Unveiling the applications of membrane proteins from oil bodies: leading the way in artificial oil body technology and other biotechnological advancements.

Oil bodies (OBs) function as organelles that store lipids in plant seeds. An oil body (OB) is encased by a membrane composed of proteins (e.g., oleosins, caleosins, and steroleosins) and a phospholipid monolayer. The distinctive protein-phospholipid membrane architecture of OBs imparts exceptional stability even in extreme environments, thereby sparking increasing interest in their structure and properties. However, a comprehensive understanding of the structure-activity relationships determining the stability and properties of oil bodies requires a more profound exploration of the associated membrane proteins, an aspect that remains relatively unexplored. In this review, we aim to summarize and discuss the structural attributes, biological functions, and properties of OB membrane proteins. From a commercial perspective, an in-depth understanding of the structural and functional properties of OBs is important for the expansion of their applications by producing artificial oil bodies (AOB). Besides exploring their structural intricacies, we describe various methods that are used for purifying and isolating OB membrane proteins. These insights may provide a foundational framework for the practical utilization of OB membrane proteins in diverse applications within the realm of AOB technology, including biological and probiotic delivery, protein purification, enzyme immobilization, astringency detection, and antibody production.

Genome-Wide Association Study of Cuticle and Lipid Droplet Properties of Cucumber (Cucumis sativus L.) Fruit.

The fruit surface is a critical first line of defense against environmental stress. Overlaying the fruit epidermis is the cuticle, comprising a matrix of cutin monomers and waxes that provides protection and mechanical support throughout development. The epidermal layer of the cucumber (Cucumis sativus L.) fruit also contains prominent lipid droplets, which have recently been recognized as dynamic organelles involved in lipid storage and metabolism, stress response, and the accumulation of specialized metabolites. Our objective was to genetically characterize natural variations for traits associated with the cuticle and lipid droplets in cucumber fruit. Phenotypic characterization and genome-wide association studies (GWAS) were performed using a resequenced cucumber core collection accounting for >96% of the allelic diversity present in the U.S. National Plant Germplasm System collection. The collection was grown in the field, and fruit were harvested at 16-20 days post-anthesis, an age when the cuticle thickness and the number and size of lipid droplets have stabilized. Fresh fruit tissue sections were prepared to measure cuticle thickness and lipid droplet size and number. The collection showed extensive variation for the measured traits. GWAS identified several QTLs corresponding with genes previously implicated in cuticle or lipid biosynthesis, including the transcription factor SHINE1/WIN1, as well as suggesting new candidate genes, including a potential lipid-transfer domain containing protein found in association with isolated lipid droplets.

Genome-wide analysis of the GDSL esterase/lipase family genes in Physcomitrium patens and the involvement of GELP31 in spore germination.

In plants, the ability to produce hydrophobic substances that would provide protection from dehydration was required for the transition to land. This genome-wide investigation outlines the evolution of GDSL-type esterase/lipase (GELP) proteins in the moss Physcomitrium patens and suggests possible functions of some genes. GELP proteins play roles in the formation of hydrophobic polymers such as cutin and suberin that protect against dehydration and pathogen attack. GELP proteins are also implicated in processes such as pollen development and seed metabolism and germination. The P. patens GELP gene family comprises 48 genes and 14 pseudogenes. Phylogenetic analysis of all P. patens GELP sequences along with vascular plant GELP proteins with reported functions revealed that the P. patens genes clustered within previously identified A, B and C clades. A duplication model predicting the expansion of the GELP gene family within the P. patens lineage was constructed. Expression analysis combined with phylogenetic analysis suggested candidate genes for functions such as defence against pathogens, cutin metabolism, spore development and spore germination. The presence of relatively fewer GELP genes in P. patens may reduce the occurrence of functional redundancy that complicates the characterization of vascular plant GELP genes. Knockout lines of GELP31, which is highly expressed in sporophytes, were constructed. Gelp31 spores contained amorphous oil bodies and germinated late, suggesting (a) role(s) of GELP31 in lipid metabolism in spore development or germination. Future knockout studies of other candidate GELP genes will further elucidate the relationship between expansion of the family and the ability to withstand the harsh land environment.

Impact of processing on the oxidative stability of oil bodies.

