Brassinosteroid-regulated bHLH transcription factor CESTA induces the gibberellin 2-oxidase GA2ox7

Abstract

Brassinosteroids (BRs) are plant steroids that have growth-promoting capacities, which are partly enabled by an ability to induce biosynthesis of gibberellins (GAs), a second class of plant hormones. In addition, BRs can also activate GA catabolism; here we show that in Arabidopsis (Arabidopsis thaliana) the basic helix-loop-helix transcription factor CESTA (CES) and its homologues BRASSINOSTEROID-ENHANCED EXPRESSION (BEE) 1 and 3 contribute to this activity. CES and the BEEs are BR-regulated at the transcriptional and posttranslational level and participate in different physiological processes, including vegetative and reproduction development, shade avoidance, and cold stress responses. We show that CES/BEEs can induce the expression of the class III GA 2-oxidase GA2ox7 and that this activity is increased by BRs. In BR signaling - and CES/BEE-deficient mutants, GA2ox7 expression decreased, yielding reduced levels of GA110, a product of GA2ox7 activity. In plants that over-express CES, GA2ox7 expression is hyper-responsive to BR, GA110 levels are elevated and amounts of bioactive GA are reduced. We provide evidence that CES directly binds to the GA2ox7 promoter and is activated by BRs, but can also act by BR-independent means. Based on these results, we propose a model for CES activity in GA catabolism where CES can be recruited for GA2ox7 induction not only by BR, but also by other factors.

Figure 1

BL increases GA2ox7 expression in a CES/BEE-dependent manner. A and B, Relative expression changes of GA2ox7 in bri1 (A) and ces (B) mutants as compared to wt. Plants were grown for 10 d on 1/2 MS medium without (c) or with 1 µM 24-epiBL (+BL) and GA2ox7 expression was analyzed by RT-qPCR. The means and standard deviations of three biological replicates measured in four technical repeats are shown. Statistical significance was determined with a Student’s t test and log2-fold changes are given. C, Heat-map showing the fold of relative expression changes in a set of GA biosynthetic and catabolizing genes in CES mutants as compared to wt following BL treatments. The experiment was performed as in A and B. The mean of three biological replicates measured in four technical repeats is shown (for bar diagrams with standard deviations and statistical significance tests, see Figure 1B;  Supplemental Figure S2). The color-code highlights fold expression changes (red: increase; blue: decrease).

Figure 2

GA₁₁₀ levels are altered in BR and CES mutants. Gas chromatography–mass spectrometry measurements of GA₁₁₀ and GA₄ in aerial parts of 10-d-old CES and BR mutants. The values are given in nanogram/gram fresh weight. The mean and standard deviations of three biological replicates is shown. Significant differences to wt were calculate with a Student’s t test and are indicated through underlining.

Figure 3

Aspects of CES mutant phenotypes can be rescued with GA. CES mutant plants and wt were grown either in LDs or SDs in soil and sprayed trice a week with 10 µM GA_4 + 7 (GA) or DMSO as a control, before different parameters were assessed. The bar charts show means and standard deviations of at least 10 plants. Statistically significant differences at P ≤ 0.01 are indicated with different letters and were determined with one-way ANOVA with a post-hoc Tukey HSD test. A and B, Flowering time of plants grown in LDs (A) or SDs (B). C, Rosette morphologies in SDs at 42 d after germination. Left: composite figure with photos of representative plants, showing top and side views. Right: quantification of rosette diameters. D, Number of secondary rosettes in SDs. The number of secondary rosettes formed by wt and all mutants, apart from ces-D, was counted at bolting. ces-D was evaluated at 100 d after germination, since it does not flower in SDs.

