Catherine A. Preston

Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore.

Abstract. Herbivory induces both direct and indirect defenses in plants; however, some combinations of these defenses may not be compatible. The jasmonate signal cascade activated both direct (nicotine accumulations) and indirect (mono- and sesquiterpene emissions) whole-plant defense responses in the native tobacco Nicotiana attenuata Torr. Ex Wats. Nicotine accumulations were proportional to the amount of leaf wounding and the resulting increases in jasmonic acid (JA) concentrations. However, when larvae of the nicotine-tolerant herbivore, Manduca sexta, fed on plants or their oral secretions were applied to leaf punctures, the normal wound response was dramatically altered, as evidenced by large (4- to 10-fold) increases in the release of (i) volatile terpenoids and (ii) ethylene, (iii) increased (4- to 30-fold) accumulations of endogenous JA pools, but (iv) decreased or unchanged nicotine accumulations. The ethylene release, which was insensitive to inhibitors of induced JA accumulation, was sufficient to account for the attenuated nicotine response. Applications of ethylene and ethephon suppressed the induced nicotine response and pre-treatment of plants with a competitive inhibitor of ethylene receptors, 1-methylcyclopropene, restored the full nicotine response. This ethylene burst, however, did not inhibit the release of volatile terpenoids. Because parasitoids of Manduca larvae are sensitive to the dietary intake of nicotine by their hosts, this ethylene-mediated switching from direct to a putative indirect defense may represent an adaptive tailoring of a plants defense response.

The eco-physiological complexity of plant responses to insect herbivores

Plants must fend off a myriad of aggressive biotic agents and this biotic onslaught occurs across the complete spectrum of spatial scales from pathogens that attack single cells (Somssich and Hahlbrock 1998) to herbi vores that select their hosts by characteristics of the community in which a plant grows (Hay 1986). The recent advances in understanding plant defense respons es that function on the cellular level against pathogens have been awe-inspiring, particularly with regard to the mechanisms of pathogen recognition (Baker et al. 1997). In contrast, our understanding of how plants recognize attack from herbivores is in its infancy; the first herbivore-specific elicitor (volicitin) was only just re cently characterized (Alborn et al. 1997). While the mechanisms of many plant-herbivore interactions are not well understood, the ecological complexity of plant responses against herbivores is well recognized, as the defense response elicited by volicitin underscores. Vol icitin [N-(17-hydroxylinolenoyl)-L-glutamine], isolated from the oral secretions of beet armyworm (Spodoptera exigua) larvae, increases the emission of volatile organic compounds when applied to corn (Alborn et al. 1997). How these volatile emissions function as a defense highlights a key difference between plant defenses against pathogens and herbivores: the physiological autonomy of herbivores, which allow plants to utilize components of their ecological community for defense.

Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants

Abstract. Jasmonic acid (JA) and salicylic acid (SA) have both been implicated as important signal molecules mediating induced defenses of Nicotiana tabacum L. against herbivores and pathogens. Since the application of SA to a wound site can inhibit both wound-induced JA and a defense response that it elicits, namely nicotine production, we determined if tobacco mosaic virus (TMV) inoculation, with its associated endogenous systemic increase in SA, reduces a plants ability to increase JA and nicotine levels in response to mechanical damage, and evaluated the consequences of these interactions for the amount of tissue removed by a nicotine-tolerant herbivore, Manduca sexta. Additionally, we determined whether the release of volatile methyl salicylic acid (MeSA) from inoculated plants can reduce wound-induced JA and nicotine responses in uninoculated plants sharing the same chamber. The TMV-inoculated plants, though capable of inducing nicotine normally in response to methyl jasmonate applications, had attenuated wound-induced JA and nicotine responses. Moreover, larvae consumed 1.7- to 2.7-times more leaf tissue from TMV-inoculated plants than from mock-inoculated plants. Uninoculated plants growing in chambers downwind of either TMV-inoculated plants or vials releasing MeSA at 83- to 643-times the amount TMV-inoculated plants release, exhibited normal wound-induced responses. We conclude that tobacco plants, when inoculated with TMV, are unable to elicit normal wound responses, due likely to the inhibition of JA production by the systemic increase in SA induced by virus-inoculation. The release of volatile MeSA from inoculated plants is not sufficient to influence the wound-induced responses of neighboring plants.