Plant lipids are stored as emulsified lipid droplets also called lipid bodies, spherosomes, oleosomes or oil bodies. Oil bodies are found in many seeds such as cereals, legumes, or in microorganisms such as microalgae, bacteria or yeast. Oil Bodies are unique subcellular organelles with sizes ranging from 0.2 to 2.5 µm and are made of a triacylglycerols hydrophobic core that is surrounded by a unique monolayer membrane made of phospholipids and anchored proteins. Due to their unique properties, in particular their resistance to coalescence and aggregation, oil bodies have an interest in food formulations as they can constitute natural emulsified systems that does not need the addition of external emulsifier. This manuscript focuses on how extraction processes and other factors impact the oxidative stability of isolated oil bodies. The potential role of oil bodies in the oxidative stability of intact foods is also discussed. In particular, we discuss how constitutive components of oil bodies membranes are associated in a strong network that may have an antioxidant effect either by physical phenomenon or by chemical reactivities. Moreover, the importance of the selected process to extract oil bodies is discussed in terms of oxidative stability of the recovered oil bodies.

Liverwort oil bodies: diversity, biochemistry, and molecular cell biology of the earliest secretory structure of land plants.

Liverworts are known for their large chemical diversity. Much of this diversity is synthesized and enclosed within oil bodies (OBs), a synapomorphy of the lineage. OBs contain the enzymes to biosynthesize and store large quantities of sesquiterpenoids and other compounds while limiting their cytotoxicity. Recent important biochemical and molecular discoveries related to OB formation, diversity, and biochemistry allow comparison with other secretory structures of land plants from an evo-devo perspective. This review addresses and discusses the most recent advances in OB origin, development, and function towards understanding the importance of these organelles in liverwort physiology and adaptation to changing environments. Our mapping of OB types and chemical compounds to the current liverwort phylogeny suggests that OBs were present in the most recent common ancestor of liverworts, supporting that OBs evolved as the first secretory structures in land plants. Yet, we require better sampling to define the macroevolutionary pattern governing the ancestral type of OB. We conclude that current efforts to find molecular mechanisms responsible for the morphological and chemical diversity of secretory structures will help understand the evolution of each major group of land plants, and open new avenues in biochemical research on bioactive compounds in bryophytes and vascular plants.

Exploring The Relative Astringency of Tea Catechins and Distinct Astringent Sensation of Catechins and Flavonol Glycosides via an In Vitro Assay Composed of Artificial Oil Bodies.

Artificial oil bodies covered by a recombinant surface protein, caleosin fused with histatin 3 (a major human salivary peptide), were employed to explore the relative astringency of eight tea catechins. The results showed that gallate-type catechins were more astringent than non-gallate-type catechins, with an astringency order of epicatechin gallate > epigallocatechin gallate > gallocatechin gallate > catechin gallate > epigallocatechin > epicatechin > gallocatechin > catechin. As expected, the extension of brewing time led to an increase in catechin content in the tea infusion, thus elevating tea astringency. Detailed analysis showed that the enhanced proportion of gallate-type catechins was significantly higher than that of non-gallate-type catechins, indicating that tea astringency was elevated exponentially, rather than proportionally, when brewing time was extended. Rough surfaces were observed on artificial oil bodies when they were complexed with epigallocatechin gallate (a catechin), while a smooth surface was observed on those complexed with rutin (a flavonol glycoside) under an atomic force microscope and a scanning electron microscope. The results indicate that catechins and flavonol glycosides induce the sensation of rough (puckering) and smooth (velvety) astringency in tea, respectively.

NaCl induces flocculation and lipid oxidation of soybean oil body emulsions recovered by neutral aqueous extraction.

BACKGROUND: Soybean oil bodies (SOB) are naturally pre-emulsified lipid droplets recovered directly from soybean seeds. Almost all food emulsions contain salts. However, it was not clear how the incorporation of salts affected the physicochemical stability of SOB. RESULTS: This study investigated the effect of NaCl (0-1.2%) on the physical and oxidative stability of SOB emulsions under neutral (pH 7) and acidic (pH 3) conditions. In the presence of NaCl, the SOB emulsion (pH 7) showed strong flocculation during storage due to electrostatic screening. The NaCl-induced flocculation of SOB was attenuated at pH 3, which may be due to the difference in conformation or interaction of the protein interfaces covering SOB at different pH values. The increase in ionic strength or acid conditioning treatment resulted in a remarkable increase in the stability of SOB emulsions against coalescence. The confocal laser scanning microscopy images also confirmed the NaCl-induced changes in the flocculation/coalescence properties of SOB. The oxidative behavior tests indicated that SOB emulsions containing NaCl were more susceptible to lipid oxidation but protein oxidation was inhibited due to electrostatic screening, which reduced pro-oxidant accessibility of unadsorbed proteins in the emulsion. This oxidative behavior was attenuated at pH 3. CONCLUSION: The incorporation of NaCl significantly reduced the physical and oxidative stability of the SOB emulsion, and acidic pH mitigated NaCl-induced flocculation and lipid oxidation of SOB. © 2021 Society of Chemical Industry.

