The DELLA proteins interact with MYB21 and MYB24 to regulate filament elongation in Arabidopsis

Abstract

Background: Gibberellin (GA) and jasmonate (JA) are two essential phytohormones for filament elongation in Arabidopsis. GA and JA trigger degradation of DELLAs and JASMONATE ZIM-domain (JAZ) proteins through SCF^SLY1 and SCF^COI1 separately to activate filament elongation. In JA pathway, JAZs interact with MYB21 and MYB24 to control filament elongation. However, little is known how DELLAs regulate filament elongation.

Results: Here we showed that DELLAs interact with MYB21 and MYB24, and that R2R3 domains of MYB21 and MYB24 are responsible for interaction with DELLAs. Furthermore, we demonstrated that DELLA and JAZ proteins coordinately repress the transcriptional function of MYB21 and MYB24 to inhibit filament elongation.

Conclusion: We discovered that DELLAs interact with MYB21 and MYB24, and that DELLAs and JAZs attenuate the transcriptional function of MYB21 and MYB24 to control filament elongation. This study reveals a novel cross-talk mechanism of GA and JA in the regulation of filament elongation in Arabidopsis.

Keywords: Filament elongation; Gibberellin; Jasmonate; MYB21; MYB24.

Fig. 1

Interactions of DELLAs with MYB21 and MYB24. a Schematic diagram of MYB21 and MYB24 domain constructs. The conserved R2R3 domain, and the NYW^G/_S^M/_VDD^I/_LW^S/_P motif are indicated by yellow and blue respectively. The numbers indicate positions of the first and the last amino acid of the domain constructs. b Yeast Two-Hybrid (Y2H) assay to detect interactions of MYB21NT and MYB24NT with DELLAs and their derivatives. MYB21NT and MYB24NT were individually fused with the LexA DNA binding domain (BD) in pLexA. DELLAs and their derivatives were individually fused with the activation domain (AD) in pB42AD. Interactions of MYB21NT and MYB24NT with the AD domain in pB42AD empty vector were used as negative controls. Interactions (represented by blue color) were assessed on 2% Gal/1% raffinose/SD/−Ura/−His/−Trp/−Leu/X-β-Gal medium. c Schematic diagram of DELLAs domain constructs. The R part of DELLAs contain the conserved DELLA domain (green). The numbers indicate positions of the first and the last amino acid of the domain constructs. d Y2H assay to detect interactions of R domains of DELLAs with MYB21, MYB24, and their derivatives. R domains of DELLAs were individually fused with BD domain in pLexA. MYB21, MYB24 and their derivatives were individually fused with the AD domain in pB42AD. Interactions of R domains of DELLAs with the AD domain in pB42AD empty vector were used as negative controls. Interactions were assessed on 2% Gal/1% raffinose/SD/−Ura/−His/−Trp/−Leu/X-β-Gal medium. e-g In vitro pull-down assay to detect the interactions of RGA (e), RGL1 (f), RGL2 (g) with MYB21 and MYB24. Purified MBP, MBP-MYB21, and MBP-MYB24 fusion proteins were incubated with the TAP-RGA, TAP-RGL1, or TAP-RGL2 expressed in TAP-RGA, TAP-RGL1, or TAP-RGL2 transgenic Arabidopsis plants. Bound proteins were washed, separated on SDS-PAGE, and immunoblotted with the anti-c-myc antibody (α-myc). The input lane shows the expression level of TAP-RGA, TAP-RGL1, or TAP-RGL2 expressed in TAP-RGA, TAP-RGL1, or TAP-RGL2 transgenic plants. The positions of purified MBP, MBP-MYB21 and MBP-MYB24 on SDS-PAGE are indicated with asterisks (stained by Coomassie blue). The original data can be viewed from Additional file 4: Figure S3a-d

Fig. 2

DELLAs and JAZs coordinately inhibit the transcriptional function of MYB21. a The schematic diagram shows the constructs used in the transient expression assays of b and c. b and c Transient expression assays show that RGA and RGL2 inhibit transcriptional function of MYB21 b and MYB24 c. The GUS reporter and the internal control luciferase (LUC) were cotransformed with the indicated constructs. Data are means (±SE) of three biological replicates. Asterisks represent Student’s t-test significance compared with the MYB21 (**, P < 0.01). d Schematic diagram of the constructs used in transient expression assays in e. e Transient expression assays show that both RGL2 and JAZ1 proteins could significantly repress transcriptional function of MYB21. Data are means (±SE) of three biological replicates. Asterisks represent Student’s t-test significance between pairs indicated with brackets (*, P < 0.05; **, P < 0.01). f and g Effects of jasmonate and gibberellin on the protein stability of MYB21. MYB21 (myc-MYB21) transiently expressed in N. benthamiana was extracted from tobacco leaves, and then incubated without (Mock) or with methyl jasmonate (JA) f, gibberellin (GA) g, for the indicated time (hours). Then myc-MYB21 was detected by immunoblot using anti-c-myc antibody. The PVDF membrane was stained with Memstain to serve as loading control. The original data can be viewed from Additional file 4: Figure S3e-f. h and i Quantitative analysis of myc-MYB21 protein level in f and g. Data are means (±SE) of three biological replicates. Asterisks represent Student’s t-test significance between pairs indicated with brackets (*, P < 0.05; **, P < 0.01). The abundance of myc-MYB21 with mock treatment for 0 h was set to 1.0

Fig. 3

DELLAs and JAZs converge on MYB21 and MYB24 to regulate filament elongation. a Comparison of flowers at floral stage 13 in Col-0 and opr3 treated without (Mock) or with methyl jasmonate (JA), gibberellin (GA), or JA plus GA for the indicated concentration. b The ratio of filament length and pistil length at floral stage 13 in the indicated genotypes. Data are means (±SE) of three biological replicates. c Comparison of flowers at floral stage 13 in Landsberg erecta (Ler) wild-type and ga1–3 gai-t6 rga-t2 rgl1–1 (Q3) treated without (Mock) or with methyl jasmonate (JA), gibberellin (GA), or JA plus GA for the indicated concentration. d The ratio of filament length and pistil length at floral stage 13 in the indicated genotypes. Data are means (±SE) of three biological replicates. e A simplified model for the crosstalk between jasmonate and gibberellin in regulating filament elongation. JAZs interact with and inhibit the transcriptional function of MYB21 and MYB24 to suppress filament elongation (Song et al. 2011; Qi et al. 2015). DELLAs inhibit the expression of JA-biosynthesis gene DAD1 and LOX1 (Cheng et al. 2009), and as well as interact with and attenuate MYB21 and MYB24 to inactivate downstream genes and repress filament elongation. JA and GA signal respectively induce degradation of JAZs and DELLAs to derepress MYB21 and MYB24, and synergistically modulate filament elongation in Arabidopsis