Maize leaves were treated with W+W or W+OS, and samples were collected at 1.5 and 6 h (for simplicity, named 1.5 W, 1.5 OS, 6 W, and 6 OS); non-treated ones severed as controls (Con). Venn diagrams of the numbers of genes up-regulated by treatment of (a) 1.5 W and 1.5 OS and (b) 6 W and 6 OS. (c) Overall up-regulated numbers of genes after W+W and W+OS treatment. (d) Venn diagram of the gene numbers further induced (FI) 1.5 and 6 h after W+OS treatment compared with W+W treatment. (e) Heatmap of the relative expression levels (fold change after log10 transformation) of the 60 most up-regulated genes at 1.5 and 6 h (30 genes for each time point) induced by W+OS treatment (detailed descriptions of these genes can be found in ; NA = not annotated; *: name abbreviated due to length).

Maize leaves were treated with W+W or W+OS, and samples were collected at 1.5 and 6 h (for simplicity, named 1.5 W, 1.5 OS, 6 W, and 6 OS); non-treated ones severed as controls (Con). Venn diagrams of the numbers of genes down-regulated by treatment of (a) 1.5 W and 1.5 OS (a) and (b) 6 W and 6 OS. (c) Overall down-regulated gene numbers after W+W and W+OS treatment. (d) Venn diagram of the gene numbers further repressed (FR) 1.5 and 6 h after W+OS treatment compared with W+W treatment. (e) Heatmap of the relative expression levels (fold change after log10 transformation) of the 60 most down-regulated genes at 1.5 and 6 h (30 genes for each time point) after W+OS treatment (detailed descriptions of these genes can be found in ; NA = not annotated; *: name abbreviated due to length).

Maize leaves were treated with W+W or W+OS, and samples were collected at 1.5 and 6 h (for simplicity, named 1.5 W, 1.5 OS, 6 W, and 6 OS); leaves from untreated plants severed as controls (Con). Venn diagrams indicate the numbers of proteins up-regulated by (a) 1.5 W and 1.5 OS and (b) 6 W and 6 OS. (c) Overall up-regulated protein numbers. (d) Venn diagram of the numbers of proteins further induced (FI) 1.5 and 6 h after W+OS treatment compared with W+Wtreatment. (e) Relatively protein levels (fold change after log2 transformation) of the 60 most up-regulated genes at 1.5 and 6 h (30 proteins for each time point) induced by W+OS treatment (detailed descriptions of these proteins can be found in ; NA = not annotated; *: name abbreviated due to length).

Maize leaves were treated with W+W or W+OS, and samples were collected at 1.5 and 6 h (for simplicity, named 1.5 W, 1.5 OS, 6 W, and 6 OS); leaves from untreated plants severed as controls (Con). Venn diagram of the numbers of proteins down-regulated by (a) 1.5 W and 1.5 OS and (b) 6 W and 6 OS. (c) Overall down-regulated protein numbers. (d) Venn diagram of the numbers of proteins further repressed (FR) 1.5 and 6 h after W+OS treatment compared with W+W treatment. (e) Relative protein levels (fold change after log2 transformation) of the 60 most down-regulated genes at 1.5 and 6 h (49 proteins for 1.5 h and 11 proteins for 6 h) by W+OS treatment (detailed descriptions of these proteins can be found in ; NA = not annotated; *: name abbreviated due to length).

(a) Venn diagram of the numbers of all detected genes, regulated genes, all detected proteins, and regulated proteins. Numbers of up- and down-regulated genes having no changes in their protein levels (b), numbers of regulated proteins, whose transcript levels were unchanged after indicated treatments (c), and numbers of proteins up- or down-regulated, whose transcript levels increased after indicated treatments (d).

Mean levels (± SE, n = 5) of JA (a), JA-Ile (b), SA (c), ABA (d) and ethylene (e) induced by W+W and W+OS treatment (Con = control). Asterisks represent significant differences between the levels in samples treated with W+OS and W+W at the indicated times (Student’s t-test; *, P < 0.05; **, P < 0.01). “a” and “b” indicate significant differences of ET level (one-way ANOVA, Duncan’s multiple range test). (F) Relative levels (fold change after log10 transformation) of hormone biosynthesis-related genes for JA (LOX, AOS, AOC and OPR), SA (ICS and PAL), ET (ACS) and ABA (NCED, AO, and SDR) and levels (fold change after log2 transformation) of JA biosynthesis-related proteins LOX and AOS detected in the transcriptome and proteome data (detailed descriptions of these genes and proteins can be found in ).

Maize leaves were treated with W+W or W+OS, and samples were collected at 48 h; non-treated leaves severed as controls (n = 5); Venn diagram of the number of induced (a) or repressed (b) non-volatile metabolites; (c) relative changes of Bxs (n = 5); transcript (average ± SE; n = 3) and protein (average ± SE; n = 3) levels of the Bx biosynthesis-related (d) or terpene biosynthesis-related (e) genes or proteins in W+W- and W+OS-treated samples (control levels in plot c-e were normalized to 1, and the data were retrieved from the metabolites, transcriptome and proteome data, respectively. Note: the Y-axis has been log10 transformed. Bxs having the same molecular masses and cannot be distinguished by MS are indicated by suffixes “-1” and “-2” after the names of the two Bxs separated by a slash). Letters indicate significant differences between different treatment groups (one-way ANOVA, Duncan’s multiple range test). Detail IDs of Bx biosynthesis-related and TPS biosynthesis-related genes can be found in .

Maize leaves were treated with W+W or W+OS, and samples were collected at 1.5 and 6 h (named 1.5 W, 1.5 OS, 6 W, and 6 OS); non-treated leaves severed as controls (Con). Venn diagram of the numbers of up-regulated TF genes at 1.5 h (a) and 6 h (b), and the numbers of down-regulated TF genes at 1.5 h (c) and 6 h (d). (E, F) Relative expression levels (log10 transformed) of the 60 most up-regulated TFs (30 genes at each time point) (e) and all the down-regulated TFs (F) at 1.5 and 6 h after W+OS treatment. Detailed descriptions of these TFs can be found in ).

The components of the OS of M. separata are perceived by unknown receptors, and H₂O₂ is produced by GOX activity in the OS. In turn, initial early signaling events, such as Ca²⁺ influxes and MAPK activation are triggered, leading to the earliest proteome reconfigurations, including activation of certain transcription factors (TFs) and protein activity changes resulted from posttranslational modifications. Changes in the levels of transcripts and proteins follow rapidly, resulting in reconfiguration of the transcriptome, proteome, and phytohormone levels. The crosstalk among transcriptome, proteome, and phytohormones continue to reshape each and finally lead to augmented levels of defense metabolite biosynthesis enzymes and in turn accumulation of defense metabolites, such as Bxs and terpenes, counteracting M. separata feeding. Although mechanical wounding generates similar responses, it generally induces much less changes on the transcriptomic, proteomic, metabolomic, and phytohormone levels. Red and blue arrows, circles, and rectangles represent OS- and wounding-elicited responses, respectively. Up and down arrows in the rectangles or circles indicate up- or down-regulation, and the thickness of these arrows represent the number of genes, proteins, metabolites, or hormones.