Identification of quantitative trait loci controlling linolenic acid concentration in PI483463 (Glycine soja)

Key message

The QTLs controlling alpha-linolenic acid concentration from wild soybean were mapped on nine soybean chromosomes with various phenotypic variations. New QTLs for alpha-linolenic acid were detected in wild soybean.

Abstract

Alpha-linolenic acid (ALA) is a polyunsaturated fatty acid desired in human and animal diets. Some wild soybean (Glycine soja) genotypes are high in ALA. The objective of this study was to identify quantitative trait loci (QTLs) controlling ALA concentration in a wild soybean accession, PI483463. In total, 188 recombinant inbred lines of F_5:6, F_5:7, and F_5:8 generations derived from a cross of wild soybean PI483463 (~15 % ALA) and cultivar Hutcheson (~9 % ALA) were planted in four environments. Harvested seeds were used to measure fatty acid concentration. Single nucleotide polymorphism markers of the universal soybean linkage panel (USLP 1.0) and simple sequence repeat markers were used for molecular genotyping. Nine putative QTLs were identified that controlled ALA concentration by model-based composite interval mapping and mapped to different soybean chromosomes. The QTLs detected in four environments explained 2.4–7.9 % of the total phenotypic variation (PV). Five QTLs, qALA5_3, qALA6_1, qALA14_1, qALA15_1, and qALA17_1, located on chromosomes 5, 6, 14, 15, and 17 were identified by model-based composite interval mapping and composite interval mapping in two individual environments. Among them, qALA6_1 showed the highest contribution to the PV with 10.0–10.2 % in two environments. The total detected QTLs for additive and epistatic effects explained 52.4 % of the PV for ALA concentration. These findings will provide useful information for understanding genetic structure and marker-assisted breeding programs to increase ALA concentration in seeds derived from wild soybean PI483463.     

4.5S ribonucleic acid, a novel ribosome component in the chloroplasts of flowering plants.

A species of low-molecular-weight ribosomal RNA, referred to as '4.5S rRNA', was found in addition to 5S rRNA in the large subunit of chloroplast ribosomes of a wide range of flowering plants. It was shown by sequence analysis that several variants of this RNA may occur in a plant. Furthermore, although in most flowering plants the predominant variant contains about 100 nucleotides, in the broad bean it has less than 80. It seems, therefore, to be much more diverse in size and sequence than the other ribosomal RNA species. Like 5S rRNA , it does not contain modified nucleotides and it is also unusual in having an unphosphorylated 5'-end. It is apparently neither a homologue of cytosol 5.8S rRNA nor a fragment of 23S rRNA.     

Development of free radical products during the greening reaction of caffeic acid esters (or chlorogenic acid) and a primary amino compound.

ESR spectra were measured directly on a marked greening reaction mixture of Et-caffeate and a primary amino compound in alkali solution under aeration. A clear hyperfine structure was commonly detected early in the greening reaction with different amino compounds. Its hyperfine spectrum split into seven peaks was analyzed and found to be due to the oxidized free radical product of the Et-caffeate using an authentic sample system. Another type of hyperfine ESR spectrum was observed later in the reaction, and was altered with different amino compounds. The hyperfine structure for n-butylamine split into 12 lines. The latter type of free radical products were assumed to be a semiquinone type radical compound of the trihydroxy benzacridine derivative, which was identified as the principal structure of the green and yellow pigments formed by this greening reaction system. A formation mechanism of the green pigment and related products involving these free radical products is proposed.     

Identification of Escherichia coli ZapC (YcbW) as a component of the division apparatus that binds and bundles FtsZ polymers

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified in Escherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission process per se but contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring in E. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.     

Identification and partial characterization of the human erythrocyte membrane component(s) that express the antigens of the LW blood-group system.

Rhnull human erythrocytes lack the antigens of the Rhesus blood-group system, have an abnormal shape, have an increased osmotic fragility, and are associated with mild chronic haemolytic anaemia. Rhnull erythrocytes also lack all antigens of the LW blood-group system, but the functional significance of this deficiency is unknown. We have identified, by immunoblotting with two mouse monoclonal antibodies (BS46 and BS56), the LW-active component(s) in normal human erythrocytes as a broad band of Mr 37 000-47 000 on SDS/polyacrylamide-gel electrophoresis. Treatment of intact human erythrocytes with endoglycosidase F preparation destroyed the epitopes recognized by antibodies BS46 and BS56, suggesting that one or more N-glycosidically linked oligosaccharides are required for the formation of the LW antigens. Estimation of the number of LW antigen sites per erythrocyte by using radioiodinated purified antibody BS46 gave average values of 4400 molecules/cell for Rh(D)-positive adult erythrocytes and 2835 molecules/cell for Rh(D)-negative adult erythrocytes. Like the Rh(D) polypeptide, the LW polypeptide(s) is (are) associated with the cytoskeleton of normal erythrocytes. These results suggest the possibility that the absence of the LW polypeptide may also contribute to the functional and/or morphological abnormalities of Rhnull erythrocytes.     

