Nitrogen supply influences herbivore-induced direct and indirect defenses and transcriptional responses in Nicotiana attenuata

Abstract

Although nitrogen (N) availability is known to alter constitutive resistance against herbivores, its influence on herbivore-induced responses, including signaling pathways, transcriptional signatures, and the subsequently elicited chemical defenses is poorly understood. We used the native tobacco, Nicotiana attenuata, which germinates in the postfire environment and copes with large changes in soil N during postfire succession, to compare a suite of Manduca sexta- and elicitor-induced responses in plants grown under high- and low-N (LN) supply rates. LN supply decreased relative growth rates and biomass by 35% at 40 d compared to high-N plants; furthermore, it also attenuated (by 39 and 60%) the elicitor-induced jasmonate and salicylate bursts, two N-intensive direct defenses (nicotine and trypsin proteinase inhibitors, albeit by different mechanisms), and carbon-containing nonvolatile defenses (rutin, chlorogenic acid, and diterpene glycosides), but did not affect the induced release of volatiles (cis-alpha-bergamotene and germacrene A), which function as indirect defenses. M. sexta and methyl jasmonate-induced transcriptional responses measured with a microarray enriched in herbivore-induced genes were also substantially reduced in plants grown under LN supply rates. In M. sexta-attacked LN plants, only 36 (45%) up-regulated and 46 (58%) down-regulated genes showed the same regulation as those in attacked high-N plants. However, transcriptional responses frequently directly countered the observed metabolic changes. Changes in a leaf's sensitivity to elicitation, an attacked leaf's waning ability to export oxylipin wound signals, and/or resource limitations in LN plants can account for the observed results, underscoring the conclusion that defense activation is a resource-intensive response.

Figure 1.

Mean (±se) WP fresh mass (lines) and accumulated nitrogen (N) (bars) per plant, and relative growth rate (RGR: inset) of N. attenuata plants grown under low nitrogen (LN) and high nitrogen (HN) supply rates. Asterisks indicate significant differences between members of a pair (P < 0.05, t test).

Figure 2.

Mean (±se) jasmonic acid (JA) and salicylic acid (SA, insets) concentrations in leaves of 40-d-old N. attenuata plants grown under high nitrogen (HN) and low nitrogen (LN) supply rates. Leaves were wounded with a fabric pattern wheel and the resulting puncture wounds immediately treated with either 20 μL of M. sexta oral secretions (OS) or deionized water (W) at time 0. Asterisks indicate significant differences between members of a pair (P < 0.05, t test).

Figure 3.

Mean (±se) nicotine (A, μg/g fresh mass) and trypsin proteinase inhibitor activity (B, TrypPI) from 35-d-old N. attenuata plants grown under low nitrogen (LN) and high nitrogen (HN) supply rates. Leaves growing at nodes 0 (systemic) and 2 (local) were analyzed 3 d after leaves at nodes 2 and 3 were either treated with 20 μL of lanolin containing 150 μg MeJA (MeJA), in 20 μL of pure lanolin (LC), or wounded and treated with 40 μL of M. sexta oral secretions (OS), 40 μL of deionized water (W), or were left unwounded and untreated (C). Asterisks indicate significant differences between members of a treatment and control pair (LC versus MeJA; OS versus W; P < 0.05, t test).

Figure 4.

Mean (±se) caffeoylputrescine (A), chlorogenic acid (B), rutin (C) and diterpene glycosides (DTGs) from 35-d-old N. attenuata plants grown under low nitrogen (LN) and high nitrogen (HN) supply rates. Leaves growing at nodes 0 (systemic) and 2 (local) were analyzed 3 d after leaves at nodes 2 and 3 were either treated with 20 μL of lanolin containing 150 μg MeJA (MeJA) in 20 μL of pure lanolin (LC), or wounded and treated with 40 μL of M. sexta oral secretions (OS), 40 μL of deionized water (W), or left unwounded and untreated (C). Asterisks indicate significant differences between members of a treatment and control pair (LC versus MeJA; OS versus W; P < 0.05, t test).

Figure 5.

Mean (±se) peak areas of limonene (1); cis-α-bergamotene (2); germacrene A (3) (percent, relative to internal standard [IS]) in the headspace of 36-d-old N. attenuata plants grown under low nitrogen (LN) and high nitrogen (HN) supply rates. Headspace samples were collected for 7 h, starting 24 h after treatment with 20 μL of lanolin containing 150 μg MeJA (MeJA), in 20 μL of pure lanolin (LC), or after wounding and treatment with 40 μL of M. sexta oral secretions (OS) or with 40 μL of deionized water (W) to leaves at nodes 2 and 3. For each compound, letters indicate significant differences among treatments (P < 0.05, Fisher post hoc test).

Figure 6.

PCA (top) and number of up-regulated (↑), down-regulated (↓) or not-regulated ( ) genes (bottom) observed in a microarray analysis of 40-d-old N. attenuata plants grown under low nitrogen (LN) and high nitrogen (HN) supply rates, 24 h after application of 30 μL of lanolin containing 225 μg methyl jasmonate (MeJA) to leaves growing at nodes 1, 2, and 3 or attacked by eight M. sexta larvae (one second instar larva per leaf, Cat). Each array was hybridized with Cy3-labeled cDNA generated from MeJA- or Cat-treated plants and Cy5-labeled cDNA from their corresponding controls (LC or C). Principal component axis 1 accounted for 45.9% and axis 2, 32.5% of the total variation in the transcriptional responses.

Figure 7.

Comparison mean (±se) ERs of four replicate oligonucleotides of selected signaling- and secondary-metabolism-related genes of 40-d-old N. attenuata plants grown under low nitrogen (LN) and high nitrogen (HN) supply rates, 24 h after application of 30 μL of lanolin containing 225 μg methyl jasmonate (MeJA) to leaves growing at nodes 1, 2, and 3, or attacked by eight M. sexta larvae (one second instar larva per leaf, Cat). Gene names and accession numbers: LOX1, NaLOX1 (AY254347); LOX2, NaLOX2 (AY254348); LOX3, NaLOX3 (AY254349); AOS, NaAOS (AJ295274); HPL, NaHPL (AJ414400); DOX, Naα-dioxygenase (AW191821); ACO, NaACO (AY426756); RALF, NaRALF, rapid alkalinization factor (AF407278); TD, NaTD, threonine deaminase (AW191811); TPI, NaTPI, trypsin proteinase inhibitor (AY426751); THI, Na-thionin (BU494528); γTHI, Na-γ-thionin, (AY456268); PAL, Na-Phe ammonia lyase (M90692); PPO, polyphenyl oxidase (A27686); C4H, cinnamic acid 4-hydroxylase (AF212318); 4CL, 4-coumarate-CoA ligase (M62755). Asterisks indicate significant differences between members of a treatment and control pair (LC versus MeJA; OS versus W; P < 0.05, t test).