Eating the evidence? Manduca sexta larvae can not disrupt specific jasmonate induction in Nicotiana attenuata by rapid consumption

Abstract. As feeding by the tobacco specialist Manduca sexta L. is known to result in significantly higher jasmonate (JA) concentrations in Nicotiana plants than do mechanical simulations of the larval damage, we investigated whether M. sexta, which is known to rapidly consume large quantities of leaf material, can impair this “recognition” response by consuming the leaf tissue before it can respond with amplified JA levels. We report that oral secretions (OS) from M. sexta, but not from the cabbage specialist Pieris rapae, amplified the wound-induced JA response of Nicotiana attenuata Torr. Ex Wats., regardless of larval diet, instar and molting stage, and were still active after boiling and when diluted to 1/1000. The largest JA response occurred within 40 min in tissues adjacent to the OS application site. When 3 mm of leaf tissue immediately adjacent to the OS application site was excised within 40 s, the signal that elicits JA amplification was found to travel rapidly into the leaf, beyond the mandibular reach of the larvae. We conclude that M. sexta is not able to consume the evidence of feeding activity.

POSITIVE AND NEGATIVE SIGNALS REGULATE GERMINATION IN THE POST-FIRE ANNUAL, NICOTIANA ATTENUATA

The litter of many plant species is known to inhibit germination, and this phenomenon is commonly interpreted as allelopathic inhibition of one species by another. However, an alternative interpretation is that seeds may be using environmental signals to inform the timing of their germination and thereby use dormancy as a mechanism of habitat choice. Nicotiana attenuata Torr. ex Wats. (Solanaceae) is typically found for less than three years after fire in the sagebrush, blackbrush, and pinyon–juniper forests of the Great Basin desert of North America. A dormant seed bank is established during this ephemeral post-fire period, and pyrolysis products of α-cellulose (containing only C, H, and O) in wood smoke are known to initiate germination in dormant seeds of this species. We demonstrated in a glasshouse experiment that germination into burned soils (as compared to unburned) results in a 12-fold increase in lifetime seed production, which reflects a minimum estimate of the fitness benefit of making accurat...

Patterns and consequences of benzyl acetone floral emissions from Nicotiana attenuata plants

The emission of a single compound, benzyl acetone (BA, 4-phenyl-2-butanone), is barely detectable during the day in the headspace of flowers of the self-compatible disturbance species Nicotiana attenuata, but it increases dramatically (50×) in the evening, becoming the dominant component in the floral headspace. This striking temporal pattern of emission may be sculpted by its potential ecological roles (e.g., synomonal and kairomonal), which we examine here. We measured the nightly BA emissions from individual flowers at six different branch positions on plants receiving either self-pollen or pollen from another genotype and calculated the nightly whole-plant emission. The first flowers produced on a branch have a lower rate of emission than flowers produced later on the same branch; however, cross pollination did not influence the quantity of BA emitted from subsequently produced flowers. Informed by these measures of whole-plant emission, we constructed a device that released BA at a constant rate equivalent to that of a plant with 240 open flowers (an approximate 10× increase in emissions). This device and a control device were attached to 50 matched pairs of plants growing in a native population in Utah to estimate the fitness consequences of enhanced, constant BA emission. Plants with elevated BA emissions in the field were browsed more frequently than control plants and produced fewer capsules, so that lifetime seed production was reduced by 3.1%. However, both treatment and control plants were heavily attacked by negro bugs (Cormelina spp.) and produced light seeds with low viabilities, representing 47% and 23% of the mass per seed and viability, respectively, of unmanipulated plants, which flowered two weeks later in the same population. From glasshouse experiments, we estimated the consequences of out-crossing and attack by negro bugs on seed production. Out-crossing did not significantly affect seed production, seed mass or viability. In contrast, negro bug infestation dramatically decreased seed mass and viability. We conclude that while the phenological variation in attack rates might have obscured our ability to estimate the fitness consequences of enhanced BA emission, the effects are likely to be dominated by kairomonal rather than synomonal interactions for this self-compatible species.

