcium content due to salinity in intercellular space is less than reduction in the cytoplasm of cortical cells. It seems that sodium inhibits the radial movement of calcium in symplastic pathway more than in the apoplastic pathway. The cell wall seems to have an important role in providing calcium for the apoplastic pathway. Redistribution of calcium from the cell wall to intercellular space is because of its tendency towards xylem through the apoplastic pathway. This might be a strategy to enhance loading of calcium to xylem elements and to reduce calcium deficiency in young leaves under salinity. This phenomenon may be able to increase salt tolerance in sunflower plants. Supplemental calcium has been found to be effective in reducing radial transport of Na+ across the root cells and their loading into the xylem, but not sodium absorption. Supplemental calcium enhanced Ca2+ uptake and influx into roots and transport to stele.     

Regulation of gametangial formation in bryophytes

Under in vitro conditions gametangial formation in bryophytes can be regulated by a variety of physical and chemical factors. Liverworts are relatively more sensitive to photoperiod than are mosses. With the exception ofRiccia crystallina all species of liverworts thus far investigated are either long-day or short-day plants. Among mosses, all butSphagnum plumulosum appear to be independent of photoperiod for the onset of the reproductive phase. The photoperiodic response in liverworts increases with increasing light intensity. Mosses, on the other hand, exhibit differential behavior.Leptobryum pyriforme andBartramidula bartramioides exhibit a linear relationship with light intensity for this response, whereasBryum argenteum, B. coronatum andBarbula gregaria have a specific light intensity requirement for optimal response. The photoperiodic effect in bryophytes is operative within certain temperature limits. Most bryophytes do not require a low temperature pretreatment for gametangial induction. Some become fertile in a broad temperature range, whereas others require a specific temperature. Among chemical factors, carbohydrates in general enhance gametangial formation.Riccia crystallina andBartramidula bartramioides develop gametangia in the presence of a carbohydrate, butBryum spp. (B. argenteum andB. coronatum) andBarbula gregaria respond even in its absence. High carbohydrate-nitrogen ratio seems more favorable for the onset of reproductive phase. Bryophytes respond differentially to various forms of nitrogen. Depletion of inorganic nitrogen (nitrate or ammonium) significantly favors gametangial induction. On the other hand, organic nitrogen (such as amino acids, peptone and urea) has differential effects on the enhancement of antheridial and archegonial formation in liverworts. Growth hormones have variable effects on gametangial induction. Indole-3-acetic acid increases archegonial formation inRiccia crystallina, but it is more favorable for antheridial production in mosses likeBryum coronatum, B. argenteum andBarbula gregaria. Gibberellins enhance antheridial formation in all the investigated bryophytes. Cytokinins stimulate induction of archegonia and inhibit antheridial formation inRiccia crystallina andBryum argenteum. Auxins, gibberellins and cytokinins also interact in eliciting the response. Iron and copper chelating agents such as EDTA and EDDHA favor vegetative growth and gametangial formation. InRiccia these chelates enhance archegonial production more than antheridial formation, but inBryum argenteum their effect is just the reverse. Salicylic acid, known to chelate iron and copper in certain animal systems, inhibits gametangial formation in most bryophytes, except inBartramidula in which it enhances vegetative growth as well as gametangial formation. InBryum argenteum the effect of these chelates is accompanied with marked changes in the endogenous levels of iron and copper. Iron appears to favor the onset of reproductive phase, whereas copper is inhibitory. Cyclic AMP, a well known mediator of hormone action in animal systems, enhances gametangial formation inBryum argenteum. The response is specific and is mimicked by phosphodiesterase inhibitors. Cyclic AMP also increases antheridial production inBryum coronatum andBarbula gregaria. InBryum argenteum it overcomes the inhibitory effect of ammonium ions and high concentrations of sucrose on gametangial formation. In addition to the above factors, pH and nutritional status of the medium also affect the onset of reproductive phase. Bryophytes exhibit maximum response in a definite pH range. Moreover, a specific change in pH of the medium is observed during gametangial initiation. Nutritional status, as affected by the concentration of nutrients in the medium, has varied effects on gametangial formation. In most instances dilution of the medium is more favorable for the response. Certain other factors, such as yeast extract and animal sex hormones, also enhance the formation of antheridia and archegonia. The onset of the reproductive phase involves metabolic changes in the differentiating tissues. Archegonial initiation is accomplished by intense metabolic activities, and in certain liverworts there is an increase in the content of carbohydrates, auxins, RNA and proteins, whereas the level of total nitrogen drops. Besides this a number of enzymes and phenolic compounds also exhibit marked qualitative and quantitative changes. Once gametangia are induced under in vitro conditions, fertilization can be accomplished by flooding the cultures bearing mature gametangia with sterile, distilled water. Development of sporophytes is markedly affected by temperature and nutritional status of the medium.     

