The Responses of the Lipoxygenase Gene Family to Salt and Drought Stress in Foxtail Millet (Setaria italica)

Plant lipoxygenases (LOXs), a kind of non-heme iron-containing dioxygenases, participate plant physiological activities (especially in response to biotic and abiotic stresses) through oxidizing various lipids. However, there was few investigations on LOXs in foxtail millet (Setaria italica). In this study, we identified the LOX gene family in foxtail millet, and divided the total 12 members into three sub-families on the basis of their phylogenetic relationships. Under salt and drought stress, LOX genes showed different expression patterns. Among them, only SiLOX7 showed up-regulated expression in Yugu1 (YG1) and Qinhuang2 (QH2), two stress-tolerant varieties, indicating that SiLOX7 may play an important role in responses to abiotic stress. Our research provides a basis for further investigation of the role of LOX genes in the adaptation to abiotic stresses and other possible biological functions in foxtail millet.

Loss of ZmLIPOXYGENASE4 Decreases Fusarium verticillioides Resistance in Maize Seedlings

Fusarium verticillioides is one of the most relevant fungal species in maize responsible for ear, stalk and seedling rot, as well as the fumonisin contamination of kernels. Plant lipoxygenases (LOX) synthesize oxylipins that play a crucial role in the regulation of defense mechanisms against pathogens and influence the outcome of pathogenesis. To better uncover the role of these signaling molecules in maize resistance against F. verticillioides, the functional characterization of the 9-LOX gene, ZmLOX4, was carried out in this study by employing mutants carrying Mu insertions in this gene (named as UFMulox4). In this regard, the genotyping of five UFMulox4 identified the mutant UFMu10924 as the only one having an insertion in the coding region of the gene. The impact of ZmLOX4 mutagenesis on kernel defense against F. verticillioides and fumonisin accumulation were investigated, resulting in an increased fungal susceptibility compared to the inbred lines W22 and Tzi18. Moreover, the expression of most of the genes involved in the LOX, jasmonic acid (JA) and green leaf volatiles (GLV) pathways, as well as LOX enzymatic activity, decreased or were unaffected by fungal inoculation in the mutant UFMu10924. These results confirm the strategic role of ZmLOX4 in controlling defense against F. verticillioides and its influence on the expression of several LOX, JA and GLV genes.

Exogenous Pathogen and Plant 15-Lipoxygenase Initiate Endogenous Lipoxin A4 Biosynthesis

Lipoxin A4 (LXA4) is a potent endogenous lipoxygenase-derived eicosanoid with antiinflammatory and proresolving properties. Supraphysiological levels of LXA4 are generated during infection by Toxoplasma gondii, which in turn reduces interleukin (IL) 12 production by dendritic cells, thus dampening Th1-type cell-mediated immune responses and host immunopathology. In the present work, we sought evidence for the structural basis of T. gondii's ability to activate LXA4 biosynthesis. Proteomic analysis of T. gondii extract (soluble tachyzoite antigen [STAg]), which preserves the immunosuppressive and antiinflammatory activity of the parasite, yielded several peptide matches to known plant lipoxygenases. Hence, we incubated STAg itself with arachidonic acid and found using LC-UV-MS-MS–based lipidomics that STAg produced both 15-HETE and 5,15-diHETE, indicating that T. gondii carries 15-lipoxygenase activity. In addition, T. gondii tachyzoites (the rapidly multiplying and invasive stage of the parasite) generated LXA4 when provided with arachidonic acid. Local administration of a plant (soybean) lipoxygenase itself reduced neutrophilic infiltration in murine peritonitis, demonstrating that 15-lipoxygenase possesses antiinflammatory properties. Administration of plant 15-lipoxygenase generated endogenous LXA4 and mimicked the suppression of IL-12 production by splenic dendritic cells observed after T. gondii infection or STAg administration. Together, these results indicate that 15-lipoxygenase expressed by a pathogen as well as exogenously administered 15-lipoxygenase can interact with host biosynthetic circuits for endogenous “stop signals” that divert the host immune response and limit acute inflammation.

Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

Mapping of Cotton-Melon Aphid Resistance in Melon

Resistance to cotton-melon aphid (Aphis gossypii Glover) segregated as a single dominant gene in a melon (Cucumis melo L.) mapping population derived from the cross `Top Mark' × PI 414723. Sixty-four F2-derived F3 families were used to map the aphid resistance locus, Vat, with respect to randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers. RFLP markers NBS-2 and AC-39 flanked Vat at distances of 3.1 cM and 6.4 cM, respectively. NBS-2 is homologous to the nucleotide binding site-leucine-rich repeat (NBS-LRR) superfamily of plant resistance genes. Another homolog of this superfamily, NBS-5, was positioned ≈16.8 cM from Vat, raising the possibility that Vat resides in a cluster of NBS-LRR paralogs. RFLP marker AC-8, which has similarity to plant lipoxygenases, was positioned at ≈5.5 cM from Vat. Monogenic resistance to A. gossypii has been identified in two sources of melon germplasm, Indian accession PI 371795 (progenitor of PI 414723) and Korean accession PI 161375. To test for an allelic relation between the genes controlling aphid resistance in these two distinct germplasm sources, melon plants of a backcross population from a cross between two resistant lines having Indian- or Korean-derived resistance were infested with aphids. At least 90 out of 92 segregating progeny were aphid resistant, suggesting that the same resistance gene, Vat, is present in both sources of melon germplasm.

Creating lipoxygenases with new positional specificities by site-directed mutagenesis

In order to analyse the amino acid determinants which alter the positional specificity of plant lipoxygenases (LOXs), multiple LOX sequence alignments and structural modelling of the enzyme-substrate interactions were carried out. These alignments suggested three amino acid residues as the primary determinants of positional specificity. Here we show the generation of two plant LOXs with new positional specificities, a Δ-linoleneate 6-LOX and an arachidonate 11-LOX, by altering only one of these determinants within the active site of two plant LOXs. In the past, site-directed-mutagenesis studies have mainly been carried out with mammalian lipoxygenases (LOXs) [1]. In these experiments two regions have been identified in the primary structure containing sequence determinants for positional specificity. Amino acids aligning with the Sloane determinants [2] are highly conserved among plant LOXs. In contrast, there is amino acid hetero-geneity among plant LOXs at the position that aligns with P353 of the rabbit reticulocyte 15-LOX (Borngräber determinants) [3].