Identification of a 55-kDa ezrin-related protein that induces cytoskeletal changes and localizes to the nucleolus.

Normal and transformed human cells when stained for ezrin, an F-actin-binding ERM (ezrin/radixin/moesin) family protein, revealed a faint and intense immunofluorescence, respectively. Surprisingly, nuclear staining that was assigned to the nucleolus by confocal laser and immunoelectron microscopy was detected in both cell types and was more prominent in normal cells due to the absence of glistering cytoplasmic fluorescence. By Western analysis the nuclear fraction was seen to have a 55-kDa ezrin-reactive protein that did not react to the antibodies raised against the C-terminus of the protein, suggesting that it may correspond to an endogenously cleaved N-terminus of the protein. Transfections of cells with a cDNA encoding full-length ezrin tagged with green fluorescent protein (GFP) at its N-terminus indeed resulted in two GFP-tagged products corresponding to full-length and 55-kDa endogenously cleaved forms. Transfection with a cDNA encoding approximately 55 kDa of the ezrin N-terminus (N-ezrin) showed that it can translocate to the nucleus. N-ezrin transfected cells exhibited irregular cell edges and collapse of actin fibers. Similar changes were seen following microinjection of anti-p81/ezrin antibody, suggesting that N-ezrin may function as a dominant negative competitor of ezrin. These data demonstrate the existence of an N-terminal cleavage form of ezrin that localizes to the nucleolus and that its overexpression induces cytoskeletal changes.     

Identification of an allene oxide synthase (CYP74C) that leads to formation of alpha-ketols from 9-hydroperoxides of linoleic and linolenic acid in below-ground organs of potato

Allene oxide synthase (AOS) enzymes are members of the cytochrome P450 enzyme family, sub-family CYP74. Here we describe the isolation of three cDNAs encoding AOS from potato (StAOS1-3). Based on sequence comparisons, they represent members of either the CYP74A (StAOS1 and 2) or the CYP74C (StAOS3) sub-families. StAOS3 is distinguished from the other two AOS isoforms in potato by its high substrate specificity for 9-hydroperoxides of linoleic and linolenic acid, compared with 13-hydroperoxides, which are only poor substrates. The highest activity was shown with (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) as a substrate. This hydroperoxide was metabolized in vitro to alpha- and gamma-ketols as well as to the cyclopentenone compound 10-oxo-11-phytoenoic acid. They represent hydrolysis products of the initial StAOS3 product 9,10-epoxyoctadecadienoic acid, an unstable allene oxide. By RNA gel hybridization blot analysis, StAOS3 was shown to be expressed in sprouting eyes, stolons, tubers and roots, but not in leaves. StAOS3 protein was found in all organs tested, but mainly in stems, stolons, sprouting eyes and tubers. As in vivo reaction products, the alpha-ketols derived from 9-hydroperoxides of linoleic and linolenic acid were only found in roots, tubers and sprouting eyes. Immunolocalization showed that StAOS3 was associated with amyloplasts and leucoplasts.     

n-Hexanal and (Z)-3-hexenal are generated from arachidonic acid and linolenic acid by a lipoxygenase in Marchantia polymorpha L.

Most terrestrial plants form green leaf volatiles (GLVs), which are mainly composed of six-carbon (C6) compounds. In our effort to study the distribution of the ability of lipoxygenase (LOX) to form GLVs, we found that a liverwort, Marchantia polymorpha, formed n-hexanal and (Z)-3-hexenal. Some LOXs execute a secondary reaction to form short chain volatiles. One of the LOXs from M. polymorpha (MpLOX7) oxygenized arachidonic and α-linolenic acids at almost equivalent efficiency and formed C6-aldehydes during its catalysis; these are likely formed from hydroperoxides of arachidonic and α-linolenic acids, with a cleavage of the bond between carbon at the base of the hydroperoxy group and carbon of double bond, which is energetically unfavorable. These lines of evidence suggest that one of the LOXs in liverwort employs an unprecedented reaction to form C6 aldehydes as by-products of its reaction with fatty acid substrates.     

