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• W2049580786 abstract "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. 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. Symbiotic nitrogen fixation in legumes takes place in specialized organs called nodules that develop from root cortical cells following contact with soil bacteria of the family Rhizobiaceae. The symbiosis is mutualistic; the plant receives a crucial supply of reduced nitrogen from the bacteria in exchange for reduced carbon and other nutrients (1Udvardi M.K. Day D.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 493-523Crossref PubMed Scopus (297) Google Scholar). The interaction between legumes and rhizobia is generally specific; different rhizobial strains are capable of infecting one or a few legume species. For example, soybean (Glycine max) is preferentially infected by Bradyrhizobium japonicum, whereas Mesorhizobium loti establishes a symbiosis with Lotus japonicus (2Sullivan J.T. Patrick H.N. Lowther W.L. Scott D.B. Ronson C.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8985-8989Crossref PubMed Scopus (336) Google Scholar). Apart from being an important crop plant, soybean has been employed as a model for biochemical and physiological studies of symbiotic nitrogen fixation for decades. L. japonicus, on the other hand, has emerged as a model species for genetics and genomics studies of symbiotic nitrogen fixation and other legume-specific processes (3Handberg K. Stougaard J. Plant J. 1992; 2: 487-496Crossref Scopus (487) Google Scholar, 4Jiang Q. Gresshoff P.M. Mol. Plant-Microbe Interact. 1997; 10: 59-68Crossref PubMed Scopus (99) Google Scholar, 5Udvardi M.K. Mol. Plant-Microbe Interact. 2001; 14: 6-9Crossref PubMed Scopus (17) Google Scholar).Following a series of initial signal exchanges (6Schultze M. Kondorosi A. Annu. Rev. Genet. 1998; 32: 33-57Crossref PubMed Scopus (367) Google Scholar, 7Stougaard J. Plant Physiol. 2000; 124: 531-540Crossref PubMed Scopus (169) Google Scholar, 8Long S.R. Plant Physiol. 2001; 125: 69-72Crossref PubMed Scopus (207) Google Scholar), rhizobia enter root hair and underlying cells via an infection thread (9Rae A.L. Bonfante-Fasolo P. Brewin N.J. Plant J. 1992; 2: 385-395Crossref Scopus (100) Google Scholar, 10Brewin N.J. Semin. Cell Biol. 1993; 4: 149-156Crossref PubMed Scopus (8) Google Scholar) from which they are eventually released into cortical cells via endocytosis. This series of events results in a unique cytoplasmic “organelle” called the symbiosome, which consists of bacteria surrounded by a membrane of plant origin, called the symbiosome or peribacteroid membrane (PBM) 1The abbreviations used are: PBM, peribacteroid membrane; DGDG, digalactosyldiacylglycerol; EST, expressed sequence tag; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SL, sulfolipid; GC, gas chromatography; MES, 4-morpholineethanesulfonic acid; RACE, rapid amplification of cDNA ends. 1The abbreviations used are: PBM, peribacteroid membrane; DGDG, digalactosyldiacylglycerol; EST, expressed sequence tag; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SL, sulfolipid; GC, gas chromatography; MES, 4-morpholineethanesulfonic acid; RACE, rapid amplification of cDNA ends. (11Verma D.P. Hong Z. Trends Microbiol. 1996; 4: 364-368Abstract Full Text PDF PubMed Scopus (56) Google Scholar). The PBM is both the structural and functional interface between the plant and bacterial cells, and its lipid and protein composition contribute significantly to its biological roles. Growth and division of rhizobia, matched by synthesis of PBM, continues until thousands of symbiosomes, each containing one or more bacteria, pack the infected cells. Rhizobia within symbiosomes eventually differentiate into nitrogen-fixing bacteroids, in response to lowered oxygen concentrations and possibly other physiological changes that accompany nodule development. Nodulation is also accompanied by a reprogramming of plant metabolism, which becomes specialized for the provision of reduced carbon and other nutrients to the bacteroids and for the assimilation of ammonium and other nitrogen compounds from the bacteroids (1Udvardi M.K. Day D.