MYB21 and MYB24 Interact with DELLA Proteins
We fused MYB21 with LexA DNA binding domain (BD), and found that BD-MYB21 showed strong auto-activation in yeast (Supplemental Figure 1a). We further truncated MYB21 into MYB21NT containing R2R3 DNA binding domain and MYB21CT including NYWG/SM/VDDI/LWS/P motif (Figure 1a), and found that MYB21NT lost strong auto-activation (Supplemental Figure 1a). MYB21NT was used as bait to screen MYB21 interaction proteins in Arabidopsis cDNA library in Y2H system. The DELLA protein RGA is one of the putative interaction clones.
We further detected the interactions of MYB21NT with the five Arabidopsis DELLAs in Y2H system, and found that BD-fused MYB21NT interacted with activation domain (AD)-fused RGA, GAI, RGL1, RGL2, and RGL3 (Figure 1b). MYB21 and MYB24 are homologs with 67.7% identity at amino acid level [52]. We next investigated whether MYB24 can also interact with DELLAs. BD-fused MYB24 showed strong auto-activation in yeast (Supplemental Figure 1b). We truncated MYB24 into MYB24NT (Figure 1a), which did not show auto-activation (Supplemental Figure 1b). As shown in Figure 1b, MYB24NT also interacted with RGA, GAI, RGL1, RGL2, and RGL3 in yeast.
Full-length of DELLAs with BD domain showed strong auto-activation in yeast, therefore, we truncated DELLAs into L and R domains (RGA-L and RGL2-L; RGA-R, GAI-R, RGL1-R, RGL2-R, and RGL3-R) (Figure 1c), and R domains lost strong auto-activation. MYB21 and MYB24 were fused with AD domain, respectively. The Y2H results in Figure 1d showed that RGA-R, GAI-R, RGL1-R, RGL2-R, and RGL3-R interact with MYB21 and MYB24 respectively in yeast.
We next performed pull-down assay to verify the interactions of MYB21 and MYB24 with DELLAs in vitro. Maltose binding protein (MBP), MBP-fused MYB21 (MBP-MYB21) and MYB24 (MBP-MYB24) were expressed in Escherichia coli and purified by amylose resin. DELLA proteins RGA, RGL1 and RGL2 were extracted from transgenic Arabidopsis expressing tandem affinity purification (TAP) tag-fused DELLA proteins (TAP-RGA, TAP-RGL1 and TAP-RGL2) [41]. MBP, MBP-MYB21 and MBP-MYB24 were incubated with TAP-RGA extracts expressing TAP-RGA, and then separated on SDS-page for immunoblotting with anti-c-myc antibody. The result showed that MBP-MYB21 and MBP-MYB24 could efficiently pull down TAP-RGA, but the negative control MBP could not (Figure 1e), indicating that RGA interacts with MYB21 and MYB24. In addition, we also observed that DELLA proteins RGL1 and RGL2 interact with MYB21 and MYB24 in pull -down assay (Figures 1f and 1g).
Taken together, the Y2H assay, and pull-down assay consistently demonstrate that DELLAs interact with the R2R3 MYB transcription factors (MYB21 and MYB24), implying that these two transcription factors function as direct targets of DELLA proteins.
R2R3 Domains of MYB21 and MYB24 Are Involved in Interactions with DELLAs
We further investigated interactions of DELLA R fragments with MYB21NT, MYB24NT, MYB21CT, and MYB24CT. As shown in Figure 1d, RGA-R, GAI-R, RGL1-R, RGL2-R, and RGL3-R interacted with MYB21NT and MYB24NT, but not with MYB21CT and MYB24CT, indicating that DELLAs interact with R2R3 DNA binding domains of MYB21 and MYB24.
We also examined whether L or R domains of DELLA proteins interact with MYB21NT and MYB24NT. Y2H results suggested that MYB21NT and MYB24NT interact with both L parts and R parts of RGA and RGL2 (Figure 1b), indicating that both N-terminus and C-terminus of DELLAs interact with MYB21 and MYB24.
DELLA and JAZ Proteins Synergistically Inhibit Transcriptional Function of MYB21 and MYB24
Having shown that DELLAs interact with MYB21 and MYB24, we then performed an Arabidopsis protoplast transient expression assay [53] using the GAL4 DNA binding domain (GAL4DB) and its binding sites [GAL4(4X)-D1-3(4X)] to test whether DELLAs could influence the transcriptional function of MYB21 and MYB24.
