Crystal structures of vegetative soybean lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms LOX-1 and LOX-3

The lipoxygenase family of lipid-peroxidizing, nonheme iron dioxygenases form products that are precursors for diverse physiological processes in both plants and animals. In soybean (Glycine max), five vegetative isoforms, VLX-A, VLX-B, VLX-C, VLX-D, VLX-E, and four seed isoforms LOX-1, LOX-2, LOX-3a, LOX-3b have been identified. In this study, we determined the crystal structures of the substrate-free forms of two major vegetative isoforms, with distinct enzymatic characteristics, VLX-B and VLX-D. Their structures are similar to the two seed isoforms, LOX-1 and LOX-3, having two domains with similar secondary structural elements: a beta-barrel N-terminal domain containing highly flexible loops and an alpha-helix-rich C-terminal catalytic domain. Detailed comparison of the structures of these two vegetative isoforms with the structures of LOX-1 and LOX-3 reveals important differences that help explain distinct aspects of the activity and positional specificity of these enzymes. In particular, the shape of the three branches of the internal subcavity, corresponding to substrate-binding and O(2) access, differs among the isoforms in a manner that reflects the differences in positional specificities.     

The LOX1 Gene of Arabidopsis Is Temporally and Spatially Regulated in Germinating Seedlings

We examined the temporal and spatial expression patterns of the LOX1 gene during the development of Arabidopsis thaliana seedlings. Measurements of steady-state LOX1 mRNA levels indicated that this gene is transiently expressed during germination. LOX1 mRNA was not detected in seed that had imbibed (T0) but reached a maximum level by 1 d in both light- and dark-grown seedlings. The induction of the LOX1 gene was not light dependent; however, mRNA levels were 4-fold greater in light-grown seedlings. Immunoblot analysis of lipoxygenase protein levels and measurements of enzyme activity suggested that the induction of the LOX1 gene resulted in the production of functional lipoxygenase enzyme. Lipoxygenase protein was not present in dry seed or seed that had imbibed, but was first detected by immunoblot analysis after 1 and 2 d of growth in the light and dark, respectively. In both cases, lipoxygenase protein levels remained high for 2 d and then declined. Lipoxygenase activity paralleled the changes in protein levels. In situ hybridization studies revealed that the LOX1 gene is transiently expressed in the epidermis and the aleurone layer during germination. LOX1 mRNA levels were particularly high in the epidermis of the radicle and the adaxial side of the cotyledons. These results suggest that the LOX1 gene product is produced specifically during early germination and plays a role in the functioning of the epidermis.     

Changes in Lipoxygenase Components of Rice Seedlings during Germination

Changes in lipoxygenase (LOX) activity were followed duringthe germination of rice seeds. The enzyme activity of 3-day-oldseedlings was 20 times higher than that of ungerminated seeds.Sixty per cent of the increased activity was found in shoots.The increase in LOX activity was mainly due to an increase inlipoxygenase-2 (LOX-2), a minor component in ungerminated seeds;this increase was inhibited by cycloheximide. LOX-2 was isolatedfrom the 3-day-old seedlings and compared for its enzymologicalproperties with rice lipoxygenase-3 (LOX-3), a major componentin ungerminated seeds. Both LOX-2 and LOX-3 were stable at pH5 to 8, but LOX-2 was more heatstable than LOX-3. Apparent K_mvalues of LOX-2 and LOX-3 for linoleic acid were 170 and 59µM, and those for linolenic acid were 5,300 and 88 µM,respectively. Both LOXs were inhibited by some metal ions andantioxidants. (Received February 5, 1986; Accepted May 9, 1986)     

Patterns of Starchy Endosperm Acidification and Protease Gene Expression in Wheat Grains following Germination

The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process.     

Stability of immobilized soybean lipoxygenases: influence of coupling conditions on the ionization state of the active site Fe

The potential application of lipoxygenase as a versatile biocatalyst in enzyme technology is limited by its poor stability. Two types of soybean lipoxygenases, lipoxygenase-1 and -2 (LOX-1 and LOX-2) were purified by a two step anion exchange chromatography. Four different commercially available supports: CNBr Sepharose 4B, Fractogel((R)) EMD Azlactone, Fractogel((R)) EMD Epoxy, and Eupergit((R)) C were tested for immobilization and stabilization of the purified isoenzymes. Both isoenzymes gave good yields in enzyme activity and good stability after immobilization on CNBr Sepharose 4B and Fractogel((R)) EMD Azlactone. Rapid decay in activity associated with change in the ionization state of Fe, as shown by EPR measurements was observed within the first 5 days after immobilization on epoxy activated supports (Eupergit((R)) C and Fractogel((R)) EMD Epoxy) in high ionic strength buffers. Stabilization of the biocatalyst on these supports was achieved by careful adjustment of the immobilization conditions. When immobilized in phosphate buffer of pH 7.5 and low ionic strength (0.05 M), the half-life time of the immobilized enzyme increased 20 fold. The dependence of the stability of LOX immobilized on epoxy activated supports on the coupling conditions was attributed to a modulation of the ligand environment of the iron in the active site and consequently its reactivity.     