Methyl jasmonate as an allelopathic agent: Sagebrush inhibits germination of a neighboring tobacco, Nicotiana attenuata

Artemisia tridentata ssp. tridentata is the dominant and defining shrub in the Great Basin Desert, with well-documented allelopathic tendencies that have generally been ascribed to its most abundantly released secondary metabolites. However, as a minor component, sagebrush releases a highly biologically active substance, methyl jasmonate (MeJA), which is known to function as both a germination inhibitor and promoter in laboratory studies. Nicotiana attenuata is a tobacco species native to the Great Basin Desert and grows in newly burned juniper–sagebrush habitats for 2–3 yr following a fire. With a combination of field and laboratory studies, we examined the role of MeJA release from sagebrush by both air and water transport in inhibiting N. attenuata seed germination. We demonstrated that sagebrush interacts allelopathically with the seed bank of N. attenuata through its release of MeJA. In the field, seeds buried 0–40 cm from sagebrush plants for 4 months in net bags had significantly reduced germination compared to seeds buried similarly but protected in plastic bags. Moreover, germination on soils collected from underneath sagebrush plants was reduced by 60% compared to seeds placed on soils collected between sagebrush plants or outside of the sagebrush population. Exposure to A. tridentata seeds and seedlings did not affect N. attenuata germination, suggesting that established sagebrush plants only influence the tobaccos seed bank. In the laboratory, exposure of seeds to sagebrush emissions resulted in germination delays of up to 6 d. Exposure to volatile and aqueous MeJA also inhibited germination of N. attenuata seeds at quantities that are released naturally by sagebrush: 3.5 μg/hr and 1.12 μg/seed cup (56 ng/seed), respectively. A. tridentata seeds were significantly more resistant to MeJA, being inhibited at 336 μg MeJA (16.8 μg/seed), 300 times greater than the level of aqueous MeJA required to inhibit N. attenuata seeds. MeJA inhibited N. attenuata germination regardless of the seeds dormancy state and the specific epimer (trans- or cis-) of MeJA. Germination on sagebrush chaff that had been heated to reduce MeJA content was negatively correlated with the amount of MeJA remaining in the chaff. Germination of a nondormant, conspecific tobacco, N. trigonophylla, which grows in the same area but is not associated with fire, is less sensitive than N. attenuata to the extracts of sagebrush litter, but similarly sensitive to MeJA. Additionally, four of five other tobacco species that are not known to be associated with sagebrush are less sensitive to MeJA, suggesting an evolved sensitivity to MeJA. To determine the proportion of germination inhibition of a sagebrush extract that could be attributed to MeJA, we serially diluted sagebrush extracts with water and restored the quantity of MeJA of the original extract by adding appropriate quantities of synthetic MeJA; 16–60% of the inhibitory activity of the original extract could be attributed to the MeJA. We conclude that MeJA release from sagebrush plays an allelopathic role for N. attenuata seed banks, but other unidentified compounds are also involved.