Fungal-Type Terpene Synthases in Marchantia polymorpha Are Involved in Sesquiterpene Biosynthesis in Oil Body Cells.

The liverwort Marchantia polymorpha possesses oil bodies in idioblastic oil body cells scattered in its thallus. Oil bodies are subcellular organelles in which specific sesquiterpenes and bisbibenzyls are accumulated. Therefore, a specialized system for the biosynthesis and accumulation of these defense compounds specifically in oil bodies has been implied. A recent study on M. polymorpha genome sequencing revealed 10 genes that shared high similarities with fungal-type terpene synthases (TPSs). Eight of these fungal-type TPS-like genes in M. polymorpha (MpFTPSL1-6, -9 and -10) are located within a 376-kb stretch on chromosome 6 and share similarities of over 94% at the nucleotide level. Therefore, these genes have likely originated from recent gene duplication events. The expression of a subset of MpFTPSLs was induced under non-axenic growth on vermiculite, which increased the amounts of sesquiterpenes and number of oil bodies. The tdTomato fluorescent protein-based in-fusion reporter assay with MpFTPSL2 promoter revealed fluorescent signals specifically in oil body cells of the thallus, indicating that MpFTPSL2 functions in oil body cells. Recombinant MpFTPSL2 expression in Escherichia coli led to sesquiterpene synthesis from farnesyl pyrophosphate. Moreover, suppression of a subset of MpFTPSLs through RNA interference reduced sesquiterpene accumulation in thalli grown on vermiculite. Taken together, these results suggest that at least a subset of MpFTPSLs is involved in sesquiterpene synthesis in oil body cells.

A pex1 missense mutation improves peroxisome function in a subset of Arabidopsis pex6 mutants without restoring PEX5 recycling.

Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid ß-oxidation. The interacting ATPases PEX1 and PEX6 contribute to peroxisome function by recycling PEX5, a cytosolic receptor needed to import proteins targeted to the peroxisomal matrix. Arabidopsis pex6 mutants exhibit low PEX5 levels and defects in peroxisomal matrix protein import, oil body utilization, peroxisomal metabolism, and seedling growth. These defects are hypothesized to stem from impaired PEX5 retrotranslocation leading to PEX5 polyubiquitination and consequent degradation of PEX5 via the proteasome or of the entire organelle via autophagy. We recovered a pex1 missense mutation in a screen for second-site suppressors that restore growth to the pex6-1 mutant. Surprisingly, this pex1-1 mutation ameliorated the metabolic and physiological defects of pex6-1 without restoring PEX5 levels. Similarly, preventing autophagy by introducing an atg7-null allele partially rescued pex6-1 physiological defects without restoring PEX5 levels. atg7 synergistically improved matrix protein import in pex1-1 pex6-1, implying that pex1-1 improves peroxisome function in pex6-1 without impeding autophagy of peroxisomes (i.e., pexophagy). pex1-1 differentially improved peroxisome function in various pex6 alleles but worsened the physiological and molecular defects of a pex26 mutant, which is defective in the tether anchoring the PEX1-PEX6 hexamer to the peroxisome. Our results support the hypothesis that, beyond PEX5 recycling, PEX1 and PEX6 have additional functions in peroxisome homeostasis and perhaps in oil body utilization.

Standardized methods for testing the quality attributes of plant-based foods: Milk and cream alternatives.

The food industry is creating a diverse range of plant-based alternatives to dairy products, such as milks, creams, yogurts, and cheeses due to the increasing demand from consumers for more sustainable, healthy, and ethical products. These dairy alternatives are often designed to mimic the desirable physicochemical, functional, and sensory properties of real dairy products, such as their appearance, texture, mouthfeel, flavor, and shelf-life. At present, there is a lack of systematic testing methods to characterize the properties of plant-based dairy alternatives. The purpose of this review is to critically evaluate existing methods and recommend a series of standardized tests that could be used to quantify the properties of fluid plant-based milk alternatives (milk and cream). These methods could then be used to facilitate the design of milk alternatives with somewhat similar attributes as real dairy milk by comparing their properties under standardized conditions. Moreover, they could be used to facilitate comparison of the properties of milk alternatives developed in different laboratories.

The wheat ABC transporter Lr34 modifies the lipid environment at the plasma membrane.

