Similar metabolic changes induced by HIPVs exposure as herbivore in Ammopiptanthus mongolicus

Abstract

Herbivore-induced plant volatiles (HIPVs) are important compounds to prim neighboring undamaged plants; however, the mechanism for this priming process remains unclear. To reveal metabolic changes in plants exposed to HIPVs, metabolism of leaves and roots of Ammopiptanthus mongolicus seedlings exposed to HIPVs released from conspecific plants infested with larvae of Orgyia ericae were analyzed together with control and infested seedlings using nuclear magnetic resonance (NMR)-based metabolic technology and multi variate data analysis. Results presented showed that HIPVs exposure led to similar but specific metabolic changes compared with those induced by infestation in both leaves and roots. Furthermore, both HIPVs exposure and herbivore attack resulted in metabolic changes involving a series of primary and secondary metabolites in both leaves and roots. Taken together, these results suggested that priming of yet-damaged plants may be achieved by reconfiguring metabolic pathways in leaves and roots to make similar concentrations for all metabolites as those in seedlings infested. Therefore, we propose that improved readiness of defense induction of primed plants toward subsequent herbivore attack may be based on the similar metabolic profiling induced by HIPVs exposure as those caused by herbivore.

Figure 1. Typical ¹H NMR spectra of leaves.

a, ¹H NMR spectra of leaves from control plants for 12 h; b, ¹H NMR spectra of leaves from seedlings infested for 12 h; c, ¹H NMR spectra of leaves from seedlings exposed to HIPVs for 12 h. 1: α-Linoleic acid analogues, 2: ethanol, 3: threonine, 4: acetic acid, 5: oxalacetic acid, 6: glycolate, 7: α-hydroxyisobutyric acid, 8: glutaric acid, 9: succinic acid, 10: citric acid, 11: acetone, 12: choline, 13: alanine, 14: valine, 15: aspartic acid, 16: asparagin, 17: serine, 18: glycine, 19: phenylalanine, 20: sucrose, 21: α-Glucose, 22: pinitol, 23: inositol, 24: hydroxybenzene derivative 1, 25: 4-hydroxyphenylacetic acid, 26: trigonelline, 27: adenine.

Figure 2. PCA score plots of leaves and roots.

A, PCA score plot of all leaves obtained from binned ¹H NMR data scaled to intensityof TMSP; B, PCA score plot of all leaves obtained from binned ¹H NMR data scaled to total area of the corresponding spectra; C, PCA score plot of roots from control seedlings, infested seedlings and HIPVs induced seedlings at 6 h obtained from binned ¹H NMR data scaled to intensity of TMSP; D, PCA score plot of roots from control seedlings, infested seedlings and HIPVs induced seedlings at 12 h obtained from binned ¹H NMR data scaled to intensity of TMSP; E, PCA score plot of roots from control seedlings, infested seedlings and HIPVs induced seedlings at 24 h obtained from binned ¹H NMR data scaled to intensity of TMSP; leaf samples were symbolized as follows: ▴, control for 6 h; ▪, control for 12 h; ♦, control for 24 h; △, leaves infested for 6 h; □, leaves infested for 12 h; ◊, leaves infested for 24 h; ▾, leaves exposed to HIPVs for 6 h; •, leaves exposed to HIPVs for 12 h; *, leaves exposed to HIPVs for 24 h. Symbolizations of root samples were corresponded with the symbols of leaves. Percents of the total variances for the evaluated samples explained by the first two principal components were labeled following t and t respectively.

Figure 3. OPLS-DA score plots (left) and corresponding loading plots (right) of roots.

A, B: OPLS-DA score and loading plots of roots from seedlings infested for 12 h versus control; C, D: OPLS-DA score and loading plots of roots from seedlings exposed to HIPVs for 12 h versus control; E, F: OPLS-DA score and loading plots of roots from seedlings infested for 24 h versus control; G, H: OPLS-DA score and loading plots of roots from seedlings exposed to HIPVs for 24 h versus control. Symbolization of samples in score plots were the same with figure 2. For coefficient-coded loading plots, horizontal axis corresponds to the integrated regions of 0.4–10.0 ppm in ¹H NMR spectra; vertical axis are the back transformed loading value and colors for variables (compounds) are coded by square of coefficients (r²).

Figure 4. Herbivore and HIPVs exposure induced metabolic changes in leaves or roots.

Metabolites identified were shown in bold letters. Dashed lines: multiple-step reactions; solid lines: one-step reactions. Abbreviations for metabolites: AA, acetic acid; Ade, adenine; Ala, alanine; ALA, α-linoleic acid analogues; Asn, asparagines; Asp, aspartic acid; Cho, choline; Cit, citrate; Eth, ethanol; F-6-P, fructose-6-phosphate; Fru, fructose; Fum, fumarate; Glc, glucose; Gln, glutamine; Glu, glutamate; GLVs, green leaf volatiles; GLY, glycolate; Ici, isocitrate; Ile, isoleucine; JAs, Jasmonates Mal, malate; MI, myo-Inositol; NAD⁺, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide; OAA, oxaloacetate; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; Phe, phenylalanine; Pin, pinitol; PRPP, 5-phosphoribosyl 1-pyrophosphate; Pyr, pyruvate; Ser, serine; Shik, shikimate; Suc, sucrose; Succ, succinate Thr, threonine; TRG, trigonelline; Val,valine; α-KG, alpha-ketoglutarate.