Caroline C. von Dahl

Deciphering the Role of Ethylene in Plant–Herbivore Interactions

Most plants emit ethylene in response to herbivory by insects from many different feeding guilds. The elicitors of these ethylene emissions are thought to be microorganisms or oral secretion-specific compounds that are transferred when the attacking insect feeds. To find the receptors for these elicitors and describe the signaling cascades that are subsequently activated will be the challenge of future research. Past experiments on the function of herbivore-induced ethylene, which were biased toward the use of chemical treatments to manipulate ethylene, identified seven ethylene-dependent defense responses. In contrast, a genetic toolbox that consists of several mutants has rarely been used and to date, mutants have helped to identify only one additional ethylene-dependent defense response. Ethylene-dependent responses include the emission of specific volatile organic compounds as indirect defense, the accumulation of phenolic compounds, and proteinase inhibitor activity. Besides being ethylene regulated, these defenses depend strongly on the wound-hormone jasmonic acid (JA). That ethylene requires the concomitant induction of JA, or other signals, appears to be decisive. Rather than being the principal elicitor of defense responses, ethylene modulates the sensitivity to a second signal and its downstream responses. Given this modulator role, and the artifacts associated with the use of chemical treatments to manipulate ethylene production and perception, future advances in the study of ethylene’s function in plant–herbivore interactions will likely come from the use of signaling mutants or transgenic plants. It will be exciting to see if adaptive phenotypic plasticity is largely an ethylene-mediated response.

The extent to which methyl salicylate is required for signaling systemic acquired resistance is dependent on exposure to light after infection

Systemic acquired resistance (SAR) is a state of heightened defense to a broad spectrum of pathogens that is activated throughout a plant following local infection. Development of SAR requires the translocation of one or more mobile signals from the site of infection through the vascular system to distal (systemic) tissues. The first such signal identified was methyl salicylate (MeSA) in tobacco (Nicotiana tabacum). Subsequent studies demonstrated that MeSA also serves as a SAR signal in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum). By contrast, another study suggested that MeSA is not required for SAR in Arabidopsis and raised questions regarding its signaling role in tobacco. Differences in experimental design, including the developmental age of the plants, the light intensity, and/or the strain of bacterial pathogen, were proposed to explain these conflicting results. Here, we demonstrate that the length of light exposure that plants receive after the primary infection determines the extent to which MeSA is required for SAR signaling. When the primary infection occurred late in the day and as a result infected plants received very little light exposure before entering the night/dark period, MeSA and its metabolizing enzymes were essential for SAR development. In contrast, when infection was done in the morning followed by 3.5 h or more of exposure to light, SAR developed in the absence of MeSA. However, MeSA was generally required for optimal SAR development. In addition to resolving the conflicting results concerning MeSA and SAR, this study underscores the importance of environmental factors on the plant’s response to infection.

Interconnection between Methyl Salicylate and Lipid-Based Long-Distance Signaling during the Development of Systemic Acquired Resistance in Arabidopsis and Tobacco

Systemic acquired resistance (SAR) is a salicylic acid (SA)-dependent heightened state of resistance against a broad spectrum of pathogens activated in the uninoculated systemic tissue of a pathogen-infected plant. For systemic protection to be initiated, a mobile signal that is produced at the site

Jasmonic Acid and Ethylene Modulate Local Responses to Wounding and Simulated Herbivory in Nicotiana attenuata Leaves

Jasmonic acid (JA) and ethylene (ET) are known to play important roles in mediating plant defense against herbivores, but how they affect development in herbivore-attacked plants is unknown. We used JA-deficient (silenced in LIPOXYGENASE3 [asLOX3]) and ET-insensitive (expressing a mutated dominant negative form of ETHYLENE RESPONSE1 [mETR1]) Nicotiana attenuata plants, and their genetic cross (mETR1asLOX3), to examine growth and development of these plants under simulated herbivory conditions. At the whole plant level, both hormones suppressed leaf expansion after the plants had been wounded and the wounds had been immediately treated with Manduca sexta oral secretions (OS). In addition, ectopic cell expansion was observed around both water- and OS-treated wounds in mETR1asLOX3 leaves but not in mETR1, asLOX3, or wild-type leaves. Pretreating asLOX3 leaves with the ET receptor antagonist 1-methylcyclopropane resulted in local cell expansion that closely mimicked the mETR1asLOX3 phenotype. We found higher auxin (indole-3-acetic acid) levels in the elicited leaves of mETR1asLOX3 plants, a trait that is putatively associated with enhanced cell expansion and leaf growth in this genotype. Transcript profiling of OS-elicited mETR1asLOX3 leaves revealed a preferential accumulation of transcripts known to function in cell wall remodeling, suggesting that both JA and ET act as negative regulators of these genes. We propose that in N. attenuata, JA-ET cross talk restrains local cell expansion and growth after herbivore attack, allowing more resources to be allocated to induced defenses against herbivores.

Sebacina vermifera promotes the growth and fitness of Nicotiana attenuata by inhibiting ethylene signaling.