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 493-523Crossref PubMed Scopus (297) Google Scholar, 12.Day, D. A., Kaiser, B. N., Thomson, R., Udvardi, M. K., Moreau, S., and Puppo, A. (2001) 28, 667–674Google Scholar, 13Lodwig E.M. Hosie A.H.F. Bourdes A. Findlay K. Allaway D. Karunakaran R. Downie J.A. Poole P.S. Nature. 2003; 422: 722-726Crossref PubMed Scopus (344) Google Scholar).The lipid composition of the PBM has been analyzed on a qualitative level. The PBM contains saturated (16:0, palmitic acid; 18:0, stearic acid) and unsaturated fatty acids (16:1Δ3trans, palmitoleic acid; 18:1Δ9cis, oleic acid 18:2Δ9,12, linoleic acid; 18:3Δ9,12,15, α-linolenic acid) typically found in higher plants. In addition to phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI)), sterol lipids and glycolipids of unknown structure were detected previously (14Hernández L.E. Cooke D.T. Phytochemistry. 1996; 42: 341-346Crossref Scopus (19) Google Scholar, 15Whitehead L.F. Day D.A. Physiol. Plant. 1997; 100: 30-44Crossref Google Scholar). The origin of PBM lipids presumably is the endoplasmic reticulum, where they are synthesized and transported to the PBM via the Golgi apparatus (16Brewin N.J. Larsson C. Møller I.M. The Plant Plasma Membrane. Springer-Verlag, Berlin, Germany1990: 551-555Google Scholar).Under normal growth conditions, galactolipids (monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)) are restricted to chloroplast membranes in plants, where they are important in maintaining the structural integrity of photosynthetic complexes. The enzymes involved in galactolipid synthesis (MGDG synthases and DGDG synthases) have been isolated from Arabidopsis thaliana (17Awai K. Maréchal E. Block M.A. Brun D. Masuda T. Shimada H. Takamiya I.-I. Ohta H. Joyard J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10960-10965Crossref PubMed Scopus (231) Google Scholar, 18Dörmann P. Balbo I. Benning C. Science. 1999; 284: 2181-2184Crossref PubMed Scopus (160) Google Scholar, 19Kelly A.A. Dörmann P. J. Biol. Chem. 2002; 277: 1166-1173Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). During phosphate deprivation, the amount of DGDG increases greatly in Arabidopsis (20Essigmann B. Güler S. Narang R.A. Linke D. Benning C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1950-1955Crossref PubMed Scopus (268) Google Scholar, 21Härtel H. Dörmann P. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10649-10654Crossref PubMed Scopus (289) Google Scholar). Under these conditions, phospholipids are replaced by glycolipids to save phosphorus for other essential processes (for a review see Ref. 22Dörmann P. Benning C. Trends Plant Sci. 2002; 7: 112-118Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Interestingly, a fraction of DGDG synthesized during phosphate deprivation is localized to extraplastidic membranes, e.g. the plasma membrane (21Härtel H. Dörmann P. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10649-10654Crossref PubMed Scopus (289) Google Scholar, 23Andersson M.X. Stridh M.H. Larsson K.E. Liljenberg C. Sandelius A.S. FEBS Lett. 2003; 537: 128-132Crossref PubMed Scopus (206) Google Scholar). However, under normal growth conditions, galactolipids are of very low abundance in extraplastidic membranes.Rhizobia contain a different set of fatty acids as compared with higher plants, e.g. with double bonds at different positions or with methyl or cyclopropane groups. The predominant lipids in Rhizobia are phospholipids (PC, PE, PG, and cardiolipin; Refs. 24Miller K.J. Shon B.C. Gore R.S. Hunt W.P. Curr. Microbiol. 