Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea

Seasonal changes in the fatty acid composition of phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FA) and triglycerides (TG) separated from oleosomes (lipid bodies) of perennial root nodules of beach pea (Lathyrus maritimus) were analysed. Thin layer chromatography (TLC) revealed that PL and MG are the major lipids in nodule oleosomes. The fatty acid profile and overall double bond index (DBI) varied among lipid classes depending upon the season. High DBI in PL and MG found during late winter and early spring indicated that they may play a major role in winter survival and regeneration of perennial nodules. The DBI of DG was high at the end of the fall season and the DBI of FA and TG was high in summer months. The dominant fatty acids are C16:0 followed by C18:0 and C18:1. The levels of many unsaturated fatty acids such as C18:1, C18:2 and C18:3 increased while saturated fatty acid C18:0 decreased during winter. These unsaturated fatty acids possibly play an important role in the protection of nodule cells from cold stress. Nodules seem to retain some fatty acids and selectively utilize specific fatty acids to survive the winter and regenerate in spring.     

Symbiotic nitrogen-fixing root nodules of Lathyrus maritimus (L.) Bigel (beach pea) from Newfoundland shore lines with special reference to oleosomes (lipid bodies)

Lathyrus maritimus (L.) Bigel, commonly known as beach pea, grows along the shorelines of Newfoundland, Canada. Rhizobia have been isolated from the subterranean root nodules and the cultures were induced to nodulate seedlings raised in the laboratory. The nodules collected from the field were elongated and sometimes branched with proximal ends thickened. Histological characteristics revealed their indeterminate perennial form. Oleosomes (lipid bodies) were present in the nodule tissues. Morphometric analysis showed their presence in the nodule cortex cells throughout all developmental stages, but they were found in the infected cells only during the early stages of infection and symbiosis. It is suggested that oleosomes are utilized for membrane and rhizobial proliferation during establishment of symbiosis in the infected cells. The role of oleosomes in the cortical cells remains unclear.     

Oleosomes in flag leaves of wheat; their distribution,composition and fate during senesence and rust-infection

Oleosomes, up to 14m in diameter, were found in mesophyll and bundle sheath cells of the flag and lower leaves of wheat cv Professeur Marchal. They develop in flag leaves at least 10 d before anthesis, possibly from fatty acids secreted by the plastids, and persist in mature and senescing leaf tissue. Oleosomes are bordered with an osmiophilic layer rather than a unit membrane. The major lipids of oleosomes, isolated 20 d after anthesis, are triacylglycerols (50%) and sterol or wax exter (34%). The dominant fatty acids of both lipid classes are plamitic (16:0) and stearic (18:0) acids which accounts for the low osmiophilia of the oleosomes. The function of the oleosomes is unknown but they may act as short-term energy reserves. Oleosomes persist in leaves infected with brown rust, even in cells penetrated by haustoria. Yellowish-brown oleosomes found in senescing and rust-infected leaves may be formed by the release and coalescence of pigmented plastoglobuli.     

Fine structure of nitrogen-fixing leguminous root nodules from the Canadian Arctic

Effective (nitrogen-fixing) root nodules of Oxytropis maydelliana Trautv., O. arctobia Bunge and Astragulus alpinus L. were collected in the high Arctic tundra and subsequently processed for structural studies. The cylindrically-shaped perennial nodules consisted of the following tissues: nodule cortex, nodule meristem, nodular vascular bundles, an active central region with uninfected and infected cells at various stages of development, and a proximal region of senescent cells. The active central region was dark red-coloured due to the presence of the pigment leghemoglobin. The host cells became infected by the growth of infection threads into cells recently derived from the nodule meristem and the subsequent endocytotic release of rhizobia from unwalled membrane-bound regions of the infection thread. The host plasma membrane adjacent to the unwalled regions of infection thread gave rise to the peribacteroid membrane which surrounded the released bacteria. Thus, nodule development and the basic tissue arrangement of the arctic nodules was similar to that of cylindrically-shaped nodules formed on temperate species of legumes.
The arctic legume nodules are unique in having large numbers of lipid droplets present in the cytoplasm of the nodule cortex and uninfected cells of the central active region. Newly infected cells also have lipid droplets. More developed infected cells lack lipid droplets but often contain amyloplasts. Mature differentiated bacteria were spherically-shaped and contained electron-dense inclusions. Electron-dense material was also present in vesicles formed from dilated endoplasmic reticulum and in the peribacteroid space. The lipid droplets present in the host cytoplasm of the nodule cortex and uninfected cells of the central tissue may be storage products which are used to support nitrogen-fixation in nodules growing under cool temperatures of this harsh environment.     

Electron-dense material in the cell sall/plasma membrane interface of specialized cortical cells of peanut nodules.

