Accumulation of 5-hydroxynorvaline in maize (Zea mays) leaves is induced by insect feeding and abiotic stress

Abstract

Plants produce a wide variety of defensive metabolites to protect themselves against herbivores and pathogens. Non-protein amino acids, which are present in many plant species, can have a defensive function through their mis-incorporation during protein synthesis and/or inhibition of biosynthetic pathways in primary metabolism. 5-Hydroxynorvaline was identified in a targeted search for previously unknown non-protein amino acids in the leaves of maize (Zea mays) inbred line B73. Accumulation of this compound increases during herbivory by aphids (Rhopalosiphum maidis, corn leaf aphid) and caterpillars (Spodoptera exigua, beet armyworm), as well as in response to treatment with the plant signalling molecules methyl jasmonate, salicylic acid and abscisic acid. In contrast, ethylene signalling reduced 5-hydroxynorvaline abundance. Drought stress induced 5-hydroxynorvaline accumulation to a higher level than insect feeding or treatment with defence signalling molecules. In field-grown plants, the 5-hydroxynorvaline concentration was highest in above-ground vegetative tissue, but it was also detectable in roots and dry seeds. When 5-hydroxynorvaline was added to aphid artificial diet at concentrations similar to those found in maize leaves and stems, R. maidis reproduction was reduced, indicating that this maize metabolite may have a defensive function. Among 27 tested maize inbred lines there was a greater than 10-fold range in the accumulation of foliar 5-hydroxynorvaline. Genetic mapping populations derived from a subset of these inbred lines were used to map quantitative trait loci for 5-hydroxynorvaline accumulation to maize chromosomes 5 and 7.

Keywords: 5-hydroxynorvaline; Aphid; Rhopalosiphum maidis.; drought; maize.

Fig. 1.

(A) HPLC-fluorescence detection chromatogram of maize amino acids. Black = uninduced; Red = induced with methyl jasmonate. The arrow indicates the induced peak of interest. (B) Structure of 5-hydroxynorvaline and the fluorescent derivative that is detected in A.

Fig. 2.

Induction and localization of 5-hydroxynorvaline in maize. Abundance of 5-hydroxynorvaline in maize seedlings after treatment with (A) 0.45mM methyl jasmonate, (B) 0.1mM abscisic acid, (C) 0.45mM salicylic acid or (D) 0.45mM 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor). N = 3 for A–D; *P < 0.05, t test relative to day 0 sample. 5-Hydroxynorvaline accumulation in maize seedlings treated with (E) R. maidis (N = 3), (F) S. exigua (N = 3) and (G) mock-infected or infected with C. heterostrophus (southern leaf blight; N = 4). *P < 0.05 relative to controls, t test. (H) Abundance of 5-hydroxynorvaline in dry seeds and different parts of field-grown maize inbred line B73 (N =4).

Fig. 3.

Effects of 5-hydroxynorvaline on R. maidis and S. exigua. (A) Concentration of 5-hydroxynorvaline in aphids feeding from artificial diet for 4 days (N = 3–5; *P < 0.05 compared to plant-fed control aphids, t test). (B) S. exigua larval weight after 9 days on artificial diet containing 5-hydroxynorvaline (N = 11–13). (C) Reproduction of R. maidis over 4 days with different concentrations of 5-hydroxynorvaline in the diet (N = 3–5; *P < 0.05 compared to 0mM control sample, t test).

Fig. 4.

Induction of 5-hydroxynorvaline accumulation by abiotic stress. (A) Comparison of well-watered plants and plants that were not watered (N = 3; *P < 0.05, t test). (B) 5-Hydroxynorvaline accumulation in leaves that were removed from the plant and left at 23 °C. 5-Hydroxynorvaline concentration over time is expressed relative to the original wet weight of the leaves (N = 3; line was place by linear regression, R ² = 0.96). (C) Concentration of 5-hydroxynorvaline in maize plants placed at 4 °C (N = 3; *P < 0.05, t test relative to 0 day or 0h controls). Means ± S.E. are shown.

Fig. 5.

Incorporation of isotope-labelled amino acids into 5-hydroxynorvaline. (A) Structures of isotope-labelled glutamine (Gln), glutamate (Glu), proline (Pro), ornithine (Orn) and arginine (Arg) that were tested as precursors for the synthesis of 5-hydroxynorvaline. (B) Uptake of isotope-labelled amino acids via the petiole. Leaves were inserted into vials containing the indicated amino acids at 5mM concentration. Shown is the fraction of each free amino acid that is isotope-labelled. (C) Accumulation of m/z 350 (mass of trimethylsilyl-derivatized compound) and m/z 351 (m+1) 5-hydroxynorvaline (with incorporation of ¹⁵N, see inset) in maize leaves after uptake of unlabelled and isotope-labelled amino acid precursors via the petioles. Control samples received unlabelled amino acids. Shown is the fraction of 5-hydroxynorvaline that is m+1 (m/z 351) relative to the total 5-hydroxynorvaline in the leaves. Mean ± S.E. of N = 3. Different letters indicate significant differences, P < 0.05, ANOVA followed by Tukey’s HSD.

Fig. 6.

Natural variation in 5-hydroxynorvaline accumulation. Abundance of 5-hydroxynorvaline in parental lines of the maize NAM population after induction with 0.45mM methyl jasmonate. Mean ± S.E. of N = 3, *P < 0.05, t test relative to the B73 control.