1990; 21: 205-210Crossref Scopus (21) Google Scholar and 25Geiger O. Spaink H.P. Kondorosi A. Hooykaas P.J.J. The Rhizobiaceae: Molecular Biology of Model Plant-associated Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 56-80Google Scholar). A diglycosyldiacylglycerol lipid was detected in B. japonicum USDA110, which was believed to be structurally identical with DGDG of higher plants (26Tan Y. Hollingsworth R.I. Glycobiology. 1997; 7: 935-942Crossref PubMed Scopus (9) Google Scholar). Interestingly, the Bradyrhizobium lipid was restricted to the bacteroid form but was not detected when bacteria were grown in liquid culture. The occurrence of DGDG in rhizobia raised many important questions regarding its function and origin, because this glycolipid was believed to be restricted to higher plants and Cyanobacteria but absent from nonphotosynthetic bacteria. To improve our understanding of the role of membrane lipids in the PBM, subcellular fractions were isolated from soybean and Lotus, and the glycerolipid composition was determined on a quantitative level. From these studies, it became clear that DGDG is indeed localized to the symbiosomes of legume nodules. However, we obtained strong evidence that this glycolipid is derived from plant lipid biosynthesis and is localized to the PBM but not to the bacteroids. Furthermore, our data demonstrate that the accumulation of DGDG in extraplastidic membranes is not restricted to the plasma membrane nor to plants deprived of external phosphate.EXPERIMENTAL PROCEDURESPlant Material and Growth Conditions—Soybean seeds (G. max cv. Stevens) were incubated overnight in water for swelling. The seeds were transferred into silica sand and grown under controlled conditions (16 h of light/day; day and night temperatures, 25 and 20 °C, respectively) and 65% humidity. B. japonicum USDA110 was grown in liquid culture (YT medium; 5 g of tryptone, 3 g of yeast extract, 10 ml of 0.45 m CaCl2 in 1000 ml of water) for 5 days at 28 °C and diluted 1:10 with water before inoculation of 7-day-old soybean seedlings.Prior to germination, L. japonicus seeds were treated with liquid nitrogen and incubated overnight in water. The plants were grown in silica sand at 16 h of light/day with day and night temperatures of 21 and 17 °C, respectively. M. loti strain R7a was grown in liquid culture (YT medium) to an A600 of 1.3 (5 days, 28 °C), and the cells were diluted 1:10 and used for the inoculation of 3-day-old Lotus seedlings.After infection with bacteria, soybean and Lotus plants were watered two times/week with complete mineral mixture excluding KNO3 (27Pavkovsky R. Fuller G. Physiol. Plant. 1988; 72: 733-746Crossref Scopus (59) Google Scholar). All of the soybean plants grown under +Pi conditions were additionally watered with 100 ml of 2.5 mm phosphate two times/week. For phosphate deprivation experiments, soybean plants were grown for 2 weeks without nodulation in the presence of complete mineral mix including 5 mm KNO3 and phosphate (27Pavkovsky R. Fuller G. Physiol. Plant. 1988; 72: 733-746Crossref Scopus (59) Google Scholar). The plants were subsequently grown with mineral mix excluding phosphate for another 10 weeks before sampling.Isolation of Symbiosomes and Peribacteroid Membranes from L. japonicus and G. max—Approximately 20 g of fresh nodules from 12-week-old soybean or L. japonicus plants were used for the isolation of symbiosomes and PBMs via three-step Percoll density gradient centrifugation (28Price G.D. Day D.A. Gresshoff P.M. J. Plant Physiol. 1987; 130: 157-164Crossref Scopus (48) Google Scholar, 29Panter S. Thomson R. de Bruxelles G. Laver D. Trevaskis B. Udvardi M. Mol. Plant-Microbe Interact. 