The nitrogen-fixing peanut root nodules have 2-3 layers of specialized cortical cells surrounding the infected zone. These cells are morphologically distinct from the other cortical cells due to the presence of prominent amyloplasts, lipid bodies (oleosomes), large microbodies and localized pockets of electron-dense material at the plasma membrane-cell wall interface. The material is resistant to solubilization by hexane and chloroform. Electron density is enhanced by p-phenylenediamine staining and heavy metal ions such as iron, uranium and osmium. The silver proteinate staining method for suberin has shown positive results. Peroxidative activity can be demonstrated by diaminobenzidine reaction. The material appears to be proteinaceous, but could be complexed with suberin or phenolics. Although the exact nature of this material remains to be clarified, its unique position in the cells of specialized tissue of the cortex relates it to the regulation of bidirectional transport of gases and solutes during symbiosis in the root nodules.     

Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules

Summary Nitrogen-fixing peanut root nodules are characterized by their unique structural organization, distinct from other legume nodules. The focus of this study has been in and around the hostsymbiont interface, where the bacterioid and the host cell surface (peribacteroid membrane envelope) interact during symbiosis. The infected nodule cells have revealed the presence of lipid bodies (oleosomes) in intimate association with the peribacteroid membrane, which encloses the large spherical bacteroids with a relatively narrow peribacteroid space. Electron dense structures, referred to as dense bodies have been found attached to the bacteroid outer membranes at the host-symbiont interface. The dense bodies are osmiophilic, amorphous and 3,3-diaminobenzidine positive. The isolated intact bacteroids with dense bodies attached to their cell wall showed significant catalase activity. Many microbodies showing DAB-positive reaction have been found in the host cytoplasm, associated closely with the peribacteroid membrane. These ultrastructural and cytochemical characteristics of peanut root nodules suggest that lipids are utilized during symbiosis and the dense bodies and microbodies may be involved in the catabolic process.Abbreviation DAB 3,3-diaminobenzidine     

OXYGEN CONCENTRATION WITHIN THE NITROGEN-FIXING ROOT NODULES OF MYRICA GALE L.

Microelectrodes were used to study the oxygen concentration within Myrica gale L. nodules. Low oxygen concentrations were found only in the region of the mature, nitrogen-fixing endophyte, and appeared to correspond to clusters of infected host cells. The oxygen concentration in the remainder of the nodule was much higher. Interconnected intercellular air spaces were demonstrated by infiltration with India ink. Infiltration of the spaces with water greatly reduced oxygen concentration throughout the nodule, indicating that they function in supplying oxygen to the infected cells and remainder of the nodule. These results differ from those found previously for soybean nodules and provide evidence that legume and actinorhizal nodules have different mechanisms for protecting nitrogenase from oxygen.     

Lipid composition of somatic and zygotic embryos from Prunus avium. Effect of a cold treatment on somatic embryo quality

In order to evaluate the quality of Prunus avium somatic embryos, a comparison of lipid composition between somatic and zygotic embryos was undertaken. In both zygotic and somatic embryos, neutral glycerolipids (NL) and phosphatidylcholine (PC) were the 2 major lipid classes. The content of NL increased over the course of development in zygotic embryos and reached 490 μg per embryo, while the PC content reached 100 μg per embryo. However, the contents of NL and PC in somatic embryos were similar to immature zygotic embryos at stage 3. Fatty acid composition of NL from both zygotic and somatic embryos revealed more unsaturated than saturated fatty acids. In somatic embryos, the saturated/unsaturated fatty acid ratios of NL and phosphatidylinositol (PI) were similar to those observed in immature zygotic embryos up to stage 6. Conversely, in phosphatidylethanolamine (PE) the ratio was similar to the ratio observed in mature zygotic embryos, at stage 7. Histological studies confirmed the immaturity of somatic embryos: no protein or lipid reserves were observed in the vacuolated cotyledonary cells. Maturation of somatic embryos was improved by a 2-month cold period. In cold-treated somatic embryos, both NL and PC increased to levels comparable to those observed in mature zygotic embryos, and the PE content reached 10 times the level of that in mature zygotic embryos. The cold treatment induced a large increase in the saturated/unsaturated fatty acid ratio in phospholipids but only a slight increase in that of neutral glycerolipids. Histological studies revealed a lipid accumulation at cellular level. Lipid bodies surrounded by protein bodies were observed in cotyledonary cells of cold-treated somatic embryos. Furthermore, the cold-treated somatic embryos developed into plantlets with a frequency of 14%, whereas no development was obtained with the non-treated somatic embryos.     

Cytoskeletal arrays in the cells of soybean root nodules: The role of actin microfilaments in the organisation of symbiosomes

