Substrate specificity of potato lipoxygenase

The substrate specificity of potato lipoxygenase was examined using a partially purified enzyme preparation from tubers of a potato variety with low lipolytic acyl hydrolase activity. Potato lipoxygenase is fully active only on free linoleic acid or linolenic acid, and only acts directly on more complex glyceride moieties in the absence of any significant endogenous lipolytic acyl hydrolase activity.     

Epirrita autumnata induced VOC emission of silver birch differ from emission induced by leaf fungal pathogen

The production of volatile organic compounds (VOCs) through the activation of different signal-transduction pathways may be induced in various biotic and abiotic stress situations having importance e.g. in insect and disease resistance. We compared the emission of VOCs emitted from silver birch Betula pendula Roth (clones 4 and 80) twigs damaged either by larvae of Epirrita autumnata, or infected with pathogenic leaf spot causing fungus Marssonina betulae. We also analysed whether local herbivore damage can systemically induce the release of VOCs from the undamaged top of same sapling. The emissions of methylsalicylate (MeSA), (Z)-ocimene, (E)-β-ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and linalool were induced from the twigs after 72 h feeding damage by E. autumnata larvae. However, 48 h feeding damage did not induce rapid systemic release of VOCs from undamaged top leaves of the same twigs. Pathogen-infected birch twigs had significantly greater emission of (Z)-ocimene and (E)-β-ocimene than intact control twigs. The emission of DMNT was not significantly induced and MeSA was not found at all after pathogen infection, both being significantly different from herbivore damaged twigs. According to our results leaf fungal pathogen induces VOC emission profile differs from that of arthropod herbivore-damaged leaves, suggesting that birch is able to transmit parasite-specific information via VOC emissions to conspecifics and natural enemies of herbivores. Handling editor: Yvan Rahbé     

Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection

The peptide snakin-2 (StSN2) has been isolated from potato (Solanum tuberosum cv Jaerla) tubers and found to be active (EC(50) = 1-20 microM) against fungal and bacterial plant pathogens. It causes a rapid aggregation of both Gram-positive and Gram-negative bacteria. The corresponding StSN2 cDNA encodes a signal sequence followed by a 15-residue acidic sequence that precedes the mature StSN2 peptide, which is basic (isoelectric point = 9.16) and 66 amino acid residues long (molecular weight of 7,025). The StSN2 gene is developmentally expressed in tubers, stems, flowers, shoot apex, and leaves, but not in roots, or stolons, and is locally up-regulated by wounding and by abscisic acid treatment. Expression of this gene is also up-regulated after infection of potato tubers with the compatible fungus Botritys cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum and Erwinia chrysanthemi. These observations are congruent with the hypothesis that the StSN2 is a component of both constitutive and inducible defense barriers.     

Inactivation of potato lipoxygenase by hydroperoxy acids as suicide substrates

The preincubation of potato lipoxygenase with 9(S)-hydroperoxyoctadecatrienoic acid, 15(S)-hydroperoxyeicosatetraenoic acid or 5(S)-hydroperoxyeicosatetraenoic acid which can be subjected to further lipoxygenation led to the gradual inactivation of the lipoxygenase activity, whereas 13(S)-hydroperoxy-9,13,15-octadecatrienoic acid or 15(S)-hydroperoxy-11,13,17-eicosatrienoic acid had no significant effect. The inhibitory effect of the peroxy acids was abolished by hemoglobin. Based on these observations, it is proposed that the unstable epoxide intermediates from the respective peroxy acids may be responsible for the inactivation of potato lipoxygenase. In the comparative study, it was found that 15(S)-hydroperoxyeicosatetraenoic acid possessed more effective inhibitory role than the other acids with Ki value of 250 microM.     