2000; 13: 325-333Crossref PubMed Scopus (116) Google Scholar). Intact symbiosomes were harvested from the 60/80% Percoll interface. After washing with washing buffer (350 mm mannitol, 25 mm MES-KOH, 3 mm MgSO4, pH 7.0), the suspension was centrifuged at 4000 × g for 5 min at 4 °C. The pellet containing symbiosomes was resuspended in washing buffer and vortexed vigorously for 5 min. The broken symbiosomes were centrifuged at 20,000 × g for 10 min, and the bacteroids were recovered from the pellet. The PBM was recovered from the supernatant by ultracentrifugation at 300,000 × g for 60 min.Lipid Extraction and Quantification of Polar Lipids, Fatty Acids, and Sugars—The lipids were isolated from frozen plant material, rhizobia (isolated from liquid culture), symbiosomes, or peribacteroid membranes (30Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42153) Google Scholar). The separation of the polar lipids was done by thin layer chromatography (31Dörmann P. Hoffmann-Benning S. Balbo I. Benning C. Plant Cell. 1995; 7: 1801-1810Crossref PubMed Scopus (233) Google Scholar). For quantification by gas chromatography, the lipids were isolated from the plates and converted to fatty acid methyl esters (32Browse J. McCourt P.J. Sommervile C.R. Anal. Biochem. 1986; 152: 141-145Crossref PubMed Scopus (395) Google Scholar). Pentadecanoic acid (15:0) was used as internal standard. The sugar head group composition of DGDG was determined after hydrolysis by quantifying alditol acetates using gas chromatography (33Reiter W.-D. Chapple C. Somerville C.R. Plant J. 1997; 12: 335-345Crossref PubMed Scopus (198) Google Scholar).Northern Analysis—Total RNA was isolated from 1 g of soybean tissue (34Logemann J. Schell J. Willmitzer L. Anal. Biochem. 1987; 163: 16-20Crossref PubMed Scopus (1607) Google Scholar). 10 μg of total RNA was separated on an agarose gel and blotted onto a neutral nylon membrane (Parablot Ny amp; Macherey-Nagel, Düren, Germany) in 10× SSC. The RiboScribe RNA probe synthesis kit with T7 polymerase (Epicenter Technologies, Madison, WI) was used for labeling probes with [32P]dUTP (EST clones sre-pk0035-e2 and BE021559 representing soybean DGD1 and DGD2).Isolation of Full-length DGDG Synthase cDNAs from Soybean by 5′-RACE—Rapid amplification of cDNA ends was done to obtain the 5′ ends of the partial cDNA clones of soybean DGDG synthases. Poly(A) mRNA was isolated from total leaf RNA by oligo(dT) affinity purification (PolyATtract; Promega, Mannheim, Germany). The first part of the missing 5′ end of soybean DGD1 was amplified from cDNA synthesized with the SMARTII oligonucleotide (AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG) and the gene-specific primer PD227 (CCT AGC TCC CTC TCT GCA GCA ATC T) using the SMART Race cDNA amplification kit (Clontech). A 1200-bp fragment was amplified from this cDNA with universal primer mix (long, CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA ACA ACG CAG AGT; short, CTA ATA CGA CTC ACT ATA GGG C) and the primer PD250 (CGA AGA ACC TTG TGG CAG TAT GCT C). A second round of PCR was done after cDNA synthesis with the primer PD250 and the GeneRace RNA oligonucleotide (CGA CUG GAG CAC GAG GAC ACU GAC AUG GAC UGA AGG AGU AGA AA) using the GeneRace kit (Invitrogen). PCR amplification with the primers PD277 (TCC GCC GAC ACA ATC GCG TTC TTA) and the Gene-5′-primer yielded a 500-bp DGD1 fragment containing the missing start codon. 5′-RACE for soybean DGD2 was done after cDNA synthesis using the primer PD226 (TAA TCC TGG GTG GCG GCA GAT AAT C) and SMARTII oligonucleotide (AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG) with the SMART RACE cDNA amplification kit (Clontech). PCR amplification with the universal primer mix (see above) and the gene-specific primer PD251 (GCT TGC ATG ACT CCA TTC TTC TCT C) yielded a 600-bp fragment containing the missing start codon.Western Analysis—Total protein was isolated from leaves, roots, and PBM with phenol (35Cahoon E.B. Shanklin J. Ohlrogge J.