MYB21 and MYB24 were respectively fused with GAL4DB vector and served as effectors. The reporter was the GUS (β-glucuronidase) gene controlled by four copies of GAL4 DNA binding site [GAL4(4x)-D1-3(4x)], and the internal control was 35S promoter-driven firefly luciferase (LUC) gene (Figure 2a). The DELLA genes RGA and RGL2 were respectively cloned into pGreen62 vector (Figure 2a). As shown in Figure 2b, expression of GAL4DB-MYB21 together with the GUS reporter could enhance the GUS/LUC ratio, while coexpression of RGA or RGL2 with GAL4DB-MYB21 repressed the transcriptional function of MYB21. We also observed that RGA and RGL2 inhibit the transcriptional function of MYB24 (Figure 2c).
As both JAZs and DELLAs interact with MYB21 and MYB24 (Figure 1) [32], and DELLAs could repress these two factors transcriptional function (Figures 2b and 2c), we further investigated whether JAZs or DELLAs and JAZs synergistically regulate the transcriptional function of MYB21 and MYB24. We used MYB21 as the representative and found that RGL2 or JAZ1 alone repressed the transcriptional activity of MYB21 (Figure 2e). We further discovered that coexpression of RGL2 and JAZ1 exhibited a much stronger inhibition of the transcriptional activity of MYB21 compared with RGL2 or JAZ1 alone (Figure 2e). These results revealed that DELLAs and JAZs coordinately repress the transcription activity of MYB21.
We also explored a cell-free assay system [54] to discuss whether the MYB21 protein level is regulated by GA or JA through analysis of N. benthamiana leaves transiently expressed myc-taged MYB21 (myc-MYB21). As the results shown in Figures 2f and 2h, the myc-MYB21 protein level was decreased to ~33% after 2 hours treatment with mock, while JA treatment promoted the degradation of myc-MYB21 protein (~24% of the myc-MYB21 level without treatment). However, GA could delay the degradation of myc-fused MYB21 (~46% of the myc-MYB21 level without treatment) (Figures 2g and 2i). These results indicated that GA and JA may play an opposite role in regulating the stability of MYB21 protein.
DELLAs and JAZs Converge on MYB21 and MYB24 to Regulate Filament Elongation
As both JAZs and DELLAs target MYB21 and MYB24, we further explore the cross-talk of JA and GA in the regulation of filament elongation.
Filaments in JA-deficient mutant opr3 at floral stage 13 are much shorter than that in wild type, indicating that stabilized JAZ proteins attenuate the function of MYB21 and MYB24 to repress filament elongation, and JA treatment could restore the filament elongation of opr3 (Figures 3a and 3b). Previous studies showed that GA could induce degradation of DELLA proteins [46, 55]. We tested whether GA treatment could release DELLA-targeted MYB21 and MYB24 to enhance filament growth in opr3. We further measured the ratio of filament to pistil length of opr3 treated with GA. As shown in Figures 3a and 3b, GA treatment could only very slightly rescue the filament/pistil ratio of opr3, indicating that majority of MYB21 and MYB24 are inhibited by JAZs in opr3, and that the released MYB21 and MYB24 by GA treatment were not enough to rescue the filament elongation of opr3, whereas treatment with both JA and GA recovered filament elongation of opr3 (Figures 3a and 3b).
RGL2 protein alone could repress the function of MYB21 and MYB24 to control filament elongation in Q3 (ga1-3 gai-t6 rgl1-1 rga-t2, wild type for RGL2 and RGL3) [52]. Application of GA could induce RGL2 degradation and restored the filament elongation of Q3 (Figures 3c and 3d). We further explored whether degradation of JAZs by JA treatment could release enough MYB21 and MYB24 to rescue the filament growth of Q3. As shown in Figures 3c and 3d, JA treatment could very weakly recover the ratio of filament and pistil length of Q3, indicating that RGL2 alone in Q3 is able to dominantly inhibit the function of MYB21 and MYB24 to repress the filament elongation. Exogenous application of both GA and JA could also restore the filament elongation of Q3 (Figures 3c and 3d).