Soil chemistry determines whether defensive plant secondary metabolites promote or suppress herbivore growth

Abstract

Plant secondary (or specialized) metabolites mediate important interactions in both the rhizosphere and the phyllosphere. If and how such compartmentalized functions interact to determine plant-environment interactions is not well understood. Here, we investigated how the dual role of maize benzoxazinoids as leaf defenses and root siderophores shapes the interaction between maize and a major global insect pest, the fall armyworm. We find that benzoxazinoids suppress fall armyworm growth when plants are grown in soils with very low available iron but enhance growth in soils with higher available iron. Manipulation experiments confirm that benzoxazinoids suppress herbivore growth under iron-deficient conditions and in the presence of chelated iron but enhance herbivore growth in the presence of free iron in the growth medium. This reversal of the protective effect of benzoxazinoids is not associated with major changes in plant primary metabolism. Plant defense activation is modulated by the interplay between soil iron and benzoxazinoids but does not explain fall armyworm performance. Instead, increased iron supply to the fall armyworm by benzoxazinoids in the presence of free iron enhances larval performance. This work identifies soil chemistry as a decisive factor for the impact of plant secondary metabolites on herbivore growth. It also demonstrates how the multifunctionality of plant secondary metabolites drives interactions between abiotic and biotic factors, with potential consequences for plant resistance in variable environments.

Keywords: benzoxazinoids; herbivore resistance; maize; plant herbivore interactions; plant secondary metabolites.

Fig. 1.

The effect of benzoxazinoids on herbivore performance depends on the soil type. (Center) Map depicting soil collection sites around Yixing (China). Gray boxes (A–H): Growth of S. frugiperda caterpillars on WT and benzoxazinoid-deficient bx1 mutant plants growing in the different soils (+SE, n = 10), together with respective soil properties. Soil properties are depicted as fold change relative to the average across all tested soils. Refer to SI Appendix, Fig. S1 for absolute values. Soils 1 through 4 are anthrosols, and soils 5 through 8 are ferrosols. Asterisks indicate significant differences between plant genotypes (ANOVA; *P < 0.05). (I) PCA of field soil properties. Green triangles represent soils on which caterpillars grow better on WT plants. Yellow squares represent soils on which caterpillars grow better on bx1 mutant plants. Vectors of soil parameters are shown as gray arrows. (J) Iron contents in the leaves of WT and bx1 plants grown in the different soils (+SE, n = 3, with three to four individual plants pooled per replicate). For full elemental analysis, refer to SI Appendix, Fig. S4. DW, dry weight. O.C., organic carbon. Two-way ANOVA results testing for genotype and soil effects are shown (**P < 0.01; ***P < 0.001). Asterisks indicate significant differences between genotypes within the same soil (pairwise comparisons through FDR-corrected LSMeans; *P < 0.05; **P < 0.01).

Fig. 2.

Soil-dependent benzoxazinoid resistance is driven by root iron supply. (A) Growth of S. frugiperda feeding on WT and bx1 plants supplied with different iron sources (+SE, n = 14 to 15). (B) Consumed leaf area (+SE, n = 14 to 15). (C) Growth of S. frugiperda feeding on WT and bx1 plants complemented with pure DIMBOA added to the rhizosphere under different iron sources (+SE, n = 14 to 15). “None” nutrient solutions received either NaCl or Na₂SO₄. “Complex” nutrient solutions received Fe-EDTA. “Free” nutrient solutions received FeCl₃ or Fe₂(SO₄)₃. For full results showing genotype effects of all individual nutrient solutions, refer to SI Appendix, Fig. S7. Two-way ANOVA results testing for genotype and iron source effects are shown (n.s., not significant; ***P < 0.001). Asterisks indicate significant differences between genotypes within the same soil (pairwise comparisons through FDR-corrected LSMeans; *P < 0.05; **P < 0.01).

Fig. 3.

