Intracellular Localization of Lipoxygenases-1 and -2 in Germinating Soybean Seeds by Indirect Labeling with Protein A-Colloidal Gold Complexes

Vernooy-Gerritsen, Marjan; Leunissen, J. L. M.; Veldink, Gerrit A.; Vliegenthart, Johannes F.G.

doi:10.1104/pp.76.4.1070

Cited by 48 publications

(13 citation statements)

References 21 publications

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“…Vernooy-Gerritsen et al (28,29) have examined the subcellular distribution of soybean seed lipoxygenases and suggest that during germination they are initially present in the storage tissues of etiolated cotyledons and later appear in the cytoplasm and protein bodies. Our results, on the other hand, show that the vast majority of lipoxygenase that cross-reacts with our antiserum is present in the vacuoles, both before and after exposure to methyl jasmonate.…”

Section: Discussionmentioning

confidence: 99%

Expression, Activity, and Cellular Accumulation of Methyl Jasmonate-Responsive Lipoxygenase in Soybean Seedlings

Grimes

Koetje²,

Franceschi³

1992

Plant Physiol.

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Exposure of soybean (Glycine max) seedlings to low levels of atmospheric methyl jasmonate induced the expression and accumulation of one or more lipoxygenase(s) in the primary leaves, hypocotyls, epicotyls, and cotyledons. In the primary leaf, the major site of lipoxygenase accumulation in response to methyl jasmonate was in the vacuoles of paraveinal mesophyll cells. In the other organs, however, most of the methyl jasmonate-responsive lipoxygenase(s) were associated with both the epidermal and cortical cells and were present in both vacuoles and plastids. In plastids, the methyl jasmonate-responsive lipoxygenase was sequestered into protein inclusion bodies; no lipoxygenase was evident in either the thylakoids or the stroma. Both spectrophotometric measurement of conjugated diene formation and thin layer chromatography of lipoxygenase product formation indicated that methyl jasmonate caused an increase in the amount of lipoxygenase activity. Electron microscopy of the methyl jasmonate-responsive lipoxygenase protein in the vacuoles showed that it was arranged into a stellate, paracrystalline structure in various cell types other than the paraveinal mesophyll cells. The paracrystals appeared to be composed of tubular elements of between 5 and 8 nm in diameter, were of variable length, and were observed in most cell types of the seedling organs. Jasmonic acid and its methyl ester, methyl jasmonate, are rapidly emerging as strong candidates for intracellular or intercellular messengers in higher plants. When applied directly to plants or suspension cultures, they produce a number of phytohormone-like effects (1,19,26,31 (27). In mammals, eicosanoids are synthesized following the release of arachidonic acid into the cytoplasm and function as intracellular stress messengers (27). Analogously, plant cells are able to utilize linolenic acid as a substrate for the lipoxygenase-dependent synthesis of jasmonic acid (30). Although it has yet to be definitively established that jasmonic acid or methyl jasmonate are intracellular messengers in plant systems, the results of Farmer and Ryan (3) suggest that they may be involved in a wound-responsive signal cascade.The expression and accumulation of soybean VSPs is regulated by jasmonic acid and methyl jasmonate (1,7,10,14,22,23). The VSPs consist of three proteins of 27, 29, and 94 kD (vsp27 or vspa, vsp29 or vsp#, and vsp94, respectively), which accumulate during vegetative growth in a specialized cell layer termed the PVM layer (5,6,8,11,13). The PVM layer apparently serves as a direct conduit from the palisade parenchyma to the phloem (4-6, 8). VSP transcripts and their proteins respond to the immediate need to store nitrogen or amino acids (22,34) and to the removal of sink tissue or pods (20,25,32,33). The VSPs decrease in abundance during seed development (21, 34). Apparently, the VSPs have been recruited for temporary storage of nitrogen during vegetative growth, and this nitrogen is later contributed to the developing seed.In soybean (Glycine max) seedlings, ...

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Section: Discussionmentioning

confidence: 99%

Expression, Activity, and Cellular Accumulation of Methyl Jasmonate-Responsive Lipoxygenase in Soybean Seedlings

Grimes

Koetje²,

Franceschi³

1992

Plant Physiol.

