Differential Expression of Lipoxygenase Isoenzymes in Embryos of Germinating Barley

Expression of lipoxygenase was studied in barley (Hordeum distichum L.) embryos during germination. Total lipoxygenase activity was high in quiescent grains, dropped during the 1st d of germination, and subsequently increased to a level similar to that in quiescent grains. The contribution of two isoenzymes, lipoxygenases 1 (LOX-1) and 2 (LOX-2), was studied at the activity, protein, and mRNA levels. Activity ratios of the two isoforms were determined via the ratio of 9- and 13-hydroperoxides, which are formed from linoleic acid. Isoenzyme protein levels were determined using specific monoclonal antibodies. mRNA levels were studied using the specific cDNA probes LoxA and LoxC, which correspond to LOX-1 and LOX-2, respectively. The major difference in temporal expression of LOX-1 and LOX-2 was observed in quiescent grains. At this stage, LOX-1 contributed almost exclusively to total lipoxygenase activity. LOX-2 activity rapidly increased until d 2 of germination. From this time point onward, LOX-1 and LOX-2 showed similar patterns at both activity and protein levels. The tissue distribution of the two isoenzymes in the germinating embryo was closely similar, with the highest expression levels in leaves and roots. The levels of LOX-1 and LOX-2 may be regulated mainly pretranslationally, as suggested by the similarity of the protein and mRNA patterns corresponding to the two isoforms.     

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.     

Effects of exogenous auxins on expression of lipoxygenases in cultured soybean embryos

The expression of lipoxygenases (LOXs) is known to be developmentally regulated in soybeans (Glycine max. [L.] Merr.). Hormones have been firmly established as being involved in the growth and developmental processes of a number of plant species. Correlation between the expression of LOXs and the development and germination of soybean embryos suggests that plant hormones may affect the expression of LOXs. The present studies were conducted to investigate the effects of exogenous auxins on the expression of LOX isozymes and LOX activities in cultured cotyledon tissues of immature soybean seeds. The results revealed that at least one of the more acidic nonembryo LOX isozymes was induced by either α-naphthaleneacetic acid or indoleacetic acid but not by 2,4-dichlorophenoxyacetic acid after 4 days' exposure. Levels of LOX-1, -2, and -3 proteins and activities were significantly decreased by 2,4-dichlorophenoxyacetic acid 10 days after explanting. S1 analysis showed that embryo LOX messenger RNAs were detectable in the tissues treated with each of the auxins. The reduced levels of the embryo LOX proteins may, therefore, be regulated at the levels of translation, posttranslational modification, or degradation. The more acidic isozymes induced by α-naphthaleneacetic acid showed enzymatic activity and shared the same molecular mass and isoelectric point values as the germination-associated LOX isozymes found in hypocotyls and radicles, suggesting that those LOXs are involved in germination competency of soybean embryos.     

大豆黄酮对衰老小鼠脑组织SOD、LDH同工酶的影响

利用SOD和LDH同工酶电泳分析,研究大豆黄酮对衰老小鼠的抗氧化作用。结果显示大豆黄酮没有改变SOD和LDH同工酶谱的特征,但对因衰老引起的小鼠脑组织LDH和SOD同工酶活性、各组分的相对活性和比活力的变化有不同程度的改善作用,即LDH同工酶中LDH-2、LDH-3的活性明显下降,LDH-1的活性下降最为明显,而LDH-4的活性有所下降,但不显著,LDH-5的活性几乎没有变化,SOD同工酶的SOD-1和SOD-2的活性有不同程度的升高。这表明大豆黄酮是通过抑制LDH同工酶H亚基的合成来降低LDH的活性,而对M亚基的合成没有影响,并且能够促进SOD同工酶SOD-1和SOD-2的合成,不影响其遗传稳定性。     

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).     