Sodium chloride stress induces nitric oxide accumulation in root tips and oil body surface accompanying slower oleosin degradation in sunflower seedlings

Present work highlights the involvement of endogenous nitric oxide (NO) in sodium chloride (NaCl)‐induced biochemical regulation of seedling growth in sunflower (Helianthus annuus L., cv. Morden). The growth response is dependent on NaCl concentration to which seedlings are exposed, they being tolerant to 40 mM NaCl and showing a reduction in extension growth at 120 mM NaCl. NaCl sensitivity of sunflower seedlings accompanies a fourfold increase in Na⁺/K⁺ ratio in roots (as compared to that in cotyledons) and rapid transport of Na⁺ to the cotyledons, thereby enhancing Na⁺/K⁺ ratio in cotyledons as well. A transient increase in endogenous NO content, primarily contributed by putative NOS activity in roots of 4‐day‐old seedlings subjected to NaCl stress and the relative reduction in Na⁺/K⁺ ratio after 4 days, indicates that NO regulates Na⁺ accumulation, probably by affecting the associated transporter proteins. Root tips exhibit an early and transient enhanced expression of 4,5‐diaminofluorescein diacetate (DAF‐2DA) positive NO signal in the presence of 120 mM NaCl. Oil bodies from 2‐day‐old seedling cotyledons exhibit enhanced localization of NO signal in response to 120 mM NaCl treatment, coinciding with a greater retention of the principal oil body membrane proteins, i.e. oleosins. Abolition of DAF positive fluorescence by the application of specific NO scavenger [2‐phenyl‐4,4,5,5‐tetramethyllimidazoline‐1‐oxyl‐3‐oxide (PTIO)] authenticates the presence of endogenous NO. These novel findings provide evidence for a possible protective role of NO during proteolytic degradation of oleosins prior to/accompanying lipolysis.     

Observation of polarity induction by cytochemical localization of phenylalkylamine-binding sites in regenerating protoplasts of the moss Physcomitrella patens

Different external (e.g., light) and internal (e.g., auxin and calcium gradients) factors control differentiation of the moss protonema. The present investigations demonstrate that exogenously applied auxin, the pharmacological blockade of auxin efflux by naphthylphthalamic acid, and treatment with (-)bepridil, a calcium channel antagonist, inhibit protoplast division without affecting protoplast viability in the moss Physcomitrella patens. A fluorescently labelled phenylalkylamine (DM-Bodipy PAA), another calcium channel antagonist, was used as a probe for in vivo labelling of phenylalkylamine(PAA)-binding sites. The specificity of this binding was demonstrated by competition with (-)bepridil. Confocal laser scanning microscopy visualized PAA-binding sites on the plasma membrane and along the nuclear membrane as uniformly distributed clusters. During asymmetric division of P. patens protoplasts, however, fluorescence labelling particularly increases at the membrane invagination and later along the plate separating the new cells. Intracellular localization of PAA-binding sites, probably at the membranes of vesicles and vacuoles, significantly increases in the smaller daughter cell, destined to later form a polar outgrowth, the first chloronema cell. Thus, a system was established to visualize early events in P. patens protoplast polarization at the subcellular level.     

Accumulation and scavenging of reactive oxygen species and nitric oxide correlate with stigma maturation and pollen–stigma interaction in sunflower

During the course of stigma development in sunflower (bud, staminate and pistillate stages), correlation is evident among the accumulation of reactive oxygen species (ROS), nitric oxide (NO), and the activities of ROS scavenging enzymes [superoxide dismutase (SOD) and peroxidase (POD)]. Confocal image analysis shows a gradual increase in ROS and NO accumulation in the stigmatic papillae, which may provide immunity to the developing stigma and function as signalling molecules. A novel, NO-specific probe (MNIP-Cu) has been employed for the detection and quantification of intracellular NO. Mn-SOD (mitochondrial) and Cu/Zn-SOD (cytoplasmic) exhibit differential expression during the staminate stage of stigma development. An increase in total SOD activity at the staminate stage is followed by a peak of POD activity during pistillate stage, thereby indicating the sequential action of the two enzymes in scavenging ROS. An increase in the number of POD isoforms is observed with the passage of stigma development (from three at bud stage to seven at pistillate stage), and two POD isoforms are unique to pistillate stage, thereby highlighting their role in ROS scavenging mechanism. ROS and NO accumulation exhibit reverse trends during pollen–stigma interaction.     