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11184-11188Crossref PubMed Scopus (172) Google Scholar). Protein (30 μg) was separated in 8% polyacrylamide gels and blotted onto nitrocellulose (Protran; Schleicher & Schuell) in a semi-dry blotting apparatus (Biometra, Göttingen, Germany) following the manufacturer's protocol. Antibodies (antiserum from rabbits) raised against a component of the protein import complex of the inner envelope membrane (TIC40) and against the protein import complex of the outer envelope membrane of plastids (TOC75) were provided by J. Soll (Ludwig Maximilians University of Munich, Munich, Germany). The antibody raised against the PBM-specific aquaporin protein NOD26 (36Fortin M.G. Morrison N.S. Verma D.P. Nucleic Acids Res. 1987; 15: 813-824Crossref PubMed Scopus (175) Google Scholar) (antiserum from rabbits) was obtained from D. M. Roberts (Department of Biochemistry, University of Tennessee, Knoxville). The immunoreactions were visualized after incubation with secondary goat anti-rabbit antibodies (Kirkegaard & Perry, Gaithersburg, MD) coupled to alkaline phosphatase, using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate p-toluidine salt (Roche Applied Science).Quantification of Protein, Fatty Acids, and Inorganic Phosphate in Symbiosomes—Protein was measured according to Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar). Total fatty acids were quantified using pentadecanoic acid (15:0) as an internal standard after transmethylation of lipids in whole tissues by GC. The yield of symbiosome isolation from nodules was calculated using 19:011,12cyclo as a bacteroid-specific marker fatty acid.Total phosphate in soybean tissues was determined after hydrolysis with 65% (w/v) nitric acid (Suprapur; Merck). The concentration of total phosphate was determined by inductively coupled plasma optical emission spectroscopy using IRIS Advantage Duo ER/S (Thermo Jarell Ash, Franklin, MA) (38Becher M. Talke I.N. Krall L. Krämer U. Plant J. 2004; 37: 251-258Crossref PubMed Scopus (450) Google Scholar).In Situ Hybridization—In situ hybridization experiments were performed as previously described (39Scheres B. van de Wiel C. Zalensky A. Horvath B. Spaink H.P. van Eck H. Zwartkruis F. Wolters A. Gloudemans T. van Kammen A. Bisseling T. Cell. 1988; 60: 281-294Abstract Full Text PDF Scopus (197) Google Scholar, 40Flemetakis E. Dimou M. Cotzur D. Aivalakis G. Efrose R.-C. Kenoutis C. Udvardi M. Katinakis P. Biochim. Biophys. Acta. 2003; 1628: 186-194Crossref PubMed Scopus (26) Google Scholar). L. japonicus nodules harvested at 21 days post-infection with M. loti (strain E1R.pMP2112) were fixed in 4% paraformaldehyde supplemented with 0.25% glutaraldehyde in 10 mm sodium phosphate buffer, pH 7.4, for 4 h in a vacuum aspirator. Fixed nodules were block-stained in 0.5% safranin, dehydrated through ethanol series, and embedded in paraffin, and 8–10-μm-thick sections were cut. Antisense and sense RNA probes labeled with digoxigenin-11-rUTP (Roche Applied Science) were transcribed from the LjDGD1 and LjDGD2 cDNA clones. The probes were partially degraded to an average length of 150 nucleotides to improve probe penetration into the tissue. The sections were prepared for hybridization (39Scheres B. van de Wiel C. Zalensky A. Horvath B. Spaink H.P. van Eck H. Zwartkruis F. Wolters A. Gloudemans T. van Kammen A. Bisseling T. Cell. 1988; 60: 281-294Abstract Full Text PDF Scopus (197) Google Scholar) and hybridized overnight at 42 °C in 50% formamide, 300 mm NaCl, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.025% bovine serum albumin, 10% dextran sulfate, and 60 mm dithiothreitol. Hybridization signals were visualized with anti-digoxigenin antibodies conjugated with alkaline phosphatase.RESULTSThe Galactolipid DGDG Is a Major Constituent of the Peribacteroid Membrane of Soybean and Lotus Nodules—The nodules harbor large numbers of symbiosomes in the cytosol of the