The Mediator complex subunits MED25/PFT1 and MED8 are required for transcriptional responses to changes in cell wall arabinose composition and glucose treatment in Arabidopsis thaliana

Abstract

Background: Plant cell walls are dynamic structures involved in all aspects of plant growth, environmental interactions and defense responses, and are the most abundant renewable source of carbon-containing polymers on the planet. To balance rigidity and extensibility, the composition and integrity of cell wall components need to be tightly regulated, for example during cell elongation.

Results: We show that mutations in the MED25/PFT1 and MED8 subunits of the Mediator transcription complex suppressed the sugar-hypersensitive hypocotyl elongation phenotype of the hsr8-1 mutant, which has cell wall defects due to arabinose deficiency that do not permit normal cell elongation. This suppression occurred independently of light and jasmonic acid (JA) signaling. Gene expression analyses revealed that the expression of genes induced in hsr8-1 that encode enzymes and proteins that are involved in cell expansion and cell wall strengthening is reduced in the pft1-2 mutant line, and the expression of genes encoding transcription factors involved in reducing hypocotyl cell elongation, genes encoding cell wall associated enzymes and proteins is up-regulated in pft1-2. PFT1 was also required for the expression of several glucose-induced genes, including those encoding cell wall components and enzymes, regulatory and enzymatic components of anthocyanin biosynthesis, and flavonoid and glucosinolate biosynthetic pathways.

Conclusions: These results establish that MED25 and MED8 subunits of the Mediator transcriptional complex are required for the transcriptional regulation of genes involved in cell elongation and cell wall composition in response to defective cell walls and in sugar- responsive gene expression.

Fig. 1

Identification of a suppressor of hsr8-1 sugar-hypersensitive hypocotyl elongation in the dark. a Image of sugar hypersensitive hypocotyl elongation of Col, hsr8-1 and hsr8-1soh715 grown on 1 % glucose on vertical plates in the dark. b Quantitative measurements of hypocotyl lengths of Col, hsr8-1 and hsr8-1soh715. Seedlings were grown vertically in the dark for 14 days on MS medium with 1 % Glucose. Errors bars represent SD (n > 30). ***, p < 0.001 comparing Col to hsr8-1 and hsr8-1 to soh715 hsr8-1 (Student’s t- test). Data shown is representative of three independent experiments. c Quantitative Real-time PCR analysis of β-Amylase mRNA levels in Col, hsr8-1 and the hsr8-1soh715 repressor in response to glucose. Seedlings were grown on MS medium supplemented with 0.5 % glucose in constant light. After 7 days, seedlings were transferred 24 h in a MS glucose-free liquid medium and then treated for 6 h with 3 % MS medium containing 3 % glucose. Errors bars represent SD from three biological replicates. Data shown is representative of three independent experiments. **, p < 0.01 comparing Col to hsr8-1; ***, p < 0.001 comparing hsr8-1 to soh715 hsr8-1 (Student’s t- test). Relative transcript levels (RTL) were calculated using transcript levels of the reference gene TUB6 (At5g12250). d Anthocyanin accumulation in response to glucose in Col, hsr8-1 and hsr8-1soh715. Seedlings were grown in continuous light for 7 days on MS medium containing 1 % glucose (solid bars) or 3 % glucose (dashed bars). Errors bars represent SD from three biological replicates. **, p < 0.01 comparing Col to hsr8-1; ***, p < 0.001 comparing hsr8-1 to soh715 hsr8-1 (Student’s t- test). Data shown is representative of two independent experiments

Fig. 2

hsr8-1 sugar hypersensitive phenotypes are suppressed by the pft1-2 mutation. a Identification by microarray analysis of a cluster of six genes that are down regulated in the suppressor line soh715hsr8-1 compared to hsr8-1. Values on the Y-axis are those obtained after normalization of the entire microarray data set. Dark grey bars and light grey bars represent values obtained for the hsr8-1 mutant and the hsr8-1soh715 suppressor line respectively. b Sugar hypersensitive dark development of Col, hsr8-1, soh715hsr8-1 and soh715hsr8-1 complemented with each of the 6 genes of the deletion. Seedlings were grown vertically in the dark for 14 days on MS medium containing 1 % Glucose. Only the genomic fragment containing the At1g25540 gene rescued the dark development phenotype. c Sugar hypersensitive dark development of Col, hsr8-1, the double mutant hsr8-1pft1-2 and pft1-2. Seedlings were grown as described in (B) above. d Quantitative Real-time PCR analysis of β-Amylase mRNA levels in Col, hsr8-1 and the double mutant hsr8-1pft1-2 in response to glucose. Seedlings were grown on MS medium supplemented with 0.5 % glucose in constant light. After 7 days, the seedlings were transferred for 24 h to MS glucose-free liquid medium and then treated for 6 h with MS medium containing 3 % glucose. Errors bars represent SD from three biological replicates. Data shown is representative of three independent experiments. **, p < 0.01 comparing Col to hsr8-1; ***, p < 0.001 comparing hsr8-1 to hsr8-1 pft1-2 (Student’s t- test). Relative transcript levels (RTL) were calculated using transcript levels of the reference gene TUB6 (At5g12250). e Anthocyanin accumulation in response to glucose in Col, hsr8-1 and the double mutant hsr8-1pft1-2. Seedlings were grown in continuous light for 7 days on MS medium containing 1 % glucose (solid bars) or 3 % glucose (dashed bars). Errors bars represent SD from three biological replicates. Data shown is representative of two independent experiments. **, p < 0.01 comparing Col to hsr8-1; ***, p < 0.001 comparing hsr8-1 to hsr8-1 pft1-1 (Student’s t- test). f Quantitative Real-time PCR analysis of the sugar-responsive APL3 gene mRNA levels in Col, hsr3, pft1-2hsr3, hsr4, pft1-2hsr4 and pft1-2 in response to glucose. Hsr3 and hsr4 are sugar-hypersensitive mutations in subunits of the ARP2/3 complex [18]. Seedlings were grown on MS medium supplemented with 0.5 % glucose in constant light. After 7 days, the seedlings were transferred to glucose-free liquid MS medium for 24 h and then treated for 6 h with MS medium containing either 0 % glucose (solid bars) or 3 % glucose (dashed bars). Errors bars represent SD from three biological replicates. **, p < 0.01 comparing Col to hsr3 and Col to hsr4; ***, p < 0.001 comparing hsr3 to hsr3 pft1-2 and hsr4 to hsr4 pft1-2 (Student’s t- test). Relative transcript levels (RTL) were calculated using transcript levels of the reference gene TUB6 (At5g12250)

