An ABA-increased interaction of the PYL6 ABA receptor with MYC2 Transcription Factor: A putative link of ABA and JA signaling

Abstract

Abscisic acid (ABA) is a plant hormone that mediates abiotic stress tolerance and regulates growth and development. ABA binds to members of the PYL/RCAR ABA receptor family that initiate signal transduction inhibiting type 2C protein phosphatases. Although crosstalk between ABA and the hormone Jasmonic Acid (JA) has been shown, the molecular entities that mediate this interaction have yet to be fully elucidated. We report a link between ABA and JA signaling through a direct interaction of the ABA receptor PYL6 (RCAR9) with the basic helix-loop-helix transcription factor MYC2. PYL6 and MYC2 interact in yeast two hybrid assays and the interaction is enhanced in the presence of ABA. PYL6 and MYC2 interact in planta based on bimolecular fluorescence complementation and co-immunoprecipitation of the proteins. Furthermore, PYL6 was able to modify transcription driven by MYC2 using JAZ6 and JAZ8 DNA promoter elements in yeast one hybrid assays. Finally, pyl6 T-DNA mutant plants show an increased sensitivity to the addition of JA along with ABA in cotyledon expansion experiments. Overall, the present study identifies a direct mechanism for transcriptional modulation mediated by an ABA receptor different from the core ABA signaling pathway, and a putative mechanistic link connecting ABA and JA signaling pathways.

Figure 1. PYL6 interacts with MYC2 in yeast and the interaction is enhanced by ABA.

(A) Drop test assays show growth of yeast after 3 days in the presence of 10 μM ABA for the PYL6-MYC2 interaction pair and the positive control PYL6-ABI1 (presence of ABA (+ABA) is indicated on top of the panel; number of days of plate growth is indicated on the bottom). No interaction was found with the closest homologues of MYC2 (MYC3-PYL6) and PYL6 (MYC2-PYL5), suggesting a specific interaction. After 10 days of growth (right panel) PYL6-MYC2 interaction can be observed in the absence of ABA. (B) Using truncated versions of MYC2, the required region for MYC2-PYL6 interaction was mapped. Left side shows a cartoon with the different MYC2 truncated versions investigated in Y2H experiments. Only the truncated version MYC2 141–623 (amino acid numbers) was able to interact with PYL6. The enhancement by ABA was also observed for the truncated MYC2 (141–623). Negative controls with empty vector are shown in Figure S1.

Figure 2. MYC2 and PYL6 co-immunoprecipitate in planta.

N. benthamiana leaves were co-infiltrated with Flag-MYC2 and Venus-PYL6 or Venus alone. Protein extractions from leaves were split in 2 halves and either ABA-treated or mock-treated (indicated at the bottom of the panels). Immunoprecipitation was performed with anti-Flag beads against MYC2 (IP lanes indicated on the top right of each panel). (A) Left panel shows MYC2 detection with anti-Flag antibody suggesting low MYC2 expression levels before the purification. (B) Venus or Venus-PYL6 protein were detected using an anti-YFP antibody, and both can be observed in the input samples. Venus-PYL6, and not Venus alone, was co-immunoprecipitated with MYC2 (IP lanes).

Figure 3. PYL6 interacts with MYC2 in the nucleus.

(A,B) Subcellular localization of (A) PYL6 and (B) MYC2 tagged with Venus fluorescence protein. Venus-PYL6 was localized in the cytoplasm and nucleus and Venus-MYC2 was localized exclusively in the nucleus. (C,D) BiFC experiments show that (C) Yc-PYL6 interacts with Yn-ABI1 in the cytoplasm and nucleus of N. benthamiana leaves cells while (D) the interaction of Yc-PYL6 with Yn-MYC2 was only observed in the nucleus.

Figure 4. pyl6 mutant plants are more sensitive to JA + ABA.

pyl6 seedlings are more sensitive to ABA and to the combination of ABA and JA than WT (Col-0). (A) 5 days after sowing expanded and green cotyledons were quantified per number of plants analyzed (%). Data are represented as mean ± SD and **Indicates p-value <0.01 after a two-tailed t-test. Images of the seedlings at this stage can be found in Supplementary Figure S4. (B) In 11 day-old seedlings, the synergistic action of JA over the ABA effect was enhanced in pyl6 mutants compared to WT. Right panels show 0.5 μM ABA plus 10 μM Me-JA.

Figure 5. PYL6 modifies the transcriptional activity of MYC2 in yeast one hybrid (Y1H) analyses.

(A) MYC2 alone induced transcription driven by JAZ6 promoter. The presence of PYL6 and ABA reduced MYC2 transcriptional activity. (B) MYC2 alone did not induce transcription driven by JAZ8 promoter fragment. PYL6 strongly activates MYC2 transcription activity with a JAZ8 promoter fragment. Error bars indicate standard deviation of at least 3 independent transformants. ***Indicate the results of a two-tailed t-test with p values ≤0.001 and **Represent p value ≤0.01. BD indicates the empty vector where PYL6 was cloned.

Figure 6. Proposed working model.

Thick arrows indicate active transcription and dashed arrows weak transcription. Left: In the absence of ABA, PYL6 weakly interacts with MYC2, allowing the TF to bind a set of gene promoters such as JAZ6. When ABA is present, PYL6 strongly binds to MYC2 modifying its transcriptional activity, promoting the expression of JAZ8, whereas transcription via pJAZ6 is reduced. We propose that PYL6 acts as a transcriptional modulator upon interaction with MYC2.