Final Report Summary - CELLWALLSIGN (Genetic analysis of mechanisms linking cell wall integrity with growth control in Arabidopsis)
Project context and objectives
The high sugar response8 mutant altered in cell wall arabinose content displays a range of sugar hypersensitivity phenotypes (Li et al., 2008). Among these, dark grown hsr8 seedlings show reduced hypocotyl elongation in response to glucose in comparison to wild-type plants, and light-grown seedlings show increased sugar-regulated gene expression and anthocyanin content. To identify molecular components that may coordinate cell-wall composition and sugar responses, we screened for suppressors of the sugar hypersensitive hsr8 phenotypes. We isolated eight suppressors of hsr8 (soh), and the soh715 recessive mutant was selected for further analysis.
Because we used fast neutron mutagenesis, we used a transcript-based cloning approach to map the soh715 mutation. We compared gene expression in hsr8 and soh175 seedlings by using the ATH1 gene chip. The microarray analysis revealed that six consecutive genes on chromosomes 1 showed strongly reduced RNA levels in soh715 compared to hsr8. We complemented the soh715 mutant with six genomic fragments, each containing one gene in the deletion. Only the genomic fragment containing the At1g25540 gene, coding for the PFT1 protein, restored the hsr8 phenotypes. We crossed the hsr8 mutant with the insertion line pft1-2 and the hsr8pft1-2 double mutant displayed the same phenotypes than the soh715 mutant. Therefore, loss of PFT1 gene function suppresses hsr8 sugar hypersensitivity.
Project results
PFT1, PHYTOCHROME AND FLOWERING TIME 1, was identified as a positive regulator of flowering in response to suboptimal light conditions (Cerdan and Chory, 2003) and as an important key regulator of jasmonate signalling pathway (Kidd et al., 2009). We crossed the hsr8 mutant with phya, phyb and coi1-16 mutants and none of these mutations suppressed hsr8 dark development phenotype. We concluded that the pft1 mutation suppresses the hrs8 sugar hypersensitivity independently of the phya, phyb and jasmonate signalling pathways.
Because pft1 suppressed sugar hypersensitivity in the cell wall defective mutant hsr8, we wanted to know if pft1 could suppress sugar hypersensitivity in mutants that are sugar hypersensitive but have a normal cell-wall composition. To do so, we crossed pft1-2 mutant with two hsr mutants, hsr3 and hsr4, isolated from the same genetic screen as hsr8 (Baier et al., 2004). hsr3 and hsr4 display an enhanced expression of the APL3 gene in response to a sugar treatment in comparison to wild-type but are mutated in genes that are not related to cell-wall composition (Thodey et al., Ms submitted). The hsr3 and hsr4 mutants have significantly higher APL3 gene expression in comparison to wild-type and the pft1-2 mutation suppressed this higher expression. This result shows that pft1 suppresses sugar hypersensitivity in mutants that are not defective in their cell-wall composition. Therefore, pft1 seems to be involved in the sugar signalling pathway rather than in the cell-wall integrity pathway.
PFT1 has been shown to encode the subunit 25 of the Mediator Complex, a central regulator of transcription in eukaryotes (Backstrom et al., 2007). In order to assess PFT1 function in the sugar signalling pathway, we therefore chose to analyse gene expression in the pft1-2 mutant in response to a sugar treatment. We selected a set of 11 sugar-regulated genes from microarrays experiments previously published in the lab (Li et al., 2006). The sugar upregulated genes APL3, ?-Amylase, At1g32900, At1g61800, At4g33070, all involved in sugar primary metabolism, showed a lower transcript accumulation in the pft1-2 mutant compared to wild-type. Similarly, three genes, encoding enzymes of the anthocyanin synthesis pathway FLS, CHS and TT6, upregulated by a sugar treatment, showed lower expression levels in the pft1-2 mutant. Consistent with this down-regulation of expression, the levels of anthocyanin in response to sugar were decreased by 45% in the pft1-2 mutant compared to wild-type. Our results show that PFT1 is necessary for high expression level of several sugar induced genes, including genes of the anthocyanin pathway.
In order to have a broader view of PFT1 implication in the sugar signalling pathway, we performed a microarray experiment using the Affymetrix ATH1 Genome Array. Initially, we could confirm the results we obtained for the small set of sugar-regulated genes previously described; we could also show that an important number of genes involved in primary metabolism are affected in the pft1 mutant. PFT1 is a member of a large multi-protein complex. We tested if other subunits of the mediator complex could play a role in sugar signalling and growth. We found that a mutation in the subunit number 8 of the mediator complex also suppresses the short hypocotyl phenotype of hsr8, but the MED8 subunit does not play the same role in sugar signalling as PFT1.
Project outcome
A publication presenting all the results described above is currently in preparation and will be submitted in July in PNAS. In parallel to the results described in this report, we performed other experiments to unravel the function of PFT1 in transcription. Indeed, we developed a two-hybrid screen to isolate transcription factors interacting with PFT1 and also developed transgenic lines expressing a tagged version of the PFT1 protein to perform chromatin ImmunoPrecipitation.