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.     

Iron homeostasis regulates maturation of tomato (climacteric) and capsicum (non-climacteric) fruits

Journal of Plant Biochemistry and Biotechnology - Endogenous iron quickly interchanges its oxidation states (Fe III and Fe II), which modulate production of reactive oxygen species (ROS) and nitric...     

A novel fluorescence imaging approach to monitor salt stress‐induced modulation of ouabain‐sensitive ATPase activity in sunflower seedling roots

Seedlings exposed to salt stress are expected to show modulation of intracellular accumulation of sodium ions through a variety of mechanisms. Using a new methodology, this work demonstrates ouabain (OU)‐sensitive ATPase activity in the roots of sunflower seedlings subjected to salt stress (120 mM NaCl). 9‐Anthroylouabain (a derivative of ouabain known to inhibit Na⁺,K⁺‐ATPase activity in animal systems, EC 3.6.3.9) has been used as a probe to analyze OU‐sensitive ATPase activity in sunflower (Helianthus annuus) seedling roots by spectrofluorometric estimation and localization of its spatial distribution using confocal laser scanning microscopy. Salt stress for 48 h leads to a significant induction of OU‐sensitive ATPase activity in the meristematic region of the seedling roots. Calcium ions (10 mM) significantly inhibit enzyme activity and a parallel accumulation of sodium ions in the cytosol of the columella cells, epidermis and in the cells of the meristematic region of the roots is evident. As a rapid response to NaCl stress, the activity of OU‐sensitive ATPase gets localized in the nuclear membrane of root protoplasts and it gets inhibited after treatment with calcium ions. Nuclear membrane localization of the OU‐sensitive ATPase activity highlights a possible mechanism to efflux sodium ions from the nucleus. Thus, a correlation between OU‐sensitive ATPase activity, its modulation by calcium ions and accumulation of sodium ions in various regions of the seedling roots, has been demonstrated using a novel approach in a plant system.     

Nitric oxide modulates specific steps of auxin-induced adventitious rooting in sunflower

