Atsushi Muroi

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

BackgroundPlant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable.ResultsTo investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response.ConclusionsThese results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.

Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana

Hydroxycinnamic acid amides (HCAAs) are secondary metabolites involved in the defense of plants against pathogens. Here, we report the first identification of HCAAs, p-coumaroylagmatine, feruloylagmatine, p-coumaroylputrescine and feruloylputrescine, in Arabidopsis thaliana rosette leaves infected with Alternaria brassicicola and the assignment of At5g61160 as the agmatine coumaroyltransferase (AtACT) that catalyzes the last reaction in the biosynthesis of the HCAAs. Feeding experiments with putative labeled precursors revealed that the four HCAAs were synthesized from hydroxycinnamic acids and agmatine or putrescine. AtACT gene function was identified from an analysis of a mutant that did not accumulate HCAAs. In wild-type Arabidopsis, AtACT transcripts markedly increased in response to A. brassicicola infection. Enzymatic activity that catalyzes the synthesis of the HCAAs was confirmed in vitro by using a recombinant AtACT expressed in Escherichia coli. The Atact mutant was susceptible to infection by A. brassicicola, indicating that HCAAs are responsible for defense against pathogens in A.thaliana.

The Composite Effect of Transgenic Plant Volatiles for Acquired Immunity to Herbivory Caused by Inter-Plant Communications

A blend of volatile organic compounds (VOCs) emitted from plants induced by herbivory enables the priming of defensive responses in neighboring plants. These effects may provide insights useful for pest control achieved with transgenic-plant-emitted volatiles. We therefore investigated, under both laboratory and greenhouse conditions, the priming of defense responses in plants (lima bean and corn) by exposing them to transgenic-plant-volatiles (VOCos) including (E)-β-ocimene, emitted from transgenic tobacco plants (NtOS2) that were constitutively overexpressing (E)-β-ocimene synthase. When lima bean plants that had previously been placed downwind of NtOS2 in an open-flow tunnel were infested by spider mites, they were more defensive to spider mites and more attractive to predatory mites, in comparison to the infested plants that had been placed downwind of wild-type tobacco plants. This was similarly observed when the NtOS2-downwind maize plants were infested with Mythimna separata larvae, resulting in reduced larval growth and greater attraction of parasitic wasps (Cotesia kariyai). In a greenhouse experiment, we also found that lima bean plants (VOCos-receiver plants) placed near NtOS2 were more attractive when damaged by spider mites, in comparison to the infested plants that had been placed near the wild-type plants. More intriguingly, VOCs emitted from infested VOCos-receiver plants affected their conspecific neighboring plants to prime indirect defenses in response to herbivory. Altogether, these data suggest that transgenic-plant-emitted volatiles can enhance the ability to prime indirect defenses via both plant-plant and plant-plant-plant communications.

Exogenous Polyamines Elicit Herbivore-Induced Volatiles in Lima Bean Leaves: Involvement of Calcium, H2O2 and Jasmonic Acid

We investigated the role of polyamines (PAs) in lima bean (Phaseolus lunatus) leaves on the production of herbivorous mite (Tetranychus urticae)-induced plant volatiles that attract carnivorous natural enemies of the herbivores. To do this, we focused on the effects of the exogenous PAs [cadaverine, putrescine, spermidine and spermine (Spm)] on the production of volatiles, H(2)O(2) and jasmonic acid (JA) and the levels of defensive genes, cytosolic calcium and reactive oxygen species (ROS). Among the tested PAs, Spm was the most active in inducing the production of volatile terpenoids known to be induced by T. urticae. An increase in JA levels was also found after Spm treatment, indicating that Spm induces the biosynthesis of JA, which has been shown elsewhere to regulate the production of some volatile terpenoids. Further, treatment with JA and Spm together resulted in greater volatile emission than that with JA alone. In a Y-tube olfactometer, leaves treated with Spm + JA attracted more predatory mites (Phytoseiulus persimilis) than those treated with JA alone. After treatment with Spm + JA, no effects were found on the enzyme activity of polyamine oxidase and copper amine oxidase. However, induction of calcium influx and ROS production, and increased enzyme activities and gene expression for NADPH oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase were found after treatment with Spm + JA. These results indicate that Spm plays an important role in the production of T. urticae-induced lima bean leaf volatiles.