一种新发生的油桐叶枯病病原真菌

报道了中国广西田林县油桐主产区一种新的叶片真菌病害,定名为油桐叶枯病。该病主要为害叶片,病菌侵染后叶片呈现灰褐色病斑,后期扩至整个叶片,引起过早脱落,影响油桐产量。从采集的带病标本中分离到16个纯培养物,隶属5个分类单元。依据柯赫法则和致病性测定,证明只有菌株VT-04为油桐叶枯病的病原物。在病斑组织中观察不到有性或无性繁殖结构,而该病原菌在诱导培养（含无菌松针的水琼脂+近紫外照射）条件下可产生子实体。比较培养性状、产孢结构特征并结合分析核糖体rDNA基因转录间隔区（ITS）和RNA聚合酶次大亚基基因（RPB2）序列和系统发育关系,将该病原菌鉴定为葡萄座腔菌科Botryosphaeriaceae中的一种无性型菌物,即小新壳梭孢Neofusicoccum parvum。这是首次在油桐叶片上发现由该病菌引起的病害。     

白粉菌侵染对小麦叶片显微及超微结构的影响

通过半薄及超薄切片,比较了正常和受白粉菌感染的小麦叶片细胞的显微及超微结构的差异。观察结果发现：（1）受感染小麦叶肉细胞的细胞壁上局部沉积大量团状电子致密颗粒;（2）叶绿体形状由原来的椭圆形转变成圆形,叶绿体膜破裂;类囊体膨大,基粒片层排列疏松,同时,叶绿体内嗜饿性颗粒数量增加;（3）线粒体膜解体,内含物分散到了细胞质中。     

A cell membrane alteration specifically induced by SV40 transformation.

Transformation of mouse 3T3 cells by SV40 results in a specific membrane modification which renders cells resistant to the killing action of the lipophilic antibiotic Amphotericin B. This alteration is under genetic cellular control and is specific for SV40 transformation since transformation with polyoma or mouse sarcoma viruses does not confer resistance to the antibiotic. Analogous resistance is induced by SV40 transformation of primary human fibroblast cells. The acquired resistance is not due to decreased binding of Amphotericin B and is partially reversed if cells are grown in the presence of cholesterol. The results are interpreted as a specific change of sterol structure of the membrane or the loss of a minor cholesterol fraction responsible for the killing action of the antibiotic.     

Enzyme-catalyzed and enzyme-triggered pathways in dioxygenation of 1-monolinoleoyl-rac-glycerol by potato tuber lipoxygenase

It was shown for the first time that potato tuber lipoxygenase (ptLOX) catalyzed the aerobic oxidation of 1-monolinoleoyl-rac-glycerol (mLG) in a mixed micellar reaction solution with the non-ionic detergent monododecyl ether of decaoxyethylene glycol. No hydrolysis of mLG occurred during the reaction. The four major reaction products obtained at 23 degrees C were identified as 1-[9-hydroperoxy-10E,12Z-octadecadienoyl]-rac-glycerol (9-(E,Z)HPODE-GE, 41%), 1-[13-hydroperoxy-9Z,11E-octadecadienoyl]-rac-glycerol (13-(Z,E)-HPODE-GE, 17%), and their all-trans isomers ( approximately 21% each). The molar fraction of all-trans isomers depended on the temperature of the reaction solution; it was found that at 0 degrees C their molar fractions were approximately 15.5% each, while 9-(E,Z)HPODE-GE and 13-(Z,E)-HPODE-GE gave 42% and 27%, respectively, of the overall product. A free radical scavenger, 4-hydroxy-TEMPO, dramatically increased the molar fraction of 9-(E,Z)HPODE-GE, yielding 83% at 23 degrees C, at the expense of all other products. Chiral HPLC of 9-(E,Z)HPODE-GE formed in the presence of 4-hydroxy-TEMPO revealed that it was composed of approximately 94% S and approximately 6% (R) isomers. This assures largely a uniform orientation of mLG molecules in the ptLOX active center, with their methyl end most likely deepened into the protein globule. The second major product, 13-(Z,E)-HPODE-GE, which yielded approximately 9% of the total product formed in the presence of 4-hydroxy-TEMPO, was racemic, and so were the all-trans isomers. Therefore, the last three cannot be considered the true products of the enzyme reaction, which is known to be stereospecific. It appears that they were formed as a result of (i) leakage of the pentadienyl radicals from the ptLOX active center and their subsequent non-enzymatic dioxygenation, and/or (ii) leakage of the peroxyl radicals leading to a free radical chain reaction affording all positional, geometrical and stereoisomers of the products. This reaction resembles ptLOX oxidation of another non-ionizable substrate, linoleyl alcohol [I.A. Butovich, S.M. Luk'yanova, C.C. Reddy, Arch. Biochem. Biophys. 378 (2000) 65-77], and differed substantially from oxidation of ionizable linoleic acid. Consequently, formation of large amounts of the non-specific oxidation products might be considered a universal characteristic of ptLOX oxidation of non-ionizable compounds.     

