The 9-lipoxygenase GhLOX1 gene is associated with the hypersensitive reaction of cotton Gossypium hirsutum to Xanthomonas campestris pv malvacearum.

Hypersensitive reaction (HR) cell death of cotton to the incompatible race 18 from Xanthomonas campestris pathovar malvacearum (Xcm) is associated with 9S-lipoxygenase activity (LOX) responsible for lipid peroxidation. Here, we report the cloning of cotton (Gossypium hirsutum L.) LOX gene (GhLOX1) and the sequencing of its promoter. GhLOX1 was found to be highly expressed during Xcm induced HR. Sequence analysis showed that GhLOX1 is a putative 9-LOX, and GhLOX1 promoter contains SA and JA responsive elements. Investigation on LOX signalisation on cotyledons infiltrated with salicylic acid (SA), or incubated with methyl-jasmonate (MeJA) revealed that both treatments induced LOX activity and GhLOX1 gene expression. HR-like symptoms were observed when LOX substrates were then injected in treated (MeJA and SA) cotyledons or when Xcm compatible race 20 was inoculated on MeJA treated cotyledons. Together these results support the fact that GhLOX1 encodes a 9 LOX whose activity would be involved in cell death during cotton HR.     

Differential expression pattern of an acidic 9/13-lipoxygenase in flower opening and senescence and in leaf response to phloem feeders in the tea plant

Background  

Lipoxygenase (LOXs) is a large family of plant enzymes that catalyse the hydroperoxidation of free polyunsaturated fatty acids into diverse biologically active compounds, collectively named phyto-oxylipins. Although multiple isoforms of LOXs have been identified in a wide range of annual herbaceous plants, the genes encoding these enzymes in perennial woody plants have not received as much attention. In Camellia sinensis (L.) O. Kuntze, no LOX gene of any type has been isolated, and its possible role in tea plant development, senescence, and defence reaction remains unknown. The present study describes the isolation, characterization, and expression of the first tea plant LOX isoform, namely CsLOX1, and seeks to clarify the pattern of its expression in the plant's defence response as well as in flower opening and senescence.     

The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.     

Oxylipins from both pathogen and host antagonize jasmonic acid‐mediated defence via the 9‐lipoxygenase pathway in Fusarium verticillioides infection of maize

Oxylipins are a newly emerging group of signals that serve defence roles or promote virulence. To identify specific host and fungal genes and oxylipins governing the interactions between maize and Fusarium verticillioides, maize wild‐type and lipoxygenase3 (lox3) mutant were inoculated with either F. verticillioides wild‐type or linoleate‐diol‐synthase 1‐deleted mutant (ΔFvlds1D). The results showed that lox3 mutants were more resistant to F. verticillioides. The reduced colonization on lox3 was associated with reduced fumonisin production and with a stronger and earlier induction of ZmLOX4, ZmLOX5 and ZmLOX12. In addition to the reported defence function of ZmLOX12, we showed that lox4 and lox5 mutants were more susceptible to F. verticillioides and possessed decreased jasmonate levels during infection, suggesting that these genes are essential for jasmonic acid (JA)‐mediated defence. Oxylipin profiling revealed a dramatic reduction in fungal linoleate diol synthase 1 (LDS1)‐derived oxylipins, especially 8‐HpODE (8‐hydroperoxyoctadecenoic acid), in infected lox3 kernels, indicating the importance of this molecule in virulence. Collectively, we make the following conclusions: (1) LOX3 is a major susceptibility factor induced by fungal LDS1‐derived oxylipins to suppress JA‐stimulating 9‐LOXs; (2) LOX3‐mediated signalling promotes the biosynthesis of virulence‐promoting oxylipins in the fungus; and (3) both fungal LDS1‐ and host LOX3‐produced oxylipins are essential for the normal infection and colonization processes of maize seed by F. verticillioides.     

