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. 2012 Jun;24(6):2635-48.
doi: 10.1105/tpc.112.098749. Epub 2012 Jun 5.

Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression

Gao-Jie Hong  1 , Xue-Yi Xue, Ying-Bo Mao, Ling-Jian Wang, Xiao-Ya Chen
Affiliations

Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression

Gao-Jie Hong et al. Plant Cell. 2012 Jun.

Abstract

Arabidopsis thaliana flowers emit volatile terpenes, which may function in plant-insect interactions. Here, we report that Arabidopsis MYC2, a basic helix-loop-helix transcription factor, directly binds to promoters of the sesquiterpene synthase genes TPS21 and TPS11 and activates their expression. Expression of TPS21 and TPS11 can be induced by the phytohormones gibberellin (GA) and jasmonate (JA), and both inductions require MYC2. The induction of TPS21 and TPS11 results in increased emission of sesquiterpene, especially (E)-β-caryophyllene. DELLAs, the GA signaling repressors, negatively affect sesquiterpene biosynthesis, as the sesquiterpene synthase genes were repressed in plants overaccumulating REPRESSOR OF GA1-3 (RGA), one of the Arabidopsis DELLAs, and upregulated in a penta DELLA-deficient mutant. Yeast two-hybrid and coimmunoprecipitation assays demonstrated that DELLAs, represented by RGA, directly interact with MYC2. In yeast cells, the N terminus of MYC2 was responsible for binding to RGA. MYC2 has been proposed as a major mediator of JA signaling and crosstalk with abscisic acid, ethylene, and light signaling pathways. Our results demonstrate that MYC2 is also connected to GA signaling in regulating a subset of genes. In Arabidopsis inflorescences, it integrates both GA and JA signals into transcriptional regulation of sesquiterpene synthase genes and promotes sesquiterpene production.