Phospholipids (PLs) are emerging as important factors that initiate signal transduction cascades at the plasma membrane. Their distribution within biological membranes is tightly regulated, e.g. by ATP-binding cassette (ABC) transporters, which preferably translocate PLs from the cytoplasmic to the exoplasmic membrane leaflet and are therefore called PL-floppases. Here, we demonstrate that a plant ABC transporter, Lr34 from wheat (Triticum aestivum), is involved in plasma membrane remodeling characterized by an intracellular accumulation of phosphatidic acid and enhanced outward translocation of phosphatidylserine. In addition, the content of phosphatidylinositol 4,5-bisphosphate in the cytoplasmic leaflet of the plasma membrane was reduced in the presence of the ABC transporter. When heterologously expressed in Saccharomyces cerevisiae, Lr34 promoted oil body formation in a mutant defective in PL-transfer in the secretory pathway. Our results suggest that PL redistribution by Lr34 potentially affects the membrane-bound proteome and contributes to the previously reported stimuli-independent activation of biotic and abiotic stress responses and neutral lipid accumulation in transgenic Lr34-expressing barley plants.

Improving the Stability of Oil Body Emulsions from Diverse Plant Seeds Using Sodium Alginate.

In this study, peanut, sesame, and rapeseed oil bodies (OBs) were extracted by the aqueous medium method. The surface protein composition, microstructure, average particle size d 4 ,   3 , ζ-potential of the extracted OBs in aqueous emulsion were characterized. The stability of the OB emulsions was investigated. It was found that different OB emulsions contained different types and contents of endogenous and exogenous proteins. Aggregation at low pHs (<6) and creaming at high pHs (7 and 8) both occurred for all of three OB emulsions. Sodium alginate (ALG) was used to solve the instability of OB emulsions under different conditions-low concentration of ALG improved the stability of OB emulsions below and near the isoelectric point of the OBs, through electrostatic interaction. While a high concentration of ALG improved the OB emulsion stability through the viscosity effect at pH 7. The OB emulsions stabilized by ALG were salt-tolerant and freeze-thaw resistant.

Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies.

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

Disparate peroxisome-related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization.

Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail-anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling ß-oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome-associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.

Peroxisome extensions deliver the Arabidopsis SDP1 lipase to oil bodies.

Lipid droplets/oil bodies (OBs) are lipid-storage organelles that play a crucial role as an energy resource in a variety of eukaryotic cells. Lipid stores are mobilized in the case of food deprivation or high energy demands--for example, during certain developmental processes in animals and plants. OB degradation is achieved by lipases that hydrolyze triacylglycerols (TAGs) into free fatty acids and glycerol. In the model plant Arabidopsis thaliana, Sugar-Dependent 1 (SDP1) was identified as the major TAG lipase involved in lipid reserve mobilization during seedling establishment. Although the enzymatic activity of SDP1 is associated with the membrane of OBs, its targeting to the OB surface remains uncharacterized. Here we demonstrate that the core retromer, a complex involved in protein trafficking, participates in OB biogenesis, lipid store degradation, and SDP1 localization to OBs. We also report an as-yet-undescribed mechanism for lipase transport in eukaryotic cells, with SDP1 being first localized to the peroxisome membrane at early stages of seedling growth and then possibly moving to the OB surface through peroxisome tubulations. Finally, we show that the timely transfer of SDP1 to the OB membrane requires a functional core retromer. In addition to revealing previously unidentified functions of the retromer complex in plant cells, our work provides unanticipated evidence for the role of peroxisome dynamics in interorganelle communication and protein transport.

Phytosterol Composition of Arachis hypogaea Seeds from Different Maturity Classes.

The seeds of cultivated peanut, Arachis hypogaea, are an agronomically important crop produced for human nutrition, oilseed and feed stock. Peanut seed is the single most expensive variable input cost and thus producers require seed with excellent performance in terms of germination efficiency. During the maturation process, triglycerides are stored in oil bodies as an energy resource during germination and seedling development. The stability of oil body membranes is essential for nutrient mobilization during germination. This study focused on evaluating the phytosterol composition in seed components including the kernel, embryo (heart), and seed coat or skin. Samples of different maturity classes were analyzed for macronutrient and phytosterol content. The three biosynthetic end products in the phytosterol pathway, ß-sitosterol, campesterol and stigmasterol, comprised 82.29%, 86.39% and 94.25% of seed hearts, kernels and seed coats, respectively. Stigmasterol concentration was highest in the seed kernel, providing an excellent source of this sterol known to have beneficial effects on human health. Peanut hearts contained the highest concentration of sterols by mass, potentially providing protection and resources for the developing seedling. The amount of α-tocopherol increases in peanut hearts during the maturation process, providing protection from temperature stress, as well as stability required for seedling vigor. These results suggest that phytosterols may play a significant role in the performance of seeds, and provide a possible explanation for the poor germination efficiency of immature seeds.