Resolution of the isoenzymes of soybean lipoxygenase using isoelectric focusing and chromatofocusing

Isoelectric focusing in thin-layer polyacrylamide gels has been applied to the analysis of the enzymes involved in the formation and destruction of peroxides in soybeans [Glycine max (L.)], lipoxygenases and peroxidases, respectively. As a result of differences in pH optima for catalytic activity, lipoxygenases were selectively detected by adjusting the pH employed for activity-specific staining. Type-1 lipoxygenase was revealed not only by staining based on the conversion of linoleic acid to hydroperoxide but also by two stains based on the reduction of the hydroperoxide. These methods were found to be suitable for the analysis and characterization of isoenzyme patterns in different soybean cultivars. A substantial difference in the distribution of lipoxygenases maximally active near pH 7 was observed for cultivars Provar and Vickery. A similar degree of separation of the isoenzymes was achieved on a larger scale using chromato-focusing in the pH range 7.4-5.0.     

The differential effect of disulfiram on lipoxygenases from Glycine max

A kinetic analysis of the effect of disulfiram on the isoenzymes of lipoxygenase from soybean has been carried out. The compound is an effective inhibitor of type-2 isoenzymes but has no effect on the type-1 isoenzyme under the conditions employed in this study. The inhibitory effect is reversible and therefore does not result from covalent modification of the enzyme. The inhibition is manifest as a prolongation of the lag phase commonly seen in progress curves for lipoxygenases rather than as a reduction of the catalysed rate. A variety of structurally related and unrelated compounds have been investigated to identify the nature of the inhibitory effect. The antioxidant properties of disulfiram account for its ability to inhibit type-2 lipoxygenases. The inhibitory effect of antioxidants on type-2 lipoxygenase is only partly reversed when product hydroperoxide is included in the incubation mixture. These observations support the conclusion that free radical intermediates are integral to the catalytic mechanism of type-2 lipoxygenases.     

Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability

Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a approximately 30 kDa N-terminal domain and a approximately 60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a approximately 60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of alpha-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 x 10(6) and 10.9 x 10(6) M(-1) x s(-1), respectively), the activation energy of the reaction being 4.5 +/- 0.5 and 8.3 +/- 0.9 kJ x mol(-1) for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, alpha-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio- and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inhibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio(patho)logical conditions.     

Differential Expression of Two Soybean (Glycine max L.) Proline-Rich Protein Genes after Wounding

We have investigated the wound-induced expression of two members of the soybean (Glycine max L.) proline-rich cell wall protein gene family and show that SbPRP1 and SbPRP2 exhibit unique patterns of expression after physical damage. SbPRP1 mRNA can be detected in the hook of soybean seedlings within 2 h after wounding and is present at high levels in the hook and elongating hypocotyl 20 h after wounding. In contrast, SbPRP2 mRNA increases transiently and rapidly throughout the soybean seedling after wounding. SbPRP2 is also induced by wounding in soybean leaves, but the pattern of mRNA accumulation in leaves is distinct from that seen in seedlings and reaches high levels of expression 20 h after physical damage. SbPRP2 mRNA levels were also found to increase in the mature hypocotyl and roots of seedlings in response to treatment with 10 [mu]M indoleacetic acid and naphthalene-1-acetic acid. These data indicate that the wound-induced expression of PRPs in soybean is tissue specific and that the regulation of these genes after physical damage may operate through different signal transduction pathways.     

Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.     

Light-responsive subtilisin-related protease in soybean seedling leaves.

Protease C1 (E.C. 3.4.21.25), the soybean (Glycine max L. Merrill) proteolytic enzyme responsible for initiating the degradation of soybean storage proteins in seedling cotyledons appears at even higher levels in seedling leaves. This was manifested at the mRNA level through northern blot analysis, at the protein level through western blot analysis, through determination of enzyme activity, and also through isolation and partial sequencing of active leaf enzyme. Comparison of cDNA and amino acid sequences, as well as characterization of enzyme activity, is consistent with the leaf enzyme being identical to or highly similar to the cotyledon enzyme. Protease C1 mRNA and protein are also present in stems of soybean seedlings, but is very low to absent in the roots. This presence in the aerial tissues is consistent with the higher steady state level of gene expression at both the mRNA and protein levels when the seedlings are grown in a 12-h light: 12-h dark photoperiod as compared to seedlings grown in continuous darkness. Transfer of dark-grown seedlings to light is followed by marked elevation in protease C1 protein as seen in western blots.     