Cotton GhSSI2 isoforms from the stearoyl acyl carrier protein fatty acid desaturase family regulate Verticillium wilt resistance

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl-ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2-A, GhSSI2-B, and GhSSI2-C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia-lyase-mediated salicylic acid (SA) accumulation and activated a SA-independent defence response in GhSSI2s-silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2-dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance-related genes and nitric oxide (NO) signal were activated in GhSSI2s-silenced cotton. Taken together, these results indicate that SA-independent defence response, multiple resistance-related proteins, and elevated NO level play an important role in GhSSI2s-regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance.     

Long noncoding RNAs involve in resistance to Verticillium dahliae,a fungal disease in cotton

Long noncoding RNAs (lncRNAs) have several known functions in plant development, but their possible roles in responding to plant disease remain largely unresolved. In this study, we described a comprehensive disease‐responding lncRNA profiles in defence against a cotton fungal disease Verticillium dahliae. We further revealed the conserved and specific characters of disease‐responding process between two cotton species. Conservatively for two cotton species, we found the expression dominance of induced lncRNAs in the Dt subgenome, indicating a biased induction pattern in the co‐existing subgenomes of allotetraploid cotton. Comparative analysis of lncRNA expression and their proposed functions in resistant Gossypium barbadense cv. ‘7124’ versus susceptible Gossypium hirsutum cv. ‘YZ1’ revealed their distinct disease response mechanisms. Species‐specific (LS) lncRNAs containing more SNPs displayed a fiercer inducing level postinfection than the species‐conserved (core) lncRNAs. Gene Ontology enrichment of LS lncRNAs and core lncRNAs indicates distinct roles in the process of biotic stimulus. Further functional analysis showed that two core lncRNAs, GhlncNAT‐ANX2‐ and GhlncNAT‐RLP7‐silenced seedlings, displayed an enhanced resistance towards V. dahliae and Botrytis cinerea, possibly associated with the increased expression of LOX1 and LOX2. This study represents the first characterization of lncRNAs involved in resistance to fungal disease and provides new clues to elucidate cotton disease response mechanism.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.     

Cunxi Wang · Kevan P.C. Croft · David F. Hildebrand

 Auxin [α-naphthaleneacetic acid (NAA) or indole-3-acetic acid] can induce the expression of lipoxygenases (LOXs) in cultured immature zygotic embryo cotyledons of soybean [Glycine max. (L.) Merr]. These auxin-induced LOXs are different from those normally expressed in seeds but have the same isoelectric points (pI) as those found in seedlings. The pIs of the two seedling LOXs were determined to be 5.09 and 5.23. One of the auxin-induced LOXs has the same pI (5.09) and molecular mass (94 kDa) as seedling LOX4. The partial amino acid sequences from the purified NAA-induced pI-5.09 LOX are identical to those of LOX4. RNA protection assays showed that NAA induces the expression of LOX4 and LOX5 mRNAs in cultured embryo cotyledons where they are not normally expressed. Soybean genotypes with a polymorphic variant of LOX4 in hypocotyls showed the same variation as NAA-induced LOXs in the embryo cotyledons. These results demonstrate that the NAA-induced pI-5.09 LOX is seedling LOX4 and also suggest that auxin might be directly or indirectly involved in seedling LOX expression during seed germination. Received: 10 January 2000 / Revision received: 16 June 2000 / Accepted: 29 June 2000     

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.     