Figure 4

Emission ratios of nlsGPS1 in hypocotyls of ces-D as compared to wt. A, Three dimensional images of light-grown hypocotyls 3 d post sowing. nlsGPS1 in wt Col-0 was compared to two independent lines of nlsGPS1 in ces-D. Image processing and analysis were performed with IMARIS version 8.3.1. Surfaces were segmented based on the AxAm channel and with the background subtraction set to 3 μm and thresholding set as default. Three more hypocotyls from individual seedlings of each line are presented in Supplemental Figure S4A. B, Violin plots of nlsGPS1 emission ratios for nuclei of the measured region 60–700 µM from the shoot apical meristem. nlsGPS1 emission ratios were statistically different in ces-D compared to wt (Student’s t test ^****P < 0.0001 for wt versus ces-D). Violin plots from individual seedlings are presented in Supplemental Figure 4B. Complete experiments were repeated three times with similar results. C and D, Hypocotyls of 10-d-old light-grown plants used to determine nlsGPS1 reporter expression in wt and ces-D as in A and B. Violin plots of nlsGPS1 emission ratios for nuclei of the measured region 100–1,400 µM from the shoot apical meristem.

Figure 5

CES can directly bind to the GA2ox7 promoter. A, Illustration of the GA2ox7 promoter, to show regulatory motifs and the promoter regions that were tested in in vivo and in vitro DNA-binding studies. B, Chromatin immunoprecipitation assays using CES-YFP as a bait. Three-week-old 35S:CES-YFP (CES-YFP) and wt plants were sprayed with 10 μM 24-epiBL and CES-YFP was immuno-precipitated with anti-GFP beads. Enrichment of CES-YFP on the indicated fragments (C0, C1, C3, C8, and Cx) was determined by qPCR and calculating the ratio between samples with antibody and samples without antibody. TA3 was used for normalization. Fragment C0 served as an internal control for the experiment. Error bars show the standard error of four biological replicates measured in three technical repeats. Asterisks indicate significant differences in the fold enrichment of CES-YFP compared with the wt (t test, ^*P < 0.05, ^**P < 0.01). C, Electrophoretic mobility shift assays with products of DNA-binding reactions, that used recombinant, MBP-tagged CES (CES-MBP) and 100-bp fragments (P1–P8), that span a 716-bp region upstream of the ATG. Two concentrations of CES-MBP or no protein as a control were used.

Figure 6

BRI1 is required for the ces-D-facilitated BL-induction of GA2ox7. A, Root length of 10-d-old ces-Dxbri1-1 plants, grown for 10 d on 1/2 MS medium without (c) or with 1-µM 24-epiBL (+BL). Left: Means and standard deviations of 30 plants. Right: photos of representative plants. Bar = 1 cm. B, GA2ox7 expression in ces-Dxbri1-1 as compared to its parents and wt. Plants were grown for 10 d on 1/2 MS medium without (c) or with 1 µM 24-epiBL (+BL) and GA2ox7 expression was analyzed by RT-qPCR. The means and standard deviations of three biological replicates measured in four technical repeats are shown. Statistically significant difference at P ≤ 0.05 is indicated in both panels with different letters and was determined with one-way ANOVA with a Bonferroni post-hoc analysis.

Figure 7

A loss of GA2ox7 function yields BR hypersensitivity. A and B, ga2ox7 knock-out plants and wt were grown on 1/2 MS medium containing 0.5 μM epiBL (+BL) or DMSO as a control (c) and hypocotyl elongation (A) and root length (B) was measured 7 d after germination. C–E, ga2ox7 knockout plants and wt were grown in SDs and sprayed trice a week with DMSO as a control or with 10 µM epiBL and the rosette diameter at 9 weeks after germination (C) as well as the number of days (D) and leaves (E) until bolting were determined. The data for A–D stems from 10 to 20 plants; means and standard deviations are shown. Statistically significant difference at P ≤ 0.05 is indicated with different letters and was determined with one-way ANOVA with a Bonferroni post hoc analysis. F, Working model for BR activity in GA metabolism in A. thaliana. When an induction of GA biosynthesis is required, BRI1 induces BES1/BZR1 for increased GA3ox1 and GA20ox1 expression (a). This removes the DELLAs, promoting BES1/BZR1 and PIF4 activity on downstream targets (b). When GA catabolism is required, CES/BEE are activated to induce GA2ox7 expression (c). Since CES also induces CBF expression, and CBFs increase GA catabolism via activation of other types of GA2oxs, CBF induction could further feed into the response (d). Moreover, since CES/BEEs can also act by BRI1-independent means, additional stimuli may recruit the module for an inactivation of GAs.