Regulation of growth-defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module

Abstract

The plant hormone jasmonate (JA) promotes the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins to relieve repression on diverse transcription factors (TFs) that execute JA responses. However, little is known about how combinatorial complexity among JAZ-TF interactions maintains control over myriad aspects of growth, development, reproduction, and immunity. We used loss-of-function mutations to define epistatic interactions within the core JA signaling pathway and to investigate the contribution of MYC TFs to JA responses in Arabidopsis thaliana. Constitutive JA signaling in a jaz quintuple mutant (jazQ) was largely eliminated by mutations that block JA synthesis or perception. Comparison of jazQ and a jazQ myc2 myc3 myc4 octuple mutant validated known functions of MYC2/3/4 in root growth, chlorophyll degradation, and susceptibility to the pathogen Pseudomonas syringae. We found that MYC TFs also control both the enhanced resistance of jazQ leaves to insect herbivory and restricted leaf growth of jazQ. Epistatic transcriptional profiles mirrored these phenotypes and further showed that triterpenoid biosynthetic and glucosinolate catabolic genes are up-regulated in jazQ independently of MYC TFs. Our study highlights the utility of genetic epistasis to unravel the complexities of JAZ-TF interactions and demonstrates that MYC TFs exert master control over a JAZ-repressible transcriptional hierarchy that governs growth-defense balance.

Keywords: gene cluster; glucosinolate; growth-defense tradeoffs; jasmonate (JA); plant defense; plant hormone; plant-insect interaction; triterpenoid.

Figure 1

Genetic interaction between mutations affecting the core jasmonate (JA) response pathway. (a) Perturbations (shown in red) used in this study to manipulate JA responses included treatment with exogenous JA and loss‐of‐function mutations affecting JA biosynthesis (allene oxide synthase (aos)), the JA co‐receptor (coronatine insensitive1 (coi1)), five JASMONATE ZIM‐DOMAIN (JAZ) repressors (jazQ), or three MYC transcription factors (mycT). (b, e, g) Root growth inhibition assays of Arabidopsis sextuple mutants of jazQ combined with (b) coi1 or (e) aos, and of (g) jaz1, jaz3, jaz4, jaz9 and jaz10 single mutants. Root lengths were determined from seedlings grown on plates supplemented (closed bars) or not supplemented (open bars) with 25 μM methyl jasmonate (MeJA). Bars are means ± SD (n = 7–24 seedlings per genotype). Per cent inhibition by MeJA is shown for each genotype in parentheses. Different letters represent significant differences at P < 0.05 determined by two‐way ANOVA with Tukey's honest significant difference (HSD) test. Experiments were repeated twice with similar results. (c, d, f) Male sterility of Arabidopsis sextuple mutants of jazQ combined with (c, d) coi1 or (f) aos. Anther filaments elongate and anthers dehisce in Col‐0 and jazQ flowers, but filaments do not elongate fully and anthers fail to dehisce in the sterile coi1 and jazQ coi1 flowers (c). Flowers of (d) coi1 and jazQ coi1 and of (f) aos and jazQ aos are sterile.

Figure 2

Jasmonate (JA) hypersensitivity of the jasmonate zim‐domain quintuple mutant (jazQ) depends on MYC2/3/4 transcription factors. (a) Root length of Arabidopsis seedlings grown on plates supplemented (closed bars) or not supplemented (open bars) with 25 μM methyl jasmonate (MeJA). Bars are means ± SD (n = 33–48 seedlings per genotype). Per cent inhibition by MeJA is shown for each genotype in parentheses. (b) Photograph showing genotype‐dependent loss of chlorophyll in detached Arabidopsis leaves treated with 0 μM (mock) or 100 μM MeJA in the dark for 4 d. (c) Measurement of total chlorophyll levels in leaves treated as described in (b). Bars are means ± SD (n = 3 leaves per genotype). Different letters represent significant differences at P < 0.05 determined by two‐way ANOVA with Tukey's honest significant difference (HSD) test. Experiments were repeated three times with similar results.

Figure 3

Growth suppression of jasmonate zim‐domain quintuple mutant (jazQ) rosette leaves is mediated by MYC transcription factors. (a) Photograph of Col‐0, jazQ, mycT and jazQ mycT rosettes of 21‐d‐old Arabidopsis plants. (b–e). Rosette growth at 21 d was assessed by measuring (b) biomass, (c) leaf area, (d) number of leaves, and (e) petiole length. Bars are means ± SD (n = 14–15 plants per genotype). Different letters represent significant differences at P < 0.05 determined by two‐way ANOVA with Tukey's honest significant difference (HSD) test. The experiment was repeated four times with similar results.