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

BACKGROUND: Lobosphaera 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. RESULTS: Here, 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. CONCLUSIONS: Here 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.

Oil body biogenesis and biotechnology in legume seeds.

The seeds of many legume species including soybean, Pongamia pinnata and the model legume Medicago truncatula store considerable oil, apart from protein, in their cotyledons. However, as a group, legume storage strategies are quite variable and provide opportunities for better understanding of carbon partitioning into different storage products. Legumes with their ability to fix nitrogen can also increase the sustainability of agricultural systems. This review integrates the cell biology, biochemistry and molecular biology of oil body biogenesis before considering biotechnology strategies to enhance oil body biosynthesis. Cellular aspects of packaging triacylglycerol (TAG) into oil bodies are emphasized. Enhancing seed oil content has successfully focused on the up-regulation of the TAG biosynthesis pathways using overexpression of enzymes such as diacylglycerol acyltransferase1 and transcription factors such as WRINKLE1 and LEAFY COTYLEDON1. While these strategies are central, decreasing carbon flow into other storage products and maximizing the packaging of oil bodies into the cytoplasm are other strategies that need further examination. Overall there is much potential for integrating carbon partitioning, up-regulation of fatty acid and TAG synthesis and oil body packaging, for enhancing oil levels. In addition to the potential for integrated strategies to improving oil yields, the capacity to modify fatty acid composition and use of oil bodies as platforms for the production of recombinant proteins in seed of transgenic legumes provide other opportunities for legume biotechnology.

Oil Secretory System in Vegetative Organs of Three Arnica Taxa: Essential Oil Synthesis, Distribution and Accumulation.

Arnica, a genus including the medicinal species A. montana, in its Arbo variety, and A. chamissonis, is among the plants richest in essential oils used as pharmaceutical materials. Despite its extensive use, the role of anatomy and histochemistry in the internal secretory system producing the essential oil is poorly understood. Anatomical sections allowed differentiation between two forms of secretory structures which differ according to their distribution in plants. The first axial type is connected to the vascular system of all vegetative organs and forms canals lined with epithelial cells. The second cortical type is represented by elongated intercellular spaces filled with oil formed only between the cortex cells of roots and rhizomes at maturity, with canals lacking an epithelial layer.Only in A. montana rhizomes do secretory structures form huge characteristic reservoirs. Computed tomography illustrates their spatial distribution and fusiform shape. The axial type of root secretory canals is formed at the interface between the endodermis and cortex parenchyma, while, in the stem, they are located in direct contact with veinal parenchyma. The peripheral phloem parenchyma cells are arranged in strands around sieve tube elements which possess a unique ability to accumulate large amounts of oil bodies. The cells of phloem parenchyma give rise to the aforementioned secretory structures while the lipid components (triacylglycerols) stored there support the biosynthesis of essential oils by later becoming a medium in which these oils are dissolved. The results indicate the integrity of axial secretory structures forming a continuous system in vegetative plant organs.

Nitric oxide accumulation and protein tyrosine nitration as a rapid and long distance signalling response to salt stress in sunflower seedlings.

Sensing of salt stress by sunflower seedlings accompanies temporal and spatial modulation of intracellular nitric oxide (NO) accumulation and protein tyrosine nitration as markers of nitrosative stress. Employing a novel NO-specific probe for NO localization (a copper derivative of 4-methoxy-2-(1H-naphtho(2,3-d)imidazol-2-yl)phenol; MNIP-Cu) synthesized in author's laboratory, immunological analysis of tyrosine nitrated proteins by confocal laser scanning microscopy (CLSM) and Western blot analysis, these rapid signalling events have been investigated. MNIP-Cu reveals the distribution of NO in whole seedlings. Preferential and enhanced NO localization around oil bodies (OBs) in cotyledons within 48 h of salt-stressed seedlings exhibits rapid transport of nitrosative stress signal from roots to the cotyledons. Immunological analysis reveals enhanced gradient of tyrosine nitrated proteins in salt-stressed roots from tip to the differentiating zone and from columella to the deep-seated cells. Western blot analysis shows that at least eight major cytosolic proteins exhibit enhanced tyrosine nitration in seedling roots in response to salt stress. Present observations provide strong evidence for rapid NO accumulation in salt stressed sunflower seedling roots and cotyledons and its impact on enhanced tyrosine nitration of cytosolic and OB proteins, as a mechanism to provide longevity to OBs for seedling survival under the salt stress.