Role of Tomato Lipoxygenase D in Wound-Induced Jasmonate Biosynthesis and Plant Immunity to Insect Herbivores

In response to insect attack and mechanical wounding, plants activate the expression of genes involved in various defense-related processes. A fascinating feature of these inducible defenses is their occurrence both locally at the wounding site and systemically in undamaged leaves throughout the plant. Wound-inducible proteinase inhibitors (PIs) in tomato (Solanum lycopersicum) provide an attractive model to understand the signal transduction events leading from localized injury to the systemic expression of defense-related genes. Among the identified intercellular molecules in regulating systemic wound response of tomato are the peptide signal systemin and the oxylipin signal jasmonic acid (JA). The systemin/JA signaling pathway provides a unique opportunity to investigate, in a single experimental system, the mechanism by which peptide and oxylipin signals interact to coordinate plant systemic immunity. Here we describe the characterization of the tomato suppressor of prosystemin-mediated responses8 (spr8) mutant, which was isolated as a suppressor of (pro)systemin-mediated signaling. spr8 plants exhibit a series of JA-dependent immune deficiencies, including the inability to express wound-responsive genes, abnormal development of glandular trichomes, and severely compromised resistance to cotton bollworm (Helicoverpa armigera) and Botrytis cinerea. Map-based cloning studies demonstrate that the spr8 mutant phenotype results from a point mutation in the catalytic domain of TomLoxD, a chloroplast-localized lipoxygenase involved in JA biosynthesis. We present evidence that overexpression of TomLoxD leads to elevated wound-induced JA biosynthesis, increased expression of wound-responsive genes and, therefore, enhanced resistance to insect herbivory attack and necrotrophic pathogen infection. These results indicate that TomLoxD is involved in wound-induced JA biosynthesis and highlight the application potential of this gene for crop protection against insects and pathogens.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function

Herbivory results in an array of physiological changes in the host that are separable from the associated physical damage. We have made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins is defective in some, but not all, of the typical plant responses to herbivory. We tested the responses of plasma transmembrane potential (Vm) depolarization, voltage gated K⁺ channel activity, cytosolic calcium [Ca²⁺]_cyt and reactive oxygen species (ROS) (H₂O₂ and NO) release, shoot‐to‐root signaling, biosynthesis of the phytohormone jasmonic acid (JA) and the elicitation of volatile organic compounds (VOCs). Following herbivory and the release of factors present in insect oral secretions (including a putative β‐galactofuranose polysaccharide), both the pdko3 and wild type (WT) plants showed a increased accumulation of [Ca²⁺]_cyt, NO and H₂O₂. In contrast, unlike WT plants, the mutant line showed an almost complete loss of voltage gated K⁺ channel activity and Vm depolarization, a loss of shoot‐induced root‐Vm depolarization, a loss of activation and regulation of gene expression of the JA defense pathway, and a much diminished release and altered profile of VOCs. The mutations in genes for plasmodesmal proteins have provided valuable genetic tools for the dissection of the complex spectrum of responses to herbivory and shown us that the responses to herbivory can be separated into a calcium‐activated oxidative response and a K⁺‐dependent Vm‐activated jasmonate response associated with the release of VOCs.     

Estrus synchronization and ovarian hyper‐stimulation treatments have negligible effects on cumulus oocyte complex gene expression whereas induction of ovulation causes major expression changes