Interactions between root iron supply and benzoxazinoids determine leaf iron homeostasis. Relative expression of genes involved in iron homeostasis in the leaves of WT and bx1 mutant plants supplied with different iron sources (+SE, n = 7 to 8). “None” nutrient solutions received either NaCl or Na₂SO₄. “Complex” nutrient solutions received Fe-EDTA. “Free” nutrient solutions received FeCl₃ or Fe₂(SO₄)₃. For full results showing genotype effects of all individual nutrient solutions, refer to SI Appendix, Fig. S8. Two-way ANOVA results testing for genotype and iron source effects are shown (n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001). Asterisks indicate significant differences between genotypes within the same soil (pairwise comparisons through FDR-corrected LSMeans; ***P < 0.001).

Fig. 4.

Changes in leaf herbivore performance are not explained by changes in leaf primary metabolism. Contents of soluble protein (A), hydrolysable amino acids (B), sugars (C), and starch (D) in the leaves of WT and bx1 mutant plants supplied with different iron sources (+SE, n = 14 to 15). “None” nutrient solutions received either NaCl or Na₂SO₄. “Complex” nutrient solutions received Fe-EDTA. “Free” nutrient solutions received FeCl₃ or Fe₂(SO₄)₃. For full results showing genotype effects of all individual nutrient solutions, refer to SI Appendix, Fig. S9. Two-way ANOVA results testing for genotype and iron source effects are shown (n.s., not significant; *P < 0.05; ***P < 0.001). No significant differences between genotypes within the same soil were observed (pairwise comparisons through FDR-corrected LSMeans).

Fig. 5.

Soil iron and benzoxazinoids interactively reprogram a subset of leaf defenses. (A–D) Concentrations of chlorogenic acid (A), chlorogenic acid isomer (B), rutin (C), and maysin (D) in the leaves of WT and bx1 mutant plants supplied with different iron sources (+SE, n = 8). FW, fresh weight. (E and F) Expression levels of ZmMPI (E) and ZmRIP2 (F) in the leaves of WT and bx1 plants supplied with different iron sources (+SE, n = 8). “None” nutrient solutions received either NaCl or Na₂SO₄. “Complex” nutrient solutions received Fe-EDTA. “Free” nutrient solutions received FeCl₃ or Fe₂(SO₄)₃. For full results showing genotype effects of all individual nutrient solutions, refer to SI Appendix, Fig. S10. Two-way ANOVA results testing for genotype and iron source effects are shown (*P < 0.05; ***P < 0.001). Asterisks indicate significant differences between genotypes within the same soil (pairwise comparisons through FDR-corrected LSMeans; ***P < 0.001).

Fig. 6.

Herbivore iron supply is associated with soil-dependent benzoxazinoid resistance. (A) Iron contents of S. frugiperda larvae feeding on WT and bx1 plants grown in field soils (+SE, n = 3, with three to four individual larvae pooled per replicate). Soils 1 through 4 are anthrosols, and soils 5 through 8 are ferrosols. Twoway ANOVA results testing for genotype and soil effects are shown (*P < 0.05; **P < 0.01; ***P < 0.001). Asterisks indicate significant differences between genotypes within the same soil (pairwise comparisons through FDR-corrected LSMeans; *P < 0.05; **P < 0.01). (B and C) Iron contents of S. frugiperda larvae feeding on WT and bx1 plants (B), with DIMBOA rhizosphere complementation (C) under different iron source treatments (+SE, n = 5, with three individual larvae pooled per replicate). “None” nutrient solutions received either NaCl or Na₂SO₄. “Complex” nutrient solutions received Fe-EDTA. “Free” nutrient solutions received FeCl₃ or Fe₂(SO₄)₃. For full results showing genotype effects of all individual nutrient solutions, refer to SI Appendix, Fig. S13. Two-way ANOVA results testing for genotype and iron source effects are shown (***P < 0.001). Asterisks indicate significant differences between genotypes within the same soil (pairwise comparisons through FDR-corrected LSMeans; ***P < 0.001). (D) Growth of S. frugiperda larvae feeding on B73 and ys1 mutants (+SE, n = 13 to 16). Note that the ys1 mutant is in an undefined background. Asterisks indicate significant differences between plant genotypes (ANOVA; *P < 0.05). (E) Growth of S. frugiperda larvae feeding on the artificial diets supplemented with different iron sources (+SE, n = 12). Asterisks and letters indicate significant differences between genotypes or iron sources (one-way ANOVA followed by pairwise comparisons through FDR-corrected LSMeans; P < 0.05; ***P < 0.001).