The role of jasmonic acid and ethylene crosstalk in direct defense of Nicotiana attenuata plants against chewing herbivores

Abstract

We examined performance of herbivores on plants lacking either jasmonate (JA, asLOX3) or ethylene (ET, mETR1) signaling or both (mETR1asLOX3). Plant defenses against Manduca sexta caterpillars were strongly impaired in JA-deficient asLOX3 plants; however, making asLOX3 plants ethylene insensitive did not further increase the performance of the larvae on a mETR1asLOX3 genetic cross. This result demonstrates the dominant role of JA over ET in the regulation of plant defenses against herbivores. However, ET-insensitivity combined with otherwise normal levels of JA in mETR1 plants promoted faster caterpillar growth, which correlated with reduced accumulation of the alkaloidal direct defense nicotine in mETR1 compared to WT plants. Our data points to an important accessory function of ET in the activation of JA-regulated plant defenses against herbivores at the level of alkaloid biosynthesis in the roots and/or accumulation in the leaves.

Jasmonate and Ethylene Signaling in Defense Against Herbivores

Jasmonic acid (JA) is an important plant defense signal mediating resistance to herbivores.^1–4 Silencing LOX3, a rate-limiting enzyme in JA biosynthesis in Nicotiana attenuata, inhibits resistance against Manduca sexta and other herbivores by decreasing the production of defensive metabolites (e.g., nicotine and TPIs; direct defenses) and volatile organic compounds (VOCs; indirect defenses) in the glasshouse and in the natural environment.^3,4 While the release of ethylene (ET) during herbivory has been shown in several species,⁵ the direct role of ET and its crosstalk with JA in defense against herbivores remains ambiguous. In maize, the inhibition of ET biosynthesis or perception increased herbivore performance and damage in treated plants;⁶ however, in experiments using ET-insensitive plants (etr1) in Arabidopsis, insect performance on these plants was unexpectedly diminished,⁷ suggesting that the role and outcome of ET in defense may depend on the plant-herbivore system being studied.

Regulation of Local Growth by JA and ET

In Onkokesung et al.⁸ we described a new genetic cross (mETR1asLOX3) that was created by crossing a single hormone-manipulated, JA-deficient (asLOX3 - ♂) plants with ethylene insensitive (mETR1 - ♀) plants. The plants were used to examine local responses of defense signaling-impaired plants to wounding and simulated herbivory treatments; while mETR1asLOX3 cross responded to wounding with abnormal ectopic cell expansion around wounds on the leaves, the single hormone-deficient plants and wild-type (WT) plants showed no cell expansion in wounded area. The cell expansion phenotype around wounds in mETR1asLOX3 plants was associated with higher levels of auxin (growth hormone) and increased transcript levels of cell wall loosening enzymes (e.g., expansin) in the leaves of mETR1asLOX plants.⁸ While the local restriction of plant growth by JA and ET suggested a response of plants leading to conservation of resources after herbivore attack, it remained unknown how JA and ET crosstalk modulated direct defense responses in N. attenuata. To this end, we used wild-type, asLOX3, mETR1 and mETR1asLOX3 plants and examined defense capacity of these genotypes by directly placing neonates of chewing specialist herbivore M. sexta on the leaves.

Role of JA and ET in Direct Defense against M. sexta Caterpillars

Freshly hatched M. sexta neonates were placed on rosette stage WT, mETR1, asLOX3 and mETR1asLOX3 plants and caterpillar mass was determined after 4, 6, 8 and 10 d. Caterpillars that fed on mETR1, asLOX3 and mETR1asLOX3 plants gained more weight than those feeding on WT plants (Fig. 1A), suggesting that both ET and JA significantly contributed to resistance against herbivores. At all measured time points, the average mass of caterpillars that fed on asLOX3 was equivalent to the caterpillar masses on mETR1asLOX3 plants. However, the mass of caterpillars that fed on mETR1 plants was intermediate between the mass of caterpillars that fed on asLOX3 and WT plants. This result suggests that while ET alone contributes to defense of N. attenuata plants, JA is a key factor that determines the total strength of defense in the mETR1asLOX3 cross plants, regardless of ET action.

Figure 1.