infected cells. As a consequence, the nodules contain more lipid/unit mass than other organs, and a large fraction of lipids is contained within symbiosomes (Fig. 1A). The glycerolipid composition of soybean nodules is similar to that of roots, with phospholipids (PC, PE, and PG) representing the most abundant lipid classes (Fig. 2A). Galactolipids (MGDG and DGDG), which are the predominant lipids in leaves, are less abundant in roots and nodules (Fig. 2A). The symbiosomes were isolated from soybean nodules and, after rupture by osmotic shock, separated into bacteroids and PBM. In addition to phospholipids (PC, PE, and PG), the PBM contained another lipid co-migrating with a leaf DGDG standard. This lipid stained with α-naphthol, indicating that it contains sugar in its head group. It was absent from bacteroids (Fig. 2A) and was also not found in Bradyrhizobium cells grown in liquid culture (data not shown). The PBM fraction was devoid of MGDG, another galactolipid highly abundant in plastids. Therefore, it is unlikely that DGDG in the PBM was derived from contamination with plastid membranes. Quantification of membrane lipids in different soybean tissues demonstrated that the amount of DGDG in symbiosomes was ∼7–8 mol %, about three times as high as in roots (∼2.5 mol %; Table I).Fig. 2Lipid composition in soybean, Lotus, and rhizobia. The lipids were isolated from soybean (G. max) and L. japonicus, separated by TLC, and stained with iodine. Galactolipids that positively stained with α-naphthol for the presence of sugar in the head groups are indicated by dashed circles. Other lipid bands were not stained with α-naphthol and thus might represent sugar-free phospholipids or sterol lipids. A, lipids from soybean (Bradyrhizobium bacteroids were isolated from nodules). B, lipids isolated from Lotus (Mesorhizobium bacteria were derived from liquid culture).View Large Image Figure ViewerDownload (PPT)Table IPolar lipid composition of soybean tissuesLipidLeafRootNodulesSymbiosomes+Pi-Pi+Pi-Pi+Pi-Pi+Pi-Pimol %mol %mol %mol %MGDG45.0 ± 3.748.0 ± 2.06.5 ± 0.37.7 ± 1.610.4 ± 0.76.8 ± 2.4NDaND, not detected.NDPG9.8 ± 1.63.5 ± 0.15.1 ± 1.45.2 ± 0.95.1 ± 0.43.5 ± 0.79.9 ± 1.511.4 ± 1.2DGDG20.9 ± 3.238.4 ± 2.82.5 ± 0.336.6 ± 2.93.4 ± 0.211.1 ± 1.47.6 ± 1.310.6 ± 2.3SL/PIbSulfolipid (SL) and phosphatidylinositol (PI) were not separated.3.2 ± 0.34.4 ± 1.76.7 ± 0.49.9 ± 1.11.9 ± 0.45.9 ± 1.024.6 ± 1.426.7 ± 2.2PE9.6 ± 2.41.7 ± 0.241.4 ± 0.311.7 ± 2.641.4 ± 3.434.1 ± 1.627.1 ± 2.519.2 ± 3.0PC11.7 ± 3.04.0 ± 0.337.8 ± 2.029.5 ± 3.337.8 ± 4.638.7 ± 3.230.7 ± 0.932.1 ± 2.9a ND, not detected.b Sulfolipid (SL) and phosphatidylinositol (PI) were not separated. Open table in a new tab To address the question of whether the presence of DGDG in the PBM is unique to soybean, we included a second legume species, L. japonicus, in our analysis. Lotus nodules were obtained after inoculation with M. loti and used for symbiosome and PBM preparation. Similarly to soybean, a lipid co-migrating with DGDG was detected by TLC of Lotus PBM fractions (Fig. 2B). This lipid was also stained with α-naphthol and therefore tentatively identified as DGDG. It was absent from Mesorhizobium bacteria, indicating that similarly to soybean, DGDG is an abundant lipid in Lotus PBM, but not in bacteria or bacteroids (Fig. 2B).DGDG Is an Authentic Plant-derived Lipid of the PBM and Does Not Originate from Bacterial Lipid Biosynthesis—The fact that DGDG but not MGDG was detected in the soybean PBM suggested that the PBM fraction was largely devoid of plastid contamination. Western blot analysis was done to confirm the absence of plastid membranes in the isolated PBM fraction (Fig. 3). The NOD26 protein, a marker for symbiosome membranes (41Miao G.