Sebacina vermifera, a growth-promoting endophytic fungus, significantly increases Nicotiana attenuatas growth but impairs both its herbivore resistance and its accumulation of the costly, jasmonic acid (JA)-regulated defense protein, trypsin proteinase inhibitor (TPI). To determine if the fungis growth-promoting effects can be attributed to lower TPI-related defense costs, we inoculated transformed N. attenuata plants silenced in their ability to synthesize JA, JA-isoleucine, and TPI by antisense (lipoxygenase 3 [as-lox3] and Thr deaminase [as-td]) and inverted repeat (ir-tpi) expression, and found that inoculation promoted plant growth as in untransformed wild-type plants. Moreover, herbivore-elicited increases in JA and JA-isoleucine concentrations did not differ between inoculated and uninoculated wild-type plants. However, inoculation significantly reduced the morphological effect of 1-aminocyclopropane-1-carboxylic acid on wild-type seedlings in a triple response assay, suggesting that ethylene signaling was impaired. Furthermore, S. vermifera failed to promote the growth of N. attenuata plants transformed to silence ethylene production (1-aminocyclopropane-1-carboxylic acid oxidase [ir-aco]). Inoculating wild-type plants with S. vermifera decreased the ethylene burst elicited by applying Manduca sexta oral secretions to mechanical wounds. Accordingly, oral secretion-elicited transcript levels of the ethylene synthesis genes NaACS3, NaACO1, and NaACO3 in inoculated plants were significantly lower compared to these levels in uninoculated wild-type plants. Inoculation accelerated germination in wild-type seeds; however, uninoculated wild-type seeds germinated as rapidly as inoculated seeds in the presence of the ethylene scrubber KMnO4. In contrast, neither inoculation nor KMnO4 exposure influenced the germination of ir-aco seeds. We conclude that S. vermifera increases plant growth by impairing ethylene production independently of JA signaling and TPI production.

Identification of multiple salicylic acid-binding proteins using two high throughput screens

Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays.

Pectin methylesterase NaPME1 contributes to the emission of methanol during insect herbivory and to the elicitation of defence responses in Nicotiana attenuata

Pectin methylesterases (PMEs) catalyse the demethylation of pectin within plant cell walls, releasing methanol (MeOH) in the process. Thus far, PMEs have been found to be involved in diverse processes such as plant growth and development and defence responses against pathogens. Herbivore attack increases PME expression and activity and MeOH emissions in several plant species. To gain further insights into the role of PMEs in defence responses against herbivores, the expression of a Manduca sexta oral secretion (OS)-inducible PME in Nicotiana attenuata (NaPME1) was silenced by RNA interference (RNAi)-mediated gene silencing. Silenced lines (ir-pme) showed 50% reduced PME activity in leaves and 70% reduced MeOH emissions after OS elicitation compared with the wild type (WT), demonstrating that the herbivore-induced MeOH emissions originate from the demethylation of pectin by PME. In the initial phase of the OS-induced jasmonic acid (JA) burst (first 30 min), ir-pme lines produced WT levels of this hormone and of jasmonyl-isoleucine (JA-Ile); however, these levels were significantly reduced in the later phase (60-120 min) of the burst. Similarly, suppressed levels of the salicylic acid (SA) burst induced by OS elicitation were observed in ir-pme lines even though wounded ir-pme leaves contained slightly increased amounts of SA. This genotype also presented reduced levels of OS-induced trypsin proteinase inhibitor activity in leaves and consistently increased M. sexta larvae performance compared with WT plants. These latter responses could not be recovered by application of exogenous MeOH. Together, these results indicated that PME contributes, probably indirectly by affecting cell wall properties, to the induction of anti-herbivore responses.

The combined use of photoaffinity labeling and surface plasmon resonance-based technology identifies multiple salicylic acid-binding proteins

Salicylic acid (SA) is a small phenolic molecule that not only is the active ingredient in the multi-functional drug aspirin, but also serves as a plant hormone that affects diverse processes during growth, development, responses to abiotic stresses and disease resistance. Although a number of SA-binding proteins (SABPs) have been identified, the underlying mechanisms of action of SA remain largely unknown. Efforts to identify additional SA targets, and thereby elucidate the complex SA signaling network in plants, have been hindered by the lack of effective approaches. Here, we report two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SABPs from Arabidopsis. Using these approaches, multiple proteins, including the E2 subunit of α-ketoglutarate dehydrogenase and the glutathione S-transferases GSTF2, GSTF8, GSTF10 and GSTF11, were identified as SABPs. Their association with SA was further substantiated by the ability of SA to inhibit their enzymatic activity. The photoaffinity labeling and surface plasmon resonance-based approaches appear to be more sensitive than the traditional approach for identifying plant SABPs using size-exclusion chromatography with radiolabeled SA, as these proteins exhibited little to no SA-binding activity in such an assay. The development of these approaches therefore complements conventional techniques and helps dissect the SA signaling network in plants, and may also help elucidate the mechanisms through which SA acts as a multi-functional drug in mammalian systems.

Use of silenced plants in allelopathy bioassays: a novel approach

Volatile phytohormones or other chemicals can affect processes in distal plant parts but may also influence neighboring plants, and thereby function allelopathically. While this hypothesis has been widely discussed, rigorous tests are lacking. Transgenic plants, silenced in the production of an emitted chemical, are ideal tools to test the hypothesis that the release of a chemical can negatively influence the growth of neighbors (allelopathy). We used isogenic wild type (WT) and genetically transformed plants that lacked the ability to produce ethylene (ir-aco), as both “emitters” and “receivers” of this volatile phytohormone in experiments where receiver plants were only exposed to the headspace of WT or ir-aco emitters, in order to evaluate if natural ethylene releases can function allelopathically. Root growth (a proxy of plant fitness) of WT receivers correlated negatively with the number of WT emitters and headspace ethylene concentrations. Reducing ethylene concentrations in the headspace with the ethylene scrubber, KMnO4, and using ir-aco seedlings as emitters restored root growth of WT receiver seedlings. 1-Aminocyclopropane-1-carboxylic acid (ethylene biosynthesis substrate) supplementation to WT but not ir-aco emitters inhibited root growth of ir-aco, but not WT receivers, suggesting increased sensitivity to exogenous ethylene of ir-aco seedlings. We conclude that plants genetically silenced in the production of a putative allelochemical are useful in determining if the emitted chemical functions allelopathically.