Plant–Plant Signaling: Application of trans- or cis-Methyl Jasmonate Equivalent to Sagebrush Releases Does Not Elicit Direct Defenses in Native Tobacco

Nicotiana attenuata plants growing in close proximity to damaged sagebrush (Artemisia tridentata ssp. tridentata) suffer less herbivory than plants near undamaged sagebrush. Sagebrush constitutively releases methyl jasmonate (MeJA), a compound that when applied directly to N. attenuata, elicits herbivore resistance and the direct defense traits [protease inhibitors (PIs), nicotine]. Damage increases the release of volatile MeJA, primarily in the cis epimer, suggesting that cis-MeJA may mediate this apparent interplant signaling. We characterized sagebrushs MeJA plume before and after damage in nature and in the laboratory, and compared the activity of trans- and cis-MeJA in inducing PIs, nicotine, and Manduca sexta resistance in N. attenuata. We used both lanolin applications and aqueous sprays that mimic natural exposures, and we determined the amount of volatilized MeJA required to elicit a nicotine response in open-grown plants. Wounding rapidly and transiently increased cis-MeJA emissions from damaged parts (but not systemically), and the released plume did not rapidly dissipate in nature. cis-MeJA was not consistently more active than trans-MeJA, and the order of exposure (trans- then cis-) did not influence activity. We conclude that volatile MeJA, either trans- or cis-, when applied at levels consistent with those released by sagebrush does not elicit direct defenses in N. attenuata.

Fast track to the trichome : induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda

Abstract. Nicotiana repanda Wildenow ex Lehmann acylates nornicotine in its trichomes to produce N-acyl-nornicotine (NacNN) alkaloids which are dramatically more toxic than nicotine is to the nicotine-adapted herbivore, Manduca sexta. These NacNNs, like nicotine, were induced by methyl jasmonate (MeJA) and wounding, but the 2-fold increase in NacNN pools was much faster (within 6 h) than the MeJA-induced increase in nornicotine pools (24 h to 4 d), its parent substrate. When 15NO3− pulse-chase experiments with intact and induced plants were used to follow the incorporation of 15N into alkaloids in different plant parts over the plants lifetime, it was found that the root nicotine pool was most rapidly labeled, followed by the shoot nornicotine and NacNN pools. After 3 d, 3.12% of 15N acquired was in nicotine (0.93%), nornicotine (0.32%) and NacNNs (1.73%) while only 0.14% was in anabasine. Once NacNNs are externalized to the leaf surface, they are not readily re-distributed within the plant and are lost with senescing leaves. The wound- and MeJA-induced N-acylation of nornicotine is independent of induced changes in nornicotine pools and the rapidity of the response suggests its importance in defense against herbivores.

ISSR and AFLP analysis of the temporal and spatial population structure of the post-fire annual, Nicotiana attenuata , in SW Utah

BackgroundThe native annual tobacco, Nicotiana attenuata, is found primarily in large ephemeral populations (typically for less than 3 growing seasons) after fires in sagebrush and pinyon-juniper ecosystems and in small persistent populations (for many growing seasons) in isolated washes typically along roadsides throughout the Great Basin Desert of the SW USA. This distribution pattern is due to its unusual germination behavior. Ephemeral populations are produced by the germination of dormant seeds from long-lived seed banks which are stimulated to germinate by a combination of unidentified positive cues found in wood smoke and the removal of inhibitors leached from the unburned litter of the dominant vegetation. Persistent populations may result where these inhibitors do not exist, as in washes or along disturbed roadsides. To determine if this germination behavior has influenced population structure, we conducted an AFLP (244 individuals), ISSR (175 individuals) and ISSR+ AFLP (175 individuals) analysis on plants originating from seed collected from populations growing in 11 wash and burns over 11 years from the SW USA.ResultsGenetic variance as measured by both ISSR and AFLP markers was low among sites and comparatively higher within populations. Cluster analysis of the Utah samples with samples collected from Arizona, California, and Oregon as out-groups also did not reveal patterns. AMOVA analysis of the combined AFLP and ISSR data sets yielded significantly low genetic differentiation among sites (Φct), moderate among populations within sites (Φsc) and higher genetic differentiation within populations (Φst).ConclusionsWe conclude that the seed dormancy of this post-fire annual and its resulting age structure in conjunction with natural selection processes are responsible for significantly low among sites and comparatively high within-population genetic variation observed in this species.