The effects of exogenous hormones, used for estrus synchronization and ovarian hyper stimulation, on cumulus oocyte complexes (COCs) gene expression in sexually mature rats were determined using microarrays. Gene expression in COCs collected from GnRH (G_trt), GnRH + eCG (G + E_trt), and GnRH + eCG + hCG (G + E + H_trt) treatments were compared to COCs from naturally cycling (NC) rats before the preovulatory luteninizing hormone surge. There was no significant difference in gene expression among NC, G_trt, and G + E_trt; however, over 2,600 genes were significantly different between NC and G + E + H_trt (P < 0.05). Genes upregulated in G + E + H_trt encode for: proteins that are involved in prostaglandin synthesis (Ptgs2, Pla2g4a, and Runx1) and cholesterol biosynthesis (Hmgcr, Sc4mol, and Dhcr24); receptors that allow cholesterol uptake (Ldlr and Scarb1), regulate progesterone synthesis (Star), and inactivate estrogen (Sult1e1); and downstream effectors of LH signal (Pgr, Cebpb, Creb3l1, Areg, Ereg, and Adamts1). Conversely, G + E + H_trt downregulated genes encoding proteins involved in: DNA replication and cell cycle progression (Ccne2, Orc5l, Rad50, and Mcm6); reproductive developmental process; and granulosa cell expansion (Gdf9, Bmp15, Amh, Amhr2, Bmpr1b, Tgfb2, Foxl2, Pde3a, Esr2, Fshr, Ybx2, Ccnd2, Ccnb1ip1, and Zp3); maternal effect genes required for embryo development (Zar1, Npm2, Nlrp5, Dnmt1, H1foo, and Zfp57); amino acid degradation; and ketogenesis (Hmgcs2, and Cpt1b). These results from the rat show that hormones used for estrus synchronization (G_trt) and ovarian hyper stimulation (G + E_trt) had minimal effects on gene expression, whereas induction of ovulation (G + E + H_trt) caused major changes in gene expression of rat COCs. This study provides comprehensive information about regulated genes during late follicle development and ovulation induction. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

Induction of some defense-related genes and oxidative burst is required for the establishment of systemic acquired resistance in <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The inoculation of primary pepper leaves with an avirulent strain of Xanthomonas campestris pv. vesicatoria induced systemic acquired resistance (SAR) in the non-inoculated, secondary leaves. This SAR response was accompanied by the systemic expression of the defense-related genes, a systemic microoxidative burst generating H₂O₂, and the systemic induction of both ion-leakage and callose deposition in the non-inoculated, secondary leaves. Some defense-related genes including those encoding PR-1, chitinase, osmotin, peroxidase, PR10, thionin, and SAR8.2 were markedly induced in the systemic leaves. The conspicuous systemic accumulation of H₂O₂ and the strong increase in peroxidase activity in the pepper leaves was suggested to play a role in the cell death process in the systemic micro-hypersensitive responses (HR), leading to the induction of the SAR. Treatment of the primary leaves with diphenylene iodinium (DPI), an inhibitor of oxidative burst, substantially reduced the induction of some of the defense-related genes, and lowered the activation of the oxidative bursts in the systemic leaves distant from the site of the avirulent pathogen inoculation and subsequently SAR. Overall, these results suggest that the induction of some defense-related genes as well as a rapid increase in oxidative burst is essential for establishing SAR in pepper plants.     

Microarray analysis of the interaction between the aphid <Emphasis Type="Italic">Rhopalosiphum padi</Emphasis> and host plants reveals both differences and similarities between susceptible and partially resistant barley lines

The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H₂O₂ and NO. Scavenging of H₂O₂ or NO in upper leaves blocked BR‐induced systemic virus resistance. BR‐induced systemic H₂O₂ accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite‐dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR‐triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR‐activated H₂O₂ is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H₂O₂ generation blocked BR‐induced systemic NO production, but BR‐induced H₂O₂ production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR‐induced systemic virus defense in NbRBOHB‐silenced plants, but H₂O₂ did not reverse the effect of NbNR silencing on BR‐induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR‐mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR‐induced H₂O₂ and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR‐mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)‐dependent H₂O₂ production and subsequent systemic NR‐dependent NO generation.     

Local and systemic effects of two herbivores with different feeding mechanisms on primary metabolism of cotton leaves