. M. sexta caterpillar performance and defense metabolite acumulation in JA and ET signaling-deficient plants. (A) Mean (± SE) mass of M. sexta caterpillars that fed on WT, mETR1, asLOX3 and mETR1asLOX3 plants for 4, 6, 8 and 10 d. Freshly hatched neonates were placed on the leaves and caterpillars were allowed to feed on the plants. After weighings, the caterpillars were placed on a new previously un-damaged leaf of the same plant. (B) Mean (±) relative transcript abundances of trypsin proteinase inhibitor (TPI) gene during simulated herbivory in N. attenuata. A fully expanded rosette leaf was wounded with a pattern wheel (W) and diluted M. sexta oral secretions (OS) were applied to the wounds (W + OS). Transcript abundances were normalized against housekeeping elongation factor 1-alpha (EF1α) gene expression. (C) Mean (±) nicotine contents determined at 0, 20 and 72 h after induction of the leaves with W + OS. Nicotine content in methanolic tissue extracts was determined by reverse phase (RP)-HPLC coupled to Agilent 1100 Series diode-array detector and UV absorbance at 254 nm against external nicotine calibration curve.

Accumulation of Direct Defense Metabolites in Hormone-Deficient Plants

To examine possible cause behind differential performance of caterpillars on transformed plants, we determined the levels of two direct defense metabolites in the plants after treating them with larval oral secretions (OS) to simulate herbivory. The accumulation of transcripts belonging to trypsin proteinase inhibitors (TPIs) that function as a strong anti-herbivore defenses in N. attenuata plants,⁹ was examined before and 6, 20 and 60 h after simulated herbivory. Relative to WT plants, the levels of TPI transcripts were strongly reduced in asLOX3 and mETR1asLOX3 plants but not in mETR1 plants (Fig. 1B). This result suggested that ET alone or in combination with JA does not interfere with the accumulation of TPI transcripts and defense. In contrast, the accumulation pattern of nicotine determined at 0, 20 and 72 h after challenging the leaves with simulated herbivory suggested a direct role of both JA and ET in alkaloid accumulation in the leaves (Fig. 1C). At 3 d after elicitation, the leaves of mETR1, asLOX3 and mETR1asLOX3 plants contained significantly lower levels of induced nicotine compared to WT plants. In our experiments, the constitutive levels of nicotine were not influenced by hormone deficiency, which is in contrast to previously found lower constitutive levels of nicotine in mETR1 plants.¹⁰

Multilevel Control of Defense against Herbivores

Our results demonstrated that a majority of defense responses against chewing herbivores in N. attenuata are directly subordinate to JA regulation, which strongly dominates the induction of defense response. However, a subset of JA-regulated defenses seems to be controlled and/or require ET for their full elicitation, which is demonstrated by clearly lower nicotine levels in ET-insensitive plants (Fig. 1C). It should be noted that mETR1 plants possess otherwise normal levels of induced JA.⁸ This double regulation mechanism (Fig. 2) may not be essential for defense of N. attenuata against chewing herbivore M. sexta caterpillars (Fig. 1A). However, it may have important role in defense against herbivores from other feeding guilds (e.g., piercing-sucking insects), which elicit a different profile of hormonal responses in plants. Indeed, a crosstalk between phytohormones has been proposed to tune plant defenses against herbivores with variable feeding strategies, increasing plants' chances of surviving the attack.^11–15

Figure 2.

Schematic representation of JA and ET function in herbivore-attacked tobacco plants. Herbivore attack, composed of wound signal and release of insect elicitors to the wounds, induces a strong burst of defense hormones, JA and ethylene. While both signals suppress local growth in attacked leaves, JA and ET are also required to trigger accumulation of root-produced nicotine alkaloid in local (attacked) and systemic (undamaged) tobacco leaves. In contrast, trypsin proteinase inhibitor (TPI) expression and protein activity in the leaves requires only JA signaling pathway that is independent of ET.

Future Perspectives

Previously, ETHYLENE RESPONSE FACTOR1 (ERF1) and OCTANO-DECANOID RESPONSIVE ARABIDOPSIS AP2/ERF 59 (ORA59) have been implicated in integrating JA and ET signals in plant-pathogen interactions.^16–18 In addition, the jasmonate signaling-associated MYC2 transcription factor is required for efficient JA-ET crosstalk in plants attacked by pathogens.¹⁹ Therefore, detailed analysis of genes such as ERF1, ORA59 or MYC2, together with discovery of novel regulators of hormonal crosstalk is required before we can fully understand the role of hormones and their crosstalk in plant defense against herbivores, pathogens and ubiquitously present abiotic stress factors.

Addendum to: Onkokesung N, Galis I, von Dahl CC, Matsuoka K, Saluz HP, Baldwind IT. Jasmonic acid and ethylene modulate local responses to wounding and simulated herbivory in Nicotiana attenuata leaves. Plant Physiol. 2010;153:785–798. doi: 10.1104/pp.110.156232.