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“…Because this distribution is observed after 2 or 3 d of germination and shortly before intracellular organization leading to photosynthetic capability the authors suggested that LOX may regulate cellular processes. Recently, however, intracellular labelling with protein A-colloidal gold complexes (60) revealed that neither LOX isozyme was associated with lipid bodies, mitochondria or any organelle in the hypodermis arid vascular bundle sheath but rather that these tissues contained LOX in aberrant protein bodies. In storage parenchyma LOX was found in the cytoplasm (60).…”

Section: Introductionmentioning

confidence: 99%

Two soybean seed lipoxygenase nulls accumulate reduced levels of lipoxygenase transcripts

Start

Polacco

et al. 1986

Plant Mol Biol

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Soybean (Glycine max L. [Merrill]) seed lipoxygenase cDNA clones were recovered from two cDNA libraries: a size-selected library in pBR322 and an expression library in pUC8. The pUC8 library was made with total poly(A)(+) embryo RNA and was screened with antiserum to lipoxygenase-1, one of 3 seed lipoxygenase isozymes. Three lipoxygenase antigen-producing clones were identified: two with identical cDNA inserts of 977 nucleotides representing an open-reading frame and a third truncated clone bearing a 3' end common to the longer clones. A long clone, pAL-134, was chosen for further study and was used to screen the size-selected cDNA library from which sixteen clones were identified. They fall into two homology classes represented by pLX-10 (ca. 1360 bp) and pLX-65 (2047 bp).The lipoxygenase expression clone pAL-134 hybridized much more strongly to pLX-65 than to pLX-10. pAL-134 and pLX-65 share 89% nucleotide homology and 75% deduced amino acid homology along their common sequence. Their deduced amino acid sequences each show 80% homology to sequences determined for isolated peptides of the lipoxygenase-1 isozyme.pAL-134 hybridizes poorly with a 3.8 kb RNA from LOX-1 null (lx1) embryos while pLX-65 hybridizes more strongly, but still to a lesser extent than its hybridization to standard embryo RNA or to RNA from embryos lacking lipoxygenase-2 (lx2) or lipoxygenase-3 (lx3) protein.The lx3 null lacks almost all embryo 3.8 kb RNA homologous to pLX-10. This hybridization pattern suggests that pLX-10 encodes LOX-3. Thus, the lx1 and lx3 genotypes accumulate little, if any, mRNA for the lipoxygenase-1 and lipoxygenase-3 isozymes, respectively.

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“…Fractionation studies designed to establish the subcellular localization(s) of lipoxygenase have proven problematic in part because soluble lipoxygenase tends to adhere nonspecifically to membranes (29). In situ immunogold labeling has shown that lipoxygenase is randomly distributed throughout the cytoplasm in storage parenchyma cells of germinating soybean (31). As well, biochemical and immunological measurements have indicated that the activity of lipoxygenase is highest during the early stages of soybean germination suggesting that the enzyme may play a role in reserve mobilization (25).…”

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confidence: 99%

Characteristics of a Membrane-Associated Lipoxygenase in Tomato Fruit

Todd¹,

Paliyath²,

Thompson³

1990

Plant Physiol.

131

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Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22°C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2* phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 300C, the formation of fatty acid oxidation products from 16:0/18:2* phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 220C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2* phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same pattems of metabolite formation from radiolabeled linoleic acid and 16:0/18:2* phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent Km of 0.52 millimolar and an apparent V,,. of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons.Lipoxygenase (EC 1.13.11.12) is a dioxygenase that catalyzes the peroxidation of fatty acids containing a cis,cis-l1,4-pentadiene configuration. Distinguishable isozymes of lipoxygenase have been described for a number of tissues in different plant species (8) and the best characterized of these are the three isozymes of soybean known as lipoxygenase-1, -2, and -3 (18 94, 1990 from lipoxygenase-1 of soybean (26). In the present study, we describe a lipoxygenase activity associated with microsomal membranes from tomato fruit that comprises 38% ofthe total (microsomal plus soluble) lipoxygenase activity in the tissue, appears to utilize free fatty acid released from membrane phospholipids as substrate and is immunologically related to its soluble counterpart and to soybean lipoxygenase-1. MATERIALS AND METHODS Plant Material and FractionationTomato fruit (Lycopersicon esculentum L. cv Caruso) were grown under greenhouse conditions and harvested at the mature-green stage. Pericarp tissue was cut into small pieces (~10 mm3) and suspended (-1 g/mL) in cold homogenizing buffer (100 mm Mops, 10 mM EGTA, and 7% sucrose at pH 7...

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Intracellular Localization of Lipoxygenases-1 and -2 in Germinating Soybean Seeds by Indirect Labeling with Protein A-Colloidal Gold Complexes

Cited by 48 publications

References 21 publications

Expression, Activity, and Cellular Accumulation of Methyl Jasmonate-Responsive Lipoxygenase in Soybean Seedlings

Expression, Activity, and Cellular Accumulation of Methyl Jasmonate-Responsive Lipoxygenase in Soybean Seedlings

Two soybean seed lipoxygenase nulls accumulate reduced levels of lipoxygenase transcripts

Characteristics of a Membrane-Associated Lipoxygenase in Tomato Fruit

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