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Figures

Figure 1.
Figure 1.
MYC2 Mutations Lead to Reduced Sesquiterpene Emission and Sesquiterpene Synthase Gene Expression. (A) (E)-β-Caryophyllene emission (nanograms/gram fresh weight and hour [ng/gFW·h]) from the wild-type (Col-0), myc2-1, and myc2-2 inflorescences. Error bars indicate sd of three biological replicates. The letters “a” and “b” are relative to the corresponding Col-0 line. aP < 0.01, very significant difference; bP < 0.05, significant difference. (B) Expression of sesquiterpene synthase genes of TPS21 and TPS11 in inflorescences of wild-type (Col-0), myc2-1, and myc2-2 mutants. The transcripts were analyzed by qRT-PCR, and β-TUBULIN2 was used as the internal standard. Error bars indicate sd of three biological replicates.
Figure 2.
Figure 2.
Overexpression of MYC2 Results in Enhanced Sesquiterpene Emission and Higher Expression of Sesquiterpene Synthase Genes. (A) (E)-β-Caryophyllene emission (ng/gFW·h) from the wild-type (Col-0) and Pro35S:cMyc-MYC2 inflorescences. Error bars indicate sd of three biological replicates. The letters “a” and “b” are relative to the corresponding Col-0 line. aP < 0.01, very significant difference; bP < 0.05, significant difference. (B) Expression of sesquiterpene synthase genes of TPS21 and TPS11 in inflorescences of the wild-type (Col-0) and the Pro35S:cMyc-MYC2 (MYC2OX-3) plants. The transcripts were analyzed by qRT-PCR. Error bars indicate sd of three biological replicates. (C) Expression of TPS21 and TPS11 in ProAlcA:MYC2 inflorescences. The plants were sprayed with 1% ethanol, and the total RNAs were isolated for analysis 4 h later. Error bars indicate sd of three biological replicates.
Figure 3.
Figure 3.
Analysis of E-box Elements in TPS21 and TPS11 Promoters. (A) to (D) GUS staining of inflorescences of ProTPS21:GUS and ProTPS11:GUS in the wild-type (Col-0) and myc2-2 backgrounds. (E) Schematic diagrams of the ProTPS21:GUS and ProTPS11:GUS constructs; triangles indicate E-box cis-elements. A 1899-bp 5′-upstream fragment of TPS21 and a 2254-bp 5′-upstream fragment of TPS11 were fused to GUS, respectively. The amplicons I to IV of TPS21 and TPS11 were used for ChIP analyses. (F) to (J) GUS staining of inflorescences of the plants expressing ProTPS21:GUS and its mutated versions, mu1 to mu4. Note that the staining of mu2 was clearly fainted (G). (K) and (L) ChIP enrichment of TPS21 (K) and TPS11 (L) promoter regions bound by cMyc-MYC2. The 2-week-old Pro35S:cMyc-MYC2 and the wild-type (Col-0) seedlings were used; DNA fragments were analyzed by quantitative PCR, with the β-TUBULIN2 promoter as a reference. Enrichments are referred to the Pro35S:cMyc-MYC2 against wild-type seedlings. Error bars indicate sd of three PCR repeats of four separate samples.
Figure 4.
Figure 4.
Effects of Red Light on Expression of Sesquiterpene Synthase Genes and (E)-β-Caryophyllene Emission. (A) Expression of sesquiterpene synthase genes in inflorescences in response to white, red, and blue light. The wild-type (Col-0) plants were placed in dark conditions for 72 h, followed by a 4-h light exposure. Error bars indicate sd of three biological replicates. (B) Expression of sesquiterpene synthase genes in the wild-type (Col-0) and myc2-2 mutant inflorescences after a 4-h red light treatment. The plants had been placed in dark conditions for 72 h prior to the treatments. Error bars indicate sd of three biological replicates, (C) (E)-β-caryophyllene emission (ng/gFW·h) of the wild-type (Col-0) and myc2-2 mutant inflorescences after a 6-h red light treatment. The plants were placed in dark conditions for 24 h prior to the treatments. Error bars indicate sd of three biological replicates. The letter “a” is relative to the corresponding line in dark conditions. aP < 0.01, very significant difference.
Figure 5.
Figure 5.
Induction of Sesquiterpene Synthase Genes by MeJA. (A) Expression of sesquiterpene synthase genes in the wild-type (Col-0) and myc2-2 mutant inflorescences after a 4-h MeJA treatment. Error bars indicate sd of three biological replicates. (B) Expression of sesquiterpene synthase genes of TPS21 and TPS11 and the JA-responsive gene VSP1 in the wild-type (Col-0) inflorescences after a 4-h MeJA treatment. The plants were placed in dark conditions for 72 h prior to the JA treatment in darkness. Error bars indicate sd of three biological replicates.
Figure 6.
Figure 6.
