Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis

Abstract

The phytohormones, brassinosteroids (BRs), play important roles in regulating cell elongation and cell size, and BR-related mutants in Arabidopsis display significant dwarf phenotypes. Cellulose is a biopolymer which has a major contribution to cell wall formation during cell expansion and elongation. However, whether BRs regulate cellulose synthesis, and if so, what the underlying mechanism of cell elongation induced by BRs is, is unknown. The content of cellulose and the expression levels of the cellulose synthase genes (CESAs) was measured in BR-related mutants and their wild-type counterpart. The chromatin immunoprecipitation (CHIP) experiments and genetic analysis were used to demonstrate that BRs regulate CESA genes. It was found here that the BR-deficient or BR-perceptional mutants contain less cellulose than the wild type. The expression of CESA genes, especially those related to primary cell wall synthesis, was reduced in det2-1 and bri1-301, and was only inducible by BRs in the BR-deficient mutant det2-1. CHIP experiments show that the BR-activated transcription factor BES1 can associate with upstream elements of most CESA genes particularly those related with the primary cell wall. Furthermore, over-expression of the BR receptor BRI1 in CESA1, 3, and 6 mutants can only partially rescue the dwarf phenotypes. Our findings provide potential insights into the mechanism that BRs regulate cellulose synthesis to accomplish the cell elongation process in plant development.

Fig. 1.

BRs regulate biomass accumulation in aerial parts of Arabidopsis. (A) Biomass accumulation in aerial parts of BRs mutants at different developmental stages: I, having nine rosette leaves, the stage with vigorous vegetative growth; II, initiation of bolting; III, having one primary inflorescence with four nodes; IV, having three side shoots on the primary inflorescence stalk; V, having two or more secondary inflorescences, the end of plant growth. (B) Cellulose content in primary inflorescence stems of BR-related mutants at stages V. Data represent means (±SE) of four independent experiments. The asterisks indicate significant levels of *P <0.05 and **P <0.01, respectively.

Fig. 2.

The exogenously applied epiBL induces expression of CESA genes in Arabidopsis. These CESA genes include CESA1, at4g32410; CESA2, at4g39350; CESA3, at5g05170; CESA4, at5g44030; CESA5, at5g09870; CESA6, at5g64740; CESA7, at5g17420; CESA8, at4g18780; CESA9, at2g21770; and CESA10, at2g25540. (A) Relative expression levels of CESA genes in BR-deficient and BR-insensitive mutants. Quantitative real-time RT-PCR was conduced with total RNA from 9-d-old light-grown shoots with or without the treatment of 5 μM epiBL for 2 h. (B) Relative expression levels of CESA genes in the BRI1-GFP line. (C) Expression levels of CESA4, 7, and 8 in the first internode of primary inflorescence stems of the BRI1-GFP line. Quantitative real-time RT-PCR was performed using total RNAs from 9-d-old light-grown seedlings.

Fig. 3.

The GUS staining in transgenic seedlings with CESA promoter-driven GUS reporters is enhanced on the medium containing epiBL. The expression pattern and regulation by BRs of CESA genes are shown by GUS-staining of CESA promoter::GUS reporters in 5-d-old light-grown seedlings on the 1/2 MS medium (A–G), or on the 1/2 MS medium containing 10 nm epiBL (H–N).

Fig. 4.

BES1 can associate with the promoter regions of most CESA genes. The BES1 antibody and the GFP antibody (as a negative control) were used to immunoprecipitate chromatin prepared from 14-d-old light-grown Col-0 seedlings. Quantitative real-time PCR was performed using primers from the indicated positions of CESA promoters. The fold changes were calculated based on the change for anti-BES1 relative to anti-GFP, after normalization to 5S rRNA, an internal control. (A) CESA1; (B) CESA2; (C) CESA3; (D) CESA4; (E) CESA5; (F) CESA6; (G) CESA8; (H) CESA9; (I) CESA10; (J) the positive control at3g23770.

Fig. 5.

The CESA mutant enhances the dwarf phenotype of bri1-301, and over-expression of BRI1 in CESA mutants cannot completely rescue their dwarf phenotype. (A) Col-0; (B) the BR receptor mutant bri1-301; (C) the BRI1-GFP over-expression line; (D) rsw1-1, a CESA1 mutant (Ala549Val); (E) rsw1-1 bri1-301 double mutant; (F) the over-expression line of BRI1-GFP in rsw1-1; (G) ixr1-1, a CESA3 mutant (Gly998Asp); (H) ixr1-1 bri1-301 double mutant; (I) the over-expression line of BRI1-GFP in ixr1-1; (J) prc1-1, a CESA6 mutant (Q720STOP); (K) prc1-1 bri1-301 double mutant; (L) the over-expression line of BRI1-GFP in prc1-1. (This figure is available in colour at JXB online.)

Fig. 6.

CESA mutants show an altered BR response. (A) Hypocotyl length of 7-d-old light-grown seedlings of rsw1-1, CT-2 (SALK_091570), ixr1-1, CT-5 (SALK_023353), prc1-1, ixr2-1,CT-9 (SALK_049129), Col-0, and det2-1 grown on the 1/2 MS medium with or without 10 nM epiBL. Means and standard deviations were calculated from 30–45 seedlings. (B) The relative value of hypocotyl elongation of 4-d-old dark-grown seedlings in the absence or presence of 10 nM epiBL. (C) The fold changes of CESA2 and CESA5 expression in the light, compared to that in the dark. Quantitative real-time PCR was performed using 7-d-old light-grown seedlings and 4-d-old dark-grown seedlings of prc1-1 and the wild type.

Fig. 7.

A model to illustrate the mechanism of BR-regulated CESA expression in plant growth. The BR signalling activates the transcription factor BES1 and promotes its binding to the E-boxes of CESA1, CESA3, and CESA6 promoters to enhance their expression during the primary growth. BES1 can also promote the expression of CESA4 and CESA8 for the secondary growth. These CESAs provide more cellulose to sustain the architecture of enlarged cells in cell elongation. BR signalling can regulate the expression of CESA2, CESA5, or CESA9 and CESA10, which partially substituted for the function of CESA6 and rescued the dwarf phenotype of prc1-1 in the light.