Present work on indole-3-acetic acid (IAA)-induced adventitious rooting in sunflower hypocotyl highlights a clear demarcation of nitric oxide (NO)-dependent and NO-independent roles of auxin in this developmental process. Of the three phases of adventitious rooting, induction is strictly auxin-dependent though initiation and extension are regulated by an interaction of IAA with NO. A vital role of auxin-efflux transporters (PIN) is also evident from 1-napthylphthalamic acid (NPA)-triggered suppression of adventitious roots (AR). Use of actin depolymerizing agent, latrunculin B (Lat B), has demonstrated the necessity of functional actin filaments in auxin-induced AR response, possibly through its effect on actin-mediated recycling of auxin transporter proteins. Thus, evidence for a linkage between IAA, NO and actin during AR formation has been established.Key words: adventitious roots, auxin, sunflowerAdventitious roots (AR) are post-embryonic roots known to originate from stem, leaf petiole and non-pericycle tissue of old roots. In young stem, AR commonly arise from the interfascicular parenchyma while they appear from vascular rays near the cambium in older stem. Formation of AR begins with re-differentiation of predetermined cells which switch from their morphogenetic path to act as mother cells for initiation of root primordia.¹ The process of AR formation consists of three physiologically interdependent phases: induction, initiation and extension.¹ Induction phase comprises of various molecular and biochemical events but no morphologically visible changes appear during this phase. Formation of multilayered cells and conception of root primordia occurs during initiation phase. During expression phase, root primordia exhibit intra-stem growth and their emergence through epidermis. Various environmental and endogenous factors, such as temperature, light, hormones (particularly auxin), sugars and mineral salts, act as cues for promoting redifferentiation of predetermined cells resulting in root induction.The three phases of AR formation are known to be regulated by alterations in the endogenous level of auxin.² A transient increase in auxin concentration has been reported during induction phase, which is followed by a decrease and again an increase during expression phase.³ Auxin transport to and from the responding region is essential for root organogenesis. Acropetal transport of auxin occurs through the vascular cylinder and the basipetal transport takes place through the epidermal and subtending cortical cells.⁴ Polar transport of auxin from the shoot apical meristem to the rooting region is primarily facilitated by auxin-influx (AUX1) and -efflux (PIN) transporters. Asymmetric localization of these transporter proteins in the vascular cambium cells is responsible for differential distribution of auxin in a particular zone of cells.⁵ Polar auxin transport is known to be inhibited by 1-napthylphthalamic acid (NPA, a phytotropin). This inhibition is mediated through a binding of NPA molecule to putative NPA-binding protein (NBP), which is functionally associated with PIN proteins.⁶ Efflux transporters exhibit rapid turnover in plasma membrane.⁷ High affinity of NBP for actin filaments,^8,9 suggests its involvement in the cycling and polar distribution of PIN proteins.¹⁰ Organization of actin filaments is known to be rapidly, reversibly and specifically disrupted by Latrunculin B (Lat B), a macrolide toxin isolated from Latrunculia magnificia, a red sea sponge.¹¹ Lat B associates with actin monomers in 1:1 ratio, thereby preventing their repolymerization into filaments, resulting in a complete shift from F-actin to G-actin.¹² Owing to its well-understood and simple mode of action and low effective dosage, Lat B has supplanted the classic actin-depolymerizing drug cytochalasin D¹³ in pharmacological investigations. In the past few years, significant work has been done on NO as a signaling molecule in a variety of plant developmental processes.¹⁴ Nitric oxide is known to play a crucial role in root development.¹⁵Using sunflower as a model system, present work has been undertaken to investigate the possible role of NO during IAA-induced adventitious rooting in hypocotyl explants. Since auxin action is principally based on PIN-regulated polar transport of IAA molecules, and PIN proteins are known to exhibit actin-asssisted rapid recycling in the target cells, attempts have been made in the present work to find a correlation between auxin transport, actin and NO, using specific pharmacological agents. Additionally, effect of Cyclosporin A (CsA), an inhibitor of nitric oxide synthase (NOS) in animal systems,¹⁶ has also been investigated. These specific agents have been used to monitor root initiation at the target sites in our attempts to decipher a signaling cascade for AR.Seeds of Sunflower (Helianthus annuus L. cv. Morden) were germinated on moist germination sheets at 25 ± 2°C under continuous illumination of 4.3 Wm⁻². Hypocotyls from 4 d old seedlings and with similar growth rate, were excised up to 6 cm below the cotyledonary node. Hypocotyl explants with apical meristem intact but cotyledons excised were selected for the present work with a view to provide a continuity of the endogenous auxin source. Similar explants were also recently used by Huang et al.¹⁷ to investigate indole-3-butyric acid (IBA)-induced AR formation in mung bean (Phaseolus radiatus L.). Using IAA instead of IBA for such investigations was preferred for the present work keeping in view that the two auxins seem to employ different transport proteins for their polar transport.