The synthetic cationic lipid diC14 activates a sector of the Arabidopsis defence network requiring endogenous signalling components

Natural and synthetic elicitors have contributed significantly to the study of plant immunity. Pathogen‐derived proteins and carbohydrates that bind to immune receptors, allow the fine dissection of certain defence pathways. Lipids of a different nature that act as defence elicitors, have also been studied, but their specific effects have been less well characterized, and their receptors have not been identified. In animal cells, nanoliposomes of the synthetic cationic lipid 3‐tetradecylamino‐tert‐butyl‐N‐tetradecylpropionamidine (diC14) activate the TLR4‐dependent immune cascade. Here, we have investigated whether this lipid induces Arabidopsis defence responses. At the local level, diC14 activated early and late defence gene markers (FRK1, WRKY29, ICS1 and PR1), acting in a dose‐dependent manner. This lipid induced the salicylic acid (SA)‐dependent, but not jasmonic acid (JA)‐dependent, pathway and protected plants against Pseudomonas syringae pv. tomato (Pst), but not Botrytis cinerea. diC14 was not toxic to plant or pathogen, and potentiated pathogen‐induced callose deposition. At the systemic level, diC14 induced PR1 expression and conferred resistance against Pst. diC14‐induced defence responses required the signalling protein EDS1, but not NDR1. Curiously, the lipid‐induced defence gene expression was lower in the fls2/efr/cerk1 triple mutant, but still unchanged in the single mutants. The amidine headgroup and chain length were important for its activity. Given the robustness of the responses triggered by diC14, its specific action on a defence pathway and the requirement for well‐known defence components, this synthetic lipid is emerging as a useful tool to investigate the initial events involved in plant innate immunity.     

Duplicate maize 13-lipoxygenase genes are differentially regulated by circadian rhythm, cold stress, wounding, pathogen infection, and hormonal treatments

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defence responses to pathogens and pests. By contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. Two maize LOXs, ZmLOX10 and ZmLOX11 that share >90% amino acid sequence identity but are localized on different chromosomes, were cloned and characterized. Phylogenetic analysis revealed that ZmLOX10 and ZmLOX11 cluster together with well-characterized plastidic type 2 linoleate 13-LOXs from diverse plant species. Regio-specificity analysis of recombinant ZmLOX10 protein overexpressed in Escherichia coli proved it to be a linoleate 13-LOX with a pH optimum at approximately pH 8.0. Both predicted proteins contain putative transit peptides for chloroplast import. ZmLOX10 was preferentially expressed in leaves and was induced in response to wounding, cold stress, defence-related hormones jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA), and inoculation with an avirulent strain of Cochliobolus carbonum. These data suggested a role for this gene in maize adaptation to abiotic stresses and defence responses against pathogens and pests. ZmLOX11 was preferentially expressed in silks and was induced in leaves only by ABA, indicating its possible involvement in responses to osmotic stress. In leaves, mRNA accumulation of ZmLOX10 is strictly regulated by a circadian rhythm, with maximal expression coinciding temporally with the highest photosynthetic activity. This study reveals the evolutionary divergence of physiological roles for relatively recently duplicated genes. Possible physiological functions of these 13-LOXs are suggested.     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

The green microalga Lobosphaera incisa harbours an arachidonate 15S‐lipoxygenase

• The green microalga Lobosphaera incisa is an oleaginous eukaryotic alga that is rich in arachidonic acid (20:4). Being rich in this polyunsaturated fatty acid (PUFA), however, makes it sensitive to oxidation. In plants, lipoxygenases (LOXs) are the major enzymes that oxidise these molecules.
• Here, we describe, to our best knowledge, the first characterisation of a cDNA encoding a LOX (LiLOX) from a green alga. To obtain first insights into its function, we expressed it in E. coli, purified the recombinant enzyme and analysed its enzyme activity.
• The protein sequence suggests that LiLOX and plastidic LOXs from bryophytes and flowering plants may share a common ancestor. The fact that LiLOX oxidises all PUFAs tested with a consistent oxidation on the carbon n‐6, suggests that PUFAs enter the substrate channel through their methyl group first (tail first). Additionally, LiLOX form the fatty acid hydroperoxide in strict S configuration.
• LiLOX may represent a good model to study plastid LOX, because it is stable after heterologous expression in E. coli and highly active in vitro. Moreover, as the first characterised LOX from green microalgae, it opens the possibility to study endogenous LOX pathways in these organisms.