Hydrogen sulfide protects soybean seedlings against drought-induced oxidative stress

Increasing evidence indicates that hydrogen sulfide (H₂S) is the third “gas signal molecule” after NO and CO in animal. In the present study, we found that soybean (Glycine max L.) seedlings sprayed with exogenous H₂S donor NaHS prolonged the longer survival time of life, and enlarged higher biomass of both leaf and root than in non-sprayed controls under continuous drought stress. With the continuous drought stress, the content of chlorophyll in the leaves of both Xu-1 and Xu-6 cultivar of soybean decreased dramatically. The drought-induced decrease in chlorophyll could be alleviated by spraying H₂S donor. It was also shown that spraying with H₂S donor dramatically retained higher activities of superoxide dismutase (SOD, EC 1.1.5.1.1), catalase (CAT, EC1.11.1.6) and lower activity of lipoxygenases (LOX, EC 1.13.11.12), delayed excessive accumulation of malondialdehyde, hydrogen peroxide, and superoxide anion (O₂^·−) compared with the control. These results suggest that H₂S can increase drought tolerance in soybean seedlings by acting as an antioxidant signal molecule for the response.     

Protein dynamics, activity and cellular localization of soybean lipoxygenases indicate distinct functional roles for individual isoforms

Vegetative lipoxygenases (VLXs) in soybean are hypothesized to function in nitrogen storage and partitioning. Isoform-specific antibodies for four of the five known VLX isoenzymes were used to investigate the influence of source-sink status on protein levels, as well as to analyze the tissue and subcellular localization of the different isoforms. VLXD responded most strongly to sink limitation, although the levels of VLXA, B and C increased as well. After sink limitation, VLXD and the vegetative storage protein, VSPalpha, accumulated in the vacuoles of bundle sheath and paraveinal mesophyll cells, while VLXA, B and C localized to the cytosol of these cells. All five known VLX isoenzymes were active with both linoleic and linolenic acid substrates after expression in Escherichia coli. The strong upregulation of VLXD levels after sink limitation as well as the localization of this isoform to the vacuoles of paraveinal mesophyll and bundle sheath cells (where VSPs are found) strongly suggest that VLXD should be considered as a major storage protein in soybean leaves. Furthermore, since VLXA, B and C also accumulate in sink-limited soybean leaves, are located in the cytosol of paraveinal mesophyll cells and are active at pH values typically found in this compartment, their activities may well contribute to lipid metabolism in this tissue. This multi-gene family is thus ideally poised to play a pivotal role in the balance of N deposition relative to lipid-based storage, defense or signaling, by modulating contributions to these processes in the transient storage cells of the paraveinal mesophyll.     

Immunological comparison of lipoxygenase isozymes-1 and -2 with soybean seedling lipoxygenases

An affinity-purified polyclonal antibody against soybean seed lipoxygenase-2 was prepared and used to characterize the immunological relatedness of lipoxygenase isozymes 1 and 2 and lipoxygenases from soybean seedling roots, hypocotyls, leaves, and cotyledons. All soybean lipoxygenases tested cross-reacted with the anti-lipoxygenase-2. Cross-reactivity of seed-derived lipoxygenases was evidenced by formation of a line of identity in double-diffusion tests, by positive results on an immunoblot, and by antibody precipitation of enzyme activity. Levels of anti-lipoxygenase-2, which inhibited lipoxygenase-2 activity, had no effect on lipoxygenase-1 activity. Root, hypocotyl, and leaf lipoxygenases did not form precipitation lines in double-diffusion tests but the anti-lipoxygenase-2 did inhibit and precipitate lipoxygenase activity from these sources as well as cross-react on immunoblots. All the cross-reactive lipoxygenases examined were found to have the same apparent molecular weight. Lipoxygenase activity found in soybean seedling roots, hypocotyls, leaves, and cotyledons is associated with proteins which are all immunologically related to the seed-derived enzymes.