Summary Within the infected cells of root nodules there is evidence of stratification and organisation of symbiosomes and other organelles. This organisation is likely to be important for the efficient exchange of nutrients and metabolites during functioning of the nodules. Using immunocytochemical labelling and confocal microscopy we have determined the organisation of cytoskeletal elements, micro tubules and actin microfilaments in soybean nodule cells, with a view to assessing their possible role in organelle distribution. Most microtubule arrays occurred in the cell cortex where they formed disorganised arrays in both uninfected and infected cells from mature nodules. In infected cells from developing nodules, parallel arrays of microtubules, transverse to the long axis of the cell, were observed. In incipient nodules, before release of rhizobia into the plant cells, the cells also had an array of microtubules which radiated from the nucleus into the cytoplasm. Three actin arrays were identified in the infected cells of mature nodules: an aster-like array which emanated from the surface of the nucleus, a cortical array which had an arrangement similar to that of the cortical microtubules, and, throughout the cytoplasm, an array of fine filaments which had a honeycomb arrangement consistent with a distribution between adjacent symbiosomes. Uninfected cells from mature nodules had only a random cortical array of actin filaments. In incipient nodules, the density of actin microfilaments associated with the nucleus and radiating through the cytoplasm was much less than that seen in mature infected cells. The cortical array of actin also differed, being composed of swirling configurations of filaments. After invasion of nodule cells by the rhizobia, the number of actin filaments emanating from the nucleus increased markedly and formed a network through the cytoplasm. Conversely, the cytoplasmic array in uninfected cells of developing nodules was identical to that in the cells of incipient nodules. The cytoplasmic network in infected cells of developing nodules is likely to be the precursor of the honeycomb array seen in mature nodule cells. We propose that this actin array plays a role in the spatial organisation of symbiosomes and that the microtubules are involved in the localisation of mitochondria and plastids at the cell periphery in the infected cells of root nodules.     

Oleosomes (Spherosomes) from Daucus carota suspension culture cells

Isolated oleosomes from Daucus carota L. cells are lipid droplets consisting mainly of triacylglycerols (>97%) and very little protein (1–2%). The boundary between the lipid phase and the cytosol, which is visible on electron micrographs, is not built up by a true phospholipid-containing unit or half unit membrane. Enzymatic activities of lipid metabolism were not found to be associated with oleosomes with the exception of very low (contaminating) acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) and relatively high acyl-CoA hydrolase (EC 3.1.2.2) activities. The triacylglycerols exhibited a half life time of about 70 h, which is below the generation time of the cells (80–90 h). The fatty acid pattern of triacylglycerols was very similar to that of polar cellular membrane lipids.     

Differences in properties of peanut seed lectin and purified galactose- and mannose-binding lectins from nodules of peanut

Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal galactose - Gle glucose - GNL galactose-binding nodule lectin - Fru fructose - MNL mannosebinding nodule lectin - M _r rerative molecular mass - PBS phosphate-buffered saline - PSL peanut seed lectin - SDS sodium dodecyl sulphate - Sorb sorbitol     

Sequential expression of two late nodulin genes in the infected cells of alfalfa root nodules.

The Nms-22 and leghemoglobin (Lb) genes are expressed exclusively in the infected cells of alfalfa root nodules. Expression of these two late nodulin genes originated at distinct cellular boundaries within the symbiotic region of the nodule. The Nms-22 gene was expressed in all infected cells, including those just adjacent to the meristematic region. Lb gene expression was induced in older infected cells and was most prominent in the mature region of the nodule. Despite this temporal separation of gene expression, both the Nms-22 and Lb genes were expressed in nodules elicited by bacA mutants in which bacteroid development has been blocked just after release from the infection thread.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.     

Liposomal formulations from phospholipids of Greek almond oil. Properties and biological activity

The seeds of the almond tree [(Prunus dulcis (Mill.) D. A. Webb. (syn. Prunus amygdalus)] were collected in two different periods of maturity and were studied for their lipid content. The total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated by chromatographic techniques and were analyzed by HPTLC coupled with a flame ionization detector (HPTLC/FID) and GC-MS. The oils were found to be rich in neutral lipids (89.9% and 96.3% of total lipids) and low in polar lipids (10.1% and 3.7% of total lipids) for the immature and mature seed oils, respectively. The neutral lipid fraction consisted mainly of triacylglycerides whereas the polar lipids mainly consisted of phospholipids. GC-MS data showed that the main fatty acid for both oils was 9-octadecenoic acid (oleic acid). The unsaturated fatty acids were found as high as 89.4% and 89.7%, while the percentage of the saturated fatty acids was found 10.6% and 10.3% for the immature and mature seed oils, respectively. Liposomes were prepared from the isolated phospholipids using the thin lipid film methodology, and their physical properties were characterized. Cytotoxicity was found absent when assayed against normal and cancerous cell lines. These new formulations may have future applications for encapsulation and delivery of drugs and cosmetically active ingredients.     

Cell and endophyte structure of the nitrogen-fixing root nodules ofCeanothus integerrimus H.and A.

Summary The nitrogen fixing root nodules ofCeanothus integerrimus were very similar in appearance to other non-legume nodules. Each nodule was a cluster of small lobes. Each lobe in cross section had a central vascular cylinder and a hypertrophied cortex. The cortex contained very large infected cells, with large nuclei; among these infected cells were scattered small, normal-appearing cortex cells. The actinomycete endophyte consisted of wavy hyphae 0.4 m in diameter which terminated in pear-shaped vesicles 1.6 m×2 m. The vesicles were not septate. The function of the vesicles was unknown. The infected cells had apparently normal nuclei, chloroplasts and mitochondria and were probably alive, except at the base of the nodule where both infected cells and the endophyte they contained were dead.