Effects of GA/DELLAs on Expression of Sesquiterpene Synthase Genes and (E)-β-Caryophyllene Emission. (A) Expression of sesquiterpene synthase genes in the wild-type (Col-0) and myc2-2 mutant inflorescences after a 4-h GA treatment. The plants had been placed in dark condition for 72 h prior to the GA treatment in darkness. Error bars indicate sd of three biological replicates. (B) Expression of sesquiterpene synthase genes in the wild-type (Col-0) and myc2-2 mutant inflorescences after a 4-h treatment with PAC, a GA biosynthesis inhibitor. Error bars indicate sd of three biological replicates. (C) (E)-β-caryophyllene emission (ng/gFW·h) of the wild-type (Col-0) and the myc2-2 mutant inflorescences after a 6-h PAC treatment. Error bars indicate sd of three biological replicates. The letter “a” is relative to the corresponding line treated with MOCK. aP < 0.01, very significant difference. (D) Expression of sesquiterpene synthase genes in inflorescences of the wild-type (Col-0) and the transgenic ProRGA:RGA-HA (RGAOX) plants. Error bars indicate sd of three biological replicates. (E) GC-MS chromatogram of volatile sesquiterpenes collected from the wild-type (WT; Col-0) and the transgenic ProRGA:RGA-HA (RGAOX) plants.
Figure 7.
Figure 7.
GA and JA Jointly Regulate Sesquiterpene Synthase Genes. (A) Expression of sesquiterpene synthase genes in inflorescences of the wild-type (Col-0) and ProRGA:RGA-HA (RGAOX) transgenic plants after a 4-h MeJA treatment. Error bars indicate sd of three biological replicates. (B) (E)-β-caryophyllene emission from inflorescences of the wild-type (Col-0) and ProRGA:RGA-HA (RGAOX) transgenic plants after a 6-h MeJA treatment. Error bars indicate sd of three biological replicates. The letter “a” is relative to the corresponding mock-treated line. aP < 0.01, very significant difference. (C) Expression of sesquiterpene synthase genes in inflorescences of the wild-type (Col-0) and the penta DELLA-deficient mutant (della) after a 4-h MeJA treatment. The plants were placed in dark conditions for 72 h prior to the treatment in darkness. Error bars indicate sd of three biological replicates. (D) Expression of sesquiterpene synthase genes in the wild-type (Col-0) inflorescences after treatments by MeJA, GA, and MeJA plus GA for 4 h. The plants were placed in dark conditions for 72 h prior to the treatments in darkness. Note that MeJA further induced the gene expression in the presence of GA. Error bars indicate sd of three biological replicates. (E) Expression of sesquiterpene synthase genes in the wild-type (Col-0) and myc2-2 mutant inflorescences after a 4-h treatment with GA plus JA. The plants were placed in dark conditions for 72 h prior to the treatments in darkness. Error bars indicate sd of three biological replicates. (F) (E)-β-caryophyllene emission (ng/gFW·h) from inflorescences of the wild-type (Col-0) and myc2-2 mutant plants after treatments with MeJA, GA, and MeJA plus GA for 6 h. The plants were placed in dark conditions for 24 h prior to the treatments in darkness. Error bars indicate sd of three biological replicates. The letter “a” is relative to the corresponding mock-treated line. aP < 0.01, very significant difference.
Figure 8.
Figure 8.
MYC2 Interacts with DELLAs. (A) Yeast two-hybrid assays of the interactions between MYC2 and DELLA proteins of RGA, GAI, RGL1, RGL2, and RGL3. The empty vectors of pGBKT7 and pGADT7 were used as negative controls. The concentration of 3-amino-1,2,4-triazole was 25 mM. (B) Schematic diagrams of MYC2 and RGA truncated versions (left) and their interactions in the yeast two-hybrid system. The concentration of 3-amino-1,2,4-triazole was 25 mM. aa, amino acids. (C) Coimmunoprecipitation of the Arabidopsis cMyc-MYC2 and RGA proteins. Protein extracts of the p35S:cMyc-MYC2 and the wild-type (WT; Col-0) seedlings before (Input) and after (IP) immunoprecipitation with the anti-cMyc antibody-conjugated beads were detected by protein gel blots using an anti-RGA antibody. (D) Yeast three-hybrid assays of the influences of RGA on MYC2-JAZ3 interaction. The MYC2-JZA3 binding activities are represented by β-galactosidase activity, and the promoter driving RGA expression was suppressed by increasing concentrations of Met. Error bars indicate sd of three technical replicates, and the results were consistent in three biological replicates.
Figure 9.
Figure 9.
Regulation of Sesquiterpene Synthase Genes by GA and JA Signaling Pathways. A model for the role of GA and JA in promoting biosynthesis of sesquiterpenes in Arabidopsis inflorescences. The transcription factor MYC2 positively regulates the expression of sesquiterpene synthase genes, such as TPS21 and TPS11. JAZ and DELLA proteins are negative regulators of JA and GA signaling pathways, respectively, and both of them repress sesquiterpene synthase genes through interacting with MYC2. Increased levels of JA and GA result in decreased levels of JAZs and DELLAs, releasing MYC2. Arrows indicate positive regulation, blunt ends indicate negative regulation, and the dashed line indicates the unidentified pathway.
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