Fig. 3

Comparison of cell elongation and cell wall composition in Col, hsr8-1, pft1-2 and hsr8-1pft1-2. a Hypocotyl cell length was measured from scanning electron micrograph images. n = 10 cells each from 5 hypocotyls. **, p < 0.01 comparing hsr8-1 to Col, and hsr8-1 to pft1-2 and hsr8-1 pft1-2 (Student’s t- test). b Monosaccharide composition of cell wall material isolated from 14 day old light grown seedlings (5 biological replicates). Fuc fucose; Rha rhamnose; Ara arabinose; Xyl xylose; Man mannose. . ***, p < 0.001 comparing arabinose levels in Col to hsr8-1, and pft1-2 to hsr8-1 pft1-2 (Student’s t- test). c Compositional analysis of cell wall material isolated from dark grown hypocotyl tissues using FTIR. The data are represented as differences in relative absorbance from wild-type Col. d Principal Components Analyses of FTIR data. Score loadings of PC1, PC2 and PC3 are plotted against the range of wavelengths to show the major variance

Fig. 4

PFT1 acts independently of phyA and phyB and the jasmonate response pathway in the suppression of the hsr8-1 hypocotyl elongation phenotype. a Sugar-hypersensitive dark development of Col, hsr8-1, hsr8-1pft1-2, phyA-201hsr8-1, phyB-1hsr8-1 mutants. Seedlings were grown vertically in the dark for 14 days on MS medium containing 1 % glucose. b Hypocotyl phenotypes of Col, hsr8-1, hsr8-1pft1-2, phyA-201hsr8-1, phyB-1hsr8-1 mutants grown in white light. Seedlings were grown 7 days on MS sugar free medium under constant white light. c Hypocotyl phenotypes in far-red light of Col, hsr8-1, hsr8-1pft1-2, phyA-201hsr8-1, phyB-1hsr8-1 mutants. Seedlings were grown 4 days on MS sugar free medium under constant far-red light. d Sugar hypersensitive hypocotyl elongation of Col, hsr8-1, coi1-16hsr8-1 mutants. Seedlings were grown as in (a)

Fig. 5

Microarray analyses of gene expression in Col, hsr8-1, pft1-2 and hsr8-1pft1-2 seedlings. a Venn diagram of glucose- induced genes in Col and pft1-2. b Hierarchical clustering of 19 genes showing no induction in response to glucose in pft1-2 compared to Col. c Hierarchical clustering of 28 genes showing reduced induction in response to glucose in pft1-2 compared to Col. d Hierarchical clustering of 15 genes that were down- regulated in hsr8-1 compared to Col, and up- regulated in hsr8-1pft1-2 compared to hsr8-1 in dark grown seedlings. These genes require PFT1 for repression in response to hsr8-1. e Hierarchical clustering of 14 genes that were up- regulated in hsr8-1 compared to Col, and down- regulated in hsr8-1pft1-2 compared to hsr8-1 in dark grown seedlings. These genes require PFT1 for induction in response to hsr8-1

Fig. 6

The MED8 subunit plays a role in sugar responsive growth and gene expression. a Sugar hypersensitive dark development of Col, hsr8-1, med8hsr8-1 and med8 mutants. Seedlings were grown vertically in the dark for 14 days on MS medium containing 1 % Glucose. b and c Quantitative Real-time PCR analysis of mRNA levels of cell wall modifying encoding genes PME17 and AtPME41 in Col, hsr8-1, med8hsr8-1 and med8. Seedlings are grown as described in (a) above. Errors bars represent SD from three biological replicates. **, p < 0.01 comparing Col to hsr8-1, and med8 hsr8-1 to med8 (Student’s t- test). Relative transcript levels (RTL) were calculated relative to the transcript level of the reference gene TUB6 (At5g12250). d to f Quantitative Real-time PCR analysis of BAM, APL3, and CHS mRNA levels in Col, med8, pft1-2 and the double mutant med8pft1-2 in response to glucose. Seedlings were grown on MS medium supplemented with 0.5 % glucose in constant light. After 7 days, the seedlings were transferred to glucose-free liquid MS medium for 24 h and then treated for 6 h with 3 % Glucose. Errors bars represent SD from three biological replicates. **, p < 0.01 comparing Col to med8 and pft1-2 to med8 pft1-2 (D); **, p < 0.01 comparing pft1-2 to med8 pft1-2 (E); **, p < 0.01 comparing Col to med8 and pft1-2 to med8 pft1-2 (F) (Student’s t- test). Relative transcript levels (RTL) were calculated using transcript levels of the reference gene TUB6 (At5g12250)