Figure 4

MYC2/3/4 are required for increased resistance of the jasmonate zim‐domain quintuple mutant (jazQ) to a lepidopteran herbivore. Arabidopsis plants of the indicated genotype were challenged with neonate Trichoplusia ni larvae. Larval weights were measured 10 d later. (a) Photograph of representative T. ni larvae at the end of the feeding trial. (b) Larval weight at the end of the feeding trial. Bars are means ± SD (n = 12, where each sample is the mean of four larvae per plant). Different letters represent significant differences at P < 0.05 with Tukey's honest significant difference (HSD) test. (c) Photograph of control (Con) and insect‐challenged plants at the end of the feeding trial. The experiment was repeated three times with similar results.

Figure 5

Enhanced susceptibility of the jasmonate zim‐domain quintuple mutant (jazQ) to bacterial infection requires MYC2/3/4. Five‐week‐old Arabidopsis plants of the indicated genotype were dip‐inoculated with Pseudomonas syringae pv. DC3000 (Pst DC3000) at 1 × 10⁸ colony forming units (CFUs) ml⁻¹. (a) Bacterial populations, represented as CFUs, in fully expanded leaves were determined 3 d after inoculation. Data show the mean ± SD (n = 4 technical replicates). Different letters represent significant differences at P < 0.05 with Tukey's honest significant difference (HSD) test. The experiment was repeated three times with similar results. (b) Photograph of plants taken 6 d after inoculation with Pst DC3000. (c) Zoom‐in images to show increased symptom development on young leaves of jazQ plants 4 d after inoculation.

Figure 6

Delayed flowering of the jasmonate zim‐domain quintuple mutant (jazQ) is not dependent on MYC transcription factors. (a) Photograph of Col‐0, jazQ, mycT and jazQ mycT inflorescence in 45‐d‐old Arabidopsis plants. (b, c) Flowering time was assessed by counting the days required for (b) bolting and (c) flowering. (d) Quantification of the number of leaves at the time of bolting. Bars are means ± SD (n = 29–32 plants per genotype). Different letters represent significant differences at P < 0.05 with Tukey's honest significant difference (HSD) test. The experiment was repeated four times with similar results.

Figure 7

JASMONATE ZIM‐DOMAIN (JAZ) proteins coordinate gene expression through MYC‐dependent and ‐independent mechanisms in Arabidopsis. (a) Conceptual framework of genes up‐regulated in jazQ. Genes controlled by the JAZ‐MYC module are up‐regulated in jazQ but not jazQ mycT, whereas genes controlled by other (MYC‐independent) JAZ‐transcription factor (TF) modules are up‐regulated in both jazQ and jazQ mycT. (b) Gene ontology (GO) terms of genes up‐regulated in jazQ only (top) or in jazQ and jazQ mycT (bottom). (c) MYC‐dependent expression of selected genes associated with jasmonate biosynthesis, glucosinolate biosynthesis, and the wound response. lipoxygenase 2 (LOX2), branched‐chain aminotransferase 4 (BCAT4), isopropyl malate dehydrogenase 1 (IPMD1), CYP79B3 and TAT1 promoters are reported targets of MYC2. (d) MYC‐independent expression of genes associated with triterpenoid metabolism from the marneral and thalianol clusters (shaded genes), with genes flanking these clusters shown for comparison. Errors bars are ± SD of the mean of three biological replicates. Expression levels are transcripts per million (TPM).

Figure 8

Conceptual model of how jasmonate (JA) signaling controls multiple JASMONATE ZIM‐DOMAIN (JAZ)‐transcription factor modules to mediate diverse physiological responses. The model depicts specific processes that are either dependent (light purple) or not dependent (blue) on MYC transcription factors (TFs). Some processes, including glucosinolate and anthocyanin metabolism, are controlled by JAZ‐mediated repression of both MYC and non‐MYC TFs. See the Discussion section for details. COI1, coronatine insensitive 1; FT, flowering locus T; TOE, target of eat; TT8, transparent testa 8; GL3, glabrous 3; GL1, glabrous 1; PAP1, production of anthocyanin pigment 1; EGL3, enhancer of glabra 3; TF, transcription factor; PLT1/2, plethora 1 and 2.