Caterpillars and spider mites are herbivores with different feeding mechanisms. Spider mites feed on the cell content via stylets, while caterpillars, as chewing herbivores, remove larger amounts of photosynthetically active tissue. We investigated local and systemic effects of short-term caterpillar and spider mite herbivory on cotton in terms of primary metabolism and growth processes. After short-term caterpillar feeding, leaf growth and water content were decreased in damaged leaves. The glutamate/glutamine ratio increased and other free amino acids were also affected. In contrast, mild spider mite infestation did not affect leaf growth or amino acid composition, but led to an increase in total nitrogen and sucrose concentrations. Both herbivores induced locally increased dark respiration, suggesting an increased mobilization of storage compounds potentially available for synthesis of defensive substances, but did not affect assimilation and transpiration. Systemically induced leaves were not significantly affected by the treatments performed in this study. The results show that cotton plants do not compensate the loss of photosynthetic tissue with higher photosynthetic efficiency of the remaining tissue. However, early plant responses to different herbivores leave their signature in primary metabolism, affecting leaf growth. Changes in amino acid concentrations, total nitrogen and sucrose content may affect subsequent herbivore performance.     

Induction of plant responses by a sequestering insect: Relationship of glucosinolate concentration and myrosinase activity

Induction of plant allelochemicals is of particular ecological importance for interactions with herbivores that can make use of induced metabolites by incorporating them for their own defence. Induction patterns in white mustard, Sinapis alba, were investigated following herbivory of the turnip sawfly, Athalia rosae, which sequesters plant glucosinolates. Larvae of different age were allowed to feed for 24 h on young leaves of premature, non-flowering plants. Changes in primary and secondary metabolites were recorded in the damaged leaves (local) and in the adjacent leaves and stems (systemic) for several days. Organ- and time-specific patterns were evident. Local responses included increases in glucosinolate concentrations, soluble and insoluble myrosinase activity and glucose levels, while systemic responses in leaves were restricted to increases in myrosinase activities and glucose. All effects were strongest immediately after feeding and declined mostly within a day. Stems had overall lower constitutive levels of glucosinolates and myrosinase activities than leaves. Feeding by one large larva had a greater impact on the plant's physiology than feeding by three small ones, even though both treatments resulted in quantitatively similar leaf destruction. Local increase in glucosinolate concentration could be beneficial for larvae, while conspecifics feeding on induced adjacent leaves might be negatively affected due to higher myrosinase activity levels. The results are discussed in the context of the ‘optimal defence theory’ and the ‘lethal plant defence paradox’.     

Nitric oxide is induced by wounding and influences jasmonic acid signaling in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Nitric oxide (NO) has been associated with plant defense responses during microbial attack, and with induction and/or regulation of programmed cell death. Here, we addressed whether NO participates in wound responses in Arabidopsis thaliana (L.) Heynh.. Real-time imaging by confocal laser-scanning microscopy in conjunction with the NO-selective fluorescence indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) uncovered a strong NO burst after wounding or after treatment with JA. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive responses. Furthermore, we were able to detect NO in plants (here induced by wounding) by means of electron paramagnetic resonance measurements using diethyldithiocarbamate as a spin trap. When plants were treated with NO, Northern analyses revealed that NO strongly induces key enzymes of jasmonic acid (JA) biosynthesis such as allene oxide synthase (AOS) and lipoxygenase (LOX2). On the other hand, wound-induced AOS gene expression was independent of NO. Furthermore, JA-responsive genes such as defensin (PDF1.2) were not induced, and NO induction of JA-biosynthesis enzymes did not result in elevated levels of JA. However, treatment with NO resulted in accumulation of salicylic acid (SA). In transgenic NahG plants (impaired in SA accumulation and/or signaling), NO did induce JA production and expression of JA-responsive genes. Altogether, the presented data demonstrate that wounding in Arabidopsis induces a fast accumulation of NO, and that NO may be involved in JA-associated defense responses and adjustments.Abbreviations AOS Allene oxide synthase - cPTIO Carboxy-2-phenyl-4,4,5,5-tetramethylimidazolinone-3-oxide-1-oxyl - DAF-2 DA 4,5-Diaminofluorescein diacetate - DETC Diethyldithiocarbamate - EPR Electron paramagnetic resonance - iNOS Inducible nitric oxide synthase - JA Jasmonic acid - JIP Jasmonic acid-induced protein - LOX2 Lipoxygenase 2 - NO Nitric oxide - OPR3 12-Oxophytodienoate reductase - PDF1.2 Plant defensin - ROS Reactive oxygen species - SA Salicylic acid - SNP Sodium nitroprusside