Characteristic expression of wheat PR5 gene in response to infection by the leaf rust pathogen,Puccinia triticina

TaLr35PR5 gene was obtained from the gDNA and cDNA of TcLr35 wheat. It was induced by Puccinia triticina, ABA and SA, but TaLr35PR5 was induced earlier and its expression level was higher in the incompatible interaction than that in the compatible interaction. In addition, the accumulations of TaLr35PR5 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blottings were conducted to confirm that TaLr35PR5 be induced by P. triticina infection at the protein expression level. Similar to the expression patterns of TaLr35PR5 at RNA levels, the accumulations of TaLr35PR5 protein were weak in the seedling stage, then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene.     

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.     

A novel plastidial lipoxygenase of maize (Zea mays) ZmLOX6 encodes for a fatty acid hydroperoxide lyase and is uniquely regulated by phytohormones and pathogen infection

Lipoxygenases (LOXs) are members of a large enzyme family that catalyze oxygenation of free polyunsaturated fatty acids into diverse hydroperoxide compounds, collectively called oxylipins. Although LOXs have been well studied in dicot species, reports of the genes encoding these enzymes are scarce for monocots, especially maize. Herein, we reported the cloning, characterization and molecular functional analysis of a novel maize LOX gene, ZmLOX6. The ZmLOX6 nucleotide sequence encodes a deduced translation product of 892 amino acids. Phylogenetic analysis showed that ZmLOX6 is distantly related to previously reported 9- or 13-LOXs from maize and other plant species, including rice and Arabidopsis. Although sequence prediction suggested cytoplasmic localization of this protein, ZmLOX6 protein has been reportedly isolated from mesophyll cell chloroplasts, emphasizing the unique features of this protein. Plastidial localization was confirmed by chloroplast uptake experiments with the in vitro translated protein. Analysis of recombinant protein revealed that ZmLOX6 has lost fatty acid hydroperoxide forming activity but 13-LOX-derived fatty acid hydroperoxides were cleaved into odd-chain ω-oxo fatty acids and as yet not identified C5-compound. In line with its reported abundance in mesophyll cells, ZmLOX6 was predominantly expressed in leaf tissue. Northern blot analysis demonstrated that ZmLOX6 was induced by jasmonic acid, but repressed by abscisic acid, salicylic acid and ethylene and was not responsive to wounding or insects. Further, this gene was strongly induced by the fungal pathogen Cochliobolus carbonum during compatible interactions, suggesting that ZmLOX6 may contribute to susceptibility to this pathogen. The potential involvement of ZmLOX6 in maize interactions with pathogens is discussed.     

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides . A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.     

Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection

The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.     

浙江番茄灰叶斑病病原菌

近年浙江省杭州市余杭区温室大棚内番茄灰叶斑病发生为害严重,对当地番茄生产造成了较大的经济损失,但该病害的病原菌种类尚未在浙江报道。本文通过对病原菌形态学、分子系统学（rDNA-ITS、gpd和EF-1α基因的PCR扩增、序列分析和系统进化树的构建）和致病性测定相结合的研究,将侵染番茄的病原菌鉴定为番茄匍柄霉菌Stemphylium lycopersici。研究结果对制定该病害的防治决策和有效防治提供了科学理论依据。     

Fungal pathogen protection in potato by expression of a plant defensin peptide

Defensins are small cysteine-rich peptides with antimicrobial activity. We demonstrate that the alfalfa antifungal peptide (alfAFP) defensin isolated from seeds of Medicago sativa displays strong activity against the agronomically important fungal pathogen Verticillium dahliae. Expression of the alfAFP peptide in transgenic potato plants provides robust resistance in the greenhouse. Importantly, this resistance is maintained under field conditions. There have been no previous demonstrations of a single transgene imparting a disease resistance phenotype that is at least equivalent to those achieved through current practices using fumigants.