Lipoxygenase isoenzymes: a spectroscopic and structural characterization of soybean seed enzymes

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.     

Lipoxygenase activity in soybean is modulated by enzyme-substrate ratio

The off-flavour development in soybean based food and oil industry is considered as a serious problem. In soybean three lipoxygenase isozymes namely LOX-1, LOX-2 and LOX-3 which contribute to about 1 % of storage protein have been reported and are the major culprits for the generation of volatile compounds causing the off-flavour. The present study showed that the 3 lipoxygenase isozymes isolated from defatted soybean flour exhibited inhibition potential by modulating the enzyme to substrate ratio. LOX-2 was the most inhibition prone enzyme. Defatting the flour may help in reducing off-flavour generation.     

Angiotensin II increases mRNA levels of all TGF‐β isoforms in quiescent and activated rat hepatic stellate cells

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF‐β (transforming growth factor‐β). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF‐β actions. Here, we studied the effect of AII on the mRNA levels of TGF‐β isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF‐β1, TGF‐β2 and TGF‐β3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF‐β isoforms, particularly TGF‐β2 and TGF‐β3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF‐β protein after AII treatment. The mRNA expression of TGF‐β isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF‐β expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.     

Effect of drought on proteins and isoenzymes in rice during germination

Two drought tolerant varieties TKM-1 and TKM-2 and two drought susceptible varieties Jaya and Improved Sabarmati of rice were studied for soluble protein pattern and isoenzymes of malate dehydrogenase, glutamate dehydrogenase, esterase and peroxidase during germination at different water stress. MDH, GDH and esterase patterns were not affected, but the soluble proteins were changed. Peroxidase isoenzyme pattern from drought tolerant and susceptible varieties showed characteristic differences. The intensity of bands with higher electrophoretic mobility decreased in Jaya and Improved Sabarmati while in TKM-1 and TKM-2 the intensity of these bands did not change much after 72 hr water stress. In shoots of Jaya and Improved Sabarmati, the activity of the peroxidase isoenzymes decreased more than in TKM-1 and TKM-2 shoots with increase in water stress.     

Profile of peroxidase isoforms influenced by spongy tissue disorder in Alphonso mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) fruits

This paper describes the profile of peroxidase (POX) isoenzymes induced due to the natural infection of Staphylococcus xylosus in spongy Alphonso mango fruits. Very low levels of protein and POX activity was observed in non-spongy unripe fruits, and when these fruits turned table-ripe, the levels of both the protein content and POX activity increased several fold. The spongy fruits, however, showed further 2-fold increase in POX activity; although drastic decrease in protein content was observed. Anionic and cationic PAGE, and isoelectric focusing (IEF), resulted in separation of various isoenzymes of POX. Both, anionic and cationic PAGE indicated that, at unripe stage, only basic isoforms were present in trace amounts. In non-spongy ripe fruits, increased levels of both anionic and cationic isoforms were observed after staining the gel with o-dianisidine, the POX substrate. In spongy fruits, however, an anionic PAGE showed appearance of four acidic isoforms with relative electrophoretic mobility (REM) of 0.52, 0.73, 0.78, and 0.84 and an isoenzyme (REM 0.52), showed further activation, as indicated by the intense dark color formation. Cationic PAGE also indicated higher levels of two basic isoforms (REM 0.56 and 0.62), in the spongy fruits. Isoelectric focusing resolved these isoenzymes in acidic, neutral, and basic isoforms. Two acidic isoforms in the pI range of 2–3.5 were detected toward the anode region and two cationic isoforms of pI 7.8 and 8.7, toward the cathode, giving visible indication of increased levels of these isoforms. The increased intensities of the POX bands observed in anionic and cationic PAGE, and IEF, gave confirmatory evidence for the up regulation of anionic and cationic isoforms in spongy fruits. These isoenzymes could have been overexpressed as a defense response of the spongy fruits against the Staphylococcus infection.     

The N-terminal beta-barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies.

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post-translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A beta-barrel-forming N-terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the beta-barrel represents a membrane-binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact beta-barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX beta-barrel with glutathione S-transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST-fusion protein confirmed the role of the beta-barrel as the membrane-targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX-1. When the beta-barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.     

Light-induced Changes in Expression of Pathogenesis-related Anionic Peroxidase in Cucumber Seedlings

The effect of irradiance on the expression of the Cucumis sativus pathogenesis-related (srPRX) anionic peroxidase was studied in germinating seeds at the period when seedlings start to be photosynthetically active. The diversity in the expression patterns of srPRX was observed in both dark- and light-grown seedlings using activity staining and immunoblotting: beside the three srPRX isoenzymes also other three, serologically unrelated, peroxidase isoforms were accumulated in dark-grown seedlings and one in light-grown seedlings. Furthermore, in light-grown seedlings, it was observed a marked difference in the expression of particular srPRX isoenzymes at higher irradiance (up to 260 W m-2, 400 - 700 nm) in comparison to low irradiance. By tissue printing on nitrocellulose paper it was demonstrated that irradiance during germination induced changes in the temporal and spatial distribution of the srPRX.     

The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.     

Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.