Fe homeostasis in plant cells: Does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.     

TMEM165 deficiency causes a congenital disorder of glycosylation

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

The cellular structure of the Hartig net: coenocytic and transfer cell-like organization

The organization of the Hartig net in the mature state was studied in mycorrhizas of Amanita muscaria (Pers. ex. Fries) Hook. Picea abies (L.) Karst. grown in vitro. The tips of the fan-like branched hyphae contain dense cytoplasm with a large number of mitochondria and rER frequently stretched in the direction of the hyphal growth, indicating that active transfer of solutes between host and fungus is localized here. Lack of septation of the hyphae and their intimate juxtaposition, leaving no space in between, result in a coenocytic, transfer cell-like structure of the Hartig net. The multinucleate status of the cells is shown. The advantage of this organization for nutrient exchange between host and fungus and for nutrient transport within the hyphal system is discussed.     

Metabolism of apolipoprotein E-containing human plasma lipoproteins by rat and human cells in culture.

Cultured preadipocytes from rat epididymal fat pads were able to bind, internalize, and degrade human plasma very-low-density lipoproteins (VLDL) more efficiently than low-density lipoproteins (LDL). VLDL, but not LDL, activated acyl-CoA: cholesterol acyltransferase (ACAT) and increased cholesterol accumulation in these cells. However, trypsin-treated VLDL (T-VLDL) lost the capacity to bind, activate ACAT, and increase cholesterol accumulation. After the treatment of VLDL with trypsin, SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that apolipoprotein E (apo E) was completely degraded, whereas apolipoprotein CII (apo C-II) was preserved. ApoE complexed with dimyristoyl phosphatidylcholine (DMPC) was able to complete with VLDL for binding to the cells. Although T-VLDL did not bind to the preadipocytes, these cells accumulate triacylglycerols from T-VLDL, presumably after lipolysis, as efficiently as from native VLDL. Rat smooth muscle cells and skin fibroblasts also bind and metabolize human VLDL better than LDL. However, human skin fibroblasts and omental preadipocytes metabolized LDL better than VLDL. These studies indicate that rat tissues can recognize and metabolize apoE-containing human plasma VLDL although they cannot recognize human LDL.     

Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower

Background

More than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources – PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome.

Results

Eight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences.

Conclusion

The new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.

     

Zur Kompartimentierung der Synthese von Mono- und Sesqui-Terpenen des ätherischen Öls beiPoncirus trifoliata

Zusammenfassung In der Frucht vonPoncirus trifoliata liegen in der Außenschale Drüsenzellkomplexe, die ein monoterpenreiches ätherisches Öl mit geringem Anteil an Sesquiterpenen und O-haltigen Substanzen produzieren. Ähnlich aussehende Exkretzellkomplexe aus den Saftschläuchen enthalten hauptsächlich Sesquiterpenkohlenwasserstoffe (STKW) und O-haltige Komponenten und sehr wenig Monoterpenkohlenwasserstoffe (MTKW). Im Schalenöl konnten nach gaschromatographischer Trennung mit Hilfe der Massenspektrometrie 19 Komponenten identifiziert werden, im Saftschlauchöl 25.Elektronenmikroskopische Aufnahmen der jüngsten Drüsenzellen beider Drüsenkomplexe lassen erkennen, daß beide Terpenklassen wahrscheinlich hauptsächlich bzw. ausschließlich plastidär entstehen.Exogen angebotenes¹⁴CO₂ wird zunächst überwiegend in die MTKW eingebaut, erst später nimmt die Markierung der STKW und O-haltigen Komponenten stark zu. Über den Ferntransportweg angebotenes¹⁴C-Leucin führt anfangs zu einer starken Markierung der STKW und O-haltigen Komponenten, erst später verschiebt sich der Einbau etwas mehr in Richtung MTKW. Als Hauptursache für den differenten Einbau wird das Vorhandensein zweier Typen von Drüsenzellkomplexen mit unterschiedlichen Syntheseleistungen angesehen.Die aus dem¹⁴CO₂ in der Außenrinde gebildeten Assimilate werden zuerst in das MTKW-reiche Öl der Schalenexkretbehälter eingebaut. Die überwiegend STKW erzeugenden Saftschlauchbehälter werden erst später beliefert. Beim Leucinangebot über die Fruchtstiele scheint es gerade umgekehrt zu verlaufen. Die aufeinanderfolgenden Maxima der Ölproduktion in den beiden Drüsenzellkomplex-Typen und die Änderung des Komponentenspektrums ihres ätherischen Öls im Verlauf der Vegetationsperiode tragen ebenfalls zu einem je nach Jahreszeit unterschiedlichen Einbau in die MTKW und STKW bei.

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Compartmentation of mono- and sesqui-terpene biosynthesis of the essential oil inPoncirus trifoliata

Summary The fruit ofPoncirus trifoliata shows glandular cell complexes in the exocarp, which produce a volatile oil rich in monoterpenes but poor in sesquiterpenes and oxigenated compounds. The juice vesicles of the endocarp possess similar cell complexes mainly containing sesquiterpenes and oxigenated compounds, whereas monoterpenes only occur in small amounts. By the use of combined gas chromatography-mass spectrometry 19 components of the rind oil and 15 compounds of the endocarp oil could be identified.As demonstrated by electron microscopy the terpenes most probably are synthesized predominantly, if not exclusively in plastids. As shown by gasradiochromatography radioactive precursors (¹⁴CO₂ and¹⁴C-leucine) are incorporated into mono- and sesqui-terpenes to a different extent.This is due to two gland types producing essential oils of different composition with regard to their mono- and sesqui-terpene percentage. In fruit development the exocarp glands differentiate earlier than the endocarp glands do. The activity of exogenously applied¹⁴CO₂ first reaches the peripheral glands and later on appears in the interior glands. Depending upon the growth season, labelled leucine transported by the conducting tissues from lower plant parts leads to a high specific activity of the sesqui-terpenes and oxigenated compounds. It could be argued that in this instance the glands of the pulp are better provided with precursors than the exocarp glands. The successive maxima of essential oil production in both glandular complexes, and the changes in the concentration of individual oil constituents during the ontogeny of the fruit also contribute to different incorporation ratios of radioactive precursors into mono- and sesqui-terpenes.