The high sugar response8 mutant altered in cell wall arabinose content displays a range of sugar hypersensitivity phenotypes (Li et al., 2008). Among these, dark grown hsr8 seedlings show reduced hypocotyl elongation in response to glucose in comparison to wild-type plants, and light-grown seedlings show increased sugar-regulated gene expression and anthocyanin content. To identify molecular components that may coordinate cell-wall composition and sugar responses, we screened for suppressors of the sugar hypersensitive hsr8 phenotypes. We isolated eight suppressors of hsr8 (soh), and the soh715 recessive mutant was selected for further analysis.
Because we used fast neutron mutagenesis, we used a transcript-based cloning approach to map the soh715 mutation. We compared gene expression in hsr8 and soh175 seedlings by using the ATH1 gene chip. The microarray analysis revealed that six consecutive genes on chromosomes 1 showed strongly reduced RNA levels in soh715 compared to hsr8. We complemented the soh715 mutant with six genomic fragments, each containing one gene in the deletion. Only the genomic fragment containing the At1g25540 gene, coding for the PFT1 protein, restored the hsr8 phenotypes. We crossed the hsr8 mutant with the insertion line pft1-2 and the hsr8pft1-2 double mutant displayed the same phenotypes than the soh715 mutant. Therefore, loss of PFT1 gene function suppresses hsr8 sugar hypersensitivity.
Project results
PFT1, PHYTOCHROME AND FLOWERING TIME 1, was identified as a positive regulator of flowering in response to suboptimal light conditions (Cerdan and Chory, 2003) and as an important key regulator of jasmonate signalling pathway (Kidd et al., 2009). We crossed the hsr8 mutant with phya, phyb and coi1-16 mutants and none of these mutations suppressed hsr8 dark development phenotype. We concluded that the pft1 mutation suppresses the hrs8 sugar hypersensitivity independently of the phya, phyb and jasmonate signalling pathways.
Because pft1 suppressed sugar hypersensitivity in the cell wall defective mutant hsr8, we wanted to know if pft1 could suppress sugar hypersensitivity in mutants that are sugar hypersensitive but have a normal cell-wall composition. To do so, we crossed pft1-2 mutant with two hsr mutants, hsr3 and hsr4, isolated from the same genetic screen as hsr8 (Baier et al., 2004). hsr3 and hsr4 display an enhanced expression of the APL3 gene in response to a sugar treatment in comparison to wild-type but are mutated in genes that are not related to cell-wall composition (Thodey et al., Ms submitted). The hsr3 and hsr4 mutants have significantly higher APL3 gene expression in comparison to wild-type and the pft1-2 mutation suppressed this higher expression. This result shows that pft1 suppresses sugar hypersensitivity in mutants that are not defective in their cell-wall composition. Therefore, pft1 seems to be involved in the sugar signalling pathway rather than in the cell-wall integrity pathway.
PFT1 has been shown to encode the subunit 25 of the Mediator Complex, a central regulator of transcription in eukaryotes (Backstrom et al., 2007). In order to assess PFT1 function in the sugar signalling pathway, we therefore chose to analyse gene expression in the pft1-2 mutant in response to a sugar treatment. We selected a set of 11 sugar-regulated genes from microarrays experiments previously published in the lab (Li et al., 2006). The sugar upregulated genes APL3, ?-Amylase, At1g32900, At1g61800, At4g33070, all involved in sugar primary metabolism, showed a lower transcript accumulation in the pft1-2 mutant compared to wild-type. Similarly, three genes, encoding enzymes of the anthocyanin synthesis pathway FLS, CHS and TT6, upregulated by a sugar treatment, showed lower expression levels in the pft1-2 mutant. Consistent with this down-regulation of expression, the levels of anthocyanin in response to sugar were decreased by 45% in the pft1-2 mutant compared to wild-type. Our results show that PFT1 is necessary for high expression level of several sugar induced genes, including genes of the anthocyanin pathway.
In order to have a broader view of PFT1 implication in the sugar signalling pathway, we performed a microarray experiment using the Affymetrix ATH1 Genome Array. Initially, we could confirm the results we obtained for the small set of sugar-regulated genes previously described; we could also show that an important number of genes involved in primary metabolism are affected in the pft1 mutant. PFT1 is a member of a large multi-protein complex. We tested if other subunits of the mediator complex could play a role in sugar signalling and growth. We found that a mutation in the subunit number 8 of the mediator complex also suppresses the short hypocotyl phenotype of hsr8, but the MED8 subunit does not play the same role in sugar signalling as PFT1.
Project outcome
A publication presenting all the results described above is currently in preparation and will be submitted in July in PNAS. In parallel to the results described in this report, we performed other experiments to unravel the function of PFT1 in transcription. Indeed, we developed a two-hybrid screen to isolate transcription factors interacting with PFT1 and also developed transgenic lines expressing a tagged version of the PFT1 protein to perform chromatin ImmunoPrecipitation.