¹⁸ Freshly harvested explants were put upright in glass vials with their proximal cut ends dipped in 1 ml of different concentrations (1, 5, 10 and 15 µM) of IAA, thus bathing the hypocotyls up to 5 mm of their lower ends. Explants were maintained in dark during the course of experiments. The number of AR visible on hypocotyl surface was recorded daily up to 4 days of incubation. Concentration of IAA thus observed optimal for rooting (10 µM) was used for all further experiments. Similarly, various concentrations of other test solutions, namely NPA (auxin efflux blocker; 1 and 10 µM), Lat B (an inducer of actin depolymerization; 25, 50 and 100 nM), CsA (an inhibitor of cyclophilins; 1, 5 and 10 µM), sodium nitroprusside (SNP; NO donor; 1, 5, 10 and 100 µM) and 2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO; NO scavenger; 1 and 1.5 mM), were initially used to select their respective optimal concentrations. Based on these preliminary experiments NPA, Lat B, CsA, SNP and PTIO were used at 10 µM, 100 nM, 10 µM, 100 µM and 1.5 mM, respectively, for all subsequent experiments. Some other treatment combinations, namely NPA (10 µM) + IAA (10 µM); LatB (100 nM) + IAA (10 µM); CsA (10 µM) + IAA (10 µM); SNP (100 µM) + NPA (10 µM) and PTIO (1.5 mM) + IAA (10 µM) were also used to investigate their effects on adventitious rooting. Hypocotyl explants incubated in distilled water served as control. Morphological observations of rooting response were imaged after 7 days of incubation, using Nikon digital camera fitted on a stereomicroscope (Stemi 2000, Zeiss, Germany). Detailed evaluation of root initiation was observed after clearing by immersing the explants in a 3:1 solution of ethanol: acetic acid overnight. They were then transferred to 2 N NaOH solution, left overnight, washed once with distilled water and stained with safranin solution for 2–3 min. Excess stain was removed by repeated washing in distilled water. The lower 2 cm region of hypocotyl explants was then cut and mounted on a glass slide to examine endogenous root initials, using a stereomicroscope (Stemi 2000, Zeiss, Germany) fitted with a Nikon camera.Root initiation and extension in the basal region of hypocotyl explants maintained in distilled water indicates the expected basipetal transport of the inducing factor (endogenous IAA) from the intact meristem, as also reported earlier.¹⁹ Treatment with IAA (10 µM) elicited two effects on hypocotyl explants in comparison to those subjected to distilled water treatment: (1) Formation of greater number of root initials, (2) Greater extension of the initiated roots (Figs. 1 and ​and22). A response similar to that evoked by IAA is also evident in hypocotyl explants treated with 100 µM of SNP (Figs. 1 and ​and22). Recently SNP (NO donor) has been reported to evoke dose-dependent response on AR formation in marigold.²⁰ In the present work, treatment with variable concentrations of SNP, ranging from 1–100 µM, lead to a gradual increase in the number and extension growth of AR till 100 µM. Pagnussat et al.²¹ and Liao et al.²⁰ have used 10 µM and 50 µM as effective SNP concentration in cucumber and marigold, respectively. Thus, optimal concentration of SNP for AR formation is species-dependent. In presence of PTIO (1.5 mM; a specific NO scavenger), complete suppression of AR was evident in sunflower, as also reported earlier in mung bean.¹⁷ Combination of PTIO with IAA lead to root initiation only (no extension growth). NPA (10 µM) blocked AR initiation by endogenous (distilled water treatment) and exogenous IAA (Fig. 1). Application of NPA inhibits polar auxin transport, thus reducing the optimal concentration of IAA required for AR formation at the hypocotyl base (zone of AR formation). Thus, no evidence of root initials was evident in presence of NPA, which is also reported in cucumber²² and loblolly pine,²³ respectively. Though NO is expected to act downstream of IAA²⁴ but a treatment of SNP in combination with NPA (present work) lead to complete suppression of AR formation. Our unpublished observations have indicated the expression of NO in the interfascicular cells after induction phase (i.e., during AR iniation and extension). Thus, it can be proposed that IAA is involved in induction phase of adventitious rooting independent of NO, while initiation and extension phases appear to involve IAA-NO interaction. CsA-cyclophilin complex is known to inhibit calcineurin (a protein phosphatase) and NOS activity in animal systems.²⁵ Treatment of hypocotyl explants with CsA (10 µM) lead to formation of fewer number of roots which exhibited extension growth. Oh et al.²⁶ reported a significant reduction in the number of roots in the presence of CsA in hypocotyl explants from tomato. Subjecting hypocotyl explants with a combination of CsA and IAA lead to formation of fewer number of root initials, reaffirming the involvement of NO in auxin action in the developmental process under investigation (AR). However, further investigations on the role of cyclophilins and NOS in auxin-modulated AR formation are required to pinpoint their specific sites of action in this developmental process. Treatment with Lat B (+ and − IAA) lead to complete AR suppression in sunflower hypocotyl explants. Actin-mediated polar localization of PIN proteins is responsible for polar auxin transport and disruption of microfilaments by Lat-B would thus, directly affect IAA transport leading to the observed AR suppression. These observations indicate a convergence of the effect of IAA with that of NO and a role of a well organized actin in the responding cells.Open in a separate windowFigure 1Effect of IAA and various other pharmacological agents on adventitious rooting in hypocotyl explants. Morphological observations of rooting response (A and C). Evaluation of endogenous root initiation and elongation observed in cleared explants stained with safranin (B and D). Scale bar represents 3 mm.Open in a separate windowFigure 2Quantitative analysis of AR initiation in presence of distilled water, IAA and various pharmacological agents in hypocotyl explants of sunflower. Each datum presents a mean and standard error from at least three observations.To sum up, present investigations provide evidence for a linkage between auxin-induced AR response in seedling hypocotyls and NO (Fig. 3). Both endogenous and exogenous IAA-mediated AR induction seem to depend on actin. Significance of actin in this developmental response has become evident via its role in the cycling of auxin efflux proteins (PIN). The three phases of AR formation can be differentiated from each other in terms of their sensitivities to IAA and NO. AR induction phase seems to be governed by auxin alone, independent of NO. NO seems to become operative in this auxin-modulated response (AR) during initiation and extension phase only. Investigations are being undertaken in the author''s laboratory to visualize and quantitate the NO signal in the IAA-responding hypocotyl explants so that the phasing of the role of NO during AR formation can be precisely predicted.Open in a separate windowFigure 3Schematic presentation of the probable events associated with NO-mediated adventitious rooting.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.