Effects of feeding Spodoptera littoralis on lima bean leaves: IV. Diurnal and nocturnal damage differentially initiate plant volatile emission

Abstract

Continuous mechanical damage initiates the rhythmic emission of volatiles in lima bean (Phaseolus lunatus) leaves; the emission resembles that induced by herbivore damage. The effect of diurnal versus nocturnal damage on the initiation of plant defense responses was investigated using MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximal levels of beta-ocimene and (Z)-3-hexenyl acetate in the late photophase. Leaves damaged during the dark phase responded with the nocturnal emission of (Z)-3-hexenyl acetate, but with only low amounts of beta-ocimene; this emission was followed by an emission burst directly after the onset of light. In the presence of (13)CO(2), this light-dependent synthesis of beta-ocimene resulted in incorporation of 75% to 85% of (13)C, demonstrating that biosynthesis of beta-ocimene is almost exclusively fueled by the photosynthetic fixation of CO(2) along the plastidial 2-C-methyl-D-erythritol 4-P pathway. Jasmonic acid (JA) accumulated locally in direct response to the damage and led to immediate up-regulation of the P. lunatus beta-ocimene synthase gene (PlOS) independent of the phase, that is, light or dark. Nocturnal damage caused significantly higher concentrations of JA (approximately 2-3 times) along with enhanced expression levels of PlOS. Transgenic Arabidopsis thaliana transformed with PlOS promoter :: beta-glucuronidase fusion constructs confirmed expression of the enzyme at the wounded sites. In summary, damage-dependent JA levels directly control the expression level of PlOS, regardless of light or dark conditions, and photosynthesis is the major source for the early precursors of the 2-C-methyl-D-erythritol 4-P pathway.

Figure 1.

Gas chromatographic separation of the β-ocimene isomers formed by the recombinant PlOS enzyme with GDP as substrate.

Figure 2.

Emission of the volatiles β-ocimene and Hex-Ac and transcript levels of PlOS in damaged lima bean leaves. Damage conditions: larvae of S. littoralis under a 14:10 LD cycle (A) and under a LD cycle set with 4 h of additional darkness (AD) during the second (25–29 h) and third (49–53 h) photoperiods (B). Time of day is shown across the top x axis, and total elapsed time is shown on the bottom x axis. Headspace analyses were performed in triplicate. Emission is expressed as ng min⁻¹ g⁻¹ FW. Data regarding to the transcript levels represent the mean ± se (n = 4–5).

Figure 3.

Emissions of β-ocimene and Hex-Ac (A and B) and levels of JA and PlOS transcripts (C and D) in MecWorm-treated leaves. Damage program: 6 h during either the photophase (A and C) or the dark phase (B and D) according to a 14:10 LD cycle. Damage periods started 2 h after the onset of the light or dark phase with punching every 5 s for 6 h (9 am–3 pm during the photophase and from 11 pm–5 am during the night). Headspace analyses were performed in triplicate. Emission is expressed as ng min⁻¹ g⁻¹ FW. JA and transcript levels represent the mean ± se (n = 3–4).

Figure 4.

Pulse labeling of (E)-β-ocimene after administration of ¹³CO₂. Ambient air in the cabinet was exchanged by synthetic air containing ¹³CO₂ gas (380 μg mL⁻¹) 0.5 h before the onset of the light phase. Headspace analysis was started at its onset. The shaded box corresponds to the presence of ¹³CO₂. One hour after the onset of the photophase, the cabinet was purged with ambient air to remove ¹³CO₂. Collection periods for volatiles are indicated. Mass spectra of β-ocimene from leaves in ambient air or ¹³CO₂-containing air are shown on top. Data represent the mean ± se (n = 4).

Figure 5.

Effect of herbivory, wounding, and chemical treatment on the expression level of PlOS in lima bean leaves. Chemical treatments: JA (0.5 mm), salicylic acid (SA; 0.5 mm), and alamethicin (ALA; 1 mm). Means with small letters are significantly different according to ANOVA followed by Scheffe's test (P < 0.05). Data represent the mean ± se (n = 4).

Figure 6.

Localization of PlOS transcript and accumulation of JA in mechanically damaged lima bean leaves. A, Leaf damaged for 6 h by MecWorm during the first and second dark phases. See Figure 3D for details. Leaf segments were harvested 32 h after initiation of the first MecWorm damage. Sections 2 and 3 correspond to the first and the second nocturnal damage. Undamaged sections 1 and 4 served as control. Data represent the mean ± se (n = 3–4). Scale bar = 10 mm. Means followed with small letters are significantly different according to ANOVA followed by Scheffe's test (P < 0.05). B and C, Wound-induced expression of PlOS promoter∷GUS reporter gene constructs in leaves of 6-week-old Arabidopsis plants. Wounding by a needle (B) or MecWorm treatment (C) induced GUS activity after 2 h in areas in close proximity to wounding sites. Histochemical GUS assays were performed with two independent lines. Scale bar = 2 mm.

Figure 7.

Schematic representation of the signaling and metabolic pathways required for herbivore-induced β-ocimene and Hex-Ac emissions in lima bean leaves in a daily cycle. LOX, Lipoxygenase.