Herbivore-induced maize volatiles serve as priming cues for resistance against post-attack by the specialist armyworm Mythimna separata

Abstract Plants show a diversity of defense strategies against a wide range of herbivores. The emission of a specific blend of volatiles in response to herbivory (HIPVs – herbivore-induced plant volatiles) plays a great ecological role in indirect protection of the plants by attracting natural enemies of herbivores and priming their neighboring plants that can cause poorer foraging habitats on the ‘receiver’ plants. Maize plants showed induction of a blend of volatiles including monoterpenes, sesequiterpenes, homoterpenes, green leaf volatiles, and indole, in response to the specialist herbivore Mythimna separata. Conspecific plants placed downwind of infested maize plants showed reduced larval development after exposure to HIPVs, compared to those exposed to volatiles from uninfested plants. These findings indicate that exposure of maize plants to HIPVs primed these plants for enhanced defense responses to specialist herbivores of Poaceae.

Biochemical and Spectroscopic Properties of Cyanide-Insensitive Quinol Oxidase from Gluconobacter oxydans

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.

Temperature‐dependent, behavioural, and transcriptional variability of a tritrophic interaction consisting of bean, herbivorous mite, and predator

Different organisms compensate for, and adapt to, environmental changes in different ways. In this way, environmental changes affect animal–plant interactions. In this study, we assessed the effect of temperature on a tritrophic system of the lima bean, the herbivorous spider mite Tetranychus urticae and the predatory mite Phytoseiulus persimilis. In this system, the plant defends itself against T. urticae by emitting volatiles that attract P. persimilis. Over 20–40 °C, the emission of volatiles by infested plants and the subsequent attraction of P. persimilis peaked at 30 °C, but the number of eggs laid by T. urticae adults and the number of eggs consumed by P. persimilis peaked at 35 °C. This indicates that the spider mites and predatory mites performed best at a higher temperature than that at which most volatile attractants were produced. Our data from transcriptome pyrosequencing of the mites found that P. persimilis up‐regulated gene families for heat shock proteins (HSPs) and ubiquitin‐associated proteins, whereas T. urticae did not. RNA interference‐mediated gene suppression in P. persimilis revealed differences in temperature responses. Predation on T. urticae eggs by P. persimilis that had been fed PpHsp70‐1 dsRNA was low at 35 °C but not at 25 °C when PpHsp70‐1 expression was very high. Overall, our molecular and behavioural approaches revealed that the mode and tolerance of lima bean, T. urticae and P. persimilis are distinctly affected by temperature variability, thereby making their tritrophic interactions temperature dependent.

Polyamines and jasmonic acid induce plasma membrane potential variations in Lima bean.

Exogenous polyamines (PAs) [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces of the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone.5 JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex. 5 Here, we report that a plasma membrane potential (Vm) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter Vm but the perfusion of JA and the polyamines significantly increased Cad and Put Vm depolarization. When JA was perfused with polyamines, a negative correlation was found between Vm depolarization and the number of amino group of the polyamines tested.

Exposing Arabidopsis seedlings to borneol and bornyl acetate affects root growth: Specificity due to the chemical and optical structures of the compounds

Abstract Root growth of Arabidopsis seedlings on the surface of agar plates was measured after the seedlings were exposed to volatile organic compounds. Similar to the roots of unexposed seedlings, the roots of seedlings exposed to volatile methanol (control) grew straight down. On the other hand, seedlings exposed to volatile bornyl acetate produced wavy roots. Interestingly, the wavy roots from seedlings exposed to (+)-bornyl acetate were significantly longer than those from seedlings exposed to (−)-bornyl acetate. Exposure to either (+)- or (−)-borneol resulted in thick root tips and reduced root growth. The roots from seedlings treated with (+)-borneol were significantly longer than those from seedlings exposed to (−)-borneol. The interactions between root length and the concentrations of (+)- or (−)-borneol were significantly different, showing that the Arabidopsis seedlings specifically responded to the molecular configuration of the borneol.

Future prospects of GM plant-based plant–plant communications

Abstract Volatile organic compounds (VOCs) emitted from plants function as airborne signals for communications between plants. We discuss possible uses of plant–plant communication systems using genetically modified plants emitting VOCs for pest control.