-H. Verma D.P.S. Plant Cell. 1993; 5: 781-794PubMed Google Scholar), was specifically detected in the PBM. Immunoblots with antibodies raised against membrane components of the plastid protein import apparatus (TIC40, inner envelope; TOC75, outer envelope) showed weak or no cross-reaction with the PBM fraction but strong bands in the plastid fractions (Fig. 3). Therefore, the PBM fraction was indeed devoid of contamination by plastid membranes.Fig. 3The PBM fraction from soybean is devoid of plastidic membranes. Membrane proteins isolated from plastids of leaves and roots or from PBM isolated from nodules were subjected to Western analysis with antibodies raised to TIC40 (marker for plastid inner envelope), TOC75 (marker for plastid outer envelope), and NOD26 (marker for PBM).View Large Image Figure ViewerDownload (PPT)To assess the purity of the PBM fraction with regard to possible contaminations by bacterial lipids, total fatty acid composition was determined by GC (Table II). In contrast to soybean tissues that are rich in unsaturated fatty acids (18:1Δ9cis, oleic acid; 18:2Δ9cis,12cis, linoleic acid; and 18:3Δ9cis,12cis,15cis, α-linolenic acid), B. japonicum contains large amounts of the monounsaturated fatty acid 18:1Δ11cis (vaccenic acid) and of the cyclopropane fatty acid 19:011,12cyclo (Ref. 42Boumahdi M. Mary P. Horzez J.-P. J. Appl. Microbiol. 1999; 89: 611-619Crossref Scopus (29) Google Scholar and Table II). Oleic acid, linoleic acid, and α-linolenic acid were predominant fatty acids in the PBM, but no bacteria-specific fatty acids were detected (Table II). Therefore, the PBM was devoid of contamination by bacterial lipids. DGDG was isolated from soybean PBM and subjected to fatty acid and sugar analysis by GC. As shown in Table III, the PBM-derived DGDG contains large amounts of typical plant fatty acids (oleic acid, linoleic acid, and α-linolenic acid) but no bacteria-specific fatty acids. Furthermore, galactose was the predominant sugar in the head group of this glycolipid. (Table III). In conclusion, these findings strongly suggest that the glycolipid detected in the PBM fraction of soybean nodules represents authentic DGDG originating from plant lipid metabolism.Table IIFatty acid composition of soybean tissues and BradyrhizobiumFatty acidLeafRootNoduleSymbiosomePBMBacteriaa17:0 and 17:09,10cyclo are present in bacteria and bacteroids in trace amounts (below 1 mol%).Bacteroidsa17:0 and 17:09,10cyclo are present in bacteria and bacteroids in trace amounts (below 1 mol%).mol %mol %mol %mol %mol %mol %mol %16:011.0 ± 0.518.9 ± 2.412.9 ± 0.412.5 ± 0.734.3 ± 4.79.4 ± 1.69.9 ± 0.216:1bContains 16:1Δ3trans (derived from plant) and 16:1Δ9cis (from bacteria/bacteroids).2.4 ± 0.21.3 ± 0.51.0 ± 0.11.0 ± 0.13.3 ± 2.12.3 ± 1.9NDcND, not detected.16:20.3 ± 0.10.8 ± 0.30.5 ± 0.10.6 ± 0.1NDNDND18:02.5 ± 0.42.4 ± 0.40.3 ± 0.11.7 ± 0.37.6 ± 1.61.1 ± 0.21.5 ± 0.118:1dContains 18:1 Δ9cis (derived from plant) and 18:1Δ11cis (from bacteria/bacteroids).1.4 ± 0.52.0 ± 0.422.2 ± 0.451.4 ± 2.510.6 ± 0.780.5 ± 3.764.4 ± 0.118:2Δ9,1210.6 ± 2.828.6 ± 1.930.5 ± 0.29.0 ± 2.522.8 ± 8.3NDND18:3Δ9,12,1571.3 ± 3.444.7 ± 5.728.5 ± 0.14.6 ± 2.021.4 ± 1.5NDND19:011,12cycloNDND3.4 ± 0.616.9 ± 0.9ND1.5 ± 1.322.5 ± 0.4a 17:0 and 17:09,10cyclo are present in bacteria and bacteroids in trace amounts (below 1 mol%).b Contains 16:1Δ3trans (derived from plant) and 16:1Δ9cis (from bacteria/bacteroids).c ND, not detected.d Contains 18:1 Δ9cis (derived from plant) and 18:1Δ11cis (from bacteria/bacteroids). Open table in a new tab Table IIIFatty acid and sugar composition of DGDG from soybean symbiosomesFatty acidLeafNodulesRootSymbiosomesmol %mol %mol %mol %16:011.7 ± 1.621.9 ± 1.329.4 ± 3.425.1 ± 4.018" @default.
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• W2049580786 title "The Galactolipid Digalactosyldiacylglycerol Accumulates in the Peribacteroid Membrane of Nitrogen-fixing Nodules of Soybean and Lotus" @default.
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