Identification of a hyaluronic acid (HA) binding domain in the PH-20 protein that may function in cell signaling.

The macaque sperm surface protein PH-20 is a hyaluronidase, but it also interacts with hyaluronic acid (HA) to increase internal calcium ( [Ca(2+)](i) ) in the sperm cell. A region of the PH-20 molecule, termed Peptide 2 (aa 205-235), has amino acid charge homology with other HA binding proteins. The Peptide 2 sequence was synthesized and two recombinant PH-20 proteins were developed, one containing the Peptide 2 region (G3, aa 143-510) and one without it (E12, aa 291-510). On Western blots, affinity-purified anti-Peptide 2 IgG recognized the 64 kDa band corresponding to PH-20 in acrosome intact sperm and, under reducing conditions, recognized the whole 67 kDa PH-20 and the endoproteolyzed N-terminal fragment of PH-20. HA conjugated to a photoaffinity substrate specifically bound to sperm surface PH-20. Indirect immunofluorescence demonstrated that Fab fragments of anti-Peptide 2 IgG bound to the head of live sperm. Biotinylated HA was bound by Peptide 2 and by sperm extracts in a microplate binding assay, and this binding was inhibited by Fab fragments of anti-Peptide 2 IgG. Biotinylated HA bound to the G3 protein and this binding was inhibited by anti-Peptide 2 Fab, but HA did not bind to the E12 protein. Fab fragments of anti-Peptide 2 IgG inhibited the increase in [Ca(2+)](i) induced in macaque sperm by HA. Our results suggest that the Peptide 2 region of PH-20 is involved in binding HA, which results in the cell signaling events related to the elevation of [Ca(2+)](i) during sperm penetration of the cumulus.     

Inhibition of flowering in the long-day plant Lemna gibba G3 by Hutner's medium and its reversal by salicylic acid

Flowering in the long-day plant Lemna gibba L., strain G3 ispoor or absent in Hutner's medium even under continuous light,an effect generally ascribed to the ammonium content of themedium. However, flowering is also inhibited in ammonium-freemodifications of Hutner's medium, particularly in the presenceof sucrose, but is restored to high levels by the presence of10 µu salicylic acid. These results link two of the leastunderstood chemical effects in Lemnaceae flowering, and theyprovide a system in which large effects of salicylic acid canbe readily obtained. ²Present address: Lab. of Applied Bot., Fac. of Agric, KyotoUniv., Kyoto 606, Japan. (Received January 27, 1979; )     

Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide.

A phosphorylated 3-deoxy-manno-octulosonic acid (KDO) was released from the lipopolysaccharide (LPS) of the deep rough mutant (Rb+169) of Haemophilus influenzae by acid hydrolysis. Both phosphorylated and dephosphorylated KDO, produced by treatment with alkaline phosphatase, were identified by gas chromatography-mass spectrometry after trimethylsilylation. This technique provides a rapid and reliable method for the identification of phosphorylated KDO in LPS.     

Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA.

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.     

Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm

A monoclonal antibody, J18/29, induces the acrosome reaction (AR) in spermatozoa of the sea urchin Strongylocentrotus purpuratus. J18/29 induces increases in both intracellular Ca2+ and intracellular pH similar to those occurring upon induction of the AR by the natural inducer, the fucose sulfate-rich glycoconjugate of egg jelly. Lowering the Ca2+ concentration or the pH of the seawater inhibits the J18/29-induced AR, as does treatment with Co2+, an inhibitor of Ca2+ channels. The J18/29-induced AR is also inhibited by verapamil, tetraethylammonium chloride, and elevated K+. All these treatments cause similar inhibition of the egg jelly-induced AR. J18/29 reacts with a group of membrane proteins ranging in molecular mass from 340 to 25 kD, as shown by immunoprecipitation of lysates of 125I-labeled sperm and Western blots. The most prominent reacting proteins are of molecular masses of 320, 240, 170, and 58 kD. The basis of the multiple reactivity appears to reside in the polypeptide chains of these proteins, as J18/29 binding is sensitive to protease digestion but resistant to periodate oxidation. There are approximately 570,000 sites per cell for J18/29 binding. J18/29 is the only reagent of known binding specificity that induces the AR; it identifies a subset of sperm membrane proteins whose individual characterization may lead to the isolation of the receptors involved in the triggering of the AR at fertilization.